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| Nusantara Biosci | vol. 9 | no. 3 | pp.

 237‐337 | August 2017|
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Mungbean flowers photo  by  Dinesh  Valke 
 

 
| Nusantara Biosci | vol. 9 | no. 2 | pp. 111‐235 |May 2017| 
|ISSN 2087‐3948| E‐ISSN 2087‐3956| 
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N U SAN TA RA B IOSC IE NC E ISSN: 2087-3948
Vol. 9, No. 3, pp. 237-242 E-ISSN: 2087-3956
August 2017 DOI: 10.13057/nusbiosci/n090301

Characterization and clustering of agronomic characters of several


soybean genotypes

M. MUCHLISH ADIE♥, AYDA KRISNAWATI♥♥


Indonesian Legumes and Tuber Crops Research Institutes, Jl. Raya Kendalpayak Km 8, Malang 65101, East Java, Indonesia. Tel./Fax. +62-341-
801468/+62-341-801496, ♥email: mm_adie@yahoo.com, ♥♥email: my_ayda@yahoo.com.

Manuscript received: 17 November 2016. Revision accepted: 5 June 2017.

Abstract. Adie MM, Krisnawati A. 2016. Characterization and clustering of agronomic characters of several soybean genotypes.
Nusantara Bioscience 9: 237-242. The development of improved soybean variety in Indonesia is mostly derived from crossing
(51.19%), which requires the availability of gene source to serve as the parental stock. Characterization of 150 soybean genotypes was
conducted in Probolinggo, Indonesia, from February to May 2016. The research was arranged in a randomized block design with two
replicates. Clustering of agronomic characters was carried out using cluster analysis. The grouping was based on plant age characters
(vegetative phase duration, generative phase duration, generative and vegetative ratio, days to maturity), growth characters (plant height,
number of filled pods, number of empty pods, number of branches, number of nodes), and seed characters (100-seed weight and seed
yield plant-1), which resulted in 10 groups/clusters. Cluster I, II and III consisted of 47, 80, and 13 genotypes, respectively; whereas
other clusters consisted of one up three genotypes, respectively. Each cluster represents specific characters. Cluster IV that consisted of
three genotypes showed a late maturity (83.5-88.5 days), high number of pods (81.8-83.3 pods plant-1) and high yield plant-1 (21.76-
25.78 g plant-1), but have small seed size (12.24-13.75 g 100 seed-1). Cluster VI consisted of one genotype, and characterized by large
seed size (16.79 g 100 seed-1) and high yield plant-1 (15.76 g). Cluster IX (1 genotype) was characterized by early maturity (73 days),
unbranched, but produced relatively low yield. The preference of soybean consumer in Indonesia is high yield, in addition to early
maturity (< 80 days), and large seed size (>14 g 100 seed-1). Soybean genotypes within cluster IV, VI, and IX are valuable as gene
source in the development of superior soybean varieties in Indonesia.

Keywords: Agronomic character, cluster, seed yield

INTRODUCTION increase resistance to diseases, pests, and stresses imposed


by natural environments.
High yielding varieties, as a component of cultivation The seed yield of soybean, which consists of several
technologies, are still become the most preferred and most components, including the number of plants per unit area,
rapidly adopted by users. In addition to high yield, pods number per plant, seeds per pod, seed number and
development of soybean varieties in Indonesia is intended seed size, depends mainly on the soybean genotype used
to produce varieties with general and specific objectives. (Sumarno and Zuraida 2006; Susanto and Adie 2006;
The general soybean breeding objectives are to develop Showkat and Tyagi 2010). Genetic variability can be
large seed size and early maturing soybean varieties, while analyzed using various methods such as agronomic and
the specific objectives are to generate soybean varieties biochemical traits, and molecular marker polymorphisms.
with good adaptation to abiotic stress, biotic stress, and Valliyodan et al. (2015) performed a landscape analysis of
high nutrients content (protein, fat, isoflavones, etc.). genomic diversity and trait discovery of 106 soybean
The development of improved variety requires the genomes representing the wild, landraces, and elite lines,
availability and diversity of genetic resources. Moreover, and obtained 10 million high-quality SNPs. This approach
the Indonesian government has released as many as 84 is very useful in the genomic aspects. Information from this
soybean varieties, of which 51.19% were generated study provides a valuable resource for understanding
through crossbreeding processes. This indicates that soybean genome structure and evolution, and can also
genetic diversity plays an important role as gene sources in facilitate trait dissection leading to sequencing-based
varietal development. Efforts to broaden the genetic molecular breeding. Characterization of several soybean
diversity can be achieved through the characterization of genotypes in terms of abiotic stresses revealed PI 416937
soybean genotypes from various sources. In the USA, more as a drought-tolerant genotype. The drought-tolerant
than 70% released variety was originated from seven genotype was reported to be able to maintain its leaf
crossbreeding using nine unique parents (Chen et al. 2004). turgidity as a result of efficient extraction of soil water by a
These genetic resources play a major role in soybean larger and fibrous root system (Patterson and Hudak 1996).
improvement and serve as the basis for the introduction of Hao et al. (2012) performed a genome-wide association
new genes to improve productivity, crop quality, and to analysis detecting significant single nucleotide
polymorphisms for chlorophyll and chlorophyll
238 N U S A N T A R A B I O S C I E N C E 9 (3): 237-242, August 2017

fluorescence parameters in soybean (Glycine max) filled pods, number of empty pods, number of branches,
landraces. The previous study by Vollmann (2000) showed number of nodes, 100 seed weight, and seed yield plant-1.
that selection of early maturing soybean genotypes with The vegetative phase duration was the period started from
improved seed protein content appears to be feasible and is the seed emergence until the opening of the first complete
only limited by the moderately negative correlation flower while the generative phase duration was the period
between protein content and seed yield. started from the emergence of the first complete flower
A previous study by Navabi et al. (2014) on the genetic until pod maturation.
diversity in dry bean varieties released between 1930 and
2010 revealed that the pedigree-based estimate of genetic Data analysis
diversity for all dry bean varieties was 0.93. This result Data were analyzed using an ANOVA for a randomized
indicated a narrow genetic diversity among the Canadian block design (Gomez and Gomez 1984). Accessions
beans of Andean origin (kidney and cranberry beans) diversity was determined by Principal Component Analysis
suggesting that the breeding efforts for these market classes (PCA) to identify the principal traits. Furthermore, the PCA
would benefit from the introduction of new genetic values were used for cluster analysis. PCA and cluster
diversity. In kidney bean (Phaseolus vulgaris L.), Khanal analysis were performed using Minitab 14 program
et al. (2015) obtained an accession named Dynasty, with (McKenzie and Goldman 2005).
the characteristic as a mid to late season maturity cultivar
with excellent yield potential, superior seed size, and good
cooking quality. RESULTS AND DISCUSSION
Genetic diversity is important for crop improvement
(Malek et al. 2014). Variations among agronomic and Result
yield-related characters in soybean offer the most effective Analysis of variance showed significant
selection for particular characters. The objectives of the variation among the 150 soybean genotypes. The CV
present study were to characterize and classify 150 soybean values ranged from 3.96 to 23.61% (Table 1). This
genotypes based on agronomic characters. indicates that there were variations in particular agronomic
characters, thus allowing us to perform grouping of the
genotypes based on their agronomic characters.
MATERIALS AND METHODS The evaluated 150 soybean genotypes were grouped
into characters associated with plant duration (vegetative
Research material phase duration, generative phase duration, generative and
A total of 150 soybean genotypes which consisted of vegetative ratio, days to maturity), growth characters (plant
145 soybean genotypes and five soybean popular varieties height, number of filled pods, number of empty pods,
in Indonesia (Burangrang, Argomulyo, Grobogan, number of branches, number of nodes), and seed characters
Anjasmoro, and Dega 1) were used in this study. These (100 seed weight, seed yield plant-1) (Table 2). The average
soybean genotypes were derived from a selection of vegetative phase duration was 32.54 days, generative phase
various soybean crossing with different genetic duration was 45.01 days, generative and vegetative ratio
background. was 1.39, days to maturity was 77.5 days, plant height was
48.31 cm, number of filled pods plant-1 was 42.65 pods,
Field research number of empty pods plant-1 was 2.06 pods, number of
The experiment was conducted in dryland in branches plant-1 was 2.38, number of nodes plant-1 was
Probolinggo (East Java, Indonesia) during the dry season 10.07, 100 seed weight was 15.25 g, and seed dry weight
(February-May 2016) at an elevation of 10 m asl, climate plant-1 was 13.41 g. Skewness value for all characters was
type C2 based on Oldeman system, and Alfisol soil type. positive, except for number of branches, which indicates
The experiment was arranged in randomized completely that most of the tested genotypes were centralized on the
block design, with 150 treatments and two replicates. Each right side, thus have an opportunity to perform better than
genotype was planted in 1.2 m × 4.5 m, plant spacing was the average value. On the contrary, most genotypes tended
40 cm × 15 cm, two plants hill-1. The soil management was to have relatively a few number of branches.
optimally performed and drainage channel was made. The grouping of 150 soybean genotypes based on
Planting system was done using individually planted agronomic characters resulted in 10 clusters (Table 3,
(tugal) direct seeded, 2-3 seeds hole-1 and only two plants Figure 1). Cluster I consisted of 47 genotypes,
hole-1 were retained at 12 days after planting onward. characterized by the relatively short stem and large seed
Fertilizer of 250 kg Phonska ha-1 and 100 kg SP 36 ha-1 size. Cluster II, as the largest cluster members (80
was applied prior to sowing. Crop maintenance consisted genotypes), had characteristics of a short stem and a high
of optimal irrigation, pest and disease control, and number of nodes. Cluster III (13 genotypes) had a medium
weeding. stem and heavier seed per plant. Cluster IV (three
genotypes) had characteristics of long duration of the
Observation vegetative phase, late maturity, high number of filled pods
The observed data consisted of vegetative phase plant-1 and also high yield plant-1. Cluster V with only two
duration, generative phase duration, vegetative and genotype members had characteristics of long duration of
generative ratio, days to maturity, plant height, number of the generative phase, late maturity, and medium seed size.
ADIE & KRISNAWATI – Characterization and clustering soybean’s agronomic characters 239

Cluster VI (one genotype) was characterized by the long (one genotype) was characterized by the short duration of
duration of generative phase and large seed size. Cluster the vegetative phase, high G/V, very early days to maturity,
VII (one genotype) was characterized by a high number of and a few number of branches. The last cluster, cluster X
filled pods, a few empty pods, and high yield. Cluster VIII (one genotype) was characterized by a long stem and a high
(one genotype) had a low number of filled pods. Cluster IX number of nodes (Table 3).

Table 1. Analysis of variance of 150 soybean genotypes in 2016

Mean square
Observed variables CV (%)
Replication Genotype
Vegetative phase duration (V) (day) 74.00333** 9.08682** 5.25
*
Generative phase duration (G) (day) 19.76333 5.99644** 3.99
**
G/V ratio 0.05796 0.00974** 4.59
Days to maturity (day) 170.25333** 24.89360** 3.96
Plant height (cm) 38.30613ns 73.51535** 10.59
Number of filled pods plant-1 282.65813ns 238.28127** 21.83
Number of empty pods plant-1 0.05205ns 0.21088** 27.55
-1 ns
Number of branches plant 1.28053 1.13594** 27.77
Number of nodes plant-1 3.63000ns 2.01104** 9.35
**
100 seed weight (g) 18.72001 5.02741** 8.78
Seed weight plant-1 (g) 72.42253** 21.47234** 23.61
Note: ** = significant at 1% probability level (p<0.01), * = significant at 5% probability level (p<0.05).

Table 2. Descriptive statistic of 150 soybean genotypes in 2016

Observed variables Range Mean Std Skewness


Vegetative phase duration (V) (day) 29.00-38.50 32.54 2.131 0.407
Generative phase duration (G) (day) 42.00-51.50 45.01 1.731 1.784
G/V ratio 1.23-1.55 1.39 0.069 0.082
Days to maturity (day) 73.00-88.50 77.55 3.528 1.163
Plant height (cm) 33.40-67.80 48.31 6.063 0.268
Number of filled pods plant-1 22.00-83.30 42.65 10.915 1.222
Number of empty pods plant-1 0.20-5.40 2.06 1.039 0.684
Number of branches plant-1 0.50-3.90 2.38 0.754 -0.244
Number of nodes plant-1 7.50-14.30 10.07 1.003 0.452
100 seed weight (g) 11.59-21.23 15.25 1.585 0.708
Seed weight plant-1 (g) 6.48-25.78 13.41 3.277 0.882
Note: Std = standard deviation

Table 3. Grouping of 150 soybean genotypes based on agronomic characters in 2016

Cluster Number of Characteristic


genotypes
I 47 Short plant, large seed size
II 80 Short plant, high number of nodes
III 13 Medium plant height, heavy seed weight
IV 3 Long duration of vegetative phase, late maturity, high number of filled pods, heavy seed weight
V 2 Long duration of generative phase, late maturity, medium seed size
VI 1 Long duration of generative phase, large seed size
VII 1 High number of filled pods, a low number of empty pods, heavy seed weight
VIII 1 A low number of filled pods
IX 1 Short duration of vegetative phase, high G/V, early days to maturity, a few number of branches
X 1 High plant, high number of nodes
240 N U S A N T A R A B I O S C I E N C E 9 (3): 237-242, August 2017

Table 4. Range of agronomic characters of each cluster in 2016

Range
No. of
Vegetative Generative Days to Plant No. of No. of No. of
Cluster empty 100 seed Seed weight
phase phase G/V ratio maturity height filled pods branches nodes
-1 pods weight (g) plant-1 (g)
duration duration (day) (cm) plant plant-1 plant-1
plant-1
I 29.0-36.5 43.0-49.0 1.28-1.52 73.5-85.5 33.4-48.0 27.0-37.8 0.3-4.0 0.7-3.0 7.5-10.5 13.67-19.65 6.48-13.6
II 29.0-36.5 44.0-49.0 1.28-1.52 74.0-84.5 42.7-56.5 34.0-52.1 0.2-4.7 1.3-3.9 8.8-12.3 11.59-19.98 7.26-17.32
III 33.0-37.0 42.0-48.0 1.24-1.49 73.5-85.0 47.8-59.8 55.6-63.6 0.5-5.0 2.6-3.6 10.3-11.9 13.09-16.01 14.80-20.51
IV 36.0-38.5 47.5-51.0 1.27-1.36 83.5-88.5 50.4-56.9 81.8-83.3 0.5-2.6 3.5-3.7 11.2-12.5 13.42-14.78 21.76-25.78
V 36.0-37.5 51.0-51.5 1.36-1.43 87.5-88.5 48.5-48.6 55.0-59.7 0.8-1.6 2.9-3.5 10.1-10.9 12.24-13.75 15.03-16.54
VI 37.0 51.0 1.38 88.0 56.5 49.3 1.6 2.7 11.2 16.79 15.76
VII 34.0 48.0 1.42 82.0 56.0 71.9 0.3 3.4 11.1 13.48 21.53
VIII 35.5 47.5 1.34 83.0 53.6 27.8 1.1 1.1 10.0 15.28 9.87
IX 29.0 44.0 1.52 73.0 58.4 28.1 1.3 0.5 11.8 15.01 8.1
X 31.5 44.0 1.44 74.5 61.8 50.3 0.8 1.7 14.3 15.85 14.41

Similarity
50

40

30

20

10

Genotypes

Figure 1. Grouping 150 soybean genotypes based on agronomic characters

Discussion seven different clusters at similarity coefficient of 0.52 by


Plant age, growth, and seed characters of the 150 using Unweighted Pair Group Method with Arithmetic
soybean genotypes were genetically greatly varied. By Mean (UPGMA) (Moe 2012). By using cluster analysis,
using cluster analysis, the 150 soybean genotypes were Salimi et al. (2012) also successfully identified genotypes
grouped into 10 clusters using 11 morphological traits. TNH56 and BP which are drought tolerant, and these
Morphological characterization of the existing genetic genotypes could be used as a source of germplasm for
variation has been used in various researches. For example, breeding for drought tolerance.
Dayaman (2007) investigated 45 soybean accessions from In this study, 150 soybean genotypes were grouped into
different geographical areas and screened for their genetic 10 clusters. Cluster I, II, and III consisted of 47, 80, and 13
diversity using 22 morphological traits. The investigation genotypes, respectively; or about 93.33% of the total tested
revealed out the accessions into six clusters. Another study genotypes. Meanwhile, the other eight clusters consisted of
by Diazcarrasco et al. (1986) grouped seventeen soybean only one up to three genotypes each. This indicates that
varieties based on days to maturity, plant height, and seed some agronomic characters of the 150 genotypes have a
yield plant-1. Rasaily et al. (1986) characterized twenty narrow diversity. This finding is quite different from that of
soybean genotype characters based on plant height, number Malik et al. (2011), who found a high level of diversity on
of branches, pods plant-1 and seed yield plant-1. For agro- morphological traits (leaf area, pods plant-1, branches plant-
1
morphological traits, 94 accessions were grouped into , 100-seed weight and grain yield plant-1) on 92 soybean
ADIE & KRISNAWATI – Characterization and clustering soybean’s agronomic characters 241

genotypes evaluated in Pakistan. Kaga et al. (2012) also (83.5-88.5 days) and relatively small seeded-size, i.e.
found highly heterogeneous accessions and comprised of 12.24-13.75 g 100 seeds-1. On the contrary, cluster VI was
accessions from Nepal, Pakistan, Myanmar and China. characterized by days to maturity of 88 days but had large
These accessions generally flowered very late in the seed size, i.e. 16.79 g 100 seeds-1. This implies that days to
experimental field and had primitive characters such as maturity within those clusters was similar, except the seed
twining, elongated stem and small seeds. By using RAPDs, size. Those characters could serve as gene source for
Li and Nelson (2001) found that the mean genetic distance soybean's genetic improvement since studies on genetic
within accession from China was much larger than that diversity not only serve as a basis for understanding the
within Japan or South Korea, but smaller than that between genetic basis of soybean of different gene pools, but also to
China and Japan or South Korea. Meanwhile, Oda et al. help identify new sources of genes to increase the
(2015) evaluated the combination of six microsatellite productivity and quality of the soybean (Fu et al. 2007).
primers, which were able to distinguish the 21 cultivars The identified character for each cluster depends on the
used in that study; and those microsatellite markers showed genetic material used or is also determined by the origin of
less biased estimates compared to the estimates obtained by the gene source used. According to Peric et al. (2014), a
the parentage coefficient and phenotypic characters in great similarity was usually found, primarily between the
studies on genetic diversity. varieties come from the same institution because these
Study of genetic diversity is important to determine the varieties, generally, were developed from the same crosses.
genetic diversity of a population and to explore the superior In Indonesia, soybean was categorized as a secondary
character in order to support the development of improved crop. Hence, most of the cultivars were planted after paddy.
varieties. Similarly, Paterson et al. (1991) stated that In such condition, the plant age preferred by farmers is
evaluation of genetic diversity would promote the efficient early days to maturity (under 80 days). In addition,
use of genetic variations in the breeding program. Genetic soybeans with large seeded size (>14 g/100 seeds) are used
diversity and relationships among breeding materials are as raw material for tempeh. Thus, improvement of
also of essential information for a plant breeder to improve soybean’s genetic potency in Indonesia could use the
an efficient crop variety. The genetic diversity study is superior characters existed in each cluster identified in the
important not only for crop improvement but also for present study.
efficient management and conservation of germplasm We concluded from the present study that based on
resources (Tahir and Karim 2011). The detection of genetic agronomic characters, the 150 genotypes were successfully
diversity can be done through analysis of morphological clustered into 10 clusters, each cluster has its own
characterization and genetic markers. In this research, agronomic characteristics related to days to maturity,
morphological characters (vegetative phase duration, growth, and seed. The soybean users in Indonesia prefer
generative phase duration, vegetative and generative ratio, soybeans with high yield, early maturity, and large seed
days to maturity, plant height, number of filled pods, size. Genotypes in the group IV, VI, and IX were
number of empty pods, number of branches, number of potentially used as gene sources for the development of
nodes, 100 seed weight, and seed yield plant-1) were used to improved soybean variety in Indonesia.
reveal the genetic diversity among 150 soybean genotypes.
Some researchers stated that plant morphology-based
approach is easier and cheaper, but the phenotypic ACKNOWLEDGEMENTS
appearance of the characters is strongly affected by the
environment (Bohn et al. 1999; Maric et al. 2004), on the We thank Arifin who has assisted in data acquisition
contrary, Ozkaya et al. (2006) stated that genetic marker during the field research. This research was supported by
approach such as random amplified polymorphic DNA Indonesian Agency for Agricultural Research and
(RAPD), amplified fragment length polymorphism Development (IAARD), the Indonesian Ministry of
(AFLP), simple sequence repeat (SSR), and inter-simple Agriculture, Jakarta.
sequence repeat (ISSR) are not much affected by the
environment.
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N U SAN TA RA B IOSC IE NC E ISSN: 2087-3948
Vol. 9, No. 3, pp. 243-250 E-ISSN: 2087-3956
August 2017 DOI: 10.13057/nusbiosci/n090302

The effect of growing season on growth rate, pod partitioning,


phenology and yield variations of mungbean varieties

HERDINA PRATIWI♥, AGUSTINA ASRI RAHMIANNA


1
Indonesian Legumes and Tuber Crops Research Institutes, Jl. Raya Kendalpayak Km 8, Malang 65101, East Java, Indonesia. Tel.: +62-341-801468,
Fax.: +62-341-801496, ♥email: herdina_p@mail.com

Manuscript received: 18 November 2016. Revision accepted: 7 June 2017.

Abstract. Pratiwi H, Rahmianna AA. 2016. The effect of growing season on growth rate, pod partitioning, phenology and yield
variations of mungbean varieties. Nusantara Bioscience 9: 243-250. The primary objective of mungbean improvement program is the
production of high yielding varieties, which is influenced by physiological trait of variety or genotype and environment. The present
research aimed to study the effect of growing season on growth rate, pods partitioning, phenology and yield variations of several
mungbean varieties. Field experiments were conducted at Muneng Experimental Station of the Indonesian Legume and Tuber Crops
Research Institute (ILETRI), Probolinggo District, East Java, Indonesia during rainy season (October to December 2011), and dry
season (May to July 2012). A randomized complete block design was employed in the present study consisted of mungbean varieties as
treatments in each growing season, and three replicates. The treatments were superior mungbean varieties, i.e. Vima 1, Sriti, Murai,
Kutilang and Fore Belu. The observed variables were plant phenology, plant growth rate, seed yield and yield attributes. The results
indicated the growing season affected plant growth rate, pod partitioning, phenology and seed yield and yield components with rainy
season influenced more parameters of plant growth rate, crop phenology, seed yield and its components as compared to dry season.
Conversely, dry season had more effect on pod partitioning coefficient, total dry biomass rate, and seed size. There was significant
interaction effect of genotype by growing season on total dry matter and pod growth rates, days to flowering, days to pod maturity, plant
height, seed yield, plant population, pod weight, and 1000-seed weight. Seed yield varied among seasons and varieties with the average
seed yield in rainy season was 75 to 119% higher than those in dry season except Fore Belu that had the same yield in both seasons.

Keywords: Growth rate, mungbean varieties, phenology, pod partitioning, season

INTRODUCTION 2015 was only 1.18 t ha-1 (Central Bureau of Statistic


Indonesia 2015) with a large yield variation among
Mungbean (Vigna radiata) is the fifth leading food production centers. This indicates a significant interaction
crops in Indonesia with a high protein content. Mungbean of genotype by the environment on the mungbean yield
is usually planted in the dry season as the second crop after (Trustinah 2013). Kuo (1998) reported that mungbean yield
paddy rice, maize or peanut because of its short duration per unit area is determined by the characters of variety,
and drought tolerance. Until recently, 25 superior environment, and cultivation technology. Meanwhile,
mungbean varieties had been released by The Indonesian Chauhan et al. (2010) revealed that establishment of crop
Ministry of Agriculture since 1945 up to 2016. Each of yield could be explained by the capture and management of
these varieties has specific characteristics despite their high environmental resources such as solar radiation, soil water,
protein content that ranges from 18-28% (Iletri 2016). and nutrients for the production of biomass. According to
Efforts to obtain high mungbean yield can be carried out Atwell et al. (1999), the balance between vegetative and
through appropriate selection of superior variety that is reproductive growths is very important in determining the
suitable to the climate and environmental condition of the final yield. Vijaylaxmi and Bhattacharya (2006) reported
growing sites. However, farmers in some regions are still that mungbean seed yield is positively affected by total dry
continuously cultivating local varieties because they are not matter production, dry matter production per day, dry
aware of the existence of the above mentioned superior matter partitioning per day, and negatively affected by dry
varieties (Badal et al. 2007). Vima 1, Sriti, Murai and matter allocated to pod wall. Mondal et al. (2011) stated
Kutilang are superior varieties that are widely distributed that leaf area index contributes to total dry biomass, while
and grown in Indonesia (Iletri 2011). These varieties have number of racemes, flowers, and pods per plant contribute
specific characters and yield potential. Vima 1 variety is a mostly to seed yield. Furthermore, mungbean genotypes
short duration variety with yield potential of 1.76 t ha-1. with high leaf area, high total dry biomass, and high crop
Sriti and Murai are high yielding varieties with yield growth rate produce higher seed yield (Mondal et al. 2012).
potential of 2.45 t and 2.5 t ha-1, respectively. Kutilang Plant growth is affected by the interaction of various
variety has large seed size with yield potential of 1.96 t ha-1 environment elements on certain temperature range, while
(Iletri 2012). Despite their high yield potential, the actual plant development is affected by temperature changes
yields of these varieties in the farmers’ fields are still low. (Gordon and Bootsma, 1993). According to Miller et al.
The average mungbean productivity at the national level in (2001), plants need a certain amount of temperature units
244 N U S A N T A R A B I O S C I E N C E 9 (3): 243-250, August 2017

or growing degree days to develop from one to the next was introduced from the Philippines, and Fore Belu was a
growth phase and the different heat requirement of each local variety from Belu, East Nusa Tenggara, Indonesia.
genotype will cause variation in growth and yield. The experimental areas were previously grown with corn.
The atmospheric temperature change relates to climate Land preparation was done properly to obtain friable soils,
change. Unpredictable climate change at many regions in free from any debris of previous crops and weeds. The
the world generates global climate change, which causes fertilizer used was composite NPKS (250 kg ha-1), applied
prolonged dry season (El Nino) or rainy season (La Nina). at planting time. Plant spacing was 40 cm between rows
As a result, farmers face difficulties in determining the and 10 cm within row. The irrigation was applied once (at
most appropriate planting schedule (Jennings and Magrath 0 days after sowing, DAS) during the rainy season
2009). Unpredictable season changes affect plant cropping, and four times (at 0, 12, 32, and 45 DAS) during
phenology and productivity (White et al. 2003; Cleland et the dry season cropping. Weeding was done twice and pest
al. 2007; Rai 2015). Hence, it is necessary to evaluate control was done depending on the incidence of pests and
mungbean varieties in different growing seasons to obtain diseases.
the factors that affect the variation of growth and yield and Data was collected on Growth Degree Days (GDD),
to observe the more potential variety. The objective of this plant phenology, plant growth, seed yield, and yield
research was to study the effect of growing season on attributes. The observed plant phenology covered days to
growth rate, pods partitioning, phenology and yield 50% flowering and 50% pod maturity. Growth analysis
variations of several mungbean varieties. included growth rate and pod partitioning, where the
observations were started at 10 DAS in a 10-day interval,
and finished at 60 and 70 DAS for rainy and dry seasons,
MATERIALS AND METHODS respectively. Growth analysis was done by destructive
sampling of all plants in the area of 0.8 m2 (2 rows, 1 m
Field experiments were conducted at Muneng long) outside the harvesting plot. The plants were separated
Experimental Station of the Indonesian Legume and Tuber into roots, stems, leaves, and pods and the corresponding
Crops Research Institute (Iletri), Probolinggo District, East dry weights were recorded after oven drying at 80 oC for 72
Java Province, Indonesia (7.7oS, 11.2oE, 10 m above sea hours. At harvesting time, all pods in the plots were
level) during the rainy season (October to December 2011), harvested. Harvesting time of mungbean varieties in the
and dry season (May to July 2012). Indonesia has two main rainy season was started at 60 DAS for Vima 1, Sriti,
seasons i.e. rainy/wet season and dry season; each lasts for Kutilang, Murai varieties except for Fore Belu variety (at
six months. The rainy season starts from the end of 70 DAS). In the dry season, the pod harvesting of Vima 1,
October/early November to the end of March/early April, Sriti, Kutilang, Murai varieties was started at 70 DAS, and
and dry season starts at the end of March/early April and that of Fore Belu variety was at 80 DAS. Vima 1 was
finishes at the end of October/early November. The climate harvested once while the other three varieties were
data during the research period was presented in Table 1. harvested three times. The pods were then sun-dried until
A split plot design was employed in the present study the seeds reached 10% water content, and unshelled to
with seasons as the main plots, and mungbean varieties as separate the seeds from the pods prior to weighing. The
subplots. The mungbean varieties consisted of five superior number of harvested plants in each plot was recorded. The
varieties, namely Vima 1, Sriti, Murai, Kutilang and Fore observations were undertaken on plant yield and yield
Belu. Each variety was grown in a 5 m x 6 m plot size. In components including 1000 seed weight, number of pods
each season, the treatments were laid out in a randomized plant-1, pods and seeds weight harvested plot-1.
complete block design, and three replications. Vima 1 was Growth Degree Days (GDD) is an accumulation of
originated from the crossing of VC2750A and VC 1973A; daily temperature minus base temperature of mungbean.
Sriti and Kutilang were introduced from Taiwan; Murai Daily GDD were calculated based on Tzudir et al. (2014):

Table 1. Temperature, rainfall, and relative humidity on research site during growing seasons of mungbean

Air temperature (oC) Relative Rainfall Number of rainy


Month
Minimum Maximum Average humidity (%) (mm) days

Rainy season (2011)


October 23.2 34.7 28.0 55.6 0 0
November 23.1 28.5 25.8 65.8 133 5
December 23.5 29.0 26.2 65.0 122 12

Dry season (2012)


May 22.6 33.6 27.5 72.8 16 2
June 20.7 33.4 26.3 51.6 22 1
July 20.1 32.9 26.1 51.4 0 0

Source: weather station of Muneng Experimental Station, Probolinggo District, East Java Province, Indonesia
PRATIWI & RAHMIANNA – Effect of growing season on mungbean yield 245

GDD=[ ( (Tmax+Tmin)/2)-Tbase] Vima 1 was the shortest in both seasons (Table 3).
Where, Table 3 shows that Fore Belu had the lowest pod
Tbase= base temperature below which the crop cannot growth rate and the highest plant height in both seasons.
thrive, Tbase for mungbean=10 oC This low pod growth rate was due to its dominant
Tmax=daily maximum temperature vegetative growth especially the plant height. Conversely,
Tmin=daily minimum temperature Vima 1 variety was a short plant but had a dominant pod
growth rate. These differences are presumably due to the
Plant growth rate is the gradient of regression equation different genetic background of the two varieties. Fore
between the plant age and plant dry matter (Manshuri Belu had a semi-determinate growth type (Iletri 2012), and
2011). The coefficient of pod partitioning is the ratio of therefore the plant is still continuing its vegetative growth
pod growth rate with crop growth rate (Duncan et al. 1978). when its generative growth has been commenced. Vima 1,
The collected data were statistically analyzed following the on the other hand, has a determinate growth type (Iletri
analysis of variance, and the mean differences were 2012) where its vegetative growth has stopped when the
assessed by using LSD. The correlation analysis was plant enters the reproductive phase. The semi-determinate
performed to find out the relation between parameters trait enables the pods to grow optimally as “sink” while the
observed. leaves and stems as “source” continuously grow (Atwell
1999). In other words, there is a competition between pods
and leaves + stems in using the assimilate. The other reason
RESULTS AND DISCUSSION for this slow pod growth rate of Fore Belu could be due to
shorter pod formation period as a result of the slow/late
Plant growth rate and pod partitioning initiation of the flowering period (Vijaylaxmi and
Growing season affected leaf and stem growth rate, as Bhattacharya 2006).
well as pod partitioning coefficient, whereas varieties
affected only stem growth rate (Table 2). Leaf and stem Table 2. Leaves growth rate, stem growth rate, and pod
partitioning coefficient of mungbean varieties in different
growth rates were superior in the dry season. Conversely,
growing seasons.
pod partitioning coefficients were superior in the rainy
season. Leaf growth rate in the dry season was 39% higher Leaf growth Stem Coefficient of
than that in the rainy season; however, there was no Treatment rate growth rate pod
significant variation among varieties. Stem growth rate in (g m-2 day-1) (g m-2 day-1) partitioning
the dry season was 163% higher than that in the rainy
season and significantly varied among varieties. The Growing season
highest stem growth rate was observed in Fore Belu and Rainy 1.68 b 1.14 b 1.60 a
Sriti while the lowest one was observed in Vima 1. Dry 2.33 a 3.00 a 0.82 b
The pod partitioning coefficient in the rainy season was
Varieties
two times higher than that in the dry season. The
Vima 1 1.76 a 1.69 b 1.35 a
coefficient was >1 in the rainy season and <1 in the dry Sriti 2.34 a 2.25 a 1.17 a
season. According to Purnawati and Manshuri (2015), pod Murai 1.88 a 2.01 ab 1.24 a
partitioning coefficient above 1 indicates that the increase Kutilang 1.75 a 2.05 ab 1.27 a
of pod dry weight is higher than the increase of plant dry Fore Belu 2.29 a 2.35 a 1.02 a
biomass rate. A higher pod partitioning coefficient Note: Values in the same treatment that followed by the same
indicates that a higher amount of assimilates is distributed letter (s) were not significantly different at LSD 5%
to the economic parts of the crop. All varieties possessed
the same capability in distributing assimilates to the pods
as shown by their non-significantly different pod Table 3. Pod and total dry biomass growth rate, plant height of
partitioning coefficients. mungbean varieties on different growing seasons
There was an interaction effect of growing season and
Growing Varieties
mungbean varieties on total dry biomass, pod growth rate,
season Vima 1 Sriti Murai Kutilang Fore Belu
and plant height (Table 3). Total dry biomass growth rate
of all varieties in the dry season was 40-222% higher than Total dry biomass growth rate (g m-2 day-1)
those in rainy season except Vima 1, which exhibited the Rainy 6.48 c 6.58 c 6.26 c 5.73 c 2.89 d
same rate in both seasons. Fore Belu produced the lowest Dry 7.11 bc 9.22 a 8.24 ab 8.10 ab 9.30 a
as well as the highest total dry biomass growth rates in the
rainy and dry season, respectively. All varieties had 27- Pod growth rate (g m-2 day-1)
92% higher pod growth rate in the rainy season than those Rainy 11.81 a 9.76 b 9.87 b 9.38 bc 3.98 f
in dry season except Fore Belu, which on the contrary, had Dry 6.15 de 7.65 cd 7.31 de 7.09 de 5.83 e
32% lower pod growth rate in the rainy season than in dry
Plant height at harvest (cm)
season. Vima 1 and Fore Belu had the highest and the
Rainy 75.55 cd 103.75 b 83.20 c 85.60 c 170.20 a
lowest pod growth rate, respectively. All varieties grew Dry 45.30 f 48.35 f 55.90 ef 51.75 f 64.35 de
1.7-2.6 times taller in the rainy season than those in dry Note: Values in one variable followed by the same letter (s) were
season. Fore Belu was the tallest in both seasons, while not significantly different at LSD 5%
246 N U S A N T A R A B I O S C I E N C E 9 (3): 243-250, August 2017

Total dry biomass production of all varieties gradually to senescence. In the dry season, total dry biomass rapidly
increased in rainy season (Fig 1A), but it then sharply increased from 20 until 60 DAS (Figure 1 B). This was in
increased in dry season (Fig 1B). The dry season crops are accordance to Mondal et al. (2012), who stated that plant
supposed to be harvested earlier than the crops grown in dry biomass increased rapidly after the reproductive stage.
rainy season. The figures, however, showed that rainy Total dry biomass of Sriti and Fore Belu tended to increase
season crops were harvested 10 days earlier than those of after 60 DAS, which resulted in a higher total dry biomass
dry season ones. This might have happened because all growth rate than other varieties (Figure 1B).
varieties were attacked by the bean flower thrips Figure 2A and 2B showed pod dry weight at 40-70
(Megalurothrips usitatus) during the vegetative phase in DAS in rainy and dry seasons, respectively. The figures
the dry season. The infected plants then successfully indicated that the observation on pod weight was
recovered their growth and established new and healthy commenced at 40 DAS. Pod weight in 40 DAS was very
leaves after being sprayed with chemical insecticides. As a low i.e., below 5 g m-2 and then increased until reached
result, the plants in the dry season had a longer maturity point above 100 g m-2 in the last observation. Pods yield in
than that in the rainy season. In the rainy season, total dry the rainy season was higher than that in the dry season. In
biomass was low at early growth (10-20 DAS), which then the rainy season, Vima 1, Sriti, Murai and Kutilang had the
increased rapidly at the onset of the reproductive stage (20- same pods weight, while Fore Belu had the lowest due to
30 DAS). The total dry biomass of the plants grown in the its dominant vegetative growth (Figure 2A). In the dry
rainy season was constant at 30-40 DAS, and then rapidly season, pod dry weights among five varieties were slightly
increased after 50 DAS following the pod filling period different, and Fore Belu had the lowest pod weight. Water
(Figure 1 A). The longer duration of Fore Belu made the shortage in dry season inhibited pod formation and resulted
dry biomass of the variety observed until 70 DAS. lower pod dry weight as compared to that in the rainy
However, the total dry biomass of this variety decreased season.
sharply in the last observation as many leaves dropped due

Figure 1. Total dry biomass on 10, 20, 30, 40, 50, 60 and 70 days after sowing in rainy season (A) and dry season (B)

Figure 2. Pod dry weight on 10, 20, 30, 40, 50, 60, and 70 days after sowing in rainy season (A), and dry season (B)
PRATIWI & RAHMIANNA – Effect of growing season on mungbean yield 247

Plant phenology season except Fore Belu that produced higher seed and pod
The present study results revealed significant yields in the dry season. Kutilang produced the highest
interaction effect of season and variety on days to grains and pods yields, while Fore Belu produced the
flowering, GDD of flowering, days to mature, and GDD of lowest. The grains and pods yields of all varieties more
maturing (Table 4). Across seasons, the longest flowering fluctuated in the rainy season than those in the dry season.
date was observed in Fore Belu i.e. 38 DAS, while the Table 5 showed that the seed yield of Vima 1 and Kutilang
shortest was observed in Vima 1 grown in dry season i.e. in rainy season i.e. 2.04 t and 2.48 t ha-1 successfully
34 DAS. The same patterns were observed on GDD of exceeded its yield potentials of 1.76 t and 1.96 ha-1,
flowering, maturing date, and GDD of maturing. The data respectively. The seed yield of Fore Belu in both seasons,
pointed out that the longer the flowering date, the longer indeed, was higher than its yield potential (1.076 t ha-1).
was the maturing date. For example, Vima 1 and Kutilang Climate, especially the amount of rainfall, affects
had the shortest flowering and maturing dates, while Fore mungbean yield, and the productivity increases along with
Belu had the longest flowering and maturing dates (Table the increase of rainfall (Ariyanto 2010). Four times of
4). The average maturing dates for Vima 1, Sriti, Murai, irrigations during the growing period of mungbean planted
Kutilang, and Fore Belu across the production center areas in dry season could not supply water as much as a number
in Indonesia were 53 days, 60-65 days, 63 days, 60-67 of rainfalls in rainy season (Table 1). In other words,
days, 60 to 67 days, respectively (Ministry of Agriculture mungbean crops received more water in the rainy season
Republic of Indonesia 2005; Iletri 2012). In this research, for their growth as compared to that in the dry season. Fore
all mungbean varieties had a maturing date earlier than Belu variety, however, produced lower yield in the rainy
those mentioned in their varietal descriptions. In addition, season. This lower yield was supported by Trustinah et al.
the flowering and maturing dates of all varieties in dry (2014) who reported that Fore Belu produced low yield
season were shorter than those in rainy season. One likely when planted in soil with excessive/abundant water and, on
reason for this finding was the air temperature differences the contrary, it produced higher yield when grown in less
between the two seasons. Menzel et al. (2006) mentioned water condition. Abundant water from rains caused Fore
that the rising of temperature accelerates flowering and Belu had a longer vegetative growth than the generative
maturing dates and the effects varies between the plants. As one.
reported in Table 1 that the mean air temperatures during To some extent, the weight of mature pods of
the experiment in the dry season were 0.3 oC higher than mungbean determines its grains yield. The higher pod
that in the rainy season. weight of Sriti, Murai, and Kutilang was followed by the
Growing degree days (GDD) required by mungbean for higher seed yields of these varieties in rainy season. In the
flower development in the rainy season was generally dry season, all varieties produced the same mature pod
higher than that in the dry season. However, GDD required weights as well as grain yields.
for pod maturity in the rainy season was lower than in dry Seed size is generally illustrated by the weight of 1000
season. The exception was for Fore Belu that required the seeds. Kutilang had the biggest seed size while Vima 1 had
same GDD forflower development and required higher the smallest in both seasons. The seed sizes of Vima 1, Sriti
GDD for pod maturity in the rainy season than in dry and Kutilang were not substantially different either in rainy
season. According to Tzudir et al. (2014), the rising of air or dry season planting. Meanwhile, Murai and Fore Belu
temperature increased the GDD requirement by the plants. had significantly bigger seed size in the dry season (Table
At the first month of the rainy season, the air temperature 5). This indicates that the seed size of Murai and Fore Belu
was higher than that for the first month of the dry season, was influenced by environment i.e. growing season.
and there was no rainfall (Table 1). Therefore, the amount According to Hampton et al. (2016), the variation of
of GDD for flowering in the rainy season was higher than climate as expressed in the different growing season could
that in the dry season. At the second month, when the plant affect the seed mass because of the changes of seed growth
entered the pod formation until pod maturity stages, GDD rate and seed filling duration.
required in the rainy season was generally lower than that There was a higher number of plants at harvesting time
in the dry season. This presumably caused by the lower in the rainy season than that in the dry season. Vima 1 had
temperature in the rainy season due to precipitation. In the highest plant population in the rainy season, while
addition, the higher GDD in dry season during pod set was Murai, Kutilang, and Sriti had the highest numbers in a dry
caused by a higher difference between the minimum and season among the tested varieties.
maximum temperature (Table 1). Vima 1 had the lowest Number of pods plant-1 was only influenced by growing
GDD while Fore Belu had the highest GDD for flowering season, where rainy season resulted in 56.6% higher pod
and pod maturity in both rainy season and dry season. numbers than the dry season. All five varieties had the
same amount of pods plant-1 (Table 6). In more detail, the
Yield and yield attributes differences in pod numbers plant-1 between the growing
Variety by growing season interaction affected grain seasons were likely caused by the differences in pod
yield, the weight of mature pods, plant population, and growth rates as discussed in Table 3. The higher pod
1000 seed weight (Table 5). All varieties produced higher growth rate accelerated the pod formation of mungbean
grains and pod yields in the rainy season than those in dry plant.
248 N U S A N T A R A B I O S C I E N C E 9 (3): 243-250, August 2017

Table 4. Flowering date, growing degree days (GDD) of flowering, maturing date and growing degree days (GDD) of maturing of
mungbean varieties on different growing seasons

Varieties
Growing season
Vima 1 Sriti Murai Kutilang Fore Belu

Flowering date (DAS)


Rainy 36.00 d 37.00 bc 37.50 a 36.50 cd 38.00 a
Dry 34.00 f 35.00 e 37.50 a 36.50 cd 38.00 a

GDD of flowering (oCd)


Rainy 645.953d 660.778 bc 669.478 ab 653.795 cd 677.319 a
Dry 608.420 f 626.517 e 645.594 626.517 e 677.997 a

Maturing date (DAS)


Rainy 55.00 d 59.00 b 59.00 b 55.00 d 65.00 a
Dry 55.50 d 56.50 c 56.75 c 56.50 c 58.50 b

GDD of maturing (oCd)


Rainy 900.750 e 964.093d 964.093 900.750 e 1067.576 a
Dry 968.873d 985.198 c 989.233 c 985.198 c 1016.364 b
Note: Values inone variable followed by the same letter (s) were not significantly different at LSD 5%. DAS=days after sowing,
GDD=growing degree days

Table 5. Grain yield, weight of mature pods, plant population, and 1000 seed weight of mungbean varieties on different growing seasons

Varieties
Growing season
Vima 1 Sriti Murai Kutilang Fore Belu

Grain yield (t ha-1)


Rainy 2.04 c 2.15 bc 2.35 ab 2.48 a 1.10 e
Dry 1.02 e 1.22 de 1.34 d 1.13 de 1.23 de

Weight of mature pods (t ha-1)


Rainy 2.62 b 3.58 a 3.51 a 3.56 a 1.34 e
Dry 1.91 d 2.33 bcd 2.35 bc 2.00 cd 2.08 cd

Plant population ha-1


Rainy 491.250 a 453.500 ab 452.256 ab 413.000 bc 455.250 ab
Dry 303.250 d 363.000 c 359.750 c 363.000 c 297.500 d

1000 seed weight (g)


Rainy 55.65 ef 63.53 bcd 52.60 f 71.88 a 55.20 ef
Dry 58.25 def 60.63 bcde 58.78 cde 66.10 ab 64.65 bc
Note: Values inone variable followed by the same letter (s) were not significantly different at LSD 5%

Table 6. Number of pods per plant of mungbean varieties in Correlation coefficient


different growing seasons The relationship between growth rate, pod partitioning,
phenology traits, yield components and seed yield was
Treatments Number of pods plant-1 determined by the correlation coefficient. Growing season
caused a different effect on the observed parameters (Table
Growing season 7). Rainy season emphasized the significant role of
Rainy season 23.10 a phenological traits on seed yield, yield component (number
Dry season 14.75 b of pods plant-1), and growth rate (pod growth rate and plant
height). Dry season, however, born the significant role of
Varieties phenological traits on different parameters i.e. pod
Vima 1 16.25
Sriti 19.75
partitioning, growth rate (stem growth rate, leaves growth
Murai 19.38 rate, and plant height).
Kutilang 17.75 In the rainy season, seed yield was positively correlated
Fore belu 21.50 to the growth rate (pod growth rate, total dry biomass
Note: Values in the same column and treatment that followed by growth rate), and yield component (mature pod weight),
the same letter (s) were not significantly different at LSD 5% but negatively correlated to phenology traits (flowering
PRATIWI & RAHMIANNA – Effect of growing season on mungbean yield 249

date, maturing date, plant height) and a number of pods in the rainy season. Based on correlation coefficients, a
plant-1. Pod growth rate declined when the pod numbers factor that mostly contributed to seed yield was the weight
increased. The high pod growth rate was characterized by of mature pods. Meanwhile, Ahmad et al. (2015) reported
the low plant height, the short flowering and maturing that pod number plant-1 was positively correlated to seed
dates. In the dry season, seed yield was positively yield, but the parameters of plant height and number of
correlated to the growth rate (pod growth rate, total dry branch per plant have a maximum direct effect on seed
biomass growth rate), and yield component (mature pod yield.
weight, plant population) while phenology traits had In summary, the growing season affected plant growth
significant correlation with seed yield. rate, pod partitioning, phenology and seed yield and yield
In both seasons, the main components that were components with rainy season influenced more parameters
positively correlated to seed yield included pod growth of plant growth rate, crop phenology, seed yield and its
rate, total dry biomass growth rate, and mature pod weight. components. Conversely, dry season had more effect on
These results were in line with the results of Mondal et al. pod partitioning coefficient, total dry biomass rate, and
(2011) and Mondal et al. (2012) who found that seed yield seed size. Yield variation among five varieties showed that
was positively correlated to total dry biomass. Mature pods average yield in the rainy season was 75 to 119% higher
weight was positively and significantly related to pod than those in dry season except Fore Belu that had the same
growth rate, and total dry biomass growth rate. The yield in both seasons. There was significant interaction
increase of pod growth rate would increase the pod weight effect of variety and growing season on the total dry matter
and pod partitioning, therefore, increased the seed yield. In and pod growth rates, days to flowering, days to maturing
these two growing seasons, the seed yield did not correlate pod, plant height, seed yield, plant population, the weight
to seed size, and seed size was not correlated to any single of mature pods, and 1000-seed weight. The variation of
parameter observed (Table 7). However, seed size of Vima growth rate, phenology, and yield of mungbean between
1, Sriti, Kutilang was similar either in rainy or dry season growing seasons were affected by the differences between
except for Murai and Fore Belu (Table 5). Table 5 shows minimum and maximum temperatures and the amount of
that the larger seed was not always resulted in higher seed rainfall. Despite its short duration type, high pod growth
yield. The absence of correlation between seed size and rate, and simultaneous harvest, Vima 1 had reasonably high
seed yield in this study was in line with a report by Gul et seed yield.
al. (2008) who stated that seed size of mungbean was not
significantly correlated to seed yield. This means that
genetic factor is more dominant in controlling seed size ACKNOWLEDGEMENTS
rather than the environmental factors. Even though the
1000-seed weight was more genetically controlled trait, it The authors gratefully acknowledge the ACIAR
could be affected by the growing season. It is apparent in (Australia Centre for International Agricultural Research)
the field that seed size could be smaller or larger than that Project No. SMAR/2007/068 on “Productivity and
mentioned in the description list. To certain varieties, the Profitability Enhancement of Tropical Pulse in Indonesia
seed size changed when the plants were grown in different and Australia” which funded the experiment, and also Ir.
seasons. For examples: the seed size of Murai and Fore Abdullah Taufiq, MP and the technicians for their great
Belu was, respectively, 10% and 15% lower when planted assistance in conducting the trials.

Table 7. Coefficient of correlation of parameters in two growing seasons

Para Rainy season


meters FD MT PH LGR SGR PGR TDM PT PW SS NP PP Y
FD 0.73** 0.56** 0.06 0.50* -0.47* -0.29 -0.32 -0.40 -0.19 0.58** -0.22 -0.53*
MT 0.88** 0.88** -0.10 0.10 -0.80** -0.67* -3.30 -0.64** -0.40 0.67** 0.02 -0.79**
PH 0.87** 0.87** -0.19 -0.04 -0.80** -0.77** -0.18 -0.69** -0.14 0.66* -0.11 -0.81**
LGR 0.65** 0.65** 0.57** 0.50* 0.27 0.67** -0.43 0.02 -0.03 0.01 -0.32 0.07
SGR 0.51* 0.49* 0.40 0.94** 0.03 0.38 -0.42 0.20 0.09 0.31 -0.29 0.14
PGR -0.22 -0.33 -0.31 -0.05 0.08 0.85** 0.50* 0.60** 0.11 -0.6** 0.19 0.67**
TDM 0.46* 0.40 0.34 0.88** 0.92** 0.41 0.02 0.58** 0.13 -0.45* -0.05 0.65**
PT -0.58** -0.64** -0.54* -0.79** -0.70** 0.62** -0.45* 0.29 0.04 -0.34 0.31 0.21
PW 0.16 0.15 0.11 0.31 0.29 0.63** 0.56* 0.13 0.41 -0.43 -0.13 0.90**
SS 0.30 0.36 0.16 0.16 0.21 -0.05 0.12 -0.19 -0.09 0.31 -0.41 0.43
NP 0.00 0.07 -0.10 -0.14 -0.22 -0.20 -0.22 -0.04 0.02 -0.23 0.17 -0.49*
PP -0.20 -0.03 0.00 0.03 0.08 0.34 0.18 0.17 0.57** 0.04 -0.06 -0.12
Y 0.35 0.32 0.33 0.30 0.22 0.50* 0.48* 0.09 0.88** -0.07 -0.04 0.45*
Dry season
Values above shading cells are correlation coefficients in rainy season, and those below are correlation coefficients in dry season. FD=
flowering days, MT=maturing days, PH=plant height, LGR=leaves growth rate, SGR=stem growth rate, TDM=total dry mass growth
rate, PGR=pod growth rate, PT=pod partitioning, PW=weight of maturing pods, SS=1000 seed weight, NP=number of pod per plant,
PP=plant population, Y=yield, *=significant at 0.05 probability level, **= significant at 0.01 probability level.
250 N U S A N T A R A B I O S C I E N C E 9 (3): 243-250, August 2017

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N U SAN TA RA B IOSC IE NC E ISSN: 2087-3948
Vol. 9, No. 3, pp. 251-259 E-ISSN: 2087-3956
August 2017 DOI: 10.13057/nusbiosci/n090303

Assessment of the successfulness of mangrove plantation program


through the use of open source software and freely available satellite
images

ILYAS NURSAMSI1,♥, WULAN RATNA KOMALA2


1
Bird Conservation Society (BICONS). Bandung 40184, West Java, Indonesia.♥email: ilyasnursyamsi05@gmail.com
2
Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Padjadjaran. Jatinangor, Sumedang 45363, West Java, Indonesia.

Manuscript received: 10 May 2016. Revision accepted: 13 June 2017.

Abstract. Nursamsi I, Komala WR.2017. Assessment of the successfulness of mangrove plantation program through the use of open
source software and freely available satellite images. Nusantara Bioscience 9: 251-259. Mangrove forest has a major role in the process
of human-environment interaction, but almost every mangrove forest in the world is under threat. In Indonesia alone, 25% of South East
Asia’s mangroves are at a risk. The continued decline in mangrove forest induced by anthropogenic activity has made all the
stakeholders who have the concern at the mangrove forest preservation worried, including the government. There were several programs
have been performed by the government to preserve the mangrove forest. One of the programs was “Mangrove Rehabilitation Program
in three districts: Ciamis, Indramayu, and Subang” held by Forestry Department of West Java Province in 2007. The aims of this study
were to assess the changes in mangrove forest area before the program performed and to evaluate the successfulness of the program,
using the increasing of mangrove forest area as a parameter. This study was conducted only in Subang and Indramayu Districts of West
Java, Indonesia. The assessment was conducted using Landsat 4-5 TM, Landsat 7 ETM+, and Landsat 8 OLI acquired in 1996, 2006,
and 2016 respectively. For each image, a supervised classification method was performed using open source GRASS GIS software. The
resulting maps were then compared to quantify the changes. Field work activity conducted and confirmed the changes that occurred in
the study areas. Our study shows that all of the two districts exhibit successfulness of the plantation program. Ground truth survey
confirmed that the successfulness of the plantation program is due to the participation of communities in the area of study. This study
also shows that by using open source software and freely available satellite images, the fast, robust, and reliable data as an initial step to
monitor both short-term and long-term plantation program can be collected effectively and inexpensively.

Keywords: Classification, Indramayu, Landsat TM/ETM+/OLI, mangroves, remote sensing, Subang

Abbreviations: TM = Thematic Mapper, ETM+ = Enhanced Thematic Mapper, OLI = Operational Land Imagery, DN = Digital Number

INTRODUCTION 2015). The destruction of mangroves is usually related to


human population density (Donato et al. 2011).
Mangroves are coastal forests found in sheltered The continued decline in mangrove forests due to
estuaries and along river banks and lagoons in the tropics various anthropogenic activities in various regions in
and subtropics. The term ‘mangrove’ describes both the Indonesia has increased the concern of the stakeholders in
ecosystem and the plant families that have developed mangrove forests conservation field. Various efforts have
specialized adaptations living in this tidal environmental been made by these stakeholders, including by the
(FAO 2007). Mangrove forest ecosystems fulfill some of government. The Forestry Department has been, is, and
important functions in terms of providing wood and non- will carry out activities in the form of technical operational
wood forest products (Hussain and Badola 2010), coastal activities, both the field and in the concept (Forestry
protection (Alongi 2008), conservation of biological Department 2003).
diversity and provision of habitat (Barbier et al. 2011), In 2007, Forestry Department of West Java Province
spawning grounds and nutrients for a variety of fish and performed a rehabilitation program of mangrove forest in a
shellfish (FAO 2007). total of 750 ha areas (outside the forest area) spread in
The total area of mangroves in the year 2011 Indramayu, Ciamis, and Subang Districts. The program
approximately were 10,872,000 ha spread in 118 countries was implemented by using a green belt and ditch pond
(IUCN I-IV). Indonesia is the country with the largest pattern. To achieve the expected goals, the implementation
extent of mangrove in the world amounted to 3,112,989 ha, of such activity need to be monitored and evaluated
representing 22.6% of the world's total mangrove routinely (Forestry Department of West Java Province
ecosystems (Giri et al. 2011). On the basis of FAO data, 2008). In the implementation, monitoring efforts of
cumulatively, Indonesia has lost 30% of its mangrove mangroves rehabilitation program are less intensive
forests between 1980 and 2005; this is equivalent to an because of a direct monitoring over a large area requires an
annual deforestation rate of 1.24% (Murdiyarso et al. enormous number of human resources, funds, and time.
252 N U S A N T A R A B I O S C I E N C E 9 (4): 251-259, August 2017

Thus, a different approach is needed. The most possible Landsat 8 OLI and 8 spectral bands for Landsat 4-5 TM
approach is to conduct the monitoring effort by using and Landsat 7 ETM+. The selected imageries were
remote sensing technology. acquired over the Subang and Indramayu Districts of West
Remote Sensing (RS) refers to obtaining information Java Province, Indonesia (Figure 1).
about objects or areas at the Earth’s surface without being
in direct contact. Remote sensing uses a part or several Procedures
parts of the electromagnetic spectrum. It records the Landsat image pre-processing
electromagnetic energy reflected or emitted by the earth's The first necessary step to analyze vegetation indices
surface (Aggarwal 2007). Remote sensing can be and land use/land cover is to perform image Pre-
performed with limited funds (cost) by using open source processing. The Landsat 8 OLI and 7 ETM+ images were
software (e.g. GRASS GIS and QGIS) and Satellite images geometry-corrected by the U.S Geological Survey (USGS),
that have been provided for free (e.g. Landsat Programs). while the Landsat 4-5 TM was corrected by using the GCP
Through this study, we want to help the evaluation and the points data collected from USGS (USGS 2013).
monitoring of mangrove rehabilitation program undertaken Landsat Image Pre-processing means to convert the
by the government and to promote remote sensing as an digital number (DN) values to the surface reflectance (Top
inexpensive method to perform these activities with of Canopy) values by using a two-step process. This level
reliable results. Several studies, for example, have applied of data correction is the proper level of correction to
remote sensing technology for monitoring, evaluating, and perform vegetation indices calculation and land use/land
conserving mangrove ecosystems by Huang et al. (2009), cover analysis (GrassGIS Development Team 2013). The
Lee and Yeh (2009), Giri et al (2011), Kuenzer et al. first step is to convert the DN values into radiance values
(2011), and Ramdani et al. (2015). using the Lmin and Lmax spectral radiance scaling factors
(USGS 2013). The values are specific to the individual
scene and are produced in the image header file (Ramdani
MATERIALS AND METHODS et al. 2015). The gain and bias for TOA radiance
calibration in the Landsat 4-5 TM and Landsat 7 ETM+
Study area data could be read in the metadata(.MTL) file as “mult”
Multispectral images of Landsat 4-5 TM (1996), and “add” values that are also similar to Landsat 8 OLI
Landsat 7 ETM+ (2006), and Landsat 8 OLI (2016) were (USGS 2016). The ”mult” is a multiplicative rescaling
collected from the United States Geological Survey factor, while “add” is an additive rescaling factor for each
(USGS) website (http://earthexplorer.usgs.gov). The band (Ramdani et al. 2015).
multispectral imagery consists of 11 spectral bands for

Figure 1. The study areas were located in Cantigi sub-District, Indramayu and Pusakanagara sub-District, Subang, West Java Province,
Indonesia. Images were taken from Google Earth. The red polygon represents the area of the program.
NURSAMSI & KOMALA – Mangrove forest change assessment 253

The second step is to convert the radiance values into Where, NIR is stood for Near Infrared channel
Surface reflectance values using the 6S (Second Simulation corresponds to Band 4 (0.77 – 0.90 μm) for Landsat 7
of Satellite Signal in the Solar Spectrum) algorithm. The ETM+ and Landsat 4-5 TM, and to Band 5 (0.85 – 0.88
atmospheric correction process is essential when the multi- μm) for Landsat 8 OLI. Red channel is a visible
temporal analysis is conducted (Fichera et al. 2012). The wavelength channel at Band 3 (0.63 - 0.69 μm) for TM and
effect of atmosphere can prevent the proper interpretation ETM+ and at Band 4 (0.63 - 0.68 μm) for OLI images.
of the image if it is not taken into account (Martinez et al. NDWI is sensitive to the changes in the water content
2008; Abrams et al 2009; Hadjimitsis et al. 2010; of vegetation canopies. It is considered as an independent
Srivastava et al. 2014; Hagolle et al. 2015; Raab et al. vegetation index that was developed to delineate vegetation
2015). The USGS currently provides a higher-level data water content features and to enhance their presence in
product which is already converted into surface reflectance. remotely sensed digital imagery (Alsaaideh et al. 2013;
However, to calculate Landsat 4-5 TM, 7 ETM+, and Ramdani et al. 2015). In the command console of GRASS
Landsat 8 OLI surface reflectance values the USGS used GIS, the formula to calculate the NDWI is written as
two different Algorithms, namely: (i) LEDAPS (Landsat follows (equation 2). Every formula that contains and will
Ecosystem Disturbance Adaptive Processing System) for result in float values calculated in GRASS GIS needs to be
Landsat 4-5 TM and Landsat 7 ETM+. (ii) LaSRC (Landsat multiplied by 1.0 to ensure that the results of the data are in
Surface Reflectance Code) for Landsat 8 OLI (USGS the same float values. Multiplying the equation by 1.0 will
2016). Furthermore, a study of the continuity of reflectance give GRASS GIS information it needed to handle or
data between Landsat-7 ETM and Landsat 8 OLI by Flood operate the equation in the manner of floating value. Thus,
(2014) exhibited that there were slight differences in the the result will come out as a float value rather than an
reflectance measurements of both Series of Landsat. Thus, integer value if such information is not included in the
the standardization of surface reflectance algorithm is equation (GrassGIS Development Team 2016).
essential by using the 6S algorithm. All of the 6S parameter
values needed to perform the algorithm were obtained from r.mapcalc NDWI = 1.0×(Green −SWIR)/(Green +
the USGS (https://landsat.usgs.gov/calibration). After these
SWIR) (equation 2)
steps had finished, the pixel values of each scene were
atmospherically corrected as surface reflectance.
To convert the radiance values into surface reflectance Where, SWIR (Short Wavelength Infrared) is the
values in GRASS GIS software, we used the “r.atcorr” tool reflectance in a short wavelength Infrared and corresponds
with the requirement of nine parameters. The nine 6S to Band 5 (1.55 – 1.75 μm) for Landsat 7 ETM+ and
Parameters required were : (i) Geometrical conditions, (ii) Landsat 4-5 TM and to Band 6 (1.57 – 1.65 μm) for
Acquiring date of the image (month, day, decimal hours Landsat 8 OLI.
GMT), (iii) Longitude and latitude in the middle of the
image, (iv) Atmospheric model, (v) Aerosol model, (vi) RGB composite
Visibility in Kilometer(s) (Aerosol model concentration), RGB (Red-Green-Blue) composite was performed by
(vii) Mean target elevation above sea level in Kilometer(s), composing three different band channels to produce a new
(viii) Sensor height (Sensor on board satellite) in image that shows the different nature of land surface color
Kilometer(s), and (ix) Selected Sensor band. based on its response to an electromagnetic impulse from
the sun (Xie et al. 2008). The red channel in RGB
NDVI and NDWI composite was made by using the Blue Band in Landsat 7
The images that were atmospherically corrected then ETM+ and Landsat 4-5 TM, while in Landsat 8 OLI the
could be used to perform the NDVI (Normalize Difference deep blue coastal band was used. NDVI image was used as
Vegetation Index) (Sharma et al. 2009) and NDWI the Green channel, and NDWI image as the Blue channel
(Normalize Difference Water Index) (Yilmaz et al. 2008) (Ramdani et al. 2015). In the command console of GRASS
calculation. The purpose of this step is to provide the GIS, the formula to calculate the RGB composite is written
necessary indices images that will be used as classification as follows (equation 3). Because of the wide distribution of
factors. fish pond in the study areas, NDWI was then used to
NDVI is a numerical indicator that uses the visible and accurately differentiate the waterbody and non-waterbody
near-infrared bands of the electromagnetic spectrum. NDVI in those areas since the utilization of green and NIR
is adopted to analyze the density of vegetation and also to wavelengths are advisable to monitor water content in
separate the healthy vegetation and unhealthy or sparse water bodies (Li et al. 2013; Koet al. 2015; Mishra and
vegetation, (Devadas2008; Genc et al. 2008; Szilárd et al Rama 2015; Gulcan and Mehmet 2016; Yun et al. 2016).
2016) by the use of density as a base of hypothetical Several studies by Xu (2006), Li et al. (2013), Szabo et al.
measurement. In the command console of GRASS GIS, the (2016), and Gao et al. (2016) have been conducted in an
formula to calculate the NDVI is written as follows attempt to utilize the NDWI as a tool to extract the
(equation 1). waterbody content in order to map the land surface water
with acceptable results. The NDVI was used to indicate the
r.mapcalc NDVI = 1.0×(NIR – Red)/(NIR + Red) green vegetation presents in the pixel to classify vegetated
(equation 1) and non-vegetated areas (Verrelst et al. 2008; Bhandari et
al. 2012; Pujiono et al 2013).
254 N U S A N T A R A B I O S C I E N C E 9 (4): 251-259, August 2017

i.pansharpen equation : output previx =

(equation 5)

Where,
ms1 = Name of input raster map (Green: B2).
ms2 = Name of input raster map (NIR: B4).
ms3 = Name of input raster map (MIR: B5).
Pan = Name of input raster map (Panchromatic: B8).

Output prefix = Name for output raster map prefix (e.g.


"fusedimage").
in = The name of the raster image to be rescaled.
Out = The resulting raster map name.
From = min, max (the input data range to be rescaled).
To = min,max (the output data range).

Image classification
Image classification was performed on the fused
images. To extract the mangrove forest area from the
Figure 2. Screenshot of the r.composite tool in Grass GIS ver.
7.0.2. images, we used a maximum likelihood decision rule
method with two classes that were defined for mangrove
r.composite r = Blue g = NDVI b = NDWI output = forests map: mangrove forest and non-mangrove forest
Mangrove_compo (equation 3) (e.g. built-up area, fishpond, and water).
Generating supervised classification map using the
Where : maximum likelihood method can be conducted after
r = Red Channel creating training area. Training area represents the spectral
g = Green Channel information of each land use/land cover class (Perumal and
b = Blue Channel Bhaskaran 2010; Ahmad 2012). A vector layer map has to
be created to the layer manager before training areas can be
Image fusion digitized. The process is available from the Graphical User
Since Landsat 4-5 TM does not has a panchromatic Interface (GUI) of GRASS GIS by choosing "develop
band, the image fusion technique was performed only on vector map" in the "vector" menu tab and then click "create
Landsat 7 ETM+ and Landsat 8 OLI. An image fusion vector map”. We created 100 training areas for each of fused
technique was conducted using the panchromatic band of image. Fifty training areas for every land cover class
Landsat 7 ETM+ and Landsat 8 OLI and the RGB image. (mangrove forest and non-mangrove forest) were evenly
This technique was performed to enhance the image distributed and mixed pixels were avoided carefully since
resolution for a better visual interpretation and to enhance the selection of training area can significantly influence the
the classification result by fusing the high spatial resolution performance of image classification algorithm (Kavzoglu
of the panchromatic image with the multispectral content of and Yildiz 2011; Colditz 2015). The vector training area
multiband images (RGB image) into a single band (Cetin then must be converted into raster format using the
and Musaoglu 2009; Butt et al. 2015; Muhsin and Salih “v.to.rast” expression (equation 6). To select all relevant
2015). Several examples of the study conducted by Han et bands (blue band of Landsat 4-5 TM and Landsat 7 ETM+,
al. (2008), Sarup and Singhai (2011), and Yuhendra et al. a deep blue coastal band of Landsat 8 OLI, NDVI images,
(2011) have illustrated the advancement utilization of and NDWI images) of raster images we used the “i.group”
image fusion with reliable results. Landsat 8 OLI stores expression (equation 7). To generate statistics from the
values as "DN" from zero to 65,535. Thus, we need an training area, the “i.gensig” expression (Equation 8) was
additional step to rescale it into zero to 255 to get the same used. The inputs into the “i.gensig” for the creation of
acceptable color-balanced composite image as the Landsat signature statistics are the training area images in raster
7 ETM+ after pan-sharpening (Ramdani et al. 2015). format, which its format conversion has been performed
Rescaling those values can be done using the "r.rescale" priory.
expression on GRASS GIS command console (equation 4). The maximum classification likelihood algorithm was
The Brovey spectral sharpening technique was used in this performed using the “i.maxlik” expression (equation 9) in
study using the "i.pansharpen" expression in GRASS GIS GRASS GIS. The results were then converted into vector
software (equation 5). format using the “r.to.vect” expression (equation 10). The
final analysis and cartography design were then performed
r.rescale in = B(i)_DNs from = 0,65535 out = using QuantumGIS software ver. 2.16.2.
B(i)_DNs_255 to = 0,255 (equation 4)
NURSAMSI & KOMALA – Mangrove forest change assessment 255

v.to.rast input = training _ vector output = training _ reference = Name of raster map containing reference
raster (equation 6) classes.
i.group group = Landsat7 _ group subgroup = Landsat7 output = Name for output file containing error matrix
_ subgroup input = B1,NDVI,NDWI (equation 7) and kappa.

i.gensigtrainingmap= training _ raster group = Landsat7


_ group subgroup = Landsat7 _ subgroup RESULTS AND DISCUSSION
signaturefile= Landsat7 _ signature (equation 8)
RGB color composite and supervised classification
i.maxlik group = Landsat7 _ group subgroup = using Maximum Likelihood decision rule
Landsat7 _ subgroup RGB (Red-Green-Blue) color composite was produced
signaturefile= Landsat7 _ signature class = mangrove _ by using the Blue Band (Landsat 5 TM and Landsat 7
class7 (equation 9) ETM+) and the deep blue coastal in Landsat 8 OLI as red
channel, NDVI as Green channel, and NDWI as Blue
r.to.vect -s input = mangrove _ class7 output = channel. This combination will produce an image that
mangrove _ class7 feature = area (equation 10) highlights the striking differences of mangrove forests
among another landcover classes.
Field work and accuracy assessment Maximum likelihood decision rule is a classification
Field work was carried out in 2 villages from 2 districts tool that considers both the variances and co-variances of
namely: (i) Cangkring Village, Cantigi Sub-District, the class signatures when assigning each pixel to one of the
Indramayu. (ii) Patimban Village, Pusakanagara Sub- classes represented in the signature file (Myint et al. 2014;
District, Subang. Field data were collected using Garmin Srivastava et al. 2014) which was made from training area.
Oregon 550 GPS. For accuracy assessment, Google Earth Therefore, the training area needs to be carefully selected;
Imagery was used to help visual interpretation from above otherwise, the classification result may introduce error
the ground. We transformed the image of study area taken (Ramdani et al 2015). Maximum likelihood utilizes the
from Google Earth into KML format to be imported into class signature as a representative means and variance
the GPS so we are able to performed landcover validation. values of each land cover class which are then used to
Two hundred validation points were selected randomly for estimate the probabilities. Maximum likelihood decision
each study area. The Google Earth image was also used to rule considers not only the mean or average values in
assist the selection of those validation points so that all of assigning classification, but also the variability of the
the points were distributed evenly between two land cover brightness values of each class (Campbell 2011).
classes (mangrove and non-mangrove class). Several points
from each village were randomly selected to be validated Mangrove forest change detection
directly in the field (Hansen and Loveland 2012). Mangrove forest change analysis in this study is used to
Accuracy assessment calculation was performed by measure the successfulness of mangrove forest plantation
generating a kappa value (Liu et al. 2007; Mas 2012; program performed by the Forestry Department of West
Ramdani et al. 2015). To generate a kappa value we need Java in 2007. In addition, this study also measures the
to import shapefile data of the ground truth and the mangrove forest change before the program performed.
classified map (GrassGIS Development Team 2016) into Therefore, we can present a quantitative measurement why
GRASS GIS with “v.in.ogr” command (equation 11), and such a program was needed.
then convert it again to a raster with “v.to.rast” command After overlaying the mangrove forest extraction from
(equation 12). We then employed the “r.kappa” command classification result in 1996 with the one from 2006 also
(equation 13) in GRASS GIS command console. overlaying the mangrove forest extraction in 2006 with the
one from 2016, clearly we can see the changes in the
v.in.ogrdsn /home / user / shape _ data layer survey _ mangrove forest area before and after the program
data = output = survey _ area performed.
v.in.ogr input=/home/user/shape_data/test_shape.shp The kappa analysis and subsequent accuracy of the
output=grass_map (equation 11) classification using RGB composite color are presented in
Table 1. The kappa statistic of 0.90 provides strong
v.to.rast input = survey _ area output = survey _ area confidence to the results of this study (Gwet 2008; Foody
(equation 12) 2009; McHugh 2012).
r.kappa classification = mangrove _ class7 reference =
Table 1. Classification accuracy of Landsat 8 OLI
survey _ area output = result _ kappa (equation 13)
Mangrove Fishpond
Where, Class/Region Water (%)
(%) (%)
layer = Name for input shapefile vector data. Water 97.00 0.00 6.00
input = Name for input vector map. Mangrove 0.00 97.00 0.00
output = Name for output vector/raster map. Fishpond 1.00 0.00 93.00
classification = Name of raster map containing Overall Kappa = 0.90
classification result. Overall classification accuracy = 93.02%
256 N U S A N T A R A B I O S C I E N C E 9 (4): 251-259, August 2017

Figure 3. RGB color composite and classification results of Figure 4. RGB color composite and classification results of
mangrove forests in Indramayu District. Group A (from left to mangrove forests in Subang District. Group A shows the RGB
right) is the RGB color composite that shows the different color color composite that shows a different color for different types of
for different types of land surface. Group B (from left to right) land surface. Group B shows the Mangrove forests extraction
shows the Mangrove forests extraction derived from the derived from a supervised classification method. The red polygon
supervised classification method. The red polygon represents the represents the area of the program.
area of the program.

Figure 6. The mangrove forest change detection in Subang


District derived from the classification method. The year 1996-
2006 shows the change before the mangrove forest plantation
program was performed in 2007 and the year 2006-2016 shows
Figure 5. The mangrove forest change detection in Indramayu the change in mangrove forests after the program performed. The
District derived from the classification method. The year 1996- red polygon represents the area of the program.The red polygon
2006 shows the change before the mangrove forest plantation represents the area of the program.
program was performed in 2007 and the year 2006-2016 shows
the change in mangrove forests after the program performed. The
red polygon represents the area of the program.

Discussion program in 2007 was initiated. The mangrove forest


Mangrove forest change detection over the ten-years plantation program in 2007 was conducted in a total of 400
period before the program performed in Indramayu was ha area in Cangkring Village, Cantigi Sub-District,
conducted to analyze the general description about why Indramayu. The program was performed with the help of
such a program was needed. Figure 3 illustrates the the community since they are well-informed about the
enormous decline in mangrove forest area due to the ecological advantage of mangrove forest (Forestry
expansion of fishpond and salt pond activities, as shown in Department 2013).
the figure where the sky blue color of mangrove forest Mangrove forest change detection in the same period of
declined in conjunction with the increased of the dark blue time before the program performed in Subang District
color of water that found in the fish pond. The shows a different pattern from the result shown in
quantification of the mangrove forest declining is presented Indramayu. From 1996 to 2006, there was a significant
in Table 2. This change was also detected by the Forestry increase in mangrove forest area due to the wide area of
Department; therefore, the mangrove forest plantation abandoned fishpond that remains unused; therefore, it
NURSAMSI & KOMALA – Mangrove forest change assessment 257

occupied by the mangroves (Figure 5 and Table 3). Lots of


fishponds were abandoned as the sudden increase of sea Year Area (ha) Changes (%)
water level flooded their fishpond and released their fishes 1996 1060.83 0.00
into the ocean. The farmers then have no more fund to 2006 258.75 -75.6
continue their business, so they prefer to search opportunity 2016 719.46 +174.2
in another field of business (Forestry Department 2008).
The after-program mangrove forest change detection in
the applied area of program in Indramayu shows an Table 3. Temporal changes of mangrove forest area in applied
increasing area of mangrove forest (Figure 3 and Table 2). area of program in Subang District, West Java Province,
The increasing of mangrove forest area at approximately Indonesia
17.8% year-1 indicates the successfulness of the program.
The successfulness of the program also detected in the Year Area (ha) Changes (%)
applied area of program in Subang District by the 1996 60.93 0.00
increasing of approximately 16.3% year-1 of mangrove 2006 123.12 +102.1
forests area. All of these achievements were due to the 2016 336.47 +173.3
community participation in rehabilitation, conservation,
and management of mangrove forest through the program.
However, the community also asked the government to The increasing of mangrove forest areas both in
accommodate their need of income from aquaculture Indramayu and Subang Districts indicates that the main
activities by increasing their business knowledge through purpose of the mangrove plantation program has been
technical training and supporting of seaweed seedlings to achieved. The sustainability of the mangrove forest in these
diversify the aquaculture activities. In addition, They also areas is another goal that can be ensured by maintaining the
demand a clear policy to protect both the sustainable of positive growth rate of mangrove forest. To achieve this
mangrove forest and the sustainable of their income from goal, the government must not abandon the participation of
aquaculture activities. the community. The community will gladly participate in
Another approach to accommodate those purposes is to such a program if they can gain some profits in return.
re-introduce a system that combines the fishpond (for fish Thus, the idea of ensuring sustainability of both the
farming and/or shrimp) with mangrove trees in the pond, mangrove forests and the income from aquaculture
called silvofishery (Pujiono et al. 2013). In Indonesia, a activities must become a long-term program of the
silvofishery system has been applied since the early 1990s government. To monitor both the short and long-term
(Wetland International-The Green Coast Project 2006). The mangrove rehabilitation program, remote sensing is
Southeast Asian Fisheries Development Center proposed to be used widely to become such an initial step
(SEAFDEC) stated that the model of silvofishery consists to collect fast, robust, and reliable data.
of a ratio of 60–80% mangroves and 20–40% pond canal This study also tried to highlight the importance of
for aquaculture. This system has a great chance to be freely available geospatial software such as GRASS GIS
rejected by the people if the re-introduction does not and QGIS software and satellite images that are freely
conduct properly. People will assume that the silvofishery available in the public domain such as Landsat data.
system will reduce their fish productivity because of the Several studies have been conducted by implementing the
high proportion of land that must be occupied by mangrove inexpensive method of remote sensing to remotely observe
plants. They will apparently need some proof and warranty the change and the conversion of mangrove forest area; for
that silvofishery system will not reduce their productivity. example, the application of Landsat image to assess the
To answer this question, the study conducted by Kupang decreased of mangrove forest area in Al-Dakhira, The State
Forest Research Institute in 2009 found that the fish weight of Qatar (Balakrishnan 2012). This study presented that the
in the silvofishery system is greater than that of the application of an inexpensive method of remote sensing is
traditional fishpond (Pujiono et al. 2013). In addition, extremely useful to understand the coastal habitats,
during the first two years of observation, the growth rate of especially mangroves.
mangrove species (Rhizophora mucronata Lam.) reached The application of Landsat images and open source
16.01 cm year-1 and the growth rate of diameter reached software also implemented in the study of mangrove
1.02 cm year-1 (Njurumana et al. 2010). Another study, mapping and change detection in HaiPhong City, Vietnam
focusing on assessing the advantage of silvofishery system, (Dat-Pham 2015). This study highlights the mangrove
was conducted in Vietnam in a two-years period of time by forests declining throughout the year 1989 to 2013 with the
Food and Agriculture Organization of the United Nations overall accuracy of 83% and the Kappa coefficient of 0.81.
(FAO). FAO reported an increasing of the shrimp In Indonesia, Ramdani et al. (2015) also promote the use of
productivity by 10% year-1 after the silvofishery program inexpensive method of remote sensing to be used widely in
was initiated. This implementation of silvofishery system an attempt to help monitor the mangrove forest areas in
in Cha Mau Province, Vietnam, project will be held for the Indonesia without encumbering the government financially.
next 20 years. Thus, the silvofishery system has been In addition, this study also pursued the same goals with the
proofed to be a suitable method to be implemented as an specification in evaluating and monitoring the mangrove
addition program of mangrove rehabilitation. plantation program held by the government.
Table 2. Temporal changes of mangrove area in applied area of
program in Indramayu District, West Java Province, Indonesia
258 N U S A N T A R A B I O S C I E N C E 9 (4): 251-259, August 2017

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N U SAN TA RA B IOSC IE NC E ISSN: 2087-3948
Vol. 9, No. 3, pp. 260-267 E-ISSN: 2087-3956
August 2017 DOI: 10.13057/nusbiosci/n090304

Development of shrimp shells-based compost and plant-based pesticide


using bio-activators from Golden Apple Snails and their effects on the
kenaf plant growth and pest population

RUSMINI♥, RIAMA RITA MANULLANG, DARYONO


Politeknik Pertanian Negeri Samarinda. Jl. Samratulangi Gunung Panjang, Samarinda 75131, P.O. Box 192, East Kalimantan, Indonesia. Tel.: +62-541-
260421, Fax.: +62-541-260680, ♥email: mini9964@rocketmail.com

Manuscript received: 13 November 2016. Revision accepted: 13 June 2017.

Abstract. Rusmini, Manullang RR, Daryono. 2017. Development of shrimp shells-based compost and plant-based pesticide using bio-
activators from Golden Apple Snails and their effects on the kenaf plant growth and pest population. Nusantara Bioscience 9: 260-267.
Kenaf (Hibiscus cannabinus L) cultivation generally uses chemical fertilizers and pesticides to increase production despite the adverse
effects of those chemicals toward environmental ecosystem. Meanwhile, there are a lot of unutilized wastes produced from agriculture
and fishery. This study aimed to develop the best bio-activator from Golden Apple Snails and spices to decompose the shrimp shells
waste. The bioactivator was utilized for the production of plant-based pesticide to control pests in kenaf plant, and shrimps-shells-based
composts to improve kenaf plant growth. This study employed a Randomized Block Design with two factorials. The first factor was the
shrimp shells-based composts (K) which consist of three levels, and the second factor was the plant-based pesticide (P) which also
consists of three levels. Each of experiment was repeated twice. Thus, in total there were 18 treatments. The shrimp shells-based
composts had a pH value of 8.79, Potassium (K) 8.13 %, organic carbon (C) 17.45%, Nitrogen (N) 3.62%, Phosphorus (P) 2.27%;
Magnesium (Mg) 0.59%, and Calcium (Ca) 7.64%; and a C/N ratio of 4.82. Kenaf plant height at 6 and 9 weeks after planting (WAP)
showed significant differences upon the shrimp shells-based composts treatments. At 6 weeks after planting, kenaf plant diameter
showed significant differences upon the composts treatment, while at 9 weeks after planting, the plant diameter indicated significant
differences upon both the compost and plant-based pesticide applications. Pest populations that were present in kenaf plants were
caterpillars, bugs, and beetles.

Keywords: Golden Apple Snails, kenaf, shrimp shells

INTRODUCTION effects from its residue that affect the environment and the
safety of agricultural products (Huang et al. 2014).
Kenaf (Hibiscus cannabinus L.) is a plant well known Another issue is the increasingly high fertilizer prices
for producing natural fibers that are eco-friendly because of because of the chemical fertilizers distributed in Indonesian
its readily degradable nature and its capacity for absorbing market is highly dependable on the imported raw materials
a significant amount of CO2. Besides as a raw material for whose prices rise following the US Dollar rate in the
agricultural/estate crops product packaging, there is a vast international currency market. The use of inorganic
array of diversified products of the kenaf fiber, such as: fertilizers dramatically increases because of the farmer
paper, wallpaper, car interior material, geotextile, soil safer, fanaticism and the growth in the agricultural field area,
fiber drain, particle board, reinforced plastic, and raw whereas the use of organic fertilizers does not expand.
material in biofuel industry (Lips and Dam 2013; Ryu et al. Development of organic fertilizer was based on the adverse
2013; Saba et al. 2015; Ryu et al. 2016; Bourguignon et al. effects exerted by the chemical fertilizer towards soil and
2017). plants. Organic fertilizer is developed to solve the negative
Kenaf cultivation generally involves the use of effects of chemical fertilizer that can drain a nutrient rich
chemical fertilizers to boost its production, despite the soil and turn it into a nutrient-deprived soil. Plants growing
harm exerted by the chemical to the environment. The on the nutrient-deficient soil suffer malnutrition and less
continuous application of fertilizers, pesticides, and other vigorous (Maheswari et al. 2014; Cheng et al. 2014;
chemicals can harm organisms in the soil, induce pest and Nkbiwe et al. 2016; Thonar et al. 2017).
pathogen resistance, and change the vitamin and mineral Bodies of water in Indonesia are rich with various
content of crops. Indeed, if this practice is left as is, it will invertebrates such as shrimps. Shrimps are commonly
deleteriously affect the cycle of life survival (Xu et al. consumed seafood among Indonesian people. However,
2012; Maheswari et al. 2014; Nkbiwe et al. 2016; Thonar et shrimp shells often time are wasted without being utilized.
al. 2017). However, agricultural farming is impossible to A pile of shrimp shells waste produces a putrid odor that
be undertaken without involving pesticides. Pesticide pollutes the air and disturbs people activities (Prakash et al.
controls plant diseases and pests as well as grain insects, 2011). One alternative to solve this issue is to utilize the
while at the same time pesticide causes a lot of adverse shrimp shells waste as organic fertilizer by composting.
RUSMINI et al. – Development of compost and biopesticide using Golden Apple Snails 261

According to Jamaluddin et al. (2013; Osman (2012); Instruments and materials


Nathand Singh (2016), production of organic fertilizer in Instruments employed in this research were a water
the form of liquid is less effective if the fermentation time plastic drum (capacity 200 l), scale, grinding machine, a
is short and the cellulose content of the raw material is wooden container for fermentation, and stirrer. Materials
hard. These two conditions cause an incomplete used in this study were Golden Apple Snails, cattle urine,
fermentation of the waste material, as indicated by the low rice-washing water, coconut water, palm sugar, kencur
nutrient content of the analyzed material sample. (Kaempferia galanga), turmeric, temulawak (Curcuma
The addition of an activator in the form of zanthorrhiza), shrimp shells, rice bran, chicken manure and
microorganism can accelerate decomposition of organic Kenaf variety KR-11 seeds.
waste. The activator microorganism can be from a local
microorganism (MOL). MOL is a liquid fermentation Experimental design
product generated from easy-to-obtain local resources. One This study was a two factorials experiment conducted
of the natural resources that can be utilized in MOL using a randomized block design. The first factor was the
production is Golden Apple Snails (Pomacea canaliculata shrimp shells-based composts (K) which consist of three
and Pomacea maculata). Golden Apple Snails, a levels, namely: K1 = 10 ton ha-1 compost application (1
disconcerting pest to farmers, is an excellent source of kg/plot), K2 = 20 ton ha-1 compost application (2 kg/plot),
bacteria for compost activator. MOL of the snail includes K3= 30 ton ha-1 compost (3 kg/plot); the second factor was
organic matter bacteria decomposer, plant growth the plant-based pesticide using a diluted bio-activator from
regulators, pest and pathogen control agents, and nutrients Golden Snails (P) which consists of three levels, namely:
for plants. MOL of Golden snails contains Pseudomonas P0 = control, P1 = 50 ml pesticide /plant pesticide, P2 = 100
fluorescence (Rusmini et al. 2016). The bacteria were able ml pesticide/plant
to suppress the population of pathogens and associated with Each experiment was repeated twice. Thus there were
protected plant roots from soil pathogen infection via root 18 units of experiment/plot. Each plot consisted of 16 kenaf
surface colonization, secretion of chemical compounds plants. Hence in total, there were 288 kenaf plants. Each
such as antifungal and antibiotics, and competition in Fe sampling was done on four plants in the center of the plot.
cation absorption (Gajendran et al. 2016). Previous studies
report that Pseudomonas fluorescence was able to control Preparation of bio-activator
Anthracnose disease in banana (Peeran et al. 2014), and All materials were prepared with specified amount as
Fusarium wilt disease in onion and tomato (Gajendran et al. follows: 75 kg Golden Apple Snails, 45 l cattle urine, 30 l
2016; Khan et al. 2011). Furthermore, Pseudomonas rice-washing water, 22.5 L coconut water, 37.5 kg palm
fluorescens was able to solubilize phosphate from its fixed sugar, 22.5 kg kencur (Kaempferia galanga L.), 3.75 kg
and unavailable form, thus become available and turmeric, 7.5 kg temulawak (Curcuma zanthorrhiza Roxb.).
absorbable to plants (Khan et al. 2011). Furthermore, The Golden Apple Snails were pulverized into a fine
Subashri et al. (2012) reported that Pseudomonas ground by using grinding machine. Palm sugar, kencur,
fluorescens was also able to improve plant production. turmeric, and temulawak were blended into fine pieces
The low productivity of kenaf plant is caused by several using a blender. The ground snail material and other
hindrances, one of them is biotic stress including pest, materials were put into the plastic drum and then mixed
pathogen, and weed attack. Therefore, to tackle this issue, well. The drum containing the mixture was closed with a
we try to develop a plant-based pesticide using a diluted lid that had a small hole in it. A plastic hose was attached
bio-activator from Golden Snails to control pests that attack into the drum lid hole on the one end, and into a 1500 ml
Kenaf plant effectively. This study aimed to produce a bio- plastic bottle containing 500 ml water on the other end. The
activator from Golden Snails and the best spices and herbs mixture was stirred daily and fermented for 20-25 days.
as a decomposer of shrimp shells waste. The product will The final fermentation product or MOL would indicate
serve as a plant-based pesticide and generate composts certain characteristics, including no gas remaining, and
from shrimp shells waste for the best interest of kenaf plant smelled like tape (fermented rice or cassava). For
growth. composting, 500 ml MOL of golden snails was added into
1 kg organic matter and mixed thoroughly (a thoroughly
mixed organic matter will not break upon grasping). As for
the application of MOL as a plant-based pesticide, 1 l MOL
MATERIALS AND METHODS was mixed with 5 l water and sprayed on kenaf plants to
control the plant’s pest. Furthermore, the activator from
This study was conducted in Center for Agricultural Golden Snails was analyzed for its nutrient and bacterial
Technology Assessment (BPTP) of East Kalimantan, contents.
Indonesia for nutrient content analysis; and State
Agricultural Polytechnic of Samarinda, Indonesia for bio- Composts production from shrimp shells
activator development, nutrient content analysis, and All the necessary materials were prepared with
composts and plant-based pesticide application on fenaf specified amount as follows: 50 kg fresh shrimp shells, 20
plants. kg rice bran, 20 kg chicken manure. All Shrimp shells were
cut into fine pieces by a chopping machine. Shrimp shells
were mixed thoroughly with the rice bran, chicken manure
262 N U S A N T A R A B I O S C I E N C E 9 (4): 260-267, August 2017

and the MOL of the golden snails. The mixture was then al. (2017) that suggested that the high pH and Ca level in
transferred into a fermentation container. The mixture was crab shells-based composts was due to the alkaline mineral
flip once a week to maintain the ideal condition for comprising the crab shells. Crabs and shrimps are
composting. The mixture was then analyzed for its Crustacean whose shells contain protein, calcium
macronutrient level and C/N ratio. For application as plant carbonate, and chitin (Weidema et al. 2013).
fertilizers, the composts were applied 2 weeks before kenaf Nutrient analysis revealed that the composts N content
plantation. For the second application, the composts were was 3.62%, which was higher than that of the previous
used when the plants reached 1,5 month old. The plant- study (0.12 %) by Rusmini et al. (2015). This N level was
based pesticide was applied to the plants three times: still below the standard stipulated by the Regulation of
firstly, 6 day old after planting; secondly, 30 days after the Minister of Agriculture Republic Indonesia (Permentan).
first application; and thirdly, 30 days after the second This substandard N content was probably due to the MOL
application. of Golden Apple Snails used in this research only contain
Pseudomonas fluorescent. The addition of rice bran in the
Observations composting material did not seem to increase the N
Multiple observations were conducted on the MOL of content. The low N level could also be due to the chitin and
the Golden Apple Snails, including color, odor, and CaCO3 content of the shrimp shells. Shrimp shells
temperature observations. Whereas, with regards to shrimp consisted of protein (25%-40%), chitin ( (C8H13NO5)n)
shells-based composts, in addition to observations of color, (15%-20%), and calcium carbonate (CaCO3) (45%-50%)
odor, temperature, the compost humidity was also (Kandra et al. 2012 ; Radwan et al. 2012;Paul et al. 2015).
monitored. Moreover, the nutrient content analysis was Chitin can be further processed into chitosan (
carried out on the mature composts. Observations on plants (C6H11NO4)n) and glucosamine (C6H13NO5). Chitosan is an
included plant height and diameter measurements. Both antibacterial compound that can inhibit bacterial growth.
plant height and diameter were measured once every three The natural polycation of chitosan was able to inhibit the
weeks for three months. Pest observation was done through growth of bacteria and mold (Sarasam et al. 2008; Baherei
direct monitoring of the kenaf plants on a daily basis. et al. 2009; Kingkaew et al. 2014; Chudinova et al. 2016;
Kulikov et al. 2016; Fardioui et al. 2017).
Data analysis Shrimp shells contained chitin that inhibited
A difference in the nutrient content variable between decomposer microorganism development, thus hindering
each shrimp shells composting treatment was analyzed the composting process. Menurut Zhou and Haynes (2012).
using a parametric statistical method of Least Significant Kaya et al. (2014), Mohanasrinivasan et al. (2014),
Different (LSD) test. Nidheesh and Suresh (2015), chitin and its derivatives were
highly potential for antimicrobial materials.The low N
content in the composts could also because shrimp shells
RESULTS AND DISCUSSION contain 15%-20% CaCO3. The high calcium carbonate
content made N volatilize. According to Manurung (2011),
Bioactivator (MOL) from Golden Apple Snails application of lime on composts was not recommended.
Observation on the preparation of MOL of Golden Liming caused nitrogen to volatilize into gaseous ammonia.
Apple Snails indicated that the MOL did not produce smell In comparison, the N level of the composts produced in this
at day 28 after the onset of fermentation. This result study was lower than that of chicken-feather-based
indicated that each of the MOL raw material worked compost (7.23 %) supplemented with manure and rice bran
properly. In the fermentation mixture used in this study, the (Sorathiya et al. 2014), but higher than that of the compost
energy source was adequately supplied such as by the palm generated by Jamaluddin et al. (2013), Azim et al. (2015),
sugar. In addition, the carbohydrate supply in the mix also and Manullang and Rusmini (2015). Kusmiadi et al. (2014)
satisfied the need of the bacteria that were originated from reported a decrease in N level of the composts
the Golden Apple Snails. According to Rusmini et al. supplemented with crab shells because of chitin and CaCO3
(2015), the nutrient content in the MOL of the Golden of the shells. Khan (2009) suggested that addition of rice
Apple Snails was higher than those from fruits and banana bran and hull waste to the composting material as a
rhizome (Manullang and Rusmini, 2015). Bacteria species carbohydrate and nutrition source for the activator
in the MOL of Golden Apple Snails was also different from microorganisms led to composts with high N content.
those of fruits and banana rhizome. Bacteria in the MOL of Suwastika et al. (2005), Partey et al. (2013) and Sutanto et
Golden Apple Snails, fruits, and banana rhizome were al. (2016) suggested that addition of composted rice bran
Bacillus sp., Enterobacter sp., and Pseudomonas was able to increase the N content of the fertilizer. This
fluorescence, respectively (Rusmini et al. 2016). increase in the compost N content was due to the presence
of organic compounds.
Nutrient content of shrimp shells-based composts The phosphorus content of the shrimp shells-based
Nutrient content of the shrimp shells-based composts composts was 3.62%, which was higher than those of
was shown in Table 1. The compost's pH was 8.79 (basic). composts produced by Rusmini et al. (2015) (1.42%),
The high pH of the composts was likely due to the alkaline Azim et al. (2015), and Manullang and Rusmini (2015).
mineral content in the shrimp shells. This argument was in The P level indicated in this study met the Indonesian
agreement with Vandecastlee et al. (2016), and Munoz et National Standard (SNI) Number 19-7030-2004 on
RUSMINI et al. – Development of compost and biopesticide using Golden Apple Snails 263

composts, which stated the minimum P content is 0.10%. Table 1. Nutrient content of shrimp shells-based composts
The low P level in the composts was probably due to the
consumption of the P contained in the activator by Shrimp
SNI range*
microorganisms during composting (Vaneeckhaute et al. shells- Per-
Parameter Unit
based Min Max mentan**
2014; Ratna et al. 2015; Vandecasteele et al. 2016).
composts
The Potassium (K) content of the shrimp shells-based pH - 8.79 6.8 7.49 4-9
composts was 8.1%, which was higher than the minimum N total % 3.62 0.4 - min.4
Permentan standard and SNI for K of 4% and 0.20%, Water content % 31.83 - 50 15-25
respectively. The high K content of the composts was Organic C % 17.45 9.8 32 min.15
likely contributed by its raw material of shrimp shells. The C/N ratio - 4.82 4.82 0.10 15-25
K level of the composts produced in this study was greater P % 2.27 0.10 - min.4
than those of the composts reported by Manullang and K % 8.13 0.20 - min.4
Rusmini (2015) and Azim et al. (2015). Microorganism Ca % 7.64 - 25.50 -
Mg % 0.59 - 0.60 -
availability significantly affected Kalium content in
Note: *SNI (Indonesian National Standard) 19-7030-2004 and
composts. Ratna et al. (2015) stated that K element was
**Permentan (Regulation of Minister of Agriculture Republic
obtained from organic material decomposition by the Indonesia) Number 70/Permentan/SR. 140/10/2011
microorganism. Hence, the decline in K content was highly
influenced by the microorganism nutrition factor.
Organic carbon content identified in the shrimp shells The compost exhibited a low C/N ratio probably
composts was 17.45%, which satisfied the standard C because, during decomposition, microorganism preferred
content for organic fertilizer according to Permentan (15%) carbon as their energy source. This idea was corroborated
and SNI on composts (9.8-32%). The shrimp shells-based by Zaman and Endro (2007) who suggested that during
composts resulted in this study showed a lower organic C composting, microorganism decomposed organic matter
content than that of the previous study (27.3240%) aerobically and thus releasing CO2 and H2O and in turn
(Rusmini et al. 2015). This phenomenon was likely due to diminishing carbon content in the composting material.
the high chitin content of the shrimp shells. Upon such high Yuniawati et al. (2012) suggested that the decrease in C
chitin environment, the decomposer microorganism content during composting was due to the conversion of
required a greater amount of energy to break down the organic matters into gaseous CO2 and CH4, thereby
material. Microorganisms used carbon as energy sources, lowering the C/N ratio.
thus during composting the carbon content of the compost Widaryanto (2013); Partey et al. (2013) and Sutanto et
might gradually deplete. Manurung (2011) suggested that al. (2016) stated that the C/N ratio corresponded to the
the decline in the compost organic C content was due to the organic matters decomposition intensity. Higher C/N ratio
utilization of carbon as an energy source by the indicates that material was recalcitrant to decomposition,
decomposing agents to power their metabolism activities. whereas lower C/N ratio means that the material was
Laboratory analysis on the shrimp shells-based compost readily decomposed. Also, a higher C/N ratio was indicative
indicated a C/N ratio of 4.82, which failed to meet the C/N of cellulose and lignin-rich material, i.e. a recalcitrant
standard range of SNI 19-7030-2004 (10-20) and that of material. Conversely, a lower C/N ratio was indicative of
Permentan (15-25). The low C/N ratio was probably due to cellulose and lignin-poor material which was readily
the complete decomposition of the shrimp shells. The decomposed. The decrease in C content during composting
compost C/N ratio achieved in this research was better than was due to the conversion of organic matters into gaseous
that of the previous study (Rusmini et al. 2015). In that CO2 and CH4, thereby lowering the C/N ratio. Another
earlier study by Rusmini et al. (2015), the interaction value factor that accelerates a decomposition is the size of the
between MOL (a2) and the shrimp shells (b3) was 41.52, raw material. Kusmiadi et al. (2014) suggested that the low
which indicated that the compost was not completely C/N ratio in compost was also likely caused by the CaCO3
decomposed. The low C/N ratio showed that organic content in the compost material. Calcium carbonate in the
material in the two composting treatments was actually composting material will increase the decomposition rate.
decomposed. Depletion and maturation of the compost With regards to the Mg content of the compost in this
material were indicated by compost flaking, color change study, its level reached 0,59% which was below the
into soil color, no odor, and low water content in the maximum Mg level dictated by SNI 19-7030-2004 for
bokashi and composts. Composting rate depends on the compost fertilizer (0.60 %). The Mg level of the compost in
availability of C and N elements in the bokashi/compost this study was slightly higher than that of the compost
material. Microorganisms require a balance amount of C produced by Rusmini et al. (2015) (0.5121%). Meanwhile,
and N to decompose organic materials during composting the Ca content of the shrimp shells-based composts was
process optimally. Organic matters will be oxidized by 7.64% or in other words was below the maximum compost
microorganisms, thereby reducing the C/N ratio of the Ca level of 25.5% as recommended by SNI for compost
matters and devouring all its N element. Supplementation fertilizer. The compost Ca level higher compared with that
of the organic matter with an N-rich material such as rice of the previous report by Rusmini et al. (2015) (1.10%).
bran can alleviate the N loss. Probably, the significant amount of calcium carbonate
constituting the shrimp shells contributed to the high Ca
content of the resulted composts.
264 N U S A N T A R A B I O S C I E N C E 9 (4): 260-267, August 2017

Table 2. Mean of kenaf plant height (cm) 3 weeks after planting composts gave significant effects on kenaf plant height. K3
(WAP) upon plant-based pesticideapplication (P) and shrimp treatment resulted in the highest plant height of 165.7083
shells-based compost (K) cm. The plant height upon K3 treatment (164.7083 cm)
was significantly different from that of K1 treatment
Treatment K1 K2 K3 Mean
(133.6667 cm) but was not from that of K2 treatment
P0 21.1250 20.8150 21.1900 21.0433
P1 23.4400 20.0000 25.3150 22.9183
(155.8333 cm). This result indicated that at 6 weeks after
P2 22.2550 25.5050 20.8150 22.8583 planting, the shrimp shells-based composts were maximally
Mean 22.2733 22.1067 22.4400 absorbed by the kenaf plants. In agreement with this result,
Note: Mean followed by the same letter indicated a non- Daryono (2016) reported that at 75 days after planting,
significant difference (α = 5%) plant growth increase showed significant differences in
response to fertilizer treatments applied in the experiment,
whereas at 15, 30, 45, and 60 days after planting, such
Table 3. Mean of kenaf plant height (cm) 6weeks after planting significant differences in plant growth increase were
(WAP) upon plant-based pesticide application (P) and shrimp absent.
shells-based compost (K) Table 4 indicated the plant height upon pesticide (P)
and composts (K) treatments on kenaf plant 9 weeks after
Treatment K1 K2 K3 RMean
130.6250 141.1250 168.0000 146.5833
planting. The shrimp shells-based compost treatments
P0
P1 139.0000 165.5000 162.7500 155.7500 significantly affected kenaf plant height. K3 treatment
P2 131.3750 160.8750 163.3750 151.8750 which represented the highest amount of compost applied
Mean 133.6667 b 155.8333 a 164.7083 a to the plants resulted in the highest plant height of
Note: Mean followed by the same letter indicated a non- 223.1667 cm. This plant height upon K3 treatment was
significant difference (α = 5%) using LSD test (K) 16.00 significantly different from that of the lowest concentration
of compost applied, the K1 treatment (185.3750 cm) but
was not from that of K2 treatment (213.7917 cm). Again,
Table 4. Mean of kenaf plant height (cm) 9weeks after planting this result was in agreement with Daryono (2016) who
(WAP) upon plant-basedpesticide application (P) and shrimp
reported that at 75 days after planting, plant growth
shells-based compost (K)
increase showed significant differences in response to
Treatment K1 K2 K3 Mean fertilizer treatments applied in the experiment, whereas at
P0 172.5000 196.3750 220.8750 196.5833 15, 30, 45, and 60 days after planting, the growth increases
P1 185.3750 224.1250 217.7500 209.0833 were not significantly different. Rusmini and Nurlaila
P2 198.2500 220.8750 230.8750 216.6667 (2016) pointed out that application of liquid fertilizer from
Averange 185.3750 b 213.7917 a 223.1667 a three types of agricultural wastes did not give significant
Note: Mean followed by the same letter indicated a non- effect on the kenaf plant height. Meanwhile, Khodijah et al.
significant difference (α = 5%) using LSD test (K) 23,31 (2015) reported that application of solid and liquid
formulations of bioinsecticide supplemented with an active
ingredient of entomopathogenic B. bassiana fungi together
Table 5. Mean of kenaf plant diameter (mm) 3weeks after
with a liquid formulation of bioinsecticide with a shrimp
planting (WAP) upon plant-based pesticide application (P) and
shrimp shells-based compost (K)
shells-based carrier material as well as with a solid
formulation of compost enriched with Trichoderma sp.
Treatment K1 K2 K3 Mean fungi on rice plants was able to positively affect rice plant
P0 2.6600 2.9450 3.6100 3.0717 vigor such as stem height, productive tiller number, grain
P1 2.5350 2.8350 2.7050 2.6917 weight per clump, grain number per panicle. Furthermore,
P2 2.0550 2.5200 2.5900 2.3883 a study by Yenanni (2013) on the effect of shrimp shells-
Mean 2.4167 2.7667 2.9683 based compost media on the growth of ornamental plant,
Note: Mean followed by the same letter indicated a non- Aglaonema costatum, identified that 4% shrimp shells
significant difference (α = 5%) waste powder treatment resulted in the optimum growth of
the Aglaonema costatum, with a mean of total leaf number
of 6.25 and an average of plant height of 21.5 cm.
Table 2 showed the analysis of variance on kenaf plant Table 5 presented the analysis of variance of kenaf
height 3 weeks after planting (WAP) indicated that there plant diameter at 3 weeks after planting. There were no
were no significant differences among pesticide application significant differences in the plant diameter in response to
treatments (P) and shrimp shells-based compost application the plant-based pesticide (P) and the shrimp shells-based
treatments (K). This result was probably due to the composts (K) treatments. It was likely that at 3 weeks after
suboptimum absorption of the composts and pesticides by planting, both the pesticide and the compost were not
the kenaf plants at 3 weeks after planting, thereby affecting optimally absorbed by the plant, resulting in no significant
plant height. effect on the plant diameter. In line with this present result,
Table 3 showed the analysis of variance of Kenaf plants Rusmini and Nurlaila (2016) have also shown that
height treated with plant-based pesticide treatments (P) and application of liquid fertilizers produced from three
shrimp shells-based compost treatments (K) at 6 weeks different agricultural wastes did not significantly affect the
after kenaf planting. Applications of shrimp shells-based content of kenaf fiber.
RUSMINI et al. – Development of compost and biopesticide using Golden Apple Snails 265

Tabel 6. Mean of kenaf plant diameter (mm) 6weeks after diameter of 22.4050 mm. The plant diameter upon K3
planting (WAP) upon plant-based pesticide application (P) and treatment differed significantly from that of the lowest
shrimp shells-based compost (K) concentration of compost applied, the K1 treatment
(15.5917 mm ) but did not so from that of the K2 treatment
Treatment K1 K2 K3 Mean
(20.0300 mm). This result indicated that at 6 weeks after
P0 11.3000 13.5000 16.4650 13.7550
P1 10.9950 15.7150 16.8100 14.5067
planting, the applied composts were optimally absorbed.
P2 13.2850 15.7500 16.9650 15.3333 With regards to the effect of the plant-based pesticide, the
Mean 11.8600 c 14.9883 b 16.7467 a application of P2 dose (100 ml pesticide/plant) showed a
Note: Mean followed by the same letter indicated a non- significant effect on plant diameter compared with that of
significant difference (α = 5 %) with LSD test (K) 1.23 control treatment (P0). Meanwhile, there were not
significant differences in the resulting plant diameter
between the P¬1 with those of the control and the P2
treatments. As stated earlier, this finding was in agreement
Tabel 7. Mean of kenaf plant diameter (mm) 9weeks after with Daryono (2016). Previously, Daryono (2016) reported
planting (WAP) upon plant-based pesticide application (P) and
that plant height increase exhibited significant differences
shrimp shells-based compost (K)
in response to fertilizer treatments applied in the
Treatment K1 K2 K3 Mean experiment at 75 days after planting, whereas such
P0 13.0650 17.3550 22.4400 17.6200 b significant differences were not observed at 15, 30, 45, and
P1 15.2200 21.7450 21.3150 19.4267 ab 60 days after planting.
P2 18.4900 20.9900 23.4600 20.9800 a Table 8 showed demonstrated that there were three pest
Mean 15.5917 b 20.0300 a 22.4050 a populations infesting the kenaf plants. The total individuals
Note: Mean followed by the same letter indicated a non- constituted each pest population of each pest was still
significant difference (α = 5 %) LSD test (K) 1.23 ; LSD test (P) below the economic threshold. This observation indicated
2.40 that the plant-based pesticide supplemented with diluted
MOL of the Golden Apple Snails was able to control the
pests as well as to provide nutrients required for the plant
diameter growth and increase. Furthermore, it appeared
Table 8. Pest population of kenaf plants during 9 weeks of
observation that the application of shrimp shells-based composts was
able to control the pests that attacked the plants. This
Pest type Amount present finding was in agreement with Syahril et al. (2014)
Caterpillar 15 that reported that an extract of shrimp shells compost
Bugs 5 effectively suppressed the severity of leaf rust disease in
Beetle 4 long beans (Vigna unguiculata) with a suppression rate of
59.4%, and rotten leaf disease in angled luffa (Luffa
acutangula) and cucumber with a suppression rate of
10.31% and 14.85%, respectively. In addition, the
Table 6 showed the plant diameter upon pesticide (P) application of the extract of shrimp shells compost was also
and composts (K) treatments on kenaf plant at 6 weeks able to increase oyong and yardlong bean plants production
after planting. The shrimp shells-based compost treatments up to 17.40% and 246.67%, respectively. Moreover,
significantly affected kenaf plant height. K3 treatment Khodijah (2012) stated that utilization of shrimp shells-
which represented the highest amount of compost applied based compost extract as a carrier material for liquid
to the plants resulted in the largest kenaf plant diameter of formulation bioinsecticide with an active ingredient of
16.7467 mm. This plant diameter upon K3 treatment was Beauveria bassiana had been proved to be effective in
significantly different from that of K2 treatment (213.7917 controlling the rice stem borer pest in the swamp and non-
cm), and that of the lowest compost concentration applied, tidal marsh areas. In addition to containing chitin which
the K1 treatment (185.3750 cm). The significant difference was required by B. bassiana fungi in order to maintain the
in the plant diameter in response to the compost treatments fungi virulence during storage, the extract of the shrimp
might indicate that at 6 weeks after planting, the shrimp shells-based compost also included other elements for
shells-based compost was already maximally absorbed by improving rice plant production.
the plant. This result, again, was in line with the study To conclude, this present study concluded that the
conducted by Daryono (2016) who reported that at 75 days shrimp shells-based composts exhibited a pH value of 8.79;
after planting, plant height increase showed significant nutrient content: Potassium (K) 8.13 %, organic carbon (C)
differences in response to fertilizer treatments applied in 17.45%, Nitrogen (N) 3.62%, Phosphorus (P) 2.27%;
the experiment, whereas at 15, 30, 45, and 60 days after Magnesium (Mg) 0.59%, and Calcium (Ca) 7.64%; and a
planting, the height increases were not significantly C/N ratio of 4.82. Kenaf plant height at 6 and 9 weeks after
different. planting (WAP) showed significant differences upon the
Table 7 showed that both the plant-based pesticide (P) shrimp shells-based composts treatments. At 6 weeks after
and the shrimp shells-based composts (K) applications planting, kenaf plant diameter showed significant
gave significant effects on the plant diameter at 9 weeks differences upon the composts treatment, while at 9 weeks
after planting. In response to K3, the highest amount of after planting, the plant diameter indicated significant
composts applied, the kenaf plants showed the largest plant
266 N U S A N T A R A B I O S C I E N C E 9 (4): 260-267, August 2017

differences upon both the compost and plant-based Khodijah, Meidalima D, Nunilahwati H et al. 2015. The Performance of
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10.1134/S0003683816050100.
Competitive Research Grants 2015 and 2016 no.SK Kusmiadi R, Khodijah N, Akbar A. 2014. The Utilization Chicken
24/PM/2015 that funded this study. Authors also thank Feather and Crab Shells for improving the Physical and Chemical
Head of P2M Unit, State Agricultural Polytechnic Institute Quality Compost. Enviagro 7: 33-42.
of Samarinda and all parties that helped the completion of Lips SJJ, Dam JEGV. 2013. Kenaf fibre crop for bioeconomic industrial
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this research. Purpose Crop for Several Industrial Applications. Springer, London
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DOI: 10.1007/s12665-011-1321-4
N U SAN TA RA B IOSC IE NC E ISSN: 2087-3948
Vol. 9, No. 3, pp. 268-274 E-ISSN: 2087-3956
August 2017 DOI: 10.13057/nusbiosci/n090305

The appearance of rabbit skin tissue (Oryctolagus cuniculus) after


supplementation of Aloe vera and Spirulina fusiformis

YASMI PURNAMASARI KUNTANA1,♥, HUSMY YURMIATI2, ASRI PENI WULANDARI1,


FARIDA SYAFITRI3, RUHYAT PARTASASMITA1,♥♥
1
Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Padjadjaran. Jl. Ir. Soekarno Km 21, Jatinangor, Sumedang 45363,
West Java, Indonesia. Tel. +62-22-7796412 line 104, Fax. +62-22-7794545, email: y.purnamasari@unpad.ac.id; ruhyat.partasasmita@unpad.ac.id
2
Departmenf of Animal husbandry production. Faculty of Animal Husbandry, Universitas Padjadjaran. Jl. Ir. Soekarno Km 21, Jatinangor, Sumedang
45363, West Java, Indonesia
3
Program of Biology, Faculty of Mathematic and Natural Sciences, Universitas Padjadjaran. Jl. Ir. Soekarno Km 21, Jatinangor, Sumedang 45363, West
Java, Indonesia

Manuscript received: 30 March 2017. Revision accepted: 14 June 2017.

Abstract. Kuntana YP, Yurmiati H, Wulandari AP, Syafitri F, Partasasmita R. 2017. The appearance of rabbit skin tissue (Oryctolagus
cuniculus) after supplementation of Aloe vera and Spirulina fusiformis. Nusantara Bioscience 9: 268-274. The research is about the
appearance of rabbit skin tissue (Oryctolagus cuniculus L.) after supplementation of Aloe vera L. and Spirulina fusiformis Vor. has been
done. This study was carried out to find the most effective formulation ofsupplement of A. vera and S. fusiformis as a natural supplement
that can generate the best appearance of rabbit skin tissue. The method in this research was a single Complete Random Design (CRD) on
28 male New Zealand White strains rabbits with the age of 16 weeks. The treatments were divided into seven groups with four
replications, namely, P0 (negative control ), P1(positive control, vitamin C19 mg/kg BW), P2 (A. vera 74 mg/kg BW), P3 (S. fusiformis
296 mg/kg BW), P4 (A. vera: S. fusiformis, 74: 148 mg/kg BW), P5 (A. vera: S. fusiformis, 74: 296 mg/kg BW), and P6 (A. vera: S.
fusiformis, 74: 593 mg/kg BW). The parameters observed were production aspect (hair texture, slaughter weight, skin width, and skin
weight percentage) and histological aspect (thickness of skin tissue and the amount of hair follicle). All of the data was analyzed using
ANOVA test (P ˃ 95%) and Duncan test (P ˃ 95%). The result showed that the giving of supplement with the basis of the formulation
of A. vera 74 mg/kg bw and S. fusiformis 296 mg/kg bw was effective in generating the best appearance of rabbit skin tissue.

Keywords: Aloe vera, Appearance of skin tissue, Rabbit, Spirulina fusiformis

INTRODUCTION pellet with high protein (16%), thus it gives sufficient


natural supplement for rabbits. Spirulina was chosen as one
The skin raw materials commonly used for various of the alternative natural supplement because it has several
types of clothing and food products are derived from cattle, advantages such as high protein which is up to 60-70% of
buffalo, sheep, and goats. The skin produced from cattle the entire dry weight, containing essential fatty acids,
has good quality but, has relatively low reproductive rate polysaccharides, carotenoids, vitamins, and minerals,
and high production costs lead to limited provision. One especially vitamin B12 (Bourges et al. 1971; Anusuya et al.
attempt to solve this problem is to develop a type of 1981; Kabinawa 2014; El-Tantawy 2015). The contents of
livestock that has high reproduction and low production minerals and vitamins in Spirulina are potassium (15,400
costs, such as rabbit. This time, the rabbit skin is just a mg/kg), calcium (1,315 mg/kg), zinc (39 mg/kg),
waste of the ranch. The rabbit skin is one alternative that magnesium (1915 mg/kg), manganese (25 mg/kg), iron
has great potential to be processed into useful products (580 mg/kg), selenium (0.40 ppm), phosphorus (8942
such as jackets, bags, carpets and toys, in addition, the hair mg/kg), as well as vitamins A, B1 (thiamine), B2
fibers can be developed into wool. (riboflavin), B3 (nicotinamide), B6 (pyridoxine), B9 (sour
The skin quality is closely related to feeding and folate), B12, vitamin C, vitamin D and vitamin E. The
enclosure management. The skin width covering the body complete and balanced of Spirulina nutritional content has
surface will increase with weight gain. The increase of been used optimally in some countries to overcome
body volume will be followed by increasing of chest size malnutrition and immune system. The supplementation of
circumference and body length so that it can affect the Spirulina dose of 800 mg/kg body weight (BW) of mice is
width and length of the skin (pelt). Pelt is a fresh skin proven to improve liver function and to repair kidney and
tissue of furry animals that has been skinned. Pelt thickness testicular damage due to mercury exposition. The dosage is
is related to fat content, the layers of epidermis, dermis and converted to rabbits consumption and it becomes 296
connective tissue. mg/kg BW (Hermosillo et al. 2011; Henrikson 2009;
Food is needed by rabbit for the production of wool and Susanna et al. 2007.
pelts like Angora and Rex which need 120 g/day with 15% The plant of A.vera is one of the herbs. This plant
of crude protein. Nowadays, rabbit ranch uses expensive contains two types of liquids, namely a clear liquid which
KUNTANA et al. – Effect of Aloe vera and Spirulina fusiformis on rabbit skin tissue 269

is jelly and a yellowish fluid which is aloin. Jelly contains Research procedure
antibacterial and antifungal agents stimulating the growth Determination of dose
of fibroblast, which is a component of skin tissue Aloe vera dose is based on Tansar (2011) research
functioning in the wound healing process. Aloin can act as namely 200 mg/kg BW mice which is converted to 74
a laxative. A. vera have 72 essential substances needed by mg/kg BW rabbit. S. fusiformis with dose of 400 mg/kg
body. 18 out of 72 substances are amino acids, BW, 800 mg/kg BW and 1600 mg/kg BW mice
carbohydrates, fats, water, vitamins (A, B1, B2, B3, B12, (Hermosillo et al., 2011) were converted to 148 mg/kg BW,
C, E), minerals (calcium (Ca), magnesium (Mg), potassium 296 mg/kg BW and 593 mg/kg BW rabbit. The dose of
(K), sodium (Na), iron (Fe), zinc (Zn), chromium (Cr)) and vitamin C is 400 mg for men (Sandjaja A et al. 2009) and it
enzymes. The A. vera has salicylate which is effective as an is converted to 19 mg/kg BW rabbit.
anti-inflammatory like aspirin. The folic acid is also useful
for the regeneration of skin by producing new cells, Preparation of extract Aloe vera and Spirulina fusiformis
whereas the inositol and chromium can reduce hair loss. The meat of A. vera which has been cleared from
The administration of A. vera in a dose of 200 mg/kg BW exudate was cut into pieces and then crushed with a
mice was proven to help prevent damage of hair follicles blender. Samples were put in macerator by maceration in
exposed to etoposide (Sandjaja et al. 2009; Tansar 2011; 96% ethanol until they were completely submerged. This
Yuliarti 2008). process is carried out for 3 × 24 hours and every 24 hours
Thus, the natural supplement of Spirulina and A. vera the macerator is fit into the bottle and a solution of ethanol
can improve the skin tissue appearance and keep the is added to the macerator. The whole results from
immunity of rabbit so as to reduce production costs such as macerator is evaporated in an evaporator until all the
vaccines and fodder. The study on the quality of the skin solvent evaporates and resulting pasta of A. vera. After a
tissue of rabbits (Oryctolagus cuniculus) after week, the S. fusiformis culture in Zarrouk medium is
supplementation of A. vera and S. fusiformis has not been filtered using Monel cloth and dried by a fan to obtain the
widely reported, so that the study was conducted for further dry weight. The dried S. fusiformis is pulverized to a
research. powder and then is weighed to obtain the desired dose
which is 148 mg, 296 mg, and 593 mg. This powder is
dissolved in water for rabbits suited to their needs namely
MATERIALS AND METHODS 150 ml per day (daily).

Research methods Preparation of animal test


The method is an experimental method with a A total of 28 male rabbits New Zealand White
completely randomized design (CRD) on single male crossbreed of 12 weeks old were weighed and separated
rabbits aged 16 weeks. The test animals were randomly according to the normal distribution of data (coefficient of
divided into seven treatments with four replications, variation <10%), the rabbits were grouped into seven
namely: (i) P0: negative control, (ii) P1: positive control treatments with four replications. Cage habituation is done
(vitamin C dose of 19 mg/kg BW rabbit), (iii) P2: A. vera for one month. Slowly the rabbits are initially only given
dose of 74 mg/kg BW rabbit, (iv) P3: S. fusiformis dose of forage gradually replaced by pellets. Feed provided ad-
296 mg/kg BW rabbit, (v) P4: A. vera dose of 74 mg/kg libitum. Water was given twice daily (morning and
BW + S. fusiformis dose of 148 mg/kg BW, (vi) P5: A. vera evening) for total administration of 300 mL. The cage and
dose of 74 mg/kg BW + S. fusiformis dose of 296 mg/kg eating, as well as drinking equipment, are cleaned every
BW, (vii) P6: A. vera dose of 74 mg/kg BW + S. fusiformis morning. The treatment has been given when the rabbit is
dose of 593 mg/kg BW. 16 weeks old.
The number of rabbits in this study was 28 with ± 1 kg
bw. The parameters are aspects of livestock production Experimental treatment
(hair texture, slaughter weight, wide skin, and skin weight The treatment is given through drinking in every
percentage) and aspects of histology (thickness of skin morning for four weeks. Rabbits of 20 weeks old were
tissue and the amount of hair follicle). The collection of weighed after being put in 12 hours of fasting to calculate
data on hair texture (smoothness and brightness of the hair) slaughter weight. The percentages of width and skin weight
is performed at the Center for Textile Testing Laboratory are calculated and skin tissue sections are taken a bit and
and the Laboratory of Chemical Physics Bandung College stored in Bouin fixative solution for ± 24 hours for
of Textiles Textile Technology Bandung. The data of skin histological preparations.
width is obtained using Hegenaur (1977). The percentage
of Skin weight is calculated after the slaughter weight and Data analysis
skin weight is performed. The collection of data on Production aspects
thickness of skin and the amount of hair follicle is carried Hair fineness (denier) is calculated after a few strands
out under a lighted microscope using histological of hair are cut for as many as 225 pieces at ± 2cm long
preparations with Haemotoxylin-eosin staining of paraffin (SNI 08-1111-1989). The calculation of hair fineness
method. All data were statistically tested using test (denier) is as follows: 9000 X (Weight A strand of hair
Analysis of Variance (ANOVA) (P ˃ 95%) and Duncan's (mg))/(A x B) where A = the number of hair and B = length
Multiple Range Test (P ˃ 95%). of hair. The brightness of hair was tested using a
270 N U S A N T A R A B I O S C I E N C E 9 (4): 268-274, August 2017

spectrophotometer SS6200. One bundle of hair is arranged highest brightness value exceeding P1 as a positive control
in a spectrophotometer until it is dense and opaque. Hair and P0 as a negative control.
brightness was measured at a wavelength of 650. The The brightness of hair of rabbit is influenced by feeding
results are recorded as the value W = brightness hair. and enclosure management. The good feed will affect the
The width of skin (cm 2) was calculated by the appearance of the brightness of hair. The proteins and
Hegenaur method (1977) by measuring the length of the essential fats will produce bright shiny hair. Thus the
skin from head to tail churned drawn vertically. The width protein content in the A. vera and S. fusiformis can produce
of the skin was measured by an auxiliary line drawn from a good quality of hair brightness. Dirty hutch of rabbit will
the tip of the left front part to right rear part. This line cause the hair to become dirty so the values of hair
crosses the line of skin length, and it creates a meeting brightness will be low.
point. This meeting point is used as the basis for drawing a Anova test results to a slaughter weight of rabbits
vertical line to show the width of the skin. At last, the showed that the supplementation of A. vera and S.
number of skin length is multiplied by the number of skin fusiformis affect rabbit slaughter weight. Table 3 is the
width to determine the number of skin breadth (Figure 1). result of Duncan Multiple Range Test.
The percentage of skin weight obtained from the Duncan's Multiple Range Test results show that the P5
calculation: (Weight Skin)/(Weight Cut) X 100% treatment has the highest slaughter weight, which is 1769
grams and is not significantly different from P6 which is
Histological aspects 1721.25-gram. The P5 and P6 treatment are significantly
Observation of the thickness of the skin tissue was done different from the treatment of P2, P3, P4, P0 and P1. From
under lighted microscope with 400X magnification. these data, the treatment of P5 has the highest slaughter
Measurement of tissue thickness uses a micrometer in the weight exceeding P1 as a positive control and P0 as a
epidermis, dermis, and hypodermis. negative control.
The percentage calculation of hair follicles was The average slaughter weight is obtained in accordance
measured by counting each visible follicle (primary with the data of rabbits in West Java at age of 3-5 months,
follicles and secondary follicles) in the skin tissue area of namely from 1.5 to 2.1 kg. A. vera and S. fusiformis as
the back (dorsal), calculated to reach the number 100. Each supplement contain amino acids. Amino acids are
number of primary and secondary follicles is the necessary to the principal needs in rabbit’s life and growth.
percentage to the appropriate formula: The intake of protein in the body is a source of energy and
(primary follicles)/100 X 100%, at a certain degree, it can increase body weight (Susanna
(secondary follicles)/100 X 100% 2007).
The results of the ANOVA test on the skin width of
rabbit show that the supplement of A. vera and S. fusiformis
RESULTS AND DISCUSSION affect the skin width of the rabbit. Table 4 is the result of
Duncan Multiple Range Test.
Production aspect
The result of the analysis of variance (ANOVA) on the
smoothness of hair show that the supplementation of A.
vera and S. fusiformis affect the fineness of hair. Table 1 is
the result of Duncan's Multiple Range Test.
Duncan's Multiple Range Test results show that the
lowest value is in P5 treatment, i.e., 6.765 deniers but it is
not significantly different from the treatment of P2 and P3.
The values of P5 treatment is significantly different from
the treatment of P0 (12.025 deniers) and P1 (8.985
deniers). From these data, the treatment of P5 has the
highest level of refinement exceeding P1 as a positive
control and P0 as a negative control. The lighter the weight
of the hair, the more subtle the hair is. Treatment of P5 has
the lowest fineness value among the other treatments,
which means treatment of P5 brings on the smoothest hair
among treatment.
Anova test results on the brightness of the hair show
that the supplement administration of A. vera and S.
fusiformis affects the brightness of rabbit hair. Table 2 is
the result of Duncan's Multiple Range Test.
Duncan's Multiple Range Test results show that the
treatment of P6 brings on the highest value, which is
88.458%. The Value of treatment of P6 is significantly Figure 1. How to calculate the area of skin. Description: AB =
different from P0 treatment (80.280%) and P1 treatment length of the skin, EF = Width of the skin, CD = Lines aid to EF
(80.025%). From these data, the treatment of P6 has the
KUNTANA et al. – Effect of Aloe vera and Spirulina fusiformis on rabbit skin tissue 271

Table 1. The average hair fineness of rabbit after administration Table 5. The average skin weight percentage of rabbit after
of supplement of A. vera and S. fusiformis administration of supplement of A. vera and S. fusiformis

Treat- Replications Hair fineness Treat- Replications Skin weight


ment 1 2 3 4 (denier) x ± SD* ment 1 2 3 4 (%) x ± SD*
P0 10.52 10.26 9.38 11.48 10.50 ± 1.02cd P0 10.53 8.54 9.28 8.62 9,24 ± 0,92bc
P1 10.36 7.20 11.68 6.70 8.985 ± 2.42bc P1 9.05 8.85 8.04 8.53 8,62 ± 0,44b
P2 6.9 8 6.48 7.9 7.32 ± 0.75ab P2 6.94 6.48 6.07 7 6,62 ± 0,44a
P3 8.14 8.64 6.84 7.78 7.85 ± 0.76ab P3 8.93 9.28 7.84 8.08 8,53 ± 0,68b
P4 10.38 11.38 11.5 14.84 12.025 ± 1.94d P4 9.45 9.05 9.4 8.77 9,17 ± 0,32bc
P5 7.36 6.58 6.78 6.34 6.765 ± 0.44a P5 9.77 9.87 10.14 10.07 9,96 ± 0,17c
6
P6 11.16 9.36 10.16 11.16 10.46 ± 0.87cd P 8.95 9.25 9.54 8.68 9,11 ± 0,37b
Note: The different letters in the same column are indicating Note: The different letters in the same column are indicating
significant differences of Duncan test results (P˃ 95%) significant differences of Duncan test results (P˃ 95%)

The P3, P4, P5 and P6 treatment have the average skin


Table 2. The average brightness of hair of rabbit after width of rabbit which are higher than the average of P0, P1,
administration of supplement of A. vera and S. fusiformis and P2 treatment. The P6 treatment provides the highest
effect on the skin width of rabbit because it has the highest
Replications Hair skin width, i.e., 1401.51 cm2. From these data, the
Treat-
brightness
ment 1 2 3 4 treatment of P6 has exceeded the skin width of P1 as a
x ± SD*
P0 80.624 80.231 79.869 80.376 80.280 ± 0.32b
positive control and P0 as a negative control.
P1 81.015 78.37 80.73 79.985 80.025 ± 1.19b Crude fiber and nutrients affects the skin width of
P2 80.222 78.225 79.458 81.733 79.910 ± 1.47b rabbits. Nutrients in A. vera and S. fusiformis namely
P3 73.162 76.679 75.632 74.358 74.958 ± 1.53a protein, vitamins, and minerals are good for the growth of
P4 84.893 80.241 82.667 81.437 82.310 ± 1.99c rabbit. The process of growth is the increase in the number
P5 80.465 82.214 82.712 81.305 81.674 ± 0.99bc and size of the body cell. The process takes place in line
P6 87.834 89.6 88.956 87.443 88.458 ± 1.00d with the age and condition of the rabbit. The increasing
Note: The different letters in the same column are indicating volume of the body can increase the skin width covering
significant differences of Duncan test results (P˃ 95%) the surface of the body so that different weight will
produce a different skin width (Sandjaja et al. 2009;
Yuliarti 2008).
Table 3. The Average slaughter weight of rabbit after Anova test results to the skin weight percentage of
administration of supplement of A. vera and S. fusiformis rabbit show that the supplementation of A. vera and S.
fusiformis affect skin weight percentage of the rabbit. Table
Replication Slaughter weight 5 is the result of Duncan's Multiple Range Test.
Treatment The rabbits in P5 treatment have greater average skin
1 2 3 4 (gr) x ± SD*
P0 1434 1745 1414 1648 1560,25 ± 162,44a weight percentage than the other treatments, i.e., 9.96%,
P1 1414 1423 1629 1512 1494,5 ± 99,98a but it is not significantly different from P0 which has an
P2 1426 1482 1549 1457 1478,5 ± 52,28a average of 9.24%. The lowest average of skin weight
P3 1635 1497 1544 1571 1561,75 ± 57,62a percentage is found in the treatment of P2 (6.62%). This
P4 1672 1524 1574 1483 1563,25 ± 81,49a value is below the average of the percentage of rabbits’
P5 1780 1804 1794 1698 1769 ± 48,35b
P6 1844 1708 1688 1647 1721,75 ± 85,36b
skin weight in the treatment of P0 and P1 (9.24% and
Note: The different letters in the same column are indicating
8.62%). The results show that body weight has an influence
significant differences of Duncan test results (P˃ 95%) on skin weight. The skin weight is 8-10% of body weight
(Sandjaja et al. 2009; Yuliarti 2008). From these data, the
P5 treatment has the highest skin weight percentage
compared to P1 as a positive control.
Table 4. The Average skin width of rabbit after the administration
of Supplement of A. vera and S.fusiformis Histologic aspect
The results of Anova test on the thickness of the skin
Treat- Replications Skin width (cm2) tissue of rabbit show that the supplementation of A. vera
ment 1 2 3 4 x ± SD*
and S. fusiformis affect the thickness of the skin tissue of
P0 1042.48 1171.74 1033.32 1142.25 1097,45 ± 69,93a
P1 1025.88 1054.56 1087.56 1051.68 1054,92 ± 25,29a
rabbit. Table 6 is the result of Duncan's Multiple Range
P2 1017.52 1046.4 1081.92 1152.54 1074,60 ± 58,26a Test. The P4 treatment has the highest thickness of skin
P3 1481.61 1171.74 1285.2 1321.32 1314,97 ± 128,07b tissue, i.e., 1090.63 μm. It is significantly different from the
P4 1339.34 1272.08 1267.67 1356.32 1308,85 ± 45,57b negative control (P0) and the positive control (P1) which
P5 1426.36 1386.51 1216.44 1275.39 1326,18 ± 97,12b only has an average thickness of 575 μm and 631.25 μm.
P6 1503.04 1346.63 1472.56 1283.81 1401,51 ± 103,63b The P4 treatment does not differ significantly from P5 and
Note: The different letters in the same column are indicating P6 treatment. From these data, the P4 treatment has higher
significant differences of Duncan test results (P˃ 95%) thickness of skin than the P1 treatment as a positive control.
272 N U S A N T A R A B I O S C I E N C E 9 (4): 268-274, August 2017

Table 6. The average thickness skin of rabbit after administration The results of Anova test on hair follicles show that the
of Supplement of A. vera and S. fusiformis supplementation of A. vera and S. fusiformis affects the
amount of hair follicles percentage of the rabbit. Table 7 is
Treat- Replications Thickness skin the result of Duncan Multiple Range Test.
ment 1 2 3 4 tissue (µm) x ± SD*
The Duncan test result is about the average amount of
P0 481.25 818.75 443.75 556.25 575 ± 169,10ab
hair follicles percentage of the rabbit after administration of
P1 743.75 481.25 637.5 662.5 631,25 ± 109,81ab
P2 493.75 325 612.5 431.25 465,625 ± 120,17a supplement of A. vera and S. fusiformis showing that the
P3 431.25 525 406.25 550 478,125 ± 70,06a percentage of primary rabbit hair follicle is inversely
P4 756.25 1625 1156.25 825 1090,63 ± 396,75c proportional to the percentage of secondary rabbit hair
P5 637.5 512.5 1000 1100.25 812,563 ± 282,01abc follicles. This is because the primary rabbit hair follicle is
P6 981.25 668.75 718.75 1100 857,188 ± 207,06bc surrounded by several secondary hair follicles. Cheeke
Note: The different letters in the same column are indicating (1987) said the number of secondary hair follicles/unit area
significant differences of Duncan test results (P˃ 95%) varies depending on the season. In the winter, the number
of secondary hair follicles multiplies to produce thicker
hair in order to maintain body heat. Conversely, in summer,
Table 7. The average percentage of total hair follicles of rabbit
after administration of supplement of A. vera and S. fusiformis
the number of secondary hair follicles was reduced to allow
heat dissipation mechanism of the body so that body heat
% Total primary % Total scondary balance is maintained.
Treatment The most average value of percentage secondary hair
follicles x ± SD* follicles x ± SD*
P0 11,17 ± 2,62cd 88,83 ± 2,62ab follicles is the treatment of P5, 97.67%, while the control
P1 11,75 ± 0,92d 88,25 ± 0,92a has the lowest number of secondary hair follicles, which is
P2 9,04 ± 2,95c 90,96 ± 2,95b 88.25% for P1 ( positive control) and 88.83% for P0
P3 5,75 ± 0,42b 94,25 ± 0,42c (negative control). The study was conducted during the
P4 3,665 ± 0,81ab 96,335 ± 0,81cd rainy season which has a low environmental temperature so
a
P5 2,3325 ± 0,27 97,6675 ± 0,27d that the number of secondary hair follicles of rabbit
b
P6 5,415 ± 1,07 94,585 ± 1,07c
multiplies to maintain body heat. Thus, in addition to low
Note: The different letters in the same column are indicating
temperatures, the supplement of A. vera and S. fusiformis also
significant differences of Duncan test results (P˃ 95%)
can spur the growth of fine hair (own hair) so the percentage
of secondary rabbit hair follicles will be much more.
Tansar (2011) showed that A. vera potentially prevent
The results of the study of the thickness of skin tissue damage of hair follicles of mice after the administration of
range from 465.625 to 1090.63 μm. It was larger than the etoposide. Mice given by A. vera with a dose of 200 mg/kg
BW mice (74 mg/kg BW rabbits) have 41.31 hair follicles
range of skin thickness of rabbit generally ranging from
380-840 μm. The thickness of skin is closely related to fat while apoptosis frequency is decreased by 90.94%.
content, feed quality, and growth rate. Animals which were Substances of aloin in A. vera is used to treat hair loss and
nourish hair. Vitamin E along with linoleic acid and
fed on low nutritious will produce the low quality of skin
tissue (Wibowo 2008). Figure 2 is the preparation of arachidonic acid contained in Spirulina is a fatty acid
histological thickness of skin tissue of rabbit after which is important for maintaining healthy of hair,
especially hair roots and for maintaining the integrity of
treatment.
The weight gain is closely related to the development of overall hair. The observation of histological preparations of
muscle that forms meat and skin tissue, through increasing the rabbit hair follicles with HE staining can be seen in
Figure 3.
the content of the subcutaneous fat tissue; therefore it
contributes to the thickness of the skin. The thickness of The nutrition in A. vera and S. fusiformis such as
protein is needed by rabbit for basic living needs and
skin is dominated more by the subcutaneous fat and has
little relationship with the slaughter weight, as in the growth. Proteins that enter the body of the rabbit will be
statement of Wibowo (2008), that the increase in the converted into amino acids and absorbed by the small
intestine and carried by the blood throughout the body to
subcutaneous fat tissue will give rise to the thickness of
subcutaneous layer. form the body's tissues, especially muscle tissue. Muscle
Aloe vera or S. fusiformis contains vitamins A, C and E tissue/rabbit meat is a major component in the muscle
tissue production. The more muscle tissues are formed, the
that are good for the skin. Vitamin A can stimulate the
formation of collagen thus spurring the epithelialization. production of the meat, the slaughter weight and also the
The function of vitamin C relates to the synthesis of production of skin will increase(Sandjaja et al. 2009;
Wibowo 2008; Yuliarti 2008).
collagen, a protein found in connective tissue. This tissue
consists of insoluble collagen fibers that are stored in a Rabbits have special characteristics in the efficiency of
matrix called the basic substance. This tissue is found in protein utilization for their lives. Rabbits need protein
between 12-18%. In this study, the given ration already contains
the skin, cartilage, tendons, ligaments, bones and blood
vessels. Vitamin E is fat soluble and is absorbed by the skin of 15% protein. An extra supplement containing high
efficacious as an antioxidant to suppress the formation of protein would make rabbits experience excess of protein.
Excess of protein will be absorbed by the body for other
free radicals, preventing damage to skin cells (Bajwa et al.
2007; Widagdo 2004). production purposes, namely fur production (Cheeke 1987).
KUNTANA et al. – Effect of Aloe vera and Spirulina fusiformis on rabbit skin tissue 273

x x x x

A B C D

x x x

E F G

Figure 2. The preparation of histological thickness of skin tissue of rabbit after treatment, the incision in the lengthwise direction. Note:
X: thick skin tissues of rabbits, lighted microscope, 400 × magnification, HE staining. A. P0 negative control, B. P1 positive control, C.
P2A. vera 74 mg/kg BW, D. P3S. fusiformis 296 mg/kg BW, E, P4A. vera: S. fusiformis, 74 mg/kg BW: 148 mg/kg BW, F. P5A. vera:
S. fusiformis, 74 mg/kg BW: 296 mg/kg BW, G. P6A. vera: S. fusiformis, 74 mg/kg BW: 593 mg/kg BW

A B C D

E F G

Figure 3. The histological preparations of rabbit hair follicles. Note: Lighted microscope, magnification 400X, HE Staining, direction
transverse incision. A. P0 negative control, B. P1 positive control, C. P2A. vera 74 mg/kg BW, D. P3S. fusiformis 296 mg/kg BW, E,
P4A. vera: S. fusiformis, 74 mg/kg BW: 148 mg/kg BW, F. P5A. vera: S. fusiformis, 74 mg/kg BW: 296 mg/kg BW, G. P6A. vera: S.
fusiformis, 74 mg/kg BW: 593 mg/kg BW

The number of ration consumed depends on the animal Conversely, if the protein content of the ration is too high it
concerned, ambient temperature, palpability ration, the will decrease digestibility of other food substances (Cheeke
ration of energy levels, the physical form of ration, the 1987). The rations in this study are so much that a number
production function, and age of cattle. Feed consumption of nutrients are obtained from the same ration. The addition
will be lower when the protein level is low so the of supplement of A. vera and S. fusiformis increases the
unbalance metabolism of connective tissue will occur. amount of protein digested. On the other hand, the shortage
274 N U S A N T A R A B I O S C I E N C E 9 (4): 268-274, August 2017

and excess of protein are not good for the metabolism of REFERENCES
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indispensable. Studies on the proteins of mass-cultivated, blue-green alga (Spirulina
platensis). J Agric Food Chem 29: 522-525.
Aloe vera or S. fusiformis contains vitamins A, C and E Bajwa R, Shafique S, Shafique S. 2007. Appraisal of antifungal activity of
which are good for the skin. Vitamin A can stimulate the Aloe vera. Mycopath 5 (1): 5-9.
formation of collagen thus spurring epithelialization. The Bourges H, Sotomayor A, Mendoza E, Chavez A. 1971. Utilization of the
function of vitamin C relates to the synthesis of collagen, a algae Spirulina as a protein source. Nutr Rep Int 4: 31-43.
Cheeke PR.1987. Rabbit Feeding and Nutrition. Corvallis: Department of
protein found in connective tissue. This tissue consists of Animal Science. Academic Press, Inc., Oregon.
insoluble collagen fibers that are stored in a matrix called El-Tantawy WH. 2015. Antioxidant effects of Spirulina supplement
the basic substance. This tissue is found in the skin, against lead acetate-induced hepatic injury in rats. J Trad Compl Med.
cartilage, tendons, ligaments, bones and blood vessels. DOI: 10.1016/j.jtcme.2015.02.001
Hegenauer H. 1977. Fachlunde Fur Lederverabeitende Berufe.Verlag
Vitamin E is fat-soluble, which is absorbed by the skin Ernst Heyer, Essen, Nuremberg.
efficacious as an antioxidant useful to suppress the Henrikson R. 2009. Earth Food Spirulina: How this Remarkable Blue-
formation of free radicals and to prevent cell damage of the green Algae Can Transform Your Health and Our Planet. Ronore
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Hermosillo FA, Torres D, Shamosh H, Juarez O. 2011. Biological Effects
The results show that in this research, the use of dose of Spirulina and Current Research on its Antioxidant Activity.
combination of A. vera with 74 mg/kg BW and S. Univesity of Mexico, Mexico City.
fusiformis with 296 mg/kg BW (P5) is the appropriate dose Kabinawa INK. 2014. Healthy foods and herbs healthy from microalgae
for a supplement of the rabbit. The combination of two Spirulina. J Food Tech Appl 3 (3): 103-109 [Indonesian]
Sandjaja A, Basuki B, Rina H, Nurfi A, Moesijanti S, Gustina S,
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seen from the improved production aspects such as hair Susanna D, Zakianis, Ema H, Haryo KA. 2007. The Utilization of
texture, slaughter weight, skin width, and skin weight Spirulina platensis as supplement of Single Cell Protein (PST) of
mice (Mus musculus). Makara Health 11: 44-49. [Indonesian]
percentage and the improved histological aspect such as the Tansar IA. 2011. The Potential of Aloe Eels and Aloe Tigers in Preventing
thickness of skin tissue and the amount of hair follicle. Damage Hair Follicles of Swiss Webster mice after administration of
Etoposide. [Hon. Thesis]. Universitas Padjadjaran, Jatinangor.
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Wibowo RY. 2008. The Effect administration of Dregs (Camellia
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N U SAN TA RA B IOSC IE NC E ISSN: 2087-3948
Vol. 9, No. 3, pp. 275-281 E-ISSN: 2087-3956
August 2017 DOI: 10.13057/nusbiosci/n090306

Evaluation of orange-fleshed sweetpotato genotypes for yield and yield


contributing parameters in two environments

WIWIT RAHAJENG♥, ST. A. RAHAYUNINGSIH


Indonesian Legumes and Tuber Crops Research Institute. Jl. Raya Kendalpayak Km 8, Po Box 66, Malang 65101, East Java, Indonesia.
Tel.: +62-341-801468, Fax.: +62-341-801496, ♥email: wiwit.rahajeng@gmail.com

Manuscript received: 6 November 2016. Revision accepted: 19 June 2017.

Abstract. Rahajeng W, Rahayuningsih SA. 2017. Evaluation for yield and yield contributing parameters of orange-fleshed sweetpotato
genotypes in two environments. Nusantara Bioscience 9: 275-281. Orange-fleshed sweetpotatoe (OFSP) is one of the most important
sources of betacaroten and carbohydrates. OFSP showed varying responses to different environments, depending on the adaptability of
the genotypes. The objective of this study was to evaluate the correlation between yield and yield component of OFSP genotypes at two
different agroclimatological environments (Mojokerto and Blitar) from May to September 2013. Twenty four OFSP genotypes (twenty
two clones and two varieties as the check) were used in this study and arranged in a randomized complete block design with three
replications. Results showed that responses of OFSP genotypes to the environments varied and genotypes had highly significantly
affected all parameters. Based on the criteria of high yield, amylum content, and dry matter production four clones were selected (MSU
09008-54, MSU 07009-113, MSU 07025-45, and MSU 09025-71) from Blitar and two clones (MSU 07009-113 and MSU 070-64) from
Mojokerto. Clone MSU 07009-113 showed good performance in both environments for yield, amylum content, and dry matter
production parameters. The result of the correlation analysis reveals that root yield is highly significant and positively correlated with
dry matter production, number of roots, diameter of root, and harvest index in two locations (Blitar and Mojokerto). Based on the
correlation and path analysis, dry matter production, root diameter, and harvest index can be used as an effective indicators selection for
high yield OFSP genotypes.

Keywords: Environment, correlation, orange-fleshed sweetpotatoes, path analysis, yield

INTRODUCTION the shortage of vitamin A because it is cheap and easily


cultivated. According to Sindi and Low (2016) 125 grams
Sweetpotato (Ipomoea batatas) is one of the crops that of most OFSP varieties can supply the recommended daily
is potentially good enough to be developed in Indonesia for allowance of vitamin A for children and non-lactating
a food security program. Sweetpotato roots have a fairly women.
high carbohydrate content so it can be used as food, The OFSP varieties option in Indonesia is still limited.
industrial, and bioethanol. Globally sweetpotato is an So far, there are only three OFSP varieties with high beta-
important food crop in the world after wheat, rice, maize, carotene levels that have been released; Beta1, Beta 2, and
potatoes, barley, and cassava (FAOSTAT 2012; Teshome- Beta 3. To fulfill the need of food and industry, the breeding
Abdissa and Nigussie-Dechassa 2012; Wera et al. 2014). of OFSP varieties which have high beta-carotene levels and
Ambarsari et al. (2009), state that in Indonesia sweetpotato high yield is still necessary to be conducted. Root yield is a
ranks fourth after rice, maize, and cassava. Besides carbo- very complex character which associating with many
hydrates, sweetpotatoes are also rich in fiber, minerals, interconnected components. Therefore, the breeding
vitamins, and antioxidants (Kure et al. 2012; Mbah and program to gain high yield potency varieties should be
Eke-Okoro 2015; Rodriguez-Bonilla et al. 2014). Orange- supported by information about the relationship between
fleshed sweetpotatoes (OFSP) is one of the most important the yield and the parameters contributing to the yield to
sources of carbohydrates and vitamins (beta carotene). determine the selection criteria (Tsegaye et al. 2006).
The OFSP varieties contain beta-carotene which is The information required to determine the selection
useful for the body to produce vitamin A (Agili et al. 2012; criteria is the correlation coefficient and path analysis
Burri 2011; Wariboko and Ogidi 2014). Vitamin A is an between yield and yield components. According to
essential nutrient, which has many roles in the regulation of Mohanty et al. (2016), correlation analysis provides
genetic, visual cycle, normal growth, and development, as information about the relationship between the important
well as for immune function. According to Kapil and characters and as a useful index to predict the yield potency
Sachdev (2013), vitamin A deficiency can cause blindness based on the value change of certain characters. The
and reduced immune system so that it can easily get relationship between yield and yield components in
infections causing death. Children are easier to suffer from correlation analysis still shows a very complex chain.
Vitamin A deficiency than adults. The need of vitamin A in Therefore, path analysis is done to explain the direct and
children is high because of their physical growth and low indirect associations and to identify the most prominent
food intake. Therefore, OFSP has the potency to overcome character which contributes to the increasing yield.
276 N U S A N T A R A B I O S C I E N C E 9 (4): 275-281, August 2017

A previous study of the correlation and path analysis

Rainfall (mm/month) 
showed that stem diameter, root number and weight of big
and medium roots are the indicators for the selection of
high-yield potency (Wang et al. 2012). The research of
Sasmal et al. (2015) reported that fresh root weight, root
number plot-1, root diameter, length of vines, and leaf size
were correlated with the root yield. Meanwhile, the
research of Egbe et al. (2012) and Tsegaye et al. (2006)
showed that the characters of root weight per plant and
harvest index were appropriate characters used as selection
criteria to obtain high yield potency. Month 
Besides information on the correlation and path
Figure 1. Graphic rainfall in Blitar and Mojokerto, East Java,
analysis, evaluation of the interaction of genotype and
Indonesia from January to December 2013
environment also needs to be done, due to the unstable
response of sweetpotato genotypes in many environments
and also the environmental differences which greatly affect
yield and yield-related trait. Therefore, the objectives of RESULTS AND DISCUSSION
this study were to evaluate the root yield potency and the
correlation between yield and yield component of OFSP Analysis of variance and average of genotype
genotypes in two different environments (Mojokerto and performance in two locations
Blitar). The combined analysis of variance in two locations
(Blitar and Mojokerto) showed the result of interaction
between genotype and an environment that was highly
significant for all the observed characters (Table 1). It
MATERIALS AND METHODS
means that the 24 tested genotypes give different responses
in the different growing environment so that the selection
Study area and procedures
can be done in each environment. The tested genotypes
This study was conducted at Pacet (Mojokerto) and
also showed significantly different in all the observed
Srengat (Blitar), from May to September 2013. The
characters. Moussa et al. (2011); Kathabwalika et al.
experiment was arranged in a randomized complete block
(2013) and Rahajeng et al. (2014) reported that they gained
design with three replications. A total 24 OFSP genotypes
almost the same results on the conducted research. It
which consisted of twenty two OFSP selected clones and
indicates that each tested genotype showed and had a
two released OFSP varieties (Beta-1 dan Beta-2) as the
different genetical character, it also had a significant
check varieties. Each genotype was planted on a 3 m wide
impact, especially for the yield and yield components
x 5 m long plot (3 rows) with the spacing between rows
character. At the analysis of variance for location, almost
and plants was 100 cm x 25 cm. 2/3 dose of fertilizer
all characters showed significantly different results, except
(300kg/ha NPK Phonska) was applied at planting, and the
in the root diameter. The growth and the yield of crops are
remaining was applied 5 weeks after planting. Weeding
affected by characteristics of the two different
was done at 4, 7, and 10 weeks after planting. Earthing
environments (Blitar and Mojokerto). According to
down was done at 4 weeks after planting, earthing up was
Pimsaen et al. (2010), the environment has a considerable
done at 7 or 8 weeks after planting. Irrigation, pest, and
contribution to the diversity of production factors such as
disease control was applied as needed. Harvesting was
the weight and the size of the root.
done at 4.5 months. Rainfall data is presented in Figure 1.
Based on the character of the root yield, dry matter
The graph showed that the rainfall in Mojokerto was higher
production, root length and root diameter, sweetpotato
than the rainfall in Blitar, especially during the study period
genotypes in Mojokerto had a higher average than the
(May to September 2013).
average in Blitar. On the characters of amylum content, dry
matter content, number of roots plot-1, and harvest index,
Data analysis sweetpotato genotypes in Blitar showed higher average
The observations were recorded for number of root plot- than in Blitar (Figure 2, Table 2 and 3). High root yield did
1
, root length and diameter, dry matter content, harvest not always show high yield components as well, especially
index, root yield, dry matter production, and amylum for the characters of amylum content and dry matter
content. Data were processed by combined analysis of content. It was indicated from almost all the sweetpotato
variance procedures for two environments to determine the genotype in Mojokerto having higher result than those in
interaction of genotype and environment by using Blitar. In the other hand, they were lower for the amylum
MINITAB 14 program. Least significant differences (LSD) content and dry matter contents. It confirms the results of
at 5% level of probability were used to detect differences correlation analysis which showed that the character of
between treatment means. Analyzes of correlation were starch content and the root yield did not correlate (r = -
conducted to determine the relationship among yield and 0.252ns) while the root dry matter content and the root yield
yield components. Direct and indirect contributions of was negatively correlated (r = -0.609**) (Table 4). Based
various characters to yield were calculated through path on the correlation value, the root yield is more determined
coefficient analysis according to Singh and Chaudary (1985). by the root size, especially the root diameter.
RAHAJENG & RAHAYUNINGSIH – Evaluation of orange-fleshed sweetpotato genotypes in two environment 277

Blitar  Mojokerto
108.79

87.71

77.92

Mean 
64.05

36.62
28.11
25.98 26.09
14.93
17.17
12.14 12.75
3.35 4.31 5.53 5.74

Amylum Root yield Dry matter Dry matter Number of Root length Root diameter Harvest index
-1 -1
(%) (t ha ) production content root plot (cm) (cm) (%)
-1
(t ha ) (%)

Figure 2. Comparison of the average values of the quantitative characters in Blitar and Mojokerto, East Java, Indonesia

Tabel 1. Analysis of variance and coefficient of variance of sweetpotato genotypes. Mojokerto and Blitar, East Java, Indonesia in 2013.

Mean
Characters CV (%)
Location (L) Genotype (G) GxL
Amylum content 6,925.43** 6.47** 36.56** 0.42
Root yield 910.83** 230.25** 72.39** 20.34
Dry matter production 33.30* 13.17** 5.51** 20.85
Dry matter content 163.39** 50.91** 24.75** 2.83
Harvest index 3,988.03** 856.84** 202.90** 15.34
Number of rootplot-1 16,000.14* 5,107.61** 2,489.46** 19.51
Root length 172.11** 43.91** 11.15** 10.91
Root diameter 1.71ns 9.04** 2.65** 11.79
Note: ** significant at p< 0.01, * significant at p< 0.05, ns= non significant

Tabel 2. Number of root plot-1, root length, and root diameter of 24 OFSP genotypes in two environment (Blitar and Mojokerto, East
Java, Indonesia), 2013

Number of root plot-1 Root length Root diameter


Genotypes
Blitar Mojokerto Blitar Mojokerto Blitar Mojokerto
MSU 09008-54 137.67 100.00 17.11 20.07 4.89 5.40
MSU 09008-92 93.67 170.00 9.22 16.00 6.66 7.77
MSU 09013-10 90.33 63.00 14.22 17.53 5.63 5.53
MSU 09013-48 78.67 69.67 9.44 14.53 6.11 5.57
MSU 09016-80 123.33 29.33 13.33 12.13 4.60 2.20
MSU 09016-87 73.67 78.33 15.11 17.10 5.00 5.37
MSU 09017-11 100.33 86.33 13.89 16.00 5.32 6.07
MSU 09017-32 95.00 65.67 13.55 18.00 4.33 5.57
MSU 09018-27 92.67 31.67 14.56 11.07 4.47 2.33
MSU 07023-27 106.00 101.67 9.11 12.03 7.33 7.77
MSU 07025-05 93.00 83.33 9.34 15.10 6.61 7.53
MSU 070-64 115.67 121.33 17.45 17.83 3.56 5.70
MSU 07009-149 106.33 19.70 8.99 8.33 5.49 2.67
MSU 07024-82 120.67 52.67 8.67 8.43 5.67 3.77
MSU 07024-43 104.00 82.33 10.78 11.33 5.53 6.30
MSU 07021-56 48.67 49.67 13.67 16.97 4.94 5.50
MSU 07009-112 130.00 64.00 15.22 14.33 3.95 5.93
MSU 07009-123 123.33 105.00 17.56 18.27 3.83 5.87
MSU 07009-113 176.67 145.67 13.44 16.57 5.83 6.33
MSU 07024-36 161.33 122.33 11.45 14.70 6.00 6.17
MSU 07025-45 131.00 96.00 10.67 13.77 7.10 7.90
MSU 09025-71 110.67 84.00 11.33 14.07 6.95 7.10
Beta 1 77.00 90.67 17.33 16.10 5.43 5.63
Beta 2 121.33 192.67 10.45 18.10 7.39 7.90

Average 108.79 87.71 12.75 14.93 5.53 5.75


CV (%) 19.51 10.91 11.79
LSD 5% Environment 15.91 1.20 0.31
Genotypes 21.98 1.73 0.76
278 N U S A N T A R A B I O S C I E N C E 9 (4): 275-281, August 2017

Tabel 3. Root yield, amylum content, dry matter production, dry matter content, and harvest index of 24 OFSP genotypes in two
environment (Blitar and Mojokerto), 2013

Dry matter
Root yield Amylum content Dry matter
production Harvest index
Genotypes (t ha -1) (%) content (%)
(t ha -1)
Blitar Mojokerto Blitar Mojokerto Blitar Mojokerto Blitar Mojokerto Blitar Mojokerto
MSU 09008-54 15.11 24.05 78.43 61.09 4.32 5.50 28.60 22.90 48.61 32.95
MSU 09008-92 12.55 31.48 79.24 61.13 3.70 8.79 29.51 27.91 48.14 47.28
MSU 09013-10 11.75 14.95 78.28 61.98 3.28 3.67 27.91 24.53 35.54 20.20
MSU 09013-48 7.08 11.19 76.52 65.51 2.59 2.90 36.54 25.93 30.06 17.92
MSU 09016-80 11.09 2.07 76.44 66.52 3.58 0.62 32.05 29.90 31.62 3.01
MSU 09016-87 7.76 15.58 75.90 68.10 2.66 4.50 34.27 28.89 23.17 20.73
MSU 09017-11 14.76 22.19 77.02 65.44 3.80 5.86 25.89 26.39 36.85 19.40
MSU 09017-32 7.61 11.06 75.67 67.44 1.98 3.13 26.05 28.30 24.67 13.14
MSU 09018-27 7.31 2.36 76.90 67.94 1.98 0.88 27.06 37.28 23.46 3.28
MSU 07023-27 14.06 16.54 75.81 68.56 3.38 3.95 24.06 23.88 43.04 26.60
MSU 07025-05 9.27 25.48 77.56 65.15 2.47 6.39 26.61 25.08 37.17 35.50
MSU 070-64 10.03 23.36 76.85 66.16 2.48 5.62 24.75 24.00 31.24 38.56
MSU 07009-149 7.41 2.65 78.83 62.85 2.27 0.68 30.39 25.76 23.37 3.71
MSU 07024-82 9.65 3.57 79.42 61.36 3.04 0.84 31.56 23.76 30.09 8.10
MSU 07024-43 13.00 17.49 79.69 61.25 3.64 4.53 28.03 25.88 45.72 43.43
MSU 07021-56 4.20 11.20 77.84 65.40 1.35 3.43 31.90 30.58 19.41 14.02
MSU 07009-112 9.02 13.43 77.44 66.37 2.28 3.89 26.14 28.94 29.12 19.04
MSU 07009-123 11.04 21.21 76.19 67.56 2.66 4.72 24.07 22.23 32.08 50.17
MSU 07009-113 21.57 30.92 78.26 64.90 5.50 5.96 25.50 19.28 53.02 49.11
MSU 07024-36 17.64 17.96 77.29 66.03 4.90 4.87 27.70 27.11 47.00 37.40
MSU 07025-45 18.82 22.77 80.44 58.33 4.88 4.67 25.89 20.49 57.37 25.53
MSU 09025-71 17.01 19.40 80.99 56.16 4.73 5.00 27.88 25.74 45.64 22.84
Beta 1 14.66 18.12 80.12 57.81 3.55 3.85 24.23 21.06 33.27 20.91
Beta 2 18.96 33.07 79.04 64.20 5.33 9.20 28.13 27.81 49.20 53.43

Average 12.14 17.17 77.92 64.05 3.35 4.31 28.11 25.98 36.62 26.09
CV (%) 20.34 0.42 20.85 2.83 15.34
LSD 5% Environment 2.56 0.04 0.70 0.53 1.48
Genotypes 3.42 0.34 0.92 0.88 5.52

Based on the root yield character, nine clones (MSU 09008-54, 07009-113 MSU, MSU 07025-45, and MSU
09008-54, 09008-92 MSU, MSU 09017-11, 07023-27 09025-71) from Blitar and two clones (MSU 07009-113
MSU, MSU 07024-43, 07009-113 MSU, MSU 07024-36, and MSU 070-64) were selected from Mojokerto. Clone
07025-45 MSU and MSU 09025-71) gained in Blitar had a MSU 07009-113 showed good performance in both
higher value than the average of yield roots, one clone environments for yield (21.57 and 30.92 t ha -1), amylum
(MSU 07009-113) had higher root yield than check variety content (78.26 and 64.90%), and dry matter production
(Beta 2). In Mojokerto, there were 11 clones (MSU 09008- (5.50 and 5.96).
54, MSU 09008-92, 09017-11 MSU, MSU 07025-05, MSU
070-64, MSU 07024-43, MSU 07009-123, 07009-113 Correlation and path analysis
MSU, MSU 07024-36, 07025-45 MSU and MSU 09025- The result of the correlation analysis in Blitar and
71) having root yield higher than average, but there was no Mojokerto (Table 4) revealed that the root yield was highly
clone having a value above of Beta 2 (check variety) (Table significant and positively correlated with the dry matter
3). The average of root yields in Blitar was lower than in production (r = 0.966** in Blitar and r = 0.962** in
Mojokerto. It was likely due to the differences in Mojokerto), number of root (r = 0.685** in Blitar and r =
environmental conditions at each study site. Based on 0.906** in Mojokerto), diameter of root (r = 0.524** in
rainfall data during the study from May to September 2013 Blitar and r = 0.827**), and harvest index (r = 0.903** in
(Figure 1), it can be seen that the condition in Blitar was Blitar and r = 0.886**). The root yield was also highly
drier than Mojokerto, especially during planting and 2 significant and positively correlated with amylum content
months after planting. Lack of water during this period led in Blitar, while in Mojokerto the root yield was also highly
to a negative impact on growth and root yield because that significant and positively correlated with the root length.
period was the early stage of the growth and the root This result means that the increase in the value of the
formation stage. characters will increase the root yield. The previous studies
Based on the criteria of high yield, starch content, and also showed similar results, where the root yield was
dry matter production, four clones were selected (MSU positively correlated with the total number of roots (Afuape
RAHAJENG & RAHAYUNINGSIH – Evaluation of orange-fleshed sweetpotato genotypes in two environment 279

et al. 2011; Demelie and Aragaw 2015; Egbe et al. 2012; Mbah and Eke-Okoro (2015) got a positive and significant
Mekonnen et al. 2015), root diameter (Gedamu et al. 2010; correlation between root yield and dry matter content of
Jha 2012), harvest index (Jha 2012; Rahajeng and roots. Root yield was not correlated with root length (in
Rahayuningsih 2015; Tirkey 2011; Tsegaye et al. 2006). Blitar) and amylum content (in Mojokerto). The research of
According to Mekonnen et al. (2015), the significant and Mohanty et al. (2016) also showed a similar result, where
the positive correlation indicates that characters the tuber yield was also not correlated with root length and
significantly correlated with root yields can be used as an amylum content.
indicator of the adaptability of sweetpotatoes in the study To study the direct and the indirect effect between the
area to increase root yields (as indirect selection tool), so root yield and the yield components, the path analysis was
that the sweetpotato breeding program with the purpose of conducted. Based on the direct and the indirect
increasing root yields should use these characters as a relationships between the yield components and the root
selection criteria. yield, dry matter production and root diameter in Blitar had
On the other hand, the negative and significant a correlation coefficient of significant positive to the root
correlation was shown between the root yield and the root yield, with a value of 0.966** and 0.524** (Table 4). The
dry matter content in both locations (r = -0.446* in Blitar value of that correlation coefficient is equivalent to path
and r = -0.470* in Mojokerto). Tsegaye et al. (2006) and coefficient of its direct effects, ie 0.868 and 0.098 (Table
Gedamu et al. (2010) also got the root yield significantly 5).
negatively correlating with dry matter content of roots. But,

Tabel 4. Coefficient correlation between yield and yield component of OFSP genotypes in Blitar and Mojokerto, East Java, Indonesia,
2013

RY AC DMP DMC RL RD
Characters NRP
(t ha-1) (%) (t ha-1) (%) (cm) (cm)
Amylum Content (AC, %) 0.461*
-0.212ns
Dry matter production (DMP, t ha-1) 0.966** 0.488*
0.962** -0.150ns
Dry matter content (DMC, %) -0.446* -0.072ns -0.214ns
-0.470* 0.438* -0.268ns
Number of root plot-1 (NRP) 0.685** 0.112ns 0.663** -0.360ns
0.906** -0.107ns 0.904** -0.357ns
Root length (RL,cm) -0.063ns -0.260ns -0.164ns -0.330ns 0.009ns
0.631** 0.097ns 0.627** -0.189ns 0.521**
Root diameter (RD, cm) 0.524** 0.475* 0.578** 0.057ns 0.053ns -0.749**
0.827** -0.231ns 0.829** -0.428* 0.741** 0.440*
Harvest index (HI) 0.903** 0.498* 0.897** -0.340ns 0.611** -0.255ns 0.633**
0.886** -0.063ns 0.850** -0.340* 0.887** 0.508* 0.712**
Note: Upper = Blitar, Lower = Mojokerto

Tabel 5. Path-coefficient of the direct and indirect effects of yield components characters on the yield of OFSP genotypes, Blitar and
Mojokerto, East Java, Indonesia, 2013

AC DMP DMC NRP RL RD HI


Characters
(%) (t ha-1) (%) (cm) (cm)
Amylum Content (AC, %) -0.004 -0.002 0.000 -0.000 0.001 -0.002 -0.002
0.003 -0.000 0.001 -0.000 0.000 -0.001 -0.000
Dry matter production (DMP, t ha-1) 0.424 0.868 -0.186 0.576 -0.142 0.502 0.779
-0.125 0.834 -0.223 0.753 0.523 0.691 0.709
Dry matter content (DMC, %) 0.017 0.051 -0.240 0.086 0.079 -0.014 0.082
-0.096 0.059 -0.219 0.078 0.041 0.094 0.095
Number of root plot-1 (NRP) 0.003 0.019 -0.010 0.029 0.000 0.002 0.018
-0.002 0.018 -0.007 0.020 0.011 0.015 0.018
Root length (RL, cm) -0.017 -0.011 -0.022 0.001 0.067 -0.050 -0.017
0.003 0.023 -0.007 0.019 0.036 0.016 0.018
Root diameter (RD, cm) 0.047 0.057 0.006 0.005 -0.074 0.098 0.062
0.010 -0.034 0.018 -0.031 -0.018 -0.041 -0.029
Harvest index (HI) -0.009 -0.017 0.006 -0.005 0.012 -0.012 -0.019
-0.005 0.064 -0.033 0.067 0.038 0.054 0.075
Note: Residual effect (R)= 0.0736170 , Bold font denote direct effect, Upper = Blitar, Lower = Mojokerto
280 N U S A N T A R A B I O S C I E N C E 9 (4): 275-281, August 2017

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N U SAN TA RA B IOSC IE NC E ISSN: 2087-3948
Vol. 9, No. 3, pp. 282-287 E-ISSN: 2087-3956
August 2017 DOI: 10.13057/nusbiosci/n090307

Isolation of anti-idiotype minor capsid Human papillomavirus type 16


(HPV 16 L2) IgY from egg yolk as immunogen of HPV vaccine

EKA NONENG NAWANGSIH1, SAYU PUTU YUNI PARYATI1, JUSUF S. EFFENDY2,


SUNARJATI SUDIGDOADI3, EDHYANA SAHIRATMADJA4, DIKI HILMI5, EVI SOVIA6
1
Department of Microbiology, Faculty of Medicine, Universitas Jenderal Achmad Yani. Jl. Terusan Jendral Sudirman, PO Box 148, Cimahi 40525, West
Java, Indonesia. Tel.: +62-22-6642781, ♥email: eka.n.nawangsih@gmail.com
2
Department Obstetric and Gynaecology, Faculty of Medicine, Universitas Padjajaran. Jatinangor, Sumedang 45363, West Java, Indonesia
3
Department of Microbiology, Faculty of Medicine, Universitas Padjajaran. Jatinangor, Sumedang 45363, West Java, Indonesia
4
Department of Biochemistry and Molecular Biology, Faculty of Medicine, Universitas Padjajaran. Jatinangor, Sumedang 45363, West Java, Indonesia
5
Depatment of Immunology, STIKES Jenderal Achmad Yani. Cimahi 40525, West Java, Indonesia
6
Department of Pharmacology, Faculty of Medicine, Universitas Jenderal Achmad Yani. Cimahi 40525, West Java, Indonesia

Manuscript received: 12 March 2017. Revision accepted: 4 July 2017.

Abstract. Nawangsih EN, Paryati SPY, Effendy JS, Sudigdoadi S, Sahiratmadja E, Hilmi D, Sovia E. 2017. Isolation of anti-idiotype
minor capsid Human papillomavirus type 16 (HPV 16 L2) IgY from egg yolk as immunogen of HPV vaccine. Nusantara Bioscience 9:
282-287. A potential strategy for the production of safe protective vaccines for human papillomavirus (HPV) infection is to utilize anti-
ids. Meanwhile, hyperimmunized hens could provide a convenient of specific immunoglobulin in their yolks (IgY). This study aimed to
isolate IgY specific to anti-idiotype HPV 16 L2 from egg yolk which could be used for future alternative immunogen for HPV vaccine.
Antibody anti-idiotype derived from chicken egg yolk that chickens immunized by HPV 16 L2 antibody. The antibodies were purified
from immunized chicken egg yolk, then electrophoresis it using SDS-PAGE method to determine the molecular weight of IgY. To
determine an amount of protein, we used fluorometer method and to confirm an existence of specific IgY using ELISA method. The
results showed that the IgY preparation dissociated into three protein major bands with molecular weights of 180; 65; and 25 kD. The
IgY and IgY specific concentration in eggs yolk increased until 8th week. After 8th week the levels decreased gradually. Samples
derived from chickens immunized showed significantly higher concentrations of IgY and IgY specifics than control (p<0.05). These
results suggested that chicken IgY could be a practical strategy in large-scale production of specific antibody anti-idiotype HPV 16 L2
for HPV Vaccine.

Keywords: Antibody anti-idiotype, egg yolk, HPV 16 L2, isolation

INTRODUCTION high-titer antibody but type-restricted neutralizing


antibodies. Meanwhile, L2 of genital HPV types contain
Cervical cancer, the most common cancer affecting broadly cross-neutralizing epitopes but low
women in developing countries, is caused by persistent immunogenicity (Karanaam et al. 2009). To enhance
infection with “high-risk” genotypes of human immunogenicity IgY in eggs yolk was used as a carrier.
papillomaviruses (HPV) (Castellsagué 2008). Two virus- The laying hens are an excellent source for the large-scale
like particles HPV VLP (virus-like-particle) vaccines antibody production. Moreover, the phylogenetic distance
aimed to prevent infection by high-risk HPV types are of chicken from mammals caused it can produce high-titer
currently available: bivalent and quadrivalent vaccine (Cho antibodies against conserved mammalian antigens
et al. 2011; Clutt et al. 2007 ). They were a recombinant compared to other experimental animals commonly used
vaccine. Another potential strategy for the production of (da Silva 2010).
safe protective vaccines for HPV infection is utilized anti- This study aimed to produce IgY specific to anti-
ids method. This approach arose from Jerne’s idiotypic idiotype HPV 16 L2 from egg yolk which could be used for
network theory (Ladjemi 2011). The theory stated that future alternative immunogen for HPV vaccine.
antigenic epitope elicits an immune response, resulting in
the production of Ab1 antibody. The ab1 antibody can, in
turn, trigger an anti-idiotypic response consisting of distinct MATERIALS AND METHODS
subsets of Ab2 antibodies: α, β, γ, and ε. The β subtype of
anti-Id Abs express the internal image of the Ag recognized Materials
by the Ab1 Ab and can, therefore, be used as Ag surrogates The study used two specific pathogenic free white
(Jerne 1974). The ab2β have successfully induced anti- leghorn hens from Bio Farma as eggs source, one hen
pathogen and anti-tumor B-cell and T-cell response in injected by antibody HPV 16 L2 (Gmab #2 from Santa
several different species (Ladjemi 2012). The existing Cruz) and another hen injected with physiological NaCl.
vaccines that made of capsid HPV major (L1) can induce Freund's adjuvant from Sigma-Aldrich used to increase
NAWANGSIH et al. – Isolation of antibody anti-idiotype HPV IgY 283

immunogenicity and for purification using solution A and 5min at RT. Spin for 10min at 10000g/11500rpm 4°C.
solution B diagnostic kit from Promega (2004). Discard the supernatant and dissolve the IgY pellet in 3 mL
Fluorometer reagent from Invitrogen used to measure IgY TBS by stirring on a magnetic stirrer with a small stir bar at
levels and to measure IgY specific using KPL reagent. RT. Filter the purified IgY through a 0.2μm filter.

Methods Sodium dodecyl sulfate-polyacrylamide gel electrophoresis


Hen immunization (SDS-PAGE)
Immunization procedure based on Thermo Fisher Sodium dodecyl sulfate-PAGE was done under
Scientific Inc (2012) method. The hen was immunized denaturing conditions using Mini-PROTEAN II Cell
subcutaneously with antibody HPV 16 L2 to generate IgY (BioRad 2014) according to the instruction of the
of anti-idiotype HPV 16 L2 in eggs yolk. Booster injections manufacturer. The purity of various IgY preparations was
performed at week 0, 2, 4 and 5 to raise the antibody anti- estimated using 10% SDSPAGE, and Coomassie brilliant
idiotype levels in eggs yolk (0.2 mg/dose/animal). For the blue R-250 (BioRad) was used to visualize the protein
first injection, the antibody was emulsified in the complete bands. Broad-range SDS-PAGE molecular weight
Freund's adjuvant and for three subsequent boosters in the standards of 15 to 225 kDa (BioRad) were used as markers.
incomplete adjuvant. Eggs were collected daily beginning
before and after the fourth immunizations and stored at 4oC Fluorometer
to be immediately purified with a diagnostic kit. By using fluorometric detectors, sample concentrations
can be measured without the interference of contaminants
Egg Yolk preparation by only emitting fluorescence when bound to the specific
Allow eggs to warm at room temperature before target molecules. Qubit fluorometer used because the Qubit
starting the preparation. Break the shell carefully and drain Fluorometer accurately detects very low concentrations
most ofalbumen. Transfer the yolk into a petri dish and protein. Measurement of IgY levels according to manual
remove the residual albumen with a pipette and with tissue part no. MP32866 MAN0003231 Qubit®2.0 fluorometer
or use the Egg Separator (Promega). Do not break the yolk from Invitrogen.
sack. When the yolk was clean, puncture the yolk sack with
a pipette tip and lets the yolk fluid drip into a tared 100 mL Enzyme-linked immunosorbent assay (ELISA)
beaker glass. Hold back the yolk sack with the pipette tip. The titer of IgY against HPV 16 L2 virus was measured
Determine the weight of the yolk to get the volume (1g = 1 by an indirect noncompetitive enzyme-linked
mL). A yolk of an average egg was 10-15g. immunosorbent assay (ELISA) according to previously
methods with modifications (Fischer and Hlinak 1996).
Precipitation of egg by diagnostic kit First, microtiter plates were coated with the catching
The antibodies were purified from immunized chicken antibody. Monoclonal antibody HPV 16 L2 (G mab#2
eggs yolk by A and B solution diagnostic kit from Promega Santacruz) was diluted 1: 800 in coating buffer and 50 µI
according to the established manual procedures from the per well was added. The plates were incubated for 2 hours
manufacturer. The antibodies were purified from at room temperature and after that overnight at 4°C. The
immunized chicken eggs yolk by diagnostic kit from plates were washed three times with washing buffer (8,0 g
Promega. Place the beaker with the yolk on a magnetic NaCl; 0,2 g KH2P04; 2,9 g Na2HP04 x 12 H20; 0,2 g
stirrer and mix for 1-2min to get a homogenous suspension. KCL; 0,5 mL Tween20; add 1000,0 mL Aquadest., pH
Added three volumes of precipitation A Solution (30 mL 7,4). All further washing steps were carried out with this
per 10 mL yolk) slowly and continue stirring for 5 min at buffer. Then plate were added blocking solution from KPL.
room temperature (RT). Transfer to 50 mL centrifuge tubes The plates were washed three times with washing
and spin 10min 10000g/11500rpm at 4°C. Filter the buffer.Samples containing the IgY were added in well
supernatant through a 0.45μm filter into a tared 100 mL following 4-hour incubation at 37°C, the plates were
glass beaker and determine the weight to get the volume washed three times, and 50 µI per well POD-labeled anti-
(1g = 1 mL). Discard the pellet. Added a stir bar and place chicken IgY (Abcam)were added. Plates were incubated for
on a magnetic stirrer. Added 1/3 volume Solution B 3 hours at 37°C. After this time the plates were washed and
precipitation (10 mL per 30 mL) filtered supernatant and 50 µL of substrate chromogen solution were pipetted into
continued stirring for 5 min at RT. Transfer to 50 mL each well. The reaction was stopped solution (50 ul/well)
centrifuge tubes and spin 10 min 10000g/11500rpm at 4°C. after 15 min. The absorption was measured at 450 nm
Discard the supernatant and dissolve the IgY pellet in 12 using ELISA reader. The reproducibility of the experiment
mL TBS by stirring on a magnetic stirrer with a small stir was ascertained by including a blank control (PBS) and a
bar at RT. At this point, the protein concentration will be negative control (IgY derived from hen immunized by
10 mg/mL and IgY will be ca. 75% pure. This preparation NaCl) in each plate. Positive results are determined based
of IgY was suitable for most applications and could be on three times the value of the negative control. The
stored at -20°C or -75°C. For further purifications of IgY, substance concentration contained in the sample is
place the centrifuge tube containing the redissolved IgY expressed by optical density. Measuring the optical density
from the first precipitation with Solution B on a magnetic (OD) is a common method to quantify the concentration of
stirrer. Add 1/3 volume B solution precipitation slowly (4 substances (Beer-Lambert law), since the absorbance is
mL per 12 mL redissolved pellet) and continue stirring for
284 N U S A N T A R A B I O S C I E N C E 9 (4): 282-287, August 2017

proportional to the concentration of the absorbing molecules are considerably enhanced which separate them
substances in the sample (David W, 2001). from the other proteins of the suspensions (Shafique
1996).
Data analysis The IgY concentration in eggs yolk determined by
All the values will be expressed as the mean±standard fluorometer method. By using fluorometric detectors,
error mean and analyzed by unpaired Student’s T test. The sample concentrations can be measured without the
level of statistical significance will be set at p<0.05 interference of contaminants by only emitting fluorescence
when bound to the specific target molecules (Qubit 2010).
The IgY concentration in eggs yolk increased during the
RESULTS AND DISCUSSION immunization period and the titer began to rise
dramatically in the 4th week. Antibody levels increased
Nowadays, the anti-idiotypic vaccine approach has been until the 8th week, after 8th week the levels decreased
successfully used for various aspects of vaccinology, gradually. The results were accordance with the
especially for tumor immunotherapy (Thomas 2012). recommendations of Thermo Fisher Scientific Inc protocol
Recently, immunoglobulins obtained from avian eggs yolk that the eggs collection was done at 4th week and harvested
were preferred to offer new fields of IgY application, at 8th week. The maximum concentration reached at the 8th
particularly for therapeutic and/or prophylactic use in week is 6,942 ± 0,041 mg/mL and the average of 12nd week
human and veterinary medicine. The use of chickens for is 6,076 ± 0,935 mg/mL. The results were significantly
the production of polyclonal antibodies provide several different (p<0.05) compared with the control group which
advantages over the traditional method of producing the levels were stable relatively with the average value in
antibodies in mammals (Schade et al. 2005). Chicken eggs 12 weeks was 4,348 ± 0,167 mg/mL. The statistical test
yolk as a source for antibody production represents a results showed a significant difference between the
reduction in animal use since chickens produce larger treatment group and negative control group (p<0,05).
amounts of antibodies than laboratory mammals. It also Serum IgG antibodies of immunized chicken were
possible to eliminate the collection of blood, which is transported and accumulated in the egg yolk efficiently
painful for the animal. The European Centre for the (Bar-Joseph and Malkinson 1980). Previous studies
Validation of Alternative Methods (ECVAM) recommends showed that isolation of IgY dengue results obtained 5,77
that yolk antibodies should be used instead of mammalian mg/mL (Sudjarwo et al. 2012), Other studies that isolated
antibodies for animal welfare reasons (Schade et al. 1996). antibody anti-idiotype rabies IgY obtained 9,40 mg/mL
There were several methods for purifying IgY based on (Paryati and Soejoedono, 2006). The concentration of
the strategy of separation of proteins from lipoproteins and isolated antibodies was varied. The variations of antibodies
the rest of the yolk lipids. Purification methods based on formation were influenced by several factors, including
organic solvents like chloroform remain in use. Other animal age, molecular antigen size, complexity of antigenic
methods are based on affinity chromatography or on chemical structure, genetic constitution, the methods of
dilution of the yolk followed by a freezing-thawing antigen insertions and doses of antigen (Leeddell and
process. Ion exchange is also often used for purification Weeks 1995). To confirm the existence of specific IgY
and is usually combined with the number of salts against HPV, ELISA examination was performed. Specific
precipitation method e.g. polyethylene glycol (PEG), antibody anti-idiotype HPV 16 L2 IgY were detected
dextran sulfate, dextran blue, sodium sulfate, ammonium positive result at 4th week after immunization with Optical
sulfate, caprylic acid or sodium citrate (Akita and Nakai Density value 0,575 ± 0,106. It is based on the value of the
1992). Method selection was defended on the yield and cut-off 0,475 calculated from 3 times the average value of
purity desired, final use of the IgY as well as material cost the negative control. Following reimmunization, the level
and labor skills (Carlender 2002). In this study, diagnostic of specific antibodies continued to increase at 8th weeks
kits from Promega for purifying the harvested yolk were after immunization and achieve maximum value with the
used, it contains 3,5% and 12% PEG .The purity level was value of OD 2,564 ± 0,168. After 8th week the levels
70-90%. The Polyethylene glycol (PEG) that used in this decreased gradually to reach a level of 2,051 ± 0,014 (table
process have low toxicity and widely used in 2). The decreasing of IgY levels in chicken eggs is a
pharmaceutical production. PEG is an excellent method to reflection of the loss of plasma cell populations that
precipitate a specific protein from a complex mixture produce specific antibodies. Once fully differentiated,
proteins (Goldring and Coetzer 2003). In the current study, plasma cells die after three to six days and the resulting
PEG precipitation technique was found to be simple, easy level of immunoglobulin would slowly decrease due to this
and economical (Polson 1990) for purification of process of catabolism (Tizard 2013). Meanwhile, on
Immunoglobulins. PEG has multiple hydroxyl groups, chicken that is not injected antibody HPV 16 L2 (control)
which become highly reactive in an aqueous phase. In the showed negative results consistently. The statistical test
aqueous phase, PEG acts as an anion nucleophile and can results showed a significant difference between the
attract positively charged substances, thereby this treatment group and negative control group (p<0,05). The
electrostatic produces changes in water. Therefore, in a pattern of increased levels of specific antibodies in
PEG medium, the hydrophobic interaction of antibody accordance with elevated levels of IgY (Table 1).
NAWANGSIH et al. – Isolation of antibody anti-idiotype HPV IgY 285

Table 1. Concentration of IgY in egg yolks Table 2. Optical density of antibody anti-idiotype of HPV 16 L2 IgY

Concentration of IgY Concentration of IgY Optical Optical


Week Inter- Inter-
sample (mg /mL) control (mg /mL) Week density (OD) density (OD)
pretation pretation
X ± SD X ± SD sample control
0 4.061 ± 0.098 4.010 ± 0.099 X ± SD X ± SD
1 4.386 ± 0.076 4.280 ± 0.071 0 0.172 ± 0.056 - 0.178 ± 0.020 -
2 4.511 ± 0.044 4.190 ± 0.099 1 0.206 ± 0.089 - 0.176 ± 0.014 -
3 4.766 ± 0.062 4.410 ± 0.099 2 0.273 ± 0.107 - 0.175 ± 0.010 -
4 5.054 ± 0.076 4.320 ± 0.035 3 0.46 ± 0.106 - 0.171 ± 0.016 -
5 6.050 ± 0.057 4.278 ± 0.088 4 0.575 ± 0.106 + 0.171 ± 0.004 -
6 6.314 ± 0.079 4.530 ± 0.071 5 0.865 ± 0.104 + 0.175 ± 0.010 -
7 6.410 ± 0.071 4.590 ± 0.085 6 1.401 ±0.087 + 0.175 ± 0.011 -
8 6.942 ± 0.041 4.540 ± 0.057 7 2.158 ± 0.281 + 0.178 ± 0.010 -
9 6.546 ± 0.079 4.420 ± 0.085 8 2.564 ± 0.168 + 0.177 ± 0.011 -
10 6.040 ± 0.028 4.490 ± 0.099 9 2.254 ± 0.116 + 0.179 ± 0.015 -
11 5.995 ± 0.640 4.240 ± 0.071 10 2.142 ± 0.103 + 0.116 ± 0.011 -
12 5.841 ± 0.960 4.220 ± 0.099 11 2.137± 0.202 + 0.115 ± 0.014 -
Average 6.076 ± 0.935 4.348 ± 0.167 12 2.051 ± 0.014 + 0.115 ± 0.012 -
Average 1.424 + 0.158 -
SD 0.890 0.026
Cut off
(3x control
average) 0.475

Figure 1. IgY titer pattern Figure 2. IgY specific for anti-idiotype HPV 16 L2 pattern

IgY and specific IgY were successfully obtained by most common adjuvants used for parenteral immunization.
immunizing the hens with antibody HPV 16 L2 emulsified These adjuvants are a mixture of mineral oil, surfactant,
in Freund’s adjuvant. The use of adjuvants to stimulate the and heat-killed Mycobacterium tuberculosis or M. butyric
immune system and to enhance the immune response is (FCA) or without mycobacteria (FIA) (Freund et al.
routine in antibody production. Most protein antigens, 1937).The water-in-oil emulsion is prepared by mixing one
especially small polypeptides (<10 kDa) and non-protein volume of the adjuvant (FCA or FIA) to one volume
antigens, usually need to be conjugated to a large aqueous antigen solution. In the emulsion, the antigen is
immunogenic carrier molecule to become good distributed in oil droplets which disperse widely after
immunogens. The administration of these antigens would injection in the body, hence increasing the potential for
need an adjuvant especially if it administered in small interaction with relevant cells. Antibody production was
quantity, to assure a high quality/high quantity antibody enhanced by Freund’s adjuvant primarily because of the
response by the immunized animal (Mayo 2009). depot effect, a depot of antigen forms at the injection site
According to Schade et al. (2005) experiment, Freund’s resulting sustained release of small quantities of the antigen
complete adjuvant (FCA) and Freund’s incomplete over a long period of time and non-specific
adjuvant (FIA) remain the most effective adjuvant to immunopotentiation of macrophages by surfactants and the
enhance antibody levels. Freund's Complete Adjuvant mycobacteria (Stills 2005). FIA is frequently used to boost
(FCA) and Freund's Incomplete Adjuvant (FIA) are the animals that received a primary antigen injection with FCA
286 N U S A N T A R A B I O S C I E N C E 9 (4): 282-287, August 2017

but it also can be used as an adjuvant for primary injection.


It has adjuvant properties that favor humoral immunity
without cell-mediated-immunity, although less potently
than FCA. Our experiment used subcutaneous (s.c.)
injection method. The most common route for antigen
injection in hens is the intramuscular (i.m.) route, but the
extensive study by Swarzkoft et al. (2000) showed that the
s.c. injection method provokes higher titer than the
intramuscular (i.m.) injection.
Positive results in the ELISA examination showed
chicken’s immune response to HPV 16 L2 antibody (Ab1).
Antibodies that produced are anti-idiotype that are
expected to have the same serological characteristics with
the original antigen and can be used to replace antigens in
immunization. The ability to mimic the structure of the
original antigen (internal image) is the cornerstone of its
use as a replacement antigen. As with antigens, anti-
idiotype antibodies have the ability to bind competitively
with specific antibodies to the original antigen (Fields et al.
2002).
SDS-PAGE electrophoresis performed to illustrate the
presence of IgY in the sample based on their molecular
weight. Samples at 8th week the highest titer is checked by Figure 3. Proteins isolation of immunoglobulin(IgY) from egg
electrophoresis. There were three major bands of 180, 65 yolk analyzed on SDS-PAGE. Patterns of sample fractions
and 25 kDa, and three minor bands of 100; 70 and 35 kDa. obtained from solution A and solution B purification
The electrophoresis pattern was in accordance with the
standard IgY (figure 2). Narat (2003) stated that IgY had
the molecular weight greater than IgG, which is about 180
kDa or greater. ). IgY is the major low molecular weight
immunoglobulin in egg yolk (Michael 2010). The general ACKNOWLEDGEMENTS
structure of the IgY molecule is the same as the IgG
molecule with two heavy (H) chains and two light (L) This study financially supported by Department of
chains. The molecular mass of the H chain in IgY is larger Ristekdikti Indonesia. The authors gratefully acknowledge
than the H chain from mammals. The greater molecular the help given by experts and staff of the Research and
mass of IgY is due to an increased number of heavy-chain Development Department of Microbiology Faculty of
constant domains and carbohydrate chains (Alexander et al. Medicine Jenderal Achmad Yani University and
2009). IgG has 3 C regions (Cγ1-Cγ3), while IgY has 4 C Laboratory of Genetic and Biomolecular Faculty of
regions (Cυ1-Cυ4) and the presence of one additional C Medicine Padjajaran University.
region with its two corresponding carbohydrate chains
(Charlender 2002) logically results in a greater molecular
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N U SAN TA RA B IOSC IE NC E ISSN: 2087-3948
Vol. 9, No. 3, pp. 288-294 E-ISSN: 2087-3956
August 2017 DOI: 10.13057/nusbiosci/n090308

Yield and Zn content of biofortified rice genotypes in an


Indonesian rice agro-ecosystem

UNTUNG SUSANTO1,♥, UMI BAROKAH1, AKHMAD HIDAYATULLAH1, SATOTO1, MALIKARJUNA SWAMY2


1
Indonesian Center for Rice Research. Jl. Raya 9, Sukamandi, Subang 41256, West Java, Indonesia. Tel. +62-260-520157, Fax: +62-260-521104,

email: untungsus2011@gmail.com
2
Plant Breeding Division, International Rice Research Institute. Los Banos, Laguna, The Philippines, Philippines

Manuscript received: 27 September 2016. Revision accepted: 21 July 2017.

Abstract. Susanto U, Barokah U, Hidayatullah A, Satoto, Swamy M. 2017. Yield and Zn content of biofortified rice genotypes in an
Indonesian rice agro-ecosystem. Nusantara Bioscience 9: 288-294. Approximately one-third of the world's population suffer from Zn
deficiency causing significant socio-economic losses as a result of stunting and compromised immune system function. One strategy to
overcome the problem is by developing rice cultivars with high grain Zn content (Zn Rice) to improve dietary intake.This study reports
the yield and Zn content of 22 rice genotypes developed at the International Rice Research Institute (IRRI) and the Indonesian Centre
for Rice Research (ICRR).The experiment was conducted in the Cirebon district of West Java province during the 2013 wet season
(2013WS). Yield and grain Zn content (using an XRF machine) were measured. Five lines demonstrated higher yield (ranging from 7.0
to 8.9 t/ha) than the check variety Ciherang (5.2 t/ha), but had similar grain Zn content to the check variety Ciherang (23.4 ppm),
ranging from 19.0 to 24.8 ppm. On the other hand, seven lines had higher grain Zn content (ranging from 30.0 to 34.2 ppm) compared to
Ciherang, and five of these lines had comparable yield to Ciherang. The selected lines had acceptable agronomic traits, and are suitable
for further testing and utilization, in addition to providing a foundation for future improvement in the dual goals of increasing the yield
and nutritional value of rice.

Keywords: Indonesia, micronutrients, rice genotype, yield, Zn content

INTRODUCTION Philippines, which was reported as 39.1% during 2000


(Guno 2004).
The prevalence of micronutrient malnutrition, Rice is consumed by almost a half the world's
especially Zn, is recognized as a significant public health population. Nevertheless, it does not provide enough Zn to
problem across much of the world (Mc Clean et al. 2009; match human requirements, and there is a high prevalence
Wessells and Brown, 2012), and this includes Indonesia. of Zn deficiency in countries having rice as a staple food
Zinc deficiency is more extensive in developing countries (Impa and Johnson-Beebot, 2012). Efforts have been
where more than 60 per cent of the population is at risk directed at overcoming this Zn deficiency, such as
(www.harvestplus.org). Approximately one-third of the supplementation, fortification, and biofortification. Zn
world's population are at risk of suffering Zn deficiency fortification efforts have also been conducted in Indonesia
due to low dietary intake of Zn (Hotz and Brown 2004; (Herman et al. 2002). Nevertheless, the coverage of Zn
Myers et al. 2015). Among them, 2 billion people are in fortification in the world is very low (Bhutta et al. 2013). A
Asia and 400 million are in sub-Saharan Africa (IRRI, further complementary effort to overcome this nutrient
2006). The prevalence of Zn deficiency in Indonesia ranges deficiency is by biofortification (Bouis 2004, Bouis et al.
from 10 to 90% according to varying parameters (such as 2011), i.e. developing plant varieties with increased
demographic groups) used in the study of the prevalence micronutrient content, including Zn. This approach is
level of Zn deficiency (Herman 2009). In particular, the sustainable and economically viable (Nakandalage et al.
prevalence of Zn deficiency in Indonesia for children under 2016). Improving Zn content in rice grains is believed to be
five years during 2006 was 31.6% (Herman 2007). one of the most feasible, sustainable, and economical
Zn is an important micronutrient for humans. Zn is a approach to combat Zn deficiency in the world (Salunke et
key component of more than 300 enzymes needed to repair al. 2011; Atiqueur-Rehman et al. 2014).
wounds, maintain fertility, synthesize protein, and boost Breeding efforts to conduct Zn biofortification for rice
immunity among the many functions important to human has been initiated at IRRI either by using conventional
health and productivity (Mares-Perlman et al.1995). Zn is breeding approaches (Graham et al. 1999; Gregorio et al.
also important in vitamin A metabolism, and one effect of 2000; Slamet-Leodin et al. 2015) or transgenic approaches
Zn deficiency is xeropthalmia (Morrison et al. 1978). (Trijatmiko et al. 2016), and efforts continue on
Another effect of Zn deficiency is stunting during both.Promising breeding materials have been developed
childhood (Herman 2007). The prevalence of stunting in and shared with collaborating countries, including
Indonesia was around 45% in the1990s and 36.2% in 2006 Indonesia. Bangladesh and Philippines had just released Zn
(Taufiqurrahman et al. 2009). It is also prevalent than in the rice varieties for the certain countries.
SUSANTO et al. – Zn content of various rice genotypes 289

Some promising lines had been shared with Indonesia Table 1. Source of variance and expected value of Randomized
and initially screened under irrigated lowland conditions. Complete Block Design
Promising lines have been selected for further testing.
Nevertheless, the stability of yield and grain Zn content Source of Expected
dF Mean square
requires evaluation following each round of selection to variance value
Block r-1 -
ensure the dual goals of improved productivity and
Genotype g-1 Mean Square of σ2e + r σ2g
improved nutrition are met. This research aimed to test the Genotype (MSG)
yield and Zn content of 22 rice genotypes originating from Error (r-1) (g-1) Mean Square of Error σ2e
IRRI and ICRR, Indonesia. (MSE)
Total gt-1
Note: σ2g = Genetic Variance; σ2e = Variance of environment.
MATERIALS AND METHODS
Heritability was calculated by the formula:
Study area
This study was focused on twenty-two selected rice σ2e= MSE
lines consisted of genotypes introduced from IRRI along
with Indonesian varieties. The experiment was conducted σ2g = (MSG-MSE)/r
in Palimanan of Cirebon District, West Java Province,
Indonesia during Wet Season of 2013. It is located in- σ2P = σ2g+ (σ2e/r)
6.705825, 108.435025 at around 15 m asl. The experiment
was conducted under irrigated conditions.

Procedures
Field experiment Genetic variability is defined as the tendency of
The trial was designed as a Randomized Complete individual genetic characteristics in a population to vary
Block Design with two replications.Seedlings (21 days from one another (biology online, http://www.biology-
after sowing) were transplanted at a spacing of 20cm x online.org/bodict/index.php?title=Genetic_variability&acti
20cm of in plots measuring 2m x 3m. Crop establishment on=edit). Genetic variability could be classified as wide or
and fertilizer applications followed local recommendations narrow, calculated by the comparison of genetic variability
(equal to each 300 kg Urea, 50 kg SP-36 and 50 kg KCl for and standard deviation of genetic variability. Genetic
one ha, referred to Ministry of Agriculture Decree No. variability was considered as wide if σ2g> 2 σσ2g, and
40/Permentan/OT.140/04//2007), while pests and diseases otherwise was narrow (Pinaria et al. 1995). Standard
were managed according to Integrated Crop Protection deviation of genetic variability was calculated as follows
principles. The main traits to be measured were yield, (Hallauer and Miranda 1995):
which was converted into t/ha at 14% moisture content, and
Zn content in rice grains. Some basic agronomic traits were
also measured, i.e. heading date, plant height, tiller number,
number of filled and unfilled grains/panicle, seed set, and
1000 grain weight (g).

Zn content measurement RESULTS AND DISCUSSION


The Zn content (ppm) of dehulled (brown rice) grains
sample was measured using an XRF machine (Oxford Variances and genetic variability
Instrument X-Supreme) that had been validated by ICP The trial was conducted during wet season, and was
method. The machine was located in the Plant Breeding fully irrigated during the complete plant growth cycle.
Laboratory of ICRR (Indonesian Center for Rice Research) There were no serious biotic stresses such as pests and
of IAARD (Indonesian Agency of Agricultural Research diseases during the plant establishment.However, at the
and Development) in Sukamandi, Subang District, West early vegetative stage irrigation water containing waste
Java. Approximately 50g grain samples from each plot from spirt oil factory was accidentally used to irrigate the
were de-hulled using Satake THU Testing Husker. Brown field, but did not significantly affect the plants.
rice samples were then sorted to get only healthy and fully Variance analysis showed that at P threshold of 0.05
filled grain then used for Zn content measurement using the there was significant variation among genotypes for all the
XRF machine. observed traits, except for seed set (Table 2). At the
Data analysis minimum, two genotypes had significant differences for all
Data analysis was executed using Excel and CropStat traits except seed set. Further analysis allowed the
Ver 6.1. (IRRI 2007). Variance components were analysis partitioning of genetic variance, demonstrating variation
based on Randomized Complete Block Design model among the genotypes, with some traits having larger
(Table 1) and the analysis of heritability and genetic genetic variances e.g., plant height, 1000 grain weight, and
variability were calculated following (Pinaria et al. 1995; yield. On the other hand, heading date, tiller number/plant,
Yuwono et al. 2015). panicle number / plant, filled grain/panicle, unfilled grain/
290 N U S A N T A R A B I O S C I E N C E 9 (4): 288-294, August 2017

Table 2. Variance analysis and Heriability of agronomic traits of 22 Rice Genotypes, Cirebon, WS 2013/2014

Grand Var. Var. St. Dev. Gen.


Character MS Gen MS E P Gen H2 (%) CV (%)
mean Gen Phe. Gen. Var.
Heading date (DAS) 87.50 51.33 16.47 0.01 11.62. 56.8239 8.11 Narrow 20.45 4.60
Plant height (cm) 115.40 643.83 33.43 0.00 203.47 654.974 95.11 Wide 31.06 5.00
Tiller number/plant 23.82 66.23 27.55 0.03 12.89 75.4121 10.91 Narrow 17.10 22.00
Panicle number/plant 19.77 62.57 24.53 0.03 12.68 70.7482 10.19 Narrow 17.93 24.60
Filled grain/panicle 80.10 652.58 298.10 0.05 118.16 751.943 109.70 Narrow 15.71 21.80
Unfilled grain/panicle 52.59 483.18 167.04 0.01 105.38 538.862 77.12 Narrow 19.56 24.80
Seed set (%) 60.45 191.78 95.49 0.07 32.10 223.614 32.93 Narrow 14.35 16.20
1000 grains weight (g) 24.77 21.06 1.21 0.00 6.62 21.4653 32.35 Wide 30.82 4.50
Yield (t/ha) 5.14 7.41 0.74 0.00 2.22 7.65509 3.11 Wide 29.02 17.30
Zn content (ppm) 26.56 51.21 7.97 0.00 14.41 53.8654 1.10 Narrow 26.76 22.60
Note: MS Gen = Mean Square of Genotype; MS E = Mean Square of Error; P Gen = Probability of Genotype; H2 = Broad Sense
Heritability; CV = Coefficient of Variation; DAS = Days After Sowing; Seed Set = proportion of filled grains over total grain per
panicle

panicle, and Zn content had smaller genetic variance weight, and yield (Table 2). This highlights the
components. The greater the genetic variability the greater opportunities for selection of lines which match the yield of
the opportunity to improve the trait through selection locally adapted types, such as Ciherang, and which match
among genotypes included in the study. Lower genetic for agronomic characteristics such as plant height and grain
variability for a trait indicates uniformity across the studied weight.
genotypes, thus little chance to select outstanding lines
among the genotypes (Ruchjaniningsih 2006). Yield and Zn content
Based on yield performance, IR64 (8.87 t/ha), BR28
Heritability (8.14 t/ha), IR91152AC-81 (8.03 t/ha), NSICRc222 (7.29
Heritability represents the proportion of variation due to t/ha), NSICRc238 (7.04 t/ha) had higher yield compared to
genetic effects compared to the total variation in the Ciherang (popular variety; 5.16 t/ha) (Figure 1).The variety
expression of a trait (Sleper and Poehlman 2006).Broad IR64 in this research originated from IRRI, and had the
sense heritability in the trial ranged from 15 to 31%. highest yield in this trial, reflecting its adaptability and
Among the traits, plant height, 1000 grain weight, yield, productivity. IR64 is commonly referred to as a mega
and Zn content had the highest levels of heritability ranging variety in the tropical rice growing areas of the world. The
from 26.8% to 31.1% (Table 2).It indicated that for the genotypes which had relatively high yield were mostly
performance of genotypes for these traits, genetic factors recently developed varieties, not local varieties. Many of
were responsible for around one-third of the variation these modern varieties have IR64 as one of the parents in
noted. Thus, selection for those traits is warranted, despite the pedigree, or have the parents of IR64 in the pedigree.
the fact that genetic variation is a minor portion of the total The plant architecture and physiological characteristics of
variation noted. the modern varieties and their derivatives had been
On the basis of testing these genotypes, selection optimized for high yield, including elements such as large
among them to develop high yielding with high Zn content panicle size and appropriate tillering capacity. Those traits
variety is feasible. Further, management options are important in increasing yield capacity of the plant
(agronomic management) to maximize yield and Zn (Peng et al. 2008).
content may also be beneficial. Slaton et al. (2001) reported Nevertheless, in this study, those high yielding
that either Zn seed treatment or soil Zn fertilizer increase genotypes had relatively low Zn content (19.00-24.80
Zn content in rice plant tissue compared to control. It is a ppm). For further efforts, these genotypes should be used
different case with Iron, which crop Fe fertilization is not as recurrent or recipient parents to combine high grain Zn
very effective due to Fe soil insolubility (Sperotto et al. content in combination with high yield. Harvest Plus is an
2012). On the other hand, organic farming system international program which developed initiatives to
decreases Fe and Zn content (Sakagami et al. 2016). While address human micronutrient malnutrition through
it was suggested that plant height, heading time, or grain improving the micro nutrient concentration of staple foods.
shape are not of primary importance in controlling This program has targeted Zn level of brown and polished
economic variation (include Zn) in rice grain (Pinson et al. rice up to 30 ppm and 28 ppm respectively (Johnson-
2015). Selection for specific growth duration best suited to Beebout et al. 2009, Trijatmiko et al. 2016).
the production environment and specific plant stature is Based on grain Zn content, seven lines had higher Zn
also possible, given the variation demonstrated. content compared to the current most popular variety in
The genotypes tested in this study demonstrated that all Indonesia, i.e. Ciherang (23.35 ppm). Among the seven,
the traits demonstrated a range of genetic variability, with five lines had comparable yield to Ciherang, i.e. BR7840-
the greatest genetic variation for plant height, 1000 grain 54-2-5-1 (33.08 ppm; 5.75 t/ha), IR68144-2B-2-2-3-1-166
SUSANTO et al. – Zn content of various rice genotypes 291

(34.22 ppm; 5.08 t/ha), IR84020-84-2-3-2 (29.9 ppm; 5.70 best genotypes among the tested materials in each traits and
t/ha), Vanjakohonandiana (31.73 ppm; 4.39 t/ha), combine the best traits might be useful to get the ideal
IR10M195 (IR84842-35-3-1-1-2-2) (35.68 ppm; 3.95 plants which have a combination of high yielding traits,
t/ha;). While these lines had relatively high grain Zn such as more tiller number, more filled grain per panicle,
content, this was combined with modest yield performance higer seed set, and bigger grain size consequencing heavier
compared to the best conventional check variety (Table 3). of 1000 grain weight. Grain number, panicle seed setting
However, two lines, i.e. IR10M196 (IR84842-131-1-2-1-1- rate, panicle number and grain weight are the most
3) (34.48 ppm; 2.74 t/ha) and BR7840-54-1-2-5 (29.98 important components of rice grain yield (Li et al. 2013).
ppm; 3.29 t/ha) had relatively low yield. It is possible that
these lines could hopefully be utilized as donors for high Discussion
Zn content in the breeding program, proving that the The target level of breeding for high Zn content is 24-
relatively higher grain Zn content was not driven by the 28 ppm Zn content in polished rice grains which is
lower yield. essential to attain 30% of the estimated average
Correlation between yield and grain Zn content in this requirement (EAR) for humans (Bouis et al. 2011).
trial was significantly negative (-0.46) with t value of - Nevertheless, studies have reported that for current
2.286 and t5%=1.73. This contrasts with the results reported varieties, the polished rice grains supply only one fifth of
by Sala and Geetha (2015) in which grain Zn content and daily Zn requirements (Prom-uthai et al. 2010, Sharma et
yield were positively correlated. They used F4 lines al. 2013).
developed from two cross combinations, viz., ADT37 × The highest Zn content identified in this study was
IR68144-3B-2-2-3 and TRY(R)2 × Mapillaisamba. They 35.68 ppm achieved byIR10M195 (IR84842-35-3-1-1-2-2).
noted the importance of choosing appropriate parental It was measured on brown rice, in which the bran and the
genotypes to dissect the relationship between Zn content germ remain intact, and naturally, carry higher Zn and
and grain yield, and concluded that the combination of high other nutrients. The Fe and Zn are mostly located in
yield and high grain Zn was possible. aleurone layer, but the percentage of Zn in polished rice
(endosperm only) varies from around 75-84 % of the total
Agronomic characteristics of selected genotypes Zn content including the aleurone layer. The case for Fe is
Agronomic characteristics among the lines were varied only around 19-30% (Johnson et al. 2011). Assuming 75%
however the genotypes mostly had similar characteristics to of the Zn is located in endosperm, IR10M195 (IR84842-
modern varieties in Indonesia such as Ciherang. The 35-3-1-1-2-2) is predicted to have Zn content in polished
genotypes had growth duration of around 115 days, plant grain of around 26.76 ppm which is within the range of the
height of around 100 cm, and erect leaves (ICRR 2015). targeted levels. Nevertheless, the line had a relatively low
Heading dates ranged from 76 days (Pokkali) to 96 days yield (3.95 t/ha).
(IR83286-22-1-2-1-1), and mostly the high Zn materials
had a longer time to flower compared to Ciherang (85
days). Plant height ranged from 76.70 cm (IR68144-2B-2-
2-3-1-166) to 158.80 cm (Pokkali), while Ciherang was
113.20 cm. Tiller number ranged from 14 (IR10M195
(IR84842-35-3-1-1-2-2)) to 41 (IR68144-2B-2-2-3-1-166)
and Ciherang had an average of 19 tillers. Most of the high
Zn materials had more tillers than Ciherang. Filled
grains/panicle varied between 43 (IR83286-22-1-2-1-1) to
124 (IR69428-6-1-1-3-3), while Ciherang had 81 filled
grains per panicle. Seed set ranged from 35.27 %
(IR83286-22-1-2-1-1) to 74.62 % (IR91152AC-819), while
Ciherang had 64.46 %. Weight of 1000 grains ranged from
15.80 g (IR68144-2B-2-2-3-1-166) to 30.90 g (Pokkali),
while Ciherang had 26.30 g (Figure 2). Selection of the Figure 1. Yield (solid) and Zn Content (vertical lines) of 22 rice
genotypes grown at Cirebon, WS 2013/2014

Table 3. Agronomic traits of Five selected lines based on Zn content and yield, Cirebon, WS 2013/2014

1000
Heading Plant Filled Unfilled Seed
Tiller Panicle grain Yield Zn
Genotype date height grains grain/ Set
number number weight (t/ha) (ppm)
(DAS) (cm) /panicle panicle (%)
(g)
BR7840-54-2-5-1 89 104.50 26 16 86 57 61.60 21.80 5.75 33.08
IR84020-84-2-3-2 89 103.10 31 28 68 37 65.04 21.45 5.69 29.90
IR10M195 (IR84842-35-3-1-1-2-2) 93 107.30 14 12 92 56 62.43 26.59 3.95 35.68
IR68144-2B-2-2-3-1-166 85 76.70 41 32 66 24 73.73 15.80 5.08 34.22
Vanjakohonandian 83 148.90 29 15 46 80 36.38 28.95 4.39 31.73
Ciherang 85 113.20 19 17 81 44 64.46 26.30 5.16 23.35
292 N U S A N T A R A B I O S C I E N C E 9 (4): 288-294, August 2017

A B

C D

E F

Figure 2. Agronomic traits of 22 Rice Genotypes, Cirebon, WS 2013/2014. A. Heading date (days After Sowing), B.Plant Height (cm),
C. Tiller Number, D. Filled Grain/Panicle, E. Seed Set (%), F. 1000 Grain Weight (g)

Zn content in rice grains in a quantitative trait which is also affects heavy metals (including Fe, Zn, and Cd)
either directly or indirectly affected by some other traits availability for uptake by the plants. Flooding practice
simultaneously. It had medium heritability, reflecting that increases Fe availability but decreases Zn and Cd
environmental effect is presented in expressing Zn content. availability (Slamet-Leodin et al. 2015).
Regarding this one, crop genetic and management practices Zn accumulation incorporates many processes, such as
improvement should be conducted simultaneously uptake, remobilization, transport in the plant, and
considering the environmental change. The interaction of environmental interaction. It is important to study genetic
environmental and genetic factors on Zn homeostasis properties of each of these component traits. Increasing the
should be taken into account. Different processing efficiency of each of these processes affecting Zn uptake
technologies, promoters, and inhibitors of Zn from roots to its accumulation in endosperm should allow
bioavailability in rice grains is also important the maximum accumulation of Zn in the grain. Zn-efficient
(Nakandalage et al. 2016). Achieving the target of 30 ppm and non-efficient varieties should be tested under Zn
Zn content in brown rice requires strategic use of Zn sufficient and deficient conditions at various growth stages
fertilizers as many as rice fields have low available Zn to define the genetic capacity of Zn uptake (Nakandalage et
(Johnson-Beebout et al. 2009).Fertilizer N and P al. 2016). On the other hand, transgenic approaches by
applications during grain filling also promote Zn uptake modifying genes controlling those traits may result in
and remobilization (Khan et al. 2015). Soil redox potential greater grain Zn content compared to using conventional
SUSANTO et al. – Zn content of various rice genotypes 293

breeding, however, this introduces further complexity in improve Zn intake among those at risk from micronutrient
gaining regulatory approval for such varieties to be grown. malnutrition. This is contingent on developing varieties
Global climate change increases CO2 concentration in with high Zn content in the rice grain while maintaining or
the air. Some studies indicated that increasing CO2 affects increasing yield. Once the variety is disseminated and
plant growth, yield, and quality of cereals, including rice. adopted, this becomes a sustainable method of improving
Without any other limitations, increasing the CO2 level nutrition among the population of rice consumers, provided
increased photosynthesis and thus yield. Nevertheless, for that seed purity (and thus nutritional benefit) is maintained,
grain quality, increased CO2 level reduces all micro and farmers elect to use the variety.
nutrient content, including Zn content in rice (Nakandalage
et al. 2016; Myers et al. 2014). Thus, as CO2 rises, greater
efforts may be needed to retain Zn levels at sufficient levels ACKNOWLEDGEMENTS
to supply Zn requirement of rice consumers in the future.
In this study, five lines, i.e. IR64 (8.87 t/ha), BR28 This research was collaboratively conducted by IRRI
(8.14 t/ha), IR91152AC-81 (8.03 t/ha), NSICRc222 (7.29 (International Rice Research Institute) and Indonesian
t/ha), NSICRc238 (7.04t/ha) had higher yield compared to Agency for Agricultural Research (IAARD) through
Ciherang (popular variety; 5.16 t/ha). Nevertheless, The Indonesian Center for Rice Research (ICRR).
lines had relatively low Zn content (19.00-24.80 ppm). On
the other hand, seven lines had higher Zn content compared
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field areas of Jatinangor. Jurnal Hortikultura. 16 (4): 290-298.
[Indonesian]
N U SAN TA RA B IOSC IE NC E ISSN: 2087-3948
Vol. 9, No. 3, pp. 295-299 E-ISSN: 2087-3956
August 2017 DOI: 10.13057/nusbiosci/n090309

Short Communication:
Augmentation of cardioprotective effect of captopril by Costus speciosus
against isoproterenol induced myocardial toxicity in rats

ABDULRAHMAN A.I. AL-YAHYA1,♥, MOHAMMED ASAD1, ABDULMONIEM SADABY1,


KHALID ELFAKKI IBRAHIM2
1
College of Applied Medical Sciences, Shaqra University. P O Box 1383, Shaqra 11961, Saudi Arabia, Tel.: +966-531339880,

email: alya7ya@su.edu.sa, alyahyashaqra@gmail.com
2
Department of Zoology, College of Science, King Saud University. Riyadh 12371, Saudi Arabia

Manuscript received: 12 July 2017. Revision accepted: 21 August 2017.

Abstract. Al-Yahya AAI, Asad M, Sadaby A, Ibrahim KE. 2017. Short Communication: Augmentation of cardioprotective effect of
captopril by Costus speciosus against isoproterenol induced myocardial toxicity in rats. Nusantara Bioscience 9: 295-299. This study
determined the pharmacodynamic interaction of captopril, a known cardioprotective effect with methanolic extract of Costus speciosus
rhizomes (Costaceae) that is known for its powerful antioxidant action during isoproterenol induced cardiac toxicity in rats. A
methanolic extract of the rhizomes was prepared by maceration. Rats were administered the methanolic extract at two different doses of
200 mg/k or 400 mg/kg orally and captopril was administered orally at a dose of 30 mg/kg. All the drugs alone or in combination were
once daily for two weeks. At the end of treatment period, two doses of isoproterenol (150 mg/kg, s.c) were administered to rats at 24 hr
interval. Blood was withdrawn to estimate creatinine kinase-MB (CK-MB) activities. The heart tissue was subjected to histological
examinations to determine the extent of damage. Isoproterenol induced severe damage to the myocardium that was indicated through an
elevation in serum CK-MB activity and the same was confirmed by histological examinations. Costus speciosus at both the tested doses
attenuated the damage produced by isoproterenol. Both the doses caused a decrease in the biomarker activity as well as reduced the
myocardial damage as observed in histological examination. A similar effect was observed with captopril. The co-administration of
captopril with either dose of Costus speciosus demonstrated excellent cardioprotection suggesting that combination of this herb with
captopril augments its cardioprotective action. It was concluded that Costus speciosus shows dose-dependent cardioprotection and
augments the cardioprotective effect of captopril during isoproterenol induced cardiotoxicity in rats.

Keywords: Acute myocardial injury, Costus speciosus extract, captopril, isoproterenol, interaction, methanolic extract

INTRODUCTION action, the rhizomes is believed to have very strong


antioxidant effect that can attenuate the Fenton reaction-
Costus speciosus (Koen ex. Retz.) belongs to the family mediated oxidation of biological lipid substrates (Pai
Costaceae (Zingiberaceae). The plant possesses many Kotebagilu et al. 2015). It was rated as third plant with
pharmacological activities and is used traditionally in the potent antioxidant effect among eighteen commonly used
treatment of several diseases. The plant is so widely used antioxidant plants (Lee et al. 2015). It is also reported to
that is considered as endangered if its use continues to reduce hydrogen peroxide induced oxidative damage on
exceed its cultivation. Some of the pharmacological biological substrates (Pai Kotebagilu et al. 2014) and also
activities reported for this plant includes anti-inflammatory, prevent development of cancer due to its antioxidant action
antiangiogenic, antioxidant, antibacterial and antifungal (Baskar et al. 2012). The antioxidant effect of this plant is
effects due to presence of susquiterpines (Duraipandiyan et directly related to its phenolic contents (Vijayalakshmi and
al. 2012; Al-Attas et al. 2015; Selim and Al Jaouni 2016), Sarada 2008; Nehete et al. 2010).
antihyperglycemic activity that is mediated through It is well known from several studies that plants having
inhibitory effect on α-glucosidase and glycation (Perera et phenolic compounds and exhibiting antioxidant effects
al. 2016), anticholinesterase (Bhattacharya et al. 1972), protects heart from different types of damage such as
antipyretic, anti-inflammatory and analgesic activities ischemia reperfusion and isoproterenol induced cardiac
(Srivastava et al. 2013) among other activities that includes injury (Panda et al. 2016; Wang et al. 2017; Cao et al.
antidiuretic, larvicidal, antistress, estrogenic, astringent, 2017; Garjani et al. 2017; Rasines-Perea et al. 2017).
aphrodisiac, purgative, anthelminthic, expectorant Furthermore, the rhizomes of Costus speciosus known as
activities. It also has anti-fertility, anabolic properties Ru-rta are believed to regulate blood pressure and correct
(Pawar and Pawar 2012). heart problems (Clifford 2001). Hence, we studied earlier
Of all these activities of Costus speciosus, the most its cardioprotective effect and demonstrated that it has good
interesting and widely studied effect is its antioxidant cardioprotective action (unpublished data). However, the
effect. Though many plants are known to have antioxidant trend of using herbal plants alone is declining and use of
296 N U S A N T A R A B I O S C I E N C E 9 (4): 295-299, August 2017

herbal plants as supplements along with modern medicine speciosus extract- 200 mg/kg and 400 mg/kg, along with
is on the rise. Hence, the present study was carried out to captopril (30 mg/kg) respectively for two weeks.
determine if the coadministration of methanolic extract of All treatment was given daily for 2 weeks. At the end of
rhizomes of Costus speciosus can augment the the treatment period, ISO (150 mg /kg, s.c) was
cardioprotective effect of captopril on myocardial damage administered to all the animals (except group I) for two
induced by isoproterenol in rats. consecutive days (Asdaq et al. 2008). Forty eight hours
after the first dose of ISO, animals were anesthetized using
ether and blood was withdrawn and the serum was used for
MATERIALS AND METHODS the estimation of creatinine kinase-MB (CK-MB) using
commercially available kits. Thereafter, all the animals
Preparation of the extract were sacrificed, and the hearts were used for histological
The rhizomes of Costus speciosus were purchased from examinations using H&E stain. The myocardial damage
a local market in Riyadh, Saudi Arabia. It was was determined by giving scores depending on the intensity
authenticated by Prof A.M. Sadaby (College of Applied as follows (Karthikeyan et al. 2007); no changes—score 00;
Medical Sciences, Shaqra University, Saudi Arabia) and a mild—score 01 (focal myocytes damage or small
voucher specimen (CAMS/rh03/09-2016) has been multifocal degeneration with slight degree of inflammatory
preserved in the college for future reference. The rhizomes process); moderate—score 02 (extensive myofibrillar
were finely powdered and extracted using methanol in a degeneration and/or diffuse inflammatory process);
closed glass jar for 72 hr initially. The extraction of the marked—score 03 (necrosis with diffuse inflammatory
solid residue (marc) was further done twice for 48 hr each. process).
The extract obtained was evaporated under reduced
pressure. The final extract obtained after evaporation was Statistical analysis
stored in a refrigerator till use. Results are given as mean ± SEM. Statistical
significance was determined through one-way analysis of
Preliminary chemical analysis variance (ANOVA) followed by Tukey-Kramer multiple
The preliminary chemical analysis revealed the comparison tests. For comparison of histological scores,
presence of carbohydrates, saponins, flavovoids and amino Kruskal-Wallis test with Dunn post test was used. P < 0.05
acids. indicated statistically significant difference.

Experimental animals
Laboratory bred male Wistar rats (180-220 g) were RESULTS AND DISCUSSION
housed in a controlled environment at a temperature of 25
± 2 °C under 12: 12 hr light dark cycle. The animals were Effect on serum CK-MB levels
maintained under standard conditions in an animal house Administration of ISO to rats induced myocardial
and all the experimental procedures were approved by the damage that was confirmed by an increase in serum CK-
university scientific committee. MB levels compared to normal animals. The methanolic
extract of Costus speciosus significantly reduced the serum
Dose selection CK-MB levels in a dose dependent manner when compared
The methanolic extract of Costus speciosus was to ISO administered control group. (p<0.001). As
administered at two doses of 200 mg/kg and 400 mg/kg expected, captopril (30 mg/kg) also reduced the serum CK-
orally. The doses were selected after doing preliminary MB levels when compared to ISO control group (p<0.001)
experiments in our laboratory and from previous reports indicating cardioprotective effect. The coadministration of
(Ali et al. 2014). Captopril was administered at a dose of either dose of methanolic extract of Costus speciosus with
30 mg/kg orally (Milanez et al. 1997). The extract and captopril (30 mg/kg) showed the maximum effect (Figure
captopril were suspended in distilled water using 2% 1).
acacia. The control animals received only vehicle, which as
2% acacia in water. Effect on histological scores
Heart damage was determined by loss of cellular
Isoproterenol induced myocardial damage in rats architecture, nuclear duplication, increased infiltration of
Animals were divided into seven groups of six animals leucocytes and prominent hyperchromasia. These changes
each, i.e. (i) Group I had normal animals and they received were prominent in control animals treated with ISO (Figure
only vehicle (2 ml/kg, p.o). (ii) Group II animals were 3). The histological scores that were given based on the
treated with vehicle (2 ml/kg, p.o) for two weeks and they severity of damage was significantly less in Costus
received isoproterenol (ISO). (iii) Groups III and IV speciosus (400 mg/kg, p.o) and captopril (30 mg/kg, p.o)
received orally Costus speciosus extract- 200 mg/kg and treated groups compared to ISO treated control while the
400 mg/kg, respectively for two weeks and followed by lower dose of Costus speciosus (200 mg/kg, p.o) did not
administration of ISO. (iv) Group V was treated with show any significant reduction in severity scores (Figure
captopril at a dose of 30 mg/kg orally for two weeks 2). Sections of heart tissue from animals treated with lower
followed by ISO. (v) Group VI and VII received Costus dose of Costus speciosus (200 mg/kg, p.o) showed mild to
moderate damage ranging from loss of cellular architecture
AL-YAHYA et al. – Interaction of captopril with Costus speciosus 297

to nuclear duplication and increased infiltration of our laboratory. In our study, we observed that
leucocytes (Figure 4) while those treated with higher dose administration of higher doses of extract such as 500 mg/kg
of Costus speciosus (400 mg/kg, p.o) showed no or very or 1000 mg/kg orally produced toxic effects in the animals.
mild damage such as some loss of cellular architecture The animals started to show weakness after 4-5 days of
(Figure 5). Captopril (30 mg/kg, p.o) almost prevented the drug administration and continuing the administration for
ISO induced myocardial damage with sections from 3-4 days more resulted in deaths of some animals. Hence,
animals showing almost no damage (Figure 6) while the doses more than 400 mg/kg orally were not used. On the
maximum effect was observed in animals treated with other hand, administration of doses less than 200 mg/kg
either dose of Costus speciosus plus captopril (Figure 7). such as 100 mg/kg or 50 mg/kg did not produce any
significant effect.
Discussion The rhizomes of Costus speciosus are reported to
The present study was undertaken to determine the contain a number of chemical constituents that includes
interaction of methanolic extract of Costus speciosus saponin such as tigogenin, diosgenin, sapogenin along with
rhizomes with captopril on the ISO induced myocardial steroids and alkaloids (Muniyandi et al. 2013). It is also
infarction in rats. The result of the study suggests that known to contain aliphatic hydroxyl ketones, triterpenes,
Costus speciosus rhizomes augment the cardioprotective starch mucilage, oxa-acids, fatty acids, abscisic acid and
activity of captopril during myocardial damage induced by corticosteroids (Rajesh et al. 2009). An important
ISO in rats. constituent with respect to the antioxidant effect of Costus
As mentioned earlier, Costus speciosus is so widely speciosus rhizomes are flavonoids, that are a subclass of
used traditionally that it is on the verge of extinction if its polyphenols, which are further divided into subclasses such
cultivation is not increased indicating its therapeutic as flavones, flavonols, isoflavones, anthocyanins, flavanols,
potential. As mentioned earlier, the rhizomes of Costus proanthocyanidins and other plant phenolics (Nehete et al.
speciosus are the most widely part of the plant; hence, this 2010; Chang et al. 2012). As mentioned earlier
was used in the present study. The doses of the extract for (Vijayalakshmi and Sarada, 2008; Nehete et al. 2010),
administration to rats were selected in earlier literature (Ali these polyphenols are responsible for the antioxidant effect
et al. 2014) and also by preliminary studies carried out in of Costus speciosus.

aaa
Figure 1: Effect of Costus speciosus extract and captopril on serum CK-MB levels. Note: All values are mean±SEM, n=6, p<0.001
compared to normal animals, *p<0.05, **p<0.01, ***p<0.001 compared to ISO treated control

aa
Figure 2: Effect of Costus speciosus extract and captopril on histological scores. Note: All values are mean±SEM, n=6, p<0.01
compared to normal animals, *p<0.05, **p<0.01, ***p<0.001 compared to ISO treated control
298 N U S A N T A R A B I O S C I E N C E 9 (4): 295-299, August 2017

Figure 3: Microscopic section of heart from isoproterenol (ISO) Figure 6: Microscopic section of heart from captopril (30 mg/kg,
control animals (H&E stained, ×400) showing severe damage to p.o) + isoproterenol (ISO) treated animals (H&E stained, ×400)
heart tissue. There is loss of cellular architecture (a), nuclear showing almost normal cytoarchitechture
duplication (b) , increased infiltration of leucocytes (c) and
prominent hyperchromasia (d).

Figure 4: Microscopic section of heart from Costus speciosus Figure 7: Microscopic section of heart from Costus speciosus
(200 mg/kg, p.o) + isoproterenol (ISO) treated animals (H&E (400 mg/kg, p.o) + captopril (30 mg/kg, p.o) + isoproterenol
stained, ×400). . There is loss of cellular architecture (a), nuclear (ISO) treated animals (H&E stained, ×400) showing almost
duplication (b) and increased infiltration of leucocytes (c) normal cytoarchitechture.

ISO induces myocardial through reduction of


endogenous antioxidants such as superoxide dismutase
(SOD) and catalase which leads to myocardial damage due
to oxidative stress (Yang et al. 2011). Once the
myocardium is damages due to oxidative stress, it causes
release of CK-MB present in the myocytes to the plasma
leading to an increase in serum CK-MB levels. An increase
in levels of CK-MB is an indication of myocardial damage.
In the present study, ISO administration led to an increase
in serum CK-MB levels indicating damage to myocardium.
Administration of methanolic extract of Costus speciosus
rhizomes and captopril attenuated the increase in serum
CK-MB levels suggesting cardioprotective effect. The
cardioprotective effect was further confirmed by
Figure 5: Microscopic section of heart from Costus speciosus histological studies, wherein changes in cellular
(400 mg/kg, p.o) + isoproterenol (ISO) treated animals (H&E
architecture inflammation and necrosis were taken as
stained, ×400) showing almost normal cytoarchitechture.
AL-YAHYA et al. – Interaction of captopril with Costus speciosus 299

parameters for determination of myocardial damage. The injury in isolated rat heart. Folia Morphol (Warsz). DOI:
10.5603/FM.a2017.0011.
results of the histological studies supported the biochemical Karthikeyan K, SaralaBai BR, Devaraj N. 2007. Cardioprotective effect of
findings. grape seed proanthocyanidins on isoproterenol-induced myocardial
The exact mechanism for augmentation of injury in rats. Intl J Cardiol 115: 326-333.
cardioprotective effect of captopril by Costus speciosus Lee YH, Choo C, Watawana MI, Jayawardena N, Waisundara VY. 2015.
An appraisal of eighteen commonly consumed edible plants as
rhizomes cannot be explained with the present data. functional food based on their antioxidant and starch hydrolase
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N U S AN T AR A BIO S C IEN C E ISSN: 2087-3948
Vol. 9, No. 3, pp. 300-305 E-ISSN: 2087-3956
August 2017 DOI: 10.13057/nusbiosci/n090310

Microbe-enriched compost application on germination substrates of


Beilschmiedia roxburghiana, Bouea oppositifolia and Syzygium polycephalum

HENDRA HELMANTO♥, FRISCA DAMAYANTI, DIAN LATIFAH


Center for Plant Conservation, Botanic Garden, Indonesian Institute of Sciences. Jl. Ir. H. Juanda No. 13, Bogor 16122, West Java, Indonesia.
Tel.: +62-251-8322187, Fax.: +62-215-8322187, email: hendraforestugm@gmail.com

Manuscript received: 30 January 2017. Revision accepted: 22 August 2017.

Abstract. Helmanto H, Damayanti F, Latifah D. 2017. Microbe-enriched compost application on germination substrates of
Beilschmiedia roxburghiana, Bouea oppositifolia and Syzygium polycephalum. Nusantara Bioscience 9: 300-305. The success of
germination has been an important issue in many forest restoration programs; that is mainly affected by the quality of sowing media.
Application of Bioposka compost (microbe-enriched compost) in the sowing media was hypothesized to increase the success of the
germinating and transplanting also. The research aimed at investigating the effect of Bioposka compost to the germination and the
seedling growth of the study species. The complete-randomized research design was used for three sowing media, i.e., sand, Bioposka
compost and the mixture of sand: compost (1: 1 ratio) of tree species i.e., Syzygium polycephalum, Bouea oppositifolia and Beilsmedia
roxburghiana. The variables observed were total and normal germination capacity, first germination, final germination, germination
rate, germination simultaneity and seedling growth. The results were analyzed using STAR (Statistical Tool for Agricultural Research).
In addition, the microbial abundance of bacteria, fungi, and yeast in each media were calculated using the spread-plate methods. The
germination and seedling growth responses were varied between the different media. The germination capacity and seedling growth of
Syzygium polycephalum and Bouea oppositifolia were lower by microbe-enriched compost. By contrast, the germination capacity and
the seedling growth of Beilsmedia roxburghiana were not significantly different. Moreover, the microbe-enriched compost application
increased the abundance of bacteria, fungi, and yeast in the media.

Keywords: Beilschmiedia roxburghiana, Bouea oppositifolia, compost, microbe, Syzygium polycephalum

INTRODUCTION for the species. Bouea oppositifolia (Anacardiaceae)


famous namely burmese plum, marian plum, marian tree,
Germination and early seedling growth are an important gandaria (Indonesia). This species is indigenous to the
phase of a plant lifecycle. According to Koornneef et al. Andaman Islands, Myanmar, Laos, Cambodia, Thailand to
(2002), germination and seed dormancy are an adaptive Vietnam and South-China (Yunnan, Hainan), Indonesia
strategy of tree plant regeneration. Germination substrates (Sumatera, Java, Borneo) and Malaysia (Lim 2012) and
are essential for sowing seeds in nurseries (Akbar 1992; become an extremely rare mango taxon from the South
Lakitan 2004; Fahmi 2013). Deforestation and plant over- Andaman (Damodaran et al. 2013). Indonesian people use
exploitation made many species lost and become rare. this fruits for seasoning and some food. Syzygium
Conservation strategy also needs technology applied and polycephalum (Myrtaceae) is one of many kinds species
innovation to enhance the impact of sustainability. which developed in pharmacology. It was reported to lower
Developing germination process must be applied, high blood pressure and high cholesterol level and it
especially on species which uncultivated like Beilschmiedia exhibits antioxidant activity (Florido and Cortiguerra
roxburghiana, Bouea oppositifolia, Syzygium 2003). A recent study reported that a decoction of the bark
polycephalum, etc. because uncultivated species are more is used for the treatment of dysentery (Roosita 2008).
susceptible than cultivated species to be extinct. Sands is a common germination substrate for sowing
Beilschmiedia roxburghiana (Lauraceae) is a seeds in nurseries (ISTA 2015). However, it lacks nutrients
subtropical tree, which lives at 200-400 m altitude (Long that is immediately required after the seedlings were early
1985; Turner 1995). This species spread in China, Bhutan, established before transplanting. Furthermore, sands are
India, Myanmar, Nepal, Thailand (Li et al. 2008; Liu 2013) porous which had good aeration and drainage (Fahmi
and Indonesia. Its wood is hard and has multiple usages in 2013). These characteristics of sands may dry out quickly
construction, boat and paper industries. In pharmacology, in some cases that may lead to decrease the seedling quality
the terpenoid compound α-amirin from B. roxburghiana for transplanting. Therefore, a compost may be added to
stem bark exhibits insecticidal and cytotoxic activities sowing/germination substrates to improve the quality of
(Zetra and Prasetya 2007). Liu (2013) said populations of early-growth seedlings. This compost is normally used for
B. roxburghiana have become increasingly fragmented in fertilizing the Garden’s living collections. Ekasari (1994)
recent years due to deforestation and environmental reported that the compost application promoted the
deterioration caused by economic development in China. germination of candlenut seeds (Aleurites moluccana).
Therefore, need to design effective conservation programs Quassia indica and Clausena excavata exhibited the
HELMANTO et al. – Microbe-enriched on germination 301

highest normal and total germination capacities using 𝑛 (𝑛𝑜𝑟𝑚𝑎𝑙)


Normal germination capacity = 𝑋 100%
compost and sand+compost mix (Damayanti and Helmanto 𝑁

2015; Helmanto et al. 2015). The mixture of soil and Σn


compost had also shown higher germination capacity of Germination rate =
𝑛𝑥𝑡
Morinda citrifolia (88.7%) than those sown in sand i.e.
74.7% (Muniarti and Suminar 2006). 𝛴n
This research aimed to investigate the effect of Germination Simultaneity =
𝛴[(𝑇−𝑡)2 𝑥 𝑛]
𝑥 100
microbe-enriched compost Bioposka application on the
germination and seedling growth of three tree species Where,
Syzygium polycephalum (Myrtaceae), Bouea oppositifolia
(Anacardiaceae), Beilschmiedia roxburghiana (Lauraceae). 𝛴n Σ (t x n)
T=
Σ[(T − t)2 x n] Σn

MATERIALS AND METHODS N = number of germinated seeds


n = number of survived seedling
Germination n x t = n germinated seeds on day-t (t = 1, 2, 3,....)
This research was conducted in the glass house of Seed
Bank Unit, Center for Plant Conservation Bogor Botanic Microbial analysis
Gardens LIPI on 29th October 2014-29th January 2015. Microbial analysis was conducted in Research Center
Materials used were the seeds of Syzygium polycephalum for Biology (Department of Microbiology) Indonesian
(Myrtaceae), Bouea oppositifolia (Anacardiaceae), and Institute of Sciences. The abundance of bacteria, fungi, and
Beilschmiedia roxburghiana (Lauraceae).The research was yeast was analyzed using the spread-plate methods. The
designed in a completely randomized design. Three of the bacteria abundance was determined by using 10 g samples
treatments used a compost i.e. Bioposka which is enriched of the three sowing media in an Erlenmeyer flask which
by a certain amount of nutrients (N total 0,66-0,84 %; P2O5 was diluted by adding 90 mL purified water (10 -1 dilution)
0,15-0,18 %; K2O 0,17-0,21 %), Beyonic Startmik© and homogenized by using a vortex; this dilution method
produced by Center for Plant Conservation Botanic was repeated until 10-6 dilution. The 10 µL suspension of
Gardens-LIPI in collaboration with Research Center for the 10-4, 10-5 and 10-6 dilution was taken and spread into
Biology). Bioposka is produced by Bogor Botanic Gardens the frozen Nutrient Agar (NA) growth media in a petri dish
Compost Unit using litter collected across the garden. The (three replicates). After 48-72-hour incubation, the number
experiments applied to the germination substrates of the bacteria colony was calculated then multiplied by the
treatments (the microbe enrichment criteria were based on dilution factor. The abundance of fungi was examined by
Table 1-3) as follows: less moist, no microbe media (5.1 % spread method and calculated by applying TPC (Total Plate
moisture, pH 7.0), moist, microbe enriched media (the mix Count) method. The fungal colony of each seed-sowing
of sand and compost 1: 1 ratio, 24.4 % moisture, pH 6.9), media was sown in rose bengal chloramphenicol agar
and very moist, microbe enriched media (42.6 % moisture, medium; then the average density of colonies (Colony
pH 7.0) and; with 3 species trees, each species using two Forming Unit per milliliter; CFU/mL) developed in each
replicates and10 seeds in every experimental replication agar medium on the 10-2, 10-3,10-4 dilution (three replicates
unit due to the limited availability of ripe fruit. each) was calculated. The yeast abundance was also
Moisture levels, acidity levels, and microbial determined using spread method and calculated by
abundance were used to characterize the sowing media undertaking TPC (Total Plate Count) method. Yeast colony
properties. The moisture levels of the sowing media were from each seed-sowing media were grown in rose bengal
determined based on the media-sample percentage of chloramphenicol agar medium and the average density of
volume on the 8.6 µ pore diameter (pF 2.54). Acidity levels colonies (Colony Forming Unit per milliliter in CFU/mL)
were measured using a soil tester (Demetra, Japan). on the 10-1, 10-3,10-5 dilution with three replicates was
The variables of germination observed were the total calculated.
germination capacity, normal germination capacity, first
germination, final germination, germination rate and
germination simultaneity (Bewley and Black 1994; ISTA
2015). Seedling vigor determined at the end of the RESULTS AND DISCUSSION
observation period (29th January 2015). The data were
analyzed by analysis of variance (ANOVA) using STAR Results
(Statistic Tool for Agricultural Research). The formulas The responses of the three species varied to the sowing
used to calculate total germination capacity, normal media with and without microbe-enriched supplementation
germination capacity, germination rate and germination (Table 1 and Figure 1). Analyze by compile all species
simultaneity are as follows (Draper et al. 1985): shown that the different responses are demonstrated by the
𝑛
normal germination capacities, first germination,
Total germination capacity = 𝑋 100% germination simultaneity and seedling height of S.
𝑁
polycephalum. The sand had the highest normal
germination in S. polycephalum, i.e. 40% compared to 20%
302 N U S A N T A R A B I O S C I E N C E 9 (4): 300-305, August 2017

germination with the addition of microbe-enriched compost moist, no microbe-enriched media is the lowest value on
(Table 2). On the other hand, the compost had the lowest bacterial abundance i.e. 3.1 x 106 CFU/mL. Table 6
germination in S. polycephalum, i.e. 5% total germination presented the fungal colony densities from all media. The
capacities, then the germinating seeds died after 40 days moist, microbe-enriched media have the highest fungal
i.e. 0% normal germination capacity. The first germination colony is i.e. 85388,89 CFU/mL, and the lowest one is in
of seed sown in the sand was also earlier (19 day after the very moist, microbe enriched media. Yeast content
sowing) with higher seedlings. The germination showing at table 7, by comparing each media, the less
simultaneity was not significantly different between the moist no microbe treatment has biggest yeast abundance
sand and compost application. i.e. 446.67 CFU/mL and the lowest one is moist, microbe
Although the differences were not significant, through enriched 16,67 CFU/mL.
tabulation data calculation, microbe-enrichment media Microbe content analysis was done to determine the
show increased the total germination capacity and normal fungi, bacteria and yeast content in each sample media.
germination capacity value Bouea oppositifolia. The effects This analysis showed the variation of microorganism
of sowing media on the germination of B. oppositifolia contains in media. The very moist, microbe-enriched media
were various (Table 3). The microbe-enriched gave the has highest bacterial content compare to other media i.e.
highest germination capacity of B. oppositifolia, i.e. 65%. 8.1 x 106 (CFU1/mL). In other hands, this media has the
Normal germination capacity was also not different (Table low fungi and yeast colony number. Moist, microbe
3). By contrast, the seedling had shown the highest height enriched media has highest fungi colony contain i.e.,
in the sand (lest moist-microbe media) i.e. 13.93 cm after 85388.89 CFU1. Less moist, no microbe media has highest
90 days [F (2,5) = 12.42, p<0.05]. yeast colony contenti.e. 446.67 CFU1.
The highest total and normal germination capacity of
Beilschmiedia roxburghiana were 85% and 80%, that Discussions
means there is 5% abnormal germinating seed, respectively The three species studied responded differently to the
in the microbe-enriched compost; although the results were sowing media with and without microbe-enriched
not significantly different among treatments (Table 4). On supplementation (Table 1). The normal germination
the other hand, moist-microbe enrichment media showed capacities, first germination, germination simultaneity and
lower total germination capacity, i.e. 70% which was seedling height of S. polycephalum were significantly
similar to the normal germination capacity. different among the varied sowing media. The sand may
The result of microbial analysis reported in Table 5. promote the normal germination of S. polycephalum i.e.,
The very moist, microbe-enriched media has the highest 40% compared to 20% germination with the addition of
abundance of bacterial i.e. 8.1 x 106 CFU/mL. The less microbe-enriched compost (Table 2).

Table 1. The recapitulation analyses of variant result by various treatment effect sowing media on the germination of Syzygium
polycephalum, Bouea oppositifolia, and Beilsmedia roxburghiana

Total Normal First Final Germ. Germ. Seedling


Species
germ. capacity germ. capacity germ. germ. rate simultaneity height

S. polycephalum ns ns ns ns ns ns ns
B. oppositifolia
B. roxburghiana

Each species analyzing result


S. polycephalum ns * * ns ns ** **
B. oppositifolia ns ns ns ns ns ns *
B. roxburghiana ns ns ns ns ns ns ns
Note: *= significant (p<0,05); **= significant (p<0,01); ns= not significant (p>0,05)

Table 2. The treatment effect of various sowing media on the germination of Syzygium polycephalum*

Normal germination First germination Germination Seedling height


Treatments
capacity (%) (day) simultaneity (cm)
Less moist, no microbe 40 19 a 0.22 b 8.22 a
Moist, microbe enriched 20 68 c 0.13 b 5.7 b
Very moist, microbe enriched 5 40 b 2.49 a 0c
Note: *Means with the same letters are not significantly different (p > 0.05)
HELMANTO et al. – Microbe-enriched on germination 303

Table 3. The treatment effect of various sowing media on the may indicate the microbe-enriched compost seemed to
germination of Bouea oppositifolia* inhibit the seedling survival after 40 days. The sand also
had stimulated the seeds to germinate earlier (19 days after
Total germ. Seedling sowing) and resulted in higher seedlings. The sand and
Treatments
capacity (%) height (cm)
compost application did not affect the germination
Less moist, no microbe 40 13.93 a
Moist, microbe enriched 45 10.18 b
simultaneity. Mudiana (2006) found that genus Syzygium
Very moist, microbe enriched 65 8.85 b had a low germination which was 53.33% total germination
Note: *Means with the same letters are not significantly different capacity and less germinating seeds survived resulting in
(p > 0.05) 6.67% normal germination capacity using the sand and soil
mix (1: 1).
From all media which used, there are significant
differences in high growth rate. Less moist, no microbe-
Table 4. The treatment effect of various sowing media on enriched media gave the best results in high indicator.
germination of Beilsmedia roxburghiana* During the research time (3 months) seedling can reach
high average 8.22 cm on this media. In other hands, on the
Total germ. Seedling
Treatments
capacity (%) height (cm)
moist, microbe-enriched media gave high average 5.7 cm.
Less moist, no microbe 75 18.25 (Table 2)
Moist, microbe enriched 70 16.31 The microbe-enriched compost appeared to stimulate
Very moist, microbe enriched 85 13.41 the germination of B. oppositifolia i.e., 65% total
Note: *Means with the same letters are not significantly different germination capacity (Table 3). The moist, microbe-
(p > 0.05) enriched media shown 40% total germination capacity, it is
mean increased 12.5% from total germination on lest
moist-microbe media which usually used. Also on very
moist-microbe enriched shown 65% total germination
Table 5. Bacterial abundance of various types sowing media*
capacity, it is mean increased 62.5% from lest moist-
microbe media. However, the sand (lest moist-microbe
Samples Abundance (CFU1/mL) media) may promote the seedling growth.
Less moist, no microbe 3.1 x 106 The microbe-enriched compost resulted in the highest
Moist, microbe enriched 3.7 x 106 total and normal germination capacity of Beilschmiedia
Very moist, microbe enriched 8.1 x 106 roxburghiana i.e., 85% and 80% respectively (Table 4). It
Negative control2 0
means the total germination increased on very moist-
Note: *CFU = Colony Forming Unit; 2Negative control =
microbe enriched media 13,3% compared to the less moist
sterilized-purified water used in dilution process
media. However, the compost application may not enhance
the seedling growth; although the differences were not
significant. By contrast, the moist-microbe enrichment
Table 6. Fungal colony densities of various types sowing media media shown to decrease total germination capacity until
6.6% which was almost similar to the normal germination
N Colony CFU* capacity indicating the seedling survival was maintained.
Samples
density* The three study species responded differently to the
Less moist, no microbe 9 7,11 a 15555.56 a microbe-enriched sowing media. Thus the microbe-
Moist, microbe enriched 9 56,78 b 85388.89 b enriched compost can be a good sowing media in some
Very moist, microbe enriched 9 4 ab 17000.00 ab
species for transplanting success. Furthermore, the
Note: *Means with the same letters are not significantly different
(p > 0.05) moisture level of sowing media tends to affect the
germination rather than the seedling growth. The moisture
level is essential in imbibition process preceded the onset
of germination. Imbibition is an increase of seed water
Table 7. Yeast abundance of various types sowing media content required to metabolism for seed germination
(Hartman et al. 1990; Asiedu et al. 2000). This imbibition
Samples N Abundance1 CFU* process occurs as the seed water potential is lower than
Less moist, no microbe 3 44,67 a 446.67 a those seed surroundings, therefore the seeds absorb water
Moist, microbe enriched 3 1,67 b 16.67 b to run metabolism for germination (Benech and Sanchez
Very moist, microbe enriched 3 13,33 b 133.33 b 2004; Soemardi et al. 2009). Germination substrates are
Note: *Means with the same letters are not significantly different essential for seed germination in nurseries including
(p > 0.05) shading houses or greenhouse (Akbar 1992; Lakitan 2004;
Fahmi 2013). Growing media useful to buttress the
seedling for growing; however, Syzygium polycephalum
germination was low in very moist media. The high
However, the compost may inhibit the germination of S. moisture level of sowing media may attract diseases caused
polycephalum i.e., 5% total germination capacities. This by fungi and or pests attack (Lakitan 1995).
304 N U S A N T A R A B I O S C I E N C E 9 (4): 300-305, August 2017

A B C

Figure 1. Microbe content analysis. A. Less moist, no microbe media sample microbe counting after 48-72 hour incubation; B. Moist,
microbe enriched media sample microbe counting; C. Very moist, microbe enriched media sample microbe counting

On this research, microbe enriched media preferred by DIPA (thematic program entitled: Seed Conservation on
slower high growth comparing than no microbe media. Indonesiam Rare Plants, Orchids, and Economical-
That is show there is an indication, which enriched media Potentially Wild Plants) of Center for Plant Conservation
potentially inhibit germination process or early growth. Botanic Gardens, Indonesian Institute of Sciences (LIPI).
This may be caused by the cotyledon presence. These We also thank the staffs of Seed Bank of Botanic Gardens
cotyledons may cause the nutrition provided by the LIPI and Compost Bioposka Unit for their assistance.
microbe-enriched compost had not been utilized optimally Thanks, Research Center for Biology (Department of
for the early growth of the seedlings. Microorganism Microbiology) LIPI for the assistance in the microbial
presence will inhibit germination process also reported by analysis as well as to Center for Soil Research and
several researches before. Dormancy can be overcome by Development, Ministry of Agriculture, Republic of
gibberellins (Wareing and Saunders 1971), which is Indonesia.
claimed to be common microbial metabolite (Brown 1974).
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N U SAN TA RA B IOSC IE NC E ISSN: 2087-3948
Vol. 9, No. 3, pp. 306-311 E-ISSN: 2087-3956
August 2017 DOI: 10.13057/nusbiosci/n090311

Topical treatment of ointment containing ethanol extract of


Archidendron pauciflorum fruit peel on the wound healing in
streptozotocin-induced diabetic mice

DESAK MADE MALINI1,♥, MADIHAH1, FITRI KAMILAWATI1, NINING RATNINGSIH1,


KARTIAWATI ALIPIN1, JOHAN ISKANDAR1
1
Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Padjadjaran. Jl. Raya Bandung-Sumedang Km. 21 Jatinangor,
Sumedang 45363, West Java, Indonesia. Tel./Fax. +62-22-7796412, ♥email: desak.made@unpad.ac.id

Manuscript received: 15 July 2017. Revision accepted: 25 August 2017.

Abstract. Malini DM, Madihah, Kamilawati F, Ratningsih N, Alipin K, Iskandar J. 2017. Topical treatment of ointment containing
ethanol extract of Archidendron pauciflorum fruit peel on the wound healing in streptozotocin-induced diabetic mice. Nusantara
Bioscience 9: 306-311. Diabetic wounds lead to severe tissue damage and are difficult to cure. One alternative medicine known well by
local Indonesian communities to treat diabetic wounds is the fruit peel of djengkol. This study aimed to evaluate the ointment containing
ethanol extract of djengkol fruit peel to accelerate wound healing process in the skin of streptozotocin-induced diabetic mice. The
method was experimental using completely randomized design with six treatments and four replications. Diabetes was induced by
intraperitoneal injection of streptozotocin 180 mg/kg BW. Mice with blood glucose level ≥150 mg/dL were used for diabetic mice
models. The incision wound created at dorsolateral region of shaven skin at ±1 cm2 using sterile scissors. The ointment containing
extract was applied topically to the diabetic mice wounds at concentration of 5%, 10% and 15%, as well as Betadine® as the reference
group. The ointment basis was applied to the wound of diabetic mice as a positive control and to the wound of non-diabetic mice as a
negative control. The treatment was done twice a day for 14 days. The results showed that topical application of ointment containing
ethanol extract of djengkol fruit peel at concentration of 10% gave significant recovery (p<0.05) on the wounded skin by the
enhancement of re-epithelization and granulation tissue, as well as the increase of capillary number and collagen density which were
higher than other treatments and comparable to negative control group. It was concluded that the topical application of ointment
containing ethanol extract of djengkol fruit peel can support the healing of diabetic wounds.

Keywords: Archidendron pauciflorum, diabetic, djengkol, skin, streptozotocin, topical application

Abbreviations: DM = Diabetes Mellitus, DFUs = Diabetic Foot Ulcers, EPC = Endothelial Progenitor Cells, EEJFP = Ethanol Extract
of Djengkol Fruit Peel, STZ = Streptozotocin

INTRODUCTION these processes (Baltzis et al. 2014). Diabetic wounds


estimated to occur in 15% of diabetic patients and often
Diabetes Mellitus (DM) is a chronic disease in which cause pain and could reduce productivity. A series of
individuals lose the ability to regulate the level of sugar in multiple mechanisms, including decreased cell and growth
their blood caused by the unability of pancreas to produce factor response, leads to diminished peripheral blood flow
insulin or the body cannot use it effectively which leads to and decreased local angiogenesis, all of which can
raised glucose levels in the blood (hyperglycemia). Over contribute to the lack of healing in person with diabetic
the long-term high glucose levels are associated with wounds (Brem and Tomic-Canic 2007).
damage to the body and failure of various organs and Diabetic wounds have to be treated with the right
tissues (International Diabetes Federation 2017). Many medicine to accelerate wound healing and prevent infection
diabetic patients have difficulty in healing wounds, which in wounds, including by using herbal medicine. Herbal
in particular, wounds affecting the feet are common and medicines are being used by about 80% of the population
called diabetic foot ulcers (DFUs). DFUs constitute one of in developing countries for primary health care, including
the most common complications of this disease. About treating wounds, infectious and metabolic diseases, due to
58% of ulcers become clinically infected often leading to their efficacy, safety, cultural acceptability, and fewer side
amputation (Mendes and Neves 2012). effects (Agyare et al. 2015). The phytomedicines for
Wound healing is a dynamic and complex biological wound healing are not only cheap and affordable but are
process that can be divided into four partly overlapping also purportedly safe as hyper sensitive reactions are rarely
phases: hemostasis, inflammation, proliferative and encountered with the use of these agents (Singh et al.
remodeling. These phases involve a large number of cell 2014). One of the herbal medicines to treat wounds is
types, extracellular components, growth factors, and djengkol (Archidendron pauciflorum (Benth.) I.C.
cytokines. Diabetes mellitus causes impaired wound Nielsen). In order to treat wounds, ashes of burnt young
healing by affecting one or more biological mechanisms of leaves are applied onto the injured area, whereas raw-eaten
MALINI et al. – Djengkols’s fruit peel extract on diabetic wound 307

seeds are used to purify the blood and to serve as obtain a paste extract. The ointment was made by mixing
antidiabetic agent (Bunawan et al. 2013). Our previous petroleum jelly and ethanol extract of djengkol fruit peel at
study showed that local people in Karangwangi village, concentration of 5%, 10%, and 15% (w/w).
Cianjur District, West Java used the water decoction of
dried djengkol fruit peels to decrease blood sugar levels. Experimental design
Djengkol fruit peel is known to contain saponin, Twenty-four male Swiss-Webster mice (8-12 weeks
glycosides, and steroids (triterpenoids) (Wahyuni et al. old, 30-40 g of weight) were obtained from the Faculty of
2012) as well as alkaloids, flavonoids, tannin, quinones, Animal Husbandry University of Padjadjaran. They were
and polyphenols (Syafnir et al. 2014; Sihombing et al. housed in standard environmental condition and fed with
2015). piglet standard diets (CP-551, PT. Charoen Pokphand) and
Djengkol fruit peel has pharmacological activities as water ad libitum. The animals were acclimatized for seven
anti-hyperglycemia, antimicrobial and antioxidant due to days before the experiment. The experiment used
the phytochemical contents (Bunawan et al. 2013). This completely randomized design with six treatments and four
study aimed to evaluate the ointment containing ethanol replications (Table 1).
extract of djengkol fruit peel (EEJFP) to accelerate wound
healing process in the skin of streptozotocin-induced Induction of diabetes
diabetic mice. Streptozotocin (STZ; 2-deoxy-2-[3- (3- The animals fasted for 4-6 hours, and their baseline
methyl-nitrosourea)-D-gluco piranose]) is a permanent fasting blood glucose level were measured using a
diabetogenic agent which are toxic molecules causing glucometer, by collecting blood via tail cut before
damage to pancreatic beta cells (Szkudelski 2001). Rodent induction of diabetes. Diabetes was induced by
models of diabetic wound healing have received a great intraperitoneal injection of a freshly prepared solution of
deal of focus due to ease of maintenance, cost and STZ (Nacalai Tesque, Inc.) with a dose of 180 mg/kg BW
availability of genetically modified lines. A full-thickness in 10 mM citrate buffer solution pH 4.5 of five groups,
wound model is a standard approach which removed both while the negative control rats were injected with the
epidermal and dermal tissues, allowing evaluation of re- vehicle. The mice were provided by 10% of sucrose
epithelialization, granulation, and angiogenesis, all key solution for three constitutive days to prevent
processes during physiological wound healing (Suckow et hypoglycemia after STZ induction. Four days after
al. 2017). In this study, the EEJFP is in the form of an administration of STZ, the mice fasted and the blood was
ointment, thus the active ingredient in the extract could collected via tail cut for measuring their fasting glucose
persist longer on the surfaces and penetrate optimally into levels. The animals which have glucose level more than
the skin (Yanhendri and Yenny 2012). 150 mg/dL were used for further experiment and
categorized as diabetic mice (Wu and Huan 2008; Furman,
2015)
MATERIALS AND METHODS
Procedure of the wound creation and treatment with
Preparations of the ointment the ointment
Djengkol fruit peel was collected from Karangwangi The mice were euthanized with inhaled ether, and then
village, Cianjur District, West Java Province, Indonesia. the hair on right side of cutting area was shaved before the
The samples were identified in Taxonomy laboratory in creation of wound. Incision wounds were created on all
Biology Department, Faculty of Mathematics and Sciences, mice by cutting the dorsolateral surface of skin ± 1,5 cm
University of Padjadjaran. The samples were air-dried to a from the shaven area using sterile scissors (full-thickness
constant weight and blend to a coarse powder. The dried type extending up to the adipose tissue). The ointment
powder was soaked and macerated on ethanol 70% (ratio treatment was given twice a day in the morning and
1:2) for 72 hours and every 24 hours the macerate was afternoon for 14 days. The animals were allowed to
collected. The macerate was then evaporated using a rotary consume food and water ad libitum (Chen et al. 2005;
evaporator at a temperature of 40-50oC, and freeze-dried to Winarsih et al. 2012).

Table 1. Experimental design of the treatment

Treatments
S/N ID Induction of diabetes by Incision wound
Treatment with the ointment
STZ 180 mg/kg BW creation
A Negative control (NC) - + Petroleum jelly
B Positive control (PC) + + Petroleum jelly
C Reference (REF) + + Betadine® ointment
D Ointment containing extract + + EEJFP ointment at concentration
treatment 1 (EO1) 5% (w/w)
E Ointment containing extract + + EEJFP ointment at concentration
treatment 2 (EO2) 10% (w/w)
F Ointment containing extract + + EEJFP ointment at concentration
treatment 3 (EO3) 15% (w/w)
308 NUSANTARA BIOSCIENCE 9 (4): 306-311, August 2017

Histological preparation and examination accelerated on the closure of the wound. Histological
On the 15th days, after an overnight fast, the animals observation also showed that the diabetic mice (PC) with
were weighted and sacrificed by cervical dislocation, the most extensive granulation tissue indicated slower
wounds were harvested and fixed in 10% of neutral healing process than the non-diabetic (NC) and other
buffered formalin. The tissue was dried using a serial of n- treatments. In ointment containing EEJFP-treated mice, the
butanol-xylene solution, inserted in paraffin, sectioned at 5- granulation tissue area became narrow along with the
7 μm thickness, mounted on glass slides, stained with increase of the concentration (EO1, EO2, EO3). From all of
hematoxylin and eosin as well as Trchrome Heidenhains the treatments, the mice given by ointment containing
Azan. Histological examination included percentage of re- EEJFP at a concentration of 10% (EO2) had the narrowest
epithelization, formation of the granulation tissue, number granulation tissue when it was compared to the PC and the
of blood capillaries, and density of collagen in five areas reference (REF) groups, and was almost similar to NC
per slide. Percentage of re-epithelization was determined group. It showed that the ointment containing ethanol
by dividing the length of new epithelium layer by the total extract of djengkol fruit peel at concentration of 10% has a
length of the wound; the formation of granulation tissue potential to accelerate healing process of diabetic wounds.
and the number of blood capillaries were examined in the
dermis layer under the new epithelium layer. The density of The effect of treatment on number of capillaries and
collagen in the granulation tissue was examined by using a density of collagen in wound site
scoring method from 0-5 for no collagen until a very dense Proliferative phase in wound healing process was
collagen was found in the wound site (Adriani et al. 2012). characterized by the formation of epithelium (re-
epithelization) as well as blood capillaries
Data analysis (neovascularization) and collagen deposition in dermis
Results were expressed as mean ± standard deviation layer. The phase was undertookon day 5th until 21st of
(S.D). The formation of collagen was analyzed healing process. Histological of wound area that showed
descriptively. Statistical significance for the percentage of the blood capillary and collagen deposition in the dermis
re-epithelization and the number of blood capillaries was after treatment for 14 days and stained with Heidenhain's
analyzed using one-way ANOVA followed by Duncan Azan Trichrome was represented in Figure 2, while the
multiple range test, whereas the density of collagen was examination results were represented in Table 1. In
analyzed by Kruskal Wallis test. P values less than 0.05 histological examination, wound area of NC and EO2
were considered as significant. groups showed higher number of blood capillaries as well
as density of collagen that were significantly different from
PC, EO1, EO2, and REF groups (p<0.05). Ointment
RESULTS AND DISCUSSION containing ethanol extract of djengkol fruit peel at a
concentration of 10% (EO2) was effective to increase the
The effect of treatment on re-epithelization and number of blood capillaries and the density of collagen,
formation of granulation tissue in wound area and the value was not significantly different from that of
The histological examination of wound area in day 15th non-diabetic mice (NC). These results indicated that the
after treatment showed 100% re-epithelization on all mice ointment containing ethanol extract of djengkol fruit peel at
(Figure 1). It was characterized by the formation of new concentration of 10% could accelerate wound healing
epidermis layer above granulation tissue in wound area. process in diabetic model mice.
This result cannot confirm which one of the treatments has

Table 2. Effect of treatment groups in a number of blood capillaries of skin wounds

Number of blood Score number of


S/N Treatments
capillaries* collagen density**
NC Wounded, non-diabetic mice and treated by petroleum jelly 19.00 ± 1.15 (b) 4,00 ± 0,00 (4)
PC Wounded, diabetic mice, and treated by petroleum jelly 12.50 ± 1.73 (a) 1,75 ± 0,96 (1)
REF Wounded, diabetic mice, and treated by Betadine® ointment 13.50 ± 0.58 (a) 2,00 ± 0,82 (2)
EO1 Wounded, diabetic mice, and treated by ointment containing EEJFP at 13.75 ± 1.26 (a) 3,75 ± 0,50 (3)
concentration 5% (w/w)
EO2 Wounded, diabetic mice, and treated by ointment containing EEJFP at 17.75 ± 0.96 (b) 4,00 ± 0,00 (4)
concentration 10% (w/w)
EO3 Wounded, diabetic mice, and treated by ointment containing EEJFP at 12.5 ± 0.58 (a) 3,75 ± 0,00 (3)
concentration 15% (w/w)
Note: The value is expressed as mean ± standard deviation (n=3). *Data were analyzed using one-way ANOVA followed by Duncan
multiple range test; difference alphabet in the same column showed P values less than 0.05 and considered significant. ** Data were
analyzed using Kruskal Wallis test; higher number showed increased rank of collagen density
MALINI et al. – Djengkols’s fruit peel extract on diabetic wound 309

NC  PC  REF 

EO1  EO2  EO3

Figure 1. Photomicrograph of the cross section of mice skin from each treatment groups after treatments for 14 days. Hematoxylin-
Eosin stain. M.100×. Note: Epidermis in wound site indicated re-epithelization (arrow) and granulation tissue area in dermis (red
arrowhead). NC: Negative control, non-diabetic mice; PC: Positive control, diabetic mice; REF: Reference, Betadine ointment-treated
diabetic mice; EO: Diabetic mice treated with ointment containing ethanol extract of djengkol fruit peel at concentration 5 % (EO1), 10
5 (EO2), and 15 % (EO3)

NC  PC  REF 

EO1  EO2  EO3

Figure 2 Photomicrograph of the cross section of mice skin from each treatment groups after treatments for 14 days. Heidenhain’s
Trichrome Azan stain. M.100×. Note: Blood capillary (arrow); collagen in blue color. NC: Negative control, non-diabetic mice;
PC: Positive control, diabetic mice; REF: Reference, Betadine ointment-treated diabetic mice; EO: Diabetic mice treated with ointment
containing ethanol extract of djengkol fruit peel at concentration 5 % (EO1), 10 5 (EO2), and 15 % (EO3)

Discussion healing processes. Most often, it is triggered by


Diabetes mellitus causes impaired wound healing by hyperglycemia, chronic inflammation, angiogenesis and
affecting one or more biological mechanisms of these vasculogenesis impairment, micro- and macro-circulatory
310 NUSANTARA BIOSCIENCE 9 (4): 306-311, August 2017

dysfunction, hypoxia, autonomic and sensory neuropathy, the level of antioxidant enzymes in granuloma tissue
and impaired neuropeptide signaling (Baltzis et al. 2014). (Singh et al. 2014; Singh et al. 2017). Saponin also has
Diabetes in animal models occured as the results of properties of precipitating and coagulating red blood cells
experimental manipulation, for example by administration and also has cholesterol binding properties and supports the
of streptozotocin that produces reliable diabetic mice, as formation of foams in aqueous solutions and hemolytic
shown in this study and others (Wu and Huan 2008; Singh activity (Okwu 2004).
et al. 2014; Furman, 2015; Singh et al. 2017). The Triterpenoids and flavonoids promote wound healing
induction of streptozotocin leads to the condition of due to their astringent and antimicrobial property.
hyperglycemia that affects the wound healing process. Flavonoids have also a potent of antioxidant and have free
Hyperglycemia can lead to glycation of collagen and others radical-scavenging effect, thus it enhances the level of
protein and to formation of glycation end products, which antioxidant enzymes in granulation tissue (Singh et al.
alter the inflammatory phase. Chronic hyperglycemia- 2014; Singh et al. 2017). Flavonoid compounds can act as
induced inflammation further increases oxidative stress vascular-protection agent or as agent to improve blood
because of the abundance of neutrophils, which can circulation. The action is by affecting chemical factors in
produce a large number of reactive oxygen species and neovascularization and increasing vascular tone, while
proteases, and macrophages in wound area. In addition, triterpenoids and alkaloid compounds have the ability to
fibroblasts in diabetic wounds have been found to exhibit increase collagen synthesis by increasing fibroblast
decreased proliferation, increased apoptosis and decreased proliferation (Hasanoglu et al. 2001). Flavonoid also has
migration ability, whereas keratinocytes had an increased been known to increase collagen synthesis, support the
proliferation but decreased differentiation and impaired cross-linking of collagen, and decrease the degradation of
migration ability. Hyperglycemia and chronic inflammation soluble collagen. This accelerates the conversion of soluble
are also considered as the cause of Endothelial Progenitor collagen to insoluble collagen, and inhibit the catabolism of
Cells (EPC) dysfunction and impaired EPC recruitment soluble collagen. From clinical aspects, collagen deposition
from the bone marrow, therefore non-enzymatic glycation in the wound is the most essential phase of healing.
of vascular basement membranes results in impaired Facilitating oxygen diffusion, diminishing oxygen free
homing of EPCs and hence impaired blood vessel radical overproduction, and increasing collagen synthesis
regeneration (Baltzis et al. 2014). Thus, in diabetic mice were found collectively for healing improvement (Lodhi et
(PC group), it showed narrower granulation tissue, al. 2016). Collagen is the predominant extracellular protein
decreased blood capillaries number and collagen density as in the granulation tissue of a healing wound and there is a
shown in this study. rapid increase in the synthesis of this protein in the wound
Wound healing occurs as a cellular response to area soon after an injury. In addition to providing strength
injury and involves activation of keratinocytes, and integrity to a tissue matrix, collagen also plays an
fibroblasts, endothelial cells, macrophages, and important role in homeostasis. Subsequent epithelialization
platelets. Many growth factors and cytokines released also requires collagen (Chitra et al. 1998; Chen et al. 2005).
by these cell types are needed to coordinate and From this study, it showed that topical application of
maintain healing (Brem and Tomic-Canic 2007). The the ointment containing EEJFP with concentration of 10%
results of this study showed that the topical treatment with was effective to accelerate wound healing in diabetic mice
ointment containing ethanol extract of djengkol fruit peel models, and it was better than the Betadine® ointment-
(A. pauciflorum) affects re-epithelization, granulation treated group as reference. Concentration of 10% may be
tissue formation, blood capillaries number and collagen the best concentration because according to Douglas and
density. Medicinal plant plays a key role in the indigenous Soejarto (2002), the low concentration of plant extracts will
system of medicine for diabetes and in the related contain only active compounds in small amounts, and then
complications wound healing due to presence of natural the biological function becomes inferior, but at high
compound. Several studies have shown that djengkol fruit concentrations, these could be toxic and give no optimal
peel contains saponin, glycosides, steroids (triterpenoids), therapeutic effect.
alkaloids, flavonoids, tannin, quinones, and polyphenols Betadine® ointment contains 10% povidone iodine
(Wahyuni et al. 2012; Syafnir et al. 2014; Sihombing et al. which has bactericidal properties, but in a long term use,
2015). this compound can disrupt the synthesis of fibroblasts and
Phenolic compounds like tannins. phenolic acids, and keratinocytes (Balin et al. 2002). Fibroblasts are the cells
flavonoids are important plant metabolites that play synthesizing collagen, thus, diminishing the healing
significant role in the diabetic wound healing. Tannins process. Povidone iodine also has side effects such as
promotes the wound healing through several cellular irritation, an extension of the inflammatory phase, and
mechanisms, chelating the free radicals and reactive cytotoxic on the wound tissue, which could decrease the
species of oxygen, promoting contraction of the wound and wound healing process (Sabiston 1995). Wounds treatment
increasing the formation of capillary vessels and of diabetic mice with the ointment containing EEJFP at
fibroblasts. Saponin promotes wound healing due to their concentration of 10% could be more effective than other
antioxidant and antimicrobial property which appear to be extract treatments because according to Douglas and
responsible for wound contraction and elevated rate of re- Soejarto (2002), the low concentration of plant extracts will
epithelialization. Flavonoids also possess potential of contain only active compounds in small amount, and then
antioxidant and free radical-scavenging effect, enhancing the biological function becomes inferior, but at high
MALINI et al. – Djengkols’s fruit peel extract on diabetic wound 311

concentrations, these could be toxic and give no optimal as a link between wounding and tissue repair. Diabetes 54 (7): 2143-
2154.
therapeutic effect. It is concluded that the treatment of Chithra P, Sajithlal GB, Chandrakasan G. 1998. Influence of aloe vera on
diabetic rats with ointment containing ethanol extract of the healing of dermal wounds in diabetic rats. J Ethnopharmacol 59:
djengkol fruit peel may have beneficial influence on wound 195-201.
healing process because it enhances re-epithelization and Furman BL. 2015. Streptozotocin-induced diabetic models in mice and
rats. Curr Prot Pharmacol 70:5.47.1-5.47.20. DOI:
granulation tissue, as well as increases capillary number 10.1002/0471141755.ph0547s70.
and collagen density. Hasanoglu A. Ara C, Ozen S, Kali K, Senol M, Ertas E. 2001. Efficacy of
micronized flavonoid fraction in healing of clean and infected
wounds. Intl J Angilogy 10 (1): 41-44.
International Diabetes Federation. 2017. Promoting Diabetes Care,
ACKNOWLEDGEMENTS Prevention and a Cure Worldwide. https://www.idf.org/
Lodhi S, Jain AP, Rai G, Yadav AK. 2016. Preliminary investigation for
The research was supported by Academic Leadership wound healing and anti-inflammatory effects of Bambusa vulgaris
Grant (ALG), the University of Padjadjaran on behalf of leaves in rats. J Ayurveda Integr Med. DOI:
10.1016/j.jaim.2015.07.001.
Prof. Johan Iskandar. Therefore, in this opportunity, we Mendes JJ, Neves J. 2012. Diabetic foot infections: Current diagnosis and
gratefully acknowledge the financial support from the treatment. J Diabet Foot Compl 4 (2): 26-45.
Rector of Universitas Padjadjaran through Directorate of Okwu DE. 2004. Phytochemicals and vitamin content of indigenous
Research, Community Service and Innovation (DRPMI) spices of Southeastern Nigeria. J Sustain Agric Environ 6 (1): 30-37.
Sabiston DC. 1995. Buku Ajar Bedah Bagian 1. Penerj: Adrianto P,
which enables us to conduct the research. The authors Timan IS. Penerbit Buku Kedokteran EG, Jakarta. [Indonesian]
would also like to acknowledge the technical assistance of Sihombing JR, Dharma A, Chaidir Z, Almahdy, Fachrial E, Munaf E.
Deden Deni. 2015. Phytochemical screening and antioxidant activities of 31 fruit
peel extract from Sumatera, Indonesia. J Chem Pharmaceut Res 7
(11): 190-196.
Singh A, Singh PK, Singh RK. 2014. Antidiabetic and wound healing
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N U S AN T AR A BIO S C IEN C E ISSN: 2087-3948
Vol. 9, No. 3, pp. 312-317 E-ISSN: 2087-3956
August 2017 DOI: 10.13057/nusbiosci/n090312

Kidney histology in streptozotocin-induced diabetic male Wistar rats


treated with combined extract of temulawak rhizome and belimbing
wuluh fruit

KARTIAWATI ALIPIN♥, EMA PURNAMA SARI, MADIHAH, TIA SETIAWATI,


NINING RATNINGSIH, DESAK MADE MALINI
Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Padjadjaran. Jl. Raya Bandung-Sumedang Km. 21 Jatinangor,
Sumedang 45363, West Java, Indonesia. Tel./Fax. +62-22-7796412, email: kartiawati@unpad.ac.id

Manuscript received: 15 Juni 2017. Revision accepted: 29 August 2017.

Abstract. Alipin K, Sari EP, Madihah, Setiawati T, Ratningsih N, Malini DM. 2017. Kidney histology in streptozotocin-induced diabetic
male Wistar rats treated with combined extract of temulawak rhizome and belimbing wuluh fruit. Nusantara Bioscience 9: 312-317.
Complications that are occurred in patients with Diabetes Mellitus usually followed by kidney damage. Temulawak (Curcuma
xanthorrhiza Roxb.) and belimbing wuluh (Averrhoa bilimbi L.) were traditionally used to decrease blood glucose level. Thus, they
were potential as antidiabetic drugs. This study aimed to evaluate the combination of ethanol extracts of temulawak rhizome and
belimbing wuluh fruit in repairing kidney damage in diabetic male Wistar rats induced by streptozotocin (STZ). An experimental
method using a completely randomized design that consist of seven treatments with three replications. Six treatment groups were
injected intraperitoneally with a dose of 60 mg/kg BW STZ, and one group served as a control. The animals which have blood glucose
level ≥200 mg/dl were stated as diabetic. Furthermore, the animals were treated orally with single extract i.e. temulawak 17.5 mg/kg
BW or belimbing wuluh 750 mg/kg BW and combined extracts 383.75 or 767.5 mg/kg BW, as well as glibenclamide 0.45 mg/kg BW as
reference, including diabetic rat as positive control and non-diabetic rat as negative control. The results showed that combine extract at
dose of 383.75 mg/kg BW treatment repaired the kidney histology, i.e., glomerular diameter and Bowman space width, as well as
significantly decreased the necrosis percentage of proximal tubular in diabetic rat compared with positive control group (p<0.05). In
conclusion, the combined extract of temulawak rhizome and belimbing wuluh fruit has potent to cure renal failure in diabetic rats
induced by streptozotocin.

Keywords: Averrhoa bilimbi, Curcuma xanthorrhiza, kidney, streptozotocin

INTRODUCTION primarily in the glomerulus that results in kidney structure


changes and disruption of its function (Ritz et al. 2000).
Diabetes Mellitus (DM) is a metabolic disease caused International Diabetes Federation (IDF) in 2012 stated that
by the inability to produce any or enough insulin which 4.8 % of Indonesians were diagnosed with diabetes.
results in elevated blood glucose level. In a diabetic Diabetic nephropathy is one of the most important DM
condition, the body does not properly process food to be complications, which is marked with histological kidney
used as energy, because there were combinations of various lesions that may vary from nodular or diffuse
disturbances in the body's metabolic system due to chronic glomerulosclerosis to tubulointerstisial and/or vascular
hyperglycemia conditions associated with abnormal lesion (Martinez-Castelao et al. 2015).
metabolism of carbohydrates, fats, and proteins (World Research in the use of traditional medicine, including
Health Organization 1999). The typical symptoms of herbs develops rapidly, as it is recommended by the World
diabetes are polyuria, polydipsia, polyphagia, which are Health Organization (WHO) to maintain public health, to
accompanied by hyperglycemia condition in which the prevent and treat diseases, especially degenerative diseases,
plasma glucose concentration is greater than 200 mg/dL such as diabetes and cancer (Patwardhan 2005). There are
(11.1 mmol/L), and the fasting glucose concentration is 126 many Indonesian native plants, which are used empirically
mg/dL (7.0 mmol/L) (Cooke and Plotnick 2008). Diabetes to treat diabetes. Belimbing wuluh (Averrhoa bilimbi L.) is
caused serious complications including retinopathy that a medicinal plant belonging to the family of Oxalidaceae
leads to blindness, nephropathy which leads to kidney and mainly used as a folk medicine to treat DM,
failure, neuropathy which leads to impotence, diabetic foot hypertension, and as an antimicrobial agent (Alhassan and
disorder, as well as macrovascular complications including Ahmed 2016). The extract of belimbing wuluh fruits with
cardiovascular disease (WHO 2016). In parallel with the as dose of 750 mg/kg body weight (BW) was effective to
increase of diabetes, an increase in the prevalence of lower blood glucose level in alloxan-induced diabetic rats
diabetic nephropathy has been noted (Ritz and Zeng 2011). (Candra 2012). Temulawak (Curcuma xanthorrhiza Roxb.,
Damage to the kidneys (diabetic nephropathy) occurs Zingiberaceae) is used widely as traditional medicine to
because of the damage to the blood vessels in the kidney, treat various diseases such as cancer, gastric, and diabetes
ALIPIN et al. – Combination of temulawak and belimbing wuluh as antidiabetic 313

(Hassan and Fadzilah 2014). The extract of temulawak peritoneally injecting six rat groups with streptozotocin
rhizomes with a dose of 17.5 mg/kg BW was effective to (STZ) (60 mg/kg BW) that was freshly prepared in 10 mM
lower blood glucose level in alloxan-induced diabetic rats citrate buffer solution, pH 4.5. Negative control rats were
(Cahyani 2014). injected with citrate buffer. To prevent hypoglycemia after
Based on the potential hypoglycemic effect of the STZ induction, the rats were supplemented with 10%
plants, this study aimed to evaluate the effect of ethanol sucrose solution for three days (Wu and Huan 2008). Three
extract combination of temulawak rhizomes and belimbing days after the administration of STZ, the rats were fasted
wuluh fruit in repairing kidney damage in streptozotocin- again and the blood was collected from their tail cut to
induced diabetic Wistar rat. Streptozotocin (2-deoxy-2-[3- determine their fasting glucose levels. Rats which have
(3-methyl-nitrosourea)-D-glucopyranose]) is a permanent glucose level more than 200 mg/dL were categorized as
diabetogenic agent which are toxic molecules that cause diabetic and used for the further experiments.
damage to pancreatic beta cells (Szkudelski 2001).
Streptozotocin is widely used as a diabetogenic agent as it Treatments of the extract
could be utilized at low doses and has a higher success rate Treatment for each of the group was showed in Table 2.
(up to 95%) than other inducers substances, such as alloxan The single or combined extract was administered orally
(up to 70%) (Junod et al. 1967). Streptozotocin-induced using an intragastric tube for 21 days. The reference group
rats showed the occurrence of nephropathy, i.e. the were administered with glibenclamide at a dose of 0.45
expansion of glomerular extracellular matrix, thickening of mg/kg BW, while the negative control group were
the glomerular basal membrane, increased deposition of administered 0.5% CMC solution. The animals were
type IV collagen in mesangial cells (Gambaro et al. 1999), allowed to consume food and water ad libitum.
as well as necrosis in proximal tubules with the loss of On the 22nd days, after an overnight fast, the animals
brush borders (Zafar et al. 2009). were weighed and sacrificed by cervical dislocation, and
necropsies were done on them. Furthermore, kidneys were
isolated from the rats and weighed, then examined for their
MATERIALS AND METHODS morphology, i.e., the colors and textures. Last, the organs
were preserved in Bouin’s solution for histopathological
Collection and extraction of plant materials examination. The kidneys were embedded in paraffin, and
Belimbing wuluh (Averrhoa bilimbi L.) fruits were then sectioned, stained with hematoxylin and eosin, then
collected from Arboretum, University of Padjadjaran in were examined microscopically and analyzed using ImageJ
Jatinangor, Sumedang, Indonesia, whereas temulawak software as described by Kotyk et al. (2015).
(Curcuma xanthorrhiza Roxb.) rhizomes were collected
from Indonesian Medicinal and Aromatic Crops Research Parameters observed
Institute, Lembang, Bandung, Indonesia. The samples were
identified in Taxonomy Laboratory in Department of Relative weight observation of kidney organs
Biology, Faculty of Mathematics and Natural Sciences, Relative kidney weight was calculated using formula as
University of Padjadjaran. The samples were air-dried to a follows (Mossa et al. 2015):
constant weight and blend to a coarse powder. The dried
powder was soaked and macerated on 95% ethanol for 72
hours and every 24 hours the macerate was collected. The
macerate was then evaporated using a rotary evaporator,
resulting in a paste extract. Morphological observation of kidney organs
The morphological observation of kidney organ is done
Experimental animals directly (descriptive) with a scoring on color parameters
Twenty-one male Wistar rat (160-180 g) were obtained and organ surface based on Leeson et al. (1996) as Table 1.
from the animal house of the Laboratory of Biosystematics,
Department of Biology, University of Padjadjaran, Histological observation of kidney
Indonesia. The rats were weighed and sorted into seven Histological observations of the kidneys were
groups (Table 1) of three animals each so that their average performed under a microscope by measuring the
weight approximately equal. They were housed in a glomerular diameter, Bowman space, and percentage of
standardized environmental condition and fed with piglet proximal tubular necrosis in 5 different planes (four angles
standard diets (CP-551, PT. Charoen Pokphand) and water of the upper and lower and one center angle) using 400
which were given ad libitum. Animal care and handling times magnification with 1 replication (Rohmah 2014).
conformed to accepted guidelines.
Data analysis
Induction of diabetes and determination of blood Results were expressed as mean ± standard deviation
glucose level (S.D). Statistical significance was analyzed using one-way
After one-week of acclimatization period, the animals ANOVA followed by Duncan multiple range test. P values
were fasted overnight, and their baseline fasting glucose less than 0.05 were considered significant.
levels were determined using a glucometer, by collecting
blood via tail cut. Diabetes was induced by intra-
314 N U S A N T A R A B I O S C I E N C E 9 (4): 312-317, August 2017

Table 1. Morphological kidney organ score

Parameters
Score Damage level
Colors Surface
1 Brownish red Smooth and no abnormalities Normal
2 Brownish red Coarse and there are abnormalities Medium damaged
3 Pale red Coarse and there are abnormalities Badly damaged

Table 2. Experimental design of the treatment

S/N ID Treatment
A Negative control (NC) Citrate buffer and CMC 0.5%
B Positive control (PC) STZ 60 mg/kg BW and CMC 0.5%
C T STZ 60 mg/kg BW and temulawak rhizomes extract 17.5 mg/kg BW
D BW STZ 60 mg/kg BW and belimbing wuluh fruit extract 750 mg/kg BW
E TBW1 STZ 60 mg/kg BW and combination extracts 767.5 mg/kg BW
F TBW2 STZ 60 mg/kg BW and combination extracts 383.75 mg/kg BW
G Reference STZ 60 mg/kg BW and glibenclamide 0.45 mg/kg BW

RESULTS AND DISCUSSION width and number of necrotized proximal tubules in PC


and reference groups was significantly different compared
Effect of treatments on relative weight of kidney with NC (p<0.05). The treatment of single or combined
The relative kidney weight showed the percentage of extract, however, significantly decreased the
kidneys-to-body weight at the end of treatment period histopathological parameters compared with KP group
(Table 3). The result demonstrated that treatment of 60 (P<0.05), but did not different with KN group. Application
mg/kg BW STZ (PC), the single and the combined extracts of combined extract of temulawak rhizomes and belimbing
of temulawak rhizomes and belimbing wuluh fruit (T, BW, wuluh fruit extracts at dose of 385.75 mg/kg BW (TBW2)
TBW1, TBW2), as well as 0.45 mg/kg BW glibenclamide showed the lowest number of necrotized proximal tubules
(Reference) significantly increased the relative kidney and significantly differed with other treatments (p<0.05).
weight of compared with the negative control (NC)
(p<0.05). The treatment with 767.5 mg/kg BW combined Discussion
extract of temulawak rhizomes and belimbing wuluh fruit Organ weight is an important index of physiological
(TBW2) result in the highest relative weight of kidney. and pathological status in animals. The relative organ
weight is fundamental to diagnose whether the organ was
Effect of treatments on kidney morphology exposed to the injury or not. Changes in kidney weight may
The aim of the morphological examination was to reflect renal toxicity, tubular hypertrophy or chronic
evaluate the effect of the treatments on the color and progressive nephropathy (Sellers et al., 2007). According
texture of kidneys (Figure 1). The result showed that to Al-Samawy (2012), the weight of the right kidney was
treatment with 60 mg/kg BW STZ dose (PC), single and 1.1 g and the left kidney is 0.96 g, with mean about 1.06 g
combined extracts of temulawak rhizomes and belimbing in albino rats. This study showed an increasing kidney
wuluh fruit (T, BW, TBW1, TBW2), as well as 0.45 mg/kg weight and relative weight of kidney presumably due to
BW glibenclamide (reference), did not caused difference in directly or indirectly toxicity of streptozotocin which
the kidney morphology compared with the NC group. caused diabetic condition in rats. Both single and combined
extract treatments of temulawak rhizomes and belimbing
Effect of treatment on kidney histopathology wuluh fruit did not affect relative weight of kidney possibly
The histopathological examinations of kidney included due to the insufficient treatment time. This result is
measurement of glomerulus diameter, Bowman’s space supported by the research of Usoh et al. (2015), in which
width, and the number of necrotized proximal tubules diabetic rats treated with a minimum dose (200 mg/kg BW)
(Figure 2, Table 4). The kidneys of NC (Figure 2A) and of Gongronema latifolium and Ocimum gratissimum
TBW2 rats (Figure 2.F) showed normal glomerulus, combined extract showed a marked difference in the
Bowman’s space, and proximal tubules, whereas the relative kidney weight compared with the non-treated rats
kidneys of PC, T, BW, TBW1, and reference groups after 28 days of treatment. Glibenclamide treatment did not
showed architectural damages, i.e., increase in glomerulus improve the relative weight of kidney due to drug
diameter and Bowman’s space width, as well as increased pharmacokinetic changes in diabetic models. The reasons
number of proximal tubules with necrosis nuclei (Figure for these pharmacokinetic changes are diverse and
2.B, C, D, E, and G). complex, such as changes in transport, metabolism and
Increasing of glomerulus diameter, Bowman’s space drug elimination (Li et al. 2012).
ALIPIN et al. – Combination of temulawak and belimbing wuluh as antidiabetic 315

Table 3. Mean of relative weight of kidneys after 21 days of treatment

Relative
Body weight Kidneys
ID Treatment weight of
(g) weight (g)
kidney (%)
NC Citrate buffer and CMC 0.5% 228.0 ± 29.10 1.750 ± 0.24 0.760 ± 0.02a
PC STZ 60 mg/kg BW and CMC 0.5% 179.0 ± 18.52 1.910 ± 0.09 1.067 ± 0.08bc
T STZ 60 mg/kg BW and temulawak rhizomes extract 17.5 mg/kg BW 179.0 ± 4.35 1.950 ± 0.04 1.087 ± 0.04bc
BW STZ 60 mg/kg BW and belimbing wuluh fruit extract 750 mg/kg BW 172.6 ± 21.93 1.883 ± 0.12 1.090 ± 0.07bc
TBW1 STZ 60 mg/kg BW and combination extracts 767.5 mg/kg BW 176.3 ± 14.84 2.026 ± 0.10 1.147 ± 0.06c
TBW2 STZ 60 mg/kg BW and combination extracts 383.75 mg/kg BW 183.1 ± 5.50 1.886 ± 0.04 1.027 ± 0.03b
Reference STZ 60 mg/kg BW and glibenclamide 0.45 mg/kg BW 177.0 ± 13.07 1.913 ± 0.04 1.080 ± 0.05bc
Note: The value is expressed as the mean ± standard deviation (n=3). Data were analyzed using one-way ANOVA followed by Duncan
multiple range test. Difference alphabet in the same column showed P values less than 0.05 and considered significant

A B C D

E F G

Figure 1. Morphology of kidneys after treatment for 21 days. Note: A. NC, B. PC, C. T, D. BW, E. TBW1, F. TBW2, G. Reference

A B C D

E F G

Figure 4. Photomicrograph of histological cross section of kidneys after treatment for 21 consecutive days. Heamatoxylin-Eosin stain.
Note: A. NC, B. PC, C. T, D. BW, E. TBW1, F. TBW2, G. Reference. ( ) Glomerulus, ( ) Bowman’s space, ( ) Normal proximal
tubules, ( ) Necrotized proximal tubules, ( ) Mesangial matrix expansion, ( ) Glomerular basal membrane.
316 N U S A N T A R A B I O S C I E N C E 9 (4): 312-317, August 2017

Table 2. Histopathological parameters measurement of kidneys after 21 days of treatment

Number of
Glomerulus Bowman’s
necrotized
ID Treatment diameter space width
proximal
(µm) (µm)
tubules
NC Citrate buffer and CMC 0.5% 75.25 ± 1.58a 2.22 ± 1.29a 15.33 ± 3.46a
PC STZ 60 mg/kg BW and CMC 0.5% 92.20 ± 4.72c 12.09 ± 2.68b 64.33 ± 3.05e
ab a
T STZ 60 mg/kg BW and temulawak rhizomes extract 17.5 mg/kg BW 80.33 ± 1.99 5.28 ± 2.74 56.33 ± 2.88d
BW STZ 60 mg/kg BW and belimbing wuluh fruit extract 750 mg/kg BW 74.68 ± 3.57a 5.93 ± 0.88a 49.00 ± 5.29c
ab a
TBW1 STZ 60 mg/kg BW and combination extracts 767.5 mg/kg BW 78.42 ± 1.79 6.14 ± 2.42 51.66 ± 5.50cd
ab a
TBW2 STZ 60 mg/kg BW and combination extracts 383.75 mg/kg BW 77.95 ± 4.38 5.06 ± 0.20 34.00 ± 1.00b
Reference STZ 60 mg/kg BW and glibenclamide 0.45 mg/kg BW 85.64 ± 7.77bc 10.98 ± 1.72b 63.66 ± 5.68e
Note: The value is expressed as mean ± standard deviation (n=3). Data were analyzed using one-way ANOVA followed by Duncan
multiple range test. Different alphabets in the same column indicate P < 0.05 and was considered significantly different.

Changes in organ weights should always be interpreted increased absorption and accumulation of glycogen in
in conjunction with morphological and histopathological kidneys (Mahood 2012) which caused nephropathy in
findings because of the inherent variability. All treatments diabetic disease model.
in this study did not affect the kidney morphology, which The percentage of proximal tubular with necrosis nuclei
shown a bean–shaped and dark-red bodies with smooth and in diabetic rats also greater than in the non-diabetic rats.
possess convex and concave borders. The results of this Necrosis of proximal tubules is indicated by the brush
study is agrees with Al-Samawy (2012). border deficiency, epithelial cell changes in the proximal
The histopathological examination result in this study tubules (such as closed lumen caused by swollen
agrees with Zafar and Naqvi (2010) who found that the cytoplasm), cryolysis (dissolved chromatin in the cell
relative kidney weight in STZ-induced rat for 12 weeks nucleus) and picnosis (thickening and shrinking of the cell
was significantly higher than that of the non-induced rat. nucleus due to chromosomal dissolution and condensation).
Vallon and Thomson (2012) stated that diabetes mellitus This finding is consistent with the result of Zafar et al.
affected the kidneys gradually. The onset of damage in (2009) that found that necrosis with loss of brush borders in
diabetic is marked by the increase in kidney size and the the proximal tubular area occurred in the kidneys of rats
glomerular filtration rate (GFR), leading to the induced with 45 mg/kg BW STZ. STZ-induced kidney
development of kidney failure in diabetic diseases. damage is presumably mediated by Glut2 uptake in the
The mean of glomerular diameter in diabetic rats was proximal tubular cells, which stimulates tubular necrosis
greater than that of non-diabetic rats indicates that and then alters kidney function (Tay et al. 2005).
streptozotocin induced glomerular enlargement Treatment with single extract of temulawak at dose of
(hypertrophy) in kidney. Glomerular hypertrophy is an 17,5 mg/kg BW showed improvement in kidney
initial event in the development of glomerular damage, histological structure as indicated by decreased of
which occurs as a result of the expansion of mesangial glomerular diameter, narrowed of Bowman space, and
matrix and the thickening of the glomerular basement decreased of the necrotized proximal tubules. Temulawak
membrane (Zafar et al. 2009). The expansion of mesangial contains potent curcumin that serves as an antioxidant
matrix and the thickening of glomerular membrane were which capable of improving kidney damage caused by
also observed in this study. Glomerular hypertrophy reactive oxygen species (ROS). However, this treatment
associated with the increase in GFR which is caused by the has a less effect in improving kidney structure compared
increase of plasma flow and the pressure of glomerular with other treatments. The kidneys structure upon treatment
capillary hydrostatic. Glomerular hemodynamic changes with single extract of belimbing wuluh at dose of 750
are associated with growth processes (O'Bryan and mg/kg BW (BW) better than the diabetic and reference
Hostetter, 1997). Growth factors and cytokines involved in groups. The fruit of Belimbing wuluh contains saponin and
the mesangial or hypertensive expansion, such as flavonoid. Both compounds are known to be helpful in
Transforming growth factor-β1 (TGF -β1) and angiotensin improving kidney condition as shown by the study of
II, are found to participate in the development of diabetic Firdous et al. (2009) who stated that saponin from
nephropathy (Lehman and Schleicher 2000). Momordica cymbalaria extract increased insulin secretion
In this study, the Bowman's space in kidneys of diabetic by regenerating pancreatic beta cells in streptozotocin-
rats was wider than of non-diabetic rats. This result is induced diabetic rat. An increasing insulin level has a role
similar to the results of Kotyk et al. (2015) that reported the in glucose metabolism, so that the blood entering the
widening of Bowman’s space in rat upon fructose kidney was normal and in turn, this would facilitate the
treatment. BoTman's space widening is caused by the process of kidney filtration.
expansion of the Bowman capsule when glucose enters The histological structure of kidney after the combined
kidneys (Zhang et al. 2015), which also associated with extract treatment with temulawak and belimbing wuluh at
ALIPIN et al. – Combination of temulawak and belimbing wuluh as antidiabetic 317

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N U S AN T AR A BIO S C IEN C E ISSN: 2087-3948
Vol. 9, No. 3, pp. 318-321 E-ISSN: 2087-3956
August 2017 DOI: 10.13057/nusbiosci/n090313

Short Communication:
Infraspecific variations in essential oil compositions of Nepeta fissa from
Iran

SEYED MEHDI TALEBI1,♥, MAJID GHORBANI NOHOOJI2, MAHBOOBEH YARMOHAMMADI1


1
Department of Biology, Faculty of Sciences, Arak University. Arak, 38156-8-8349, Iran. Tel.: +98-863-4173317.

email: seyedmehdi_talebi@yahoo.com
2
Medicinal Plants Research Center, Institute of Medicinal Plants, ACECR, Karaj, Iran

Manuscript received: 7 May 2017. Revision accepted: 29 August 2017.

Abstract. Talebi SM, Nohooji MG,Yarmohammadi M. 2017. Short Communication: Infraspecific variations in essential oil compositions
of Nepeta fissa from Iran. Nusantara Bioscience 9: 318-321. Nepeta fissa C.A. Mey. is a member of Mint family (Lamiaceae) and
distributed naturally in different regions of Iran. The aim of this investigation was to provide more information about the influence of
environmental conditions on the essential oil composition of two populations, Polor and Dizin, of this species. The dry plant samples
were submitted to hydrodistillation for 2.5 h, using a Clevenger-type apparatus. The highest amount of essential oil yields (0.2%) were
obtained for Dizin population. Forty-nine compounds, representing 85.9 to 97.80% of the total essential oils, were separated and
identified. In Dizin and Polor populations, the essential oils were characterized by the very high percentage of oxygenated monoterpenes
(79.1%), oxygenated diterpenes (20.01%) that constituted the predominant classes, respectively. From the analyzed populations, the
major compounds in Dizin population were 1, 8-Cineole, 4 a-α, 7-α, 7 a-α-Nepetalactone and 2E, 4E-nonadienal. While, phytol,
caryophyllene oxide, E-caryophyllene, and thymol were the core compounds of essential in Polor population. The obtained results
showed that environmental conditions are the important factors influencing the yield and also the chemical compositions of essential oils
in N. fissa. Knowledge of the essential oil chemical composition in relation to ecological factors is a most prominent quality criterion for
its marketing and contributes to its valorization as a functional ingredient in food industry.

Keywords: Ecological factors, essential oil, Nepeta fissa, population

INTRODUCTION Zouari (2013) stated various factors, such as climatic,


geographic conditions, and ontogeny of selected plants may
Nepeta L. is a genus of Labiatae (Lamiaceae) family, severely affect the yield of essential oil, its composition,
which grows naturally in various regions of the world. Its and its biological qualities. For these reasons, examinations
name has been taken of the ancient Italian city Nephi. This of the chemical variability of essential oil in relation to
genus has about 280 taxa occurs all over the world but ecological factors can provide data on what determines
extensively grown in various parts of Africa, Asia, North chemical polymorphism of essential oil. Moreover,
America, Central and also the southern part of Europe understanding of the essential oil chemotype in various
(Pojarkova 1954). Recently, Jamzad (2012) listed seventy- plant taxa is a very prominent property criterion for its
nine species of the genus from Iran. Nepeta fissa C.A marketing and contributes to its commercialization as
Mey is a widespread species of the genus. This species has functional ingredients in different industries like food and
various synonym names, such as N. microphylla Stapf, N. phytopharmacy.
trautvetteri Boiss. & Buhse., N. carmanica Bornm. and N. Yet, different infraspecific studied were carried out on
teucriifolia sensu Boiss. (Mozaffarian 1996). essential oil variations. For example, Marčetić et al. (2013)
The essential oils that found in aromatic herbs are a examined the variability of root essential oil in seven
mixture of various compounds such as monoterpenes, natural populations of Seseli rigidum Waldst. & Kit. They
sesquiterpenes and also phenylpropanoids. The great observed three groups of populations based on climatic
diversity of terpenes in plant species is partly attributable to differences and found that biosynthesis of terpenes is
the existence of terpene synthases, which can manufacture influenced by different environmental factors.
different compounds from a single substrate (Degenhardt et As far as we could search, we could not find any
al. 2009; Dudareva et al. 2013). Former studies (e.g. infraspecific study in the essential oil compositions of N.
Figueiredo et al. 2008; Lakusic et al. 2012) demonstrated fissa. Therefore, the aims of this work were: A) to study the
populations of the same species collected from various geographical variability of essential oil and define the
habitats have different essential oil composition. Therefore, chemotypes; and B) to explore the impact of climate on
it seems that essential oils biosynthesis is under the compositions and amount of essential oil.
influence of multitude abiotic as well as biotic agents.
TALEBI et al. – Infraspecific variations in Nepeta fissa 319

MATERIALS AND METHODS RESULTS AND DISCUSSION

Populations sampling Results


Two populations of N. fissa collected from different GC/MS analysis of the essential oils of N. fissa
localities and altitudes in Iran, i.e. Polor, Mazandaran collected from Dizin and Polor populations led to the
(2400 m asl.) and Dizin, Tehran (2250 m asl.) (Table 1). identification of a total of 49 constituents (Table 2). In the
Twenty individuals from each population were sampled essential oil obtained from Dizin population, 32
over the entire area of the population at their flowering compounds amounting to 97.80% of the total oil content
stage (May 2016). The distance among the selected were identified. Monoterpenes accounted for 82.44% of the
individuals exceeded 30 m, to avoid selection from close total oil composition, 79.1% of that were attributed to
parents. Then, the fresh plant material was dried in the oxygenated monoterpenes. Among the oxygenated
shade, until mass constancy (14 days). monoterpenes detected, 1, 8-cineole was the major part
(55.98%) of the total oil. Other constituents identified in
Essential oil extraction this category were 4 a-α,7-α,7 a-α-nepetalactone (6.54%),
The dry aerial parts of plants were submitted to 1,4-cineole ( 3.93%) and 4 a-α,7-α,7 a-β,-nepetalactone
hydrodistillation for 2.5 h, using a Clevenger-type (2.97%).Sesquiterpene hydrocarbons were detected in this
apparatus. The obtained essential oils were dried over with population (5.97% of the total oil content) with (Z) α-
anhydrous sodium sulfate and stored in sealed vials at bisabolene (3.17%) and E-caryophyllene (0.87%) were the
−25°C until analysis. main compounds. Oxygenated sesquiterpenes accounted
only for 2.69% of the total oil content and were represented
Volatile compounds identification spathulenol (1.49%) and caryophyllene oxide (1.20%).
The essential oil compositions of N. fissa were However, in Polor population the chemical composition
identified by comparing the data of mass spectra with varied greatly. A total of 26 constituents were identified
spectra available from the Wiley 275 mass spectra comprising 85.34% of the total oil compounds. Oxygenated
libraries). Further identification confirmations were made diterpenes had the highest contribution at this population,
referring to data of retention indices (RI) created a series of accounting for 20.01% of the total oil content. The major
identified standards of the n-alkanes mixture (C7-C25). contributor to this category was phytol (20.01%). The
Identification of the compounds of essential oil was concentration of oxygenated monoterpene decreased
based on a gas chromatography (Younglin Acme 6000) compared to the Dizin population (13.91%, 79.01%,
Plus instrument equipped with a GC-QP 2010 Plus respectively).
(Agilent) series mass selective detector in the electron Thymol (7.26%) was the major component between the
impact ionization mode at 70 eV. Components separation seven oxygenated monoterpenes detected from the Polor
was performed on fused silica (100% dimethyl population. The amount of sesquiterpene hydrocarbons
polysiloxane) column BP5 (30 m × 0.25 µ ID × 0.25 mm increased to 14.43% of the total oil content, with E-
film thickness) (Restek, USA), splitless injection; helium caryophyllene (7.76%) and germacrene D (2.15%) being
was carrier gas at a flow rate of 1 ml/min, injector and FID detected as main constituents of the fraction. Dibutyl
detector temperatures 300◦C. The temperature of GC oven phthalate and benzyl benzoate were the only esters detected
programmed as follows: initially temperature 50◦C in the essential oil Polor population at concentrations of
(isothermal for 5 min) maximized to 240ºC at the rate 4.29 and 5.31%, respectively.
3◦C/min to (isothermal for 5 min) and further maximized at
the rate 15◦C/min to 300◦C, the final temperature kept for 3 Discussion
min. Total response time was 75 min. The amount of yielded essential oil varied between the
The silica capillary column BP5 with 30 m × 0.25 µ ID studied populations. Although, its amount in population
× 0.25 mm film thickness (Thermo Electron Corporation, Dizin was equal to that was reported by Sefidkon et al.
USA) under the same chromatographic conditions. The (2002), the essential oil value in Polor population was very
percentage amounts of the investigated compounds were low like those were recorded by Baser et al. (2000). The
recalculated according to the areas of the FID Polor population growing in a higher altitude than Dizin
chromatographic peaks assuming that all constituents of the population. It seems that, plants grow at lower altitude
essential oil comprise 100%. yield more essential oil compared with those grown at
higher altitude. These conditions were seen in different
species. For example, Haider et al. (2009) found that
Table 1. Locality address of studied populations of N. fissa populations of Artemisia roxburghiana grow at higher
altitudes yield the lowest amount of essential oil.
Populations Localities Moreover, Thymus praecox growing in higher altitudes
Polor Mazandaran province, Haraz road, Polor, 2400 m. have lower essential oil than plants grown in lower
Dizin Tehran province, Albourz mountain, Protected altitudes (Avci 2011). While, Zouari et al. (2014) in
area, 2250 m asl. Artemisia saharae (Asteraceae), observed that populations
of higher altitudes had the highest amount of essential oil.
320 N U S A N T A R A B I O S C I E N C E 9 (4): 318-321, August 2017

Table 2. Essential oil composition of the studied populations of analyzed it by GC/MS. They reported caryophyllene oxide
N. fissa (24%) and P-caryophyllene (8.3%), spathulenol (7.4%), β-
pinene (6.8%) and α-pinene (6.7%) as the main
Compounds RT Polor Dizin constituents.
1,8-Cineole 16.94 0.6 55.98 The compositions of essential oil greatly differed
Linalool 20.50 0.4 0 between our studied populations. When the obtained results
2-Methoxy-P-Cresol 26.54 1.53 0 were compared with previous studies (Sefidkon et al.,
Thymol 30.12 7.26 0 2002; Baser et al., 2000) on this species, it was observed
β-Bourbonene 33.88 0.72 0.85 that the essential oil compositions of the Polor population
4 a-α,7-β,7 a-α-Nepetalactone 34.78 0.79 0 were more similar to the mentioned investigations than
E-Caryophyllene 35.47 7.76 0.87
Dizin samples. For example, caryophyllene oxide and E-
(Z)-β-Farnesene 36.68 0.89 0.35
Germacrene D 38.09 2.15 0.73 caryophyllene were among the core constituents of Polor
Bicylcogermacrene 38.71 0.77 0 population essential oil. These conditions were seen in
β-Bisabolene 39.08 0.87 0 Sefidkon et al. (2002) as well as Baser et al. (2000) works.
δ-Cadinene 39.43 0.50 0 But, in the mentioned works, their percentages were lower
β-Sesquiphellandrene 39.79 0.77 0 than Dizin population.
Spathulenol 42.19 7.49 1.49 Although, a relative likeness was seen between
Caryophyllene oxide 42.37 8.27 1.20 essential oil compositions of the Polor population with
Hexadecane 42.88 0.41 0 previous works, the main constituents of essential oil of
caryophylla-4(14),8(15)-dien-5.α-ol 44.50 0.33 0
Polor and also Dizin populations highly differed with
epi-α-Cadinol 44.64 2.63 0
Heptadecane 46.24 0.79 0 results of those studies. There are several reasons for these
Benzyl Benzoate 49.54 5.31 0 variations, but the edaphoclimatic condition is one of the
Octadecane 49.81 1.23 0 possible reasons. The plant samples of Turkey grown at an
6,10,14-trimethyl-2-oentadecanone 51.42 6.71 0 altitude of 1300-1400 m (Baser et al. 2000), while the
Dibutyl phthalate 55.53 4.29 0 habitats of our studied populations had more than 2100 m
Phytol 60.07 20.01 0 elevations. Kofidis et al. (2003) demonstrated altitude as
3E-Hexanal 8.28 0 0.23 one of the abiotic stresses that are associated with changes
α-Pinene 11.68 0 0.43 in a wide range of ecological factors. Furthermore, on the
β-Pinene 14.04 0 0.63
basis of different examinations (e.g. Rahimmalek et al.
1,4-Cineole 16.17 0 3.93
ortho-Cymene 16.66 0 0.82 2009; Zouari et al. 2012) the altitude induces high
δ-Terpinene 18.30 0 0.33 variations on essential oils to yield as well as chemical
α-Campholenal 22.05 0 0.37 compounds according to the plant species.
trans-Pinocarveol 22.76 0 1.73 Beside, the geographical locations of our studied
cis-Verbenol 23.00 0 1.14 populations had many differences. Angioni et al. (2006)
Pinocarveon 23.90 0 0.56 stated the composition of the essential oil has been varying
δ-Terpineol 24.28 0 1.13 according to the region, soil type and environmental
p-Mentha-1,5-dien-8-ol 24.38 0 0.53 conditions from where the plant species have been
Menthol 24.60 0 0.28
collected. For example, studies showed that the essential oil
Terpinene-4-ol 24.72 0 1.12
α-Terpineol 25.52 2.32 2.32 compositions of Artemisia herba-alba varied between the
Verbenone 26.19 0.54 0.54 populations, therefore the geographical locations and
2E,4E-Nonadienal 26.59 0 5.01 climatic conditions have a strong effect on it (Mighri et al.
E-Anethole 29.91 0 0.42 2010; Belhattab et al. 2014).
4 a-α,7-α,7 a-α-Nepetalactone 33.16 0 6.54 1, 8-Cineole is the main compound of essential oil of
4 a-α,7-α,7 a-β-Nepetalactone 33.48 0 2.97 Dizin population. Its percentage was more than 55% of
Geranyl Acetate 33.66 0 0.58 essential oil compositions. The compound was seen as a
(Z)-a-Bisabolene 38.79 0 3.12 major or minor constituent occurring in different aromatic
4 a-α,7-β,7 a-α-Nepetalactone 34.78 0 0.79
6,10,14-Trimethyl-2-Pentadecanone 51.39 0 0.36
herbs, such as Mentha longifolia L., Origanum vulgare L.,
Manoyl oxide 56.79 0 0.45 Rosmarinus officinalis L., Thymus vulgaris L. (Lamiaceae),
Artemisia dracunculus L. (Compositae), Coriandrum
sativum L. (Umbelliferae), and also Zingiber officinale
Rosc. (Zingiberaceae) (van Vuuren and Viljoen 2007).
Sefidkon et al. (2002) studied the essential oil Therefore, changes in essential oil compositions leading
compositions of aerial parts of N. fissa collected from to variations in medicinal properties of this species.
Tehran Province, Iran. The major compounds of essential Because, previous reports showed that 1, 8-Cineole had
oil in their study were; β-caryophyllene (17.4%), multiple usages in food as well as medicinal industries. For
caryophyllene oxide (12.3%), γ-muurolene (7.9%), example, the compound employed as a flavoring agent for
valencene (6.6%), β-pinene (6.0%) α-pinene (5.8%), food products and treatment of infectious respiratory
bicyclogermacrene (4.9%), spathulenol (4.1%). disorders in folk medicine. Moreover, the compound is
Furthermore, Baser et al. (2000) extracted the essential oil used in the pharmaceutical industry for formulations of
of aerial parts of Turkish population of this species and drug (Santos and Rao 2001). Investigations have proved
TALEBI et al. – Infraspecific variations in Nepeta fissa 321

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N U S AN T AR A BIO S C IEN C E ISSN: 2087-3948
Vol. 9, No. 3, pp. 322-329 E-ISSN: 2087-3956
August 2017 DOI: 10.13057/nusbiosci/n090314

Visual symptoms and control of the Red Palm Weevil (Rhynchophorus


ferrugineus) in the Gaza Strip, Palestine

ABDEL FATTAH N. ABD RABOU♥, EQBAL S. RADWAN


Department of Biology, Faculty of Science, Islamic University of Gaza, P.O.Box 108, Gaza Strip, Palestine. Tel.: (+970-8-2860-700, Fax.: +970-8-2860-
800), ♥email: arabou@iugaza.edu.ps

Manuscript received: 19 June 2017. Revision accepted: 31 August 2017.

Abstract. Abd Rabou AN, Radwan ES. 2017. Visual symptoms and control of the Red Palm Weevil (Rhynchophorus ferrugineus) in the
Gaza Strip, Palestine. Nusantara Bioscience 9: 322-329. The Red Palm Weevil – RPW (Rhynchophorus ferrugineus Olivier)
(Coleoptera: Curculionidae) has recently become one of the most destructive pests of Date Palms in the Gaza Strip and the Middle East.
It is a serious pest threatening the Date Palm health and production, with the larva is the most destructive stage. The current study aims
at introducing the visual symptoms and control techniques of the RPW in the Gaza Strip, Palestine. Field surveys and institutional visits
were applied to fulfill the purpose of the study. The current study documented that first local infestation of Date Palm trees with the
RPW was discovered in late 2011. The introduction of infected offshoots from Egypt through earth tunnel trade and the ability of the
adult RPW to fly long distance and cross borders seem to be main causes of the local infestations with the pest. Different control
techniques have been adopted by the responsible parties to combat the RPW; with the integrated pest management (IPM) program was
tracked and respected. Finally, the study recommends the cooperation of different parties and authorities to adopt appropriate policies to
eliminate the RPW and to support farmers with the necessary pesticides and equipment to control this painful pest.

Keywords: Red palm weevil, date palm, control, IPM, Gaza Strip, Palestine

INTRODUCTION The existing methods for managing and controlling the


RPW depend on its stage of attack (Al-Saqer and Hassan
Date Palm (Phoenix dactylifera) is the most important 2011). The Integrated Pest Management (IPM) program is
fruit tree of arid, tropical and sub-tropical regions of the most successful in controlling and managing this serious
world. It is a holy tree because of its commercial, pest. It ensures a regular monitoring and treating of infested
nutritional, environmental, social, health and religious Date Palm trees. Other actions are included and respected
values (Chao and Krueger 2007). The Date Palm is such as the detection of the RPW at early stage, trapping of
commonly attacked by different pests, but in the recent the adult insects, treatment of wounds, eradication of
years the Red Palm Weevil – RPW (Rhynchophorus infested trees, and education of farmers on the best
ferrugineus, Olivier) (Coleoptera: Curculionidae), became management ways to deal with the RPW (Al-Saqer and
one of the most dangerous threats to the Date Palm trees in Hassan 2011; Faleiro et al. 2012; Al-Dosary et al. 2016).
most parts of the world including the U.S.A. (Nisson et al. Multi-purpose surveys regarding the RPW were carried
2010). The spread of RPW comes mainly from transporting out in different countries worldwide. Abe et al. (2009)
of infested Date Palm trees and offshoots from infected highlighted the life history of the RPW in 17 infested
regions to other places (Ferry and Gommez 2002). Phoenix canariensis trees in southern Japan. In developing
Nowadays, the date palm crop is under a real threat in the countries, many researchers focused on the ecological
Arab countries including the Palestinian Territories. aspects and pattern of RPW infestation in Date Palm
The RPW is known to cause significant ecological and orchards in addition to the methods engaged in the RPW
economic damages to farmers (Hoddel et al. 2015). It is control and management (Azam et al. 2001; Kaakeh et al.
responsible for the death of a large number of Date Palm 2001; Bertone et al. 2010; Abdel-Wahed et al. 2014; Mazza
trees and consequent yield losses (Vidyasagar and Aldosari et al. 2014).
2011; Vidyasagar et al. 2016). According to Abraham et al. In the Palestinian Territories, many studies have been
(1998), RPW mostly attacks young Date Palm trees under conducted concerning the status and perspective of the
the age of 20 years. The length of the RPW life cycle varies Date Palm tree and its pests including the RPW (Abu-
depending on the environmental and geographical Qaoud 2015; Al-Agha 2016; Radwan 2017; Abd Rabou
conditions (Murphy and Briscoe 1999). This pest hide and and Radwan 2017). Abu-Qaoud (2015) pointed out that the
remains inside the palm during larval development and productivity of Date Palms has declined as a result of pests
makes tunnels and pupates. Because of the concealed including the RPW and the death of a great number of Date
nature of RPW larvae (The main stage cause the injury), Palm trees infested. Ferry and Gomez (2002) mentioned
effective methods to control this pest have been difficult to that an early detection of the pest in the Palestinian
develop. Territories resulted very quickly in the establishment of an
ABD RABOU & RADWAN – Rhynchophorus ferrugineus in the Gaza Strip, Palestine 323

IPM program. In the Gaza Strip, work on the RPW is


restricted to a few studies. Al-Jaghoub et al. (2003),
Ministry of Agriculture – MOA (2011 and 2012), Earth and
Human Center for Research and Study – EHCRS (2011),
and Al-Ahlyah Association for the Development of Date
Palm – ASDPD (2012) described the nature of the RPW,
reasons and symptoms of infection in addition to the
methods of early detection and control levels.
Moreover, El-Hindi (2017) and El Kichaoui et al.
(2017) revealed that the use of Metarhizium anisopliae and
Beauveria bassiana fungi can be useful as a preventive and
curative tool for the protection of Date Palm tree against
the RPW in the Gaza Strip. Because of the critical situation
dictated by the RPW on Date Palm sector locally, and the
real shortage of studies treating this painful pest, the
current study comes to introduce descriptive data on the
occurrence of the RPW (Rhynchophorus ferrugineus,
Olivier) in the Gaza Strip in terms of infestation and
control techniques adopted by the responsible parties. This
study tries to raise the Gazans' awareness towards the RPW
that threatens the Date Palm as a strategic and resistant
crop in the Gaza Strip.

MATERIALS AND METHODS

Study area
The Gaza Strip (Figure 1) is very populated (about 2.0
million) with an area of 365 km². It lies on the eastern coast
Figure 1. The geographic location of the Gaza Strip, Palestine
of the Mediterranean Sea that borders Egypt on the
southwest for 11 km and the Occupied Palestinian
Territories on the east and north along a 51 km border
(PCBS, 2016). The Gaza Strip has the semi-arid
RESULTS AND DISCUSSION
Mediterranean climate. The average daily mean
temperature ranges from 25ºC in summer to 13ºC in winter.
Discovery and origin of the RPW pest
The daily relative humidity fluctuates between 60% to 85%
First detection of RPW was noticed in September 2011
throughout the year depending on seasons (UNEP 2003).
in a Date Palm nursery in the Middle Governorate of the
Gaza Strip. It has been assumed by the farmers and the
Site and institutional visits specialized authorities that the origin of RPW was the
During the course of the present study, frequent visits
infested Date Palms of the Egyptian area bordering Gaza
were carried out to different Date Palm orchards in order to
Strip, and the introduction of infested offshoots from Egypt
investigate the current status of the RPW and its control
through the earth tunnel trade. Many zones in the Date
and management methods in the Gaza Strip. Meetings and
Palm are usually infested by the RPW. These include the
discussions with farmers and owners were of utmost
aerial offshoots and apical region, offshoot near to the soil
priority to fill the gaps needed in data collection. Moreover,
surface, zone infected with Palm Stalk Borer and modern
vital visits were carried out to the MOA, EHCRS, ASDPD
wounds, holes, and tunnels caused boring fauna. With
and the Palestinian Al-Nakheel Association for Progress
regard to the reasons promoting the injury of Date Palms
and Development (PNAPD). A set of close and open ended
with RPW, they were numerous as follows: (i) Removal of
questions were developed and used during the structured
offshoots in periods of insect activity. (ii) Over-pruning of
and semi-structured interviews conducted.
Date Palm trees (iii) Ignorance of wound treatment. (iv)
Prior infection with Date Palm borers. (v) Wounds, holes,
Photography and data analysis and tunnels caused by boring fauna. It is commonly known
For documentary purposes, a digital camera was used to that the holes made by the Fruit Stalk Borer may provide
take photos covering the stages of RPW infestation and appropriate places for the RPW to lay its eggs. (vi) The use
control in the Gaza Strip. The data collected throughout the of flood irrigation system.
course of the study were plotted using Microsoft Excel
program 2010.
324 NUSANTARA BIOSCIENCE 9 (4): 322-329, August 2017

Symptoms of infection with RPW distribution as well as to combat the insect.


There were many symptoms noted and commonly
photographed in the infested Date Palm orchards visited in Control levels of the RPW
the southern parts of the Gaza Strip as follows: (i) The The most successful ways of control and management
presence of tunnels and cavities on the trunk or at the bases of the RPW in the Gaza Strip were represented by the
of leaf petiole (Figure 2.A). (ii) Hearing of gnawing sounds application of IPM program. The IPM includes vital steps
due to feeding by grubs. (iii) Oozing out of thick brown such as the regular monitoring of Date Palms, quick
fluid from the tunnels (Figure 2.B). (iv) The presence of treatment of cuts and infections, early detection and
chewed plant tissues with a typical fermented odor in and treatment of infested trees, trapping of adult weevils,
around the openings of the tunnels (Figure 2.C and D). (v) eradication of infested plants, proper pruning of fronds in
Breaking of the trunk or toppling of the crown in case of addition to the conduction of good training programs
severe and prolonged infestation (Figure 2.E). (vi) Drying targeting both farmers and agricultural officials. The
and/or death of Date Palm offshoots in case of severe following control levels were tracked in the Gaza Strip:
injuries (Figure 2.F). (vii) Drying of Date Palm fruits and
sometimes the death of leaves in the heart of trees (Figure Regulation level
2.G). (viii) The presence of any of the RPW phases (Figure It includes enacted laws and decisions by the
2.H). responsible authorities to protect Date Palms from pest
infestation and to prevent the spread within the borders of
The different stages of RPW noted in Date Palm Gaza Strip. When the first infested palms were discovered
orchards in 2011, the MOA has enacted a law that established the
During field visits conducted to Date Palm orchards, all RPW as a quarantine pest having potential economic
the four stages of the RPW were monitored and importance, and as a result, the transport of Date Palm trees
photographed inside the infested Date Palm trees as and offshoots has been prohibited. This point is crucial
follows: (i) Egg stage: The eggs (Figure 3.A) are usually from the beginning because no success was achieved in
found hidden inside the palm. The female RPW deposits its controlling the RPW pest. Both farmers and responsible
eggs inside the wounds of the leaves and trunks, cavities, or associations have been informed not to transport such trees
the roots of offshoots. (ii) Larval stage: The larva (Figure till the RPW is to be eradicated.
3.B) is the most destructive stage to Date Palms when it
feeds on the soft succulent tissues. When feeding by boring
inside the trunk, it eliminates stinking excrements or
residues that get mixed with a thick slimy liquid.
Subsequently, the palm tree falls down during 6-12 months.
(iii) Pupal stage (cocoon): The pupa (Figure 3.C), which
has an average length of 6 cm and width of 3 cm, prefers its
cocoon to be surrounded by high humidity to prevent
drying out and death. The cocoon was commonly found
inside the trunks or inside the leafstalk of the infested Date
Palms. (iv) Adult weevil: The ridden brown adult insect or
weevil (Figure 3.D) is large and has a total length up to 42
mm.

The magnitude of infested Date Palms


Figure 4. The magnitude of infested Date Palms in the Gaza Strip
The number of infested Date Palms increased year by
(2011-2015) (MOA 2016)
year. Figure 4 show that the magnitude of infested Date
Palms in the Gaza Strip increased from 2011 to 2014. In
2015, there was a decline of the infested Date Palms.

Trapping of the RPW


With regard to the trapping of the RPW, figure 5 shows
that there is a direct proportional concerning the number of
traps employed and the number of adult weevils caught
(2012–2015). It is worth mentioning that the trapping of the
RPW mostly occurred in the southern Gaza Strip. The
young Date Palms (Hayani, Barhee or other cultivars) that
have been planted during the last 10-15 years are very
attractive to and easily infested by the RPW as they have a
lot of offshoots and their leaf bases are not dry enough to
prevent egg lying of the adult insects. The aim of the
deployment of traps, one trap per 50 trees in 2012 till 2015
is given to indicate the source of infection and its Figure 5. The number of traps and caught weevils (MOA 2016)
ABD RABOU & RADWAN – Rhynchophorus ferrugineus in the Gaza Strip, Palestine 325

A B

C D

E F

G H

Figure 2. Symptoms of infection with RPW: A. Presence of tunnels on the trunk, B. Oozing out of thick brown fluid from the tunnels,
C-D. Presence of chewed plant tissues, E. Breaking of the trunk, F. Drying and/or death of offshoots, G. Drying of fruits, H. Presence of
any of RPW phases.
326 NUSANTARA BIOSCIENCE 9 (4): 322-329, August 2017

A B

C D

Figure 3. Life cycle of the Red Palm Weevil: A. Egg stage, B. Larval stage, C. Pupal stage, D. Adult weevil

Organization level Such cultural, chemical, biological, mechanical, quarantine


Once the infestation by the RPW occurred in 2011, the control techniques have been applied:
MOA cooperated with the FAO and other associations Agricultural control: The farming practices adopted
including ASDPD, PNAPD, ICRC and Palestinian Non- by farmers play an important role in the health of Date
Governmental Organizations Network (PNGOs) to Palms and the infestation levels. Local farmers applied on a
establish the RPW Committee in order to control and scheduled basis various agricultural control methods such
combat the RPW pest. A team from MOA has realized an as the use the right irrigation, proper fertilization, cleaning
important work of detection, sanitation, and monitoring of and pruning of palms and their offshoots. Such operations
the RPW. The team distributed pesticides and other tools to can remove the potential sources of infestation by pests.
farmers and brought a bulldozer to eliminate the infested Mechanical control: This method depends on the use
Date Palms. In the Gaza Strip, farmers have been involved of manual or mechanical means to eliminate the insect. The
in the early detection and control process of the RPW pest. most important means noticed in Date Palm orchards were:
Many activities and interventions adopted and held by the Removal of the infested or dead trees and the pruning
related associations and institutions regarding the products in neglected farms (Figure 7), the use of mud to
seriousness of the RPW pest. These included: (i) close the holes of trunk resulted from offshoot removal, the
Workshops to train the local farmers on the suitable control cover of roots of small trees with soil to a height of 20 cm
methods of the RPW pest were organized by the RPW to prevent insect attack, and the burning of old fronds and
Committee (Figure 6). (ii) A total number of 1500 infected offshoots to prevent the spread of infection to
brochures prepared by FAO and ASDPD were edited and healthy trees. When severely damaged and dead palms are
distributed by MOA. (iii) A technical booklet concerning noticed, they were removed and disposed of properly. In
the RPW was edited by ASDPD with the assistance of this case, the palms are cut to smaller bits and treated with
GEF/SGP. (iv) A leaflet concerning the RPW was prepared pesticide and then burned. In large scale removals, heavy
and distributed by the EHCRS. machinery like bulldozers was used to transport the
infested trees to a dumping site far from the infested farm
Technical level to be completely burnt in order to kill any residual
It was mandatory to judiciously mix various methods of populations of the RPW.
control to successfully control the RPW in the Gaza Strip.
ABD RABOU & RADWAN – Rhynchophorus ferrugineus in the Gaza Strip, Palestine 327

Biological control: In spite of its importance as an IPM Egyptian Territories is a capital factor enhanced the
technique, the biological control means have never been occurrence of the pest locally. It is well known that the
applied by the responsible authorities to combat the RPW adult RPW has the ability to fly relatively long distances (~
in the Gaza Strip. 1 km per day), and this may interpret the quick and
Behavioral control using the aggregation pheromone widespread infestation among Date Palm trees coming
traps: The efficient and cheap aggregation pheromone from Egypt into the Gaza Strip in the recent years. In this
traps (Figure 8) used in the Gaza Strip are either home- context, FAO recommended the establishment of a buffer
made or imported from abroad. They are commonly put zone with the Egyptian borders to prevent the entry of adult
one trap per 50 trees to attract the adult insects. The Earth weevils into Gaza Strip. This proposed recommendation
and Human Center for Research and Study (EHCRS) relied on the capability of the adult RPW to fly for long
experienced home-made traps with different colors; red, distances as mentioned previously.
white, black and yellow. The preliminary results showed The life cycle of RPW is reported to vary
that the black traps were the most efficient in attracting the chronologically depending on the environmental and
adult RPW. geographical conditions (Al-Saqer and Hassan 2011). The
Chemical control: This method depends on both larva stage is considered the main damaging stage because
preventive and therapeutic programs. The preventive it bores into Date Palm trees, feeds on the succulent plant
control included the spraying or dusting of chemical material and remains hidden through most of the life cycle.
pesticides such as Dursban, Rogor, and Confidor on the It is responsible for the most symptoms encountered and
trunks and leaves of the intact trees (Figure 9). The seen on the infested trees during the field visits conducted
therapeutic control included the spraying or dusting of by the authors to Date Palm orchards. Similar symptoms
pesticides on the trunks and leaves of the infected trees by were reported by Vidyasagar and Aldosari (2011), Al-
using of injection devices and fumigation. Saqer and Hassan (2011) and Faleiro et al. (2012) who
Injection: The injection is taken place through making confirmed more or less symptoms in the Date Palms
three holes rising 20 cm from the place of infestation. infested by the RPW in various geographical areas.
Injection devices (Figure 10) are commonly used to inject The local control of the RPW depended on the
the appropriate pesticides into the infected tree. Such an application of IPM programs. IPM seeks to reduce
injection treatment may weaken the tree and allow other chemical input through the inclusion of a range of methods
pests to invade the tree itself. Cotonion, Diazinon, which are environmentally compatible. Different
Metasystox, Marshal, Sybrein, Dursban, Rogor, and approaches such as agricultural, chemical, behavioral,
Confidor are commonly used pesticides in this method. mechanical and quarantine control means are more or less
Fumigation: This method is used in severe infections applied in the Gaza Strip. Agricultural and mechanical
causing tunnels in the trunks. Fumigation tablets called controls are sometimes the commonest because of the non-
Phostoxin have been applied against the RPW infestation availability or high costs of chemical pesticides. Murphy
(Figure 11). To fumigate the pest, three holes have to be and Briscoe (1999), Vidyasagar and Aldosari (2011), Aleid
drilled into the tree: one at the point of infestation, the et al. (2015) and Dembilio and Jaques (2015) indicated that
second 20 cm above and the third 20 cm below the first the concealed nature of the larvae of RPW requires
hole. The part of the trunk that oozes is to be cleaned and effective methods for its management including the IPM
then, the decaying tissues and grubs are to be removed as strategy.
much as possible. One tablet of Phostoxin is usually placed Despite its importance, the biological control methods
in the drilled hole. Sealing with moistened clay is then are not applied in the Gaza Strip due to several reasons
followed in order to prevent the leak of evaporated gas including the shortage or unavailability of qualified staff,
resulting from Phostoxin. specialized research, specialized equipment and funding
sources. The political, military and socioeconomic
Discussion circumstances of the Gaza Strip could not be ignored in this
The RPW is considered one of the most destructive regard. The crucial benefit of biological control comes
pests of the Date Palm trees all over the world. The from its relative safety for human health and the
findings of the current study revealed that the RPW is the environment, compared to widespread use of different
main threat facing Date Palm cultivation and making pesticides. In Egypt, many researcher gained good results
considerable losses in the Gaza Strip, which represents a when they used different biological agent to control the
microscopic zone in the Middle East. The severity of the RPW (Abbas et al. 2000; Abdullah 2009 and Abdel-Samad
pest comes from the fact that it is the most common 2011). Locally, El-Hindi (2017) and El Kichaoui et al.
dangerous pest threatening the Date Palm sector in the (2017) achieved good laboratory results regarding the
Middle East. It is responsible for the death of a large effectiveness of the fungi Metarhizium anisopliae and
number of Date Palms and consequent yield losses in the Beauveria bassiana against the RPW in the Gaza Strip.
area as indicated by Vidyasagar et al. (2016). Finally, the study recommends the cooperation of different
The current survey revealed that the number of infested parties and authorities to adopt appropriate policies to
Date Palm trees is increasing year by year and causes eliminate the RPW and to support farmers with necessary
severe losses to Gazan farmers. The absence of inspection control means and modern equipment to combat this
and surveillance of the new offshoots entering the Gaza painful pest.
Strip through the earth tunnels connecting it with the
328 NUSANTARA BIOSCIENCE 9 (4): 322-329, August 2017

Figure 6. Organization of workshops to train the local farmers on Figure 9. The Dursban pesticide is commonly used to combat the
the suitable control methods of the RPW pest RPW pest

Figure 7. Removal of Date Palms infected by RPW Figure 10. Injection device used to inject pesticides inside Date
Palms infested by RPW

Figure 8. The components of RPW traps Figure 11. Phostoxin fumigation tablets applied on Date Palms
infested by RPW
ABD RABOU & RADWAN – Rhynchophorus ferrugineus in the Gaza Strip, Palestine 329

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N U S AN T AR A BIO S C IEN C E ISSN: 2087-3948
Vol. 9, No. 3, pp. 330-337 E-ISSN: 2087-3956
August 2017 DOI: 10.13057/nusbiosci/n090315

Growth, biomass production and nutrient accumulation of Macaranga


gigantea in response to NPK fertilizer application

DWI SUSANTO1,♥, SRI MULYATI1, HERI PURNOMO1, DADDY RUHIYAT2, RUDIANTO AMIRTA2
1
Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Mulawarman. Jl. Barong Tongkok No. 4, Gunung Kelua, Samarinda-
75123, East Kalimantan, Indonesia. Tel./Fax.: +62-541-749140, 749152, 749153, ♥email: susantodwiki@yahoo.com
2
Faculty of Forestry, Universitas Mulawarman. Jl. Ki Hajar Dewantara Kampus Gunung Kelua, Samarinda-75123, East Kalimantan, Indonesia

Manuscript received: 23 November 2016. Revision accepted: 31 August 2017.

Abstract. Susanto D, Mulyati S, Purnomo H, Ruhiyat D, Amirta R. 2017. Growth, biomass production and nutrient accumulation of
Macaranga gigantea in response to NPK fertilizer application. Nusantara Bioscience 9: 330-337. Research described in this paper
investigated the effect of fertilizer application on the growth, biomass production and nutrient accumulation of mahang (Macaranga
gigantea) plant components. The experiment was carried out in a research area of the Faculty of Forestry, Mulawarman University,
Samarinda, East Kalimantan. The effects of five dose levels of an NPK (16-16-16) fertilizer on the growth rate of mahang seedlings
planted in field conditions were compared. The treatments consisted of a control group (with no fertilizer), and dosages of 40 g, 80 g,
120 g and 160 g per plant. Fertilization at those levels was applied twice: the first application at four weeks after the planting and the
second application at 6 months after the first. Growth rates between treatments were compared over a twelve month period. The research
findings revealed that as the dosages of fertilizer increased, so the growth, biomass production and nutrient accumulation in components
of the plants also increased. The best growth was found in the treatment in which fertilizer was applied (twice) at a dosage of 120 g per
plant; at this dose level, by twelve months of age, the stem basal diameter had reached 45.24.9 cm, stem height 219.239.1 cm, number
of leaves per plant 17.31.2, and canopy diameter 246.725.0 cm. The highest production of above-ground biomass was also found in
the fertilizer treatment of 120 g per plant; the largest proportion of the biomass was in the leaves, followed by the wood, and then bark.
The nutrient element that had accumulated most in plants at 1 year of age was potassium, followed by phosphorus and then nitrogen.
The highest nitrogen uptake of stands was found in the NPK fertilization with a dosage of 160 g per tree, while the highest phosphorus
and potassium uptake was found at a fertilizer dosage of 120 g per tree. The amount of nutrients accumulated in producing one tonne of
above-ground biomass increased in response to different fertilization treatments. The fertilizer treatment of 120 g per plant (applied
twice; the first soon after planting and the next after 6 months) resulted in the accumulation within the 1 year old plants of 2.38 kg of
nitrogen, 6.36 kg of phosporus and 17.83 kg of potassium, with an N:P:K ratio of 13.3 : 35.6 : 100. The availability of the element
potassium needs special attention when this species is cultivated.

Keywords: Macaranga gigantea, pioneer species, growth, biomass production and nutrient accumulation

INTRODUCTION highest reduced sugar (82.47%) if compared with other


types of wood which are potential to develop as raw
The global fuel crisis has stimulated the development of material for ethanol (Amirta et al. 2010).
alternative renewable biofuel energy sources. Utilization of Information about the cultivation of this plant is rare.
biofuels, derived from agricultural crops like sugarcane, M. gigantea grows naturally in lowland tropical rainforests,
corn, cassava, palm wine, and oil palm, converted into especially in gaps that result from timber harvesting, forest
ethanol, presents challenges as well as opportunities. fires, and shifting cultivation (Lawrence 2005; Eichorn
Various efforts made to optimize these biofuel industries 2006; Silk 2008). This plant is a pioneer species which
have implications for agricultural production and on prices requires high levels of light in extensive forest gaps and
of food materials. Liquid bioethanol derived from wood is secondary forest habitats (Davies et al. 1998; Romell et al.
an alternative to the use of agricultural crops in biofuel 2008). The percentage and rate of germination of seeds
production and could be sustainable as long as the forests extracted through the wet extraction process are higher than
are managed sustainably. those extracted through the dry extraction process. Highest
One type of soft wood that could be used for this rates of germination (65%) have been reported from seeds
purpose is produced by the forest tree species Macaranga extracted through the wet extraction process and grown on
gigantea (Rchb.f. & Zoll.) Müll.Arg. The production of compost media (Susanto et al. 2016a). Nussbaum at al.
wood from this tree varies from 320 kg3m-1 (low), to 370 (1995) states that nutrient deficiency is an important factor
kg3m-1 (medium), to 460 kg3 m-1 (high), with a water that hinders the initial growth of Dipterocarpaceae plants
content of 35% (Suzuki 1999). Based on chemical analysis (Dryobalonops lanceolata and Shorea leprosula) planted
of M gigantea biomass, it is found to contain 24.4% lignin. simultaneously with pioneer species (M. gigantea and M.
71.14% holocellulose, and 46.67% -cellulose (Amirta et hipoleuca) at the age of 6 months in the degraded lands of
al. 2016b). Enzymatic hydrolysis of this plant produces the unused log stock piling areas and roadworks in Malaysia.
SUSANTO et al. – Nutrient accumulation of Macaranga gigantea 331

Susanto et al. (2016a) investigated the relative growth rate exchange capacity: 7.06 meq.100g-1 , base saturation:
of seedlings planted on different planting media and found 7.45%, and bulk density: 1.16 g. ml-1, and soil nutrients
that mushroom spawn waste media resulted in the highest concentration are C Organic: 1.45%, N: 0.12%, P: 3.61 ppm,
rate (0.36 ±0.42%), followed by compost media K: 67.48 ppm, Ca: 1.17 cmol. kg-1, Mg: 0.13 cmol. kg-1.
(0.15±0.09%), top soil media (0.10±0.04%) and sand
media (0.10±0.07%). According to Okuda (1996), Procedures
seedlings of M. gigantea produce their leaves and shed Preparing seedlings
them every 18.86 and 16.75 days respectively, while their Seedling were obtained by germinating M. gigantea
leaves reach their full extent in 20.8 days and their twigs seed collected from ripe fruits of parent trees in the
their full length in 91.8 days. Photosynthetic capacity Botanical Garden of Mulawarman University, Samarinda.
increases rapidly during the development of leaves, The seeds obtained from the parent trees were spread on
reaching a maximum as soon as the leaves reach their full soil beds. When the seeds were germinated and 2-3 leaves
extent and then decreasing with increasing age. Effective had appeared, the seedlings were then planted in polybags
placement of young leaves on the plant creates a high filled with growing media in the form of a mixture of
capacity for photosynthesis to increase the capture of topsoil, rice husks, and chicken manure (4:1:1). Seedlings
carbon by the whole plant. The seedlings of M. gigantea aged 3 months, with average height 2573.8 mm, basal
reduce self-shading of their leaves by increase in the stem diameter 5.21.3 mm and with number of leaves
petiole length and in the angle between petiole and the 4.72.4, were ready to plant in the research plots.
upright main stem, from youngest to oldest leaves on the
plant (Okuda et al. 1996; Yamada et al. 2000). Land preparation
Seedlings of M. gigantea grow rapidly over the first 18 Initial vegetation found in the research location was in
weeks when planted in polybags if supplied with a the form of bushes dominated by Melastoma sp and
combination of nitrogen and phosphorus fertilizers saplings of pioneer plants such as Macaranga. The land
(Lawrence 2001). Ishida et al. (2005) reported that the was prepared by clearcutting the area and this was carried
lowest total photosynthesis rate per unit leaf area in out manually by exploring, cutting, slashing and burning.
M.gigantea is found at the seedling stage and the highest at Planting holes were prepared at a spacing of 3m x 3m. The
the sapling stage. The leaves at the sapling stage have the size of each planting hole was 30 cm x 30 cm x 30 cm. The
highest nitrogen concentration, total photosynthesis rate, total size of the research plot was 50 x 90 m.
and stomatal conductance, indicating that the gradual
transition from seedling stage to sapling stage is Experimental design
accompanied by accumulating N in the plant body and The trial was set up with five randomized treatments plots
development of effiency of the plant’s water transport with 3 block (each containing 20 plants, making 20 x 5 x
system to face unpredictable environmental stresses. 3= 300 plants in the entire trial area). The treatments were
Our research reported in this paper focused on an five dosages of NPK fertilizer (16-16-16), consisting of a
experiment examining the effect of dose level of an NPK control (with no fertilizer), and either 40 g, 80 g, 120 g or
fertilizer on the growth of M. gigantea planted in a 160 g applied per plant. Fertilization was carried out twice.
monoculture system in an open field. The growth in total The first application was four weeks after the planting and
above-ground biomass and of its component plants parts the second application was applied 6 months after the first
was measured, and the accumulation of nutrient elements application. Measurement of growth performance was
N, P and K in the plant components at the age of one year carried out every three months from the time of planting up
was determined, as a practical step in optimising the to final sampling at 12 months.
potential industrial cultivation of this useful forest species.
Planting
Seedlings in polybags were placed at the sides of the
MATERIALS AND METHODS prepared planting holes. The polybags were opened
carefully with a knife, then the seedlings were placed into
Study area the planting holes which were then backfilled with soil.
This research was conducted in the research forest of Every plant was identified by a wooden stake 1 meter long,
the Faculty of Forestry, Mulawarman University, and numbered. Every treatment sub-plot and research block
Samarinda, East Kalimantan (00.44’71.11” South and was marked. One month after planting, the plants were
117.21’67.50” East) (Figure 1). Data from 2003 to 2012 fertilized using a compound NPK fertilizer (16% N: 16%
shows that the average annual rainfall over that period was P2O5: 16% K2O, 1.5% MgO, and 5% CaO), with an amount
2423 mm, and that the highest annual rainfall was 2757.5 given to each plant in accordance with the requirements of
mm in 2008. The highest monthly rainfall was in April the experimental design. The fertilizer was applied by
(288.3 mm) and the lowest in August (115.3 mm). The wet broadcasting it around the sampling stem within a circular
season varied from 9 to 12 months, and the dry season from pattern of radius 50 cm. A second round of fertilization was
0 to 3 months. Average monthly temperature was 27.50C applied six months after the first fertilization. Weeds were
and average air humidity was 82% (Anonymous 2012). removed from the research plots manually, whenever this
Chemical properties of the soil in the research plots over was needed.
the depth of 0-30 cm are as follows: pH: 4.97, cation
332 NUSANTARA BIOSCIENCE 9 (4): 330-337, August 2017

Figure 1. Map of study area in the Research Forest, Faculty of Forestry, Mulawarman University, Samarinda, East Kalimantan,
Indonesia

Soil analysis The sampling of M. gigantea biomass


The soil in the trial area was sampled at the beginning The measurement of biomass in the experiment was
of the planting period (before the application of fertilizer). limited only to plant components above the ground. The
The bulk density of a sample (0-30 cm depth) whose biomass of trees that were the sampled within one stand
volume had been determined, was calculated after the was used to calculate the biomass of all the trees within a
sample was dried in an oven at a temperature of 1500C particular plot. The trees sampled from one plot were
until constant weight was reached. A composite sample selected after all the available trees were grouped into three
was wind-dried and its pH, base saturation, cation stages based on their estimated size according to the
exchange capacity, organic carbon content, total Nitrogen formula D2H (diameter squared multiplied by height). The
(Kjeldahl), available phosphorus (Bray), and available entire stand biomass was calculated by multiplying the dry
potassium were measured. weight of the components of the sampled tree by the
number of trees in each stage, and then it was converted
into a biomass per hectare. One year after planting, 3 plants
Plant measurement were randomly sampled from each of the five fertilizer
The M. gigantea plants were measured in terms of their treatments, and then they were felled. The wet weight of
stem heights, basal stem diameters, number of leaves, every component was measured, including wood, barks and
canopy diameters, all of which were measured at the leaves (Ruhiyat 1996). Samples of the wood, barks and
beginning of the planting and every three months until the leaves were weighed wet and dry. The plant samples were
plants reached the age of 12 months. taken to the laboratory and their nutrient contents (N, P and
K) were analyzed.
SUSANTO et al. – Nutrient accumulation of Macaranga gigantea 333

Analysis of nutrient concentration of plant components Multiple Range Test (DMRT) at significance level of 95%.
The total N concentrate was measured using the Data for soil and plant nutrient concentrations were
Kjeldahl method (extraction, distillation, and titration). To analyzed descriptively.
measure the elements P and K, the plant components were
extracted using the High Pressure Digestion method at a
temperature of 1800C for 10 hours with HNO3 as the RESULTS AND DISCUSSION
reductant. Phosphorus was measured using a calorimetric
technique with nitrate-molybdate-vanadate acid as the Plant growth
coloring agent and a spectrophotometer at the wavelength Fertilizer application had a significant effect on the
of 470 nm. Potassium was measured using an Atomic growth of mahang seedlings. Best growth was when the
Absorption Spectrophotometer at wavelengths of 766.5 nm, NPK fertilizer was applied (twice) at a dosage of 120 g per
489.5 nm and 245.2 nm. To calculate the total macro plant, followed by 160 g, 80 g, 40 g and 0 g. Treatment T3
nutrient elements (N, P, K) accumulated in the tree (120 g fertilizer per plant applied at planting and then
components in the stand, the dry weights of the tree repeated at 6 months) produced a basal stem diameter of
components were multiplied by their nutrient concentrations. 45.24.9 mm, stem height of 219.239.1, number of leaves
of 17.31.2 and canopy diameter of 246.725.0 cm, twelve
Data analysis months after planting seedlings in the field (Table 1; Figure
The growth rate data were analyzed using analysis of 2-3).
variance (ANOVA) and further tested using Duncan’s

Table 1. The effect of fertilizer treatment on growth of Macaranga gigantea: basal stem diameter, stem height, leaf number per plant
and crown diameter, at 6 months and 12 months after planting seedlings in the field. Values followed by the same letter within the same
column do not differ significantly (p<0.05).

Fertilizer Basal diameter (mm) Height (cm) ∑ Leaves Crown diameter (cm)
6 month 12 month 6 month 12 month 6 month 12 month 6 month 12 month
0g 15.5±1.6a 34.1±5.3a 65.1±8.3a 175.6±34.1a 6.9±0.5a 13.8±2.1a 118.4±20.7a 210.5±33.0a
40 g 16.9±4.1a 35.4±5,7a 66.75±7.9a 172.7±28.2a 6.9±0.4a 14.2±2.2a 125.4±13.8a 219.8±23.2 ab
a
80 g 18.7±1.7 40.6±2.1b 69.8±4.8 bc
195.8±13.5ab 7.9±0.3 b
17.7±2.9b 128.1±7.0a 227.2±19.6 ab
120 g 23.1±6.1b 45.2±4.9bc 84.7±16.7c 219.2±39.1b 7.9±0.4b 17.3±1.2b 148.2±29.0b 246.7±25.0c
160g 18.8±3.6a 42.9±3.7c 77.85±13.1 b
211.4±19.4b 8.2±0.5 b
16.1±1.3ab 130.2±14.2a 236.3±6.8 b

Figure 2. Growth statistics for M. gigantea, 3, 6, 9 and 12 months after planting: basal stem diameter; stem height, number of leaves per
plant and crown diameter
334 NUSANTARA BIOSCIENCE 9 (4): 330-337, August 2017

Figure 3. M. gigantea plants in the field, 12 months after planting out the seedlings. The photographs, from left to right, are of plants
growing in treatment plots T0, T1, T2, T3 and T4, (0, 40 g, 80 g, 120 g and 160 g fertilizer per plant).

Plant biomass Discussion


The estimated above-ground biomass increased due to There is little published information about the quantity
the fertilization treatments. The highest production was of nutrient elements required for growth and biomass
found in the treatment with 120 g fertilizer, which reached production of M. gigantea plants, especially those growing
a total above-ground biomass of 2255.3 kg ha-1, consisting in plantations. In Malaysia, pioneer plants of M. gigantea
of 32.3% wood, 9.6% bark and 58.1% leaves. This was and M. hypoleuca species planted together with
followed by fertilizer treatments 160 g, 80 g, 40 g, and 0 g Dryobalonops lanceolata and Shorea leprosula
(Table 2). Leaves were the largest component of the (Dipterocarpaceae) on degraded soil were shown to
biomass, followed by stemwood and then stembark. respond to fertilization (Nusbaum et al. 1995). Seedlings
had increased in dry weight, basal diameter, and height for
Nutrient concentration in the soil and plants each species at the age of 6 months. The increase in relative
Fertilizer treatment increased the concentration of N, P, height and basal diameter of fertilized M. gigantea
K in the leaves measured twelve months after planting. The seedlings was four times higher than for unfertilized plants
highest leaf concentrations of each nutrient were all found otherwise treated the the same. Their canopy diameter
at the highest fertilizer dosage, of 160 g per plant (applied reached 100 cm in six months (Nusbaum et al. 1995). In
twice): i.e. 0.81% for leaf Nitrogen, 0.73% for leaf our study, the canopy diameter of fertilized M. gigantea at
Phosphorous, and 1.35 % for leaf Potassium.(Table 3). The the age of 12 months reached 246.725.0 cm, which was
highest soil nutrient found in 160 g (nitrogen and quite comparable with the values obtained in the Malaysian
phosphorus) and 120 g (potassium) (Table 3). study.
Research on secondary forest gaps in Malaysia has
Nutrient content of plant components shown an average increment in diameter at breast height of
Fertilizer treatments increased the accumulation of M. gigantea of 4.0 mm y-1 to 4.5 mm y-1 with a maximum
nutrient elements N, P, and K in the above-ground plant rate of 6.6 mm y-1 to 7.8 mm y-1 (Manokaran and
biomass. The highest accumulation of nitrogen was found Kochument 1992). Davies et al. (1998) reported that the
in the highest fertilization treatment 160 g per plant, growth in diameter of M. gigantea was 0.22 mm y-1 in the
reaching 7.3 kg ha-1 in the leaves. Treatment T3 (120 g seedling stage; 0.37 mm y-1 r in the sapling stage; 0.74 mm
fertilizer per plant) showed the highest accumulation of y-1 in the reproductive stage; and 0.39 mm. y-1 in the
phosphorus, reaching 7.2 kg ha-1, also in leaves. The mature stage. Hiratsuka et al. (2006) stated that the average
highest accumulation of potassium was found in T2, the 80 stem diameter of M. gigantea at the age of three years after
g fertilizer per plant treatment reaching 15.9 kg ha-1 in forest fire in East Kalimantan, Indonesia was 4.64.6±2.6
stemwood. (Table 4; Figure 4) cm. Compared with these estimates of growth of M.
The estimated nutrient elements accumulated by 1-year- gigantea in nature, in our study the increments in diameter
old M. gigantea plants in producing one tonne biomass and and height of fertilized mahang plants were far higher.
the ratio of N:P:K for annual biomass production is Research on secondary forest gaps in Malaysia has
presented in Table 5. One of the most important parameters shown that the median diameter growth rate of M. gigantea
in analyzing the nutrient accumulation and concentration in of 4.0 mm y-1 to 4.5 mm y-1 with a maximum rate of 6.6
plant biomass is the ratio of the nutrient elements of N:P:K mm y-1 to 7.8 mm y-1 (Manokaran and Kochument 1992).
accumulated in the production of one tonne of biomass. Davies et al. (1998) reported that the median diameter
Fertilization treatments created variation in the ratio of growth rate of M. gigantea was 0.22 mm y-1 in the seedling
accumulated N:P:K. Treatment T3 (120 g fertilizer per stage; 0.37 mm y-1 r in the sapling stage; 0.74 mm y-1 in the
plant applied twice) produced the largest estimated reproductive stage; and 0.39 mm. y-1 in the mature stage.
biomass, with an N:P:K ratio of 13.3:35.6:100 (Table 5). Hiratsuka et al. (2006) stated that the average stem
SUSANTO et al. – Nutrient accumulation of Macaranga gigantea 335

diameter of M. gigantea at the age of three years after Table 4. Effect of fertilizer treatment on the amount (kg ha-1) of
forest fire in East Kalimantan, Indonesia was 4.6±2.6 cm. N, P, K accumulated in the stemwood, stembark, leaves of 1 year
Compared with these estimates of growth of mahang in old Macaranga gigantea.
nature, in our study the increments in diameter and height
Fertilizer Nutrient content (kg ha-1)
of fertilized M. gigantea plants were far higher. Tree section
treatment N P K Ca Mg
In a study by Nusbaum et al. (1995), the average dry
0g Stemwood 0.39 1.38 2.85 1.09 0.37
weight at age 6 months of M. gigantea grown on a Stembark 0.22 0.56 2.01 0.73 0.36
degraded soil without fertilization was 4.18 g, while the Petiole 0.22 0.99 3.76 0.82 0.40
average dry weight of fertilized plants reached 250 g Leaves 1.79 4.42 7.40 1.05 0.53
(above-ground biomass was 198 g). In our study, the most
effective fertilizer treatment (120 g of NPK fertilizer per 40 g Stemwood 0.54 1.97 6.09 0.72 0.44
plant) produced an above-ground biomass yield of 2255.3 Stembark 0.25 0.69 1.93 2.16 0.58
kg ha-1. Petiole 0.89 1.05 5,80 2.29 0.51
Leaves 1.35 3.57 5.18 1.82 0.56

Table 2. Estimated above-ground biomass in three components 80 g Stemwood 1.41 3.85 15.89 2.05 0.67
(wood, bark and leaves) of trees in the five different fertilizer Stembark 0.24 1.26 4.09 1.57 0.64
treatments twelve months after planting out the seedlings Petiole 0.25 1.24 7.66 1.60 0.63
Leaves 1.90 4.02 5.86 1.21 0.48
Stemwood Stembark Leaves
Fertilizer 120 g Stemwood 1.03 4.16 14.76 1.53 0.52
kg kg kg
treatment kg ha-1 kg ha-1 kg ha-1 Stembark 0.49 1.32 5.07 1.57 0.57
plot-1 plot-1 plot-1
Petiole 0.98 1.69 10.62 1.38 0.65
0g 12.3 310.7 4.6 114.7 31.9 807.6
Leaves 2.87 7.18 9.76 0.58 0.43
40 g 12.7 319.9 5.3 131.5 26.2 663.3
80 g 25.1 634.1 8.7 218.2 34.3 866.1
160 g Stemwood 0.49 3.39 8.88 0.81 0.46
120 g 28.9 728.46 8.5 216.5 51.9 1310.3
Stembark 0.99 1.48 4.17 0.88 0.43
160 g 24.9 629.4 9.6 243.3 45.1 1138.3
Petiole 0.32 1.50 7.43 0.61 0.42
Leaves 7.33 6.16 11.12 0.74 0.41

Table 5. Amounts of nutrients accumulated by M. gigantea to


Table 3. Soil nutrient at 0-30 cm deep and leaf nutrient
produce one tonne of biomass twelve months after planting,
concentration after fertilizer treatment (1 year)
together with the calculated N:P:K ratios, in response to different
fertilizer treatments
Nitrogen Phosphorus Potassium
Fertilizer
Soil Leaves Soil Leaves Soil Leaves
treatment Nutrients accumulation
(%) (%) (ppm) (%) (ppm) (%) Ratio
Fertilizer (kg ton-1)
0g 0.11 0.28 20.82 0.69 101.14 1.13
N P K N P K
40 g 0.10 0.34 22.10 0.73 124.74 1.10
0g 2.12 5.96 12.97 16.3 45.9 100
80 g 0.10 0.30 19.70 0.65 75.81 0.99
40 g 2.70 6.52 17.02 15.9 38.3 100
120 g 0.10 0.28 23.90 0.71 179.96 1.01
80 g 2.70 6.52 17.02 11.3 30.9 100
160 g 0.13 0.81 32.64 0.73 99.56 1.35
120 g 2.38 6.36 17.83 13.3 35.6 100
160 g 4.54 6.23 15.70 28.9 39.6 100

Figure 4. Amounts of N, P, and K accumulated in above-ground biomass (kg ha-1) of 1year old Macaranga gigantea
336 NUSANTARA BIOSCIENCE 9 (4): 330-337, August 2017

Information about fertilizer treatment on the seedlings Accumulation of potassium in our study consisted of
of some pioneer species, including M. gigantea in polybags 36.7% in wood, 12.6% in barks and 50.7% in leaves.
was reported by Lawrence (2001) in West Kalimantan The nutrient content of fast growing Eucaliptus
Indonesia. The study found that N had to be combined with deglupta in industrial plant forests in East Kalimantan (100
P to optimize growth over the first 18 weeks. Application m3 log and bark) was reported by Meckensen (1999) and
of N and N+P fertilizers did not significantly increase the N Meckensen et al. (2001) as N: 44. 4 kg ha-1, P: 2.3 kg ha-1
content of the plant canopy, while application of P fertilizer and K:125 kg ha-1. Rahmawati (1999) reported much less
alone significantly decreased N content of the canopy. nutrient contents for the above-ground biomass of
Moreover, the application of N+P fertilizer did not increase Eucaliptus deglupta at the age of 1 year; the contents were
the P content of the plant canopy, while the application of estimated to be N:17.83 kg ha-1, P: 1.68 kg ha-1 and K:
N fertilizer alone decreased the P content of the plant 41.16 kg ha-1. Ruhiyat (1993) reported that forests of leda
canopy, and the application of P fertilizer alone did not (Eucalyptus deglupta) and sengon (Paraseriantes
significantly increase the P content of the plant canopy. falcataria) at the age of 5-10 years in East Kalimantan had
The N concentration ranged between 15 to 20 mg g-1, while accumulate potassium in largest amounts, followed by
phosphorus concentration ranged between 1.7 and 2.7 mg calcium, nitrogen and magnesium. Therefore, the
g-1 in the seedling canopy. On the other hand, Ishida et al. conclusion has been that ensuring the availability of
(2005) reported that the highest N concentration in M. potassium is of high priority in the production of
gigantea leaves growing naturally was 2.5 mol kg-1 at the Eucalyptus stands.
sapling stage and 2.0 mol kg-1 at the sucker stage, while the Susanto et al. (2016b) and Susanto et al. (2017)
lowest nitrogen concentration was found at the seedling reported that a positive correlation was observed between
and mature stages, namely 1.5 mol kg-1. Breulmann et al. the P and K content of the leaves and the plant growth of
(2006) reported that phosphorus concentration in M. gigantea in secondary forest after shifting cultivation
Macaranga in the natural forests in Malaysia ranged and in forest gaps after selective logging.
between 0.06% to 0.09%, while potassium content was In our study, the application of fertilizer at a dose of
0.71% to 0.82% in leaves only. 120 g per plant produced the highest above-ground biomass
Susanto et al. (2014) reported that for M. gigantea, the and the highest accumulation of nutrients P and K. In
highest N, P and K concentrations occur in the leaves. In addition, the amount of nutrients accumulated in one tonne
our study, the highest nitrogen concentration in the leaves of biomass increased, with the highest amounts also being
of fertilized plants (in the 160 g fertilizer per plant found in the 120 g fertilizer per plant, namely 2.38 kg of N,
treatment) was 0.8130.53%; the highest phosphorus 6.36 kg of P, and 17.83 kg of K at an N:P:K ratio of 13.3:
concentration of 0.8400.24% was in the wood of plants 35.6:100. There is no other published information that can
fertilized with 120 g of fertilizer per plant, while the be used as a comparison with this study as to the ratio of
highest potassium concentration of 3.5130.73% was in the N:P:K required for M. gigantea to produce one tone of
bark of plants fertilized with 120 g per plant. biomass. For another plant species, Uri et al. (2003) found
There has been no information in the published literatre that the amount of nutrients accumulated by a one year old
about the accumulation of N, P, and K nutrients in M. grey alder in producing one tonne of biomass was16.7 kg
gigantea. In this study, the fertilization treatment of 120 g of N, 1.5 kg of P and 7.2 kg of K at an N:P:K ratio of
fertilizer per plant produced the highest above-ground 100:9:43.
biomass with the accumulation of N, P, K reaching 5.383 In conclution, fertilizer treatment in our study increased
kg ha-1, 14.355 kg ha-1 and 40.207 kg ha-1 respectively. growth, biomass production and nutrient accumulation in
Nitrogen accumulation was 19.2 % in wood, 9.2% in bark the components of M. gigantea plants. The best growth and
and 71.8% in leaves. The large accumulation of nitrogen in production of above-ground biomass was found in plants to
leaves was due to the high nitrogen concentration together which 120 g NPK fertiliser per plant was applied at the
with the high biomass production of leaves. This is in line time of planting seedlings in the field, and again at the
with what was reported by Ishida et al. (2005). In that same dose level, six months later. The highest amounts of
study, leaves of M. gigantea had the lowest nitrogen nutrient elements accumulated by M. gigantea at the age of
concentrate at seedling stage and the highest concentrate at one year were for potassium, followed by phosphorus,
the sapling stage. There is a gradual transition from nitrogen, calcium and magnesium in declining order.
seedling stage to sapling stage, with accumulation of plant
N and development of the hydraulic system to face
unexpected environmental stress. ACKNOWLEDGEMENTS
In this study, the accumulation of phosphorus was 29%
in wood, 9.2% in bark and 61.9% in leaves. The high We acknowledge the Forest Education, Faculty of
accumulation of phosphorus in leaves was due mainly to Forestry, Mulawarman University, Samarinda, Indonesia
the large amount of leaf biomass. The largest size for M. for providing permission to conduct the field work. The
gigantea leaves is found at the sapling stage, reaching 60 research was supported by decentralization research
cm long and 50 cm wide (Okuda 1996; Silk et al. 2000). program of Mulawarman University, Indonesia.
SUSANTO et al. – Nutrient accumulation of Macaranga gigantea 337

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| Nusantara Biosci | vol. 9 | no. 3 | pp. 237‐337| August 2017|
| ISSN 2087‐3948 | E‐ISSN 2087‐3956| 

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KARTIAWATI ALIPIN, EMA PURNAMA SARI, MADIHAH, TIA SETIAWATI, NINING 
RATNINGSIH, DESAK MADE MALINI  
Short Communication: Infraspecific variations in essential oil compositions of Nepeta fissa  318‐321 
from Iran 
SEYED MEHDI TALEBI, MAJID GHORBANI NOHOOJI , MAHBOOBEH YARMOHAMMADI 
Visual symptoms and control of the Red Palm Weevil (Rhynchophorus ferrugineus) in the  322‐329 
Gaza Strip, Palestine  
ABDEL FATTAH N. ABD RABOU, EQBAL S. RADWAN 
Growth, biomass production and nutrient accumulation of Macaranga gigantea in response  330‐337 
to NPK fertilizer application 
DWI SUSANTO, SRI MULYATI, HERI PURNOMO, DADDY RUHIYAT, RUDIANTO AMIRTA 
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the author(s) or manuscript(s). The editor is not obligated to use any change at hemic peat natural regeneration following burning; a case
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Acceptance The acceptance of a paper implies that it has been reviewed The usage of “et al.” in long author lists will also be accepted:
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Ethics Author(s) must obedient to the law and/or ethics in treating the communities. In: Carson W, Schnitzer S (eds).Tropical Forest
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Copyright Submission of a manuscript implies that the submitted work 50thAnnualSymposium of the International Association for Vegetation
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Preparing the Manuscript Balagadde FK, Song H, Ozaki J, Collins CH, Barnet M, Arnold FH,
Quake SR, You L. 2008.A synthetic Escherichia coli
Please make sure before submitting that: The manuscript is proofread predator-prey ecosystem. Mol Syst Biol 4:
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