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International Journal of Agricultural Science and Research (IJASR) ISSN 2250-0057 Vol.

2, Issue 3 Sep 2012 129-141 TJPRC Pvt. Ltd.,

PROMOTION OF GROWTH CHARACTERISTICS IN GREENHOUSE CUCUMBER AND TOMATO BY TALAROMYCES FLAVUS


1

LALEH NARAGHI, 2ASGHAR HEYDARI, 3SAEED REZAEE,


4

MOHAMMAD RAZAVI & 5HOMAYOON AFSHARI-AZAD

1,3

Department of Plant Pathology, College of Agriculture and Natural Resources, Science and Research Branch, Islamic Azad University, Tehran, Iran
2,4,5

Plant Disease Research Department, Iranian Research Institute of Plant Protection , Iran

ABSTRACT
Beneficial plant microbe interactions in the rhizosphere are primary determinants of plant health and soil fertility. Some antagonistic fungi have shown great effects towards the growth of plant crops. In this study, two major crops, tomato and greenhouse cucumber were selected to evaluate their growth promotion by antagonistic fungus, Talaromyces Flavus. For each plant, five T. flavus isolates were selected from our fungal collection which had previously shown antagonistic activities against the causal agent of wilt diseases on these plants. In next step, for every crop, five isolates were used in the greenhouse condition. For evaluation of the plant growth promotion ability of T. flavus isolates, a split plot trial was arranged in a randomized complete block design with four replications. The main-factor was methods of application of T. flavus as soil treatment, seed treatment and combining both methods. The sub-factor was the use of different fungal isolates. Measured parameters were root length, crown length, plant height, plant fresh weight and plant dry weight. Results showed that for both above mentioned crops, there were no significant differences among various levels in main factor. Results obtained from sub-factor section, showed that most effective isolates were TF-To-V-31 and TF-

Cu-V-55 in tomato and greenhouse cucumber respectively. In interaction between main factor and subfactor, TF-To-V-31 increased plant dry weight by 2.49 folds when it was applied as soil treatment. However, TF-Cu-V-55 increased plant dry weight by 2.93 folds as seed treatment or soil and seed treatment combined. The overall results of this study suggest that it may be possible to promote tomato and greenhouse cucumber growth characteristics by using the antagonistic fungus flavus. T.

KEY WORDS: Plant growth promotion, Talaromyces flavus, Tomato, Cucumber. INTRODUCTION
Environmental concerns have led to the need for sustainable use of natural resources. The conventional agriculture has caused considerable negative impacts on soil and water. It is important to

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Laleh Naraghi, Asghar Heydari, Saeed Rezaee, Mohammad Razavi & Homayoon Afshari-Azad

change certain management practices to environmentally cleaner techniques (Sudha Lakshmi et al., 2011). The sustainable agriculture employs many approaches and techniques to reduce negative effects of conventional agricultural practices on the environment. One of these strategies is the utilization of soil microorganisms for the promotion of plant growth and control of plant diseases (Botelho and Hagler, 2006). The widespread use of chemical pesticides and fertilizers have been subject of public concerns and environmental protection agencies. The harmful chemicals may affect human health, contaminate environment and negatively affect biological resources. In addition, their high production cost and appearance of resistant pests should also be considered (Zaki et al., 1998; Heydari and Misaghi, 1998 and 2003). Some antagonistic fungi play direct and indirect roles in promoting plant growth characteristics. In a direct role, metabolites of antagonistic fungi promote plant growth by providing soluble elements which are necessary for plant nutrition. Indirectly, some microorganisms can act as biological control agents and affect plant growth promotion indirectly through decreasing plant diseases (Le Floch et al., 2003). Talaromyces flavus (Klocker) Stolk and Samson, is one of the most important species of antagonistic fungi. This ascomycete, is frequently isolated from soil, although it may also occur on organic materials under going decomposition (Domsch et al., 1980). The organic soluble metabolites of this fungus include D-glucono-1,4-lacton; 5-hydroxymethylfurfural; 4,6-dihydroxy-5-methylphthaide; methyl 4-carboxy-5-hydroxyphethalal dehydrate; hexaketide; 7-hydroxy-2,5-dimethylchromone; 3hydroxymethyl-6,8-dimethoxycoumarin; altenusin; desmethyldehyroaltenusin; talaroflavone,

deoxytalaroflavone, 2-Methylsorbic acid, sorbic acid, bromomethylsorbic acid and bromosorbic acid (Ayer and Racok, 1990; Wakelin et al., 2004). Some of above-mentioned metabolites (2-Methylsorbic acid, sorbic acid, bromomethylsorbic acid and bromosorbic acid) play a fundamental role in the biogeochemical cycling of phosphorous (P) in natural and agricultural ecosystems (Wakelin et al., 2004). Harvey et al. (2009) demonstrated that microbiological activity in the rhizosphere, could dissolve sparingly soluble inorganic phosphorous and increase plant growth. On the other hand, T. flavus is a biological control agent that has been used in biological control of important soil-borne pathogens such as Verticillium dahliae, V. albo-atrum, Rhizoctonia solani and Sclerotinia sclerotiorum (Marois et al., 1984). Naraghi et al. (2007) showed that this fungus is a very effective biocontrol agent in controlling Verticillium wilt disease on cotton. T. flavus is a common mycoflora member of tomato and greenhouse cucumber which are two major crops in the world including Iran. In this study, we investigated the possible growth promotion activities of different T. flavus isolates on some characteristics of these crops including plant height, root length, crown length, plant fresh weight and plant dry weight.

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MATERIALS AND METHODS


The following experiments were conducted and executed in 2010 in the greenhouses of Iranian Research Institute of Plant Protection (IRIPP). PREPARATION OF T. FLAVUS ISOLATES For each plant (tomato and greenhouse cucumber), five T. flavus isolates were selected from our fungal collection which had shown the highest antagonistic activities against the causal agent of wilt diseases of these plants in the previous studies (Naraghi et al. 2010a and c). These isolates (TF-To-V-24, TF-To-V30, TF-To-V-31, TF-To-U-36 and TF-To-U-38) were obtained from tomato fields in Tehran (Varamin) and Western Azarbayjan (Uremia) provinces of Iran and the remaining isolates (TF-Cu-V-55, TF-Cu-V57, TF-Cu-V-58, TF-Cu-V-59 and TF-Cu-V-60) were obtained from cultivation regions of

greenhouse cucumber in Tehran (Varamin) province.

PREPARATION OF T. FLAVUS INOCULUM


Talaromyces flavus inoculum was prepared separately for every isolate as follows: Talaromyces flavus isolated from the soil was cultured in test tubes containing TF medium. After 5 days, 20 mL SDW was poured into 50 cm-wide80-cm-tall sterile plastic bags containing 250 g of peat moss mixed with 10 mL D-lactose monohydrate (20 g L-1). The plastic bags were incubated at 30C for 30 days. The contents of each plastic bag was evacuated after peat moss was completely covered with T. flavus hyphae. The number of ascospores of T. flavus in each g of peat moss was then determined using a haemocytometer. In this procedure, one g of peat moss was suspended in 10 mL SDW and the number of ascospores in one mL of this suspension was counted. Talaromyces flavus inoculum was added to the soil in the pots at 107 ascospores g-1 soil (Chet and Baker, 1981). For seed treatment, tomato and cucumber seedlings at the second true-leaf stage were floated in this inoculum for half an hour.

STUDY OF THE PROMOTION EFFECTS OF T. FLAVUS ISOLATES ON TOMATO AND GREENHOUSE CUCUMBER GROWTH CHARACTERISTICS
The experiment for studying the effect of T. flavus isolates on the growth characteristics of tomato and greenhouse cucumber was carried out as follows: Soil samples (22% sand, 68% silt and 10% clay) were collected from cultivation areas of tomato and greenhouse cucumber in Varamin region of Tehran province, and used for experimental pots after autoclaving. For every plant, the experiment was performed as a split plot arranged in a randomized complete block design with four replications. The main-factor was different types of T. flavus treatments at three levels (1 soil, 2 seed, 3 both soil and seed) and the sub-factor was different fungal isolates at six levels ( from 1 to 5: one of five T. flavus isolates, 6: without fungal inoculum). Inoculated plants were kept at 22C in the greenhouse under a 12 h light period. Plants were harvested 90 days after sowing for growth evaluation. Measured parameters included root length, crown length (the

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lower swollen section length of the stem), plant height, plant fresh weight and plant dry weight. Data were analyzed by ANOVA (Analysis of Variance) using the MS TAT C statistical software, while means were separated by Duncans multiple range test.

RESULTS
Study of the Promotion Effects of T. Flavus Isolates on Tomato and Greenhouse Cucumber Growth Characteristics The effects of the main-factor (methods of application of T. flavus), sub-factor (T. flavus isolates) and both factors on growth characteristics of tomato and greenhouse cucumber which were determined separately were as follows: The Effects of The Main Factor on Tomato Growth Characteristics The results of this experiment showed that the effects of main factor on all growth parameters (root length, crown length, plant height, plant fresh weight and plant dry weight) were significant (Table 1). According to the results, there were no significant differences among various methods of application of T. flavus in all growth parameters (Table 1). The Effects of The Sub-Factor on Tomato Growth Characteristics The results of this experiment showed that the effects of main factor on all parameters (root length, crown length, plant height, plant fresh weight and plant dry weight) was also significant (Table 2). In this study, most treatments affected by different isolates of T. flavus mediated significant increase in crown length, plant fresh weight and plant dry weight all growth characteristics except root length when compared with control. Among these treatments, maximum and minimum means related to plant dry weight were observed in treatments affected by TF-To-V-31 and TF-To-V-24 respectively (Table 2). The maximum plant height and plant fresh weight means belonged to treatments affected by TFTo-U-38 and TF-To-V-30 respectively. Maximum Crown length means were observed in treatments affected by TF-To-V-24, TF-To-U-36 and TF-To-U-38. However, there were no significant differences among T. flavus isolates in term of root length (Table 2).

THE EFFECTS OF THE BOTH FACTORS ON TOMATO GROWTH CHARACTERISTICS


The effects of the both factors on growth parameters (crown length, plant fresh weight and plant dry weight) were significant (Table 3). In this study, all treatments mediated significant increase in all growth characteristics except plant fresh weight when compared with control. The maximum increase in plant dry weight was observed when TF-To-V-31 was applied as soil treatment (Table 3).

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THE EFFECTS OF THE MAIN FACTOR ON GREENHOUSE CUCUMBER GROWTH CHARACTERISTICS


The results of this experiment showed that the effects of main factor on root length, crown length, plant fresh weight and plant dry weight were significant (Table 4). According to the results, there were no significant differences among various methods of application of T. flavus in above mentioned growth parameters (Table 4).

THE EFFECTS OF SUB-FACTOR ON GREENHOUSE CUCUMBER GROWTH CHARACTERISTICS


Results of this experiment showed that the effects of subfactor on all growth parameters were significant (Table 5). According to the table, all treatments affected by different isolates of T. flavus mediated significant increase in all growth parameters when compared with control. The maximum increase in root length and crown length was mediated by TF-Cu-V-60. However, the maximum means related to plant height and plant dry weight were observed in TF-CU-V-58 and TF-Cu-V-55 respectively (Table 5).

THE EFFECTS OF BOTH FACTORS ON POTATO GROWTH CHARACTERISTICS


The effects of both factors on all growth parameters were significant (Table 6). This experiment showed that most treatments mediated significant increase in all growth characteristics when compared with control (Table 6). The maximum increase in root length, plant height and plant dry weight was mediated by TF-Cu-V-55 as seed coating (Table 6).

DISCUSSIONS
The overall results of this study show that it may be possible to promote some plant growth characteristics by different T. flavus isolates. The results of this study reveal the potential of T. flavus for increasing plant growth in tomato and greenhouse cucumber. The type of growth promotion may be similar to that produced by the addition of Trichoderma spp. which have been found to enhance the growth of various plants (Chang et al., 1986; Paulitz et al., 1986; Windham et al., 1986; Baker, 1988; Kleifeld and Chet, 1992; Ousley et al., 1994 a,b; Phuwiwat and Soytong, 1999; Harman, 2000; Contreras-Cornejo et al., 2009; Hajieghrari, 2010; Masunaka et al., 2011). The increased plant growth induced by Trichoderma spp., has been shown to be dependent on many factors such as plant species, the strain of Trichoderma, the form of inoculum, the concentration of inoculum, and the soil environmental conditions (Paulitz et al., 1986; Baker, 1988; Kleifeld and Chet, 1992; Ousley et al., 1994a,b). In this study, T. flavus showed significant effect on the growth of tomato and greenhouse cucumber through increasing root length, crown length, plant height, plant fresh weight and plant dry weight. In this regard, there are previous reports that T. flavus and Penicillium notatum may

enhance plant growth directly (Phuwiwat and Soytong, 2001; Bewley et al., 2006). Phuwiwat and Soytong (2001) showed that P. notatum isolated from rhizospheric soil gave higher plant yields

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of Chinese mustard compared to non-treated plants. The Chinese mustard grown in sterilized planting medium mixed with P. notatum yielded highest plant growth. Plant height, root length, root diameter, fresh and dry weight of shoot, root and total plant dry weight increased gradually as the inoculum of P. notatum was applied. Penicillium species (including the teleomorphic states of Talaromyces and Eupenicillium) are considered to be a key group of soil microflora involved in P cycling (Whitelaw, 1999). This activity is generally attributable to the production of organic acids that can directly dissolve P precipitates, or chelate P-precipitating cations with the concomitant release of P into solution (Kucy et al., 1989; Gadd, 1999). Mentioned statement could be an important reason for more growth in plants such as cotton and potato observed in the present study. However, many studies have shown that T. flavus is capable to control some important soil borne pathogens such as Verticillium dahliae, Rhizoctonia solani and Sclerotinia sclerotiorum and S. rolfsii in several crops incuding cotton, potato, tomato, eggplant and bean (Dutta, 1981; Madi et al., 1997; Tjamos and Fravel, 1997; Menendez and Godeas, 1998; Naraghi et al., 2006). Interestingly, Naraghi et al. (2010a, b and c) reported that T. flavus could reduce Verticillium wilt disease caused by V. albo-atrum in potato, tomato and greenhouse cucumber. Despite the presence of T. flavus in cotton and potato fields of Iran, its population density is not enough to mediate soil nutritional processing. It should be added to the soil as inoculum until it gets active for decomposition, nutrient mobilization, mineralization, storage and release of nutrients and water (Pandya and Saraf, 2010). There is overwhelming evidence in the previous literature indicating that plant growth promoting fungi (PGPF) can be a true success story in sustainable agriculture (Sudha Lakshmi et al., 2011). In fact, through their numerous direct or indirect modes of action, PGPF can allow significant reduction in the use of pesticides and chemical fertilizers. These beneficial events producing biological control of diseases and pests, plant growth promotion, increase in crops yield and quality improvement, can take place simultaneously or sequentially. Plant age and the soil chemical, physical and biological properties will greatly influence the outcome of PGPF inoculation (Hyakumachi and Kubota, 2004; Siddiqui et al., 2008). Presently, the absence of a universal magic PGPF bioinoculant formulation for each important field crop, simply reflects the complexity of the interactions and of the molecular signal exchanges taking place in the soil-plant-organisms ecosystems (Arora, 2004). It would be interesting to test T. flavus for biological control of important plant pathogens. Further investigations are needed to determine the potential of this fungus on the growth promotion of different crop plants. The effective strains should be selected and the suitable concentrations for plant growth promotion should be investigated. Moreover, the soil environmental conditions that is suitable for promoting plant growth should also be studied.

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The overall results of the present study are promising and may be used in the biological fertilization of tomato and greenhouse cucumber in their cultivation regions. Since the tested fungal isolates have shown both antagonistic and plant growth promotion properties, their successful application in the field may result in the reduction of chemical use in the agriculture, protection of agricultural environment and biological resources which are very important factors for a sustainable agricultural system.

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APPENDICES
Table 1: The effects of main-factor (methods of application of T. flavus) on root length,

crown length, plant height, plant fresh weight and plant dry weight of tomato plants. Variable source Treatment Root length Crown length Plant height Plant fresh weight (cm) Mainfactor Seed Soil and Seed 5.38 6.06 a a 1.69 1.78 a a 72.13 85.1 a a 133.2 140.01 a a 29.18 35.66 a a Soil 5.82 a (cm) 1.86 a (cm) 71.63 a (g) 123.68 a 33.7 Plant dry weight (g) a

In each column values marked with the same letter (s) are not significantly different (p < 0.01). Table 2: The effects of sub-factor (different T. flavus isolates) on root length, Crown length, plant height, plant fresh weight and plant dry weight of tomato plants. Variable source Treatment Root length Crown length Plant height Plant fresh weight (cm) Subfactor TF-To-V24 TF-To-V30 TF-To-V31 TF-To-U36 TF-To-U38 Control 5 c 1.5 b 78.75 ab 116 c 20 f 5.5 c 2 a 85.67 a 116 c 40 ab 5.33 c 1.96 a 78.92 ab 140 ab 33 cd 5.33 c 1.63 ab 81.17 ab 140 ab 43 a 5.67 c 1.67 ab 83.17 ab 146 a 38 abc 5 c (cm) 2.04 a (cm) 78.92 ab (g) 142 ab Plant dry weight (g) 30 de

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In each column values marked by the same letter (s) are not significantly different (p< 0.01). Table 3: The effects of both factors (methods of application of T. flavus and different T. flavus isolates) on crown length, plant fresh weight and plant dry weight of tomato plants

Variable source

Treatment

Crown length

Plant fresh weight

Plant dry weight

(cm) Both factors TF-To-V24, Soil TF-To-V30, Soil TF-To-V31, Soil TF-To-U36, Soil TF-To-U38, Soil TF-To-V24, Seed TF-To-V30, Seed TF-To-V31, Seed TF-To-U36, Seed TF-To-U38, Seed TF-To-V24, Soil & Seed TF-To-V30, Soil & Seed TF-To-V31, Soil & Seed TF-To-U36, Soil & Seed TF-To-U38, Soil & Seed Control 2.25 1.36 1.75 2.25 2 1.88 2.13 1.88 1.63 1.5 2 b gh def b bcd cde bc cde efg fgh bcd 124.33 143.48 117.95 132.43 96.69 140.03 136.37 138.88 153.52 125.78 161.97

(g) fgh bc h cdefg i c cdef cd ab defgh a 28.7 33.7 49.7 33.6 45.4 25.7 32.4 36.5 28.6 32.3 35

(g) g ef a ef bc gh f de g f def

1.5

fgh

157.4

47.1

ab

1.25

163.25

43

bcd

161.89

38

2.5

97

42.1

1.5

fgh

115.8

19.9

In each column values marked by the same letter (s) are not significantly different (p<0.01

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Laleh Naraghi, Asghar Heydari, Saeed Rezaee, Mohammad Razavi & Homayoon Afshari-Azad

Table 4: The effects of main-factor (methods of application of T. flavus) on root length, crown length, plant fresh weight and plant dry weight of greenhouse cucumber plants. Variable source (cm) Mainfactor Soil Seed Soil and Seed 1.52 2.02 1.67 a a a 4.17 4.27 3.72 (cm) a a a 97.64 82.08 87.44 Treatment Root length Crown length Plant fresh weight (g) a a a 9.58 10.12 9.75 (g) a a a Plant dry weight

In each column, values marked with the same letter (s) are not significantly different (p < 0) Table 5: The effects of sub-factor (different T. flavus isolates) on root length, crown length, plant height, plant fresh weight and plant dry weight of greenhouse cucumber plants. Variable source (cm) Sub-factor TF-Cu-V55 TF-Cu-V57 TF-Cu-V58 TF-Cu-V59 TF-Cu-V60 Control 1 d 3 c 62 e 65.62 c 5.3 c 2.21 a 4.96 a 69.25 de 89.75 b 8.81 b 1.46 cd 4.13 abc 91.83 bc 87.34 b 8.32 b 1.92 abc 4.17 abc 109.58 a 103.9 a 8.17 b 1.67 bc 3.5 bc 94.92 abc 91.48 b 8.22 b 2.13 ab Treatment Root length Crown length (cm) 4.33 ab (cm) 95.08 abc Plant height Plant fresh weight (g) 94.84 ab Plant dry weight (g) 14.13 a

In each column values marked by the same letter (s) are not significantly different (p<0.01).

Promotion of Growth Characteristics in Greenhouse Cucumber and Tomato by Talaromyces Flavus

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Table 6: The effects of both factors (methods of application of T. flavus and different T. flavus isolates) on root length, crown length, plant height, plant fresh weight and plant dry weight of greenhouse cucumber plants. Variable source Treatment Root length (cm) Both factors TF-Cu-V-55, Soil TF-Cu-V-57, Soil TF-Cu-V-58, Soil TF-Cu-V-59, Soil TF-Cu-V-60, Soil TF-Cu-V-55, Seed TF-Cu-V-57, Seed TF-Cu-V-58, Seed TF-Cu-V-59, Seed TF-Cu-V-60, Seed TF-Cu-V-55, Soil & Seed TF-Cu-V-57, Soil & Seed TF-Cu-V-58, Soil & Seed TF-Cu-V-59, Soil & Seed TF-Cu-V-60, Soil & Seed Control 2.13 1.25 1.38 1.25 1.75 2.75 2.13 2.25 1.5 2.63 1.5 b fg ef fg cd a b b def a def Crown length (cm) 4.88 4.13 4.75 4 4.25 4.63 3.63 4.25 4.25 5.5 3.5 abcd defg abcde efg cdef bcde fgh cdef cdef a fghi Height plant (cm) 100 104 95.5 103 67 119 95.5 110 68.5 64.5 66 cd bc cd bc g a cd b fg g g Plant fresh weight (g) 116.26 100.5 123.48 96.65 67.96 78.88 95.08 92.87 81.3 85.43 89.37 b c a cd i h cde de gh fg ef Plant dry weight (g) 11.33 7.41 8.72 9.08 9.09 15.55 9.81 9.51 6.19 8.59 15.52 d h g fg fg a e ef i g a

1.63

de

2.75

85

78.86

7.44

2.13

3.5

fghi

123

95.35

cd

6.3

1.63

de

4.13

defg

104

bc

84.05

fgh

9.69

2.25

5.13

ab

76.3

115.86

8.74

hi

62

65.62

5.29

In each column values marked by the same letter (s) are not significantly different (p<0.01).