International Journal of Agricultural

Science and Research (IJASR)

ISSN(P): 2250-0057; ISSN(E): 2321-0087
Vol. 4, Issue 6, Dec 2014, 81-94
TJPRC Pvt. Ltd.

MICROBIAL AND NUTRIENT STUDY IN ALKALINE SOIL USED FOR CULTIVATION

OF DIFFERENT VARIETIES OF MULBERRY PLANTS
YASHVANT RAO1, NAVEEN KUMAR NIGAM 2, RAJAN VERMA3,
VENKATESH K. R4 & ANIL KUMAR5
1,5
2,3,4

Centre for Nanosciences, Central University of Gujarat, Gujarat, India

Department of Applied Animal Sciences, BBAU, Lucknow, Uttar Pradesh, India

ABSTRACT
This research work, the soil having the alkaline range from 7.9 to 9.2 pH was found to have an effect on number
of soil microbes per gram of sample. Soil sample AR-14 was found to have the highest number of bacterial colonies.
The fungal colonies observed in the sample was 9.0 10 5, represent at pH 9.2 proved the unfavorable for growth of
the soil fungi. In soil samples S-1 and BR-2 marked decrease was observed in the number of soil fungi and bacteria.
Factors such as source of Carbon, Nitrogen, Vitamins and trace elements determine the rate of spore development
under natural conditions. The high value of potassium content was measured in this study. The majorities of bacteria
isolated from soil samples were gram negative bacteria. The present study represent that soil sample high pH (9.2) and
K content (212 ppm) is capable of supporting the growth of maximum number of bacteria and soil higher concentration of
Cu favors abundant growth of soil fungi carrying out biodegradation during their secondary metabolism.

KEYWORDS: Microbes, Macronutrients, Micronutrients, Gram-Positive, Gram-Negative, PDA, Sericulture

INTRODUCTION
The microbes are playing an important role in soil, agriculture, silk industry and food sciences, microbes can
boom and decrease the production of fruits as well as silk both in the sericulture. Sericulture is an art of rearing silkworm
for the production of cocoons which is the raw material for the production of silk. Sericulture is essentially a great
industry, providing employment to a talented section of the population. Although Sericulture is considered as a subsidiary
occupation, technological innovation has made it possible to take up an intensive scale capable of generating adequate
income. Mulberry is a fast growing deciduous woody perennial plant. The leaves are simple, alternate, stipulate, petiolate,
entire or lobed. Soil matters more in the growth rate of any plant. Mulberry grows in a wide range of soils and mulberry
is a deep rooted perennial plant. Therefore, the soil with good water holding capacity and good air penetration is more
suitable. The acidic soils ranging from 6.2 - 6.8 pH reveal the healthy growth of the mulberry plant. Minerals and
nutrients have been dissolved in the soil, water contribute to the soil solution that is the nutrient lifeline for plants.
Soil Components
The soil has been composed of five major components such as inorganic matter, organic matter, soil, air, water
soluble nutrients and soil microorganisms. The organic matter content of mineral surface soils ranges from less than 0.5
percent in highly weathered, sandy soils to more than 6% in poorly drained soils. Initially, plant residues are attacked by
soil animals such as insects or worms. Carbon has changed to carbon dioxide, and complex nitrogen compounds are
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transformed into soluble forms that plants can use. Humus also having significant store house of phosphorus and sulphur.
The climate and soil of region have a great bearing on foundations, availability and degree of decomposition of organic
material in the soil while the land use pattern significantly influences of the soil organic carbon. Minerals and nutrients
that have been dissolved in the soil, water contributes to the soil solution that is the nutrient lifeline for plants.
The amount of minerals in the soil and the rate at which dissolved into water help determine the fertility of the soil. Fungi
and soil-living bacteria, instead of artificial fertilizers, are improving crop yields, boosting harvests, and saving money for
some developing world farmers.
The mulberry (Kumar R. V. et al., 2012) can grow on the saline alkali soil containing salt below 2% where the
plantation is setup on the saline alkali soil measure must be taken to build up high rigid, enticement ash the salt with fresh
water and plant such salt tolerant green manure crop as sesbania in order to lower salt content.
The soil considered as an excellent habitat for insect pathogenic fungi and other microorganisms since it is
protected from UV radiation and buffered against extreme biotic and abiotic influences. Soils typically contain 109 to
1010 microorganisms per gram dry weight (Srivastava et al., 1998) which represent more than a million bacterial
species. Soil microbiology deals with the microorganisms present in soil, important function of soil microorganisms to
decompose various kinds of organic matter and mineralization of various organic constitutions. Mineralization of organic
carbon, nitrogen, phosphorus and sulphur by soil microorganisms makes these elements available for plants and either
organism. The fertility of soil has been fixing atmospheric nitrogen into nitrogen compounds used by plants to synthesize
protein and organic nitrogenous compounds. Our basic food supply depends on the trillions of microbes that exist in the
soil, degrading organic matter, recycling nitrogen, carbon, and producing new soil. The rhizosphere, area surrounding the
roots of most plants, contains a wide variety of microorganisms that help the plant to absorb minerals and nutrients, have
nodules on their roots that contain nitrogen-fixing bacteria, which take nitrogen from the air and produce nitrogen
compounds to use in synthesis of amino acids and protein. Microbes are alive, and must have nutrition to survive,
and that nutrition comes from organic matter. Microbes create foods like nitrogen, carbon, oxygen, hydrogen, phosphorus,
potassium and trace minerals for our plants. Microbes convert the NPK and minerals in the soil into plants used to grow
and produce decent quality and quantity of foliages. Insect pathogenic fungi the genera Beauveria, Conidiobolus,
Metarhizium and Paecilomyces are all commonly found in the soil (Domsch et al., 1980). For over 80 years it has
been known that there is a large discrepancy between the number of bacterial colonies that form on solid media when
soil used an inoculum and the total number of bacterial cells actually present in that same soil (Cutler, et al., Jensen,
1968 and Wellington, et al., 1997). Conns description (Conn, H. J, 1918) of the inability of microbiologists to culture
most soil microorganisms. (Hiware C.J, 2001). The absence of pure cultures or genome sequences makes it difficult to
ascertain the roles of specific microbes in soil environments, this is particularly true for bacteria in the phylum
Acidobacteria, which are broadly distributed in soils but poorly represented in culture. Microbes are everywhere, their
populations in soil are numerous as many as one billion of up to 13,000 species can reside in a single gram of soil (David
A. Zuberer, 2008). Most microbes need organic carbon to live, they get this from eating wood chips, leaves, manures and
other organic materials added to the soil. As microbes digest organic matter, they create humus which increases soil
structure, good for root penetration and development. Microbes also get some carbon from the rhizosphere (the area
immediately around plant roots) because roots give off substances the microbes can use, like sugars and amino acids and
then the microbes convert some of it back in forms the plants can use, as minerals, vitamins, nitrogen and amino acids.
Some microbes (like some bacteria and blue-green algae) are able to fix nitrogen from the air and make it available to
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plants. Some plants and trees cannot grow if deprived of specific microbes (mycorhizal fungi) around their roots.
Microbes break down contaminants and toxins, like oil spills and toluene from gasoline leaks, and action is
bioremediation and research is ongoing to select microbes that digest other toxins in our soils. Some isolates have
recently been cultured by new culture media and extended incubation periods to increase the numbers of colonies formed
isolates from plates, receiving only small inoculate and yielding only small numbers of colonies (Janssen, P. H, 2002,
Joseph, S. J, 2003 and Sait, M. P, 2002). The phylogenetic groups of bacteria are globally distributed and abundant in
terms of the contributions of individuals of those groups to total soil bacterial communities (Buckley, et al., 2002,
Hugenholtz, et al., 1998 and Rappe, et al., 2003). They lack membrane-bound organelles, and can function and
reproduce as individual cells, but often aggregate in multicellular colonies. A group of microscopic, single-celled
organisms that inhabit virtually environments, including soil, water, organic matter, and the bodies of multicellular
animals. Bacteria are distinguished in part by their morphological and genetic features, for instance, spherical, rodlike,
or spiral shapes. They also can be divided into two main groups, gram-positive or gram-negative.
Plant Nutrients
The availability and interactions of nutrients and need for assessing the availability of micro-nutrients and
their relation with soil chemical properties in the crop field soil has been emphasized (Bongale U. D. and Lingaiah,
1998). Soil micronutrients have attained special significance in recent times and organic matter being a major source of
plant micronutrients (Bongale U. D. and Lingaiah, 1997). The plants need macro and microelements for their growth
and life cycle. The Carbon, Hydrogen, Oxygen, Nitrogen, Phosphorus, Potassium, Calcium, Magnesium, Sulphur, Iron,
Manganese, Zinc, Copper and Boron are essentially required. In addition, four more elements viz. Sodium, Cobalt,
Vanadium and Silicon have also been established as essential nutrients plants. (Bose P. C. and Majumdar M. K,
1996). Zinc, Iron, Manganese, Molybdenum, Boron, Magnesium, Sulphur are essential as macronutrients for all plants.
Importance of Soil Nutrients
Nitrogen is necessary for chlorophyll synthesis and as a part of the chlorophyll molecule, is involved in
photosynthesis. Phosphorus is essential for plant growth. In agricultural soil, Phosphorus depletion as a result of
successive crop (Morel C. et al., 1995). Phosphorus is a component of ADP, ATP, DNA and various RNA. It plays a
role in photosynthesis, respiration, energy storage and transfer, cell division, cell enlargement and several other processes
in living plants. It prevents the harmful effect of excess of nitrogen in the soil (Gupta P.K, 2003). Potassium is essential
for growth and development of plants. In mulberry potassium plays in important role in various biochemical functions,
development and yield of foliage, in addition to improvement in the leaf quality (Shankar M.A. et al., 1995). Potassium is
absorbed by Plants in ionic form. It is essential for protein synthesis. Calcium stimulates root and leaf development,
microbial activity and uptake of the other nutrient. It forms compounds which is part of cell walls. It helps to reduce
nitrate-nitrogen to activate plant enzyme to neutralize organic acid in plants. It is required in the large quantities of
nitrogen fixing bacteria (Gupta P.K, 2003). Magnesium is taken up by the plant as the Mg++ cation Mg ion is the
central atom in the chlorophyll molecules, so it is actively involved in the photosynthesis. Magnesium also aids in
phosphate metabolism, plant respiration and the activation of many enzyme systems (Gupta P.K, 2003). Sulfur is a part
of every living cell and constitute of two of the 21 amino acids which form proteins. It helps in developing enzymes and
vitamins. It is necessary in chlorophyll formation, although it is not constituted of Chlorophyll. Iron is an important
constituent of many enzymes, particularly the respiratory enzymes. It is also essential for the synthesis of chlorophyll.
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The role of Boron in mulberry growth is unknown, but it appears to be involved in the translocation of the sugar and
utilization of calcium in cell wall formation. Manganese is a part of enzyme systems in plants. It activates several
metabolic reactions and plays a direct role in photosynthesis by aiding the chlorophyll synthesis. Manganese accelerates
germination and maturity while increasing the availability of P and Ca. Zn is essential for the transformation of
carbohydrates and regulates consumption of sugars and it is part of the enzyme systems which regulate plant growth.
Copper is necessary for chlorophyll formation in the plants. This is a co-factor for many oxidation enzymes
involved in respiration.

MATERIALS AND METHODS

Collection of Samples: Soil samples was collected from campus of Babasaheb Bhimrao Ambedkar University,
Lucknow, Uttar Pradesh,.Soil samples were collected in polythene bags (Akinyanju and Fadayomi, 1989). The soil was
dug out using augers up to 0-30cm depth. The samples were collected from different mulberry varieties plots namely S-1,
BR-2, AR-14, S-1635, S-146(Kumar R. V. et al., 2012).
Soil pH Determination: 10gm of soil into a 100ml beaker with 30ml of sterile distilled water the suspension was
left to stand for 30 minutes and know the pH of settled suspension.
Preparation of Media: Where taken two media such as Nutrient Agar (NA) and Potato Dextrose Agar (PDA).
hence is the most commonly used in growth media and autoclaved at 121C for 15 minutes for the cultivation of fungi and
bacterial.
Isolation of Bacteria: Each 10gm of soil samples dissolved in 90ml of sterilized water in conical flasks.
The soil suspension was diluted in using serial dilution techniques ranging from 10 -1 to 10-5 for the isolation of
bacteria. Each time the sample transferred must be thoroughly mixed with the dilution fluid before putting the next test
tube. Marked with two plates for each tube dilution on the bottom with the dilution. From each dilution tube (but not
the 10-2) place 1 ml of dilution fluid into each of two sterile petri plates. Taken a flask for Nutrient Agar from the 45C
water bath and pour nutrient agar into each petri plate for that set and placed in the incubator at room temperature for 24
hours. One ml of each dilution was spread into petri plates with the help of spreader on the nutrient agar medium. The
plates were incubated at 300C for 24 hours for bacterial isolation.
Isolation of Fungi: Further 10gm of soil samples of mulberry varieties were transferred into 90 ml in 250 ml,
and diluted in serial dilution up to 10-2 to 10-5. 100 ml dilution blank is 10-2 and the tubes sequentially are 10-3, 10-4 and
-5

-3

-4

-4

-5

10 . transferred 1ml from the 10 dilution of the 10 dilution blank, then from the 10 to the 10 . One ml of each
dilution spread into petriplates of PDA and incubated at 280C for 24 hours for fungal isolation.
Study of Microbial Population: Isolated bacterial and fungal colonies was counted by calculation of no. of
Soil microorganisms:

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Gram Staining of Bacterial Isolates: The staining procedure is as follows-( Gram, 1884) A thin film of young
culture (smear) is fixed on a clean slide. The smear is stained for one minute with ammonium oxalate crystal violet.
The slide has been washed with tap water for not more than 2 second to remove excess stain and immersed for one
minute in Lugois iodine solution. The bacteria become deeply stained and appear deep purple in colour. Slide washed
with tap water and blot-dried. The slide is gently agitated for 30 seconds in 95% ethyl alcohol and blot-dried. Gram
negative bacteria lose in this step (i.e. decolourize).however, the gram positive ones retain deep purple colour. The slide is
now counter stained for 10 seconds in the safranin solution. The slide is washed to tap water, dried and examined under
electron microscope.
Analysis of Micro and Macronutrients: Organic carbon was estimated by Walky and Black wet oxidation
method. Macronutrients namely available P (Olsens Method), K (Flame Photometer), S (Turbidometry method) and
micro nutrients, namely Zinc, Iron, Copper and Manganese were digested with di-acid mixture (perchloric acid and nitric
acid) and estimated with the aid of Atomic Absorption Spectrophotometer.

RESULTS AND DISCUSSIONS

The pH of soil samples of different mulberry varieties ranges 7.9 to 9.2 (Table 1) which indicate the alkaline
nature of soil. The pH value was alkaline in S-146 (7.9), BR-2 (8.5), S-1635 (8.9), S-1 (8.6), and AR-14 (9.2).
The degree of alkalinity was also found in S-1635, S-1, S-146, AR-14 and BR-2 mulberry varieties. Further fungi were
isolated (Table 2) the study revealed that five soil samples of different mulberry varieties were isolated from soils.
Minimum number of colonies per plate population of fungi was counted in S-146 (45), S-1635 (11), AR-14 (9) S-1 (8) and
BR-2 (6) respectively and found maximum in S-146 (45). The least number of fungi was isolated and colony was counted
from the soil sample BR-2 (6). Bacteria were isolated as such which shown in Table 2. The data concluded that the soil
samples of different mulberry varieties were taken and bacteria were isolated from soils. Minimum colony number per
plate population of bacteria was counted in AR-14 (57), S-1635 (43), BR-2 (19), S-1 (17) and S-146 (10) respectably.
The maximum number of bacteria was isolated in AR-14 (57) and the least number of bacteria was isolated and colony was
counted in S-146 (10). In soil samples S-1 and BR-2 marked decrease and observed in the number of fungi and bacteria.
Further colony counted bacterial samples was taken for gram staining to differentiate gram positive and gram negative
bacteria. The both gram positive and gram negative found in all soil samples.
The organic carbon content in all mulberry soil samples were deficient, i.e. S-1635 (0.15%), S-1(0. 39 %),
BR-2 (0. 33 %), S-146(0.40 %) and AR-14 (0. 20%) respectively. The P content mulberry soil samples were shown in
Table 3 i.e. S-1635 (19. 8 kg/ha), S-1(18.0 kg/ha), BR-2 (13.5 kg/ha), S-146 (19.8kg/ha) and AR-14 (19.1 kg/ha).
The K content in all samples was found in a balanced diet, i.e. S-1635 (190 kg/ha), S-1(145 kg/ha), BR-2 (146 kg/ha),
S-146 (179 kg/ha) and AR-14 (212 kg/ha) and maximum in soil AR-14. The S content was analyzed in all samples, i.e.
S-1635 (15ppm), S-1(14ppm), BR-2 (16ppm), S-146 (20ppm) and AR-14 (18ppm). The Zn content was deficient in AR-14
(1.23 ppm), S-1635 (0.95ppm), BR-2 (1.27ppm), S-146 (1.71ppm) where as in S-1(1.09ppm). The Fe content was
deficient in AR-14 (5.88 ppm), S-1(6.05ppm), BR-2 (8.15ppm), S-146 (7.70ppm) whereas in a mulberry variety S-1635
(11.35 ppm) it was balanced. Mn and Cu content was present in higher concentration in all the mulberry soil samples,
i.e. S-1(8.51 ppm and 1.40 ppm), S-146(12.07 ppm and 1.41 ppm), AR-14 (9.56 ppm and 1.27 ppm), S-1635 (10.28 ppm
and 1.38 ppm), and BR-2 (9.70 ppm and 1.40 ppm) respectively.

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The present study revealed that microorganisms play a significant role in the decomposition of soil nutrients
Malik and Sandhu (1973) studied the saline soils are very low in organic matter content for the reclamation of saline
soils and maintenance of soil fertility, it is essential to augment the soil with organic matter which undergoes microbial
decomposition thus increase the humus content of the soil. According to Rajankeret. et al., (2007) fungal and bacterial
organisms were isolated from saline soil in serial dilution method on PDA (Potato Dextrose Agar) and NA (Nutrient Agar)
respectively. Isolated colonies were identified by their colony characteristic, population, microscopic observations and
isolates are maintained on Potato Dextrose Agar and Nutrient Agar starts at 4C.
Soil sample S-146 was found to have the highest number of fungi per gram of sample. High soil
carbon/phosphorus/zinc/manganese/copper in comparison to others proved that optimum growth condition of soil
fungi. The result contributes in this study, microbial population in alkaline soils according to their different pH range
which are alkaline in nature. The data in the table of microbial population indicate that Bacterial colony is higher in
S-1635 and AR-14 soil sample and low in S-1, BR-2 and S-146 according to their pH range and fungal colony is higher in
S-146 and lower in S-1.
Hence there is a need to study the microbes in alkaline soil as decomposition also their application as a
biofertilizer. Which is prepares by bacteria and fungi should be helpful to reduce the alkalinity of soil by the
nutilization phenomenon because these microorganisms play a significant role in soil decomposition of organic matter.
Distribution of microbes in soil depends on several physico-chemical factors in which soil pH plays significant
role. The present study reveals that the number of bacterial population increases with increasing in soil pH excluding
sample S-146 it may be due to the highest amount of Copper. Copper plays important role in the biodegradation of soil
organic matter by fungi. Cu is supposed to have in ducive effect on Biodegradation of litter during secondary
metabolism of fungi up to a certain level. It enhances the secretion and stability of litter (lignocellulose) degrading
enzymes like ligninases, cellulose etc. Elevated level of Cu may have an inhibitory effect on the growth of bacteria, since
bacteria do not have secondary metabolism.

CONCLUSIONS AND FUTURE PROSPECTS

The study of microbial population, soil having the alkaline pH ranging from 7.9 to 9.2 was taken. Aforesaid
range of pH was found to have a profound effect on number of soil microbes per gram of sample. Soil sample AR-14 was
found to have the highest number (57) of bacterial colonies. The fungal colonies observed in the sample was
9.0 10 5, which represent that pH 9.2 proved unfavorable for growth of the soil fungi. The highest number of bacterial
colonies in sample AR-14 indicates the alkaline nature of bacteria. In soil samples S-1 and BR-2 marked
decrease was observed in the number of soil fungi and bacteria. Factors such as source of Carbon, Nitrogen, Vitamins and
trace elements also determine the rate of spore development under natural (soil) conditions (Michael and Donald, 1996
and Ivan et al., 2008). The high value of potassium (K) content was recorded in this sample which may have an
augumentary/positive effect on bacterial growth. All soil samples were characterized by the presence of both gram
positive and gram negative. Majorities of bacteria isolated from soil sample were gram negative (cocci) bacteria.
The present study concluded that soil sample high pH (9.2) and K content (212 ppm) is capable of supporting the
growth of maximum number of bacteria and soil higher concentration of Cu fovours abundant growth of soil fungi
carrying out biodegradation during their secondary metabolism.
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Index Copernicus Value (ICV): 3.0

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Table 1: PH Analysis of Different Soil Samples

Table 2: Microbial Population of Different Soil Samples

Table 3: Analysis of Micro and Macronutrients

Table 4: Gram Staining of Bacterial Isolates/Colony

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Figure 1
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Figure 2: pH of Different Soil Samples of Mulberry Varieties

Figure 3: Microbial Population in Different Soil Samples of Mulberry Varieties

Figure 4: Carbon Content in Different Soil Samples of Mulberry Varieties

Figure 5: Phosphorus Content in Different Soil Samples of Mulberry Varieties

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Yashvant Rao, Naveen Kumar Nigam, Rajan Verma, Venkatesh K. R & Anil Kumar

Figure 6: Potassium Content in Different Soil Samples of Mulberry Varieties

Figure 7: Sulphur Content in Different Soil Samples of Mulberry Varieties

Figure 8: Zinc and Iron Content in Different Soil Samples of Mulberry Varieties

Figure 9: Manganese and Copper Content in Different Soil Samples of Mulberry Varieties
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ACKNOWLEDGEMENTS
Authors are thankful to Babasaheb Bhimrao Ambedkar (Central) University of Lucknow for infrastructural
and experimental facilities for conducting this research work.

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