Rhizosphere Effect of Microorganisms on Green Gram Cultivated Soil - A Field Study
V.JeyanthiKumari1, M.Muthukani2, S.Selvam3, V.A.Leslie4
1,2Assistant Professor of Zoology, A.P.C. Mahalaxmi College for Women, Thoothukudi, Tamil Nadu, India
3 Assistant Professor of Geology,V.O. Chidambaram College, Tuticorin,Tamil Nadu, India
4 CMFRI, Kovalam, Kerala, India
ABSTRACT
The quantitative comparative analysis of the rhizosphere effect between rhizosphere soil and non-rhizosphere soil of the green gram cultivated soil was done based on R:S ratio (Root-Soil ratio). The results revealed that high rhizosphere effect was seen in the rhizosphere soil of green gram also the quantification studies explored that the rhizosphere effect among the microbes was more in bacteria than in fungi and actinomycetes during the plate count method on various media used. The result of the isolation and enumeration of rhizosphere bacterial culture by selective and decimal dilution method revealed that among the bacterial culture, the quantity of Rhizobiumsp(140x106±2.056CFU/ml) was more followed by Phosphate Solubilizing Bacteria(PSB) (89x106±3.080CFU/ml) and Cellulolytic bacteria (54x106±3.012CFU/ml). The lowest count was observed in the TCBS agar medium).
Keywords
Rhizosphere effect, green gram soil, R:S ratio, Bacteria, fungi
INTRODUCTION
Microbial communities play a pivotal role in the functioning of plants by influencing their physiology and development and are beneficial to plant growth (Rodrigo Mendes, et al.2013).
Plants are colonized by an astounding number of microorganisms that can reach cell densities much greater than the number of plant cells (Johnson and Graham, 2013).Microbial population interacts with plants through a series of complex mechanism and the interactions can be beneficial, neutral or detrimental depending upon the nature of microbiome in the plant also the microbial activity is high due to the secretion of bioactive compounds from roots (Gaurav Yadav, et al.2017).
The root microbiota provides indirect pathogen protection, and to serve additional host functions through the acquisition of nutrients from soil for plant growth. Thus, the plant microbiota emerge mutualism through diverse biochemical mechanisms by plant growth–promoting and plant health–promoting bacteria (DavideBulgarelli, et al.2013). The study of microbiomes helps in the identification of new groups involved in plant diseases from the rhizospheremicrobiome and the number of studies have revealed that many plant-associated microorganisms have profound effects on seed germination, seedling vigor, plant growth and development, nutrition, diseases, and productivity (Inceoglu, et al.2013).
The microbial community at the seedling stage was distinct from the other developmental time and the phylum such as Acidobacteria, Actinobacteria, Bacteroidetes, Cyanobacteria and specific generaare associated with plant development and root exudation (Jacqueline Chaparro, et al.2014). Recently, it was reported that the composition of root exudates can change a plant developmental gradient (Chaparro et al., 2013). Cumulative secretion levels of sugars and sugar alcohols were higher in early time points and decreased through plant growth (Badri et al., 2013).
As the plant ages it releases specific substrates and potentially antimicrobial compounds in an
Symbiotic nitrogen-fixing Rhizobium species utilize the flavonoid compounds secreted by the roots of leguminous plants and produce nod factors that induce the formation of root nodules by the plants and the bacteria in these nodules are nourished by the plants, in turn, these microbes convert nitrogen gas to nitrate that can be used by the plant(Tian, et al.2012).With this background, this study quantified the rhizosphere bacteria, fungi, and actinomycetes with non- rhizosphere region of green gram field based on R:S ratio and various selective media were used to identify the types of bacteria present in rhizosphere region.
MATERIALS AND METHODS
Sample collection
The rhizosphere and non-rhizosphere soil were collected aseptically and separately in sterile conical flasks from the green gram field of Thiruvengadam. These collected soil samples were placed in an ice container and transported immediately to the lab for further microbiological analysis
Quantification of rhizosphere and non rhizospheremicroflora
The quantity of one gram rhizosphere and non-rhizosphere soil was dissolved in 100ml of sterile water separately and were kept in a shaker for about 15 minutes. Then one ml of sample was taken and serially diluted up to 10-7 dilutions. From the respective selected dilution, one ml of sample was plated by pour plate method in the nutrient agar medium, Rose Bengal agar medium, and Glycerol yeast agar medium separately. These plates were incubated at 37o C for 48hrs for bacteria and 7 days for fungi and actinomycetes. The following selective media were used to find out which bacteria have the highest levels in the rhizosphere region.
Table 1 Selective media and rhizospherebacteria.
Name of the Hi-media Name of the Rhizosphere bacteria YEMA (Yeast Mannitol Agar) Rhizobiumsp
Bacillus M1383 Bacillus sp
Pseudomonas Isolation Agar 406 Pseudomonassp Cellulose Powder- Peptone Medium Cellulolytic sp
Hydroxy Apatite (HA) Medium Phosphate solubilizing sp KF Streptococcal Agar M248 Streptococcussp
TCBS Agar M 189 Vibrio cholera
Phenolphthalein phosphate Agar M652 Staphylococcussp
S.S.AgarM108 Salmonellasp and Shigellasp
The amount of one ml of sample was serially diluted and was plated on the above mentioned selective agar plates adopting the pour-plating technique. The plates were incubated for 24-48 hours at 370C. Bacterial outgrowth in countable plates was selected and enumerated. The bacterial populations were expressed as the number of Colony Forming Units per ml (CFU/ml).
RESULTS
Microbial counts of rhizosphere and non-rhizosphere soil colonies were counted and the R:S value was calculated by the following formula.
R:S ratio = Number of microorganisms (bacteria or fungi) in the rhizosphere soil Number of microorganisms (bacteria or fungi) in the non-rhizosphere soil
Number of microorganisms/gram of soil=Number of colonies/plate×dilution factor The dry weight of the soil taken
The results revealed that the rhizosphere soil consists of more microbial populationthan non- rhizosphere soil (Table 1) and among the microbial population bacterial population influenced (3.2×104) more than other microbial population in the soil in both rhizosphere and non- rhizosphere soil.
Table 2: Microbial Plate Counts (CFU/ml) S.No Microbes Number of colonies present
in rhizosphere soil CFU/ml
Number of colonies present in non rhizosphere soil CFU/ml
1. Bacteria 3.2×104 ± 0.07 2.2×102±0.06
2. Fungi 6.9×106 ± 0.10 5.5×104±0.09
3. Actinomycetes 3×104 ±0.02 1.8×103±0.05
Table 3: R:S ratio of rhizosphere microbes
S.No Microbes R:S ratio
1. Bacteria 66:1±0.08
2. Fungi 22:8±0.01
3. Actinomycetes 16:6±0.06
Values in Mean±Standard Deviation
Fig 1Microbial load in both rhizosphere and non-rhizosphere green gram soil
0 2 4 6 8
Bacteria
Fungi
Actinomycetes Rhizosphere microbes Non Rhizosphere microbes
DISCUSSION
The diversity of microbes associated with plant roots are enormous, in the order of tens of thousands of species (Barata, et al.2012). This statement coincided with the present investigation that the greater microbial population were observed in the rhizosphereregion than the non- rhizosphere region also the rhizosphere effect was greater in the bacteria than in the actinomycetes and fungi (Table 2). The rhizosphere effect increased with the age of the plant and normally reached its maximum at the stage of greater vegetative growth. Following the death of the plant, the microbial population reverted gradually to the level as that of the surrounding soil.
The viable nature of Rhizobium in the soil is more when compared to other soil microbes and its sustainability mainly depends on the physical and chemical nature of the soil (Chaparro, et al.
2013). This was true in this study because, during rhizosphere bacterial analysis, the quantity of Rhizobium is more when compared to other bacterial counts (Fig 3) also, the microbial load is more in the rhizosphere region compared to the count of non-rhizosphere bacterial load (Fig2).
The flourishing nature of the rhizosphere could be increased by the addition of either biofertilizer or by carrier-based inoculum in the rhizosphere region of the plant (Owen, et al. 2015).The presence of efficient phosphate solubilizing microorganisms in the rhizosphere of crops and soil increases the availability of phosphorous from an insoluble source of phosphates and make it readily available to plants (Sahu and Brahmaprakash, 2016). In this study, next to Rhizobium the concentration of PSB is more when compared to its following microbial count. Most of the phosphates solubilizing organisms are Pseudomonassp and Bacillus sp. Rhizosphere effect not only enhances the microbial population but also acted as antierodibility by improving soil physical processes such as aggregate stability (Zhenhong, et al.2020). Fromthis study, it is concludedthat the plants and microorganisms release exudates which improve the soil environment and provide food for animalsandmicrobes in the soil.
Rhizobium sp 31%
PSB 20%
Cellulolytic sp 12%
Pseudomonas sp 11%
Bacillus sp 9%
Streptococus sp 7%
Staphylococcus sp
5% Salmonella sp 3%
Shigella sp 2%
Vibrio sp 0%
Acknowledgment: I would like to thank the Secretary, Director, and Principal of A.P.C.Mahalaxmi College for women for providing facilities and their constant encouragement to carry out this research study.
Funding: None
Data availability: All datasets and statistical report analyses during this study are included in the manuscript.
Ethics Statement: This article does not contain any studies with human participants or animals.
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