on black gram and green gram plants

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1 INT J CURR SCI 2013, 9: E 1-6 RESEARCH ARTICLE ISSN Biochemical characterization of Rhizobium and its impact Abstract on black gram and green gram plants Lalitha S a * and Sam Immanuel P b a Department of Botany, Nirmala College, Coimbatore , India b Department of Microbiology, Karpagam University, Coimbatore , India *Corresponding author: samimmanuel87@yahoo.com, lara9k@gmail.com An isolation study of Rhizobium was carried out in rhizosphere soil of leguminous plants grown in various parts of Coimbatore. Four isolates (AhV01, VmV01, VmG02 and VrV01) were isolated and their colony morphology was recorded. All of them were positive for catalase, amylase, oxidase and nitrate reductase tests. These isolates variably showed resistance to various antibiotics in antibiotic susceptibility testing. On the basis of these traits, all the four isolates were selected for an investigation of their growth promoting potential on black gram (Vigna mungo) and green gram (Vigna radiata) in a pot experiment. Plant biomass, total nitrogen content and total chlorophyll content were estimated. Soil NPK content and enzymes were also determined. VmG02 and VrV01 showed better performance compared to other isolates. Keywords: Catalase, amylase, oxidase, nitrate reductase, antibiotic susceptibility testing Received: 20 th Nov 2012; Revised: 26 th January 2013; Accepted: 04 th April; IJCS New Liberty Group 2013 Introduction Black gram (Vigna mungo) is the third important pulse crop in India. It is annual pulse crop and native to central Asia. It is also extensively grown in West Indies, Japan and other tropics/subtropical countries. Black gram seeds are highly nutritious containing higher amount of protein (24-26%) and are reported to be rich in potassium, phosphorus and calcium with good amount of sodium. Green gram (Vigna radiata L.) Wilczek (syn: Phaseolus aureus Roxb.) constitutes the important group of grain legumes which form a major source of dietary proteins of high biological value, energy, minerals and vitamins (Taylor et al., 2005). Inoculation of Rhizobium sp. causes a greater increase in growth and yield and the number of nodules per root system is significantly higher in plants inoculated with Rhizobium sp. compared to plants without Rhizobium sp. under field condition (Akhtar et al., 2009). Soil microorganisms produce quite a number of extra cellular enzymes to decompose the complex organic matter before it is absorbed as a source of energy. Seasonal variations in enzymes activities in forest soils are seen to bear correlation with the counts of fungi and bacteria (Kathiresan and Selvam, 2006; Richard et al., 2007). The present study is to isolate and characterise Rhizobium from leguminous plants and induce the growth of black and green gram by inoculation of Rhizobium as bio-fertilizer. Materials and Methods Isolation of Rhizobium from root nodules The root nodules were collected from Arachis hypogaea, Vigna mungo and Vigna radiata at various places in Coimbatore. Rhizobium was isolated from root

2 Length of the plant (cm) Lalitha and Sam Immanuel, 2013 nodules (Vincent, 1970). The isolates were subjected to various biochemical and antibiotic susceptibility tests. Plant infection and inoculation with Rhizobium Seeds of V. mungo and V. radiata were obtained from the Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu surface sterilized with 0.1% HgCl 2 and sown in earthen pots containing garden soil and sand (2:1 ratio w/w). Plant growth conditions and rhizobia inoculation were as described by Rajagopalan and Raju (1972). The plants were watered with sterile tap water and harvested at 90 th day. Post-harvest works deaminase, lysine decarboxylase and hydrogen sulphide production (Table 1). These results are in agreement with the report of Graham and Parker (1964) and show that the isolates are more related to fast-growing rhizobia. Antibiotic resistance in microbes has been successfully used to screen the competitive ability of elite strains with the indigenous strains (Brockwell et al., 1976) (Table 2). All the isolates of this study were of different identity, since each isolate had a unique innate antibiotic resistance pattern. Fig. 1. Shoot and root length of Vigna mungo 25 The chlorophyll content of leaf tissue was estimated following the method of Arnon (1949). Shoot and root length, fresh and dry matter yield (plant materials dried to constant weight) and total nitrogen content by micro-kjeldahl method (Umbriet et al., 1972). Total soil nitrogen (Subbiah and Asija, 1956), soil phosphorus (Olsen et al., 1954) and soil potassium SHOOT (in cm) ROOT (in cm) (Jackson, 1973), total bacterial and fungal population in Fig. 2. Shoot and root length of Vigna radiate soil by dilution technique and assay of amylase, chitinase (Skujins, 1976), dehydrogenase (Casida et al., 1964), phosphatase (Tabatabai and Bremner, 1969) and protease (Nannipieri et al., 1980) in the soil were carried out. Statistical analysis All experiments were performed in duplicate and the standard error of the mean values was calculated. The means were tested according to Students t-test for significant differences among the samples. A statistical significance was accepted at P< Results All the isolates were positive to catalase, amylase, and oxidase and nitrate reductase activity but negative to gelatinase, urease, caseinase, lipase, phenylalanine Discussion Four isolates are identified and subjected to biochemical analysis. Biochemical characteristics among the four selected isolates indicated that they were closely related to Rhizobium species. Antibiotic susceptibility test showed that isolates are resistant to most of the

3 Table 1. Biochemical characterization of groundnut, blackgram, green gram Rhizobium isolates; + Positive reaction; - Negative reaction Characteristics AhV01 VmV01 VmG02 VrV01 Gram staining Motility IMViC test Indole production Methyl red test Voges Proskauer Citrate utilisation Acid production in YEM broth Extra-cellular enzymes Catalase Amylase Gelatinase Oxidase Nitrate reductase Urease Caseinase Lipase Phenylalanine deaminase Lysine decarboxylase Growth at different temperatures 28 C 37 C 45 C 60 C Growth in different ph ph, 4 ph, 5 ph, 6 ph, 7 ph, 8 ph, 9 ph, Growth in different carbon source Mannitol Sucrose Dextrose Lactose Arabinose Maltose Salt tolerance (NaCl) 1% 2% 5% 10% H 2 S Produce antibiotics. Two isolates, VmG02 and VrV01, showed significant results. Total chlorophyll content in leaves of inoculated plants was higher than the uninoculated control plants. Two isolates, VmG02 and VrV01, showed insignificant difference between each other in fresh weight, dry weight, shoot and root length (Tables 3 & 5). Table 2. Antibiotic susceptibility test Antibiotic used AhV01 VmV01 VmG02 VrV01 Bacitracin R R R R Cephalexin R R R R Methicillin R R R R Metronidazole R R R R Nalidixic acid R R R R Nitrofurantonin S S S S Rifampicin R R R R Vancomycin I I I I R - Resistant; I - Intermittent; S - Sensitive Microbial inoculation induced significant changes in soil characteristics. Inoculation in black gram and green gram significantly enhanced the N (180, 170 mg/kg soil), P (6, 8.2 mg/kg soil) content of the soil and K (171, 188 mg/kg soil). However, the total bacterial (2.9 X 10 7, 3.8 X 10 7 cfu/ g soil) and fungal (3.2 X 10 5, 2.6 X 10 5 cfu/g soil) population in soil increased substantially (Tables 4, 6). The activities of amylase, protease, chitinase, dehydrogenase and phosphatase in the soil also increased significantly following microbial inoculation. For amylase and protease, the increase was much pronounced in VrV01 strain, followed by VmG02 strain. For phosphatase, dehydrogenase and chitinase, the increase was less pronounced with Rhizobium inoculation, since a positive correlation has been established between soil enzymes and soil microbial biomass (Tabatabai, 1994). Conclusion In conclusion, inoculation of black gram and green gram with Rhizobium, enhanced plant growth by

4 providing a balanced nutrient supply due to their beneficial association with root system of the host plant. Table 3. Effect of bioinoculants on fresh weight, dry weight, total nitrogen content and total chlorophyll content of Vigna mungo at 90 DAI Treatments Control AhV01 VmV01 VmG02 VrV01 Fresh weight (g/plant) 0.234± ± ± ± ±0.34 Dry weight (g/plant) 0.062± ± ± ± ±0.4 Total nitrogen (mg N/g dry 6.1± ± ± ± ±0.82 wt.) Total chlorophyll (mg chl./g fresh leaf) 1.42± ± ± ± ±1.33 Table 4. Impact on NPK content and soil enzymes by bioinoculants of Vigna mungo at 90 DAI Treatments Parameters Control AhV01 VmV01 VmG02 VrV01 Soil N (mg/kg soil) 91± ± ± ± ±4.7 Soil P (mg/kg soil) `3.6± ± ± ± ±3.2 Soil K (mg/kg soil) 34± ± ± ± ±0.86 Bacteria (cfu/g soil) Fungi (cfu / g soil) Soil amylase (μg starch degraded / h / g soil) Soil alkaline phosphatase (μg PNP formed / h/ g soil) Soil chitinase (μg glucose liberated/ h / g soil) Soil protease (μg amino acid released / h / g soil) Soil dehydrogenase (μg TNF formed / day / g soil) 1827± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±26.4 Table 5. Effect of bioinoculants on fresh weight, dry weight, total nitrogen content and total chlorophyll content of Vigna radiata at 90 DAI Treatments Control AhV01 VmV01 VmG02 VrV01 Fresh weight (g/plant) 0.224± ± ± ± ±0.84 Dry weight (g/plant) 0.023± ± ± ± ±0.34 Total nitrogen (mg N/g dry wt.) 5.1± ± ± ± ±0.66 Total chlorophyll (mg chl./g fresh leaf) 0.8± ± ± ± ±1.22 Table 6. Impact on NPK content and soil enzymes by bioinoculants of Vigna radiata at 90 DAI Treatments Parameters Control AhV01 VmV01 VmG02 VrV01 Soil N (mg/kg soil) 84±5.7 97± ± ± ±3.7 Soil P (mg/kg soil) 2.2± ± ± ± ±5.6 Soil K (mg/kg soil) 48±1.5 56± ± ± ±5.3 Bacteria (cfu/g soil)

5 Fungi (cfu / g soil) Soil amylase (μg starch degraded / h / g soil) Soil alkaline phosphatase (μg PNP formed / h/ g soil) Soil chitinase (μg glucose liberated/ h / g soil) Soil protease (μg amino acid released / h / g soil) Soil dehydrogenase (μg TNF formed / day / g soil) 2107± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±52.4 ± - Standard deviation References Taylor SR, Weaver BD, Wood WC, van Edzard S (2005). Nitrogen application increases yield and early dry matter accumulation in late-planted soybean crop. Sci J 45: Akhtar MS, Siddiqui ZA (2009). Use of plant growthpromoting rhizobacteria for the biocontrol of rootrot disease complex of chickpea. Australian Plant Pathology 38 (Suppl 1): Kathiresan K, Selvam MM (2006). Evaluation of beneficial bacteria from mangrove soil. Botanica Marina 49(1): Richard W, Brenda, LB, John CK, Teri J, Balser C., (2007). Soil microbial communities and extracellular enzyme activity in the New Jersey Pinelands. Soil Bio Biochem 39: Vincent JM (1970). A manual for the practical study of the Root Nodule Bacteria. IBP Hand Book No. 15. Blackwell scientific publications, Oxford, UK. Rajagopalan N, Raju PN (1972). The influence in infection by Dolichos enation mosaic virus on nodulation and nitrogen fixation by field bean (Dolichos lablab) Phytopath. Z 73: Arnon DI (1949). Copper enzymes in isolated chloroplasts, polyphenol oxidase in Beta vulgaris. Plant Physiol 24: Umbriet WW, RH Burris, JF Stauffer (1972). Method for nitrogen. In: Manometric and Biochemical techniques, 5 th edn, Burgess Publishing Company, Minnesota, USA, pp Subbiah BV, Asija GL (1956). A rapid method for the estimation of available nitrogen in soils. Curr Sci 25: Olsen SR, Cole CV, Watanabe FS, Dean LA (1954). Estimation of available phosphorus in soils extraction with sodium bicarbonate. USDA Circ Jackson ML (1973). Soil chemical analysis. Prentice- Hall India Pvt. ltd., New Delhi. India pp Skujins J (1976). Extracellular enzymes in soil. Critical Reviews in Microbiology 4: Casida LE Jr, Klein DA, Santoro T (1964). Soil dehydrogenase activity. Soil Sci 98: Tabatabai MA, Bremner JM (1969). Use of p-nitrophenyl phosphate for assay of soil phosphatase activity. Soil Biol Biochem 1: Nannipieri P, Ceccanti B, Cervelli S, Matarese E (1980). Extraction of phosphatase, urease, protease, organic carbon and nitrogen from soil. Soil Science Society of America Journal 44:

6 Graham PH, Parker CA (1964). Diagnostic features in the characterization of the root nodule bacteria of legumes. Plant Soil 20: Brockwell J, Schwingtiamer EA, Gault RR (1976). Ecological studies of root nodule bacteria introduced into the field environment. Soil Biol. Biochem 9: Tabatabai MA (1994). Soil enzymes. In: Methods of soil Analysis Part 2. Microbiological and Biochemical properties. Ed : Weaver RW, Angle S, Bezdicek D, Smith S, Tabatabai, MA, Wollum, A Soil Science society of America Book Series, No.5. pp