Rhizobia are vital for nitrogen input, fertility of soil and legume plant growth. Knowledge on rhizobial diversity from arid and semiarid areas is important for dry land agriculture in the context of climatic change and for economic utilization. This study provides morphological, biochemical, stress tolerance and plant growth promoting characteristics of fifteen rhizobial isolates from the nodules of same number of wild legumes and one isolate from cultivated Arachis hypogea from semi-arid region, Tirupati. The bacterial isolates were confirmed as rhizobia based on colony morphology and biochemical tests. Based on the colour change of YMA-BTB medium, eight isolates were identified as slow growers and six were fast growers. The isolates differed in growth pattern, colony morphology, antibiotic resistance at higher concentrations and uniformity in utilization of carbon and nitrogen sources. The isolates are tolerant to NaCl up to one percent, displayed normal growth at temperatures 28 ℃ - 30 ℃ , at neutral pH and poor growth at pH 5and 9. The isolates varied in the production of EPS and IAA, positive for phosphate solubilization and siderophore formation. This functional diversity displayed by the isolates can be utilised for the legume crop production by cross inoculation.
Symbiotic nitrogen fixation, involving legumes and rhizobia is of considerable economic and ecological significance. Rhizobia are a group of soil bacterial species that infect leguminous plants leading to the formation of special type of organs called nodules, where nitrogen fixation takes place. The nitrogenase enzyme complex of bacterioids i.e. symbiotic form of rhizobia supplies constant source of reduced nitrogen to the legume host and intern, receives the nutrients and energy. This symbiotic process occurs on all continents and accounts for a fourth of the nitrogen fixed annually on earth [
The nitrogen fixing genes are located in Rhizobia and symbiotic establishment and regulation of nitrogenase activity in the nodules depends on the genes of host plant. Host plant genotype compatibility and differences in nitrogen fixation potential of Rhizobial strains often results in variation in symbiotic effectiveness [
Rhizobium is also a plant growth promoting rhizobacteria (PGPR) exhibit a variety of characteristics responsible for influencing plant growth and performance. As a symbiotic partner in addition to nitrogen supply, Rhiz- obium also improves nutritional uptake by promoting the growth of plant root system through production of Indole acetic acid (IAA) [
Legume crops, which provide dietary proteins, fats and fodder, are preferred in semi-arid agriculture system due to their inherent adaptation to the existing characteristic ecological features and symbiotic nitrogen fixation potential. Climatic changes experienced during the recent years further aggravating ecological problems of arid and semiarid legume cropping areas leading to prolonged dry season, rise in temperature, increase in soil salinity and soil pH alteration. These typical environmental stresses are problematic for the establishment of effective symbiosis and survival of rhizobia in the soil [
Genetic variability for the environmental stress factors in the commercial inoculants/strains and Rhizobial populations in the agriculture fields most often very limited due to monoculture of legume crops and competition between Rhizobial populations in the soil favours colonization of certain genotypes and co-evolution of symbiotic partners.
Enormous diversity exists in rhizobia due to their wide geographical distribution, different plant hosts and niches they occupy all over the globe [
Nodulated wild legumes with their biodiversity and adaptation to arid/semi-arid climatic conditions are vital to the soil fertility and nitrogen cycle [
There were few reports on phenotypic characterization of rhizobia from wild legumes of arid/semiarid regions viz, Rajasthan, India [
The knowledge on genetic and functional diversity of root-nodule bacteria associated with wild legume flora of arid and semiarid ecosystems of the continents is largely unexplored. This study aimed at evaluating the phenotypic diversity, stress tolerance and plant growth promoting characteristics of rhizobial isolates from wild legumes of semiarid habitat, Tirupati, India.
Isolation of rhizobia: Rhizobial isolates from fifteen nodulating native wild legumes were selected to study morphological, biochemical and plant growth promoting characters. Wild legume hosts include Albizia lebbeck, Alysicarpus monilifer, Chamaecrista absus, Cassia tora, Clitoria ternatea, Crotalaria hebecarpa, Desmodium trifolium, Indigofera linnaei, Indigofera mysorensis, Indigofera trifoliata, Millettia pinnata, Mimosa pudica, Rhynchosia minima, Tephrosia purpurea and Tephrosia tinctoria and cultivated Arachis hypogea for comparison. From carefully uprooted roots, soil was removed by gentle washing with tap water, separated from the stem and placed in polythene covers, kept in cool box and brought to the laboratory. By leaving small piece of root above and below the nodules, roots were cut. Root pieces containing nodules were first subjected to surface sterilization with 0.01% HgCl2 for 30 sec. Traces of HgCl2 were removed by repeated washings with sterile distilled water and then incubated in 70% ethanol for 3 min and final washings were made with sterile distilled water for 3 times. Randomly selected ten nodules were separated from root part of each species, crushed with sterile glass rod and squeezed for white coloured milky secretion into 0.5 ml of sterile YEM broth. From this suspension loopful of inoculum was streaked onto YEMA medium (Composition per litre: Yeast extract 1 gr, Mannitol 10 gr, Dipotassium phosphate 0.5 gr, Magnesium sulphate 0.2 gr, Sodium chloride 0.1 gr, Agar 20 gr, pH 6.8) containing 0.0025% Congo red [
Morphological and Biochemical characterization: Isolates were characterized for colony and cell morphology, biochemical characteristics includes (Melanin production test, Ammonia production test, Phosphate solubilisation test, Organic acid production test, Oxidase test, Catalase test, Indole production, Gelatin hydrolysis, Starch hydrolysis) was carriedout following standard methods described by Somasegaran and Hoben [
Statistical and Cluster analysis: Cluster analysis was performed for rhizobial isolates. Analysis of variance and correlation coefficient was calculated using SPSS version 12 statistical software which was used to estimate the simple matching similarity coefficient (Ssm) of each strain and to generate a similarity matrix [
Study site: The study site, S. V. University campus is located in Tirupati (13˚62'N and 79˚40'E) at foothills of Seshachalam (Tirumala) hill range covering nearly 200 acres (
Morphological characteristics: Bacteria that form nitrogen-fixing nodules in the roots of legumes have different growth rates, morphologies, biosynthetic pathways, catabolic capabilities and habitats depending on host species, edaphic and climatic conditions of habitats. Generally morphological features of the colony i.e, form, elevation and margins vary with rhizobia species/strain [
Morphology of the colony formed by Cte, Tpu, and Tti isolates was irregular and the colonies of remaining isolates were regular. The colony elevation was flat in Che, Itr, Imy and Tti, raised in Cab, Cto, Cte, Ili, Rmi and Tpu, and convex in the remaining isolates. Margin of the colony was lobate in Cte, Che, Rmi and Tpu and in the remaining 11 isolates it was entire. The circular and irregular colonies are in agreement with characteristic colony forms of rhizobia [
S. No. | Isolate | Cultural characteristics | |||
---|---|---|---|---|---|
Form | Elevation | Margin | Growth rate | ||
1 | Albizia lebbeck (Ale) | Circular | Convex | Entire | Slow |
2 | Alysicarpus monilifer (Amo) | Circular | Convex | Entire | Fast |
3 | Arachis hypogea (Ahy) | Circular | Convex | Entire | Fast |
4 | Chamaecrista absus (Cab) | Circular | Raised | Entire | Slow |
5 | Cassia tora (Cto) | Circular | Raised | Entire | Slow |
6 | Clitoria ternatea (Cte) | Irregular | Raised | Lobate | Fast |
7 | Crotalaria hebecarpa (Che) | Circular | Flat | Lobate | Fast |
8 | Desmodium trifolium (Dtr) | Circular | Convex | Entire | Fast |
9 | Indigofera linnaei (Ili) | Circular | Raised | Entire | Slow |
10 | Indigofera mysorensis (Imy) | Circular | Flat | Entire | Slow |
11 | Indigofera trifoliata (Itr) | Circular | Flat | Entire | Slow |
12 | Millettia pinnata (Mpi) | Circular | Convex | Entire | Fast |
13 | Mimosa pudica (Mpu) | Circular | Convex | Entire | Fast |
14 | Rhynchosia minima (Rmi) | Circular | Raised | Lobate | Fast |
15 | Tephrosia purpurea (Tpu) | Irregular | Raised | Lobate | Slow |
16 | Tephrosia tinctoria (Tti) | Irregular | Flat | Entire | Slow |
Note: All isolates exhibit characteristic colony colour creamy on YEMA, white on YEMA with Congo red, blue and yellow on YEMA with BTB. Mucose in appearance, aerobic and non-spore forming in nature.
blue and yellow (
When the light transmittency of the colony is considered, it is transparent in Cab, Cto, Dtr, Imy, Rmi, Tpu and Tti, and opaque in the remaining isolates. All isolates were aerobic, rod shaped, gram negative and motile. The discriminative power of colony morphology characters in revealing genetic diversity among the isolates was realised when the combination of four characters (form, elevation, margin and opacity) were considered. The isolates formed into 11 groups of which 9 were loners; one group represented by 5 isolates and another with 2 isolates.
Biochemical characters: The isolates are positive to Ammonia, Catalase, Indole, Melanin, Oxidase and H2S production tests. Further, isolates are also positive to MR-VP test, Starch hydrolysis, and negative to gelatin hydrolysis test (
Phenotypic characteristics: Several techniques for characterization, identification and assessment of metabolic diversity of strains have been developed, such as Carbon, Nitrogen sources utilization and Intrinsic Antibiotic Resistance pattern. Of 11 sugars tested all the isolates were able to utilise fructose, galactose, glucose, mannose, sucrose, starch, succinate, rhamnose, malate, citrate and they fail to utilise dulcitol as carbon source (
Different rhizobial strains show different degrees of susceptibility to antibiotics due to genetic variation in target genes and resistance genes acquired through horizontal gene transfer. Many researchers emphasized the significance of intrinsic resistance as a phenotypic character for discrimination and identification rhizobial strains [
S. No. | Isolate | Biochemical characterization | |||||||||
---|---|---|---|---|---|---|---|---|---|---|---|
Ammonia production | Catalase production | Gelatin hydrolysis | H2S production | Indole production | Melanin production | Methyl red test | Oxidase production | Starch hydrolysis | Vogesproskauer test | ||
1 | Ale | + | + | - | + | + | + | + | + | + | + |
2 | Amo | + | + | - | + | + | + | + | + | + | + |
3 | Ahy | + | + | - | + | + | + | + | + | + | + |
4 | Cab | + | + | - | + | + | + | + | + | + | + |
5 | Cto | + | + | - | + | + | + | + | + | + | + |
6 | Cte | + | + | - | + | + | + | + | + | + | + |
7 | Che | + | + | - | + | + | + | + | + | + | + |
8 | Dtr | + | + | - | + | + | + | + | + | + | + |
9 | Ili | + | + | - | + | + | + | + | + | + | + |
10 | Imy | + | + | - | + | + | + | + | + | + | + |
11 | Itr | + | + | - | + | + | + | + | + | + | + |
12 | Mpi | + | + | - | + | + | + | + | + | + | + |
13 | Mpu | + | + | - | + | + | + | + | + | + | + |
14 | Rmi | + | + | - | + | + | + | + | + | + | + |
15 | Tpu | + | + | - | + | + | + | + | + | + | + |
16 | Tti | + | + | - | + | + | + | + | + | + | + |
+ Indicates positive-indicates negative.
Characteristic | Ale | Amo | Ahy | Cab | Cto | Cte | Che | Tti | Dtr | Ili | Imy | Tpu | Itr | Mpi | Mpu | Rmi | ||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Carbon sources | ||||||||||||||||||
Citrate | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | ||
Dulcitol | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | ||
Fructose | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | ||
Galactose | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | ||
Glucose | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | ||
Malate | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | ||
Mannitol | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | ||
Rhamnose | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | ||
Starch | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | ||
Succinate | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | ||
Sucrose | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | ||
Nitrogen sources | ||||||||||||||||||
Ammonium chloride | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | ||
Asparagine | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | ||
Casein hydrolysate | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | ||
Glycine | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | ||
Glutamine | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | ||
Potassium nitrate | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | ||
Tryptophan | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | ||
+ Indicates positive-indicates negative.
A total of 9 known antibiotics were employed to know the resistance/susceptibility of the isolates (
Salt tolerance: One of the major problems in semi-arid regions is increase in the salinity levels of the soil. Application of salinity tolerant rhizobia in legume cropping area helps in the formation of effective nodules and efficient nitrogen fixation. Symbiotic effectiveness depends on the specific combination of compatible legume and rhizobium under the saline conditions [
Antibiotic (Concentration in µg∙ml−1) | Ale | Amo | Ahy | Cab | Cto | Cte | Che | Dtr | Ili | Imy | Itr | Mpi | Mpu | Rmi | Tpu | Tti |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Carbenicillin | ||||||||||||||||
250 | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + |
350 | + | - | - | + | + | + | + | + | - | - | - | - | - | - | + | + |
500 | - | - | - | + | - | - | - | - | - | - | - | - | - | - | - | + |
Chloramphenicol | ||||||||||||||||
300 | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + |
500 | + | - | - | + | - | - | - | - | - | - | - | - | - | - | - | + |
Erythromycin | ||||||||||||||||
150 | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + |
250 | + | - | - | + | - | - | - | - | - | - | - | - | - | - | - | + |
Nalidixicacid | ||||||||||||||||
50 | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + |
75 | + | - | - | + | - | - | - | - | - | - | - | - | - | - | - | + |
Rifampicin | ||||||||||||||||
300 | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + |
500 | + | - | - | + | - | - | - | - | - | - | - | - | - | - | - | + |
Streptomycin | ||||||||||||||||
75 | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + |
100 | - | - | - | + | - | - | - | - | - | - | - | - | - | - | - | + |
Tetracycline | ||||||||||||||||
50 | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + |
100 | + | - | - | + | - | - | - | - | - | - | - | - | - | - | - | + |
Trimethoprim | ||||||||||||||||
50 | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + |
75 | + | - | - | + | - | - | - | - | - | - | - | - | - | - | - | + |
Vanomycin | ||||||||||||||||
50 | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + |
75 | + | - | - | + | + | + | + | + | - | - | - | - | - | - | + | + |
100 | - | - | - | + | - | - | - | - | - | - | - | - | - | - | - | + |
+ Indicates positive-indicates negative.
was decreased thereafter with increase in concentration upto 3.5% and no growth was observed in 4.5%. Decrease in growth of rhizobial isolates with increased salt concentration was also reported by Ali et al. [
Temperature tolerance: The growth and survival of rhizobia in soils are adversely affected by high soil temperatures [
Characteristic | Ale | Amo | Ahy | Cab | Cto | Cte | Che | Dtr | Ili | Imy | Itr | Mpi | Mpu | Rmi | Tpu | Tti |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Growth at Temperature | ||||||||||||||||
(ºC) | ||||||||||||||||
28 | ++ | ++ | ++ | ++ | ++ | ++ | ++ | ++ | ++ | ++ | ++ | ++ | ++ | ++ | ++ | ++ |
33 | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + |
38 | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + |
43 | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + |
48 | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - |
Growth at pH | ||||||||||||||||
5 | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - |
6 | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + |
7 | ++ | ++ | ++ | ++ | ++ | ++ | ++ | ++ | ++ | ++ | ++ | ++ | ++ | ++ | ++ | ++ |
8 | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + |
9 | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - |
Growth at Salinity | ||||||||||||||||
(%) | ||||||||||||||||
0.5 | ++ | ++ | ++ | ++ | ++ | ++ | ++ | ++ | ++ | ++ | ++ | ++ | ++ | ++ | ++ | ++ |
1.5 | ++ | ++ | ++ | |||||||||||||
2.5 | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + |
3.5 | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + |
4.5 | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - |
+ Indicates positive-indicates negative.
growth at 28˚C, moderate growth at 33˚C and 38˚C, poor growth at 23˚C and 43˚C (
PH tolerance: Soil pH influences the growth and survival of rhizobia through alteration in the permeability of the membrane and uptake of nutrients. Neutral pH allows the uptake of appropriate amount of nutrients and results in optimum growth of rhizobia. At neutral pH all the isolates showed maximum growth, the growth was decreased with increase in acidity or alkalinity (
Rhizobia in addition to the nitrogen supply, they promote growth of the plant as a symbiotic partner and soil microbe in a number of ways. Plant growth could be induced by rhizobia through some of their growth stimulating mechanisms such as mobilization of nutrient, enhancement in stress resistance, solubilization of phosphates, production of phytohormones and siderophores [
Rhizobial isolates varied in IAA production, a maximum of, 27 µg/ml and a minimum of 6 µg/ml produced by Cte and Ale isolates respectively (
S. No. | Isolate | Siderophore production test | Phosphate solubilization test | IAA (µg/ml) | EPS (µg/ml) |
---|---|---|---|---|---|
1 | Ale | + | + | 06a ± 1.67 | 63a ± 5.59 |
2 | Amo | + | + | 15b ± 1.16 | 38b ± 3.34 |
3 | Ahy | + | + | 19c ± 0.55 | 43b, d ± 3.74 |
4 | Cab | + | + | 12b ± 1.94 | 81c ± 3.08 |
5 | Cto | + | + | 13b ± 1.55 | 55a ± 4.64 |
6 | Cte | + | + | 27d ± 1.41 | 59a ± 3.88 |
7 | Che | + | + | 18c ± 1.41 | 56a ± 5.98 |
8 | Dtr | + | + | 20c ± 1.47 | 58a ± 3.45 |
9 | Ili | + | + | 18c ± 0.89 | 44b, d ± 3.94 |
10 | Imy | + | + | 21e ± 0.98 | 42b, d ± 5.06 |
11 | Itr | + | + | 17e ± 0.76 | 48d ± 3.45 |
12 | Mpi | + | + | 22e ± 1.16 | 34b ± 3.35 |
13 | Mpu | + | + | 23e ± 1.09 | 23e ± 4.18 |
14 | Rmi | + | + | 13b ± 0.81 | 27e ± 3.03 |
15 | Tpu | + | + | 09f ± 0.83 | 68a ± 3.41 |
16 | Tti | + | + | 08a, f ± 0.75 | 91f ± 3.74 |
Means are value of six replications; Means followed by the same letter in a column are not significantly different but by different letters are significantly different (p = 0.05). + indicates positive.
produced more than 10 µg/ml of IAA and 5 strains produced more than 20 µg/ml. The range of IAA produced by these isolates is in agreement with soybean isolates [
Variation in the production of IAA by different rhizobial species/strains and influence of cultural conditions on the amount of IAA produced reported by many workers [
Polysaccharide production is a marked characteristic of rhizobia and involved in infection process and nodule formation. Polysaccharides also protect rhizobia in the soil against deleterious biotic and abiotic stress factors [
Cluster analysis: Cluster analysis including colony morphology, biochemical characteristics, IAA and EPS production features, the 16 isolates resolved into five clusters at a distance of 0.205 (
lates from the legume hosts belonged to same genus.
The rhizobial isolates showed variation in colony morphological features including form, elevation, margin and opacity, and Intrinsic Antibiotic Resistance. The isolates tolerated moderate level of salinity, temperature and showed optimum growth at neutral pH. Majority of rhizobia are significantly high IAA producers, and all isolates displayed other plant growth promoting EPS, siderophore production and phosphate solubilisation. Molecular characterization for the species/strain identification is in progress. Functional diversity of isolates can be utilised for the improvement of nitrogen fixation in legume crops through cross inoculation.
Y.Bhargava,J. S. R.Murthy,T. V. RajeshKumar,M. NarayanaRao, (2016) Phenotypic, Stress Tolerance and Plant Growth Promoting Characteristics of Rhizobial Isolates from Selected Wild Legumes of Semiarid Region, Tirupati, India. Advances in Microbiology,06,1-12. doi: 10.4236/aim.2016.61001