Co-inoculation of biochar and arbuscular mycorrhizae for growth promotion and nutrient fortification in soybean under drought conditions.

Drought is significant abiotic stress that affects the development and yield of many crops. The present study is to investigate the effect of arbuscular mycorrhizal fungi (AMF) and biochar on root morphological traits, growth, and physiological traits in soybean under water stress. Impact of AMF and biochar on development and root morphological traits in soybean and AMF spores number and the soil enzymes' activities were studied under drought conditions. After 40 days, plant growth parameters were measured. Drought stress negatively affected soybean growth, root parameters, physiological traits, microbial biomass, and soil enzyme activities. Biochar and AMF individually increase significantly plant growth (plant height, root dry weight, and nodule number), root parameters such as root diameter, root surface area, total root length, root volume, and projected area, total chlorophyll content, and nitrogen content in soybean over to control in water stress. In drought conditions, dual applications of AMF and biochar significantly enhanced shoot and root growth parameters, total chlorophyll, and nitrogen contents in soybean than control. Combined with biochar and AMF positively affects AMF spores number, microbial biomass, and soil enzyme activities in water stress conditions. In drought stress, dual applications of biochar and AMF increase microbial biomass by 28.3%, AMF spores number by 52.0%, alkaline phosphomonoesterase by 45.9%, dehydrogenase by 46.5%, and fluorescein diacetate by 52.2%, activities. The combined application of biochar and AMF enhance growth, root parameters in soybean and soil enzyme activities, and water stress tolerance. Dual applications with biochar and AMF benefit soybean cultivation under water stress conditions.

Biochar and Arbuscular mycorrhizal fungi mediated enhanced drought tolerance in Okra (Abelmoschus esculentus) plant growth, root morphological traits and physiological properties.

Drought is a major abiotic factor limiting plant growth and crop production. There is limited information on effect of interaction between biochar and Arbuscular mycorrhizal fungi (AMF) on okra growth, root morphological traits and soil enzyme activities under drought stress. We studied the influence of biochar and AMF on the growth of Okra (Abelmoschus esculentus) in pot experiments in a net house under drought condition. The results showed that the biochar treatment significantly increased plant growth (the plant height by 14.2%, root dry weight by 30.0%) and root morphological traits (projected area by 22.3% and root diameter by 22.7%) under drought stress. In drought stress, biochar treatment significantly enhanced the chlorophyll 'a' content by 32.7%, the AMF spore number by 22.8% and the microbial biomass as compared to the control. Plant growth parameters such as plant height, shoot and root dry weights significantly increased by AMF alone, by 16.6%, 21.0% and 40.0% respectively under drought condition. Other plant biometrics viz: the total root length, the root volume, the projected area and root diameter improved significantly with the application of AMF alone by 38.3%, 60.0%,16.8% and 15.9% respectively as compared with control. Compared to the control, AMF treatment alone significantly enhanced the total chlorophyll content by 36.6%, the AMF spore number by 39.0% and the microbial biomass by 29.0% under drought condition. However, the highest values of plant growth parameters (plant height, shoot dry weight, root dry weight) and root morphological traits (the total root length, root volume, projected area, root surface area) were observed in the combined treatment of biochar and AMF treatment viz: 31.9%, 34.2%, 60.0% and 68.6%, 66.6%, 45.5%, 41.8%, respectively compared to the control under drought stress. The nitrogen content, total chlorophyll content and microbial biomass increased over un-inoculated control. The soil enzymes; alkaline phosphatase, dehydrogenase and fluorescein diacetate enzyme activities significantly increased in the combined treatment by 55.8%, 68.7% and 69.5%, respectively as compared to the control under drought stress. We conclude that biochar and AMF together is potentially beneficial for cultivation of okra in drought stress conditions.

Beneficial Features of Biochar and Arbuscular Mycorrhiza for Improving Spinach Plant Growth, Root Morphological Traits, Physiological Properties, and Soil Enzymatic Activities.

Biochar and arbuscular mycorrhizal fungi (AMF) can promote plant growth, improve soil properties, and maintain microbial activity. The effects of biochar and AMF on plant growth, root morphological traits, physiological properties, and soil enzymatic activities were studied in spinach (Spinacia oleracea L.). A pot experiment was conducted to evaluate the effect of biochar and AMF on the growth of spinach. Four treatments, a T1 control (soil without biochar), T2 biochar alone, T3 AMF alone, and T4 biochar and AMF together, were arranged in a randomized complete block design with five replications. The biochar alone had a positive effect on the growth of spinach, root morphological traits, physiological properties, and soil enzymatic activities. It significantly increased the plant growth parameters, such as the shoot length, leaf number, leaf length, leaf width, shoot fresh weight, and shoot dry weight. The root morphological traits, plant physiological attributes, and soil enzymatic activities were significantly enhanced with the biochar alone compared with the control. However, the combination of biochar and AMF had a greater impact on the increase in plant growth, root morphological traits, physiological properties, and soil enzymatic activities compared with the other treatments. The results suggested that the combined biochar and AMF led to the highest levels of spinach plant growth, microbial biomass, and soil enzymatic activity.

Microbial Community and Function-Based Synthetic Bioinoculants: A Perspective for Sustainable Agriculture.

Interactions among the plant microbiome and its host are dynamic, both spatially and temporally, leading to beneficial or pathogenic relationships in the rhizosphere, phyllosphere, and endosphere. These interactions range from cellular to molecular and genomic levels, exemplified by many complementing and coevolutionary relationships. The host plants acquire many metabolic and developmental traits such as alteration in their exudation pattern, acquisition of systemic tolerance, and coordination of signaling metabolites to interact with the microbial partners including bacteria, fungi, archaea, protists, and viruses. The microbiome responds by gaining or losing its traits to various molecular signals from the host plants and the environment. Such adaptive traits in the host and microbial partners make way for their coexistence, living together on, around, or inside the plants. The beneficial plant microbiome interactions have been exploited using traditional culturable approaches by isolating microbes with target functions, clearly contributing toward the host plants' growth, fitness, and stress resilience. The new knowledge gained on the unculturable members of the plant microbiome using metagenome research has clearly indicated the predominance of particular phyla/genera with presumptive functions. Practically, the culturable approach gives beneficial microbes in hand for direct use, whereas the unculturable approach gives the perfect theoretical information about the taxonomy and metabolic potential of well-colonized major microbial groups associated with the plants. To capitalize on such beneficial, endemic, and functionally diverse microbiome, the strategic approach of concomitant use of culture-dependent and culture-independent techniques would help in designing novel "biologicals" for various crops. The designed biologicals (or bioinoculants) should ensure the community's persistence due to their genomic and functional abilities. Here, we discuss the current paradigm on plant-microbiome-induced adaptive functions for the host and the strategies for synthesizing novel bioinoculants based on functions or phylum predominance of microbial communities using culturable and unculturable approaches. The effective crop-specific inclusive microbial community bioinoculants may lead to reduction in the cost of cultivation and improvement in soil and plant health for sustainable agriculture.

Multi-trait PGP rhizobacterial endophytes alleviate drought stress in a senescent genotype of sorghum [Sorghum bicolor (L.) Moench].

Root-tissue colonizing bacteria demonstrated with multiple PGP traits from sorghum plants were identified as Ochrobactrum sp. EB-165, Microbacterium sp. EB-65, Enterobacter sp. EB-14 and Enterobacter cloacae strain EB-48 on the basis of 16S rRNA gene sequencing. Here, the in vivo experiments using ½-MS media and ½-MS media + 15% PEG 8000 (for inducing drought stress) indicated stress tolerance imparting ability of these rhizobacterial endophytes in a non-stay green and senescent genotype (R-16) of sorghum. In the experiment with sterile soilrite mix base, seed bacterization with these isolates showed improved plant growth specifically the roots, in terms of root length (~ 44.2 to 50.8% over controls), root dry weight (~ 91.3 to 99.8% over controls) and root surface area (~ 1 to 1.5 fold over controls) under drought stress. Rhizobacterial endophytes were successful, not only in providing better cellular osmotic adjustment in leaves (≥ 1-fold increase in proline accumulation over controls), but favorable physiological responses like Relative Water Content (RWC) and cell Membrane Stability Index (MSI) in the inoculated plants during the drought stress induction. Up-regulation of drought responsive genes like sbP5CS2 and sbP5CS1 was observed in these endophytes-treated plants as compared to untreated control and Escherichia coli DH5α (negative control)-treated plants. Interestingly, the stress imparting traits of rhizobacterial endophytes, including up-regulation of specific genes, were observed during sorghum seedling growth only under drought stresses. The results of this study lead to the conclusion that the potential endophytic rhizobacterial interactions can contribute to plant growth promotion as well as induced stress tolerance in sorghum.

Enhancing the effectiveness of zinc, cadmium, and lead phytoextraction in polluted soils by using amendments and microorganisms.

For remediating polluted soils, phytoextraction of metals received considerable attention in recent years, although slow removal of metals remained a major constraint in this approach. We, therefore, studied the effect of selected organic and inorganic amendments on the solubility of zinc (Zn), cadmium (Cd), and lead (Pb) in polluted soil and enhancing the efficacy of phytoextraction of these metals by Indian mustard (Brassica juncea cv. Pusa Vijay). For this purpose, a greenhouse experiment was conducted using a metal-polluted soil to evaluate the effect of amendments, viz. green manure (T2), EDTA (T3), sulfur (S)+S oxidizing bacteria (Thiobacillus spp.) (T4), metal-solubilizing bacteria (Pseudomonas spp.) (T5), and green manure + metal-solubilizing bacteria (T6), on solubility and bioavailability of Zn, Cd, and Pb. Distribution of metals in different soil fractions revealed that Cd content in water soluble + exchangeable fraction increased to the extent of 34.1, 523, 133, 123, and 75.8% in T2, T3, T4, T5, and T6 treatments, respectively, over control (T1). Cadmium concentrations in soil solution as extracted by Rhizon sampler were recorded as 3.78, 88.1, 11.2, 6.29, and 4.27 µg L-1in T2, T3, T4, T5, and T6, respectively, whereas soil solution concentration of Cd in T1 was 0.99 µg L-1. Activities of Cd (pCd2+) in Baker soil extract were 12.2, 10.9, 6.72, 7.74, 7.67, and 7.05 for T1, T2, T3, T4, T5, and T6, respectively. Cadmium contents in shoot were recorded as 2.74, 3.12, 4.03, 4.55, 4.68, and 4.63 mg kg-1 in T1, T2, T3, T4, T5, and T6 treatments, respectively. Similar trend in Zn and Pb content with different magnitude was also observed across the different amendments. Cadmium uptake by shoot of mustard was enhanced to the extent of 125, 62.5, 175, 175, and 212% grown on T2-, T3-, T4-, T5-, and T6-treated soil, respectively, over T1. By and large, free ion activity of metals as measured by Baker soil test proved to be the most effective index for predicting Zn, Cd, and Pb content in shoot of mustard, followed by EDTA and DTPA. Among the metal fractions, only water soluble + exchangeable metal contributed positively towards plant uptake, which explained the variation in shoot Zn, Cd, and Pb content to the extent of 74, 81, and 87%, respectively, along with other soil metal fractions. Risk to human health for intake of metals through the consumption of leafy vegetable (mustard) grown on polluted soil in terms of hazard quotient (HQ) ranged from 0.64 to 1.10 for Cd and 0.11 to 0.34 for Pb, thus rendering mustard unfit for the human consumption. Novelty of the study mainly consisted of the use of natural means and microorganisms for enhancing solubility of metals in soil with the ultimate aim of hastening the phytoremediation.

Whole genome shotgun sequence of Bacillus paralicheniformis strain KMS 80, a rhizobacterial endophyte isolated from rice (Oryza sativa L.).

Bacillus paralicheniformis strain KMS 80 (MTCC No. 12704) is an isolate from the root tissues of rice (Oryza sativa L.) that displays biological nitrogen fixation and plant growth promoting abilities. Here, we report the complete genome sequence of this strain, which contains 4,566,040 bp, 4424 protein-coding genes, 8692 promoter sequences, 67 tRNAs, 20 rRNA genes with six copies of 5S rRNAs along with a single copy of 16S-23S rRNA and genome average GC-content of 45.50%. Twenty one genes involved in nitrogen metabolism pathway and two main transcriptional factor genes, glnR and tnrA responsible for regulation of nitrogen fixation in Bacillus sp. were predicted from the whole genome of strain KMS 80. Analysis of the ~ 4.57 Mb genome sequence will give support to understand the genetic determinants of host range, endophytic colonization behaviour as well as to enhance endophytic nitrogen fixation and other plant beneficial role of B. paralicheniformis in rice.

Draft Genome Sequence of Pseudomonas stutzeri Strain KMS 55, an Endophytic Diazotroph Isolated from Rice Roots.

Pseudomonas stutzeri strain KMS 55 (MTCC 12703) is an isolate from the root tissues of rice (Oryza sativa L.) that displays a high biological nitrogen fixation ability. Here, we report the complete genome sequence of this strain, which contains 4,637,820 bp, 4,289 protein-coding genes, 5,006 promoter sequences, 62 tRNAs, a single copy of 5S-16S-23S rRNA, and a genome average GC content of 51.18%. Analysis of the ~4.64-Mb genome sequence will give support to increased understanding of the genetic determinants of host range, endophytic colonization behavior, endophytic nitrogen fixation, and other plant-beneficial roles of Pseudomonas stutzeri.

Comparative conventional and phenomics approaches to assess symbiotic effectiveness of Bradyrhizobia strains in soybean (Glycine max L. Merrill) to drought.

Symbiotic effectiveness of rhizobitoxine (Rtx)-producing strains of Bradyrhizobium spp. in soybean (cultivar NRC-37/Ahilya-4) under limited soil moisture conditions was evaluated using phenomics tools such as infrared(IR) thermal and visible imaging. Red, green and blue (RGB) colour pixels were standardized to analyse a total of 1017 IR thermal and 692 visible images. Plants inoculated with the Rtx-producing strains B. elkanii USDA-61 and USDA-94 and successive inoculation by B. diazoefficiens USDA-110 resulted in cooler canopy temperatures and increased canopy greenness. The results of the image analysis of plants inoculated with Rtx-producing strains were correlated with effective nodulation, improved photosynthesis, plant nitrogen status and yield parameters. Principal component analysis (PCA) revealed the reliability of the phenomics approach over conventional destructive approaches in assessing the symbiotic effectiveness of Bradyrhizobium strains in soybean plants under watered (87.41-89.96%) and water-stressed (90.54-94.21%) conditions. Multivariate cluster analysis (MCA) revealed two distinct clusters denoting effective (Rtx) and ineffective (non-Rtx) Bradyrhizobium inoculation treatments in soybean. Furthermore, correlation analysis showed that this phenotyping approach is a dependable alternative for screening drought tolerant genotypes or drought resilience symbiosis. This is the first report on the application of non-invasive phenomics techniques, particularly RGB-based image analysis, in assessing plant-microbe symbiotic interactions to impart abiotic stress tolerance.

An Environmentally Friendly Engineered Azotobacter Strain That Replaces a Substantial Amount of Urea Fertilizer while Sustaining the Same Wheat Yield.

In our endeavor to improve the nitrogen fixation efficiency of a soil diazotroph that would be unaffected by synthetic nitrogenous fertilizers, we have deleted a part of the negative regulatory gene nifL and constitutively expressed the positive regulatory gene nifA in the chromosome of Azotobacter chroococcum CBD15, a strain isolated from the local field soil. No antibiotic resistance gene or other foreign gene was present in the chromosome of the engineered strain. Wheat seeds inoculated with this engineered strain, which we have named Azotobacter chroococcum HKD15, were tested for 3 years in pots and 1 year in the field. The yield of wheat was enhanced by ∼60% due to inoculation of seeds by A. chroococcum HKD15 in the absence of any urea application. Ammonium only marginally affected acetylene reduction by the engineered Azotobacter strain. When urea was also applied, the same wheat yield could be sustained by using seeds inoculated with A. chroococcum HKD15 and using ∼85 kg less urea (∼40 kg less nitrogen) than the usual ∼257 kg urea (∼120 kg nitrogen) per hectare. Wheat plants arising from the seeds inoculated with the engineered Azotobacter strain exhibited far superior overall performance, had much higher dry weight and nitrogen content, and assimilated molecular 15N much better. A nitrogen balance experiment also revealed much higher total nitrogen content. Indole-3-acetic acid (IAA) production by the wild type and that by the engineered strain were about the same. Inoculation of the wheat seeds with A. chroococcum HKD15 did not adversely affect the microbial population in the field rhizosphere soil.IMPORTANCE Application of synthetic nitrogenous fertilizers is a standard agricultural practice to augment crop yield. Plants, however, utilize only a fraction of the applied fertilizers, while the unutilized fertilizers cause grave environmental problems. Wild-type soil diazotrophic microorganisms cannot replace synthetic nitrogenous fertilizers, as these reduce atmospheric nitrogen very inefficiently and almost none at all in the presence of added nitrogenous fertilizers. If the nitrogen-fixing ability of soil diazotrophs could be improved and sustained even in the presence of synthetic nitrogenous fertilizers, then a mixture of the bacteria and a reduced quantity of chemical nitrogenous fertilizers could be employed to obtain the same grain yield but at a much-reduced environmental cost. The engineered Azotobacter strain that we have reported here has considerably enhanced nitrogen fixation and excretion abilities and can replace ∼85 kg of urea per hectare but sustain the same wheat yield, if the seeds are inoculated with it before sowing.

Dynamics of soil diazotrophic community structure, diversity, and functioning during the cropping period of cotton (Gossypium hirsutum).

The soil sampled at different growth stages along the cropping period of cotton were analyzed using various molecular tools: restriction fragment length polymorphism (RFLP), terminal restriction length polymorphism (T-RFLP), and cloning-sequencing. The cluster analysis of the diazotrophic community structure of early sampled soil (0, 15, and 30 days) was found to be more closely related to each other than the later sampled one. Phylogenetic and diversity analysis of sequences obtained from the first (0 Day; C0) and last soil sample (180 day; C180) confirmed the data. The phylogenetic analysis revealed that C0 was having more unique sequences than C180 (presence of γ-Proteobacteria exclusively in C0). A relatively higher richness of diazotrophic community sequences was observed in C0 (S(ACE) : 30.76; S(Chao1) : 20.94) than C180 (S(ACE) : 18.00; S(Chao1) : 18.00) while the evenness component of Shannon diversity index increased from C0 (0.97) to C180 (1.15). The impact of routine agricultural activities was more evident based on diazotrophic activity (measured by acetylene reduction assay) than its structure and diversity. The nitrogenase activity of C0 (1264.85 ± 35.7 ηmol of ethylene production g(-1) dry soil h(-1) ) was statistically higher when compared to all other values (p < 0.05). There was no correlation found between diazotrophic community structure/diversity and N2 fixation rates. Thus, considerable functional redundancy of nifH was concluded to be existing at the experimental site.

Mitigation of salt stress in wheat seedlings by halotolerant bacteria isolated from saline habitats.

Eighty four halotolerant bacterial strains were isolated from the saline habitats and screened for growth at different NaCl concentrations. All grew well at 5% NaCl, but only 25% isolates showed growth at 20% NaCl concentration. Five strains SL3, SL32, SL35, J8W and PU62 growing well in 20% NaCl concentrations were further characterized for multiple plant growth promoting traits such as indole -3- acetic acid (IAA) production, HCN and siderophore production, ACC deaminase activity and P-solubilization. None were positive for HCN production and PCR amplification of acdS, the structural gene for ACC deaminase enzyme was found negative. 16S rRNA gene sequencing analysis of the five strains showed them to belong to two genera Bacillus and Hallobacillus. In vitro experiments showed that salt concentrations had significant inhibitory effects on development of seedlings but not on the growth of the bacterial strains. Inoculation of the 5 halotolerant bacterial strains to ameliorate salt stress (80 mM, 160 mM and 320 mM) in wheat seedlings produced an increase in root length of 71.7% in comparison with uninoculated positive controls. In particular, Hallobacillus sp. SL3 and Bacillus halodenitrificans PU62 showed more than 90% increase in root elongation and 17.4% increase in dry weight when compared to uninoculated wheat seedlings at 320 mM NaCl stress indicating a significant reduction of the deleterious effects of NaCl. These results indicate that halotolerant bacteria isolated from saline environments have potential to enhance plant growth under saline stress through direct or indirect mechanisms and would be most appropriate as bioinoculants under such conditions.

Cadinene sesquiterpenes from Eupatorium adenophorum and their antifungal activity.

Bioactive constituents of Eupatorium adenophorum were investigated for antifungal activity. A structure-antifungal activity relationship of cadinene sesquiterpenes was predicted by evaluating individual derivatives. Cadinene derivatives were extracted from leaves of Eupatorium adenophorum using ethyl acetate. Five cadinene sesquiterpenes were isolated by column chromatography and Preparative Thin Layer Chromatography. Bioactivity of these cadinene sesquiterpenes were evaluated in vitro against four phytopathogenic fungi using poison food technique. Purified sesquiterpenes were spectroscopically elucidated as cadinan-3-ene-2,7-dione (1), 7-hydroxycadinan-3-ene-2-one (2), 5,6-dihydroxycadinan-3-ene-2,7-dione (3), cadinan-3,6-diene-2,7-dione (4) and 2-acetyl-cadinan-3,6-diene-7-one (5). Antifungal evaluation of these compounds against pathogenic fungi was found to be selective. Compound 1 was highly inhibitory towards S. rolfsii (ED50 181.60 ± 0.58 µgmL(-1)) and R. solani (ED50 189.74 ± 1.03 µgmL(-1)). Availability of plant material and significant antifungal activity makes the plant a potential source of antifungal agent and that can be exploited for the development of a natural fungicide.

Isolation and partial characterization of antibacterial lipopeptide produced by Paenibacillus polymyxa HKA-15 against phytopathogen Xanthomonas campestris pv. phaseoli M-5.

An antibacterial metabolite was isolated from Paenibacillus polymyxa HKA-15, a soybean bacterial endophyte. The purification of the crude metabolite from Paenibacillus polymyxa HKA-15 was done by column chromatography. In TLC, a spot with an R ( f ) value of 0.86 (±0.02) from the purified fraction showed bioactivity against Xanthomonas campestris pv. phaseoli M-5. In SDS-PAGE, the purified antibiotic was separated in the molecular weight range of 3.5 kDa. The exact molecular weight of the active compound was identified as 1,347.7 Da using MS-MS analysis. Infra red spectrum and (1)H NMR analysis showed the presence of amino acids and fatty acids in the active compound. The characterization of the antibacterial compound revealed its lipopeptide nature. In an agar diffusion assay, the crude metabolite showed a broad spectrum of activity, being able to inhibit the growth of the fungal pathogen, Rhizoctonia bataticola, Macrophomina phaseolina and Fusarium udum. A stronger inhibition was observed against bacterial pathogens viz., X. campestris pv.phaseoli M-5, X. campestris pv. phaseoli CP-1-1, Xanthomonas oryzae, Ralstonia solanacearum and Micrococcus luteus.

Isolation of Rhizobacteria from Jatropha curcas and characterization of produced ACC deaminase.

Decreased levels of ACC (1-aminocyclopropane-1-carboxylic acid) result in lower levels of endogenous ethylene, which eliminate the potentially inhibitory effects of stress-induced higher ethylene concentrations. It is worth noting the substantial ability of the bacterial species to colonize different environments, including taxonomically distinct plants cultivated in distantly separated geographical regions. For example, Enterobacter cloacae, designated as MSA1 and Enterobacter cancerogenus, designated as MSA2 were recovered from the rhizosphere of Jatropha in the present work. This study first time confirms the ACC deaminase activity in the Enterobacter cancerogenus on the preliminary basis. Several bacterial plant growth-promoting mechanisms were analyzed and detected like phosphate solubilization, siderophore production, IAA production, GA(3) (gibberellic acid) production and ACC deaminase activity in the isolated cultures. Isolates were grown until exponential growth phase to evaluate their ACC deaminase activity and the effect of pH, temperature, salt, metals and substrate concentration after the partial purification of enzyme by ion exchange chromatography. The FOURIER TRANSFORM INFRARED (FT-IR) spectra were recorded for the confirmation of α-ketobutyrate production. By using lineweaver Burk plot K(m) and V(max) value for ACC deaminase of both the organism was calculated in the different fractions. In this work, we discuss the possible implications of these bacterial mechanisms on the plant growth promotion or homeostasis regulation in natural conditions.

Isolation and characterization of ACC deaminase gene from two plant growth-promoting rhizobacteria.

Lowering of plant ethylene by deamination of its immediate precursor 1-aminocyclopropane-1-carboxylate (ACC) is a key trait found in many rhizobacteria. We isolated and screened bacteria from the rhizosphere of wheat for their ACC-degrading ability. The ACC deaminase gene (acdS) isolated from two bacterial isolates through PCR amplification was cloned and sequenced. Nucleotide sequence alignment of these genes with previously reported genes of Pseudomonas sp. strain ACP and Enterobacter cloacae strain UW4 showed variation in their sequences. In the phylogenetic analysis, distinctness of these two genes was observed as a separate cluster. 16S rDNA sequencing of two isolates identified them to be Achromobacter sp. and Pseudomonas stutzeri.

Isolation and identification of natural endophytic rhizobia from rice (Oryza sativa L.) through rDNA PCR-RFLP and sequence analysis.

Three novel endophytic rhizobial strains (RRE3, RRE5, and RRE6) were isolated from naturally growing surface-sterilized rice roots. These isolates had the ability to nodulate common bean (Phaseolus vulgaris). Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and sequencing of 16S rDNA of these isolates revealed that RRE3 and RRE5 are phylogenetically very close to Burkholderia cepacia complex, whereas RRE6 has affinity with Rhizobium leguminosarum bv. phaseoli. Plant infection test using gusA reporter gene-tagged construct of these isolates indicated that bacterial cells can go inside and colonize the rice root interiors. A significant increase in biomass and grain yield was also recorded in greenhouse-grown rice plants inoculated with these isolates.