Rhizobial Strains Research Articles

Evaluation of Root Nodulating Rhizobial Strains for Soybean Yield and Yield Components at Dangur District, North Western Ethiopia

Inoculating soybean seeds with superior rhizobia strains is necessary for nodulation and nitrogen fixation. Ironically, many commercial agricultural products (biological and chemical) claim increases in crop productivity but their efficacy cannot be guaranteed. Thus, the trial was conducted for two consecutive years during rainy seasons of 2014 and 2015 in order to determine the effectiveness of some commercial microbial inoculants and recommend the best thizobia that increase the soybean grain yield in study area (Dangur) district. The experiment consists of six strain bacteria, one inorganic fertilizer alone and negative control, totally eight treatments viz., SB-14, SB-6B1, SB-6A2, SB-Murdock, SB-12, MAR-1495, recommended rate of fertilizer (100/ha TSP) , and control treatment. The experiment was replicated three times in randomized complete block design. The results revealed that co-inoculation of SB-Murdock with the soybean variety Belesa 95, produced significantly higher grain yield per hectare (1792.05kg), nodule numbers per plant (105.7), nodules dry weight per plant (0.51g), pod numbers per plant (36.57), seed numbers per plant (95.07) than the negative control treatment. On the other hand, the lowest grain yield (1099.16kg/ha), plant biomass (3055.56kg/ha), nodule numbers per plant (4.73), nodule dry weights per plant (0.04g), pod numbers per plant (23.87) and seed numbers per plant (55.3) were obtained from negative control treatment. The overall results thus suggest that rhizobial strains SB-Murdock can be used for producing higher yield of soybean when compared with the rest strain under agro climatic conditions of Dangur district. Keywords: nitrogen fixaxion, Glycine max, nodules, inoculation. DOI: 10.7176/JBAH/10-16-01 Publication date: August 31 st 2020

Impact of Different Rhizobial Strains on Physiological Responses and Seed Yield of Mungbean [Vigna radiata (L.) Wilczek] under Field Conditions

Background: Water availability is a main controlling factor for agricultural productivity. Erratic monsoon and ever-growing population limit the water accessibility to mungbean and thus reduce its yield. Rhizobia–legume interactions are environmental friendly and are very well known for boosting the production potential of mungbean. But, the screening of new and better rhizobial isolates is essential to improve mungbean yield in sub-optimal conditions. So this study was conducted with the objectives to evaluate the rhizobial isolates for the physiological traits in mungbean and to measure the association of these traits with yield under moisture stress. Methods: In this field experiment during kharif 2016, five rhizobial strains of mungbean viz. Vigna 703 + PSB strain P-36, MR 63, MR 54, MB 17a and MH 8b2 were tested with respect and in combination with RDF at two water levels viz. control and drought. Moisture stress was created by withholding irrigations at flowering and pod formation stages. Result: Growth and physiological traits were altered under moisture stress. Rhizobial isolates MR 63 and MB 17a sustained higher yield over other rhizobial strains under both moisture regimes. These strains maintained higher RWC, leaf water potential and photosynthesis during stress and appear promising for drought tolerance.

Physiological and symbiotic variation of a long-term evolved Rhizobium strain under alkaline condition

Physiological variation and adaptation of the long-term evolved rhizobia to alkaline environments where no host plant existence and the stability of their symbiotic properties when they are reinoculated to legume host remain unclear. A highly effective N2-fixing Rhizobium yanglingense strain CCBAU 01603 was used as the ancestral strain and was cultured continuously with/without addition of extra alkaline reagent (KOH) in laboratory conditions for approximately 500 generations. Total 60 evolved clones obtained were checked for their adaptation to higher alkaline pH level and inoculated to their host plant Caragana microphylla to evaluate their symbiotic efficiencies. Most of the evolved clones showed increased adaptation to higher alkaline pH but all of them decreased symbiotic efficiencies, resulting in the formation of irregular root nodules with lower nitrogenase activity, production of abnormal bacteroids, and accumulation more starch grains in uninfected nodule cells. Further demonstration of lower symbiotic efficiencies came from the down-regulated expression of genes related to nitrogen fixation in the bacteroids by transcriptome comparison. In addition, genes related to transporters and other diverse functions were up- or down-regulated in the evolved clones in free-living conditions (like yjiS gene) or in symbiotic situations, demonstrating the significant variations in cellular physiology and symbiosis. Our study revealed that the enhancement of alkaline adaptation but loss of symbiotic efficiencies of the evolved clones had happened during the long-term evolution in alkaline environments where no selective pressures from host plant, offering new insight into the molecular mechanism and direction of rhizobial evolution in nature.

Rhizobial Microsymbionts of Kamchatka Oxytropis Species Possess Genes of the Type III and VI Secretion Systems, Which Can Affect the Development of Symbiosis.

A collection of rhizobial strains isolated from root nodules of the narrowly endemic legume species Oxytropis erecta, O. anadyrensis, O. kamtschatica, and O. pumilio originating from the Kamchatka Peninsula (Russian Federation) was obtained. Analysis of the 16S ribosomal RNA gene sequence showed a significant diversity of isolates belonging to families Rhizobiaceae (genus Rhizobium), Phyllobacteriaceae (genera Mesorhizobium, Phyllobacterium), and Bradyrhizobiaceae (genera Bosea, Tardiphaga). A plant nodulation assay showed that only strains belonging to genus Mesorhizobium could form nitrogen-fixing nodules on Oxytropis plants. The strains M. loti 582 and M. huakuii 583, in addition to symbiotic clusters, possessed genes of the type III and type VI secretion systems (T3SS and T6SS, respectively), which can influence the host specificity of strains. These strains formed nodules of two types (elongated and rounded) on O. kamtschatica roots. We suggest this phenomenon may result from Nod factor-dependent and -independent nodulation strategies. The obtained strains are of interest for further study of the T3SS and T6SS gene function and their role in the development of rhizobium-legume symbiosis. The prospects of using rhizobia having both gene systems related to symbiotic and nonsymbiotic nodulation strategies to enhance the efficiency of plant-microbe interactions by expanding the host specificity and increasing nodulation efficiency are discussed.

Bactérias diazotropicas no crescimento inicial e alterações bioquímicas de Vigna unguiculata sob deficit hídrico

O cultivo do feijão caupi vem se destacando por ser uma cultura adaptável para a região árida e semi-árida do Nordeste brasileiro. Dentre os fatores que reduzem a sua produção, o déficit hídrico e a adubação, tem destaque. Devido a sua capacidade de realizar simbiose com bactérias noduladoras, poderá optimizar a fixação do N2 atmosférico dependendo da combinação e compatibilidade das estirpes envolvidas. O objetivo do trabalho foi avaliar a resposta de plantas de feijão-caupi (cultivar BRS Marataoã) sob déficit hídrico inoculadas e coinoculadas com bactérias fixadoras de nitrogênio. O delineamento experimental utilizado foi o inteiramente casualizado em esquema de parcelas subdivididas com dois fatores (4x5), composto por: T1: BR3264 (A); T2: BR85 (B); T3: BR3264+BR85 (C); T4: controle nitrogenado (CN); T5: controle absoluto (CA), associado com as lâminas de água nas seguintes proporções: 100 %, 80%, 60% e 40% da evapotranspiração da cultura (ETc). As análises foram realizadas aos 45 dias após a emergência (DAE) e compostas de parâmetros filotécnicos (número de folhas, altura da planta, diâmetro do caule, clorofila a, b e total) e bioquímicos (teores de açúcares solúveis totais, açúcares redutores, e prolina). Os resultados obtidos demonstraram que o déficit hídrico ocasionou influência negativa no número de folhas, no diâmetro do caule, na altura das plantas, na clorofila A, na massa seca da parte aérea e na inoculação. A inoculação das cepas de rizóbio é reduzida quando submetida a plantas com estresse hídrico, se sobressaindo cepas nativas. Sendo que a inoculação cruzada favorece a inoculação nessas condições.

Towards sustainable yield improvement: field inoculation of soybean with Bradyrhizobium and co-inoculation with Azospirillum in Mozambique.

The effects of sole inoculation of soybean (Glycine max L. Merrill) with Bradyrhizobium and co-inoculation with Bradyrhizobium and Azospirillum on nodulation, plant growth and yields were investigated in the 2013/2014 and 2014/2015 cropping seasons under field conditions in Mozambique. The treatments included (1) Control (non-inoculated control, with symbiosis depending on indigenous rhizobia), (2) Urea (non-inoculated, receiving 200kgha-1 of N), (3) Sole inoculation with B. diazoefficiens strain USDA 110, and (4) Co-inoculation with B. diazoefficiens strain USDA 110 and A. brasilense strains Ab-V5 and Ab-V6, evaluated in a randomized complete block design with five replications. Nodule number and dry weight, shoot dry weight, biological and grain yields, grain dry weight, and harvest index were evaluated. In general, both sole inoculation and co-inoculation enhanced nodulation in relation to control. Sole inoculation increased grain yield by 22% (356kgha-1), the same enhancement magnitude attained under mineral N treatment, suggesting that Bradyrhizobium inoculation provides ecological and economic sustainability to the soybean crop in Mozambique or other countries with similar agro-climatic conditions. Co-inoculation did not increase grain yields in relation to neither the control nor sole inoculation, indicating that further research with adapted and high yielding soybean varieties along with effective rhizobial strains is required in Mozambique to attune the beneficial Azospirillum-plant cultivar-rhizobia interactions that have been reported in other countries for several legumes, including soybean.

The Exopolysaccharide Cepacian Plays a Role in the Establishment of the Paraburkholderia phymatum - Phaseolus vulgaris Symbiosis.

Paraburkholderia phymatum is a rhizobial strain that belongs to the beta-proteobacteria, a group known to form efficient nitrogen-fixing symbioses within root nodules of several legumes, including the agriculturally important common bean. The establishment of the symbiosis requires the exchange of rhizobial and plant signals such as lipochitooligosaccharides (Nod factors), polysaccharides, and flavonoids. Inspection of the genome of the competitive rhizobium P. phymatum revealed the presence of several polysaccharide biosynthetic gene clusters. In this study, we demonstrate that bceN, a gene encoding a GDP-D-mannose 4,6-dehydratase, which is involved in the production of the exopolysaccharide cepacian, an important component of biofilms produced by closely related opportunistic pathogens of the Burkholderia cepacia complex (Bcc), is required for efficient plant colonization. Wild-type P. phymatum was shown to produce cepacian while a bceN mutant did not. Additionally, the bceN mutant produced a significantly lower amount of biofilm and formed less root nodules compared to the wild-type strain with Phaseolus vulgaris as host plant. Finally, expression of the operon containing bceN was induced by the presence of germinated P. vulgaris seeds under nitrogen limiting conditions suggesting a role of this polysaccharide in the establishment of this ecologically important symbiosis.

In-Vitro Plant Growth Promotion of Rhizobium Strains Isolated from Lentil Root Nodules under Abiotic Stresses

Plant growth-promoting rhizobia are known to improve crop performance by multiple mechanisms. However, the interaction between host plants and Rhizobium strains is highly influenced by growing conditions, e.g., heat, cold, drought, soil salinity, nutrient scarcity, etc. The present study was undertaken to assess the use of Rhizobium as plant growth promoters under abiotic stress conditions. Fifteen Rhizobium strains isolated from lentil root nodules were tested for phosphate solubilization activity (PSA) and phytohormones production under salt and drought conditions. The results showed that 15 Rhizobium strains were significant phosphate solubilizers, and indole acedic acid (IAA) and gibberellic acid (GA3) producers based on least significant difference (LSD) analysis (p ≤ 0.05). The highest rate of PSA was attributed to three strains namely, 1145N5, 1159N11, and 1159N32 with a range of 144.6 to 205.6 P2O5 (µg/mL). The highest IAA production was recorded in the strain 686N5 with 57.68 ± 4.25 µg/mL as compared to 50.8667 ± 1.41 µg/mL and 37.32 ± 12.59 µg/mL for Rhizobium tropici CIAT 899 and Azospirillum brasilense DSM-1690, respectively. Strain 318N2111 produced 329.24 ± 7.84 µg/mL of GA3 as against 259.84 ± 25.55 µg/mL for A. brasilense DSM-1690. R. tropici CIAT 899 showed tolerance to salt (5% NaCl) and drought (ψ = −2.6 MPa) stress, whereas strain 686N5 showed an extremely high level of salt-tolerance (5% NaCl) and moderate level of drought tolerance (ψ = −0.75 MPa). These results indicate different pathways for drought and salt tolerance mechanisms. The assessment of plant growth promoting (PGP) activities of Rhizobium showed differences between bacterial viability and bacterial PGP activity in terms of abiotic stress tolerance where bacterial PGP activity is interrupted before reaching the bacterial tolerance threshold. These results integrate a new concept of PGPR screening based on PGP activity under abiotic stress.

Isolation and identification of Rhizobium from non-saline coastal soils of Bangladesh and preparation of mother culture

Nitrogen is the essential mineral macro nutrients which are required for the maximum magnification and yield of agriculturally paramount crops. Microbial inoculants may supplement and abbreviate the dependency on synthetic costly N-fertilizers in reverence of crop yield. The categorical objectives of the research works are to isolate and identify the Rhizobium from culled soils. The ability of soil microorganisms to fix atmospheric nitrogen is an important trait in promoting plant growth and increasing crop yield. The study was conducted for the isolation and identification of nitrogen fixing bacteria from saline and non-saline soils of different locations of tidal floodplain region of Bangladesh. Six Rhizobium strains were isolated and purified. The isolates were preliminary identified on the basis of their morphological and biochemical characteristics. Based on the results, it can be concluded that the isolates possess great potential to be developed as biofertilizers to enhance soil fertility and plant growth. However, their performance under green house and field conditions should be assessed afore being recommended for biofertilizer production and their applications.
 Asian J. Med. Biol. Res. June 2020, 6(2): 222-228

A PGPR-Produced Bacteriocin for Sustainable Agriculture: A Review of Thuricin 17 Characteristics and Applications.

A wide range of prokaryotes produce and excrete bacteriocins (proteins with antimicrobial activity) to reduce competition from closely related strains. Application of bacteriocins is of great importance in food industries, while little research has been focused on the agricultural potential of bacteriocins. A number of bacteriocin producing bacteria are members of the phytomicrobiome, and some strains are plant growth promoting rhizobacteria (PGPR). Thuricin 17 is a single small peptide with a molecular weight of 3.162 kDa, a subclass IId bacteriocin produced by Bacillus thuringiensis NEB17, isolated from soybean nodules. It is either cidal or static to a wide range of prokaryotes. In this way, it removes key competition from the niche space of the producer organism. B. thuringiensis NEB17 was isolated from soybean root nodules, and thus is a member of the phytomicrobiome. Interestingly, thuricin 17 is not active against a wide range of rhizobial strains involved in symbiotic nitrogen fixation with legumes or against other PGPR. In addition, it stimulates plant growth, particularly in the presence of abiotic stresses. The stresses it assists with include key ones associated with climate change (drought, high temperature, and soil salinity). Hence, in the presence of stress, it increases the size of the overall niche space, within plant roots, for B. thuringiensis NEB17. Through its anti-microbial activity, it could also enhance plant growth via control of specific plant pathogens. None of the isolated bacteriocins have been examined as broadly as thuricin 17 on plant growth promotion. Thus, this review focuses on the effect of thuricin 17 as a microbe to plant signal that assists crop plants in managing stress and making agricultural systems more climate change resilient.

The role of defecate and microbial nitrogen-fixing preparations in the formation of alfalfa variety yield in the conditions of the south of Primorsky Krai

Four-year studies revealed a positive role of the effect and aftereffect of the defecate and inoculation of seeds by virulent active strains of Rhizobium Synorhizobium meliloty on the productivity of green and dry alfalfa varieties VEGA 87. The total yield of green alfalfa has varied during four years of life (ten bites) from 142.2 t/ha in the control variant to 153.4-183.2 t/ha in the experimental variants. Total productivity of green mass in variant with application of lime increased by 18.3 %, in the variants with presowing treatment of the seeds with rhizobia strains –by 7.9-11.5 % and their combination – by 23.4-28.8 %. The yield of dry mass in the variant with application of defecate increased by 28.8 %, in the variants with presowing treatment of the seeds with rhizobia strains by 10.7-13.7 % and at their joint action by 37.6-42.0 %. The biggest increase in productivity of green and dry mass is provided by inoculation of the seeds with a promising A4 strain when applying defecate, and without the use of meliorant – inoculation of the seeds with the main production strain 425a. The application of defecate and bacterial preparations for presowing treatment of the alfalfa seeds is agronomically and economically profitable, it promotes to increase the environmental stability of the agricultural landscape and plants to stressful conditions, a balanced level of nutrition, increasing plant productivity and profitability (low cost) of their cultivation.

Plant transcriptome analysis reveals specific molecular interactions between alfalfa and its rhizobial symbionts below the species level

BackgroundLeguminous plants alter patterns of gene expression in response to symbiotic colonization and infection by their cognate rhizobial bacteria, but the extent of the transcriptomic response has rarely been examined below the species level. Here we describe the identification of 12 rhizobial biotypes of Ensifer meliloti, which form nitrogen-fixing nodules in the roots of alfalfa (Medicago sativa L.), followed by a comparative RNA-seq analysis of four alfalfa cultivars each inoculated with two E. meliloti strains varying in symbiotic performance and phylogenetic relatedness.ResultsRhizobial biotypes were identified on the basis of their symbiotic performance, particularly shoot dry weight. Differentially expressed genes (DEGs) and metabolic pathways were determined by comparing the RNA-seq data with that of the uninoculated control plant. Significant differences were found between DEGs generated in each cultivar with the inoculation of two rhizobial strains in comparison (P < 0.01). A total of 8111 genes was differentially expressed, representing ~ 17.1% of the M. sativa genome. The proportion of DEGs ranges from 0.5 to 12.2% for each alfalfa cultivar. Interestingly, genes with predicted roles in flavonoid biosynthesis and plant-pathogen interaction (NBS-LRR) were identified as the most significant DEGs. Other DEGs include Medsa002106 and genes encoding nodulins and NCR peptides whose expression is specifically induced during the development of nitrogen-fixing nodules. More importantly, strong significant positive correlations were observed between plant transcriptomes (DEGs and KEGG pathways) and phylogenetic distances between the two rhizobial inoculants.ConclusionsAlfalfa expresses significantly distinct sets of genes in response to infection by different rhizobial strains at the below-species levels (i.e. biotype or strain). Candidate genes underlying the specific interactions include Medsa002106 and those encoding nodulins and NCR peptides and proteins in the NBS-LRR family.

Evaluation of beneficial and inhibitory effects of nitrate on nodulation and nitrogen fixation in common bean (Phaseolus vulgaris)

AbstractThe effects of applied nitrate on symbiotic nitrogen fixation in legumes are complex. Both inhibition and promotion of nodulation by nitrate have been observed in a dose‐dependent manner. The objectives of this study were to determine the effects of nitrate at different concentrations on root nodulation in different genotypes in common bean (Phaseolus vulgaris). Six genotypes were inoculated with the same rhizobial strain and grown hydroponically in growth pouches in a growth chamber and exposed to six nitrate concentrations, including 0, 2.5, 5, 10, 15, and 20 mM for 4 weeks. The tested genotypes included three recombinant inbred lines (RILs, 25, 46, and 70) that differed in their responses to nitrogen (based on observations of one field growing season), their parents (Mist and Sanilac—registered varieties), which are different in N‐fixing abilities, and one nonnodulating mutant (R99). Our results showed that small amounts of nitrate (2.5 and 5 mM) promoted nodule formation and increased nodule biomass, compared with plants in the 0 nitrate control treatment. In contrast, nitrate concentrations over 10 mM inhibited nodulation, resulting in reductions in nodule number and nodule biomass. Nodulation was completely inhibited by 15‐mM nitrate in all the genotypes. Regression analyses indicated that 5‐mM nitrate is the optimum concentration for promoting nodulation as measured by the total number of nodules formed, the number of effective nodules formed, and the nodule biomass formed. In contrast, nitrogen fixation was inhibited by all levels of nitrate. No genotypic differences were observed in nodulation among the three RILs and their parental cultivars, but all were significantly different than R99, a nonnodulating mutant.

Unraveling the impact of arsenic on the redox response of peanut plants inoculated with two different Bradyrhizobium sp. strains

Arsenic (As) can be present naturally in groundwater from peanut fields, constituting a serious problem, as roots can accumulate and mobilize the metalloid to their edible parts. Understanding the redox changes in the legume exposed to As may help to detect potential risks to human health and recognize tolerance mechanisms. Thirty-days old peanut plants inoculated with Bradyrhizobium sp. strains (SEMIA6144 or C-145) were exposed to a realistic arsenate concentration, in order to unravel the redox response and characterize the oxidative stress indexes. Thus, root anatomy, reactive oxygen species detection by fluorescence microscopy and, ROS histochemical staining along with the NADPH oxidase activity were analyzed. Besides, photosynthetic pigments and damage to lipids and proteins were determined as oxidative stress indicators. Results showed that at 3 μM AsV, the cross-section areas of peanut roots were augmented; NADPH oxidase activity was significantly increased and O2˙¯and H2O2 accumulated in leaves and roots. Likewise, an increase in the lipid peroxidation and protein carbonyls was also observed throughout the plant regardless the inoculated strain, while chlorophylls and carotenes were increased only in those inoculated with Bradyrhizobium sp. C-145. Interestingly, the oxidative burst, mainly induced by the NADPH oxidase activity, and the consequent oxidative stress was strain-dependent and organ-differential. Additionally, As modifies the root anatomy, acting as a possibly first defense mechanism against the metalloid entry. All these findings allowed us to conclude that the redox response of peanut is conditioned by the rhizobial strain, which contributes to the importance of effectively formulating bioinoculants for this crop.

Phylogenetic diversity of bacterial strains from root nodules of legumes grown wild in Egypt

Abstract A total of 38 bacterial strains were isolated from root nodules of Lotus, Vicia, Trifolium, Lathyrus, Astragalus, and Hippocrepis plants grown-wild at different geographical locations in Egypt. Analysis of the almost complete sequence of the 16S rRNA gene from the bacterial strains showed that seventeen of them isolated from all the six plant species were members of the genus Rhizobium, six from Lotus, Vicia and Hippocrepis were Ensifer, and the remaining six strains from Lotus and Vicia were Neorhizobium. Sequencing of the symbiotic nodC gene from eight selected rhizobial strains confirmed their affiliation with rhizobial species. These results suggest the existence of high rhizobial diversity in wild-grown Egyptian legumes. Nodules from Lotus, Trifolium, and Astragalus also contained bacterial species of the genera Achromobacter, Ancylobacter, Bacillus, Cronobacter, Curtobacterium, Flavobacterium, Paenibacillus and Pseudomonas. These results provide information about the diversity of rhizobia associated with wild-grown legumes in Egypt, and enlarged the rhizobial spectrum of Lotus, Vicia, Trifolium, Lathyrus, Hippocrepis and Astragalus, which supported to the idea that rhizobial promiscuity could be an adaption strategy to diverse environments.

Legume-Rhizobium Strain Specificity Enhances Nutrition and Nitrogen Fixation in Faba Bean (Vicia faba L.)

This study reports the effectiveness of some selected rhizobium strains in enhancing nitrogen fixation and nutrient uptake in Vicia faba L. Multi-location field experiments were conducted for two years (2016 and 2017) using a split-plot in randomized complete block design. Treatments comprised six rhizobium strains as the main plot factor and three varieties of Vicia faba as the sub-plot factor. Non-inoculated plants with or without N fertilizer served as +N and −N controls, respectively. Peat carrier-based inoculant of each strain was applied at the rate of 10 g kg−1 seed. Data on nodulation were taken at the late-flowering stage, whereas nitrogen and phosphorus concentrations in plant parts were analyzed at physiological maturity. The total nitrogen difference method was employed to quantify nitrogen fixation. Location x rhizobium strain x variety interaction had a significant effect on nodule dry weight plant−1. Rhizobium strains significantly enhanced nodulation, nitrogen fixation, nutrient uptake and soil nitrogen balance. Inoculation with NSFBR-12 and NSFBR-15 resulted in the highest nitrogen fixed, nutrient uptake and soil nitrogen balance. Vicia faba inoculated with the two top performing strains, NSFBR-12 and NSFBR-15 fixed respectively 87.7% and 85.5% of the total nitrogen uptake. Non-inoculated plants fulfilled proportionately more of the total nitrogen uptake through nitrogen derived from the soil rather than fixed nitrogen. Soil available phosphorus and pH had appreciable influences on nitrogen and phosphorus uptake of inoculated Vicia faba. Inoculation with competitive and effective rhizobium strains can improve soil nitrogen balance, nitrogen fixation and nutrient uptake of Vicia faba.

Stress tolerant rhizobia inhabiting the root nodules of Gliricidia sepium from selected locations of Anuradhapura district, Sri Lanka

The symbiotic association between Rhizobium and leguminous species plays a significant role in sustainable agricultural systems as it contributes higher amounts of fixed nitrogen to soil through biological nitrogen fixation. Gliricidia sepium is one of the most commonly cultivated agro forestry trees in the world. This leguminous tree is widely distributed throughout Sri Lanka. A very limited number of studies are available on G. sepium–Rhizobium symbiosis in Sri Lanka. The main objective of this study is to isolate and identify the stress tolerant Rhizobium sp. in G. sepium as a preliminary approach to use rhizobial strains for cross inoculation of crop legumes in order to minimize the heavy use of chemical nitrogen fertilizers. The root nodules of G. sepium were collected from seven sites in Anuradhapura district which is located in the dry zone of Sri Lanka. A total of 34 strains were tested for their tolerance to different physiological conditions namely pH, salinity, drought and temperature. Fifteen strains showed a higher tolerance among the isolated 34 strains when grown under a wide range of physiological conditions. When these 15 stress tolerant rhizobial strains were grown under extreme physiological conditions, 12 strains could survive well as they often experience adverse environmental conditions in their natural habitat. DNA fingerprinting analysis with ERIC 1R and ERIC 2R showed that the 15 strains are genetically diverse and they belong to 9 clusters at 71% similarity level

Inoculation with Different Nitrogen-Fixing Bacteria and Arbuscular Mycorrhiza Affects Grain Protein Content and Nodule Bacterial Communities of a Fava Bean Crop

The introduction of nitrogen fixing bacteria (NFB) and arbuscular mycorrhizal fungi (AMF) into the soil is an advisable agricultural practice for the crop, since it enhances nutrient and water uptake and tolerance to biotic and abiotic stresses. The aim of this work was to study plant nutrition, biological nitrogen fixation (BNF) and crop yield and quality, after inoculating seeds with NFBs ((Rhizobium leguminosarum, Burkholderia cenocepacia, Burkholderia vietnamiensis)) and/or AMFs (Rhizophagus irregularis, Claroideoglomus etunicatum, Claroideoglomus claroideum and Funneliformis mosseae) in a fava bean crop in two seasons. The composition of the nodule bacterial community was evaluated by the high-throughput sequencing analysis of bacterial 16 S rRNA genes. It was found that microbial inoculation accompanied by a 20% decrease in mineral fertilization had no significant effect on crop yield or the nutritional characteristics compared with a non-inoculated crop, except for an increase in the grain protein content in inoculated plants. None of the inoculation treatments increased biological nitrogen fixation over a non-inoculated level. The bacterial rRNA analysis demonstrated that the genus Rhizobium predominated in all nodules, both in inoculated and non-inoculated treatments, suggesting the previous presence of these bacteria in the soil. In our study, inoculation with Rhizobium leguminosarum was the most effective treatment for increasing protein content in seeds, while Burkholderia sp. was not able to colonise the plant nodules. Inoculation techniques used in fava beans can be considered an environmentally friendly alternative, reducing the input of fertilizers, while maintaining crop yield and quality, with the additional benefit of increasing the grain protein content. However, further research is required on the selection and detection of efficient rhizobial strains under local field conditions, above all those related to pH and soil type, in order to achieve superior nitrogen-fixing bacteria.

Agronomical parameters of host and non-host legumes inoculated with Melilotus indicus-isolated rhizobial strains in desert unreclaimed soil.

In a search for identification of rhizobial strains with superior N2-fixation efficiency and improved plant agronomic characteristics upon inoculation, four strains, 4.21, 9.17, 11.2 and 14.1, isolated from root nodules of wild-grown Melilotus indicus have been used to inoculate field-grown common bean, pea, cowpea and fenugreek plants. Uninoculated plants and those inoculated with host-specific commercial inoculants were used as a control. The root length, shoot height, shoot dry weight and root dry weight and the grain yield of the plants were determined after harvest. The content of N, organic C and carbohydrates content of the grain were also recorded. The inoculation with the strains 4.21 and 14.1 increased the grain yield of the fenugreek compared both with the uninoculated plants and those inoculated with the commercial strain ARC-1. The grain yield of the common bean treated with the strains 9.17 and 14.1 was also higher than that of the uninoculated and the commercial strains ARC-301. In contrast, none of the strains increased the grain yield of the pea and cowpea plants compared to the commercial strains ARC-201 and ARC-169, respectively. Significant increases of some agronomical parameters were observed in some plant-bacterium couples, albeit nodulation was not observed. It is possible that the positive effects of rhizobial inoculation on the agronomical parameters of the non-nodule forming legumes could be due to plant growth promotion characteristic of the strains used for inoculation. Analysis of the phylogeny of the almost complete 16S rRNA sequence of the rhizobial inoculants revealed that the strains 4.21 and 9.17 clustered together with R. skierniewicense and R. rosettiformans, respectively, and that the strains 11.2 and 14.1 grouped with E. meliloti. All the four strains produced IAA, and showed biocontrol activity against Rhizotocnia solani, Fusarium oxysporum, Pythium ultimum, Alternaria alternata and Sclerotonia rolsfi, albeit to a different extent.

Biochemical, functional and molecular characterization of pigeon pea rhizobia isolated from semi-arid regions of India.

Pigeon pea (Cajanus cajan (L.) Millspaugh) is among the top ten legumes grown globally not only having high tolerance to environmental stresses along, but also has the high biomass and productivity with optimal nutritional profiles. In the present study, 55 isolates of rhizobia were identified from 22 nodule samples of pigeon pea collected from semi-arid regions of India on the basis of morphological, biochemical, plant growth promoting activities and their ability to tolerate the stress conditions viz. pH, salt, temperature and drought stress. Amongst all the 55 isolates, 37 isolates showed effective nodulation under in vitro conditions in pigeon pea. Further, five isolates having multiple PGP activities and high in vitro symbiotic efficiency were subjected to 16S rRNA sequencing and confirmed their identities as Rhizobium, Mesorhizobium, Sinorhizobium sp. Further these 37 isolates were characterized at molecular level using ARDRA and revealed significant molecular diversity. Based on UPGMA clustering analysis, these isolates showed significant molecular diversity. The high degree of molecular diversity is due to mixed cropping of legumes in the region. The assessment of genetic diversity and molecular characterization of novel strains is a very important tool for the replacement of ineffective rhizobial strains with the efficient strains for the improvement in the nodulation and pigeon pea quality. The pigeon pea isolates with multiple PGPR activities could be further used for commercial production.