Evaluation of Symbiotic Association between Various Rhizobia, Capable of Producing Plant-Growth-Promoting Biomolecules and Mung Bean for Sustainable Production

To feed the increased world population, sustainability in the production of crops is the need of the hour, and exploration of an effective symbiotic association of rhizobia with legumes may serve the purpose. A laboratory-scale experiment was conducted to evaluate the symbiotic effectiveness of twenty wild rhizobial isolates (MR1–MR20) on the growth, physiology, biochemical traits, and nodulation of mung bean to predict better crop production with higher yields. Rhizobial strain MR4 resulted in a 52% increase in shoot length and 49% increase in shoot fresh mass, while MR5 showed a 30% increase in root length, with 67% and 65% improvement in root fresh mass by MR4 and MR5, respectively, compared to uninoculated control. Total dry matter of mung bean was enhanced by 73% and 68% with strains MR4 and MR5 followed by MR1 and MR3 with 60% increase in comparison to control. Rhizobial strain MR5 produced a maximum (25 nodules) number of nodules followed by MR4, MR3, and MR1 which produced 24, 23, and 21 nodules per plant. Results related to physiological parameters showed the best performance of MR4 and MR5 compared to control among all treatments. MR4 strain helped the plants to produce the lowest values of total soluble protein (TSP) (38% less), flavonoids contents (44% less), and malondialdehyde (MDA) contents (52% less) among all treatments compared to uninoculated control plants. Total phenolics contents of mung bean plants also showed significantly variable results, with the highest value of 54.79 mg kg−1 in MR—inoculated plants, followed by MR5- and MR1-inoculated plants, while the minimum concentration of total phenolics was recorded in uninoculated control plants of mung bean. Based on the results of growth promotion, nodulation ability, and physiological and biochemical characteristics recorded in an experimental trial conducted under gnotobiotic conditions, four rhizobial isolates (MR1, MR3, MR4, and MR5) were selected using cluster and principal component analysis. Selected strains were also tested for a variety of plant-growth-promoting molecules to develop a correlation with the results of plant-based parameters, and it was concluded that these wild rhizobial strains were effective in improving sustainable production of mung bean.

Enrichment of Restraint to Endmost Salinity and Outmost pH Circumstance in Common Bean (Phaseolus vulgaris L.) Nodulating Rhizobial Isolates from Eastern Hararghe Lowlands around Babile, Eastern Ethiopia, through Mutagenesis.

This study was committed to examining the symbiotic efficacy difference between wild and mutant rhizobial isolates from lowlands of eastern Ethiopia around Babile, and at the same time to appreciate the enrichment effect of mutagenesis on restraint of common bean (Phaseolus vulgaris L.) nodulating rhizobia to Endmost Salinity and Outmost pH Circumstance. A total of 50 wild rhizobia isolate, and 8 mutant isolates; after mutagenesis were selected based on their ability to endure endmost salt and outmost pH circumstances at laboratory condition. All of the mutants were found to be symbiotically effective. Among the observed rhizobium isolates, wild isolates HUCR (3D, 3A), HUCR 2D, and mutant isolate HUCRM 2D showed the highest symbiotic efficacy. Only the mutant isolates HUCRM2D (tolerated 12% NaCl, and pH4 and pH12), HUCRM5C (tolerated 12% NaCl and pH 4), HUCRM3B (tolerated 12% NaCl) and HUCRM9C (tolerated 11% NaCl) were growing effectively at the point out utmost conditions. Therefore, based on their restraint to utmost conditions and symbiotic efficacy, the above mentioned wild and mutant rhizobia isolates were suggested as nominees for the future development of common bean biofertilizer that grown under endmost saline and outmost pH conditions.

Isolation and Assessment of the Symbiotic Efficiency of Faba bean (Vicia faba L.) Nodulating Rhizobial Isolates from Eastern Hararghe Highlands of Ethiopia

This particular work was devoted to isolate and assess the symbiotic efficiency of faba bean (Vicia faba L.)-nodulating rhizobia isolate at few faba bean growing areas of the eastern Hararghe highlands of Ethiopia. Overall 50 rhizobia isolates were obtained from soil samples of three Woredas (districts) of the eastern Hararghe highlands using the host trap method. Out of these 50 isolates, 40 were presumptively identified as rhizobia. Among these 40 rhizobia isolates, only 31 were successful to nodulate faba bean, and authenticated as true faba bean nodulating rhizobia. Concerning the symbiotic efficiency, about 52%, 35%, and 13% of the rhizobial isolates were found to be highly effective, effective, and lowly-effective, respectively. The correlation data on the sand experiment displayed that nodule dry weight was associated positively and significantly (r = 0.494, p<0.05) with shoot dry weight while shoot dry weight was associated positively and significantly (r=0.41, p<0.05) with plant total nitrogen. Positive correlations were also observed concerning shoot dry weight and dry weight of nodules (r = 0.7, p<0.05) on unsterilized soil. Among the observed rhizobium isolates, EHHFR (4A, 6A) showed the highest symbiotic efficiency above 110%, tolerated NaCl concentration ranging from 2% to 6% and 2% to 8%, respectively, and a pH range of 4.5 to 8 and 5 to 8, respectively. Thus, based on their symbiotic efficiency at the greenhouse level and relative tolerance to extreme conditions these faba bean nodulating rhizobia isolates were recommended to be used as nominees for the future development of faba bean rhizobial inoculants after being tested on field conditions.

Functional and genetic diversity of native rhizobial isolates nodulating cowpea (Vigna unguiculata L. Walp.) in Mozambican soils

Identification and symbiotic characterization of indigenous rhizobial isolates are the basis for inoculant formulations needed for sustainable grain legume production. This study screened for morpho-genetic diversity of indigenous cowpea nodulating rhizobia in farmers’ fields across two contrasting agroecological zones of Northern Mozambique. The photosynthetic function induced by the isolates in their homologous cowpea was assessed. The results showed high genetic variability among the isolates based on morphology and ERIC-PCR fingerprinting. The trap cowpea genotype did not influence the diversity of isolates collected from the two different agroecologies, suggesting that the cowpea-rhizobia compatibility may be conserved at species level. Phylogenetic analysis of the 16S rRNA gene assigned representative rhizobial isolates to species in the Bradyrhizobium and Rhizobium genera, with some isolates showing high divergence from the known reference type strains. The isolates from both agroecologies highly varied in the number and biomass of nodules induced in the homologous cowpea, resulting in variable plant growth and photosynthetic activities. A total of 72% and 83% of the isolates collected from the agroecological zones 7 and 8 were respectively classified as highly effective candidates with > 80% relative effectiveness compared to plants fertilized with nitrate, indicating that elite native strains populated the studied soils. Moreover, the top 25% of high N2-fixing isolates from the two agroecologies recorded relative effectiveness ranging from 115 to 154%, values higher than the effectiveness induced by the commercial Bradyrhizobium sp. strain CB756. These strains are considered as having potential for use in inoculant formulations. However, future studies should be done to assess the ecologically adaptive traits and symbiotic performance under field conditions.

Dominant associations of Ensifer medicae-Medicage polymorpha and Ensifer meliloti-Medicago lupulina in farmland and natural ecosystem

Aims The nitrogen-fixing rhizobia associated with Medicago polymorpha and M. lupulina in Yunnan, China have been poorly documented. This study aims to analyze the diversity of rhizobia isolated from these two Medicago species and investigate the impact of abiotic (soil properties) and biotic (plant hosts) factors on Medicago-associated rhizobia in this region. Methods 91 rhizobial isolates were characterized by RFLP of 16S rDNA and 16S–23S IGS, BOX-PCR fingerprinting, nodulation assays and phylogeny analyses based on housekeeping and symbiosis genes. The genetic diversity of the rhizobial isolates was assessed by the BOX AIR pattern and Shannon index. Additionally, the correlation of soil properties and rhizobial distribution was determined by the constrained analysis of principle coordinates (CAP) based on Bray-Curtis distance of presence/absence (PA) transformed species data. Results All the tested strains fell in the genus Ensifer and divided into two species, E. medicae and E. meliloti. Both host plants and soil properties contributed to the rhizobial diversity. For either E. meliloti or E. medicae, isolates from native host plants tended to be more genetically diverse than those of the same species from non-native hosts. The soil edaphic factor analysis elucidated that nitrogen, organic matter as well as Ca2+ and Na+ are the key factors to shape the biogeographical distribution of rhizobia. Conclusions This study evidenced the microsymbiont preference of M. polymorpha to E. medicae and M. lupulina to E. meliloti, but also revealed the considerable impacts of both plant hosts and soil factors on the rhizobial diversity and biodistribution.

Productivity Improvement of Faba Bean (Vicia faba L.) through Elite Rhizobial Inoculants in the Central Highlands of Ethiopia

This study envisioned to assess the inoculation response of faba bean to six indigenous rhizobial isolates, 100 kg ha-1 DAP as a positive control and non-inoculated negative control under farmers’ field conditions at Welmera district, Ethiopia. The results revealed the presence of significantly different treatments (p ≤ 0.05) in grain yield. The highest grain yields (3966 kg ha-1and 3694 kg ha-1) were obtained from inoculation of rhizobial isolates (FB-AR-13 and FB-1018) during the 2019/20 and 2020/21 cropping seasons. Based on the two successive year’s average grain yield response, FB-AR-13 (3966 kg ha-1) became the first most performing isolate. The partial budget analysis results also showed that FB-AR-13 was the most promising isolate that showed a higher MRR 6422 %, which is 43% higher than the MRR of the local standard check FB-1018. Therefore, isolate FB-AR-13 is the best promising candidate for the development of commercial faba bean rhizobial inoculant in the central high lands of Ethiopia after further verification over different farmers' fields at different agro-ecologies.

Genetic characterization at the species and symbiovar level of indigenous rhizobial isolates nodulating Phaseolus vulgaris in Greece

Phaseolus vulgaris (L.), commonly known as bean or common bean, is considered a promiscuous legume host since it forms nodules with diverse rhizobial species and symbiovars. Most of the common bean nodulating rhizobia are mainly affiliated to the genus Rhizobium, though strains belonging to Ensifer, Pararhizobium, Mesorhizobium, Bradyrhizobium, and Burkholderia have also been reported. This is the first report on the characterization of bean-nodulating rhizobia at the species and symbiovar level in Greece. The goals of this research were to isolate and characterize rhizobia nodulating local common bean genotypes grown in five different edaphoclimatic regions of Greece with no rhizobial inoculation history. The genetic diversity of the rhizobial isolates was assessed by BOX-PCR and the phylogenetic affiliation was assessed by multilocus sequence analysis (MLSA) of housekeeping and symbiosis-related genes. A total of fifty fast-growing rhizobial strains were isolated and representative isolates with distinct BOX-PCR fingerpriniting patterns were subjected to phylogenetic analysis. The strains were closely related to R. anhuiense, R. azibense, R. hidalgonense, R. sophoriradicis, and to a putative new genospecies which is provisionally named as Rhizobium sp. I. Most strains belonged to symbiovar phaseoli carrying the α-, γ-a and γ-b alleles of nodC gene, while some of them belonged to symbiovar gallicum. To the best of our knowledge, it is the first time that strains assigned to R. sophoriradicis and harbored the γ-b allele were found in European soils. All strains were able to re-nodulate their original host, indicating that they are true microsymbionts of common bean.

Rhizobial isolates in active layer samples of permafrost soil of Spitsbergen, Arctic

Twenty-nine strains were isolated from two samples of the permafrost active layer of the Spitsbergen archipelago. The estimated number of bacteria ranged from 4.0⋅104 to 1.7⋅107 CFU∙g-1. As a result of sequencing of the 16S rRNA (rrs) genes, the isolates were assigned to 13 genera belonging to the phyla Actinobacteria, Proteobacteria (classes α, β, and γ), Bacteroidetes, and Firmicutes. Six isolates belonged to the rhizobial genus Mesorhizobium (order Rhizobiales). A plant nodulation assay with seedlings of legume plants Astragalus norvegicus, A. frigidus, A. subpolaris and Oxytropis sordida, originated from Khibiny (Murmansk region, Russia) and inoculated with Mesorhizobium isolates, showed the inability of these strains to form nodules on plant roots. Symbiotic (sym) genes nodC and nodD were not detected in Mesorhizobium strains either.

Molecular Characteristics of Rhizobia Isolated from Arachis hypogaea Grown under Stress Environment

The phenotypic and genotypic characterization of eight rhizobial isolates obtained from Arachis hypogaea nodules grown under stress environment was performed. Isolates were screened for their ability to tolerate different abiotic stresses (high temperature (60° C), salinity (1–5% (w/v) NaCl), and pH (1–12). The genomic analysis of 16S rRNA and housekeeping genes (atpD, recA, and glnII) demonstrated that native groundnut rhizobia from these stress soils are representatives of fast growers and phylogenetically related to Rhizobium sp. The phenotypic characterization (generation time, carbon source utilization) also revealed the isolates as fast-growing rhizobia. All the isolates can tolerate NaCl up to 3% and were able to grow between 20 and 37 °C with a pH between 5 to 10, indicating that the isolates were alkali and salt-tolerant. The tested isolates effectively utilize mono and disaccharides as carbon source. Out of eight, three rhizobial isolates (BN-20, BN-23, and BN-50) were able to nodulate their host plant, exhibiting their potential to be used as native groundnut rhizobial inoculum. The plant growth promoting characterization of all isolates revealed their effectiveness to solubilize inorganic phosphate (56–290 µg mL−1), synthesize indole acetic acid (IAA) (24–71 µg mL−1), and amplification of nitrogen fixing nifH gene, exploring their ability to be used as groundnut biofertilizer to enhance yield and N2-fixation for the resource poor farmers of rainfed Pothwar region.