Competitiveness of Bradyrhizobium japonicum inoculation strain for soybean nodule occupancy

The competitiveness of Bradyrhizobium japonicum inoculation strain against indigenous rhizobia was examined in a soil pot experiment. The effect of inoculation strain was evaluated under different soil conditions: with or without previously grown soybean and applied commercial inoculant. Molecular identification of inoculation strain and investigated rhizobial isolates, obtained from nodules representing inoculated treatments, was performed based on 16S rDNA and enterobacterial repetitive intergenic consensus (ERIC) sequencing. Inoculation strain showed a significant effect on the investigated parameters in both soils. Higher nodule occupancy (45% vs. 18%), nodule number (111% vs. 5%), nodule dry weight (49% vs. 9%), shoot length (15% vs. 7%), root length (31% vs. 13%), shoot dry weight (34% vs. 11%), shoot nitrogen content (27% vs. 2%), and nodule nitrogen content (9% vs. 5%) was detected in soil without previously grown soybean and applied commercial inoculant. Soil had a significant effect on the shoot, root and nodule nitrogen content, while interaction of experimental factors significantly altered dry weight and nitrogen content of shoots, roots and nodules, as well as number of nodules. Nodulation parameters were significantly related with shoot dry weight, shoot and nodule nitrogen content. Symbiotic performance of inoculation strains in the field could be improved through co-selection for their competitiveness and effectiveness.

Bradyrhizobium japonicum FN1 produces an inhibitory substance that affects competition for nodule occupancy

Bacteriocins are narrow spectrum antibiotics of bacterial origin that can affect competition in resource-limited environments such as the rhizosphere. Therefore, bacteriocins may be good candidates for manipulation in order to generate more competitive inocula for soybean. In this study,<i> B. japonicum</i> FN1 along with other Bradyrhizobia in our culture collection were screened for bacteriocin-like activity. A total of five distinct inhibitory activities could be observed. FN1 genes putatively involved in bacteriocin production were computationally identified. These genes were mutagenized and the subsequent strains were screened for loss of inhibitory activity. Mutant strain BRJ-48, with an insert in<i> bjfn1_01204</i>, displayed a loss of the ability to inhibit an indicator strain. This loss could be complemented by the introduction of a plasmid expressing <i>bjfn1_01204 </i>in trans. The strain carrying the mutation did not affect competition in broth cultures, but was shown to be less competitive for nodule occupancy. Annotation suggests that <i>bjfn1_01204</i> encodes a carboxymuconolactone decarboxylase, however the direct contribution of how this enzyme contributes to inhibiting the tester strain remains unknown.

Competition, Nodule Occupancy, and Persistence of Inoculant Strains: Key Factors in the Rhizobium-Legume Symbioses

Biological nitrogen fixation by Rhizobium-legume symbioses represents an environmentally friendly and inexpensive alternative to the use of chemical nitrogen fertilizers in legume crops. Rhizobial inoculants, applied frequently as biofertilizers, play an important role in sustainable agriculture. However, inoculants often fail to compete for nodule occupancy against native rhizobia with inferior nitrogen-fixing abilities, resulting in low yields. Strains with excellent performance under controlled conditions are typically selected as inoculants, but the rates of nodule occupancy compared to native strains are rarely investigated. Lack of persistence in the field after agricultural cycles, usually due to the transfer of symbiotic genes from the inoculant strain to naturalized populations, also limits the suitability of commercial inoculants. When rhizobial inoculants are based on native strains with a high nitrogen fixation ability, they often have superior performance in the field due to their genetic adaptations to the local environment. Therefore, knowledge from laboratory studies assessing competition and understanding how diverse strains of rhizobia behave, together with assays done under field conditions, may allow us to exploit the effectiveness of native populations selected as elite strains and to breed specific host cultivar-rhizobial strain combinations. Here, we review current knowledge at the molecular level on competition for nodulation and the advances in molecular tools for assessing competitiveness. We then describe ongoing approaches for inoculant development based on native strains and emphasize future perspectives and applications using a multidisciplinary approach to ensure optimal performance of both symbiotic partners.

Involvement of a Novel TetR-Like Regulator (BdtR) of Bradyrhizobium diazoefficiens in the Efflux of Isoflavonoid Genistein

A wide variety of leguminous plant-released (iso)flavonoids, such as genistein, are potential inducers of the nodulation (nod) genes of endosymbiotic rhizobia for the production of Nod factors, which are vital signaling molecules for triggering the symbiotic process. However, these (iso)flavonoids are generally thought to be toxic to the bacterial partner to varying degrees. Here, a novel TetR-like regulator gene of the soybean symbiont Bradyrhizobium diazoefficiens USDA110, bdtR (systematic designation blr7023), was characterized. It was found to be rapidly and preferentially induced by genistein, and its mutation resulted in significantly increased expression of the neighboring bll7019-bll7021 genes, encoding a multidrug resistance efflux pump system, in the absence of this isoflavonoid. Then, the transcriptional start site of BdtR was determined, and it was revealed that BdtR acted as a transcriptional repressor of the above efflux system through the binding of an AT-rich operator, which could be completely prevented by genistein. In addition, the ΔbdtR deletion mutant strain showed higher accumulation of extracellular genistein and became less susceptible to the isoflavonoid. In contrast, the inactivation of BdtR led to the significantly decreased induction of a nodulation gene (nodY) independent of the expression of nodD1 and nodW and to much weaker nodulation competitiveness. Taken together, the results show that BdtR plays an early sensing role in maintaining the intracellular homeostasis of genistein, helping to alleviate its toxic effect on this bacterium by negatively regulating neighboring genes encoding an efflux pump system while being essentially required for nodule occupancy competitiveness. [Formula: see text] Copyright © 2020 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

CHARACTERIZATION OF SYMBIOTIC EFFECTIVENESS OF RHIZOBIA NODULATING FIELD PEA (PISUM SATIVUM).

Field Pea is one of the most important Legumes plants and widely grown in Ethiopia. A study was made to re- isolate, characterize, and select best rhizobia for field pea. Results showed that all the 25 isolates exhibited typical colony characteristics and presumptive reactions of fast growing rhizobia. Out of the 25 isolates, 3(KL3, BR1 andCF5) relatively superior isolates were selected in sterilized sand. All isolates characterized their morphological and physiological characteristics. All isolates formed watery and mucoid colonies on YEMA medium, their mean growth time mostly between 2 &4 hours and failed to grow on peptone glucose agar medium and to solubilize inorganic phosphate. Almost all isolates were tolerated to pH 5to 9, 2% and 3% salt concentration, and at temperature of 15oC to 35oC. The isolates were also tolerant to erythromycin, streptomycin and ampicillin, and relatively sensitive to penicillin and chloroamphenicol at concentration of 50μg/ml. All isolates utilized to sucrose, glucanate, galactose and fructose as the sole source of carbon, and almost all isolates grow on YEMA medium containing galactose (90%), fructose (88.9%) and glucanate (76.7%) and the isolates utilized many amino acids as the source of nitrogen. BR1 was the most competitive inoculant with nodule occupancy of 75%; followed by KL3 and CF5 with nodule occupancy of 60 and 50% respectively. The mean nodule number, nodule dry weight, mean shoot dry weight and N content and of the host plants inoculated with different isolates showed variations. Particularly BR1 can be recommended as inoculants and good strain for field pea in the future.

Competitiveness prediction for nodule colonization in Sinorhizobium meliloti through combined in vitro tagged strain characterization and genome-wide association analysis

ABSTRACTAssociations between leguminous plants and symbiotic nitrogen fixing bacteria (rhizobia) are a classical example of mutualism between a eukaryotic host and a specific group of prokaryotic microbes. Though being in part species-specific, different strains may colonize the same plant symbiotic structure (nodule). It is known that some rhizobial strains are better competitor than others, but detailed analyses aimed to predict from the rhizobial genome its competitive abilities are still scarce. Here we performed a bacterial genome wide association (GWAS) analysis to define the genomic determinants related to the competitive capabilities in the model rhizobial species Sinorhizobium meliloti. Thirteen tester strains were GFP-tagged and assayed against three reference competitor strains RFP-tagged (Rm1021, AK83 and BL225C) in a Medicago sativa nodule occupancy test. Competition data in combination with strains genomic sequences were used to build-up a model for GWAS based on k-mers. The model was then trained and applied for competition capabilities prediction. The model was able to well predict the competition abilities against two partners, BL225C, Rm1021 with coefficient of determination of 0.96 and 0.84, respectively. Four strains showing the highest competition phenotypes (> 60% single strain nodule occupancy; GR4, KH35c, KH46 and SM11) versus BL225C were used to identify k-mers associated with competition. The k-mers with highest scores mapped on the symbiosis-related megaplasmid pSymA and on genes coding for transporters, proteins involved in the biosynthesis of cofactors and proteins related to metabolism (i.e. glycerol, fatty acids) suggesting that competition abilities reside in multiple genetic determinants comprising several cellular components.IMPORTANCEDecoding the competitive pattern that occurs in the rhizosphere is challenging in the study of bacterial social interaction strategies. To date, single-gene approach has been mainly used to uncover the bases of nodulation, but there is still a gap about the main features that a priori turn out rhizobial strains able to outcompete indigenous rhizobia. Therefore, tracking down which traits make different rhizobial strains able to win the competition for plant infection over other indigenous rhizobia will allow ameliorating strain selection and consequently plant yield in sustainable agricultural production systems. We have proven that a k-mer based GWAS approach can effectively predict the competition abilities of a panel of strains, which were analyzed for their plant tissue occupancy by using double fluorescent labeling. The reported strategy could be used for detailed studies on the genomic aspects of the evolution of bacterial symbiosis and for an extensive evaluation of rhizobial inoculants.

Nodule Occupancy Behaviour of Bacteriocinogenic Rhizobium spp. in Mungbean (Vigna radiata)

A pot culture study was undertaken to determine the synergistic effect of bacteriocinogenic Rhizobium spp. (N8, S1, S6 and S13 isolated from mungbean fields) with Indicator Rhizobium spp. (mungbean biofertilizer) in terms of nodulation, plant growth traits and grain yield in mungbean (SML 668). Dual inoculation of bacteriocinogenic Rhizobium isolates N8 with indicator strain (indicator+N8) resulted significant increase in number of nodules (24.3 nn/plant), nodule index (1.36), nodule dry weight (23.4 mg/plant), dry weight of shoot (0.79 g/plant), dry weight of root (1.65 g/plant), chlorophyll content (1.34 mg/g of leaves), leghemoglobin content (3.43 mg/g of nodules), number of pods (15.9 /plant), number of seeds (13.8 /pods), seed set percent (86.73%), plant height (96 cm), yield (1.69 g/plant), nitrogen content of grains (4.02%) as compared to indicator strain alone. Nodule occupancy was assessed by comparing the total viable count of nodule microbiota, which demonstrated the domination of bacteriocin producers. Bacteriocinogenic Rhizobium N8 strain can be explored as potent bio-fertilizers along with indicator Rhizobium spp. in mungbean.

Increasing biological nitrogen fixation by white clover-rhizobia symbiosis

Biological nitrogen fixation (BNF) is the process of converting atmospheric nitrogen to ammonia through legume–rhizobia symbiosis. The nitrogen fixed by rhizobia in root nodules is available for plant use. This process can be harnessed to improve N fertility on farm. Field surveys across New Zealand (NZ), within a farm and within paddocks, have revealed large spatial variability of rhizobial population size and symbiotic effectiveness with white clover. These results indicate that naturalised rhizobia may not be supporting optimal BNF. Over 500 strains of clover-nodulating rhizobia were isolated from NZ pasture soils, with more than 90 demonstrating greater N-fixation capacity with white clover than the commercial inoculant strain TA1. Seven NZ isolates were tested for nodule occupancy and all seven had significantly higher occupancy rates than TA1 in an in vitro assay, indicating increased competitiveness of those strains. In addition, novel seed-coating technology improved the survival of TA1 and isolate S10N9 from 1 month to more than 4 months compared with a standard coating formulation. There is potential to increase the symbiotic capacity of white clover in pastures through use of more effective and competitive rhizobial strains, along with their improved survival on seed provided by a new coating technology.

Competitiveness among Rhizobia for Nodulation of Introduced Leguminous Tree Gliricidia sepium in a Sub-Saharan Sandy Soil

Agroforestry systems are progressively integrated by small farmer holder to mitigate agricultural production charge and contribute to sustainable agriculture by restoring and maintaining sandy soil fertility. A greenhouse experiment was carried out to test the nodulation level of introduced Gliricidia sepium tree with foreign rhizobial reference strains TAL1769 and TAL1770 against native rhizobial community using PCR/RFLP techniques. Restriction patterns of the two inoculated reference strains obtained for 16S – 23S rDNA - IGS were different to those of indigenous rhizobia detected in all remained nodules collected from root plants regarding the restriction enzymes HaeIII and MspI. Nodule occupancy rates of the reference strains and native rhizobial strains of profile F ranged from 18.36 to 22.45 and were higher compared to others rhizobial strains detected in gliricidia rhizosphere. Therefore, regarding its high competitiveness level, the native rhizobial strain may be considered as a good candidate to inoculate the introduced legume tree gliricidia.