Enhancement of soybean tolerance to drought using Rhizobium species and mycorrhizal fungi

Abstract

Food security is seriously under threat in many developing tropical countries as agriculture is becoming unsustainable due to drought stress. There is therefore an urgent need for a sustainable means to accomplish food availability in this region. Thus, the aim of this study was to evaluate the ability of new Rhizobium spp. and arbuscular mycorrhizal fungi (AMF) to enhance soybean (Glycine max L.) tolerance to drought. In this present study rhizobial strains were isolated from Bambara groundnut (Vigna subterranea) rhizospheric soil and their mechanisms in relation to enhancement of plant growth and drought tolerance were evaluated. Isolates were characterized and identified by culture based and molecular techniques as Rhizobium sp. strain R1, Rhizobium tropici strain R2, Rhizobium cellulosilyticum strain R3, Rhizobium taibaishanense strain R4 and Sinorhizobium meliloti strain R5. Rhizobial strains were positive to almost all the plant growth promoting traits (such as exopolysaccharide, siderophore and indole-acetic-acid) tested. Rhizobial strains also survived and grew under stress conditions imposed by polyethylene glycol (PEG) but Rhizobium sp. strain R1 and R. cellulosilyticum strain R3 showed the highest significant tolerance with optical density (OD) values of 1.35 and 0.32 respectively at a concentration of 30% PEG while S. meliloti had the lowest OD value of 0.17 at the same PEG concentration. Rhizobium sp. strain R1 and R. cellulosilyticum strain R3 inoculation of soybean significantly (P <0.05) enhanced seedling shoot dry weights under drought condition imposed by 4% PEG. Thus, genomic insights into Rhizobium sp. strain R1 and R. cellulosilyticum strain R3 revealed the presence of some genes with their respective proteins involved in symbiotic establishment, nitrogen fixation, drought tolerance and plant growth promotion. In particular, exoX, htrA, Nif, nodA, eptA, IAA and siderophore-producing genes were found in the two rhizobial strains. A total of 5773 contigs with a GC content of 61.91% and a total of 17,408,810 reads with a mean read length of 201.15 were obtained for Rhizobium sp. strain R1 while a total of 129 contigs with GC content of 43.59% and total of 17,794,094 reads with a mean read length of 214.18 were found in R. cellulosilyticum strain R3. Rhizobium sp. strain R1, R. cellulosilyticum strain R3 and mycorrhizal consortium (MY) were therefore used to inoculate soybean under drought conditions in the greenhouse. A gradient of watering levels ranging from 100 to 40% field capacity (FC) of soil retention capacity of water was tested on non-inoculated soybean plants and plants inoculated with rhizobia and AMF in pot cultures. It was observed that the inoculated soybean plants especially soybean dually inoculated with Rhizobium sp. strain R1 and R. cellulosilyticum strain R3 (R1+R3) as well as Rhizobium sp. strain R1, R. cellulosilyticum strain R3 and mycorrhizal consortium (R1+R3MY) had significant impacts (P < 0.05) on soybean leaf relative water content (RWC) and electrolyte leakage respectively. Also, the levels of accumulated soluble sugars and proline revealed that their concentrations increased mainly in microbially amended soybean plants exposed to drought stress (70 and 40% FC) and similar results were observed for chlorophyll content. Plants inoculated with R1+R3MY showed the highest number of spore and % mycorrhization in all the water regimes. At 40% FC, R1+R3MY treatment was found to promote soybean growth since it had significant effects (P < 0.05) on soybean shoot width, branch number, and root dry weight compared to the non-inoculated plants. Similarly, under severe drought stress (40% FC), R1+R3MY inoculum had the greatest impacts on soybean pod number, seed number, seed fresh weight, highest seed number per pod and seed dry weight while under 70% water stress, significant impacts (P<0.05) of Rhizobium sp. strain R1 and mycorrhizal (R1MY) co-inoculation were observed on pod number, pod fresh weight and seed dry weight. Rhizobium sp. strain R1, R. cellulosilyticum strain R3 and mycorrhizal fungal inoculation of soybean in semi-arid field equally enhanced soybean tolerance to drought stress. Single and dual inoculation of the Rhizobium species and mycorrhizal fungi actually increased leaf and shoot RWC and decreased electrolyte leakage and increased soluble sugars and proline accumulation in soybean plants. Rhizobial and mycorrhizal inoculation also increased below-ground and above-ground plant components (such as shoot height and width, leaf number, taproot length, root number and pod number) at different stages of soybean growth but more significant increase (P<0.05) was observed in plants dually inoculated with R1+R3MY. In particular, soybean plants amended with R1+R3MY produced seeds with 34.3 g fresh weight, 15.1 g dry weight and 23% crude fat and soybean plants singly inoculated with Rhizobium sp. strain R1 (R1) produced more large seeds with 12.03 g dry weight. The non-inoculated (control) seeds contained higher percentage of moisture content compared to the microbially amended seeds. Increase in macro and micronutrient assimilation especially N, P, Mg, S, Ca, Co, Mo, Fe and B in soybean seeds inoculated with Rhizobium species and mycorrhizal fungi was also observed. However, co-inoculation with R1MY resulted in a significant increase (P<0.05) in almost all the macronutrients (N, P, Mg and Ca). A study of the bacterial communities of soybean rhizosphere inoculated with R1+R3MY in the field using Next Generation Sequencing showed variations in the richness and abundance of bacteria during soybean growth. Stack bar/area plot and Heatmap clustering analysis unveiled variations among the rhizospheric bacterial communities at the class and order levels respectively. Particularly, Actinobacteria was the most abundant bacterial group with the highest reads counts observed at full seed (FS) stage followed by Proteobacteria which contained plant growth promoting bacterial species such as Streptomyces and Rhizobium species. Alpha-diversity and Bray Curtis Index analyses at the family, genus, species and operational taxonomic units (OTUs) levels similarly showed that bacterial composition and abundance of the rhizosphere changed significantly during soybean growth. These results revealed that rhizosphere bacterial community structured varied at different growth stages of soybean in the field. In conclusion, this study has revealed two Rhizobium species (Rhizobium sp. strain R1 and R. cellulosilyticum strain R3) with exoX, htrA, Nif, nodA, eptA, IAA and siderophore-producing genes that were able to enhance soybean tolerance to drought stress even when co-inoculated with mycorrhizal fungi. It is therefore recommended that the availability of the whole genome sequences of Rhizobium sp. strain R1 and R. cellulosilyticum strain R3 strains in public databases may further be exploited to comprehend the interaction of drought tolerant rhizobia with soybean and other legumes and the plant growth promoting ability of these rhizobial strains can also be harnessed for biotechnological application in the field especially in semiarid and arid regions of the globe.