Co-inoculation of rhizobia and AMF improves growth, nutrient uptake, and cadmium resistance of black locust grown in sand culture.

Rhizobia and arbuscular mycorrhizal fungi (AMF) are symbiotic microorganisms important for plants grown in nutrient-deficient and heavy metal-contaminated soils. However, it remains unclear how plants respond to the coupled stress by heavy metal and nitrogen (N) deficiency under co-inoculation. Here, we investigated the synergistic effect of Mesorhizobium huakuii QD9 and Funneliformis mosseae on the response of black locust (Robinia pseudoacacia L.) grown in sand culture to cadmium (Cd) under N deficiency conditions. The results showed that single inoculation of AMF improved the growth and Cd resistance of black locust, co-inoculation improved the most. Compared to non-inoculated controls, co-inoculation mediated higher biomass and antioxidant enzyme activity, reduced oxidative stress, and promoted nodulation, mycorrhizal colonization, photosynthetic capacity, and N, P, Fe and Mg acquisition when exposed to Cd. This increase was significantly higher under N deficiency compared to N sufficiency. In addition, the uptake of Cd by co-inoculated black locust roots increased, but Cd translocation to the above-ground decreased under both N deficiency and sufficiency. Thus, in the tripartite symbiotic system, not merely metabolic processes but also Cd uptake increased under N deficiency. However, enhanced Cd detoxification in the roots and reduced allocation to the shoot likely prevent Cd toxicity and rather stimulated growth under these conditions.

Physiological responses of low- and high-cadmium accumulating Robinia pseudoacacia-rhizobium symbioses to cadmium stress.

The role of rhizobia in alleviating cadmium (Cd) stress in woody legumes is still unclear. Therefore, two types of black locust (Robinia pseudoacacia L.) with high and low Cd accumulation abilities were selected from 11 genotypes in China, and the effects of rhizobium (Mesorhizobium huakuii GP1T11) inoculation on the growth, CO2 and H2O gas exchange parameters, Cd accumulation, and the absorption of mineral elements of the high (SX) and low Cd-accumulator (HB) were compared. The results showed that rhizobium-inoculation significantly increased biomass, shoot Cd contents, Cd accumulation, root-to-shoot translocation factor (TF) and the absorption and accumulation of mineral elements in both SX and HB. Rhizobium-inoculation increased chlorophyll a and carotenoid contents, and the intercellular carbon dioxide concentrations in HB plants. Under Cd exposure, the high-accumulator SX exhibited a significant decrease in photosynthetic CO2 fixation (Pn) and an enhanced accumulation of Cd in leaves, but coped with Cd exposure by increasing chlorophyll synthesis, regulating stomatal aperture (Gs), controlling transpiration (Tr), and increasing the absorption and accumulation of mineral elements. In contrast, the low-accumulator HB was more sensitive to Cd exposure despite preferential accumulation of Cd in roots, with decreased chlorophyll and carotenoid contents, but significantly increased root biomass. Compared to the low-accumulator HB, non-inoculated Cd-exposed SX plants had higher chlorophyll contents, and rhizobium-inoculated Cd-exposed SX plants had higher Pn, Tr, and Gs as well as higher levels of P, K, Fe, Ca, Zn, and Cu. In conclusion, the high- and low-Cd-accumulator exhibited different physiological responses to Cd exposure. Overall, rhizobium-inoculation of black locust promoted the growth and heavy metal absorption, providing an effective strategy for the phytoremediation of heavy metal-contaminated soils by this woody legume.

Amelioration of NaCl stress on germination, growth, and nitrogen fixation of Vicia faba at isosmotic Na-Ca combinations and Rhizobium.

MAIN CONCLUSION: The Na+/Ca2+ ratio of 1/5 ameliorated the inhibitory action of NaCl and improved the germination and growth of Vicia faba. Addition of Rhizobium also enhanced nodulation and nitrogen fixation. Casting light upon the impact of salinity stress on growth and nitrogen fixation of Vicia faba supplemented with Rhizobium has been traced in this work. How Ca2+ antagonizes Na+ toxicity and osmotic stress of NaCl was also targeted in isosmotic combinations of NaCl and CaCl2 having various Na+:Ca2+ ratios. Growth of Vicia faba (cultivar Giza 3) was studied at two stages: germination and seedling. At both experiments, seeds or seedlings were exposed to successively increasing salinity levels (0, 50, 100, 150, and 200 mM NaCl) as well as isosmotic combinations of NaCl and CaCl2 (Na+:Ca2+ of 1:1, 1:5, 1:10, 1:15, 1:18, and 1: 20), equivalent to 150 mM NaCl. Inocula of the local nitrogen-fixing bacteria, Rhizobium leguminosarum (OP715892) were supplemented at both stages. NaCl salinity exerted a negative impact on growth and metabolism of Vicia faba; inhibition was proportional with increasing salinity level up to the highest level of 200 mM. Seed germination, shoot and root lengths, fresh and dry weights, chlorophyll content, and nodules (number, weight, leghemoglobin, respiration, and nitrogenase activity) were inhibited by salinity. Ca2+ substitution for Na+, particularly at a Na/Ca ratio of 1:5, was stimulatory to almost all parameters at both stages. Statistical correlations between salinity levels and Na/Ca combinations proved one of the four levels (strong- or weak positive, strong- or weak negative) with most of the investigated parameters, depending on the parameter.

Biopolymer as an additive for effective biochar-based rhizobial inoculant.

Biochar is an efficient and inexpensive carrier for bacteria that stimulate plant development and growth. In this study, different biopolymer additives (cellulose, xanthan gum, chitin and tryptone) were tested with different addition ratios (1:0.1, 1:0.5 and 1:1) on further enhancing biochar capacity for supporting the growth and activity of Bradyrhizobium japonicum (CB1809). We utilized pine wood biochar (PWBC) pyrolyzed at 400 °C as the base inoculum carrier. The shelf life and survival rate of CB1809 were counted using the colony-forming unit (CFU) method for up to 120 days. Peat served as a standard reference material against which all treatments were compared. Subsequent experiments evaluated the ability of carrier inoculants to promote Glycine max L. (soybean) plant growth and nodulation under different watering regimes, i.e., 55 % water holding capacity (WHC) (D0), 30 % WHC (D1) and, 15 % WHC (D2) using sandy loam soil. Results revealed that among different additives; xanthan gum with 1:0.5 to PWBC [PWBC-xanthan gum(1:0.5)] was observed as a superior formulation in supporting rhizobial shelf life and survival rate of CB1809. In pot experiments, plants with PWBC-xanthan gum(1:0.5) formulation showed significant increase in various physiological characteristics (nitrogenase activity, chlorophyll pigments, membrane stability index, and relative water content), root architecture (root surface area, root average diameter, root volume, root tips, root forks and root crossings), and plant growth attributes (shoot/root dry biomass, shoot/root length, and number of nodules). Additionally, a reduced enrichment of isotopic signatures (δ13C, δ15N) was observed in plants treated with PWBC-xanthan gum(1:0.5), less enrichment of δ15N indicates an inverse link to nodulation and nitrogenase activity, while lower δ13C values indicates effective water use efficiency by plants during drought stress. These results suggest that biopolymers supplementation of the PWBC is useful in promoting shelf life or survival rate of CB1809.

Antagonistic effect of mercury and excess nitrogen exposure reveals provenance-specific phytoremediation potential of black locust-rhizobia symbiosis.

Interaction of different environmental constrains pose severe threats to plants that cannot be predicted from individual stress exposure. In this context, mercury (Hg), as a typical toxic and hazardous heavy metal, has recently attracted particular attention. Nitrogen (N2)-fixing legumes can be used for phytoremediation of Hg accumulation, whereas N availability could greatly affect its N2-fixation efficiency. However, information on the physiological responses to combined Hg exposure and excess N supply of woody legume species is still lacking. Here, we investigated the interactive effects of rhizobia inoculation, Hg exposure (+Hg), and high N (+N) supply, individually and in combination (+N*Hg), on photosynthesis and biochemical traits in Robinia pseudoacacia L. seedlings of two provenances, one from Northeast (DB) and one from Northwest (GS) China. Our results showed antagonistic effects of combined + N*Hg exposure compared to the individual treatments that were provenance-specific. Compared to individual Hg exposure, combined + N*Hg stress significantly increased foliar photosynthesis (+50.6%) of inoculated DB seedlings and resulted in more negative (-137.4%) δ15N abundance in the roots. Furthermore, combined + N*Hg stress showed 47.7% increase in amino acid N content, 39.4% increase in NR activity, and 14.8% decrease in MDA content in roots of inoculated GS seedlings. Inoculation with rhizobia significantly promoted Hg uptake in both provenances, reduced MDA contents of leaves and roots, enhanced photosynthesis and maintained the nutrient balance of Robinia. Among the two Robinia provenances investigated, DB seedlings formed more nodules, had higher biomass and Hg accumulation than GS seedlings. For example, total Hg concentrations in leaves and roots and total biomass of inoculated DB seedlings were 1.3,1.9 and 3.4 times higher than in inoculated GS seedlings under combined + N*Hg stress, respectively. Therefore, the DB provenance is considered to possess a higher potential for phytoremediation of Hg contamination compared to the GS provenance in environments subjected to N deposition.

The Rpf107 gene, a homolog of LOR, is required for the symbiotic nodulation of Robinia pseudoacacia.

MAIN CONCLUSION: Rpf107 is involved in the infection process of rhizobia and the maintenance of symbiotic nitrogen fixation in black locust root nodules. The LURP-one related (LOR) protein family plays a pivotal role in mediating plant defense responses against both biotic and abiotic stresses. However, our understanding of its function in the symbiotic interaction between legumes and rhizobia remains limited. Here, Rpf107, a homolog of LOR, was identified in Robinia pseudoacacia (black locust). The subcellular localization of Rpf107 was analyzed, and its function was investigated using RNA interference (RNAi) and overexpression techniques. The subcellular localization assay revealed that Rpf107 was mainly distributed in the plasma membrane and nucleus. Rpf107 silencing prevented rhizobial infection and hampered plant growth. The number of infected cells in the nitrogen fixation zone of the Rpf107-RNAi nodules was also noticeably lower than that in the control nodules. Notably, Rpf107 silencing resulted in bacteroid degradation and the premature aging of nodules. In contrast, the overexpression of Rpf107 delayed the senescence of nodules and prolonged the nitrogen-fixing ability of nodules. These results demonstrate that Rpf107 was involved in the infection of rhizobia and the maintenance of symbiotic nitrogen fixation in black locust root nodules. The findings reveal that a member of the LOR protein family plays a role in leguminous root nodule symbiosis, which is helpful to clarify the functions of plant LOR protein family and fully understand the molecular mechanisms underlying legume-rhizobium symbiosis.

Lessons learned from metabolic engineering in hairy roots: Transcriptome and metabolic profile changes caused by Rhizobium-mediated plant transformation in Cucurbitaceae species.

Cucurbitaceae species are used in traditional medicine around the world. Cucurbitacins are highly oxygenated triterpenoids found in Cucurbitaceae species and exhibit potent anticancer activity alone and in combination with other existing chemotherapeutic drugs. Therefore, increasing production of these specialized metabolites is of great relevance. We recently showed that hairy roots of Cucurbita pepo can be used as a platform for metabolic engineering of cucurbitacins to modify their structure and increase their production. To study the changes in cucurbitacin accumulation upon formation of hairy roots, an empty vector (EV) control and Cucurbitacin inducing bHLH transcription factor 1 (CpCUCbH1)-overexpressing hairy roots of C. pepo were compared to untransformed (WT) roots. Whilst CpCUCbH1-overexpression increased production of cucurbitacins I and B by 5-fold, and cucurbitacin E by 3-fold when compared to EV lines, this increase was not significantly different when compared to WT roots. This indicated that Rhizobium rhizogenes transformation lowered the cucurbitacins levels in hairy roots, but that increasing expression of cucurbitacin biosynthetic genes by CpCUCbH1-overexpression restored cucurbitacin production to WT levels. Subsequent metabolomic and RNA-seq analysis indicated that the metabolic profile and transcriptome of hairy roots was significantly changed when compared to WT roots. Interestingly, it was observed that 11% of the differentially expressed genes were transcription factors. It was noteworthy that the majority of transcripts showing highest Pearson correlation coefficients to the Rhizobium rhizogenes genes rolB, rolC and ORF13a, were predicted to be transcription factors. In summary, hairy roots are an excellent platform for metabolic engineering of plant specialized metabolites, but these extensive transcriptome and metabolic profile changes should be considered in subsequent studies.

VPT-like genes modulate Rhizobium-legume symbiosis and phosphorus adaptation.

Although vacuolar phosphate transporters (VPTs) are essential for plant phosphorus adaptation, their role in Rhizobium-legume symbiosis is unclear. In this study, homologous genes of VPT1 (MtVPTs) were identified in Medicago truncatula to assess their roles in Rhizobium-legume symbiosis and phosphorus adaptation. MtVPT2 and MtVPT3 mainly positively responded to low and high phosphate, respectively. However, both mtvpt2 and mtvpt3 mutants displayed shoot phenotypes with high phosphate sensitivity and low phosphate tolerance. The root-to-shoot phosphate transfer efficiency was significantly enhanced in mtvpt3 but weakened in mtvpt2, accompanied by lower and higher root cytosolic inorganic phosphate (Pi) concentration, respectively. Low phosphate induced MtVPT2 and MtVPT3 expressions in nodules. MtVPT2 and MtVPT3 mutations markedly reduced the nodule number and nitrogenase activity under different phosphate conditions. Cytosolic Pi concentration in nodules was significantly lower in mtvpt2 and mtvpt3 than in the wildtype, especially in tissues near the base of nodules, probably due to inhibition of long-distance Pi transport and cytosolic Pi supply. Also, mtvpt2 and mtvpt3 could not maintain a stable cytosolic Pi level in the nodule fixation zone as the wildtype under low phosphate stress. These findings show that MtVPT2 and MtVPT3 modulate phosphorus adaptation and rhizobia-legume symbiosis, possibly by regulating long-distance Pi transport.

Phyto-interactive impact of green synthesized iron oxide nanoparticles and Rhizobium pusense on morpho-physiological and yield components of greengram.

The iron oxide nanoparticles (IONPs) prepared by green synthesis method using Syzigium cumini leaf extract was characterized by X-ray diffraction (XRD), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). The XRD confirmed the crystalline structure of green synthesized NPs measuring around 33 nm while SEM revealed its nearly spherical shape. Rhizobium species recovered from greengram nodules, identified by 16s rRNA gene sequencing as Rhizobium pusense produced 30% more exopolysaccharides (EPS) in basal medium treated with 1000 µg IONPs/ml. Compositional variation in EPS was observed by Fourier-transform infrared spectroscopy (FTIR). There was no reduction in rhizobial viability and no damage to bacterial membrane was observed under SEM and confocal laser scanning microscopy (CLSM), respectively. Effects of IONPs and R. pusense, used alone and in combination on the growth and development of greengram plants varied considerably. Plants grown with IONPs and R. pusence, used alone and in combination, showed a significant increase in seed germination rate, length and dry biomass of plant organs and seed components compared to controls. The IONPs in the presence of rhizobial strain further increased seed germination, plant growth, seed protein and pigments. Greater protein content (442 mg/g) was observed in seeds at 250 mg/kg of IONPs compared to control. Plants raised with mixture of IONPs plus R. pusense had maximum chlorophyll content (39.2 mg/g FW) while proline content decreased by 53% relative to controls. This study confirms that the green synthesis of IONPs from S. cumini leaf possess useful plant growth promoting effects and could be developed as a nano-biofertilizer for optimizing legume production.

Pectin modifications at the symbiotic interface.

Plant cells are surrounded by a structured cell wall, which not only defines cell shape but also provides a structural barrier for protection against pathogen infection. However, the presence of this barrier does not impede the establishment of mutualistic symbioses between plants and several microbes (e.g. ectomycorrhizal fungi, arbuscular mycorrhizal fungi, and rhizobia). To establish such beneficial associations, symbiotic microbes need to colonize the plant tissues via intercellular and/or intracellular infection, a process that requires cell wall modifications. Although cell wall composition and changes during this process have interested researchers for years, the functional characterization of the molecular players involved is still limited. In this viewpoint, based on several new studies, I discuss how the PME-PL/PG pathway mediates cell wall pectin modifications at the symbiotic interface and highlight further research directions which can broaden our understanding of how beneficial root symbioses are established.

Treatment with atypical rhizobia, Pararhizobium giardinii and Ochrobactrum sp. modulate the rhizospheric bacterial community, and enhances Lens culinaris growth in fallow-soil.

Diazotrophic nodule isolates are acknowledged promoters of plant growth and rhizospheric community. Consequently, in the lentil agroecosystem, inoculation of atypical rhizobial isolates could be a viable alternative to chemical fertilizers for fallow land usage optimization. The aim of this study is to evaluate and select the rhizobial isolates of lentil nodules with plant-growth-promoting (PGP) attributes and to elucidate their application in rice-fallow soil for determining the growth of lentils and its impact on the rhizospheric bacterial community. Lentil's nodule isolates were identified and screened for their PGP attributes, biofilm, exopolysaccharide (EPS) formation, and early plant growth promotion. The pot experiment with the selected atypical rhizobial isolates Pararhizobium giardinii (P1) and Ochrobactrum sp. (42S) significantly enhanced germination, vigour index, nodule formation (P1 60%, 42S 42% increase), nodule fresh weight, shoot length (65% P1 & 35% 42S), and chlorophyll content as compared to the uninoculated control treatment. The genes for nitrogen fixation nifH and nifK were detected in both isolates. Scanning Electron Microscopy (SEM) revealed successful root and nodule colonization by both isolates, while Transmission Electron Microscopy (TEM) displayed nitrogen-fixing zones within root nodules. Proteobacteria predominated in the lentil rhizosphere of all the treatments. Whereas, application of either P1 or 42S increased Rhizobium, Mesorhizobium, and Bradyrhizobium genra, thus positively modulating rhizospheric community structure. The correlation network analysis revealed an abundance of some interdependent bacterial genera with a possible role in overall plant growth. Functional genes for siderophore biosynthesis and ABC transporter were positively modulated by application of either P1 or 42S. This study showed the significant effect of P. giardinii P1 and Ochrobactrum sp. 42S of L. culinaris on lentil growth, improving fallowsoil health for optimum usage, and modulated rhizospheric community structure which strongly manifest prospects of low-cost, eco-friendly and sustainable biofertilizers.

Collection of Hairy Roots as a Basis for Fundamental and Applied Research.

Due to population growth, instability of climatic conditions, and reduction of the areas of natural ecosystems, it becomes necessary to involve modern biotechnological approaches to obtain highly productive plant material. This statement applies both to the creation of plant varieties and the production of new pharmaceutical raw materials. Genetic transformation of valuable medicinal plants using Agrobacterium rhizogenes ensures the production of stable and rapidly growing hairy roots cultures that have a number of advantages compared with cell culture and, above all, can synthesize root-specific substances at the level of the roots of the intact plant. In this regard, special attention should be paid to the collection of hairy roots of the Institute of Plant Physiology RAS, Russian Academy of Sciences, the founder of which was Dr. Kuzovkina I.N. Currently, the collection contains 38 hairy roots lines of valuable medicinal and forage plants. The review discusses the prospects of creating a hairy roots collection as a basis for fundamental research and commercial purposes.

Potential role of rhizobia to enhance chickpea-growth and yield in low fertility-soils of Tunisia.

Plant growth-promoting rhizobacteria are bacteria that improve plant growth and reduce plant pathogen damages. In this study, 100 nodule bacteria were isolated from chickpea, screened for their plant growth-promoting (PGP) traits and then characterised by PCR-RFLP of 16 S rDNA. Results showed that most of the slow-growing isolates fixed nitrogen but those exhibiting fast-growth did not. Fourteen isolates solubilized inorganic phosphorus, 16 strains produced siderophores, and 17 strains produced indole acetic acid. Co-culture experiments identified three strains having an inhibitory effect against Fusarium oxysporum, the primary pathogenic fungus for chickpea in Tunisia. Rhizobia with PGP traits were assigned to Mesorhizobium ciceri, Mesorhizobium mediterraneum, Sinorhizobium meliloti and Agrobacterium tumefaciens. We noted that PGP activities were differentially distributed between M. ciceri and M. mediterraneum. The region of Mateur in northern Tunisia, with clay-silty soil, was the origin of 53% of PGP isolates. Interestingly, we found that S. meliloti and A. tumefaciens strains did not behave as parasitic nodule-bacteria but as PGP rhizobacteria useful for chickpea nutrition and health. In fact, S. meliloti strains could solubilize phosphorus, produce siderophore and auxin. The A. tumefaciens strains could perform the previous PGP traits and inhibit pathogen growth also. Finally, one candidate strain of M. ciceri (LL10)-selected for its highest symbiotic nitrogen fixation and phosphorus solubilization-was used for field experiment. The LL10 inoculation increased grain yield more than three-fold. These finding showed the potential role of rhizobia to be used as biofertilizers and biopesticides, representing low-cost and environment-friendly inputs for sustainable agriculture.

Rhizobial HmuSpSym as a heme-binding factor is required for optimal symbiosis between Mesorhizobium amorphae CCNWGS0123 and Robinia pseudoacacia.

Nitrogen-fixing root nodules are formed by symbiotic association of legume hosts with rhizobia in nitrogen-deprived soils. Successful symbiosis is regulated by signals from both legume hosts and their rhizobial partners. HmuS is a heme degrading factor widely distributed in bacteria, but little is known about the role of rhizobial hmuS in symbiosis with legumes. Here, we found that inactivation of hmuSpSym in the symbiotic plasmid of Mesorhizobium amorphae CCNWGS0123 disrupted rhizobial infection, primordium formation, and nitrogen fixation in symbiosis with Robinia pseudoacacia. Although there was no difference in bacteroids differentiation, infected plant cells were shrunken and bacteroids were disintegrated in nodules of plants infected by the ΔhmuSpSym mutant strain. The balance of defence reaction was also impaired in ΔhmuSpSym strain-infected root nodules. hmuSpSym was strongly expressed in the nitrogen-fixation zone of mature nodules. Furthermore, the HmuSpSym protein could bind to heme but not degrade it. Inactivation of hmuSpSym led to significantly decreased expression levels of oxygen-sensing related genes in nodules. In summary, hmuSpSym of M. amorphae CCNWGS0123 plays an essential role in nodule development and maintenance of bacteroid survival within R. pseudoacacia cells, possibly through heme-binding in symbiosis.

Pangenome Evolution Reconciles Robustness and Instability of Rhizobial Symbiosis.

Root nodulating rhizobia are nearly ubiquitous in soils and provide the critical service of nitrogen fixation to thousands of legume species, including staple crops. However, the magnitude of fixed nitrogen provided to hosts varies markedly among rhizobia strains, despite host legumes having mechanisms to selectively reward beneficial strains and to punish ones that do not fix sufficient nitrogen. Variation in the services of microbial mutualists is considered paradoxical given host mechanisms to select beneficial genotypes. Moreover, the recurrent evolution of non-fixing symbiont genotypes is predicted to destabilize symbiosis, but breakdown has rarely been observed. Here, we deconstructed hundreds of genome sequences from genotypically and phenotypically diverse Bradyrhizobium strains and revealed mechanisms that generate variation in symbiotic nitrogen fixation. We show that this trait is conferred by a modular system consisting of many extremely large integrative conjugative elements and few conjugative plasmids. Their transmissibility and propensity to reshuffle genes generate new combinations that lead to uncooperative genotypes and make individual partnerships unstable. We also demonstrate that these same properties extend beneficial associations to diverse host species and transfer symbiotic capacity among diverse strains. Hence, symbiotic nitrogen fixation is underpinned by modularity, which engenders flexibility, a feature that reconciles evolutionary robustness and instability. These results provide new insights into mechanisms driving the evolution of mobile genetic elements. Moreover, they yield a new predictive model on the evolution of rhizobial symbioses, one that informs on the health of organisms and ecosystems that are hosts to symbionts and that helps resolve the long-standing paradox. IMPORTANCE Genetic variation is fundamental to evolution yet is paradoxical in symbiosis. Symbionts exhibit extensive variation in the magnitude of services they provide despite hosts having mechanisms to select and increase the abundance of beneficial genotypes. Additionally, evolution of uncooperative symbiont genotypes is predicted to destabilize symbiosis, but breakdown has rarely been observed. We analyzed genome sequences of Bradyrhizobium, bacteria that in symbioses with legume hosts, fix nitrogen, a nutrient essential for ecosystems. We show that genes for symbiotic nitrogen fixation are within elements that can move between bacteria and reshuffle gene combinations that change host range and quality of symbiosis services. Consequently, nitrogen fixation is evolutionarily unstable for individual partnerships, but is evolutionarily stable for legume-Bradyrhizobium symbioses in general. We developed a holistic model of symbiosis evolution that reconciles robustness and instability of symbiosis and informs on applications of rhizobia in agricultural settings.

Soybean (Glycine max L. Merrill) responds to phosphorus application and rhizobium inoculation on Acrisols of the semi-deciduous forest agro-ecological zone of Ghana.

Soybean cultivation in Ghana is limited mainly to the Guinea savanna and the forest/savanna transitional agro-ecological zones. Although soybean can be cultivated in the semi-deciduous forest zone, low soil pH and limited nodulation limit its productivity in this zone. In this study, a randomized complete block design, with four replications, was used to test if rhizobia inoculation and/or p-fertilizer could improve yield of soybean in the semi-deciduous forest zone. The residual effects of the treatments were tested on maize and soybean sequentially during the 2018 and 2019 cropping seasons. The inoculation study was repeated in 2020. Phosphorus and inoculation significantly (p = 0.0009) increased soybean grain yield in the 2018 cropping season by 88% and 108%, respectively over the control. Co-application of P and inoculant increased grain yield 3-fold over the control. Maize grain yield ranged from 4.3 t ha-1 in the control plots to 5.2 t ha-1 in treated plots but did not differ significantly among treatments. In 2020, the combined application of P and inoculant produced a significantly (0.002) higher yield than any of the other treatments. This demonstrates that soybean can be grown economically in the semi-deciduous forest agro-ecological zone of Ghana. Co-application of P and inoculant appeared cost-effective, in terms of return on investment.

Biogeographic pattern and relevant environmental factors for rhizobial communities in the rhizosphere and root nodules of kudzu (Pueraria lobata).

Kudzu (Pueraria lobata) is an important medicinal plant, which can associate with rhizobia for nitrogen fixation. The mutualistic symbiosis between rhizobium and kudzu is not well understood, but it is necessary to fully utilize kudzu. Nodules and rhizosphere soils collected from 16 sampling sites were characterized based on phylogenetic analyses of the rpoB gene; 16S rRNA gene; the housekeeping genes SMc00019, truA, and thrA; and the symbiotic genes nodA and nifH. The relationships between biogeographic pattern, nitrogenase activity, and environmental factors were studied. Results indicated that a clear biogeographic pattern of rhizobial communities in the kudzu rhizosphere existed in southern China; latitude and soil pH were found to be the most important factors affecting the biogeographic pattern. Bradyrhizobium diazoefficiens and Bradyrhizobium erythrophlei were the dominant species in kudzu rhizosphere. The symbiotic rhizobia in kudzu nodules mainly belonged to B. lablabi, B. elkanii, B. pachyrhizi, and B. japonicum. Nitrogenase activities in the nodules of kudzu in the Jiangxi sampling region were significantly higher than those in the Guangxi and Hunan sampling regions, and they were significantly negatively correlated to pH and exchangeable Ca. These results constitute the first report of the existence of symbiotic genes in kudzu bradyrhizobia, which are similar to those in B. elkanii and B. pachyrhizi. Our findings could improve the understanding of kudzu-rhizobium symbiosis and could advance the application of rhizobial inoculation in medicinal legumes in terms of increasing the content of active ingredients.

Physiological responses of black locust-rhizobia symbiosis to water stress.

The present study explores the interaction of water supply and rhizobia inoculation on CO2 and H2 O gas exchange characteristics, physiological and biochemical traits in seedlings of Robinia pseudoacacia L. originating from two provenances with contrasting climate and soil backgrounds: the Gansu Province (GS) in northwest China and the Dongbei region (DB) of northeast China. Rhizobia strains were isolated from the 50-years old Robinia forest sites grown in the coastal region of east China. Robinia seedlings with and without rhizobia inoculation were exposed to normal water supply, moderate drought, and rewatering treatments, respectively. After 2 weeks of drought treatment, photosynthetic and physiological traits (net photosynthetic rate, stomatal conductance, stable isotope signature of carbon, malondialdehyde and hydrogen peroxide content) of Robinia leaves were significantly altered, but after rewatering, a general recovery was observed. Rhizobia inoculation significantly increased the drought resistance of both Robinia provenances by promoting photosynthesis, increasing the foliar N content and reducing the accumulation of malondialdehyde and hydrogen peroxide. Among the two provenances, DB plants developed more nodules than GS plants, but GS plants were more drought-tolerant than DB plants, both inoculated or noninoculated, indicated by the foliar gas exchange parameters and biochemical traits studied. Our results also show that inoculation of rhizobia could significantly improve the drought resistance of Robinia in both provenances. The present study contributes to the scientific background for the selection of drought-resistant varieties of Robinia to ensure the success of future afforestation projects in degraded terrestrial ecosystems under global climate change.

Phylogenetic diversity and plant growth-promoting activities of rhizobia nodulating fenugreek (Trigonella foenum-graecum Linn.) cultivated in different agroclimatic regions of India.

Fenugreek (Trigonella foenum-graecum Linn.), is an extensively cultivated legume crop used as a herb, spice, and traditional medicine in India. The symbiotic efficiency and plant growth-promoting potential of fenugreek rhizobia depend on the symbiont strain and environmental factors. We isolated 176 root-nodulating bacteria from fenugreek cultivated in different agroclimatic regions of India. MALDI-TOF MS-based identification and phylogenetic analyses based on 16S rRNA and five housekeeping genes classified the fenugreek-rhizobia as Ensifer (Sinorhizobium) meliloti. However, the strains represent separate sub-lineages of E. meliloti, distinct from all reported sub-lineages across the globe. We also observed the spatial distribution of fenugreek rhizobia, as the three sub-lineages of E. meliloti recorded during this study were specific to their respective agroclimatic regions. According to the symbiotic gene (nodC and nifH) phylogenies, all three sub-lineages of E. meliloti harboured symbiotic genes similar to symbiovar meliloti; as with the housekeeping genes, these also revealed a spatial distribution for different clades of sv. meliloti. The strains could nodulate fenugreek plants and they showed plant growth-promoting potential. Significant differences were found in the plant growth parameters in response to inoculation with the various strains, suggesting strain-level differences. This study demonstrates that fenugreek rhizobia in India are diverse and spatially distributed in different agro-climatic regions.

Perennial, but not annual legumes synergistically benefit from infection with arbuscular mycorrhizal fungi and rhizobia: a meta-analysis.

Many plant species simultaneously interact with multiple symbionts, which can, but do not always, generate synergistic benefits for their host. We ask if plant life history (i.e. annual vs perennial) can play an important role in the outcomes of the tripartite symbiosis of legumes, arbuscular mycorrhizal fungi (AMF), and rhizobia. We performed a meta-analysis of 88 studies examining outcomes of legume-AMF-rhizobia interactions on plant and microbial growth. Perennial legumes associating with AMF and rhizobia grew larger than expected based on their response to either symbiont alone (i.e. their response to co-inoculation was synergistic). By contrast, annual legume growth with co-inoculation did not differ from additive expectations. AMF and rhizobia differentially increased phosphorus (P) and nitrogen (N) tissue concentration. Rhizobium nodulation increased with mycorrhizal fungi inoculation, but mycorrhizal fungi colonization did not increase with rhizobium inoculation. Microbial responses to co-infection were significantly correlated with synergisms in plant growth. Our work supports a balanced plant stoichiometry mechanism for synergistic benefits. We find that synergisms are in part driven by reinvestment in complementary symbionts, and that time-lags in realizing benefits of reinvestment may limit synergisms in annuals. Optimization of microbiome composition to maximize synergisms may be critical to productivity, particularly for perennial legumes.