Characterization of soybean chitinase genes induced by rhizobacteria involved in the defense against Fusarium oxysporum.

Rhizobacteria are capable of inducing defense responses via the expression of pathogenesis-related proteins (PR-proteins) such as chitinases, and many studies have validated the functions of plant chitinases in defense responses. Soybean (Glycine max) is an economically important crop worldwide, but the functional validation of soybean chitinase in defense responses remains limited. In this study, genome-wide characterization of soybean chitinases was conducted, and the defense contribution of three chitinases (GmChi01, GmChi02, or GmChi16) was validated in Arabidopsis transgenic lines against the soil-borne pathogen Fusarium oxysporum. Compared to the Arabidopsis Col-0 and empty vector controls, the transgenic lines with GmChi02 or GmChi16 exhibited fewer chlorosis symptoms and wilting. While GmChi02 and GmChi16 enhanced defense to F. oxysporum, GmChi02 was the only one significantly induced by Burkholderia ambifaria. The observation indicated that plant chitinases may be induced by different rhizobacteria for defense responses. The survey of 37 soybean chitinase gene expressions in response to six rhizobacteria observed diverse inducibility, where only 10 genes were significantly upregulated by at least one rhizobacterium and 9 genes did not respond to any of the rhizobacteria. Motif analysis on soybean promoters further identified not only consensus but also rhizobacterium-specific transcription factor-binding sites for the inducible chitinase genes. Collectively, these results confirmed the involvement of GmChi02 and GmChi16 in defense enhancement and highlighted the diverse inducibility of 37 soybean chitinases encountering F. oxysporum and six rhizobacteria.

The divalent metal ion exporter IhpABC is required to maintain iron homeostasis under low to moderate environmental iron conditions in the bacterium Bradyrhizobium japonicum.

Bacterial iron export mitigates high iron stress, but a role for it under lower iron conditions has not been established. MbfA is the high iron stress exporter in Bradyrhizobium japonicum. Here, we identify the ihpABC genes in a selection for secondary site mutations that suppress the poor growth phenotype of feoAB mutants defective in iron acquisition. IhpABC belongs to the RND tripartite efflux pump family. High iron conditions that derepress the mbfA gene partially rescued the growth of an ihpC mutant but reverted the feoB ihpC mutant to the feoB growth phenotype. The ihpA mutant grown under low iron conditions accumulated higher levels of iron compared to the wild type, and it displayed aberrant iron-responsive gene expression. The mbfA mutant was more sensitive than the wild type to H2 O2 , but the ihpA mutant was not sensitive. The ihpA mutant accumulated more Zn, Co and Cd than was found in the wild type, and growth of the mutant was more sensitive to inhibition by ZnCl2 , CoCl2 and CdCl2 . The findings suggest that IhpABC is a divalent metal ion exporter that helps maintain iron homeostasis under low to moderate environmental iron levels. Thus, iron export is not limited to managing high iron stress.

Probing the potential of salinity-tolerant endophytic bacteria to improve the growth of mungbean [Vigna radiata (L.) Wilczek].

Soil salinity is one of the major limiting factors in plant growth regulation. Salinity-tolerant endophytic bacteria (STEB) can be used to alleviate the negative effects of salinity and promote plant growth. In this study, thirteen endophytic bacteria were isolated from mungbean roots and tested for NaCl salt-tolerance up to 4%. Six bacterial isolates, TMB2, TMB3, TMB5, TMB6, TMB7 and TMB9, demonstrated the ability to tolerate salt. Plant growth-promoting properties such as phosphate solubilization, indole-3-acetic acid (IAA) production, nitrogen fixation, zinc solubilization, biofilm formation and hydrolytic enzyme production were tested in vitro under saline conditions. Eight bacterial isolates indicated phosphate solubilization potential ranging from 5.8-17.7 µg mL-1, wherein TMB6 was found most efficient. Ten bacterial isolates exhibited IAA production ranging from 0.3-2.1 µg mL-1, where TMB7 indicated the highest potential. All the bacterial isolates except TMB13 exhibited nitrogenase activity. Three isolates, TMB6, TMB7 and TMB9, were able to solubilize zinc on tris-minimal media. All isolates were capable of forming biofilm except TMB12 and TMB13. Only TMB2, TMB6 and TMB7 exhibited cellulase activity, while TMB2 and TMB7 exhibited pectinase production. Based on in vitro testing, six efficient STEB were selected and subjected to the further studies. 16S rRNA gene sequencing of efficient STEB revealed the maximum similarity between TMB2 and Rhizobium pusense, TMB3 and Agrobacterium leguminum, TMB5 and Achromobacter denitrificans, TMB6 and Pseudomonas extremorientalis, TMB7 and Bradyrhizobium japonicum and TMB9 and Serratia quinivorans. This is the first international report on the existence of A. leguminum, A. denitrificans, P. extremorientalis and S. quinivorans inside the roots of mungbean. Under controlled-conditions, inoculation of P. extremorientalis TMB6, B. japonicum TMB7 and S. quinivorans TMB9 exhibited maximum potential to increase plant growth parameters; specifically plant dry weight was increased by up to 52%, 61% and 45%, respectively. Inoculation of B. japonicum TMB7 displayed the highest potential to increase plant proline, glycine betaine and total soluble proteins contents by 77%, 78% and 64%, respectively, compared to control under saline conditions. It is suggested that the efficient STEB could be used as biofertilizers for mungbean crop productivity under saline conditions after field-testing.

Rhizobium Soaking Promoted Maize Growth by Altering Rhizosphere Microbiomes and Associated Functional Genes.

Rhizobium is a Gram-negative bacterium, which dissolves minerals, produces growth hormones, promotes root growth, and protects plants from different soil-borne pathogens. In the present study, roots, stalks, and fresh weight of maize (Zea mays L.) were significantly increased after soaking in Bradyrhizobium japonicum compared with the control. Subsequently, transcriptome sequencing results of the whole maize plant soaked in B. japonicum showed that multiple growth and development-related genes were up-regulated more than 100-fold compared to the control. Furthermore, the abundance of plant growth promoting bacteria, such as Acidobacteria Subgroup_6 and Chloroflexi KD4-96, were increased significantly. On the contrary, the abundance of multiple pathogens, such as Curvularia, Fusarium and Mycocentrospora, were significantly decreased. Moreover, inoculation with B. japonicum could inhibit the infection of the pathogen Fusarium graminearum in maize. These results suggest that soaking seeds in B. japonicum may affect the expression of maize growth and development-related genes as the bacteria changes the soil microorganism community structure. These findings may help to expand the application of B. japonicum in crop production and provide new opportunities for food security.

Effects of Co-Inoculating Saccharomyces spp. with Bradyrhizobium japonicum on Atmospheric Nitrogen Fixation in Soybeans (Glycine max (L.)).

Crop production encounters challenges due to the dearth of nitrogen (N) and phosphorus (P), while excessive chemical fertilizer use causes environmental hazards. The use of N-fixing microbes and P-solubilizing microbes (PSMs) can be a sustainable strategy to overcome these problems. Here, we conducted a greenhouse pot experiment following a completely randomized blocked design to elucidate the influence of co-inoculating N-fixing bacteria (Bradyrhizobium japonicum) and PSMs (Saccharomyces cerevisiae and Saccharomyces exiguus) on atmospheric N2-fixation, growth, and yield. The results indicate a significant influence of interaction on Indole-3-acetic acid production, P solubilization, seedling germination, and growth. It was also found that atmospheric N2-fixation, nodule number per plant, nodule dry weight, straw, and root dry weight per plant at different growth stages were significantly increased under dual inoculation treatments relative to single inoculation or no inoculation treatment. Increased seed yield and N and P accumulation were also noticed under co-inoculation treatments. Soil available N was highest under sole bacterial inoculation and lowest under the control treatment, while soil available P was highest under co-inoculation treatments and lowest under the control treatment. We demonstrated that the co-inoculation of N-fixing bacteria and PSMs enhances P bioavailability and atmospheric N2-fixation in soybeans leading to improved soil fertility, raising crop yields, and promoting sustainable agriculture.

Silver nanoparticles inhibit nitrogen fixation in soybean (Glycine max) root nodules.

Antimicrobial silver nanoparticles (AgNPs) are popular in consumer and industrial products, leading to increasing concentrations in the environment. We tested whether exposure to AgNPs could be detrimental to a microbe, its host plant, and their symbiotic relationship. When subjected to 10 µg/mL AgNPs, growth of Bradyrhizobium japonicum USDA 110 was halted. Axenic nitrogen-fertilized Glycine max seedlings were unaffected by 2.5 µg/mL of 30 nm AgNPs, but growth was inhibited with the same dose of 16 nm AgNPs. With 2.5 µg/mL AgNPs, biomass of inoculated plants was 50% of the control. Bacteroids were not found in nodules on plants treated with 2.5 µg/mL AgNPs and plants given 0.5-2.5 µg/mL AgNPs had 40-65% decreased nitrogen fixation. In conclusion, AgNPs not only interfere with general plant and bacterial growth but also inhibit nodule development and bacterial nitrogen fixation. We should be mindful of not releasing AgNPs to the environment or to agricultural land.

Understanding plant-microbe interaction of rice and soybean with two contrasting diazotrophic bacteria through comparative transcriptome analysis.

Understanding the beneficial plant-microbe interactions is becoming extremely critical for deploying microbes imparting plant fitness and achieving sustainability in agriculture. Diazotrophic bacteria have the unique ability to survive without external sources of nitrogen and simultaneously promote host plant growth, but the mechanisms of endophytic interaction in cereals and legumes have not been studied extensively. We have studied the early interaction of two diazotrophic bacteria, Gluconacetobacter diazotrophicus (GAB) and Bradyrhizobium japonicum (BRH), in 15-day-old seedlings of rice and soybean up to 120 h after inoculation (hai) under low-nitrogen medium. Root colonization of GAB in rice was higher than that of BRH, and BRH colonization was higher in soybean roots as observed from the scanning electron microscopy at 120 hai. Peroxidase enzyme was significantly higher at 24 hai but thereafter was reduced sharply in soybean and gradually in rice. The roots of rice and soybean inoculated with GAB and BRH harvested from five time points were pooled, and transcriptome analysis was executed along with control. Two pathways, "Plant pathogen interaction" and "MAPK signaling," were specific to Rice-Gluconacetobacter (RG), whereas the pathways related to nitrogen metabolism and plant hormone signaling were specific to Rice-Bradyrhizobium (RB) in rice. Comparative transcriptome analysis of the root tissues revealed that several plant-diazotroph-specific differentially expressed genes (DEGs) and metabolic pathways of plant-diazotroph-specific transcripts, viz., chitinase, brassinosteroid, auxin, Myeloblastosis (MYB), nodulin, and nitrate transporter (NRT), were common in all plant-diazotroph combinations; three transcripts, viz., nitrate transport accessory protein (NAR), thaumatin, and thionin, were exclusive in rice and another three transcripts, viz., NAC (NAM: no apical meristem, ATAF: Arabidopsis thaliana activating factor, and CUC: cup-shaped cotyledon), ABA (abscisic acid), and ammonium transporter, were exclusive in soybean. Differential expression of these transcripts and reduction in pathogenesis-related (PR) protein expression show the early interaction. Based on the interaction, it can be inferred that the compatibility of rice and soybean is more with GAB and BRH, respectively. We propose that rice is unable to identify the diazotroph as a beneficial microorganism or a pathogen from an early response. So, it expressed the hypersensitivity-related transcripts along with PR proteins. The molecular mechanism of diazotrophic associations of GAB and BRH with rice vis-à-vis soybean will shed light on the basic understanding of host responses to beneficial microorganisms.

The Effects of an Arbuscular Mycorrhizal Fungus and Rhizobium Symbioses on Soybean Aphid Mostly Fail to Propagate to the Third Trophic Level.

The cascading effects of microbe-plant symbioses on the second trophic level, such as phytophagous insects, have been most studied. However, few studies have examined the higher third trophic level, i.e., their natural enemies. We investigated the effects of the symbiotic associations between an arbuscular mycorrhizal (AM) fungus, Rhizophagus irregularis (Glomerales: Glomeraceae), a nitrogen-fixing bacterium, Bradyrhizobium japonicum (Rhizobiales: Bradyrhizobiaceae), and soybean, Glycine max (L.) Merr. (Fabaceae) on two natural enemies of the soybean aphid, Aphis glycines Matsumura (Hemiptera: Aphididae), the ladybird beetle Coleomegilla maculata (De Geer) (Coleoptera: Coccinellidae), and the parasitoid Aphelinus certus Yasnosh (Hymenoptera: Aphelinidae). We measured the growth and survival in the predator and parasitoid reared on aphids feeding on soybean inoculated seedlings. The rhizobium symbiosis alone was affected with a decreased rate of parasitoid emergence, presumably due to decreased host quality. However, number of mummies, sex-ratio, development time, and parasitoid size were all unaffected by inoculation. AM fungus alone or co-inoculated with the rhizobium was unaffected with any of the parameters of the parasitoid. For the predator, none of the measured parameters was affected with any inoculant. Here, it appears that whatever benefits the microbe-plant symbioses confer on the second trophic level are little transferred up to the third.

Influence on Soybean Aphid by the Tripartite Interaction between Soybean, a Rhizobium Bacterium, and an Arbuscular Mycorrhizal Fungus.

The inoculation of arbuscular mycorrhizal (AM) fungi and rhizobia in legumes has been proven to increase plant growth and yield. To date, studies of the effects of these interactions on phytophagous insects have shown them to be context-dependent depending on the inoculant strain, the plant, and the insect species. Here, we document how a symbiosis involving an AM fungus, Rhizophagus irregularis; a rhizobium, Bradyrhizobium japonicum; and soybean, Glycine max, influences the soybean aphid, Aphis glycines. Soybean co-inoculated with the AM fungus-rhizobium pair increased the plant's biomass, nodulation, mycorrhizal colonization, nitrogen, and carbon concentrations, but decreased phosphorus concentration. Similar effects were observed with rhizobium alone, with the exception that root biomass was unaffected. With AM fungus alone, we only observed an increase in mycorrhizal colonization and phosphorus concentration. The aphids experienced an increased reproductive rate with the double inoculation, followed by rhizobium alone, whereas no effect was observed with the AM fungus. The size of individual aphids was not affected. Furthermore, we found positive correlation between nitrogen concentration and aphid population density. Our results confirm that co-inoculation of two symbionts can enhance both plant and phytophagous insect performance beyond what either symbiont can contribute alone.

Effects of rehydration on physiological and transcriptional responses of a water-stressed rhizobium.

As a microsymbiont of soybean, Bradyrhizobium japonicum plays an important role in symbiotic nitrogen fixation and sustainable agriculture. However, the survival of B. japonicum cells under water-deplete (e.g., drought) and water-replete (e.g., flood) conditions is a major concern affecting their nitrogen-fixing ability by establishing the symbiotic relationship with the host. In this study, we isolated a water stress tolerant rhizobium from soybean root nodules and tested its survival under water-deplete conditions. The rhizobium was identified as Bradyrhizobium japonicum and named strain 5038. Interestingly, both plate counting and live/dead fluorescence staining assays indicate that a number of viable but non-culturable cells exist in the culture medium upon the rehydration process which could cause dilution stress. Bradyrhizobium japonicum 5038 cells increased production of exopolysaccharide (EPS) and trehalose when dehydrated, suggesting that protective responses were stimulated. As expected, cells reduced their production upon the subsequent rehydration. To examine differential gene expression of B. japonicum 5038 when exposed to water-deplete and subsequent water-replete conditions, whole-genome transcriptional analysis was performed under 10% relative humidity (RH), and subsequent 100% RH, respectively. A total of 462 differentially expressed genes (DEGs, > 2.0-fold) were identified under the 10% RH condition, while 3,776 genes showed differential expression during the subsequent rehydration (100% RH) process. Genes involved in signal transduction, inorganic ion transport, energy production and metabolisms of carbohydrates, amino acids, and lipids were far more up-regulated than down-regulated in the 10% RH condition. Notably, trehalose biosynthetic genes (otsAB, treS, and treYZ), genes ligD, oprB, and a sigma factor rpoH were significantly induced by 10% RH. Under the subsequent 100% RH condition, genes involved in transcription, translation, cell membrane regulation, replication and repair, and protein processing were highly up-regulated. Interestingly, most of 10%-RH inducible genes displayed rehydration-repressed, except three genes encoding heat shock (Hsp20) proteins. Therefore, this study provides molecular evidence for the switch of gene expression of B. japonicum cells when encountered the opposite water availability from water-deplete to water-replete conditions.

Identification of the metabolites regulated in soybean-Rhizobia symbiosis through solid phase microextraction coupled with LC-MS.

Legumes provide one of the uniquely nutrient-rich food sources to the population and are one of the primary field crops that play significant roles in agricultural sustainability. Inoculation with Bradyrhizobium japonicum is necessary for the high yield of leguminous crops, i.e. soybean. Nodulation of soybean by Bradyrhizobium japonicum is a complex process that is essential for cultivation of these legumes and external stress factors, such as draught and soil acidity, that influence the nodulation and crop yield. Alterations in the nodule metabolites are known to identify the type of stress that mitigates nodulation and lowers crop yield. Current techniques aimed at understanding the metabolic activities in the symbiont, such as in the case of metabolic regulations in varying nodule growth phases, rely on exhaustive techniques based on the removal of nodules or other plant tissue. Aiming to capture a more in-depth, accurate profile of this system without quenching the metabolic activity in the nodules, or removing the nodules, a workflow was prepared for the metabolite sampling through in vivo solid phase microextraction in thin film format (TF-SPME). This technique was followed by LC-QTOF-MS instrumental analysis with subsequent metabolite annotation and reference standard validation. Our approach is unique in terms of eliminating the effects that arise due to analyte partition coefficients. We show that the symbiont undergoes metabolic regulations throughout the cultivation period, displaying the efficacy of TF-SPME as a non-exhaustive sampling method that can be used as a tool to investigate the metabolic alterations in nodules. These alterations would potentially fingerprint the environmental effects on soybean yield.

Overexpression of Terpenoid Biosynthesis Genes From Garden Sage (Salvia officinalis) Modulates Rhizobia Interaction and Nodulation in Soybean.

In legumes, many endogenous and environmental factors affect root nodule formation through several key genes, and the regulation details of the nodulation signaling pathway are yet to be fully understood. This study investigated the potential roles of terpenoids and terpene biosynthesis genes on root nodule formation in Glycine max. We characterized six terpenoid synthesis genes from Salvia officinalis by overexpressing SoTPS6, SoNEOD, SoLINS, SoSABS, SoGPS, and SoCINS in soybean hairy roots and evaluating root growth and nodulation, and the expression of strigolactone (SL) biosynthesis and early nodulation genes. Interestingly, overexpression of some of the terpenoid and terpene genes increased nodule numbers, nodule and root fresh weight, and root length, while others inhibited these phenotypes. These results suggest the potential effects of terpenoids and terpene synthesis genes on soybean root growth and nodulation. This study provides novel insights into epistatic interactions between terpenoids, root development, and nodulation in soybean root biology and open new avenues for soybean research.

Response of Soybean to Hydrochar-Based Rhizobium Inoculation in Loamy Sandy Soil.

Hydrochar is rich in nutrients and may provide a favorable habitat or shelter for bacterial proliferation and survival. Therefore, in this study, we investigate the efficiency of a hydrochar-based rhizobial inoculant (Bradyrhizobium japonicum) on the symbiotic performance of soybean under both greenhouse and field conditions. There were positive and significant effects of hydrochar-based inoculation on the root and shoot growth of soybean as compared to uninoculated plants grown under irrigated and drought conditions. The drought stress significantly inhibited the symbiotic performance of rhizobia with soybean. Soybean inoculated with hydrochar-based B. japonicum produced twofold more nodules under drought stress conditions as compared to plants inoculated with a commercial preparation/inoculant carrier B. japonicum (HISTICK). The N concentration of inoculated plants with hydrochar-based B. japonicum was by 31% higher than that of un-inoculated plants grown in pots and by 22% for HISTICK. Furthermore, the soybean treated with hydrochar-based B. japonicum showed higher grain yield of 29% under irrigated conditions and 40% higher under rainfed condition compared to un-inoculated plants. In conclusion, the obtained results proved the potential of hydrochar-based B. japonicum inoculant for soybean in terms of increased symbiotic performance and agronomic traits, especially under rainfed conditions.

Assessing the Plant Growth Promoting and Arsenic Tolerance Potential of Bradyrhizobium japonicum CB1809.

Accumulation of heavy metals in soil is of concern to the agricultural production sector, because of the potential threat to food quality and quantity. Inoculation with plant growth-promoting bacteria (PGPR) has previously been shown to alleviate heavy metal stress but the mechanisms are unclear. Potential mechanisms by which inoculation with Bradyrhizobium japonicum CB1809 affected the legume soybean (Glycine max cv. Zeus) and the non-legume sunflower (Helianthus annus cv. Hyoleic 41) were investigated in solution culture under 5 µM As stress. Adding As resulted in As tissue concentrations of up to 5 mg kg-1 (shoots) and 250 mg kg-1 (roots) in both species but did not reduce shoot or root biomass. Inoculation increased root biomass but only in the legume (soybean) and only with As. Inoculation resulted in large (up to 100%) increases in siderophore concentration but relatively small changes (±10-15%) in auxin concentration in the rhizosphere. However, the increase in siderophore concentration in the rhizosphere did not result in the expected increases in tissue N or Fe, especially in soybean, suggesting that their function was different. In conclusion, siderophores and auxins may be some of the mechanisms by which both soybean and sunflower maintained plant growth in As-contaminated media.

Field Resistance to Phakopsora pachyrhizi and Colletotrichum truncatum of Transgenic Soybean Expressing the NmDef02 Plant Defensin Gene.

Fungal diseases lead to significant losses in soybean yields and a decline in seed quality; such is the case of the Asian soybean rust and anthracnose caused by Phakopsora pachyrhizi and Colletotrichum truncatum, respectively. Currently, the development of transgenic plants carrying antifungal defensins offers an alternative for plant protection against pathogens. This paper shows the production of transgenic soybean plants expressing the NmDef02 defensin gene using the biolistic delivery system, in an attempt to improve resistance against diseases and reduce the need for chemicals. Transgenic lines were assessed in field conditions under the natural infections of P. pachyrhizi and C. truncatum. The constitutive expression of the NmDef02 gene in transgenic soybean plants was shown to enhance resistance against these important plant pathogens. The quantification of the P. pachyrhizi biomass in infected soybean leaves revealed significant differences between transgenic lines and the non-transgenic control. In certain transgenic lines there was a strong reduction of fungal biomass, revealing a less severe disease. Integration and expression of the transgenes were confirmed by PCR, Southern blot, and qRT-PCR, where the Def1 line showed a higher relative expression of defensin. It was also found that the expression of the NmDef02 defensin gene in plants of the Def1 line did not have a negative effect on the nodulation induced by Bradyrhizobium japonicum. These results indicate that transgenic soybean plants expressing the NmDef02 defensin gene have a substantially enhanced resistance to economically important diseases, providing a sound environmental approach for decreasing yield losses and lowering the burden of chemicals in agriculture.

Mechanistic insights into heme-mediated transcriptional regulation via a bacterial manganese-binding iron regulator, iron response regulator (Irr).

The transcription factor iron response regulator (Irr) is a key regulator of iron homeostasis in the nitrogen-fixating bacterium Bradyrhizobium japonicum Irr acts by binding to target genes, including the iron control element (ICE), and is degraded in response to heme binding. Here, we examined this binding activity using fluorescence anisotropy with a 6-carboxyfluorescein-labeled ICE-like oligomer (FAM-ICE). In the presence of Mn2+, Irr addition increased the fluorescence anisotropy, corresponding to formation of the Irr-ICE complex. The addition of EDTA to the Irr-ICE complex reduced fluorescence anisotropy, but fluorescence was recovered after Mn2+ addition, indicating that Mn2+ binding is a prerequisite for complex formation. Binding activity toward ICE was lost upon introduction of substitutions in a His-cluster region of Irr, revealing that Mn2+ binds to this region. We observed that the His-cluster region is also the heme binding site; results from fluorescence anisotropy and electrophoretic mobility shift analyses disclosed that the addition of a half-equivalent of heme dissociates Irr from ICE, likely because of Mn2+ release due to heme binding. We hypothesized that heme binding to another heme binding site, Cys-29, would also inhibit the formation of the Irr-ICE complex because it is proximal to the ICE binding site, which was supported by the loss of ICE binding activity in a Cys-29-mutated Irr. These results indicate that Irr requires Mn2+ binding to form the Irr-ICE complex and that the addition of heme dissociates Irr from ICE by replacing Mn2+ with heme or by heme binding to Cys-29.

Rhizobial inoculation in black wattle plantation (Acacia mearnsii De Wild.) in production systems of southern Brazil.

Black wattle (Acacia mearnsii De Wild.) is a tree legume native to southeast Australia, but present in all continents. Today it covers about 142,400 ha in Brazil, with plantations concentrated in the southern region of the country. Black wattle may form nodules and establish rhizobial symbiosis capable of fixing N2, but rhizobial inoculation is not done in commercial plantations. About 40 kg ha-1 of urea is applied during seedling transplantation. In this review, evidences by which rhizobial inoculation affects monoculture, mixed cultivation, and agroforestry black wattle production systems were searched in literature. Previous measurements in cultivated forests have indicated that biological nitrogen fixation in black wattle may provide up to 200 kg of N ha-1 year-1 to the soil. Therefore, rhizobia inoculation may bring several opportunities to improve black wattle production systems. Black wattle is not a very selective partner in the rhizobial symbiosis, but the genus Bradyrhizobium dominates the rhizobial diversity of black wattle nodules. Investigation on rhizobial diversity in soils where the crop is cultivated may represent an opportunity to find more effective rhizobia strains for inoculants. The successful history of biological nitrogen fixation in grain legumes must inspire the history of tree legumes. Microbiology applied to forestry must overcome challenges on the lack of trained professionals and the development of new application technologies.

Identification of soybean Bradyrhizobium strains used in commercial inoculants in Brazil by MALDI-TOF mass spectrometry.

Biological nitrogen fixation (BNF) with the soybean crop probably represents the major sustainable technology worldwide, saving billions of dollars in N fertilizers and decreasing water pollution and the emission of greenhouse gases. Accordingly, the identification of strains occupying nodules under field conditions represents a critical step in studies that are aimed at guaranteeing increased BNF contribution. Current methods of identification are mostly based on serology, or on DNA profiles. However, the production of antibodies is restricted to few laboratories, and to obtain DNA profiles of hundreds of isolates is costly and time-consuming. Conversely, the matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS technique might represent a golden opportunity for replacing serological and DNA-based methods. However, MALDI-TOF databases of environmental microorganisms are still limited, and, most importantly, there are concerns about the discrimination of protein profiles at the strain level. In this study, we investigated four soybean rhizobial strains carried in commercial inoculants used in over 35 million hectares in Brazil and also in other countries of South America and Africa. A supplementary MALDI-TOF database with the protein profiles of these rhizobial strains was built and allowed the identification of unique profiles statistically supported by multivariate analysis and neural networks. To test this new database, the nodule occupancy by Bradyrhizobium strains in symbiosis with soybean was characterized in a field experiment and the results were compared with serotyping of bacteria by immuno-agglutination. The results obtained by both techniques were highly correlated and confirmed the viability of using the MALDI-TOF MS technique to effectively distinguish bacteria at the strain level.

Pectin-Rich Amendment Enhances Soybean Growth Promotion and Nodulation Mediated by Bacillus Velezensis Strains.

Plant growth-promoting rhizobacteria (PGPR) are increasingly used in crops worldwide. While selected PGPR strains can reproducibly promote plant growth under controlled greenhouse conditions, their efficacy in the field is often more variable. Our overall aim was to determine if pectin or orange peel (OP) amendments to Bacillus velezensis (Bv) PGPR strains could increase soybean growth and nodulation by Bradyrhizobium japonicum in greenhouse and field experiments to reduce variability. The treatments included untreated soybean seeds planted in field soil that contained Bv PGPR strains and non-inoculated controls with and without 0.1% (w/v) pectin or (1 or 10 mg/200 µL) orange peel (OP) amendment. In greenhouse and field tests, 35 and 55 days after planting (DAP), the plants were removed from pots, washed, and analyzed for treatment effects. In greenhouse trials, the rhizobial inoculant was not added with Bv strains and pectin or OP amendment, but in the field trial, a commercial B. japonicum inoculant was used with Bv strains and pectin amendment. In the greenhouse tests, soybean seeds inoculated with Bv AP193 and pectin had significantly increased soybean shoot length, dry weight, and nodulation by indigenous Bradyrhizobium compared to AP193 without pectin. In the field trial, pectin with Bv AP193 significantly increased the shoot length, dry weight, and nodulation of a commercial Bradyrhizobium japonicum compared to Bv AP193 without pectin. In greenhouse tests, OP amendment with AP193 at 10 mg significantly increased the dry weight of shoots and roots compared to AP193 without OP amendment. The results demonstrate that pectin-rich amendments can enhance Bv-mediated soybean growth promotion and nodulation by indigenous and inoculated B. japonicum.

Genome-informed Bradyrhizobium taxonomy: where to from here?

Bradyrhizobium is thought to be the largest and most diverse rhizobial genus, but this is not reflected in the number of described species. Although it was one of the first rhizobial genera recognised, its taxonomy remains complex. Various contemporary studies are showing that genome sequence information may simplify taxonomic decisions. Therefore, the growing availability of genomes for Bradyrhizobium will likely aid in the delineation and characterization of new species. In this study, we addressed two aims: first, we reviewed the availability and quality of available genomic resources for Bradyrhizobium. This was achieved by comparing genome sequences in terms of sequencing technologies used and estimated level of completeness for inclusion in genome-based phylogenetic analyses. Secondly, we utilized these genomes to investigate the taxonomic standing of Bradyrhizobium in light of its diverse lifestyles. Although genome sequences differed in terms of their quality and completeness, our data indicate that the use of these genome sequences is adequate for taxonomic purposes. By using these resources, we inferred a fully resolved, well-supported phylogeny. It separated Bradyrhizobium into seven lineages, three of which corresponded to the so-called supergroups known for the genus. Wide distribution of key lifestyle traits such as nodulation, nitrogen fixation and photosynthesis revealed that these traits have complicated evolutionary histories. We present the first robust Bradyrhizobium species phylogeny based on genome sequence information for investigating the evolution of this important assemblage of bacteria. Furthermore, this study provides the basis for using genome sequence information as a resource to make important taxonomic decisions, particularly at the species and genus levels.