Role of a LORELEI- like gene from Phaseolus vulgaris during a mutualistic interaction with Rhizobium tropici.

Reactive oxygen species (ROS), produced by NADPH oxidases known as RBOHs in plants, play a key role in plant development, biotic and abiotic stress responses, hormone signaling, and reproduction. Among the subfamily of receptor-like kinases referred to as CrRLK, there is FERONIA (FER), a regulator of RBOHs, and FER requires a GPI-modified membrane protein produced by LORELEI (LRE) or LORELEI-like proteins (LLG) to reach the plasma membrane and generate ROS. In Arabidopsis, AtLLG1 is involved in interactions with microbes as AtLLG1 interacts with the flagellin receptor (FLS2) to trigger the innate immune response, but the role of LLGs in mutualistic interactions has not been examined. In this study, two Phaseolus vulgaris LLG genes were identified, PvLLG2 that was expressed in floral tissue and PvLLG1 that was expressed in vegetative tissue. Transcripts of PvLLG1 increased during rhizobial nodule formation peaking during the early period of well-developed nodules. Also, P. vulgaris roots expressing pPvLLG1:GFP-GUS showed that this promoter was highly active during rhizobium infections, and very similar to the subcellular localization using a construct pLLG1::PvLLG1-Neon. Compared to control plants, PvLLG1 silenced plants had less superoxide (O2-) at the root tip and elongation zone, spotty hydrogen peroxide (H2O2) in the elongation root zone, and significantly reduced root hair length, nodule number and nitrogen fixation. Unlike control plants, PvLLG1 overexpressing plants showed superoxide beyond the nodule meristem, and significantly increased nodule number and nodule diameter. PvLLG1 appears to play a key role during this mutualistic interaction, possibly due to the regulation of the production and distribution of ROS in roots.

CRK12: A Key Player in Regulating the Phaseolus vulgaris-Rhizobium tropici Symbiotic Interaction.

Cysteine-rich receptor-like kinases (CRKs) are a type of receptor-like kinases (RLKs) that are important for pathogen resistance, extracellular reactive oxygen species (ROS) signaling, and programmed cell death in plants. In a previous study, we identified 46 CRK family members in the Phaseolus vulgaris genome and found that CRK12 was highly upregulated under root nodule symbiotic conditions. To better understand the role of CRK12 in the Phaseolus-Rhizobia symbiotic interaction, we functionally characterized this gene by overexpressing (CRK12-OE) and silencing (CRK12-RNAi) it in a P. vulgaris hairy root system. We found that the constitutive expression of CRK12 led to an increase in root hair length and the expression of root hair regulatory genes, while silencing the gene had the opposite effect. During symbiosis, CRK12-RNAi resulted in a significant reduction in nodule numbers, while CRK12-OE roots showed a dramatic increase in rhizobial infection threads and the number of nodules. Nodule cross sections revealed that silenced nodules had very few infected cells, while CRK12-OE nodules had enlarged infected cells, whose numbers had increased compared to controls. As expected, CRK12-RNAi negatively affected nitrogen fixation, while CRK12-OE nodules fixed 1.5 times more nitrogen than controls. Expression levels of genes involved in symbiosis and ROS signaling, as well as nitrogen export genes, supported the nodule phenotypes. Moreover, nodule senescence was prolonged in CRK12-overexpressing roots. Subcellular localization assays showed that the PvCRK12 protein localized to the plasma membrane, and the spatiotemporal expression patterns of the CRK12-promoter::GUS-GFP analysis revealed a symbiosis-specific expression of CRK12 during the early stages of rhizobial infection and in the development of nodules. Our findings suggest that CRK12, a membrane RLK, is a novel regulator of Phaseolus vulgaris-Rhizobium tropici symbiosis.

Rhizobacteria control damping-off and promote growth of lima bean with and without co-inoculation with Rhizobium tropici CIAT899.

Rhizoctonia solani compromises the production of lima bean, an alternative and low-input food source in many tropical regions. Inoculation of bacterial strains has been used, but research on their biocontrol and growth promotion potential on lima bean is scarce. The objective of this study was to evaluate the effects of inoculation with rhizobacterial strains of the genera Bacillus, Brevibacillus, Paenibacillus, Burkholderia, Pseudomonas, and Rhizobium in combination or not with N2-fixing Rhizobium tropici on the control of damping-off disease and growth promotion in lima bean plants. Greenhouse experiments were conducted to evaluate the inoculation with bacterial strains with biocontrol potential in combination or not with R. tropici in substrate infected with R. solani CML 1846. Growth promotion of these strains was also assessed. Strains of Brevibacillus (UFLA 02-286), Pseudomonas (UFLA 02-281 and UFLA 04-885), Rhizobium (UFLA 04-195), and Burkholderia (UFLA 04-227) co-inoculated with the strain CIAT 899 (Rhizobium tropici) were the most effective in controlling R. solani, reducing the disease incidence in 47-60% on lima bean. The promising strains used in the biocontrol assays were also responsive in promoting growth of lima bean under disease and sterile conditions. A positive synergistic effect of co-inoculation of different genera contributed to plant growth, and these outcomes are important first steps to improve lima bean production.

Rhizobia exopolysaccharides: promising biopolymers for use in the formulation of plant inoculants.

Inoculants with beneficial microorganisms comprise both selected strains and carriers that ensure a favorable microenvironment for cell survival and stability. Formulations of inoculants using synthetic polymers as carriers are common. However, only a few studies are available in the literature regarding the formulation of inoculants using natural biomolecules as carriers. Exopolysaccharides (EPS) are biomolecules produced by a vast array of microbial species, including symbiotic nitrogen-fixing bacteria, commonly known as rhizobia. EPS perform several functions, such as the protection against the deleterious effects of diverse environmental soil stresses. Two Rhizobium tropici strains and one Paraburkholderia strain were selected after semiquantitative analysis by scanning electron microscopy (SEM) of their EPS production in liquid YMA medium. Their EPS were characterized through a series of analytical techniques, aiming at their use in the formulation of plant inoculants. In addition, the effect of the carbon source on EPS yield was evaluated. Multi-stage fragmentation analysis showed the presence of xylose, glucose, galactose, galacturonic acid, and glucuronic acid in EPS chemical composition, which was confirmed by FT-IR spectra and 13C NMR spectroscopy. Thermal stability (thermogravimetric) was close to 270 °C and viscosity ranged from 120 to 1053.3 mPa.s. Surface morphology (SEM) was rough and irregular, with a cross-linked spongy matrix, which, together with the hydrophilic functional groups, confers water holding capacity. The present study showed that the three EPS have potential as microorganism carriers for formulation of microbial inoculants to be applied in plants.

Evaluation of water stress and co-inoculation of Azospirillum brasilense and Rhizobium tropici in beans (Phaseolus vulgaris l) / Avaliação do estratégio de água e co-inoculação de Azospirillum brasilense e Rhizobium tropici in beans (Phaseolus vulgaris l) / Evaluación del estrés hídrico y la coinoculación de Azospirillum brasilense y Rhizobium tropici en frijoles (Phaseolus vulgaris l)

Climate change has caused major changes in abiotic factors, with water stress as the greatest threat to agricultural production. The measures aimed at alleviating the problems caused by this limiting production factor have occurred through the adoption of sustainable strategies, especially microbial biotechnology, which uses the interactions between the microorganism and the plant, ensuring productive quality and inducing plant resistance to stresses biotic and abiotic. The objective of the present work was to evaluate the biological nitrogen fixation and the development of bean seedlings, with co-inoculation of two types of inoculants, which were subjected to water stress by different pot capacities. The experiment was conducted in a greenhouse, at Universidade Paranaense - UNIPAR, from April to June 2019. The experimental design was completely randomized (DIC), with 5 replications, 16 treatments and 80 experimental units. The cultivar used was SCS Riqueza. The parameters evaluated were pot capacity (25%, 50%, 75% and 90%); small, large and total nodules, shoot and root length, dry and fresh weight, total carbon and nitrogen. The evaluation of the morphological parameters of the bean seedlings indicated that the co- inoculation technique promoted beneficial effects for the dry mass parameters of shoot, nodule and root. The analysis of the percentage of carbon and nitrogen in the tissues of the seedlings provided an increase in the concentration of these elements in treatments that involved co-inoculation (Azospirillum brasilensis and Rhizobium tropici) with pot capacities of 25 and 75% (CV), demonstrating that the association of microorganisms is beneficial in the limiting water situation.(AU)

A mudança climática tem causado grandes mudanças nos fatores abióticos, sendo o estresse hídrico a maior ameaça à produção agrícola. As medidas destinadas a aliviar os problemas causados por este fator limitante de produção ocorreram através da adoção de estratégias sustentáveis, especialmente a biotecnologia microbiana, que utiliza as interações entre o microorganismo e a planta, garantindo a qualidade produtiva e induzindo a resistência da planta ao estresse biótico e abiótico. O objetivo do presente trabalho foi avaliar a fixação biológica de nitrogênio e o desenvolvimento de mudas de feijão, com co-inoculação de dois tipos de inoculantes, que foram submetidos ao estresse hídrico por diferentes capacidades de vaso. A experiência foi realizada em uma estufa, na Universidade Paranaense - UNIPAR, de abril a junho de 2019. O projeto experimental foi completamente randomizado (DIC), com 5 réplicas, 16 tratamentos e 80 unidades experimentais. A cultivar utilizada foi a SCS Riqueza. Os parâmetros avaliados foram a capacidade do vaso (25%, 50%, 75% e 90%); nódulos pequenos, grandes e totais, comprimento do rebento e da raiz, peso seco e fresco, carbono total e nitrogênio. A avaliação dos parâmetros morfológicos das mudas de feijão indicou que a técnica de co-inoculação promoveu efeitos benéficos para os parâmetros de massa seca do turião, nódulo e raiz. A análise da porcentagem de carbono e nitrogênio nos tecidos das mudas proporcionou um aumento na concentração destes elementos nos tratamentos que envolveram a co-inoculação (Azospirillum brasilensis e Rhizobium tropici) com capacidades de vaso de 25 e 75% (CV), demonstrando que a associação de microorganismos é benéfica na situação limite da água.(AU)

El cambio climático ha provocado importantes cambios en los factores abióticos, siendo el estrés hídrico la mayor amenaza para la producción agrícola. Las medidas encaminadas a paliar los problemas causados por este factor limitante de la producción se han producido mediante la adopción de estrategias sostenibles, especialmente la biotecnología microbiana, que utiliza las interacciones entre el microorganismo y la planta, asegurando la calidad productiva e induciendo la resistencia de la planta a los estreses bióticos y abióticos. El objetivo del presente trabajo fue evaluar la fijación biológica de nitrógeno y el desarrollo de plántulas de frijol, con la co-inoculación de dos tipos de inoculantes, que fueron sometidos a estrés hídrico por diferentes capacidades de maceta. El experimento se realizó en un invernadero, en la Universidade Paranaense - UNIPAR, de abril a junio de 2019. El diseño experimental fue completamente al azar (DIC), con 5 repeticiones, 16 tratamientos y 80 unidades experimentales. El cultivar utilizado fue SCS Riqueza. Los parámetros evaluados fueron capacidad de maceta (25%, 50%, 75% y 90%); nódulos pequeños, grandes y totales, longitud de brotes y raíces, peso seco y fresco, carbono y nitrógeno total. La evaluación de los parámetros morfológicos de las plántulas de frijol indicó que la técnica de coinoculación promovió efectos beneficiosos para los parámetros de masa seca de brotes, nódulos y raíces. El análisis del porcentaje de carbono y nitrógeno en los tejidos de las plántulas proporcionó un aumento en la concentración de estos elementos en los tratamientos que involucraron la coinoculación (Azospirillum brasilensis y Rhizobium tropici) con capacidades de maceta de 25 y 75% (CV), demostrando que la asociación de microorganismos es beneficiosa en la situación de agua limitante.(AU)

The Rhizobium tropici CIAT 899 NodD2 protein promotes symbiosis and extends rhizobial nodulation range by constitutive nodulation factor synthesis.

In the symbiotic associations between rhizobia and legumes, the NodD regulators orchestrate the transcription of the specific nodulation genes. This set of genes is involved in the synthesis of nodulation factors, which are responsible for initiating the nodulation process. Rhizobium tropici CIAT 899 is the most successful symbiont of Phaseolus vulgaris and can nodulate a variety of legumes. Among the five NodD regulators present in this rhizobium, only NodD1 and NodD2 seem to have a role in the symbiotic process. However, the individual role of each NodD in the absence of the other proteins has remained elusive. In this work, we show that the CIAT 899 NodD2 does not require activation by inducers to promote the synthesis of nodulation factors. A CIAT 899 strain overexpressing nodD2, but lacking all additional nodD genes, can nodulate three different legumes as efficiently as the wild type. Interestingly, CIAT 899 NodD2-mediated gain of nodulation can be extended to another rhizobial species, since its overproduction in Sinorhizobium fredii HH103 not only increases the number of nitrogen-fixing nodules in two host legumes but also results in nodule development in incompatible legumes. These findings potentially open exciting opportunities to develop rhizobial inoculants and increase legume crop production.

Priming of defense-related genes in Brassica oleracea var. capitata using concentrated metabolites produced by Rhizobium tropici CIAT 899.

To verify the potential of metabolites extracted from Rhizobium tropici to trigger the priming of defense responses in cruciferous plants, we analyzed the expression of defense-related genes by qRT-PCR. Brassica oleracea var. capitata, susceptible to Xanthomonas campestris pv. campestris, were grown in greenhouse conditions. At 18 days after sowing, plants were inoculated with 1 mL of 1% concentrated metabolites produced by R. tropici (CM-RT) in the root. In a second experiment, leaves were sprayed with 1 mL of a solution containing 1% CM-RT. Aerial and root tissue were collected separately at 0 (non-treated control condition), 24, and 48 h after application, submitted to RNA extraction and gene expression analysis by qRT-PCR. The results showed that, after root treatment with CM-RT, most evaluated genes were upregulated at 24 h after application and downregulated at 48 h after application in roots, while in leaves, genes were downregulated both at 24 and 48 h after application. On the other hand, leaf treatment with CM-RT showed that most evaluated genes in leaves and roots were upregulated at 24 and 48 h after application. These results indicate that the effect of CM-RT applied in roots seems restricted to the applied region and is not sustained, while the application in leaves results in a more systemic response and maintenance of the effect of CM-RT for a longer period. The results obtained in this study emphasize the biotechnological potential of using metabolites of R. tropici as an elicitor of active defense responses in plants.

Phospholipid translocation captured in a bifunctional membrane protein MprF.

As a large family of membrane proteins crucial for bacterial physiology and virulence, the Multiple Peptide Resistance Factors (MprFs) utilize two separate domains to synthesize and translocate aminoacyl phospholipids to the outer leaflets of bacterial membranes. The function of MprFs enables Staphylococcus aureus and other pathogenic bacteria to acquire resistance to daptomycin and cationic antimicrobial peptides. Here we present cryo-electron microscopy structures of MprF homodimer from Rhizobium tropici (RtMprF) at two different states in complex with lysyl-phosphatidylglycerol (LysPG). RtMprF contains a membrane-embedded lipid-flippase domain with two deep cavities opening toward the inner and outer leaflets of the membrane respectively. Intriguingly, a hook-shaped LysPG molecule is trapped inside the inner cavity with its head group bent toward the outer cavity which hosts a second phospholipid-binding site. Moreover, RtMprF exhibits multiple conformational states with the synthase domain adopting distinct positions relative to the flippase domain. Our results provide a detailed framework for understanding the mechanisms of MprF-mediated modification and translocation of phospholipids.

Application of Azospirillum on seeds and leaves, associated with Rhizobium inoculation, increases growth and yield of common bean.

Association of rhizobia with other plant growth-promoting bacteria (PGPB), such as Azospirillum, have the potential to increase crop yields. This work aimed to assess how Rhizobium tropici and Azospirillum brasilense alone or in combination, affect the growth and yields of common bean grains (Phaseolus vulgaris L.). In a field experiment, R. tropici and A. brasilense were inoculated on seeds, alone or in combination, associated or not with foliar spraying of A. brasilense. Shoot biomass, nitrogen accumulation, thousand-grain weight, and grain yield were evaluated. Application of A. brasilense, on seed or by foliar spraying, and seed inoculation of R. tropici, had an additive effect, increasing biomass and accumulated nitrogen, thousand-grain weight, and grain yield.

OnfD, an AraC-Type Transcriptional Regulator Encoded by Rhizobium tropici CIAT 899 and Involved in Nod Factor Synthesis and Symbiosis.

Rhizobium tropici CIAT 899 is a broad-host-range rhizobial strain that establishes symbiotic interactions with legumes and tolerates different environmental stresses such as heat, acidity, or salinity. This rhizobial strain produces a wide variety of symbiotically active nodulation factors (NF) induced not only by the presence of plant-released flavonoids but also under osmotic stress conditions through the LysR-type transcriptional regulators NodD1 (flavonoids) and NodD2 (osmotic stress). However, the activation of NodD2 under high-osmotic-stress conditions remains elusive. Here, we have studied the role of a new AraC-type regulator (named as OnfD) in the symbiotic interaction of R. tropici CIAT 899 with Phaseolus vulgaris and Lotus plants. We determined that OnfD is required under salt stress conditions for the transcriptional activation of the nodulation genes and therefore the synthesis and export of NF, which are required for a successful symbiosis with P. vulgaris Moreover, using bacterial two-hybrid analysis, we demonstrated that the OnfD and NodD2 proteins form homodimers and OnfD/NodD2 form heterodimers, which could be involved in the production of NF in the presence of osmotic stress conditions since both regulators are required for NF synthesis in the presence of salt. A structural model of OnfD is presented and discussed.IMPORTANCE The synthesis and export of rhizobial NF are mediated by a conserved group of LysR-type regulators, the NodD proteins. Here, we have demonstrated that a non-LysR-type regulator, an AraC-type protein, is required for the transcriptional activation of symbiotic genes and for the synthesis of symbiotically active NF under salt stress conditions.

Multiple Effect of Different Plant Growth Promoting Microorganisms on Beans (Phaseolus vulgaris L. ) Crop

Abstract We evaluated the effect of combined Rhizobium tropici, Trichoderma asperellum and plant growth-promoting rhizobacteria (PGPR) in beans crop. The hypothesis that strains of T. asperullum, R. tropici and PGPR combined could improve growth, biomass accumulation and beans yield was tested under greenhouse and field conditions. The treatments consisted of control, mineral nitrogen application and inoculation, isolated and associated with the following microorganisms: Rhizobium tropici, Bacillus subtilis, Trichoderma asperellum and Burkholderia sp. 10N6. Results were evaluated by shoot dry weight (SDW) and root dry weight (RDW), number of nodules and yield components. In greenhouse environment all the microorganisms behaved similarly, and the treatments inoculated with Burkholderia sp. 10N6 (IBu) and R. tropici (IR) stood out regarding the production components. In field conditions the treatments IR and IRTBa presented the highest values of SDW and RDW. Our results suggest that inoculation with R. tropici, T. asperellum and PGPR may promote beans growth and bring benefits to shoot and root accumulation, increase the number of nodules as well as improve yield components, contributing to a sustainable agriculture.

Transcriptome analysis of the differential effect of the NADPH oxidase gene RbohB in Phaseolus vulgaris roots following Rhizobium tropici and Rhizophagus irregularis inoculation.

BACKGROUND: Reactive oxygen species (ROS) are generated by NADPH oxidases known as respiratory burst oxidase homologs (RBOHs) in plants. ROS regulate various cellular processes, including the mutualistic interactions between legumes and nitrogen-fixing bacteria or arbuscular mycorrhizal (AM) fungi. Rboh is a multigene family comprising nine members (RbohA-I) in common bean (Phaseolus vulgaris). The RNA interference-mediated silencing of RbohB (PvRbohB-RNAi) in this species diminished its ROS production and greatly impaired nodulation. By contrast, the PvRbohB-RNAi transgenic roots showed early hyphal root colonization with enlarged fungal hypopodia; therefore, we proposed that PvRbohB positively regulates rhizobial infection (Rhizobium tropici) and inhibits AM colonization by Rhizophagus irregularis in P. vulgaris. RESULTS: To corroborate this hypothesis, an RNA-Seq transcriptomic analysis was performed to identify the differentially expressed genes in the PvRbohB-RNAi roots inoculated with Rhizobium tropici or Rhizophagus irregularis. We found that, in the early stages, root nodule symbioses generated larger changes of the transcriptome than did AM symbioses in P. vulgaris. Genes related to ROS homeostasis and cell wall flexibility were markedly upregulated in the early stages of rhizobial colonization, but not during AM colonization. Compared with AM colonization, the rhizobia induced the expression of a greater number of genes encoding enzymes involved in the metabolism of auxins, cytokinins, and ethylene, which were typically repressed in the PvRbohB-RNAi roots. CONCLUSIONS: Our research provides substantial insights into the genetic interaction networks in the early stages of rhizobia and AM symbioses with P. vulgaris, as well as the differential roles that RbohB plays in processes related to ROS scavenging, cell wall remodeling, and phytohormone homeostasis during nodulation and mycorrhization in this legume.

Genetic Interaction Studies Reveal Superior Performance of Rhizobium tropici CIAT899 on a Range of Diverse East African Common Bean (Phaseolus vulgaris L.) Genotypes.

We studied symbiotic performance of factorial combinations of diverse rhizobial genotypes (GR) and East African common bean varieties (GL) that comprise Andean and Mesoamerican genetic groups. An initial wide screening in modified Leonard jars (LJ) was followed by evaluation of a subset of strains and genotypes in pots (contained the same, sterile medium) in which fixed nitrogen was also quantified. An additive main effect and multiplicative interaction (AMMI) model was used to identify the contribution of individual strains and plant genotypes to the GL × GR interaction. Strong and highly significant GL × GR interaction was found in the LJ experiment but with little evidence of a relation to genetic background or growth habits. The interaction was much weaker in the pot experiment, with all bean genotypes and Rhizobium strains having relatively stable performance. We found that R. etli strain CFN42 and R. tropici strains CIAT899 and NAK91 were effective across bean genotypes but with the latter showing evidence of positive interaction with two specific bean genotypes. This suggests that selection of bean varieties based on their response to inoculation is possible. On the other hand, we show that symbiotic performance is not predicted by any a priori grouping, limiting the scope for more general recommendations. The fact that the strength and pattern of GL × GR depended on growing conditions provides an important cautionary message for future studies.IMPORTANCE The existence of genotype-by-strain (GL × GR) interaction has implications for the expected stability of performance of legume inoculants and could represent both challenges and opportunities for improvement of nitrogen fixation. We find that significant genotype-by-strain interaction exists in common bean (Phaseolus vulgaris L.) but that the strength and direction of this interaction depends on the growing environment used to evaluate biomass. Strong genotype and strain main effects, combined with a lack of predictable patterns in GL × GR, suggests that at best individual bean genotypes and strains can be selected for superior additive performance. The observation that the screening environment may affect experimental outcome of GL × GR means that identified patterns should be corroborated under more realistic conditions.

Regulation of hsnT, nodF and nodE genes in Rhizobium tropici CIAT 899 and their roles in the synthesis of Nod factors and in the symbiosis.

Rhizobium tropici strain CIAT 899 possesses outstanding agronomic properties as it displays tolerance to environmental stresses, a broad host range and high effectiveness in fixing nitrogen with the common bean (Phaseolus vulgaris L.); in addition, it carries intriguing features such as five copies of the regulatory nodD gene, and the capacity to synthesize a variety of nodulation factors (NFs), even in a flavonoid-independent manner, when submitted to abiotic stresses. However, the roles of several nod genes of the repertoire of CIAT 899 remain to be determined. In this study, we obtained mutants for the hsnT, nodF and nodE genes of CIAT 899 and investigated their expression, NF structures and symbiotic properties. Either in the presence of the flavonoid apigenin, or of salt the expression of hsnT, nodF and nodE in wild-type CIAT 899 was highly up-regulated in comparison to the mutants of all five copies of nodD, indicating the roles that regulatory nodD genes play in the activation of hsnT, nodF and nodE; however, NodD1 was recognized as the main inducer. In total, 29 different NF structures were synthesized by wild-type CIAT 899 induced by apigenin, and 36 when induced by salt, being drastically reduced by mutations in hsnT, nodF and nodE, especially under osmotic stress, with specific changes related to each gene, indicating that the three genes participate in the synthesis of NFs. Mutations in hsnT, nodF and nodE affected differently symbiotic performance (nodule number and shoot dry weight), according to the host plant. Our results indicate that the expression of hsnT, nodF and nodE genes of CIAT 899 is mediated by nodD genes, and although these three genes do not belong to the main set of genes controlling nodulation, they contribute to the synthesis of NFs that will impact symbiotic performance and host specificity.

Rhizobium tropici CIAT 899 copA gene plays a fundamental role in copper tolerance in both free life and symbiosis with Phaseolus vulgaris.

Rhizobium tropici CIAT 899 is a facultative symbiotic diazotroph able to deal with stressful concentrations of metals. Nevertheless the molecular mechanisms involved in metal tolerance have not been elucidated. Copper (Cu2+) is a metal component essential for the heme-copper respiratory oxidases and enzymes that catalyse redox reactions, however, it is highly toxic when intracellular trace concentrations are surpassed. In this study, we report that R. tropici CIAT 899 is more tolerant to Cu2+ than other Rhizobium and Sinorhizobium species. Through Tn5 random mutagenesis we identify a R. tropici mutant strain with a severe reduction in Cu2+ tolerance. The Tn5 insertion disrupted the gene RTCIAT899_CH17575, encoding a putative heavy metal efflux P1B-1-type ATPase designated as copA. Phaseolus vulgaris plants inoculated with the copA::Tn5 mutant in the presence of toxic Cu2+ concentrations showed a drastic reduction in plant and nodule dry weight, as well as nitrogenase activity. Nodules induced by the copA::Tn5 mutant present an increase in H2O2 concentration, lipoperoxidation and accumulate 40-fold more Cu2+ than nodules formed by the wild-type strain. The copA::Tn5 mutant complemented with the copA gene recovered the wild-type symbiotic phenotypes. Therefore, the copA gene is essential for R. tropici CIAT 899 to survive in copper-rich environments in both free life and symbiosis with P. vulgaris plants.

Osmotic stress activates nif and fix genes and induces the Rhizobium tropici CIAT 899 Nod factor production via NodD2 by up-regulation of the nodA2 operon and the nodA3 gene.

The symbiosis between rhizobia and legumes is characterized by a complex molecular dialogue in which the bacterial NodD protein plays a major role due to its capacity to activate the expression of the nodulation genes in the presence of appropiate flavonoids. These genes are involved in the synthesis of molecules, the nodulation factors (NF), responsible for launching the nodulation process. Rhizobium tropici CIAT 899, a rhizobial strain that nodulates Phaseolus vulgaris, is characterized by its tolerance to multiple environmental stresses such as high temperatures, acidity or elevated osmolarity. This strain produces nodulation factors under saline stress and the same set of CIAT 899 nodulation genes activated by inducing flavonoids are also up-regulated in a process controlled by the NodD2 protein. In this paper, we have studied the effect of osmotic stress (high mannitol concentrations) on the R. tropici CIAT 899 transcriptomic response. In the same manner as with saline stress, the osmotic stress mediated NF production and export was controlled directly by NodD2. In contrast to previous reports, the nodA2FE operon and the nodA3 and nodD1 genes were up-regulated with mannitol, which correlated with an increase in the production of biologically active NF. Interestingly, in these conditions, this regulatory protein controlled not only the expression of nodulation genes but also the expression of other genes involved in protein folding and synthesis, motility, synthesis of polysaccharides and, surprinsingly, nitrogen fixation. Moreover, the non-metabolizable sugar dulcitol was also able to induce the NF production and the activation of nod genes in CIAT 899.

Respuesta del simbiosistema frijol (Phaseolus vulgaris L.) y Rhizobium tropici CIAT899 ante el efecto alelopático de Ipomoea purpurea L. Roth / Responses of the common bean (Phaseolus vulgaris L.) and Rhizobium tropici CIAT899 symbiosystem to induced allelopathy by Ipomoea purpurea L. Roth

La alelopatía es un fenómeno que involucra la producción de metabolitos secundarios que influyen en el crecimiento de las plantas, pero este potencial alelopático ha sido poco estudiado en la simbiosis rizobio-leguminosa. Esta investigación tuvo los siguientes objetivos: 1) evaluar el potencial alelopático de lixiviados acuosos de Ipomoea purpurea L. Roth en la germinación de semillas y en el crecimiento radical de plántulas de frijol (Phaseolus vulgaris L.); 2) determinar el efecto de estos lixiviados en el crecimiento in vitro de Rhizobium tropici CIAT899, y 3) evaluar el potencial alelopático de I. purpurea en el crecimiento, la fisiología y la nodulación de frijol en simbiosis con R. tropici. Tanto el lixiviado acuoso de raíz como el de la parte aérea de I. purpurea estimularon la germinación de semillas de frijol y la elongación radical. El crecimiento in vitro de R. tropici fue inhibido al aplicar los 2 tipos de lixiviado. La presencia de I. purpurea tuvo un efecto negativo en el crecimiento y en las respuestas fisiológicas de las plantas de frijol, que fue atenuado cuando las plantas fueron inoculadas con Rhizobium tropici; no obstante, la nodulación asociada a esta bacteria fue afectada en presencia de la planta alelopática. Los resultados indican que la simbiosis de rizobios en las raíces de frijol es un elemento importante en la atenuación de los danos producidos por la planta alelopática I. purpurea.

Allelopathy is a phenomenon that involves the production of secondary metabolites that influence the growth of plants and microorganisms; however, this alellopathic effect has been scarcely studied on the rhizobia-legume symbiosis. The aims of this research were 1) to assess the allelopathic potential of aqueous extracts of Ipomoea purpurea L. Roth on seed germination and root length of common bean seedlings (Phaseolus vulgaris L.), 2) to determine its effects on the in vitro growth of Rhizobium tropici CIAT899, and 3) to evaluate the allelopathic potential of I. purpurea on the growth, nodulation and physiology of common bean plants inoculated with R. tropici. After 48 h, 15% of the aqueous root extract of I. purpurea stimulated seed germination, whereas 4% of the aqueous shoot extracts stimulated such germination. Both the root or shoot extracts stimulated seed germination and e root length. In vitro growth of R. tropici was inhibited as a result of the application of both aqueous extracts. The presence of I. purpurea negatively affected both the growth and physiological responses of common bean plants, and this effect was attenuated after the inoculation of R. tropici; nevertheless, this allelopathic plant affected root nodulation. Our results suggest that the symbiosis of rhizobia and roots of common bean plants is an important element for attenuating the negative effects caused by the allelopathic plant.

Flocculating, emulsification and metal sorption properties of a partial characterized novel exopolysaccharide produced by Rhizobium tropici SRA1 isolated from Psophocarpus tetragonolobus (L) DC

A novel exopolysaccharide (EPS) was produced by a bacterium which was isolated from Psophocarpus tetragonolobus (L) D.C. and identified as 99% Rhizobium tropici SRA1 by 16S rDNA sequencing. The flocculating performances along with emulsifying activity began simultaneously with the growth and the production of EPS and reached its utmost at 28 h. EPS was purified via chilled ethanol precipitation followed by dialysis and lyophilization. The existence of hydroxyl, methoxyl, and carboxylic functional groups were confirmed by Fourier transform infrared (FT-IR) spectrum. EPS was found to be compose of 82.44% neutral sugar and 15.93% uronic acid. The average molecular weight of the exopolysaccharide was estimated as ~1.8×105. Gas-liquid chromatography indicated the presence of glucose and galactose at a molar ratio of 3:1 in EPS. In the pH range of 3-5 with EPS dosage of 15 mg/l at 30 °C, cation-independent flocculation greater than 90% was observed. Emulsification indices (E24) of EPS were observed as 86.66%, 83.33%, 76.66%, and 73.33% with olive oil, kerosene, toluene, and n-hexane respectively. Biosorption of Cu K [45.69 wt%], Cu L [05.67 wt%], Co K [15.58 wt%], and Co L [11.72 wt%] by EPS was confirmed by energy-dispersive X-ray spectroscopy (EDS). This report on the flocculating, emulsifying, and metal sorption properties of EPS produced by R. tropici SRA1 is unique in the literature

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Flocculating, emulsification and metal sorption properties of a partial characterized novel exopolysaccharide produced by Rhizobium tropici SRA1 isolated from Psophocarpus tetragonolobus (L) D.C.

A novel exopolysaccharide (EPS) was produced by a bacterium which was isolated from Psophocarpus tetragonolobus (L) D.C. and identified as 99% Rhizobium tropici SRA1 by 16S rDNA sequencing. The flocculating performances along with emulsifying activity began simultaneously with the growth and the production of EPS and reached its utmost at 28 h. EPS was purified via chilled ethanol precipitation followed by dialysis and lyophilization. The existence of hydroxyl, methoxyl, and carboxylic functional groups were confirmed by Fourier transform infrared (FT-IR) spectrum. EPS was found to be compose of 82.44% neutral sugar and 15.93% uronic acid. The average molecular weight of the exopolysaccharide was estimated as ~ 1.8 × 105. Gas-liquid chromatography indicated the presence of glucose and galactose at a molar ratio of 3:1 in EPS. In the pH range of 3-5 with EPS dosage of 15 mg/l at 30 °C, cation-independent flocculation greater than 90% was observed. Emulsification indices (E24) of EPS were observed as 86.66%, 83.33%, 76.66%, and 73.33% with olive oil, kerosene, toluene, and n-hexane respectively. Biosorption of Cu K [45.69 wt%], Cu L [05.67 wt%], Co K [15.58 wt%], and Co L [11.72 wt%] by EPS was confirmed by energy-dispersive X-ray spectroscopy (EDS). This report on the flocculating, emulsifying, and metal sorption properties of EPS produced by R. tropici SRA1 is unique in the literature.

Revealing the roles of y4wF and tidC genes in Rhizobium tropici CIAT 899: biosynthesis of indolic compounds and impact on symbiotic properties.

Rhizobium tropici CIAT 899 is a strain known by its ability to nodulate a broad range of legume species, to synthesize a variety of Nod factors, its tolerance of abiotic stresses, and its high capacity to fix atmospheric N2, especially in symbiosis with common bean (Phaseolus vulgaris L.). Genes putatively related to the synthesis of indole acetic acid (IAA) have been found in the symbiotic plasmid of CIAT 899, in the vicinity of the regulatory nodulation gene nodD5, and, in this study, we obtained mutants for two of these genes, y4wF and tidC (R. tropiciindole-3-pyruvic acid decarboxylase), and investigated their expression in the absence and presence of tryptophan (TRP) and apigenin (API). In general, mutations of both genes increased exopolysaccharide (EPS) synthesis and did not affect swimming or surface motility; mutations also delayed nodule formation, but increased competitiveness. We found that the indole-3-acetamide (IAM) pathway was active in CIAT 899 and not affected by the mutations, and-noteworthy-that API was required to activate the tryptamine (TAM) and the indol-3-pyruvic acid (IPyA) pathways in all strains, particularly in the mutants. High up-regulation of y4wF and tidC genes was observed in both the wild-type and the mutant strains in the presence of API. The results obtained revealed an intriguing relationship between IAA metabolism and nod-gene-inducing activity in R. tropici CIAT 899. We discuss the IAA pathways, and, based on our results, we attribute functions to the y4wF and tidC genes of R. tropici.