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Abstract: Sugarcane is an important Brazilian commodity, being usually cultivated in soils with low natural fertility. This study aimed to isolate diazotrophic endophytes from sugarcane tissues and evaluate the morphological and physiological characteristics of their colonies as well as their plant growth-promoting (PGP) traits in select diazotrophic endophytic bacteria. Fifty-six bacterial isolates were identified in the sugarcane tissues, and these isolates presented distinct morphological and physiological traits. A total of thirty-five bacterial isolates were biochemically evaluated. Overall, Bacillus was the dominant genus. Isolates of Methylobacterium spp. and Brevibacillus agri were present only in leaves, while Herbaspirillum seropedicae occurred only in stems. Except to IPA-CF45A, all isolates were nitrogenase positive. All endophytes exhibit production of indol 3-acetic acid. Over 50% of endophytes solubilize phosphate, release N-acyl homoserine lactones, and present the activity of 1-aminocyclopropane-1-carboxylic acid deaminase, catalase, lipase and protease. The network analysis showed that isolates belonged to Burkholderia, Herbaspirillum, and Methylobacterium interact with Bacillus. Bacterial endophytes exhibited distinct morphological, physiological, and PGP traits that are useful for sustainable agriculture, highlighting the isolates IPA-CC33, IPA-CF65, IPA-CC9 and IPA-CF27. Further studies on the effects of these diazotrophic endophytes and their potential for providing microbial inoculants for improving sugarcane fields will provide valuable information to maintain the sustainability and environment quality.

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Brazil has a long history of research with rhizobia and plant growth-promoting rhizobacteria (PGPR). Currently, the use of bio-based products in Brazil, containing microorganisms that are effective in promoting plant growth through various mechanisms, is already a consolidated reality for the cultivation of several crops of agricultural interest. This is due to the excellent results obtained over many years of research, which contributed to reinforce the use of rhizobia and PGPR by farmers. The high quality of the products offered, containing elite strains, allows the reduction and prevention in the use of mineral fertilization, contributing to low-cost and sustainable agriculture. Currently, research has turned its efforts in the search for new products that further increase the efficiency of those already available on the market and for new formulations or inoculation strategies that contribute to greater productivity and efficiency of these products. In this review, the history of biological products for main crops of agricultural interest and the new biotechnologies and research available in the agricultural market are discussed.

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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.

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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.

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We investigated the effects of Azospirillum brasilense strains Ab-V5 and Ab-V6 in the induction of mechanisms of systemic acquired resistance (SAR) and induced system resistance (ISR) on maize (Zea mays L.) plants. Under normal growth conditions, the treatments consisted of the standard inoculation of cells at sowing, and leaf spray of cells or their metabolites at the V2.5 growth stage; under saline stress (170 mM NaCl), the treatment consisted of standard single and co-inoculation of A. brasilense and Rhizobium tropici. The main compounds in the Azospirillum metabolites were identified as indole-3-acetic acid (IAA) and salicylic acid (SA). Under normal conditions, A. brasilense cells applied at sowing or by leaf spray increased the activities of catalase (CAT), superoxide dismutase (SOD), and malondialdehyde (MDA) in leaves, and of ascorbate peroxidase (APX) in roots; however, interestingly, in general the highest activities were observed by leaf spray of metabolites. Under normal conditions, the highest levels of salicylic acid (SA) and jasmonic acid (JA) were achieved in leaves by leaf spray of metabolites, of SA in roots by leaf spray of cells, and of JA in roots by standard inoculation and leaf spray of metabolites. Under saline stress, plant protection occurred via SA and abscisic acid (ABA), but not JA. In general, inoculation resulted in further increases in SA in leaves and roots, and ABA in leaves. We hypothesize that A. brasilense confers protection to maize plants by simultaneous induction of JA and SA pathways, and, under saline stressing conditions, by SA and ABA pathways.

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Plants are highly affected by salinity, but some plant growth-promoting bacteria (PGPB) may trigger induced systemic tolerance (IST), conferring protection against abiotic stresses. We investigated plant mechanisms under saline stress (170mM NaCl) when maize was singly or co-inoculated with Azospirillum brasilense strains Ab-V5 and Ab-V6 and Rhizobium tropici strain CIAT 899. Under greenhouse conditions, plants responded positively to inoculation and co-inoculation, but with differences between strains. Inoculation affected antioxidant enzymes that detoxify reactive oxygen species (ROS) - ascorbate peroxidase (APX), catalase (CAT) and superoxide dismutase (SOD) - mainly in leaves. Proline contents in leaves and roots and malondialdehyde (MDA) in leaves - plant-stress-marker molecules - were significantly reduced due to the inoculation, indicating reduced need for the synthesis of these molecules. Significant differences were attributed to inoculation in the expression of genes related to antioxidant activity, in general with upregulation of APX1, CAT1, SOD2 and SOD4 in leaves, and APX2 in roots. Pathogenesis-related genes PR1, prp2, prp4 and heat-shock protein hsp70 were downregulated in leaves and roots, indicating that inoculation with PGPB might reduce the need for this protection. Together the results indicate that inoculation with PGPB might provide protection from the negative effects of saline stress. However, differences were observed between strains, as A. brasilense Ab-V5 did not show salt tolerance, while the best inoculation treatments to mitigate saline stress were with Ab-V6 and co-inoculation with Ab-V6+CIAT 899. Inoculation with these strains may represent an effective strategy to mitigate salinity stress.

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Azospirillum brasilense is an important plant-growth promoting bacterium (PGPB) that requires several critical steps for root colonization, including biofilm and exopolysaccharide (EPS) synthesis and cell motility. In several bacteria these mechanisms are mediated by quorum sensing (QS) systems that regulate the expression of specific genes mediated by the autoinducers N-acyl-homoserine lactones (AHLs). We investigated QS mechanisms in strains Ab-V5 and Ab-V6 of A. brasilense, which are broadly used in commercial inoculants in Brazil. Neither of these strains carries a luxI gene, but there are several luxR solos that might perceive AHL molecules. By adding external AHLs we verified that biofilm and EPS production and cell motility (swimming and swarming) were regulated via QS in Ab-V5, but not in Ab-V6. Differences were observed not only between strains, but also in the specificity of LuxR-type receptors to AHL molecules. However, Ab-V6 was outstanding in indole acetic acid (IAA) synthesis and this molecule might mimic AHL signals. We also applied the quorum quenching (QQ) strategy, obtaining transconjugants of Ab-V5 and Ab-V6 carrying a plasmid with acyl-homoserine lactonase. When maize (Zea mays L.) was inoculated with the wild-type and transconjugant strains, plant growth was decreased with the transconjugant of Ab-V5-confirming the importance of an AHL-mediated QS system-but did not affect plant growth promotion by Ab-V6.

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Azospirillum spp. are plant-growth-promoting bacteria used worldwide as inoculants for a variety of crops. Among the beneficial mechanisms associated with Azospirillum inoculation, emphasis has been given to the biological nitrogen fixation process and to the synthesis of phytohormones. In Brazil, the application of inoculants containing A. brasilense strains Ab-V5 and Ab-V6 to cereals is exponentially growing and in this study we investigated the effects of maize inoculation with these two strains applied on seeds or by leaf spray at the V2.5 stage growth-a strategy to relieve incompatibility with pesticides used for seed treatment. We also investigate the effects of spraying the metabolites of these two strains at V2.5. Maize growth was promoted by the inoculation of bacteria and their metabolites. When applied via foliar spray, although A. brasilense survival on leaves was confirmed by confocal microscopy and cell recovery, few cells were detected after 24 h, indicating that the effects of bacterial leaf spray might also be related to their metabolites. The major molecules detected in the supernatants of both strains were indole-3-acetic acid, indole-3-ethanol, indole-3-lactic acid and salicylic acid. RT-PCR of genes related to oxidative stress (APX1, APX2, CAT1, SOD2, SOD4) and plant defense (pathogenesis-related PR1, prp2 and prp4) was evaluated on maize leaves and roots. Differences were observed according to the gene, plant tissue, strain and method of application, but, in general, inoculation with Azospirillum resulted in up-regulation of oxidative stress genes in leaves and down-regulation in roots; contrarily, in general, PR genes were down-regulated in leaves and up-regulated in roots. Emphasis should be given to the application of metabolites, especially of Ab-V5 + Ab-V6 that in general resulted in the highest up-regulation of oxidative-stress and PR genes both in leaves and in roots. We hypothesize that the benefits of inoculation of Azospirillum on seeds or by leaf spray, as well as of leaf spraying of Azospirillum metabolites, are strongly correlated with the synthesis of phytohormones and by eliciting genes related to plant-stress tolerance and defense against pathogens.

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BACKGROUND: Common bean (Phaseolus vulgaris L.) is the most important legume cropped worldwide for food production and its agronomic performance can be greatly improved if the benefits from symbiotic nitrogen fixation are maximized. The legume is known for its high promiscuity in nodulating with several Rhizobium species, but those belonging to the Rhizobium tropici "group" are the most successful and efficient in fixing nitrogen in tropical acid soils. Rhizobium leucaenae belongs to this group, which is abundant in the Brazilian "Cerrados" soils and frequently submitted to several environmental stresses. Here we present the first high-quality genome drafts of R. leucaenae, including the type strain CFN 299(T) and the very efficient strain CPAO 29.8. Our main objective was to identify features that explain the successful capacity of R. leucaenae in nodulating common bean under stressful environmental conditions. RESULTS: The genomes of R. leucaenae strains CFN 299(T) and CPAO 29.8 were estimated at 6.7-6.8 Mbp; 7015 and 6899 coding sequences (CDS) were predicted, respectively, 6264 of which are common to both strains. The genomes of both strains present a large number of CDS that may confer tolerance of high temperatures, acid soils, salinity and water deficiency. Types I, II, IV-pili, IV and V secretion systems were present in both strains and might help soil and host colonization as well as the symbiotic performance under stressful conditions. The symbiotic plasmid of CPAO 29.8 is highly similar to already described tropici pSyms, including five copies of nodD and three of nodA genes. R. leucaenae CFN 299(T) is capable of synthesizing Nod factors in the absence of flavonoids when submitted to osmotic stress, indicating that under abiotic stress the regulation of nod genes might be different. CONCLUSION: A detailed study of the genes putatively related to stress tolerance in R. leucaenae highlighted an intricate pattern comprising a variety of mechanisms that are probably orchestrated to tolerate the stressful conditions to which the strains are submitted on a daily basis. The capacity to synthesize Nod factors under abiotic stress might follow the same regulatory pathways as in CIAT 899(T) and may help both to improve bacterial survival and to expand host range to guarantee the perpetuation of the symbiosis.

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The rice endophyte Pantoea ananatis AMG521 shows several plant growth-promoting properties and promotes rice yield increases. Its draft genome was estimated at 4,891,568 bp with 4,704 coding sequences (CDS). The genome encodes genes for N-acylhomoserine lactone (AHL) synthases, AHL hydrolases, hyperadherence (yidQ, yidP, and yidR), fusaric acid resistance, and oxidation of lignin, highlighting its biotechnological potential.