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The production of 3-indoleacetic acid (IAA) by plant growth-promoting bacteria (PGPR) stimulates root development and plant growth. In addition, morphological changes such as an increased root ramification and root hair production improves nutrient absorption and biomass accumulation. The objective of this work was to evaluate the effect of IAA-producing strains on rice in an advanced stage of its vegetative cycle. Rice was inoculated with  Gluconacetobacter diazotrophicus  PAL 5 and its lao- mutant, deficient in auxin production,  Azospirillum baldaniorum  Sp 245, and Escherichia coli  DH10b. Both the mutant and wild-type  G. diazotrophicus  stimulated root elongation, area, volume, and diameter. However, the lao- mutant strain was the only one capable of increasing the number of roots. In turn, inoculation with  A. baldaniorum  had no significant effect on plant development. The inoculation with  E. coli  led to changes in root volume, area, and diameter, and a response that may be related to the stress caused by its presence. We conclude that the inoculation with  G. diazotrophicus  stimulates the root system’s growth independently of their IAA production ability, suggesting that a metabolite other than IAA is responsible for this effect at advanced stages of the rice’s vegetative cycle.

Background and aims Plant growth promoting bacteria (PGPB) are associated with plant roots and augment plant productivity and immunity; however, recent work by several groups has shown that PGPB also elicit the so-called “induced systemic tolerance” to drought stress. This work aimed to evaluate the effect of Gluconacetobacter diazotrophicus Pal5 inoculation on the mitigation of drought stress in red rice ( Oryza sativa L.). Methods The experiment treatments of the red rice cultivar inoculated and uninoculated with G. diazotrophicus , and cultivated with and without water restriction. Physiological, biochemical, and molecular analyses of plant tissues were carried out, along with measurements of biomass and seed yield components. Results The plants showed a positive response to inoculation with G. diazotrophicus , with promotion of growth and induction of tolerance to drought. Increase in plant biomass and higher levels of gas exchange and osmoprotectant solutes were observed in shoots. The bacterial inoculation increased the yield and positively regulated some defense genes against the water deficit in plants. Conclusions G. diazotrophicus Pal5 strain inoculation was beneficial for red rice plants, suppressing the various deleterious effects of drought stress and increasing the biomass and seed yield components.

Plant growth-promoting rhizobacteria (PGPR) increase plant growth and crop productivity. The inoculation of plants with a bacterial mixture (consortium) apparently provides greater benefits to plant growth than inoculation with a single bacterial strain. In the present work, a bacterial consortium was formulated containing four compatible and desiccation-tolerant strains with potential as PGPR. The formulation had one moderately (Pseudomonas putida KT2440) and three highly desiccation-tolerant (Sphingomonas sp. OF178, Azospirillum brasilense Sp7 and Acinetobacter sp. EMM02) strains. The four bacterial strains were able to adhere to seeds and colonize the rhizosphere of plants when applied in both mono-inoculation and multi-inoculation treatments, showing that they can also coexist without antagonistic effects in association with plants. The effects of the bacterial consortium on the growth of blue maize were evaluated. Seeds inoculated with either individual bacterial strains or the bacterial consortium were subjected to two experimental conditions before sowing: normal hydration or desiccation. In general, inoculation with the bacterial consortium increased the shoot and root dry weight, plant height and plant diameter compared to the non-inoculated control or mono-inoculation treatments. The bacterial consortium formulated in this work had greater benefits for blue maize plants even when the inoculated seeds underwent desiccation stress before germination, making this formulation attractive for future field applications.

Sugarcane interacts with particular types of beneficial nitrogen-fixing bacteria that provide fixed-nitrogen and plant growth hormones to host plants, promoting an increase in plant biomass. Other benefits, as enhanced tolerance to abiotic stresses have been reported to some diazotrophs. Here we aim to study the effects of the association between the diazotroph Gluconacetobacter diazotrophicus PAL5 and sugarcane cv. SP70-1143 during water depletion by characterizing differential transcriptome profiles of sugarcane. RNA-seq libraries were generated from roots and shoots of sugarcane plants free of endophytes that were inoculated with G. diazotrophicus and subjected to water depletion for 3 days. A sugarcane reference transcriptome was constructed and used for the identification of differentially expressed transcripts. The differential profile of non-inoculated SP70-1143 suggests that it responds to water deficit stress by the activation of drought-responsive markers and hormone pathways, as ABA and Ethylene. qRT-PCR revealed that root samples had higher levels of G. diazotrophicus 3 days after water deficit, compared to roots of inoculated plants watered normally. With prolonged drought only inoculated plants survived, indicating that SP70-1143 plants colonized with G. diazotrophicus become more tolerant to drought stress than non-inoculated plants. Strengthening this hypothesis, several gene expression responses to drought were inactivated or regulated in an opposite manner, especially in roots, when plants were colonized by the bacteria. The data suggests that colonized roots would not be suffering from stress in the same way as non-inoculated plants. On the other hand, shoots specifically activate ABA-dependent signaling genes, which could act as key elements in the drought resistance conferred by G. diazotrophicus to SP70-1143. This work reports for the first time the involvement of G. diazotrophicus in the promotion of drought-tolerance to sugarcane cv. SP70-1143, and it describes the initial molecular events that may trigger the increased drought tolerance in the host plant.

Gluconacetobacter diazotrophicus PAL5 and Azospirillum brasilense REC3 are plant growth promoting bacteria. They are able to produce hydroxamate and catechol type siderophores, respectively, when iron is not available, chelating this metal to facilitate its absorption. Iron is required by plants and is involved in physiological processes as part of many important compounds. The aim of this work was to evaluate the two siderophores producing bacteria in their contribution to iron nutrition for strawberry plants through the growth index, leaf and root area, greenness index, total soluble phenolic compounds and total iron content. Strawberry plants were grown hydroponically with a 16-h photoperiod in Hoagland nutrient solution, modified in iron sources, and inoculated with each bacterium. At day 60, the highest values of growth index, root area, greenness index, and iron content, were obtained for treatments with reduced iron, and the lowest values in treatments without iron addition. Values in treatments with oxidized iron and inoculated with bacteria were similar to those obtained with reduced iron and uninoculated plants. At day 30, phenolic compounds were higher in treatments without iron addition and uninoculated, while they decreased when plants were inoculated. For treatments with reduced iron, phenolic compounds content was low and increased when plants were inoculated. In conclusion, the siderophores produced by G. diazotrophicus PAL5 and A. brasilense REC3 can contribute to the iron nutrition of hydroponically grown strawberry plants. The participation of the hydroxamates was better than that of the catechols in the provision of iron to the plants.

Sugarcane interacts with particular types of beneficial nitrogen-fixing bacteria that provide fixed-nitrogen and plant growth hormones to host plants, promoting an increase in plant biomass. Other benefits, as enhanced tolerance to abiotic stresses have been reported to some diazotrophs. Here we aim to study the effects of the association between the diazotroph Gluconacetobacter diazotrophicus PAL5 and sugarcane cv. SP70-1143 during water depletion by characterizing differential transcriptome profiles of sugarcane. RNA-seq libraries were generated from roots and shoots of sugarcane plants free of endophytes that were inoculated with G. diazotrophicus and subjected to water depletion for 3 days. A sugarcane reference transcriptome was constructed and used for the identification of differentially expressed transcripts. The differential profile of non-inoculated SP70-1143 suggests that it responds to water deficit stress by the activation of drought-responsive markers and hormone pathways, as ABA and Ethylene. qRT-PCR revealed that root samples had higher levels of G. diazotrophicus 3 days after water deficit, compared to roots of inoculated plants watered normally. With prolonged drought only inoculated plants survived, indicating that SP70-1143 plants colonized with G. diazotrophicus become more tolerant to drought stress than non-inoculated plants. Strengthening this hypothesis, several gene expression responses to drought were inactivated or regulated in an opposite manner, especially in roots, when plants were colonized by the bacteria. The data suggests that colonized roots would not be suffering from stress in the same way as non-inoculated plants. On the other hand, shoots specifically activate ABA-dependent signaling genes, which could act as key elements in the drought resistance conferred by G. diazotrophicus to SP70-1143. This work reports for the first time the involvement of G. diazotrophicus in the promotion of drought-tolerance to sugarcane cv. SP70-1143, and it describes the initial molecular events that may trigger the increased drought tolerance in the host plant.

Background and aims This research investigates the biocontrol mechanisms of Gluconacetobacter diazotrophicus against the harmful bacterium Ralstonia pseudosolanacearum , causal agent of bacterial wilt disease. Given the ongoing need for effective control strategies, our focus lies in understanding the hormonal pathways, particularly the salicylic acid (SA) and jasmonic acid/ethylene (JA/Et) signaling pathways, involved in plant defense. Methods SA signalization deficiencies effects in plant-endophyte-pathogen interactions were observed through colonization ability, morpho-anatomical, physiological and disease severity parameters of sid2 (unable to produce SA via isochorismate synthase 1) and NahG (unable to accumulate SA) compared to wild-type Col0 plants treated with G. diazotrophicus , R. pseudosolanacearum or both bacteria. Additionally, pr1 , pr5, myc2, pdf1.2 (associated with SA or JA/Et defense response), and fitness (linked to redox homeostasis) relative expression were analyzed in Col0. Results G. diazotrophicus increased root hairs in Col0 and NahG. This correlated with a higher endophytic colonization frequency and enhanced lignification and xylem expansion. However, chlorophyll content and bacterial counts indicated an endophyte-NahG imbalance. Disease index, R. pseudosolanacearum bacterial counts and H 2 O 2 accumulation were lower in G. diazotrophicus inoculated Col0 and expression analysis revealed up-regulation of pr1 and myc2 . In co-inoculated plants, R. pseudosolanacearum increased pdf1.2 levels by 15.2-times, which was significantly higher than in those inoculated solely with pathogenic bacteria. G. diazotrophicus managed to maintain fitness low levels even after R. pseudosolanacearum infection. Conclusions The interplay G. diazotrophicus -Arabidopsis, mediated by the expression of pr , myc2 and fitness primes for subsequent infection with R. pseudosolanacearum , triggering the expression of JA/Et pathway genes.

Acetic acid fermentation involves the oxidation of ethanol to acetic acid via acetaldehyde as the intermediate and is catalyzed by the membrane-bound alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) of acetic acid bacteria. Although ADH depends on pyrroloquinoline quinone (PQQ), the prosthetic group associated with ALDH remains a matter of debate. This study aimed to address the dependency of ALDH of Gluconacetobacter diazotrophicus strain PAL5 on PQQ and the physiological role of ALDH in acetic acid fermentation. We constructed deletion mutant strains for both the ALDH gene clusters of PAL5, aldFGH and aldSLC. In addition, the adhAB operon for ADH was eliminated, since it shows ALDH activity. The triple-deletion derivative ΔaldFGH ΔaldSLC ΔadhAB failed to show ALDH activity, which suggested that ALDH activity in PAL5 is derived from these three enzyme complexes. Since the single-gene cluster deletion derivative ΔaldFGH lost most ALDH activity, and accumulated much higher acetaldehyde than wild type under acetic acid fermentation conditions, we concluded that AldFGH functions as the major ALDH in PAL5. Furthermore, deletion of the PQQ biosynthesis gene cluster (pqqABCDE) abolished ADH activity completely, but did not affect ALDH activity. Instead, the molybdopterin biosynthesis gene deletion derivatives lost ALDH activity. Thus, we concluded that the AldFGH and AldSLC complexes of Ga. diazotrophicus PAL5 require a form of molybdopterin but not PQQ for ALDH activity.Key points• AldFGH is the major aldehyde dehydrogenase in Gluconacetobacter diazotrophicus PAL5.• Acetaldehyde accumulated from ethanol in the absence of AldFGH.• Molybdopterin, rather than pyrroloquinoline quinone, is required for AldFGH.

Background Gluconacetobacter diazotrophicus Pal5 is an endophytic diazotrophic bacterium that lives in association with sugarcane plants. It has important biotechnological features such as nitrogen fixation, plant growth promotion, sugar metabolism pathways, secretion of organic acids, synthesis of auxin and the occurrence of bacteriocins. Results Gluconacetobacter diazotrophicus Pal5 is the third diazotrophic endophytic bacterium to be completely sequenced. Its genome is composed of a 3.9 Mb chromosome and 2 plasmids of 16.6 and 38.8 kb, respectively. We annotated 3,938 coding sequences which reveal several characteristics related to the endophytic lifestyle such as nitrogen fixation, plant growth promotion, sugar metabolism, transport systems, synthesis of auxin and the occurrence of bacteriocins. Genomic analysis identified a core component of 894 genes shared with phylogenetically related bacteria. Gene clusters for gum -like polysaccharide biosynthesis, tad pilus, quorum sensing, for modulation of plant growth by indole acetic acid and mechanisms involved in tolerance to acidic conditions were identified and may be related to the sugarcane endophytic and plant-growth promoting traits of G. diazotrophicus . An accessory component of at least 851 genes distributed in genome islands was identified, and was most likely acquired by horizontal gene transfer. This portion of the genome has likely contributed to adaptation to the plant habitat. Conclusion The genome data offer an important resource of information that can be used to manipulate plant/bacterium interactions with the aim of improving sugarcane crop production and other biotechnological applications.

BACKGROUND AND AIMS: Gluconacetobacter diazotrophicus is a plant growth-promoting bacteria (PGPB) that colonizes several plant species. Here, we studied the internal colonization of Arabidopsis thaliana tissues by G. diazotrophicus and analyzed its effects on physiology, growth, and activation of plant immune system during such association. METHODS: A. thaliana seedlings were inoculated with G. diazotrophicus and grown in substrate for 50 days. Effects on plant growth were estimated by quantifying number of leaves, leaf area, and fresh and dry weight. Endophytic bacterial population was determined by colony-forming unit (CFU), and its location in plant tissues was assayed by epifluorescence microscopy of red fluorescent protein-labeled bacterium. Whole canopy gas exchange (photosynthesis and transpiration) was determined using a portable photosynthesis system. RESULTS: G. diazotrophicus efficiently promoted A. thaliana plant growth at 50 days after inoculation. Inoculated plants showed higher whole canopy photosynthesis, lower whole plant transpiration, and increased water-use efficiency. The bacterium colonized preferentially root xylem. The inoculation of plants defective in systemic acquired resistance (SAR)-associated defense revealed that plant immune system plays an important role during the early association stages. CONCLUSIONS: G. diazotrophicus endophytically colonizes A. thaliana roots, promotes plant growth, and increases whole canopy photosynthesis. Our results indicate that A. thaliana is useful for molecular studies of the mechanisms involved in the interaction between plants and PGPB, especially those involving G. diazotrophicus.