Antivirulence effects of cell-free culture supernatant of endophytic bacteria against grapevine crown gall agent, Agrobacterium tumefaciens, and induction of defense responses in plantlets via intact bacterial cells.

BACKGROUND: Crown gall disease caused by Agrobacterium tumefaciens is a very destructive affliction that affects grapevines. Endophytic bacteria have been discovered to control plant diseases via the use of several mechanisms. This research examined the potential for controlling crown gall by three endophytic bacteria that were previously isolated from healthy cultivated and wild grapevines including Pseudomonas kilonensis Ba35, Pseudomonas chlororaphis Ba47, and Serratia liquefaciens Ou55. RESULT: At various degrees, three endophytic bacteria suppressed the populations of A. tumefaciens Gh1 and greatly decreased the symptoms of crown gall. Furthermore, biofilm production and motility behaviors of A. tumefaciens Gh1were greatly inhibited by the Cell-free Culture Supernatant (CFCS) of endophytic bacteria. According to our findings, CFCS may reduce the adhesion of A. tumefaciens Gh1 cells to grapevine cv. Rashe root tissues as well as their chemotaxis motility toward the extract of the roots. When compared to the untreated control, statistical analysis showed that CFCS significantly reduced the swimming, twitching, and swarming motility of A. tumefaciens Gh1. The findings demonstrated that the endophytic bacteria effectively stimulated the production of plant defensive enzymes including superoxide dismutase (SOD), polyphenol oxidase (PPO), peroxidase (POD), phenylalanine ammonia lyase (PAL), and total soluble phenols at different time intervals in grapevine inoculated with A. tumefaciens Gh1. The Ba47 strain markedly increased the expression levels of defense genes associated with plant resistance. The up-regulation of PR1, PR2, VvACO1, and GAD1 genes in grapevine leaves indicates the activation of SA and JA pathways, which play a role in enhancing resistance to pathogen invasion. The results showed that treating grapevine with Ba47 increased antioxidant defense activities and defense-related gene expression, which reduced oxidative damage caused by A. tumefaciens and decreased the incidence of crown gall disease. CONCLUSION: This is the first study on how A. tumefaciens, the grapevine crown gall agent, is affected by CFCS generated by endophytic bacteria in terms of growth and virulence features. To create safer plant disease management techniques, knowledge of the biocontrol processes mediated by CFCS during microbial interactions is crucial.

Biosynthesis of silver nanoparticles using Pseudomonas canadensis, and its antivirulence effects against Pseudomonas tolaasii, mushroom brown blotch agent.

This study reports the biosynthesis of silver nanoparticles (AgNPs) using a Pseudomonas canadensis Ma1 strain isolated from wild-growing mushrooms. Freshly prepared cells of P. canadensis Ma1 incubated at 26-28 °C with a silver nitrate solution changed to a yellowish brown color, indicating the formation of AgNPs, which was confirmed by UV-Vis spectroscopy, scanning electron microscopy (SEM), and X-ray diffraction. SEM analysis showed spherical nanoparticles with a distributed size mainly between 21 and 52 nm, and the XRD pattern revealed the crystalline nature of AgNPs. Also, it provides an evaluation of the antimicrobial activity of the biosynthesized AgNPs against Pseudomonas tolaasii Pt18, the causal agent of mushroom brown blotch disease. AgNPs were found to be bioactive at 7.8 µg/ml showing a minimum inhibitory concentration (MIC) effect against P. tolaasii Pt18 strain. AgNPs at the MIC level significantly reduced virulence traits of P. tolaasii Pt18 such as detoxification of tolaasin, various motility behavior, chemotaxis, and biofilm formation which is important for pathogenicity. Scanning electron microscopy (SEM) revealed that bacterial cells treated with AgNPs showed a significant structural abnormality. Results showed that AgNPs reduced brown blotch symptoms in vivo. This research demonstrates the first helpful use of biosynthesized AgNPs as a bactericidal agent against P. tolaasii.

Effect of volatile compounds produced by endophytic bacteria on virulence traits of grapevine crown gall pathogen, Agrobacterium tumefaciens.

The volatile organic compounds (VOCs) produced by endophytic bacteria have a significant role in the control of phytopathogens. In this research, the VOCs produced by endophytic bacteria including Serratia sp. Ba10, Pantoea sp. Sa14, Enterobacter sp. Ou80, Pseudomonas sp. Ou22, Pseudomonas sp. Sn48 and Pseudomonas sp. Ba35, which were previously isolated from healthy domesticated and wild-growing grapevine were evaluated in terms of their effects on the virulence traits of Agrobacterium tumefaciens Gh1, the causal agent of crown gall disease. Based on the gas chromatography-mass spectrometry analysis, 16, 15, 14, 7, 16, and 15 VOCs have been identified with high quality in strains of Ba10, Sa14, Ou80, Ou22, Sn48, and Ba35, respectively. All endophytic bacteria produced VOCs that significantly reduced crown gall symptoms and inhibited the populations of A. tumefaciens Gh1 at different levels. Moreover, scanning electron microscopy analysis revealed various morphological abnormalities in the A. tumefaciens cells exposed to the VOCs produced by Ba35, Ou80, and Sn48 strains. The VOCs significantly reduced swarming-, swimming-, twitching motility and biofilm formation by A. tumefaciens Gh1. Our results revealed that VOCs could reduce the attachment of A. tumefaciens Gh1 cells to root tissues of grapevine cultivars Rashe and Bidane sefid, as well as chemotaxis motility towards root extract of both cultivars. Based on our results, it was shown that the antibacterial VOCs produced by endophytic bacteria investigated in the current study can manage crown gall disease and increase our knowledge on the role of VOCs in microbial interactions.

Antibacterial Activity of Endophytic Bacteria Against Sugar Beet Root Rot Agent by Volatile Organic Compound Production and Induction of Systemic Resistance.

The volatile organic compounds (VOCs) produced by endophytic bacteria have a significant role in the control of phytopathogens. In this research, the VOCs produced by the endophytic bacteria Streptomyces sp. B86, Pantoea sp. Dez632, Pseudomonas sp. Bt851, and Stenotrophomonas sp. Sh622 isolated from healthy sugar beet (Beta vulgaris) and sea beet (Beta maritima) were evaluated for their effects on the virulence traits of Bacillus pumilus Isf19, the causal agent of harvested sugar beet root rot disease. The gas chromatographymass spectrometry (GC-MS) analysis revealed that B86, Dez632, Bt851, and Sh622 produced 15, 28, 30, and 20 VOCs, respectively, with high quality. All antagonistic endophytic bacteria produced VOCs that significantly reduced soft root symptoms and inhibited the growth of B. pumilus Isf19 at different levels. The VOCs produced by endophytic bacteria significantly reduced swarming, swimming, and twitching motility by B. pumilus Isf19, which are important to pathogenicity. Our results revealed that VOCs produced by Sh622 and Bt851 significantly reduced attachment of B. pumilus Isf19 cells to sugar beetroots, and also all endophytic bacteria tested significantly reduced chemotaxis motility of the pathogen toward root extract. The VOCs produced by Dez632 and Bt851 significantly upregulated the expression levels of defense genes related to soft rot resistance. Induction of PR1 and NBS-LRR2 genes in sugar beetroot slices suggests the involvement of SA and JA pathways, respectively, in the induction of resistance against pathogen attack. Based on our results, the antibacterial VOCs produced by endophytic bacteria investigated in this study can reduce soft rot incidence.

Characterization of Arsenic-Resistant Endophytic Bacteria From Alfalfa and Chickpea Plants.

The present investigation was carried out to isolate arsenic (As)-resistant endophytic bacteria from the roots of alfalfa and chickpea plants grown in arsenic-contamination soil, characterize their As tolerance ability, plant growth-promoting characteristics, and their role to induce As resistance by the plant. A total of four root endophytic bacteria were isolated from plants grown in As-contaminated soil (160-260-mg As kg-1 of soil). These isolates were studied for plant growth-promoting (PGP) characteristics through siderophore, phosphate solubilization, nitrogen fixation, protease, and lipase production, and the presence of the arsenate reductase (arsC) gene. Based on 16S rDNA sequence analysis, these isolates belong to the genera Acinetobacter, Pseudomonas, and Rahnella. All isolates were found As tolerant, of which one isolate, Pseudomonas sp. QNC1, showed the highest tolerance up to 350-mM concentration in the LB medium. All isolates exhibited phosphate solubilization activity. Siderophore production activity was shown by only Pseudomonas sp. QNC1, while nitrogen fixation activity was shown by only Rahnella sp. QNC2 isolate. Acinetobacter sp. QNA1, QNA2, and Rahnella sp. QNC2 exhibited lipase production, while only Pseudomonas sp. QNC1 was able to produce protease. The presence of the arsC gene was detected in all isolates. The effect of endophytic bacteria on biomass production of alfalfa and chickpea in five levels of arsenic concentrations (0-, 10-, 50-, 75-, and 100-mg kg-1 soil) was evaluated. The fresh and dry weights of roots of alfalfa and chickpea plants were decreased as the arsenic concentration of the soil was increased. Results indicate that the fresh and dry root weights of alfalfa and chickpea plants were significantly higher in endophytic bacteria-treated plants compared with non-treated plants. Inoculation of chickpea plants with Pseudomonas sp. QNC1 and Rahnella sp. QNC2 induced lower NPR3 gene expression in chickpea roots grown in soil with the final concentration of 100-mg kg-1 sodium arsenate compared with the non-endophyte-treated control. The same results were obtained in Acinetobacter sp. QNA2-treated alfalfa plants grown in the soil plus 50-mg kg-1 sodium arsenate. These results demonstrated that arsenic-resistant endophytic bacteria are potential candidates to enhance plant-growth promotion in As contamination soils. Characterization of bacterial endophytes with plant growth potential can help us apply them to improve plant yield under stress conditions.

Isolation and Identification of Endophytic Bacteria with Plant Growth Promoting Activity and Biocontrol Potential from Wild Pistachio Trees.

In this study, samples were collected from the leaves and stems of healthy wild Pistachio trees (Pistacia atlantica L.) from various locations of Baneh and Marivan regions, Iran. In total, 61 endophytic bacteria were isolated and grouped according to phenotypic properties. Ten selected isolates from each group were further identified by partial sequencing of the 16S rRNA gene. Based on the results, isolates were identified as bacteria belonging to Pseudomonas, Stenotrophomonas, Bacillus, Pantoea and Serratia genus. The ability of these isolates was evaluated to phytohormone production such as auxin and gibberellin, siderophore production, phosphate solubilization, atmospheric nitrogen fixation, protease and hydrogen cyanide production. All strains were able to produce the plant growth hormone auxin and gibberellin in different amounts. The majority of strains were able to solubilize phosphate. The results of atmospheric nitrogen fixation ability, protease and siderophore production were varied among strains. Only Ba66 could produce a low amount of hydrogen cyanide. The results of biocontrol assay showed that Pb78 and Sp15 strains had the highest and lowest inhibition effects on bacterial plant pathogens, Pseudomonas syringae pv. syringae Pss20 and Pseudomonas tolaasii Pt18 under in vitro condition. Pb3, Pb24 and Pb71 strains significantly promote root formation on carrot slices. To our knowledge this is the first report of the isolation of endophytic bacterial strains belonging to Pantoea, Bacillus, Pseudomonas, Serratia and Stenotrophomonas genus from wild pistachio trees with plant growth promoting potential and biocontrol activity.

Genetic evidence for CheB- and CheR-dependent chemotaxis system in A. tumefaciens toward acetosyringone.

Agrobacterium tumefaciens has homologues of chemotaxis genes of the alpha-subgroup of proteobacteria, which include orf1, orf2, cheY1, cheA, cheR, cheB, cheY2, orf9, and orf10 and organized in one operon [Wright et al. A chemotaxis cluster from Agrobacterium tumefaciens. Gene 1998;220:83-9]. Two mutants of A. tumefaciens were created by in-frame deletions of cheB and cheR using suicide vector, pK18mobsacB. The chemosensory behaviour of mutants studied on swarm plates and in blind-well chemotaxis assay toward acetosyringone. Under the conditions tested, both CheR and CheB were essential for normal chemotaxis.

Role of CheY1 and CheY2 in the chemotaxis of A. tumefaciens toward acetosyringone.

Agrobacterium tumefaciens has a chemtaxis operon, which includes orf1, orf2, cheY1, cheA, cheR, cheB, cheY2, orf9, and orf10. In-frame deletions of cheY1 and cheY2 were constructed and the chemosensory behavior of the mutants was examined on swarm plates and in a chemotaxis assay toward acetosyringone. The cheY2 mutant (C1/delY2) showed impaired chemotactic capabilities in both swarming and chemotaxis assays. The effect of lacking CheY1 on chemotaxis is less severe than that of CheY2, under the conditions studied.