| The environmental strain Bacillus amyloliquefaciens FZB42 promotes plant growth and suppresses plant pathogenic organisms present in the rhizosphere. FZB42 genome analysis revealed the presence of numerous gene clusters involved in synthesis of non-ribosomally synthesized cyclic lipopeptides and polyketides with distinguished antimicrobial action. Recent studies have demonstrated that Bacillus spp. can suppress the growth of harmful algal bloom species. In a previous study, we demonstrated that rice plants treated with B. amyloliquefaciens FZB42 suspensions showed significant improvement in resistance to Xanthomonas. oryzae pv. oryzae over untreated plants while the active compound(s) and mechanisms of action remain to be identified. Cyclodipeptides obtained by the condensation of two a-amino acids are relatively simple compounds and,therefore,are among the most common peptide derivatives found in nature. In this study,the effect of cyclodipeptides on the physiological responses and plant signaling pathways was also investigated. The main research results are as follows:Amongst 24 strains, B. amyloliquefaciens FZB42 showed strongest bactericidal activity against M.aeruginosa, with a kill rate of 98.78%. After treatment with FZB42, the water quality was restored. And metabolites may exist in the culture filtrates that exert anti-cyanobacterial activities. The algicidal agent was identified as a thermo-stable,pH-stable, molecular weight less than 3 kD, non-protein substance. To identify the anti-cyanobacterial agent, 2000 TnYLB-1 transposon-inserted mutants were screened after FZB42 was transformed with transposon-carrying pMarA. Two mutants, M436(bactericidal activity 2.65%) and M1125 (bactericidal activity 6.48%), were unable to inhibit the growth of M. aeruginosa. The aroA gene of M436 and aroE gene of M1125 had been disrupted by the TnYLB-1 transposon. Both genes are responsible for aromatic acid biosynthesis in many Bacillus spp. All aro mutants are deficient in bacilysin biosynthesis and it is known that the bacilysin pathway branches off the aromatic amino acid pathway at prephenate. Thus, we hypothesize that it is bacilysin produced by B. amyloliquefaciens FZB42 that has specific bactericidal activity against M. aeruginosa. Disruption of bacB, the gene in the cluster responsible for synthesizing bacilysin, or supplementation with the antagonist N-acetylglucosamine abolished the inhibitory effect, but this was restored when bacilysin synthesis was complemented. Bacilysin caused apparent changes in the algal cell wall and cell organelle membranes, and this resulted in cell lysis. Meanwhile, there was downregulated expression of glmS, psbAl, mcyB and ftsZ, which are genes involved in peptidoglycan synthesis,photosynthesis,microcystin synthesis and cell division,respectively. In addition, bacilysin suppressed the growth of other harmful algal species. In summary, bacilysin produced by B. amyloliquefaciens FZB42 has anti-cyanobacterial activity and thus could be developed as a biocontrol agent to mitigate the effects of harmful algal blooms.FZB42 was shown to possess biocontrol activity against these Xanthomonas strains by producing the antibiotic compounds difficidin and bacilysin. Analyses using fluorescence,scanning electron and transmission electron microscopy revealed difficidin and bacilysin caused changes in the cell wall and structure of Xanthomonas. Biological control experiments on rice plants demonstrated the ability of difficidin and bacilysin to suppress disease. Difficidin and bacilysin caused downregulated expression of genes involved in Xanthomonas virulence, cell division, and protein and cell wall synthesis. Taken together,our results highlight the potential of B. amyloliquefaciens FZB42 as a biocontrol agent against bacterial diseases of rice, and the utility of difficidin and bacilysin as antimicrobial compounds.B. amyloliquefaciens strains FZBREP and FZBSPA were derived from the wild-type FZB42 by replacement of the native bacilysin operon promoter with constitutive promoters PrepB and Pspac from plasmids pMK3 and pLOSS, respectively. These strains contained two antibiotic resistance genes, and markerless strains were constructed by deleting the chloramphenicol resistance cassette and promoter region bordered by two lox sites (lox71 and lox66) using Cre recombinase expressed from the temperature-sensitive vector pLOSS-cre. The vector-encoded spectinomycin resistance gene was removed by high temperature (50?) treatment. RT-PCR and qRT-PCR results indicated that PrepB and especially Pspac significantly increased expression of the bac operon, and FZBREP and FZBSPA strains produced up to 173.4% and 320.1% more bacilysin than wild type,respectively. Bacilysin overproduction was accompanied by enhancement of the antagonistic activities against Staphylococcus aureus (an indicator of bacilysin) and Clavibacter michiganense subsp. sepedonicum (the causative agent of potato ring rot). Both the size and degree of ring rot-associated necrotic tubers were decreased compared with the wild-type strain, which confirmed the protective effects and biocontrol potential of these genetically engineered strains.In this study, we also found that cyclo (L-Pro-L-Pro) and cyclo (D-Pro-D-Pro) could induce defense responses and systemic resistance in Nicotiana benthamiana. Treatment with the two cyclodipeptides led to a reduction in disease severity by Phytophthora nicotianae and tobacco mosaic virus (TMV) infections compared with the controls. Both cyclopeptides triggered stomatal closure, induced reactive oxygen species production, and stimulated cytosolic calcium ion and nitric oxide production in guard cells. Additionally,application of cyclodipeptides significantly upregulated expression of the plant defense gene PR-1? and the PR-1a protein, and increased cellular salicylic acid (SA) levels. These results suggest that the SA-dependent defense pathway is involved in cyclodipeptide-mediated pathogen resistance in N. benthamiana. We report on the systemic resistance induced by cyclodipeptides, which sheds light on the potential of cyclodipeptides in the control of plant diseases. |
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