Orfamide B

Although plant pathogens are traditionally controlled using synthetic agrochemicals, the availability of commercial bactericides is still limited. One potential control strategy could be the use of plant growth-promoting bacteria (PGPB) to suppress pathogens via resource competition or the production of antimicrobial compounds. This study aimed to conduct in vitro and in vivo screening of eight Pseudomonas strains against Ralstonia solanacearum (the causative agent of bacterial wilt) and to investigate underlying mechanisms of potential pathogen suppression. We found that inhibitory effects were Pseudomonas strain-specific, with strain CHA0 showing the highest pathogen suppression. Genomic screening identified 2,4-diacetylphloroglucinol, pyoluteorin, and orfamides A and B secondary metabolite clusters in the genomes of the most inhibitory strains, which were investigated further. Although all these compounds suppressed R. solanacearum growth, only orfamide A was produced in the growth media based on mass spectrometry. Moreover, orfamide variants extracted from Pseudomonas cultures showed high pathogen suppression. Using the "Micro-Tom" tomato cultivar, it was found that CHA0 could reduce bacterial wilt disease incidence with one of the two tested pathogen strains. Together, these findings suggest that a better understanding of Pseudomonas-Ralstonia interactions in the rhizosphere is required to successfully translate in vitro findings into agricultural applications.

Kiwifruit worldwide suffers from the devastating diseases of bacterial canker caused byPseudomonas syringaepv.actinidiae(Psa) and gray mold caused byBotrytis cinerea. Here, an endophytic bacterium XL17 isolated from a rape crown gall was screened out for its potent antagonistic activities against Psa andB. cinerea. Strain XL17 and its cell-free culture filtrate (CF) inhibited the growth of Psa andB. cinerea, Psa-associated leaf necrosis, andB. cinerea-associated kiwifruit necrosis. Electron microscopy showed that XL17 CF could damage the cell structures of Psa andB. cinerea. Genome-based taxonomy revealed that strain XL17 belongs toPseudomonas bijieensiswithin theP. corrugatasubgroup of theP. fluorescensspecies complex. Among theP. corrugatasubgroup containing 31 genomospecies, the presence of thephloperon responsible for the biosynthesis of the phenolic polyketide 2,4-diacetylphloroglucinol (DAPG) and the absence of the lipopeptide/quorum sensing island can serve as the genetic marker for the determination of a plant-protection life style. HPLC detected DAPG in extracts from XL17 CF. MALDI-TOF-MS analysis revealed that strain XL17 produced cyclic lipopeptides of the viscosin family and orfamide family. Together, phenotypic, genomic, and metabolic analyses identified thatP. bijieensisXL17 producing DAPG and cyclic lipopeptides can be used to control bacterial canker and gray mold pathogens of kiwifruit.

Orfamide-type cyclic lipopeptides (CLPs) are biosurfactants produced by Pseudomonas and involved in lysis of oomycete zoospores, biocontrol of Rhizoctonia and insecticidal activity against aphids. In this study, we compared the biosynthesis, structural diversity, in vitro and in planta activities of orfamides produced by rhizosphere-derived Pseudomonas protegens and related Pseudomonas species. Genetic characterization together with chemical identification revealed that the main orfamide compound produced by the P. protegens group is orfamide A, while the related strains Pseudomonas sp. CMR5c and CMR12a produce orfamide B. Comparison of orfamide fingerprints led to the discovery of two new orfamide homologs (orfamide F and orfamide G) in Pseudomonas sp. CMR5c. The structures of these two CLPs were determined by nuclear magnetic resonance (NMR) and mass spectrometry (MS) analysis. Mutagenesis and complementation showed that orfamides determine the swarming motility of parental Pseudomonas sp. strain CMR5c and their production was regulated by luxR type regulators. Orfamide A and orfamide B differ only in the identity of a single amino acid, while orfamide B and orfamide G share the same amino acid sequence but differ in length of the fatty acid part. The biological activities of orfamide A, orfamide B, and orfamide G were compared in further bioassays. The three compounds were equally active against Magnaporthe oryzae on rice, against Rhizoctonia solani AG 4-HGI in in vitro assays, and caused zoospore lysis of Phytophthora and Pythium. Furthermore, we could show that orfamides decrease blast severity in rice plants by blocking appressorium formation in M. oryzae. Taken all together, our study shows that orfamides produced by P. protegens and related species have potential in biological control of a broad spectrum of fungal plant pathogens.