| Lysobacter enzymogenes OH11,belonging to Xanthomonadaceae family,is an environmentally-friendly gram-negative biocontrol agent.This strain is originaly isolated from pepper rhizosphere and could secrete a broad-spectrum antifungal antibiotic HSAF(heat-stable antifungal factor)to fight against a variety of crop fungal pathogens.In our earlier works,the in vivo biosynthetic and regulatory mechanisms of HSAF in L.enzymogenes OH 11 have been well elucidated.The objective of this work is aimed to explore whether OH11’s ecologically-related biotic factors(i.e pathogenic and biocontrol microorganisms)and abiotic factors(i.e.pesticide)could affect OH11 to produce HSAF.If yes,what is the potential underlying mechanism(s)?In this work,we first test whether the pathogenic Phytophthora capsici with a same niche with strain OH 11 could affect HSAF production in strain OH11.To mimic P.capsici-L.enzymogenes interaction in niche,we artificially add culture supernatant of P.capsici into OH 11 broth to cultivate OH11 growth,followed to quantify HSAF yield.We found that the P.capsici’s supernatant(metabolites)failed to affect HSAF levels.Similarly,two well-applied pesticides in crop rhizosphere,tebuconazole and fludioxon were also ineffective in affecting HSAF production via adding to H11 culture.Surprisingly,we found that when OH 11 is co-cultivated with two niche-related biocontrol Pseudomonas strains,YL-1 and 2P24,the ability of strain OH11 in producing HSAF was significantly impaired,but the growth of strain OH11 was not inhibited in the presence of YL-1 or 2P24.We also observed that co-culture of OH 11 with a series of decreased amount of YL-1 or 2P24,the HSAF production levels were increased gradually.Via promoter activity and semi-quantitative RT-PCR assays,we found that both YL-1 and 2P24 could suppress the transcription of the HSAF biosynthesis operon genes,leading to inhibit HSAF production.These results suggest that among our test biotic and abiotic factors,two biocontrol Pseudomonas strains could affect HSAF production in strain OH11 via uncharacterized underlying mechanism(s).To understand how the test Pseudomonas strains affect HSAF production in strain OH11,we focus on the secondary metabolites as well as quorum-sensing signals,as both of them are important for to control gene expression in response to stimuli.These factors may mediate interaction to affect HSAF production in strain OH11.To test this hypothesis,we performed detailed bioinformatics analyses on the genome of strain YL-1 and identified the respective gene clusters with predicted functions to participate the production of phenazine,pyrromycin,hydrocyanic acid,a group of common metabolites in Pseudomonas.We established a genetic manipulation system of YL-1 and generated the corresponding mutants for each of the identified gene clusters.Via a co-culture approach,we did not find each of the test mutants failed to inhibit HSAF production in strain OH11,which is similar with that of the wild-type YL-1.These results raise a possibility that YL-1 inhibits HSAF synthesis in strain OH 11 is possibly not dependent on the test gene clusters responsible for secondary-metabolite biosynthesis.On the basis of the above findings,we move to investigate whether the quorum-sensing signal commonly used by Pseudomonas strains will involve in Pseudomonas-Lysobacter interaction.For this purpose,the wild-type 2P24 and its PcoI/PcoR quorum-sensing deficient mutants were selected,however,no obvious difference between 2P24 and its quorum-sensing mutants was observed in repressing HSAF biosynthesis of strain OH11.We also explore whether type VI secretion system,which is a recently identified system mediated bacterial-bacterial antagonistic interaction,could contribute to Pseudomonas-Lysobacter interaction,however,we also failed to observe an effective effect.Taken together,these results suggest that both commonly-spread quorum-sensing and type VI secretion systems are also not mediate Pseudomonas-Lysobacter interaction in repressing HSAF biosynthesis.In summary,among our test factors,we validated that niche-related biocontrol Pseudomonas strains could inhibit HSAF biosynthesis of strain OH 11 and this type of inhibition did not seem to be dependent on the gene clusters responsible for secondary-metabolite biosynthesis,quorum sensing and type VI secretion system.The detailed mechanism is current unidentified.Nevertheless,our study establishes a"competitive" Pseudomonas-Lysobacter interaction system for future deeply understanding how niche-related Pseudomonas strains affect HSAF biosynthesis of strain OH 11 and also provides fundamental insights into efficient usage of strain OH11 in field against crop fungal pathogens. |
