| Fungus-growing termites are engaged in cultivating specific basidiomycetes(Termitomyces sp.)in their nests to form a fungus garden that serves as their main food source.The termites are symbiosis with specific fungi in vitro and symbiosis with bacteria in vivo,forming a trilateral symbiosis relationship of termites,in vitro fungi and intestinal microbes.The healthy fungus garden is often threatened by Xylaria spp.and termites may be infected by entomopathogens(Metarrhizium anisopliae and Beauveria bassiana).Its unique living habits may foster unique metabolites to defend antagonistic fungi against infections.Macrotermes barneyi is fungus-growing termite of the Macrotermes subfamily and are widely distributed in southern China.Previous studies on the diversity of the intestinal microbiota of Macrotermes barneyi showed that the intestinal microbiota contained a small amount of actinomycetes.Actinomycetes,as the main producer of natural products,may produce active metabolites to help termites to defend infection by antagonistic fungi.Therefore,we selected Macrotermes barneyi as the research material and started related research from the perspective of the secondary metabolites of intestinal actinomycetes.Actinomycetes were isolated from the the gut of workers and soldiers.In addition,the antifungal compounds produced by the actinomycetes were analyzed and identified.The specific research contents are shown as bellow.Isolation and identification of intestinal actinomycetes and analysis of antifungal activityIn this study,83 strains of actinomycetes were isolated from the intestines of workers and soldiers with six selective media.The 16S rRNA sequence analysis showed that these actinomycetes belong to 8 genera,60 strains of which belong to Streptomyces and 11 strains belong to Kitasatospora.The other actinomycetes belong to Amycolyticus,Micromonospora,Cellulomonas,Verrucosporium,Tsukamurella and Protomicromonospora respectively.Additionally,a noval rare actinomycete Amycolatopsis macrotermitis GM8 was identified.Through the Actinomyces-Xylaria paired challenge assays,44 strains of actinomycetes with moderate to strong antifungal activity against Xylaria sp.were obtained.On this basis,21 strains of actinomycetes with strong antifungal activity(Streptomyces and Kitasatospora)were further analyzed for their active metabolites,identifying 4 types of typical polyenes from active metabolites of 10 Streptomyces isolates.These polyenes correspond to cyclic tetraene,cyclic pentaene,cyclic and linear heptaene compounds,respectively.It is speculated that these polyene compounds are involved in the antifungal effect on Xylaria.In addition,the study showed that the crude extract of Xylaria sp.inhibit the growth of 3 polyene-producing Streptomyces strains(HF10,GS7 and GF20).Therefore,there is an antagonistic effect between Streptomyces and Xylaria.Isolation and purification of pentaene compounds from Streptomyces sp.HF10The active metabolites of Streptomyces HF10 are adsorbed and extracted by macroporous resin.The active metabolites are sequentially separated to middle-pressure liquid chromatography column(RP18),Sephadex LH-20 column and purified by semi-preparative reverse-phase HPLC based on activity tracking,finally to purify two pentaene macrolide compounds.Through compound mass spectrometry,nuclear magnetic resonance and biosynthetic gene cluster analysis,the chemical structures of these two compounds were resolved.They are known compounds,pentamycin(1)and 1'14-dihydroxy isochainin(2).The antifungal activity of pentamycin against Xylaria sp is 4-fold higher than that of Termitomyces sp.,and its antifungal activity against entomopathogens is 1.5-fold higher than that of Termitomyces sp.While,the antifungal activity of 1'14-dihydroxyisochainin against Xylaria is twice higher than that against entomopathogens,but it has no obvious antifungal activity against Termitomyces sp.In short,pentamycin and 1'14-dihydroxyisochainin have the strongest antifungal activity against Xylaria sp.,and the weakest antifungal activity against Termitomyces sp.The results of the study indicate that these two compounds have a potential protective effect on the fungus garden and termites.Knockout of tetraene and heptaene polyketide synthase genes in Streptomyces and antifungal activity analysis of knockout mutantsStreptomyces GS7 and GF20 as the starting strains,double cross-over knockout mutants GS7?PimS0 and GF20?fscA of natamycin(tetraene)and candidin(heptene)biosynthesis gene were successfully constructed by PCR-targeting knockout method,wild-type and mutant strains were subjected to fermentation,product extraction,HPLC detection,mass spectrometry analysis,and antifungal activity determination.It was found that the tetraene or heptaene compounds that were produced by wild-type strains had stronger antifungal activity against Xylaria sp.than Metarhizium anisopliae.Besides,the inhibitory activity of the mutant strains against Xylaria sp.and Metarhizium anisopliae was much lower than that of wild-type strains.The results of the study indicate that the tetraene and heptaene compounds produced by Streptomyces GS7 and GF20 participate in the inhibitory effect on antagonistic fungi.In summary,83 actinomycetes strains were isolated from the intestinal tract of Macrotermes barneyi,and 53%of the strains could inhibit the growth of Xylaria sp.Different types of polyene compounds were identified from the crude extracts of active metabolism of 10 Streptomyces strains.Using methods of isolating and purifying monomer compounds and knocking out key biosynthesis genes,the effects of tetraene,pentaene and heptaene compounds on antagonistic fungi of garden and termites were studied respectively.The study proved that these polyene compounds exhibit stronger inhibitory activity against the antagonistic fungus of the garden than the antagonistic fungus of the termite.Therefore,the results of this study indicate that the polyene compounds produced by Streptomyces from the intestinal tract of fungus-growing termites may protect termites and fungus gardens to defend pathogen infections.In short,this study provides a basis for understanding the ecological significance of intestinal actinomycetes in this special habitat,and also lays a foundation for the further development and utilization of active metabolites of intestinal actinomycetes in the fungus-growing termite. |
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