Studies On The Secondary Metabolites Encoded By Two Actinomycetes Strains

Anthracnose of Yam is one of the major diseases that influence the growth and development of Yam production,which has caused serious economic losses to yam industry in recent years for massively outbreking in China.In this paper,two strains of actinomycetes,HNM30702 and HNM0452,which showed significant antagonism to Colletotrichum gloeosporioides Cg9 were obtained.A series of separation techniques,such as HPLC,Sephades LH-20 gel chromatography,Octa decyl-bonded silica and thin layer chromatography were used to separate and purify the active metabolites of the two actinomycetes.The structure of antimicrobial active substances were identified by Mass spectrometry,Infrared spectroscopy,NMR analysis and optical rotation analysis,and its bioactivity was evaluated.(1)Three active monomers were isolated from the fermentation extract of actinomycetes HNM30702(compounds 1-3).Compound 1 is a cyclic hexapeptide containing two piperazic acid groups with N-N bonds,compounds 2 and 3 are macrolides antibiotic azalomycin F4a and azalomycin Fsa,respectively.It was found that compound 1 had inhibitory activity against human enterovirus 71 with IC50 23.0μM,and antimicrobial activity against Staphylococcus aureus and Streptococcus acidis with MIC 8μg/ml.Compounds 2 and 3 had strong antimicrobial activity against Colletotrichum gloeosporioides Cg9,Colletotrichum asianum T04085 Colletotrichum gloeosporioides RC178,Fusarium oxysporum f.sp.cubense race 4,Magnaporthe grisea,which had great potential and application value as agricultural antibiotic,and the value of MIC were 1.25、1.25、1.25、2.5、2.5 μg/ml and 2.5、5、2.5、5、5 μg/ml,respectively.(2)Six active monomers were isolated from the fermentation extract of actinomycetes HNM0452(compound 4-9).Compound 4 and 5 were polyketone antibiotic tirandamycin A and tirandamycin B,compound 6 were photopigments,compound 7 is adenosine,compound 8 is caffeine,and compound 9 is 4-hydroxyphenyl methylester.It was found that compound 4-9 showed obvious antimicrobial activity against Streptococcus agalactiae,the value of MIC were 2.5、5、2.5、20、20、80 μg/ml;and compound 7、8 and 9 showed weak antimicrobial activity against Staphylococcus aureus,the value of MIC were 40、20、80 μg/ml,respectively.(3)HNM30702 was identified as Streptomyces solisilvae in Streptomyces violaceusniger 16S rRNA gene clade,HNM0452 was identified as Streptomyces through 16S rRNA gene sequence analysis and Multilocus sequence analysis.