| Sclerotinia rot of colza is a plant disease caused by Sclerotinia sclerotiorum,the general incidence rate is about 30% and the disease rate is as high as 80% in some situations.Sclerotinia sclerotiorum has the characteristics of strong vitality and wide spread ways,which make the disease difficult to control.At present,chemical fungicides are still used to prevent the occurrence of Sclerotinia sclerotiorum,but the long-term use of chemical fungicides not only has caused great harm to human health and ecological environment,but also increase the resistance of pathogens at the same time,therefore,it is necessary to choose safe and environment friendly measures to prevent the disease.Actinomycetes have the features of great variety and wide distribution,and the ability of producing active metabolites,it is important to use actinomycetes to control the occurrence of Sclerotinia rot of colza.In this study,six media(CPA,DPA,SSA,AAG,GS,HV)were used to isolate actinomycetes from the rhizosphere soil of oilseed rape,and the actinomycetes were classified initially.Activity screening test of actinomycetes was conducted by using Sclerotinia sclerotiorum as indicator fungus,and the control effect of antagonistic strains against Sclerotinia rot of colza was explored at the same time.Subsequently,the physiological and biochemical characteristics of the antagonistic strains were analyzed,and the secondary metabolites of one strain were isolated.In addition,this study identified a rare actinomycete by polyphasic taxtonomy,it increased the richness of species.The results are as follows:(1)There were totals of 467 actinomycetes isolated from the rhizosphere soil of oilseed rape and there were 116 strains by classification.Morphological observation and sequence analysis suggested that the primary strains belonged to Streptomyces,there were also some rare genus,such as Micromonospora,Streptacidiphilus,Nocardia,Kribbella,Nonomuraea,Glycomyces and Amycolatum.(2)The results of resistance test showed that six actinomycetes(sw3,sw15,sw23,sw41,sw66 and sw108)existed inhibitory effect to Sclerotinia sclerotiorum,the inhibition rates were55.7%,47.3%,51.4%,85.7%,88.5% and 14.3%,respectively,of which strain sw66 had the best inhibitory effect.(3)Vitro and pot test showed that the spore suspension and fermentation broth of strains sw41 and sw66 could significantly control the expansion of leaf lesions.No lesions were found in the leaves of the vitro and pot test,the control rate reached to 100% after the treatment of strain sw66 fermentation broth.The two strains were identified as Streptomyces by morphological characteristics and 16 S r RNA sequence analysis.(4)The fermentation conditions of strain sw66 were optimized,experiment results showed that the fermentation broth had the highest activity in GYM medium when the fermentation time was 7 days,inoculation amount was 5%,rotation speed was 200 rpm and liquid volume was 80 m L.Best fermentation condition was used to isolate the secondary metabolites of strain sw66,three secondary metabolites were obtained,the compounds were thymidine,N-acetyl tryptophan and4-methyl-3,4-dihydro-2H-1-naphthalenone,respectively.The inhibition rate of N-acetyl tryptophan against Sclerotinia sclerotiorum was 72.5% when the concentration was 1.0 mg/m L.(5)A rare actinomycete NEAU-SW521 T was identified by polyphasic taxonomy,the comparisons of physiological and biochemical characteristics,morphological characteristics and molecular biological experiments between NEAU-SW521 T and the closely strains showed that it was consistent with the Kribbella,but differented from the related species,NEAU-SW521 T was identified as a new species of Kribbella,the name is Kribbella monticola.The functions and metabolites of strain NEAU-SW521 T were predicted by genome informaion. |
