Identification Of The Antifungal Metabolites From Streptomyces Sp.3-10 And Evaluation Of Their Efficacy Against Plant Fungal Diseases

Streptomyces species are Gram-positive,filamentous and spore-producing actinobacteria with high G+C content in their genomes.Streptomyces is generally regarded as pharmaceutically important biological resources,because many species of Streptomyces can produce various physiological active chemicals,including antibiotics,antimicrobial enzymes,antioxidants,as well as anti-inflammatory and anti-tumor compounds.It is estimated that 70 to 80% antibiotics in clinical use are produced by Streptomyces.Che(2010)isolated Streptomyces sp.strain3-10,which showed high antifungal activity.In this study,the taxonomic status of Streptomyces sp.3-10 was re-evaluated.The antifungal efficacy of the crude extract of Streptomyces sp.3-10 was determined both in vitro and in vivo.The antifungal substances produced by Streptomyces sp.3-10 were purified and identified.Meanwhile,the antifungal activity of the purified compounds from strain 3-10 was determined.The main findings were summarized below:1.Morphological,physiological and biochemical characterizations and 16 S rDNA analysis indicated that Streptomtces sp.3-10 is a putative novel phylotype of Streptomyces yanglinensis.2.Ethyl acetate was found capable of being used as an organic solvent to extract the antifungal metabolites from cell free culture filtrates of strain 3-10,which were designated as the crude extract(CE)of 3-10.The CE at 5.0 ?g/mL had a broad antifungal spectrum with the inhibition rates higher than 80% against mycelial growth of 16 plant pathogenic fungi.The CE could inhibit conidial germination of Botrytis cinerea,and sporangiospore germination of Rhizopus stolonifer and Mucor hiemails.Mycelia malformation was also observed on the hyphal tips treated with the CE.The disease severity of B.cinerea,S.sclerotiorum,R.stolonifer,M.hiemails on strawberry fruit under the storage conditions were decreased after application of the CE.In greenhouse experiment,application of the CE at 50 ?g/mL reduced the disease incidence and disease severity of gray mold fruit rot on strawberry approximated 50%.On barley,the CE showed both protective and curative effects on infection by the powdery mildew fungus Blumeria graminis var.hordei.3.Environmental factors affecting the antifungal stability of the CE were characterized.The antifungal activity of the CE was not stable after incubation at 80°C for 10 min.Exposure to UV-C light for 1 min could significantly reduce the antifungal activity of the CE.Under the acidic condition,the CE showed stable antifungal activity.The CE in the dry and powdery form could be stored for 100 days with high antifungal activity.4.Two antifungal compounds were purified from the CE of strain of 3-10.The two compounds were white amorphous powder and could be easily dissolved in methanol.The results of LC-MS and NMR data demonstrated that these two compounds were reveromycins A and B,respectively.They effectively suppressed mycelial growth of B.cinerea,M.hiemails,R.stolonifer,and S.sclerotiorum,and spore germination of B.cinerea,M.hiemails,and R.stolonifera.The antifungal activity of reveromycin A was significantly higher than reveromycin B at the same ambient pH condition.For mycelial growth,the EC50 values of reveromycin A at pH 4.5 were 0.88 ?g/mL,0.65 ?g/mL,0.67 ?g/mL,0.74 ?g/mL against B.cinerea,S.sclerotiorum,R.stolonifer and M.hiemails respectively.The EC50 values of reveromycin B at pH 4.5 were 5.37 ?g/mL,5.49 ?g/mL,4.48 ?g/mL,4.07 ?g/mL against B.cinerea,S.sclerotiorum,R.stolonifer and M.hiemails respectively.For spore germination,the EC50 values of reveromycin A at pH 4.5 were 0.57 ?g/m L,0.1 ?g/mL,0.11 ?g/mL against B.cinerea,R.stolonifer and M.hiemails respectively.The EC50 values of reveromycin B at pH 4.5 were 4.01 ?g/mL,1.15 ?g/mL,1.59 ?g/mL against B.cinerea,R.stolonifer and M.hiemails respectively.With the increasing of ambient pH,the antifungal activity of reveromycins was gradually decreased.5.The volatile organic compounds from strain 3-10 were analysed by SPME-GC-MS.Alcohols,alkene,terpenes,aromatic alcohols,esters were found in VOCs from strain 3-10.The compound 2-methylisoborneol was the main component in VOCs from strain 3-10.Four synthetic chemicals were used in bioassay,2-methylisoborneol,methyl 2-methylbutyrate,2-phenylethanol showed antifungal activity against aflatoxins-producing agents Aspergillus flavus and Aspergillus parasiticus,whereas the other compound ?-caryophyllene showed weakly antifungal activity.6.The antifungal spectrum of VOCs from strain 3-10 was determined.Mycelial growth of 14 plant pathogenic fungi was inhibited by the rates higher than 60%.The VOCs effectively suppressed mycelial growth,sporulation and conidial germination of A.flavus and A.parasiticus in vitro.Aspergillus diseases severity on peanuts exposed to VOCs from 17g/L autoclaved wheat grain(AWG)culture of strain 3-10 was suppressed and the level of aflatoxins was significantly reduced.The peanuts fumingated by VOCs from 86 g/L culture of strain 3-10 on autoclaved wheat grains(AWG)remained healthy and no aflatoxins were detected.Scanning electron microscopic observation indicated that Aspergillus growth on peanuts was inhibited and mycelia showed malformed.When treated with the VOCs from 86 g/L AWG culture of strain 3-10,the conidia of A.flavus and A.parasiticus could hardly germinate and develop.These results suggest that the VOCs from strain 3-10 have a potential to be used as a bio-fumigant.7.The genome of strain 3-10 was sequenced by the de novo sequencing technique.The whole genome size were 8.65 Mb,which contains a chromosome(8.59 Mb)and a plasmid(55.49 Kb).The content of G+C% in genome was 73.8%.A total of 7051 coding genes were found in the genome and the gene clusters for the secondary metabolites were analyzed by using the online Antismash tool.A total of 34 secondary metabolites gene clusters were found based on database retrieval results.The gene cluster of reveromyces-producing in strain 3-10 showed 100% similarity to that in Streptomyces reveromyceticus SN-593,suggesting that strain 3-10 is capable of producing all reveromycins.These results laid a solid foundation for exploiting the metabolites of strain 3-10 to control plant fungal diseases.Meanwhile,the genome information will be useful for improving production of reveromycins by strain 3-10 through genetic approaches.