Study On Biocontrol Potential And Antifungal Mechanism Of Streptomyces Tjga-19 And Bwl-h1 Against Litchi Downy Blight

Litchi?Litchi chinensis Sonn.?is a famous fruit in the Chinese region south of the Five Ridges,which has a delicious taste and lovely shape,being desired by many people.Litchi downy blight,caused by Peronophythora litchii,is the most important diseases of litchi,which not only causes severe pre-harvest disease,but also post-harvest disease of litchi,causing numerous fruit rot.At present,chemical agents are commonly used to control this disease.But more and more shortcomings are emerging,such as pesticide residues,environmental pollution and so on.These shortcomings of pesticide affect the sustainable development of litchi industry and litchi export.Hence biological control has become the hotspot study recently.Streptomyces is one of the most important microorganism resources for agriculture production.Most of the antibiotics and other bioactive substances produced by them have been used to control plant diseases.In this study,two streptomyces strains with significant antifungal activity against P.litchii were isolated from soil under wild litchi trees of Bawangling in Changjiang County,HaiNan Province,China.They are Streptomyces abikoensis strain TJGA-19 and S.fimicarius strain BWL-H1.Based on this study,the biological classification,fermentation optimization,antifungal activity,biocontrol potential and antifungal mechanism have been conducted.The results are as follows:1.In this study,476 actinomycetes strains were isolated from 28 soil samples through plate dilution method.163 actinomycetes strains were selected for further study.Strain TJGA-19 shows better significant antifungal activity against litchi downy blight among 163strains.The antifungal spectrum of the strain was very wide,it showed significantly antagonistic activities against several plant fungal pathogens both its thallus and fermentation filtrate;double-dish chamber was used to measure antifungal activity of volatile compounds from 163 strains of actinomycetes.Volatile compounds of two strains showed significantly antifungal activity against P.litchii.volatile compounds of strain BWL-H1 showed the strongest antifungal activity against P.litchii,the inhibition rate was94.6%,follow by strain TJGA-19,the inhibition rate was 86.3%.2.Base on classic actinomycete classification principles and 16S rDNA sequence analysis,strain TJGA-19 was identified as Streptomyces abikoensis var.Bawanglingensis.And strain BWL-H1 was identified as S.fimicarius.3.Study on the optimization of fermentation conditions of TJGA-19 showed that the content of soybean powder affects most significantly the TJGA-19 antibiotic productivity,follow by soluble starch.?NH₄?₂SO₄,NaCl and yeast had a certain role in promoting antibiotic productivity of the TJGA-19.The optimum fermentation broth was as follows:2.5%soluble starch,5.5%soybean powder,0.2%?NH4?₂SO4,0.2%NaCl,0.1%yeast extract.The optimum fermentation conditions were 180 r·min^-1,temperature at 28?,medium pH at 7.0 and fermentation time is 7 d.It was suitable for 250 mL triangular flask to contain 60 mL of fermentation medium.4.To determine the physicochemical property of antibiotics,the fermentation filtrate was treated in various ways.We found that antifungal substance was stability to visible light,ultraviolet light,heat and at pH7-12.It was also stable by treated with Proteinase K at55?for 30 min.But at pH2-6,the antibacterial activity decreased significantly.5.The fermentation filtrate of strain TJGA-19 has a strong inhibitory effect on mycelial growth,production of sporangia,sporangia germination and oospores formation.Optical microscopy and scanning electron microscopy of P.litchii indicated that the mycelium treated with the 2%fermentation filtrate of strain TJGA-19 resulted in mycelium seriously deformed,over branched,grow slowly,collapsed because of cytoplasm leakage.The mycelium treated with 5%or 10%fermentation filtrate for 36 h,and the green fluorescence signal was enhanced.The results showed that the antifungal substance destroyed the permeability of cell membrane.Control effect of litchi downy blight was conducted by detached leaves;the results showed that the fermentation filtrate had a great control effect on litchi downy blight.The incidence rate was 24.1%and the lesion diameter was 2.6 mm when the sample was treated with 10%fermentation filtrate,which significantly lower than the incidence rate 97.3%and lesion diameter 18.3 mm of the control.6.The suppression mechanism against P.litchii of volatile compounds produced by strain TJGA-19 in vitro and in planta.volatile compounds showed strong antifungal activity on mycelial growth,production of sporangia,sporangia germination and oospores formation of P.litchii.The bigger the concentration of volatile substances was,the stronger the inhibition effect was.And it also could control litchi downy blight on detached leaves.But volatile compounds showed no impact on sporangia germination.To understand the mechanism of inhibition,the effect of volatile compounds on ultrastructure of P.litchii hypha was examined by scanning electron microscope?SEM?and transmission electron microscope?TEM?.The mycelia and sporangia of P.litchii exposure to volatile compounds showed considerable changes on their morphology.SEM observation showed the mycelia collapsing and shrunkening and a warty surface of sporangia.TEM revealed that organelles were digested and vacuolated.By GC-MS analysis,22 compounds were identified from the volatile compounds produced by S.abikoensis TJGA-19.Most of these compounds belonged to alkenes,alcohols,esters and alkanes.Among these chemicals,the most abundant compound was 2-Methylisoborneol.7.The inhibitory spectrum of volatile substance from S.fimicarius BWL-H1 was very wide,which could inhibit the growth of 15 plant pathogens.Among them,the inhibition rate to P.litchii,Pythium myriotylum,Alternaria alternata,Colletotrichum musarum and Pyricularia grisea was 100%.Antifungal activity against P.litchii of volatile compounds produced by S.fimicarius BWL-H1 was studied in vitro and in planta.volatile compounds showed the strong antifungal activity on mycelial growth,production of sporangia,oospores formation,and suppression of germ tube penetrate cellophane membrane.Disease incidence on fungal inoculated detached leaves,detached branch and fruits were suppressed fumigating with volatile compounds.SEM showed a sinking and shrunken of mycelia and a warty surface of sporangia.TEM revealed that organelles were digested,vacuolated and cell membrane invaginating.The infection process of P.litchii on litchi leaves was observed by SEM,the results showed invasion and expansion was suppressed significantly.By GC-MS analysis,12 and 21 compounds were identified from the volatile compounds produced by cultiving 14 d and 21 d S.fimicarius BWL-H1.Most of these compounds belonged to alkenes,alcohols,esters and alkanes.Among these,eight volatile compounds were selected for further study:Phenylethyl alcohol,Methyl salicylate,Phenylacetic acid ethyl ester,Methyl anthranilate,?-Copaene,Caryophyllene,4-ethyl-Phenol and Humulene.The results showed that,except for Humulene,the other 7 compounds had strong antibacterial activity.