| Poplar anthracnose,as a serious branch and leaf disease of poplar,is mainly caused by Colletotrichum gloeosporioides.The disease generally occurs in various poplar cultivated areas in the north and south of the country.Chemical fungicides are currently the most widespread method used to control poplar anthracnose.However,long-term use of chemical fungicides that are resistant to degradation is environmentally unfavorable and can lead to the evolution of resistance in pathogenic fungi.It is necessary to develop alternative approaches that are potent and environmentally friendly,using of biocontrol agents and their metabolic substances to manage poplar anthracnose is an effective way.The aim of this study was to screen the effective bacteria that had significant control effect against poplar anthracnose and to research the antimicrobial active substance,so as to provide effective biocontrol strain and early theoretical basis for biocontrol of poplar anthracnose.In this study,a total of 35 endophytic bacteria were isolated from 11 healthy poplar leaves that collected from Beijing.One strain named XW2 showed the best antagonistic against C.gloeosporioides by using a dual culture assay.Greenhouse studies revealed that strain XW2 had a 49.1%efficacy in controlling poplar anthracnose.Finally,strain XW2 was identified as Bacillus atrophaeus on the basis of cultural properties,morphological properties,physiological and biochemical properties as well as the sequence analysis of 16S rRNA.B.atrophaeus XW2 could produce several cell wall-degrading enzymes.The sterile culture filtrate and sterile bacterial lysate of XW2 were antagonistic against hyphal growth and spore germination of C.gloeosporioides.The average diameter of the inhibition zones of hyphal growth were 22.3 mm and 12.1 mm,respectively.The average inhibition rates of spore germination after 24 h were 94.6%and 88.8%,respectively.Both culture extracts led to vacuole formation and swelling in hyphae and germ tubes from germinated spores,and abnormalities were observed in 100%of germinated spores.Lipopeptides isolated from culture filtrate and bacterial lysate had high antifungal activities against C.gloeosporioides.The average diameter of the inhibition zones were 23.2 mm and 11.8 mm,respectively.Crude proteins isolated from culture filtrate and bacterial lysate also showed good inhibition qualities.The average diameters for the zones of inhibition were 22.3 mm and 13.4 mm,respectively.Volatiles produced by XW2 also inhibited hyphal growth and spore germination of C.gloeosporioides,the inhibition rate of against hyphal growth by 60.2%,and C.gloeosporioides appeared ramified and curved hyphae after treat with volatiles.The fermentation medium of B.atrophaeus XW2 was optimized by Response Surface Methodology(RSM).The basal medium of modified medium No.3 was screening from 8 fermentation medium by used inhibition rate of sterile cultural filtrate against C.gloeosporioides as an indicator.Plackett-Burman(PB)design revealed that among 8 components within the basal medium,glucose,peptone,and yeast extract had positive effects on inhibition rate.The steepest ascent method determined the local maximums for these important factors.Central composite design(CCD)and RSM analysis then determined the optimum levels of these factors.Finally get the optimized fermentation culture medium was glucose(22.64 g/L),NH4Cl(3.00 g/L),KH2PO4(1.00 g/L),NaZHP04(1.00 g/L),MgSO4·7H2O(0.50 g/L),yeast extract(1.85 g/L),peptone(11.93 g/L),and soybean meal(5.0 g/L).After optimization,a 52.15%increase in inhibition rate of sterile cultural filtrate.The inhibition rates of the extracellular lipopeptides and proteins were effectively increased by 44.84%and 44.83%,respectively.Moreover,sterile cultural filtrate of B.atrophaeus XW2 also displayed strong antagonism towards fungal phytopathogens belonging to various taxonomic groups,with inhibition rates ranging from 69.55%to 100%.Extracellular lipopeptides produced by B.atrophaeus XW2 had a strong inhibition on hyphae growth and spore germination of C.gloeosporioides,and led to vacuole formation and swelling in hyphae and germ tubes from germinated spores.The minimum inhibitory concentration(MIC)and concentration for 50%of maximal effect(EC50)were 0.01 mg/disc and 0.044 mg/mL,respectively.After extracellular lipopeptides treatment,cell membrane permeability and glycerol content of C.gloeosporioides increased markedly,but the content of exopolysaccharide(EPS)significantly decreased.Also,extracellular lipopeptides significantly suppressed the development of poplar anthracnose caused by C.gloeosporioides on poplar leaves and strongly inhibited 11 fungal phytopathogens belonging to various taxonomic groups,with the average diameter of the inhibition zones ranging from 12.3～19.2 mm.Moreover,the extracellular lipopeptides continued to exhibit high antifungal activity even after extreme temperature,pH,and protease treatments,UV radiation,and prolonged storage at room temperature.Extracellular lipopeptides also had powerful biosurfactant properties.Finally,we detected iturin A(C15-C17)and surfactin(C12-C17)by Ultra-high performance liquid chromatography coupled with hybrid quadrupole-orbitrap mass spectrometry(UHPLC-Q-Orbitrap MS)in the extracellular lipopeptides.Extracellular proteins produced by B.atrophaeus XW2 had a strong inhibition on hyphae growth and spore germination of C.gloeosporioides,and led to vacuole formation and swelling in hyphae and germ tubes from germinated spores.The minimum inhibitory concentration(MIC)and concentration for 50%of maximal effect(EC50)were 0.01 mg/disc and 0.036 mg/mL,respectively.Also,extracellular proteins significantly suppressed the development of poplar anthracnose caused by C.gloeosporioides on poplar leaves and strongly inhibited 11 fungal phytopathogens belonging to various taxonomic groups,with the average diameter of the inhibition zones ranging from 11.6～18.6 mm.Moreover,the extracellular proteins were stable with good antifungal activity at as high as 110℃ for 30 min or in pH 6.0～9.0,and were not sensitive to proteases(papain,pepsin,trypsin,protease K),UV radiation,metal ions(Li＋,Na＋,K＋,Ag＋,Ca2+,Mg2+,Zn2+,Cu2+),SDS,EDTA and prolonged storage at room temperature.However,Mn2+ and Fe3+ can inhibited the antifungal activity,Tween 20 and H2O2 can activated the antifungal activity.Volatiles produced by B.atrophaeus XW2 had different degrees of antagonism towards 12 fungal phytopathogens belonging to various taxonomic groups,with inhibition rates ranging from 7.0%to 88.9%.Based on Gas Chromatography-Mass Spectrometer(GC-MS)analysis,14 volatile compounds produced by B.atrophaeus XW2 were indentified,including a range of aldehydes,benzene,alcohols,ketones,alkenes,and alkanes.Accounting for 46.29%of the total volatile compounds,and 1,3-Dichlorobenzene represented one of the most abundant volatile compounds(relative content 1 1.88%).10 volatile compounds were purchased and used for the antifungal activity assay.Among them,3-Ethylbenzaldehy showed the best inhibition effects on 12 fungal phytopathogens,and completely inhibited hyphae growth in vitro at a concentration of 10μL/plate. |
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