Antifungal Activity And Preliminary Mechanism Of Active Volatiles From Endophytic Fungal Schizophyllum Commune And Diaporthe Spp.

Endophytic fungi of plants can produce various volatile organic compounds(VOCs),some of which have the ability to inhibit the growth of pathogens.At present,there are few studies on the mechanism of fungal VOCs inhibiting the growth of plant pathogens.There are still many potential VOCs to be discovered.In this study 174 endophytic fungi were isolated from Populus genus.Six endophytes with special odor among them were obtained and identified as Schizophyllum commune.Other3 endophytes endophytes with special odor(isolated from Populus in Heibei)were identified as S.commune,too.In addition,five endophytes with special odor(isolated from Leucaena leucocephala and Catharanthus roseus in Hainan)were identified as Diaporthe spp..The 9Schizophyllum commune strains and 5 Diaporthe spp.strains above were found to have antifungal ability with their VOCs.Furthermore,S.commune strain ROHY8225b2 and Diaporthe spp.(strains FPYF3052-3056)were screened for further study due to their stronger inhibitory effects on pathogens.SPEM-GC-MS technology was used to analyze and identify the components of the fungal VOCs.Fluorescent dyes were used to explore the mechanism of VOCs against pathogenic fungi and determined the effect of VOCs on the cell structure of pathogenic fungal.At the same time,the conditions affecting the production of VOCs by S.commune ROHY8225b2 were studied.The main contents and results of the research were as follows:1.S.commune ROHY8225b2 and S.commune 1-1-36 showed stronger bioactivities against pathongens among 9 strains of Schizophyllum strains.Within 24 hours,the pathogens Colletotrichum gloeosporioides,Botryosphaeria dothidea and Alternaria alternata were the most sensitive to the VOCs of S.commune ROHY8225b2 and were more than 64 % growth inhibited.The VOCs of S.commune ROHY8225b2 maximally reduced the radial growth of Botryosphaeria dothidea by 77.5% at 24 hours.The pathogens Cercospora asparagi,B.dothidea and A.alternata were the most sensitive to the VOCs of S.commune 1-1-36 were more than 74 % growth inhibited within 24 hours.The VOCs of S.commune 1-1-36 exhibited the greatest inhibition rate on Cercospora asparagi by 97.9% after 24 hours.The VOCs of D.apiculatum FPYF3052 maximally reduced the radial growth of 66.7% within 24 hours.The inhibition intensity of VOCs produced of S.commune and Diaporthe sp.decreased along with increasing tested time from 24 to 48 hours to most pathogens.2.Fifteen VOCs produced by D.apiculatum FPYF3052 were identified by the SPME-GC/MS technique.Terpenoid chemicals in the VOCs were were dominant and most diverse including ?-terpinene,?-terpinene and(-)-4-terpineol with relative areas from 8.4% to39.8%.accounting for 90.0% of the relative area.There were 31 VOCs indentified emitted by S.commune ROHY8225b2.The principal components were terpenoid,alcohols and esters.3-methyl-1-butanol,?-bisabolol,isobutyl acetate,isoamyl acetate were the most abundant components with relative area from 8.8%?32.2%.Among these single artificial components,isoamyl acetate,isobutyl acetate and(-)-4-terpineol showed the best volatile antifungal ability.While ?-bisabolol had almost no volatile inhibitory against pathogens.3.Temperature,pH,nitrogen source and carbon source are factors affecting the growth of S.commune ROHY8225b2.Temperature and nitrogen sources made a difference on the growth rate of mycelium.S.commune ROHY8225b2 grew fastest in the range of 25 °C ? 35 °C.When organic nitrogen was the nitrogen source S.commune ROHY8225b2 grew fastest.4.S.commune ROHY8225b2 had effects on the cell wall structure of the pathogens.The hyphal tips of the C.gloeosporioides in the experimental group showed obvious swelling.A.alternata in the experimental group showed lysis of hyphal tips.The pathogens with arrested growth showed a higher intensity of Congo red and intense fluorescence occurred at the apex of hypha.After staining with SYTOX Green staining solution,A.alternata of the experimental group showed strong red fluorescence comparing with the control group.Its cell membrane appeared damaged.The VOCs of S.commune ROHY8225b2 showed inhibitory effects on the spore germination of A.alternata and C.gloeosporioides.The germination rate were 0%within 12 hours.