Determination Of Volatile Organic Compounds By Trichoderma Atroviride And Their Biological Function

Trichoderma species with biocontrol and biostimulatory functions had been developed widely as microbial fungicide.It is valuable to search novel Trichoderma strains for further application.In our previous investigation,we found that there was high-level diversity of Trichoderma in the Genoderma lucidum-cultivated soil.Moreover,it is of interest that some strains had coconut-like odors.In this study,the volatile organic compounds（VOCs）from these strains were focused and determined.The biological functions of VOCs on plant growth and pathogen inhibition were analyzed.The main results were listed as following.1.Three Trichoderma strains with strong coconut-like odors,including GL01,GL02 and GL03,were seleted and identified using morphological and molecular identification methods.The colony morphology of the seleted strains is described as:mainly radial and the mycelia are white as well as cotton-like.The conidia are oval and smooth.The branches on the main axis of the conidiophores are distributed evenly,alone,paired or 3-4 whorled.A phylogenetic tree comprising the seletecd strains and other Trichoderma species was constructed based on the concatenated sequences of the ITS and RPB genes.Phylogenetic analysis results revealed the seleted strains to be more closely related to T.atroviride in the neighbor joining tree,thus named as T.atroviride GL01,T.atroviride GL02,and T.atroviride GL03.2.The VOCs produced by these three strains were assayed for antifungal activity using the plate buckle method.Four plant pathogenic fungus,including Verticillium dahlia,Botryospbaeria dothidea,Phytophthora cinnamomi,as well as Fusarium oxysporum,were selected and tested for the antifungal activity of fungal VOCs.Results showed that mycelial growth from the tested pathogens had been reduced significantly after exposure to the emitted VOCs.For instance,the inhibitory rates of GL01,GL02,and GL03 to V.dahliae were 75.16%,68.15%,and 80.25%,respectively.The fungal hyphae exhibited thinned,increased separation,less content in the hyphae or larger lipid droplets in the treaments.The results indicated that VOCs emitted by the tested T.atroviride strains were capable to inhibit the hyphal growth of other fungi.3.The VOCs produced by these three strains were assayed for plant growth promotion.It was found that VOCs produced by T.atroviride strains promoted the growth of Arabidopsis thaliana.After exposure to VOCs of GL01,GL02,and GL03,length of main root was increased by 42.04%,39.55%,and 41.59%;number of fibrous roots was increased by 42.11%,31.58%,and 36.84%,respectively.The similar effects of VOCs were found on tomato seedlings.In the Y-tube olfactometer trail,VOCs produced by T.atroviride strains positively directed the growth of the primary roots of tomato seedlings and increased the biomass of the roots.The results indicated that T.atroviride VOCs exhibited plant growth-promoting activity.4.The SPME-GC-MS was used to analysis the volatile compounds emited by T.atroviride.The results revealed the volatile compounds included alkanes,alkenes,esters and ketones.The most abundant compound is 6-pentyl-2H-pyran-2-one（6PP）,which produces the same coconut-like scents as strains.In addition,the proportion of 6PP in the three strains was different,GL01 accounted for 35.64%of the total VOCs,GL02 for 46.66%,GL03 for 37.41%.5.The antifungal activity of 6PP against plant pathogenic fungi was assayed using the fumigation method.It was found that 6PP inhibited the fungal growth of the test pathogens.The 50%effective concentrations(EC₅₀)of 6PP against V.dahlia,P.cinnamomi,F.oxysporum,B.dothidea were 25.033μL/L headspace,35.645μL/L headspace,49.025μL/L headspace,32.214μL/L headspace,respectively.The hyphal morphology of the pathogens treated with 6PP was consistent with the results of the plate buckle experiment.The fungal hyphae exhibited thinned,increased separation,less content in the hyphae or larger lipid droplets after exposure to 6PP.Meanwhile,the effect of 6PP on Bursaphelenchus xylophilus was tested.It was found that 6PP inhibited the move activity of B.xylophilus.In addition,6PP affected the growth of A.thaliana.Lower concentration of 6PP increased the root growth,while higher concentration of 6PP inhibited its growth.6.Greenhouse experiment results show that T.atroviride GL01,GL02 and GL03 could promote the growth of tomato seedlings.Moreover,the inoculation of these strains inhibited F.oxysporum and reduced effectively the occurrences of Fusarium wilt disease.The relative control efficiency of GL01,GL02 and GL03 was 80.00%,77.94%and 78.46%,respectively.In summary,T.atroviride strains which emits VOCs with multiple biological functions,is a promising agent for the biostimulation of vegetable plants and integrated disease management.The results would contribute to Trichoderma resources,and provide the insight to mechanism research of biocontrol and bio-stimulation of Trichoderma.