Screening And Mechanism Chracterization Of Trichoderma Isolates For Biological Control Of Botrytis Cinerea

Gray mold is an important plant disease caused by Botrytis and Botrytis-like fungi.B.cinerea is the most prevalent necrotrophic agent for the gray mold disease on many crops,including strawberry and tomato.It has a wide host range(more than 1400 plant species)and causes substantial economic losses at both pre-and post-harvest stages.Resistant host species against this pathogen are not available in most cultivated fruits and vegetables.As a consequence,control of gray mold largely depends on repeated use of fungicides.Although expected control efficacy can be achieved with optimal use of fungicides,their usage is not environmental friendly and repeated use of fungicides can result in generation of resistant pathogen strains.Thus,it is useful to explore other safe and sustainable control measures,including microbe-mediated biological control of pathogen organisms.Trichoderma species are groups of fungi commonly inhabiting the rhizosphere.This study was initiated to isolate and characterize Trichoderma-mediated biocontrol of pathogenic microbes and included following objectives:(1)to screen indigenous isolates of Trichoderma that can be used to control B.cinerea in central China’s Hubei Province,(2)to evaluate the biocontrol potential of the selected Trichoderma isolates,(3)to elucidate mechanism underlying Trichoderma-mediated biocontrol and(4)to investigate the identity and biology of a novel Trichoderma mycovirus in a debilitated isolate(T-70D)of Trichoderma.A total of 72 Trichoderma isolates were isolated from soil collected from Enshi,Ezhou,and Wuhan of Hubei Province.They were identified as 11 species based on ITSr DNA sequence.Three species,including T.koningiopsis(17 isolates),T.hamatum(17isolates)and T.brevicompactum(17 isolates)represented three major species.A multiple-screening strategy was established to identify Trichoderma isolates that showed biocontrol activity against B.cinerea.These parameters included adaptation to temperatures at 20℃ and 15℃,mycoparasitism on B.cinerea,antifungal activity against B.cinerea,and promotion of tomato seed germination.Four isolates,including T.koningiopsis T-35 and T-51,and T.harzianum T-21 and T-68 were selected as potential biocontrol agents.These isolates were evaluated for biocontrol of B.cinerea and for promotion of tomato growth.The results showed that all four Trichoderma isolates had a wide range of biocontrol activity and inhibited growth of many soil borne plant pathogenic fungi including Rhizoctonia solani,Sclerotinia spp.and Fusarium oxysporum.Tomato seeds soaked with conidial suspension of Trichoderma showed significantly enhanced germination and subsequent growth.The Trichoderma conidial suspensions applied to tomato roots conferred foliar resistance against B.cinerea and lowered the disease index values by 49.68% to 62.77%.Trichoderma isolates T-21,T-51 and T-68 inhibited B.cinerea sporulation when applied to tomato leaf discs.In a greenhouse trial,tomato seedlings drench-treated with the conidial suspension of Trichoderma T-51 in transplanting,together with two sprays with the antifungal substances of Streptomyces 3-10(10 mg crude extract/L)on aerial parts of tomato plants at the flowering stage showed a significant reduction in severity of gray mold on adult tomato plants.In a plastic tunnel trial,two sprays with the conidial suspension of Trichoderma T-51 on strawberry plants at the fruit-setting stage significantly reduced severity of Botrytis fruit rot and the efficacy(65%)was equivalent to that of two sprays with the fungicide pyrimethanil.The four Trichoderma showed versatile mechanisms in suppression of B.cinerea and in promotion of plant growth.For instance,they produced mycoparasism-associated enzymes,including chitinase,glucanase and protease.The mycelial extract of B.cinerea significantly enhanced production of chitinase by Trichoderma T-51.A chitinase gene Tkchit42 was cloned from T-51,sequenced and analyzed.Second,the Trichoderma isolates T-21,T-35 and T-68 showed antifungal activity against B.cinerea.Meanwhile,isolate T-51 was detected to be able to produce antifungal volatiles,which showed growth-promotion activity against Arabidopsis thanliana.Finally,the Trichoderma isolates can produce phytohormones,such as auxin and gibberellin,and isolate T-51 had the ability to dissolve phosphorus and to produce siderophores.The signaling pathway for induced systemic resistance(ISR)by T.koningiopsis induced by T-51 was investigated further and found to be dependent on jasmonate(JA),ethylene,salicylic acid(SA),pipecolic acid(Pip)and glycerol-3-phosphate(G3P).An overlap between ISR and systemic acquired resistance(SAR)was further substantiated by assaying SAR with vascular exudates obtained from ISR induced plants.Together,this study showed that T.koningiopsis T-51 is a promising biocontrol agent that can confer foliar resistance against microbial pathogens.Isolate T-70 D of T.harzianum was derived from isolate T-70.Compared to T-70,T-70 D showed slow mycelial growth and abnormal morphology.Two species of ds RNAs(ds RNA-L and ds RNA-S)were detected in T-70 D,whereas only one species of ds RNA(ds RNA-L)was detected in T-70.Sequence analysis showed that ds RNA-L is a novel hypovirus,designated as Trichoderma harzianum hypovirus 1(Th HV1),whereas ds RNA-S is a defective form of ds RNA-L,designated here as Th HV1-S.Both Th HV1 and Th HV1-S can be vertically transmitted to the conidia of T-70 D during its asexual reproduction.Both mycoviruses were successfully transmitted from T-70 D to T.koningiopsis T-51 and T.harzianum T-68 through hyphal contact in the pair cultures of T-70D/T-51 and T-70D/T-68 on PDA.The resulting four derivatives showed reduced mycelial growth and abnormal colony morphology and were significantly suppressed in colonization of the colonies of B.cinerea,compared to their parental isolates T-51 and T-68.Intriguingly,they were significantly enhanced for antifungal activity against B.cinerea.The results about the mechanisms involved in ISR induced by T-51 broadened our understanding about interaction between Trichoderma and plant.Elucidation of the Th HV1/Th HV1-S-associated mechanism involved in decline of colonization of the B.cinerea colonies in T-70 D is useful in further screening of Trichoderma isolates for control of B.cinerea.