| The Phytophthora blight,Verticillium wilt,and white mold are serious diseases that occur every year,which are caused by the soil-borne phytopathogens Phytophthora capsici,Verticillium dahliae,and Sclerotinia sclerotium,respectively,leading to severe economic loss in production of the horticulture crops worldwide.Control of these diseases is often difficult due to formation and long-term survival of their dormant structures such as oospores,microsclerotia and sclerotia in soil.To control these diseases,huge quantities of synthetic chemical fungicides are used.However,the extensive usage of chemicals causes environmental and food safety problems.By comparison,biological control has been considered as one of the most promising and environmentally friendly methods to protect plant production by constructing an integrated pest management scheme.Among biocontrol agents,Trichoderma species,which are widely distributed in soil,are now often used as biocontrol agents for the control of a large number of plant diseases.Therefore,the objective of this research was to isolate,characterize,and identify Trichoderma isolates,reveal the mechanisms of their interaction with pathogens,and produce silver nanoparticles as new approaches to their application in the control of three important soilborne plant pathogens.The main results are outlined below:1.This study identified 15 Trichoderma strains with antagonistic activity,one of which is a new species.A total of 77 isolates of Trichoderma spp.were obtained from the rhizosphere soil of bell or chilli pepper plants in the heavily infested fields by P.capsici.Among them,15 isolates showed the highest antagonistic activity with antagonism class 1 against P.capsici.Furthmore,15 candidate isolates were identified as eight species,including T.brevicompactum,T.atroviride,T.afroharzianum,T.koningiopsis,T.citrinoviride,T.virens,T.asperellum,and T.harzianum.Among them,a new species isolated was described as T.dorothopsis by a combination of the morphological characteristics and phylogenetic analysis using sequences of the r DNA internal transcribed spacers(ITS),translation elongation factor 1-α(TEF1)and RNA polymerase II subunit B(RPB2)gene.2.This study identified the antagonistic active substances of T.virens and its mechanism to control chili pepper blight.The antagonistic activity of T.virens HZA14 against P.capsici HZ07 was investigated by the dual culture technique.The antagonistic component produced by T.virens HZA14 was analyzed by Reversed-Phase High-Performance Liquid Chromatography.The in vitro antagonistic assay and analysis of metabolite fractions indicate that T.virens HZA14 could cause colony collapse and hypha degradation of P.capsici,and a high activity compound was identified as gliotoxin by spectrometric analysis.The biocontrol tests demonstrated that the T.virens HZA14 significantly delayed the occurrence of chili pepper blight and caused a 62.64%and 64.20% reduction in disease incidence and severity,respectively.Thus,isolate HZA14 could be considered for developing potential biocontrol agent for management of P.capsici in pepper.3.This study identified the microsclerotial degradation mechanism of T.virens against Verticillium wilt disease.The mechanism of T.virens HZA14 degrading microsclerotia of V.dahliae was determined by using the dual culture method and pot experiment,in combination with transcriptome data analysis.Among 15 isolates,isolate HZA14 exhibited the greatest potential for microsclerotial degradation after 15 days.The disease control tests showed that T.virens HZA14 decreased Verticillium wilt disease severity for 2.77% in eggplant seedlings 30 days after inoculation.When hyphae of T.virens HZA14 interacting with microsclerotia of V.dahliae for 6,9,12,and 15 days,transcriptome analysis indicated that compared with the control,genes of T.virens HZA14 were differentially expressed in mycoparasitism process,while the numbers of up-regulated genes were 1197,1758,1936,and1914 and the numbers of down-regulated genes were 1191,1963,2050,and 2114 for 6,9,12,and 15 days,respectively.Among these genes,enzymes associated with the degradation of microsclerotia,such as Endochitinase A1,Endochitinase 3,Endo-1,3-beta-glucanase,Alpha-Nacetylglucosaminidase,Laccase-1,and Peroxidase were predicted based on bioinformatics analysis.The expression of several genes encoding enzymes being related to the degradation of microsclerotia were confirmed by the real-time quantitative PCR(q PCR)analysis as well,being similar to the results of gene expression in the transcriptomes.4.This study biosynthesized nanoparticles with antifungal activity using T.virens HZA14 metabolites and revealed their antifungal mechanism.The 15 isolates of the Trichoderma species was screened for the biosynthesis of silver nanoparticles(Ag NPs).Among them,the highest yield occurred in the synthesis of Ag NPs using an aqueous cellfree filtrate of T.virens HZA14 producing gliotoxin.The synthetic Ag NPs were charactered by SEM,EDS,TEM,XRD,and FTIR.Electron microscopic observation showed that Ag NPs with size ranging from 5 nm to 50 nm had spherical or oval shapes with smooth surfaces.FTIR analysis revealed the interaction of Ag NPs with carbohydrate,protein and heterocyclic compound molecules,and especially,interaction patterns of Ag NPs with the gliotoxin molecule were proposed.The antifungal activity assays demonstrated that percentage inhibition of the biosynthesized Ag NPs was 100% against hyphal growth,93.8% against sclerotial formation and100% against myceliogenic germination of sclerotia at a concentration of 200 μg/ml,respectively.The direct interaction between this biosynthesized Ag NPs and Trichoderma cells revealed by SEM and EDS,includes contact with nanoparticles,accumulation and production of lamellar fragment as well as micropore or fissure formation on cell walls.These findings will greatly enriched our understanding of the action mechanisms of Ag NPs for controlling diversified fungal disease. |
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