Transcriptome Research Of Trichoderma Harzianum Antagonized Phytophthora Capsici

The abuse of chemical fertilizers and pesticides in agricultural production has becoming increasingly caused a series of problems,such as the hardening and compaction of soil,ecological environment pollution,decresing of agricultural products quality and the wares ranks high for pesticide residues.Above problems can be well solved by choosing efficient and safe biocontrol agents to replace chemical control.Trichoderma spp has been widely used as a biological control agent because of its a strong ability to secrete biological control active substances,which can antagonize a variety of plant pathogenic fungi.However,the molecular mechanism that Trichoderma spp plays an biocontrol role has not been clarified,which will greatly limit the industrialization development of Trichoderma spp.Studying the antagonistic mechanism of Trichoderma spp can help to the biological control of plant pathogens effectively and reduce agricultural economic losses.With the public of Trichoderma genome sequence,it has become an effective methods to explore Trichoderma biocontrol function genes based on transcriptome technology.In this study,we isolated and identified the Trichoderma strain form Guizhou Province,the antagonistic activity of the isolated strains and related physiological and biochemical indexes was calculated by the dual culture.The transcriptome sequencing technology was used to obtain the different stages from class 1 to class 3 of sequencing data.Class 1: Trichoderma harzianum grew on at least two-thirds of the culture medium,Class 2: each Trichoderma harzianum and Phytophthora capsici colonized nearly half of the culture medium and no organism dominated the other,Class 3: at least two-thirds of the culture medium was colonized by the Trichoderma harzianum.The genes of differential expression and alternative splicing were identified by using bioinformatics related software on three stages.To explore the regulation pathways ofmetabolism for Trichoderma harzianum of antagonistic ability for laying a foundation for further revealing the biocontrol of molecular mechanism of trichoderma spp.The results of this study were as follows:1.11 strains of trichoderma were isolated from guizhou,which were identified by morphological and molecular biological identification as four species inchuding Trichoderma virens,Trichoderma harzianum,Trichoderma hamatum and Trichoderma.asperellum species.2.All of 11 isolated strains could inhibit the growth of Phytophthora capsica,the inhibition abilities of strains were different from one another.Four strains(T.virens,T.harzianum,T.hamatum and T.virens)showed higher inhibition rates.The mycelium of Phytophthora capsici were treated with the zymotic fluid of 4 stains after detecting the changes of malondialdehyde(MDA)concentration,cellulase,polygalacturonase(PG)and β-glucosidase(β-GC)enzyme activities.The results is that the MDA and cellulase were higher than that of the control group,while the activity of PG andβ-GC was lower.It is suggested that Trichoderma may destroy the mycelial structure of Phytophthora capsici by secreting primary or secondary metabolites.3.To selecte a strain of Trichoderma harzianum with the highest comprehensive antagonistic abilities.Observing the mycelium changes of Trichoderma harzianum and Phytophthora capsici were cultured after 3,5,7 days by scanning electron microscopy.It is found that the hyphae of Trichoderma harzianum is tightly wrapped around the Phytophthora capsici,then penetrate deep into the hyphae inside caused severe distortion of the Phytophthora capsici,finally the hyphae gradually were made it down to small fragments.It is suggested that the biocontrol ability of Trichoderma harzianum is related to the recognition of the pathogen host and the degradation of the cell wall,the degradation of cell membrane.4.HiSeq X-ten was used to perform transcriptome sequencing of 15 hyphae samples of 3 stage in dual culture and the control group(Trichoderma harzianum),the sequencing data of 120 Gb was obtained.A total of 3,791 differentially expressedgenes were Identificated in the 3 periods of interaction compareing with the control group.including 2,324 up-regulated genes and 1,467 down-regulated genes.In initial stage of the interaction,there were 499 genes differentially expressed,of which 312 genes were up-regulated and 187 genes were up-regulated.1,061 genes were differentially expressed in the middle stage of the interaction,including 794 genes were up-regulated,and 267 genes were down-regulated;In the later stage of interaction,2,249 differentially expressed genes,of which 1,236 up-regulated genes and 1,013 down-regulated genes 236 up-regulations,and 1013 down-regulations were observed.There were 142 differentially expressed genes before,during and after interaction,and 58,97 and 1764 differentially expressed genes were expressed specifically.There were 142 differential genes co-expressed in 3 periods,163 differentially expressed genes were only expressed in the initial stage of interaction,only 522 expressed in the middle stage of interaction and 1851 expressed in the later stage of interaction respectively.5.Total of 14172 alternative splicing events of 3 stage were identified in dual culture 5138,5163,5581 differentially selective splicing(DAS)events including Intron retention(IR),skipping exon(SKIP),Transcription start site(TSS),Transcription terminal site(TTS),Mutually exclusive exons(MEX),where intron retention(IR)is most common(56-60%).Comparing the transcriptome data of the 3 periods of interaction finding that the number of up-regulated genes was higher than down-regulated genes,and it was speculated that the up-regulated genes played an important role in the interaction process.In the later stage of interaction,the number of differential genes,up-regulated genes,pecifically expressed genes showed a significant upward trend,it suggesting that the later stage of interaction is the most complicated and important stages of interaction process.6.Functional annotation and pathway enrichment of the differentially expressed genes were performed using Blast2 GO,GO,KEGG and other databases,It was found that the genes encoding cell wall degrading enzymes,cell membrane degrading enzymes,antibacterial peptides and other metabolites were significantly differentexpression during the process of interaction.These genes in the MAPK signaling pathway(promoting protein kinase signaling pathway)and the apoptosis-inducing pathway were up-regulated.A total of 24 biocontrol-related candidate genes were verified for qRT-PCR.The results showed that the expression trends of 24 genes were consistent with the transcriptome data analysis.