Screening Of Trichoderma And Its Induction Of Tomato Resistance To Botrytis Cinerea And Identification Of MilRNA

Tmato gray mold is a serious disease in tomato caused by Botrytis cinerea.At present,chemical agents are the main methods to control gray mold,which leads to the increase of drug resistance of germs,and the use of pesticides also brings potential risks to the environment,human and animal health.The development and utilization of microbial agents and biopesticide is an effective method for green control and is also an important means to improve the quality of agricultural products.Therefore,on the basis of screening the dominant biocontrol Trichoderma,we studied the molecular mechanism of Trichoderma induced tomato resistance and identification of Trichoderma milRNA in the process of interaction.In order to study the molecular mechanism of Trichoderma milRNA controlling tomato resistance to Botrytis cinerea.The main results are as follows:1.23 strains of Trichoderma spp.isolated from laboratory were used as materials,after seed soaking and root irrigation respectively to evaluate the effects of Trichoderma on seed germination and root elongation of tomatos.It was found that DQ-1,HN2325-2,HN2108-4,HL119 significantly promoted seed germination and root elongation.The effect of DQ-1 was the most obvious,the germination rate of seeds was 6.64% higher than that of control,the length of root was increased 37.86% after root irrigation.The inhibitory effect of these Trichoderma strains against Botrytis cinerea was compared by confrontation culture.It was found that the antibacterial rate of DQ-1 against Botrytis cinerea was as high as 88.41%.Trichoderma asperellum DQ-1 can be used as a potential biocontrol strain for the control of tomato gray mold.2.Trichoderma asperellum DQ-1 could reduce the occurrence of Botrytis cinerea in tomato leaves after root irrigation,indicating that Trichoderma asperellum DQ-1 might induce resistance of tomato.The expression of resistance gene(PR2,TPX)and ethylene jasmonate signaling pathway gene(ETR1,CTR1,LOX1,PAL)in tomato roots and leaves were detected by Real-time PCR(qRT-PCR)after DQ-1 of Trichoderma asperellum.The results showed that the expression levels of PR2 and LOX1,TPX and ETR1,PAL in tomato roots at 6 h,12 h and 24 h,CTR1 was down-regulated to the lowest level at 6 h.In tomato leaves,the expression of ETR1,PR2 and PAL,LOX1 reached the highest value at 12 h,24 h,48 h,and the expression of CTR1 decreased to the lowest at 24 h.The expression level of TPX in tomato leaves did not change significantly.The results showed that DQ-1 root irrigation could induce the expression of resistance genes and ethylene jasmonate signaling pathway genes in tomato roots and leaves.3.Trichoderma asperellum DQ-1 was challenged to inoculate Botrytis cinerea after 24 hours.The results showed that the expression of PR2,TPX,ETR1 reached peak at 24 h after inoculation,LOX1,PAL reached peak at 12 h after inoculation,and CTR1 was down-regulated to the lowest level at 24 h.The expression of PR2,ETR1,LOX1,PAL in tomato leaves reached its peak at 24 h,TPX reached its peak at 12 h,and CTR1 down-regulated to the lowest level at 12 h.The results showed that Trichoderma asperellum DQ-1 could increase the expression of disease resistance gene and ethylene jasmonate signaling pathway gene in tomato roots and leaves under the challenge of inoculation with Botrytis cinerea.The resistance of tomato to gray mold was improved.4.In order to detect the presence of milRNA in Trichoderma asperellum DQ-1 to induce tomato disease resistance.The miRNA sequencing library of DQ-1 in the interaction between DQ-1 and tomato was constructed by high throughput sequencing technique.The related milRNA expression levels were screened and identified.Total of 126 known milRNA was found,predictions were identified and 21 new milRNA were obtained.The expression of Tri-milR947-y,Tri-milR008-5p reached the peak at 24 h after the interaction between Trichoderma and tomato roots,and the expression of Tri-milR5215-y reached its lowest point at 12 h,and the expression of Tri-milR7122-x,Tri-milR001-3p reached its peak at 12 h.There was no significant change in the expression of Tri-milR004-3p in the interaction,suggesting that milRNA Tri-milR947-y,TrimilR008-5p,Tri-milR5215-y,Tri-milR7122-x,Tri-milR001-3p may be involved in the regulation of Trichoderma induced disease resistance and promoting growth of tomato.