Role Of Trichokonins In Trichoderma Longibrachiatum SMF2-Xanthomonas Oryzae Pv. Oryzae Interaction

Trichoderma spp.are filamentous fungi widely distributed in soil,water or the surface of plant root and leaf,which can effectively inhibit the growth of soil-borne plant pathogens and indirectly exert their beneficial roles in plants.In addition,Trichoderma can induce plant systemic resistance and promote plant growth to exert their direct benificial effects on plants.Therefore,Trichoderma are important biological agents in the production of biological pesticides and biological fertilizers in recent years.Secondary metabolites from Trichoderma play important roles in the process of Trichoderma-phytopathogens interaction.Among these secondary metabolites,peptaibols play important biological control functions and can effectively antagonize the growth of Gram-positive bacteria,plant pathogenic fungi,plant virusesand nematodes.Peptaibols are a class of secondary metabolites rich in αaminoisobutyric acid synthesized by non-ribosomal peptide synthetases However,up to now,there is no report that peptaibols can effectively antagonize Gram-negative bacteria.In previous study,we screened a high peptaibols-producing Trichoderma strain-Trichoderma longibrachiatum SMF2(hereafter TlSMF2),which can produce three long chain peptaibols with 20 amino acid residues-Trichokonins A(TrichokoninⅥ,Trichokonin Ⅶ and Trichokonin Ⅷ)and six medium chain peptaibols with 11 amino acid residues-Trichokonins B(Trichokonin B1-B6).Trichokonins A and Trichokonins B are synthesized by non-ribosomal peptide synthetase genes tlx1 and tlx2,respectively.Trichokonins A not only had antiviral and inducing plant resistance activities,but also could effectively inhibit the growth of Gram-positive bacteria and phytopathogenic fungi.It was not found obvious antagonistic effect on Gram-negative bacteria in our previous study.In the preliminary study,it was found that TlSMF2 to control rice diseases,it was found that TlSMF2 had antimicrobial activity on the growth of Xanthomonas oryzae pv.oryzae(Xoo),the casual angent of bacterial leaf blight on rice.Bacterial leaf blight is one of the most destructive bacterial diseases on rice,resulting in up to 50%losses in rice production and posing a great threat to food security.Currently,chemical control is the main strategy for rice bacterial blight controlling,which bring serious environmental pollution and food safety issues.Therefore,it is urgent to find new.green biological control strategies for Xoo controlling.In this study,co-culture method was used to evaluate the antimicrobial effect of TlSMF2 on Xoo.An antagonistic circle could be clearly observed around the margin of TlSMF2.Then,the secondary metabolites(SMs)produced by TlSMF2 were extracted and the anti-Xoo activity of the SMs extract was analyzed.Compared with the control,inhibition zones could be clearly seen on the test plate.To test whether Trichokonins.the main secondary metabolites from T/SMF2.involved in the process of antibiosis,the antimicrobial activities of the wild-type strain of TlSMF2 and its gene-deletion strains △tlx1,Atlx2 and △tlx1&tlx2 on Xoo were analyzed.It was found that the antimicrobial activities of Atlxl and △tlx1&tlx2 on Xoo were decreased significantly.while no significant difference could be found compared with the wildtype strain of TlSMF2.indicating that Trichokonins A played a major role in the interaction of TISMF2-Xoo interaction.Then,Trichokonins A and the three Trichokonins A components were purified by HPLC and were used to evaluate their antimicrobial activities.It was found that Trichokonins A and three Trichokonin Ⅵ.Ⅶ and Ⅷ all showed remarkable antimicrobial activity on Xoo.The MIC of Trichokonins A against Xoo was 54 μg/ml.Compared with Trichokonins A,Trichokonin VI showed the strongest antimicrobial activity on Xoo,with the MICs at 38 μg/ml.After TEM and AFM observation it was found that Xoo cells were ruptured.and the stain penetrated into the cell.Extracellular supernatant test showed that DNA and protein were released into the extracellular.Trichokonins A effectively reduced the pathogenicity of Xoo on rice IR 24.For better understanding TlSMF2-Xoo interaction,the biological control-related genes in TlSMF2 genome were analyzed and mined.The results showed that there are 1.3 genes containing secondary metabolites of PKS type.9 genes of NRPS type and 4 terpenes in TlSMF2 genome.Possible secondary metabolites produced by TlSMF2 include Squalestatin S1,trichodiene,viriditoxin,YWA1,alternariol.choline,and Ilicicolin H.Evolutionary analysis of NRPs-related genes revealed that the 5th module in TLX2 may be skipped.Moreover,TlSMF2 exocrine proteins contained 11 xylan transferases of GH16 family.16 chitinases of GH18 family,16 proteases of A01A family.18 proteases of S08A family.These predicted secondary metabolites and extracellular enzymes may play important roles in TlSMF2-Xoo interaction.In conclusion.Trichokonins A play key roles in TlSMF2-Xoo interaction.which inhibited the growth of Xoo and decreased the pathogenicity of Xoo on rice.There are tens of genes encoding secondary metabolites and extracellular proteins,which may involve in TlSMF2-Xoo interaction.These results lay a foundation for the further study of the interaction mechanism between Trichoderma and Xoo,and provide a theoretical basis for the biological control of rice bacterial blight.