Analysis Of Signal Transduction Pathway Of Clonostachys Rosea Primed Resistance To Gray Mould Disease In Tomato

Tomato gray mold disease caused by Botrytis cinerea（B.cinerea）is a serious disease that threatens tomato production in daily production.Chemical control can easily induce the resistance of B.cinerea,pollutes the environment and endangers the health of human and animals.With the unique advantages of safety and effectiveness,biological control methods have increasingly become an important way to prevent and control the gray mold disease.Clonostachys rosea（C.rosea）as a member of Clonostachys,can effectively inhibit gray mold,leaf mildew and other diseases.However,in the process of C.rosea-primed resistance mechanisms,how these different hormone pathways cooperate to regulate signal transduction pathways has not been reported in detail,and there is none in-depth study on the mechanism.In order to understand the effects of C.rosea on key defense enzymes,second messengers,key resistance genes,stomatal regulation of guard cells under different hormone signal pathways,we have done this research.And the interaction between these resistance-related signals and genes,and their regulatory mechanisms were analyzed.In this study,four hormone-deficient mutants（Spr2,nor,Nah G and Gib3）of jasmonic acid,ethylene,salicylic acid and gibberellin,and their corresponding wild-type tomato varieties（CM,AC and MM）were selected as experimental materials.In this study,the mechanism of C.rosea inducing resistance to tomato gray mold was studied at physiological,pathological and molecular levels.It provides direct evidence for the interaction of all regulatory signals and key genes in the process of C.rosea-primed resistance to gray mold disease in tomato plant,and lays a theoretical foundation for exploring the mechanism of C.rosea inducing resistance to plant disease.The main results are as follows:（1）In vitro experiments: C.rosea has a parasitic effect on B.cinerea,which have inhibition,phagocytosis and decomposite of B.cinerea colonies.C.rosea aseptic fermentation broth has strong antimicrobial susceptibility.It can inhibit B.cinerea spore germination,induce mycelial deformity,inhibit mycelial growth,inhibit B.cinerea respiration,and change living conditions of B.cinerea,resulting in B.cinerea unable to reproduce and pass on,eventually inhibited growth and gradually died.（2）Single B.cinerea treatment: JA,ET,SA and GA3 all participated in the endogenous immune response of tomato plants to gray mold disease.When any of the above hormones were absent,tomatoes were more susceptible to B.cinerea.B.cinerea stimulates the passive immunity of plant endogenous immunity and PTI basic immune model of active immunity.In this process,B.cinerea may produce effective factors to inhibit PTI,thus inactivating PTI resistance of tomato,and then the tomato plants shows susceptibility.（3）B.cinerea + C.rosea induction treatment: C.rosea significantly reduced the course of gray mold.C.rosea（and/ or its metabolites）itself or the interaction products of C.rosea with B.cinerea were used as inducers to induce non-host resistance of tomato to gray mold.The inductors activated HR and SAR,induced the activation of potential defense genes atp A and Lexyl2,and enhanced the expression of activated genes WRKY and MAPK.C.rosea can effectively alleviate the ultrastructural damage of cells and chloroplasts caused by B.cinerea infection,promote the increase of PAL,PPO,O₂^-,H₂O₂ and NO,and reduce CAT activity.Therefore more lignin,phytoprotegerin,fungicidal quinones and other substances are produced.ROS outbreak was accelerated and resistance to pathogenic bacteria was accelerated.Inductive defense response is produced through signal transduction regulation,which can effectively resist the infection of pathogens.（4）JA regulation pathway: Among the four hormone regulatory pathways in C.rosea-primed defense,JA has the most significant regulatory effect and GA has the least significant effect.JA positively regulates PAL,PPO and CAT activities,induces the accumulation of H₂O₂ and NO,inhibits the increase of O₂^-and promotes the up-regulation of MAPK,WRKY and Lexyl2 genes,but inhibits the expression of atp A genes.JA can increase the levels of H₂O₂ and NO in guard cells by up-regulating MAPK,inhibiting the scavenging system of signal molecules H₂O₂ and NO,or JA can directly promote the elevation of H₂O₂ and NO to induce stomatal closure.It was found that the regulation rhythm of stomatal opening and closing was closely related to the levels of H₂O₂,NO and MAPK.（5）ET and SA pathways: In the process of C.rosea-primed resistance,ET can effectively up-regulate PAL activity,promote the accumulation of O₂^-and down-regulate CAT activity,and inhibit the accumulation of H₂O₂ and NO.ET may regulate PPO activity both positively and negatively or not directly.SA increased the activity of PAL and CAT and inhibited the accumulation of H₂O₂.However,the regulation of NO,O₂^-and PPO levels by SA is more complex,and may be related to both positive and negative regulation or cross regulation.（6）GA pathway: During the induction of disease resistance by C.rosea,exogenous GA3 was needed for the cultivation of gibberellin-deficient mutant Gib3 tomato plants.Gib3 tomato plants were dwarf-shaped if exogenous Gibberellin was not added,and plants were prone to post-growth death if exogenous Gibberellin was added too much.Compared with other hormone-deficient mutant plants,Gib3 tomato plants had no significant regular changes in many physiological indicators in this study due to the effect of exogenous Gibberellin addition.In conclusion,this study revealed that C.rosea induced resistance to B.cinerea was regulated by many factors,including the expression of protective enzymes（PAL,PPO and CAT）,ROS,NO and key genes（MAPK,WRKY,Lexyl2 and atp A）.All these factors are affected by the regulation of hormones,especially the regulation pathway of jasmonic acid.JA,ET and SA are mutuallysynergistic and antagonistic.Under the interaction of different hormone pathways,they cooperated and participated in the resistance patterns of HR and SAR induced by C.rosea.Based on the PTI basic immune model of active immunity and passive immunity,inducible factors produced by C.rosea can make tomato plants produce more specific resistance genes and proteins.Thus,the tomato plants can recognize the specific effector factors,and the ETI immune pattern of tomato plants can be triggered.This study provides a new research direction for the regulation pathway and molecular mechanism of C.rosea-primed resistance to gray mold in tomato.