Mechanism Of Action Of Phytotoxin Tea Produced By Alternarialternata(Fr.) Keissler During Pathogen Infecting The Host Plant

Alternaria alternata(Fr.)Keissler is one of the three worst plant pathogenic fungi in nature.Fungus Alternaria from croftoweed can produce a toxin-Tenuazonic acid(TeA).The toxin can quickly kill croftoweed and most monocotyledon and dicotyledon weeds at a low concentration and have the potential to be developed into a biological herbicide.TeA blocks the electron transport from QAto QB by binding protein D1 of photosystem II,causes excessive energy,results in the outbreak of reactive oxygen species in chloroplasts,therefore,causes structural damage to chloroplast and the massive spreading of reactive oxygen throughout the cell,further causes membrane lipid peroxidation,membrane rupture,organelles disintegrated,nuclear condensation and DNA fragmentation,leading to cell death and tissue necrosis,and ultimately kills the weeds.In this study,a toxin-deficient mutant strain 001,which was obtained by restricting enzyme-mediated integration(REMI),and a wild-type strain were used to elucidate the mechanism of TeA during A.alternata's infection stage.Wild-type(WT)and mutant-type(MU)hyphas were inoculated on the intact leaves of croftonweed as materials,Mutant-type is combined with TeA.Results showed that TeA is the main toxicological factor for pathogen Alternaria to infect the host and cause plant disease.Results from Imaging-PAM show that after wild-type hyphas were inoculatd on the Crofton weed leaf for 18h,the reaction center of photosystem II was blocked;in the 36h,plant disease started to appear.In the corresponding time,PSII activity of the leaf,which was inoculatd with mutant hyphaes,had no significant decrease and disease did not emergene,but similar results with wild-type hyphae in mutant-type hyphae can be obtained by adding a certain concentration of TeA,and mutant-type pathogenicity could be restored partially.Meanwhile the expansion ability of mutant hyphae with toxin is partially restored.In the test of spores germination and growing,appressorium formation or not,infecting and spreading of hyphas in leaf cells and lesion formation,the toxin plays a role in the process of pathogens invading leaf tissue,mycelium expansion and the development of the disease.TeA is an important factor of pathogens successly invading and lesion causing.In wild-type spores or hyphas disease Eupatorium leaves,we detect the phenomenon of cell death,which means that reactive oxygen species(ROS)may participate into the process.In order to further elucidate the mechanisms of TeA functioning in the process of pathogen fungus invading the host,we explored the relationship between TeA?reactive oxygen species(ROS)generation?cell death and disease development utilizing a series of experiments.DAB and NBT staining showed that the leaves treated with wild-type pathogen for 5 hours produced significant ROS;However,Mutant pathogen treatment did not cause the generation of ROS,in the case of the presence TeA mutant treatment can also cause reactive oxygen species significantly increased.Active oxygen scavenger CAT and DPI pretreatment can significantly reduce the activity damage to blade PSII,disease development and formation of disease spot caused by the wild-type pathogen.Active oxygen scavenger has remarkable effect on diffusion of pathogens hyphaes in leaf tissues,CAT and DPI pretreatment significantly reduced the development velocity of hyphae,but has no effect on the initial invasion process of the pathogen.This suggests that role way of toxin TeA in in the process of pathogen fungus causing host disease is through the induction of reactive oxygen species generation,causing cell death,helping the pathogen rapid expansion in the organization,resulting in tissue necrosis,which provide nutrition for pathogens to breed and grow further.In order to further illustrate the mechanism of action,we use the other two compound can cause necrosis of plant leaf-AAL-toxin(another toxin produced by A.alternata)and herbicide Bentazone instead of TeA processing.We found that AAL-toxin and Bentazone,like TeA,can help mutant-type restore some pathogenic ability and obviously promote diffusion of the mutant-type hyphae in the blade and the formation of disease spot.This shows that the toxin TeA causing cell death is the important condition of pathogens A.alternata spreading in leaf tissues successfully and development of disease.In the process of plant disease devolopment caused by A.alternata,The generation of reactive oxygen species and occurrence of cell death means certain pathological signaling pathways were activated.Semi-quantitative RT-PCR was used to study singlet oxygen marker aaa-ATPase and hydrogen peroxide marker ferritin gene,Some pathological related pr1,sib1,wrky33,wrky46 gene expression in Arabidopsis thaliana retreated with A.alternata wild-type spores and mutant-type spores.Results show the gene expression level in the arabidopsis leaves retreated by wild-type spores for 12h increased rapidly,but related gene expression quantity of arabidopsis leaves retreated by the mutant spores is obviously lower than that of wild type processing plants.This study established TeA as a key virulence factor in the process of pathogen fungus A.alternata infecting the host plant,basicly explicited the main action mechanism of toxin TeA in the stage of pathogen's infection to host plants and disease development and preliminary revealed the singlet oxygen and hydrogen peroxide mediated signal pathway may be involved in the interaction process of pathogen fungus A.alternata and A.thaliana.