Study On Pathogenic Mechanism Of Phoma Medicaginis On Alfalfa

Spring black stem and leaf spot caused by Phoma medicaginis commonly occurs in alfalfa producing areas of all over the world.The symptoms of the disease are the appearance of black spots on the leaves and the stems blackening.The disease has a great negative influence on alfalfa yield,seed yield and grass quality.However,at present,most of the alfalfa varieties have no resistance to the pathogen.Therefore,it is very important to systematically elucidate the effects of P.medicaginis on physiological and metabolic activities of alfalfa for the disease control and cultivation of disease-resistant varieties.In our studies,the photosynthetic physiology,nutrient metabolism,antioxidant system and resistance-related genes and other indicators in the alfalfa leaves were determined at 2-28 days post inoculation（dpi）with portable photosynthetic apparatus,fluorescence quantitative PCR and GC-MS.The main results are as follows:1.The photosynthesis and carbon assimilation in the alfalfa leaves decreased significantly under the infection of P.medicaginis.Compared with the control groups,the following indicators reduced significantly during infection:net photosynthetic rate（Pn）,stomatal conductance（Gs）and transpiration rate（Tr）.However,the intercellular CO₂ concentration（Ci）decreased significantly in the early and late stages of infection.The following indicators reduced significantly in the late stage of infection:PSⅡreaction center of excitation energy capture efficiency（Fv’/Fm’）,the relative quantum efficiency of PSII photochemistry（ΦPSII）,electron transfer rate（ETR）,photochemical quenching（qP）and non-photochemical quenching（qN）,initial fluorescence（F0）and photochemical quantum efficiency（Fv/Fm）.The following indicators decreased significantly in the early and late satges of infection:the content of chlorophyll a,chlorophyll b,chlorophyll a/b and total chlorophyll,and the expression of small subunit ribulose-1,5-diphosphate carboxylase（rbcS）and chlorophyll a/b binding protein（cab）gene.The light compensation point（LCP）and the carboxylation efficiency（CE）decreased significantly.The CO₂ compensation point（CCP）increased significantly.2.The infection of P.medicaginis causes nutrient metabolism disorder in alfalfa leaves.Compared with the control groups,the total content of the protein decreased significantly in the middle and late stages of infection.The activity of sucrose phosphate synthase（SPS）and sucrose synthase（SS）increased significantly in the late stage of infection.The activity of adenosine phosphoglucose pyrophosphorylase（AGPase）increased significantly at different stages of infection.The content of glutamine synthase（GS）increased significantly at the early stage of infection,glutamate dehydrogenase（GDH）content decreased significantly in the early and middle stages of infection,and increased significantly in the later stages.Sucrose content decreased significantly in the middle and late stages of infection,while the content of glucose and fructose increased significantly at different stages of infection.The contents of Mn,Li,Cr,Co,Mg,Ca and Fe increased significantly,however,the content of Na decreased significantly.3.The metabolic pathway of primary metabolites changed under P.medicaginis infection.Among 70 compounds in the uninoculated and inoculated leaves identified by GC-MS,16significant different compounds associated with 6 metabolic pathways,including glycolysis,tricarboxylic acid cycle,fatty acids,glutathione,inositol phosphate and amino acid metabolic pathways.Among them,compounds of glycolysis,tricarboxylic acid cycle,fatty acid and amino acid metabolic pathways,such as,glucose,pyruvic acid,succinic acid,fumaric acid and citric acid;palmitic acid and stearic acid;5-o-proline,aspartic acid,glutamic acid and lysine increased significantly in the late stage of infection.However,glycine,leucine,isoleucine and tyrosine were lower than those of the control groups（significant or not significant）in the middle and late stages of infection.Theγ-aminobutyric acid（GABA）decreased significantly during infection of the pathogen.The content of methionine was not detected,except in the early stage of infection,had no significant difference or decreased significantly.The compounds in the inositol phosphate pathway,such as inositol phosphate and inositol-1,3,4,5,6-pentyl phosphate decreased significantly in the middle and late stages of infection.Other compounds:2-pyrrolidone decreased significantly during infection of the pathogen.The malonic acid content decreased significantly in the early stage of infection,and increased significantly in the late stage of infection.4.The antioxidant system in the alfalfa leaves disordered under the infection of P.medicaginis.Compared with the control groups,the content of malondialdehyde（MDA）and hydrogen peroxide（H₂O₂）increased significantly in the middle and late stages of infection.The activity of superoxide gasification enzymes（SOD）decreased significantly in the late stage of infection,however,the activity of catalase（CAT）and peroxidase（POD）changed irregularly.The polyphenol oxidase（PPO）activity was not detected in the leaves of the uninoculated groups,and it could be detected in the inoculated group during infection of the pathogen,the activity of it was the highest in the early stage of infection,and then decreased.5.The relative expression of the pathogenesis-related protein（PR）and disease-resistant genes delayed upregulated in the inoculated alfalfa leaves.The expression levels ofβ-1,3-glucanase and chitinase（CHI）,glutamine synthase（GS1）,phenylalanine ammonia lyase（PAL）and chalcone synthase（CHS）genes all accumulated significantly in the middle stage of infection,after that,their genes expression all decreased rapidly.In conclusion,there is a complex physiological regulation network for the pathogenic mechanism of P.medicaginis to host.The infection of pathogen destroys the antioxidant balance system in the host,which increases the reactive oxygen species,and total antioxidant enzyme activity decreased,leading to the damage of lipid peroxidation in alfalfa leaves.All of which reduces photosynthesis and carbon assimilation,and then disturbs the metabolism of sugar and nitrogen in the host,meanwhile,delays rapid and strong response of the activity of sugar and nitrogen related enzymes,and the expression of PR and disease resistant genes,which promotes plant senescence and death.