Screening Of Resistant Varieties To Alfalfa Leaf Spot Caused By Alternaria Alternatd And Study On Its Resistance Mechanism

In this study,21 varieties of alfalfa(Medicago sativa)were infected by Alternaria alternata that identified and separated from the infected alfalfa leaves.Three indexes were used to determine the resistance of different varieties to alfalfa spot disease.Phenylalanine ammonia-lyase(PAL)enzyme activity was measured by laboratory hydroponic and disease index of leaves was evaluated by leaves in vitro and filed experiment.In order to study the mechanism of disease resistance,the effect of Alfalfa response to pathogen-related enzymes and gene expression was deeply analyzed.It will lay a foundation for further research on disease resistance.1.The ITS1/ITS4 primers and the OPA2-1L/OPA2-1R primers were used for PCR amplification of the ribosomal transcribed spacers.The analysis identified the strain as Alternaria alternata.2.Using the fuzzy subordinate function method as evaluation of disease resistance among 21 alfalfa varieties.And the results showed that the disease resistance were:'Instinct'>'MT4015'>'Beilin201'>'Tango'>'Vision'>'Barcrown'>'Magna995'>'SK3010'>'Dryland'>'Spyder'>MF4020>'MagnaGraze ?'>'Magna801>'Magna601 '>'Magnum ?-Naiwang'>'Victori a'>'Magnum?'>'Zhao Dong'>'Magnum-salt'>'Zhungeer'>'Xinjiang Daye'.3.The activities of APX,POD,CAT and PAL in'Xinjiang Daye','Beilin201','Barcrown' and'Magnum-salt' during 0-18 days were studied.The results showed that 0-6 days after infection was the key time for alfalfa APX,CAT and POD antioxidant enzymes to respond to A.alternata:the activity of CAT and APX decreased,and the activity of hydrogen peroxide could accumulate in a short time to amplify signal molecules under stress;the activity of POD enzymes increased,and the excessive concentration of hydrogen peroxide was cleared to protect cells from injury.From 6 to 18 days,the activity of APX and CAT decreased,which may be due to the prolongation and intensification of environmental stress after exceeding the tolerance of cells,resulting in the deepening of lipid peroxidation of cell membranes,thus affecting the synthesis of intracellular proteins and other substances.4.The expression of APX,CuZnSOD?MnSOD?NPR1?NPRlLand PAL with recognized roles in A.alternata infection in 'Xinjiang Daye'and 'Beilin201' were analyzed by real-time quantitative PCR(RT-qPCR)technique.The results showed that the transcription levels of APX,MnSOD and PAL genes were significantly increased,and CuZnSOD,NPR1 and NPR1L genes were significantly different among alfalfa varieties.These genes were induced by A.alternata and presumed to be involved in the early resistance of alfalfa to A.alternata.It will increase our knowledge of A.alternata-alfalfa interaction and help to develop molecular and conventional breeding strategies for variety improvement.