Molecular Identification Of The Pathogen And Mechanism Of Pathogenesis Of The Black-spot Disease On Zizyphus Jujuba Cv. Dongzao

Pathogens isolated from Zizyphus Jujuba cv. Dongzao were identified according to their molecular characteristics. The three isolates, Coniothyrium fucsidulum Sacc named as 94, Phoma pomirurn Thüm named as 95, Alternaria alternata (Fr.) Keissler named as 96, were cloned and sequenced with the internal transcribed spacer(ITS) and 5.8S rDNA using the universal primers ITS1 and ITS4, and were compared with published sequences in GenBank database. Phylogenetic trees were produced based on the sequences which were high identical to ITS and 5.8S rDNA sequences of pathogens from different hosts and different places by software MrBayes3-0b4. The results indicated that ITS sequences of pathogen 94 and 95 were completely identity, and were 97% identical to Phoma species. They were identified with one genera Phoma, who was named as 95. The phylogenetic trees indicated that it is closed related to Phoma glomerata. ITS and 5.8S rDNA sequences of pathogeny 96 was 98% identical to Alternaria and clustered(96% bootstrap) with small-spored species whose morphological characteristics were similar in a clade in phylogenetic trees, including A. alternata, A. tenuissima, A. longipes, A. mali, and A. arborescens, et al. Considering its morphological identification, the pathogen 96 was identified as Alternaria alternata (Fr.) Keissler.The pathogens of the black-spot disease Zizyphus Jujuba cv. Dongzao was explored through cell-wall degrading enzymes(CWDEs) and toxins produced by the pathogens. The crude CWDEs were produced in improved Marcus's medium including different inducers to induce different kinds of CWDEs. Results indicated Polygalacturonase(PG), cellulase(Cx), pectin methylgalacturonase(PMG), polygalacturonic acid trans-eliminase (PGTE), and pectin methyl trans-eliminase(PMTE) were determined in the crude enzymes. Among the CWDEs, the activities of PG were significantly higher than the others'. Medium of Dongzao as inducer could induce PG of high activities and Cx of low activities, which showed sterilized Dongzao could induce certain amount CWDEs, and there were lots of pectin and a little cellulose in Dongzao host, which benefited lots of PG and a little Cx produced by pathogens. So pathogens produced many kinds of CWDEs induced by pectin and cellulose in host, which helped them to invade the host tissue in field. Effects of toxins in pathogenesis on cell membrane permeability, malondidehyde (MDA) content and activities of peroxidase (POD), ascorbaticperoxidase (APX), and catalase (CAT) in Dongzao pericarp tissue were tested. The results showed that the toxin of pathogens could over-oxidate cell membrane fat, augment cell membrane permeability, lead the leakage of cell electrolyte and the damage of membrane system; and the toxin could also restrain elimination system of active oxygen. Then the active oxygen accumulated plentifully in host cell, the normal metabolism was disturbed, the cell lost their physiology function. So all the results indicated that toxin is the important pathogenicity of black-spot disease Zizyphus Jujuba cv. Dongzao.