| Canola stem blight, caused by Sclerotinia sclerotiorum, is an important fungal disease worldwide. Shortage of resistant varieties resulted in severe occurrence in some years in China. Mechanism of interaction between canola and S. sclerotiorum is not clear, therefore the progress on the resistance research has been slow.Arabidopsis is a model plant with its known genomic sequence sequenced. Sciensists have been exploring interaction mechanisms between pathogens and plants with Aarabidopsis as an experiemental model, to recognize the molecular basis of plant disease resistance. These studies certainly provide the fundamental knowledge for plant molecular breeding for disease resistance.Chitinase is one of main enzymes secreted by Trichoderma fungus that can inhibit many kinds of fungi by decomposing the chitins in the cell wall. Transgenic plants with chitinase enzyme gene could improve plant resistance and reduce disease.In this paper, a new pathosystem was set up and physiological, biochemical and molecular mechanism in the interaction between Arabidopsis and S. sclerotiorum was analyzed. Effect of Trichoderma chitinase on S. sclerotiorum in the transgenic Arabidopsis was also tested.All the data above will provide knowledge, techiniques or resistant sources for control of the diseases caused by S. sclerotiorumBacterial leaf blight (Xanthomonas oryzae pv oryzae, Xoo) is an important disease in the world. Xoo can not invade the Arabidopsis thaliana (Col-0), so the nonhost resistance perhaps exists in this kind of plant spieces. Screening the mutants of Arabidopsis through loss of function will be very important in studying the nonhost resistance of Arabidopsis to Xoo.The main results were obtained as following:1. Establishment of the pathosystem of S. sclerotiorum and ArabidopsisTo study the pathosystem of S. sclerotiorum on Arabidopsis, the growth of the pathogen was observed on three kinds of media (millet, wheat and rice bran). The results showed that abundant white mycelia grew on the millet grains without dark sclerotia. Three ecotypes of Arabidopsis were highly susceptible to artificial inoculation with millet inocula. The pathosystem of S. sclerotiorum and Arabidopsis developed in this paper is as following: the pathogen growed at 25℃ in the darkness for 48-72h, andthen was transferred with ten small mycelial plugs into millet media. After 7 day-incubation (shaking once each day), the millet grains covered by white mycelia were evenly sprinkled onto pot surfaces, with 40-days-old Arabidopsis plants. The pathogens severely infected the Arabidopsis plants at 100% RH, 23-25 °C and 14h light/1 Oh darkness for 48h.2. Cytological and physiological features of S. sclerotiorum pathogenicityTrypan blue-staining showed that S. sclerotiorum mainly infected the Arabidopsis leaves with hyphae, while some ball-like or cushion-like structures formed on the tops or sides of a few hyphae. Plants began to wet-rot with yellowish-brown syptoms 24h post-inoculation, and all died 72h post inoculation. Assays with the dye-label colorimetric method indicated that cellulase was the main enzyme secreted by S. sclerotiorum and acted in a pH-dependent manner. Oxalic acid, another pathogenecity factor of S. sclerotiorum, quickly induced necrotic spots with a mechanism different from other inorganic acid, HC1 and H2SO4. It is proposed that oxalic acid acts synergistically with cellulase in the pathogenesis.3. Defense mechanism of Arabidopsis in response to S. sclerotiorumSclerotinia sclerotiorum infection strongly induced the production of pathogenesis -related proteins, chitinase and f5-l,3-glucanase. Furthermore, PDF 1.2 but not PR1 and GST1 was strongly induced 8h after inoculation, indicating that the infection of S. sclerotiorum activated the basal defense pathway -jasmonic acid/ethylene-dependent pathway. This result is consistent with the characteristics of facultative fungal pathogens. This pathosystem of Arabidopsis-S'c/erotforw/n will provide a tool for study of interactions between Sclerotinia and other plants, therefore facilitating the genetic manipulation for controlling this pathogen.4. Antagonism of Trichoderma to S. sclerotiorumThe investigation on the antagonism of Trichoderma to Sclerotinia showed that the hyphae of Trichoderma harzianum extended into Sclerotinia mycelia and formed conidia. Microscopic observation proved that the hyphae of Trichoderma firstly grew around the hyphae of the pathogen and then penetrated, indicating that mycoparasitism is tha main antagonistic mechanism of Trichoderma harzianum against S. sclerotiorum.5. Transformation of Arabidopsis thaliana mediated by Agrobacterium tumuficienct with endo-chitinase gene from Trichoderma harzianumArabidopsis thaliana (Col-0) was transformed by spraying Agrobacterium tumuficience with Trichoderma endo-chitinase gene (Then-42) at initial flower stage. 11 seedlings (transformed at about 0.22 percentage) exhibited resistance to hygromycin, which was a marker for the screening. The DNA fragment unique to endo-chitinase (Then-42) was detected from transgenic Arabidopsis leaf by PCR. Southern blotting indicated that the target gene had been inserted into the genomic DNA of arabidopsis plants.6. Overexpression of ThEn42 enhanced Arabidopsis resistance to S. sclerotiorumArabidopsis thaliana (Col-O) was transformed by Agrobacterium tumuficience-mediated method to produce eleven transformants overexpressing the Trichoderma endo-chitinase gene ThEn42. Northern blotting showed that mRNA levels in four transgenic plants were expressed at least 10 times more than control plants and other transgenic plants. The Tl generation of plants displayed sterile, dwarf and normal phenotypes. Enhanced resistance to S. sclerotiorum was also observed in the transgenic plants..7. Obtained a susceptible mutant of Arabidopsis thaliana (Col-O) to Xanthomonas oryzae pv. oryzae560 activation-tagging and 100,000 EMS mutants of Arabidopsis thaliana(C6l-0) were identified for the resistance to S. sclerotiorum and no resistant mutant was found. In a population of 200,000 arabidopsis seeds mutagenized with EMS, a mutant 99A-6-8 was found susceptible to Xanthomonas oryzae pv.oryzae (Xoo), which was named as noxl. This mutant showed yellow lesion appeared 2 day after inoculation with Xoo-99A. It was greatly different from the wildtype and exhibited dwarf, indistinct main stem and few siliques with few seeds in the phenotype. The bacterial pathogens were still alive in the leaves of the mutant plants 7 day after injection-inoculation with 99G2 carrying a reporter GUS gene, but hardly observed in the wildtype plant leaves 2 day after inoculation. This mutant will provide very useful material for the research on the nonhost resistance of Arabidopsis to Xoo. |
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