| This thesis report the expression in wheat spikelets of Tril2 gene encoding trichothecene efflux pump that is responsible for the transportation of trichothecenes out of fungal cells in Fusarium graminearum, the main toxins of F. graminearum produced and the diffusion of trichotheneces in wheat spike tissuses in relation to Tril2, and the role of trichotheneces to wheat pathogenicity, So we can defmitude the relationship of the expression of trichotheneces efflux pump gene and it's pathogenicity.Trichothecenes are secondary metabolites, some of which are pathogenicity factors in Fusarium graminearum. With the wild type strain NRRL 29169 as parent strain, we generate a Tril2 knock-out mutant strain (MT12). On potato-dextrose-agar, MT12 grew slower and produced less mycelia than NRRL 29169 (P<0.01), though macroconidia of MT12 were longer than those of NRRL 29169 (P<0.05). Virulence on wheat was determined for NRRL 29169, MT12 and GZT40, a strain with the Tri5 gene disrupted. Tri5 encodes trichothecene synthase that catalyzes the first step of trichothecene biosynthesis. Among the three strains tested, NRRL 29169 was most virulent with disease symptoms spreading from the inoculated spikelets to other spikelets, while the two mutant strains were less virulent with no spreading being observed. From these experiments, we conclude that disruption of Tri12 causes accumulation of trichothecenes in fungal mycelia instead of them being pumped into host tissues. We further believe that trichothecenes are toxic to the fungus itself and that exposure to the toxins inhibits its growth and toxin production. As a result, the pathogenicity of the pathogen may be greatly reduced.In the study of knock-out of Tri12 reduced trichotheneces production sensitivity in F.graminarum, We find that on potato-dextrose-agar containing 0-50 ppm of either deoxynivalenol (DON), 3-AcDON (3-acetyldeoxynivalenol) or 15-AcDON (15-acetyldeoxynivalenol), MT12 showed no significant difference in growth rate, indicating that MT12 is not sensitive to trichothecenes. On wheat grain or rice grain solid meida and glucose-yeast extract-peptone liquid medium, MT12 produced little or no trichothecenes. From these results, we hypothesize that the trichothecene efflux pump encoded by Tri12 is a bidirectional pump. Removal of efflux pump causes accumulation of trichothecenes within the fungal cells, which inhibits the fungal growth and toxin production, and causes failure to transport trichothecenes from outside into inside of fungal cells, which reduces fungal sensitivity to trichothecenes in surrounding environment.Diffusion of trichothecenes in wheat spike tissues in relation to an efflux pump gene Tril2 in F.graminearum indicate that MT12 was weakly pathogenic on wheat, and only a small amount of DON, 15-acetyldexynivalenol and 3-acetylnivalenol was detected in the MT12-inoculated spikelet. No diffusion of trichothecenes from the inoculated spikelet to other spikelets was observed. In a feeding experiment, a floret in a middle spikelet of a spike was fed with 4 ug of DON daily for 10 days beginning from second day after heading. The day after the last feeding DON was recovered from various parts of the fed spikes. Among the DON recovered, 97% was from the fed spikelet. Among the remaining 3% of recovered DON, 75% was from spikelets and rachis segments above the fed spikelet, and the remainder from spikelets and rachis segments below the fed spiklets and a 1 cm peduncle segment below the spike. In another feeding, experiment, a floret was fed once with 4 ug of DON and was analyzed for DON 10 days later. Among the DON recovered, 97% was from the fed spikelet. Among the remaining 3% of DON, 49% was recovered from one spikelet immediately above and one spikelet immediately below the fed spikelet, 39% from the two top spikelets and 12% from a 1 cm peduncle segment below the spike. These results indicate that Thl2 influences efflux of trichothecenes from trichothecene producing cells of F. graminearum and influx of trichothecenes into wheat spike tissues. Fa... |
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