Studies On Pathogenic Mechanism Of Citrus Sour Rot Pathogen And Antagonistic Yeast

Geotrichum citri-aurantii, one of the most important pathogenic fungi of citrus postharvest, frequently causes serious fruit decay and large economic loss. Pathogenic isolates from the naturally rotted citrus fruit were isolated on potato dextrose agar (PDA), and identified as G. citri-aurantii, the cause of citrus sour rot. In vitro sensitivity of pathogen to three fungicides was tested by inhibiting growth of mycelia in PDA medium, which were supplemented with various concentrations of prochloraz、imazalil or guazatine. And the infection process of G. citri-aurantii on artificially inoculated fruits was observed by using sanning electron microscope (SEM). For researching pathogenic mechanism, three potential factors produced by pathogen were investigated, which included well cell-degrading enzymes, organic acids and toxins. Activity of several cell wall-degrading enzymes produced in vitro and in vivo was determined by UV-Vis Spectrophotometry; HPLC was used to detect organic acids; The crude toxins in fermentation filtrate were collected by extracting with organic solvents or precipitating with (NH4)2SO4, biological activity was measured in vivo. Antagonistic yeast Wf-b-1was selected by in vitro and in vivo screening, which exhibited significantly inhibitory effect against G. citri-aurantii. Wf-b-1had been identified by lab members, and known as Torulaspora globosa. To evaluate the inhibitory effect, different concentrations of yeast cells、different inoculated-time and population dynamics of yeast in fruit wound were conducted. The main results were as follows:1. Seven isolates of G. citri-aurantii were isolated and obtained. On the basis of morphological criteria、symptoms of fruit decay and sequencing the internal transcribed spacer regions of ribosomal DNA (rDNA-ITS), they were identified as G. citri-aurantii. And phylogenetic tree of G. spp. also showed that they were closely related to G. citri-aurantii.2. In vitro sensitivity analysis showed that isolates were extremely sensitive to guazatine, but lacked sensitivity to prochloraz and imazalil. EC50values were lower than90pμg/L for guazatine, whereas those of prochloraz and imazalil were between20000and60OOOμg/L.3. SEM was used to observe the infection process of G. citri-aurantii on citrus fruit. We found that the fruit surface was attached by arthrospores of G. citri-aurantii; Spore began to germinate and germ tube started to form within6h; Growth of mycelium trend to nutrition at the wound sites; When nutrition was absent, arthrospores were formed from hyphal segmentation within36h. But they failed to invade tissue through stoma.4. Preliminary studies on pathogenic mechanism indicated that PG (polygalacturonase) and PMG (polymethylgalacturonase) showed appreciable activity, which were both produced by pathogen in vitro and in vivo. PG activity was highest at84h and PMG was60h in diseased tissue. These suggested that they play a crucial role in pathogenicity. pH decrease was significant in G. citri-aurantii-decayed citrus fruit. Production of organic acids was detected by HPLC in vitro and in vivo, and gluconic acid had a high content in rotting tissue. Biological activity of crude toxins was measured on citrus fruit. Organic solvents were less effective for toxin extraction, and high crude toxin content in water phase suggested that toxin chemical compound has higher polarity.5. Antagonistic yeast Wf-b-1was selected by in vitro and in vivo. Control of fruit decay was effective when the concentration of Wf-b-1cells was higher than which of pathogenic arthrospores; In order to decrease disease incidence, Wf-b-1should be applied simultaneously or prior to inoculation with the pathogen; Population dynamics of yeast would be more stable when pathogen was inoculated into fruit wound than when which was not. It’s necessary to evaluate bio-control efficacy and explore possible mechanism, due to the potential of Wf-b-1controlling citrus sour rot.