Study On The Life History Of Plasmodiophora Brassicae Ⅰ

Plasmodiophora brassicae Woronin is a soil-borne obligate plant pathogen causing the economically important clubroot disease of crucifers but there are very few effective ways to control this disease. In resent decades, clubroot become more serious due to scale planting of cruciferous plant. Pathogen infection research could help to find effective prevention and control measures. However, due to the obligate characteristic of P.brassicae, the study of the pathogen infection progresses slowly, which hinders the research of prevention and cure way. To solve these problems, it is very important to find an appropriate technique that entices pathogen to infect the host. But the technique hasn’t be studied out yet because it is difficult to meet the conditions of infection. In this study, we developed an improved solution culture to observe root hair infection on cabbage roots, and with the help of the technique, we have found the answers of the two important question about life history of P.brassicae, The results of the study are as follows:1. Germination of resting sporangiaResting spores of P. brassicae were obtained from3-month-old Stem lump mustard (Brassica juncea coss Var tumida Tsen et lee) galls were washed and refrigerated at24℃to thaw. Sliced the soft out layer of the galls and the hard part discarded, and then intermittently homogenizing20g of the shredded gall in100mL of distilled water at high speed for4min and filed through2,8layers of Gauze Wrap, then centrifuged. Surface sterilization of resting spores was performed according to Asano et al by freshly prepared2%chloramine-T solution (wt/vol) at room temperature (25℃) for20min and then washed twice in sterile water by centrifugation as above. The resulting pellet was suspended in a solution of antibiotics, lug/mL of colistin sulfate, lug/mL of vancomycin hydrochloride, and6ug/mL of cefotaxime sodium, in distilled water and incubated at25℃in the darkness for1d. After1day, the suspension was washed twice in sterile water by centrifugation. The spore suspension was prepared with sterile distilled water and stored at-4℃.The highest germination rate was6.4%/day at15day after cultured the resting spores in the nutrient solution contain root secretion at pH6.2.25℃2. Water culture inoculation methods and conditionsBrassica oleracea seedlings were sown directly into10mL centrifuge tubes containing half-strength Hoagland nutrient solution. Seedlings were fastened in1cm thick foam in the tube top and tubes were wrapped with black tape to block light and prevent algal growth.Maximal root hair infection was observed under conditions of pH5.5, temperature of20to25℃, and an inoculation concentration of1×10’ spores/mL. Infection efficiency was obviously higher in fresh nutrient. Under optimum infection conditions, plasmodia were found in the root hair five days after inoculation (DAI), with some undergoing cleavage and developing into round zoosporangia three days later. At ten DAI, the number of root hairs containing zoosporangia per root reached208, and the cytoplasm within the zoosporangia began to cleave and form incipient zoospores. Most zoosporangia released the zoospores and became diaphanous at ten DAI. Root hairs became swollen during the experimental trial and small galls appeared clearly on the root at20DAI. In addition, sectioned specimens showed that some cortical cells contained resting spores. We sequentially tested the germination of the resting spores in tubes from1to12DAI and the temporal dynamics of germination corresponding to the infection. Zoosporangia were found in epidermal cells at the same time as in the root hair.3. Observation of primary zoospores infectionFor test that if the primary zoospores would infect other parts of the host except for the root hair, the host have been infected at the time of most-spores-germination. It was found that primary zoospores could invade the root hair and skin cells and epithelial cells, and the secondary zoospores formed in epithelial cells could be released to outside the host and re-infect the root hair and epithelial cells.4. Asexual short cycling testThe asexual short cycling has been tested though2methods:one was infected the healthy host plant with the infected root hairs, the other was put the infected and healthy seedlings in a tube to plant together. Observing and conclusion that there exist the Short asexual cycle based on the results of the test on five kinds of cruciferous plant, that is, the secondary zoospores formed in root hairs could be released to outside the host and reinfect the root hair.