Identification Of The Verticillium Wilt Pathogens And Quick Detection Of Diseased Plants In Chinese Cabbage

Chinese cabbage [Brassica rapa L. ssp. pekinensis (Lour.) Olsson] is an economically important vegetable crop with the largest cultivated area and highest yield in China, and become popular worldwide. Now studies on Chinese cabbage diseases focus on downy mildew, soft rot and virus disease, however for the past years a new disease happened an increasing trend seemed like Verticillium wilt on Chinese cabbage in Beijing area and Yunnan Tonghai planting farms. Symptoms appeared in the beginning of the folding stages, showed yellowing, wilting of leaves, discoloration in vascular tissue of the stem even deathly, which proved to be a serious soil-born vascular disease and result in a high proportion of loss in yield and quality. Thus, for Chinese cabbage breeding, it is important to clear the mechanism of resistance to Verticillium wilt.In this study, the pathogen causing Verticillium wilt on Chinese cabbage in Beijing area was identified by morphological characteristics, pathogenicity test and the sequence of ribosomal DNA-ITS. Moreover, seedling resistance identification technology, combined with real time PCR technology for fast and accurate pathogens detection technology of Chinese cabbage Verticillium wilt were established. Results are as follows:1. Four isolates from diseased plants of four different varieties were obtained, the pathogens proved to be V. dahliae by morphological characteristics, pathogenicity test and molecular identification. Further more, hyphae showed fastest growth at22℃.2. A mixed factorial design was used for seeding stage resistant identification of Chinese cabbage Verticillium wilt through spore suspension inoculation method (root-dipping, pouring and seed-dipping method factor), inoculation concentration (106,107,108ind cell-mL-1). Root-dipping by spore suspension (107×10min) was established to be the best method.3. The high virulent strain BCHW10-2was used for host range test of pathogens. Studies reveal that the long-spored Verticillium isolates collected from Chinese cabbage causes wilt diseases on many crops, including turnip, radish, cabbage, chinese kale, broccoli, cauliflower, savoy, tomato, pepper and eggplant with different degrees of wilt.4. QPCR assays for quantification of V. dahliae DNA were conducted using the primer pair HW1designed according to rDNA-ITS sequence differences between JN564038and JN564039, which detection sensitivity reached5×10-3ng·μL-1. In present study, we quantified the fungus along with the DNA relative amount (molecules·μL-1) of V. dahliae strain in the susceptible and tolerant cvs. as by RT-PCR technology. It was further demonstrated that V. dahliae were present at a significantly higher level in susceptible cvs. than in tolerant cvs. It was finally observed that the relative amount of the pathogen in roots was higher than in stems and leaves and changed in plant tissues over time. The data constituted a valuable contribution in evaluating resistance of Chinese cabbage cultivars to V. dahliae pathotypes.