| Cucurbit blight is a destructive disease of cucurbit crops worldwide, and the disease has become one of the most serious threats to production of cucurbit crops. Multiple Phytophthora species have been reported to be associated with the disease. It is extremely difficult to control the disease because of the diversity and fungicide resistance of Phytophthora pathogens. To ascertain composition and population dynamics of the pathogens causing cucumber and wax gourd blight, Phytophthora isolates were recovered from the diseased cucumber and wax gourd plants and identified based on their morphological and biological characters as well as molecular analysis in South China during2007-2010in this dissertation. Meanwhile, the characteristcs of metalaxyl sensitivity, mating type, virulent differentiation and genetic diversity of the pathogen populations were systematically studied. Lastly, screening highly effective fungicides for chemical control of wax gourd phytophthora blight was carried out. The main results of the study are summarized as follows.1. Totally193isolates of Phytophthora sp. were isolated and purified from wax gourd and cucumber phytophthora blight samples in major vegetable growing areas among9cities of Guangxi Zhuang Autonomous Region and Guangdong province during2007-2010. Comparative studies on morphological, cultural, physiological characters and host range of10representative isolates were conducted. Moreover, sequences of ribosomal DNA-ITS (internal transcribed spacer) of89isolates were analyzed. The representative isolates grew well on OA plates, with white or yellowish colonies and moderate or abundant aerial mycelia. Hyphae transparent, aseptate,4.1-7.6μm (av.5.5μm) in diameter. Hyphal swellings were commonly found and predominantly rounded. Sporangia ovoid, ellipsoid to long ellipsoid, nonpapillate and noncaducous, with a dimension of35.4-86.7x20.4-40.8μm (av.54.4×32.3μm). Sporangia were frequently borne on unbranched sporangiophores. The length-to-width ratio of a sporangium varied from2.43to1.13(av.1.69). Heterothallic, oogonia spherical, smooth-walled,25.5-40.8μm (av.30.9μm) in diameter. Oogonial stalks were frequently club-shaped, rarely tapered. Oospores spherical, thick-walled,22.9-30.7μm (av.26.9μm) in diameter, plerotic or slightly aplerotic. Antheridia were predominantly amphigynous. The optimal temperature and pH value were25.0-30.0℃and5-7.5, respectively, for vegetative growth of the pathogen. The isolates were slightly resistant to malachite green and could utilize multiple carbon and nitrogen sources for their growth. A wide host range of the pathogen was confirmed by artificial inoculations. The rDNA-ITS sequence similarity was compared between the representative isolates causing cucumber and wax gourd blight and38isolates of other Phytophthora spp. registered in GenBank. An optimal phylogenetic tree was established using the Neighbor-Joining method under10000bootstrap replicates. The10representative isolates clustered with the isolates of P. melonis and P. sinensis registered in GenBank at99%bootstrap level, and were clearly differentiated from the remaining34isolates of the other Phytophthora spp. This indicated that the representative isolates were genetically close with P. melonis and P. sinensis. The representative isolates were identified as the members of P. melonis (synonym:P. sinensis) based on their morphological and biological characters as well as ribosomal DNA-ITS sequences.2. Virulence of193P. melonis isolates and their host preference were tested and compared by inoculating the mycelial disks on wax gourd and cucumber plants using non-wounding method. The results showed that marked difference of virulence existed in the P. melonis populations. The isolates were divided into highly virulent, moderately virulent and weakly virulent groups. The highly virulent isolates were dominant in most of the areas sampled on both host plants. At least three types (A, B and C) of host preference were found among the P. melonis isolates. The isolates of A-type host preference were highly virulent on both wax gourd and cucumber plants regardless of their original isolation hosts. The isolates of B-type host preference were highly virulent only on the wax gourd plants from which they were isolated, and weakly or moderately virulent on the cucumber plants. The isolates of C-type host preference were highly virulent only on the cucumber plants from which they were isolated, and weakly or moderately virulent on the wax gourd plants. The present study clearly indicated the existence of virulent differentiation in the P. melonis populations in South China.3. The sensitivity of193isolates of P. melonis to metalaxyl was tested using mycelial growth rate method in vitro and floating-leaf-disk method in vivo, respectively. The results showed that the sensitive, moderately resistant and resistant isolates were recorded as29.0%,18.1%and52.8%, respectively. The frequency and level of resistance of P. melonis from Guangdong were higher than that from Guangxi. The isolates from cucumber was generally more resistant to metalaxyl than those from wax gourd. The metalaxyl-resistant isolates were frequently detected as a predominant population in most of the sampling sites and the highest resistance index (4226.9) was confirmed. Metalaxyl-resistant (Mtr) mutants could be isolated from approximately60%of the sensitive wild-type isolates. The resistance level of the Mtr mutants was189-407times higher than that of their sensitive parental isolates. The EC50values of9sensitive isolates from a sampling site without a record of phenylamide fungicide application ranged from0.0429to0.5461μg/mL. Their mean EC50value (0.3200±0.1617μg/mL) was considered as the baseline sensitivity of P. melonis to metalaxyl in South China. Metalaxyl-resistant isolates universally occur in South China, especially in the vegetable-growing areas with a longer history of metalaxyl application.4. A total of193P. melonis isolates were paired with the reference mating types A1and A2of P. capsici to determine their mating types. The results showed that5mating types of P. melonis were found in9sampling cities. The type A0was predominant in Guilin city, contributing40.9%of the isolates recovered, while the type A2predominant in the other sampling cities with a frequency of37.5-66.7%. The types A0, A2and A1A2were isolated from both hosts, however, the types Al and A1,A2were obtained only from the wax gourd and cucumber plants, respectively, revealing the existence of host-specific mating types of P. melonis in nature. The ratio of types Al and A2isolated from Guilin was close to1:1, indicating that Guilin might be located at the center of geographic origin of P. melonis, and demonstrating that Asia is the geographic origin of P. melonis.5. Random amplified polymorphic DNA (RAPD) analysis was used to detect the genetic variation of P. melonis isolates derived from9cities in South China. Among180primers screened, twelve that generated consistent polymorphic bands were applied to amplify the genomic DNA of96isolates collected in South China. In total,135RAPD markers were obtained and124of them showed polymorphism, contributing to91.9%of total bands amplified. Based on the genetic distance, a dendrogram was established with the software NTSYSpc Version2.1, and the96tested isolates were divided into12RAPD groups in0.81genetic similarity coefficient. A lower genetic similarity was detected among majority of the isolates, suggesting the existence of considerably genetic variation and diversity in P. melonis populations at DNA levels in South China. No correlation was obviously found between the RAPD groups and the groups defined by geographic origin, host, virulence, mating type as well as metalaxyl resistance. The results also showed that the degree of genetic variation of the isolates from cucumber was greater than those from wax gourd.6. To screen highly effective fungicides for the P. melonis-induced wax gourd phytophthora blight management, the suppressive effects of19fungicides against P. melonis were tested in vitro. The results showed that dimethomorph (trade name:Acrobat) and flumorph+mancozeb showed the highest growth inhibition rates (89.36%and87.43%, respectively) against the pathogen. The growth inhibition rates of the pathogen by cymoxanil+mancozeb (trade name:Curzate), metalaxyl+mancozeb (trade name:Ridomil Gold), oxadixyl+mancozeb (trade name:Sandofan), mancozeb (trade name:Dithane M-45), fenaminosulf (trade name:Dexon) and fosetyl-aluminum+zineb were53.07%,51.43%,46.57%,46.00%,45.86%and40.43%, respectively. The growth inhibition rates of the pathogen by the remaining fungicides were lower than40%. No suppressive efficacy of the pathogen was observed by azoxystrobin (trade name:Amistar), iprodione (trade name: Rovral) and hymexazol. The results of potted tests indicated that both dimethomorph and flumorph+mancozeb showed100%of control efficacy in protective treatments. The control efficacies of fenaminosulf, chlorothalonil, fosetyl-aluminium+zineb and metalaxyl+propamocarb were73.53%,73.53%,64.70%and60.29%, respectively. The therapeutic treatments of dimethomorph and flumorph+mancozeb also showed higher control efficacies (71.71%and63.99%, respectively). The control efficacies of propamocarb (trade name:Previcur) and metalaxyl+propamocarb against the disease were58.00%and53.70%, respectively. The dimethomorph and flumorph+mancozeb showed higher control efficacies (84.5%and77.8%, respectively) against wax gourd phytophthora blight in field trials as compared to a lower control efficacy (30.1%) of metalaxyl. |
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