| The methods of culturing and maintenance of obligate parasite pathogen Pseudoperonospora cubensis, which causing cucumber downy mildew, were studied in this paper. The baseline-sensitivity of the pathogen to flumorph was established and the resistant risk of P. cubensis to flumorph was also assessed in both laboratory and field. The effects of flumorph on the different developing stages, as well as the respiration and membrane permeability of P. cubensis were investigated. The results are as follows.1. The method of detached cucumber leaf was suitable for the subculture preservation of the pathogen. The method of being kept at -80 ℃ in a plastic bag was suitable for the long-term preservation, and its pathogenicity to the host plant remained at least for 60 days. The method of leaf disc assay was proved to be reliable and repeatable to determine the sensibility of P. cubensis to fungicides.2. The sensitivity to flumorph of 77 bulk strains, which were collected from Beijing, Hebei and Innermongolia in 2003 and 2004, were determined by detached leaf discs method. The EC5o values rang from 0.0213 to 0.2647 ug/mL, and the average of 0.1360±0.0487ug/mL, which showed a narrow sensitivity distribution. There was no resistant subpopulation appeared among these strains, so this sensitivity baseline was suitable for the field resistance monitoring.3. In order to evaluate the potential risk of resistance development to flumorph, laboratory studies were conducted to compare the easy of isolating resistant mutants for flumorph and those for dimethomorph and azoxystrobin, whose resistance risk is well understood based on past years of commercial use. The results showed that flumorph-resistant(FR), dimethomorph-resistant(DR) and azoxystrobin-resistant (AR) strains could be all selected through fungicide adaption and ultraviolet irradiation. After selection by adaption, their resistance factors for flumorph, dimethomorph, azoxystrobin resistant mutants were 1.64~3.28, 1.64~4.10 and 16.10~29.90, respectively. But the resistance factors of mutants induced by UV were 2.24~64.94, 5.46—88.31 and 55~733, were higher than those of mutants selected by adaption. These results also indicated that it was easier to obtain AR mutants than to obtain FR and DR mutants. The pathogenicity, stability, fitness and competition of flumorph-resistant strains were decreased than parent strains, moreover, the resistance of some mutants were stable. So there is potential risk of resiatancde to flumorph in P.cubensis. The degree of resistance risk of flumorph was similar to that of dimethomorph, but lower than azoxystrobin. Data also showed that there were no cross resistance between flumorph and metalaxyl, cymoxanil or azoxystrobin.4. Field resistance of the pathogen to flumorph has also been assessed. The data indicated that the sensitivity of P.cubensis to the flumorph was decreased after flumorph and flumorph+mancozeb applied 7 times, respectively, and the higher dose and frequency of flumorph applied, the more sensitivity of P.cubensis to flumorph decreased. The sensitivity distribution of strains collected from plastic-houseswere all included in the baseline before flumorph and fiumorph+mancozeb applied, but the sensitivity distribution out of baseline after 14 times spray, which indicated that there were flumorph resistant strains appeared under both single and mixure use of flumorph. The pathogenicity, stability, fitness and competition of field flumorph resistant strains were indentical or higher than those of sensitivity strains.5. By comparing of six staining methods, it was proved that the method of alcoholic lactophenol trypan blue was appropriate for the staining of hypha and haustorium, and the calcofluor epifluorescence technique was useful for comparative analysis of the early infection process and sporulation of P.cubensis. These two methods were easy to handle, fast and reliable. The effect of flumorph on these different stages in the life cycle of P.cubensis were studied. The results showed that flumorph had no... |
