| Plasmopara viticola is a biotrophic oomycete that causes grape downy mildew. At present the main control of grape downy mildew is to use chemical fungicide, which not only causes environmental pollution, but also results in resistance of P. viticola agrochemicals. The use of biological control as an alternative management is important. In this study, the inhibitory effects of 13 strains of endophytes, Pseudomonas fluorescens strains including the wild type WCS417 r and its 12 transgenic derivatives, and three plants grinding filtrate and water extracts on grape downy mildew were tested. Based on these results, the control experiments of grape downy mildew in growth chamber and field were also performed. Finally, induction of systemic acquired resistance was determined with different concentrations of salicylic acid against grape downy mildew. The main results are summarized as follows. 1 The control effects of endophytic bacteria and 12 phl-transgenic strains on grape downy mildewThe inhibition effects of 13 endophytic bacteria strains isolated from different plants, 12 phl-transgenic strains of WCS417 r, which belongs to P. fluorescens, on sporangia germination of P. viticola were tested. The control effects in growth chamber and field were tested as well. The results indicated that Bacillus subtilis strain CN181 and 12 phltransgenic strains all had inhibitory effect on germination of sporangia, and the inhibition rate reached 92.5%. In the control experiment of growth chamber with detached grape leaves, the control effect of CN181 reached 93.1%. The 12 phl-transgenic strains of WCS417 r all showed control effect on grape downy mildew, and the L2c9 strain is the best with a morbidity of zero. The morbidities of L1d6, L2d6 and L3d6 strains were at or below 5.3%, and those of other strains were between 7% to 20%, with the control effects of over 80%. The parental strain WCS417 r exhibited no inhibition and control effect, indicating that the phl-transgenic expression product of 2,4-diacetylphloroglucinol(DAPG) has a strong inhibitory effect on grape downy mildew. The results of field experiments showed that the preventive effects of CN181, L1d6, L2d6, L3d6 and L2c9 were excellent with no diseased leaves, and their curative control effects were above 39.7%. 2 Control of grape downy mildew with the extract of branches and leaves of Cinnamomum camphoraThe inhibitory effect of grinding filtrate and water extracts from Cinnamomum camphora branches and leaves on sporangia of Plasmopara viticola was determined, and further performed control tests on downy mildew in grape leaves in growth chamber and field. The results indicated that the grinding filtrate and water extracts all had the inhibitory effects on germination of sporangia, and the inhibition rate reached 100%. The water extracts were selected for separation using organic solvents with different polarities, and the separated fraction of n-hexane, ethyl acetate, n-butanol and water were obtained, respectively. At the concentration of 1% for each fraction, the inhibition rates of sporangia germination were 58.4%, 92.4%, 32.5%, and 58.7%, respectively, indicating that the four fractions could inhibit the germination of sporangia significantly. However, the most effective compounds were in the fraction of ethyl acetate. In the control experiment of growth chamber with detached grape leaves, the control effects of 1% grinding filtrate, each extracted fraction of four solvents mentioned above at 1%, water extracts at 0.3 g/m L and 0.1 g/m L + 50% Aloe vera grinding filtrate reached 97.2%, 100%, 100%, 68.9%, 88.9%, 94.7% and 94.5%, respectively. In preventive control experiment no symptoms of grape downy mildew were observed by using 5% grinding filtrate. In the curative assays the control effects of 5% grinding filtrate reached 94.1%. The control effect of water extracts at 1 g/m L in the preventive control- and curative experiments could reach 100%. In preventive control experiment of the field no symptoms of grape downy mildew were observed by using water extracts at 0.3 g/m L and 0.6 g/m L, and 5% grinding filtrate. In the curative experiment, 5% grinding filtrate exhibited no effect on grape downy mildew. However, the curative control effects of water extracts at 0.3 g/m L and 0.6 g/m L could reach 67.2% and 60%, with significant differences compared with control. It did not significantly increase the therapeutic effect by addition of 0.1% Cu SO4 in the water extract. To the best of our knowledge, this is the first report to show the control effect of camphor extracts on grape downy mildew, laying the foundation for the future development of camphor tree branches and leaves as a botanical fungicide. 3 Control of grape downy mildew with the extract of Eucalyptus amplifolia and Portulaca oleraceaThe inhibitory effect of grinding filtrate and water extracts from E. amplifolia and P. oleracea on sporangia of P. viticola was determined, and further performed control tests on grape downy mildew in growth chamber and field. The results indicated that the E. amplifolia, P. oleracea grinding filtrate and water extracts all had the inhibitory effects on germination of sporangia, and the inhibition rate reached 100%. In the control experiment of growth chamber with detached grape leaves, the control effects of 1% E. amplifolia and P. oleracea grinding filtrate, their water extracts at 0.3 g/m L and 0.1 g/m L of both plants + 50% Aloe vera grinding filtrate reached 100%, 100%, 97.5%, 97.5%, 90.6% and 94.0%, respectively. The control effects of water extracts at 1 g/m L in the preventive control- and curative experiment could reach 100%. The curative control effects of E. amplifolia and P. oleracea water extracts at 1 g/m L reached 91.2% and 95.9%. In preventive control experiment of the field no symptoms of grape downy mildew were observed by using water extracts at 0.3 g/m L and 0.6 g/m L, and 5% grinding filtrate. In the curative experiment, 5% grinding filtrate exhibited no effect on grape downy mildew. However, the curative control effects of E. amplifolia water extracts at 0.3 g/m L and 0.6 g/m L could reach 57.9% and 74.7%, and the curative control effects of P. oleracea water extracts at 0.3 g/m L and 0.6 g/m L were 53.7% and 59.6%, with significant differences compared with control. It did not significantly increase the therapeutic effect by addition of 0.1% Cu SO4 in the water extract of both plants. 4 Systemic acquired resistace induced by salicylic acid against grape downy mildew caused by Plasmopara viticolaThe tests with different concentrations of salicylic acid(SA) at 0.1 m M, 0.5 m M, 1 m M, 3 m M, 5 m M and 7 m M were performed against grape downy mildew for their inductive effect. The inductive effect of 1 m M SA in detached leaf of Kyoho grapes was 100%, and those of other SA concentrations at 0.1 m M, 0.5 m M, 3 m M, 5 m M and 7 m M were above 95.7%, with significant differences compared with controls. The petioles of leaves treated with 7 m M SA turned black, indicating that high concentration of SA is toxic to grape leaves. In the induction experiment of disease resistance on the detached leaves of Manicure finger grapes, both concentrations of 0.1 m M and 0.5 m M SA were capable of inducing resistance against grape downy mildew, with induction effects of 82.1% and 97.9%, respectively, which showed significant difference compared with controls. The higher concentrations of 1 m M to 7 m M SA were also toxic to grape leaves and the petioles readily turned black. Application of 0.5 m M SA by leaves spraying in the field 1-7 days before inoculation of P. viticola, the inductive effects in the variety of Kyoho grape were all above 85.7%, and no obvious inductive effects in the susceptible variety, Manicure finger grape, could be observed. |
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