應用螢光假單胞菌Pseudomonas putida YLFP14防治甜椒細菌性斑點病

Abstract

螢光假單胞菌 Pseudomonas putida YLFP14 在 PDA 培養基上對測試 之 27 個甜椒細菌性斑點病菌之生長有明顯之抑制作用， 其不產生 氰酸且在含硫酸銅(CuSO4 · 5H2O) 500ppm 或三元硫酸銅 ( 銅高尚 ) 與克枯爛之培養基上可生長良好。添加展著劑與甲基纖維素有助 P. putida YLFP14 於甜椒葉表之群集。利用糖蜜、酵母粉及黃豆粉之 MYS 基質於酵槽進行 P. putida YLFP14r 之液態培養，結果顯示，於 30 ℃下培養 30 小時， 菌量可達 109 cfu/ml，在低溫 (4 ℃ )環境下，有 利於 MYS-YLFP14r 菌液中 P. putida YLFP14r 菌量之維持。 測試 P. putida YLFP14 對甜椒細菌性斑點病之防治效果，結果顯示不論先接種病 原菌或與病原菌同時接種或先處理 P. putida YLFP14 再接種病原細菌均 可降低甜椒細菌性斑點病之發生， 而以 MYS-YLFP14r 菌液處理於甜椒葉 片亦同樣有降低病害發生之效果。藉由 Biolog GN Microplate 系統測試 53 個螢光假單胞菌菌株對 95 種碳素源之利用情形，結果顯示 53 個菌 株皆可利用之碳素源有 18 種；皆無法利用的有 13 種， 而其餘 64 種碳素源之利用則因菌株之不同而有所差異。 分別添加0.5% 蛋白或 D- 葡糖酸於含 P. putida YLFP14 及細菌性斑點病菌之混合液中，接種 試驗結果顯示，添加 0.5% 蛋白並無增加 P. putida 14 對細菌性斑點 病之防治效果，而添加 0.5%D- 葡糖酸則有助於 P. putida 14 對甜椒細 菌性斑點病之防治。P. putida YLFP14r 在甜椒葉表之存活受濕度影響很 大，高濕有助於螢光假單胞菌在葉表族群之維持， 而蛋白或 D- 葡糖 酸等營養物質之添加則有助於P. putida YLFP14r 於甜椒葉表上族群之增 加。 此外 YLFP14r 在 Hoagland''s 溶液及泥炭土中之存活測試顯示， 低溫環境 (12 ℃ ) 較高溫環境下之存活 (20 及30 ℃ ) 為佳。

Pseudomonas putida YLFP14 was selected to examine its ability tocontrol bacterial spot on leaves of sweet pepper caused by Xanthomonascampestris pv. vesicatoria. Pseudomonas putida YLFP14 could inhibit the growthof all the 27 tested strains of X. c. pv. vesicatoria on PDA medium. It didnot produce hydrogen cyanide and were resistant to CuSO4.5 H2O (500ppm) andformulated chemicals tribasic copper sulfate and tecloftalam. Carboxymethylcellulose and SILWETL-77 could improve the ability of cells of P. putidaYLFP14 to colonize on leaves of sweet pepper. To grow P. putida YLFP14 infermentor using molasses, yeast extract and soybean powder (MYS) as substrate,the population of P. putida YLFP14 could reach to 109 cfu/ml 30 hr afterincubation at 30 ℃. Cells of P. putida YLFP14r in the MYS-YLFP14 liquidculture harvested from fermentor survived up to 20 weeks without dramaticdecline from the initial population (109 cfu/ml) at 4 ℃, whereas thepopulation of P. putida YLFP14 in MYS- YLFP14 liquid culture decreased to 102cfu/ml 4 weeks after storage at 30 ℃. Disease index of bacterial spot onleaves of sweet pepper inoculated with the mixture of X. c. pv. vesicatoriaand P. putida YLFP14 or MYS-YLFP14 was significantly lower than that ofcontrol plants on which the plants were inoculated with X. c. pv. vesicatoriaonly. In addition, the disease index of bacterial spot on leaves of sweetpepper sprayed with cell suspension of P. putida YLFP14 or MYS-YLFP14 liquidculture before or after being inoculated with X. c. pv. vesicatoria was alsosignificantly lower than that of control. Utilization of carbon sources by 53strains of P. putida and P. fluorescens were examined in the Biolog GNMicroplate. The results (24hr reaction time) revealed that among the 95 carbonsources teated, all these 53 strains could utilize 18 carbon sources; andvaried in utilization of 64 carbon sources; in addition, they all did notutilize the other 13 carbon sources. The effect of peptone and D-gluconic acidon improvement of P. putida YLFP14 to control of bacterial spot of sweetpepper was determined. The results showed that D-gluconic acid but not peptonecould significantly enhance the ability of P. putida YLFP14 to control thedisease. Relative humidity(RH) is important factor for the survival of P.putida YLFP14r on the leaves of sweet pepper. Pseudomonas putida YLFP14 couldsurvive much better on the leaves at high RH(100%), but the population of P.putida YLFP14 decreased rapidly at low RH(70- 85%). The effect of peptone andD-gluconic acid on the population of P. putida YLFP14 on leaves of sweetpepper was determined. The results showed that both peptone and D-gluconicacid could increase the population of P. putida YLFP14 on the leaves of sweetpepper at high RH. Survival of P. putida YLFP14r in Hoagland''s solution andpeat moss was also examined. The results showed that P. putida YLFP14 survivemuch better at low temperature(12 ℃ ) than that at higher temperature(20 and30 ℃ ).