胡瓜露菌之繁殖與保存

Propagation and preservation of pathogen of cucumber downy mildew

胡瓜露菌（Pseudoperonospora cubensis）屬於絕對寄生菌（Obligate parasite; biotrophs），無法用人工培養基培養與保存，但將露菌之單一孢囊挑起，置於葉圓片葉背之小水滴中，便能純化培養並且產生孢囊。為了大量繁殖胡瓜露菌，將露菌孢囊懸浮液噴佈接種於葉圓片葉背上，於16℃、20℃培養7–10天或於24℃培養7天之處理即形成較大量的孢囊（1.1–2.5×105 sporangia/disc）與較高的孢囊發芽率（80.3–97.7%），而12℃之處理在10–14天才有較大量孢囊（2.2–4.6×105 sporangia/disc）與較高的孢囊發芽率（85.3–91.7%）；8℃與28℃之處理在試驗期間孢囊量明顯較少，32℃之處理在試驗期間內皆未見產生孢囊。在切離葉接種相同病原濃度（5 sporangia/cm2）的試驗中，在12℃培養7–14天、16–24℃培養7天具有高發芽率（86.3–97.0%）的孢囊明顯可造成較高的發病度（83.8–91.3%），在12℃培養22天、16–28℃培養14天具有低發芽率（10.0–75.0%）的孢囊造成較低的發病度（12.5–64.5%），而培養在16–28℃、22天發芽率為0%的孢囊則無法造成病害。以葉圓片培養之新鮮孢囊在4–32℃水中皆能間接發芽產生游走子，適溫為8–24℃（最適溫為20℃），在處理30分鐘即有部分孢囊發芽，在2小時即有最大發芽率85–95%。無論是新鮮或經由乾燥處理孢囊保存在25℃、14天，4℃、60天或-30℃、120天後則無發芽能力，若保存在液態氮中1–120天，新鮮孢囊失去發芽能力，但乾燥處理的孢囊發芽率皆仍維持在20.7%以上。在保存於液態氮的試驗中，在葉圓片上的新鮮或乾燥處理24小時的孢囊置於含有無菌水、10%甘油與30%甘油的冷凍保存管內皆無法存活，然而乾燥處理0、2–8、24小時及3、7、14–28天的孢囊在無添加任何介質的冷凍保存管內保存在液態氮中1天後，發率芽分別0%、3–9.0%、20%、3.7%、0%與0%。將在繁殖在葉圓片20℃培養7天的胡瓜露菌孢囊，連帶葉圓片乾燥24小時後，置於冷凍保存管內，可於液態氮中存活至少2年。

"Single sporangium of Pseudoperonospora cubensis, causal agent of downy mildew of cucumber, was isolated from colonies on disease leaves and cultured on the leaf discs of cucumber for purification and production of sporangia. In order to culture the pathogen, sporangium suspension should be sprayed on abaxial leaf surface. When sporangia were inoculated on the leaf discs (1.5 cm in diam.) and incubated at 16 or 20℃ for 7–10 days or 24℃ for 7 days, the downy mildews produced 1.1–2.5×105 sporangia/disc with high germination rates of 80.3–97.7%. When the inoculated leaf discs were incubated at 12℃ for 10–14 days, the sporangia number could be 2.2–4.6×105 sporangia/disc with the germination rates of 85.3–91.7%. The sporangia number did not increase and the germination rates were low when the inoculated leaf discs were cultured at 12–24℃ for more than 14 days. The cultures produced fewer sporangia at 8℃ or 28℃ and no sporangia at 32℃. In the detached leaf inoculation test with same inoculum density (5 sporangia/cm2), the sporangia with high germination rates (86.3–97.0%) that produced from the cultures at 12℃ for 7–14 days and 16–24 for 7 days obviously caused high disease severity (83.8–91.3%); whereas, the sporangia with low germination rates (10.0–75.0%) from the cultures at 12℃ for 22 days and 16–28 for 14 days caused low disease severity (12.5–64.5%). Moreover, the sporangia with no germination from the cultures at 16–28℃ for 22 days could not cause disease. Fresh sporangia of P. cubensis could germinate indirectly to produce zoospores when immerged in sterilized distilled water at 4–32℃. At the suitable temperature 8–24℃ (optimal at 20℃), the fresh sporangia had the higher germination rate 85–95% 2 hours after water immerging. Both the fresh or dried sporangia on the leaf discs lost germination ability when preserved at 25℃ for 14 days, 4℃ for 60 days or -30℃ for 120 days. After being preserved in liquid nitrogen for 1–120 days, the fresh sporangia could not germinate, but the 24-hour-dry sporangia still have more than 20.7% of germination rate. In the trial of preservation in liquid nitrogen, the fresh or dried sporangia could not germinate after being preserved with sterilized distilled water, 10% glycerol or 30% glycerol. However, when sporangia on leaf discs were respectively dried for 0 , 2–8, 24 hours, as well as 3, 7 and 14–28 day and then placed in cryo-vials for preservation in liquid nitrogen, they had the germination rate of 0%, 3–9.0%, 20%, 3.7%, 0% and 0%, respectively."