松材線蟲新內部寄生菌ESTEYA VERMICOLA 之病原學,生理學及微細構造之探討(3/3)

Esteya vermicola 為松材線蟲之內部寄生性真菌天敵，具生物防治松材線蟲 之潛能，為探究E. vermicola 之生物防治機制，需瞭解其黏著、侵染松材線蟲之 致病過程及影響菌株生長、繁殖之生理、環境因子。在病原學之顯微鏡檢顯示， E. vermicola 產於瓶梗上之新月型黏性孢子具內生孢子，略為凹陷之單邊具黏著 層，可黏附至游經之線蟲體表，經過約18 小時後孢子發芽，源生於內生孢子之 侵入釘侵入穿透線蟲體壁，之後侵入釘膨大形成球形、次球形之囊狀體，後者脫 落釋放至蟲體體腔，成長為紡錘形，節狀同化菌絲，於蟲體內生長、分枝、蔓延。 黏著侵染後約48 小時，線蟲即瀕臨死亡，菌絲突破線蟲體表直接產孢，或形成 營養菌絲，向外生長再產生黏性孢子，並可黏著其他線蟲；而黏著之發生與孢子 是否著生於產孢梗上關係密切，一旦孢子脫落，在空間上就較不易進行黏著。多 數情況下，液體培養會以出芽方式產生大量芽生孢子，僅於特定條件下可產生黏 性孢子，但由於相對產量太低，未觀察到有效黏附於線蟲之情形。而芽孢移殖於 養分貧瘠之水洋菜培養基上，則可發芽產生大量之黏性孢子。芽孢亦能於松材上 定殖、生長，若將芽孢引入樹體，待其殖據後於樹體木質部中應可產孢黏附松材 線蟲，或可收生物防治之效。此外，在測試E. vermicola 僅對地上部之植物寄生 性線蟲：Aphelenchoides besseyi、Aphelenchoides sp.及Bursaphelenchus xylophilus； 此外，E. vermicola 之生長會受到一些殺菌劑之抑制，不過多為保護型，不會滲 入植株及在植物內移行，對於灌入樹體內之芽孢，應不至於造成不利影響，而對 生物防治效益發生衝擊。但殺線蟲劑Century 對E. vermicola 之抑制效應顯著， 故於防治松樹萎凋病時應避免同時使用。More recently, a new endoparasitic fungus, Esteya vermicola, has been isolated from naturally infected pinewood nematode, Bursaphelenchus xylophilus, retrieved from a wilted pine tree in Yangmingshan, Taipei City. E. vermicola possesses a potential for biocontrol of the pinewood nematode to alleviate the devastating pine wilt disease. However, fully understanding of E. vermicola , particularly in the aspects of pathogenesis, physiological characters, and ecological adaptations, appears to be compulsory, and merit for study. Ultrastructural studies show that the lunate conidium harbors an ovoid endospore, and the slightly indented side is coated with an adhesive mucilage. After adhesion to the nematode cuticle for 18 hours or more, the conidium germinates, an infection peg derived from the endospore penetrates the nematode, and forms submuscular vesicles, which later separate from the peg and flow in the body cavity of the prey. The vesicles grow, ramify, become cylindrical, septate, branched assimilative hyphae, consume and destroy the host organ and tissue, and lead to the death of the host in 48 hours. After nematode died, the trophic mycelium protrudes the cuticle, and forms a new crop of flask-shaped conidiophore and adhesive lunate conidia driectly, or generates mycelium, which grows and sporulates to form adhesive conidia. The topographic position and angle of the lunate conidium seems to be crucial for attachment to nematode. The drop-off conidia seem incapable to adhere to nematode. E. vermicola produced tremendous number of blastospores, but a trace amount of adhesive conidia in submerged culture conditions. However, a large number of adhesive conidium produced when subculture the balstospores to nutrient poor water agar. The blastospores also showed substantial growth and sporulation when inoculated onto autoclaved pinewood blocks, implicating the potential for biocontrol of pinewood nematode. This viewpoint was further strengthened by its specific infectivity to leaf and stem nematodes, i.e., Aphelenchoides besseyi, an Aphelenchoides sp., and B. xylophilus. Some protective fungicides and a nematicide, Century, are inhibitory to the growth of E. vermicola. Therefore, the coapplication of these pesticides, particularly, Century, should be avoided