| 摘要： 番石榴( Psidium guajava Linn )，為台灣重要果樹之一，其於生長期中，遭受許多植物病原菌之危害，其中尤以番石榴立枯病最為嚴重。番石榴立枯病於1926年首先由黑澤英一(E. Kurosawa)在台灣報導，其後經黑澤英一與澤田兼吉(K. Sawada)分離及鑑定，確定由不完全菌所引起，並命名為Myxosporium psidii Sawada et Kurosawa。 本病害為台灣本土性病害，除南非(Guava wilt disease)與印度(Sunken brown discolouration of guava)有類似病害的報導外，在其它番石榴分佈國家中，並無此病害之發生。為釐清該病原菌之分類地位，本研究首先針對其菌落與產孢形態及18s rDNA和ITS等輔助分子分類依據，進行分析。結果顯示，其產孢方式至少有三種，一為短橢圓形孢子，成鏈著生於分枝之分生孢子柄上；二為長橢圓形孢子，著生於不分枝之分生孢子柄上；而第三種產孢形態，孢子為卵圓形，成鏈著生於不分枝之分生孢子柄上。18s rDNA與ITS區域之增幅、選殖、定序及親緣分析之結果顯示，其與子囊菌肉座菌目( Hypocreales )中的菌株，具有高度之相似性。而分析我國主要栽培區分離菌株之ITS區域序列後發現，所有菌株之序列完全相同，無任何差異存在。進一步ITS區域序列比對發現，七株Penicillium sp.之不同分離株，ITS序列之相似度高達95%以上，其中三株分離株之ITS序列甚至完全相同，進一步查詢其來源，發現為南非Guava wilt disease之病原菌分離株。另外，由野生番石榴植株表面，分離到10株具有不同程度拮抗效力的細菌菌株，經鑑定結果，一株為Paenibacillus lentimorbus，二株為Bacillus licheniformis，並皆具產生幾丁質分解酵素(chitinase)之能力，其餘七株皆為Bacillus subtilis。利用掃描式電子顯微鏡觀察，拮抗細菌P. lentimorbus TP-2會造成M. psidii菌絲變形，而B. licheniformis TP-9則會造菌絲變形與孢子崩解之現象。在四種不同營養成份之培養基進行對峙試驗時，其中以P. lentimorbus TP-2在四種培養基上，對所有供試植物病原真菌的菌絲生長皆具有不同程度之抑制效果，並且以稀釋10倍之馬鈴薯葡萄糖培養基，所表現之抑制能力最佳。而以M. psidii與Magnaporthe grisea進行孢子發芽抑制試驗顯示，除B. licheniformis TP-9無明顯抑制效果外，P. lentimorbus TP-2及B. subtilis TP-7與TP-8皆具有不同程度之抑制效果，其中亦以P. lentimorbus TP-2抑制效果最佳，可降低M. psidii與M. grisea之孢子發芽率，分別達22%和2.5%。以光學顯微鏡觀察發現，被抑制發芽之孢子具有原生質顆粒化之現象，縱使有些孢子可以發芽，但其發芽管及菌絲有膨大且外壁不平滑之不正常情形。另一方面，應用M. psidii pXH9轉形菌株，進行生物防治技術之開發上，吾人成功地利用基因槍轉殖系統將帶有Cryphonectria parasitica dsRNA virus之 infectious cDNA的質體pXH9轉殖進入M. psidii中，並共獲得36株具有Hygromycin B抗性之轉形株，其中二株轉形株具有形態異常之情形，包括菌落顏色改變、菌絲稀疏與產孢量減少。進一步以PCR與南方雜合分析，確定pXH9已插入此二株轉形株之染色體中，但是否dsRNA存於此二轉形株中，則有待進一步之實驗分析。Guava (Psidium guajava Linn), a member of Myrtaceae, is one of the most favorite and important fruit in Taiwan. Many pathogens cause diseases on guava, however guava Myxosporium wilt or guava Li-Ku-Bin is the most devastating disease in the country. The disease was first reported by E. Kurosawa in Taiwan in 1926. Subsequently E. Kurosawa and K. Sawada isolated and identified the causal agent as a Deuteromycetous fungus, and named the fungus Myxosporium psidii Sawada et Kurosawa. The disease is indigenous, and has not been reported in other guava growing countries except in Southern Africa (Guava wilt disease) and India (Sunken brown discoloration of guava). To clarify the taxonomic status of the fungus, we isolated and reexamined its morphological features. Microscopic observation revealed that the causal fungus has three types of conidial sporulation. The first type of conidia are in short elliptical shape, and are produced in chains on the branched conidiophores; the second type of conidia are in long elliptical shape, and are produced on the unbranched conidiophores; and the third type of conidia are in oval shape, and are produced in chains on the unbranched conidiophores. Additionally, 18s rDNA and ITS sequence were used for phylogenetic study. The results indicated that M. psidii is more closely related to the fungi within the Order Hypocreales in the Ascomycetes and no sequence variation was observed in the ITS region among 10 isolates collected across the island. Blast search analysis of M. psidii ITS sequence found that top seven scorers are “Penicillium” species isolated from guavas with wilt disease in South Africa. The sequence similarity is greater than 95% when compared with M. psidii, and three of them indeed contain exactly identical sequences as M. psidii. The ultimate goal of this study is to develop novel control techniques for guava Myxosporium wilt. To achieve this goal, ten bacterial isolates (TP-1 to TP-10) with different antagonistic against M. psidii were isolated. By analyzing the fatty acid profile, TP-2 is identified as Paenibacillus lentimorbus, TP-9 and TP-10 are identified as Bacillus licheniformis with chitinase activity, and the others are all B. subtilis. Observation under the scanning electron microscope showed that P. lentimorbus TP-2 and B. licheniformis TP-9 both cause M. psidii hyphae deformed, and B. licheniformis TP-9 also results the lysis of conidia. Dual culture studies on four different media found that P. lentimorbus TP-2 displayed good but variable antagonistic activities against all plant pathogenic fungi tested. However, 10-fold diluted PDA showed the strongest effect. Spore germination tests also revealed that P. lentimorbus TP-2 and B. subtilis TP-7 and TP-8 are capable of inhibiting spore germination, and the germination rates of M. psidii and Magnaporthe grisea spores were reduced to 22% and 2.5% by P. lentimorbus TP-2. Vacuolation and granulation in the cytoplasm of conidia or hyphae were found and swelling or irregular shapes of hyphae were also observed. The other non-chemical control approach is to develop the hypovirulence strain in M. psidii using Cryphonectria parasitica dsRNA infectious cDNA plasmid pXH9. Totally 36 hygromycin resistant transformants were obtained by biolistic transformation. Two of them were found to produce less conidia, sparse mycelia and abnormal color of colony. Studies using PCR and Southern blot analysis confirmed that pXH9 is stably integrated into the chromosome of these two M. psidii transformants. Further experiments will be required to determine whether the dsRNA is present in these transformants. |
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