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1、粗肋草細菌性軟腐病病原菌之鑑定及其病害防治之研究 Etiology and control of aglaonema bacterial soft rot研究生：馮靖廷 Feng, Ching-Ting指導教授：何婉清 Ho, Wang-Ching【摘要】2003年6月於屏東縣內埔鄉中林村金獅園藝栽培場，首度發現自泰國進口之”彩虹(Sithiporn aglaonema)”品種粗肋草植株發生嚴重之軟腐病徵，由各發病植株葉片上分離出7株細菌，在完成柯霍式法則(Kochs postulates)後，經modified crystal violet pectate(CVP)選擇性培養基、馬鈴薯薯片及
2、白菜菜梗接種等步驟，進行病原細菌初步鑑定，確認所分離出之細菌皆為Pectobacterium軟腐細菌，再經傳統生理生化測定並配合專一性引子對5A/5B，以聚合酶連鎖反應(PCR)進行種的鑑定，結果顯示所分離出之7株軟腐細菌皆為P. chrysanthemi。上述分離的7株P. chrysanthemi菌株，分別以ASR1至ASR7命名，經接種至”彩虹”粗肋草，證實ASR3菌株的致病力最強，接著並以ASR3菌株接種在彩虹品種粗肋草的切離葉上以評估離葉接種法進行品種間抗病篩選之可行性，結果顯示彩虹粗肋草切離葉於接種處會產生典型的軟腐病徵，並產生與植株活體葉片接種相同的病徵，且病勢進展的速度也幾乎完
3、全一致，說明離葉接種法可做為粗肋草葉部病害抗病篩選的替代方法。溫度可影響粗肋草細菌性軟腐病之發生，20至32皆能造成細菌性軟腐病的危害，其中以28為最適發病溫度，當接種濃度在104 cfu/ml以上時，即能快速發病，病勢會隨著接種濃度之增加而越趨嚴重。以分離之7株軟腐細菌菌株進行其對不同收穫後農產品之致腐能力測試，結果顯示粗肋草軟腐細菌除對大蒜無致腐能力外，對其他供試農產品組織皆有不同之致腐能力。利用ASR3軟腐菌株進行天南星科觀賞植物之抗病篩選時，發現白鶴芋、合果芋及黃金葛等三種天南星科植物為高感病種類，而火鶴、白花海芋及黛粉葉星光、綠雪與白玉三品種則對本病原菌免疫，其他供試之天南星科植物則
4、有不同程度之感病性。進一步以ASR3菌株進行不同粗肋草品種之抗病篩選試驗，結果顯示各品種皆能感病，但呈現不同之感病性，其中以箭羽最為抗病，白馬與雪后為中抗病品種，其餘供試之15種品種則多為中感與高感病品種。測試不同寄主上分離之P. chrysanthemi菌株在不同寄主間之交互感染能力時，結果顯示粗肋草軟腐菌株ASR3較文心蘭上分離之OS2及蝴蝶蘭上分離之OS2-CHU菌株的致病力為強，其中ASR3能同時感染粗肋草、蝴蝶蘭及文心蘭，而OS2及OS2-CHU只能交互感染對方之寄主，對彩虹粗肋草則無致病性，顯示粗肋草軟腐細菌有較強的致病性。以濾紙圓盤法評估化學藥劑對軟腐細菌ASR3之抑菌效果時顯示
5、，鏈黴素類之抗生素如鏈黴素、鏈土黴素、鏈四環黴素與多保鏈黴素等對本病原細菌皆有抑制效果，而供試之銅劑類藥劑如嘉賜銅、氫氧化銅與三元硫酸銅則對本菌皆無抑制效果。進一步以離葉接種法進行藥劑的防治效果評估，發現以多保鏈黴素的預防效果最佳，而供試之四種化學藥劑則均無明顯的治療效果。利用玻璃紙抗生法篩選出能產生抗生物質並抑制軟腐細菌繁殖之木黴菌(Trichoderma spp.)菌株，並以同樣方法測試螢光假單胞菌Pseudomonas fluorescens OFP7菌株與商品化枯草桿菌混合製劑(Bacillus subtilis broth)是否能產生抑制軟腐細菌生長之抗生物質，結果顯示本研究室收集之
6、16株木黴菌菌株及供試之拮抗細菌，包括木黴菌之T1、T4、T5、T6、T13、T14、T17及螢光假單胞菌OFP7等菌株皆具備產生抗生物質之能力，其中以T1菌株之拮抗效果最佳。再以離葉接種法測試上述拮抗微生物對粗肋草細菌性軟腐病之防治效果，結果顯示木黴菌T1菌株之培養過濾液及孢子懸浮液對細菌性軟腐病並無明顯的防治效果，而拮抗細菌OFP7及商品化枯草桿菌皆能有效預防軟腐病之發生，但若於病害發生後再施用上述之拮抗菌，則無法有效減緩軟腐病病勢之進展。關鍵詞：粗肋草(Aglaonema spp.)、細菌性軟腐病、Pectobacterium chrysanthemi、抗病篩選、拮抗菌、木黴菌(Tric
7、hoderma spp.)、螢光假單胞菌(Pseudomonas fluorescens)、枯草桿菌(Bacillus subtilis)、預防、治療【Abstract】In 2003, soft rot symptoms on Sithiporn aglaonema (Aglaonema tricolor A. nitidium) leaves were found in a nursery at Neipu, Pingtung, in Taiwan. 7 isolates of bacteria from soft rot aglaonema tissues could cause the
8、 typical soft rot symptoms both on aglaonema plants and the detached aglaonema leaves by inoculating the bacterial suspensions of those 7 isolates with a concentration of 108 cfu/ml on the carborumdum wounded tissues. No symptoms on control plants and leaves were showed when they were inoculated wit
9、h sterilized distilled water. Bacteria could be reisolated from the inoculated tissues that fulfilled the Kochs postulated. All 7 isolates of the bacteria were proven belong to soft rot Pectobacterium, due to they were negative for gram stain. Could depredated the pectate on CVP medium and could cau
10、se soft rot symptoms on potato slices and Chinese cabbage petioles. According to Dickey & Kelman and Goto & Matsumoto, the physiological characters of these 7 isolates matched the characters of P. chrysanthemi. The primer pair 5A/5B also could further confirm that all isolates were P. chrysanthemi b
11、y polymerase chain reaction (PCR). All isolates were, therefore, named as ASR1 to ASR7. ASR3 was proven to be the most virulent isolates, after the detached leaves were dipped in a bacterial suspension, ASR3 was, therefore, chooser to run the further experiments. The disease progresses of ASR3 were
12、parallel on wounded aglaonema plant and wounded detached aglaonema leaves. No disease would happen on unwounded plants and detached leaves. Soft rot of aglaonema could happen between 24 to 30 at a inoculums concentration of 104 cfu/ml and above, the higher of inoculums concentration, the faster of t
13、he disease progress, the disease progress reached optimum at 28 with a inoculums concentration of 107 cfu/ml. The postharvest products, the wounded detached leaves of Araceae and the cultivars of aglaonema were inoculated with ASR3 with a concentration of 108 cfu/ml and incubated at 30 for 5 days to
14、 evaluating the rotten ability of ASR3 on product, and for disease resistant screening of plants to soft rot. Results showed that ASR3 could macerated and rotted the storage root of carrot, radish, and sweet potato, cabbage petiole, corm of taro, leaf petiole of celery, onion slice, eggplant fruit,
15、and leaves of green onion, but not rotted the bulb scales of garlic. Spathiphythum sp., Syngonium sp., and Epipremnum sp. of Araecae were highly susceptible and Anthurium sp., Diffenbachia cv. Star Bright, cv. Superba, cv. Camilla and Zantedeschia aethiopica were immune to soft rot. Among aglaonema,
16、 cv. Curtisii, cv. White Tip and cv. Snow queen were resistant or lower susceptible to ASR3. P. chrysanthemi ASR3 could cause the soft rot of Oncidium sp. and Phalaenopsis sp., but the P. chrysanthemi from Oncidium sp.(OS2) and Phalaenopsis sp.(OS2-CHU) could not infect aglaonema. It was proven that ASR3 was more virulent then OS2 and OS2-CHU. Results of filter paper disc method used on the screening test of agrochemicals showed that all three copper
