Investgation of Fusarium oxysporum f. sp. anoectochili, development of molecular marker and distribution in field
金線連莖腐病 ； F. oxysporum f. sp. anoectochili ； 病原型 ； 套袋式盆缽栽培 ； Anoectochilus stem rot ； F. oxysporum f. sp. anoectochili ； pathotype ； plastic bag cultivation method (PBCM)
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尖鐮孢菌 (Fusarium oxysporum) 是一種土棲性真菌，可引起多種作物的萎凋病，且遍佈於全世界，依其感染寄主的不同可區分為不同分化型 (forma specialis)，現今已有超過150種的分化型及生理小種有被紀錄。在台灣，由F. oxysporum f. sp. anoectochili (Foa) 引起之金線連莖腐病可依據其菌落形態區分為白色菌絲型cottony alba (CA) 及分生子座型sporodochial (S)，且依核醣體轉錄間隔區間 (IGS) 序列之差異可分成兩個獨立分子群。由於以往開放式栽培環境下造成金線連莖腐病發生相當嚴重，故現今農民多改採用塑膠袋套袋栽培法 (plastic bag cultivation method, PBCM) 減少莖腐病於種植期間之快速傳播。本研究重新於PBCM栽培環境下調查金線連病害相與發生率，調查結果顯示，套袋金線連有莖腐病、花腐病及葉斑病的發生，其中約有25%之金線連植株發生莖腐病，於冬季開花期有高達57%植株花梗出現花腐病徵。將罹病植株進行分離純化病原菌，結果顯示所分離之菌株仍以Foa為主，且形態與分子序列鑑定多數屬CA型菌株，少數為S型菌株，且CA型菌株多分離自於莖、葉、花梗等植物組織上，S型相對只於莖與葉部被分離到。病原性測試結果顯示，來自莖與葉部組織之CA型菌株毒力高於S型，然分離自花梗之CA型菌株之病原性最弱。本研究為建立快速鑑定並診斷金線連莖腐或花腐病之平台，本研究透過增幅Foa菌株之IGS全長與其他作物萎凋病菌比較其序列差異，設計出2-F/6-R及3-F/7-R兩組引子對，可個別鑑別CA與S 型菌株。於專一性測定，所設計之引子對無法增幅其他15種分化型作物萎凋病菌菌株、非病原性F. oxysporum及溫室常見之真菌(Penicillum屬、Trichoderma屬等真菌)。於靈敏度測試結果得知，兩組專一性引子對最低可偵測菌株DNA濃度為10-2 ng/ μl；此外嘗試混合兩組引子對同時增幅CA與S兩型菌株之可能性，結果得知當CA型與S型兩菌株的引子對以8: 2比例混合時，可同時偵測到CA型與S型。以新設計專一性引子於偵測溫室環境之病原菌存在，結果證實，CA 型菌株孢子懸浮於溫室空氣中及在植株體內偵測到之比例較S 型高；反之，S 型菌株在泥炭土及地面搜集沙塵存在之比例較CA 型高，而在蒐集之盆缽樣本中CA與S 型偵測到之比例約各佔一半，且使用過之盆缽皆可偵測到Foa菌株之存在。根據PCR偵測結果，病原菌潛在感染源可能主要來自於種植時所使用之盆缽，後續便以消毒處理盆缽探討對金線連種植之影響，結果顯示未處理過滅菌處理盆缽之處理組，莖腐病發生率最高，而經2000 ppm次氯酸鈣消毒後之盆缽，發生莖腐病之比例最低。綜合本研究結果，透過所設計之專一性引子對，得知造成田間莖腐病發生之主要感染源可能來自於盆缽之重複使用、病原菌孢子之空氣傳播或是低濃度之病原菌孢子殘存於未使用之泥炭土中，以次氯酸鈣消毒、滅菌器皿與栽培介質，加強田間衛生管理，可有效降低病害發生之比例。
Fusarium oxysporum (Fo) is the causal agent of Fusarium wilt disease in many crops and is well represented among the communities of soilborne fungi and in every type of soil all over the world. The pathogen has several specialized forms (known as forma specialis, f. sp.) according to the host it specifically infects. Based on the plant species and cultivars, Fo is classified into more than 150 formae speciales and races. Stem rot disease of jewel orchids (Anoectochilus formosanus) caused by F. oxysporum f. sp. anoectochili (Foa) could be separated into two colony types, cottony alba (CA) type and sporodochial (S) type, based on the morphology characteristics and the nucleotides polymorphism of intergenic spacer (IGS) region. Previous studies indicated that the Foa could disperse easily and cause serious yield loss throughout the production area in Taiwan. Therefore, the plastic bag cultivation method (PBCM) was been developed for reducing the Foa dissemination in the greenhouses. In this study, the field survey of the plant diseases on jewel orchids were investigated in PBCM greenhouses. The results indicated that 25% of jewel orchids were showed stem rot symptom and the flower rot symptom were also been observed with 57% of disease rate on flowers of jewel orchids. All the pathogens were been isolated and identified as the Foa. Among these isolates, the CA type were the dominant type of Foa isolates and could be obtained from stem, leaf and flower tissues; the S type only could be obtained from stem and leaf tissues. The pathogenicity tests indicated that the CA type isolates collected from stem and leaf tissues showed high virulence than S type isolates, but the CA type isolates collected from flower tissues were not. In order to develop a rapid diagnosis system of the jewel orchid diseases, the specific primers of 2-F/6-R and 3-F/7-R were designed successfully based on IGS sequence for identifying the CA and S type isolates, respectively. In addition, the two primer pairs (2-F/6-R and 3-F/7-R) showed highly specificity and could differentiate the target isolates from other formae speciales, non-pathogenic Fo and widespread fungal species such as Penicillium sp. and Trichoderma spp.. The sensitive tests revealed that the primers 2-F/6-R and 3-F/7-R could amplify the target fragment from as little as 10 pg (10-2 ng) template DNA. In multiplex PCR experiments, the two type primers mixed with the proportion of 8: 2 (CA+S type) could amplified the both target fragments of two type of Foa genomic DNA at the same times. These primers were then used to detect the CA and S type of Foa in environments, the results indicated that the CA type isolates were dominant in atmosphere and plant tissues samples; the S type isolates were dominant in soil and dust samples; all the reused pot samples could found the Foa pathogen, and both type of Foa could been identified and share the same rate. Among these PCR results, , the reused pots might be the major inoculation source of Foa in field. Therefore, the effect of sterilization methods on disease development of the Foa was been evaluated. The result indicated that the sterilization with 2000 ppm Ca(ClO)2 could reduce the stem rot disease effectively. According to these results, the pathogen colonized on the reused pots, Foa air-borne spores and low concentration of Foa existed in the unused peat moss might be the key factor in the disease cycle of the stem rot disease of jewel orchid. Following the good agricultural practices with the effective sterilization method could reduce the disease development in greenhouse.
生物農學 > 植物學