台灣四種木本植物癌腫病之病原分離鑑定與菌系分析

Isolation, identification, and characterization of four woody plant-infecting Agrobacterium tumefaciens in Taiwan

梁瀚心(Han-Sin Liang)

指導教授 ： 陳滄海

國立屏東科技大學/農學院/植物醫學系所/碩士(2009年)

https://doi.org/10.6346/NPUST.2009.00318

摘要

由 Agrobacterium tumefaciens 所引起的細菌性癌腫病常發生於植物的根冠、樹幹或枝條修剪處，其寄主範圍相當廣泛，至少可危害 138 科、588 屬、1,193 種以上的植物。近年來在林務局東勢林區管理處栽培之苦楝 (Melia azedarach L.)、台北的榕樹 (Ficus spp.)、南投梅峰農場的水蜜桃 (Prunus persica L. B.)、及彰化的玫瑰 (Rosa hybrida) 樹幹與枝條上發現疑似癌腫病之病徵。將患部腫瘤帶回後實驗室進行表面消毒，並切取內部組織經研磨後，以無菌棉花棒沾取研磨組織液塗抹於胡蘿蔔肉質根切片上，將胡蘿蔔切片置於保濕的培養皿中，於 25℃ 培養箱中培養 21 天以上，待胡蘿蔔形成層處長出淡綠色之癒傷組織後，切取部分癒傷組織進行研磨後再將萃取液在 nutrient agar (NA) 培養基上以劃線平板方式進行分離。分離所得之細菌在 NA 培養基上呈現圓形突起、光亮且不透明的白色菌落。從單一菌落分離的細菌為革蘭氏陰性、好氣桿菌，經 Biolog system 鑑定為 Agrobacterium tumefaciens，相似值介於0.23-0.75。分別將純化後的菌株以針刺接種至約 6 週大的健康番茄 (Solanum lycopersicum L., 農友 301 品種) 及向日葵幼苗 (Helianthus annuus L., 日輝品種) 莖部，可於接種5到7天後在接種處出現淡綠色的不規則突起，14 天後形成明顯的腫瘤，而接種無菌水的植物則無病徵，確定不同寄主來源的A. tumefaciens 菌株對番茄及向日葵均具有致病力。將採自苦楝水蜜桃的菌株分別接種至約1 m高的苦楝與水蜜桃植株莖部，接種處
在2 到 3 週後出現增生細胞，隨著接種時間增加，病徵轉變為表面粗糙之黃褐色腫瘤，而所有接種無菌水的對照植株皆無病徵產生。此外，將苦楝與水蜜桃接種植株的腫瘤剪下後，再將研磨後的組織液塗抹於胡蘿蔔切片上，3 週後可在胡蘿蔔形成層處長出癒傷組織，再從這些癒傷組織內可分離到與接種菌株相同的細菌，完成柯霍氏法則。根據以上的實驗結果，證實引起苦楝與水蜜桃細菌性癌腫病之病原菌為 A. tumefaciens，此為 A. tumefaciens 在台灣可感染苦楝與水蜜桃並引發細菌性癌腫病的首次研究報導。另外，本研究針對苦楝、水蜜桃、榕樹與玫瑰等分離菌株的生理生化特性、碳素源利用及 T-DNA 上的 6b gene 序列與其他寄主來源的 A. tumefaciens進行分析與比對，得知苦棟的菌株為 biovar 1，其 6b 的胺基酸序列與 GenBank database 中 octopine-type Ti plasmid 的 6b 序列相似度達 91% 以上，水蜜桃菌株為 biovar 1，但其6b 的胺基酸序列與 nopaline-type Ti plasmid 的 6b 序列相似 (98%-100%)，至於榕樹與玫瑰的分離菌株則均屬 biovar 3，其6b 的胺基酸序列亦與 nopaline-type Ti plasmid 的 6b 序列相似 (98%-100%)。根據本研究的結果得知分離自苦楝、水蜜桃、榕樹與玫瑰等木本植物的 A. tumefaciens 具有不同的生化特性與不同的 6b 基因序列，推測其應為不同的菌株，但菌株的來源及其感染途徑則有待進一步分析。

關鍵字

癌腫病 ； 農桿菌 ； 分離 ； 鑑定 ； 6b基因

並列摘要

Bacterial crown gall, often found on the root crown, stem, shoot, and pruned parts, is caused by Agrobacterium tumefaciens that can infect more than 1,193 plant species worldwide. In Taiwan, bacterial gall-like symptoms were recently found on the stems and branches of china berry tree (Melia azedarach L.) in Dongshih, ficus (Ficus spp.) in Taipei, peach (Prunus persica L. B.) in Nantou, and rose (Rosa hybrida) in Changhua. The gall tissues were surface-sterilized and ground in SCPAP buffer, and the resultant plant extracts were smeared onto carrot slices to detect if the slices can develop callus. At 21 days post inoculation (dpi), the developed callus was excised for bacterial isolation on nutrient agar (NA) medium, and a gram-negative, aerobic bacterium consistently formed circular, white colonies with convex elevation on NA was isolated. Several strains of the bacterium from different plant species were characterized by biochemical analyses and the Biolog system and confirmed the isolated strains as A. tumefaciens with similarity values of 0.23-0.75. Virulence assays were performed by prick-inoculating bacterial colonies into the stems of greenhouse-grown, disease-free seedlings of tomato (Solanum lycopersicum L. cv. Known-you
301) and sunflower (Helianthus annuus L. cv. Sunbright). At 5 to 7 dpi, the inoculation sites developed small, light green spongy spots that were gradually transformed into prominent galls at 14 dpi, whereas control plants inoculated with sterile water remained symptomless, indicating that the strains of A. tumefaciens originated from different plants are virulent to tomato and sunflower. Koch's postulates were fulfilled by prick-inoculating bacterial colonies into the stems of greenhouse-grown, disease-free, 1-meter-tall seedlings of china berry tree or peach tree, and symptoms were evident at 2 to 3 weeks post inoculation on all bacteria-inoculated plants but not on sterile water-inoculated ones. Elicited gall tissues were extracted and smeared onto carrot slices as described above, and the bacteria isolated from the carrot calli at 21 dpi showed morphological and biochemical similarities to the ones used for inoculation. Based on the results, the causal agent of bacterial galls on china berry tree and peach tree, the two new bacterial gall diseases reported in Taiwan, is confirmed to be A. tumefaciens. In addition, A. tumefaciens strains isolated from china berry tree, peach, ficus and rose were characterized by the physiological and biochemical characteristics, carbon source utilization, and the nucleotide sequences of 6b residing in T-DNA region. The results revealed that the A. tumefaciens strain of china berry tree is biovar 1 harboring a 6b gene whose deduced amino acid sequence is 91% similar to the 6b of octopine-type Ti plasmid found in GenBank database; the A. tumefaciens strain of peach tree belongs to biovar 1 with a 6b protein that shares 98% to 100% similarity to the 6b of nopaline-type Ti plasmid; the strains of rose and ficus belong to biovar 3, and the amino acid sequences of 6b are 98% to 100% similar to the 6b of nopaline-type Ti plasmid. In Taiwan, the A. tumefaciens strains isolated from different woody plants have different biochemical characteristics and 6b sequences, indicating the bacterial gall disease of woody plants might be elicited by different strains of A. tumefaciens, the questions of the disease transmission and the source of the pathogens remain to be elucidated.

並列關鍵字

Crown gall disease ； Agrobacterium tumefaciens ； isolation ； identification ； 6b gene

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