請用此 Handle URI 來引用此文件: http://hdl.handle.net/11455/31045
標題: 柑橘潰瘍病菌生物膜與致病性及枯草桿菌群 於其生物防治應用機制
Mechanisms of biofilm formation and patho- genicity in Xanthomonas axonopodis pv. citri and biocontrol by Bacillus subtilis group
作者: 呂冠民
Lu, Kuan-Min
關鍵字: Biofilm
生物膜
Two-component regulatory system
Pathogenicity
二元反應調控系統
致病性
出版社: 植物病理學系所
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摘要: 由Xanthomonas axonopodis pv. citri引起之柑橘潰瘍病 (citrus bacterial canker) 為影響世界柑橘生產最嚴重之細菌性病害,在美國以及中南美國家即將之列為檢疫病害,並花費不貲極力除滅,現行此病害之防治多仰賴銅劑及抗生素等化學藥劑,然已有報導指出發現抗藥性菌株。尤當今人之環境保護意識抬頭,與對安全農業之要求,發展創新、安全、有效之防治策略為時勢所趨。微生物群聚附著於物體表面形成膜狀構造,此泛稱為生物膜,已知生物膜之形成有助於微生物抵抗抗生素等不良環境,且此過程在多種微生物亦已被證實為致病、拮抗或共生作用之關鍵,因此藉由化學藥劑或生物製劑干擾或抑制生物膜形成為研發病害防治策略之嶄新標的。本研究目的即在釐清X. axonopodis pv. citri於柑橘葉表形成生物膜之分子機制及其與致病作用之關係,並嘗試應用拮抗性枯草桿菌作為柑橘潰瘍病管理策略,其中特別針對此病原與拮抗菌在葉表纏據形成生物膜之交互關係及拮抗性枯草桿菌與農藥混用之親和性進行探討,以期提供柑橘潰瘍病害管理之生物製劑與化學性農藥混合應用基礎,並降低農藥殘留及減少抗藥性問題產生。為瞭解柑橘潰瘍病菌生物膜形成機制,本研究中利用跳躍子進行菌株X. axonopodis pv. citri XW19基因體隨機突變,篩選生物膜形成能力明顯較野生型菌株為低之菌株,其中X. axonopodis pv. citri TPH1,其跳躍子插入使其喪失功能者,推測其為二元反應調控蛋白TCR,即為本研究中主要探討標的。根據序列比對分析結果顯示台灣所分離之X. axonopodis pv. citri XW19,其TCR基因序列與亦屬亞洲型潰瘍病原且已有基因體全長序列之X. axonopodis pv. citri 306菌株者具100%相同度。分析其轉譯蛋白序列亦與多種動、植物病原及環境微生物之二元反應調控蛋白具高相似性 (84-100%相同度),且亦具訊息接受 (signal receiver, REC) 特性及N端具DNA鍵結區域等高度保留序列。藉由比較親本及TCR突變與互補菌株於聚苯乙烯表面及墨西哥萊姆、臍橙與葡萄柚柑橘葉表生物膜形成能力與葉面纏據及存活能力,試驗結果顯示,TCR突變株其形成生物膜及纏存於柑橘葉表能力明顯低於親本菌株,而互補菌株此些能力則回復如親本菌株。另於墨西哥萊姆植株上之病原性測定結果亦發現,突變菌株所造成潰瘍病斑數明顯低於親本及其互補菌株。綜合上述結果證實此二元反應調控蛋白TCR在此病原於柑橘葉表形成生物膜、纏據並進而導致病害發生過程扮演重要角色。為瞭解此調控蛋白所調控之功能與上述過程之相關性,本研究分析了親本、突變及互補菌株之澱粉、脂質及卵磷脂等廣泛被報導與致病相關之胞外酵素活性,並利用反轉錄聚合酶鏈鎖反應測定hrpG、rfbC、rpfF、katE等致病基因之表現,結果顯示菌株XW19 TCR基因之突變並不影響上述胞外分解酵素活性,然在模擬細胞間質之培養基中,此二元反應調控蛋白TCR則可正調控rpfF基因之表現。由上述研究結果證實柑橘潰瘍病菌於葉表生物膜形成及纏存能力與潰瘍病徵表現呈正相關性,另邱氏 (2004) 研究中證實拮抗性枯草桿菌可抑制柑橘潰瘍病害發生,本研究中則針對枯草桿菌及潰瘍病原於葉表形成生物膜能力與抑制病害相關性進行探討,利用共軛焦雷射掃描顯微鏡配合綠螢光蛋白標定及吖啶橙 (acridine orange) 染色法觀察,結果顯示於接種潰瘍病原前一天噴灑枯草桿菌群菌株TKS1-1及WG6-14可明顯抑制潰瘍病原於墨西哥萊姆葉表生物膜形成及纏據能力,另葉表多由枯草桿菌群菌株所纏據,且枯草桿菌之應用亦可明顯降低單位葉表面積柑橘潰瘍病斑數,此結果證實枯草桿菌於柑橘潰瘍病害防治之應用潛力。因此本研究中針對常用分屬不同作用機制之殺蟲及殺菌劑,篩選與試驗中研發之拮抗性枯草桿菌生物製劑具親和性者,以供日後作為田間生物製劑與非生物藥劑混合應用參考依據。已完成測試之殺蟲劑包含:大滅松、新殺蟎、賽洛寧、益達胺及畢達本等;殺菌劑則包括:亞托敏、貝芬替、滅達樂、腈硫醌、待克利、四氯異苯腈等。結果顯示大部分所測試殺菌劑與殺蟲劑在推薦濃度下,並不會影響枯草桿菌群菌株之生長;但亦發現於添加濃度為250及500 ppm下待克利與四氯異苯腈有抑制枯草桿菌群菌株生長,而腈硫醌則會抑制柑橘潰瘍病菌生長,推測應為藥劑之劑型或作用點不專一所致。綜合上述試驗結果推論柑橘潰瘍病菌XW19於葉表形成生物膜及纏存能力為潰瘍病徵表現關鍵因子,而此過程可由二元反應調控蛋白TCR,藉由調控訊息傳遞分子DSF生合成基因rpfF路徑所調控。而篩選可抑制或影響柑橘潰瘍病原生物膜形成機制之微生物,如本研究中之拮抗性枯草桿菌之應用,或可提供柑橘潰瘍病害防治管理應用。
Citrus bacterial canker caused by Xanthomonas axonopodis pv. citri, is the most devastating bacterial disease resulted in great loss of citrus production worldwide. It is under quarantine and eradication program in the northern and southeastern Unite States. For control of the disease, copper bactericides and antibiotics are recommended, while strains resistant to these compounds has been found. The development of effective strategies for control of this disease is necessary. Microbes attaching to a surface is known as biofilm. Bacteria in biofilms allow them to survive in hostile environments. The development of biofilm has been demonstrated to play important roles in pathogenesis, antagonism or symbiosis. Thus strategies for disease control are developed by interference of biofilm formation. The objectives of this study are to understand the molecular mechanisms of biofilm formation on leaf surface by X. axonopodis pv. citri and the potential link to pathogenicity, and to investigate the efficacy of using antagonistic bacilli to interfere the biofilm formation by X. axonopodis pv. citri on surfaces of citrus leaves. In addition, the compatibility of fungicides and insecticides with Bacillus biopesticides was assessed. The information gained will provide insights for mixture application of chemical and bio-pesticides. To understand the mechanisms of biofilm formation by X. axonopodis pv. citri XW19, transposon mutagenesis was used and mutants that were defective in biofilm formation in polystyrene microplate were selected for further study. One mutant with mutation in two-component regulatory protein, TCR, was the target in this study. The tcr nucleotide sequences of X. axonopodis pv. citri strain XW19 shared 100% identity with that, locus tag XAC1284 of the strain 306. Comparison of translated protein sequences, TCR in strain XW19 shared 84-100% identity with two component regulatory proteins in various animal and plant pathogens and environmental microorganisms. In addition, XW19 TCR exhibited the REC conserved domain and the DNA-binding motif at N-terminus. Biofilm formation and colonization by TCR mutant was significantly decreased in polystyrene plates and on leaves surfaces of Mexican lime compared to that by the parental strain. In addition, TCR mutant was found to be compromised in its ability to cause canker lesions. Providing pTCRF1, pTCRF2 in trans in TCR mutant restored all the phenotypes tested to those in the parental strain. The results suggested that TCR plays important roles in biofilm formation, colonization on leaves surfaces of citrus and the development of canker disease. To determine the downstream phenotypes and genotypes that TCR regulated, the virulence-related extracellular enzymes such as amylase, lipase and lecithinase and mRNA expression of hrpG, rfbC, rpfF and katE were assessed. Data indicated that TCR did not regulate the production of tested enzymes, while positively controlled the expression of rpfF in XVM2 medium which mimics cytoplasmic fluids in planta. To determine the effect of antagonistic Bacillus subtilis group on biofilm formation by X. axonopodis pv. citri harboring green fluorescence protein expressing plasmid, biofilm formation by these strains were observed by confocal laser scanning microscopy. The data indicated that biofilm development by X. axonopodis pv. citri on leaves surfaces of Mexican lime was reduced with the spray inoculation of antagonists one day prior to inoculation of the pathogen compared to that without treatment, while biofilm development by bacilli was scattered around cells of X. axonopodis pv. citri. Data from pathogenicity assay also indicated that the application of antagonists on citrus leaves resulted in compromised symptom development, suggesting the potential of using antagonistic bacilli as biocontrol agents for control of citrus bacterial canker. To provide insights for the mixture application of biopestides and chemical pesticides, the comparability of fungicides and insecticides with Bacillus species were determined. Results indicated that most of the tested pesticides including azoxystrobin, carbendazim, metalaxyl, bromopropylate, cyhalothrin, dimethoate, imidacloprid and pyridaben did not affect the growth and antagonistic activity of Bacillus strains TKS1-1 and WG6-14 and the growth of X. axonopodis pv. citri, except for those including chlorothalonil, difenoconazole and dithianon. In conclusion, biofilm formation and epiphytic colonization and survival are important for canker development in X. axonopodis pv. citri. The processes are controlled by two-component regulatory protein TCR in X. axonopodis pv. citri XW19 through positive regulation of rpfF transcription, which is required for biosynthesis of diffusible signal factor. Application of antagonistic bacilli on citus leaves resulted in interference of biofilm formation by X. axonopodis pv. citri which may be a feasible way for the disease control of citrus bacterial canker.
URI: http://hdl.handle.net/11455/31045
其他識別: U0005-2108201122334700
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-2108201122334700
顯示於類別:植物病理學系

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