Xanthomonas屬引起之細菌性斑點病為茄科作物上之重要病害，此類病原菌可分為Xanthomonas euvesicatoria (Doidge) Jones et al.、X. vesicatoria (Doidge) Vauterin et al、X. perforans Jones et al.及X. gardneri (Sutic) Jones et al.等四種，在台灣X. perforans引起之番茄細菌性斑點病於2010年首次被報導，但X. perforans之檢測技術則尚未建立，本研究利用X. perforans之序列資料與其他Xanthomonas屬之病原細菌比對後，以hpaF基因序列設計引子對HpaF-f/r，HpaF-f/r對供試之X. perforans菌株可增幅300 bp之專一性條帶，其靈敏度可達50 pg，HpaF-f/r同時可應用於檢測田間之番茄罹病組織及人工帶菌種子。進一步利用此技術鑑定2001年至2010年自番茄主要產區分離而來之病原菌，鑑定結果顯示79%的菌株皆屬於X. perforans，本研究開發之X. perforans專一性引子HpaF-f/r未來可應用於番茄種子(苗)進出口檢疫工作之用。
茄科細菌性斑點病 (Bacterial spot of pepper and tomato)由Xanthomonas屬之病原菌所引起，為番茄及甜椒之重要病害。此類病原菌依不同鑑別品種，可區分為五個番茄生理小種及十一個甜椒生理小種，然而，針對此類複雜的菌群，仍然缺乏分子層次的快速鑑定技術。本研究目的是以rep-PCR及IS-PCR技術分析不同種茄科細菌性斑點病菌之基因指紋圖譜，並進一步配合無毒力基因設計之引子鑑定番茄生理小種，以探究X. perforans的基因多型性與生理小種間的關聯性。rep-PCR及IS-PCR之結果顯示，利用rep-PCR之BOX-PCR、ERIC-PCR、REP-PCR及IS-PCR之IS404、IS476之圖譜可區分X. euvesicatoria、X. vesicatoria 及X. perforans。其中，利用BOX-PCR圖譜分析可將田間分離而來的40個X. perforans菌株區分為兩群，其中一群菌株具有完整avrXv3基因，且大部分菌株具有avrXv4基因，應屬於番茄生理小種第三型；而另一群菌株則不具有完整無毒力基因。由rep-PCR及IS-PCR之基因圖譜可知，不同種內之菌株具基因多樣性，未來可鑑定國內茄科細菌性斑點病菌菌株之番茄及甜椒生理小種，並從多型性圖譜中篩選特殊條帶，用於進行生理小種專一性引子之設計，應用於探究國內生理小種之分布情形。 Chapter 1.Identification and detection of Xanthomonas perforans by the polymerase chain reaction technique
Bacterial spot caused by Xanthomonas is an important disease of solanaceous crops. The bacterial spot-causing xanthomonads (BSX) have been classified as four species of Xanthomonas euvesicatoria, X. vesicatoria, X. perforans and X. gardneri. Bacterial spot of tomato caused by X. perforans was first reported in Taiwan in 2010. An accurate and rapid PCR technique for identification and detection of X. perforans is not available. In order to develop a rapid and specific PCR technique for identification and detection of X. perforans, the genomic DNA of X. perforans and other Xanthomonas spp. were compared. Based on the unique sequence of hpaF of X. perforans, the primer pair HpaF-f/r specific for X. perforans was designed in this study. A 300 bp PCR product was specifically amplified from X. perforans. The detection limit of the DNA from X. perforans with primer pair HpaF-f/r was 50 pg. In this study, primer pair HpaF-f/r could specifically detect X. perforans in diseased leaves and artificially infested seeds of tomato. In addition, 195 strains of BSX isolated from tomato fields since 2001 in Taiwan were examined by PCR with primer pair HpaF-f/r. The results showed that 79% of these BSX strains were identified as X. perforans. The primer pair HpaF-f/r developed in this study can be further used to identify and detect X. perforans in tomato seeds for plant quarantine.
Chapter 2.Characterization of X. perforans strains in Taiwan by DNA polymorphism
Bacterial spot of pepper and tomato caused by Xanthomonas spp. are important disease worldwide. Bacterial spot-causing xanthomonads (BSX) could be differentiated to five races of tomato and eleven races of pepper by inoculating differential cultivars. However, a molecular tool for detection the races of BSX is still not available. The objective of this study is to clarify the relationship between the genetic diversity and races of BSX by the fingerprinting patterns assays and the PCR amplification with specific primers of races. The genetic diversity of BSX strains were studied by the rep-PCR and IS-PCR. The results showed that X. euvesicatoria, X. vesicatoria and X. perforans could be grouped to three distinct clusters by BOX-PCR, ERIC-PCR, REP-PCR of rep-PCR and IS404, IS476 of IS-PCR. The clusters analysis by BOX-PCR revealed two major clusters among 40 strains of X. perforans in Taiwan. There are full lengths of avrXv3 and avrXv4 genes in the strains of one group, so they might belong to tomato race T3, but the strains in another group aren’t. Based on the patterns of rep-PCR and IS-PCR, the intraspecific clusters are varied. In the future, we might identify the tomato and pepper races of BSX and select the specific fragment of fingerprinting patterns to design primers for races. The race-specific primers could help us to study the distribution of races in Taiwan.