柑桔潰瘍病病原菌致病基因之研究

Abstract

柑桔潰瘍病病原菌（Xanthomonas campestris pv. citri）是一種植物的致病細菌，會感染柑桔類植物的葉片，果實和枝條；造成感染部位的突起，細胞崩解，以及褐化、木栓化等潰瘍的現象，此即柑桔潰瘍病（citrus canker）。?瞭解植物致病細菌與寄主植物之間的關係，可經由突變體的篩選，分離致病基因，來探討其致病機制。 本研究是以移位子（transposon）Tn5tac1來誘導突變體的產生，並以其篩選標誌篩選到695株抗kanamycin的菌株；再以Tn5tac1?探針，經由菌落雜合實驗確認其中的692株有Tn5tac1的插入；接種至檸檬葉片，測定病原性的結果得到6株突變株，表現3種與野生型不同的病徵。分別?：XT-10和XT-122幾乎無任何病徵的表現；XT-27的病徵?類似癒合組織（calluslike）的白色突起；XT-37、XT-604和XT-606則呈現水漬狀（water soaking）的深色病斑，周圍並有黃色暈環（yellow halo），表現了部分的野生型病徵；接著以南方氏轉漬法和DNA雜合實驗進一步分析Tn5tac1插入突變株染色體的情形，發現此6株突變體，皆僅有一個Tn5tac1插入染色體中，而且插入的位置除了XT-604和XT-606相似外，其他突變株間皆不相同；營養需求試驗的結果顯示XT-27亦?營養需求突變體；生長曲線測定結果，此6株突變體的生長情形皆與野生型相似；分析各菌株分泌胞外多醣體（extracellular polysaccharide, EPS）的產量和分泌性酵素的活性，結果胞外多醣體的產量在各菌體間並無明顯差異，而XT-27的澱粉?及果膠?的活性皆明顯的低於野生型者；若將野生型與各突變體的培養液，細胞萃取物及菌體單獨或交互接種於檸檬葉片，培養液和細胞萃取物皆無致病能力，而菌體交互接種後，病徵表現的強度以野生型最強，次?水漬病斑者，再次?無病徵者，而以癒合組織突起者表現最弱。由這些結果顯示，柑桔潰瘍病的病原菌感染植物時，可能不只一種致病基因參與，而且其致病機制亦相當複雜。

Xanthomonas campestris pv. citri is a plant pathogenic bacterium, causing citrus canker on citrus plants. The disease causes canker spots on leaves, fruit and stems. To understand the mechanism of pathogenesis of citrus canker, bacterial mutants, showed the alternation in pathogenicity, were isolated. In this study, the insertion mutagenesis with Tn5tacl was used. Amoung 695 Kanamycin resistant colonies carried were detected by colony hybridization with Tn5tacl probe. To determine the pathogenicity of these mutants, these colonies were inoculation on limon leaves, and the types of symptom were determined. Base on the type of symptom, six pathogenic mutants could be classified into three types. The first type, such as XT-10 and XT-122, was nonpathogenic mutant, did not form symptom on plants; The second type, such as XT-27, showed calluslike eruption of tissue and no water soaking or necrosis; The third type, such as XT-37, XT-604 and XT-606, showed water soaking dark spots and surrounded yellow halo. Sounthern blot analysis revealed that each of the six mutants has only one Tn5tacl insertion, and the insertion occurred in at last 5 different positions. Further studies of the six mutants, only the XT-27, showed that the calluslike lesion was also an auxotrophic mutant, and produced lower levels of extracellar enzyme than that of wild type. But the amount of extracellar polysaccharide is similar to that of other mutants and wild type. In co-inoculation test, there were no wild type symptom recovered, when two kinds of mutants were co-inoculated in the same place.