引起非典型病徵之柑橘潰瘍病菌pgi基因與外源基因pthAp對其致病性之影響

Effects of Pgi and Exogenous Pthap in Pathgenicity of Atypical Symptom-inducing Strain of Xanthomonas Axonopodis Pv. citri

"5種會引起非典型病徵之柑橘潰瘍病菌（Xanthomonas axonopodis pv. citri, Xac）陸續被發表，其中Xac-A*類型菌株在葡萄柚與甜橙葉片上會引起小的扁平壞疽病斑，而非凸起的潰瘍病斑，利用Tn5跳躍子針對Xac-A*類型之XC322菌株進行嵌入突變，並篩選出在柑橘葉片上不會產生病徵缺少致病性之突變菌株TP322；經特性分析，TP322之基因體中被跳躍子所嵌入之phosphoglucose isomerase基因（pgi）是決定柑橘潰瘍病菌是否可在葉片上表現出致病性之重要因子。應用pgi基因座所設計出之專一性引子對pgiF/ pgiR進行PCR反應，可從XC322基因體中增幅出一段含有pgi基因之2.5 kb片段，並將此片段送入突變菌株TP322則可使TP322恢復原來病徵及致病性；分別將XC322及TP322利用滲透注射接種到葡萄柚葉片，則TP322菌量數在接種後第24天較XC322菌量數少105~106倍。將會引起典型潰瘍病斑之pthA基因與會引起大的具水浸狀邊緣壞疽病斑之pthAp基因轉入XC322菌株中，分別獲得轉型菌株TA1935與TA3836，TA3836在葡萄柚及甜橙葉片上會引起大的具水浸狀邊緣壞疽病斑，並且TA3836在葉片內之菌量數在接種後第24天較XC322菌量數多10倍，然而TA1935仍引起小的具水浸狀邊緣壞疽病斑，而非潰瘍病斑。由上述結果說明，pgi與pthAp基因均與XC322之毒力表現有關。"

"Five atypical symptom-producing types of Xanthomonas axonopodis pv. citri were described successively. The variants designated as Xac-A* type produce small flat necrotic lesions with water-soaked margin instead of canker lesions on leaves of grapefruit (Citrus. paradisi) and sweet orange (C. sinensis). Random mutagenesis using the transposon Tn5 in Xac-A* type strain XC322 was used to isolate a pathogenicity-deficient mutant TP322, which caused no visible symptom on citrus leaves. The gene, pgi, inactivated by the transposon was cloned and characterized showing to be responsible for the pathogenicity of XC322 on citrus leaves. Base on the sequence of pgi locus, a primer pair pgiF/pgiR was designed and could amplify a 2.5 kb DNA fragment encoded a protein homology to phosphoglucose isomerase that complemented the mutant TP322. The suspensions of XC322 and TP322 individually were inoculated into grapefruit leaves with injection-infiltration method. The bacterial populations of TP322 were 105-106 folds lower than that of XC322 24 days after inoculation. Two clones individually containing pthA and pthAp gene responsible for Xac-A type strains to produce canker lesions and Xac-Ap type strains to produce large flat necrotic lesions with water-soaked margin were transferred into XC322, respectively. The transformant TA3836 can produce large flat necrotic lesions with water-soaked margin on citrus leaves. The bacterial multiplication of TA3836 was approximately 10 folds higher than those of XC322 24 days after inoculation. While, transformant TA1935 still produced small flat necrotic lesion on citrus leaves. The results illustrated that pgi and pthAp are responsible for the virulence of XC322."