臺灣水稻白葉枯病菌菌系及其噬菌體之分析

Abstract

本研究係利用對菲律賓和日本判別品種之感染性、噬菌體之敏感性、 Biolog的生化鑑定和用一重複性DNA序列做RFLP分析，分析本省所分離的 水稻白葉枯病菌。供試之14種水稻品種對本省白葉枯病菌菌株反應，屬於 高抗病之品種有DV 85、IR 1545-339、IR 20、Wase Aikoka 3、Java 14及Chugoko 45；屬於中度抗(感)病品種有IR 8、IR 24、Kogyoku、 Tetep、Kinmaze及高雄秈 7；屬於極度感病之品種有Cas 209及Rantai Emas。以五種日本判別品種(Rantai Emas、Kinmaze、Kogyoku、Wase Aikoku 3、Java 14)之接種反應可將本省菌株分為六個病原群，其中以J1 群最多，佔測試菌捏44.2%，J2、J3、J4、J5及J6群所佔的比率分別為6.3 、8.4、5.2、18.9和12.6%，另有4菌株無法歸類。以五種菲律賓判別品 種(Cas 209、DV 85、IR 1545-339、IR 20、IR8)之接種反應可分出七個 病原群，以I1群佔最多，佔測試菌株的31.6%，I2、I3、I4、I5、I6及I7 群所佔的比率分別為22.1、15.8、3.2、4.2、4.2及14.7%，有4株菌無法 歸類。Biolog生化鑑定測試，接種細菌濃度較低時，所鑑定之結果大部份 菌株為生化型E型，較高濃度時，則轉為A型；相同菌株重複測試時，兩次 均測得相同種之準確度達91.7%，相同生化型之準確度達66.7%；所有菌株 之測試結果，屬的鑑定準率為100%，種的鑑定準確率為85.7%；多數菌株 均可利用之碳源有16種，多數菌株均不能利用之碳源有65種，多數菌株均 呈中間型反應之碳源有7種，菌株間利用情形有較大差異之碳源有7種。由 Xo 89菌株所構築之基因庫中，挑選出12株重組質體，其插入之片段經非 放射性之NEBlotTM PhototopeTM kit標定後而得測試之探針，再經直接及 次選殖後以南方墨漬法篩選，得到最專一性之探針為pXoD10，其和所有測 試之白葉枯病菌DNA皆可產生專一性之雜合反應訊號，而和23株非白葉枯 病菌DNA均不產生雜合反應訊號，另一探針pXoC3為一重複性DNA序列，以 其對本省白葉枯病菌菌株做RF分析，可分出八個RFLP群，以R7群最多， 佔40.9%，R1、R2、R3、R4、R5、R6及R8所佔之比率分別為4.3、6.5、5.4 、3.2、29.0、1.1及9.7%，親緣性分析以R8群和各群之親緣性最遠，其它 Xanthomonas spp.以和X. campestris pv. phaseoli之親緣性最近，平均 相似度為33%，X. matophilia之親緣性最遠，相似度均為0%。以噬菌體感 染反應可將本省菌株分成七群，以A群佔76.8%最多，B、C、D、E、F、G群 所佔的比率分別為2.1、2.1、2.1、1.1、15.6、1.1%。由台中縣大里市地 區所分離之白葉枯病菌噬菌體其核酸均為雙股DNA，五種噬菌體DNA均有 EcoR I、BamH I、Sac I及SalI切匈，均無Pst I切位點，以XOP24和其它 噬菌體核酸之親緣性最遠。

By using Japanese and Philippine differential varieties, Biolog identification system, RFLP analysis and phage sensitivities, 102 strains of Xanthomonas oryzae pv. oryzae , the causal organism of rice bacterial blight, isolated from Taiwan were analyzed and grouped. Among fourteen rice cultivars examined against Taiwanese isolates of X. oryzae pv. oryzae, DV 85, IR 1545-339, IR 20, Wase Aikoku 3, Java 14 and Chugoko 45 were highly resistance; IR 8, IR 24, Kogyoku, Tetep, Kinmaze and Kaoshiung sen 7 were morately resistance or susceptible, and Cas 209 and Rantai Emas were very susceptible. The Taiwanese isolates were fallen into 6 groups according the responses aganist inoculation to 5 Japanese differential cultivar (Rantai Emas, Kinmaze, Kogyoku, Wase Aikoku 3 and Java 14), the major group was J1, sharing 44.2% of the tested isolates, and 6.3, 8.4, 5.2, 18.9 and 12.6% for J2, J3, J4, J5 and J6 group, respectively, however, 4 isolates were uncertain. These isolates were classified into 7 groups according the sponses aganist inoculation to 5 Philippine differential cultivar(Cas 209, DV 85, IR 1545-339, IR 20 and IR 8), the major group was I1, sharing 31.6% of the tesed isolates, and 22.1, 15.8, 3.2, 4.2, 4.2 and 14.7% for I2, I3, I4, I5, I6 and I7 groups, respectively, but 4 isolates were uncertain. In Biolog GN Microplate examination, most of the isolates appeared to be biochemical type E when inoculated with low concentrations of inocula. However, when high concentrations of inocula were inoculated, they were nverted to biochemical type A. The repeatable accuracy of twice examinations was 91.7% for the same species, and 66.7% for same biochemical type. The accuracy of identifications was 100% for genus and 85.7% for species calculated against all tested isolates. Among 95 carbon sources, 16 were valid, 65 were invalid and 7 were borderline for most of the tested isolates. There was a large difference in utilization of 7 carbon sources by tested isolates. A genomic library of strain Xo 89 of X. oryzae pv. oryzae s constructed in the plasmid pUC18 and transformed into E. coli strain DH5α. Twelve recombinant plasmids were selected from gene library of strain Xo 89. The most specific probe pXoD10 was obtained from the insertion fragment which was labelled by non-radioactive NEBlotTM PhototopeTM kit, screened by direct and subcloning, and examined by Southern blotting. All DNA from causal bacterium of rice bacterial blight showed specific hybridization singnal aganist the probe, but not to other 23 bacterial strains tted. A repetitive DNA sequences (pXoC3) was obtained from the genome of X. oryzae pv. oryzae. Restriction fragment length polymorphism( RFLP) analysis with DNA probe pXoC3 of genomic DNA revealed hybridization profiles that separated the strains into eight groups. The major group was R7, sharing 40.9% of the tested isolates, and 4.3, 6.5, 5.4, 3.2, 29.0, 1.1 and 9.7% for R1, R2, R3, R4, R5, R6 and R8 group, respectively.The genetic distance of R8 group of X. oryzae pv. oryzae was most far from the other grou. X. oryzae pv. oryzae had the hightest similarity with X. campestris pv. phaseoli, whereas the lowest similarity with X. motophilia. The Taiwanese isolates were classified into 7 groups according their sensitive to seven phages. The major group was A sharing 76.8% of the tested isolates, and 2.1, 2.1, 2.1, 1.1, 15.6 and 1.1% for B, C, D, E, F and G group respectively. Five strains of bacteriophage of X. oryzae pv. oryzae isolated from Tali city of Taichung county were double-strain DNA. They had cleavage ses of EcoR I, BamH I, Sac I and SalI , but no cleavage site of Pst I. The genetic distance of XOP24 were the most far from the other phages.