小星辰花炭疽病菌之鑑定及其病原性測定

Identification for the Causal Agent of Caspia Anthracnose and Its Pathogenicity Tests

10.6649/PPB.200612_15(4).0004

洪爭坊(C. F. Hong)；張碧芳(P. F. L. Chang)；張景宜(J. Y. Chang)；黃振文(J. W. Huang)

小星辰花 ； 炭疽病 ； Colletotrichum gloeosporioides ； Glomerella cingulata ； Colletotrichum dematium ； caspia ； anthracnose ； Colletotrichum gloeosporioides ； Glomerella cingulata ； Colletotrichum dematium

植物病理學會刊

15卷4期（2006 / 12 / 01）

241 - 249

繁體中文

西元2002-2003年間，台灣中部地區多處花卉育苗場與栽培區發現小星辰花(Limonium bellidifolium Dumort.)葉片佈滿棕褐色圓形至不規則形之壞疽斑，嚴重時常導致小星辰花整個葉片壞疽或整株死亡。取回罹病葉片進行組織分離，共獲得八個Colletotrichum spp.菌株。經由柯霍氏法則證實各菌株均具有病原性後，取Colletotrichum sp. Cas-01、Cas-03、Cas-06及Sta-04菌株培養於馬鈴薯葡萄糖瓊脂培養基(potato dextrose agar, PDA)平板上。其中Cas-01與Cas-03菌株氣生菌絲少，菌落呈暗褐色，會產生大量剛毛(seta)，分生孢子鐮刀形或新月形；Cas-06與Sta-04菌株的菌落在PDA上呈灰白至橄欖綠色，分生孢子長橢圓形，兩端鈍圓或一端為截頭狀(truncated)。此外，Cas-06與Sta-04菌株在PDA上會產生黑色球形的子囊果，子囊果內含透明子囊，每個子囊內有八個子囊孢子，子囊孢子呈棍棒狀至紡錘形且略微彎曲。利用Mills氏等人研發之C. gloeosporioide，專一性CgInt引子配合ITS4引子進行PCR，可由Cas-04、Cas-05、Cas-06、Cas-07、Sta-04與Sta-b3菌株增幅出大小約450bp的專一性條帶，卻無法由Cas-01及Cas-03菌株增幅出任何條帶。進一步以ITS1及ITS2引子對增幅Cas-01與Cas-03菌株之ITS1與5.85rDNA部分序列，經解序後進行比對，結果它們與Colletotrichum dematium的序列相同度達99-100%。綜合上述病原菌形態特徵與分子生物學上的佐證，確定引起小星辰花苗炭疽病之病原菌為C. gloeosporioides (Penz. ) Penz, & Sacc.與Colletotrichum dematium (Pers.: Fr.)Grove，前者的有性世代為Glomerella cingulata (Stonem. ) Spauld, & Schrenk。將C. dematium Cas-01及Cas-03菌株與C. gloeosporioides Cas-06及Sta-04菌株的分生孢子分別噴霧接種於十四種植物上，C. dematium Cas-01及Cas-03菌株僅在高溫高濕（280℃，RH100%持續七天以上）下，才可造成小星辰花苗、蜀葵、筒蒿及太陽菊的壞疽病徵；至於C. gloeosporioide, Cas-06及Sta-04菌株則可感染小星辰花、藍雪花、檬果、筒蒿及太陽菊等植株。

In 2002 and 2003, plants showing symptoms of necrotic, reddish brown spots on caspia (Limonium bellidfolium) leaves were observed in central Taiwan. Eight isolates of Colletotrichum sp., Cas-01. Cas-03, Cas-04, Cas-05, Cas-06, Cas-07, Sta-04, and Sta-b3, were isolated from leaf lesions of diseased seedlings and adult plants. Conidia of the isolates were used to inoculate caspia and kept at 100% RH for one day, then grown in the growth chamber. After 7 to 14 days, numerous acervuli were produced on inoculated leaves from which the same pathogen could be consistently isolated. On potato dextrose agar (PDA), spore masses of six isolates of Colletotrichum sp. Cas-04, Cas-05, Cas-06, Cas-07, Sta-04, and Sta-b3 were pinkish or salmon red with dense to sparse grayish aerial mycelia. Conidia were hyaline, straight, cylindrical, and apices obtuse. Perithecia of Colletotrichum isolates Cas-06 and Sta-04 formed on PDA produced asci with 8 single-celled, slightly curved ascospores. Spore masses of the other two Colletotrichum isolates, Cas-01 and Cas-03, were pale buff to salmon red with sparse grayish aerial mycelia. Conidiomata produced abundant setae. Conidia were hyaline, falcate, fusiform, apices acute. Further polymerase chain reaction was conducted with specific CgInt, CcInt, and universal ITS4 primer pairs for the identification of the eight isolates of Colletotrichum spp. A DNA fragment of about 450bp was amplified from six Colletotrichum isolates, Cas-04, Cas-05, Cas-06, Cas-07, Sta-04, and Sta-b3, by using C. gloeosporioides specific CgInt primer and ITS4 universal primer, but not from isolates Cas-01 and Cas-03. The ITS1 and partial 5.8S rDNA sequences of Colletotrichum isolates Cas-01 and Cas-03 were amplified by using universal ITS1 and ITS2 primer pairs. The DNA sequences showed 99-100% identities to those of Colletotrichum dematium (Accession numbers: AB046608.1、AB042310.1、AB042311.1) deposited to NCBI GenBank database. Based on morphology, pathogenicity and molecular biological evidences, it was concluded that the disease on caspia plants was caused by C. dematium (Pers.: Fr.) Grove and C. gloeosporioides (Penz) Penz. & Sacc. with the teleomorph Glomerella cingulata (Stonem.) Spauld. & Schrenk. Fourteen plant species including African marigold, caspia, cape leadwort, Chinese cabbage, Chrysanthemum grinatum, egg plant, garland chrysanthemum, hollyhock, lettuce, mango, pepper, sweet pepper, tomato, and transvaal daisy were inoculated with conidial suspension (10^5 conidia/ml) of C. dematium isolates Cas-01 and Cas-03 and C. gloeosporioides isolates Cas-06 and Sta-04, respectively, for evaluation of pathogenicity of the pathogens on different plants. The results showed that C. dematium isolates Cas-01 and Cas-03 infected caspia, Chrysanthemum grinatum, garland chrysanthemum, and hollyhock only under high temperature (28℃) and high humidity (RH 100 % was prolonged over 7 days). On the other hand, C. gloeosporioides Cas-06 and Sta-04 could infect caspia, cape leadwort, Chrysanthemum grinatum, mango, and garland chrysanthemum under high temperature (28℃) and high humidity (RH 100 % was only kept for one day).