蓮霧果腐病菌兩種病徵型菌株之研究

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論文名稱: 蓮霧果腐病菌兩種病徵型菌株之研究
研究生姓名: 楊尹甄
指導教授姓名: 梁文進
出版年: 2004
學校名稱: 國立屏東科技大學
系所名稱: 植物保護系
關鍵字: 蓮霧果腐病;病徵;形態特徵;培養特性;核酸序列;wax-apple;Pestalotiopsis eugeniae;symptom;morphological character;nucleotide sequences
摘要: 台灣蓮霧果腐病由Pestalotiopsis eugeniae感染而引起,為蓮霧果實重要病害之一。主要危害成熟或近成熟的果實,病徵大部份出現在果實傷口處或裂開處,呈現紫色或褐色兩型病斑,病斑表面散生許多黑色分生胞子盤,直徑約為79.7~146.7 × 47.8~73.9μm,分生胞子呈紡綞形,分為5室,中間3個細胞為淡褐色或深褐色,首尾2室無色透明,頂端細胞呈三角形或圓錐形,其上長有1~3根線狀附屬絲。果腐病菌可於滅菌蓮霧葉片產生子囊果。子囊果埋生,大小為125.5 × 90μm(直徑×高度),子囊為長圓柱形具單囊壁,頂端經碘液染色可呈現藍色反應,大小為54.3~82.1×3.4~5.6 μm,每個子囊中具8個子囊胞子,子囊胞子褐色3胞,胞子中央細胞顏色較深,頭尾2胞顏色較淡,大小平均為10.5~15.0 × 3.8~5.7 μm。依據上述特徵應分類上應歸於Pestalosphaeria屬。 P. eugeniae之培養特性,依不同病徵型的菌株有如下結果:紫斑型菌株(PW-5101、 PW-5121、 PW-7102)及褐斑型菌株(PW-5212、 PW-8201)培養於PDA培養基,菌落呈白色,除菌株PW-5212在培養基上不產生色素沈澱,其餘都有明顯同心輪紋及色素形成,另一褐斑型菌株PW-5221在PDA培養基上菌落呈黃褐色有同心輪紋及色素形成,其菌落形態與其他供試菌株間有明顯差異。紫斑型菌株分生胞子在溫度24~28 ℃發芽率可達90 ﹪以上,而褐斑型菌株在24 ℃發芽率達最高峰。在核酸分析方面, PW-5101、PW-5121及PW-8101之3株紫斑病型菌株與PW-5212及PW-8201之2株褐斑型菌株的ITS序列皆相同,比對結果與已有記載的P. palmarum(AF409990)完全相符;另與番石榴等其他寄主分離之5個菌株比對ITS序列也都相似,但仍有紫斑及褐斑病徵之不同,此有寄主”適應”之分化現象;而另一褐斑型菌株PW-5221之ITS區域片段較長50 bp。 所有菌株皆以24~28 ℃為最適生長溫度,菌絲生長對酸鹼度適應性極廣,於pH 2.5~9皆可生長,以pH 4為最適生長,葡萄糖、麥芽糖為最佳碳源,可利用之氮源為硝酸鈉、硝酸銨、氯化銨、硫酸銨、甘氨酸;水分潛勢則以-1~-25 bar生長最佳,溫度高於55 ℃ 時,分生胞子不發芽;不同酸鹼值對兩型病徵菌株分生胞子發芽有相同之影響,以pH 3~7之間的發芽率最高。在乾燥環境下(R. H. 30~50 ﹪),分生胞子在載玻片上可存活60天以上,而在葉片上可殘存6個月之久。溫度24~28 ℃,相對濕度達100 ﹪時最合適分生胞子盤形成。利用選擇性培養PSM-S基偵測田間的蓮霧葉片、枝條,在3~5天後即可出現蓮霧果腐病菌菌落。The Pestalotiopsis fruit rot caused by Pestalotiopsis eugeniae is one of the most serious diseases of wax-apple fruit in Taiwan. The rot lesions in purple or brown are developed from the wounds or physiological splits on matured or maturing fruits. There are many black-points erumpent acervuli scattered on the surface of lesions. Acervuli are 79.7~146.8 × 47.8~74.0 μm in diameter. Conidia of P. eugeniae are 5-celled, elliptic or cylindrical fusiform. Intermediate three cells of conidia are vescicolorous, while apical and basal cells hyaline. The apical cell is triangular to conic with one to three setulae or appendages. Perithecia are produced on sterilized leaf of wax-apple inoculated with a isolate, PW-5221 of P. eugeniae. Perithecia are immersed at first, then erumpent, 125.5 μm in diameter, 90 μm in elevation. Asci are cylindrical, unitunicate, 50.3~82.1×3.4~5.6 μm. Every ascus has 8 ascospores. Ascospores are 3-celled, brown, 10.5~15.0×3.8~5.7 μm. Intermediate cell of ascospore is darker than the apical and basal cells. Basing on the characters of sexual reproduction, the meiosporic stage of this pathogen is classified to Pestalosphaeria genus. The isolates of P. eugeniae PW-5101, PW-5121 and PW-7102 from purple lesions and PW-5212 and PW-8201 from brown lesions, produce white holoocentric colonies on PDA. All of them can produce pigment except PW-5212 isolate. One isolate of PW-5221 from brown lesion produces light brown, holocentric colony and pigment on PDA, differing from white colonies produced by the above 5 isolates. For conidial germination, the optimal temperatures are from 24 to 28 ℃ for all purple-lesion isolates of P. eugeniae, while 24 ℃ for brown-lesion isolates. The amplified products of ITS region containing 5.8S rDNA from 6(3 purple and 3 brown) isolates of P. eugeniae using ITS 5/ ITS 4 primer pair by PCR are sequenced and compared the identities with DNA information listed in database of NCB1 GeneBank. Three purple lesion isolates of PW-5101, PW-2121 and PW-8101 and two brown-lesion isolates of PW-5212 and PW-8201 have the same ITS region sequences, and are identical to that of listed species P. palmarum(AF409990). The ITS region sequences of other 5 isolated of Pestalotiopsis sp. isolated from guava and other hosts are all similar to that of these five wax-apple pathogen islolates in sequence and in length. Howere, these 5 isolates from other hosts also induce purple and brown lesions on wax-apple or guava, or purple on wax-apple and brown on guava, individually. This phenomenon of lesion color differentiation may be due to “host adaptation”. The other brown-lesion isolate is 50 bp longer than that of 5 wax-apple isolates, and is similar to listed species Pestalotiopsis sp. EN 5 (AF409960). For mycelial growth of the tested isolates, the optimal temperatures are from 24 to 28 ℃; the suitable pH values are from 2.5 to 9, while optimal pH value 4; the optimal carbon sources are glucose and maltose, the nitrogen sources sodium nitrate、ammonium nitrate、ammonium chloride、ammonium sulfate and glycine; the suitable water potentials are from —1 to —25 bar. Conidia of all pathogen isolates can not germinate at 55 ℃. Conidia of all isolates can germinate well at pH values from 3 to 7. Conidia can survive for 60 days sprayed on glass slide stored at R.H. 30 to 50 %, for 6 months on sterilized dry leaves. The optimal relative humidity for acervuli formation is 100% at 24~28 ℃. A selective medium developed for isolating the pathogen, P. eugeniae can be used easier in pathogen isolation from leaves and twig collected from orchards within 3 or 5 days.
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