菜豆萎凋病之病原菌鑑定與其野生型和變異型菌株間致病毒力與生理特性的比較

Abstract

西元2001年，於南投縣集集鎮的菜豆（Phaseolus vulgaris L.）栽培田發生植株萎凋死亡的現象；隨後也在埤頭、魚池與頭社等地區發現菜豆植株萎凋、黃化的病徵。另外，受害植株地下部根系大多發育不良，根莖部維管束褐化，且蔓延至葉片，繼而全葉枯黃脫落。將罹病菜豆植株取回，進行病原菌的分離工作，共獲得FOP-044、FOP-061、FOP-062、FOP-071、FOP-072、FOP-081、FOP-082、FOP-083、FOP-091、FOP-092、FOP-101、FOP-102等12個菌株。各菌株在馬鈴薯葡萄糖培養基(PDA)斜面培養基上，菌落顏色為白色或粉橘紅色，可在試管壁形成細小的菌絲結。多數菌株的菌落生長基質呈深紫色；部分菌株會出現數目不等的深墨綠色 (dark bluish green)菌核體 (sclerotial bodies)。孢子有大孢子、小孢子及厚膜孢子等三種：大孢子呈鐮刀形，具典型的足細胞，大小3.0 ~ 6.0 × 25.0 ~ 35.0μm（平均4.5 × 30.0μm），以3~4個隔膜的大孢子較多；小孢子為橢圓或臘腸形，無色，大小2.0 ~ 3.8 × 3.8 ~ 11.4μm（平均2.9 × 7.6μm）；厚膜孢子大多形成於菌絲間，亦有頂生者，球形至橢圓形，大小2.0 ~ 4.0 × 6.0 ~ 15.0μm（平均 3.0 × 10.5μm）。大小分生孢子皆著生於分生孢子梗 (conidiophore) 的瓶狀枝 (phialide) 上，小孢子呈假頭狀排列。孢子堆呈橙黃色、平鋪型或粒狀型。將FOP-044與FOP-091菌株接種於16種不同作物，結果發現僅菜豆受感染。另外取F. oxysporum其他分化種的菌株接種於菜豆上，發現只有由罹病菜豆植株分離到的菌株能感染菜豆。依上述菌株之形態特徵與病原性，參照 Booth、Nelson 氏等及 Gerlach 與 Nirenberg 之 Fusarium 分類系統，確定引起菜豆萎凋病之病原菌為F. oxysporum Schlechtend.:Fr. f. sp. phaseoli J. B. Kendrick and W. C. Snyder。本菌最適生長溫度介於24至28℃之間。最適生長酸鹼值為pH6。最適發病溫度介於24至28℃之間。利用浸根接種法與病菌土接種法，發現菜豆受萎凋病危害的罹病度，會隨著接種菌量濃度的增加，越趨嚴重。菜豆品種間以扁圓莢有絲-屏東大莢最為耐病；而以圓莢無筋絲83-10-28最為感病。本菌自田間分離得到之野生菌株均為孢子堆型(sporodochial type)，但在PDA上培養一段時間後，會變異為白色菌絲型(cottony alba type)、黏膜菌絲型(ropy mycelial type)、和黏膜型(pionnotal type)。將孢子堆型FOP-061ST、FOP-062ST及FOP-071ST與黏膜型菌株FOP-061PT、FOP-062PT及FOP-071PT分別接種於菜豆幼苗，結果發現孢子堆型菌株可以引起53 ~ 67%的菜豆萎凋；而黏膜型僅引起27 ~ 33%的植株發病；此外，發現野生型菌株FOP-071ST的侵襲能力是變異型菌株FOP-071PT的2 ~3倍。取菜豆幼苗浸於野生型與變異型菌株的馬鈴薯葡萄糖煎汁(PDB)後，發現野生型菌株的培養濾液引起菜豆的萎凋指數皆高於黏膜型菌株的培養濾液。評估十三種碳素源及十四種氮素源對孢子堆型與黏膜型菌株在蔡氏液態培養基(Czapek’s solution) 生長21天之培養濾液毒性時，發現以半乳糖及甘露糖作為碳素源可使野生型的培養液毒力大於變異型；而以果糖、蔗糖和棉子糖作為碳素源可使變異型的培養液毒力大於野生型。至於氮素源則以硝酸鉀及尿素為氮素源可以使野生型的培養液毒力大於變異型；但若以天門冬醯氨酸作為氮素源可使變異型的培養液毒力大於野生型。添加碳素源對致病毒性的影響中，發現棉子糖可以顯著促進FOP-071PT病勢的擴展；而木糖與乳糖卻具有抑制野生型與變異型菌株病勢的發展。

In 2001, a disease of common bean (Phaseolus vulgaris L.) was observed for the first time in Central Taiwan fields. The fungus traversed the vascular bundles throughout almost the entire length of the stem and extends into the branches and into the petioles of the leaves. The vascular bundles were conspicuously discolored. Discolored vascular streak in yellowing leaves was extended from the root. In the early stage the color of the leaves changed to bright yellow, later to dark brown. The leaves finally dried up and dropped off. Fusarium oxysporum was consistently isolated from all affected plants. Twelve isolates of the fungus isolated from the diseased tissues were able to cause a wilt disease of common bean, but did not do any symptom in other crops and composite species when they were conducted for inoculation in greenhouse. According the morphological characteristics and pathogenicity tests, the pathogen was been identified as Fusarium oxysporum f. sp. phaseoli. The isolates FOP-044 and Fop-091 produced the blue-purple colony. Sporodochia appeared discrete or confluent, straw or orange. Sclerotial bodies were sparse to abundant, dark bluish green. Microconidia produced abundantly from monophialides only in false heads, generally single-celled, elliptical to allantoid, 2.0-3.8×3.8-11.4μm. Macroconidia produced abundantly from monophialides, 3.0-6.0×25.0-35.0μm, on branched or unbranched conidiophores, slightly sickle-shaped, thin-walled, and delicate, with a foot-shaped basal cell, mostly 3-5 septate. Chlamydospores formed mostly in hyphae, terminal or intercalary, solitary, in pairs or in chains, 2.0-4.0×6.0-15.0μm. The optimum temperature for mycelial growth and conidial germination of F. oxysporum f. sp. phaseoli isolates FOP-044 and FOP-091 was at 24 – 28 0C, and for their chlamydospore germination were at 240C. The optimum pH value for mycelial growth and conidial germination of two isolates was at pH 6.The appropriate temperature for both infection on common bean was at 24 - 280C. The common bean (Pingdon big pod ) cultivar was the most resistant to Fusarium wilt among the tested cultivars. Four morphological types or forms of the pathogen were observed in PDA slants. They were sporodochial type(ST), cottony alba , ropy mycelial , and pionnotal types(PT). Pathogenicity tests showed that the sporodochial type isolate had 20 – 40% higher in virulence than the pionnotal type. Higher percentage of common bean seedlings showed the wilt syndrome 48hr after they were dipped in culture filtrates (8-fold dilution) of ST isolates than that in culture filtrates of PT isolates. Thirteen carbohydrates and fourteen nitrogenous compounds were evaluated for their effects on toxicity of filtrates obtainded from FOP-071ST and FOP-071PT grown in Czapek’s solutions for 21 days to common bean seedlings. Among those, galactose and mannose were more effective than other carbohydrates to increase toxicity of filtrates of FOP-071ST. Otherwise, fructose、sucrose and raffinose were more effective than other carbohydrates to increase toxicity of filtrates of FOP-071PT. As to nitrogenous compounds, KNO3 and urea were more effective than other nitrogens to increase toxicity of filtrates of FOP-071ST. However, asparagine was more effective than other nitrogens to increase toxicity of filtrates of FOP-071PT. Raffinose as a carbon source could increase virulence of the FOP-071PT, but xylose and lactose decrease the virulence of the pathogen.