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標題: 評估番茄萎凋病菌厚膜孢子之逆境耐受能力
Assessing the stress resistant abilities of chlamydospores of Fusarium oxysporum f. sp. lycopersici
作者: Yuan-Yu Chen
關鍵字: 番茄萎凋病;厚膜孢子;逆境;生物防治;Fusarium wilt of tomato;Fusarium oxysporum f. sp. lycopersici;chlamydospore;stress;biocontrol
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番茄萎凋病是由病原真菌 Fusarium oxysporum f. sp. lycopersici 所造成的土壤傳播性病害,此病原菌可藉由產生分生孢子及厚膜孢子增殖。一般認為由於厚膜孢子可於土壤中長期殘存,導致根除此病害較為困難,但番茄萎凋病菌厚膜孢子對逆境條件的耐受能力之研究仍闕如。本篇研究中,利用土壤栽培介質之萃取液,作為產生病原菌株 CT-Fo-5 之厚膜孢子的基質。經測試11種在市售的土壤栽培介質,以 BVB 和 KEKKILÄ 栽培介質之萃取液添加 0.5% (w/v) 葡萄糖,分別能生成最大量的厚膜孢子,濃度可達 5×105 chlamydospores/ml 以上。此外,添加 0.03% (w/v) 的硫酸銨至BVB土壤萃取液中,可明顯提升厚膜孢子的數量,並降低分生孢子的產生。為了解厚膜孢子之逆境耐受能力,因此比較厚膜孢子與分生孢子於不同逆境下之發芽率,厚膜孢子在高溫、高pH值、高滲透壓、高過氧化物及高濃度剛果紅的逆境下,發芽率皆優於分生孢子。以超過60℃之熱處理5分鐘下,厚膜孢子與分生孢子的發芽率皆顯著降低。在四種防治萎凋病的殺菌劑處理下,厚膜孢子的發芽能力對撲克拉、賽普護汰寧較不敏感,而對貝芬替、鋅錳乃浦較為敏感,其中濃度超過 50 ppm 之鋅錳乃浦即可使厚膜孢子及分生孢子完全無法發芽,且其對孢子為殺菌作用。生物逆境方面,當厚膜孢子與分離自土壤的拮抗微生物之上清培養液或菌體共同培養一天後,其中11個拮抗微生物菌
株可以降低厚膜孢子的發芽率至40%以下,並以菌株 PL T-1 N-5.5 的效果為最佳,厚膜孢子經其上清培養液或菌體處理下幾乎無法發芽,亦能顯著減少厚膜孢子形成菌落,但其濾液處理則對厚膜孢子沒有抑制效果。經過16S rDNA、脂肪酸甲酯及Biolog鑑定,菌株PL T-1 N-5.5 鑑定為 Brevibacillus sp.。本研究證實番茄萎凋病菌之厚膜孢子確實能比分生孢子更加耐受多種不良的逆境,並顯示厚膜孢子之耐受程度作為防治此病害的參考。

Fusarium wilt of tomato caused by Fusarium oxysporum f. sp. lycopersici is a soil-borne disease. The pathogen produces macroconidia, microconidia and chlamydospores for propagation. It is wildly accepted that the disease is difficult to eradicate because its thick-walled chlamydospores can persist in soil for a long time. However, the study on stress resistance ability of F. oxysporum f. sp. lycopersici is still largely lacking. In this study, we used the potting soil broth as culture medium to generate chlamydospores of F. oxysporum f. sp. lycopersici strain CT-Fo-5 in one week. Among 11 potting soils, BVB and KEKKILÄ potting soil broths with 0.5% (w/v) glucose could induce chlamydospres in a concentration of at least 5×105 chlamydospores/ml, respectively. By adding 0.03% (w/v) (NH4)2SO4 into BVB broth, the ratio of chlamydospores/conidia significantly increased. Comparing the germination rate of chlamydospores and microconidia of strain CT-Fo-5 in different stresses, the chlamydospores had higher germination rate than conidia in alkaline pH levels and at 36℃, and could be more tolerant to higher concentration of osmotic stress, oxidative stress and Congo red. Both chlamydospores and conidia dramatically reduced the germination after a heat shock of 60℃ for 5 minutes. In fungicide tests, the germination of chlamydospores was resistant to prochloraz and cyprodinil + fludioxonil, but sensitive to carbendazim and mancozeb. When the chlamydospores of strain CT-Fo-5 were treated with the supernatants or pellets of soil microorganisms, the pellet treatments of 11 microorganisms reduced the germination rate of chlamydospores to a level below 40%. Especially, the strain PL T-1 N-5.5 almost completely inhibited the germination and dramatically decreased the number of colonies formed from chlamydospores, but the filtrate of PL T-1 N-5.5 was no inhibition effect on chlamydospores. By the analyses of 16S rDNA, fatty acid methyl ester (FAME) and Biolog, the strain PL T-1 N-5.5 was identified as Brevibacillus sp. These results consent to the notion that chlamydospores are more resistant to various stresses than conidia, and provide information for disease control.
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