二氧化氯對數種細菌性植物病原之殺菌效率及其應用於種子、切花及果實處理上之除菌效果

Abstract

二氧化氯（chlorine dioxide；以下簡稱ClO2）為強氧化劑，能有效地殺死病原微生物和病毒，可作為消毒劑和漂白劑。其應用範圍廣泛，常用於環境衛生、飲用水、游泳池、冷卻水塔之水、食品加工、廢水處理、醫療衛生等之消毒殺菌處理及紡織品、紙漿、纖維之漂白處理。ClO2在防除蔬果、農畜產品及海鮮食品上數種有害人體微生物之污染，亦有良好效果，且無安全上之疑慮。ClO2為廣效性殺細菌劑，但在植物病原細菌之應用上，研究甚少，因此廣泛評估ClO2防治細菌性植物病害之可行性，有其必要。本研究選擇台灣數種重要細菌性植物病害，探討ClO2對這些病原之殺菌能力及防治應用。主要研究內容包括ClO2溶液：1)對十字花科蔬菜黑腐病菌（Xanthomonas campestris pv. campestris）、茄科細菌性斑點病菌（X. axonopodis pv. vesicatoria）及瓜類細菌性果斑病菌（Acidovorax avenae subsp. citrulli）、文心蘭花梗軟腐病菌（Pectobacterium chrysanthemi）及柑桔潰瘍病菌（X. citri subsp. citri）之殺菌效率；2)應用於花椰菜、甜椒及西瓜種子處理對去除相關病原之效果；3)應用於文心蘭切花處理對花梗軟腐病之防治效果；及4)應用於文旦果實處理對去除潰瘍病菌之效果。結果顯示以ClO2溶液10 ppm處理黑腐病菌及5 ppm處理斑點病菌20分鐘、50 ppm處理果斑病菌30分鐘、5 ppm處理軟腐病菌5分鐘或10 ppm 處理潰瘍病菌10分鐘，皆可完全抑制所有供試病原菌菌株之生長。ClO2溶液以10 ppm浸漬處理人工污染黑腐病菌Xcc52菌株之花椰菜種子30分鐘，5 ppm浸漬處理人工污染細菌性斑點病菌XVT-28菌株之甜椒種子10分鐘及50 ppm浸漬處理人工污染或自然帶有果斑病菌之西瓜種子30分鐘，均可有效去除各病菌，獲致無病菌污染之種子，且此處理不會影響或可提高種子之發芽率。在人工污染及自然帶有果斑病菌之西瓜種子試驗上，ClO2之處理亦可完全防治幼苗果斑病之發生，使發病率由66.6(人工帶菌)或29.2(自然帶菌)降低至0%，且對幼苗之生長無影響甚或可促進。文心蘭花梗以ClO2溶液浸漬處理之濃度在50 ppm或以上時，會造成花梗基部產生褐化之藥害現象，但濃度在較低（1、5及10 ppm）時，則無明顯藥害。文心蘭切花浸漬於不同濃度（102-108 CFU ml-1）之軟腐病菌懸浮液後，花梗軟腐病之發生程度隨病菌濃度增高而增加，而在此病菌懸浮液中加入最終濃度為5 ppm之ClO2後，軟腐病之發生程度均顯著降低；若ClO2之濃度達10 ppm，則完全防止軟腐病之發生。文心蘭切花花梗以5或10 ppm ClO2溶液浸漬處理7天後，與只接種軟腐病菌者比較，可顯著減少花朵枯萎或落花數，但與單獨浸漬無菌水相比，則無顯著性差異。此結果表示浸漬處理10 ppm ClO2溶液，可防治切花花梗軟腐病，且不會影響切花之壽命。以10 ppm ClO2溶液每週一次連續噴灑處理文心蘭植株15週後，不會影響其葉片之生長，且連續噴灑處理40週後，亦不會影響其抽梗數。由文旦與白柚罹病葉分離之潰瘍病菌菌株，經病原性與PCR鑑定均屬於X. citri subsp. citri（即原先所稱之CBC A type）。以Bio-PCR檢測結果顯示，由發病或未發病果園採集之無病徵文旦與白柚果實表面未檢測出潰瘍病菌，而利用10 ppm二氧化氯溶液，浸漬處理人工污染潰瘍病菌之文旦果實10分鐘，可有效殺滅果實表面的潰瘍病菌。由本研究結果得知，ClO2溶液具有防治植物細菌性病害，尤其是種子傳播及收穫後病害之潛力。

Chlorine dioxide(ClO2)is a strong oxidizer which can effectively kill pathogenic microorganisms and viruses. It is widely used as a disinfectant in the environmental sanitation, drinking water, swimming pool, cooling tower water, foodstuffs production, sewage water, and medical sanitation, it also can be used as a bleach for bleaching of the textile products, paper, and fiber. ClO2 treatments of vegetables and fruits, agricultural and livestock products, and seafood are also highly effective in decontamination to human disease-causing microorganisms without any significant risks of chemical residue. ClO2 is a broad-spectrum bactericide, however, its application for control of bacterial plant diseases is little studied. Therefore, a more comprehensive evaluation of the effectiveness of ClO2 to control phytopathogenic bacteria is necessary. Several important bacterial plant diseases in Taiwan were selected for this study. Specifically the purposes of this study were to determine the efficacies of ClO2 solution in 1) inhibition of growth of Xanthomonas campestris pv. campestris(Xca), the causal agent of black rot of crucifers; X. axonopodis pv. vesicatoria(Xav), the causal agent of bacterial spot of tomato and pepper; Acidovorax avenae subsp. citrulli(Aac), the causal agent of bacterial fruit blotch of cucurbits; Pectobacterium chrysanthemi(Pch), the causal agent of soft rot of Oncidium ; and X. citri subsp. citri(Xci), the causal agent of citrus bacterial canker; 2) application on seed treatment for decontaminating Xca, Xav, and Aac on cauliflower, pepper, and watermelon seeds, respectively; 3) application on cut flowers of Oncidium for control of bacterial stalk soft rot; and 4) application on pomelo fruit treatment for eradication of Xci. The results showed that treatment of various strains of Xca and Xav with 10 and 5 ppm ClO2 solution, respectively, for 20 min, strains of Aac with 50 ppm ClO2 solution for 30 min, strains of Pch with 5 ppm ClO2 solution for 5 min, and strains of Xci with 10 ppm ClO2 solution for 10 min completely inhibited the growth of these bacteria on agar media. Treatments of cauliflower seeds artificially infested with Xca with 10 ppm ClO2 solution for 30 min, pepper seeds artificially infested with Xav with 5 ppm ClO2 solution for 10 min, and watermelon seeds artificially or naturally infested with Aac with 50 ppm ClO2 solution for 30 min, completely removed the contaminated bacteria from these seeds. The effective concentrations of ClO2 solution did not affect or may increase the germination rate of the seeds. In the experiments with artificially or naturally infested watermelon seeds, the ClO2 treatment not only eradicated the infested bacteria but also completely controlled the fruit blotch disease at the seedling stage, since no diseased seedlings were observed in the treated seeds in contrast to about 66.6%(artificially infested)or about 29.2 %(naturally infested)diseased seedlings in the nontreated control. The ClO2 treatment did not affect or may show better growth of the watermelon seedlings as compared with the nontreated control. Oncidium cut flower stalk showed sign of toxicity(brown discoloration at basal part) after dipping into 50 ppm or above of ClO2 solution for 5 days, but lower concentrations (1, 5 and 10 ppm) were no toxic to the cut flower stalks. Stalks of Oncidium cut flower were inoculated by dipping into various concentrations(102-108 CFU ml-1)of Pch. The severity of stalk rot increased with increasing concentrations, but when the inoculum was added with 5 ppm ClO2 solution, the disease severity was reduced significantly at all inoculum levels, and the disease was completely controlled with ClO2 added at 10 ppm. Treatment of cut flower stalks by dipping into Pch suspension containing 5 or 10 ppm ClO2 for 7 days reduced significantly the number of withered and fallen flowers as compared to the stalks treated with Pch only, but was not significantly different from those that treated with sterile water. These indicate that dipping treatment with 10 ppm ClO2 solution could control the bacterial stalk rot and did not affect the longevity of cut flower. Spraying leaves of Oncidium plants with 10 ppm ClO2 solution at weekly intervals after 15 weeks did not affect the growth of the leaves, and also did not affect the number of stalks developed after spraying for 40 weeks. The bacterial strains isolated from cankered leaves of Wentan and Pai pomelos were identified as X. citri subsp. citri(formally as citrus bacterial canker A type)by pathogenicity and PCR tests. X. citri subsp. citri was not detected by the bio-PCR technique on surfaces of bacterial canker symptomless fruits of Wentan and Pai pomelos, collected from four orchards either with or without diseased plants. Treatments of Wentan pomelo fruits artificially infested with Xci with 10 ppm ClO2 solution for 10 min, completely removed the contaminated bacteria from surfaces of the fruit. This study indicates that ClO2 solution has a potential to control bacterial plant diseases, particularly the seedborne and postharvest diseases.