臺灣博碩士論文加值系統

紅龍果莖潰瘍病的病原菌為葡萄座腔菌科之Neoscytalidium dimidiatum (Penz.) Crous & Slippers，該病原菌已在許多國家被報導為紅龍果之主要病原。N. dimidiatum會於枝條、果實上造成黃色凹陷斑點及潰瘍等病徵，使產量下降並導致果實喪失商品價值。根據田間觀察，莖潰瘍病病徵可能分布於整個枝條，或侷限於枝條上的特定段落。其中，年輕枝條似乎較容易受到病原菌侵入感染，然而，尚無研究深入探討枝條生長階段或生理狀況對病徵潛伏期或病斑擴展速度的影響。因此，本研究擬針對寄主生理因子對於病徵潛伏期與病斑擴展的影響進行深入探討。除此之外，目前管理該病害的方式仍以施用化學藥劑為主，為了尋找替代的防治方法，本研究亦篩選有益微生物，期能有助管理該病害。由於表皮為植物抵禦病原菌的第一道防線，加上表皮厚度會隨著枝條發育與年齡而增加，因此本研究假設枝條表皮厚度與相對年齡會影響接種後病徵出現的時間(病徵潛伏期)與病斑的擴展。在接種大紅(cv. Dahong)與越南白肉(cv. Vietnamese White)枝條上的不同段落後(代表不同相對年齡)，觀察記錄並分析枝條相對年齡、表皮厚度、病徵潛伏期與病斑擴展之間的關係。結果顯示，莖潰瘍病之病徵潛伏期與枝條相對年齡之間具有正相關性，但不一定與表皮厚度有正相關。年輕段落的表皮厚度通常較薄，其病徵潛伏期約5.6~7.0天，且病斑擴展較快；成熟及老化段落之病徵潛伏期則分別約為8.0~10.4天與10.6~14.7天，且病斑擴展較慢。若在枝條上製造傷口後進行接種，則會顯著的縮短病徵潛伏期至3.0~5.0天；而表皮受傷的年輕段落相較於成熟及老化的段落，仍有較短的病徵潛伏期且病斑擴展較快的趨勢，說明除了表皮的物理防禦外，不同生長階段的枝條，可能存在其他防禦反應上的差異。此外，為了尋找替代農藥的防治方法，本研究於土壤中篩選了50個菌株，其中，Bv14、Bv19、Bv22及Bv44四個菌株抑制N. dimidiatum菌絲生長效果最佳，且能抑制孢子發芽並產生抗菌之揮發性有機化合物(VOCs)，經確認其對白菜種子發芽無不良影響。經16S rDNA序列分析後初步確認四個菌株皆屬於Bacillus 屬的細菌，根據生理生化測試結果，四個菌株都能夠分泌澱粉酶、蛋白質酶、纖維素酶並具有溶磷能力。進一步評估Bacillus sp. Bv14、Bv19、Bv22、Bv44於枝條上之防治效果，結果顯示，當各菌株之培養液原液與病原菌孢子同時接種於枝條時，會使病徵潛伏期略為縮短，且會增加罹病度；提前一天施用稀釋100倍之Bv44培養液或Bv44細胞懸浮液亦無顯著之防治效果，然而添加0.5% K2HPO4的配方，則可有效延後病徵出現時間並降低枝條的罹病度，未來可再深入探討如何將拮抗菌株製劑化，作為低風險期時紅龍果莖潰瘍病的防治替代方案。

Neoscytalidium dimidiatum, a fungus belonging to the family Botryosphaeriaceae, has been documented as a major pathogen on dragon fruit (Hylocereus spp.) in many countries. The pathogen can cause yellow sunken spots and necrotic lesions on cladodes and fruits, leading to severe yield losses and a reduction of market value. Based on field observations, stem canker symptoms could distribute across the entire cladode or be limited in certain sections of infected cladodes, and the symptoms seems to appear earlier on younger cladodes. However, the effect of cladode developmental stages and physiological conditions on the incubation period of infection process or lesion expansion remains unexplored. Since the epidermis plays an important role on plant physical defense, and the epidermal thickness gradually increases during the vegetative growth of cladode, we hypothesized that the incubation period and lesion expansion rate are affected by the age of the cladode and the epidermal thickness when weather conditions are conducive. The relationship between the cladode age (represented by different sections of the cladode), epidermal thickness, and the incubation period and lesion expansion were investigated after artificial inoculating the cladodes of Dahong and Vietnamese White cultivars in a controlled environment. The results exhibited a positive association between the incubation period and relative cladode age, but not necessarily the epidermal thickness. Younger parts of the cladode with thinner epidermis tended to have a shorter incubation period of 5.6~7.0 days, along with more rapid symptom development. On contrary, longer incubation period of 8.0~10.4 days and 10.6~14.7 days and slower symptom development was observed in the mature and aged sections of the cladodes, respectively. When the cladodes were wounded, the incubation period was significantly shortened compared to unwounded ones. Nevertheless, lesion expansion rate in mature and aged sections was still slower compared to younger sections, suggesting that in addition to the physical defense provided by the epidermis, ontogenic resistance or other defense responses may also be involved in different developmental stages of the cladodes. Since the disease is primarily managed by fungicides, the second objective was to screen for potential biocontrol agents against dragon fruit stem canker. Among 50 bacterial strains isolated from soil, Bv14, Bv19, Bv22 and Bv44 showed the best in vitro antagonistic effect against N. dimidiatum mycelial growth and conidial germination. The bacterial strains could also produce antifungal volatile compounds against N. dimidiatum and had no adverse impact on pak-choi seed germination. Based on the biochemistry tests, the four bacteria could produce amylase, protease, cellulose, and showed phosphate solubilization activities, and were identified belonging to the Genus Bacillus through 16S rDNA sequence analysis. To further evaluate the efficacy of cultural broth on disease management, Bacillus spp. Bv14, Bv19, Bv22, Bv44 were respectively incubated in nutrient broth. Application of cultural broth of Bacillus spp. Bv14, Bv19, Bv22, Bv44 with the spore suspension of N. dimidiatum shortened the incubation period and increased the disease severity on the cladodes. Furthermore, applying 100-fold diluted culture broth or cell suspension of Bv44 one day before inoculation also showed no significant control effect, while adding 0.5% K2HPO4 in the formula effectively extended the incubation period and reduced the disease severity. Further studies are warranted to determine the optimal formula and application methods for applying the biocontrol agents as a fungicide alternative when there is a low risk of dragon fruit stem canker.

摘要 i
ABSTRACT iii
目錄 v
表次索引 vii
圖次索引 viii
前言 1
材料與方法 7
一、供試植株與病原菌株來源 7
(一)、供試植株來源 7
(二)、供試病原菌株來源、保存與活化 7
二、寄主生理因素對紅龍果莖潰瘍病病徵潛伏期之影響 7
(一)、供試病原菌株與接種源的製備 7
(二)、枝條相對年齡與表皮厚度對病徵潛伏期之影響 8
(三)、枝條表皮受傷對病徵潛伏期之影響 9
三、拮抗菌株之分離與篩選 9
(一)、拮抗菌之誘釣、分離及保存 9
(二)、初步篩選對N. dimidiatum具有抑制效果之拮抗菌 10
四、拮抗菌株之分子鑑定與生理特性 10
(一)、拮抗菌株之DNA萃取與初步分子鑑定 10
(二)、胞外水解酵素與溶磷能力測試 12
五、拮抗菌株之生物防治潛力評估 12
(一)、供試病原菌株 12
(二)、拮抗菌株對N. dimidiatum菌絲生長之影響 13
(三)、拮抗菌株培養濾液對N. dimidiatum孢子發芽之影響 13
(四)、拮抗菌株產生揮發性有機化合物(VOCs)之能力 14
(五)、拮抗菌株對白菜種子發芽之影響 14
六、拮抗菌株對紅龍果莖潰瘍病的影響 15
(一)、拮抗菌株培養液原液對紅龍果莖潰瘍病的影響 15
(二)、不同配方對紅龍果莖潰瘍病的影響 15
七、統計分析 16
結果 18
一、寄主生理因素對紅龍果莖潰瘍病病徵潛伏期之影響 18
(一)、不同相對年齡枝條段落的表皮厚度 18
(二)、不同相對年齡枝條段落的病徵潛伏期與病斑擴展 18
(三)、枝條相對年齡與表皮厚度對病徵潛伏期之影響 19
(四)、枝條表皮受傷對病徵潛伏期之影響 19
二、拮抗菌株之分離與初步篩選 20
三、拮抗菌株之分子鑑定與生理特性 20
(一)、拮抗菌株之初步分子鑑定結果 20
(二)、胞外水解酵素與溶磷能力測試 21
四、拮抗菌之生物防治潛力評估 22
(一)、拮抗菌株對N. dimidiatum菌絲生長之影響 22
(二)、拮抗菌株培養濾液對N. dimidiatum孢子發芽之影響 22
(三)、拮抗菌株產生之揮發性有機化合物(VOCs)對N. dimidiatum菌絲生長之影響 23
(四)、拮抗菌株對白菜種子發芽之影響 23
五、拮抗菌株對紅龍果莖潰瘍病的影響 23
(一)、拮抗菌株培養液原液對紅龍果莖潰瘍病的影響 23
(二)、不同配方對紅龍果莖潰瘍病的影響 24
討論 26
參考文獻 34
表圖 45

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