台灣番石榴瘡痂病菌之鑑定及利用 Bacillus amyloliquefaciens 防治番石榴瘡痂病之效果評估

Identification of the pathogen causing guava scab disease (GSD) in Taiwan and evaluation of effects of the Bacillus amyloliquefaciens on the control of GSD

黃湘珊(Huang Siang-Shan)

指導教授 ： 林盈宏

國立屏東科技大學/農學院/植物醫學系所/碩士(2019年)

https://doi.org/10.6346/NPUST201900346

摘要

番石榴瘡痂病菌 (Pestalotiopsis spp.) 為番石榴於生長期及採收後貯藏時造成果實產量損失之重要病原菌。針對此病原菌目前多以化學藥劑來進行防治，基於菌株抗藥性問題及環保意識抬頭，仍須有其他防治方法來降低農藥之使用，而生物防治被認為是一種相對安全且對環境較友善之防治方法。在台灣的研究記錄中，番石榴瘡痂病菌為 Pestalotiopsis psidii，但目前有學者將 Pestalotiopsis 屬重新審視後區分出另 2 個屬 (分別為 Neopestalotiopsis 及 Pseudopestalotiopsis)。因此本論文擬釐清台灣引起番石榴瘡痂病的可能病原，並擬以生物防治策略來防治番石榴瘡痂病。首先本論文自台灣南部田間採集受番石榴瘡痂病菌感染之檢體，經由菌株形態特徵並依分子生物學方式進行形態特徵與親緣關係分析，結果顯示，本論文中所採集之番石榴瘡痂病菌在型態上與 Neopestalotiopsis 屬較為相似，且藉由真核生物核糖體內轉錄區 (Internal transcribed spacer, ITS)、編碼β-微管蛋白 (β-tubulin, TUB)、真核轉譯延伸因子 (Elongation factor 1-alpha, TEF)、核糖體去氧核糖核酸大次單元 (Large subunit, LSU) 等分子親緣鑑定的結果，顯示該些菌株皆與Neopestalotiopsis 屬的親緣關係上較為相近。此外，本論文亦自田間分離並篩選出的 3 株拮抗菌 PM-SSH-B009-NP、PM-SSH-B018-NP 及 PM-SSH-B060-NP，經 16S rDNA 基因序列分析後，鑑定為 Bacillus amyloliquefaciens。且經熱處理所形成的內生孢子與拮抗菌本身皆具耐紫外線 (Ultraviolet-C, UV-C) 的能力。此 3 株拮抗菌亦能抑制番石榴瘡痂病菌菌絲生長及降低番石榴瘡痂病菌分生孢子的發芽，且於顯微鏡下可看到處理拮抗菌懸浮液或濾液的瘡痂病菌孢子發芽管出現膨大、破裂的情況，且菌絲呈腫脹等不正長現象。為了瞭解此 3 株拮抗菌實際應用於果實上是否具有預防及防治番石榴瘡痂病之效果，則分別以共同接種試驗 (同時接種病原菌及拮抗菌)、治療試驗 (先接種瘡痂病菌)、與保護試驗 (先接種拮抗菌) 等 3 種不同試驗方式進行防治效果評估。由共同接種及保護接種之試驗結果顯示，處理 PM-SSH-B018-NP 或 PM-SSH-B060-NP 可降低瘡痂病病斑大小。另於防治之試驗結果發現，僅以 PM-SSH-B018-NP 處理後的果實病斑大小與對照組相比具有顯著性差異。綜合上述，本研究自田間篩選出之 3 株拮抗菌對番石榴瘡痂病菌具有抑制菌絲生長及孢子發芽之能力，並造成處理後的分生孢子發芽管及菌絲呈現膨大且不正常現象。實際應用於果實上，其中預先處理 PM-SSH-B018-NP 或 PM-SSH-B060-NP 於番石榴果實上更能有效降低番石榴瘡痂病菌對果實造成之危害。未來將更進一步探討拮抗菌防治此病害可能之機制，藉此擬出對番石榴瘡痂病生物防治之最佳策略。

關鍵字

番石榴瘡痂病 ； 液化澱粉芽孢桿菌 ； 譜系分析 ； 生物防治

並列摘要

Guava scab disease (GSD) caused by Pestalotiopsis spp. is an important disease causing loss of fruit yield during guava growth and storage after harvest. As most diseases, chemical agents are used to control GSD. On the basis of the present of the drug-resistant pathogens and the increasing rise of environmental disruption, an alternative method to reduce the use of pesticides for disease prevention and control is still needed. Biological control is considered as a safe and environmentally friendly prevention method. The aim of this study was to screen and identify the antagonistic microorganisms against guava scab. The research record of the guava scab was caused by Pestalotiopsis psidii in Taiwan. However, Pestalotiopsis sp. have been re-distinguished into two other genus (Neopestalotiopsis and Pseudopestalotiopsis). Therefore, the aim of this study was to clarify the possible pathogens causing guava scab in Taiwan, and to screen antagonistic microorganisms against guava scab. Firstly, in this study, the samples collected from the guava scab infection in the field in southern Taiwan were identified by morphological characteristics and molecular sequencing identification methods. The results showed that the pathogens causing guava scab disease in Taiwan were identified as Neopestalotiopsis sp. based on their morphological characteristics and the results of molecular phylogenetic identification of Internal transcribed spacer (ITS), β-tubulin (TUB), Elongation factor 1-alpha (TEF), Large subunit (LSU) sequences. In addition, three isolates of endospores-forming bacteria PM-SSH-B009-NP, PM-SSH-B018-NP and PM-SSH-B060-NP were identified as Bacillus amyloliquefaciens by 16S rDNA gene sequencing analysis. The three antagonistic bacteria can inhibit the growth of mycelial and reduce the spore germination of the pathogens Neopestalotiopsis sp. The three inoculation methods with co-, pre- and post-inoculation of antagonistic bacteria and the pathogen on guava fruit were used for evaluation of the practical effects on control and preventio of GSD. The results showed that co- and pre-treatment of PM-SSH-B018-NP or PM-SSH-B060-NP could reduce the lesion size of GSD. In summary, the three isolates antagonistic bacteria have the ability to inhibit mycelium growth and spore germination spore germination of Neopestalotiopsis The spore germination tube and hyphae became swollen and abnormal after antagonistic bacteria treatments. Pretreatment of PM-SSH-B018-NP or PM-SSH-B060-NP on guava fruit can effectively reduce the damage of GSD. In the future, in order to develop the best strategy for the biological control of guava scab, we will explore the possible mechanisms of the antagonistic bacteria against GSD.

並列關鍵字

Guava scab ； Bacillus amyloliquefaciens ； Phylogenetic analysis ； Biological control

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