Bacillus amyloliquefaciens PMB05之最適發酵條件建立與其應用於檸檬潰瘍病之防治

Optimization of submerged fermentation conditions on Bacillus amyloliquefaciens PMB05 and its application on control bacterial canker of lemon

林鍇薳(Lin Kai-Wei)

指導教授 ： 林宜賢

國立屏東科技大學/農學院/植物醫學系所/碩士(2020年)

https://doi.org/10.6346/NPUST202000275

摘要

檸檬是國際上重要的經濟作物，在台灣以高雄及屏東地區為最主要的產區。在作物的生長期中，常受Xanthomonas citri所引起之檸檬潰瘍病危害而造成品質下降，因此如何防治此病害為重要的課題。本研究主要探討應用可提升植物免疫訊號之Bacillus amyloliquefaciens PMB05菌株來防治檸檬潰瘍病的發生並建立其發酵液之發酵條件。首先確認B. amyloliquefaciens PMB05菌株在檸檬葉片上是否具有提升植物免疫及防禦相關酵素之效果，在研究結果中顯示PMB05確實可強化檸檬潰瘍病菌所誘導的激活化氧產生與癒傷葡聚糖累積外，亦會提升多酚氧化酶與苯丙胺酸裂解酶之活性。進一步利用PMB05細菌懸浮液於溫室試驗之結果亦顯示對檸檬潰瘍病之防治效果顯著，罹病度可降低25.6%。之後以PMBFL配方製成之發酵液於溫室中更可降低42.3%之罹病度；在田間試驗中也能降低43.3%之罹病度。隨後，為了更進一步提升發酵液菌量及產孢率，來達到更有效防治檸檬潰瘍病之目標，分別進行500 mL搖瓶、10 L、30 L發酵槽之最佳發酵條件評估。於500 mL搖瓶不同碳氮比試驗顯示碳氮比3:1有最高之產孢率，並將此配方稱PMBFL-2A。進一步於10 L發酵槽中不同碳氮比、通氣量試驗，結果顯示，PMBFL-2A在1.5vvm下有最高之產孢率。進一步在30 L發酵槽中，評估不同轉速對於產孢率之影響上亦顯示在120 rpm條件下有最好產孢率。將30 L發酵槽經此條件下所獲得之發酵液進行儲架壽命試驗之結果顯示，在54°C處理十四天下及在4、25、37°C處理八個月都能維持90%以上的菌量。同時，我們也證明PMBFL-2A發酵培養基的配方條件所獲得之發酵液相較於細菌懸浮液可顯著增強PMB05在檸檬葉片上所提升的免疫反應，於溫室處理下100、200及500倍稀釋後分別在罹病度上減少64.4、61.0及53.0%之檸檬潰瘍病發生。在2020年所進行的田間試驗中三個月內之防治率更達100%。綜上所述，本研究已建立可增強植物免疫反應之Bacillus amyloliquefaciens PMB05之最適量產條件且具有應用在田間防治檸檬潰瘍病之潛力。

關鍵字

檸檬潰瘍病 ； PAMPs-triggered immunity (PTI) ； Bacillus amyloliquefaciens PMB05 ； 液態發酵

並列摘要

Citrus limon is crop with economic importance worldwide. In Taiwan, both Kaohsiung and Pingtung are the most important producing areas. During the growing seasons of lemon, citrus canker caused by Xanthomonas citri subsp. citri (Xcc) is the main limiting factor. Thus, strategies on how to control bacterial canker are important issues on lemon production. The main purpose in this study is how to apply a plant PAMPs-triggered immunity- intensifying Bacillus amyloliquefaciens strain PMB05 on controlling bacterial canker of lemon. First, the intensification of plant immune responses and defense-related enzymes by B. amyloliquefaciens PMB05 strain was assay on lemon leaves. Results revealed that PMB05 can indeed intensify the rapid ROS generation and callose deposition induced by Xcc. In addition, the activities of polyphenol oxidase and phenylalanine ammonia-lyase were increased by PMB05 upon inoculation of Xcc. In the inoculation assay performed by using bacterial suspension of PMB05, the disease severity of citrus canker can be reduced by 25.6% in the greenhouse. Disease control done by initial PMBFL formula in greenhouse and field were reduced 42.3% and 43.3%, respectively. To further explore the formula on increasing bacterial populations and sporulations, optimal conditions were evaluated in 500 mL shake flasks, 10 L, and 30 L fermentation bioreactors step by step. In the results from 500 mL flask showed the carbon/nitrogen of 3:1 had the highest sporulation, and this formula was named PMBFL-2A. Further, PMBFL-2A had the highest sporulation at 1.5 vvm in 10 L fermentation bioreactor. Finally, PMBFL-2A at 120 rpm had the highest sporulation in a 30 L fermentation bioreactor. The the fermentation broth obtained from the 30 L fermentation bioreactor could maintain over 95% of survival rate at 54 °C for 14 days. And, over 90% of survival rate of fermentation broth while storage at 4, 25 and 37 °C for over 8 months and. Meanwhile, the fermentation broth prepared with PMBFL-2A formula can intensify the immune response upon Xcc inoculation on lemon leaves significantly. The disease severity of citrus canker can be reduced 64.4, 61.0, and 53.0% by 100, 200, and 500-fold dilutions, respectively. The bacterial canker was complete inhibited within three months in the field trial conducted in 2020. In summary, Bacillus amyloliquefaciens PMB05 fermentation broth prepared with the PMBFL-2A formula, which can intensity plant immune response to control bacterial canker disease of lemon.

並列關鍵字

Bacterial citrus canker ； PAMPs-triggered immunity(PTI) ； Bacillus amyloliquefaciens PMB05 ； Submerged fermentation

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