應用Bacillus amyloliquefaciens PMB05發酵液防治檸檬潰瘍病

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 subsp. 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菌株所建立之發酵液是否可防治檸檬潰瘍病的發生。首先確認B. amyloliquefaciens PMB05細菌懸浮液可在檸檬葉片上強化檸檬潰瘍病菌所誘導的激活化氧產生與癒傷葡聚醣的累積等植物免疫訊號外，也能在檸檬葉片上展現良好的存活能力。進一步PMB05於溫室試驗中，利用細菌懸浮液測試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%。為了可進一步應用於田間，本研究分別利用500毫升搖瓶及30公升發酵槽來評估較佳之發酵條件，由菌量及產孢率兩項指標的結果顯示在500毫升的搖瓶培養中，碳氮比3:1能獲得最高之產孢率，並將此配方所獲得之發酵液稱PMBFL-2A。進一步於30公升發酵槽中也顯示PMBFL-2A之發酵配方在120 rpm及1.5vvm通氣量下有最高之產孢率。將30公升發酵槽所獲得之PMBFL-2A發酵液進行儲架壽命試驗之結果顯示，54°C處理二周及分別在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發酵液相較於單純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，於溫室條件下處理100、200及500倍稀釋之發酵液後分別減少檸檬潰瘍病之罹病度達64.4、61.0及53.0%。在2020年於屏東縣里港鄉所進行的田間試驗至第三個月的防治率更達100%。綜上所述，本研究已建立可增強植物免疫反應之Bacillus amyloliquefaciens PMB05之最適發酵條件，且具有應用在田間防治檸檬潰瘍病之潛力。

Lemon is an internationally important economic crop. In Taiwan, Kaohsiung and Pingtung are the main producing areas. During the growth period of crops, bacterial canker caused by Xanthomonas citri subsp. citri (Xcc) is often reduce its quality. Due to the demand on disease control, this study mainly explores whether the fermentation broth established by using Bacillus amyloliquefaciens PMB05, which can enhance plant immune signals and further contribute to control bacterial canker. Firstly, it was confirmed that the bacterial suspension of B. amyloliquefaciens PMB05 enhanced the plant immune signals such as reactive oxygen generation and callose deposition induced by Xcc on lemon leaves. B. amyloliquefaciens PMB05 also exhibited good survival ability on lemon leaves. Furthermore, in the greenhouse test of PMB05, the disease severity of bacterial canker can be significant reduced by 25.6% in the treatment with the bacterial suspension. To further develop its fermentation broth, bacteria amount and sporulation rate were used as indicators to evaluate the formula in 500 mL flasks and 30 L fermentation tanks. In the 500 mL shake flask culture, results revealed that the carbon to nitrogen ratio of 3:1 can achieve the highest cell yield and sporulation rate, and this fermentation broth is called PMBFL-2A. Furthermore, the 30 L fermentation tank also showed that PMBFL-2A had the highest sporulation rate under the 120 rpm and 1.5 vvm. The shelf life assay showed that the 54 °C treatment for two weeks and the treatment at 4, 25, and 37 °C for 8 months can maintain more than 90% of the bacterial population in PMBFL-2A. At the same time, we also proved that the PMBFL-2A fermentation broth can significantly enhance the immune response of PMB05 on lemon leaves compared to that of the bacterial suspension. After 100, 200 and 500 folds dilutions of PMBFL-2A, the occurrence of bacterial canker was reduced by 64.4, 61.0, and 53.0% in disease severity, respectively, in the greenhouse. In 2020, the field trial conducted in Ligang, Pingtung County, the control rate reached 100% to the third month. In summary, this study has established the optimal fermentation conditions for Bacillus amyloliquefaciens PMB05, which can enhance plant immune response, and has the potential to be used in the field to control bacterial canker of lemon.