評估含鈣化合物對增強植物免疫反應之Bacillus amyloliquefaciens PMB05菌株於番茄青枯病上之防治

Evaluation of calcium compounds on plant immunity intensifying Bacillus amyloliquefaciens PMB05 for disease control of tomato

楊晴安(Yang Qing-An)

指導教授 ： Lin ； Yi-Hsien

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/NPUST202000097

摘要

番茄青枯病是由Ralstonia solanacearum (RS) 所引起，在熱帶、亞熱帶及溫帶地區均會發生，為世界上極具破壞性的重要細菌性病害。此病害目前還未有單一且有效的防治方法，在防治相關的研究議題中，應用拮抗微生物的防治法為許多科學家的發展方向。已有研究顯示Bacillus amyloliquefaciens PMB05於番茄根圈可減少青枯病的發生，此防治效果可能與PMB05菌株具有提升植物PAMPs-triggered immunity (PTI) 的機制有關。PMB05菌株是否可在番茄上提升PTI仍未直接證實。因此，除釐清PMB05在PTI路徑上的功能外，因鈣離子也在PTI路徑上扮演訊息傳遞的角色，本研究亦進一步分析不同含鈣化合物的處理對PMB05所強化的PTI反應的影響與防治番茄青枯病之效果。本研究首先證明PMB05菌株之細菌懸浮液確實在二個不同栽培種番茄上均有防治青枯病發生之效果。進一步於防治效果較佳之番茄栽培種上，以青枯病菌之PopW作為誘引物，來分析PMB05菌液是否在激活化氧產生及癒傷葡聚醣累積這兩項PTI指標訊號的強度，結果顯示在PMB05確實可在番茄上強化PopW誘導的免疫訊號。進一步在含鈣化合物的試驗中，分別進行不同含鈣化合物對PMB05菌株對青枯病菌的拮抗作用、PopW在番茄葉片上所誘導的過敏性反應與PTI指標訊號及對番茄青枯病防治效果等項目進行分析。在對青枯病菌的拮抗作用的結果中顯示，氯化鈣與硝酸鈣在高濃度下仍對PMB05抑制青枯病之活性沒有影響，而僅高濃度之碳酸鈣及硫酸鈣會減少其對青枯病之活性沒有影響，氯化鈣則使PMB05之拮抗活性完全消失。在低濃度的情況下，五種含鈣化合物對PMB05抑制青枯病菌的活性皆沒有影響。在不同含鈣化合物對過敏性反應與植物免疫訊號影響的結果顯示，僅碳酸鈣可顯著提升PMB05所強化的過敏性反應與植物免疫訊號。分別利用鈣離子通道阻斷劑LaCl3及鈣離子螯合劑EGTA在不同含鈣化合物處理中均顯示在番茄葉片上會顯著降低PopW所誘導過敏性發生的影響，說明鈣離子確實參與了PTI路徑。進一步測試不同鈣離子對PMB05菌株於防治番茄青枯病的效果，結果亦顯示碳酸鈣的添加相較於僅處理PMB05細菌懸浮液的對照組可更顯著降低對番茄青枯病的罹病度。綜合上述結果，Bacillus amyloliquefaciens PMB05可在番茄上具有提升PTI免疫反應之效果，且碳酸鈣為有效增加此免疫反應之效果，且碳酸鈣為有效增加此免疫反應與對番茄青枯病防治效果的含鈣化合物。

關鍵字

不同含鈣化合物 ； Bacillus amyloliquefaciens PMB05 ； 植物免疫反應 ； 番茄青枯病

並列摘要

Bacterial wilt caused by Ralstonia solanacearum (RS) is one of the most destructive diseases on tomato in tropical, subtropical and temperate regions. This pathogen cause rapid and fatal wilting of host plants. At present, there is no completely effective method to control this disease. Many researchers are working on how to prevent and control this disease, including application of antagonistic microorganisms. Our previous study exhibits that bacterial wilt of tomato was reduced by a PAMP-trigger immunity (PTI) intensifying rhizobacterium, Bacillus amyloliquefaciens strain PMB05. The control efficacy may be related to the PAMPs-triggered immunity (PTI) intensified by strain PMB05. However, whether the PTI can be enhanced by PMB05 on tomato has not been confirmed. In addition to the calcium ion also play a role in PTI signaling pathway, its function on combining PMB05 in PTI pathway is still need to be studied. This study first revealed that the bacterial suspension of PMB05 did prevent the occurrence of bacterial wilt on two different cultivated tomatoes. Further, on tomato cultivars with better control effects, PopW from Ralstonia solanacearum was used as an PAMP to analyze whether the PMB05 bacterial solution is activating the two PTI indicators, rapid reactive oxygen generation and callose deposition. These results show that PopW-induced immune signals were enhanced by PMB05 in tomato leaves and roots. Furthermore, in the assay of calcium-containing compounds, their effects on PMB05 to the inhibitory effect against R. solanacearum, PopW-induced hypersensitive reaction (HR) and PTI signals on tomato leaves, and control efficacy on tomato bacterial wilt were assayed. The results of antagonism against Ralstonia solanacearum showed that Calcium chloride and calcium nitrate still have no effect on the bacterial wilt resistance of PMB05 at high concentrations, while only high concentrations of calcium carbonate and calcium sulfate will reduce their inhibitory effect. The inhibitory activity was abolished in the treatment with calcium chloride. At low concentrations, none of the five calcium-containing compounds had any effect on PMB05's activity against R. solanacearum. The results of different calcium-containing compounds on HR and plant immune signals showed that only the calcium carbonate can significantly enhance the HR and plant immune signals intensified by PMB05. The calcium ion channel blocker, LaCl3, and calcium ion chelate compound, EGTA, also exhibited the PopW-induced HR on tomato leaves in different calcium-containing compounds treatments were reduced significantly, indicating that calcium ions do participate in the PTI pathway. Finally, the effect of different calcium ions on the control of tomato bacterial wilt by PMB05 strains, the results also showed that the addition of calcium carbonate can significantly reduce the incidence of tomato bacterial wilt compared to the PMB05 alone treatment as control. Based on the above results, we demonstrated that Bacillus amyloliquefaciens PMB05 can improve the PTI immune response in tomato plant, and calcium carbonate is effective to increase the B. amyloliquefaciens PMB05 intensifying immune response and disease control of tomato bacterial wilt.

並列關鍵字

calcium compounds ； Bacillus amyloliquefaciens PMB05 ； plant immune response ； tomato bacterial wilt

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