臺灣博碩士論文加值系統

十字花科黑腐病 (Black rot disease, BRD) 是由 Xanthomonas campestris pv. campestris (Xcc) 所引起的病害。在全世界被認為是十字花科中最重要的病害。目前許多研究開發了各種方法來控制十字花科黑腐病，包括拮抗微生物之使用。在本研究中，利用土壤分離之 Bacillus amyloliquefaciens PMB04 及 PMB05 菌株來評估其防治甘藍黑腐病能力。首先在平板拮抗活性分析中，PMB04 及 PMB05 菌株皆對於 Xcc 不同菌株均有抑制的效果。進一步將此兩拮抗菌株分別使用預防性處理與清除性處理二種方式進行種子處理來評估其對甘藍黑腐病之防治效果。結果顯示，此兩種拮抗菌株於兩種處理中均對甘藍黑腐病有顯著的抑制效果，其中又以PMB05菌株的處理為佳。然而，在污染有黑腐病菌再處理拮抗菌的清除性處理中，黑腐病菌於幼苗上之族群卻在PMB04菌株的處理中有較好的抑制能力。進一步分析拮抗菌在植物免疫訊號上的影響，結果顯示僅PMB05菌株能夠加強植物免疫反應之訊號。由以上結果推測PMB05菌株對黑腐病之抑制效果可能與其在植物免疫訊號的提升有關。在清除性處理中，更進一步使用PMB05菌株發酵液的200倍稀釋液處理，可完全抑制甘藍黑腐病的發生。綜上所述，本研究所使用之Bacillus amyloliquefaciens PMB04 及 PMB05 菌株利用種子處理並利用不同的作用機制來防治甘藍黑腐病的發生。

Black rot disease (BRD) caused by Xanthomonas campestris pv. campestris (Xcc) is considered the most important disease of crucifers worldwide. This destructive disease causes considerable yield losses up to 50% by premature defoliation. Various strategies have been developed to control BRD on cabbage, including the use of antagonistic microorganisms. In this study, we attend to assay the possibility of applying Bacillus amyloliquefaciens strains, PMB04 and PMB05, to control BRD on cabbage. Firstly, we assay the inhibitory effect of B. amyloliquefaciens strains againsts Xcc strains on nutrient agar plate. The inhibitory assay revealed that both antagonistic strains were able to inhibit the growth of Xcc strains. Moreover, PMB04 exhibited strongerinhibitory effect against two Xcc strains. These two antagonistic strains were further used to evaluate their biocontrol efficacy of BRD by two seed coating strategies, including preventing and scavenging treatment. As a result, both two treatments exhibited that BRD on cabbage was significantly suppressed by two B. amyloliquefaciens strains. Above all, PMB05 showed stronger suppression than PMB04 in both two seed coating strategies. On the contrary, the population of Xcc in plants was strongly reduced by PMB04 rather than PMB05. Our previous studies showed that B. amyloliquefaciens PMB05 was able to intensify plant immune system for further enhancing disease resistance of crops. To gain more insight on this response, we observed the deposition of callose to evaluate the activation of PAMP-triggered immunity. Correspondingly, callose deposition on the leaf of cabbage was strongly intensified by the cells of PMB05 upon the inoculation of Xcc62 or Xcc64. In addition, the growth of cabbage was not improved by the application of B. amyloliquefaciens. To optimize the biocontrol efficacy of BRD by PMB05, the fermentation liquid of PMB05 was further applied. Results exhibited that disease incidence and disease severity were completely suppressed. In conclusion, BRD could be controlled by B. amyloliquefaciens strain PMB04 due to the antagonistic effect. Most importantly, plant immune response intensified by B. amyloliquefaciens strain PMB05 and its fermentation liquid provides strong efficacy against BRD. The strong biocontrol efficacy might be due to the intensification of plant immunity by PMB05. We concluded that the application of these two B. amyliliquefaciens strains to control BRD on cabbage would be promising in the future.

Table of Contents
摘要 .................................................................................................................... I
Abstract ........................................................................................................... III
Acknowledgment ............................................................................................. V
Table of Contents ............................................................................................ VI
List of Figure ................................................................................................... IX
List of Table ..................................................................................................... XI
1. Introduction ................................................................................................... 1
2. Literature Review .......................................................................................... 3
2.1 Black rot disease on cabbage ............................................................... 3
2.2 Beneficial microorganism as biological control agent ........................ 4
2.3 Bacillus spp. as biocontrol agents ....................................................... 4
2.4 Plant systemic resistance induced by Bacillus spp. ............................ 6
2.5 Plant growth promoting rhizobacteria ................................................. 7
3. Material and Methods.................................................................................... 9
3.1 Bacterial strains and fermentation liquid ............................................ 9
3.2 Media and buffer ................................................................................. 9
3.2.1 Nutrient broth and nutrient agar media .................................... 9

VII
3.2.2 Selective medium of Bacillus amyloliquefaciens and Xanthomonas campestris pv. campestris ................................ 9
3.2.3 Carboxymethylcellulose sodium ......................................... 10
3.2.4 Phosphate buffer .................................................................... 10
3.3 In vitro inhibitory assay of B. amyloliquefaciens against X. campestris pv. campestris ................................................................................... 10
3.4 Biocontrol efficacy of B. amyloliquefaciens to control black rot disease on cabbage ......................................................................................... 11
3.4.1 Preventing strategy of B. amyloliquefaciens on cabbage ...... 11
3.4.2 Scavenging strategy of B. amyloliquefaciens on cabbage ..... 12
3.5 B. amyloliquefaciens and X. campestris pv. campestris population assay from plants ............................................................................... 13
3.6 Callose deposition ............................................................................. 14
3.7 The effect of B. amyloliquefaciens application on the plant growth 14
3.8 Biocontrol efficacy of fermentation liquid of B. amyloliquefaciens strain PMB05 to control BRD on cabbage ....................................... 15
4. Results ......................................................................................................... 16
4.1 In vitro inhibitory assay of Bacillus amyloliquefaciens against Xanthomonas campestris pv. campestris .......................................... 16
4.2 Biocontrol efficacy of B. amyloliquefaciens to control black rot disease on cabbage ......................................................................................... 16
4.2.1 Preventing strategy of B. amyloliquefaciens on cabbage ...... 16

VIII
4.2.2 Scavenging strategy of B. amyloliquefaciens on cabbage ..... 17
4.3 Population of B. amyloliquefaciens in the seedling of cabbage ........ 17
4.4 Population of X. campestris pv. campestris in the seedling of cabbage ........................................................................................................... 17
4.5 Callose deposition on cabbage leaf ................................................... 17
4.6 The effect of application of B. amyloliquefaciens on the plant growth ........................................................................................................... 18
4.7 Biocontrol efficacy of B. amyloliquefaciens strain PMB05 fermentation liquid to control BRD on cabbage ............................... 18
5. Discussion ................................................................................................... 19
6. Conclusion ................................................................................................... 25
7. References ................................................................................................... 26
8. Figure and Table .......................................................................................... 34
Biosketch of Author ........................................................................................ 48

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