- 學位論文
探討殺真菌劑、抗病性與微生物群相於台灣大豆紅冠腐病之防治研究
Studies on the fungicide efficacy, resistance screening, and microbiome-associated resistance to control soybean red crown rot in Taiwan
摘要
大豆是台灣重要出口及經濟作物之一，栽培面積超過13,000公頃，年貿易額超過21億新台幣。自2017年起，台灣新發現由Calonectria ilicicola所引起之土傳性紅冠腐病危害逐漸嚴重，然而國內尚未有推薦之防治方法。為瞭解C. ilicicola於大豆種子發芽至幼苗階段的感染影響，本研究將0至18天共7個不同時期之幼苗移植到已接種病原菌之土壤中觀察病徵發展，結果發現播種一週以內的大豆感病性顯著高於播種18天之大豆，顯示幼苗期保護或可減少紅冠腐病之危害。因此，本研究量化11種殺菌劑對C. ilicicola對菌絲生長的抑制效果，發現得克利、撲克拉、氟派瑞、鋅錳乃浦、賽普護汰寧對菌絲抑制率可達80%以上；另建立高通量之藥劑篩選方法，利用生長曲線及Z' 統計值為標準，納入光學平面掃描模式、吸收波長、孢子濃度及測量時間等條件優化高通量操作之準確度，測得田間80個C. ilicicola分離株對得克利、氟派瑞及賽普護汰寧之EC50平均值分別為2.34、2.46及0.14 ppm，且沒有於族群內發現抗藥性菌株的存在。針對這三種測試藥劑進行種子披衣實驗，發現以賽普護汰寧對幼苗保護效果最佳，顯著減少幼苗倒伏及根腐嚴重度，故推薦以種子披衣賽普護汰寧作為紅冠腐病之即時防治方法。另針對18個大豆及毛豆品種進行紅冠腐病之種子腐敗及根部腐朽抗性篩選，結果顯示不同品種間在根腐抗性上並未發現具有高度抗性之品種，然而對種腐抗性具有差異，其中以「Ralsoy」及「台南11號」抗性最高。鑑於近期研究顯示微生物群相可提供植物抗病性，而大豆種子內的微生物是否提供種腐抗性尚不清楚，故本研究利用抗生素處理四個抗病及四個感病的大豆品種，發現處理之後抗種腐之大豆品種感病度皆顯著增加，證實種子抗性與種子內細菌有關。應用PacBio 16S rRNA基因全長定序及微生物群相分析，發現兩個Bacillus altitudinis ASVs和其他12個分屬六個細菌物種的ASVs在抗性品種中相對數量較高。後續分離得到兩株B. altitudinis TN3S3及 TN5S8，並在接種實驗中顯示B. altitudinis可以恢復種腐抗性，但僅限於五個大豆品種中。計算B. altitudinis分離株TN5S8於此五個大豆品種中發現，其細菌定殖量高於另外三個無法藉由施加B. altitudinis分離株而提升抗病性的大豆品種，顯示B. altitudinis與大豆品種的相容性在種腐抗性上扮演關鍵的角色。此外，qPCR結果顯示B. altitudinis在接種後21天仍可存在於相容品種之大豆地上部，但在接種後9天即無法於根部測得，故可解釋B. altitudinis所提供之種腐抗性之所以無法延續到同一個大豆品種的根腐抗性，並突顯未來應用種子相關細菌進行種子保護時，需考慮細菌與植物品種及組織之相容性。綜觀而論，本研究在大豆紅冠腐病之殺菌劑篩選、抗病性篩選之成果，為後續病害整合防治建立全面的學理基礎，並首次發現微生物群相介導之大豆種腐抗病性。
並列摘要
Soybean represents one of the pivotal export and economic crops in Taiwan, with a cultivation area exceeding 13,000 hectares and annual revenue of NTD 2.1 billion. Since 2017, soybean production in Taiwan has encountered an increasing threat from red crown rot (RCR), a soil-borne fungal disease caused by Calonectria ilicicola. Yet, no recommended control strategies have been established in Taiwan. To understand the susceptibility of soybean seedlings at different growth stages to RCR, C. ilicicola was inoculated on 7 distinct stages of soybeans, and disease severity and fresh weight reduction rates were evaluated 21 days post-inoculation. The results showed that seedlings less than a week old exhibited significantly higher susceptibility compared to 18-day-old seedlings, indicating that early-stage infection can increase the damage caused by RCR. Therefore, fungicide seed treatments emerge as a viable management approach. Subsequently, this study evaluated 11 fungicides for their inhibitory effects on the mycelial growth of C. ilicicola, identifying that cyprodinil+fludioxonil, fluopyram, mancozeb, prochloraz, tebuconazole exhibited over 80% of inhibition. To further comprehend fungicide resistance of field isolates, a high-throughput screening method was developed, optimized for optical surface-scanning modes, wavelength, spore concentration, and measurement timing based on growth curves and Z' factors. Utilizing this method, the mean EC50 of cyprodinil+fludioxonil, fluopyram, and tebuconazole for 80 C. ilicicola isolates were determined to be 0.14, 2.34, and 2.46 ppm, respectively, with no resistant isolates detected. Greenhouse experiments revealed that cyprodinil+fludioxonil offered the best protection when applied as seed treatment, significantly reducing post-emergence damping-off and root rot severity. Accordingly, cyprodinil+fludioxonil exhibits potential for controlling RCR through seed treatment. On another front, resistant varieties represent the most economical and environmentally friendly control method. Screening 18 soybean varieties in Taiwan revealed that no variety has high root rot resistance, but some varieties such as 'Ralsoy' and 'Tainan No.11' displaying moderate seed rot resistance to RCR. Considering recent research underscores the significance of microbiome and plant-associated microbes in plant disease resistance, antibiotic treatment of 4 susceptible and 4 resistant soybean varieties was evaluated in this study to find that seed rot resistance depends on the seed-associated bacteria. The results of PacBio 16S rRNA full-length sequencing and microbiome analysis revealed two Bacillus altitudinis ASVs and other 12 ASVs being more abundant in the four resistant varieties. Further isolation and inoculation experiments indicated that B. altitudinis isolate TN5S8 could restore seed rot resistance in 5 soybean varieties, for which the bacterial colonization was higher. For 3 other varieties, B. altitudinis TN5S8 not only colonized in a lower titer but also reduced germination rates of the seeds. Additionally, qPCR results showed that B. altitudinis persisted on the apical shoot of compatible variety until 21 days post-inoculation but was undetectable in the roots after 9 days post-inoculation. This result explained the reason that seed rot resistance cannot extend to root rot of the same soybean variety, which offers a new insight into the mechanism of seed rot resistance and highlights the importance of colonization compatibility between bacteria and plant genotypes/tissues. In summary, this study offers novel insights into the fungicides, disease resistance, and microbiome-mediated resistance to manage soybean RCR, providing a comprehensive foundation for future integrated disease management strategies.