褐根病對都市樹木根部微生物族群之影響及褐根病菌即時定量PCR偵測技術開發

Abstract

褐根病是熱帶和亞熱帶地區的重要樹木病害，由木材白腐朽真菌Phellinus noxius (Corner) G. Cunn.所引起。為釐清褐根病危害對樹木根部相關微生物族群的影響，本研究以榕樹為材料，採集被褐根病菌自然感染榕樹之五類根部相關樣本 (立地環境土、幼根根圈土、老根根圈土、幼根組織及老根組織)，對樣本中的核醣體內轉錄區間 (ribosomal internal transcribed spacer, ITS) 和16S核醣體RNA (16S rRNA) 進行次世代定序。發現在罹染褐根病樹木的立地環境土、老根根圈土及老根組織中，真菌的物種多樣性顯著降低。雖然褐根病菌的侵染對細菌多樣性沒有顯著影響，但仍造成細菌組成在健康及罹病樣本間有所差異。值得注意的是，Cosmospora是唯一與褐根病菌感染呈正相關的真菌屬。透過26個真菌屬和35個細菌屬共838個分離株與褐根病菌的對峙培養實驗，發現Bacillus、Pseudomonas、Aspergillus、Penicillium和Trichoderma對褐根病菌具有拮抗能力。透過玻璃紙覆蓋及纖維素/木質素培養實驗，發現Cosmospora對褐根病菌的分泌物具耐受性，並推測褐根病菌對木質素的分解可為Cosmospora創造適合的生長環境。進一步的顯微觀察發現，在木質素培養基上對峙培養褐根病菌和Cosmospora時，可觀察到似夾狀的菌絲特化結構。經由和其他真菌的對峙培養，推測該特化菌絲結構為褐根病菌所產生，其功能仍有待釐清。此外，由於褐根病初期的病徵和病兆常在組織廣泛被病原菌侵染後才顯現，本研究進一步發展褐根病菌即時定量聚合酶連鎖反應 (real-time quantitative PCR, qPCR) 偵測技術。透過不同褐根病菌分離株之ITS序列比對，新設計引子對Pn_ITS_F/Pn_ITS_R；另透過比較基因體分析，篩選出褐根病菌特有Nucleotide-binding-oligomerization-domain-like receptor (NLR) 基因家族，據此設計引子對Pn_NLR_F/Pn_NLR_R。將此兩組新設計的引子對和目前廣泛用於褐根病菌傳統PCR檢測的引子對G1F/G1R (吳等 2009)，應用於qPCR並進行反應條件的優化。針對61株自世界各地收集的褐根病菌菌株、5種其他Phellinus屬真菌和22種其他木材腐朽菌進行專一性測試，發現G1F/G1R之專一性最佳。三組引子對在qPCR反應中，對褐根病菌基因體核酸 (genomic DNA) 的檢測極限均可低至100 fg，其中Pn_NLR_F/Pn_NLR_R有較佳的增幅效率。為建立qPCR檢測褐根病菌的陽性檢測標準，本研究也針對各引子對分別設定檢測臨界Cq值，G1F/G1R為34，Pn_ITS_F/Pn_ITS_R為29，Pn_NLR_F/Pn_NLR_R為32。進一步使用人工接種褐根病菌的垂榕 (Ficus benjamina) 枝條，以及6種野外自然感染褐根病的樹木、根圈土和植穴土，驗證qPCR之實用性。總結來說，本研究透過分析樹木根部相關微生物族群，瞭解褐根病菌在自然生態中所扮演的角色，而新建立具高靈敏度及專一性的qPCR檢測技術，可實際應用於褐根病菌的檢測和定量，有助於褐根病的長期監測和防治。

Brown root rot disease (BRRD), caused by the white rot fungus Phellinus noxius (Corner) G. Cunn., is a destructive tree disease prevalent in tropical and subtropical regions. To clarify the influence of BRRD on the root-associated microbiota of trees, the five root-associated compartments (i.e., bulk soil, young root rhizosphere soil, old root rhizosphere soil, young root tissue, and old root tissue) of Ficus trees naturally infected by P. noxius were analyzed using next generation sequencing (NGS) of the ribosomal internal transcribed spacer (ITS) and 16S rRNA. The results showed that fungal diversity significantly decreased in the bulk soil, old root rhizosphere soil, and old root tissue of BRRD samples. In contrast, bacterial diversity was not significantly affected by P. noxius-infection, although the bacterial composition differed between healthy and BRRD samples. Notably, Cosmospora was the only fungal genus positively correlated with infection by P. noxius. Dual culture assays with 838 isolates from 26 fungal and 35 bacterial genera showed antagonistic activities against P. noxius for isolates of Bacillus, Pseudomonas, Aspergillus, Penicillium, and Trichoderma. Cellophane overlay and cellulose/lignin utilization assays suggested that Cosmospora could tolerate P. noxius secretions, and P. noxius might create suitable conditions for Cosmospora growth through lignin degradation. Microscopic observation of the interaction between P. noxius and Cosmospora sp. on the lignin medium revealed the formation of the specialized clamp-like hyphal structure. Dual culture assays with other fungal species suggested that this specialized hyphal structure was produced by P. noxius. However, its function needs further investigation. Additionally, because the first disease symptoms and signs of BRRD are often observed only after extensive tissue colonization, this study aimed to develop real-time quantitative PCR (qPCR) assays for P. noxius. By comparing ITS sequences of different P. noxius isolates, a new primer pair Pn_ITS_F/Pn_ITS_R was designed. Through comparative genome analysis, the nucleotide-binding-oligomerization-domain-like receptor (NLR) gene family unique to P. noxius was identified, and the primer pair Pn_NLR_F/Pn_NLR_R was designed accordingly. The two newly designed primer pairs and G1F/G1R, a currently widely used primer pair for P. noxius detection by conventional PCR (Wu et al. 2009), were optimized for qPCR. Specificity tests using 61 P. noxius isolates collected worldwide, five other species of Phellinus, and 22 wood-decay fungal species indicated that G1F/G1R showed the highest specificity. All three primer pairs could detect as little as 100 fg of P. noxius genomic DNA, with Pn_NLR_F/Pn_NLR_R exhibiting the highest amplification efficiency. To establish criteria for positive detection of P. noxius by qPCR, cutoff Cq values were assigned for each primer pair, with 34 for G1F/G1R, 29 for Pn_ITS_F/Pn_ITS_R, and 32 for Pn_NLR_F/Pn_NLR_R. The practicality of the qPCR assays was further validated using samples from Ficus benjamina seedlings artificially inoculated with P. noxius, six tree species naturally infected by P. noxius, rhizosphere soil, and bulk soil. In conclusion, analysis of the root-associated microbiota of trees enhanced our knowledge of the ecological role of P. noxius in the natural environment. The newly developed qPCR assays with high sensitivity and specificity can be applied to detect and quantify P. noxius, which are helpful for the long-term monitoring and control of BRRD.