Development and evaluation of real-time quantitative PCR assays for detection of Phellinus noxius causing brown root rot disease

• Published in: Plant disease
• Main Authors: Liu, Tse-Yen, Chen, Chao-Han, Ko, Yi-Chun, Wu, Zong-Chi, Liao, Ting-Zhi, Lee, Hsin-Han, Tsai, Isheng Jason, Chang, Tun-Tschu, Wu, Meng-Ling, Tsai, Jyh-Nong, Klopfenstein, Ned B, Kim, Mee-Sook, Stewart, Jane E, Atibalentja, Ndeme, Brooks, Fred E, Cannon, Phil, Mohd Farid, Ahmad, Hattori, Tsutomu, Kwan, Hoi-Shan, Lam, Yau-Ching Regent, Ota, Yuko, Sahashi, Norio, Schlub, Robert L, Shuey, Louise S, Tang, Alvin M C, Chung, Chia-Lin
• Format: Journal Article
• Language: English
• Published: United States 29.06.2024
• Subjects: internal transcribed spacer (ITS); comparative genomics analysis; nucleotide-binding-oligomerization-domain-like receptor (NLR); molecular diagnosis
• ISSN: 0191-2917
• DOI: 10.1094/PDIS-01-24-0238-RE

Brown root rot disease (BRRD) is a highly destructive tree disease. Early diagnosis of BRRD has been challenging because the first symptoms and signs are often observed after extensive tissue colonization. Existing molecular detection methods, all based on the internal transcribed spacer (ITS) region, were developed without testing against global isolates, other wood decay fungi, or host plant tissues. This study developed SYBR Green real-time quantitative PCR (qPCR) assays for . The primer pair Pn_ITS_F/Pn_ITS_R targets the ITS, and the primer pair Pn_NLR_F/Pn_NLR_R targets a -unique group of homologous genes identified through a comparative genomics analysis. The homologous genes belong to the nucleotide-binding-oligomerization-domain-like receptor (NLR) superfamily. The new primer pairs and a previous primer pair G1F/G1R were optimized for qPCR conditions and tested for specificity using 61 global isolates, five other species, and 22 non- wood decay fungal species. While all three primer pairs could detect as little as 100 fg (about 2.99 copies) of genomic DNA, G1F/G1R had the highest specificity and Pn_NLR_F/Pn_NLR_R had the highest efficiency. To avoid false positives, the cutoff Cq values were determined as 34 for G1F/G1R, 29 for Pn_ITS_F/Pn_ITS_R, and 32 for Pn_NLR_F/Pn_NLR_R. We further validated these qPCR assays using seedlings artificially inoculated with , six tree species naturally infected by , rhizosphere soil, and bulk soil. The newly developed qPCR assays provide sensitive detection and quantification of , which is useful for long-term monitoring of BRRD status.