Development and Evaluation of Real-Time Quantitative PCR Assays for Detection of Phellinus noxius Causing Brown Root Rot Disease
Journal
Plant Disease
Journal Volume
108
Journal Issue
11
Pages
3288-3299
Date Issued
2024-10
Author(s)
Tse-Yen Liu
Chao-Han Chen
Yi-Chun Ko
Zong-Chi Wu
Ting-Zhi Liao
Hsin-Han Lee
Isheng Jason Tsai
Tun-Tschu Chang
Meng-Ling Wu
Ned B. Klopfenstein
Mee-Sook Kim
Jane E. Stewart
Ndeme Atibalentja
Fred E. Brooks
Philip G. Cannon
A. Mohd Farid
Tsutomu Hattori
Hoi-Shan Kwan
Regent Yau Ching Lam
Yuko Ota
Norio Sahashi
Robert L. Schlub
Louise S. Shuey
Alvin M. C. Tang
Chia-Lin Chung
Abstract
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 Phellinus noxius isolates, other wood-decay fungi, or host plant tissues. This study aimed to develop SYBR Green real-time quantitative PCR (qPCR) assays for P. noxius. The primer pair Pn_ITS_F/Pn_ITS_R targets the ITS, and the primer pair Pn_NLR_F/Pn_NLR_R targets a P. noxius–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 P. noxius isolates, 5 other Phellinus species, and 22 non-Phellinus wood-decay fungal species. Although all three primer pairs could detect as little as 100 fg (approximately 2.99 copies) of P. noxius 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 quantification cycle 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 Ficus benjamina seedlings artificially inoculated with P. noxius, six tree species naturally infected by P. noxius, rhizosphere soil, and bulk soil. The newly developed qPCR assays provide sensitive detection and quantification of P. noxius, which is useful for long-term monitoring of BRRD status.
Subjects
comparative genomics analysis
internal transcribed spacer (its)
molecular diagnosis
nucleotide-binding-oligomerization-domain-like receptor (NLR)
Publisher
American Phytopathological Society
Type
journal article