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

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 developed 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, five other Phellinus species, and 22 non-Phellinus wood decay fungal species. While all three primer pairs could detect as little as 100 fg (about 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 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 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.

Comparative analysis of mitochondrial genomes in Ceratocystis fimbriata complex across diverse hosts.

The decline ofAcacia mangiumWilld. in Malaysia, especially in Sabah since 2010, is primarily due to Ceratocystiswilt and canker disease (CWCD) caused by theCeratocystis fimbriataEllis & Halst. complex. This study was aimed to investigate the mitochondrial genome architecture of two differentC. fimbriatacomplex isolates from Malaysia: one fromA. mangiumin Pahang (FRIM1162) and another fromEucalyptus pellitain Sarawak (FRIM1441). This research employed Next-Generation Sequencing (NGS) to contrast genomes from diverse hosts with nine additional mitochondrial sequences, identifying significant genetic diversity and mutational hotspots in the mitochondrial genome alignment. The mitochondrial genome-based phylogenetic analysis revealed a significant genetic relationship between the studied isolates and theC. fimbriatacomplex in the South American Subclade, indicating that theC. fimbriatacomplex discovered in Malaysia isC. manginecans. The comparative mitochondrial genome demonstrates the adaptability of the complex due to mobile genetic components and genomic rearrangements in the studiedfungal isolates. This research enhances our knowledge of the genetic diversity and evolutionary patterns within theC. fimbriatacomplex, aiding in a deeper understanding of fungal disease development and host adaption processes. The acquired insights are crucial for creating specific management strategies for CWCD, improving the overall understanding of fungal disease evolution and control.