Emerging plant pathogens have been increasing exponentially over the last century. To address this issue, it is critical to determine whether these pathogens are native to ecosystems or have been recently introduced. Understanding the ecological and evolutionary processes fostering emergence can help to manage their spread and predict epidemics/epiphytotics. Using restriction site-associated DNA sequencing data, we studied genetic relationships, pathways of spread, and evolutionary history of Phellinus noxius, an emerging root-rotting fungus of unknown origin, in eastern Asia, Australia, and the Pacific Islands. We analyzed patterns of genetic variation using Bayesian inference, maximum likelihood phylogeny, populations splits and mixtures measuring correlations in allele frequencies and genetic drift, and finally applied coalescent based theory using Approximate Bayesian computation (ABC) with supervised machine learning. Population structure analyses revealed five genetic groups with signatures of complex recent and ancient migration histories. The most probable scenario of ancient pathogen spread is movement from an unsampled population to Malaysia and the Pacific Islands, with subsequent spread to Taiwan and Australia. Furthermore, ABC analyses indicate P. noxius spread occurred thousands of generations ago, contradicting previous assumptions that this pathogen was recently introduced to multiple geographic regions. Our results suggest that recent emergence of P. noxius in eastern Asia, Australia, and the Pacific Islands is likely driven by anthropogenic and natural disturbances, such as deforestation, land-use change, severe weather events, and/or introduction of exotic plants. This study provides a novel example of applying genome-wide allele frequency data to unravel dynamics of pathogen emergence under changing ecosystem conditions.
Brown root rot disease (BRRD), caused by Phellinus noxius, is an important tree disease in tropical and subtropical areas. To improve chemical control of BRRD and deter emergence of fungicide resistance in P. noxius, this study investigated control efficacies and systemic activities of fungicides with different modes of action. Fourteen fungicides with 11 different modes of action were tested for inhibitory effects in vitro on 39 P. noxius isolates from Taiwan, Hong Kong, Malaysia, Australia, and Pacific Islands. Cyproconazole, epoxiconazole, and tebuconazole (Fungicide Resistance Action Committee [FRAC] 3, target-site G1) inhibited colony growth of P. noxius by 99.9 to 100% at 10 ppm and 97.7 to 99.8% at 1 ppm. The other effective fungicide was cyprodinil + fludioxonil (FRAC 9 + 12, target-site D1 + E2), which showed growth inhibition of 96.9% at 10 ppm and 88.6% at 1 ppm. Acropetal translocation of six selected fungicides was evaluated in bishop wood (Bischofia javanica) seedlings by immersion of the root tips in each fungicide at 100 ppm, followed by liquid or gas chromatography tandem mass spectrometry analyses of consecutive segments of root, stem, and leaf tissues at 7 and 21 days posttreatment. Bidirectional translocation of the fungicides was also evaluated by stem injection of fungicide stock solutions. Cyproconazole and tebuconazole were the most readily absorbed by roots and efficiently transported acropetally. Greenhouse experiments suggested that cyproconazole, tebuconazole, and epoxiconazole have a slightly higher potential for controlling BRRD than mepronil, prochloraz, and cyprodinil + fludioxonil. Because all tested fungicides lacked basipetal translocation, soil drenching should be considered instead of trunk injection for their use in BRRD control.
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.