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.

Translocation of Fungicides and Their Efficacy in Controlling Phellinus noxius, the Cause of Brown Root Rot Disease.

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.

Next-generation sequencing dataset of bacterial communities of Microcerotermes crassus workers associated with Ironwood trees (Casuarina equisetifolia) in Guam.

Ironwood trees (Casuarina equisetifolia) in Guam have been suffering from Ironwood Tree Decline (IWTD) since 2002. Putative plant pathogenic bacteria such as Ralstonia solanacearum and Klebsiella species were identified in the ooze of declining trees and considered to be linked to IWTD. In addition, termites were found to be significantly associated with IWTD. Microcerotermes crassus Snyder (Blattodea: Termitidae) was identified as a termite species that attacks ironwood trees in Guam. Since termites harbor a diverse community of symbiotic and environmental bacteria, we sequenced the microbiome of M. crassus workers attacking ironwood trees in Guam to assess the presence of IWTD-associated pathogens in termite bodies. This dataset contains 652,571 raw sequencing reads present in M. crassus worker samples collected from six ironwood trees in Guam obtained via sequencing the V4 region of the16S rRNA gene on the Illumina NovaSeq (2 × 250bp) platform. Sequences were taxonomically assigned in QIIME2 using SILVA 132 and NCBI GenBank as reference databases. Spirochaetes and Fibrobacteres were the most dominant phyla in M. crassus workers. No putative plant pathogens of the genera Ralstonia or Klebsiella were found in the M. crassus samples. The dataset has been made publicly available through NCBI GenBank under BioProject ID PRJNA883256. This dataset can be used to compare the bacterial taxa present in M. crassus workers in Guam to bacteria communities of related termite species from other geographical locations. In addition, this dataset can also be used to investigate the relationship between termite microbiomes and the microbiomes of ironwood trees they attack and of the surrounding soil.

Taxonomic profiling of Nasutitermes takasagoensis microbiota to investigate the role of termites as vectors of bacteria linked to ironwood tree decline in Guam.

The ironwood tree (Casuarina equisetifolia, family Casuarinaceae), an indigenous agroforestry species in Guam, has been threatened by ironwood tree decline (IWTD) since 2002. Formation of bacterial ooze by the wilt pathogen from the Ralstonia solanacearum species complex and wetwood bacteria (primarily Klebsiella species) has been linked to IWTD. In addition, termite infestation of trees was statistically associated with IWTD. Termites are known carriers of a diverse microbiome. Therefore, we hypothesized that termites could be vectors of bacteria linked to IWTD. To investigate the potential role of termites as pathogen vectors, we employed next-generation 16S rRNA gene sequencing to describe the bacteria diversity of Nasutitermes takasagoensis (Family Termitidae) workers collected from 42 ironwood trees of different disease stages in Guam in association with tree-, plot-, and location-related factors. Nasutitermes takasagoensis workers account for the majority of termite infestations of ironwood trees. The bacterial phyla composition of N. takasagoensis workers was typical for wood-feeding higher termites consisting mainly of Spirochaetes and Fibrobacteres. However, Ralstonia species were not detected and Klebsiella species were rare even in termites collected from trees infected with Ralstonia and wetwood bacteria. Feeding experiments suggested that termites prefer to consume wood with low pathogen content over wood with high pathogen load. Termites were able to ingest Ralstonia but Ralstonia could not establish itself in healthy termite bodies. We concluded that N. takasagoensis workers are not vectors for Ralstonia spp. or the bacterial endophytes associated with wetwood (Klebsiella, Pantoea, Enterobacter, Citrobacter, and Erwinia) that were previously observed in IWTD-infested trees. The bacterial diversity in termite samples was significantly influenced by various factors, including Tree Health, Site Management, Plot Average Decline Severity, Proportion of Dead Trees in the Plot, Proportion of Trees with Termite Damage in the Plot, Presence of Ralstonia, and Altitude.

Complete Genome Sequences of Tomato Leaf Curl Guam Virus, a Novel Tomato-Infecting Begomovirus from Guam, USA.

Genome sequences of a novel begomovirus infecting tomato on Guam were obtained using primer-walking and sequencing. The complete genome sequences are 2,750 nucleotides long with a typical monopartite organization and display less than 91% nucleotide sequence identity to other begomoviruses. A provisional name, tomato leaf curl Guam virus (ToLCGuV), is proposed.

Morphological and Molecular Species Identification of Termites Attacking Ironwood Trees, Casuarina equisetifolia (Fagales: Casuarinaceae), in Guam.

Ironwood trees (Casuarina equisetifolia subsp. equisetifolia L.) are ecologically and economically important trees in tropical and subtropical regions of the Indo-Pacific. Ironwood is one of the dominant tree species in Guam, but since 2002, this tree has been declining dramatically. A previous study showed that numerous sick or dead trees were under termite attack. However, the species of termites were not identified. As a first step to investigate causal relationships between termites and ironwood tree death, we assigned termites collected from ironwood trees to species using a combination of morphological characters and DNA barcoding of the 12S, 16S, COI, COII, and ITS2 regions. Based on morphology and comparisons to reference sequences in NCBI GenBank, the most likely species assignments were Nasutitermes takasagoensis (Nawa) (Blattodea: Termitidae) found to infest 45 trees, followed by Coptotermes gestroi (Wasmann) (Blattodea: Rhinotermitidae) (2 trees), Microcerotermes crassus Snyder (Blattodea: Termitidae) (2 trees), and an additional unidentified Microcerotermes species (1 tree) with no close sequence match to identified species in NCBI GenBank. However, taxonomic revisions and broader representation of DNA markers of well-curated specimen in public databases are clearly needed, especially for the N. takasagoensis species complex.