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.

Morpho-molecular characterization of Discosia ravennica sp. nov. and a new host record for Sporocadus rosigena.

Collections of fungal samples from two dead leaf specimens from Italy were subjected to morphological examination and phylogenetic analyses. Two coelomycetous taxa belonging to two different genera in Xylariomycetidae, Sordariomycetes, namely Discosia and Sporocadus, were identified. The Discosia taxon is revealed as a new species and is herein introduced as Discosia ravennica sp. nov. while the Sporocadus taxon is identified as Sporocadus rosigena. Multi-locus phylogeny based on DNA sequence data of the large subunit (LSU) and internal transcribed spacer (ITS) of nuclear ribosomal genes, ß-tubulin (ß-tub) and RNA polymerase II second largest subunit (rpb2) showed that D. ravennica is related to D. neofraxinea but it forms an independent lineage that supports its new species status. The new taxon also differs from other Discosia species by its unilocular to bilocular, superficial and applanate conidiomata with basal stroma composed of cells of textura angularis, elongate-ampulliform conidiogenous cells and conidia smaller in size. Sporocadus rosigena is here reported as a new host record from Quercus ilex from Italy. Descriptions, illustrations and molecular data for both species are provided in this paper.

Identification of endophytic fungi from leaves of Pandanaceae based on their morphotypes and DNA sequence data from southern Thailand.

The authors established the taxonomic status of endophytic fungi associated with leaves of Pandanaceae collected from southern Thailand. Morphotypes were initially identified based on their characteristics in culture and species level identification was done based on both morphological characteristics and phylogenetic analyses of DNA sequence data. Twenty-two isolates from healthy leaves were categorised into eight morphotypes. Appropriate universal primers were used to amplify specific gene regions and phylogenetic analyses were performed to identify these endophytes and established relationships with extant fungi. The authors identified both ascomycete and basidiomycete species, including one new genus, seven new species and nine known species. Morphological descriptions, colour plates and phylogenies are given for each taxon.

Phylogenetic relationships of Nemania plumbea sp. nov. and related taxa based on ribosomal ITS and RPB2 sequences.

During a survey fungal diversity of xylariaceous fungi in Thailand, a new Nemania species, N. plumbea, was identified. Nemania plumbea is characterized by soft-textured grey stromata on a persistent mat of white hyphae, pale brown ascospores with a short germ-slit on the more convex side. It also produces a Geniculosporium-like anamorph in culture. In order to evaluate its phylogenetic relationships among related species and genera, ITS-5.8S rDNA and RPB2 were analysed separately and simultaneously. Results from the phylogenetic analyses indicate that there is close phylogenetic association between N. plumbea and N. aenea. A preliminary account into the natural grouping of Xylariaceae based on ITS-5.8S rDNA and RPB2 sequences is also discussed.

Phylogenetic utility of protein (RPB2, beta-tubulin) and ribosomal (LSU, SSU) gene sequences in the systematics of Sordariomycetes (Ascomycota, Fungi).

The Sordariomycetes is an important group of fungi whose taxonomic relationships and classification is obscure. There is presently no multi-gene molecular phylogeny that addresses evolutionary relationships among different classes and orders. In this study, phylogenetic analyses with a broad taxon sampling of the Sordariomycetes were conducted to evaluate the utility of four gene regions (LSU rDNA, SSU rDNA, beta-tubulin and RPB2) for inferring evolutionary relationships at different taxonomic ranks. Single and multi-gene genealogies inferred from Bayesian and Maximum Parsimony analyses were compared in individual and combined datasets. At the subclass level, SSU rDNA phylogenies demonstrate their utility as a marker to infer phylogenetic relationships at higher levels. All analyses with SSU rDNA alone, combined LSU rDNA and SSU rDNA, and the combined 28 S rDNA, SSU rDNA and RPB2 datasets resulted in three subclasses: Hypocreomycetidae, Sordariomycetidae and Xylariomycetidae, which correspond well to established morphological classification schemes. At the ordinal level, the best resolved phylogeny was obtained from the combined LSU rDNA and SSU rDNA datasets. Individually, the RPB2 gene dataset resulted in significantly higher number of parsimony informative characters. Our results supported the recent separation of Boliniaceae, Chaetosphaeriaceae and Coniochaetaceae from Sordariales and placement of Coronophorales in Hypocreomycetidae. Microascales was found to be paraphyletic and Ceratocystis is phylogenetically associated to Faurelina, while Microascus and Petriella formed another clade and basal to other members of Halosphaeriales. In addition, the order Lulworthiales does not appear to fit in any of the three subclasses. Congruence between morphological and molecular classification schemes is discussed.

Succession of microfungal communities on decaying leaves of Castanopsis fissa.

A total of 38 fungal taxa were identified on senescent untreated and autoclaved Castanopsis fissa leaves during a 4-month study period. Seventy-six percent of the fungal genera found in this survey have not previously been recorded from Castanopsis. Frequency and time of occurrence of fungal taxa occurring on untreated senescent leaves were clustered into four groups when analyzed by cluster analysis, suggesting the replacement of microfungi in stages of succession on naturally senescent leaves. Autoclaved leaves revealed significantly different fungal communities, with only 26% of overlap with the natural ones and no clear patterns of replacement of fungal communities. Factors regulating the rates of decomposition are also discussed.

The persistence of ripe fleshy fruits in the presence and absence of frugivores.

A trade-off between antimicrobial defences and palatability to dispersers may place limits on fruit persistence in nature. The retention times of ripe fruits on 34 wild plant species under natural conditions (unbagged persistence) and when fruits had been bagged with nylon mesh to exclude frugivores (bagged persistence) were compared in Hong Kong, China (22 degrees N). Bagged persistence is a measure of the effectiveness of fruit defence while unbagged persistence is an inverse measure of attractiveness to vertebrate frugivores. Bagged fruits persisted significantly longer than unbagged fruits in 30 species, with half the species tested persisting for more than 2 months. There was a significant positive relationship between the median persistence times of bagged and unbagged fruits, suggesting that species with a high resistance to microbial infection are also less attractive to frugivores. Both bagged and unbagged fruits persisted significantly longer at lower temperatures. There was a significant positive relationship between bagged persistence time and fibre content of the fruit pulp, but no significant relationships between unbagged persistence and the six fruit traits tested (diameter, pulp as a percentage of fruit fresh weight, and lipid, total soluble carbohydrate, nitrogen and fibre as percentages of pulp dry weight). Mechanical damage significantly decreased the bagged persistence time for half of the species. Although some fruits decayed or dried up while attached to the plant, fruits of 53% of the species remained visually attractive until they fell off.