Micromelanconis kaihuiae gen. et sp. nov., a new diaporthalean fungus from Chinese chestnut branches in southern China.

Melanconis-like fungi are distributed in several families of Diaporthales, mainly Juglanconidaceae, Melanconidaceae, Melanconiellaceae and Pseudomelanconidaceae. A new Melanconis-like genus of Pseudomelanconidaceae was discovered on branches of Chinese chestnut (Castanea mollissima) in southern China, which was confirmed by both morphology and phylogenetic analysis of combined ITS, LSU, tef1a and rpb2 sequences. The new genus Micromelanconis is characterized by two types of conidia from natural substrate and manual media of PDA, respectively. Conidia from Chinese chestnut branches are pale brown, ellipsoid, multiguttulate, aseptate with hyaline sheath. While conidia from PDA plates are pale brown, long dumbbell-shaped, narrow at the middle and wide at both ends, multiguttulate, aseptate, and also with hyaline sheath. All Pseudomelanconidaceae species were only reported on tree branches in China until now. More interesting taxa may be discovered if detailed surveys on tree-inhabiting fungi are carried out in East Asia in the future.

New species and records of Diaporthe from Jiangxi Province, China.

Diaporthe species have often been reported as important plant pathogens, saprobes and endophytes on a wide range of plant hosts. Although several Diaporthe species have been recorded, little is known about species able to infect forest trees in Jiangxi Province. Hence, extensive surveys were recently conducted in Jiangxi Province, China. A total of 24 isolates were identified and analysed using comparisons of DNA sequence data for the nuclear ribosomal internal transcribed spacer (ITS), calmodulin (cal), histone H3 (his3), partial translation elongation factor-1α (tef1) and ß-tubulin (tub2) gene regions, as well as their morphological features. Results revealed five novel taxa, D. bauhiniae, D. ganzhouensis, D. schimae, D. verniciicola, D. xunwuensis spp. nov. and three known species, D. apiculatum, D. citri and D. multigutullata.

Tree inhabiting gnomoniaceous species from China, with Cryphogonomonia gen. nov. proposed.

Species of Gnomoniaceae are commonly associated with leaf spot diseases of a wide range of plant hosts worldwide. During our investigation of fungi associated with tree diseases in China, several gnomoniaceous isolates were recovered from symptomatic branches and leaves on different woody plants in the Fagaceae, Pinaceae, and Salicaceae families. These isolates were studied by applying a polyphasic approach including morphological, cultural data, and phylogenetic analyses of partial ITS, LSU, tef1, rpb2 and tub2 gene sequences. As a result, three species were identified with characters fitting into the family Gnomoniaceae. One of these species is described herein as Cryphognomonia pini gen. et sp. nov., characterized by developed pseudostromata and ascospores with obvious hyaline sheath; Gnomoniopsis xunwuensis sp. nov. is illustrated showing sympodially branched conidiophore, oval or fusiform conidia; and one known species, Plagiostoma populinum. The current study improves the understanding of gnomoniaceous species causing diebacks and leaf spot on ecological and economic forest trees.

Gnomoniopsis chinensis (Gnomoniaceae, Diaporthales), a new fungus causing canker of Chinese chestnut in Hebei Province, China.

Chinese chestnut (Castanea mollissima) is an important crop tree species in China. However, branch canker and fruit rot are two kinds of severe diseases, which weaken the host and decrease chestnut production. During our investigations into chestnut diseases in China, several fungi have been confirmed as casual agents in previous studies, namely Aurantiosacculus castaneae, Cryphonectria neoparasitica, Cry. parasitica, Endothia chinensis and Gnomoniopsis daii. In this study, a new canker pathogen is introduced based on morphology, phylogeny and pathogenicity. Typical Gnomoniopsis canker sign of wide, orange tendrils emerging from hosts' glaucous lenticels were obvious on the diseased trees in the field. Symptomatic branches or bark on stems from different chestnut plantations were sampled and isolated, then strains were identified by comparisons of DNA sequence data for the nuclear ribosomal internal transcribed spacer (ITS), partial translation elongation factor-1α (tef1) and ß-tubulin (tub2) gene regions as well as morphological features. As a result, these strains appeared different from any known Gnomoniopsis species. Hence, we propose a novel species named Gnomoniopsis chinensis. Pathogenicity was further tested using the ex-type strain (CFCC 52286) and another strain (CFCC 52288) on both detached branches and 3-year-old chestnut seedlings. The inoculation results showed that Gnomoniopsis chinensis is mildly pathogenic to Chinese chestnut. However, further studies are required to confirm its pathogenicity to the other cultivated Castanea species in America, Europe and Japan.

Three new Diaporthe species from Shaanxi Province, China.

Diaporthe species (Sordariomycetes, Diaporthales) are often reported as important plant pathogens, saprobes and endophytes on a wide range of plant hosts. In this study, Diaporthe specimens were collected from symptomatic twigs and branches at the Huoditang Forest Farm in Shaanxi Province, China. Identification was done using a combination of morphology and comparison of DNA sequence data of the nuclear ribosomal internal transcribed spacer (ITS), calmodulin (cal), histone H3 (his3), partial translation elongation factor-1α (tef1) and ß-tubulin (tub2) gene regions. Three new Diaporthe species are proposed: D. albosinensis, D. coryli and D. shaanxiensis. All species are illustrated and their morphology and phylogenetic relationships with other Diaporthe species are discussed.

Identification of six Cytospora species on Chinese chestnut in China.

Chinese chestnut (Castanea mollissima) is an important crop tree species in China. In the present study, Cytospora specimens were collected from Chinese chestnut trees and identified using molecular data of combined ITS, LSU, ACT and RPB2 loci, as well as morphological features. As a result, two new Cytospora species and four new host records were confirmed, viz. C. kuanchengensis sp. nov., C. xinglongensis sp. nov., C. ceratospermopsis, C. leucostoma, C. myrtagena and C. schulzeri.

Taxonomy of two synnematal fungal species from Rhus chinensis, with Flavignomonia gen. nov. described.

Rhus chinensis represents a commercially and ecologically important tree species in China, but suffers from canker diseases in Jiangxi Province. Synnemata, pycnidia and ascomata were discovered on cankered tissues. Strains were obtained from single ascospore or conidium within the fruiting bodies and identified based on morphological comparison and the phylogenetic analyses of partial ITS, LSU, tef1 and rpb2 gene sequences. As a result, two species were confirmed to represent two kinds of synnemata. One of these species is described herein as Flavignomonia rhoigena gen. et sp. nov.; and Synnemasporella aculeans is illustrated showing ascomata, pycnidia and synnemata. Flavignomonia is distinguished from Synnemasporella by the colour of the synnematal tips. Additionally, Flavignomonia can be distinguished from the other gnomoniaceous genera by the formation of synnemata.

Nectria-related fungi causing dieback and canker diseases in China, with Neothyronectriacitri sp. nov. described.

To clarify phylogenetic relationships amongst Nectria, Neothyronectria and Thyronectria in Nectriaceae, we examined detailed morphological characters and performed phylogenetic analyses of a concatenated dataset, based on the ITS, LSU, tef1 and tub2 DNA sequences of fungal specimens in China. Four species of nectria-related fungi were identified, i.e. Nectriadematiosa, N.pseudotrichia, Neothyronectriacitri and Thyronectriapinicola. The newly described species, Neothyronectriacitri, is characterised by its ascomatal wall with bright yellow scurf, unitunicate asci, each with 4-spored and ascospores allantoid to short-cylindrical, uniseriate, muriform, hyaline to slightly yellowish-brown. This species has affinities with other one known species of Neothyronectria and can be distinguished by molecular data.

Morphology and phylogeny reveal two novel Coryneum species from China.

Coryneum is currently the sole genus of Coryneaceae in Diaporthales, distinguished from other diaporthalean genera by transversely distoseptate brown conidia. However, Coryneum species are presently difficult to identify because of variability and overlap of morphological characters and the lack of sequence data for most described species. During fungal collection trips in China, 13 Coryneum isolates were obtained from cankered branches of Ilex and Quercus. Morphological and phylogenetic analyses (ITS, LSU, TEF1-α and RPB2) revealed that these strains belong to two new species (viz. Coryneumilicis sp. nov. and C.songshanense sp. nov.), and three known species, C.gigasporum, C.sinense, and C.suttonii. Coryneumilicis has larger conidia and more distosepta than most Coryneum species. Coryneumsongshanense was similar to C.sinense from the same host genus, Quercus, in conidial length, but distinct in conidial width and by molecular data.

Re-collection of Dermeaprunus in China, with a description of D.chinensis sp. nov.

Dermea was protected against its synonym, Foveostroma, due to its well-circumscribed generic concept and more frequent use. We describe and illustrate Dermeachinensis sp. nov. based on its morphological characteristics and a molecular analysis of the internal transcribed spacer (ITS) and large subunit (LSU) sequence data. Dermeachinensis is isolated from Betulaalbosinensis with sexual and asexual morphs and can be distinguished from D.molliuscula on Betula trees by its aseptate and wider ascospores. The connection between the two morphs is proved based on sequence data. Here, we describe the asexual morph of D.pruni for the first time based on morphological and molecular data from the same host and country of origin, and compare it with other species of Prunus.

Species of Dendrostoma (Erythrogloeaceae, Diaporthales) associated with chestnut and oak canker diseases in China.

Dendrostoma was recently proposed in Erythrogloeaceae (Diaporthales, Sordariomycetes), with all known members recorded as being plant pathogenic on economically important tree hosts. During our collections of Dendrostoma species in China, mild to severe canker symptoms were observed on sweet chestnut (Castaneamollissima) and oak (Quercus spp.) trees. Dead and dying plant tissues exhibiting Dendrostoma canker symptoms were sampled for fungal isolation. A total of 37 Dendrostoma isolates were obtained and analysed using morphological characteristics and molecular data (ITS, LSU, RPB2, TEF1-α). Based on these data, 10 novel clades could be distinguished, which also proved to represent morphologically distinct species described here as Dendrostomaaurorae, D.castaneae, D.castaneicola, D.chinense, D.dispersum, D. parasiticum, D.qinlingense, D.quercus, D.shaanxiense and D.shandongense spp. nov. A key to species of the genus is also provided.

High diversity of Diaporthe species associated with dieback diseases in China, with twelve new species described.

Diaporthe species have often been reported as important plant pathogens, saprobes and endophytes on a wide range of plant hosts. Although several Diaporthe species have been recorded in China, little is known about species able to infect forest trees. Therefore, extensive surveys were recently conducted in Beijing, Heilongjiang, Jiangsu, Jiangxi, Shaanxi and Zhejiang Provinces. The current results emphasised on 15 species from 42 representative isolates involving 16 host genera using comparisons of DNA sequence data for the nuclear ribosomal internal transcribed spacer (ITS), calmodulin (cal), histone H3 (his3), partial translation elongation factor-1α (tef1) and ß-tubulin (tub2) gene regions, as well as their morphological features. Three known species, D.biguttulata, D.eres and D.unshiuensis, were identified. In addition, twelve novel taxa were collected and are described as D.acerigena, D.alangii, D.betulina, D.caryae, D.cercidis, D.chensiensis, D.cinnamomi, D.conica, D.fraxinicola, D.kadsurae, D.padina and D.ukurunduensis. The current study improves the understanding of species causing diebacks on ecological and economic forest trees and provides useful information for the effective disease management of these hosts in China.

Development and Characterization of Novel Genic-SSR Markers in Apple-Juniper Rust Pathogen Gymnosporangium yamadae (Pucciniales: Pucciniaceae) Using Next-Generation Sequencing.

The Apple-Juniper rust, Gymnosporangium yamadae, is an economically important pathogen of apples and junipers in Asia. The absence of markers has hampered the study of the genetic diversity of this widespread pathogen. In our study, we developed twenty-two novel microsatellite markers for G. yamadae from randomly sequenced regions of the transcriptome, using next-generation sequencing methods. These polymorphic markers were also tested on 96 G. yamadae individuals from two geographical populations. The allele numbers ranged from 2 to 9 with an average value of 6 per locus. The polymorphism information content (PIC) values ranged from 0.099 to 0.782 with an average value of 0.48. Furthermore, the observed (HO) and expected (HE) heterozygosity ranged from 0.000 to 0.683 and 0.04 to 0.820, respectively. These novel developed microsatellites provide abundant molecular markers for investigating the genetic structure and genetic diversity of G. yamadae, which will help us to better understand disease epidemics and the origin and migration routes of the Apple-Juniper rust pathogen. Further studies will also be completed to dissect how human activities influence the formation of current population structures. Furthermore, these SSR (simple sequence repeat) markers can also be used as tools to identify virulence by mapping the whole genomes of different virulent populations. These markers will, thus, assist the development of effective risk-assessment models and management systems for the Apple-Juniper rust pathogen.

Comparative transcriptome analysis and identification of candidate effectors in two related rust species (Gymnosporangium yamadae and Gymnosporangium asiaticum).

BACKGROUND: Rust fungi constitute the largest group of plant fungal pathogens. However, a paucity of data, including genomic sequences, transcriptome sequences, and associated molecular markers, hinders the development of inhibitory compounds and prevents their analysis from an evolutionary perspective. Gymnosporangium yamadae and G. asiaticum are two closely related rust fungal species, which are ecologically and economically important pathogens that cause apple rust and pear rust, respectively, proved to be devastating to orchards. In this study, we investigated the transcriptomes of these two Gymnosporangium species during the telial stage of their lifecycles. The aim of this study was to understand the evolutionary patterns of these two related fungi and to identify genes that developed by selection. RESULTS: The transcriptomes of G. yamadae and G. asiaticum were generated from a mixture of RNA from three biological replicates of each species. We obtained 49,318 and 54,742 transcripts, with N50 values of 1957 and 1664, for G. yamadae and G. asiaticum, respectively. We also identified a repertoire of candidate effectors and other gene families associated with pathogenicity. A total of 4947 pairs of putative orthologues between the two species were identified. Estimation of the non-synonymous/synonymous substitution rate ratios for these orthologues identified 116 pairs with Ka/Ks values greater than1 that are under positive selection and 170 pairs with Ka/Ks values of 1 that are under neutral selection, whereas the remaining 4661 genes are subjected to purifying selection. We estimate that the divergence time between the two species is approximately 5.2 Mya. CONCLUSION: This study constitutes a de novo assembly and comparative analysis between the transcriptomes of the two rust species G. yamadae and G. asiaticum. The results identified several orthologous genes, and many expressed genes were identified by annotation. Our analysis of Ka/Ks ratios identified orthologous genes subjected to positive or purifying selection. An evolutionary analysis of these two species provided a relatively precise divergence time. Overall, the information obtained in this study increases the genetic resources available for research on the genetic diversity of the Gymnosporangium genus.

Integrating a Numerical Taxonomic Method and Molecular Phylogeny for Species Delimitation of Melampsora Species (Melampsoraceae, Pucciniales) on Willows in China.

The species in genus Melampsora are the causal agents of leaf rust diseases on willows in natural habitats and plantations. However, the classification and recognition of species diversity are challenging because morphological characteristics are scant and morphological variation in Melampsora on willows has not been thoroughly evaluated. Thus, the taxonomy of Melampsora species on willows remains confused, especially in China where 31 species were reported based on either European or Japanese taxonomic systems. To clarify the species boundaries of Melampsora species on willows in China, we tested two approaches for species delimitation inferred from morphological and molecular variations. Morphological species boundaries were determined based on numerical taxonomic analyses of morphological characteristics in the uredinial and telial stages by cluster analysis and one-way analysis of variance. Phylogenetic species boundaries were delineated based on the generalized mixed Yule-coalescent (GMYC) model analysis of the sequences of the internal transcribed spacer (ITS1 and ITS2) regions including the 5.8S and D1/D2 regions of the large nuclear subunit of the ribosomal RNA gene. Numerical taxonomic analyses of 14 morphological characteristics recognized in the uredinial-telial stages revealed 22 morphological species, whereas the GMYC results recovered 29 phylogenetic species. In total, 17 morphological species were in concordance with the phylogenetic species and 5 morphological species were in concordance with 12 phylogenetic species. Both the morphological and molecular data supported 14 morphological characteristics, including 5 newly recognized characteristics and 9 traditionally emphasized characteristics, as effective for the differentiation of Melampsora species on willows in China. Based on the concordance and discordance of the two species delimitation approaches, we concluded that integrative taxonomy by using both morphological and molecular variations was an effective approach for delimitating Melampsora species on willows in China.