Ying-Mei Liang

Cytospora species associated with walnut canker disease in China, with description of a new species C. gigalocus.

Cytospora species associated with canker disease are presently difficult to identify because of lack of ex-types cultures with molecular data, few distinguishable characters, and only Internal transcribed spacer (ITS) sequence data is available for most Cytospora strains in GenBank. We report on Cytospora species from the walnut tree in China. Collections were subjected to morphological and phylogenetic study. The relatedness of species associated with walnut canker were established using combined ITS, nrLSU, β-tubulin, and actin gene sequence data. Cytospora atrocirrhata, Cytospora chrysosperma, Cytospora sacculus, and a new species, Cytospora gigalocus, were identified causing canker disease of walnut. Cytospora gigalocus is formally described and compared with most similar species. Cytospora chrysosperma and C. sacculus have previously been recorded from walnut, whereas C. atrocirrhata is reported as associated with walnut canker for the first time. This is the first study that has established the Cytospora species causing walnut canker in China using a multi-phasic approach. All species are recorded as being associated with walnut canker disease in China for the first time.

Cytospora from Ulmus pumila in Northern China

Cytospora species are cosmopolitan, and commonly associated with dieback and canker diseases of numerous hosts. In the present study, isolates were collected and identified from diseased branches or twigs of Ulmus pumila in northern China. The morphological characteristics and multilocus phylogeny (act1, ITS, LSU, tefA and tubB) indicate four distinct lineages with high branch support, i.e., C. carbonacea, C. chrysosperma, C. ribis and C. pruinopsis sp. nov. Cytospora pruinopsis is distinguishable from the other Cytospora spp. on Ulmus by its single conidiomatal locule with one ostiole per disc, and its smaller conidia. This study represents the first attempt to clarify the taxonomy of Cytospora spp. associated with canker and dieback symptoms of Ulmus pumila in northern China.

Melanconis (Melanconidaceae) associated with Betula spp. in China

Melanconis is a monotypic genus in Melanconidaceae and mostly restricted to hardwood trees in Betulaceae, as endophytes and plant pathogens. During a routine forest pathogens investigation of canker disease in China, some melanconidaceous fungi were observed on Betula spp. with various symptoms. The primary objective of the present study was to identify species of Melanconidaceae associated with canker and dieback of Betula spp. in China. In the present study, we investigated 45 isolates using morphology and multi-gene phylogenetic analyses based on the internal transcribed spacers (ITS), nuclear large subunit rDNA (LSU), RNA polymerase II subunit 2 (rpb2), translation elongation factor 1-α (tef1-α) and β-tubulin (tub). Three melanconidaceous taxa were identified: Melanconis betulae sp. nov., M. itoana and M. stilbostoma. This is the first phylogenetic study evaluating species of Melanconis associated with canker and dieback disease of Betula spp. in China, resulting in the description of a novel species and the report of M. itoana as a new record.

Phragmidium species parasitizing species of Rosaceae in Tibet, China, with descriptions of three new species

Phragmidum spp. are rust fungi that are serious pathogens of plants in the Rosaceae. We characterized 15 Phragmidium species from Tibet, including 12 previously described and 3 new species. All the taxa, including the three new species (Ph. chayuensis, Ph. cibanum, Ph. zangdongii) are described and illustrated based on morphological characteristics. These taxa have multi-celled teliospores with firm pedicels. Phragmidium chayuensis is characterized by (5)7–9-celled teliospores with short papillae. Phragmidium cibanum is characterized by smooth teliospores that are distinctly constricted at the septa. Phragmidium zangdongii is characterized by large uredinia and 11–13-celled teliospores. Five types of urediniospore-surface structures were identified based on the gross shape of ornamentations and their distribution on the urediniospore wall. Molecular sequence data from the LSU rDNA analysis showed that the new taxa formed distinct clades independent from previously recorded species represented by LSU rDNA sequence data. The phylogenetic tree indicated that Phragmidium species are highly host specific.

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)

BackgroundRust 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.ResultsThe 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.ConclusionThis 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.