Jiye Yan

One stop shop: backbones trees for important phytopathogenic genera: I (2014)

Many fungi are pathogenic on plants and cause significant damage in agriculture and forestry. They are also part of the natural ecosystem and may play a role in regulating plant numbers/density. Morphological identification and analysis of plant pathogenic fungi, while important, is often hampered by the scarcity of discriminatory taxonomic characters and the endophytic or inconspicuous nature of these fungi. Molecular (DNA sequence) data for plant pathogenic fungi have emerged as key information for diagnostic and classification studies, although hampered in part by non-standard laboratory practices and analytical methods. To facilitate current and future research, this study provides phylogenetic synopses for 25 groups of plant pathogenic fungi in the Ascomycota, Basidiomycota, Mucormycotina (Fungi), and Oomycota, using recent molecular data, up-to-date names, and the latest taxonomic insights. Lineage-specific laboratory protocols together with advice on their application, as well as general observations, are also provided. We hope to maintain updated backbone trees of these fungal lineages over time and to publish them jointly as new data emerge. Researchers of plant pathogenic fungi not covered by the present study are invited to join this future effort. Bipolaris, Botryosphaeriaceae, Botryosphaeria, Botrytis, Choanephora, Colletotrichum, Curvularia, Diaporthe, Diplodia, Dothiorella, Fusarium, Gilbertella, Lasiodiplodia, Mucor, Neofusicoccum, Pestalotiopsis, Phyllosticta, Phytophthora, Puccinia, Pyrenophora, Pythium, Rhizopus, Stagonosporopsis, Ustilago and Verticillium are dealt with in this paper.

Improving ITS sequence data for identification of plant pathogenic fungi

SummaryPlant pathogenic fungi are a large and diverse assemblage of eukaryotes with substantial impacts on natural ecosystems and human endeavours. These taxa often have complex and poorly understood life cycles, lack observable, discriminatory morphological characters, and may not be amenable to in vitro culturing. As a result, species identification is frequently difficult. Molecular (DNA sequence) data have emerged as crucial information for the taxonomic identification of plant pathogenic fungi, with the nuclear ribosomal internal transcribed spacer (ITS) region being the most popular marker. However, international nucleotide sequence databases are accumulating numerous sequences of compromised or low-resolution taxonomic annotations and substandard technical quality, making their use in the molecular identification of plant pathogenic fungi problematic. Here we report on a concerted effort to identify high-quality reference sequences for various plant pathogenic fungi and to re-annotate incorrectly or insufficiently annotated public ITS sequences from these fungal lineages. A third objective was to enrich the sequences with geographical and ecological metadata. The results – a total of 31,954 changes – are incorporated in and made available through the UNITE database for molecular identification of fungi (http://unite.ut.ee), including standalone FASTA files of sequence data for local BLAST searches, use in the next-generation sequencing analysis platforms QIIME and mothur, and related applications. The present initiative is just a beginning to cover the wide spectrum of plant pathogenic fungi, and we invite all researchers with pertinent expertise to join the annotation effort.

Species of Botryosphaeriaceae involved in grapevine dieback in China

Botryosphaeria dieback is a serious disease problem for table and grape wine production worldwide. The disease however, has been less well-studied in China. In this study, we surveyed Botryosphaeria dieback in 72 vineyards of 20 grape-growing regions in China and found that Botryosphaeria dieback occurs in 18 out of 20 provinces. Morphological and multi-gene phylogenetic analyses confirmed that Botryosphaeria dothidea, Diplodia seriata, Lasiodiplodia theobromae and Neofusicoccum parvum are associated with different grapevine dieback symptoms. This study also revealed considerable differences in the geographical distribution of Botryosphaeriaceae species in China with Lasiodiplodia theobromae and Neofusicoccum parvum occurring only in subtropical monsoon climate regions, Diplodia seriata occurring only in temperate monsoon climate regions, and Botryosphaeria dothidea occurring in both temperate and subtropical monsoon climate regions. Analysis of 26 isolates showed that there is little genetic variation within species. Koch’s postulates were satisfied for all species, and pathogenicity testing showed that among the 25 major cultivars growing in China, none was resistant to the four taxa. The current paper represents the first detailed report on Botryosphaeria dieback of grapevines in China.

Diverse species of Colletotrichum associated with grapevine anthracnose in China

Grapevine anthracnose is an important disease, responsible for mild to severe yield losses in grape production, and is also an important post harvest disease. The disease was studied in vineyards in six provinces in China, with 34 isolates obtained from diseased grapes. Multi-gene (ACT, ITS, GAPDH, TUB2 and CHS) analysis coupled with morphology showed that Colletotrichum aenigma, C. hebeiense sp. nov. and C. viniferum were associated with grapevine anthracnose in China. Colletotrichum aenigma is reported for the first time as associated with grapevine anthracnose. Colletotrichum hebeiense is a new species introduced here. Pathogenicity testing showed that all species can infect grapes, causing anthracnose however, virulence of species and isolates showed great variation. Phylogenetic analysis showed that C. viniferum is a cryptic species and its taxonomy needs to be resolved in the future.

Morphological and molecular characterisation of Diaporthe species associated with grapevine trunk disease in China

Trunk diseases in grapevine (Vitis spp.) are major problems in the wine and table-grape industries reducing the productivity, quality and longevity of vineyards. Species of Diaporthe are important fungal pathogens of grapevine trunk disease worldwide. A survey of 14 grape vineyards located in different provinces of China was yielded Diaporthe isolates associated with symptomatic grapevine wood. These isolates were identified based on morphology and a combined data matrix of rDNA ITS, partial sequences of translation elongation factor 1-α (EF 1-α), β-tubulin (TUB) and calmodulin (CAL) gene regions. Four species of Diaporthe were identified, which included Diaporthe eres, Diaporthe hongkongensis, Diaporthe phaseolorum and Diaporthe sojae. All isolates of Diaporthe caused disease on detached grape shoots in pathogenicity experiments but differed in virulence. The incidence in local vineyards and the pathogenicity results indicate that D. eres is an important pathogen of grapevine in Chinese vineyards, where it may significantly limit grape production. This is the first detailed report of Diaporthe species associated with grapevine trunk diseases in China with morphology, pathogenicity and molecular data.

Identification and characterization of Pestalotiopsis- like fungi related to grapevine diseases in China

Pestalotiopsis-like fungi are an important plant pathogenic genus causing postharvest fruit rot and trunk diseases in grapevine in many countries. Pestalotiopsis-like fungi diseases were studied in vineyards in nine provinces across China. Multi-gene (ITS, β-tubulin and tef1) analysis coupled with morphology showed that a Neopestalotiopsis sp. and Pestalotiopsis trachicarpicola are associated in causing grapevine fruit rot and trunk diseases in China. Pestalotiopsis trachicarpicola is reported as the causative agent of grapevine diseases in the world for the first time. Neopestalotiopsis sp. caused significantly longer lesions than the other taxon present. This study represents the first attempt to identify and characterize the Pestalotiopsis-like fungi causing grapevine diseases in China using both morphological and molecular approaches.

A new Alternaria species from grapevine in China

Twelve Alternaria strains were isolated from pedicels and rachis from different parts of grapevines in China. In a phylogenetic analysis, nine showed a close relationship with Alternaria longipes, and are described as a new species, A. viniferae sp. nov., but the other four clustered with A. alternata and A. arborescens within the alternata species-group. The new species can clearly be separated from other related small-spored Alternaria species based on sequences of portions of the glyceraldehyde-3-phosphate dehydrogenase (gpd) and Alternaria major allergen (Alt a 1) genes as well as by distinctive morphology.

Characterization of Botryosphaeria dothidea, the causal agent of grapevine canker in China

Grapevine canker caused by Botryosphaeriaceae is currently one of most important trunk diseases in the world. This study identified one of the pathogens-B. dothidea that causes grapevine canker in most grapevine growing areas of China and verified its pathogenicity and geographical distribution. In total, 320 grapevine canker sympotoms samples were collected from 17 provinces between 2009 and 2011, and 4261 isolates were isolated from diseased samples. Among these, 1172 isolates purified from 150 samples were identified as B. dothidea by morphological and molecular characterizations. Three representative isolates were used to determine both optimal temperature and optimal nutrition medium for growth and germination. Pathogenicity of B. dothidea was determined in 25 grapevine cultivars. Pathogenicity results showed that most of the main grapevine cultivars growing in China are susceptible to the pathogen.

Occurrence of Grapevine Trunk Disease Caused by Botryosphaeria rhodina in China

In the early summer of 2009, grapevine (Vitis vinifera), an important fruit crop in China, declined in most of the vineyards in Hunan, Hubei, and Zhejiang provinces. Characteristic symptoms of Botryosphaeria canker were apparent, including trunk cankers (visible in cross-section), leaf drop, shriveling and drying of fruit clusters, and berry rot (1). To identify the causal pathogen, we tested 126 samples by attempting to culture the pathogen from a small piece of tissue from the canker margin between the necrotic and apparently healthy tissue. Plant tissue was surface sterilized by placing it in 75% ethanol for 1 min and rinsed with sterilized water three times before culturing to potato dextrose agar (PDA) at 28°C. Five days later, the cultures were hyphal-tip purified and then single-spore isolates were used for identification. On the basis of colony characteristics in PDA, these colonies were identified as Botryosphaeria spp. (2). They were grayish white, becoming dark brown with age, and pycnidia were formed after incubation for approximately 9 days. Conidia measured 11 to 15 × 22 to 28 μm. A subset of isolates were used for rDNA ITS (internal transcribed spacer) sequence analysis with primers ITS1 and ITS4 (3). PCR products were separated by electrophoresis and bands were purified (Qiagen Plasmid Mini Kit; Qiagen, Valencia, CA) for sequencing (Sunbiotech Company, Beijing). BLAST searches of three ITS sequences (Accession Nos. GU226851, GU226853, and GU226856) had 100% identity to B. rhodina. EF1-α and β-tubulin sequence analysis gave similar results. Kochs postulates were completed in the laboratory on grape shoots inoculated with two isolates of B. rhodina, originally isolated from plants in the field with symptoms of Botryosphaeria canker. Isolates were incubated on PDA at 25°C for 1 week. Inoculations were made on green shoots of V. vinifera cvs. Muscat Hamburg and Crimson Seedless. Five shoots per cultivar were inoculated per isolate by wounding with a 4-mm cork borer (2 mm deep), placing a colonized agar plug on the wound, and wrapping the wound with Parafilm. Controls were mock inoculated with an agar plug from sterile PDA. Inoculated shoots were incubated in the laboratory in the dark under moist conditions for 10 days at 25°C. Inoculated shoots had necrotic cankers after 10 days and B. rhodina was recovered from each canker margin. The results suggest that some grapevines in China with symptoms of Botryosphaeria canker were indeed infected by B. rhodina. To our knowledge, this is the first report of this pathogen on grapevine in China. References: (1) J. Luque et al. Mycologia 97:1111, 2005. (2) J. M. Niekerk et al. Mycologia 96:781, 2004. (3) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990.

First Report of Grapevine Trunk Disease Caused by Botryosphaeria obtusa in China

In September 2010, grapevine (Vitis vinifera) trunk diseases were observed in several vineyards of Yantai District in Shandong Provinces and Changli County of Hebei Provinces of China. Characteristic symptoms of Botryosphaeria canker were apparent, including dark brown discoloration on the trunk (visible in cross-section), cob base shriveling, drying of fruit clusters, and berry falling (2). To identify the causal pathogen, culturing of fungi was attempted from 387 small pieces of tissue from the canker margins of 43 diseased plants. Samples were surface disinfected by placing them in 75% ethanol for 1 min and rinsing with sterilized water three times before culturing on potato dextrose agar (PDA) at 28°C for 7 to 10 days. Fungi isolated were single spored to obtain pure cultures. On the basis of colony characteristics on PDA, 18 isolates from the 387 tissue pieces were eventually identified as Botryosphaeria obtusa (1), Most of the other fungi isolated were B. dothidea. B. obtusa colonies were grayish white, becoming dark brown with age, and pycnidia were formed after incubation for approximately 7 days. Conidia measured 8 to 11 × 17 to 26 μm (n= 50). Two isolates were used for rDNA internal transcribed spacer (ITS) sequence analysis with primers ITS1 and ITS4 (3). PCR products were separated by electrophoresis and bands were purified for legation with PMD-18T (Takara Company, Dalian, China) vector for sequencing. BLAST searches of two ITS sequences had 99 to 100% identity to B. obtusa. EF1-α and β-tubulin sequence analysis gave similar results. Kochs postulates were completed in the greenhouse on grape shoots inoculated with two isolates of B. obtusa originally isolated from diseased plants in the field. Inoculations were made on green shoots of V. vinifera cv. Dunkelfelder T. Six shoots were inoculated per isolate by wounding with a 4-mm cork borer (2 mm deep) and placing a colonized agar plug from a 5-day-old culture on the wound and wrapping it with Parafilm. Controls were mock inoculated with an agar plug from sterile PDA. Inoculated shoots were incubated in the dark under moist conditions in the laboratory for 8 to 10 days at 25°C. Inoculated shoots had necrotic cankers after 8 to 10 days and B. obtusa was recovered from each canker margin. The results indicated that some grapevines in China with symptoms of Botryosphaeria canker were infected by B. obtusa. To our knowledge, this is the first report of this pathogen causing trunk disease on grapevine in China. References: (1) A. Taylor et al. Australas. Plant Pathol. 34:187, 2005. (2) J. R. Úrbez-Torres et al. Plant Dis. 92:519, 2008. (3) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990.