Genome Resources for Seven Fungal Isolates That Cause Dollar Spot Disease in Turfgrass, Including Clarireedia jacksonii and C. monteithiana.

Fungi in the genus Clarireedia are widespread and destructive pathogens of grasses worldwide, and are best known as the causal agents of dollar spot disease in turfgrass. Here, we report genome assemblies of seven Clarireedia isolates, including ex-types of the two most widespread species, Clarireedia jacksonii and C. monteithiana. These datasets provide a valuable resource for ongoing studies of the dollar spot pathogens that include population diversity, host-pathogen interactions, marker development, and disease control.

Clarireedia: A new fungal genus comprising four pathogenic species responsible for dollar spot disease of turfgrass.

Dollar spot is one of the most destructive and economically important fungal diseases of amenity turfgrasses. The causal agent was first described in 1937 as the ascomycete Sclerotinia homoeocarpa. However, the genus-level taxonomic placement of this fungus has been the subject of an ongoing debate for over 75 y. Existing morphological and rDNA sequence evidence indicates that this organism is more appropriately placed in the family Rutstroemiaceae rather than the Sclerotiniaceae. Here we use DNA sequence data from samples of the dollar spot fungus and other members of the Rutstroemiaceae (e.g. Rutstroemia, Lanzia, Lambertella) collected throughout the world to determine the generic identity of the turfgrass dollar spot pathogen. Phylogenetic evidence from three nucleotide sequence markers (CaM, ITS and Mcm7; 1810-bp) confirmed that S. homoeocarpa is not a species of Sclerotinia; nor is it a member of any known genus in the Rutstroemiaceae. These data support the establishment of a new genus, which we describe here as Clarireedia gen. nov. The type species for the genus, Clarireedia homoeocarpa comb. nov., is described to accommodate the dollar spot fungus, and a neotype is designated. Three new species in this clade, Clarireedia bennettii sp. nov., Clarireedia jacksonii sp. nov., and Clarireedia monteithiana sp. nov. that also cause dollar spot disease are described. Clarireedia homoeocarpa and C. bennettii occur primarily on Festuca rubra (C3 grass) hosts and appear to be restricted to the United Kingdom. Clarireedia jacksonii and C. monteithiana occur on a variety of C3 and C4 grass hosts, respectively, and appear to be globally distributed. This resolved taxonomy puts to rest a major controversy amongst plant pathologists and provides a foundation for better understanding the nature and biology of these destructive pathogens.

Development of a greenhouse-based inoculation protocol for the fungus Colletotrichum cereale pathogenic to annual bluegrass (Poa annua).

The fungus Colletotrichum cereale incites anthracnose disease on Poa annua (annual bluegrass) turfgrass. Anthracnose disease is geographically widespread throughout the world and highly destructive to cool-season turfgrasses, with infections by C. cereale resulting in extensive turf loss. Comprehensive research aimed at controlling turfgrass anthracnose has been performed in the field, but knowledge of the causal organism and its basic biology is still needed. In particular, the lack of a reliable greenhouse-based inoculation protocol performed under controlled environmental conditions is an obstacle to the study of C. cereale and anthracnose disease. Our objective was to develop a consistent and reproducible inoculation protocol for the two major genetic lineages of C. cereale. By adapting previously successful field-based protocols and combining with components of existing inoculation procedures, the method we developed consistently produced C. cereale infection on two susceptible P. annua biotypes. Approximately 7 to 10 days post-inoculation, plants exhibited chlorosis and thinning consistent with anthracnose disease symptomology. Morphological inspection of inoculated plants revealed visual signs of the fungus (appressoria and acervuli), although acervuli were not always present. After stringent surface sterilization of inoculated host tissue, C. cereale was consistently re-isolated from symptomatic tissue. Real-time PCR detection analysis based on the Apn2 marker confirmed the presence of the pathogen in host tissue, with both lineages of C. cereale detected from all inoculated plants. When a humidifier was not used, no infection developed for any biotypes or fungal isolates tested. The inoculation protocol described here marks significant progress for in planta studies of C. cereale, and will enable scientifically reproducible investigations of the biology, infectivity and lifestyle of this important grass pathogen.

IMA Genome-F 4: Draft genome sequences of Chrysoporthe austroafricana, Diplodia scrobiculata, Fusarium nygamai, Leptographium lundbergii, Limonomyces culmigenus, Stagonosporopsis tanaceti, and Thielaviopsis punctulata.

The genomes of Chrysoporthe austroafricana, Diplodia scrobiculata, Fusarium nygami, Leptographium lundbergii, Limonomyces culmigenus, Stagonosporopsis tanaceti, and Thielaviopsis punctulata are presented in this genome announcement. These seven genomes are from endophytes, plant pathogens and economically important fungal species. The genome sizes range from 26.6 Mb in the case of Leptographium lundbergii to 44 Mb for Chrysoporthe austroafricana. The availability of these genome data will provide opportunities to resolve longstanding questions regarding the taxonomy of species in these genera, and may contribute to our understanding of the lifestyles through comparative studies with closely related organisms.

Influence of host and geographic locale on the distribution of Colletotrichum cereale lineages.

Colletotrichum cereale is an ascomycete inhabitant of cool-season Pooideae grasses. The fungus has increased in frequency over the past decade as a destructive pathogen of Poa annua and Agrostis stolonifera turfgrass. Colletotrichum cereale exists as two lineages, designated clades A and B, but little is known about the distribution of these clades in natural environments, or what role these subdivisions may play in the trajectory of disease outbreaks. In this study, our objective was to determine the frequency of C. cereale clades A and B. To rapidly discriminate between the two C. cereale clades, a real-time PCR assay was developed based on the Apn2 gene. A collection of 700 C. cereale pathogens and endophytes from twenty Pooideae grass genera were genotyped. 87% of the collection was identifed as part of clade A, 11.7% as part of clade B, and 1.3% was a mixture. Colletotrichum cereale from turfgrass hosts in North America were most commonly members of clade A (78%). The overabundance of clade A in turfgrass isolates was directly attributable to the dominance of this lineage from southern sampling sites, irrespective of host. In contrast, 111 C. cereale turfgrass isolates collected from northern sampling sites were evenly distributed between clades A and B. Only 28% of C. cereale from A. stolonifera at northern sampling sites were part of clade A. These data show that environmental factors such as geographic location and host identity likely played a role in the distribution of the major C. cereale clades in North American turfgrass.

IMA Genome-F 3: Draft genomes of Amanita jacksonii, Ceratocystis albifundus, Fusarium circinatum, Huntiella omanensis, Leptographium procerum, Rutstroemia sydowiana, and Sclerotinia echinophila.

The genomes of fungi provide an important resource to resolve issues pertaining to their taxonomy, biology, and evolution. The genomes of Amanita jacksonii, Ceratocystis albifundus, a Fusarium circinatum variant, Huntiella omanensis, Leptographium procerum, Sclerotinia echinophila, and Rutstroemia sydowiana are presented in this genome announcement. These seven genomes are from a number of fungal pathogens and economically important species. The genome sizes range from 27 Mb in the case of Ceratocystis albifundus to 51.9 Mb for Rutstroemia sydowiana. The latter also encodes for a predicted 17 350 genes, more than double that of Ceratocystis albifundus. These genomes will add to the growing body of knowledge of these fungi and provide a value resource to researchers studying these fungi.

Molecular Analysis of Turfgrass Rusts Reveals the Widespread Distribution of Puccinia coronata as a Pathogen of Kentucky Bluegrass in the United States.

Over the past 10 years, rust diseases have become increasingly prevalent on certain cultivars of Kentucky bluegrass. This pattern suggests that new races or new species of rust fungi may have emerged. To test this hypothesis, 66 samples of turfgrass rust fungi collected from across the United States were evaluated based on sequences of the internal transcribed spacer (ITS)-5.8S rDNA region. Phylogenetic analysis revealed three species: Puccinia coronata, P. graminis, and P. striiformis, comprising 67, 28, and 5% of the samples, respectively. P. coronata was frequently found in association with Kentucky bluegrass, a host-pathogen relationship that has not been previously reported. Comparison of molecular analyses with the use of standard field identification techniques-host association and pustule pigmentation-showed that 58% of the Kentucky bluegrass samples would have been incorrectly diagnosed using nonmolecular criteria. To avoid such misidentifications, a real-time polymerase chain reaction diagnostic protocol was developed for turfgrass-associated P. graminis, P. coronata, and P. striiformis using ITS sequences. Accurate, reproducible, species-specific identifications were made using as few as 50 to 150 urediniospores, even in mixed infections. This study represents the first DNA-based evaluation of turfgrass rust fungi and provides a quick and reliable sequence-based protocol as an alternative to less reliable field-based identification techniques.

Anthracnose disease of switchgrass caused by the novel fungal species Colletotrichum navitas.

In recent years perennial grasses such as the native tallgrass prairie plant Panicum virgatum (switchgrass) have taken on a new role in the North American landscape as a plant-based source of renewable energy. Because switchgrass is a native plant, it has been suggested that disease problems will be minimal, but little research in this area has been conducted. Recently, outbreaks of switchgrass anthracnose disease have been reported from the northeastern United States. Incidences of switchgrass anthracnose are known in North America since 1886 through herbarium specimens and disease reports, but the causal agent of this disease has never been experimentally determined or taxonomically evaluated. In the present work, we evaluate the causal agent of switchgrass anthracnose, a new species we describe as Colletotrichum navitas (navitas=Latin for energy). Multilocus molecular phylogenetics and morphological characters show C. navitas is a novel species in the falcate-spored graminicolous group of the genus Colletotrichum; it is most closely related to the corn anthracnose pathogen Colletotrichum graminicola. We present a formal description and illustrations for C. navitas and provide experimental confirmation that this organism is responsible for switchgrass anthracnose disease.