Bruce B. Clarke

Unraveling evolutionary relationships among the divergent lineages of colletotrichum causing anthracnose disease in turfgrass and corn.

ABSTRACT Colletotrichum species cause anthracnose diseases on a number of grass hosts and are common inhabitants of many others. They are divided into four species: C. sublineolum is pathogenic to Sorghum spp.; C. caudatum is found on C4 grasses such as indiangrass and big bluestem; C. falcatum causes red rot of sugarcane; and C. graminicola sensu lato is a broadly defined species including isolates that attack maize, wheat, oats, and many forage, turf, and amenity grasses of the subfamily Pooideae. In this paper, a combination of hierarchal- and nonhierarchal-based analyses were employed to examine evolutionary relationships among the grass-infecting Colletotrichum species, with special emphasis on isolates from turf and other grasses in the subfamily Pooideae. Reconstructions performed with data sets from over 100 Colletotrichum isolates at three variable loci using phylogenetic and network-based methodologies unambiguously supported the taxonomic separation of maize-infecting isolates of C. graminicola from the pooid-infecting strains of Colletotrichum. To reflect the evolutionary relationships that exist between these distinct lineages, we propose the resurrection of the species name C. cereale to describe the pooid-infecting isolates. There was also support for further subdivision of C. cereale, but the current data are insufficient to confidently subdivide the species, as there was some evidence of recombination between lineages of this species.

Isolation of the chitinolytic bacteria Xanthomonas maltophilia and Serratia marcescens as biological control agents for summer patch disease of turfgrass

Abstract A mycelial baiting method was used to isolate chitinolytic bacteria from soils known to harbor Magnaporthe poae, the causal agent of summer patch on Kentucky bluegrass. Two bacterial isolates, identified as Xanthomonas maltophilia 34S1 and Serratia marcescens 9M5, suppressed summer patch symptom development in Kentucky bluegrass cv. Baron by more than 70 and 50%, respectively, when compared to untreated control plants in growth chamber studies. Further studies indicated that time of application and concentration of bacteria influenced the degree of disease suppression. In general, bacteria were least effective in suppressing disease when they were applied to plants at dates closest to planting, and were most effective when applied up to 4 wk after planting. In bacterial concentration experiments, the highest degree of disease suppression was observed when plants were treated with the highest concentration of X. maltophilia 34S1. In contrast, the highest concentration of S. marcescens 9M5 did not provide the highest amount of disease control. Instead, less disease suppression was observed for plants treated with bacterial concentrations greater than the observed optimal concentration of 109 cfu ml−1. Survival of both bacteria was monitored in soil and the rhizosphere after application of three different bacterial concentrations. Populations of both bacteria in the rhizosphere stabilized 18 days after the final bacterial application, regardless of the concentration. For both bacteria, however, significant differences in non-rhizosphere soil populations, which steadily declined over time, were observed between the highest and lowest concentrations used throughout the sampling period.

What is the value of ITS sequence data in Colletotrichum systematics and species diagnosis? A case study using the falcate-spored graminicolous Colletotrichum group

Because the genus Colletotrichum is among the most important groups of plant pathogenic fungi worldwide, the ability to accurately diagnose species is vital for the implementation of effective disease control and quarantine measures. Although the long-standing, unresolved taxonomic issues in the genus have recently begun to be addressed through multi-locus phylogenetic research, the tools most commonly used for Colletotrichum species identification are either insufficiently variable (e.g. morphology), or homoplasic (e.g. morphology and host range criteria). In this study, using the systematically well-defined falcate-spored, grass-associated group (FG) of Colletotrichum as a model, we test the utility of ITS sequence data to diagnose species affiliations through similarity-based searches of the NCBI GenBank database or by means of gene trees constructed using phylogenetic methods. 43% of all Colletotrichum sequences accessioned by GenBank are from the ITS region, making it the single most common sequence curated by the community; however, 34% of the ITS accessions existed only as sequence data in the database, with no associatedPUBLICation. Using Colletotrichum ITS sequence data from 53 FG defined isolates and 16 falcate-spored, non-graminicolous isolates to perform GenBank BLASTN searches, we found that erroneous identifications occurred for 86% of the 14 species tested. In contrast, the phylogenetic tree generated by the ITS sequence data, although poorly supported by bootstrap values, correctly grouped most of the species, but 10% of the individual isolates were incorrectly placed. From this study, we conclude that the currently available infrastructure of Colletotrichum ITS sequence data may yield unreliable species diagnoses, particularly if sequence similarity alone is the only criterion applied.

Evaluation of bacterial antagonists for reduction of summer patch symptoms in Kentucky bluegrass

Ten bacterial strains previously identified for their ability to control soilborne pathogens on agronomic crops were evaluated for their ability to suppress summer patch disease caused by Magnaporthe poae in Kentucky bluegrass (Poa pratensis L). Bacterial strains varied in the ability to inhibit the growth of M. poae in agar plate bioassays, although most strains inhibited the fungus to some degree. Three strains originally isolated from wheat, Pseudomonas fluorescens 2-79, P. fluorescens 13-79, and Bacillus subtilis D-39Sr, and two strains originally isolated from cotton, Enterobacter cloacae EcH-1 and EcCT-501, significantly reduced summer patch symptoms by between 29 and 46% compared to untreated control plants after a 5-week period in the growth chamber. The five strains did not reduce summer patch disease in field trials in 1990. However, in 1991, B. subtilis D-39Sr and E. cloacae EcH-I reduced summer patch severity by 53 and 49%, respectively, over the entire season compared to untreated control plots. In 1994, both B. subtilis D-39Sr and E. cloacae EcH-1 also reduced summer patch severity over the entire season by up to 39 and 34%, respectively, compared to the untreated control in field plots. All five bacteria that reduced summer patch in growth chamber studies were present in the rhizosphere of greenhouse/growth chamber grown turfgrass at 10 4 to 10 6 CFU/g of fresh weight sample 2 weeks after application to plants. A single sampling of field plots during 1991 and four separate samplings of field plots during 1994 indicated that introduced bacteria were present within the turf at populations above 10 3 CFU/g of sample.

Phylogenetic and population genetic divergence correspond with habitat for the pathogen Colletotrichum cereale and allied taxa across diverse grass communities.

Over the past decade, the emergence of anthracnose disease has newly challenged the health of turfgrasses on North American golf courses, resulting in considerable economic loss. The fungus responsible for the outbreaks, Colletotrichum cereale, has also been identified from numerous natural grasses and cereal crops, although disease symptoms are generally absent. Here we utilize phylogenetic and population genetic analyses to determine the role of ecosystem in the advancement of turfgrass anthracnose and assess whether natural grass and/or cereal inhabitants are implicated in the epidemics. Using a four‐gene nucleotide data set to diagnose the limits of phylogenetic species and population boundaries, we find that the graminicolous Colletotrichum diverged from a common ancestor into distinct lineages correspondent with host physiology (C3 or C4 photosynthetic pathways). In the C4 lineage, which includes the important cereal pathogens Colletotrichum graminicola, C. sublineolum, C. falcatum, C. eleusines, C. caudatum and several novel species, host specialization predominates, with host‐associated lineages corresponding to isolated sibling species. Although the C3 lineage —C. cereale— is comprised of one wide host‐range species, it is divided into 10 highly specialized populations corresponding to ecosystem and/or host plant, along with a single generalist population spread across multiple habitat types. Extreme differentiation between the specialized C. cereale populations suggests that asymptomatic nonturfgrass hosts are unlikely reservoirs of infectious disease propagules, but gene flow between the generalist population and the specialized genotypes provides an indirect mechanism for genetic exchange between otherwise isolated populations and ecosystems.

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.

Systematic analysis of the falcate-spored graminicolous Colletotrichum and a description of six new species from warm-season grasses

Species limits in the fungal genus Colletotrichum are traditionally distinguished by appressorial and/or conidial morphology or through host plant association, but both criteria are criticized for their inability to resolve distinct taxa. In previous research eight novel falcate-spored Colletotrichum species were identified from graminicolous hosts using multilocus molecular phylogenetic analysis. In the present work formal descriptions and illustrations are provided for six of the new taxa: C. hanaui sp. nov., C. nicholsonii sp. nov., C. paspali sp. nov., C. jacksonii sp. nov., C. miscanthi sp. nov. and C. axonopodi sp. nov.; and an emended description with epitypification is provided for C. eleusines. Comparison of hyphopodial appressoria and host association against phylogenetic species boundaries and evolutionary relationships in the graminicolous Colletotrichum group demonstrate that, while these characters can be useful in combination for the purpose of species diagnosis, erroneous identification is possible and species boundaries might be underestimated if these characters are used independently, as exemplified by the polyphyletic taxa C. falcatum. Appressoria have been subject to convergent evolution and were not predictive of phylogenetic relationships. Despite these limitations, the results of this work establish that in combination appressorial and host range characters could be used to generate informative dichotomous identification keys for Colletotrichum species groups when an underlying framework of evolutionary relationships, taxonomic criteria and nomenclature have been satisfactorily derived from molecular systematic treatments.

Phylogenetic relationships within and between Epichloë and Neotyphodium endophytes as estimated by AFLP markers and rDNA sequences

Analysis of Amplified Fragment Length Polymorphisms (AFLP) in a sample population of Epichloe and Neotyphodium was conducted to evaluate the usefulness of this technique in estimating phylogenetic relationships among these fungi. A total of 963 unique restriction fragments were identified with 13 selective primer combinations. Neighbour-joining and parsimony analysis produced phylogenetic trees with high bootstrap support. All Epichloe festucae isolates were monophyletic, and clades of E. festucae were identified that correspond to the host species from which they were isolated. These groups may indicate the importance of coevolution in the development of the fine fescue- E. festucae symbiosis. The grouping of E. festucae isolates from hard fescue and Chewings fescue into one clade conflicts with the current taxonomy of these host grasses. Sequencing of ribosomal DNA spacer regions ITS1 and ITS2 was conducted for comparisons to the phylogenetic relationships estimated by AFLP. Using the maximum likelihood test, a significant difference was detected between AFLP and rDNA trees due to discrepancies in the association of anamorphs ( Neotyphodium ) and teleomorphs ( Epichloe ). This discrepancy may be due to some mechanism of genetic recombination, such as hybridization or parasexual recombination, that occurs in Neotyphodium populations. It is concluded that AFLP markers are useful for producing meaningful phylogenies among a diverse group of fungi and for identifying groups of closely related individuals that are genetically and biologically similar. Further investigation is needed to determine how AFLP markers change over short periods of time in populations and over evolutionary time.

Nitrogen Form and Rate of Nitrogen and Chloride Application for the Control of Summer Patch in Kentucky Bluegrass.

The influence of nitrogen form and rate of nitrogen and chloride application on turf quality and summer patch severity was assessed in Kentucky bluegrass cv. Fylking at one site for 2 yr. Plots were artificially inoculated with a five-isolate mixture of Magnaporthe poae in 1990. Every 3 wk, varying rates of ammonium sulfate or calcium nitrate (to supply 0, 98, or 196 kg N ha −1 yr −1 ) and combinations of potassium sulfate and potassium chloride (to provide 0, 122, or 244 kg Cl ha −1 yr −1 ) were applied from May to October during 1990 and 1991. In 1990, patch diameter, patch severity, and rhizosphere pH were slightly reduced by the application of ammonium sulfate. In 1991, onset of summer patch symptoms was delayed, and patch development and pH of the rhizosphere and bulk soil were greatly reduced where ammonium sulfate was applied. The high (196 kg N ha −1 yr −1 ) rate of ammonium sulfate reduced summer patch severity up to 75% compared with the same rate of calcium nitrate. Chloride application did not influence disease severity, turf quality, or soil pH. In general, turf quality was not significantly influenced by the form of nitrogen or the rate of nitrogen application

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.