Detached Maize Sheaths for Live-Cell Imaging of Infection by Fungal Foliar Maize Pathogens.

We have optimized a protocol to inoculate maize leaf sheaths with hemibiotrophic and necrotrophic foliar pathogenic fungi. The method is modified from one originally applied to rice leaf sheaths and allows direct microscopic observation of fungal growth and development in living plant cells. Leaf sheaths collected from maize seedlings with two fully emerged leaf collars are inoculated with 20 µL drops of 5 x 105 spores/mL fungal spore suspensions and incubated in humidity chambers at 23 °C under continuous fluorescent light. After 24-72 h, excess tissue is removed with a razor blade to leave a single layer of epidermal cells, an optically clear sample that can be imaged directly without the necessity for chemical fixation or clearing. Plant and fungal cells remain alive for the duration of the experiment and interactions can be visualized in real-time. Sheaths can be stained or subjected to plasmolysis to study the developmental cytology and viability of host and pathogen cells during infection and colonization. Fungal strains transformed to express fluorescent proteins can be inoculated or co-inoculated on the sheaths for increased resolution and to facilitate the evaluation of competitive or synergistic interactions. Fungal strains expressing fluorescent fusion proteins can be used to track and quantify the production and targeting of these individual proteins in planta. Inoculated sheath tissues can be extracted to characterize nucleic acids, proteins, or metabolites. The use of these sheath assays has greatly advanced the detailed studies of the mechanisms of fungal pathogenicity in maize and also of fungal protein effectors and secondary metabolites contributing to pathogenicity.

Identification of Quinone Outside Inhibitor Fungicide-Resistant Isolates of Parastagonospora nodorum from Illinois and Kentucky.

Stagonospora leaf and glume blotch, caused by Parastagonospora nodorum, is a major disease of winter wheat (Triticum aestivum) in the United States capable of significantly reducing grain yield and quality. Pathogens such as P. nodorum that overwinter in crop residue are often an increased concern in cropping systems that utilize no-till farming. In addition, the lack of wheat cultivars with complete resistance to P. nodorum has led to the reliance on foliar fungicides for disease management. Quinone outside inhibitor (QoI) fungicides (Fungicide Resistance Action Committee group 11) are one of the major classes used to manage foliar diseases in wheat. Use of the QoI class of fungicides tends to select isolates of fungal pathogens with resistance due to mutations in the fungal cytochrome b gene. Isolates of P. nodorum were collected from Illinois in 2014 and Kentucky in 2018, 2019, and 2020. Amplification and sequencing of a segment of the cytochrome b gene from these isolates revealed a mutation at codon 143 that confers a change from glycine to alanine in the amino acid sequence (known as the G143A mutation). In vitro plate assays and greenhouse trials were used to confirm and characterize the QoI resistance caused by the G143A mutation. The frequency of the tested isolates with the G143A mutation was 46% (57 of 123 isolates) and 5% (3 of 60 isolates) for Kentucky and Illinois, respectively. This research is the first to identify the G143A mutation in P. nodorum isolates with resistance to QoI fungicides in Illinois and Kentucky.

Maize Anthracnose Stalk Rot in the Genomic Era.

Anthracnose stalk rot (ASR) of maize results in millions of dollars in losses annually in the United States. ASR, together with anthracnose leaf blight and anthracnose top dieback, is caused by the fungus Colletotrichum graminicola. Current ASR management recommendations emphasize host resistance and reduction of plant stressors (e.g., drought, heat, low fertility, or soil acidity). Stress reduction may be more difficult to achieve in the future due to more high-intensity production protocols and climate change. Moreover, cultural and chemical management practices may conflict with other important goals, including environmental sustainability and maximization of yield potential. Thus, future ASR management may rely more heavily on host resistance, for which there are relatively few highly effective sources. The last comprehensive review of C. graminicola and maize anthracnose was written over two decades ago. The genomic age has brought important new insights into mechanisms governing the host-pathogen interaction from the application of molecular and cytological technologies. This review provides a summary of our current model of maize anthracnose etiology, including how increased knowledge of molecular and cellular events could contribute to better ASR management. Improved understanding of C. graminicola taxonomy has confirmed that the fungus is specific to Zea mays, and that it colonizes living maize tissues via a critical biotrophic phase. Successful biotrophic establishment relies on an array of secreted protein effectors and secondary metabolites produced at different stages of infection and dispersed to multiple locations. These molecules could provide therapeutic targets for the next generation of transgenic or gene-edited ASR-resistant hybrids.

Fusarium graminearum Species Complex: A Bibliographic Analysis and Web-Accessible Database for Global Mapping of Species and Trichothecene Toxin Chemotypes.

Fusarium graminearum is ranked among the five most destructive fungal pathogens that affect agroecosystems. It causes floral diseases in small grain cereals including wheat, barley, and oats, as well as maize and rice. We conducted a systematic review of peer-reviewed studies reporting species within the F. graminearum species complex (FGSC) and created two main data tables. The first contained summarized data from the articles including bibliographic, geographic, methodological (ID methods), host of origin and species, while the second data table contains information about the described strains such as publication, isolate code(s), host/substrate, year of isolation, geographical coordinates, species and trichothecene genotype. Analyses of the bibliographic data obtained from 123 publications from 2000 to 2021 by 498 unique authors and published in 40 journals are summarized. We describe the frequency of species and chemotypes for 16,274 strains for which geographical information was available, either provided as raw data or extracted from the publications, and sampled across six continents and 32 countries. The database and interactive interface are publicly available, allowing for searches, summarization, and mapping of strains according to several criteria including article, country, host, species and trichothecene genotype. The database will be updated as new articles are published and should be useful for guiding future surveys and exploring factors associated with species distribution such as climate and land use. Authors are encouraged to submit data at the strain level to the database, which is accessible at https://fgsc.netlify.app.

Aggressiveness and Mycotoxin Production by Fusarium meridionale Compared with F. graminearum on Maize Ears and Stalks in the Field.

Fusarium meridionale and F. graminearum both cause Gibberella ear rot (GER) and Gibberella stalk rot (GSR) of maize in Brazil, but the former is much more common. Recent work with two isolates of each from maize suggested this dominance could be caused by greater aggressiveness and competitiveness of F. meridionale on maize. We evaluated pathogenicity and toxigenicity of 16 isolates of F. graminearum and 24 isolates of F. meridionale recovered from both wheat and maize. Strains were individually inoculated into ears of four maize hybrids in field trials. GER severity varied significantly between isolates within each species. Although ranges overlapped, the average GER severity induced by F. meridionale (25.2%) was two times as high overall as that induced by F. graminearum (12.8%) for isolates obtained from maize but was similar for those isolated from wheat (19.9 and 21.4%, respectively). In contrast, severity of GSR was slightly higher for F. graminearum (22.2%) than for F. meridionale (19.8%), with no effect of the host of origin. Deoxynivalenol and its acetylated form 15ADON were the main mycotoxins produced by F. graminearum (7/16 strains), and nivalenol toxin was produced by F. meridionale (17/24 strains). Six isolates of F. graminearum and three of F. meridionale also produced zearalenone. Results confirmed that F. meridionale from maize is, on average, more aggressive on maize but also suggested greater complexity related to diversity among the isolates within each species and their interactions with different hybrids. Further studies involving other components of the disease cycle are needed to more fully explain observed patterns of host dominance.

First Report of Fusarium graminearum Causing Flower Blight On Hemp (Cannabis sativa) in Kentucky.

In October of 2020, a grower in Boyle County, KY, reported mold and blight symptoms on flowers of field-grown hemp. Plants were approaching harvest, and the mold was affecting 100% of the cultivar 'White CBG' being grown for cannabinoid (CBD) extraction. Mycelium colonized the flower heads and any seeds within bracts. Affected flower bracts were necrotic, and mycelium and necrosis in the most severe cases also encompassed adjacent (sugar) leaves. Necrotic symptomatic tissue was collected, disinfested in 10% bleach for one minute, and cultured on acidified potato dextrose agar (APDA). Each isolate was single-spored, transferred to PDA, stored in 15% glycerol at -80°C and maintained at room temperature under blacklight blue and fluorescent bulbs on a 12-hour light-dark cycle. Colonies produced white-pink mycelia with a dark red pigment on the undersides. Conidia collected after 7-9 days were falcate and septate (5 to 6). No microconidia were produced. Macroconidia measured 35.4-49.7 µm x 3.4-5.8 µm (n=50). The strains produced blue-black fertile perithecia on carrot agar when induced according to the method of (Bowden and Leslie, 1999). To confirm pathogenicity, flowers of hemp cultivars 'Lifter', 'Trump Towers', 'Wife' and 'White CBG' were inoculated in the greenhouse with a representative fungal strain (20Hemp010). Plants were inoculated at two different stages: when the styles were still green or after they had become senescent. Macroconidia were collected from 7- to 9-day-old cultures grown under a 12-hour light-dark cycle. Plants were spray-inoculated with a 5 x 105 per ml conidial suspension in 0.05% Tween 20 until runoff. Flower heads were individually covered with clear plastic bags and incubated for 72 h at 95-100% humidity under greenhouse benches to avoid direct light. Bags were removed after 72 h and returned to the bench. Greenhouse conditions were 23-25°C with a 14-hour photoperiod and 50% RH. Symptoms developed 7 dai in 1% of the flowers inoculated when styles were green, and 36% of the flowers that had senescent styles. Symptoms were similar to those initially noticed in Boyle County, including necrotic flower bracts and sugar leaves, and visible fungal growth. Symptoms were more severe on plants inoculated when styles were necrotic. Recovered fungi were morphologically similar to 20Hemp010. Genomic DNA was extracted from the mycelium with the Zymo Research Quick-DNA Fungal/Bacterial Miniprep Kit. A fragment of the translation elongation factor 1-alpha 1 gene was amplified with primers EF1 and EF2 as described by (O'Donnell et al. 1998). Amplicons were sequenced and the consensus (MZ407909) was compared with the NCBI GenBank Refseq database by BLASTn. The top hit was Fusarium graminearum with 100% identity (JF270185.1). Pairwise alignments via MycoBank Fusarium MLST and Fusarium-ID also revealed a top hit of F. graminearum with 100% identity (AY452957.1). Conidial and colony morphology were also consistent with F. graminearum (Leslie and Summerell, 2006), thus we conclude that this species was the causal agent of the flower blight and mold. The same disease was subsequently confirmed on hemp in Breathitt and Franklin Counties in KY in 2020. This is the first report of this disease in KY, although F. graminearum has been reported previously causing a similar flower blight on hemp in NY and NC (Bergstrom et al., 2019, Thiessen et al. 2020). Fusarium graminearum is common in KY as a cause of Fusarium head blight on wheat and Gibberella ear rot on corn. In cereals, fungal infection is facilitated by the production of the mycotoxin deoxynivalenol (DON), which is harmful to humans and livestock (Desjardins and Hohn, 1997). As hemp production in Kentucky continues to rise for production of CBD products and edible grains, accumulation and concentration of DON in these products could become a concern.

The Dominance of Fusarium meridionale Over F. graminearum Causing Gibberella Ear Rot in Brazil May Be Due to Increased Aggressiveness and Competitiveness.

In Brazil, Gibberella ear rot (GER) of maize is caused mainly by Fusarium meridionale, whereas F. graminearum is a minor contributor. To test the hypothesis that F. meridionale is more aggressive than F. graminearum on maize, six experiments were conducted in the south (summer) and one in the central-south (winter), totaling seven conditions (year × location × hybrid). Treatments consisted of F. graminearum or F. meridionale (two isolates of each) inoculated once 4 days after silk, inoculated sequentially and alternately (F. graminearum → F. meridionale or F. meridionale → F. graminearum) 6 days apart, or (in the central-south) inoculated sequentially without alternating species (F. meridionale → F. meridionale or F. graminearum → F. graminearum). Overall, severity was two times greater in the south (37.0%), where summer temperatures were warmer (20 to 25°C) than in central-south. In the south, severity was greatest in F. meridionale treatments (67.8%); followed by F. meridionale → F. graminearum (41.1%), then F. graminearum → F. meridionale (19.4%), and lowest in F. graminearum (2.1%), suggesting an antagonistic relationship. In the central-south (15 to 20°C), severity was generally higher in the sequential nonalternating inoculation treatments (F. meridionale → F. meridionale or F. graminearum → F. graminearum) than when either species was inoculated only once. Only nivalenol (NIV) or deoxynivalenol was detected when F. meridionale or F. graminearum, respectively, was inoculated singly, or sequentially with no alternation. Both toxins were found in grains harvested from the F. meridionale → F. graminearum treatment, whereas only NIV was found in kernels from the F. graminearum → F. meridionale treatment, suggesting that F. meridionale was more competitive than F. graminearum in coinoculations. The dominance of F. meridionale as a cause of GER in Brazil may be due in part to its higher aggressiveness and competitiveness compared with F. graminearum.

Use of Telomere Fingerprinting to Identify Clonal Lineages of Colletotrichum fioriniae in Kentucky Mixed-Fruit Orchards.

Multiple species in the fungal genus Colletotrichum cause anthracnose fruit rot diseases that are responsible for major yield losses of as much as 100%. Individual species of Colletotrichum typically have broad host ranges and can infect multiple fruit species. Colletotrichum fioriniae causes anthracnose fruit rots of apples, blueberries, and strawberries in Kentucky orchards where these fruits grow in close proximity. This raises the possibility of cross-infection, which may have significant management implications. The potential occurrence of cross-infection was investigated by using telomere fingerprinting to identify C. fioriniae clones in several mixed-fruit orchards. Telomere fingerprints were highly polymorphic among a test group of C. fioriniae strains and effectively defined clonal lineages. Fingerprints were compared among apple, blueberry, and strawberry isolates of C. fioriniae from three different orchards and similarity matrices were calculated to build phylograms for each orchard group. Multiple clonal lineages of C. fioriniae were identified within each orchard on the same fruit host. Related lineages were found among isolates from different hosts, but the results did not provide direct evidence for cross-infection of different fruit species by the same clones. Recovery of the same clonal lineages within orchards across multiple years suggested that local dispersal was important in pathogen population structure and that C. fioriniae strains persisted within orchards over time. Isolates from blueberry were less diverse than isolates from apple, perhaps related to more intensive anthracnose management protocols on apple versus blueberry. Telomere fingerprinting is a valuable tool for understanding population dynamics of Colletotrichum fruit rot fungi.

Diversity and Cross-Infection Potential of Colletotrichum Causing Fruit Rots in Mixed-Fruit Orchards in Kentucky.

Fungi in the genus Colletotrichum cause apple, blueberry, and strawberry fruit rots, which can result in significant losses. Accurate identification is important because species differ in aggressiveness, fungicide sensitivity, and other factors affecting management. Multiple Colletotrichum species can cause similar symptoms on the same host, and more than one fruit type can be infected by a single Colletotrichum species. Mixed-fruit orchards may facilitate cross-infection, with significant management implications. Colletotrichum isolates from small fruits in Kentucky orchards were characterized and compared with apple isolates via a combination of morphotyping, sequencing of voucher loci and whole genomes, and cross-inoculation assays. Seven morphotypes representing two species complexes (C. acutatum and C. gloeosporioides) were identified. Morphotypes corresponded with phylogenetic species C. fioriniae, C. fructicola, C. nymphaeae, and C. siamense, identified by TUB2 or GAPDH barcodes. Phylogenetic trees built from nine single-gene sequences matched barcoding results with one exception, later determined to belong to an undescribed species. Comparison of single-gene trees with representative whole genome sequences revealed that CHS and ApMat were the most informative for diagnosis of fruit rot species and individual morphotypes within the C. acutatum or C. gloeosporioides complexes, respectively. All blueberry isolates belonged to C. fioriniae, and most strawberry isolates were C. nymphaeae, with a few C. siamense and C. fioriniae also recovered. All three species cause fruit rot on apples in Kentucky. Cross-inoculation assays on detached apple, blueberry, and strawberry fruits showed that all species were pathogenic on all three hosts but with species-specific differences in aggressiveness.

A Colletotrichum graminicola mutant deficient in the establishment of biotrophy reveals early transcriptional events in the maize anthracnose disease interaction.

BACKGROUND: Colletotrichum graminicola is a hemibiotrophic fungal pathogen that causes maize anthracnose disease. It progresses through three recognizable phases of pathogenic development in planta: melanized appressoria on the host surface prior to penetration; biotrophy, characterized by intracellular colonization of living host cells; and necrotrophy, characterized by host cell death and symptom development. A "Mixed Effects" Generalized Linear Model (GLM) was developed and applied to an existing Illumina transcriptome dataset, substantially increasing the statistical power of the analysis of C. graminicola gene expression during infection and colonization. Additionally, the in planta transcriptome of the wild-type was compared with that of a mutant strain impaired in the establishment of biotrophy, allowing detailed dissection of events occurring specifically during penetration, and during early versus late biotrophy. RESULTS: More than 2000 fungal genes were differentially transcribed during appressorial maturation, penetration, and colonization. Secreted proteins, secondary metabolism genes, and membrane receptors were over-represented among the differentially expressed genes, suggesting that the fungus engages in an intimate and dynamic conversation with the host, beginning prior to penetration. This communication process probably involves reception of plant signals triggering subsequent developmental progress in the fungus, as well as production of signals that induce responses in the host. Later phases of biotrophy were more similar to necrotrophy, with increased production of secreted proteases, inducers of plant cell death, hydrolases, and membrane bound transporters for the uptake and egress of potential toxins, signals, and nutrients. CONCLUSIONS: This approach revealed, in unprecedented detail, fungal genes specifically expressed during critical phases of host penetration and biotrophic establishment. Many encoded secreted proteins, secondary metabolism enzymes, and receptors that may play roles in host-pathogen communication necessary to promote susceptibility, and thus may provide targets for chemical or biological controls to manage this important disease. The differentially expressed genes could be used as 'landmarks' to more accurately identify developmental progress in compatible versus incompatible interactions involving genetic variants of both host and pathogen.

Characterization of Glomerella strains recovered from anthracnose lesions on common bean plants in Brazil.

Anthracnose caused by Colletotrichum lindemuthianum is an important disease of common bean, resulting in major economic losses worldwide. Genetic diversity of the C. lindemuthianum population contributes to its ability to adapt rapidly to new sources of host resistance. The origin of this diversity is unknown, but sexual recombination, via the Glomerella teleomorph, is one possibility. This study tested the hypothesis that Glomerella strains that are frequently recovered from bean anthracnose lesions represent the teleomorph of C. lindemuthianum. A large collection of Glomerella isolates could be separated into two groups based on phylogenetic analysis, morphology, and pathogenicity to beans. Both groups were unrelated to C. lindemuthianum. One group clustered with the C. gloeosporioides species complex and produced mild symptoms on bean tissues. The other group, which belonged to a clade that included the cucurbit anthracnose pathogen C. magna, caused no symptoms. Individual ascospores recovered from Glomerella perithecia gave rise to either fertile (perithecial) or infertile (conidial) colonies. Some pairings of perithecial and conidial strains resulted in induced homothallism in the conidial partner, while others led to apparent heterothallic matings. Pairings involving two perithecial, or two conidial, colonies produced neither outcome. Conidia efficiently formed conidial anastomosis tubes (CATs), but ascospores never formed CATs. The Glomerella strains formed appressoria and hyphae on the plant surface, but did not penetrate or form infection structures within the tissues. Their behavior was similar whether the beans were susceptible or resistant to anthracnose. These same Glomerella strains produced thick intracellular hyphae, and eventually acervuli, if host cell death was induced. When Glomerella was co-inoculated with C. lindemuthianum, it readily invaded anthracnose lesions. Thus, the hypothesis was not supported: Glomerella strains from anthracnose lesions do not represent the teleomorphic phase of C. lindemuthianum, and instead appear to be bean epiphytes that opportunistically invade and sporulate in the lesions.

Evidence for a diffusible factor that induces susceptibility in the Colletotrichum-maize disease interaction.

Colletotrichum graminicola, the causal agent of maize anthracnose, is a hemibiotrophic fungus that initially infects living host cells via primary hyphae surrounded by a membrane. A nonpathogenic mutant disrupted in a gene encoding a component of the signal peptidase complex, and believed to be deficient in protein processing and secretion, regained pathogenicity when it was inoculated onto maize leaf sheaths close to the wild-type fungus. Evidence is presented suggesting that the wild-type produces a diffusible factor(s) that induces the localized susceptibility of host cells at the borders of expanding colonies, causing them to become receptive to biotrophic invasion. The induced susceptibility effect is limited to a distance of approximately eight cells from the edge of the wild-type colony, is dosage dependent and is specific to C. graminicola.

The common metabolite glycerol-3-phosphate is a novel regulator of plant defense signaling.

Conversion of glycerol to glycerol-3-phosphate (G3P) is one of the highly conserved steps of glycerol metabolism in evolutionary diverse organisms. In plants, G3P is produced either via the glycerol kinase (GK)-mediated phosphorylation of glycerol, or via G3P dehydrogenase (G3Pdh)-mediated reduction of dihydroxyacetone phosphate (DHAP). We have recently shown that G3P levels contribute to basal resistance against the hemibiotrophic pathogen, Colletotrichum higginsianum. Since a mutation in the GLY1-encoded G3Pdh conferred more susceptibility compared to a mutation in the GLI1-encoded GK, we proposed that GLY1 is the major contributor of the total G3P pool that participates in defense against C. higginsianum.

Glycerol-3-phosphate levels are associated with basal resistance to the hemibiotrophic fungus Colletotrichum higginsianum in Arabidopsis.

Glycerol-3-phosphate (G3P) is an important component of carbohydrate and lipid metabolic processes. In this article, we provide evidence that G3P levels in plants are associated with defense to a hemibiotrophic fungal pathogen Colletotrichum higginsianum. Inoculation of Arabidopsis (Arabidopsis thaliana) with C. higginsianum was correlated with an increase in G3P levels and a concomitant decrease in glycerol levels in the host. Plants impaired in utilization of plastidial G3P (act1) accumulated elevated levels of pathogen-induced G3P and displayed enhanced resistance. Furthermore, overexpression of the host GLY1 gene, which encodes a G3P dehydrogenase (G3Pdh), conferred enhanced resistance. In contrast, the gly1 mutant accumulated reduced levels of G3P after pathogen inoculation and showed enhanced susceptibility to C. higginsianum. Unlike gly1, a mutation in a cytosolic isoform of G3Pdh did not alter basal resistance to C. higginsianum. Furthermore, act1 gly1 double-mutant plants were as susceptible as the gly1 plants. Increased resistance or susceptibility of act1 and gly1 plants to C. higginsianum, respectively, was not due to effects of these mutations on salicylic acid- or ethylene-mediated defense pathways. The act1 mutation restored a wild-type-like response in camalexin-deficient pad3 plants, which were hypersusceptible to C. higginsianum. These data suggest that G3P-associated resistance to C. higginsianum occurs independently or downstream of the camalexin pathway. Together, these results suggest a novel and specific link between G3P metabolism and plant defense.

Parameters affecting the efficiency of Agrobacterium tumefaciens-mediated transformation of Colletotrichum graminicola.

We have developed an Agrobacterium tumefaciens-mediated transformation (ATMT) protocol for the plant pathogenic fungus Colletotrichum graminicola, the cause of anthracnose leaf blight and stalk rot of corn. The ATMT results in higher transformation efficiencies than previously available polyethylene glycol-mediated protocols, and falcate spores can be used instead of protoplasts for transformation. Various experimental parameters were tested for their effects on transformation efficiencies. The parameters with the greatest influence were the A. tumefaciens strain used and the Ti-plasmid it carried, the ratio of bacterium to fungus during cocultivation, and the length of cocultivation. Southern analysis demonstrated that most transformants (80%) contained tandem integrations of plasmid sequences, and at least 36% had integrations at multiple sites in the genome. In a majority of cases (70%), the whole Ti-plasmid, and not just the T-DNA, had integrated as a series of tandem repeats. Tandem integrations, especially of the whole plasmid, make it difficult to rescue DNA from both flanks of the integrations with standard PCR-based approaches. Thus, ATMT may be unsuitable for insertional mutagenesis of C. graminicola without further modification.

Using mating-type gene sequences for improved phylogenetic resolution of Collectotrichum species complexes.

Colletotrichum species are defined primarily on the basis of host preference and morphology of the organism in planta and in culture. However the genus contains several species complexes that encompass such a broad range of morphological and pathological variation that the species name is of relatively little use either to the taxonomist or plant pathologist. Phylogenetic analyses, primarily based on variable regions of the ribosomal DNA (rDNA) sequences, have indicated that these species complexes comprise a variable number of identifiable monophyletic clades. However rDNA sequences often are insufficiently diverse to fully resolve such closely related lineages. A group of isolates representing three species complexes (C. graminicola, C. gloeosporioides and C. acutatum) were analyzed by using the high mobility group (HMG)-encoding sequence of the MAT1-2 mating type sequence, which has been shown in other fungi to be especially suitable for distinguishing relationships among closely related groups. Results were compared with those obtained from analysis of variable regions of the rDNA as well as from standard morphological classification methods. Results achieved through analysis of MAT1-2 sequences correlated well with those obtained by analysis of rDNA sequences but provided significantly better resolution among the various lineages. Morphological traits, including hyphopodia size, colony appearance, spore size, appresorial shape and size and host preference, frequently were unreliable as indicators of phylogenetic association. Spore shape and hyphopodia shape more often were useful for this purpose.