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.

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.

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.