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ARS Home » Northeast Area » Beltsville, Maryland (BARC) » Beltsville Agricultural Research Center » Mycology and Nematology Genetic Diversity and Biology Laboratory » Research » Research Project #432548

Research Project: Enhancing Plant Protection through Fungal Systematics

Location: Mycology and Nematology Genetic Diversity and Biology Laboratory

2018 Annual Report

Objective 1: Identify genotypes and species limits of emerging, invasive or other pathogenic fungal species associated with plants and plant pests. [NP303, C1, PS1] Objective 2: Develop systematic resources for agriculturally important fungi, including diagnostic methods, genome sequences and phylogenies. [NP303, C1, PS1]

Emerging, insect, and quarantine-significant pathogenic fungi in historically understudied groups important to U.S. agriculture will be studied using cutting edge molecular technologies to determine species boundaries, phylogenetic relationships and gene regions most useful for diagnostic methods development. Fungi to be studied include smut species closely related to the flag smut of wheat pathogen (Urocystis tritici), rust species on imported plants, and downy mildew species in the genera Peronospora and Plasmopora. Anthracnose, canker, and leaf spot fungi in various ascomycete genera will be studied as they emerge in pathogenic situations. For determining initial identities and superficial relationships of non-insect associated fungi, the ITS regions of the nuclear ribosomal repeat unit will be sequenced, compared with existing data, and correlated with morphological data. Insect-associated fungi will be screened utilizing nuclear intergenic regions Bloc for Beauveria, MzIGS3 for Metarhizium, and RPB1 for Lecanicillium and Simplicillium. ITS, LSU, tef1-a, beta-tub, and rpb gene regions will be analyzed to determine species boundaries and phylogenetic relationships for canker and anthracnose fungi. The genetic diversity of insect-pathogenic species in Beauveria, Lecanicillium, and Metarhizium in soil and other habitats will be determined through both culture and direct DNA sequencing methodologies. Soils will be sampled for insect-associated fungi from agricultural fields and natural habitats from the mid-Atlantic region. Candidate intergenic sequences that minimize both amplicon length (500-700 bp) and percentage gapped sites while simultaneously maximizing the average pairwise divergence and the proportion of parsimony informative sites will be evaluated for utility. Genomic libraries for agriculturally important fungi including Diaporthe, Plasmopara, Pseudoperonospora, Neonectria, Sclerotinia, Microdochium and Urocystis will be prepared utilizing current kit-based technologies and sequenced on an Illumina MiSeq sequencer or other platforms as they become available. Sequence reads will be processed and assembled using bioinformatics software packages or custom-developed software as appropriate. New markers will be identified using a comparative genomic approach or through whole genome-scale comparative analysis of all single copy orthologous proteins. It is expected that species previously unknown to science will be described, illustrated, and characterized. Expected outputs include diagnostic assays, DNA sequences, digital images, monographs, and phylogenies for fungi important as insect or plant pathogens. Knowledge gained will enable development and enhancement of resources critical for effective disease management strategies and for making sound plant quarantine and biocontrol decisions.

Progress Report
This report is for Project 8042-22000-298-00D Enhancing Plant Protection through Fungal Systematics. Progress was made on both objectives, which fall under Component 1, Problem Statement 1. Under Objective 1, significant progress was made in identifying and describing invasive and emerging plant pathogens and characterizing the genotypes of disease-causing fungi and oomycetes. Several accounts of plant pathogenic fungi were published, including new reports of downy mildews on ornamental plant hosts. A new smut fungus related to and potentially confused with the flag smut of wheat pathogen was described and characterized with DNA sequences. 664 samples of the impatiens downy mildew pathogen were genotyped using SSR markers. A new genus of turfgrass fungi and four species associated with the dollar spot disease were also identified using morphological features and DNA sequences. New collections of fungal pathogens causing boxwood blight and volutella blight of boxwood, sarcococca and pachysandra were made. 150 strains of insect and fungal pathogens Lecanicillium and Simplicillium were characterized using DNA sequences, further refining species and population level limits of these fungi. Multilocus microsatellite genotyping of Puerto Rican Beauveria bassiana isolates infecting coffee berry borer and population genetic structure of the insect pathogen Metarhizium robertsii in western North America were also completed. Accurately identifying plant pathogens will enable better methods of control and tracking of disease outbreaks and the genotyping of insect pathogenic fungi will enhance the development of integrated and sustainable pest management strategies, minimizing the need to apply chemical pesticides. Under Objective 2, genome sequence assemblies were generated for several key plant pathogenic fungi: one isolate each of Coccinonectria pachysandricola (pachysandra blight) Corinonectria fuckeliana (pine canker); Neonectria ditissima (willow canker), N. hederae (canker of English Ivy), N. punicea (buckthorn canker); Pseudonectria buxi and P. foliicola (volutella blight of boxwood); and 27 isolates of Calonectria henricotiae and C. pseudonaviculata (boxwood blight). Comparative analyses were completed between genomes of fungal phytopathogens causing chestnut blight, rice blast and turfgrass dollar spot diseases (Cryphonectria parasitica, Magnaporthe oryzae, Clarireedia spp.) and related fungi to identify genes and origins uniquely associated with the plant pathogens. A DNA-sequence based identification method for the insect and fungal pathogens in Lecanicillium and Simplicillium was validated. These resource will be used to enable development and enhancement of resources critical for effective disease management strategies and for making sound plant quarantine and biocontrol decisions.

1. New smut pathogen of a native plant discovered. Smut diseases of crops and wild plants can be destructive and are often difficult to identify. Using DNA sequences and spore characteristics, ARS researchers in Beltsville, Maryland, in collaboration with scientists at Washington State University, discovered and described a new fungal species on a native plant in Arizona that is related to and could be confused with the flag smut of wheat pathogen. Flag smut diseases can be destructive pathogens of cereal crops such as wheat, as well as quarantine pathogens that threaten the export of U.S. crops. This research will be used by extension agents, plant breeders, plant pathologists, and plant quarantine officials to accurately identify these pathogens, preventing potential misidentifications that could disrupt the export of crop plants.

2. New genotypes cause impatiens downy mildew disease epidemics. Downy mildew disease attacking impatiens has recently become a significant threat to the horticulture industry in the U.S. and globally, causing an estimated $72 million in wholesale losses per year. ARS scientists in Beltsville, Maryland, discovered the two genotypes currently causing epidemics in the U.S. differ from the preserved historical samples originally collected up to 148 years ago and that the same DNA types infected both impatiens and closely related native jewelweed plants. This information is critical for breeding efforts and the horticulture industry for the development of disease resistant plants. This research will help growers, extension personnel and plant health professionals minimize the transmission and impact of the disease on economically important impatiens.

3. Dollar spot of turfgrass caused by multiple fungal species. Dollar spot disease is a constant threat to turfgrasses covering millions of acres of lawns, parks, recreational fields and golf courses and is the most economically important disease of turfgrass worldwide. The $70 billion golf course industry spends more money on fungicides to control dollar spot than any other disease. ARS scientists in Beltsville, Maryland, working with researchers from Rutgers University, North Carolina State University and Ohio State University, discovered that dollar spot is caused by an entirely new and undescribed group of fungi, which was named Clarireedia, and that at least four different species cause the disease on different grasses in different locations. This research will help plant health practitioners and turfgrass breeders and growers to develop control measures that are specifically targeted against each disease-causing agent and to reduce both the cost and amount of fungicides needed to control this disease.

Review Publications
Wallace, E., Daughtery, M., Rane, K., Salgado-Salazar, C., Crouch, J. 2018. First report of Peronospora sp. causing downy mildew disease on Geum sp. in the northeastern United States. Plant Disease. 102(7):1463.
Wallace, E., Crouch, J. 2017. First Report of Peronospora digitalidis causing downy mildew disease on foxglove in Oregon. Plant Disease. 102:827.
Mueller, U.G., Ishak, H.D., Bruschi, S.M., Smith, C.C., Herman, J.J., Mikheyev, A.S., Rabeling, C., Scott, J.J., Cooper, M., Rodrigues, A., Brandao, C.R., Lattke, J.E., Pagnocca, F.C., Rehner, S.A., Schultz, T.R., Vasconcelos, H.L., Adams, R.M., Bollazi, M., Clark, R.M., Himler, A.G., Lapolla, J.S., Leal, I.R., Johnson, R.A., Roces, F., Sosa-Calvo, J., Wirth, R., Bacci, M., Solomon, Scott E., Ortiz, A. 2017. Biogeography of mutualistic fungi cultivated by leafcutter ants. Molecular Ecology. 26:6921-6937.
Salgado-Salazar, C., Ismaiel, A., Crouch, J. 2018. Downy mildew of Double Knock Out® rose caused by Peronospora sparsa in Maryland. Plant Disease. 102(7):1464.
Leblanc, N., Salgado-Salazar, C., Crouch, J.A. 2018. Boxwood blight: an ongoing threat to ornamental and native boxwood. Applied Microbiology and Biotechnology. 102(10):4371-4380.
Kepler, R., Luangsa-Ard, J.J., Hywel-Jones, N.L., Quandt, A.C., Sung, G., Rehner, S.A., Aime, M.C., Henkel, T.W., Sanjuan, T., Rasoul, Z., Mingjun, C., Li, Z., Rossman, A.Y., Spatafora, J.W., Shrestha, B. 2017. A phylogenetically-based nomenclature for Cordycipitaceae (Hypocreales). IMA Fungus. 8(2):335-353.
Aime, M.C., Castlebury, L.A., Abbasi, M., Begerow, D., Berndt, R., Kirschner, R., Marvanova, L., Ono, Y., Padamsee, M., Scholler, M., Thines, M., Rossman, A. 2018. Competing sexual and asexual generic names in Pucciniomycotina and Ustilaginomycotina (Basidiomycota) and recommendations for use. IMA Fungus. 9(1):75-90.
Espevig, T., Brurberg, M., Usolteseva, M., Dahl, A., Normann, K., Kvalbein, A., Crouch, J. 2017. First report of dollar spot disease, caused by Sclerotinia homoeocarpa, of Agrostis stolonifera in Sweden. Crop Science. 57:S-349-S-353. https://doi:10.2135/cropsci2016.10.0835.
Crouch, J.A., Carter, Z., Ismaiel, A., Roberts, J.A. 2017. The U.S. National Mall microbiome: A census of rhizosphere bacteria inhabiting landscape turf. Crop Science. 57:S341-S348.
Salgado-Salazar, C., Beirn, L.A., Ismaiel, A.A., Boehm, M.J., Carbone, I., Putnam, A.I., Tredway, L.P., Clarke, B.B., Crouch, J.A. 2018. Clarireedia: A new fungal genus comprising four pathogenic species responsible for dollar spot disease of turfgrass. Fungal Biology. 122(8):761-773.
Salgado-Salazar, C., LeBlanc, N., Ismaiel, A.A., Rivera, Y., Warfield, C., Crouch, J.A. 2018. Genetic variation of the pathogen causing impatiens downy mildew pre-dating and including 21st century epidemics on Impatiens walleriana. Plant Disease. 102(12):2411-2420.
Salgado-Salazar, C., Shishkoff, N., Daughtrey, M.L., Palmer, C., Crouch, J. 2018. Downy mildew: a serious disease threat to rose health worldwide. Plant Disease.
Wallace, E.C., Crouch, J.A. 2018. Downy mildew disease of New England aster (Symphyotrichum novae-angliae) caused by Basidiophora simplex in New York. Plant Disease. 102(11):2382.
Zhang, N., Cai, G., Price, D.C., Crouch, J., Gladieux, P., Hillman, B., Khang, C.H., Lebrun, M., Lee, Y., Luo, J., Qiu, H., Veltri, D., Wisecarver, J.H., Zhu, J., Bhattacharya, D. 2018. Genome wide analysis of the transition to pathogenic lifestyles in Magnaporthales fungi. Scientific Reports. 8:5862.