Project : USDA ARS

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Research Project: Enhancing Plant Protection through Fungal Systematics

Location: Mycology and Nematology Genetic Diversity and Biology Laboratory

2021 Annual Report

Objectives
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]

Approach
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 2, Problem Statement 1. Under Objective 1, substantial progress was made in identifying and describing invasive and emerging plant pathogens and characterizing the genotypes of disease-causing fungi and oomycetes. Downy mildew nomenclatural and taxonomic resources were compiled, curated, and summarized for species of Hyaloperonospora, Peronospora, Plasmopara, and species causing graminicolous downy mildew diseases (>550 species), and United States distribution maps of available herbarium specimens were compiled. North American collections of 9838 unique downy mildew herbarium specimens originating from the United States, Canada, and Mexico were analyzed to develop a baseline census of species diversity throughout the continent. Downy mildew host mycorrhizal associations and photosynthetic pathways were assessed to test hypotheses about the factors underlying downy mildew evolution and diversification. Approximately 1000 DNA sequences of molecular markers (five or more markers/fungal samples) were generated using Sanger technology for a variety of fungi causing canker, leaf spots, and wilts of a diverse range of plant host species. Sequenced fungi include: 1) Alternaria sp., Cercospora sp., Pseudocercospora sp. and Septoria sp. isolated from different host plants intercepted at the borders or sent by collaborators; 2) 50 isolates of Anthostomella fungi isolated from chestnut leaves; 3) Calonectria hawksworthii from avocado in California; 4) Dactylonectria sp. isolated from Quercus sp. in California; 5) various unidentified fungi isolated from leaf spots of corn in Illinois; 6) Coniochaeta sp. isolated from iris plants with potential for producing biofungicides. Digital images for approximately 50 type species of rust fungi were captured and made available to USDA-APHIS with relevant metadata. A Fusarium oxysporum species complex isolates associated with soybean cyst nematodes were identified. Three new species of fungi causing leaf spot diseases on corn and ornamental grasses were identified. Genome sequences for 17 Lecanicillium species were completed, and multilocus phylogenetic analyses were performed. Fifty-two isolates of Lecanicillium spp. parasites of coffee leaf rust were sequence-typed, resulting in the discovery of four new species. Field sites were established with collaborators for the application of selected strains to assess their ability to suppress coffee leaf rust disease, with surveys for new isolates ongoing. Accurately identifying and genotyping fungal, plant, and insect pathogens will enable better methods of control and tracking disease outbreaks. This will enhance the development of integrated and sustainable pest management strategies, minimizing the need to apply chemical pesticides. Under Objective 2, genomes sequences of 19 strains of the Colletotrichum gloeosporioides complex of plant pathogens were assembled and annotated. Genome assemblies of insect pathogens Metarhizium and Beauveria were completed, and gene models were annotated. A genome sequence was generated for the first reported isolate of Calonectria hawksworthii occurring in the United States on avocado grafting seedlings. Genome sequences were generated for 20 isolates of Calonectria pseudonaviculata causing boxwood blight disease and 14 additional Calonectria species. Genome assemblies of 88 boxwood blight strains were annotated. New genome sequences and assemblies were completed for seven nectriaceous fungal pathogens: Corinectria fuckeliana, Chrysoporthe doradensis, Cyanonectria buxi, Nectria mariae, Neonectria coccinea, N. ditissima, and N. faginata. Comparative analyses of single copy ortholog datasets revealed informative genetic markers for phylogenetic research and diagnostic marker development. Genome sequences of 17 Colletotrichum pathogens of monocots and dicots covering the diversity of the genus were annotated and analyzed, and comparative genomic approaches were used to identify pan- and core-genomes, lineage-specific genes, and expansions/contractions of gene families. DNA extracts from 21 strains of boxwood blight fungi were submitted for PacBio long read sequencing. These resources will be used to enable the development and enhancement of resources critical for effective disease and pest management strategies and for making sound plant quarantine and biocontrol decisions.

Accomplishments
1. Pathogens causing tar spot disease of corn and grasses are unexpectedly diverse in the Americas. Tar spot is a destructive disease of corn caused by the fungus Phyllachora maydis. The fungus is endemic to Central and South America and the Caribbean but has recently emerged as a pathogen across major corn-growing regions in the midwestern U.S. ARS scientists in Beltsville, Maryland, in collaboration with ARS scientists in Peoria, Illinois, and several universities conducted a DNA-based assessment of 189 samples of tar spot fungi collected from corn and other grasses in North, South and Central America. DNA sequences identified three distinct populations of P. maydis among five distinct groups of Phyllachora in the U.S., including at least two previously unknown species. These results will be used by extension agents and plant pathologists to develop effective disease management strategies and by corn, breeders to develop disease-resistant varieties.

2. The spread of one clonal lineage drives 2. Expansion of boxwood blight in the United States. Boxwood blight disease has spread rapidly across the United States, causing widespread damage to millions of dollars of plants in landscapes and nurseries since 2011. Early United States outbreaks of the boxwood blight have been characterized, but the genetic composition of the fungus as it has spread since 2014 is unknown. Using custom DNA markers called SSRs to genotype populations collected from 2014 to the present day, ARS scientists in Beltsville, Maryland, discovered that just one major genetic group is responsible for the expansion of boxwood blight in the United States, although a second major genetic group is also present at low frequency. The boxwood blight fungi multiply by making clones of themselves, but new genetic types have evolved from the dominant genetic type since 2014. This research shows how the spread of blight fungus has occurred and suggests how it will continue to move in the United States.

3. An old fungal pathogen returns to infect hemp. Industrial hemp is increasingly grown in the Midwest across larger acreages. Because of past restrictions on the growth and study of hemp, the fungal pathogens associated with hemp are poorly known. Using DNA sequences and microscopic features, ARS researchers in Beltsville, Maryland, and the University of Kentucky discovered that a fungal pathogen, Septoria cannabina, has re-emerged to cause a leaf spot disease on hemp. This research is important for plant pathologists, plant breeders and other plant disease management professionals who need to identify plant pathogens and implement plant disease management strategies.

4. New biological tools for control of coffee rust. With an estimated annual value of $100 million, coffee production in Puerto Rico is under significant threat from coffee rust, the most serious disease of coffee worldwide. ARS scientists in Beltsville, Maryland, in collaboration with the University of Puerto Rico scientists, conducted field surveys in Puerto Rico and discovered six species of the fungi Lecanicillium and Simplicillium parasitizing the fungi that cause coffee rust disease, including some which are new to science. These findings suggest that indigenous fungi may play a role in reducing rust populations and may help minimize the impact of coffee rust disease. This information will be vital to developing sustainable fungal-based biological control measures for coffee rust and may contribute to improving the yield and quality of coffee crops in Puerto Rico.

5. Genome resources for beech tree pathogens. Beech trees are an important component of forest ecosystems and support diverse populations of other organisms. Several fungi cause destructive diseases of beech, including the recently emerged pathogens that cause beech bark disease, Neonectria coccinea, N. ditissima and N. faginata. ARS scientists in Beltsville, Maryland, generated high-quality genome sequences for N. coccinea and N. faginata, providing the first genomes for these two pathogenic species. These resources will be useful for developing rapid and efficient assays for the targeted detection of these pathogens. These datasets may also be instrumental in developing strategies to control the spread and prevent economic losses associated with these and related fungi.

6. New diagnostic guides for diseases of boxwood, pachysandra and sweetbox. Boxwood, pachysandra, and sweetbox plants worldwide are threatened by a deadly fungal disease known as boxwood blight. Accurate diagnosis of boxwood blight is one of the best ways to reduce the spread and reduce damage, but differences in di7407316sease symptoms or sometimes the lack of symptoms can make disease diagnosis difficult. The disease also may be confused with more common diseases such as Volutella or anthracnose. Therefore, ARS scientists in Beltsville, Maryland, Frederick, Maryland, and Corvallis, Oregon, in partnership with university collaborators across the United States, developed two illustrated guides to aid in their diagnosis. Diagnosticians will use these two practical diagnostic guides, regulatory officials, plant pathologists, extension personnel, and growers to minimize the spread and impact of boxwood blight to discriminate blight from less destructive diseases.

7. New tropical pathogens found on grasses. Fungal pathogens on tropical grasses are understudied and poorly known. Because of changing weather patterns, these pathogens may become problematic in new areas, affecting both crops and the natural environment. Using DNA sequences and microscopic characteristics, ARS scientists in Beltsville, Maryland, in collaboration with scientists in Sri Lanka, discovered three new pathogenic species of Curvularia on grasses in Sri Lanka. These findings are important for plant pathologists, plant breeders and other plant disease management professionals who need to identify plant pathogens and implement plant disease management strategies.

8. Downy mildew killer of endangered Hawaiian plants identified and named. Efforts to repopulate the endangered native Hawaiian plantain plant known as "ale" have been greatly hindered by destructive downy mildew disease outbreaks since 2014. However, the identity of the pathogen that causes the disease is not known, which makes it difficult to develop effective disease control measures. In a collaborative effort between USDA-ARS scientists in Beltsville, Maryland, USDA-APHIS, and the Hawaii Department of Agriculture, the downy mildew pathogen was identified as a new species, different from all other known downy mildews, and was given the new scientific name Peronospora kuewa. The research includes identifying DNA marker sequences that will make it possible for regulatory agencies to detect possible movement of the pathogen and prevent its introduction into new regions. Identifying the downy mildew pathogen of the endangered Hawaiian ale plant is also the first step towards developing control measures that can be specifically targeted against the pathogen.

9. Five new genera of insect-killing fungi discovered. Many fungi used for biological control of insect pests are members of the Cordycipitaceae family of fungi, including the genus Lecanicillium. Many family members are well studied because of their use as commercial biocontrol agents of insect pests, but numerous other members of the family are obscure and have not been studied by scientists. Using comparative genomic approaches, ARS scientists in Beltsville, Maryland, and Ithaca, New York, in collaboration with researchers at Oregon State University, identified five new genera in this fungal family. These new genera are related to Lecanicillium and share many of the same genes, including secondary metabolite gene clusters that may serve as a rich source of new biological resources useful for agricultural, medical, and industrial applications.

U.S. DEPARTMENT OF AGRICULTURE

Mycology and Nematology Genetic Diversity and Biology Laboratory: Beltsville, MD