Project : USDA ARS

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Research Project: Enhancing Plant Protection through Fungal Systematics

Location: Mycology and Nematology Genetic Diversity and Biology Laboratory

2022 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 serves as the final report for Project 8042-22000-298-00D titled “Enhancing Plant Protection through Fungal Systematics.” It has been replaced by Project 8042-22000-323-00D titled “Fungal Systematics and Diagnostic Resource Development for Safeguarding Plant Health.” Significant 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. Accounts of plant pathogenic fungi were published, including new reports of downy mildews on ornamental plant hosts. New collections of downy mildews continued through the life of the project especially from impatiens, which experienced an upsurge in impatiens downy mildew in 2020 in both industry standard, highly susceptible, impatiens varieties and newly released cultivars that were reportedly resistant. Downy mildew pathogens collected were analyzed using molecular and morphological characters. Collections of an unknown downy mildew from an endangered native Hawaiian plantain conservation nursery were identified as a new species, Peronospora kuewa. A downy mildew infecting seaside nehe (Lipochaeta succulenta), an endangered, endemic Hawaiian plant, was identified as Peronsopora spagneticolae, the first time that this pathogen has been identified outside of Australia. Plasmopara ampelopsidis and P. carveri infecting grape and related hosts were described for the first time. Other downy mildews infecting bee balm, hyssops, candytuft, and cleome were described as the new species Peronospora bergamotii, Hyaloperonospora daughtreyae and H. iberidis, respectively. 664 samples of the impatiens downy mildew pathogen were genotyped using simple sequence repeat (SSR) markers. 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). North American collections of 9,838 downy mildew specimens originating from the United States, Canada, and Mexico were analyzed to develop a baseline census of species diversity throughout the continent. The new disease of the woody ornamental sarcococca discovered in FY19 was described using molecular and molecular characteristics and symptomology. Next generation sequencing techniques were used to sequence 11 SSR markers from 218 new Calonectria pseudonaviculata (boxwood blight pathogen) collected across 21 states in the U.S. and the resulting dataset was analyzed. Several hundred isolates of plant-associated fungi were isolated from hybrid chestnut trees, several which were completely new to science or new records for chestnut. Efforts continue to identify these fungi and determine if any are known to have beneficial effects against pathogens in other systems. Canker and blight fungi isolated from strawberry were characterized and analyzed. A new genus was proposed to accommodate a well-known pathogen (Phomopsis obscurans) that has been misidentified for over 100 years. Mating type genes were characterized for the devastating beech bark disease pathogens, Neonectria faginata and N. coccinea, which will enhance our ability to distinguish these two closely related species. A new genus of turfgrass fungi and four species associated with the dollar spot disease were also identified using morphological features and DNA sequences. Approximately 1,000 molecular marker DNA sequences (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 isolates from different host plants intercepted at the borders or sent by collaborators, 50 isolates of Anthostomella fungi isolated from chestnut leaves, Calonectria hawksworthii from avocado in California, Dactylonectria sp. isolated from Quercus sp. in California, various unidentified fungi isolated from leaf spots of corn in Illinois, and Coniochaeta sp. isolated from walking iris and potato plants with potential for producing biofungicides. A Fusarium oxysporum species complex associated with soybean cyst nematodes was identified along with three new species of fungi causing leaf spot diseases on corn and ornamental grasses. Thesequence diversity of 186 single stroma isolates of Phyllachora maydis collected from 16 hosts representing 15 countries was assessed and represents the first molecular characterization of Phyllachora species infecting maize and other grass hosts. A rust pathogen on hemp was identified and characterized for the first time in the United States and various smut fungi have been collected for characterization with molecular and morphological data. A new smut fungus related to and potentially confused with the flag smut of wheat pathogen was describe and characterized with DNA sequences. Digital images for approximately 50 type species of rust fungi were captured and made available to USDA-APHIS with relevant metadata. Approximately 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 for discerning the population genetic structure of the insect pathogen Metarhizium robertsii in western North America were also completed. Multilocus microsatellite genotyping was performed to verify identities and evaluate Beauveria bassiana strains applied as experimental biological control agents against crape myrtle bark scale insects infesting crape myrtle in Baton Rouge, Louisiana. 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. Nuclear and mitochondrial genome sequence annotations completed for 23 Metarhizium and 76 Beauveria isolates, and GenBank submissions for these are in revision pending final acceptance. Microsatellite and mating type markers developed and validated for Beauveria psesudobassiana. In silico surveys of ultra-conserved elements and flanking regions demonstrate utility for phylogenetic and population genetic analysis of Beauveria, Metarhizium, and Lecanicillium. Under Objective 2, genome sequence assemblies were generated for several key plant pathogenic fungi: one isolate each of Coccinonectria pachysandricola (pachysandra blight), Corinectria fuckeliana (pine canker); Neonectria ditissima (willow canker), N. hederae (canker of English Ivy), N. punicea (buckthorn canker); Pseudonectria buxi, P. foliicola (volutella blight of boxwood) and Thelonectria rubi (canker of caneberries); and 27 isolates of Calonectria henricotiae and C. pseudonaviculata (boxwood blight). The genomes of 80 isolates of the boxwood blight pathogens Calonectria pseudonaviculata and C.henricotiae were analyzed. Global migration analyses for the boxwood blight pathogens Calonectria pseudonaviculata and C. henricotiae were completed using comparative genome analyses.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. Genome sequences of 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. Genome sequences for isolates of beech bark canker disease, a potentially insect-associated smut fungus, several species of canker-causing Diaporthe infecting a variety of plant hosts, and isolates of the destructive dogwood anthracnose fungus were preliminarily assembled and analyzed. A genome sequence was generated for the first reported isolate of Calonectria hawksworthii occurring in the United States on avocado and 14 additional Calonectria species. New genome sequences and assemblies were completed for seven nectriaceous fungal pathogens of forest and fruit-producing trees and shrubs. Comparative analyses of single copy ortholog datasets revealed informative genetic markers for phylogenetic research and diagnostic marker development. A data pipeline to annotate genome sequences of the biocontrol fungus Beauveria bassiana was developed and validated for a population genomics sample from Puerto Rico. Comparative analyses of genetic variation in their mitochondrial genomes and mating loci revealed informative genetic markers for strain identification and population genetic analysis. Genome assemblies of insect pathogens Metarhizium and Beauveria were completed, and gene models were annotated. A DNA-sequence based identification method for the insect and fungal pathogens in Lecanicillium and Simplicillium was validated. 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. Accurately identifying and genotyping fungal, plant, and insect pathogens will enable better methods of control and tracking of disease outbreaks. This will also enhance the development of integrated and sustainable pest management strategies, minimizing the need to apply chemical pesticides.

Accomplishments
1. New downy mildew discovered on wild grapes in the United States. Downy mildew diseases are widespread, pervasive and can result in damage so great that the host’s value is reduced and/or the commodity is not marketable, yield is reduced, or the host is killed outright. Grape downy mildew caused by Plasmopara viticola is one of the most severe and destructive diseases of grapes, impacting both the yield and the quality of the harvested fruits and making it a major constraint for the grape industry. ARS scientists in Beltsville, Maryland, in collaboration with research scientists in Germany, identified the existence of different downy mildew in wild relatives of the cultivated grapes. The results of this study have important implications for viticulture, including breeding for resistance and disease management.

2. Genomic resources for Ceratocystis manginecans and allied species were developed. The threat of exotic pests and diseases of mango is one of the major constraints for the development of expanded trade between the U.S. and foreign markets. One such disease is caused by Ceratocystis manginecans. ARS scientists in Beltsville, Maryland, in collaboration with USDA APHIS scientists also in Beltsville, Maryland, sequenced whole genomes of Ceratocystis manginecans isolates to be used for the development species-specific DNA-based diagnostic assays. This work will be crucial in the identification of infected material and in preventing the entry and spread of this pathogen in the United States and protection of the U.S. mango industry.

3. New species discovered in the fungal genus Coniochaeta. Coniochaeta is a diverse group of fungi that includes many undescribed species, some of which produce chemicals with antimicrobial activity. ARS scientists in Beltsville, Maryland, and the University of Arizona identified three new species of fungi in the genus Coniochaeta isolated from plants and lichens. This work will enhance the ability to identify isolates or species with the potential for antimicrobial activity.

4. Exotic pathogen of the ornamental plant liriope discovered in United States. The ornamental grass liriope (monkey grass or spidergrass) is widely used in landscapes and susceptible to a number of diseases. One disease caused by the fungus Colletotrichum liriope was not known to occur on liriope in the United States, resulting the rejection of imports of this ornamental plant at ports of entry. Diseased material was either destroyed or returned to its original port of departure, resulting in millions of dollars in losses to both importers and exporters. ARS scientists in Beltsville, Maryland, examined numerous samples of diseased liriope collected in the U.S. and discovered that this fungus is relatively widespread. This knowledge will be used by quarantine officials and result in policy changes that save both importers and exporters of liriope millions of dollars each year.

5. The insect biocontrol fungus Beauveria bassiana is a complex of distinct genetic species. The insect biocontrol fungus Beauveria bassiana is used for the control of a wide range of pest insect species of significance to agriculture, horticulture, domestic animals, and humans. Genetic studies indicate that isolates identified as Beauveria bassiana comprise many very similar species that may attack different insects. ARS scientists in Beltsville, Maryland, and Ithaca, New York, in collaboration with researchers at Louisiana State University, tested a new approach for analysis of large numbers of DNA sequences that can distinguish very similar species. This method enables discrimination of previously unrecognized species with probable distinct insect host specificities. This work will be used by pest management professionals to develop new insect biocontrol methods, which will lessen the use of pesticides and result in lower costs and better management for insect control.

U.S. DEPARTMENT OF AGRICULTURE

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