Research Project: Enhancing Plant Protection through Fungal Systematics

Location: Mycology and Nematology Genetic Diversity and Biology Laboratory

2019 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, 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. Collections of downy mildew pathogens collected during FY18 were analyzed using molecular and morphological characters. New collections of downy mildews continued in FY19. New collections of an unknown downy mildew were made from an endangered native Hawaiian Plantago conservation nursery, where the disease has severely limited efforts to repopulate this plant in the wild. New collections of fungal pathogen isolates causing boxwood blight and Volutella blight of boxwood, sarcococca and pachysandra were made from across the U.S., including several new boxwood blight locations, with a total of 161 new cultures established 2018-2019. A new disease of the woody ornamental plant sweetbox was characterized as a Volutella blight, caused by the same fungal pathogen that induces Volutella blight of pachysandra. Three new species of Diaporthe infecting soybeans were discovered through DNA sequencing methods. 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. Over 200 strains of the entomopathogen and fungal hyperparasite Lecanicillium were classified in Cordycipitaceae using multilocus phylogenetics as part of a systematic revision of this genus. Multilocus microsatellite genotyping was performed for >400 Beauveria bassiana strains isolated from coffee berry borer in Puerto Rico to identify and characterize an epizootic of coffee berry borer on coffee farms in Puerto Rico. Under Objective 2, genome sequence assemblies were generated for several species of key plant pathogenic fungi. The genomes of 24 isolates of boxwood blight fungi were sequenced using Illumina technology, adding to the 35 previously sequenced isolates. 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 generated. To better detect the cucurbit downy mildew pathogen, two new diagnostic methods were developed. Pathogen-specific gene clusters were identified to develop a rapid diagnostic assay to discriminate between fungal pathogens of boxwood. Candidate loci for species-specific multiplexed PCR assay for distinguishing between Metarhizium anisopliae, M. brunneum, M. pingshaense and M. robertsii all of which have insect biocontrol capabilities, have been identified and validated. The genomes of 50 Beauveria, 20 Metarhizium and 20 Colletotrichum were assembled and partitioned for analysis. These resources will be used to enable development and enhancement of resources critical for effective disease and pest management strategies and for making sound plant quarantine and biocontrol decisions.

Accomplishments
1. Limited genetic diversity exists in boxwood blight pathogens. Boxwood blight disease has rapidly spread across the U.S., causing widespread damage of plants in landscapes and across the nursery industry since its arrival in 2011. ARS scientists in Beltsville, Maryland, in collaboration with Belgian scientists, used DNA markers to determine the DNA fingerprints of the isolates causing boxwood blight. They discovered that the boxwood blight fungi currently in the United States belong to the two most common international DNA groups. This information is critical for efforts to breed resistant plants and will help growers, extension personnel and plant health professionals track and manage this disease.

2. New foliar blight affects popular ornamental plants. Woody plants such as boxwood, pachysandra, and sweet box are among the most popular and widely grown evergreen shrubs and groundcovers, representing millions of dollars in annual sales in the United States. ARS scientists in Beltsville, Maryland, and Frederick, Maryland, recently discovered that a new fungal disease is causing severe damage to sweet box plants at the U.S. National Arboretum in Washington, District of Columbia. Using DNA sequence profiles, the scientists showed that Volutella blight affecting sweet box is caused by the same fungus that infects pachysandra. Knowledge of this new disease and the conditions that allow the disease to occur will be useful to growers, horticulturalists, plant pathologists, disease diagnostic clinics, and regulatory agencies working to control diseases of these important landscape plants.

3. New species of fungi isolated from soybeans. Soybean canker diseases can limit yields, reducing the volume available for domestic use and trade and income for producers and processors. Soybean canker diseases are caused by multiple species of fungi and knowing which fungi are present is critical to the management of the disease. ARS scientists in Beltsville, Maryland, in collaboration with university researchers throughout the United States, surveyed the fungi present in soybeans and discovered three new species of Diaporthe. Soybeans are one of the largest commodity exports, valued at approximately $20 billion per year. Knowing the identity of the disease-causing fungi will aid in the management of soybean diseases, thereby increasing yields, and help maintain export markets.

4. Genetic diversity of coffee berry borer determined. Coffee production in Puerto Rico, with an estimated annual value of $100 million, is under significant threat from the coffee berry borer. ARS scientists in Beltsville, Maryland, in collaboration with university researchers in Puerto Rico analyzed the genetic diversity of the coffee berry borer using DNA analyses and found evidence of numerous genotypes, although some were much more abundant than others. Understanding the genetic diversity of this invasive and destructive insect will aid in the management and control of the coffee berry borer, thereby reducing the damage to coffee and increasing the yields for growers.

Review Publications
Wallace, E., Salgado-Salazar, C., Gregory, N., Crouch, J. 2018. Basidiophora delawarensis, a new downy mildew species that infects cultivated goldenrod (Solidago sphacelata) in the United States. Mycological Progress. 17(12):1283-1291. https://doi.org/10.1007/s11557-018-1444-z.
Salgado-Salazar, C., Bauchan, G.R., Wallace, E.C., Crouch, J.A. 2018. Detection and visualization of the impatiens downy mildew pathogen using fluorescence in situ hybridization (FISH). Plant Methods. 14:92. https://doi.org/10.1186/s13007-018-0362-z.
Mayerhofer, J., Lutz, A., Dennert, F., Rehner, S.A., Kepler, R.M., Widmer, F., Enkerli, J. 2019. A species-specific multiplexed PCR amplicon assay for distinguishing between Metarhizium anisopliae, M. brunneum, M. pingshaense and M. robertsii. Journal of Invertebrate Pathology. 161(1):23-28.
Salgado-Salazar, C., Creswell, T., Ruhl, G.E., Crouch, J. 2018. First report of Plasmopara halstedii on Coreopsis grandiflora in the United States. Plant Disease. https://doi.org/10.1094/PDIS-09-18-1501-PDN.
Leblanc, N., Gehesquière, B., Salgado-Salazar, C., Heungens, K., Crouch, J.A. 2019. SSRs identify limited genetic diversity across pathogen populations responsible for the global emergence of boxwood blight. Plant Pathology. 68(5):861-868. https://doi.org/10.1111/ppa.13003.
Salgado-Salazar, C., Crouch, J. 2018. Genome resources for the stem and bark canker pathogens Corinectria fuckeliana, Neonectria hederae and N. punicea. Plant Disease. 103(1):389-391. https://doi.org/10.1094/PDIS-05-18-0904-A.
Rivera, Y., Salgado-Salazar, C., Veltri, D., Malapi-Wight, M., Crouch, J. 2018. Genome analysis of the ubiquitous boxwood pathogen Pseudonectria foliicola. PeerJ. 6:e5401. https://doi.org/10.7717/peerj.5401.
Salgado-Salazar, C., Shishkoff, N., LeBlanc, N., Ismaiel, A.A., Collins, M., Cubeta, M.A., Crouch, J. 2018. Coccinonectria pachysandricola, causal agent of a new foliar blight disease of Sarcococca hookeriana. Plant Disease. 103(6):1337-1346. https://doi.org/10.1094/PDIS-09-18-1676-RE.
Duellman, K.M., Mathew, F., Markell, S.G., Castlebury, L.A. 2019. Diaporthe gulyae: the new pathogen on common buckwheat (Fagopyrum esculentum). Plant Health Progress. 1(1):70-72. https://doi.org/10.1094/PHP-12-18-0078-RS.
Wight-Malapi, M., Veltri, D., Gehesquière, B., Heungens, K., Rivera, Y., Salgado-Salazar, C., Crouch, J. 2019. Global distribution of mating types shows limited opportunities for mating across populations of fungi causing boxwood blight disease. Fungal Genetics and Biology. https://doi.org/10.1016/j.fgb.2019.103246.