Research Project: Enhancing Plant Protection through Fungal Systematics

Location: Mycology and Nematology Genetic Diversity and Biology Laboratory

2020 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. New collections of downy mildews continued in FY20 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. Downy mildew pathogens collected during FY19 were analyzed using molecular and morphological characters. Collections of an unknown downy mildew from an endangered native Hawaiian plantago conservation nursery, were identified as a new species of Peronospora and named P. kuewa. New collections of a downy mildew infecting seaside nehe (Lipochaeta succulenta), an endangered, endemic Hawaiian plant, were identified as Peronsopora spagneticolae, the first time that this pathogen has been identified outside of Australia where it was previously known to parasitize the invasive weed Sphagneticola trilobata (creeping oxeye). Collections of downy mildews infecting bee balm, hyssops, candytuft and cleome were described as the new species Peronospora bergamotii, Hyaloperonospora daughtreyae and H. iberidis. The new disease of the woody ornamental plant sweetbox 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, a number of which are completely new to science or new records for chestnut. Efforts are ongoing 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 have been characterized and analyzed. A new genus has been proposed to accommodate a well-known pathogen (Phomopsis obscurans) that has been misidentified for over 100 years. Mating type genes have been 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 rust pathogen on hemp has been 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. Multilocus microsatellite genotyping was performed to verify identities and evaluate Beauveria bassiana strains applied as experimental biological control agents against crapemyrtle bark scale insects infesting crapemyrtle in Baton Rouge, Louisiana. Accurately identifying plant and insect 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, the genomes of 80 isolates of the boxwood blight pathogens Calonectria pseudonaviculata and C. henricotiae were analyzed. Genome sequences of Cryphonectria parasitica, Clarireedia jacksonii, Clarireedia monteithiana, Clarireedia spp., and Coccinonectria pachysandricola were analyzed. 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 generated in FY19 were preliminarily assembled and analyzed in FY20. Global migration analyses for the boxwood blight pathogens Calonectria pseudonaviculata and C. henricotiae were completed using comparative genome analyses. A 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. 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. A new genus for a strawberry killer fungus described. Strawberry leaf blight has been known as a destructive pathogen for over 100 years. However, because of its nondescript appearance, this fungal pathogen has been known under at least seven different names. Using DNA sequences, ARS scientists in Beltsville, Maryland, in collaboration with scientists in Sri Lanka, determined that this fungus represents a new genus of fungi closely related to a fungus causing a dieback disease on grapes and provided updated descriptions for two other strawberry pathogens. 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.

2. Hidden fungal guardians in chestnut trees. Chestnut blight, a fungal disease, has devastated the American chestnut tree over the last century. However, the impact of fungal endophytes, which often provide protection from pathogens and other stressors, has not been evaluated in newly developed hybrid trees. ARS scientists in Beltsville, Maryland, in collaboration with the Maryland Chapter of the American Chestnut Foundation (ACF), isolated approximately 50 new fungal species from leaves to assess the diversity of fungi found in healthy trees. This work will provide critical knowledge for tree breeders and forest health professionals in their efforts to restore this valuable hardwood forest tree in North American forests.

3. Genomic resources for berry pathogens. The fungal family Nectriaceae harbors a variety of widespread and destructive plant pathogens affecting ornamental plants, forest trees, grapevines, and fruit canes and trees. ARS scientists in Beltsville, Maryland, generated a high-quality genome sequence for the fungus Thelonectria rubi, which infects blackberry and raspberry crops valued at approximately $500 million per year. This research will be useful for mycologists and plant pathologists to develop assays for its targeted detection and to develop strategies to control the spread and prevent the economic losses associated with the disease and those caused by related fungi.

4. New downy mildew diseases affect valuable ornamental plants. Herbaceous perennial plants represent millions of dollars in annual sales in the United States and many are susceptible to downy mildew pathogens. ARS scientists in Beltsville, Maryland, recently discovered three new species of downy mildew pathogens causing diseases of bee balm, cleome, candytuft, and hyssops using DNA sequence profiles. Knowledge of these new diseases is useful to growers, horticulturalists, plant pathologists, disease diagnostic clinics, and regulatory agencies working to control diseases of these important landscape plants.

5. Invasive boxwood blight fungus came to the U.S. from two foreign sources. Boxwood blight disease has rapidly spread across the U.S., causing widespread damage to millions of dollars of plants in landscapes and nurseries since 2011. ARS scientists in Beltsville, Maryland, developed a new comparative genomics approach to trace the origins of the boxwood blight fungi in the U.S. More than 2,000 DNA differences were discovered, showing the pathogen entered the U.S. twice from different sources. This research is important because it shows how the blight fungus is moving around the world and is critical for efforts to manage this disease.

6. Biological control of coffee berry borer in Puerto Rico. Coffee production in Puerto Rico, with an estimated annual value of $100 million, is under significant threat from the invasive coffee berry borer. ARS scientists in Beltsville, Maryland, in collaboration with university researchers in Puerto Rico, conducted field experiments comparing the effectiveness of a commercial strain (Mycotrol) and local strains of the insect pathogenic fungus Beauveria bassiana to reduce populations of coffee berry borer. Two local strains, applied individually and as a mixture, were either equally or more effective than Mycotrol in suppressing coffee berry borer infestations and generally persisted in the environment longer than the commercial strain. Clarifying the potential of local strains of Beauveria bassiana to regulate populations of this invasive, economically destructive insect will contribute to improving yield and quality of coffee crops in Puerto Rico.

7. Insect killing chemicals produced by the fungus Metarhizium. Insect pests of plants cause billions of dollars of losses to agricultural and ornamental crops and can be devastating to forest systems. Insect pathogenic fungi are one way these pests can be controlled, and the mechanisms can, in part, be attributed to the production of chemicals that kill or disable insects. ARS scientists in Beltsville, Maryland, in collaboration with researchers at West Virginia University, showed that Metarhizium species produce ergot alkaloids when infecting insects but not when cultured independently or in the presence of plant roots. This suggests a role for these compounds in insect pathogenicity. This information will be vital to the development of fungal-based control measures for plant pests and better methods of production for these chemical compounds.

Review Publications
Savchenko, K.G., Harper, S.R., Carris, L.M., Castlebury, L.A. 2020. Urocystis cumminsii sp. nov., a smut fungus on Themidaceae from Arizona. Mycotaxon. 134:591-599.
Doherty, J.R., Crouch, J., Roberts, J.A. 2020. Elucidating the influence of resident seed and soil microbiota on the developing creeping bentgrass microbiome. Agrosystems, Geosciences & Environment. 3(1):3:e20038. https://doi.org/10.1002/agg2.20038.
Stravoradis, S., Leblanc, N., Marra, R.E., Crouch, J., Hulvey, J.P. 2019. Widespread occurrence of a CYP51A pseudogene in Calonectria pseudonaviculata. Mycobiology. 48(1):44-50. https://doi.org/10.1080/12298093.2019.1689600.
Crouch, J.A., Dawe, A., Aerts, A., Churchill, A.C., Grimwood, J., Hillman, B.I., Milgroom, M.G., Smith, M., Salamov, A., Schmutz, J., Yadav, J., Gregoriev, I., Nuss, D. 2020. Genome sequence of the chestnut blight fungus Cryphonectria parasitica EP155: a fundamental resource for an archetypical invasive plant pathogen. Phytopathology. 110(6):1180-1188. https://doi.org/10.1094/PHYTO-12-19-0478-A.
Skaltsas, D.N., Salgado-Salazar, C. 2020. Genome resources for Thelonectria rubi, the causal agent of Nectria canker of caneberry. Phytopathology. 110(4):723-725. https://doi.org/10.1094/PHYTO-12-19-0466-A.
Leblanc, N., Crouch, J. 2019. Prokaryotic taxa play keystone roles in the soil microbiome associated with ornamental boxwood accessions at the U.S. National Arboretum. Ecology and Evolution. 9(19):11102-11111. https://doi.org/10.1002/ece3.5614.
Salgado-Salazar, C., Leblanc, N., Wallace, E., Daughtery, M., Crouch, J. 2020. Peronospora bergamotii, Hyaloperonospora daughtreyae and H. iberidis: new species associated with downy mildew diseases affecting ornamental plants in the United States. European Journal of Plant Pathology. 157(1):311-326. https://doi.org/10.1007/s10658-020-01989-9.
Skaltsas, D.N., Badotti, F., Martins, A.B., Da Silva, F.F., Gazis, R., Wurdack, K., Castlebury, L.A., Góes-Neto, A., Chaverri, P. 2019. Exploration of stem endophytic communities revealed developmental stage as one of the drivers of fungal endophytic community assemblages in two Amazonian hardwood genera. Scientific Reports. 9(1):12685. https://doi.org/10.1038/s41598-019-48943-2.
Berry, D., Mace, W., Rehner, S.A., Grage, K., Dijkwel, P.P., Young, C.A., Scott, B. 2018. Orthologous peramine and pyrrolopyrazine-producing biosynthetic gene clusters in Metarhizium rileyi, Metarhizium majus and Cladonia grayi. Environmental Microbiology. 21(3):928-939. https://doi.org/10.1111/1462-2920.14483.
Emanuel, I.B., Farinas, C., Lin, S., Pierzynski, J., Crouch, J., Hand, F.P. 2019. Occurrence of boxwood blight caused by Calonectria pseudonaviculata in Ohio landscapes. Plant Disease. 103(10):2670. https://doi.org/10.1094/PDIS-03-19-0520-PDN.
Groben, G., Clarke, B., Murphy, J., Koch, P., Crouch, J., Lee, S., Zhang, N. 2020. Quantitative real-time PCR detection of Clarireedia spp., the causal agents of dollar spot in turfgrasses. Plant Disease. 104:3118-3123. https://doi.org/10.1094/PDIS-04-20-0726-RE.