Source: AGRICULTURAL RESEARCH SERVICE submitted to
ARIS LOG: 0068708. TITLE: LEVERAGING POPULATION GENOMICS TO UNDERSTAND THE MIGRATION AND EVOLUTION OF THE CAUSAL PATHOGENS OF BOXWOOD BLIGHT
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1026758
Grant No.
2019-67012-34934
Cumulative Award Amt.
$117,450.05
Proposal No.
2021-06284
Multistate No.
(N/A)
Project Start Date
Jul 1, 2021
Project End Date
Jun 30, 2024
Grant Year
2021
Program Code
[A7201]- AFRI Post Doctoral Fellowships
Project Director
LeBlanc, N.
Recipient Organization
AGRICULTURAL RESEARCH SERVICE
800 BUCHANAN ST, RM 2020
BERKELEY,CA 94710-1105
Performing Department
(N/A)
Non Technical Summary
Fungal plant pathogens in the genus Calonectria cause devastating diseases on a wide range of economically important horticultural and agronomic crops. In particular, the recent global emergence and spread of the fungal pathogens Calonectria henricotiae and C, pseudonaviculata has become a limiting factor for the production of high-value boxwood plants. A major constraint in controlling these fungal plant pathogens is that the genetic basis of plant infection is very poorly understood. The purpose of this project is to analyze genomic data from fungal plant pathogens in the genus Calonectria to identify genes that play a role in plant host infection, with particular emphasis on the two pathogens C. henricotiae and C. pseudonaviculata. This new knowledge has potential to reduce negative impacts of diseases caused by these fungi by improving basic understanding of pathogen biology and providing breeders new tools to develop disease resistance plants.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
21206701102100%
Knowledge Area
212 - Pathogens and Nematodes Affecting Plants;

Subject Of Investigation
0670 - Short rotation woody crops, including holiday trees;

Field Of Science
1102 - Mycology;
Goals / Objectives
The goal of this project is to characterize evolutionary changes in the genomes of fungal plant pathogens in the genus Calonectria to identify gene families or individual genes that play a role in host plant infection by the target pathogen species C. henricotiae and C. pseudonaviculata. Specific project objectives are: 1) characterize evolutionary relationships among target fungal plant pathogens in the genus Calonectria and closely related fungi in the family Nectriaceae through generation of a time-calibrated phylogeny using previously identified single-copy protein sequence data, 2) identify rapidly evolving gene families by measuring gene family expansion and contraction across the time-calibrated phylogeny, 3) identify gene families or genes that play a role in host plant infection through functional annotation of protein sequence data from rapidly evolving gene families in target pathogens.
Project Methods
The goal and objectives of this project will be accomplished through bioinformatic analysis of genomic data from fungal plant pathogens in the genus Calonectria and family Nectriaceae. Previously identified single copy protein sequence data from genomes of target fungal plant pathogens will be used to generate a time-calibrated phylogeny. These phylogenomic data will used to measure evolutionary changes in previously identified gene families to determine which families are rapidly evolving in the target fungal pathogens. Protein sequence data from rapidly evolving gene families will be annotated using established bioinformatic pipelines. Annotation will include broad classification based on the Cluster of Orthologous Genes (COGs) scheme and additional specific annotation. Specific annotation will include bioinformatic identification of secondary metabolite producing enzymes, secreted effectors, and carbohydrate active enzymes. Protein sequence data from target gene families will also be compared to the Pathogen Host Interactions database to identify genes with homologs in other plant pathogens that have been functionally validated as pathogenicity or virulence factors. Completing key milestones and the primary research objectives will be used to evaluate progress of the project. Progress will also be evaluated based on communication of research results to stakeholders in the research community through publication of conference abstracts and peer reviewed manuscripts.

Progress 07/01/21 to 06/30/24

Outputs
Target Audience:Target audience:Audiences reached during this reporting period included government and academic research scientists working in the biological sciences as well as graduate and undergraduate students pursuing careers in science. Research results were communicated through presentation ofconference abstracts at the 2024 Annual Meeting of the Ecological Society of America and the 32ndFungal Genetics Conference as well as a seminar presentation at the USDA-ARS Crop Improvement and Protection Research Unit in Salinas, CA. Results were also communicated through release of a genomic sequence data and submission of a peer reviewed manuscript. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Training and professional development opportunities were provided for the project director and support scientist during this reporting period. Science communication was improved through attendance and presentation of research at the annual the Fungal Genetics Conference and Ecological Society of America Annual Conference by the support scientist. The professional network of the support scientist was expanded by attending these conferences and participating in bi-monthly virtual meetings with a USDA-ARS scientist in Beltsville, MD. Teaching and science communication was also improved for the project director through mentorship and bioinformatics training of the support scientist contributing to the project. How have the results been disseminated to communities of interest?Results were disseminated to communities of interest through presentation and publication. The support scientist on the project presented a research abstract at theFungal Genetics Conference and Ecological Society of America Conference. Research results were further communicated through submission of a peer reviewed manuscript in thejournal Plant Disease. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? The goal of this project is to characterize evolutionary changes in the genomes of fungal plant pathogens in the genusCalonectriato identify gene families or individual genes that play a role in host plant infection by the target pathogen speciesC. henricotiaeandC. pseudonaviculata. Specific project objectives are: 1) characterize evolutionary relationships among target fungal plant pathogens in the genusCalonectriaand closely related fungi in the family Nectriaceae through generation of a time-calibrated phylogeny using previously identified single-copy protein sequence data, 2) identify rapidly evolving gene families by measuring gene family expansion and contraction across the time-calibrated phylogeny, 3) identify gene families or genes that play a role in host plant infection through functional annotation of protein sequence data from rapidly evolving gene families in target pathogens. Accomplishments for Objectives 1 and 2 have already been completed and described in the prior progress reports for this project. To expand knowledge of potential virulence and pathogenicity factors (Objective 3), secondary metabolite biosynthetic clusters were predicted in the genomes ofC. pseudonaviculataandC. henricotiaeusing bioinformatic methods. These analyses tentatively identified 74 biosynthetic clusters inC. henricotiaeand 72 clusters inC. pseudonaviculata. Further analysis of the 74 biosynthetic clusters in theCalonectriaspp. identified a partial cluster for production of trichothecenes which are known virulence factors ofFusariumgraminearumcausing disease on cereal crops. Identification of genes and primary proteins present in theCalonectriasp. suggest these fungi can complete a substantial part of the trichothecene pathway and potentially produce calonectrin, which is a precursor to trichothecene secondary metabolites. These results provide a basis for future research on the potential role of calonectrin as a virulence or pathogenicity factor inCalonectria pseudonaviculataandC. henricotiaeor other pathogenicCalonectriaspp.

Publications

  • Type: Journal Articles Status: Under Review Year Published: 2024 Citation: Harrigian F, LeBlanc N, Eriksen R, Bush E, Rodriguez Salamanca L, Salgado Salazar C. 2024. Uncovering the fungus responsible for stem and root rot of false indigo: pathogen identification, new disease description, and genome analyses. Plant Disease (in review).
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2024 Citation: Harrigian F, Salgado-Salazar C, LeBlanc N. 2024. Goldmine of ecological interactions in plant microbiomes: what can be learned from close observation and following your curiosity. Ecological Society of America Annual Meeting, Long Beach, CA.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2024 Citation: Harrigian F, Bush E, Rodriguez Salamanca L, Salgado-Salazar C, LeBlanc N. 2024. Polishing a pathogen: Annotation of a hybrid genome assembly reveals putative virulence and pathogenicity factors of Calonectria sp. 134-2022, a novel pathogen of Baptisia australis. 32nd Fungal Genetics Conference, Pacific Grove, CA.


Progress 07/01/22 to 06/30/23

Outputs
Target Audience:Audiences reached during this reporting period included government and academic research scientists working in the biological sciences as well as graduate and undergraduate students pursuing careers in science. Research results were communicated through oral presentation of aconference abstract at the 2023 Annual Meeting of the Mycological Society of America and publication of a peer-reviewed manuscript focused onCalonectriapathogens impacting ornamental horticulture in the journal Plant Disease. Results were also communicated through release of genome annotation data forCalonectria henricotiaeandC. pseudonaviculataused for genomic analyses in prior publications. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Training and professional development opportunities were provided for the PrincipalInvestigator and recently hired support scientist during this reporting period. Science communication was improved through attendance and presentation of research at the annual Mycological Society of America conference by the support scientist. The professional network of the support scientist was expanded by participating in bi-monthly virtual meetings with a USDA-ARS scientist in Beltsville, MD. Teaching and science communication was also improved for the PrincipalInvestigatorthrough mentorship and bioinformatics training of the support scientist contributing to the project. How have the results been disseminated to communities of interest?Results were disseminated to communities of interest through presentation and publication. The support scientist on the project presented a research abstract at the2023 Annual Meeting of the Mycological Society of America. Research results were further communicated through publication of a peer reviewed manuscript in thejournal Plant Disease by the Principal Investigator. What do you plan to do during the next reporting period to accomplish the goals?Existing genomic data and additional genome assemblies generated during the current reporting period will be further annotated and analyzed. Three types of genes previously identified as rapidly expanding inC. henricotiaeorC. pseudonaviculatawill be compared to other fungal genomes through phylogenetic analyses and domain searches to determine putative functional roles of predicted proteins and relevance to host plant infection. In addition, putative secondary metabolite biosynthetic clusters identified in this reporting period will be annotated to complement existing data and determine if both of these fungi produce secondary metabolites that are known virulence or pathogenicity factors in other fungal plant pathogens.

Impacts
What was accomplished under these goals? Accomplishments for Objectives 1 and 2 have already been completed and described in the prior progress reports for this project. To expand knowledge of potential virulence and pathogenicity factors (Objective 3), secondary metabolite biosynthetic clusters were predicted in the genomes ofC. pseudonaviculataandC. henricotiaeusing bioinformatic methods. These analyses tentatively identified 74 biosynthetic clusters inC. henricotiaeand 72 clusters inC. pseudonaviculata. These data will provide a basis for further annotation and potential identification of metabolites secreted by these pathogens that enable them to infect host plants and cause disease.

Publications

  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Aiello D, Guarnaccia V, Vitale A, LeBlanc N, Shishkoff N, Polizzi G. 2022. Impact of Calonectria diseases on ornamental horticulture: diagnosis and control strategies. Plant Disease 106:1773-1787.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Harrigian F, Bush E, LeBlanc N, Salgado-Salazar C. 2023. Nanopore sequencing generates a high-quality genome assembly of Calonectria colhounii 134-2022, a novel pathogen of false indigo (Baptisia australis Pink Truffles). 91st Annual Meeting of the Mycological Society of America.
  • Type: Other Status: Published Year Published: 2023 Citation: LeBlanc N. 2023. Genome annotation data from Calonectria species. Ag Data Commons. https://doi.org/10.15482/USDA.ADC/1529351. Accessed 2023-07-27.


Progress 07/01/21 to 06/30/22

Outputs
Target Audience:Audiences reached during this reporting period included government and academic research scientists working in the plant sciences as well as graduate and undergraduate students pursuing careers in science. Primary research results were communicated through publication of a peer reviewed manuscript in the journal BMC Ecology and Evolution.Research results were also communicated to target audiences as a conference abstract at the 2021 joint meeting between the Mycological Society of America and Botanical Society of America. Research was also communicated through three invited seminars presented by the Principal Investigator. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?Results were disseminated to communities of interest through presentation and publication. A Masters student mentored by the Principal Investigator presented a research abstract at the 2021 Joint meeting between the Mycological Society of America and Botanical Society of America Conference. The principal investigator presented research results as part of three invited seminars. Research results were further communicated through publication of a peer reviewed manuscript in the journal BMC Ecology and Evolution What do you plan to do during the next reporting period to accomplish the goals?Genomic data will be further annotated to determine putative functional roles of rapidly evolving gene families and secondary metabolism biosynthetic clusters in C. henricotiae and C. pseudonaviculata.

Impacts
What was accomplished under these goals? The evolutionary relationships among Calonectria henricotiae, C. pseudonaviculata, and 22 additional closely related fungi were characterized using phylogenomic methods. A time-calibrated phylogeny was generated using protein sequence data from 2,154 single-copy genes shared among the 24 taxa. Each branch of the phylogeny received complete statistical support demonstrating high confidence in the topology of the phylogeny and the inferred evolutionary relationships among the 24 fungal taxa. Outcomes of from this research objective provide data required for further analysis of gene family expansion and contraction among the target fungal plant pathogens (Objectives 2-3). Rapidly evolving gene families and their expansion or contraction in individual fungal taxa were identified. Out of 19,759 gene families identified, 422 gene families were rapidly expanding or contracting in at least one of the 24 fungal taxa. Forty-three gene families were rapidly evolving in C. henricotiae and 35 in C. pseudonaviculata. Over 75 percent of these rapidly evolving gene families in C. henricotiae and C. pseudonaviculata were rapidly contracting. In contrast, more than 70% of rapidly evolving gene families identified in other Calonectria spp. were expanding. These results show that most rapidly evolving gene families in C. henricotiae and C. pseudonaviculata have contracted during the evolution of these species. Outcomes from this research objective demonstrate the evolution of gene families in the target boxwood blight pathogen species is significantly different from closely related fungal taxa, potentially explaining the narrow host range of these fungal plant pathogens. Gene families that play a role in host infection have partially been identified through functional annotation of protein sequence data representing rapidly evolving gene families. Protein sequence data were annotated using broad classification schemes based on Clusters of Orthologous Groups (COGs) and Pfam domains. Three rapidly evolving gene families were identified in each of the target species that were not rapidly evolving in non-target taxa. Among these families, one in C. pseudonaviculata was annotated as containing a CFEM protein domain, which has previously been shown to play a role in fungal pathogenesis. This research provides a list of candidate gene families and genes that can be further analyzed using functional genetics to evaluate their role in host-pathogen interactions and develop novel genetic approaches to manage the boxwood blight disease.

Publications

  • Type: Journal Articles Status: Accepted Year Published: 2022 Citation: Rogers LW, Koehler AM, Crouch JA, Cubeta MA, LeBlanc N. 2022. Comparative genomic analysis reveals contraction of gene families with putative roles in pathogenesis in the fungal boxwood pathogens Calonectria henricotiae and C. pseudonaviculata. BMC Ecology and Evolution 22:79. https://doi.org/10.1186/s12862-022-02035-4.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2021 Citation: Rogers L, Cubeta MA, LeBlanc N. 2021. Characterization of rapidly evolving gene family diversity among apathogenic and pathogenic species of Calonectria. Mycological Society of America and Botanical Society of America Joint Conference.