COORDINATED PATHOGEN SURVEILLANCE AND RESISTANCE DELIVERY

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: ACTIVE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1025284
• Grant No.: 2021-68013-33758
• Cumulative Award Amt.: $950,000.00
• Proposal No.: 2020-06498
• Project Start Date: Feb 1, 2021
• Project End Date: Jan 31, 2026
• Grant Year: 2021
• Program Code: [A1181]- Tactical Sciences for Agricultural Biosecurity

Project Director
Huguet-Tapia, J. C.

Recipient Organization
UNIVERSITY OF FLORIDA
G022 MCCARTY HALL
GAINESVILLE,FL 32611

Performing Department
(N/A)

Non Technical Summary
The project will develop systems for pathogen classification and the prediction of disease resistance in plant breeding populations. A sequencing based approach will be developed for pathogen classification and identification. Probes targeting genes that have traditionally been used for diagnostics, highly conserved loci and genes associated with pathogenicity will be designed and bioinformatically validated using existing genomes. The accuracy and universality of the pathogen classification models will then be quantified using samples from the USDA Fungal Collection and the National Plant Diagnostic Network (NPDN). The system will be evaluated by the UF NPDN laboratory for in-planta pathogen identification. Practical laboratories will be delivered for bacterial, fungal and viral identification and a course on molecular identification of pathogens will be developed. Germplasm from pine and maize breeding programs will be screened for tolerance to infection by Fusarium pathogens. Controlled inoculation and assessment of clonally replicated pine populations and maize varieties will provide phenotype that will be paired with genotype data to create genomic prediction models for Fusarium tolerance. Standard genomic prediction models will be compared with alternative models to evaluate the accuracy of predictions.

Animal Health Component

20%

Research Effort Categories

Basic

20%

Applied

20%

Developmental

60%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
202	0611	1081	30%
202	1510	1081	20%
212	2410	1160	50%

Knowledge Area
212 - Pathogens and Nematodes Affecting Plants; 202 - Plant Genetic Resources;

Subject Of Investigation
1510 - Corn; 0611 - Conifer forests of the South; 2410 - Cross-commodity research--multiple crops;

Field Of Science
1160 - Pathology; 1081 - Breeding;

Keywords

classifying

pathogens

and

predicting

disease

resistance

Goals / Objectives
The two major goals of the project are: 1) Develop a pathogen identification system based onenrichment and sequencingof traditionaldiagnosticloci and other conserved loci; 2) Develop genomic prediction models that incorporate phenotype data from controlled inoculation studies withgenotype data from genes regulating disease development. Methods for sequence-based identification of pathogens will be tested in the UF laboratory of the National Plant Diagnostics Network and shared with the plant pathology community via workshops and by the development of formal laboratories and a course on molecular diagnostics. Methods for phenotyping, genotyping and developing genomic prediction models will be shared with the plant breeding community and results of screening for disease resistance will be delivered to the pine and maize breeding programs.

Project Methods
Coordinated pathogen diagnostics - Diagnostics initially focuses on bacterial identification using well developed 16-S kits and software while biotinylatedprobes are developed for fungal pathogen identification. A diverse set of fungi will be selected for the the in-silico development of probes to capture and isolate conserved regions from within traditional barcode loci as well as alternative 'Tree of Life' barcodes for sequencing. Diagnostic probes will be designed using a selection of the 556 sequenced fungal isolates currently available from the Department of Energy Joint Genome Institute MycoCosm database and/or other public repositories. Probes, initially evaluated in-silico, will be synthesized, and used to capture and sequence hundreds of diagnostic and alternative (highly conserved) loci from pathogen isolates identified by the project steering committee.A database of variants within these loci will be created and used for the development of a classification model for fungal pathogens. The model will be validated using isolates from the USDA Fungal Collection and other sources to estimate classification accuracy and universality. Probes will also be designed for the enrichment of plant viruses.Developing disease resistant populations - Pine and maize breeding populations will be evaluated for tolerance to Fusarium infection using controlled inoculation and the phenotype will be combined with genotype data to develop genomic prediction models. Genes associated with disease will be bioinformatically identified in Fusarium, pine and maize genomes and probes will be designed for enrichment and subsequent sequencing of 100 isolates and pine breeding populations. Maize varieties with existing genome sequences and pine clones that will be genotyped will be screened for damage following inoculation with diverse sets of Fusarium. Genomic prediction models will be used to evaluate the accuracy of predictions and identify individuals for validation.Teaching molecular diagnostics - Teaching laboratories will be developed for bacterial and fungal diagnostics using existing kits and the enrichment and sequencing platform that will be developed. The teaching objective will develop content for a molecular pathogen diagnostics platform that is generalizable for use with plant, animal and human health systems.

Progress 02/01/23 to 01/31/24

Outputs
Target Audience:Plant disease diagnosticians;Mycologists;Plant breeders; Forest tree seed orchard managers; Forest managers Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project has supported a PhD student develop disease screening and genomic prediction models for agronomic crops, and a PhD student developing disease screening and genomic prediction models for a pine breeding program has completed study. A post-doctoral associate has been supported to develop sequencing based pathogen identification methods and to present the protocol to professional diagnosticians and students. How have the results been disseminated to communities of interest?Formal traning ofNational Plant Diagnostics Network staff and others interested in diagnostics has been provided with hands on training. Disease resistant germplasm has been identified for the pine breeding community and this information has been shared via the UF pine breeding cooperatives genetic evaluation database. Disease resistant maize varieties have been identified for the UF sweetcorn breeding program. What do you plan to do during the next reporting period to accomplish the goals?Extending the sequencing methods to natural systems, forests, and other crops is underway. The database is being expanded to include new genomes from fungal pathogens that have been added to NCBI and DOE repositories. Comparative genomics publications are underway for two pathogenpopulations and a genome wide association analysis is underway.

Impacts
What was accomplished under these goals? Protocols for sequence-based identification of fungal pathogens were provided to participants in the National Plant Diagnostics Network and the plant pathology community via a workshop and the seminars. Analysis of resulting data using traditional phylogenetic trees and k-mer based classification has been completed and documented in publications. Methods for phenotyping and developing genomic prediction models have been formally shared with the plant breeding community via publications.

Publications

• Type: Journal Articles Status: Published Year Published: 2023 Citation: Pasche J, Brito JA, Vallad GE, Brawner JT, Snyder SL, Fleming EA, Yang J, Terra WC, Martins SJ. 2023. Assessing the impact of successive soil cultivation on Meloidogyne enterolobii infection and on soil bacterial assemblages. Plant Pathology. Online 00, 1 9. https://doi.org/10.1111/ppa.13742
• Type: Journal Articles Status: Published Year Published: 2023 Citation: Owen Hudson, Dylan Hudson, Colin Brahmstedt and Jeremy Brawner. 2023 The Ear Unwrapper: A Maize Ear Image Acquisition Pipeline for Disease Severity Phenotyping. AgriEngineering 5(3), 1216-1225 https://doi.org/10.3390/agriengineering5030077
• Type: Journal Articles Status: Published Year Published: 2023 Citation: Andrew D. Sims, Gary F. Peter, Katherine Smith, W. Patrick Cumbie, Dominic Kain and Jeremy T. Brawner. 2023. Genetic Control of Pitch Canker Response in Southern Pine and Southern Pine Hybrids. Forests. https://doi.org/10.3390/f14030554
• Type: Journal Articles Status: Published Year Published: 2023 Citation: Pei-Ling Yu, James C. Fulton, Owen H. Hudson, Jose C. Huguet-Tapia, and Jeremy T. Brawner. 2023. Next-Generation Fungal Identification Using Target Enrichment and Nanopore Sequencing. BMC Genomics. 24, 581. https://doi.org/10.1186/s12864-023-09691-w.
• Type: Journal Articles Status: Published Year Published: 2023 Citation: Andrea Karina Suazo Tejada, Pei-Ling Yu, Katherine Smith, Jose Huguet-Tapia, Joseph Carrillo, Jeremy Brawner, and Gary Vallad. 2023. Genome Sequencing and De Novo Assembly of Trichoderma longibrachiatum Isolate collected from Florida agricultural soils. Microbiology Resource Announcements. https://doi.org/10.1128/mra.00906-23.
• Type: Journal Articles Status: Submitted Year Published: 2024 Citation: Owen Hudson, Marcio Resende, Carlos Messina, James Holland, Jeremy Brawner, 2023, Prediction of resistance, virulence, and host-by-pathogen interactions using dual genome prediction models. Theoretical and Applied Genetics

Progress 02/01/22 to 01/31/23

Outputs
Target Audience:The main audience reached during this period has been associated with the National Plant Diagnostics Network and the forest health community to develop our pathogen surveillance network. Interested labs have applied to join the workshop we will deliver in March to train network participants to use the 'Universal Fungal Identification Tool' we have developed for this project. For the resistance breeding part of the project, the UF sweet corn and USDA field corn breeding programs as well as the USDA fungal collection facility have been engaged and have supplied germplasm for disease screening. Forest industry genetic improvement programmanagersproducing seed and seedlings for reforestation have provided input for pine the screening program and are preparing to use results to guide deployment to areas where disease pressure is high. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?A PhD student has been engaged to work on the sweet corn screening program and two post-docs have been employed to develop the pathogen surveillance tool.The workshop we will provide in March will provide formal training to use this tool. How have the results been disseminated to communities of interest?Results of the disease screening activities have been reported to the relevant breeding programs. What do you plan to do during the next reporting period to accomplish the goals?A workshop to train collaborators to use the pathogen surveillance tool will be delivered in March. Results from the workshop will be prepared for publication. The sweet corn screening will be completed in the spring of 2023 and the pine screening will be completed in the fall of 2023. Prediction models have been developed and will be used to provide breeding programs with recommendations to deliver improvements via the breeding programs.

Impacts
What was accomplished under these goals? The 'Universal Fungal Identification Tool' has been created and tested with various samples. A workshop with forest health and National Plant Diagnostics Network collaborators has been organized and 16 attendees have confirmed attendance for mid-March. Publication of results from the tool is underway. Two seasons of disease screening havebeen completed for maize and the pine population is being produced for screening in August with the US Forest Service. Genomic prediction models have been developed for these populations.

Publications

• Type: Journal Articles Status: Published Year Published: 2021 Citation: James Fulton, Jeremy Brawner, Jose Huguet-Tapia, Katherine E Smith, Randy Fernandez, and Nicholas S Dufault. 2021. Six de novo assemblies from pathogenic and non-pathogenic strains of Fusarium oxysporum f. sp. niveum. PhytoFrontiers. 2(1) https://doi.org/10.1094/PHYTOFR-04-21-0031-SC
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Pei-Ling Yu, James Fulton, Sandra Carmona Guti�rrez, Diana Burbano-David, Luz Barrero, Jose Huguet-Tapia, Jeremy Brawner, and Mauricio Soto-Suarez. 2022. Draft Genome and de novo Assembly of Fusarium oxysporum f. sp. lycopersici Isolate Collected from the Andean Region in Colombia Microbiology Resource Announcements, Dec 2021 https://doi.org/10.1128/mra.00980-21
• Type: Journal Articles Status: Published Year Published: 2022 Citation: James C. Fulton, Pei-Ling Yu, Katherine E. Smith, Jose C. Huguet-Tapia, April Meeks, Tania Quesada, Kathleen McKeever, Jeremy T. Brawner. 2022. Comparative genomics of Fusarium circinatum isolates used to screen southern pines for pitch canker resistance. Molecular Plant-Microbe Interactions. https://doi.10.1094/MPMI-10-21-0247-R

Progress 02/01/21 to 01/31/22

Outputs
Target Audience:The project team hasdeveloped linkages with the National Plant Diagnostics Network (NPDN) laboratory, which employs some of the end users that will use the platform we are developing. The team is drafting a concept note with the director of the NPDN describing the technology for distrubution to the wider Plant Pathology community via the American Phytopathology Society (APS) special issue on diagnostics. The team has applied and been accpeted to provide a workshop to demonstrate the platform at the APS annual meeting in August. The project director has presented the resistance breeding workplans for the project to 25 research and technical professionals participating in the Forest Biology Reserach Program. The project has worked with 5 landowners and 3 companies to identify the fusarium pathogen that is the focus of the project and visited a large nursery that will deploy the germplasm we develop. Over 20 pathologist have received a brief summary of the project as part of our request for isolates for screening. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Post doctoral fellows and PhD students have contributed to three publications for this project and are developing two new publications for presentations at the 2022 APS annual meeting. We are developing a new diagnostics focused publication as well as a comparative genomics publication for the pathogen we will use for disease resistance screening. How have the results been disseminated to communities of interest?Members of the pine breeding cooperative have been provided provisional disease resistance breeding values for pine families evaluated and enganged in additional screening to support validation of prediciton models. What do you plan to do during the next reporting period to accomplish the goals?We are focused on providing validation data from a closely related clade of fungi and a diverse set of fungi in multiplexed samples. Two probe sets have been synthesized and we are evaluating different protocols for each set of probes to finalize the enrichement and sequencing protocols. The main focus will be on devloping classification pipelines for pathogens. All F. circinatum have been collected and will be grown on for screening of the clonally replicated progeny trials. Whole genome sequences of 50 isolates from both the maize and pine pathogens will be produced for association with host DNA sequences and subsequently used to predict disease progression. Experiments for disease sceening have been designed and timelines are in place to produce data as planned.

Impacts
What was accomplished under these goals? For Goal 1, we have compiled genomes for all fungi available on NCBI and identified 2 sets of 13 genes as targets for sequencing, the diagnostic and BUSCO sets. Pipelines were created to extract these genes from 400 available fungal genomes and convert protein sequence to nucleotide sequence for each. Data was submitted to 3 companies that synthesize oligonucleotide probes and none provided acceptable probe designs. We developed an in-house probe design pipeline to clustered each gene into sets containing 120bpk-mers with 85% similarity and then identify the most commong 120bp motif within each cluster. These designs were provided totwo companies and probes have been synthesized. Two separate reactions, one contaiing two barcoded samples,were run on the nanopore sequencer and enrichment was achieved. Long-reads have been assembled and post-processing classification was completed with 4different pipelines. Additional fungal samples have been collected and two new experiments are underway to evaluate efficiencies from greater multiplexing of samples. A CRISPR based enrichment platform has been created to capture and sequence the entire ITS region from fusarium to provide another example of third generation sequencing's application to pathogen diagnostics. The PI has obtained permits from the USDA APHIS to managed pathogens in the laband has traveled across the SE US collecting Fusarium isolates for the pine resistance breeding work. Over 100 samples have been processed to isolate Fusarium, single spore isolates were generated and PCR has been used to validate these samples. Seedlings have been produced from the CFGRP pine breeding program and seedlings have been provided to a commercial pine nursery for propagation of the clones needed for the screening experiments. The department has allocated greenhouse space for the screening that is to take place August to November 2022. The department is is supporting a PhD student to work on maize resistance breeding and requests to provide Fusarium isolates from accross the US have been sent to collaborators. USDA scientists and UF faculty have provided germplasm for the maize screening and have agreed to establish field trials for the project. All activities are proceeding as planned.

Publications

• Type: Journal Articles Status: Published Year Published: 2021 Citation: Ence, D., Smith, K.E., Fan, S., Neves, L.G., Paul, R., Wegrzyn, J., Kirst, M. Brawner, J.T., Peter, G. Nelson, C.D., Davis, J.M. 2021. NLR Diversity and Candidate Fusiform Rust Resistance Genes in Loblolly Pine. G3 Genes|Genomes|Genetics Online first paper #: G3-2021-402533Announcements. 9(30).
• Type: Journal Articles Status: Published Year Published: 2021 Citation: James Fulton, Jeremy Brawner, Jose Huguet-Tapia, Katherine E Smith, Randy Fernandez, and Nicholas S Dufault. 2021. Six de novo assemblies from pathogenic and non-pathogenic strains of Fusarium oxysporum f. sp. niveum. PhytoFrontiers. Published Online:21 Jul 2021 https://doi.org/10.1094/PHYTOFR-04-21-0031-SC
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Pei-Ling Yu, James Fulton, Sandra Carmona Guti�rrez, Diana Burbano-David, Luz Barrero, Jose Huguet-Tapia, Jeremy Brawner, and Mauricio Soto-Suarez. 2022. Draft Genome and de novo Assembly of Fusarium oxysporum f. sp. lycopersici Isolate Collected from the Andean Region in Colombia Microbiology Resource Announcements, Dec 2021 https://doi.org/10.1128/mra.00980-21