(MC) DEVELOPING AND SCALING UP THE NEXT GENERATION OF HEALTHY FORESTS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: ACTIVE
• Funding Source: MCINTIRE-STENNIS
• Reporting Frequency: Annual
• Accession No.: 1022115
• Project Start Date: Feb 5, 2020
• Project End Date: Jan 31, 2025
• Program Code: [(N/A)]- (N/A)

Project Director
Brawner, JE, T.

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

Performing Department
Plant Pathology

Non Technical Summary
Southern pines (loblolly (Pinus taeda) and slash (Pinus elliottii) pine) are severely impacted by fungal pathogens such as fusiform rust (Cronartium quercuum (Berk.) Miyabe ex Shirai f. sp. Fusiforme or Cqf) and pine pitch canker (Fusarium circinatum or FC). These endemic diseases cause significant economic damage to forests each year (Powers et al. 1974). Losses from rust have been estimated to be as high as $578 per hectare (Brawner et al. 1999). Decades of tree improvement research designed to identify rust and pitch canker resistant families have led to more productive plantations (Walker and McKeand 2017) and improved financial returns for investments in planted forests (Cubbage et al. 2000). Controlled inoculation systems for rust and pitch canker have been developed through collaboration among US Forest Service's Resistance Screening Center (RSC) in conjunction with university and industry stakeholders (Cowling and Young 2013). Results from tests performed at the RSC have been supported by networks of field trials established across the SE US (Spitzer et al. 2017). Research by the University of Florida, North Carolina State University and Texas A&M University has demonstrated strong evidence for the genetic control of disease resistance and has provided a better understanding of the genes regulating resistance (Resende et al. 2012, Quesada et al. 2010, Quesada et al. 2014). Recent project work has utilized methods developed to reduce the complexity of the pine genome (Neves et al. 2013) to target resistance genes for sequencing of transcripts in infected pine tissue following inoculation at the US Forest Service Resistance Screening Center.The proposed project will develop a better understanding of molecular interactions between pathogen and pine genomes to validate hypotheses implying gene-for-gene or polygenic genetic architectures for resistance or tolerance to pathogens. Damage from pine pathogens will be less predictable as climate variability increases (Quesada et al. 2019) and a better understanding of the regulation of disease would be useful for achieving both industrial and restoration goals in future forests. The well-developed phenotyping systems for screening pine seedlings for resistance to rust and pitch canker will be used to provide empirical data to validate data analysis systems to identify disease resistant reforestation populations so that these methods may be used to address other forest health problems. For example, the system used to characterize Cqf resistance genes in loblolly pine may be extended to develop disease resistant breeds of longleaf pine (Pinus palustris) and reduce replanting requirements following Cqf-induced mortality in restoration plantings or develop disease resistant breeds of slash pine for areas where FC severely reduces timber quality.

Animal Health Component

50%

Research Effort Categories

Basic

50%

Applied

50%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
202	0611	1081	50%
201	0699	1160	50%

Knowledge Area
202 - Plant Genetic Resources; 201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
0611 - Conifer forests of the South; 0699 - Trees, forests, and forest products, general;

Field Of Science
1160 - Pathology; 1081 - Breeding;

Keywords

healthy

forests

Goals / Objectives
The goal of the research is to develop analytical systems to connect data from disease phenotyping and genotyping platforms in order to identify diseaes resistant germplasm for reforestation .

Project Methods
The project will use Pinus elliottii and Fusarium circinatum as models to produce empirical evidence to address the debate over the relevance of polygenic and omnigenic models of gene architecture in explaining disease phenotypes (Boyle el al. 2017, Wray et al. 2018). We will screen large pine populations at the US Forest Service RSC to provide phenotype data that may be used to target individuals for genotyping. Disease screening of large numbers of pedigreed seedlings with various isolates of the FC pathogen were developed in 2019 and will be applied to a diverse set of slash pine families from the University of Florida Cooperative Forest Genetics Research Program (CFGRP) in 2020. Screening with multiple isolates will provide the required host-by-pathogen interaction data for the evaluation of alternative genomic prediction models that may be used to identify disease resistant individuals and markers within putative disease tolerance genes.Two approaches will be used to identify disease tolerance genes that will be differentially weighted in genomic prediction models: dual-RNAseq and genome wide association studies (GWAS). The transcriptomics experiments will identify differentially expressed genes in relatively healthy and diseased plants following pathogen challenge. A 2019 RNAseq experiment provides data from relatively tolerant and relatively susceptible individuals within the most tolerant and least tolerant families of slash pine that were inoculated with several isolates of FC. Samples of FC-infected slash pine tissue were genotyped in a dual-RNAseq experiment (Westermann et al. 2017) that targeted sequences of R-genes (RenSeq) (Arora et al. 2019), coding sequences from FC genes (exome capture) (Mercer et al. 2014, Neves et al 2013) and additional neutral markers. These RNAseq data are to be processed in Q1 of 2020 to guide the development of capture probes for the pathogen that will be used for pathogenicity gene enrichment sequencing (PenSeq) (Thilliez et al. 2019) of the many FC isolates available in the UF Forest Pathology lab (Quesada et al. 2019). Capture probe design will be further facilitated by a Plant Pathology department-funded PhD student to sequence, assemble and compare genomes of the FC isolates used in the screening.The 2020 screening of CFGRP families will provide pine samples for genotyping, developing prediction equations for FC tolerance and identifying markers in gene coding sequence that are significantly associated with FC tolerance. Concurrently, 1,000 slash pine seedlings are being clonally replicated by a CFGRP collaborator for additional screening of cuttings by the RSC. This population will be inoculated with different strains of FC to provide a validation of the genomic prediction models developed using the RenSeq genotyping of the population screened in 2020 and also provide for estimates of pathogen-by-host interactions that may be used to estimate tolerance repeatability. The 2019 dual-RNAseq experiment will be used to design and synthesize new capture probes for the PenSeq component of a second dual-RNAseq experiment using the validation population. Further work will focus on integrating phenotype and genotype data to evaluate the accuracy of genomic selection models for prediction of general and pathogen-specific tolerance.Standard Genomic Selection (GS) models will provide baseline estimates of model accuracy for comparisons with the alternative GS models we propose. Selected genes may be differentially weighted in Bayesian or expanded GS models that utilize different kernels to parse relatedness and major gene effects. The use of dual variance components (Moser et al. 2015, Tucker et al. 2015) to partition relatedness and resistance gene network effects will be the initial focus for GS model comparisons. Expanding on concepts developed by Brawner et al. (2012), multivariate GS models that include both phenotype and markers from genes of major effect as additional traits will be further developed and compared with standard GS models. Multivariate models will be further expanded to incorporate markers within genes of major effect in both the host and pathogen. The dual-RNAseq and GWAS approaches will be used to identify these tolerance genes and the multivariate framework will be used to quantify correlations among tolerance phenotype, pathogen and host gene content (Legarra & Vitezica 2015). We will provide direct estimates of host and pathogen gene interactions as genetic correlations between markers estimating R-gene and Vir-gene dosage and disease rating. Expanding the multivariate models that include phenotype and markers in several genes to include phenotype and markers in hundreds of genes underlying R-gene networks will be used to dissect the genic control of tolerance to FC infection.

Progress 10/01/20 to 09/30/21

Outputs
Target Audience:Tree breeders in the Southern US have been engaged to discuss disease screening methods and associated genomic prediction methods. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Training provided by workshop to optimize crossing programs for breeding programs and development of protocols with Industy collaborators and US Forest Service. How have the results been disseminated to communities of interest?Breeding values for disease resistance have been provided to collaborators following screening. What do you plan to do during the next reporting period to accomplish the goals?Delivery of two NIFA projects that support this project will faciliate accomplishment of the project goals.

Impacts
What was accomplished under these goals? Collaborations between the UF Plant Pathology and School of Forest Resources and Conservation haveled to USDA NIFA projects on two forest pathosystems that intend to identify genes associated with disease resistance.Projects follow collaboration with USFS to provide provisional data on host-pathogen interactions.

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 12(2) https://doi.org/10.1093/g3journal/jkab421

Progress 02/05/20 to 09/30/20

Outputs
Target Audience:Plantation forest owners have been engaged with site visits to forests and planning of collaborative project. The development of a successfull USDA NIFA AFRI grant has allowed a team from UF, US Forest Service and industry to coordinate pathogen detection and the development of disease resistant germplasm. Comparisons between forest and agricultural crop resistant mechanisms is underway to broaden the scope of the reserach project. Experiments have been run to develop screening methods that are being used to identify disease resistant pines. These experiments were managed in collaboration and results are being delivered to landowners by targetting individuals for seed collection that may be used to reduced disease incidence in newly established plantations. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Three graduate students have been engaged to work on these pine focused projects. More general development of disease resistance that supports this project is being conducted by two other post-doctoral candidates. An additional project has been initiated with a masters student using the methods from this project to screen endangered species (Taxus torreya) for resistance to a similar pathogen. How have the results been disseminated to communities of interest?Publication of the results of the genome sequences are in the literature. The software for optimizing diversity and gain was provided to workshop attendees. Results were communicated in presentations to the Cooperative Forest Genetics Reserach Cooperative and the Forest Biology Reserach Cooperativeannual meetings. What do you plan to do during the next reporting period to accomplish the goals?The AFRI sponsored project (FLA-PLP-005931) provides a four-year plan to expand upon this project.

Impacts
What was accomplished under these goals? Screening methods have been developed for the pathosystem of interest that allow a diversity of host and pathogens to be screened. The results have been provided to orchard managers to develop seedlots of more resistant pines. The genomes of the pathogen isolates used for screening have be sequenced and published. This work directly supported the recently approved AFRI project that will supprot this project. Methods were also expanded to the Fusiform rust pathogen and another proposal is under development.

Publications

• Type: Journal Articles Status: Published Year Published: 2020 Citation: Fulton JC, Huguet-Tapia JC, Adams SM, Dufault NS, Quesada T, Brawner JT. 2020. Draft genome sequences of three Fusarium circinatum isolates used to inoculate a pedigreed population of Pinus elliottii seedlings. Microbiology Resource Announcements. 9(30).