TRACKING THE MOVEMENT OF CYTOSPORA PATHOGENS IN ORCHARDS TO DEVELOP NOVEL IPM STRATEGIES

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: ACTIVE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1025405
• Grant No.: 2021-68008-34116
• Cumulative Award Amt.: $298,794.00
• Proposal No.: 2020-04969
• Project Start Date: Jan 15, 2021
• Project End Date: Jan 14, 2026
• Grant Year: 2021
• Program Code: [A1701]- Critical Agricultural Research and Extension: CARE

Recipient Organization
COLORADO STATE UNIVERSITY
(N/A)
FORT COLLINS,CO 80523

Performing Department
Agricultural Biology

Non Technical Summary
The long-term goal of this research is to develop management strategies for canker pathogens based on sound understandings of pathogen epidemiology across stone fruit orchards. Canker pathogens are difficult to manage because disease symptoms are hard to identify early in disease process, and trees produce a crop in initial phases of infection, and this delays growers' decisions about IPM treatments. Pathogens to spread and result in new economically destructive infections. In Colorado, Cytospora canker is the most damaging disease faced by fruit growers. Several Cytospora spp. are documented to cause disease on peach, apple and cherry, yet management for Cytospora occurs mainly in peach. The importance of host specificity and the role apple and cherry orchards play in the epidemiological cycle has not been demonstrated. The goals of this integrated research and extension CARE project are to: 1. Survey peach, cherry, and apple orchards in Colorado to estimate Cytospora spp. incidence and severity, 2. Delineate Cytospora species distribution in fruit orchards with molecular and morphological techniques and elucidate movement of Cytospora spp. within and among fruit orchards, 3. Perform pathogenicity assays to determine fruit crop host range of each Cytospora species, and 4. Develop conceptual scenarios to illustrate the importance of early implementation of best management practices to reduce Cytospora canker. The project will be completed in collaboration with the Cytospora Working Group and Tri Area Extension. Developing novel methods could revolutionize the sustainable management for canker pathogens in orchards which are an integral part of the culture and economy.

Animal Health Component

80%

Research Effort Categories

Basic

20%

Applied

80%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
212	1114	1160	100%

Knowledge Area
212 - Pathogens and Nematodes Affecting Plants;

Subject Of Investigation
1114 - Peach;

Field Of Science
1160 - Pathology;

Keywords

canker pathogens

fruit crops

management

population genetics

Goals / Objectives
The long-term goal of this research is to develop management strategies for canker pathogens based on sound understandings of pathogen epidemiology across stone fruit orchards. Canker pathogens are difficult to manage because disease symptoms are hard to identify early in disease process, and trees produce a crop in initial phases of infection, and this delays growers' decisions about IPM treatments. Pathogens to spread and result in new economically destructive infections. In Colorado, Cytospora canker is the most damaging disease faced by fruit growers. Several Cytospora spp. are documented to cause disease on peach, apple and cherry, yet management for Cytospora occurs mainly in peach. The importance of host specificity and the role apple and cherry orchards play in the epidemiological cycle has not been demonstrated. The goals of this integrated research and extension CARE project are to: 1. Survey peach, cherry, and apple orchards in Colorado to estimate Cytospora spp. incidence and severity, 2. Delineate Cytospora species distribution in fruit orchards with molecular and morphological techniques and elucidate movement of Cytospora spp. within and among fruit orchards, 3. Perform pathogenicity assays to determine fruit crop host range of each Cytospora species, and 4. Develop conceptual scenarios to illustrate the importance of early implementation of best management practices to reduce Cytospora canker. The project will be completed in collaboration with the Cytospora Working Group and Tri Area Extension. Developing novel methods could revolutionize the sustainable management for canker pathogens in orchards which are an integral part of the culture and economy.

Project Methods
Objective 1:Survey peach, cherry, and apple orchards in Colorado to estimate Cytospora spp. incidence and severity and collect isolates from each orchard surveyed.Selection of orchards. Dr. Stewart and her lab will work with growers in western Colorado to identify orchards for investigation. We will target 10 each of peach, apple, and cherry orchards grown in Orchard Mesa (Grand Junction, CO, USA) and Rogers Mesa (Hotchkiss, CO, USA). Many growers on the western slope of Colorado typically grow multiple fruit crops, therefore, most of our surveyed orchards will likely be adjacent to other fruit crops. Care will be given to record surrounding areas and adjacent fruits. Surveys will be performed in conjunction with growers and extension personnel.Isolate collections. Isolates will be collected from symptomatic peach, apple and cherry tree samples from orchards in Orchard Mesa (Grand Junction, CO, USA) and Rogers Mesa (Hotchkiss, CO, USA) from the same surveyed orchards. Samples will include branches, twigs and pieces of bark of variable aged trees, including newly planted and well established trees. We currently have 130 C. plurivora isolates from peach orchards from western Colorado in our collections. Therefore, our efforts will target samples from apple and cherry orchards to determine if new Cytospora species, new genotypes of C. plurivora, or C. parasitica are present are in the orchards.Objective 2: Delineate Cytospora species distribution in fruit orchards on the western slope of Colorado using molecular and morphological techniques, elucidate genetic diversity of each Cytospora spp. within and among orchards, and develop an epidemiological model of the spread of the pathogen.Sequencing of housekeeping loci for Cytospora species identification. Single hyphal-tipped isolates will be grown in 250 mL flasks containing 50 mL modified V8 juice medium and placed on a rotary shaker at 100 rpm for 7 days at room temperature. Collected mycelia will then be lyophilized and stored at −20°C. DNA will be extracted from lyophilized mycelia using ZR Fungal/Bacterial DNA MiniPreps (Zymo Research Corporation, Irvine, CA).SNP discovery using sequenced genomes. Single nucleotide polymorphisms will be identified with Illumina sequencing of the ddRADseq libraries for Cytospora spp. isolates collected from growers' fields of peach, apple, and cherry. Libraries will be prepared following the methods of Graham et al. (2015) with slight modifications. A restriction digest of 100ng DNA from each sample will be completed using 1x Cutsmart Buffer (New England Biolabs, Ipswich, MA; NEB) 5.0 mL of dH2O, 0.5ml of each enzyme; XbaI; NEB (20 units), EcoRI-HF; NEB (20 units) and NheI-HF; NEB (10 units), and samples will be incubated for 1 h at 37°C for digestion. Internal barcode adapters will be ligated onto each cut site using 0.75 ATP mM (NEB), 0.25x Ligase Buffer (NEB) and 100 units of DNA Ligase (NEB) and samples will be incubated at 22°C for 20 min, 37°C for 10 min for 2, followed by 22 °C for 20 min to halt enzyme activity. Samples will then be purified with NaCl-PEG diluted 1.2x SpeedBeads and then resuspended with 20 ml of dH2O.Data analyses. For SNP identification, Illumina reads from demultiplexed reads will be mapped to a 100x coverage genome of Cytospora plurivora using BWA (Li & Durbin 2009). Comparisons among isolates will be conducted using STACKS (Catchen etal. 2013), ref map (Catchen etal. 2013) and populations (Catchen etal. 2013) pipelines used to call and analyze SNP data. Additionally, population structure, genetic diversity of populations, and migration of isolates will be estimated using Structure (Pritchard et al. 2000), Poppr implemented in R (Kamvar et al. 2014). These data will help us elucidate the genetic profiles of populations of C. plurivora and other Cytospora spp. found in individual apple, cherry and peach orchards. Migration of genotypes will be estimated among isolates collected from growers' fields within and among host crops. We expect to genotype 150 isolates of each species collected. Species with less than 80 individuals will not be genotyped. We will use C. plurivora as a case study to characterize the movement of Cytospora spp. from other sources besides nearby infected trees, through rain splash.Objective 3: Perform pathogenicity assays to determine host range within fruit crops of each of the identified Cytospora species.Virulence Assays. We will test pathogenicity/virulence of the most prevalent Cytospora spp. collected and identified in Objective 2. Each Cytospora spp. will be inoculated on apple, cherry and peach. For each identified Cytospora spp., 3 genetically distinct isolates will be inoculated onto 2 varieties each of apple, cherry and peach. The experiment will likely include four Cytospora spp. (identified from apple, cherry and peach) * 3 isolates of each species * 5 replicates for each, therefore, we will need 60 branches (20cm) for each cultivar/host type for the inoculations. We will include 5 replicates per each isolate/host type.Analysis of the virulence data will be completed using the lme4, pbkrtest, and lmerTest packages with R statistical software, a linear mixed-effect model will be created and tested using ANOVA (Bates et al., 2015; Halekoh & Højsgaard, 2014; Kuznetsova et al. 2016). This mixed-effects model will account for fixed effects such as species, and for random effects such as isolate and run. Likely, our data will need to be square root transformed to better fit the ANOVA assumption of equal variances. The lsmeans package will be used to return pairwise comparisons among groups with a Tukey adjustment, HSD post-hoc test (Lenth, 2016). For the tests used, a P- value < .05 will be considered significant evidence to reject the null hypothesis that mean differences are equal to 0.Objective 4: Develop conceptual scenarios to illustrate to growers the importance of implementing developed Best Management Practice for reduction of Cytospora canker.For field days and workshops, we will develop a package of slide shows and informational materials that highlight the movement of the pathogen within and across orchards under difference management scenarios. The main focus of this outreach development is to insure wider impact among growers. We will also develop these resources as tools that extension personnel can utilize for general canker management. We will first highlight the documented ways Cytospora spp. can move in orchards (rain splash, air, humans), the importance of Cytospora spp. in other apple and cherry orchards based on results of this proposed work, and how different management strategies could reduce incidence and severity. We will also include cost estimates based on results from a Colorado Specialty Crop funded proposal that was highlighted in the preliminary work.

Progress 01/15/24 to 01/14/25

Outputs
Target Audience:During the reporting period, we shared the knowledge gained through this research with growers, extension agents and with K12 and college students. We presented research findings to growers and extension agents in the peach growing areas of Colorado and Utah. Presentations were given at field days, summer 2024, at the annual pest and disease workshop, and at the Western Colorado Horticultural Conference in 2024. This work is vital to the vitality of peaches and tree fruit grown in high deserts of the west, including Colorado, Idaho, Utah and eastern Washington. The work that we are doing is also important to Extension, researchers, and growers in peach producing areas around the country as well. States in dry areas, like Colorado, are dealing with a high incidence and severity of Cytopsora canker, but all states have Cytospora canker that is difficult to manage. Our work has shown that all tree fruit grown in Colorado have incidences of Cytospora infection, but that species of Cytospora can differ on different hosts. This is important knowledge for tree fruit growers because we found that the main causal agent Cytospora plurivora is pathogenic to cherries as well, and there are also Cytospora species on cherry and apple. Therefore, management should be similar across all tree fruit grown in areas where incidence and severity of Cytospora is high. We also presented these results in classroom setting at K12 and at the college level. For student presentations, we discussed the importance of integrated pest management for farmers producing food. The college students were also involved in setting up mock experiments to understand host specificity in plant pathogens and why this matters for management of pathogens and understanding the importance of Koch's postulates to identify causal agents of disease. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project has funded 1 post-doctoral scholar, 1 master student and 3 undergraduate students. In addition to the research tasks, the post-doctoral scholar has the opportunity to present at a grower meeting and a research conference. The graduate student alsohad experience working with growers and presenting his research findings. The undergraduates had a broad range of experiences, including performing field work (surveys and sample collections), lab work (isolating fungi and performing molecular biology techniques), and presenting to growers. We also partnered with CSU Extension to also provide our undergraduate students with experience in extension as well. How have the results been disseminated to communities of interest?The results have been disseminated primarily through extension presentations and field days. We are also working on several peer reviewed manuscripts and a CSU Extension bulletin that will highlight best management practices for growers in Colorado and other tree fruit growing regions. What do you plan to do during the next reporting period to accomplish the goals?Dissemination to growers and fellow researchers are our top goals for the next period. We plan to continue presenting results at field days and at grower conferences, as new data is analyzed, and results are finalized.We will also finish the manuscripts and the CSU Extension bulletin.

Impacts
What was accomplished under these goals? We surveyed growers' tree fruit orchards in western Coloradoand sampled symptomatic trees to determine if Cytospora canker was present across tree fruit crops, and if so, we determined the Cytospora spp. associated with each host species. We sampled a total of 1,630 trees and collected 428 Cytospora isolates. Of those, we identified 15 Cytospora species associated with 6 tree fruit species (apple, apricot, cherry, nectarine, peach and plum). We then performed pathogenicity tests on the most commonly found Cytospora species, including C. plurivora (on peach), C. amygdali (on apple) and C. sorbicola (on cherry) across peach, apple and cherry. We identified that some Cytospora species are broad host pathogens, whereas others, like C. amygdali was host specific on the host it was collected from. From the C. plurivora isolates we collected, we also examine population genetics to determine if multilocus haplotypes (MLST) are found within or across fields. We observed that MLSTs were shared among fields that were over 100 miles apart, suggesting that C. plurivora is spread via a human mediated mechanism. We are now working on three manuscripts to highlight our survey results that show that peach and cherry have high incidence and severity of Cytospora canker, whereas apple is minimally affected, the genetic diversity of C. plurivora across western Colorado orchards, and results from our cross-pathogenicity trials. These manuscripts will be published prior to the end of the funding period. We have presented our results at multiple growers' meetings, and we are working on developing best-management practices that will be outlined in a CSU Extension Factsheet.

Publications

Progress 01/15/23 to 01/14/24

Outputs
Target Audience:The target audiences include extension personnel, growers, and fellow researchers. This research is important to the extension community because Cytospora canker is an important pathogen not only in orchards, but also in urban and forest trees in Colorado.The results have been disseminated to Colorado State University extension personnel and growers as several recent conferences, including the Western Colorado Horticultural Conference in January 2023 and the Rocky Mountain International Society of Arboriculture Conference in June 2023. We also presented the research to researchers at the Pacific Division of American Phytopathological Society conference in March 2023. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?We have hired an undergraduate Colorado State University extension intern on the project. She, with mentorship by a graduate student, was able to help survey, collect isolates, and train an additional technician. From this work, she gained interest inextension activities and was able to perform onsite visits with our Tri-State extension collaborators. This undergraduate presented her work at a CSU extension conference, and at the Western Colorado Horticultural Conference in 2023. We also hired two post docs to work on the project in 2023. One of the post docs was working on the population genetics of Cytosporaplurivora, speciating the Cytospora species, and performing Cytospora spp. inoculations. The otherpost doc has been analyzing the survey data. The post docs and undergraduate students attended and presented at the Western Colorado Horticulture Conference and the National American Phytopathological Society Conference that was held in Denver. How have the results been disseminated to communities of interest?The results have been disseminated to Colorado State University extension personnel and growers as several recent conferences, including the Western Colorado Horticultural Conference in January 2023 and the Rocky Mountain International Society of Arboriculture Conference in June 2023. We also presented the research to researchers at the Pacific Division of American Phytopathological Society conference in March 2023. What do you plan to do during the next reporting period to accomplish the goals?This next reporting period, we will finish analyzing the population genetics data and will work on getting 2 manuscripts published, and start the 3rd manuscript focused on the population structure of C. plurivora across western Colorado.

Impacts
What was accomplished under these goals? We have been performing surveys and collecting Cytospora and other canker pathogens from symptomatic tissues from apple, cherry,peach, apricot, pear and plumorchards. Over the past 2 years, we have surveyed 26 orchards and 3,497 trees. From the Cytospora collections, we have speciated Cytospora spp. across the different hosts bysequencing the internal transcribed spacer region (ITS) and elongation factor 1 alpha (EF) loci. We have identified 14 Cytospora species in tree fruit in Western Colorado and C. plurivora, the most damaging species on peaches has been found on all the other surveyed hosts. We also performed pathogenicity assays of the 2 main species collected and found that C. plurivora in pathogenic on cherry, peach, and apple, but that apple is the most tolerant. Some of the Cytospora spp. have a broad host range, like C. plurivora, whereas others, like C. sorbicola have a narrow host range just on cherries. We are now performing population genetics across the C. plurivora isolates collected from different orchards and hosts. With the survey data, we have been able to associate Cytospora incidence and severity with orchard management practices, field locations, and host tree age and cultivar. Several manuscripts will be published in 2024.

Publications

Progress 01/15/22 to 01/14/23

Outputs
Target Audience:Our target audience includesgrowers, extension personnel, students, and researchers. We hacseveral grower meeting presentations this past year at the Western Colorado Horticultural Conference and to the Cytospora Working group, which is a group comprised of growers, extension personnel and researchers from Colorado, Idaho and Utah. We also presented some of our work at the American Phytopathogical Society meetings in Pittsburgh last August. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?One post-doctoral scholar has been working on this project. He has learned new skills in population genetics and genomics both in lab skill and analysis skills. Also, 2 undergraduate students have trained with the post-doc and helped with field surveys, isolations, DNA extractions and Sanger sequencing for identifying the Cytospora species. We also have a technician who has helped on the project. Prior to this work, she has little experience in tree fruit so she has learned about fruit tree systems and disease management. How have the results been disseminated to communities of interest?We have given presentations both at conferences and growers groups. What do you plan to do during the next reporting period to accomplish the goals?During this next year, we plan to finish our population genetic studies, pathogenicity assays, and will start to develop the conceptual scenarios to present to growers. We also plan to continue teaching undergraduates molecular biology skills and give them applied disease IPM experiences.

Impacts
What was accomplished under these goals? We have accomplished much of the work for Objective 1 focused on surveys peach, cherry and apple orchards (and additional tree fruit) for incidence and severity of Cytospora spp. We also collected roughly 450 Cytospora isolates and speciated these. We found that multiple species were associated with all fruit crops, but that C. plurivora was the most dominated species found on peach and cherry whereas C. parastica was the most dominate species found on apple. We are currently working on objective 2 and 3. We have extracted the DNA of all Cytospora isolates and are currently running RADseq on these isolates to examine epidemiology. We have also purchased cherry, peach and apple trees to conduct pathogenicity assays of the 10 Cytospora collected across the multiple hosts.

Publications

Progress 01/15/21 to 01/14/22

Outputs
Target Audience:The focus audience for this project is aimed at fruit growers in western states, including Colorado, Utah and Washington. Since the start of the project during this first year, we have met with the Cytospora Working Group. This group was established in 2015 when the Stewart lab begun working on efforts to combat diseases in peaches caused by canker fungi, particularly Cytospora canker or gummosis canker. The group is comprised of peach and cherry growers from Colorado and Utah, extension personnel, and CSU faculty. We have met with the group several times to discuss methods and techniques and to establish connections with growers interested in being involved in the Cytospora canker surveys. We have also presented preliminary results at the Colorado State University Extension conference. We will also be presenting our preliminary results at the Western Colorado Horticultural Conference in 2022. Changes/Problems:Due to COVID, hiring personnel has been difficult. However, there will be post-doc starting on the project March 2022. I have also hired the undergraduate extension intern and a field person to perform surveys and collections and to speciate the cultures. What opportunities for training and professional development has the project provided?We have hired an undergraduate Colorado State Universityextension intern on the project. She, with mentorship by a graduate student, was able to help survey, collect isolates,and train an additional technician. From this work, she gained interestinto extension activities and was able to perform onsite visits with our Tri-State extension collaborators. This undergraduate also presented her work at a CSU extension conference, and will present at the Western Colorado Horticultural Conference in several weeks. How have the results been disseminated to communities of interest?The results have been disseminated to Colorado State University extension personnel via a poster at a recent conference. This research is important to the extension community because Cytospora canker is an important pathogen not only in orchards, but also in urban and forest trees in Colorado. We will also be presenting preliminary results to growers at the upcoming Western Colorado Horticultural Conference in January 2022. What do you plan to do during the next reporting period to accomplish the goals?This next reporting period, we will finish with our surveys, analyze results and report to the growers how canker survey differs across the orchard species. We will also finish with our Cytospora collections, and speciate each culture. Lastly, we will perform genetic analyses of isolates to determine the gene flow across apple, cherry, and peachorchards.

Impacts
What was accomplished under these goals? For the first year of the project, we were able to begin performing surveys and collecting Cytospora and other canker pathogens from apple, cherry, and peach orchards. We have performed surveys on 5 apple, cherry, peach orchards, and have included isolations from canker symptomatic tissues. In addition, species identification of the collected isolates is underway with sequencing of the internal transcribed spacer region (ITS) and elongation factor 1 alpha (EF) loci. These two gene regions together will provide insight into the species found within crop species.

Publications