Source: CLEMSON UNIVERSITY submitted to
SOLUTIONS TO THE ARMILLARIA ROOT ROT THREAT AFFECTING THE U.S. STONE FRUIT INDUSTRY
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
OTHER GRANTS
Reporting Frequency
Annual
Accession No.
1023452
Grant No.
2020-51181-32142
Cumulative Award Amt.
$4,972,730.00
Proposal No.
2020-02650
Multistate No.
(N/A)
Project Start Date
Sep 1, 2020
Project End Date
Aug 31, 2025
Grant Year
2020
Program Code
[SCRI]- Specialty Crop Research Initiative
Recipient Organization
CLEMSON UNIVERSITY
(N/A)
CLEMSON,SC 29634
Performing Department
Plant & Environmental Sciences
Non Technical Summary
Stone fruit and nut crops (peach, almond, and cherry) provide vital contributions to human health, communities, and economies across the U.S. These crops are under serious threat of rapid collapse and eventual extinction due to a devastating fungal threat called Armillaria Root Rot (ARR). ARR presents a major challenge to the U.S. stone fruit and nut industry. Growers with ARR - infected orchards are faced with a persistent problem, as both the host and the disease are long-lived. Producers cannot rotate to other sites due to the lack of un-infected land for new plantings, especially critical in SC peach and MI cherry industries. They cannot fumigate the soil, and once an entire orchard is replanted, they are left with the prospects of having to replant again not long after having invested in the high orchard establishment costs. With this same scenario in peaches in the Southeast, cherries in Michigan, and peach, cherry, and almond in California, ARR is a threat of national importance. Without practices to eradicate the pathogens or methods to counteract reduced productivity, infected sites are unsuitable for replanting. The pathogen is long-lived, with inoculum that can persist below ground for many years. As such, leaving the ground fallow before replanting would require up to a decade and have an unacceptable economic burden on producers. Furthermore, planting another horticultural crop other than a Prunus species is not an economically viable option, given the broad host range of ARR (500+ woody plant species). Without solutions, this loss of production will not only have devastating impacts on the affected producers and rural communities but will also contribute to a reduction in the supply of these products and potentially increased costs for consumers.U.S. stakeholders and industries that span these crops have given ARR the highest priority for the development of short and long-term solutions to preserving these critical crops and industries. The most sustainable and effective approach to mitigate the ARR threat to peach, cherry, and almond production in the U.S. is to develop genetically durable ARR-resistant rootstocks. While long-term solutions are required, in the interim, short- and medium-termed solutions are necessary, which is why this project encompasses both. This project will identify resistant germplasm, characterize the mode of resistance and understand the genetic base of the resistance by linking the genome with phenome through systems-scale datasets, such as transcriptomics, metabolomics, etc., along with sophisticated statistical methods, to enable genomic solutions and translational breeding of natural (local and exotic) sources of ARR resistance to deliver ARR resistant rootstocks in the longer term. The industry will perceive an immediate benefit with the change in cultural practice and direct utilization of new sources of resistance in nurseries. Above ground root collar excavation cultural practice (AGRCE), that extends the lifespan of orchards on ARR infected sites, and nursery and producer evaluation of horticultural acceptance of the resistant material, provide short- and medium-term solutions, respectively, as the work on genetic resistance progresses. Cultural practices have the potential to reduce the financial burden of ARR to producers by extending the long-term productivity of Prunus orchards, therefore this project will implement AGRCE for peach and cherry and explore the use of sectoral chimera rootstocks in almond as a strategy to increase tree longevity until ARR resistant compatible rootstocks are developed. Socio-economic research will determine economic effects of ARR and impact of proposed solutions to provide guidelines for growers, while outreach and extension activities will target peach, cherry, and almond growers affected by ARR and tree-fruit nurseries, as the key partners in delivering genetic solutions to producers, so that new knowledge can be quickly disseminated and feedback obtained. In this way, a range of consumers will benefit from sustainable stone fruit and nut production, which would increase consumption and improve human health and physical well-being.
Animal Health Component
40%
Research Effort Categories
Basic
30%
Applied
40%
Developmental
30%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2011119108040%
2011119108125%
2051119116015%
6011119301020%
Knowledge Area
601 - Economics of Agricultural Production and Farm Management; 205 - Plant Management Systems; 201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1119 - Deciduous tree fruits, general/other;

Field Of Science
1160 - Pathology; 1081 - Breeding; 1080 - Genetics; 3010 - Economics;
Goals / Objectives
The long-term goal of the project is to improve the profitability and sustainability of the U.S. stone fruit and nut industries through the use of Armillaria Root Rot - resistant and horticulturally acceptable rootstocks. This proposal will provide the 'building blocks' needed to enable and accelerate on-going Prunus breeding programs, as well as support the testing of cultural practices as short-term solutions to increase tree longevity on replant sites. These goals will be achieved through the following objectives:Identify, characterize, and utilize sources of resistance to Armillaria/DesarmillariaDiscover genomics to enable breeding solutions for ARR resistance/toleranceImplement short- and long-term system approaches to manage ARRDetermine economic impact of ARR and cost-benefit of adopting cultural practicesConduct outreach activities to facilitate the adoption of results
Project Methods
Response to Armillaria/Desarmillaria infection in Prunus germplasm, will be evaluated using in vitro method of Baumgartner et al. (2018) followed by co-culture in liquid medium-infused Oasis® IVE phenolic resin for germplasm with resistant response confirmed by root micorscopy. Anatomical and biochemical responses of root tissue to infection will be characterized using freshly excavated and surface sterilized roots (6 cm length × 0.5 - 1.5 cm diameter) of resistant germplasm using Devkota and Hammerschmidt (2019) protocol. Sterile roots (3-cm) sealed with paraffin at both ends and placed on the top of a 14-d culture of Armillaria on 1.5 % malt extract agar (MEA), incubated at 23 ºC in the dark. After 21 days, fungal penetration and the extent of colonization will be assessed. Tissue (10-mm thick) surrounding the inoculation point will be cut and fixed in formalin-acetic alcohol (FAA), and after 48 hours processed with an alcohol:xylene series, embedded in melted paraffin wax (54-55 ºC) overnight, cut with a rotary microtome in 6 µm thick sections, stained with 0.5 % aqueous Toluidine blue and visualized under light microscope. Biochemical response will be evaluated by incorporating ground periderm of resistant germplasm into MEA at a 1 % concentration (1 g/100 L). A 5 mm plug of fungus will be placed at the center of the plate and incubated at 23 ºC. In 21 days, the relative growth of fungi on each media type will be measured. Accessions that exhibit activity against Armillaria spp. will be tested using two different bioassays: 1) periderm tissues will be extracted overnight in 80 % acetone (1 ml per 0.1 g of dried periderm), and 10 µl of each extract will be spotted onto silica gel thin layer chromatography (TLC) plates, following evaporation of the solvent, the plate will be sprayed with a dense spore suspension of 7-day-old Cladosporium cucumerinum; 2) 4 g of dried Prunus periderm will be placed into 200 ml of 80 % acetone, extracted overnight at room temperature, filtered and re-extracted 2X with 100 % acetone and lyophilized to dryness, separated by a thin layer of chromatography in Silica gel and observed under UV light (365 nm). Putative antifungal compounds will be semi-purified and analyzed by the CycloGraphTM and preparative TLC29. Unknown compounds will be structurally characterized by liquid chromatography-quadrupole-time of flight mass spectrometry analysis following standard protocols and/or with two-dimensional nuclear magnetic resonance spectroscopy (2D NMR). De novo genome sequencing and a haplotype-phased, chromosomal-scale assembly for each resistant accession will be constructed using 100X coverage obtained from the Pacific Biosciences Sequel II instrument and the MECAT3 algorithm and/or FALCON-Phase approach; error polished with 2 rounds of Arrow and 1 round with 100X Illumina and Pilon; base analyzed for single-copy orthologs with BUSCO and primary assembly created with HaploMerger2; aligned and scaffolded with BioNano maps and the RefAligner tool. Chromosome-scale references will be created with deep-coverage HiC; final curation for gap filling and polishing with PBjelly. RNAseq from various tissues over developmental stages (root, meristem, flower, and leaf) will be used as evidence for annotation by GenSAS40 coupled with InterproScan, BLAST(P/X), SignalP/TargetP, GO, KEGG, and DIAMOND. For transcriptome analyses: the total RNA will be purified from the root samples using the Ambion PureLink mini kit following the manufacturer's recommendation, qualitatively analyzed using an Agilent Bioanalyzer 2100, stranded mRNA sequencing libraries prepared using standard TruSeq kits and sequenced to a depth of at least 40M reads (2x150bp PE) per replicate sample using the latest chemistry release on an Illumina NovaSeq. Raw sequence reads will be preprocessed for adapters and low-quality bases with the Trimmomati, aligned to the respective reference genome assembly with the latest version of the HiSat2; short read and transcript quantification determined with the latest version RSEM with the HiSAT2. Pairwise comparisons of gene expression over infection time course within candidate genotypes will be conducted using EdgeR differential analysis tool. Data will be analyzed using a variety of approaches including Gene Set Enrichment Analysis (GSEA), pairwise comparisons, GO enrichment, mapping to KEGG pathways, and hierarchical /k-means clustering. Root metabolites will be extracted using methanol and partitioned to polar and non-polar metabolites using chloroform partitioning (Suseela et al. 2015). The suberin content of the roots will be characterized following base hydrolysis of the methanol extracted tissue (Suseela et al. 2016). Targeted and non-targeted metabolomic studies will be used to detect and quantify the major metabolites and stable intermediaries that originate from the major biosynthetic pathways. General screening of the samples will be done using GC-MS and GC-MS/MS; then liquid chromatography mass spectrometry with electrospray ionization interface (both positive and negative ionization mode) will be used for the targeted analysis of polar metabolites. The polar metabolites will be separated using HILIC, and less-polar compounds will be separated via C18-reverse phase chromatography. Following LC separation, the compounds will be analyzed using a quadruple-ion trap orbitrap platform coupled to an ESI in both positive and negative ionization modes. Resistant germplasm will be included in rootstock trials or trials on peach tree short life (PTSL) and ARR sites to evaluate its potential as a rootstock for peach, almond and cherry. Newly discovered resistant material will be included directly as a rootstock and or interstem to test their horticultural performance in a nursery setting and in the field. Binary, sectoral-rootstocks will be propagated through approach-grafting at the seedling, semi-hardwood and/or hardwood stages and planted in the nursery and on ARR sites in MI and SC. Resistant germplasm will be combined with peach standard rootstock Guardian® and cherry standard rootstock P. mahaleb. Root Collar Excavation (RCE) efficiency will be evaluated on established field trials in SC and GA for peach and MI for cherry on ARR-infested replant sites, with RCE and grower standard (GS) treatments arranged in a complete block randomized trial with three or more replicates per trial. Soil removal will be performed in the second year after planting using newly developed mechanical prototype. An industry-wide survey of the effects of ARR on affected regions will be obtained from a survey instrument designed and pretested by the economics team and administered to stone fruit farm managers through USDA NASS with the target sample of 1,200 surveys sent equally across the four states. The questionnaire will include sections related to: i) demographic characteristics, and risk tolerance levels of the producer; ii) agronomic characteristics of the field; iii) orchard operation characteristics; iv) the impact of ARR on production; iv) current management practices to control ARR; and v) an evaluation of producer preferences and willingness to pay for a newly developed rootstock and alternative methods to eliminate or mitigate the impacts of ARR. Results will be shared with growers at field days, regional and annual producer meetings, personal communication through county agents and our outreach collaborators, publication in scientific and trade journals, extension bulletins and on the project website (https://www.clemson.edu/cafls/arr-solutions/), the University of Georgia Peach website (https://blog.extension.uga.edu/peaches/), individual cultivars web pages (http://www.clemsonpeach.org), the National Peach Council web page (www.nationalpeachcouncil.org/), almond board (www.almonds.com) and cherry Marketing Institute (http://www.choosecherries.com/) websites.

Progress 09/01/23 to 08/31/24

Outputs
Target Audience:Target audiences reached during this period are almond, cherry and peach growers in CA, MI and SC and GA, respectively, via virtual and in person (when possible) extension and outreach. Fruit Tree nurseries and commodity boards were also included in this years effort to share the accomplishments generated by the project. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Undergraduate students were trained in the in vitro screening pipeline, field experiment set up and rootstock tolerance evaluation. How have the results been disseminated to communities of interest?Results of the project have been disseminated via peer reviewed publications, regional and national scientific conferences, growers or commodity boards meetings. All activities and accomplishments are regularly updated on the project website. What do you plan to do during the next reporting period to accomplish the goals?Obj. 1. Cherry pollen has been collected and stored for plum x cherry crosses that will be conducted in 2025 spring. Additional crosses utilizing pollination tents will be performed between peach and plum and generated hybrids evaluated for ARR tolerance. Obj. 2. Genes detected in transcriptome and metabolome analyses of ARR fungi and ARR tolerant and susceptible plants will be used to develop molecular testing to determine the tissue specificity of their expression and to test germplasm from Prunus National Clonal Germplasm Repository in Davis, CA for potential sources of ARR tolerance that escaped our in vitro testing conducted in year 1 of the project. These qPCR gene expression tests will be used also on all plum germplasm available in germplasm collections of the project participants, and newly released stone fruit rootstocks avaialbe for nurseries in the Unites States and those housed at our Advisory Board members form Spain and Chile. In addition, USDA rootstock breeding program at Byron GA will also screen germplasm using newly developed molecular tools. Obj. 3. Continue evaluating field performance and survival of experimental field at the PTSL and ARR infested sites, and rate for bacterial canker incidence. Evaluate horticultural performance and effect on scion of newly identified ARR tolerant rootstock candidates grafted with peach. Complete field testing of the sensors and functions of new debermer on artificial berms and research plots. Obj. 4. Continue with the investigating ARR related tree death using field images and confirming ARR as the death cause. Develop economic impact of ARR on peach industry and simulatingcost effects of applying cultural practices developed by the project. Obj. 5. Continue to disseminate information regarding the rootstock development to the target audience as well as publish the results in peer reviewed journals.

Impacts
What was accomplished under these goals? Obj. 1. Wild Prunus seeds collected in MI, germinated in vitro and tested for ARR tolerance. None showed tolerance in vitro. Cherry x plum cross conducted in spring 2024 resulted in no hybrid seedlings. Initial hybridization showed more than 200 fruitlets, but all of them dropped by the end of May. Pollination tent was used for peach x plum crosses, resulting in 3 seed, and bees were used as pollinators in plum x peach crosses, resulting in 27 seeds. Seeds from 2024 crossing season have completed the stratification and are germinating in vitro. The interspecific origin will be confirmed using molecular markers before the seedlings are included in the in vitro testing for tolerance. Sixteenhybrids from '21 and '22 crosses with detected ARR resistance profileshave been multiplied in vitro. Out of 16, 3 putatively resistant hybrids have passed the second stage IVE screen and additional 4 hybrids are undergoing second stage IVE testing. Clonal propagation (>100) performed for four putatively resistant hybrids screened for co-culture. Characterizing tolerant responses Chitinase and peroxidase assays were conducted to understand the presence of these enzymes in defense. Chitinase and peroxidase activity was elevated in tissue treated with chitin fragments and Armillaria solidipes. The results correlate with our prior findings on anatomical defenses as they may aid in the formation of structural polymers. Various flavonoids such as naringenin, taxifolin, and chrysin were identified as the antifungal metabolites from Prunus maackii and they were found to inhibit the growth of Armillaria solidipes as compared to the controls. Obj. 2. Transcriptome and metabolome profiling of Armillaria fungi, A. melea, and Desarmillaria caespitosa, revealed differential response to the hosts with different tolerance levels. Transcriptome data revealed transcripts encoding for extracellular cell wall degrading enzymes (CWDEs) and for fungal effector genes. Both fungi exhibited time and host dependent gene expression. Similarly, both fungi exhibit significant metabolic reprogramming early in the infection process with the most affected chemical classes including amino acids, organic acids, and carbohydrates. Comparative metabolic analyses suggested that A. mellea employs a sustained metabolic response, including oxidative stress management and biosynthesis of secondary metabolites, while D. caespitosa takes a more direct approach, focusing on rapid tissue breakdown and nutrient acquisition through catabolic pathways. Obj. 3. Survival of new rootstocks and selections wereevaluated at the three field screening trials established in 2021 on Armillaria and PTSL replant sites at the Sandhill Research and Education Center in Pontiac, South Carolina and at Titan Farms (2 trials) in Ridge Spring, South Carolina. One of the Ridge Spring trials included new rootstocks from private breeding programs in California. Third year survival was excellent at Titan Farms (only one dead) for the plum selections, but some California rootstocks had high mortality except for 'Krymsk® 86'. A new ARR rootstock screening trial was established near Johnston, SC in 2024. At the Sandhill REC, tree death was mostly several weak trees succumbing from the initial year establishment problems, and tree growth was much better in 2023. In addition, at Titan Farms plum genotypes (used as rootstocks) that survived 10 years on a severe ARR and PTSL orchard site were successfully propagated by budding for future testing and breeding. Trees are being monitored to assess horticultural performance and mortality associated with Armillaria root rot. Peach trees on 'Krymsk® 86'and 'MP-29' rootstocks planted in west central Michigan in spring 2023 do not show ARR related mortality. A few trees were dead, but tree death was not related to Armillaria root rot. Peaches on 'MP-29' rootstock have reduced growth compared to peaches on 'Krymsk® 86'rootstocks. Cherry trees grafted via interstem 'Adara' on 'MP-29' were dissected at graft union to understand the cause of tree death. Formation of necrotic layer was detected at graft interface, incomplete vascular connections and disorganized vascular tissue suggests graft incompatibility. Evaluation of root collar excavation experimental orchards showed no signs of winter injury in the root and root collar area in MI. From July 2022 to May 2024 about 30% of the Montmorency/Mahaleb trees under grower's standard planting and 35% of the trees planted on the unexcavated berm were dead due to ARR. Only 1% of the trees planted on berm with root-collar excavated died due to ARR. A new in-row orchard weeder was purchased and modified to make it suitable for deberming. A LiDAR-based sensing system was developed to detect tree trunks within tree row. This system is designed to control the position of the paddle wheel, moving it in and out of the row based on the proximity to the trees. After satisfactory laboratory testing, the system has been installed on the new in-row weeder, and field trials are currently underway. Another sensing system was developed using infrared sensor to perform the function of detecting tree trunks. Laboratory tests have been completed, and this system has also been installed on the in-row weeder. Field data collection is also in progress. The profitability analysis of berm removal machines for peach growers has been completed. The evaluation considered various scenarios, including different peach prices, yields, and levels of ARR disease pressure. Obj. 4. ARR infection foci expansion in peach orchards using the United States Department of Agriculture (USDA)'s National Agriculture Imagery Program (NAIP) aerial imagery data were identified. Monte Carlo analysis was used to estimate peach producer profit under different scenarios with and without disease spread. The maximum amount a profit-maximizing producer would pay for preventative measures based on differences in simulated profits under alternate scenarios were estimated. The focus area for this work was Titan Farms. Specifically, we selected 20 orchards for the analysis. The findings for part I were verified both by the farm Manager and from experts. Obj. 5. Results from the studies were presented to growers, stakeholders, and other scientists at regional and national meetings. Production of a 4-minute animated video documentary on root collar excavation. The video is in the last stages of production at the time this report is being written and will be available on the project website and YouTube shortly. Round table discussion conducted with peach growers in the southeastern U.S. (Savannah, GA. Jan. 12, 2024). Outreach information updated in MyIPM app. Project was presented at regional and national producer, stakeholder, and Extension meetings, including state producer meetings (Lyman, SC. Jan. 26, 2024; Edgefield, SC. Feb. 8, 2024), as well as national and international scientific meetings such as the National Association of Plant Breeders Conference, Southern Region - ASHS Annual Conference, American Society for Horticultural Science Annual Conference, American Phytopathology Plant Health Annual Conference, and the International Plant Protection Congress, among others. Project website was updated with the current information.

Publications

  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Reighard, G.L., L. Cai, A. Calle, J. Adelberg, C. Saski and K. Gasic.. 2024. Prunus rootstock breeding, gene discovery and field testing for Armillaria root rot resistance in South Carolina. Vth European Horticulture Congress Online abstract. May 16, 2024.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Reighard, G.L., D. Ouellette and I. Minas. 2023. Field testing Prunus rootstocks in South Carolina for peach tree short life and Armillaria tolerance. XXXI ISHS HortCongress, Angers, France. Acta Hortic. 1366:69-74. https://doi.org/10.17660/ActaHortic.2023.1366.8
  • Type: Conference Papers and Presentations Status: Other Year Published: 2024 Citation: Singh, S., A.Bulent Koc, Michael Vassalos, Guido Schnabel, Juan Carlos Melgar. 2024. Development and Economics of a LiDAR and Inductive Proximity Sensor-Based System for Intra-row Berm Removal in Peach Orchards. Poster, presented at the American Society of Agricultural and Biological Engineers (ASABE) Annual International Meeting. July 28-31 Anaheim CA.
  • Type: Theses/Dissertations Status: Published Year Published: 2024 Citation: Jared Weaver (2024) Transcriptomic Profile of Armillaria Root Rot Fungi Affecting the U.S. Stone Fruit Industry
  • Type: Other Status: Published Year Published: 2024 Citation: Alejandro Calle, Jeffrey Adelberg, Guido Schnabel, Jacqueline Naylor-Adelberg, Jhulia Gelain, Yeter Karakoc, Jared Weaver, Christopher Saski, Ksenija Gasic 2024. In vitro co-culture system using a fiber-supported liquid approach. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzj56xlx1/v1
  • Type: Journal Articles Status: Awaiting Publication Year Published: 2024 Citation: Calle, A., Adelberg, J., Schnabel, G., Naylor-Adelberg, J., Gelain, J., Karakoc, J., Weaver, J., Saski, C. and K. Gasic 2024.In vitro co-culture system for investigating Armillaria root rot in Prunus spp. using a fiber-supported liquid approach. PLOS ONE
  • Type: Conference Papers and Presentations Status: Other Year Published: 2024 Citation: Schnabel, G. and B. Koc 07/2024. Cultural management of Armillaria root rot in peach orchards. IPPC, Athens, Greece.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2024 Citation: Schnabel, G. 02/2024. Peach Disease Management. Peach Production Meeting, Clemson Extension, Edgefield SC.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2024 Citation: Schnabel, G. 01/2024 Peach Disease Control Update. 2024 Upstate peach grower production meeting, Lyman SC
  • Type: Conference Papers and Presentations Status: Other Year Published: 2023 Citation: Schnabel, G. 09/2023. Introduction to Disease Management for Fruit and Vegetable Crops. IPM systems workshop for beginning farmers. Zoom Presentation
  • Type: Conference Papers and Presentations Status: Other Year Published: 2024 Citation: Reighard, G.L., L. Cai, A. Calle, J. Adelberg, C. Saski and K. Gasic.. 2024. Prunus rootstock breeding, gene discovery and field testing for Armillaria root rot resistance in South Carolina. Vth European Horticulture Congress. Bucharest, Romania. May 12-16, 2024.
  • Type: Other Status: Other Year Published: 2024 Citation: Reighard, G.L. 2024. Peach rootstocks and Peach Tree Short Life and Desarmillaria caespitosa. Lecture for University of Bologna fruit course. Bologna, Italy, May 22, 2024.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2023 Citation: Devkota P. Michigan cherry committee meeting, Grand Rapids, MI, December 4, 2023. Update on Armillaria root rot research in Michigan.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2024 Citation: Devkota P. Mycological society of America, Markham, Ontario, Canada, June 9-12, 2024 Towards understanding virulence factors in Armillaria solidipes
  • Type: Conference Papers and Presentations Status: Other Year Published: 2024 Citation: Devkota P. American Phytopathological Society Annual Meeting, Plant Health 2024, Memphis, Tennessee, July 27-30, 2024 Unveiling the battle at the bark: Exploring Prunus periderm defense strategies against Armillaria


Progress 09/01/22 to 08/31/23

Outputs
Target Audience:Target audiences reached during this period are almond, cherry and peach growers in CA, MI and SC and GA, respectively, via virtual and in person (when possible) extension and outreach. Fruit Tree nurseries and commodity boards were also included in this years effort to share the accomplishments generated by the project. Changes/Problems:Obj1: Obtaining cherry × plum hybrid seed and then viable seedlings from the putatively hybrid seed. Seed germination rates are below optimal and attempts at culturing shoot buds through tissue culture result in limited success. In 2023 strategies were put into place to do the cross in the reciprocal direction and attempt embryo culture. Peach × plum crosses performed at Clemson were not successful due to late spring frost damage. These crosses will be continued in 2024 and beyond. Inoculation assays attempted using explants with the different growing rate may bias the results. Modified inoculation assay is being developed for screening individual rootstocks to accommodate for differential growth rates. Establishing wild cherry in vitro cultures from dormant budwood of accessions collected in MI was not successful due to the poor bud growth and contamination. We will repeat the attempt with actively growing shoots as explants. Postdoc A. Calle left the program in June to start permanent breeding position. Temporary replacement for resistance screening was secured until new postdoc is hired to start in January 2024. ARR inoculation was conducted on live plant roots in the growth chamber and proved unsuccessful due to challenges related to inoculum desiccation. Obj 2: Alternate defense responses seem to exist based on the pathogen species. A likely scenario is that the pathogen excretes different effectors that trigger plant defense in different ways. One species forms rhizomorphs while another does not which corroborates this observation. To resolve this challenge, a study is ongoing to detect fungal species-specific effectors. MP-29 is a peach × plum hybrid whose genome is comprised of multiple plum progenitor species that are distantly related to cultivated Prunus germplasm which results in a significant number of uncharacterized metabolite profiles detected. Obj 3:PTSL experimental planting at the Sandhill REC experienced a series of unfortunate events with recent herbicide drift from the nearby crop fields. The extent of the herbicide drift damage and non-PTSL induced stress will be monitored and decision made if the results from this experiment can be used. In case the PTSL study was jeopardized we will establish future experiment(s) on the grower land. The RCE using air spade is difficult and has not been implemented by GA growers as it is currently proposed. Obj. 4: Despite our best efforts (working with extension agents, attending many producer conferences, etc.) the data collection from the surveys is relatively slow. A potential reason for that is many producers (especially for almonds) currently do not face ARR problems thus they may not see a benefit in completing the questionnaires. We will continue working with the extension agents to collect data. We will also utilize Qualtrics to supplement our survey responses. What opportunities for training and professional development has the project provided?Postdoc, graduate and undergraduate students were trained in the in vitro screening pipeline, microscopy, field experiment set up and rootstock tolerance evaluation. Postdoc and graduate student were also trained in the transcriptome data analyses. How have the results been disseminated to communities of interest?Results of the project have been disseminated via peer reviewed publications, regional and national scientific conferences, growers or commodity boards meetings. All activities and accomplishments are regularly updated on the project website as well. More is described under objective 5 accomplishments. What do you plan to do during the next reporting period to accomplish the goals?Obj 1. Y4 plans:Generate plum × cherry hybrids and attempt embryo rescue to breed for Armillaria tolerance and graft compatibility with cherry. Screen more Prunus genotypes in the screening pipeline to understand their susceptibility to Armillaria mellea, A. solidipes and Desarmillaria caespitosa. Test the consistency of the modified inoculation assay. Explore possibility of ARR screening approach using whole live plant growing in soil under controlled conditions in growth chamber to better understand the fungal defense response. Obj 2. Y4 Plans: Finalize genome assemblies for MP-29 and Guardian®, annotate genes and determine function. Collect additional transcriptomic data (2b) paired with metabolomic data from 2c to focus on biochemical pathways and genes driving ARR resistance. Collect fungal transcriptomes during infection in A. mellea and D. caespitosa to annotate fungal effectors and signaling during infection of resistant and susceptible germplasm. Translate current metabolomic results into first version candidate biomarkers for testing and validation. Integrate the metabolomic profiles with gene expression profiles collected in Y3 to establish a working integrated disease resistance mechanism in MP-29. Obj 3. Y4 Plans: Evaluate early ARR mortality in the three field screening trials of new rootstocks and selections and rate for bacterial canker resistance (i.e., PTSL). Evaluate new selected genotypes to determine their compatibility with peach for future orchard trials on PTSL and ARR sites. Continue to monitor Armillaria tolerance and horticultural properties of peach and sweet cherry scions on 'MP-29' and 'Krymsk® 86' in MI. Establish experimental fields on ARR/PTSL sites with 'Augustprince' grafted on the four ARR putatively tolerant rootstocks (14-4, 20-3, 20-4 and 59-1). Continue with deployment of the levee plow for planting in berms across the GA peach production area and with data collection in ARR demonstration trial at Southern Orchards. Three different treatments are being tested: 1) non-bedded, 2) bedded without collar soil removal, and 3) bedded with collar soil removal (done 2020). Identify new demonstration plots across the Georgia peach producing area. Evaluate the effectiveness of the de-bermer and brush mechanism in diverse soil conditions, and assess the economic effect of using the de-bermer. Obj 4. Y4Plans: Analyze data from the survey(s), completion of the simulation model to evaluate the economic impact of RCE. Continue collecting survey data and updating the simulation models based on that information and updated budgets. Specifically for peaches we are going to use Google Earth data to have a more accurate economic impact based on our simulations. Continue working with producers to establish the benefits of RCE techniques. Obj 5. Y4 Plans: Continue producing video documentaries and newsletters; collect grower feedback at regional and roundtable meetings; present accomplishments at the regional growers' meetings; organize field demonstration days; obtain grower feedback through discussions at stakeholder meetings.

Impacts
What was accomplished under these goals? Obj. 1: Six new interspecific hybrids, with stage 1 putative resistance micropropagated on one common medium that facilitates rapid multiplication, are in the 2nd round of in vitro testing using phenolic foam to confirm the putative resistance. Out of 79 tested, four new tolerant genotypes, with confirmed no fungal penetration (2201G-6, -49, -59 and -83) and one P. pumila obtained. Plum × peach crosses, 69 putative hybrids are maintained on rooting media and await first stage screening. Matured embryo seeds from four fruits of plum × cherry crosses established in tissue culture, one went immediately into multiplication, the other three are in cold stratification. Anatomical defensive mechanisms in putative Armillaria tolerant accessions 14-4, 59-1, 20-4, and 20-3. showed the root periderm in all four exhibited the formation of a barrier zone, effectively deterring fungal invasion. No signs of fungal damage were detected beyond this zone for a duration of 21 days after Armillaria inoculation. Obj. 2: Reference-grade, haplotype-resolved genome sequence assembly of MP-29 and Guardian® and Illumina whole-genome sequencing of 6 Prunus species were obtained. A list of 10 candidate Armillaria resistance genes, such as Glutathione S-transferase, calcium activated genes that coordinate an early defense response, genes involved in chitin degradation, and genes involved in biofortification, detected in the MP-29 rootstock. Development of a first-generation ARR molecular diagnostic test(s) is in progress. Different responses at the genetic level (gene expression) to A. mellea and D. caespitosa observed in MP-29 and 14-4 accession, with tolerance of both accessions to both fungi observed, and MP-29 showing higher tolerance to A. mellea, while 14-4 to D. caespitosa. The defense/immune response happens within the first 72 hours after infection and persists at least for 8 weeks. A co-culture slurry system was developed to generate tissue for metabolomic studies that provides a more uniform inoculum interaction with roots in a liquid medium. Baseline metabolic profiles were obtained for the three fungi: A. mellea, A. ostoyae and D. caespitosa. Obj. 3: Three field screening trials of new rootstocks and selections, established in 2021 on ARR and Peach Tree Short Life (PTSL) replant sites were evaluated for survival. Second year survival was excellent at Titan Farms. At Sandhill REC, tree death was mostly due to the initial year establishment problems. Tree browsing was noticeable on the Titan Farms site. Plum genotypes (used as rootstocks) that survived 10 years on a severe ARR and PTSL orchard site were propagated by budding for future testing and breeding. Peach trees on K-86 and MP-29 planted in west central MI in spring 2022 are doing well, assessment of horticultural performance and mortality associated with ARR is ongoing. No instances of ARR related mortality were observed in RCE trees during the first year after excavation. Cumberland Valley Nursery (CVN) grafted clones of four ARR putatively tolerant selections: 14-4, 20-3, 20-4 and 59-1 with 'Augustprince' and evaluated nursery related horticultural characteristics. Success in grafting ranged from 42-45% for all except 59-1, that had 24%. Field-evaluations in a high ARR infection site established in CA to assess the level of resistance/tolerance for candidate species and species-graft combinations using traditional approach-grafted sectoral rootstocks were performed. Some of the controls at this plot have become infected with ARR though no disease is currently apparent in the shoot growth of sectoral rootstocks with some of the remaining susceptible standards also remaining free of disease. Methods for high-throughput grafting to develop sectoral rootstocks are being developed using micro-grafting at the seedling stage with the most promising results to date using breathable surgical tape to constrict seedling collar expansion leading to interspecies rootstock fusion. A rapid seed germination method allowing seedling development without the need for multi-month stratification (developed in Chile and modified at CU for peach) has been further modified to allow non-stratified germination for related species including P. cerasifera and P. davidiana. A key modification was nighttime exposure to temperatures below 20 °C to prevent seedling rosetting. Field trials at grower locations and at CU research station were maintained and data was collected. Trees in the bermed and RCE treatments continue to decline at a slower pace compared to the grower standard. Approximately 1000 acres of peaches were planted in 2023 in berms across Georgia at five different locations. At each farm RCE is being performed to set-up demonstration of excavated rows versus non-excavated rows. Soil excavation equipment (de-bermer) has been improved with addition of tree trunk sensing mechanism, the feeler rod-based proximity sensor, and a solenoid controlled directional valve with a relay. The functionalities of the added components were tested in laboratory conditions and the de-bermer with the installed sensing mechanism was demonstrated to the TitanFarms farm crew where its functionality was confirmed. Obj. 4: Completed the analysis of the peach survey data (55 telephone surveys organized by Qualtrics) using appropriate statistical techniques. During this process, we realized that a yield curve for peaches was missing. An Applied Economics and Statistics Master student (Gracie Herrin) worked closely with Chalmers Carr (Titan Farms) to create a yield curve. Completed simulations for the potential impact of ARR on peaches, cherries, and almonds. Obj. 5: Production of a 4.5-minute animated video documentary on Armillaria root rot. The video is available on YouTube (https://www.youtube.com/watch?v=5I5efeIu0po) and collected 725 views in the first two months after being published. Newsletter was shared with stakeholders, Extension specialists and researchers in March 2023. Project was presented at regional and national grower, stakeholder, and scientific meetings, including state grower meetings, SE Regional Fruit & Vegetable Conference, National Association of Plant Breeders, American Society for Horticultural Science Annual Conference, and APS Plant Health Annual Conference, among others. Outreach information and pictures of ARR of cherry were included in MyIPM app. Project website (https://blogs.clemson.edu/arrsolutions/) was updated with the current information. Michigan cherry growers, Michigan Tree Fruit Commission, other cherry stakeholders, and scientists were informed about the use of ARR tolerant rootstocks in Michigan and methods and challenges of RCE study conducted in Michigan.

Publications

  • Type: Conference Papers and Presentations Status: Other Year Published: 2022 Citation: P. Devkota & R. Hammerschmidt (2022). Identification of virulence determinants of Armillaria solidipes. In Phytopathology (vol. 112, no. 11, pp. 197-197). 3340 Pilot Knob Road, St Paul, MN 55121 USA: Amer Phytopathological Soc. Abstract submitted
  • Type: Journal Articles Status: Awaiting Publication Year Published: 2023 Citation: P. Devkota, A. Iezzoni, K. Gasic, G. Reighard, and R. Hammerschmidt. Rapid In Vitro Screening of Prunus Genotypes for Resistance to Armillaria Root Rot Using Roots of Young Rootstocks (HortScience 2023, Accepted)
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Cai, L., Adelberg, J. W., Naylor-Adelberg, J., Schnabel, G., Calle, A., Li, Z., Reighard, G. L., Gasic, K. and Saski, C. A. (2023). Transcriptomics reveal the genetic coordination of early defense to Armillaria root rot (ARR) in Prunus spp. Frontiers in Plant Science, 14. https://www.frontiersin.org/articles/10.3389/fpls.2023.1181153
  • Type: Conference Papers and Presentations Status: Other Year Published: 2022 Citation: P. Devkota, A. Iezzoni, & R. Hammerschmidt. Rapid in vitro method utilizing roots of Prunus species seedlings to screen against Armillaria root rot. In Phytopathology (Vol. 112, No. 11, pp. 147-147). 3340 Pilot Knob Road, St Paul, MN 55121 USA: Amer Phytopathological Soc. Abstract submitted.
  • Type: Conference Papers and Presentations Status: Awaiting Publication Year Published: 2023 Citation: Reighard, G.L., D. Ouellette and I. Minas. 2023. Field Testing Prunus Rootstocks in South Carolina for Peach Tree Short Life and Armillaria Tolerance. XXXI ISHS HortCongress, Angers, France. Acta Hort (in press).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Gasic, K., J. Adelberg, K. Baumgartner, P. Brannen, L. Cai, A. Calle, D. Chavez, P. Devkota, T. Gradziel, R. Hammerschmidt, A. Iezzoni, B. Koc, T. Mark, J.C. Melgar, G. Reighard, C. Saski, G. Schnabel, N. Tharayil, and M. Vassalos. 2022. Solutions to the Armillaria Root Rot affecting the U.S. stone fruit industry. Acta Hort. 1352:501-507. https://doi.org10.17660/ActaHortic.2022.1352.68
  • Type: Conference Papers and Presentations Status: Other Year Published: 2023 Citation: Calle, A.C., Adelberg, J., Schnabel G., Naylor-Adleberg, J., Saski, C., Gelain, J., Karaloc, Y., Weaver, J., Cai, L., Gasic, K. An In Vitro Co-Culture System for Armillaria/Desarmillaria and Prunus Species Using a Fiber-Supported Liquid Approach. 2023 Annual ASHS conference, Orlando, FL, United States. (August 1, 2023).
  • Type: Conference Papers and Presentations Status: Other Year Published: 2022 Citation: P. Devkota, R. Nikki, R. Hammerschmidt, A. Iezzoni, G. Schnabel, K. Gasic, M. Vassalos, and T. Mark. Measures to control Armillaria root rot in Prunus species and determine the economic impact and cost benefit of adopting cultural practices. Great Lakes Expo, December 6, 2022, Grand Rapids, Michigan
  • Type: Conference Papers and Presentations Status: Other Year Published: 2023 Citation: P. Devkota and R. Hammerschmidt. Fractionation and identification of antifungal phytoanticipins from Prunus maackii periderm using liquid chromatography-mass spectrometry. American Phytopathological Society Annual meeting, Plant Health 2023 held in Denver, Colorado (August 12-16).
  • Type: Conference Papers and Presentations Status: Other Year Published: 2022 Citation: Reighard, G. and D. Ouellette. 2022. Evaluation of semi-dwarfing peach rootstocks for intensive orchard systems in South Carolina. 98th Cumberland-Shenandoah Fruit Workers Conference, Winchester, VA. Dec. 1, 2022.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2022 Citation: Reighard, G.L. 2022. Peach rootstock performance on Peach Tree Short Life and Desarmillaria Replant Sites. Southeastern Professional Fruit Workers Conference, Nov. 14, 2022.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2023 Citation: Reighard, G.L. and T. Mark. Solutions to Armillaria root rot replant disease & its economic impact on stone fruit. February 2, 2023. Mid-Atlantic Horticulture Convention. Hershey, PA.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2023 Citation: Herrin, G., M. Vassalos, F. Silva, A. Thayer, N. Smith, and G. Reighard. An Economic Estimation of the benefits of Root Collar Excavation (RCE) Method in the Peach Industry Paper Presented at the Southern Agricultural Economics Annual Meeting, February 4-8, 2023. Oklahoma City, OK.,
  • Type: Conference Papers and Presentations Status: Other Year Published: 2023 Citation: Herrin, G., M. Vassalos, T. Mark, G. Reighard, G. Schnabel, K. Gasic, and N. Smith. What is the Economic Value of Root Collar Excavation? An Analysis Using Simulation Techniques. Paper Presented at the Agricultural and Applied Economics Annual Meeting. Washington, D.C. July 23-25, 2023.


Progress 09/01/21 to 08/31/22

Outputs
Target Audience:Target audiences reached during this period were almond, cherry and peach growers and extension agents in CA, MI and SC and GA, respectively,via virtual and in person extension and outreach. Fruit Tree nurseries and commodity boards were alsoinlcuded in this year's effort to share the accomplishmentsgenerated by the project. Changes/Problems:Obj. 1: KASP DNA test developed to confirm peach-plum hybrids revealed none of seedlings from 'Guardian®' trees used as mother were hybrids. In 2022, we repeated the pollination and caging the trees with flagging single branch on each mother tree that was emasculated but not pollinated. Fruit development on unpollinated branches confirmed wind pollination through the net used for preventing bee entrance to the cage. We will use pollination bags in 2023 to cover targeted branches to prevent pollination with undesirable pollen. Low or no germination was observed for some interspecific crosses between P. cerasifera and P. avium. Embryo rescue will be attempted next season to try to obtain hybrid plants from those crosses. Obj. 2: Many genes discovered to be involved in the resistance response Genetic resistance seems to be rare in Prunus germplasm which complicates traditional genetic mapping approaches Instrument break down in the Clemson University metabolome facility caused prolonged times for obtaining metabolomic data. Samples are in queue, and we expect to have the results of the sample preparation test run by the end of the October 2022. Obj. 3: Sweet cherry grafted on 'MP-29' and 'K-86' with Adara interstem are not viable commercial options due to weak, dying trees. Therefore, the sweet cherry trial was discontinued. This indicates that finding a tolerant rootstock for cherry will require new genetics - either cherry × plum hybrid or tolerant cherry species. The peaches on 'MP-29' and 'K-86' are showing good compatibility and will help answer the question of whether these rootstocks are tolerant to Armillaria spp. in Michigan. Discussions to find possible approach for grafting cherries to 'K-86'; and 'MP-29' with experts have been initiated, in case these rootstocks show tolerance to Armillaria species in Michigan. Shipping live or dormant Prunus from South Carolina to California is very difficult therefore the establishment of the potted trees was initiated to use as source of material (USDA). We will explore shipment of invitro unrooted plants as an alternative. At one RCE location in SC the grower had pushed up a significant number of the trees and a comparison between treatments was no longer possible. Identifying locations with Armillaria incidence in Georgia was problematic. We are currently working to make sure that we can access those locations to set demonstration plots. The soil removal using air spade is not being implemented by growers in GA. Due to sandy soil, trees at MI experimental plots had to be staked up for stability as they began falling to the ground after RCE. Obj. 4: Originally, we were planning to distribute the survey in year 2 using USDA NAAS services. During survey development we realized that even though USDA NAAS might reach more participants we had no control over who those participants would be, and that responses collected might not be relevant. The decision was made to re-budget the consultant funds to support travel of the socio economics team to the key grower meetings for each of the crops to administer the survey. As most of the regional growers' meetings are happening during fall and winter and the discussion with the focus groups and pre-testing survey instruments were completed in February of 2022, we decided to distribute the survey in person during the 2023 meetings. During the pre-test phase we noticed limitation in the peach yield data availability. Will work with producers to get an estimate of yields as the model is developed. Obj. 5: Frist Annual project meeting was organized in the fall of 2021 during second project year as we were waiting for Covid-19 restrictions to pass. Since the situation did not improve the meeting was virtual over several days. That reduced the opportunity for commmunication with the project PI and Advisory panel members. We plan on organizing the annual project meeting in Davis, CA in person. What opportunities for training and professional development has the project provided?Postdoc, graduate and undergraduate students were trained in the in vitro screening pipeline, field experiment set up and rootstock tolerance evaluation. Postdoc was also trained in the transcriptome data analyses. Extension agents were trained to explain and demonstrate the process of berm removal and root collar excavation. How have the results been disseminated to communities of interest?Results of the project have been disseminated via newsletter, peer reviewed publications, regional and national scientific conferences, growers or commodity boardmeetings. All activities and accomplishments are regularly updated on the project website and shared via social platforms (Tweeter) as well. What do you plan to do during the next reporting period to accomplish the goals?Obj. 1 Y2 Plans:Confirmation of resistance with 40 in vitro plants of each genotype will be conducted on the 4 hybrids. Clonal plants would be available for field nursery planting if resistance were confirmed. Generate cherry × plum hybrids to breed for Armillaria tolerance and graft compatibility with cherry. Screen more Prunus genotypes in the screening pipeline to characterize the anatomical and biochemical responses. Obj 2. Y2 Plans: Finalize genome assemblies for 'MP-29' and 'Guardian®', annotate genes and determine function. Genomes will be released to the public domain with a scientific publication. Collect additional transcriptome data paired with metabolomic data from to further hone in on biochemical pathways and genes driving ARR resistance. Obj 3. Y2 Plans: Continue evaluating field trials. Equip the debermer system with sensing mechanism to automate the paddle wheel extension and retraction motions to avoid tree trunks while deberming. Integrate an electronic flow control valve to adjust the flow rate of the hydraulic fluid to the paddle wheel to vary the paddle wheel RPM in different operating conditions (soil type and soil conditions). Experiment brush mechanisms for soil removal around tree trunks. Identify new demonstration plots across the Georgia peach producing area. Target locations with known Armillaria presence, availability of multiple rootstocks, and planting of plots has occurred 2 years ago. Obj 4. Y2 Plans: Analyze data from the survey(s), completion of the simulation model to evaluate the economic impact of Root Collar Excavation. Administer grower surveys with direct engagement of extension agents and commodity boards. Organize annual project meeting in Davis CA. Obj 5. Y2 Plans: Continue producing video documentaries and newsletters; collect grower feedback at regional and roundtable meetings; present at the regional growers' meetings; organize field demonstration days; deploy RCE in demonstration plots; provide updates and presentations via project website, organize annual meeting.

Impacts
What was accomplished under these goals? Rootstocks resistant to Armillaria root rot (ARR) for peach, almond and cherry are lacking. The only ARR resistant rootstock available for peach, MP-29, is interspecific, requires vegetative propagation, has not been extensively tested and is not readily available. This project found new sources of resistance in plum germplasm from the Prunus National Clonal Germplasm Repository, determined the mode of tolerance in and uses the tolerant material in breeding with the standard peach, almond and cherry rootstocks to introduce tolerance to ARR. Participating nurseries are testing discovered material for their horticultural acceptability as rootstocks. Cultural practice root collar excavation (RCE) ability to extend the life of the orchard on ARR infested sites is documented for peach and is being evaluated on cherry. Similarly, short term approach with using sectoral chimera rootstocks between ARR resistant material and standard rootstocks are being evaluated for almond. New equipment is developed to assist growers with berm removal between and around the trees. Effect of Armillaria replant disease on stone fruit industry, and potential costs and benefits of applying cultural practices is being investigated. Results of the project are shared with the stakeholders via project website, field days, presentations at national and regional grower conferences, and direct contact via extension agents. Obj. 1 accomplishments: 8 new plants with confirmed absence of fungus penetration by microscopy have been discovered. Four of them have been multiplied in vitro and are being tested for tolerance with A. mellea and Desarmillaria tabescens in the screening pipeline. Crosses of ARR resistant genotypes [P. munsoniana; Prunus cerasifera DPRU.2101 (cv. Coheco), and DPRU. 2314] with standard rootstocks 'Guardian®', Prunus avium NY54 were performed, and hybrid seed tested for Armillaria resistance. Crosses between cherry and plum are difficult and hybrid seed germination unsuccessful. Embryo rescue will be implemented. Antifungal assays revealed constitutive antifungal compounds, barrier zone formation and host susceptibility to ARR in tolerant material. Rapid in-vitro screening of tolerant material with Armillaria solidipes, Desarmillaria tabescens, and A. mellea showed less susceptibility, no genotype and fungal interaction, and root periderm tissue forming barrier zone to deter fungal invasion. Obj. 2 accomplishments: A high quality(haplotype resolved) genome assembly was produced from the ARR resistant peach/plum hybrid rootstock, MP-29, and the peach tree short life (PTSL) resistant (ARR susceptible) rootstock 'Guardian®'. De novo transcriptomes were assembled, and candidate genes underlying the infection response were determined. A slurry-based inoculation approach for transcriptomic/metabolomics analyses was developed and inoculum density tested to maximize root contact with pathogen (A. mellea and D. tabescens) in the co-culture system. Number of plants per vessel was adjusted in preliminary tests to get enough amount of root tissue for metabolomics and transcriptomic analyses. Phenotypic information and reproductive compatibility on A. mellea isolates were gathered. Obj. 3 accomplishments: 3 field screening trials of new rootstocks and selections were planted on Armillaria and PTSL replant sites at the Sandhill Research and Education Center in Pontiac, South Carolina and at Titan Farms (2 trials) in Ridge Spring, South Carolina. One trial included new rootstocks from private breeding programs in California. First year survival was excellent. Peach trees on 'Krymsk® 86' and 'MP-29' rootstocks were planted in nursery at the Southwestern Michigan Research and Extension Center (SWMREC) and in Armillaria infected grower's location in west central Michigan. ARR disease evaluation plot is established at the University of California Davis (UCD) Wolfskill Experimental Orchard in Winters California with replicated plantings of resistant species and/or interspecies hybrids of a diverse germplasm including Prunus dulcis, Prunus persica, Prunus davidiana, Prunus cerasifera, Prunus americana, Prunus tangutica and almond as susceptible controls. Evaluated rootstock selections include sectoral chimeras generated through modified approach grafting techniques. Field trials at locations in South Carolina were monitored for the impact of berming and root collar excavation (RCE) on tree health on replant sites with a history of ARR problems. No difference in tree health was detected. Equipment for soil removal was developed and tested. Obj. 4: Survey instrument pre-test, completed with the focus groups cherries and peaches, was distributed to 55 peach growers across the U.S., using a phone survey. The survey was conducted by professional interviewers from Qualtrics. A simulation model to evaluate the economic impact of Root Collar Excavation for peach orchards is in process. Obj. 5 accomplishments: Video documentaries related to ARR management, were shown at the Southeastern Professional Fruit Workers Conference. First newsletter (spring 2022) was developed and shared with stakeholders, Extension specialists and researchers. Project was presented at regional and national grower, stakeholder, and scientific meetings. Round table and lunch discussions with growers as well as demonstrations and trainings were carried out at these meetings. Project website was updated with the current information. More footage of activities for different objectives has been collected. Extension agents were trained to explain and demonstrate the process of berm removal and root collar excavation. The root collar excavation system was promoted to SC growers and students as one strategy to manage the ARR disease. Annual project meetingwas organized virtually with all project participants and Advisory panel members.

Publications

  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: Gasic, K., J. Adelberg, K. Baumgartner, P. Brannen, L. Cai, A. Calle, D. Chavez, P. Devkota, T. Gradziel, R. Hammerschmidt, A. Iezzoni, B. Koc, T. Mark, J.C. Melgar, G. Reighard, C. Saski, G. Schnabel, N. Tharayil, and M. Vassalos. 2022. Solutions to the Armillaria Root Rot affecting the U.S. stone fruit industry. 10th International ISHS Peach Symposium, Naoussas Greece. Acta Hort (accepted)
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: Reighard, G.L., D. Ouellette and I. Minas. 2022. Field Testing Prunus Rootstocks in South Carolina for Peach Tree Short Life and Armillaria Tolerance. XXXI ISHS HortCongress, Angers, France. Acta Hort (submitted).
  • Type: Theses/Dissertations Status: Other Year Published: 2022 Citation: Scroggs, C. 2022.Mechanizing the removal of soil between peach trees planted on berms. MSc Thesis.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2022 Citation: Calle, Alejandro. Transcriptome response in roots following infection by Desarmillaria tabescens and Armillaria mellea in resistant and susceptible Prunus genotypes. ASHS 2022 Annual Conference (Chicago, IL).
  • Type: Conference Papers and Presentations Status: Other Year Published: 2022 Citation: Pratima Devkota, Amy Iezzoni, and Raymond Hammerschmidt. Rapid in vitro method utilizing roots of Prunus species seedlings to screen against Armillaria root rot Plant Health 2022, Pittsburgh, PA (August 6-10).
  • Type: Conference Papers and Presentations Status: Other Year Published: 2022 Citation: Pratima Devkota and Raymond Hammerschmidt. Identification of virulence determinants of Armillaria solidipes. Plant Health 2022, Pittsburgh, PA (August 6-10).
  • Type: Conference Papers and Presentations Status: Other Year Published: 2022 Citation: Reighard, G.L. Performance of Semi-dwarfing Peach Rootstocks for High-density Plantings in the Southeastern States. January 7, 2022. Southeastern Regional Fruit & Vegetable Conference, Savannah, Georgia.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2022 Citation: Reighard, G.L. Performance of New Rootstock Cultivars in South Carolina. October 27, 2021. Southeastern Professional fruit Workers, Clemson, South Carolina.
  • Type: Other Status: Other Year Published: 2022 Citation: Schnabel, G. 03/2022. Disease management in peach. Production meeting; Clemson Extension. Walhalla, SC
  • Type: Other Status: Other Year Published: 2022 Citation: Schnabel, G. 01/2022. Disease management in peach. Production meeting; Clemson Extension. Lyman, SC
  • Type: Conference Papers and Presentations Status: Other Year Published: 2022 Citation: Scroggs, Coleman. 2021. Mechanizing Removal of Soil Between Peach Trees Planted on Berms. Southeastern Professional Fruit Workers Conference, Virtual, Hosted by Clemson Peach Team. October 26-28, 2021.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2022 Citation: Scroggs, C., A.B. Koc, G. Schnabel, J-C. Melgar, M. Vassalos. 2022. Mechanizing the Removal of Soil Between Peach Trees Planted on Berms. Southeast Regional Fruit & Vegetable Conference, Savannah, GA. The Savannah Convention Center on January 6-9, 2022.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2022 Citation: Scroggs, C., A.B. Koc, G. Schnabel, J-C. Melgar, M. Vassalos. 2022. Removal of Soil Between Peach Trees Planted on Berms. American Society of Agricultural and Biological Engineers (ASABE) Annual International Meeting, Houston TX, July 17-20, 2022.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: Reighard, G.L., I. Minas and D. Ouellette. 2022. Field performance of Peach Rootstock Cultivars in South Carolina. 10th International ISHS Peach Symposium, Naoussas Greece. Acta Hort (accepted)


Progress 09/01/20 to 08/31/21

Outputs
Target Audience:Target audiences reached during this period are almond, cherry and peach growers in CA, MI and SC and GA, repsectively, via virtual and in person (when possible) extension and outreach. Fruit Tree nurseries and commodity boards were also inlcuded in this years effort to share the accomplishmentsgenerated by the project. Changes/Problems:Obj. 1:Delay in using P. cerasifera cv. Coheco in crossing due to frost damage in spring 2021. To prevent this from happening in the future, a cage was constructed over the 'Coheco' trees to hold netting to keep out bees to facilitate crossing in 2022, and to hold tarps in case of freezing temperatures. Obj. 2:DNA extraction was delayed due to insufficient amount and quality of young leaf tissue needed for genome sequence from some of the material generated in vitro in obj. 1a.Root contact of the pathogen in the co-culture system is uneven. We are addressing this challenge by developing a 'slurry' inoculation approach for collecting metabolome time series data.Genomic resources for Armillaria mellea are lacking. Obj 3:Sweet cherry on 'MP-29' and 'Krimsk-86' with Adara interstem are not viable commercial options due to weak, dying trees. Therefore, the sweet cherry trial was discontinued. Shipping live or dormant Prunus from South Carolina to California is very difficult therefore the establishment of the potted trees was initiated to use as a source of material (USDA).The trees of one of the grower demonstration trials in SC were partially pushed up by the grower.Identifying locations with Armillaria incidence in Georgia was problematic. We are currently working to make sure that we can access those locations to set demonstration plots. Obj. 5:Pandemic prevented in person meetings and field day activities;Annual meeting was moved to year 2 in anticipation of in person meeting; Field demonstration days were postponed. What opportunities for training and professional development has the project provided?Postdoc, graduate and undergraduate students were trained in the in vitro screening pipline, field experiment set up and rootstock tolerance evaluation. Postdoc was also trained in the transcriptome data analyses. How have the results been disseminated to communities of interest?Results ofthe project have been disseminated via peer reviewed publictations, regional and national scientific conferences, growers or commodity boards meetings. All activities and accomplishments are regulary updated on the project website as well. What do you plan to do during the next reporting period to accomplish the goals?Obj. 1 Y2 plan:Repeat crossing scheme from previous year and initiate crossing of P. cerasifera cv. Coheco trees and sweet cherry. Continue to evaluate for disease incidence/severity in vitro and in field and add additional material to the pipeline. Screen additional Prunus genotypesto characterize the anatomical and biochemical responses.Conduct antifungal assays for the genotypes provided by Fowler Nursery. Conduct thin layer antifungal bioassay of commercially available possible antifungal compounds identified from Prunus maackii. Obj. 2 Y2 plan:Develop DNA diagnostic markers (KASP/SSR/Other) for distinguishing the origin of the hybrid material, PTSL and ARR tolerance using the whole genome sequences and validate these markers on known and unknown PTSL/ARR resistant germplasm.Analyze the transcriptome sequences for SNP markers and corroborate these markers with the whole genome sequences collected in Obj. 2a. Release transcriptome data as a peer reviewed scientific journal article.Test a slurry-based inoculation approach to maximize root contact with the pathogen in the co-culture system.Collect primary and secondary metabolites from at least 7 putative resistant genotypes and integrate with available RNAseq data.Generate reference genomes and trancriptomes of A. mellea. Obj. 3 Y2 plan:Continue evaluation of current rootstock trials and seek/obtain additional promising germplasm to test as rootstocks. Establish peach field trialsin two Armillaria infected grower's locations in northwestern and west central MI, respectively. Plant ten peach trees each on rootstock 'MP-29' and 'Krymsk® 86' on a non-ARR site at SWMREC to assess horticultural performance.Work with Foundation Plant Science virologists at UCD to modify their Prunus axillary meristems culture techniques for application to epicormic meristems.Optimize conditions for in-vitro culturing of isolated epicormic meristems.Develop additional binary sectoral rootstocks using proven approach graft technique and test strategies for tissue culture in-vitro approach grafting of isolated epicormic meristems from different species. Evaluate tree root establishment to decide the best timing for RCE treatment in MI.Collect field data (tree mortality, trunk cross sectional area, and canopy size) from the demonstration plot at Southern Orchards.Identify new demonstration plots across theGA peach producing area. Target locations with known Armillaria presence, 2nd year orchards and on multiple rootstocks to deploy RCE and compare with non-excavated controls at different locations.Determine efficiency and performance of newly developed tool by conducting experiments at producers' fields in smoothing the berms between the trees.Determine the amounts of torque, power, fuel consumption and labor required in varying soil and field conditions using electronics.Analyze and summarize tree health and mortality data from 2021 and collect additional data in 2022. Obj. 4 Y2 plan: Distribute the survey on the effect of Armillaria root rot on the stone fruit industry and collect the data Obj. 5 Y2 plan: Initiate video documentaries; collect grower feedback via round table discussions (in person if possible); present accomplishmetns at the regional grower's meetings and organize field demonstration days; and provide access to video documentaries and presentationsvia project website. Organize annual project meeting.

Impacts
What was accomplished under these goals? Obj. 1 accomplishments: 157 hybrid seedlings generated in spring 2020 from 12 crosses between standard peach and cherry rootstocks with 3 accessions showing tolerance to Armillaria (P. cerasifera DRPRU.2101 and DPRU.2314, and P. munsoniana #4) were screened with A. mellea. Root tissue of 15 seedlings showing tolerance in vitro submitted for root histology Crosses have been repeated in spring 2021 and seeds stratified in vitro of: P. cerasifera DPRU.2101 × Guardian® (108); P. mahaleb × DPRU.2101, Montmorency × DPRU.2101, and Montmorency × DPRU.2341 (23); Guardian® × P. munsoniana; P. munsoniana × Guardian®; Guardian® × DPRU.2101, and Guardian × DPRU.2314 (743). 58 seeds from P. dulcis × P. cerasifera DPRU.2101 Seeds were collected from 23 trees representing five Prunus species (P. cerasus, P. pensylvanica, P. serotina, P. virginiana, and P. pumila) from woodlots near infected orchards and infected forest sites in MI. 101 seeds are stratified in vitro at Clemson. P. cerasifera cv. Coheco trees were purchased and planted at MSU's Clarksville research station to facilitate crossing. Second in vitro screening stage has been initiated for one interspecific hybrid with tolerance confirmed by root histology. Ten interspecific hybrids are being multiplied while awaiting root histology results. Co-culture methodology for existing sources of ARR tolerance is being improved. Clones of four resistant plum accessions were propagated for PTSL, ARR, and graft compatibility field experiments (objective 3). An ARR field evaluation plot has been established at the UCD Wolfskill Experimental Orchard in Winters California (UC Davis) with replicated plantings comprising reportedly resistant species and/or interspecies hybrids of diverse germplasm including P. dulcis, P. persica, P. davidiana, P. cerasifera, P. americana, P. tangutica and almond as susceptible controls. Susceptible controls are starting to show disease, but no disease symptoms are evident yet in the experimental plantings. In vitro assay developed to screen roots of seedling Prunus genotypes to A. mellea, A. solidipes and D. tabescens. Twelve different Prunus genotypes provided by Fowler Nursery and Prunus cerasifera were screened for ARR. None of the screened Prunus genotypes were resistant to tested Armillaria and D. tabescens isolates. Obj. 2 accomplishments: Total genomic DNA was extracted from 6 ARR resistant accessions used in breeding in objective 1a to obtain genomic sequences. The DNA from four seedlings (DPRU.2101_20-3, DPRU. 2101_20-4, DPRU.2314_14-4 and P. munsoniana #4_59-1) and two accessions (DPRU.2101 and DPRU.2314) is currently being sequenced at a 3rd party service provider. A denovo transcriptome has been assembled and annotated for each of the 3 Prunus species. Pairwise comparisons between inoculated and non-inoculated experiment for each species has been completed. Candidate genes have been identified that confer resistance to Armillaria Transcriptome analyses indicate a high degree of variation in early time points in the root co-culture system. Obj. 3 accomplishments: Three field screening trials of new rootstocks and selections were planted on ARR and PTSL replant sites at the Sandhill Research and Education Center in Pontiac, South Carolina and at Titan Farms (2 trials) in Ridge Spring, SC. One of the Ridge Spring trials included new rootstocks from private breeding programs in CA. First year survival was excellent at all sites. Clonally propagated (at least 50 each) of four ARR resistant accession from Obj. 1a were grafted with peach scion and finished trees will be planted in 2021/2022 winter to evaluate graft compatibility and other horticultural characteristics. Cherry and peach trees provided by Sierra Gold Nursery were received in MI in April 2021. Sweet cherry trees were grafted with Adara interstem as a "bridge" to attempt to achieve graft compatibility. Field trial establishment was postponed until 2022 as the nursery trees were too small for field planting and instead were planted in a nursery at the Southwestern Michigan Research and Extension Center (SWMREC) in 2021. P. cerasifera accessions DPRU.2101 ('Coheco') and DPRU.2314 ('AL 097') were propagated to enable establishment of tissue cultures of both accessions in USDA lab (USDA). Potted plants are obtained. DPRU2314 had notably better rooting than DPRU2101 Binary sectoral rootstocks were developed using modified approach and wedge-grafting techniques between:Prunus dulcis and an interspecies hybrid with P. cerasifera;Nemaguard rootstock with Krymsk 86 rootstock;and P. dulcis and Krymsk 86 rootstoc Methods were developed for identifying and isolating epicormic meristems at an emergent developmental state Tart cherry root collar excavation field trials were establishedon an ARR-infested orchard site in Manistee County (MI). A total of 1220 acres of peaches were planted in 2021 in berms across Georgia A tractor-powered orchard implement was modified to flatten the soil between trees planted on raised beds. A modified paddle wheel with larger diameter and height was fabricated and mounted on the implement Obj. 4 accomplishments: The survey instrument was developed and approved by Clemson's IRB

Publications

  • Type: Websites Status: Published Year Published: 2020 Citation: Armillaria Solutions project website has been redesigned and moved to new platform. https://blogs.clemson.edu/arrsolutions/
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Adelberg, J., Naylor-Adelberg, J., Miller, S., Gasic, K., Schnabel, G., Bryson, P., Saski, C., Parris, S. and Reighard, G. (2021)In vitro co-culture system for Prunus spp. and Armillaria mellea in phenolic foam rooting matric. In Vitro Cell.Dev.Biol.-Plant 57, 387397 https://doi.org/10.1007/s11627-020-10136-2
  • Type: Journal Articles Status: Under Review Year Published: 2021 Citation: Devkota P., and Hammerschmidt R. (2021). How do woody hosts respond to Armillaria spp. infection? (Review article)
  • Type: Conference Papers and Presentations Status: Other Year Published: 2020 Citation: Gasic, K., Saski, C., Adelberg, J., Baumgartner, K., Brannen, P., Chavez, D., Gradziel, T., Hammerschmidt, R., Iezzoni, A., Koc, B., Mark, T., Melgar, J.C., Reighard, G., Schnabel, G., Tharayil, N., & M. Vassalos (2021) Solutions to the Armillaria root rot affecting the U.S. stone fruit industry. Presented at the IFTA virtual annual conference, February 22-24, 2021
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Gasic, K., C.A. Saski, J. Adelberg, K. Baumgartner, P. Brannen, D. Chavez, T. Gradziel, R. Hammerschmidt, A. Iezzoni, B. Koc, T. Mark, J.C. Melgar, G.L. Reighard, G. Schnabel, N. Tharayil, and M. Vassalos. 2021. Solutions to the Armillaria Root Rot Affecting the U.S. Stone Fruit Industry. HortSci. 56(9):S
  • Type: Conference Papers and Presentations Status: Other Year Published: 2021 Citation: Reighard, G.L, K. Gasic, C.A. Saski, J. Adelberg, K. Baumgartner, P. Brannen, D. Chavez, T. Gradziel, R. Hammerschmidt, A. Iezzoni, B. Koc, T. Mark, J.C. Melgar, G. Schnabel, N. Tharayil, and M. Vassalos. 2021. Solutions to the Armillaria Root Rot Affecting the U.S. Stone Fruit Industry. ISHS XII International Symposium on Integrating Canopy, Rootstock and Environmental Physiology in Orchard Systems. Poster Abstracts. P. 33.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2021 Citation: Reighard, G.L, K. Gasic, C.A. Saski, J. Adelberg, K. Baumgartner, P. Brannen, D. Chavez, T. Gradziel, R. Hammerschmidt, A. Iezzoni, B. Koc, T. Mark, J.C. Melgar, G. Schnabel, N. Tharayil, and M. Vassalos. 2021. Solutions to the Armillaria Root Rot Affecting the U.S. Stone Fruit Industry. ISHS XII International Symposium on Integrating Canopy, Rootstock and Environmental Physiology in Orchard Systems. Virtual. July 26, 2021.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2021 Citation: Minas, I.S., G.L. Reighard, B. Black, J.A. Cline, D.J. Chavez, E. Coneva, G. Lang, M. Parker, T. Robinson, J. Schupp, P. Francescato, J. Lordan, T. Beckman, W. Shane, J. Pieper, D. Sterle, C. Bakker, B. Clark, D. Ouellette, A. Swain, and H. E. Winzeler. 2021. Establishment performance of the 2017 NC-140 semi-dwarf peach rootstock trial across 10 sites in North America. ISHS XII International Symposium on Integrating Canopy, Rootstock and Environmental Physiology in Orchard Systems. Oral Abstracts. P. 57.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Reighard, G.L., W.G. Henderson and D. Ouellette. 2020. Screening peach rootstocks for adaptability, productivity, and tolerance to Armillaria and bacterial canker. Annual Peach Research Report, Vol. 20:36-42.