Source: CLEMSON UNIVERSITY submitted to
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
Funding Source
Reporting Frequency
Accession No.
Grant No.
Project No.
Proposal No.
Multistate No.
Program Code
Project Start Date
Sep 1, 2020
Project End Date
Aug 31, 2024
Grant Year
Project Director
Gasic, K.
Recipient Organization
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
Research Effort Categories

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
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 (, the University of Georgia Peach website (, individual cultivars web pages (, the National Peach Council web page (, almond board ( and cherry Marketing Institute ( websites.

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

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.

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


  • Type: Websites Status: Published Year Published: 2020 Citation: Armillaria Solutions project website has been redesigned and moved to new platform.
  • 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
  • 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.