Location: Physiology and Pathology of Tree Fruits Research
2025 Annual Report
Objectives
The long-term objective of this program is to formulate effective and economically sustainable methods for management of tree fruit diseases in high value production systems. Utilization of host resistance presents an economically effective, ecologically desirable, and durable disease control strategy. Additionally, horticultural traits that allow for disease avoidance, such as dwarfing of the scion that allows for better spray and light penetration of canopies, have yet to be widely incorporated into pear rootstocks. For this purpose, it is a necessary first step to understand the molecular mechanisms underlying resistance/avoidance traits in apple and pear roots by identifying key genes regulating root resistance responses and other relevant physiological processes. This research plan will make it possible to develop molecular tools for accurately and efficiently incorporating resistance traits into new apple and pear rootstocks.
Objective 1: Discover rootstock resistance traits and architectural features in deciduous tree fruits involved in disease avoidance.
Sub-objective 1.A: Characterize genotype-specific variations in biochemical and metabolic features linked to apple root resistance to P. ultimum.
Sub-objective 1.B: Determine the connection between pear root architecture, disease resistance, and dwarfing.
Sub-objective 1.C: Identify genetic components involved in rootstock-mediated dwarfing of pear scions.
Sub-objective 1.D: Develop a rapid-cycle breeding tool for use in breeding pear rootstocks.
Objective 2: Identify genotype-specific expression patterns of candidate genes underlying disease resistance and disease avoidance traits.
Sub-objective 2.A: Conduct bioinformatic analysis of the sequence features of selected candidate genes identified in previous transcriptome analyses.
Sub-objective 2.B: Characterize genotype-specific expression patterns for selected candidate genes between resistant and susceptible genotype groups.
Sub-objective 2.C: Transgenic manipulation of selected apple candidate genes using CRISPR/Cas9 tool and in planta expression analysis.
Approach
A combination of current and emerging genetic and genomic techniques will be applied in this research plan. Various methodologies in phenotyping and molecular analysis including genomics, transcriptomics genetics, biochemistry, breeding and microscopy will be utilized to identify the genetic elements and unravel the regulation mechanisms underlying apple root disease resistance and pear architectural features. Such studies will improve knowledge of molecular mechanisms underlying important traits and genome annotation, facilitate the development of germplasm and establishment of a rapid-cycle breeding tool to enhance our understanding of how rootstocks confer traits to scions.
Experiments will be conducted using previously identified apple rootstock germplasm pairs with contrasting resistant versus susceptible phenotypes, such as O3R5-#161 vs # 132; #58 vs #47 and/or #164 vs #1, respectively. Expression analysis of selected candidate genes and biochemical or enzymatic assays will provide experimental evidence connecting genes and trait during apple root response to P. ultimum infection. De novo sequencing of specific genomic fragments containing the genes of interest will identify variations at gene structure and sequences between resistant and susceptible O3R5 genotype groups, which may provide valuable reference for their functional roles in defense activation. The knockout (KO) transgenic line will be generated using established in-house protocols including plasmid construction, transformation of E. coli and Agrobacterium, subsequent callus induction and individual transgenic line establishment/propagation by tissue culture.
For research related to pear architectural traits, parental genotypes will be established in tissue culture, rooted, acclimated, and grown to similar sizes before moving to different phenotyping methods during the first year. Data analysis will compare differential gene expression between the most and least dwarfing individuals across tissue type and time, allowing us to understand more about dynamic gene activity on the scion and rootstock sides of the graft union, as well as in the root system and how the rootstock is affecting scion leaf tissue. A major limitation for breeding new rootstocks is the long juvenility period of pear (reaching up to ~10 years in European pear), especially when stacking multiple traits is the desired goal. DNA-informed breeding speeds the selection process, but juvenility periods remain long, meaning variety releases are 20-40 years from initial crosses, depending on species and breeding scheme. To shorten this process, development of a rapid-cycle breeding tools by modifying flowering gene expression can greatly reduce amount of time allows for stacking multiple loci or traits.
Progress Report
This report documents FY 25 progress of project 2094-21220-003-000D which began in March 2022.
In support of Sub-objective 1A, ARS researchers continued to evaluate underexplored apple root phytoalexins for their inhibitory activity against key apple replant pathogens including: Pythium irreglurare, Pythium ultimum, Rhizoctonia solani and Cylindrocarpon destructans. This year, progress was made toward development of a high-throughput, quantitative, liquid-based growth inhibition assay for use with filamentous fungi/oomycetes, including those which do not produce spores (e.g., R. solani). This approach differs from existing ones which require spore suspension preparation and/or absorbance-dry weight calibrations. While developing the procedure, several technical challenges were encountered. Ensuring detection of small changes in fungal biomass and standardizing inoculum concentrations required troubleshooting. In the current procedure, very small changes (down to 0.001 mg) in fungal biomass can be directly measured over time. Efforts are currently underway to validate the procedure using reference inhibitory molecules. This methodology will streamline investigation into the roles of apple phytoalexins in assembling root-associated microbial communities and/or mitigating apple replant disease (ARD). Ultimately, this knowledge is expected to facilitate the development of strategies to improve productivity and sustainability in orchard systems.
For Sub-objective 1B, research continued towards understanding replant disease in pear. The previous rootstock trial (summer/fall of FY23) failed due to root systems being too small when dug from nursery ground. This year, the experiment was successfully repeated with new pear rootstocks (received from a different nursery). Experimental results provided preliminary support for the hypothesis that OHxF97 may be less susceptible to replant disease than other commercially available pear rootstock genotypes including Bartlett and OHxF87. OHxF97 was the only rootstock that showed a lack of growth reduction (relative to the pasteurized control) when cultivated in replant-affected soil from two different orchard locations. This lack of response was consistent in assays with tissue-cultured plantlets and in experimental trials with nursery-derived rootstocks. Both Bartlett (micropropagated plantlets) and OHxF87 (nursery-derived rootstocks) showed growth responses in replant-affected soil with relatively high nematode pressure (Pratylenchus penetrans). Pathogen-specific qPCR assays designed to quantify the abundance of other replant pathogens in root tissue are currently in progress. Sequence-based analysis of bacterial and fungal communities associated with OHxF87 and OHxF97 in replant-affected soils is also in progress and is expected to reveal soil- and genotype-specific differences (including potentially beneficial components) of the pear rhizobiome.
In addition, work continued to test root system architecture measurement and analysis methods. Root systems architectures of 4 pear genotypes receiving 8 different micropropagation strategies were compared 1, 2, and 3 months after transfer to soil, using Rhizovision Explorer. Manual validation is underway. Early results suggest a strong initial effect of micropropagation strategy on early root system architecture. Seven genotypes from the pear diverse germplasm collection were budded with a common scion (Bartlett) last year and are beginning to successfully grow out. This will allow for study of rootstock genotype effect on scion architecture and correlation with root system architecture. Development of rooting protocols and assessment of root development responses to auxin and cytokinin treatments across a diverse pear germplasm have continued.
In support of Sub-objective 1C, selected trees from a pear population segregating for a dwarfing trait were received, this time in good health. These trees will continue to grow through the season and be budded with a common scion (Bartlett) in the early fall. Buds will be allowed to establish, go dormant, and in the spring, tissue collections will begin and continue throughout the season, to compare the effects of sibling dwarfing and vigorous rootstocks on the scion transcriptome.
Characterization of architecture- and hormone-related genes has continued, including analysis of IGT protein-protein interactions in other rosaceous fruit trees (peach) and development of yeast-2-hybrid tools for analysis of pear IGT protein interactions. Further, testing of pear auxin-signaling protein functions has begun with collaborators at Whitman College, assessing protein degradation rates of pear Aux/IAA repressors from different genotypes. This will be important to connect with the root development auxin responses assessed in Sub-objective 1C for better understanding and predictability of hormone responses across genotypes. Tissue previously collected for the pear tissue-specific gene expression atlas was further used for Pac-Bio Iso-seq RNA-seq analysis and development of an improved genome reannotation for Bartlett pear. This allowed for the improvement of gene models and identification of splice variants, as many gene models were previously incorrect and difficult to determine. This Bartlett genome reannotation marks a tool that will be important for the entire pear molecular biology and genomics communities to use.
For Sub-objective 1D, significant progress towards improving and understanding adventitious shoot regeneration and Agrobacterium-mediated callus transformation was made. Adventitious shoot regeneration in the absence if Agrobacterium was optimized and improved in 3 genotypes: Bartlett, the most common scion genotypes in the U.S. pear industry, and OHxF 87 and 97, the two most common rootstock genotypes in the industry. Findings included optimal hormone treatments, regeneration media, micropropagation media for plants prior to tissue excision, and methods to avoid oxidation of cut leaf tissue. Callus transformation via Agrobacterium was also significantly improved, especially by changing the type of media that plants were micro propagated on prior to leaf excision. Multiple trials were carried out using the altruistic Agrobacterium strain from collaborators at Oregon State University, leading to improved callus transformation with the rapid-cycle-breeding construct. Efficiency was largely affected by micropropagation media and ratio of Agrobacterium. Thus far, two transformed leaves have been identified, but were not able to survive long-term, partially due to Agrobacterium overgrowth. ARS collaborators in Kearneysville, West Virgnia, recently had early success transforming a rapid-cycle-breeding construct that uses overexpression, rather than inducibility of flowering genes, and using tissue from pear genotypes that are readily regenerated. Plans are underway to receive this tissue and attempt transformation of the inducible-flowering construct into more easily regenerable plants.
The collaborative Long-Term Agricultural Research and Extension (LTARE) project between ARS and Washington State University scientists continues. This year, soil and microbiome composition were assessed in responses to different soil amendments and minirhizotron tubes were placed in the orchard to begin measurement of root system architecture responses to these same amendments. This project extends our abilities to assess relationships between root system architecture and the microbiome.
Last year, ARS researchers refocused efforts to ensure progress towards NP Action Plan Problem Statement 1A. Through a collaboration with researchers at the University of Turin, Italy, ARS researchers continue research on potential similarities in Kiwivine decline syndrome (KVDS) and apple replant disease (ARD) pathosystems. This year, ARS researchers and collaborators characterized the transcriptomic response of kiwifruit over time under biotic stress (inoculation with Phyotpythium vexans), abiotic stress (root flooding), and their combination, mimicking disease symptoms under controlled conditions. The transcriptomic profile of the multifactorial condition was distinct and could not be predicted from individual stress responses. Notably, key biotic defense mechanisms were suppressed in the presence of flooding (including phenylpropanoid biosynthesis and auxin signaling) despite their upregulation during pathogen infection alone. The analysis highlighted the importance of flavonoid biosynthesis in the kiwifruit defense response, mirroring previous findings in apple. These results support the hypothesis that plant stress adaptation mechanisms may come at the cost of defense capabilities and provide novel insights into the adaptive strategies of woody perennial plants under multifactorial/combined stress conditions. Manuscript preparation is in progress. In addition, ARS researchers collaborated with researchers at the University of Turin, Italy, to help validate detection/quantification of P. vexans in apple root tissue and orchard soil. The method, published in Plant Disease, is a useful tool for monitoring the distribution, spread and impact of the pathogen in woody perennial crops. In support of Objective 1, ARS researchers also continued to conduct research on the use of arbuscular mycorrhizal fungi (AMF) to enhance agricultural practices in apple production systems. Compatible apple rootstock/AMF species combinations previously reported by ARS researchers were used in subsequent experiments designed to assess specific functional benefits. Experiments provided clear evidence of AMF species directly functioning in beneficial roles with commercially available apple rootstock genotypes, including specific AMF-rootstock relationships that could be harnessed to improve drought tolerance. Manuscript preparation is in progress.
Accomplishments
1. Beyond Borders: Improving Rapid Detection of Soilborne Pathogens in Woody Perennials. Phytopythium vexans is a soil-borne plant pathogen primarily affecting woody perennial crops, such as kiwifruit, pear, apple and grapevine. Its impact, particularly in kiwifruit, has risen in the last decade, and there is evidence suggesting that Phytopythium spp. could gain an advantage over other microorganisms in areas with higher soil temperatures. To date, most molecular assays for the detection of the pathogen target a genetic region which is ill-suited for accurate species identification. In collaboration with researchers at the University of Turin (Turin, Italy), an ARS scientist from Wenatchee, Washington, helped develop a molecular diagnostic tool for early detection and quantification of the pathogen. The assay, validated in kiwifruit and apple, was found to be highly sensitive and specific to the target pathogen, regardless of sample matrix (soil or root tissue) or cropping system. The method is a useful tool for monitoring the pathogen load and spread.
Review Publications
Zhou, Z., Zhang, H., Yao, J., Gao, Q., Wang, Y., Liu, Z., Zhang, Y., Tian, Y., Yan, Z., Zhu, Y., Zhang, H. 2024. The MdERF61-mdm-miR397b-MdLAC7b module regulates apple resistance to Fusarium solani via lignin biosynthesis. Plant Physiology. 197(1). Article kiae518. https://doi.org/10.1093/plphys/kiae518.
Zhang, H., Wang, W., Honaas, L.A., Mazzola, M., Somera, T.S. 2024. Evaluating the stability of nursery-established arbuscular mycorrhizal fungal associations in apple rootstocks. Applied and Environmental Microbiology. 91(1). Article e01937. https://doi.org/10.1128/aem.01937-24. [Corrigendum: Applied and Environmental Microbiology, 91(1), Article e01937, https://doi.org/10.1128/aem.00198-25].
Guaschino, M., Prencipe, S., Somera, T.S., Tabone, G., Spadaro, D. 2025. Assay for early detection and quantification of Phytopythium vexans in kiwifruit plant and soil affected by vine decline syndrome. Plant Disease. 109(7):1489-1495. https://doi.org/10.1094/PDIS-09-24-2044-RE.
Zhu, Y., Ortiz Uriarte, B.P., Rainbow, J.L., Zhou, Z. 2024. The expression and sequence analysis of MdMATE52 in apple roots during activation of defense against Pythium ultimum infection. Horticulturae. 10(11). Article 1204. https://doi.org/10.3390/horticulturae10111204.
Waite, J.M., Burchard, E.A., Dardick, C.D., Hollender, C.A. 2025. Peach (Prunus persica) TAC1 protein interaction with a Light Harvesting Chlorophyll A/B Binding (LHCB) homolog and transcriptomic analyses reveal connections to photosynthesis. microPublication Biology. https://doi.org/10.17912/micropub.biology.001371.
Galimba, K.D., Denew, M., Waite, J.M. 2025. Application of ABA and ACC in autumn affects dormancy and cold hardiness of pear (Pyrus communis). HortTechnology. 35(4):385-391. https://doi.org/10.21273/HORTTECH05618-25.