Location: Physiology and Pathology of Tree Fruits Research
2024 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 2024 progress for project 2094-21220-003-000D, “Uncovering Rootstock Disease Resistance Mechanisms in Deciduous Tree Fruit Crops and Development of Genetics-Informed Breeding Tools for Resistant Germplasm”, which began in March 2022.
In support of Sub-objective 1A, ARS researchers in Wenatchee, Washington, continued to identify apple and pear rootstock resistance traits and architectural features in deciduous tree fruits involved in disease avoidance. Histochemical and microscopic methods were developed to test the hypothesis that induced lignin deposition plays a vital role in disease resistance trait in apple roots. Features of induced lignin output in response P. ultimum infection were systematically assessed between resistant and susceptible apple rootstock genotypes. Maule and Wiesner lignin-staining methods were compared, and the Maule test was found to be more efficient for non-fixed root tissue. Both brightfield and fluorescence microscopic images were analyzed to compare the intensity of lignin deposition in the parenchyma cells of infected root cortex tissues. Results suggest intrinsic differences in lignin richness and/or monolignol composition between individual O3R5 genotypes and during P. ultimum infection. These findings provide valuable perspective for future breeding efforts and new management strategies for soilborne diseases of fruit trees. Nine different apple root phytoalexins (obtained via a previous research collaboration with chemists at Technische Universität, Braunschweig, Germany) were evaluated for their inhibitory activity against a panel of key apple replant pathogens in Washington State including Pythium irreglurare, Pythium ultimum, Rhizoctonia solani AG-5 and Cylindrocarpon destructans. Each compound was tested at three different “ecologically relevant” concentrations (based on natural volumetric concentrations in fresh root tissue) in triplicate using the disk diffusion method. Five of the compounds demonstrated inhibitory activity toward the apple root pathogens tested. The growth of P. ultimum and P. irregulare was inhibited by noraucuparin, noreriobofuran and 2-hydroxy-4-methoxydibenzofuran. Growth of C. destructans was reduced by 2'-3-dihydroxy-5-methoxybiphenyl and 2'-hydroxyaucuparin. R. solani AG-5 was inhibited by aucuparin, 2'-hydroxyaucuparin and 2-hydroxy-4-methoxydibenzofuran. These analyses imply that a diversity of compounds in the root zone, many of which are underexplored, have the capacity to mitigate the activity of ARD pathogens. Active compounds were selected for use in a subsequent liquid-based assay designed for quantitative determination of fungal/oomycete growth inhibition (i.e., IC50 value and MIC). These assays are currently underway.
For Sub-objective 1B, efforts towards building infrastructure and measuring root system architecture in pears have continued. In addition to growth responses in agar-based media, growth in prototypes for mesocosms (allowing for 3D visualization of root growth in soil) and rhizotron boxes (allowing for 2D visualization) has begun. Determination and improvement of rooting protocols for diverse germplasm is also ongoing. Wenatchee scientists also made headway towards exploring connections between root system architecture and the root-associated microbiome, a research area which is not well developed or understood. The selective capacity of pear rootstock genotype on the composition of the rhizosphere microbiome was assessed using Pyrus Old Home x Farmingdale (OHxF) 97 and 87 rootstocks (received from Willamette Nurseries, Oregon, in March of 2023) planted into replant soils from different orchard locations (previously planted to apple). Trees were received in poor condition. Due to lack of growth, new pear rootstocks were ordered in October of 2023 and received in March of 2024 (Raintree Nursery, Washington). The repeat experiment was started in June of 2024. In addition, a variety of pear genotypes (including OHxF 87 and 97) were micropropagated on location for use in bioassays with orchard replant soil. In order to propagate these trees, different media formulations and inputs were tested to optimize micropropagation and rooting conditions. Bioassay experiments are planned for July of 2024.
Through a collaboration with researchers at the University of Turin, Italy, a visiting PhD student worked in a scientist’s lab for six months (11/2023-5/2024) on a project designed to explore potential similarities in kiwivine decline syndrome (KVDS) and apple replant disease (ARD) pathosystems, disease systems which are similar in temporal and biological attributes. Differentially expressed genes (relative to uninfected controls) were identified in apple and in kiwifruit based on the analysis of high-quality transcriptomic datasets. Validation of kiwifruit RNA Seq data is currently in progress. ARS scientists also continue research on the use of arbuscular mycorrhizal fungi (AMF) to enhance agricultural practices in apple production systems. Compatible apple rootstock/AMF species combinations were previously reported by ARS scientists. Subsequent experiments designed to assess specific functional benefits resulting from compatible associations, including control of replant pathogens, are currently in progress. Further, ARS scientists are participating in collaborative research with Washington State University on a Long-Term Agricultural Research and Extension (LTARE) site focused on understanding soil health, soil-borne disease, and root architecture responses to different soil amendments over time. ARS scientists will analyze soil for microbiome composition, assess apple replant responses and measure root architecture via minirhizotrons.
Under Sub-objective 1C, sibling germplasm from a population segregating for a dwarfing trait was received in the previous year and transitioned to soil. Trees were received in poor health, and new trees have been ordered, to be received in the coming year spring. These trees will be further along in their growth cycle and will be able to be grafted with Bartlett scions during the same spring. Samples from across Bartlett and Anjou tissues have been sequenced and analysis has begun for the development of a gene expression atlas, which will be useful for obtaining correct gene models and understanding expression patterns. This will allow for determination of the correct gene sequences to use for complementation and subcellular localization experiments. Additionally, ARS scientists worked with the Washington State University pear rootstock breeding program to determine potentially important genes within a recently identified Quantitative Trait Locus (QTL) for dwarfing. In the previous year, samples from a breeding population were sent for low-pass sequencing. This year, analysis began to determine genomic sequences associated with the dwarfing trait.
In support of Sub-objective 1D, optimization of callus transformation has been significantly improved, which is observable through the fluorescent marker in the inducible Rapid Cycle Breeding (RCB) construct. Adventitious shoot regeneration remains difficult, and efforts are ongoing to optimize this process. This year, an ARS scientist obtained a strain of agrobacterium from a collaborating lab at Oregon State University that, when co-inoculated with agrobacterium containing a construct of interest, has shown to effectively signal to callus to differentiate and regenerate shoots. Current experiments with this agrobacterium are underway and many more shoots have been regenerated and will soon be genotyped. Through this troubleshooting process, much has been learned about transformation and regeneration in pears. In addition, a second, non-inducible RCB construct has been developed to test in transformations.
For Objective 2, several groups of apple candidate genes, including members of two gene families, MATE (Multidrug and Toxic Compound Extrusion) and MLO, were selected for further analyses on bioinformatics features, expression profiles and genotype-specific sequence variations. Genes encoding MATE transporters represent a well conserved family which exists from microorganism to plant and human (Sub-objective 2A,). Based on our previous transcriptome data, the expression profiles for each and all members in MATE gene family were contrasted and summarized between a resistant and a susceptible genotype during P. ultimum infection. Subsequently, an in-depth study was carried out for MdMATE52, which showed differential induction between resistant and susceptible rootstock genotypes. The sequence features (binding sites for potential transcription factors at the promoter region and mutations within coding regions) were compared through targeted cloning and sequencing of resistant and susceptible genotypes. The results from these analyses provided essential information for designing precise guide RNA for subsequent CRISPR-cas9-mediated transgenic studies (Sub-objective 2B).
In support of Sub-objective 2C, three apple genes were selected for transgenic study based on current and earlier investigation of genotype-specific expression analysis and sequence features. These three genes are: MdWRKY33 (MD04G1167700) which encodes a transcription factor, MdMATE52) which encodes a transporter and MdMLO1 (MD12G1121200) which is homolog to a mildew resistance locus-like gene. Several steps in generating the transgenic plants have been completed for all three candidate genes, including 1. cloning the genomic copy from specific genotypes and designing the guide RNA; 2. constructing binary and Agrobacterium-specific plasmid; and 3. performing the initial transgenic procedure, i.e., infect apple leaf tissue using an agrobacterium strain carrying designated construct. Currently, potential transgenic callus from these infected leaf tissues is being induced to form bona fide transgenic plants.
Accomplishments
Review Publications
Somera, T.S., Mazzola, M., Cook, C. 2023. Directing the apple rhizobiome toward resiliency post-fumigation. Agriculture. 13(11). Article 2104. https://doi.org/10.3390/agriculture13112104.
Waite, J.M., Hollender, C.H., Eilers, J.R., Burchard, E.A., Dardick, C.D. 2024. Peach LAZY1 and DRO1 protein-protein interactions and co-expression with PRAF/RLD family support conserved gravity-related protein interactions across plants. microPublication Biology. Article 000995. https://doi.org/10.17912/micropub.biology.000995.
Zhu, Y., Rainbow, J.L., Zhou, Z. 2024. Microscopic features of lignin deposition patterns in young apple roots using brightfield and fluorescence imaging. Fruit Research. 4. Article e007. https://doi.org/10.48130/frures-0023-0045.
Zhu, Y., Zhou, Z. 2023. Challenges and progress in evaluating apple root resistance responses to Pythium ultimum infection. American Journal of Plant Sciences. 14(12):1410-1429. https://doi.org/10.4236/ajps.2023.1412095.
Cook, C., Huskey, D.A., Mazzola, M., Somera, T.S. 2024. Effect of rootstock genotype and arbuscular mycorrhizal fungal (AMF) species on early colonization of apple. Plants. 13(10). Article 1388. https://doi.org/10.3390/plants13101388.
Waite, J.M., Dardick, C.D. 2024. IGT/LAZY genes are differentially influenced by light and required for light-induced change to organ angle. BMC Biology. 22. Article 8. https://doi.org/10.1186/s12915-024-01813-4.
Waite, J.M., Gottschalk, C.C., Reinhold Aboosaie, L.A., Bassil, N.V., Volk, G.M., Postman, J., Elkins, R., Bell, R. 2024. Vulnerability of pear (Pyrus) genetic resources in the U.S. Genetic Resources and Crop Evolution. https://doi.org/10.1007/s10722-024-01990-9.