Location: Physiology and Pathology of Tree Fruits Research
2022 Annual Report
Objectives
This project will investigate the effect of host genotype on composition and
activity of the rhizosphere microbiome, in concert with host resistance attributes and organic soil amendment strategies, as a means to manage soil-borne diseases of fruit crops incited by diverse pathogen complexes.
Objective 1: Define the metabolic and biological constituents functional in soil-borne disease suppression attained via organic input methodologies. [NP303, C3, PS3A]
• Subobjective 1A: Determine the spectrum of metabolites produced during Anaerobic Soil Disinfestation (ASD) as affected by carbon input.
• Subobjective 1B: Characterize shifts in soil/rhizosphere microbiome associated with ASD and correlate with suppression of apple and strawberry soil-borne pathogens.
• Subobjective 1C: Characterize the effect of management practices on soil inoculum density of potential post-harvest pathogens and subsequent colonization of the phyllosphere and/or carposphere by these potential pathogens.
Objective 2: Assess plant genotype specificity for composition of the root microbiome and its relationship to disease susceptibility/tolerance. [NP303, C3, PS3A]
• Subobjective 2A: Conduct microbial profiling (NextGen sequencing) to determine relative differences in composition of the microbiome recruited by tolerant and susceptible apple rootstocks.
• Subobjective 2B: Determine the effect of apple rootstock genotype on efficacy of reduced rate Brassica seed meal amendments or ASD for control of replant disease.
Objective 3: Determine the metabolic composition of exudates from disease tolerant and susceptible rootstocks and assess their effect on rhizosphere microbial recruitment. [NP303, C3, PS3B]
• Subobjective 3A: Define differences in apple root exudate metabolite profiles produced by rootstock cultivars that differ in susceptibility to soil-borne plant pathogens.
• Subobjective 3B: Test the impacts of apple root exudate metabolites, alone or in combination, on components/entirety of the soil microbiome.
Objective 4: Identify genetic sources of pathogen resistance and contribute to improved pest-resistant, size-controlling rootstocks to enhance orchard efficiency in pears. [NP303, C3, PS3A] Benefits will include availability of dwarfing, precocious, cold hardy, disease-resistant, and easily propagated rootstocks adapted to various U.S. production areas and enhanced genetic understanding of host-pathogen-environment interactions for sustainable and profitable pear orchard systems.
Approach
Objective 1: ASD will be applied using different carbon inputs and soils sampled on a periodic basis. Metabolites will be extracted from soil and analyzed using GCMS and LC-MS methods. Concurrently, the effect of the ASD process on pathogen viability will be determined. Effect of ASD on pathogen density will be determined using qPCR protocols. Profiling of the microbiome using NextGen sequencing will be conducted to associate specific microbial taxa with changes in the soil metabolome, and ultimately relationship to observed pathogen suppression. OTU taxonomic counts from soil microbial community analysis and relative metabolite amounts will be subjected to ANOVA-simultaneous component analysis. Network analysis will be used to correlate metabolic and microbial activity unique to ASD treatment, potentially indicating metabolites produced in relation to activity of certain microbial taxa.
Objective 2: A series of susceptible and tolerant rootstocks will be evaluated to assess the effect of genotype on the root microbiome and its influence on disease development. Pathogen root infestation will be determined by qPCR and composition of the rhizosphere and endophytic microbiome will be determined by amplicon sequence analysis. Greenhouse and field trials will assess the influence of rootstock genotype on efficacy of ASD and Brassica seed meal amendments for the control of apple replant disease. Disease control efficacy of soil treatments will be assessed by monitoring the replant disease pathogen complex using qPCR methods.
Objective 3: The interaction of the rhizosphere and orchard soil eventually determines composition of orchard soil and rhizosphere associated microbial communities that regulate numerous processes. Root exudates among genotypes will be evaluated for the presence of potentially antimicrobial exudates or symbiotic/mutualistic recruitment signaling molecules. Collected root exudates will be analyzed by LC-MS. Exudates will be assayed for capacity to inhibit the growth of soil-borne pathogens. Exudates will also be applied directly to orchard soils and their effect on pathogen population dynamics and composition of the soil microbiome will be assessed.
Objective 4: Rootstock genotypes will be phenotypically analyzed for susceptibility to apple replant disease. Susceptible and potentially resistant genotypes will be utilized in studies to assess the function of selected apple candidate genes to infer their roles in activating defense responses. Tissue culture generated plants will be exposed individually to one of the target pathogens for a select period of time. Plant RNA will be isolated to assess relative expression of the target genes. Based on gene expression pattern analysis, selected genes showing robust association with resistance phenotypes will be subject to in planta expression manipulation to further characterize the potential role of these genes in observed host resistance.
Objective 5: Using available plant resources, quantitative genetic and genomics will be used to identify the genetic underpinning of phenotypic traits of pear such as resistance to biotic and abiotic stresses, precocity, dwarfing and cold hardiness.
Progress Report
This is the final report for project 2094-21220-002-000D, which has been replaced by new project 2094-21220-003-000D, titled, “Uncovering Rootstock Disease Resistance Mechanisms in Deciduous Tree Fruit Crops and Development of Genetics-Informed Breeding Tools for Resistant Germplasm”. All objectives and associated sub-objectives for this project have been completed, all of which fall under National Program 303, Component 1 Plant Health Management. Progress on this project focuses on Problem A Development and deployment of host resistance, Problem B, Development of biologically based and integrated disease management practices, and Problem C, Development of alternatives to pre-plant methyl bromide soil fumigation.
In support of Objective 1, research on identification and manipulation of microbial and metabolic factors that influence the efficacy of anaerobic soil disinfestation (ASD) for the control of soil-borne diseases in apple and strawberry production systems was completed. Novel (previously unreported) metabolic mechanisms of pathogen suppression were identified in response to ASD conducted using rice bran as the carbon source. Correspondingly, bacterial species possessing the capacity to produce these novel metabolites were identified based upon dynamic changes in relative microbial abundance and metabolite accumulation. Physical attributes of the carbon source utilized in ASD were found to have significant effect on generation of biologically active (pathogen suppressive) metabolites. Long-term strawberry field trials using 2-year or 4-year broccoli or lettuce crop rotations in combination with ASD or mustard seed meal amendment were completed. ASD using rice bran as the carbon input and ASD with a subsequent compost amendment induced significant changes in the soil and strawberry rhizosphere microbiome (Sub-objective 1A). The altered structure of the rhizosphere microbiome in ASD treatments was maintained throughout the strawberry growing season and was associated with significant increases in strawberry yield relative to all other soil treatments (Sub-objective 1B). Although all treatments significantly increased strawberry yield relative to a no treatment control, use of broccoli as the cover crop resulted in elevated levels of strawberry crown infection by the fungal pathogen Macrophomina phaseolina (Sub-objective 1C).
For Objective 2, research that focused on examination of genotype effects on composition of the rhizosphere and endophytic microbiome recruited by different apple rootstocks and identification of important characteristics with potential to influence plant productivity was completed. In multiple controlled environment and field experiments, rootstock genotype was demonstrated to have a significant effect on composition of the rhizosphere microbiome. Notably, genotype also determined relative endophytic colonization of apple rootstocks by mycorrhizal fungi. Among rootstock endophytic fungal communities detected across a diversity of disease tolerant and susceptible apple rootstocks, the disease tolerant rootstock G.890 consistently harbored the highest percentage of arbuscular mycorrhizal fungal species (greater than 5% of total). The effect of Brassica seed meal (BSM) amendments on apple rootstock physiology in M.26 vs. G.210 was explored/assessed via transcriptomic analysis. The temporal dynamics of gene expression indicated that the seed meal (SM) amendment altered the trajectory of the root transcriptome in a genotype-specific manner (Sub-objective 2A). Research to assess the efficacy of ASD and mustard seed meal (MSM) amendment for control of replant disease was completed through the monitoring of three orchard replant field trials established in 2017. MSM and ASD were as effective as soil fumigation in improving tree growth and yield at 3 of 3 and 2 of 3 commercial scale field trials, respectively. At all three trial sites, depending on rootstock genotype, the seed meal treatment continued to perform as well or better than soil fumigation in terms of fruit yield and tree growth. MSM was superior to soil fumigation for suppressing root populations of lesion nematode, Pratylenchus penetrans. Lesion nematode suppression persisted for three years in MSM treated soil while numbers were elevated in fumigated soil within one year of treatment to levels that were greater than that observed in the absence of preplant soil treatment. The consistent results obtained in numerous field trials over a decade demonstrate that MSM treatment is an effective measure for the control of apple replant disease and promotion of tree growth and yield (Sub-objective 2B).
For Objective 3, research on Sub-objective 3A that focused on examination of differences in root exudate composition as affected by apple rootstock genotype was completed. Studies were conducted to examine the effect of differentially abundant root exudate metabolites on composition of the soil microbiome. Metabolites that differed significantly between a disease tolerant (G.935) and susceptible (M.26) rootstock genotype included sorbitol, myo-inositol, malic acid, benzoic acid, hydroxy-benzoic acid, several triterpenoids and phloridzin. Myo-inositol was more abundant in root exudates from G.935 than M.26; interestingly this compound was evaded by the plant parasitic nematode, Pratylenchus penetrans, when evaluated using in vitro choice assays. This corresponds with the fact that M.26 supports significantly higher root populations of P. penetrans than does G.935 resulting in a greater level of damage to the susceptible rootstock. Research on Sub-objective 3B focused on benzoic acid, which was found at higher concentrations in root exudates of the tolerant rootstocks G.935 and G.41 than the susceptible rootstocks M.9 and M.26. The results showed benzoic acid significantly reduced in vitro growth of the apple root pathogens Phytophthora cactorum, Pythium ultimum and Rhizoctonia solani. In total, these findings indicate that differential metabolic composition of root exudates among apple rootstock genotypes may contribute to the relative tolerance or avoidance of rootstocks to infection by a diverse array of plant pathogens that contribute to apple replant disease.
For Objective 4, a new scientist came on board in March of 2020 and began planning and working towards this objective. With the goal of contributing to improved pest-resistant, size-controlling rootstocks to enhance orchard efficiency in pears, a collaboration with other scientists in Wenatchee, Washington, was initiated to improve genomic resources in pear. This work led to the identification and genomic characterization of dwarfing-and architecture-related genes in the Bartlett and Anjou genomes, as well as a much-improved Bartlett genome and a workflow for finding gene families in future pear genomes. Pear tissue samples across developmental stages and tissue types have been collected for the purpose of developing an atlas of gene expression for European pear, as knowledge of gene expression outside of fruit is extremely lacking and will be important for studies focusing on stem- and root-expressed genes in pear. Further, connections with scientists in Wenatchee, and Pullman, Washington, Hood River, Oregon, Davis, California, and Kearneysville, West Virginia, were established to begin the development of projects and sharing of germplasm resources related to dwarfing, architecture, and fire-blight resistance in pears. Two sets of germplasm have been received and planted, including a population of aneuploid pears and a diverse germplasm set with potential and variation of dwarfing characteristics. These will be phenotyped and used to study dwarfing and precocity in the coming years. In addition, steps were taken towards pear biotechnology capabilities: the new scientist began establishing a tissue culture lab space, establishing key common pear rootstock accessions (OHxF 87 and 97), as well as common cultivars that have been used for genomics studies (Bartlett and Anjou). Collaborations and connections were established with scientists in Riverside, California, and Kearneysville, West Virginia, to explore rapid-cycle breeding history and options and obtain constructs for inducible flowering to be introduced into pears, with the goal of drastically shortening breeding cycles. Her group has begun optimizing pear transformation protocols for successful establishment of this inducible flowering tool, as well as for future studies necessitating transgenic plants.
Accomplishments
1. Optimal conditions for soil amendment-based strategies for the control of apple replant disease (ARD) were determined. ARS researchers in Wenatchee, Washington, determined the following strategies to control ARD: 1) Brassicaceae seed meal (BSM) soil amendment and 2) anaerobic soil disinfestation (ASD). Both strategies are specifically designed to harness the potential of commercially available apple rootstock genotypes and their root-associated microbiomes, which develop in response to the amendment, to tolerate and/or suppress replant pathogens. This research helped to define optimal conditions (e.g., lowest effective amendment rate for BSM, most effective formulation of BSM, best carbon source for ASD) for integrating organic inputs with commercially available apple rootstock genotypes for the control of soil-borne pathogens. The scope of this work also demonstrated the breadth of knowledge required to attain optimal efficacy when utilizing host plant genetics in combination with amendment-based control of the soil and root microbiome as part of an integrated approach to mitigating the detrimental effects of ARD.
2. Soil amendment-based strategies prove consistent efficacy in the control of apple replant disease (ARD) and the promotion of tree growth and yield. Apple replant disease has been commercially controlled with the use of pre-plant fumigants that can harm the atmosphere. ARS researchers in Wenatchee, Washington, conducted multiple field trials to evaluate the efficacy of soil amendment-based (BSM) strategies for control of ARD. Disease control along with apple growth and yield observed with soil amendment treatment was equivalent to or exceeded that attained in response to pre-plant soil fumigation. These results demonstrated that regardless of soil type or orchard location, BSM can be utilized as an effective alternative to fumigation to improve productivity while also helping to protect the environment.
3. Gene expression analysis in commercially available apple rootstocks revealed genotype-specific responses indicative of different defense strategies. ARS researchers in Wenatchee, Washington, demonstrated that commercially available apple rootstocks may be susceptible or tolerant to different soil-borne pathogens for different reasons. For example, shorter root lifespan (higher turn-over) appears to be an important mechanism promoting replant tolerance in the rootstock G.210. In addition, expression patterns of defense-related genes were temporally associated with changes in rhizosphere microbiome density and composition. Besides opening many new avenues for further exploration, these findings add to our understanding of defense-related changes occurring under pathogenic pressure in susceptible versus resistant genotypes.
4. Specific apple root exudates that may contribute to replant tolerance or susceptibility were identified. Plants affect the microbial communities in their root zone via the release of compounds into the surrounding soil. The mix of compounds being exuded into the rhizosphere by apple plants differs according to rootstock genotype. Metabolomic studies conducted by ARS researchers in Wenatchee, Washington, showed that replant “tolerant” rootstocks share similar exudates, including sorbitol, myo-inositol, and malic acid, thought to play roles in limiting plant susceptibility to replant pathogens. Some of these metabolites were shown to inhibit the growth and/or activity of a diverse array of apple replant pathogens. These findings were recently incorporated into the National Apple Rootstock Breeding program in Geneva, New York. Furthermore, work on apple root exudates is extremely limited. These studies expand the foundation that researchers have in terms of targeting specific compounds that contribute to disease tolerance or susceptibility.
5. The genome-wide defense activation patterns in apple root to Pythium ultimum infection were determined. To date, there has been limited information regarding the molecular underpinnings regulating defense responses of apple roots to infection by necrotrophic pathogens, which are organisms that first destroy cells of their plant host and then derive their nutrition from dead host cells. Using three consecutive RNA-seq based transcriptome analyses, ARS scientists in Wenatchee, Washington, provided a panoramic and high-resolution depiction of defense activation in apple root tissue in response to P. ultimum infection. Analysis of global gene expression data between resistant and susceptible genotypes reveals a multi-phased and multi-layered molecular regulation scheme, which was the first reported and significantly expanded our knowledge about this pathosystem. These findings are crucial for subsequent hypothesis-driven research to pinpoint specific apple genes contributing to apple root disease resistance traits.
Review Publications
Liu, J., Abdelfattah, A., Wasserman, B., Wisniewski, M., Droby, S., Fazio, G., Mazzola, M., Wu, X. 2022. Contrasting effects of genotype and root size on the fungal and bacterial communities associated with apple rootstocks. Horticulture Research. 9. Article uhab013. https://doi.org/10.1093/hr/uhab013.
Zhu, Y., Zhou, Z. 2021. The genotype-specific laccase gene expression and lignin deposition patterns in apple root during Pythium ultimum infection. Fruit Research. 1. Article 12. https://doi.org/10.48130/FruRes-2021-0012.
Zhu, Y., Li, G., Singh, J., Khan, A., Fazio, G., Saltzgiver, M., Xia, R. 2021. Laccase directed lignification is one of the major processes associated with the defense response against Pythium ultimum infection in apple roots. Frontiers in Plant Science. 12. Article 629776. https://doi.org/10.3389/fpls.2021.629776.
Somera, T.S., Mazzola, M. 2022. Toward a holistic view of orchard ecosystem dynamics: A comprehensive review of the multiple factors governing development or suppression of apple replant disease. Frontiers in Microbiology. 13. Article 949404. https://doi.org/10.3389/fmicb.2022.949404.
