1a. Objectives (from AD-416):
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. 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: Define the functional roles of candidate genes conferring resistance to apple replant pathogens. [NP303, C3, PS3A] • Subobjective 4A: Evaluate apple root resistance phenotypes for genotypes in an apple rootstock cross population during their interactions with apple replant disease (ARD) pathogens. • Subobjective 4B: Analyze the function of selected apple candidate genes to infer their roles in activating defense responses and conferring ARD resistance. • Subobjective 4C: Examine the sequence features of genomic DNAs containing functionally analyzed apple genes. Objective 5: 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.
1b. Approach (from AD-416):
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.
3. Progress Report:
Progress was made on all four objectives and sub-objectives, 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. Objective 1: Studies were completed to document shifts in the complete soil metabolome resulting from the application of anaerobic soil disinfestation (ASD) to an orchard replant soil. The time course study demonstrated that dramatic transformation in soil metabolite composition occurred during the ASD process with changes continuously detected over a four-week period in both consumption and production of metabolites. Co-abundance correlation indicated that production of certain biologically active metabolites during the study period was associated with corresponding changes in composition of the soil microbiome. Progress was made in identifying important components of the soil microbiome and metabolome that operate to suppress plant disease through the ASD process. Amplification of these microbial and metabolic elements through modification of the ASD implementation process should be targeted to enhance overall efficacy of this disease control method. Objective 2: Significant progress was made toward the goal of reducing overall production systems input of a Brassica seed meal (SM) amendment for control of apple replant disease by integrating the treatment with the appropriate apple rootstock genotype. In greenhouse trials, a SM rate 1/3 (2.2 t ha-1) of the conventional application rate (6.6 t ha-1) resulted in significant suppression of apple replant disease symptoms and overall pathogen root infection when the treatment was employed in conjunction with apple rootstocks G.41 and G.210, but not M.9 and MM.106. When SM was applied at 2/3 (4.4 t ha-1) the conventional rate, overall disease control efficacy and tree growth was equivalent or superior to that obtained relative to the full-rate treatment when employed with rootstocks G.41 and G210. Field trials are ongoing, and at this time a 1/3 reduction in the SM amendment rate (4.4 t ha-1) has resulted in tree growth equivalent to the full-rate SM treatment for rootstocks M.26 and G.41. The 4.4 t ha-1 treatment has resulted in tree growth that is superior to pre-plant soil fumigation when implemented with rootstock G.41. This significant reduction in SM amendment rate may accelerate the commercial adoption of this technique and ensure economic benefits to growers by reducing orchard establishment costs. Objective 3: Additional progress was made toward defining differences among apple rootstock genotypes in terms of the metabolic composition of root exudates/rhizodeposits and its impact on biotic and abiotic attributes of the rhizosphere soil habitat. In a greenhouse experiment assessing the impact of rootstock genotype exudates on a spatially separated orchard soil, the soil microbiome did not differ consistently among rootstock genotypes after 6 weeks of exudate percolation. However, rootstock cultivar-specific differences in the soil microbiome were apparent at 12 weeks demonstrating the temporal fashion at which changes in the microbial community are established in response to root exudates. Cultivation of an apple rootstock in an orchard soil lowered the soil pH relative to the control soils where no trees were planted. However, rhizosphere soil pH did not differ significantly among rootstock cultivars. Sub-objective 4a: An additional 30 genotypes derived from a cross between the rootstocks ‘Ottawa 3’ and ‘Robusta 5’ were subjected to careful phenotypic analysis of resistance responses to root pathogens. As a result, a panel of 12 genotypes were selected for their highly reproducible resistance or susceptible phenotype to infection by Pythium ultimum. In a comparison of highly resistant and susceptible genotypes, the overall plant survival rates ranged from 10-30 percent for susceptible genotypes and from 80-100 percent for resistant genotypes when inoculated with P. ultimum. Root and shoot biomass of the seedlings was positively correlated with the overall plant survival rates among tested genotypes. The distinguishable and revealing patterns of necrotic progression between resistant and susceptible genotypes were documented for the first time by non-interruptive, non-destructive and minimally-invasive observations under dissection microscope. The characterized panel of genotypes identified here will serve as valuable material in studies seeking to dissect the molecular regulation network underlying apple rootstock resistance traits. Furthermore, this material will be of use in developing future diagnostic tools for marker-assisted breeding of disease resistant apple rootstocks. Preliminary data also indicated an association between resistance to the oomycete P. ultimum and the fungal pathogen Rhizoctonia solani; but no relationship with relative resistance of these genotypes to root infestation by the lesion nematode, Pratylenchus penetrans. Sub-objective 4B: A panel of apple rootstock genotypes were selected for their distinct resistance levels to P. ultimum infection from the on-going phenotyping effort. Progress from the phenotype assessment allowed for the design of experiments to validate the roles of specific candidate genes in contribution to the observed resistance response in apple roots. Based on transcriptome data analysis, 20 apple genes were selected as primary candidates for gene expression pattern analysis among the resistant and susceptible rootstock genotypes. The expression patterns for selected apple genes which demonstrated an association with resistance level among six pairs of rootstocks are considered elite candidate genes for subsequent analysis. Their full-length DNA sequences of these genes have been obtained from the genome database for rosaceae (www.gdr.org); and gene-specific primers have been designed using web-based software. Plant root tissues, both mock-inoculated and P. ultimum infected at four timepoints, from six pairs of resistant and susceptible O3R5 genotypes have been collected. Preliminary expression data for most of the targeted 20 genes has been obtained and the relationship between gene expression patterns and resistance levels are currently under analysis. The protocol for validating the gene functions by transgenic approach using Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9 (CRISPR/cas9) technology has been fine-tuned and is ready for implementation once target genes have been identified.
1. Brassica seed meal soil amendment results in up-regulation of defense genes in apple root tissue. A long-held model formerly recognized glycosylate hydrolysis products as a primary determinant of pest suppression incurred in response to soil incorporation of Brassica plant residues. ARS scientists in Wenatchee, Washington, subsequently demonstrated that multiple mechanisms of action, both biological and chemical, contribute to seed meal derived soil-borne disease control and recently established that roots of apple trees planted in seed meal amended soil exhibit enhanced expression of several plant defense and stress response genes. Changes in composition of the rhizosphere microbiome were temporally associated with initiation of plant defense responses in the seed meal amended soil and the corresponding reduction in apple root infection by plant pathogens. Bacterial groups involved in production of nitric oxide, a metabolite previously shown to trigger plant defenses, or functioning in direct pathogen suppression were more abundant in the rhizosphere of trees cultivated in the seed meal amended soil. Changes in gene expression in roots of apple when trees were planted in seed meal amended soil were more robust and prolonged for apple rootstock G.210 than for rootstock M.26. Optimal performance of the Brassica seed meal treatment for control of soil-borne pathogens of apple will be obtained when growers utilize the treatment in combination with the appropriate responsive rootstock genotype.
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