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.
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 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. Research in support of Sub-objective 1A, continued 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. 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. 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). Research on Sub-objective 2A, continued 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. In multiple orchard replant soils, rootstock genotype was demonstrated to have a significant effect on composition of the endophytic microbiome. Notably, genotype determined composition of the mycorrhizal fungal community that resided in plant roots. Among rootstock endophytic fungal communities detected across a diversity of disease tolerant and susceptible apple rootstocks, and the disease tolerant rootstock G.890 consistently harbored the highest percentage of arbuscular mycorrhizal fungal species (>5% of total). Interestingly, rootstock genotype did not influence the composition of the mycorrhizal community detected in the rhizosphere of apple rootstocks. This finding indicates that factors expressed at the rootstock genotype level influence the relative ability of these fungi to effectively form association with this plant host. Ilyonectria robusta, which functions as a pathogen of apple, was detected at high relative abundance in the endosphere of all genotypes, with the highest relative abundance in moderately tolerant rootstock G.41, and in the susceptible rootstocks M.26 and M.9. I. robusta was present at relatively lower abundance in the tolerant Geneva series apple rootstocks. Interestingly, although G.41 rootstock is considered disease ‘tolerant’, unlike other Geneva rootstocks, it was derived from a cross that contained a Malling rootstock parent, that being M.9. These results provide further insight into rhizosphere and endophytic microbial communities of apple rootstocks, which could be exploited or manipulated to improve tree fruit agricultural management practices with respect to plant nutrition and disease control. Research in support of Sub-objective 2B, continued through the monitoring of three orchard replant field trials established in 2017 to assess the efficacy of ASD and mustard seed meal (MSM) amendment for control of replant disease. 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 pre-plant 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. Research on Sub-objective 3A, continued on examination of differences in root exudate composition as affected by apple rootstock genotype. In addition, studies were conducted to examine the effect of differentially abundant root exudate compounds on composition of the soil microbiome. A number of compounds differed significantly between a disease tolerant (G.935) and susceptible (M.26) rootstock genotype. A compound that was higher in G.935 is known to repel plant parasitic nematodes and this difference corresponds with the fact that M.26 supports significantly higher root populations of nematodes 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 it significantly reduced in vitro growth of the apple root pathogens Phytophthora cactorum, Pythium ultimum and Rhizoctonia solani. Phloridzin, which is found at higher quantities in exudates from susceptible apple rootstocks, and previously has been reported as having anti-microbial properties, did not have any effect on the growth of these pathogenic fungi and oomycetes. 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. With regard to research progress on Objective 4, a new SY came on board March 23, 2020 who will be addressing this objective. Research activity planning is ongoing, and necessary personnel and material resources are being secured.
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