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.
1b. Approach (from AD-416):
Obj 1: Anaerobic soil disinfestation (ASD) will be applied using different carbon inputs and soils sampled on a periodic basis. Metabolites extracted from soil will be analyzed using GC-MS and LC-MS methods. Concurrently, the effect of the ASD process on pathogen viability will be determined. Studies will be conducted in soils artificially infested with the target pathogens, and pathogen density will be determined using qPCR protocols. Community profiling using NextGen sequencing will be be used to associate specific microbial taxa with changes in the metabolome, and ultimately relationship to observed pathogen suppression. Operation Taxonomic Unit counts from soil microbial community analysis and relative metabolite amounts will be subjected to ANOVA-simultaneous component analysis. Network analysis can then indicate correlated metabolic and microbial activity unique to ASD treatment, potentially indicating metabolites produced in relation to activity of certain microbial taxa. Obj 2: A series of susceptible and tolerant apple rootstocks will be evaluated to asses the effect of genotype on the root microbiome and its influence on disease development. Root infestation by an introduced pathogen will be determined by qPCR and composition of the rhizosphere microbiome as well as the endophyte community will be determined by NextGen sequence analysis. Greenhouse and field trials will be performed to determine the influence of rootstock genotype on the efficacy of anaerobic soil disinfestation and Brassica seed meal amendments for the control of apple replant disease. Relative disease control efficacy of soil treatments will be assessed by monitoring components of the replant disease pathogen complex using qPCR methods. Obj 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. Obj 4: Rootstock genotypes will be phenotypically analyzed for susceptibility to apple replant disease. A number of 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 reponses. Plants will be propagated through tissue culture and exposed individually to one of the target pathogens for a select period of time. Plants will be sampled and total RNA isolated to assess relative expression of the target genes. Based on gene expression pattern analysis, five to eight 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.
3. Progress Report:
This is the first report for this project which began in March of 2017. Please see the report for the previous project, 2094-21220-001-00D, "Integration of Host-Genotype and Manipulation of Soil Biology for Soil-borne Disease Control in Agro-Ecosystems”, for additional information. A field trial was established in the conduct of studies associated with objective 2 to determine potential of anaerobic soil disinfestation (ASD) as an alternative method for the control of apple replant disease in a tree fruit nursery setting, and whether rootstock genotype would influence ASD disease control efficacy. ASD was conducted using orchard grass as the carbon input at a rate of twenty ton per hectare. The trial employed three apple rootstock genotypes varying in tolerance/susceptibility to apple replant disease; including the rootstocks M.9 (susceptible), G.41 (moderately tolerant), and G.935 (tolerant). Rootstock growth in ASD treated soils was comparable to that attained in response to 1,3-dichloropropene/chloropicrin (Telone-C35) pre-plant soil fumigation. Apple rootstock growth in ASD treated soil was superior to that obtained in control soil irrespective of rootstock genotype. Contrary to previous reports on assumed host susceptibility, under field conditions quantity of the replant pathogens Pythium ultimum and Rhizoctonia solani AG-5 DNA detected in root tissue from the ‘susceptible’ M.9 rootstock was significantly lower than that detected in roots of moderately tolerant G.41 rootstock. ASD improved soil nutrient levels, especially NO3- N, compared to the fumigation and control treatments and significantly suppressed post-treatment orchard weed biomass. ASD and soil fumigation treatments significantly altered composition of the soil microbiome and this effect was prolonged in ASD treated soils. Modification of the microbiome may have been beneficial in terms of increasing nutrient availability and pathogen suppression in orchard soil thereby improving rootstock growth in general, while enhancing even the growth of the apple replant disease (ARD) susceptible M.9 rootstock. Hence ASD using orchard grass as the carbon input can serve as an economically viable method for control of ARD in tree fruit nursery operations. In conduct of studies associated with Objective 1 and 3, collection, extraction, and liquid chromotography-mass spectrometry procedures for the recovery and analysis of the soil metabolome and exudates from apple roots were successfully developed and implemented. Profiles of phenolic apple root exudate metabolites were compared across four commonly utilized apple rootstock genotypes; G.41, G.935, M.9Nic29, and M.26. G.41 and G.935 have generally demonstrated field tolerance to apple replant disease while M.9Nic29 and M.26 are highly susceptible. Levels of fifteen phenolic compounds, including benzoic acid, kaempferol, rutin, phloridzin, phloretin, and 4-hydroxybenzoic acid and numerous other unidentified compounds were compared. On a root weight adjusted basis, benzoic acid was higher in exudates from G.935 than M.9Nic29 and M.26, while kaempferol was higher for M.26 than G.935 and G.41, and concentration of rutin in root exudates was higher for M.26 than all rootstock genotypes. Phloridzin and phloretin were higher in exudates from M.9Nic29 than G.41 and G.935 at the inception of the experiment but did not differ at later time points over the two-month assessment period. Differentiation in metabolite levels between G.41/G.935 versus M.9Nic29/M.26 rootstocks was also apparent in the levels of other unidentified compounds. In studies associated with Objective 3, separation between G.41/G.395 and M.9Nic29/M.26 rootstocks was also apparent in the levels of other unidentified compounds. Assessment of the effects of bulk root exudates on the soil microbiome did not reveal consistent transformation in the soil microbiome developing according to rootstock genotype in a short time frame (2 months or less). However, previous work conducted on rhizosphere (root zone) soil over a longer time frame has demonstrated that rootstock genotypes develop distinct functional microbial communities in the zone of soil immediately surrounding the roots. On-going assessment of natural biochemical compounds recovered from the apple root zone indicates that the compounds 4-hydroxybenzoic acid, benzoic acid, and phloridzin can inhibit Pythium ultimum, a pathogen which contributes to apple replant disease. In addition, 4-hydroxybenzoic acid and benzoic acid were shown to inhibit growth of Phytophthora cactorum, causal agent of collar rot and root rot of apple, in a concentration-dependent manner. These results potentially contribute to explanation of differential tolerance to apple replant disease among rootstock genotypes. In studies associated with Objective 4, progeny from a cross between the cultivars ‘Ottawa 3’ and ‘Robusta 5’ (O3 X R5) were used to identify potential mechanisms of resistance to apple root pathogens. It was proposed and partially confirmed that a wide spectrum of resistance phenotypes exist among the cross population. P. ultimum was used as a model ARD pathogen for defining the resistance responses in roots of these germplasm. A major obstacle in the phenotyping of apple root resistance to plant pathogens is the lack of genetically uniform plant material. This impediment was overcome by implementing a tissue culture based micropropagation procedure enabling generation of apple rootstock plants with uniform genetic background of equivalent developmental stage. Among an initial twenty O3 x R5 progenies, differential resistance to P. ultimum was observed with plant survival rate varying from ten to ninety percent. In addition, microscopic observation of infected roots from tolerant germplasm often exhibited a “defined zone” that clearly demarcated healthy from infected root tissue, a response that may indicate pathogen inhibition. In contrast, profuse growth of the pathogen with no limitation to progression through root tissue was observed in susceptible germplasm resulting in wide-spread tissue necrosis and collapse. This germplasm possessing defined phenotyping data will be fundamental for functional validation of the role of candidate genes identified from previous transcriptome analysis in host resistance responses.
1. Anaerobic soil disinfestation (ASD) effectively controls tree fruit nursery replant disease. Apple replant disease (ARD) is a major impediment to the economic viability of orchard and nursery production systems. ARS scientists at Wenatchee, Washington, demonstrated that ASD conducted using orchard grass as an economically acceptable carbon input resulted in effective replant disease control. Multi-year apple rootstock growth in a replant nursery environment was equivalent to that attained using the standard treatment of pre-plant soil fumigation. The ASD treatment growth response was comparable to that attained in response to soil fumigation even when the comparison was made concerning the disease susceptible rootstock genotype M.9. Although pre-plant 1,3-dichloropropene/chloropicrin soil fumigation did not affect weed emergence and growth, ASD significantly reduced weed biomass in this field trial. This ASD protocol may serve as a viable non-fumigant alternative for the control of ARD in nursery production systems.
Leisso, R.S., Rudell, D.R., Mazzola, M. 2017. Metabolic composition of apple rootstock rhizodeposits differs in a genotype-specific manner and affects growth of subsequent plantings. Soil Biology and Biochemistry. 113:201-214.
Zhu, Y., Shao, J.Y., Zhou, Z., Davis, R.E. 2017. Comparative transcriptome analysis reveals a preformed defense system in apple root of a resistant genotype of G.935 in the absence of pathogen. International Journal of Plant Genomics. doi: 10.1155/2017/8950746.