Research Project: Utilization of the Rhizosphere Microbiome and Host Genetics to Manage Soil-borne Diseases

Location: Physiology and Pathology of Tree Fruits Research

2019 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. 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.

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
In support of Objective 1, research 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. 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 as well as documentation in the literature of metabolic production pathways linked to a specific bacterial species. Physical attributes of the carbon source utilized in ASD were found to have significant effect on generation of biologically active (pathogen-suppressive) metabolites. For instance, both plant developmental stage and genotype had significant effects on the generation of fungal inhibitory volatiles generated during ASD when triticale residues were used as the ASD carbon source. In addition, among three species in the same Gramineae plant family, volatiles produced during ASD conducted using wheat bran (ASD-WH) or orchard grass (ASD-GR) inhibited fungal growth to a greater degree than did volatiles generated when rice bran was used as the carbon input. Correspondingly, ASD-WH and ASD-GR were more effective than ASD-RB for the control of Macrophomina crown rot of strawberry. In support of Sub-objective 2A, research 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 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; a group that plays a significant role in plant nutrition through acquisition and transport of phosphorous to plant roots. Although abundance in the rhizosphere of mycorrhizal fungi belonging to the genera Glomus and Paraglomus did not differ between rootstock genotypes G.890 and M.26, both fungal genera were documented in G.890 roots at significantly greater abundance than in roots of M.26. 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. Other fungal genera such as Terfezia and Serendipita, previously reported to form mycorrhizal associations in other plant systems, were detected at high frequencies as endophytes of apple and were detected at significantly different levels among apple rootstock genotypes. The role of these fungi in association with apple has not previously been examined but will be evaluated in forthcoming experiments. Research on Sub-objective 2B continued through the establishment of three new orchard replant field trials to assess the efficacy of anaerobic soil disinfestation and mustard seed meal amendment for control of replant disease. Monitoring of an orchard trial planted in 2016 continued and, 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. Research on Sub-objectives 3A and 3B continued to examine 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 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 conducting 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. 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, 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. In support of Objective 4, research continued to assess apple rootstock germplasm for resistance to the oomycete pathogen Pythium ultimum, which is a component of the pathogen complex that incites apple replant disease. Under Sub-objective 4A, screening of 60 genotypes resulting from a breeding population (Ottawa 3 x Robusta 5) resulted in the identification of ten resistant genotypes. The resistance trait was characterized by delayed progression of pathogen-induced root necrosis, which suggested the existence of an effective defense mechanism. Under Sub-objective 4B, twenty-five candidate genes were previously selected based upon comparative transcriptome analysis in resistant and susceptible rootstock genotypes. Two of these genes, MdCERK1 (a receptor possibly involved in pathogen detection) and MdWRKY33 (a transcription factor involved in plant defense), were selected for transgenic expression manipulation. Transgenic plants, using CRISPR/cas9 technology and Agrobacterium-mediated genetic transformation, have been generated and are being analyzed for the potential altered resistance due to gene knock-out. The germplasm resulting from the Ottawa3 x Robusta5 (O3/R5) cross is likely to possess a certain degree of divergence from the publicly available apple genome sequences. Therefore, under Sub-objective 4C, genomic fragments from the O3/R5 germplasm corresponding to the MdWRKY33 and MdCERK1 genes were cloned and sequenced. This sequence data was used to design "guide RNA" (gRNA) for transgenic manipulation using CRISPR/Cas9 based gene knockout technology.

Accomplishments

Review Publications
Garton, W., Mazzola, M., Dasgupta, N., Alexander, T.R., Miles, C.A. 2018. Efficacy of excision, cauterization, and fungicides for management of apple anthracnose canker in maritime climate. HortTechnology. 28(6):728-736. https://doi.org/10.21273/HORTTECH04148-18.
Aguilar, C., Mazzola, M., Xiao, C. 2019. Timing of perennial canker development in apple trees caused by Neofabraea perennans and Neofabraea kienholzii. Plant Disease. 103(3):555-562. https://doi.org/10.1094/PDIS-06-18-0935-RE.
Wang, L., Mazzola, M. 2019. Interaction of Brassicaceae seed meal soil amendment and apple rootstock genotype on microbiome structure and replant disease suppression. Phytopathology. 109(4):607-614. https://doi.org/10.1094/PHYTO-07-18-0230-R.
Garton, W., Mazzola, M., Alexander, T.R., Miles, C.A. 2019. Efficacy of fungicide treatments for control of anthracnose canker in young cider apple trees in Western Washington. HortTechnology. 29(1):35-40. https://doi.org/10.21273/HORTTECH04201-18.
Nyoni, M., Lotze, E., Mazzola, M., Wessels, J.B., McLeod, A. 2019. Evaluating different approaches in the application of phosphonates for the control of apple root diseases. Australasian Plant Pathology. 48(5):461-472. https://doi.org/10.1007/s13313-019-00647-x.
Nyoni, M., Mazzola, M., Wessels, J.B., McLeod, A. 2019. The efficacy of semiselective chemicals and chloropicrin/1,3-dichloropropene-containing fumigants in managing apple replant disease in South Africa. Plant Disease. 103(6):1363-1373. https://doi.org/10.1094/PDIS-10-18-1844-RE.
Wang, L., Mazzola, M. 2019. Effect of soil physical conditions on emission of allyl isothiocyanate and subsequent microbial inhibition in response to Brassicaceae seed meal amendment. Plant Disease. 103:846-852. https://doi.org/10.1094/PDIS-08-18-1389-RE.
Sikdar, P., Mazzola, M., Xiao, C. 2019. Genetic and pathogenic characterization of Phacidiopycnis washingtonensis from apple and Pacific madrone from western United States. Phytopathology. 109(3):469-479. https://doi.org/10.1094/PHYTO-10-17-0358-R.
Wang, L., Mazzola, M. 2019. Field evaluation of reduced rate Brassicaceae seed meal amendment and rootstock genotype on the microbiome and control of apple replant disease. Phytopathology. 109(8):1378-1391. https://doi.org/10.1094/PHYTO-02-19-0045-R.
Leisso, R.S., Rudell Jr, D.R., Mazzola, M. 2018. Targeted metabolic profiling indicates apple rootstock genotype-specific differences in primary and secondary metabolite production and validate quantitative contribution from vegetative growth. Frontiers in Plant Science. 9:1336. https://doi.org/10.3389/fpls.2018.01336.
Mazzola, M., Hewavitharana, S.S. 2019. Advances in understanding tree fruit-rhizosphere microbiome relationships for enhanced plant health. In: Lang, G., editor. Achieving Sustainable Cultivation of Temperate Zone Free Fruits and Berries. Volume 1: Physiology, Genetics and Cultivation. Cambridge, UK: Burleigh Dodds Science, p. 3-30. https://doi.org/10.19103/AS.2018.0040.01.
Zhu, Y., Saltzgiver, M.J., Zhao, J. 2018. A phenotyping protocol for detailed evaluation of apple root resistance responses utilizing tissue culture micropropagated apple plants. American Journal of Plant Sciences. 9(11):2183-2204. https://doi.org/10.4236/ajps.2018.911158.
Zhu, Y., Zhao, J., Zhou, Z. 2018. Identifying an elite panel of apple rootstock germplasm with contrasting root resistance to Pythium ultimum. Journal of Plant Pathology & Microbiology. 9(11):1000461. https://doi.org/10.4172/2157-7471.1000461.
Zhu, Y., Shao, J.Y., Zhou, Z., Davis, R.E. 2019. Genotype-specific suppression of multiple defense pathways in apple root during infection by Pythium ultimum. Horticulture Research. 6:10. https://doi.org/10.1038/s41438-018-0087-1.