Characterization of soils properties associated with suppression of Fusarium wilt in spinach seed crops, and development of a quantitative molecular assay for Fusarium oxysporum f. sp. spinaciae.

Project Overview

Project Type: Graduate Student
Funds awarded in 2008: $19,960.00
Projected End Date: 12/31/2010
Region: Western
State: Washington
Graduate Student:
Major Professor:
Dr. Lindsey du Toit
Washington State University


  • Vegetables: greens (leafy)


  • Crop Production: foliar feeding, tissue analysis
  • Education and Training: extension, on-farm/ranch research, participatory research
  • Farm Business Management: risk management
  • Pest Management: cultural control, economic threshold, integrated pest management, physical control
  • Soil Management: soil analysis

    Project objectives from proposal:

    Project Description

    (a) Statement of Problem
    The long, dry summer days with mild temperatures in the maritime Pacific Northwest (PNW) make this the only region of the U.S. suitable for spinach seed production (5). As a result, Washington and Oregon seed growers produce up to 50% of the U.S. and 25% of the world spinach seed supply annually (5). However, the acidic soils of this region are highly conducive to Fusarium wilt of spinach (5). The disease is caused by the soilborne fungus Fusarium oxysporum f. sp. spinaciae, and has become the most limiting factor for spinach seed production in the U.S. (5). Management of the disease by soil fumigation is cost-prohibitive, so growers avoid significant yield losses from this disease by crop rotations of 12-15 years for susceptible parent lines, and 8-10 years for partially resistant lines. Although cultivars with partial resistance are available, most carry little or no resistance. Also, growers have no choice of the lines they are contracted to grow for seed. In contrast, spinach seed is grown on alkaline, calcareous soils in Denmark on 4- to 5-year rotations without losses to Fusarium wilt (3). Lime amendments have been shown to decrease significantly the severity of diseases caused by formae speciales of F. oxysporum (4,8,9) and other soilborne pathogens (7,12). This includes research by du Toit et al. (4), funded by the Puget Sound Seed Growers’ Assoc., on calcitic lime (CaCO3) applications for control of spinach Fusarium wilt. The mechanism(s) of control have been associated with a complex of factors, including micronutrient deficiencies incurred by the pathogen under alkaline soils and/or greater capacity of antagonistic bacterial populations in the soil (1,2). Higher soil pH affects availability of nutrients in the soil, including zinc, manganese, and iron, which affects aggressiveness of some plant pathogens (2). In addition, growth of bacterial populations antagonistic to F. oxysporum is favored at neutral soil pH and adequate soil moisture (1). Spinach seed growers in western Washington do not know until March 1st each year into which fields their crops will be planted, because the locations of the seed crops are finalized on March 1st to avoid unwanted cross-pollination among crops. Therefore, rapidly dissolving hydrated lime, Ca(OH)2, should be evaluated as a tool for soil pH change and disease control.

    Formae speciales of F. oxysporum and nonpathogenic Fusaria are ubiquitous in soils worldwide (6). In general, subspecific strains are morphologically indistinguishable, and formae speciales are only reliably identified by time-consuming pathogenicity tests. A quick and reliable DNA-based detection method for F. oxysporum f. sp. spinaciae has yet to be developed, but would allow differentiation of F. oxysporum f. sp. spinaciae from other Fusaria, and could be used to accurately quantify DNA of the pathogen in soil and plant material. Such a tool would greatly facilitate research and development of disease management tools for Fusarium wilt.

    (b) Relevance to Western SARE Goals
    1. Soil amendment with lime may be a sustainable, site-specific method of rendering acid soils of the maritime PNW less conducive to Fusarium wilt of spinach.
    2. The ability to grow spinach seed crops on a 5 vs. a 10- to 15-year rotation will facilitate an increase in acreage planted to high-value spinach seed crops in Washington and Oregon. This will increase profitability and sustainability of the PNW spinach seed industry.
    3. Lime amendments and improved selection of fertilizers to render soils less conducive to Fusarium wilt will optimize the ability of seed growers in the PNW to utilize non-toxic materials to enhance biological suppressiveness of soils to Fusarium wilt.
    4. This project is on high-value spinach seed crops, and may also be pertinent to Fusarium wilts of other important crops in the PNW, e.g., tulips, daffodils, radish seed, and pea seed.
    5. Economic implications of using high rates of lime application to soils and optimum choice of fertilizers as sustainable management practices for Fusarium wilt will be assessed for benefits to crops grown in rotation with spinach in the PNW, e.g., peas, brassica seed, and ornamental bulb crops.

    (c) Objectives
    1. Develop a real-time PCR assay for specific detection of F. oxysporum f. sp. spinaciae to quantify populations of the spinach Fusarium wilt pathogen and determine soilborne inoculum threshold(s) for identifying fields suitable for planting spinach seed crops.
    2. Assess the potential of rapidly-dissolving hydrated lime applications to raise soil pH in western Washington and Oregon to suppress Fusarium wilt of spinach.
    3. Assess the effects of available calcium and micronutrients (specifically Zn, Mn, and Fe) on aggressiveness of F. oxysporum f. sp. spinaciae to spinach.
    4. Monitor changes in populations of soilborne bacteria in the rhizosphere of spinach that may be antagonistic to F. oxysporum, following lime applications in the lime trial in Objective 2, and correlate changes in the soil microflora with changes in soil moisture and pH.
    Accomplishing these objectives is expected to help optimize management of spinach Fusarium wilt using sustainable soil amendment practices, to enable reduced crop rotation intervals from 10+ to 5 years without increasing losses to spinach Fusarium wilt in the PNW.

    (d) Methods
    1. In preliminary research, Harrison used ribosomal DNA intergenic spacer (IGS) region-specific primers to attain IGS sequence data from isolates of F. oxysporum f. sp. spinaciae, and a collection of nonpathogenic and other pathogenic isolates from the PNW. IGS sequences were obtained for 23 additional formae speciales from the NCBI GenBank database. Sequence alignments revealed a single nucleotide polymorphism (SNP) unique to F. oxysporum f. sp. spinaciae. A Taqman® fluorochrome-labeled minor groove binding (MGB) probe and primers were designed for the SNP. Efficacy of the probe is being evaluated for a real-time PCR assay. Sensitivity of the assay for soilborne inoculum will be tested on a range of soil types. Each soil will be inoculated with the pathogen, planted with spinach lines susceptible or partially resistant to Fusarium wilt, and monitored for wilt. Inoculum concentration will be confirmed by dilution plating and real-time PCR and correlated with incidence or severity of wilt to identify inoculum threshold(s) of each soil type for planting spinach seed crops of parent lines with different levels of susceptibility to Fusarium wilt. This bioassay will be assessed as a diagnostic tool for seed growers.

    2. In each of two seasons, a field trial consisting of a randomized complete block design with 5 replicates will be established in a grower-cooperator field in Skagit Co., WA in acidic soil where a spinach seed crop was grown 4-5 years previously. Hydrated lime (0 to ~8 tons/acre) will be applied based on soil buffering capacity, and incorporated ~1 month before planting. Soil sampled from each plot 1, 2, and 3 months after planting will be tested for pH and nutrient status. Plots will be monitored for stand, incidence/severity of wilt, crop development, and nutrient deficiencies. Seed will be harvested, threshed, and germination and health assays completed. Results will be analyzed by GLM and regression analysis using SAS (SAS Institute, Cary, NC). The trial will be repeated with modifications based on results, e.g., foliar micronutrient feeds to counter deficiencies induced by alkaline soils.

    3. The effects of pH will be evaluated separately from effects of available calcium and micronutrients on pathogen virulence, by amending potato dextrose agar (PDA) with a range in rates of each factor. The effects will be evaluated for each of two isolates of F. oxysporum f. sp. spinaciae that originate from western Washington. A gypsum amendment (Ca(SO4)) will be used to increase available calcium without decrease in acidity, and magnesium carbonate (MgCO3) will be used to assess the effect of increase in pH without increases in available calcium. Separate experiments will be carried out with amendments of the micronutrients zinc, manganese, and iron. The influence of each factor will be assessed on growth of the fungus, spore production, germination, and aggressiveness of the fungus on spinach. Analyses of variance will be used to identify factors that suppress the pathogen.

    4. Changes in soil bacterial populations in the rhizosphere of spinach will be monitored during the lime trial described in Objective 1. Three plants will be sampled from each lime plot at monthly intervals from emergence to harvest. A root wash will be used isolate rhizosphere bacteria, and total DNA will be extracted. Universal bacterial primers will be used to amplify the 16S ribosomal DNA, and populations of specific bacterial groups will be quantified by fluorescence-tagged amplified rDNA restriction analysis (FT-ARDRA) (11). Fluorescent Pseudomonas spp. contribute to suppression of Fusarium wilt (10), so populations of these bacteria will be enumerated by dilution plating on Pseudomonas agar F (PAF), and correlated with results by FT-ARDRA. Changes in the populations will be correlated with changes in soil conditions and the incidence/severity of wilt. Bacterial groups contributing to suppression of Fusarium wilt will be identified by multivariate analysis.

    (e) Outreach Plan
    Results will be demonstrated to seed growers and industry personnel at the WSU Vegetable Seed Field Days (2009 and 2010). The graduate student and supervisor will present results at the Western WA Hort. Assoc./Puget Sound Seed Growers’ Assoc. meeting each season, with results published as proceedings articles. Results will also be posted on the PNW Vegetable Extension Group website (

    (g) Literature Review
    The originality/significance of this research is described in the “statement of problem,” and the contribution to sustainable agriculture is noted in the “relevance to Western SARE goals.” References are listed below. The National SARE projects, National Agric. Library, and USDA CSREES-CRIS research databases list the following projects that support this project: LNC03-234 (Calcium inputs for soil quality improvement), GS04-034 (control of soilborne fungi with biofumigation), GS01-011 (suppression of soilborne phytopathogenic fungi of tomatoes via integrated production systems that utilize biofumigation, composted amendments, solarization, and chemical fumigants); FLA-HOM-03402 (integrated pest management as an alternative for control of soilborne pests of vegetable crops); IND-055035 (nutrient management practices on pests, pathogens, and beneficial organisms in soil ecosystems).

    1. Baker, K.F., and Cook, R.J. 1974. Biological Control of Plant Pathogens. Freeman Co., San Francisco, CA.
    2. Datnoff, L.E., Elmer, W.H., and Huber, D.M. 2007. Mineral Nutrition and Plant Disease. APS Press, St. Paul, MN.
    3. Deleuran, L.C., and Boelt, B. 2006. Spinach seed production in Denmark. Pages 13-15, in: Proc. 2006 Int. Spinach Conf., 13-14 July 2006, La Conner, WA.
    4. du Toit, L.J., Derie, M.L., Brissey, L.M., and Cummings, J.A. 2007. Evaluation of limestone amendments for control of Fusarium wilt in a spinach seed crop, 2006. Plant Dis. Management Reports 1:V091.
    5. Foss, C.R., and Jones, L.J. 2005. Crop Profile for Spinach Seed in Washington. U.S. Dep. Agric. National Pest Management Centers.
    6. Gordon, T.R., and Martyn, R.D. 1997. The evolutionary biology of Fusarium oxysporum. Annu. Rev. Phytopathol. 35:111-128.
    7. Ingemarsson, A. 2004. Effects of lime and organic amendments on soilborne pathogens, Aphanomyces spp. of sugarbeet and spinach. M.S. thesis. Swedish Univ. of Agric. Sciences.
    8. Jones, J.P., and Woltz, S.S. 1970. Fusarium wilt (race 2) of tomato: Calcium, pH, and micronutrient effects on disease development. Plant Dis. Rep. 53:276-279.
    9. Kommedahl, T., Christensen, J.J., and Fredericksen, R.A. 1970. A half century of research in Minnesota on flax wilt caused by Fusarium oxysporum. Minn. Agric. Exp. Stn. Bull. 273:35.
    10. Lemanceau, P., and Alabouvette, C. 1993. Suppression of Fusarium wilt by fluorescent pseudomonads—mechanisms and applications. Biocont. Sci. 3:219-34.
    11. McSpadden Gardener, B.B., and Weller, D.M. 2001. Changes in populations of rhizosphere bacteria associated with take-all disease in wheat. Appl. Environ. Microbiol. 67:4414-25.
    12. Murray, T.D., Walter, C.C., and Anderegg, J.C. 1992. Control of Cephalosporium stripe of winter wheat by liming. Plant Dis. 76:282-86.

    (h) Scholarly and Educational products
    Annual results will be published as proceedings of the WWHA/PSSGA meeting, and final results in Plant Disease and Phytopathology. Results will be posted on the PNW Vegetable Extension Group website (

    Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture or SARE.