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.

Final Report for GW08-005

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
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Project Information

Summary:

The maritime Pacific Northwest (PNW) is the only region of the U.S. suitable for spinach seed production. Washington and Oregon seed growers produce up to 50% of the U.S.and 25% of the world’s spinach seed supply annually. However, acidic soils of this region are highly conducive to spinach Fusarium wilt caused by Fusarium oxysporum f. sp. spinaciae, which has become the most limiting factor for spinach seed production in the U.S. This project demonstrates the potential for limestone applications to reduce losses to Fusarium wilt and a soil bioassay for growers to identify low-risk fields for spinach seed production.

Introduction

Background:

Spinach is a widely-consumed leafy vegetable with relatively high concentrations of vitamins, minerals, proteins and antioxidants that are important for prevention of human disorders such as age-related macular degeneration (Lucier, 2000; Pratt, 1998). Spinach is grown on approximately 56,800 acres in the U.S. annually for fresh and processed markets [USDA National Agricultural Statistics Service, (NASS)]. The primary states for spinach production are Arizona, Arkansas, California, Colorado, Maryland, New Jersey, Oklahoma, Texas and Virginia (USDA NASS). Spinach crops are planted at populations ranging from 30,000 to >2 million seed/acre, with the higher populations used for the expanding ‘baby leaf’ bagged spinach market (Holloway et al., 2003; LeStrange et al., 1996). The dense populations, particularly for ‘baby leaf’ crops harvested 30-45 days after planting, necessitate production of high quality spinach seed and have increased the demand for spinach seed.

Spinach seed crops in the mild maritime region of western Washington and western Oregon produce up to 50% of the U.S. and up to 25% of the world’s supply of spinach seed on 3,000-4,000 acres annually. Few areas of the world have the climatic conditions of the coastal Pacific Northwest (PNW) region of the U.S. that are required for production of high quality spinach seed, i.e., long summer day length (>16 hours) to trigger uniform flowering of this day length-sensitive species, dry summers to minimize pathogen infections on developing seed and mild summer temperatures for this heat-sensitive species (Metzger and Zeevaart, 1985). The few other countries with regions that meet these climatic requirements, and in which spinach seed is produced, include Denmark, France, Italy, Holland and New Zealand (Deleuran and Boelt, 2006; Ben Baerends, Seminis Vegetable Seeds, personal communication). Spinach seed growers in these countries compete directly with U.S. spinach seed growers.

Many vascular wilt diseases caused by formae speciales of Fusarium oxysporum are economically significant, limiting the productivity and sustainability of susceptible crops in numerous areas of the world (Beckman, 1987). In the maritime PNW, depletion of virgin ground for spinach has resulted in Fusarium wilt, caused by the soilborne fungus F. oxysporum f. sp. spinaciae, becoming the limiting factor for high-yielding spinach seed crops in the U.S. (Foss and Jones, 2005). Seed treatments do not provide protection against infection of spinach plants by this pathogen after the seedling phase of growth, and soil fumigation is neither economically nor environmentally sound. Management tools are needed, particularly through the extended season for spinach seed production.

Current efforts to manage spinach Fusarium wilt in the PNW are based on planting resistant cultivars and using very extensive crop rotations (Foss and Jones, 2005), but both these approaches have inherent limitations. Spinach cultivars with partial resistance are available, but many carry little resistance to this pathogen (Correll et al., 1994). In addition, seed crops are grown on a contract basis, so growers have little choice of the parent (inbred) lines they grow. Once introduced into soil, F. oxysporum f. sp. spinaciae can survive many years as a saprophyte and/or a pathogen on spinach and related asymptomatic host crops (e.g., beet and chard) (Armstrong and Armstrong, 1976; Reyes, 1979). When spinach Fusarium wilt was first identified in the PNW in the 1960s, the disease was managed by growing seed crops in fields not previously planted to spinach. Subsequent depletion of virgin ground for spinach led to Fusarium wilt becoming the main factor limiting production of high yielding spinach seed crops in this region (Foss and Jones, 2005). Losses to Fusarium wilt now necessitate rotation intervals of six to ten years for spinach lines with partial resistance and at least 12 to 15 years for susceptible lines. Confounding this is the need to isolate seed crops to avoid unwanted cross pollination of this wind-pollinated species. Therefore, selection of fields for spinach seed production is an increasingly complex and limiting factor for growers.

Development of effective soil-based management approaches for F. oxysporum f. sp. spinaciae requires an intimate understanding of the effects of soil chemical and biological properties on growth and survival of these fungi. Soils differ widely in the capacity to suppress or promote these and other soilborne plant pathogens (Hornby, 1983). Some naturally-conducive soils can be rendered more suppressive to particular diseases, to various degrees and durations, using practices such as soil amendments, cover crops and selection of fertilizers (Beckman, 1987; Cook and Baker, 1983; Datnoff et al., 2007; Hornby, 1983; Stone et al., 2004; Ochiai et al., 2008). In general, Fusarium wilts tend to be promoted by acid soils, ammonium forms of nitrogen and high available concentrations of micronutrients such as iron, manganese and zinc (Datnoff et al., 2007). The relationship of these soil properties to growth and pathogenicity of the causal agent of Fusarium wilt of spinach has not been investigated and remains an important gap in our understanding of how to manage this soilborne disease and reduce the extensive rotation intervals required for production of high quality spinach seed.

Goal 1: Management practices

Over the past eight years, with >$52,000 in funding from the Puget Sound Seed Growers’ Association (PSSGA) and matching support from the state-funded Washington State Commission for Pesticide Registration (WSCPR), we have initiated a series of research trials to evaluate potential management tools for Fusarium wilt in spinach seed crops in the PNW. Based on evidence that alkaline and calcareous soils are naturally-suppressive to spinach Fusarium wilt in Denmark and Texas, we initiated field trials in western Washington in 2006 to 2008 to evaluate the potential efficacy of limestone amendments for suppression of Fusarium wilt (du Toit et al., 2007 and 2008). Agricultural limestone applied at >1.4 tons/acre suppressed Fusarium wilt significantly, and plant biomass was significantly greater in all plots treated with limestone compared to non-treated plots. For the Fusarium wilt-susceptible spinach female line evaluated, wilt was reduced 45% and seed yield increased 318% in plots amended at 3.5 tons/acre compared to control plots (du Toit et al., 2007). For the moderately susceptible spinach female line, seed yield was greatest on plots amended at 2.8 tons/acre, and decreased at higher and lower rates. This illustrates the need for research to optimize limestone amendments for suppression of spinach Fusarium wilt and to assess additional tools that may further enhance this suppression. For example, NO3- vs. NH4+ forms of N have been shown to suppress vs. promote, respectively, Fusarium wilt in other species (Datnoff et al., 2007; Huber and Watson, 1974). Woltz and Jones (1973) found that NH4+-N actually reversed the beneficial effects of limestone on Fusarium wilt management in tomato, demonstrating the need to determine whether the potential benefits of limestone applications in spinach seed production may be mitigated by the types of fertilizers used by spinach seed growers.

Goal 2: Field site risk assessment

Presently, the only factors used by growers to assess the risk of a field site for spinach Fusarium wilt is the duration of crop rotation between spinach seed crops and the level of partial resistance of the parent lines to Fusarium wilt. The latter is often not known for many of the parent lines used in spinach seed crops, and growers typically have no choice of the specific parent lines they are contracted to grow for seed companies. These two pieces of information are not foolproof, however, as some fields that had been out of spinach for as long as 12-16 years in northwestern Washington have developed severe Fusarium wilt (project director’s personal observations from 2000-2010). A better understanding of the relationships among soil properties and pathogen population, host-pathogen interactions and soil microbial communities could be used to develop a more effective tool for assessing the relative risk of Fusarium wilt in specific fields. A soil bioassay that correlates Fusarium wilt development on a set of standard parent lines to inoculum density, microbial diversity and fertility status of a representative soil sample from a field could be a valuable improvement in Fusarium wilt risk assessment and subsequent field site selection by spinach seed growers.

Summary

Development of effective risk assessment tools and sustainable management practices for Fusarium wilt are essential for the U.S. spinach seed industry to remain competitive with the few other countries that have suitable climates for spinach seed crops. U.S. growers currently do not know how to reduce rotation intervals to periods comparable to those in Denmark without incurring severe losses to spinach Fusarium wilt. Lack of such knowledge is important because, until it becomes available, U.S. farmers will continue to be restricted in their capacity to produce spinach seed on the limited acreage in the U.S. suitable for this crop. The research is expected to improve our understanding of the relationships of soil properties to suppression of spinach Fusarium wilt. This is expected to improve the ability of U.S. spinach seed growers to manipulate soil properties using cultural practices to render naturally-conducive soils of the PNW more suppressive to Fusarium wilt.

We expect the outcome of the proposed research to contribute to reduced rotation intervals for spinach seed crops in the U.S. This contribution is significant because application of the new knowledge is expected to increase the capacity of each acre in the U.S. that can be used to produce spinach seed. This, in turn, will increase the sustainability and profitability of spinach seed as a U.S. crop, potentially equating to a larger slice of the world market for spinach seed, and decreased dependence on other countries for spinach seed by the many states that produce fresh and processed spinach. Furthermore, results generated by this research are expected to have a broader impact by providing information relevant to suppression and management of other Fusarium wilts, e.g., radish, onion, pea, and ornamental bulbs crops.

Project Objectives:

Long-term survival of the Fusarium wilt pathogen in soils necessitates a rotation interval of 10 to 15 years, or longer, for spinach seed crops in the PNW. The overall objective is to reduce this rotation interval in order to increase the carrying capacity of PNW acreage for spinach seed production, based on an understanding of how soil chemical and microbial properties affect conduciveness of soils in this region to Fusarium wilt.

Specific objectives are to:

a. 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.

b. Assess the potential of rapidly-dissolving hydrated lime applications to raise soil pH in western Washington and Oregon to suppress Fusarium wilt of spinach.

c. Assess the effects of available calcium and micronutrients (specifically Zn, Mn, and Fe) on aggressiveness of F. oxysporum f. sp. spinaciae to spinach.

As described in previous progress reports, I (Gatch) started on this project in 2008, first assisting du Toit’s program with a field trial evaluating rates of limestone amendments and parent lines with different levels of susceptibility to Fusarium wilt for suppressing this disease in spinach seed crops. Based on the results of this and previous experiments in du Toit’s program, the objectives listed above were modified from those of the original Western SARE Graduate Student Fellow Grant, as well as the Robert MacDonald Vegetable Seed Memorial Fund proposals. Additional federal funding was obtained (see Milestones section) to support progress toward these objectives.

The modified objectives of this SARE project are as follows:

a. Assess the potential for enhancing suppression of spinach Fusarium wilt using annual applications of limestone and nitrate-N fertilizers and monitor microbial and chemical changes in the soil associated with these practices over four years (modified objectives ‘b’ and ‘d’ of the original Western SARE proposal)

b. Determine the effects of micronutrients, pH, calcium and form of nitrogen on F. oxysporum f. sp. spinaciae and on interactions of the pathogen with spinach (objective ‘c’ of the original Western SARE proposal)

c. Develop a soil bioassay for spinach seed growers to determine the relative risk of field sites for Fusarium wilt (complementary to objective ‘a’ in the original Western SARE proposal on a real-time PCR assay).

d. Monitor changes in populations of soilborne bacteria, particularly in the rhizosphere of spinach plants, that may be antagonistic to F. oxysporum f. sp. spinaciae, following limestone applications in the field trial in objective a, and correlate changes in soil microflora with other changes in soil properties such as pH.

Research

Materials and methods:
Application of limestone and nitrate-N fertilizers

To assess the practical and economic feasibility of longer-term management of Fusarium wilt using low rates of limestone applied annually over four to five years in the same field, along with non-acidifying fertilizers (nitrate-N instead of less expensive ammonium-N fertilizers most commonly used in this area), a five-year field trial in the Skagit Valley was established in a grower-cooperator site in spring 2009. Each year from 2009-2013, limestone was/will be applied at 0, 1, and 2 tons/acre to the same plots. The 2009 trial was planted to a spinach seed crop trial (described below). The grower-cooperator is proceeding with typical rotation crops in 2010-2012, and the same spinach seed crop trial will be repeated in 2013. This will enable us to assess the degree to which annual limestone applications may enable (practically and economically) a reduction in spinach seed crop rotations to as little as five years without significant losses to Fusarium wilt. Soil from each plot will be sampled each season and analyzed for shifts in nutrient availability and microbial community structure (latter using 454-pyrosequencing in collaboration with Drs. Paulitz and Hulbert in Pullman) and assessed for reduction in conduciveness to Fusarium wilt using a greenhouse soil bioassay I am developing for spinach Fusarium wilt (new objective ‘c’).

For the 2009 spinach trial, a split-split plot randomized complete block design with five replications of a 2 x 3 x 3 factorial treatment design was used. The field had been planted to a spinach seed crop five years previously and had a pH of 5.8 in March 2009. The field was prepared and limestone incorporated at 0, 1 and 2 tons/acre in April. Spinach seeds of three female lines (susceptible, moderately resistant and partially resistant to Fusarium wilt) were planted in split plots along with a male line for pollination. At planting, two nitrogen fertilizer programs were applied in-furrow to main plots:

1) nitrate-N (15:5:0 calcium nitrate [Tropicote] at ~100 lb/acre

2) ammonium-N (11-52-0 at ~270 lb/acre)

Soil samples were collected from each of the 15 limestone plots (three rates x five replications) every three weeks for nutrient analysis to monitor changes in soil properties. Plants in each plot were rated for stand and wilt at three-week intervals. Spinach seed was harvested in August. In addition, soil samples were collected for nutrient analyses at three-week intervals. At the final collection, soil was sampled between plants within rows. A subsample from each plot was frozen at -20C to be assayed using molecular methods for differences in microbial populations associated with treatments. An additional subsample of soil from each plot was assayed by dilution plating on semi-selective agar media to quantify Verticillium and F. oxysporum. Whole plants were sampled in late June and July for biomass measurements, and plants from the first biomass were subjected to nutrient analyses. Harvested seed was cleaned, sized and weighed and samples tested using the AOSA germination blotter assay and a freeze-blotter seed health assay. Results were subjected to analysis of variance for significance at (p < 0.05) and means separation with Fisher’s least significant difference test.

In spring of 2010, prior to the grower-cooperator planting a potato crop in the rotational sequence, the same rates of limestone were applied to the same plots and incorporated by rototilling. Soils were sampled three weeks and three months after limestone incorporation and again in August 2010. Soils were subjected to nutrient analysis and assayed for F. oxysporum populations, as in 2009, and will also be assessed for shifts in microbial community structure. The potato crop was monitored visually for potential effects of limestone rates of application on crop growth as well as tuber yield and diseases. The annual application of limestone will occur again in 2011, 2012 and 2013, when another spinach seed crop will be planted mirroring the 2009 trial to determine whether these annual applications can reduce the conduciveness of soils to Fusarium wilt in an economically-justifiable manner.

Effects of micronutrients, pH, calcium and form of nitrogen on F. oxysporum f. sp. spinaciae and host-pathogen interactions

Experiments for this objective are currently in progress. The influence of each of these factors is being assessed on growth of the fungus, spore production and germination, polygalacturonase (plant cell wall-degrading enzyme) activity and aggressiveness of the fungus on spinach. These experiments will improve our understanding of how soil properties associated with suppression or promotion of spinach Fusarium wilt might specifically affect F. oxysporum f. sp. spinaciae. We recognize the limitations of assessing each factor in isolation, unlike the complex soil environment, but this research is expected to increase our understanding of the potential to manipulate specific factors related to the suppression of spinach Fusarium wilt. The trials are expected to help determine whether concentrations of available Fe, Mn, Zn and/or Ca, which are affected by limestone amendments, suppress or promote mycelial growth, spore production, spore germination, enzyme production and/or aggressiveness of the fungus on spinach. Similarly, the trials will clarify whether NO3- or NH4+ may suppress or promote growth and aggressiveness of the fungus on spinach. This will clarify the importance of selecting specific types of nitrogen fertilizers for suppressing Fusarium wilt of spinach. Finally, the results are expected to improve our understanding of how development and aggressiveness of F. oxysporum f. sp. spinaciae are influenced by pH.

Develop a soil bioassay for spinach seed growers to determine the relative risk of field sites for Fusarium wilt

The former student on this project, Harrison, did preliminary work on development of a Taqman real-time PCR assay for the spinach Fusarium wilt pathogen in cooperation with Drs. Okubara and Schroeder, USDA ARS plant pathologists in Pullman. The molecular assay was based on a putatively unique single nucleotide polymorphism (SNP) in the ribosomal DNA intergenic spacer (IGS) region of a regional collection of isolates of F. oxysporum f. sp. spinaciae, as well as other formae speciales of this species. Sensitivity and robustness of the molecular assay were evaluated in 2008-2010 with >20 isolates of the spinach pathogen from the U.S. and Japan (courtesy of Dr. Correll, University of Arkansas) and >20 strains of other pathogenic and non-pathogenic formae speciales (courtesy of multiple collaborators). I also spent winter of 2008-2009 assaying soils for F. oxysporum on a semi-selective agar medium to compare with results of the real-time PCR assay for the spinach Fusarium wilt pathogen, in cooperation with Drs. Okubara and Schroeder. Recent comparisons of the putative SNP for this target pathogen with DNA sequences in GenBank and in published papers for additional isolates of F. oxysporum suggest the real-time PCR assay may cross-react with a small number of strains of closely related fungi, and there may be strains of the spinach pathogen that do not have this SNP, i.e., the latter isolates will not be detected by the real-time PCR assay. Confounding this further is the fact that when the authors of these papers and/or those who submitted the DNA sequences to GenBank were contacted, we discovered that some of the isolates that did not have the correct SNP had not been tested for pathogenicity on the reported hosts (spinach, cucumber, lily and others) to confirm the formae speciales designation assigned to those isolates. We are attempting to clarify this by requesting a USDA APHIS PPQ permit to receive cultures of the isolates to test pathogenicity or lack of pathogenicity of the isolates on spinach in greenhouse evaluations. In addition, sensitivity of the assay for soilborne inoculum was tested using soil samples collected in summer 2008 from growers’ fields in the Skagit Valley of Washington. The fields had all been rotated out of spinach seed crops for durations ranging from 1 to 15 years. One field that had not been planted to spinach since 1994, and that tested negative using the real-time PCR assay, was planted to a 50-acre spinach seed crop in 2009. The crop was severely affected by Fusarium wilt (yields were reduced almost 10-fold), demonstrating a significant current limitation of the real-time PCR assay for assaying soil samples accurately for the risk of Fusarium wilt. A very major hurdle for using molecular assays like the real-time PCR assay to assess the risk of a field for a soilborne pathogen like F. oxysporum f. sp. spinaciae is the very small amount of soil that can, realistically, be assayed, i.e., one to five grams soil per sample. Soilborne pathogens are never uniformly distributed in a field, so testing such small samples of soil, even with as many as 10 replicate samples per field (which becomes prohibitively expensive because of the cost of DNA extraction kits/processing), is unlikely to give a realistic assessment of the level of risk for a field. We are continuing to collaborate with Okubara to address these aspects of the real-time PCR assay. It is possible the primary practical application of the molecular assay may be for confirming the formae speciales of pure cultures of F. oxysporum obtained from soil samples and spinach plants, thereby eliminating the need for time-consuming pathogenicity tests that are currently the only means of accurately identifying isolates of this pathogen.

Given the hurdles encountered with the real-time PCR assay, and the urgency with which growers have requested a means of assessing the relative risk of spinach Fusarium wilt in order to avoid major losses to Fusarium wilt, we developed a soil bioassay for quantifying the risk of Fusarium wilt in fields. This soil bioassay is expected to be complementary to the real-time PCR assay, if the latter can be optimized. Soil samples used for developing a soil bioassay were collected in 2009 from fields planted to spinach seed crops. Given the wide range in quantitative levels of susceptibility of spinach parent lines to Fusarium wilt, a major component of accurate risk assessment is the relative susceptibility of the specific parent lines a seed grower is contracted to grow. Therefore, a series of preliminary soil bioassays were completed in a greenhouse in fall of 2009 using three female spinach lines with different levels of resistance to Fusarium wilt. Based on this preliminary work, a protocol for a quantitative soil bioassay was developed that can be completed in six to eight weeks. Demonstration of the preliminary soil bioassay in a greenhouse trial to stakeholders (regional growers and seed industry personnel) led to my setting up a large grower-cooperator soil bioassay trial in the winter of 2009-2010. For this bioassay, spinach seed growers voluntarily submitted soil samples (~eight gallon/field) from each of 26 fields in Skagit and Snohomish Counties in late December 2009. The soil for each field was partially dried, sieved and mixed thoroughly. Seeds of the three standard parent lines (susceptible, moderate and partially resistant to Fusarium wilt) were planted into each soil sample in January 2010 and into each of three control soils that represented fields with high, moderate and low- to no-risk of Fusarium wilt (control soils were tested in preliminary trials in 2009). The replicated bioassay was demonstrated to participating stakeholders in late February 2010, prior to the regional seed crop ‘pinning meeting’ on March 1, 2010, at which specific fields were allocated to particular spinach seed crops for planting in spring 2010. The bioassay results were used by stakeholders to assess the risk of Fusarium wilt in each field relative to the susceptibility of the three standard parent lines.

I also conducted a preliminary test of the susceptibility of seven additional spinach parent lines to Fusarium wilt using the soil bioassay. Seeds of these lines were submitted by seed companies in December 2009 and represented a subset of parent lines to be contracted to seed growers in 2010. The susceptibility of these parent lines to Fusarium wilt was unknown prior to this test. The test enabled seed company personnel and growers to assess the susceptibility of these seven lines relative to the three standard lines. Based on empirical evidence as well as results of the 2009-2010 soil bioassay, the susceptibility of parent lines to Fusarium wilt is a critical aspect of accurately assessing the risk of Fusarium wilt for spinach seed crops. I am currently monitoring fields that were tested with this soil bioassay in winter 2009-2010 and subsequently planted to spinach seed crops in spring 2010. This will provide an additional means of assessing the bioassay. The bioassay will be modified as needed based on these validation steps.

The next stage in the development of a reliable, accurate and quantitative soil bioassay for spinach seed growers and seed companies is to test soil samples in January to February 2011 for a large number of grower-cooperators’ fields, so that these stakeholders can more accurately select fields for spinach seed crops with minimal risk of losses to Fusarium wilt than currently possible based on number of years rotated out of spinach. If the soil bioassay continues to prove accurate and reliable, I will investigate the possibility of regional soil testing labs and other diagnostic labs providing the bioassay as a commercial diagnostic tool for spinach seed growers (similar to soil fertility analyses). Ultimately, the bioassay could be combined with the real-time PCR assay for F. oxysporum f. sp. spinaciae, if the latter can be optimized adequately, for a more rapid means of testing soils.

Research results and discussion:
Application of limestone and nitrate-N fertilizers

Results of the 2009 field trial demonstrated the potential for limestone amendments to render the acid soils of western Washington less conducive to Fusarium wilt and the significant influence of the susceptibility of spinach parent lines on the degree to which limestone amendments can reduce the risk of losses to this disease (see detailed results in Gatch et al., to be submitted to Plant Disease Management Reports Vol. 5 in December 2010).

Effects of limestone:

Plots with two tons limestone/acre had fewer wilted plants (5.3% incidence of wilt) than plots with one ton/acre (12.9%) or no limestone (33.6%). By mid-season, spinach biomass in plots with two tons limestone/acre was >200% that of plots with no limestone and 31% more than in plots with one ton/acre. Incidence of vascular discoloration was significantly less in plots with two tons limestone/acre vs. zero or one tons/acre. Application of two tons limestone/acre increased seed yield to 906 lb/acre compared to 337 lb/acre in plots with no limestone. Limestone increased soil pH significantly within three weeks of application, from 5.9 in control plots to 6.4 and 6.6 with one and two tons/acre, respectively. Although soil pH declined gradually through the season, significant differences in pH among limestone treatments were detected through harvest, by which time soil pH was 5.2, 5.8 and 6.3 for plots with zero, one and two tons/acre respectively. Available soil calcium increased significantly with increasing rate of limestone amendment. Plant calcium, phosphorus and sulfur were significantly lower in plots with no limestone than the amended plots, whereas soil zinc and manganese were significantly greater in plots with no limestone compared to those amended with one or two tons/acre.

Effects of spinach parent line:

The susceptible and moderately resistant spinach parent lines had significantly more wilt (>90% incidence) than the resistant line (80.4%) by mid-July. The moderate and resistant parent lines had 62% and 50% more marketable seed yield, respectively, than the susceptible parent. The incidence of harvested seeds infected with Fusarium spp. was significantly greater for the susceptible parent than the moderate or resistant parents. Soil sampled from plots with the susceptible and moderate parent lines had significantly more F. oxysporum compared to plots with the resistant parent. This suggests that planting a spinach seed crop with parent lines susceptible to Fusarium wilt could result in higher levels of soilborne inoculum compared to planting more resistant parent lines. This could potentially affect the minimum rotation interval needed between spinach seed crops to minimize losses to Fusarium wilt in the next spinach seed crop in a particular field.

Effects of form of nitrogen fertilizer:

Heavy rainfall shortly after planting caused leaching of nitrate from the plots fertilized with nitrate-N. Spinach in these plots remained nitrogen-deficient (pale green and stunted) for the duration of the trial. This was a valuable learning experience; it is not uncommon to get extensive precipitation in the maritime Pacific Northwest in the spring, so application of a nitrate-N fertilizer at planting or during side-dressing in spinach seed crops may not be advisable. Early in the season, plots fertilized with ammonium-N had a significantly lower soil pH than plots fertilized with nitrate-N, as expected. In plots with zero and one ton limestone/acre, nitrate-N fertilizer significantly increased wilt compared to ammonium-N, but presumably due to the added stress of nutrient deficiency, not a direct effect of the type of fertilizer. The two types of N fertilizers had no significant effect on spinach in plots with two tons limestone/acre. A similar effect was observed with the interaction between limestone and nitrogen form on spinach dry biomass. This suggests that if the timing of nitrate-N fertilizer applications can be optimized, using slow-release or organic nitrogen sources could potentially be used as a less acidifying form of nitrogen to help suppress Fusarium wilt.

Long-term effects of limestone application:

As noted in the Materials and Methods, the same plots in this field site will be amended annually for the next three years with limestone at the same rates and monitored. Results of this portion of the first objective will be compiled and prepared for publication in 2012-2013. We successfully procured a three-year grant from the USDA Western Region IPM ($99,482) for this project, as well as funding ($50,961) for the 2009 field season from the Puget Sound Seed Growers’ Association (PSSGA) and the Washington State Commission for Pesticide Registration (WSCPR). The federal, stakeholder and state funding have facilitated longer-term research to address the goals of this project.

Effects of micronutrients, pH, calcium and form of nitrogen on F. oxysporum f. sp. spinaciae and host-pathogen interactions

As noted in the Materials and Methods section, experiments for this objective are currently underway and will be completed in the winter of 2010-2011. Preliminary results show significant effects on growth of the pathogen in vitro, particularly on pigment formation. This phenomenon will be investigated further in greenhouse trials for potential association with pathogenicity to spinach.

Develop a soil bioassay for spinach seed growers to determine the relative risk of field sites for Fusarium wilt

The purpose of the preliminary bioassay was to develop planting and rating protocols so that a reliable index of Fusarium wilt severity could be used to differentiate the disease potential (= risk) associated with different fields. Upon successful completion of this preliminary work (repeated in spring 2010 with relatively consistent results to the first bioassay), a grower-cooperator soil bioassay was completed in 2009-2010, as described above. Field site, spinach parent line and the interaction between these factors significantly affected severity of Fusarium wilt. Disease ratings 28 and 35 days after planting providied optimum differentiation of Fusarium wilt risk among the soils. A wide range in severity of Fusarium wilt was observed among the 26 fields and the three control soils. The accuracy of the soil bioassay for assessing Fusarium wilt risk is being assessed by evaluating Fusarium wilt severity in spinach seed crops grown in those assayed fields that were planted to spinach seed crops in 2010. Some fields tested with the bioassay in winter 2009-2010 were not planted to spinach seed crops in 2010 because the bioassay demonstrated a very high risk of Fusarium wilt. For some of these soils the risk was high even with the moderately resistant parent line. A complicating factor affecting the ability to assess accuracy of the bioassay results based on Fusarium wilt in the seed crops planted into those fields in 2010 is the unknown relative resistance of the parent lines planted in the fields. Therefore, plans are being developed in cooperation with stakeholders to address this in the next round of soil bioassays to be completed in 2010-2011. An unanticipated benefit of the bioassay was the ability to observe the risks of other adverse factors associated with specific fields, e.g., residual herbicide injury (spinach is very sensitive to numerous herbicides used in various other crops grown in this region) and insect pests (severe damage from cranefly larvae was observed in a few fields that had been planted to pasture prior to being considered for spinach seed crops in 2010).

Participation Summary

Educational & Outreach Activities

Participation Summary

Education/outreach description:

I presented a progress report on this project at the WSU Mount Vernon NWREC Field Day on July 9, 2009, which was attended by ~75 growers, field reps, consultants, researchers, extension educators, graduate students and interns. An update on this report was presented again at the NWREC Field Day on July 8, 2010.

The 2009 spinach limestone/nitrogen field trial was demonstrated to members (stakeholders) of the Puget Sound Seed Growers’ Association and the Western Washington Small Seed Advisory Committee (seed industry) on July 9, 2009. We received very enthusiastic and appreciative comments from stakeholders. We also showed the 2009 field trial to participants in the Washington State Pest Control Tour that visited the Skagit Valley on July 23, 2009. The latter included state/federal regulators (from the U.S. EPA, Washington State Dept. of Energy, Washington State Dept. of Health, Washington State Dept. of Labor & Industries, USDA, and WSDA), industry representatives, consultants, legislators and researchers.

I presented a poster and oral presentation on this project at the Graduate Student Symposium on Sustainability at the WSU Mount Vernon NWREC on November 10, 2009, which was attended by graduate students from four universities in Washington State, as well as researchers and private industry.

A newsletter article on my project was published in the October issue of The Western Front, the newsletter of the USDA Western IPM Center: du Toit, L.J., and Gatch, E.W. 2009. Increasing the capacity for spinach seed production in the United States by promoting soil suppression of Fusarium wilt. The Western Front, October 2009: Page 7. http://www.wripmc.org/Newsletter/October%202009%2010-6-09%20for%20posting.pdf

I submitted an abstract and gave an oral presentation entitled “Development of a soil bioassay to assess the risk of spinach Fusarium wilt” at the American Phytopathological Society (APS) Pacific Division Meeting in Vancouver, BC, Canada on June 23,2010. I received a $500 student travel award from the APS Pacific Division to present this paper.

A publication entitled “Effect of agricultural limestone and nitrogen fertilizers on Fusarium wilt and Verticillium wilt in a spinach seed crop, 2009” by Gatch et al. has been prepared for submission to Vol. 5 of Plant Disease Management Reports, an online journal of the American Phytopathological Society (the next call for submission of manuscripts is in December 2010).

Project Outcomes

Project outcomes:

We have received very enthusiastic grower participation in the spinach Fusarium wilt soil bioassay. In the first grower-cooperator bioassay completed in winter 2009-2010, soils were voluntarily submitted from 26 grower-cooperator fields in Skagit and Snohomish Counties. These fields had tentatively been selected for growing spinach seed crops in 2010. Results of the bioassay helped stakeholders assess the potential risk of those 26 fields for Fusarium wilt on spinach parent lines that ranged from highly susceptible to partially resistant to Fusarium wilt. The results influenced the specific fields some growers and contracting seed companies selected for planting particular spinach parent lines in 2010, based on their observations of the bioassay results and, if the proprietary information was known to the stakeholders, the susceptibility of the parent lines being considered for specific grower contracts. Given the high value of spinach seed crops both to growers and seed companies, and the current frequency with which spinach seed crops are significantly affected by Fusarium wilt, seed company field representatives and growers indicated they would be willing to pay up to $250/field for a soil bioassay that reliably identifies higher risk fields for spinach Fusarium wilt, in order to reduce the risk of significant losses to this disease.

Soil amendment with limestone may be a sustainable, site-specific method of rendering acid soils of the maritime PNW less conducive to Fusarium wilt of spinach. The ability to grow spinach seed crops on a five-eight year rotation vs. a 10-15 year rotation will facilitate an increase in acreage planted to high-value spinach seed crops in Washington and Oregon. This is expected to increase profitability and sustainability of the PNW spinach seed industry and could potentially double the capacity for spinach seed production in the sole region of the U.S. suitable for producing spinach seed. Limestone amendments to render soils less conducive to Fusarium wilt is expected to optimize the ability of seed growers in the PNW to utilize non-toxic materials to enhance biological suppressiveness of soils to Fusarium wilt. This project may also be pertinent to Fusarium wilts of other specialty crops in the PNW, e.g., tulips, daffodils, radish seed and pea seed. Economic implications of using high rates of limestone application to soils, combined with a soil bioassay for identifying and avoiding high risk fields as sustainable management practices for Fusarium wilt, are being calculated. The expenses of these practices will also need to be assessed for potential benefits to crops grown in rotation with spinach, e.g., peas, brassica seed crops, pumpkin, potato and ornamental bulb crops.

Economic Analysis

Spinach Fusarium wilt limestone field trials:

The current estimated cost of applying one ton agricultural limestone/acre, including application: $63/acre + $20/A for field work required to incorporate the limestone.

Current estimated cost of two tons per acre: $146 [= (2 x $63) + $20)].

Average spinach seed crop production costs for western WA: $1,400–$1,600/acre.

Average price a spinach seed grower is paid for spinach seed: $1.80–$2.00/lb seed.

Typical spinach seed yield (varies widely with parent lines): 1,200 lb/acre.

Conservative estimated net income from a spinach seed crop: $560/acre.

Minimum increase in seed yield needed to justify the cost of applying one ton limestone/acre: 46 lb/A (a 4% increase over an ‘average’ yield).

Minimum increase in seed yield needed to justify the cost of applying two tons limestone/acre: 81 lb (a 7% increase over an ‘average’ yield).

If a grower applied one ton limestone/acre annually with a four year rotation between spinach seed crops: four years x $83/ton/acre = $332/acre increase in costs (vs. $83/acre for a single application the spring that the spinach seed crop is planted).

If a grower applied two tons limestone/acre annually with a four year rotation between spinach seed crops: four years x $146/acre = $584/acre increase in costs (vs. $83/acre for a single application the spring that the spinach seed crop is planted).

Tables 1 to 4 show estimated economic returns and spinach seed yield increases associated with different rates of limestone applied in a field trial completed in the Skagit Valley, WA in each of 2006, 2007, 2008 and 2009. Each year, the trial was on a different grower-cooperator field site and subject to the weather conditions of that season and site. Field sites were chosen, in part, based on a rotation interval of four-five years since the previous spinach seed crop to ensure high Fusarium wilt pressure. The low yields obtained for certain year/parent line combinations reflect this very short rotation, which spinach seed growers in the U.S. avoid (typical rotations are 10 to >15 years) but which were used in this series of trials to investigate “worst case” scenarios for spinach Fusarium wilt potential.

Seed yields differed depending on the trial, spinach parent line and rate of limestone amendment. The increase in spinach seed yield from applying two tons limestone/acre ranged from 14% (in 2008) to 190% (in 2006) of the yield in plots not amended with limestone and averaged 98% for all four trials. Generally, the more susceptible the spinach parent line, the greater the benefit of applying one to two tons limestone/acre, which underlines the importance of parent line susceptibility as a factor in the decision about how much limestone to apply for Fusarium wilt management.

Spinach Fusarium wilt soil bioassay:

An economic analysis of the soil bioassay must be framed by the goal of this objective, which is to help spinach seed growers avoid losses to Fusarium wilt due to high inoculum potential in field sites that were presumed to be low-risk based solely on years of rotation out of spinach seed crops. We anticipate the bioassay will prevent catastrophic losses that occur when a susceptible parent line is planted in a field that, unexpectedly, turns out to be highly conducive to Fusarium wilt due to factors that growers currently are not equipped to assess. While expenses associated with completing the soil bioassay can be estimated for a single field site, it is challenging to assign a precise value to losses associated with higher risk field sites, because losses can range from negligible to almost 100%.

With this caveat, the following are estimated costs of completing the soil Fusarium wilt bioassay for each field site (soil sample):

– 120 minutes to partially dry, sieve, crush and thoroughly mix five to ten gallon soil.

– 15 minutes to fill pots or flats (replicate pots or flats for each of three spinach parent lines).

– 15 minutes to plant seed in pots or flats.

– 15 minutes to rate x three weekly ratings = 45 minutes.

– 30 minutes for maintenance of the greenhouse bioassay (watering, fertilizing, clean-up).

Total labor: ? 220 minutes = ~3-4 hours x $15.00/hour = $45-$60 per soil sample.

Costs for pots, greenhouse infrastructure and maintenance, and propagative inputs (fertilizers, pesticide sprays as necessary, etc.) vary widely depending on the facility where the bioassay is completed but represent a small fraction of the total expense per sample compared to labor costs.

Spinach seed growers and seed industry personnel in the PNW have indicated they are willing to pay up to $250/field for an effective soil bioassay that will help them avoid significant crop losses by reducing the chance of planting spinach seed crops in fields with high risks of Fusarium wilt. We anticipate a fee of ~$200/field site (soil sample) should cover the expenses for a Fusarium wilt soil bioassay.

Farmer Adoption

The significant stakeholder involvement in this project, evidenced by >five years of grower-cooperator field trials and widespread stakeholder participation in the soil bioassay, is a testimony to the investment stakeholders have placed in this research project. According to feedback we have received from growers and seed company representatives, there has been widespread adoption of increased rates of limestone application for spinach seed production in the maritime Pacific Northwest based on results of the 2006-2009 field trials on limestone-mediated suppression of spinach Fusarium wilt. Application of one ton limestone/acre was a standard practice because of the acidic nature of soils in this region, but application of two tons limestone/acre is becoming routine after stakeholders observed the benefit of this higher rate of application, particularly for spinach parent lines highly susceptible to Fusarium wilt. Fields with a soil pH <5.8 are common in western WA, for which application of two tons limestone/acre is necessary for any significant change in soil pH given the highly buffered nature of these soils. Part of this decision-making process has been the understanding that applying limestone and raising the pH and calcium levels in highly-leached, acid soils of this region is also beneficial for many of the crops grown in rotation with spinach, including small grains, cabbage seed, corn, cucurbits and grass seed. There is evidence that limestone application may enhance potato yields as well. Spinach seed growers in other countries, such as Holland and New Zealand, are also evaluating limestone amendments based on the evidence from this research.

For years, spinach seed growers and industry representatives have asked researchers at Washington State University, the Land Grant university for Washington State, to help develop tools to predict Fusarium wilt risk so they can select “safe” fields with low risk of Fusarium wilt for spinach seed crops, more effectively than currently possible based solely on rotation interval between spinach seed crops. Active stakeholder participation in development and evaluation of the soil bioassay, even in the preliminary stages of development, has demonstrated widespread interest in adopting this diagnostic tool. Almost every spinach seed producer (or contracting seed company) in western Washington submitted two five-gallon buckets of soil in December 2009 from potential field sites to be assayed for the risk of spinach Fusarium wilt. Many of these stakeholders visited the bioassay in the greenhouse in February and March to monitor progress of the bioassay with their soils, particularly the response of different spinach parent lines, in anticipation of making decisions where to locate spinach seed crops prior to the March ‘pinning’ meeting at which seed crop locations were finalized for the 2010 season. As we visited these fields over the summer 2010 to assess Fusarium wilt, we received queries from stakeholders about whether the bioassay will be repeated in 2011 and subsequent years. Stakeholders have acknowledged the significant time and resources required for the bioassay and shared what they would be willing to pay for a diagnostic bioassay should the method be made available through private agencies such as soil testing labs. This feedback from stakeholders illustrates the urgent need for this disease prediction tool.

Recommendations:

Areas needing additional study

1. Timing of and/or other types of nitrate-N fertilizers.

2. Effect of pH on Verticillium infection of spinach seeds, another soilborne pathogen of spinach.

3. Effect of limestone/elevated pH on incidence of diseases of crops commonly rotated with spinach seed crops in the PNW, e.g., scab of potato and clubroot of cabbage.

4. Screening for resistance to Fusarium wilt in spinach parent lines, given how critical this information is to an accurate bioassay for assessing the risk of Fusarium wilt. Without information on parent line susceptibility, stakeholders will need to assume a ‘worst case’ scenario of highly susceptible parent lines, which could increase the chance of fields being rated as greater risk than would occur if less susceptible parent lines were planted. This is complicated by the difficulty of screening diverse spinach germplasm given the highly proprietary nature of parent lines and the inability to set up a field screening site at the WSU Mount Vernon NWREC given the location of this facility in the center of the seed crop production area of northwestern Washington.

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.