Fusarium & Verticillium Wilts in Spinach - WSU Mount Vernon ...

Fusarium & Verticillium Wiltsin Spinach: Research UpdateWilbur-Ellis/Alf Christianson Seed Co. Seed Growers’ Mtg6 February 2013, Burlington, WALindsey du Toit, Mike Derie, Emily Gatch, & Barbara HolmesWSU Mount Vernon NWREC

Emily Gatch’s PhD Research Objectives1. Assess potential for enhancing suppression of Fusariumwilt using annual applications of limestone and nitrate-Nfertilizers, and the effects of limestone on Verticilliumdahliae.2. Develop a soil bioassay for spinach seed growers todetermine the relative risk of a field for Fusarium wilt,using spinach parent lines with different levels ofresistance.3. Determine the effects of micronutrients and pH on F.oxysporum f. sp. spinaciae and host-pathogeninteractions.

Rep 1Rep 2NH 4 NO 3 NO 3 NH 42009 Field TrialRep 3NO 3 NH 4Rep 4NH 4 NO 3Rep 5NO 3 NH 42 tons limestone0 tons1 ton0 tons2 tons1 ton limestone1 ton0 tons2 tons1 tonS R M S R MO tons limestoneR M S R M S M R S M R S2 tons2 tonsR S M R S M1 tonS M R S M R0 tons

2009 2010 2011 2012spinach potato winter wheat spinachLimestone applications

2012 Field TrialRep 1 Rep 2 Rep 3 Rep 4Rep 5Proline2 tons limestone0 tons1 ton0 tons2 tonsProlineProlineProline1 ton limestone1 ton0 tons2 tons1 tonProlineS M R S M R0 tons limestoneM R S M R S R M S R M S2 tons2 tonsM S R M S R1 tonS R M S R M0 tons

2 tons limestone/acre each year1 ton limestone/acre each year0 tons limestone/acre each year0 tons limestone/acre each year+ 2 tons/acre in spring 2012

Soil pHEffect of limestone rate on soil pH: 2009-20128.07.06.05.00120/24.02009 2010 2011 2012spinach potato winter wheat spinachLimestone applications

Soil pHEffect of limestone rate on soil pH: 2009-20128.07.06.05.00120/24.02009 2010 2011 2012spinach potato winter wheat spinachLimestone applications

Soil pHEffect of limestone rate on soil pH: 2009-20128.07.06.05.00120/24.02009 2010 2011 2012spinach potato winter wheat spinachLimestone applications

Soil pHEffect of limestone rate on soil pH: 2009-20128.07.06.05.00120/24.02009 2010 2011 2012spinach potato winter wheat spinachLimestone applications

Incidence of wilt (%)Wilt incidence1008024 June 2009SusceptibleModerateResistant19 June 201260Male402000 1 20x4 1x4 2x4 0-0-0-2Limestone rate (t/A)

Incidence of wilt (%)Wilt incidence10014 July 20099 July 2012806040200SusceptibleModerateResistantMale0 1 20x4 1x4 2x4 0-0-0-2Limestone rate (t/A)

Severity of wilt (0 to 5 scale)Wilt severity5439 July 2012 31July 2012SusceptibleModerateResistantMale2100 1 20x4 1x4 2x4 0-0-0-2Limestone rate (t/A)

Dried plant weight (g)Spinach biomass (dry plant weight)250Susceptible22 July 2009 11 July 2012200150ModerateResistantMale1005000 1 20x4 1x4 2x4 0-0-0-2Limestone rate (t/A)

Seed yield (lb/A)Seed yield1600Susceptible2009 20121200Moderate800Resistant40000 1 20x4 1x4 2x4 0-0-0-2Limestone rate (t/A)

Fusarium-infected seed (%)Harvested seed (%) infected with Fusarium252009 20122015SusceptibleModerateResistant10500 1 2Limestone rate (t/A)0x4 1x4 2x4 0-0-0-2

V. dahliae infected seed (%)Harvested seed (%) infected with Verticillium dahliae252009 20122015SusceptibleModerateResistantMale10500 1 20x4 1x4 2x4 0-0-0-2Limestone rate (t/A)

2. Develop a soil bioassay for spinach seed growers todetermine the relative risk of a field site for Fusariumwilt, using spinach lines with different levels ofresistance.- Wilt ratingsResearch Objectives- Spinach biomass- Quantify F. oxysporum and V. dahliae on selective media- Soil nutrient analyses: macro & micronutrients, pH, OM, CEC- Texture analysis- # of years out of spinach seed crops- Quantify F. oxysporum f. sp. spinaciae with a real-time PCRassay

2009-10: 26 soils submitted2010-11: 42 soils submitted2011-12: 39 soils submitted2012-13: 40 soils submittedControl soils included each year:Low (no), medium, and high Fusarium wilt risk

RSoil AMSoil CSRMRSMSSoil B

Fusarium wilt indexRisk assessment: Spinach Fusarium wilt soil bioassayPartially resistant line Moderately-susceptible line Susceptible lineA. 2009-10 bioassayField (soil) sampleB. 2010-11 bioassayField (soil) sample

Risk assessment: Spinach Fusarium wilt soil bioassayPartially resistant line Moderately-susceptible line Susceptible lineC. 2011-12 bioassayField (soil) sample

Bioassay validation

AcknowledgementsPuget Sound Seed Growers’ AssociationWashington State Commission for Pesticide RegistrationAlfred Christianson Family Endowment, Robert MacDonald FundUSDA Western Region IPM, Western SAREParticipating growers and seed companiesTodd GordonDan Nelson, Russ DuckworthMike Derie, Barbara Holmes,Avi Alcala,Sarah Meagher,Ron Dralle, Jonathan Roozen,Martha Sudermann,John Kuhn, Anita da Costa,Shawn Watkinson, Tammy Ennen,Nathan McCartney, Tyler VanderPol

Evaluation of Proline for Management ofFusarium Wilt in Spinach Seed Crops

Proline and Fusarium WiltsProline 480 SC = prothioconazole (triazole, Group 3)Spinach seed crops in DK: empirical observations?Transplanted watermelon Fusarium wilt: drench, drip trialsZhou & Everts. 2009. PDMR 3:V092; Zhou et al. 2010. PDMR 4:V065Everts et al. 2011. Phytopathology 101:S50-S51 (Abstract)120100806040200eabcabcdbcdaedbaeabcdcdbcabca abbcdaabcdebc cd cd• 3 drenchapplications• 2-weekintervals fromtransplantingWilt incidence 17 JulPlot vigor 7 AugYield (kg/plot)

2012 Spinach Seed Crop Fusarium Wilt Field TrialRep 1 Rep 2 Rep 3 Rep 4Rep 5Proline2 tons limestone0 tons1 ton0 tons2 tonsProlineProlineProline1 ton limestone1 ton0 tons2 tons1 tonProlineS M R S M R0 tons limestoneM R S M R S R M S R M S2 tons2 tonsM S R M S R1 tonS R M S R M0 tons

Banded Proline applications (CO 2 -backpack sprayer)10 May = 2 days after planting spinach31 May = 3 weeks after planting21 June = 6 weeks after planting

Severity of wilt (0 to 5 scale)Severity of wilt (0 to 5 scale)Effect of Proline on severity of wilt54ProlineNo Proline54ProlineNo Proline* 5.1% * 8.2%* 6.3%33221109 July 31 JulyWilt assessment date0Susceptible Moderate ResistantSpinach inbred line* Denotes significant difference between Proline vs. no Proline plots at P

Seed yield (lb/acre)Effect of Proline on seed yield15001000500ProlineNo Proline* 18.4% * 11.4% 6.9%0Susceptible Moderate ResistantSpinach inbred line* Denotes significant difference between Proline vs. no Proline at P

Incidence of seed with Fusarium (%)Incidence of seed with Verticillium (%)Effect of Proline on incidence of harvested seedinfected with Fusarium and Verticillium10FusariumProline15VerticilliumProline8No ProlineNo Proline6104520Susceptible Moderate Resistant0Susceptible Moderate ResistantSpinach inbred lineSpinach inbred line

2013 Spinach Seed Crop Fusarium WiltProline Field Trial• Field: acid soil, spinach seed crop last 4-5 yrs,proximity to NWREC• Treatments:• Proline vs. no Proline• 3 female spinach inbred lines (S, M, R)• 2 tons limestone/acre (all plots)• Apron + Thiram + Topsin seed treatment• Proline applications:• Method: In-furrow drench + banded soil applications• Number: 3 = maximum (sugar beet)• Interval: 14-21 days

2013 Spinach Fusarium WiltProline Field TrialSuggestions? Questions?dutoit@wsu.edu or 360-848-6140

Management of Verticilliumin SpinachLindsey du Toit & Mike Derie, Washington State UniversityJim Correll, University of Arkansas

Verticillium associated with spinach seed1. Prevalence ofVerticillium incommercial spinachseed lots (90% of lotstested: US, EU, NZ,Chile)2. Pathogenicity of seedisolates to spinach3. Seed-to-seedtransmissionPlant Disease 89:4-11Plant Disease 89:1305-1312PDMR 1:ST003; PDMR 3:ST020;PDMR 4:ST038

Spinach/lettuce connection?S. T. Koike?S. T. KoikeAtallah et al. 2010. Fungal Genetics & Biology.Gene flow among V. dahliae isolates from CA lettuce, pepper, strawberry,artichoke & weeds, spinach seed, & WI ornamentals (not tomato)Relative significance of infested spinach seed?

Evaluation of spinach seed treatmentsA. Hot water & chlorine (du Toit & Hernandez-Perez, 2005.Plant Disease 89:1305-1312)B. Efficacy of spinach seed treatments for managingseedborne Verticillium (du Toit et al., 2009. PDMR 3:ST020,du Toit et al., 2010. PDMR 4:ST038)1. Reduce incidence of seedinfected/infested2. Reduce rate of seedtransmission3. Reduce rate of soilinfestation from plantinginfested seedC. Influence of soil fumigation

% seed with Verticillium dahliaeGH Trials: % Seeds with Verticillium - Trial 170605040302010aaababcbccdConventionalOrganicd deefef efg fggh0ControlACX802ACX801Coronet 200 mlVortexCoronet 400 mlACX804Exp. IIncotec IACX803Incotec II(du Toit et al., 2009. Plant Dis. Mgmt. Reports 3:ST020)Exp. IICaptan 400CThiram 42-SSeed Support Ihi ij kl ij kl lSeed Support IISeedgardBAS 595 XGFMertect 340FFarm.D300+MertectlTopsin M70WP

CFUs V. dahliae /100 seedlings140000120000100000GH Trials: Seed transmission assay - Trial 2800006000040000200000abControlACX802Not evaluatedACX801Coronet 200 mlVortexCoronet 400 ml(du Toit et al. 2010. PDMR 4:ST038)acdACX804eExp. IIncotec IACX803bcIncotec IIabExp. IIabCaptan 400CcdThiram 42-SConventionalOrganicSeed Support IcdSeed Support IIdSeedgardef fgh eBAS 595 XGFMertect 340FFarm.D300+MertecthTopsin M70WPghTopsin 4.5FLControl, soilefgControl, pastzd soil

Influence of soil fumigation/pH on Verticilliumseed transmission & soil infestation2010 WA field trial of baby leaf spinach & head lettuce1. Main plots:MeBr:Chloropicrin (57:43% at 350 gpa)No soil fumigation2. Split plots:Acid soil pH (~5.7-5.9)5 tons limestone/ha (pH 6.6-6.8)3. Spinach split-split plots:No spinach seedBaby leaf spinach: No seed treatmentBaby leaf spinach: Apron + ThiramBaby leaf spinach: Apron + Thiram + Topsin 4.5 FLBaby leaf spinach: Farmore D300 + Mertect 340FFollowed by head lettuce in each plot (‘Salinas’ type)

1 June 201014 June 2010

2011 & 2012 Field trialsMount Vernon, WA (2011) & Salinas, CA (2011, 2012)Baby leaf spinach & head lettuce, RCBD, 5 reps1. Main plots:Fumigated vs. non-fumigated2. Spinach split plots:No spinachNon-treated seedApron + ThiramFarmore D300 + Mertect 340FApron + Thiram + Topsin 4.5 FL (WA 2011)Seed Support (WA 2011)ProGro (WA & CA, 2011), ProGro + Apron (CA, 2012)Followed by head lettuce in each plot

Salinas, CA: 2011 & 2012Ocean Mist Farms (2011)Christensen & Giannini (2012)New FreshSchafer Ag ServicesHoladay SeedsGermain’s Technology GroupRachel Yim Bobby Eugenia

Mount Vernon, WA: 2010, 2011Effect of seed treatment & soil fumigation onseed transmission of V. dahliaeCFUs V. dahliae/100 spinach plants after harvest of leavesSignificant fumigation & seed treatment effects

Height (mm) of spinach plantson 16 June 2011Mount Vernon, WA: 2011Effect of seed treatment on spinach plant height1412108642abcbcabcdaNon-treated seedApron+ThiramApron+Thiram+TopsinFarmoreD300+MertectProGroSeed Support0Soil fumigation treatment

Mount Vernon, WA: 2010, 2011% of lettuce stems infected with V. dahliae at harvestNo significant seed treatment or fumigation effects,& no symptoms of Verticillium wilt on lettuce.

Mount Vernon, WA: 2010, 2011CFU of V. dahliae in soil at spinach plantingSignificant fumigation effect, but no seed treatment effect

Mount Vernon, WA: 2010, 2011CFU of V. dahliae in soil at lettuce plantingSignificant fumigation & seed treatment effects in 2010,significant seed treatment effects in 2011.

Mount Vernon, WA: 2010, 2011CFU of V. dahliae in soil 2-5 weeks after harvest oflettuce & incorporation of lettuce residuesNo significant fumigation or seed treatment effects in 2010.

Salinas, CA: 2011, 2012Effect of seed treatment & soil fumigation onseed transmission of V. dahliaeCFUs V. dahliae/100 spinach plants after harvest of leavesSignificant seed treatment effects,no significant fumigation effects

Salinas, CA: 2011, 2012% of lettuce stems infected with V. dahliae at harvestNo significant seed treatment or fumigation effects,& no symptoms of Verticillium wilt on lettuce plants.

Salinas, CA: 2011, 2012CFU of V. dahliae in soil at spinach plantingNo significant fumigation or seed treatment effects

Salinas, CA: 2011, 2012CFU of V. dahliae in soil at lettuce plantingNo significant fumigation or seed treatment effects

Salinas, CA: 2010, 2011CFU of V. dahliae in soil 2-5 weeks after harvest oflettuce & incorporation of lettuce residuesNo significant fumigation or seed treatment effects

Summary• Soil fumigation increased colonization of spinach plantsby V. dahliae 7-fold in 2010 in WA & 3-fold in 2012 in CA(not in 2011 in WA or CA sites)• Seed treatments reduced seed transmission effectivelyin all 4 trials• Best seed treatments = not planting spinach seed• Mertect 340F: federal registration spring 2014• Rancona Spinach (ipconazole): registered Jan. 2013• Topsin M 70WP: SLN spinach stock seed in WA• Planting infested spinach seed did not result inVerticillium wilt in head lettuce in all 4 trials, & had nosignificant effect on soilborne V. dahliae detected 2-5weeks after incorporating lettuce residues

Characterization of Verticillium dahliae fromspinach seed, and screening for resistance toVerticillium wilt in spinach.Villarroel-Zeballos, M.I., Feng, C., Iglesias, A., du Toit, L.J., & Correll, J.C. 2012.HortScience 47:1297–1303.Pathogenicity, virulence, &vegetative compatibility grouping ofVerticillium isolates from spinach seedIglesias-Garcia, A.M., Villarroel-Zeballos, M.I., Feng, C., du Toit, L.J.,& Correll, J.C. 201_.Plant Disease: submitted January 2013.

AcknowledgementsAmerican Seed Research Foundation of ASTAPuget Sound Seed Growers’ Assoc.WA State Comm. for Pesticide RegistrationCA Leafy Greens Research BoardAlfred Christianson EndowmentParticipating seed & chemical companiesInternational Seed Health Initiative – Root, Bulb & Leafy VegetableInternational Technical GroupMike Derie, Barbara Holmes,Emily Gatch, Ana Vida Alcala, Ron Dralle, others …Angela Iglesias-Garcia (MS), Maria Villarroel-Zeballos (MS)

Incidence of wilt (%)Wilt incidence100804 June 2009 29 May 2012SusceptibleModerate60ResistantMale402000 1 20x4 1x4 2x4 0-0-0-2Limestone rate (t/A)