ProductClass(es):Methodtypedescribed:Dateofprotocol:ProvenforShouldbesuitablefor Version comments iniatriticina(P.recondita) SBIfungicides,alsosuitedforotherfungicideclasses leafsegmenttest(detachedleafmethod) 2006-05 Tebuconazole,Prothioconazole otherSBI-fungicidesandactiveingredientsfromotherclasses.Protocoladjustmentsmaybeneededduetothepoundcharacteristics.
1 •validatedroutinemethodforlabsequippedwithclimatechambersand/orgreenhouses •provenmethodologyfortheactiveingredientslistedabove.Othersnotmentionedhavetobeevaluatedcarefullytoensurevalidresults. •themethodwasdevelopedandkindlyprovidedbypanyEpiLogic,Germany(Dr.F.G.Felsenstein) Method:
1.Toobtainrepresentativedatafromdifferentrustpopulations,samplesofuredo-/teleuto-sporescanbetakenbysporetraps(mobileorstationary)(a)orbyrandomsamplingofinfectedleaves(b).a)Uredo-/teleuto-sporesarecollected(trapped)ontosegmentsofprimaryleavesfromahighlysusceptiblevarietyplacedinPetridishesonwateragar(0.6%agar,35mg/lbenzimidazole).Sampleshavetobetakenduringthepeakphaseoftheepidemicwheneverpossible.Letthesampledsporesgrowtosinglecolonyisolates(climatechamber:18°C,10-20µmol/m²scontinuouslight)andtransferthemontofreshleafsegmentsonwateragarformaintenanceandforpropagationbeforetesting.b)Uredo-/teleuto-sporesfromsampledleavesshouldbepropagatedonfreshleafsegmentsplacedinPetridishesonwateragarasmentionedina).
2.DeterminetheSBIsensitivityofeachsinglesporeprogenyonatestsetofleafsegmentswhichareplacedonbenzimidazoleagar(6mg/lagar-agar,35mg/lbenzimidazole).Onedaybeforecuttingtheleafsegments,treattendayoldplants(firsttosecondleafstage)withpoundtorunoffconditions.FungicidetreatmentsshouldbegradedlogarithmicallybyarelativelysmallfactoroftwoorthreeinordertoobtainanoptimalEC50evaluationforthesensitivitytowardstheactivesubstance(dependingonpound). Examples: oTebuconazole:0,0.04,0.08,0.16,0.32,0.64,1.28,2.56,5.12,10.24,20.48mg/l oProthioconazole:0,0.64,1.28,2.56,5.12,10.24,20.48,40.96,81.92,163.84mg/l Keepdifferentlytreatedplantsstrictlyseparatedtoavoidgasphaseinteractions.Adayafterthespraytreatmentoftheplants,cutouttheleafsegments.Toavoidgasphaseinteractionsbetweendifferentlytreatedleafsegmentsinthetestassortment,useseparatedisposablePetridishesforeachfungicideconcentrationineverysingletestset.Asrepetition,eachPetridishshouldcontainseveralsegmentsofdifferentplantstreatedwiththesameconcentration.AtestassortmentforasingleisolatewithtenconcentrationsofthepoundthereforeconsistsoftenPetridishesincludingtheuntreatedcontrol.
3.Thedishesofatestsetshouldbeonlyplacednexttoeachotherduringtheinoculationphase.Inoculatetheleafsegmentswiththesinglesporeprogenybyairpressure(dryconditions-noaqueoussporesolution).
4.Afteranincubationperiod(climatechamber:18°C,10-20µmol/m²scontinuouslight)oftendays,scoreeachtestassortmentmacroscopicallyregardingdiseasecoverage/developmentparisontotheuntreatedcontrol(percentage).Then,calculateEC50ofeachtestisolatebyProbitanalysis. References: FELSENSTEINFG,1996.Responseofiniareconditaf.sp.triticitotherecentselectionpressureofDMIfungicideswithinwesternEurope.Proc.Ofthe9thEuropeanandMediterraneanCerealRusts&PowderyMildewsConference,2-6September1996,Lunteren,TheNetherlands,P2.27,155. FELSENSTEINFG,1997.Brownrustofwheat–aEuropeanwidemonitoringprogrammetostudyitsfugicidesensitivity.Resistance´97:IntegratedApproachtoCombatingResistance,IACR-Rothamsted,Harpenden,UK,70. FELSENSTEINFG,PARKRF,ZELLERFJ,1998.TheuseofdetachedseedlingleavesofTriticumaestivumtostudypathogenicityininiareconditaf.sp.tritici.J.athology146,115-121. author Dr.F.G.Felsenstein,EpiLogicGmbH,Hohenbachernstrasse19-21,85354Freising-Weihenstephan,Germany