(1):36-41 Philippine Journalof CropScience(PJCS)P9ri12012,37 2Q12,CropScienceSocietyof the Philippines Copyright
An Optimized MicrochamberMethod for Rapid Screening of Rice for S h e a t hB l i g h tR e s i s t a n c ei n t h e P h i l i p p i n e s Jean J. Someral and Antonio A. Alfonso2' lMonsanto Phils., Inc., Research & Der,elopmentStation,Conel Road, Lagao, Generalsantos CiV 9500; 2Plant Breedingand BiotechnologyDiMsion,PhilippineRice ResearchInstitute,Maligaya,Science City of Mu6oz 31'19, Nuera Ecija; *Correspondingauthor,
[email protected] Breedingfor resistanceto sheath blight (caused by Rhizoctoniasolani KUhn)in rice is constrainedby the lack of highly resistant donors and a rapid, simple but effective screening rnethod. The use of microchamberssuch as plasticsoda bottles provides bettercontrol of humidityand temperaturenecessary for growth and establishrnentof the causal organism. In this study, the microchamber protocol was o p t i m i z e df o r r a p i d a n d e f f e c t i v es c r e e n i n g T . h e f a c t o r sc o n s i d e r e da r e : 1 ) k i n d o f i n o c u l u mc a r r i e r , 2 ) seedling age for inoculation,3) length of incubation period prior to disease evaluation, and 4) disease scoring system. Results indicatethat R. solani grows better using mycelialdisks as inoculum carrier as comparedto rice hull substrate. Seedlingscan be inoculatedas early as 14 d after sowing and disease scoringcan be done at 5-7 d after inoculation.Using the StandardEvaluationSystemfor Rict(lRR 2002)as basis, a modified scoring system that takes into account seedling age during inoculation and use of qualitative and quantitative disease indices was also developed. Using these optimized parameters, screening results can be obtained in less than a month after seed germination.In a subsequent experiment, 103breedinglines were evaluatedusingthe optimizedmicrochambermethod.The reactionsof 13 advanced lines rnatchedwith the previous results of induced field screening,Moreover,the resistantto moderately resistantreactionsreported for l0 rice accessionswere also confirmedusing the microchamberrnethod. The optimizedmicrochambermethod is fast, simple, reliableand economicalcomparedto the traditional rnethodof screeningfor sheath blight resistance. Keywords: actual disease index, induced screening, inoculum carrier, microchamber method, optimized protocol,rice sheath blight,Msualdisease index
INTRODUCTION
was the medianlesionlengthin the first 1 to 3 leaves from the base of the plant The number of infection Sheath blight, caused by the fungus Rhizoctonia cushionsand lesion type also harc been used to so/anl Kuhn, is a rnajordiseaseof rice that reduces differentiate the lerclof sheathblightresistance in rice yield and grain quality. Screening rice breeding linesby someresearchers.Othershave rateddisease materials againstsheathblightis routinelydone under reactionsbased on the ratio of the lesionarea to the field conditionusuallywith inducedinoculationusing total leaf area. localisolateof the pathogen.Plots for inoculationare densely planted Wth the test entries to allow more In the Universityof Stuttgart,Georgia,Eizengaet al. fawrable growth of the pathogen Entries are then (2002)deleloped a standardizedscreeningtechnique ratedfor selerityof diseaseafterat least21 d or when called the 'microchambermethod'for a ouick and the susceptible check starts to manifest severe accuratedetectionof sheathblightin seedlingsThis symptoms.Such screeningprocesstypicallyentails techniqueuses plastic bottles to create a humidity one full season, and is therefore, labor intensive, chamber that promotes disease development.This costlyand requiresa lot of seeds(Pinson2007). allowsthe researchersto measureseedlings'disease reaction in just 7 d accelerating the process of While differentinoculationsourcesand methodsused identifyingnornelresistantsourcesfrom cultivatedand for the analysisof sheathblight resistancehave been wrldrelatircsof rice. used and studied,differenttechniquesfor eraluating the severityof sheathblight infectionhave also been Pinson (2007) has been studying gene-mapping applied.Grothet al. (1990)firstestablished the Vsual populations from recombinantinbred lines (RlLs) of ratingof a 0 to 9 scalewth lesionlengthestimatedas domesticrice cultivar"Lemont'and Chinesecultilar a fractionof the total plant height. Eizengaet al "TeQing".Using microchambermethod,it was found (2002) used two ewluation methodsto quantify the that '18 chromosomalregions in these RlLs have sewrity of sheath blight. The first was a modified genes that can help rice plantsresistdamagefrom vrsual rating scale of 0 to I, with the number sheathblight. One such region was qShB9-2.The correspondingto the percentageof leaves co\ered same region was verifiedto haw a major effect on with lesions.The secondewluation techniqueused controllingthe disease(Jia et al. 2007) Prasadand
Eizenga(20OA) screened 73 Oryza accessionsand reportedthat the microchambermethodgave a more uniform,reproducibleresponse,and was easierto use undergreenhouse conditionas comparedto detached leaf and toothpick inoculation methods. They identifiedse\,en test entries with moderateresistance to sheathblight
screenhouse
condilion
at
the
Philippine
Rice
Research Institute (PhilRice), Munoz, Nueva Ecija, Philippines in 2010 dry season. Three seedlings were sown in each earlhen pot and pots u/ereplaced insideseedboxeswith shallow standingwater (Figure 1). lnoculatronwith the pathogentook place 14lo21 d after sowing by placing R. so/ani infecbd inoculum carrier at the base of each seedling.At 5-10 d after ln the microchamber methodof screeningwhichwas inoculation, symptoms \ €re rated hrough visual firstconductedin Bangladesh, morstureis kappedby diseaseindex and actual measurementof the lesions, soda bottlesduring incubationperiod, creatingand The optimizationset-ups lrrere carried out in 3 x 3 m a i n t a i n i n ga h u m i d ' m i c r o - e n v i r o n m e nw t ' h e r e factorialexperirnenlarranged in RCBD that includes seedlingsfor testing are grown. The set-up is more two inoculum types (PDA mycelialdisk and rice hull conducivefor growth and establishmentof the substrate), three test enbies (TN1, Lemont and pathogen.This process requires less seeds, less Jasmine85) replicatedthreetirnes. labor, less screeningcosts and shorter plant-growth time needed to come uD with a result lt is also Inoculumpreparation conductedunder controlledscreenhousecondrtions, Pure culture ol R. solani was lirst obtained usirg thus allowng researchers to conduct disease sclerotia from disease-infectedplants in the field. screeningindependentof the field growingseason Collectedsclerotiawere disinfectedwith 10% Clorox (Pinson2007). for 1 min and rinsed with three changes of sterile distilledwater. Sclerotiar €re then plated into potato Validationand impro\€mentof the microchamber dextroseagar (PDA) slants and incubatedfor 4-5 d, method of screeningagainst sheath blight under The pure cultures r €re transferredinto petri plates Philiopinescreenhouseconditionallow breedersto fast-track the dev-.lopment of elite lines with containingPDA incubatedfor 7-10 d, and used as the sourceof inoculumgrown on differentcarriers. resistance/tolerance to the fungaldisease.With this new method,screeningfor early generationbreeding Tvrc types of inoculumcarriers $/eretesbd, the PDA which usuallyhavefew seeds,can also be materials, mycelialdisk and rice hull subsEate.For the rnycelial conducted disks, sclerotiaof R. so/ani were grorr/nin petri plates This study was conductedto test, modifyand develop containingpotato defose agar (PDA) for 3 d. One a screeningprotocolfor sheath blight in rice using liter of distilled water was mi)€d with 39 g PDA, methodunder screenhousecondition, dissol\€d using microffaveoven, and sterilizedfor t h microchamber and identify resistantand susceptiblechecks in at 121 "C. The sterilizedPDA was then set aside to screening sheathblightin rice and possibledonorsfor cool down, 30 mL was poured onto each sterile dry petri platesand allowedto solidify.The substratewas sheathblightresistance. preparedby miing 3 parb rice hull wih 1 part rice bran and a littletao water to moistenthe mi)dure. The substratewas then autocla\€d for t h at 121'C.When MATERIALSAND METHODS the sterilized substrate was cooled down, slices of PDA wih R so/anlgrowh were placedin the mixture. Test materials Seedsof sheathblightsusceptible varietyLemontand The inoculated substrate was incubated at room for 2-3 uik Driorto use resistant\ariety Jasmine85 were obtainedfrom the temDerature T.T ChangGeneticResourcesCenterat International Rice ResearchInstitute(lRRl), Los Baflos,Laguna lnoculation For inoculaton using mycelial disk as inoculum PhilippinesTaichungNative 1 (TNl) was used as susceptiblecontrol One hundred three breeding carrier,disksabout 1cm in diarneterrrrErecut out from materialswere initiallyscreenedagainstthe disease a 3-d PDA cultureof R solanl in peti dishes.A single usingthe modifiedprotocol.Theseincludebackcross disk was placed at he base of every seedling.For deri\atives of Oryza rufipogon Griff and PSB Rc18, inoculation using rice hqll substrate as carrier, a ad\ancedlRRl lines, traditionalculti\€rs,and some thumb-sizepinch of 2-!r,ftoH culture of the subsfate wild rices like Oryza barthil A.Chev., Oryza nivara was also placed at the base of e\,ery seedling.Each S.D.Sharma& Shastry, Oryza meridionalis Ng and pot was then co\erd wih a plastic soda bottle (bottomand cap removed)and pushedinto the soil to Oryza officinaliswall ex G Watt createa seal. The soda bottle createsa humid microenvironmentallowingthe fungi to grow and infect the Experimentalset-up Following the basic protocol developed by Pinson olants. (2007), the microchambermethod of screening against sheath blight was conducted under JJSomera& AA Alfonso
37
with 6 seedlingsper replication.The seedlingswere grownin snnll pots (3 seedlingsper pot at 2 potsper replication),
Figure 1. Experimentalset-upfor the optimizationof microchamber method conducted at the PhilRice screenhouse
DiseaseEvaluation At S10 d post inoculation(DPl) or when the susceptible materials already showed disease symptons, the seedlingsu,ere evafuatedon the progress of the disease. Using the Standard EvaluationSystem (SES) for Rice (lRRl 2002) as basis,a rnodifiedscoringsystemfor Msualdisease indexwas recordedas follovre: G^-,Jcale
1 3 5 7 9
Descriptive Rating
Description Based on Relative Lesion Height
Resistant
Lesion limitedto lower 20% of the sheath 2Oto 3oo/o 31 to 45o/o 46 to 65% more than 65%
Resistant lntermediate Susceptible Susceptible
lvloreover,actual measurernentof lesions (actual diseaseindex)on the sheathwas alsorecordedusing the forrrulausedby Jia et al. (2007)with rnodification so that sheath height was used instead of plant height: Actual Diseaselndex = (Lesim length / Sheathheight) x 9
Lesionlengthwas rneasured fromthe lowestlesionat the base of the seedlingup to the topnrcstlesion dewlopingwithinthe entireseedling.Sheathheight u/asmeasured fromthe baseof the seedlingup to the collarusinga meterstick.DiseaseIndex\€lueswere interpreted descriptively as follows:
ProtocolOptimization/Va lidation Fourimportant factorswereconsidered in thisinduced screenhouse screening: 1) inmulumcarrier,wherein theuseof PDAmycelial disksas inoculum carrierwas compared withthe usualpractice of usinga mixtureof rice hulland branat 3:1 ratio;2) theage of seedlings for inoculation;3) the duration of disease establishment or numberof days after inoculation before the susceptiblecontrol exhibits disease symptoms, and 4) data gatheredfor diseaserating. During optimization, temperature inside the screenhouseand inside the microchamber were monitored andrecorded. RESULTS ANDDISCUSSION Testfor SuitableInoculum Twc preliminary set-upswere madeduringthe 2010 dry seasonto optimizethe four importantfactorsin microchamber screening.In these set-ups, TN1 (standardsusceptiblecheck), Lemont(susceptible entry)"and entry)wereusedas Jasmine85 (resistant test materials. Thefirstset-upincludedPDA mycelial disk and rice hull substrateas inoculumcarriers. Visualdiseaseindexand the actualdiseaseindex resulting fromthefirstpreliminary set-upare shownin Figure2. Jasmine85 had a susceptiblerating reactionin bothset-upscontraryto a preMousreport (Jiaet al.2007). Whenthe two inoculum carriersu/erecompared, the useof PDA mycelialdisksgavea moredifferentiated reaction between the resistantand susceptible at 10 controls.The differencein sheathappearance DPIusingmycelial diskas inoculum carrieras wellas in overallseedlingstand was very much eMdent between Lemont (susceptible)and Jasmine 85 (reported resistant) as seenin Figure3.
OptimumConditionsfor Microchamber Screening Similar results were obtained in a follow-up erperimentintendedto verify the results of the preliminarystudy. Jasmine85 had more healthy seedlingscomparedto Lernont(Figure4). Results Descriptive Rating Scale confirmedthat the useof PDA mycelialdisksis nrcre Resistant 0-3 carrier.Moreover, effective thanricehullas inoculum ModeratelyResistant 3.1- 4.9 can be used for inoculation.ln 14-day-old seedlings 5.0- 5.9 lntermediate time, it is also necessary sinceolder addition to saving 6.0- 6.9 ModeratelyS usceptible not totally by the seedlings become tall and covered 7.0- 9.0 Susceotible plasticbottlesduringthe durationof the screening (Figure5). Resultsalso indicatedthat the post Screening of Breeding Materials periodpriorto diseasescoringcanbefrom inoculation One hundred three genotypes from rarious sources 5-10 d only sincethe susceptible entriesstartedto rirrerescreened using the modified microchamber nranifestdiseasesymptomsas early as 5 d after protocol. Screening was done in three replications, inoculation (Figure3). 38
RapidScreeningMethodfor RiceSheathBlight
Figure2. Reactions of Jasmine 85,TNI andLemontagainstR. so/arlasdetermined throughmicrochamber screening usirE (A)visualdisease PDAdisks andricehullsubstrates as inoculum carriers: index:fB)actualdisease index.
Figure 3. Differentialdisease severityon the sheaths of TN1 , Lemont,and Jasmine 85 when inoculatedwith substrateand myceliadisk,incubatedusingmicro-chamber period.Arrowsshow the good methodat '10days post inoculation seedlingstandof Jasmine85 and susceptibility of Lemontwhen inoculated with mycelialdisk.
100 ;80 !p
E40 -3 z
30 l0 0 slbrirate
Myce|al Oirk
subsrr.rc
Myceli.l oirk
J a s m r n8e5
Figure 4. Reactionot Jasmine 85 and Lemontto sheath blightinoculationusrngmicrochamber method a;rdwith rice hullsubstrateor mycelialdisk as inoculumcarriers.The use of mycelialdisk produced a more distinct and consistent susceptible reactionin Lemont JJ Somera E AA Alfonso
Figure 5. Inoculation at l4 DAS (left)allowsthe whole seedling to be covered with plastic soda bottlewhile at 2'1 DAS (right)seedlingsare already so tall makingit difficultto impose the micro-environment condition that promotesdiseasedevelopment.
39
Finally, either Msual or actual cjisease index measurementmethodscan be used to evaluate diseasesymptomsusing the nrcdifiedscale as reflectedin the methods,In evaluating the disease development, a very highcorrelation valueof 0.951 wiasestablishedbetweenvisualestinration(usinga rnodified rating scale) and that of the actual rneasurerrent of diseaselesionsto determinedisease index(Figure2). Thissuggeststhat eitherof the tu,rc diseasescoring methodscan be used in data gathering.However,thoughvisualdiseaseindexis faster to obtain, the actual disease index was for a nrcrequantifiable considered data. Microchamber Screening of Breeding Materials With the establishrnent of the variouspararneters in microchamber the ewluationof breeding screening, nraterials in searchfor possible donorsfor resistance was conducted.Sercral batchesof screeningswere rnadeto evaluate103 breedingmaterialscomposed of 72 backcrossderivativesof PSB Rc18 and O. rufipopn includingparentalgenotypes,and 31 IRGC collections. Two experimental set-ups were conductedto checkthe consistencyof the reactions. genotypeswerefoundto haveresistant Twenty-sevren to interrnediatereactionsbased on the rnodified 1R84422-B-10 scoringsystem(Figure6). Interestingly, -2, |R8/422-B-35-1 have shown and |RB6170-14-B resistantreactionsunderfield screenings, and such reactionswere confirmedusing the microchamber screeningmethod(Alfonsoet al. 2010).Also, 12 of the 27 rnaterialswere previouslyreportedin other studies to have resistant to nnderately resistant reactions under screenhousescreeningsusing microchamber methodas shownin Table1 (Eizenga etal.2002;PrasadandEizenga 2008).
oMltrlrRffl@rlil In86ll6 t{ tl B O @'didrir Jjffi 163&l D Dlda/O rd'rd lltG( 100*4ll !Rt01l64 6 a tqlnl JNt6tA _Slo8 :RA61 /{i t rS { :8861/Stl/ I o fl/r@|trd l6n4
rB8E17Ftr0 r{N6llt + 5t t 1il6tn2+t2St rl! lCtB 16 EtJ[
U f,,@ fiM( ll}4d43l
R&r], R lt I o ffio tri{lt lo4rtFl .dtbeE o iiK lfrntl 0 olll&'tAtt tUAtsl
tm
2m
i|I)
4m
AwflgaArt|J3lDbrlratdr|
Figure 6. AverageActualDiseaseIndexof 27 materials showing either resistant or intermediate reactionsagainst R. solani. The resistant to moderately resistant reaclions of materials indicatedby a red bar have been confirmedin field screenings. Table 1. Reactions of different accessions against R. " solanias reportedby variousworkers.
Te Qing LSBR33 L S B R5 O. barthii(IRGC100223) O. nivara(IRGC100898 O. nivaralO.satlva(IRGC 6 100943) 7 O. nivara(IRGC 104443) I O. nivara(IRGC 104705) O. rufipogon (IRGC I 104785) O. meridiondis (IRGC 10 105306) Khao nok: O. rufipogon 1 1 ( | R G C1 0 5 7 5 7 ) O. otrictnalis(IRGC 12 105979) 1 2 3 4 5
MR R R R MR
MR* R to MR* R to MR* MRT MRi
R
MRt
MRT MR ln 2008, Prasadand Eizengaeraluated73 Oryza MRi MR genotypesusingthreedifferentmethodsconductedin the greenhouse(microchambermethod), groMh R I inoculation method), or laboratory chamber(toothpick MRi R (detached-leaf method).Asidefrom the sevenOryza (tRGC104705, I RGC100898, accessrons R R rRGC104443, tRGC100223. rRGC105306. 1RGC100943, that were identified and 1RGC105979) MR! I to be moderatelyresistant,it was also found that ' lhis ; ' Eizengaet al (2002);t Prasadand Eizenga (2008). microchamber methodwas the bestfor screeningwild R - Resisbnt - tvloderablyResistant and toothpick MR Oryzaspp. comparedto detached-leaf l- lntermediats methodsof screening.The seven accessionswere also screenedin this study using the optimized microchamber methodand resultsshowedthat the CONCLUSION ANDRECOMMENDATIONS accessionshave either resistant or moderately (intermediate screening resistant reactions) againstthe disease Thisstudyconfirmed thatthe microchamber can be used under (Table1). Moreorer,the resistantor moderately methodwith specificparameters conditionto rapidlyidentify screenhouse resistantreactionsof genotypesTe Qing, LSBR 33 Philippine to sheathblightin rice sourcesof resistance andLSBR5 reportedby G, Eizengaet al. (2002)were possible can Screening andto evaluatebreedingpopulations. in thisstudy. alsoconfirmed the following month using than a be done in less
40
RapidScreeningMethodfor RiceSheathBlight
'actors. use of PDA mycelialdisksas a moreeffective aoculumcarrier comparedto the more commonly .sed rice hull substrate because it shows wider :rfferentiation in the reactionof the tests materials, roculation of younger seedlings at 14 DAS, e'raluation of diseaseat 5-10 d after inoculationand -se of the actualdiseaseindex scale in additionto ..sualor qualitativescoring.Using these optimized :arameters for microchamberscreening, results :cincidedwith the results from severalseasonsof ':rlier field eperiments. The screening results :cnfirmedoreMousresultsof otherstudiesconducted -^der field condition,thus, establishing a degreeof -eliabilitv on the method optimized lt is -ecommendedthat breeders consider using the :cbmizedmicrochambermethodto effectivelyidentify 'esistancedonors,evaluatethe reactionof derived :'cgenies,and identifyresistantbreedinglines in a 'acid,simoleand efficientmanner. ACKNOWLEDGEMENT
LITERATURE CITED AlfonsoAA, MirandaRT, AvellanozaES, SomeraJJ, RillonJP, DucaMSV 2010 Alien C'eneTransfer for Resistance to Stemborerand SheathBlightin Rice PhilRice R&DHighlights 2010p 26-27 EizengaGC, Lee FN, RutgerJN 2OO2 Screening Oryza species plants for rice sheath blight resistance PlantDis 86:808-812 DE Groth, Rush MC, LindbergGD. 1990 Foliar fungicides for controlof ricediseasesin the United States In: Grayson,BT,Green,MB, and Copping, L G. (Eds.),Pest Managementin Rice. Elsevier, London,pp. 31-52. HashibaT. 1984. Estimatingmethod of severity for resistanceto rice sheath blight disease. Bull. HokurikuNat Agric E4c.Stn.26.115-164 lnternationalRice Research Institute(lRRl) 2002 Page 19 in: StandardEwluationSystemfor Rice (SES). InternationalRice Research Institute, Manila,Philippines
-s work was part of the study Alien Gene Transfer Jia, Y, Correa-Victoria F, McClungA, Zhu L, Liu G, ':. Resistance to Stemborerand SheathBlightin Rice '-'ded by the PhilippineRice ResearchInstitute WamisheY, Xie J, MarchettiMA, PinsonSRM, J N Rutger J N, Correll JC. 2007 Rapid -^3er the FavorableEnMronmentProgram Also determination of rice cultivarresponsesto sheath a:
