MARDI Res. Bul l . (1986) I4( l ) , l -5

IN VITRO BLAST SCREENING TECHNIQUE FOR GENETICSTUDIES OF RICE

B.H. CHEW*

Keywords: Blast screentng, In vitro, Price, Genetic studies.

RINGKASAN

Teknik inokulasi berganda Yokoo dan Hujimaki yang diubahsuai serta kebaikannya telahdibentangkan. Reaksi daun yang normal dan daun yang mana permukaannya dicederakan dalambeberapa genotip terhadap jenis-jenis kulat yang tertentu telah diketengahkan. Dua jenis mekanismaketahanan dicadangkan iaitu ketahanan yang berpunca daripada sifat-sifat kerintangan fizik daun danketahanan yang semula jadi, sama ada berupa biokimia atau fisiologi. Teknik yang telah diperbaiki iniadalah sesuai digunakan dalam kajian-kajian genetik ketahanan penyakit karah pada tanaman padi. Ialuga menj imatkan masa. ruang dan tenaga.

INTRODUCTION

An accurate and efficient massscreening technique is a prerequisite of anydisease resistance breeding in cropimprovement programmes. Rice blastcaused by Pyricularia oryzae is a devastatingand rampant disease. Upland blast nurseryscreening techniques are available for theident i f icat ion of res is tanl . genotypes orindividuals. Nevertheless, the virulence ofthe fungal populations in these nurseriesvaries seasonally (Axnve, 1978), giving riseto inconsistent results and making selectionof resistant individuals less effective.

In Malaysia, as many as 54 races basedon the reactions to International andJapanese Blast Differentials have beenclassified by Arnnan in 1978. It is notpossible to relate host response to aparticular fungal race from screening resultsof upland blast nurseries, as the number ofraces present would inevitably confound theresponses of the host plants. For geneticstudy of disease resistance where accuratephenotypic classification of plant individualis essential, a more precise technique toidentify the reaction of a single host plant toa specific fungal race is mandatory. Thispaper describes and discusses the multipleinoculation technique of Yoxoo andHu:rverr (1973), modified to suit the localconditions, for use in genetic studies.

MATERIALS AND METHODS

Six genotypes namely, Raminad Strain3, NP125, Usen, Dular , Kanto 51 andCaloro of the International Rice BlastDifferentials were used. Their origins andreactions to blast are given in Table l.

Seedlings were raised in pots inisolation to avoid contamination from aerialspores. At f ive to six-leaf stages, the fullyexpanded uppermost leaf was cut intolengths of approximately 2.0-2.5 centi-metres. These detached leaf segments wereplaced in a square polystyrene boxmeasuring 15 x 15 x 4 cm containing 100ppm of benzimidazole dissolved in 0.8Vo

Table 1. The or ig ins of the 6. Internat ionalBlast Differentials and their reactions to

3 fungal races as determined byArnvn (1978)

Genotype Orig in Blast react ion to

R293 R3r7 R485

RaminadS t ra i n3 Bu rma /Ch ina R R R

NP 125 I nd ia S R R

U s e n C h i n a S S S

D u l a r I n d i a S S R

Kan to 5 l Japan S S R

C a l o r o U . S . A . S S R

R : ResistantS : Suscept ib le

*Central Research Laboratories Division, MARDI, Serdang, Selangor, Malaysia.

agar (w/v), f i l led to the depth of about 5

mill imetres. Each genotype had 20 leaf

segments, ten of which were wounded by

rubbing several t imes with a piece of cotton

wool in the direction towards the leaf t ip to

cause surface abrasion. Each box

accommodated 60 leaf segments, six rows of

ten leaf segments each. Two boxes were

required for each fungal race, one containingleaf segments of a l l the s ix genotypes, ten toa row per genotype and the other boxcontained the treated leaf segments of thesame. The second set of boxes was preparedfor control check.

Three fungal races (Source: RiceDiv is ion, Bumbong L ima) were used: Race293, Race 317 and Race 485, representingthe most virulent, virulent and fairly virulentraces respectively (Axnvr, 1978). Race 293was isolated from RC7 planted in a farmer'sfield. RC7 (now MR 7 or Sekencang, varietyreleased in 1979) was a blast resistant varietywhich derived its resistance from Tadukan(CHIN and CHEw, 1977). Tadukan, a var ietyfrom Phil ippines, is known to have a verywide spectrum of resistance to blast (Ou,1972). Race 317 was isolated from BumbongLima Rice Research Station's Upland Blast

Nurser ies, and Race 485 was commonlyfound in the farmers' f ields. The inoculumpotential was adjusted to approximately 2.t)x 105 spores/mill i l i tre. One per cent sodiumcarboxyl methyl cellulose was added to theinoculum which was then homogenized bybubbling air through it for half an hour usingan aquarium air pump. The inoculum was

delivered as very fine mist using an aerosolspray uni t (Camag Uni-Spray) held aboutone meter from the boxes. The rate of spraywas regulated to avoid blowing away theleaf segments in the boxes. Immediate lyafter inoculation, the l ids were replaced onthe boxes and then placed in incubator(Convi ron G30) regulated at 28"C andunder continuous l ighting (Phil ips TL 40W/

54 ) .Pe rcen tage a rea cove red by b las tlesions on the leaf segments was recordedon the 4th, 5th and 6th day after inoculation(DAI) . The contro l set was t reated s imi lar lybut sprayed with 1Vc sodium carboxylmethyl ce l lu lose in d is t i l led water .

RESULTS

The responses of the six genotypes tothe three fungal races are presented in Table2. The typical spindle-shaped blast lesions

Table 2. Percentage area covered by b last les ions on leaf segments of 6 Internat ional Blast

Di f ferent ia l genotypes inoculated wi th 3 fungal races (Mean of l0 leaf scgments)

Genotypc4th DAI 5 I h D A I 6 th DA I

R29-l R+ti5 R-l l7 R293 R-185 R317 R1E-s R3 l7

I 2 0 N II 50N 5N

5 1 0 N I O N20 30N -50N-+0 ;l0N 30N70 80 6{)

10 I l08 0 I 6 0

6 0 I 2 05 0 r 8 0

6ON I 2ON50 lON 30

II

I20N

50N6(l

550

20N50

20N30

l 0 N.+0N

5N1 0 N

30N-s0

II

10N5

l 0 Nl 0

20,10

5030

6L}N,10

I I II .5ON I

5 N I I50N 10N -5N

ION I 2ON50N 50 -50

30N I 5N5 1 5

5ON I lON40N I -s

5ON I I5N10 r 30N

B

B

B

B

B

B

Raminad Stra in 3

N P 1 2 5

Uscn

Du la r

Kanto -51

Calorol 0

DAI : Day af ter inoculat ion

A : Untreated leaf scgments

B : Trcatcd leaf segmcnts (surface abrasion)

I : ImmuncN : Percentage arcas covered by necrot ic spots

appeared on the 4th DAI in susceptiblegenotypes; by the 6th DAI many of the blastlesions-had coalesced to form elongatedbrownish blotches (Plate 1). Mycelia couldeasily be seen under low power dissectingmicroscope; a few had sporulated. On theother hand, no lesion was observed in thecontrol set at 6th day after inoculation.

Some genotypes had initial symptomof many tiny brown necrotic spots at 4thDAI, and blast lesions appeared only on the5th day after inoculation. Resistance wasexpressed either as lesion-free (immune)such as in Raminad Strain 3 in response toRace 293 and Race 317 or as many tinybrown spots (hypersensitive reaction) suchas in Raminad Strain 3 and NP125 inresponse to Race 485. Leaf segments withimmune reaction remained clean at the endof the last recording (i.e. at 6th DAI); butthose with brown spots normally increasedin number as time progressed.

One apparent difference between theuntreated and treated (abraded) leafsegments was that the latter was moresusceptible to attack and proliferation of thefungus, leading to higher percentage area ofleaf covered by the blast lesions. Further-more in genotypes such as Usen (in

Fungal race used: 293Bottom right: Mahsuri (susceptible parent)Bottom left: Zenith (resistant parent)

Plate 1. Screening segregating population of(Zenith x Mahsuri) F2.

response to Race 485 and Race 317) andCaloro (in response to Race 293), theuntreated leaves showed typical hypersensitivereactions, whereas highly susceptible blastlesions were observed on the treated leaves.Even in some of those genotypes showingimmune reaction, the treated leaves of thesame genotypes showed certain degree ofhypersensitive spots which increased as timeprogressed (e.9. NP125 in response to Race317 and Caloro in response to Race 485).

DISCUSSION

The results of the responses of the sixgenotypes inoculated with the three fungalraces agree with those obtained by Arnuein 1978 (Tables 1 and 2). Akama usedseedlings for inoculation. A large space wasrequired for the seedlings raised for screeningand generally only one fungal race can beinoculated to any one seedling plant. In thepresent in vitro screening technique, if oneleaf segment were to be taken from oneplant, one polystyrene box measuring L5 x15 x 4 cm can easily accommodate 60 plants.In addition, many fungal races can beinoculated at any one time into a singleplant, limited only by the number of leafsegments that can be obtained from theplant. Yokoo and Hujimaki's multipleinoculation technique (1973) differed in thatthe inocula of different fungal races wereintroduced on the wounded detached leafsegments floated on 100 ppm benzimidazolesolution. Inocula of three to four fungalraces were administered as drops to a single-leaf segment by fine pipettes. Susceptibilityappeared only as a single lesion. As the leafsegments were floated on benzimidazolesolution, handling was cumbersome. Thisdifficulty is overcome presently by dissolvingthe 100 ppm benzimidazole in O.8Vo agar toprovide a solid medium for the leafsegments to rest upon. Similar techniquewas also used by CuIN and Sup,q,^co (1983) toscreen detached organs (leaf segments andpanicle axes) of rice plant for resistance toblast disease. However. inoculation methoddiffered in that the leaf was inoculatedbefore detachment.

Different responses were observed insome genotypes on treated and untreatedleaves. Untreated leaves of Usen wereresistant to two of the three fungal races(Race 485 and Race 317) but were highlysusceptible with treated leaves. This showedthat some resistance barrier had beenremoved in the treated leaves and that theleaves had subsequently succumbed to theattack by the fungus. Two types ofresistance mechanism are implied here:firstly, the resistance is being conferred byphysical barriers on the leaf surface, e.g.waxy substances, thick cuticle, residentantagonistic microflora which act as aprotective covering etc. When this barrier isremoved, e.g. by rubbing the leaf withcotton wool, wounding occurs and infectiongets in. Secondly, the resistance is intrinsic,probably biochemical or physiological innature, and cannot be removed by anyphysical means. Caution is needed inhandling the disease data to avoid spuriousinterpretation with regard to the nature ofhost resistance to the disease in question. Itis therefore suggested that in typing hostresistance. both untreated and treatedleaves should be tested. Wounding of leavescan also be accomplished by brushing theleaf blade with a moderately hard oil paintingbrush.

This lzr vitro screening technique wasfound to be ideal for screening segregat ingpopulation (such as F2) in genetic studies of

AKAMA, Y. (1978). Study on r ice breeding technology.Report submitted to MARDI, 85 pp. (mimeo).

CurN, K.M. and Cntw, B.H. (1977). Recent studies onthe blast resistance of RCi.Mal. Appl. Biol.

disease resistance. The bottom row of thebox was divided into two longitudinal halvesoccupied by the two parents. one res is tantand the other susceptible as checks. Theremaining rows above were the segregatingplant population (Plate 1).

Each box can easily accommodate 40-60 leaf segments depending on the width ofthe leaf blade as this determines the numberof segments that can be put within a row.Each leaf segment in the box was from asingle plant. A segregating population of300 individuals needs only five boxes.assuming each box can accommodate 60 leafsegments. More than one fungal race can beinoculated on different sets of leaf segmentsthus making it possible to study the hostresponses to different fungal racessimultaneously with less space and labourput in to i t .

ACKNOWLEDGEMENTS

The author wishes to express hisgrat i tude to Dr. B.S. Lee and Dr. Mohd.Senawi Dato' Mohd. Tamin who havecrit ically gone through the manuscript, andMr. K.W. Sze and Ms. Noraini Moeslim fortheir technical assistance during this study.The author a lso wishes to thank Dr. K.M.Chin for supplying the three fungal racesand Dr. Othman Omar for providing therice seeds in this studv.

6(2) ,189-92.

CrrrN. K.M. and Surreo, M. A. (19S3). Resistance ofdetached organs of the rice plant to the blastdisease. MARDI Res. Bul l . t1(3), 385-8.

ABSTRACT

A modified Yokoo and Hujimaki's multiple inoculation technique was described and its meritsdiscussed. It had been brought to light the contrastic reaction of leaf and leaf which had been surfacewounded in some genotypes in response to certain fungal races. Two types of resistance mechanismswere suggested, namely, the one that was conferred by the physical barriers ofthe leaf, and the one thatwas of intrinsic nature, either biochemically or physiologically. This improved technique was suitable foruse in genetic studies of blast resistance in rice. It was time, space and labour saving too.

REFERENCES

Ou, H.S. (1972). Studies on stable resistance to r ice inoculat ion method for gene analvsis of r ice

bfastdisease. lnRice Breedingpp.22T 36. Los blast resistance. Jap. J. Breed. 23.109-11 ( in

Banos, Phi l ippines: IRRL Japanese).

Yoxoo . M .H . and Hu . rn tex t , H . ( 1973 ) . Mu l t i p l e

Accepted for publication on 15 January 1986

@Copyright Malaysian Agricultural Research and Development Institute, 1986.

First Published 1986"

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