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Identification and validation of sheath blight resistancein rice (Oryza sativa L.) cultivars against RhizoctoniasolaniMd Kamal Hossaina, Ong Shin Tzeb, Kalaivani Nadarajahb, Kshirod Jenac, Md Atiqur RahmanBhuiyana & Wickneswari Ratnama

a School of Environmental and Natural Resource Sciences, Faculty of Science andTechnology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysiab School of Bioscience and Biotechnology, Faculty of Science and Technology, UniversitiKebangsaan Malaysia, 43600 Bangi, Selangor, Malaysiac Plant Breeding, Genetics and Biotechnology Division, International Rice Research Institute,Los Banos, Metro Manila, PhilippinesAccepted author version posted online: 30 Sep 2014.Published online: 16 Oct 2014.

To cite this article: Md Kamal Hossain, Ong Shin Tze, Kalaivani Nadarajah, Kshirod Jena, Md Atiqur Rahman Bhuiyan &Wickneswari Ratnam (2014): Identification and validation of sheath blight resistance in rice (Oryza sativa L.) cultivars againstRhizoctonia solani, Canadian Journal of Plant Pathology, DOI: 10.1080/07060661.2014.970577

To link to this article: http://dx.doi.org/10.1080/07060661.2014.970577

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Genetics and resistance / Génétique et résistance

Identification and validation of sheath blight resistance in rice(Oryza sativa L.) cultivars against Rhizoctonia solani

MD KAMAL HOSSAIN1, ONG SHIN TZE2, KALAIVANI NADARAJAH2, KSHIROD JENA3,MD ATIQUR RAHMAN BHUIYAN1 AND WICKNESWARI RATNAM1

1School of Environmental and Natural Resource Sciences, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi,Selangor, Malaysia2School of Bioscience and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor,Malaysia3Plant Breeding, Genetics and Biotechnology Division, International Rice Research Institute, Los Banos, Metro Manila, Philippines

(Accepted 25 September 2014)

Abstract: Rice sheath blight, caused by Rhizoctonia solani, is a devastating disease of rice which causes major yield loss in most rice growingregions of the world. Hence, identification and subsequent development of disease resistance in rice cultivars is crucial. Six moderatelyresistant cultivars, namely ‘Teqing’, ‘Jasmine85’, ‘Tetep’, ‘Pecos’, ‘Azucena’ and ‘Taducan’, one susceptible local cultivar, ‘MR 219’, and twonew advanced breeding lines, ‘UKMRC 2’ and ‘UKMRC 9’, were screened using micro-chamber and mist-chamber methods. The fungalisolate was confirmed as R. solani using ITS-rDNA sequencing. Severe sheath blight was recorded following inoculation with R. solani undermicro-chamber conditions. The most resistant cultivar was ‘Tetep’, followed by ‘Teqing’. In mist-chamber screening, ‘UKMRC 2’ showed thehighest level of susceptibility with a disease severity index (DSI) of 6.67, while ‘MR 219’ produced the highest DSI of 7.22 in the micro-chamber. Significant correlation of plant height and disease was obtained with relative lesion height (RLH) indices. Significant correlationswere also observed among diseased plant affected area (DPAA), VRT (visual rating) and RLH, with VRT being the most accurate. On the basisof the disease reactions, ‘Tetep’ and ‘Teqing’ were identified as suitable donors to improve resistance in ‘UKMRC 2’ and ‘MR 219’.Mist-chamber screening method was more reliable to evaluate sheath blight under greenhouse conditions than the micro-chamber method.

Keywords: disease resistance, Rhizoctonia solani, rice cultivar, screening methods, sheath blight

Résumé: Le flétrissement de la gaine du riz, causé par Rhizoctonia solani, est une maladie dévastatrice qui engendre des pertes de rendement majeuresdans la plupart des régions productrices de riz de la planète. Par conséquent, la détermination des facteurs de résistance et le développement subséquentde cultivars résistants à la maladie sont cruciaux. Six cultivars modérément résistants, dont ‘Teqing’, ‘Jasmine85’, ‘Tetep’, ‘Pecos’, ‘Azucena’ et‘Taducan’, un cultivar local réceptif, ‘MR 219’, et deux nouvelles lignées généalogiques avancées, ‘UKMRC 2’ et ‘UKMRC 9’, ont été criblés à l’aidede méthodes basées sur la microchambre et la chambre à brouillard. Grâce au séquençage de l’ITS de l’ADNr, l’isolat fongique a été identifié comme R.solani. Un grave flétrissement de la gaine a été noté dans la microchambre à la suite de l’inoculation avec R. solani. Le cultivar le plus résistant était‘Tetep’, suivi de ‘Teqing’. Dans la chambre à brouillard, ‘UKMRC 2’ a affiché le plus haut degré de réceptivité avec un indice de gravité de la maladie(IGM) de 6.67, tandis que, dans lamicrochambre, ‘MR219’ a produit l’IGM le plus élevé (7.22). Une corrélation significative entre la hauteur du plant etla maladie a été obtenue avec des indices de hauteur relative des lésions (HRL). Des corrélations significatives ont également été observées entre la partietouchée du plant malade, l’évaluation visuelle et la HRL, l’évaluation visuelle ayant été la plus précise. En fonction des réactions à la maladie, ‘Tetep’ et‘Teqing’ ont été désignés comme donneurs appropriés pour améliorer la résistance chez ‘UKMRC 2’ etMR 219’. Le criblage recourant à la méthode dela chambre à brouillard s’est avéré plus fiable quant à l’évaluation du flétrissement de la gaine en serre que celui basé sur la méthode de la microchambre.

Mots clés: Flétrissement de la gaine, méthodes de criblage, résistance à la maladie, Rhizoctonia solani

Correspondence to: Wickneswari Ratnam. E-mail: wicki@ukm.edu.my

Can. J. Plant Pathol., 2014http://dx.doi.org/10.1080/07060661.2014.970577

© 2014 The Canadian Phytopathological Society

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Introduction

Rice sheath blight (ShB), caused by the soilborne necro-trophic fungal pathogen Rhizoctonia solani Kühn [tele-omorph: Thanatephorus cucumeris (A.B. Frank)], is aneconomically important disease in rice (Ou 1985). Itcauses substantial losses in intensive rice production sys-tems worldwide (Teng et al. 1990; Savary et al. 2006).Isolates of R. solani are genetically diverse, and vary intheir cultural, morphological and physiological character-istics, as well as in their pathogenicity on a range of hostplants, including rice (Lee & Rush 1983; Gonzӓlez et al.2006; Zeng et al. 2011). The fungus is separated into 14anastomosis groups (AGs) (Zheng et al. 2013), using acombination of traditional methods (vegetative hyphalfusion with tester isolates) as well as ribosomal DNA(rDNA)-internal transcribed spacer (ITS) sequence analy-sis (Sharon et al. 2008).

Previous studies on ShB resistance indicated that thereare no rice varieties or lines with resistance to this diseasein rice landraces (Jia et al. 2007; Liu et al. 2009; Zenget al. 2011). However, certain varieties were identified as‘moderately resistant’, such as ‘Teqing’ (Pan et al. 1999;Pinson et al. 2005), ‘Tetep’ (Sato et al. 2004;Channamallikarjuna et al. 2010), ‘Jasmine85’ (Liu et al.2009), and ‘Pecos’ (Bollich et al. 1985; Sharma et al.2009). A number of QTLs have been detected, linked tothe provision of disease resistance in rice using mappingpopulations derived from indica or japonica-type rice(Pan et al. 1999). The polygenic nature of ShB resistancecorresponds to the observations made with regard to thewide variation in susceptibility levels among rice culti-vars (Khush 1977; Sha & Zhu 1990; Pinson et al. 2005).So far, 60 QTLs (Zeng et al. 2011) associated with ShBresistance have been identified and 10 of these are co-localized with QTLs in terms of morphological attributes,especially plant height or days to heading (Groth &Nowick 1992). Of these QTLs, 16 have been mapped atleast twice from the same or different genetic back-grounds. Nine of the 16 QTLs were independent ofmorphological traits and days to heading, leading to thehypothesis that ShB resistance may be attributed to phy-siological resistance factors, as well to disease escape(Singh et al. 2002). However, the mechanism underlyingthis quantitative resistance to sheath blight in rice is stillunclear.

Besides the genetic mechanisms, the phenotypic var-iation in disease resistance may be influenced by anumber of environmental factors, such as sunlight,humidity, temperature (Lee & Rush 1983; Rush &Lee 1992), soil fertility (Rush & Lee 1992) and the

silicon level in the soil (Rodrigues et al. 2003). Inaddition, morphological traits such as plant height,days to heading, tiller angle and leaf morphologyhave been reported to affect the resistance of ricecultivars to sheath blight (Zou et al. 2000; Pinsonet al. 2005). The multitude of factors influencing thedisease epidemics makes the evaluation of the diseaseincidence and severity difficult. This may be whydeveloping ShB resistant cultivars through conven-tional breeding has attained only limited success, dueto the lack of resistance-adapted germplasm, the limitedefficiency and effectiveness of available screeningmethods and the polygenic nature of the resistance(Jia et al. 2007).

A number of inoculation methods were reported forthe precise detection of ShB resistance in rice germ-plasm, such as micro-chamber method (Jia et al. 2007;Nelson et al. 2012), mist-chamber method (Park et al.2008; Nelson et al. 2012), detached leaf method(Prasad & Eizenga 2008), detached tiller method(Willocquet et al. 2011) and field screening (Jia et al.2007). The micro-chamber method has been used inseveral ShB disease screenings and evaluation pro-grammes and is considered a rapid, efficient, cost-and time-effective sheath blight screening method (Jiaet al. 2007).

Therefore, considering all the possible factors con-tributing to ShB resistance in rice, this study aimed to:(1) validate the suitability of the reported rice cultivarsas sheath blight resistance donors for introgression intohigh-yielding susceptible cultivars in Malaysia againsta local R. solani field isolate; (2) establish a rapid andreproducible ShB screening system under greenhouseconditions; and (3) confirm the identity of the local R.solani field isolate using a taxon-specific molecularanalysis. This study confirmed the sheath blight resis-tance potential of the cultivars ‘Tetep’ and ‘Teqing’against R. solani, providing opportunities for resistancebreeding of susceptible local elite cultivars.

Materials and methods

Plant materials

A total of nine rice cultivars – ‘Teqing’ (IRGC117912),‘Jasmine 85’ (IRGC32591), ‘Tetep’ (PI20070424), ‘Pecos’(IRGC66758), ‘Azucena’ (IRGC117264), ‘Taducan’(PI20080727) – all moderately resistant to sheath blight –and ‘UKMRC2’, ‘UKMRC 9’ (unknown reaction tosheath blight) and ‘MR 219’ (a susceptible check) wereused in this study.

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R. solani field isolate

The origin of the R. solani isolate, hereafter referred to asKB1802, used in this study was the Malaysian AgriculturalResearch and Development Institute’s (MARDI) riceexperimental field in Kota Baharu, Kelantan. KB1802 wasisolated from a few host plants of the popularMalaysian ricecultivar ‘MR219’, which is considered to be highly suscep-tible to sheath blight. Severely affected tillers showingsheath blight symptoms were selected for sampling withthe help of a rice pathologist. Diseased plant tissues weredipped in 70% ethanol and cut into 1.5–2.0 cm pieces. Thepieces were then surface-sterilized with 10% Clorox solu-tion for 30 s. The tissues were cultured on rose bengal agar(RBA) to facilitate fungal growth while inhibiting bacterialcontaminants. After 1 week of incubation in the dark at 28 °C, the Petri dishes were examined for R. solani growth,which was then confirmed by observation under a com-pound microscope (Olympus TH4-200, OlympusAustralia Pty Ltd). Verified isolates of R. solani were sub-cultured by transferring a 5–7 mm RBA block onto potatodextrose agar (PDA, Difco) that contained tetracycline(0.005%, w/v) and incubated in the dark for 3 days at 28 °C. Single tips of mycelia were then transferred onto freshPDA and maintained at 27 to 30 ºC (Jia et al. 2007).

PCR amplification of ITS–rDNA

Total genomic DNA of KB1802 was isolated usingDNeasy plant mini kit (Qiagen, USA) following themanufacturer’s instructions. Internal transcribed spacerregions, including the ribosomal 5.8S RNA gene, wereamplified as one amplicon using the universal ITS1 (5ʹ-TCCGTAGGTGAACCTGCGG-3ʹ) and the ITS4 (5ʹ-TCCTCCGCTTATTGATATGC-3ʹ) primers that annealedto the flanking 18S and 28S rRNA genes (White et al.1990). Each PCR reaction mixture contained 5–10 ng ofgenomic DNA, 1 μM each of the primers ITS1 and ITS4, areaction buffer (50 mM KCl, 50 mM Tris-HCl; [pH 8.3]0.1 mg mL−1 bovine serum albumin), 3 mM MgCl2,200 μM each of dNTP and 2.5 U of Taq DNA polymerase(Promega, Mannheim, Germany) in a total volume of 50 μL(White et al. 1990). Gradient PCR reaction was conductedvia Mastercycler Gradient (Eppendorf). The temperatureprofile used for the PCR reaction is as follows: 2 min at94 °C, initial denaturation cycle; 1 min at 94 °C, 1 min at 58°C, 1 min at 72 °C, 24 cycles; 2 min at 72 °C, 1 cycle(Pascual et al. 2000). An aliquot (5 µL) of the PCR productwas separated in an agarose gel [1.0 g L–1 agarose in Tris-acetate-ethylene-di-amine-tetra-acetic acid (TAE) electro-phoresis buffer] supplemented with ethidium bromide(0.1 mg L–1) by electrophoresis and viewed under UV light.

ITS–rDNA sequence comparisons

PCR products were purified via a Qiagen PCR purificationkit, following the manufacturer’s instructions (Qiagen,USA) and the purified PCR products were subjected todirect sequencing in both directions using the ITS1 andITS4 primers at First Base Sdn. Bhd. Malaysia. The con-sensus sequence was obtained using the BioEdit program(http://www.mbio.ncsu.edu/bioedit/bioedit.html). The con-sensus sequence was then used as the query and was com-pared with all nucleotide sequences in GenBank via NCBIBLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi).

Disease evaluation

Micro-chamber method

Plant growth conditions. The micro-chamber method of Jiaet al. (2007) was used in this study. Plastic pots (15-cmdiameter) were filled with steam-sterilized soils, whichensured the absence of soilborne R. solani inocula. Riceseeds were germinated on moist filter paper in Petri dishesat 30 ºC, with 12 h dark and 12 h light conditions for 7 days.Three germinated seedlings were planted in the pots, main-taining at least a 3 cm distance from each other. Threereplicate pots of each cultivar were arranged in a completelyrandomized design (CRD) in a walk-in humidity chamber.Urea (N), triple super phosphate (P2O5), and potassiumchloride (K2O) were applied at 310 mg, 230 mg and205 mg per pot, respectively. Fertilizers were applied thrice –at planting, 15 days after planting and 1 day before inocula-tion. Pots were placed in a humidity chamber (4 × 3 × 2 m),prepared with a wooden frame, and covered with white clothand black plastic netting to reduce temperatures. The green-house temperature sometimes reached 40–42 °Cwhich couldaffect the experimental results. Therefore, to maintain opti-mum humidity and temperature, a 3–5 cm water level on thefloor of the walk-in humidity-chamber was ensured. Thehigh temperature and humidity in the micro-chamber gener-ally favours pest attack of the plants, especially by brownplant hoppers. Hence, Furadan 3 G (Carbofuran 3% w/w)insecticide was used at 50 mg per pot during pot preparationand at 15 days after planting. Temperature and humidity wererecorded by using a hygro-thermometer (Dual Temperature-Humidity/clock, Smart Sensor, Model AR 867). The studywas repeated twice as two different experiments i.e. test 1 andtest 2. Test 1 was conducted from 20March to 28 April 2012and test 2 from 27 April to 6 June 2012.

Pathogen inoculation. Each seedling was inoculated witha 5 mm mycelia block, excised from the edge of 3-day-old cultures, pressed up to the base of the stem and

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ensuring that the mycelia were in contact with the plant.After inoculation, each pot was covered with a 2-litre softdrink bottle, with the cap removed and the bottom pusheddown into the soil to create a sealed micro-humiditychamber (Fig. 2a).

At 10 days post inoculation, diseased seedlings werecut at the base. The four-leafed seedlings were subjectedto visual inspection for disease using the percentage ofDPAA (diseased plant affected area), as described by Liuet al. (2009). A value of 30% was assigned to the oldestleaf; a maximum disease severity of 20% was assigned toeach of the following two leaves; a maximum of 10%was assigned to the remaining leaf, and a maximum of20% was attributed to the stem, giving a possible total of100%. The sum of total DPAA for each plant was thenconverted into a 0–9 scale (Jia et al. 2007; Prasad &Eizenga 2008), where 0 = no lesion observed and9 = 90–100% plant parts affected by the pathogen. Forexample, if the oldest leaf was half covered with ShBlesions, a disease severity of 15% was assigned to thisleaf. The ShB disease indices were then categorized as<4 = moderately resistant, 5 to 6 = moderately susceptibleand 7 to 9 = highly susceptible (Prasad & Eizenga 2008).

Mist-chamber method

Seeds were germinated as per the procedure followed inmicro-chamber method. Slight modifications of the methoddescribed by Nelson et al. (2012) were used in this screen-ing. Plastic pots of 25-cm-diameter were filled with ster-ilized soil. Three 7-day-old germinated seedlings fromeach entry were planted 10 cm apart. Urea (N), triplesuper phosphate (P2O5) and potassium chloride (K2O)were applied at 850 mg, 650 mg and 575 mg per pot,respectively. Fertilizers were applied with four equal por-tions during pot preparation, 21 days and 42 days afterplanting, and 1 day before inoculation. Plants were grownon the greenhouse bench until inoculation. The averagetemperature and humidity in the greenhouse was 35 °C and65%, respectively during experimental periods. Inoculawere prepared using the same protocol as described inthe micro-chamber method. Three tillers of each plant,i.e. nine tillers in each pot per replication, on 60-day-oldplants, were inoculated. Five to seven millimetres PDAblocks were placed at the base of each stem and coveredwith aluminium foil. A single pot with three plants of eachcultivar was used as a control. Pots were arranged follow-ing the CRD design, with three replications. The alumi-nium foil was removed 3–5 days after inoculation, whendisease lesions appeared. The inoculated plants were keptfor 2 weeks inside the walk-in humidity chamber andreturned to the greenhouse bench for 1 week to maintain

normal growth conditions of plants. Pots were again placedin the walk-in humidity chamber for another week toprovide the favourable conditions for infection. The aver-age day-night temperatures and relative humidity in thescreening chambers were 28–34 °C and >80%, respec-tively. Plants were evaluated 4 weeks after inoculation.RLH was calculated using the following formula: (lesionlength/plant height)*100 (Sharma et al. 1990). RLH wasthen converted into sheath blight disease index based on a0–9 scale, where 0 = no lesion and 9 = lesion visible up tothe tip of the longest leaf (Prasad & Eizenga 2008). VRTswere also assigned to the infected plants based on theactual visible damage on the plant tissues. VRTs werescored according to the sheath blight disease index of 0to 9 scale, where 0 = no lesion, 9 = complete damage ofthe plant, and <4 was considered moderate resistance (Jiaet al. 2007; Prasad & Eizenga 2008). Days to heading wascalculated from the date of soaking of seeds to the initia-tion of the first flowering of the main culm, and plantheight from the soil surface to the tip of the longest leaf(IRRI 2002).

Statistical analysis

Disease ratings from three individual plants (micro-chambermethod) and nine individual plants (mist-chamber method)in each individual pot were averaged as a single replication.Analysis of variance (ANOVA) and least significant differ-ence (LSD) values were calculated using the generalizedlinear model procedure (PROC GLM) in SAS (version 9.1for Windows, SAS, Cary, NC). Correlation coefficient andmultiple comparisons were computed for morphological data(plant height and days to heading) and disease severityindexes based on DPAA, VRT and RLH using PROCCORR in SAS.

Results

Identification of R. solani isolate KB1802

Using ITS1/ITS4 specific primer pair, a single PCR pro-duct of 716 bp was produced (Fig. 1). The product sizewas well within the range reported by Johanson et al.(1998) and Pannecoucque and Hofte (2009). BLASTanalysis of the 716 bp consensus sequence (GenBankAccession No.: KF312465) revealed four hits, with amaximum identity of 100% and an E-value of 0.0, forisolates from rice plants identified as R. solani AG1-1A.We therefore conclude that R. solani KB strain 1802belongs to anastomosis group AG1-1A. The R. solaniKB strain ‘1802’ strain was also more closely related toother Asian strains of R. solani (GenBank Accession Nos:

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JF701748.1, JF701746.1, KC285893.1 and JF701728.1)particularly those from India and China, thus indicating apossible geographic relationship to the isolated R. solanistrain.

Pathogenicity of R. solani

The KB1802 isolate of R. solani was tested by infectingrice plants at both seedling (micro-chamber, (Fig. 2a))and at late tillering stages (mist-chamber, Fig. 2b). Sheath

blight pathogenicity tests were conducted using threedifferent rice cultivars, namely ‘MR 219’, ‘UKMRC 9’and Oryza rufipogon (IRGC105491). In both seedlingand late tillering stages, sufficient disease severity wasrecorded (Fig. 2b). Diseased plant parts from the testedentries were collected and the pathogen was re-isolatedfollowing the same protocol used for the first isolationfrom the field samples. Colonies of the isolate showedcharacteristic growth morphology (Fig. 2c) and confirmedto be R. solani by microscopic examination of the pre-sence of septation in the hyphae and the perpendicularbranching pattern of hyphae (Figs 2d and 3).

Micro-chamber method

All five different evaluation measurements such as DPAA,VRT, RLH, plant height and days to heading showed highlysignificant variances (Table 1). The DPAA-based diseaseindex for test 1 ranged from 4.89 to 6.44 (Table 2). Thelowest index was found in ‘Tetep’, followed by ‘UKMRC9’, which was significantly lower than that of the susceptible

Fig. 3 (Colour online) Symptoms of sheath blight disease detectedin nine rice cultivars using the micro-chamber method.

Fig. 1 PCR product of ‘KB1802’ strain using ITS1/ITS4 primers.Lane M: DNA ladder (Promega).

Fig. 2 (Colour online) a, Sheath blight screening using micro-cham-ber method in greenhouse. b, Typical ShB symptoms produced frommist-chamber method on mature plant. c,Mycelia of 3-day culture ofR. solani on PDA. d,Microscopic view of mycelia of R. solani.

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check, ‘MR 219’. Most of the exotic rice cultivars referred toas ‘moderately resistant’ showed an average disease indexbetween 5.89 and 6.33 except for ‘Tetep’. Similarly, for test 2,the mean DPAA index varied from 4.67 to 7.22 (Table 2).‘UKMRC 9’ showed the lowest DPAA index, followed by‘Teqing’, which was significantly different (P < 0.005) fromthe susceptible check, ‘MR 219’.

Mist-chamber method

Using the mist-chamber method, ShB disease severitywas evaluated based on VRT and RLH. Results showed

that the VRT-based disease index ranged from 3.67 to6.67, with a highly significant difference of P < 0.000. Inthis study, ‘Tetep’ had the lowest score followed by‘Teqing’ and ‘Jasmine85’ (Table 2). The other testedcultivars exhibited an average disease index, which ran-ged from 5.67 to 6.67 with no significant differences.However, for RLH, the disease index ranged from 3.00to 5.67. ‘Tetep’ had the lowest score which was signifi-cantly different (P < 0.002) from the highest one,‘UKMRC 2’.

Plant height and days to heading

All the nine tested cultivars were found to have highlysignificant differences for both plant height and days toheading (Table 2). The shortest plant height was ‘Jasmine85’, followed by ‘Pecos’, whereas the tallest were ‘Tetep’and ‘Taducan’. ‘Pecos’ was the early maturing cultivarfollowed closely by ‘Teqing’, with ‘UKMRC 9’ being ofintermediate growth duration.

We investigated the correlation between ShB resistanceand morphological traits. Correlation between plantheight and VRTs, and plant height and RLH were sig-nificant. DPAA1 index was positively correlated to VRT-and RLH-based disease scores. The VRT-based diseasescore was highly significant and positively correlated toRLH (Table 3).

Discussion

Our results confirmed that the field isolate KB1802 wasR. solani causing sheath blight in rice. Genetic variationamong different Rhizoctonia species can be distinguishedthrough ITS-rDNA nucleotide sequence comparisons.According to Pascual et al. (2000) and Pannecoucqueand Hofte (2009), the total length of the ITS amplifiedregion could vary from 600–750 bp. The consensussequence obtained through the ITS-rDNA amplification

Table 1. ANOVA for days to heading (days), plant height (cm) andthree different sheath blight disease indexes of nine rice cultivars.

Parameter DTHa PHTb

Disease severity index based on

DPAA1c DPAA2d VRTe RLHf

MSg 653.73 1149.06 0.90 1.58 3.59 1.56F 289.36 100.47 4.42 2.78 3.94 5.20Pr>F <0.0001 <0.0001 <0.0056 <0.0390 <0.0095 <0.0025

aDays to heading.bPlant height.cDiseased plant affected area test 1.dDiseased plant affected area test 2.eVisual rating.fRelative lesion height.gMean sum of square.

Table 2. Mean value of days to heading (days), plant height (cm)and three different sheath blight disease indexes of nine ricecultivars.

Cultivar DTH PHT

Disease severity index based on

DPAA1 DPAA2 VRT RLH

‘Teqing’ 79.00f 110.63c 5.57abc 5.56bc 4.00c 4.00c

‘Jasmine’ 97.67e 96.78d 6.33a 5.89bc 5.00bc 4.33bc

‘Tetep’ 96.67e 145.96a 4.89c 6.44bc 3.67c 3.00d

‘Pecos’ 73.00g 100.37d 6.33a 6.56bc 6.33ab 5.00bc

‘Azucena’ 107.67c 140.56a 6.11a 6.44bc 6.67a 4.33bc

‘Taducan’ 111.33b 145.78a 5.88ab 6.22bc 5.67ab 4.40bc

‘MR 219’ 111.00b 116.85b 6.44a 7.22a 6.60ab 4.67bc

‘UKMRC 2’ 105.00d 107.45c 6.00d 5.78bc 6.67a 5.67a

‘UKMRC 9’ 115.00a 109.00c 5.11bc 4.67c 5.67ab 4.33bch LSD 0.05 2.601 5.853 0.783 1.306 1.653 0.949

Note: The numbers followed by the same letter are not significantlydifferent.aDays to heading.bPlant height.cDiseased plant affected area test 1.dDiseased plant affected area test 2.eVisual rating.fRelative lesion height.gMean sum of square.hLeast significant difference.

Table 3. Correlation co-efficient of days to heading, plant heightand sheath blight disease index.

Parameter

Disease severity index based on

DPAA2 VRT RLH

DPAA1 0.522ns 0.592* 0.647**DPAA2 - 0.164ns −0.001ns

DTH - 0.319ns 0.047ns

PHT - −0.164ns −0.551*VRT - - 0.823***

*, ** and ***, significant at P < 0.1, P < 0.05 and 0.01% level ofprobability, respectively.nsnot significant.

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used for BLAST analysis showed 100% sequence simi-larity to R. solani AG1-1A from four different geographicregions and hosts including rice.

A number of ShB disease evaluation systems havebeen used to find a potentially stable source of sheathblight resistance. In our study, nine rice cultivars, includ-ing six with resistance to sheath blight (referred to as‘moderately resistant’) were identified for inclusion in aresistance breeding programme. Results from the mist-chamber method were more consistent in disease indexbased on the VRT and RLH indexing. ‘Tetep’ producedthe lowest disease score followed by ‘Teqing’ indicatingresistant disease reactions. In considering the diseaseevaluation methods, significant variations in DSI wereobserved across the cultivars tested (Table 2). Therewere some inconsistencies observed among the cultivars(Fig. 4). For example, ‘Tetep’ and ‘Teqing’ were found tohave resistance in mist-chamber screening. But most ofthe reported resistant materials including ‘Tetep’ and‘Teqing’ showed higher disease scores in micro-chambermethod where humidity conditions were very high anddisease was enhanced. This could explain the differencesin the cultivar rankings. Similar results were reported for‘Teqing’ and ‘Cocodrie’ (Jia et al. 2007), where theseresistant cultivars had susceptible disease reaction underfield inoculation conditions in Arkansas, USA. On thecontrary, ‘Cypress’, which is a susceptible cultivar,showed resistant disease reaction in Arkansas. Diseasereactions may also differ between tests and locationsdue to the plant age and genotypic response to localsheath blight isolates. For example, ‘Cocodrie’, a USresistant cultivar, exhibited a susceptible reaction inColombia when inoculated plants were 50 days old (Jiaet al. 2007). We also noted that the susceptible check

‘MR219’ did not show a significant difference for RLHcompared with resistant cultivar ‘Teqing’, but VRT scorewas significantly different (Table 2). This inconsistencyoccurred due to the differences in scoring system i.e. theRLH-based evaluation system refers to the relative esti-mation of the disease lesion, found on the leaf sheathacross the plant height where overall plant damage wasnot considered during evaluation. On the other hand, theVRT system is based on the total plant damage by patho-gen infection. In the case of ‘MR219’, the plant wasaffected significantly at the lower part rather than at thehigher part. Hence, some susceptible plants showed lowerRLH scores close to those of the resistant cultivars.

The correlation of sheath blight resistance and morpho-logical traits, such as plant height and days to heading,have been frequently observed in many previous studies.Groth and Nowick (1992) reported that the ShB resis-tance detected in rice cultivars might be due to the varia-tion in morphological and physiological characters. Fromour observation, highly significant and positive correla-tion between days to heading and VRT was detected(Table 3) which indicated that long duration cultivarsare more vulnerable to disease infestation. In our results,it has been confirmed that short duration cultivars such as‘Teqing’, ‘Jasmine85’ and ‘Tetep’ had higher resistancepotential than that of long duration cultivars, for example‘MR219’. Similar findings were also reported by Zouet al. (2000). On the other hand, significant and negativecorrelation between plant height and VRT, and plantheight and RLH were recorded from our findings whichimplies that a shorter plant is more likely to be attackedby sheath blight. ‘Tetep’ is a tall cultivar and had thehighest resistance in this study. These results were sup-ported by Loan et al. (2004) and Channamallikarjuna

Fig. 4 (Colour online) Comparison of four different sheath blight disease indices of nine rice cultivars inoculated with local isolate of R.solani. DPAA1, Diseased plant affected area test 1; DPAA2, Diseased plant affected area test 2; VRT, Visual rating, RLH = Relative lesionheight, S = susceptible, R = Resistant. Bar at the column head indicates standard error.

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et al. (2010). However, ‘Teqing’ is a short cultivar thatshowed improved resistance in our findings.

The inconsistent relationship between morphologicaltraits and sheath blight resistance has been reported byseveral previous authors. The localization of the quantita-tive traits loci (QTLs) conferring resistance as well asrelated to the morphological traits might be the majorreasons for this inconsistent relationship. The QTLs ofmorphological traits co-localized with those of sheathblight resistant QTLs could be dependent on such morpho-logical traits and vice versa. Zou et al. (2000) mappedthree QTLs (qPH-3, qPH-4 and qPH-11) controlling plantheight and four QTLs (qHD-2, qHD-3, qHD-5 and qHD-7) for days to heading; two characteristics in the sameplant population. Of these QTLs, qPH-11was detected at18.6 cm from qSB-11 on chromosome 11, meaning thatthe two loci may have incomplete linkage. However,neither days to heading QTLs nor plant height QTLswere identified near other disease resistance QTLs.Similar results were also reported by Loan et al. (2004),where QHd4 for days to heading was mapped with sheathblight resistance QTLs (QSbr4a) at the same chromosomalregion on chromosome 4. Moreover, QSbr3a, QSbr8a andQSbr9a were mapped to approximately the same locationsas the major QTLs, QHd3a, QHd8a and QHd9a, for daysto heading, corresponding to the QTLs QPh3a, QPh8a andQPh9a (Li et al. 1995) for plant height.

Correlation coefficients among the three different eva-luation methods were also determined. The DAPP indexwas significantly and positively correlated to VRT andRLH. Likewise, a highly significant and positive correla-tion was detected between the VRT and RLH. A signifi-cant and positive correlation between micro-chamberdisease index, the visual score (detached-leaf), the visualscore (micro-chamber) and lesion length (toothpick) werereported by Prasad and Eizenga (2008).

We conclude that, although comparatively higher diseaseindices were observed in the micro-chamber method than inthe mist-chamber method, the results were consistent.Screening rice cultivars using the mist-chamber method atthe mature stage produced more realistic disease reactionswhich confirmed the validation of the resistant materials.We found ‘Teqing’ and ‘Tetep’ to be the most resistantcultivars, followed by ‘UKMRC 9’, ‘Azucena’, ‘Taducan’,‘Pecos’, and ‘Jasmine85’, ‘UKMRC 2’ and ‘MR 219’.

Taller plants and shorter growth duration have beenrecorded in ‘Tetet’ and ‘Teqing’, respectively. Moreover,the presence of major QTLs, qSB9 in ‘Teqing’ (Zuo et al.2008) and qSB11-1 in ‘Tetep’ (Channamallikarjuna et al.2010), validates the resistance potentialities of those cul-tivars favoured in our selection as donors.

Acknowledgements

This work was supported by the Ministry of HigherEducation, Malaysia, under the Long-term ResearchGrant Scheme for Food Security (Project code LRGS/TD/2011/UPM-UKM/KM/01). The authors would alsolike to thank individuals who enabled the collection ofthe R. solani isolate from rice fields.

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