characterization by conventional techniques and pcr of rhizoctonia solani isolates causing banded...

Characterization by conventional techniques and PCR ofRhizoctonia solani isolates causing banded leaf sheathblight in maize

C. B. Pascuala,b, T. Todab, A. D. Raymondoc and M. Hyakumachib*aPlant Pathology Laboratory, Institute of Plant Breeding, University of the Philippines at Los Banos, College, Laguna 4031, Philippines;bLaboratory of Plant Disease Science, Faculty of Agriculture, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan; and cDepartmentof Plant Pathology, University of the Philippines at Los Banos, College, Laguna 4031, Philippines

Rhizoctonia-diseased specimens were collected from various host species growing in or near maize fields in differentgeographic regions of the Philippines. A greater range of host species, with varying types of disease symptoms, wasfound in Mindanao than in Luzon. Fifty-two isolates belonged to anastomosis group AG1-IA and caused banded leafand sheath blight in maize (Zea mays), but they showed considerable variation in virulence. The most and leastvirulent isolates recovered from maize were both collected from Mindanao. Isolates from necrotic spots/foliar blight ofdurian and coffee, which were collected from the same region, showed the lowest lesion heights. UPGMA-SAHNclustering analysis from RAPD fingerprint data of 30 haplotypes of R. solani AG1-IA isolates from the Philippines andJapan resolved seven groups of AG1-IA at the 75% similarity level. Variation among isolates from upland cropsseemed to be partially correlated with geographical origin and virulence. In the case of paddy rice isolates from Japanand the Philippines, some were closely related, with over 75% similarity, suggesting a common origin. In PCR-RFLPanalysis of the rDNA internal transcribed spacer region, no polymorphism was observed among the AG1-IA isolatesbut they were differentiated from subgroups AG1-IB and AG1-IC using the endonucleases EcoRI, MboI and HinfI.

Keywords: anastomosis groups, cluster analysis, PCR-RFLP analysis, Rhizoctonia solani, Zea mays

Introduction

Rhizoctonia solani (teleomorph: Thanatephorus cucu-meris) is a destructive soilborne pathogen of many cropsworldwide, and can survive saprophytically as sterilemycelium (hyphae or sclerotia) associated with organicdebris in soil. Hyphae, sclerotia and/or basidiosporeshave been reported as possible sources of inoculum forthe diseases caused by R. solani (Baker & Martinson,1970). In the Philippines, this fungus causes banded leafand sheath blight in maize (Zea mays), rice andsorghum, damping-off in cotton, aerial blight and stemrot in mungbean and soybean, sheath rot in sugarcane,heart rot in cabbage, black scurf and sprout canker inpotato, and foliar blights of fruits and plantation crops(Tangonan & Quebral, 1992). However, very limitedstudies on the classification and characterization of theisolates from these crops (apart from rice) have beencarried out so far in tropical countries of South-EastAsia.

In the past few years, banded leaf and sheath blight(BLSB) has become a production constraint in maize-growing areas not only in the Philippines but also inIndonesia and Vietnam (De Sharma et al., 1993). In afield survey conducted in the 1998 wet season in SouthCotabato, a maize-producing region in Mindanao,Philippines, an 80–90% incidence of BLSB wasobserved, with more than 50% of the plants havinginfected ears at the hard-dough stage (IPB, 1998).Damage is less serious in Japan. Sources of resistanceto BLSB in maize have been elusive in Asian countrieswhere BLSB is an important disease (De Sharma et al.,1993). Chemical control is neither economic norenvironmentally friendly, and alternative control mea-sures based on the ecology of the pathogen are beingstudied in many research projects on Rhizoctonia(Schneider et al., 1997).

As a collective species, R. solani is genetically diverse.A method based on anastomosis groups (AGs) has beenused for its identification and classification (Parmeteret al., 1969; Ogoshi, 1987). Although AG and patho-genicity are related to some extent, evidence fromseveral studies indicates considerable pathogenic varia-tion between strains from within an AG (Ogoshi, 1987;Vilgalys & Gonzales, 1990). Among the 11 AGs of

Plant Pathology (2000) 49, 108–118

108 Q 2000 BSPP

*To whom correspondence should be addressed.

Email: [email protected].

Accepted 13 September 1999.

multinucleate Rhizoctonia, AG1 comprises many plant-pathogenic isolates recovered from a range of hosts (Liu& Sinclair, 1993). Isolates within AG1 have been dividedinto three subgroups based on host origin, symptomsand cultural characteristics: AG1-IA (sheath blight);AG1-IB (web blight); and AG1-IC (damping-off).However, the host ranges of AG1-IA and AG1-IBoverlap (Ogoshi, 1987; Sneh et al., 1991; Liu & Sinclair,1993). Although isolates of AG1 (which includesR. solani causing BLSB) have been reported worldwide(Ogoshi, 1987), information on the occurrence ofanastomosis groups is sparse in many Asian countries,including the Philippines. Knowledge of anastomosisgroupings of isolates is important because antagonists,the popular method of control against many AGs ofRhizoctonia, can be specific in their effectiveness againsta particular anastomosis group (Olsen & Baker, 1968;Schisler et al., 1994).

Isozyme and DNA analyses have advanced ourunderstanding of the structure of R. solani populations.These molecular tools have easily and distinctly groupedR. solani into subgroups of an AG. Simplified techniquessuch as PCR-RFLP of the rDNA internal transcribedspacer (ITS) region, and random amplified polymorphicDNA (RAPD), have been utilized for rapid detection ofvariation in Rhizoctonia spp. and other fungi (Williamset al., 1990; Liu et al., 1993; Manaut et al., 1998). Theuse of RFLP in DNA analysis, which focuses on rDNA,has also been successful. Ribosomal RNA genes in fungiare conserved and contain sequence components reflect-ing different evolutionary rates which are phylogeneti-cally and taxonomically informative (Bruns et al., 1991;Liu & Sinclair, 1993). Furthermore, the variationobserved in the length of PCR fragments of the rDNAITS region was not random and could be used as agrouping characteristic (Liu et al., 1993). The use ofRAPD markers has great potential in populationanalysis (Welsh & McClelland, 1990; Williams et al.,1990). The RAPD technique was successfully utilized byDuncan et al. (1993) in the analysis of variation ofseveral AGs of R. solani.

The experiments in this study were designed toinvestigate the anastomosis groupings of isolates ofR. solani recovered from various host species grown inor near maize fields in different geographic locations inthe Philippines. The isolates were also examined forsymptoms produced on the host from which they wereisolated, and for their virulence on maize. Geneticvariation among isolates belonging to AG1-IA from thePhilippines and Japan, using RAPD and PCR-RFLP ofthe rDNA ITS region, was also studied.

Materials and methods

Collection of R. solani isolates

Rhizoctonia-diseased specimens were collected fromvarious host species in or near maize fields in themaize-growing areas of Luzon and Mindanao in the

Philippines. Luzon, in the northern part, is characterizedby distinct dry and wet seasons, while Mindanao in thesouth is a region with prolonged, light rains and warmconditions throughout the year.

Characteristic symptoms on the diseased plantscollected were observed, and R. solani isolates wererecovered from infected tissues by the tissue plating(Riker & Riker, 1936). Small sections from an advancinglesion on each diseased sample were cut (0·5–1·0 cmlong) and surface sterilized for 10 min with 2% sodiumhypochlorite, rinsed in three changes of sterile distilledwater, plated on 2% acidified water agar and incubatedat room temperature (288C). After 48 h, fungal coloniesfrom the tissue pieces were examined with a lightmicroscope at ×100 magnification. Colonies withmycelial characteristics of Rhizoctonia spp. were trans-ferred to PDA. Nuclear number of the isolates wasdetermined by safranin staining (Bandoni, 1979).

Ten AG1-IA isolates from maize, sorghum, rice andweeds were requested from the National GrasslandResearch Institute, Japan, for genetic comparison withthe Philippine strains. All isolates used in this study arelisted in Table 1.

Anastomosis grouping

Hyphal anastomosis of 52 Philippine isolates with testerstrains (AG1-IA to AG-8) was examined according tothe procedure of Parmeter et al. (1969). Discs (5 mm) ofthe isolate and the tester were placed in pairs, 2–3 cmapart, on 2% water agar in Petri dishes. The Petri disheswere incubated at 258C in the dark until the advancinghyphae from opposite discs overlapped. The overlappedportion was stained with 0·5% aniline blue in lactophe-nol and examined under the microscope at ×100magnification for hyphal fusion.

Virulence determination

Fifty-two Philippine R. solani AG1-IA isolates wereinoculated into 1-month-old plants of the commercialmaize hybrid IPB 949, using the leaf sheath inoculationmethod (Pascual & Raymundo, 1989). Autoclavedsugarcane leaves, cut into 2-3 cm lengths and colonizedby each R. solani AG1-IA isolate, were used asinoculum. Inoculum was prepared by aseptically placingfive 5 mm discs of pure culture of the isolate inautoclaved sugarcane substrate in 1 L bottles. Cultureswere incubated for 10 days with occasional shaking toensure even colonization. Inoculation was carried out byinserting five pieces of inoculum substrate into thesecond basal leaf sheath of the plant. High relativehumidity was maintained by frequently misting theplants each day. Lesion length and plant height weremeasured at anthesis on 20 plants in the inner 4 m of theplot. The experiment was repeated once and wasconducted in plots with 5 m rows, 20 cm betweenplants and 75 cm between rows, using a randomizedcomplete block design with two replications. Relative

Rhizoctonia solani on maize 109

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lesion height was determined using the formula: (lengthof lesion/plant height) ×100. Analysis of variance andmean separations were performed using the SAS compu-ter program (SAS Institute, 1988).

DNA extraction

A pure culture of each of the 62 Philippine and JapanAG1-IA isolates was grown in potato dextrose broth for2–3 days, and the mycelial mat harvested by vacuumfiltration and blotted dry. The dried mycelial mat of eachisolate was stored at ¹808C until used for DNAextraction. One AG1-IB and two AG1-IC isolates werealso included as checks.

Genomic DNA was extracted according to theprocedure of Lee & Taylor (1990) with some modifica-tions. Dried frozen mycelial mat (50 mg) was ground in asterile mortar and pestle, suspended in extraction buffer(100 mM LiCl, 10 mM EDTA, 10 mM Tris-HCl at 7·5 pHand 0·5% SDS) and heated at 658C for 30 min. Thesupernatant obtained after centrifugation (5 000 g for10 min) was extracted twice with phenol–chloroform–isoamyl alcohol (25 : 24 : 1 v/v/v) and precipitated withan equal volume of isopropanol. The genomic DNApellet was washed with 70% ethanol, dried undervacuum, dissolved in TE buffer (10 mM Tris–HCl,pH 7·5, 1 mM EDTA) and stored at 48C overnight.After treatment with RNase A (50 mg mL¹1) for 1 h at378C, the DNA was extracted sequentially with equalvolumes of Tris–HCl saturated phenol, phenol–chloro-form–isoamyl alcohol (25 : 24 : 1 v/v/v), chloroform–isoamyl alcohol (24 : 1 v/v) and finally with diethylether. The DNA extract was precipitated with isopro-panol and the pellet washed with 70% ethanol, driedunder vacuum, dissolved in TE buffer and stored at¹308C until use. DNA was quantified using a spec-trophotometer (RNA/DNA Calculator, Gene Quant,Pharmacia).

RAPD fingerprinting

A 50 mL volume of PCR reaction mixture containing38·5 mL distilled water, 5 mL 10× PCR buffer, 4 mLdNTPs (2·5 mM each), 1·25 mL primer (40 mM), 0·25 mLTaq polymerase (5 U) and 1 mL template DNA(1 ng mL¹1) was prepared for each sample. GenomicDNA was amplified in a Perkin Elmer DNA thermalcycler using three primers: P14 (50-CCACAGCACG-30),RCO9 (50-GATAACGCAC-30) and R28 (50-ATG-GATCCGC-30), which were earlier used in detectingpolymorphisms in fungi including R. solani (Crowhurstet al., 1991; Haemmerli et al., 1992; Duncan et al.,1993). The temperature profile was programmed to 40cycles of 948C for 1 min (2 min at first cycle), 358C for1 min and 728C for 3 min (10 min at final cycle). Theamplified products were loaded into 1% agarose gelstained with ethidium bromide. Electrophoresis wascarried out at 50 V for 1 h and photographed under UVlight. Each amplification reaction was performed at least

twice to verify the reproducibility of the bandingpattern.

The RAPD DNA fingerprints generated from differentisolates were compared visually to generate haplotypegroupings for the three primers. Isolates showing 100%similarity in banding pattern were classified in onehaplotype group. The bands in each haplotype groupwere scored as either 1 for presence or 0 for absence ofthe character bands in a continuous pattern for allprimers, and pooled in a single matrix. The data wereanalysed using the unweighted pair group method witharithmetic mean (UPGMA) and sequential agglomera-tive, hierarchical and nested (SAHN) clustering methodsof the Numerical Taxonomy System (NTSYS PC version1·8) computer program. A matrix of genetic distancevalues was calculated and a tree drawn in phenogramform. In addition, bootstrap analysis was carried outusing the WINBOOT program (Yap & Nelson, 1996) todetermine the robustness of the cluster.

PCR-RFLP of rDNA ITS region

Genomic DNA of the isolates was used for restrictionanalysis. The rDNA of internal transcribed spacerregions including the 5·8 S gene was amplified using apair of primers, ITS1 (50-TCCGTAGGTGAACCTG-CGG-30) and ITS4 (50-TCCTCCGCTTATTGATAT-GC-30) (White et al., 1990). A 50 mL volume of PCRreaction mixture containing 5 mL 10× PCR buffer, 4 mLdNTPs (2·5 mM each), 1·25 mL each of primers ITS1and ITS4 (100 mM each), 0·25 mL Taq polymerase(5 U mL¹1), 1 mL (1 ng mL¹1) DNA and 37·25 mL steriledistilled water was prepared for each sample and addedwith one drop of light mineral oil (Sigma). Themixtures were amplified in a Perkin Elmer DNAThermal Cycler with a temperature profile as follows:25 cycles of 948C for 1 min (2 min for the first cycle),598C for 1 min and 728C for 1 min (2 min for the finalcycle). After chloroform extraction, the amplifiedproducts, together with the DNA marker, were resolvedby gel electrophoresis on 1% agarose stained withethidium bromide. Electrophoresis was run at 50 V for1 h. The amplified products were visualized under UVlight.

The amplified DNA samples were precipitated with amixture of 125 mL 99·5% ethanol, 5 mL 3 M NaOAc and1 mL glycogen (20 mg mL¹1), stored at ¹808C for 30 min,dried under vacuum, and the pellets then dissolved in25 mL TE. Restriction analysis of amplified rDNA ITSregion was done using three restriction enzymes, EcoRI,MboI and HinfI. A volume of 12 mL reaction mixturecontaining 1·2 mL 10× buffer, 1 mL restriction enzyme,4 mL amplified DNA and 5·8 mL sterile distilled waterwas prepared for each sample and stored overnight atoptimal temperature (378C) as suggested by the manu-facturers (Toyobo Co. Ltd and Takara Biomedicals). Thedigested products, together with the marker, wereresolved by gel electrophoresis in a 2% NuSieve (3 : 1)gel stained with ethidium bromide. Electrophoresis was

C. B. Pascual et al.110




Isolate namea Host Origin AG Sourceb

M1-L Mungbean Luzon 1-IA 1Sb-L Soybean Luzon 1-IA 1C1-L Maize Luzon 1-IA 1C2-L Maize Luzon 1-IA 1C3-L Maize Luzon 1-IA 1C4-L Maize Luzon 1-IA 1C5-L Maize Luzon 1-IA 1C6-L Maize Luzon 1-IA 1W-M Wheat Mindanao 1-IA 1C9-L Maize Luzon 1-IA 1Ct2-M Cotton Mindanao 1-IA 1I1-M Itchgrass Mindanao 1-IA 1

(Rottboellia exaltata)I2-M Itchgrass Mindanao 1-IA 1I3-M Itchgrass Mindanao 1-IA 1I5-M Itchgrass Mindanao 1-IA 1Su1-M Sugarcane Mindanao 1-IA 1Su2-M Sugarcane Mindanao 1-IA 1Su3-M Sugarcane Mindanao 1-IA 1So1-M Sorghum Mindanao 1-IA 1C10-M Maize Mindanao 1-IA 1C11-M Maize Mindanao 1-IA 1C12-M Maize Mindanao 1-IA 1C13-M Maize Mindanao 1-IA 1C7-L Maize Luzon 1-IA 1C8-L Maize Luzon 1-IA 1M2-L Mungbean Luzon 1-IA 1Ct1-L Cotton Luzon 1-IA 1Cf1-M Coffee Mindanao 1-IA 1We1-M Broadleaf weed Mindanao 1-IA 1

(unknown)C14-M Maize Mindanao 1-IA 1Cf2-M Maize Mindanao 1-IA 1C15-M Maize Mindanao 1-IA 1Cf3-M Coffee Mindanao 1-IA 1Su4-M Sugarcane Mindanao 1-IA 1Cp-M Cowpea Mindanao 1-IA 1Cg1-M Cogon Mindanao 1-IA 1

(Imperata cylindrica)Su5-M Sugarcane Mindanao 1-IA 1Cg2-M Cogon Mindanao 1-IA 1C16-M Maize Mindanao 1-IA 1C17-M Maize Mindanao 1-IA 1C18-M Maize Mindanao 1-IA 1D-M Durian Mindanao 1-IA 1C19-M Coffee Mindanao 1-IA 1We2-M Broadleaf weed Mindanao 1-IA 1

(unidentified)Cf4-M Coffee Mindanao 1-IA 1We3-M Grass weed Mindanao 1-IA 1I4-M Itchgrass Mindanao 1-IA 1Su6-M Sugarcane Mindanao 1-IA 1C20-M Maize Mindanao 1-IA 1R1-J Rice Japan 1-IA 2R2-J Rice Japan 1-IA 2R3-J Rice Japan 1-IA 2R5-L Rice Luzon 1-IA 1R6-L Rice Luzon 1-IA 1R7-L Rice Luzon 1-IA 1R4-J Rice Japan 1-IA 3B8-J Bluegrass Japan 1-IA 3C21-J Maize Japan 1-IA 3

Table 1 Rhizoctonia solani isolates used inthe study

run at 100 V for 1 h. Banding patterns were visualizedunder a UV transilluminator.

Results

Characterization of R. solani isolates

Rhizoctonia-infected specimens from maize, sorghum,rice, wheat, sugarcane, cotton, soybean, cowpea, mung-bean, durian, coffee and weed species showed differentcharacteristic symptoms (Table 2), for example bandedleaf and sheath blight in Gramineae, foliar leaf blight indurian and coffee, dry decay of stem tissues in legumesand aerial blight in cotton. More host species, withvarying types of AG1 symptoms, were found in or nearmaize fields in Mindanao than were found in Luzon. Thetissue transplant method yielded growth of Rhizoctoniasp. which fitted the description of R. solani AG1 type 1(AG1-IA) by Sherwood (1969). The 52 isolates recov-ered from the diseased tissues had multinucleate hyphalcells with four to nine nuclei per cell; the number ofnuclei varied from cell to cell and from one isolate to

another. All isolates anastomosed with the AG1-IA testerisolate and with the other subgroups of AG1, IB and IC.AG1-IA isolates formed sclerotia 2–4 mm long on PDAafter 5–7 days’ incubation at room temperature (288C),exhibiting pinkish brown (durian isolate) to dark brown(rice and coffee isolates) colour types. Sclerotia fromother crop species were light brown to brown.

Virulence on maize

All isolates were pathogenic to maize and caused similarsymptoms on maize within 48 h of inoculation. Initially,irregular circular lesions developed at the inoculationsite. Lesions were water-soaked at first and thendeveloped a bleached or straw-coloured centre with abrownish border. These enlarged and produced tan orbrown discoloured areas alternating with darker brownbands on the infected leaf sheaths. However, there wasconsiderable variation in virulence among isolates onmaize. C15-M, which was isolated from maize collectedin Mindanao, was the most virulent, with a relativelesion height [(length of lesion / plant height) ×100] of



Table 1 continuedIsolate namea Host Origin AG Sourceb

C22-J Maize Japan 1-IA 3C23-J Maize Japan 1-IA 3So2-J Sorghum Japan 1-IA 3So3-J Sorghum Japan 1-IA 3IB-J Weed Japan 1-IB 3IC-J Buckwheat Japan 1-IC 2IC-US Cauliflower USA (ATCC) 1-IC 2

aLetters denote host origin–geographic origin.bIsolates from 1, Institute of Plant Breeding, UP Los Banos, Philippines; 2, Gifu University, Japan;3, National Grassland Research Institute, Japan.

Table 2 Symptom appearance under natural conditions of Rhizoctonia-diseased samples collected from various host species in or near maizefields in different geographic areas in the Philippines

Host species Symptom description

Maize Sheath blight having straw-coloured centre with brown to dark brown border; wilted leaves attached to theinfected sheath; usually with sclerotial bodiesattached to the old part of the lesion; other samples with leaves showing irregular banded pattern ofalternating straw- and brown-coloured lesions

Rice Sheath blight having straw-coloured centre and thin, dark brown margins; some samples with sclerotialbodies attached to the older lesion

Sorghum Sheath blight having lesions with tan centre and irregular reddish brown marginsSugarcane Sheath blight with patches of reddish brown lesion having straw-coloured centreItchgrass Blighted brown tissues from soil line upward (from about half of the plant to whole plant)Cogon/weed grass Sheath blight, light brown lesions with brown marginsUnknown broadleaf weed Blighted from soil line to the crown with sclerotial bodies at the basal part of the stemSoybean, cowpea and mungbean Reddish brown, dry, decayed stem tissues from the soil line upward; with sclerotial bodies (1–2 mmdiameter)Durian Foliar blight; pinkish brown leaf; necrotic spots (2–4 cm diameter) with irregular margin; with sclerotia on

lesionsCoffee Foliar blight; brown leaf; necrotic spots (1–5 cm diameter) of irregular shape; no sclerotia on infected tissuesCotton (i) Aerial blight; dry boll rot and blighted leaves with plenty of 2–3 cm sclerotial bodies on the infected bolls

(isolate from Mindanao); (ii) dry rotted boll adhering to soil surface and with plenty of sclerotia (isolate fromLuzon)

80·0% (Table 3). In general, isolates recovered frommaize at Mindanao caused more severe infection on IPB949 than maize isolates from Luzon. However, the maizeisolate (C13-M) causing the lowest relative lesion heightwas also from Mindanao. Isolates recovered from otherhost species, such as durian (D-M), itchgrass (I4-M) andcoffee (Cf1-M and Cf4-M) also produced low diseaseseverity (Table 3).

RAPD fingerprinting

DNA of 62 AG1-IA isolates from the Philippines andJapan and of the three checks (one AG1-IB and twoAG1-IC) was purified, and PCR reaction with the threeprimers gave amplification products that generatedreproducible polymorphisms (Fig. 1). All AG1-IAisolates had one or two fragments in common for eachprimer. Haplotype groupings were produced based onvisual comparison of the banding patterns (Table 4) forall primers. The relationship between isolates is shownin the dendrogram (Fig. 2), which was derived from atotal of 28 reproducible polymorphic bands using thethree primers. UPGMA–SAHN clustering analysis of 30haplotypes of R. solani isolates allowed grouping ofAG1-IA, IB and IC into main clusters and resolved sevengroups of AG1-IA at the 75% similarity level. It is clearthat Japanese isolates from upland crops and one fromrice clustered in one group (G4). However, lowland riceisolates (R2-J, R3-J and R1-J) and a bluegrass isolate(B8-J) from Japan were closely related to each other andto other rice and upland crop isolates from thePhilippines (clustered in G2), while a rice isolate fromthe Philippines (R7-L) was closely related to upland cropisolates in Japan. A very distinct cluster, G1, includesisolates from mungbean, soybean and maize that werecollected from one Institute of Plant Breeding experi-mental station in Luzon. Another distinct cluster,corresponding to G7, was specific to isolates fromnecrotic spots of durian and coffee leaves (D-M, Cf1-M,Cf2-M, Cf3-M and Cf4-M) that were collected fromMindanao and were less virulent on maize. G6, whichincludes the most virulent, C15-M, and the other highlyvirulent isolates from Mindanao, were not closelyrelated to the other groups (F ¼ 70%). The rest of theupland crop isolates from Luzon and Mindanao wereclosely related to one another at the 75% similarity leveland were clustered in G3 and G5.

Restriction analysis of rDNA ITS region

The rDNA ITS region, including 5·8 S for 62 isolates ofAG1-IA and the tester isolates of AG1-IB (1) and AG1-IC (2) was amplified and digested using three restrictionenzymes. The amplified fragment length of AG1-IA wasestimated to be 720 bp long but showed no restrictionsite variation among AG1-IA isolates. However, thethree subgroups (AG1-IA, IB and IC) differed from eachother in the amplified fragment length (720, 730 and690 bp, respectively) and restriction sites.



Table 3 Relative lesion height caused by Rhizo-ctonia solani isolates on the maize hybrid IPB949 using leaf sheath inoculation method

Isolate Percentage meanrelative lesion heighta

C15-M 80·0 ab

C20-M 78·5 abC11-M 76·0 abcCg2-M 74·8 abcC10-M 73·7 abcdC16-M 72·2 abcdeC2-L 71·0 abcdefC8-L 67·0 abcdefgC14-M 66·0 abcdefghC3-L 63·5 abcdefghC12-M 61·1 abcdefghC17-M 60·2 abcdefghM1-L 58·3 abcdefghSu5-M 56·5 abcdefghI1-M 56·1 abcdefghSu2-M 56·0 abcdefghC1-L 54·5 abcdefghSb-L 54·2 abcdefghSu4-M 53·0 abcdefghC19-M 52·8 abcdefghR5-L 51·2 abcdefghCg1-M 51·2 abcdefghSu6-M 50·6 abcdefghSo1-M 50·5 abcdefghC9-L 49·2 abcdefghC7-L 49·2 abcdefghCt1-L 48·6 abcdefghC5-L 47.5 abcdefghWe1-M 46·6 abcdefghC4-L 46·4 abcdefghCp-M 44·5 abcdefghM2-L 42·9 abcdefghW-M 42·7 abcdefghC6-L 42·7 abcdefghI3-M 42·5 abcdefghWe3-M 42·1 abcdefghR7-L 41·3 bcdefghCt2-M 40·2 bcdefghR6-L 40·0 bcdefghSu3-M 39·7 bcdefghWe1-M 38·0 cdefghSu1-L 37·5 cdefghC18-M 37·5 cdefghI2-M 36·8 defghCf2-M 36·5 defghI5-M 36·3 defghCf3-M 34·0 efghD-M 33·7 efghCf4-M 32·3 fghI4-M 30·5 ghC13-M 30·0 ghCf1-M 27·1 h

aPercentage relative lesion height ¼ (length oflesion/plant height) ×100.bMeans in a column followed by the same letterare not significantly different according to DMRT(P ¼ 0·05).

Discussion

The results of this study indicate that multinucleateR. solani AG1-IA was the established, and perhaps thepredominant, AG1 subgroup in the warm, humid maize-

production areas of the Philippines. Although theybelong to the same subgroup, the isolates caused diversesymptoms on the host plants from which they wererecovered. AG1-IA isolates were found on more diversehost species in Mindanao than in Luzon (Table 1).



Figure 1 Random amplified polymorphic DNA banding patterns of Rhizoctonia solani AG1-IA isolates and checks (AG1-IB and IC) collectedfrom various hosts in or near maize fields at different geographic locations generated with primers P14 (a), R28 (b) and RCO9 (c). Lanes, M1,marker from stylI digestion of bacteriophage lambda; M2, marker (100 bp ladder) and representative R. solani isolates listed in Table 1.

Sheath blight, stem blight and cotton boll rot (collectedfrom bolls adhering to the soil) were the only symptomsobserved in Luzon, suggesting that the primary source ofinoculum for these infections was either sclerotia orhyphae in soil organic debris. In addition to thesesymptoms, leaf necrotic spots or foliar blight on cotton,coffee and durian trees, which were far above theground, were present in Mindanao. This suggests thatbasidiospores could also be an important source ofinfection in this region. Basidiospores of T. cucumerishave been reported as sources of inoculum for aerialblight diseases on cotton and other trees (Baker, 1970;Baker & Martinson, 1970). Basidiospore production ismore likely to occur abundantly in Mindanao because ofits warm, humid conditions throughout the year,whereas in Luzon there are distinct dry and wet seasons.Echandi (1965) reported that rapid spread of aerial

blight infection caused by T. cucumeris under warm,humid conditions might be explained by the efficientproduction and dissemination of basidiospores.

Virulence tests showed that all isolates, regardless ofhost origin, caused sheath blight on maize, but there wasconsiderable variation in virulence. It is noteworthy thatisolates from Mindanao produced the highest and lowestrelative lesion height, and that isolates from foliarblights or leaf necrotic spots from coffee and durianalso caused least disease severity on maize. As R. solaniis multinucleate, variation can occur through recombi-nation, hyphal fusion and mutation. The rapid growthor spread of its mycelia, and/or the likely abundantoccurrence of basidiospores due to environmentalconditions conducive for their growth and production,may have caused the great variation of AG1-IA isolatesin Mindanao. Baker & Martinson (1970) pointed out



Figure 2 Dendrogram obtained from percentage similarity coefficients after UPGMA–SAHN clustering of band data generated using threeprimers (P14, R28 and RCO9) in RAPD assay of 30 Rhizoctonia solani AG1-IA haplotype groups (Table 4), and one IB and two IC isolateslisted in Table 1. G1–G7 are seven groups of AG1-IA isolates resolved at the 75% similarity level. Bootstrap values obtained using the WINBOOT

program are written on the cluster branches.

that production of basidiospores is important inincreasing the probability of genetic recombinationamong wild-type isolates, and this may result in theproduction of new types with a high degree of variationin virulence. Attempts in this study to induce basidios-pore formation in vitro of some AG1-IA isolates usingthe techniques of Oniki et al. (1986) were unsuccessful,but further investigation of basidiospore productionunder natural conditions in the Luzon and Mindanaoareas needs to be done. This information is relevant tothe application of chemical control measures, whichmay have to be directed to the soil or to the plants, andto the evaluation of resistance of maize breeding lines todifferent virulence types of R. solani AG1-IA.

There was a considerable polymorphism within AG1-IA of R. solani, as detected by RAPD analysis. Thisconfirms the significant inter-isolate variability withinsubgroups of R. solani that has been demonstrated usingisozyme and DNA analysis (Vilgalys & Gonzales, 1990;Duncan et al., 1993; Liu & Sinclair, 1993). Variationwas evident even among isolates virulent to a commonhost cultivar such as maize. Cluster analysis using theUPGMA–SAHN method shows that variation amongisolates seems to be correlated with geographical originand virulence. Upland crop isolates from Japan (G4)

were distinct from upland crop isolates in the Philippines(Fig. 2). Isolates recovered from different hosts (maize,mungbean, soybean) but collected from the same area inLuzon were mostly classified into the same haplotypegroup, for example A and B, and were all related at morethan the 80% similarity level (G1). The same result wasobserved in some haplotype groups. This supports theassertion of Jabaji-Hare et al. (1990), who emphasizedthat within an AG in R. solani, genetic relatedness wasgreater among isolates from a common geographicorigin than among isolates from distant geographicareas.

Polymorphisms evident from the dendrogram basedon RAPD banding patterns of the isolates (Fig. 1) arealso in line to some extent with the variation observed inthe virulence test. The highly virulent isolates fromMindanao, including the most virulent one, weregrouped in one cluster at the 75% similarity level(Fig. 2), and the least virulent were discretely separatedin another group. Whether this variation was triggeredby the teleomorph or by the heterokaryotic nature ofR. solani (Bolkan & Butler, 1973; Puhalla & Carter,1976; Julian et al., 1996) remains to be determined.

Isolates from lowland rice were not related to oneanother. Three rice isolates from Japan (R1-J, R2-J and



Table 4 Haplotype and dendrogram group-ings of Rhizoctonia solani AG1-IA isolatesbased on visual comparison of their RAPDbanding patterns using three primers (P14,R28 and RCO9) and on RAPD banding pat-terns resolved at the 75% similarity level,respectively

Dendrogramgroup Haplotype Isolate

G1 A M1-L, Sb-L, C1-L, C2-L and C3-LG1 B M2-L and Ct1-LG2 C C4-L, C5-L and C6-LG2 D C10-M, C11-M, Ct2-M, Su1-M, Su2-M, Su3-M, I1-M,

I2-M, I3-M, So1-M and W-MG2 E C12-MG2 F R2-JG2 G R3-JG2 H R1-JG2 I C13-MG2 J C7-L and C9-LG2 K I5-M, C16-M, C17-M, C18-M and C20-MG2 L R5-LG2 M B8-JG3 N R6-LG3 O We1-M, We2-M and We3-MG3 P Cp-M, Cg1-M, Su4-M and Su5-MG4 Q R7-LG4 R R4-JG4 S C21-JG4 T C22-JG4 U C23-JG4 V So2-JG4 W So3-JG5 X C8-LG5 Y I4-M and Su6-MG5 Z C19-MG6 AA C15-M and C14-MG6 BB Cg2-MG7 CC Cf2-M, Cf1-M and D-MG7 DD Cf3-M and Cf4-M

R3-J) were closely related (F ¼>80%) to some isolatesfrom rice and upland crops in the Philippines, while arice isolate from the Philippines (R7-L) was closelyrelated to isolates from upland crops in Japan. Theaquatic environment involved in lowland rice produc-tion supports diversity in populations of Rhizoctoniaspp. (Jones & Belmar, 1989) and can be influenced byproduction practices. Germplasm exchange and/orimportation of contaminated seeds may also causevariation, as rice is highly cultivated and intensivelystudied in both countries.

Using three endonucleases, the fragment size of therDNA ITS region within AG1 subgroups and restrictionsites was the same as that found by Liu & Sinclair(1993). However, no genetic differences in the ITS regionwere observed among AG1-IA isolates. PCR-RFLP wasuseful only in differentiating the three subgroups of AG1in this experiment. This may be because only threerestriction enzymes were used. Liu & Sinclair (1993)used different restriction enzymes to those tested here,some of which generated greater polymorphism. The ITSregion is highly conserved and covers relatively few loci,thus additional endonucleases may be needed to detectvariation between isolates. In contrast, the RAPDtechnique analyses multiple loci with each primer, sodifferences between genotypes were more easily detected(Duncan et al., 1993).

The experiments from this study have increasedknowledge of the spread of R. solani AG1-IA in thePhilippines and added new insights into the nature ofvariation generated in this pathogen population.

Acknowledgements

This work was supported by the Institute of PlantBreeding-UPLB Basic Research Program, University ofthe Philippines at Los Banos, Philippines; Japan Societyfor the Promotion of Science through a RONPAKUFellowship given to the first author, and Gifu University,Japan. The authors wish to thank Dr Naomi Tangonanand the University of Southern Mindanao, Philippinesfor their support during collection of diseased samples,and Dr Koji Kageyama and Ms Nobuyo Koike for theirtechnical assistance.

References

Baker KF, 1970. Types of Rhizoctonia diseases and theiroccurrence. In: Parmeter Jr JR, ed. Rhizoctonia solani, Biol-ogy and Pathology. Berkeley, California, USA: University ofCalifornia Press, 125–33.

Baker R, Martinson CA, 1970. Epidemiology and diseasescaused by Rhizoctonia solani. In: Parmeter Jr JR, ed. Rhi-zoctonia solani, Biology and Pathology. Berkeley, Califor-nia, USA: University of California Press, 172–8.

Bandoni RJ, 1979. Safranin O as a rapid nuclear stain forfungi. Mycologia 71, 873–4.

Bolkan HA, Butler EE, 1973. Studies on heterokaryosis andvirulence of Rhizoctonia solani. Phytopathology 64, 515–22.

Bruns TD, White TJ, Taylor JW, 1991. Fungal molecular sys-tematics. Annual Review of Ecology and Systematics 22,525–64.

Crowhurst RN, Hawthorne BT, Rikkerink EHA, TempletonMD, 1991. Differentiation of Fusarium solani f. sp. cucur-bitae races 1 and 2 by random amplification of poly-morphic DNA. Current Genetics 20, 391–6.

De Sharma RC, Leon C, Payak MM, 1993. Diseases of maizein South and Southeast Asia: problems and progress. CropProtection 12, 414–22.

Duncan S, Barton JE, O’Brien PA, 1993. Analysis of variationin isolates of Rhizoctonia solani by random amplifiedpolymorphic DNA assay. Mycological Research 97, 1075–82.

Echandi E, 1965. Basidiospore infection by Pellicularia fila-mentosa (¼Corticium microsclerotia), the incitant of webblight of common bean. Phytopathology 55, 698–9.

Haemmerli UA, Brandle UE, Petrini O, McDermott JM,1992. Differentiation of isolates of Discula umbrinella(teleomorph: Apiognomnia errambunda) from beech, chest-nut and oak using random amplified polymorphic DNAmarkers. Molecular Plant Microbe Interactions 5, 479–83.

IPB, 1998. Annual Report, 1998. Los Banos, Philippines:Institute of Plant Breeding, University of the Philippines atLos Banos, 169–71.

Jabaji-Hare SH, Meller Y, Gill S, Charest PM, 1990. Investi-gation of genetic relatedness among anastomosis groups ofRhizoctonia solani using cloned DNA probes. CanadianJournal of Plant Pathology 12, 393–404.

Jones RK, Belmar SB, 1989. Characterization and pathogeni-city of Rhizoctonia spp. isolated from rice, soybean andother crops grown in rotation with rice in Texas. Plant Dis-ease 73, 1004–10.

Julian MC, Debets F, Keijer J, 1996. Independence of sexualand vegetative incompatibility mechanisms of Thanate-phorus cucumeris (Rhizoctonia solani) anastomosis group1. Phytopathology 86, 566–74.

Lee SB, Taylor JW, 1990. Isolation of DNA from fungal myce-lia and single cells. In: Innis MA, Gelfand DH, Sninsky JJ,White TJ, eds. PCR Protocols, a Guide to Methods andApplications. San Diego, USA: Academic Press, 282–7.

Liu ZL, Sinclair JB, 1993. Differentiation of intraspecificgroups within anastomosis group 1 of Rhizoctonia solaniusing ribosomal DNA internal transcribed spacer and iso-zyme comparisons. Canadian Journal of Pant Pathology15, 272–80.

Liu ZL, Domier LL, Sinclair JB, 1993. ISG-specific ribosomalDNA polymorphism of the Rhizoctonia solani species com-plex. Mycologia 85, 795–800.

Manaut F, Hamaide N, Vander Stappen J, Maraite H, 1998.Genetic relationships among isolates of Colletotrichumgloeosporioides from Stylosanthes spp. in Africa and Aus-tralia using RAPD and ribosomal DNA markers. PlantPathology 47, 641–8.

Ogoshi A, 1987. Ecology and pathogenicity of anastomosisand intraspecific groups of Rhizoctonia solani Kuhn.Annual Review of Phytopathology 25, 125–43.

Olsen CM, Baker KF, 1968. Selective heat treatment of soiland its effect on the inhibition of Rhizoctonia solani byBacillus subtilis. Phytopathology 58, 79–87.

Oniki M, Ogoshi A, Araki T, 1986. Development of the



perfect state of Rhizoctonia solani Kuhn AG1. Annals ofPhytopathological Society of Japan 52, 169–74.

Parmeter Jr JR, Sherwood RT, Platt WD, 1969. Anastomosisgrouping among isolates of Thanatephorus cucumeris. Phy-topathology 59, 1270–8.

Pascual CB, Raymundo AD, 1989. Evaluation of resistanceand yield loss in sorghum due to Rhizoctonia sheath blight.Philippine Journal of Crop Science 14, 133–5.

Puhalla JE, Carter WW, 1976. The role of the H locus in hetero-karyosis in Rhizoctonia solani. Phytopathology 66, 1348–53.

Riker AJ, Riker RS, 1936. Introduction to Research on PlantDiseases. St Louis, USA: JJS Swift.

SAS Institute Inc., 1988. SAS/STAT User’s Guide. ED 6.03.Cary, NC, USA: SAS Institute.

Schisler DA, Neate SM, Masuhara G, 1994. The occurrenceand pathogenicity of Rhizoctonia fungi in South Australianplant nurseries. Mycological Research 98, 77–82.

Schneider JHM, Salazar O, Rubio V, Keijer J, 1997. Identifi-cation of Rhizoctonia solani associated with field growntulips using ITS rDNA polymorphism and pectic zymo-grams. European Journal of Plant Pathology 103, 607–22.

Sherwood RT, 1969. Morphology and physiology in four ana-stomosis groups of Thanatephorus cucumeris. Phytopathol-ogy 59, 1924–9.

Sneh B, Burpee L, Ogoshi A, 1991. Identification of

Rhizoctonia Species. St Paul, MN, USA: American Phyto-pathological Society.

Tangonan NG, Quebral FC, 1992. Host Index of Plant Dis-eases in the Philippines, 2nd edn. Los Banos, Philippines:University of the Philippines at Los Banos.

Vilgalys R, Gonzales D, 1990. Ribosomal DNA restrictionfragment length polymorphisms in Rhizoctonia solani. Phy-topathology 80, 151–8.

Welsh JH, McClelland M, 1990. Fingerprinting genomesusing PCR with arbitrary primers. Nucleic Acid Research18, 7213–8.

White TJ, Bruns T, Lee S, Taylor J, 1990. Amplification anddirect sequencing of fungal ribosomal RNA genes for phy-logenetics. In: Innis MA, Gelfand DH, Sninsky JJ, WhiteTJ, eds. PCR Protocols: A Guide to Methods and Applica-tions. San Diego, USA: Academic Press, 315–22.

Williams JGK, Kubeilik AR, Livak KJ, Rafalski JA, TingeySV, 1990. DNA polymorphisms amplified by arbitrary pri-mers are useful as genetic markers. Nucleic Acid Research18, 6531–5.

Yap IV, Nelson RJ, 1996. WINBOOT: A Program for PerformingBootstrap Analysis of Binary Data to Determine the Confi-dence Limits of UPGMA-Based Dendrograms. IRRI Dis-cussion Paper Series No. 14. Manila, Philippines:International Rice Research Institute.



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