Isozyme and PCR-based genotyping of epidemic Phytophthora colocasiae associated with taro leaf blight

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Isozyme and PCR-based genotyping of epidemic Phytophthora colocasiaeassociated with taro leaf blightAjay Kumar Mishraa; Kamal Sharmaa; Raj Shekhar Misraa

a Central Tuber Crops Research Institute, Thiruvananthapuram, Kerala, India

Online publication date: 16 September 2010

To cite this Article Mishra, Ajay Kumar , Sharma, Kamal and Misra, Raj Shekhar(2010) 'Isozyme and PCR-basedgenotyping of epidemic Phytophthora colocasiae associated with taro leaf blight', Archives Of Phytopathology AndPlant Protection, 43: 14, 1367 — 1380To link to this Article: DOI: 10.1080/03235400802476450URL: http://dx.doi.org/10.1080/03235400802476450

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Isozyme and PCR-based genotyping of epidemic Phytophthora colocasiaeassociated with taro leaf blight

Ajay Kumar Mishra, Kamal Sharma and Raj Shekhar Misra*

Central Tuber Crops Research Institute, Thiruvananthapuram, Kerala-695017, India

(Received 14 August 2008; final version received 28 August 2008)

The Oomycetous fungus Phytophthora colocasiae causing leaf blight of taro is widelydistributed in India. Wide geographic range or sexual recombination provides geneticdifferentiation within this species. To determine how genetic variation is partitioned inP. colocasiae, 14 isolates were isolated from different regions of India, where theincidence of leaf blight is great. Molecular and biochemical techniques were employedfor assessing and exploiting the genetic variability among isolates of P. colocasiae. Sevenpolymorphic enzyme systems revealed 23 isozyme patterns, each uniquely characterisedby the presence or absence of electromorphs. Further, 10 oligodeoxynucleotide primerswere selected for random amplified polymorphic DNA (RAPD) assays, which resultedin 123 polymorphic bands for 10 isolates of P. colocasiae. The data were entered into abinary matrix and a similarity matrix was constructed using a DICE similarity (SD)index. A UPGMA cluster based on SD values was generated using a NTSYS computerprogram. Shannon’s index was used to partition genetic diversity. Similarly, isozymesand RAPDs yielded high estimates of genetic variability. Genetic diversity estimates viaisozyme and RAPD pattern indicated 78.26% and 100%, respectively, total diversityamong populations. This type of genetic variation in P. colocasiae indicates thatvariation due to asexual and/or possibly infrequent sexual mechanisms is possible andthat genetic differentiation has taken place as a result of geographic isolation. Thepresence of larger than expected RAPD variation in isolates of P. colocasiae and thepresence of distinct different zymotypes among these isolates suggests that geneticrecombination (or less likely hybridisation) is at least possible in this fungus and thatgeographic differentiation has taken place. Even isolates obtained from the same habitathave different RAPD patterns, indicating that many populations of this fungus aremade up of more than one genet and that few are derived clonally.

Keywords: taro leaf blight; Phytophthora colocasiae; phylogenetic relationships; isozyme;RAPD

Introduction

Taro (Colocasia esculenta (L.) Schott), a member of the Araceae family, is a traditionalroot crop of the tropics grown for its edible corms and leaves, and is believed to be one ofthe earliest cultivated root crops in the world (Plucknett 1976; Kuruvilla and Singh 1981).Worldwide production is on the increase, with Food and Agriculture Organisation (FAO)records indicating that taro production has doubled over the past decades (FAOSTAT2000), and taro is now the fifth most consumed root vegetable worldwide. Taro leaf blightcaused by oomycetous pathogen Phytophthora colocasiae Raciborski is the most

*Corresponding author. Email: [email protected]

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Vol. 43, No. 14, 20 September 2010, 1367–1380

ISSN 0323-5408 print/ISSN 1477-2906 online

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DOI: 10.1080/03235400802476450

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destructive disease of taro. It was first reported from Java in 1900 and this disease alonebrought a 30–50% decline to taro cultivation and production in the Solomon Islands,Papua New Guinea, Philippines, Indonesia, China, Malaysia, Japan, India and countriesof Africa and the Caribbean. In India, this disease is more prominent in northern andeastern parts, which are potential areas for taro production. In addition, P. colocasiaecauses serious post-harvest decay of corms. It penetrates through the mycelium present inseed corms and crop residues and the secondary spread in the fields takes place mainlythrough elongated, lemon or pear shaped sporangia carried to the healthy plants by windor rain splashes. Small circular lesions, brown on the upper surface of the leaf, are the firstsymptoms of disease infection. With the advancement of the disease, lesions enlarge andbecome irregular in shape and dark brown in colour with yellow margins. The infectedleaves die within 20 days and yield losses of 30–50% are common during favourableconditions (intermittent rain and cloudy weather). Effective means of reducing the spreadof the pathogen are quarantine, crop cultivation during dry seasons, and adherence tohygiene with a high level of public cooperation (Brandis and Batini 1985; Shearer andTippett 1989; Colquhoun and Hardy 2000). The possibility of introducing naturallyresistant lines of some susceptible plant species is being investigated (McComb et al. 1994;Colqu-houn et al. 2000). It seems that the basis of the adaptive variability of P. colocasiaeis that it has a large amount of genetic variation in pathogenesis characters relative toother characters. In order to identify genetic diversity and relationships, the developmentof markers is a prerequisite to discriminate amongst strains and make comparisons amonglocal, regional and national isolates of P. colocasiae. Unequivocal markers are also aprerequisite for examining mechanisms of genetic exchange, determining the extent ofgenetic variation due to asexual and sexual reproduction and providing distinct characterslinked to quantitative traits. Among several efficient methods for revealing geneticvariability within and among Phytophthora species, some of the most widely appliedmethods are isozyme electrophoresis (Old et al. 1988) and random amplified DNApolymorphism-RAPD (Dobrowolski and O’Brien 1993). The isozymes represent allelicexpression of same locus, but fragments produced by RAPD are independent geneticmarkers (Ochiai et al. 2001) with a lower proportion of non-neutral markers than former(Bartish et al. 2000). Therefore, RAPD and isozyme analysis often give discordantpatterns suggesting the importance of using multiple molecular marker system in studies ofpopulation structure (Wendel and Doyle 1998; Bartish et al. 2000; Lebot et al. 2003). Inthis study, 14 isolates of P. colocasiae collected from different regions of India, where theincidence of disease is high, were used. We compared isozyme and RAPD techniqueadequacy as tools for providing genetic markers useful for (1) evaluating the levels ofgenetic variations among the P. colocasiae isolates; (2) examining the genetic differentia-tion by cluster analysis based on allele frequencies of gene loci; (3) analysing relationshipsamong these markers and climatic and geographical variation. The cryptic nature of lesiondevelopment by some isolates and the host–pathogen interaction variation induced bysome environmental conditions are a particular challenge to experimental design. Thiswork could assist in experimental design of breeding and selecting resistant variety of taro.

Materials and methods

Fungal strains

Isolates of P. colocasiae used in this study were isolated from mature leaves of taroshowing typical symptoms of taro blight. The regions of India representing the knownrange of this fungus were given preference for this study (Table 1). Phytophthora colocasiae

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was confirmed in all 14 isolates by comparing their morphology with several otheraccessions of P. colocasiae maintained in the collections of the Indian Institute ofSpices Research (IISR), Calicut, India. Typically, P. colocasiae is characterised by theproduction of ovoid, ellipsoid or fusiform, semipapillate sporangia that are caducousand with a medium pedicel (3.5–10 mm). For isolation, leaf tissue segments of 2–3 cmfrom leaf blight infected area were excised from lesion margins. The leaf segments weresterilised in 1% sodium hypochlorite for 2 min, rinsed twice with sterile distilled waterand placed onto Phytophthora selective media (rye agar amended with 20 mg/lrifamycin, 200 mg/l vancomycin, 200 mg/l ampicillin, 68 mg/l pentachloronitrobenzeneand 50 mg/l 50% benlate). Segments were incubated in Petri dishes for 4–5 days at208C and mycelia were then transferred and maintained on potato dextrose agarmedium (PDA; 250 g/l potato, 20 g/l dextrose and 20 g/l agar). All voucher specimenswere deposited in IISR.

Determination of mating type

The mating type of isolates was determined by paring each unknown with two isolates of aknown A1 mating type (98-35a and 98-111) on V8 agar medium (50 g/l V-8 juice, 0.2 g/lCaCO3 and 20 g/l agar). Plates were placed in an incubator at 158C in the dark andexamined for the presence of oospores after four weeks. Isolates that produced oosporeswhen paired with A1 tester strain were designated A2 mating type.

Preparation of mycelium and genomic DNA extraction

P. colocasiae mycelium was grown on potato dextrose agar medium (containing 250 gpotato, 20 g dextrose and 20 g agar per litre of water) at 258C for one week.Erlenmeyer flasks (250 ml) containing 100 ml of potato dextrose broth were inoculatedwith two 1-cm discs removed from actively growing cultures. The cultures were placedon a rotary shaker (100 rev/min) and incubated at 278C for 4–5 days. Mycelia wereharvested by filtration through cheesecloth, blotted dry with sterile paper towels andused immediately for DNA extraction. Genomic DNA was extracted using the method

Table 1. Phytophthora colocasiae strains used in isozyme and RAPD analysis along with theirregion of collection.

No. IISR number Place of collection

1 02-03 RC CTCRI, Bhubaneswar, Orissa2 02-04 Salepur, Bhubaneswar, Orissa3 02-05 Malikpur, Faizabad, Uttar Pradesh4 02-06 Gopigand, Varanasi, Uttar Pradesh5 02-07 Nayagarh, Orissa6 02-08 Nadia, West Bengal7 02-09 Anandpur, Orissa8 02-10 Khandpara, Orissa9 98-35a Chelavoor, Calicut, Kerala10 98-35b Chelavoor, Calicut, Kerala11 PC-53 Jaipur, Orissa12 PC-71 Khairabad, Uttar Pradesh13 98-111 Sikkim, Assam14 PC-Tvm. CTCRI, Trivandrum, Kerala

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of Mishra et al. (2008). Proteins were extracted by centrifugation at 13,000 rpm for15 min in 25:24:1 (by volume) phenol-chloroform-isoamyl alcohol at room temperature.Nucleic acid in aqueous phase was precipitated with 0.25 volume of 7.5 M ammoniumacetate and separately with 2.5 volume of ice-cold ethanol. The samples werecentrifuged at 13,000 rpm for 15 min and the pellets were washed with 70% ethanol,air dried, and suspended in 50 ml of TE buffer (10 mM tris-HCl [pH 8], 1 mM EDTA).The nucleic acid dissolved in TE buffer was treated with 3 ml of RNase A (20 mg/ml),incubated at 378C, and stored at 7208C until use. DNA was quantified viaspectrophotometric measurement of UV absorption at 260 nm (Shimadzu UV-260).DNA was also quantified by means of 0.8% agarose gel electrophoresis followed byethidium bromide visualisation using 1-kbp DNA ladder (Fermentas) as DNA sizemarker.

Primer screening

Twenty-two 10-mer primers, corresponding to kits A, B,D andN fromOperonTechnologies(Alameda, California), were initially screened using four isolates to determine the suitabilityof each primer for the study. Primers were selected for further analysis based on their abilityto detect distinct, clearly resolved and polymorphic amplified products within the isolates ofP. colocasiae. To ensure reproducibility, the primers generating no, weak, or complexpatterns were discarded.

RAPD analysis

A set of 10 screened random decamer oligonucleotides primers were used for RAPDanalysis (Table 2). Each 25 ml of polymerase chain reaction (PCR) consisted of 10 ng oftemplate DNA, 100 mM each deoxynucleotide triphosphate, 20 ng of decanucleotideprimers (integrated DNA Technologies, Coralville, USA), 1.5 mM MgCl2, 16Taqbuffer (10 mM tris-HCl pH 9.0, 50 mM KCl, 0.01% gelatin), and 1U of Taq DNApolymerase (Promega). Amplifications were performed in a Techne progene thermal cycler(Techne (Cambridge) Ltd.). The PCR mixtures were heated at an initial step of 948C for2 min and then subjected to 35 cycles of the following programme: 948C for 30 s, 378C for

Table 2. Total number of amplified fragments and number of polymorphic fragments generated byPCR using selected random decamers in 14 isolates of Phytophthora colocasiae.

Name of primer Sequence of primer

Total no. ofamplificationproducts

No. of polymorphicproducts

OPA–1 50-CAGGCCCTTC-30 10 10OPA-2 50-TGCCGAGCTG-30 13 13OPA-3 50-AGTCAGCCAC-30 13 13OPA-4 50-AATCGGGCT-30 14 14OPA-5 50-AGGGGTCTTG-30 12 12OPA-6 50-GGTCCCTGAC-30 09 09OPA-7 50-GAAACGGGTG-30 10 10OPA-8 50-GTGACGTAGG -30 13 13OPA-9 50-GGGTAACGCC-30 13 13OPA-10 50-GTGATCGCAG-30 16 16

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1 min, 728C for 1 min 45 s. After the last cycle, the temperature was maintained at 728Cfor 8 min. Amplified products were resolved on a 1.5% agarose gel containing 0.5 mg/mlethidium bromide and visualised under UV light. Gel photographs were scanned throughGel Doc System (Alpha imager, Alpha Innotech, USA). Clear bands were revealed andwere scored for their presence (1) or absence (0). All profiles were reproducible and gaveclear and easy to score bands.

Isozyme analysis

Approximately 100 mg of the mycelia were ground in liquid nitrogen andextracted with 1.5 ml of extraction buffer (100 mM tris-HCl pH 7.5, 1mM disodiumEDTA, 10 mM KCl, 10 mM MgCl2.6H2O, 350 mM polyvinylpyrrolidone, 0.1%b-mercaptoethanol, 10% dimethyl sulfoxide). After centrifugation at 10,000 rpm for10 min at 48C, aliquots of supernatant were transferred to microcentrifuge tubes andstored at 7708C until used for electrophoresis. Protein extracts were subjected tovertical slab polyacrylamide gel electrophoresis as described by Iglesias et al. (1974).The following enzyme systems were screened: esterase (EST, 3.1.1.1), malatedehydrogenase (MDH, 1.1.1.37), phosphoglucomutase (PGM, 2.7.5.1), hexokinase(HEX, 2.7.1.1), b-glucosidase (b-GLU, 3.2.1.21), acid phosphatase (ACP, 3.1.3.2),malic enzyme (ME, 1.1.1.40). After electrophoresis, gels were stained with specificsubstrate as described by Selander et al. (1986). For each isolate, isozyme bands werescored. Loci were numbered consecutively and alleles at each locus were labeledalphabetically, beginning from the most anodal form.

Data analysis

Allelic frequencies from both isoenzymes and RAPD marker were used separately toestimate the percentage of polymorphic loci (P), mean number of alleles per locus (A0),effective number of alleles (AE), observed hetrozygosity (HO), and expected meanhetrozygosity (HE) with respect to the Hardy–Weinberg equilibrium (Hedrick 2000) usingthe computational program POPGENE 32 (Yeh and Yang 1999). Loci were consideredpolymorphic if more than one allele was detected. Fixation indices (F), reflecting deviationfrom the Hardy–Weinberg equilibrium, were calculated and out crossing rate (t) wereestimated using t ¼ (1 7 F)/(1 þ F) (Weir 1990). The portioning of genetic diversitywithin and among the cultivar of taro were analysed using F-statistics (Nei 1973)according to the equations of Weir and Cockerham (1984). Cluster analysis of the binaryRAPD and isozyme data were performed separately with the assistance of the SIMQUALprogram of NTSYS software, version 2.10 (Applied Biostatistics Inc. Setauket, NY,USA). Similarity matrices were generated using DICE and simple matching coefficients.An unweighted pair grouping by mathematical averaging (UPGMA) cluster analysis wasproduced from similarity matrices constructed for isozyme and RAPD data and resultingdendrograms were compared.

Results

Mating type identification

A set of 12 isolates of P. colocasiae were tested for mating type by pairing with two knownA1 testers. Only two of the isolates 98-35b and PC-Tvm formed abundant, typicaloospores in these pairings. The isolates were thus designated as A2 mating group.

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Isozyme analysis

Enzyme electrophoresis resulted in clear and consistent staining for seven enzymesencoded by 23 putative loci: Gluco1, Gluco2, Gluco3, Gluco4, APH1, APH2, APH3,PGM1, PGM2, PGM3, HK1, HK2, HK3, ME1, ME2, ME3, ME4, EST1, EST2, EST3,MDH1, MDH2 and MDH3. Five loci EST3, PGM1, Gluco1, HK1 and MDH3 werefound monomorphic in all isolates and thus showed diagnostic alleles for the P. colocasiaepopulation. All enzymes migrated anodally and were polymorphic. Eighteen loci (78.26%)were polymorphic in the isolates of Phytophthora colocasiae selected for study and differedin allelic frequencies (Table 3). The mean number of allele per locus (A0) was 1.8696.Observed mean hetrozygosity (HO) and expected mean hetrozygosity (HE) were 0.3043and 0.3370, respectively. The value of observed mean hetrozygosity was less than that ofexpected mean hetrozygosity. Mean fixation indices (F) were 0.0970, indicating an overallconformance to the Hardy–Weinberg equilibria. The F value was significantly greater thanzero and positive, indicating an excess of homozygotes. The outcrossing rate (t) based onfixation indices was 0.8321. The genetic variation parameters for all the loci provided anindication of the overall variation that existed among the electrophoretic types producedby this study. Cluster analysis was valuable for determining the relationships among 14isolates of P. colocasiae. The dendrogram obtained by the UPGMA clustering methodrevealed the genetic relationship of 14 isolates of P. colocasiae (Figure 1). The copheneticcorrelation coefficient was high (r ¼ 0.92), indicating that the UPGMA phenogramaccurately portrays the original data matrix. The isolates examined were divided into twogroups: the first group comprising 12 isolates, the second group comprising two isolatesand was in close agreement with the cluster analysis.

Table 3. Allelic frequencies of polymorphic loci studied in 14 isolates of Phytophthora colocasiae,sample size (N ¼ 28).

Locus Allele Allelic frequency Locus Allele Allelic frequency

Gluco1 a 1.0000 HK 3 a 0.4286Gluco2 a 0.6786 b 0.5714

b 0.3214 ME1 a 0.9286Gluco3 a 0.2500 b 0.0714

b 0.7500 ME 2 a 0.6429Gluco4 a 0.2857 b 0.3571

b 0.5000 ME 3 a 0.5714c 0.2143 b 0.4286

APH1 a 0.6429 ME 4 a 0.3929b 0.3571 b 0.2500

APH2 a 0.5357 c 0.3571b 0.4643 EST1 a 0.8929

APH3 a 0.6071 b 0.1071b 0.3929 EST 2 a 0.9286

PGM1 a 1.0000 b 0.0714PGM2 a 0.5000 EST 3 b 1.0000

b 0.5000 MDH1 a 0.7143PGM3 a 0.2500 b 0.2857

b 0.7500 MDH 2 a 0.6786HK1 a 1.0000 b 0.3214HK2 a 0.7857 MDH 3 a 1.0000

b 0.2143

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RAPD analysis

The 10 arbitrary primers chosen for the present analysis revealed 123 polymorphic bandsthat were consistently and unambiguously scorable in 14 isolates of P. colocasiae studied.These bands were treated as genetic loci. All of the 123 RAPD loci were polymorphic. Themaximum numbers of bands was produced by primer OPA–10 (Figure 2). Evaluation of

Figure 1. Dendrogram based on UPGMA analysis of genetic diversity obtained from isozymedata, showing relationship among 14 isolates of P. colocasiae.

Figure 2. Random amplified DNA polymorphisms of Phytophthora colocasiae isolates withrandom primers OPA 10. Lanes 1–14 represent isolates (1–14 as shown in table 1). Lane M indicatesthe 1 kbp molecular weight marker.

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RAPD-PCR data produced infrahost genetic distance values ranging from 0.113 to 0.658.The RAPD data matrix of band presence and absence was transformed into an allelicfrequency table (Table 4). The mean number of alleles per locus (AE) was 2.000. Observedmean hetrozygosity (HO) and expected mean hetrozygosity was (HE) 0.3380 and 0.3706,respectively. The value of observed mean hetrozygosity was less than that of expected

Table 4. Allelic frequencies of polymorphic loci studied in 14 isolates of Phytophthora colocasiae,sample size (N ¼ 14).

Locus AlleleAllelic

frequency Locus AlleleAllelic

frequency Locus AlleleAllelic

frequency

OPA1-1 0 0.8452 OPA4-6 0 0.9636 OPA8-2 0 0.65471 0.1548 1 0.0364 1 0.3453

OPA1-2 0 0.7559 OPA4-7 0 0.2673 OPA8-3 0 0.59761 0.2441 1 0.7327 1 0.4024

OPA1-3 0 0.5345 OPA4-8 0 0.6547 OPA8-4 0 0.37801 0.4655 1 0.3453 1 0.6220

OPA1-4 0 0.8452 OPA4-9 0 0.5345 OPA8-5 0 0.88641 0.1548 1 0.4655 1 0.1136

OPA1-5 0 0.5976 OPA4-10 0 0.7559 OPA8-6 0 0.92581 0.4024 1 0.2441 1 0.0742

OPA1-6 0 0.5976 OPA4-11 0 0.8452 OPA8-7 0 0.92581 0.4024 1 0.1548 1 0.0742

OPA1-7 0 0.5345 OPA4-12 0 0.8864 OPA8-8 0 0.53451 0.4655 1 0.1136 1 0.4655

OPA1-8 0 0.8452 OPA4-13 0 0.7559 OPA8-9 0 0.59761 0.1548 1 0.2441 1 0.4024

OPA1-9 0 0.5976 OPA4-14 0 0.9636 OPA8-10 0 0.88641 0.4024 1 0.0364 1 0.1136

OPA1-10 0 0.8018 OPA5-1 0 0.9258 OPA8-11 0 0.88641 0.1982 1 0.0742 1 0.1136

OPA2-1 0 0.7071 OPA5-2 0 0.8864 OPA8-12 0 0.92581 0.2929 1 0.1136 1 0.0742

OPA2-2 0 0.3780 OPA5-3 0 0.7071 OPA8-13 0 0.92581 0.6220 1 0.2929 1 0.0742

OPA2-3 0 0.8864 OPA5-4 0 0.8864 OPA9-1 0 0.88641 0.1136 1 0.1136 1 0.1136

OPA2-4 0 0.5976 OPA5-5 0 0.9636 OPA9-2 0 0.65471 0.4024 1 0.0364 1 0.3453

OPA2-5 0 0.7559 OPA5-6 0 0.4629 OPA9-3 0 0.70711 0.2441 1 0.5371 1 0.2929

OPA2-6 0 0.7559 OPA5-7 0 0.8864 OPA9-4 0 0.59761 0.2441 1 0.1136 1 0.4024

OPA2-7 0 0.9258 OPA5-8 0 0.8864 OPA9-5 0 0.59761 0.0742 1 0.1136 1 0.4024

OPA2-8 0 0.4629 OPA5-9 0 0.8864 OPA9-6 0 0.53451 0.5371 1 0.1136 1 0.4655

OPA2-9 0 0.8864 OPA5-10 0 0.7071 OPA9-7 0 0.37801 0.1136 1 0.2929 1 0.6220

OPA2-10 0 0.5345 OPA5-11 0 0.8452 OPA9-8 0 0.53451 0.4655 1 0.1548 1 0.4655

OPA2-11 0 0.9258 OPA5-12 0 0.3780 OPA9-9 0 0.80181 0.0742 1 0.6220 1 0.1982

(continued)

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mean hetrozygosity. Mean fixation indices (F) were 0.0879, indicating an overallconformance to Hardy–Weinberg equilibria. The F value was significantly greater thanzero and positive, indicating an excess of homozygotes. Outcrossing rate (t) based onfixation indices was 0.8348. Both isozyme and RAPD yielded almost similar geneticvariability estimates (Table 5). The dendrograms constructed through the UPGMAmethod are presented in Figure 3. Considerable variation was observed among theisolates. Cluster analysis showed a distinct separation of the isolates and formed twomajor groups. Isolates collected from the northern part of India formed a separate groupand clustered together, while isolates collected from the southern part of India formed a

Table 4. (Continued).

Locus AlleleAllelic

frequency Locus AlleleAllelic

frequency Locus AlleleAllelic

frequency

OPA2-12 0 0.6547 OPA6-1 0 0.9636 OPA9-10 0 0.70711 0.3453 1 0.0364 1 0.2929

OPA2-13 0 0.6547 OPA6-2 0 0.2673 OPA9-11 0 0.84521 0.3453 1 0.7327 1 0.1548

OPA3-1 0 0.9636 OPA6-3 0 0.3780 OPA9-12 0 0.84521 0.0364 1 0.6220 1 0.1548

OPA3-2 0 0.9258 OPA6-4 0 0.9258 OPA9-13 0 0.70711 0.0742 1 0.0742 1 0.2929

OPA3-3 0 0.3780 OPA6-5 0 0.8864 OPA10-1 0 0.88641 0.6220 1 0.1136 1 0.1136

OPA3-4 0 0.7559 OPA6-6 0 0.2673 OPA10-2 0 0.88641 0.2441 1 0.7327 1 0.1136

OPA3-5 0 0.4629 OPA6-7 0 0.7559 OPA10-3 0 0.26731 0.5371 1 0.2441 1 0.7327

OPA3-6 0 0.8452 OPA6-8 0 0.3780 OPA10-4 0 0.92581 0.1548 1 0.6220 1 0.0742

OPA3-7 0 0.8864 OPA6-9 0 0.8864 OPA10-5 0 0.92581 0.1136 1 0.1136 1 0.0742

OPA3-8 0 0.8864 OPA7-1 0 0.8864 OPA10-6 0 0.59761 0.1136 1 0.1136 1 0.4024

OPA3-9 0 0.4629 OPA7-2 0 0.8452 OPA10-7 0 0.37801 0.5371 1 0.1548 1 0.6220

OPA3-10 0 0.6547 OPA7-3 0 0.3780 OPA10-8 0 0.80181 0.3453 1 0.6220 1 0.1982

OPA3-11 0 0.8452 OPA7-4 0 0.2673 OPA10-9 0 0.80181 0.1548 1 0.7327 1 0.1982

OPA3-12 0 0.6547 OPA7-5 0 0.5976 OPA10-10 0 0.37801 0.3453 1 0.4024 1 0.6220

OPA3-13 0 0.7071 OPA7-6 0 0.5345 OPA10-11 0 0.37801 0.2929 1 0.4655 1 0.6220

OPA4-1 0 0.4629 OPA7-7 0 0.4629 OPA10-12 0 0.59761 0.5371 1 0.5371 1 0.4024

OPA4-2 0 0.3780 OPA7-8 0 0.8864 OPA10-13 0 0.53451 0.6220 1 0.1136 1 0.4655

OPA4-3 0 0.7071 OPA7-9 0 0.7071 OPA10-14 0 0.46291 0.2929 1 0.2929 1 0.5371

OPA4-4 0 0.8864 OPA7-10 0 0.9636 OPA10-15 0 0.65471 0.1136 1 0.0364 1 0.3453

OPA4-5 0 0.2673 OPA8-1 0 0.9258 OPA10-16 0 0.88641 0.7327 1 0.0742 1 0.1136

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separate group and were separated alone except isolate 98-111, indicating the possibilityfor the isolate to evolve from the southern part of India. Genetic differentiation betweenthe mating types isolated from the same location (Chelavoor, Calicut, Kerala) was notsignificantly high, indicating that hybridisation occurred between A1 (98-35a) and A2 (98-35b) mating types in that region.

Discussion

The objective of this study was to use isozyme and RAPD markers to characterise thepopulation structure of P. colocasiae isolates isolated from India. To the best of ourknowledge this study represents the first eco-geographical survey of genetic variationexisting among P. colocasiae isolates collected from different regions of India. Plant

Table 5. Genetic variation parameters of Phytophthora colocasiae based on isozyme and RAPDdata.

P Ao AE Ho HE F t

Isozyme 78.26 1.8696 1.6330 0.3043 0.3370 0.0970 0.8321RAPD 100.00 2.0000 1.5771 0.3380 0.3706 0.0879 0.8348

P ¼ percentage of polymorphic loci; A0 ¼ mean number of alleles per locus; AE ¼ mean effective number ofalleles; Ho ¼ mean observed hetrozygosity; HE ¼ mean expected hetrozygosity; F ¼ Wright’s fixation index;t ¼ out crossing rate.

Figure 3. Dendrogram based on UPGMA analysis of genetic diversity obtained from RAPD data,showing relationship among 14 isolates of P. colocasiae.

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breeding programmes selecting for resistance to P. colocasiae should take account of theconsiderable genetic and pathogenic phenotypes in the pathogen. If resistance is sufficientto enable reseeding and regeneration, then not only will rehabilitation be improved but thepossibility of natural selection for enhanced resistance by gene flow through thecommunity will be enhanced. Fundamental factors to disease management and controlare (i) how much genetic variability is in a pathogen population, (ii) how and when thatvariability flows through the population and (iii) how it affects the pathogen’s capacity tocause disease, survive and reproduce. Both isozyme and RAPD markers appear to beuseful to study the extent of molecular variation existing within this population. The use ofseven isozyme systems revealed significant differences among isolates of P. colocasiae,while RAPD markers demonstrated that it was possible to distinguish further between theisolates showing identical zymotypes. An isozyme study on 14 isolates of P. colocasiaerevealed variation between isolates collected from the same region. This isozyme variationmost probably appears as a result of sexual reproduction. The present estimates of geneticvariability with both techniques resemble each other. The clustering of UPGMA based onRAPD distance (Figure 2) is consistent with the geographical distribution of P. colocasiae.However, this congruence was not observed in isozyme clustering analysis (Figure 1).Isolates collected from the same region or sites are divided into different groups. Thediscordance of genetic distance measured with two molecular markers has been reported(Wendel and Doyle 1998; Bartish et al. 2000). Differences between results from these twodifferent techniques cannot be attributed to sampling error because the coefficient ofvariance (CV) was similar for both the techniques. A possible explanation for thedifferences found among these dendrograms might be based on the kind of informationprovided by each type of marker. Isozyme variation only reflects differences in protein-coding genes. Coding sequences are under greater selection pressure to maintainfunctional sequences. RAPDs can detect variation in both coding and non-codingregions. Small, repeated, random sequence mutations would be accumulated in non-coding sequences, and the diversity can be revealed by RAPD. Another factor which needsto be considered for RAPD analysis is that bands of identical mobility may occasionallycorrespond to non-homologous fragments (Chalmers et al. 1992; Tinker et al. 1993).Considering the above factors, the incongruence of genetic diversity trees derived fromthese methods was not surprising. An additional factor affecting genetic diversity assayedby different marker techniques is the number of markers or probes used in an analysis(Messmer et al. 1991; Smith et al. 1992). Generally, precision improves as more probes ormarker loci are detected in the analysis (Moser and Lee 1994). Generally, 10 RAPDprimers appear to be necessary in order to develop reliable estimates of relatedness amongisolates (Brummer et al. 1995). RAPD markers provide useful information because theydetect length polymorphisms arising from base sequence changes, insertions, deletions andsubstitutions either at or between priming sites (Cooke et al. 1996). According to thepresent results, RAPD markers tend to show higher value of genetic differentiation amongisolates than those obtained from isozymes, and might be more useful in screening geneticvariability. It is becoming clear from recent molecular studies that fungi assumed to beexclusively clonal actually are capable of recombination in nature (Taylor et al. 2000), andthis appears to be the case with P. colocasiae as well. The presence of larger than expectedRAPD variation in isolates of P. colocasiae and the presence of distinct differentzymotypes among these isolates suggests that genetic recombination (or less likelyhybridisation) is at least possible in this oomycetes and that geographic differentiation hastaken place. Even isolates obtained from the same habitat have different RAPD patterns,indicating that many populations of this omycetes are made up of more than one genet

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and that few are derived clonally. The results from mating studies proved the presence ofintercompatibility mating group (98-35a, 98-35b and 98-111, PC-Tvm) in the studiedpopulation of P. colocasiae. The designated mating status was in conjunction with themolecular evidence furnished by this work, demonstrating the relatively high variability ofisolate 98-111 from the other isolates of the northern region of India. This could probablybe explained by the restricted gene exchange of 98-111 with the other P. colocasiae isolatesand grouping of 98-111 with PC-Tvm. A mean fixation index (F) and outcrossing rates (t)based on fixation index obtained on the basis of 23 isozymatic loci was comparable withthose of RAPD analysis of P. colocasiae studied population and indicated an overallconformance to Hardy–Weinberg equilibria. The F value was significantly greater thanzero and positive, indicating an excess of homozygotes. The population genetic analysisdata further provides ample evidence for the above stated fact that recombination eventshave occurred in the P. colocasiae isolates. In the case of P. colocasiae, there is noindication of a significant genetic separation caused by host switching, as is sometimesobserved in fungal plant pathogens (e.g. Harvey et al. 2001; Brem and Leuchtmann 2003).Genetic studies of other widely distributed fungal species demonstrate that geneticdifferentiation may take place without either host switching or geographic separation. Forexample, a study of cryptic species in Stachybotrys chartarum (Cruse et al. 2002), whichhas been implicated as a possible cause of sick-building syndrome, indicated littledifferentiation caused by geographic separation. Steenkamp et al. (2002) similarly foundevidence for genetic differentiation, even speciation, in various groups within Fusariumsubglutinans, but the cause of this differentiation could not be attributed to either hostswitching or geographic distance. The existence of sexual reproduction in P. colocasiae hasalready been reported (Narula and Mehrotra 1981; Zhang et al. 1994). Based on theseobservations, it has been suggested that mycelial incompatibility maintains the geneticidentity of genotypes; genetic exchange between certain genotypes during sexualreproduction and geographical isolation might be factors for rapid evolution and geneticdrift of P. colocasiae isolates. Taro is a clonally propagated species and cultivars sharea narrow genetic base in most countries (Lebot and Aradhya 1991; Lebot et al. 2000). Tarois a clonally propagated species and shows a variable degree of susceptibility againstP. colocasiae. This study may be further beneficial for breeders to breed local taro cultivarsfor resistance against strains of P. colocasiae.

Acknowledgements

The funding provided for conducting the research work by the Indian Council of AgriculturalResearch, New Delhi, is gratefully acknowledged. The authors thank the director of the CentralTuber Crops Research Institute (Thiruvananthapuram) for providing infrastructure facilities.

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