molecular variability among the isolates of sclerotium...

Saddala Durga Prasad, Shaik Thahir Basha*, Narreddy Peddanarappa Gari Eswara Reddy

Department of Plant Pathology, S.V. Agricultural College, Tirupati, 517502 A.P.,India

*Corresponding Author: [email protected]

Molecular variability among the isolates ofSclerotium rolfsii causing stem rot of groundnut byRAPD, ITS-PCR and RFLP

AbstractGenetic variability among the virulent isolates of Sclerotium rolfsii was studied using moleculartechniques like RAPD, ITS-PCR and RFLP. The RAPD banding pattern reflected the geneticdiversity among the isolates by formation of two clusters. A total of 221 reproducible andscorable polymorphic bands ranging approximately as low as 100 bp to as high as 2500 bpwere generated with five RAPD primers. ITS region of rDNA amplification with specific ITS1and ITS4 universal primers produced approximately 650 to 700 bp in all the isolates confirmedthat all the isolates obtained are Sclerotium rolfsii. Studies by ITS-RFLP indicated that therewas no polymorphism in restriction banding pattern among the isolates with the restrictionendonucleases used.

Keywords: Groundnut, molecular variability, Sclerotium rolfsii, stem rot.

Prasad SD, Basha ST, Reddy NPGE (2010) Molecular variability among the isolates ofSclerotium rolfsii causing stem rot of groundnut by RAPD, ITS-PCR and RFLP. EurAsia JBioSci 4: 80-87.DOI:10.5053/ejobios.2010.4.0.10

©EurAsian Journal of BioSciences, 2010 80

EurAsian Journal of BioSciences EurAsia J BioSci 4, 80-87 (2010)DOI:10.5053/ejobios.2010.4.0.10

Groundnut (Arachis hypogea L.) is a majorlegume and important oil seed crop in Indiawhich is grown over an area of 6.74 m.ha.with an annual production and productivity of7.99 m.t. and 1185 kg/ha respectively. InAndhra Pradesh, it is grown to the extent of1.87 m. ha. with 1.64 m.t. production andwith a productivity of 728 kg/ha. Sclerotiumrolfsii Sacc. which is a serious soil bornepathogen common in tropical and sub-tropicalregions of the world where high temperaturecoupled with high humidity is prevalent duringthe rainy season causing severe damage tothe crop with yield losses of over 25 per cent(Mayee and Datur 1998). The symptoms of S.rolfsii include yellowing and wilting ofbranches, presence of mustard seed likesclerotia at the site of infection (Asghari andMayee 1991). To understand the presentplant disease situations and for effectivemanagement, it is essential to study as muchas possible about the genetic variability inplant pathogenic fungi. The information on

genetic variability among the groundnutisolates of S. rolfsii is limited. Thus, thepresent study was undertaken to assess thesignificant genetic variations by nucleic acidbased marker techniques using ITS-PCR,RAPD and RFLP to distinguish differentvirulent isolates of S. rolfsii.

The pathogenic variability and pathogenicpotential of S. rolfsii isolates was tested onthe groundnut variety TGCS888 as describedby Shokes et al. 1996. The molecularvariability among the isolates of S. rolfsii wasstudied by using ITS region of rDNA, RandomAmplified Polymorphic DNA (RAPD) andInternal Transcribed Spacer - RestrictionFragment Length Polymorphism (ITS-RFLP).

Fungal culturesDiscs of 5 mm diameter S. rolfsii isolates

Received: May 2010Accepted: July 2010

Printed : August 2010

INTRODUCTION

MATERIAL AND METHODS

were cut from periphery of an activelygrowing 7 d old culture on PDA andinoculated into 250 mL conical flaskcontaining 100 mL of sterile potato dextrosebroth. The flasks were kept rotated constantlyat a speed of 125 rpm in an orbital shaker at28±2°C. The resultant growth of mycelialmat was harvested and excess moisture wascompletely removed through sterile blottingpaper and used for DNA extraction.

DNA extractionThe total genomic DNA of S. rolfsii isolates

was extracted from vegetative myceliumusing the procedure Murray and Thompson(1980) with slight modifications. Fungal matof 0.5 g grounded to fine powder in liquidnitrogen and transferred to sterile eppendorftube. To this, 1 mL of extraction buffer (1 MTris-5.0 mL; 5 M Nacl-14.0 mL; 0.5 M EDTA-2.0 mL; 0.1% Mercaptoethanol-50.0 μL; 2%CTAB-1.0 g; 1% PVP-0.5 g) was added andincubated for 1 h in water bath at 65°C. Thenthe tubes were centrifuged at 10,000 rpm for10 min at room temperature. The supernatantwas transferred into other tubes. To thesupernatant equal mL of chloroform andisoamyl alcohol (24:1) and RNase (1 μL/100μL) was added and incubated at roomtemperature for 10-20 min. The tubes werecentrifuged at 10,000 rpm for 10 min,separated the supernatant and added 0.6 volof ice cold isopropanol + 0.1 vol of sodiumacetate and incubated at -20°C for overnight.Next day, the tubes were centrifuged at13,000 rpm for 20 min at 4°C, thesupernatant was discarded and the pelletwashed with 70 per cent ethanol andcentrifuged at 13,000 rpm for 20 min at 4°C.Again the supernatant discarded, the pellet airdried and dissolved in 100 μL of steriledistilled water or TE. The DNA samples werestored at -20°C for further studies.

Qualitative and quantitative verification ofDNA

The quality and quantity of DNA wasanalyzed by running 2 μL of each samplemixed with 2 μL of 10x loading dye in 1%agarose gel. The DNA from all isolatesproduced clear sharp bands in one per centagarose gel indicating the good quality of

DNA. The DNA has been quantified bycomparing with the 1 kb size marker (Genei,Bangalore) and by spectrophotometer(Nanodrop ND1000).

RAPD profiles through Polymerized ChainReaction (PCR)

Five random primers belong to Operon "A"series viz., OPA-01, OPA-12, OPA-17, OPA-18 and OPA-20 (Operon technologies Inc.,)were screened for generating polymorphismamong the isolates under the study. Theexperiment was repeated thrice and resultswere reproducible. The Oligonucleotide primersequences used in RAPD technique are givenbelow:

PCR amplifications were carried out in 0.2mL eppendorf tubes with 25 μL reactionmixture which consists of 2.5 μL of 10x Taqbuffer, 2.5 μL of 25 mM MgCl2, 2.5 μL ofprimer (1 picomolar/μL), 0.5 μl of 100 mMdNTP mix, 0.2 μL of Taq polymerase enzyme(conc. 1 U μL-1) and 16.8 μL of sterile PCRwater (Genei, Bangalore) and 2 μL (40-50 ng)of DNA sample. Amplification was carried outby 5 min of initial denaturation at 94°Cfollowed by 40 cycles of denaturation of94°C for 1 min; annealing at 37°C for 1 min;extension at 72°C for 2 min with finalelongation at 72°C for 5 min. Amplified PCRproducts were subjected to 1.0 per centagarose gel electrophoresis with 1.0 x TBE asrunning buffer. The banding patterns werevisualized under UV trans-illuminator withethidium bromide (10 mg mL-1) staining. TheDNA banding profiles were documented in thegel documentation system (Alpha Innotech)and compared with 1 kb DNA ladder (Genei,Bangalore).

Scoring and data analysisEach amplified band was considered as

RAPD marker and recorded for all samples.

Prasad et al.

©EurAsian Journal of BioSciences, 201081

EurAsian Journal of BioSciences

Data was entered using a matrix in which allobserved bands or characters were listed. TheRAPD pattern of each isolate was evaluated,assigning character state 'I' to all the bandsthat could be reproducible and detected in thegel and 'O' for the absence of band. The datamatrix thus generated was used to calculateJaccard similarity coefficient for each pairwise comparison. The coefficients werecalculated In Silico following Jaccard (1908).The similarity coefficients were subjected toUnweighted Pair-Group Method on ArithmeticAverage (UPGMA) cluster analysis to groupthe isolates based on their overall similarities.Statistical Package for Social Science (SPSS)package was used for the cluster analysis andsubsequent dendogram preparation.

PCR amplification of ITS regionPCR amplification of Internal Transcribed

Spacers (ITS) region of rDNA was performedusing universal primers ITS-1 (5' - TCC GTAGGT GGA CCT GCG G - 3') as forward primerand ITS-4 (5' - TCC TCC GCT TAT TGA TATGC - 3') as reverse primer (White et al. 1990)in eppendorf PCR master cycler. Amplificationwas carried out in 0.2 mL eppendorf tubeswith 25 μL reaction mixture containing 2.5 μLof 10x Taq buffer, 2.5 μL of 25 mM MgCl2,2.0 μL of ITS1 primer (0.6 picomolar/μL), 2.0μL of ITS-4 primer (0.6 picomolar/μL), 0.5 μLof 100 mM dNTP mix, 0.125 μL of Taqpolymerase (0.5 u/μL) and 14.37 μL of sterilePCR water (Genei, Bangalore) and 3 μL (40-50ng) of DNA sample. The PCR amplificationwas carried out by 35 cycles, of whichdenaturation at 94°C for 1 min, annealing at56°C for 1 min and extension at 72°C for 1.5min with initial denaturation at 94°C for 4 minbefore cycling and final extension at 72°C for6 min after cycling. Amplified PCR productswere observed in 1.0 per cent agarose gel in1% TBE buffer and visualized under UV trans-illuminator with ethidium bromide staining.The size of the PCR product was estimated bycomparison with known DNA marker of 1 kbDNA ladder. The banding profiles of ITS-PCRproducts were documented in geldocumentation system (Alpha Innotech).

ITS-RFLP PCR products of ITS region of the isolates

under the study were digested with threedifferent restriction enzymes viz., AluI, HinfIand MseI. Digestion was carried out with 20μL reaction mixture containing 7 μL of ITS-PCR product, 1 μL of enzyme (10 U/μL), 2 μLof 10X enzyme buffer and 10 μL of sterilePCR water. The digestion was carried outovernight at 37°C for AluI, HinfI and 65°C forMseI in a water bath. Restricted productswere analyzed on 2.0 per cent agarose gel.

The pathogenic variability among thetwenty Sclerotium rolfsii isolates was testedon groundnut variety TGCS888, and theresults revealed that the isolates CSr1, CSr2,CSr9, CSr10, KSr15, KSr17, KSr19 werevirulent with regard to disease severityresulting in different degrees of percentdisease incidence (PDI) (Table. 1)(Durgaprasad et al. 2009). Based on PDI, thehigh, moderate and low virulent eight isolateswere selected for molecular characterization.Total genomic DNA extracted from the eightisolates viz., CSr1, CSr2, CSr9, CSr10,KSr15, KSr17, KSr19, and KSr20 havingvarying degrees of virulence and all the eightisolates produced clear sharp bands indicatingthe good quality of DNA.

Characterization of S. rolfsii isolates byRAPD

Five random primers viz., OPA-01, OPA-12, OPA-17, OPA-18 and OPA-20 generatedreproducible polymorphism among the eightisolates. Amplified products with all theprimers have showed polymorphic anddistinguishable banding pattern indicating thegenetic diversity among all isolates of S.rolfsii. A total of 221 reproducible andscorable polymorphic bands rangingapproximately as low as 100 bp to as high as2500 bp were generated with five primersamong the eight isolates characterized. Theprimer OPA-01 amplified two uniquefragments of approximately 100 bp and 250bp in case of KSr19 isolate (Fig. 1A). PrimerOPA-12 amplified specific bands of 1400 bpand 1500 bp in case of KSr15 isolate (Fig.1B). Moreover, 600 bp fragment was absent


Prasad et al.EurAsian Journal of BioSciences

RESULTS AND DISCUSSION

in case of CSr1 isolate compared to otherisolates. OPA-17 primer yielded a specificfragment approximately 200 bp in case ofisolates CSr9 and KSr17 and absent in allother isolates. The 700 and 650 bp bandswere specifically amplified in case of isolateKSr20 showing the polymorphism among theisolates (Fig. 1C). The primer OPA-18amplified a unique band of approximately 500bp in case of CSr1, 575 bp in case of CSr9and 750 bp in case of KSr20 (Fig. 1D). PrimerOPA-20 amplified a fragment of 1000 bp and1050 bp both in case of KSr15 and KSr17isolates (Fig. 1E). Almeida et al. (2001)studied variability among 30 isolates of S.rolfsii from different hosts of Brazil usingRAPD profiles and identified 11 haplotypes.

Relationships among the isolates wereevaluated by cluster analysis of data based onsimilarity matrix. The dendogram wasgenerated using UPGMA package based onWard's Squared Equalidean Distance method(Fig. 2). Based on the results obtained, all theeight isolates were grouped into two mainclusturs. Cluster I contains four isolates viz.,CSr9, KSr17, CSr1 and KSr19 of which thefirst two and last two formed two separate

sub-clusters viz., Ia (CSr9and KSr17); Ib(CSr1 and KSr19). Cluster II contains fourisolates viz., CSr2, CSr10, KSr15 and KSr20.Jaccard's similarity co-efficient among theeight isolates were calculated to establishtheir genetic relationships. The similarity indexvalues ranged from 0.00 to 100 per centindicating the presence of a high range ofvariability at nucleic acid level among theeight S. rolfsii isolates. In the presentinvestigation, no correlation was observedamong the clusters formed based on theRAPD and pathogenic virulence amongisolates of S. rolfsii. Punja and Sun (2001)studied the extent of genetic diversity amongMCG of S. rolfsii and S. delphinii by theirunique banding patterns using six primerswhich were genetically diverse but sharedgreater numbers of common bands andclustered together.

The RAPD study clearly indicated thegenetic variability among the S. rolfsiiisolates. The 650 bp and 700 bp bands whichhas been amplified with OPA-17 primers maybe used to develop Sequence CharacterizedAmplified Region (SCAR) marker for thedetection of virulent isolates of S. rolfsii anddevelopment of correlation between bandingpattern and virulence.

Characterization of S. rolfsii isolates byITS-PCR

The structure of rDNA cluster and theexpected amplified products with ITS-1 andITS-4 primers are shown in Fig. 3. Theseprimers viz., ITS-1 and ITS-4 were used forPCR amplification of ITS region of rDNAcluster which includes ITS-1, 5.8S and ITS-2regions of all eight isolates. Both the primersproduced amplified product size of 650-700bp in all the eight isolates (Fig. 4). Theseresults confirmed that all the isolates belongto genus Sclerotium. Harlton et al. (1995)screened a worldwide collection of S. rolfsiiwhich revealed variation in ITS regions of 12sub-groups of S. rolfsii. Almeida et al. (2001)studied variability among 30 isolates of S.rolfsii by RAPD and were differentiated intodistinct groups by ITS-PCR.

Characterization of isolates by ITS-RFLPThe ITS region of rDNA amplified with



Table 1. Pathogenicity of different isolates of S. rolfsii at 15 DAI on groundnut variety TCGS-888.

*Mean of three replications

(ITS-1 and ITS-4) primer yielded the singleband of approximately 650-700 bp werefurther subjected to restriction analysis withdifferent endonucleases in order to observethe polymorphism among the S. rolfsii

isolates. The products were digested withthree different restriction endonucleases viz.,AluI, HinfI, and MseI and the results depictedin Fig. 5. The AluI enzyme yielded 5fragments for all isolates approximately with



Fig 1. Random Amplified Polymorphic DNA (RAPD) patterns of S. rolfsii isolates with random primers. M: 1kb DNA ladder; A: OPA-01; B: OPA-12; C: OPA-17; D: OPA-18; E: OPA-20. Lane 1-8 are S. rolfsii isolates, CSr1, CSr2, CSr9, CSr10, KSr15, KSr17, KSr19 and KSr20 respectively.



molecular weight ranging from 102 bp to 512bp. The HinfI enzyme yielded 5 fragments forall the isolates approximately with molecularweight ranging from 50 bp to 390 bp and theMseI enzyme yielded 4 fragments for all theisolates approximately with molecular weightranging from 88 bp to 336 bp. Okabe et al.(1998) divided 67 isolates of the southernblight fungus from Japan into five groupsbased on ITS-RFLP analysis of nuclear rDNA.Three groups were reidentified as S. rolfsiiand two resembled S. delphinii in RFLPpatterns. In the present investigation, all theenzymes above have shown differentrestriction pattern but the each enzyme hasthe same restriction sites among all theisolates under the study. It is worthwhile to

use more number of enzymes to study thepolymorphism.

Prof. N.P. Eswara Reddy gratefullyacknowledge financial grant and supportprovided by the Department of Biotechnology,Government of India, New Delhi.

Fig 2. Dendogram generated using UPGMA analysis showing polymorphism between isolates of S. rolfsii using RAPD markers.

Fig 3. Structure of rDNA cluster and the position of ITS primers used in the PCR amplification of S. rolfsii isolates.

Fig 4. Amplification product of Internal Transcribed Spacer (ITS) with ITS-1 and ITS-4 ribosomal DNA primers; M=1 kb DNA ladder. Lanes 1to 8represents S. rolfsii isolates, CSr1, CSr2, CSr9, CSr10,KSr15, KSr17, KSr19 and KSr20 respectively.

Fig 5. Restriction enzyme internal transcribed spacer (ITS) region of different isolates of S. rolfsii;A: Alu I; B: Hinf I; C: Mse I; M: 100 bp DNA ladder. Lanes 1-8 are S. rolfsii isolates, CSr1, CSr2, CSr9, CSr10, KSr15, KSr17, KSr19 aand KSr20 respectively.

ACKNOWLEDGEMENT



Almeida AMR, Abdelnoor RV, Calvo ES, Tessnman D, Yorinori JT (2001) Genotypic diversityamong Brazilian isolates of Sclerotium rolfsii. Journal of Phytopathology 149: 493-502.

Asghari MA, Mayee CD (1991) Comparative efficacy of management practices on stem and podrots of groundnut. Indian Phytopathology 44: 328-332.

Durgaprasad S, Eswarareddy NP, Bhaskarareddy BV, Hemalatha TM, Sudhakar P (2009)Cultural, morphological and pathological variability among the isolates of Sclerotium rolfsiiSacc. from Groundnut. Geobios 36(2): 169-174.

Harlton CE, Levesque CA, Punja ZK (1995) Genetic diversity in Sclerotium (Athelia) rolfsii andrelated species. Phytopathology 85: 1269-1281.

Jaccard P (1908) Nouvelles recherches sur la distribution florale. Bulletin de la Société vaudoisedes sciences naturelles 44: 223-270.

Mayee CD, Datur VV (1998) Diseases of groundnut in the tropics. Review of Tropical PlantPathology 5: 85-118.

Murray M, Thompson W (1980) The isolation of high weight plant DNA. Nucleic Acid Research8: 4321-4325.

Okabe I, Morikawa C, Matsumoto N, Yokoyama (1998) Variation in Sclerotium rolfsii isolates inJapan. Mycoscience 39: 399-407.

Punja ZK, Sun LJ (2001) Genetic diversity among the mycelial compatibility groups ofSclerotium rolfsii (telemorph Athlia rolfsii) and S. delphini. Mycological Research 105: 537-546.

Shokes FM, Rhogalski K, Gorbet DW, Brenneman TB, Berger DA (1996) Techniques forinoculation of peanut with Sclerotium rolfsii in the greenhouse and field. Peanut Science 23:124-128.

White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and direct sequencing of fungalribosomal RNA genes for phytogenetics in PCR protocols. Academic Press, London.

REFERENCES



Yer Fistigi'nda Kök Cürümesine Yol Açan Sclerotium rolfsii'nin IzolatlariArasindaki Moleküler Degiskenligin RAPD, ITS-PCR ve RFLP ile Incelenmesi

Özet

Sclerotium rolfsii'nin virulent izolatlari arasindaki genetik çesitlilik RAPD, ITS-PCR ve RFLP gibi moleküler

teknikler kullanilarak çalisildi. RAPD bantlari, iki küme olusturmak suretiyle izolatlar arasindaki genetik

çesitliligi gösterdi. Bes RAPD primeri kullanilarak, yaklasik 100 bp ile 2500 bp arasinda degisiklik gösteren

toplam 221 adet tekrarlanabilir ve ölçülebilir bant elde edildi. Spesifik ITS1 ve ITS4 universal primerleri ile

rDNA'nin ITS bölgesinin çogatilmasi, bütün izolatlarda yaklasik 650 ila 700 bp üretti ve bu durum bütün

izolatlarin S. rolfsii'den elde edildigini teyit etti. ITS-RFLP çalismalari, kullanilan restrüksiyon

endonükleazlarla izolatlar arasindaki restrüksiyon bantlarinda her hangi bir polimorfizm olmadigini gösterdi.

Anahtar Kelimeler: Moleküler çesitlilik, kök çürümesi, Sclerotium rolfsii, yer fistigi.

molecular variability among the isolates of sclerotium...

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