Post on 30-Oct-2020
1396
30-11
DNAAG-4Rhizoctonia solani
rDNA RFLP
*123
:24/2/95 :16/7/95
DNArRNA
.ITS
DNA
.s28s18
Rhizoctonia solani4)AG-4(28 .
DNAPCR .
ITS1ITS4DNA700 .
Taq IXba I, Hae III, Bam HI, Hind III, Sac I, Pst I, Nde I, Xho I, Hinc II, Hinf I, Eco
RI, .Xba I, Bam HI, Hind III, Sac I, Pst I, Nde I, Xho I .
Rhizoctonia solani AG4ITS-RFLPHinc II
AG4-HG IAG4-HG II.
:(ITS)Rhizoctonia solani
4 .
1- .2-.3- .*-:badpafarzaneh@yahoo.com
12DNAAG-4Rhizoctonia solani
Rhizoctonia11815
crocorumRhizoctonia)Menzies, 1970 .(Parmeter
Whitney1970 -
.
Rhizoctonia .
Rhizoctoni
.
)Anderson, 1982; Sneh et al., 1991 .(
.
.
(R. solani complex)(Moore, 1996) .
.
.DNA
Kuniniga (1996)»
« .R.solani
)Lübeck, 2004 .( -
AG 1AG 2AG 3AG 4)Marcus et al., 1986 .(
)AG 4 (
).(Banville, 1989; Pitt, 1964, Sneh et al., 1991Rhizoctonia solani2AG4)Rahimian, 1989 .(
)(AGsAG 1- AG 13AG-BI)Carling et al., 1994 .(
)(AG 1, AG 2, AG 3, AG 4, AG 6, AG 8, AG 9
)Ogoshi, 1987 .(
AG4AG6AG9DNA/DNAAG8
.
1 De Candol2 Anastomosis group
/ / /139613
)Lübeck,
2004.(DNA-DNA)b1984 a,1984 ()1988(
AG4AG6 .-
G4 HGIHGIIAG447 -30AG6 HGIAG6 GV66 -55
.(Kuninaga, 1996) AG41DNA
AG4-HG I AG4-HG II .AG4- HG I2PDADNA93-89 %
AG4- HG IIDNA100 -93 %
)Sneh et al., 1991.(
.
.
Rhizoctonia .
Rhizoctonia)Jabaji-Hare,
1996 ()Cubeta et al., 1996; González et al., 2001, 2002 (.Jabaji-Hare
(1996)345
678
.
RhizoctoniaR. solani .
.DNA
RNA
)Bruns et al., 1991 .()rDNA (DNA
)White et al., 1990( .18s
)SSU(95.8s28s)LSU(10 .
1 Autotroph2 Potato Dextrose Agar3 soluble protein patterns4 isoenzymes5 pectic zymograms6 fatty acids profiles7 lectins8 Monoclonal and polyclonal antibody9 Small subunit10 Large subunit
14DNAAG-4Rhizoctonia solani
18s28s1)ITS1ITS2 (5.8s)Cubeta et al.,
1996.(Rhizoctonia
rDNA-ITS.
5.8s rDNAITS rDNA
.ITS96 %
100 -66 %
96 -55 %)Lübeck, 2004( .2)2010 (rDNA-ITS114
R. solani AG4
.Rhizoctonia
.3
.
)McDonald and Rovira, 1985( .
R. solani .
.
)R. solani
AG4 (rDNA RFLP.
DNA
18R. solani AG4
10 .
1 Internal transcribed spacer2 Kiliço lu and Özkoç
AG-BIAG6AG3 AG2AG8
.
/ / /139615
1 .
)Parmeter et al., 1969.(
1 -Rhizoctonia solanirDNA
AG 4/HG II bean1
AG 4/HG II bean2
AG 4/HG II bean3
AG 4/HG II bean6
AG 4/HG II bean9
AG 4/HG II bean11
AG 4/HG II bean12
AG 4/HG II bean13
)( AG 4/HG II white-bean
AG 4/HG II tomato
AG 4/HG II alfalfa1
AG 4/HG II alfalfa2
AG 4/HG II alfalfa4
AG 4/HG II potato4
AG 4/ HG I potato2-6
AG 4/HG II sunflower
AG 4/HG II radish
AG 4/HG II pepper
- AG 4/HG I 137
- AG 4/HG II 168
- AG 4/HG I 243
- AG 4/HG I 260
AG 4/HG II 134H
- AG 4/HG II CB2
AG 4/HG II LR7
AG 4/HG II E-K-R-14
AG 4/HG II KH-3-S-6
)( AG 4/HG II M-KH-S-3
16DNAAG-4Rhizoctonia solani
PDA25
.
250100) - ( .
25
.
.
DNA70 -.
DNA
3/0
)ml5/1 (150 ml Tris Hcl(1M) PH=8.5, 25 ml
Nacl(5M), 25 ml EDTA(0.5M), 1.25gr SDS, add dd H2O to 250 ml) .2
.
.)lµ750 (5/1
)lµ750 (
. .6014000
.
-)1 :24 (
.1014000 .
54/0 .
DNA1020 --
14000 .700
1 SDS buffer
/ / /139617
70214000
.50100)1ml Tris Hcl(1M) PH =8,TE 200µl Na2-EDTA pH=8
add dd H2O to 100 ml( .3037
.20 - .DNA
.
)PCR(
PCR25
2 .)ITS15’-TCCGTAGGTGAACCTGCG
G-3’ ()ITS45’-TCCTCCGCTTATTGATATGC-3’(
.PCR94
39430
60307250
727.
2 -¬¬PCR25
PCRX10µl5/2X1MgCl2mM50µl5/1mM3
dNTP mixmM10µl5/0mM2/0
pmol10µl3pmol2/1
pmol10µl3pmol2/1
DNAng/µl20-40µl3ng6/3Taq DNA polymerase5 u/µlµl3/0u5/1
-µl25-
PCR
PCR .
TBE
90 .bp100
18DNAAG-4Rhizoctonia solani
. )ml/µg5/0 (20
Gel Document1UV.
PCR
Eco RI, Hinf I, Xho I, Hae III, Hinc II, Taq I, Bam HI, Hind III, Sac I Pst I,
Nde I, Xba IDNA .4
PCR42X10
)1u / µl ( .C°37
)Taq IC°65 .(C°20 -
.
µl2%2
752 . bp100
..
137CB2
JX965000JX965001Gene BankGene Bank, NCBI
)3(.MEGA42
.
3 -¬GenBank¬
GenBank
697 bpHQ629873.1AG4-HGII ND13678 bpHQ629872.1AG4-HGII ND12684 bpHQ629868.1AG4-HGII ND8684 bpHQ629866.1AG4-HGII ND6679 bpJX965001AG4-HGII FB1(CB2)656 bpJX965000AG4-HGI FB2(137)654 bpAY154307.1AG4-HGI665 bpJQ343830.1AG4-HG-1
1 Ultra Violet2 Nearest Neibor
/ / /139619
)PCR(
DNA¬ITS1ITS4
DNA700)1.(
a1-PCRITS rDNA .19
alfalfa1bean3bean2bean1alfalfa4alfalfa2bean13bean6alfalfa2M)100 bp
DNA Ladder (.
b1-. PCR M)100 bp DNA Ladder(1
1372.CB2
20DNAAG-4Rhizoctonia solani
DNA
PCREcoRIHincIITaqIHinfIHaeIII
700BamHIHindIIIXhoISacIPstI
NdeIXbaI .
:
Taq I
DNATaq IPCR .
.
)2.(
2- PCRTaqI%2
.14243260M-KH-S-3137Mbp100
.
EcoRI
PCR)3.(
/ / /139621
a3-PCREcoRI%2
.111243260M-KH-s-3137134 HCB2E-K-R-14168
LR7KH-3-S-6 bean12M)100 bp DNA Ladder(.
b3-111potato2-6bean3potato4radishbean9alfalfa1bean11
bean2bean1alfalfa4alfalfa2 .
HaeIII
PCRHae III
)4.(
22DNAAG-4Rhizoctonia solani
a4- PCRHaeIII%2
.111243260M-KH-s-3137134 HCB2 E-K-R-14
168LR710KH-3-s-6 bean12M)100 bp DNA Ladder (.
b4-111potato2-6bean3potato4radishbean9alfalfa1
bean11bean2bean1alfalfa4alfalfa2M)100 bp DNA Ladder (.
c4-17alfalfa4alfalfa2bean13bean6peppertomatowhite
beanM)100 (.
/ / /139623
HincII
PCRHincIIAG4 HGI .
425266 .AG4 HGII)5.(
a5- PCRHincII%2
.14137CB2260 243M )100
bp DNA Ladder (.
b5-110bean12potato2-6bean3potato4radishbean9
alfalfa1bean11bean2KH-3-S-6M)100 bp DNA Ladder (.
HinfI
6 .
24DNAAG-4Rhizoctonia solani
6-PCRHinfI%2
.111) (CB2243260M-KH-S-3137E-K-
R-141688LR7KH-3-S-6bean12 potato2-6M)100 bp DNA Ladder (.
NdeI, XbaI, BamHI, XhoI, HindIII, SacI, PstI,.
R. solani AG4¬¬ rDNA ITS
¬47 .¬R. solani
AG4¬rDNA ITS-RFLPSimple matching (SM)8
.
7-R. solani AG4¬¬ rDNA ITS
¬4 .137 CB2Hinc II
AG4 HGIAG4 HGIIGene Bank, NCBI
.
/ / /139625
8UPGMA¬R. solani AG4¬rDNA ITS-
RFLPSimple matching (SM)NTSYS .
AG4 HGIAG4 HGII.
ITSR. solani AG4
)Kiliço lu and Özkoç, 2010( .)1996(1ITS
AG4 )HG I, HG II,HG III ()Kuninaga, 1997(.
AG 423
)Kuninaga, 1997 .( R. solani AG4
)Kiliço lu and Özkoç, 2010( .
)42012.(
4
.
1 Boysen2 Ecological habitat3 Virulence4 Haratian
AG4 HG II
AG4 HG I
26DNAAG-4Rhizoctonia solani
)Meinhardt et al., 2002; Balali and Kowsari, 2004; Kiliço lu and
Özkoç, 2010 .(Kiliço lu and Özkoç)2010(
R. solani AG4AG4HG I .
2002AG4HG I .
R. solani AG41997
AG4 HG II .1)2008 (
AG4AG4 HGII .
AG4H -
AG4)(Puhalla and Carter, 1976 .
PDA)(HG I (HG II))Kiliço lu and
Özkoç, 2010( .AG4)HG I, HG II (
C250 -30R. solani AG4
)Kiliço lu and Özkoç, 2010( .AG4
)Lübeck, 2004.(
)Tylor et al., 1999 .(
.
)Rosewich et al., 1999( .
R. solani AG4
)Anderson, 1982( .RFLPITSDNA
)Guillemaut et al., 2003.(Hinc II
AG4)Guillemaut et al., 2003( .Mse I
AG4HG IAG4HG IIRFLPAG4HG I
(Meinhardt et al., 2002) .Ava IIAG4
)Kiliço lu and Özkoç, 2010( .rDNA-ITS28R. solani AG4
PCR-RFLP .ITS1
ITS4ITS1-5.8S-ITS2700 .
Taq I, Xba I, Hae III, Bam HI, Hind III, Sac I, Pst I, Nde I, Xho I, Hinc II,
Hinf I, Eco RITaq I, Hinc II, Hinf I, Hae III, Eco R I-
1 Pannecoucque
/ / /139627
AG4Hinc IIAG4 HGIAG4 HGII
.Hinc IIRhizoctonia solani AG4 HGI
266425AG4 HG II .
Hinc II12003.Xba I, Bam HI,
Hind III, Sac I, Pst I, Nde I, Xho IAG4 .
.
, Taq IHinf I, Hae IIIEco RIR. solani AG4)
(Hinc II .
Mse IAva II
.
.CB2137
. .
.
.Hinf IHae III
)Schneider et al., 1997 (
. .2
.ITS
)Schneider et al., 1997 .()2012(ITS-
rDNA
-1 (
.
1 Guillemaut2 Bridging phenomenon
28DNAAG-4Rhizoctonia solani
.rDNA ITS
.
.
References1. Anderson NA. 1982. The genetics and pathology of Rhizoctonia solani. Annual
Review of Phytopathology 20: 329–347.2. Balali G and Kowsari M, 2004. Pectic zymogram variation and pathogenicity of
Rhizoctonia solani AG-4 to bean (Phaseolus vulgaris) isolates in Isfahan, Iran.Mycopathologia 158: 377–384.
3. Boysen M, Borja M, Del Moral C, Salazar O and Rubio V. 1996. Identification atstrain level of Rhizoctonia solani AG4 isolates by direct sequence of asymmetricPCR products of the ITS regions. Current Genetics 29: 174–181.
4. Bruns TD, White TJ and Taylor JW. 1991. Fungal molecular systematics. AnnualReview of Ecology, Evolution and Systematics 22: 525–564.
5. Cubeta MA, Vilgalys R and González D. 1996. Molecular analysis of ribosomalRNA genes in Rhizoctonia fungi. pp. 81–86, In B Sneh, S Jabaji-Hare, S Neate andG Dijst (eds). Rhizoctonia Species: Taxonomy, Molecular Biology, Ecology,Pathology and Disease Control. Dordrecht: Kluwer Academic Publishers.
6. González D, Carling DE, Kuninaga S, Vilgalys R and Cubeta MA. 2001.Ribosomal DNA systematics of Ceratobasidium and Thanatephorus withRhizoctonia anamorphs. Mycologia 93: 1138–1150.
7. González D, Cubeta MA and Vilgalys R. 2006. Phylogenetic utility of indels withinribosomal DNA and -tubulin sequences from fungi in the Rhizoctonia solanispecies complex. Molecular Phylogenetics and Evolution 40: 459–470.
8. González V, Salazar O, Julián MC, Acero J, Portal MA, Muñoz R, López-CórcolesH, Gómez-Acebo E, López-Fuster P and Rubio V. 2002. Ceratobasidiumalbasitensis, a new Rhizoctonia-like fungus isolated in Spain. Persoonia 17: 601–614.
9. Guillemaut C, Edel-Herman V, Comporota P, Alabouvette C, Richard-Molard Mand Steinberg C. 2003. Typing of anastomosis groups of Rhizoctonia solani byrestriction analysis of ribosomal DNA. Canadian Journal of Microbiology 49: 556–568.
10. Haratian M. 2012. Evaluation genetic structure populations Rhizoctonia solani AG4in Iran [PhD]. [Tehran]: Tarbiat Modares University.
/ / /139629
11. Jabaji-Hare S. 1996. Biochemical methods. pp. 65–71, In B Sneh, S Jabaji-Hare, SNeate and G Dijst (eds). Rhizoctonia Species: Taxonomy, Molecular Biology,Ecology, Pathology and Disease Control. Dordrecht: Kluwer Academic Publishers.
12. Kiliço lu Mc and Özkoç I. 2010. Molecular characterization of Rhizoctonia solaniAG4 using PCR-RFLP of the rDNA-ITS region. Turkish. Journal of Biology 34:261–269.
13. Kuninaga S 1996. DNA base sequence relatedness. pp. 74–78, In RhizoctoniaSpecies: Taxonomy, Molecular Biology, Ecology, Pathology and Disease Control.(B Sneh, S Jabaji-Hare, S Neate and G Dijst (eds). Dordrecht: Kluwer AcademicPublishers.
14. Kuninaga S and Yokosawa R. 1984a. DNA base sequence homology inRhizoctonia solani Ktihn. IV. Genetic relatedness within AG4. Annals of thePhytopathological society of Japan 50: 322–330.
15. Kuninaga S and Yokosawa R. 1984b. DNA base sequence homology inRhizoctonia solani Ktihn. V. Genetic relatedness within AG6. Annals of thePhytopathological society of Japan 50: 346–352.
16. Lübeck M. 2004. Molecular characterization of Rhizoctonia solani. AppliedMycology and Biotechnology 4: 205–224.
17. McDonald HJ and Rovira AD. 1985. Development of inoculation technique forRhizoctonia solani and its application to screening cereal cultivars for resistance.pp. 174–176, In CA Parker, AD Rovira, KJ Moore, PTW Wong and JF Kollmorgen(eds.). Ecology and Management of Soilborne Plant Pathogens. St. Paul (MN):American Phytopathological Society.
18. Meinhardt LW, Wulff NA, Bellato CM and Tsai SM. 2002. Genetic analyses ofRhizoctonia solani isolates from Phaseolus vulgaris grown in the Atlanticrainforest region of Sao Paulo, Brazil. Fitopatologia Brasileira 27: 259–267.
19. Menzies JD. 1970 Introduction: The first century of Rhizoctonia solani. pp. 3–5 InParmeter JR (ed.). Rhizoctonia solani: Biology and Pathology. Berkeley (CA):University of California Press.
20. Moore RT. 1996. The dolipore/parenthesome septum in modern taxonomy. pp. 13–35, In B Sneh, S Jabaji-Hare, S Neate and G Dijst (eds). Rhizoctonia Species:Taxonomy, Molecular Biology, Ecology, Pathology and Disease Control.Dordrecht: Kluwer Academic Publishers.
21. Pannecoucque J, Van Beneden S and Hofte M. 2008. Characterization andpathogenicity of Rhizoctonia isolates associated with cauliflower in Belgium. PlantPathology 57: 737–746.
22. Parmeter JR and Whitney HS. 1970. Taxonomy and nomenclature of the perfectstate. pp. 7–19 In Parmeter JR (ed.). Rhizoctonia solani: Biology and Pathology.Berkeley (CA): University of California Press.
23. Parmeter JR, Sherwood, RT and Platt WD. 1969. Anastomosis grouping amongisolates of Thanatephorus cucumeris. Phytopathology 59: 1270–1278.
24. Puhalla J and Carter W. 1976. The role of the H locus in heterokaryosis inRhizoctonia solani [from soil of a cotton field, fungal diseases]. Phytopathology 66:1348–1353.
25. Rahimian H. 1986. Rhizoctonia soil rot of tomato. Paper presented at: 8th IranianPlant Protection Congress; 30 August-4 September; Isfahan, Iran.
30DNAAG-4Rhizoctonia solani
26. Rosewich UL, Pettway RE, McDonald BA and Kistler HC. 1999. High levels ofgene flow and heterozygote excess characterize Rhizoctonia solani AG 1-1A(Thanatephorus cucumeris) from Texas. Fungal Genetics Biology 28: 148–159.
27. Schneider J, Salazar O, Rubio V and Keijer J. 1997. Identification of Rhizoctoniasolani associated with field-grown tulips using ITS rDNA polymorphism and pecticzymograms. European Journal of Plant Pathology 103: 607–622.
28. Sneh B, Burpee I and Ogoshi A (eds). 1991. Identification of Rhizoctonia species.St. Paul (Mn): American Phytopathology Society Press.
29. Taylor JW, Jacobsen DJ and Fisher MC. 1999. The evolution of asexual fungi:reproduction, speciation and classification. Annual Review Phytopathology 37:197–246.
30. Vilgalys R and Cubeta M. 1994. Molecular systematics and population biology ofRhizoctonia. Annual Review of Phytopathology 32: 135–155.
31. Vilgalys R and Gonzalez D. 1990. Ribosomal DNA restriction fragment lengthpolymorphisms in Rhizoctonia solani. Phytopathology 80: 151–158.
32. Vilgalys R. 1988. Genetic relatedness among anastomosis groups in Rhizoctonia asmeasured by DNA/DNA hybridization. Phytopathology 78: 698–702.
33. Visscher H, Sephton MA and Looy CV. 2011. Fungal virulence at the time of theend-Permian biosphere crisis? Geology 39: 883–886.
34. White TJ, Burns T, Lee S and Talor J. 1990. Amplification and direct sequencing offungal ribosomal RNA genes for phylogenetics. pp 315–322, In MA Innis (ed).PCR Protocols: A Guide to Methods and Applications. San Diego (CA): AcademicPress.
Research in Plant Pathology/Vol. 5/No. 1/Spring and Summer 2017 2
Study on DNA polymorphism in populations of Rhizoctonia solanianastomosis group 4 using rDNA RFLP
F. Badpa*1, G.R. Balali 2, B. Sharifnabi3
AbstractRibosomal DNA (rDNA) sequences have been widely used to study the phylogenetic
relationships in different fungi. Fungal nuclear rRNA genes are arranged as tandem repeatswith several hundred copies per genome. These spacer regions are considerably more variablethan the subunit sequences and have been widely used in studies on the relationships amongspecies within a single genus or among intraspecific populations. To evaluate thepolymorphism between 18S and 28S genes, 28 isolates of Rhizoctonia solani anastomosisgroup 4 were examined. Genomic DNA was extracted from the isolates and prepared for PCRreaction. The amplification was performed using internal transcribed spacer (ITS) ITS1 andITS4 primers. A DNA fragment of 700 bp in size was detected in PCR products of all testedisolates. To assess existence of any further polymorphism in the ITS region, the PCR productswere digested with restriction endonucleases. Although there were restriction sites for XbaI,HaeIII, BamHI, HindIII, SacI, PstI, and TaqI endonucleases, there were no restriction sites forNdeI, XhoI, HincII, HinfI, EcoRI and XhoI endonucleases. The endonuclease HincIIrecognized a restriction site on PCR products that discriminated the isolates belonging toAG4-HGII form isolates of AG4-HGI. Based on the results, it has been concluded that AG-4isolates of Rhizoctonia solani were heterogenic.
Keywords: Genetic diversity, ITS-rDNA, PCR, Rhizoctonia solani AG4.
1 - Former MSc student, Department of Biology, Isfahan University, Isfahan, Iran.2- Associate professor, Department of Biology, Isfahan University, Isfahan, Iran.3- Professor, Department of Plant Protection, College of Agriculture, Isfahan University of Technology,
Isfahan, Iran.*Corresponding author: badpafarzaneh@yahoo.com