SUITABILITY OF INTERGENIC SPACER OR INTERNAL TRANSCRIBED SPACER MICROSATELLITE-PRIMED PCR FOR THE IDENTIFICATION OF RHIZOCTONIA SOLANI AND SOME PHYTOF

suitability of intergenic spacer or internal transcribed spacer microsatellite primed pcr for the identification of rhizoctonia solani and some phytofungi k a abd elsalam1 3 j r guo2 m a moslem1 a h bahkali1 and j a verreet2 1botany and microbiology department college of science king saud university po box 2455 riyadh 1145 saudi arabia 2institute of phytopathology university of kiel kiel germany accepted for publication january 5 2008 abstract the intergenic spacer igs region or internal transcribed spacer its region were used in pair combinations with microsatellite primed polymerase chain reaction mp pcr primers to establish whether additional polymor phisms can be yielded a total of 24 rhizoctonia solani isolates representing 13 anstomosis groups and 9 different fungal species isolate were recovered fromdifferentareasandhosts fortydifferentprimercombinationsweretested for their ability to provide discrete bands and individual isolates readily interpretable and reproducible igs its mp pcr profi les both approaches produced highly reproducible and complex genomic fi ngerprints with frag ments ranging in size from 100 to 2 000 bp igs mp pcr and 50 to 2 000 bp its mp pcr mp pcr markers yielded more bands than igs its mp pcr because of their higher redundancy in the fungal genome the number of fragments generated by both techniques varied according to the fungal species and also with the primer combination used each primer used could differen tiate all of the fungal isolates examined in this study the profi les generated were identical and reproducible between repeated pcr experiments 3corresponding author tel 00966566581006 fax 0096014675834 email abdelsalamka journal of rapid methods guillemaut et al 2003 polymerase chain reaction pcr based techniques have also proved to be very helpful by providing important new taxonomic information on these fungi which may assist the development of species specifi c molecular diagnostic methods several pcr based markers are available varying in complexity reliability and information generating capacity the choice of the most suitable technique for each one s specifi c use will depend upon the material being studied and the nature of the ques tions being addressed the discriminatory power of some methods appears insuffi cient when these methods are tested alone these techniques include random amplifi ed polymorphic dna rapd amplifi ed fragment length polymorphisms aflps and microsatellites or simple sequence repeats ssrs le guen et al 2004 rubini et al 2004 steimel et al 2004 each technique has its own advantages and disadvantages for instance microsatellite markers are co dominant multi allelic highly polymorphic genetic markers requiring small amounts of dna for straightforward pcr and gel electrophoresis analysis ellegren 2004 mic rosatellites have found application in variability analyses in fungi descenzo and harrington 1994 such as ascochyta rabei geistlinger et al 1997a b fusarium oxysporum f sp ciceri races brave et al 2001 f oxysporum f sp radicis lycopersici phoma tracheiphila balmas et al 2005a b and rhizoctoni solani mwang ombe et al 2007 fungal rrna genes are tandemly repeated with each repeat encoding 18s small subunit 5 8s and 26s large subunit genes two other regions exist in each repeat the internal transcribed spacer its region and the intergenic spacer igs 384k a abd elsalam et al region ribosomal dna rdna has been widely utilized for sequence comparisons for phylogenetic analyses of a variety of fungal groups white et al 1990 henrion et al 1992 the its domain is a molecular marker for comparing different species of the same genus of fungi paule and lofquist 1996 the identifi cation of pcr based markers that can be used for analysis of genetic diversity in rhizoctonia has resulted in an improved understand ing of the basis of habitat and host specifi city of member species and subgroups of r solani such markers have provided useful tools for dis criminating isolates within an anastomosis group ag and those belonging to different ags emery et al 2003 balali et al 2007 at the moment nucleotide sequences of the igs or its region alone cannot clearly differentiate between the species and it is necessary to combine the analysis of this region with partial sequencing of ssrs the idea to combine igs or its and microsatellites technique evolved after consideration of the shortcomings and advantages of both methods the aim of this study was to test the fi tness of a method based on the selective amplifi cation of the its or the igs region of the rdna in combination with ssrs to characterize the diversity of r solaniags and some plant pathogenic fungi the approach involves two primer amplifi cation the principle and the procedure of igs its mp pcr technique are outlined in fig 1 to our knowledge igs its mp pcr molecular typing method for fungal species has never been reported before fig 1 diagrammatic presentation of the intergenic spacer and rdna internal transcribed spacer regions arrows indicate the annealing sites of the primers used for pcr amplifi cation also the potential pcr products for its mp pcr and igs mp pcr techniques are indicated 385identification of r solani and some phytofungi materials and methods fungal isolates and genomic dna preparation the fungal isolates used in this study are listed in table 1 twenty four r solani isolates representing 13 ags and 9 single representatives of different fungal species were used most isolates were maintained in dried infested wheat grain at 10c fungal mycelium was produced in 20 ml of table 1 fungal isolates included in the study fungal isolatesisolate code and ag group supplierhostlocation fusarium oxysporum f sp vasinfectum fovm r omarcottonegypt f solanifsm r omarcottonegypt f graminearumfgf schniederwheatgermany macrophomina phaseolinampm r omarcottonegypt chaetomium olivaceumch1m r omarcottonegypt c cochliodesch2m r omarcottonegypt septoria triticistf schniederwheatgermany stagonospora nodorumsnf schniederwheatgermany cercospora beticolacbf schniederwheatgermany rhizoctonia solani ag1 r groschsugar beetjapan rhizoctonia solani ag2 1 r groschpeajapan r solani ag2 2 r groschsugar beetgermany r solani ag2 iiib r groschpotatogermany r solani ag3 r groschpotatogermany r solani ag3 s1 current studypotatosaudi r solani ag3 s2 current studypotatosaudi r solani ag3 s3 current studypotatosaudi r solani ag3 s4 current studypotatosaudi r solani ag4 m r omarcottonegypt r solani ag4 s1 current studypotatosaudi r solani ag4 hgi r groschsugar beetjapan r solani ag4 hgii r groschsugar beetjapan r solani ag5 r groschsoybeanjapan r solani ag5 s1 current studypotatosaudi r solani ag6 r groschsoiljapan r solani ag7 r groschsoiljapan r solani ag8 r groschwheatjapan r solani ag9 r groschpotatojapan r solani ag9 r groschpotatojapan r solani ag10 r groschwheatjapan r solani ag12 r groschorchidjapan r solani ag12 r groschorchidjapan r solani ag13 tomaso petersoncottonusa 386k a abd elsalam et al liquid medium 24 g l of potato dextrose broth pdb difco laboratories detroit mi mycelium was harvested by fi ltration through mesh sieves 40 mm washed with sterile water and deposited onto whatman paper to remove excess water mycelium was ground to a fi ne powder in liquid nitrogen using a mortar and dnawas extracted by the method of le cam et al 2001 and abd elsalam et al 2007 preliminary screen of igs or its primers combined with microsatellite primers forty primer combinations were tested on four isolates ag2 ag4 fov and fs selected from different populations primers were synthesized and supplied by mwg biotech ebersberg germany the fi ngerprints generated were evaluated for overall clearness of the banding pattern and the number of polymorphic markers present was recorded igs or its microsatellite amplifi cation pcr amplifi cations were carried out in pcr reactions in a total volume of 25 ml containing 20 ng genomic dna 1x pcr buffer 20 mm tris hcl 10 mm nh4 2so4 10 mm kcl 2 mm mgso4 0 1 triton x 100 0 2 units taq polymerase roche germany 0 2 mm of each dntps and 10 pmol of microsatellite primer pcr amplifi cation was carried out according to the following temperature profi le an initial step of 2 min at 94c 40 cycles of 60 s at 94c 90 s at 52c and 2 min at 72c as well as a fi nal step of 7 min at 72c the reproducibility of dna patterns was tested by repeating the pcr ampli fi cation with each of the selected primers all isolates were tested at least twice with each procedure to ensure the reproducibility of the results the reproduc ibility of the igs mp pcr and its mp pcr fi ngerprinting methods was proved by comparing the banding patterns resulting from different extractions and amplifi cations of the same isolates microsatellite amplifi cation to evaluate the effi cacy of the igs its mp pcr assay with standard mp pcr assay pcr amplifi cation was performed in peltier thermal cycler 200 mj research inc waltham ma pcr was performed using 20 ng of template dna in 25 ml reaction volume using 0 2 unit of taq polymerase with reaction buffer containing buffer 20 mm tris hcl 10 mm nh4 2so4 10 mm kcl 2 mm mgso4 0 1 triton x 100 ph 8 8 0 2 units taq polymerase roche 0 2 mm of each dntps and 10 pmol of microsatellite primer the pcr program consisted of one cycle of 2 min at 94c followed by 387identification of r solani and some phytofungi 40 cycles of denaturation at 94c for 1 min annealing at 52c for 90 s and extension at 72c for 2 min the thermal profi le ended with a fi nal extension at 72c for 7 min gel electrophoresis and documentation electrophoresis of pcr amplifi ed products was performed in 1 5 agarose gels for 1 5 h at 7 0 v cm2 sambrook et al 2001 pcr products were stained with 0 5 g ml of ethidium bromide and visualized with 305 nm ultra violet light pictures of the gels were scanned using the gelsystem flexi biostep labor und systemtechnik gmbh jahnsdorf germany sizes of dna fragments amplifi ed by pcr were analyzed by direct comparison with the dna marker 100 bp ladder england biolab beverly ma fingerprints were considered highly similar when all visible bands obtained had the same migration distance for each isolate variations in intensity and shape of bands among isolates were not considered as differences while the presence or absence of one distinct band was considered as a difference results primer selection variousprimersweretestedfor effi cacyintheigs its mp pcr methods and the primers listed in table 2 were selected because they gave readily interpretable and reproducible results initial testing with a smaller set of isolates was performed with 40 primer combinations to deter mine their ability to generate dna fi ngerprints and to evaluate the repro ducibility of both methods in the current study 17 primer combinations were the most informative the most effective primers for identifi cation were lr12r and cag 5 since they detected the greatest number of poly morphic bands table 3 regardless of the tested primers the mp pcr technique gave rise to more complex dna fi ngerprints than the igs its mp pcr method reproducibility of igs its mp pcr assay molecular typing of r solani isolates from different ags by igs its mp pcr generated from one to fi ve major amplifi cation products ranging in size from 2 to 0 5 kb on the other side the mp pcr yielded a variable number of amplifi cation products ranging from 2 000 to 400 bp with various intensities fig 2 388k a abd elsalam et al with 26 selected primers the pcr fi ngerprints of different isolates belonging to the same species showed different degrees of intraspecies varia tion fig 3 the numbers of scorable bands were between one to nine depending on the primer combination and about 60 of the scored bands ranged polymorphic between different fungal isolates igs its mp pcr fi ngerprinting technique clearly distinguished all tested fungal species similar but not identical pcr fi ngerprints were produced table 2 list of mp its and igs primers used in experiments and their nucleotide sequences pcr method primerssequence 5 3 reference its pcrits4tcctccgcttattgatatgcwhite et al 1990 its5ggaagtaaaagtcgtaac aagg its fov2 rtggcattcatcatttactgthis study igs pcrcnl12ctgaacgcctctaagtcagappel and gordon 1995 cns1gagacaagcatatgactactg lr12rgaacgcctctaagtcagaatcc harrington and wingfi eld 1995 igs 5sa fcagagtcctatggccgtggat seq5s rcagatcagacgggatgcggt mp pcr cag 5cagcagcagcagcagalves et al 2007 gtg 5gtggtggtggtggtg gtgc 4gtgcgtgcgtgcgtgc m13gagggtggcggttct t3baggtcgcgggttcgaatcc table 3 the degree of informativity of combined primers used in this study igs or its primersmp primers t3b gtgc 4m13 gtg 5 cag 5 intra inter intra inter intra inter intra inter intra inter its primersits4 its5 its fov2 r igs primerscns1 lr12r igs 5sa f seq5s r cnl12 degree of primer informativity weak middle and high 389identification of r solani and some phytofungi by chaetomium spp isolates ch1 and ch2 with lr12r cag 5 and with its4 cag 5 fig 4 as can be seen from fig 6 in this experiment we attempted to use igs or its mp pcr analysis for direct detection and iden tifi cation of 13 ags in r solani igs its mp pcr provided a higher degree of discrimination by visual analysis within and between fungal species than microsatellite primed pcr mp pcr alone fig 2 representative fingerprints obtained for the isolates of r solani ags ag2 1 ag9 ag10 and ag12 respectively with igs mp pcr lr12r cag 5 or with its mp pcr its4 cag 5 and mp pcr cag m amplisize molecular ruler 100 bp ladder molecular weight bands are indicated on the left fig 3 representative fingerprints obtained for the isolates of r solani ags ag2 1 ag4 fusarium oxysporum f sp vasinfectum fov and f solani fs respectively with igs mp pcr igs 5s f cag 5 and igs 5s f m13 or with its mp pcr its fov2 r m13 and its fov2 r gtg 5 m amplisize molecular ruler 100 bp ladder molecular weight bands are indicated on the left 390k a abd elsalam et al fig 4 representative fingerprints obtained for different fungal species isolates with igs mp pcr lr12r cag 5 or with its mp pcr its4 cag 5 m amplisize molecular ruler 100 bp ladder molecular weight bands are indicated on the left fig 5 representative fingerprints obtained for the isolates of r solani ags ag1 ag2 1 ag3 ag4 ag5 ag6 ag7 ag8 ag9 ag10 ag12 and ag13 respectively with lr12r t3b primers m amplisize molecular ruler 100 bp ladder molecular weight bands are indicated on the left 391identification of r solani and some phytofungi inter and intra group genetic diversity in r solani because igs its mp pcr technique is very sensitive it is a method of choice for detecting band profi les generated by igs its mp pcr in the present study were unaffected by replicated pcrs amplifi cation fig 5 poly morphic markers in closely related populations that are otherwise diffi cult to discriminate by using other pcr based marker systems band profi les gener ated by igs its mp pcr in the present study were unaffected by replicated pcrs amplifi cation fig 5 we also used this technique successfully to amplify the genomic dna extracted from different subgroups of r solani for example ag1 ag2 1 ag2 2 ag2 iiib ag 4 ag4 hgi and ag4 hgii igs its mp pcr have easily and distinctly grouped r solani into subgroups of an ag by comparing specifi c pcr fi ngerprints of unidentifi ed r solani isolates with those ofag testers r solani isolates could be identifi ed to the groups and subgroups level even if they could not be identifi ed by routine morphological methods data not shown in all the cases very clear easily scorable and highly reproducible igs its mp pcr markers could be resolved depending on the type of primers combination used fig 6 reproducibility of pcr amplifications of dna extracted from rhizoctonia solani isolates by intergenic spacer microsatellite primer lr12r cag 5 genomic dna was amplified at 2 different times a first amplifi cation b second amplifi cation 392k a abd elsalam et al discussion dna based techniques provide markers suitable for molecular typing of a range of species of fungi these methods are commonly used as tools in fungal taxonomy allowing the discrimination of isolates from intrageneric to strain levels soll 2000 the retrotransposon microsatellite amplifi ed poly morphism technique has been proposed as a new marker system for genetic diversity assessment in magnaporthe grisea chadha and gopalakrishna 2005 the inter ssr pcr issr pcr has been used to study the genetic diversity of r solani both within and between the various ags and to examine the relationships of the ags to one another stodart 2002 stodart et al 2007 in the current research we have established a new method to amplify and detect simple microsatellite sequence related polymorphisms by com bining the concept of igs its microsatellite technique a total of 40 primer combinations were screened for polymorphisms in the pilot study from which 26 primers produced reproducible clear igs its mp pcr bands and were used in further analysis the size of amplifi ed fragments ranged from 50 to 2 000 bp all 26 primers revealed polymorphisms useful for classifying isolates particularly lr12r t3b primers that revealed distinct polymor phisms among fungal species isolates and between r solani isolates from different ags and subgroups the presented fi ndings suggest that igs its mp pcr fi ngerprints are specifi c enough for the rapid differentiation of dif ferent fungal species and r solani ags at the level of subgroups also igs its mp pcr revealed a higher level of polymorphism than mp pcr alone fungal species banding patterns were obtained with proposed tech nique with different degrees of intraspecifi c differentiation depending on the method microsatellite regions are highly mutable and thus are able to dif ferentiate between related taxa even at the level of individual isolates in a single species simple dna profi ling methods based on microsatellite variability provide possibilities to identify individual genotypes for