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Internal transcribed spacer 2 amplicon as a molecular marker for identification of Peronospora parasitica (crucifer downy mildew)

2004; Oxford University Press; Volume: 96; Issue: 3 Linguagem: Inglês

10.1111/j.1365-2672.2004.02193.x

1365-2672

Sandra Casimiro, Maria M. Moura, Líbia Zé‐Zé, Rogério Tenreiro, A. Monteiro,

Plant-Microbe Interactions and Immunity

Journal of Applied MicrobiologyVolume 96, Issue 3 p. 579-587 Free Access Internal transcribed spacer 2 amplicon as a molecular marker for identification of Peronospora parasitica (crucifer downy mildew) S. Casimiro, S. Casimiro Instituto Superior de Agronomia, Universidade Técnica de Lisboa, Tapada da Ajuda, Lisboa, Portugal Departamento de Biologia Vegetal and Centro de Genética e Biologia Molecular, Faculdade de Ciências da Universidade de Lisboa, Campo Grande, Lisboa, PortugalSearch for more papers by this authorM. Moura, M. Moura Instituto Superior de Agronomia, Universidade Técnica de Lisboa, Tapada da Ajuda, Lisboa, Portugal Departamento de Biologia Vegetal and Centro de Genética e Biologia Molecular, Faculdade de Ciências da Universidade de Lisboa, Campo Grande, Lisboa, PortugalSearch for more papers by this authorL. Zé-Zé, L. Zé-Zé Departamento de Biologia Vegetal and Centro de Genética e Biologia Molecular, Faculdade de Ciências da Universidade de Lisboa, Campo Grande, Lisboa, PortugalSearch for more papers by this authorR. Tenreiro, R. Tenreiro Departamento de Biologia Vegetal and Centro de Genética e Biologia Molecular, Faculdade de Ciências da Universidade de Lisboa, Campo Grande, Lisboa, PortugalSearch for more papers by this authorA.A. Monteiro, A.A. Monteiro Instituto Superior de Agronomia, Universidade Técnica de Lisboa, Tapada da Ajuda, Lisboa, PortugalSearch for more papers by this author S. Casimiro, S. Casimiro Instituto Superior de Agronomia, Universidade Técnica de Lisboa, Tapada da Ajuda, Lisboa, Portugal Departamento de Biologia Vegetal and Centro de Genética e Biologia Molecular, Faculdade de Ciências da Universidade de Lisboa, Campo Grande, Lisboa, PortugalSearch for more papers by this authorM. Moura, M. Moura Instituto Superior de Agronomia, Universidade Técnica de Lisboa, Tapada da Ajuda, Lisboa, Portugal Departamento de Biologia Vegetal and Centro de Genética e Biologia Molecular, Faculdade de Ciências da Universidade de Lisboa, Campo Grande, Lisboa, PortugalSearch for more papers by this authorL. Zé-Zé, L. Zé-Zé Departamento de Biologia Vegetal and Centro de Genética e Biologia Molecular, Faculdade de Ciências da Universidade de Lisboa, Campo Grande, Lisboa, PortugalSearch for more papers by this authorR. Tenreiro, R. Tenreiro Departamento de Biologia Vegetal and Centro de Genética e Biologia Molecular, Faculdade de Ciências da Universidade de Lisboa, Campo Grande, Lisboa, PortugalSearch for more papers by this authorA.A. Monteiro, A.A. Monteiro Instituto Superior de Agronomia, Universidade Técnica de Lisboa, Tapada da Ajuda, Lisboa, PortugalSearch for more papers by this author First published: 11 February 2004 https://doi.org/10.1111/j.1365-2672.2004.02193.xCitations: 12 Rogério Tenreiro, Departamento de Biologia Vegetal, Faculdade de Ciências da Universidade de Lisboa, R. Ernesto de Vasconcelos, Edificio C2, Piso 4, Campo Grande, 1749-016 Lisboa, Portugal (e-mail: rptenreiro@fc.ul.pt). AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Abstract Aims: The purpose of the study was to characterize the internal transcribed spacer (ITS) regions of Peronospora parasitica (crucifer downy mildew) in order to evaluate their potential as molecular markers for pathogen identification. Methods and Results: PCR amplification of ribosomal RNA gene block (rDNA) spacers (ITS1 and ITS2) performed in 44 P. parasitica isolates from different Brassica oleracea cultivars and distinct geographic origins, revealed no length polymorphisms. ITS restriction analysis with three endonucleases, confirmed by sequencing, showed no fragment length polymorphisms among isolates. Furthermore, ITS amplification with DNA isolated from infected host tissues also allowed the detection of the fungus in incompatible interactions. The combination of the universal ITS4 and ITS5 primers, for amplification of full ITS, with a new specific forward internal primer for ITS2 (PpITS2F), originates a P. parasitica specific amplicon, suitable for diagnosis. Conclusions: As ITS2 regions of P. parasitica, B. oleracea, other B. oleracea fungal pathogens and other Peronospora species are clearly distinct, a fast and reliable molecular identification method based on multiplex PCR amplification of full ITS and P. parasitica ITS2 is proposed for the diagnosis of crucifer downy mildew. Significance and Impact of the Study: The method can be applied to diagnose the disease in the absence of fungal reproductive structures, thus being useful to detect nonsporulating interactions, early stages of infection on seedlings, and infected young leaves packed in sealed plastic bags. Screening of seed stocks in sanitary control is also a major application of this diagnostic method. Introduction Peronospora parasitica is an exclusively biotrophic oomycete responsible for crucifer downy mildew, one of the most important diseases of brassica crops worldwide (Channon 1981). Brassicas are economically very important all over the world, Brassica oleracea being the most cultivated species in the Western Hemisphere, whereas B. campestris predominates Asia (Monteiro and Lunn 1999). Between 1989 and 1991, world brassica crops represented 16 69 000 ha, increasing to 19 83 000 ha in 1999 (FAO 1999). Although field plants are also severely affected, crucifer downy mildew damages are particularly important in the nursery, when the infection may kill the seedlings, retard their development or cause lack of uniformity and quality (Coelho et al. 1999). As the identification of P. parasitica is based on morphological characteristics of conidia and conidiophores (Dickinson and Greenhalgh 1977), downy mildew diagnosis on infected seedlings is delayed until sporulation occurs. The ineffectiveness of conventional identification methods is also a major concern in incompatible interactions with resistant hosts, where no pathogen reproduction occurs but tissue damage is observed (Leckie et al. 1999), and in the detection of infected cabbage seeds, embedding intact or germinating oospores, mycelium and conidia (Kluczewski and Lucas 1983; Badul and Achar 1998). Conventional methods also cannot detect infected young brassica leaves packed in sealed plastic bags, which may develop sporulating lesions before reaching the consumer. Therefore, new sensitive and reliable diagnostic methods are needed to reduce seedling losses, detect pathogen reservoirs and perform an efficient sanitary control. In fungal genomes, the highly conserved rRNA genes are separated by two less conserved internal transcribed regions, the internal transcribed spacers 1 and 2 (ITS1 and ITS2), which are therefore suitable for polymorphism studies among species or even at infra-specific level (Duncan et al. 1998; Mills et al. 1998). Amplified ribosomal DNA restriction analysis (ARDRA), using PCR primers based on conserved regions of the rRNA genes (White et al. 1990), followed by restriction with frequently cutting endonucleases, allows the easy assessment of sequence differences in ITS regions without length polymorphisms (Buscot et al. 1996; Lanfranco et al. 1998). Although fungal ITS1 has been shown to be more polymorphic at sequence level than ITS2 (Duncan et al. 1998), analysis of P. parasitica ITS1 sequences showed no differences among isolates collected from the same host species (B. oleracea and A. thaliana) and only 85% similarity between isolates from different hosts (Rehmany et al. 2000). In fact, host range must be associated with genetic differences of the isolates, classified as belonging to the same species, and these data point to the potential of ITS regions as molecular markers, both at species and forma specialis levels. There are, however, no available data on ITS2 variability and taxonomic relevance. The objective of this work was to characterize the ITS regions of P. parasitica isolates, from different B. oleracea crops and distinct geographic origins, in order to evaluate their potential as molecular markers for identification purposes. Materials and methods P. parasitica isolates and conidia isolation Forty-four P. parasitica isolates, collected from B. oleracea plants, were grown on seedlings of B. oleracea hosts '1' or '2' (Table 1). For each isolate, ca 50 1-week-old seedlings were inoculated with two droplets of a conidia suspension (5 × 104conidia ml−1) per cotyledon and maintained in the dark, at 16°C for 24 h. Then the inoculated seedlings were transferred to a growth room and maintained at 20 ± 1°C, under a 20-h photoperiod. After 6 days of incubation, the seedlings were transferred to a dark room for 24 h, to induce sporulation. Cotyledons with sporulation were harvested and shaken in 50 ml of sterile distilled water to dislodge conidia. The conidial suspension was gauze filtered and centrifuged at 2600 g for 3 min. The pellet was re-suspended in 7·5 ml of sterile distilled water, aliquoted in 1·5-ml fractions and stored at −20°C until use. Table 1. Peronospora parasitica isolates used in this study Isolate Original host crop type (Brassica oleracea) Geographical origin Lab host* P501, P522 Tronchuda cabbage Batalha, Portugal 1 P502 Tronchuda cabbage Póvoa do Varzim, Portugal 1 P503 Kale cabbage Oliveira do Hospital, Portugal 2 P504 Tronchuda cabbage Castelo Branco, Portugal 2 P505 Tronchuda cabbage Évora, Portugal 1 P506 Unknown Odemira, Portugal 2 P507 Unknown Batalha, Portugal 2 P508, P515 Tronchuda cabbage Condeixa, Portugal 2 P509 Tronchuda cabbage Vila Real, Portugal 2 P510 Tronchuda cabbage Faro, Portugal 2 P511 Tronchuda cabbage Lourinhã, Portugal 2 P512†, P516† Cauliflower, Broccoli, Tronchuda cabbage Batalha, Portugal 2 P513 Tronchuda cabbage Pombal, Portugal 2 P514 Galega kale Condeixa, Portugal 2 P517† Cauliflower, Broccoli and Tronchuda cabbage 'Murciana' Batalha, Portugal 1 P518† Tronchuda cabbage and Broccoli Batalha, Portugal 2 P519 Cauliflower Batalha, Portugal 2 P520, P521, P527 Broccoli Batalha, Portugal 1 P523† Broccoli, Tronchuda cabbage Batalha, Portugal 1 P524 Tronchuda cabbage 'Murciana' Batalha, Portugal 1 P525† Broccoli, Tronchuda cabbage 'Murciana' Batalha, Portugal 1 P526 Broccoli Batalha, Portugal 2 P528 Tronchuda cabbage 'Murciana' Batalha, Portugal 2 P529 Unknown German 1 P531 Tronchuda cabbage Ameal, Coimbra, Portugal 2 P532 Tronchuda cabbage 'Algarvia' Ameal, Coimbra, Portugal 2 P533, P534 Tronchuda cabbage Casconha, Coimbra, Portugal 2 P535 cabbage 'Coração-de-boi' Casconha, Coimbra, Portugal 2 P536 Broccoli Coimbra, Portugal 1 P537, P538 Broccoli Coimbra, Portugal 2 P539 Broccoli Eira Pedrinha, Coimbra, Portugal 2 FP06, FP09 Unknown France 1 P005a, P005b, P006a, P006b Cauliflower HRI-England 1 *Host 1 – CrGC3·1, short-cycle B. oleracea, Crucifer Genetics Cooperative, University of Wisconsin, Madison, WI, USA; Host 2 – Cabbage 'Coração-de-boi' (B. oleracea). †Mixture of isolates collected from different hosts. B. oleracea fungal pathogens' selection and growth Ten isolates of B. oleracea fungal pathogenic species or related species of the same genera were selected, namely Fusarium culmorum, Trichoderma sp., Alternaria sp., Phoma sp., Phytophtora cinnamomi, Sordaria sp., from our collection, and from CECT (Collecion Espanola de Cultivos Tipo) F. oxysporum (CECT 2154), Scerotinia sclerotiorum (CECT 2882), Mycosphaerella tassiana (CECT 2665) and Diaporthe phaseolorum (CECT 2022). All fungi were grown on potato dextrose agar medium, with exception of Ph. cinnamomi which was grown on corn meal agar, at 28°C for 7 days. DNA isolation DNA was isolated using an adaptation of the Ferreira and Grattapaglia (1995) method. An aliquot of each P. parasitica conidial suspension was centrifuged at 6400 g for 3 min. The pellet, or 100 mg of each fungal mycelium (obtained by colony scraping), was macerated with 200 μl of glass beads (425–600 microns), and 500 μl of extraction buffer (CTAB 2%, 1·4 mol l−1 NaCl, 0·02 mol l−1 EDTA, 0·01 mol l−1 Tris-HCl pH 8·0, 1% PVP, 0·2%β-mercaptoethanol, 0·1% Proteinase K), at 65°C, were added. The suspension was incubated at 65°C for 45 min, with mixing by inversion each 15 min. After cooling to room temperature, 500 μl of chloroform : isoamyl alcohol (24 : 1) were added, the tube was mixed by inversion and centrifuged at 16 700 g for 10 min. The upper aqueous phase was collected and the DNA was precipitated with 600 μl of isopropanol (−20°C) for 1 h at −70°C. After a 10-min centrifugation at 16 700 g, the pellet was washed with 500 μl of washing buffer (ethanol 70%, 0·15 mol l−1 NaCl) and centrifuged at 16 700 g for 5 min. The pellet was re-suspended in 25 μl of TE (0·01 mol l−1 Tris-HCl pH 8·0, 0·001 mol l−1 EDTA) and stored at 4°C until utilization. After maceration with liquid nitrogen and using the method above, DNA was extracted from 100 mg of short-cycle B. oleracea CrGC3·1 and cabbage 'Coração-de-boi' seedling tissue, which were not infected with P. parasitica, and from 100 mg of Tronchuda cabbage 'Algarvia' seedling tissue, either infected with the isolate P501 or uninfected. ITS amplification To amplify ITS1, ITS2 and full ITS regions, either from the fungus or the hosts, the following primers were used (White et al. 1990): ITS2 and ITS5 to amplify ITS1 region; ITS3 and ITS4 to amplify ITS2 region; and ITS4 and ITS5 to amplify full ITS. Each reaction mixture contained 2 μl DNA, PCR buffer 1X (GibcoBRL, Paisley, UK), 0·0025 mol l−1 MgCl2, 0·05% W1 (GibcoBRL), 0·0002 mol l−1 of each dNTP (GibcoBRL), 0·001 mol l−1 of each primer and 2 U of Taq DNA Polymerase (GibcoBRL), in a final volume of 50 μl. To each PCR tube, ca 50 μl of mineral oil were added and amplification occurred in a RoboCycler 96 (Stratagene, La Jolla, CA, USA), according to the following amplification programme: 4 min at 95°C; 35 cycles of 1 min at 95°C, 1 min at 56°C and 2 min at 72°C; 4 min at 72°C. Each reaction sample was run on a 1·5% agarose gel, in 0·5 X TBE (0·05 mol l−1 Tris, 0·045 mol l−1 boric acid, 0·001 mol l−1 EDTA) at 90 V for 2 h 30 min, using 1 kb Plus standard (GibcoBRL) as molecular size marker. After ethidium bromide staining, the gels were analysed with KODAK 1D 2·0 software (GibcoBRL). For each isolate or host ITS regions, amplification was performed two to three times in order to assess the reproducibility of the method. The molecular sizes of P. parasitica ITS regions were estimated using the arithmetic average and standard error of the 44 isolates. Molecular sizes of host ITS regions were calculated as the average value of two replicates. ITS restriction assay To perform restriction digestion of amplified ITS regions, 10 μl samples of each PCR product, not purified, were digested with 5 U of each one of three restriction endonucleases, RsaI (Nbl, Northumberland, UK), HaeIII (Biolabs, Beverly, MA, USA) and Sau3AI (GibcoBRL), in a final volume of 15 μl, according to manufacturer instructions. After a 3-h incubation period at 37°C, 1·5 μl of bromophenol blue solution (0·25% bromophenol blue, 0·25% xylene cyanol, 15% Ficoll in water) was added to each sample to stop the reaction. Each reaction sample was run on a 1·5% agarose gel, in 0·5 X TBE at 90 V for 3 h, using a 100 bp standard (GibcoBRL) as a molecular size marker. After ethidium bromide staining, the gels were analysed with KODAK 1D 2·0 software. Reproducibility of the method was assessed with duplicate reactions. Molecular sizes of individual restriction fragments produced from P. parasitica ITS regions were estimated using the arithmetic average and standard error of the 44 isolates. For host ITS regions, molecular size estimations were based on two replications. ITS2 sequencing In order to sequence the ITS2 region, 15 μl of the PCR reaction of isolate P524 were run in 1% agarose gel, in 0·5 X TBE at 90 V for 1 h 30 min. After ethidium bromide staining, the ITS2 band was extracted with a sterile scalpel and purified with the Concert Rapid Gel Extraction Systems kit (GibcoBRL). The purified product was cloned using the pGEM-T Easy Vector Systems kit (Promega, Madison, WI, USA), with the following adaptations: 3 μl of the purified PCR product in the ligation reaction; JM109 competent cells, after inoculation in TSS medium (1 X LB (1% tryptone, 0·5% yeast extract, 0·5% NaCl, pH 7·0), 10% PEG 6000, 5% DMSO, 0·05 mol l−1 MgSO4, pH 6·5); SOC medium replaced by LB medium in JM109 transformation. The recombinant cells were plated in LB medium with 0·15 g l−1 ampicilin, 0·04 g l−1 IPTG and 0·04 g l−1 X-GAL. Screening of recombinant white colonies was performed after an overnight incubation of each colony in 2 ml of LB medium with 0·15 g l−1 ampicilin. Each cell suspension was centrifuged at 18 000 g for 1 min and the pellet was resuspended in 150 μl TEG (0·05 mol l−1 glucose, 0·025 mol l−1 Tris-HCl, 0·01 mol l−1 EDTA, pH 8·0), followed by the addition of 200 μl 0·2 mol l−1 NaOH, 1% SDS. The suspension was mixed by inversion and chilled on ice, 200 μl 3 mol l−1 potassium acetate (pH 4·8) were added and the suspension was centrifuged at 18 000 g for 10 min. To the collected upper phase, 500 μl of isopropanol (−20°C) were added. After a 30 min centrifugation at 18 000 g, the pellet was washed with 500 μl of 70% ethanol and centrifuged at 18 000 g for 5 min. The final pellet was resuspended in 50 μl TE with RNase (0·05 g l−1). Restriction analysis of putative recombinants with the endonuclease PvuII (Biolabs) occurred for 2 h at 37°C, according to the manufacturer instructions, in a final volume of 30 μl. Restriction products were resolved by electrophoresis in a 1% agarose gel, in 0·5 X TBE at 90 V for 1 h 30 min. The gel was stained with ethidium bromide and fragment molecular size was estimated with KODAK 1D 2·0 software. Recombinant colonies containing the insert were re-inoculated in LB medium with 0·15 g l−1 ampicilin and incubated overnight at 37°C. Recombinant plasmid DNA was extracted with the Concert High Purity Plasmid Miniprep System kit (GibcoBRL). Sequencing was performed using the CEQ2000 Dye Terminator Cycle Sequencing kit (Beckman, Fullerton, CA, USA) and a capilary electrophoresis CEQ2000-XL (Beckman) sequencer, both directly from purified PCR product and from the cloned fragment. Both DNA strands were sequenced, with the primers T7 and SP6. BLASTN (Altschul et al. 1997) of the two sequences was performed in the GenBank database. Internal primer design and multiplex PCR ITS sequences of Peronospora (26 from P. parasitica and 15 from other Peronospora spp.), Albugo (5), Botrytis (2), Alternaria (3), Leptosphaeria (2), Plasmodiophora (4), Fusarium (3), Cladosporium (2), Trichoderma (1), Phoma (1), Diaporthe (1), Phytophthora (1), Sclerotinia (1), Mycospharella (2) and B. oleracea (4) available in the GenBank database (http://www.ncbi.nlm.nih.gov) were aligned with hierarchical clustering (Corpet 1988) at INRA website (http://prodes.toulouse.fr/multialign/multialign.html). Based on this alignment, internal primers were designed for specific amplification of full ITS and ITS2 regions of P. parasitica: PpITS1F (5′-CAAYTWTAATTGGGGG TCGTGATCTT-3′), PpITS2F (5′-AAGCGTGACG ATACTAATTTG-3′) and PpITS2R (5′-TGAAGTG CGGCCGAAGCTT-3′. Three multiplex PCR amplifications were performed using the following combinations of primers: ITS3 and ITS4 (to amplify any ITS2 region) plus PpITS2F and PpITS2R (to specifically amplify P. parasitica ITS2 region); ITS5 and ITS4 (to amplify any full ITS region) and PpITS1F and PpITS2R (to specifically amplify P. parasitica full ITS region); and ITS5 and ITS4 plus PpITS2F to amplify any full ITS region and P. parasitica specific ITS2 region. Selectivity of internal primers was tested with samples corresponding to uninfected 'Algarvia' cabbage; the same host infected with P. parasitica; the infected host DNA combined with Alternaria sp. and Ph. cinnamomi DNA; P. parasitica DNA free from host DNA contamination and each one of the 10 B. oleracea fungal pathogens. Each reaction mixture contained 2 μl DNA, PCR buffer 1X (GibcoBRL), 0·0025 mol l−1 MgCl2, 0·05% W1 (GibcoBRL), 0·0004 mol l−1 of each dNTP (GibcoBRL), 0·001 mol l−1 of each primer and 2 U of Taq DNA Polymerase (GibcoBRL), in a final volume of 50 μl. To each PCR tube, ca 50 μl of mineral oil were added and amplification occurred in a RoboCycler 96 (Stratagene), according to the following amplification programme: 4 min at 95°C; 35 cycles of 1 min at 95°C, 1 min at 54°C and 2 min at 72°C; 4 min at 72°C. Each reaction sample was run on a 1·5% agarose gel, in 0·5 X TBE (0·05 mol l−1 Tris, 0·045 mol l−1 boric acid, 0·001 mol l−1 EDTA) at 90 V for 2 h 30 min, using 1 kb Plus standard as molecular size marker. After ethidium bromide staining, the gels were analysed with KODAK 1D 2·0 software. For each sample, amplification was performed two to three times in order to assess the reproducibility of the method. Molecular sizes of ITS regions were calculated as the average value of two replications. Results ITS analysis The amplification of ITS1, ITS2 and full ITS regions (Fig. 1) revealed a common PCR product for all the 44 P. parasitica isolates with 323 ± 0·9 bp, 684 ± 2·1 bp and 987 ± 3·0 bp, respectively. Isolates P512, P516, P517, P518, P523 and P525, representing mixtures collected from different hosts, also have equivalent mean amplicon sizes. Figure 1Open in figure viewerPowerPoint Internal transcribed spacer amplification profiles of Peronospora parasitica isolates. (a) ITS1. (b) ITS2. (c) Full ITS. Lanes 1, 16: 1 kb Plus DNA ladder. Lanes 2–15: isolates P501, P502, P505, P517, P519, P520, P521, P522, P523, P524, P525, P526, P527 and P528 Other amplification products, usually in lower abundance, were observed in most of the isolates (Fig. 1). The number of these additional amplicons was higher for ITS1, pointing to a better specificity of the primers used to amplify the ITS2 region. Comparison of amplification reactions of isolates with amplifications from B. oleracea noninfected seedlings allowed the identification of host ITS amplicons among the additional products. PCR products of ITS1 and ITS2 regions of plant hosts had the same length that was estimated as 388 ± 0·1 bp and 380 ± 0·1 bp for CrGC3·1 and cabbage 'Coração-de-boi', respectively. Total ITS of CrGC3·1 and cabbage 'Coração-de-boi' was estimated as 756 ± 0·1 bp and 740 ± 0·1 bp, respectively. Nevertheless, when host and P. parasitica ITS regions were co-amplified, their distinction was evident for ITS2 and full ITS (Fig. 1), because of significant differences of molecular sizes. As the biotrophic nature of P. parasitica prevents the use of axenic cultures, the remaining unidentified products presumably resulted from secondary pathogens infecting the seedlings after tissue necrosis. In fact, the presence of biological contaminants in conidial suspensions was detected by microscopic observation during this work. ARDRA analysis The restriction profiles of ITS regions, obtained with the three endonucleases (Table 2), showed that only Sau3AI recognized a restriction sequence in P. parasitica ITS1 region, producing two fragments. Conversely, all three enzymes recognized two restriction sites in the ITS2 region. No restriction fragment length polymorphisms were observed in ITS regions among P. parasitica isolates. Table 2. Internal transcribed spacer (ITS) restriction fragments obtained with the endonucleases RsaI, HaeIII and Sau3AI for Peronospora parasitica and Brassica oleracea CrGC3·1 and cabbage 'Coração-de-boi' Enzyme Peronospora parasitica Brassica oleracea CrGC3·1 Cabbage 'Coração-de-boi' ITS1 (bp) ITS2 (bp) Total ITS (bp) ITS1 (bp) ITS2 (bp) ITS1 (bp) ITS2 (bp) RsaI No restriction 157 ± 0·5* 157 ± 0·6 194 ± 0·1† No restriction No restriction No restriction 242 ± 0·6 285 ± 0·7 285 ± 0·6 545 ± 1·1 HaeIII No restriction 71 ± 1·5 71 ± 0·8 48 ± 0·1 66 ± 0·1 37 ± 0·1 100 ± 0·1† 248 ± 4·5 248 ± 0·3 127 ± 0·1 109 ± 0·1 83 ± 0·1 180 ± 0·1 365 ± 3·7 668 ± 0·7 213 ± 0·1 213 ± 0·1 260 ± 0·1 Sau3AI 85 ± 0·8 71 ± 0·9 71 ± 0·6 68 ± 0·1 40 ± 0·1 54 ± 0·1 36 ± 0·1 238 ± 1·8 223 ± 1·3 85 ± 1·3 320 ± 0·1 348 ± 0·1 142 ± 0·1 344 ± 0·1 390 ± 1·0 223 ± 1·3 184 ± 0·1 608 ± 2·3 *Values refer to average ± s.e. The number of determinations was 44 for P. parasitica and two for CrGC3·1 and cabbage 'Coração-de-boi'. †Double co-migrating fragments. To reinforce these results and to contribute to restriction site location within the internal transcribed spacers, ARDRA analysis was also performed with full ITS (Table 2). Although a small variation in fragment molecular size estimations was observed, ITS1 and ITS2 restriction profiles were confirmed and the majority of restriction sites could be located in the physical map displayed in Fig. 2. Only the relative order of the two 3′ terminal RsaI and HaeIII fragments could not be assessed. Figure 2Open in figure viewerPowerPoint Physical map of Peronospora parasitica rDNA cluster. R = RsaI restriction sites (positions 242 and 527 within ITS2 amplicon); H = HaeIII restriction sites (positions 365 and 436 within ITS2 amplicon); S = Sau3AI restriction sites (position 85 within ITS1 amplicon; positions 390 and 461 within ITS2 amplicon) Beyond the fragments resulting from P. parasitica ITS restriction, others could be seen after electrophoresis. Some fragments resulted from partial or incomplete restriction, and provided an additional tool to locate the restriction sites within the ITS regions. ARDRA analysis of brassica ITS regions (Table 2) also confirmed the host origin of other fragments. The few remaining unidentified fragments, less frequent in ITS2 restriction analysis, possibly resulted from the additional amplicons referred in ITS analysis. ITS2 sequence of isolate P524 Although sequencing was directly attempted from the PCR product, fully reliable sequence data were only obtained from the cloned PCR product. In this case, a product of 684 bp was sequenced for both DNA strands. Homology sites for ITS3 and ITS4 primers were detected in the 3′–5′ and 5′–3′ DNA strands, respectively, and the BLASTN (Altschul et al. 1997) of both sequences revealed a total complementarity between them. Homology search against 16 available GenBank sequences of Peronospora spp. (one from P. parasitica, eight from P. sparsa, two from P. destructor and one from P. farinosa, P. rumicis, P. niessleana, P. arborescens and P. manshurica) showed that nucleotides 1–128 correspond to the 5·8S rRNA gene terminal sequence, nucleotides 129–624 represent the complete ITS2 spacer sequence (496 bp) and nucleotides 625–684 correspond to the 28S rRNA gene initial sequence. The complete sequence of the ITS2 of P. parasitica, isolate P524, is available in the GenBank database (accession number AY029235). The ITS2 sequence obtained, confirmed the ARDRA results and the physical map presented in Fig. 2. ARDRA analysis in incompatible host–pathogen interactions Cabbage 'Algarvia' is resistant to P. parasitica isolate P501, despite some growth of intercellular mycelium. This incompatible host–pathogen system was selected to amplify ITS2 and full ITS, using DNA isolated from a small amount of cabbage tissue both infected and not infected. Amplification of P. parasitica ITS regions could be achieved from infected tissue, whereas amplification of host ITS regions occurred in both cases (Fig. 3a). As ITS regions of cabbage 'Algarvia' and CrGC3·1 have similar sizes, distinction of pathogen and host products was also more evident with ITS2 amplification. Figure 3Open in figure viewerPowerPoint (a) Amplification of internal transcribed spacer 2 and full ITS from Tronchuda cabbage 'Algarvia' infected with Peronospora parasitica isolate P501 (lanes 2 and 4, respectively) and from noninfected tissues of the same Brassica oleracea (lanes 3 and 5, respectively). P. parasitica amplicons are indicated with arrows. Lanes 1, 6: 100 bp DNA ladder. (b) Amplified ribosomal DNA restriction analysis profiles with RsaI, from noninfected Tronchuda cabbage 'Algarvia' (lane 2: ITS2; lane 4: full ITS) and the same B. oleracea infected with P. parasitica isolate P501 (lane 3: ITS2; lane 5: full ITS). Lanes 1, 6: 100 bp DNA ladder ARDRA with RsaI applied to these PCR samples (Fig. 3b) revealed that this enzyme does not recognize any sequence within ITS regions of cabbage 'Algarvia', as observed with cabbage 'Coração-de-boi', and produced the expected restriction fragments from P. parasitica. Multiplex PCR The amplification of ITS2 region with primers ITS3 and ITS4, simultaneously with primers PpITS2F and PpITS2R, revealed the expected amplicons of host and P. parasitica, with 380 bp and 684 bp, respectively. Also, as expected was the internal PCR product with 381 bp, amplified only in P. parasitica. The molecular sizes of ITS amplicons of the other samples matched the expected values (data not shown). The amplification of full ITS with primers ITS5 and ITS4, simultaneously with primers PpITS1F and PpITS2R, also revealed the expected amplicons of host and P. parasitica, with 760 bp and 987 bp, respectively. The internal PCR product, specific for P. parasitica, pre