Detection and Quantification of Spongospora subterranea f. sp. subterranea in Soils and on Tubers Using Specific PCR Primers

PCR-based methods were developed for the detection and quantification of the potato pathogen Spongospora subterranea f. sp. subterranea (S. subterranea) in peel, tuber washings and soil. A partial sequence was obtained for S. subterranea ribosomal DNA and specific PCR primers (Sps1 and Sps2) were chosen from the internal transcribed spacer regions. These primers amplified a 391bp product from S. subterranea DNA but did not amplify DNA from potato or a range of soil-borne microbes, including related species. Diluted S. subterranea DNA was detected at a concentration equivalent to 25×10^–5 cystosori or 1 zoospore per PCR. Amplification was detected from peel and washings of infected and apparently healthy tubers, but not from peel of Scottish classified seed potatoes or axenically micropropagated potatoes. A rapid method for extracting S. subterranea DNA from soils was developed. This yielded DNA pure enough for PCR within 3h and facilitated the detection of 1–5 cystosori per gram of soil. A PCR quantification technique was developed involving comparison of product ratios obtained after co-amplification of S. subterranea DNA along with an internal standard (competitor DNA fragment). This quantitative technique was also adapted for use in soil. PCR detection of S. subterranea in soil was considerably more sensitive than previously reported immunoassays and was quicker and easier than conventional bait plant bioassays. Such an assay could be useful for developing disease risk assessments for field soils and seed potato stocks and for future studies on the ecology and control of S. subterranea.     

Detection of Colletotrichum coccodes from soil and potato tubers by conventional and quantitative real-time PCR

Colletotrichum coccodes is the causal agent of the potato blemish disease black dot. Two PCR primer sets were designed to sequences of the ribosomal internal transcribed spacer (ITS1 and ITS2) regions for use in a nested PCR. The genus-specific outer primers (Cc1F1/Cc2R1) were designed to regions common to Colletotrichum spp., and the species-specific nested primers (Cc1NF1/Cc2NR1) were designed to sequences unique to C . coccodes . The primer sets amplified single products of 447 bp (Cc1F1/Cc2R1) and 349 bp (Cc1NF1/Cc2NR1) with DNA extracted from 33 European and North American isolates of C. coccodes. The specificity of primers Cc1NF1/Cc2NR1 was confirmed by the absence of amplified product with DNA of other species representing the six phylogenetic groups of the genus Colletotrichum and 46 other eukaryotic and prokaryotic plant pathogenic species. A rapid procedure for the direct extraction of DNA from soil and potato tubers was used to verify the PCR assay for detecting C. coccodes in environmental samples. The limit of sensitivity of PCR for the specific detection of C. coccodes when inoculum was added to soils was 3·0 spores per g, or the equivalent of 0·06 microsclerotia per g soil, the lowest level of inoculum tested. Colletotrichum coccodes was also detected by PCR in naturally infested soil and from both potato peel and peel extract from infected and apparently healthy tubers. Specific primers and a TaqMan fluorogenic probe were designed to perform quantitative real-time (TaqMan) PCR to obtain the same levels of sensitivity for detection of C. coccodes in soil and tubers during a first-round PCR as with conventional nested PCR and gel electrophoresis. This rapid and quantitative PCR diagnostic assay allows an accurate estimation of tuber and soil contamination by C. coccodes .     

Characterisation of <Emphasis Type="Italic">Phoma tracheiphila</Emphasis> by RAPD-PCR,microsatellite-primed PCR and ITS rDNA sequencing and development of specific primers for <Emphasis Type="Italic">in planta</Emphasis> PCR detection

Thirty six isolates of Phoma tracheiphila from Italy, the causal agent of the mal secco disease on Citrus species, were characterised by different molecular tools in comparison with representative isolates of other phytopathogenic Phoma species. These included analysis of the distribution of RAPD and microsatellite markers and sequencing of the internal transcribed spacer (ITS) region of the nuclear rRNA genes. The results obtained with 12 RAPD primers (92 markers) and 7 microsatellite primers (56 markers) suggest that Italian isolates of P. tracheiphila are genetically homogeneous, leading to identical patterns upon amplification with all the tested primers. Accordingly, ITSI-5.8S-ITS2 sequences were highly conserved (98–100% identity along a 544-characters alignment) among all the isolates of P. tracheiphila. A neighbor-joining analysis of ITS sequences of P. tracheiphila in comparison with those of other Phoma species, as well as with alignable sequences from anamorphic and teleomorphic taxa retrieved in BLAST searches, revealed a close relationship between P. tracheiphila and Leptosphaeria congesta. A pair of P. tracheiphila-specific primers was designed on the consensus sequence (555 residues) obtained from the alignment of the newly generated P. tracheiphila ITS sequences. A PCR-based specific assay coupled to electrophoretic separation of amplicons made it possible to detect P. tracheiphila in naturally infected Citrus wood tissue collected from both symptomatic and symptomless plants. The limit of detection was 10 pg of genomic DNA and 5 fg of the ITS target sequence.     

The β-tubulin Gene is a Useful Target for PCR-based Detection of an Albino Ophiostoma piliferum Used in Biological Control of Sapstain

The potential of Cartapip, an albino Ophiostoma piliferum, as a biocontrol agent against sapstain in logs has been tested in Germany. To detect the albino strain in field-tested wood, the usefulness of the -tubulin gene as a target region for developing PCR-based assays was evaluated with 102 strains of O. piliferum and 31 strains of other wood-inhabiting species. A partial -tubulin gene sequence of O. piliferum strains from different geographic origins was amplified by PCR and analyzed by restriction enzyme digestions and DNA sequencing. Variation in size and nucleotide sequences was found in intron regions indicating that intraspecific variation is present in the -tubulin gene. Consequently, -tubulin gene-derived PCR methods using PCR–RFLP patterns generated by HinfI and SpeI and sequence-specific primers Cat1 and Cat2, were developed and their specificity for Cartapip was accessed with field-tested logs and lumber. The -tubulin gene-based PCR methods were found to be valuable tools for rapid and reliable identification of Cartapip in field-tested logs and lumber in Germany. Specificity tests against other wood-inhabiting species and wild type O. piliferum strains from diverse nations showed that the Cat1 and Cat2 primers have potential to be used in other European countries, New Zealand, Alberta and British Columbia.     

Detection of Phytophthora nicotianae and P. citrophthora in Citrus Roots and Soils by Nested PCR

The polymerase chain reaction (PCR) was used for the specific detection of Phytophthora nicotianae and P. citrophthora in citrus roots and soils. Primers were based on the nucleotide sequences of the internal transcribed space regions (ITS1 and ITS2) of 16 different species of Phytophthora. Two primer pairs, Pn5B–Pn6 and Pc2B–Pc7, were designed specifically to amplify DNA from P. nicotianae and P. citrophthora, respectively. Another primer pair (Ph2–ITS4) was designed to amplify DNA from many Phytophthora species. All primer pairs were assessed for specificity and absence of cross-reactivity, using DNA from 118 isolates of Phytophthora and 82 of other common soil fungi. In conventional PCR, with a 10-fold dilution series of template DNA, the limit of detection was of 1pgl^–1 DNA for all the primer pairs (Ph2–ITS4, Pn5B–Pn6, and Pc2B–Pc7). In nested PCR, with primers Ph2–ITS4 in the first round, the detection limit was of 1fgl^–1 for both the primer sets (Pn5B–Pn6 and Pc2B–Pc7). Simple, inexpensive and rapid procedures for direct extraction of DNA from soil and roots were developed. The method yielded DNA of a purity and quality suitable for PCR within 2–3h. DNA extracted from soil and roots was amplified by nested PCR utilizing primers Ph2–ITS4 in the first round. In the second round the primer pairs Pn5B–Pn6 and Pc2B–Pc7 were utilized to detect P. nicotianae and P. citrophthora, respectively. Comparison between the molecular method and pathogen isolation by means of a selective medium did not show any significant differences in sensitivity.     

A Combination of Baiting and Different PCR Formats, Including Measurement of Real-Time Quantitative Fluorescence, For the Detection of Phytophthora fragariae in Strawberry Plants

Phytophthora fragariae, the cause of strawberry red stele disease, is a quarantine pathogen in Europe. Detecting low levels of infection requires sensitive and specific methods. In the past, Dutch and English inspection services have used bait plants to test strawberry propagation stocks destined for export. Increasingly though, PCR is being incorporated into these testing procedures in an effort to increase sensitivity and speed. Various combinations of baiting and PCR assays were compared with existing testing procedures. Water and root samples from the bait test were screened by nested PCR and the PCR amplicon was detected by several methods, including fluorescent labelled probes (TaqMan and Molecular Beacon). PCR amplification was monitored in real-time and semi-quantitative detection was possible. Because PCR reactions are sensitive to inhibitors present in extracted DNA samples, an internal control containing the primer sequences specific for P. fragariae was developed to avoid false negatives.     

Asymmetric PCR ELISA: Increased Sensitivity and Reduced Costs for the Detection of Plant Viruses

PCR ELISA is the immunodetection of the products of a polymerase chain reaction (PCR). It is effective for detecting and differentiating plant viral nucleic acids, but as currently performed, it is laborious and expensive. The procedure has been modified and simplified by using asymmetric PCR. This eliminated the need to denature and neutralize samples prior to hybridization. It also increased the relative concentration of the target DNA species, making PCR ELISA more sensitive than TaqMan, a fluorescence-based detection method. Reducing the reaction volumes to half and the concentration of the dNTPs and the digoxigenin label by tenfold significantly reduced the costs of PCR ELISA without reducing its sensitivity. The usefulness of these modifications was demonstrated for the detection of Citrus tristeza virus and Rupestris stem pitting-associated virus. We expect that with only minor modifications asymmetric PCR ELISA could be used effectively for the detection of most nucleic acid molecules of interest.     

Detection of Plasmodiophora Brassicae By PCR in Naturally Infested Soils

A nested polymerase chain reaction (PCR) method was developed for detection of DNA from Plasmodiophora brassicae in naturally infested field soil samples. The target sequences 389 bp and 507 bp were amplified from Swedish populations of P. brassicae. The protocols described enabled detection of DNA in various soil classes with an inoculum level of P. brassicae corresponding to a disease severity index (DSI) higher than 21 in a greenhouse bioassay. Three sequenced Swedish P. brassicae isolates had identical sequence in the 18S/ITS 1 region, but differed by a few nucleotides from an isolate sequenced in the UK. The results indicate that the primers used are general for P. brassicae, and consequently the nested PCR assay has a potential to be developed as a routine diagnostic test.     

Development of Specific PCR Primers for Identification and Detection of Rhizopycnis vagum

Rhizopycnis vagum is a recently described coelomycete known to belong to the complex of root rot pathogens contributing to vine decline of cucurbits in several parts of the world. However, the fungus has also been reported to infect tomato, and as an endophytic associate of mycorrhizal roots of wild, asymptomatic Pinus halepensis and Rosmarinus officinalis plants in Italy. To accelerate epidemiological and ecological investigations on this fungus, a PCR primer pair was developed. Primers Rv1-F and Rv1-R were designed, based on alignment of internal transcribed spacer (ITS) sequences (ITS1-5.8S-ITS2), which amplified a 396-bp fragment from all R. vagum isolates tested, including isolates pathogenic to melons and endophytic isolates from mycorrhizae. Specificity of the primer pair was verified both in silico (BLAST searches using each primer string as a query) and in PCR assays, where the primers failed to amplify DNA from any isolate of fungi taxonomically related to R. vagum (e.g. Massarina walkeri and Stagonospora spp.) and other vine decline and common soilborne pathogens (e.g. Monasporascus cannonballus, Acremonium cucurbitacearum, Fusarium spp. and Rhizoctonia solani). Under optimum conditions, detectable amplification of the specific sequence required 0.05 pg of target DNA. Amplification of the expected 369-bp fragment was also obtained from DNA root extracts of nearly asymptomatic Cucumis melo plants inoculated with R. vagum under greenhouse conditions.     

Real-time detection of <Emphasis Type="Italic">Phytophthora nicotianae</Emphasis> and <Emphasis Type="Italic">P. citrophthora</Emphasis>in citrus roots and soil

Two primers, specific for Phytophthora nicotianae (Pn6) and P. citrophthora (Pc2B), were modified to obtain Scorpion primers for real-time identification and detection of both pathogens in citrus nursery soils and roots. Multiplex PCR with dual-labelled fluorogenic probes allowed concurrent identification of both species ofPhytophthora among 150 fungal isolates, including 14 species of Phytophthora. Using P. nicotianaespecific primers a delayed and lower fluorescence increase was also obtained from P. cactorumDNA. However, in separate real-time amplifications, the aspecific increase of fluorescence from P. cactorum was avoided by increasing the annealing temperature. In multiplex PCR, with a series of 10-fold DNA dilutions, the detection limit was 10 pg l^-1 for P. nicotianaeand 100 pg l^–1 for P. citrophthora, whereas in separate reaction DNA up to 1 pg l^-1 was detected for both pathogens.Simple and rapid procedures for direct DNA extraction from soil and roots were utilised to yield DNA whose purity and quality was suitable for PCR assays. By combining these protocols with a double amplification (nested Scorpion-PCR) using primers Ph2-ITS4 amplifying DNA from the main Phytophthora species (first round) and PnB5-Pn6 Scorpion and Pc2B Scorpion-Pc7 (second round), it was possible to achieve real-time detection of P. nicotianaeand P. citrophthora from roots and soil. The degree of sensitivity was similar to that of traditional detection methods based on the use of selective media. The analyses of artificially and naturally infested soil showed a high and significant correlation between the concentration of pathogen propagules and the real-time PCR cycle threshold.     

Specific Oligonucleotide Primers for the Rapid Identification and Detection of the Agent of Tomato Pith Necrosis, Pseudomonas corrugata, by PCR Amplification: Evidence for two Distinct Genomic Groups

Unique DNA bands from strains representative of two groups of Pseudomonas corrugata, as shown by amplification of their genomic DNA by polymerase chain reaction using short random sequence oligonucleotide primers (RAPD-PCR), were isolated, cloned and sequenced. Two pairs of specific primer sequences, based on the ends of the cloned unique DNA bands from strains IPVCT10.3 and IPVCT8.1, were used in multiplex PCR with a range of P. corrugata strains. All strains produced one of the two specific bands, 1100bp (from the IPVCT10.3-based primers) and 600bp (from the IPVCT8.1-based primers), representing groups designated I and II, respectively. The primers were also tested on a wider range of Pseudomonas species, including the closely-related fluorescent Pseudomonas genomospecies FP1, FP2 and FP3: none of these bacteria produced any bands following amplification by PCR with these primers. The primer sets detected P. corrugata in tomato pith necrosis-infected plants providing a useful tool for rapid identification and epidemiological studies.     

A new quantitative real-time PCR assay for Rhizoctonia solani AG3-PT and the detection of AGs of Rhizoctonia solani associated with potato in soil and tuber samples in Great Britain

Rhizoctonia solani is an important pathogen of potatoes causing stem canker and black scurf. The fungus is a species complex comprised of 13 known anastomosis groups (AGs). AG3-PT is the anastomosis group frequently associated with disease in potatoes. A real-time PCR assay was designed to the rDNA ITS region of AG3-PT isolates to enable the pathogen to be detected directly in tuber and soil samples. The resulting assay was highly specific for AG3-PT, and did not amplify DNA from isolates from other AGs or subgroups of AG3. Using a bulk DNA extraction method capable of extracting from up to 250 g of soil, the assay could detect one individual sclerotium of AG3-PT (weighing 200 μg) in 250 g of soil. The AG3-PT assay was used, with assays for AG2-1, AG5 and AG8 to determine the prevalence of those AGs in UK potato soils and tubers. AG2-1 and AG3-PT were the predominant groups in tubers and soils, although AG3-PT was more frequently isolated from tubers, highlighting its importance as a potato pathogen. AG3-PT was also detected in more than half of the tuber samples tested suggesting the importance of seed borne inoculum.     

Pathogenicity, Electrophoretic Characterisation and In planta Detection of the Cocoyam Root Rot Disease Pathogen, Pythium myriotylum

Cocoyam (Xanthosoma sagittifolium), an important staple food crop for many people in the tropics and subtropics, suffers great losses from a root rot disease which is most probably caused by Pythium myriotylum, although it has been claimed that a complex of three root pathogens is needed to cause the disease. In this study, we compared two Pythium isolates from diseased cocoyam roots, CRPm and Bokwai, with other putative P. myriotylum isolates from culture collections and from Cameroonian soil, with respect to host range and isozyme patterns. Pathogenicity was tested on tomato, bean, cowpea, tobacco and cocoyam. CRPm and Bokwai were only pathogenic to tobacco and cocoyam. On cocoyam, these isolates caused typical symptoms within 48h on 100% of the inoculated plantlets. Only two other isolates of P. myriotylum from culture collections were moderately to weakly pathogenic to cocoyam. Isolates of P. myriotylum were very variable in their pathogenicity to bean, cowpea, tomato and tobacco. Isozyme patterns of - and -esterases were used to differentiate CRPm and Bokwai from all other isolates. Unlike the other P. myriotylum strains, cocoyam isolates were unable to grow at 37°C. Malate dehydrogenase isozyme bands originating from CRPm were consistently detected in CRPm-infected cocoyam roots grown in vitro and in vivo. These findings indicate that CRPm can penetrate cocoyam roots and cause disease in the absence of other root pathogens. This study also indicates that P. myriotylum from cocoyam developed a certain degree of host specialisation.     

PCR-based Assays to Assess Wheat Varietal Resistance to Blotch (Septoria Tritici and Stagonospora Nodorum) and Rust (Puccinia Striiformis and Puccinia Recondita) Diseases

A multiplex Polymerase Chain Reaction (PCR) assay was developed to detect and quantify four fungal foliar pathogens in wheat. For Septoria tritici (leaf blotch) and Stagonospora nodorum (leaf and glume blotch), the -tubulin gene was used as the target region. Diagnostic targets for Puccinia striiformis (stripe or yellow rust) and P. recondita (brown rust) were obtained from PCR products amplified with -tubulin primer sequences. Final primer sets were designed and selected after being tested against several fungi, and against DNA of infected and healthy wheat leaves. For detection of the four pathogens, PCR products of different sizes were amplified simultaneously, whereas no products were generated from wheat DNA or other non-target fungi tested. The presence of each of the diseases was wheat tissue- and cultivar specific. Using real-time PCR measurements with the fluorescent dye SYBR Green I, PCR-amplified products could be quantified individually, by reference to a standard curve generated by adding known amounts of target DNA. Infection levels for each of the diseases were measured in the flag leaf of 19 cultivars at Growth Stage (GS) 60–64 in both 1998 and 1999. The infection levels for the cultivars were ranked, and showed, with a few exceptions, a good correlation with the NIAB Recommended List for winter wheat, which is based on visual assessment of symptoms. With PCR, the presence of the different pathogens was accurately diagnosed and quantification of pre-symptomatic infection levels was possible. Although sampling and DNA detection methods need further optimisation, the results show that multiplex PCR and quantitative real-time PCR assays can be used in resistance screening to measure the interaction between different pathogens and their hosts at different growth stages, and in specific tissues. This should enable an earlier identification of specific resistance mechanisms in both early-stage breeding material and field trials.     

Development of specific PCR primers for identification and detection of <Emphasis Type="Italic">Phytophthora capsici</Emphasis> Leon

A PCR-based method was developed for the identification and detection of Phytophthora capsici in pepper plants. Three PCR primers (CAPFW, CAPRV1 and CAPRV2) specific for P. capsiciwere designed based on the sequence of its internal transcribed spacer regions. CAPFW/CAPRV1 amplify a 452 bp product from P. capsici DNA whereas CAPFW/CAPRV2 a 595 bp fragment; neither set amplifies DNA from pepper or several fungi pathogenic to pepper. In conventional (single-round) PCR, the limit of detection was 5 pg DNA for both primer sets, whereas in nested PCR the detection limit for both was of 0.5 fg. However, when the dilution series of target DNA were spiked with plant DNA, amplification declined two-fold in both conventional and nested PCR. The CAPFW/CAPRV2 set in conventional PCR was used to detect P. capsici DNA in inoculated plants. Detection occurred as soon as 8h post-inoculation in stem samples from infected but still symptomless plants. The method was also tested to detect fungal DNA in infected soils.     

Identification and Detection of Rosellinia Necatrix by Conventional and Real-time Scorpion-PCR

Several polymerase chain reaction (PCR) primers were designed from the internal transcribed spacer (ITS) regions of the rDNA genes of Rosellinia necatrix to develop a PCR-based identification method. Screening the primers against two isolates of R. necatrix and six other Rosellinia species resulted in the amplification of a single specific product from R. necatrix for most of the primer pairs. Two primer pairs (R2-R8 and R10-R7) confirmed their specificity when tested against 72 isolates of R. necatrix and 93 other fungi from different hosts and geographic areas. The R10 primer was modified to obtain a Scorpion primer for detecting a specific 112bp amplicon by fluorescence emitted from a fluorophore in a self-probing PCR assay. This assay specifically recognised the target sequence of R. necatrix over a large number of other fungal species. In conventional PCR, with primer pairs R2-R8 and R10-R7, 10-fold dilutions of R. necatrix DNA indicated a detection limit of 10pgul^-1 using a single set of primers and 10fgl^-1 in nested-PCR. For Scorpion-PCR, the detection limit was 1pgl^-1 and 1fgl^-1 in nested Scorpion-PCR, i.e. 10 times more sensitive than conventional PCR. A simple and rapid procedure for DNA extraction directly from soil was modified and developed to yield DNA of purity and quality suitable for PCR assays. Combining this protocol with the nested Scorpion-PCR procedure it has been possible to specifically detect R. necatrix from artificially inoculated soils in approximately 6h.     

Differentiation of Cotton-defoliating and Nondefoliating Pathotypes of Verticillium dahliae by RAPD and Specific PCR Analyses

Severe Verticillium wilt of cotton in southern Spain is associated with the spread of a highly virulent, defoliating (D) pathotype of Verticillium dahliae. Eleven of the D and 15 of a mildly virulent, nondefoliating (ND) pathotype were analyzed by random amplified polymorphic DNA (RAPD) using the polymerase chain reaction (PCR). Six of 21 primers tested generated pathotype-associated RAPD bands. Another 21 V. dahliae isolates were compared in blind trials both by RAPD-PCR using the six selected primers and pathogenicity tests on cotton cultivars. There was a 100% correlation between pathotype characterization by each method. Unweighted paired group method with arithmetic averages cluster analysis was used to divide the 47 V. dahliae isolates into two clusters that correlated with the D or ND pathotypes. There was more diversity among ND isolates than among D isolates, these latter isolates being almost identical. ND- and D-associated RAPD bands of 2.0 and 1.0kb, respectively, were cloned, sequenced, and used to design specific primers for the D and ND pathotypes. These pathotype-associated RAPD bands were present only in the genome of the pathotype from which they were amplified, as shown by Southern hybridization. The specific primers amplified only one DNA band of the expected size, and in the correct pathotype, when used for PCR with high annealing temperature. These specific primers successfully characterized V. dahliae cotton isolates from China and California as to D or ND pathotypes, thus demonstrating the validity and wide applicability of the results.     

A comparison of potato seed-tuber sampling strategies using visual and DNA analyses to estimate incidence of major seed tuber-borne pathogens

Potato seed certification is a disease management tool that minimises the risk of spreading seed tuber-borne inoculum of infectious diseases. Traditionally, certification sampling strategies have relied upon visual assessment of a seedlot from samples taken at one or two points within the load of seed tubers. However methodologies in selection of tuber samples have not been critically assessed for their precision in estimating disease load. This study presents an analysis of 37 potato seedlots over a 3 year period. Analysis of sample data using receiver operating curves (ROCs) indicates that point sampling taking two samples of 100 tubers at the beginning and end of a seedlot gives equivalent disease estimation as a continuous sampling strategy taking ten samples of 20 tubers randomly throughout the seedlot, although at lower statistical precision. This was confirmed both by visual assessment of tuber-borne disease and by analysis of pathogen DNA content from tuber peel. Across the 3 years of study, powdery scab and black scurf were the major seed tuber-borne diseases recognised and this corresponded with high levels of pathogen DNA from peel analysis for both Spongospora subterranea and Rhizoctonia solani AG3 respectively.     

A TaqMan real-time PCR assay for <Emphasis Type="Italic">Rhizoctonia cerealis</Emphasis> and its use in wheat and soil

Rhizoctonia cerealis causes sharp eyespot in cereals and the pathogen survives as mycelia or sclerotia in soil. Real-time Polymerase Chain Reaction (qPCR) assays based on TaqMan chemistry are highly suitable for use on DNA extracted from soil. We report here the first qPCR assay for R. cerealis using TaqMan primers and a probe based on a unique Sequence Characterised Amplified Region (SCAR). The assay is highly specific and did not amplify DNA from a range of other binucleate Rhizoctonia species or isolates of anastomosis groups of Rhizoctonia solani. The high sensitivity of the assay was demonstrated in soils using a bulk DNA extraction method where 200 μg sclerotia in 50 g of soil were detected. DNA of the pathogen could also be amplified from asymptomatic wheat plants. Using the assay on soil samples from fields under different crop rotations, R. cerealis was most frequently detected in soils where wheat was grown or soil under pasture. It was detected least frequently in fields where potatoes were grown. This study demonstrates that assays derived from SCAR sequences can produce specific and sensitive qPCR assays.     

PCR assays for the sugarcane rust pathogens Puccinia kuehnii and P. melanocephala and detection of a SNP associated with geographical distribution in P. kuehnii

Puccinia kuehnii and P. melanocephala cause orange and brown rust of sugarcane, respectively. Puccinia kuehnii has been confirmed in Asia, Australia and recently, the Caribbean basin, whereas P. melanocephala is distributed among the majority of sugarcane growing regions. Differentiating these two economically significant pathogens visually is problematic and limited to material exhibiting mature disease symptoms or spores. Partial ITS1, ITS2 and complete 5·8S sequences were generated from P. kuehnii and P. melanocephala isolates from around the world. PCR primers and dual labelled hydrolysis probes were designed for each pathogen for use in real‐time PCR and optimized using locked nucleic acids (LNA). The primers amplified DNA from their target pathogens and not from other species of Puccinia or fungal species isolated from sugarcane leaves. Optimized real‐time PCR conditions allowed the detection of 0·19 pg of P. kuehnii or P. melanocephala genomic DNA and differentiated the pathogens on sugarcane leaves prior to observing typical symptoms in the field. Primer‐introduced restriction analysis‐PCR (PIRA‐PCR) was used to detect a single nucleotide polymorphism (Pk ITS1 183A>G) in ITS1 of P. kuehnii. Allele 183A was observed in all samples, whereas 183G was detected in 52% of samples from Asia and Australia yet absent from all Caribbean basin samples. Long distance spore dispersal, dispersal through an intermediate location or improper movement of contaminated material could explain the introduction of P. kuehnii to the Western hemisphere. However, the current proliferation of the pathogen in the Americas is limited to isolates which contain only the 183A allele.