DNA-based characterisation and identification
Index
Our method of DNA preparation, (Ward et al., 1994) is routinely used in our laboratory for preparations from Polymyxa, Plasmodiophora, Spongospora and Ligniera. We have put full details on a separate page. It was adapted from Lee and Taylor (1990) and can be used for preparation of DNA from zoospores, resting spores and infected roots. We have also used the DNA extraction method of Zolan and Pukkila, 1986 after pre-treatment of samples as described below (see Mutasa et al., 1993). More recently, we have also used successfully the method of Volossiouk et al., for extraction of Polymyxa DNA from soil and from Spongospora spore balls. Bell et al., 1999 reported methods for the extraction of Spongospora DNA from potato peel, tuber washings and soil. Other papers describing DNA extraction protocols for plasmodiophorids are Bryngelsson et al. (1988), Pilgeram and Duffus (1993), Ito et al. (1994), Buhariwalla et al. (1995), Arnold et al. (1996) and Möller and Harling (1996).
Back to  |
|
Back to  |
1. Specific probe for Polymyxa betae (pPbKES-1)
References
Mutasa et al., 1993; 1995; Mutasa-Göttgens et al., 1996.
Details
A repetitive DNA sequence from Polymyxa betae was used as a probe to specifically detect P.betae in sugar beet roots. It did not detect any other organisms including Polymyxa graminis. Construction of the probe and information about conditions used for non-radioactive (DIG) labelling are given in Mutasa et al., 1993. The partial sequence of the probe is given in Mutasa-Göttgens et al., 1996 and EMBL Accession No X83745. Sequence information from the probe was used to design specific PCR primers for P.betae (see below).
2. Specific PCR assays for P. betae
Specific primers were developed after sequencing the P. betae-specific probe (pPbKES-1) described above. These tests will not detect P. graminis or any other organism.
- A two-stage, nested PCR using gel-based detection of the PCR product was described by Mutasa et al., 1995 and Mutasa-Göttgens et al., 1996. Details of the primers and PCR conditions are available here.
- One tube nested PCR using gel based or colorimetric detection of the product was described by Mutasa et al., 1996 and Mutasa-Göttgens et al., 1996. Details of the primers and PCR conditions are available here.
3. Specific PCR assays for Polymyxa species and P. graminis
Specific primers were developed after sequencing the ITS ribosomal DNA of isolates of Polymyxa and other plasmodiophorids (Ward & Adams, 1998, Morales et al., 1999). Two tests are described - one that detects both Polymyxa species and one that detects Polymyxa graminis only. Both tests can also discriminate between some of the subgroups of Polymyxa graminis on the basis of amplicon size. Details of the primers and PCR conditions are given here.
4. Discrimination of Polymyxa species and isolates using RFLP analysis of PCR-amplified ribosomal DNA (rDNA)
Nuclear rDNA, amplified from samples using 'universal fungal' primers, was digested with restriction enzymes DdeI and CfoI (HhaI) to produce restriction fragment length polymorphism (RFLP) patterns which are diagnostic of Polymyxa species. (Ward et al., 1994; Adams et al., 1994; Legrève et al., 1996; Ward and Adams, 1996; Morales et al., 1999).
Polymyxa graminis and Polymyxa betae gave different RFLP patterns and P. graminis isolates could be divided into 5 subgroups using this method. The method was also used to analyse isolates of Plasmodiophora, Ligniera and Spongospora (E. Ward, unpublished results). Details of the primers and PCR conditions are available here.
5. DNA profiling methods used on Polymyxa species
- Pilgeram and Duffus (1993) tried RAPD analysis of P.betae, but the banding patterns were highly variable and difficult to interpret.
- Mutasa-Göttgens et al. (1996) used single copy probes to show polymorphisms between isolates.
| Back to |
|
Back to |
1. Specific PCR assay for Plasmodiophora brassicae
A PCR assay specific for Plasmodiophora brassicae was developed by Ito et al., 1997. The route by which it was developed was as follows. PCR using primers PBTZS-1 and PBTZS-2, designed to amplify ipt genes (coding for isopentenyl transferase, a key enzyme in cytokinin biosynthesis) produced a 1.4kb fragment in P. brassicae isolates. However, when this was cloned and sequenced (available as D85819), the PBTZS-1 primer sequence was not present on the fragment and it was shown that the product had arisen from primer PBTZS-2 only. Also the sequence did not show significant homology to other ipt genes nor to anything else in sequence databases. Primers (PBTZS-3 and PBTZS-4) were designed from the sequence that could be used in a PCR assay that was specific for P. brassicae. It may also be possible to use PBTZS-3/4 in a nested PCR after first amplifying with PBTZS-1/2. Details of the primers and PCR conditions are available here.
2. DNA profiling studies of Plasmodiophora brassicae
Buhariwalla et al. (1995) used SG (sequence generated) primers and RAPDs to study variability of P.brassicae isolates. The SG primers gave clearer results than the RAPD primers and were useful for discriminating between isolates of P. brassicae.
- The SG primers were derived by sequencing repetitive/ high copy number elements isolated by shotgun cloning of P. brassicae DNA.
Details of the primers and PCR conditions are available here.
- RAPDs - 3 Operon primers were tested (A03, A09 and A20), but the results were difficult to interpret. This was possibly due to contamination of resting spores during the extraction process or uptake of host DNA by P. brassicae.
Möller and Harling (1996) tested RAPDs as a means of strain differentiation in P. brassicae using 3 single spore isolates. Out of 40 primers tested (Operon sets A and R), 23 gave amplified products, 3 (OPR02, OPA10 and OPR12) gave isolate- specific profiles and one (OPA07) a profile that corresponded with the ECD (European Clubroot Differential) race classification of the isolates.
Yano et al. (1997) also tested RAPD primers on P. brassicae (16 isolates from Japan). They reported that almost all of the 32 primers tested (Operon kit A and DNA Oligomer set A-1) gave reproducible banding patterns and showed polymorphisms among the P. brassicae isolates in the study.
| Back to |
|
Back to |
1. Specific PCR assay for Spongospora subterranea (Bulman and Marshall, 1998)
Specific PCR primers were designed using ITS ribosomal DNA sequences Details of the primers and PCR conditions are available here. The assay is specific for Spongospora subterrnanea isolates from potato (formerly described as Spongospora subterranea fsp. subterranea and will not detect Spongospora nasturtii from watercress (formerly known as Spongospora subterranea fsp. nasturtii).
2. Specific PCR assay for Spongospora subterranea (Bell et al., 1999)
Specific PCR primers were designed using ITS ribosomal DNA sequences. The assay is specific for Spongospora subterrnanea isolates from potato (formerly described as Spongospora subterranea fsp. subterranea and will not detect Spongospora nasturtii from watercress (formerly known as Spongospora subterranea fsp. nasturtii).
3. Specific PCR assay for Spongospora nasturtii sequences (Down & Clarkson, 2002)
Specific PCR primers were designed using ITS ribosomal DNA sequences. Details of the primers and PCR conditions are available here. The assay is specific for Spongospora nasturtii from watercress (formerly known as Spongospora subterranea fsp. nasturtii) and will not detect Spongospora subterranea isolates from potato (formerly described as Spongospora subterranea fsp. subterranea).
| Back to |
|
Back to |
PFGE is a method used to separate large DNA molecules, including some fungus chromosomes. Some results have been achieved using the plasmodiophorids.
Gel images as published by Bryan et al., 1996:-
Other papers describing PFGE:-
Ward et al. (1993) - Polymyxa graminis and P. betae.
Ito et al. (1994) - Plasmodiophora brassicae.
Graf et al. (2001) - Plasmodiophora brassicae.
| Back to |
|
Back to |
