Molecular Diagnostic Techniques

Different isolates of P. chlamydosporia that are impossible to differentiate by morphology can be discriminated using a variety of molecular methods based on PCR. Restriction digest fingerprints of the products from PCR amplification of the ribosomal intergenic spacers (IGS), and the patterns obtained using PCR with arbitrary ERIC and REP primers, could differentiate isolates although digests of the ribosomal internal transcribed spacers (ITS) did not (Arora et al. 1996). Subsequently, ERIC fingerprinting was used to infer a phylogenetic tree of isolates, which grouped according to both their geographical and the host nematode origin (Morton et al. 2003b). PCR fingerprinting has been used to identify fungal colonies re-isolated from soil and plants during mixed infection experiments (Mauchline et al. 2004; Flores-Camacho et al. 2008).

The b-tubulin gene of P. chlamydosporia var. chlamydosporia was found to have an intron not present in other fungi, which provided the basis for designing specific primers to positively identify fungal cultures (Hirsch et al. 2001). Subsequently, these were used for quantitative PCR (qPCR) estimates of fungal abundance in the rhizosphere, using DNA extracted from soil, roots and nematode galls rather than from fungal colonies re-isolated on culture media (Mauchline et al. 2002). Methods such as qPCR that avoid the need for culture are valuable in ecological studies but may potentially overestimate fungal abundance as they will detect DNA from cells that are no longer viable. However their use alongside direct visualisation (Macia-Vicente et al. 2009) might resolve this problem in the future.

The b-tubulin gene primers did not amplify P. chlamydosporia var. catenulata, which also differs in ITS sequence compared to P. c. var. chlamydosporia, providing the basis for a discriminatory real-time qPCR assay (Atkins et al. 2009) to measure the relative abundance of the two varieties that had been co-inoculated on nema-tode-infected plants. A real-time PCR assay using a set of molecular beacons specific for P. c. var. chlamydosporia has also been developed (Ciancio et al. 2005). These methods allow the quantification of different Pochonia species and varieties in rhizosphere-extracted DNA but it is more challenging to estimate the relative proportions of different biotypes and isolates without resorting to culture. To discriminate between two different P. c. var. chlamydosporia isolates, a polymorphic region of the genome was identified as the basis of primers for discriminatory qPCR (Atkins et al. 2009). This approach, using "Sequence Characterised Amplified Polymorphic Regions" (SCARs) offers a general strategy for estimating the relative abundance of several closely-related individuals in soil, root or gall-extracted DNA using qPCR.

The VCP1 gene polymorphism associated with nematode host-preference has also been used to design primers to characterise P. chlamydosporia isolates (Siddiqui et al. 2009; Manzanilla-Lopez et al. 2009) and could act as the basis for qPCR assays in the future.

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