Genetic diversity in the blackberry rust pathogen, Phragmidium violaceum, in Europe and Australasia as revealed by analysis of SAMPL
Introduction
Population genetics is being applied increasingly to understand the dynamics of plant diseases in time and space (Milgroom & Peever 2003). The selection and deliberate release of exotic plant pathogens, especially rust fungi (Uredinales), for successful biological control of introduced weeds relies on a sound knowledge of epidemiology and host–pathogen interactions. Molecular techniques aid investigation of relationships between weed and pathogen diversity, and can be applied for identifying and monitoring the fate of the released pathogen genotypes with certainty (Evans & Gomez 2004). In the case of European blackberry (Rubus fruticosus aggr.) in Australia, efficient DNA markers will allow the study of evolutionary processes in Phragmidium violaceum, before and after the release of additional strains of this biological control agent.
At least 15 species of European blackberry are naturalised in Australia, where it is considered a ‘weed of national significance’ (Thorp and Lynch, 2000, Evans et al., 2004). It is also considered an important weed in New Zealand, South Africa, North America and Chile (Amor et al. 1998). Phragmidium violaceum, the cause of leaf rust of European blackberry, was first reported in Australia in 1984 and subsequently in New Zealand in 1990, following an unauthorised introduction first identified in Victoria, Australia (Marks et al., 1984, Pennycook, 1998). Subsequently, there were two authorised introductions of P. violaceum for the biological control of European blackberry in Australia. First, strain F15 from France was released throughout south eastern and south western Australia in 1991 and 1992 (Bruzzese & Lane 1996). Second, eight strains of P. violaceum were released in New South Wales, Western Australia and Victoria in 2004 after their collection in France in 2000 (Scott et al. 2002). All spore states of this autoecious, macrocyclic rust fungus can form under Australian conditions (Washington 1985).
Before authorised releases of additional strains of P. violaceum in Australia in 2004, Evans et al. (2000) identified RFLPs using M13 DNA to probe genomic DNA of Australian isolates of P. violaceum digested with the restriction enzyme HaeIII. The robustness of this technique was demonstrated when 13 DNA phenotypes were identified among 18 single-uredinium isolates of P. violaceum from various locations in mainland Australia between 1997 and 1999 (Evans et al. 2000). This DNA marker also identified strain F15 of P. violaceum as being genetically distinct from the 18 isolates screened in the same study. The disadvantage of the RFLP protocol is that it uses relatively large quantities of DNA when compared with a PCR-based marker system. Furthermore, the M13 RFLP technique of Evans et al. (2000) was limited to analysis of restriction fragments in the range 4–9 kbp and additional markers were deemed necessary to expand the proportion of the genome analysed for genetic diversity.
AFLPs are used frequently in studies of genetic diversity in plants and fungi due to their ease of development, efficiency, high multiplex ratio and reproducibility (Zabeau and Vos, 1993, Majer et al., 1996). AFLPs have been demonstrated in at least seven species of Uredinales (Pei and Ruiz, 2000, Samils et al., 2001, Hovmøller et al., 2002, Keiper et al., 2003, Yourman and Luster, 2004). A modification of the AFLP procedure called ‘selective amplification of microsatellite polymorphic loci’ (SAMPL, Morgante & Vogel 1994) combines the advantages of AFLP with the analysis of highly variable microsatellite regions of eukaryotic genomes. SAMPL has been reported to be more powerful than AFLPs in discriminating between closely related individuals in several plant complexes (Paglia and Morgante, 1998, Roy et al., 2002, Singh et al., 2002, Tosti and Negri, 2002) and in the rust pathogen, Puccinia striiformis f. sp. tritici, which is strictly clonal (Stubbs, 1985, Keiper et al., 2003).
We report the development of a modified SAMPL technique using adapters linked to the blunt end restriction enzyme, HaeIII, following on from the useful resolution identified by RFLPs using this same enzyme (Evans et al. 2000). The utility of SAMPL, relative to AFLP, in describing genetic differentiation within and among populations of P. violaceum from Europe and Australasia, is demonstrated.
Section snippets
Origin of isolates
Throughout this paper, individual collections of Phragmidium violaceum will be called isolates, whereas isolates that have been characterised for the purpose of biological control will be called strains. A total of 44 single uredinium-derived isolates of P. violaceum were analysed through the course of developing markers for the fungus (Table 1). Eighteen single uredinium-derived isolates of P. violaceum from Australia originated from collections made by Evans et al. (2000). For the purpose of
AFLP and modified AFLP
No polymorphic loci were observed among DNA preparations of Phragmidium violaceum isolates SA1, V1 and V2 using 20 MseI/EcoRI AFLP primer pairs, each of which generated at least 25 scoreable loci. Loci were also monomorphic among DNA from the seven reference isolates of P. violaceum (F15, SA1, V1, V2, WA9, WA12 and WP69) using primer pairs E–AC + M–C, E–0 + M–0 or E–0 + M–T. The use of primer pair E–0 + M–0 to detect AFLPs among 44 European and Australasian isolates revealed a single 300 bp polymorphic
Discussion
This study highlights the importance not only of screening AFLP primers for polymorphisms but also of choosing restriction enzymes that are polymorphic for the species being studied, as practised during development of RFLP markers. In contrast to the application of AFLP for genetic analysis of other Uredinales by means of commonly-used linkers and primers (e.g. Hovmøller et al. 2002), genetic diversity was not detected in Phragmidium violaceum using AFLP. This result was inconsistent with our
Acknowledgements
The Cooperative Research Centre for Australian Weed Management supported this research and the PhD scholarship of D.R.G. Research in New Zealand (NZ) was funded by the NZ Forest Health Research Collaborative and the Bay of Plenty Regional Council, NZ. Special thanks go to staff of Landcare Research, NZ, including Paula Wilkie for technical assistance, Duckchul Park for extraction of DNA and David Glenny for identification of blackberry species in NZ. We also thank John K. Scott and Andy
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