be found on the expanding genomic databases that are facilitating this type of data exploration.
Emerging technologies
High-quality sequence data are likely to be a prerequisite for emerging technolo- gies that utilize high-density DNA arrays or microarrays that merge DNA and silicon chips (Lipshutz et al., 1999). The so-called ‘lab on a chip’ (Lévesque, 2001) is a substrate of thousands of ‘spotted’ oligonucleotides that can be hybridized to labelled DNA (Maughan et al., 2001). The detection of multiple DNA hybrids can be used, among many other things, for genotyping and simultaneous moni- toring of multiple genetic variants (Dalma-Weiszhausz et al., 2002). This tech- nology was applied recently to examine relationships among closely related microbial species (Murray et al., 2001). The ability to genotype thousands of indi- viduals will generate overwhelming amounts of data, which will require appro- priate analytical skills for making valid conclusions about complex population dynamics. As always, adoption of new technology will depend on accuracy, sen- sitivity, reproducibility and cost effectiveness.
in defining what is an ‘adequate’ genetic composition for the introduced rust pathogen.
The skeleton rust story
The skeleton rust biocontrol programme provides an instructive example of the impact of genetic diversity in an apomictic weed on the success of biocontrol. It also provides a conceptual framework for the selection of rust strains. Despite early success in controlling the ‘narrow-leaf ’ form of C.junceain Australia, the other two clones have since colonized wheat crops in areas previously occupied by the ‘narrow-leaf ’ form. The origin ofC.junceaclones in Australia is unknown, although the centre of origin of the genus Chrondrillawas assumed to be eastern Europe. Following the identification of the isozyme variants of C.juncea(Burdon et al., 1980), the first strategy employed to find suitable strains of P.chondrillinafor the other forms ofC.juncearelied on the existence of a strong correlation between isozyme variants and plant disease resistance phenotypes. Subsequently, Chaboudez (1989, 1994) used isozyme marker systems to identify western Turkey as the source of the diploid progenitor of the triploid apomicts of C.juncea. Not surprisingly, there is a concentration of triploid apomicts in this region (Chaboudez, 1994). In a parallel evolutionary story, sexual stages of P.chondrilli- naare known to occur in the general region of Turkey where apomicts ofC.juncea arise (Hasan and Wapshere, 1973). Climatic conditions in western Europe are generally unfavourable for the sexual stage, consequently clonal reproduction is the principal mode of reproduction. The current hypothesis is that virulent forms of P.chondrillina originate in eastern Europe and, by wind dispersal of asexual spores, infect susceptible clones of C.junceaas they migrate away from the centre of origin. In western Europe, where P.chondrillinaappears to be predominantly clonal, the outcome of this selection process is that clones of the plant are matched to clones of the pathogen (Chaboudez, 1989). The practical outcome of this research was that Hasan et al. (1995) established a field planting or ‘trap garden’ of the Australian and North American forms of C.junceain Turkey, the
‘centre of diversity’, in the hope that suitable pathotypes of P.chondrillinawould infect the target weed biotypes. Suitable pathotypes were found for clones of C.
junceain the USA (Hasan et al., 1995) and potential matches were found for the
‘intermediate form’ in Australia. As the CSIRO’s work on skeleton weed ceased in 1996, the impact of the release of additional strains of P.chondrillina on C.
junceain Australia has never been assessed.
In a later study, Espiau et al. (1998) determined that there was only 58% con- gruence between host resistance phenotype and multi-locus isozyme variants in a population of C.junceafrom Turkey. Assuming sexual recombination in P.chon- drillinais common in this region, linkages among virulence loci would not always be maintained. Therefore, the disease response of an individual clone ofC.juncea would be dependent on the genetic structure of the local pathogen population at the time of infection. This information, plus the high level of isozyme variability
Genetic Markers in Rust Fungi 79
in Turkey, adds to the complexity of recognizing forms ofC.junceain Europe that might provide appropriate pathotypes of P.chondrillinafor the introduced weedy biotypes. In summary, the population genetics of this pathosystem suggest that the selection of P.chondrillina strains by the trap garden approach is a more effi- cient strategy than searching for the weedy biotypes of C. juncea in the native range in the hope of collecting a virulent rust strain. If the source populations of the clones in Australia and the USA had become extinct in Europe, then the trap garden approach would offer the only hope of finding matching rust strains.
The blackberry rust story
The increasing availability of molecular tools and the lessons learned from research on skeleton weed were the main reasons for the revival of the biocontrol programme for weedy blackberry in Australia, some 12 years after Phragmidium violaceumwas first reported in Australia in 1984. European blackberry, compris- ing closely related taxa of the Rubus fruticosusaggregate, is an important weed of agriculture, forestry and natural ecosystems in Australia. Identification of the many apomictic taxa can be difficult, as phenotypic plasticity may be high and morphological variants can arise by hybridization between taxa. A consensus in the taxonomic treatment of Rubusin Europe, let alone in Australia, has not been reached.
A strain of P.violaceum, F15, was released as a biological control agent in Australia in 1991 and 1992. Despite the spectacular success of biocontrol in a number of blackberry infestations, there appear to be some blackberry biotypes that are escaping severe disease in locations where the weather is mostly favourable for the development of rust disease. Clones of the R.fruticosusagg., a facultative apomict, were collected for identification of biotypes resistant to disease. By using M13 DNA phenotyping, we were able to identify each Rubus clone propagated for use in pathogenicity studies with P.violaceum(Evans et al., 2000, 2001b). A timely interaction with Rubus taxonomists, D.E. Symon (Australia), H.E. Weber (Germany) and A. Newton (UK), enabled us to widen the application of this DNA marker to clarify some taxonomic problems in the R.fru- ticosusagg.
Evans et al. (1998 and unpublished data) identified 33 M13 DNA phenotypes that were correlated to 13 taxa of the R.fruticosusagg. and one undetermined taxon. A further 16 DNA phenotypes were undetermined, based on morpholo- gy, or determined with only a moderate level of confidence. These undetermined DNA phenotypes are new biotypes, biotypes that have not yet been recognized and characterized in Europe, or biotypes that no longer exist in Europe. Exotic Rubus spp. have had over 150 years to evolve in Australia, and it is conceivable that new biotypes may have arisen by hybridization or somatic mutation.
An unexpected outcome of the taxonomic research was the identification of the most common and widespread weedy blackberry in Australia. This taxon, previously misnamed Rubus procerus, Rubus discolor or Rubus affin. armeniacus,
80 K.J. Evans and D.R. Gomez
appears to exist as a clonal lineage with greater than 97% of samples (n= 76) col- lected across Australia representing a single DNA phenotype. With relatively little effort we were able to sample the same DNA phenotype from England among a population of Rubus anglocandicans that is morphologically similar to the Australian material and also uniform in DNA phenotype. The weedy taxon in Australia has been renamed R.anglocandicansand can now be distinguished from R. armeniacus, the common weedy European blackberry in the pacific north- western region of the USA and in some parts of New Zealand (Evans and Weber, 2003). It has long been assumed by weed managers that Australia, New Zealand and north-western America shared their most widespread biotype of the R.fruti- cosusagg.: this new finding may have implications for the selection of biocontrol agents in the respective countries.
Disease resistance in the R. fruticosus agg. and physiological specialization among three Australian isolates of P. violaceum was identified in pathogenicity assays of 26 Rubusclones representing 17 DNA phenotypes and 14 taxa (Table 4.1). Physiological specialization in P.violaceumwas detected readily, as it was for seven P.chondrillinaisolates tested over six populations of C.junceafrom the USA (Emge et al., 1981). M13 DNA phenotyping of the rust strains used in the black- berry bioassays confirmed that genetically different rust strains were being tested (Fig. 4.2).
Table 4.1.Physiological specialization in Phragmidium violaceum. Twenty-six clones of the Rubus fruticosusagg. (European blackberry) were grouped according to patterns of susceptibility (S) or resistance (R) when inoculated with each of four strains of P.violaceum. Isolates V1, V2 and SA1 were collected in Australia between 1997 and 1999.
Strain of Group 1 Group 2 Group 3
P.violaceum (n= 22)a,b (n= 1)a,c (n = 3)a,d
F15, France S R S
V1, western Victoria S S S
V2, eastern Victoria S S R
SA1, Adelaide Hills S S R
anis the number of Rubusclones identified in each group.
b Group 1 includes the most common weedy taxon of blackberry in Australia, R.anglocan- dicans.
cA clone of R.laciniatus.
dClones representing taxa of R.erythrops, R.leucostachysand R. sp. (not determined).
In other tests, a clone of R.cissburiensiswas found to be resistant to strain SA1.
P.violaceumstrain V1, isolated from western Victoria, produced a susceptible disease response in all Rubusclones tested (Table 4.1) but many questions remain as to why some blackberry biotypes are escaping severe disease at some locations.
Genetic Markers in Rust Fungi 81
It may be that a rust strain with the corresponding virulence does arrive on the
‘resistant’ host biotype, but that it arrives too late (Burdon et al., 1996); this would delay the initiation of the epidemic and reduce disease levels at critical times in the growing season. Another explanation relates to the fact that blackberry plants exhibit leaf-age-related disease resistance (Evans and Bruzzese, 2003). Two black- berry biotypes susceptible to a particular strain ofP.violaceumand growing adja- cent to each other may have different growth rates and/or cane densities (Amor, 1975). Different growth characteristics result in blackberry canopies with differ- ent leaf-age profiles and differences in the proportion of the canopy that is sus- ceptible to disease at any given time. Indeed,P.violaceumstrain V1 was isolated from Rubusclone EB19 growing adjacent to Rubusclone EB18, and clone EB19 appeared more severely diseased than clone EB18. Both of these Rubusclones were characterized as ‘susceptible’ when inoculated with strain V1 under con- trolled-environment conditions (Evans, 2001b, and unpublished data), which sug- gests that leaf-age-related disease resistance might have been the factor most limiting rust disease on Rubus clone EB18 in situ.
While this research was being conducted, a complementary research pro- gramme was initiated by CSIRO and the CRC for Australian Weed Management to search for additional strains of P.violaceumin Europe for release in Australia. DNA phenotyping of Rubus clones enabled characterized plant
82 K.J. Evans and D.R. Gomez
Fig. 4.2.Schematic representation of Southern hybridization of probe M13 to total Phragmidium violaceumDNA, digested with the restriction enzyme HaeIII: lane 1, strain SA1, Adelaide Hills; Lane 2, strain V1, western Victoria; Lane 3, strain V2, eastern Victoria; Lane 4, strain F15 from France.
material to be shipped to Europe as in vitrocultures. These pest- and disease-free clones were established in an outdoor trap garden at the CSIRO laboratory in Montpellier. Urediniospores ofP.violaceum, in air currents over the Montpellier region, were trapped on the plants and incited disease in all 21 Rubus clones, rep- resenting 19 DNA phenotypes, planted in the garden. Rust strains were then iso- lated and multiplied from the infected plants. Prior to importation in Australia, strains of P.violaceumcollected from the Montpellier trap garden are being eval- uated for their genetic similarity to the existing population of P. violaceum in Australia, using DNA and virulence phenotyping ( J.K. Scott, M. Jourdan and K.J. Evans, unpublished data).
This approach demonstrates the use of DNA typing in streamlining the col- lection of rust strains through a well-characterized trap garden. Unlike the skele- ton rust pathosystem, lack of knowledge about the origin of P. violaceum in Europe or the Middle East meant that we were unable to determine the best loca- tion to place a stationary trap garden. If necessary, additional trap gardens could be placed in regions of high diversity of Rubus species, for example southern England, where the clone ofR.anglocandicanswas found. Alternatively, a mobile trap garden could be developed, whereby target plants are placed on the roof- rack of a car so that urediniospores are sampled from a wide range of air cur- rents. Infection of trap plants would be promoted by covering plants in moist chambers during overnight stopovers at blackberry-infested but tourist-friendly destinations!
Identifying the released pathogen with certainty
Responsible risk assessment associated with releasing an exotic organism into the environment should include identification, with certainty, of the released agent.
Separation of closely related rust species can sometimes be problematic, as illus- trated by the development of rust fungi as biocontrol agents for musk thistle (Carduus thoermeri).
The musk thistle rust story
In North America, seven species of Carduus (thistles) are introduced weeds (McCarty, 1982). Rust fungi of the genus Pucciniawere evaluated as candidates for biological control of the widespread musk thistle (C.thoermeri), but the selec- tion process was confounded by unclear relationships and morphological simi- larity among Carduus rusts. Urediniospore morphologies, host ranges and isozyme patterns of rust isolates from C.thoermeri, C.tenuiflorus and C.pycnocephaluswere compared (Bruckart and Peterson, 1991). The isozyme data supported the hypothesis that all rust isolates were P.carduorumand could be distinguished from P.carthami, a morphologically similar rust species which infects the economically important crop, safflower (C.tinctorius). Morphological differences and differences
Genetic Markers in Rust Fungi 83
in host preference among isolates of P.carduorumfrom the different Carduushosts were observed. These relationships were explored further by RFLP analysis of internal transcribed spacer (ITS) regions of rDNA (Berthier et al., 1996), which allowed P.carduorumto be separated into two groups, each group being correlat- ed to particular Carduushosts. Eight years following this release of an isolate of P.
carduorumin the USA, eight samples of urediniospores were collected across the USA from infected C.thoermeri. DNA was extracted from each sample of uredin- iospores, and the ITS2 region of rDNA was amplified. Given that each collection of urediniospores might have been genetically heterogeneous, Luster and Bruckart (1998) omitted the time-consuming step of isolating single rust pustules prior to extraction of DNA. The population of amplified products was cloned and positive clones purified before being sequenced. Therefore, each DNA clone represented an individual in the population of amplified products. The sequences of clones representing the field samples were compared with ITS2 DNA sequences of the original rust isolate released in Virginia and those of the closely related rust species, P. cyani, P. jaceae and P. chondrillina. All rust isolates from infected C.thoermerihad identical ITS2 nucleotide sequences and could be dis- tinguished from the other rust species. This information, combined with analysis of urediniospore morphology, confirmed the identity of the pathogen on musk thistle as P.carduorum.
Identifying and monitoring the fate of the released pathogen strain with certainty
Following identification of the released pathogen strain, its fate should be moni- tored, primarily to assess the impact of the release. If the pathogen strain is being released in an existing or resident population of the pathogen species, then genetic markers are essential for distinguishing the ‘immigrant’ from the resident population. DNA markers can provide sufficient resolution to separate fungal genets. Characterization of a rust strain at the level of genet should provide the most robust ‘fingerprint’ of the foreign entity being released into the environ- ment. Microsatellite loci would be ideal for this purpose, but their development is time consuming. DNA fingerprints generated with multiple AFLP primer sets should provide enough polymorphic loci to reduce the chance of detecting the same DNA phenotype by chance. Even higher levels of variation might be detected by techniques such as single-strand conformation polymorphism (Orita et al., 1989) or by heteroduplex mobility assay (Wang and Hiruki, 2000). It is unlikely that such a high genetic resolution would be required unless the rust strain being released was clonally related to the resident pathogen population.
In the past, thorough characterization of pathogen strains prior to their release as a biocontrol agent has rarely been attempted. In the case of the black- berry rust pathosystem, the virulence phenotype of strain F15 of P.violaceumwas determined partially during tests for host specificity (Bruzzese and Hasan, 1986).
The establishment and fate of strain F15 ofP.violaceumwas not monitored imme-
84 K.J. Evans and D.R. Gomez
diately after it was released in 1991. In the late 1990s, M13 DNA phenotyping was used to identify genetic variation in the Australian population ofP.violaceum (Evans et al., 2000). Thirteen DNA phenotypes of P. violaceum were identified among 18 strains collected from various locations in mainland Australia between 1997 and 1999. The restriction fragment patterns and DNA band-sharing indices of similarity suggest that the so-called ‘illegal’ strains predominate in the population of P.violaceum, and that strain F15 did not become well established in Australia. In order to verify the paucity of strain F15 or its descendants in Australia, DNA markers developed for the mitochondrial genome could define lines of descent of P. violaceum, regardless of its mode of reproduction.
Otherwise, larger sample sizes, additional DNA markers and phylogeographic analyses could be used to separate current population structure from historical events.
Morin et al. (2001) combined RAPD data with sequence determination of the ITS region of rDNA to examine genetic variation among a collection of P.
myrsiphylli strains isolated in South Africa for the biocontrol of Asparagus asparagoides (bridal creeper) in Australia. Three rust strains collected from the winter rainfall region of Western Cape Province were found to be genetically dis- tinct from three rust strains collected from the ‘even’ rainfall region of the same province. Given that the sample size is small, this genetic difference appears to be correlated to the susceptibility of Australian accessions of bridal creeper to disease caused by strains of P.myrsiphylli from each region. One strain of P.myr- siphyllifrom the winter rainfall region was released in Australia in 2000 and severe rust disease has been observed near release sites in the 2002 growing season (Morin et al., 2003). This work provides one of the first examples where a rust bio- control agent has been characterized molecularly before being released in a new environment.
Population genetics of rust fungi in relation to strain selection
Having highlighted the importance of matching weed and pathogen diversity, the question remains about what constitutes an ‘adequate’ genetic composition of the introduced agent. More precisely, what is the appropriate genetic structure of the introduced pathogen that will have the greatest impact on the target weed?
The genetic structure of a population refers to the amount and distribution of genetic variation within and between populations (McDonald, 1997). The genetic structure of a population reflects its evolutionary history and its potential to evolve. Studies of genetic diversity as described previously are an obvious start- ing point for exploring the question of whether or not the search strategy has exhausted the potential genetic variation available in the native range. How the genetic diversity is distributed within and between populations, and on what spatial scale, is the next question. For weed biocontrol, we might ask how the genetic structure and evolution of the pathogen population varies from popula- tions in their native range. An understanding of the evolutionary processes in
Genetic Markers in Rust Fungi 85