Molecular Diversity in Puccinia triticina Isolates from Ethiopia and Germany

A total of 43 isolates of the wheat leaf rust fungus, Puccinia triticina Eriks, collected from Ethiopia and Germany were analysed for their genetic diversity using the amplified fragment length polymorphism (AFLP) technique. Out of 18 EcoRI/MseI primer combinations screened, 15 produced 219 highly polymorphic fragments. The average AFLP difference between pairs of the leaf rust isolates (26 from Ethiopia, 17 from Germany) was calculated using Dice’s genetic similarity (GS) coefficient. The overall GS for the 43 isolates was 0.67 ± 0.13. The Ethiopian leaf rust isolates had lower average GS (0.63 ± 0.13) than the German ones (0.76 ± 0.10). A cluster analysis and a two-dimensional principal coordinate analysis (PCoA) grouped the 43 isolates into two significantly different (P £ 0.01) clusters. Isolates in cluster I (35 isolates) had an average GS of 0.76 ± 0.06 while the isolates in cluster II (8 isolates) had an average GS of 0.55 ± 0.12. Isolates were also grouped into three regions of collection, central Ethiopia, south and south-east Ethiopia, and Germany. The regions were significantly different at P £ 0.01 indicating regional variation in terms of molecular diversity of the leaf rust isolates studied. Each isolate, however, had a unique AFLP fingerprint. The results indicated that the leaf rust population in central Ethiopia is genetically distinct and this might be related to the predominant cultivation of durum wheat cultivars in this area. Introduction The leaf (brown) rust fungus Puccinia triticina occurs almost everywhere where wheat is grown and is considered to be one of the most important pathogens in wheat production worldwide (Dehne and Oerke, 1998). Leaf rust causes significant yield losses every year; the disease can reduce yield by 1% for every 1% increase in the infection level (Khan et al., 1997). Although effective fungicides are available, the use of host resistance is the most economical and environment-friendly method to control the disease. Especially in developing countries where fungicides are not available or their use in low input systems is economically not justified, successful crop production depends on the use of disease-resistant cultivars. Effective breeding for disease resistance requires extensive information on the incidence and virulence of endemic pathogens; which is of high importance for rust fungi as they are able to rapidly evolve new virulent races. Knowledge on the diversity of the leaf rust fungus in Ethiopia is limited; however, information on the diversity of P. triticina isolates regularly occurring in Germany is also rare; few reports on the incidence and frequency of isolates differing in virulence genes have been published for Germany (Von Kröcher et al., 1992) and Western Europe (Park and Felsenstein, 1998; Park et al., 2000, 2001) respectively. To study the molecular diversity of Ethiopian and German P. triticina isolates, amplified fragment length polymorphism (AFLP) analysis was chosen because AFLPs (i) generate more information per analysis than other molecular techniques, (ii) require no prior knowledge about the genetic make-up of the organism under study, (iii) are highly reproducible, and (iv) allow discrimination among closely related individuals within a species (Vos et al., 1995; Majer et al., 1996; Mueller and Wolfenbarger, 1999). AFLP markers have been successfully used in mycology and plant pathology for the differentiation of species within genera (Keiper et al., 2003; Menzies et al., 2003; Schmidt et al., 2004; Leisova et al., 2005b) as well as of isolates within species from straminopiles, ascomycetes and basidiomycetes (Schnieder et al., 2001; Rau et al., www.blackwell-synergy.com J. Phytopathology 154, 701–710 (2006) 2006 The Authors Journal compilation 2006 Blackwell Verlag, Berlin 2002, 2005; Abd-Elsalam et al., 2003; Radisek et al., 2003; Singru et al., 2003; Martinez et al., 2004; Leisova et al., 2005a,b; Kauserud et al., 2006). Molecular techniques such as AFLP produce neutral markers in contrast to resistance-specific virulence, which is subject to strong host selection (Kolmer, 1993; Brown, 1996) and environmental conditions. Such drawbacks limit the value of virulence markers as tools for population genetics studies (Leung et al., 1993; McDonald and McDermott, 1993). Hence, only AFLP markers were used for this study with the objective of investigating the genetic variability of leaf rust isolates collected from Germany and Ethiopia. Our general hypothesis was that genetic similarity within populations of the two countries would be considerably higher than between populations. This hypothesis might have been nullified by the existence of isolates of P. triticina specifically adapted to durum wheat in Central Ethiopia. Viennot-Bourgin (1941) and Ezzahiri et al. (1992) reported leaf rust pathogen genotypes specifically adapted to durum wheat [Triticum turgidum var. durum (Desf.) Husn.] in the Mediterranean region and such specificity was used to classify those pathogen genotypes into a different species, P. tritici-duri V.-Bourgin. As the importance of this pathogen is limited to the Mediterranean region, P. triticina has been widely accepted as the leaf rust fungal pathogen of cultivated wheat. Our objective was, however, to study the general molecular diversity of P. triticina isolates collected from Ethiopia and Germany. Our results, showing the genetic variability of isolates in central Ethiopia where durum wheat is predominantly cultivated, could be used as baseline information for further studies related to pathogen classification. Materials and Methods Sources of pathogen isolates Ethiopian isolates of leaf rust (Puccinia triticina Eriks.) were collected from farmers wheat (Triticum spp.) fields in the main cropping season (summer) of 2003. The collection was made in 10 km intervals along the roadsides. The German P. triticina isolates were kindly provided by Dr Kerstin Flath, Federal Biological Research Center for Agriculture and Forestry (BBA), Kleinmachnow, Germany (Table 1). Production of monopustule isolates Bulk collections of each sample were sparsely inoculated on detached leaf segments placed on 5% water agar in Petri dishes. The inoculated leaf segments were incubated in a chamber adjusted to 20 C with continuous white light. A single pustule was isolated with a moistened cotton swab and inoculated on leaf segments for multiplication. The spores harvested from the leaf segments were used to inoculate 7to 9-dayold seedlings of the susceptible cv. Monopol, with no known leaf rust resistance gene described, for further spore production. For each isolate, six to eight pots (7 · 7 · 8 cm) thickly sown with the susceptible cultivar were used for inoculation. The inoculated plants were kept in a moist chamber for 24 h under a dark system at ambient temperature. Then each pot was covered with a cellophane bag (145 · 235 mm) and tied with a rubber band at the base of the pot to prevent cross-contamination. While preventing contaminants, the cellophane bag allows air exchange for use by the plants inside. The inoculated seedlings were transferred to a growth chamber with 16 h/8 h light/ dark system and a temperature of 20–22 C. Spores of each isolate were harvested from infected plants 10–12 days after inoculation by tapping over aluminium foil. A total of 43 monopustule isolates were developed from bulk samples collected from the two countries.