Isolation and genotypic diversity of two Mycosphaerella graminicola populations from Australia

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1 Research Collection Student Paper Isolation and genotypic diversity of two Mycosphaerella graminicola populations from Australia Author(s): Levy, Lilia Publication Date: 2003 Permanent Link: Rights / License: In Copyright - Non-Commercial Use Permitted This page was generated automatically upon download from the ETH Zurich Research Collection. For more information please consult the Terms of use. ETH Library

2 Isolation and genotypic diversity of two Mycosphaerella graminicola populations from Australia ETH Phytopathology Group, Institute of Plant Sciences Semester Project from Lilia Levy Supervised by Marcello Zala, Dr. Celeste Linde and Prof. Bruce A. McDonald. 1

3 Introduction The pathogen Mycosphaerella graminicola is an ascomycetous fungus that causes leaf blotch on wheat. M. graminicola is the teleomorph (sexual) stage of the better-known anamorph (asexual) stage called Septoria tritici. It is a heterothallic fungus with two compatible mating types, Mat1-1 and Mat1-2 (Waalwijk et al., 2002). The sexual stage contributes to genetic recombination (Chen and McDonald, 1996) and produces airborne ascospores with the potential to be dispersed over several kilometers (primary inoculum) whereas the asexual phase, S. tritici has limited spore dispersal because it produces pycnidiospores that move only by splash dispersal (Shaw and Royle, 1989; McDonald and Martinez, 1990). Leaf blotch symptoms develop throughout the growing season on all aerial plant parts. Initial symptoms are chlorotic flecks, usually on lower leaves in contact with soil. The flecks expand into irregular leasions, normally 1-5 mm x 4-15 mm. Lesions caused by M. graminicola tend to be restricted laterally and assume parallel sides (McDonald et al., 2001). It has recently been shown that the evolutionary potential of pathogens can be determined by their genetic structure (McDonald and Linde, 2002a, b). It has been proposed that pathogens which pose the greatest risk of breaking down resistance genes are those which have a mixed reproduction system (both sexual and asexual reproduction), a potential for long distance genotype flow, large effective population sizes and high mutation rates (McDonald and Linde, 2002a, b). Because M. graminicola has a mixed reproduction system, high effective population sizes, and intermediate potential for genotype flow, it is placed in a relatively high risk category (McDonald and Linde, 2002a, b). Fig. 1. Mycosphaerella graminicola infections on wheat leaves. Fig. 2. Mycosphaerella graminicola infected wheat leaf with necrotic lesions and pycnidia within the lesions. 2

4 Introduction to mating type Sexual reproduction in heterothallic fungi involves the temporary joining of two fungal strains carrying compatible mating types, followed by meiosis and exchange of genetic information between the individuals. For these fungi, sexual reproduction is possible only when two compatible mating types are available at the same geographic location at the same point in time. When two fungal strains of opposite mating type come together, they detect each other s presence in response to the mating pheromone produced by unlike mating types (Coppin et al., 1997). Given the central role of the geographic distribution of mating types to the life cycle of heterothallic fungi and of mating systems to the evolution and population biology of fungi, this knowledge is important in understanding and predicting the population genetics, dynamics, and evolutionary potential of fungi (Zhan et al., 2002; McDonald and Linde, 2002 b). M. graminicola is a heterothallic filamentous fungus that causes Septoria leaf blotch on wheat. The mating types of M. graminicola are determined by two different alleles at a single locus (Kema et al., 1996). As studies showed (McDonald et al., 1999), the asexual reproduction of M. graminicola may have an important impact over an area of a few square meters, but sexual reproduction has much greater consequences for the population genetics and biology of the fungi. It has been shown that the two mating types are distributed in equal frequencies in most populations studied (Zhan et al., 2002). Both mating types are also often co-occurring in the same lesions, providing ample opportunities for isolates to encounter the opposite mating type and reproduce sexually (Linde et al., 2002). Introduction to Rep-PCR (Polymerase Chain Reaction) Rep-PCR is a DNA amplification based technique, which has been found to be extremely reliable, reproducible, rapid and highly discriminatory (Versalovic et al., 1994, Louws et al., 1996). Rep-PCR genomic fingerprinting makes use of DNA primers complementary to naturally occurring, highly conserved, repetitive DNA sequences, present in multiple copies in the genomes (Lupski and Weinstock, 1992). Three families of repetitive sequences have been identified, including the bp repetitive extragenic palindromic (REP) sequence, the bp enterobacterial repetitive intergenic consensus (ERIC) sequence, and the 154 bp BOX element (Versalovic et al., 1994). The use of these primer(s) and PCR leads to the selective amplification of distinct genomic regions located between REP, ERIC or BOX elements. The corresponding protocols are referred to as REP-PCR, ERIC-PCR and BOX-PCR genetic fingerprinting respectively, and Rep-PCR genomic fingerprinting collectively (Versalovic et al. 1991, 1994). The amplified fragments can be resolved in a gel matrix, yielding a profile referred to as a Rep-PCR genomic fingerprint. The Rep- PCR genomic fingerprints generated from fungal isolates permit differentiation within the species, subspecies and isolate level. In M. graminicola, DNA fingerprinting has been used to measure genotypic diversity (Chen et al., 1994), to monitor the movement of individual clones over time and space (Chen et al., 1994), and to demonstrate the distribution of genetic variation on a micro-geographical scale (Boeger et al., 1993). 3

5 Goal of the semester project This semester work is part of a major project that conducts a phylogeographical study on M. graminicola. This project attempts to answer two elementary questions regarding the population genetics of the fungi. How much genetic diversity is present within populations? How is genetic diversity distributed within and among populations? As the knowledge of the genetic structure of the pathogen increased, more complex questions were addressed. How stable are populations over time? Does selection for specific pathogen genotypes occur on particular host genotypes (McDonald et al., 1999)? To answer some of those questions, infected plant material was collected all over the world for different analyses. Comparisons of gene diversities across populations revealed some interesting patterns (McDonald et al., 1999). The collection of Israel, for example, showed a high gene and genotypic diversity in comparison to the fungal population of Mexico. This was interpreted as evidence, that the Middle East is a centre of diversity of M. graminicola, and the likely centre of origin for this fungus (McDonald et al., 1999). Although a lot of information regarding the population genetic structure of M. graminicola is available from countries around the world, there is only a limited amount of information available on Australian M. graminicola populations. Therefore infected wheat leaves were collected in 2001 in two different fields in Australia. The first goal of this semester project was to build an Australian collection of isolates. Then analyse those isolates for mating frequencies and types, prepare them for Southern blots by digesting and blotting. The final goal was to make a preliminary analysis of genotype diversity with Rep-PCR. This will broaden our knowledge on the genotypic diversity of M. graminicola in Australia. 4

6 Material and Methods Fungal and Plant Material Wheat leaves infected with M. graminicola were collected from two geographic locations in Wagga Wagga, Australia in 2001, by Bruce McDonald and Andrew Milgate. The leaves in each population were taken from a naturally infected field at a single point in time. Each infected leaf was sampled from a different plant or tiller. The hierarchical strategy (McDonald et al., 1999) was used to sample the field populations. Hierarchical sampling offers a powerful method to determine the spatial scale on which most of the genetic diversity is distributed for pathogens which have not been studied (McDonald et al., 1999). This sampling allows gene diversity to be partitioned into two levels in a hierarchy: among leaves within a site and among sites within a field. The hierarchy can be extended by sampling additional fields at different spatial scales (McDonald et al., 1999). Within each field, 80 leaves were collected in an area of ~1 m 2 for each of eight sights located along parallel transects separated by 10 m. The total geographic area covered in each of these fields was approximately 400 m 2, thought only ~8 m 2 was represented by the collection of leaves taken from each field. Collection 1 and 2 were sampled in October 2001 in Wagga Wagga, Australia (location of Collection 1: trade 186, GPS coordinates SSH , UTM ). Collection 2 was done by Mascot Poddode on Peter Sherman property. The distance between these fields was 2 km. Isolation and growth of Mycosphaerella graminicola strains Leaves were placed in paper envelopes and allowed to air-dry at room temperature for at least 48 h. Dried leaves were wet for 10 s in 70% ethanol, surface-sterilized for 90s in a 0.5% sodium hypochlorite solution, and rinsed twofold in distilled autoclaved water for 20 s. Wet leaves were pressed dry between paper towels and placed on a plastic screen that rested on rubber bands above damp filter paper in a Petri dish. Petri dishes were incubated at 20 C for 20 to 24 h to induce fungal growth and sporulation. Fine metal needles were used to remove the cirrus that emerged from the open pycnidium. Only one lesion was sampled from the same leaf and just one isolation was made from each lesion. Spores were transferred to Petri dishes (four samples per Petri dish) containing Yeast Maltose Agar (YMA) amended with 50 µg/µl of kanamycin. During the cultivation, visual control was made to eliminate possible contamination from the isolates. After incubating at 20 C for 6 days, one colony per sample was spread across fresh YMA plates in a three-dimensional trace and incubated for additional 6 days at 20 C. A single-spore-colony was harvested from each isolate by scraping gently across the mycelium with a sterile toothpick. The resulting slurry of spores was used to inoculate 100-ml flasks containing 40 ml of Yeast Sucrose Broth (YSB) amended with 50 µg/µl of kanamycin. Additionally 150 µl of the suspension were transferred into silica gel tubes for long-term storage. Inoculated flasks were incubated on a liquid shaker (135 rpm -1 ) at 20 C for 10 to 11 days. The spores were harvested by centrifugation, cold down during one hour 5

7 at -85 C and lyophilised for 2 to 3 days. Sixty-nine isolates were obtained from each collection (from a total of 80 leaves per collection). DNA- Extraction and Quantification Total DNA from each strain was extracted using the DNeasy Plant Mini Kit (Qiagen GmbH, Germany) according to the specification of the manufacturer. From this procedure we obtained a 200 µl DNA-TE buffer solution. Quantification of DNA was made using the SPECTRA Fluor Plus robot (TECAN AG, Switzerland) and the PicoGreen dsdna Quantitation Reagent (Molecular Probes) according to the recommended assay protocol by the manufacturer. Restriction digestion and Southern Blotting An enzyme buffer mastermix was prepared mixing 3.5 µl Pst I (15 u/µl) and 20 µl reaction buffer per reaction. 5 µg of DNA was used for each isolate. The amount of ddh 2 O was calculated to get a total volume of 200 µl. The mastermix and the ddh 2 O water were added to the chosen DNA into Eppendorf tubes. The samples were incubated at 37 C overnight for restriction enzyme digestion. Test gels (small gels) were made to confirm complete restriction enzyme digestions and to confirm equal DNA concentrations. DNA was electrophoresed on 0.8% agarose gels on 1 X TBE (Tris-borate-EDTA). The big gel ran at 72 volts overnight for 16 hours. It was stained in EtBr - (7 µg/l) for 20 min and destained in distilled water for 20 min. Afterwards the gel was photographed with UV light (312 nm ultraviolet light gel translumination) to visualize the nucleic acids that had been subjected to electrophoresis, and to confirm no partial digestion. The DNA in the gel was depurinated by soaking the gel in 1 litre 0.25M HCl for minutes until the blue dye changed to a yellow colour, indicating a ph change. In order to make a capillary blot, a platform was constructed and covered with a wick made from three sheets of 3MM paper saturated with 0.4N NaOH. A glass pipette was used to roll out air bubbles that would have impaired the upward flow of the DNA fragments to the Hybond-N + Nylon Transfer membrane (Amersham Pharmacia Biotech). To avoid contact between paper towels and transfer buffer a plastic ring was used to cover the platform. At the end of the depurination step, the wells were cut off and the gel was placed on top of the platform. Air bubbles were rolled out with a glass pipette. The membrane was immersed in ddh 2 O. The bottom edge of the membrane was aligned with the bottom edge of the gel. The membrane was then slowly lowered onto the gel. Air bubbles were rolled out. Three pieces of 3MM paper 20 cm x 24 cm were drawn through 0.4M NaOH and placed on top of the membrane. Air bubbles were rolled out. A stack of absorbent paper towels was placed on top of the 3MM paper and a plexiglass plate was placed on top of the towels. Finally, a 1 kg weight was put on top. After 20 hours the southern blot construct was dismantled. The membrane was washed in 2 x SSC for 5-10 minutes with agitation. The DNA was fixed to the membrane by baking it at 80 C for 2 hours. 6

8 Mating type determination The mating type of fungal strains was determined by PCR amplification of the two mating type idiomorphs (Waalwijk et al., 2002). The primer sequences used to amplify the MAT 1-1 idiomorph were 5 -CCGCTTTCTGGCTTCTTCGCACTG-3 (forward primer) and 5 -GATGCGGTTCTGGACTGGAG-3 (reverse primer), generating a 340-bp fragment in MAT 1-1 isolates. The primer sequences used to amplify the MAT 1-2 idiomorph were 5 -GGCGCCTCCGAAGCAACT-3 (forward primer) and 5 -GATGCGGTTCTGGACTGGAG-3 (reverse primer), generating a 660-bp fragment in MAT 1-2 isolates. Each PCR reaction mix (total reaction volume 20 µl) contained: 0.5 µm of each primer (all four together), 0.1 mm of datp, dctp, dttp and dgtp; buffer (100mM Tris-HCl, ph9; 15 mm MgCl 2 ; 500 mm KCl); 1 unit of Taq Polymerase (Amersham Pharmacia Biotech) and 5-20 ng of template. PCR amplification was carried out using a Biometra Termocycler (Germany). The PCR reaction conditions were: denaturation at 96 C for 2 min followed by 35 cycles of denaturation at 96 C for 1 min, annealing at 61 C for 1 min and elongation at 72 C for 1 min with a final extension step at 72 C for 10 min. PCR reaction products were separated in a 1% agarose gel (0.5 X TBE) electrophoresis at 80 volts for 1.5 hours and visualised by UV irradiation at 312 nm. Genomic Fingerprints: primers, PCR conditions and scoring The genotype of the fungal strain was determined using the Rep-PCR technology. The primer sequences used to amplify the genomic region of interest were BOX A1R as reverse primer (5 -CTACGGCAAGGCGACGCTGACG-3 ) and ERIC 2L as forward primer (5 -AAGTAAGTGACTGGGGTGAGCG-3 ). Those primers were used as they are the most suitable for M. graminicola (Sommerhalder et al., 2001). Rep-PCR reaction mix (total reaction volume 20 µl) contained: 0.5 µm of each primer, 0.1 mm of datp, dctp, dttp and dgtp; buffer (100mM Tris-HCl, ph9;15 mm MgCl 2 ; 500 mm KCl); 1 unit of Taq Polymerase (Amersham Pharmacia Biotech) and 5-20 ng of template. PCR amplification was carried out using a Biometra Termocycler (Germany). The PCR reaction conditions were: denaturation at 96 C for 2 min followed by 35 cycles of denaturation at 94 C for 30 s, annealing at 52 C for 1 min and elongation at 65 C for 5 min with a final extension step at 65 C for 8 min. PCR reaction products were separated in a 1% agarose gel (1 X TBE) electrophoresis. The gel ran at 72 volts overnight for about 16 hours, stained in EtBr - (7 µg/l) for 20 minutes and was then visualised by UV irradiation (312 nm). The scoring of the PCR amplified segments of DNA from all isolates was made based on the presence or absence of amplicons (Fig. 5). Twenty-four of the amplified segments ranging from 470 bp to 2850 bp that were clearly present were scored with 1, not well-defined bands were scored with 0 (appendix, tables 5 and 6). As the dataset of amplicons larger than 1400 bp and smaller than 700 bp was not consistent, those bands were excluded from the final analysis. Twelve amplicons of this segment were chosen: 1400 bp, 1350 bp, 1280 bp, 1200 bp, 1110 bp, 1050 bp, 1000 bp, "960 bp, 800 bp, 780 bp, "740 bp and 700 bp. Isolates with the same multilocus fingerprint pattern were considered clonal. Genotypic diversity was calculated using the genotype diversity measure of Stoddart and Taylor (1988). 7

9 Results Southern Blots From a total of 138 isolates obtained from the two Australian collections, four southern blots were made by digesting 5 µg DNA per isolate with 3.5 µl Pst I (15 u/µl). This four blots (Fig. 3; appendix Table 2) comprising a total of 96 isolates were chosen according to their DNA concentration. The remaining 42 isolates did not contain enough DNA for this analysis. Fig. 3. An example of a big gel of Mycosphaerella graminicola after a run of 16 h at 72 V. The vertical lines represent 5 µg DNA of M. graminicola digested with 3.5 µl Pst I (15 u/µl). λ- Hind III size standard is shown in the first line on the left. The further lines are from Collection 1, Blot 2, samples 1 to 24 from the left to the right. For database see appendix Table 2. 8

10 Mating type determination A total of 138 fungal strains (STAus01 Collections 1 and 2; appendix Table 1) were assayed using PCR amplification with mating type specific primers; 131 of these strains produced single bands of the expected size, corresponding to either the MAT 1-1 or MAT 1-2 band (Fig. 4). The remaining 7 samples did not produce a visible band (e.g. Fig. 4, Line 10, 2 nd isolate from the left). From the 131 fungal strain samples 65 were found to be of the MAT 1-1 and 66 of the MAT 1-2 (Table 1). In four cases there where two bands, one stronger and one weaker (Fig. 4; Line 6, 4 th and 5 th isolate from the left, Line 8, 7 th isolate and Line 10, 5 th isolate). The isolates where counted as being from the mating type corresponding to the stronger one. This could be due to a mixed culture. Line 1 Line 2 Line bp 340 bp Line 4 Line 5 Line bp 340 bp Line 7 Line 8 Line bp 340 bp Line 10 Line 11 Line bp 340 bp Fig. 4. An example showing the PCR products of 96 isolates of Mycosphaerella graminicola DNA, amplified with MAT 1-1 and MAT 1-2 specific primers. On the right sight, bands corresponding to MAT 1-1 and MAT 1-2 (340 bp and 660 bp respectively) are indicated by an arrow. The upper data stem from Workbox 10, lines 1 to 12. Line 1 corresponds to the isolates of Workbox 10 A1 to H1, line 2 to those of B1 to H1 and so on. In the Collection 1, 33 samples were found to be of the mating type MAT 1-1 and also 33 of MAT 1-2. Collection 2 showed a proportion of 32 MAT 1-1 to 33 MAT 1-2 (Table 1; appendix Table 1). 9

11 Table 1. Distribution of mating types within two Australian collections of Mycosphaerella graminicola. Both MAT 1-1 and MAT 1-2 have a nearby equal frequency in the Collections 1 and 2. PCR- products Collection Number of isolates Proportion MAT MAT No products MAT MAT No products Genotype determination by Rep- PCR (BOX and ERIC DNA fingerprints) In this semester project we used Rep- PCR, with BOX and ERIC primers to study the genotype diversity of 138 isolates of Mycosphaerella graminicola. PCR amplified segments of DNA from all isolates when resolved by electrophoresis resulted in a pattern of multiple bands (Fig. 5; appendix Fig.1-5). Amplified segments ranged from 470 bp to 2850 bp. Twenty-four bands were scored in an excel-sheet (appendix Tables 3 and 4) using 1 when a well-defined band was present and 0 when no band could be distinguished. As the dataset of amplicons larger than 1400 bp and smaller than 700 bp was not consistent and to ensure that only reliable bands were scored, data analysis was done based on well-defined, resolved bands ranging in size from 1400 bp to approximately 700 bp. Marker bp 1200bp 1031bp 900bp 700bp 1400bp 1110bp 960bp 780bp 700bp 500bp Fig. 5. Rep-PCR genomic Fingerprinting of 24 M. graminicola isolates. The primer sequences used to amplify the genomic region of interest were BOX A1R as reverse primer and ERIC 2L as forward primer. The gel ran at 72 volts overnight for about 16 hours, stained in EtBr - (7 µg/l) for 20 minutes and was then visualised by UV irradiation (312 nm). On the left sight the 100 bp marker. Arrows are indicating some of its amplicons. From the left to the right isolates 1 to 24 of the Collection 1 (appendix Table 3). Twelve bands ranging from 1400 bp to 700 bp were scored for further analysis (arrows are indicating some of its amplicons). 10

12 The following bands were used for data analysis: 1400 bp, 1350 bp, 1280 bp, 1200 bp, 1110 bp, 1050 bp, 1000 bp, "960 bp, 800 bp, "780 bp, "740 bp and 700 bp. In the Australia field population, 30 different genotypes were found among the 108 isolates in the sample. Some genotypes were present at a high frequency. Two genotypes made up over 37% of the collection (Table 2) and one of these genotypes was found 25 times in the collection (e.g. Fig. 5, sample 21). The pattern of this most common genotype was using the code described above was in Collection 2 the second and in Collection 1 the third most frequent pattern (e.g. Fig. 5, sample16). The sequence took the second place in Collection 1. The average number of amplicons obtained in Collections 1 and 2 was 6.00 and did not differ significantly (standard deviation ranging from ) between populations (Table 2). The number of amplicons scored for each isolate ranged from 3 to 8 (Table 2), distinguishing 30 genotypes from 108 isolates. Genotypic diversity for both populations was low (Ĝ 1 = 6.9; Ĝ 2 = 10.4) and represent 12.2% of the theoretical maximum of Collection 1 respectively 20.3% of the Collection 2. The average of both populations was 8.3%. A larger number of genotypes could be distinguished when all the amplicons were considered. Table 2. Genotypic diversity and other parameters in Mycosphaerella graminicola populations. Parameters Collection 1 Collection 2 Collections 1 & 2 Average number of amplicons (Std. dev.) 6.09 (0.86) 5.90 (1.07) 6.00 (0.97) Minimum number of amplicons Maximum number of amplicons Total number of isolates Number of isolates with data Number of genotypes Frequency of the 2 most common genotypes 43.9% 35.3% 37.0% Genotypic diversity (Ĝ) Theoretical maximum (Ĝ/N)% 12.2% 20.3% 8.3% 11

13 Discussion Mycosphaerella graminicola infected wheat leaves of two different fields were collected in Australia in The fungi were isolated, cultivated and a single spore colony was obtained. The DNA of these isolates was extracted, quantified and used for further analysis. Southern Blotting was made after digesting the DNA, but as we did not have enough time to hybridize the blots, it isn t possible to make conclusions on this topic. PCR of the MAT 1-1 and MAT 1-2 idiomorphs were used to determine the frequency of mating types in the Australian population. Collection 1 showed exactly the same number of MAT1-1 as of MAT1-2 strains. The proportion in the Collection 2 lies with 50.38% MAT1-1 to 49.62% MAT1-2 close to the 50% mark. Thus it appears that both populations in Australia are undergoing regularly sexual reproduction. In comparison, a study with Stagonospora nodorum revealed an unequal proportion of 92% MAT1-1 to 8% MAT1-2, regarding the isolates that could be identified (Halama, 2002). The remaining 7 samples that did not produce a visible band (e.g. Fig.4, Line 10, 2 nd isolate from the left) where observed on their Rep-PCR characteristics. As no Rep-PCR amplicons where obtained, we conclude that there was no fungal DNA in these samples (compare DNA Quantification, Appendix, Table 6). The PCR-Mating type results corresponded to the expectance of a 50% frequency of each mating type. Because the two mating types occur in approximately equal frequency and are evenly distributed in both collections, this fungus regularly experiences the optimum conditions needed for compatible fungal strains to meet and mate (Zhan et al., 2002). An advantage of sex to the pathogen is that new combinations of genes can come together through sexual reproduction each generation, leading to a high degree of genotype diversity that may enable some component of the pathogen population to survive in a threatening environment (McDonald and Linde, 2002 b). Rep-PCR was used to screen the dataset for polymorphisms. The section with more consistent dataset was chosen for evaluation. The two most common genotypes made up thirty seven per cent of the isolates, considering only this mentioned section. These data suggest that a significant fraction of the population was clonal. This fact is confirmed by the genotypic diversity measure (Stoddart and Taylor, 1988). The obtained values ranging from 6.9% to 10.4% of its maximum value are very low and indicate a high degree of asexual reproduction. However, if the whole dataset including very large and small amplicons (2850 bp to 470 bp) are considered, noticeable differences among the samples are visible. In this case isolates could be resolved into many more genotypes (79). However, these amplicons were not consistent and were excluded from the analysis. In consideration of the fact, that there is a nearly equal frequency of both compatible mating types, it is possible, that a large number of different genotypes should be present. Genotype diversity was relatively low compared to other M. graminicola populations studied previously (Linde et al., 2002). This could indicate that the Australian populations studied here, represent a founder population with low genotypic diversity. These results are confirmed by other studies (McDonald et al., 1999) and by preliminary RFLP results of our Australian blots (T. Jürgens, personal communication). 12

14 Two explanations for the large number of clones are plausible. Firstly, the way the scoring was done. As only well defined amplicons were scored, weaker uncertain bands were not counted. Consequently some samples were identified as clonal, although there would be discriminating bands on the non regarded area. Another reason for low genotypic diversity could be that in the previous studies, genotypes were identified by a combination of single copy and multicopy RFLP markers, giving a greater resolution of genotypes compared to using only one marker, Rep-PCR. This would suggest that Rep-PCR alone is not sufficient to distinguish clones in M. graminicola. This work offered additional information about the M. graminicola population in Australia. As wheat was introduced relatively late to this continent we expected to find a relatively low genotypic diversity, because of a founder event. We ascertain that in conjuction with the wheat, both compatible mating types were introduced to Australia. According to the equal frequencies of mating types of Mycosphaerella graminicola in the fields, M. graminicola must be undergoing a frequent sexual reproduction in this continent, pushing pathogen evolution and raising its polymorphism, although we found a high percentage of clonal isolates. Asexual reproduction may have an important impact over an area of a few square meters, but sexual reproduction has much greater consequences for the population genetics and biology of the fungus. Nevertheless it is still imperative to carry out further research to confirm these results and reconstruct pathogen evolution and predict best way of crop protection. Acknowledgements I would like to thank Mr. Marcello Zala, who gave me the instructions during the month I worked in the Phytopathology Department and afterwards in the writing period. Dr. Celeste Linde who supported me and made helpful comments and suggestions on the script, and finally Mr. Bruce A. McDonald for having offered me this semester project. 13

15 Literature Boeger, J.M., R.S. Chen, B.A. McDonald (1993): Gene flow between geographic populations of Myosphaerella graminicola (anamorph Septoria tritici) detected with RFLP markers. Phytopathology. 83, Chen, R.S., J.M. Boeger, B.A. McDonald (1994): Genetic stability in a population of a plant pathogenic fungus over time. Mol. Ecol. 3, Chen, R.S., B.A. McDonald (1996): Sexual reproduction plays a major role in the genetic structure of populations of the fungus Mycosphorella graminicola. Genetics 142, Coppin, E., R. Debuchy, S. Arnaise, M. Picard (1997): Mating types and sexual development in filamentous ascomycetes. Microbiol. Mol. Biol. Rew. 61, (Coppin et al., 1997) Halama, P. (2002). Mating relationships between isolates of Phaeosphaeria nodorum, (anamorph Stagonospora nodorum) from geographical location. European Journal of Plant Pathology 108, Kema, G.H.J., E.C.P. Verstappen, M. Todorova, C. Waalwijk (1996): Successful crosses and molecular tetrad and progeny analyses demonstrate heterothallism in Mycosphaerella graminicola. Curr. Genet. 30, Linde, C.C., J. Zhan, B.A. McDonald (2002). Population structure of Mycosphaerella graminicola: From lesions to continents. Phytopathology 92, Louws, F.J., M. Schneider, F.J. de Bruijn (1996): Nucleic Acid Amplification Methods for the Analysis of Environmental Samples. Toranzos G (ed), Technomic Publishing Co Lupski, J.R., G.M. Weinstock (1992): J. Bacteriol. 174, McDonald, B.A., J. Zahn, O. Yarden, K. Hogan, J. Garton, R.E. Pettway (1999): The Population Genetics of Mycosphaerella graminicola and Stagonospora nodorum. In Lucas, J.A., P. Bowyer, H.M. Anderson (Eds.). Septoria on Cereals: a Study of Pathosystems, CAB, Wallingford UK, McDonald, B.A. (2001) : Septoria diseases on wheat caused by Mycosphaerella graminicola (anamorph Septoria tritici) and Phaeosphaeria nodorum (anamorph Stagonospora nodorum). Plant Pathology, Small grain pathology I. McDonald B.A., C. Linde (2002 a): Pathogen population genetics and the durability of disease resistance. Annu. Rev. Phytopathol. 40, McDonald B.A., C. Linde (2002 b): The population genetics of plant pahtogens and breeding strategies for durable resistance. Euphytica. 124, McDonald B.A., J.P. Martinez (1990): Restriction fragment length polymorphisms in Septoria tritici occur at a high frequency. Curr. Genet. 17,

16 Shaw, M.W., D.J. Royle (1989): Airborne inoculum as a major source of Septoria tritici (Mycosphaerella graminicola) infections in winter wheat crops in the UK. Plant Pathol. 38, Sommerhalder, R., S. Banke, B.A. McDonald (2001): Rep fingerprinting of the cereal pathogens Rynchosporium secalis, Mycosphaerella graminicola and Satgonospora nodorum. ETH Phytopathology Group, Semester Project, Institute of Plant Sciences. Stoddart, J.A., F.F. Taylor (1988): Genotypic diversity: Estimation and prediction in samples. Genetics. 118, Versalovic, J., T. Koeuth, J.R. Lupski (1991): Nucleic Acids Res. 19, Versalovic, J., M. Schneider, F.J. de Bruijn, J.R. Lupski (1994): Meth. Cell. Mol. Biol. 5, Waalwijk, C., O. Mendes, E.D.P. Verstappen, M.A. de Waard, G.H.J. Kema (2002): Isolation and characterization of the mating-type idiomorphs from the wheat septoria leaf blotch fungus Mycosphaerella graminicola. Fungal Genet. Biol. 35, Zhan, J., G.H.J. Kema, C. Waalwijk, B.A. McDonald (2002): Distribution of mating type alleles in the wheat pathogen Mycosphaerella graminicola over spatial scales from lesions to continents. Fungal Genet. Biol. 36,

17 Appendix Table 1. Mating types and genotypes of Mycosphaerella graminicola isolates from two different fields in Wagga Wagga, Australia (2001). Collection 1 Collection 2 Leaf number Isolate Mating type Genotype Leaf number Isolate Mating type Genotype 1 STAus01-1A2 MAT STAus01-2A1 MAT STAus01-1A3 MAT STAus01-2A2 MAT STAus01-1A4 MAT STAus01-2A3 MAT STAus01-1A5 MAT STAus01-2A4 MAT STAus01-1A6 MAT STAus01-2A STAus01-1A7 MAT STAus01-2A6 MAT STAus01-1A9 MAT STAus01-2A7 MAT STAus01-1A10 MAT STAus01-2A8 MAT STAus01-1A11 MAT STAus01-2A9 MAT STAus01-1B1 MAT STAus01-2A10 MAT STAus01-1B2 MAT STAus01-2B1 MAT STAus01-1B3 MAT STAus01-2B2 MAT STAus01-1B4 MAT STAus01-2B3 MAT STAus01-1B5 MAT STAus01-2B4 MAT STAus01-1B6 MAT STAus01-2B5 MAT STAus01-1B7 MAT STAus01-2B6 MAT STAus01-1B8 MAT STAus01-2B7 MAT STAus01-1C1 MAT STAus01-2B9 MAT STAus01-1C2 MAT STAus01-2C1 MAT STAus01-1C3 MAT STAus01-2C2 MAT STAus01-1C4 MAT STAus01-2C3 MAT STAus01-1C5 MAT STAus01-2C4 MAT STAus01-1C6 MAT STAus01-2C6 MAT STAus01-1C7 MAT STAus01-2C7 MAT STAus01-1C8 MAT STAus01-2D1 MAT STAus01-1D STAus01-2D2 MAT STAus01-1D STAus01-2D3 MAT STAus01-1D4 MAT STAus01-2D4 MAT STAus01-1D5 MAT STAus01-2D5 MAT STAus01-1D7 MAT STAus01-2D6 MAT STAus01-1D8 MAT STAus01-2D8 MAT STAus01-1D9 MAT STAus01-2D9 MAT STAus01-1E1 MAT STAus01-2D10 MAT STAus01-1E2 MAT STAus01-2E1 MAT STAus01-1E3 MAT STAus01-2E2 MAT STAus01-1E4 MAT STAus01-2E3 MAT STAus01-1E5 MAT STAus01-2E4 MAT STAus01-1E6 MAT STAus01-2E6 MAT STAus01-1E7 MAT STAus01-2E7 MAT STAus01-1E8 MAT STAus01-2E8 MAT STAus01-1E9 MAT STAus01-2E9 MAT STAus01-1E10 MAT STAus01-2E10 MAT STAus01-1F1 MAT STAus01-2F1 MAT STAus01-1F2 MAT STAus01-2F4 MAT STAus01-1F3 MAT STAus01-2F5 MAT STAus01-1F4 MAT STAus01-2F6 MAT STAus01-1F STAus01-2F7 MAT STAus01-1F6 MAT STAus01-2F8 MAT STAus01-1F7 MAT STAus01-2F9 MAT STAus01-1F8 MAT STAus01-2F STAus01-1F9 MAT STAus01-2G1 MAT STAus01-1F10 MAT STAus01-2G3 MAT STAus01-1G1 MAT STAus01-2G4 MAT STAus01-1G2 MAT STAus01-2G STAus01-1G3 MAT STAus01-2G6 MAT STAus01-1G4 MAT STAus01-2G7 MAT STAus01-1G5 MAT STAus01-2G8 MAT STAus01-1G6 MAT STAus01-2G9 MAT STAus01-1G7 MAT STAus01-2G10 MAT STAus01-1G8 MAT STAus01-2H1 MAT STAus01-1G10 MAT STAus01-2H2 MAT STAus01-1H1 MAT STAus01-2H3 MAT STAus01-1H2 MAT STAus01-2H STAus01-1H3 MAT STAus01-2H5 MAT STAus01-1H6 MAT STAus01-2H6 MAT STAus01-1H7 MAT STAus01-2H7 MAT STAus01-1H8 MAT STAus01-2H8 MAT STAus01-1H9 MAT STAus01-2H9 MAT STAus01-1H10 MAT STAus01-2H10 MAT

18 Table 2. Isolates of Mycosphaerella graminicola used for Southern Blotting. Besides the consecutive numeration in each blot you can see the respective isolate with its DNA concentration. The DNA concentration was a criterion for selection of 96 isolates out of 138 for further analysis. Blot 1 (Collection 1) Blot 2 (Collection 1) Consecutive Isolate DNA concentration Consecutive Isolate DNA concentration number (ng/ul) number (ng/ul) 1 STAus01-1A STAus01-1D STAus01-1A STAus01-1E STAus01-1A STAus01-1E STAus01-1A STAus01-1E STAus01-1A STAus01-1E STAus01-1A STAus01-1E STAus01-1A STAus01-1F STAus01-1A STAus01-1F STAus01-1A STAus01-1F STAus01-1B STAus01-1F STAus01-1B STAus01-1F STAus01-1B STAus01-1F STAus01-1B STAus01-1G STAus01-1B STAus01-1G STAus01-1B STAus01-1G STAus01-1C STAus01-1G STAus01-1C STAus01-1H STAus01-1C STAus01-1H STAus01-1C STAus01-1H STAus01-1C STAus01-1H STAus01-1C STAus01-1E STAus01-1C STAus01-1E STAus01-1D STAus01-1G STAus01-1D STAus01-1H Blot 3 (Collection 2) Blot 4 (Collection 2, mainly) Consecutive Isolate DNA concentration Consecutive Isolate DNA concentration number (ng/ul) number (ng/ul) 1 STAus01-2A STAus01-2E STAus01-2A STAus01-2E STAus01-2A STAus01-2F STAus01-2A STAus01-2F STAus01-2A STAus01-2F STAus01-2B STAus01-2F STAus01-2B STAus01-2F STAus01-2B STAus01-2G STAus01-2B STAus01-2G STAus01-2B STAus01-2G STAus01-2B STAus01-2H STAus01-2C STAus01-2H STAus01-2C STAus01-2H STAus01-2D STAus01-2C STAus01-2D STAus01-2E STAus01-2D STAus01-2A STAus01-2D STAus01-2C STAus01-2D STAus01-2D STAus01-2D STAus01-2H STAus01-2E STAus01-1H STAus01-2E STAus01-1H STAus01-2E STAus01-2A STAus01-2E STAus01-2E STAus01-2E STAus01-2G

19 Table 3. Scoring of the genomic fingerprint of Mycosphaerella graminicola strains, Collection 1 from Australia in 2001, with BOX and ERIC-PCR patterns. Twenty-four bands ranging from 2850 bp to 470 bp were scored manually using 1 when a well- defined band was present and 0 when no band could be distinguished. The following bands were used for further analysis: 1400 bp, 1350 bp, 1280 bp, 1200 bp, 1110 bp, 1050 bp, 1000 bp, 960 bp, 800 bp, 780 bp, 740 bp and 700 bp. Collection 1 No Isolate Genotype 1 STAus01-1A STAus01-1A STAus01-1A STAus01-1A STAus01-1A STAus01-1A STAus01-1A STAus01-1A STAus01-1A STAus01-1B STAus01-1B STAus01-1B STAus01-1B STAus01-1B STAus01-1B STAus01-1B STAus01-1B STAus01-1C STAus01-1C STAus01-1C STAus01-1C STAus01-1C STAus01-1C STAus01-1C STAus01-1C STAus01-1D STAus01-1D STAus01-1D STAus01-1D STAus01-1D STAus01-1D STAus01-1D STAus01-1E STAus01-1E STAus01-1E STAus01-1E STAus01-1E STAus01-1E STAus01-1E STAus01-1E STAus01-1E STAus01-1E STAus01-1F STAus01-1F STAus01-1F STAus01-1F STAus01-1F STAus01-1F STAus01-1F STAus01-1F STAus01-1F STAus01-1F STAus01-1G STAus01-1G STAus01-1G STAus01-1G STAus01-1G STAus01-1G STAus01-1G STAus01-1G STAus01-1G STAus01-1H STAus01-1H STAus01-1H STAus01-1H STAus01-1H STAus01-1H STAus01-1H STAus01-1H

20 Table 4. Scoring of the genomic fingerprint of Mycosphaerella graminicola strains, Collection 2 from Australia in 2001, with BOX and ERIC-PCR patterns. Twenty-four bands ranging from 2850 bp to 470 bp were scored manually using 1 when a well- defined band was present and 0 when no band could be distinguished. The following bands were used for further analysis: 1400 bp, 1350 bp, 1280 bp, 1200 bp, 1110 bp, 1050 bp, 1000 bp, 960 bp, 800 bp, 780 bp, 740 bp and 700 bp. Collection 2 No Isolate Genotype 1 STAus01-2A STAus01-2A STAus01-2A STAus01-2A STAus01-2A STAus01-2A STAus01-2A STAus01-2A STAus01-2A STAus01-2A STAus01-2B STAus01-2B STAus01-2B STAus01-2B STAus01-2B STAus01-2B STAus01-2B STAus01-2B STAus01-2C STAus01-2C STAus01-2C STAus01-2C STAus01-2C STAus01-2C STAus01-2D STAus01-2D STAus01-2D STAus01-2D STAus01-2D STAus01-2D STAus01-2D STAus01-2D STAus01-2D STAus01-2E STAus01-2E STAus01-2E STAus01-2E STAus01-2E STAus01-2E STAus01-2E STAus01-2E STAus01-2E STAus01-2F STAus01-2F STAus01-2F STAus01-2F STAus01-2F STAus01-2F STAus01-2F STAus01-2F STAus01-2G STAus01-2G STAus01-2G STAus01-2G STAus01-2G STAus01-2G STAus01-2G STAus01-2G STAus01-2G STAus01-2H STAus01-2H STAus01-2H STAus01-2H STAus01-2H STAus01-2H STAus01-2H STAus01-2H STAus01-2H STAus01-2H

21 Table 5. The following table shows the plate set up for DNA Quantification on TECAN. The samples were collected in Australia in Collection 1 goes from A1 (1A2) to E9 (1H10) from the first box and Collection 2 follows from G9 (2A1) from the first box to B7 (2H10) from the second box A 1A2 1A11 1B8 1C8 1E1 1E9 1F7 1G5 1H6 2A4 2B2 C B 1A3 1B1 1C1 1D2 1E2 1E10 1F8 1G6 1H7 2A5 2B3 O C 1A4 1B2 1C2 1D3 1E3 1F1 1F9 1G7 1H8 2A6 2B4 N D 1A5 1B3 1C3 1D4 1E4 1F2 1F10 1G8 1H9 2A7 2B5 T E 1A6 1B4 1C4 1D5 1E5 1F3 1G1 1G10 1H10 2A8 2B6 R F 1A7 1B5 1C5 1D7 1E6 1F4 1G2 1H1 2A1 2A9 2B7 O G 1A9 1B6 1C6 1D8 1E7 1F5 1G3 1H2 2A2 2A10 2B9 L H 1A10 1B7 1C7 1D9 1E8 1F6 1G4 1H3 2A3 2B1 2C A 2C2 2D4 2E3 2F4 2G3 2H1 2H9 C B 2C3 2D5 2E4 2F5 2G4 2H2 2H10 O C 2C4 2D6 2E6 2F6 2G5 2H3 N D 2C6 2D8 2E7 2F7 2G6 2H4 T E 2C7 2D9 2E8 2F8 2G7 2H5 R F 2D1 2D10 2E9 2F9 2G8 2H6 O G 2D2 2E1 2E10 2F10 2G9 2H7 L H 2D3 2E2 2F1 2G1 2G10 2H8 Table 6. The following tables correspond to the upper two tables (see attachment table 7). They show the results of the DNA Quantification on TECAN : total DNA in µg per isolate. These samples of Mycosphaerella graminicola collected in Australia in 2001, Collection 1 and 2, have an average content of µg of fungal DNA A C B O C N D T E R F O G L H A C B O C N D T E R F O G L H

22 Fig. 1. Rep- PCR fingerprint of Mycosphaerella graminicola strains with BOX and ERIC- PCR patterns. The 100 bp marker can be seen on the very left side followed by 24 samples. The samples correspond to the isolates 25 (STAus01-1C8) to 48 (STAus01-1F6) of Collection 1 from the left to the right. Fig. 2. Rep- PCR fingerprint of Mycosphaerella graminicola strains with BOX and ERIC- PCR patterns. The 100 bp marker can be seen on the very left side followed by 24 samples. The samples correspond to the isolates 49 (STAus01-1F7) to 69 (STAus01-1H10) of Collection 1 and to the isolates 1 (STAus01-2A1) to 3 (STAus01-2A3) of Collection 2 from the left to the right. 21

23 Fig. 3. Rep- PCR fingerprint of Mycosphaerella graminicola strains with BOX and ERIC- PCR patterns. The 100 bp marker can be seen on the very left side followed by 24 samples. The samples correspond to the isolates 4 (STAus01-2A4) to 27 (STAus01-2D3) of Collection 2 from the left to the right. Fig. 4. Rep- PCR fingerprint of Mycosphaerella graminicola strains with BOX and ERIC- PCR patterns. The 100 bp marker can be seen on the very left side followed by 24 samples. The samples correspond to the isolates 28 (STAus01-2D4) to 51 (STAus01-2G1) of Collection 2 from the left to the right. 22

24 Fig. 5. Rep- PCR fingerprint of Mycosphaerella graminicola strains with BOX and ERIC- PCR patterns. The 100 bp marker can be seen on the very left side followed by 18 samples. The samples correspond to the isolates 52 (STAus01-2G3) to 69 (STAus01-2H10) of Collection 2 from the left to the right. 23