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Rep fingerprinting of the cereal pathogens Rhynchosporiumsecalis, Mycosphaerella graminicola and Stagonospora nodorumSemesterarbeit, Summer 2001

Author(s): Sommerhalder, Rubik Jaffarou

Publication Date: 2001

Permanent Link: https://doi.org/10.3929/ethz-a-004303418

Rights / License: In Copyright - Non-Commercial Use Permitted

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Semesterarbeit Rubik Sommerhalder, Summer 2001: Rep-PCR fingerprinting of plant pathogenic fungi 1

Rep fingerprinting of the cereal pathogens Rhynchosporium secalis,Mycosphaerella graminicola and Stagonospora nodorum

ETH Phytopathology Group, Institute of Plant Sciences

Supervised by Dr. Soren Banke and Prof. Bruce A. McDonald

Introduction

Introduction to Rep-PCR (Polymerase Chain Reaction) Rep-PCR is a relatively new technique for measuring genotype diversity in fungal populations, which cancomplement the existing DNA-based methods of RFLP fingerprinting and multilocus analysis of total DNA afterhybridization with a series of random DNA probes. Rep-PCR (repetitive element based polymerase chainreaction, George et al. 1998) generates DNA fingerprints by amplifying sequences between randomlydispersed copies of a repeated DNA element in the genome. The rep-PCR technique was first applied tobacteria and only recently was applied to fungi to understand the population biology of fungal plant pathogens.

The goal in this project was to define primer-pairs, which could work efficiently for rep-PCR-based DNAfingerprinting of several fungi that are significant pathogens on cereals. The DNA fingerprints will be used todifferentiate clones in order to measure genotype diversity within pathogen populations and gauge theirgenetic structure and evolutionary potential.

Rep-PCR is based on the fact that repetitive DNA sequences are found in the genome of the majority ofeucaryotes and prokaryotes. The function of these repetitive elements is not often known, but it seems thatthey often have no importance or effect on phenotype. We could compare these repetitive elements toparasites that replicate within an organism’s genome (parasitic or junk DNA). Particular repetitive elementsexist as families that have a specific conserved sequence conformation. DNA primers can be designed tomatch the conserved sequence elements for particular families of repetitive elements. These primers can beused to amplify the sections of DNA that exist between adjacent copies of the repetitive elements. Some of theprimers work in the forward direction (5’ to 3’) and other primers work in the reverse direction (3’ to 5’) on adouble-stranded DNA molecule. Taq polymerase is a heat-stable enzyme needed to polymerize DNA and is acritical component of the polymerase chain reaction.

Why do we consider Rep-PCR as a method of DNA fingerprinting?

We test the Rep-PCR method because the current methods (RFLP fingerprinting of digested DNA andmultilocus PCR or RFLP methods) are too time- and resource-consuming and therefore make it prohibitive toanalyze a large number of samples in population studies. In particular we plan to apply a series of candidaterep-PCR primers to the cereal pathogens Rhynchosporium secalis, Stagonosopora nodorum andMycosphaerella graminicola to differentiate the different clones that exist in farmer’s fields. I will test differentprimer combinations for good amplification and usefulness against populations of all three fungi.

The project can be divided into three parts. In the first part, primer PCR conditions will be optimized. In thesecond stage, data will be collected from different populations. In the third part, the data will be analyzed anda report summarizing the results will be written.

Population Biology:

Mycosphaerella graminicola is an ascomycete fungus that causes septoria tritici leaf blotch on wheat. M.graminicola is the teleomorph (sexual) stage of the better-known anamorph (asexual) stage called Septoriatritici. The sexual stage contributes to genetic recombination (Chen and McDonald 1996) and has airbornespores with the potential to be dispersed over several km (primary inoculum) whereas the asexual phase,Septoria tritici has limited spore dispersal because it produces pycnidiospores that move only by splashdispersal.

One sample of the wheat pathogen M. graminicola consisted of a set of 48 isolates (Swiss isolates from asingle leaf lesion) covering an area of a few square centimeters. A second collection was composed of three

Semesterarbeit Rubik Sommerhalder, Summer 2001: Rep-PCR fingerprinting of plant pathogenic fungi 2populations (96 isolates total) originating from Switzerland, Oregon, and Israel. The fields sampled for thesepopulations had roughly the same dimensions, with different fields separated by distances from 20 up toseveral thousand km. In the first collection of isolates we expected to find little diversity, or none at all, becausemany of the isolates within a lesion were expected to be the same clone. For the second set of isolates, whichoriginated from three different regions in the world, we expected more differences would exist betweenpopulations and within populations because these isolates didn`t come from the same leaf.

Rhynchosporium secalisThis fungal pathogen causes the scald disease on barley. It is not known to have a sexual stage so we expectto find lower diversity, and more clones than the sexual pathogen M. graminicola. But there are suggestionsfrom previous population genetic studies that this fungus undergoes regular sexual recombination inagricultural populations (Salamati et al. 2000). The known sources of primary inoculum include conidiadispersed by rainsplash and mycelium present in infected seeds. In this case, the sample included in theexperiments was a collection of 76 Swiss isolates collected from the same field in the year 2000.

Stagonospora nodorumStagonospora nodorum (Berk,Castellani) is an ascomycete fungus. This pathogen affects mainly wheat, butalso can infect barley. Like M. graminicola, it is known to have two reproduction stages. The anamorph(asexual stage) is S. nodorum and the teleomorph (sexual stage) is called Phaeosphaeria nodorum (E.Müller,Hedjaroude). The sexual stage contributes to genetic recombination. The primary inoculum for S. nodorumare the splash or windblown rain dispersed pycnidiospores and the mycelium on contaminated seeds.Ascospores are also thought to play a major role as primary inoculum, but this is uncertain (McDonald et al.1999; Keller et al. 1997). The samples used in this experiment were collected from several fields inSwitzerland in 1999 with several km distance between the fields. Therefore we expected to find a relativelyhigh degree of polymorphism.

Materials and Methods

Table 1: Isolates of M. graminicola in the Swiss collection taken from the same lesion.1 2 3 4 5 6

A 4.20 9A4W1 7.3 9A5Y3 7.13 9A5Z5 7.24 9A8C 4.1 9A3Y7 2.14 9A2X4

B 4.21.9A4W2 7.4 9A5Y4 7.14 9A5Z6 4.2 9A3X9 4.13 9A3Z1 2.15 9A2X5

C 4.22 9A4W3 7.6 9A5Y7 7.15 9A5Z7 4.3 9A3X10 4.15 9A3Z3 2.16 9A2X6

D 4.23 9A4W4 7.7 9A5Y8 7.16 9A5Z8 4.5 9A3Y2 4.16 9A3Z6 2.2O 9A2Y1

E 4.24 9A4W5 7.8 9A5Y9 7.17 9A5Z10 4.6 9A3Y3 4.17 9A3Z8 2.22 9A2Y4

F 6.24 9A5X10 7.10 9A5Z1 7.18 9A6A 4.7 9A3Y4 4.18 9A3Z9 2.12 9A2X2

G 7.1 9A5Y1 9.11 9A5Z3 7.20 9A6C 4.8 9A3Y5 2.8 9A2W4 2.13 9A2X3

H 7.2 9A5Y2 7.12 9A5Z4 4.9 9A3Y6 2.11 9A2X1 2.21 9A2Y3

Table 2: Isolates of Mycosphaerella graminicola from Swiss, Oregon, and Israel populations. This DNA was arrayed as shown in the microtiter plate called Workbox No.1.

1 2 3 4 5 6A CH 1D5 CH 8.5a CH 8.22a CH 8.36a OR 176

a1-3B.19OR 185a1-4A.7

B CH 1E3 CH 8.8a CH 8.24a CH 8.37a OR 177a1-3B.20

OR 186a1-4A.8

C CH 1E5 CH 8.10a CH 8.27a CH 8.40a OR 178a1-3B.21

OR 188a1-4A.11

D CH 1E10 CH 8.13a CH 8.29a CH 8.42a OR 179 a1-4A.1

OR 189a1-4A.12

E CH 1G3 CH 8.14a CH 8.31a CH 8.43B OR 180a1-4A.2

OR 190a1-4A.13

F CH 1H1 CH 8.16B CH 8.32a CH 8.44a OR 181 a1-4A.3

OR 191a1-4A.14

G CH 1H8 CH 8.20 CH 8.33a CH 8.46a OR 182a1-4A.4

OR 193a1-4A.16

H CH 8.4B CH 8.21a CH8.35B CH 8.49a OR 184a1-4A.6

OR 194a1-4A.17

Semesterarbeit Rubik Sommerhalder, Summer 2001: Rep-PCR fingerprinting of plant pathogenic fungi 3

7 8 9 10 11 12A OR 195

a1-4A.18OR 283a3-4A.8

IS 2ISYD3a.1

IS 43ISZE2c.2

IS 82ISYAR2b

IS 94ISYAR4g

B OR 196 a1-4A.19

OR 801a12-3B.3

IS 17ISZB3b.3

IS 48ISZE3d.2

IS 83ISYAR2c

IS 102ISYAR5d

C OR 277a3-4A.1

OR 802a12-3B.4

IS 20ISZD2a.2

IS 67ISZH1a.2

IS 84ISYAR2d

IS 103ISYAR5e

D OR 278a3-4A.2

OR 810a12-3B.12

IS 22ISZC2b.2

IS 71ISZH2c.2

IS 86ISYAR2f

IS 104ISYA5Rf

E OR 279a3-4A.3

OR 812a12-3B.14

IS 27IDZC3a.2

IS 75ISYAR1c

IS 90ISYAR4b

IS 118ISYAR9g

F OR 280a3-4A.4

OR 813a12-3B.15

IS 32ISZD1c.3

IS 77ISYAR1h

IS 91ISYAR4c

IS 123ISYAR11g

G OR 281a3-4A.5

OR 815a12-3B.17

IS 34ISZD1d.2

IS 80ISYAR1k

IS 92ISYAR4e

IS 125ISYAR11i

H OR 282a3-4A.7

OR 816a12-3B.18

IS 39ISZE2a.2

IS 81ISYAR2a

IS 93ISYAR4f

IS 128ISYAR12b

CH = Switzerland OR = Oregon IS = Israel Date: January 2001

Table 3: Rhynchosporium secalis isolates sampled from a single field included in the 2000 Swiss collection.1 2 3 4 5 6 7 8 9 10

A A1a A5a C1a D1a E4a E8b E12b G1a G6a H2aB A1b A6a C1b D2b E4b E9a F1a G1b G6b H2bC A2a A7a C2a D3b E5a E9b F2a G2a G7a H3aD A2b A8a C3a D4a E5b E10a F3a G3a G7b H4aE A3a A8b C3b D4b E6a E10b F4a G3b G8aF A3b B1a C4a E1a E6b E11a F4b G4a G8bG A4a B1b C4b E1b E7a E11b F5a G4b G9aH A4b B2a C5a E3a E7b E12a F6a G5a H1a

Table 4: Stagonospora nodorum isolates collected from across Switzerland in 1999 from several fieldsseparated by many kilometers.

1 2 3A 1H2a 34a 5H10B 5C4 4D7a 341bC 1F9a 4F7C 1C6aD 86b 236b 28aE 4G1b 1A5bF 4E7b 1D9aG 5F5 5E9H 5A7 4B2b

1-5 refers to field number; B3c2 means site B, leaf 3, lesion c, isolate 2

PCR Protocols:

PCR amplifications were performed in 96 well plates using a Biometra thermocycler. Total PCR reactionvolume per well was 20 �l, which contained 10 microliters of water, 2 �l of buffer (10X PCR buffer = 5 M KCL,0.15 Mm MgCl2 and 1mM Tris-HCl pH=9), 1 �l of each primer, 0.2 �l Taq Polymerase (the equivalent to oneTaq unit) and 5�l of sample DNA. Sample DNA was diluted to between 1 and 3% of the starting concentration.The starting solution contained between 100-200 �g/l.

Semesterarbeit Rubik Sommerhalder, Summer 2001: Rep-PCR fingerprinting of plant pathogenic fungi 4

Table 5. Different primer combinations that were tested in these experiments.Combination Forward

primerReverse primer

1 ERIC 2 L ERIC 1 R2 ERIC 2 L BOX 1 R3 ERIC 2 L REP 1 R4 REP 2 L ERIC 1 R5 REP 2 L BOX 1 R6 REP 2 L REP 1 R

Thermal cycling reactions and conditions:Three major steps were repeated for 30-40 cycles. This was done on an automated thermal cycler, which canheat and cool the tubes with the reaction mixture in a very short time.

Denaturation at 94°CHere the double-stranded DNA molecule opens up to form two single stranded DNA molecules. All enzymaticreactions stop.

Annealing at 52°C:Add a large amount of primers relative to the amount of DNA being amplified and cool the reaction mixture toallow double-stranded DNA to form again. Because of the large amount of primers the two strands will alwaysbind to the primers instead of with each other.

Extension at 65°C:Add Taq polymerase (a heat-stable enzyme) to the reaction mixture (of nucleotides etc.). Taq polymerasereads the opposing strands as a template and extends the DNA fragment by hooking nucleotides together inan order defined by the template so they pair across from one another A:T, C:G. Now we have Taqpolymerase synthesizing new DNA in opposite directions but only for this particular region of DNA. After manycycles of amplification, we will have many copies of the DNA sequences that lie between two adjacent primersites.

Figure 1. Here represented are the various steps of the thermal cycling reactions that occurin the thermocycler, explained before. (Andy Vierstraete, http://allserv.rug.ac.be/~avierstr/)

Semesterarbeit Rubik Sommerhalder, Summer 2001: Rep-PCR fingerprinting of plant pathogenic fungi 5Exact cycling parameters used: In our case we performed six different types of cycles. The first, the fifth,and the sixth type were performed one time; the second, the third and the fourth type were repeated 35 times.

1st at 96°C for 2 min ------------------------------------------initial melting step2nd at 94°C for 30 sec --------------------------------------- melting step during cycles3rd at 52°C for 1 min ----------------------------------------- annealing step4th at 65°C for 5 min (34 times back to step 2) ------- extension step5th at 65°C for 8min ------------------------------------------ final extension step6th at 15°C pause --------------------------------------------- pause until sample is removed

Gel Electrophoresis:Before loading the samples into a gel, we added 4 �l (=1/6 volume) of 6X blue juice, a loading buffer. Aftermixing the blue juice, we loaded the PCR product on an agarose gel and let it run in an electric field. As DNAhas a negative charge, it migrates to the positive charged end of the gel. Depending on the size of theamplified DNA molecules (number of base pairs in the amplified fragment, or amplicon) they will move throughthe gel with different speeds. Smaller fragments move faster than large fragments. As a result, the DNAfragments will run to a different location on the gel according to size. Next we stained the gel with ethidiumbromide and put the gel under a UV light and visualized the amplicon (band) patterns. Because the differentamplicons have different positions on the gel according to their size we can see the genetic diversity amongindividuals on the same gel. The gel was left running for about 3 hours with a voltage at 100 V and 4h with avoltage at 80 V. Choice of the size of the gel and its corresponding wells was important. When the large gelwas used in combination with the larger 25 well combs, 10 �l of PCR product could be loaded. When the smallgel was used with its corresponding smaller 25 well combs, 8 �l of PCR product was loaded to avoidoverloading problems. The rest of the running parameters were kept the same for both gels. Better resultswere achieved with the small gel, in my opinion, because it was easier to score. In this time period (3 h or 4 h)the bands migrated about 8 cm from the original loading point on the gel. The amplified DNA fragments wererecorded using a 2000 Gel Documentation System by Bio Rad. The sizes of the amplicons were determinedwith the help of a DNA ladder (peqGOLD bp D N A-Leiter Plus) with known base lengths included on each gel.

Results

Table 6: Results from preliminary screening of combinations of PCR primers shown in Table 5.

Primercombination

1 2 3 4 5 6

M. graminicola + + - - - -R. secalis - - - + + - -S. nodorum -- - - + - -/++ = scoreable amplicons - = not scoreable -- = no or very few amplicons showed.

M. graminicola results

Figure 2. Primer combinations 1 and 2 were used for M. graminicola (16 isolates per combination). Isolatenumbers correspond to Table 1.

1A 1B 1C 1D 1E 1F 1G 1H 2A 2B 2C 2D 2E 2F 2G 2H 1A 1B 1C 1D 1E 1F 1G 1H

Semesterarbeit Rubik Sommerhalder, Summer 2001: Rep-PCR fingerprinting of plant pathogenic fungi 6

Figure 3. Primer combinations 2 (first 8 lanes), and 3 (last 16 lanes).

2A 2B 2C 2D 2E 2F 2G 2H 1A 1B 1C 1D 1E 1F 1G 1H 2A 2B 2C 2D 2E 2F 2G 2H

Figure 4. Primer combinations 4 (first 16 lanes), and 5 (next 8 lanes).

1A 1B 1C 1D 1E 1F 1G 1H 2A 2B 2C 2D 2E 2F 2G 2H 1A 1B 1C 1D 1E 1F 1G 1H

Figure 5. Primer combinations 5 (first 8 isolates), and 6 (next 16 lanes).

2A 2B 2C 2D 2E 2F 2G 2H 1A 1B 1C 1D 1E 1F 1G 1H 2A 2B 2C 2D 2E 2F 2G 2H

Summary of results with M. graminicola: Primer combinations 1 and 2 were more informative than the others,and had a larger number of amplicons. Primer combination 3 had fewer amplicons and combination 4 did notproduce a good amplification. Combination 5 produced some blank isolates (no amplicons) and combination 6was difficult to score.

R. secalis results.

Figure 6. Primer combination test on R.secalis. Primer combinations 1 and 2 (last 8 isolates). Isolate numberscorrespond to Table 3.

1A 1B 1C 1D 1E 1F 1G 1H 2A 2B 2C 2D 2E 2F 2G 2H 1A 1B 1C 1D 1E 1F 1G 1H 3000bp

1500bp

900bp

Semesterarbeit Rubik Sommerhalder, Summer 2001: Rep-PCR fingerprinting of plant pathogenic fungi 7

Figure 7. Primer combination 2 (first 8 isolates) and 3. Isolate numbers correspond to Table 3.

2A 2B 2C 2D 2E 2F 2G 2H 1A 1B 1C 1D 1E 1F 1G 1H 2A 2B 2C 2D 2E 2F 2G 2H

Figure 8. Primer combinations 4 (first 8 isolates) and 5. Isolate numbers correspond to Table 3.

1A 1B 1C 1D 1E 1F 1G 1H 2A 2B 2C 2D 2E 2F 2G 2H 1A 1B 1C 1D 1E 1F 1G 1H

Summary of results with R. secalis: Primer combination 1 was unscoreable due to amplification problems (noamplicons). Combination two showed too little valid data. Combination 3 gave good results. In combination 4the amplicons were a little weak but good discrimination between isolates was possible. Combination 5 did notallow a good discrimination among isolates. Combination 6 did not show any valid data.

S. nodorum results

Figure 9. Primer combinations 1 (first 16 lanes) and 2 (last 8 lanes) for S. nodorum. Isolate numberscorrespond toTable 4.

1A 1B 1C 1D 1E 1F 1G 1H 2A 2B 2C 2D 2E 2F 2G 2H 1A 1B 1C 1D 1E 1F 1G 1H

2000bp

500bp

3000bp

1500bp

1031bp

600bp

Semesterarbeit Rubik Sommerhalder, Summer 2001: Rep-PCR fingerprinting of plant pathogenic fungi 8

Figure 10. Primer combination 2 (first 8 lanes) and 3 (last 16 lanes). Isolate numbers correspond to Table 4.

2A 2B 2C 2D 2E 2F 2G 2H 1A 1B 1C 1D 1E 1F 1G 1H 2A 2B 2C 2D 2E 2F 2G 2H

Figure 11. Primer combination 4 (first 16 lanes) and 5 (last 8 lanes). Isolate numbers correspond to Table 4.

1A 1B 1C 1D 1E 1F 1G 1H 2A 2B 2C 2D 2E 2F 2G 2H 1A 1B 1C 1D 1E 1F 1G 1H

Figure 12. Primer combinations 5 (first 8 lanes) and 6 (last 16 lanes). Isolate numbers correspond to Table 4.

2A 2B 2C 2D 2E 2F 2G 2H 1A 1B 1C 1D 1E 1F 1G 1H 2A 2B 2C 2D 2E 2F 2G 2H

Summary of results with S. nodorum: Primer combination 4 clearly gave the best results. Combination 1 didnot amplify. Combination 2 amplified very weakly. Combination 3 amplified the same repetitive elements, andgave no differences between isolates. Combinations 5 and 6 also showed no differences.

Quantification of results from Mycosphaerella graminicola:

Table 7. Summary of results from the Swiss population of M. graminicola out of Workbox 1 with Primercombination 1.Average # amplicons 4.63# min 2# max 8Standard deviation 2.91Tot. isolates 32# with data 31

Semesterarbeit Rubik Sommerhalder, Summer 2001: Rep-PCR fingerprinting of plant pathogenic fungi 9# genotypes 25 (3 clones with 2 individuals each)

Table 8. Summary of results from the Oregon population of M. graminicola out of Workbox 1 with Primercombination 1.Average # amplicons 2.38# min 1# max 9Standard deviation 4.11Tot. isolates 32# with data 21# genotypes 5 which were clearly scorable

Table 9. Summary of results from the Israeli population of M. graminicola out of Workbox 1 with Primercombination 1.Average # amplicons 3.88# min 1# max 7Standard deviation 3.44Tot. isolates 32# with data 27# genotypes 21 which were detectable

Table 10. Summary of results from the Swiss population of M. graminicola out of Workbox No.1 with Primercombination 2Average # amplicons 4.45# min 2# max 8Standard deviation 2.06Tot. isolates 32# with data 31# genotypes 22 with 3 clone pairs

Table 11. Summary of results from the Oregon population of M. graminicola out of Workbox 1 with Primercombination 2Average # amplicons 2.91# min 1# max 7Standard deviation 3.53Tot. isolates 32# with data 23# genotypes 12 that were scorable. With 2 clones

Table 12. Summary of results from the Israeli population of M. graminicola out of Workbox 1 with Primercombination 2.Average amplicons 2.89# min 1# max 7Standard deviation 2.4Tot. isolates 32# with data 19# genotypes 16 (no clones), rest didn’t have enough data to

be able to score the isolates.

Semesterarbeit Rubik Sommerhalder, Summer 2001: Rep-PCR fingerprinting of plant pathogenic fungi 10Good results were obtained with primer combinations 1 (ERIC 2L -ERIC 1R) and 2 (ERIC 2L -BOX 1R).Combinations 3, 4, 5 and 6 didn’t give any useful results. Combination 1 showed useful DNA fingerprints withgood discriminating power between the various isolates, suggesting that there were few places where thedistance between adjacent repetitive elements was identical. Combination one showed clear bandsrepresenting the conserved repetitive elements. Primer combination 2 amplified a smaller number of bands,but they were more reproducible. Combinations 3, 4 and 5 either amplified repetitive elements of the samesize or gave no amplicons at all.

Combination 1 enabled us to see 7-8 amplicons with different degrees of clarity and with amplicon sizesranging from 100 bp up to 3400 bp. The amplicons were clearer in the Swiss populations collected from thesame lesion and for the Swiss samples collected from various field populations separated by many kilometers.For samples from Oregon and Israel, the amplifications didn’t work as well although they were run under thesame PCR conditions and gel electrophoresis conditions. DNA from the Swiss isolates was extracted usingQiagen columns, while all other isolates were extracted using a CTAB based protocol.

Results with the collection from the same leaf were as expected. In fact they clearly showed isolates havingthe same fingerprint (clones) and therefore few polymorphisms. Only 3 clones types were identified amongthese isolates. These results are in agreement with findings of C.linde et al. (2002) who also found the samethree genotypes with RFLP fingerprints. In this case we found some very stable repetitive elements whichshowed 4 very clear bands, with sizes ranging from 325 bp and 1250 and 4-5 additional amplicons that did notamplify as well.

Figure 13. Primer combination 1 on collection of Swiss isolates of M. graminicola from different fieldpopulations. Isolate numbers correspond to Table 2.

1A 1B 1C 1D 1E 1F 1G 1H 2A 2B 2C 2D 2E 2F 2G 2H 1A 1B 1C 1D 1E 1F 1G 1H

With primer combination number two (ERIC 2L -BOX 1R) we visualized a smaller number of bands but eachband was better amplified. Here, too we found the same results, confirming that most isolates from the samelesion were clones. Swiss isolates collected from different field populations showed a high level ofpolymorphism, although the fingerprints were very similar. It is interesting to notice that they differed by only afew major bands.

500

3000

2000

1031

100bp

Semesterarbeit Rubik Sommerhalder, Summer 2001: Rep-PCR fingerprinting of plant pathogenic fungi 11Figure 14. . Primer combination 2 on Swiss M. graminicola isolates originating from the same lesion. It seemslike we have two clone types here (type 1 and 2 represented in 1D, 1H, 2A, 2B and 2F). Isolate numberscorrespond to Table 1

1A 1B 1C 1D 1E 1F 1G 1H 2A 2B 2C 2D 2E 2F 2G 2H 3A 3B 3C 3D 3E 3F 3G 3H

With primer combination 1 you can see up to 8 amplicons with good discriminating power. The result is thatthere were three clones (plus 19 different genotypes) identified in this Swiss collection. As already mentioned,the Swiss isolates had better amplifications than the foreign isolates even after several re-amplifications.

The Oregon isolates proved to be less polymorphic than the Swiss isolates as they presented five clones eachrepresented by two individuals (= 5/36 are clones). Here too, the fingerprints were very similar to each otherand the differences among isolates were due to a couple of amplicons... Out of 32 Oregon isolates assayed,only two clone types with three individuals each were found.

Figure 15. Primer combination 1 on gel 2 24.07.01 (Israeli population of M. graminicola). Isolate numberscorrespond to Table 2.

9A 9B 9C 9D 9E 9F 9G 9H 10A10B 10C 10D 10E 10F 10G 10H 11A11B 11C 11D 11E 11F 11G 11H

Considering all three populations together, there were two amplicons that were conserved and were amplifiedin all three populations with lengths of 1200 bp and 1900 bp.

Results with Rhynchosporium secalis:

Table 13. Summary of results from the Swiss population of R. secalis using Primer combination 3.

Average # amplicons 6.29# min 1# max 10Standard deviation 5.1Tot. isolates 76# with data 37# genotypes 2

3000bp

1500bp

800bp

400bp

100bp

3000bp

1031bp

500bp

Semesterarbeit Rubik Sommerhalder, Summer 2001: Rep-PCR fingerprinting of plant pathogenic fungi 12

Table 14. Summary of results from the Swiss population of R. secalis using primer combination 4.

Average # amplicons 6.1# min 1# max 8Standard deviation 3.36Tot. isolates 76# with data 30# genotypes 2

For R. secalis, good amplifications were obtained with primer combinations 3 (ERIC 2L - REP 1R) and 4 (REP2L - ERIC 1R), but only for the second part of the population. Significant problems were encountered with thefirst part of the population tested. These DNAs never amplified completely or didn’t amplify at all. So it wasimpossible to score all 48 isolates. We tried to overcome this problem by varying the DNA concentration in themaster mix (10 �l sample) and the amount of PCR product loaded on the gel (8,10,12 �l). Combination 4enabled us to differentiate two amplification patterns (type 1, 15 isolates and type 2, 9 isolates) on the secondgel (=last 28 isolates). These isolates differed from each other by only a single band. Combination 4 gave goodamplifications if only the very clear bands were considered, but the weak bands had a low degree ofreproducibility. The weak bands showed up in some amplifications but not others, so we decided to score onlythe very clear amplicons.

Figure 16. Primer combination 4 on Swiss R. secalis isolates, giving fewer but more reproducible amplicons.Isolate numbers correspond to Table 3.

7A 7B 7C 7D 7E 7F 7G 7H 8A 8B 8C 8D 8E 8F 8G 8H 9A9B 9C9D 9E 9F 9G 9H

Results with S. nodorum

Table 15. Summary of results from the Swiss population of P. nodorum using primer combination 4.

Average amplicons 4.47# min 1# max 11Standard deviation 5.39Tot. isolates 20# with data 19# genotypes 4

Results with S. nodorum weren`t as satisfying as with the other fungi. The best primer combination tested wasnumber 4 (REP 2L - ERIC 1R) where up to 7 bands were amplified. The reproducibility of this combination

3000bp

500bp

Semesterarbeit Rubik Sommerhalder, Summer 2001: Rep-PCR fingerprinting of plant pathogenic fungi 13remained questionable, as there were some bands that weren’t always amplified. Therefore I would advise torepeat the PCR more than once to achieve reproducible results. Combination 6 showed some promise, butthe DNA concentrations had to be higher because the bands were so weak that it was practically impossible toscore them.

Scoring the S. nodorum gels showed that we had four clones patterns out of twenty samples. The rest of theisolates were either difficult to score (they gave few clear amplicons) or they showed polymorphic fingerprintpatterns. These Swiss populations had one very conserved amplicon in common, the 650-bp amplicon.

Figure 17. Fingerprints of all S. nodorum isolates with primer combination 4.

1H2a 5C4 1F9a 86b 4G1b 4E7b 5F5 5A7 34a 4D7a 4F7c 236b 1A5b 1D9a 5E9 4B2b 5H10 341b 1C6a 28a

Discussion

The goal of this study was to define primer combinations based on rep-PCR, which could show a certaindegree of polymorphism, and would offer a good substitute for the more time consuming RFLP fingerprintingtechnique. The goal was partially achieved, meaning that rep-PCR did show polymorphisms betweenindividuals, but the procedures must be improved to become a valid substitute to the more informative RFLPfingerprinting method. Because the rep-PCR technique was developed to make fingerprints of bacterial DNA,the application and potential usefulness for fungi was unknown. This should be considered only a starting pointto develop rep-PCR for these fungi. Many details must be worked out. The most satisfying results wereobtained with M. graminicola. But even here to achieve reproducible results the various steps must beimproved (optimized) as follows: DNA concentration (we worked with concentrations containing between 100-200 �g/l that were subsequently diluted 1:100), gel running time (we obtained good results with 80 V for 4h).We used gels of two different sizes, one 25x30 cm and the other one 20x 25 cm. Personally I preferredworking with the smaller gels because the bands were easier to score. Running distance was about 9-10 cmfor the larger gel and 7-8 cm for the smaller gel. In my opinion, the results with primer combination number twowere the most promising because of better reproducibility, that means that the fingerprints were always similarin the isolates taken from one lesion. An interesting result was found with the Israeli population where a largerdegree of polymorphism (compared to the Swiss and the Oregon populations) was found. This may offer someadditional support to the theory that M. graminicola originated in Mesopotamia and therefore the degree ofpolymorphism should be larger in the center of origin.

More significant problems were encountered with R. secalis. Difficulties were encountered with the first 48isolates because they amplified very poorly or didn`t amplify at all. The problem did not appear to be related tothe DNA concentrations as the first 48 isolates (from 1A to 6H corresponding to Table 2) had similarconcentrations to the last 28 isolates tested (from 9E to 12 H referring to table 2). Tests were carried out toconfirm this hypothesis. We tested 8 isolates that never amplified and 3 isolates that always amplified withdifferent DNA concentrations in the master mix (1/2, 1.5 and 2 times the starting amount of DNA were put inthe master mix) but the results did not change.

3000bp

500bp

Semesterarbeit Rubik Sommerhalder, Summer 2001: Rep-PCR fingerprinting of plant pathogenic fungi 14

Figure 18. DNA concentration tests with R. secalis and primer combination 4.

0.5 x initial amount of DNA 2 x initial amount of DNA

1.5 x initial amount of DNA 1x initial amount of DNA

After discussing this problem we hypothesized that there could be some impurities in the DNA of the first 48isolates that prevented it from amplifying.

Rep-PCR primers used on the last 28 isolates often amplified highly conserved bands with the result thatdifferentiation capability was reduced, especially for primer combination 3. I would advise to see if there is anycorrelation between the RFLP technique and the rep-PCR to see if the problematic amplicons obtained withthe rep-PCR create problems using the other method or if the problem resides in the rep-PCR.

The same interpretation can be drawn for S. nodorum. The same repetitive elements are often amplifiedleading to the same problem of lack of differentiation among strains.

Conclusions

RFLP-fingerprints representing clones or isolates that were very similar to each other were compared with thecorresponding rep-PCR fingerprints. Results showed that there was a good correlation between the twomethods for M. graminicola, although the rep-PCR amplified a smaller number of bands than RFLPfingerprints. For example, Israeli isolates ISYAR1h and ISYAR5d, which had 9/10 of the bands in common withthe RFLP method, were fairly well-correlated with the rep-PCR results, although the rep-PCR amplified fewerbands and therefore it might be less informative. In fact the bands that were amplified in both isolates with therep-PCR corresponded completely (4 out of 4 amplicons matched). The same can be said for ISYAR4e andISYAR4g which had 8/10 of the bands in common with the RFLP-method, and showed nearly the samefingerprints with the rep-PCR method (3 out of 3 amplicons were the same). Sometimes problems occurredwith few and weak rep-PCR amplicons this made it difficult to compare between the RFLP`s and rep-PCRmethod. Comparisons between the Oregon isolates were more difficult and less informative because of the difficultiesthat resulted during amplification of these isolates with the rep-PCR method. Oregon isolates OR 176, OR 178,OR 279, are practically clones if analyzed with the RFLP technique. These isolates could not be compared

Semesterarbeit Rubik Sommerhalder, Summer 2001: Rep-PCR fingerprinting of plant pathogenic fungi 15with the rep-PCR method because there was no available data. The same can be said for isolates OR 278 andOR 816. For isolates OR 802 and OR 810, that had 12/12 bands in common with the RFLP technique, therewas a high degree of correlation between the results as the 4 scoreable amplicons with the rep-PCR matchedperfectly. In my opinion these two techniques are fairly well correlated, but the rep-PCR has limits inapplication because of the relatively low number of bands that were amplified. The low number of ampliconscan lead to scoring and comparison problems. If the number of amplicons can be increase, perhaps bychanging or improving the primers, it would be great. Because as already mentioned this system is less timeand material consuming than the RFLP-technique, and plus it is fairly easy to carry out and therefore you arenot so limited in the personnel that can use this procedure.

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