PCR-based detection of Rhizoctonia cerealis

The present invention relates to the use of species-specific primers in polymerase chain reaction assays for the detection of Rhizoctonia cerealis , a fungal pathogen of wheat. The use of these primers enables the monitoring of disease development in plant populations.

Diseases in plants cause considerable crop loss from year to year resulting both in economic deprivation to farmers and, in many parts of the world, to shortfalls in the nutritional provision for local populations. The widespread use of fungicides has provided considerable security against plant pathogen attack. However, despite 1 billion worth of expenditure on fungicides, worldwide crop losses amounted to approximately 10% of crop value in 1981 (James, 1981 ; Seed Sci. & Technol . 9: 679-685).

The severity of the destructive process of disease depends on the aggressiveness of the pathogen and the response of the host. One aim of most plant breeding programs is to increase the resistance of host plants to disease. Typically, different races of pathogens interact with different varieties of the same crop species differentially, and many sources of host resistance only protect against specific pathogen races. Furthermore, some pathogen races show early signs of disease symptoms, but cause little damage to the crop. Jones and Clifford (1983; Cereal Diseases, John Wiley) report that virulent forms of the pathogen are expected to emerge in the pathogen population in response to the introduction of resistance into host cultivars and that it is therefore necessary to monitor pathogen populations. In addition, there are several documented cases of the evolution of fungal strains that are resistant to particular fungicides. As early as 1981, Fletcher and Wolfe (1981 ; Proc . 1981 Brit. Crop Prot. Conf .) contended that 24% of the powdery mildew populations from spring barley and 53% from winter barley showed considerable variation in response to the fungicide triadimenol and that the distribution of these populations varied between varieties, with the most susceptible variety also giving the highest incidence of less susceptible types. Similar variation in the sensitivity of fungi to fungicides has been documented for wheat mildew (also to triadimenol), Botrytis (to benomyl), Pyrenophora (to organomercury), Pseudocercosporella (to MBC-type fungicides) and Mycosphaerella fijiensis to triazoles to mention just a few (Jones and Clifford; Cereal Diseases, John Wiley, 1983).

Wheat is currently the most important agricultural commodity in international markets and occupies about 20% of the world's farmed land (1977; Compendium of Wheat Diseases, Amer. Phytopath. Soc. page 1). Eightly percent of the world's supply of wheat is grown in North America, Europe, China, and the Soviet Union. Approximately 20% of the worldwide production of wheat is lost to disease annually.

Sharp eyespot is caused by Rhizoctonia cerealis van der Hoeven (teleomorph Ceratobasidium cereale Murray & Burpee) and occurs on wheat, barley, oat and rye (1977; Compendium of Wheat Diseases, Amer. Phytopath. Soc. Page 50). Some isolates of the pathogen are also capable of infecting turfgrass causing yellow patch. Severe wheat infections cause premature ripening and lodging, thereby effecting yield. There are presently no known sharp eyespot-resistant cultivars.

In view of the above, there is a real need for the development of technology that will allow the identification of specific fungal pathogens early in the infection process. By identifying the specific pathogen before disease symptoms become evident in the crop stand, the agriculturist can assess the likely effects of further development of the pathogen in the crop variety in which it has been identified and can choose an appropriate fungicide if such application is deemed necessary.

The present invention is drawn to methods of identification of different pathotypes of plant pathogenic fungi. The invention provides Internal Transcribed Spacer (ITS) DNA sequences that show variability between different fungal pathotypes. Such DNA sequences are useful in the method of the invention as they can be used to derive primers for use in polymerase chain reaction (PCR)-based diagnostic assays. These primers generate unique fragments in PCR reactions in which the DNA template is provided by specific fungal pathotypes and can thus be used to identify the presence or absence of specific pathotypes in host plant material before the onset of disease symptoms.

In a preferred embodiment, the invention provides ITS1 and ITS2 DNA sequences (e.g., SEQ ID NO:17-26) for the pathogen Rhizoctonia cerealis . In another preferred embodiment, the invention provides ITS-derived diagnostic primers (e.g., SEQ ID NO:7-16) for the detection of Rhizoctonia cerealis.

This invention provides the possibility of assessing potential damage in a specific crop variety-pathogen strain relationship and of utilizing judiciously the diverse armory of fungicides that is available. Furthermore, the invention can be used to provide detailed information on the development and spread of specific pathogen races over extended geographical areas. The invention provides a method of detection that is especially suitable for diseases with a long latent phase.

Kits useful in the practice of the invention are also provided. The kits find particular use in the identification of the fungal pathogen Rhizoctonia cerealis.

SEQ ID NO:1 Oligonucleotide Primer ITS 1.

SEQ ID NO:2 Oligonucleotide Primer ITS2.

SEQ ID NO:3 Oligonucleotide Primer ITS3.

SEQ ID NO:4 Oligonucleotide Primer ITS4.

SEQ ID NO:5 M13 Universal-20 Primer.

SEQ ID NO:6 Reverse Primer used in Example 2.

SEQ ID NO:7 Oligonucleotide Primer JB643.

SEQ ID NO:8 Oligonucleotide Primer JB644.

SEQ ID NO:9 Oligonucleotide Primer JB645.

SEQ ID NO:10 Oligonucleotide Primer JB646.

SEQ ID NO:11 Oligonucleotide Primer JB647.

SEQ ID NO:12 Oligonucleotide Primer JB648.

SEQ ID NO:13 Oligonucleotide Primer JB649.

SEQ ID NO:14 Oligonucleotide Primer JB650.

SEQ ID NO:15 Oligonucleotide Primer JB687.

SEQ ID NO:16 Oligonucleotide Primer JB688.

SEQ ID NO:17 DNA sequence of the ITS region PCR-amplified from R. cerealis isolate 44235, comprising in the 5′ to 3′ direction: 3′ end of the small subunit rRNA gene (nucleotides 1-30), Internal Transcribed Spacer 1 (nucleotides 31-242), 5.8S rRNA gene (nucleotides 244-395), Internal Transcribed Spacer 2 (nucleotides 397-630), and 5′ end of the large subunit rRNA gene (nucleotides 631-687).

SEQ ID NO:18 DNA sequence of the ITS region PCR-amplified from R. cerealis isolate AGDC57, comprising in the 5′ to 3′ direction: 3′ end of the small subunit rRNA gene (nucleotides 1-30), Internal Transcribed Spacer 1 (nucleotides 31-242), 5.8S rRNA gene (nucleotides 243-395), Internal Transcribed Spacer 2 (nucleotides 396-629), and 5′ end of the large subunit rRNA gene (nucleotides 630-686).

SEQ ID NO:19 DNA sequence of the ITS region PCR-amplified from R. cerealis isolate CAG1BN1, comprising in the 5′ to 3′ direction: 3′ end of the small subunit rRNA gene (nucleotides 1-30), Internal Transcribed Spacer 1 (nucleotides 31-243), 5.8S rRNA gene (nucleotides 244-396), Internal Transcribed Spacer 2 (nucleotides 397-630), and 5′ end of the large subunit rRNA gene (nucleotides 631-687).

SEQ ID NO:20 DNA sequence of the ITS region PCR-amplified from R. cerealis isolate AGDC73, comprising in the 5′ to 3′ direction: 3′ end of the small subunit rRNA gene (nucleotides 1-30), Internal Transcribed Spacer 1 (nucleotides 31-241), 5.8S rRNA gene (nucleotides 242-394), Internal Transcribed Spacer 2 (nucleotides 395-628), and 5′ end of the large subunit rRNA gene (nucleotides 629-685).

SEQ ID NO:21 DNA sequence of the ITS region PCR-amplified from R. cerealis isolate 52182, comprising in the 5′ to 3′ direction: 3′ end of the small subunit rRNA gene (nucleotides 1-30), Internal Transcribed Spacer 1 (nucleotides 31-243), 5.8S rRNA gene (nucleotides 244-396), Internal Transcribed Spacer 2 (nucleotides 397-630), and 5′ end of the large subunit rRNA gene (nucleotides 631-687).

SEQ ID NO:22 DNA sequence of the ITS region PCR-amplified from R. cerealis isolate Bn505, comprising in the 5′ to 3′ direction: 3′ end of the small subunit rRNA gene (nucleotides 1-30), Internal Transcribed Spacer I (nucleotides 31-243), 5.8S rRNA gene (nucleotides 244-396), Internal Transcribed Spacer 2 (nucleotides 397-630), and 5′ end of the large subunit rRNA gene (nucleotides 631-686).

SEQ ID NO:23 DNA sequence of the ITS region PCR-amplified from R. cerealis isolate R88-303, comprising in the 5′ to 3′ direction: 3′ end of the small subunit rRNA gene (nucleotides 1-30), Internal Transcribed Spacer 1 (nucleotides 31-243), 5.8S rRNA gene (nucleotides 244-396), Internal Transcribed Spacer 2 (nucleotides 397-630), and 5′ end of the large subunit rRNA gene (nucleotides 631-687).

SEQ ID NO:24 DNA sequence of the ITS region PCR-amplified from R. cerealis isolate 52184, comprising in the 5′ to 3′ direction: 3′ end of the small subunit rRNA gene (nucleotides 1-30), Internal Transcribed Spacer 1 (nucleotides 31-240), 5.8S rRNA gene (nucleotides 241-393), Internal Transcribed Spacer 2 (nucleotides 394-627), and 5′ end of the large subunit rRNA gene (nucleotides 628-684).

SEQ ID NO:25 DNA sequence of the ITS region PCR-amplified from R. cerealis isolate 62063, comprising in the 5′ to 3′ direction: 3′ end of the small subunit rRNA gene (nucleotides 1-30), Internal Transcribed Spacer I (nucleotides 31-242), 5.8S rRNA gene (nucleotides 243-395), Internal Transcribed Spacer 2 (nucleotides 396-629), and 5′ end of the large subunit rRNA gene (nucleotides 630-686).

SEQ ID NO:26 DNA sequence of the ITS region PCR-amplified from R. cerealis isolate 52183, comprising in the 5′ to 3′ direction: 3′ end of the small subunit rRNA gene (nucleotides 1-30), Internal Transcribed Spacer I (nucleotides 31-242), 5.8S rRNA gene (nucleotides 243-395), Internal Transcribed Spacer 2 (nucleotides 396-629), and 5′ end of the large subunit rRNA gene (nucleotides 630-686).

SEQ ID NO:27 GenBank sequence (accession #AF063019) listing of DNA sequence of the ITS region from R. cerealis , comprising in the 5′ to 3′ direction: 3′ end of the small subunit rRNA gene (nucleotides 1-29), Internal Transcribed Spacer 1 (nucleotides 30-241), 5.8S rRNA gene (nucleotides 242-394), Internal Transcribed Spacer 2 (nucleotides 395-628), and 5′ end of the large subunit rRNA gene (nucleotides 629-685).

SEQ ID NO:28 DNA sequence of the ITS region PCR-amplified from P. herpotrichoides isolate R1, comprising in the 5′ to 3′ direction: 3′ end of the small subunit rRNA gene, Internal Transcribed Spacer 1, 5.8S rRNA gene, Internal Transcribed Spacer 2, and 5′ end of the large subunit rRNA gene.

SEQ ID NO:29 DNA sequence of the ITS region PCR-amplified from S. nodorum isolate 24425, comprising in the 5′ to 3′ direction: 3′ end of the small subunit rRNA gene, Internal Transcribed Spacer 1, 5.8S rRNA gene, Internal Transcribed Spacer 2, and 5′ end of the large subunit rRNA gene.

SEQ ID NO:30 DNA sequence of the ITS region PCR-amplified from S. tritici isolate 26517, comprising in the 5′ to 3′ direction: 3′ end of the small subunit rRNA gene, Internal Transcribed Spacer 1, 5.8S rRNA gene, Internal Transcribed Spacer 2, and 5′ end of the large subunit rRNA gene.

SEQ ID NO:31 DNA sequence of the ITS region PCR-amplified from P. tritici-repentis isolate 6715, comprising in the 5′ to 3′ direction: 3′ end of the small subunit rRNA gene, Internal Transcribed Spacer 1, 5.8S rRNA gene, Internal Transcribed Spacer 2, and 5′ end of the large subunit rRNA gene.

SEQ ID NO:32 DNA sequence of the ITS region PCR-amplified from F. culmorum isolate 62215, comprising in the 5′ to 3′ direction: 3′ end of the small subunit rRNA gene, Internal Transcribed Spacer 1, 5.8S rRNA gene, Internal Transcribed Spacer 2, and 5′ end of the large subunit rRNA gene.

SEQ ID NO:33 DNA sequence of the ITS region PCR-amplified from M. nivale isolate 520, comprising in the 5′ to 3′ direction: 3′ end of the small subunit rRNA gene, Internal Transcribed Spacer 1, 5.8S rRNA gene, Internal Transcribed Spacer 2, and 5′ end of the large subunit rRNA gene.

The present invention provides unique DNA sequences that are useful in identifying different pathotypes of plant pathogenic fungi. Particularly, the DNA sequences can be used as primers in PCR-based analysis for the identification of fungal pathotypes. The DNA sequences of the invention include the Internal Transcribed Spacer (ITS) sequences of the ribosomal RNA gene regions of particular fungal pathogens as well as primers derived from these regions that are capable of identifying the particular pathogen. These ITS DNA sequences from different pathotypes within a pathogen species or genus, which vary between the different members of the species or genus, can be used to identify those specific members.

Biomedical researchers have used PCR-based techniques for some time and with moderate success to detect pathogens in infected animal tissues. Only recently, however, has this technique been applied to detect plant pathogens. The presence of Gaumannomyces graminis in infected wheat has been detected using PCR of sequences specific to the pathogen mitochondrial genome (Schlesser et al., 1991 ; Applied and Environ. Microbiol . 57: 553-556), and random amplified polymorphic DNA (i.e. RAPD) markers were able to distinguish numerous races of Gremmeniella abietina , the causal agent of scleroderris canker in conifers. U.S. Pat. No. 5,585,238 (incorporated herein by reference in its entirety) describes primers derived from the ITS sequences of the ribosomal RNA gene region of strains of Septoria, Pseudocercosporella , and Mycosphaerella and their use in the identification of these fungal isolates using PCR-based techniques. In addition, WO 95/29260 (herein incorporated by reference in its entirety) describes primers derived from the ITS sequences of the ribosomal RNA gene region of strains of Fusarium and their use in the identification of these fungal isolates using PCR-based techniques. Furthermore, U.S. Pat. No. 5,800,997 (incorporated herein by reference in its entirety) describes primers derived from the ITS sequences of the ribosomal RNA gene region of strains of Cercospora, Helminthosporium, Kabatiella, and Puccinia and their use in the identification of these fungal isolates using PCR-based techniques.

Ribosomal genes are suitable for use as molecular probe targets because of their high copy number. Despite the high conservation between mature rRNA sequences, the non-transcribed and transcribed spacer sequences are usually poorly conserved and are thus suitable as target sequences for the detection of recent evolutionary divergence. Fungal rRNA genes are organized in units, each of which encodes three mature subunits of 18S (small subunit), 5.8S, and 28S (large subunit). These subunits are separated by two Internal Transcribed Spacers, ITS1 and ITS2, of around 300 bp (White et al., 1990; In: PCR Protocols; Eds.: Innes et al.; pages 315-322). In addition, the transcriptional units are separated by non-transcribed spacer sequences (NTSs). The ITS and NTS sequences are particularly suitable for the detection of specific pathotypes of different fungal pathogens.

The DNA sequences of the invention are from the Internal Transcribed Spacer sequences of the ribosomal RNA gene region of different plant pathogens. The ITS DNA sequences from different pathotypes within a pathogen species or genus vary among the different members of the species or genus. Once having determined the ITS sequences of a pathogen, these sequences can be aligned with other ITS sequences. In this manner, primers can be derived from the ITS sequences. That is, primers can be designed based on regions within the ITS sequences that contain the greatest differences in sequence among the fungal pathotypes. These sequences and primers based on these sequences can be used to identify specific pathogens.

Particular DNA sequences of interest include ITS DNA sequences from Rhizoctonia cerealis . Such ITS DNA sequences are disclosed in SEQ ID NOs: 17-26. Sequences of representative oligonucleotide primers derived from these ITS sequences are disclosed in SEQ ID NOs: 7-16. The sequences find use in the PCR-based identification of the pathogen of interest.

Methods for the use of the primer sequences of the invention in PCR analysis are well known in the art. For example, see U.S. Pat. Nos. 4,683,195 and 4,683,202, as well as Schlesser et al. (1991) Applied and Environ. Microbiol . 57:553-556. See also, Nazar et al. (1991 ; Physiol. and Molec. Plant Pathol . 39: 1-11), which used PCR amplification to exploit differences in the ITS regions of Verticillium albo-atrum and Verticillium dahliae and therefore distinguish between the two species; and Johanson and Jeger (1993 ; Mycol. Res . 97: 670-674), who used similar techniques to distinguish the banana pathogens Mycosphaerella fijiensis and Mycospharella musicola.

The ITS DNA sequences of the invention can be cloned from fungal pathogens by methods known in the art. In general, the methods for the isolation of DNA from fungal isolates are known. See, Raeder & Broda (1985) Letters in Applied Microbiology 2:17-20; Lee et al. (1990) Fungal Genetics Newsletter 35:23-24; and Lee and Taylor (1990) In: PCR Protocols: A Guide to Methods and Applications , Innes et al. (Eds.); pages 282-287.

Alternatively, the ITS sequences of interest can be determined by PCR amplification. In an exemplified embodiment, primers to amplify the entire ITS region are designed according to White et al. (1990; In: PCR Protocols; Eds.: Innes et al. pages 315-322), and the amplified ITS sequence are subcloned into the pCR2.1 cloning vector. The subcloned sequence include the left hand ITS (ITS 1), the right hand ITS (ITS2), as well as the centrally located 5.8S rRNA gene. This is undertaken for several isolates of Rhizoctonia cerealis.

The determined ITS sequences are compared within each pathogen group to locate divergences that might be useful to test in PCR to distinguish the different species and/or strains. Exemplary determined ITS DNA sequences are shown in SEQ ID NOs: 17-26. A comparative alignment is made of these ITS DNA sequences. From the identification of divergences, numerous primers are synthesized and tested in PCR-amplification. Templates used for PCR-amplification testing are firstly purified pathogen DNA, and subsequently DNA isolated from infected host plant tissue. Thus, it is possible to identify pairs of primers that are diagnostic, i.e. that identify one particular pathogen species or strain but not another species or strain of the same pathogen.

Preferred primer combinations are able to distinguish between the different species or strains in infected host tissue, i.e. host tissue that has previously been infected with a specific pathogen species or strain. This invention provides numerous primer combinations that fulfill this criterion for detection of Rhizoctonia cerealis . The primers of the invention are designed based on sequence differences among the fungal ITS regions. A minimum of one base pair difference between sequences can permit design of a discriminatory primer. Primers designed to a specific fungal DNA's ITS region can be used in combination with a primer made to a conserved sequence region within the ribosomal DNA's coding region to amplify species-specific PCR fragments. In general, primers should have a theoretical melting temperature between about 60 to about 70 degree ° C. to achieve good sensitivity and should be void of significant secondary structure and 3′ overlaps between primer combinations. Primers generally have 100% sequence identity with at least about 5-10 contiguous nucleotide bases of ITS 1 or ITS2. In preferred embodiments, primers are anywhere from approximately 5-30 nucleotide bases long.

The present invention lends itself readily to the preparation of “kits” containing the elements necessary to carry out the process. Such a kit may comprise a carrier being compartmentalized to receive in close confinement therein one or more container, such as tubes or vials. One of the containers may contain unlabeled or detectably labeled DNA primers. The labeled DNA primers may be present in lyophilized form or in an appropriate buffer as necessary. One or more containers may contain one or more enzymes or reagents to be utilized in PCR reactions. These enzymes may be present by themselves or in admixtures, in lyophilized form or in appropriate buffers.

Finally, the kit may contain all of the additional elements necessary to carry out the technique of the invention, such as buffers, extraction reagents, enzymes, pipettes, plates, nucleic acids, nucleoside triphosphates, filter paper, gel materials, transfer materials, autoradiography supplies, and the like.

The examples below show typical experimental protocols that can be used in the isolation of ITS sequences, the selection of suitable primer sequences, the testing of primers for selective and diagnostic efficacy, and the use of such primers for disease and fungal isolate detection. Such examples are provided by way of illustration and not by way of limitation.

Standard recombinant DNA and molecular cloning techniques used here are well known in the art and are described by J. Sambrook, E. F. Fritsch and T. Maniatis, Molecular ( 1989) and by T. J. Silhavy, M. L. Berman, and L. W. Enquist, Experiments with Gene Fusions , Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1984) and by Ausubel, F. M. et al., Current Protocols in Molecular Biology , pub. by Greene Publishing Assoc. and Wiley-Interscience (1987).

Artificial Sequence

Description of Artificial Sequence ITS1

1

tccgtaggtg aacctgcgg 19

2

20

Artificial Sequence

Description of Artificial Sequence ITS2

2

gctgcgttct tcatcgatgc 20

3

20

Artificial Sequence

Description of Artificial Sequence ITS3

3

gcatcgatga agaacgcagc 20

4

20

Artificial Sequence

Description of Artificial Sequence ITS4

4

tcctccgctt attgatatgc 20

5

17

Artificial Sequence

Description of Artificial Sequence M13

Universal 20 Primer

5

gtaaaacgac ggccagt 17

6

16

Artificial Sequence

Description of Artificial Sequence Reverse

Primer

6

aacagctatg accatg 16

7

18

Artificial Sequence

Description of Artificial Sequence JB643

7

gcgagagaga ggctggct 18

8

21

Artificial Sequence

Description of Artificial Sequence JB644

8

ctcgcgagag agaggctggc t 21

9

21

Artificial Sequence

Description of Artificial Sequence JB645

9

gagatcagat cataaagtgt g 21

10

25

Artificial Sequence

Description of Artificial Sequence JB646

10

gagatcagat cataaagtgt gtttg 25

11

23

Artificial Sequence

Description of Artificial Sequence JB647

11

ctgtgcaact gtttagacgg tcg 23

12

20

Artificial Sequence

Description of Artificial Sequence JB648

12

tgcaactgtt tagacggtcg 20

13

23

Artificial Sequence

Description of Artificial Sequence JB649

13

accgttagaa gcggttcgtc cat 23

14

20

Artificial Sequence

Description of Artificial Sequence JB650

14

gttagaagcg gttcgtccat 20

15

20

Artificial Sequence

Description of Artificial Sequence JB687

15

tgcacctgtt tagacggttg 20

16

20

Artificial Sequence

Description of Artificial Sequence JB688

16

tgtgcacctg tttagacggt 20

17

687

Rhizoctonia cerealis

17

tccgtaggtg aacctgcgga aggatcatta atgaaatgaa tgtagagtcg gttgtagctg 60

ggtcttttaa tcgaggccat gtgcacacct tctctttcat ccactcacac ctgtgcacct 120

gtttagacgg ttgaaggaaa aagtctttct cgcgagagag agggccggct ccttttcccg 180

tccaatacat aaaatcttat atatttaatc agaatgtaat cgatgtaaac gcatctataa 240

actaagtttc aacaacggat ctcttggctc tcgcatcgat gaagaacgca gcgaaatgcg 300

ataagtaatg tgaattgcag aattcagtga atcatcgaat ctttgaacgc accttgcgct 360

ccttggtatt cctcggagca cgcctgtttg agtatcatga aattctcaaa gcaagtcttt 420

tgttaattca actggctttt gttttggatt tggaggtttt gcagattcac gtctgctcct 480

cttaaatgca ttagctggat ctctataaaa ccggttccac tcggcgtgat aagtatcact 540

cgctgaggac actcttgaaa aagggtggcc ggattcatgg atgaaccgct tctaacggtc 600

tattagatta gacaaacaca ctttatgatc tgatctcaaa tcaggtggga ctacccgctg 660

aacttaagca tatcaataag cggagga 687

18

686

Rhizoctonia cerealis

18

tccgtaggtg aacctgcgga aggatcatta atgaaatgaa tgtagagtcg gttgtagctg 60

ggtcttttaa tcgaggccat gtgcacacct tctctttcat ccactcacac ctgtgcacct 120

gtttagacgg ttgaaggaaa aagtctttct cgcgagagag aggccggctc cttttcccgt 180

ccaatacata aaatcttata tatttaatca gaatgtaatc gatgtaaacg catctataaa 240

ctaagtttca acaacggatc tcttggctct cgcatcgatg aagaacgcag cgaaatgcga 300

taagtaatgt gaattgcaga attcagtgaa tcatcgaatc tttgaacgca ccttgcgctc 360

cttggtattc ctcggagcac gcctgtttga gtatcatgaa attctcaaag caagtctttt 420

gttaattcaa ctggcttttg ttttggattt ggaggttttg cagattcacg tctgctcctc 480

ttaaatgcat tagctggatc tctataaaac cggttccact cggcgtgata agtatcactc 540

gctgaggaca ctcttgaaaa agggtggccg gattcatgga tgaaccgctt ctaacggtct 600

attagattag acaaacacac tttatgatct gatctcaaat caggtgggac tacccgctga 660

acttaagcat atcaataagc ggagga 686

19

687

Rhizoctonia cerealis

19

tccgtaggtg aacctgcgga aggatcatta atgaaatgaa tgtagagtcg gttgtagctg 60

ggtcttttaa tcgaggccat gtgcacacct tctctttcat ccactcacac ctgtgcacct 120

gtttagacgg ttgaaggaaa aagtctttct cgcgagagag agggccggct ccttttcccg 180

tccaatacat aaaatcttat atatttaatc agaatgtaat cgatgtaaac gcatctataa 240

actaagtttc aacaacggat ctcttggctc tcgcatcgat gaagaacgca gcgaaatgcg 300

ataagtaatg tgaattgcag aattcagtga atcatcgaat ctttgaacgc accttgcgct 360

ccttggtatt cctcggagca cgcctgtttg agtatcatga aattctcaaa gcaagtcttt 420

tgttgattca actggctttt gttttggatt tggaggtttt gcagattcac gtctgctcct 480

cttaaatgca ttagctggat ctctataaaa ccggttccac tcggcgtgat aagtatcact 540

cgctgaggac actcttgaaa aagggtggcc ggattcatgg atgaaccgct tctaacggtc 600

tattagatta gacaaacaca ctttatgatc tgatctcaaa tcaggtggga ctacccgctg 660

aacttaagca tatcaataag cggagga 687

20

685

Rhizoctonia cerealis

20

tccgtaggtg aacctgcgga aggatcatta atgaatgaat gtagagtcgg ttgtagctgg 60

gtcttttaat cgaggccatg tgcacacctt ctctttcatc cactcacacc tgtgcacctg 120

tttagacggt tgaaggaaaa agtctttctc gcgagagaga ggccggctcc ttttcccgtc 180

caatacataa aatcttatat atttaatcag aatgtaatcg atgtaaacgc atctataaac 240

taagtttcaa caacggatct cttggctctc gcatcgatga agaacgcagc gaaatgcgat 300

aagtaatgtg aattgcagaa ttcagtgaat catcgaatct ttgaacgcac cttgcgctcc 360

ttggtattcc tcggagcacg cctgtttgag tatcatgaaa ttctcaaagc aagtcttttg 420

ttaattcaac tggcttttgt tttggatttg gaggttttgc agattcacgt ctgctcctct 480

taaatgcatt agctggatct ctataaaacc ggttccactc ggcgtgataa gtatcactcg 540

ctgaggacac tcttgaaaaa gggtggccgg attcatggat gaaccgcttc taacggtcta 600

ttagattaga caaacacact ttatgatctg atctcaaatc aggtgggact acccgctgaa 660

cttaagcata tcaataagcg gagga 685

21

687

Rhizoctonia cerealis

21

tcctccgctt attgatatgc ttaagttcag cgggtagtcc cacctgattt gagatcagat 60

cataaagtgt gtttgtctaa tctaatagac cgttagaagc ggttcgtcca tgaatccggc 120

cacccttttg caagagtgtc ctcagcgagt gatacttatc acgccgagtg gaaccggttt 180

tatagagatc cagctaatgc atttaagagg agcagacgtg aatctgcaag acctccaaat 240

ccaaagcaaa agccagttga attaacaaaa gacttgcttt gagaatttca tgatactcaa 300

acaggcgtgc tccgaggaat accaaggagc gcaaggtgcg ttcaaagatt cgatgattca 360

ctgaattctg caattcacat tacttatcgc atttcgctgc gttcttcatc gatgcgagag 420

ccaagagatc cgttgttgaa acttagttta tagatgcgtt tacatcgatt acattctgat 480

taaatatata agattttatg tattggacgg gaaaaggagc cggccctctc tctcgcgaga 540

aagacttttt ccttcaaccg tctaaacagg tgcacaggtg tgagtggatg aaagagaagg 600

tgtgcacatg gcctcgatta aaagacccag ctacaaccga ctctacattc atttcattaa 660

tgatccttcc gcaggttcac ctacgga 687

22

686

Rhizoctonia cerealis

22

tccgtaggtg aacctgcgga aggatcatta atgaaatgaa tgtagagtcg gttgtagctg 60

ggtcttttaa tcgaggccat gtgcacacct tctctttcat ccactcacac ctgtgcacct 120

gtttagacgg ttgaaggaaa aagtctttct cgcgagagag agggccggct ccttttcccg 180

tccaatacat aaaatcttat atatttaatc agaatgtaat cgatgtaaac gcatctataa 240

actaagtttc aacaacggat ctcttggctc tcgcatcgat gaagaacgca gcgaaatgcg 300

ataagtaatg tgaattgcag aattcagtga atcatcgaat ctttgaacgc accttgcgct 360

ccttggtatt cctcggagca cacctgtttg agtatcatga aattctcaaa gcaagtcttt 420

tgttaattca actggctttt gttttggatt tggaggtttt gcagattcac gtctgctcct 480

cttaaatgca ttagctggat ctctataaaa ccggttccac tcggcgtgat aagtatcact 540

cgccgaggac actcttgaaa aagggtggcc ggattcatgg atgaaccgct tctaacggtc 600

tattagatta gacaaacaca ctttatgatc tgatctcaaa tcaggtggga ctacccgctg 660

aacttaagca ttcaataagc ggagga 686

23

687

Rhizoctonia cerealis

23

tccgtaggtg aacctgcgga aggatcatta atgaaatgaa tgtagagtcg gttgtagctg 60

ggtcttttaa tcgaggccat gtgcacacct tctctttcat ccactcacac ctgtgcacct 120

gtttagacgg ttgaaggaaa aagtctttct cgcgagagag agggccggct ccttttcccg 180

tccaatacat aaaatcttat atatttaatc agaatgtaat cgatgtaaac gcatctataa 240

actaagtttc aacaacggat ctcttggctc tcgcatcgat gaagaacgca gcgaaatgcg 300

ataagtaatg tgaattgcag aattcagtga atcatcgaat ctttgaacgc accttgcgct 360

ccttggtatt cctcggagca cacctgtttg agtatcatga aattctcaaa gcaagtcttt 420

tgttaattca actggctttt gttttggatt tggaggtctt gcagattcac gtctgctcct 480

cttaaatgca ttagctggat ctctataaaa ccggttccac tcggcgtgat aagtatcact 540

cgccgaggac actcttgcaa aagggtggcc ggattcatgg acgaaccgct tctaacggtc 600

tattagatta gacaaacaca ctttatgatc tgatctcaaa tcaggtggga ctacccgctg 660

aacttaagca tatcaataag cggagga 687

24

684

Rhizoctonia cerealis

24

tccgtaggtg aacctgcgga aggatcatta atgaatgaat gtagagtcgg ttgtagctgg 60

gtcttttaat cgaggccatg tgcacgcctt ctctttcatc cacacacacc tgtgcacctg 120

tttagacggt cgaaggaaaa agtctttctc gcgagagaga ggctggctcc ttttccgtcc 180

aatacataaa atcttatata tttaatcaga atgtaatcga tgtaaacgca tctataaact 240

aagtttcaac aacggatctc ttggctctcg catcgatgaa gaacgcagcg aaatgcgata 300

agtaatgtga attgcagaat tcagtgaatc atcgaatctt tgaacgcacc ttgcgctcct 360

tggtattcct cggagcacgc ctgtttgagt atcatgaaat tctcaaagca agtcttttgt 420

taattcaact ggcttttgtt ttggatttgg aggtcttgca gattcacgtc tgctcctctt 480

aaatgcatta gctggatctc tataaaatcg gttccactcg gcgtgataag tatcactcgc 540

tgaggacact cttgcaaaag ggtggccgga ttcatggacg aaccgcttct aacggtctat 600

tagattagac aaacacactt tatgatctga tctcaaatca ggtgggacta cccgctgaac 660

ttaagcatat caataagcgg agga 684

25

686

Rhizoctonia cerealis

25

tccgtaggtg aacctgcgga aggatcatta atgaaatgaa tgtagagtcg gttgtagctg 60

ggtcttttga tcgaggccat gtgcacacct tctctttcat ccactcacac ctgtgcacct 120

gtttagacgg tcgaaggaaa aagtctttct cgcgagagag aggccggctc cttttcccgt 180

ccaatacata aaatcttata tatttaatca gaatgtaatc gatgtaaacg catctataaa 240

ctaagtttca acaacggatc tcttggctct cgcatcgatg aagaacgcag cgaaatgcga 300

taagtaatgt gaattgcaga attcagtgaa tcatcgaatc tttgaacgca ccttgcgctc 360

cttggtattc ctcggagcac gcctgtttga gtatcatgaa attctcaaag caagtctttt 420

gttaattcaa ctggcttttg ttttggattt ggaggttttg cagattcacg tctgctcctc 480

ttaaatgcat tagctggatc tctataaaac cggttccact cggcgtgata agtatcactc 540

gctgaggaca ctcttgaaaa agggtggccg gattcatgga tgaaccgctt ctaacggtct 600

attagattag acaaacacac tttatgatct gatctcaaat caggtgggac tacccgctga 660

acttaagcat atcaataagc ggagga 686

26

686

Rhizoctonia cerealis

26

tccgtaggtg aacctgcgga aggatcatta atgaatgaat gtagagatcg gttgtagctg 60

ggtcttttaa tcgaggccat gtgcacgcct tctctttcat ccacacacac ctgtgcacct 120

gtttagacgg tcgaaggaaa aagtctatct cgagagagag aggccggctc cttttccgtc 180

caatacataa aatccttata tatttaatca gaatgtaatc gatgtaaacg catctataaa 240

ctaagtttca acaacggatc tcttggctct cgcatcgatg aagaacgcag cgaaatgcga 300

taagtaatgt gaattgcaga attcagtgaa tcatcgaatc tttgaacgca ccttgcgctc 360

cttggtattc ctcggagcac gcctgtttga gtatcatgaa attctcaaag caagtctttt 420

gttaattcaa ctggcttttg ctttggattt ggaggtcttg cagattcacg tctgctcctc 480

ttaaatgcat tagctggatc tctataaaac cggttccact cggcgtgata agtatcactc 540

gctgaggaca ctcttgcaaa agggtggccg gattcatgga cgaaccgctt ctaacggtct 600

attagattag acaaacacac tttatgatct gatctcaaat caggtgggac tacccgctga 660

acttaagcat atcaataagc ggagga 686

27

685

Rhizoctonia cerealis

27

tccgtaggtg aacctgcgga agatcattaa tgaaatgaat gtagagtcgg ttgtagctgg 60

gtcttttaat cgaggccatg tgcacacctt ctctttcatc cactcacacc tgtgcacctg 120

tttagacggt tgaaggaaaa agtctttctc gcgagagaga ggccggctcc ttttcccgtc 180

caatacataa aatcttatat atttaatcag aatgtaatcg atgtaaacgc atctataaac 240

taagtttcaa caacggatct cttggctctc gcatcgatga agaacgcagc gaaatgcgat 300

aagtaatgtg aattgcagaa ttcagtgaat catcgaatct ttgaacgcac cttgcgctcc 360

ttggtattcc tcggagcacg cctgtttgag tatcatgaaa ttctcaaagc aagtcttttg 420

ttaattcaac tggcttttgt tttggatttg gaggttttgc agattcacgt ctgctcctct 480

taaatgcatt agctggatct ctataaaacc ggttccactc ggcgtgataa gtatcactcg 540

ctgaggacac tcttgaaaaa gggtggccgg attcatggat gaaccgcttc taacggtcta 600

ttagattaga caaacacact ttatgatctg acctcaaatc aggtgggact acccgctgaa 660

cttaagcata tcaataagcg gagga 685

28

627

Pseudocercosporella herpotrichoides

28

tccgtaggtg aacctgcgga aggatcatta atagagcaat ggatagacag cgccccggga 60

gaaatcctgg gggccaccct acttcggtaa ggtttagagt cgtcgggcct ctcggagaag 120

cctggtccag acctccaccc ttgaataaat tacctttgtt gctttggcag ggcgcctcgc 180

gccagcggct tcggctgttg agtacctgcc agaggaccac aactcttgtt tttagtgatg 240

tctgagtact atataatagt taaaactttc aacaacggat ctcttggttc tggcatcgat 300

gaagaacgca gcgaaatgcg ataagtaatg tgaattgcag aattcagtga atcatcgaat 360

ctttgaacgc acattgcgcc ctctggtatt ccggggggca tgcctgttcg agcgtcatta 420

taaccactca agctctcgct tggtattggg gttcgcgtct tcgcggcctc taaaatcagt 480

ggcggtgcct gtcggctcta cgcgtagtaa tactcctcgc gattgagtcc ggtaggttta 540

cttgccagca acccccaatt ttttacaggt tgacctcgga tcaggtaggg atacccgctg 600

aacttaagca tatcaataag cggagga 627

29

583

Septoria nodorum

29

tccgtaggtg aacctgcgga aggatcatta cactcagtag tttactactg taaaaggggc 60

tgttagtctg tatagcgcaa gctgatgagc agctggcctc ttttatccac ccttgtcttt 120

tgcgtaccca cgtttcctcg gcaggcttgc ctgccggttg gacaaattta taaccttttt 180

aattttcaat cagcgtctga aaaacttaat aattacaact ttcaacaacg gatctcttgg 240

ttctggcatc gatgaagaac gcagcgaaat gcgataagta gtgtgaattg cagaattcag 300

tgaatcatcg aatctttgaa cgcacattgc gccccttggt attccatggg gcatgcctgt 360

tcgagcgtca tttgtaccct caagctctgc ttggtgttgg gtgtttgtcc tctccctagt 420

gtttggactc gccttaaaat aattggcagc cagtgttttg gtattgaagc gcagcacaag 480

tcgcgattcg taacaaacac ttgcgtccac aagccttttt aacttttgac ctcggatcag 540

gtagggatac ccgctgaact taagcatatc aataagcgga gga 583

30

548

Septoria tritici

30

tccgtaggtg aacctgcgga gggatcatta ccgagcgagg gcctccgggt ccgacctcca 60

accctttgtg aacacatccc gttgcttcgg gggcgaccct gccgggcgcc cccggaggac 120

caccaaaaaa cactgcatct ctgcgtcgga gtttacgagt aaatcgaaac aaaactttca 180

acaacggatc tcttggttct ggcatcgatg aagaacgcag cgaaatgcga taagtaatgt 240

gaattgcaga attcagtgaa tcatcgaatc tttgaacgca cattgcgccc cctggtattc 300

cggggggcat gcccgttcga gcgtcattac accactccag cctcgctggg tattgggcgt 360

cttttcgcgg gggatcactc ccccgcgcgc ctcaaagtct ccggctgagc ggtctcgtct 420

cccagcgttg tggcatcacg tctcgccgcg gagttcacga gccctcacgg ccgttaaatc 480

acacctcagg ttgacctcgg atcgggtagg gatacccgct gaacttaagc atatcaataa 540

gcggagga 548

31

579

Pyrenophora tritici-repentis

31

tccgtaggtg aacctgcgga gggatcatta cacaaatatg aagccggact gggatagggc 60

ctcgctgcct tgcccgtctg gcgccatatt cacccatgtc tttttgcgta ctacttgttt 120

ccttggcggg tccgcccgcc aattggacct tattcaaacc tttttttcag ttgcaatcag 180

cgtcagcaaa acaaatgtaa tcaattacaa ctttcaacaa cggatctctt ggttctggca 240

tcgatgaaga acgcagcgaa atgcgataag tagtgtgaat tgcagaattc agtgaatcat 300

cgaatctttg aacgcacatt gcgccctttg gtattccaaa gggcatgcct gttcgagcgt 360

catttgtacc ctcaagcttt gcttggtgtt gggcgtcttg tctctctccc gagactcgcc 420

ttaaaatcat tggcagccgg cctactggtt tcggagcgca gcacattatt tgcgctcttg 480

tccagccgcg gtcgcgcgtc catgaagctt ctttcaacct tttgacctcg gatcaggtag 540

ggatacccgc tgaacttaag catatcaata agcggagga 579

32

504

Fusarium culmorum

32

tgcggaggga tcattaccga gtttacaact cccaaacccc tgtgaacata ccttatgttg 60

cctcggcgga tcagcccgcg ccccgtaaaa agggacggcc cgccgcagga accttaaact 120

ctgtttttag tggaacttct gagtataaaa aacaaataaa tcaawacttt caacaacgga 180

tctcttggtt ctggcatcga tgaagaacgc agcaaaatgc gataagtaat gtgaattgca 240

gaattcagtg aatcatcgaa tctttgaacg cacattgcgc ccgccagtat tctggcgggc 300

atgcctgttc gagcgtcatt tcaaccctca agcccagctt ggtgttggga gctgcagtcc 360

tgctgcactc cccaaataca ttggcggtca cgtcgagctt ccatarcgta gtaatttaca 420

tatcgttact ggtaatcgtc gcggccacgc cgttaaaccc caacttctga atgttgacct 480

cggatcaggt aggaataccc gctg 504

33

526

Microdochium nivale

33

ggtgaacctg cggagggatc atwactgagt ttwtaactct ccaaaccatg tgaacttacc 60

actgttgcct cggtggatgg tgytgtctct cgggacggtg ccacckcsgg tggacwacct 120

aaactctgtt aatttttgtc aatctgaatc aaactaagma ataagtrarr actwtcwacw 180

acggatctct tggttctggc atcgatgaag aacgcakcga aatgcgataa gtaatgtgaa 240

tygyagaatt cagtgaatca tcgaatcttt gaacgcacat tgcgcccaty agtattctag 300

tgggcatrcc tgttcgagcg tcatttcaac ccttaagcct agcttagtgt tggragactg 360

cctaatacgc agctcctcaa aaccagtggc ggagtcggtt cgtkctctga gcgtagtaat 420

tttttatctc gcttctgcaa gccggactgs caacagccat aaaccgcacc cttcsggggc 480

actttttaat ggttgacctc ggatcaggta ggaatacccg ctgaac 526