Abstract:
The present invention relates to the use of at least one nucleic acid comprising or consisting of: 
     
       
         
               
             
                 (i) 
               
                 (SEQ ID NO: 1) 
               
                 CATCGATGAAGAACGCWGCRAAHTGCGATAMGTARTGYGAATTGCAGR 
               
                   
               
                 ATTCAGTGARTCATCGAAWYTTTGAACGCAYMTTGCRC, 
               
           
              
              
              
              
              
             
          
         
       
       
         
           
             wherein: 
             R represents A or G 
             Y represents C or T 
             M represents A or C 
             W represents A or T 
             H represents A or C or T 
             (ii) a portion of SEQ ID NO: 1, provided said nucleic acid binds under stringent conditions to a nucleic acid comprising or consisting of the complementary sequence of SEQ ID NO: 1, or 
             (iii) complementary sequences of (i) and (ii); for the detection of nucleic acids from one or more fungi in a sample.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
       [0001]    The present application is a divisional of U.S. application Ser. No. 12/737,006, filed Dec. 1, 2010, which is a U.S. National Phase of PCT Application No. PCT/EP2009/051689, filed May 20, 2009, which claims the benefit of EP Application No. 08104225.1 and U.S. Provisional Application No. 61/059,862, which are incorporated herein by reference. 
     
    
     FIELD OF INVENTION 
       [0002]    The present invention relates to nucleic acids and methods for detecting organic substrate pathogenic fungi. 
       BACKGROUND OF THE INVENTION 
       [0003]    Disease in lawngrasses or turfgrasses develops from an interaction among a susceptible plant, an environment favorable for disease development, and a pathogenic organism, usually a fungus. Such fungi may also develop on decorative grasses, plants and crops; indeed, they may appear on any suitable organic substrate. Thus, treatment of a diseased substrate, especially turfgrass, usually consists in applying fungicides that will either kill the fungus or keep it from growing. 
         [0004]    However, the first step in disease management, and especially turfgrass disease management, should always consist in identifying the causative pathogenic agent. Indeed, it is important to have identified the disease correctly, so that an appropriate fungicide can be selected. Arbitrary selection and application of fungicides without knowledge of the disease cause can do as much harm as good. Using the wrong fungicide wastes money and may involve the risk of exacerbating the disease, as well as causing other unwanted side effects. 
         [0005]    Classical methods for the identification of the causative pathogenic agent essentially rely on the symptoms which can be observed on the individual plant and on the turf stand, as well as on the fungal structures, such as mycelia or spores, which can be found in the vicinity of the diseased turfgrass. 
         [0006]    However, these methods may require a long time to be implemented, since they often involve the isolation and the culture of the fungi in a laboratory. Besides, differentiating closely related fungal species can prove difficult. 
         [0007]    Accordingly, molecular biology methods have been developed which circumvent these difficulties. One of the most popular fungal detection methods relies on the PCR amplification of the internal transcribed spacers (1, 2) and the 5.8S rRNA gene (ITS1-5.8S-ITS2) from the fungal rRNA operon (Goodwin et al. (1995)  Plant Pathology  44:384-391; Ranjard et al. (2001)  Applied and Environmental Microbiology  67:4479-4487). However, a specific primer pair is often necessary for the identification of a given fungal species, which renders this method cumbersome where the identity of pathogenic fungus is unknown and is sought for. Furthermore, some fungal species can not be differentiated using the primers currently available which target this region. Accordingly, this method is not used in routine for determining the antifungal agent most adapted to treat a given turfgrass disease. 
         [0008]    Japanese patent application No. 2008005760 discloses 458 probes for detecting molds that can be found in food. These 458 probes are designed for detecting molds by a hybridization-based method involving the use of a microarray. 
         [0009]    It is therefore an object of the invention to provide a method which allows the rapid specific detection of nucleic acids from the fungi most commonly involved in turfgrass diseases. 
       SUMMARY OF THE INVENTION 
       [0010]    The present invention arises from the identification, by the inventors, of a conserved region within the rRNA operon of the genome of pathogenic fungi generally affecting turfgrasses, but also possibly other organic substrates, which is liable to be used as a target in the frame of nucleic acid amplification-based detection method of most of such pathogenic fungi. 
         [0011]    Thus the present invention relates to the use of at least one nucleic acid comprising or consisting of: 
         [0000]    
       
         
               
             
           
               
                 (i) 
               
               
                 (SEQ ID NO: 1) 
               
               
                 CATCGATGAAGAACGCWGCRAAHTGCGATAMGTARTGYGAATTGCAGR 
               
               
                   
               
               
                 ATTCAGTGARTCATCGAAWYTTTGAACGCAYMTTGCRC, 
               
             
          
         
       
       
         
           
             wherein: 
             R represents A or G 
             Y represents C or T 
             M represents A or C 
             W represents A or T 
             H represents A or C or T 
             (ii) a portion of SEQ ID NO: 1, provided said nucleic acid binds under stringent conditions to a nucleic acid comprising or consisting of the complementary sequence of SEQ ID NO: 1, or 
             (iii) complementary sequences of (i) and (ii);
 
for the detection of nucleic acids from one or more fungi in a sample.
 
           
         
       
     
         [0020]    In a preferred embodiment, the present invention more particularly relates to the use of at least one nucleic acid comprising or consisting of: 
         [0000]    
       
         
               
               
             
           
               
                   
                 (i) 
               
               
                   
                 (SEQ ID NO: 2) 
               
               
                   
                 GTGARTCATCGAAWYTTTGAACGCA, 
               
             
          
         
       
       
         
           
             wherein: 
             R represents A or G 
             Y represents C or T 
             W represents A or T 
             (ii) a portion of SEQ ID NO: 2, provided said nucleic acid binds under stringent conditions to a nucleic acid comprising or consisting of the complementary sequence of SEQ ID NO: 1, or 
             (iii) complementary sequences of (i) and (ii);
 
for the detection of nucleic acids from one or more fungi in a sample.
 
           
         
       
     
         [0027]    The present invention also relates to a method for detecting nucleic acids from one or more fungi in a sample, wherein at least one nucleic acid comprising or consisting of: 
         [0000]    
       
         
               
             
           
               
                 (i) 
               
               
                 (SEQ ID NO: 1) 
               
               
                 CATCGATGAAGAACGCWGCRAAHTGCGATAMGTARTGYGAATTGCAGR 
               
               
                   
               
               
                 ATTCAGTGARTCATCGAAWYTTTGAACGCAYMTTGCRC, 
               
             
          
         
       
       
         
           
             wherein: 
             R represents A or G 
             Y represents C or T 
             M represents A or C 
             W represents A or T 
             H represents A or C or T 
             (ii) a portion of SEQ ID NO: 1, provided said nucleic acid binds under stringent conditions to a nucleic acid comprising or consisting of the complementary sequence of SEQ ID NO: 1, or 
             (iii) complementary sequences of (i) and (ii);
 
is contacted with the sample.
 
           
         
       
     
         [0036]    In a preferred embodiment, the invention more particularly relates to a method for detecting nucleic acids from one or more fungi in a sample, wherein at least one nucleic acid comprising or consisting of: 
         [0000]    
       
         
               
               
             
           
               
                   
                 (i) 
               
               
                   
                 (SEQ ID NO: 2) 
               
               
                   
                 GTGARTCATCGAAWYTTTGAACGCA, 
               
             
          
         
       
       
         
           
             wherein: 
             R represents A or G 
             Y represents C or T 
             W represents A or T 
             (ii) a portion of SEQ ID NO: 2, provided said nucleic acid binds under stringent conditions to a nucleic acid comprising or consisting of the complementary sequence of SEQ ID NO: 1, or 
             (iii) complementary sequences of (i) and (ii);
 
is contacted with the sample.
 
           
         
       
     
         [0043]    The present invention also relates to a method for treating a diseased organic substrate, which comprises the steps of: 
         [0000]    a) detecting the absence or the presence of nucleic acids from at least one pathogenic fungus in a sample of the substrate, with at least one nucleic acid according to the invention as defined above;
 
b) if nucleic acids from one or more pathogenic fungi have been detected in step a), selecting one or more antifungal agents which target the one or more pathogenic fungi from which nucleic acids have been detected;
 
c) applying the selected one or more antifungal agents of step b) to the diseased substrate.
 
         [0044]    The present invention also relates to a kit for the detection of fungi, comprising each one of the nucleic acids represented by: 
         [0000]    
       
         
               
             
           
               
                 (i) 
               
               
                 (SEQ ID NO: 1) 
               
               
                 CATCGATGAAGAACGCWGCRAAHTGCGATAMGTARTGYGAATTGCAGR 
               
               
                   
               
               
                 ATTCAGTGARTCATCGAAWYTTTGAACGCAYMTTGCRC, 
               
             
          
         
       
       
         
           
             wherein: 
             R represents A or G 
             Y represents C or T 
             M represents A or C 
             W represents A or T 
             H represents A or C or T 
             (ii) a portion of SEQ ID NO: 1, provided said nucleic acid binds under stringent conditions to a nucleic acid comprising or consisting of the complementary sequence of SEQ ID NO: 1, or 
             (iii) complementary sequences of (i) and (ii). 
           
         
       
     
         [0053]    In a preferred embodiment, the invention more particularly relates to a kit for the detection of fungi, comprising each one of the nucleic acids represented by: 
         [0000]    
       
         
               
               
             
           
               
                   
                 (i) 
               
               
                   
                 (SEQ ID NO: 2) 
               
               
                   
                 GTGARTCATCGAAWYTTTGAACGCA, 
               
             
          
         
       
       
         
           
             wherein 
             R represents A or G 
             Y represents C or T 
             W represents A or T 
             (ii) a portion of SEQ ID NO: 2, provided said nucleic acid binds under stringent conditions to a nucleic acid comprising or consisting of the complementary sequence of SEQ ID NO: 1, or 
             (iii) complementary sequences of (i) and (ii). 
           
         
       
     
         [0060]    The present invention also relates to a nucleic acid comprising or consisting of: 
         [0000]    
       
         
               
             
           
               
                 (i) 
               
               
                 (SEQ ID NO: 1) 
               
               
                 CATCGATGAAGAACGCWGCRAAHTGCGATAMGTARTGYGAATTGCAGR 
               
               
                   
               
               
                 ATTCAGTGARTCATCGAAWYTTTGAACGCAYMTTGCRC, 
               
             
          
         
       
       
         
           
             wherein: 
             R represents A or G 
             Y represents C or T 
             M represents A or C 
             W represents A or T 
             H represents A or C or T 
             (ii) a portion of SEQ ID NO: 1, provided the said nucleic acid binds under stringent conditions to a nucleic acid comprising or consisting of the complementary sequence of SEQ ID NO: 1, or 
             (iii) complementary sequences of (i) and (ii). 
           
         
       
     
         [0069]    In a preferred embodiment, the invention more particularly relates to a nucleic acid comprising or consisting of: 
         [0000]    
       
         
               
               
             
           
               
                   
                 (i) 
               
               
                   
                 (SEQ ID NO: 2) 
               
               
                   
                 GTGARTCATCGAAWYTTTGAACGCA, 
               
             
          
         
       
       
         
           
             wherein: 
             R represents A or G 
             Y represents C or T 
             W represents A or T 
             (ii) a portion of SEQ ID NO: 2, provided said nucleic acid binds under stringent conditions to a nucleic acid comprising or consisting of the complementary sequence of SEQ ID NO: 1, or 
             complementary sequences of (i) and (ii). 
           
         
       
     
         [0076]    In another preferred embodiment, the invention more particularly relates to a nucleic acid as defined above, comprising or consisting of a sequence selected from the group consisting of SEQ ID NO: 1 to 38, or complementary sequences thereof. 
     
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0077]    Nucleic acids as intended herein can be of any type, however it is preferred that they be DNA. 
         [0078]    “Stringent conditions” can be easily be defined by the man skilled in the art using common knowledge. If necessary, guidance for defining such conditions can be found in numerous textbooks, such as Tijssen, 1993, Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2 “Overview of principles of hybridization and the strategy of nucleic acid probe assays”, Elsevier, New York. Preferably, stringent conditions according to the invention are constituted of an annealing temperature of 60° C. carried out in a PCR reaction medium comprising, e.g. 50 mM KCl, 1.5 mM MgCl 2  and 10 mM Tris pH 8.3. 
         [0079]    As intended herein a “portion” of nucleic acid preferably comprises a number of nucleotides sufficient to provide for a specific hybridisation to the nucleic acid comprising or consisting of the complementary sequence of SEQ ID NO: 1. Thus, the portion of nucleic acid preferably comprises at least 9 nucleotides, more preferably at least 15 nucleotides, even more preferably at least 20 nucleotides, and most preferably at least 25 nucleotides. As intended herein, it is preferred that the maximum length of the nucleic acids according to the invention is less than 500 nucleotides. 
         [0080]    Preferably, the at least one nucleic acid as defined above is used as a primer in a nucleic acid amplification-based detection method, such as Amplification Fragment Length Polymorphism (AFLP) or Terminal Restriction Fragment Length Polymorphism (TRFLP or T-RFLP). 
         [0081]    Nucleic acid amplification-based detection methods are particularly well known to one of skill in the art. Terminal Restriction Fragment Length Polymorphism (TRFLP or T-RFLP), for instance, is notably described by Formey et al. (1997)  Applied and Environmental Microbiology  63:4516-4522. 
         [0082]    Where the at least one nucleic acid as defined above is used as a primer, it is preferably used in association with at least one other primer. The other primer can be any primer targeting a sequence comprised within the genome of the one or more fungi. However, it is preferred that the other primer targets the 18S rDNA/ITS1 region or the ITS2/28S rDNA region of the one or more fungi. 
         [0083]    The fungal rRNA operon comprises the 18S rRNA gene, the Internal Transcribed Spacer 1 (ITS1), the 5.8S rRNA gene, the Internal Transcribed Spacer 2 (ITS2), and the 28S rRNA gene. The 18S rDNA/ITS1 and ITS2/28S rDNA regions thus relate to the regions of the genome of the one or more fungi in the vicinity respectively of the junction of the 18S rRNA gene and ITS1, and of the junction of TIS2 and the 28S rRNA gene. As will be clear to anyone of skill in the art, where the at least one nucleic acid as defined above is used as a forward primer, the primer which targets the 18S rDNA/ITS1 region or the ITS2/28S rDNA region will be a reverse primer, and vice versa. The sequences of the 18S rDNA/ITS1 and ITS2/28S rDNA regions are well known to one of skill in the art and can usually be accessed from public sequence databases. Where sequences of the 18S rDNA/ITS1 and ITS2/28S rDNA regions would not be publicly available for a particular fungus species, they can be routinely sequenced. Besides, it is well within the common knowledge of anyone of skill in the art to select primers within the known sequences. 
         [0084]    By way of example of a primer targeting the 18S rDNA/ITS1 region, one can cite the so-called “ITS1-F primer” of sequence TCCGTAGGTGAACCTGCGG (SEQ ID NO: 39). Conversely, by way of example of a primer targeting the ITS2/28S rDNA region one can cite the so-called “ITS4 primer” of sequence TCCTCCGCTTATTGATATGC (SEQ ID NO: 40). Other examples of primers liable to be used with the primers according to the invention include the “ITS5 primer” of sequence GGAAGTAAAAGTCGTAACAAGG (SEQ ID NO: 41) and the “SR6R primer” of sequence AAGWAAAAGTCGTAACAAGG (SEQ ID NO: 42). Still other Examples are available from http://www.biology.duke.edu/fungi/mycolab/primers.htm. 
         [0085]    As intended herein the primers to be used may be unmodified or modified nucleic acids, in particular DNA. Where the primers are modified nucleic acids they can notably be labelled nucleic acids, in particular fluorescently labelled nucleic acids. 
         [0086]    Where TRFLP is used, the one of skill in the art knows how to design primers and to select restriction enzymes so that the nucleic acid generated and fragmented by the enzyme presents a fragment length that can be detected. In this regard, it should be noted that detection of a fragment is dependant on the instrumentation and detection techniques used, but in general detection is possible for sequences featuring at least 15 nucleotides. Besides, the length of the fragment should preferably be such that it is distinguishable from fragment lengths generated by the same primer pair/enzyme in other fungi (in general, fragment lengths differing by more than 1, and preferably more than 2, nucleotides are distinguished by current techniques). Numerous databases and tools are available to one of skill in the art for selecting primers and restriction enzymes, such as the REBASE database. 
         [0087]    In the case where nucleic acids according to the invention include nucleotides featuring a “wobble” position, that is to say they include nucleotides that may be selected from among two or more possible nucleotides, one of skill in the art knows that it is usually advantageous to use a mixture of primers such that all of the nucleotide possibilities are represented. 
         [0088]    Thus, by way of example, each one of the nucleic acids represented by GTGARTCATCGAAWYTTTGAACGCA (SEQ ID NO: 2), are as follows: 
         [0000]    
       
         
               
               
             
           
               
                   
                 (SEQ ID NO: 3) 
               
               
                   
                 GTGAATCATCGAAACTTTGAACGCA; 
               
               
                   
                   
               
               
                   
                 (SEQ ID NO: 4) 
               
               
                   
                 GTGAGTCATCGAAACTTTGAACGCA; 
               
               
                   
                   
               
               
                   
                 (SEQ ID NO: 5) 
               
               
                   
                 GTGAATCATCGAATCTTTGAACGCA; 
               
               
                   
                   
               
               
                   
                 (SEQ ID NO: 6) 
               
               
                   
                 GTGAGTCATCGAATCTTTGAACGCA; 
               
               
                   
                   
               
               
                   
                 (SEQ ID NO: 7) 
               
               
                   
                 GTGAATCATCGAAATTTTGAACGCA; 
               
               
                   
                   
               
               
                   
                 (SEQ ID NO: 8) 
               
               
                   
                 GTGAGTCATCGAAATTTTGAACGCA; 
               
               
                   
                   
               
               
                   
                 (SEQ ID NO: 9) 
               
               
                   
                 GTGAATCATCGAATTTTTGAACGCA; 
               
               
                   
                   
               
               
                   
                 (SEQ ID NO: 10) 
               
               
                   
                 GTGAGTCATCGAATTTTTGAACGCA; 
               
               
                   
                   
               
               
                   
                 (SEQ ID NO: 11) 
               
               
                   
                 GTGAATCATCGAAACTTTGAACGCA; 
               
               
                   
                   
               
               
                   
                 (SEQ ID NO: 12) 
               
               
                   
                 GTGAGTCATCGAAACTTTGAACGCA; 
               
               
                   
                   
               
               
                   
                 (SEQ ID NO: 13) 
               
               
                   
                 GTGAATCATCGAATCTTTGAACGCA; 
               
               
                   
                   
               
               
                   
                 (SEQ ID NO: 14) 
               
               
                   
                 GTGAGTCATCGAATCTTTGAACGCA; 
               
               
                   
                   
               
               
                   
                 (SEQ ID NO: 15) 
               
               
                   
                 GTGAATCATCGAAATTTTGAACGCA; 
               
               
                   
                   
               
               
                   
                 (SEQ ID NO: 16) 
               
               
                   
                 GTGAGTCATCGAAATTTTGAACGCA; 
               
               
                   
                   
               
               
                   
                 (SEQ ID NO: 17) 
               
               
                   
                 GTGAATCATCGAATTTTTGAACGCA; 
               
               
                   
                   
               
               
                   
                 (SEQ ID NO: 18) 
               
               
                   
                 GTGAGTCATCGAATTTTTGAACGCA. 
               
             
          
         
       
     
         [0089]    As intended herein, it is to be understood that the invention aims at the detection of nucleic acids of fungi of any type, however it is preferred that the fungus is a plant pathogenic fungus, in particular a turfgrass pathogenic fungus, such as a fungus selected from the group constituted of  Ascochyta phleina, Curvularia affinis, Glomerella graminicola, Thanatephorus cucumeris, Pythium ultimum, Gaeumannomyces graminis, Marasmius oreades, Corticium fuciforme, Phytophthora nicotianae. Fusarium culmorum, Bipolaris sorokiniana, Microdochium nivale, Rhizoctonia cerealis, Pythium graminicola, Rhynchosporium secalis, Sclerotinia homoeocarpa, Typhula incarnate, Ustilago striiformis, Septoria macropoda.    
         [0090]    As intended herein the sample in which nucleic acids are to be detected can be of any type of organic substrate liable to contain nucleic acids from fungi. However, it is preferred that the sample be a turfgrass sample or a soil sample. 
         [0091]    Where the sample is a turfgrass sample, it can be a sample obtained from the turfgrass as a whole or from or a sample of a part of the turfgrass, such as the root. 
         [0092]    Where the sample is a soil sample, it is preferably taken directly under the diseased turfgrass or in the vicinity of the diseased turfgrass. 
         [0093]    The sample can be obtained directly from turfgrass or soil, or be obtained after treatment steps, such as grinding or extraction, in particular nucleic acid extraction, steps. 
         [0094]    As intended herein, any diseased turfgrass can be subjected the use or methods as defined above. Preferred turfgrasses to be considered within the frame of the present invention are notably described in http://www.ars-grin.gov/cgi-bin/npgs/html/index.pl, from the Germplasm Resources Information Network, National Germplasm Resources Laboratory, Beltsville, Md., or in the  Compendium of Turfgrass Diseases, Third Edition  (2005) by the American Phytopathological Society. Most preferably, the diseased turfgrass according to the invention is selected from the group consisting of the Festaceae, Aveneae, Triticeae, Chlorideae, Zoysieae, Paniceae and Andropogoneae Tribe. 
         [0095]    In a specific embodiment, nucleic acids comprising or consisting of the sequences of SEQ ID Nos. 159, 188, 198, 221, 263 and/or 416 of Japanese patent application No. 2008005760 are excluded from the scope of the present invention. 
       EXAMPLE 
       [0096]    The following turfgrass pathogenic fungus species were sequenced by the inventors:  Ascochyta phleina, Curvularia affinis, Glomerella graminicola, Thanatephorus cucumeris, Pythium ultimum, Gaeumannomyces graminis, Marasmius oreades, Corticium fuciforme, Phytophthora nicotianae. Fusarium culmorum, Bipolaris sorokiniana, Microdochium nivale, Rhizoctonia cerealis, Pythium graminicola, Rhynchosporium secalis, Sclerotinia homoeocarpa, Typhula incarnate, Ustilago striiformis, Septoria macropoda.    
         [0097]    Briefly, the well-known sequencing method developed by Fred Sanger—chain termination method—was used using Applied Biosystem BigDye® Terminator Cycle Sequencing v1 or v3.1 chemistry on an Applied Biosystems Genetic Analyzer 3130. The sequences were gathered by using the ITS1 forward primer (ITS1-F, TCCGTAGGTGAACCTGCGG, SEQ ID NO: 39). 
         [0098]    The obtained sequences are represented by SEQ ID NO: 19 to 37. 
         [0099]    From these sequences a particular consensus sequence could be determined by the inventors: 
         [0000]    
       
         
               
             
           
               
                 (SEQ ID NO: 1) 
               
               
                 CATCGATGAAGAACGCWGCRAAHTGCGATAMGTARTGYGAATTGCAGRA 
               
               
                   
               
               
                 TTCAGTGARTCATCGAAWYTTTGAACGCAYMTTGCRC 
               
             
          
         
       
     
         [0100]    The discriminative potential of this region was then evidenced by in silico Terminal Restriction Fragment Length Polymorphism (TRFLP or T-RFLP) by using the primer ITSOMYAr (TGCGTTCAAARWTTCGATGAYTCAC, SEQ ID NO: 38, the complementary of SEQ ID NO: 2), which hybridizes to the above consensus sequence, in association with the above ITS1-F primer. 
         [0101]    The applied enzymes for the T-RFLP analysis were TaqI (T ▾ CG ▴ A) and Tsp509I ( ▾ AATT ▴ ). 
         [0102]    The following tables show the PCR fragment length of both primer combination ITS1-F/ITS4 (full length ITS) (ITS4, TCCTCCGCTTATTGATATGC, SEQ ID NO: 40) and ITS1/ITSOMYAr (partial ITS length) (Table 1), as well as the corresponding in silico and in vitro digested partial ITS (ITS1/ITSOMYAr) fragments by TaqI (Table 2) and Tsp509I (Table 3) (mean and standard deviation (StDev) were calculated from triplicate measurements of two independent PCR assays). 
         [0000]    
       
         
               
               
               
             
               
               
               
               
             
           
               
                   
                 TABLE 1 
               
             
             
               
                   
                   
               
               
                   
                 Fragments [bps] 
                   
               
             
          
           
               
                   
                   
                 Full ITS 
                 Partial ITS 
               
               
                   
                   
                 length 
                 length 
               
               
                   
                 Fungal name 
                 ITS1/ITS4 
                 ITS1/ITSOMYAr 
               
               
                   
                   
               
               
                   
                 
                   Ascochyta phleina 
                 
                 588 
                 326 
               
               
                   
                 
                   Curvularia affinis 
                 
                 584 
                 316 
               
               
                   
                 
                   Glomerella graminicola 
                 
                 587 
                 320 
               
               
                   
                 
                   Thanatephorus cucumeris 
                 
                 710 
                 360 
               
               
                   
                 
                   Pythium ultimum 
                 
                 914 
                 360 
               
               
                   
                 
                   Gaeumannomyces graminis 
                 
                 558 
                 280 
               
               
                   
                 
                   Marasmius oreades 
                 
                 672 
                 355 
               
               
                   
                 
                   Corticium fuciforme 
                 
                 716 
                 353 
               
               
                   
                 
                   Phytophthora nicotianae 
                 
                 892 
                 367 
               
               
                   
                 
                   Fusarium culmorum 
                 
                 545 
                 286 
               
               
                   
                 
                   Bipolaris sorokiniana 
                 
                 586 
                 309 
               
               
                   
                 
                   Microdochium nivale 
                 
                 556 
                 283 
               
               
                   
                 
                   Rhizoctonia cerealis 
                 
                 684 
                 348 
               
               
                   
                 
                   Pythium graminicola 
                 
                 872 
                 319 
               
               
                   
                 
                   Rhynchosporium secalis 
                 
                 627 
                 371 
               
               
                   
                 
                   Sclerotinia homoeocarpa 
                 
                 586 
                 320 
               
               
                   
                 
                   Typhula incarnata 
                 
                 825 
                 410 
               
               
                   
                 
                   Ustilago striiformis 
                 
                 780 
                 380 
               
               
                   
                 
                   Septoria macropoda 
                 
                 540 
                 277 
               
               
                   
                   
               
             
          
         
       
     
         [0000]    
       
         
               
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                   
                 TABLE 2 
               
             
             
               
                   
                   
               
               
                   
                 TaqI fragments [bps] 
               
             
          
           
               
                   
                   
                   
                 measured 
                   
               
               
                   
                 Fungal name 
                 in-silico 
                 mean 
                 StDev 
               
               
                   
                   
               
             
          
           
               
                   
                 
                   Ascochyta phleina 
                 
                 252 
                 247.0 
                 0.022 
               
               
                   
                 
                   Curvularia affinis 
                 
                 242 
                 236.4 
                 0.048 
               
               
                   
                 
                   Glomerella graminicola 
                 
                 246 
                 239.3 
                 0.113 
               
               
                   
                 
                   Thanatephorus cucumeris 
                 
                 286 
                 284.4 
                 0.123 
               
               
                   
                 
                   Pythium ultimum 
                 
                 286 
                 284.0 
                 0.043 
               
               
                   
                 
                   Gaeumannomyces graminis 
                 
                 206 
                 198.9 
                 0.030 
               
               
                   
                 
                   Marasmius oreades 
                 
                 281 
                 279.6 
                 0.057 
               
               
                   
                 
                   Corticium fuciforme 
                 
                 279 
                 275.7 
                 0.054 
               
               
                   
                 
                   Phytophthora nicotianae 
                 
                 293 
                 290.9 
                 0.056 
               
               
                   
                 
                   Fusarium culmorum 
                 
                 212 
                 207.9 
                 0.033 
               
               
                   
                 
                   Bipolaris sorokiniana 
                 
                 235 
                 230.2 
                 0.054 
               
               
                   
                 
                   Microdochium nivale 
                 
                 209 
                 206.5 
                 0.247 
               
               
                   
                 
                   Rhizoctonia cerealis 
                 
                 71 
                 67.7 
                 0.049 
               
               
                   
                 
                   Pythium graminicola 
                 
                 245 
                 242.8 
                 0.033 
               
               
                   
                 
                   Rhynchosporium secalis 
                 
                 113 
                 108.5 
                 0.043 
               
               
                   
                 
                   Sclerotinia homoeocarpa 
                 
                 102 
                 98.1 
                 0.043 
               
               
                   
                 
                   Typhula incarnata 
                 
                 32 
                 29.9 
                 0.182 
               
               
                   
                 
                   Ustilago striiformis 
                 
                 31 
                 29.6 
                 0.130 
               
               
                   
                 
                   Septoria macropoda 
                 
                 162 
                 147.2 
                 0.161 
               
               
                   
                   
               
             
          
         
       
     
         [0000]    
       
         
               
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                   
                 TABLE 3 
               
             
             
               
                   
                   
               
               
                   
                 Tsp509I fragments [bps] 
               
             
          
           
               
                   
                   
                   
                 measured 
                   
               
               
                   
                 Fungal name 
                 in-silico 
                 mean 
                 StDev 
               
               
                   
                   
               
             
          
           
               
                   
                 
                   Ascochyta phleina 
                 
                 167 
                 163.5 
                 0.041 
               
               
                   
                 
                   Curvularia affinis 
                 
                 278 
                 272.1 
                 0.044 
               
               
                   
                 
                   Glomerella graminicola 
                 
                 207 
                 201.2 
                 0.034 
               
               
                   
                 
                   Thanatephorus cucumeris 
                 
                 34 
                 32.3 
                 0.091 
               
               
                   
                 
                   Pythium ultimum 
                 
                 322 
                 321.4 
                 0.040 
               
               
                   
                 
                   Gaeumannomyces graminis 
                 
                 134 
                 125.7 
                 0.096 
               
               
                   
                 
                   Marasmius oreades 
                 
                 225 
                 223.9 
                 0.056 
               
               
                   
                 
                   Corticium fuciforme 
                 
                 315 
                 312.8 
                 0.042 
               
               
                   
                 
                   Phytophthora nicotianae 
                 
                 108 
                 104.1 
                 0.026 
               
               
                   
                 
                   Fusarium culmorum 
                 
                 248 
                 243.3 
                 0.029 
               
               
                   
                 
                   Bipolaris sorokiniana 
                 
                 193 
                 189.0 
                 0.032 
               
               
                   
                 
                   Microdochium nivale 
                 
                 137 
                 133.5 
                 0.015 
               
               
                   
                 
                   Rhizoctonia cerealis 
                 
                 310 
                 307.6 
                 0.032 
               
               
                   
                 
                   Pythium graminicola 
                 
                 64 
                 58.6 
                 0.048 
               
               
                   
                 
                   Rhynchosporium secalis 
                 
                 102 
                 329.7 
                 0.030 
               
               
                   
                 
                   Sclerotinia homoeocarpa 
                 
                 175 
                 172.0 
                 0.066 
               
               
                   
                 
                   Typhula incarnata 
                 
                 34 
                 31.9 
                 0.112 
               
               
                   
                 
                   Ustilago striiformis 
                 
                 143 
                 140.7 
                 0.013 
               
               
                   
                 
                   Septoria macropoda 
                 
                 239 
                 222.4 
                 0.169 
               
               
                   
                   
               
             
          
         
       
     
         [0103]    In general, since the length of the fragments yielded by the ITS1/ITSOMYAr combination, either in AFLP or in T-RFLP, is usually smaller than the corresponding fragment obtained using the ITS1/ITS4 combination, the fragments can be separated with a higher resolution. 
         [0104]    Furthermore, it can be seen that the ITS1/ITSOMYAr combination provides, either in AFLP or in T-RFLP, a good alternative to the ITS1/ITS4 combination in AFLP since it notably enables to discriminate between species which discrimination was either impossible or very difficult to carry out with the ITS1/ITS4 combination (e.g.  Ascochyta phleina, Curvularia affinis, Glomerella graminicola, Bipolaris sorokiniana, Sclerotinia homoeocarpa ).