WO2004065627A1

WO2004065627A1 - Method and kit for early diagnosis of fungal infection (pyricularia oryzae) in rice

Info

Publication number: WO2004065627A1
Application number: PCT/EP2004/000496
Authority: WO
Inventors: Francesco Sala; Ana Maria Picco
Original assignee: REGIONE LOMBARDIA; Universita degli Studi di Pavia
Current assignee: REGIONE LOMBARDIA; Universita degli Studi di Pavia
Priority date: 2003-01-22
Filing date: 2004-01-22
Publication date: 2004-08-05
Anticipated expiration: 2005-07-22
Also published as: ITMI20030088A1

Abstract

The present invention describes a method and oligonucleotide primers for early diagnosis of fungal infection in rice.

Description

METHOD AND KIT FOR EARLY DIAGNOSIS OF FUNGAL INFECTION (PYRICULARIA ORYZAE) IN RICE Field of the invention

The field of the invention is the early molecular detection of fungal infection in plants.

State of the art

Several biotic and abiotic agents can damage rice cultures. Adverse climatic conditions, nutritional deficiencies, weeds, algae, bacteria, viruses and nematodes are among the common causes of disturbance or weakening of the plant during the growth phase. In particular, rice diseases caused by pathogenic fungi are especially investigated due to the heavy consequences on crop yield and also to the complexity of disease presentation.

Epidemics of blast are constantly reported in Italian regions characterized by a good rice production. This is considered among the most serious pathologies world-wide, and is present wherever rice is cultivated on large scale. Blast disease is caused by the fungus Pyricularia oryzae Cavara. This term designates the vegetative form of the fungus, while the perfect form, characterized by sexual reproduction, is termed Magnaporthe grisea (Hebert) (Yaegashi & Udagawa). Infection of rice plants causes leaf-blast, neck-blast and culm-blast. In phytopathological terms, rice blast must be included in the group of toxic or necrotoxic diseases, wherein cells of invaded tissues are quickly killed by pathogens that eat the cellular material. The evident incompatibility between host and pathogen defines such diseases as "disadaptive". Fungal pathogens are characterized by a remarkable genetic variability; the mutant strains give rise to sub-species. The fungus Pyricularia oryzae is capable of autonomous life, thus can be cultured in the laboratory.

P. oryzae is able to produce at least two toxins harmful to the host: alpha-picolinic acid, that alters cell permeability, and pyricularin that, in addition to carrying out a critical role in leaf necrosis, stimulates the accumulation of coumarin, a substance that is as toxic for rice (Tamari, K., J. Kaji, 1954, Journal of the Agricultural Chemical Society of Japan 28: 254-258; Tamari, K., J. Kaji, 1959, Journal of the Agricultural Chemical Society of Japan 33: 181-183). The disease can occur at any stage of plant development the plant, however, in Italy, it is particularly severe in the adult plant, especially in July, when the temperature approaches the optimum for pathogen growth. Round spots (2-3 millimeters) of a red-tawny colour appear on the basal leaves. In susceptible rice cultivars, such spots enlarge and elongate along the leaf nerves, acquiring a fusiform appearance, with the central part of light grey colour, a first tawny ring and a second yellowish margin. When spots converge, the leaf sheets acquire a yellow-reddish colour and dry up, conferring to the plant the typical burnt appearance, from which the term blast arise. Culm nodes necrotic areas are characterized by an intense black or tawny colour.

When the infection occurs at an early stage, the upper part of the culm, being no longer fed, dries up and dies. If the panicle already came out, it remains with empty spikelets and white glumes. In case the fungal infection occurs at a later stage, only few and malformed grains reach maturity. The leaf symptoms are frequently aggravated due to invasion by other saprophytic fungi.

The problem of fighting the blast infection is still of difficult solution, mainly because its typical symptomatic appearance, that is dark spots on leaves, nodes and parts of the panicle, can be confused with that of brown spot a rice disease πd caused by Bipolaris oryzae (Ou, S. H. 1985. Rice Diseases, 2 ed., CAB Int.

Mycol. Inst.). Also other pathogenic fungi, such as Alterna a spp., Epicoccum sp. and Fusarium spp., can cause necrotic leaf spots that, when observed with naked eye and without specific mycological surveying, are often erroneously interpreted as an alert sign of non-existent epidemics of blast. Interventions to protect rice production from blast are based essentially on plant treatment with fungicides preventing fungal action. Moreover, it is possible to obtain a partial prevention by applying specific agronomic practices, mainly aimed at controlling nitrogenous fertilization (Matsuyama, N., 1975, Annals of the Phytopathological Society of Japan 41 : 56-61 ), or selecting, through breeding programs, rice cultivars that are partially resistant to the fungus. However, the fear of a financial damage often leads the rice farmer to make inappropriate chemical treatments, in case of wrong diagnosis, or anyhow excessive and useless treatments, in case of late identification of the disease.

During the latest years; the high sensitivity and selectivity of PCR-based tests, together with their ease of use, led to the development of diagnostic tests for different systems of host-parasite infection. The technical issue is to design primers that are so specific that they amplify only DNA regions of the parasite but not of the host, to obtain a certain diagnosis. In fact, there are several studies on PCR (Ferreira, M. A. S, V et al., 1996, Appl. Environ. Microbiol. 62: 87-93) and industrial applications also in the plant field. As an example, US 5827695 describes the development of a PCR based test for diagnosis of wheat fungal pathogens belonging to the genus Fusarium and Microdochium. Analogously, US 5776686 describes the development of specific oligonucleotides and of a PCR test for the wheat pathogen belonging to the genus Tilleiia.

However, molecular tests for the diagnosis of Pyricularia oryzae infection in rice were not available until now. Field studies have shown that fungal spore of P. oryzae are source of inoculum for the blast disease. The events preceding the penetration of the pathogen are represented by: spore germination, germ tube growth and formation of the appressorium that penetrates plant tissues. At this stage, the disease is irreversible. However, the infection can be reduced by a timely and specific chemical treatment aimed at protecting the rice and preventing the secondary inoculum, that is responsible for spreading of the pathogen and for most damage of the culture. Therefore, it is evident the extreme usefulness of a method for early diagnosis of the infection, that can be performed directly on the plant and unambiguously identifies the responsible pathogen. Currently, it is possible to monitor corridia air dispersion. However, this method does not allow assessment of the actual penetration of the pathogen in plant tissues in order to establish the stage of fungal infection. Summary ©f th© invention The invention describes oligonucleotides for identification of the fungus Pyricularia oryzae that are useful for the diagnosis of fungal infections of rice {Oryza sativa L) which cause the pathology known as blast disease. In particular, the oligonucleotides of the present invention are used as "primers" in in vitro DNA amplification reactions (PCR). Moreover, the invention relates to a method for identifying the fungus using DNA extracted from strains of P. oryzae isolated from plant infected organs (leaves, culms, necks), and a kit for its execution. Description of the figures Figure 1 : Gel electrophoresis in 1% agarose of fragments PWL2 obtained by PCR amplification, with primers MG15 and MG16, of DNA from P. oryzae strains isolated from cultivars of rice Drago and Padano (lanes 1 and 2), of DNA from the cultivar of Arborio rice (lane 3) and of DNA from fungi Alternaria alternata, Bipolaris oryzae, Cladosporium sp., Epicoccum nigrum (lane 4, 5, 6, 7 respectively). MW: molecular weight marker (100 bp ladder, MBI Fermentas).

Figure 2: Gel electrophoresis in 1% agarose of fragments PWL2 obtained by PCR amplification, with primers MG15 and MG16, of DNA from P. oryzae strains^' isolated from necks of cultivar of Perla rice (lanes 1 and 3) and Ghibli (lanes 2 and 4), that turned out to be not infected from previous microscopical analysis, from leaves of cultivars of Perseo and Perla rice (lanes 5 and 6), and from necks of cultivars of Ghibli and Perla rice that turned out to be infected from previous microscopical analysis (lanes 7 and 8). The positive control is represented by P. oryzae DNA isolated from cultivar of Drago rice (lane 9), amplified under the same conditions. MW: molecular weight marker (100 bp ladder, MBI Fermentas). Detailed description of the invention

The invention describes oligonucleotides useful for identification in a plant sample of fungal infections caused by the fungus P. oryzae. The oligonucleotides according to the present invention are used as oligonucleotide "primers" in DNA amplification chain reactions by means of PCR (Polymerase Chain Reaction). In particular, the oligonucleotides according to the present invention are capable of generating unique fragments when used in PCR reactions carried out in presence of DNA from P. oryzae strains. In the rice (O. saliva), the fungal pathogen P. oryzae is responsible for the so-called blast disease and is capable of infecting various rice cultivars such as: Arborio, Adelio, Ariete, Argo, Balilla, Carnaroli, Drago, Gange, Lamone, Padano, S. Andrea, Taibonet, Teyo, Vialone Nano. The oligonucleotide primers according to the present invention are -capable of amplifying DNA fragments from P. oryzae strains derived from various rice cultivars, as for example those just mentioned.

The oligonucleotide primers according to the invention are: (1) selective, as they only amplify fungal preferably P. oryzae DNA and not rice DNA, (2) specific for the selected gene, as they give rise to a single amplification product, and (3) of optimal size, as they amplify a fragment of approximately 1000 base pairs, even in presence of excess host DNA.

Therefore, in a preferred embodiment the invention provides oligonucleotide primers comprising a sequence of at least 12 consecutive bases that are identical to at least one of the oligonucleotides defined in the group of sequences from ID NO: 1 to ID NO: 8. A brief description of the oligonucleotides according to the present invention is therefore provided and is further reported in the Sequence

Listing: sequence ID NO: 1

P. oryzae cutinase gene MG1 TGTGTGTACCACGTGAACC sequence ID NO: 2

P.oryzae cutinase gene

MG2 CGGCAACAGTACGCATTACC sequence ID NO: 3 P.oryzae MAP kinase gene

MG11 TTAACACGAAGACGGATC sequence ID NO: 4

P.oryzae MAP kinase gene

MG12 TACCACACGATACCGTAGC sequence ID NO: 5

P.oryzae PWL2 gene

MG15 TTTCTCATTCCCTTAACG sequence ID NO: 6

P.oryzae PWL2 gene MG 16 TCCCTCACACTTAAGTTAAC sequence ID NO: 7

P.oryzae PWL2 gene MG17 TAAATTACATCCCTTACTC sequence ID NO: 8 P.oryzae PWL2 gene MG18 GCTTATAAATTAGCCAACC According to a further embodiment, the invention comprises oligonucleotide primers chosen within the group consisting of sequences from ID NO: 1 to ID NO: 8. Said oligonucleotides have a length ranging between 18 and 22 bases, have a Tm ranging between 55°C and 60°C, with optimal average value of 58°C and a G+C content of approximately 40-50%. Based on the oligonucleotides of the present invention, the expert in the field is able to design further oligonucleotides with the same recognition specificity by introducing minimal variations of the sequence. Examples of oligonucleotides that can be derived from those described in the present invention are those comprising only partially the sequences from ID NO: 1 to 8, even though they amplify the same region of the corresponding P. oryzae gene or cDNA. An example of oligonucleotides according to this embodiment are those comprising at least 12 consecutive bases that are identical to at least one of the oligonucleotides in the group composed of sequences from ID NO: 1 to ID NO: 8, and sequences immediately upstream and/or downstream of the sequence as reported in the database. Moreover, even more preferably these oligonucleotides comprise 2 to 10 bases upstream (5') and/or downstream (3') the described sequences.

Moreover, the oligonucleotides according to the invention can be chemically modified following methods known in the art. An example of oligonucleotide modification is the so-called «labeling» that can be carried out with radioactive isotopes or with molecules suitable for detection, as for example by chemiluminescence, or other molecules used for labeling, such as biotin. Therefore, the invention provides oligonucleotide sequences useful for identification of the fungal pathogen P. oryzae. These sequences are derived from already known genes coding the proteins: cutinase, MAP kinase, host-species specificity (PWL2), respectively identified in the database with the following accession numbers: cutinase (EMBL ACN° MGCUT1), MAP kinase Mps1 (MPS1) (EMBL ACN° AFO20316), host-species specificity (PWL2) (EMBL ACN° MG26313).

According to a preferred embodiment, the oligonucleotides are used in pair in the DNA amplification reaction, the Polymerase Chain Reaction (PCR) in presence of P. oryzae DNA template. According to a preferred embodiment, the oligonucleotide pairs consist of independent oligonucleotide primers comprising the following sequences: SEQ ID NO: 1 + SEQ ID NO: 2 (MG1 +MG3), SEQ ID NO: 3 + SEQ ID NO: 4 (MG11+MG12), SEQ ID NO: 5 + SEQ ID NO: 6 (MG15+MG16), SEQ ID NO: 7 + SEQ ID NO: 8 (MG17+MG18). Oligonucleotides with sequence complementary to the one described are included in the present invention, and are used for amplifications from genomic DNA of P. oryzae. Particularly preferred is the pair of oligonucleotide primers constituted by SEQ ID NO: 5 + SEQ ID NO: 6 (MG15+MG16) or by oligonucleotides comprising said sequences. This pair of oligonucleotide primers recognizes the PWL2 gene. When used in PCR according to the preferred embodiment, these pairs of oligonucleotide primers produce amplified fragments not exceeding 1000 bases in length, having a melting temperature (Tm) ranging between 75°C and 85°C. Particularly preferred is the pair of oligonucleotide primers consisting of SEQ ID NO: 5 + SEQ ID NO: 6, that amplifies a fragment of the PWL2 gene of approximately 1000 base pairs, whose sequence is reported in SEQ ID NO: 9. The PWL2 gene controls the ability of the fungus to infect a different species of rice, Eragrostis curvula (Weeping lovegrass). The PWL2 locus is highly polymorphic (Swegard et al. Plant Cell, 1995, 7:1221-1233); in fact, as it is known in the art, different alleles of the PWL2 gene have been identified in fungal isolates from different geographic areas. By RAPD analysis of the P. oryzae genome, it has been possible for the authors of the present invention to identify at least 5 different strains of the fungus. The pairs of oligonucleotide primers according to the present invention are capable of amplifying, by the PCR method, a single band from all the different strains of P. oryzae that have been isolated. Therefore these pairs of oligonucleotide primers are particularly useful for the diagnosis of fungal infections regardless of the infecting strain. In a further embodiment, the invention refers to the use of the pairs of oligonucleotide primers described in the sequence list for the early diagnosis of P. oryzae infection in a plant sample. Preferably the plant sample, consisting of parts of the plant or its organs, as for example, leaves, culms, necks, is rice (O. sativa). Even more preferably the rice is chosen among cultivars identified as: Arborio, Adelio, Ariete, Argo, Balilla, Carnaroli, Drago, Gange, Lamone, Padano, S.Andrea, Taibonet, Teyo, Vialone Nano.

According to a further embodiment, the invention also refers to a process for identification of P. oryzae infection directly from the total DNA isolated from organs or tissues or parts of plant, preferably rice (leaves, culms, necks), or from isolated and fungal purified DNA, comprising the steps: optional in vitro culture of the fungus isolation of genomic DNA from a plant sample composed of organs or parts of the plant or by the fungal culture amplification of a fungal DNA fragment that corresponds to the fragment identified by the pair of oligonucleotide primers used in the polymerization chain reaction (PCR) and chosen among those previously defined detection of P. oryzae infection by visualization of the amplified product.

The culture of the fungus isolated from infected rice is carried out in solid or liquid medium according to methods that are known to the expert in the field. For instance, it is carried out on solid PDA medium (potato 200 g, dextrose 20 g, agar

15 g, to one liter with bi-distilled H2O) and subsequently transferred to liquid PDB medium (SIGMA, MO, USA), in order to obtain a mycelial growth sufficient for

DNA extraction. Other media, known to the expert in the field, can be used for in vitro culture of the fungus. The isolation of total DNA from the infected plant, parts of the plant (leaves, culms, necks) comprising also the fungal DNA or the DNA of the fungus isolated and grown in culture, is carried out according to methods known in the art and described, for example, in Sambrook, J., E.F. Fritsch and T. nd Maniatis. 1989. Molecular cloning: a laboratory manual, 2 edition, Cold Spring

Harbor Laboratory Press, N.Y. According to a preferred embodiment, the genomic DNA must be purified according to established standards, for instance, by means of commercial purification kits, such as "Dneasy Plant Mini Kit for DNA Isolation"

(Qiagen, CA, USA), or NucleoSpin Nucleic Acid Purification Kit (Clontech, CA, SA). Alternatively, the DNA can be also purified by the boiling method. The purified DNA is used as template for the Polymerization Chain Reaction (PCR) under conditions known to the expert in the field, which can be found in specific handbooks, and are described, for example in "PCR Protocols: A guide to Methods and Applications ", Innis, Gelfand, Sninsky and White eds., Academic Press, 1990.

When used for PCR according to the method of the invention, the described pairs of oligonucleotide primers amplify a fragment whose length does not exceed 1000 base pairs and with a melting temperature (Tm) ranging between 75°C and 85°C. Particularly preferred is the pair composed of SEQ ID NO: 5 + SEQ ID NO: 6 that amplifies a fragment of the PWL2 gene of approximately 1000 base pairs, whose sequence is reported in SEQ ID NO: 9.

In this last preferred embodiment (use of the pair of oligonucleotide primers with SEQ ID NO: 5+ SEQ ID NO: 6), the method according to the invention is performed under the following conditions. The standard amplification mixture comprises 10 ng of DNA purified from plant parts, 100 ng of each oligonucleotide, 0.2 mM dNTPs, 1.5 mM MgCl2 and other components, including spermidine, pH

8.8, in 10 μl final volume. The amplification cycles can comprise for example a step of incubation at 94°C for sample denaturation followed by 30-35 cycles each consisting of 15" at 92°C, 30" at 58°C and 90" at 72°C, and a final extension step for 10' at 72°C. Modifications of the aforesaid conditions, leading to a single amplified product specific for the organism P. oryzae, can be easily obtained by an expert in the field. In particular, variations of the reaction temperature are defined depending on the pair of oligonucleotide primers chosen for amplification. When if is obtained as result of the preferred embodiment (pair of oligonucleotide primers consisting of SEQ ID NO: 5 + SEQ ID NO: 6), the amplification product is a PWL2 gene fragment of 999 or 948 bp (SEQ ID NO: 9 or SEQ ID NO: 10). According to the method of the invention, the amplification product is visualized by horizontal electrophoresis in 1% agarose gel, in presence of a molecular weight marker. Both fragments of 999 and 948 bp length can be amplified in the same PCR reaction. Alternatively, it is visualized either only the 999 fragment or only the 948 bp fragment. The 948 bp fragment corresponds to a 50 nucleotide deletion in position 173-222 (numbering relative, to the 999 bp fragment). Finally, in a further embodiment, the invention provides a kit to perform the method according to the invention, comprising the oligonucleotides described in the present invention. In a preferred embodiment, the kit comprises at least the pair of oligonucleotide primers SEQ ID NO: 5 and SEQ ID NO: 6, separately, either as buffered solution or lyophilized. These oligonucleotides are mixed with a solution containing the sample DNA or the template DNA, and the fragment of interest can be amplified only if both oligonucleotides are present. Moreover, the kit may include solutions for DNA extraction from P. oryzae and reagents for performing the PCR reaction, such as buffers, enzymes, nucleotide triphosphates or co- factors (e.g. MgCl2) useful for PCR execution. Oligonucleotides could be modified, for example they could be labeled or not.

The examples provided in the experimental part describe some of the possible embodiments of the invention, only with the intent to better clarify the invention without being limited thereto. EXPERIMENTAL PART Example 1

Isolation and characterization of P. oryzae strains. Fungal isolates were grown in amounts useful for the study, according to the following treatment:

1) washing: roots, leaves and culms have been subjected to washing in running water and subsequently in sterile physiological solution (NaCI 9%);

2) sterilization: the washed material has been incubated in 3% sodium hypochloride aqueous solution; 3) seeding on a humidified substrate that allows development of the fungal

2 2 conidiophores: fragments of leaves (1 cm ) and culms (2 cm ) have been arranged under sterile conditions in plates containing agar (1.5%) in water.

Incubation was for 24 and 48 hours at 25°C. Conidiophores and developed conidia, following observation at the stereomicroscope, have been isolated in sterile PDA culture medium (potato 200 g, dextrose 20 g, agar 15 g, 1000 ml distilled H2O). The fungal isolates were constantly controlled and then transferred to fresh PDA medium. All strains were morphologically characterized by the technique of fungal growth on glass slide (Revalier).

DNA extraction from P oryzae

Fungal isolates were obtained from rice samples collected in the period of greater infection (July-August). in cultivations of the rice cultivars reported in Tab. 3. These isolates were cultured in liquid medium PDB (SIGMA, MO, USA) with shaking at

25°C for 7 days. Mycelia were filtered, washed with sterile physiological solution and used for DNA extraction by the Kit "Dneasy Plant Mini Kit for DNA Isolation"

(Qiagen, CA, USA).

The sampled plants from cultivars reported in table 1 , originated from paddy fields of Lombardia and were collected during the summery months, optimal for fungal infection.

Table 1. Rice varieties used for isolation of P. oryzae strains (year of harvest).

Fungal strains isolated from the 17 rice cultivars were characterized by RAPD analysis (Random Amplified Poiymorfhic DNA), that can evidentiate DNA polymorphisms randomly distributed in the genome. Appropriately selected oligonucleotide primers (decameric oligonucleotides) gave reproducible electrophoretic profiles. These electrophoretic profiles showed variability among

DNAs of the various fungal isolates, thus making possible to discriminate P. oryzae strains.

From the RAPD analysis it turns out that isolates are genetically distinguishable in the case of at least 5 different P. oryzae strains.

Example 2

Choice of primers for PCR amplification. A computer-assisted search of the EMBL database was performed to identify various P. oryzae sequences potentially usable for amplification of specific fragments. Among those, primers were synthesized that correspond to the genes: cutinase (EMBL ACN° MGCUT1), xylanase (EMBL ACN° MGXYN22A), hydrophobin-like protein (EMBL ACN° MGMPG1A), MAP kinase Mps1 (MPS1 ) gene (EMBL ACN° AFO20316), host-species specificity (PWL2) gene (EMBL ACN° MG26313) and xylanase (EMBL ACN° MGXYN33A), as listed in Tab. 2.

Table 2: Primer sequences designed by the software Gene works 2.3 (IntelliGenetics, Inc., Ca, USA.). Sequences are 5'->3'.

P. oryzae gene Primer 5' Primer 3'

MGCUT1 MG1 : MG2:

(cutinase) TGTGTGTACCACGTGAACC CGGCAACAGTACGCATTACC

MG3:

ACGGGATATGAGATCGTTGG

MG4:

ACTGCGTTGAACATGACTGC

MGXYN22A MG5: MG6:

(xylanase) TCGGACATCCAGTCTCAAC TGTACTCGCCGTAGTTCTCG

MG7:

ACTCTCAAGGAGGACTTGCG

MGMPG1A MG8: MG9:

TGCTCACCATAGCAATAGC CACTGGATGTTGACCAGACC

(Hydrophobin- MG10: like protein) ATTGTGTGCCTAGCCATTCC

AFO20316 G11: MG12:

TTAACACGAAGACGGATC TACCACACGATACCGTAGC

(MAP kinase MG13: ps1 ) TGGATACAAGGACACCAAG P. oryzae gene Primer 5' Primer 3'

MG14:

GTGCCAGATGTGGAGATAG

MG26313 MG15: MG16: (PWL2 gene) TTTCTCATTCCCTTAACG TCCCTCACACTTAAGTTAAC MG17: MG18: TAAATTACATCCCTTACTC GCTTATAAATTAGCCAACC

MG19:

ATTGTTTGTTAGTCCACC

MGXYN33A MG20: MG21 : (xylanase) ATCGATGCTCTCATGAAC ATCGATGCTCTCATGAAGC

MG22:

TTGGTTAGTGGCTTGGATG

MG23:

TTGCTGTCGAACAACAGAGG

Primers selected in table 1 were then synthesized (GIBCO BRL, UK) and used for amplification of P. oryzae DNA (isolated from the rice cultivar Arborio) and of rice DNA from the cultivar Arborio.

The "standard mixture" for PCR amplification contained: 10 ng fungal or rice DNA, 100 ng each oligonucleotide, 0.2 mM dNTPs, Fynazyme buffer (10 mM Tris-HCI, pH 8.8 at 25°C, 1.5 mM MgCl2, 50 mM KCI, 0.1% Triton X-100), 0.25 units of

Dynazyme II, 0.5 mM Spermidine, and H2O to 10 μl). In some cases DNA concentration was varied, as described in the results. The amplification cycles were carried out in the Thermocycler Techne (Mod. Genius) and included one step of incubation for 3' at 94°C followed by 35 cycles of 15" at 92°C, 30" at 58°C, 90" at 72°C. The final incubation was for 10' at 72°C. The analysis of the amplified fragment was done by electrophoresis in 1% agarose gel with 3 μl of reaction mixture. Electrophoresis was for 90 minutes at 75 Volts.

All pairs of oligonucleotide primers had a length ranging between 18 and 22 bases, a Tm ranging between 55°C and 60°C, with optimal value of 58°C, a G+C content of 40-50% and, in addition, produced an amplified fragment of maximum length of 1000 base pairs, with a melting temperature (Tm) between 75°C and

85°C.

Results of PCR amplification are summarized in table 3.

Table 3: Amplification of P. oryzae DNA by PCR with the selected pairs of oligonucleotide primers.

EMBL Pair of Length DNA DNA bands accession n Primers (bp) P.oryzae Rice (n°)

MGCUT1 MG1-MG2 336 YES 1

MG1-MG3 891 YES YES 1

MG1-MG4 1464 NOT II

MGXYN22A MG5-MG6 597 NOT II

MG5-MG7 1138 NOT II

MGMPG1A MG8-MG9 502 YES 5

MG8-MG10 731 YES YES 1

AFO20316 MG11-MG12 375 YES 1

MG11-MG13 1334 YES 4

MG11-MG14 1679 YES 3

MG26313 MG15-MG16 999 YES NOT 1

MG17-MG18 376 YES 1

MG17-MG19 616 YES YES 1

MGXYN33A MG20-MG21 404 YES 2 .

MG20-MG22 616 YES YES 2

MG20-MG23 1204 NOT II

(YES: amplification products are obtained; NOT: amplification products are not obtained; // : not tested). From the data presented in table 3, it can be concluded that only some pairs of oligonucleotide primers, among those selected by the GeneWorks software, were: (1) selective, as they only amplified fungal and not rice DNA, (2) specific for the selected gene, as they gave rise to a single amplification product, and (3) of optimal size, as they amplified a fragment of less than 1000 base pair length. In particular, the pair of primers MG15 and MG16 (SEQ ID NO: 5 and SEQ ID NO: 6), turned out to be particularly suitable and its specificity was thus tested also for other fungal pathogens in rice. In figure 1 are shown the results of amplification with the pair of primers MG15-16 (SEQ ID NO: 5 and SEQ ID NO: 6) from DNA of rice (rice cultivar Drago and Padano) infected by P. oryzae, from DNA of Arborio rice and DNA of fungi: Alternaria alternata, Bypolaris oryzae, Cladosporium sp, Epicoccum nigrum. As it can be seen in Figure 1 , the bands corresponding to the 999 and 948 bp fragments of the PWL2 gene are present only in lanes corresponding to P.oryzae DNA isolated from infected rice cultivars, but not from non infected cultivars of Arborio rice or from DNA of other fungal pathogens.

The extreme sensitivity of the PCR test has been verified in presence of excess rice DNA that was isolated from two different cultivars (Drago and Arborio). In particular, it has been observed that the specificity of the test remains optimal even in presence of 2000 fold weight excess of rice DNA versus P.oryzae DNA (5- 10 ng versus 5 pg). Example 3

Amplification of the PWL2 fragment from leaves and necks of rice DNA manipulations were according to methodologies known in the art and described for example in: Sambrook, J., E. F. Fritsch and T. Maniatis. 1989. nrl

Molecular cloning: a laboratory manual, 2 edition, Cold Spring Harbor

Laboratory Press, N.Y.

Extraction of P. oryzae DNA from rice.

Genomic DNA was extracted from leaves and necks of infected rice by the NucleoSpin Nucleic Acid Purification Kit (Clontech, CA, USA), according to the protocol recommended by the manufacturer.

Alternatively, DNA was extracted by boiling, according to the following protocol: fragments of leaves and neck (3 millimeters side) were boiled for 10 minutes in

100 μl of Tris HCI 10 mM solution, pH 8. The sample was incubated for 3 minutes at 37°C in presence of Rnase A at concentration of 20 μg/ml. The solution was incubated for 10 minutes at 50°C in presence of 2 μg of Proteinase K. Finally the

Proteinase K was inactivated by incubation for 5 minutes at 100°C. 10 μl of the solution were used in a PCR reaction according to the above described protocol. In figure 2 are shown the results of PCR amplification with the primer pair MG15- 16 (SEQ ID NO: 5 and SEQ ID NO: 6), obtained directly from parts of the infected rice plant. In particular it has been demonstrated that PCR can diagnose the P. oryzae infection in rice even when this appears to be negative by accurate microscopic analysis, demonstrating that the test is able to diagnose P. oryzae infection at a very early stage. Example 4. Characterization of the allele PWL2 948 bp. Materials and Methods.

The fragments amplified with the pair of oligonucleotide primers MG15 and MG16 (SEQ ID NO: 5 and SEQ ID NO: 6) (see Table 1) were used in a ligation reaction with plasmid pGEM-T vector (Promega, Wl, USA). The product of this reaction was used to transform competent Escherichia coli DHδαcells. Recombinant plasmids were extracted by alkaline liysis (Ausubel, F. M. et al., 1992, Short nd Protocols in Molecular Biology, 2 edition, Harvard) and subsequently purified by

Sephadex G-50 mini-columns (Sambrook, J., E. F. Fritsch and T. Maniatis. 1989. nd Molecular cloning: a laboratory manual, 2 edition, Cold Spring Harbor

Laboratory Press, N.Y). It was observed that PCR amplification of the PWL2 gene with primer MG15-16

(SEQ ID NO: 5 and SEQ ID NO: 6) produced two fragments of different length, in particular two bands of approximately 1000 and 950 base pairs, when the DNA was amplified as described.

Table 4: Presence of 948 and 999 bp fragments in amplification products obtained with the pair of oligonucleotide primers MG15 and MG16 (SEQ ID NO: 5 and SEQ

ID NO: 6) from P. oryzae DNA isolated from various rice cultivars. Rice cultivar Amplified fragments (bp)

Adelio (1999) 948

Arborio (1998) 948

Ariete (1999) 948

Argo (1999) 948 and 999

Balilla (1998) 948 and 999

Balilla (1999) 948 and 999

Camaroli (1998) 948 and 999

Drago (1998) 948 and 999

Gange (1999) 948 and 999

Lamone (1999) 999

Padano (1998) 948

Padano (1999) 948 and 999

S. Andrea (1999) 948 and 999

Taibonet (1998) 999

Teyo (1999) 948

Vialone Nano (1998) 948 and 999

Vialone Nano (1999) 948 and 999

As it can be seen in table 4, fragments can be amplified either individually or simultaneously from different rice cultivars. Both fragments were then sequenced by the company MWG-BIOTECH (GE), using primers MG15, SP6 and T7. Their sequence corresponds respectively to the SEQ ID NO: 9 (999 bp fragment) and SEQ ID NO: 10 (948 bp fragment).

The size difference is due to deletion of nucleotides 173-222 according to fragment 999 (that is numbering of fragment 999, SEQ ID NO: 9).

Claims

1. Oligonucleotide primer for identification of the pathogenic fungus Pyricularia oryzae.

2. Oligonucleotide primer according to claim 1 wherein said identification is accomplished by means of chain amplification of DNA (PCR).

3. Oligonucleotide primer according to claim 2 wherein said identification is performed on a plant sample.

4. Oligonucleotide primer according to claim 3 wherein said plant sample is rice (O. sativa)

5. Oligonucleotide primer according to claim 1 wherein the rice belongs to cultivars selected from: Arborio,' Adelio, Ariete, Argo, Balilla, Carnaroli, Drago, Gange, Lamone, Padano, S. Andrea, Taibonet, Teyo, Vialone Nano.

6. Oligonucleotide primer according to claim 1 comprising a sequence of at least 12 consecutive bases identical to at least one of the oligonucleotides of the group consisting of sequences from ID NO: 1 to ID NO: 8.

7. Oligonucleotide primer according to claim 6 selected from the group consisting of sequences from ID NO: 1 to ID NO: 8.

8. A pair of oligonucleotide primers selected from the group consisting of: a primer compri sing SEQ ID NO: 1 and a primer comprising SEQ ID NO: 2, or a primer compri sing SEQ ID NO: 3 and a primer comprising SEQ ID NO: 4, or primer compri sing SEQ ID NO: 5 and a primer comprising SEQ ID NO: 6, or primer comprising SEQ ID NO: 7 and a primer comprising SEQ ID NO: 8.

9. A pair of oligonucleotide primers selected from the group consisting of: SEQ ID NO: 1 + SEQ ID NO: 2; SEQ ID NO: 3 + SEQ ID NO: 4; SEQ ID NO: 5 + SEQ ID NO: 6; SEQ ID NO: 7 + SEQ ID NO: 8.

10. A method for identification of infection by Pyricularia oryzae, comprising the following steps: a) optional culture of the fungus Pyricularia oryzae; b) DNA isolation from a plant sample or from the optional culture of the fungus; c) amplification of a DNA fragment from Pyricularia oryzae by means of polymerase chain reaction (PCR) in presence of at least one of the oligonucleotide primers or of the pairs of oligonucleotide primers according to claims from 1 to 9; d) visualization of the amplification product

11. A method according to claim 10, wherein said amplification occurs in presence of at least one of the pairs of oligonucleotide primers according to claims 8 and 9.

12. A method according to claim 11 , wherein said oligonucleotide primer pair is amplified by means of SEQ ID NO: 5 + SEQ ID NO: 6

13. A method according to claim 10, wherein said plant sample is represented by parts of the plant of rice (O.sativa).

14. A method according to claim 13, wherein the rice is selected from the following cultivars: Arborio, Adelio, Ariete, Argo, Balilla, Carnaroli, Drago, Gange, Lamone,

Padano, S.Andrea, Taibonet, Teyo, Vialone Nano.

15. A method according to claim 10, wherein said visualization is made by means of electrophoresis of the amplification product.

16. Use of oligonucleotide primers or of pairs of oligonucleotide primers according to claims 1-9 for diagnosis of infection by Pyricularia oryzae in a plant sample.

17. Use according to claim 16, wherein said sample comprises organs of the plant of rice (O.sativa).

18. Use according to claim 17, wherein the rice is chosen from the following cultivars: Arborio, Adelio, Ariete, Argo, Balilla, Carnaroli, Drago, Gange, Lamone, Padano, S.Andrea, Taibonet, Teyo, Vialone Nano.

19. A kit for diagnosis of infection by Pyricularia oryzae in a plant sample comprising one more tubes containing separately at least one of the oligonucleotide primers or of the pairs of oligonucleotide primers according to claims 1-9.

20. A kit according to claim 19 wherein said oligonucleotide primers are SEQ ID NO: 5 and SEQ ID NO:6.