A method of genotyping fusarium spp

Abstract

The present invention is generally concerned with the field microbial phytopathogens. The present invention describes a quick, accurate and reliable molecular genetic method and different techniques which are designed for and provide an accurate and consistent detection, classification, identification and differentiation of Fusarium spp. in order to enable early and effective intervention and / or treatment. Furthermore, the present invention provides a quick, accurate and reliable molecular genetic method and specific nucleotide primers which are designed for and provide an accurate and consistent detection, classification, identification and differentiation of Fusarium spp.. The present invention also provides methods which can be used to characterise the mycotoxin profile of the newly detected Fusarium spp..

Description

[0001] Attorney Ref: 6666 / P005-SAR-PCT

[0002] Title: A METHOD OF GENOTYPING FUSARIUM SPP

[0003] INTRODUCTION

[0004] [1] Cereal crops, grown worldwide, represent a major global industry. Maize (Zea mays L.) is recognised among the most important of the cereals. Maize has a key place in global food systems and plays a crucial role in the agricultural and broader commercial sectors. It is an essential and versatile cereal crop that is relied on for grain as feed and food, silage, green fodder, ethanol production and other applications. Sugar-rich varieties of maize are a staple food in many parts of the world, particularly in North and South America, and field com is a major carbohydrate feed for animals due to its high starch content. Global maize production and cultivation areas have been increasing in recent decades and in the not too distant future, maize is expected to become the most widely grown and traded crop worldwide (https: / / fas.usda.gov / ).

[0005] [2] Unfortunately, like many commercially farmed cereal crops, maize farming is affected by various devastating diseases worldwide such as microscopic fungi being the primary phytopathogenic microorganisms. Of these microorganisms, representatives of Fusarium spp. are considered the most destructive fungal pathogens on cereals such as maize, and this trend is observed across all continents (Khokhar et al., 2013).

[0006] [3] Phytopathogenic Fusarium representatives are considered responsible for a number of diseases such as seedling blight, stem root rod (FSR), ear rot (FER) and others. Fusarium-related diseases are caused by individual species or more frequently by co-occurring species in the field. The genus’s predominant phytopathogenic members differ according on the variety, geographic region, treatment methods and climate during the growing season. Spore formation of asexual and / or sexual spores is an essential part of the life cycle of the genus Fusarium, and some can persist in soil for many years (Goncharov et al., 2020).

[0007] [4] In order to better adapt to and colonise new ecological niches, Fusarium spp. have evolved their biosynthetic metabolism to produce a wide range of secondary metabolites known as mycotoxins, which vary in chemical structure and level of toxicity. Fusarium spp. mycotoxins with the greatest impact on public health is attributed to - fumonis (FUMs), zearalenone (ZEA) and type A trichothecenes: T-2 toxin (T-2) and HT-2 toxin (HT-2).

[0008] [5] The kernels resulting from infected spikes are known as “tombstones” because they are light in weight, shrivelled, discoloured with a pinkish or chalky appearance, and poor quality. Mycotoxins contaminating animal feed could accumulate to high levels and have toxic effects on animal health, and subsequently transfer to animal products such as meat, milk and eggs, compromising consumer safety. A number of Fusarium mycotoxins can alter various intestinal defence mechanisms, such as epithelial integrity, cell proliferation, mucus layer, cytokine and immunoglobulin production. Oral intake of Attorney Ref: 6666 / P005-SAR-PCT mycotoxin-contaminated food / feed has multiple toxic effects in both animals and humans. Above certain concentrations, mycotoxins lead to carcinogenic, neurotoxic, mutagenic, teratogenic, estrogenic, hepatotoxic and immunosuppressive effects.

[0009] [6] The International Agency for Research on Cancer (IARC) classifies fumonisin Bi (FBi) produced mainly by F. verticillioides and F. proliferatum to group 2B as possibly carcinogenic to humans. Deoxynivalenol (DON) and ZEA have been placed by IARC in group 3, due to limited data and evidence available of their effects on humans and animals (IARC, 2012). The most common contaminant of maize and maize products worldwide, especially in regions with warm climates, are fumonisins (FUMs).

[0010] [7] The pathogenic strains, F. verticillioides, F. proliferatum, F. subglutinans, F. graminearum, F. oxysporum and F. temperatum cause seedling diseases that inhibit seed germination and emergence in cereal crops. F. verticillioides is thought to be the producer of the highest levels of FUM and a major cause of leukoencephalomalacia in horses fed contaminated cereals such as maize, pulmonary edema in pigs, and esophageal cancer in humans. Diseases induced by FUMs most likely result from the capability of the toxins to inhibit the metabolism of sphingolipids.

[0011] [8] In Northern and Central Europe, Fusarium diseases of maize are caused by several companion species, for example F. graminearum, F. culmorum, F. avenaceum, favoured by temperate and humid environmental conditions, while F. verticillioides, F. proliferatum, F. temperatum are more common in the warm and dry climates in southern Europe. The species composition of pathogenic species responsible for these diseases in crops can vary over the years and depends on geographical and climatic factors as well as agrotechnical practices.

[0012] [9] The production of a certain type of mycotoxins is a specific characteristic not only of the species but also of individual strains. Therefore, their accurate and reliable identification is crucial in order to assess the impact of these ascomycete fungi in agriculture, animal husbandry and on public health.

[0013]

[0010] However, due to the great similarity of some species and the variations in mycelial colors and conidial morphology of different strains, the most common and widely used morphological approach for Fusarium spp. identification is challenging to apply. This is particularly evident among very closely related Fusarium species, such as for instance members of the F. avenaceum, F. acuminatum, F. tricinctum species complex.

[0014]

[0011] In particular, F. avenaceum and F. acuminatum can be difficult to distinguish based on morphological characteristics alone and are often misidentified.

[0015]

[0012] Furthermore, F. avenaceum and F. tricinctum have similar colony morphology and therefore, relying on morphological assessments or even microscopic considerations presents a challenge which could Attorney Ref: 6666 / P005-SAR-PCT lead to inconsistent and inaccurate classification, wrong diagnosis, treatment with impactful commercial or even health consequences. These processes are challenging because they can be expensive, time-consuming and laborious.

[0016]

[0013] Therefore, there exists a real and urgent need to develop a quick and reliable method for accurate and consistent detection, classification and identification of Fusarium spp. in order to enable early and effective treatment and reduce any potentially devastating impact on agriculture, animal husbandry and threat on public health by these versatile and highly adaptive phytopathogenic microorganisms.

[0017]

[0014] The above problems are at least in part solved by the present invention as described below.

[0018] SUMMARY OF THE INVENTION

[0019]

[0015] In the broadest sense, the present invention is concerned with the field of microbial pathogens such as phytopathogens. The present invention is also generally concerned with phytopathogenic microorganisms which can target cereal plants. The present invention is also concerned with phytopathogenic microscopic fungi which can efficiently infect cereal plants and cause a disease with devastating consequences to crops. Of these phytopathogenic microorganisms, representatives of Fusarium spp. are considered the most destructive fungal pathogens of cereal crops such as maize, wheat, barley and others, and this trend can seasonally be observed across all continents.

[0020]

[0016] The present invention provides a quick, accurate and reliable molecular method and different techniques which are designed for and provide an accurate and consistent detection, classification and identification of Fusarium spp. in order to enable early and effective treatment. Furthermore, the present invention provides a quick, accurate and reliable molecular genetic method and specific nucleotide primers which are designed for and provide an accurate and consistent detection, classification and identification of Fusarium spp..

[0021]

[0017] According to an aspect, there is provided a method for identifying a genotype of Fusarium spp. from a cereal plant, the method comprising the steps of:

[0022] (a) obtaining a sample from a cereal plant to be tested for Fusarium spp.;

[0023] (b) isolating and identifying Fusarium spp. from the step (a) using morphological assessments and / or microscopy analysis;

[0024] (c) extracting genomic DNA from the isolated and identified Fusarium spp.;

[0025] (c) replicating a desired region in a DNA template by binding a specific oligonucleotide primer set selected from the primer sets comprising SEQ ID: 1; SEQ ID:2; SEQ. ID:3 and SEQ ID:4 to the DNA template; where the primer set is designed to give rise to a test PCR product containing at least one nucleotide variance compared to a control sequence; and Attorney Ref: 6666 / P005-SAR-PCT

[0026] (e) determining whether the test PCR product is an amplified Fusarium spp. product from a cereal plant.

[0027]

[0018] In some embodiments of the present invention, the cereal plant is selected from the group comprising rice, barley, wheat, maize, oat, teff, sorghum, millet.

[0028]

[0019] In some embodiments of the present invention, the Fusarium spp. is a pathogenic Fusarium spp.. In embodiments of the present invention, the pathogenic Fusarium spp. is selected from F. proliferatum, F. subglutinans, F. fujikuroi, F. verticillioides, or F. oxysporum.

[0029]

[0020] In some embodiments of the present invention, the PCR primer set is designed to give rise to PCR products used for differentiating between Fusarium spp. In some embodiments of the present invention, the differentiation between Fusarium spp. uses the technique of barcoding the genotype of pathogenic Fusarium spp. from a cereal plant.

[0030]

[0021] In some embodiments of the present invention, the differentiation between Fusarium spp. relies on oligonucleotide primer set selected from the primer sets comprising SEQ ID: 1; SEQ ID: 2; SEQ. ID: 3 and SEQ ID: 4 which primer sets are designed to enable the amplification of a test PCR product from a genomic DNA corresponding to a gene sequence or a fragment thereof selected from Internal Transcribed Spacer region (nrDNA- / 7'.S'). rDNA intergenic spacer region (IGS), Translation elongation factor 1-a (TEFl-a) and fi-tub. In some embodiments of the present invention, the pathogenic Fusarium spp. is associated with or is the causative agent of a cereal plant disease.

[0031]

[0022] In some embodiments, the primer sets comprising SEQ ID: 1; SEQ ID: 2; SEQ. ID: 3 and SEQ ID: 4 lead to a quicker or more accurate differentiation between Fusarium spp.. In some embodiments, the primer sets comprising SEQ ID: 1; SEQ ID: 2; SEQ. ID: 3 and SEQ ID: 4, lead to at least 0.01%, at least 0.05%, at least 0.1%, at least 0.5%, at least 1%, at least 2.5%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more, quicker or more accurate differentiation between Fusarium spp..

[0032]

[0023] In some embodiments of the present invention, the cereal plant disease is selected from at least one of seedling blight, stem rot, root rot, ear rot, stalk rot, head blight and crown rot.

[0033]

[0024] In some embodiments of the present invention, the method comprises the steps of:

[0034] (a) obtaining a sample from a cereal plant to be tested for Fusarium spp.;

[0035] (b) isolating and identifying Fusarium spp. from the step (a) using morphological assessments and / or microscopy analysis;

[0036] (c) extracting genomic DNA from the isolated and identified Fusarium spp.; Attorney Ref: 6666 / P005-SAR-PCT

[0037] (c) amplifying genomic DNA by conducting the polymerase chain reaction from about 30 cycles to about 35 cycles to produce a test PCR product; and

[0038] (e) determining whether the test PCR product is an amplified Fusarium spp. product from a cereal plant.

[0039]

[0025] In some embodiments of the present invention, the oligonucleotide primer set, as selected from the primer sets, comprising SEQ ID: 1; SEQ ID: 2; SEQ. ID: 3 and SEQ ID: 4 amplify a test PCR product from genomic DNA corresponding to a gene sequence or a fragment thereof selected for Internal Transcribed Spacer region (nrDNA- / 7'.S'). rDNA intergenic spacer region (IGS), Translation elongation factor 1-a (TEF1- a) and fl-tub.

[0040]

[0026] In some embodiments of the present invention, the annealing temperature of the primer sets is in the range of from 51.1 °C to 61.2°C. In some embodiments of the present invention, the annealing temperature of the primer set for nrDNA- / 7'.S' is 52.5°C, for TEF-la primers is 54°C, for the fl-tub primers is 51.1°C, and forthe IGS primers 56.6°C.

[0041] [1] In some embodiments of the present invention, the PCR product is sequenced and the nucleotide sequence of the product generates a DNA barcode signature that is unique to the Fusarium spp. from which the genomic DNA template was extracted. In some embodiments of the present invention, the DNA barcode signature is compared to reference DNA barcodes sequence for reliable identification of Fusarium spp. such as pathogenic Fusarium spp.. In some embodiments of the present invention, the DNA barcode signature is a unique DNA barcode signature which is compared to reference DNA barcodes sequence for reliable identification of Fusarium spp. such as pathogenic Fusarium spp.. In some embodiments of the present invention, the Fusarium spp. is a pathogenic Fusarium spp.. In some embodiments of the present invention, the pathogenic Fusarium spp. is identified as F. proliferatum, F. subglutinans, F. fujikuroi, F. verticillioides, or F. oxysporum.

[0042] [2] In some embodiments, the DNA barcode signatures lead to a quicker or more accurate differentiation between Fusarium spp.. In some embodiments, the DNA barcode signatures lead to at least 0.01%, at least 0.05%, at least 0.1%, at least 0.5%, at least 1%, at least 2.5%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more, quicker or more accurate differentiation between Fusarium spp..

[0043]

[0027] According to an aspect of the present invention, there is provided a Fusarium spp. as identified by the methods, primers, kits, primers and techniques described herein throughout. In some embodiments of the present invention, there is provided a pathogenic Fusarium spp., such as a pathogenic Fusarium spp. selected from F. proliferatum, F. subglutinans, F. fujikuroi, F. verticillioides, or F. oxysporum. In some embodiments of the present invention, the pathogenic Fusarium spp., selected from F. proliferatum, F. Attorney Ref: 6666 / P005-SAR-PCT subglutinans, F. fujikuroi, F. verticillioides, or F. oxysporum produces trichothecene mycotoxin and / or zearalenone mycotoxin.

[0044]

[0028] According to an aspect of the present invention, there is provided a kit for identifying a genotype of Fusarium spp., the kit comprising a primer sets comprising SEQ ID: 1; SEQ ID:2; SEQ. ID:3 and SEQ IDA as designed to amplify a genomic DNA template which template is extracted from isolated and identified Fusarium spp., and optionally a leaflet of instructions for use of the primers to identify the genotype of Fusarium spp..

[0045] BRIEF DESCRIPTION OF THE DRAWINGS

[0046]

[0029] FIG. 1. Experimental design scheme for DNA barcoding in maize.

[0047]

[0030] FIG. 2 Number of samples with different percentage of Fusarium spp. contamination

[0048]

[0031] FIG. 3. Electrophoretic analysis in 1% agarose. Amplification of the 500 bp fragment by PCR with BT tub 2 F and BT tub 2 R specific primers to detect Fusarium spp. Lane 1 - Gene Ruler 1Kb DNA Ladder; lanes 2-12 - Fusarium DNA obtained from maize collected in different Bulgarian regions.

[0049]

[0032] FIG. 4. Nucleotide sequencing data for amplified gene regions

[0050]

[0033] FIG. 5. Fragment with TEF-la region sequences from 17 Fusarium spp. isolates in MEGA software v. 11, aligned by CLUSTALW; * A, C, G, T - nucleotides

[0051]

[0034] FIG. 6. Neighbour-joining - dendrogram built on the base of sequences resulted of the region rDNA-ITS of 17 Fusarium isolates from maize samples in accordance with their reference sequences obtained from NCBI. Numbers at nodes indicate levels of bootstrap support (percentage) based on 1000 resampled datasets; only values > 50% are presented

[0052]

[0035] FIG. 7. Neighbour-joining-dendrogram built on the base of sequences resulted of the region TEF- la of 17 Fusarium isolates from maize samples in accordance with their reference sequences obtained from NCBI. Numbers at nodes indicate levels of bootstrap support (percentage) based on 1000 resampled datasets; there were a total of 545 positions in the final dataset

[0053]

[0036] FIG. 8. Species composition by region based on aggregated data from targeted 4 loci.

[0054]

[0037] FIG. 9. Mycotoxin profile of Fusarium spp.

[0055] DETAILED DESCRIPTION OF THE INVENTION

[0056]

[0038] Throughout this disclosure, various scientific publications, internet links, patents and published patent application or granted patents are referenced by an identifying citation or number. The disclosures of these publications, content in the internet links, patents and published patent application or granted patents Attorney Ref: 6666 / P005-SAR-PCT are hereby incorporated by reference into the present disclosure to more fully describe the state of the art and field to which this present disclosure and intention pertains.

[0057] [3] As used herein, certain terms may have the following defined meanings unless stated otherwise.

[0058] [4] As used herein, the term "about" includes the recited number or number and + / - 10% from the recited numeral or number. By way of non-limiting example, the term "about ten (10)" would encompass nine (9) to eleven (11) or 9-11.

[0059] [5] The disclosure illustratively described herein can suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms “comprising”, “including,” containing”, etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognised that various modifications are possible within the scope of the disclosure claimed.

[0060] [6] The terms “replicating” and “amplifying” are used interchangeably here and refer to the increase in the number of copies of a target nucleotide sequence such as a desired region in a DNA template, using a PCR technique.

[0061]

[0039] As used in the specification and claims, the singular form “a,” “an” and “the” include singular and plural references unless the context clearly dictates otherwise. For example, the term “microorganism”, “pathogenic microorganisms ”, “pathogenic microorganisms belonging to the genus Fusarium”, “pathogenic Fusarium spp. ”, “virulent pathogenic Fusarium spp. ”, “phytopathogenic microorganisms”, “phytopathogenic microorganisms belonging to the genus Fusarium ” , “Phytopathogenic Fusarium spp. ”, “mycotoxin producing Fusarium spp.” includes a single or plurality of microorganisms, phytopathogenic microorganism, phytopathogenic microorganism belonging to the genus Fusarium, pathogenic Fusarium spp., virulent pathogenic Fusarium spp., phytopathogenic Fusarium spp., mycotoxin producing Fusarium spp. such as newly classified Fusarium spp. as described, identified or differentiated by the present invention using the methods, primers such as nucleotide primers for PCR or protein techniques for mycotoxin detection, and techniques and experiments according to embodiments of the invention. In some embodiments, the pathogenic Fusarium spp. include but are not limited to F. proliferatum, F. subglutinans, F. fujikuroi, F. verticillioides, or F. oxysporum.

[0062]

[0040] Trichothecene Attorney Ref: 6666 / P005-SAR-PCT

[0063]

[0041] Trichothecene is a type of mycotoxin produced by different pathogenic Fusarium spp. Trichothecene is one of the most notorious mycotoxins not only because trichothecenes are extremely toxic but also because they are difficult to eliminate.

[0064]

[0042] Trichothecene Types

[0065]

[0043] There are 60 known types of trichothecene mycotoxins. Some of them include: Deoxynivalenol, Diacetoxyscirpenol, HT-2 mycotoxins, Neosolaniol, Nivalenol, Satratoxin-H, T-2 mycotoxins, Verrucarin A, and Vomitoxin.

[0066]

[0044] Trichothecene Toxicity

[0067]

[0045] Trichothecene is amongst the most toxic types of mycotoxins. The LD50 rate (the dosage level that caused 50% of the group to die) for laboratory mice given trichothecene mycotoxins is between 1 and 7 mg / kg depending on the specific type of trichothecene and the method of exposure.

[0068]

[0046] Destroying Trichothecene Mycotoxins

[0069]

[0047] Trichothecene mycotoxins can remain toxic for years in a normal environment. Ultraviolet light does not destroy trichothecenes and they are not soluble in water.

[0070]

[0048] Trichothecenes can also withstand freezing. Temperatures of 500 degrees Fahrenheit for 30 minutes or 500 degrees Fahrenheit for 10 minutes is needed to destroy them. A solution of 5% sodium hydrochloride, the active ingredient in bleach, can destroy trichothecene mycotoxins.

[0071]

[0049] T-2 Trichothecene Mycotoxins

[0072]

[0050] T-2 trichothecene mycotoxins are the only mycotoxins that have been used in biological weapons. These trichothecene mycotoxins have the advantages of being highly stable in the air, not degrading under ultraviolet light and being able to withstand heat.

[0073]

[0051] T-2 mycotoxins are also the only substances used in biological warfare that can be absorbed through a subject’s skin.

[0074]

[0052] Fusarium spp. produced mycotoxins and their metabolites which exert their pathogenic potentially devastating toxic impact by affecting various intestinal defence mechanisms of a subject, such as epithelial integrity, cell proliferation, mucus layer, cytokine and immunoglobulin production. Oral intake of mycotoxin-contaminated food / feed has multiple toxic effects in both animals and humans. Above certain concentrations, mycotoxins lead to carcinogenic, neurotoxic, mutagenic, teratogenic, estrogenic, hepatotoxic and immunosuppressive effects. Due to the highly toxic effect of these mycotoxins and their Attorney Ref: 6666 / P005-SAR-PCT metabolites on human and animal health, it is important to rapidly and effectively detect and quantify these toxins in food and feed.

[0075]

[0053] As used herein the terms “pathogenic” or “pathogenic Fusarium spp.” are used interchangeably and refer to a microorganism capable of exerting some form of harm to health such as a condition, symptom or disease on a subject. The harm to health such as a condition, symptom or disease on a subject can be for example impaired epithelial integrity, abnormal cell proliferation, mucus layer, cytokine and immunoglobulin production, carcinogenic, neurotoxic, mutagenic, teratogenic, estrogenic, hepatotoxic and immunosuppressive effects.

[0076]

[0054] As used herein, the term “subject” means any animal, such as a vertebrate, preferably a mammal such as farmed mammal or human, exposed to pathogenic Fusarium spp.” as described, identified or differentiated by the present invention using the methods, primers such as nucleotide primers for PCR or protein primers for mycotoxin detection, and techniques and experiments according to embodiments of the present invention.

[0077]

[0055] As used herein the term “phytopathogenic” or “phytopathogenic Fusarium spp.” are used interchangeably and refer to a microorganism capable of exerting some form of physiological harm to a cereal plant such as a condition, symptom or disease. The condition, symptom or disease of a cereal plant can be one or more selected from seedling blight, stem rot, root rot, ear rot, stalk rot, head blight and crown rot.

[0078]

[0056] The present invention is generally directed to a quick, accurate and reliable method and different techniques which are designed for and provide an accurate and consistent detection, classification and identification of Fusarium spp., The method and different techniques of the present invention facilitate an early and effective treatment and management of Fusarium spp. pathogenicity. Furthermore, the present invention provides a quick, accurate and reliable molecular genetic method and specific nucleotide primers which are designed for and provide an accurate and consistent detection, classification and identification of Fusarium spp.. The present invention is therefore generally directed to a quick, accurate and reliable molecular method and different techniques which are designed for and provide an accurate and consistent detection and identification of Fusarium spp. produced mycotoxins, in order to enable early and timely prevention of harmful effects on subjects and / or cereals. Furthermore, the present invention provides a quick, accurate and reliable method and specific protein primers which are designed for and provide an accurate and consistent detection and identification of Fusarium spp. produced mycotoxins.

[0079]

[0057] According to an aspect of the present invention, there is provided a method for identifying a Fusarium spp., the method comprising the steps of: Attorney Ref: 6666 / P005-SAR-PCT

[0080] (a) obtaining a sample to be tested for Fusarium spp.;

[0081] (b) isolating and identifying Fusarium spp. from the step (a) using morphological assessments and / or microscopy analysis;

[0082] (c) extracting genomic DNA from the isolated and identified Fusarium spp.;

[0083] (c) replicating a desired region in a DNA template by binding a specific oligonucleotide primer set selected from the primer sets comprising SEQ ID: 1; SEQ ID:2; SEQ. ID:3 and SEQ ID: 4 to the DNA template; where the primer set is designed to give rise to a test PCR product containing at least one nucleotide variance compared to a control sequence; and

[0084] (e) determining whether the test PCR product is an amplified Fusarium spp..

[0085]

[0058] As used herein the term “primer set SEQ ID No: 1” refers to Forward (F) primer (ID) 1 and Reverse (R) primer (ID) 2 as shown in Table 1 and the enclosed sequence listings. As used herein the term “primer set SEQ ID No: 2” refers to Forward (F) primer (ID) 7 and Reverse (R) primer (ID) 8 as shown in Table 1 and the enclosed sequence listings. As used herein the term “primer set SEQ ID No: 3” refers to Forward (F) primer (ID) 3 and Reverse (R) primer (ID) 4 as shown in Table 1 and the enclosed sequence listings. As used herein the term “primer set SEQ ID No: 4” refers to Forward (F) primer (ID) 5 and Reverse (R) primer (ID) 6 as shown in Table 1 and the enclosed sequence listings.

[0086]

[0059] As used herein the abbreviation “F” stands for forward primer and abbreviation “R” stands for reverse primer.

[0087]

[0060] In further embodiments, the sample to be tested for Fusarium spp. is derived from a subject. In further embodiments, the sample to be tested for Fusarium spp. is derived from a plant. In further embodiments, the sample to be tested for Fusarium spp. is derived from a cereal crop or cereal plant. In further embodiments, the sample to be tested for Fusarium spp. is derived from rice, barley, wheat, maize, oat, teff, sorghum, millet. In some embodiments the sample is derived from seedling, stem, root, ear, stalk, head and crown.

[0088]

[0061] In an aspect of the present invention, there is provided a method for identifying a genotype of Fusarium spp. from a cereal plant, the method comprising the steps of:

[0089] (a) obtaining a sample from a cereal plant to be tested for Fusarium spp.;

[0090] (b) isolating and identifying Fusarium spp. from the step (a) using morphological assessments and / or microscopy analysis;

[0091] (c) extracting genomic DNA from the isolated and identified Fusarium spp.;

[0092] (c) replicating a desired region in a DNA template by binding a specific oligonucleotide primer set selected from the primer sets comprising SEQ ID: 1; SEQ ID:2; SEQ. ID:3 and SEQ ID: 4 Attorney Ref: 6666 / P005-SAR-PCT to the DNA template; where the primer set is designed to give rise to a test PCR product containing at least one nucleotide variance compared to a control sequence; and

[0093] (e) determining whether the test PCR product is an amplified Fusarium spp. product from a cereal plant.

[0094]

[0062] In some embodiments, the cereal plant is selected from the group comprising rice, barley, wheat, maize, oat, teff, sorghum, millet. In some embodiments the cereal plant is a commercially cultivated. In some embodiments the cereal plant is a commercially cultivated for grain, silage, green fodder, ethanol production and other applications. In some embodiments the cereal plant is maize.

[0095]

[0063] The skilled person would be familiar with different techniques of isolating and identifying different microorganisms such as for example using classic microbiology materials and protocols (Bernard D. Davis, Renato Dulbecco, Herman N. Eisen, Harold S. Ginsberg, Lippincott Williams & Wilkins; Subsequent edition, 1 January, 1990). The skilled person would also be familiar with different techniques for characterisation approaches such as phenotypic characterisation of different microorganisms.

[0096]

[0064] By way of example, phenotypic characterisation can be based on formation, appearance, description and morphology, wherever applicable, of pathology, a colony (e.g. single cell colony), mycelia, microconidia, spores and microscopic demarcation and analysis. In some embodiments, as in step a) of the method of the present invention, Fusarium spp., is isolated and identified using morphological assessments and / or microscopy analysis.

[0097]

[0065] In some embodiments of the present invention, genomic DNA (gDNA) comprises complementary DNA (cDNA) and plasmid DNA (pDNA). In some embodiments of the present invention, gDNA comprises the DNA template. In some embodiments of the present invention, gDNA comprises a desired region of a DNA template. In some embodiments of the present invention, gDNA comprises the desired region of the target DNA template. In some embodiments of the present invention, the desired region in a DNA template is replicated. In some embodiments of the present invention, the desired region in the DNA template is replicated by PCR.

[0098]

[0066] The method of the present invention uses polymerase chain reaction (PCR) techniques, whereby for example total DNA (such as genomic DNA) is extracted from a sample such as from plant tissues, cells, and seed, and is then amplified using specific oligonucleotide primers to target desired DNA regions for replication and amplification. If the DNA sequence complimentary to the oligonucleotide primers is found in the samples to be analysed, a test PCR product is generated that can be visualised using non-specific (ethidium bromide staining, FIG. 3). Attorney Ref: 6666 / P005-SAR-PCT

[0099]

[0067] The present method involves a step of isolating nucleotide sequences such as genomic DNA of Fusarium spp. using techniques well known to those skilled in the art. Standard molecular biology textbooks, such as MOLECULAR CLONING: A LABORATORY MANUAL, 2nd ed„ (Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989) and METHODS FOR GENERAL AND MOLECULAR BACTERIOLOGY (eds., Philipp Gerhardt et al., American Society for Microbiology, Washington, DC, 1994) may be consulted for procedures to isolate DNA, without undue experimentation by those of ordinary skill in the art.

[0100]

[0068] The method of the present invention relies on unique DNA primers for a rapid assay using the polymerase chain reaction (PCR) techniques. The skilled person would be familiar with PCR as a technique by which for example different size nucleotide fragments of isolated nucleotide sequences such as genomic deoxyribonucleic acid (DNA) isolated from Fusarium spp. (i.e. test nucleotide), can be rapidly duplicated, or cloned, to produce multiple DNA copies.

[0101]

[0069] It is contemplated by the present inventors that the strength of the PCR technique is that it can be used to identify genetically diverse Fusarium spp. even where there is a single nucleotide difference between the test sample and the control. The PCR amplified Fusarium spp. DNA sequence produces a test PCR product. In some embodiments the test PCR product is subjected to detailed sequencing. In some embodiments the sequenced test PCR product which is subjected to DNA barcoding. In some embodiments each sequenced test PCT product is allocated a unique DNA barcode. In some embodiments of the present invention, the unique DNA barcoded test PCR products are compared relative to control DNA barcodes.

[0102]

[0070] Across the globe and in particular across Northern and Central Europe, Fusarium spp. diseases of cereals such as maize, wheat, barley and others, are caused by several species such as companion species, for example F. graminearum, F. culmorum, F. avenaceum as favoured by temperate and humid environmental conditions, while F. verticillioides, F. proliferatum, F. temperatum are more common in the warm and dry climates. The species composition of pathogenic Fusarium spp. responsible for these diseases in crops can vary and can depend on geographical and climatic factors as well as agrotechnical practices and habits.

[0103]

[0071] Without wishing to be bound by theory, the production of a certain types of mycotoxins is a specific characteristic not only of the species but also of individual Fusarium strains. Therefore, their accurate and reliable identification is crucial in order to assess the impact of these ascomycete fungi in agriculture and on public health. Furthermore, accurate and reliable identification of individual Fusarium strains is crucial in order to assess effective management as well as treatment protocols or regimens. Attorney Ref: 6666 / P005-SAR-PCT

[0104]

[0072] Again, without wishing to be bound by theory, it is suggested that due to the great similarity of some species and the variations in mycelial colors and conidial morphology of different strains also referred to as morphological assessments and / or microscopy analysis, the most common and widely used morphological approach for Fusarium spp. identification is challenging and unreliable. This lack of consistency and reliability of morphological assessments and / or microscopy analysis, is particularly evident among very closely related Fusarium species, such as members of the F. avenaceum / F. acuminatum / F. tricinctum species complex. In particular, F. avenaceum and F. acuminatum can be difficult to distinguish based on morphological characteristics and are often misidentified as a result. F. avenaceum and F. tricinctum also have similar colony morphology. Using this method alone is a serious challenge for scientists and could lead to inaccurate data with unreliable differentiation between Fusarium spp.

[0105]

[0073] In some embodiments of the present invention, the differentiation between Fusarium spp. relies on oligonucleotide primer set selected from the primer sets comprising SEQ ID: 1; SEQ ID: 2; SEQ ID: 3 and SEQ ID: 4.

[0106]

[0074] In some embodiments of the present invention, the differentiation between Fusarium spp. relies on oligonucleotide primer set selected from the primer sets comprising SEQ ID: 1; SEQ ID: 2; SEQ ID: 3 and SEQ ID: 4, wherein primer sets are designed to enable the amplification of a test PCR product from a genomic DNA corresponding to a gene sequence or a fragment thereof selected from Internal Transcribed Spacer region (nrDNA- / 7'.S). rDNA intergenic spacer region (IGS), Translation elongation factor 1-a (TEF1- a) and fl-tub.

[0107]

[0075] In some embodiments of the present invention primer set SEQ ID No: 1 comprises Forward (F) primer (ID) 1 and Reverse (R) primer (ID) 2 which are designed to PCR amplify a 544 base pair size fragment from the nrDN A- / 7.S' gene.

[0108]

[0076] In some embodiments of the present invention, primer set SEQ ID No: 2 refers to Forward (F) primer (ID) 7 and Reverse (R) primer (ID) 8 which are designed to PCR amplify a 200 base pair size fragment from the rDNA intergenic spacer region (IGS) gene.

[0109]

[0077] In some embodiments of the present invention, primer set SEQ ID No: 3 refers to Forward (F) primer (ID) 3 and Reverse (R) primer (ID) 4 which are designed to PCR amplify a 700 base pair size fragment from the Translation elongation factor 1-a (TEFl-a) gene.

[0110]

[0078] In some embodiments of the present invention, primer set SEQ ID No: 4 refers to Forward (F) primer (ID) 5 and Reverse (R) primer (ID) 6 which are designed to PCR amplify a 600 base pair size fragment from the fl-tub gene. Attorney Ref: 6666 / P005-SAR-PCT

[0111]

[0079] In some embodiments of the present invention, the Fusarium spp. is a pathogenic Fusarium spp. selected from F. proliferatum, F. subglutinans, F. fujikuroi, F. verticillioides, or F. oxysporum. In some embodiments of the present invention, the PCR primer set is designed to give rise to PCR products, such as test PCT products, used for differentiating between Fusarium spp.

[0112]

[0080] In some embodiments of the present invention, the differentiation between Fusarium spp. uses the technique of barcoding the genotype of pathogenic Fusarium spp. from a cereal plant.

[0113]

[0081] The skilled person would be familiar with DNA barcoding for classification and differentiation between different genomes. The skilled person would also be aware that species can share identical barcodes making it challenging for barcode DNA when using established barcode approach such as relying on single barcode.

[0114]

[0082] The present inventors surprising and unexpectedly observed that using DNA barcoding based on a plurality, of specifically selected target Fusarium spp. genomic sequences was able to provide a quick and reliable approach for accurate and consistent detection, classification and identification of Fusarium spp.

[0115]

[0083] In addition, the present inventors surprising and unexpectedly observed that using DNA barcoding based on at least four (4) or more specifically selected target Fusarium spp. genomic sequences was able to provide a quick and reliable approach for accurate and consistent detection, classification and identification of Fusarium spp.

[0116]

[0084] Moreover, it was unexpectedly observed that using DNA barcoding based on four (4) particular target pathogenic Fusarium spp. genomic sequences, was able to provide an optimal, quick and consistently reliable method for accurate and reproducible detection, classification and wherever necessary differentiation or identification between hard to distinguish pathogenic Fusarium spp. compared to previous methods such as conventional methods or standard DNA barcoding approaches.

[0117]

[0085] By matching the presently described plurality of DNA barcodes against reference DNA barcodes comprising databases, accurate and reliable identification and differentiation of Fusarium spp. can consistently be achieved. In some embodiments, matching the unique DNA barcodes described in the present invention against reference DNA barcodes comprising databases, accurate and reliable identification is achieved.

[0118]

[0086] One of the desired regions according to the method of the present invention is the Internal Transcribed Spacer region or fragments thereof. The ITS region is very stable under evolutionary pressure and is generally conserved within species. The spacer region (ISG), which separates repetitive regions of rDNA, has rapid evolutionary rate, expands that of other rDNA regions. Attorney Ref: 6666 / P005-SAR-PCT

[0119]

[0087] Closely related Fusarium spp., often exhibit considerable divergence in this region, manifested as variations in length and nucleotide sequence, along with the occurrence of restriction site variation in some molds. In some embodiments of the present invention the desired target region is Internal Transcribed Spacer region (nrDN A- / 7.S) or fragments thereof. In some embodiments of the present invention, the desired target region Internal Transcribed Spacer region (nrDN A- / 7.S) or fragments thereof, is amplified using PCR primer set SEQ ID NO: 1. Sequencing determined the exact structure of the nucleotides in the amplified genes, producing a nrDNA- / 7.S' unique DNA barcode specific to the strain studied. Difference sequencing techniques would be familiar to the skilled person and these are encompassed by the present invention.

[0120]

[0088] A further desired region according to the method of the present invention is rDNA intergenic spacer region or a fragment thereof. In some embodiments of the present invention, the desired region is rDNA intergenic spacer region (IGS). In some embodiments of the present invention, the desired region is rDNA intergenic spacer region (IGS) is amplified using PCT primer set SEQ ID NO: 2. Sequencing determined the exact structure of the nucleotides in the amplified genes, producing a rDNA intergenic spacer region (IGS) unique DNA barcode specific to the strain studied.

[0121]

[0089] A further desired region according to the method of the present invention is translation elongation factor 1-a or fragments thereof. In some embodiments of the present invention, the desired region is translation elongation factor 1-a or fragment thereof (TEFl-a). In some embodiments of the present invention, the desired target region translation elongation factor 1-a or fragment thereof, is amplified using PCR primer set SEQ ID NO: 3. Sequencing determined the exact structure ofthe nucleotides in the amplified genes, producing a TEFl-a unique DNA barcode specific to the strain studied.

[0122]

[0090] A further desired region according to the method of the present invention is beta-tubulin or fragments thereof.

[0123]

[0091] In some embodiments of the present invention, the desired region is beta-tubulin ([3-tub) or fragments thereof. In some embodiments of the present invention, the desired region is beta-tubulin which is amplified using PCT primer set SEQ ID NO: 4. Sequencing can be used to determine the exact structure of the contiguous nucleotides in the amplified gene, such as gene fragment and be used to produce or generate a [3-tub unique DNA barcode that is specific to the strain under investigation or sequencing study.

[0124]

[0092] In some embodiment, the nuclear gene RPB2 encoding the second largest subunit of RNA polymerase II can also be used in the methods of the present invention. Sequencing determined the exact structure of the nucleotides in the amplified genes, producing a RPB2 unique DNA barcode specific to the strain studied such as the strain that is under investigation or sequencing study.

[0125]

[0093] In some embodiments of the present invention, the method comprising the steps of: Attorney Ref: 6666 / P005-SAR-PCT

[0126] (a) obtaining a sample from a cereal plant to be tested for Fusarium spp.;

[0127] (b) isolating and identifying Fusarium spp. from the step (a) using morphological assessments and / or microscopy analysis;

[0128] (c) extracting genomic DNA from the isolated and identified Fusarium spp.;

[0129] (c) amplifying genomic DNA by conducting the polymerase chain reaction from about 30 cycles to about 35 cycles to produce a test PCR product; and

[0130] (e) determining whether the test PCR product is an amplified Fusarium spp. product from a cereal plant.

[0131]

[0094] In some embodiments of the present invention, the oligonucleotide primer set selected from the primer sets comprising SEQ ID: 1; SEQ ID: 2; SEQ. ID: 3 and SEQ ID: 4 amplify a test PCR product from genomic DNA corresponding to a gene sequence or a fragment thereof selected for Internal Transcribed Spacer region (nrDNA-77'.S'). rDNA intergenic spacer region (IGS), Translation elongation factor 1-a (TEF1- a) and fl-tub.

[0132]

[0095] In some embodiments of the present invention, the annealing temperature of the primer sets is in the range of from 51 °C to 61 ,5°C. In some embodiments of the present invention, the annealing temperature of the primer sets is in the range of from 51.1 °C to 61.2°C.

[0133]

[0096] In some embodiments of the present invention, the annealing temperature of the primer set 1 for nrDNA-77'.S' comprising sequence ID1 and sequence ID2 is 52.5°C.

[0134]

[0097] In some embodiments of the present invention, the annealing temperature of the primer set 2 for the IGS primers comprising sequence ID7 and sequence ID8 is 56.6°C.

[0135]

[0098] In some embodiments of the present invention, the annealing temperature of the primer set 3 for TEF-la comprising sequence ID3 and sequence ID4 is 54°C.

[0136]

[0099] In some embodiments of the present invention, the annealing temperature of the primer set 4 for the fl-tub comprising sequence ID5 and sequence ID6 is 51.1 °C.

[0137]

[0100] In some embodiments of the present invention, the PCR product is sequenced and the nucleotide sequence of the product generates a DNA barcode signature that is unique to the Fusarium spp. from which the genomic DNA template was extracted or the strain that was studied or tested.

[0138]

[0101] In some embodiments of the present invention, the unique DNA barcode signature is compared to reference DNA barcodes sequence for reliable identification of Fusarium spp. such as pathogenic Fusarium spp.. Attorney Ref: 6666 / P005-SAR-PCT

[0139]

[0102] DNA barcoding is a novel method of species identification that uses a short DNA sequence of a specific gene or genes, or "barcodes". The technique is based on comparing each individual sequence or fragment such as a test sequence of Fusarium spp. to a reference DNA database. In some embodiments of the present invention, the method of the present invention leads to an alteration of Fusarium spp differentiation or classification.

[0140]

[0103] As used herein, the term “alteration” may be used interchangeably with the terms, “alter” or “modify” such as increase or decrease in the level of confidence of the differentiation or classification between different Fusarium spp., In some embodiments, the increase in the confidence of the determinations is at least 0.1%, 0.5%, 1%, 2%, 2.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% or greater compared to control. In some embodiments the alteration may be at least 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10-fold or greater compared to control.

[0141]

[0104] In some embodiments, the primer sets comprising SEQ ID: 1; SEQ ID: 2; SEQ. ID: 3 and SEQ ID: 4 lead to a quicker or more accurate differentiation or classification between Fusarium spp.. In some embodiments, the primer sets comprising SEQ ID: 1; SEQ ID: 2; SEQ. ID: 3 and SEQ ID: 4, lead to at least 0.01%, at least 0.05%, at least 0.1%, at least 0.5%, at least 1%, at least 2.5%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more, quicker or more accurate differentiation or classification between Fusarium spp..

[0142]

[0105] In some embodiments, the quicker or more accurate differentiation or classification between Fusarium spp. can be reliant on or use a single nucleotide difference between the test sample and compared that of the control. In some embodiments, the single nucleotide difference is detected within a gene sequence of Internal Transcribed Spacer region (nrDNA- / 7'.S). rDNA intergenic spacer region (IGS), Translation elongation factor 1-a (TEFl-a) or fi-tub, compared to that of the control sequence.

[0143]

[0106] In some embodiments of the present invention, pathogenic Fusarium spp., is selected from F. proliferatum, F. subglutinans, F. fujikuroi, F. verticillioides, or F. oxysporum. In some embodiments of the present invention, the selected from F. proliferatum, F. subglutinans, F. fujikuroi, F. verticillioides, or F. oxysporum produces trichothecene mycotoxin and / or zearalenone mycotoxin.

[0144]

[0107] In some embodiments, the present invention contemplates different techniques which can detect different Fusarium spp. mycotoxins. The mycotoxin techniques may include primers such as protein detecting primers, spectroscopy, antibodies (such as ELISA) or different protein tags which are capable of detecting and differentiating between different Fusarium spp. mycotoxins. Attorney Ref: 6666 / P005-SAR-PCT

[0145]

[0108] In some embodiments of the present invention, the primers can identify Fusarium spp. mycotoxins selected form the group comprising fumonis (FUMs), zearalenone (ZEA) and type A trichothecenes: T-2 toxin (T-2) and HT-2 toxin (HT-2), trichothecene mycotoxin and zearalenone mycotoxin. In some embodiment the mycotoxin techniques are mycotoxin detection techniques. In some embodiment, the mycotoxin techniques provide qualitative characterisation of the Fusarium spp. mycotoxin. In some embodiments, the mycotoxin techniques provide quantitative determination of the Fusarium spp. mycotoxin.

[0146]

[0109] The skilled person would be familiar with different techniques for detecting and characterising mycotoxins. Example Fusarium spp. mycotoxin detecting techniques include and are not limited to visual imaging, infrared (IR) / thermal imaging, laser techniques, and other physico-chemical and / or chemical parameters which can deploy devices or techniques such as HPLC, MALDI TOF (MS), Energy Dispersive X-ray Spectroscopy, EDX analysis and SEM (Scanning Electron Microscopy) studies using FESEM (Field Emission Scanning Electron Microscope).

[0147] [HO] In an aspect of the present invention, there is provided a. Fusarium spp. as identified by the methods, primers, kits, primers and techniques described herein throughout. In some embodiments of the present invention, there is provided a pathogenic Fusarium spp., such as a pathogenic Fusarium spp. selected from F. proliferatum, F. subglutinans, F. fujikuroi, F. verticillioides, or F. oxysporum. In some embodiments of the present invention, the pathogenic Fusarium spp., selected from F. proliferatum, F. subglutinans, F. fujikuroi, F. verticillioides, or F. oxysporum produces trichothecene mycotoxin and / or zearalenone mycotoxin.

[0148]

[0111] In an aspect of the present invention there is provided a kit for identifying a genotype of Fusarium spp., the kit comprising a primer sets comprising SEQ ID: 1; SEQ ID: 2; SEQ. ID: 3 and SEQ IDA as designed to amplify a genomic DNA template which template is extracted from isolated and identified Fusarium spp., and optionally a leaflet of instructions for use of the primers to identify the genotype of Fusarium spp..

[0149] Reports and Data Transmission

[0150]

[0112] In some embodiments, the methods disclosed herein further comprise generating one or more reports such as primer information, PCR conditions, test DNA sequencing information, DNA barcoding information, or differentiation of Fusarium spp. data.

[0151]

[0113] In some embodiments, the methods disclosed herein further comprise storing one or more reports.

[0152]

[0114] In some embodiments, the methods disclosed herein further comprise transmitting one or more reports. Attorney Ref: 6666 / P005-SAR-PCT

[0153]

[0115] In some embodiments, the report includes information on the pathogenic capability of a differentiated or classified new Fusarium spp. as determined according to the methods of the present invention.

[0154]

[0116] In some embodiments, the report includes information on the mycotoxin profile of the identified pathogenic Fusarium spp.

[0155]

[0117] In some embodiments, the report includes information on gene sequences of pathogenic Fusarium spp. In some embodiments, the report includes information on phylogenetic tree.

[0156]

[0118] In further embodiments, the report may be implemented in the form of a recording medium including instructions executable by a computer, such as a program module executed by the computer.

[0157]

[0119] Computer-readable media may be any available media that may be accessed by a computer and include both volatile and nonvolatile media and removable and non-removable media. In addition, the computer-readable media may include all computer storage media. The computer storage media includes both volatile and nonvolatile media and removable and non-removable media implemented by any method or technology of storing information, such as a computer readable instruction, a data structure, a program module, and other data. The storage may be in the iCloud.

[0158]

[0120] In order that the invention may be readily understood and put into practical effect, particular embodiments will now be described by way of the following non-limiting examples.

[0159] MATERIALS & METHODS and EXPERIMENTAL EXAMPLES

[0160]

[0121] Materials and Methods

[0161]

[0122] 1.1. Samples for analysis

[0162]

[0123] The present inventors analysed a total of 50 maize samples.

[0163]

[0124] These 50 samples were subjected to a series of tests and experimental assessments in order to establish the Fusarium species. Furthermore, the 50 samples were subjected to a series experiments in order to establish which of the identified Fusarium species were pathogenic. Further experiments where conducted to determine the type of mycotoxin produced or secreted by the Fusarium species. In addition, these same samples were exposed to a series of tests and experimental assessments in order to establish the degree of contamination.

[0164]

[0125] The maize kernels samples were collected in the period September-November 2023, from 3 main commercial maize-growing regions in Bulgaria (Figure 1) - Pleven (26 samples), Shumen (16 samples), and Stara Zagora (8 samples), from storage facilities according to according to ISO 24333:2010 and ISO 24333:2009. Attorney Ref: 6666 / P005-SAR-PCT

[0165]

[0126] Briefly, 1 kg spot samples of maize with no visible disease symptoms were taken with a bulk profile sampler from 10 points throughout the bulk lot (lots ranged from 5 to 100 tons). Multiple samples from appropriate sites were aseptically poured into a sterile container (paper bag) and manually mixed to obtain a homogeneous composite sample.

[0166]

[0127] Following their complete mixing, a bulk sample weighing 10 kg was formed, which was manually reduced to 0.5 kg and labeled. The entire experimental design scheme of for DNA barcoding in maize is shown in Figure 1.

[0167]

[0128] 1.2. Culture media and reagents

[0168]

[0129] Samples for mycological examination were inoculated on Czapek Dox medium (HiMedia©, India) and on a selective culture medium (SNA).

[0169]

[0130] Czapek Dox medium contains NaNOs - 3.0 g, K2HPO4 - 1.0 g, MgSC VFFO - 0.5 g, sucrose - 30 g, KC1- 0.5 g, FeSO4.7H2O - 0.01 g, agar - 15 g; distilled water - 1000 ml (pH=7.2). SNA contains KNO3 - 1.0 g; K2HPO4 - 1.0 g, MgSC>4.7H2O - 0.500 g, sucrose - 0,2 g, glucose - 0,2 g, agar - 20 g. FIG. 3.

[0170]

[0131] All used reagents were of analytical and reagent grade using manufacturer’s instructions.

[0171]

[0132] VWR Red Taq DNA Polymerase Master Mix 2x Master Mix Kit (1,5 mM MgCT) contains Tris- HC1 pH 8,5, (NH4)2S04, 3,0 or 4,0 mM MgCl2, 0,2 % Tween® 20, 0,4 mM of each dNTP, 0,2 units / pl VWR, Taq polymerase, Inert red dye and a stabiliser.

[0172]

[0133] 1.3. Isolation and identification of fungi using cultural methods

[0173]

[0134] One hundred whole maize kernels from each sample were randomly selected and were superficially sterilized with 70% ethanol for 5 min, followed by three rinses with distilled water to eliminate surface microflora. The kernels were dried with sterile filter paper and placed in 4 petri dishes (d=I5cm) (25 grains in each) on Czapek Dox medium.

[0174]

[0135] Inoculated plates were incubated at 22-25°C for 7 days (BG / EU National Standard 11374 / 86), followed by calculation the number of growing colonies belonging to the Fusarium spp. The identification of Fusarium spp. was confirmed using keys known in the art such as those published by Gerlach and Nirenberg (1982), and Leslie and Summerall (2006).

[0175]

[0136] All suspected Fusarium spp. colonies were subcultured, using the single spore technique (Pitt and Hocking, 2009). A spore suspension was prepared in a 10 ml sterile water sample to contain 1 to 10 spores.

[0176]

[0137] The prepared water agar medium (WA) plates was inoculated with 0.1 ml of suspension and incubated for 18-20 h at 25°C. For further molecular identification, the resulting single spore cultures were Attorney Ref: 6666 / P005-SAR-PCT transferred onto SNA and incubated at 25 °C for 7 days. Each isolate's spores were examined under a microscope to identify distinctive characteristic unique to Fusarium spp.

[0177]

[0138] 1.4. DNA extraction and PCR amplification

[0178]

[0139] Genomic DNA of 5 -day-old mycelium (1.5±2 cm in diameter) of the Fusarium spp. strains on SNA plates was extracted. To degrade the fungi's polysaccharide cell wall, the mycelium was initially frozen for 24 hours at -20°C and then pulverized in conical Eppendorf tubs with quartz sand.

[0179]

[0140] In order to extract DNA from the samples, DNA isolation kit (Jena Bio-science, Germany) was used according to the manufacturer's instructions.

[0180]

[0141] The concentration of extracted genomic DNA of each sample was measured using a NanoView Plus spectrophotometer (GE Healthcare) at a 260-280 nm wavelength. The DNA concentration of all samples was adjusted to 10 ng / pl, in a working volume of 70 pl and the genomic DNA-extracted samples were then stored at -18°C. The gene regions used, the primers sequence, the annealing temperature, and the expected fragments length are listed in Table 1.

[0181] Table 1. Summarised information for DNA barcodes and primer sets 1, 2, 3 and 4. There is also described the anticipated size of the test PCR product.

[0182] Attorney Ref: 6666 / P005-SAR-PCT

[0183]

[0142] ‘ ‘F” stands for forward primer and “R” stands for reverse primer.

[0184]

[0143] Each primer anneals to the complementary nucleotide strand for PCT amplification. The stringency of the annealing and the fidelity of the PCT reaction would depend of factors such as for example temperature, salt concentration, C to G ratio in the primer set as well as the selected for the PCT amplification reaction polymerase.

[0185]

[0144] The optimal annealing temperature was determined based on gradient PCR. Attorney Ref: 6666 / P005-SAR-PCT

[0186]

[0145] The PCR reaction mixture was 25 pl volume and contained 10 l of the extracted DNA, 10 pl RedTaq 2x Master Mix, 1,5 mM MgCT (VWR, Belgium), 0.8 pl of each primer, and 3.4 pl of nuclease-free water.

[0187]

[0146] The primers’ optimal annealing temperature was previously determined by multiple PCR amplification of the same sample in the temperature range 51.1-61.2°C - it was 52.5°C for the ITS primers, 54°C for TEF-la primers, 51.1°C for the fl-tub primers, and 56.6°C for the IGS primers. Amplification was performed in athermal cycler (QB-96, Quanta Biotech), under the following conditions: initial denaturation at 95°C for 5 min, followed by 30 cycles of amplification (denaturation at 95°C for 0.30 min, annealing at specific temperature for 0.45 min, extension at 72°C for 1 min, ending with a final extension at 72°C for 9 min.

[0188]

[0147] The obtained PCR products were identified by horizontal electrophoresis on a 1% agarose gel (TopVision agarose, Fermentas, USA), using 1*TBE buffer, at 70 V / 70 min and stained with 10,000*GelRed™ (Cat. N° 41003, Biotium Inc, USA). The fragments size was determined using Gene- Ruler™ 100 bp Ladder Plus (Cat. N° SM0323, Thermo Fisher Scientific Inc., Waltham, MA, USA) and the fragments were observed on a MiniBis photodocumentation system using a transilluminator (ECX-15M BioImaging Systems).

[0189]

[0148] The obtained products were visualized through GelCapture image acquisition software.

[0190]

[0149] 1.5. Nucleotide sequence analysis

[0191]

[0150] Before sequence analysis the amplified products (such as test PCR products) were purified by a PCR purification kit (Gene Matrix, PCR clean-up kit, Cat. No. E3520; EURx Ltd., Gdansk, Poland) according to the manufacturer instructions. The purified DNA was stored at -20°C until it was used in any follow up assays and sequencing analysis such as a PCR assay. In FIG. 4. There is shown nucleotide sequencing data for amplified gene regions.

[0192]

[0151] The successfully amplified and purified 68 products for Fusarium spp. were sequenced by a PlateSeq kit in Eurofins Genomics laboratory, Ebersberg, Germany.

[0193]

[0152] 1.6. Statistical Analysis

[0194]

[0153] Identification of isolates was performed by comparing each sequence to the corresponding reference from the NCBI database via Nucleotide Blast (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). DNA Sequence Assembler v4. (2013) was used to convert sequences from “txt” to “Fasta” format. All 68 obtained DNA sequences were manually edited and aligned with a reference sequence using the MUSCLE algorithm (Edgar, 2004) as implemented in the MEGA v. software. Attorney Ref: 6666 / P005-SAR-PCT

[0195]

[0154] The established sequences from analyzed samples were manually edited, assembled and aligned by CLUSTALW tool using MEGA software v. 11. The same software was used to determine genetic distances between the studied fungal isolates and the phylogenetic tree was reconstructed by Neighbor-joining method. The evolutionary distances were computed using the p-distance method (Saitou etal., 1987; Tamura et al., 2021). Identification of isolates was performed by comparison of each sequence to respective reference from the NCBI database through Nucleotide Blast (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi).

[0196]

[0155] 2. Results

[0197]

[0156] 2.1. Mycological examination analysis

[0198]

[0157] The mycological studies of 50 maize samples, from 3 regions in Bulgaria, harvested in 2022, included the following 8 types of microscopic fungi: Fusarium spp.. Allernaria spp.. Mucor spp., Aspergillus spp., Penicillium spp., Rhizopus spp., Nigrospora spp., and Epicoccum spp.

[0199]

[0158] Based on morphological characteristics of the fungal isolates such as characteristics and growth rate of mycelium, microscopic observation of macro- and microconidia, conidiogenous cells, mono- and polyphialides, basal cell morphology, presence and absence of chlamydospores, etc., as well as based on the identification key 17 strains were found to belong to Fusarium spp.

[0200]

[0159] The isolates possessed a white, pinkish to purple cottony aerial mycelium. Changes in pigmentation from white to light purple or from purple to a darker shade of purple were observed. The macroconidia were thin and had a serrate basal cell and a curved apical cell. The microconidia of all isolates had an oval shape and were arranged in a chain. A number of authors have reported that the use of morphological characters alone in the species identification of molds can lead to inaccuracies in reported results.

[0201]

[0160] It should be noted that in 2022, a period characterized by low soil and air humidity during the maize growing season, in the Stara Zagora and Shumen regions, an expected lower presence of genus Fusarium members was identified, compared to the Pleven region (Figure 2). Of the 50 maize samples (5 000 grains) examined, 32 samples (64%) were positive for Fusarium spp., while the remaining 18 samples (36%) were not infected with these fungi. The highest percentage (69%) was found in the Pleven region - 12 samples (46%) from were contaminated with up to 10% Fusarium spp.

[0202]

[0161] In 5 samples (19%) insemination was up to 20% and in only 1 sample (0.04%) insemination reached 50%. In the Shumen region, only 3 samples were contaminated up to 20% with Fusarium spp. representatives, and none were found in the Stara Zagora region. Based on the identification key, 17 strains were isolated and identified. Distinctive mycelial morphological features, growth rate, pigment formation, and macroconidia species were used to determine both similarities and differences between isolates, but all isolates were assigned to the genus. Attorney Ref: 6666 / P005-SAR-PCT

[0203]

[0162] 2.1.1. Mycotoxin tests, methods and techniques

[0204]

[0163] The inventors were aware that mycotoxins represent a wide range of secondary, naturally occurring and practically unavoidable fungal metabolites with low molecular weight of -700 Da. In our tests we relied on Chromatographic techniques such as LC, TLC, HPLC, LC-MS / MS, GC-MS / MS and immunological methods such as ELISA. The LC methods we used enabled us to detect simultaneous determination of several mycotoxins, regardless of their chemical structure and biological activity. Data show in Figure 9. ELISA enabled us to test simultaneous multiple samples and its detection is precise and reliable (data show in Figure 9 was confirmed using ELISA. Data not shown).

[0205]

[0164] 2.2. Data analysis from fungal isolates PCR amplification

[0206]

[0165] Seventeen fungal strains isolated from maize were selected and successfully amplified along rDNA-ITS, TEF-la, fl-iith and IGS regions by conventional PCR. The determined amplicon lengths for Fusarium isolates were 540 bp for ITS, 700 bp for TEF-la, 500 bp for -tub and 200 bp for IGS, respectively. The results of the molecular genetic analysis showed that all strains were representatives of Fusarium spp. (data not shown).

[0207]

[0166] All 4-mark amplified isolates were sequenced by Eurofins MWG Operon (Ebersberg, Germany) and compared to the annotated sequence data for each species in the database (Figures 5 and 6).

[0208]

[0167] By manual BLAST alignment in NCBI, all isolates were proven to belong to the genus Fusarium and different species were identified (Table 2).

[0209] Table 2. Species affiliation of isolates according to ITS, TEF, ft-tub, and IGS regions after manual alignment at NCBI

[0210] * Isolates from several regions such as commercially grown cereals areas around the following cities: P- Pleven, Sh-Shumen, SZ-Stara Zagora Attorney Ref: 6666 / P005-SAR-PCT

[0211]

[0168] The length of phylogenetic tree branches is shown on a Figure 6 corresponding to genetic distances between studied strains. Neighbour-joining dendrogram was built on the base of obtained sequences of the region rDNA-ITS of 17 Fusarium isolates from maize samples in accordance with their reference sequences obtained from NCBI database: GQ167233. 1 for F. proliferatum, JF499679 for F. subglutinans, OR884165. 1 Attorney Ref: 6666 / P005-SAR-PCT for F. oxysporum. OM955976.1 forF. verticillioides and MH266059.1 for F sporotrichioides . The analysis involved 22 nucleotide sequences. All positions containing gaps and missing data were eliminated (complete deletion option). There were a total of 483 positions in the final dataset.

[0212]

[0169] The dendrogram (Figure 6) showed that fungal isolates were grouped in two main clusters (Cluster I and II). Cluster I is composed of isolates belonging to 2 species complexes - F. fujikuroi species complex (FFSC) n F. oxysporum species complex (FOSC). The F. oxysporum species complex (FOSC) includes the following 4 F. oxysporum isolates: ITS 351, ITS 361 from the Stara Zagora region, ITS 421 from the Pleven region, and ITS 381 from the Shumen region. Eight F. proliferatum strains were assigned to the FFSC based on phylogenetic relationships.

[0213]

[0170] The most widespread were in Pleven - 4 isolates (ITS 341, ITS 371, ITS 190, ITS 401), followed by Shumen- 3 isolates (ITS 391, ITS 431, ITS 189), and Stara Zagora - 1 isolate (ITS 41c). The only isolate (ITS45) identified as F. subglutinans was from the Shumen region. Cluster II is composed of 4 isolates from the Pleven region - strain ITS 191 belonging to the species F. sporotrichioides, assigned to the Fusarium samhuchinum species complex (FSSC) and the three strains ITS 44, ITS 187, and ITS 188 identified as F. verticillioides, assigned to FFSC.

[0214]

[0171] Additionally, a phylogenetic tree based on the TEF-la locus was built where the Fusarium spp. strains were categorized into two large clusters (Cluster I and II) (Figure 7). Cluster I was divided into two groups (group 1 and 2) belonging to the 2 species complexes FFSC and FOSC. A total of 11 strains were identified as F proliferatum - TEF80, TEF81, TEF86 from the Stara Zagora region; TEF84, TEF88, TEF200 - from the Shumen region, and TEF85, TEF87, TEF199, TEF201, TEF202 - from the Pleven region, all assigned to group 1 along with 2 F. fujikuroi strains TEF79 and TEF198 from Pleven region. Isolate TEF901 was identified as F. subglutinans .

[0215]

[0172] Species with the following numbers were selected for reference: MN861773 - forF. Proliferatum, OQ511505 for F. fujikuroi, MN193870 for F. Subglutinans, LC745428.1 for F. oxysporum, and FN179340.1 for the only isolate separated in the phylogenetic tree, identified as F. verticillioides and belonging to the FFSC.

[0216]

[0173] 2.3. Species composition by region based on aggregated data from 4 loci

[0217]

[0174] Data from fours loci namely Internal Transcribed Spacer region (nrDNA- / 7'.S). rDNA intergenic spacer region (IGS), Translation elongation factor 1-a (TEFl-a) and fi-tub is summarised in Figure 8.

[0218]

[0175] We are able to develop a quick and reliable method of differentiating between Fusarium spp., such as pathogenic Fusarium spp., by targeting four loci. This new approach produced surprisingly reproducible Attorney Ref: 6666 / P005-SAR-PCT and reliable results which could be safely used for classification and differentiation assessments of different strains of Fusarium fungi.

[0219]

[0176] 2.4. Mycotoxin characterisation and profile of Fusarium spp.

[0220]

[0177] The pathogenic Fusarium spp., selected from F. proliferatum, F. subglutinans, F. fujikuroi, F. verticillioides, or F. oxysporum as identified by the methods of the present invention were tested for production of mycotoxins.

[0221]

[0178] The data is summarised in Figure 9 shows that Fusarium spp., selected from F. proliferatum, F. subglutinans, F. fujikuroi, F. verticillioides, or F. oxysporum produce trichothecene mycotoxin and / or zearalenone mycotoxin.

[0222]

[0179] 2.4.1. Testing for Trichothecenes

[0223]

[0180] Solid PDA medium was used as follows: test samples comprising 1 g of mycelium admixed with medium were extracted using acetonitrile / water at different rations but we found that approximately 82: 18 worked well in our hands. The test samples were then purified using Myco Sep 227 Trich column. Group B trichothecenes (DON, NIV, 3AcDON, 15AcDON, FUS-X) were analysed as trimethylsilylsilyl ethers derivatives. After sililation, the test samples were extracted with isooctane and 1 u\ of sample was injected on a GC / MS system. The analyses were run on a gas chromatograph (GC) (Hewlett Packard GC 6890, Waldbronn, Germany) hyphenated to a mass spectrometer (MS) (Hewlett Packard 5972 A, Waldbronn, Germany), using an HP-5MS, 0-25 mm x 30 m capillary column. The injection port temperature and the transfer line temperature were set at 280°C. Initial temperature was 80°C was maintained for 1 min, from 80°C to 200°C at 15°C min1was maintained for 6 min and from 200°C to 280°C at 10°C min the final temperature was maintained for 3 min. Helium flow rate was maintained constant at 0-7 ml min ' . In the quantitative analysis we relied on a single ion monitored mode while for the qualitative analysis we used SCAN mode (100-700 amu). Recoveries for analysed toxins were for example: DON 84 ± 3 -8%; 3AcDON 78 ± 4-8%; 15AcDON 74 ± 2-2%; FUS X 87%±5-9%; NIV 81 ± 3 -8%. Limit of detection was 0 01 mg kg ' . Some of the results are presented in Figure 9.

[0224]

[0181] 2.4.2. Testing for Zearalenone

[0225]

[0182] Solid PDA medium was used as follows: Test samples comprising 1 g of mycelium with medium were extracted using acetonitrile / water at different rations but we found that approximately 82: 18 worked well in our hands. The test samples were then purified using Zearala affinity columns. Prepared samples were analysed by HPLC consisting of a Waters HPLC 2695 apparatus with a Waters 2475 Multi 2 Fluorescence Detector and a Waters 2996 Array Detector (Waters, Milford, MA). Separation was achieved on a 150 mm length x 3-9 mm diameter Nova Pak C-18, 4- / .zm particle size column and eluted with Attorney Ref: 6666 / P005-SAR-PCT acetonitrile-water-methanol (46:46:8, v / v / v) at a flow rate of 0-5 ml min ' . ZEA was detected with a Waters 2475 Multi ! Fluorescence Detector, and the excitation and emission wavelengths were 274 and 440 nm, respectively. Estimation of ZEA was performed by a comparison of peak areas with those of an external standard (>95%; Sigma-Aldrich) or by co-injection with the standard. The detection limit of ZEA was 3 ng g ' . Some of the results are presented in Figure 9.

[0226]

[0183] CONCLUSION

[0227]

[0184] In the present invention as exemplified in by the present non-limiting study, the degree of contamination of maize samples and the potential and applicability of the different loci used as DNA barcodes to detect the presence of the most common mycotoxigenic Fusarium spp. in cereals was evaluated. It should be noted that the universal fungal ITS marker does not always provide species-level resolution, especially for the genera Aspergillus, Penicillium, Fusarium, ('ladosporium. and Trichoderma. necessitating the use of additional regions of the Fusarium genome. The TEFl-a and i-iub genes show similar interspecific divergence variation in Fusarium spp., which is higher than that of IGS and ITS. Therefore, as the best barcode for the differentiation of Fusarium spp. as developed in this study, we advantageously observed that selecting the fours (4) loci namely Internal Transcribed Spacer region (nrDNA- / 7'.S). rDNA intergenic spacer region (IGS), Translation elongation factor 1-a (TEFl-a) and / 3-tub provided the quickest and most reproducible detection, classification, identification and differentiation between Fusarium spp. with the greatest confidence levels.

[0228] * * *

[0229]

[0185] Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the disclosure claimed. It will also be appreciated that the device(s), method(s), use(s), detector(s), sensor(s), physiological parameters(s) such as test parameters, may be subject to numerous rearrangements, modifications and substitutions without departing from the scope of the present disclosure as set forth and defined by the following claims.

[0230]

[0186] REFERENCES

[0231] 1. Goncharov A. A., A. A. Glebova, A. V. Tiunov (2020). Trophic interactions between Fusarium species and soil fauna: A meta-analysis of experimental studies. Appl.Soil Ecol. 145: 103302. https: / / doi.Org / 10.1016 / j.apsoil.2019.06.005 Atorney Ref: 6666 / P005-SAR-PCT . Bernard D. Davis, Renato Dulbecco, Herman N. Eisen, Harold S. Ginsberg, Lippincott Williams & Wilkins; Subsequent edition, 1 January, 1990

[0232] 3. Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989

[0233] 4. Philipp Gerhardt et al., American Society for Microbiology, Washington, DC, 1994

[0234] 5. Gerlach W., H. Nirenberg (1982). The Genus Fusarium - a Pictorial Atlas. Mitt. Bit. Bundesanst. Lor Stwirtsch.Berlin-Dahlem, Germany. 406 p. Vol. 209, . Berlin: Kommissionsverlag P. Parey.

[0235] 6. Leslie, J. L., B. A. Summerall. (2006). The Fusarium Laboratory Manual. Blackwell Publishing, IOWA, USA.388 p. htp: / / doi.org / 10.1002 / 978047Q278376

[0236] 7. Pit J. I., Hocking, A.D. (2009) Lungi and Pood Spoilage. 3rd Edition, Springer Dordrecht Heidelberg London New York Cambridge, 519 p. https: / / doi.org / 10.1007 / 978-0-387-922Q7-2

[0237] 8. Protocol: Using DNA Barcodes to Identify and Classify Living Things. 2023. Cold Spring Harbor Laboratory, DNA Learning Center, https: / / dnabarcodin l01.org / fdes / using-dna-barcodes.pdf

[0238] 9. O'Donnell K., E. Cigelnik (1997). Two divergent intragenomic rDNA ITS2 types within a monophyletic lineage of the fungus Fusarium are non-orthologous. Mol. Phylogenet. Evol. 7: 103- 116. htps: / / doi.org / 10.1006 / mpev.1996.Q376

[0239] 10. Wulff E. G., J. L. Sorensen, M. Lubeck, K. F. Nielsen, U.Thrane, J. Torp (2010). Fusarium spp. associated with rice Bakanae: ecology, genetic diversity, pathogenicity and toxigenicity. Environ. Microbiol. 12: 649-657. https: / / doi.org / 10, 1111 / j , 1462-2920.2009.02105.X

[0240] 11. Kim Y., R. B. Hutmacher, R. M. Davis (2005). Characterization of California isolates of Fusarium oxysporum f. sp. vasinfectum. Plant Dis. 89: 366-372. htps: / / doi.org / 10.1094 / PD-89-Q366

[0241] 12. Jurado M., C. Vazquez, S. Marin, V. Sanchis, M. T. Gonzalez-Jaen (2006). PCR-based strategy to detect contamination with mycotoxigenic Fusarium species in maize. Syst. Appl. Microb. 29: 681- 689. htps: / / doi.Org / 10.1016 / j.syapm.2006.01.014

[0242] 13. Saitou N., M. Nei (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4: 406-425. htps: / / doi.org / 10.1093 / oxfordjoumals.molbev.a040454

[0243] 14. Tamura K., G. Stecher, S. Kumar (2021). MEGA11: Molecular evolutionary genetics analysis version 11. Mol. Biol. Evol. 38: 3022-3027. htps: / / doi.org / 10.1093 / molbev / msabl2Q

Claims

1. Attorney Ref: 6666 / P005-SAR-PCTCLAIMS1. A method for identifying a genotype of Fusarium spp. from a cereal plant, the method comprising the steps of:(a) obtaining a sample from a cereal plant to be tested for Fusarium spp.;(b) isolating and identifying Fusarium spp. from the step (a) using morphological assessments and / or microscopy analysis;(c) extracting genomic DNA from the isolated and identified Fusarium spp.;(c) replicating a desired region in a DNA template by binding a specific oligonucleotide primer set selected from the primer sets comprising SEQ ID: 1; SEQ ID: 2; SEQ. ID: 3 and SEQ ID: 4 to the DNA template; where the primer set is designed to give rise to a test PCR product containing at least one nucleotide variance compared to a control sequence; and(e) determining whether the test PCR product is an amplified Fusarium spp. product from a cereal plant.

2. A method according to claim 1, wherein the cereal plant is selected from the group comprising rice, barley, wheat, maize, oat, teff, sorghum, millet.

3. A method according to anyone of claims 1 or 2, wherein the cereal plant is maize.

4. A method according to anyone of the preceding claims, wherein the Fusarium spp. is a pathogenic Fusarium spp. selected from F. proliferatum, F. subglutinans, F. fujikuroi, F. verticillioides, or F. oxysporum.

5. A method according to anyone of the preceding claims, where the PCR primer set is designed to give rise to PCR products used for differentiating between Fusarium spp.

6. A method according to claim 5, wherein the differentiation between Fusarium spp. uses the technique of barcoding the genotype of pathogenic Fusarium spp. from a cereal plant.

7. A method according to claim 5 or claim 6, wherein the differentiation between Fusarium spp. relies on oligonucleotide primer set selected from the primer sets comprising SEQ ID: 1; SEQ ID: 2; SEQ ID: 3 and SEQ ID: 4, wherein primer sets are designed to enable the amplification of a test PCR product from a genomic DNA corresponding to a gene sequence or a fragmentAttorney Ref: 6666 / P005-SAR-PCT thereof selected from Internal Transcribed Spacer region (nrDNA-77'.S'). rDNA intergenic spacer region (IGS), Translation elongation factor 1-a (TEFl-a) and fl-tub.

8. A method according to anyone of claims 5 to 7, wherein the pathogenic Fusarium spp. is associated with or is the causative agent of a cereal plant disease.

9. A method according to claim 8, wherein the cereal plant disease is selected from at least one of seedling blight, stem rot, root rot, ear rot, stalk rot, head blight and crown rot.

10. A method according to anyone of the preceding claims, the method comprising the steps of:(a) obtaining a sample from a cereal plant to be tested for Fusarium spp.;(b) isolating and identifying Fusarium spp. from the step (a) using morphological assessments and / or microscopy analysis;(c) extracting genomic DNA from the isolated and identified Fusarium spp.;(c) amplifying genomic DNA by conducting the polymerase chain reaction from about 30 cycles to about 35 cycles to produce a test PCR product; and(e) determining whether the test PCR product is an amplified Fusarium spp. product from a cereal plant.

11. A method according to claim 10, wherein the oligonucleotide primer set selected from the primer sets comprising SEQ ID: 1; SEQ ID: 2; SEQ. ID: 3 and SEQ ID: 4 amplify a test PCR product from genomic DNA corresponding to a gene sequence or a fragment thereof selected for Internal Transcribed Spacer region (nrDNA-77'.S'). rDNA intergenic spacer region (IGS), Translation elongation factor 1-a (TEFl-a) and fl-tub.

12. A method according to anyone of claims 9 or 10, wherein the annealing temperature of the primer sets is in the range of from 51.1°C to 61.2°C.

13. A method according to anyone of claims 9 to 11, wherein the annealing temperature of the primer set for nrDNA-77'.S' is 52.5°C, for TEF-la primer set is 54°C, for the fl-tub primer set is 51.1°C, and for the IGS primer set is 56.6°C.

14. A method according to anyone of claims 6 to 13, wherein the PCR product is sequenced and the nucleotide sequence of the product generates a DNA barcode signature that is unique to the Fusarium spp. from which the genomic DNA template was extracted.Attorney Ref: 6666 / P005-SAR-PCT15. A method according to claim 14, wherein the unique DNA barcode signature is compared to reference DNA barcodes sequence for reliable identification of Fusarium spp. such as pathogenic Fusarium spp..

16. A method according to anyone of claims 14 or 15, wherein the Fusarium spp. such as pathogenic Fusarium spp. is identified as F. proliferatum, F. subglutinans, F. fujikuroi, F. verticillioides, or F. oxysporum.

17. A method according to anyone of the preceding claims, wherein the method leads to an increase in the confidence in the determinations eF usarium spp. by at least 0.1%, 0.5%, 1%, 2%, 2.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% compared to control.

18. A Fusarium spp. such as pathogenic Fusarium spp., selected from F. proliferatum, F. subglutinans, F. fujikuroi, F. verticillioides, or F. oxysporum as identified by the method of any one of claims 1 to 17.

19. A Fusarium spp. according to claim 18, wherein the pathogenic Fusarium spp., selected from F. proliferatum, F. subglutinans, F. fujikuroi, F. verticillioides, or F. oxysporum produces trichothecene mycotoxin and / or zearalenone mycotoxin.

20. Use of primer sets for quick and reliable identification of Fusarium spp. such as pathogenic Fusarium spp., the primer sets comprising SEQ ID: 1; SEQ ID: 2; SEQ. ID: 3 and SEQ ID: 4 as designed to amplify a genomic DNA template which template is extracted from isolated and identified Fusarium spp. and generate a test PCR product containing at least one nucleotide variance compared to a control sequence.

21. Use of primer sets according to claim 20, wherein the PCR product is sequenced and the nucleotide sequence of the product used to generate a DNA barcode signature that is unique to the Fusarium spp. from which the genomic DNA template was extracted.

22. A kit for identifying a genotype of Fusarium spp., the kit comprising a primer sets comprising SEQ ID: 1; SEQ ID: 2; SEQ. ID: 3 and SEQ ID: 4 as designed to amplify a genomic DNA template which template is extracted from isolated and identified Fusarium spp., and optionally a leaflet of instructions for use of the primers to identify the genotype of Fusarium spp..

Images