Classifications

• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
  • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
  • A01H1/04—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
  • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
  • A01H1/12—Processes for modifying agronomic input traits, e.g. crop yield
  • A01H1/122—Processes for modifying agronomic input traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
  • A01H1/1245—Processes for modifying agronomic input traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, e.g. pathogen, pest or disease resistance
  • A01H1/1255—Processes for modifying agronomic input traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, e.g. pathogen, pest or disease resistance for fungal resistance
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
  • A01H3/00—Processes for modifying phenotypes, e.g. symbiosis with bacteria
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/0098—Plants or trees
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
  • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/37—Assays involving biological materials from specific organisms or of a specific nature from fungi
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/415—Assays involving biological materials from specific organisms or of a specific nature from plants

Definitions

• the present disclosure relates to a phenotype-based method for screening and identifying plants which exhibit resistance phenotypes to plant pathogens, and the use of same in plant breeding.
• Classical phenotyping methods are designed to mimic natural infection processes and cannot distinguish when a pathogen is recognized by the plant or how it then acts to prevent or slow infection and disease.
• the inventors have invented a phenotyping method which allows plant reactions against plant pathogens (such as fungal pathogens) to be categorized into different classes of defence.
• plant pathogens such as fungal pathogens
• the new method described in this disclosure dissects the infection process into two main phases, pre and post entry of the pathogen past the leaf external layers.
• Plants can trigger defence responses to a pathogen at different points along the infection pathway.
• the recognition of the presence of the pathogen by the host is the first step in this process and when this occurs the plant triggers a defence response.
• This recognition step can occur at the earliest point of a spore landing on a leaf or at any point of the following stages of infection and disease development.
• the inventors have taken a novel approach by combining two different inoculation techniques which allows the identification and separation of when defence is triggered by the plant.
• the phenotyping method represents a large step forward in developing crop varieties with multiple layers of defence against pathogens, such as fungi, particularly for species of plant where there is little or no knowledge of the genetic background which confers disease resistance or susceptibility.
• the present disclosure provides a method of identifying a plant which exhibits a resistance phenotype to a plant pathogen, the method comprising: (a) performing a first screening method comprising:
• step (iii) determining one or more responses of the plant(s) or plant part(s) to the pathogen at a plurality of time points during step (ii);
• step (iii) determining one or more responses of the plant(s) or plant part(s) to the pathogen at a plurality of time points during step (ii);
• the method comprises determining whether or not the plant species or cultivar exhibits a resistance phenotype to the plant pathogen prior to and/or during entry of the pathogen into the plant or part thereof, and/or after entry of the plant pathogen into the plant or part thereof, based on the one or more responses of the plants(s) or plant part(s) to the pathogen in the first and second methods.
• the method may comprise determining that the plant species or cultivar exhibits a resistance phenotype to the plant pathogen prior to and/or during entry of the pathogen into the plant or part thereof, but not after entry of the plant pathogen into the plant or part thereof, based on the one or more responses of the plants(s) or plant part(s) to the pathogen in the first and second methods.
• the method may comprise determining that the plant species or cultivar exhibits a resistance phenotype to the plant pathogen after entry of the plant pathogen into the plant or part thereof, but not prior to and/or during entry of the pathogen into the plant or part thereof, based on the one or more responses of the plants(s) or plant part(s) to the pathogen in the first and second methods.
• the method may comprise determining that the plant species or cultivar exhibits a resistance phenotype to the plant pathogen prior to and/or during entry of the pathogen into the plant or part thereof, as well as after entry of the plant pathogen into the plant or part thereof, based on the one or more responses of the plants(s) or plant part(s) to the pathogen in the first and second methods.
• the first screening method comprises contacting the surface of the first plant(s) or plant part(s) with the inoculum by spraying, swiping and/or swabbing the inoculum onto the surface of the plant(s) or plant part(s), and/or dipping or soaking the plant(s) or plant part(s) in the inoculum.
• the first screening method may comprise contacting the surface of the first plant(s) or plant part(s) with the inoculum by spraying the plant(s) or plant part(s) with the inoculum.
• the first screening method may comprise contacting the surface of the first plant(s) or plant part(s) with the inoculum by swiping and/or swabbing the surface of the plant(s) or plant part(s) with the inoculum.
• the first screening method may comprise contacting the surface of the first plant(s) or plant part(s) with the inoculum by dipping, immersing and/or soaking the plant(s) or plant part(s) in the inoculum. Any one or more of the fore-mentioned methods may be used for contacting the surface of the first plant(s) or plant part(s) with the inoculum.
• the second screening method comprises infiltrating the tissue of the second plant(s) or plant part(s) with the inoculum by syringe infiltration, syringe pressure infiltration and/or vacuum infiltration.
• the second screening method may comprise infiltrating the tissue of the second plant(s) or plant part(s) with the inoculum by syringe infiltration.
• the second screening method may comprise infiltrating the tissue of the second plant(s) or plant part(s) with the inoculum by syringe pressure infiltration.
• the second screening method may comprise infiltrating the tissue of the second plant(s) or plant part(s) with the inoculum by vacuum infiltration.
• the one or more responses of the plant(s) or plant part(s) to the pathogen may comprise one or more phenotypic responses to the pathogen.
• the method may comprise classifying the plant(s) or plant part(s) into response phenotypes comprising:
• a Class 1 response phenotype characterised by an absence of chlorosis, necrosis and pathogen fruiting structures on the plant(s) or plant part(s) following performance of the first screening method, and chlorosis, necrosis and/or formation of pathogen fruiting structures on the plant(s) or plant part(s) following performance of the second screening method;
• a Class 3 response phenotype characterised by chlorosis, necrosis and/or formation of pathogen fruiting structures on the plant(s) or plant part(s) following performance of the first screening method, and an absence of chlorosis, necrosis and pathogen fruiting bodies form on the plant(s) or plant part(s) following performance of the second screening method;
• a Class 4 response phenotype characterised by colonization of the plant(s) or plant part(s) by the pathogen, but infection is halted with minimal chlorosis, following performance of the first screening method and/or the second screening method;
• a plant or plant part which exhibits a Class 1 response phenotype elicits a defence response to the pathogen prior to or during entry of the pathogen into the plant tissue, but not post entry.
• a plant or plant part which exhibits a Class 2 response phenotype elicits a defence response to the pathogen prior to, during and post entry of the pathogen into the plant tissue.
• a plant or plant part which exhibits a Class 3 response phenotype elicits a defence response to the pathogen post entry of the pathogen into the plant tissue, but not prior to or during entry of the pathogen into the plant tissue.
• a plant or plant part which exhibits a Class 4 response phenotype does not elicit a defence response to the pathogen prior to or during entry of the pathogen into the plant tissue, but does elicit a defence response to the pathogen post entry of the pathogen into the plant tissue and colonisation of the plant by the pathogen still occurs.
• a plant or plant part which exhibits a Class 5 response phenotype does not elicit a defence response to the pathogen prior to, during or post entry of the pathogen into the plant tissue, but shows evidence of resistance to the pathogen represented by a reduced number of lesion and/or reduced lesion area compared to a plant or plant part in which there is no evidence of host resistance (e.g., compared to a plant or plant part which exhibits a Class 6 response phenotype).
• a plant or plant part which exhibits a Class 6 response phenotype does not elicit a defence response to the pathogen prior to, during or post entry of the pathogen into the plant tissue, and shows no evidence of resistance to the pathogen.
• the method may comprise selecting one or more plants or plant parts classified as being resistant to the plant pathogen based on a defence response to the pathogen prior to or during entry of the plant pathogen to the plant tissue.
• the method may comprise selecting one or more plants or plant parts classified as being resistant to the plant pathogen based on a defence response to the pathogen post entry of the plant pathogen to the plant tissue.
• the method may comprise selecting one or more plants or plant parts classified as being resistant to the plant pathogen based on a defence response to the pathogen prior to, during and post entry of the plant pathogen to the plant tissue.
• the one or more responses of the plant(s) or plant part(s) to the pathogen comprises a change of expression in one or more immune response genes.
• the method may further comprise determining an expression profile of one or more immune response genes in the plant(s) or plant part(s) prior to and following inoculation with the pathogen to determine a change of expression in one or more immune response genes.
• the plant pathogen may be a fungal pathogen or an oomycete pathogen.
• the plant pathogen is a fungal pathogen.
• the fungal pathogen may be selected from the group consisting of Magnaporthe oryz.ae, Botrytis cinerea, Puccinia spp., Blume ria graminis, Mycosphaerella graminicola (Zymoseptoria tritici), Colletotrichum spp., Melampsora lint, Phyrenophora tritici-repentis, Leptosphaeria maculans, Phakopsora pachyrhizi (Asian Soybean Rust), Phakopsora meibomiae (New world Soybean Rust), Puccinia striiformis f.
• the fungal pathogen may be Mycosphaerella graminicola (Zymoseptoria tritici).
• the fungal pathogen may be Phyrenophora tritici-repentis.
• the fungal pathogen may be Puccinia striiformis f. sp. tritici.
• the fungal pathogen may be Rhynchosporium commune.
• the fungal pathogen may be Septoria apiicola.
• the fungal pathogen may be Thekospora minima.
• the fungal pathogen may be Leptosphaeria maculans.
• the fungal pathogen may be Septoria lycopersici.
• plant pathogen is a oomycete pathogen.
• the oomycete pathogen may be selected from the group consisting of Phytophthora infestans, Hyaloperonospora arabidopsidis, Phytophthora ramorum, Phytophthora sojae, Phytophthora capsici, Plasmopara viticola, Phytophthora parasitica, and Albugo Candida.
• the plants or plant parts which are screened in the method of the disclosure may be of any species or variety.
• the plant may be a gymnosperm, a monocot or a dicot.
• the plant is selected from the group consisting of a fruiting plant, a leguminous plant, an oil plant, a vegetable plant, a cereal plant, a fibre plant, an ornamental plant, a forestry plant, an aquatic plant, a medicinal plant and a noxious plant or weed.
• the plant is a cereal plant.
• the cereal plant may be selected from the group consisting of wheat, durum, barley, oats, rye, triticale, rice, millet, sorghum, corn (maize), quinoa and buckwheat.
• the cereal plant is wheat.
• the plant is wheat and the plant pathogen is Mycosphaerella graminicola (Zymoseptoria tritici), Pyrenophora tritici-repentis or Puccinia striiformis f. sp. tritici.
• the plant is barley and the plant pathogen is Rhyncosporium commune.
• the plant is canola and the plant pathogen is Leptosphaeria maculans.
• the plant is blueberry and the plant pathogen is Thekospora minima.
• the plant is tomato and the plant pathogen is Septoria lycopersici.
• the plant is celery or celeriac and the plant pathogen is Septoria apiicola.
• the method of identifying a plant which exhibits a resistance phenotype or a susceptibility phenotype to a plant pathogen may further comprise identifying one or more molecular markers associated with resistance to the plant pathogen and/or identifying one or more molecular markers associated with susceptibility to the plant pathogen, comprising:
• a method of identifying one or more molecular markers associated with resistance of a plant to a plant pathogen and/or identifying one or more molecular markers associated with susceptibility of a plant to a plant pathogen comprising: (a) performing the screening method described herein to identify one or more plants which exhibit a resistance phenotype to a plant pathogen and one or more plants which exhibit a susceptibility phenotype to the plant pathogen;
• identifying one or more molecular markers associated with resistance to the plant pathogen and/or identifying one or more molecular markers associated with susceptibility to the plant pathogen comprising:
• the polynucleotide sequence data may be DNA sequence data and/or RNA sequence data.
• the polynucleotide sequence data is DNA sequence data.
• the polynucleotide sequence data is RNA sequence data.
• the one or more molecular markers identified are associated with a resistance phenotype prior to and/or during entry of the pathogen into the plant or part thereof.
• the one or more molecular markers identified may be associated with a resistance phenotype prior to entry of the pathogen into the plant or part thereof.
• the one or more molecular markers identified may be associated with a resistance phenotype during entry of the pathogen into the plant or part thereof.
• the one or more molecular markers identified are associated with a resistance phenotype after entry of the pathogen into the plant or part thereof. In one example, the one or more molecular markers identified are associated with a susceptibility phenotype prior to and/or during entry of the pathogen into the plant or part thereof. For example, the one or more molecular markers identified may be associated with a susceptibility phenotype prior to entry of the pathogen into the plant or part thereof. Alternatively, or in addition, the one or more molecular markers identified may be associated with a susceptibility phenotype during entry of the pathogen into the plant or part thereof.
• the one or more molecular markers identified are associated with a susceptibility phenotype after entry of the pathogen into the plant or part thereof.
• the one or more molecular markers may be selected from the group consisting of a single nucleotide polymorphism (SNP) marker, an amplified fragment length polymorphism (AFLP) marker, a DNA amplification fingerprinting (DAF) marker, a random amplified polymorphic DNA (RAPD) marker, micro satellite markers (e.g. simple sequence repeats (SSRs)), insertion mutation markers, sequence-characterized amplified region (SCAR) markers, cleaved amplified polymorphic sequence (CAPS) markers, kompetitive allele specific PCR (KASP) markers and any combinations thereof.
• SNP single nucleotide polymorphism
• AFLP amplified fragment length polymorphism
• DAF DNA amplification fingerprinting
• RAPD random amplified polymorphic DNA
• micro satellite markers e.g. simple sequence repeats (SSRs)
• SSRs sequence-characterized amplified region
• CAS cleaved amplified polymorphic sequence
• comparing the sequence data at (iii) comprises performing one or more sequence alignments to identify the one or more molecular markers.
• the method comprises generating the polynucleotide sequence for the plant of the species or cultivar by performing one or more assays selected from a nucleic acid isolation assay, a nucleic acid purification assay, a digestion assay, an assay for enrichment of molecular markers, a nucleic acid amplification assay, nucleic acid sequencing and combinations thereof.
• the present disclosure also provides a method of breeding a plant which is resistant to a plant pathogen, comprising crossing or selfing a plant identified as being resistant to the plant pathogen using the method described herein.
• the method may comprise one or more selection steps to select for progeny which exhibit resistance to the plant pathogen.
• the method of breeding a plant which is resistant to a plant pathogen comprises selecting the plant to be crossed or selfed based on the presence of one or more molecular markers associated with resistance to the plant pathogen and/or the absence of one or more molecular markers associated with susceptibility to the plant pathogen, wherein the one or more molecular markers are identified using the method described herein.
• the plant to be crossed or selfed is selected based on the presence of one or more molecular markers associated with resistance to the plant pathogen.
• the plant to be crossed or selfed is selected based on the absence of one or more molecular markers associated with susceptibility to the plant pathogen.
• the present disclosure also provides a method of breeding a plant which is resistant to a plant pathogen, comprising:
• the method of breeding the plant comprising performing the method of identifying a plant which exhibits a resistance phenotype to the plant pathogen as described herein.
• the plants which are bred in the method of the disclosure may be of any species or variety.
• the plant may be a gymnosperm, a monocot or a dicot.
• the plant which is bred is selected from the group consisting of a fruiting plant, a leguminous plant, an oil plant, a vegetable plant, a cereal plant, a fibre plant, an ornamental plant, a forestry plant, an aquatic plant and a noxious plant or weed.
• the plant which is bred is a cereal plant.
• the cereal plant may be selected from the group consisting of wheat, durum, barley, oats, rye, triticale, rice, millet, sorghum, corn (maize), quinoa and buckwheat.
• the cereal plant is wheat.
• the present disclosure also provides a plant or plant part which has been bred by performing the method of breeding a plant as described herein.
• the plant part is a plant propagation material.
• the plant propagation material may be a seed, a plant cutting, callus or scion.
• Figure 1 is a schematic illustrating the Defend system, whereby two inoculation techniques (topical spray and infiltration) are used to identify plant genotypes with different defence trigger mechanisms.
• the upper panel represents the infiltration method, whereby spores are forced into the leaf.
• the lower panel represent a traditional topical spray in which spores are applied to the leaf surface.
• R resistant phenotype
• I intermediate phenotype
• S susceptible phenotype.
• Figure 2 shows comparative results obtained using the first screening method (i.e., spray method) and the second screening method (i.e., infiltration method) for three varieties of wheat, ‘Lorikeet’ , ‘Currawong’ and ‘Summit’. On the left of each paired leaves, it shows the result from the spray method, on the right of each pair, it shows the result from the infiltration method.
• first screening method i.e., spray method
• infiltration method i.e., infiltration method
• Figure 3 shows asexual fruiting body of called pycnidium (pl. pycnidia) which appear as tiny black spots on the surface of infected leaves.
• Figure 4 illustrates the difference in phenotype following performance of the spray method (left hand side) and the infiltration method (right hand side) for the celery varieties ‘Jive’ (A), ‘Stringless Organic’ (B), ‘Tall Utah’ (C), ‘Amsterdam’ (D), ‘Peppermint Stick’ (E), ‘Balena’ (F), ‘Giant Pascal’ (G) and ‘Laboni’ (H) with S. apiicola spores.
• Figure 5 illustrates the difference in phenotype following performance of the spray and infiltration methods for wheat cultivars ‘Lancer’, ‘McVey’ and ‘Tammarian Rock’ with Psi spores.
• Figure 6 illustrates the exemplary ‘Defend’ classes identified in tomato varieties following challenge with S. lycopersici.
• Figure 7 illustrates the difference in phenotype following performance of the spray and infiltration methods for Blueberry cultivars ‘Peach Sorbent’ and ‘Pink Icing’ with T. minima spores. Red circles indicate the sites where the infiltration was conducted.
• the Defend Class was categorized according to Table 3.
• Figure 8 illustrates the difference in phenotype following performance of the infiltration and wound methods for canola varieties ‘Westar’, ‘Egra’ and ‘ATR-Beacon’ with L. maculans.
• Figure 9 illustrates the difference in phenotype following performance of the spray method (left hand side) and the infiltration method (right hand side) for the barley varieties ‘Briar’ (A), ‘Atlas 46’ (B), ‘Franklin’ (C), and ‘ICARD A4’ (D), with R. ses spores.
• Figure 10 illustrates the difference in phenotype following performance of the spray and infiltration methods for wheat cultivars ‘ISIS’, ‘Oasis’ and ‘Spear’ with the isolate YES WAI240 of P. tritici-repentis.
• the Defend Class is categorized according to Table 3.
• the recombinant DNA, recombinant protein, cell culture, and immunological techniques utilized in the present disclosure are standard procedures, well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989), T.A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D.M. Glover and B.D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F.M.
• the term about refers to +/- 10%, more preferably +/- 5%, more preferably +/- 1% of the designated value.
• the present disclosure relates to a method of identifying a plant which exhibits a resistance phenotype to a plant pathogen, comprising the steps of: (a) performing a first screening method comprising:
• step (iii) determining one or more responses of the plant(s) or plant part(s) to the pathogen at a plurality of time points during step (ii);
• step (ii) growing the plant(s) or plant part(s) for a time and under conditions suitable for the plant pathogen to infect the plant(s) or plant part(s) and (ill) determining one or more responses of the plant(s) or plant part(s) to the pathogen at a plurality of time points during step (ii);
• a "cultivar” or a “variety” refers to a group of similar plants that belong to the same species and that, by structural features and performance, may be distinguished from other varieties within the same species.
• Two essential characteristics of a variety are identity and reproducibility. Identity is necessary so that the variety may be recognized and distinguished from other varieties within the crop species. The distinguishing features may be morphological characteristics, molecular markers, colour markings, physiological functions, disease reaction, or performance. Most agricultural varieties are pure for the characteristic or for those characteristics that identify the variety; per se. Reproducibility is needed in order that the characteristic(s) by which the variety is identified will be reproduced in the progeny.
• the terms “cultivar” and “variety” are used interchangeably to refer to a group of plants within a species that share certain constant characters which separate them from the typical form and from other possible varieties within that species. While possessing at least the distinctive trait, a “variety” of the invention also may be characterized by a substantial amount of overall variation between individuals within the variety, based primarily on the Mendelian segregation of traits among the progeny of succeeding generations. On the other hand, “cultivar” or “variety” also can denote a clone, since some plant cultivars may individually be reproduced asexually, via stem cuttings, and all of the clones would be essentially identical genetically.
• One exemplary method for applying an inoculum to the surface of a plant is by spraying the inoculum using any conventional means for spraying liquids, such as spray nozzles or spinning-disk atomizers or hand held spray bottles.
• inoculum may be applied to the surface of a plant or plant part by swiping or swabbing the inoculum onto the surface of the plant or plant part, or dipping, soaking or otherwise immersing the plant or plant part in the inoculum.
• inoculum comprising a pathogen (e.g., pathogenic spores or mycelial fragments) is infiltrated into the plants tissue (e.g., leaf, stem or fruit), or into the intercellular air spaces of the plant tissue (e.g., leaf, stem of fruit) or the sub- stromal cavity, with the help of pressure using a syringe or other device known in the art (Chincinska 2021).
• a syringe or other device known in the art Choincinska 2021
• Several factors might influence the successful entry of spores or other inoculum (e.g., mycelial fragments) into the sub- stromal cavity and intracellular spaces without causing physical damage or eliciting a hypertensive response from the plant tissue.
• the second screening method comprise infiltrating the tissue of the second plant(s) or plant part(s) with the inoculum by syringe infiltration. In one example, the second screening method comprises infiltrating the tissue of the second plant(s) or plant part(s) with the inoculum by syringe pressure infiltration. In one example, the second screening method comprises infiltrating the tissue of the second plant(s) or plant part(s) with the inoculum by vacuum infiltration.
• the term "inoculum” refers to a preparation suitable for use in inoculation of plant tissue with a plant pathogen of interest.
• the inoculum may comprise spores derived from the plant pathogen.
• an “inoculum” may comprise any part of the pathogen that can initiate infection.
• an inoculum may comprise spores, sclerotia (i.e., a compact mass of mycelium), or fragments of mycelium.
• an inoculum refers to whole individuals of bacteria, mollicutes, protozoa, viruses, and viroids, respectively.
• An inoculum may comprise a single individual of a pathogen, e.g., one spore or one multicellular sclerotium, or may comprise a plurality of individual pathogens, such as millions of individuals of a pathogen, e.g., bacteria carried in a drop of water (Agrios GN (2005) Fifth Edition Plant Pathology, Fifth edn. Elsevier, London).
• the inoculum may be provided in any form which is convenient for the method of application.
• the inoculum is in the form of a liquid.
• the inoculum may comprise spores of the plant pathogen and a diluent, carrier and/or excipient. Suitable carriers include but not limited to beta glucan, sugar, Tween 20, gelatin or agar.
• the inoculum may comprise spores of the plant pathogen in a suitable storage and/or growth media.
• the inoculum may comprise spores of the plant pathogen provided in the form of a dry powder suspended in an oil.
• the inoculum may comprise spores of the plant pathogen suspended in an oil-in-water emulsion.
• the inoculum may comprise spores at a concentration of at least 10 2 spores/mL, or at a concentration of at least 10 3 spores/mL, or at a concentration of at least 10 4 spores/mL, or at a concentration of at least 10 5 spores/mL, or at a concentration of at least 10 6 spores/mL, or at a concentration of at least 10 7 spores/mL.
• the composition comprises spores at a concentration of at least IxlO 4 spores/mL.
• the composition comprises spores at a concentration of at least 1x10 s spores/mL. In one example, the composition comprises spores at a concentration of at least IxlO 6 spores/mL. In another example, the composition comprises spores at a concentration of IxlO 7 spores/mL or more.
• the term “response” shall be understood to mean any change in a plant or plant part following exposure to a stimuli, such as an environmental stimuli or a plant pathogen of the disclosure.
• the response of the plant or plant part may manifest as observable changes in one or more traits of the plant or plant part (e.g., a change in a plant phenotype) and/or may occur at a molecular or cellular level without resulting in any observable change in the plant appearance or plant phenotype.
• a “phenotypic response”, “response phenotype” or similar shall therefore be understood to mean one or more changes in the phenotype of the plant or plant part in response to the plant pathogen.
• phenotypic response of a plant or plant part to a pathogen will depend on a range of factors, including the type of plant pathogen, the species of plant and its stage of development, the part of the plant affected by the plant pathogen, susceptibility or resistance of the plant to the plant pathogen, and any plant defences to the pathogen.
• some exemplary phenotypic responses of plants to plant pathogens may include, but are not limited to, no change in phenotype (resistance to pathogen), chlorosis, necrosis of plant tissue, leaf curl, leave wrinkling or a change in rate of growth of the plant or plant part (e.g., stunting), fungal fruiting structures forming (e.g., acervulus, apothecium, ascocarp, ascoma, basidiocarp, coremium, pycnidium, perithecium, sporcarp, spordochium, etc.), hypersensitive response, melanism or programmed cell death.
• phenotype resistance to pathogen
• chlorosis e.g., chlorosis, necrosis of plant tissue, leaf curl, leave wrinkling or a change in rate of growth of the plant or plant part (e.g., stunting), fungal fruiting structures forming (e.g., acervulus, apo
• the response phenotype of the plant or plant part may be indicative of a plant’s susceptibility to the plant pathogen or the plant’s ability to resist the pathogen at different stages of the pathogen’s lifecycle or stage of infection.
• the response phenotype may also be a reflection of a plant’s inherent defences against the particular plant pathogen.
• plants have developed a wide variety of constitutive and inducible defences in order to protect themselves from damage (including from pathogens).
• Constitutive (continuous or structural) defences include many preformed barriers such as cell walls, waxy epidermal cuticles, and bark. These substances not only protect the plant from invasion, they also give the plant strength and rigidity.
• PAMPs/MAMPs pathogen/microbe-associated molecular patterns
• PAMPs/MAMPs pathogen/microbe-associated molecular patterns
• PAMP-triggered immunity a series of defence responses is induced, collectively known as P AMP-triggered immunity (PTI).
• PTI P AMP-triggered immunity
• specific pathogens have acquired the ability to suppress PTI in plants.
• These pathogens secrete various PTI- interfering effectors in the host plants.
• R (resistance) proteins effector-triggered immunity (ETI) is induced, which involves stronger and longer- lasting responses than PTI.
• ETI effector-triggered immunity
• the method may comprise classifying the plant(s) or plant part(s) based on their phenotypic response to the plant pathogen following inoculation using the first and second screening methods. These classifications (Classes 1-6) are described below.
• Class 1 response phenotype A plant or plant part which exhibits a Class 1 response phenotype elicits a defence response to the pathogen prior to or during entry of the pathogen into the plant tissue, but not post entry.
• a Class 1 response phenotype may be characterised by an absence of chlorosis, necrosis and pathogen fruiting structures on the plant(s) or plant part(s) following performance of the first screening method, and chlorosis, necrosis and/or formation of pathogen fruiting structures on the plant(s) or plant part(s) following performance of the second screening method.
• Class 2 response phenotype A plant or plant part which exhibits a Class 2 response phenotype elicits a defence response to the pathogen prior to, during and post entry of the pathogen into the plant tissue.
• a Class 2 response phenotype may be characterised by an absence of chlorosis, necrosis and pathogen fruiting structures on the plant(s) or plant part(s) following performance of the first screening method and the second screening method.
• Class 3 response phenotype A plant or plant part which exhibits a Class 3 response phenotype elicits a defence response to the pathogen post entry of the pathogen into the plant tissue, but not prior to or during entry of the pathogen into the plant tissue.
• a Class 3 response phenotype may be characterised by chlorosis, necrosis and/or formation of pathogen fruiting structures on the plant(s) or plant part(s) following performance of the first screening method, and an absence of chlorosis, necrosis and pathogen fruiting bodies form on the plant(s) or plant part(s) following performance of the second screening method.
• Class 4 response phenotype A plant or plant part which exhibits a Class 4 response phenotype does not elicit a defence response to the pathogen prior to or during entry of the pathogen into the plant tissue, but does elicit a defence response to the pathogen post entry of the pathogen into the plant tissue and colonisation of the plant by the pathogen still occurs.
• a Class 4 response phenotype may be characterised by colonization of the plant(s) or plant part(s) by the pathogen, but infection is halted with minimal chlorosis, following performance of the first screening method and/or the second screening method.
• Class 5 response phenotype A plant or plant part which exhibits a Class 5 response phenotype does not elicit a defence response to the pathogen prior to, during or post entry of the pathogen into the plant tissue, but shows evidence of resistance to the pathogen represented by a reduced number of lesion and/or reduced lesion area compared to a plant or plant part in which there is no evidence of host resistance (e.g., compared to a plant or plant part which exhibits a Class 6 response phenotype).
• a Class 5 response phenotype may be characterised by chlorosis, necrosis and formation of pathogenic fruiting structures with evidence of host resistance, wherein host resistance is characterised by a reduction in number of lesion and/or lesion area compared to a plant or plant part in which there is no evidence of host resistance
• Class 6 response phenotype A plant or plant part which exhibits a Class 6 response phenotype does not elicit a defence response to the pathogen prior to, during or post entry of the pathogen into the plant tissue, and shows no evidence of resistance to the pathogen.
• a Class 6 response phenotype may be characterised by chlorosis, necrosis and formation of pathogen fruiting structures with no evidence of host resistance.
• Plants screened using the phenotyping method of the disclosure may categorised into one of Classes 1-6 based on the ability (or inability) of the plant to trigger a defence response to a pathogen at different points along the infection pathway, as determined by the plant’s response phenotype at different points along the infection pathway. Based on these classifications, it may be possible to identify varieties and/or specific plants capable of mounting a defence response to the plant pathogen at a desired time point within the infection lifecycle. Alternatively, it may be possible to identify varieties and/or specific plants which mount a poor defence response or no defence response to the plant pathogen at one or more desired time points within the infection lifecycle if that were desirable.
• a plant may be categorised as exhibiting a resistance phenotype at one or more time points along the infection pathway.
• resistance phenotype refers to a phenotype which is indicative of a level of resistance of a plant to the plant pathogen.
• the resistance phenotype may be characterised in terms of morphological features of the plant or plant part and/or in term of functional characteristics.
• a resistance phenotype may be one in which there are few or no disease symptoms (e.g., chlorosis or necrosis) exhibited by the plant following exposure to a pathogen.
• a resistance phenotype will differ according to the plant species and plant pathogen, for example.
• resistance refers to the ability of a plant to mount a defence response to the plant pathogen prior to, during or post entry of the pathogen into the plant tissue, such that the plant develops no disease symptoms or few disease symptoms following exposure of the plant to the plant pathogen.
• resistance may be determined relative to a plant that is susceptible to the pathogen. For example, a plant that exhibits resistance may exhibit fewer disease symptoms than a plant that is susceptible to the pathogen and which does not exhibit disease resistance. “Resistance” includes complete resistance to the disease and also varying degrees of resistance manifested as decreased or weakening disease symptoms, longer survival, recovery or weakening of other disease parameters.
• the term “susceptible”, “susceptible phenotype” or similar as used herein, shall be understood to mean a plant that does not elicit a defence response to the pathogen prior to, during or post entry of the pathogen into the plant tissue, and shows no evidence of resistance to the pathogen.
• a plant which is determined to be susceptible to a plant pathogen following performance of the method of the disclosure will exhibit a Class 6 response phenotype.
• One or more plants exhibiting a desired response phenotype may then be selected for downstream breeding activities and/or genetic characterisation based on the response phenotype classification attributed to the plant.
• the method may comprise selecting a plant or part thereof which is capable of eliciting a defence response to the pathogen prior to or during entry of the pathogen into the plant tissue (e.g., a plant exhibiting a Class 1 or Class 2 response phenotype).
• the method may comprise selecting a plant or part thereof which is capable of eliciting a defence response to the pathogen post entry of the pathogen into the plant tissue (e.g., a plant exhibiting a Class 2, Class 3 or Class 4 response phenotype).
• the method may comprise selecting a plant or part thereof which is capable of eliciting a defence response to the pathogen prior to, during and post entry of the pathogen into the plant tissue (e.g., a plant exhibiting a Class 2 response phenotype).
• the method may comprise selecting a plant or part thereof which does not elicit a defence response to the pathogen prior to, during or post entry of the pathogen into the plant tissue but which still shows evidence of resistance to the pathogen (e.g., a plant exhibiting a Class 5 response phenotype).
• the method may comprise selecting a plant or part thereof which is susceptible to the plant pathogen (e.g., a plant exhibiting a Class 6 response phenotype). Genetic characterisation studies may be performed to identify genes and/or gene variants involved in the plant-pathogen interaction pathway which confer the response phenotype of interest.
• the one or more responses of the plant(s) or plant part(s) to the pathogen may comprise a change of expression in one or more immune response genes.
• the method may further comprise determining an expression profile of one or more immune response genes in the plant(s) or plant part(s) prior to and following inoculation with the pathogen to determine a change of expression in one or more immune response genes.
• an “immune response gene” (also referred to as a “resistance gene”) shall be understood to mean a nucleic acid encoding a protein that is directly or indirectly involved in the detection of the pathogen or in the induction of a signal transduction pathway leading to a plant defence response against the plant pathogen following contact of the plant with that particular pathogen.
• immune response genes include genes involved in P AMP-triggered immunity (PTI) and effector-triggered immunity (ETI).
• the first and second screening methods comprise determining one or more responses of the plant(s) or plant part(s) to the plant pathogen at a plurality of time points, including pre-inoculation and at least 5 days (e.g., at least 5, or at least 10, or at least 15, or at least 20, or at least 25, or at least 30 days, or at least 35 days, or at least 40 days, or at least 45 days, or at least 50 days, or at least 55 days, or at least 60 days or more) post inoculation.
• 5 days e.g., at least 5, or at least 10, or at least 15, or at least 20, or at least 25, or at least 30 days, or at least 35 days, or at least 40 days, or at least 45 days, or at least 50 days, or at least 55 days, or at least 60 days or more
• the methods comprise monitoring the plants and determining one or more responses of the plant(s) or plant part(s) to the plant pathogen for at least about day 5 days post inoculation. In some examples, the methods comprise monitoring the plants and determining one or more responses of the plant(s) or plant part(s) to the plant pathogen for at least about day 10 days post inoculation. In some examples, the methods comprise monitoring the plants and determining one or more responses of the plant(s) or plant part(s) to the plant pathogen for at least about day 15 days post inoculation. In some examples, the methods comprise monitoring the plants and determining one or more responses of the plant(s) or plant part(s) to the plant pathogen for at least about day 20 days post inoculation.
• the methods comprise monitoring the plants and determining one or more responses of the plant(s) or plant part(s) to the plant pathogen for at least about day 25 days post inoculation. In some examples, the methods comprise monitoring the plants and determining one or more responses of the plant(s) or plant part(s) to the plant pathogen for at least about day 30 days post inoculation. In some examples, the methods comprise monitoring the plants and determining one or more responses of the plant(s) or plant part(s) to the plant pathogen for at least about day 35 days post inoculation. In some examples, the methods comprise monitoring the plants and determining one or more responses of the plant(s) or plant part(s) to the plant pathogen for at least about day 40 days post inoculation.
• the methods comprise monitoring the plants and determining one or more responses of the plant(s) or plant part(s) to the plant pathogen for at least about day 45 days post inoculation. In some examples, the methods comprise monitoring the plants and determining one or more responses of the plant(s) or plant part(s) to the plant pathogen for at least about day 50 days post inoculation. In some examples, the methods comprise monitoring the plants and determining one or more responses of the plant(s) or plant part(s) to the plant pathogen for at least about day 55 days post inoculation. In some examples, the methods comprise monitoring the plants and determining one or more responses of the plant(s) or plant part(s) to the plant pathogen for at least about day 60 days post inoculation. In some examples, the methods comprise monitoring the plants and determining one or more responses of the plant(s) or plant part(s) to the plant pathogen for more than 60 days post inoculation.
• a “pathogen” of plant refers to an organism which can infect a plant, or which can cause a disease in a plant.
• Pathogens which can infect a plant, or which can cause a disease in a plant include but not limited to fungi, oomycetes, bacteria, viruses, viroids, virus-like organisms, phytoplasmas, protozoa, and nematodes.
• a “plant pathogen” shall be understood to be a fungal pathogen or fungus-like pathogen (such as, for example, Chromista) belonging to the group comprising Plasmodiophoramycota, Oomycota, Ascomycota, Chytridiomycetes, Zygomycetes, Basidiomycota and Deuteromycetes (Fungi imperfecti).
• Plant pathogens include, by way of example, but not by limitation, those detailed in Tables 1 and 2, and the diseases which are associated with them.
• Rust (tropical corn) Physopella pallescen Angiopsora zeae
• Anthracnose leaf blight Colletotrichum graminicola (teleomorph: Glomerella graminicola Politis);
• Curvularia leaf spot Curvularia clavata, C. eragrostidis, C. maculans (teleomorph: Cochliobolus eragrostidis), Curvularia inaequalis, C. intermedia (teleomorph: Cochliobolus intermedins), Curvularia lunata (teleomorph: Cochliobolus lunatus), Curvularia pallescens (teleomorph: Cochliobolus pallescens), Curvularia senegalensis, C. tuberculata (teleomorph:
• Green ear downy mildew (graminicola Sclerospora graminicola downy mildew)
• Exserohilum prolatum Drechslera prolata (teleomorph: Setosphaeria prolata) Graphium penicillioides, Leptosphaeria maydis, Leptothyrium zeae, Ophiosphaerella herpotricha, (anamorph: Scolecosporiella sp. ),Paraphaeosphaeria michotii, Phoma sp., Septoria zeae, S. zeicola, S. zeina
• Phaeosphaeria leaf spot Phaeosphaeria maydis Sphaerulina maydis
• Ro stratum leaf spot Helmin- thosporium Setosphaeria rostrata, (anamorph: leaf disease, ear and stalk rot)
• xserohilum rostratum Helminthosporium rostratum
• Java downy mildew Perono sclerospora maydis Sclerospora maydis
• the plant pathogen may belong to the group Plasmodiophoromycota, such as, for example, Plasmodiophora brassicae (clubroot of crucifers), Spongospora subterranea, or Polymyxa graminis.
• Plasmodiophoromycota such as, for example, Plasmodiophora brassicae (clubroot of crucifers), Spongospora subterranea, or Polymyxa graminis.
• the plant pathogen may belong to the group Oomycota, such as, for example, Bremia lactucae (downy mildew of lettuce), Peronospora (downy mildew) in snapdragon (P. anlirrhini). onion (P. destructor), spinach (P. effusa), soybean (P. manchurica), tobacco ("blue mold”; P. tabacina) alfalfa and clover (P. trifolium), Pseudoperonospora humuli (downy mildew of hops), Plasmopara (downy mildew in grapevines) (P. viticola) and sunflower (P.
• the plant pathogen may belong to the group Ascomycota, such as, for example, Microdochium nivale (snow mold of rye and wheat), Fusarium, Fusarium graminearum, Fusarium culmorum (partial ear sterility mainly in wheat), Fusarium oxysporum (Fusarium wilt of tomato), Blumeria graminis (powdery mildew of barley (f.sp. hordei) and wheat (f.sp.
• Ascomycota such as, for example, Microdochium nivale (snow mold of rye and wheat), Fusarium, Fusarium graminearum, Fusarium culmorum (partial ear sterility mainly in wheat), Fusarium oxysporum (Fusarium wilt of tomato), Blumeria graminis (powdery mildew of barley (f.sp. hordei) and wheat (f
• the plant pathogen may belong to the group Basidiomycetes, such as, for example, Typhula incarnata (typhula blight on barley, rye, wheat), Ustilago maydis (blister smut on maize), Ustilago nuda (loose smut on barley), Ustilago tritici (loose smut on wheat, spelt), Ustilago avenae (loose smut on oats), Rhizoctonia solani (rhizoctonia root rot of potato), Sphacelotheca spp.
• Basidiomycetes such as, for example, Typhula incarnata (typhula blight on barley, rye, wheat), Ustilago maydis (blister smut on maize), Ustilago nuda (loose smut on barley), Ustilago tritici (loose smut on wheat, spelt), Ustilago aven
• the plant pathogen may belong to the group Deuteromycetes (Fungi imperfecti), such as, for example, Septoria (Stagonospora) nodorum (glume blotch) of wheat (Septoria tritici), Pseudocercosporella herpotrichoides (eyespot of wheat, barley, rye), Rynchosporium secalis (leaf spot on rye and barley), Alternaria solani (early blight of potato, tomato), Phoma betae (blackleg on Beta beet), Cercospora beticola (leaf spot on Beta beet), Alternaria brassicae (black spot on oilseed rape, cabbage and other crucifers), Verticillium dahliae (verticillium wilt), Colletotrichum, Colletotrichum lindemuthianum (bean anthracnose), Phoma lingam (blackleg of cabbage and oilseed rape), or Botrytis
• the plant pathogen is a fungal pathogen.
• the fungal pathogen may be selected from the group consisting of Magnaporthe oryzae, Botrytis cinerea, Puccinia spp., Blumeria graminis, Mycosphaerella graminicola (Zymoseptoria tritici), Colletotrichum spp., Melampsora lini, Phakopsora pachyrhizi, Phyrenophora tritici-repentis, Leptosphaeria maculans, Phakopsora pachyrhizi, Phakopsora meibomiae, Puccinia striiformis f. sp.
• the fungal pathogen may be Mycosphaerella graminicola. (Zymoseptoria tritici).
• the fungal pathogen may be Phyrenophora tritici-repentis.
• the fungal pathogen may be Puccinia striiformis f.
• the fungal pathogen may be Rhynchosporium commune.
• the fungal pathogen may be Septoria apiicola.
• the fungal pathogen may be Thekospora minima.
• the fungal pathogen may be Leptosphaeria maculans.
• the fungal pathogen may be Septoria lycopersici.
• plant pathogen is a oomycete pathogen.
• the oomycete pathogen may be selected from the group consisting of Phytophthora inf estans, Hyaloperonospora arabidopsidis, Phytophthora ramorum, Phytophthora sojae, Phytophthora capsici, Plasmopara viticola, Phytophthora parasitica, and Albugo Candida.
• the term “plant” refers to any living organism belonging to the kingdom Plantae (i.e., any genus/ species in the Plant Kingdom). This includes familiar organisms such as but not limited to trees, herbs, bushes, grasses, veins, ferns, mosses and green algae. The term refers to both monocotyledonous plants, also called monocots, and dicotyledonous plants, also called dicots.
• the plant may be a fruiting plant, a leguminous plant, an oil plant, a vegetable plant, a cereal plant, a fibre plant, an ornamental plant, a forestry plant, an aquatic plant, a medicinal plant and a noxious plant or weed. In this regard, the choice of plant may vary depending on the plant pathogen of interest, geographic location and season.
• the plant may be a cereal plant selected from the group consisting of wheat, durum, barley, oats, rye, triticale, rice, millet, sorghum, corn (maize), quinoa and buckwheat.
• plants include but are not limited to potatoes, roses, apple trees, sunflowers, bananas, tomatoes, opo, pumpkins, squash, lettuce, cabbage, oak trees, guzmania, geraniums, hibiscus, clematis, poinsettias, sugarcane, taro, duck weed, pine trees, Kentucky blue grass, zoysia, coconut trees, brassica leafy vegetables (e.g., broccoli, broccoli raab, Brussels sprouts, cabbage, Chinese cabbage (Bok Choy and Napa), cauliflower, cavalo, collards, kale, kohlrabi, mustard greens, rape greens, and other brassica leafy vegetable crops), bulb vegetables (e.g., garlic, leek, onion (dry bulb, green, and Welsh), shallot, and other bulb vegetable crops), citrus fruits (e.g., grapefruit, lemon, lime, orange, tangerine, citrus hybrids, pummelo, and other citrus fruit crops), cucurbit vegetables (e
• the crop plant is wheat.
• the crop plant is barley.
• the crop plant is canola.
• the crop plant is blueberry.
• the crop plant is tomato.
• the crop plant is celery or celeriac.
• plant in a broad sense refers to differentiated plants as well as undifferentiated plant material, such as protoplasts, plant cells, seeds, plantlets, etc., that under appropriate conditions can develop into mature plants, the progeny thereof, and parts thereof. “Progeny” comprises any subsequent generation of a plant, plant cell, plant tissue, or plant organ.
• Ornamental and horticulture plants include, for example, flowering plants, shrubs, grasses and trees.
• Examples of Ornamental plants include, but not limited to, Frangipani Tree, Gardenia, Bougainvillea, Hibiscus Tree, Blue Water Lily, Ylang Ylang, Madagascar Rosy Periwinkle, Orchid, Croton, Spanish Jasmine, Red Ginger, Firecracker, Indian Coleus, Porcelain Rose, Geranium, Petunia, Begonia, Pelargonium, Fuchsia etc.
• any ornamental or horticultural plants may be screened using the method of the disclosure.
• Forestry plants are ones that are used for conserving and repairing forests, woodlands.
• Non-limiting examples include gum tree, oak, Acacia, Callitris, Casuarina, Eucalyptus, Pinus, Mangrove, Melaleuca etc.
• Aquatic plants include those that, under normal conditions, germinate and grow with at least their bases in the water and are large enough to be seen with the naked eye.
• Non-limiting examples include parrot feather, creeping primrose, water mint, water hyacinth, duckweed, hydrilla etc.
• Medicinal plants include those plants that possess therapeutic properties or exert beneficial pharmacological effect on the human or animal body.
• Medicinal plants typically comprise phytochemicals and other active compounds which have the potential to have pharmacological activity, including alkaloids, glycosides, polyphenols and terpenes, for example. Examples of medicinal plants will be known to a person skilled in the art.
• Noxious plants or weed can be monocotyledonous and dicotyledonous.
• Examples of weeds in crop fields include but not limited to Echinochloa, Sagittaria, Alisma, Eleocharis, Scirpus and Cyperus.
• plant part refers to any part of a plant including but not limited to the shoot, root, stem, seeds, stipules, leaves, petals, flowers, ovules, bracts, branches, petioles, internodes, bark, pubescence, tillers, rhizomes, fronds, blades, pollen, stamen, and the like.
• DNA-based molecular markers are useful for detecting the genetic variation available in germplasm collections and/or breeding lines. These markers have been used extensively for the development of saturated molecular genetic maps and physical maps and for the identification of genes or quantitative trait loci (QTLs) controlling traits of economic importance which are in turns can be used for marker- assisted selection.
• QTLs quantitative trait loci
• a number of new next-generation sequencing (NGS) technologies have been developed and subsequently deployed to generate DNA sequence data inexpensively and at a rate that is several orders of magnitude faster than that of traditional technologies.
• NGS next-generation sequencing
• genomics-assisted breeding approaches include marker assisted selection (MAS), genome selection (GS), and genome wide association studies (GW AS) (for review see, e.g. , Varshney et al. ,(2009) Trends Biotechnol. 27 (9):522-530).
• GW AS methods in particular have attracted significant interest in plant breeders. The GW AS method typically involves an examination of many common genetic variants in different individuals to see if any variant is associated with a trait.
• GW AS technology focuses on associations between single- nucleotide polymorphisms (SNPs) and specific traits such as increased yield or disease resistance. These studies typically compare the DNA sequence information of two groups of plants that either displays or lacks the target trait (i.e., controls).
• Each plant gives a sample of DNA, from which millions of genetic variants are read using SNP arrays. If one type of the variant (one allele) is more frequent in plants with the disease resistance, the SNP is said to be "associated" with the disease resistance. The associated SNPs are then considered to mark a region of the genome which influences the risk of disease, for example. In contrast to methods which specifically test one or a few genetic regions, the GW AS studies investigate the entire genome. This approach is therefore considered to be non-candidate-driven in contrast to gene- specific candidate-driven studies.
• GW AS identifies SNPs and other variants in DNA (described herein) which may be associated with a trait (such as a resistance or susceptibility to a plant pathogen), but which may not on their own specify which genes or genetic elements are causal.
• the present disclosure contemplates the use of such methods to identify molecular markers associated with resistance or susceptibility to plant pathogens present in plants identified as being resistant or susceptible to plant pathogens using the phenotypic screening method described herein. These molecular markers can then be used as tools for selection and breeding of plant varieties which are resistant or susceptible to plant pathogens, as desired.
• the present disclosure also provides a method of identifying one or more molecular markers associated with resistance of a plant to a plant pathogen and/or identifying one or more molecular markers associated with susceptibility of a plant to a plant pathogen, comprising:
• identifying one or more molecular markers associated with resistance to the plant pathogen and/or identifying one or more molecular markers associated with susceptibility to the plant pathogen comprising:
• the polynucleotide sequence data may be DNA sequence data and/or RNA sequence data.
• the polynucleotide sequence data is DNA sequence data.
• the polynucleotide sequence data is RNA sequence data.
• molecular marker and “genetic marker” are used interchangeably herein and refer to a region of a nucleotide sequence (e.g. , in a chromosome) that is subject to variability (i.e. , the region can be polymorphic for a variety of alleles). Genetic markers are typically used in methods for visualizing differences in characteristics of nucleic acid sequences.
• RFLP restriction fragment length polymorphism
• AFLP amplified fragment length polymorphism
• SNPs single nucleotide polymorphisms
• INDEE insertion/deletion
• SSRs or micro satellite sequence-characterized amplified regions
• SCARs sequence-characterized amplified regions
• RFLPs restriction fragment length polymorphisms
• RAPDs randomly amplified polymorphic DNAs
• isozyme markers arbitrarily primed polymerase chain reaction (AP-PCR), DNA amplification fingerprinting (DAF), , kompetitive allele specific PCR (KASP) markers or combinations of the markers described herein, which define a specific genetic and chromosomal region or chromosomal location.
• AP-PCR arbitrarily primed polymerase chain reaction
• DAF DNA amplification fingerprinting
• KASP kompetitive allele specific PCR
• Genetic markers can, for example, be used to locate genetic loci containing alleles on a chromosome that contribute to variability of phenotypic traits, such as resistance or susceptibility to a plant pathogen.
• the phrase "genetic marker” can also refer to a polynucleotide sequence complementary to a genomic sequence, such as a sequence of a nucleic acid used as probes.
• a genetic or molecular marker can be physically located in a position on a chromosome that is distal or proximal to the genetic loci with which it is linked (i.e. is intragenic or extragenic, respectively).
• Genetic or molecular markers can be used during the breeding process for the selection of qualitative and/or quantitative traits. For example, markers closely linked to alleles, or markers containing sequences within the actual alleles of interest, can be used to select plants that contain the alleles of interest during a backcrossing breeding program. The markers can also be used to select for the genome of the recurrent parent and against the genome of the donor parent. Using this procedure can minimize the amount of genome from the donor parent that remains in the selected plants. It can also be used to reduce the number of crosses back to the recurrent parent needed in a backcrossing program. The use of genetic markers in the selection process is often called genetic marker enhanced selection or marker-assisted selection (MAS). Genetic molecular markers may also be used to identify and exclude certain sources of germplasm as parental varieties or ancestors of a plant by providing a means of tracking genetic profiles through crosses.
• MAS genetic marker enhanced selection
• Molecular tools e.g., oligonucleotide primers and/or detection probes
• molecular assays may be designed to determine the genotype of a plant at the one or more molecular markers associated with resistance or susceptibility of a plant to a plant pathogen.
• genotype refers to the genetic constitution of a cell or organism.
• An individual's "genotype” for one or more molecular markers includes the specific alleles for the one or more molecular markers present in the individual's haplotype.
• a genotype can relate to a single locus (e.g., a single molecular marker) or to multiple loci (e.g., multiple molecular marker), whether the loci are related or unrelated and/or are linked or unlinked.
• a plant’s genotype relates to one or more molecular markers within or associated with genes that are involved in the expression of the phenotype of interest (e.g. a resistance or susceptibility to a plant pathogen).
• the one or more molecular markers associated with the resistance or susceptibility phenotype may comprise between one and ten markers. In some examples, the one or more molecular markers associated, with the resistance or susceptibility phenotype comprise more than ten genetic markers. In one example, the one or more molecular markers identified are associated with a resistance phenotype prior to and/or during entry of the pathogen into the plant or part thereof. For example, the one or more molecular markers identified may be associated with a resistance phenotype prior to entry of the pathogen into the plant or part thereof. Alternatively, or in addition, the one or more molecular markers identified may be associated with a resistance phenotype during entry of the pathogen into the plant or part thereof.
• the one or more molecular markers identified are associated with a resistance phenotype after entry of the pathogen into the plant or part thereof.
• the one or more molecular markers identified are associated with a susceptibility phenotype prior to and/or during entry of the pathogen into the plant or part thereof.
• the one or more molecular markers identified may be associated with a susceptibility phenotype prior to entry of the pathogen into the plant or part thereof.
• the one or more molecular markers identified may be associated with a susceptibility phenotype during entry of the pathogen into the plant or part thereof.
• the one or more molecular markers identified are associated with a susceptibility phenotype after entry of the pathogen into the plant or part thereof.
• the one or more molecular markers may be selected from the group consisting of a single nucleotide polymorphism (SNP) marker, an amplified fragment length polymorphism (AFLP) marker, a DNA amplification fingerprinting (DAF) marker, a random amplified polymorphic DNA (RAPD) marker, micro satellite markers (e.g. simple sequence repeats (SSRs)), insertion mutation markers, sequence-characterized amplified region (SCAR) markers, cleaved amplified polymorphic sequence (CAPS) markers, kompetitive allele specific PCR (KASP) and any combinations thereof.
• SNP single nucleotide polymorphism
• AFLP amplified fragment length polymorphism
• DAF DNA amplification fingerprinting
• RAPD random amplified polymorphic DNA
• micro satellite markers e.g. simple sequence repeats (SSRs)
• SSRs sequence-characterized amplified region
• CAS cleaved amplified polymorphic sequence
• comparing the sequence data at (iii) comprises performing one or more sequence alignment steps to identify the one or more molecular markers or sequence variants.
• Methods of alignment of sequences for comparison are well-known in the ait. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman, (1981 ) Adv. Appl. Math. 2: 482; by the homology alignment algorithm of Needleman and Wunsch, (1970) J. Mol. Biol. 48: 443; by the search for similarity method of Pearson and Lipman, (1988) Proc. Natl. Acad. Set.
• the BLAST family of programs which can be used for database similarity searches includes: BLASTN for nucleotide query sequences against nucleotide database sequences: BLASTX for nucleotide query sequences against protein database sequences; BLASTP for protein query sequences against protein database sequences: TBLASTN for protein query sequences against nucleotide database sequences; and TBLASTX for nucleotide querysequences against nucleotide database sequences.
• HSPs high scoring sequence pairs
• the word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always ⁇ 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negativescoring residue alignments; or the end of either sequence is reached.
• the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
• W wordlength
• E expectation
• BLOSUM62 scoring matrix see Henikoff & Henikoff (1989) Proc. Nall. Acad. Sd. USA 89:10915).
• the BLAST algorithm In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences [see e.g., Karlin & Altschul, (1993) Proc. Nat. Acad. Sd. USA 90:5873-5877).
• One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
• One or more informative molecular bio markers may be determined between corresponding polynucleotide sequences obtained from resistant and susceptible plants in the alignment. The alignment can be made such that the sequences derived from the resistant and susceptible plants can be compared. Differences can then be identified which are informative for a resistant phenotype or a susceptible phenotype (e.g., polymorphisms). In some examples, sequence differences at one or more molecular markers are associate with differing phenotypes of Classes 1-6 identified in the method of the disclosure.
• Molecular selection tools may then be designed to discriminated between marker variants.
• detection probes may be designed for detection of marker variants in a sample, for example, by hybridization on DNA chips or a beads-based detection platform.
• Detection probes may be designed such that a polymorphism or variant is reflected therein.
• the detection probes typically contain the variant SNP alleles at the central position such as to maximize allele discrimination.
• SNPs single nucleotide polymorphisms
• the probes can be synthesized using any method known in the art.
• the probes are typically designed such that they are suitable for high throughput screening methods e.g., such as the Affymetrix platform using chip-based detection of SNPs or bead technology provided by Illumina.
• the method comprises generating one or more polynucleotides sequences for the plant of the species or cultivar by performing one or more assays selected from a nucleic acid isolation assay, a nucleic acid purification assay, a digestion assay, an assay for enrichment of molecular markers, a nucleic acid amplification assay, nucleic acid sequencing and combinations thereof.
• the present disclosure also provides a method of breeding a plant which is resistant or susceptible to a plant pathogen (as desired).
• the method comprises crossing or selfing a plant identified as being resistant or susceptible to the plant pathogen (as appropriate or desired) using the phenotyping method described herein to thereby produce one or more progeny plants which are resistant or susceptible to the plant pathogen.
• the method may comprise: (i) obtaining a plant identified as being resistant or susceptible to the plant pathogen (as desired) using the method described herein; and (ii) introducing one or more foreign nucleic acids to the plant and/or modifying or editing one or more polynucleotides within the plant’s genome, to produce a plant which is resistant or susceptible to the plant pathogen.
• breeding and grammatical variants thereof, refer to any process that generates a progeny individual. Breeding can be sexual or asexual, or any combination thereof. Exemplary non-limiting types of breeding include crossing, selfing, doubled haploid derivative generation, and combinations thereof.
• the method of breeding a plant as described herein may comprise performance of the method described herein for identifying the plant which exhibits the resistance or susceptibility to the plant pathogen (as desired), and then selfing, crossing, backcrossing or genetically modifying the plant identified as exhibiting the resistance or susceptibility to the plant pathogen (as desired).
• selfing refers to fusion of male and female gametes from a plant identified as being resistant or susceptible to the plant pathogen (as appropriate or desired) using the phenotyping method described herein.
• crossing refers to crossing a plant identified as being resistant or susceptible to the plant pathogen (as appropriate or desired) using the phenotyping method described herein with another plant of the same species and/or variety.
• backcross refers to a process in which a breeder crosses a progeny (e.g., a hybrid progeny) back to one of the parents, for example, a first generation hybrid Fl with one of the parental genotypes of the Fl hybrid.
• a backcross is performed repeatedly, with a progeny individual of one backcross being itself backcrossed to the same parental genotype.
• progeny or “progeny plants” or similar includes the descendants of a plant identified as being resistant or susceptible to the plant pathogen (as appropriate or desired) using the phenotyping method of the disclosure.
• the term “progeny” is intended to encompass “direct progeny” and “indirect progeny”.
• direct progeny refers to plants that derive from the seed (or, sometimes, other tissue) of a plant identified as being resistant or susceptible to the plant pathogen (as appropriate or desired) using the phenotyping method described herein and is in the immediately subsequent generation. For instance, for a given lineage, a T2 plant is the direct progeny of a T1 plant.
• the "indirect progeny” refers to plants that derive from the seed (or other tissue) of the direct plant progeny produced by the method of breeding a plant as described herein, or from the seed (or other tissue) of subsequent generations in that lineage; for instance, a T3 plant is the indirect progeny of a T1 plant.
• the method of breeding a plant which is resistant to a plant pathogen comprises selecting the plant to be crossed or selfed based on the presence of one or more molecular markers associated with resistance to the plant pathogen and/or the absence of one or more molecular markers associated with susceptibility to the plant pathogen, wherein the one or more molecular markers are identified using the method described herein.
• the plant to be crossed or selfed is selected based on the presence of one or more molecular markers associated with resistance to the plant pathogen.
• the plant to be crossed or selfed is selected based on the absence of one or more molecular markers associated with susceptibility to the plant pathogen.
• the method of breeding a plant which is resistant to a plant pathogen of interest comprises: (i) obtaining a plant which has been identified by the method of the disclosure as being capable of eliciting a defence response to the pathogen prior to or during entry of the pathogen into the plant tissue (e.g., a plant exhibiting a Class 1 or Class 2 response phenotype); and (ii) producing a plant which is resistant to the plant pathogen of interest by selfing, crossing or genetically modifying the plant obtained at (i).
• the method of breeding a plant which is resistant to a plant pathogen of interest comprises: (i) obtaining a plant which has been identified by the method of the disclosure as being capable of eliciting a defence response to the pathogen post entry of the pathogen into the plant tissue (e.g., a plant exhibiting a Class 2, Class 3 or Class 4 response phenotype); and (ii) producing a plant which is resistant to the plant pathogen of interest by selfing, crossing or genetically modifying the plant obtained at (i).
• a plant which has been identified by the method of the disclosure as being capable of eliciting a defence response to the pathogen post entry of the pathogen into the plant tissue e.g., a plant exhibiting a Class 2, Class 3 or Class 4 response phenotype
• producing a plant which is resistant to the plant pathogen of interest by selfing, crossing or genetically modifying the plant obtained at (i).
• the method of breeding a plant which is resistant to a plant pathogen of interest comprises: (i) obtaining a plant which has been identified by the method of the disclosure as being capable of eliciting a defence response to the pathogen prior to, during and post entry of the pathogen into the plant tissue (e.g., a plant exhibiting a Class 2 response phenotype); and (ii) producing a plant which is resistant to the plant pathogen of interest by selfing, crossing or genetically modifying the plant obtained at (i).
• the method of breeding a plant which is resistant to a plant pathogen of interest comprises: (i) obtaining a plant which has been identified by the method of the disclosure as not being capable of eliciting a defence response to the pathogen prior to, during or post entry of the pathogen into the plant tissue but which still shows evidence of resistance to the pathogen (e.g., a plant exhibiting a Class 5 response phenotype); and (ii) producing a plant which is resistant to the plant pathogen of interest by selfing, crossing or genetically modifying the plant obtained at (i).
• the method of breeding a plant which is susceptible to a plant pathogen of interest comprises: (i) obtaining a plant which has been identified by the method of the disclosure as being susceptible to the plant pathogen (e.g., a plant exhibiting a Class 6 response phenotype); and (ii) producing a plant which is resistant to the plant pathogen of interest by selfing, crossing or genetically modifying the plant obtained at (i).
• a plant which has been identified by the method of the disclosure as being susceptible to the plant pathogen e.g., a plant exhibiting a Class 6 response phenotype
• producing a plant which is resistant to the plant pathogen of interest by selfing, crossing or genetically modifying the plant obtained at (i).
• the plant obtained at (i) may be crossed with a plant which has been identified by the method of the disclosure as having a response phenotype to the pathogen which is different to the plant obtained at (i).
• the plant obtained at (i) may be crossed with a plant which has been identified by the method of the disclosure as having a response phenotype to the pathogen which is the same as that of the plant obtained at (i).
• the plant obtained at (i) may be crossed with a plant whose response phenotype to the pathogen is unknown.
• introducing means presenting the nucleic acid to plant tissue in such a manner that the nucleic acid gains access to the interior of a cell in the plant tissue.
• these nucleic acids can be assembled as part of a single nucleic acid construct, or as separate nucleic acid constructs, and can be located on the same or different transformation vectors. Accordingly, multiple nucleic acid can be introduced into the plant tissue in a single transformation event, in separate transformation events, or, e.g., as part of a breeding protocol.
• transformation refers to the introduction of a nucleic acid into plant tissue. Transformation of the cell may be stable or transient.
• Acceleration methods include, for example, microprojectile bombardment and the like.
• microprojectile bombardment in addition to it being an effective means of reproducibly transforming monocots, is that neither the isolation of protoplasts, nor the susceptibility of Agrobacterium infection are required.
• plastids can be stably transformed.
• Methods disclosed for plastid transformation in higher plants include particle gun delivery of DNA containing a selectable marker and targeting of the DNA to the plastid genome through homologous recombination (U.S. 5, 451,513, U.S. 5,545,818, U.S. 5,877,402, U.S. 5,932479, and WO 99/05265).
• AgroZzac/erzizm-mediated transfer is another widely applicable system for introducing nucleic acids into plant cells because the DNA can be introduced into whole plant tissues, thereby bypassing the need for regeneration of an intact plant from a protoplast.
• the use of AgroZzac/erzizm-mediated plant integrating vectors to introduce DNA into plant cells is well known in the art (see, for example, US 5,177,010, US 5,104,310, US 5,004,863, US 5,159,135). Further, the integration of the T-DNA is a relatively precise process resulting in few rearrangements.
• the region of DNA to be transferred is defined by the border sequences, and intervening DNA is usually inserted into the plant genome.
• the nucleic acid molecule introduced to the plant tissue is stably introduced.
• stably introducing or “stably introduced”, in the context of a nucleic acid introduced into plant tissue, is intended to mean that the introduced nucleic acid is stably incorporated into the genome of the plant, and thus the plant cells are stably transformed with the nucleic acid.
• the integrated nucleic acid is capable of being inherited by the progeny thereof, more particularly, by the progeny of multiple successive generations.
• the term “genome” includes the nuclear and the plastid genome, and therefore includes integration of a nucleic acid into, for example, the chloroplast genome.
• Stable transformation as used herein can also refer to a nucleic acid that is maintained extrachromasomally, for example, as a minichromosome.
• nucleic acid as used herein shall be understood to mean a polynucleotide such as, for example, DNA, RNA or oligonucleotides.
• the one or more nucleic acids introduced to the plant tissue may be present in one or more nucleic acid constructs, such as expression cassettes, capable of directing expression of a particular polynucleotide sequence in the nucleic acid in the plant, and will generally comprise a promoter operatively -linked to the polynucleotide sequence and/or one or more other regulatory elements required for proper expression and translation of a protein or polypeptide encoded thereby.
• the nucleic acid introduced into the plant tissue comprises at least one gene of interest.
• the gene of interest may increase or decrease the endogenous level of activity of a protein in the genetically modified plant, or may introduce a new protein to the plant.
• the gene of interest may encode a protein or functional polynucleotide which; increases yield, confers enhanced animal and/or human nutrition, confers herbicide tolerance (e.g., glyphosate resistance or glufosinate resistance), affects carbohydrate biosynthesis or modification (e.g., starch branching enzyme, starch debranching enzyme, starch synthases, ADP- glucose pyrophosphorylase), is involved in fatty acid biosynthesis or modification (e.g., a desaturase, elongase, hydroxylase, epoxidase, conjugase, acetylase, TAG assembly), confers insect resistance (e.g.
• crystal toxin protein of Bacillus thuringiensis' confers viral resistance (e.g. viral coat protein); confers fungal resistance (e.g., chitinase, 0-1,3-glucanase, moricin-related peptides or phytoalexins), alters sucrose metabolism (e.g., invertase or sucrose synthase), confers reduced allergenicity, increases digestibility, confers environmental stress tolerance, confers nematode resistance, is a gene encoding a pharmaceutical (e.g., antibiotics, antibodies, secondary metabolites, pharmaceutical peptides or vaccines), is a gene encoding an industrial enzyme, or increases the use of the sorghum plant or part thereof as a biofuel.
• viral resistance e.g. viral coat protein
• fungal resistance e.g., chitinase, 0-1,3-glucanase, moricin-related peptides or phytoalexins
• alters sucrose metabolism
• CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
• the method of breeding a plant as described herein may comprise introducing one or more nucleic acid(s) related to the clustered regulatory interspaced short palindromic repeats (CRISPR)ZCas system for inducing targeted genetic alterations.
• the CRISPR system can be portable to plant cells by co-delivery of plasmids expressing the Cas endonuclease and the necessary crRNA components.
• the Cas endonuclease may be converted into a nickase to provide additional control over the mechanism of DNA repair (Cong et al., 2013).
• loci of the CRISPR system may be transformed into the plant to enable CRISPR mediated gene editing thereof.
• the plants which are bred in the method of the disclosure may be of any species or variety. Exemplary groups of plants and plant species are described herein in the context of the phenotypic screening method of the disclosure and shall be taken to apply mutatis mutandis to each of the following and foregoing examples describing methods of breeding a plant, unless specifically stated otherwise
• the plant which is bred is selected from the group consisting of a fruiting plant, a leguminous plant, an oil plant, a vegetable plant, a cereal plant, a fibre plant, an ornamental plant, a forestry plant, an aquatic plant and a noxious plant or weed.
• the plant which is bred is a cereal plant.
• the cereal plant may be selected from the group consisting of wheat, barley, oats, rye, triticale, rice, millet, sorghum, corn (maize), quinoa and buckwheat.
• the cereal plant is wheat.
• the cereal plant is barley.
• the plant which is bred is an oil plant.
• oil plants are described herein in the context of screening methods and shall be taken to apply mutatis mutandis to each and every example describing plant breeding methods.
• the oil plant may be canola, soybean, sunflower, olive, coconut or palm.
• the oil plant is canola.
• the plant which is bred is a vegetable plant.
• Exemplary vegetable plants are described herein in the context of screening methods and shall be taken to apply mutatis mutandis to each and every example describing plant breeding methods.
• the vegetable plant is a tomato plant, or celery or celeriac plant.
• the plant which is bred is a fruiting plant.
• Exemplary fruiting plants are described herein in the context of screening methods and shall be taken to apply mutatis mutandis to each and every example describing plant breeding methods.
• the fruiting plant is a blueberry plant.
• the plant is an ornamental plant.
• Exemplary ornamental plants are described herein in the context of screening methods and shall be taken to apply mutatis mutandis to each and every example describing plant breeding methods.
• the plant is a forestry plant.
• Exemplary forestry plants are described herein in the context of screening methods and shall be taken to apply mutatis mutandis to each and every example describing plant breeding methods.
• the plant is a fibre plant.
• Exemplary fibre plants are described herein in the context of screening methods and shall be taken to apply mutatis mutandis to each and every example describing plant breeding methods.
• the plant is an aquatic plant.
• Exemplary aquatic plants are described herein in the context of screening methods and shall be taken to apply mutatis mutandis to each and every example describing plant breeding methods.
• the plant is an noxious plant or weed.
• noxious plants and weeds are described herein in the context of screening methods and shall be taken to apply mutatis mutandis to each and every example describing plant breeding methods.
• the present disclosure also provides a plant or plant part which has been bred by performing the method of breeding a plant as described herein.
• plant as used herein as a noun refers to whole plants, but as used as an adjective refers to any substance which is present in, obtained from, derived from, or related to a plant, such as for example, plant organs (e.g., leaves, stems, roots, flowers), single cells (e.g., pollen), seeds, plant cells and the like.
• plant part refers to all plant parts that comprise the plant DNA, including vegetative structures such as, for example, leaves or stems, roots, floral organs or structures, pollen, seed, seed parts such as an embryo, endosperm, scutellum or seed coat, plant tissue such as, for example, vascular tissue, cells and progeny of the same, as long as the plant part is the product of a method of the disclosure.
• the plant part is a plant propagation material.
• the plant propagation material may be a seed, a plant cutting or scion.
• YMS (Yeast Malt Sucrose) medium was prepared as a solid medium with agar on petri dishes (90 x 15 mm, Thermo Fisher), or liquid without agar in flasks. 1 litre of YMS medium contained: 4 g yeast extract, 4 g malt extract, 4 g sucrose, 15 g technical agar, and 25 mg Gentamicin. Generally, liquid YMS is a more convenient inoculum preparation for large number of plants, while solid YMS agar is better for small number of plants. Since the infiltration method requires a minimal amount of inoculum, the solid YMS agar was prepared. Isolation of pathogen and growth of inoculum
• pycnidium The asexual fruiting body of Z. tritici is called pycnidium (pl. pycnidia). These fruiting bodies appear as tiny black spots on the surface of infected leaves (see Figure 2). Infected pieces of leaf were collected, then surface sterilized with 70% ethanol and placed flat on a piece of wet Whatman® Filter Paper in a petri dish humid chamber. Leaf pieces were incubated at 25°C for 3 hrs or overnight (16 hrs). Infected leaves from different host plants were placed in different petri dishes and marked with identifiers to allow the tracking of individual plants. The pycnidium on the infected leaf pieces produces conidia as a cirrhus or ooze-like structure.
• All conidia belonging to a sporulating pycnidium are genetically identical, so they are recognized as an isolate with a designated individual name.
• the cirrhus of the sporulating pycnidium were picked out with a sterilized needle and sub-cultured on solid YMS agar petri dishes under aseptic conditions in a Laminar Air Flow cabinet. After 4-10 days, each sub-cultured isolate grew into approximate 1 mm pinkish cream cluster of spores (blastospores).
• the pinkish isolate clusters were also sub-cultured for future experiments. Briefly, 500 pl of sterile water was added to the solid YMS petri dish agar surface using a sterile pipette to keep the solid YMS petri dish agar surface moist. This step was repeated, as required to maintain a moist agar surface.
• the petri dishes were incubated under white fluorescent lights (800-1000 lumens) at 18°C. After 5-6 days, a pinkish cream growth was observed in the plate. Spores can be maintained in this form under cool moist conditions with frequent subculturing, ensuring that the petri dish agar surface does not dry out. Petri dishes were also monitored constantly for contaminants. A fresh culture was started every 30 days to minimise the risk of contamination and loss of pathogenicity.
• Blastospores collected from the YMS agar surface can also be mixed with a solution of 0.5% glycerol. This spore solution is added to a 2 ml cryogenic tube containing 1-2 mm diameter sterilised glass or plastic beads, and stored at -80 °C for up to 5 years. Blastospores can be recovered from cryogenic storage and regenerated on solid YMS agar petri dishes.
• a haemocytometer Nebauer, Counting Chambers, BLAUBRAND®, Cell culture
• the chamber was approximately 0.1 millimetres deep.
• the spore culture was diluted 5x-10x and added to the haemocytometer using a Pasteur pipette and checked to see if the spores were evenly distributed.
• the ‘L’ squares were used for counting the number of cells. If the count was less than 200 cells in a selected square, then all four squares were counted and the mean cell count determined.
• the Z. tritici isolates in the liquid YMS medium were diluted to the desired concentration with the addition of gelatin (0.5%), which acts as a surfactant helping spores to adhere to the leaf surface.
• gelatin 0.5%), which acts as a surfactant helping spores to adhere to the leaf surface.
• the spore concentration used was approximately 4 x 10 6 spores/ml.
• Gelatin solution was prepared by dissolving 5 g of gelatin (Mckenzie’s, AUS) in 100 ml of hot water, for every litre of final inoculum. The water and gelatin powder were placed on a magnetic stirrer and stirred until dissolved. Once the solution has cooled to less than 30°C (a large amount of hot water can decrease viability of inoculum), the gelatin solution was added to the inoculum suspension.
• the glasshouse temperature was controlled using an Evaporative Cooler/Gas Heater Air conditioning system (Bonaire, AUS), with temperatures ranging from 18°C ( ⁇ 3°C) to 22°C ( ⁇ 3°C). Plants reached the two-leaf stage 10-16 days after sowing. To maintain the healthy growth of plants, regularly watering them is required until the finish of the experiments.
• AUS Evaporative Cooler/Gas Heater Air conditioning system
• the final spore concentrations were adjusted to 4 x 10 6 spores/ml, supplemented with 0.5% gelatin.
• the spore suspension was applied as a fine mist to the leaf surface of 2-3 weeks-old plants with two fully emerged leaves, using an airbrush (Anest Iwata, Japan).
• an airbrush Adest Iwata, Japan.
• similar application of the spore suspension to the leaf surface could be achieved by the spray method using a pressurised spray can or spray bottle, or by applying the spore suspension to the leaf surface as a thin film using a paint brush, cotton swab or by immersion.
• Inoculated seedlings were placed in a dark chamber at 90-100% relative humidity at 16 °C for 48 hrs in the dark prior to being transferred to the glasshouse (18-22 °C).
• a negative control (mock) treatment was included for the spray which consisted of the carrier liquid for the spray method applied without fungal spores to ensure the response phenotypes were not due to background effects. This was applied to all varieties sown in the experiment.
• leaf infiltration was carried out 4-7 days after the emergence of the 2nd leaf, this allowed the spore suspension to more easily enter via the stomata and spread within the intercellular space minimizing variations of the amount of inoculum that were infiltrated into the leaves of different genotypes, as it is very common that leaves of some genotypes are much more difficult to infiltration than others.
• a thin mist of water was sprayed on the plants within the first 24 hours.
• Plants were scored 4-42 days after the inoculation, depending on the progress of experiments. Septoria tritici blotch (STB) scores (1-5) were then assigned to each plant according to the method described in Zwart et al (2010) Molecular Breeding, 26:107-124.
• STB Septoria tritici blotch
• Example 3 Different Stages of infection in plants resulting from Z. tritici spores
• This example describes the nine stages of interaction between Z. tritici and wheat plants which have been used in the phenotyping method of the disclosure to classify plants according to their defence and resistance phenotypes.
• Stage 1 -Attachment of spores to plant surface e.g., leaves (0-12 hr)
• Primary infection can be caused by ascospores or pycnidia spores, which attach themselves to the surface of a plant (e.g., a plant leaf). Ascospores are, in general, responsible for primary infection at the start of a growing season. Some plants possess a physical barrier, comprised of a thin waxy cuticle and epidermis, which restricts attachment of spores to the host plant leaves.
• the spores degrade the epidermis in anticipation of germination. Water acts as an important factor for the successful pathogen infection and surfactants (e.g., Tween 20) are frequently added to inoculum buffer to assist in this process.
• the plant initiates the first stage of host defence i.e., effector triggered immunity (ETI).
• ETI effector triggered immunity
• PR Plant pathogenesis-related
• Hyphae enlarges in the substomatal cavity and branch into intercellular space. Symptoms are typically not evident at this stage and the latent period varies depending on the plant genotype and environmental conditions. Expressions of PR genes increases at this stage. As the inoculum directly infiltrates into the leaf surface, the infection process using the second screening method (i.e., the infiltration method) starts at this point.
• the second screening method i.e., the infiltration method
• Hyphae elongate through the mesophyll to the surrounding substomatal cavities. Hyphae are still growing slowly at this point of the infection, and fungal biomass is very low. No macro symptoms are observable. Stage 6 - Host cell death (7-11 day)
• a hypersensitive response (HR)-like reaction starts at this point, which is characterized by rapid cell death localised to the area of infection in order to restrict the growth and spread of hyphae to other leaf areas.
• Resistance genes such as NBS-LRR and cysteine kinase, may function here as receptors and interact with the effectors from the pathogen. The plant can induce another stage of defence, ‘FTP, if the appropriate resistant genes are present.
• the leaf loses its physical integrity and collapses, establishing the necrotic leaf area where the asexual pycnidiospores are produced to form small, black pycnidia,. Sexual ascospores may also be produced in pseudothecium.
• a new cycle of infection commences. With the assistance of water splash dispersal, pycnidiospores disperse and spread the disease up through the leaf layers of the host.
• Example 4 A phenotyping selection tool to classify plant defence responses to fungal pathogens
• This example relates to a multi-modal screening method for classifying plants based on their defence response phenotypes to fungal pathogens (designated the ‘Defend system’).
• the Defend system is able to distinguish between events occurring during the early phases of infection (i.e., pre-entry or during entry) versus those occurring after the pathogen has successfully entered into the leaf or other plant tissue (i.e., post-entry).
• the method is capable of identifying when a plant defence (to a plant pathogen) is being triggered at different points along the infection pathway by separating those events which occur during the early phases of infection, to those which occur after the pathogen has successfully entered into the plant tissue.
• _ Class 1 a response phenotype characterised by an absence of chlorosis, necrosis and pathogen fruiting structures on the plant(s) or plant part(s) following performance of the first screening method (i.e., the contact inoculation method), and chlorosis, necrosis and/or formation of pathogen fruiting structures on the plant(s) or plant part(s) following performance of the second screening method (i.e., the infiltration inoculation method).
• a plant which exhibits a Class 1 response phenotype elicits a defence response to the pathogen prior to or during entry of the pathogen into the plant tissue, but not post entry This response phenotype is only effective during the very early stages of the infection cycle.
• Class 2 a response phenotype characterised by an absence of chlorosis, necrosis and pathogen fruiting structures on the plant(s) or plant part(s) following performance of the first screening method and the second screening method.
• the plant’s defence response prevents/restricts the colonisation phase from proceeding to chlorosis and prevents fruiting structures from forming.
• a plant which exhibits a Class 2 response phenotype elicits a defence response to the pathogen prior to or during and post entry of the pathogen into the plant tissue
• Class 3 a response phenotype characterised by chlorosis, necrosis and/or formation of pathogen fruiting structures on the plant(s) or plant part(s) following performance of the first screening method, and an absence of chlorosis, necrosis and pathogen fruiting bodies form on the plant(s) or plant part(s) following performance of the second screening method.
• the plant’s defence response prevents/restricts the colonisation phase from proceeding to chlorosis and prevents fruiting structures from forming.
• a plant which exhibits a Class 3 response phenotype elicits a defence response to the pathogen post entry of the pathogen into the plant tissue, but not prior to or during entry.
• Class 4 a response phenotype characterised by colonization of the plant(s) or plant part(s) by the pathogen, but infection is halted with minimal chlorosis, following performance of the first screening method and/or the second screening method.
• a plant which exhibits a Class 4 response phenotype does not elicit a defence response to the pathogen prior to entry or during of the pathogen into the plant tissue, but does elicit a defence response to the pathogen post entry of the pathogen into the plant tissue and colonisation of the plant by the pathogen still occurs.
• Class 4 is typically identified in the second screening method (i.e., the infiltration inoculation method) during the latent period and at the end of the experiment.
• Class 5 a response phenotype characterised by chlorosis, necrosis and formation of pathogenic fruiting structures with evidence of host resistance, wherein host resistance is characterised by a reduction in number of lesion and/or lesion area compared to a plant or plant part in which there is no evidence of host resistance.
• a plant which exhibits a Class 5 response phenotype does not elicit a defence response to the pathogen prior to or post entry of the pathogen into the plant tissue, but shows evidence of resistance to the pathogen represented by a reduced number of lesion and/or reduced lesion area compared to a plant or plant part in which there is no evidence of host resistance (e.g., a plant classified as Class 6).
• Class 5 is typically identified in the second screening method (i.e., the infiltration inoculation method) during the latent period and at the end of the experiment.
• Class 6 a response phenotype characterised by chlorosis, necrosis and formation of pathogen fruiting structures with no evidence of host resistance. A plant which exhibits a Class 6 response phenotype does not elicit a defence response to the pathogen prior to or post entry of the pathogen into the plant tissue, and shows no evidence of resistance to the pathogen. Class 6 is typically identified following completion of the first screening method (i.e., the contact inoculation method) and the second screening method (i.e., the infiltration inoculation method), at the end of the experiment.
• the first screening method i.e., the contact inoculation method
• the second screening method i.e., the infiltration inoculation method
• the critical step for the ‘Defend’ system is the distinction between Class 1 and Classes 2- 5 phenotypes. This allows for the selection and combination of genotypes from plants which trigger defence responses during different stages of the infection process.
• the ‘Defend’ pheno typing system is illustrated in Figure 1 and Table 3.
• ‘Defend’ uses a comparison of eight phases during the infection of leaves by a fungal pathogen using two different inoculation methods (i.e., the contact inoculation method and the infiltration inoculation method). The system combines the phenotypes observed in both phenotyping methods to reveal information that would not otherwise be available if either inoculation method was used on its own. The first separation occurs between defence being triggered at or before the pathogen enters the leaf (Class 1).
• Effective means if the pathogen is detected by the host at this stage disease is stopped or slowed.
• Detected means this phenotype can be separated from other stages of the infection process using this method alone.
• the new ‘Defend’ method has been used to discover new sources of resistance in plants.
• Example 5 Evaluation of ‘Defend’ system and traditional screening methods for selecting wheat cultivars resistant and susceptible to Z. tritici
• the inventors evaluated the new ‘Defend’ phenotypic screening method described in Example 4 relative to a traditional spray method of phenotypic screening.
• An experiment on several wheat varieties with genotypes with known resistance to Z. tritici was designed to, inter alia, determine whether the ‘Defend’ method could differentiate plants harbouring genes that are known to be resistant to Z. tritici.
• Twenty bread wheat cultivars were selected for inclusion in the experiment, including ‘Lorikeet’ with the gene Stbl9, ‘Currawong’ with the genes Stb7 and Stbl2, and ‘Summit’ which is a susceptible cultivar. Briefly, the twenty cultivars were sown in a fixed order of 5 x 4 tubes in a rack, each rack was replicated four times and arranged in a zig-zag format on the same bench. Experiment conditions using the spray method and the infiltrated method were followed as described above in Example 2. The Z. tritici isolate used for the experiment was WAI332.
• V8-PDA Potato-Dextrose Agar
• V8-PDA medium 1 litre of V8-PDA medium contained: 150 ml Campbells V8 juice, 10 g PDA, 1.5 g calcium carbonate (CaCCL), 15 g technical agar, 850 ml of distilled water and 25 mg Gentamicin.
• the asexual fruiting body of S. apiicola is called pycnidium (pl. pycnidia). These fruiting bodies appear as tiny black spots on the surface of infected leaves (see Figure 3). Infected pieces of leaves were collected, then surface sterilized with 70% ethanol and placed flat on a piece of wet Whatman® Filter Paper in a petri dish for 3 hrs or overnight (16 hrs). Infected leaves from different host plants were placed in different petri dishes and marked with identifiers to allow the tracking of individual plants. The pycnidium produces conidia as a cirrhus or ooze-like structure.
• the spores from each isolate were also sub-cultured for future experiments. Briefly, sterile water was added to the V8-PDA plate surface using a sterile pipette to keep the V8 plate surface moist. This step was repeated, whenever necessary. The plates were incubated under the white fluorescent lights (800-1000 lumens) at 18°C. After 5-6 days, a dark hyphae with spore masses was observed in the plate. Spores can be maintained in this form under cool moist conditions with frequent subculturing, ensuring that the plates do not dry out. Plates were also monitored constantly for contaminants. A fresh culture was started once a month to minimise the risk of contamination and loss of pathogenicity.
• Spores can also be mixed with 0.5% glycerol, and collected with 1-2 mm plastic beads in the 2 ml tube, and stored at -80 °C for long term storage.
• a haemocytometer was used to count and adjust the spore concentration.
• the chamber was approximately 0.1 millimetres deep. Briefly, the cover-glass was pushed on the slide firmly by rubbing along the cover-glass supports using moist fingers.
• the spore culture was diluted 5x-10x and added to the haemocytometer using a Pasteur pipette and checked to see if the spores were evenly distributed.
• the ‘L’ squares were used for counting the number of cells. If the count was less than 200 cells in a selected square, then all four squares were counted and the mean cell count determined.
• Example 7 Experimental procedures for S. apiicola inoculation
• the S. apiicola isolates on the solid V8 medium were flushed with sterile water and the agar surface was gently agitated to facilitate dislodgement of spores. Approximately 5 pl of spores was scraped from the petri dish into 50 ml of clean distilled water and adjusted for a final concentration of approximately 7 x 10 5 spores/ml. Inoculation preparation was the same for the spray and the infiltration method,
• the glasshouse temperature was controlled using an Evaporative Cooler/Gas Heater Air conditioning system (Bonaire, AUS), with temperatures ranging from 18°C ( ⁇ 3°C) to 22°C ( ⁇ 3°C). Plants reached harvest maturity 90 days after sowing. Septoria inoculation procedures
• the final spore concentrations were adjusted to 7 x 10 5 spores/ml.
• the spore suspension was applied as a fine mist to the leaf surface of 90-100 day old plants using an airbrush.
• similar application of the spore suspension to the leaf surface could be achieved by the spray method using a pressurised spray can or spray bottle, or by applying the spore suspension to the leaf surface as a thin film using a paint brush, cotton swab or by immersion.
• Inoculated plants were placed in a dark chamber at 90-100% relative humidity at 16 °C for 48 hrs in the dark prior to being transferred to the glasshouse (18-22 °C).
• Plants were scored 14-28 days after the inoculation, depending on the progress of experiments. Scores were assigned to each plant using a modified version of the Septoria tritici blotch (STB) scoring (1-5) method described in Zwart et al (2010) Molecular Breeding, 26: 107- 124.
• STB Septoria tritici blotch
• Example 8 Different Stages of infection in plants resulting from pathogenic S. apiicola spores
• Example 9 Evaluation of the ‘Defend’ system and traditional screening methods for selecting celery and celeriac varieties resistant and susceptible to S. apiicola
• the inventors evaluated the new ‘Defend’ phenotypic screening method described in Example 4 relative to a traditional spray method of phenotypic screening.
• An experiment on eighteen celery and celeriac varieties with genotypes with unknown resistance to S. apiicola was designed to, inter alia, determine whether the ‘Defend’ method could differentiate plants harbouring genes that are unknown to be resistant to S. apiicola.
• Table 4 Celery and celeriac varieties (Apium graveolens) used to demonstrate the ‘Defend’ method.
• Example 10 Protocol for the preparation of Puccinia striiformis f. sp. tritici inoculum
• the asexual fruiting body of Pst is called a uredinium (pustules) which produce asexual uredinio spores.
• These are thin-walled spores produced by the uredium on the surface of infected leaves (see Figure 5).
• the fresh rust spores from a single pustule which can be recognized a single race with a designated name, are carefully picked up and mixed with 0.05% Tween 20, and then applied on young susceptible cultivars, i.e. ‘Tammarin Rock’.
• the spore suspension was gently painted on the fully emerged leaf blades of 2-3 weeks-old plants, using a cotton swab or brush.
• Inoculated seedlings were placed in a dark chamber at 90-100% relative humidity at 8-12 °C for 48 hrs in the dark prior to being transferred to the glasshouse (18-22 °C). After 14 days, new cluster of pustules should be observed on the plant leaf blades. New pustules were collected from the plant. Then repeat the process described above on more plants, in order to obtain sufficient amount of rust spores for future experiments, or to collect sufficient amount of spores for long-term storage. Rust spores are collected in 2 ml tubes, dried with silica gel at 4 °C for 2-3 days then into -80 °C , and stored at -80 °C for long term usage.
• a haemocytometer was used to count and adjust the spore concentration in suspended cultures.
• the chamber was approximately 0.1 millimetres deep. Briefly, the cover-glass was pushed on the slide firmly by rubbing along the cover-glass supports using moist fingers.
• the spore culture was diluted 5x-10x and added to the haemocytometer using a Pasteur pipette and checked to see if the spores were evenly distributed.
• the ‘L’ squares were used for counting the number of cells. If the count was less than 200 cells in a selected square, then all four squares were counted and the mean cell count determined.
• the spore concentration of Pst used for the spray method was approximately 6 x 10 5 spores/ml.
• Dissolvents used for the spray method can be distilled water with Tween 20 or light mineral oil such as Isopar.
• a final concentration was adjusted to 10 4 spores/ml.
• 50 pl of Tween 20 was dissolved in 100 ml of water.
• the final spore concentrations were adjusted to 6 x 10 5 spores/ml, supplemented with 0.05% Tween 20.
• the spore suspension was applied as a fine mist to the leaf surface of 2-3 weeks-old plants with two fully emerged leaves, using an airbrush.
• similar application of the spore suspension to the leaf surface could be achieved by the spray method using a pressurised spray can or spray bottle, or by applying the spore suspension to the leaf surface as a thin film using a paint brush, cotton swab or by immersion.
• Inoculated seedlings were placed in a dark chamber at 90-100% relative humidity at 8-12 °C for 48 hrs in the dark prior to being transferred to the glasshouse (18-22 °C).
• leaf infiltration was carried out 4-7 days after the emergence of the 2nd leaf, this allowed the spore suspension to more easily enter via the stomata and spread within the intercellular space minimizing variations of the amount of inoculum that were infiltrated into the leaves of different genotypes, as it is very common that leaves of some genotypes are much tougher to infiltrate than others.
• a thin mist of water was sprayed on the plants within the first 24 hours.
• Example 12 Different Stages of infection in plants resulting from pathogenic spores
• This example describes the nine stages of interaction between Pst and wheat plants which have been used in the phenotyping method of the disclosure to classify plants according to their defence and resistance phenotypes.
• Stage 1 -Attachment of spores to plant surface e.g., leaves (0-3 h)
• teliospores or urediniospores, which attach themselves to the surface of a plant (e.g., a plant leaf). Teliospores are, in general, responsible for primary infection at the start of a growing season, where the alternate host is present allowing full disease life cycle to occur. Some plants possess a physical barrier, comprised of a thin waxy cuticle and epidermis, which restricts attachment of spores to the host plant leaves.
• the spores degrade the epidermis in anticipation of germination. Water acts as an important factor for the successful pathogen infection and surfactants (e.g., tween-20) are frequently added to inoculum buffer to assist in this process.
• the plant initiates the first stage of host defence i.e., effector triggered immunity (ETI).
• ETI effector triggered immunity
• Plant pathogenesis-related (PR) genes are typically expressed at this stage of the infection cycle.
• Stage 4 Apoplastic colonization (2-4 day) Fungal growth develops from the vesicle and proceeds intercellularly in the mesophyll layer. Symptoms are typically not evident at this stage. Expressions of PR genes increases at this stage. As the inoculum directly infiltrates into the leaf surface, the infection process using the second screening method (i.e., the infiltration method) starts at this point.
• the second screening method i.e., the infiltration method
• Hyphae grows and proliferates in plasmodesmata and mesophyll cells. No macro symptoms or weak sign of infection may be observed.
• Damage of cellular organelles beyond the advancing hyphae implies the involvement of a toxin in the infection process. Small brown- yellow pustules may be evident on the leaf surface.
• a hypersensitive response (HR)-like reaction starts at this point, which is characterized by rapid cell death localised to the area of infection to restrict the growth and spread of hyphae to other leaf areas.
• Resistance genes such as NBS-LRR and cysteine kinase, may function here as receptors and interact with the effectors from the pathogen. The plant can induce another stage of defence, ‘ETF, if the appropriate resistant genes are present.
• a new cycle of infection commences. Coalescing chlorotic or necrotic leaf area are otherwise extended larger and larger. With the assistance of wind and water splash dispersal, more teliospores or uredinio spores disperse and spread the disease up through the leaf layers of the host.
• Example 13 Evaluation of the ‘Defend’ system and traditional screening methods for selecting wheat varieties resistant and susceptible to Puccinia striiformis f. sp. tritici
• the inventors evaluated the new ‘Defend’ phenotypic screening method described in Example 4 relative to a traditional spray method of phenotypic screening.
• An experiment on several wheat varieties with genotypes with known resistance to was designed to Puccinia striiformis f. sp. tritici, inter alia, determine whether the ‘Defend’ method could differentiate plants harbouring genes that are known to be resistant to Puccinia striiformis f. sp. tritici.
• Example 14 Protocol for the preparation of Septoria lycopersici inoculum
• V8-PDA medium was prepared as a solid medium with agar on polystyrene petri dishes (90 x 15 mm, Thermo Fisher). 1 litre of V8-PDA medium contained: 150 ml Campbells V8 juice, 10 g PDA, 1.5 g calcium carbonate (CaCCL), 15 g technical agar, 850 ml of distilled water and 25 mg Gentamicin.
• pycnidium The asexual fruiting body of S. lycopersici is called pycnidium (pl. pycnidia). These fruiting bodies appear as tiny black spots on the surface of infected leaves (see Figure 3). Infected pieces of leaves were collected, then surface sterilized with 70% ethanol and placed flat on a piece of wet Whatman® Filter Paper in a petri dish for 3 hrs or overnight (16 hrs). Infected leaves from different host plants were placed in different petri dishes and marked with identifiers to allow the tracking of individual plants. The pycnidium produces conidia as a cirrhus or ooze-like structure.
• the conidia isolate were also sub-cultured for future experiments.
• the plates were incubated under the white fluorescent lights (800-1000 lumens) at 18°C. After 7-30 days, mycelium covered the plate and pycnidia were observed forming. Spores can be maintained in this form under cool moist conditions with frequent subculturing, ensuring that the plates do not dry out. Plates were also monitored constantly for contaminants. A fresh culture was started once a month to minimise the risk of contamination and loss of pathogenicity.
• Conidia spores can also be mixed with 0.5% glycerol, and collected with 1-2 mm plastic beads in the 2 ml tube, and stored at -80 °C for long term storage.
• a haemocytometer was used to count and adjust the spore concentration.
• the chamber was approximately 0.1 millimetres deep. Briefly, the cover-glass was pushed on the slide firmly by rubbing along the cover-glass supports using moist fingers.
• the spore culture was diluted 5x-10x and added to the haemocytometer using a Pasteur pipette and checked to see if the spores were evenly distributed.
• the ‘L’ squares were used for counting the number of cells. If the count was less than 200 cells in a selected square, then all four squares were counted and the mean cell count determined.
• Example 15 Experimental procedures for S. lycopersici inoculation
• the S. lycopersici isolates on the V8-PDA medium were diluted to the desired concentration.
• the spore concentration used was approximately 3 x 10 6 spores/ml for spray and 3 x 10 5 spores/ml for infiltration.
• plates were flooded with approximately 3 ml of water conidia spores was scraped from the petri dish into 50 ml of clean distilled water and adjusted for a final concentration of approximately 3 x 10 5 and 3 x 10 6 .
• the final spore concentrations were adjusted to 3 x 10 6 spores/ml, supplemented with 0.1% Tween20.
• the spore suspension was applied as a fine mist to the leaf surface of 3-5 weeks-old plants with fully emerged leaves, using an airbrush.
• similar application of the spore suspension to the leaf surface could be achieved by the spray method using a pressurised spray can or spray bottle, or by applying the spore suspension to the leaf surface as a thin film using a paint brush, cotton swab or by immersion.
• Inoculated seedlings were placed in a growth chamber at 90-100% relative humidity at 25 °C for 48 hrs in with 12 hr light/dark period prior to being transferred to the glasshouse (25-29 °C).
• leaf infiltration was carried out 4-7 days after the emergence of the 2nd vegetative leaf, this allowed the spore suspension to more easily enter via the stomata and spread within the intercellular space minimizing variations of the amount of inoculum that were infiltrated into the leaves of different genotypes, as it is very common that leaves of some genotypes are much tougher to infiltrate than others.
• Example 16 Different Stages of infection in plants resulting from pathogenic spores
• Example 4 the inventors evaluated the new ‘Defend’ phenotypic screening method described in Example 4 relative to a traditional spray method of phenotypic screening.
• An experiment on fifteen tomato varieties with genotypes with unknown resistance to S. lycopersici was designed to, inter alia, determine whether the ‘Defend’ method could differentiate plants harbouring genes that are unknown to be resistant to S. lycopersici.
• Table 7 Tomato varieties (Solarium lycopersicum) used to demonstrate the ‘Defend’ method.
• the asexual fruiting body of T. minima is called a uredinium (pustules) which produce asexual uredinio spores. These are thin-walled spores produced by the uredium on the surface of infected leaves (see Figure 7).
• the fresh rust spores from a single pustule which can be recognized a single race with a designated name, are carefully picked up and mixed with 0.05% Tween 20, and then applied on young susceptible cultivars, i.e. ‘Pink Icing’. Briefly, the spore suspension was gently painted on the back of young fully emerged leaf surface, using a cotton swab or brush.
• Inoculated seedlings were placed in a dark chamber at 90-100% relative humidity at 17 °C for 48 hrs in the dark prior to being transferred to the glasshouse (18-22 °C). After 14 days, new cluster of pustules should be observed on the back of plant leaf. New pustules were collected from the plant. Then repeat the process described above on more plants, in order to obtain sufficient amount of rust spores for future experiments, or to collect sufficient amount of spores for long-term storage. Rust spores are collected in 2 ml tubes, dried with silica gel at 4 °C for 2-3 days then into -80 °C , and stored at -80 °C for long term usage.
• a haemocytometer was used to count and adjust the spore concentration in suspended cultures.
• the chamber was approximately 0.1 millimetres deep. Briefly, the cover-glass was pushed on the slide firmly by rubbing along the cover-glass supports using moist fingers.
• the spore culture was diluted 5x-10x and added to the haemocytometer using a Pasteur pipette and checked to see if the spores were evenly distributed.
• the ‘L’ squares were used for counting the number of cells. If the count was less than 200 cells in a selected square, then all four squares were counted and the mean cell count determined.
• the spore concentration of T. minima used for the spray method was approximately 3 x 10 5 spores/ml.
• Dissolvents used for the spray method can be distilled water with Tween 20 or light mineral oil such as Isopar. In preparation of inoculum for the infiltration method, a final concentration was adjusted to 10 4 spores/ml. To mix the rust spores well in the liquid, 50 pl of Tween 20 was dissolved in 100 ml of water.
• the final spore concentrations were adjusted to 3 x 10 5 spores/ml, supplemented with 0.05% Tween 20.
• the spore suspension was gently painted on the back of leaf surface, using a paint brush.
• similar application of the spore suspension to the leaf surface could be achieved by the spray method using an air brush or spray bottle, or by applying the spore suspension to the leaf surface as a thin film using a paint brush, cotton swab or by immersion.
• Inoculated seedlings were placed in a dark chamber at 90-100% relative humidity at 17 °C for 48 hrs in the dark prior to being transferred to the glasshouse (18-22 °C).
• leaf infiltration is more easily applied on young leaves other than old leaves, this allowed the spore suspension to more easily enter via the stomata and spread within the intercellular space minimizing variations of the amount of inoculum that were infiltrated into the leaves of different genotypes. After the infiltration, a thin mist of water was sprayed on the plants within the first 24 hours.
• Example 20 Different Stages of infection in plants resulting from pathogenic spores
• Example 21 Evaluation of the ‘Defend’ system and traditional screening methods for selecting blueberry varieties resistant and susceptible to T. minima
• the inventors evaluated the new ‘Defend’ phenotypic screening method as described in Example 4 relative to a traditional spray method of phenotypic screening.
• An experiment on several blueberry varieties was designed to T. minima, inter alia, determine whether the ‘Defend’ method could differentiate plants harbouring genes that are resistant to T. minima.
• V8-PDA medium was prepared as a solid medium with agar on polystyrene petri dishes (90 x 15 mm, Thermo Fisher). 1 litre of V8-PDA medium contained: 150 ml Campbells V8 juice, 10 g PDA, 1.5 g calcium carbonate (CaCCE), 15 g technical agar, 850 ml of distilled water and 25 mg Gentamicin.
• the asexual fruiting body of L. maculans is called pycnidium (pl. pycnidia). These fruiting bodies appear as tiny black spots on the surface of infected leaves (see Figure 3). Infected pieces of leaves were collected, then surface sterilized with 70% ethanol and placed flat on a piece of wet Whatman® Filter Paper in a petri dish for 3 hrs or overnight (16 hrs). Infected leaves from different host plants were placed in different petri dishes and marked with identifiers to allow the tracking of individual plants. The pycnidium produces conidia as a cirrhus or ooze-like structure.
• the conidia isolate were also sub-cultured for future experiments.
• the plates were incubated under the white fluorescent lights (800-1000 lumens) at 18°C. After 7-30 days, mycelium covered the plate and pycnidia were observed forming. Spores can be maintained in this form under cool moist conditions with frequent subculturing, ensuring that the plates do not dry out. Plates were also monitored constantly for contaminants. A fresh culture was started once a month to minimise the risk of contamination and loss of pathogenicity.
• Conidia spores can also be mixed with 0.5% glycerol and collected with 1-2 mm plastic beads in the 2 ml tube and stored at -80 °C for long term storage.
• a haemocytometer was used to count and adjust the spore concentration.
• the chamber was approximately 0.1 millimetres deep. Briefly, the cover-glass was pushed on the slide firmly by rubbing along the cover-glass supports using moist fingers.
• the spore culture was diluted 5x-10x and added to the haemocytometer using a Pasteur pipette and checked to see if the spores were evenly distributed.
• the ‘L’ squares were used for counting the number of cells. If the count was less than 200 cells in a selected square, then all four squares were counted and the mean cell count determined.
• the L. maculans isolates on the V8-PDA medium were diluted to the desired concentration.
• the spore concentration used was approximately 3.5 x 10 5 spores/ml for wound, spray and infiltration.
• plates were flooded with approximately 3 ml of water conidia spores was scraped from the petri dish into 50 ml of clean distilled water and adjusted for a final concentration of approximately 3.5 x 10 5 .
• the final spore concentrations were adjusted to 3.5 x 10 5 spores/ml.
• Wounding of the cotyledons was performed using a sterile needle, pin or punch to create a hole of approximately 1 mm diameter through the cotyledon at approximately the centre of the left and right sides.
• the spore suspension was applied as a droplet of inoculum to the wound site on cotyledons of 2-3 weeks-old plants with two fully emerged cotyledons, using an pipette.
• spore suspension to the surface of the wound site could be achieved by the wound method using an eye dropper, or a syringe, or by applying the spore suspension to the leaf surface as a thin film using a paint brush, cotton swab or by immersion.
• Inoculated seedlings were placed in a dark chamber at 90-100% relative humidity at 16 °C for 48 hrs in the dark prior to being transferred to the glasshouse (18-22 °C).
• cotyledon infiltration was carried out 4-7 days after the cotyledons reached full expansion , this allowed the spore suspension to more easily enter via the stomata and spread within the intercellular space minimizing variations of the amount of inoculum that were infiltrated into the cotyledons of different genotypes, as it is very common that cotyledons of some genotypes are much tougher to infiltrate than others.
• Example 24 Different Stages of infection in plants resulting from pathogenic L. maculans spores
• Example 25 Evaluation of the ‘Defend’ system and traditional screening methods for selecting canola varieties resistant and susceptible to L. maculans
• the inventors evaluated the new ‘Defend’ phenotypic screening method as described in Example 4 relative to a traditional wounding method of phenotypic screening.
• An experiment on several canola varieties with genotypes with known resistance to L. maculans was designed to, inter alia, determine whether the ‘Defend’ method could differentiate plants harbouring genes that are known to be resistant to L. maculans.
• Eight canola cultivars were selected for inclusion in the experiment, including ‘Bristol’ with the genes Rlm2 and Rlm9, ‘ATR-Beacon’ with the genes Rlm3, Rlm4 and Rlm9, and “Westar’ which is a susceptible cultivar (Table 10). Briefly, the eight cultivars were sown in a fixed order of 8 x 4 pots, each variety was replicated four times and arranged in a zig-zag format on the same bench. Experiment conditions using the wound method and the infiltrated method were followed as described above in Example 23. The L. maculans isolate used for the experiment was WAI4364 which has at least virulence on Rlml/LepR3, LepRl using the wounding method. Table 10: Canola varieties used to demonstrate the ‘Defend’ method and resistance genes present where known.
• LB A medium was prepared as a solid medium with agar on polystyrene petri dishes (90 x 15 mm, Thermo Fisher). 1 litre of LB A medium contained: 500 g dried Lima bean extract, 15 g technical agar, 1000 ml of distilled water and 25 mg Gentamicin.
• Infected pieces of barley leaves were collected, then surface sterilized for 15 sec with 70% ethanol followed by 30 sec in 0.5% Sodium Hypochlorite solution.
• Leaf pieces were placed flat in petri dishes containing water agar (15 g technical agar, 1000 ml distilled water) and sealed with parafilm.
• Petri dishes were incubated for 5 days (120 hrs) at 15 °C in the dark.
• Infected leaves from different host plants were placed in different petri dishes and marked with identifiers to allow the tracking of individual plants. After incubation for 2-10 days scald lesions on the surface sterilised leaf pieces produce conidia that exuded in small clear whitish droplets.
• the conidiospores were picked up with a sterilized needle and sub-cultured on a petri dish containing LBA medium under aseptic conditions in a Laminar Air Flow cabinet. After 15-30 days culture at 15 °C in the dark, the sub-cultured isolate grew into approximate 5-7 mm cluster of sporulating hyphae. Conidiospores from the sub-cultured isolate were streaked on a fresh petri dish containing LBA medium under aseptic conditions in a Laminar Air Flow cabinet and after 3-7 days culture at 15 °C in the dark individual germinated spores were cut from the agar and transferred to a fresh petri dish containing LBA medium. Because all conidia derived from a single spore are genetically identical, cultures derived from a single germinated spore are recognized as an isolate with a designated name.
• the spores from each isolate were also sub-cultured for future experiments. Briefly, sterile water was added to the LBA plate surface using a sterile pipette to keep the LBA plate surface moist. This step was repeated, whenever necessary. The plates were incubated in the dark at 15 °C. After 10-20 days, a white to pinkish spore mass with hyphae was observed in the plate. Spores can be maintained in this form under cool moist conditions with frequent subculturing, ensuring that the plates do not dry out. Plates were also monitored constantly for contaminants. A fresh culture was started every 14-21 days to minimise the risk of contamination and loss of pathogenicity.
• Spores can also be mixed with 0.5% glycerol, and collected with 1-2 mm plastic beads in the 2 ml tube, and stored at -80 °C for long term storage. Calculation of the spore concentration using the haemocytometer
• a haemocytometer was used to count and adjust the spore concentration.
• the chamber was approximately 0.1 millimetres deep. Briefly, the cover-glass was pushed on the slide firmly by rubbing along the cover-glass supports using moist fingers.
• the spore culture was diluted 5x-10x and added to the haemocytometer using a Pasteur pipette and checked to see if the spores were evenly distributed.
• the ‘L’ squares were used for counting the number of cells. If the count was less than 200 cells in a selected square, then all four squares were counted and the mean cell count determined.
• the R. sesames on the solid LBA medium were flushed with sterile water and the agar surface was gently agitated to facilitate dislodgement of spores. Approximately 5 pl of spores was scraped from the petri dish into 50 ml of clean distilled water and adjusted for a final concentration of approximately 5 x 10 5 spores/ml. Inoculation preparation was the same for the spray and the infiltration method,
• the final spore concentrations were adjusted to 5 x 10 5 spores/ml.
• the spore suspension was applied as a fine mist to the leaf surface of 2-3 weeks-old plants with two fully emerged leaves using an airbrush.
• similar application of the spore suspension to the leaf surface could be achieved by the spray method using a pressurised spray can or spray bottle, or by applying the spore suspension to the leaf surface as a thin film using a paint brush, cotton swab or by immersion.
• Inoculated plants were placed in a dark chamber at 90-100% relative humidity at 16 °C for 48 hrs in the dark prior to being transferred to the glasshouse (18- 22 °C).
• Example 28 Different Stages of infection in plants resulting from pathogenic R. ses spores
• the inventors evaluated the new ‘Defend’ phenotypic screening method as described in Example 4 relative to a traditional spray method of phenotypic screening.
• An experiment on fifteen tomato varieties with genotypes with unknown resistance to R. ses was designed to, inter alia, determine whether the ‘Defend’ method could differentiate plants harbouring genes that are unknown to be resistant to R. ses.
• Table 12 Barley varieties (Hordeum vulgare) used to demonstrate the ‘Defend’ method.
• V8-PDA medium was prepared as a solid medium with agar on polystyrene petri dishes (90 x 15 mm, Thermo Fisher), containing 150 ml/1 Campbells V8 juice, 410 g/1 PDA, 41.5 g/1 sucrose calcium carbonate (CaCO3), 15 g/1 technical agar, 850 ml/1 of distilled water and 25 mg/1 Gentamicin.
• conidium The asexual fruiting body of P. tritici-repentis is called conidium (pl. conidia). Infected pieces of leaves were collected, then surface sterilized with 70% ethanol and placed flat on a piece of wet Whatman® Filter Paper in a petri dish for 3 hrs or overnight (16 hrs). Infected leaves from different host plants were placed in different petri dishes and marked with identifiers to allow the tracking of individual plants.
• the conidiophore is erect, simple (not branched) and may have one to nine transverse septa, with a swollen and olive-black base.
• the conidia spore grows and forms a white to light gray, fluffy mycelium which does not produce conidia, which can be recognized as an isolate with a designated name.
• the dense mycelium produces conidiophores and conidia after exposure to near-ultraviolet light for 12 to 24 hours followed by 12 to 24 hours of darkness.
• 0.5 cm-diameter-mycelial plugs can be carved from the growth plate, freeze-dried, and stored in the 2 ml tube at -80 °C for long term storage. Otherwise, a mycelial plug is sub-cultured for the experiment. Briefly, a single plug was placed in the middle of V8-PDA plate and incubated under the white fluorescent lights (800-1000 lumens) at 22-24 °C. After 4-7 days, a light gray, fluffy mycelium was observed in the plate. 8-10 agar plugs were carved from the plate and placed upside down on a new V8-PDA plate. The mycelium produces conidiophores and conidia after continuous exposure to near-ultraviolet light for 3-4 days.
• a haemocytometer was used to count and adjust the spore concentration.
• the chamber was approximately 0.1 millimetres deep. Briefly, the cover-glass was pushed on the slide firmly by rubbing along the cover-glass supports using moist fingers.
• the spore culture was diluted 5x-10x and added to the haemocytometer using a Pasteur pipette and checked to see if the spores were evenly distributed.
• the ‘L’ squares were used for counting the number of cells. If the count was less than 200 cells in a selected square, then all four squares were counted and the mean cell count determined.
• the P. tritici-repentis inoculum in the sterile water was diluted to the desired concentration in addition with Tween 20 (0.02%), approximately 1 x 10 4 spores/ml for both the spray method and the infiltration method. In particularly for the infiltration method, only sterile water was added to adjust the working concentration of inoculum.
• the final spore concentrations were adjusted to 1 x 10 4 spores/ml, supplemented with 0.02% Tween 20.
• the spore suspension was applied as a fine mist to the leaf surface of 2-3 weeks-old plants with two fully emerged leaves, using an airbrush.
• similar application of the spore suspension to the leaf surface could be achieved by the spray method using a pressurised spray can or spray bottle, or by applying the spore suspension to the leaf surface as a thin film using a paint brush, cotton swab or by immersion.
• Inoculated seedlings were placed in a dark chamber at 90-100% relative humidity at 16 °C for 24-48 hrs in the dark prior to being transferred to the glasshouse (18-22 °C).
• leaf infiltration was carried out 4-7 days after the emergence of the 2nd leaf, this allowed the spore suspension to more easily enter via the stomata and spread within the intercellular space minimizing variations of the amount of inoculum that were infiltrated into the leaves of different genotypes, as it is very common that leaves of some genotypes are much tougher to infiltrate than others.
• a thin mist of water was sprayed on the plants within the first 24 hours.
• Plants were scored 10-14 days after the inoculation, depending on the progress of experiments.
• P. tritici-repentis (Ptr) scores (1-5) were then assigned to each plant according to the method described in Lamari and Bernier (1989) Can. J. Plant Pathol.
• Example 32 Different Stages of infection in plants resulting from P. tritici-repentis spores
• This example describes the nine stages of interaction between P. tritici-repentis and wheat plants which have been used in the phenotyping method of the disclosure to classify plants according to their defence and resistance phenotypes.
• Stage 1 -Attachment of spores to plant surface e.g., leaves
• Primary infection can be caused by ascospores or conidia spores, which attach themselves to the surface of a plant (e.g., a plant leaf).
• Ascospores are, in general, responsible for primary infection at the start of a growing season.
• the spores degrade the epidermis in anticipation of germination. Water acts as an important factor for the successful pathogen infection and surfactants (e.g., Tween 20) are frequently added to inoculum buffer to assist in this process.
• the plant initiates the first stage of host defence i.e., effector triggered immunity (ETI).
• ETI effector triggered immunity
• Plant pathogenesis-related (PR) genes are typically expressed at this stage of the infection cycle.
• Fungal growth develops from the vesicle and proceeds intercellularly in the mesophyll layer. Symptoms are typically not evident at this stage. Expressions of PR genes increases at this stage. As the inoculum directly infiltrates into the leaf surface, the infection process using the second screening method (i.e., the infiltration method) starts at this point.
• the second screening method i.e., the infiltration method
• Hyphae grows and proliferates via plasmodesmata . No macro symptoms or weak sign of infection may be observed.
• a hypersensitive response (HR)-like reaction starts at this point, which is characterized by rapid cell death localised to the area of infection in order to restrict the growth and spread of hyphae to other leaf areas.
• Resistance genes such as NBS-LRR and cysteine kinase, may function here as receptors and interact with the effectors from the pathogen. The plant can induce another stage of defence, ‘ETI’, if the appropriate resistant genes are present. Stage 7 - Increase in fungal biomass (5-10 day)
• a new cycle of infection commences. Coalescing chlorotic or necrotic leaf area are otherwise extended larger and larger. With the assistance of water splash dispersal, more conidia spores disperse and spread the disease up through the leaf layers of the host.
• Example 33 Evaluation of ‘Defend’ system and traditional screening methods for selecting wheat cultivars resistant and susceptible to P. tritici-repentis
• the inventors evaluated the new ‘Defend’ phenotypic screening method as described in Example 4 relative to a traditional spray method of phenotypic screening.
• An experiment on several wheat varieties with genotypes with known resistance to P. tritici-repentis was designed to, inter alia, determine whether the ‘Defend’ method could differentiate plants harbouring genes that are known to be resistant to P. tritici-repentis.

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