WO2023118547A1

WO2023118547A1 - Panama disease resistance in banana

Info

Publication number: WO2023118547A1
Application number: PCT/EP2022/087674
Authority: WO
Inventors: Fernando Alexander GARCÍA BASTIDAS
Original assignee: Keygene N.V.
Priority date: 2021-12-23
Filing date: 2022-12-23
Publication date: 2023-06-29

Abstract

The invention pertains to composition for increasing resistance to Panama disease in a banana, preferably improving resistance to Panama disease in a Cavendish banana. The composition preferably comprises inactivated Fusarium fungal material, preferably inactivated material from Fusarium oxysporum f. sp. Cubense Race 1. The invention further pertains to a method for increasing resistance to Panama disease in a banana, a method for producing a banana having increased resistance as well as a banana obtained by such method.

Description

Panama disease resistance in banana

Field of the invention

The invention is in the field of improving pathogen resistance in plants. In particular, the invention pertains to compositions and methods that result in an increased resistance to Fusarium in banana.

Background

Banana (Musa spp.) is the fourth most important crop after rice, wheat, and corn (Heslop-Harrison and Schwarzacher 2007, FAOSTAT 2016). The Cavendish banana is the most widely-grown banana cultivar. Plantations devoted to this banana can be found in e.g. Latin America, Africa, and Southeast Asia. Their cultivation expanded globally and currently represents approximately 50% of the production area and dominates the export trade.

The so-called “fusarium wilt also known as “Panama disease" is a disease infecting banana plants, caused by the fungus Fusarium oxysporum. In the early 1960s the disease nearly whipped out the production of the, at that time dominant, cultivar Gros Michel, inflicting enormous costs and forcing the producers to switch to other, more resistant, cultivars. Indeed, Panama disease is one of the largest threats of the industry. Nowadays, a new outbreak of Panama disease caused by the strain Tropical Race 4 (TR4) (also known as Fusarium odoratissimurri) threatens the production of the presently dominant cultivar, the Cavendish banana. At the moment, there is no commercially available replacement for the Cavendish bananas.

In the art, several studies are available attempting to establish a mechanism for pathogen resistance in plants, such as induced systemic resistance (ISR) and systemically acquired resistance (SAR). ISR to nematodes (Vu et al. Nematology, 2006; 8(6): 847-852), the banana bunchy top virus (Kavino et al. Soil Biology and Biochemistry, 2007; 39(5): 1087-1098), and Fusarium strains (Thangavelu et al. Biologia Plantarum, 2003; 46: 107-112) are described in the art. More recently, Wu et al. (Journal of Plant Physiology, 2013; 170(1 1): 1039-1046) showed the effect of a non-pathogenic strain as activator of SAR on Cavendish in vitro plants.

In addition, several biotechnological approaches have been proposed in order to obtain plants showing resistance to fungal pathogens. For example, WO2020263561 discloses plant genes that may be involved in susceptibility for Fusarium. However, methods of producing transgenic plants are time-consuming, often unsuccessful and transgenic plants may give rise to regulatory hurdles.

Therefore, there still remains a strong need in the art for cost-effective and simple methodology for inducing resistance to Fusarium in banana plants

Summary

The invention may be summarized in the following embodiments:

Embodiment 1 . A composition for increasing resistance to Panama disease in a banana, preferably a Cavendish banana, wherein the composition comprises inactivated Fusarium fungal material. Embodiment 2. A composition according to embodiment 1 , wherein the inactivated material comprises inactivated cells, preferably inactivated spores of a Fusarium fungal strain, preferably inactivated conidia of a Fusarium fungal strain.

Embodiment 3. A composition according to embodiment 1 or 2, wherein at least about 60%, 70%, 80%, 90% or about 100% of the total amount of Fusarium fungal material in the composition is inactivated.

Embodiment 4. A composition according to any one of the preceding embodiments, wherein the Fusarium fungal material is from the fungal strain Fusarium oxysporum f. sp. cubense Race 1 (R1).

Embodiment 5. A composition according to any one of embodiments 2 - 4, wherein the composition comprises at least a concentration of about 1 x 10⁴ inactivated cells I ml, preferably 1 x 10⁴ - 1 x 10⁹ inactivated cells/ml, preferably wherein the composition comprises about 1 x 10⁵ - 1 x 10⁷ inactivated cells I ml.

Embodiment 6. A composition according to any one of embodiments 2 - 5, wherein the composition comprises a lysis buffer for inactivating the cells of the Fusarium fungal strain.

Embodiment 7. A composition according to embodiment 6, wherein the lysis buffer comprises the following components: i) an agent for pH control; ii) a chelating agent; and iii) a surfactant, and wherein the lysis buffer optionally comprises at least one of the following components: iv) an agent to remove phenolic compounds; v) a salt; vi) a reducing agent; and vii) a protease.

Embodiment 8. A composition according to embodiment 7 wherein at least one of: i) the agent for pH control is Tris-HCI, preferably at a concentration of about 10 mM - 100mM; ii) the chelating agent is EDTA, preferably at a concentration of about 1 mM - 20 mM; and iii) the surfactant is at least one of SDS, CTAB, CDA and Sarkosyl, preferably at a concentration of about 0.1 % - 2% (w/v), and optionally at least one of: iv) the agent to remove phenolic compounds is PVP, preferably at a concentration of about 1 % - 2% (w/v); v) the salt is NaCI, preferably at a concentration of about 0.1 M - 2M; vi) the reducing agent is at least one of DTT, preferably at a concentration of about 9 mM - 12 mM, and p-mercaptoethanol, preferably at a concentration of about 0.1 % - 0.2% (v/v); and vii) the protease is Proteinase K, preferably at a concentration of about 50 pg/ml - 150 pg/ml.

Embodiment 9. A method for increasing resistance to Panama disease in a banana, preferably a Cavendish banana, comprising the step of exposing at least one plant part of the banana to a composition as defined in any one of embodiments 1 - 8.

Embodiment 10. A method for producing a banana, preferably a Cavendish banana, that has increased resistance to Panama disease, comprising a step of exposing at least one plant part of the banana to a composition as defined in any one of embodiments 1 - 8.

Embodiment 11. The method according any one of the preceding claims, wherein the Panama disease is caused by the fungal strain Fusarium oxysporum f. sp. cubense Tropical Race 4 (TR4).

Embodiment 12. The method according to any one of embodiments 9 - 11 , wherein the at least one plant part is exposed to the composition for at least about 5, 10, 15, 20, 25 or 30 minutes.

Embodiment 13. Use of a composition according to any one of embodiments 1 - 8 for increasing resistance to Panama disease in a banana, preferably a Cavendish banana.

Embodiment 14. A banana obtainable by from the method of any one of embodiments 10 - 13.

Definitions

Various terms relating to the methods, compositions, uses and other aspects of the present invention are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art to which the invention pertains, unless otherwise indicated. Other specifically defined terms are to be construed in a manner consistent with the definition provided herein.

It is clear for the skilled person that any methods and materials similar or equivalent to those described herein can be used for practicing the present invention.

Methods of carrying out the conventional techniques used in methods of the invention will be evident to the skilled worker. The practice of conventional techniques in molecular biology, biochemistry, computational chemistry, cell culture, recombinant DNA, bioinformatics, genomics, sequencing and related fields are well-known to those of skill in the art and are discussed, for example, in the following literature references: Sambrook et al. Molecular Cloning. A Laboratory Manual, 4th Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., 2012; Ausubel et al.. Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1987 and periodic updates; the series Methods in Enzymology, Academic Press, San Diego and JM Walker, the series Methods in Molecular Biology, Springer Protocols. The singular terms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a cell” includes a combination of two or more cells, and the like. The indefinite article "a" or "an" thus usually means "at least one".

The term “and/or” refers to a situation wherein one or more of the stated cases may occur, alone or in combination with at least one of the stated cases, up to with all of the stated cases.

As used herein, the term “about” is used to describe and account for small variations. For example, the term can refer to less than or equal to ± (+ or -) 10%, such as less than or equal to ±5%, less than or equal to ±4%, less than or equal to ±3%, less than or equal to ±2%, less than or equal to ±1 %, less than or equal to ±0.5%, less than or equal to ±0.1 %, or less than or equal to ±0.05%. Additionally, amounts, ratios, and other numerical values are sometimes presented herein in a range format. It is to be understood that such range format is used for convenience and brevity and should be understood flexibly to include numerical values explicitly specified as limits of a range, but also to include all individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly specified. For example, a ratio in the range of about 1 to about 200 should be understood to include the explicitly recited limits of about 1 and about 200, but also to include individual ratios such as about 2, about 3, and about 4, and sub-ranges such as about 10 to about 50, about 20 to about 100, and so forth.

The term “comprising” is construed as being inclusive and open ended, and not exclusive. Specifically, the term and variations thereof mean the specified features, steps or components are included. These terms are not to be interpreted to exclude the presence of other features, steps or components.

“Increased resistance” refers to an increase in resistance of a plant or plant tissue compared to a suitable control plant. Increased resistance may be measured and optionally quantified by comparison of disease caused symptoms relative to those seen in susceptible control plants when grown under identical disease pressure. Such disease bioassays can be carried out using known methods. Both a qualitative increase (e.g. from susceptible to resistant) and a quantitative increase (e.g. a reduction of disease symptoms) are encompassed herein. Also encompassed is both a reduction of disease incidence (percentage of plants becoming infected), a delay in disease onset and/or reduction of disease severity. Preferably, a plant having improved resistance is a plant comprising at least 1 %, 2%, 5%, 10%, 15%, 20%, 30%, 50%, 70%, 80%, 90%, or even 100% higher levels of resistance to said Panama disease than the control plant, using suitable bioassays and/or field assays for assessing disease resistance under similar conditions. For example, a systemic response, preferably detectable by visual inspection, developed in response to the Fusarium oxysporum f. sp. cubense infection may be determined in both a plant as taught herein and a control plant, and may be compared, e.g. as shown in the Examples section. In addition or alternatively, a plant part, such as e.g. systemic leaves or roots, or a plant as taught herein may be quantitatively analyzed, preferably by quantitative PCR, for the presence of Fusarium nucleic acids in response to infection and preferably compared to such data of a control plant in response to the same infection.

The terms “inducing”, increasing” or “improving” resistance to Panama disease can be used interchangeable herein and refers to the delay in onset, severity and/or the prevention of Panama disease caused by the fungus Fusarium oxysporum f. sp. Cubense, preferably caused by the strain Race 4, preferably Tropical Race 4 (TR4).

A “nucleic acid” or “polynucleotide” according to the present invention may include any polymer or oligomer of pyrimidine and purine bases, preferably cytosine, thymine, and uracil, and adenine and guanine, respectively (See Albert L. Lehninger, Principles of Biochemistry, at 793-800 (Worth Pub. 1982) which is herein incorporated by reference in its entirety for all purposes). The present invention contemplates any deoxyribonucleotide, ribonucleotide or nucleic acid component, and any chemical variants thereof, such as methylated, hydroxy methylated or glycosylated forms of these bases, and the like. The polymers or oligomers may be heterogeneous or homogenous in composition, and may be isolated from naturally occurring sources or may be artificially or synthetically produced. In addition, the nucleic acids may be DNA (optionally cDNA) or RNA, or a mixture thereof, and may exist permanently or transitionally in single-stranded or double-stranded form, including homoduplex, heteroduplex, and hybrid states. An “isolated nucleic acid” is used to refer to a nucleic acid which is no longer in its natural environment, for example in vitro or in a recombinant bacterial or plant cell.

The terms “protein” or “polypeptide” are used interchangeably and refer to molecules consisting of a chain of amino acids, without reference to a specific mode of action, size, 3 dimensional structure or origin. A “fragment” or “portion” of a protein may thus still be referred to as a “protein.” An “isolated protein” is used to refer to a protein which is no longer in its natural environment, for example in vitro or in a recombinant bacterial or plant cell.

"Plant" refers to either the whole plant or to parts of a plant, calli, tissue, rootstock, scion, organs (e.g. embryos pollen, ovules, seeds, gametes, roots, leaves, flowers, flower buds, anthers, fruit, etc.) obtainable from the plant, as well as derivatives of any of these and progeny derived from such a plant by selfing or crossing.

"Plant cell(s)" include protoplasts, gametes, suspension cultures, microspores, pollen grains, etc., either in isolation or within a tissue, organ or organism. The plant cell can e.g. be part of a multicellular structure, such as a callus, meristem, plant organ or an explant.

“Similar conditions” for culturing the plant / plant cells means among other things the use of a similar temperature, humidity, nutrition and light conditions, and similar irrigation and day/night rhythm.

The term “cDNA” means complementary DNA. Complementary DNA is made by reverse transcribing RNA into a complementary DNA sequence. cDNA sequences thus correspond to RNA sequences that are expressed from genes. As mRNA sequences when expressed from the genome can undergo splicing, i.e. introns are spliced out of the mRNA and exons are joined together, before being translated in the cytoplasm into proteins, it is understood that expression of a cDNA means expression of the mRNA that encodes for the cDNA. The cDNA sequence thus may not be identical to the genomic DNA sequence to which it corresponds as cDNA may encode only the complete open reading frame, consisting of the joined exons, for a protein, whereas the genomic DNA encodes and exons interspersed by intron sequences. Genetically modifying a gene which encodes the cDNA may thus not only relate to modifying the sequences corresponding to the cDNA, but may also involve mutating intronic sequences of the genomic DNA and/or other gene regulatory sequences of that gene, as long as it results in the impairment of gene expression.

“Panama disease caused symptoms” include any symptoms of Panama disease. The first visible symptoms of Panama disease are stunted growth, leaf distortion and yellowing especially of the oldest leaves, and wilt along the edges of mature, lower leaves. The leaves may gradually collapse and droop from the plant, eventually drying up completely. The yellowing of the oldest leaves may progress to the newest ones. The symptoms may be divided into symptoms of the growing point, leaves, roots and stems. A disease-symptom of the growing point may be a dead heart, a disease-symptom of the leaves may be at least one of abnormal colours, abnormal leaf fall, necrotic areas, wilting, yellowed or dead leaves, and a disease-symptom of the roots may be at least one of rot of wood, soft rot of cortex, discoloration of bark, distortion, internal discoloration, stunting or resetting of a root. A disease-symptom of the pseudostem is breaking of the pseudostem also known as pseudostem splitting. Internally, red-brown discoloration of vascular areas and rhizome is observed with necrotic areas. Eventually this causes collapse of the plant.

The term “regeneration” is herein defined as the formation of a new tissue and/or a new organ from a single plant cell, a callus, an explant, a tissue or from an organ. The regeneration pathway can be somatic embryogenesis or organogenesis Somatic embryogenesis is understood herein as the formation of somatic embryos, which can be grown to regenerate whole plants. Organogenesis is understood herein as the formation of new organs from (undifferentiated) cells. Preferably, the regeneration is at least one of ectopic apical meristem formation, shoot regeneration and root regeneration. The regeneration as defined herein can preferably concern at least de novo shoot formation. For example, regeneration can be the regeneration of a(n) (elongated) hypocotyl explant towards a(n) (inflorescence) shoot. Regeneration may further include the formation of a new plant from a single plant cell or from e.g. a callus, an explant, a tissue or an organ. The regeneration process can occur directly from parental tissues or indirectly, e.g. via the formation of a callus.

“Resistance to Panama disease” refers herein to various levels of resistance or tolerance of a plant, including moderate resistance and high resistance or complete resistance to Panama disease. Resistance to Panama disease can be indirectly measured as higher yield of resistant plants compared to susceptible plants when grown under disease pressure, such as after inoculation with the fungus Fusarium oxysporum f. sp. cubense, preferably strain Race 4, preferably the strain Tropical Race 4 (TR4).

The term “rootstock” as used herein refers to part of a plant, often an underground part, from which new above-ground growth can be produced. For instance, it may refer to a rhizome or underground stem. In grafting, it refers to a plant, sometimes just a stump, which already has an established, healthy root system, onto which a cutting, a bud, or a scion from another plant is grafted.

The term “scion” as used herein refers to part of a plant, often an upper-ground part, that will produce the plant shoots. It will give rise to the plant’s leaves, stems, flowers and/or fruits. The scion is typically the top part of a grafted plant. A “control plant” as referred to herein is a plant of the same species and preferably same genetic background as the plant that is, or is a progeny of, a plant (or “putative test plant” or “test plant”) that has been subjected to a method as taught herein, i.e. a method for increasing resistance to Panama disease. The control plant is preferably susceptible to Panama disease, preferably Panama disease caused by fungal strain Fusarium oxysporum f. sp. cubense Tropical Race 4 (TR4). The control plant preferably develops symptoms of Panama disease or has a low resistance to Panama disease, e.g. the fungi can successfully invade, establish themselves and spread in the control plant. Preferably, the control plant only differs from the putative test plant in that the control plant is not exposed to a composition of the invention as defined herein. Preferably the control plant is grown under the same conditions as the plant of the invention as defined herein.

Detailed description

The inventors discovered an effective method for inducing resistance to Panama disease in banana plants. More in particular, the inventors discovered a composition that can be applied to the plant part, resulting in increased resistance to Panama disease, without exposing the environment to live spores.

Panama disease is a wilting disease caused by the fungus Fusarium oxysporum f. sp. cubense. This fungus produces three types of asexual spores (macroconidia, microconidia and chlamydospores) and can be divided into four different races, Race 1 , Race 2, Race 3 and Race 4. Race 4 may be subdivided in Tropical Race 4 (TR4) and Subtropical Race 4 (STR4). In the 1950s - 1960s, Race 1 destroyed a significant part of the Gros Michel banana plantations. The Cavendish banana is resistant to Race 1 and is nowadays the predominant commercial cultivar. The Cavendish banana is however not resistant to Race 4, which is the causal agent of the current Panama disease.

In an aspect, the invention pertains to a composition for increasing resistance to Panama disease in a plant, preferably in a banana, wherein the composition comprises inactivated Fusarium fungal material. The inactivated material is preferably at least one of inactivated cells of a Fusarium fungal strain and a nucleic acid of a Fusarium fungal strain. A preferred banana is a Cavendish banana. Therefore, preferably the composition of the invention increases resistance to Panama disease in a plant, preferably a Cavendish banana, after contacting at least part of said plant with the composition. Optionally, said plant part is one or more roots of said plant.

The composition of the invention may result in the delay in onset, severity or prevention of one or more of these symptoms of Panama disease.

As a non-limiting example, the conferred or improved resistance to Panama disease can be determined by comparing a control plant with a plant of the invention, under controlled conditions chosen such that in the control plant at least one sign of infection can be observed after a certain period. Such controlled conditions include e.g. infection or infestation of plants with Fusarium oxysporum f. sp. cubense, preferably Race 4, preferably Fusarium strain Tropical Race 4. Under the controlled conditions chosen, the control plant shows preferably at least one disease sign (i.e. one symptom) upon infection as known in the art and/or as exemplified herein. The control plant can show one or more of the symptoms of Panama disease as indicated herein, preferably at least two, three, four or five the signs of the disease after a certain period of time.

Said certain period is preferably a time period normal for a (control) plant to develop Panama disease after inoculation with a Fusarium strain. Such period can be easily determined by the person skilled in the art. Preferably, such period before observing at least one, two or three signs of disease is at least about 2, 3, 4, 5, 10, 20, 30, 35, 40, 45, 50, 60 or about 70 days, preferably at least about 1 , 2, 3, 4, 5, 6 or 7 weeks. Preferably, the plant of the invention will show less, reduced and/or no signs of disease as compared to the control plant after the certain period as defined herein.

Alternatively or in addition, the plant of the invention may show one or more signs of disease as defined herein at a similar or same severity as the control plant, however the one or more signs of disease will be at a later time period as compared to the control plant, e.g. there will be a delay in onset of one or more symptoms of the disease. As a non-limiting example, there can be a delay of at least 1 , 2, 3, 4, 5, 6, 7 or 8 weeks as compared to the control plants. The skilled person knows how to select suitable conditions.

When a plant has an improved resistance to Panama disease, it is preferably capable of sustaining a normal growth and/or a normal development when the plant is subjected to infection with Race 4, preferably tropical race 4 (TR4), which infection would otherwise have resulted in reduced growth and/or reduced development of the plant. Hence, an improved Panama disease resistance can be determined by comparing plants. As a non-limiting example, one plant of the invention may be compared with one control plant. Alternatively or in addition, a group of plants of the invention may be compared with a group of control plants. Each group can comprise e.g. at least about 2 ,3, 4, 5, 10, 15, 20, 25, 50 or 100 individual plants. The skilled person is well aware how to select appropriate conditions to determine improved Panama disease resistance and how to measure signs of infection.

The invention pertains to a composition for increasing resistance to Panama disease in a banana, wherein the composition comprises inactivated Fusarium fungal material. The inactivated fungal material may be one or more inactivated cells or fragments thereof. Inactivated Fusarium fungal material is understood herein as material that is unable to propagate and/or is nonviable. Preferably, inactivated Fusarium fungal material has lost the ability to grow on PDA (Potato Dextrose Agar) plates, when incubated at 25°C in the dark for 5 days.

Inactivated Fusarium fungal material may comprise inactivated Fusarium fungal cells, e.g. cell debris, or one or more cellular components of a Fusarium fungal cell. Such cellular components include, but are not limited to, components of (part of) a cell wall, cell membrane, organelle, a (glyco)protein, a nucleic acid and/or an (oligo)saccharide.

The composition of the invention preferably comprises inactivated cells of a Fusarium fungal strain. The cells may be inactivated by any conventional method known in the art for inactivating fungal cells. Preferably, the inactivation denatures and/or hydrolysis at least part of the of one or more fungal (glyco)proteins. Preferably, the inactivation denatures at least part of the fungal cell wall and cell membrane. The inactivation may retain at least part of the integrity of one or more fungal (oligoZpoly)saccharides. The inactivation method may retain at least part of the integrity of the fungal nucleic acids. Preferably, the inactivation does not, or not substantially, degrade nucleic acids of the fungus. Preferably, the inactivation method does not, or does not substantially, degrade the transcriptome of the fungus. Preferably, the inactivation method does not, or does not substantially, degrade transcripts having a length of less than about 10 kb, 8 kb, 6kb, 4kb, 3kb, 2kb, or less than about 1 kb.

Preferably the composition comprises one or more Fusarium fungal nucleic acids, preferably one or more isolated Fusarium fungal nucleic acids, wherein said one or more nucleic acids preferably are from Fusarium oxysporum f. sp. cubense, even more preferably from Fusarium oxysporum f. sp. Cubense Race 1 (R1). The fungal nucleic acid preferably has a length of about 0.1 kb - 10 kb, 0.5 kb - 8 kb, 1 kb - 6 kb or about 2 kb - 4 kb. The nucleic acid may be genomic DNA, a transcribed nucleic acid, or a fragment thereof. The nucleic acid may comprise or consist of DNA or RNA. The nucleic acid may be a naturally derived nucleic acid or a synthetic nucleic acid. The synthetic nucleic acid preferably comprises a nucleotide sequence corresponding to a nucleotide sequence present in the Fusarium fungal cell. Preferably, the sequence of the synthetic nucleic acid has at least about 70%, 80%, 90%, 95%, 98% or 100% sequence identity with a nucleic acid present in the Fusarium fungal cell. Preferably, the nucleic acid has 100% sequence identity with a nucleic acid obtainable from a Fusarium fungal strain, preferably the nucleic acid in the composition has 100% sequence identity with a nucleic acid obtainable from Fusarium oxysporum f. sp. cubense, preferably the nucleic acid has 100% sequence identity with a nucleic acid obtainable from Fusarium oxysporum f. sp. cubense Race 1 (R1).

The composition of the invention preferably comprises inactivated cells of a Fusarium fungal strain. Preferably the cells are spores, preferably asexual spores (conidia), and the composition thus preferably comprises inactivated Fusarium conidia, preferably inactivated conidia from the fungal strain Fusarium oxysporum f. sp. cubense Race 1 (R1). Part of the composition may still comprise active, or viable, cells, preferably spores. Preferably, the percentage of cells, preferably spores, in the composition that is still viable is preferably less than about 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 3%, or less than about 1 %. Preferably, there are no viable spores in the composition. Equally, the percentage of spores in the composition that is inactivated is preferably more than about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98% or about 100%.

Preferably, the composition of the invention comprises inactivated Fusarium spores, preferably inactivated Fusarium oxysporum f. sp. cubense spores. Preferably, the composition comprises inactivated Fusarium oxysporum f. sp. cubense Race 1 (R1) spores.

The amount of (inactivated) Fusarium cells, preferably spores, preferably Fusarium oxysporum f. sp. cubense Race 1 (R1) spores, in the composition is an amount that preferably results in an increased resistance to Panama disease when used in a method of the invention as detailed herein.

The concentration of inactivated Fusarium cells in the composition may be quantitatively determined, e.g. by measuring the concentration of one or more Fusarium proteins and/or one or more nucleic acids in the composition. As a non-limiting example, the determined protein and/or nucleic acid concentration may e.g. be compared to a calibration curve to retrieve the concentration inactivated Fusarium cells in the composition.

Alternatively or in addition, the concentration viable cells may be determined, followed by an inactivation step. The concentration viable cells may be determined using any method known in the art for the skilled person, e.g. by cell counting or absorbance measurements, e.g. at 530 nm as described in Pablo F. Caligiore-Gei and Jorge G. Valdez, Revista Argentina de Microbiologia, 47 (2), p152-154). After determining the concentration viable Fusarium cells, the cells may be inactivation.

The Fusarium cells may be inactivated using any method known in the art. Preferably, the Fusarium cells are inactivated using cell lysis. The Fusarium cells preferably not inactivated using heating, preferably the Fusarium cells are not inactivated by heating to about 120°C for at least about 20 minutes. Preferably, the inactivation does not comprise heating above about 95°C, 100°C, 110°C or 120°C, wherein said heating takes place for at least about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20 min. Preferably, inactivation does not comprise heating above about 95°C for at least about 10, 15 or 20 min, or above about 100°C for at least about 2, 5 or 10 min, or above about 120°C for at least about 1 , 2 or 5 min.

The amount of (inactivated) Fusarium cells in the composition, preferably the amount of (inactivated) Fusarium oxysporum f. sp. cubense Race 1 (R1) spores, is preferably at least about 1 x 10³ inactivated cells I ml, preferably at least about 5 x 10³ cells/ml, 1 x 10⁴ cells/ml, 5 x 10⁴ cells/ml, 1 x 10⁵ cells/ml, 5 x 10⁵ cells/ml, 1 x 10⁶ cells/ml, 5 x 10⁶ cells/ml, 1 x 10⁷ cells/ml, 5 x 10⁷ cells/ml, 1 x 10⁸ cells/ml, 5 x 10⁸ cells/ml or at least about 1 x 10⁹ inactivated cells/ml. Optionally, there are less than about 1 x 10⁹ inactivated cells/ml in the composition, preferably less than about 5 x 10⁸ cells/ml, 1 x 10⁸ cells/ml, 5 x 10⁷ cells/ml, or less than about 1 x 10⁷ inactivated cells/ml. Preferably, the concentration (inactivated) Fusarium cells in the composition is about 1 x 10³ - 1 x 10⁹ cells/ml, about 5 x 10³ - 5 x 10⁸ cells/ml, about 1 x 10⁴ - 1 x 10⁸ cells/ml, about 5 x 10⁴ - 5 x 10⁷ cells/ml, about 1 x 10⁵ - 1 x 10⁷ cells/ml, 1 x 10⁵ - 5 x 10⁶ cells/ml, 1 x 10⁵ - 1 x 10⁶ cells/ml, or about 5 x 10⁵ - 5 x 10⁶ inactivated cells/ml, preferably around about 1 x 10⁶ inactivated cells/ml.

For inactivation, active (viable) Fusarium cells, preferably active Fusarium oxysporum f. sp. cubense Race 1 (R1) spores, may be resuspended in a lysis buffer, causing their inactivation e.g. after heating the composition to about 65°C for about 30 minutes. In the method of the invention, a plant part, preferably a root of a banana, may be directly exposed to the composition comprising a lysis buffer and inactivated Fusarium cells. The composition of the invention therefore preferably comprises i) a lysis buffer and ii) inactivated Fusarium cells, preferably inactivated Fusarium oxysporum f. sp. cubense Race 1 (R1) spores. The composition may comprise a lysis buffer as defined herein in addition to inactivated Fusarium cells. The composition may consist of a lysis buffer, preferably a lysis buffer as defined herein, in addition to the inactivated Fusarium cells. Alternatively the composition may comprise a dilution of the lysis buffer as defined herein. After dilution, the amount of Fusarium cells is preferably an amount as defined herein. As a non-limiting example, lysis buffer comprising 5 x 10⁷ spores/mL may be diluted 50 times, such that the composition of the invention comprises 1 x 10⁶ spores/mL and 2% lysis buffer, wherein the lysis buffer preferably comprises the components as defined herein. The lysis buffer may be diluted with e.g. water, such as e.g. tab water, Milli-Q water and/or distilled water. The composition may thus comprise 1) lysis buffer, 2) inactivated Fusarium cells, and 3) water.

Optionally, at least about 0.05%, 1 %, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or about 100% (v/v) of the composition is a lysis buffer. Optionally 100%, or at most 98%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 2%, 1 % or at most about 0.05% (v/v) of the composition is a lysis buffer. Optionally, about 0.05% (v/v) - 100% (v/v), about 1 % (v/v) - 95% (v/v), about 2% (v/v) - 90% (v/v), about 5% (v/v) - 80% (v/v), about 10% (v/v) - 70% (v/v), about 20% (v/v) - 60% (v/v), about 30% (v/v) - 65% (v/v), or about 40% (v/v) - 60% (v/v) of the composition is lysis buffer. Preferably, the composition is a lysis buffer comprising inactivated Fusarium cells as defined herein. Preferably, the lysis buffer is a lysis buffer as defined herein.

The lysis buffer may be any conventional lysis buffer known in the art that is suitable for the lysis of fungal cells, preferably suitable for the lysis of Fusarium oxysporum f. sp. cubense Race 1 (R1) spores. The lysis buffer may be a commercially available lysis buffer. Components for lysing fungal cells are generally known in the art. The invention is not limited to any specific lysis buffer.

Preferably, the lysis buffer comprises at least a surfactant as defined herein. Preferably, the lysis buffer comprises a least a chelating agent as defined herein. Preferably, the lysis buffer comprises at least a surfactant and chelating agent as defined herein. Preferably, the lysis buffer comprises at least the following components: i) an agent for pH control; ii) a chelating agent; and iii) a surfactant.

A non-limiting example of i) an agent for pH control is Tris HCI. Preferably the lysis buffer comprises Tris HCI, The concentration of the pH controlling agent, preferably Tris-HCI, in the lysis buffer is preferably in between about 1 mM - 1000 mM, in between about 3 mM - 500 mM, in between about 5 mM - 200 mM or in between about 10 mM - 100 mM. The concentration of the pH controlling agent, preferably Tris-HCI, in the lysis buffer is preferably in between about 10 mM - 100mM. The concentration of the pH controlling agent, preferably Tris-HCI, in the lysis buffer is preferably about 10 mM or 100 mM.

A non-limiting example of ii) a chelating agent is at least one of EDTA and EGTA, preferably is EDTA, preferably Na-EDTA. The concentration of the chelating agent, preferably EDTA, in the lysis buffer is preferably in between about 0.1 mM - 100 mM, in between about 0.5 mM - 50 mM, in between about 0.8 mM - 25 mM, in between about 1 mM - 20 mM or in between about 1 mM - 10 mM. The concentration of the chelating agent, preferably EDTA, is in the lysis buffer is preferably in between about 1 mM - 20 mM. The concentration of the chelating agent, preferably EDTA, in the lysis buffer is preferably about 1 mM, 10 mM or 20 mM.

A non-limiting example of iii) a surfactant is at least one of SDS, CTAB, CDA, Triton X-100 and Sarkosyl. Preferably, the surfactant is at least one of SDS, CTAB, CDA and Sarkosyl. Optionally, a combination of surfactants is used, e.g. a combination of CDA and Sarkosyl or a combination of e.g. CTAB and Sarkosyl. The concentration of the surfactant in the lysis buffer is preferably in between about 0.01 % - 10%, in between about 0.05% - 5% (w/v), in between about 0.1 % - 3% (w/v), or in between about 0.5% - 2.5% (w/v). Preferably the concentration of the surfactant, preferably the concentration of at least one of SDS, CTAB, CDA and Sarkosyl, or a combination thereof, in the lysis buffer is in between about 0.1 % - 2.0% (w/v). The concentration of the surfactant in the lysis buffer, preferably the concentration of at least one of SDS, CDA, CTAB and Sarkosyl, or a combination thereof, is preferably about 0.1 %, 1 .5% or 2% (w/v).

If the surfactant in the lysis buffer is SDS (Sodium dodecyl sulfate) , the concentration is preferably in between about 0.01 % - 10%, in between about 0.05% - 5% (w/v), in between about 0.1 % - 3% (w/v), or in between about 0.5% - 2.5% (w/v). Preferably the SDS concentration in the lysis buffer is in between about 0.1 % - 2.0% (w/v). Preferably, the SDS concentration in the lysis buffer, is about 0.1 %, 1 .5% or 2% (w/v).

If the surfactant in the lysis buffer is CTAB (Cetyltrimethylammonium bromide), the concentration is preferably in between about 0.01 % - 10%, in between about 0.05% - 5% (w/v), in between about 0.1 % - 3% (w/v), or in between about 0.5% - 2.5% (w/v). Preferably the CTAB concentration in the lysis buffer is in between about 0.1 % - 2.0% (w/v). Preferably, the CTAB concentration in the lysis buffer, is about 0.1 %, 1 .5% or 2% (w/v).

If the surfactant in the lysis buffer is CDA (ethylhexadecyldimethylammonium bromide), the concentration is preferably in between about 0.01 % - 10%, in between about 0.05% - 5% (w/v), in between about 0.1 % - 3% (w/v), or in between about 0.5% - 2.5% (w/v). Preferably the CDA concentration in the lysis buffer is in between about 0.1 % - 2.0% (w/v). Preferably, the CDA concentration in the lysis buffer, is about 0.1 %, 1 .5% or 2% (w/v).

If the surfactant in the lysis buffer is Sarkosyl (N-Lauroylsarcosine sodium salt), the concentration is preferably in between about 0.01 % - 10%, in between about 0.05% - 5% (w/v), in between about 0.1 % - 3% (w/v), or in between about 0.5% - 2.5% (w/v). Preferably the Sarkosyl concentration in the lysis buffer is in between about 0.1 % - 2.0% (w/v). Preferably, the Sarkosyl concentration in the lysis buffer, is about 0.1 %, 1 .5% or 2% (w/v).

A preferred agent for pH control is Tris HCI. A preferred chelating agent is EDTA. A preferred surfactant is selected from the group consisting of SDS, CDA, CTAB and Sarkosyl.

In addition to components i) - iii) as indicated above, the lysis buffer for lysing the Fusarium fungal cells may additionally comprise at least one of the following components: iv) an agent to remove phenolic compounds; v) a salt; vi) a reducing agent; and vii) A protease.

A non-limiting example of iv) an agent for removing phenolic compounds is PVP (Polyvinylpyrrolidone), preferably at least one of PVP-10 and PVP-40, preferably PVP-10. Preferably the lysis buffer comprises PVP, preferably PVP-10. The concentration of the agent for removing phenolic compounds, preferably PVP, preferably PVP-10, in the lysis buffer is preferably in between about 0.1 % - 10% (w/v), in between about 0.5% - 5% (w/v), or between about 1 % - 2% (w/v). The concentration of the agent for removing phenolic compounds, preferably PVP, preferably PVP-10, is preferably about 1 % - 2% (w/v). The concentration of the agent for removing phenolic compounds, preferably PVP, preferably PVP-10, is preferably about 1% or about 2% (w/v). A preferred agent for removing phenolic compounds is PVP.

A non-limiting example of v) a salt is NaCI. Preferably the lysis buffer comprises NaCI. The concentration of the salt, preferably the concentration of NaCI, in the lysis buffer is preferably between about 0.01 m - 10 M, between about 0.05M - 5M, between about 0.1 M - 2 M or between about 1 M - 1 .5 M. The concentration of the salt, preferably NaCI, in the lysis buffer is preferably in between about 0.1 M - 2M. The concentration of the salt, preferably NaCI, in the lysis buffer is preferably about 0.1 M, 1 ,2M or 1 ,4M.

A non-limiting example of vi) a reducing agent is DTT (1 ,4-Dithiothreitol) or p- mercaptoethanol. Preferably the lysis buffer comprises at least one of is DTT and p- mercaptoethanol. The reducing agent in the lysis buffer may be DTT. The concentration DTT in the lysis buffer is preferably between about 1 mM - 100 mM, between about 5 - 50 mM, between about 7 mM - 30 mM, between about 8 mM - 20 mM, or between about 9 mM - 12 mM. The concentration DTT is preferably about 10 mM. The reducing agent in the lysis buffer may be p-mercaptoethanol. The concentration p-mercaptoethanol in the lysis buffer is preferable between about 0.01 % - 1 % (v/v), between about 0.05% - 0.5% (v/v) or between about 0.1 % - 0.2% (v/v). The concentration p- mercaptoethanol in the lysis buffer is preferably between about 0.1 % - 0.2% (v/v), preferably about 0.1 % or about 0.2%.

A non-limiting example of vii) a protease is Proteinase K. Preferably, the protease in the lysis buffer is Proteinase K. The concentration of the protease, preferably the concentration of Proteinase K, in the lysis buffer is preferably between about 10 pg/ml - 1000 pg/ml, between about 50 pg/ml - 500 pg/ml, between about 75 pg/ml - 200 pg/ml, between about 80 pg/ml - 120 pg/ml. The concentration of the protease, preferably Proteinase K, in the lysis buffer is preferably between about 50 pg/ml - 150 pg/ml. The concentration of the protease, preferably proteinase K, is preferably about 100 pg/ml.

A preferred salt is NaCI, a preferred reducing agent is at least one of DTT and p-mercaptoethanol and a preferred protease is Proteinase K.

Preferably, the lysis buffer comprises the surfactant CDA, preferably in a concentration as defined herein. Preferably, the lysis buffer comprises the chelating agent EDTA, preferably in a concentration as defined herein. Preferably, the lysis buffer comprises both the surfactant CDA and the chelating agent EDTA, preferably in concentrations as defined herein.

Preferably, the lysis buffer comprises the following components: i) Tris HCI; ii) EDTA; and iii) at least one of SDS, CTAB, CDA and Sarkosyl,

The lysis buffer may further comprise one or more components selected from the group consisting of iv) PVP; v) NaCI; vi) at least one of DTT or p-mercaptoethanol; and vii) Proteinase K

Optionally the lysis buffer comprises the components of Buffer 1 , 2, 3, 4 or 5 as shown in Table 1 . Optionally, the lysis buffer comprises the components and concentrations of Buffer 1 , 2, 3, 4 or 5 as shown in Table 1 .

In the embodiment wherein the composition comprises diluted lysis buffer, the skilled person readily understands how the adjust the concentrations of the components as defined herein to determine the final concentration of the components in the composition. As a non-limiting example, if the composition comprises 50% lysis buffer, the concentrations of the components of the lysis buffer may be divided by 2 to determine the final concentration of the components in the composition. Equally, if the composition comprises 2% lysis buffer, the concentrations of the components of the lysis buffer may be divided 50 times to determine the final concentration of the components in the composition.

Table 1. Exemplified lysis buffers and their components

¹The PVP is preferably at least one of PVP-10 and PVP-40, preferably PVP-10.

² The buffer may additionally comprise 0.1 -0.2 % (v/v) p-mercaptoethanol

³ or CTAB (Cetyltrimethylammonium bromide)

Method for inducing resistance

In an aspect, the invention further pertains to a method for increasing resistance to Panama disease in a plant.

A preferred plant for use in the invention is a cultivar, preferably a cultivar of the genus Musa, Heliconia or the genus Ensete. Preferably, the cultivar plant of the genus Musa is at least one of a Musa acuminata, a Musa balbisiana, a Musa coccinea, a Musa velutina and a Musa x troglodytarum. A preferred cultivar plant of the genus Ensete is an Ensete glaucum or an Ensete ventricosum.

Preferably the plant for use in the invention is a banana. A preferred banana for use in the invention is a dessert banana or a plantains. A preferred banana may be a plant from the AA group (diploid Musa acuminata), AAA group (triploid Musa acuminata), AAAA group (tetrapioid Musa acuminate), AAAB Group (tetrapioid Musa balbisiana x Musa balbisiana), AAB group (triploid Musa ), AABB group (tetrapioid Musa, AB group, ABB group (triploid Musa x paradisiaca), ABBB group (tetrapioid Musa x paradisiaca) or BB group (diploid Musa balbisiana). Preferably, the banana plant for use in the invention is a plant from the AAA group. A particularly preferred plant for use in the invention is a plant of a subgroup of Musa acuminata, preferably a Musa cavendishii or “Cavendish banana".

The Cavendish banana for use in the invention may be a Dwarf Cavendish, Grande Naine, Extra Dwarf Cavendish, a Giant Cavendish, a Poyo, a Valery, a Robusta, a Williams, a Double and a Pisang Masak Hijau (syn 'Lacatan').

The resistance is preferably increased as compared to a control banana plant that is not exposed to a composition as defined herein.

The method comprises a step of exposing at least a part of the plant to a composition as defined herein. Optionally, the complete plant is exposed to the composition. Preferably at least part of the shoot system or root system is exposed to the composition, preferably at least part of the root system is exposed to the composition as defined herein.

The composition may be sprayed onto the plant, painted onto the plant, the plant may be partly or fully submerged (“dipped”) into a composition as defined herein and/or the composition may be added to the soil wherein the plant is growing. Preferably, at least part of the plant is dipped into a composition as defined herein.

Preferably, the composition is added to the soil wherein the plant is planted and/or growing. The amount of the composition added to the soil is sufficient to increase resistance to Panama disease in the plant that is grown in said soil. The skilled artisan can straightforwardly determine the amount of composition that should be added to the soil to increase resistance to Panama disease. The composition may be sprayed or poured onto the soil. Alternatively or in addition, the composition is injected into the soil. Hence in an embodiment, provided herein is a combination of i) a composition as described herein and ii) soil. The soil is preferably a soil suitable for planting and growing a banana plant. Preferably, the soil is a soil that is optimal for planting and growing a banana plant. The soil is optionally enriched with nutrients for plants, preferably nutrients for banana plants. Preferably one or more roots of the banana plant is exposed to a composition as defined herein. Preferably, at least about 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% of the roots of the plant are exposed to the composition comprising inactivated Fusarium fungal material. Optionally about 50% of the roots are exposed to a composition as defined herein.

Optionally the composition is exposed to one or more cells that are subsequently regenerated into a banana plant. Optionally, one or more roots are exposed to a composition as defined herein, wherein the roots are part of a rootstock and a scion may be grafted onto the rootstock. Optionally a seed of a banana plant is exposed to a composition as defined herein.

Optionally, the plant is a sprout, a seedling, a plant at the vegetative stage, a plant at the budding stage, a flowering plant or a plant at the ripening stage. The plant preferably as at least one or more real leaves, such as at least 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 real leaves. Preferably, the plant is about 0.5 - 12 months old, preferably about 1 - 6 months, preferably about 2 - 3 months old.

The plant or plant part, preferably one or more roots, may be exposed once to the composition or more may be repeatedly exposed to a composition of the invention. The plant may optionally be re-exposed to a composition in case the resistance to Panama disease decreases. The plant may be re-exposed to the composition once every week, once every month or once every year. The plant may be exposed to the composition about 1x, 2x, 3x, 4x, 5x, 6x, 7x, 8x,9x,10x, 11x or 12x per year. Preferably, there are at least 4, 6, 8, 10, 12, 14, 16, 18, 20 or more weeks in between each treatment. Preferably, there are at least 6 weeks in between each treatment.

The plant or plant part may be exposed to a composition as defined herein for a sufficiently long period to allow for an increase in resistance to Panama disease. Preferably, the plant or plant part is exposed to the composition for at least about 1 , 2, 3, 4 or 5 minutes. Optionally, the plant or plant part is exposed to the composition for at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60 minutes. Optionally, the plant or plant part is exposed to the composition for at least about 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 hours. Preferably, the plant or plant part is exposed to the composition for at least about 30 minutes.

Method for producing a banana having increased resistance to Panama disease

In an aspect, the invention further pertains to a method for producing a plant, wherein the plant has an increased resistance to Panama disease, preferably as compared to a control plant. Preferably said plant is a plant as defined in the aspect relating to the method for increasing resistance to Panama disease. Preferably the plant is a banana. The produced banana having an increased resistance to Panama disease is preferably a Cavendish banana. The method preferably comprises the step of exposing part of a banana, preferably one or more roots, to a composition as defined herein. Preferably the banana having an creased resistance to Panama disease is produced using a method for increasing resistance to Panama disease as outlined above. The method preferably comprises the steps of exposing at least part of the plant, preferably one or more roots of a banana, to a composition of the invention. The invention further pertains to a plant, preferably a banana, even more preferably a Cavendish banana, obtainable by the method of the invention. The plant, preferably the banana, of the invention has an increased resistance to Panama disease, preferably as compared to a control plant. The plant of the invention preferably differs at least from a plant occurring in nature, in that it has an increased resistance to Panama disease. Preferably, the plant of the invention is not, or is not exclusively, obtained by an essentially biological process.

Further aspects

In a further aspect, the invention pertains to the use of a composition as defined herein for increasing resistance to Panama disease in a banana.

In another aspect, the invention pertains to a composition comprising lyophilized and inactivated Fusarium fungal material, preferably lyophilized inactivated spores of Fusarium oxysporum f. sp. Cubense Race 1 (R1). The composition may be reconstituted prior to use in a method of the invention as detailed herein. Upon reconstitution, the composition may comprise the inactivated Fusarium fungal material at an amount as described herein above. Upon reconstitution, the composition may be a composition of the invention as described herein above. The lyophilized Fusarium fungal material may be reconstituted using any appropriate solution, preferably a solution that is not toxic to the plants. Preferably the solution for reconstituting the lyophilized and inactivated Fusarium fungal material is water.

In an aspect, provided herein is a combination of i) a composition as described herein and ii) soil, preferably a soil as described herein. Optionally, the combination comprises a reconstituted and/or a diluted composition, wherein the composition is as described herein. Optionally, the soil of the combination is enriched with nutrients for plants, preferably nutrients for banana plants.

The present invention has been described above with reference to a number of exemplary embodiments. Modifications and alternative implementations of some parts or elements are possible, and are included in the scope of protection as defined in the appended claims

Figure legends

Figure 1. Split root system. Plant roots were cleaned and divided in two parts. One part was immersed in a container comprising a spore suspension of Race 1 and the other part was immersed in a container comprising a spore suspension of Tropical Race 4.

Figure 2 Race 1 (R1) protects against Tropical Race 4 (TR4) using a split root system. Results are shown 5 weeks after inoculation. Inoculation with TR4 results in clear symptoms of Panama disease (column 3 “Tropical Race 4”), while these symptoms are absent when part of the roots are first inoculated with R1 (column 4 “Race 1 + TR4”) using a split root system.

Figure 3 Cell lysis inactivates Race 1 (R1) spores. The dark color indicates growth of the surviving spores. Cell lysis results in complete inactivation of the spores. The control “R1B" are untreated R1 spores. The experiment was performed in duplicate and the ratio lysis buffer / spore solution is indicated.

Figure 4 Active and inactive Race 1 protects against TR4 infection. Evaluation of individual plants using scale of 0 to 5; 0=no disease, 1-2= HR like response (no disease), 3= minimal infection, 4 = more than 50% affected, 5 = totally decayed. The different treatments are shown on the y-axis. Each bar represents a single plant.

Figure 5 Inactivated Race 1 (R1) protects against Tropical Race 4 (TR4), for all lysis buffer compositions tested for the lysis of Race 1 . Inoculation with TR4 results in clear symptoms of Panama disease (TR4), while these symptoms are significantly reduced or absent when the roots are first inoculated with inactivated R1 , when lysed in lysis buffer AP1 , LB-1 , LB-2 or LB-3.

Examples

Example 1. Inoculation with active R1

Plant material

Tissue cultured Cavendish “Grand Naine” banana plants were obtained from Rahan Meristem Ltd. (Western Galilee, Israel) and grown for approximately three months until 30 cm in height under greenhouse conditions (temperature 28 ± 2°C, 16h light, 85% relativity humidity).

Fusarium species

Fusarium oxysporum f. sp. cubense Race 1 (R1) and Fusarium oxysporum f. sp. cubense Tropical Race 4 (TR4) were purchased from Stichting Wageningen Research (The Netherlands).

Spore R1 and TR4 production

For fungal spore production, 500 mL of Mung bean medium was autoclaved. Four 0.5 cm2 mycelium plugs of R1 or TR4 were taken from a freshly grown PDA (Potato Dextrose Agar) plate and were transferred sterilely into the autoclaved Mung bean medium. The inoculated Erlenmeyer flasks were incubated on a rotary shaker at 130 rpm at 28°C. Spores were collected after 6 days of incubation in the Mung bean medium on the shaker. Spores were isolated by straining the medium through two layers of sterile cheesecloth. Subseguently, 200 MQ was added to reach a concentration of 1 x 10⁶ spores/mL. Inoculation of Banana plants

Prior to inoculation, banana plants as described above of 2.5 months were taken out of pots and cleaned by rinsing the roots with water. Root systems of banana plants as described above were inoculated:

(1) for 30 minutes with a water control;

(2) for 30 minutes with a spore suspension of TR4;

(3) for 30 minutes with a spore suspension of the non-pathogenic Fusarium strain R1 ; or

(4) for 30 minutes with a spore suspension of the non-pathogenic Fusarium strain R1 and subsequently for 30 minutes with a spore suspension of TR4.

For inoculation, plants were taken out of pots and cleaned by rinsing the roots with water and then immersed in a spore suspension at a concentration of 1 x 10⁶ conidia/mL (produced as indicated above). After inoculation, that the plants were potted into sterile sand in 2L pots, and located at the climate chamber.

The plants were grown under proper climatic conditions in the growth cabins for 5 weeks under controlled conditions (75% humidity, temperature 28°C and 12-hours light). After 5 weeks plants were evaluated individually in order to verify the presence or absence of the fungus and the damage. It was found that 5 weeks after the inoculations, only plants that were given the treatment indicated above as (2) inoculation with TR4 spore suspension only, showed severe disease symptoms, while the other three treatments did not (data not shown).

“Split-root” inoculation

The above experiment proofs that pre-inoculation with the non-pathogenic Fusarium strain R1 renders the plant resistant to subsequent inoculation with the pathogenenic Fusarium strain TR4. In order to investigate that a ‘resistant signal’ is capable of traveling through the plant tissue, a ‘split-root’ inoculation experiment was designed.

For this, the same experiment as indicated above was repeated with the exception that for treatment (4), after cleaning the roots by rinsing them with water, the root system was divided in two equal parts as shown in Figure 1 . One part of the root system was immersed in a container with a spore suspension of R1 at a concentration of 1 x 10⁶ conidia/mL (produced as indicated above) for 30 minutes in order to induce resistance, and after 5 minutes the other part of the roots was immersed in a spore suspension of TR4 at the same concentration (produced as indicated above) for 30 mins.

After inoculation, both parts were kept in separate pots containing sterilized sand to prevent contamination, and located at the climate chamber. After that the plants were grown under proper climatic conditions in the growth cabins for 5 weeks under controlled conditions (75% humidity, temperature 28°C and 12-hours light). After 5 weeks plants were evaluated individually in order to verify the presence or absence of the fungus and the damage. As shown in Figure 2, also using the split root inoculation resulted in R1 triggered significant immunity to TR4 treatment.

Example 2. Inoculation with inactivated R1 Experiments were carried out to define whether R1 as living microorganism was necessary to promote induced resistance or if an inactivated version of the microorganism can replicate this effect. To this end, spores produced as indicated in Example 1 were treated by lysing the cells using AP1 lysis buffer from Qiagen DNeasy Kit (a tissue lysis buffer suitable for the lysis of plant and fungal tissue). Of the known inactivation methods, lysis is expected to cause limited damage and preserves the integrity of e.g. nucleic acids.

Different amounts of R1 spore solution (100 pL, 200 pL and 400 pL) was treated with AP1 buffer from the DNeasy Plant Mini Kit (QIAGEN). The lysis buffer was heated to 65°C and shaken until a clear solution showed and then added to the samples in a 2:1 , 1 :1 or 1 :2 ratio. After that the samples were inverted and put into a 65°C incubator for 30 minutes. Inversion of samples was performed at 10-minute intervals during incubation.

R1 inactivation

In order to guarantee that all spores were effectively inactivated or killed, 100 pL homogenized concentration of lysed spores were plated on PDA plates and incubated for at 25°C in the dark. Results are presented in Figure 3 that shows that lysis treatment completely kills off the spores in the solution.

Inoculation

Banana plants (as described in Example 1 and 2.5 months old) were inoculated conform Experiment 1 , wherein in treatment (3) either an untreated R1 spore solution was used, or an R1 spore solution that was lysed as indicated above. The treatment was performed in quadruplicate using 4 plants per treatment into deep trays to prevent cross contamination between the treatment groups, and located at the climate chamber. After that the plants were put under proper climatic conditions in the growth cabins for 6 weeks under controlled conditions (75% humidity, temperature 28°C and 12-hours light). After 6 weeks plants were evaluated individually in order to verify the presence or absence of the fungus and the disease symptoms.

It was observed that R1 spore solutions inactivated by lysis were capable of preventing disease symptoms.

Example 3 Inactivation using lysed R1 solution

To verify the effect of lysed R1 spores, further experimentation was performed.

Lysis R1 spores

In this experiment, for inactivation of R1 , two different spore solutions were used for lysis using AP1 , i.e. either (Method 1) the R1 spore solution as produced in Example 1 , i.e. 1 x 10⁶ spores/mL, or (Method 2) a 50x concentrated spore solution, i.e. 5 x 10⁷ spores/mL.

Both R1 solutions were treated with lysis buffer by diluting 1 :1 in AP1 lysis buffer from the DNeasy Plant Mini Kit (QIAGEN). In case of Method 1 , 100 mL AP1 and 100 mL R1 spore solution were admixed in 250 mL bottles. In case of Method 2, 20 mL AP1 and 20 mL R1 spore solution were admixed in 50 mL tubes. Prior to admixing with the R1 spore solution, the lysis buffer was heated to 65°C and shaken until a clear solution showed. After that the samples were inverted and put into a 65°C incubator for 30 minutes. Inversion of samples was performed at 10-minute intervals during incubation.

Samples of 1000 pL were taken before and after in order to verify if the R1 spores were inactivated which was tested by placing the samples on Petri dishes containing PDA, and growth was verified after 5 days. It was confirmed that while untreated R1 spores were viable, no growth was observed using lysed R1 spore samples.

Prior to use of the R1 spores solution for inoculation as indicated below, the R1 spore solution lysed using Method 2 was diluted 50 times using tap water in order to arrive at the same concentration of R1 spore material as the R1 spore solution lysed using method 1 .

Inoculation

Plants as described in Example 1 , of about 30 cm height and having at least 5 real leaves, were inoculated/treated as described in Example 1 and 2, but according to the scheme of Table 2. In brief, their root systems were rinsed with tap water in order to remove the soil and plants were placed on water until inoculation. For each treatment as indicated in Table 2, the root systems were immersed in the inoculation solution for 30 minutes, with 5 minutes between treatment 1 and 2.

After this period, plants were immediately potted into 1 L pots containing sand. A set of 4 plants per treatment were divided by treatment into deep trays to prevent cross contamination and located at the climate chamber. After that the plants were put under proper climatic conditions in the growth cabins for 6 weeks under controlled conditions (75% humidity, temperature 28°C and 12-hours light). After 6 weeks plants were evaluated individually in order to verify the presence or absence of the fungus and the damage. The individual disease scores are presented in Figure 4. The overall results are summarized in Table 2.

Table 2. Overall results

R1⁺ R1 untreated (active)

TR4⁺ TR4 active

R1- R1 inactivated (Method 1)

Buffer AP1 from Qiagen kit

R1* R1 Inactivated (Method 2)

TR4* TR4 Inactivated (Method 2)

The results confirmed that the cross-protection effect was due to the presence of inactivated R1 , and not e.g. due to the buffer. This is supported by results of treatments 6 and 8. As expected, no disease was observed on water control plants, plants treated with only R1 (active or inactivated), plants treated with buffer, or with the inactivated TR4 (see treatments 1 , 7, 8, 9, 10). Contrary to this, plants inoculated with active TR4 showed high disease scores as well as when the TR4 was used in combination with buffer (see treatments 3 and 6 as shown on Fig. 2 and in Table 1). Low levels or no disease was observed when plants were primed with R1 (active or inactivated) prior inoculation with active TR4 (see treatment 4 (R1 + + TR4+) and treatment 5 (R1-+TR4+), and treatment 11 (R1*+ TR4+)).

Conclusions

Experiments were carried out comprising pre-treatments with active and inactive R1 prior to inoculations with active TR4. It was observed that pre-treatments with R1 significantly reduced development of TR4. It was also observed that the priming effect was long-lasting, and maintained at various nutritional and pH conditions (data not shown). The Split-root data and the data on lysed R1 corroborate the likeliness that an active component of the R1 spores, not affected by the AP1 lysis buffer and capable of traveling to live plants, suggesting this component to be an R1 derived oligonucleotide, is responsible for the hampering of TR4 development in banana plants.

Example 4. Inactivation using a variety of lysed R1 solutions

To determine that the protective effect of inactivated R1 was not limited to the use of a specific lysis buffer, the following lysis buffers were tested:

Table 3. Lysis buffers

Ethylhexadecyldimethylammonium bromide 2% 1% (CDA)

N-Lauroylsarcosine sodium salt (Sarkosyl) 2% 0.5%

NaCI 1.2 M 1.2 M

Proteinase K 100 pg/mL 100 pg/mL 100 pg/mL

Race 1 was inactivated as indicated above. Briefly, spores of Race 1 were produced and diluted to a concentration of 1 x 10⁶ spores/ml. Then, spores of Race 1 were inactivated by using a volume of 1 :1 with the three buffers described above (LB-1 , LB-2 or LB-3). As a control, the AP1 lysis solution from Qiagen was used (/.e. the same buffer as used in Example 3).

The virulent strain TR4 was produced and diluted to a concentration of 1 x 10⁶ spores/ml. Groups of three plants were treated as indicated in Table 4.

Table 4. Inoculation scheme

R1⁺ R1 untreated (active)

TR4⁺ TR4 active

R1- R1 inactivated (Method^

Buffer AP1 from Qiagen kit

LB-1 Lysis Buffer 1

LB-2 Lysis Buffer 2

LB-3 Lysis Buffer 3

As an additional control, roots of (a separate group of) banana plants were immersed into buffer only, followed by exposure to TR4, or only in buffer, to determine whether any of the buffers would have an effect.

Plants were inoculated as described in example 3. Briefly, plants were uprooted and roots were cleaned and rinsed. The roots were subsequently immersed in a spore suspension or water, according to the scheme of Table 4. After 30 min, plants were immersed again in a spore suspension with active spore suspension of TR4. Simultaneously, samples were taken from the spore suspensions before and after the treatments and plated in PDA media to confirm that the fungal strains were effectively inactivated. It was confirmed that fungal growth was indeed still absent after 15 days (data not shown).

Results

Six weeks after inoculation, typical symptoms of Fusarium TR4 appeared in those plants that were not inoculated with Race 1 . Plants were cut in halves and visual observation was performed. Strong symptoms were observed in the TR4 group (the complete plant was rotten), but no symptoms were visible in plants treated with water, Race 1 , or just buffer and reduced or no symptoms were observed in plants treated with the inactivated Race 1 prior TR4 inoculation (Figure 5). For none of the buffers tested, buffer treatment alone prior to TR4 inoculation protected against disease symptoms.

In conclusion, a variety of lysis buffers may be used for lysing (inactivating) Race 1 and the lysed Fusarium Race 1 is responsible for the hampering of TR4 development in banana plants.

Claims

26 Claims

1. A composition for increasing resistance to Panama disease in a banana, preferably a Cavendish banana, wherein the composition comprises inactivated Fusarium fungal material.

2. A composition according to claim 1 , wherein the inactivated material are inactivated cells, preferably inactivated spores of a Fusarium fungal strain, preferably inactivated conidia of a Fusarium fungal strain.

3. A composition according to claim 1 or 2, wherein at least about 60%, 70%, 80%, 90% or about 100% of the Fusarium fungal material in the composition is inactivated.

4. A composition according to any one of the preceding claims, wherein the Fusarium fungal material is from the fungal strain Fusarium oxysporum f. sp. Cubense Race 1 (R1).

5. A composition according to any one of claims 2 - 4, wherein the composition comprises at least a concentration of about 1 x 10⁴ inactivated cells I ml, preferably 1 x 10⁴ - 1 x 10⁹ inactivated cells/ml, preferably wherein the composition comprises about 1 x 10⁵ - 1 x 10⁷ inactivated cells I ml.

6. A composition according to any one of claims 2 - 5, wherein the composition comprises a lysis buffer for inactivating the cells of the Fusarium fungal strain, wherein preferably the composition is a lysis buffer comprising inactivated cells of the Fusarium fungal strain.

7. A composition according to claim 6, wherein the lysis buffer comprises the following components: i) an agent for pH control; ii) a chelating agent; and iii) a surfactant, and wherein the lysis buffer optionally comprises at least one of the following components: iv) an agent to remove phenolic compounds; v) a salt; vi) a reducing agent; and vii) A protease.

8. A composition according to claim 7 wherein at least one of: i) the agent for pH control is Tris-HCI, preferably at a concentration of about 10 mM - 100mM; ii) the chelating agent is EDTA, preferably at a concentration of about 1 mM - 20 mM; and iii) the surfactant is at least one of SDS, CTAB, CDA and Sarkosyl, preferably at a concentration of about 0.1 % - 2% (w/v), and optionally at least one of: iv) the agent to remove phenolic compounds is PVP, preferably at a concentration of about 1 % - 2% (w/v), v) the salt is NaCI, preferably at a concentration of about 0.1 M - 2M; vi) the reducing agent is at least one of DTT, preferably at a concentration of about 9 mM - 12 mM, and p-mercaptoethanol, preferably at a concentration of about 0.1 % - 0.2% (v/v); and vii) the protease is Proteinase K, preferably at a concentration of about 50 pg/ml - 150 pg/ml.

9. A method for increasing resistance to Panama disease in a banana, preferably a Cavendish banana, comprising the step of exposing one or more roots of the banana to a composition as defined in any one of claims 1 - 8.

10. A method for producing a banana, preferably a Cavendish banana, that has increased resistance to Panama disease, comprising a step of exposing one or more roots of the banana to a composition as defined in any one of claims 1 - 8.

11. The method according any one of the preceding claims, wherein the Panama disease is caused by the fungal strain Fusarium oxysporum f. sp. Cubense Tropical Race 4 (TR4).

12. The method according to any one of claims 9 - 11 , wherein the one or more roots are exposed to the composition for at least about 5, 10, 15, 20, 25 or 30 minutes.

13. Use of a composition according to any one of claims 1 - 8 for increasing resistance to Panama disease in a banana, preferably a Cavendish banana.

14. A banana obtainable by the method of any one of claims 10 - 13.