WO2016010439A1

WO2016010439A1 - Controlling microbial infection in plants

Info

Publication number: WO2016010439A1
Application number: PCT/NZ2015/050092
Authority: WO
Inventors: Margaret Isobelle BUTLER; Iain Lowell LAMONT; Russell Tony Maskell Poulter
Original assignee: Seeka Kiwifruit Industries Limited
Priority date: 2014-07-16
Filing date: 2015-07-16
Publication date: 2016-01-21

Abstract

The present invention relates to inhibiting the iron uptake pathways of bacteria that infect plants, and in particular, to methods of utilizing iron chelating compounds to inhibit the growth of such bacteria. In addition, the invention relates to the induction by iron- chelating compounds of plant systemic acquired resistance to microorganisms.

Description

CONTROLLING MICROBIAL INFECTION IN PLANTS

FIELD OF INVENTION

BACKGROUND

Iron is an essential element in a variety of metabolic and informational pathways for almost all microbes. In many environments, the amount of free iron is below the concentration required by most microorganisms for growth. Numerous bacteria and fungi overcome iron limitation by secreting siderophores: low molecular weight, high affinity chelators of iron. Siderophores chelate iron in the extracellular environment and the resulting ferric siderophore complex is recognized by siderophore-specific membrane receptors, enabling uptake of iron by the bacterial cells. Siderophore mediated iron uptake makes a significant contribution to the pathogenesis of many Gram-positive and Gram- negative bacterial pathogens, including Pseudomonas syringae. Siderophores are also able to trigger the systemic acquired resistance response of plants.

The species Pseudomonas syringae comprises a diverse group of Gram-negative bacteria, including many plant pathogens. Strains of Pseudomonas syringae infect a wide range of plants, many of which are economically important. Strains of P. syringae are described by reference to the plant from which they were isolated. For example, the designation Pseudomonas syringae pv. actinidiae (PSA) could be applied to a P. syringae strain that was isolated from (was a pathovar of) the kiwifruit, Actinidia spp. Within the PSA group, there is considerable diversity (Butler et al., 2013). PSA produces bacterial cancer of kiwifruit, characterized by leaf spots and systemic infection resulting in death of shoots or whole plants. There is at present a worldwide pandemic of virulent PSA affecting kiwifruit. Similar diseases are described from other woody plants, for example P. syringae pv. avellanae on hazelnuts and P. syringae pv. theae on tea plants.

Some P. syringae pathovars produce abundant siderophores in culture, for example P. syringae pv. tomato produces abundant quantities of the fluorescent siderophore pyoverdin. PSA and some other P. syringae pathovars produce very little or no siderophores. For example, on a variety of media, PSA produces minimal or no fluorescent siderophores. This characteristic may make these strains vulnerable to inhibition by compounds that chelate iron. There are several synthetic chelators that have a very high affinity for iron. These include EDDHA; ethylendiamine di(o-hydroxyphenyl-acetic) acid. The IUPAC name is 2-[2-[ [2-Hydroxy-l-(2-hydroxyphenyl)-2- oxoethyl]amino]ethylamino]-2-(2- hydroxyphenyl) acetic acid.

EDDHA

EDDHA can exist as several isomers; ortho, ortho-EDDHA has a Ki (log stability constant) of 35.09 for ferric iron.

An even stronger iron chelating agent is HBED, hydroxybenzyl ethylenediamine. HBED has a Ki of 39.01 for ferric iron.

HBED

Several of these synthetic chelators (including EDDHA and HBED) are used in agriculture in the form of metal chelates, for example, Fe-EDDHA or Fe-HBED. Both these compounds are approved for use on agricultural crops according to the EU regulation 223/2012. It is generally accepted that these chelators pose no safety risk.

There are no apparent studies or claims establishing that EDDHA, HBED or other iron chelators are able to directly inhibit any bacteria growing in or on plants. This is possibly due to many bacterial pathogens of plants being resistant to inhibition by iron chelators as a result of their production of abundant siderophores.

The present invention provides a novel approach to the management of bacteria infection in plants by targeting bacterial species that are susceptible to growth inhibition using iron chelators. SUMMARY OF THE INVENTION

The inventions described and claimed herein have many attributes and embodiments including, but not limited to, those set forth or described or referenced in this Summary of the Invention. It is not intended to be all-inclusive and the inventions described and claimed herein are not limited to or by the features or embodiments identified in this Summary of the Invention, which is included for purposes of illustration only and not restriction.

In one aspect of the present invention there is provided a method to control a microbial infection in a plant, the method comprising applying to the plant infected by, or predisposed to infection by, one or more microorganisms, of a composition comprising an iron chelating compound,

wherein, the one or more microorganisms is susceptible to growth inhibition or reproduction by the iron chelating compound,

and wherein, the iron chelating compound is applied in an amount sufficient to inhibit the growth or reproduction of the microorganism.

In another aspect of the present invention there is provided a method for inducing systemic acquired resistance in a plant against infection by any microorganism, the method comprising applying to the plant infected by, or predisposed to infection by, one or more microorganisms, of a composition comprising an iron chelating compound,

In a further aspect of the present invention there is provided a composition comprising an iron chelating compound for use in controlling microbial infection in plants.

In yet another aspect of the present invention there is provided a pesticide comprising an iron chelating compound for use in controlling microbial infection in plants

In yet a further aspect of the present i nvention there is provided a kit comprising an iron chelating compound together with instructions for how to apply the iron chelating compound to a plant to control a microorganism infection.

In yet a further aspect of the present invention there is provided a kit comprising a pesticide composition comprising an iron chelating compound together with instructions for how to apply the iron chelating compound to a plant to control a microorganism infection.

In yet another aspect of the present invention there is provided a kit comprising a pesticide composition comprising an iron chelating compound selected from ethylendiamine di(o-hydroxyphenyl-acetic) acid (EDDHA) and hydroxybenzyl ethylenediamine (HBED), together with instructions for how to apply the iron chelating compound to a plant to control a bacteria infection. These and other aspects of the invention are described in the detailed description that follows which includes reference to the figures.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows growth of ICMP18708 (PSA) in King's B supplemented with EDDHA. Growth was measured as OD at 600nm. Series 1 & 2 = no EDDHA, Series 3 & 4 = 15 mg/L EDDHA, Series 5 & 6 = 50 mg/L EDDHA.

Figure 2 shows growth of Pseudomonas syringae in the presence of EDDHA. (A) growth of PSA, strains ICMP18708 (NZ) and ICMP9853 (Japan), on King's B medium with no EDDHA (left) or 21.75 ug/ml EDDHA (right). (B) growth of P. syringae PsD (ICMP18804) on King's B medium with no EDDHA (left), with 21.75 ug/ml (centre) or 43.5ug/ml EDDHA (right).

Figure 3 shows lack of EDDHA phytotoxicity. Kiwifruit plants (Actinidia chinensis) were sprayed with EDDHA at 1 g/L. A kiwifruit plant two hours after spraying (left) and 15 days after spraying (right).

Figure 4 shows persistence of EDDHA on sprayed kiwifruit leaves. Discs were cut from kiwifruit leaves that had been sprayed with 10 mg/mL EDDHA. Sprayfix and placed onto plates spread with a dense culture of PSA (ICMP18708, NZ). Disc cut on the day of spraying (T=0 h, left) and disc cut from a leaf four days after spraying (T= 100 h, right). Plates show no decrease in the zone of inhibition four days after EDDHA application.

Figure 5 shows the response of a kiwifruit leaf following the application of 100 uL of 0.05% Pulse Penetrant plus O. lg/L EDDHA. The insert illustrates the extent of the compound spreading with false colour.

Figure 6 shows a potted kiwifruit plant sprayed three times at weekly intervals with 0.05% Pulse Penetrant plus 0.1 g/L EDDHA, indicating no phytotoxic effect.

GENERAL DEFINITIONS

Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (for example, in immunology, immunohistochemistry, protein chemistry, and biochemistry).

It is intended that reference to a range of numbers disclosed herein (e.g. 1 to 10) also incorporates reference to all related numbers within that range (e.g. 1, 1.1, 2, 3, 3.9, 4, 5, 6, 6.5, 7, 8, 9 and 10) and also any range of rational numbers within that range (for example 2 to 8, 1.5 to 5.5 and 3.1 to 4.7) and, therefore, all sub-ranges of all ranges expressly disclosed herein are expressly disclosed . These are only examples of what is specifically intended and all possible combinations of numerical values between the lowest value and the highest value enumerated are to be considered to be expressly stated in this application in a similar manner. The term "and/or", e.g., "X and/or Y" shall be understood to mean either "X and Y" or "X or Y" and shall be taken to provide explicit support for both meanings or for either meaning.

DETAILED DESCRIPTION OF EMBODIMENTS

The present invention is predicated on the surprising and unexpected discovery that certain microorganisms are susceptible to growth inhibition when exposed to a n iron chelating compound. In particular, the present invention is concerned with methods, compositions and kits for controlling bacteria infection in plants (e.g.) kiwifruit, cherries, plums, hazelnuts, chestnuts, tea, pears and apples, through application of an iron chelating compound which specifically targets bacterial species that are susceptible to growth inhibition when exposed to such compounds.

The present invention is also predicated on the surprising an unexpected discovery that acquired systemic immunity by plants to pathogens, such as microorganisms including bacteria, may be achieved by targeting iron uptake pathways in the plant. Applicants have surprisingly discovered that application of iron chelating directly to plants, or into the surrounding soil, may be used to invoke such immunity.

Accordingly in one aspect of the present invention there is provided a method to control a microbial infection in a plant, the method comprising applying to the plant infected by, or predisposed to infection by, one or more microorganisms, of a composition comprising an iron chelating compound,

The iron chelating compound is any compound capable of sequestering iron. Examples of iron chelating compound according to the present invention includes, but is not limited to, ethylendiamine di(o-hydroxyphenyl-acetic) acid (EDDHA) and hydroxybenzyl ethylenediamine (HBED). As is discernable from the Examples which follow, the iron chelating agent, (e.g.) EDDHA or HBED, may be applied to the plant or plant surrounds in an amount that is sufficient to inhibit microorganism growth and/or reproduction. This will ultimately depend on the size of the plant and extent of canopy cover. In one example according to the present invention, EDDHA or HBED is applied in the range of 5 mg/L to 500 mg/L. In a related example, the EDDHA or HBED is applied to the plant at a rate of 50 mg/L.

Examples of plants in which methods, compositions and kits according to the present invention are useful include, but are not limited to kiwifruit, cherries, plums, hazelnuts, chestnuts, tea, pears and apples.

The methods according to the present invention are particularly useful in controlling bacteria infection in plants.

Examples of bacteria in which the methods, compositions and kits according to the present invention are useful include, but are not limited to, Pseudomonas syringae pathovar or Erwinia amylovora.

In a related example, the Pseudomonas syringae pathovar may be selected from the group consisting of P. syringae pv. actinidiae, P. syringae pv. avellanae, P. syringae pv. aesculi, P. syringae pv. theae and P. syringae pv. Morsprunorum. For example, direct inhibition of infection of kiwifruit plants by P. syringae pv. Actinidiae is shown in Examples 2-11 which follow. Also refer to Figures 1-6 accompanying this specification.

A person skilled in the art will recognize that the composition or pesticide comprising the iron chelating compound may be 'applied' to the plant directly (e.g. by spraying or injection) or applying the iron chelating compound to the soil or growth environment surrounding the plant. A person skilled in the relevant art will appreciate that any application method is possible, provided that the iron chelating compound is applied to the plant in amount sufficient to inhibit microorganism (e.g. bacteria) growth and/or reproduction or to induce the desired systemic immunity to later infection challenge.

According to the present invention, application of the iron chelating compound, or composition comprising the iron chelating compound, may be achieved by mixing the compound or composition in an aqueous spray and applying the spray directly to the foliage, trunk or soil surrounding the plant. In another example, the compound or composition may be applied to the plant through existing irrigation means.

The compositions comprising the iron chelating compounds according to the present invention may be formulated using standard art known techniques. In one example, and to achieve the desired application, the iron chelating compound is formulated together with a surfactant.

Surfactants (surface active agents) are a type of adjuvant designed to enhance the absorbing, emulsifying, dispersing, spreading, sticking, wetting, or penetrating properties of pesticides. Surfactants are most often used with herbicides to help a pesticide spread over a leaf surface and penetrate the waxy cuticle of a leaf or to penetrate through the small hairs present on a leaf surface. Examples of surfactants according to the present invention include non-ironic surfactants, crop oil concentrates, nitrogen-surfactant blends, esterified seed oils and organo-silicones (refer to Adjuvant & Surfactant Guide. 1998. Wilfarm L.L.C., Gladstone, MO; Miller & Westra (1996); Miller & Westra (1998); Petroff (1999)).

Non-ionic surfactants are composed of alcohols and/or fatty acids and are compatible with most pesticides. This class of surfactant reduces surface tension and improves spreading, sticking, and pesticide uptake.

Crop oil concentrates are composed of paraffin-based petroleum oil and surfactants. Crop oil concentrates reduce surface tension and improve herbicide uptake and leaf surface spreading.

Nitrogen-surfactant blends consist of premix combinations of various forms of nitrogen and surfactants. They generally are used with herbicides recommending the addition of ammonium sulfate or 28% nitrogen. These surfactants reduce surface tension and improve leaf surface spreading.

Esterified seed oils are produced by reacting fatty acids from seed oils with an alcohol to form esters. The methyl or ethyl esters produced by this reaction are combined with surfactants/ emulsifiers to form an esterified seed oil. These surfactants reduce surface tension and improve herbicide uptake by improving herbicide distribution on the leaf surface.

Organo-silicones are usually silicone/surfactant blends of silicone to non-ionic or other surfactants: a few within this classification are composed entirely of silicone. These surfactants provide a tremendous reduction in surface tension and spread more than conventional surfactants. In addition, this class of surfactants provide improved effectiveness through maximum rainfastness.

The present invention also contemplates an iron chelating compound or composition for use in controlling microorganism infection in plants. In one example, the microorganism is a bacteria and the plant is selected from the group consisting of kiwifruit, cherries, plums, hazelnuts, chestnuts, tea, pears and apples. In a related example, the bacteria is a Pseudomonas syringae pathovar and the plant in kiwifruit.

Further, the present invention also contemplates kits and articles for manufacture, particularly for use by orchardists and the like, in controlling crop infection.

Accordingly, in yet a further aspect of the present invention there is provided kit comprising an iron chelating compound together with instructions for how to apply the iron chelating compound to a plant to control a microorganism infection.

In yet another aspect of the present invention there is provided a kit comprising a pesticide composition comprising an iron chelating compound together with instructions for how to apply the iron chelating compound to a plant to control a microorganism infection. In yet another aspect of the present invention there is provided a kit comprising a pesticide composition comprising an iron chelating compound selected from ethylendiamine di(o-hydroxyphenyl-acetic) acid (EDDHA) and hydroxybenzyl ethylenediamine (HBED), together with instructions for how to apply the iron chelating compound to a plant to control a bacteria infection

Embodiments of the invention are described by reference to the following specific examples that are not to be construed as limiting.

EXAMPLES

Example 1: Materials and Methods

List of Strains

Strains of Pseudomonas syringae used in the study of EDDHA effects

Species Strain Host species Country of name isolation

P. syringae pv. actinidiae ICMP18708 Actinidia (kiwifruit) NZ

P. syringae pv. actinidiae ICMP18744 Actinidia (kiwifruit) Italy

P. syringae pv. actinidiae ICMP19455 Actinidia (kiwifruit) Chile

P. syringae pv. actinidiae Haxa4 Actinidia (kiwifruit) China

P. syringae pv. actinidiae ICMP9853 Actinidia (kiwifruit) Japan

P. syringae pv. Actinidiae 23663 Actinidia (kiwifruit) Korea

P. syringae pv. actinidiae K4 Actinidia (kiwifruit) Korea

P. syringae pv. actinidiae 134KBS Actinidia (kiwifruit) Korea

P. syringae (PsD) ICMP18804 Actinidia (kiwifruit) NZ

P. syringae pv. avellanae ICMP 9746 Cory 1 us avellanae Greece

(hazel)

P. syringae pv. aesculi ICMP8947 Aesculus indica India

(chestnut)

P. syringae pv. ICMP18416 Prunus cerasus (sour France morsprunorum cherry)

P. syringae pv. ICMP 5332 Prunus avium (wild cherry) NZ morsprunorum

P. syringae pv. theae ICMP 3939 Camellia sinensis (tea) Japan

P. syringae pv. theae ICMP 3923 Camellia sinensis (tea) Japan Description of culture media

King's B

20g/L Bacto Peptone 20g/L glycerol

1.5g/L MgS04

1.5g/L K2HP04 solidified with 15g/L Difco agar

Modifications to this medium may include:

1 Use of Cas-amino acids (20g/L) to replace the peptone. Cas-amino acids has reduced iron content compared with Bacto peptone.

2 Use of 1% Difco Noble agar to replace the 1.5% Difco agar. Noble agar has reduced iron content compared to Difco agar.

Sequencing technology and data accessions

Genomic DNA for whole genome sequencing was isolated from PSA using the Mo BioTM Microbial DNA Isolation Kit. Sequencing libraries were prepared using the Illumina TruSeq DNA Sample Preparation kit and sequenced either with a HiSeq 2000 or a MiSeq system. De novo genome assembly used the Edena assembler vl 10920 with the default settings. Comparisons were made between sequences in the genomes using various BLAST programmes at the BLAST server at NCBI (http://blast.st-va.ncbi.nlm.nih.gov/Blast.cgi).

Table of whole genome sequence accession data for fully sequenced Pseudomonas syringae pv. actinidiae (PSA) strains, two PsD strains and one PsHa.

Isolate Country of isolation Genbank Accession 1

ICMP9853 Japan ANJB00000000

M7 China ANJJ00000000

M2282 China ANJI00000000

ICMP 18744 Italy ANGD00000000

ICMP 18800 NZ ANJD00000000

ICMP 18708 NZ ANJC00000000

TP1 NZ ANJG00000000

6.1 NZ ANJH00000000 ICMP 19439 Chile ANJMOOOOOOOO

ICMP 19455 Chile ANJK00000000

ICMP 188043 NZ ANJE00000000

ICMP 188063 NZ ANJF00000000

ICMP 188074 NZ ANJL00000000

1. These Whole Genome Shotgun projects have been deposited at DDBJ/EMBL/GenBank under the accessions listed above. The versions of the assemblies described in this paper are the first versions.

2. The sequence of M228 was generated from a limited quantity of genomic DNA.

3. P. syringae PsD

4. P. syringae PsHa

Chelates described in these examples

(i) EDDHA: ethylendiamine di(o-hydroxyphenyl-acetic) acid.

The structure of this compound is given above. The EDDHA used in this study was provided by Tradecorp (Madrid) (www.tradecorp.com.es). The compound was synthesised by Tradecorp. It is used commercially in Europe as a fertilizer, especially on alkaline soils. The fertilizer used in the EU consists of EDDHA together with appropriate chelated metals, such as iron. The EDDHA manufactured by Tradecorp is approved under the EU regulation 223/2012. This reflects the quality control of the manufacturing process, the purity of the product and the proven safety of EDDHA. The study was performed using both pure ortho, ortho-EDDHA and a mixture of ortho, ortho-EDDHA and ortho, para-EDDHA isomers.

(ii) HBED: hydroxybenzyl ethylenediamine.

The structure of this compound is given above. The HBED used in this study was provided by ADOB, Poland (http://en.adob.eu/zobacz/pobierz-wizytowke/). The compound was synthesised by ADOB. It is used commercially in Europe as a fertilizer chelated with iron or zinc, especially on alkaline soils. The synthesis of HBED is the subject of a patent, owned by ADOB. By the EU's approval this chelate was included in EU regulation 223/2012. This reflects the quality control of the manufacturing process, the purity of the product and the proven safety of HBED. HBED exists as a single isomer. Example 2

EDDHA at a concentration of 50 mg/L inhibits the growth of all strains of PSA that we have tested. This includes strains from New Zealand, Italy, Chile, China, Korea and Japan. Some strains of PSA are inhibited by much lower concentrations, for example ICMP9853 (Japan) and 23663 (Korea) are inhibited at 5mg/L EDDHA. This inhibition is demonstrated in liquid culture (Figure 1) and on media solidified with agar (Figure 2). This inhibition is overcome by adding ferrous or ferric salts to the medium, demonstrating that inhibition is due to the chelating agent sequestering iron. When performing this example, it is important to limit the amount of iron in the test medium. A standard medium was King's B, which contains limited iron. For some tests, medium made with Bacto Cas-amino acids was used because of its low iron content. For some tests, medium was solidified with Difco Noble agar, because of its negligible iron content.

Example 3

EDDHA and HBED have a particularly high affinity for iron. These compounds inhibited growth of PSA in the presence of divalent cations such as magnesium in the media. In particular it was noted that cuprous and cupric ions did not interfere with the chelation of iron. This is important, because copper is widely used as a protectant in many horticultural contexts, including the culture of kiwifruit.

Example 4

EDDHA was effective in inhibiting a diversity of Pseudomonas syringae pathovars that infect woody plants, including P. syringae pv. theae, P. syringae pv. morsprunorum, P. syringae pv. aesculi and P. syringae pv. avellanae. A level of 50 mg/L was sufficient to inhibit these strains in vitro.

Example 5

A number of pseudomonads isolated from kiwifruit plants seem to be non-pathogenic or only weak, opportunistic pathogens. For example, P. syringae PsD, a strain found widely in New Zealand. The non-pathogenic strains are not inhibited by EDDHA or HBED (Figure 2). The resistance to inhibition may be due to the production by strains such as P. syringae PsD of abundant siderophores. This is an important observation, because it establishes that the chelating agents will be selective in inhibiting only the pathogenic pseudomonads, leaving the rest of the phyllosphere undisturbed.

Example 6

Prolonged incubation of PSA with EDDHA in liquid culture or on aga r has not produced any bacteria resistant to EDDHA. Application of most inhibitory compounds (for example, Streptomycin and copper) gives rise to resistance, in the laboratory and in the field. It is therefore of great significance that the iron limitation generated by the chelators cannot be corrected by any mutation in the PSA.

Example 7

Co-culturing of PSA together with fluorescent pseudomonads such as P. syringae PsD on the same agar plate containing 50 mg/L EDDHA shows either no cross feeding of the PSA by the fluorescent pseudomonad or very weak cross- feeding. This is important because, on the plant, the PSA will exist as one component of a mixed bacterial population. The EDDHA selectively inhibits PSA even in the presence of fluorescent pseudomonads.

Example 8

EDDHA and HBED combined with various cations a re used routinely worldwide, including in the EU, as fertilizers. Specifically, they are used on kiwifruit in Europe. These chelators are not reported to have any phytotoxic effects. Kiwifruit plants were sprayed with lOg/L EDDHA (200x the PSA inhibitory concentration) and monitored for phytotoxicity. No effect could be detected (Figure 3).

Example 9

Kiwifruit plants were grown outside exposed to normal weather conditions. Leaf discs were cut from kiwifruit sprayed with lOg/L EDDHA. The leaf discs were tested for the presence of EDDHA by placing the disc on an agar plate inoculated with a lawn of PSA. Discs were cut from the sprayed plants at 24h intervals for a period of weeks. The zone of PSA growth inhibition around the discs remained essentially unchanged over this time period (Figure 4) . This demonstrates that EDDHA is not removed by rain, UV-induced degradation or plant or bacterial processes. This persistence is important if the chelator is to be commercially useful.

Example 10

The chelator can be applied without phytotoxic effect at any stage of plant growth. This includes budburst, flowering (EDDHA is non-toxic to bees), fruit set, harvest and pruning.

Example 11

To better understand the way in which PSA is acquiring iron the sequences of multiple strains of PSA and other P. syringae such as P. syringae PsD were obtained using Illumina whole genome sequencing. The genes required for pyoverdin synthesis are present in PSA. To explore this situation further the levels of expression of these genes were quantified using RNA-seq. The pyoverdin genes are all expressed, but at a much lower level than that encountered in other, fluorescent pseudomonads. To increase its iron acquisition, the PSA would have to not only increase its pyoverdin synthesis, but also increase its ability to take up the secreted pyoverdin once it has acquired the iron. This provides a sound explanation of the failure of PSA to escape from inhibition by the chelating agents.

Example 12

Systemic acquired resistance (SAR) is a complex response by the plant, involving the increased expression of several, distinct biosynthetic pathways: the salicylic acid pathway, the jasmonic acid pathway and the ethylene pathway. This increased expression is dependent on the regulation of a number of i nteracting genes and the production of increased levels of various messenger RNAs. It has been known for some time that microbial siderophores can induce this response. It has recently been shown that EDDHA can also induce the response in Arabidopsis. It is, therefore, concluded that the trigger for induction is not the specific siderophore or synthetic compound, but the depletion of free iron produced by these elicitors. The iron-depletion induced SAR response is likely to occur in a wide range of plants, including, but not limited to, kiwifruit. The SAR response is likely to confer resistance to a wide range of bacterial, fungal and oomycete pathogens. This will include PSA, which has already been shown to be inhibited by ActiGuard (acibenzolar-S- methyl, an elicitor of the salicylic acid pathway).

The SAR response will occur, after a delay of several days, in plants exposed to EDDHA, HBED or other chelators of iron. The response will be systemic and occur not just in the tissue exposed to the chelator, but more widely in the plant (possibly including the stems and roots). The SAR response is likely to be sustained for an extended period until the chelator is depleted and iron levels restored to normal.

Example 13

The chelator could be applied by spraying or by injection. The effectiveness of the spray may be enhanced by the use of surfactant spreaders or other adjuvants. In order to reduce the frequency of PSA on the leaf surface, a spreader such as Sprayfix, an alkylaryl polyglycol could be used. To achieve penetration of the leaf tissue via stomatal flooding, a penetrant such as Pulse Penetrant (an organo-modified polysiloxane) could be used. Penetrants such as Pulse Penetrant are widely used as components of fertilizer or fungicide sprays. They are not phytotoxic when used at the correct concentration. They achieve the penetration of the leaf tissue via the stomata that makes the spray rain-fast. In addition, the use of such penetrants will deliver the chelator to the maximum internal leaf tissue, optimizing the elicitation of the general systemic acquired resistance response. Figure 5 demonstrates the response following the application of lOOuL of 0.05% Pulse Penetrant plus 0.1g/L EDDHA to a kiwifruit leaf. The picture was taken 60 seconds after application of the Pulse Penetrant to the lower surface of the leaf and indicates the influx and spreading of the compounds in the leaf tissue. An hour after the picture was taken, the leaf appearance had returned to normal. Figure 6 shows a potted kiwifruit plant that has been sprayed three times at weekly intervals with 0.05% Pulse Penetrant plus O. lg/L EDDHA, indicating no phytotoxic effect.

Example 14

The induction of systemic acquired resistance plants can be detected and quantified, and the duration of the SAR response measured by analysing the transcriptome of the plant (the quantitative analysis of the various RNA species present in the plant). This can be used to determine the optimal concentration of chelator/elicitor, the preferred surfactant or penetrant, the delay between application and SAR response, the extent to which the SAR response is widely systemic (within a leaf or a shoot, or even more widely), the duration of the SAR response and the response of the plant to subsequent exposure to the chelator/elicitor. This type of analysis can be applied to kiwifruit, because the complete genome sequence of the species has been described. Similarly, this analysis could be applied to a wide range of important horticultural crops and other valued plants (such as ornamentals). The analysis can also be applied to model plants such as Arabidopsis and Nicotiana. It is achieved by isolating RNA, constructing a derived DNA library by reverse transcription and the quantitative sequencing of such libraries by Illumina high throughput sequencing or similar technologies. The response of Arabidopsis to infiltration with EDDHA has been described (Aznar et al., 2014), but not the response to the intended spray application.

Example 15

In a further example, the chelator/elicitor with or without surfactant/penetrant, is combined with a carrier component to facilitate retention of the sprayed compounds on the plant leaves. Retention is unlikely to be a major problem with kiwifruit as the lower leaf surface is deeply sculpted and densely pilose (hairy). While some other plants of interest are similarly likely to retain sprayed compounds efficiently, others have smooth and waxy leaf surfaces (for example citrus) . To retain the spray on such leaves, a carrier component such as kaolin, or better, diatomaceous earth can be used. Diatomaceous earth can be sourced from geological deposits laid down in freshwater; such deposits are preferable. Freshwater diatomaceous earth contains no silica shards and is acceptable as a component of foodstuffs and is safe to handle. It is biologically inert. The highest grade diatomaceous earth is white or pale beige and contains no significant iron content. The combination of such diatomaceous earth with EDDHA does not reduce the effectiveness of the chelator as an inhibitor of PSA growth in agar medium.

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All patents, publications, scientific articles, web sites, and other documents and materials referenced or mentioned herein are indicative of the levels of skill of those skilled in the art to which the invention pertains, and each such referenced document and material is hereby incorporated by reference to the same extent as if it had been incorporated by reference in its entirety individually or set forth herein in its entirety. Applicants reserve the right to physically incorporate into this specification any and all materials and information from any such patents, publications, scientific articles, web sites, electronically available information, and other referenced materials or documents. Although the invention has been described by way of example, it should be appreciated that variations and modifications may be made without departing from the scope of the invention as defined in the claims. Furthermore, where known equivalents exist to specific features, such equivalents are incorporated as if specifically referred in this specification.

The invention illustratively described herein suitably may be practiced in the absence of any element or elements, or limitation or limitations, which is not specifically disclosed as essential. Thus, for example, in each instance described or used herein, in embodiments or examples of the present invention, any of the terms "comprising", "consisting essentially of", and "consisting of" may be replaced with either of the other two terms in the specification. Also, the terms "comprising", "including", containing", etc. are to be read expansively and without limitation. The assays and methods illustratively described herein suitably may be practiced in differing orders of steps, and that they are not necessarily restricted to the orders of steps indicated herein or in the claims. Further, as used or described herein and in the appended claims, the singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise. Under no circumstances may the patent be interpreted to be limited to the specific examples or embodiments or methods specifically disclosed herein.

The terms and expressions that have been employed are used as terms of description and not of limitation, and there is no intent in the use of such terms and expressions to exclude any equivalent of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention as claimed. Thus, it will be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts disclosed herein may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as described herein, and as defined by the appended claims.

The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

Other embodiments are within the following claims. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

REFERENCES

1. Butler et al., 2013, PLoS ONE, 8(2) : e57464

2. Aznar et al., 2014, Plant Physiology 164: 2167-2183

3. Adjuvant & Surfactant Guide. 1998. Wilfarm L.L.C., Gladstone, MO.

4. Miller, P and Westra, P. 1996. Herbicide Surfactants and Adjuvants. No. 0.559, Crop Series Fact Sheet, Colorado State University Cooperative Extension, Fort Collins, CO.

5. Miller, P and Westra, P. 1998. How Surfactants Work. No. 0.564, Crop Series Fact Sheet, Colorado State University Cooperative Extension, Fort Collins, CO.

6. Petroff, R. 1999. Pesticide Adjuvants and Surfactants. Montana State University Extension, Bozeman, MT.

Claims

1. A method to control a microbial infection in a plant, the method comprising applying to the plant infected by, or predisposed to infection by, one or more microorganisms, of a composition comprising an iron chelating compound,

2. A method for inducing systemic acquired resistance in a plant against infection by any microorganism, the method comprising applying to the plant infected by, or predisposed to infection by, one or more microorganisms, of a composition comprising an iron chelating compound,

3. A method according to claim 1 or claim 2, wherein the iron chelating compound is selected from the group consisting of ethylendiamine di(o-hydroxyphenyl-acetic) acid (EDDHA) and hydroxybenzyl ethylenediamine (HBED).

4. A method according to any one of claims 1 to 3, wherein the microorganism is a bacterium.

5. A method according to any one of claims 1 to 4, wherein the bacterium is Pseudomonas syringae pathovar or Erwinia amylovora.

6. A method according to claim 5, wherein the Pseudomonas syringae pathovar is selected from the group consisting of P. syringae pv. actinidiae, P. syringae pv. avellanae, P. syringae pv. aesculi, P. syringae pv. theae and P. syringae pv. morsprunorum

7. A method according to any one of claims 1 to 6, wherein the plant is selected from the group consisting of kiwifruit, cherries, plums, hazelnuts, chestnuts, tea, pears and apples.

8. A method according to any one of claims 1 to 7, wherein applying the iron chelating compound to the plant comprises applying the iron chelating compound directly to the plant or applying the iron chelating compound to the soil or growth environment surrounding the plant.

9. A method according to any one of claims 1 to 8, wherein the iron chelating compound is applied to the plant by aqueous spray or by injection.

10. A method according to claim 9, wherein application by aqueous spray is performed by irrigation.

11. A composition comprising an iron chelating compound for use in controlling microbial infection in plants.

12. A composition according to claim 11, further comprising a surfactant.

13. A kit comprising an iron chelating compound together with instructions for how to apply the iron chelating compound to a plant to control a microorganism infection.

14. A kit comprising a pesticide composition comprising an iron chelating compound together with instructions for how to apply the iron chelating compound to a plant to control a microorganism infection.

15. A kit comprising a pesticide composition comprising an iron chelating compound selected from ethylendiamine di(o-hydroxyphenyl-acetic) acid (EDDHA) and hydroxybenzyl ethylenediamine (HBED), together with instructions for how to apply the iron chelating compound to a plant to control a bacteria infection.