WO2011025395A1 - Anti-phytopathogenic compositions - Google Patents

Abstract

A composition for controlling phytopathogens comprising at least one strain of Actinomycetes bacteria, such as an Actinomyces or a Streptomyces strain, and (i) one or more sulphur containing amino acids; or (ii) one or more flavins; or (iii) one or more stimulators of systemic acquired resistance; or (iv) any combination of two or more of (i) to (iv).

Description

ANTI-PHYTOPATHOGENIC COMPOSITIONS

FIELD OF THE INVENTION

This invention relates to anti-phytopathogenic compositions comprising anti- phytopathogenic bacteria, and the use of such compositions and bacteria as biological control agents. Methods for the biological control of phytopathogens, including phytopathogenic bacteria, proto2oa, fungi, and insects, using the compositions of the invention are also provided.

BACKGROUND OF THE INVENTION

Plant disease caused by pathogens such as bacteria, protozoans, fungi, and insects are a significant economic cost to plant-based agriculture and industries. Losses may arise through spoilage of produce both pre and post harvest, loss of plants themselves, or through reduction in growth and production abilities.

Traditionally, control of plant pathogens (phytopathogens) has been pursued through the application of chemical pesticides. The use of chemicals is subject to a number of disadvantages. The pathogens can and have developed tolerance to chemicals to over time, producing resistant populations. Indeed, resistance to pesticides is the greatest challenge to the viability of the horticultural industry.

The problem is particularly illustrated with reference to a number of economically important phytopathogenic insects. Populations of western flower thrips worldwide are reported to be resistant to most groups of pesticides including the following examples; acephate, abamectin, chlorpyrifos, endosulfan, methomyl, methiocarb, omethoate, pyrazophos and tau-fluvalinate. Populations of onion thrips in New Zealand have developed resistance to deltamethrin, and local populations have been reported to be resistance to diazinon and dichlorvos. Onion thrips in the United States have been reported to be resistant to many pesticides (Grossman, 1994). Greenhouse whitefly has reportedly developed resistance to organochlorine, organophosphate, carbamate and pyrethroid insecticides (e.g. Georghiou 1981, Anis & Brennan 1982, Elhag & Horn 1983, Wardlow 1985 and Hommes 1986). Resistance has also been reported in newer insecticides, buprofezin and teflubenzuron (Gorman et al. 2000). For other

phytopathogens, for example phytopathogenic bacteria of the genera Phytoplasma or Candidatus, effective chemical pesticides are not available.

Chemical residues may also pose environmental hazards, and raise health concerns.

The revival of interest in biological control such as microbial fungicides over the last 20 years has come directly from public pressure in response to concerns about chemical toxicities. Biological control presents an alternative means of controlling plant pathogens which is potentially more effective and specific than current methods, as well as reducing dependence on chemicals. Such biological control methods are perceived as a "natural" alternative to chemical pesticides with the advantage of greater public acceptance, reduced environmental contamination, and increased sustainability.

Mechanisms of biological control are diverse. One mechanism which has been demonstrated to be effective is the use of antagonistic microorganisms such as bacteria to control phytopathogenic insects. For example, the large scale production of Bacillus thuήngϊensis enabled the use of this bacterio-insecticide to control painted apple moth in Auckland, New Zealand.

There is however little information on the successful application of other anti- phytopathogenic bacteria and their industrial production is still relatively unsophisticated. Applications of anti-phytopathogenic bacteria as biological control agents (BCAs) do not appear to have met with significant grower acceptance, and may be perceived to be uneconomic.

There is therefore a need for pesticides, particularly insecticides and antibiotics, that act faster, have increased efficacy in controlling phytopathogens, require less frequent or less intensive application, have lower cost, or lower resulting toxicity than the currently- available pesticides.

It is therefore an object of the present invention to provide anti-phytopathogenic compositions useful in the biological control of phytopathogens, including

phytopathogenic insects, or at least to provide the public with a useful choice.

SUMMARY OF THE INVENTION

Accordingly, in one aspect the present invention provides a composition comprising at least one strain of Actinomycetes bacteria, and

(i) one or more sulphur-containing amino acids, or

(ϋ) one or more flavins, or

(ϋi) one or more stimulator of systemic acquired resistance; or

(iv) any combination of two or more of (i) to (iii).

In one embodiment, the at least one strain of Actinomycetes spp. is a strain of Actinomyces. In another embodiment, the at least one strain of Λctinomycetes is a strain of

Streptomyces, preferably a strain selected from Streptomyces rimosus, Streptomyces aureofaάens, or Streptomyces vene^uelae.

In one embodiment, the sulphur-containing amino acid is selected from the group comprising methionine, cysteine, and derivatives or precursors thereof, such as

homocysteine. In certain embodiments, mediionine is preferred.

In one embodiment, the flavin is riboflavin.

In one embodiment, the stimulator of acquired resistance is selected from the group comprising salicylic acid, jasmonic acid, cis jasmone, and arachidonic acid. In certain embodiments, salicylic acid is preferred.

In one embodiment, the composition additionally comprises one or more of the following: one or more carriers, one or more polysaccharides, or one or more trace elements.

In one embodiment, the carrier is an agriculturally acceptable carrier.

Preferably, the carrier is selected from the group consisting of a filler stimulant, an anti-caking agent, a wetting agent, an emulsifier, and an antioxidant, more preferably said composition comprises at least one of each of a filler stimulant, an anti-caking agent, a wetting agent, an emulsifier, and an antioxidant.

Preferably, said filler stimulant is a carbohydrate source, such as a disaccharide including, for example, sucrose, fructose, glucose, or dextrose, said anti-caking agent is selected from talc, silicon dioxide, calcium silicate, or kaelin clay, said wetting agent is skimmed milk powder, said emulsifier is a soy-based emulsifier such as lecithin or a vegetable-based emulsifier such as monodiglyceride, and said antioxidant is sodium glutamate or citric acid.

Preferably, the one or more trace elements is selected from the group comprising nitrogen, phosphorous, or potassium. Preferably the composition comprises each of nitrogen, phosphorous, and potassium.

In one embodiment, the composition is a stable composition capable of supporting reproductive viability of the Λctinomycetes strain for a period greater than about two weeks, preferably greater than about one month, about two months, about three months, about four months, about five months, more preferably greater than about six months.

In certain embodiments, the composition comprises a single strain of Λctinomycetes. ^■

Alternatively, the composition comprises multiple strains of Λctinomycetes. In one embodiment, the composition comprises at least one strain of Λctinomycetes bacteria, and one or more sulphur-containing amino acids, and one br more flavins, and one or more stimulator of systemic acquired resistance.

In one embodiment, the composition comprises at least one strain of Λctinomycetes bacteria, riboflavin, and methionine. In one embodiment, the composition additionally comprises nitrogen, phosphorous, and potassium.

In one embodiment, the at least one strain of Λctinomycetes bacteria is present in the composition as spores, for example as endospores.

In various embodiments, the composition comprises from about 1 x 10² to about 1 x 10¹² spores per ml, from about 1 x 10² to about 1 x 10^π spores per ml, from about 1 x 10² to about 1 x 10¹⁰ spores per ml, from about 1 x 10² to about 1 x 10⁹ spores per ml, from about 1 x 10³ to about 1 x 10 spores per ml, from about 1 x 10⁴ to about 1 x 10⁹ spores per ml, preferably from about 1 x 10⁵ to about 5 x-10⁸, and more preferably about 1 x 10⁶ to about 2 x 10⁸ spores per ml. In certain embodiments, the composition comprises at least 5 x 10⁷ spores per millilitre at application, or at least 10⁸ spores per ml at application.

In various embodiments, the composition comprises from about 10g/L to about 1000g/L methionine, from about 10g/L to about 900g/L methionine, from about 10g/L to about 800g/L methionine, from about 10g/L to about 700g/L methionine, from about 1 Og/L to about 600g/L methionine, from about 10g/L to about 500g/L methionine, from about 10g/L to -about 400g/L methionine, from about 10g/L to about 300g/L methionine, from about 10g/L to about 200g/L methionine, from about 10g/L to about 100g/L methionine, from about 50g/L to about 500g/L methionine, from about 50g/L to about 450g/L methionine, from about 50g/L to about 400g/L methionine, from about 50g/L to about 350g/L methionine, from about 50g/L to about 300g/L methionine, from about 50g/L to about 250g/L methionine, from about 100g/L to about 300g/L methionine, from about 150g/L to about 300g/L methionine, or from about 200g/L to about 300g/L methionine. In certain embodiments, the composition comprises at least about 50g/L methionine, at least about 100g/L methionine, at least about 150g/L methionine, at least about 175g/L methionine, or at least about 200g/L methionine. In one embodiment the composition comprises about 250g/L methionine.

In various embodiments, the composition comprises from about 0.1 g/L to about 10g/L riboflavin, from about 0.1g/L to about 9g/L riboflavin, from about 0.1g/L to about 8g/L riboflavin, from about 0.1 g/L to about 7g/L riboflavin, from about 0.1 g/L to about 6g/L riboflavin, from about 0.1 g/L to about 5g/L riboflavin, from about 0.1 g/L to about 4g/L riboflavin, from about O.lg/L to about 3g/L riboflavin, from about O.lg/L to about 2g/L riboflavin, from about O.lg/L to about 1.9g/L riboflavin, from about O.lg/L to about 1.8g/L riboflavin, from about O.lg/L to about 1.7g/L riboflavin, from about O.lg/L to about 1.6g/L riboflavin, from about O.lg/L to about 1.5g/L riboflavin, from about 0.5g/L to about 5g/L riboflavin, from about 0.5g/L to about 4.5g/L riboflavin, from about 0.5g/L to about 4g/L riboflavin, from about 0.5g/L to about 3.5g/L riboflavin, from about 0.5g/L to about 3g/L riboflavin, from about 0.5g/L to about 2.5g/L riboflavin, from about lg/L to about 3g/L riboflavin, from about 1.5g/L to about 3g/L riboflavin, or from about 1.5g/L to about 2g/L riboflavin. In certain embodiments, the composition comprises at least about 0.5g/L riboflavin, at least about lg/L riboflavin, at least about l.lg/L riboflavin, at least about 1.2g/L riboflavin, at least about 1.3g/L riboflavin, at least about 1.4g/L riboflavin, at least about 1.5g/L riboflavin, at least about 1.6g/L riboflavin, or at least about 1.65g/L riboflavin. In one embodiment the

composition comprises about 1.6g/L riboflavin.

In various embodiments, the composition comprises from about lg/L to about

300g/L nitrogen, from about lg/L to about 250g/L nitrogen, from about lg/L to about 200g/L nitrogen, from about lg/L to about 150g/L nitrogen, from about lg/L to about 100g/L nitrogen, from about 5g/L to about 100g/L nitrogen, from about 1 Og/L to about 100g/L nitrogen, from about 15g/L to about 100g/L nitrogen, from about 20g/L to about 100g/L nitrogen, from about 25g/L to about 100g/L nitrogen, from about 30g/L to about 100g/L nitrogen, from about 35g/L to about 100g/L nitrogen, from about 40g/L to about 100g/L nitrogen; from about 45g/L to about 100g/L nitrogen, from about 50g/L to about 100g/L nitrogen, from about 55g/L to about 100g/L nitrogen, from about 60g/L to about 100g/L nitrogen, from about 65g/L to about 100g/L nitrogen, or from about 70g/L to about 100g/L nitrogen. In certain embodiments, the composition comprises at least about 50g/L nitrogen, at least about 55g/L nitrogen, at least about 60g/L nitrogen, at least about 65g/L nitrogen, or at least about 70g/L nitrogen. In one embodiment the composition comprises about 75g/L nitrogen.

In various embodiments, the composition comprises from about lg/L to about 100g/L phosphorous, from about lg/L to about 90g/L phosphorous, from about lg/L to about 80g/L phosphorous, from about lg/L to about 70g/L phosphorous, from about lg/L to about 60g/L phosphorous, from about lg/L to about 50g/L phosphorous, from about lg/L to about 40g/L phosphorous, from about lg/L to about 30g/L phosphorous, from about lg/L to about 20g/L phosphorous, from about lg/L to about 10g/L phosphorous, from about 5g/L to about 50g/L phosphorous, from about 5g/L to about 45g/L phosphorous, from about 5g/L to about 40g/L phosphorous, from about 5g/L to about 35g/L phosphorous, from about 5g/L to about 30g/L phosphorous, from about 5g/L to about 25g/L phosphorous, from about 10g/L to about 30g/L phosphorous, from about 15g/L to about 30g/L phosphorous, or from about 20g/L to about 30g/L phosphorous. In certain embodiments, the composition comprises at least about 5g/L phosphorous, at least about 1 Og/L phosphorous, at least about 15g/L phosphorous, at • least about 17.5g/L phosphorous, or at least about 20g/L phosphorous. In one

embodiment the composition comprises about 25g/L phosphorous.

In various embodiments, the composition comprises from about lg/L to about

100g/L potassium, from about lg/L to about 90g/L potassium, from about lg/L to about 80g/L potassium, from about lg/L to about 70g/L potassium, from about lg/L to about 60g/L potassium, from about lg/L to about 50g/L potassium, from about lg/L to about 40g/L potassium, from about lg/L to about 30g/L potassium, from about lg/L tb about 20g/L potassium, from about lg/L to about 10g/L potassium, from about 5g/L to about 50g/L potassium, from about 5g/L to about 45g/L potassium, from about 5g/L to about 40g/L potassium, from about 5g/L to about 35g/L potassium, from about 5g/L to about 30g/L potassium, from about 5g/L to about 25g/L potassium, from about 1 Og/L to about 30g/L potassium, from about 15g/L to about 30g/L potassium, or from about 20g/L to about 30g/L potassium. In certain embodiments, the composition comprises at least about 5g/L potassium, at least about 10g/L potassium, at least about 15g/L potassium, at least about 17.5g/L potassium, or at least about 20g/L potassium. In one embodiment the composition comprises about 25g/L potassium.

In another aspect, the present invention provides the use of at least one strain of Λctinomycetes together with at least one of the following

(i) one or more sulphur-containing amino acids, or

(ii) one or more flavins, or

(iii) one or more stimulator of systemic acquired resistance,

in the preparation of a composition.

Preferably, said at least one strain of Λctinomycetes is in a reproductively viable form and amount.

In still a further aspect, the invention provides a method for producing a biological control composition, the method comprising:

providing a culture of at least one strain of Λctinomycetes bacteria, maintaining the culture in media under conditions suitable for growth of the at least one strain of Actinomyceter, and

i) combining the culture or the media with one or more sulphur-containing amino acids, or

ϋ) combining the culture or the media with one or more flavins, or iii) combining the culture or the media with one or more stimulator of systemic acquired resistance, or

iv) any combination of two or more of (i) to (iii).

In a further aspect the present invention provides a method for controlling one or more phytopathogens, the method comprising applying to a plant or its surroundings a composition of the invention.

In one embodiment, the composition is applied prophylactically, typically before the plant is infected by or exposed to the phytopathogen. In other embodiments, the composition is applied when the plant is infected by or exposed to the phytopathogen, or when the phytopathogen is present on or in the plant or its surroundings.

In one embodiment, the one or more phytopathogens are one or more

phytopathogenic insects, including one or more phytopathogenic insects selected from the group comprising aphids, psyllids, leaf hoppers, caterpillers, dirips and cicadas.

In one embodiment, die one or more phytopathogens are one or more

phytopathogenic bacteria, including one or more phytopathogenic bacteria selected from Candidata spp., such as Candidatus libeήbacter solanacearum or Candidatus phytoplasma australiense. C. Phytoplasma allocasuaήnae ,C. Phytoplasma asteήs, C. Phytoplasma aurantifolia, C. Phytoplasma brasiliense, C. Phytoplasma castaneae, C. Phytoplasma cocostan^aniae, C. Phytoplasma cocosnigeήae, C. Phytoplasma cynodontis, C. Phytoplasma fraxini, C. Phytoplasma japonicum, C. Phytoplasma luffae, C. Phytoplasma malt, C. Phytoplasma ory^ae, C. Phytoplasma palmae, C. Phytoplasma phoeniάum, C. Phytoplasma pruni, C. Phytoplasma prunorum, C. Phytoplasma pyri,C. Phytoplasma rhamni, C. Phytoplasma solani, C. Phytoplasma spartii, C. Phytoplasma tήfolii C. Phytoplasma ulmi,

C. Phytoplasma vitis, C. Phytoplasma

In various embodiments, the compositions of the invention may be applied at a rate of from about 1 x 10⁸ to about 1 x 10¹⁵ infectious units (IU) per hectare, from about 1 x 10⁹ to about 1 x 10¹⁵ IU per hectare, from about 1 x 1O¹⁰ to about 1 x 10¹⁵ IU per hectare, from about 1 x 10¹¹ to about 1 x 10¹⁵ IU per hectare, preferably from about 1 x 10¹" to about 1 x 10¹⁴ IU per hectare, more preferably from about 5 x 10¹" to about 1 x 10¹⁴ IU per hectare, more preferably about 1 x 10¹¹ to about 5 x 10" IU per hectare. In one embodiment, the infectious unit is a spore, such as an endospore, and the composition is applied at a rate of from about 1 x 10⁸ to about 1 x 10¹⁵ spores per hectare, from about 1 x 10⁹ to about 1 x 10¹⁵ spores per hectare, from about 1 x 10¹" to about 1 x 10¹⁵ spores per hectare, from about 1 x 10ⁿ to about 1 x 10¹⁵ spores per hectare, preferably from about 1 x 1O^UI to about 1 x 10¹⁴ spores per hectare, more preferably from about 5 x

10¹⁰ to about 1 x 10¹⁴ spores per hectare, more preferably about 1 x 10^π to about 5 x 10¹¹ spores per hectare.

Conveniently, such a rate of application can be achieved by formulating said composition at about 10 spores per millilitre or more, and applying said composition at a rate of about IL per hectare. As discussed herein, such an application rate can be conveniendy achieved by dissolution of the composition in a larger volume of agriculturally acceptable solvent, for example, water.

Preferably, the composition is admixed with water prior to application. In one embodiment, the composition is admixed with water and applied in at least about 10OL water/Ha, in at least about 150L/Ha, in at least about 200L/Ha, in at least about 250L/Ha, in at least about 300L/Ha, in at least about 350L/Ha, in at least about 400L/Ha, in at least about 450L/Ha, or in at least about 500L/Ha. In a preferred embodiment, the

composition is admixed with water to a final concentration of about 1 x 10¹¹ to about 5 x

10¹¹ spores per 500L water prior to application and applied at a rate of 500L/hectare.

In one embodiment a desiccation protection agent, preferably Fortune Plus™, is admixed to a final concentration of about lml/L prior to application.

In one embodiment a spreader is admixed to a final concentration of about lml/L prior to application.

In various embodiments, application is in furrow or by spraying.

To those skilled in the art to which the invention relates, many changes in construction and differing embodiments and applications of the invention will suggest themselves without departing from the scope of the invention as defined in the appended claims. The disclosures and the descriptions herein are purely illustrative and are not intended to be in any sense limiting.

In this specification where reference has been made to patent specifications, other external documents, or other sources of information, this is generally for the purpose of providing a context for discussing the features of the invention. Unless specifically stated otherwise, reference to such external documents is not to be construed as an admission that such documents, or such sources of information, in any jurisdiction, are prior art, or form part of the common general knowledge in the art.

It is intended that reference to a range of numbers disclosed herein (for example, 1 to 10) also incorporates reference to all rational numbers within that range (for example, 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, dierefore, all sub-ranges of all ranges expressly disclosed herein are hereby expressly disclosed. These are only examples of what is specifically intended and all possible combinations of numerical values between the lowest value and die highest value enumerated are to be considered to be expressly stated in this application in a similar manner.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is in part directed to compositions comprising Actinomycetes bacteria having efficacy against phytopathogenic insects, and the use of such compositions in controlling said phytopathogenic insects. 1. Definitions

-The phrases "anti-phytopathogenic activity" and "anti-phytopathogenic efficacy" are used interchangeably herein and refer to the ability of certain agents, such as certain microorganisms, to antagonise one or more phytopathogens.

Preferably, said anti-phytopathogenic efficacy is the ability to parasitise and incapacitate, render infertile, impede the growth of, or kill one or more phytopathogens, such as a phytopathogenic fungi, preferably within 14 days of contact with the phytopathogen, more preferably within 7 days, more preferably still the ability to kill one or more

phytopathogens witiiin 7 days, or to kill one or more phytopathogens within 6 days, 5 days, 4 days, 3 days, 2 days, or within a single day.

The term "anti-bacterial" means an ability to antagonise one or more bacteria, particularly one or more phytopathogenic bacteria. According an anti-bacterial agent, such as an anti-fungal bacterial strain, is an agent that is an antagonist of one or more fungi, preferably of one or more phytopathogenic fungi. Such an agent is herein considered to have anti-fungal efficacy.

The term "biological control agent" (BCA) as used herein refers to a biological agent which acts as an antagonist of one or more phytopathogens, such as a

phytopathogenic insects, a phytopathogenic bacteria, or a phytopathogenic protozoa, or is able to control one or more phytopathogens. Antagonism may take a number of forms. In one form, the biological control agent may simply act as a repellent. In another form, the biological control agent may render the environment unfavourable for the phytopathogen. In a further, preferred form, the biological control agent may parasitise, incapacitate, render infertile, impeded the growth of, and/or kill the phytopathogen.

Accordingly, the antagonistic mechanisms include but are not limited to antibiosis, parasitism, infertility, and toxicity. Therefore, agents which act as antagonists of one or more phytopathogens can be said to have anti-phytopathogenic efficacy. For example, an agent that is an antagonist of a phytopathogenic insects can be said to have

mycopathogenic efficacy.

As used herein, a "biological control composition" is a composition comprising or including at least one biological control agent that is an antagonist of one or more phytopathogens. Such control agents include, but are not limited to, agents that act as repellents, agents that render the environment unfavourable for the pathogen, and agents that incapacitate, render infertile, and/or kill the pathogen. Accordingly, such a composition is herein considered to have anti-phytopathogenic efficacy.

Accordingly, as used herein an "anti-phytopathogenic composition" is a composition which comprises or includes at least one agent that is an antagonist of one or more phytopathogens. Such a composition is herein considered to have anti- phytopathogenic efficacy.

The term "comprising" as used in this specification means "consisting at least in part of. When interpreting each statement in this specification that includes the term "comprising", features other than that or those prefaced by the term may also be present. Related terms such as "comprise" and "comprises" are to be interpreted in the same manner.

The term "control" or "controlling" as used herein generally comprehends preventing, reducing, or eradicating phytopathogen infection or inhibiting the rate and extent of such infection, or reducing the phytopathogen population in or on a plant or its surroundings, wherein such prevention or reduction in the infection(s) or population(s) is statistically significant with respect to untreated infection(s) or populatiori(s). Curative treatment is also contemplated. Preferably, such control is achieved by increased mortality amongst the phytopathogen population.

The term "plant" as used herein encompasses not only whole plants, but extends to plant parts, cuttings as well as plant products including roots, leaves, flowers, seeds, stems, callus tissue, nuts and fruit, bulbs, tubers, corms, grains, cuttings, root stock, or scions, and includes any plant material whether pre-planting, during growth, and at or post harvest.

Plants that may benefit from the application of the present invention cover a broad range of agricultural and horticultural crops. The compositions of the present invention are also especially suitable for application in organic production systems.

When used in respect of an anti-phytopathogenic agent, such as an anti- phytopathogenic bacterial strain, or in respect of an anti-phytopathogenic composition, the phrase "retaining anti-phytopathogenic efficacy" and grammatical equivalents and derivatives thereof is intended to mean that the agent or composition still has useful anti- phytopathogenic activity. Preferably, the retained activity is at least about 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99 or 100% of the original activity, and useful ranges may be selected between any of these values (for example, from about 35 to about 100%, from about 50 to about 100%, from about 60 to about 100%, from about 70 to about 100%, from about 80 to about 100%, and from about 90 to about 100%). For example, to be useful in the present invention a strain having the identifying characteristics of a specified strain should retain anti-phytopathogenic activity, that is, retain at least about 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99 or 100% of the anti-phytopathogenic activity of the specified strain. Accordingly, preferred compositions of the invention are capable of supporting the maintenance of useful anti-phytopathogenic activity of the anti-pathogenic agent(s) they comprise, and can be said to retain anti-phytopathogenic activity, ideally until applied using the methods contemplated herein.

As used herein, the term "stable" when used in relation to a composition of the invention means a composition capable of supporting reproductive viability of the anti- phytopathogenic bacteria or of retaining anti-phytopathogenic activity of the composition for several weeks, preferably about one, about two, about three, about four, preferably about five, more preferably about six months, or longer.

The term "surroundings" when used in reference to a plant subject to the methods and compositions of the present invention includes soil, water, leaf litter, and/or growth media adjacent to or around the plant or the roots, tubers or the like thereof, adjacent plants, supports, water to be administered to the plant, and coatings including seed coatings. It further includes storage, packaging or processing materials such as protective coatings, boxes and wrappers, and planting, maintenance or harvesting equipment. 2. Control of phytopathogens

The present invention recognises that the horticultural sectors of many countries, including for example that of the United States of America, of New Zealand, and many states of Europe, are faced with the problem of increasing resistance amongst

phytopathogens. This is compounded under some regulatory regimes by a reduction in the availability of new chemical pesticides due to regulatory barriers or increased holding times following application.

The use of anti-phytopathogenic compositions as biological control agents presents a solution to this problem. Effective biological control agents can be selected according their ability to incapacitate or kill one or more target phytopathogens or phytopathogen populations. Under conducive conditions, phytopathogens such as phytopathogenic insects or phytopathogenic bacteria may infect plants and their surroundings including soil, leaf litter, adjacent plants, and supports. Anti-phytopathogenic compositions may be applied so as to incapacitate and/or kill the phytopathogens, thereby preventing or limiting the disease-causing capability of the pathogen. The effectiveness of these anti- phytopathogenic compositions in the field is in turn dependent on their ability to survive varying climatic conditions, such as interrupted wet periods and desiccation.

Methods to determine growth of anti-phytopathogenic bacteria, such as one or more strains of Λctinomjcetes, under different conditions, including different temperatures and on different media or other substrates, are well known in the art. Examples of methods to determine the ability of anti-phytopathogenic bacteria to grow at various temperatures are described herein, as are methods to determine whether a given isolate is dead or dormant at a given temperature.

Similarly, methods to establish whether an isolate is able to grow on a given artificial medium are exemplified herein. The use of such methods recognises that an isolate must be capable of being grown in sufficient quantity for it to be suitable for use as a biological control agent. Methods of growing sufficient amounts of bacteria of the invention are discussed further herein.

A strain of anti-phytopathogenic bacteria, for example a strain of anti- phytopathogenic Actinomycetes, suitable for use in accordance with the invention, is identified as one which is effective at reducing the population of the target phytopathogen species by a statistically significant amount with respect to the control treatment against which the strains are compared. Such strains can be considered as having anti- phytopathogenic efficacy. As described herein, the reduction in the population of the target phytopathogen may be by various antagonistic mechanisms. For example, the anti- phytopathogenic compositions may parasitise, incapacitate, render infertile, and/or preferably kill the phytopathogen. The anti-phytopathogenic compositions may also reduce the population of die target phytopathogen by rendering the environment, for example the plant to which the anti-phytopathogenic compositions is applied or its surroundings, unfavourable for the phytopathogen. In this embodiment, the anti- phytopathogenic compositions may be considered to be acting as a repellent, and reducing the effective population of the target phytopathogen in the vicinity of the plant or its surroundings.

■ Preferably, suitable strains exhibit at least about 5% anti-phytopathogenic efficacy, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, more preferably at least about50% anti-phytopathogenic efficacy expressed as a percentage reduction of the population of the relevant phytopathogen species compared to the control treatment. By way of illustration, the methodology described herein may be employed to identify

Jictinom_jcetes isolates effective against a variety of target phytopathogens, whereas procedures analogous to diose described herein can be employed in relation to other phytopadiogens and other anti-phytopathogenic strains.

Although anti-phytopathogenic efficacy is a principal requisite for an isolate to be considered suitable for use as a biological control agent, the bacterial isolate should have additional characteristics to be suitable for use as a biological control agent.

For example, the bacterial strain must be able to be stored in a viable form for a reasonable period, ultimately so as to allow it to be applied to the target plant or its surroundings in a form and concentration that is effective as a biological control agent.

The bacterial strain should also be able to achieve infection threshold when applied to a plant or its surroundings for it to be suitable for use as a biological control agent. As used herein, infection threshold refers to the concentration of bacteria required for the bacteria to become established on the target plant or its surroundings so as to then have anti-phytopathogenic efficacy. As will be appreciated, in order to achieve infection threshold, some isolates of bacteria may require application at such a high rate as to be impractical or unviable. Furthermore, some bacterial isolates may not be able to achieve infection threshold irrespective of the concentration or rate at which they are applied.

Suitable anti-phytopathogenic compositions are able to achieve infection threshold when applied at a rate of about 10¹⁰ to about 10¹² spores per hectare, or applied at a concentration of about 10⁷ to about 10⁹ spores per millilitre of composition when said composition is applied at a rate of about 1L/500L water/hectare.

Methods to determine infection threshold are well known in die art, and examples of such methods are presented herein. In certain embodiments, infection direshold can be determined directly, for example by analysing one or more samples obtained from a target plant, its surroundings, and/or a pathogen of said plant, and determining die presence or amount of anti-phytopathogenic compositions on or in said sample. In other

embodiments, infection threshold can be determined indirectly, for example by observing a reduction in the population of one or more phytopadiogens. Combinations of such mediods are also envisaged.

3. Bacterial strains

Streptomyces, the largest genus of Actinobacteria, are gram-positive bacteria with genomes having high GC-content and the ability to form a tough, protective endospore, allowing die organism to tolerate extreme environmental conditions. In excess of 500 species of Streptomyces bacteria have been described. Many important antibiotics are produced by (and were originally identified from) species of Streptomyces, including

Neomycin from S.fradiae, Streptomycin from S. griseus, Tetracycline from S. ήmosus, Vancomycin from S. oήentalis, Daptomycin from S. roseosporus, Rifamycin from S.

mediterranei, Chloramphenicol from S. veneψ- elae, Puromycin from S. alboniger, and

Lincomycin from S. lincolnensis.

Streptomyces useful in the present invention will produce one or more antibiotic compounds, such as one or more tetracyclines or one or more chloramphenicols, or may produce one or more other bacteriocidal or bacteriostatic compounds. Exemplary producers of tetracyclines include Streptomyces aureofaάens and Streptomyces rimosus, while Streptomyces vene^uele is a well known producer of chloramphenicol. Streptomyces useful in the invention are preferably capable of surviving interrupted wet periods or desiccation, and of controlling one or more phytopathogens, for example one or more phytopathogenic insects such as, but not limited to, psyllids, aphids, and leaf hoppers, or one or more phytopadiogenic bacteria, such as but not limited to Candidata, such as Candidatus libeήbacter solanacearum and Candidatus phytoplasma australiense, in the field. The degree of control of these phytopathogens using a composition of die invention comprising Streptomyces is generally as good as or better than die commonly used chemical fungicides employed by growers. Useful strains of Streptomyces may be characterised by the functional attributes described herein, including its particular anti-phytopathogenic activity against the specified phytopathogens described herein, and other phenotypic characteristics such as the morphological, biochemical and growth characteristics described herein. It will be appreciated that there are a wide variety of methods known and available to the skilled artisan that can be used to confirm the identity of Streptomyces, wherein exemplary methods include molecular biological methods including DNA fingerprinting, genomic analysis, sequencing, and related genomic and proteomic techniques. In particular, methods for the identification of bacterial strains using one or more analyses of ribosomal RNA (rRNA) are well established and are amenable to application in identifying Streptomyces and strains having the identifying characteristics thereof.

Actinomycetes are a diverse order of gram-positive Actinobacteria. A number of species oi Actinomycetes are able to form a tough, protective endospore, allowing the organism to tolerate extreme environmental conditions. Actinomycetes useful in the present invention may produce one or more antibiotic compounds, such as actinomycin, or produce one or more other bacteriocidal or bacteriostatic compounds. Actinomycetes useful in the invention are preferably capable of surviving interrupted wet periods or desiccation, and of controlling one or more phytopathogens, for example one or more phytopathogenic insects such as, but not limited to, psyllids, aphids, and leaf hoppers, or one or more phytopathogenic bacteria, such as but not limited to Candidata, such as Candidates liberibacter solanacearum and Candidates phytoplasma australiense, in the field. The degree of control of these phytopathogens using a composition of the invention comprising Actinomycetes is generally as good as or better than the commonly used chemical fungicides employed by growers.

It will be appreciated that methods suitable for identifying strains of Streptomyces suitable for use in the methods and compositions of the present invention, such as those described above, are similarly suitable for identifying strains of other Actinomycetes that are similarly useful in the present invention.

It is apparent that many phytopathogens, such as phytopathogenic insects and

phytopathogenic bacteria have developed resistance to a number of chemical fungicides; in these and other instances, compositions of the invention comprising one or more strains of Actinomycetes, such as one or more strains of Streptomyces, provide an effective alternative for phytopathogen control. This potent activity in the control of plant disease coupled with the absence of any observations of plant pathogenicity induced by the Actinomycetes or Streptomyces demonstrate that the compositions of the invention have desirable attributes for use as biological control agents.

The compositions of the invention may be used singly, or in combination with other anti-phytopathogenic agents, including other anti-phytopathogenic compositions, as described herein. Examples of other anti-phytopathogenic compositions are described in more detail below.

4. Compositions

The present invention provides compositions comprising at least one strain of Λctinomycetes, such as at least one strain of Streptomyces, and one or more sulphur-containing amino acids, or one or more flavins, or one or more stimulator of systemic acquired resistance.

Sulphur-containing amino adds

The one or more sulphur-containing amino acids useful in the present invention may be selected from the group comprising the naturally-occuring amino acids methionine, cysteine, and derivatives or precursors thereof, such as S-adenosylmethionine, S- adenosylhomocysteine, and homocysteine. In certain embodiments, methionine is preferred.

Stimulators of acquired systemic resistance

Systemic acquired resistance (SAR) is a whole-organism resistance response that occurs in plants after exposure, including localised exposure, to a pathogen. As such, SAR may be considered as analogous to the innate immune response of vertebrates. It is widely recognised that SAR not only helps plants to resist, but also to recover from, pathogenic disease. SAR may be induced by a wide range of pathogens. It has been reported that induction of SAR in response to exposure to one pathogen may confer resistance to other padiogens— the so-called "broad spectrum" response. SAR has been associated with the induction of a number of genes and with the accumulation of endogenous salicylic acid.

Accordingly, a stimulator of systemic acquired resistance is an agent that is able to induce SAR in a plant, or an agent that is able to induce one or more of the genes associated with induction of SAR in a plant. In certain embodiments, the stimulator of SAR may be an attenuated, killed or non-infective phytopathogen. For example, phytopathogenic bacteria may be rendered suitable for use as a stimulator of SAR in the compositions of the present invention by heat-killing, lysing, fractionation, pressure-killing, irradiation, or UV- or light-treatment, or material derived from the bacteria including but not limited to bacterial cell wall compositions, bacterial cell lysates, lyophilised bacteria, and the like, as well as bacterial fermentates and fractions thereof, may be used. In other embodiments, the stimulator of systemic acquired resistance is a compound that modulates the induction of SAR, or modulates the expression of one or more genes involved in the induction of SAR.

In one embodiment, the stimulator of systemic acquired resistance is selected from the group comprising salicylic acid, jasmonic acid, cis jasmone, and arachidonic acid. In certain embodiments, salicylic acid is preferred.

Flawns

Flavins are organic compounds based on pteridine and comprising the tricyclic ring isoalloxazine. In biological systems, flavin moieties will usually be present as flavin adenine dinucleotide (FAD), or as flavin mononucleotide (FMN). Riboflavin (Vitamin B2) is the common source for flavins in biological systems.

The composition may include multiple strains of anti-phytopathogenic Actinomycetes or Streptomyces, and in certain embodiments, multiple strains may be utilised to target a number of phytopathogenic species, or a number of different developmental stages of a single phytopathogen, or indeed a combination of same. For example, the larval form of a phytopathogenic insect may be targeted with one bacterial strain, while the adult form may be targeted with another bacterial strain, wherein both strains are included in a composition of the invention.

Examples of compositions comprising bacteria as biological control agents are well known in the art. To be suitable for application to a plant or its surroundings, a

composition of the invention will usefully comprise at least one carrier. Typically, said at least one carrier is an agriculturally acceptable carrier, more preferably is selected from the group consisting of a filler stimulant, an anti-caking agent, a wetting agent, an emulsifϊer, and an antioxidant, more preferably said composition comprises at least one of each of a filler stimulant, an anti-caking agent, a wetting agent, an emulsifier, and an antioxidant.

Preferably, said filler stimulant is a carbohydrate source, such as a disaccharide including, for example, sucrose, fructose, glucose, or dextrose, said anti-caking agent is selected from talc, silicon dioxide, calcium silicate, or kaelin clay, said wetting agent is skimmed milk powder, said emulsifϊer is a soy-based emulsifier such as lecithin or a vegetable-based emulsifϊer such as monodiglyceride, and said antioxidant is sodium glutamate or citric acid. However, other examples well known in the art may be substituted, provided the ability of the composition to support fungal viability is maintained. Preferably, said composition is a biological control composition. The concentration of the components present in the composition, such as the anti-phytopathogenic

Λctinomycetes or Streptomyces bacteria, that is required to be effective as biological control agents may vary depending on the end use, physiological condition of the plant; type (including bacterial species), concentration and degree of pathogen infection; temperature, season, humidity, stage in the growing season and the age of plant; number and type of conventional pesticides or other treatments (including insecticides or antibiotics) being applied; and plant treatments (such as deleafϊng and pruning) may all be taken into account in formulating the composition.

For use as a biological control agent, the anti-phytopathogenic bacteria present in the composition should be in a reproductively viable form. The term reproductively viable as used herein includes motile and spore forms (such as endospore forms) of the anti- phytopathogenic compositions. For example, for most purposes, bacterial strains are desirably incorporated into the composition in the form of spores. Spores are obtainable from the bacterial strains of the invention, and may be produced using known art techniques. The concentration of the bacterial spores in the composition will depend on the utility to which the composition is to be put. An exemplary concentration range is from about 1 x 10² to about 1 x 10¹² spores per ml, from about 1 x 10^" to about 1 x 10" spores per ml, from about 1 x 10 to about 1 x 10^lϋ spores per ml, from about 1 x 10 to about 1 x 10⁹ spores per ml, from about 1 x 10³ to about 1 x 10⁹ spores per ml, from about 1 x 10⁴ to about 1 x 10⁹ spores per ml, preferably from about 1 x 10⁵ to about 5 x 10⁸, and more preferably about 1 x 10^f) to about 2 x 10⁸ spores per ml. In certain embodiments, the composition comprises at least 10⁸ spores per ml at application.

In certain embodiments, compositions of the invention are applied at a rate of from about I x 10⁸ to about 1 x 10¹⁵ spores per hectare, from about 1 x 10⁹ to about 1 x 10¹⁵ spores per hectare, from about 1 x 10¹⁰ to about 1 x 10¹⁵ spores per hectare, from about 1 x 10¹¹ to about 1 x 10¹⁵ spores per hectare, preferably from about 1 x 10ⁱⁿ to about 1 x 10¹⁴ spores per hectare, more preferably from about 5 x 10¹⁰ to about 1 x 10¹⁴ spores per hectare, more preferably about 1 x 10¹ ' to about 5 x 10 ' spores per hectare.

It will be appreciated that the rate of application will frequendy depend on the crop to which the compositions are being applied, and the stage of development of the crop.

In theory one infective unit should be sufficient to infect a host but in actual situations a minimum number of infective units are required to initiate an infection. The concepts of lethal dose (LD) regularly used with chemical pesticides are inappropriate for microbial pesticides. Concepts of infective dose (ID) or infective concentration (IC) are more precise or applicable. ID or IC refer to die actual number of infective units needed to initiate infection or the number of infective units exposed to the pathogen to cause death. Therefore, the number of infective units applied in the field or greenhouse against a pahtogen will affect the degree of control. It is important to apply the desired

concentration of the anti-phytopathogenic compositions, property placed and at the right time, to obtain good control of the pest: this is known as the "infection threshold".

It will be apparent that the concentration of bacteria, for example, bacterial spores, in a composition formulated for application may be less than that in a composition formulated for, for example, storage. The Applicants have determined that with the anti- phytopathogenic compositions of the present invention, infection threshold occurs at about 1 x 10⁸ spores per ml of solution, when applied at a rate of about IL to about 3L per hectare.

Accordingly, in one embodiment, a composition formulated for application will preferably have a concentration of at least about 5 x 10⁷ units (such as spores) per ml. In another example, a composition formulated for storage (for example, a composition such as a wettable powder capable of formulation into a composition suitable for application) will preferably have a concentration of about 10ⁱⁿ units per gram. It will be apparent that the concentration of a composition formulated for storage and subsequent formulation into a composition suitable for application must be adequate to allow said composition for application to also be sufficiently concentrated so as to be able to be applied to reach infection direshold.

Preferably, the composition is a stable composition capable of supporting reproductive viability of said anti-phytopathogenic bacteria or anti-phytopathogenic efficacy of the compositions for a period greater than about two weeks, preferably greater than about one month, about two months, about three months, about four months, about five months, more preferably greater than about six months. To be suitable for use as a biological control composition, the composition preferably is able to support reproductive viability of the bacteria for a period greater than about six months.

Using conventional solid substrate and liquid fermentation technologies well known in the art, the anti-phytopathogenic strains of the invention can be grown in sufficient amounts to allow use as biological control agents. For example, spores from selected strains can be produced in bulk for field application using nutrient film, submerged culture, and rice substrate growing techniques. Actinomycete growth media, such as Arginine glycerol mineral salt agar, Chitin agar, or Dextrose nitrate agar, are typically preferred, particularly for initial growth. Cultures may be transferred to other media, such as Yeast malt extract broth, as required. Growth is generally effected under aerobic conditions at any temperature satisfactory for growth of the organism. For example, for Λctinomycetes, a temperature range of from 10 to 37°C, preferably 20 to 30°C, and most preferably about 26°C, is preferred. The pH of the growth medium is slightly acid to neutral, that is, about 5.0 to 7.0, and most preferably 5.5. Incubation time is sufficient for the isolate to reach a stationary growth phase and will occur in normal photoperiod as is known in the art.

The spores may be harvested by methods well known in the art, for example, by conventional filtering or sedimentation methodologies (eg. centrifugation) or harvested dry using a cyclone system. Spores can be used immediately or stored, chilled at 0° to 6°C, preferably 2°C, for as long as they remain reproductively viable. It is however generally preferred that when not incorporated into a composition of the invention, use occurs within two weeks of harvesting.

The compositions of the invention may also include one or more carrier, preferably one or more agriculturally acceptable carrier. In one embodiment the carrier, such as an agriculturally acceptable carrier, can be solid or liquid. Carriers useful herein include any substance typically used to formulate agricultural composition.

In one embodiment the agriculturally acceptable carrier maybe selected from the group comprising fillers, solvents, excipients, surfactants, suspending agents,

speaders/stickers (adhesives), antifoaming agents, dispersants, wetting agents, drift reducing agents, auxiliaries, adjuvants or a mixture thereof.

Compositions of the invention may be formulated as, for example, concentrates, solutions, sprays, aerosols, immersion baths, dips, emulsions, wettable powders, soluble powders, suspension concentrates, dusts, granules, water dispersible granules,

microcapsules, pastes, gels and other formulation types by well-established procedures.

These procedures include mixing and/or milling of the active ingredients with agriculturally acceptable carrier substances, such as fillers, solvents, excipients, surfactants, suspending agents, speaders/stickers (adhesives), antifoaming agents, dispersants, wetting agents, drift reducing agents, auxiliaries and adjuvants.

In one embodiment solid carriers include but are not limited to mineral earths such as silicic acids, silica gels, silicates, talc, kaolin, attapulgus clay, limestone, lime, chalk, bole, loess, clay, dolomite, diatomaceous earth, aluminas calcium sulfate, magnesium sulfate, magnesium oxide, ground plastics, fertilizers such as ammonium sulfate, ammonium phosphate, ammonium nitrate, and ureas, and vegetable products such as grain meals, bark meal, wood meal, and nutshell meal, cellulosic powders and the like. As solid carriers for granules the following are suitable: crushed or fractionated natural rocks such as calcite, marble, pumice, sepiolite and dolomite; synthetic granules of inorganic or organic meals; granules of organic material such as sawdust, coconut shells, corn cobs, corn husks or tobacco stalks; kieselguhr, tricalcium phosphate, powdered cork, or absorbent carbon black; water soluble polymers, resins, waxes; or solid fertilizers. Such solid compositions may, if desired, contain one or more compatible wetting, dispersing, emulsifying or coloring agents which, when solid, may also serve as a diluent.

In one embodiment the carrier may also be liquid, for example, water; alcohols, particularly butanol or glycol, as well as their ethers or esters, particularly methylglycol acetate; ketones, particularly acetone, cyclohexanone, methylethyl ketone,

methylisobutylketone, or isophorone; petroleum fractions such as paraffinic or aromatic hydrocarbons, particularly xylenes or alkyl naphthalenes; mineral or vegetable oils; aliphatic chlorinated hydrocarbons, particularly trichloroethane or methylene chloride; aromatic chlorinated hydrocarbons, particularly chlorobenzenes; water-soluble or strongly polar solvents such as dimethylformamide, dimethyl sulfoxide, or N-methylpyrrolidone; liquefied gases; or the like or a mixture thereof.

In one embodiment surfactants include nonionic surfactants, anionic surfactants, cationic surfactants and/or amphoteric surfactants and promote the ability of aggregates to remain in solution during spraying.

Spreaders /stickers promote the ability of the compositions of the invention to adhere to plant surfaces. Examples of surfactants, spreaders/stickers include but are not limited to Tween and Triton (Rhom and Hass Company), Fortune®, Pulse, C. Daxoil®, Codacide oil®, D-C. Tate®, Supamet Oil, Bond®, Penetrant, Glowelt® and Freeway, Citowett®, Fortune Plus™, Fortune Plus Lite, Fruimec, Fruimec lite, alkali metal, alkaline earth metal and ammonium salts of aromatic sulfonic acids, e.g., ligninsulfonic acid, phenolsulfonic acid, naphthalenesulfonic acid and dibutylnaphthalenesulfonic acid, and of fatty acids, alkyl and alkylaryl sulfonates, and alkyl, lauryl ether and fatty alcohol sulfates, and salts of sulfated hexadecanols, heptadecanols, and octadecanols, salts of fatty alcohol glycol ethers, condensation products of sulfonated naphthalene and naphthalene derivatives with formaldehyde, condensation products of naphthalene or

naphthalenesulfonic acids with phenol and formaldehyde, polyoxyethylene octylphenol ethers, ethoxylated isooctylphenol, ethoxylated octylphenol and ethoxylated nonylphenol, alkylphenol polyglycol ethers, tributylphenyl polyglycol ethers, alkylaryl polyether alcohols, isotridecyl alcohol, fatty alcohol ethylene oxide condensates, ethoxylated castor oil, polyoxyethylene alkyl ethers, ethoxylated polyoxypropylene, lauryl alcohol polyglycol ether acetal, sorbitol esters, lignin-sulfite waste liquors and methyl cellulose. Where selected for inclusion, one or more agricultural surfactants, such as Tween are desirably included in the composition according to known protocols.

Wetting agents reduce surface tension of water in the composition and thus increase the surface area over which a given amount of the composition may be applied. Examples of wetting agents include but are not limited to salts of polyacrylic acids, salts of lignosulfonic acids, salts of phenolsulfonic or naphthalenesulfonic acids, polycondensates of ethylene oxide with fatty alcohols or fatty acids or fatty esters or fatty amines, substituted phenols (particularly alkylphenols or arylphenols), salts of sulfosuccinic acid esters, taurine derivatives (particularly alkyltaurates), phosphoric esters of alcohols or of polycondensates of ethylene oxide with phenols, esters of fatty acids with polyols, or sulfate, sulfonate or phosphate functional derivatives of the above compounds.

In one embodiment the preferred method of applying the composition of the invention is to spray a dilute or concentrated solution by handgun or commercial airblast.

In another embodiment the preferred method of applying the composition of the invention is to apply a dilute or concentrated solution in furrow prior to or at planting.

As described above, the compositions of the present invention may be used alone or in combination with one or more other agricultural agents, including pesticides, insecticides, acaracides, additional fungicides, bactericides, herbicides, antibiotics, antimicrobials, nemacides, rodenticides, entomopathogens, pheromones, attractants, plant growth regulators, plant hormones, insect growth regulators, chemosterilants, microbial pest control agents, repellents, viruses, phagostimulents, plant nutrients, plant fertilisers and biological controls. When used in combination with other agricultural agents the administration of the two agents may be separate, simultaneous or sequential. Specific examples of these agricultural agents are known to those skilled in the art, and many are readily commercially available.

Examples of plant nutrients include but are not limited to nitrogen, magnesium, calcium, boron, potassium, copper, iron, phosphorus, manganese, molybdenum, cobalt, boron, copper, silicon, selenium, nickel, aluminum, chromium and zinc. Examples of antibiotics include but are not limited to oxytetracyline and streptomycin.

Examples of fungicides include but are not limited to the following classes of fungicides: carboxamides, benzimidazoles, triazoles, hydroxypyridines, dicarboxamides, phenylamides, thiadiazoles, carbamates, cyano-oximes, cinnamic acid derivatives, morpholines, imidazoles, beta-methoxy acrylates and pyridines/pyrimidines.

Further examples of fungicides include but are not limited to natural fungicides, organic fungicides, sulphur-based fungicides, copper/calcium fungicides and elicitors of plant host defences.

Examples of natural fungicides include but are not limited to whole milk, whey, fatty acids or esterified fatty acids.

Examples of organic fungicides include but are not limited to any fungicide which passes an organic certification standard such as biocontrol agents, natural products, elicitors (some of may also be classed as natural products), and sulphur and copper fungicides (limited to restricted use).

An example of a sulphur-based fungicide is Kumulus™ DF (BASF, Germany).

An example of a copper fungicide is Kocide® 2000 DF (Griffin Corporation, USA).

Examples of elicitors include but are not limited to chitosan, Bion™, BABA (DL-3- amino-n-butanoic acid, β-aminobutync acid) and Milsana™ (Western Farm Service, Inc., USA).

In some embodiments non-organic fungicides may be employed. Examples of non-organic fungicides include but are not limited to Bravo™ (for control of PM on cucurbits); Supershield™ (Yates, NZ) (for control of Botrytis and PM on roses); Topas® 200EW (for control of PM on grapes and cucurbits); Flint™ (for control of PM on apples and cucurbits); Amistar® WG (for control of rust and PM on cereals); and Captan™, Dithane™, Euparen™, Rovtal™, Scala™, Shirlan™, Switch™ and Teldor™ (for control of Botrytis on grapes).

Examples of pesticides include but are not limited to azoxystrobin, bitertanol, carboxin, Cu₂O, cymoxanil, cyproconazole, cyprodinil, dichlofluamid, difenoconazole, diniconazole, epoxiconazole, fenpiclonil, fludioxonil, fluquiconazole, flusilazole, flutriafol, furalaxyl, guazatin, hexaconazole, hymexazol, imazalil, imibenconazole, ipconazole, kresoxim-methyl, mancozeb, metalaxyl, R-metalaxyl, metconazole, oxadixyl, pefurazoate, penconazole, pencycuron, prochloraz, propiconazole, pyroquilone, SSF-109, spiroxamin, tebuconazole, thiabendazole, tolifluamid, triazoxide, triadimefon, triadimenol, triflumizole, triticonazole and uniconazole.

An example of a biological control agent other than a composition of the present invention is the BotryZen™ biological control agent comprising Ulocladium oudemansii. The compositions may also comprise a broad range of additives such as stablisers and penetrants used to enhance the active ingredients and so-called 'stressing' additives to improve spore vigor, germination and survivability such as potassium chloride, glycerol, sodium chloride and glucose. Additives may also include compositions which assist in maintaining microorganism viability in long term storage, for example unrefined corn oil and so called invert emulsions containing a mixture of oils and waxes on the outside and water, sodium alginate and conidia on the inside.

It is important that any additives used are present in amounts that do not interfere with the effectiveness of the biological control agents.

Examples of suitable compositions including carriers, preservations, surfactants and wetting agents, spreaders, and nutrients are provided in US 5780023, incorporated herein in its entirety by reference.

Preferred compositions may comprise trace elements, such as but not limited to nitrogen, phosphorous, manganese, magnesium, zinc, potassium, sodium, and iron;

carbohydrates, such as but not limited to molasses; one or more gums, such as but not limited to guar gum, xanthan gum, locust bean gum, cassia gum, konjac flour, beta-glucan, tara gum, gum arabic, gellan gum, carboxymethylcellulose, methylcellulose, hydroxypropyl methylcellulose, tragacanth gum, karaya gum, gum acacia, chitosan, arabinoglactins, alginate, pectin, carrageenan, or psyllium; and other ingredients, such as one or more algae, seaweed, or extracts thereof.

A particularly preferred composition for use in the invention comprises Streptomyces rimosus spores 10ⁿ/L, Nitrogen 75g/L, Potassium 25g/L, Phosphorous 25g/L, Riboflavin 1.67g/L, and Methionine 250g/L.

It will be apparent that when one or more of the many commonly used pesticides do not adversely affect the anti-phytopathogenic compositions of the invention, the compositions of the invention may therefore also include such additional pesticides, for example, one or more additional anti-phytopathogens, including one or more additional anti-phytopathogenic bacteria. Alternatively, the compositions may be used separately but in conjunction with such pesticides in control programmes. The invention also provides a method of producing a composition comprising one or more anti-phytopathogenic compositions of the invention, said method comprising obtaining a reproductively viable form of an anti-phytopathogenic strain oϊActinomycetes, and combining the reproductively viable form of the anti-phytopathogenic Actinomycetes with one or more sulphur-containing amino acids, or one or more flavins, or one or more stimulator of systemic acquired resistance.

The compositions may be prepared in a number of forms. One preparation comprises a powdered form of a composition of the invention which may be dusted on to a plant or its surroundings. In a further form, the composition is mixed with a diluent such as water to form a spray, foam, gel or dip and applied appropriately using known protocols.

In a presently preferred embodiment, a composition formulated as described herein is mixed with water, for example using a pressurised sprayer, at about IL to about 3L/500L water/hectare. Preferably, a spreader is added to the composition to improve uptake or penetrance into the plant (eg. via the root system) at about 500ml/500L water.

Compositions formulated for other methods of application such as injection, rubbing or brushing, may also be used, as indeed may any known art method. Indirect applications of the composition to the plant surroundings or environment such as soil, water, or as seed coatings are specifically contemplated.

As discussed above, the concentration at which the compositions comprising anti- phytopathogenic compositions of the invention are to be applied so as to be effective biological control agents may vary depending on the end use, physiological condition of the plant; type (including bacterial species), concentration and degree of pathogen infection; temperature, season, humidity, stage in the growing season and the age of plant; number and type of conventional pesticides or other treatments (including fungicides) being applied; and plant treatments (such as leaf plucking and pruning).

The present invention also provides a method for controlling one or more phytopathogens, the method comprising applying to a plant or its surroundings a composition as herein described.

Again, while multiple strains of the anti-phytopathogenic compositions of the invention with activity against one or more phytopathogenic species may be employed in the control process, usually three strains or less are used in the method.

Repeated applications at the same or different times in a crop cycle are

contemplated. The anti-phytopathogenic compositions of the invention may be applied either earlier or later in the season. This may be over flowering or during fruiting, tuber emergence, and the like. The anti-phytopathogenic compositions of the invention may also be applied immediately prior to harvest, or after harvest to rapidly colonise necrotic or senescing leaves, fruit, stems, machine harvested stalks and the like to prevent fungi colonisation. The anti-phytopathogenic compositions of the invention may also be applied to dormant plants in winter to slow phytopathogen growth on dormant tissues.

Application may be at a time before or after bud burst and before and after harvest. However, treatment preferably occurs between flowering and harvest. To increase efficacy, multiple applications (for example, 2 to 6 applications over the stages of flowering through fruiting) of the anti-phytopathogenic compositions of the invention or a composition of the invention is contemplated.

Reapplication of the anti-phytopathogenic compositions of the invention or composition should also be considered after rain. Using pathogen infectivity prediction models or infection analysis data, application of the composition can also be timed to account for infection risk periods.

In the presently preferred embodiments, the anti-phytopathogenic compositions of the invention is applied in a solution, for example as described above, using a pressurised sprayer or in furrow. The plant parts should be lightly sprayed until just before run off. Applications may be made to any part of the plant and/or its surroundings, for example to the whole plant canopy, to the area in the canopy where the flowers and developing fruit are concentrated, or to the plant stem and/or soil, water or growth media adjacent to or surrounding the roots, tubers or the like.

Preferably the anti-phytopathogenic composition is stable, including a composition capable of supporting reproductive viability of the anti-phytopathogenic bacteria for several weeks, preferably about one, about two, about three, about four, preferably about five, more preferably about six months, or longer. Preferably, the composition is stable without a requirement for storage under special conditions, such as, for example, refrigeration or freezing.

The applied compositions control phytopathogens, including phytopathogenic insects and bacteria. Phytopathogenic insects and bacteria are responsible for many of the pre- and post-harvest diseases which attack plant parts and reduce growth rate, flowering, fruiting, production and may cause death of afflicted plants. As used herein,

phytopathogens include organisms which are themselves plant pathogens, and organisms which may act as a vector for other plant pathogens. For example, phytopathogenic insects such as psyllids are commonly vectors for phytopathogenic bacteria, such as phytoplasma. It will be appreciated that by controlling host organisms which act as vectors for other phytopathogens, the incidence and/or severity of plant disease can be minimised. The anti-phytopathogenic compositions of the invention may also be used in the control of pathogenic organisms that exhibit their primary pathogenicity against non-plant species.

Such pathogens include those which have a plant species as a vector or host

(including a secondary host) but which exhibit their pathogenic effect, or their main pathogenic effect, against a non-plant species. For example, the bacteria of the invention may be used to control pathogens such as Pythomyces spp. or Staphylococcus aureus, both of which are resident on plant species, but which cause their primary detrimental effect on grazing stock. Those skilled in the art will recognise that the present invention has utility in the control of such pathogens, despite their primary pathogenicity being directed at a non- plant species.

The compositions of the present invention may be applied to a wide variety of crops, including crops of the solanaceae family such as potato, tomato, capsicum, chilli, eggplant, kumara, petunia, and tamarillo.

Control of psyllids and of Liberibacter (Candidatus libeήbacter solanacearum) in the crops outlined above using the compositions and method of the present invention is particularly contemplated.

The process of the invention has particular application to plants and plant products, either pre- or post-harvest. For example, the composition of the invention may be applied to stored products of the type listed above including fruits, vegetables, cut flowers and seeds. Suitable application techniques encompass those identified above, particularly spraying.

The composition can potentially be used to treat or pretreat soils or seeds, as opposed to direct application to a plant. The composition may find use in plant processing materials such as protective coatings, boxes and wrappers.

Also encompassed by the present invention are plants, plant products, soils and seeds treated directly with an active strain of the bacteria of the invention or a composition of the invention.

In a further aspect, the present invention extends to the use of anti- phytopathogenic compositions of the invention in a composition of the invention.

The invention consists in the foregoing and also envisages constructions of which the following gives examples only and in no way limit the scope thereof. EXAMPLE 1 - COMPOSITION COMPRISING Streptomyces rimosus

This example describes the preparation of an anti-phytopathogenic composition comprising a strain of Streptomyces ήmosus.

Streptomyces rimosus was obtained from Landcare Research New Zealand

(http://nzfungi.landcareresearch.co.nz/icmp/results cultures.aspPID. Streptomyces rimosus, ICMP Number: 919, Contributor: T, R. Vernon, Number: N82, Via: ATCC Number: 10970) and initially grown at 26°C on Chitin agar, then transferred to Yeast malt extract broth for large scale production. The Streptomyces rimosus was allowed to sporulate, and endospores were collected by centrifugation. The spores were combined with the following ingredients at the indicated concentrations:

Streptomyces rimosus spores 10ⁿ/L

Nitrogen 75g/L

Potassium 25g/L

Phosphorous 25g/L

Riboflavin 1.67g/L

Methionine 250g/L.

The composition was prepared in 1OL or 2OL drums for distribution to growers, or larger volumes can be prepared as required.

EXAMPLE 2 - ANTI-PHYTOPATHOGENIC EFFICACY OF A

COMPOSITION OF THE INVENTION

This example describes an analysis of the anti-phytopathogenic efficacy of a composition comprising Streptomyces rimosus against phytopathogens.

Methods

The biological control composition described in Example 1 above is prepared. Field trials are conducted to assess the efficacy of the composition as a biological control agent of psyllid, and comparing same to established chemical treatment procedures and to untreated controls.

A plot is divided into three sections, and prior to planting the biological control composition is applied in furrow to the first section (at a rate of 3L/500L water/hectare), water alone is applied to the second section, and chemical insecticide with streptomycin is applied to the third section according to the manufacturers' instructions. The plot is then planted with potato. The biological control composition (2L/500L water/hectare), water alone, and chemical insecticide/antibiotic are reapplied at emergence, and again at tuber initiation.

Liberibacter infection rate is determined quantitatively by counting the number of potato plants living at 1 week after planting, 2 weeks after planting, 4 weeks after planting, at emergence, at tuber initiation, and at harvest. Psyllid number is assessed by counting the number of psyllid (both adult and juvenile) on a representative fraction of plants (typically 10 plants) in each section. Total harvest (in kilograms) is also determined, and the crop is graded for quality.

Results and Discussion

Results showing that liberibacter infection rate is diminished, and that survival, emergence, and growth of the plants and crop yield and quality is greater amongst potato plants to which the composition is applied, compared to untreated and chemically-treated controls supports the efficacy of the composition in the control of liberibacter infection and the control of pathogenesis resulting from psyllid infestation. >

EXAMPLE 3 -ANTI-PHYTOPATHOGENIC EFFICACY OF A

COMPOSITION OF THE INVENTION

This example describes an analysis of the anti-phytopathogenic efficacy of a composition comprising Streptomyces ήmosus against phytopathogens.

Methods

The biological control composition described in Example 1 above is prepared. Field trials are conducted to assess the efficacy of the composition as a biological control agent of psyllid, and comparing same to established chemical treatment procedures and to untreated controls.

A plot is divided into three sections, and prior to planting the biological control composition is applied in water carts to the first section (at a rate of 3L/500L

water/hectare), water alone is applied to the second section, and chemical insecticide with streptomycin is applied to the third section according to the manufacturers' instructions.

The plot is then planted with kumara. The biological control composition

(2L/500L water /hectare), water alone, and chemical insecticide/antibiotic are reapplied at at tuber initiation.

Liberibacter infection rate is determined quantitatively by counting the number of kumara plants living at 1 week after planting, 2 weeks after planting, 4 weeks after planting, at emergence, at tuber initiation, and at harvest. Psyllid number is assessed by counting the number of psyllid (both adult and juvenile) on a representative fraction of plants (typically 10 plants) in each section. Total harvest (in kilograms) is also determined, and the crop is graded for quality.

Results and Discussion

Results showing that liberibacter infection rate is diminished, and that survival, emergence, and growth of die plants and crop yield and quality is greater amongst kumara plants to which the composition is applied, compared to untreated and chemically-treated controls supports the efficacy of the composition in die control of liberibacter infection and the control of pathogenesis resulting from psyllid infestation.

EXAMPLE 4 - ANTI-PHYTOPATHOGENIC EFFICACY OF A

COMPOSITION OF THE INVENTION

This example describes an analysis of the anti-phytopadiogenic efficacy of a composition comprising Streptomyces rimosus against phytopadiogens.

Methods

The biological control composition described in Example 1 above is prepared. Field trials are conducted to assess the efficacy of the composition as a biological control agent of psyllid, and comparing same to established chemical treatment procedures and to untreated controls.

A tomato glasshouse is divided into three sections., and prior to planting the biological control composition is applied to plantlet steep of the first section (at a rate of 1L/100L plandet steep), water alone is applied to the second section, and chemical insecticide with streptomycin is applied to the third section according to the manufacturers' instructions.

The tomatoes are then planted out, and the biological control composition

(2L/500L water/hectare), water alone, and chemical insecticide/antibiotic are reapplied at at fruit set and at colour expression.

Liberibacter infection rate is determined quantitatively by counting the number of kumara plants living at 1 week after planting, 2 weeks after planting, 4 weeks after planting, at emergence, at fruit set, at colour expression, and at harvest. Psyllid number is assessed by counting the number of psyllid (both adult and juvenile) on a representative fraction of plants (typically 10 plants) in each section. Total harvest (in kilograms) is also determined, and the crop is graded for quality. Results and Discussion

Results showing that liberibacter infection rate is diminished, and that survival, emergence, and growth of the plants and crop yield and quality is greater amongst tomato plants to which the composition is applied, compared to untreated and chemically-treated controls supports the efficacy of the composition in the control of liberibacter infection and the control of pathogenesis resulting from psyllid infestation.

INDUSTRIAL APPLICATION

As will be evident from the above description, the present invention provides anti- phytopathogenic compositions that are useful for the control of phytopathogens, and particularly phytopathogenic insects and phytopathogenic bacteria. The use of these compositions in the control of phytopathogens, and methods to control phytopathogens, are also provided.

Claims

1. A composition comprising at least one strain of ActLnomycetes bacteria, and

(i) one or more sulphur-containing amino acids, or

(ii) one or more flavins, or

(iϋ) one or more stimulator of systemic acquired resistance; or

(iv) any combination of two or more of (i) to (ϋi).

2. The composition of claim 1, wherein the at least one strain of Actinomycetes is a strain of Actinomyces.

3. The composition of claim 1, wherein, the at least one strain of Actinomycetes is a strain of Streptomyces.

4. The composition of claim 3 wherein the at least one strain of Streptomyces is a strain selected from Streptomyces ήmosus, Streptomyces aureofaciens, or Streptomyces venetζuelae.

5. The composition according to any one of claims 1 to 4 wherein the sulphur-containing amino acid is selected from the group comprising methionine, cysteine, and derivatives or precursors thereof including S-adenosylnethionine, S-adenosylhomocysteine, and homocysteine.

6. The composition according to any one of claims 1 to 5 wherein the flavin is riboflavin.

7. The composition according to any one of claims 1 to 6 wherein the stimulator of acquired resistance is selected from the group comprising salicylic acid, jasmonic acid, cis jasmone, and arachidonic acid.

8. The composition according to any one of claims 1 to 7 wherein the composition

additionally comprises one or more of the following: one or more carriers, one or more polysaccharides, or one or more trace elements.

9. The composition according to any one of claims 1 to 8 wherein the composition is a stable composition capable of supporting reproductive viability of the Actinomycetes strain for a period greater than about two weeks.

10. The composition according to any one of claims 1 to 9 wherein the composition comprises a single strain of Actinomycetes.

11. The composition according to any one of claims 1 to 9 wherein the composition comprises multiple strains of Actinomycetes.

12. The composition according to any one of claims 1 to 11 wherein the composition

comprises at least one strain of Actinomycetes bacteria, and one or more sulphur-containing amino acids, and one or more flavins, and one or more stimulator of systemic acquired resistance.

13. The composition according to any one of claims 1 to 12 wherein the composition comprises at least one strain of Λctinomycetes bacteria, riboflavin, and methionine.

14. The composition according to any one of claims 1 to 13 wherein the composition

additionally comprises nitrogen, phosphorous, and potassium.

15. The composition according to any one of claims 1 to 14 wherein the at least one strain of Λctinomycetes bacteria is present in the composition as spores.

16. The composition according to any one of claims 1 to 15 wherein the composition

comprises from about 1 x 10² to about 1 x 10¹² spores per ml.

17. The composition according to any one of claims 1 to 16 wherein the composition

comprises from about 10g/L to about 1000g/L methionine.

18. The composition according to any one of claims 1 to 17 wherein the composition

comprises from about 0.1 g/L to about 10g/L riboflavin.

19. The composition according to any one of claims 1 to 18 wherein the composition

comprises from about lg/L to about 300g/L nitrogen.

20. The composition according to any one of claims 1 to 19 wherein the composition

comprises from about lg/L to about 100g/L phosphorous.

21. The composition according to any one of claims 1 to 20 wherein the composition

comprises from about lg/L to about 100g/L potassium.

22. A method for producing a biological control composition, the method comprising:

providing a culture of at least one strain of Λctinomycetes bacteria, maintaining the culture in media under conditions suitable for growth of the at least one strain of Actinomyceter, and i) combining the culture or the media with one or more sulphur-containing amino acids, or

ii) combining the culture or the media with one or more flavins, or

iii) combining the culture or the media with one or more stimulator of systemic acquired resistance, or

iv) any combination of two or more of (i) to (iii).

23. A method for controlling one or more phytopathogens, the method comprising applying to a plant or its surroundings a composition comprising at least one strain of Λctinomycetes bacteria and

i) one or more sulphur-containing amino acids, or

ii) one or more flavins, or

iii) one or more stimulator of systemic acquired resistance; or

iv) any combination of two or more of (i) to (iii).

24. The method according to claim to 22 wherein the application is prophylactic.

25. The method according to claim 24 wherein the application is before the plant is infected by or exposed to die phytopathogen.

26. The method according to claim 23 wherein the composition is applied when the plant is infected by or exposed to the phytopathogen, or when the phytopathogen is present on or in the plant or its surroundings.

27. The method according to any one of claims 23 to 26 wherein the one or more

phytopathogens are one or more phytopathogenic insects.

28. The method according to claim 27 wherein the one or more phytopathogenic insects is one or more phytopathogenic insects selected from the group comprising aphids, psyllids, leaf hoppers, caterpillers, thrips and cicadas.

29. The method according to any one of claims 23 to 26 wherein the one or more

phytopathogens are one or more phytopathogenic bacteria, including one or more phytopathogenic bacteria selected from Candidata spp., such as Candidatus libeήbacter solanacearum or Candidatus phytoplasma australiense. C. Phytoplasma allocasuaήnae ,C Phytoplasma asteris, C. Phytoplasma aurantifolia, C. Phytoplasma brasiliense, C Phytoplasma castaneae,

C. Phytoplasma cocostan^aniae, C. Phytoplasma cocosnigeriae, C Phytoplasma cynodontis,

C. Phytoplasma fraxini, C. Phytoplasma japonicum, C. Phytoplasma lujfae, C. Phytoplasma mali,

C. Phytoplasma ory^ae, C. Phytoplasma palmae, C. Phytoplasma phoeniάum, C. Phytoplasma pruni,

C. Phytoplasma prunorum, C. Phytoplasma pyri, C. Phytoplasma rhamni, C. Phytoplasma so lam,

C. Phytoplasma spartii, C. Phytoplasma trifolii, C. Phytoplasma ulmi, C. Phytoplasma Ms,

C. Phytoplasma ^i^iphi

30. The method according to any one of claims 23 to 29 wherein the application is at a rate of from about 1 x 10⁸ to about 1 x 10¹⁵ infectious units (IU) per hectare.

31. The method according to any one of claims 23 to 30 wherein when the infectious unit is a spore the application is at a rate of from about 1 x 10⁸ to about 1 x 10¹⁵ spores per hectare.

32. The method according to claim 31 wherein the application is at a rate of from about 1 x 10¹¹ to about 5 x 10" spores per hectare.

33. The method according to any one of claims 23 to 32 wherein the application is in furrow or by spraying.