WO2015142186A1 - Procédés de criblage visant à sélectionner des micro-organismes capables de conférer une propriété bénéfique à une plante - Google Patents

Procédés de criblage visant à sélectionner des micro-organismes capables de conférer une propriété bénéfique à une plante Download PDF

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Publication number
WO2015142186A1
WO2015142186A1 PCT/NZ2014/000045 NZ2014000045W WO2015142186A1 WO 2015142186 A1 WO2015142186 A1 WO 2015142186A1 NZ 2014000045 W NZ2014000045 W NZ 2014000045W WO 2015142186 A1 WO2015142186 A1 WO 2015142186A1
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WIPO (PCT)
Prior art keywords
plant
microorganisms
composition
plants
microorganism
Prior art date
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PCT/NZ2014/000045
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English (en)
Inventor
Susan Jane Turner
Peter John WIGLEY
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Biodiscovery New Zealand Limited
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Publication date
Application filed by Biodiscovery New Zealand Limited filed Critical Biodiscovery New Zealand Limited
Priority to PCT/NZ2014/000045 priority Critical patent/WO2015142186A1/fr
Priority to NZ630880A priority patent/NZ630880A/en
Publication of WO2015142186A1 publication Critical patent/WO2015142186A1/fr

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Classifications

    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H3/00—Processes for modifying phenotypes, e.g. symbiosis with bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms

Definitions

  • the present invention relates to methods for the screening, identification and/or application of microorganisms of use in imparting beneficial properties to plants.
  • Geography, environmental conditions, disease and attack by insects are major factors influencing the ability to grow and cultivate different species of plant. Such factors can have a significant downstream economic and social impact on communities around the world. There would be benefit in identifying products and methods which might impart beneficial properties to a plant species to allow it to grow in a variety of geographical locations, in different weather conditions, to survive disease and to be resistant to attack by insects, for example.
  • Tins may be the result of solubilisation of minerals from the soil biomass itself, or from colonization of the plants with microorganisms in endophytic, epiphytic or rhizospheric associations leading to nitrogen fixation, resistance to pests and diseases through direct microbial competition within the plant, or the elicitation of plant defence reactions.
  • the science community has produced literature on the diverse mechanisms of endophytic, epiphytic and rhizospheric plant microorganism associations, largely in relation to crop plants and their soils. The nature of some associations is known, encompassing the genetic basis of plant-induced metabolite production by specific organisms and the reverse influence of the microbe on gene expression in the plant (e.g.
  • Neotyphod im spp Neotyphod im spp
  • increases in plant growth following microbial application to certain crop plants or seeds has been documented.
  • commercial success is limited to a relatively small range of specific microbial applications e.g. Rhizobium spp. to legume seeds, or the use of products resulting from "uncontrolled" microbial fermentations e.g. compost teas, seaweed fermentations, fish waste fermentations etc.
  • a method for the selection of one or more microorganisms capable of imparting one or more beneficial property to a plant comprising at least the steps of:
  • step b) selecting one or more plant following step a);
  • step b) acquiring a second set of one or more microorganisms associated with said one or more plant selected in step b);
  • step d) repeating steps a) to c) one or more times, wherein the second set of one or more microorganisms acquired in step c) is used as the first set of microorganisms in step a) of any successive repeat;
  • the first set of one or more microorganisms has been subjected to one or more of a conditioning and a directed evolution process prior to step a) and/or the second set of one or more microorganisms acquired in step c) is subjected to one or more of a conditioning and a directed evolution process prior to being used in step a) of any successive repeat,
  • the second set of one or more microorganisms are isolated from said one or more plant in step c).
  • the method further comprises the steps of separating the two or more microorganisms into individual isolates, selecting two or more individual isolates, and then combining the selected two or more isolates.
  • the method further comprises repeating steps a) to c) one or more times, wherein where two or more microorganisms are acquired in step c), the two or more microorganisms are separated into individual isolates, two or more individual isolates are selected and then combined, and the combined isolates are used in step a) of the successive repeat. Accordingly, where reference is made to using the one or more microorganisms acquired in step c) in step a) of the method, it should be taken to include using the combined isolates of this embodiment of the invention.
  • two or more methods of the invention may be performed separately and the one or more microorganisms acquired in step c) of each separate method combined.
  • the combined microorganisms are used in step a) when performing a further method of the invention.
  • a method for the selection of one or more microorganisms capable of imparting one or more beneficial property to a plant comprising at least the steps of:
  • step b) applying one or more selective pressure during step a);
  • step c) acquiring a second set of one or more microorganisms associated with said one or more plant selected in step c);
  • step a) wherein the first set of one or more microorganisms has been subjected to one or more of a conditioning and a directed evolution process prior to step a) and/or the second set of one or more microorganisms acquired in step d) are subjected to one or more of a conditioning and a directed evolution process.
  • a method for the selection of one or more microorganisms capable of imparting one or more beneficial property to a plant comprising at least the steps of; a) subjecting one or more plant (including for example seeds, seedlings, cuttings, and/or propagules thereof) to a growth medium in the presence of a first set of one or more microorganisms;
  • step b) applying one or more selective pressure during step a);
  • step c) acquiring a second set of one or more microorganisms associated with said one or more plant selected in step c);
  • step e) repeating steps a) to d) one or more times, wherein the second set of one or more microorganisms in step d) is used as the first set of one or more microorganisms in step a) of any successive repeat;
  • step a) wherein the first set of one or more microorganisms has been subjected to one or more of a conditioning and a directed evolution process prior to step a) and/or the second set of one or more microorganisms acquired in step d) are subjected to one or more of a conditioning and a directed evolution process.
  • the second set of one or more microorganisms acquired in step d) may be subjected to one or more of a conditioning and a directed evolution process prior to being used in step a) of any successive repeat of the method.
  • the second set of one or more microorganisms are isolated from said one or more plant in step d).
  • one selective pressure is applied in step b).
  • the selective pressure is biotic and includes but is not limited to exposure to one or more organisms that are detrimental to the plant.
  • the organisms include fungi, bacteria, viruses, insects, mites and nematodes.
  • the selective pressure is abiotic.
  • Abiotic selective pressures include, but are not limited to, exposure to or changes in the level of salt concentration, temperature, H, water, minerals, organic nutrients, inorganic nutrients, organic toxins, inorganic toxins, and metals.
  • the selective pressure is applied during substantially the whole time during which the one or more plant is subjected to the growth medium and one or more microorganisms.
  • the selective pressure is applied during substantially the whole growth period of the one or more plant.
  • the selective pressure is applied at a discrete time point.
  • the method further comprises the steps of separating the two or more microorganisms into individual isolates, selecting two or more individual isolates, and then combining the selected two or more isolates.
  • the method further comprises repeating steps a) to d) one or more times, wherein the one or more microorganisms acquired in step d) are used in step a) of any successive repeat.
  • the method further comprises repeating steps a) to d) one or more times, wherein where two or more microorganisms are acquired in step d), the two or more microorganisms are separated into individual isolates, two or more individual isolates are selected and then combined, and the combined isolates are used in step a) of any successive repeat. Accordingly, where reference is made to using the one or more microorganisms acquired in step d) in step a) of the method, it should be taken to include using the combined isolates of this embodiment of the invention.
  • one or more selective pressure applied in any successive repeats of steps a) to d) is different. In another embodiment, the selective pressure(s) applied in any successive repeats of steps a) to d) is the same.
  • the method further comprises the following step which is conducted prior to step a): subjecting the one or more plant (including for example seeds, seedlings, cuttings, and/or propagules thereof) to a growth medium in the presence of one or more microorganisms, and after a desired period, acquiring one or more microorganisms associated with said one or more plant.
  • the acquired one or more microorganisms are used in step a) of the process.
  • this step may be conducted two or more times prior to step a).
  • two or more methods of the invention may be performed separately and the one or more microorganisms acquired in step d) of each separate method combined.
  • the combined microorganisms are used in step a) when performing a further method of the invention.
  • the methods are combined, in any order or combinations.
  • the method comprises:
  • step b) selecting one or more plant following step a);
  • step b) acquiring a second set of one or more microorganisms associated with said one or more plant selected in step b);
  • step 1 c) subjecting one or more plant (including for example seeds, seedlings, cuttings, and/or propagules thereof) to a growth medium in the presence of a the second set of one or more microorganisms acquired in step 1 c);
  • step 2 b applying one or more selective pressure during step 2 a);
  • step 2 c acquiring a second set of one or more microorganisms associated with said one or more plant selected in step 2 c).
  • Such a method may be repeated any number of times, wherein the one or more
  • microorganisms acquired in step 2 d) are used in step 1 a) of any successive repeats of the method.
  • the method comprises:
  • step b) applying one or more selective pressure during step a);
  • step d) acquiring a second set of one or more microorganisms associated with said one or more plant selected in step c); and, 2. a) subjecting one or more plant (including for example seeds, seedlings, cuttings, and/or propagules thereof) to a growth medium in the presence of the second set of microorganisms acquired in step 1 d);
  • step 2 b) selecting one or more plant following step 2 a);
  • step 2 b acquiring a second set of one or more microorganisms associated with said one or more plant selected in step 2 b).
  • Such a method may be repeated any number of times, wherein the one or more
  • microorganisms acquired in step 2 c) are used in step 1 a) of any successive repeats of the method.
  • steps 1 or 2 may be repeated one or more times before the microorganisms acquired in those steps are used in step 1 a) or step 2 a), as the case may be.
  • the methods may further comprise the step of separating the two or more microorganisms into individual isolates, selecting two or more individual isolates, and then combining the selected two or more isolates. The combined isolates may then be used in any successive repeats of the method or any part thereof.
  • two or more methods or parts thereof may be performed separately and the one or more microorganisms acquired in steps 1 and/or 2 of the method combined.
  • the combined microorganisms are used in any successive rounds of the method or part thereof.
  • the first set and/or second set of one or more microorganisms are selected from the microorganisms detailed herein after.
  • the growth medium is selected from the growth media detailed herein after.
  • the step of subjecting one or more plant to a growth medium involves growing or multiplying the plant.
  • two or more plants are subjected to a growth medium in the presence of one or more microorganisms.
  • 10 to 20 plants are subjected to a growth medium in the presence of one or more microorganisms.
  • 20 or more, 100 or more, 300 or more, 500 or more, or 1000 or more plants are subjected to a growth medium in the presence of one or more microorganisms.
  • the one or more plant is selected on the basis of one or more selection criterion. In one embodiment, the one or more plant is selected on the basis of one or more phenotypic trait. In one preferred embodiment, the one or more plant is selected based on the presence of a desirable phenotypic trait. In one embodiment, the phenotypic trait is one of those detailed herein after. In one embodiment, the one or more plant is selected on the basis of one or more genotypic trait. In one preferred embodiment, the one or more plant is selected based on the presence of a desirable genotypic trait. In one embodiment, the one or more plant is selected based on a combination of one or more genotypic and one or more phenotypic traits. In one embodiment, different selection criteria may be used in each iteration of a method of the invention.
  • the second set of one or more microorganisms are isolated from the root, stem and/or foliar (including
  • the second set of one or more microorganisms are isolated from whole plant tissue of the one or more plants selected.
  • the plant tissues may be surface sterilised and then one or more microorganisms isolated from any tissue of the one or more plants, This embodiment allows for the targeted selection of endophytic microorganisms.
  • the second set of one or more microorganisms may be isolated from the growth medium surrounding selected plants.
  • the second set of one or more microorganisms are acquired in crude form and may be used in used in step a) of any successive repeat.
  • the one or more microorganisms are acquired any time after germination of the one or more plants.
  • the methods of the first or second (and/or related) aspect of the invention may also be useful in identifying and/or selecting one or more endophytic microorganism capable of imparting one or more beneficial property to a plant.
  • plant material including for example seeds, seedlings, cuttings, and/or propagules thereof
  • the plant material used as a source for microorganisms in step a) is seed material.
  • the plant material is surface sterilised.
  • the methods of the first or second (and/or related) aspect of the invention may be useful in identifying and/or selecting one or more unculturable microorganism capable of imparting one or more beneficial property to a plant.
  • plant material including for example seeds, seedlings, cuttings, and/or propagules thereof
  • the plant material used as a source for microorganisms in step a) is explant material (for example, plant cuttings).
  • the plant material is surface sterilised.
  • a method for assisting in the improvement of one or more plants comprising arranging for the evaluation of said plant(s) in the presence of one or more microorganisms and/or compositions.
  • the method preferably comprises at least the steps of a method of the first, second (and/or related), eighth (and/or related) and/or ninth (and/or related) aspects of the invention.
  • the plant(s) are for growing in a first region.
  • the microorganism(s) may or may not (or at least to a significant extent) be present in the first region.
  • Regular and “first region” are to be interpreted broadly as meaning one or more areas of land.
  • the land areas may be defined by geographical / political / private land boundaries or by land areas having similar properties such as climate, soil properties, presence of a particular pest etc.
  • the evaluation is performed in a second region in which the microorganism(s) are present, but this is in no way essential.
  • Microorganisms may be obtained from other sources including microorganism depositaries and artificially associated with plant material and/or soil.
  • plant(s) may be cultivated in essentially a conventional manner (but, in one particular embodiment in a region having
  • microorganisms not normally associated with the plant(s) at least in the first region, artificial growing environments may alternatively be used as would be appreciated by those skilled in the art.
  • possible beneficial microorganism/plant relationships may be identified that would not necessarily normally be utilised.
  • the step of arranging comprises arranging for one or more of:
  • the method comprises evaluating (or arranging for said evaluation of) said plant(s) in the presence of said lnicroorganism(s) and/or composition(s).
  • the step of evaluating preferably comprises performing one or more of the steps of a method described herein, in particular embodiments a method of the first aspect, second (and/or related) aspect, eighth (and/or related) aspect or ninth (and/or related) aspect of the invention.
  • the various steps identified above may be performed by a single entity although it is preferred that at least two parties are involved, a first which makes a request and a second which actions the request.
  • various agents may act for one or both parties and that varying levels of automation may be used.
  • the microorganism(s) may be selected by a processor querying a database based on known microorganism associations for that or similar plant(s) with little or no input required from an operator.
  • the evaluation may be performed by the requesting party and / or in the first region. Performing the evaluation in the first region better ensures that the evaluation is accurate and that no unforseen environmental factors that may impact on the plant(s) or the microorganism(s) are not considered.
  • the method preferably further comprises one or more of:
  • composition(s) to the first region including in combination with plant material;
  • the method of the third aspect may be embodied by a first party:
  • the step of receiving is preferably performed following or as a result of an assessment of plant/microorganism and/or plant/composition associations.
  • the assessment is made using a method as described herein, in particular embodiments a method of the first aspect, second aspect (and/or related), the eighth (and/or related) aspect or the ninth (and/or related) aspect.
  • the method of the third aspect may additionally or alternatively be embodied by a second party:
  • the step of sending is preferably performed following or as a result of an assessment of plant / microorganism and/or plant/composition associations.
  • the assessment is made using a method described herein, in particular embodiments a method of the second aspect (and/or related), the eighth (and/or related) aspect or the ninth (and/or related) aspect.
  • a system for implementing the method of the third aspect is provided.
  • the system of the fourth aspect preferably includes one or more of:
  • Means known to those skilled in the art may be used to provide the functionality required in the system of the third aspect.
  • conventional communication means including the internet, may be used to convey the identities of plants / microorganisms;
  • conventional carrier means may be used to convey the plant material / microorganisms/ composition(s);
  • conventional means and processes maybe used to associate a
  • microorganism and/or composition with plant material and conventional means for evaluating said plant(s) and / or the plant / microorganism and/or plant/composition associations may be used.
  • the system of the invention is embodied by a facility configured to transmit request(s) for an improvement in a plant(s) and subsequently to receive plant material and / or one or more microorganisms and / or the identities thereof, preferably following or as a result of an assessment of plant / microorganism associations.
  • the assessment is made using a method described herein, in particular embodiments a method of the first aspect, the second aspect (and/or related), the eighth (and/or related) aspect, or the ninth (and/or related) aspect.
  • the system of the fourth aspect may additionally or alternatively be embodied by a facility configured to receive an identity of a plant(s) and / or relevant plant material from together with any relevant information; and send plant material and / or one or more
  • the assessment is made using a method described herein, in particular embodiments a method of the first aspect, the second aspect (and/or related), the eighth (and/or related) aspect or the ninth (and/or related) aspect.
  • a microorganism acquired, selected or isolated by a method as herein before described.
  • the microorganism is an endophyte.
  • the microorganism is unculturable.
  • a method for the production of a composition to support plant growth, quality and/or health, or a composition to suppress or inhibit the growth, quality and/or health of a plant comprising the steps of a method herein before described and the additional step of combining the one or more microorganisms selected by the method with one or more additional ingredients.
  • a composition comprising one or more microorganism of the fifth broad aspect or as prepared by a method of the sixth broad aspect.
  • a method for the selection of a composition which is capable of imparting one or more beneficial property to a plant comprising at least the steps of:
  • step b) subjecting one or more plant (including for example seeds, seedlings, cuttings, and/or propagules thereof) to the one or more composition of step b);
  • step d) selecting one or more composition from step c) if it is observed to impart one or more beneficial property to the one or more plants.
  • step b) inactivating the one or more culture of step a) to provide one or more composition containing one or more inactivated microorganisms;
  • step b) subjecting one or more plant (including for example seeds, seedlings, cuttings, and/or propagules thereof) to the one or more composition of step b);
  • step d) selecting one or more composition from step c) if it is observed to impart one or more beneficial property to the one or more plants.
  • a ninth broad aspect of the invention there is provided a method for the selection of one or more microorganisms which are capable of producing a composition which is capable of imparting one or more beneficial property to a plant, the method comprising at least the steps of:
  • step b) separating the one or more microorganism from the one or more media in the one or more culture from step a) after a period of time to provide one or more composition substantially free of microorganisms;
  • step b) subjecting one or more plant (including for example seeds, seedlings, cuttings, and/or propagules thereof) to the one or more composition from step b);
  • step b) subjecting one or more plant (including for example seeds, seedlings, cuttings, and/or propagules thereof) to the one or more composition of step b);
  • step e) using the one or more microorganisms selected in step d) in step a) of a method of the first or second or eighth or ninth aspects of the invention.
  • step b) of the method of the ninth (and/or related) aspect could be substituted with the step of b) inactivating the one or more culture of step a) to provide one or more composition containing one or more inactivated microorganisms, and then using this composition in step c) of the process,
  • first, second (and/or related), eighth (and/or related) and ninth (and/or related) aspects may be combined in any combination, including the methods being nan concurrently or sequentially in any number of iterations, with compositions and/or microorganisms selected, acquired or isolated from the methods being used individually or combined and used in iterative rounds of any one of the methods.
  • a method of the eighth (and/or related) aspect may be performed and a composition selected.
  • the selection of a composition indicates that the one or more microorganism separated from the media in step b) is desirable for imparting beneficial properties to the one or more plant (as the one or more microorganism is capable of producing a selected composition).
  • the one or more microorganism may then be used in another round of a method of the first aspect, second (and/or related) aspect, eighth (and/or related) aspect or ninth (and/or related) aspect.
  • the combination of methods could be run in reverse. This could be repeated any number of times in any order and combination.
  • the invention provides for the use of one or more microorganism, composition or plant acquired, selected or isolated by a method of the invention in any other method of the invention.
  • composition obtained as a result of the methods of the eighth (and/or related) or ninth (and/or related) broad aspects of the invention.
  • a combination of two or more microorganisms acquired, selected or isolated by a method as herein before described.
  • the invention provides the use of one or more composition and/or microorganism acquired, selected or isolated by a method of the invention for imparting one or more beneficial property to one or more plant.
  • methods of the invention may also involve applying steps a) to c) (first aspect) or steps a) to d) (second and/or related aspect) of a method of the invention to two or more different species of plant so as to identify combinations of microorganisms that may impart a positive benefit to one species and a negative benefit to another species simultaneously.
  • steps a) to c) (first aspect) or steps a) to d) (second and/or related aspect) of a method of the invention to two or more different species of plant so as to identify combinations of microorganisms that may impart a positive benefit to one species and a negative benefit to another species simultaneously.
  • steps a) to c) first aspect
  • the invention also provides the use of a method of the invention in a plant breeding programme, and a plant breeding programme comprising conducting a method of the invention.
  • the invention may also be said broadly to consist in the parts, elements and features referred to or indicated in the specification of the application, individually or collectively, in any or all combinations of two or more of said parts, elements or features, and where specific integers are mentioned herein which have known equivalents in the art to which the invention relates, such known equivalents are deemed to be incorporated herein as if individually set forth.
  • Figure 1 shows a system according to an embodiment of the invention
  • Figure 2 shows the process flow of a method of an embodiment of the invention.
  • the following is a description of the preferred forms of the present invention given in general terms.
  • the invention will be further elucidated from the Examples provided hereafter.
  • the inventor(s) have found that one can readily identify microorganisms capable of imparting one or more beneficial property to one or more plants through use of a method of the invention.
  • the method is broadly based on the presence of variability (such as genetic variability, or variability in the phenotype, for example) in the plants and microbial populations used.
  • variability such as genetic variability, or variability in the phenotype, for example
  • the inventors have identified that this variability can be used to support a directed process of selection of one or more microorganisms of use to a plant and for identifying particular plant/microbe combinations which are of benefit for a particular purpose, and which may never have been recognised using conventional techniques.
  • the methods of the invention may be used as a part of a plant breeding programme.
  • the methods may allow for, or at least assist with, the selection of plants which have a particular genotype/phenotype which is influenced by the microbial flora, in addition to identifying microorganisms and/or compositions that are capable of imparting one or more property to one or more plants.
  • the invention relates to a method for the selection of one or more
  • microorganism(s) which are capable of imparting one or more beneficial property to a plant.
  • a "beneficial property to a plant” should be interpreted broadly to mean any property which is beneficial for any particular purpose including properties which may be beneficial to human beings, other animals, the environment, a habitat, an ecosystem, the economy, of commercial benefit, or of any other benefit to any entity or system. Accordingly, the term should be taken to include properties which may suppress, decrease or block one or more characteristic of a plant, including suppressing, decreasing or inhibiting the growth or growth rate of a plant.
  • the invention maybe described herein, by way of example only, in terms of identifying positive benefits to one or more plants or improving plants. However, it should be appreciated that the invention is equally applicable to identifying negative benefits that can be conferred to plants.
  • beneficial properties include, but are not limited to, for example: improved growth, health and/or survival characteristics, resistance to pests and/or diseases, tolerance to growth in different geographical locations and/or different environmental biological and/or physical conditions, suitability or quality of a plant for a particular purpose, stmcture, colour, chemical composition or profile, taste, smell, improved quality.
  • the invention may allow for the identification of microorganisms which allow a plant to grow in a variety of different temperatures (including extreme temperatures), pH, salt concentrations, mineral concentrations, in the presence of toxins, and/or to respond to a greater extent to the presence of organic and/or inorganic fertilisers.
  • temperatures including extreme temperatures
  • beneficial properties include, but are not limited to, for example; decreasing, suppressing or inhibiting the growth of a plant identified to be a weed; constraining the height and width of a plant to a desirable ornamental size; limiting the height of plants used in ground cover applications such as motorway and roadside banks and erosion control projects; slowing the growth of plants used in turf applications such as lawns, bowling greens and golf courses to reduce the necessity of mowing; reducing ratio of foliage/flowers in ornamental flowering shrubs; regulate production of and/or response to plant pheromones (resulting in increased tannin production in surrounding plant community and decreased appeal to foraging species).
  • improved should be taken broadly to encompass improvement of a characteristic of a plant which may already exist in a plant or plants prior to application of the invention, or the presence of a characteristic which did not exist in a plant or plants prior to application of the invention.
  • improved growth should be taken to include growth of a plant where the plant was not previously known to grow under the relevant conditions.
  • inhibiting and suppressing should be taken broadly and should not be construed to require complete inhibition or suppression, although this may be desired in some embodiments.
  • first set of one or more microorganisms and a “second set of one or more microorganisms” may be used herein to distinguish the set or group of microorganism(s) applied in step a) and the set or group of
  • the sets of microorganisms will be distinct; for example, the second set may be a subset of the first set, as a result of combining the first set with the plant and then selecting one or more plant based on one or more selection criterion.
  • the use of this terminology should not be construed in such a limited manner
  • methods of the invention relate to selecting one or more microorganisms which are capable of imparting one or more beneficial property to a plant.
  • microorganism(s) may be contained within a plant, on a plant, and/or within the plant rhizosphere. Accordingly, where reference is made herein to acquiring a second set of one or more microorganisms "from" a plant, unless the context requires otherwise, it should be taken to include reference to acquiring a second set of microorganisms contained within a plant, on a plant and/or within the plant rhizosphere.
  • the wording "associated with" may be used
  • a method of the invention broadly comprises at least the steps of a) growing one or more plant in a growth medium in the presence of a first set of one or more microorganisms; b) selecting one or more plant following step a); and, c) acquiring a second set of one or more microorganisms associated with said one or more plant selected in step b).
  • the one or more plants, growth medium and one or more microorganisms may be provided separately and combined in any appropriate order prior to step a).
  • the invention provides an iterative method in which steps a) to c) may be repeated one or more times, wherein the one or more microorganisms acquired in step c) are used in step a) of a subsequent run or cycle of the method.
  • steps a) to c) are repeated once.
  • steps a) to c) are repeated twice.
  • steps a) to c) are repeated three times.
  • steps a) to c) are repeated at least until a desired beneficial property is observed.
  • the invention comprises a step of applying a selective pressure.
  • this method comprises at least the steps of a) growing one or more plant in a growth medium in the presence of one or more microorganisms; b) applying one or more selective pressure during step a); c) selecting one or more plant following step b); and, d) acquiring one or more microorganisms associated with said one or more plant selected in step c).
  • the one or more plants, growth medium and one or more microorganisms may be provided separately and combined in any appropriate order prior to step a).
  • the invention provides an iterative method in which steps a) to d) may be repeated one or more times, wherein the one or more microorganisms acquired in step d) are used in step a) of the next cycle of the method.
  • steps a) to d) are repeated once.
  • steps a) to d) are repeated twice.
  • steps a) to d) are repeated three times.
  • steps a) to d) are repeated at least at least until a desired beneficial property is observed. Whilst the same selective pressure(s) may be applied in each iteration of the method, different selective pressures may be applied in each iteration.
  • the method may comprise a further step (or steps) which are conducted prior to step a) and include subjecting the one or more plant to a growth medium in the presence of one or more microorganisms but without a selective pressure. After a desired period, one or more microorganisms maybe acquired from said one or more plant and can be used in step a) of the process. It should also be appreciated that successive rounds of this step may be conducted, with the microorganisms acquired being used in the subsequent round of the process, before embarking on steps a) to d) as described above.
  • the inventors envisage an iterative method in which steps a) to d) are repeated one or more times, but interspersed with the step of subjecting the plants to a growth medium and one or more microorganisms and isolating the microorganisms, without applying a selective pressure.
  • the methods of the invention include the steps of subjecting the first and/or second set of one or more microorganisms to a conditioning and/or directed evolution process prior to using these microorganisms in step a) of a process of the invention.
  • the methods also envisage subjecting the second set of one or more microorganisms acquired at the end of the method to a conditioning and/or directed evolution process.
  • the conditioning and directed evolution processes are described in more detail hereinafter.
  • the method may conclude with the acquisition of a set of one or more microorganisms from step c) or step d) (as the case may be) of any such method.
  • the method may further involve subjecting the set of one or more microorganisms to a conditioning and/or directed evolution process. It should be appreciated that the methods do not require the identification of the microorganisms in the population acquired in the method (for example in step c) of the first aspect or step d) of the second and/or related aspect) nor do they require a
  • step a) is conducted using at least two plants, h other embodiments 10 to 20 plants are used. In yet other embodiments, 20 or more, 50 or more, 100 or more, 300 or more, 500 or more or 1000 or more plants are used. As noted herein before, where two or more plants are used in a particular method of the invention they need not be the same variety or species. For example, in one embodiment it may be desirable to select microorganisms that can impart a positive benefit to one plant variety or species and a negative benefit to another plant variety or species.
  • the method may further comprise the steps of separating the two or more microorganisms into individual isolates, selecting two or more individual isolates, and then combining the selected two or more isolates. This may result in the set of microorganisms acquired at the conclusion of a method of the invention. However, in one embodiment, the combined isolates may then be used in step a) of successive rounds of the method. By way of example, from two, three, four, five, six, seven, eight, nine or ten individual isolates may be combined.
  • the inventors envisage an iterative method in which steps a) to d) (second and/or related aspect) or steps a) to c) (first aspect) are repeated one or more times, utilising these additional steps of separating, selecting and combining with each repeat of the method, or interspersed or otherwise combined with a method in which individual isolates are not selected and combined.
  • Selection of individual isolates may occur on the basis of any appropriate selection criteria. For example, it may be random, it may be based on the beneficial property or properties observed by performing a method of the invention or, where information about the identity of the microorganism is known, it may be on the basis that the microorganism has previously been recognised to have a particular beneficial property.
  • two or more methods of the invention may be performed separately or in parallel and the microorganisms that result from each method combined into a single composition.
  • two separate methods maybe performed, one to identify microorganisms capable of imparting one or more first beneficial property, and a second to identify microorganisms capable of imparting one or more second beneficial property.
  • the separate methods may be directed to identifying microorganisms having the same beneficial property or having distinct beneficial properties.
  • the selective pressure in the separate methods may be the same or different.
  • the microorganisms and plants used in the separate methods may be the same or different.
  • the single composition of microorganisms may be applied to one or more further rounds of a method of the invention.
  • the single composition of microorganisms may be used, as desired, to confer the relevant properties to plant crops, without further optimisation. Combining two or more methods of the invention in this way allows for the selection and combination of microorganisms which may ordinarily be separated by time and/or space in a particular environment.
  • the methods may comprise growing or propagating one or more plants selected in step c) (first aspect) or step d) (second and/or related aspect), to grow the population of the second set of one or more microorganisms associated with the selected one or more plants, either at the conclusion of a method of the invention, or prior to using the second set of one or more microorganisms in step a) of any successive repeat of the method. If the one or more plants (with associated microorganisms) are grown or propagated at the conclusion of a method of the invention they may then be used or sold in that form.
  • one or more microorganisms may be isolated from the one or more plants, or one or more plant tissue and/or one or more plant part with associated microorganisms may be used as a crude source of the one or more microorganisms in any successive repeat of the invention, or for any other purpose at the conclusion of the method.
  • the seeds (with associated microorganisms) of one or more plant that has been grown or propagated may be obtained and used as a source of the one or more microorganisms in any successive repeat of the method.
  • the seeds and associated microorganisms may be sold or used for any other purpose.
  • the one or more microorganisms selected or acquired by the present methods can be formulated as a composition, as described herein after.
  • Such compositions may, for example, be utilised to support plant growth, quality and/or health, or to suppress or inhibit plant growth, quality or health.
  • a composition is utilized as a seed coating for commercially important agricultural crops, for example.
  • the compositions comprise one or more microorganisms combined with one or more additional ingredients.
  • the one or more microorganisms can be combined with known actives available in the agricultural space, such as: pesticide, herbicide, bactericide, fungicide, insecticide, virucide, miticide, nemataicide, acaricide, plant growth regulator, rodenticide, or anti-algae agent.
  • the one or more microorganisms can be combined with known fertilizers.
  • an active chemical agent one witnesses an additive effect on a plant phenotypic trait of interest.
  • the one or more microorganisms when the one or more microorganisms is combined with a fertilizer one witnesses an additive effect on a plant phenotypic trait of interest.
  • the one or more microorganisms when the one or more microorganisms is combined with an active chemical agent one witnesses a synergistic effect.
  • S. "Calculating Synergistic and Antagonistic Responses of Herbicide Combinations", 1 67 Weeds, vol. 15, pp. 20-22, incorporated herein by reference in its entirety.
  • by “synergistic” is intended a component which, by virtue of its presence, increases the desired effect by more than an additive amount.
  • the one or more microorganisms selected or acquired by the methods disclosed herein can synergistically increase the effectiveness of agricultural
  • composition comprising the one or more microorganisms can be formulated with certain auxiliaries in order to improve the activity of a known active agricultural compound.
  • This has the advantage that the amounts of active ingredient in the formulation may be reduced while maintaining the efficacy of the active compound, thus allowing costs to be kept as low as possible and any official regulations to be followed.
  • it may also be possible to widen the spectrum of action of the active compound since plants, where the treatment with a particular active ingredient without addition was insufficiently successful, can indeed be treated successfully by the addition of certain auxiliaries along with the one or more microorganisms selected or acquired by the methods disclosed herein.
  • the performance of the active may be increased in individual cases by a suitable formulation when the environmental conditions are not favorable.
  • auxiliaries that can be used in a composition comprising an active agricultural compound and one or more microorganism selected or acquired according to the disclsosed methods can be an adjuvant.
  • adjuvants take the form of surface- active or salt-like compounds. Depending on their mode of action, they can roughly be classified as modifiers, activators, fertilizers, pH buffers, and the like. Modifiers affect the wetting, sticking, and spreading properties of a formulation. Activators break up the waxy cuticle of the plant and improve the penetration of the active ingredient into the cuticle, both short-term (over minutes) and long-term (over hours).
  • Fertilizers such as ammonium sulfate, ammonium nitrate or urea improve the absorption and solubility of the active ingredient and may reduce the antagonistic behavior of active ingredients.
  • pH buffers are conventionally used for bringing the formulation to an optimal pH. Further methods and aspects of the invention are described herein after.
  • microorganism should be taken broadly. It includes but is not limited to the two prokaryotic domains, Bacteria and Archaea, as well as eukaryotic fungi and protists.
  • the microorganisms may include Proteobacte ia (such as Pseudomonas, Enterobacter, Stenotrophomonas, Burkholderia, Rhizobium,
  • the microorganism is an endophyte or an epiphyte or a microorganism inhabiting the plant rhizosphere.
  • the microorganism is an endophyte or an epiphyte or a microorganism inhabiting the plant rhizosphere.
  • microorganism is a seed-borne endophyte.
  • the microorganism is unculturable. This should be taken to mean that the microorganism is not known to he culturable or is difficult to culture using methods known to one skilled in the art.
  • Microorganisms of use in the methods of the present invention may be collected or obtained from any source or contained within and/or associated with material collected from any source.
  • the first set of one or more microorganisms are obtained from any general terrestrial environment, including its soils, plants, fungi, animals (including invertebrates) and other biota, including the sediments, water and biota of lakes and rivers; from the marine environment, its biota and sediments (for example sea water, marine muds, marine plants, marine invertebrates (for example sponges), marine vertebrates (for example, fish)); the terrestrial and marine geosphere (regolith and rock, for example crushed subterranean rocks, sand and clays); the cryosphere and its meltwater; the atmosphere (for example, filtered aerial dusts, cloud and rain droplets); urban, industrial and other man-made environments (for example, accumulated organic and mineral matter on concrete, roadside gutters, roof surfaces, road surfaces).
  • the atmosphere for example, filtered aerial dusts, cloud and rain droplets
  • urban, industrial and other man-made environments for example, accumulated organic and mineral matter on concrete, roadside gutters, roof surfaces, road surfaces).
  • the first set of one or more microorganisms are collected from a source likely to favour the selection of appropriate microorganisms.
  • the source may be a particular environment in which it is desirable for other plants to grow, or which is thought to be associated with terroir.
  • the source may be a plant having one or more desirable traits, for example a plant which naturally grows in a particular environment or under certain conditions of interest.
  • a certain plant may naturally grow in sandy soil or sand of high salinity, or under extreme temperatures, or with little water, or it may be resistant to certain pests or disease present in the environment, and it may be desirable for a commercial crop to be grown in such conditions, particularly if they are, for example, the only conditions available in a particular geographic location.
  • the microorganisms may be collected from commercial crops grown in such environments, or more specifically from individual crop plants best displaying a trait of interest amongst a crop grown in any specific environment, for example the fastest-growing plants amongst a crop grown in saline-limiting soils, or the least damaged plants in crops exposed to severe insect damage or disease epidemic, or plants having desired quantities of certain metabolites and other compounds, including fibre content, oil content, and the like, or plants displaying desirable colours, taste or smell.
  • the microorganisms may be collected from a plant of interest or any material occurring in the environment of interest, including fungi and other animal and plant biota, soil, water, sediments, and other elements of the environment as referred to previously.
  • the microorganisms are sourced from previously performed methods of the invention (for example, the microorganisms acquired in step d) (second aspect) or step c) (first aspect) of the method), including combinations of individual isolates separated from the second set of microorganisms acquired in step c) (first aspect) or step d) (second and/or related aspect) or combinations of microorganisms resulting from two or more separately performed methods of the invention.
  • a microorganism or a combination of microorganisms of use in the methods of the invention may be selected from a pre-existing collection of individual microbial species or strains based on some knowledge of their likely or predicted benefit to a plant.
  • the microorganism may be predicted to: improve nitrogen fixation; release phosphate from the soil organic matter; release phosphate from the inorganic forms of phosphate (e.g.
  • a microorganism or combination of microorganisms (the first set of one or more microorganisms) is selected from a pre-existing collection of individual microbial species or strains that provides no knowledge of their likely or predicted benefit to a plant. For example, a collection of unidentified microorganisms isolated from plant tissues without any knowledge of their ability to improve plant growth or health, or a collection of microorganisms collected to explore their potential for producing compounds that could lead to the development of pharmaceutical drugs.
  • the microorganisms are isolated from the source material (for example, soil, rock, water, air, dust, plant or other organism) in which they naturally reside.
  • the microorganisms may be provided in any appropriate form, having regard to its intended use in the methods of the invention. However, by way of example only, the microorganisms maybe provided as an aqueous suspension, gel, homogenate, granule, powder, slurry, live organism or dried material.
  • the microorganisms may be isolated in substantially pure or mixed cultures. They may be concentrated, diluted or provided in the natural concentrations in which they are found in the source material.
  • microorganisms from saline sediments may be isolated for use in this invention by suspending the sediment in fresh water and allowing the sediment to fall to the bottom.
  • the water containing the bulk of the microorganisms may be removed by decantation after a suitable period of settling and either applied directly to the plant growth medium, or concentrated by filtering or centrifugation, diluted to an appropriate concentration and applied to the plant growth medium with the bulk of the salt removed.
  • microorganisms from mineralized or toxic sources may be similarly treated to recover the microbes for application to the plant growth material to minimise the potential for damage to the plant.
  • the microorganisms (including the first set of one or more microorganisms and/or the second set of one or more microorganisms) are used in a crude form, in which they are not isolated from the source material in which they naturally reside.
  • the microorganisms are provided in combination with the source material in which they reside; for example, as soil, or the roots, seed or foliage of a plant.
  • the source material may include one or more species of
  • microorganisms are preferred. It is preferred that a mixed population of microorganisms is used in the methods of the invention.
  • any one or a combination of a number of standard techniques which will be readily known to skilled persons may be used.
  • these in general employ processes by which a solid or liquid culture of a single microorganism can be obtained in a substantially pure form, usually by physical separation on the surface of a solid microbial growth medium or by volumetric dilutive isolation into a liquid microbial growth medium.
  • These processes may include isolation from dry material, liquid suspension, slurries or homogenates in which the material is spread in a thin layer over an appropriate solid gel growth medium, or serial dilutions of the material made into a sterile medium and inoculated into liquid or solid culture media.
  • the material containing the microorganisms may be pre-treated prior to the isolation process in order to either multiply all microorganisms in the material, or select portions of the microbial population, either by enriching the material with microbial nutrients (for example, nitrates, sugars, or vegetable, microbial or animal extracts), or by applying a means of ensuring the selective survival of only a portion of the microbial diversity within the material (for example, by pasteurising the sample at 60°C-80°C for 10 -20 minutes to select for microorganisms resistant to heat exposure (for example, bacilli), or by exposing the sample to low concentrations of an organic solvent or sterilant (for example, 25% ethanol for 10 minutes) to enhance the survival of actinomycetes and spore-forming or solvent-resistant microorganisms).
  • microbial nutrients for example, nitrates, sugars, or vegetable, microbial or animal extracts
  • Microorganisms can then be isolated from the enriched materials or materials treated for selective survival, as above.
  • endophytic or epiphytic microorganisms are isolated from plant material. Any number of standard techniques known in the art may be used and the microorganisms may be isolated from any appropriate tissue in the plant, including for example root, stem and leaves, and plant reproductive tissues.
  • conventional methods for isolation from plants typically include the sterile excision of the plant material of interest (e.g. root or stem lengths, leaves), surface sterilisation with an appropriate solution (e.g. 2% sodium hypochlorite), after which the plant material is placed on nutrient medium for microbial growth (see, for example, Strobel G and Daisy B (2003) Bioprospecting for microbial endophytes and their natural products.
  • microorganisms are isolated from root tissue. Further methodology for isolating microorganisms from plant material are detailed herein after. As used herein, "isolate”, “isolated” and like terms should be taken broadly. These terms are intended to mean that the one or more microorganism(s) has been separated at least partially from at least one of the materials with which it is associated in a particular environment (for example soil, water, plant tissue).
  • isolated should not be taken to indicate the extent to which the microorganism(s) has been purified.
  • individual isolates should be taken to mean a composition or culture comprising a predominance of a single genera, species or strain of microorganism, following separation from one or more other microorganisms. The phrase should not be taken to indicate the extent to which the microorganism has been isolated or purified. However, “individual isolates” preferably comprise substantially only one genus, species or strain of microorganism.
  • the microbial population is exposed (prior to the method or at any stage of the method) to a selective pressure to enhance the probability that the eventually- selected plants will have microbial assemblages likely to have desired properties.
  • a selective pressure to enhance the probability that the eventually- selected plants will have microbial assemblages likely to have desired properties.
  • exposure of the microorganisms to pasteurisation before their addition to a plant growth medium (preferably sterile) is likely to enhance the probability that the plants selected for a desired trait will be associated with spore-forming microbes that can more easily survive in adverse conditions, in commercial storage, or if applied to seed as a coating, in an adverse environment.
  • the second set of microorganisms acquired in a method of the invention may be isolated from a plant or plant material, surface or growth media associated with a selected plant using any appropriate techniques known in the art, including but not limited to those techniques described herein.
  • the microorganism(s) may be used in crude form and need not be isolated from a plant or a media.
  • plant material or growth media which includes the microorganisms identified to be of benefit to a selected plant may be obtained and used as a crude source of microorganisms for the next round of the method or as a crude source of microorganisms at the conclusion of the method.
  • whole plant material could be obtained and optionally processed, such as mulched or crushed.
  • individual tissues or parts of selected plants such as leaves, stems, roots, seeds
  • one or more part of a plant which is associated with the second set of one or more microorganisms may be removed from one or more selected plants and, where any successive repeat of the method is to be conducted, grafted on to one or more plant used in step a).
  • the methods of the invention may be described herein in terms of the second set of one or more microorganisms being isolated from its source material. However, unless the context requires otherwise, this should also be taken to include reference to the use of
  • microorganisms in crude form in which they have not been isolated from the source material
  • the plants have economic, social and/or environmental value.
  • the plants may include those of use: as food crops; as fibre crops; as oil crops; in the forestry industry; in the pulp and paper industry; as a feedstock for biofuel production; and/or, as ornamental plants.
  • the plants may be economically, socially and/or environmentally undesirable, such as weeds.
  • the following is a list of non-limiting examples of the types of plants the methods of the invention may be applied to :
  • leafy vegetables (brassicaceous plants such as cabbages, broccoli, bok choy, rocket; salad greens such as spinach, cress, lettuce);
  • - fruiting and flowering vegetables e.g. avocado, sweet com, artichokes,
  • curcubits e.g. squash, cucumbers, melons, courgettes , pumpkins;
  • solononaceous vegetables/fruits e.g. tomatoes, eggplant, capsicums
  • - podded vegetables groundnuts, peanuts, peas, soybeans, beans, lentils
  • sugar crops including sugar beet ⁇ Beta vulgaris), sugar cane (Saccharum
  • offlcinarum - crops grown for the production of non-alcoholic beverages and stimulants (coffee, black, herbal and green teas, cocoa, tobacco);
  • - fruit crops such as true berry fruits (e.g. kiwifruit, grape, currants, gooseberry, guava, feijoa, pomegranate), citrus fruits (e.g. oranges, lemons, limes, grapefruit), epigynous fruits (e.g. bananas, cranberries, blueberries), aggregate fruit (blackberry, raspberry, boysenberry), multiple fruits (e.g. pineapple, fig), stone fruit crops (e.g. apricot, peach, cherry, plum), pip-fruit (e.g. apples, pears) and others such as strawberries, sunflower seeds;
  • true berry fruits e.g. kiwifruit, grape, currants, gooseberry, guava, feijoa, pomegranate
  • citrus fruits e.g. oranges, lemons, limes, grapefruit
  • epigynous fruits e.g. bananas, cranberries, blueberries
  • aggregate fruit blackberry, raspberry, boysenberry
  • spices e.g. black pepper, cumin cinnamon, nutmeg, ginger, cloves, saffron, cardamom, mace, paprika, masalas, star anise;
  • nuts e.g. almonds and walnuts, Brazil nut, cashew nuts, coconuts, chestnut, macadamia nut, pistachio nuts; peanuts, pecan nuts;
  • oilseed crops e.g. soybean, peanuts, cotton, olives, sunflower, sesame, lupin species and brassicaeous crops (e.g. canola/oilseed rape); and,
  • Trifolium species Trifolium species, Medicago species, and Lotus species
  • White clover T.repens
  • Red clover ⁇ T.pratense Red clover ⁇ T.pratense
  • Caucasian clover T, ambigum
  • subterranean clover T.subterraneum
  • Alfalfa/Lucerne ⁇ Medicago sativum annual medics
  • barrel medic black medic
  • Sainfoin Onobrychis viciifolia
  • Birdsfoot trefoil Litus corniculatus
  • Greater Birdsfoot trefoil Litus pedunculatus
  • Peas Peas (Pisum sativum), Common bean (Phaseolus vulgaris), Broad beans (Vicia faba), Mung bean (Vigna radiata), Cowpea
  • Temperate grasses such as Lolium species
  • Festuca species Agrostis spp., Perennial ryegrass (Lolium perenne); hybrid ryegrass ⁇ Lolium hybridum); annual ryegrass ⁇ Lolium multiflonim), tall fescue ⁇ Festuca arundinacea); meadow fescue ⁇ Festuca pratensis); red fescue ⁇ Festuca rubra); Festuca ovina; Festuloliums ⁇ Lolium X Festuca crosses); Cocksfoot ⁇ Dactylis glomerata); Kentucky bluegrass Poa pratensis; Poa palustris; Poa nemoralis; Poa trivialis; Poa compresa; Bromus species;
  • Tree and shrub species used in plantation forestry and bio-fuel crops are:
  • Cypress ⁇ Cupressus species Wattle ⁇ Acacia species); Alder ⁇ Alnus species); Oak species ⁇ Quercus species); Redwood ⁇ Sequoiadendron species); willow ⁇ Salix species); birch ⁇ Betula species); Cedar ⁇ Cedurus species); Ash ⁇ Fraxinus species); Larch ⁇ Larix species); Eucalyptus species; bamboo (Bambuseae species) and Poplars ⁇ Populus species).
  • Oil-producing plants such as oil palm, jatropha, soybean, cotton, linseed; - Latex-producing plants such as the Para Rubber tree, Hevea brasiliensis and the Panama Rubber Tree Castilla elastica
  • biofuels e.g. ethanol or butanol, propane diols, or other fuel or industrial material
  • sugar crops e.g. beet, sugar cane
  • starch- producing crops e.g. C3 and C4 cereal crops and tuberous crops
  • cellulosic crops such as forest trees (e.g. Pines, Eucalypts) and Graminaceous and
  • Poaceous plants such as bamboo, switch grass, miscanthus
  • biochar e.g. biomass crops such as coniferous, eucalypt, tropical or broadleaf forest trees, graminaceous and poaceous crops such as bamboo, switch grass, miscanthus, sugar cane, or hemp or softwoods such as poplars, willows; and,
  • - crops producing pharmaceutical precursors or compounds or nutraceutical and cosmeceutical compounds and materials for example, star anise (shikimic acid), Japanese knotweed (resveratrol), kiwifruit (soluble fibre, proteolytic enzymes);
  • Plants grown for bioremediation Helianthus, Brassica, Salix, Populus, Eucalyptus
  • a plant may be provided in the form of a seed, seedling, cutting, propagule, or any other plant material or tissue capable of growing.
  • the seed may surface-sterilised with a material such as sodium hypochlorite or mercuric chloride to remove surface-contaminating microorganisms.
  • the propagule is grown in axenic culture before being placed in the plant growth medium, for example as sterile plantlets in tissue culture. Growth Medium
  • growth medium should be taken broadly to mean any medium which is suitable to support growth of a plant.
  • the media may be natural or artificial including, but not limited to, soil, potting mixes, bark, vermiculite, hydroponic solutions alone and applied to solid plant support systems, and tissue culture gels. It should be appreciated that the media may be used alone or in combination with one or more other media. It may also be used with or without the addition of exogenous nutrients and physical support systems for roots and foliage.
  • the growth medium is a naturally occurring medium such as soil, sand, mud, clay, humus, regolith, rock, or water.
  • the growth medium is aitificial. Such an artificial growth medium may be constructed to mimic the conditions of a naturally occurring medium, however, this is not necessary.
  • Artificial growth media can be made from one or more of any number and combination of materials including sand, minerals, glass, rock, water, metals, salts, nutrients, water.
  • the growth medium is sterile. In another embodiment, the growth medium is not sterile.
  • the medium maybe amended or enriched with additional compounds or components, for example, a component which may assist in the interaction and/or selection of specific groups of microorganisms with the plant and each other.
  • antibiotics such as penicillin
  • sterilants for example, quaternary ammonium salts and oxidizing agents
  • the physical conditions such as salinity, plant nutrients (for example organic and inorganic minerals (such as phosphorus, nitrogenous salts, ammonia, potassium and micronutrients such as cobalt and magnesium), pH, and/or temperature) could be amended.
  • the growth medium may be pre-treated to assist in the survival and/or selection of certain microorganisms.
  • the medium may be pre-treated by incubating in an emichment media to encourage the multiplication of endogenous microbes that may be present therein.
  • the medium may be pre-treated by incubating in a selective medium to encourage the multiplication of specific groups of microorganisms.
  • a further example includes the growth medium being pre-treated to exclude a specific element of the microbial assemblage therein; for example pasteurization (to remove spore-forming bacteria and fungi) or treatment with organic solvents such as various alcohols to remove microorganisms sensitive to these materials but allow the survival of actinomycetes and spore-forming bacteria, for example.
  • Methods for pre-treating or enriching may be informed by culture independent microbial community profiling techniques that provide information on the identity of microbes or groups of microbes present. These methods may include, but are not limited to, sequencing techniques including high throughput sequencing and phylogenetic analysis, or microarray-based screening of nucleic acids coding for components of rRNA operons or other taxonomically informative loci. Growth Conditions
  • one or more plant is subjected to one or more microorganism and a growth medium.
  • the plant is preferably grown or allowed to multiply in the presence of the one or more microorganism(s) and growth medium.
  • the crOorganism(s) may be present in the growth medium naturally without the addition of further microorganisms, for example in a natural soil.
  • the growth medium, plant and microorganisms maybe combined or exposed to one another in any appropriate order.
  • the plant, seed, seedling, cutting, propagule or the like is planted or sown into the growth medium which has been previously inoculated with the one or more microorganisms.
  • the one or more microorganisms may be applied to the plant, seed, seedling, cutting, propagule or the like which is then planted or sown into the growth medium (which may or may not contain further microorganisms).
  • the plant, seed, seedling, cutting, propagule or the like is first planted or sown into the growth medium, allowed to grow, and at a later time the one or more microorganisms are applied to the plant, seed, seedling, cutting, propagule or the like and/or the growth medium itself is inoculated with the one or more microorgamsms.
  • the microorganisms maybe applied to the plant, seedling, cutting, propagule or the like and/or the growth medium using any appropriate techniques known in the art.
  • the one or more microorganisms are applied to the plant, seedling, cutting, propagule or the like by spraying or dusting.
  • the microorganisms are applied directly to seeds (for example as a coating) prior to sowing.
  • microorganisms are formulated into granules and are applied alongside seeds during sowing.
  • microorganisms may be inoculated into a plant by cutting the roots or stems and exposing the plant surface to the microorganisms by spraying, dipping or otherwise applying a liquid microbial suspension, or gel, or powder,
  • the microorganism(s) maybe injected directly into foliar or root tissue, or otherwise inoculated directly into or onto a foliar or root cut, or else into an excised embryo, or radicle or coleoptile. These inoculated plants may then be further exposed to a growth media containing further microorganisms, however, this is not necessary.
  • the microorganisms are applied to the plant, seedling, cutting, propagule or the like and/or growth medium in association with plant material (for example, plant material with which the microorganisms are associated).
  • the microorganisms may be transferred to a plant by any one or a combination of grafting, insertion of explants, aspiration, electroporation, wounding, root pruning, induction of stomatal opening, or any physical, chemical or biological treatment that provides the opportunity for microbes to enter plant cells or the intercellular space.
  • grafting any one or a combination of grafting, insertion of explants, aspiration, electroporation, wounding, root pruning, induction of stomatal opening, or any physical, chemical or biological treatment that provides the opportunity for microbes to enter plant cells or the intercellular space.
  • microorganisms infiltrate parts of the plant such as the roots, stems, leaves and/or reproductive plant parts (become endophytic), and/or grow upon the surface of roots, stems, leaves and/or reproductive plant parts (become epiphytic) and/or grow in the plant rhizosphere.
  • microorganism(s) form a symbiotic relationship with the plant.
  • the growth conditions used may be varied depending on the species of plant, as will be appreciated by persons skilled in the art.
  • fertilisers typically containing nitrates, phosphates, potassium and magnesium salts and micronutrients and at a pH of between 6 and 7.
  • fertilisers typically containing nitrates, phosphates, potassium and magnesium salts and micronutrients and at a pH of between 6 and 7.
  • the plants may be grown at a temperature between 22-24°C in an 16:8 period of daylight:darkness, and watered automatically.
  • a selective pressure may be applied at a desired time during the period within which the plant may be subjected to one or more microorganism and a growth medium.
  • the selective pressure may be any biotic or abiotic factor or element which may have an impact on the health, growth, and/or survival of a particular plant, including environmental conditions and elements which plants may be exposed to in their natural environment or a commercial situation.
  • biotic selective pressures include but are not limited to organisms that are detrimental to the plant, for example, fungi, bacteria, viruses, insects, mites, nematodes, animals.
  • Abiotic selective pressures include for example any chemical and physical factors in the environment; for example, water availability, soil mineral composition, salt, temperature, alterations in light spectrum (e.g. increased UV light), pH, organic and inorganic toxins (for example, exposure to or changes in the level of toxins), metals, organic nutrients, inorganic nutrients, air quality, atmospheric gas composition, air flow, rain fall, and hail.
  • the plant/microorganisms maybe exposed to a change in or extreme salt concentrations, temperature, pH, higher than normal levels of atmospheric gases such as C0 2 , water levels (including drought conditions or flood conditions), low nitrogen levels, provision of phosphorus in a form only available to the plant after microbial degradation, exposure to or changes in the level of toxins in the environment, soils with nearly toxic levels of certain minerals such as amminates, or high winds.
  • the selective pressure is applied directly to the plant, the
  • the selective pressure is applied indirectly to the plant, the microorganisms and/or the growth medium, via the suiTounding environment; for example, a gaseous toxin in the air or a flying insect.
  • the selective pressure may be applied at any time, preferably during the time the plant is subjected to the one or more microorganism and growth medium.
  • the selective pressure is applied during for substantially the whole time during which a plant is growing and/or multiplying.
  • the selective pressure is applied at a discrete time point during growth and/or multiplication.
  • the selective pressure may be applied at different growth phases of the one or more plants which simulate a potential stress on the plant that might occur in a natural or commercial setting.
  • the inventor has observed that some pests attack plants only at specific stages of the plant's life.
  • the inventor has observed that different populations of potentially beneficial microorganisms can associate with plants at different points in the plant's life. Simulating a pest attack on the plant at the relevant time point, may allow for the identification and isolation of microorganisms which may protect the plant from attack at that particular life stage.
  • the selective pressure may be present in the growth medium or in the general environment at the time the plant, seed, seedling, cutting, propagule or the like is planted or sown.
  • the microbial population is exposed (prior to the method or at any stage of the method) to a selective pressure to enhance the probability that the eventually- selected plants will have microbial assemblages likely to have desired properties.
  • a selective pressure to enhance the probability that the eventually- selected plants will have microbial assemblages likely to have desired properties.
  • exposure of the microorganisms to pasteurisation before their addition to a plant growth medium (preferably sterile) is likely to enhance the probability that the plants selected for a desired trait will be associated with spore-forming microbes that can more easily survive in adverse conditions, in commercial storage, or if applied to seed as a coating, in an adverse environment.
  • Example 1 IB in which microorganisms were subjected to a media which allowed for selection of phosphate solubilising microbes.
  • Such a step of applying a selective pressure to the microbial population may be referred to herein as an "enrichment step".
  • the plants may be grown and subjected to the selective pressure for any appropriate length of time before they are selected and harvested.
  • the plants and any microorganisms associated with them may be selected and harvested at any time during the growth period of a plant, in one embodiment, any time after germination of the plant.
  • the plants are grown or allowed to multiply for a period which allows one to distinguish between plants having desirable phenotypic features and those that do not.
  • wheat may be selected for improvements in the speed of foliar growth say after one month, but equally may be selected for superior grain yield on maturity of the seed head.
  • the length of time a plant is grown depends on the timing required to express the plant trait that is desired to be improved by the invention, or the time required to express a trait correlated with the desired trait.
  • a tree species may be selected for improved growth and health at 4-6 months as these traits are related to the health and growth rate and size of trees of 10 years later, an impractical period for product development using this invention.
  • the methods of the invention may involve applying two or more selective pressures simultaneously or successively in step b).
  • one or more plant is selected based on one or more selection criterion.
  • the plants are selected on the basis of one or more phenotypic traits. Skilled persons will readily appreciate that such traits include any observable characteristic of the plant, including for example growth rate, height, weight, colour, taste, smell, changes in the production of one or more compounds by the plant (including for example, metabolites, proteins, drugs, carbohydrates, oils, and any other compounds). Selecting plants based on genotypic information is also envisaged (for example, including the pattern of plant gene expression in response to the microorganisms, genotype, presence of genetic markers). It should be appreciated that in certain embodiments, plants may be selected based on the absence, suppression or inhibition of a certain feature or trait (such as an undesirable feature or trait) as opposed to the presence of a certain feature or trait (such as a desirable feature or trait).
  • the one or more marker may already be known and/or associated with a particular characteristic of a plant; for example, a marker or markers associated with an increased growth rate or metabolite profile.
  • This information could be used in combination with assessment based on other characteristics in a method of the invention to select for a combination of different plant characteristics that may be desirable.
  • Such tecliniques may be used to identify novel QTLs which link desirable plant traits with a specific microbial flora - for example matching plant genotype to the microbiome type.
  • plants may be selected based on growth rate, size (including but not limited to weight, height, leaf size, stem size, branching pattern, or the size of any part of the plant), general health, survival, tolerance to adverse physical environments and/or any other characteristic, as described herein before. Further non-limiting examples include selecting plants based on: speed of seed germination; quantity of biomass produced;
  • Selection of plants based on phenotypic or genotypic information may be performed using techniques such as, but not limited to: high through-put screening of chemical components of plant origin, sequencing techniques including high through-put sequencing of genetic material, differential display techniques (including DDRT-PCR, and DD-PCR), nucleic acid microarray techniques, RNA-seq (Whole Transcriptome Shotgun Sequencing), qRT- PCR (quantitative real time PCR).
  • selection for a combination of plant traits may be desired; for example, in the embodiments of the invention which involve repeating the basic method steps and applying different selective pressures with each iteration of the method.
  • This can be achieved in a number of ways.
  • multiple rounds of iterative improvement for one trait e.g. superior growth in nematode infested soils, are maintained until an acceptable level of nematode resistance is attained.
  • Similar, but completely separate rounds of selection are undertaken to identify microorganisms that can confer at least different desirable traits, for example simply for improved growth or other characteristics, or improved growth resulting from improved microbial soil phosphate utilisation, or improved growth resulting from increased tolerance to sucking insect pests.
  • Such separate rounds of selection may be performed using an iterative or stacking approach or a combination of separate methods could be used, with the microorganisms that result from those separate rounds or methods being combined into a single composition.
  • the microorganism(s) could be developed into a product containing combinations of separately-fermented microorganisms each shown to improve a different plant attribute.
  • the separately selected sets of microorganisms may be combined in sets of two or more and used in further methods of the invention.
  • the separately selected sets of microorganisms may be separated into individual isolates and then individual isolates combined in sets of two or more and used in further methods of the invention.
  • the combined microorganisms are applied to the plant and/or growth medium for the application of two or more selection pressures in the same iterative cycle.
  • microorganisms able to improve plant growth in a medium containing low-levels of plant- available phosphorus are combined with microorganisms able to enhance plant growth in soils infested with plant parasitic nematodes.
  • the combined microorganisms are then added to a plant growth medium with low levels of available phosphorus in which the plants are grown for a suitable period, nematodes applied and the plants are further grown until nematode damage can be expressed.
  • the degree of nematode root damage and plant biomass is assessed non-destructively and microbes are isolated from the best-performing plants for use in a succeeding iteration. Similar iterative rounds may be continued until an acceptable level of plant growth is attained under both selective pressures.
  • This approach will aid the selection of microbes that synergistically improve plant performance i.e. improve plant growth and nematode resistance to a degree better than that achieved if the microorganisms are applied simply as a combination of two separately-selected sets.
  • the same approach could be used in methods of the invention which do not employ the application of one or more selective pressure.
  • microorganisms acquired from separate runs of a method of the invention each shown to be associated with different desirable plant characteristics (by way of non-limiting example, sugar or fibre content and growth rate, or flower colour and height) could be combined into a single composition and applied to one or more plants in a further round of the method for selection of plants having both characteristics or yet a further characteristic.
  • desirable plant characteristics by way of non-limiting example, sugar or fibre content and growth rate, or flower colour and height
  • one or more plants are harvested and plant tissues may be examined to detect microorganisms forming associations with the plants (for example, endophytic, epiphytic or rhizospheric associations).
  • microorganisms forming associations with the plants (for example, endophytic, epiphytic or rhizospheric associations).
  • the techniques described in this section may be used in acquiring a second set of microorganisms at the conclusion of a method of the invention or for use in any successive repeat of the methods of the invention.
  • the one or more microorganisms may be isolated from any appropriate tissue of the plants selected; for example, whole plant, foliar tissue, stem tissue, root tissue, and/or seeds.
  • the microorganisms are isolated from the root tissue, stem or foliar tissues and/or seeds of the one or more plants selected.
  • the microorganisms may be acquired in crude form, in which they are not isolated from the source material in which they reside (such as a plant, plant tissue or part or growth media).
  • isolation of the microorganisms occurs, they may be isolated from the plants using any appropriate methods known in the art.
  • methods for isolating endophytic microbes may include the sterile excision of the plant material of interest (e.g. root, stem lengths, seed), surface sterilisation with an appropriate solution (e.g. 2% sodium hypochlorite), after which the plant material is placed on nutrient medium for microbial outgrowth, especially filamentous fungi.
  • the surface-sterilised plant material can be crushed in a sterile liquid (usually water) and the liquid suspension, including small pieces of the crushed plant material spread over the surface of a suitable solid agar medium, or media, which may or may not be selective (e.g.
  • the plant root or foliage samples may not be surface sterilised but only washed gently thus including surface-dwelling epiphytic microorganisms in the isolation process, or the epiphytic microbes can be isolated separately, by imprinting and lifting off pieces of plant roots, stem of leaves on to the surface of an agar medium and then isolating individual colonies as above.
  • This approach is especially useful for bacteria and yeasts, for example.
  • the roots may be processed without washing off small quantities of soil attached to the roots, thus including microbes that colonise the plant rhizosphere. Otherwise, soil adhering to the roots can be removed, diluted and spread out onto agar of suitable selective and non-selective media to isolate individual colonies of rhizospheric microbes.
  • Further exemplary methodology can be found in: Strobel G and Daisy B (2003) Bioprospecting for microbial endophytes and their natural products. Microbiology and Molecular Biology Reviews 67 (4): 491-502; Zinniel DK et al. (2002) Isolation and characterisation of endophytic colonising bacteria from agronomic crops and prairie plants. Applied and Environmental Microbiology 68 (5): 2198-2208; Manual of Environmental Microbiology, Hurst et al., ASM Press, Washington DC.
  • Methods for isolation may be informed by culture independent community profiling techniques that provide information on the identity and activity of microbes present in a given sample. These methods may include, but are not limited to, sequencing techniques including high throughput sequencing and phylogenetic analysis, or microarray-based screening of nucleic acids coding for components of rRNA operons or other taxonomically informative loci.
  • any appropriate methodology for separating one or more microorganism from each other may be used.
  • microbial extracts prepared from plant material could be spread on agar plates, grown at an appropriate temperature for a suitable period of time and the resulting microbial colonies subsequently selected and grown in an appropriate media (for example, streaked onto fresh plates or grown in a liquid medium).
  • the colonies may be selected based on morphology or any other appropriate selection criteria as will be understood in the art.
  • selective media could be used.
  • the one or more microorganisms maybe harvested (including in isolated or crude form) from the plants (including the rhizosphere as described herein before) at any appropriate time point. In one embodiment they are harvested at any time after germination of the plant. For example, they can be isolated from the period shortly after germination (where survival in the first few days after germination is an issue, for example with bacterial and fungal root and collar rots), then at any stage after that, depending on the timing required for a plant to grow in order to evidence a discriminatory benefit that enables it's selection from the plant population (for example, to discriminate say the top 10 of 200 plants). The inventor(s) has observed that different microorganisms may associate with a plant at different stages of the plant's life.
  • harvesting a plant at different time points may result in selection of a different population of microorganisms.
  • Such microorganisms may be of particular benefit in improving plant condition, survival and growth at critical times during its life: by way of example, as mentioned herein before, a plant may be susceptible to attack by nematodes at discrete time points during its life and the invention may be used to identify and isolate a population of microorganisms which may increase resistance to such attack at that particular life stage.
  • the microorganisms in the case of microorganisms that form an association with a plant that allows vertical transmission from one generation or propagule to the next (for example seed-endophytic or -epiphytic associations, or endophytic and epiphytic associations with plants/propagules multiplied vegetatively) the microorganisms may not be isolated from the plant(s).
  • a target or selected plant itself may be multiplied by seed or vegetatively (along with the associated microorganisms) to confer the benefit(s) to "daughter" plants of the next generation or multiplicative phase.
  • plant material whole plant, plant tissue, part of the plant
  • step a) of any successive repeat can be used in step a) of any successive repeat.
  • the inventor(s) envisage advantages being obtained by stacking selective pressures in repeated rounds of the method of the invention. This may allow for acquiring a population of microorganisms that may assist a plant in surviving in a number of different environmental conditions, resisting a number of different diseases and attack by a number of different organisms, for example.
  • the inventor(s) envisage advantages being obtained by stacking the means of selection (or the selection criteria) of plants in repeated rounds of the method of the invention. This may allow for the acquisition of a population of microorganisms that may assist a plant in having a number of different desirable traits, for example.
  • the one or more microorganisms acquired from the one or more plants selected following exposure to a selective pressure, as previously described, is used in a second round or cycle of the method; ie the microorganisms from the selected plants are provided, along with one or more plants and a growth medium, a selective pressure is applied, plants are selected at a desired time and microorganisms are isolated from the selected plants.
  • the microorganisms acquired from the second round of the method may then be used in a subsequent round, and so on and so on.
  • the selective pressure applied in each repeat of the method is different.
  • the pressure may be a particular soil pH and in the second round the pressure may be nematode attack.
  • the selective pressure applied in each round may be the same. It could also be the same but applied at differing intensities with each round.
  • the selective pressure in the first round the selective pressure may be a particular concentration of salt present in the soil.
  • the selective pressure in the second round, the selective pressure may be a higher concentration of salt present in the soil.
  • the selective pressure is increased in successive rounds in a pattern that may be linear, stepped or curvilinear.
  • wheat plus microorganisms may be exposed to 100 mM NaCl, in the second to 110 mM salt, in the third to 120 mM salt, thus increasing the selective pressure on the plants as adaptation occurs via improved plant/microorganism associations.
  • a selective pressure may be separated disjunctively from a specific step of the iterative process, particularly the first round of an iterative cycle. For example in round one the selective pressure may not be applied at all. But after the microorganisms have been isolated from the selected plants after exposure for a relevant period to a growth medium and microorganisms in round 1, they are applied to the plant growth medium along with the plant, seed, seedling, cutting, propagule or the like for round 2. After an appropriate time a selective pressure is applied in round 2 and in successive rounds. This type of selection may be especially relevant for selection factors that severely diminish the plant tissue that is the target of the selection.
  • nematodes are especially destructive of root tissue and it may be advantageous to allow particular microbes to multiply to high levels on, in, or around the roots in roundl to allow high concentrations of microorganisms from the roots of plants selected in round 1 to be applied to the growth medium in round 2.
  • the one or more microorganisms acquired from the one or more plants selected, as previously described, is used in a second round or cycle of the method, where a different selection criterion is used.
  • a different selection criterion is used in the first round.
  • one or more plants may have been selected based on biomass.
  • one or more plants may be selected based on production of a particular compound.
  • the microorganisms from the second round of the method may then be used in a subsequent round, and so on and so on. Any number of different selection criteria may be employed in successive rounds of the method, as desired or appropriate.
  • the selection criteria applied in each repeat of the method is different.
  • the selection criteria applied in each round may be the same. It could also be the same but applied at differing intensities with each round.
  • the selection criteria may be fibre levels and level of fibre required for a plant to be selected may increase with successive rounds of the method.
  • the selective criteria may increase or decrease in successive rounds in a pattern that may be linear, stepped or curvilinear.
  • the methods of the first and second (and/or related) aspects of the invention may be combined such that they alternate. For example, a method of the first aspect is performed and the microorganisms acquired in that method used in step a) of a method of the second (and/or related) aspect of the invention.
  • microorganisms acquired in step d) of a method of the second (and/or related) aspect of the invention may then be used in step a) of a method of the first aspect. This may be repeated any number of times as necessary or desired. It should be appreciated that the methods may be combined in any order and in any combination or permutation. For example, they need not alternate on a one by one basis. Any number of repeats of one a method of one aspect may be performed before switching to one or more round of a method of the other aspect.
  • the methods of the first and second (and/or related) aspect may also be run independently and then the microorganisms acquired in the methods combined and used in a further round of one or both methods, and so on. It should also be appreciated that in certain embodiments of the invention, where one or more microorganism(s) forms an endophytic or epiphytic relationship with a plant that allows vertical transmission from one generation or propagule to the next the
  • microorganisms need not be isolated from the plant(s).
  • a target or selected plant itself may be multiplied by seed or vegetatively (along with the associated microorganisms) to confer the benefit(s) to "daughter" plants of the next generation or multiplicative phase.
  • plant material whole plant, plant tissue, part of the plant
  • two or more selective pressures and/or two or more selection criterion may be applied with each iteration of a method of the invention.
  • the methods of the invention include a step of subjecting either or both of the first set of one or more microorganisms and the second set of one or more microorganisms to a conditioning and/or directed evolution process. This may occur prior to step a) at the beginning of a method of the invention and/or after step d) and prior to step a) of any successive repeat of the method steps. It may also occur after step d) at the conclusion of a method of the invention. It may also be repeated two or more times if appropriate.
  • a "conditioning process” as used herein should be taken broadly to include any process which promotes a microorganism to grow, proliferate, improve colonisation of plant tissues (including the rhizosphere), assume a specified morphology or physiological status, express one or more protein, produce one or more metabolite or other compound which may assist the one or more microorganism in imparting one or more beneficial property to one or more plant.
  • a conditioning process may involve subjecting the one or more microorganism to one or more media containing one or more ingredient that is known to promote a microorganism to grow, proliferate, or promote or activate a particular pathway for the production of one or more compound or the like.
  • a microorganism maybe conditioned for improved colonization of plant tissues of the rhizosphere through growth on specialised media.
  • microorganisms may be conditioned by growth on plant tissue culture media such as Murashige and Skoog medium (Murashige T and Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15(3): 473-497). Culture may be performed with or without co-culture of plant material.
  • a conditioning process may also involve manipulation of growth conditions to elicit a specific physiological trait such as heat shock or cold shock response, sporulation, stationary phase induction or germination, or to coordinate the growth status of a population for example through continuous culture.
  • a "directed evolution process” as used herein should be taken broadly to include any process which promotes the evolution of the one or more microorganism. Such evolution may be controlled or random. For example, the microorganism may be subjected to targeted genetic modification or random genetic modification.
  • microorganisms maybe genetically modified to include specific heterologous nucleic acids or knock out nucleic acids native to the microorganisms to affect a specific change; such as expression of a particular gene or suppression of a particular gene, or to affect a change to the gene product.
  • the directed evolution may allow for the generation of microorganisms having superior properties of use in the invention.
  • the directed evolution and/or conditioning processes may involve a step of selecting and/or enriching for a subset of one or more microorganisms from the original population of microorganisms subjected to either or both processes.
  • selecting and enrichment steps may include, by way of example, growth of the microorganisms on particular selective media, pasteurisation steps, analysis of one or more phenotypic and/or genotypic traits including levels of one or more compounds produced by the microorganisms, including metabolites, the nature and or expression levels of one or more proteins, presence of one or more genetic markers.
  • a selective process may also involve growing cultures under a selective pressure designed to select for spontaneous mutations that confer a physiological trait. For example selection for microbes that are able to use a component of plant root exudates, such as glycerol, as a sole carbon source.
  • phenotypic and/or genotypic traits of the microorganisms may be completed using any number of known techniques.
  • the following techniques may be employed: including but not limited to high through-put screening of chemical components of plant, sequencing techniques, including high through-put sequencing of genetic material, differential display techniques (including DDRT-PCR, and DD-PCR), nucleic acid microarray techniques, RNA-seq (Whole Transcriptome Shotgun Sequencing), qRT-PCR (quantitative real time PCR).
  • results of the analysis of phenotypic and/or genotypic traits of the microorganisms may be compared to information contained on one or more database, which may assist in the selection of a subset of microorganisms of use in a method of the invention.
  • certain genetic profiles, expression profiles, metabolic profiles and the like may be known to be associated with certain properties and one may be able to select the best microbial candidates on basis of this information.
  • the invention relates to microorganisms selected, acquired or isolated by such methods and compositions comprising such microorganisms.
  • a composition comprising one or more
  • microorganisms includes a culture of living microorganism, or microorganisms in a live but inactive state(s), including frozen, lyophilised or dried cultures.
  • the compositions may comprise other ingredients, as discussed below.
  • the invention should also be understood to comprise methods for the production of a composition to support plant growth, quality and/or health, or a composition to suppress or inhibit plant growth, quality and/or health, the method comprising the steps of a method herein before described and the additional step of combining the one or more
  • microorganisms with one or more additional ingredients.
  • composition to support plant growth, health, and/or quality should be taken broadly to include compositions which may assist the growth, general health and/or survival of a plant, the condition of a plant, or assist in the maintaining or promoting any desired characteristic, quality, and/or trait. It should be taken to include maintaining or altering the production of one or more metabolite or other compound by a plant as well altering gene expression and the like. The phrase should not be taken to imply that the composition is able to support plant growth, quality and/or health on its own. However, in one embodiment the compositions are suitable for this purpose.
  • compositions of this aspect of the invention include but are not limited to plant growth media, plant mineral supplements and micronutrients, composts, fertilisers, potting mixes, insecticides, fungicides, media to protect against infection or infestation of pests and diseases, tissue culture media, see coatings, hydroponic media, compositions that impart tolerance to drought or abiotic stress such as metal toxicity, compositions that modify soil pH.
  • composition to inhibit or suppress plant growth, health, and/or quality should be taken broadly to include compositions which may assist in suppressing or inhibiting one or more characteristic or quality and/or trait of a plant, including its growth, general health and/or survival. It should be taken to include maintaining or altering the production of one or more metabolite or other compounds by a plant as well altering gene expression and the like. The phrase should not be taken to imply that the composition is able to suppress or inhibit plant growth, quality and/or health on its own. However, in one embodiment the compositions are suitable for this purpose.
  • compositions of this aspect of the invention include but are not limited to plant growth suppression media, weed killer, fertilisers, potting mixes, plant mineral supplements and micronutrients, composts, mixes, insecticides, fungicides, tissue culture media, seed coatings, hydroponic media, compositions that impart tolerance to drought or abiotic stress such as metal toxicity, compositions that modify soil pH.
  • the ingredients may include liquid and/or solid carriers, microbial preservatives, microbial activators that induce specific metabolic activities, additives to prolong microbial life (such as gels and clays), wettable powders, granulated carriers, soil, sand, agents known to be of benefit to microbial survival and the growth and general health of a plant, peat, organic matter, organic and inorganic fillers, other microorganisms, wetting agents, organic and inorganic nutrients, and minerals.
  • liquid and/or solid carriers such as liquid and/or solid carriers, microbial preservatives, microbial activators that induce specific metabolic activities, additives to prolong microbial life (such as gels and clays), wettable powders, granulated carriers, soil, sand, agents known to be of benefit to microbial survival and the growth and general health of a plant, peat, organic matter, organic and inorganic fillers, other microorganisms, wetting agents, organic and inorganic nutrients, and minerals.
  • compositions can be made using standard methodology having regard to the nature of the ingredients to be used.
  • Compositions developed from the methods of the invention may be applied to a plant by any number of methods known to those skilled in the art. These include for example: sprays; dusts; granules; seed-coating; seed spraying or dusting upon application;
  • inoculation into plant tissues under axenic conditions via injection of compositions or otherwise inoculated via a cut in such tissues, for the subsequent establishment of an endophytic relationship with the plant that extends to the seed, or propagative tissues, such that the plant can be multiplied via conventional agronomic practice, along with the endophytic microbe providing a benefit(s) to the plant.
  • microorganisms When microorganisms are cultured they may produce one or more metabolites and which are passed into the media in which they reside. Such metabolites may confer beneficial properties to plants.
  • the invention also provides a method of for selecting or producing a composition capable of imparting one or more beneficial property to a plant, for example to support plant growth, quality and/or health, or for example to suppress or inhibit growth, quality and/or health of a plant, or to identify microorganisms that are capable of producing such a composition.
  • the composition is substantially free of microorganisms.
  • the method is for the selection of a composition the method comprising at least the steps of:
  • step b) subjecting one or more plant (including for example seeds, seedlings, cuttings, and/or propagules thereof) to the one or more composition of step b);
  • step d) selecting one or more composition from step c) if it is observed to impart one or more beneficial property to the one or more plants.
  • the method is for the selection of a composition and comprises at least the steps of:
  • step b) subjecting one or more plant (including for example seeds, seedlings, cuttings, and/or propagules thereof) to the one or more composition of step b);
  • step d) selecting one or more composition from step c) if it is observed to impart one or more beneficial property to the one or more plants.
  • the method is for the selection of one or more microorganisms which are capable of producing a composition and comprises at least the steps of:
  • step b) separating the one or more microorganism from the one or more media in the one or more culture from step a) after a period of time to provide one or more composition substantially free of microorganisms;
  • step b) subjecting one or more plant (including for example seeds, seedlings, cuttings, and/or propagules thereof) to the one or more composition from step b);
  • Another method of the invention comprises at least the steps of:
  • step b) subjecting one or more plant (including for example seeds, seedlings, cuttings, and/or propagules thereof) to the one or more composition of step b);
  • step b) of the method of the previous two paragraphs could be substituted with the step of b) inactivating the one or more culture of step a) to provide one or more composition containing one or more inactivated microorganisms, and then using this composition in step c) of the process.
  • inactivating the one or more culture and “inactivated microorganisms” and like terms should be taken broadly to mean that the microorganisms are substantially inactivated, fixed, killed or otherwise destroyed. The term should not be taken to mean that all microorganisms are inactivated, killed or destroyed, however, this may be preferable.
  • the microorganisms are inactivated, fixed, killed or otherwise destroyed to the extent that self-sustained replication is no longer measurable using techniques known to one skilled in the art.
  • the microorganisms can be inactivated, killed, fixed or destroyed using any appropriate techniques known in the ait. However, by way of example, one may use chemical agents and/or physical means to do so.
  • the cells are lysed. In another embodiment cells are fixed by chemical means, so as to render the organisms non-viable,, but retaining their structural integrity.
  • a "composition substantially free of microorganisms" should be taken broadly and not be construed to mean that no microorganisms are present, although this may be preferred.
  • microorganisms are cultured in two or more (preferably a large number, for example, from at least approximately 10 to up to approximately 1000) mixed cultures using media that can support the growth of a wide variety of microorganisms. Any appropriate media known in the art may be used.
  • growth media may include TSB (tryptic soy broth), Luria- Bertani (LB) broth, or R2A broth.
  • selective or enrichment media which are able to support the growth of microorganisms with an array of separate but desirable properties may be used.
  • the enrichment media referred to elsewhere herein may be used.
  • the microorganisms may be cultured in the media for any desired period. Following culture, the microorganisms are separated from the media and stored for later use, A separate composition also results.
  • One or more plants in a suitable growth medium are then subjected to the composition (using any known methodology, or methodology as described herein before). After a period of time, growth of plants is assessed and plants selected (as described herein before, for example). Plants are preferably selected on the basis of size. However, other selection criteria as referred to herein may be used.
  • the microorganism(s) producing the subset of compositions associated with the selected plants are recovered from storage. Two or more separate cultures of the microorganisms may then be mixed together and separated into two or more sub-cultures grown in two or more different media.
  • This process can be repeated iteratively as many times as is deemed efficacious, with progressive steps refining down to fewer media and a narrower diversity of
  • compositions of this aspect of the invention may be used or formulated on their own or combined with one or more additional ingredients.
  • the following methodology may be applied to a method of the invention for identifying one or more microorganisms as herein before described.
  • Figure 1 shows a system 10 according to an embodiment of the invention.
  • System 10 includes requestors 11, request processor 12, growing facility 13, database or library 14 and depository 15.
  • Figure 2 provides a flow chart illustrating a method 20 according to an embodiment of the invention. The steps shown in Figure 2 will be described with reference to the system 10 shown in Figure 1.
  • microorganism that may impart one or more desired properties to one or more plants, with particular reference to the first, second, eighth (and/or related) and/or ninth (and/or related) aspects of the invention.
  • reference to the first aspect of the invention should be taken to also include reference to the second (and/or related), eighth (and/or related) and/or ninth (and/or related) aspects of the invention, and reference to one or more microorganism should be taken to include reference to one or more composition.
  • the method begins at step 21 with a requestor 11 identifying a plant (or a class or group of plants).
  • a requestor 11 identifying a plant (or a class or group of plants).
  • Reasons why particular plants or types of plants may be identified will be apparent to those skilled in the art. However, by way of example, it may have been found that a plant noted in general for having a high growth rate is growing at lower rates or not at all, there may simply be a desire to improve on existing growth rates or there may be a desire to introduce a plant to a different climate / environment / geographical region. The invention is not limited to conferring improvements to particular plant(s) and may be used to inhibit growth or otherwise adversely affect the plant(s).
  • the requestor 11 sends the plant and / or the identity thereof to a request processor 12.
  • the requestor 11 may provide further relevant information such as why or what properties they are seeking to improve. While only one request processor 12 is shown, it will be appreciated that more than one may be provided in the system 10. Where a requestor 11 identifies a class or group of plants, more than one plant variety may be evaluated. Alternatively or additionally, selection of a one or more plant variety may be made elsewhere within the system 10 based on the group or class identified, including following evaluation of different varieties including using different microorganisms in accordance with methods of the invention.
  • Requests may conveniently be received over the internet via a web browser, although the invention is not limited thereto.
  • Use of a web browser may additionally or alternatively be used to enable a requestor 11 to view reports on the progress being made in response to their request. For example, measures of growth may be provided.
  • the request processor 12 receives and processes the request, essentially by initiating the performance of the method for the selection of one or more microorganism according to the first aspect of the invention.
  • the request processor 12 may or may not actively perform the method of the first aspect, or may only perform parts thereof.
  • the request processor 12 may act as an intermediary or agent between the requestor 11 and the parties able to perform the method of the first aspect.
  • different arrangements may be made in response to different requests. For example, for one request, the environment around the request processor 12 may be suitable for evaluating a particular plant but unsuitable for another, requiring the assistance of a third party facility. This could be due to a desire to test in a particular soil type, altitude or climate.
  • a computer processor selects parameters or conditions for a study based on data input by a requestor 11. As will be appreciated, providing a structured information request may help to effect this, and where necessary, reference may be made to databases including database 14,
  • parameters of the evaluation process are selected. For example, reference may be made to database 14 for microorganisms that may provide the desired improvement in the plant(s). While little data to date has been provided in the art on microorganisms having beneficial associations with particular plant varieties, this will be improved upon through ongoing operation of the methods of the invention and stored in database 14. Other parameters such as plant type(s) and environmental conditions may also be selected.
  • the request (or portions thereof) and evaluation parameters are sent to growing facility 13 which may obtain suitable microorganisms from depository 15. These may or may not have been previously identified. While only one growing facility 13 and one depository 15 are shown, it will be appreciated that the invention is not so limited.
  • any two or more of request processor 12, growing facility 13, database 14 and depository 15 maybe co-located and/or under the same control.
  • a selection process is performed., preferably according to the selection method of the first aspect.
  • a response is sent to the request.
  • a response may be sent to the requestor 11 and / or to a third party and preferably includes at least one of at least a subset of the results generated at step 26, identification of plant(s), plant(s), identification of microorganism(s), microorganism(s), or plant(s) provided in association with
  • microorganisms namely those that have been shown to provide benefits at step 26.
  • database 14 may be updated with results of the selection process of step 26. This step may be performed prior to step 27, including periodically or at other various stages which the selection process is conducted. Preferably, at least details of new beneficial associations between plant(s) and microorganisms are recorded. It will be appreciated that incompatible or less beneficial associations will also preferably be recorded, thereby over time building a knowledge framework of plants and
  • the invention provides means and methods to improve plant(s) (or growth or other characteristics thereof). This is achieved by enabling a requestor 11 in a first geographical region (e.g. country) or otherwise defined environment (e.g. by parameters or
  • the invention may enable a requestor to obtain the beneficial effects of a particular microorganism(s) on a particular plant(s) in a first region, even though such
  • microorganism(s) may not be present or are of limited presence in the first region.
  • the overseas company agrees to send seeds, cuttings or other plant propagules (foreign cultivar) to the home company from plant cultivars adapted to the environment(s) in its own, or other foreign countries, in order to gain access to elements of New Zealand's terrestrial and marine microbial biodiversity that are able to form beneficial plant-microorganism associations with the foreign cultivar.
  • the nature of the benefit may encompass increased plant productivity, for example through any one or more of but not limited to: increased root or foliar mass, or through an increase in efficiency in nutrient utilisation through nitrogen fixation by diazatrophs such as Klebsiella or Rhizobium, or through release of plant nutrients from the soil, such as phosphates released soil through the production of microbial phytases, or through improved resistance to attack from pests and diseases spanning a broad range of nematodes, insects, microbial and virus diseases, or through improvements in the ability of the plant to resist adverse environmental conditions such as drought, salinity, extreme temperatures, toxic soil minerals, or through improvements in plant phenotype for example date of flowering, or changes in physical form e.g. colour frequency of root or foliar branching, or changes in chemical profile including compounds associated with taste, smell or properties which make the plant suitable for a particular purpose.
  • diazatrophs such as Klebsiella or Rhizobium
  • plant nutrients from the soil such as phosphates released soil
  • the home company identifies which indigenous microorganisms can form an association with the foreign plant by exposing the seed to the microorganisms, with or without knowledge of their likely effects on the plant, by the method of germinating the seed and growing the plant in a growing material that ensures contact of the plant during its growth with indigenous microorganisms via seed coating, direct inoculation into the seed or germinating seedling and/or contamination of the growing medium.
  • the invention is not limited to this arrangement or methodology. For example, it may be apparent that
  • microorganisms present in soil other than in New Zealand may provide benefits and testing may be conducted in such regions in addition to or instead of New Zealand.
  • artificial environments maybe created. Referring to the immediately prior example, this may be achieved by obtaining soil and / or microorganisms from such regions and conducting the tests in say New Zealand.
  • such embodiments may include provision for artificial control of climatic conditions among other parameters.
  • the invention is not limited to conducting testing in a region based on its indigenous microorganisms - the microorganisms maybe artificially introduced so as to conduct the testing elsewhere than in the microorganisms' natural environment.
  • the period of growth and the physical conditions under which they take place may vary widely according to plant species and specific plant improvement traits, including based on parameters desired or specified by the overseas company. After the relevant period of plant growth the nature of possible plant- microorganisms associations may be determined by microbiological assessment to determine whether microorganisms have formed an endophytic, epiphytic or rhizospheric association with the foreign crop.
  • microorganisms form a collection of (say New Zealand indigenous) microorganisms able to associate with the (say foreign) crop or plant.
  • microbial isolates of the collection may, for example, be coated on to seeds, inoculated into seeds or seedlings, or inoculated into a growing medium that may or may not be sterile.
  • the plants are assessed for improved root and foliar growth or other desired characteristics and/ or they may be exposed to environmental stressors designed to identify the plant-microorganism associations most able to provide benefit to the plant in the manner desired by the overseas company.
  • the invention also includes introducing foreign material or creating otherwise artificial conditions in the home or test region.
  • the steps involving growing one or more plant in the presence of one or more microorganism, selecting one or more plants with desired characteristics, and acquiring the microorganism(s) forming an association with the plant will be repeated one or more time.
  • One or more conditioning and/or directed evolution step in accordance with the invention is performed at the appropriate time (such as after selection of plants and acquisition of the one or more microorganism(s) forming an association with the one or more plants) by any appropriate party.
  • the overseas company will agree that microorganisms found on, or in, the seed, cuttings or propagules of the foreign cultivar will be added to the collection of the home company to enlarge the collection for use both on that cultivar or on other foreign cultivars received for similar testing from other companies.
  • the microbial isolates able to form plant-microorganism associations with the foreign cultivar i.e., the collection are sent to the second company for testing and selection, such that items 8-11 above are performed by and / or in the grounds of the second company. This may be performed by or under the control of the first company.
  • the home company may simply expose the seed to indigenous microorganism(s) of a collection.
  • microorganisms with or without knowledge of their likely effects on the plant, for example by germinating the seed and growing the plant in a growing material that ensures contact of the plant during its growth with indigenous microorganisms via seed coating, direct inoculation into the seed or germinating seedling and / or contamination of the growing medium or otherwise.
  • the home company may additionally or alternatively arrange for similar testing in other regions, where the same or different microorganisms may be present.
  • the period of growth and the physical conditions under which they take place may vary widely according to plant species and specific plant traits desired by the overseas company. After a period of plant growth the nature of possible plant-microorganism associations may be determined in a similar manner to that described above.
  • Transgenic plants expressing beneficial traits such as insect-resistance or herbicide resistance have been widely adopted by both farmers in North and South America and Asia. It would be desirable to extend the benefits of traits similar to those expressed by transgenic crops, as well as other beneficial crop traits, without the need for engineering the plant genome.
  • beneficial traits such as insect-resistance or herbicide resistance
  • Example 1 Use of a method of the invention combined with whole genome shuffling to select corn-adapted bacteria conferring resistance to the corn root worm.
  • Step 1 Library acquisition
  • Whole genome shuffling can accelerate directed evolution by facilitating recombination between the members of a selected population of microbes.
  • Diverse strains of Bacillus thuringiensis containing Cry3 genes and other ciy genes with activity against the com root worm (CRW) have been found in numerous habitats.
  • CRW com root worm
  • multiple species and strains of Bacillus, including B. thuringiensis have been found in endo-, epi- or rhizospheric association with many crop species, including com.
  • Multiple strains from both of these sources, i.e. both CRW active and corn-root associative are combined to form a single parental library.
  • Step 2 recombination of bacterial genomes
  • Combinatorial libraries of new strains are generated from the parental library by intraspecific fusion and interspecific hybridization by methods familiar to those skilled in the art, including protoplast fusion, electroporation or any other method inducing competence.
  • strains from the parental library may be mutagenized to create additional variability.
  • Recombinant strains are then regenerated, resulting in a combinatorial strain library. This library may then be pooled and subjected to further rounds of recombination and regeneration to create still further variability.
  • Regenerated combinatorial strains resulting from step 2 are subjected to an iterative process to select strains that are both adapted to corn roots and express the anti-CRW trait.
  • strains are preferably pooled and applied to a plant growth medium or soil into which corn seeds are planted.
  • the microorganism(s) are mixed into a suitable seed coating material e.g. a gel, and coated onto seeds before being planted into a similar plant medium.
  • the seeds are geminated and then exposed to the microorganisms for a short period (usually between 1 - 24 hours to maximise the chance that the microbes may form an endophytic or epiphytic association with the germinating plant) and then planted into a similar growth medium.
  • the growing medium may be initially sterile, although this is not essential and further microorganisms applied to the growth medium and/or plant.
  • SS step 2 After a suitable period of time, preferably 2-4 weeks, the plants are harvested and the roots removed. Preferably, roots are surface sterilized using low concentrations of ethanol and/or sodium hypochlorite and other procedures well known to those skilled in the art. Endophytic bacterial strains are then isolated from root tissue and either microscopically examined for the desired phenotype i.e. presence of characteristic Cry3 protein crystals, or characterised genetically for the possession of genetic markers linked to Cry3 genes. Alternatively, isolated strains are grown in pure culture and assayed in vivo for bioactivity against CRW larvae.
  • endophytic strains selected for desired properties may be subjected to a further round(s) of recombination, regeneration and selection to create a new library of strains enriched for an improved likelihood of colonising root tissue and killing CRW larvae.
  • a materially different approach to strain selection does not require microbial isolation. Instead in this example the inventor(s) create bacterial suspensions from root-crushes of surface-sterilised roots (endophytes) or washed roots (epi-and endo-phytes) or from suspensions from rhizospheric microbes. Small fractions are assayed comparatively for activity against CRW larvae. Preparations represented by the most active fractions are selected use in a further round(s) of selection.
  • the com plants may not be harvested, but exposed to a uniform CRW selection pressure by the addition of standard numbers of CRW larvae to the plant growth medium. After a suitable period of time the plants may be removed from the growth medium and the degree of root damage assessed. Plants displaying the least damage are selected and bacteria associated with the roots are either isolated and assessed for CRW activity as above (or not), or prepared as root crushes from the least damaged plants.
  • SS step 3 microbial isolates and/or root crushes selected in step 2 are added to the plant growth medium, either individually or pooled (preferably) into which, preferably, surface- sterilized corn seeds are planted.
  • the microorganism(s) are mixed into a suitable seed coating material e.g.
  • the seeds are geminated and then exposed to the microorganisms for a short period (usually between 1 - 24 hours to maximise the chance that the microbes may form an endophytic or epiphytic association with the germinating plant) and then planted into a similar growth medium.
  • the growing medium may be initially sterile, although this is not essential and further microorganisms applied to the growth medium and/or plant.
  • step 2 to step 3 may then be repeated iteratively, with or without modification of the selection criteria for CRW.
  • Step 4 After this iterative process has been conducted to the point at which improvement of root-associated (either endophytic, epiphytic or rhizospheric) microbial activity against the CRW is deemed to be sufficient, the best-performing strains are selected again as in step 2, or bacteria are finally isolated as in step 2 from the best performing root crushes or similarly isolated from roots of the plants most resistant to CRW attack.
  • These microorganisms are both active against CRW larvae and adapted to close association with corn roots and can be used to develop commercial products such as seed coatings or granules applied at planting that impart a trait for com root worm resistance to corn .
  • Example 2 Use of a method of the invention with directed molecular evolution to select corn-adapted bacteria conferring desirable crop output traits.
  • Directed evolution of single genes or loci through random or directed mutagenesis techniques and other recombinatorial procedures known to those skilled in the art, can be used to create libraries of microbes exhibiting targeted genetic variation.
  • the process outlined in the invention is applied as a series of iterative steps following creation of the library, to generate crop-adapted strains of microbes conferring the desired phenotype.
  • the methodology is as generally described above with the difference being that specific genes or loci associated with a desired phenotype are targeted.
  • Recombinant libraries may be used separately or combined with naturally-occurring microbes derived from any material or step in a process of the invention.
  • Example 3 Use of process to select seed-borne endophytes conveying a beneficial crop trait
  • Step 1 Untreated ryegrass seeds are planted in a wide variety of soils in small pots. Soils may include additional amendments comprising pure cultures of microorganisms, mixtures of microorganisms or materials containing microorganisms that derived from other sources, including those outlined in examples 1 and 2 above.
  • Step 2 After a suitable period of growth, the plants are washed out of the soil, surface sterilised with a combination of ethanol and sodium hypochlorite or other methods Icnown to people skilled in the art, and the endophytic microorganisms (endophytes) isolated from internal tissues of roots and stems/foliage and seeds, either as individual isolates in pure culture, or as mixed populations e.g. as a microbial suspension from an aqueous root cmsh and/or a stem/foliar cmsh,
  • Step 3 The endophytic microorganisms are then added to a plant growth medium into which pre-germinated surface-sterilised ryegrass seeds are planted (seeds checked for sterility by germinating on nutrient agar plates).
  • the microorganism(s) are mixed into a suitable seed coating material e.g. a gel, and coated onto surface-sterilised seeds before being planted into a similar plant medium.
  • the surface- sterilised seeds are geminated on nutrient agar plates, checked for sterility and then exposed to the microorganisms for a short period (usually between 1 - 24 hours to maximise the chance that the microbes may form an endophytic or epiphytic association with the germinating plant) and then planted into a similar growth medium.
  • the growing medium may be initially sterile, although this is not essential and further microorganisms may be applied to the growth medium and/or plant.
  • Phenotypes may include relative resistance to abiotic selection pressures such as relative growth in nutrient deficient conditions e.g. nitrogen or phosphorus, growth in a medium high in salinity or deficient in water.
  • the selection pressures could also be biotic such as exposure to insect pests, plant parasitic nematodes, or plant diseases.
  • no selection pressure may be applied and the plants selected merely on a phenotypic attribute such as improved colour, plant form, metabolite expression, or the like.
  • Step 5 Selected plants are permitted to grow onward to the point of seed set. At this stage a subset of seeds from each plant may be screened for endophyte carriage using culture dependent or independent methods. The remaining seeds from plants yielding positive results in the screen are germinated and planted without microbial addition in a further round of selection to enrich for endophyte carriage and the ability to transmit the desired phenotype as described in steps 3 - 5.
  • endophytic microbes may be acquired from a subset of seeds from each plant either as isolates from surface sterilised seeds or as explants, or as a microbial suspension prepared, for example, by crushing the surface sterilised seed in aqueous solution. Isolates and preparations are used as an inoculum for plants arising from surface sterilised seeds as described in step3.
  • the selection for seed transmission of the trait may take place in the following generation by surface sterilising a subset of seeds (with or without prior screening) from the selected plants of the prior generation and allowing them to germinate and grow on for the period at which point phenotypic screening is conducted as generally described in steps 3 and 4 (i.e. prior to seed set).
  • Plants exhibiting the desired phenotype in this generation are selected and either tissue explants are prepared, and/or microbes isolated from plant tissues, and/or crude microbial suspensions made by crushing the surface foliage or roots in an aqueous solution.
  • tissue explants are prepared, and/or microbes isolated from plant tissues, and/or crude microbial suspensions made by crushing the surface foliage or roots in an aqueous solution.
  • One or a combination of these preparations are used as an inoculum for further iterative rounds of growth and selection and seed harvest, as described in steps 3 -5.
  • the remaining seeds of plants exhibiting the desired seed-borne trait may be germinated and planted without microbial addition in a further round of selection to enrich for endophyte carriage and the ability to transmit the desired phenotype as described in steps 3 - 5.
  • Step 6 At the end of successive rounds of this iterative process, as determined by the generation of a desired seed-borne phenotype, the best seed lines are selected for commercial assessment and cultivar development.
  • conditioning and/or directed evolution steps are employed at one or more appropriate stage of the method in accordance with the invention described herein.

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Abstract

Cette invention concerne des procédés de criblage, d'identification et/ou d'application de micro-organismes pour conférer des propriétés bénéfiques à des plantes. Dans un mode de réalisation, le procédé implique la soumission d'une ou de plusieurs plantes à un milieu de croissance en présence d'un ensemble de micro-organismes, la sélection d'une ou de plusieurs plantes, l'acquisition d'un ou de plusieurs micro-organismes associés à la plante ou aux plantes sélectionnée(s) et la répétition du procédé une ou plusieurs fois. Le procédé implique en outre l'étape de soumission du ou des micro-organismes à un procédé de conditionnement et/ou d'évolution dirigée.
PCT/NZ2014/000045 2014-03-19 2014-03-19 Procédés de criblage visant à sélectionner des micro-organismes capables de conférer une propriété bénéfique à une plante WO2015142186A1 (fr)

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WO2017207752A1 (fr) * 2016-06-02 2017-12-07 Danstar Ferment Ag Composition et méthode pour améliorer le développement des plantes
FR3052021A1 (fr) * 2016-06-02 2017-12-08 Danstar Ferment Ag Composition et methode pour ameliorer le developpement des plantes
AU2017272860B2 (en) * 2016-06-02 2022-02-24 Danstar Ferment Ag Composition and method for improving the development of plants
US20200078841A1 (en) * 2017-04-27 2020-03-12 Khanh Le Microbial soil enhancements
CN113194728A (zh) * 2018-07-25 2021-07-30 明尼苏达大学董事会 用于开发土传植物病原体抑制微生物菌群的平台

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