SEQUENCE LISTING

The instant application contains a Sequence Listing with 10 sequences which has been submitted via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Apr. 12, 2017 named 35212_US_CRF_sequencelisting.txt, and is 17,111 bytes in size.

PRIORITY CLAIM

This application is the National Stage of International Application No. PCT/AU2015/000360, filed on Jun. 19, 2015, published in English under PCT Article 21(2), which claims the benefit of and priority to Australian Patent Application Serial No. 2014902374, filed on Jun. 20, 2014, the contents of which are incorporated by reference in their entirety.

FIELD

The present invention relates to methods for enhancing at least one growth parameter of a leguminous plant via co-inoculation of a leguminous plant with at least one rhizobial microorganism together with at least one actinobacterial microorganism. In further aspects, the present invention also relates to leguminous plants co-inoculated with at least one rhizobial microorganism together with at least one actinobacterial microorganism, as well as specific actinobacterial strains and inoculant compositions which are useful in accordance with the present invention.

BACKGROUND

There are around 44 to 66 million tonnes of nitrogen fixed from atmospheric nitrogen by symbiosis of rhizobial microorganisms and legumes every year, which is nearly half of the nitrogen used in agriculture around the world.

The interaction of legume and rhizobia is highly specific and each rhizobial species has a distinct range of leguminous plant hosts for forming nodules and fixing nitrogen. Different steps of the nodulation process requires the exchange of various signals between host legumes and rhizobia.

Actinobacteria are a large group that includes different genera of Gram-positive bacteria with a high G-C content in their DNA. Actinobacteria are widely distributed in terrestrial environments and some, like the nitrogen-fixing symbiontsFrankia, are known to form associations with plants through symbiotic relationships. Recent studies have also found that endophytic actinobacteria produced plant growth-promoting compounds such as indole-3-acetic acid (IAA) and siderophores.

Effects of actinobacteria on rhizobia and symbiosis with legumes have been noticed but not many studies have investigated this complex combination, although several studies have suggested antagonism occurring between actinobacteria and rhizobia.

For example, Antoun et al. (Canadian Journal of Microbiology24: 558-562, 1978) disclosed antagonism tests between actinobacteria isolated from different soils and effective strains of rhizobia. These data demonstrated that some actinobacteria inhibit the growth of rhizobia in vitro and in planta. Specifically, thirty one percent of the 481 actinobacteria investigated inhibited two efficient rhizobia strains,Rhizobium melilotiA2 and S14.

In a further study, Damirgi and Johnson (Agronomy Journal58: 223-224, 1966) disclosed that the number of nodules on soybeans inoculated withRhizobium japonicumstrains 122 and 123 in autoclaved soil were reduced by up to 35% and 53%, respectively, by treatment with the actinobacterium E8. They also isolated about 60 actinobacteria from one soil sample where there had been poor nodulation of clovers. However, 20 of 24 actinobacteria isolated from an experimental soybean field did not inhibit eight sensitiveR. japonicumstrains in in vitro tests.

Antagonism was also examined between actinobacteria and 12 strains of rhizobia from five soil samples by Patel (Plant and Soil41: 395-402, 1974). It was reported that about 23-70% of the actinobacteria inhibited the rhizobia strains.

In light of the above, identification of actinobacterial inoculants that are compatible with rhizobial microorganisms and that can enhance the growth and development of leguminous plants would be desirable.

DESCRIPTION

Nucleotide and amino acid sequences are referred to herein by a sequence identifier number (SEQ ID NO:). A summary of the sequence identifiers is provided below:

Sequence IdentifierDescriptionSEQ ID NO: 127f primer nucleotide sequenceSEQ ID NO: 21465r primer nucleotide sequenceSEQ ID NO: 3LuP3 16S rRNA gene nucleotide sequenceSEQ ID NO: 4LuP12A 16S rRNA gene nucleotide sequenceSEQ ID NO: 5LuP30 16S rRNA gene nucleotide sequenceSEQ ID NO: 6LuP47B 16S rRNA gene nucleotide sequenceSEQ ID NO: 7EN23 16S rRNA gene nucleotide sequenceSEQ ID NO: 8EN27 16S rRNA gene nucleotide sequenceSEQ ID NO: 9LuP8 16S rRNA gene nucleotide sequenceSEQ ID NO: 10LuP44 16S rRNA gene nucleotide sequence

A sequence listing is also provided at the end of the specification.

In a first aspect, the present invention provides a method for enhancing at least one growth parameter of a leguminous plant, the method comprising co-inoculating the leguminous plant with:at least one rhizobial microorganism; andat least one actinobacterial microorganism;

wherein the co-inoculated leguminous plant has at least one enhanced growth parameter relative to a leguminous plant of the same taxon that has not been co-inoculated.

An “actinobacterial microorganism” as referred to herein should be understood to include any microorganism of the phylum Actinobacteria.

In some embodiments the actinobacterial microorganism is an endophytic actinobacterial microorganism. An “endophytic” actinobacterial organism should be understood to include any actinobacterial organism that is able to live within a plant for at least a part of its lifecycle without causing apparent disease. In some embodiments, an endophyte may also be able to complete its lifecycle in the absence of a plant host, and thus only be an opportunistic endophyte. In some embodiments, an endophytic actinobacterial microorganism refers to an actinobacterial microorganism which may be isolated from surface-sterilised healthy plant tissue. For reference, an example of isolation of actinobacterial endophytes from surface sterilised plant tissue is set out in Coombs & Franco (Appl. Environ. Micro.69(9): 5603-5608, 2003).

In some embodiments, the actinobacterial microorganism is from a genus selected fromStreptomyces, MicrobisporaorMicromonospora.

In some embodiments, the actinobacterial microorganism is from the genusStreptomyces.

In some embodiments, the actinobacterial microorganism is from a species selected from:Streptomyces drozdowiczii;Streptomyces ciscaucasicus;Streptomyces canus;Streptomyces rishiriensis;Streptomyces badius; orStreptomyces parvus

In some embodiments, the actinobacterial microorganism is from a species selected fromStreptomyces ciscaucasicus, Streptomyces canusorStreptomyces rishiriensis.

In some embodiments, the actinobacterial microorganism comprises a 16S rRNA gene nucleotide sequence which is at least 90% identical to one or more of: SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6; SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and/or SEQ ID NO: 10.

In some embodiments, the actinobacterial microorganism comprises a 16S rRNA gene nucleotide sequence which is at least 90% identical to one or more of SEQ ID NO: 5 and/or SEQ ID NO: 6.

In some embodiments the actinobacterial microorganism comprises a 16S rRNA gene nucleotide sequence which is at least 90%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.1%, at least 98.2% at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99%, at least 99.1%, at least 99.2% at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% at least 99.9% or 100% sequence identity to a comparison window of one or more of: SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and/or SEQ ID NO: 10.

When comparing nucleic acid sequences to calculate a percentage identity, the compared nucleic acid sequences should be compared over a comparison window of, for example, at least 100 nucleotide residues, at least 300 nucleotide residues, at least 600 nucleotide residues, at least 1000 nucleotide residues, at least 1100 nucleotide residues, at least 1200 nucleotide residues, at least 1300 nucleotide residues or at least 1400 nucleotide residues. In some embodiments, the comparison window may comprise the region in each of the compared nucleotide sequences between and including the binding sites of the 27f primer (SEQ ID NO: 1) and the 1465r primer (SEQ ID NO: 2) on the compared nucleotide sequences.

The comparison window may comprise additions or deletions (ie. gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by computerized implementations of algorithms such as the BLAST family of programs as, for example, disclosed by Altschul et al. (Nucl. Acids Res.25: 3389-3402, 1997). A detailed discussion of sequence analysis can be found in Unit 19. 3 of Ausubel et al. (Current Protocols in Molecular Biology, John Wiley & Sons Inc, 1994-1998, Chapter 15, 1998).

A number of particularly useful actinobacterial microorganisms of the present invention have been deposited in accordance with the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.

The first deposited microorganism, referred to herein asStreptomycessp. LuP30, has been deposited at the National Measurement Institute (NMI), Australia on 12 Dec. 2013 under accession number V13/030101.

Accordingly, some embodiments, the actinobacterial microorganism isStreptomycessp. LuP30 as deposited with the National Measurement Institute, Australia under accession number V13/030101; or a mutant or derivative of said microorganism that retains the ability to enhance at least one growth parameter of a leguminous plant when the actinobacterial microorganism co-inoculated onto a leguminous plant with with a rhizobial microorganism.

The second deposited microorganism, referred to herein asStreptomycessp. LuP47B, has been deposited at the National Measurement Institute (NMI), Australia on 12 Dec. 2013 under accession number V13/030100.

Accordingly, in some embodiments, the actinobacterial microorganism isStreptomycessp. LuP47B as deposited with the National Measurement Institute, Australia under accession number V13/030100; or a mutant or derivative of said microorganism that retains the ability to enhance at least one growth parameter of a leguminous plant when the actinobacterial microorganism is co-inoculated onto a leguminous plant with a rhizobial microorganism.

In addition, in accordance with the present invention, two previously known organisms were also surprisingly identified to be particularly useful in accordance with the method of the present invention:

In some embodiments the actinobacterial microorganism isStreptomycessp. EN23 as described in PCT publication WO/2005/003328 and deposited as AGAL Deposit No. NM03/35605, or a mutant or derivative of said microorganism that retains the ability to enhance at least one growth parameter of a leguminous plant when the actinobacterial microorganism is co-inoculated onto a leguminous plant with a rhizobial microorganism.

In some embodiments the actinobacterial microorganism isStreptomycessp. EN27 as described in PCT publication WO/2005/003328 and deposited as AGAL Deposit No. NM03/35606, or a mutant or derivative of said microorganism that retains the ability to enhance at least one growth parameter of a leguminous plant when the actinobacterial microorganism is co-inoculated onto a leguminous plant with a rhizobial microorganism.

A “mutant or derivative” of the subject actinobacterial microorganisms should be understood to encompass, for example, any spontaneous or induced mutant, conjugation progeny or genetically modified form of the deposited strains which retains the ability to enhance at least one growth parameter of a leguminous plant when the actinobacterial microorganism is co-inoculated onto a leguminous plant with a rhizobial microorganism.

Mutagenisation techniques that may be used to generate derivatives or mutants include, for example, chemical mutagenesis (eg. EMS mutagenesis), ionising radiation-induced mutagenesis (eg. X-ray mutagenesis, γ-ray mutagenesis and UV mutagenesis), genetic insertion mutagenesis methods (eg. transposon mutagenesis) and the like.

As set out above, the present invention contemplates a method for enhancing at least one growth parameter of a leguminous plant.

A “growth parameter” of a leguminous plant, as referred to herein, can include any measurable characteristic of the leguminous plant.

In some embodiments, the growth parameter is a length and/or mass of a shoot of the leguminous plant.

In some embodiments, the growth parameter is a length and/or mass of a root of the leguminous plant.

In some embodiments, the growth parameter is a number and/or mass of nodules of the leguminous plant.

In some embodiments, the growth parameter is a number and/or mass of seed pods and/or seed produced by the leguminous plant.

In some embodiments, the growth parameter is a concentration and/or amount of a nutrient in the leguminous plant.

In some embodiments, the nutrient is selected from: Boron, Calcium, Copper, Magnesium, Manganese, Phosphorous, Sodium, Sulphur, Nitrogen and/or Zinc.

The concentration and/or amount of the nutrient may be measured using any method known in the art to be suitable for the relevant nutrient. Such methods may include, for example, the methods described by: Kirsten (Organic Elemental Analysis—Ultramicro, Micro and Traces Methods. Academic Press, New York, 1984); Horwath (Instrumental Organic Analylsis. Academic Press, New York, 1977); Colombo and Giazzi (American Laboratory38-45, 1982); Fraisse and Schmidt (J. Microchem.22: 109-130, 1977); Hegedus (Microchim. Acta441-446, 1977); and Baur and Dirscherl (Microchim. Acta1: 299-244, 1980).

In some embodiments, the nutrient is Nitrogen.

In some embodiments, the growth parameter is a germination rate of a leguminous plant seed. In some embodiments, “germination rate” may refer to the proportion of seeds of a leguminous plant that successfully germinate. In some embodiments “germination rate” may refer to a speed of germination of a leguminous plant seed, and/or a proportion of seeds of a leguminous plant that successfully germinate per unit time, for example the proportion of seeds of a leguminous plant that successfully germinate per 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 17, 18, 19 or 20 days.

The “germination rate” of a leguminous plant seed may be assessed using any suitable laboratory based or field based method, as would be readily ascertained by those skilled in the art.

As set out above, the present invention contemplates “enhancement” of the one or more growth parameters of the leguminous plant. “Enhancement” of a growth parameter should be understood to include any improvement in a growth parameter in a co-inoculated leguminous plant relative to a leguminous plant of the same taxon that has not been co-inoculated in accordance with the method of the present invention.

In some embodiments, enhancement of a growth parameter will include an increase in the measured value of the growth parameter. For example, an increase in any of:a length and/or mass of a shoot;a length and/or mass of a root;a number and/or mass of nodules;a number and/or mass of seed pods and/or seed;a concentration and/or amount of a nutrient; ora germination rate.

should be considered an enhancement of such growth parameters.

In some embodiments, enhancement of a growth parameter may comprise enhancement within a particular time period. For example, in some embodiments, enhancement of the growth parameter may comprise enhancement over a time period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90 or 100 days.

In some embodiments, an “increase” in a growth parameter may include, for example, a 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 5-fold, 10-fold, 20 fold, 50-fold, 100-fold increase in the growth parameter in a co-inoculated leguminous plant relative to a leguminous plant of the same taxon that has not been co-inoculated.

In some embodiments, however, “enhancement” of the growth parameter may include a decrease in the measured value of the growth parameter. For example a decrease in the concentration and/or amount of a pathogen, disease symptom and/or toxin in the plant, and or a decrease in the time of germination of a leguminous plant seed, may be considered “enhancement” of such growth parameters.

In some embodiments, an “decrease” in a growth parameter may include, for example, a 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% decrease in the growth parameter in a co-inoculated leguminous plant relative to a leguminous plant of the same taxon that has not been co-inoculated.

As set out above, the present invention contemplates co-inoculating the leguminous plant with at least one rhizobial microorganism and at least one actinobacterial microorganism.

A “rhizobial microorganism” as referred to herein may include any microorganism that is capable of fixing nitrogen after becoming established in a root nodule of a leguminous plant.

Rhizobial microorganisms are a paraphyletic group that generally fall into two classes of the proteobacteria, the alpha- and beta-proteobacteria. Most rhizobial microorganisms belong to the order Rhizobiales, but several rhizobia occur in distinct bacterial orders of the proteobacteria.

Examples of rhizobial microorganisms include:Bradyrhizobiumspp., such asB. canariense, B. elkanii, B. japonicum, B. liaoningenseandB. yuanmingense;Ochrobactrumspp., such asO. cytisiandO. lupini;Azorhizobiumspp., such asA. caulinodansandA. doebereinerae;Devosiaspp., such asD. neptuniae;Methylobacteriumspp., such asM. nodulans;Mesorhizobiumspp., such asM. albiziae, M. amorphae, M. chacoense, M. cicero, M. huakuii, M. loti, M. mediterraneum, M. plurifarium, M. septentrionale, M. temperatum, andM. tianshanense;Phyllobacteriumspp., such asP. ifriqiyense, P. leguminum, andP. trifoli;Rhizobiumspp., such asR. cellulosilyticum, R. daejeonense, R. etli, R. galegae, R. gallicum, R. giardinii, R. hainanense, R. huautlense, R. indigoferae, R. leguminosarum, R. loessense, R. lupini, R. lusitanum, R. mongolense, R. miluonense, R. sullae, R. tropici, R. undicolaandR. yanglingense;Sinorhizobiumspp. such asS. abri, S. adhaerens, S. americanum, S. arboris, S. fredii, S. indiaense, S. kostiense, S. kummerowiae, S. medicae, S. meliloti, S. mexicanus, S. morelense, S. saheli, S. terangaeandS. xinjiangense;Ensiferspp.;Burkholderiaspp., such asB. caribensis, B. dolosa, B. mimosarum, B. phymatumandB. tuberum,Cupriavidusspp., such asC. taiwanensis; andHerbaspirillumspp., such asH. lusitanum.

In some embodiments, the rhizobial microorganism is aRhizobiumspp., aSinorhizobiumspp. orEnsiferspp., or aBradyrhizobiumspp.

In some embodiments, the rhizobial microorganism is of the speciesSinorhizobium melilotiorSinorhizobium medicae. In some embodiments, the rhizobial microorganism is selected from the list of:S. melilotistrain RRI128 (referred to hereafter as ‘RRI128’),Sinorhizobiumstrain SRDI736 (referred to hereafter as ‘SRDI736’) orS. medicaestrain WSM1115G (referred to hereafter as WSM1115G′).

In some embodiments, the rhizobial microorganism is of the speciesRhizobium leguminosarum. In some embodiments, the rhizobial microorganism is selected from the list of:R. leguminosarumbv.viciae(referred to hereafter as ‘WSM1455’) orR. leguminosarumbv.trifolii(referred to hereafter as WSM1325′).

In some embodiments, the rhizobial microorganism is of the speciesBradyrhizobiumsp.lupiniorBradyrhizobium japonicum. In some embodiments, the rhizobial microorganism is selected from the list of:Bradyrhizobiumsp.lupinistrain WSM471 (referred to hereafter as ‘WSM471’) orBradyrhizobium japonicumstrain CB1809 (referred to hereafter as ‘CB1809’).

A range of rhizobial microorganisms are available from a range of commercial culture collections, as would be readily ascertained by those skilled in the art. In relation to a range of the rhizobial microorganisms described herein, these organisms can be accessed from therhizobiumculture collection of the South Australian Research & Development Institute (Plant Research Centre, Hartley Grove, Urrbrae SA 5064, Australia; www.sardi.sa.gov.au).

As set out above, the present invention contemplates a method for enhancing at least one growth parameter of a leguminous plant.

A “leguminous plant” as referred to herein should be understood as any member of the Fabaceae (or Leguminosae) that can form nodules when infected with a rhizobial microorganism.

Examples of leguminous plants include:Medicagospp., such asMedicago sativa(also referred to as lucerne or alfalfa);Pisumspp., such asPisum abyssinicum(syn.P. sativumsubsp.abyssinicum),Pisum fulvum, Pisum sativum, Pisum sativumsubsp.elatius(syn.P. elatius, P. syriacum) andPisum sativumsubsp.sativum;Glycinespp., such asGlycine max, Glycine albicans, Glycine aphyonota, Glycine arenaria, Glycine argyrea, Glycine canescens, Glycine clandestine, Glycine curvata, Glycine cyrtoloba, Glycine falcate, Glycine gracei, Glycine hirticaulis, Glycine hirticaulissubsp.leptosa, Glycine lactovirens, Glycine latifolia, Glycine latrobeana, Glycine microphylla, Glycine montis-douglas,Glycine peratosa, Glycine pescadrensis, Glycine pindanica, Glycine pullenii, Glycine rubiginosa, Glycine stenophita, Glycine syndetika, Glycine tabacina, Glycine tomentellaandGlycine soja;Cicerspp., such asCicer arietinum,Viciaspp., such asV. fabs;Vignaspp., such asV. aconitifolia, V. angularis, V. mungo, V. radiate, V. subterranean, V. umbellattaorV. unguiculataLathyrusspp., such asLathyrus sativusorLathyrus tuberosus,Lensspp., such asL. culinarisLablabspp., such asL. purpureus;Phaseolusspp., such asP. acutifolius, P. coccineus, P. lunatus, P. vulgaris, P. polyanthusorP. dumosus;Psophocarpusspp., such asP. tetragonolobus;Cajanusspp., such asC. cajan;Stizolobiumspp.;Cyamopsisspp., such asC. tetragonoloba;Canavaliaspp., such asC. ensiformisorC. gladiata;Macrotylomaspp., such asM. uniflorum;Lupinusspp., such asL. mutabilisorL. albus, orErythrinaspp., such asE. herbacea.Trifoliumspp., such asTrifolium subterraneum

In some embodiments, the leguminous plant is aMedicagospp. plant. In some embodiments, the leguminous plant is aMedicago sativa, lucerne or alfalfa plant.

In some embodiments, the leguminous plant is aTrifoliumsp. In some embodiments, the leguminous plant is aTrifolium subterraneumplant.

In some embodiments, the leguminous plant isPisumsp. In some embodiments, the leguminous plant is aPisum sativumplant.

In some embodiments, the leguminous plant is aGlycinesp. In some embodiments, the leguminous plant is aGlycine maxplant.

As set out above, the present invention contemplates co-inoculating a leguminous plant with a rhizobial microorganism and an actinobacterial microorganism. As referred to herein, “co-inoculating” should be understood to include any method or process wherein a leguminous plant (including a leguminous plant seed) is brought into contact with a rhizobial microorganism and an actinobacterial microorganism. In some embodiments co-inoculation may comprise the rhizobial microorganism and/or actinobacterial microorganism being applied to a leguminous plant seed. Examples of leguminous plant seed inoculation are described by Hartley et al. (Crop and Pasture Science63: 858-865, 2012). In some embodiments co-inoculation may comprise the rhizobial microorganism and/or actinobacterial microorganism being applied to soil in which a leguminous plant is growing. In some embodiments, co-inoculation may comprise the rhizobial microorganism and/or actinobacterial microorganism being applied to root and/or shoot tissue of a leguminous plant.

In some embodiments, “co-inoculating” may also comprise where the actinobacterial microorganism or rhizobial microorganism is pre-existing in the environment (eg. soil) into which a leguminous plant is grown. For example, co-inoculation may comprise application of an actinobacterial microorganism to a plant or soil and wherein a natural or pre-existing rhizobial microorganism in the soil co-inoculates the plant.

In some embodiments, the leguminous plant is exposed to a pathogen and, when exposed to the pathogen, the co-inoculated leguminous plant has at least one enhanced growth parameter relative to a leguminous plant of the same taxon that has not been co-inoculated.

In some embodiments, the pathogen is a root pathogen. A “root pathogen” as referred to herein should be understood to include any leguminous plant pathogen that infects and/or damages the roots of the leguminous plant. Examples of root pathogens of leguminous plants include fungal, oomycete, bacterial and/or nematode pathogens.

In some embodiments, the root pathogen is a nematode root pathogen such as aPratylenchusspp., such asP. neglectusorP. thornei.

In some embodiments, the root pathogen is an oomycete root pathogen such as aPhytophthoraspp., such asP. sojae.

In some embodiments, the pathogen is a fungal pathogen. Leguminous plants may be subject to attack by a range of fungal pathogens, including fungal root pathogens. Examples of such pathogens includeRhizoctoniaspp.,Pythiumspp. orAphanomycesspp.

In some embodiments, the pathogen is aRhizoctoniasp. In some embodiments, the pathogen isRhizoctonia solani. In some embodiments the pathogen isRhizoctonia solaniAGB.

In a second aspect, the present invention provides a leguminous plant, a leguminous plant part or leguminous plant reproductive material, co-inoculated with at least one rhizobial microorganism and at least one actinobacterial microorganism.

The leguminous plant contemplated in the second aspect of the invention may be any leguminous plant as hereinbefore described with reference to the first aspect of the invention.

Reference herein to a plant, plant part or plant reproductive material should be understood to encompass tissues, organs, whole organisms and parts thereof. In some embodiments, the term plant, plant part, or plant reproductive material should also be understood to encompass multicellular aggregations of cultured cells such as colonies, plant calli, suspension cultures and the like.

In some embodiments, the leguminous plant part or plant reproductive material may include a leguminous plant seed. As referred to herein, a plant “seed” should be understood to refer to a mature or immature plant seed. As such, the term “seed” includes, for example, immature seed carried by a maternal plant or seed released from the maternal plant. In some embodiments, the term “seed” may encompass any seed plant sporophyte between the developmental stages of fertilisation and germination.

In some embodiments, the actinobacterial microorganism contemplated in accordance with the second aspect of the invention is an actinobacterial microorganism as hereinbefore described with reference to the first aspect of the invention.

In some embodiments, the rhizobial microorganism contemplated in accordance with the second aspect of the invention is a rhizobial microorganism as hereinbefore described with reference to the first aspect of the invention.

In a third aspect, the present invention provides an isolated actinobacterial microorganism as deposited with the National Measurement Institute, Australia under accession number V13/030101; or a mutant or derivative of said microorganism that retains the ability to enhance at least one growth parameter of a leguminous plant when the actinobacterial microorganism is co-inoculated onto a leguminous plant with a rhizobial microorganism.

In a fourth aspect, the present invention provides an isolated actinobacterial microorganism as deposited with the National Measurement Institute, Australia under accession number V13/030100; or a mutant or derivative of said microorganism that retains the ability to enhance at least one growth parameter of a leguminous plant when the actinobacterial microorganism is co-inoculated onto a leguminous plant with a rhizobial microorganism.

In some embodiments, the present invention provides any of the hereinbefore described isolated actinobacterial microorganisms when used according to the method of the first aspect of the invention.

In a fifth aspect, the present invention also provides an inoculant composition comprising an actinobacterial microorganism.

In some embodiments, the actinobacterial microorganism comprises an actinobacterial microorganism as hereinbefore described with respect to the first aspect of the invention.

In some embodiments, the inoculant composition further comprises a rhizobial microorganism. In some embodiments, the rhizobial microorganism comprises a rhizobial microorganism as hereinbefore described with respect to the first aspect of the invention.

In some embodiments, the inoculant composition comprises a carrier or additive. The carrier or additives used will depend on the nature of the inoculant composition. For example, the inoculant composition may be in the form of a liquid composition, a solid composition (such as a powder, pellet or granular composition) a seed coating or the like.

The inoculant compositions of the present invention may be adapted to be applied to a leguminous plant in any suitable way. For example, the inoculant composition could be adapted to be applied as a seed coating, applied as a solid or liquid composition to the foliage or roots of a plant, or applied as a solid or liquid composition to soil before, during or after sowing of a leguminous plant.

A range of useful carriers or additives would be readily apparent to those of skill in the art and may include, for example: one or more gums (including xanthan gum), clay or peat based carriers, one or more nutrients including carbon or nitrogen sources, one or more antifungal or antibacterial agents, one or more seed coating agents, one or more wetting agents and the like.

In some embodiments, the present invention provides the hereinbefore described inoculant composition when used according to the method of the first aspect of the invention.

The present invention is further described with reference to the following non-limiting examples:

