EP3723474A1 - Endophytes from wild populations of barley increase crop yield - Google Patents
Endophytes from wild populations of barley increase crop yieldInfo
- Publication number
- EP3723474A1 EP3723474A1 EP18830412.5A EP18830412A EP3723474A1 EP 3723474 A1 EP3723474 A1 EP 3723474A1 EP 18830412 A EP18830412 A EP 18830412A EP 3723474 A1 EP3723474 A1 EP 3723474A1
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- EP
- European Patent Office
- Prior art keywords
- seed
- endophytes
- plant
- cladosporium
- seq
- Prior art date
- Legal status (The legal status 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 status listed.)
- Pending
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Classifications
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- 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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/30—Microbial fungi; Substances produced thereby or obtained therefrom
Definitions
- This invention relates to the uses of isolated and cultured endophytes from wild populations of barley.
- the invention relates to isolated and cultured endophytes from wild populations of barley who are vertical transmission (VT) competent and use of those VT competent endophytes in a method for improving dry shoot weight, mean dry grain weight, and suppression of seed-borne infection in a crop by inoculation with at least one or a combination of the isolated VT competent endophytes.
- VT vertical transmission
- Endophytes are one group of bioactive agents that have shown the greatest promise. Endophytes are microorganisms that spend all or part of their lives inside plant tissue without causing disease symptoms, they have great potential as beneficial crop inoculants (Murphy et al. 2014). While much of the experimental work with endophytes has focused on a relatively limited range of endophytic species, endophytes recovered from wild relatives of crops have been less studied and offer a largely untapped and vast potential for improving agronomic traits in crop cultivars (Murphy et at. 2015; Murphy et at.
- Crop wild relatives represent a valuable repository of symbiotic microorganisms which can be beneficial for modern crop relatives (Murphy et al. 2014a). Genes from crop wild relatives have proved to be of great utility to breeders who wish to mine this resource for useful genetic traits that will improve crop performance, and there is every reason to believe that the diversity and functional traits of endophytes in crop wild relatives will be just as useful.
- the added benefit of using microbial crop inoculants is that they are easier to apply and develop compared to the often expensive and lengthy process of developing a genetically modified (GM) product, and they may also be more acceptable to regulatory authorities and the general public in jurisdictions where GM crops are not widely grown.
- GM genetically modified
- Endophytes isolated and cultured from wild plant populations have proved to be extremely diverse, and these are often relatively novel and previously undescribed organisms with unknown potential as crop inoculants, even within a single host species (Murphy et at. 2015b).
- Previous work by the inventors has demonstrated that these endophytes can enhance barley yield and other agronomic traits under a range of stressful environments, and they have recently demonstrated that these beneficial results translate to field-grown barley (Murphy et at. 2017b).
- Those endophytes are applied to crops as seed-coating compositions and cannot be passed on to the next generation of seeds produced by the plant sprouted from those coated seeds.
- endophytes are described as playing a role in increasing grain yield and dry shoot weight in plants, and improving tolerance to development of seed-borne fungal infections on germinated and ungerminated seeds.
- endophytes are described as playing a role in plant biomass production and yield in plants such as barley, and also enhances tolerance to environment stresses.
- the patent only claims increases of seed germination vigour and the fresh weight and yield of seedlings, but not the mature plants or seed (i.e. grain in the case of cereals).
- compositions are described which increased seed vigour and growth.
- the compositions are organic wax coatings used to coat the seeds, which enhance growth and vigour in monocots by acting as a protectant from environmental stress. The document makes no specific mention of using endophytes as a component of the coating.
- Endophytes isolated from natural populations of plants stimulate an increase in cultivated crop yield without the use of chemical inputs (such as nitrogenous fertilisers, manures) when inoculated onto seeds of a crop cultivar.
- a formulation containing spores of the endophyte(s) is applied to the crop cultivar seed before sowing.
- the formulation increases crop yield (such as grain dry weight) and biomass, and promotes biotic and abiotic stress resistance in crops.
- the endophytes of the claimed invention were isolated from the seeds of wild barley ( Hordeum murinum), not the root tissue of the plant as is the source of endophytes accepted by those working in the field.
- the source of the endophytes shows that they have seed tissue specific localisation, which is a trait shown by agents who have vertical transmission properties.
- a seed coating composition comprising a fungal endophyte isolated from a tissue of a plant, such as Hordeum murinum, and a carrier medium for application of said composition to a target seed, wherein at least one of the fungal endophytes is vertical transmission competent.
- the fungal endophyte is isolated using primers defined by SEQ ID NOs 1 and 2.
- the endophyte is characterised as having a nuclear ribosomal internal transcribed spacer (nrlTS) selected from SEQ ID NOs: 3 to 12, or variants thereof, or a combination thereof.
- nrlTS nuclear ribosomal internal transcribed spacer
- the endophyte is characterised as having a nuclear ribosomal internal transcribed spacer (nrlTS) selected from SEQ ID NOs: 3, 6, 1 1 or 12, or variants thereof, or a combination thereof.
- nrlTS nuclear ribosomal internal transcribed spacer
- a seed coating composition comprising one or more fungal endophytes isolated from a seed or a husk of the seed of Hordeum murinum and a carrier medium for application of said composition to a target seed, wherein the fungal endophytes are vertical transmission competent, and in which the endophytes are characterised as being selected from an Alternaria sp., a Penicillium sp., or a Cladosporium sp. and having a nuclear ribosomal internal transcribed spacer (nrlTS) selected from SEQ ID NOs: 3, 4, 5, 10, 1 1 or 12.
- the endophytes are characterised as being Alternaria alternata, Penicillium brevicompactum , Cladosporium ramontenellum, or Cladosporium herbarum.
- the one or more endophytes selected are isolated from the seed and are characterised as being Cladosporium herbarum and having a nuclear ribosomal internal transcribed spacer (nrlTS) defined by SEQ ID NOs: 1 1 or 12.
- the composition further comprises one or more endophytes isolated from the husk of the seed, and in which the endophytes are characterised as being Penicillium brevicompactum , Cladosporium ramontenellum or Alternaria alternata and having a nuclear ribosomal internal transcribed spacer (nrlTS) selected from SEQ ID NOs: 3, 4 or 5.
- the composition further comprises one or more fungal endophytes isolated from a tissue of Hordeum murinum other than the seed or the seed husk, in which the endophyte is characterised as having a nuclear ribosomal internal transcribed spacer (nrlTS) selected from SEQ ID NOs: 6 to 9 and selected from a Penicillium sp., a Cladosporium sp., a Humicola sp., or a Talaromyces sp..
- the endophyte is selected from Penicillium glabrum, Cladosporium ramontenellum, Humicola grisea, and/or Talaromyces purpurogenus.
- the composition comprises endophytes characterised as having a nuclear ribosomal internal transcribed spacer (nrlTS) selected from SEQ ID NOs: 6, 8, 1 1 and 12, and classified as being Cladosporium herbarum, Humicola grisea, and/or
- nrlTS nuclear ribosomal internal transcribed spacer
- a seed coating composition comprising one or more fungal endophytes isolated from a tissue of a plant, such as Hordeum murinum, and a carrier medium for application of said composition to a target seed, wherein at least one of the fungal endophytes is vertical transmission competent; in which the endophyte is characterised as having a nuclear ribosomal internal transcribed spacer (nrlTS) selected from SEQ ID NOs: 3 to 12.
- nrlTS nuclear ribosomal internal transcribed spacer
- the one or more endophytes are classified as Alternaria alternata, Penicillium brevicompactum, Penicillium glabrum, Cladosporium ramontenellum, Cladosporium herbarum, Humicola grisea, and/or
- the composition comprises the endophytes are characterised as having a nuclear ribosomal internal transcribed spacer (nrlTS) selected from SEQ ID NOs: 6, 8, 1 1 and 12, and classified as being Cladosporium herbarum, Humicola grisea, and/or Talaromyces purpurogenus.
- nrlTS nuclear ribosomal internal transcribed spacer
- a seed coating composition comprising one more fungal endophytes isolated from a root of Hordeum murinum and a carrier medium for application of said composition to a target seed, and in which the endophyte is characterised as having a nuclear ribosomal internal transcribed spacer (nrlTS) selected from SEQ ID NOs: 6, 7, 8 or 9.
- the one or more endophyte is Alternaria alternata, Penicillium brevicompactum , Penicillium glabrum, Cladosporium ramontenellum, Cladosporium herbarum, Humicola grisea, and/or Talaromyces purpurogenus.
- the plant sprouted from the coated target seed exhibits a trait selected from one or more of an increased grain dry weight, an increase in the number of heads, an increase number of grains and shoot dry weight.
- the plant is Hordeum murinum subsp. murinum L (Hmm).
- the plant Hordeum murinum is in low-nutrient conditions.
- the plant Hordeum murinum is in drought-stressed conditions. In one embodiment, the plant Hordeum murinum is in light-stressed conditions. In one embodiment, the plant Hordeum murinum is in temperature-stressed conditions. In one embodiment, the Hordeum murinum plant is in multiple-stressed conditions.
- the plant sprouted from the coated target seed exhibits an increased grain weight, an increase in the number of heads, an increase number of grains and shoot dry weight.
- the concentration of the fungal endophyte isolate in the composition is between 0.001% (w/v) to 1.0% (w/v).
- the carrier medium is selected from water, distilled water, sterilised water, sterilised distilled water, an emulsified suspension, wettable powder, encapsulation of spores of the fungal endophyte in alginate beads, or a film-coating.
- a plant grown from a seed coated with a seed-coating composition comprising a fungal endophyte isolated from a tissue of the plant Hordeum murinum and a carrier medium for application of said composition to a target seed, wherein at least one of the fungal endophytes is vertical transmission competent, and wherein the plant grown from the coated seed comprises at least one feature selected from the group consisting of (i) an endophyte isolated using primers defined by SEQ ID NOs 1 and 2; (ii) an endophyte characterised in having a nuclear ribosomal internal transcribed spacer (nrlTS) selected from SEQ ID NOs: 3 to 12, or variants thereof, or a combination thereof.
- nrlTS nuclear ribosomal internal transcribed spacer
- the one or more endophytes are Alternaria alternata, Penicillium brevicompactum , Penicillium glabrum, Cladosporium ramontenellum, Cladosporium herbarum, Humicola grisea, and/or Talaromyces purpurogenus.
- the plant grown from the coated seed exhibits a trait selected from disease resistance, drought tolerance, heat tolerance, cold tolerance, salinity tolerance, tolerance to a nutrient-deficient environment, increase in biomass, increase in crop yield, increase in seed weight, an enhanced seed oil composition, an increase in photosynthetic efficiency, an increase in growth rate, an increase in root mass.
- the plant grown from the coated seed is from a grass.
- the grass is a cereal crop selected from barley ( Hordeum vulgare), wheat ( Triticum aestivum), oats ( Avena sativa), maize ( Zea mays), rye ( Secale cereale), spelt ( Triticum spelta), rice ( Oryza sativa), millet ( Panicum miliaceum, Eleusine coracana, Setaria italica, Pennisetum glaucum), sorghum ( Sorghum bicolor), triticale (x Triticosecale), teff ( Eragrostis tef), fonio ( Digitaria exilis), wild rice ( Zizania spp.), canary grass ( Phalaris sp.), quinoa ( Chenopodium quinoa), amaranth ( Amaranthus spp.), buckwheat ( Fagopyrum esculentum).
- barley Hordeum vulgare
- wheat Triticum aestiv
- a seed comprising one or more fungal endophytes selected from a Cladosporium strain, a Penicillium strain, a Humicola strain or a Talaromyces strain, or a combination thereof, and wherein the endophytes are originally isolated from a tissue of the plant Hordeum murinum.
- the endophytes are isolated using primers defined by SEQ ID NOs 1 and 2.
- the endophyte is characterised as having a nuclear ribosomal internal transcribed spacer (nrlTS) according to any one of SEQ ID NOs: 3 to 12, or variants thereof.
- a seed comprising one or more fungal endophytes selected from an Alternaria strain, a Cladosporium strain, a Penicillium strain, a Humicola strain or a Talaromyces strain, and wherein the endophytes are originally isolated from a tissue of the plant Hordeum murinum, and are characterised as having a nuclear ribosomal internal transcribed spacer (nrlTS) selected from SEQ ID NOs: 3 to 12.
- nrlTS nuclear ribosomal internal transcribed spacer
- the one or more endophytes are Alternaria alternata, Penicillium brevicompactum , Penicillium glabrum, Cladosporium ramontenellum, Cladosporium herbarum, Humicola grisea, and/or Talaromyces purpurogenus.
- the endophyte is characterised as having a nuclear ribosomal internal transcribed spacer (nrlTS) selected from one of SEQ ID NOs: 3, 6, 8, 1 1 or 12, or variants thereof, or a combination thereof.
- nrlTS nuclear ribosomal internal transcribed spacer
- the carrier medium is alginate beads.
- a method of producing a plant from a target seed comprising the steps of coating the target seed with the composition described above; planting the coated seed in an appropriate medium; growing the plant from the seed and harvesting a crop from the plant.
- the medium is a soil-based medium, an artificial growth medium, moss peat and moss peat mixes, or mixes thereof of all medium.
- the soil- based medium is a low-nutrient soil.
- the seed would be planted in a field of soil using standard agricultural equipment. In one embodiment, where the method is for controlled environmental use, the seed is manually planted at a depth of 30-40mm in the chosen medium.
- a method of increasing one or more traits selected from grain dry weight, the number of heads, the number of grains per head, and shoot dry weight in a plant grown from a target seed comprising applying a composition comprising a fungal endophyte isolated from a tissue of the plant Hordeum murinum and a carrier medium for application of said composition to the target seed; planting the coated target seed in an appropriate medium and allowing the plant to grow.
- the endophytes are isolated using primers defined by SEQ ID NOs 1 and 2.
- the endophyte is characterised as having a nuclear ribosomal internal transcribed spacer (nrlTS) according to any one of SEQ ID NOs: 3 to 12, or variants thereof.
- nrlTS nuclear ribosomal internal transcribed spacer
- the endophyte is characterised as having a nuclear ribosomal internal transcribed spacer (nrlTS) selected from one of SEQ ID NOs: 3, 6, 8, 1 1 or 12, or variants thereof, or a combination thereof.
- nrlTS nuclear ribosomal internal transcribed spacer
- a method of increasing one or more traits selected from grain dry weight, the number of heads, the number of grains per head, and shoot dry weight in a plant grown from a target seed comprising applying a composition comprising a fungal endophyte isolated from a seed or a husk of the seed of the plant Hordeum murinum and a carrier medium for application of said composition to the target seed, wherein the fungal endophytes are vertical transmission competent, and in which the endophytes are characterised as being selected from an Alternaria sp. a Penicillium sp., or a Cladosporium sp. and having a nuclear ribosomal internal transcribed spacer (nrlTS) selected from SEQ ID NOs: 3, 4, 5, 10, 1 1 or 12; planting the coated target seed in an appropriate medium and allowing the plant to grow.
- nrlTS nuclear ribosomal internal transcribed spacer
- the medium is soil, an artificial growth medium, moss peat and moss peat mixes, or mixes thereof of.
- the soil is a low-nutrient soil.
- an endophyte characterised as having a nuclear ribosomal internal transcribed spacer (nrlTS) according to any one of SEQ ID NOs: 3 to 12, or variants thereof.
- the endophyte is isolated from a tissue of the plant Hordeum murinum.
- the endophytes are isolated using primers defined by SEQ ID NOs 1 and 2.
- an endophyte isolated from Hordeum murinum and characterised as having a nuclear ribosomal internal transcribed spacer (nrlTS) according to any one of SEQ ID NOs: 3 to 12 and in which the endophyte is Alternaria alternata, Penicillium brevicom pactum , Penicillium glabrum, Cladosporium ramontenellum, Cladosporium herbarum, Humicola grisea, and/or Talaromyces purpurogenus.
- nrlTS nuclear ribosomal internal transcribed spacer
- the fungal endophyte is isolated from a tissue of the plant Hordeum murinum.
- the tissue can be the root, seed, seed husk or other appropriate parts of the plant, such as a shoot, leaf tissue and meristem.
- the fungal endophyte is isolated from a root of the plant Hordeum murinum.
- the fungal endophyte is isolated from a seed husk of the plant Hordeum murinum.
- the root endophyte is isolated from a seed of the plant Hordeum murinum.
- the plant is Hordeum murinum subsp. murinum L. (Hmm).
- the fungal endophyte is selected based on having a nuclear ribosomal internal transcribed spacer selected from one of SEQ ID NOs 3 to 12, or variants thereof, or any combination thereof.
- the fungal endophyte is selected based on having a nuclear ribosomal internal transcribed spacer with at least 90% sequence identity to any one of SEQ ID NOs 3 to 12, or any combination thereof.
- the term“ruderal” should be understood to mean a plant species that is first to colonize disturbed lands.
- the disturbance may be natural or a consequence of human activity.
- the term“isolated endophyte” should be understood to mean fungal emergents from tissue of the plant Hordeum murinum, which have been inoculated onto culture medium and subsequently individually sub-cultured from single spores or mycelia to obtain a single uncontaminated organism.
- the tissue is a root piece, a seed or a seed husk of the plant.
- the plant is Hordeum murinum subsp. murinum L. ( Hmm ).
- the term “variants thereof”, should be understood to mean polynucleotides sequences which are substantially identical to the full nuclear ribosomal internal transcribed spacer of an isolated endophyte.
- the term should be taken to include polynucleotides that are altered in respect of one or more nucleic acids.
- such alterations involve the insertion, addition, deletion and/or substitution of 15 or fewer nucleic acids, more preferably of 12 or fewer, even more preferably of 9 or fewer, most preferably of 3 or 6 nucleic acids only.
- the variant may have conservative nucleic acid changes, wherein the nucleic acid being introduced does not change the structure or activity of the translated nucleic acid sequence.
- any sequence which has been altered by substitution or deletion of catalytically-important nucleic acids will be excluded from the term“variant”.
- the variant will have at least 70% sequence identity, preferably at least 80% sequence identity, more preferably at least 90% sequence identity, and ideally at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the wild-type nuclear ribosomal internal transcribed spacer of the isolated endophyte as depicted in SEQ ID NOs 3 to 12; and wherein the variant is capable of increasing crop yield (such as grain yield/grain dry weight) and/or dry shoot weight and/or biomass and/or tolerance to the development of seed-borne fungal infections on germinated and ungerminated seeds and/or can have its genetic material moved by the vertical transmission of DNA from a parent plant (via plant grown from a coated target seed) to its offspring.
- the endophytes are passed from F 0 to Fi to F 2 and future generation of plants
- film-coated should be understood by those skilled in the art to mean a thin polymer-based coat applied to a solid form such as a seed or a tablet.
- the coating can be aqueous or non-aqueous based.
- the thickness of such a coating is usually between 20-100 pm.
- Film coating formulations generally comprise a polymer (for example, cellulose derivatives or acrylic polymers and copolymers such as-Hypromellose,-Hydroxyethyl cellulose, Hydroxyethyl methyl cellulose, Carboxymethylcellulose sodium, Hydroxypropyl cellulose, Polyethylene glycol, ethyl cellulose, ⁇ Hypromellose phthalate, Polyvinyl acetate phthalate, Cellulose acetate phthalate, ⁇ Polymethacrylates, -Shellac), a plasticiser (such as, for example, glycerol, propylene glycol, PEG (Polyethylene glycol), phthalate esters, dibutyl sebacete, citrate esters, triacetin, castor oil, acetylated, monoglycerides, fractionated coconut oil) and a carrier (generally, water or organic solvents such as alcohols, ketones, esters and chlorinated hydrocarbons). Water is the preferable carrier.
- a carrier
- the term“light-stressed” conditions should be understood to mean where the seeds and plants have been stressed by either too much or too little light.
- a control‘ideal’ light intensity would be about 420 pmol nr 2 s 1 at leaf level; so less than this is low light-stressed and greater than this is high light-stressed.
- temperature-stressed conditions should be understood to mean where the seeds and plants have been stressed by temperatures under 5 °C) or over 25 °C). This is in controlled conditions where availability of water and uptake by the plant can be controlled. The degree of temperature stress is related to the availability of water and its uptake in the plant, and the atmospheric humidity.
- multiple-stressed should be understood to mean where plants have been stressed by a number of factors such as one or more of drought, nutrient-depleted or nutrient-poor soils, low or too much light, pathogen infection, cold and heat.
- the term“vertically transmitted infection” or“vertical transmission competent” as applied to an endophyte means that the endophyte when coated on to a seed of a parent plant is capable of vertical transmission from the coated seed of the parent plant (F 0 ) to the next generation of seeds (Fi).
- Examples include the endophytes having a wild-type nuclear ribosomal internal transcribed spacer as depicted in SEQ ID NOs 3 to 12, or variants thereof which are also vertical transmission competent. A test for identifying an endophyte capable of vertically transmitted infection is described below.
- Figure 1 illustrates the inferred evolutionary history of the five Cladosporium strains using the Maximum Parsimony method.
- the consistency index is 0.855634 (0.677165)
- the retention index is 0.405797 (0.405797)
- the composite index is 0.347214 (0.274792) for all sites and parsimony-informative sites (in parentheses).
- Yield-enhancing endophyte strains are outlined in bold dash.
- Hordeum murinum subsp. murinum L. ( Hmm ) is an annual grass species and a ruderal of roadsides, rough grassland and waste places. As the species generally grows in stressed environments, it may have evolved symbiotically-conferred stress tolerance associated with endophyte infection, and these endophytes may help the plants to grow in stressed conditions. Endophytes isolated from Hmm may have the potential to benefit cultivated barley in low-nutrient, drought-stressed and multiple-stressed conditions.
- Roots, seeds and seed husks were separated from whole plants and surface-sterilised by immersing in 70% EtOH for one minute, then placed in 5% NaCIO for 5 minutes, immersed for another minute in 70% EtOH then rinsed five times with sterile water.
- Ten root pieces of 5 mm length and 10 halved seeds and halved husks from each plant were inoculated onto culture plates of potato dextrose agar (Oxoid CM0139) and incubated in the dark at 21 °C for 28 days.
- the powdered medium was mixed to half- strength of the manufacturers’ recommendations (to avoid osmotic shock to the endophytes) using pure water, then sterilised by autoclaving.
- RNA nuclear ribosomal DNA (rDNA) internal transcribed spacer (ITS) primers ITS1 (forward primer) (SEQ ID NO: 1 - 5’
- the PCR reaction contained 1 mI of DNA extract (ca. 20 ng mI-1 ), 5 mI of 5x buffer (Promega), 0.5 mI of 10 mM dNTPs, 0.25 mI at 20 pmol, 0.25 mI of each primer at 20 pmol mI-1 , 2 mI of 25 mM MgCI2, and 0.125 mI of Go Taq Flexi DNA Polymerase (Promega).
- Thermal cycling in an Applied Biosystems Proflex® thermal cycler included a premelt of 94 ' ⁇ for 30 seconds followed by 32 cycles of 94 ' ⁇ for 2 mins, 57 °C for 1 min and 72‘Q for 1 min, followed by a final extension of 7 mins at 72 q C.
- a further PCR was carried out to increase DNA yield and the products were sent for sequencing to an outside service supplier (Macrogen Europe, Netherlands).
- the full nuclear ribosomal internal transcribed spacer (nrlTS) sequences for the ten endophyte isolates can be found as SEQ ID NOs: 3 to 12, as follows: SEQ ID NO. 3 >161024-038_G12_195EZAA186. abl 1973
- Every recovered sequence was combined into a matrix and analysed to detect recombination events using the RAT Recombination Analysis Tool, which uses the distance-based method of recombination detection.
- Window size was set to 93 characters (0.1 of sequence length) and Increment size was 46 characters (0.5 of Window size).
- the evolutionary history of the five Cladosporium strains was inferred using the Maximum Parsimony (MP) method.
- the MP tree was obtained using the Subtree- Pruning-Regrafting (SPR) algorithm with search level 0 in which the initial trees were obtained by the random addition of sequences (10 replicates).
- SPR Subtree- Pruning-Regrafting
- the analysis involved 5 nucleotide sequences. All positions containing gaps and missing data were eliminated. There were a total of 1015 positions in the final dataset. Evolutionary analyses were conducted in MEGA7 (Kumar et al. 2016).
- F 0 seeds seeds from the original seeds or seeds coated with the composition described herein (termed‘F 0 seeds’) and subsequent generations are sampled.
- the steps of the test are: sample F 0 seeds for endophyte presence, sub-culture recovered endophytes from single-spores, extract and sequence nrlTS gene and compare with the nrlTS sequences described herein. If they are similar (at least 90% identical), then the isolated endophytes are vertical transmission competent.
- the F 0 seeds are planted and grown, the seeds produced from the plant generated from the F0 seeds (F1 seeds) are tested as described above. If they are similar (at least 90% identical), then the isolated endophytes are vertical transmission competent. This confirms that the endophyte in the seed-coating composition has been passed on from the F 0 generation to the Fi generation, that is, the endophyte is vertical transmission competent.
- Seeds of the barley cultivar Propino (Goldcrop Ltd., Cork, Ireland) were soaked in warm water for 3 hours. Three seeds per pot were sown into John Innes No. 2 compost (Westland Garden Health) at 20 mm depth into 3 litre plastic pots. Five pots per treatment were sown and the pots were labelled with a reference number (reference numbers were anonymous to the plant grower). Seeds were inoculated with either 200 mI (-200,000 spores/seed) of the endophyte inoculant (10 individual strains and one combination of all strains) or 200 mI of pure water for the controls, and the seeds covered with the compost. Pots were randomly distributed in two blocks in the glasshouse and repositioned every week during the experimental period.
- the barley plants were harvested after Zadoks growth stage 89 (Zadoks et at. 1974) was reached (maturity). The number of tillers, number of heads and number of grains per plant were recorded. After drying to 15% moisture content the grain and shoot dry weight were measured.
- the barley plants can also be considered as somewhat moisture-stressed at times as they were only watered when the growing compost reached a moisture level of 15%, and similarly, previous work has also shown endophyte-associated benefits for moisture-stressed barley (Murphy et al. 2015b).
- Singh and Kapoor (1999) studied the effect of inoculation with the AMF Glomus sp.88 and two phosphate (P043-)-solubilizing microorganisms (PSM), Bacillus circulans and Cladosporium herbarum, in the presence or absence of rock phosphate in a natural P- deficient sandy soil on wheat crops.
- PSM phosphate-solubilizing microorganisms
- Bacillus circulans and Cladosporium herbarum in the presence or absence of rock phosphate in a natural P- deficient sandy soil on wheat crops.
- the significant increase in grain and straw yields due to inoculation with the consortia could be attributed to a high absorption of nutrients.
- a seed dressing combining fungal endophyte spores and fungicides improves seedling survival and early growth in barley and oat. Symbiosis 71 (1 ):69-76.
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Abstract
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Application Number | Priority Date | Filing Date | Title |
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EP17207710 | 2017-12-15 | ||
PCT/EP2018/084472 WO2019115582A1 (en) | 2017-12-15 | 2018-12-12 | Endophytes from wild populations of barley increase crop yield |
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EP3723474A1 true EP3723474A1 (en) | 2020-10-21 |
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CN (1) | CN111801005A (en) |
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US10813359B2 (en) * | 2013-11-06 | 2020-10-27 | The Texas A & M University System | Fungal endophytes for improved crop yields and protection from pests |
CA2959736A1 (en) | 2014-08-29 | 2016-03-03 | Brian Murphy | A method for improving the mean dry shoot weight, mean dry grain weight, and suppressing seed-borne infection in a cereal crop |
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BR112020012028A2 (en) | 2020-11-24 |
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