RELATED APPLICATIONS

This application derives priority from New Zealand patent application number 717094 incorporated herein by reference.

TECHNICAL FIELD

Described herein are methods for enhancing plant tolerance to stress. More specifically, methods are described whereby a plant bio-stimulant composition is applied to plants that enhance plant tolerance to stress, the stress in selected embodiments being abiotic stress, transplantation and temperature stress.

BACKGROUND ART

When plants are grown in stress inducing conditions, such as reduced water content in the growth media, (due for example to a shortfall in rainfall or irrigation), high wind, excessive salt or high or low temperatures, or the act of transplantation; the plant can undergo stress which can lead to a deterioration or breakdown of physiological functions in the plant cells. This in turn can lead to decreased growth compared to similar plants grown under non-stress inducing conditions. Decreased plant growth and productivity in crop plants for example, may represent a significant financial loss to the grower.

Breeding of stress-tolerant crop cultivars can represent a successful strategy to minimise reduced plant growth under unfavourable growing conditions. Conventional breeding is however a slow process for generating new crop varieties with improved tolerance to stress conditions and new cultivars may have limitation around stability over successive plant generations.

Transgenic approaches have demonstrated that genetic overexpression of compatible, low molecular weight osmolytes such as sugar alcohols, special amino acids, and glycine betaine (Annu Rev Plant

Physiol 31: 149-190 1980; Science 217: 1214-1222 1982) may eventually lead to production of stress- tolerant plants. The use of genetically modified plant varieties however can also involve a long development period and may not be permitted or be difficult to commercialise in some countries due to the (perceived or otherwise) environmental risk.

The application of external individual chemical substances (often synthetically produced) such as phytohormones or plant growth regulators have been shown to reduce water stress such as drought stress or excessive moisture stress (Journal of Plant Growth Regulation (2010) 29: 366- 374). These chemical substances however may have other side effects which are not necessarily satisfactory in use plus they too may have perceived or otherwise environmental risks. As may be appreciated from the above, there remains a need for new methods for improving plant tolerance to stress which overcomes the disadvantages mentioned above. In addition there remains a need for new methods for improving plant tolerance to stress while obviating the negative effects of excessive use of nitrogen fertilisers applied to pasture. Excessive nitrogen fertiliser application may lead to an increase in the level of nitrates that are leached into groundwater and pollute waterways as well as lead to an increase in the level of denitrification and higher levels of nitrous oxide emissions (a potent greenhouse gas). Attenuating the amount of nitrogen applied to plants may help to address art problems.

It is an object of the method described herein to at least provide a useful alternative to other methods of improving plant tolerance to abiotic stress or at least to provide a useful alternative to the public.

Further aspects and advantages of the method will become apparent from the ensuing description that is given by way of example only.

SUMMARY

Described herein is a method of improving plant tolerance to stress by application of a plant bio- stimulant composition. The plant bio-stimulant composition may be applied in combination with urea and/or other agricultural compounds.

The inventors have found that a composition comprising a mixture of cellular components from at least one strain of bacteria which has been grown in a growth media to at least about 10 ^s cfu/ml and/or at least one strain of yeast which have been grown in the growth media to at least about 10 ^s cfu/ml, wherein the microorganisms may be lysed in the growth media, optionally diluted in a carrier (such as water) and applied to at least one plant or part thereof; can result in improved plant growth when the at least one plant is subjected to stress (such as abiotic stress) relative to at least one plant grown without stress.

In a first aspect, there is provided a method for enhancing plant tolerance to stress comprising a step of: applying, to at least one plant or part thereof, a composition comprising:

a mixture of lysed cellular components from at least one strain of bacteria which has been grown in a growth media to at least about 10 ^s cfu/ml and/or at least one strain of yeast which have been grown in a growth media to at least about 10 ^s cfu/ml.

In a second aspect, there is provided a method for enhancing plant tolerance to abiotic stress comprising a step of:

applying, to at least one plant or part thereof, a composition comprising:

a mixture of lysed cellular components from at least one strain of bacteria which has been grown in a growth media to at least about 10 ⁶ cfu/ml and/or at least one strain of yeast which have been grown in a growth media to at least about 10 ^s cfu/ml.

In a third aspect, there is provided a method of enhancing plant seedling tolerance to wilting during transplantation comprising a step of:

applying, to at least one plant or part thereof, a composition comprising:

a mixture of lysed cellular components from at least one strain of bacteria which has been grown in a growth media to at least about 10 ⁶ cfu/ml and/or at least one strain of yeast which have been grown in a growth media to at least about 10 ^s cfu/ml.

In a fourth aspect, there is provided a method of increasing the rate of pollination in flowering crops in ambient temperatures normally associated with reduced flowering and pollination comprising a step of: applying, to at least one plant or part thereof, a composition comprising:

a mixture of lysed cellular components from at least one strain of bacteria which has been grown in a growth media to at least about 10 ^s cfu/ml and/or at least one strain of yeast which have been grown in a growth media to at least about 10 ^s cfu/ml.

In a fifth aspect, there is provided a method of improving frost resistance and fruit set in ambient temperatures normally associated with reduced fruit set and frost damage comprising a step of:

applying, to at least one plant or part thereof, a composition comprising:

a mixture of lysed cellular components from at least one strain of bacteria which has been grown in a growth media to at least about 10 ⁶ cfu/ml and/or at least one strain of yeast which have been grown in a growth media to at least about 10 ⁵ cfu/ml.

An advantage of the above noted methods is the enhancement of plant growth in the face of various plant growth challenges/stress factors, examples stress factors including abiotic stress, temperature extremes and transplantation. As may be realised from the description below, the methods described are 'natural' or 'naturally produced' methods, the term 'natural' referring to the methods using naturally occurring biochemical metabolic pathways from naturally occurring organisms to produce the composition used in the methods. Synthetically produced chemicals are not used or applied to the plants nor is there the need to wait for plant breeding techniques (traditional or genetic modification) to achieve the desired results. The inventor has also realised additional advantages that will become apparent from the description below.

BRIEF DESCRIPTION OF THE DRAWINGS

Further aspects of the methods will become apparent from the following description that is given by way of example only and with reference to the accompanying drawings in which:

Figure 1 is a bar graph illustrating field testing results using the above described method for enhancing plant tolerance to abiotic stress of water potential of white clover plants grown under two different water regimes and three fertiliser levels (no added urea (control), urea applied at a rate of 20kg/ha (U) and urea applied at a rate of 20kg/ha plus (U+) the lysed broth as described herein). Error bars are ± SE;

Figure 2 is a bar graph illustrating field testing results measuring solute potential of white clover plants grown under two different water regimes and three fertiliser levels (no added urea (control), urea applied at a rate of 20kg/ha (U) and urea applied at a rate of 20kg/ha plus (U+) the lysed broth as described herein). Error bars are ± SE;

Figure 3 is a bar graph illustrating field testing results of adjusted solute potential of white clover plants grown under two different water regimes and three fertiliser levels (no added urea (control), urea applied at a rate of 20kg/ha (U) and urea applied at a rate of 20kg/ha plus (U+) the lysed broth as described herein). Error bars are ± SE;

Figure 4 is a bar graph illustrating field testing results of total plant dry mass of white clover plants grown under two different water regimes and three fertiliser levels (no added urea (control), urea applied at a rate of 20kg/ha (U) and urea applied at a rate of 20kg/ha plus (U+) the lysed broth as described herein). Error bars are ± SE;

Figure 5 is a bar graph illustrating field testing results of total aboveground dry mass of white clover plants grown under two different water regimes and three fertiliser levels (no added urea (control), urea applied at a rate of 20kg/ha (U) and urea applied at a rate of 20kg/ha plus (U+) the lysed broth as described herein). Error bars are ± SE;

Figure 6 is a bar graph illustrating field testing results of total leaf dry matter (DM) of white clover plants grown under two different water regimes and three fertiliser levels (no added urea (control), urea applied at a rate of 20kg/ha (U) and urea applied at a rate of 20kg/ha plus (U+) the lysed broth as described herein). Error bars are ± SE;

Figure 7 is a bar graph illustrating field testing results of leaf size of white clover plants grown under two different water regimes and three fertiliser levels (no added urea (control), urea applied at a rate of 20kg/ha (U) and urea applied at a rate of 20kg/ha plus (U+) the lysed broth as described herein). Error bars are + SE; and

Figure 8 is a bar graph illustrating field testing results of stolon elongation rate of white clover plants grown under two different water regimes and three fertiliser levels (no added urea (control), urea applied at a rate of 20kg/ha (U) and urea applied at a rate of 20kg/ha plus (U+) the lysed broth as described herein). Error bars are ± SE.

DETAILED DESCRIPTION

As noted above, described herein is a method of improving plant tolerance to stress by application of a plant bio-stimulant composition. The plant bio-stimulant composition may be applied in combination with urea and/or other agricultural compounds.

For the purposes of this specification, the term 'about' or 'approximately' and grammatical variations thereof mean a quantity, level, degree, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% to a reference quantity, level, degree, value, number, frequency, percentage, dimension, size, amount, weight or length.

The term 'substantially' or grammatical variations thereof refers to at least 50%, for example 75%, 85%, 95% or 98%.

The term 'comprise' and grammatical variations thereof shall have an inclusive meaning - i.e. that it will be taken to mean an inclusion of not only the listed components it directly references, but also other non-specified components or elements.

The term 'stress' or grammatical variations thereof refers interchangeably to plant stress, plant stress factors, challenges, or growth challenges that impede or halt plant growth from a normal rate of plant growth.

The term 'abiotic stress' or grammatical variations thereof refers to environmental factors that impede or halt plant growth comprising but not limited to: drought, soil salinity, air humidity, wind speed, soil flooding, soil nutrient deficiencies, or extremes of ambient temperature in which the plant grows at a rate that differs to a normal rate for the plant.

The term 'normal rate' or grammatical variations thereof refers to a reduction of plant growth of at least about 10% when compared to the growth of a plant without being subjected

The term 'lysing' or grammatical variations thereof refers to breaking down the membrane of the cell in a substantial portion of the bacteria and yeast to produce a mixture of cellular components.

In a first aspect, there is provided a method for enhancing plant tolerance to stress comprising a step of: applying, to at least one plant or part thereof, a composition comprising:

a mixture of lysed cellular components from at least one strain of bacteria which has been grown in a growth media to at least about 10 ^s cfu/ml and/or at least one strain of yeast which have been grown in a growth media to at least about 10 ^s cfu/ml.

In a second aspect, there is provided a method for enhancing plant tolerance to abiotic stress comprising a step of:

applying, to at least one plant or part thereof, a composition comprising:

a mixture of lysed cellular components from at least one strain of bacteria which has been grown in a growth media to at least about 10 ^s cfu/ml and/or at least one strain of yeast which have been grown in a growth media to at least about 10 ^s cfu/ml. The abiotic stress may be a water stress such as that caused by drought or lack of water/moisture over a period of time. For the purposes of the specification the phrase 'water stress' means an effective osmotic stress to the plant over an extended period of time such as reduced water levels in the soil or other growth media or a high level of solutes such as salt. Osmotic stress described herein may refer for example to a reduction in leaf water potential from normal levels for a well watered plant (e.g. around 0 to -1 MPa for well watered plants to less than -0.4 MPa and particularly less than -1 MPa. The term 'high level of solutes' refers to a salt stress inducing level higher than 100 mM (milli molar) NaCI, however, as may be appreciated, for some plants, 50 mM NaCI would induce plant stress while for other plants 200 mM NaCI would be a problem and salt tolerant plants can address higher solute levels.

As should be appreciated, the time period may vary between plant types, some plants being adapted to low or no water whilst others requiring regular water hence, the time period without water and when stress results may vary considerably.

In a third aspect, there is provided a method of enhancing plant seedling tolerance to wilting during transplantation comprising a step of:

applying, to at least one plant or part thereof, a composition comprising:

a mixture of lysed cellular components from at least one strain of bacteria which has been grown in a growth media to at least about 10 ⁶ cfu/ml and/or at least one strain of yeast which have been grown in a growth media to at least about 10 ^s cfu/ml.

As noted above, the stress may be wilting on transplantation and the composition may be applied to plant seedlings.

In a fourth aspect, there is provided a method of increasing the rate of pollination and flowering in plants in ambient temperatures normally associated with reduced flowering and pollination comprising a step of:

applying, to at least one plant or part thereof, a composition comprising:

a mixture of lysed cellular components from at least one strain of bacteria which has been grown in a growth media to at least about 10 ^s cfu/ml and/or at least one strain of yeast which have been grown in a growth media to at least about 10 ^s cfu/ml.

As noted above, the stress may be ambient temperatures normally associated with reduced flowering and pollination.

The ambient temperature in the above aspect may be at least 30 ^* C, or 31'C, or 32"C, or 33 ^" C, or 34 ^* C, or 35 ^° C for a sufficiently long time period to impede or halt the rate of pollination and flowering of the plant if the method were not completed.

In a fifth aspect, there is provided a method of improving frost resistance and/or fruit set in ambient temperatures normally associated with reduced fruit set and frost damage comprising a step of: applying, to at least one plant or part thereof, a composition comprising:

a mixture of lysed cellular components from at least one strain of bacteria which has been grown in a growth media to at least about 10 ^s cfu/ml and/or at least one strain of yeast which have been grown in a growth media to at least about 10 ^s cfu/ml.

As noted above, the stress may be ambient temperatures normally associated with reduced fruit set and/or frost damage.

The ambient temperature may be less than lS ^' C, or 14"C, or 13 ^' C, or 12 ^° C, or ll ^' C, or 10° C, or 9'C, or 8'C, or 7 ^' C, or 6 ^* C, or 5 ^' C, or 4 ^' C, or 3 ^° C, or 2 ^° C, or 1 ^" C, or 0 ^* C for a sufficiently long time period to impede or halt the rate of fruit set and/or cause frost damage to the plant if the method were not completed.

In summary, the composition may be produced by fermentation of a single species or combination of microorganisms comprising but not limited to lactic acid bacteria and/or yeasts that are then substantially lysed. Any microorganism or combinations of microorganisms capable of fermentation may be used. The fermentation process itself may involve growing the microbes in a growth media such as a liquid broth that includes carbohydrate and mineral sources for the microorganisms. Any fermentation media may be used, and many suitable media materials may be used as are well known in the art.

The bacteria may be selected from the genera group comprising: Lactobacillus, Streptococcus,

Propionibacter, and combinations thereof. The bacteria species may comprise but are not limited to at least one of: Lactobacillus plantarum, Streptococcus thermophilus (also called Streptococcus salivarius), Propionibacter freudenreichii, and combinations thereof. As further examples, the bacteria species may further comprise at least one of: Lactobacillus acidophilus, Lactobacillus buchneri, Lactobacillus johnsonii, Lactobacillus murinus, Lactobacillus paraplantarum, Lactobacillus pentosus, Lactobacillus delbrueckii, Lactococcus lactis, Leuconostoc oenos, Bifidobacter bifidus, Propionibacter shermani, Propionibacter pelophilus, and Propionivibrio limicola.

The yeast may be selected from the genera group comprising: Saccharomyces, Candida, Pichia, Hanseniaspora, Metschnikowia, Issatchenkia, Kloeckeral, and combinations thereof. In one embodiment, the yeast species may be Saccharomyces cerevisiae. As further examples, the yeast species may comprise at least one of: Saccharomyces pastorianus, Saccharomyces boulardii,

Saccharomyces bayanus, Saccharomyces exiguous, Saccharomyces pombe, as well as species of: Candida, Pichia, Hanseniaspora, Metschnikowia, Issatchenkia, Kluyveromyces, and Kloeckera.

The microorganisms may be grown in growth media to concentrations of at least about 10 ^s cfu/ml for the at least one species of yeast, at least about 10 ^s cfu/ml for the at least one bacterial species, or for both the bacteria and/or yeast, about 10 ⁷ cfu/ml, or about 10 ^s cfu/ml, or about 10 ⁹ cfu/ml, or about 10 ¹⁰ cfu/ml, or about 10 ⁿ cfu/ml, or about 10 ¹² cfu/ml, or about 10 ¹³ cfu/ml, or about 10 ¹⁴ cfu/ml.

The growth media may be a liquid growth media. The growth media may be a broth. Growing in the growth media may occur prior to lysing. This may be completed to maximise the number of cells prior to lysing.

The microorganisms may produce a range of growth promoting compounds including cytokinins, betaines, oligopeptides, and related compounds. Lysing as noted above breaks cell walls and releases cell wall and internal cell compounds. Cytokinin is a microbial and plant hormone responsible for promoting cell division and growth. Betaines are substances used by microbial and plant cells for protection against osmotic stress, drought, high salinity or high temperature. Oligopeptides are short chains of amino acids that improve nutrient uptake through cell membranes. Typically plants produce their own cytokinin so the fact that the plants can still respond to cytokinin-like stimulation was unexpected, particularly since production of endogenous plant cytokinin may be restricted when the plant is under stress. As is described further below, the compounds in the lysed growth media appear to confer various beneficial effects as described herein that are not readily apparent from the art.

As may also be appreciated, the compounds in the lysed composition are naturally occurring compounds - that is, they are produced based on normal biochemical pathways and are not synthetically produced or concentrated. In addition, a range of compounds are produced and not one specific compound increasing or mimicking nature plus allowing various natural synergies to occur. The nature of the composition described herein is entirely different to that of a single compound and is also entirely different to synthetically produced compounds.

The at least one strain of bacteria and/or at least one strain of yeast may be lysed in the growth media. That is, the cellular material is not separated from the growth media prior to lysing occurring.

Lysing as noted above may be achieved by various means. For example, lysing may be completed by steps such as freezing, heating, bead beating, high osmotic pressure from use of salts, detergents including non-ionic and zwitterionic detergents, low pH treatment including by hydrochloric, hydrofluoric and sulphuric acids, and high pH treatment including by sodium hydroxide. Also included is enzymatic lysis including but not limited to one or more of types of cellulase, glycanase, lysozyme, lysostaphin, mannase, mutanolysin, protease and zymolase enzymes. Included also is solvent treatment such as with sodium dodecyl sulphate treatment followed by acetone solvent use, or ultrasonic treatment. Further included are means which increase pressure followed by a rapid decrease in pressure such as is achievable with a pressure bomb, cell bomb, or with processors that provide high shear pressure such as valve type processors including but not limited to French pressure cell press or rotor-stator processors or fixed geometry fluid processors. As should be appreciated from the above, lysing may be completed using a wide variety of methods and the list provided above should not be seen as limiting.

Substantially all of the bacteria and/or yeast cells may be made non-viable by lysing. It should be noted however that the proportion of the cells in which membranes are broken down and the extent of break down may vary.

In one embodiment, the methods described further comprise the step of mixing the composition with a carrier before the mixture is applied to the at least one plant. The carrier may for example be water however; other liquid or dry carrier materials may be used such as an organic solvent like ethanol. Water is useful since it is biologically safe, easy to handle and is generally readily available.

Mixing with a carrier may be completed after lysing. This may be done to minimise the volume of material to be processed. Note however that carrier mixing could occur post lysing or part before and part after lysing.

Mixing with a carrier may occur prior to, or at, the time of application to the plant or plants. To minimise transport costs, it is envisaged that the composition would be transported and sold as a concentrate and diluted at the time of application. Dilution at the time of application may also be important so as to ensure that a safe or cost effective dose loading is applied to the plant or plants.

The composition may be applied to the foliar growth of at least one plant. For the purposes of the specification the term "foliar" refers to the leaves, stem, flowers and fruits of the plant. However, for seedlings or container plants, the composition may be applied to the seeds or roots via seed dips, root dips or seedling tray immersion. As may be appreciated, the concentration of the composition after mixing with a carrier noted above may be varied/increased in a dip/immersion step compared to foliar spraying to suit the specific plant growth requirements and degree of abiotic stress present or likely.

The composition used in the above methods may be applied to a plant or plants by various means, examples comprising: sprays, sprinklers, drips, dips, drenches, dressings, oils, and via any type of irrigation system. As non-limiting examples, the main administration methods encompass foliar sprays, turf sprays, in-furrow sprays, root dips, root drenches, stem drenches, seedling drenches, tuber drenches, fruit drenches, soil drenches, soil drips, and soil injections.

In alternative embodiments, the composition may be applied in dry form, e.g., granules, microgranules, powders, pellets, sticks, flakes, crystals, and crumbles.

The diluted or undiluted composition used in the above methods may be applied to the at least one plant or part thereof at a rate of at least: 0.1 litres (L) per hectare (ha). For example, at a rate of at least: 0.1 L/ha, or 0.2 L/ha, or 0.5 L/ha, or 1.0 L/ha, or 1.5 L/ha, or 3.0 L/ha, or 6.0 L/ha, or 10 L/ha.

In one embodiment, where the composition is diluted with a carrier and applied by spraying or irrigation, the lysed growth media may be diluted by a factor of at least one part growth media to at least 5, or 6, or 7, or 8, or 9, or 10 parts carrier by volume.

The methods may be completed as a preventative measure before stress conditions are present. For example, the composition may be applied at a time when soil temperatures are conducive to pasture or crop growth response. Despite the above, the methods may also be completed when stress conditions are already present.

As noted above, by completing the above described methods, the plant or part thereof to which the method is completed has great tolerance to plant stress or growth challenges. As may appreciated, a variety of measures of plant tolerance may be used however, by way of illustration, the methods described may achieve one or more of the following:

5-25% greater physiological water use potential;

20-25% more efficient photosynthetic carbon fixation per unit of water lost in transpiration; - A 15% or higher dry matter increase;

30% or lower water potential values;

15% or greater leaf solute potential;

A 5% or greater leaf size;

Leaf water potential to be over -1 MPa.

The compositions used in the above methods may also be combined with urea and/or other agricultural compounds. Using urea as an example, the inventor has unexpectedly found that the composition also acts to reduce the amount of urea fertiliser needed (nitrogen input) for the same growth response and the composition described appears to enhance plant uptake of nitrogen from the urea thereby reducing potential waste and nitrogen leeching from the ground or substrate in which the plants grow.

The level of reduction of fertiliser needed may be relative and plant dependent. For example, in clover based pasture, it may be possible to reduce the fertiliser required by at least 10%, or 20%, or 30%, or 40%, or 50% to achieve a similar response as that seen from urea application alone at a full dose.

Reducing by at least 10% would be meaningful in terms of cost and commercial advantage.

Other agricultural compounds besides urea may be selected from at least one of: fertilisers, foliar fertilisers, herbicides, insecticides, fungicides, or mineral solutions. Nitrogen containing agricultural compounds may be particularly relevant.

In one embodiment, the method may comprise dissolving urea in water and adding the above described composition to the urea solution to form the combined mixture applied to a plant or plants.

In embodiments where urea is used in the above methods, the concentration of urea as applied to the plant or plants may be at about: 0.025 kg/L, or 0.05 kg/L (the lowest two rates suitable for some plants with urea sensitive foliage), or 0.1 kg/L, or 0.12 kg/L, or 0.15 kg/L, or 0.18 kg/L, or 0.2 kg/L, or 0.22 kg/L, or 0.25 kg/L, or 0.28 kg/L, or 0.3 kg/L, or 0.35 kg/L, or 0.38 kg/L, or 0.4 kg/L, or 0.42 kg/L, or 0.45 kg/L, or 0.48 kg/L, or 0.50 kg/L. The urea concentration as applied to the plant or plants may be in a range of about 0.15 kg/L to about 0.25 kg/L, or about 0.18 kg/L to about 0.22 kg/L, or about 0.35 kg/L to about 0.45 kg/L, or about 0.38 kg/L to about 0.42 kg/L.

The plant or plants may be selected from crop plants. The plants may be selected from: soybean, wheat, corn, rice, potato, sugarcane, pumpkin, cassava, cotton, and combinations thereof.

The plant or plants may be selected from pasture crops. The plants may be selected from: grass (fescue, ryegrass or the like), pasture legumes (one example being clover), fodder brassicas (one example being kale), and combinations thereof. The plant or plants may be selected from flowering crop plants. The plants may be selected from: maize, rice, sorghum, wheat, cereals and combinations thereof.

Without being bound by theory, it is understood by the inventor that the presence of the lysed microbes and their compounds, when applied to a plant, may assist with the plant stress tolerance. The process of growing and then lysing the mixture appears to release these compounds in a bioactive state able to be utilised, at least in part, by the plants to which the mixture is applied. Further, the compounds released from lysing appear to stimulate plant growth and the plant immune system perhaps by assisting or supplementing the plant to achieve more efficient nutrient utilisation, stimulation of plant

photosynthesis, proliferation of the fine feeder roots and subsequent increased nutrient uptake. Note that other mechanisms may occur not yet measured or envisaged and the above explanation should not be seen as limiting but merely to give context and help understand one possible theory of action.

As may be appreciated from the above description, an advantage of the above noted methods is the enhancement of plant growth in the face of various plant growth challenges/stress factors, example stress factors including abiotic stress, temperature extremes and transplantation. As may be realised from the above description, the methods described are 'natural' or 'naturally produced' methods, the term 'natural' referring to the methods using naturally occurring biochemical metabolic pathways from naturally occurring organisms to produce the composition used in the methods. Synthetically produced chemicals are not used or applied to the plants nor is there the need to wait for plant breeding techniques (traditional or genetic modification) to achieve the desired results.

Among the above noted enhanced growth characteristics, the methods have also been found to encourage pasture legume (e.g. white clover) growth relative to perennial ryegrass in forage applications. The pasture legume growth may be at least 5%, or 10%, or 15%, or 20% greater than the ryegrass crop in experiments completed by the inventor. This relative growth difference may have benefits because of the high feed value of white clover and the importance of root nodules of this plant in fixing atmospheric nitrogen so that more nitrogen is available for use by the plant itself and other pasture plants.

Further, the methods described have in the inventors experience reduced the amount of nitrogen fertiliser that is needed to achieve a desired rate of growth with the method described appearing to enhance plant nitrogen utilisation even in the face of stresses and challenges.

The embodiments described above 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, and any or all combinations of any two or more said parts, elements or features.

Further, where specific integers are mentioned herein which have known equivalents in the art to which the embodiments relate, such known equivalents are deemed to be incorporated herein as of individually set forth. WORKING EXAMPLES

The above described methods are now described by reference to specific examples.

EXAM PLE 1

A greenhouse experiment was completed utilising the lysed composition with dissolved urea fertiliser to assess the effect of the method on white clover plants under simulated drought stress compared to a control and urea only application.

Introduction: The field trial's objective was to identify if the above described method of using the lysed composition applied at low rates sprayed on white clover, would affect plant response to drought stress. Biomass production and drought stress parameters were measured. Low rates of application were used to set a reasonable challenge for the above described methods - naturally greater rates could be used to enhance the method results as desired/required.

Methodology: Plants of the white clover cultivar Grasslands Kopu II were grown in pots in a factorial, split plot design for nine weeks. For the fertiliser treatments, urea was applied as a foliar spray at a rate of 0 (no urea being a control in Figure 1), 20kg urea/ha (Urea or U in Figure 1) and 20kg urea/ha plus the lysed broth composition at a water diluted rate of 1.5 L/ha (Urea plus composition or U+ in Figure 1). Simulated drought conditions were applied during the last six weeks of the experiment. Measurements included a number of morphological, physiological parameters, as well as traits of relevance for plant feed value and nutrition.

Plants were exposed to two water treatments and three fertiliser treatments in a 2 x 3 factorial, split plot design. Pots were arranged in five blocks, each subdivided into two subplots, containing either well- watered or drought treatments. The three fertiliser treatment pots in each subplot were re-randomised every week.

The high-yielding white clover (Trifolium repens L.) cultivar Grasslands Kopu II was chosen for this study White clover was chosen for the trial as this plant is a major component of pasture systems in many countries providing nitrogen fixation and good feed quality for grazing animals. A further reason for choosing white clover in comparison to grass is the more limited root system of clover which makes it more prone to drought stress than grasses. Further, in drought conditions, it is white clover growth that typically suffers most before grass growth suffers hence the trial sets a significant challenge to the method tested. Finally, white clover is a representative plant in terms of (like most plants), not being a high salt tolerant or strong drought tolerant plant.

The white clover plants were exposed to well-watered or drought conditions for the purposes of the trial. Well-watered treatment was daily irrigation to 2% below field capacity of the soil. Drought treatment plants were irrigated daily to 2% above permanent wilting point of the soil.

Plants were grown in 8.5 L pots (5 seedlings per pot) and allowed to establish for 2 ½ months. The soil medium consisted of 75 % Wakanui silt loam, sourced from the Horticultural Research Area at Lincoln University and 25 % mortar sand (0.1-3 mm diameter). The soil medium also contained 2 g/L Osmocote Exact™, a 3-4 months slow-release fertiliser (N-P-K 16-5.0-9.2 + 1.8 mg trace elements + 1.8% Mg), as well as 1 g/L Hydraflo™ wetting agent and 30 g/L gypsum to prevent excessive cracking of the soil under subsequent drought conditions.

The total duration of the study was nine weeks. Fertiliser treatments were supplied by leaf spray application at the beginning of the experiment, and three weeks later, before the onset of the drought treatment. Fertiliser levels were 0 (control), 20kg/ha urea and 20kg/ha urea plus the lysed composition. Soil water levels were determined gravimetrically. Plants in the well-watered treatment were irrigated daily to 2% below field capacity of the soil. In the drought treatment, plants were irrigated daily to 2% above permanent wilting point of the soil.

A number of traits of plant morphology (Table 1) and physiology (Tables 2 and 3) were assessed with details as shown below.

Table 1. Whole plant morphology, yield and leaf morphology traits investigated.

mass (per leaf) unfolded leaves per pot, oven-dried @ 80"C for 48 hrs

Specific leaf area cm ² Ratio of lamina area / lamina dry weight of two randomly chosen young, (SLA) mg ¹ fully unfolded leaves per pot

Specific leaf mass mg Ratio of lamina dry weight / lamina area of two randomly chosen young, (SLM) cm fully unfolded leaves per pot

% leaf dry mass % % ratio of lamina dry weight / lamina fresh weight of two randomly (PDM) chosen young, fully unfolded leaves per pot, calculated as 100 * DM/FM

Petiole length Cm Determination of the average petiole length of two randomly chosen young, fully unfolded leaves per pot

Leaf appearance d ^"1 Two young, fully unfolded leaves were marked in each pot at the onset rate of the drought treatment. Determination of the number of fully

unfolded leaves from the marked leaf towards the tip of the shoot

Stolon elongation mm Two young, fully unfolded leaves were marked in each pot at the onset rate d ^"1 of the drought treatment. Determination of the elongation rate of the corresponding white clover stolons per pot

Table 2. Water relations traits investigated.

Trait Unit Materials & Methods

Water potential MPa Determination of V _w of a young, fully unfolded leaf per pot with a pressure bomb (Soil moisture co., US)

Osmotic potential MPa Osmotic potential was determined in a Wescor Osmometer, using this equation: <F _S =-RTc _j

• Where RT = 0.002437 m ³ MPa-mol ^"1 (constant value at 20°C room temperature), and q is the total solute concentration or osmolality (measured by the osmometer in mol-kg ^'1 )

Relative water % Determination of the average value of two randomly chosen young, fully content (RWC) unfolded leaves per pot:

• Lamina fresh mass (FM)

• Lamina turgid mass (TM, after saturation in water overnight)

• Lamina dry mass (DM, after oven-drying @ 80°C for 48 hrs)

Formula: RWC (%) = 100 * [(FM-DM)/(TM-DM)] Adjusted osmotic MPa OA was calculated as the difference in adjusted osmotic potential ¾ooi potential V _s(10 oj between control and stressed plants

and osmotic • The formula for calculating adjusted osmotic potential is:

adjustment (OA)

Ψ _% ( WC-RWCa)

fs (loo) ~

1-RWCa

• Where RWCa is the correction factor for dilution by apoplastic water. This is an estimated value and is thought to be approximately 0.1 for most plants in most situations

Table 3. Photosynthetic and gas exchange traits investigated.

Statistical Analysis: Data analysis was performed with the GENSTAT General AN OVA Procedure.

Results: The statistical summaries of morphological and physiological observations (Table 4 below) consist of five columns. These columns list the traits investigated and provide information on the statistical significance of the treatment factors (drought, fertiliser and the interaction of drought and fertiliser). The fifth column in Table 4 shows the fertiliser effect, when it was dependent on water availability. The latter is based on the interaction LSD at P < 0.05 from ANOVA, which can still reveal significant fertiliser effects in dependence on water treatments, even though the overall drought x fertiliser interaction term may be non-significant (e.g. total leaf DM, Table 4) (Saville, 2003).

There were several treatment interactions, showing that, in some cases, the fertiliser effect was dependent on water availability. Compared to drought-exposed plants receiving no urea, plants receiving U+ increased leaf dry matter production and total aboveground dry matter production by 16% (Figures 5 and 6), while leaving root production unaffected. Under drought, this is likely to result in a larger draw on the limited water resource, and was reflected in the collected data by 27% lower water potential values in U+-grown, drought-exposed plants, when compared with drought-exposed plants receiving no fertiliser (Figure 1). Under well-watered conditions, this effect of U+ was not observed.

Also in contrast to well-watered plants, there was a trend for both fertiliser treatments to result in osmotic adjustment (OA) under drought: the drought x fertiliser interaction term was marginally significant for adjusted solute potential (Table 1, Figure 3). It is possible that this was facilitated by a positive fertiliser effect on the production of osmotically active compatible solutes under drought.

The drought x fertiliser interaction term was also marginally significant for increased physiological water use efficiency (WUE, Table 1 and Figure 4). Thus, plants exposed to drought and receiving fertiliser showed 20% more efficient photosynthetic carbon fixation per unit of water lost in transpiration. This effect was even more pronounced in drought-exposed plants that received the U+ treatment, showing 25% higher WUE (Figure 4). Higher physiological WUE may have contributed towards the increased aboveground dry matter production in drought-exposed white clover plants receiving U+ (Figure 5).

Leaf size was reduced in plants exposed to drought and was reduced significantly further by 6% with the application of urea alone (Figure 7). The 6% reduction was avoided in the U+ treatment. Leaf size is important for photosynthetic capacity of plants and greater leaf size is aligned with greater photosynthetic capacity and hence greater plant tolerance to stress.

Compared to drought conditions, fewer fertiliser treatment effects could be observed under well- watered conditions. However, it was notable that under well-watered conditions, the U+ treatment increased stolen elongation rate (SER) by 27% (Figure 8), highlighting the growth-promoting attributes of the lysed composition for stem elongation. Both solute potential measurements increased in response to the fertiliser treatments. Unadjusted and adjusted leaf solute potential (Figures 2 and 3) showed increases of 15% and 18% respectively in well-watered plants treated with U+ when compared to well- watered plants receiving no fertiliser. This could suggest an increased turnover of cellular solutes in response to fertiliser application.

Table 4. Statistical summary for morphological and physiological observations. Treatments and effects: DR Drought; WW Well-watered; U Urea; U+ Urea+Lysed Composition ;† Significant increase; /

Significant decrease; Ns Non-significant.

Fertiliser effect,

Drought dependent on x water

Trait Drought Fertiliser Fertiliser availability ¹

Leaf water potential 4,78%, P < 0.001 Ns P = 0.082 DR: U+ 27%

Leaf solute potential 29%, P < 0.01 Ns P = 0.08 WW: U 8% and U+ l4%

WW: U l8%

Adjusted leaf solute potential 4-17%, P < 0.05 Ns P = 0.05 and U+ l8%

Total plant DM 4,29%, P < 0.01 Ns Ns

Total aboveground DM 4-34%, P < 0.001 Ns Ns DR: U+ l6%

Total leaf DM 4,40%, P < 0.01 Ns Ns DR: U+ l6%

Stolon DM 4, 16%, P < 0.05 Ns Ns

Root DM Ns Ns Ns

Root:shoot ratio Ή8%, P < 0.0S Ns Ns

Leaf size 4,40%, P < 0.05 Ns Ns

U 4-6%, P

Specific leaf mass ^• T-18%, P < 0.01 = 0.08 P = 0.094

U 6%, P

Specific leaf area 16%, P < 0.01 = 0.05 P = 0.075

Petiole length Ns Ns Ns

Relative leaf water content 4,8%, P < 0.05 Ns Ns

Leaf damage †15%, P = 0.1 Ns Ns

WW: U+

Stolon elongation rate 4,35%, P < 0.05 Ns P = 0.077 t27%

Leaf appearance rate 4,23%, P < 0.05 Ns Ns

Photosynthesis Ns Ns Ns

Conductance Ns Ns Ns

Transpiration Ns Ns Ns

DR: U -T-20%

Water use efficiency Ns Ns P = 0.089 and U+ 25%

SPAD 1^21%, P < 0.05 Ns Ns

Based on the drought x fertiliser interaction LSDs at P < 0.05

Aspects of the methods have been described by way of example only and it should be appreciated that modifications and additions may be made thereto without departing from the scope of the claims herein.