Improvements in and relating to treatment methods and treatment compositions for decreasing / preventing growth of fungus, Hymenoscyphus fraxineus and

Abstract

A treatment composition comprising jasmonic acid (JA) and salicylic acid (SA) effective in inhibiting the growth of the fungus Hymenoscyphus fraxineus (Ash dieback, Chalara fraxinea) in vivo and preve

Description

The Common Ash tree (Fraxinus Excelsior) is a tree that grows in Ireland, the United Kingdom and in Europe. The Common Ash tree (Fraxinus Excelsior), one of Ireland's tallest, most common native trees and is currently under threat due to the pathogenic fungus, Hymenoscyphus Fraxineus, causing a disease known as Ash Dieback. Ash trees play a large role in carbon sequestration due to their large population within woodlands and hedgerows. They are the primary source of wood for hurley manufacturing, leading them to play an important role in Ireland’s culture. Several species of flora and fauna depend on the Ash tree for survival, meaning they play an immense role in supporting biodiversity in Ireland’s ecosystems. Ash Dieback is a chronic fungal disease that causes leaf loss, crown dieback, and bark lesions in affected trees. Eventually, the tree will succumb to the disease affecting commercial production and lead to the felling of hedgerows and woodlands. Not only does it impact local biodiversity but it also impacts the environment as a whole. The Ash trees lose their leaves while they are green meaning the surrounding soil becomes more nutrient-dense when the leaves fall. Ash trees are known for the rapid rate of decomposition of the leaves, which leads to a high rate of nutrient recycling. With the decrease in population, there will be inevitable changes in soil formation which will impact the function of the ecosystem as a whole. While also decreasing levels of carbon sequestration. Ultimately, Ash Dieback causes serious implications for timber production, amenities, biodiversity, carbon sequestration, and the landscape. The infection first begins when the fungal spores are carried through the air and land on healthy leaves during the Summer. The spores then germinate allowing the fungus to grow on the leaves and the leaf petiole. This is an airborne disease and, in the Autumn, and Winter months the fruiting bodies release spores into the air, once again continuing the cycle. It is produced when the leaves fall to the ground and are colonised by the fungus. The Ash tree is an essential habitat for a number of different species of wildlife. The bark is alkaline and supports rich lichen communities, as the bark is eaten by caterpillars and moths. Ash trees support 955 species, 45 of which only use Ash as support and 62 of which are highly associated with Ash. These 107 species are most at risk due to Ash decline. A total of 1058 species including birds, mammals, bryophytes, fungi, invertebrates and lichens are associated with the Common Ash tree. 536 of the lichens that rely on the Ash to grow are nationally rare or scarce, some of which are categorised as under threat. There is currently no cure for Ash dieback, and no treatment method for stopping its spread. Known Research from the UK and Europe has found that 7-8 out of every 10 Ash trees may die (although there are some local variations), but some trees do show some levels of “genetic tolerance” to Ash Dieback. Much of the current research focuses on producing different genotypes of Ash trees that are resistant to the disease. The present invention seeks to alleviate or prevent the disadvantages associated with Ash Dieback disease. BRIEF SUMMARY OF THE PRESENT INVENTION The inventors have surprisingly found that the treatment composition of the present invention comprising Jasmonic acid (JA) and Salicylic acid (SA) decreases / prevents the growth of the Fungus Hymenoscyphus Fraxineus. Features of the invention are set out in the appended Claims. The present invention accordingly provides a treatment composition comprising Jasmonic acid (JA) and Salicylic acid (SA) effective in inhibiting the growth of the fungus, Hymenoscyphus Fraxineus, in-vitro, as well as preventing / delaying the symptoms which are presented due to the infection of trees by this fungus. In another aspect, the present invention provides a method for the application of this treatment, in order to receive the desired effect, in preventing symptoms of infection of trees by the fungus, Hymenoscyphus Fraxineus. An advantage of the treatment composition of the present invention is that it has no negative impact on the health and growth of the tree as demonstrated by having no negative effect on height growth, leaf and branch growth while at the same time, preventing the presentation of Ash Dieback symptoms. In another aspect, the present invention provides a method for decreasing / preventing the growth of the fungus, Hymenoscyphus Fraxineus, the method comprising the following steps: Applying a treatment composition comprising Jasmonic acid and Salicylic acid to a leaf of a tree using known application methods. Preferably, the composition is applied through topical application, by either direct application, or through the use of canopy spraying. An advantage of this method is it directly addresses the contact site of the fungus and is not reliant on the treatment travelling through the vascular system. Preferably, the ratio of JA:SA in the treatment compositions of the present invention is in the range of 1: 1.25 - 1:2.5. Most preferably, in a preferred embodiment of the treatment composition of the present invention, the ratio is about 1:2 of JA:SA. Thus, most preferably, the treatment composition of the present invention comprises a 1:2 ratio of JA:SA. In a most preferred embodiment, the JA:SA ratio of 1:2 was found to be particularly effective at relative concentrations of molar amounts of 2.3779 x 10-4 : 4.7558 x 10-4 JA:SA. The treatment composition in this most preferred embodiment was found to successfully inhibit up to 92.2% and on average of 82% of the fungal growth in an experiment carried out by the inventors consisting of 576 Petri dishes, with a sample size of 24 dishes per treatment / control group, repeating across 3 genotypes, which were monitored over a span of 10 collections (20 weeks). The following Table 1 shows the solutions that were investigated by the inventors and their representing the names, concentrations, and ratios of each treatment composition: Table 1 Treatment Concentration M:M Ratio A 2.3779 X 10“3 :0 1:0 JA:SA (higher cone.) B 2.3779 x 10“3 : 4.7558 x 10“3 1:2 JA:SA (higher cone.) C 0: 2.3779 x 10 :i 0:1 JA:SA (higher cone.) d 2.3779 X 10-4 :0 1:0 JA:SA (lower cone.) e 2.3779 x 10“4 : 4.7558 x 10“4 1:2 JA:SA (lower cone.) f 0:2.3779 x 10"1 0:1 JA:SA (lower cone.) Accordingly, it was found that in with the present invention, treatment ‘e’ was most effective across all three genotypes, decreasing the fungus up to 92.2% and on average 82%. Treatment e being the lower concentration 1:2 ratio, JA:SA at a molar concentration of about of 2.3779 x io-4: 4.7558 x io-4 JA:SA (can also be written as follows: 2.3779x10A(-4 ): 4.7558 xioA(-4) JA:SA). The term, About' as used herein refers to the measurable value of the molar concentration of the solutions and is meant to encompass variations of + / - 50% or less, + / - 20% or less, in particular + / -10% or less , more in particular + / -5% or less, even more in particular + / -1% or less, and still more in particular +1- 0.1 % or less of and from these specified concentration, (this is to say that for e.g 2.3779x10a(-4 ) can be increased / decreased on the basis that it does not exceed 3.56685x10A(-4 )and lies no smaller 1.1890x10A(-4)at the 50% barrier, the concentration of JA and SA need not increase and or decrease at the same percentages provided that the ratios in terms of small whole numbers remains within the boundaries set below) such variations are appropriate to perform similar inhibition rates on H. fraxineus in vitro. However, the value of the concentrations that the modifier 'about' refers to 3 is itself specifically disclosed as the concentration invention to the treatment to decrease growth of H. fraxineus to the most significant extent. It is also important to consider the ratios of the two hormones within the treatment solution. The amount of treatment applied is dependent on the circumference of the tree, as per the ash whips within this study (with a circumference of 2.5-3 cm) the required amount was about 2 ml of the treatment so 1 ml :1 ml. The amount of treatment required per 1 cm of circumference is about 0.85 ml (0.425 ml: 0.425 ml).About as used herein referring to the measurable volume of the amount of treatment required and is meant to encompass variations of + / - 20% or less, in particular + / -10% or less , more in particular + / -5% or less, even more in particular + / -1 % or less, and still more in particular + / - 0.1% or less of and from these specified amounts, such variations line within the acceptable range by which to apply the treatment to a tree, while ensuring its health and natural growth patterns. However, the value of the volume of treatment that the modifier 'about' refers to is itself specifically disclosed as the volume by which it is appropriate to apply such treatments to a live specimen. It was determined by the inventors that the treatment composition of the present invention can decrease the growth of the fungus, Hymenoscyphus Fraxineus, by up to 92%, with no biological or environmental impacts. The treatment composition of the present invention has been visually observed to have prevented the presentation of the disease, in-vivo during a 20 week period. The function of plant hormones is to coordinate the growth and development of plants. However, they also play a role in controlling plant immune response to microbial pathogens. Heretofore, it has been understood that the signalling pathways of the defence hormones SA and JA have a negative interplay, and act in opposite directions. So, if a plant were to produce more SA, it is generally expected that the plant levels of JA would be decreased and vice versa. Salicylic acid (SA) (2-hydroxybenzoic acid) is one of many phenolic compounds (defined as compounds containing a benzene ring bearing one or more hydroxyl groups) that are synthesised by plants. SA plays an integral role within a plant. It regulates aspects of plant growth and development, as well as thermogenesis and resistance to abiotic stress (stress in relation to non-living factors for example drought or high soil salinity) and biotic stress (stress which is caused by living organisms such as fungi or bacteria). SA is one of many plant hormones involved in signalling the defences of a plant against microbial pathogens. The SA-mediated defence signalling pathway is activated following infection by biotrophic pathogens, which require living host tissue. JA and its derivative, an amino acid conjugate of JA (jasmonoyl isoleucine: JA-lle), are responsible for signalling compounds involved in the regulation of cellular defence and development in plants. JA is recognised as a stress hormone that regulates plant responses to biotic stresses, such as those elicited by pathogens. In the experiments carried out by the inventors, the inventors used leaf extracts extracted from three different genotypes of trees. The extract imitates the ‘tree’ in the inventors’ experiments. Each genotype of tree had a varying level of resistance to the disease. This also allowed the inventors to accurately measure the impact that the applied treatments had on the fungus, Hymenoscyphus Fraxineus, by measuring the colony diameter within the petri dishes, and then comparing them to the media and negative controls which contained only the fungus itself and leaf extract. Having done so, the inventors found great success by applying the treatment compositions of the present invention in preventing / alleviating growth of the fungus, with significant differences being found by the preferred embodiments of the present invention relative to the controls, as well as functioning the most consistently across genotypes. The inventors have found that the treatment composition of the present invention was able to successfully decrease up to 92.2% of the fungal growth in-vitro. Within the in-vivo experiment carried out by the inventors, it was found that the treatment consisting of the same composition showed statistical significance in maintaining positive leaf quality fora number of weeks, through which no symptoms of Ash Dieback presented. As well as sustaining a positive bark quality (no signs of lesions, discoloration or abnormalities) in relation to the controls, and same application method of other investigated treatments. During the 20 week experimental period there was found to be no negative impacts on the biodiversity within the surrounding areas. BRIEF DESCRIPTION OF THE DRAWINGS Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which: Figure 1 is a pictorial representation showing each of the three genotypes collected and used in the experiments carried out by the inventors to demonstrate the technical advantages of the present invention; Figure 2 is a pictorial representation showing the grading system which was used in relation to the in-vivo experiments carried out by the inventors to allow the statistical analysis of numerical data; Figure 3A is an image depicting the layout of the petri dish which was used to test the colony growth of the treatment; Figure 3B is an image depicting the layout of the control petri dish which was used to compare the treated petri dishes, in order for the inventors to determine the percent growth inhibition rate of the treatment compositions; Figures 4 (a) (b) and (c) are graphs of results referred to hereinbelow showing the mean colony growth for each of genotype 1, genotype 2 and genotype 3; Figures 5 (a), 5(b) and 5 (c) are graphs of mean percentage growth inhibitions graphed over the time span, relating to Genotype 1, Genotype 2 and Genotype 3, respectively; and Figures 6 (a) and 6 (b) are graphs of the mean leaf count; and Figures 7 (a) and 7 (b) are graphs of the mean bark and leaf qualities in order to get a visual representation of the effect the treatments had over the 10 collections carried out by the inventors. DETAILED DESCRIPTION The present invention will now be described more particularly, with reference to the Examples and Figures. Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of them mean “including but not limited to”, and they are not intended to (and do not) exclude other moieties, additives, components, integers or steps. Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating pluralarity as well as singularity, unless the context requires otherwise. DETAILED DESCRIPTION OF THE DRAWINGS Examples Exemplary Compositions and methods of the present invention will now be set out in the following Examples: Experimental Method In relation to tree application methods, fungicides can be applied in two ways, topical and penetrant. For example, leaf application (Canopy spraying) or microinjection. The benefits of microinjection is that it is a precise method of application. Fungicides typically require application of a 12 -14 ml of solution for a tree trunk which is 15.25cm in circumference; and with injection sites being placed every 3cm. The inventors chose to apply approximately 2 ml of treatment to the injection sites as each of the trunks were approximately 2.5 - 3 cm in circumference. As the inventors chose to apply approximately 2 ml of treatment through microinjection, the inventors also chose to apply approximately 2 ml through topical application. This was done every 14 days, as is the case for other Ash diseases such as Ash Anthracnose. The inventors collected leaves from three different genotypes of Ash trees. To clarify these definitions of each genotype, example reference images can be seen in Figure 1. With reference to Figure 1, the genotypes can be summarised as having the following features of Ash Dieback disease: These leaves were then used throughout the in-vitro experiment, to imitate the tree, as they include the necessary hormones sufficient to maintain the immune response, this is due to the presence of metabolites within the sample (amount specific on genotype and DNA of plant). Genotype 1 Genotype 2 Genotype 3 More severe signs of Ash Dieback. Leaf loss, wilted and darkened leaves. Leaves ‘dying back’. Severe bark lesions and decaying branches. Moderate signs of Ash Dieback. Some leaf loss, and spotting on the leaves. Shows evidence of bark lesions. Little signs of Ash Dieback. Leaves appear healthy with very little black spotting. Little to no lesions on the bark. There were two separate aspects to the experiment: The first being an in-vitro experiment which tested the effect of the inventors treatments on the growth of H.fraxineus. The second is an in-vivo experiment to test whether the treatments had an impact the overall growth and health of the tree. The inventors’ in-vitro experiments required a nutrient medium on which to grow the fungus. This process began with the leaf collection. Leaf Collection 1. The inventors collected Leaf material which would be used to make the leaf extract and leaf agar from the Teagasc’s Ash gene bank. 2. The leaf media used by the inventors for the extracts were collected using leaves from three genotypes. Two trees from each genotype were used. To ensure no cross-contamination occurred between the variables we collected and stored the leaves separately. 3. A surplus of Ash leaves was collected from separate trees of many different genotypes, which was used to make the nutrient media. The genotype did not have an effect on the nutrient medium as the leaves used for this were blended in a blender which breaks up the DNA that is responsible for the immune response of separate genotypes. 4. To prevent degradation of the leaf material, once stored in their separate bags they were stored in the freezer until they were needed for use. Agar plates Two elements of the inventors’ Experiments were carried out in-vitro meaning that the H.fraxineus was grown on a sterile growth medium, outside of the Ash tree. As the fungus grows on Ash trees, it requires a medium with Ash nutrients in order to thrive. The Ash nutrient agar was made from a surplus of leaf material. This process was carried out in the inventors’ lab until all needed plates were made. 1. An antiseptic solution was used to sterilise the workplace. Bunsen burners were lit to kill any bacteria in the air and to purify it. Many of these Bunsen burners were set up around the workplace to significantly decrease the risk of contamination, while of course, observing the usual safety precautions regarding open flames. All equipment was cleaned and sterilised prior to making the plates. 2. To start, 25g of leaf material was blended in 500ml of deionised water (which was measured using a graduated cylinder) until only fine particles were left. This mixture was then added to a 1L conical flask, another 500 ml was added to make it to 1 litre. 3. 9g of agar-agar powder which is an agar medium that does not contain any nutrients was added to the conical flask using a spatula and then stirred using a stirring rod. The agar- agar was used as the only nutrients required to ensure the growth of fungus is that of the Ash tree. 4. To prepare the conical flask for the heating process, it was sealed using a piece of cotton (this prevents evaporation of the mixture upon heating) and tin foil, to reduce any possible spillage. 5. Autoclave tape was placed on top of the conical flask, this shows us that the conical flask was heated to the correct temperature of 121 °C by turning from light to dark green. 6. The water bath was used to heat the conical flask to 121 °C, this is the required temperature to kill any bacteria within the mixture and to prepare it for the later stages in the agar making process. 7. Once the heating process was completed, the conical flask was removed and left to cool for up to 15 minutes until it was safe to hold, this ensures the safety during the process. 8. The Petri dishes were prepared in a sterile environment. The agar was poured into each dish, at approximately 1 / 4 of an inch thick. The dish would then be closed and left to cool, to prevent condensation from covering the plate before they were sealed completely. 9. Once all of the Petri dishes were filled, They were sealed using parafilm to prevent contamination and evaporation of the agar within the plate. They were stored in the fridge (kept at 1-4°C) the front side down until they were inoculated. Obtaining Leaf Extract Leaf material was obtained from two trees within each of the three different genotypes. Using these six different batches of leaf extract were obtained. Two trees from each genotype were used to ensure that the effects of the genotype were not a fluke and could be replicable. The leaf extract will then be added to the dishes to introduce the variable of how the interplay reacts concerning different genotypes, essentially enabling the leaf extract to act as the tree within each plate. To obtain the leaf extract, the leaf material from a specific tree was first defrosted. 1. 1,3g of leaf material was measured using a mass balance and 10 ml of distilled water to it which was obtained using a graduated cylinder. The mixture was ground into a mulch consistency using a mortar and pestle. The paste was then strained into a separate sterilised beaker through a muslin cloth. 2. This process was repeated until 15ml of the leaf extract was obtained. The 15 ml were then added to a clean dry beaker. 3. The mixture was then heated to kill any bacterial contamination. To do so a piece of cotton and tin foil were placed on the beaker to seal it preventing loss of liquid both through evaporation and spillage. Sealing the beaker also ensured that no water from the water bath contaminated the leaf extract sample. 4. Autoclave tape was placed on top of the tinfoil, over the mouth of the beaker. This is to ensure it remained in the water bath until it reached a temperature of 121°C, as required to sterilise the mixture (killing bacterial and fungal contaminants present) 5. As stated, the beaker is placed in the water bath and heated until the autoclave tape presents a positive result for the temperature required. The beaker is removed using heat proof gloves and goggles as a safety precaution. 6. The mixture was then left to cool until it was a safe handling temperature, ensuring that no injuries were sustained. Once the mixture was of reasonable temperature the tin foil and cotton wool were removed in a sterilised environment. Heating the mixture does not only kill any contaminants but also separates the leaf extract containing the genetic make-up and the leftover leaf debris that passed through the first filtration of the muslin cloth. 7. A syringe and syringe top filter are used to separate the leaf extract and debris. The mixture is strained into a separate clean dry, sterilised container where it is stored till it is needed for use. 8. To prevent the leaf extract from degrading it is frozen (kept at -20°C) until needed for inoculation. 9. This process was repeated for every tree (2 per genotype) individually, with the working place being sterilised in between to prevent unwanted bacteria and contamination of the leaf extract. An interesting observation that was made throughout this process was that the colour of the leaf extract varied between genotypes, with the most affected tree being a rich amber colour and the partially resistant tree a warm-honey colour. The tree that was partially affected fell in between the two colours. Jasmonic and Salicylic acid solutions The aim of the Experiments was to test the molar ratio of 1:2 of jasmonic acid to salicylic acid at a lower concentration so as to assess the effectiveness of the resulting treatment composition on decreasing the growth of the fungus and the consequential effect as to how the treatment affects / sustains the health of the tree. Accordingly, six treatments were tested. The inventors tested a composition comprising the 1:2 ratio Jasmonic acid to Salicylic acid at both 2.3779 x 10:i : 4.7558 x 10-3 and 2.3779 x 10-4 : 4.7558 x 10-4, which differed by a factor of 10. These compositions refer to treatment ‘B’ and ‘e’ which were tested by the inventors. The inventors also tested both hormones separately, at the concentrations they were being used in the ratios of the two treatments, this meaning that Salicylic acid was tested at molar concentrations of 2.3779 x io-3 and 2.3779 X IO '1 , while Jasmonic acid was tested at molar concentrations of 2.3779 x IO3 and 2.3779 x IO4 hormones. Salicylic acid is commercially available in its crystallised form so before perfecting the concentration, it was necessary to make the Salicylic acid into a solution. This was completed as follows: 1. First ensure all surfaces and equipment that will be in use are sterilised using an aseptic solution, this prevents contamination of the solution; 2. Using a clean dry clock glass and a mass balance which weighs to 4 decimal points, measure 0.6569 grams of salicylic acid crystals; 3. Add the measured amount of salicylic acid into a 1000 ml beaker; 4. Measure 500ml of deionised water using a 500ml graduated cylinder; 5. Rinsing any washings from the clock glass into the beaker using the deionised water measured in the graduated cylinder, to ensure the solution is as accurate as possible; 6. Pour the remaining deionised water from the graduated cylinder into the beaker containing the salicylic acid; 7. Once again, measure 500 ml of deionised water using the same graduated cylinder and add this to the beaker containing the solution; 8. Salicylic acid has a low aqueous solubility at room temperature (21 °C). To ensure all the crystals have dissolved the mixture is heated gently using a hot plate, stirring continuously to ensure the mixture is heated evenly and that all the Salicylic acid crystals are completely dissolved; 9. The solution is heated until all crystals have dissolved; 10. The hotplate is turned off, and the solution is left to cool completely without disturbance. This prevents recrystallisation of the Salicylic acid; 11. This is the higher concentration solution that was used on the Treatment plates, Media interaction plates and the Ash whips. The solution has a molar concentration of 4.7558 x 10-3M. This solution was used as the inventors’ stock solution. The next dilution needed is the 2.3779 x 10-3M solution for the 0:1 JA:SA. This was used in the treatment plates that are fortesting the effects of the higher cone, of SA on its own. The 2.3779 x 10 :iM solution for the 0:1 JA:SA was prepared as follows: 1. Using a clean dry 10 ml graduated cylinder, measure 2 ml of the stock solution. 2. Add this to the container by which it will be stored. Ensure it is clean, dry and sterilised before use. 3. Using the same graduated cylinder measure 2 ml of deionised water. 4. Pour the 2 ml of deionised water into the container which holds the 2ml of the stock solution. 5. Ensure the last drop of water has been poured from the graduated cylinder. This is to make the solution as accurate as possible. 6. The container which holds the solution is then stopped and inverted 20 times, to ensure a homogeneous solution. 7. The concentration of this solution has been made up to 2.3779 x 10-3 M. The stock solution is then again used to make the lower concentration of the SA solution. The concentration of this solution is the concentration of the stock solution, diluted by a factor of 10. The process by which this was completed is as follows: 1. Sterilise the workspace using an antiseptic solution. 2. Rinse a 50 ml pipette with deionised water. 3. Pour some of the stock solutions into a clean dry beaker. 4. Rinse the pipette with the stock solution within the beaker. 5. Using a pipette filler, draw up the solution until the bottom of the meniscus is above the graduation mark. 6. Allow the solution to slowly drain into a waste beaker until the bottom of the meniscus is resting on the graduation mark, reading at eye level. 7. Then transfer the remaining solution to a clean, dry 500 ml volumetric flask, touching the pipette off the inner glass. Do not blow out the last drop. 8. Add deionized water to the volumetric flask, until near the calibration mark, using a funnel. 9. Remove the funnel and use a dropper to make the solution up to the calibration mark, reading the bottom of the meniscus at eye level. 10. Stopper to volumetric flask and invert 20 times to ensure a homogenous solution. 11. This is then the lower concentration SA solution, which has a molar concentration of 4.7558 x 10-4M. It is used as the lower concentration solution, for the treatment plates, media interaction plates and Ash whips. The final SA solution that was tested by the inventors is the lower concentration 0:1 JA:SA, for the treatment plates which contain only Salicylic acid. The process to complete this is the same as the indicated already above and includes the following steps: 1. Using a clean dry 10 ml graduated cylinder, measure 2 ml of the lower concentration SA solution. 2. Add this to the container by which it will be stored in. Ensure it is clean, dry and sterilised before use. 3. Using the same graduated cylinder measure 2 ml of deionised water. 4. Pour the 2 ml of deionised water into the container which holds the 2ml of the stock solution. 5. Ensure the last drop of water has been poured from the graduated cylinder. This is to make the solution as accurate as possible. 6. The container which holds the solution is then stoppered and inverted 20 times, to ensure a homogeneous solution. 7. The concentration of this solution has been made up to 2.3779 x 1() '1 M. The next step was to make the Jasmonic acid solutions. Jasmonic acid is distributed in liquid form. However, the key component to mention is that it is a volatile liquid and must be kept between 4<!C and 8°C. the jasmonic acid was received in a sealed vacuum container to prevent evaporation of the liquid. The vacuum sealed container held 250 mg of jasmonic acid. Which had a molar concentration of 4.755790175M. The first step in the process was to dilute the concentrated JA to the required higher concentration. This was carried out as follows: 1. A clean dry, 500 ml volumetric flask was placed in an ice bath, held in an upright position using a retort stand. 2. The seal on the Jasmonic acid was then broken (this had been chilled in the freezer for a period of time to ensure the liquid was cold but not frozen). 3. Using a dropper, chilled deionised water was added to the bottle of JA, closed and then inverted 20 times. 4. The same dropper was then used to add the emulsion within the container, to the volumetric flask. 5. Steps 3 and 4 were repeated until there was no residue left in the original JA container. 6. Add chilled deionized water to the volumetric flask, until near the calibration mark, using a funnel. 7. Remove the funnel and use a dropper to make the solution up to just below the calibration mark, reading the bottom of the meniscus at eye level. 8. As the solution currently resides at 4’C, the solution is left to warm slightly until it sits at 8°C. 9. Water expands at 4°C, so the solution is left to sit to ensure the solution is accurately measured on the calibration mark. 10. Once it has warmed slightly, the dropper is used to add deionised water to the volumetric flask until the bottom of the meniscus sits on the calibration mark, reading at eye level. 11. Stopper to volumetric flask and invert 20 times to ensure a homogenous solution. 12. This is then the higher concentration JA solution, which has the molar concentration of 2.3779 x 10-3M. It is used as a higher concentration solution, for the treatment plates, media interaction plates and the Ash whips. The lower concentration JA solution is the higher concentration solution diluted by a factor of 10. This process was completed as follows: 1. Rinse a 50 ml pipette with deionised water. 2. pour some of the Higher concentration solution into a clean dry beaker. 3. rinse the pipette with the solution within the beaker. 4. Using a pipette filler, draw up the solution until the bottom of the meniscus is above the graduation mark. 5. Allow the solution to slowly drain into a waste beaker until the bottom of the meniscus is resting on the graduation mark, reading at eye level. 6. Then transfer the remaining solution to a clean, dry 500 ml volumetric flask, touching the pipette off the inner glass. Do not blow out the last drop. 7. Add deionized water to the volumetric flask, until near the calibration mark, using a funnel. 8. Remove the funnel and use a dropper to make the solution up to the calibration mark, reading the bottom of the meniscus at eye level. 9. Stopper to volumetric flask and invert 20 times to ensure a homogenous solution. 10. This is then the lower concentration JA solution, which has the molar concentration of 2.3779 x 10-4 M. It is used as the lower concentration solution, for the treatment plates, media interaction plates and Ash whips The solutions of JA and SA at both higher and lower concentrations were then divided into clean sterilised containers of roughly 50 ml each. This is to ensure the solution is mainly maintained at optimal temperatures when part of it is in use. The proportions were divided in that manner to control both the temperature and the sterilisation of the solutions. The containers were then sealed using parafilm to prevent any evaporation of the solution. The following Table (Table 2) shows the solutions that were investigated by the inventors and their representing the names, concentrations and ratios of each treatment composition: Table 2 Treatment Concentration M:M Ratio A 0.0023778950880 : 0.0000000000000 1:0 JA:SA (higher cone.) B 0.0023778950880 : 0.0047557895000 1:2 JA:SA (higher cone.) C 0.0000000000000 : 0.0023778950880 0:1 JA:SA (higher cone.) d 0.0002377895088 : 0.0000000000000 1:0 JA:SA (lower cone.) e 0.0002377895088 : 0.0004755789500 1:2 JA:SA (lower cone.) f 0.0000000000000 : 0.0002377895088 0:1 JA:SA (lower cone.) Inoculation of Petri dishes The inoculation of the Petri dishes was completed in Teagasc Research Centre in Ashtown. This was to ensure complete safety when working with the fungus, and to decrease contamination rates of the dishes as opening them under the laminar flow lowers the contaminants within the air surrounding the dish. Here all of the Petri dishes were assembled with the specific components depending on which treatment or control group they belonged to and if required for the variable were then inoculated with the fungal plugs of H. fraxineus. The inventors were provided with fungal cultures , as within the time frame for the project it would not have been possible to grow their own as well as having enough time to conduct a thorough study. As stated, this work was carried out in a laminar flow, which is a sterile environment that has consistent regular airflow throughout. This prevents contamination once the dish has been opened. Even though the dish is within the laminar flow it is necessary to still work relatively quickly and to not open the Petri dish completely. For the inoculation of the plates for the second element of the inventors’ Experimental work, the media interaction. 24 plates were inoculated with each of the treatments (A-f) and 24 were left empty to act as the control. Referring now to the dishes which tested treatment effect, the following steps were carried out: 1. Each Petri dish was opened in the laminar flow. 2. Using a sterilised scalpel, the fungal cultures were cut into approximately 4mm x 4mm pieces. The fungus plug was placed onto the centre of each plate using sterile forceps. 3. For each treatment, six sterilised filter paper discs were placed around the edge of the Petri dish in a circular pattern. 4. Three of which had been soaked with 2.5 pL of the relevant leaf extract dependent on genotype .and the other three were soaked with 2.5 pL of the relevant solutions depending on treatment variable. 5. The plate was then labelled with its genotype and treatment sample number. 6. The dishes were then sealed using parafilm to prevent contamination. 7. As per the media control and negative control, the fungal plugs were placed in the centre as replicated in the treatment dishes. The media control is then sealed. 8. The negative control also contains leaf extract, three filter paper discs are places surrounding the fungal plug. They are soaked in 2.5 pL of the relevant leaf extract each dependent on genotype. They are then sealed with parafilm, similarly to the rest of the plates. 9. All dishes were placed in cardboard boxes and closed. They were then safely transported back to the school for data collection. Refer to figures 4A., 4B., 4C., and 4D. to see the visual representation of the inoculation layout of the petri dishes used within the in-vitro experiment. Data collection of in-vitro studies 1. The plates were stored in sterile conditions within the school lab, the room is maintained void of light this is to ensure the media remains safe and uncorrupted. The plates were incubated between 20 and 40°C using both heaters and heat mats to evenly heat the room - the optimum temperature for the fungus to grow. 2. The plates were arranged according to genotype and treatment / control group. This helped us to keep things organised and to prevent any crosscontamination. 3. The door was kept closed and access was restricted. Entrance to the room was only permitted during data collections. 4. The dishes were left in these conditions for 2 weeks to allow for the fungus to begin showing signs of growth before they collected the first set of data. Due to the heightened risk of cross-contamination, if the dishes were opened, the inventors decided the best method was to collect data with the dishes closed. To accurately capture the data, the plate was placed on a light box which clearly highlighted any ring of colony growth and a 1 mm square grid was placed over it to gather the correct measurements. 1. The inventors placed an acetate sheet on which they had printed a 1 mm grid on top of the Petri dish. The grid gave us an accurate measurement of the largest diameter of the fungal colony on that dish. 2. An image was then taken and saved to the inventors’ computer drive. 3. This was repeated for all the inventors’ dishes every two weeks, this process was completed a total often times over the course of 20 weeks. 4. The data collected of colony diameter of fungal growth, per dish, perweek was then inputted into the Percent growth inhibition formula. This is the percent at which the treatment composition of the present invention inhibited the growth of the fungus compared to the control sample. The formula used to calculate the percent growth inhibition is shown below: (C - T) / C x 100 = % growth inhibition Where C is the colony diameter of H. fraxineus in the control dish and T is the colony diameter of H. fraxineus in the treated Petri dish. The control being used in this case is the media control that contains only a fungal plug of H. fraxineus. The percent growth inhibition data is the data being used to conduct the statistical analysis, the control now becomes the percent growth inhibition between the media control and the negative control, and the six treatments are also being compared to the media control to maintain a fair result. Trees (in-vivo Study) The final component to the experiment consisted of the in-vivo study, which was performed to monitor the effect of the treatment on the growth and health of the tree. This was completed once again over the course of 20 weeks and consisted of 90 replicate Ash whips. The Ash whips were stored in cold stores in Waterford. So, before the experiment could begin the necessary equipment and components were collected. 1. Since the trees were taken directly out of the cold store the first step in preparation is to soak them to allow them to regain moisture. To do so all of the whips were placed in buckets full of water, fully submerged for 2 hours. 2. After soaking all of the Ash whips, they were then sorted and the top 100 were selected to continue the process of being planted. The 100 whips were chosen according to root health, size range, and absence of abnormalities. 3. The chosen whips were then trenched in damp soil. Further continuing the process of them gaining excess moisture. This is vital to ensure the trees do not dry -drown within the soil once they have been planted. This means that if they have not taken in enough moisture before potting into the compact soil, the roots will find it difficult to aid in the process of transpiration and the tree in question will die from lack of water. 4. After being trenched for 12 hrs , the whips were then individually potted into 3L pots leaving an excess 5-7 cm of the root above soil to prevent root rot. 5. The trees were watered in excess for the following days to ensure successful germination. 6. Once germination had occurred, the most successful 90 whips were chosen as part of the experiment. For the experiment, the inventors chose to test the effects of treatments ‘B’ and ‘e’ which are the 1:2 ratio of JA:SA at both higher and lower concentration. These were tested using two separate methods of application. The first method of application tested was the leaf application method, this comprised the steps of: the JA and SA solutions for the corresponding tree being applied to three separate leaflets each on the same Ash whip. This is to allow the treatment to be received through the stoma on the leaves of the tree, which then transports the treatment to the xylem of the vascular system of the tree. Allowing the treatment to reach the affected areas of the tree. The second application method consists of cutting a thin slice into the base of the tree using a sterilised scalpel. The slice is cut at an angle of approximately 15° to the vertical of the tree. The goal of the cut was not to harm the tree but to allow the solutions to reach straight to the vascular system at a faster rate. The solution would then be applied using a separate dropper for each of the required solutions. Add approximately 1 ml of each solution to the opening before sealing it back over. 7. Each of the 90 whips were labelled with the corresponding treatment, application and replicate number. There are 15 replicates per variable within this experiment. For each treatment there is 15 leaf application (LA), 15 injection treatment (I) and 15 control (C). 8. Once labelled and laid out using the latent square technique for randomisation of the samples, to prevent unwanted variable e.g. area / shaded disposition, the trees were left to grow for 2 weeks. Only disturbance was being watered when necessary. 9. After this 2 week period the first set of data was then collected. This was done as follows. 17 • Numberof leaflets were counted. • Number of branches were counted. • Height of tree was recorded using a metre stick. • pH of soil (to ensure it remained constant / within the acceptable range of 6-7) • The leaf and bark quality of the tree was also recorded. This was done using a grading system which can be seen below. Each of the trees were treated with their corresponding treatment using their correct application method once every 2 weeks, as well as results being taken at this time. Results and Analysis To begin analysing the results, the inventors graphed the median colony diameter of each treatment and control group for the inventors’ Collection 10 data. This could be carried out as an average across all 3 genotypes as for the Collection 10 data there was no significant difference across treatment or control group. This showed the inventors a simplified result of their data and allowed them to easily see the visual difference in colony growth before they moved onto statistical analysis across each genotype to determine which treatment worked best for which genotype. This showed the inventors, visually, that across the 3 genotypes treatment ‘e’ worked best - the 1:2 JA:SA at a lower concentration. As well as this, the inventors graphed the mean colony growth for each of genotype 1, genotype 2 and genotype 3, across the 10 collections to obtain an overview of the growth trend across the 20 week period for each of the treatments, before the inventors began comparing the percentage growth inhibition of the treatments. These graphs of the mean colony growth are shown in attached Figures 4 (a) (b) and (c) for genotype 1, 2 and 3, respectively. When doing so, the inventors observed an unexpected peak within each of the groupings following their 8th collection, the inventors believe this was due to a sudden temperature change, as issues occurred with the heater for a period of time (approx. 1 week). The time at which the graph spikes at collection 9 is in occurrence with the return of the sufficient temperature (ranging from 20-40 ’C) during that 2 week period. While this would be conclusive if it was only the controls which had spiked at this stage and or if they all had spiked at the same rate, but due to the increase in growth at different rates with different treatments it led us to research further into what had occurred. Upon doing so they discovered a research paper in relation to the formation of Methyl Jasmonate between Jasmonic acid and Absisic acid. This stated that at increased temperatures and increased concentrations the resulting proximity of the two acids leads to the production of the ester. While the mechanism is not fully understood, the inventors hypothesise that a similar mechanism occurs between the Jasmonic acid and the Salicylic acid, due to both abscisic and Salicylic acid being 18 aromatic organic compounds which consist of nonpolar covalent molecules. However, it is the Jasmonic acid that begins this mechanism, this can be stated as in comparison with the research paper found and the knowledge of the relative molecular mass (Mr) of each compound, it is consistent that the reaction occurs on the basis of there being enough of the other acid to react with the JA. The Mr of abscisic acid is significantly higher than that of salicylic acid at 264 to 138.12, this means a higher temperature was required to break apart the bonds within abscisic acid than that or a higher concentration to increase rate of reaction by providing more molecules which increases the chance of a collision and in turn a successful collision. Using this knowledge a similar result was observed within the graphs. When comparing the slopes of the rate of growth across treatment / control groups we can see that the slope of the control along with the slopes of treatments A,B, and C is significantly steeper than that of treatments d, e, and f. This can be observed due to the formation of the methyl jasmonate within the higher concentration dishes, upon increase in temperature. Which would then result in the increased growth of fungus as there are no longer the treatments required to slow / decrease its growth. While with the lower concentration treatments the Jasmonic acid concentration is not high enough to trigger this reaction. Similar results can be seen within the analysis of the in-vivo study, which will be discussed further. Before statistical analysis, the distribution of the percent growth inhibition data was tested using the Shapiro-Wilk test for normality. The results for the Shapiro wilk tests show that at an alpha level of 0.05 there is a significance of <0.0000001, which shows that the data was severely skewed and allowed us to continue with non parametric methods. Due to the uneven distribution of the data, the inventors decided that the best methods of analysis were to use the Kruskal Wallis-H test, and the Mann Whitney-U test. The Kruskal Wallis-H test would tell us if there was a significant difference between at least one pair of the samples. The Maximum number of samples that can be analysed using the Kruskal Wallis-H test is 5 samples, meaning that it could only be carried out across a set group of the samples when comparing across genotypes in order to determine which treatment was most significant forthat genotype. When comparing across genotype, we decided it was best to carry out a Kruskal Wallis-H test with the treatments of a higher concentration A’, ‘B’, and ‘C’ against the control and again for the treatments of lower concentration ‘d’, ‘e’, and T. When comparing across treatment / control groups we were able to use the Kruskal Wallis- H test with a sample size of 3. If there was a statistical significance amongst any of the Kruskal Wallis-H tests we went on to carry out a Mann Whitney-U test for each pair within the samples, as well as for any of the pairings which was neglected across genotype for example treatment A’ against T. This led us to carry out a total of 6 Kruskal Wallis-H tests, for each of the ten collections when comparing across genotype and a further, 7 when comparing across treatment / control groups. The Bonferroni correction was added to the alpha values of the Mann Whitney-U test to bring down the percentage error chance. Because the multiple samples that were compared to each other individually increases the percentage error beyond a reasonable level to accept significance. To resolve this the original alpha level of 0.05 is divided by the number of samples being compared to each other. In the case across treatment / control groups, the new alpha level which the p-value must fall under to show significance is 0.016 and the alpha level for comparing across genotypes to 0.0073. Collection 10 Results The first statistical analysis for collection ten was conducted across treatment / control groups. Table 3 >> Si $s<.$ $$■ ><£>$> $. >$7'7 <&>> / 7$ $ : $W Tha H-mt we rmweh for the WA the wosrth values let1,. 2 and 3 is 4<<$- The eh; sqm ir 7« for try? test to whe it fower than the e&ha wlw fo foot st 0.7 thT Ths® «sew that at a pwafoe CWW thaifMstt st between the cmfofo grwpa. Similarly, across each of the treatment comparisons we found that the H-stats fell below the chi square critical of 9.2103, meaning that there was no significant statistical difference across any of the treatments with p-values of 0.23441,0.39159, 0.04671,0.2651,0.76369 and 0.63843 ranging from A to f. This meant that after the 22 week period the treatments worked to the same degree independent of genotype. Table 4 '5-^^ if SUss- $ S3 * < uusiu >S3 S ?■ u.rurs SU su aw- xss V .. X U « "x sv. 1 . - n-v "• ** <,x ❖ a ;• x' .....■ S» s «■: '7UUV‘ .UUJ U : --- M ■» 6. &3W a ■" »: O63 W uuus •$ st US Mas < ? usu U X<-. '•XvXv.X WW U U •SOMU SU U u u was U •as ss 'SUU UUU u ■US U u iJ: .■ a,...- fwx . a SSU .<<• 1. 2 3 &3,5353,s:hs square eritkaj st most W ohow few wt to wks ft tew ths a-phs vakM of is This of 876» that Wo? is m sigsifkw® hsswasts mss SS- W U:$ u.uss$ ss •S^ ?u:$s U UU uusu u .«S U.UStU US S$ SS ^USU u Sj.SSSS...... iwa- U.USSS- •US ......SS......... S'4 $S U / ¾ as ww U- SU aw ■U USUU SU aso u ___________________________ aW S! After carrying out the statistical analysis across the 3 genotypes and determining that there was no significant difference across any of the treatments we went on to compare across the different treatment I control groups to determine which treatment was most significant for that genotype. The H-stat that the inventors received for the Kruskal Wallis comparing the higher concentration treatments and control is 43.161. The chi square critical it must fall above for the test to make it lower than the alpha value of 0.01 is 11.345. This means that at a p-value of <0.00001 that there is a significance between the treatments of higher concentration and the control, for genotype 1. Similarly when comparing the treatments of a lower concentration with the control we found there to be a statistical significance with the H-stat of 66.9253, and the p-value of <0.00001. This tells us that there is significance amongst at least one of the sample pairings. As there was a statistical significance amongst both of the Kruskal Wallis-H tests we went on to compare each of the pairings using a Mann Whitney-U test, as well as the pairings which are neglected within the Kruskal Wallis-H test. The required critical value differed from test to test due to a change in sample size, taking this into account it was determined that there was a significant difference between the following pairings. The inventors found a significant statistical difference between each of the controls and the treatments meaning each of the treatments had a significant effect on the inhibition of the fungal growth. As well as this, the inventors found a significant difference between each of the treatments and treatment e meaning that treatment e had the most significant effect in terms of inhibiting the fungal growth and that there was significant statistical difference between the performance of treatment e and the other treatments. The H-stat that the inventors received for the Kruskal Wallis comparing the higher concentration treatments and control for genotype 2 is 53.7249. The chi square critical it must fall above for the test to make it lower than the alpha value of 0.01 is 11.345. This means that at a p-value of <0.00001 that there is a significance between the treatments of higher concentration and the control. Similarly when comparing the treatments of a lower concentration with the control we found there to be a statistical significance with the H-stat of 74.8006, and the p-value of <0.00001. This tells us that there is significance amongst at least one of the sample pairings. As there was a statistical significance amongst both of the Kruskal Wallis-H tests we went on once again to compare each of the pairings using a Mann-Whitney U test, as well as the pairings which are neglected within the Kruskal Wallis-H test. The inventors once again saw a significant statistical difference between each of the controls and the treatments meaning each of the treatments had a significant effect on the inhibition of the fungal growth for genotype 2. As well as this we found a significant difference between each of the treatments and treatment e meaning that treatment e had the most significant effect in terms of inhibiting the fungal growth and that there was significant statistical difference between the performance of treatment e and the other treatments. Treatment C had a significant statistical difference in comparison to treatment C showing us the treatment C had a more significant effect than treatment d, however there was no other significance found between C and A,B, or f. The H-stat that the inventors received for the Kruskal Wallis comparing the higher concentration treatments and control for genotype 3 is 50.6916. The chi square critical it must fall above for the test to make it lower than the alpha value of 0.01 is 11.345. This means that at a p-value of <0.00001 that there is a significance between the treatments of higher concentration and the control. Similarly when comparing the treatments of a lower concentration with the control we found there to be a statistical significance with the H-stat of 72.488, and the p-value of <0.00001. This tells us that there is significance amongst at least one of the sample pairings. As there was a statistical significance amongst both of the Kruskal Wallis-H tests the inventors went on once again to compare each of the pairings using a Mann-Whitney U test, as well as the pairings which are neglected within the Kruskal Wallis-H test. The inventors found there to be a statistical significance between the controls and each of the treatments for genotype 3 with p-values of <0.00001. The inventors also found there to be a statistical significance between treatment e and each of the other treatments. This once again showed that the treatment e had a more significant impact on the growth of the fungus as well as showing the consistency of the treatment amongst each of the genotype. Table 5 To summarise the inventors’ results for Collection 10, the inventors found there to be no significant statistical difference between the treatments in relation to genotype. While having found for each of the genotypes there was a statistical significance between each treatment and the control meaning that all of the treatments had a significant effect, on the growth of the fungus. However, treatment e was significant in comparison to the other treatments and shows that treatment e, a 1:2 JA:SA at a lower concentration was the most effective treatment after the 22 week, study carried out by the inventors. Collection 1 This method of statistical analysis was repeated for each collection allowing the examination of the effectiveness of the treatments progressed across the time span of 20 weeks. For the inventors’ 1st Collection, the inventors found there to be a statistical difference between the control groups as well as no significant difference between the treatments. This means after the first 2 week period the fungus had begun to grow at an irregular rate with Collection 2 having a higher percentage growth inhibition due to the slower growth of the negative control. However, at this point, all of the treatments were performing the same as their counterpart in relation to a different genotype. After comparing within each of the genotypes with a Kruskal Wallis H test in order to determine if there was a significant difference between any of the pairings, the inventors found there to be a significant difference between the following pairings: This meant that for the lower concentration comparisons for both genotypes 1 and 2 were then compared using Kruskal Wallis-H test. In doing a significant difference between the control and treatment e, as well as when comparing treatment e to treatment A, B, and f for genotype 1 was found, showing that for the worst affected tree, treatment e had a significant effect on the growth of the fungus from as early as the first collection and worked significantly better than treatment A, B, and f. For genotype 2, the inventors found there to be a statistical significance between treatment e and each of the other treatments showing that treatment e was inhibiting the fungus at a more significant rate than the other treatments however there was still no significance amongst the treatments and the control. We also found a significant difference between treatments C and d, with treatment C having a more significant effect than treatment d. As for genotype 3, after comparing the samples which are neglected within the Kruskal Wallis-H test no significant difference between any of the treatments for the first collection were found. Collection 5 Half-way through the experiment at collection 5 it was found that the controls were not significantly different when compared to one another, as is the case from the second collection, 4 weeks after the inoculation date. This means the inventors went on to compare across the treatments with assurance for accurate readings. However, a significant difference between treatment d across the different genotypes was found, this is seen across multiple of the collection. Treatment d worked significantly better for genotype 1, the worst affected tree, than it did for the other genotypes. The inventors believe this is due to increased levels of JA and SA already occurring within the leaf extract aiding in the assistance of the inhibition. After carrying out the Kruskal Wallis-H test within the genotypes we found there to be a significance amongst each of the tests, and went on to determine which pairings were significantly different using the Mann- Whitney U test. For Genotype 1, the inventors found there to be a significant difference between each of the treatments against the control, as is the case beginning at Collection 3 of the data and for each of the genotypes at this stage. It was found that there was still a significant difference between e and each of the other treatments. Meaning that treatment e consistently has higher inhibition rates when compared to the other treatments, although all have inhibited the growth of the fungus to a significant extent when compared to the control. Table 6 A vs VC hl A * X A 0 X A vsAv X? ■- 5- wvm < «1®$ V*' ' >4. x 4 £ r > •* ::^.v A .V<: '' X? 5 After analysing genotype 2, the inventors once again found there to be a significant difference between treatment e and each of the other treatments .As well as a significant statistical difference between treatments B and C, and treatment d. This shows us that after 12 weeks (5 data collections) treatments B and C, worked significantly better than treatment d for genotype 2. However, there is still a significant difference between Treatments B and C, and treatment e, meaning that e had the most 10 significant effect on the fungal growth. Similar results were observed with regards to genotype 3 as they were found in genotype 2, As well as treatment A also having a significant difference in relation to treatment d. Table 7 C&WA$S<XSS SA i'S VC c; Corios <• X v ■: .<sm; s SWtsX S W&J A >Wt d :> <• -i'X'AX »i / A «W¥ < §^5. a w>s A s'* is'' X m K • V WA & •s •• H-*. 'SmslfKcnt •;xxXxx- X / A c <-s'.X>sX'* .S SAX l-xv-Sw -s 3 .^4- £ :>.vxw X* Is? <? >VvXX« SA : X sXXs-.Y SVA SSg^'<«s\;A toWi'A ,<A(WSsS. Collection 9 When comparing across treatment / control groups for collection 9 a significant statistical difference was found between the three control groups and treatment d. As stated previously there was a heat change between collections 8 and 9, and as heat returned to normal (20cC-40°C) the significant difference then occurred. We believe this is due to a reaction between the jasmonic acid and salicylic acid to form the ester methyl jasmonate. Evidence to support this can be seen through the progression of the results. For collection 9 the control group for genotype 1 was significantly smaller than genotype 2, and genotype 2 significantly smaller than genotype 3. When reviewing this data it is important to consider the impact of genotype. As genotype 1 is the genotype most affected by Ash dieback, its immune system has naturally higher levels of salicylic acid and jasmonic acid (the hormones responsible for cellular response and defence) than genotype 2 which is less affected and even more so than genotype 3 which is moderately resistant. Such immune systems were introduced through the addition of leaf extract and so the inventors believe with the addition of heat that the formation of methyl jasmonate, while occurring on a minute scale, impacts the growth of the fungus in vitro. The genotype impacts the growth as different levels of methyl jasmonate are produced depending on genotype. Similar results can be seen amongst treatment d however is not seen amongst the rest of the treatments. This leads us to believe that jasmonic acid is the limiting reagent within this reaction as on the small scale slightly increased levels of both hormones on treatment d, led to it having an effect on rate of fungal growth. It can be hypothesised however that this reaction is not observed within treatment A (the higher concentration of just JA) as at the already high cone, the production of minute levels of methyl jasmonate does not have as great an impact. Each of the Kruskal Wallis-H tests were significant when comparing across genotype meaning Mann Whitney U tests were then carried out. The significance between pairings were consistent across treatments which were significant for Collection 8 however they are now being compared to controls which now are significantly different across genotypes, which no longer makes it a fair test. However sit can now be said that within each genotype with regards to their controls treatment e still significantly has a greater inhibition rate on the fungus than the other treatments. Graphs of mean percentage growth inhibitions graphed over the time span: Figures 5 (a), 5(b) and 5 (c ) relating to Genotype 1, Genotype 2 and Genotype 3, respectively. As Shown in attached Figures 5(a), 5 (b) and 5 (c), respectively, the mean percentage growth inhibitions are graphed over the time span of the 10 collections this allows us to clearly see the consistency of treatment e, as well as allowing us to visualise the drastic change which occurred at collection 9 of the inventors’ data. In-vivo study Due to the inventors’ being unable to state that their Ash whips were from the same mother tree, as well as the impact of outside conditions such as wind, the inventors chose to compare their data of leaf and branch count by comparing the trend of the averages per grouping. The graphs representing these trends can be seen in Figures 6(a) and (b). No drastic change in shape of the trends can be seen, ensuring that the treatments had no ill effect on the leaf or branch count of the tree. Similarly, the inventors observed no drastic change in the height of each tree over the course of the experiment. When comparing bark and leaf quality, the inventors compared both leaf and branch quality using the same statistical methods as the percentage growth inhibition of the treated dishes, due to the fact that their data was non-normally distributed. This was seen after carrying out a Shapiro-Wilk test for normality on the quality ratings of the trees, at an alpha level of 0.05. A significance level of <0.00000001 was found showing the data was severely skewed. The inventors compared across treatments, as well as any combination of application and treatments that are neglected when comparing across treatment. This was done by carrying out 6 Kruskal Wallis-H tests and continuing with several Mann-Whitney U tests if a significance was found, in order to determine which pair of samples are significantly different. Following on they compared across application methods / control groups using a Mann-Whitney U test to determine if the application method impacted the effect the treatment had on the tree. Collection 1 For the inventors’ first collection, the inventors found there to be no significant difference amongst any of the pairings, for either leaf or bark quality with each of the H-stats falling below the chi square critical of 9.2103. In order for it to be significant the alpha value of 0.01, the H-stat must fall above the chi square critical. When comparing across the application methods we found there to be no significant difference for the leaf or bark quality, with all of the U values falling above the critical value of U at p<0.1. This showed the inventors at the beginning of the in-vivo experiment before applying the first treatment there was no difference between any of the groupings, which indicated a fair trial. Collection 5 For the inventors’ fifth collection, the inventors found there to be a significance between the following groupings for the leaf quality of the Ash whips and therefore went on to carry out Mann-Whitney U tests in order to determine which pairs had a significant difference. In doing so we found a significant difference between LAte and , CTB and ITB. meaning that there was a significant difference between the grading received by the groupings. In this case, LAte had significantly more favourable gradings then both CTB and ITB for the fifth collection. As well as showing varying significance from collections 4 to 6. The inventors however found no significance when comparing the application methods to each other, showing that neither application method had an effect on the effect of the treatment at this point. With regards to the bark quality, at this point there was no significant difference between any of the treatment or control groups, there is however groupings which are beginning to receive less favourable ratings than prior collection, and they observed a change in several of the groups. Beginning at Collection 7, the inventors no longer saw a significant difference between the leaf quality of any of the groupings as the change in weather had begun to take effect and the Ash whip’s leaves began to yellow in some cases and lose their leaves, while others wilted and became brown in colour. All of which are symptoms of Ash Dieback. Due to the fact that the control whips experienced the same change in leaf quality that was not attributed to the change in weather, they began to observe lesions on the bark, as well as it having been around the time of year that Ash Dieback symptoms begin to present. They believe that the Ash Whips had become infected with the disease. This is highly probable as other Ash trees in the local area are also affected by the disease. If they were to take this as the case, it shows that LAte slowed the onset of the symptoms I prevented symptoms depending on whether the decrease in leaf quality seen was due to the weather or the disease. Collection 10 Collection 10 was the inventors’ final collection of data. At this point the majority of the Ash trees had lost their leaves or were severely discoloured resulting in no significant difference amongst the leaf qualities. The inventors also found there to be no significant difference across application methods. However, with regards to the bark quality for collection 10 it can be stated that there was a significance between the following pairings. The inventors found that LAte showed the most significance with regards to the other treatments and application methods. There was no significance found between LAte and the two Injection applications, yet the inventors believe over a longer time period a significance would follow, as they had begun to observe a change in the bark quality of both injection treatments. As well as this for early collections these two groups were found to have no significant effect on the leaf quality with them obtaining poor ratings for a number of the data collections. It was found that there was a significant difference between leaf applications due to the different treatments, showing that for the method of leaf application treatment e was more effective with regards to the positive health of the tree. Therefore, the inventors can state that LAte showed the most significance throughout the 20 week study, having had a significant positive effect on both leaf and bark quality for a number of weeks. They believe if this study was carried out over a longer period they would have begun to see even more significant results for example LAte showing a significant difference between the injection treatments. Table 8 In attached Figures 7 (a) and 7 (b), the inventors graphed the mean bark and leaf qualities in order to get a visual representation of the effect the treatments had over the 10 collections. These graphs support the inventors’ statistical analysis, with LAte showing a significant difference for a number of weeks for both leaf and bark quality. Treated dishes To conclude, with regards to the inventors’ treatment Experiments, the inventors found that treatment e (1:2,JA:SA, at a lower concentration) worked most significantly at inhibiting the growth of the fungus when compared to the other treatments and all control groups. The statistical significance for its results can be tracked from collection 2 onwards, meaning that in-vitro this treatment begins to exhibit significant inhibition results after 4 weeks. A significant difference can be observed within the controls at week 9, and the inventors hypothesise this is due to the increase in temperature after a heater malfunction contributing to the formation of methyl jasmonate which in itself has been proven to have inhibiting factors in vitro At collection 10 , the inventors saw no significance between controls. This means it is once again a fair comparison against treatments. We observed no statistical significance between all treatments, the only exception to this being treatment e in comparison to the other treatments. While all treatments were significant against the control meaning they all inhibited growth of the fungus significantly, there was no difference between the performance of the treatments discluding treatment e. The only exception to this statement being the comparison between treatments C and d for genotype 2. This means that there was a significant difference between treatment C and it worked significantly better than treatment d at inhibiting fungal growth after 20 weeks. Overall, treatment e was most effective across all three genotypes, decreasing the fungus up to 92.2% and on average 82%. Treatment e being the lower concentration 1:2 ratio, JA:SA at a molar concentration of about 2.3779 x 10-4:4.7558 x 10 - 4 . 'About' as used herein referring to the measurable value of the molar concentration of the solutions and is meant to encompass variations of + / - 20% or less, in particular + / -10% or less , more in particular + / -5% or less, even more in particular+ / -1% or less, and still more in particular + / - 0.1% or less of and from these specified concentration, such variations are appropriate to perform to similar inhibition rates on H. fraxineus in vitro. However, the value of the concentrations that the modifier 'about' refers to is itself specifically disclosed as the concentration invention to the treatment to decrease growth of H. fraxineus to the most significant extent. It is also important to consider the ratios of the two hormones within the treatment solution. In accordance with the present invention, the preferred composition comprises a ratio that is optimal at 1:2 of JA:SA. In-vivo study After analysing the results of the inventors’ in-vivo study for the 10 Collections, the inventors have concluded that the Leaf application of treatment e (LAte) had the most significant effect across the 10 collections. It was the first to show a significance with regards to leaf quality and upheld the quality for a period of 4 collections before we started to see a decline, most likely due to the change in weather conditions. It was also the most significant with regards to bark quality for the inventors’ final collection. The inventors found there to be a significance between LAte and both controls as well as the Leaf application of treatment B meaning that treatment e is more effective as a leaf application treatment. While there was no significant difference between the bark quality of LAte, and ITB and Ite, we had begun to observe a decline in quality for these groupings and believe if we had continued the experiment over a longer period we would have begun to see a significant difference between the groups. Based on the Analysis of this data we would recommend the leaf application of treatment e, as it had no ill effect on the overall effect on the growth or the health of the Ash whips as we saw no abnormalities with regards to the trend in leaf and branch count between the groupings. The amount of treatment applied is dependent on the circumference of the tree , as per the Ash whips within this study (with a circumference of 2.5-3 cm) the required amount was about 2 ml of the treatment so 1 ml :1 ml. The amount of treatment required per 1 cm of circumference is about 0.85 ml (0.425 ml: 0.425 ml). The term, “About” as used herein refers to the measurable volume of the amount of treatment required and is meant to encompass variations of + / - 20% or less, in particular + / -10% or less , more in particular + / -5% or less, even more in particular + / -1% or less, and still more in particular + / - 0.1 % or less of and from these specified amounts, such variations line within the acceptable range by which to apply the treatment to a tree, while ensuring its health and natural growth patterns. However, the value of the volume of treatment that the modifier 'about' refers to is itself specifically disclosed as the volume by which it is appropriate to apply such treatments to a live specimen. Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and / or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive. The invention is not restricted to the details of any foregoing embodiments. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

Claims

1. A treatment composition comprising Jasmonic acid (JA) and Salicylic acid (SA) effective in inhibiting the growth of the fungus, Hymenoscyphus Fraxineus , in-vivo, as well as preventing / delaying the symptoms which are presented due to the infection of trees by this fungus.

2. A treatment composition as claimed in claim 1 wherein the ratio of JA:SA in the treatment composition of the present invention is in the range of 1: 1.25 - 1:2.5.

3. A treatment composition as claimed in claim 1 or claim 2 wherein the ratio is about 1:2 of JA:SA.

4. A treatment composition as claimed in claim 3 wherein the JA:SA ratio of 1:2 is provided at relative concentrations of molar amounts of 2.3779 x 10-4 : 4.7558 x 10-4 JA:SA.

5. A treatment method for inhibiting the growth of the fungus, Hymenoscyphus Fraxineus, in-vitro, as well as preventing / delaying the symptoms which are presented due to the infection of trees by this fungus comprising the step of applying the composition of any of the preceding claims to a tree.

6. A treatment method as claimed in claim 5 , comprising the step of applying the composition of any of claims 1 to 4 toto leaves or to a canopy of a tree, in order to achieve the desired effect, in preventing symptoms of infection of trees by the fungus, Hymenoscyphus Fraxineus.

7. A treatment method as claimed in claim 5, comprising the step of applying the composition of any of claims 1 to 4 to the collar / root region of the treein order to achieve the desired effect, in preventing symptoms of infection of trees by the fungus, Hymenoscyphus Fraxineus.

8. A treatment method as claimed in claim 7 wherein the composition is applied by delivering the composition by injection of the composition into the tree in the region of the collar / root of the tree.33

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