Non-nematicidal composition and use thereof

Abstract

A non-nematicidal composition comprises at least one glucan and/or at least one fucan which act individually or synergistically with mannitol to reduce losses in crop yield and marketable grade caused by the infestation of growth media with plant pathogenic nematodes, to levels equivalent to those achieved with commercial nematicides, but without posing a risk to the ecosystem or user. In some cases the composition comprises at least one glucan, at least one fucan and at least one mannitol which may be in a weight/weight ratio of approximately 1:2:3 of at least one glucan:at least one fucan:at least one mannitol or between approximately 1:1:1 to 1:1:3 of at least one glucan:at least one fucan:at least one mannitol.

Claims

1. A method for controlling plant pathogenic nematode populations and/or improving soil microbial dynamics whilst maintaining or increasing beneficial nematode populations, without requiring the use of any chemical pesticides or nematicides, comprising the step of: applying a composition comprising only one active ingredient to a plant or plant growth medium which is infested with plant pathogenic nematodes, wherein the active ingredient consists of glucan, wherein: the composition is at least as effective as commercially available synthetic nematicides in terms of reducing crop yield losses and symptoms of nematode infection, and wherein the composition does not pose a risk to the ecosystem; and does not pose a risk to an individual applying the composition.

2. The method as claimed in claim 1, wherein population dynamics or population density of beneficial nematodes are maintained or altered to levels which enhance the overall soil, soil ecosystem, soil fertility, levels of soil biota and microbiota and/or to levels which reduce numbers of other pathogens and/or pests.

3. The method as claimed in claim 1, wherein the beneficial nematodes are selected from beneficial bacteria/fungal feeding nematodes, entomopathogenic nematodes, grazing species, predatory species, colonizers or persisters.

4. The method as claimed in claim 1, wherein the glucan: is isolated from a brown macroalga of the class Phaeophyceae which may be derived from one or more of the families Laminariaceae, Fucaceae or Lessoniaceae; is isolated from a brown macroalga of the Ascophyllum species; is isolated from a brown macroalga of the Laminaria species; is isolated from a brown macroalga of the Sargassum species; is derived from red alga of the class Florideophyceae; or is produced by means of synthetic chemistry and/or biotechnology approaches.

5. The method as claimed in claim 1, wherein the composition is applied in an amount such that greater than 60 grams/Hectare of the glucan is applied to the growing area.

6. The method as claimed in claim 1, wherein the composition is applied at different stages of crop or plant growth, including: pre-planting stage; planting stage; sowing stage; at regular intervals throughout the plant life cycle; during key developmental stages including seed germination, vegetative and root growth, flowering, blooming and fruiting; post-harvest; during the off season period; or during periods of crop rotation.

7. The method as claimed in claim 1, wherein the composition is applied by one or more of the following means: seed treatment; soil or growth media application; irrigation; drip irrigation; foliar application; fertigation; root application; tuber application; or plant application.

8. The method as claimed in claim 1, wherein the composition is in a liquid, powder or granular form.

9. The method as claimed in claim 1, wherein the composition is applied on a regular basis at a minimum rate of approximately 60 g/Hectare.

10. The method as claimed in claim 1, wherein the plants are selected from: families of non-flowering, seed producing plants belonging to the Gymnospermae division; families of flowering plants belonging to the Angiospermae division; and plants selected from the group consisting of (a) grain legume crops, vegetable crops, cereal crops, root and tuber crops, plantation, tree and cash crops, fruit and nut crops, ornamental, nursery and flower crops, grass and turf grass, cool season crops and cover crop or (b) annuals, biennials and perennials.

11. The method as claimed in claim 1, wherein enhanced growth is conferred in vegetative tissues or reproductive plant organs selected from root, rhizoid, stem, leaves, flower, seed, fruit, cones, strobili or spores.

12. The method as claimed in claim 1, wherein increments in growth, yield or marketable-grade of plants are achieved by enhancing tolerance to biotic stress and secondary diseases, altering food supply or favourably interfering with the nematode life-cycle, fecundity, development or digestive system in a direction of decreased pathogenicity.

13. The method as claimed in claim 1, wherein crop yield and marketable grade are enhanced in conditions known to otherwise negatively impact on the efficacy of commercial nematicides.

14. The method as claimed in claim 1, wherein conditions known to otherwise negatively impact on the efficacy of commercial nematicides include non-favourable weather or climatic conditions.

15. The method as claimed in claim 1, wherein the composition does not pose a danger to bees or harm bee populations.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The invention will be more clearly understood from the following description of an embodiment thereof, given by way of example only, with reference to the accompanying drawings in which:

(2) FIGS. 1A and B are graphs illustrating the positive relationship between Potato Cyst Nematode (PCN) numbers and potato yield and marketable grade following application of Composition 2;

(3) FIG. 2 is a graph which illustrates the population dynamics of free living nematodes (FLN) throughout the growing period for each composition; and

(4) FIGS. 3A-D are graphs demonstrating the efficacy of Composition 2 in reducing localised and systemically induced fungal lesions in oilseed rape plants grown under glasshouse conditions.

DETAILED DESCRIPTION

(5) We describe a composition which is shown to improve growth, yield and marketable-grade of plants grown in media infested with plant-pathogenic nematodes, to levels comparable with those otherwise achieved through treatment with nematicide alone. Moreover, this composition is naturally-derived and shown not to be nematicidal, as nematode population numbers are not significantly reduced by the composition. Hence, this invention provides a natural, effective, safe alternative and economically viable means of enhancing the growth and yield of crops grown in a nematode infested growth media.

(6) Seaweed Extracts

(7) There is considerable interest in the possible plant health and growth promoting properties of seaweed extracts (Russo 1990 and Chojnacka et al., 2012). Evidence suggests that a number of compounds from seaweed may improve plant growth in the presence of nematodes. For example, some studies indicate that seaweed extracts can increase growth of Arabidopsis thaliana and tomato, while in some cases also impacting on nematode populations (Wu Y et al., 1998; Featonby-Smith B. C. and Van Staden J. et al.; 1983; Crouch et al. 1993A; Whapman et al., 1994). These effects are frequently attributed to the presence of compounds such as betaines and growth hormones such as cytokinins and auxins (Wu Y et al., 1998, Crouch and Van Staden 1993B, Stirk and Van Staden, 1997, Jenkins T et al., 1998). However, the levels of growth hormones in extracts of seaweeds used in these studies are often unclear, as methodologies used are frequently based on bioassays of growth hormone effects rather than taking direct quantitative measures (e.g. mung bean rooting bioassay). This approach is still widely applied today, despite its inherent limitations.

(8) Also emerging from these early studies was an apparent impact of seaweed extracts on nematode fecundity. A study on tomato plants by Whapham et al., (1994) demonstrated reductions in the number of eggs produced by Meloidogyne javanica females after one generation post-treatment with a commercial extract of Ascophyllum nodosum, ‘Maxicrop’. Further study by this group demonstrates a reduction in J2 numbers on treatment with this product, effects which they attribute to the betaine components of seaweed (Wu et al., 1997). However, much of this work was performed in laboratory conditions, for example, hatching of eggs in beakers containing seaweed media or water at a temperature of 20° C., and inoculation of plant with the second-stage juvenile (J2) infective stage. Furthermore, the ‘Maxicrop’ ash treatment also gave rise to significant reductions in J2, 63 days post-inoculation, which was not explained (Wu et al., 1997). Reductions in the number of Meloidogyne javanica females and egg recovery from plants treated with an extract of A. nodosum was reported by Wu et al., (1998) and attributed to levels of betaines in the composition. However, this study was carried out on Arabidopsis thaliana under controlled conditions. As the authors contend, these effects may be insufficient under normal agricultural conditions and furthermore, would have to be incorporated into other control measures, such as nematicides (Wu et al., 1998). Such studies are limited by several factors, such as a lack of comparison with commercial-grade nematicides, and in some cases, conclusions are based on findings from in-vitro work, effects which may not be observed in vivo, or in field conditions.

(9) Other approaches to examining effects of seaweeds on fungi and nematodes involve extraction with solvents including n-hexane, chloroform and methanol and ethanol. Sutlana V et al., 2008 demonstrate an effect of solvent (n-hexane, chloroform and methanol) and ethanol extracts of seaweed on increasing mortality of Meloidogyne javanica juveniles, while also suppressing infection of chilli roots in screen-house experiments and field plots. Nematicidal and antifungal effects of such extracts have also been attributed to oily fractions containing various fatty acid esters, obtained through ethanol extraction (Ara J et al., 2005). However, the potential for extraction agents to account for some of these effects may be significant, for example, methanol is known to enhance crop growth and performance (Nonomura et al., 1992, Li, Y et al., 1995). Furthermore, the effectiveness of these extracts have not been examined in large-scale trials and compared with current and effective commercial nematicides. While Sutlana V et al., 2011, report an effect of Solieria robusta, on reducing nematode gall numbers to levels similar to carbofuran, dried seaweed was used. Properties of dried seaweed meal are highly variable both regionally and between species and without clearly defined extraction procedures, it may be difficult to standardise or even repeat such effects. Indeed, many of the early studies into seaweed and crop growth in presence of nematodes have not been repeated. Where positive results have been found, extracts from a large number of species have been shown to be ineffective (Paracer S et al., 1987) and in some cases, extracts are found to negatively affect plant growth and reduce tolerance to nematode attack (De Waele D et al., 1988). Recent efforts have also failed to identify an extract of seaweed which can provide the protection and yield increases to levels otherwise achieved by commercial nematicides (Martin T J et al., 2007). Thus, there remains no seaweed-based composition to-date which can provide benefits to crops to levels currently achieved through use of registered nematicides.

(10) In conclusion, while the evidence from the 1980s to present day suggests that seaweeds may promote plant growth in the presence of nematodes, 30 years have passed without an effective or viable seaweed-based alternative to the use of commercial nematicidal treatments being developed. In spite of their dangers to the environment and other hazards, commercial chemical-based nematicides remain the only practical method of counteracting the effects of nematode infestation on crop performance. For a natural alternative to nematicides to be viable in agriculture, it must demonstrate effects which reach equivalence to or greater to those achieved using commercial nematicides alone. The lack of safe and viable alternatives to use of dangerous nematicides is addressed by the present invention.

(11) Laminarin, Fucoidan and Mannitol

(12) The effects of polysaccharides derived from brown algae in enhancing plant performance in nematode infested grounds, particularly laminarin, fucoidan and mannitol, have not been explored. In mammals, there is considerable evidence to support a role for laminarin and fucoidan in enhancing various physiological and immunological parameters (Reilly et al., 2008, Novak et al., 2009, Leonard S G et al., 2011A and 2011B, Smith et al., 2011). There are also studies which suggest a role for glucans in eliciting plant defence responses (Klarzynski O, et al., 2000 and references therein, Wolskia et al., 2006). Also, mannitol has also been cited for use in osmotic priming (Dursun et al., 2012).

(13) However, these findings cannot be extrapolated to plant-parasitic nematodes. Besides the obvious differences between animal and plant morphology and physiology, findings relating to plant responses to bacteria, fungal and viral pathogen do not necessarily to apply to Animalia such as nematodes or insects. In particular, the processes involved in parasitism between nematode and plant host contrasts with mechanism employed by other pathogens. For example, several species of nematodes must transform root cells into feeding sites (syncytia), a process requiring the secretion of a complex array of effector proteins, the roles of which are still being elucidated (Hamamouch N et al., 2012). Thus, while induction of SARs may provide benefits against certain bacterial or fungal pathogens, defensive responses to nematodes are likely to be considerably more complex. Indeed, a study by Chinnasri B et al., (2006) shows that SAR inducers varied in their ability to reduce nematode reproduction on pineapple, with variation in potency likely due to different activation points along the signal transduction pathway of SAR. While acibenzolar showed a broader spectrum of control than BABA and riboflavin, increases in crop yield were not achieved, rather, adverse effects in pineapple growth were reported (Chinnasri B et al., 2006). Also, while early studies indicate that plant growth promoting rhizobacteria (PGPR) may be used in inducing systemic resistance in plants against pests and diseases (Ramamoorthy V et al., 2001 and references therein), these approaches have had limited success with nematodes.

(14) Overall, the effectiveness in using elicitors of systemic resistance in plants against nematodes is poorly understood. There are no known inducers of systemic resistance in plants which can provide benefits to crops to levels currently achieved through use of registered nematicides. While glucans are known elicitors of plant defence, it is unclear if such properties impart effective responses against nematode infestation or whether or not significant increases in crop performance can be achieved. Indeed, while recent attempts to develop compositions which are effective against nematodes have made reference to glucans as elicitors of plant defence (US 2009/0104222 (also published as EP2012591A2), EP1135026 A4 (also published as U.S. Pat. No. 6,582,961), US 2002/0004458 A1, U.S. Pat. Nos. 7,927,635 and 8,246,965), in no instance do glucans represent the active component of these compositions. Rather, glucans are generally cited merely as ‘enhancers’ for the composition being described. Thus, there has been no disclosure to date on the use of laminarin or fucoidan (or related glucan or fucan) or mannitol compounds as active ingredients in enhancing plant performance parameters in nematode infested grounds, nor is there any evidence of such effects reported in the scientific literature to date.

(15) A suitable source of the active ingredients in the compositions described herein is seaweed, in particular, species of brown algae. Methods of producing laminarin with anti-cancer application are disclosed in US 2003119780, US 20050065114 and US 20050095250, via extraction from seaweed as the raw material or by means of synthesis of anaologues. The method of extraction typically involves acid hydrolysis followed by centrifugation and then ultra-filtration, thereby giving rise to a purified fraction of laminarin. Species of seaweed such as Laminaria digitata contain water soluble forms of laminarin, thus negating the use of solubilisation steps in this process.

(16) For the compositions of the invention, there is no requirement for a specific conformation of laminarin/glucan as its three-dimensional conformation is not deemed to determine it mode of action, but rather the length of the chain and the nature of the bond. In some cases there is not a requirement for the laminarin to be separated from other algal sugars such as fucoidan or sugar alcohols such as mannitol, as these molecules impact distinct biological actions of their own and act in synergy with laminarin to enhance plant performance in the face of pathogenic challenge.

(17) The distinctive nutritional characteristics of seaweed includes a category of nutrients called sulphated polysaccharides. These are carbohydrate-related nutrients, also referred to as fucans, have been examined for their properties in mammals, of which include anti-inflammatory properties and inhibition of human complement activation in vitro (Blonden et al., 1995). Biological properties of these molecules in plants are unclear.

(18) The immunological properties of laminarin, both in its naturally extracted and synthethic form, have been investigated extensively. US2005095250, US 20030119780, US 20050065114 and US 20040127457 discuss anti-cancer and anti-inflammatory properties of laminarin. Since the 1970s, β-glucans have been recognised as playing a role plant-pathogen interactions. In modern times, elicitor properties of glucans has also been demonstrated in tobacco plants (Klarzynski O, et al., 2000 and references therein) and Wolskia et al., (2006) found an enhancement in protection conferred against Fusarium solani f. sp. eumartii and Rhizoctonia solani AG3 in plants and tubers. However, the extract used by Wolskia et al., 2006 was from a rhizoctonia isolate and it is difficult to extrapolate studies on fungi to organisms such as nematodes. As discussed herein, plant defence responses which are effective against one pathogen do not necessarily indicate effectiveness against another. Nor do defensive responses which are effective against nematodes necessarily correspond to increases in crop growth. While U.S. Pat. No. 5,750,472 disclose the use of laminarin in seed germination, there is no disclosure on plant growth and performance in the presence pathogenic nematodes.

(19) The use of β-glucans or α-fucans or mannitol, singly or in combination for improved plant performance and marketable grade is not taught or suggested by the prior art. Furthermore, none of the above references refer to the use of β-glucans or α-fucans or mannitol, singly or in combination for use in enhancing tolerance to biotic stress, altering food supply or favourably interfering with the nematode life-cycle, fecundity, development or digestive system in the direction of decreased pathogenicity. Nor do they refer to the benefits of mannitol, β-glucans or α-fucans, particularly those derived from seaweeds, as a means to improving plant growth, performance and marketable grade in nematode infested ground and act as replacements for registered, commercial nematicides.

(20) The invention will be more clearly understood from the following examples.

EXAMPLES

(21) The examples given are the results of investigative research on the effects of compositions containing laminarin, fucoidan and mannitol on potato and grass species in the face of challenge against G. pallida (cyst nematode) and Meloidogyne minor (root knot nematode), as a model for all flowering plants including families belonging to Solanaceae, Poaceae, Brassicacea and Amaranthaceae and all plant parasitic nematode species including families belonging to Heteroderidae, Anguinidae, Pratylenchidae, Tylenchulidae, Hoplolaimidae, Trichodoridae, Belonolaimidae, Longidoridae, Criconematidae, Aphelenchoididae, Dolichodoridae or Parasitaphelenchidae. The examples shown include field trials carried out on potatoes using seaweed extract containing laminarin, fucoidan and mannitol in combination in two compositions, Composition 1 and Composition 2. Fucoidan is also examined individually. In addition, glucan-alone, mannitol-alone, glucan+fucan, glucan+mannitol and fucan+mannitol are also examined as separate treatments. Comparisons with a commercially available nematicide, namely DuPont™ Vydate® (oxamyl) is also provided.

Example 1

(22) Aims: To develop a composition for use in increasing growth, yield or marketable-grade of plants grown in media or soil infested with plant-pathogenic nematodes, to levels comparable with DuPont™ Vydate® (oxamyl).

(23) Materials and Methods:

(24) Field trials were undertaken in 2008, 2009, 2011 and 2012 on soils known to contain pure Globodera pallida cyst nematode populations. Free living species of pathogenic nematodes are also known to be present within these soils, including the genera: Heterodera/Globodera (cyst), Pratylenchus (lesion), Rotylenchus (spiral), Tylenchus, Tylenchorhynchus (stunt), Paratylenchus (pin), Helicotylenchus (spiral). The trials were designed to examine the efficacy of compositions containing glucan, fucan and mannitol, in enhancing potato tuber yield and marketable grade as compared with a commercial nematicide, Vydate, and untreated controls. In each trial, the forms of glucan and fucan used were laminarin and fucoidan respectively. The optimal application rates and bioactive content of the compositions required to achieve these targets was determined throughout the five year field trial period. Each treatment was fully randomized within four plots. Each plot contained six 0.7 m wide drills with a length of 4.0 m in 2008 and 3.7 m in 2009, 2011 and 2012. Twelve tubers per drill were planted at 33 cm spacing in May of 2008 and 2011 and in the first week of June in 2009 and 2012. The cultivar Navan, (Solanum tuberosum L. ev. Navan) planted in 2008, 2011 and 2012 with Désirée planted in 2009. The four centre drills were used for data collection with outer drills serving as guard rows. Compositions were applied by foliar spray at 50% post-emergence and at approximately seven day intervals thereafter. Foliar spray was stopped after senescence was noticed and crops harvested in autumn. Compound fertilizers and fungicides were also applied at recommended rates and intervals, the latter in order to prevent against potato blight, Phytophthora infestans.

(25) In the 2008 trial, two compositions (Composition 1 and 2) were applied at a rate corresponding to a bioactive ratio of 1:2:3 of laminarin:fucoidan:mannitol. The application rate of bioactives per hectare was 73 g for Composition 1. The rate of bioactives applied in the Composition 2 treatment, termed ‘Composition 2A’, corresponded to a total of 67 g per hectare. Composition 1 was selected for a replicate trial in the following year on the basis of increased performance over Composition 2 in 2008. The bioactive ratio of Composition 1 was maintained at a standard ratio of 1:2:3 in 2009 and in all future trials in which Composition 1 was assessed. In the 2009 trial, Composition 1 was applied according to the same rate and bioactive ratio as in 2008. However, as Composition 1 did not provide benefits comparable to Vydate in 2009, further experiments were undertaken in the laboratory setting in 2010, with the aim of examining the potential benefits associated with increasing composition application rates (see Example 2).

(26) Application rates and bioactive content of the compositions were increased and examined in a field trial in nematode infested grounds in 2011. For Composition 1, the application rate was increased 2.5 fold compared to 2008, corresponding to 182.5 g/Ha, according to the same bioactive content and ratio as in 2008. The total bioactive content of the Composition 2 treatment in the 2011 trial, termed ‘Composition 2B’, was increased from 2008 to achieve an application rate of 410 g of bioactives per Ha. Composition 2B represents a re-formulated version of Composition 2A used in 2008, being applied according to a ratio of 1:1:2 of laminarin, fucoidan and mannitol rather than a ratio of 1:2:3. A further trial was undertaken in 2012 to examine the reproducibility of the increases achieved over Vydate® with Composition 2 in 2011. Composition 1 was also included in the trial, according to the same bioactive rates and ratio as in 2011. In 2012, the bioactive ratio of Composition 2 was adjusted and applied according to a ratio of 1:1:3 of laminarin, fucoidan and mannitol rather than 1:1:2 in the previous year. This corresponded to an application of 493 g of total bioactives per hectare (termed ‘Composition 2C’). Additionally, seed treatment with Composition 2C was assessed in 2012 which involved the application of a solution containing the composition to the seed potatoes and allowing to dry before planting. Planting took place within 24 hours of drying.

(27) In each trial, application of the foliar sprays were stopped after senescence was noticed as beginning to occur. Each block was comprised of: 1. Control 1: A fallow plot where no plants were grown or allowed to grow 2. Control 2: A plot planted with potatoes but without any treatment. 3. Control 3: A plot with the nematicide ‘Vydate’ (Oxamyl) incorporated into the soil at full rate and planted with potatoes. 4. Potatoes planted and Composition 1 foliar spray applied. 5. Potatoes planted and Composition 2 foliar spray applied.* * Composition 2 was not assessed in 2009.

(28) The harvested tubers were graded according to size as follows in 2008 and 2011: <45 mm: small, table potato grade; 45-65 mm: Table potato grade; >65 mm: Large baking type grade potatoes. The numbers and weight of tubers were recorded for each grade, with “marketable yield” defined as tubers (weight and number) which fall into the >45 mm category. In the 2012 trial, a similar grading system was used: <35 mm, 35-55 mm and >55 mm; with the >35 mm category defining “marketable yield”. The potential impact of applications on Potato Cyst nematode (PCN) numbers was also examined. This involved the collecting of soil samples from each plot immediately before planting to calculate the initial PCN population (Pi). This was repeated on the day before harvesting, giving the final population density (Pf). The PCN were extracted from the soil using a standard protocol for the ‘Fenwick Can’ process. The number of cysts per gram of soil and the number of eggs per cyst were calculated, thereby giving the number of PCN eggs per gram of soil for both pre planting (Pi) and pre harvest (Pf). These figures were then used to calculate the multiplication rate of the nematode (Pf/Pi). One-way ANOVA was used to test for differences between groups. Linear regression analysis was used to determine the correlation between PCN on yield parameters.

(29) Results:

(30) Performance and Marketable Grade

(31) In the initial field trial in 2008, Composition 1 significantly increased the total yield per plot compared to untreated controls (22444 g versus 19334 g, p-value=0.018*, Table 1), while increases achieved with Composition 2A (21488 g) were not statistically significant. Treatment with Composition 1 was also associated with a significant increase in marketable yield versus controls (21091 g versus 18055 g, p-value=0.029*). Both Composition 1 and 2A were associated with significant yield increases in the 45-65 mm marketable grade category (11253 g and 11438 g) compared to untreated controls (8094 g; p-values 0.027 and 0.021* respectively). Composition 2A also significantly increased numbers of tubers/plot in the 45-65 mm category compared to untreated controls (n=83 versus 60, p-value=0.031*). In contrast to Composition 1 and 2A, application of Vydate® (oxamyl) did not increase yield or marketable grade of potatoes. This was attributed to the high levels of rainfall occurring during this season, a factor known to limit the effectiveness of this nematicide. In conclusion, Composition 1 enhanced both total yield and marketable grade to levels greater than Vydate® or untreated controls, while Composition 2A achieved increases in marketable grade but not in total yield. On the basis of increased performance over Composition 2A, Composition 1 was therefore selected for a second trial in 2009.

(32) In 2009, a substantial increase in average yield per drill was achieved on treatment with Composition 1 compared to untreated controls (5528 g versus 3801 g, p-value=0.052, Table 2), corresponding to a 45% increase in yield over control. In contrast however, Vydate® (oxamyl) was associated with a considerably higher total increase in yield over controls (12817 g versus 3801 g, p-value>0.0001****). This trial confirmed that application of Composition 1 is associated with enhancements in potato yield on nematode infested ground. However, given the failure to provide increases in yield to levels comparable to Vydate®, the application rates and bioactive content of the composition were re-examined in the laboratory setting in 2010 (see Example 2) and adjusted thereafter to enhance efficacy ahead of the forthcoming field trial planned for 2011.

(33) The aims of the 2011 trial were to examine the potential impact of increased bioactive application on enhancing yield and marketable grade as compared to Vydate® (oxamyl). In this trial, application of Composition 2B alone achieved an increase in average yield per drill statistically indistinguishable to that achieved with Vydate® (9284 g versus 10556 g, p>0.05) and representing a substantial 37% increase over untreated controls (6775 g, p-value<0.011*, Table 3). Increases in overall marketable yield (>45 mm tuber size) were also achieved with Composition 2B to levels comparable to Vydate (7888 g versus 9342 g, p-value>0.05) and significantly higher than untreated controls (7888 g versus 5349 g, p=0.006**). Significant increases in the overall numbers of higher marketable grade potatoes (>45 mm) were achieved with Composition 2B compared with untreated controls (n=49 versus 35, p-value=0.026*), statistically indistinguishable to Vydate (n=56). While Composition 1 also achieved increases in the numbers of high marketable grade potatoes (47 versus 35, p-value=0.049*; Table 3), it was not associated with increases in total yield to levels comparable to Vydate. Composition 1 was associated with an increase in total yield versus controls (7664 g versus 6775 g), however this did not reach statistical significance and was substantially less than that achieved with Vydate® (10556 g).

(34) In conclusion, this trial demonstrates that treatment with Composition 2B is associated with significant increases in yield and marketable grade to levels comparable and statistically indistinguishable to Vydate® (oxamyl). The effectiveness of Composition 2 alone in enhancing yield and marketable grade to levels comparable to Vydate® was examined again in the field trial setting in 2012.

(35) In 2012, application of Composition 2C was associated with significantly higher yield per drill than untreated controls (6695 g versus 4929 g, p-value=0.001**; Table 4), with increases statistically indistinguishable to those achieved Vydate® (6695 g versus 6479 g respectively, p-value>0.05). Likewise, overall marketable yield (>35 mm) per drill was significantly increased on treatment with Composition 2C compared to untreated controls (6449 g versus 4654 g, p-value=0.001*) and to levels comparable to Vydate (6300 g). While Composition 2C had no effect on low and middle marketable grade, a substantial increase in the large weight grade category (>55 mm) was achieved over controls (4571 g versus 2780 g, p-value=0.001*) and to levels comparable to Vydate (4551 g). This effect was also observed at the level of tuber number (p-value=0.004**). Composition 1 did not increase yield or marketable grade significantly over untreated controls. However, Composition 1 was associated with a significant increase in yield in the lowest weight category both in terms of tuber number (n=19 versus 13, p-value=0.009**) and weight (<35 mm; 383 versus 274, p-value=0.014*; Table 4). Seed treatment with Composition 2C was also associated with a marginal total increase in yield over control and slight increase within the 35-55 grade.

(36) Overall, the findings of the 2012 trial demonstrate a significant increase in total yield and marketable grade on application of Composition 2, statistically higher than untreated controls and to levels comparable and statistically indistinguishable to those achieved with Vydate. Since similar effects were achieved in the trial undertaken the previous year, the effectiveness of Composition 2 on enhancing overall performance and marketable grade to levels comparable to Vydate, are shown to be reproducible. Overall, increases in yield and marketable grade were observed to be largely associated with the levels of bioactives applied per hectare. While application of 67-73 g/Ha is shown to be effective in enhancing yield and marketable grade (2008 trial) and also increased total yield in 2009, composition efficacy was enhanced at higher rates of bioactive application (2011 and 2012 trials). This is in line with observations from laboratory experiments showing the substantial effects of these type of compositions when applied at higher levels (see Example 2).

(37) Potato Cyst Nematode (PCN) Populations:

(38) Increases in tuber yield are achieved with compositions without negatively impacting upon nematode population numbers as demonstrated in Table 5. The multiplication rate of PCN (Pf/Pi) was not significantly affected on treatment with either Composition 1 or Composition 2 (Table 5). In contrast, treatment with nematicide (Vydate) is associated with a significant reduction in the nematode multiplication rate compared to untreated controls (Pf/Pi=0.62 and 17.28 respectively, p-value<0.05; Table 5), to levels similar to the fallow treatment. While a reduction in Pf/Pi was observed through use of Composition 1 in 2011 this was not significantly lower than untreated controls (7.04 versus 17.28, p-value>0.05, Table 5). Thus, in contrast to nematicide treatment, Compositions 1 and 2 do not significantly reduce nematode numbers. This demonstrates that the compositions do not act as nematicidal treatments when enhancing in crop growth and marketable grade. Moreover, in the case of Composition 2, drills with the highest levels of PCN were also associated with the greatest increase in overall yield and numbers of high-grade marketable tubers (r.sup.2=0.919, p-value=0.041*, and r.sup.2=0.986, p-value=0.007**, respectively, FIG. 1). This shows that Composition 2 achieves increases in yield and marketable grade without reducing parasitic nematode numbers, and moreover, is highly effective in the plots containing high levels of PCN.

(39) Free Living Nematodes (FLN):

(40) In 2012, the effect of compositions on free living nematode (FLN) was examined throughout the growing period. Plots were assessed for counts of FLN at selected time points. Nematicide treatment was associated with significant reduction in FLN counts compared to the untreated control during the growth phase (p-value<0.05, FIG. 2). In contrast, Composition 1 and Composition 2C were associated with FLN counts similar to the untreated control throughout the crop growth phase. This demonstrates that the Compositions do not negatively affect the overall soil ecosystem (FIG. 2). FLN counts were reduced for all treatments and the control close to harvest.

(41) Discussion

(42) These trials demonstrate that treatment of potatoes with Composition 2 is associated with significant increases in total yield and marketable grade, to levels comparable with a commercial nematicide DuPont™ Vydate® (oxamyl). In contrast to Vydate® (oxamyl) these increases were achieved without reducing nematode population numbers or having the nematicidal effects as are typically required in order for most commercial nematicides to be effective. In this manner, Composition 2, provides a means of enhancing agricultural output in the face of pathogenic nematode infestation. Composition 1 and 2 were also effective in non-favourable weather conditions known to otherwise negatively impact on the efficacy of commercial nematicides, such as the very high levels of rainfall which occurred in 2008, In contrast to commercial nematicides, Composition 1 and 2 do not posing any health risk or hazard to the individual applying the composition nor does they pose a danger to bees or harming bee populations. Furthermore, Composition 1 and 2 do not negatively affect the soil ecosystem.

(43) Optimal Rates of Application:

(44) Initial field trials demonstrated that the compositions could reach a level of efficacy equivalent to nematicide when applied at a rate of ≥67 g of bioactives per Ha (Composition 1 and Composition 2A). Therefore, 67 g represents the lower limit in which efficacy can be obtained from the compositions. However, ≥67 g/Ha did not produce reproducible results in the following year. By applying bioactives at a total rate of ≥400 g/ha, reproducible results comparable with nematicide were achieved in the 2011 and 2012 trials (Composition 2B and 2C). Overall, these trials indicated that while efficacy can be achieved at minimum rate of between 67 g/ha, higher rates of ≥400 g of bioactives per ha is required to ensure consistency year-on-year and achieve maximal yields. The higher rate of ≥400 g/ha refers to either the sum total of all three bioactives together, the sum total of dual synergistic combinations of the bioactives or the amount applied per hectare when applying the bioactives as individual, singular applications. While increases in yield can be achieved at a ratio of 1:2:3 of laminarin:fucoidan:mannitol (Composition 1 and 2A), a ratio of between 1:1:2 and 1:1:3 (Composition 2B and C respectively) achieves consistent increases in yield year-on-year.

(45) In conclusion, application of Composition 2 was associated with significant increases in yield and marketable grade in nematode infested soils, to levels comparable with nematicidal treatments, but without requiring direct nematicidal effects. The differences in efficacy between Composition 1 and Composition 2 points to differences in the levels of bioactives present in the two compositions.

(46) Further analysis of the efficacy of Composition 2 demonstrates an effect in reducing localised and systemically induced necrotrophic fungal lesions in oilseed rape plants (n=10 per treatment) grown under glasshouse conditions (minimum temperature: 12° C.). Localised Sclerotinia sclerotiorum lesion diameter was reduced by over 35% at the highest application rate (1.5%; p-value<0.0001****; FIG. 3(a), while lesion size was reduced by over 43% in systemically infected leaves at the same rate of application (S+2, systemic infection; p-value<0.0001****; (b). Similarly, localised Alternaria brassicae lesions were reduced by over 49% (p-value<0.0001****; FIG. 3(c) with a greater than 36% reduction observed in systemically infected leaves (S+2); p-value<0.0001****; FIG. 3(d). This trial demonstrates that Composition 2 is effective in reducing the size of fungal lesions induced by necrotrophic fungal pathogens Alternaria brassicae and Sclerotinia sclerotiorum. In particular, Composition 2 treatment achieves reductions in size of lesions arising from both localised and systemic infection. This systemic reduction is most notable in the case of Sclerotinia sclerotiorum infections, in which a greater reduction in lesion size is achieved on leaves S+2 (systemic infection), compared to Leaf ‘S’ (local infection). These effects are to likely apply to other fungal pathogens including biotrophic species.

(47) TABLE-US-00001 TABLE 1 Effect of compositions on total yield and marketable yield in 2008. Average yield per plot (11.2 m.sup.2) Tuber Numbers Tuber Weights (g) Yield increase Vs. controls (%) 45 Total 45 Total Marketable >65 mm-65 tuber Marketable mm-65 Total Marketable Tuber Tuber Treatment mm mm <45 mm No. yield >65 mm mm <45 mm yield yield no. Weight no. Weight Control 33 60 32 125 93 9961 8094 1279 19334 18055 n/a n/a n/a n/a Nematicide 34 62 26 123 96 9394 9386 1249 20029 18780 −2% 4% 3% 4% (Vydate) Composition 33 80 28 141 113 9837 11253* 1353  22444*  21091* 13% 16%* 22% 17%* 1 Composition 30 83* 34 147 113 8598 11438* 1452 21488 20036 18% 11%  22% 11%  2A Potato yields achieved in 2008 on treatment with Composition1, Composition 2A and Nematicide (Vydate) are described in the table above and classified according to marketable grade. Asterisks denote statistically significant increases in yield and marketable grade over untreated controls. Level of statistical significance denoted as follows: *P ≤ 0.05.

(48) TABLE-US-00002 TABLE 2 Effect of Composition 1 on total yield yield in 2009 Average yield per drill (2.59 m.sup.2) Total Yield Yield increase Vs. Treatment (Weight, g) controls (%) Control 3801.44 n/a Nematicide 12817.38**** +131.8%**** (Vydate) Composition 1 5527.81.sup.# +45%.sup.#  Total potato yields achieved in 2009 on treatment with Composition1 and Nematicide (Vydate). Asterisks denote statistically significant increases in yield over untreated controls. Level of statistical significance denoted as follows: .sup.#P = 0.09-0.05, ****P ≤ 0.0001.

(49) TABLE-US-00003 TABLE 3 Effect of compositions on total yield and marketable yield in 2011. Average yield per drill (2.59 m.sup.2) Tuber Numbers Tuber Weights (g) Yield increase Vs. controls (%) 45 Total 45 Total Marketable >65 mm-65 <45 tuber Marketable mm-65 <45 Marketable Tuber Tuber Treatment mm mm mm No. yield >65 mm mm mm Total yield yield no. Weight no. Weight Control 2 33 35 69.8 35 488 4860 1427  6775 5349 n/a n/a n/a n/a Nematicide  8* 48 36 91.5 56** 2216* 7125* 1215 10556*** 9342*** 31% 56%*** 61%** 75%**** (Vydate) Composition 2 45 32 78.3 47* 571 5842 1252  7664 6412 12% 13% 34%* 20% 1 Composition 7 42 29 77.5 49* 1804  6084 1397  9284* 7888** 11% 37%* 40%* 47%** 2B Potato yields achieved in 2011 on treatment with Composition1, Composition 2B and Nematicide (Vydate) are described in the table above and classified according to marketable grade. Asterisks denote statistically significant increases in yield and marketable grade over untreated controls. Levels of statistical significance denoted as follows: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.

(50) TABLE-US-00004 TABLE 4 Effect of compositions on total yield and marketable yield in 2012. Average yield per drill (2.59 m.sup.2) Tuber Numbers Tuber Weights (g) Yield increase Vs. controls (%) Total 35 Total Marketable >55 35 mm- tuber Marketable mm-55 Total Marketable Tuber Tuber Treatment mm 55 mm <35 mm No. yield >55 mm mm <35 mm yield yield no. Weight no. Weight Control 25 42 13 79 66 2780 1874 274 4929 4654 n/a n/a n/a n/a Nematicide 34* 35  8 77 69 4551** 1749 179* 6479** 6300** −2% 31%** 5% 35%** (Vydate) Composition 1 24 47 19** 90 71 2760 2208 383* 5351 4968 14%  9% 8%  7% Composition 35** 38 12 85 74 4571** 1878 246 6695** 6449** 8% 36%** 12% 39%** 2C Seed treatment 23 47 14 84 70 2627 2232 299 5158 4695 6% 5% 6%  1% Potato yields achieved in 2012 on treatment with Composition1, Composition 2C and Nematicide (Vydate) are described in the table above and classified according to marketable grade. Asterisks denote statistically significant increases in yield and marketable grade over untreated controls. Levels of statistical significance denoted as follows: *P ≤ 0.05, **P ≤ 0.01.

(51) TABLE-US-00005 TABLE 5 Effect of compositions on multiplication rate, Pf/Pi, of PCN. Multiplication rate Treatment (Pf/Pi) Significance Fallow 0.75 a Control (no Treatment) 17.28 b Full rate Nematicide 0.62 a (Vydate) Composition 1 7.04 b Composition 2B 18.23 b Within columns means sharing the same letter are not significantly different (significance at the p < 0.05 level). This trial was undertaken in 2011.

Example 2

(52) Aims: To examine the effects of Composition 1 and fucoidan on plant root growth in nematode infested growth media.

(53) Materials and Methods:

(54) The effects of Composition 1 and fucoidan on Creeping bentgrass and Perennial ryegrass root growth were examined in the laboratory setting in the presence or absence of plant parasitic nematodes in 2010. Seeds were pre-soaked in test materials for 16 hours each and left to germinate on wet USGA sand at 16° C. Roots were analysed using WinRHIZO image analysis system, following a 7 day period to assess mean root length following applications (n=10 seedlings per treatment). Experiments were also undertaken to assess the efficacy of Composition 1 and fucoidan in enhancing root length in the presence or absence of plant pathogenic nematodes. Seedlings (×7 replication) were established in sand columns containing USGA specification sand and sprayed on a weekly basis with either Composition 1 or fucoidan. Composition 1 was applied according to the same bioactive ratio as in 2008, but at a substantially increased rate corresponding to 1460 g of bioactives applied per hectare. Fucoidan was applied at a rate equivalent to 600 g/Ha. Root knot nematodes of the species Meloidogyne minor were inoculated around the roots in week 1, as second stage juveniles. Roots were analysed using WinRHIZO image analysis system following a 25 days period.

(55) Results:

(56) While Composition 1 had no effect on early root growth of either species of grass, a significant increase in mean root length of perennial ryegrass was achieved by soaking seeds in fucoidan versus untreated controls (48.9 mm versus 43.0 mm, p-value<0.05*, Table 6). In the absence of Meloidogyne minor infestation, Composition 1 was associated with a significant increase in Perennial ryegrass seedling root growth compared to untreated controls (79.6 mm versus 69.4 mm, p-value<0.05*, Table 7). A substantial increase was observed in the presence of nematodes (97.4 mm versus 42.3 mm, p-value<0.05), suggesting enhanced efficacy of Composition 1 in the presence of nematodes. Treatment with fucoidan was highly effective in increasing root growth in the absence of nematodes, thus suggesting a high level of efficacy for fucoidan in enhancing perennial ryegrass root growth in non-nematode infested growth media. In the presence of nematodes, efficacy was also highly effective.

(57) Significant increases in root growth were also observed on treating Creeping bentgrass with Composition 1 and fucoidan. In the absence of Meloidogyne minor infestation, Composition 1 resulted in a significant increase in root growth compared to the control (22.8 mm versus 17.4 mm, p-value<0.05*, Table 7). Likewise, a significant increase in root-length on application of Composition 1 was achieved in the presence of nematodes as compared with untreated controls (18.7 mm versus 12.4 mm, p-value<0.05*). Treatment with fucoidan yielded comparable results.

(58) TABLE-US-00006 TABLE 6 Effect of pre-soaking seeds with Composition 1 and fucoidan on early root growth. Mean Root length (mm) Perennial ryegrass Creeping bentgrass Treatment (n = 10) (n = 10) Control 43.0 .sup.a 37.9 .sup.a Composition 1 38.1 .sup.a 37.7 .sup.a Fucoidan 48.9 .sup.b 39.1 .sup.a Within columns means sharing the same letter are not significantly different (significance at the p < 0.05 level).

(59) Within columns means sharing the same letter are not significantly different (significance at the p<0.05 level).

(60) TABLE-US-00007 TABLE 7 Effects of Composition 1 and fucoidan on Perennial ryegrass and Creeping bentgrass mean root length (mm) in the presence and absence of plant pathogenic nemtodes. Perennial ryegrass Creeping bentgrass (n = 7) (n = 7) Application −Mm +Mm Application −Mm +Mm Control 69.4.sup.a 42.3.sup.a Control 17.4.sup.a 12.4.sup.a Composition 1 79.6.sup.b 97.4.sup.b Composition 1 22.8.sup.b 18.7.sup.b Fucoidan 129.2.sup.c 102.2.sup.b Fucoidan 27.1.sup.b 20.2.sup.b

(61) Within columns means sharing the same letter are not significantly different (significance at the p<0.05 level). ‘+Mm’, denotes inoculation with Meloidogyne minor; ‘−Mm’, denotes conditions where inoculation with Meloidogyne minor did not take place.

(62) Discussion

(63) These experiments demonstrate that Composition 1 and a purified component thereof (fucoidan) significantly increases root growth of creeping bentgrass and perennial ryegrass in growth media infested with root knot nematodes, of the species Meloidogyne minor. Moreover, Composition 1 was shown to be as effective as fucoidan in the presence of nematodes, in spite of the composition containing lower levels of this bioactive. This suggests that the significant effects imparted by Composition 1 may be attributed to the presence of additional bioactives in the product which may be acting in synergy with fucoidan to stimulate plant growth in the presence of nematodes (laminarin and/or mannitol). Moreover, these findings indicate that the efficacy of such compositions may be increased when applied at rates which ensure high levels of bioactives are applied per hectare. This hypothesis was tested in field trials in 2011, 2012 (see Example 1) and in 2013 (Experiment 3).

(64) Composition 1 was less effective than fucoidan in the absence of nematodes, with fucoidan also having significant effects in both nematode infested and nematode-free media alike (Perennial ryegrass experiment). This indicates that fucoidan is highly effective in both a stressed and non-stressed environments. The efficacy of Composition 1 which contains lower levels of fucoidan may be enhanced in the presence of additional molecules (e.g. laminarin, mannitol) which act synergistically to obtain greater effects in nematode infested media. In addition, fucoidan was also effective as a treatment to Perennial ryegrass at pre-germination stages, with Composition 1 only effective in enhancing root growth when applied to established seedlings. Thus, while fucoidan represents an effective growth promoting composition when applied at the seed treatment stage, Composition 1 is as effective as fucoidan as a treatment once seedlings are established. Again, this points to synergistic effects between bioactives such as laminarin and/mannitol and fucoidan in Composition 1, effects which may become more apparent once seeds are established but less so before germination.

(65) In conclusion, treatment with Composition 1 and fucoidan are found to significantly increase creeping bentgrass and perennial ryegrass root growth in the presence of nematode infestation. From an economical point of view, Composition 1 would provide greater return on investment to growers due to the lower costs associated with it's production, while fucoidan would be less viable given the costs associated with purification. The significant effects observed with Composition 1 suggests a synergistic mode of action between fucoidan and other bioactives contained in the composition, such as laminarin and/or mannitol. Moreover, these findings indicate that composition efficacy is enhanced by ensuring high levels of bioactives are applied per hectare, an effect confirmed in field trials in 2011 and 2012 (see Example 1). The hypothesis that interactions between bioactives are potentially synergistic in their effects, was investigated in a field trial in 2013 (Example 3).

Example 3

(66) Aims: To determine the efficacy of glucan-alone, mannitol-alone, glucan+fucan, glucan+mannitol and fucan+mannitol in enhancing yield of potatoes grown in soil infested with plant-pathogenic nematodes, as compared with DuPont™ Vydate® (oxamyl).

(67) Materials and Methods:

(68) Differences in efficacy achieved with Composition 1 and Composition 2 in field trials between 2008-2012 pointed to a differential effect of bioactive levels within the two compositions (Example 1). Laboratory trials on grass species grown in the presence of Meloidogyne minor identified fucan (fucoidan) as one of the bioactives responsible for enhancing growth (Example 2). Despite lower levels of fucoidan, Composition 1 also achieved equivalent increases in root growth in the same trial as compared with the purified fucoidan. This suggested a synergistic effect of fucoidan with other bioactives within the composition, such as glucan and/or mannitol. A field trial was undertaken in 2013 to further determine which bioactives or combinations of bioactives are responsible for conferring enhancements in total and marketable yield in nematode infested growth media. As in previous trials, the forms of glucan and fucan used were laminarin and fucoidan respectively.

(69) Bioactives were isolated and stock solutions prepared to contain glucan alone, mannitol alone, glucan+mannitol, fucan+mannitol and glucan+fucan. The efficacy of the bioactive fractions were assessed in plots known to be infested with Globodera pallida. Treatments were fully randomized with plots of approximately 16 m.sup.2 in area and 6 drills wide. Twelve tubers were planted per drill. One metre spacing was placed between blocks with two drills fallow between treatments. The dimensions of the drills were 0.7 meters wide and 3.7 meters in length. The cultivar Navan, (Solanum tuberosum L. ev. Navan) was planted and compositions applied by foliar spray at 50% post-emergence and at approximately seven day intervals thereafter. Foliar spray ceased once senescence was noticed. For each of the 5 treatments, a total of 621 g of bioactives was applied per Ha. A nematicidal treatment, Vydate, was applied at full rate as a separate treatment. Standard compound fertilizers and fungicides were applied at recommended rates and intervals. The crop was harvested in November. Total harvest weight and marketable yield (>35 mm category) was measured, with comparisons between bioactive treatments and nematicide made by means of One-Way ANOVA.

(70) Results:

(71) Glucan-Alone

(72) Total yield in the glucan-alone treatment was statistically indistinguishable to Vydate (10524 g vs. 11337 g, p-value=0.21). Marketable yield with glucan-alone was also statistically indistinguishable to Vydate (10218 g vs. 11009 g, p-value=0.22). This demonstrates that yields achieved on treatment with glucan-alone are comparable to those achieved with Vydate.

(73) Mannitol-Alone

(74) Total yield in the mannitol-alone treatment was significantly lower than that achieved with Vydate (9652 g vs. 11337 g, p-value=0.009**). This was also observed at the level of total marketable yield (9324 vs. 11009 g, p-value=0.01*). This demonstrates that mannitol-alone does not achieve yields comparable to Vydate.

(75) Glucan+Mannitol

(76) Treatment with glucan+mannitol was associated with an almost 6% increase in total yield compared to that achieved with full rate Vydate (12028 g vs. 11337 g, p-value=0.28). Similarly, marketable yield was 6.7% higher for the glucan+mannitol treatment compared to Vydate (11748 g vs. 11009 g, p-value=0.25). This demonstrates that yields achieved on treatment with glucan+mannitol are comparable to those achieved with Vydate.

(77) Mannitol+Fucan

(78) Treatment with mannitol+fucan was associated with a total yield statistically indistinguishable to Vydate (11549 g vs. 11337 g, p-value=0.74). Marketable yield was also marginally higher and statistically indistinguishable from Vydate (11352 g vs. 11009, p-value=0.59). This demonstrates that yields achieved on treatment with mannitol+fucan are comparable to those achieved with Vydate.

(79) Glucan+Fucan

(80) Total yield achieved with glucan+fucan treatment was statistically indistinguishable from that achieved with Vydate (10650 g vs. 11337, p-value=0.29) and similar to that achieved with glucan alone (10524 g). Likewise, marketable yield in both treatments were also statistically indistinguishable (10433 g vs. 11009 g, p-value=0.37). This demonstrates that yields achieved on treatment with glucan+fucan are comparable to those achieved with Vydate.

(81) Discussion

(82) The aim of this trial was to compare yields achieved with glucan-alone, mannitol-alone, glucan+mannitol, fucan+mannitol and glucan+fucan with those achieved with a commercial nematicide, Vydate. In most cases, yields were found to be comparable and statistically indistinguishable to Vydate. Yield with mannitol-alone, however, was significantly lower than Vydate. Glucan-alone in contrast, provided a yield comparable to Vydate.

(83) In contrast to their effects as single bioactive fractions, the effectiveness of glucan-alone and mannitol-alone were substantially enhanced by the presence of additional molecules. Of note, glucan+mannitol and mannitol+fucan treatments were associated with the highest marketable yields in the entire trial, and in both cases reached levels marginally higher than Vydate, albeit not significantly so. This points to a striking level of synergy between the bioactives. While mannitol-alone did not provide yields comparable to nematicide, the yield achieved with mannitol in the presence of fucoidan was comparable to both glucan+mannitol and Vydate. In addition, the glucan+fucan treatment was also associated with yields statistically indistinguishable to Vydate and marginally higher than glucan-alone.

(84) This trial demonstrates the efficacy of individual and synergistic combinations of laminarin, laminarin & mannitol, laminarin & fucoidan and fucoidan and mannitol. Optimal application rates of individual bioactives and their synergistic combinations are as specified in Example 1. The rate of ≥400 g/ha refers to either the sum total of all three bioactives together, the sum total of dual synergistic combinations of the bioactives or the amount applied per hectare when applying the bioactives as individual, singular applications.

(85) TABLE-US-00008 TABLE 8 Effect of bioactives on total yield and marketable yield in 2013. Average yield per drill (2.59 m.sup.2) Treatment Total Weight (g) Marketable weight (g) Full rate Nematicide 11337 11009 Glucan-alone 10524 10218 Mannitol-alone   9652**  9324* Glucan + Mannitol 12028 11748 Mannitol + Fucan 11549 11352 Glucan + Fucan 10650 10433 Potato yields achieved in 2013 on treatment with glucan-alone, mannitol-alone, glucan + fucan, glucan + mannitol, fucan + mannitol and nematicide (Vydate) are described in the table above. Asterisks denote statistically significant differences in yield versus full rate of nematicide. Levels of statistical significance are denoted as follows: *P ≤ 0.05, **P ≤ 0.01.

(86) Conclusions:

(87) In conclusion, this trial demonstrates that glucan-alone, glucan+mannitol, mannitol+fucan and fucan+glucan, but not mannitol-alone, achieve yields to levels which are statistically indistinguishable to Vydate. Furthermore this study demonstrates that the efficacy of these bioactives are enhanced when present in synergistic combinations of glucan+mannitol, fucan+mannitol and glucan+fucan, each of which increased marketable yield to levels statistically equivalent to nematicide.

(88) Modification and additions can be made to the embodiments of the invention described herein without departing from the scope of the invention. For example, while the embodiments described herein refer to particular features, the invention includes embodiments having different combinations of features. The invention also includes embodiments that do not include all of the specific features described.

(89) The invention is not limited to the embodiments hereinbefore described which may be varied in construction and detail.

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