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Crop Protection by Natural Raw Material Derived Biomolecules

Final Report Summary - NATUCROP (Crop Protection by Natural Raw Material Derived Biomolecules)

Executive Summary:
The NatuCrop consortium came together to address a global need for effective and sustainable solutions for reducing crop stress in order to deliver greater crop yields. The NatuCrop consortium consisted of 4 Small to Medium Enterprises (SME's) and 3 Research Institutes from 5 European countries, each of which brought significant knowledge and expertise to the consortium. The consortium was funded under the FP7 instrument "Research for the Benefit of SME's" which meant that technology development was strongly guided by end-user and market requirements. The consortium primarily focussed on developing next generation plant biostimulant based solutions for plant abiotic stresses such as heat, drought and salinity. Plant biostimulants contain substance(s) and/or micro-organisms whose function when applied to plants or the rhizosphere is to stimulate natural processes to enhance/benefit nutrient uptake, nutrient efficiency, tolerance to abiotic stress, and crop quality.

During the 2 years of the NatuCrop project a significant amount of new knowledge was generated on the impact of biostimulants and their combinations on plant biotic and abiotic stress. The project was primarily focussed on alleviating abiotic stress in plants with the primary indicator of success being increased height, biomass or fruit/grain yield. The consortium utilised 5 different plants/crops to evaluate efficacy, these included; Tomato, Barley, Wheat, Potato and Arabidopsis Thaliana. A library of biostimulant formulations was screened for efficacy to reduce a range of biotic stresses such as: Phytophthora infestans; Rhynchosporium commune; B. graminis (powdery mildew) and Fusarium Oxysporum and abiotic stresses including; salt, heat, cold and drought.

The project led to the development of a number of tools to allow manipulation of natural biomolecules by changing their molecular mass, degree of substitution and solubility characteristics. In addition an analytical toolbox was developed to allow a more extensive characterisation of seaweed derived biostimulants. This knowledge allows for better control of product composition and enhancement of the efficacy of biostimulants to deliver more yield with better performance consistency.

The performance of the developed biostimulants exceeded expectations in growth room, glasshouse and field trials. In some instances performance far exceeded the original targets of the project. In a tomato field trial in Spain the best performing biostimulant delivered a 33% increase in fruit yield over conventional farm practice. An additional benefit of this biostimulant was that the performance was delivered with 50% of the normal fungicide rate. This reduced fungicide feature provides an important benefit in a sustainable agriculture context. A field trial in winter Barley in Scotland reported the best performing biostimulant delivered a 9% increase in yield even when little biotic or abiotic stress was experienced by the crop. These results illustrate the potential of NatuCrop developed biostimulants to consistently deliver enhanced crop yields in a number of crop systems when subjected to varying degrees of stress. Validation of the NatuCrop biostimulants in alleviating specific abiotic stresses (salinity, heat, and drought) was achieved using trials in controlled environments. The results were extremely encouraging with consistent dry matter yield increases of >10% being obtained in tomato plants for the best performing biostimulants. Molecular tools allowed the establishment of the mode of action for the biostimulants in alleviating stress and have provided important information on how to further optimise the effect for further increases in yield. Gene microarrays provided results on the unique contribution of different biostimulants to the dysregulation of genes expressed in barley.

The NatuCrop project has allowed development of a biostimulant platform which provides scientifically tailored biostimulant formulations to meet specific crop productivity challenges and deliver the increased crop yields required to feed a growing world population.

Project Context and Objectives:
Global demand for food is expected to increase by 50% by 2030 and to double by 2050, due to population growth, urbanisation and increasing affluence in the developing world [1]. However, non-food use of land is increasing; key synthetic pesticides are being removed from many markets for safety reasons; and the climate is increasingly unpredictable. A re-surgence in biotic stress (pests, plant diseases, etc.) and abiotic stress (weather, drought, salinity) is imminent. A new approach to plant stimulation and protection is required, which combines consistent effectiveness with environmental friendliness. The aim of the NatuCrop consortium was to develop a 3rd generation biostimulant product consisting of an optimal blend of naturally derived biomolecules, with a well defined formulation that provides consistent stress protection to tomato, barley and wheat crops. The NatuCrop formulations focussed on different combinations of naturally derived biomolecules from seaweed extracts, carboxylic acids and chitosans. The target for the final product(s) was to have a consistent composition and a defined mode of action with excellent stress protection. Our goal was to obtain a 10% increase in yield of target crops (barley, wheat and tomato).

If record yields can be assumed to represent plant growth under ideal conditions then the losses associated with biotic and abiotic stresses can reduce the yield potential by 50 - 82% [2, 3]. The influence of biotic and abiotic stress are the principal contributors to this yield loss. Reduction of the impact of these stresses on the crop can lead to increased yield. Approximately 25% of this loss can be attributed to biotic stress which is the primary focus of synthetic agrochemicals. The future opportunity to increase yields is to decrease abiotic stress in tandem with biotic stress. The NatuCrop consortium focussed on providing a solution to this market need with the goal of increasing crop yield for the end user.

The SME participants in the project are all involved in the supply chain of naturally derived 2nd generation plant biostimulants to the agricultural and horticultural market. A plant biostimulant is defined by the European Biostimulant Industry Consortium (EBIC) as a product that contains substance(s) and/or micro-organisms whose function when applied to plants or the rhizosphere is to stimulate natural processes to enhance/benefit nutrient uptake, nutrient efficiency, tolerance to abiotic stress, and crop quality. Biostimulants have no direct action against pests, and therefore do not fall within the regulatory framework of pesticides.

• Brandon Products specialise in the manufacture and distribution of a range of seaweed derived 2nd generation plant biostimulants for the agricultural and horticultural sectors.
• Hebridean Seaweed Company harvest seaweed from the ocean and process it into a dried seaweed meal for the manufacture of seaweed biostimulants.
• The Glenside Group distributes 2nd generation biostimulants and anti-crop stress products to end users in the agricultural and horticultural market.
• Carbotecnia manufacture carboxylic acids for plant supplementation.

2nd generation biostimulants (crude liquid extracts of natural materials) have been demonstrated by European and International end users to have a positive effect on plant health and yield. For the three biostimulant products (Seaweed extract, Chitosan and Carboxylic acids) the rate of efficacy against biotic and abiotic stress is too low to replace other environmentally harmful products. In addition the inconsistency of effect of 2nd generation biostimulants on crops is also an issue for end users who desire high crop yields on a consistent basis. These problems can be attributed to an inconsistent formulation (varying levels of biomolecules from batch to batch) and inadequate research into their mode of action [4, 5]. In the case of 2nd generation seaweed biostimulants little is understood on the mechanism of action. The use of “natural agents” for plant immune system stimulation and stress protection is an attractive proposition from environmental, human health and industry perspectives. Natural products have not been extensively targeted to-date for development due to a deficit in skills in dealing with and understanding the natural raw materials.

The overall objective of the NatuCrop consortium is to develop a product which uses a combination of biomolecules derived from seaweed, chitin/chitosan and carboxylic acids to “switch on” robust plant defences to biotic and abiotic stresses. Validating the biomolecules ability to switch on defences in a model plant (Arabidopsis thaliana) and barley is achieved using rt-PCR and Micro-array technology. Prior to beginning the project it was postulated that several different biomolecules would be required to switch on different defences against a spectrum of stresses. Therefore these biomolecules are well characterised, so that a product which consistently turns on the plant defences and increases yield is developed. The mechanism of action of the biomolecules needs to be understood in order to ensure that selected biomolecules do not overlap with regard to functionality and that all ingredients contribute to plant stress reduction. The transition from lab-scale experiments to glasshouse trials and field trials requires scale-up of the processes for extraction/modification of the selected biomolecules. The scaled up processes need to be validated and this can be achieved through field trial data on the NatuCrop product(s).

NatuCrop product(s) provide the consortium with a number of clear advantages over the competition in the market place which include:
• Consistency of formulation
• Higher efficacy
• Defined mode of action
• Broad spectrum of activity for biotic and abiotic stress
• The product(s) was to allow the practice of sustainable agriculture while enhancing crop yields and maintaining viability of enterprises.

The combinatorial product allows the consortium SMEs (Brandon Products, The Glenside Group and Carbotecnia) to be early responders to a growing market demand. For the end users in the agricultural and horticultural industry the NatuCrop product will substantially reduce the impact of de-registration of current agro-chemicals, will stimulate the plants to maintain/increase current yields and provide a more environmentally friendly and sustainable approach to maintaining the output levels and economic viability of their enterprises. The Glenside Group and Brandon Products have a major role in demonstrating the efficacy of the developed product at glasshouse and field trial level. This will inform product positioning relative to competitors in the market. The initial market positioning of NatuCrop 3G biostimulant will be as illustrated in Figure A.

Large agro-chemical multi-nationals have focused on GM crops to overcome issues being presented by biotic and abiotic stress and deregistration of effective agro-chemical products. GM crops have a number of acceptance and technical issues [6]. There are several competing natural biostimulants. However, they are chemically ill-defined crude extracts of natural materials and suffer from poor compositional uniformity, low efficacy and inconsistency of effect. There is little clarity regarding the mode of action for these products. Biocontrol agents are another category of “natural” product. However, these competitors also have issues of consistency and efficacy; these are significant hurdles to their mainstream adoption in crop management. Synthetic inducers of Induced Systemic Resistance (ISR) are a further category of potential competitors. The development of these products to-date has shown that they have a narrow spectrum of activity in protecting against biotic stress and they have not displayed robust performance at field level to-date [7, 8].

Figure A. NatuCrop’s Market Positioning Relative to other Crop Management Inputs
SWEs: seaweed extracts, CTSs: chitosans, BCAs: Biocontrol agents, ISRs: induced systemic resistance, R-COOH: carboxylic acids

Soundness of NatuCrop Concept
Following appropriate induction, plants are capable of mounting an enhanced defence capability, commonly referred to as induced resistance [9]. This type of induced resistance results in reduced damage due to biotic and abiotic stress [10]. Different naturally derived materials are capable of inducing ISR but little coherent information is currently available to demonstrate this. A major issue with the use of ISR as a management strategy is the consistency and magnitude of the response elicited and the spectrum of protection that it provides. These aspects of ISR are currently poorly understood for some elicitors or where knowledge is available the spectrum of protection and response consistency is poor. Data from initial research presented in Figure B confirms that these natural materials in combination have a significant effect in reducing symptoms of biotic stress and stimulating enhanced biomass accumulation.

The effect of specific chitin and chitosan structures has also been investigated by the RTD’s, ITT and JHI who have shown a strong influence for chitin and chitosan structure characteristics (acetylation and molecular mass) protecting the plant against biotic and abiotic stress. SME Carbotecnia have shown that carboxylic acids can carry out the function of improving parameters such as production level, fruit external and internal quality, aerial and radicular growth, crop adaptation to stress conditions or environmental milieus.

Research for SMEs
NatuCrop is an SME-driven project, and it fits with all of the core requirements of the ‘CAPACITIES-Research for the benefit of SMEs’ Call. The problems to be solved have been identified by the SMEs and their industry clients; the RTD performers have the capacity to solve these problems on behalf of the SME partners. The NatuCrop consortium involves seven partners with four SMEs and three RTD performers. The project idea was conceived by Brandon Products and the Glenside Group through their customer feedback (Horticultural and Agricultural end users). As none of the four SMEs in the consortium has the capacities or resources to conduct the necessary research, they have selected three RTD performers to develop the product and meet the needs of the end-users. The RTD performers are active in the scientific areas in the proposal and have the in-depth knowledge required to develop the requisite technology.

Scientific and Technological Objectives
In order to successfully develop the NatuCrop product, the consortium has the following measurable scientific and technological objectives:
(List of Figures)

[1] FAO, 2008. Food and Agriculture organization of the United nations. Tomato Production statistic.
[2] Boyer, JS. 1982. Plant productivity and environment. Science, 218: 443-448.
[3] Bray, EA., Bailey, SJ. and Weretilnyk, E. 2000. Responses to abiotic stresses. In (W Gruissem, B Buchannan, R Jones, eds,) Biochemistry and Molecular Biology of Plants. American Society of Plant Physiologists, Rockville, MD, pp 1158-1249.
[4] Norrie, J., Branson, T. and Keathley, PE. 2002. Marine plants extracts impact on grape yield and quality. Acta Hort. (ISHS), 594, 315-319.
[5] Colapietra, M. and Alexander, A. 2006. Effect of foliar fertilization on yield and quality of table grapes. Acta Hortic. (ISHS), 721, 213-218.
[6] Franks, JR. 1999. The status and prospects for genetically modified crops in Europe. Food Policy, 24, 565-584.
[7] Charudattan, R. and Dinoor, A. 2000. Biological control of weeds using plant pathogens: accomplishments and limitations. Crop Protection, 19, 691-695.
[8] Walters, D., Walsh, D., Newton, A. and Lyon, G. 2005. Induced resistance for plant disease control: maximising the efficacy of resistance elicitors. Phytopathology, 95, 1368-1373.
[9] Ton, J., Pieterse, CMJ. and Van Loon, LC. 2006. The relationship between basal and induced resistance in Arabidopsis. In: Multigenic and Induced Systemic Resistance in Plants (S. Tuzun and E. Bent, eds), Springer Science + Business Media, New York, pp. 197-224.
[10] Hammerschmidt, R. 1999. Induced disease resistance: how do induced plants stop pathogens? Physiological and Molecular Plant Pathology, 55, 77-84.

Project Results:
1.1 Introduction
The objectives of the NatuCrop project outlined in the previous section were achieved through the completion of 9 integrated and inter-dependant workpackages (Table 1). The outputs of these workpackages were in the form of deliverables with progress on the workpackages being monitored through the achievement of milestones.

Table 1: List of Workpackages Completed as part of the NatuCrop Project

All 20 deliverables targeted in the proposal were achieved during the 2 years of the project. The foreground knowledge generated in the project was reported in 11 of these deliverables and the specific results are detailed in Table 2. The detail on the results obtained is outlined in the subsequent sections.

Table 2: List of Results Generated from the NatuCrop Project

Seaweed Extraction, Fractionation and Compositional Characterisation (Result 2.1 and 5.1)

Ascophyllum nodosum (AN) was the species of seaweed investigated during this research programme as it is the species primarily used for biostimulant manufacture due to its excellent performance. There are a number of processing steps involved in the manufacture of biostimulants from Ascophyllum which may impact on their composition. In addition the geographical location of Ascophyllum harvesting has been reported to impact on the composition of the raw seaweed and the products manufactured from it. The NatuCrop consortium focussed on the compositional relationship between freshly harvested Ascophyllum nodosum, dried Ascophyllum nodosum meal and Ascophyllum nodosum based biostimulants. A number of key biomolecules were identified as being essential to deliver potent biostimulant effects, these biomolecules were measured using assays reported in the peer-reviewed literature with the levels being used to establish the relationship between the different forms of Ascophyllum nodosum (fresh versus dried) and the biostimulants generated from them.

Figure 1: The biomolecule and ash content of Fresh weed received from Location 1.
All values are expressed as a % of dry weight (i.e. total solid content). N = 3

It is evident from the data presented in figures 1 to 3 that a significant proportion of the fresh weed and dry meal is unaccounted for (other). This may be composed of protein, fat, cellulose, etc. Protein can be up to 10% of dry meal ( The major biomolecule component in fresh weed and dry meal is alginate. Literature values for alginate suggest 16 to 30% w/w, which is consistent with the values obtained in this study. Interestingly the levels of fucoidan and laminarin are lower than that reported in the literature. Fucoidan can range from 4 to 10 % (w/w) for Ascophyllum nodosum. Laminarin content for Ascophyllum nodosum can range from 2 to 3% in dry seaweed meal. It must be noted that location can be important and the FAO figures are for Canadian Ascophyllum nodosum. In addition the season/month of harvesting can also be important for the level of biomolecules in the raw material so perhaps this is influencing the results presented here.

Figure 2: The biomolecule and ash content of Dry Meal received from Location 1.
All values are expressed as a % of dry weight (i.e. total solid content). N = 3

Figure 3: The biomolecule and ash content of Dry Meal received from Location 2.
All values are expressed as a % of dry weight (i.e. total solid content). N = 3

The data presented in figures 1 and 2 suggest that there is an impact on composition by drying the meal with an increase in ash content and a reduction on polyphenol content. It is evident from the data presented in figures 2 and 3 that there is an impact for location on the ash content of the dry AN meal. All other variations were not found to be statistically significant.

The data presented in table 3 illustrates the importance of extraction conditions (pH, temperature and time) on the biostimulant composition. A relationship is evident between extraction conditions and the concentration of some key biomolecules such as uronics and polyphenolics. Optimising and controlling extraction conditions is key to delivering a third generation biostimulant with optimal and consistent performance.

Table 3: Impact of Ascophyllum nodosum extraction conditions on biostimulant composition

The major mineral components of the ash fraction from the 2 different extraction methods is presented in figure 4. The mineral composition was determined after aqua regia digestion using both Inductively Coupled Plasma – Mass Spectrometry (ICP-MS) and Inductively Coupled Plasma – Optical Emission Spectrometry (ICP-OES). It is evident again here that extraction conditions do have an impact on the mineral composition. Ext 1 is higher in Fe and K while Ext 2 is higher in As, Sr, Zn, B, Ca, Mg, Mn, Na, P, S, and Ti.

Figure 4: The Impact of extraction conditions on the mineral content of seaweed biostimulants
All values are expressed as a mg/Kg of dry weight (i.e. total solid content). N = 3

Further more detailed analysis was performed on the small molecule content of the 2 different Ascophyllum nodosum biostimulants (EXT 1 and EXT 2) manufactured from the seaweed using different conditions. This small molecule analysis initially focussed on the carotenoid content as these molecules are secondary metabolites which may have a significant impact on the growth and stress responses of plants. The results of the analysis of EXT 1, EXT 2 and dry seaweed meal is presented in figure 5. This data suggests that there is a relationship between biostimulant manufacturing conditions and carotenoid content with EXT 1 having 2.5 times more carotenoids than EXT 2. In addition the amounts of the different types of carotenoids appears to be influenced by the manufacturing conditions e.g. zeaxanthin in EXT 1 represents 8.1% of total carotenoids while it only accounts for 5.3% of the carotenoid content of EXT 2.

Figure 5: Carotenoid content analysis of Ascophyllum nodosum seaweed and biostimulants manufactured from it using different extraction conditions.
The seaweed meal had a carotenoid content of 24.4 ± 1.1 μg carotenes/ g DW whereas EXT 1 yielded 9.5 ± 0.2 and EXT 2 had 3.8 ± 0.2 μg carotenes/mL product.

More indepth analysis of the small molecule components which primarily reside in the polyphenol-rich fraction of the ascophyllum nodosum raw material and the biostimulants was performed in order to further understand the chemical complexity. Due to poor separation of major polyphenol components (i.e. phlorotannins) using standard chromatography methods, the consortium attempted to develop novel chromatographic means (Hydrophobic Interaction Liquid Chromatography) to better separate the complex polyphenols formed from the original seaweed tannins during preparation of the seaweed biostimulants. Despite following procedures established for analysis of similar components in other seaweeds satisfactory and reproducible chromatographic procedures could not be established. This outcome led to development of a non-targeted metabolic profiling approach which sought to discover which metabolites could be determined in the original seaweed material and the two biostimulant products. An initial composition of these mixtures after analysis using TOF MS revealed that there were substantial differences between the source material (ASCO) and the biostimulant products (figure 6).

Figure 6: HILIC separation of components from seaweed and seaweed biostimulants and analysis using TOF-MS The expanded section shows differences more clearly. The mannitol peak and a potential phlorotannin derivative (m/z 563) are denoted for illustration of differences.

Components were further separated and analysed using HILIC LCMSn and their mass spectrometric (MS) properties were gathered using an Agilent time-of-flight mass spectrometer (MS-TOF). The MS-TOF gives exact mass data (accurate to four decimal places at ppm levels) from which putative molecular (structural) formula can be obtained. Using resident MassHunter and MassProfiler software, putative identities of metabolites can be predicted from available databases. A condensed version of the original data set is shown in Table 4. Various classes of components are seen including phenolic components, fatty acid derivatives, sugars but also a large number of unknowns.

It is intriguing that certain small metabolites (especially phenolic components) were present in the product extracts but not the original seaweed; which suggests they have been released during the production procedures, or perhaps have arisen as breakdown products of other original components (e.g. phloroglucinol present in the products may arise from phlorotannin breakdown). After detailed examination of the data to assign other possible metabolites by cross-referencing with published sources, further MS data was obtained to confirm and validate the putative matches. This involved examination of the samples by re-running them using the same chromatographic system but on a MS system that could provide both exact mass capability but also provide high quality MS/MS fragmentation data. The Orbitrap MS data obtained helped provide further confirmation that certain components were apparently correctly identified but other components yielded common non-conclusive fragmentations (e.g. loss of water molecules or CO2) were insufficient for confirmation. A selection of components that passed this selection criteria and can be confidently identified are shown in Table 4. These amount to approximately one third of the potential metabolites identified. However this untargeted approach, which is novel in seaweed metabolite analysis, has uncovered tantalising evidence for the presence of a range of potentially interesting components including omega fatty acids and PUFAs etc.

Table 4 Components putatively identified by metabolite profiling approach

Fourier Transform Infrared Spectroscopy (FT-IR) was used as a technique to fingerprint the seaweed biostimulants obtained using the 2 different extraction conditions. The spectra obtained are presented in figure 7. The spectra do not tell us why the extractions are different but it does provide a rapid tool to show that they are different. This tool would prove useful in ensuring biostimulant compositional consistency.

Figure 7: FTIR spectra of seaweed biostimulants produced using two different extraction conditions.

Enrichment of specific biomolecules to enhance biostimulant efficacy was considered a logical step in the development process of a 3rd generation biostimulant. Enrichment was achieved by the NatuCrop consortium using a range of methods exploiting the solubility of the different biomolecules. These enriched fractions provide another level of diversity to enhance efficacy and provide information on what combinations are optimal for biostimulant effects in particular plant stresses. The composition of the enriched fractions is presented in Table 4.

Table 4: The effect of fractionation methodologies on the enrichment of key biomolecules

1.2 Production and Characterisation of Chitosan Oligomers (Results 3.1 3.2 and 3.3)
Chitin and chitosan have attracted much attention over recent time for their biostimulant activity and their ability to elicit effective defence responses in plants. Chitin is a naturally occurring acetylated linear polysaccharide composed of α1-4 linked N-acetyl-D-glucosamine units. It is primarily isolated from the exoskeleton of crustaceans, in particular shrimp. Chitosan is a derivative of chitin and can be generated by de-acetylating chitin to yield an α1-4 linked D-glucosamine polysaccharide. Chitin and chitosan produced at industrial scale can have a wide range of different specifications with the chain length, which is reported in terms of dynamic viscosity and the degree of de-acetylation the principal specification parameters. Additional parameters of quality include heavy metal content, moisture and particle size. Chitin is classified in product specifications as <50% de-acetylation while chitosan products have >50% de-acetylation. As the objective of NatuCrop was to develop biostimulants with combinations of different natural materials which could be applied to crops through foliar spray solubility was an important consideration for material selection. Chitosan is water soluble while chitin is not and this was one of the reasons chitosan was selected for representing this important class of biostimulant material in the NatuCrop formulations.

The initial work on chitosan involved validating the specification of material obtained from a number of commercial suppliers. This was considered an important step to ensure that adequate diversity was included in the study materials with a wide range of dynamic viscosities and degree of de-acetylation key attributes. Additional processed chitosan products were also obtained for development of a NatuCrop biostimulant formulation. This type of product had been hydrolysed by chemical means to reduce the molecular mass of the chitosan polymers. It has been widely reported in the peer-reviewed literature that low molecular mass chitosan oligosaccharides are more effective in eliciting biostimulant effects than high molecular mass chitosans. However on mixing chemically processed chitosan with Ascophyllum nodosum biostimulants it was found that they were not compatible as an insoluble precipitate formed. This result led to the investigation of other methods for reducing the molecular mass of chitosan polymers in order to make Ascophyllum nodosum compatible low molecular mass chitosan oligomers (CHOS).

In an attempt to develop an efficient process for the production of CHOS on a large scale, we studied the hydrolysis of chitosan catalysed by several commercial enzymes. All the enzymes assayed showed an appreciable level of activity in reducing molecular mass under standard reaction conditions as determined by viscometry (Table 5).

Table 5. Chitosan hydrolysis catalysed by different commercial enzymes

All the commercial enzymes evaluated showed a comparable chitosan hydrolysing activity, reaching a viscosity decrease of 78-88% in 24 h. The effects of enzyme action could be observed early, with the greatest viscosity decreases occurring in the first hour of hydrolysis. These viscosity changes are a result of the endo-type action of the enzymes on the polymer. The hydrolysis of the initially large chitosan chains cause greater viscosity decreases than the subsequent degradation of their shorter hydrolysis products.

The effect of temperature and pH on chitosan hydrolysis by the commercial enzymes was evaluated in order to establish optimal hydrolysis conditions. The chitosan hydrolysis rate increased with rising temperatures, reaching a maximum at around 40ºC for enzyme 1, enzyme 2 and enzyme 4. On the other hand, for all the enzymes assayed, apart from enzyme 3, higher temperatures lead to inactivation of the enzyme. Enzyme 3 showed a high thermal stability up to 50ºC, where no loss in enzyme activity was observed after 24h of incubation. Activity was not measured at pH values higher than 5.0 as a result of the low solubility of chitosan at pH values ≥ 6.0. Enzymes were active in pH range between 3 and 5. The optimum pH of the chitosanolytic activity of enzyme 1 (pH 4.5) markedly differed from those of the other commercial enzymes (pH 3.5).

The hydrolysis products resulting from enzyme 1P, 2P and 3B action upon chitosans with Degree of de-acetylation (DD) ranging from 78 to 90% were first divided into an insoluble fraction (LMWC) and a soluble one (CHOS). The insoluble fraction, containing low molecular weight chitosan was separated and weighed after lyophilisation. The soluble fraction composed of a mixture of oligosaccharides was analysed for yield (Table 6) and its degree of de-acetylation (DD) was calculated based on its corresponding IR spectra (figure 9).

Table 6. Product yield from enzymatic hydrolysis of chitosan for 24 h

The product distribution after a 24h hydrolysis showed some interesting features. Probably the most important was that the percentage yield of the CHOS fraction was 58-85%, 45-87% and 48-87% for enzyme 1P, 2P and 3B catalysed reactions, respectively. Chitosans with lower deacetylation degree were more prone to hydrolysis by these enzymes (Table 6).

Proportion of oligomer and the degree of polymerization are very important parameters for commercial applications of CHOS. These parameters have been evaluated by analysing the HPLC profiles of the 26 CHOS mixtures produced and using a standard curve to estimate the concentration of each oligomer (figure 8).

Figure 8. HPLC profiles of standard monomers and chito-oligosaccharides (Polyamine-II YMC column; isocratic gradient 68 % acetonitrile / 32 % water; 1 mL/min; 35°C; RI detection).

Typical chromatograms of the soluble CHOS fraction obtained with enzyme 1P showed, in addition to monomer peaks, the appearance of one major peak (trimer) and several smaller peaks corresponding to oligomers of DP 2, 4, 5 and 6 (Table 7). The percentage yield of individual chitosan-oligosaccharides (Tables 7-9) showed the presence of a moderate amount of unquantified oligosaccharides.

On the other hand, enzyme 2P mainly caused release of monomer, dimer and tetramer (Table 8). Although several small peaks were tentatively assigned to specific oligosaccharides species, such as DP5 and DP6, the extremely low levels of high DP oligomers released were near the detection threshold of the HPLC RI detector employed (Table 8). Likewise, the hydrolytic action of the enzyme on the chitosan samples resulted in a large yield of unquantified oligosaccharides (16-45 % w/w).

The product distribution after a 24h enzyme 3B hydrolysis showed some interesting features. Probably the most important was the detection of a higher amount of DP > 4 oligomers in these CHOS mixtures, ranging from 10 to 30 % of the total product weight (Table 9). This particular property of enzyme 3B allowed it to produce a significantly higher quantity of chitosan oligosaccharides capable of eliciting plant defence responses.

Table 7. Oligosaccharide composition from Enzyme 1P hydrolysis of chitosan for 24 h

Table 8. Oligosaccharide composition from Enzyme 2P hydrolysis of chitosan for 24 h

Table 9. Oligosaccharide composition from Enzyme 3B hydrolysis of chitosan for 24 h

Several procedures and equations are described in the literature for calculation of the degree of deacetylation (DD) using FT-IR spectroscopy. These equations were derived on the basis of calibration curves, where the calibration values of DD were determined by absolute methods like NMR. Calculation procedures are based on absorbance ratios of two spectral bands: a reference band and a probe band related to the amide group in chitosan molecules. A reliable calibration curve was obtained according to the skeletal vibration (reference band: 1030 cm-1), the Amide III band (probe band: 1320 cm-1) and the absolute DD values from chitosan standards.

Figure 9. FT-IR spectrum of chitosan. Reference, probe bands and corresponding baselines are indicated.The equation used in this procedure was:
A_1320⁄A_1030 =0.0037×DA+0.0852 r2 = 0.9599

The analysis of the results showed that the enzymatic hydrolysis of medium-high deacetylated chitosans (10-25 % DA or 75-90 % DD) with enzyme 1P, 2P and 3B released mixtures of short chitosan-oligosaccharides with similar average degree of N-acetylation. However, a lower DA is shown in those fractions hydrolysed with enzyme 2P. These values could be related to a higher presence of monomers, presumably deacetylated D-glucosamine (40-70 % DP1 content, Table 7).

The degree of N-acetylation of particularly physiologically active oligosaccharide pentamers were measured (Table 10). On the contrary to the average DA values most of the oligomers prepared by enzymatic hydrolysis with a DP>5 were highly-moderately N-acetylated (DA>50 %). These results are interesting mechanistically because original chitosans showed DA values lower than 25 %. Thus, it could be concluded that the endo-action of enzyme 1P, enzyme 2P and enzyme 3B cleaved only the glycosidic bonds leaving the N-acetyl groups intact, thus confirming their depolymerizing action rather than a de-N-acetylating effect on DP>5 oligomers.

On the other hand, although both chitin oligosaccharides (DA>50 %) and chitosan oligosaccharides (DA< 50 %) work as plant elicitors, the signalling and plant defence responses by oligomers of chitin and chitosan take different forms and intensities.

Table 10. Degree of N-acetylation values of DP5 oligomers determined by FT-IR

The biological activity of chitosan-oligosaccharides (CHOS) is known to depend on their degree of polymerisation and DD. Further analysis of the CHOS generated showed that a mixture of short chitosan-oligosaccharides, enriched in oligomers with a medium to high DD (74-85 %) could be easily produced from chitosan by enzymatic degradation using commercial enzymes.

1.3 Plant Growth Trials of Individual Biostimulants and Combinations (Result 2.2 6.1)
1.3.1 Tomato Trials
In order to establish the effect of biostimulant composition on plant growth and stress reduction a number of plant trials were performed. The results presented in this section primarily relate to Tomato plants with the results from Barley, Potato and Arabidopsis being presented in subsequent sections. Table 11 outlines the number of trials performed to assess the efficacy of biostimulants in reducing a number of abiotic and biotic Tomato plant stresses.

Table 11 Summary of the type and number of Tomato plant trials completed during the NatuCrop project.

The initial trials in tomato focussed on identifying the best chitosan oligosaccharide (from those studied in section 1.2) for inclusion in the NatuCrop biostimulant formulation. The fusarium oxysporum stress model was used for this selection and the results of the trials are presented in table 12.

Table 12 Summary of CHOS treatments and their effect on alleviating fusarium oxysporum stress based on plant biomass changes relative to healthy controls (that have and have not been exposed to pathogen).

The results of the CHOS trials in Tomato led to the selection of CHOS 231 as the best biostimulant to include in the initial NatuCrop formulations. The selection was based on performance (average 28% increase over stressed and non-stressed controls) and the cost of the raw material. For the best performing CHOS the severity of Fusarium Oxysporum disease symptoms and disease incidence were significantly reduced with no visible symptoms evident in some trials. The impact of the disease on plant size and health is evident from figure 10.

Figure 10: Effect of Race 1 fusarium oxysporum on Tomato Plants (var Money Maker). Left = Healthy control (no treatment, no pathogen); Right = Pathogen control (no treatment, + pathogen)

Once the best performing CHOS was selected, trials were carried out to evaluate the best combinations of biostimulants for inclusion in the NatuCrop formulation targeted at Tomato plants in order to alleviate the range of biotic and abiotic stresses outlined in Table 11. The effect of heat and salt stress on plant growth and biomass is presented in Figure 11, with statistically significant differences between the control plants, the system was accepted as a valid and reproducible method for evaluating biostimulant treatments. All combinations of Ascophyllum nodosum extract (A), CHOS (2), Carboxylic acid (C) and Phosphite (F) were evaluated. A summary of the results obtained are presented in Table 13.

Figure 11: (A) Effect of Heat stress on Tomato Plants (var Money Maker). Right = Healthy control (no treatment, no Heat); Left = Heat control (no treatment, + Heat). (B) Effect of Salinity stress on Tomato Plants (var Money Maker). Left = Healthy control (no treatment, no Saly); Right = Salt control (no treatment, + Salt)

Table 13 Summary of the performance of biostimulant treatments in alleviating stress based on Tomato plant biomass changes relative to stress controls and controls with no stress.

It is evident from the results that there is no specific formulation optimal for alleviating all Tomato plant stresses but rather specific formulations are effective at significantly reducing the plant stress leading to increases in biomass.

1.3.2 Potato Trials
To determine if treatment with individual biostimulants has an impact on the potato plants susceptibility to Phytophthora infestans, detached leaf and whole plants assays were carried out. No significant change in susceptibility to blight was observed over 3 replicate detached leaf experiments in response to XT50 or carboxylic acid. A small reduction in blight symptoms was seen in response to AQ36 treatment, however by far the most significant effect was seen in response to potassium phosphite. Potassium phosphite provided constantly strong protection against blight in Desiree plants, with the lesion area around 15% of that of mock treated leaves, making phosphite the most effective of all the treatments assessed. However, this treatment has previously been shown to be directly toxic to P. infestans and therefore is not suitable for use in NatuCrop.

Plants and detached leaves treated with CHOS were also assessed for susceptibility to P. infestans, lesion sizes on detached leaf segments treated with 231, 232, 75/20, 85/10 and 85/40 were significantly smaller than on mock treated control leaves, however, leaves treated with 231_LG and CPS010 showed an increase in disease symptoms (Figure 12).

Figure 12 Impact of CHOS treatment on potato blight. Detached leaves form 35 d old Potato plants (Desiree) were treated with CHOS and inoculated with Phytophthora infestans (Blue 13) spore solution. The Mean lesion area relative to a mock treated control is shown ± standard error (n=24). Significant differences between control and treated samples at a 5% (*) and 0.5% (**) confidence interval are marked with asterisks.

The second most effective treatment was the chitosan 85/400/A1. However, treatment with this biomolecule still resulted in a lesion with an area 70% of that of the mock treated control. The seaweed XT50 and 2 other chitosans (85/10/A1 and 85/60/A1) also provided a small (≈85%) but significant level of protection.

To determine if treatment with a combination of biostimulants had an impact on the potato plants’ susceptibility to P. infestans, detached leaf plants assays were carried out. The severity of the infection was assessed based of the area of the lesion formed on the detached leaves one week after inoculation (Figure 13). Treatment with CHOS preparations alone resulted in significantly smaller lesions compared to the mock treated controls. This effect was not observed in some earlier experiments looking at the effects of individual biomolecule treatment, however, the initial experiments only allowed 48 hours post-treatment before inoculation while these experiments had 7 days post-treatment before inoculation. In addition, some treatments that resulted in a significant reduction of infection in the original experiments had no effect during this experiment indicating that different biomolecules may require different lengths of time to elicit detectable activity, therefore correct timing of application is vital. It is apparent that many of the combined biomolecule treatments actually increased apparent infection levels above the controls.

Figure 13. Impact of combinations of biostimulants on potato blight.
Detached leaves from 6 week old potato plants (c.v. Desiree) were treated with seaweed extracts (XT50 and AQ36), carboxylic acid (COOH), CHOS (231 or 75/200) and combinations of these biostimulants and inoculated with Phytophthora infestans (Blue 13) spore solution 7 days after treatment. MeJA and Bion are commercial positive controls. The mean lesion area at 7dpi is shown ± standard error (n=24). Significant differences between control and treated samples (either reduced or enhanced infection) at a 5% confidence interval are marked with asterisks (*).

1.3.3 Barley Trials
Detached leaf assays demonstrated a reduction in R. commune lesion size in plants treated with AQ36, Carboxylic acid and potassium phosphite (Figure 14). XT50 had no significant effect on Rhynchosporium infection.

Figure 14 Impact of elicitor treatment on Rhynchosporium commune in barley. Detached leaves form 21 d old barley plants were treated with BABA, Cis-JA, Seaweed extracts (XT50 and AQ36), Carboxylic acid (COOH) and Potassium Phosphite (K Pho) solutions 24 h before cutting were inoculated with R. commune (214) spore solution 24 h after preparation. The Mean lesion area 10 dpi relative to a mock treated control is shown ± standard error (n=54). Significant differences between control and treated samples at a 5% (*) and 0.5% (**) confidence interval are marked with asterisks.

When the plants treated with CHOS solutions prior to detached leaf preparation and inoculation with R. commune, leaf segments from plants treated with 231, 231_LG, 85/100, 85/200, 85/400, 90/60, 90/100 and CPS010 had significantly smaller lesions than the mock treated control plants (Figure 15).

Figure 15 Impact of CHOS treatment on Rhynchosporium commune in barley. Detached leaves form 21 d old barley plants were treated with chitosan solutions 24 h before cutting were inoculated with R. commune (214) spore solution 24 h after preparation. The Mean lesion area 10 dpi relative to a mock treated control for 3 replicate experiments is show ± standard error (n=54). Significant differences between control and treated samples at a 5% (*) and 0.5% (**) confidence interval are marked with asterisks.

Detached leaf assays demonstrated a reduction in powdery mildew on detached leaves from plants treated with XT50 and potassium phosphite (Figure 16).

Figure 16 Impact of elicitor treatment on powdery mildew in barley. Detached leaves form 21 d old barley plants were treated with BABA, Cis-JA, Seaweed extracts (XT50 and AQ36), Carboxylic acid (COOH) and Potassium Phosphite (K Pho) solutions 24 h before cutting were inoculated with B. graminis spores 24 h after preparation. The mean number of colonies per leaf segment relative to a mock treated control is show ± standard error (n=18). Significant differences between control and treated samples at a 5% (*) confidence interval are marked with asterisks.

Barley plants grown in compost for 2 weeks were treated with biostimulant. Treatments included both seaweed extracts, carboxylic acid, Potassium Phosphite and 2 CHOS (231 and 75/20). 7 days post-treatment the plants were inoculated with powdery mildew, and the second leaf was scored for percentage leaf area with mildew colonies at 7 days post inoculation (Figure 17). No significant effect on B. graminis infection was observed in response to any of the other elicitor treatments. No significant effect on powdery mildew infection was observed in response to any of the chitosan treatments.

Figure 17. Impact of biostimulant treatment on susceptibility to powdery mildew in barley.
2 week old barley plants were treated with biostimulants, and challenged with powdery mildew after 7 days. Mean area of the 1st and 2nd leaves covered in powdery mildew colonies, 7 days post-inoculation is shown with ± standard errors (3 replicates, 24 plants).

Treatment of barley plants with the individual biostimulants did not provide any strong resistance to powdery mildew. Only CHOS 75/20 had a small effect on the levels of powdery mildew infection in this experiment and this was only statistically significant on the second leaf. These results were significantly different to that obtained in the detached leaf assays.

Experiments were also carried out looking at the effect of salt stress in barley (figure 18). Plants were grown in trays (24 plants per tray) for 2 weeks before treatment with biostimulants and combinations thereof. A week later, the plants were exposed to 10 ml 1M NaCl. The effect of the salt stress was assessed by taking chlorophyll fluorescence measurements and thermal imaging 48h post-stress application alongside analysis of fresh/dry weight and visual scoring at 7 days after stressing. In barley plants treated with salt, a reduction in biomass was seen in all treatments compared to the unstressed controls. Although, in some treatments this reduction was very small, minimal effects were seen in plants treated with XT50, AQ36/231 and AQ36/75-200. None of the treatments appeared to have a negative effect on biomass accumulation under salt stress conditions. Although all plants had some level of damage, this was universally higher in the plant expressed to salt stress. There was very little variation in level of damage with treatments.

Figure 18. Biomolecule combinations as protectants against salt stress in Barley. 2 week old golden promise plants were treated with biostimulants and exposed to salt (10ml of 1M per plant, Black bars) or water control (grey bars) 7 days later. 7 days after salt stress plants were assessed for fresh weight (A), dry weight (B) and visually scored (C). The mean weight of the salt treated plants relative to the control plants ± standard error is shown (3 replicates, 20 plants were pooled per replicate). Chlorophyll fluoresce measurements were taken 48h days after treatment (D), the mean Fv/Fm value ± standard error shown (3 replicates, 1 leaf from 10 plants per replicate). Thermal images were taken and analysed 48 h (E), the temperature relative to a constant control is shown (3 replicates, 1 leaf, 10 plants per replicate).

A reduction in chlorophyll fluorescence was observed 48 h post-salt stress application compared to the unstressed control plants. Once again XT50 and the AQ36/231 performed well and the reduction in plant treated with these compounds was only very slight. Combinations of 3 biomolecules also performed well in this test. A slight increase in leaf temperature was observed in the thermal image analysis of plants exposed to salt stress 48 h post-stress. However, some treatments lead to a reduction in this increase, with chitosan 75-200 the best performer.

1.3.4 Arabidopsis Abiotic Stress Trials
The effectiveness of biostimulants and their combinations in protecting Arabidopsis plants against abiotic stress was assessed. Two forms of abiotic stress were selected, salt stress and heat stress. A number of techniques were utilized to assess the effects of stress on the Arabidopsis plants. Traditional methods of assessing plant health were used in addition to the more novel techniques of thermal imaging and chloroplast development. Eight plants were assessed in each of 3 replicate experiments for each technique (figure 19).

Figure 19 Biostimulants as protectants against salt stress in Arabidopsis. 3 week old Arabidopsis plants were treated with biostimulants and exposed to salt (20ml of 0.5M per plant, Black bars) or water control (grey bars) 7 days post elicitor application. 7 days after salt stress, plants were harvested for fresh weight analysis (A) and visual scores (B) were assessed. The mean weight of the salt treated plants relative to the control plants ± standard error is shown (3 replicates, 8 plants were pooled per replicate). Chlorophyll fluorescence measurements taken 48h and 7 days after treatment (C, D respectively), are expressed as the mean Fv/Fm value ± standard error (3 replicates, 1 leaf from 3 plants per replicate). Thermal images were taken and analysed 48 h (E), the temperature relative to a constant control is shown (3 replicates, 3 leaves, 8 plants per replicate). Arabidopsis plants (col-0) were grown in compost for 3 weeks, before treatment with biostimulant solutions. One week post-treatment, 8 plants from each treatment were exposed to 0.5M salt solution, while a further 8 were treated with water as a control (Figure 12).

Salt treatment lead to a significant reduction in fresh weight in Arabidopsis plants (p<0.001) compared to those treated with water. However, none of the biomolecule treatments had a significant effect on fresh weight of the plants compared to the mock treated control (p = 0.504). Salt application also lead to a significant increase to the level of damage in all treatments (p<0.001) and no treatments had any significant effect on the level of visual damage to the plant in response to stress if compared to the mock stressed control. By 48h post-stress application the chlorophyll fluorescence in the salt stress plants was significantly lower than in the unstressed plants (p<0.001) and this had not been recovered by the 7 day time point (p<0.001). None of the treatments had any significant impact on the change in the chlorophyll fluorescence in response to salt stress at either time point.

Figure 20. Biostimulant combinations as protectants against heat stress in Arabidopsis. 3 week old Arabidopsis plants were treated with biostimulants and exposed to heat (8h at 45°C, Black bars) or control (grey bars) 7 days later. 7 days after heat stress plants were harvested for fresh weight (A), dry weight (B) and visual scores (C) were assessed. The mean weight of the heat treated plants relative to the control plants ± standard error is shown (3 replicates, 8 plants were pooled per replicate). Chlorophyll fluoresce measurements were taken 48h after treatment (D), the mean Fv/Fm value ± standard error shown (1 replicate, 1 leaf from 3 plants).

Both fresh and dry weights were greatly reduced in the heat stressed plants compared to the unstressed contra (figure 20)l. However, there was little difference in either fresh or dry weight between treatments. Visual damage was increased in response to heat stress, in all treatments, however, some biostimulant treated plants appeared to perform better than others, with AQ36/COOH, AQ36/75-200, XT50/COOH/75-200 and AQ36/COOH/75-200 the best performers. These biomolecule combinations also led to a reduction in the impact of heat stress on chlorophyll fluorescence, with XT50/75-200, COOH/231 and AQ36/COOH/231 also performing well in this test. However, there was large inter-experiment variation in the response of the Arabidopsis plants to the heat stress (see Figure 20).

Carboxylic acid treatment offered the best overall protection against freezing temperatures with the damage levels remaining low throughout the experiment (Figure 21). Initially AQ36 appeared to also offer good protection, however, by 72 h after exposure the plants started to show more signs of stress. XT50 and Potassium phosphite offered less protection initially but the recovery was rapid for these plants.

Figure 21 Protection from freezing temperatures by biostimulants. 3 week old Arabidopsis plants were treated with BABA, Cis-JA, Seaweed extracts (XT50 and AQ36), Carboxylic acid (COOH) and Potassium Phosphite (K Pho) solutions 24 h before exposure to -7°C for 24 h. Plants were scored for % damage 24, 48 and 72 h after exposure. The mean number of score is show ± standard error (n=12). Significant differences between control and treated samples at a 5% (*) confidence interval are marked with asterisks.

Several of the chitosan solutions offered protection against the freezing stress compared to the mock inoculated control (figure 22). 231, 85/200 and 90/60 offered the best overall protection. However, treatment with 232 resulted in an increase in damage in response to freezing temperature and showed little recovery over the course of the experiment.

Figure 22 Protection from freezing temperatures by chitosan solutions. 21 d old Arabidopsis plants were treated with CHOS 24 h before exposure to -7°C for 24 h. Plants were scored for % damage 24, 48 and 72 h after exposure. The mean number of score is shown ± standard error (n=12). Significant differences between control and treated samples at a 5% (*) confidence interval are marked with asterisks.

1.4 Effects of Biostimulants on plant signaling (Result 4.1)
Plants can respond to stresses in a variety of ways including physiological and metabolic changes. Plants have a complex signalling network which they use to trigger, coordinate and fine-tune these changes. Key components to this signal network are plant phytohormones, including salicylic acid (SA), jasmonate (JA) and ethylene (ET). It is often said that SA and JA are antagonistic and JA and ET work in union, however this is a very simplistic view and in reality all 3 hormones can work in union or be antagonistic to the others. In addition, other hormones such as abscisic acid (ABA) and brassinosteroids are also involved in stress signalling responses (Figure 1).

During this project we aimed to ascertain which of these pathways could be involved in the downstream signalling following application of the 3G biostimulant products. In order to achieve this we used molecular biology techniques, such as qRT-PCR, to investigate changes in transcript levels of classic marker genes for the involvement of the three key plant stress-related hormones (SA, JA and ET). Arabidopsis plants were chosen for the treatments with 3G biostimulant products and subsequent evaluation under abiotic stresses. All individual elicitors were assessed and the best preforming combinations in phenotype experiments were selected.

Figure 23.Stress-responsive network involving the JA, ET, SA and ABA signalling pathways. Different types of biotic or abiotic stress, such as pathogen infection or wounding, induce the synthesis and subsequent activation of several hormonal pathways (i.e. JA, ET, SA and ABA, shown in dark grey circles) (modified from Lorenzo and Solano, 2005). These hormones interact with one another via a variety of different signalling molecules (shown as white squares) and transcription factors (shown in pale grey) in either a synergistic or antagonistic manner ultimately leading to the induction of genes involved in a variety of stress responses. Abbreviations: ET: ethylene, ABA: abscisic acid, JA: Jasmonate, SA: salicylic acid, ERF1: ethylene responsive factor 1, MPK4: MAP kinase 4, NPR1: Natriuretic peptide receptor 1, MYC2: bHLH-zip transcription factor, PDF1.2: plant defensin, Thi2.1: JA-related gene expression, VSP: vegetative storage protein, LOX2: Lipoxygenase 2 and PR1: Pathogen-responsive 1.

Initial microarray studies carried out before the start of this objective indicated that treatment of Arabidopsis with seaweed extract components led to an increase in ET and JA signalling 2 weeks post-treatment, while transcript levels of SA-related genes were lower than in the untreated plants. This observation was especially prominent in plants treated with one specific seaweed extract referred to as AQ36. Initial studies looking at the effect of biostimulant treatment on defence-related hormones in barley plants were carried out. Two week old plants were treated with 0.33% v/v seaweed extracts (XT50/AQ36), 0.3% v/v carboxylic acid, 0.3% v/v phosphite or 0.125% w/v CHOS and 3 plants sampled at 1h and 24h after treatment. RNA was extracted and qPCR was carried out.

Figure 24. Response of defence hormone-related genes to elicitor treatment
The transcript levels of 2 JA-related genes (LOX2 and JIP23) and a SA-related gene (PAL) relative to the constitutive gene α-tubulin are shown for 14 day old barley plants at 1 h (V) and at 24 h ( V) after treatment with elicitor solutions. The mean of 3 replicates (± standard error) are shown.

Few significant changes in expression of any gene were observed in response to any of the biostimulant solutions. Only PAL displayed statistically significant differential expression in response to phosphite application at 1 h post-treatment. This indicates that potentially the SA pathways could be induced in response to treatments.

Relative gene expression was investigated for Arabidopsis plants one week after biostimulant treatment. qPCR was used to assess the relative transcript levels of a number of marker genes for plant stress-related hormones.

Figure 25. Effect of biostimulant treatment on hormone-related gene expression in Arabidopsis. 3 week old Arabidopsis plants were treated with biostimulant treatments and 1 week later above ground tissue was sampled. qRT-PCR was used to assess the transcript levels of hormone-related genes relative to Ubiquitin (3 replicates, 1 plant per replicate).

The jasmonate (JA) responsive gene LOX2 was down regulated in all samples compared to the mock treated control plant, but this was not significant in any of the treatments (p = 0.316). The JA responsive gene VSP was up-regulated in all treatments except 231, but this was also not significant (p = 0.479). It is worth noting that LOX2 has a negative regulatory effect on itself and therefore the low levels of LOX2 may be due to feedback from an earlier peak in expression. No significant change in ERF1 was observed (p = 0.773) but it is worth noting that the expression level in response to XT50 was reduced while an increase in expression was seen in all other samples. No significant impact on transcript levels was observed for either SA-related gene, NPR1 (p = 0.589) and PR1 (p = 0.892).

Relative gene expression was investigated for Arabidopsis plants 8h and 48h post-salt-stress application when treated with biostimulant combinations 1 week earlier. It was not practical to test all possible combinations as only limited effects had been observed previously. Only combinations of XT50, COOH and 231 where tested using qPCR. Relative transcript levels of a number of marker genes for plant stress-related hormones were assessed in plants exposed to 0.5 M NaCl and unstressed plants. The response of the hormone marker genes that were selected was very variable between replicates even in the unstressed plants, but more so in those exposed to salt stress. There are a number of possibilities as to the reason for this variation. At the time of sampling many of the plants were in an advanced stage in their life cycle and were already starting to flower. As plant hormones are heavily involved in all stages of growth and many changes in hormone signalling are involved with the flowering process it seems likely that variations in plant growth stage may be responsible for the variability in transcript levels. The variation observed in the gene expression of reporter genes post salt treatment are most likely a reflection of the various degrees of damage inflicted through the stress application in the independent replicates.

Samples taken 8h post-salt application were analysed using qPCR to assess the levels of key defence hormone-related marker genes. Transcript levels of LOX2, which is involved in JA biosynthesis, were down-regulated in response to all biostimulant treatments except XT50/COOH, which was very variable. However, as before, the JA responsive gene VSP was up-regulated in response to all treatments except the chitosan 231. Both Jasmonate-marker genes, LOX2 and VSP were down-regulated in response to salt stress in the untreated plants, indicating that jasmonate may not play a major role in the plants response to salt stress.

Figure 26. Effect of biostimulant treatments on hormone marker genes in response to salt stress. 3 week old Arabidopsis plants were treated with biostimulants and 1 week later exposed to salt stress (black bars) or water as a control (grey bars). Tissue samples were taken 8h post-salt stress application. qRT-PCR was used to assess the transcript levels of hormone-related genes relative to Ubiquitin (3 replicates, 1 plant per replicate).

The ET and JA-related gene ERF1 was up-regulated in all treatments with the exception of XT50 application but this was very variable. ERF1 was up-regulated in response to salt stress in the mock treated control and in the majority of treatments was either unchanged or up-regulated in response to stress. The SA-related gene NPR1 was up-regulated in response to all biostimulant treatments and there was a small response to salt stress by this gene. There was, however very little alteration in PR1 expression (an additional SA marker genes) in response to the biostimulants. It is possible that NPR1 expression could be an indicator of SA and redox potential changes as the formation of NPR1 monomers and polymers that display different activities, is dependent on the cell redox-state.

Tissue samples were also taken 48h after salt stress was applied, and RNA extraction carried out. However despite repeated attempts to extract RNA from some tissues no usable RNA was obtained, this may have been due to the high level of damage to the plants by 48h post-salt stress application. RNA was not obtained from a number of replicates

Figure 27. Effect of biostimulant treatments on hormone marker genes in response to salt stress. 3 week old Arabidopsis plants were treated with biostimulants and 1 week later exposed to salt stress (black bars) or water as a control (grey bars). Tissue samples taken 48h post-salt stress application. qRT-PCR was used to assess the transcript levels of hormone-related genes relative to Ubiquitin (3 replicates, 1 plant per replicate).
The missing data points make it difficult to draw any substantial conclusions from the results, however, there seems to be a general trend towards up regulation of JA/ET-related genes in response to biostimulant treatments. JA/ET-related genes also appear to be up-regulated in response to salt stress, with all 3 genes (LOX2, VSP and ERF1) being up-regulated in the salt stressed plants compared to the unstressed plants. An additive effect was also observed in seaweed extract and carboxylic acid treated plants with an additional increase in JA-related gene expression when salt stress is applied. As observed before, the response of SA-related genes to the biostimulant treatments appears more complicated, with no consistent response between the 2 marker genes.

1.4.1 Barley Microarray
Microarray experiments were carried out in barley to assess the effect of selected biostimulants and their combinations on the whole transcriptome and identify classes of genes altered in response to these treatment that may be responsible for phenotypic changes in these plants. The 2 biostimulants with the most potential (XT50 & 231) and their combination were selected for the analysis.

Plants were grown in trays containing compost, 24 plants per tray. 14 day old plants were treated with XT50, 231 or XT50/231 and tissue samples were taken 8h and 48h post-treatments, 4 replicates of 3 plants were pooled per treatment. A week after this sampling, phenotypic analysis was carried out on the remaining plants, 4 replicates of 3 plants were analysed.

The effect of the biostimulant treatment on plant phenotype a week post-treatment was assessed, with biomass, tiller number, length of longest leaf and chlorophyll fluorescence measurements taken.

Figure 28. Phenotypic response to biostimulant treatments in barley. 2 week old barley (c.v. Optic) plants were treated with 0.33% v/v XT50, 0.125% w/v 231 or a combination of the two. After 7 days treatment, plants were harvested for fresh weight (A), dry weight (B) analysis and number of tillers (C), longest shoot length (D) were assessed. The mean ± standard error is shown (4 replicates, 3 plants per replicate). Chlorophyll fluorescence measurements (E) were also taken, the mean Fv/Fm value ± standard error shown (1 replicate, 1 leaf from 10 plants).

A small increase in fresh weight was observed in the plants treated with the biostimulants and combined biostimulants, and this was more evident in the dry weight, with the combination performing the best in this test. XT50 had the biggest effect on tiller number of the individual biostimulants, while 231 resulted in the longest leaves, the combination of treatments performed as well as its best constituent component, for both parameters. The CHOS 231 and the combined treatment resulted in a reduction in chlorophyll fluorescence compared to the mock treated negative control. This measurement detects variable fluorescence within the plant photosystem II and lower values are indicative of the plant being stressed. One interpretation could be that the application of the biostimulants initiated some defense responses that yielded reduced fluorescence values as a trade-off.

Microarray analysis of the 48h samples identified a number of genes with altered expression levels in response to XT50 and Chitosan 231 as well as the combination of the two biomolecules (Figure 29). Although in the majority of cases, genes responded in the same manner to all treatments, there are cases of differential expression. However, it is worth noting that expression levels were low in many of the samples.

Figure 29. Transcriptional response to biomolecule treatment in barley.
Visual representation of transcriptional response to biomolecules XT50 and 231 and their combination, 48 h post-treatment, analysed using GeneSpring software. A line diagram (A) and heat map (B, red = up-regulated, green = down-regulated genes) show changes in gene expression in response to treatment.

Volcano plot analysis (figure 30) was carried out in order to obtained lists of genes differentially expressed in response to the biostimulants compared to the control treatment with a p-value above 0.05 and at least a 2-fold change. Treatment with XT50 resulted in 256, the chitosan 231 had 169 differentially expressed genes and the combination had 105 significantly altered genes. Relatively low overlap was seen between treatments, with just 6 genes being significantly altered by all 3 treatments, and little overlap between the individual biostimulants and the combined treatment were also observed (XT50 = 6, 231=22). This is confirmation that the individual bio-stimulants operate, as anticipated, independently of one another and thus differentially regulate different sets of plant genes. To identify pathways that are stimulated or perturbed by the bio-stimulants, a GO-term analysis was conducted.

Figure 30. Number of genes with altered expression in response to biomolecules.

Initial analysis was carried out using a list of all genes altered in any treatment. Analysis was with AgriGO software. Genes involved in development, regulation and multicellular organismal processes were all over-represented compared to the back ground list, but, perhaps most interestingly, genes involved in response to stimulus and immune system processes were also over represented in our list. When investigated more it was found that the majority of the genes identified were involved in response to stress, chemical stimulus and defence.

Figure 31. GO-term analysis of all genes altered in response to XT50.
Closest matches to Arabidopsis genes were used to carry out GO term analysis using AgriGO on a list containing genes responding response to XT50. Bar chart showing relative representation of gene categories compared to the reference gene set Tair10 (A) and a flow diagram showing GO-term classification for the data (B) are shown.

As observed in the list of all changed genes, XT50 treatment lead to over representation of genes involved in response to stimulus and immune system processes, and genes involved in localization. However, in contrast to the overall list, genes related to regulation of biological processes and biological regulation were under-represented in this list compared to the reference gene list. The only category of genes strongly enough represented in the list of be included in the flow diagram were those involved in defence response (Figure 31). Go-term analysis with the DAVID analysis tool, showed that genes involved in death and salt/osmotic stress were highly over-represented.

GO-term analysis showed the category of genes altered in response to chitosan over-represented by the largest factor is those involved in cell death (Figure 32 A). Genes involved in response to stimuli, cellular processes and metabolic processes were also over-represented in the list. The flow diagram identified those genes involved in response to stimuli, involved in response to stress and defence. DAVID analysis also identified genes involved in death and defence as being highly represented in the list of genes altered in response to 231.

Figure 32. GO-term analysis of all genes altered in response to 231.
Closest matches to Arabidopsis genes were used to carry out GO term analysis using AgriGO on a list containing genes responding to 231. Bar chart showing relative representation of gene categories compared to the reference gene set Tair10 (A) and a flow diagram showing GO-term classification for the data (B) are shown.

The list of genes changed in response to XT50 and 231 in combination were subjected to GO-term analysis using AgriGO (Figure 33). The most strongly over-represented categories of genes in this list where those involved in response to stimulus and multi-organism processes. None of the categories were strongly enough represented for a flow diagram to be produced, however, DAVID analysis of the same list of genes identified over-representation of genes involved in response to bacteria, organic substances and chitin, as well as innate defence response.

Figure 33. GO-term analysis of all genes altered in response to XT50 and 231.
Closest matches to Arabidopsis genes were used to carry out GO-term analysis using AgriGO on a list containing genes responding to a combination of XT50 and 231. Bar chart showing relative representation of gene categories compared to the reference gene set Tair10 (A).

1.5 Biostimulant Formulation and Scale-Up (Result 7.1 7.2 and 7.3)
The aim of the NatuCrop project was to produce a new 3rd generation biostimulant able to enhance yield through biotic and abiotic stress reduction. Scale-up of production of the biostimulant combinations was required in order to have adequate amounts of biostimulant material available for initial field trials in tomato, wheat and barley crops (section 1.6). Once the most robust biostimulant combination had been identified the next task for commercial exploitation was to be able to move the production of a NatuCrop 3G biostimulant formulation from lab scale to pilot scale and further to industrial scale in readiness for commercial exploitation.

Three different formulations were initially evaluated as being representative of all other combinations in terms of raw material properties (A2C, X2C and XTO). As an initial step in formulation development, the stability of individual components was analysed in order to determine the need for stabilizers or preservatives in the formulation. Results showed the need to use preservative in formulations containing carboxylic acid and CHOS biostimulants to be able to ensure adequate stability and shelf-life. The developed formulation led to a physico-chemically stable NatuCrop biostmulant formulation which met microbiological and physico-chemical acceptance criteria. Once the formulation was accepted as stable, the production of 231 CHOS as well as the Natucrop Biostimulant A2C were moved to pilot scale in order to produce 100 L of final product for future field trials. All unit operations in the production process were scaled-up with adequate performance and yield being obtained. The final product was shown to be comparable in specification and performance as that produced at lab-scale. Finally, equipment specifications and recommendations were made to be able to move the production to industrial level in the future.

1.6 Field Trials
1.6.1 Barley and Wheat Trials
All trials generally established well and were subject to low levels of both biotic and abiotic stress. No phytotoxic or developmental effects were observed by eye in any plots. For wheat there was no significant cultivar or treatment x cultivar interaction effects but treatments were significantly different (P<0.001). However, no treatment exceeded the fungicide treated yield (figure 34) but both the 231 chitosan and COOH carboxylic acid treatments were significantly reduced. For disease (AUDPC) there were significant treatment and cultivar effects (P<0.001) but no treatment x cultivar interactions. However, the dominant effect was attributable to the untreated plots as might be expected and as all other treatments included fungicides there was no biologically significant difference between the remaining treatments. For the vegetation index (NDVI) again there were significant treatment effects (P<0.001) but no cultivar or treatment x cultivar interactions. The fungicide treatment had the highest NDVI and the XT50 was also high possibly indicating some correlation with yield.

Figure 34. Winter wheat yield, LSD = 0.62.

For barley there was no significant treatment x cultivar interaction effects but both treatments and cultivars were significantly different (P<0.03 and P<0.001 respectively). Again no treatment exceeded the fungicide treated yield but both AQ36 and XT50 had higher values that were significantly greater than the untreated yield whereas the fungicide treatment was not. All other values lay between the untreated and the XT50 / AQ36 values. For disease (AUDPC) there was no significant treatment x cultivar interaction effects but both treatments and cultivars were significantly different (P<0.011 and P<0.001 respectively). AQ36 and XT50 both had significantly less disease than the untreated, as did XT+COOH+231. However, no treatments were significantly different from the fungicide treated alone and disease levels were generally very low so it is difficult to put much biological significance on these differences. For the vegetation index (NDVI) only cultivar differences were significant so no significance can be attributed to the treatment values.

Figure 35. Winter barley yield, LSD = 0.43

1.6.2 Tomato Trials
All plots were reported to establish well and develop along expected growth stages with the exception of one corner (6 plots on top right two rows) of the trial plots. These plots were excluded from analysis. As the primary focus of the trial was to establish the efficacy of biostimulants in reducing biotic and abiotic stress assessment of the stress pressure was initially determined. Unfortunately the stress pressure was minimal during the growing period.

It is evident from the data presented in Table 14 that significant treatment effects were observed with the 100 fungicide regime. Treatment 2F delivered a statistically significant 8% increase in yield with 100% fungicide. The phosphite treatment on its own (F) provide a >10% increase in yield although this was not statistically significant versus the untreated control at a P=0.05. A significant negative effect, with a 19% decrease in productivity, was obtained for treatment A2C. This treatment had been the best performer in growth room trials without any fungicide treatment so it might suggest an interaction is at play.

Table 14: Tomato yield and fruit quality attributes after treatment with biostimulants and 100% fungicide regime

It is evident from the data presented in Table 15 for the 50% fungicide trial that there is no commonality between the best and worst performers from the 100% fungicide trial. The biostimulant 2C with 50% fungicide regime significantly (p=0.05) outperformed the untreated control and the best performer from 100% fungicide regime with a 33% increase in marketable yield. This is an encouraging result.

Table 15: Tomato yield and fruit quality attributes after treatment with biostimulants and 50% fungicide regime

Figure 37: Statistical analysis and ranking of treatments with 50% fungicide regime. Ranking is based on % increase in tomato fruit yield.

Table 16 provides a summary of the performance of the biostimulants versus each other in order to determine the most potent formulation. These results suggest that a number of the formulations are not statistically worse than the best performer. This provides scope for further development of the other formulations for particular crop challenges.

Table 16: Summary of Tomato Trial Top Performing Biostimulant Formulations

1.7 Summary and Conclusions
1.7.1 Biostimulant Composition
The NatuCrop project has made important advances to current state of the art in processing and compositional analysis of natural materials (seaweed and chitin/chitosan) for use as biostimulants. The research findings on the compositional relationship between freshly harvested Ascophyllum nodosum and the processed dried meal provides an opportunity to better control this initial processing step in order to provide maximum recovery of bioactive biomolecules. The relationship between the compositions of dried meal and biostimulants manufactured from it, should prove extremely useful for processing optimisation and ensuring processing consistency. However results on compositional diversity in biostimulants due to varying extraction conditions is a significant advancement beyond current state of the art with many of the identified compounds not previously reported to be present in these Ascophyllum nodosum biostimulant formulations.

A significant amount of data was generated on the enzymatic processing of chitosan. A novel process for the formulation of chitosan with seaweed derived biostimulants has provided advancement beyond current state of the art. The data from this aspect of the project highlighted the diversity of chitosans available commercially and the impact of chitosan specification on chito-oligosaccharide (CHOS) yields from enzymatic processes. The identification and characterisation of relatively inexpensive commercially available enzymes for the processing of chitosan to potent CHOS for inclusion in biostimulants was an important outcome from a commercial exploitation perspective. Overall the data highlights the importance of biostimulant ingredient compositional characterisation and the impact of processing on it. These findings provide important support data for developing biostimulant performance consistency and additional research in this area is very likely to deliver the desired outcome.

1.7.2 Plant Trials
A robust and comprehensive plant trial programme was carried out over the course of the research programme. The majority of the trials were carried out on 3 different plant types (Barley, tomato and Arabidopsis thaliana) which provided biostimulant performance data on agronomic crops as well as a model plant. The data generated from the trials further re-enforced the impact of biostimulant compositional variation on plant biomass when subjected to various biotic and abiotic stresses. It was evident for a number of the biotic and abiotic stresses evaluated that the biostimulants delivered significant improvements in plant biomass and in some cases this effect was observed to translate into fruit/grain yield in the subsequent field trials. Of the 4 biotic stresses investigated during the project (Potato Blight, Powdery Mildew, Fusarium and Rhynchosporium) the investigated biostimulants provided significant levels of control in all cases and exceeded the performance of current commercial defence elicitors (BABA, BION and Cis-JA) in the majority of trials. The performance of the biostimulants with regard to abiotic stresses (heat, salt, drought and cold) was similar with some very encouraging performances in a number of plant health indicators such as dry biomass accumulation, % damage, relative water content and chlorophyll fluoroscence. Further optimisation of the lead biostimulants for each stress should be completed and is very likely to yield dividends in terms of plant/crop performance given the trends in the data obtained to date. This optimisation should focus on both the composition and the timing of the application of the biostimulant to the plants.

1.7.3 Signal Induction
Understanding how biostimulants deliver their beneficial effects in the plant is key in order to improve efficacy. Plant hormone signalling pathways are involved in initiating and controlling all plant responses whether growth or defence related. Biostimulants have been shown to impact these pathways. Elucidation of the key pathways influenced by biostimulants would allow tailoring of biostimulant composition to further stimulate pathways associated with protection against a particular biotic or abiotic stress. Salt stress was the model stress for this aspect of the NatuCrop programme and the focus was on Salicylic acid, Ethylene and Jasmonate signalling pathways. The data generated provided evidence of the differential regulation of these pathways by different biostimulant formulations with and without salt stress. This data provides an excellent baseline to further explore these pathways and the influence of biostimulants on them.

In order to gain a more global view of the impact of biostimulants on plant signalling and gene expression, microarray studies were performed using Barley as a model crop. This research provided data which is new to the current state of the art. The data clearly illustrated the impact of using biostimulants singly and in combination on the Barley genome and showed that each component provided a unique effect on gene expression when used in combination, therefore providing additional potential for plant productivity enhancement. This dataset provides some exciting opportunities to build on the biostimulant composition and plant gene induction relationship.

1.7.4 Field trials
On barley none of the treatments was detrimental to yield or disease resistance and the seaweed-based XT50 and AQ36 treatments alone with fungicides showed some potential for enhancing yield. This might be associated with biotic stress tolerance indicated by the trend in AUDPC data and these treatments were associated with the lowest but not significantly different NDVI values. These data are a good indication that these treatments alone have optimisation potential in future trials, particularly investigating dose rate and environment interactions. No treatment was beneficial to wheat and some showed detrimental effects. It seems unlikely therefore that further trials would give strong beneficial interactions in wheat, at least not for soft wheats under cool, near optimal environments. Both trials were grown on good grade 2 agricultural land following a leguminous crop so were under near optimal environments for these crops. Under sub-optimal environments we might expect differences between treatments to be enhanced as the crops might then be deficient in requirements that these treatments can supply. Similarly these trials were under minimal abiotic and biotic stress so stress-alleviation components of the treatments would not be strongly expressed. Therefore it would be advisable to carry out further trials, especially on barley, on crops grown under reduced nutrient conditions and across multiple trial sites to be exposed to a range of biotic and abiotic stress factors.

The tomato trial did provide useful data on fungicide reduction benefits and on productivity of a tomato crop. The performance of the biostimulants in a half rate fungicide regime was starkly different to that observed for the 100% fungicide regime. The biostimulant 2C with 50% fungicide regime significantly (p=0.05) outperformed the untreated control and the best performer from 100% fungicide regime with a 33% increase in marketable yield. This is an encouraging result. Further field trials in other locations and under other conditions will assist in building a matrix of efficacy for these biostimulants. Further manipulation of the best performing formulation may provide enhanced yield benefits beyond the 10% targeted in this project. The interaction with fungicides and the ability to reduce fungicide application rates without yield penalty is worthy of significant further evaluation given the yield benefits in the tomato trial.

1.7.5 Overall Conclusion
The research performed during the course of the NatuCrop project has provided data which has advanced current state of the art in the field of biostimulants. The data has provided the basis for commercial development of a platform of products to solve crop productivity challenges when faced with biotic and abiotic stresses. The data generated provides a compelling case for the benefits of effective biostimulants in sustainable agricultural practices. Additional research to optimise processing conditions for production of the biostimulants will be required in order to further enhance their efficacy. Additional investigation of the biostimulant composition and its impact on plant signalling pathways also offers opportunity for enhanced efficacy which will deliver more yield for growers.

Potential Impact:
Agriculture faces a challenge to find effective, sustainable and ecologically sound crop nutrition and pesticide solutions. The growing world population will require more food, while the prospect of global warming threatens to make agricultural growing conditions more demanding. NatuCrop SMEs have a vision of bringing a natural crop enhancement biostimulant to the agricultural industry to combat the “Perfect Storm” that is brewing. NatuCrop SMEs will market and commercialise an ecologically sound broad spectrum third generation (3G) biostimulant that improves crop yields by reducing plant stresses caused by temperature, salinity, drought and disease. The NatuCrop SMEs’ can carve out a distinct market niche that builds a bridge between plant nutrition and plant protection. The connection between these two areas will be the NatuCrop 3G biostimulant that will make crops more resistant to biotic and abiotic stress factors, which in turn will improve productivity. This project is an excellent example for the Innovation Union, one of the flagship programmes of Europes 2020 strategy to deliver “a smart, sustainable and inclusive economy” to sustain Europe’s competitiveness by delivering; smart growth, sustainable growth and inclusive growth.

Dissemination of the NatuCrop project will be aimed at developing the commercial exploitation of the project’s results and the communication of acquired knowledge from the project (Table 17). Dissemination will be conducted along internal and external channels with two specific paths;

1. Marketing to enhance the commercial potential of the product(s).
2. The wider informing of the project outcomes to the scientific and EC community.

The exploitation manager is responsible for the commercial exploitation and dissemination of the foreground project results. It is important to note that, any plans to publish information from the project will require prior approval of the Project Management Committee, with the needs of the SME partners a priority. Dissemination of the scientific results will need the formal written approval of the SMEs. Once IP protection measures are in place, dissemination of the project’s foreground under exploitation manager guidance will proceed. The participants plan to attend a series of exhibition events with technical presentations at international conferences and smaller focused workshops. The SMEs will participate in major horticulture/agriculture trade events (e.g. New Ag,) in order to demonstrate the product developed within NatuCrop. The promotional activities at these events will result in creating interest among potential users, direct contacts to major decision makers and stakeholders such as distributors and suppliers of plant biostimulants and professional associations including European Plant Pathology Organisation (EPPO), European Plant Science Organisation (EPSO), Australian Plant Pathology Society (APPS) and International Society of Plant Pathology (ISPP).

Smaller workshops provided by the SMEs will support on-site advice for companies concerned about incorporating the technology into their routine crop management practices. The consortium will bring its knowledge and experience to these events as a way of providing support and hands-on assistance to these customers. Seminars and on-line tutorial access will also be organised to raise awareness about the product and act as a training vehicle for disseminating instructional material as required.

Table 17: NatuCrop Dissemination Activities

The RTD performers will also publish scientific knowledge directly gained from the research work performed during the project. This dissemination will take the form of presentation at national and international conferences (e.g. International Congress of Plant Pathology) and publications in high quality peer reviewed journals such as Phytopathology, Molecular Plant-Microbe Interactions and Molecular Plant Pathology. The SMEs will demonstrate similar dissemination to the industry in the form of adverts in trade journals. Brochures, presenting the project’s achievements resulting in a validated working technology will constitute the SMEs main promotional tool documenting its concrete results. As mentioned, permission to disseminate scientific project results will be obtained in advance from the Project Management Committee so as not to compromise commercially sensitive activities. The other participants must be given at least 45 days prior notice in writing of any planned dissemination activity to allow them to assess whether their legitimate interests could suffer disproportionate harm in relation to their foreground or background, in which case they would have 30 days after that notification to object to such dissemination (Article II. 30.3 of ECGA)". The dissemination activities will enable the NatuCrop SMEs to establish direct links to some of the major international companies outside of the current market territories through on-site presentations and demonstration and to set up co-operations and partnerships with same.

Exploitation plans will be in full accordance with the IP Agreement and Consortium Agreement that has been signed by all partners at the beginning of the project. The Exploitation Plan will be developed and continuously updated as the lifecycle of the technology developed proceeds. The exploitation activities will consist of promoting, advertising and commercialising the product developed within NatuCrop. Over the short-term (<1 year), the SME partners will elaborate a common business development strategy at the end of the project where Brandon Products, Carbotecnia and Glenside Group will lead product marketing and sales of the NatuCrop technology. These SMEs are active in the biostimulant and plant protection markets and will liaise with their clients, all of whom are potential end-users for the technology, to gauge the best approach to take for product launch in specific markets and territories.

The NatuCrop project has delivered a platform of solutions for a number of crop productivity challenges. To-date these solutions have been proven at growth room level and in some cases in the field (Tomato (fungicide reduction), Barley and Wheat (productivity gains)). A programme of targeted technology demonstrations is now required to further validate the technology and build credibility in the marketplace. This will be achieved by the SMEs targeting customers in specific territories with specific productivity challenges to initially undertake small demonstration trials followed by larger trials if success is obtained. The typical sales cycle would see a customer adopt the technology into routine practice following 2 successful trial periods. This customer then becomes an early adopter and a marketing tool for the further exploitation of the technology in the market place. This strategy will be considered a work in progress throughout the commercialisation of NatuCrop products as it will need to be responsive to the changing market needs. However the plan sets the baseline for the management of project results in terms of capturing IP and disseminating results for the optimal benefit of all SME and RTD partners. In addition it broadly outlines the future requirements for technology demonstration for commercialisation of the NatuCrop technology.

The commercialisation of NatuCrop will be based on sound scientific product support and careful channel partner selection. Products brought to market as a result of this project will be technical products requiring a sales channel that can and will support the sale of technical products. Technical selling involves considerable training and education, both of the channel and the end users to communicate the value proposition and justification for using the product in the field. The market penetration strategy will need to reflect this and simply putting the product on shop shelves will not achieve significant market penetration. Through careful channel selection this awareness will be used to develop markets using the principle of "SEE THE DIFFERENCE" - growers will be encouraged to use the product on 10-20% of their acreage in year 1 and see for themselves the positive impact of using the product.

Based on results to date it is envisaged that a number of products will be possible from the NatuCrop discovery and validation process. Market penetration will be product specific due primarily for the need to register products in the target markets. Products will be formulated and optimised for a particular crop with a particular stress. The initial validation process has focussed on Wheat, Barley, Potato and Tomato under specific biotic and abiotic crop productivity challenges. The abiotic productivity challenges have been prioritised for product development and further validation of these products is currently underway in field trials.

The market penetration process will have three phases prior to product launch. The phases will run concurrently and will impact each other and have a major influence on product launch dates. Each of the SME partners in NatuCrop will adopt a similar three phase strategy for market penetration. The selection of crops, application protocols and value propositions will vary in emphasis depending on the market needs in which each SME is planning to launch products. Phase one will consist of the generation of the base information required to take a product to market. This base information includes the generation of a product specification, product safety data sheet, product information bulletin and supporting claims data sheets. Phase two will consist of fleshing out the claims that can be made for the product(s) being taken to market and the establishment of a clear and substantiated benefit statement. Claims can be made based on peer review literature that is published or specific trials that have been conducted. In most markets the local market demands local trials for a successful launch – this is not always a legal requirement but is what the local users require to satisfy them that the product will function in their climate, with their soil type and seed variety. Phase three will be in conjunction with key distributors in major markets – EU, Brazil, India, USA and Asia. An examination of the regulatory framework within each market will be required to determine the best approach for product launch. The regulatory environment is not uniform across all markets and this may necessitate a regional approach to market penetration. This phase will have a major influence regarding the composition of the first products from the NatuCrop platform.

Clear differences in efficacy have been demonstrated by the various combinations of materials tested and products targeted at specific crop growth challenges are emerging from this work. The distinction between abiotic and biotic effects is very evident and the market penetration strategy for these two effects will be significantly different for phase three.
The regulatory framework within the EU for instance is divided between plant products marketed with plant protection claims and products marketed with fertiliser claims. A third sector of products marketed as biostimulants is proposed as part of the current review of the EU fertiliser directive and this will be of major importance in determining the strategy for market penetration in the EU.

Launching products targeted at abiotic growth challenges will be simpler than launching products targeted at biotic challenges. The level of data required for products specifically targeted for biotic use will be significantly greater and the likelihood of licensing products with efficacy in this area to specialist companies will be explored to determine if this is feasible and more likely to generate commercial success quicker.

Prior to market launch a series of seminars will be developed and given by each SME to their distributor network to ensure clear and consistent communication of what NatuCrop products are, how they are used and the marketing strategy with regard to communicating this to the end user community. This education process will be an important step in market penetration and take into account all the regulatory, local grower and target crop issues that have been identified.

Product promotion will be the responsibility of each individual SME in conjunction with their distributors and will include promotional handouts (caps, pens etc), point of sale materials (leaflets, posters etc.) advertising and communication through grower meetings and roadshows as appropriate for individual markets. Initial briefings relating to the NatuCrop program and the anticipated product pipeline have already been conducted with key distributors. Through this process an expectation and anticipation among the SME’s existing distributor network has been developed and they are primed and eager to take the Natucrop products to market.

It is envisaged that the NatuCrop products will be publicised at appropriate conferences and exhibitions through 2015. Current plan is to present a research report at the New Ag International conference in New Delhi in March 2015. Posters and a presentation of the NatuCrop research will also be presented at the second world biostimulant conference scheduled for Florence in November 2015. A product launch of NatuCrop (dependent on the registration process) is also envisaged for the Hortiec exhibition held every June in Holambra, Sao Paulo State, Brazil.

To facilitate the market penetration strategy it is planned to produce a small batch of the NatuCrop Product #1 at the earliest possible date to get a quantity to the major distributors in the target markets to facilitate some field trials. This step will involve the distributors at an early date, allow a scale up of the number of market lead field trials and build confidence in the product prior to launch. In addition the data gathered will feed back into phase two above, giving additional claims substantiation.

A number of small production batches will be manufactured and distributed among the SME partners so they can organise appropriate trials in their target markets to allow for efficient and effective product launch in 2015. In addition the small batch manufacturing will facilitate scale up planning and troubleshooting which is a critical prerequisite for product launch. A number of additional activities (design, planning and purchasing) have been completed to put plant and machinery required for NatuCrop product production in place. It is envisaged that the commissioning of this equipment will occur as market demand dictates.

NatuCrop business justification is modelled on the tomato crop in a number of named countries. The total land area under cultivation in the designated countries is approximately 1.6 million Hectares and the target is to apply 2 lt on 1.8% of this land. The tomato market can be broken down into sub divisions - the two main being table and industrial. Table tomatoes are those used by consumers whereas industrial tomatoes are those used by processing industry in a variety of ways - sauces, juices, pastes etc. The field trial results will be critical in shaping the market strategy and will involve highlighting how the NatuCrop products reduce the grower pain (major yield loss drivers) and improve yield (both quality and volume). Early indications are that the products will increase the solids content of tomatoes which means fewer tomatoes to produce a kilo of sauce or paste. For table tomatoes size uniformity has been identified as a strong value driver. Within the market structure farm sizes vary from 1 acre in India to 4000 hectares in Brazil. This size range will require very different strategies which will be developed with the market partners. The overall market strategy has a common theme - scientific data supporting positive field trial data. The value proposition will focus on the increased grower profitability resulting from using the product.

IP arising from the NatuCrop program will be subject to ownership as set out in the consortium agreement. Prior to the release of information relating to the products a decision will be made on how to protect the IP arising from the project that is relevant to each product offering. It is anticipated that professional advice will be sought in relation to the IP developed to determine how best to protect the IP for the benefit of the NatuCrop SME partners.

As the NatuCrop project was funded under the research for the benefit of SME’s programme all research activity was focussed on delivering technically important outcomes for product development. A summary of the exploitable results generated during the project are outlined in Table 18. These results are broadly in line with what was targeted at the proposal stage of the project.

Table 18: Summary of Knowledge Generated

There are a number of project results which have potential to create novel intellectual property (IP). The initial capture of the knowledge through laboratory notebooks and completion of NatuCrop Invention Disclosure Forms (IDF’s) have been completed where appropriate. This exercise has assisted in segregating project results and outcomes into baskets which are aligned to table 2 for exploitation by the SME’s. This knowledge coherently contributes to meeting the original objective of the NatuCrop project which was to enhance crop yields by development of a third generation biostimulant. This outcome has been demonstrated during the course of the project through field trials which accurately reflects end user requirements.

During the course of the research programme undertaken by the RTDs (ITT, JHI and IRIS) on behalf of the SMEs a significant body of knowledge and data has been generated. This knowledge and data in many instances may have been generated by techniques and methods which were new to and not previously known by the SME’s. On account of this knowledge transfer within the project was an organic process from the very beginning. The RTD partners within the consortium participated actively in the knowledge transfer tasks which led to the building of a technical knowledge base within the SME’s on the areas of enzymology, plant pathology and induced resistance. The knowledge transfer was achieved through a number of mechanisms including:

• Seamless exchange of information on the Natucrop project management platform using discussion boards
• Presentations on technology basics at Project Meetings
• Reports on results with detailed background knowledge and literature review
• Distribution of reviews and research papers through project management tools for SME use.
• Demonstrations of the technology and how it works in laboratory/growth room based demonstrations.
• Informal telephone conversations
• On-line tutorial delivery

A number of technology demonstration sessions were delivered by RTD’s to SME’s throughout the duration of the project. This allowed the SME’s to gain experience on how the product is manufactured, applied to crops and its effectiveness in reducing abiotic/biotic stress. These technology transfer events will continue beyond completion of the project in order to ensure the SME’s are fully trained on the developed technology and can transfer it to commercial exploitation. The content of future demonstrations will be based on consultation with the SMEs and what their expected learning outcomes are to allow exploitation of the knowledge.

Additional workshops will be organised as the need arises to transfer technical knowledge to the SME staff involved in the presentation of the product to customers. Technical knowledge will involve the dissemination of the capabilities and limitations of the product and how best to use the product on the customers crop. Workshops and an interactive seminar will be organized to discuss trial results carried out over the duration of the project and to identify future trial requirements.

Formal arrangements were in place for regular project reporting during the project which allowed detailed explanation of results and questioning of any ambiguous findings. In addition, the Exploitation Manager has full access to all the project results and raw data generated during the project. Should additional review and evaluation be required the exploitation manager will request assistance from the RTD’s to clarify/explain these results.

Scale-up of NatuCrop biostimulant production will be a particular aspect of the knowledge transfer process which will require intensive support. This is due to the variation in processing parameters likely to be experienced during the transfer of the process from 100l scale to 5000l scale. Technical support and scaled down processing investigations will be provided by the RTDs to allow a smooth transition of the knowledge and know-how to the SMEs.

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