Nitrogen Fixing Corn and 199 Other Crops

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1 Nitrogen Fixing Corn and 199 Other Crops Dr David Dent Founder/Technical Director Azotic Technologies Ltd 1 In my dream I see green, vigorous, high yielding fields of wheat, rice, maize, sorghum and millet which are obtaining, free of expense, 100 kilogram of nitrogen per hectare from nodule forming, nitrogen fixing bacteria This scientific discovery has revolutionized agricultural production for the hundreds of millions of humble farmers throughout the world, for they now receive much of the needed fertilizer for their crops directly from these little wondrous microbes that are taking nitrogen from the air and fixing it without cost in the roots of cereals, from which it is transformed into grain 2 2 1

2 World renowned scientist in plant cell and tissue culture pioneered the development of nitrogen fixation. Established an International Network for research on Nitrogen Fixation in the World's major non legume crops Fellow of the Royal Society (1983); Fellow World Innovation Foundation (2003); Fellow of the Indian Academy of Agricultural Sciences (2000); Member Hungarian Academy of Sciences (1995) & Academia Europaea (1993) D.Sc. University of Bristol; Plant cell biology & nitrogen metabolism Former Chairman of the International Advisory Board of The Norman Borlaug Institute Father of the Green Revolution 4 2

3 Peter Blezard CEO Mike Panteli CFO/COO Dr David Dent CTO Allen Sheena CMO Strategic focus on sustainable agriculture Founder of Plant Impact Plc International market development expertise Chartered Management Accountant Former CFO of Plant Impact Plc Strong in company management and financial control A scientist with over 30 years experience in agriculture Vice President of Parliamentary and Scientific Committee Manager of the 12 million LUBILOSA Programme in Africa Former Managing Director of CABI Bioscience Marketing professional with over 30 years experience International experience in agriculture Focus on strategic marketing and business development 5 Azotic Technologies have licensed the nitrogen fixing technology for 200 crops from Nottingham University Strong international track record in developing new technologies and collaboration with industry Number 1 ranking out of the top 100 UK Universities for agriculture and food sciences Past successes include MRI scanner technology (Nobel prize winning research), novel vaccines, medical device for heart rate monitoring, nanoparticle production spin out company, antimicrobial catheters & others 6 3

4 Eco systems are highly sensitive to reactive Nitrogen Nitrogen pollution is costing each person in Europe around $ 200 1,000 a year More than double the extra income gained from using nitrogen fertilizers in European agriculture Annual cost of damage caused by nitrogen across Europe is $ billion The European Nitrogen Assessment INRA Research April World Nitrogen Fertiliser Consumption 104 m tonnes / $85bn 2011 Nitrogen Fertiliser Application Plants only take up 20% 30% of tonnage applied Nitrogen Pollution Food security/water pollution/global warming/human health issues Current World Fertilizer Trends and Outlook to 2015 FAO 8 4

5 9 N Fix nitrogen fixing crop seed inoculation Applicable to over 200 crops Nitrogen fixation by bacterial endophytes occurs within cells of nodules in legumes N However, can occur within cells without nodule formation, as in the nonlegume plant Gunnera This requires the formation of a nitrogen fixing symbiosome 10 5

6 Diazotroph an organism able to grow without external sources of fixed nitrogen Endophyte micro organism living within a plant Endosymbiont lives within a plant and provides benefits to both the plant and the micro organism Symbiosome membrane bound compartment (an organelle) located in the cell cytoplasm Inoculants soil treatment $130m to $255m Seed coatings greater than 660 MT per annum $100m to $165m 11 Isolated from sugar cane in 1988 Natural host range includes high sugar content crop species: sweet potato, pineapple, sweet sorghum and mango An extraordinary endophytic, diazotophic, acid tolerant bacteria Produces Indole Acetic Acid (IAA) which promotes plant growth Strains of this Gd normally found in intercellular spaces Strain IMI isolated from sugarcane has under certain conditions (patented technology) been shown to colonise a range of crop species intracellularly: wheat, rice, maize, cotton, oilseed rape, white clover, tomato and grass Seed coatings greater than Plant cell wall penetration facilitated by bacterial genes encoding a 660 MT per annum β expansin and an endo 1, 4 β glucanase i.e. an $100m innate to capacity $165m of the strain 12 6

7 Gd possesses no nitrate reductase, hence needs to fix atmospheric nitrogen Nitrogen fixation is not inhibited by presence of nitrate; NH4+ shows partial inhibition Gd excretes half of the nitrogen fixed in a form available to plants. Nitrogen fixation only possible in absence of oxygen Nitrogenase activity protected from excessive oxygen by production from sucrose, of a mucoid levan fructan which inhibits Seed coatings oxygen diffusion greater than The mucoid enables fixation of nitrogen in a wide 660 range MT per of annum oxygen concentrations $100m to $165m 13 Representation of the interaction of G. diazotrophicus (G.d.)with roots: (a) meristematic zone of the elongating root (b) G.d. penetrates the epidermal cell wall by secretion of cellulose enzymes (c) the plasma membrane pinched off via endocytosis forms a membrane surrounding vesicles containing G.d. (d) vesicles with G.d. are surrounded by a membrane analogous to the symbiosome membrane of rhizobia 14 7

8 15 IMI tagged with the Beta glucoronidase (GUS) gene enables the histochemical localization of blue staining of Gd No intracellular colonization using a population of 10 6 bacteria Colonisation observed only when very low numbers of Gd ( optimum around 5 individual bacteria) Gd produces large amounts of IAA; at low concentrations IAA probably functions as a reciprocal signaling molecule in bacterial plant interaction may explain why small numbers of Gd needed for initial colonization In all non legume species inoculated, detailed light Seed and coatings electron greater than microscopic analysis demonstrated the intracellular colonization of 660 MT per annum meristematic root cells $100m to $165m Within root meristem cells Gd was shown to be present in membranebound vesicles in the cytoplasm 16 8

9 Section of edge of maize root tip showing blue stained G. diazotrophicus bacteria within cells (scale bar = 10 μm) 17 Section of region of maize leaf showing blue stained G. diazotrophicus bacteria within cells and closely associated with chloroplasts 18 9

10 19 Presence of gene (using the marker nifh Beta glucoronidase) for N fixation in membrane bound vesicles demonstrates conditions suitable for nitrogenase gene expression By direct measurement of the nitrogenase enzyme activity of Gd wheat seedlings using the acetylene reduction gas chromatography assay, and an activity level per wheat seedling of 50% of that of clover inoculated with rhizobia was recorded. Recent results confirm N fixation in grass also. Gd colonised maize using the heavy isotope of nitrogen (15N) to show that up to 30% of total nitrogen per plant was derived from atmospheric nitrogen by symbiotic N fixation in young plants Seed coatings Oilseed rape and grass inoculated with Gd growing for greater many than months in absence of fixed nitrogen synthetic fertilizer, and 660 forming MT per green annum shoots $100m to $165m and leaves 20 10

11 21 Gd transmitted cytoplasmically to daughter cells in root meristems, spreading systemically from the root meristem into cells of the elongating region of the root and leaf cells of the shoot systems Microscopy of the leaves of inoculated plants showed blue stained Gd very closely associated with chloroplasts within leaf cells Raises possibility that energy for N fixation might be supplied directly from chloroplasts to nitrogen fixing endosymbiotic bacteria within the same cell 91% of inoculated maize had intracellularly colonized Inoculants Seed root coatings tips with 36% of their cells soil intracellularly treatment colonized by Gd greater than $130m to $255m 660 MT per annum $100m to $165m 22 11

12 Will the bacteria work in a field situation? Does the bacteria cause a yield loss because it uses up sucrose in order to produce nitrogen? Is nitrogen fixing activity inhibited by nitrogen fertiliser? Does a sub tropical species survive and function in low temperatures? Can the Gd be easily and cheaply produced? Will the bacteria survive on the shelf and still retain viability? How will the product be formulated? Inoculants Seed coatings Are there safety, soil treatment environmental or regulatory issues greater associated than with Gd? $130m to $255m 660 MT per annum Is there a route to market for the N Fix product? $100m to $165m Cob dry weight (g) of inoculated and un-inoculated maize (A&B different varieties) 140 dry weight (g) A Control A inoc B Contol B Inoc TREATMENT 24 12

13 Mean Dry Weight of Lolium perenne grown in compost with untreated and N Fix treated seed Mean above ground dry weight (g) Control N-Fix 25 Fermentation Shelf life and storage N Fix nitrogen fixing Production crop seed and inoculation scale up readily achieved at low cost Applicable to over 200 crops > 3 months as a liquid live suspension of bacteria with no loss of viability. Freeze dried preparation > 3years Formulation Field trials Regulation Currently a liquid inoculant optimised to ensure survival and colonisation of roots when applied to seed prior to sowing. Freeze dried powder seed coat formulation in development Pilot field trial in maize in UK demonstrated yield benefits 2013 trials to be undertaken in UK on grass, wheat, canola and in Canada on corn Gd is ranked as a Biosafety Level 1 micro organism and is not known to be invasive in any recorded territories 26 13

14 Trial programme Distribution Targets 27 Azotic has licensed a nitrogen fixing technology from Nottingham University with the potential to revolutionise fertiliser use in agriculture N N Fix technology is ubiquitous to all crops unique to Azotic Proof of concept of intracellular colonisation and nitrogen fixation has been established in a range of crops An inoculant formulation has been developed for field trials to be carried out in the UK and Canada in 2013 Links to distribution partners will ensure swift rollout of the technology on a crop by crop basis 28 14

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