AU2005262253A1 - Method and system for promoting microbial nitrogen fixation activity - Google Patents

Description

WO 2006/005100 PCT/AU2005/000785 METHOD AND SYSTEM FOR PROMOTING MICROBIAL NITROGEN FIXATION ACTIVITY FIELD OF INVENTION 5 The present invention relates to monitoring and promoting microbial nitrogen fixation activity during plant growth. The present invention has particular but not exclusive application for monitoring and promoting microbial nitrogen fixation activity in the field during the cultivation of a non-leguminous or leguminous crop. 10 PRIOR ART Plants need nitrogen for growth. The amount of nitrogen in the soils is limited and with the demand for increased crop yields, farmers have added nitrogenous fertilizers to meet requirements. The use of nitrogenous fertilizers however, suppress the microbial nitrogen fixing activities in the soil resulting in the need to add 15 progressively more nitrogenous fertilizer with time. Further the ongoing use of nitrogenous fertilizers causes environmental concerns. For legumes such as peas, beans and clover, there is a nitrogen fixing symbiosis between these plants and rhizobial bacteria which form root nodules. These symbioses in rhizobial bacteria infect the plant via root hairs or crack entry 20 sites that exist intercellularly between epidermal cells and cause the formation of a nodule by inducing localized proliferation of the plant host cells. Subsequently the rhizobial bacteria infect the cells of the nodule by penetrating the cell wall and being engulfed by invaginations from the plasma membrane during endocytosis. Subsequently within the cells of the nodule the rhizobial bacteria form membrane 25 bound vesicles which fix nitrogen, utilizing products of plant photosynthesis using carbon as an energy sources, and supply biologically fixed nitrogen to the plant for WO 2006/005100 PCT/AU2005/000785 -2 growth and development. These bacteria form an endosymbiosis relationship with their plant host cells. Non-legume plants which include the main cereals of the world such as wheat, corn, maize and rice comprise the major economically important plants. Non 5 legume plants do not form nodules and are dependent on fixed nitrogen from the soil for their nutrition. The non-legume crops are routinely provided with nitrogenous fertilizers to supplement the nitrogen in the soil. In contrast to infection by rhizobial bacteria that form nodules, strains of the nitrogen fixing Azorhizobia species have been shown to invade leguminous plant 10 roots by entering lateral root cracks which are the natural epidermal fissures that form around emergent lateral roots, and that infection by Azorhizobium caulinodans is independent of nodulation factors (Webster et al, 1997, Plant Soil 194: 115 - 122; Gough et al, 1996, Biology of plant-microbe interactions, International Society for Plant-Microbe Interactions, Saint Paul, Minn). It has subsequently been shown that 15 the roots of oilseed rape which is a non-leguminous plant from the family Brassicaceae (Cruciferae) can be colonized by Azorhizobium cauilnodans (O'Callaghan et al, 2000, Applied and Environmental Microbiology 66: 2185 - 2191). Strains of Azorhizobium caulinodans are able to live endosymbiotically using plant derived energy sources and fixing and providing nitrogen to the plant. 20 Prepared cultures of Azorhizobium species and rhizobial strains have been produced and sprayed onto fields to promote the microbial fixing of nitrogen for use by growing crops. There is often observed a moderate and relatively immediate effect but this effect diminishes with time. Furthermore the results taken over a number of field sprayings has produced inconsistent results and long-term beneficial 25 effects have been questioned.

WO 2006/005100 PCT/AU2005/000785 -3 OBJECT OF THE INVENTION It is an object of the present invention to provide a system and method of monitoring and promoting microbial nitrogen fixation in the cultivation of non 5 leguminous crops. STATEMENT OF THE INVENTION In one aspect the present invention broadly resides in a system for promoting microbiological nitrogen fixing activity during cultivation of a plant including 10 application of a microbial culture to the plant and or surrounding soil, said microbial culture includes nitrogen fixing strains capable of infecting the plant; application of a carbon based medium to the plant and or surrounding soil for the promotion of microbial growth; periodic monitoring for the presence of endophytic or symbiotic microbes 15 within the plant or surrounding soil or for the presence of nitrate or nitrogen within the plant; reapplication of the carbon based medium when the nitrogen in the plant reaches a predetermined level or the concentration of the endophytic or symbiotic microbes reaches a predetermined level. 20 In another aspect the present invention broadly resides in a method for promoting microbiological nitrogen fixing activity during cultivation of a plant including applying a microbial culture to the plant and or surrounding soil, said microbial culture includes nitrogen fixing strains capable of infecting the plant; applying a carbon based medium to the plant and or surrounding soil for the 25 promotion of microbial growth; WO 2006/005100 PCT/AU2005/000785 -4 periodic monitoring for the presence of endophytic or symbiotic microbes within the plant or surrounding soil or for the presence of nitrogen or nitrate within the plant; reapplying the carbon based medium when the nitrogen or nitrate in the plant 5 reaches a predetermined level or the concentration of the endophytic or symbiotic microbes reaches a predetermined level. The method and system is suitable for use with promoting growth and nitrogen fixation with non-leguminous plants. Applying a microbial culture to the plant and or surrounding soil also includes 10 applying free living nitrogen fixing bacteria that can colonize the soil and root zone. The microbial culture preferably includes a mixed culture of diazotrophic bacteria. The nitrogen fixing strains preferably include strains of Azorhizobium species and more preferably strains of Azorhizobium caulinodans. The microbial culture preferably includes one or more strains of Azospirillum brasilense, 15 Azospirillum lipoferum, Azotobacter bejerinckii, Azotobacter chroococcum, Azorhizobium caulinodans, Azotobacter vinelandii, Azoarcus indigens, Clostridium pasteuranium, Bacillus megatarium, Bacillus subtilis, Gluconacetobacter diazotrophicus, Burkholderia species, Herbaspirillum species, Klebsiella species, cyanobacteria, Nostoc species, Anabaena species, Pseudomonas species, Bacillus 20 species, enterobacter species, and Zoogloea species. Each bacterial species is preferably cultured individually prior to their application. Each bacterial strain in the preferred formulation is preferably cultured individually and preserved prior to their application. Each bacterial strain in the preferred formulation is preferably cultured individually and freeze dried prior to their 25 application. Prior to application the freeze dried cultures are preferably resuspended WO 2006/005100 PCT/AU2005/000785 -5 and allowed to multiply. The freeze dried cultures are preferably added together prior to incubation. Alternatively the freeze dried cultures are resuspended separately and combined after incubation and prior to application. The bacterial species may also be entrapped within a gel polymer to effect 5 slow release and protect against adverse environmental conditions such as osmotic stress. The bacterial culture may be entrapped by using one or more or a chemical combination of the following products, arabic gum, perodextrin, starch granules, gelatine, gellan gum, alginate, K-carrageenan and polyacrylamide. The microbial culture may be specifically formulated for application to soils or 10 to plant foliage. The formulation for soils preferably contains nitrogen fixing microorganisms that can subsist in the rhizosphere and within plants. Where the microbial culture is for application to soil, the culture would preferably include Azospirillum brasilense, Azospirillum lipoferum, Azotobacter beijerincki, Azotobacter chroococcum, Azorhizobium caulinodans, and Azotobacter vinelandii. Where the 15 microbial culture is for application to foliage, the culture would preferably include Azospirillum brasilense, Azospirillum lipoferum, Azorhizobium caulinodans, Azoarcus indigens, and Gluconacetobacter diazotrophicus. The carbon based medium is preferably formulated to provide a slow release of the products. At the time of planting, the microbial culture, carbon based medium 20 and seed may be mixed together and planted. The carbon based medium may be liquid such as a suitable combination of molasses, humic acid and liquefied solids. Alternatively the carbon based medium may be solid including manure, mulch and straw. However for quick release products to promote bacterial replication and increased nitrogen fixing activity, low molecular weight carbon products such as 25 fulvic acid are preferably used.

WO 2006/005100 PCT/AU2005/000785 -6 The carbon based medium in one preferred form is formulated to be selective for the strains of the microbial culture. The carbon based medium preferably includes caustic potash, phosphoric acid, native crude humic acid and soluble trace elements. The carbon based 5 medium may also include fulvic acid. Trace elements preferably include boron, iron, zinc, manganese, copper, molybdenum and cobalt. The monitoring for the presence of endophytic or symbiotic microbes may be performed with the use of test kits that detect characteristic properties of the microbes or microbe - specific antibody test kits. The kits preferably can be used to 10 measure or detect the endophytic or symbiotic microbes from the soil or from plant tissue or fluid. The kits preferably can be used in the field. In one preferred form the test kit is an immunoblot or ELISA type assay that relies on the conjugation of antibodies to specific endophytic or symbiotic microbial antigens. Preferably the test kit is an immunoblot assay with antibodies specific for Azorhizobium caulinodans 15 antigens. The method and system for promoting microbiological nitrogen fixing activity during cultivation of a non-leguminous plant is preferably performed as a dynamic ongoing program with continual monitoring. In another aspect the invention broadly resides in a test kit for the quantitative 20 measurement or detection for the endophytic or symbiotic microbes described above. The kits preferably can be used to measure or detect the endophytic or symbiotic microbes from the soil or from plant tissue or fluid. The kits preferably can be used in the field. In one preferred form the test kit is an immunoblot or ELISA type assay that relies on the conjugation of antibodies to specific endophytic or WO 2006/005100 PCT/AU2005/000785 -7 symbiotic microbial antigens. Preferably the test kit is an immunoblot assay with antibodies specific for Azorhizobium caulinodans antigens. In a further aspect the invention broadly resides in a device for determining the concentration of a microbial culture including 5 a measuring tube; and an indicator means moveable within the measuring tube, wherein a sample of the microbial culture is decanted into the measuring tube and the indicator means is lowered until it becomes no longer visible to the eye while viewing the indicator means from the top of the measuring tube, then the depth of the indicator means is 10 recorded. The measuring tube is preferably a measuring cylinder. The indicator means is preferably a disk attached to a connecting handle. In a preferred embodiment the device is a simple photometer where the eye measures optical density. The concentration of the microbial culture may be determined relative to other 15 determinations. In one preferred embodiment the growth curve can be determined from the comparison of recorded depths at different incubation times. As a consequence it can be determined when the incubating culture approaches late log phase. The device for determining the concentration of a microbial culture may be 20 used in the preparation of the microbial inoculant for application to a field and plants. An inoculant may be made from a master stock and incubated until the microbes reach late log growth phase before it is applied to the field and plants. Late log phase occurs when the recorded depths from consecutive samples has not substantially changed. 25 WO 2006/005100 PCT/AU2005/000785 -8 BRIEF DESCRIPTION OF THE DRAWINGS In order that the present invention be more readily understood and put into practical effect, reference will now be made to the accompanying drawings wherein: Figure 1 is a diagrammatic view of a simple photometer for measuring 5 microbial growth; Figure 2 is a graph indicating the nitrogen demand for rice and when carbon based medium should be applied with respect to nitrogen demands; and Figure 3 is a graph indicating the nitrogen demand for corn and when carbon based medium should be applied with respect to nitrogen demands. For both figures 10 2 and 3 carbon based medium should be applied before the nitrogen demand peaks. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT The method and system for promoting microbiological nitrogen fixing activity includes the preparation of a suitable microbiological culture, preparation of a 15 suitable carbon based medium, application of the carbon based medium at different stages during the cultivation of the plant and monitoring for the presence of nitrogen fixing microbes. The method and system for promoting microbiological nitrogen fixing activity during the cultivation of non-leguminous and leguminous plants is an ongoing dynamic program where carbon based growth medium is applied in 20 response to monitoring results. The amount of carbon based media added will vary according to the soil characteristics, microorganisms added, plant type, rate and stage of growth of the plant and weather and other environmental conditions. Microbiological Culture WO 2006/005100 PCT/AU2005/000785 -9 The microbiological culture includes one or more strains of Azorhizobium caulinodans and optionally one or more strains of Azospirillum brasilense, Azospirillum lipoferum, Azotobacter chroococcum, Pseudomonas species, Bacillus species, enterobacter species, and Zoogloea species. 5 A more preferred culture includes a mixture of strains from Azorhizobium caulinodans, Gluconacetobacter diazotrophicus, Azoarcus indigens, Azosprillium brasilense, Azosprillium lipoferum, Azotobacter vinelandii, Azotobacter chroococcum, and Azotobacter beUerinckii. A culture of each strain is prepared separately and combined to form a master 10 stock. The master stock is appropriately diluted with water or with a suitable medium and allowed to incubate as a batch culture with aeration until the microbes reach a combined late log phase of growth. The dilution and incubation is performed on site such as in the farmer's property. Late log phase growth of the microbes is determined by a simple photometer which measures microbial growth by light 15 opacity. The photometer includes a measuring cylinder and a black and white disk attached to a rod that can move up and down the cylinder. A sample of the incubating culture is placed into the cylinder and the disk is lowered into the sample by the rod until the disk is no longer visible to the naked eye when a person views the disk from the top. The depth of the disk is then recorded. The recorded depths 20 can be compared and where the rate of comparative opacity slows down then the culture enters late log phase and approaches stationary phase. This is illustrated in Figure 1. When the microbial culture reaches late log phase it can then be used as an inoculant for application to the field and plants. To simplify the use, another preferred method is to provide a freeze dried 25 formulation which is rehydrated and then applied with or without a short incubation WO 2006/005100 PCT/AU2005/000785 -10 period. The culture is rehydrated in a buffered solution with fulvic acid to provide a buffering effect and a readily accessible carbon source. A preferred culture product, termed Twin N (soil) includes Azospirillum brasilense, Azospirillum lipoferum, Azotobacter bejerinckii, Azotobacter 5 chroococcum, Azorhizobium caulinodans, and Azotobacter vinelandii. A preferred culture product termed Twin N (foliar) includes Azospirillum brasilense, Azospirillum lipoferum, Azorhizobium caulinodans, Azoarcus indigens, and Gluconacetobacter diazotrophicus. To prepare the Twin N culture, each microbe is fermented individually to a 10 concentration of 108/109 CFU/ml under sterile conditions. Tryptone soy broth is the preferred nutrient medium for fermentation. Once fermentation is complete, each isolate is combined and a homogenous mixed culture is formed. The combined mixed culture is then centrifuged to concentrate the organisms and remove excess water in preparation for freeze drying. 15 The centrifuged mixed culture has a concentration of 1011/1012 CFU/ml and is subsequently freeze dried in 20 ml vials. Standard methods of freeze drying are followed. The freeze drying may take several days. The freeze-drying process results in a stable uncontaminated product that has equal proportions of selected bacteria. Each unit contains a total viability between 1011/1012 CFU/ vial. 20 Twin N Application Instructions (Re-hydration process on farm) 1 vial of concentrated Twin N organisms contains 1011/1012 CFU/Vial. The concentration of organisms in the vial can provide a coverage of 10 CFU/mm 2 over 10 hectares.

WO 2006/005100 PCT/AU2005/000785 - 11 To prepare the application culture 5 litres of water is added to a container. Approximately 5 grams of fulvic acid is then added to the 5 litres of water. The contents of the freeze dried vial is then added to the solution. The culture is allowed to stand for 2 to 3 hours to promote re-hydration of the microorganisms. The culture 5 is then applied to the crop and or adjacent soil. For foliar application, a minimum of 5 litres of mixed culture is added to 50 to 100 litres of water and applied to each hectare. For fertigation (drip irrigation) or liquid inject system, the application culture was added at 5OLitres of water / ha. Rehydration is a process necessary to activate the freeze dried material. The 10 steeping time is used for activation only. This prevents competition between various strains of nitrogen fixing bacteria as well as minimizing contamination. Carbon Based Media The carbon based media contains nutrients that promote the growth of the microbes from the inoculant culture. The preferred medium termed X-Cell contains 15 crude humic/fulvic acid (Agro Lig 22.68 kg), caustic potash (potassium hydroxide) (25 kg), soluble trace elements (Trace L 4 kg), 83% phosphoric acid (20 litres) in 1000 litres of water (pH 6.5). The rate that X-Cell is applied to fields and plants will vary depending on the amount of organic carbon in the soil, the concentration and type of microbes in the 20 inoculant. Typically X-Cell is added at a concentration of 2 litres of X-Cell per 100 litres of water per hectare. X-Cell is preferably added at time of planting, germination, during rapid growth such as stem elongation, flowering and or fruit/grain formation. X-Cell may also be added during periods of stress for the plant / crop such as flooding, temperature 25 fluctuations, insect infestation, low levels of nitrogen within the plant tissues and sap.

WO 2006/005100 PCT/AU2005/000785 - 12 X-Cell may be applied by any one of commonly used methods and include boomspray, aerial application, backpack application, pivot irrigation, bucket or watercan, drip irrigation, water injection at time of planting and knifing at post germination. 5 Suitable soil conditions for the microbiological fixing of nitrogen and the application of X-Cell include a soil temperature of 200C to 30'C, moisture content of 25% to 50%, pH of 7, carbon/nitrogen ratio of 30:1 to 20:1, and organic carbon of 1% to 3%. Microorganisms may be added to X-Cell for subsequent application in 10 circumstances where little or no microbiological nitrogen fixation has been detected. Similar cultures and microbiological concentrations to those applied at the time of planting can be used for subsequent applications. A foliar application of X-Cell with a mixed culture of Azorhizobium caulinodans and Acetobacter diazotrophicus may be used at different stages particularly during stem elongation. 15 Monitoring Monitoring for the presence of nitrogen fixing activity can be achieved by a number of methods. 1. An immunoblot test kit can be used in the field for the rapid detection 20 of the presence of nitrogen fixing endophytic or symbiotic bacteria in or associated with the plant. The test kit may also be used to provide a quantitative analysis. The test kit preferably detects Azorhizobium caulinodans antigens. The assay uses tagged antibodies specific for Azorhizobium caulinodans antigens. The test involves obtaining a sample of plant sap preferably from the growth tips, introducing 25 the sap sample to the tagged antibodies specific for Azorhizobium caulinodans to WO 2006/005100 PCT/AU2005/000785 - 13 form conjugated complexes and exposing the conjugated complexes to indicator reactants to produce a detectable result. For example enzyme labeled antibody such as horseradish peroxidase labeled antibody can be employed to provide a detectable result when the conjugated antibody is exposed to an enzyme substrate 5 and a colour change occurs as a result. ELISA or sandwich ELISA tests may be performed using enzyme labeled antigen-specific antibodies under standard conditions. The detection of low levels of the microbe would indicate that X-Cell needs to be added. If low or no target organisms are detected it may be necessary to provide additional applications of the microorganisms. In some circumstances it 10 may be necessary to incubate the target organisms from the sap in order for them to reach detectable levels before an ELISA or sandwich ELISA test is carried out. An advantage of using the test kit is the specificity of the antibody molecules for the Azorhizobium caulinodans antigens and sensitivity of the assay. A further advantage is that results can be obtained immediately and a farmer can quickly 15 respond to the results. 2. Nitrate or nitrogen analysis using standard methods can be performed on the plant sap to determine the presence and amount of nitrate or nitrogen respectively. Low levels of nitrate or nitrogen would indicate that X-Cell needs to be applied. 20 3. Testing by the observation of the yellowing of mature leaves is an indication that X-Cell needs to be applied. As an alternative a chlorophyll meter can be used to measure any change in the colour of the leaves. 4. Acetylene reduction assays may be carried out prior to inoculation to determine the capacity of a particular soil type to support free living nitrogen fixing 25 bacteria and determine the concentration of X-Cell and the frequency of application.

WO 2006/005100 PCT/AU2005/000785 - 14 These assays can be used to determine the rate of fixing atmospheric nitrogen (with and without accelerants). Acetylene reduction assays are performed using standard procedures. Acetylene reduction assays however must be performed in a laboratory and it may take several days before a farmer obtains a result. 5 EXAMPLE 1: Cultivation of Rice (T1 41) X-Cell is applied to a crop of rice at planting, 10 to 30 days after planting during rapid growth and again at 70 to 80 days during ear initiation. A microbial mixed culture containing one or more strains of Azorhizobium caulinodans and 10 incubated to late log phase is diluted to a concentration of10 3 - 107 organisms per ml (and preferably 106 organisms per ml) and applied at 50 - 100 litres per hectare (preferably 100 litres per hectare). The microbiological mixed culture preferably contains of Azospirillum brasilense, Azospirillum lipoferum, Azotobacter chroococcum, Azorhizobium caulinodans, Azotobacter vinelandii, Clostridium 15 pasteuranium, Bacillus megatarium, Bacillus subtilis, Gluconacetobacter diazotrophicus, Burkholderia species, Herbaspirillum species, Klebsiella species, cyanobacteria, Nostoc species, Anabaena species, Pseudomonas species, Bacillus species, enterobacter species, and Zoogloea species. Figure 2 shows the timing of X-Cell application relative to nitrogen demand by rice. X-Cell was added at 20 days 20 and 70 days. Because soil types differ and crop requirements differ in respect to nitrogen demand, periodic assays must be performed to determine the needs of the crop. EXAMPLE 2: Cultivation of Corn WO 2006/005100 PCT/AU2005/000785 - 15 The trial field was conducted on the Darling Downs in Queensland. The soil type was clay loam. The previous crop was organic wheat. The crop that was planted was organic corn. Irrigation was by means of drip irrigation. The soil had 1.5% organic carbon (good nitrogen fixation suitability), 0.13% total nitrogen, 11.5:1 5 carbon/nitrogen ratio (poor nitrogen fixation suitability), 7.5pH (average nitrogen fixation suitability), and soil temperature of 250C (good nitrogen fixation suitability). X-Cell was applied at a rate of 2 litres/hectare by water injection at planting with the inoculant. At the time of planting, a microbial mixed culture containing one or more strains of Azorhizobium caulinodans and incubated to late log phase was 10 diluted to a concentration of 106 organisms per ml and applied at 100 litres per hectare. The microbiological mixed culture contained strains of Azospirillum brasilense, Azospirillum lipoferum, Azotobacter chroococcum, Azorhizobium caulinodans, Azotobacter vinelandi, Clostridium pasteuranium, Bacillus megatarium, Bacillus subtilis, Gluconacetobacter diazotrophicus, Burkholderia species, 15 Herbaspirillum species, Klebsiella species, cyanobacteria, Nostoc species, Anabaena species, Pseudomonas species, Bacillus species, enterobacter species, and Zoogloea species. X-Cell was further applied at a rate of 2 litres/hectare by drip irrigation at 20 to 30 days after planting and during stem elongation. 20 X-Cell was further applied at a rate of 2 litres/hectare by drip irrigation at 60 to 70 days after planting and during ear emergence. (Further application of X-Cell was made at a rate of 2 litres/hectare by drip irrigation if monitoring revealed nitrogen deficiency.) Figure 3 shows the timing of carbon application with respect to corn nitrogen 25 demand.

WO 2006/005100 PCT/AU2005/000785 - 16 EXAMPLE 3: Application Of Twin N Product To A Cotton Crop The method and systems for promoting nitrogen fixation is a dynamic and flexible program. Initially, Twin N (soil) and fulvic acid was applied at planting with 5 various combinations of biological materials. To determine the effect of Twin N (soil) on the nitrogen status in the cotton crop, leaf analysis was carried out. Leaf analysis was performed during rapid vegetative growth and when nitrogen demand was critical. Based on the leaf analysis results, Twin N (foliar) was applied to the cotton crop to promote plant growth and nitrogen fixation. 10 Trial Specifications Crop: Cotton Soil Type: Black shelf-mulching clay Location: Collarenebri Planting date: 19/12/05 15 Planting Treatments T1: Conventional N - side dress N26 @ 180 kg/ha + 40L Biological T2: Control - nil N + 40L/ha biological T3: Twin N @ 1 OL/ha + 40L/ha biological + VAM (Vesicular Arbuscular Mycorrhiza at 200g/ha) 20 T4: Twin N @ 1OL/ha + 40L/ha biological The 40 L/ha biological was a combination of humic acid and KFF (Kelp, Fish and Fulvic acid). Each planting treatment was repeated three times on different 1.2 ha blocks to identify repeatable results. Block design was randomized.

WO 2006/005100 PCT/AU2005/000785 - 17 Time Line Planting and Twin N (soil) occurred on week one (19/12/04), first leaf analysis occurred on week four (10/01/05), Twin N (foliar) occurred on week six (29/01/05) and second leaf analysis occurred on week eight(09/02/05). 5 Twin N Planting Application Twin N (Soil), which contained selected species of Azotobacter spp (108 CFU/mi) in cyst form), was applied by water injection at a rate of I OL/ha. In addition, fulvic acid was mixed with the Twin N (Soil), at a rate of 1L/ha. Both components were mixed in 1 OOL of water per hectare just prior to planting. 10 Twin N Foliar Application Twin N (Foliar) was applied mid season on the 29/01/05 on all Twin N blocks. This product contained a range of free-living microorganisms that could subsist within plants or in the soil. Twin N (Foliar) (8x10 11 CFU/ml) was applied at a rate of I sachet/Ha along with fulvic acid at a rate of 1 L/ha. The product was diluted in 1 OOL 15 of water/ha and delivered by boom-spray. Results First Leaf Analysis From the leaf analysis, it was determined that percentage of total nitrogen was 3.04, 3.07, 2.91 and 3.01 for planting treatments T1, T2, T3 and T4 respectively. 20 It was also determined that amount of nitrate nitrogen per kg of foliage was 7.57, 7.40, 7.90 and 8.17 for planting treatments T1, T2, T3 and T4 respectively. The first fully mature expanded leaf was collected for testing. Second Leaf Analysis From the leaf analysis, it was determined that percentage of total nitrogen 25 was 3.8, 4.19, 4.32 and 3.79 for planting treatments T1, T2, T3 and T4 respectively.

WO 2006/005100 PCT/AU2005/000785 - 18 It was also determined that amount of nitrate nitrogen per kg of foliage was 7.1, 6.3, 6.6 and 7.5 for planting treatments T1, T2, T3 and T4 respectively. Conclusions - With the first leaf analysis, all samples appear to be low in total N% 5 compared with optimum total N% of 3.5 to 4.5. All samples, excluding the control, appear to have similar total N% values. Planting treatments T3 (Twin N and VAM) and T4 (Twin N) appear to have higher nitrate levels than the control and conventional cotton. Twin N + VAM may have caused temporary nitrogen draw down, causing initial low N status. 10 After foliar application, a second leaf analysis was carried out revealing that a sample from each treatment demonstrated increased nitrogen levels. Twin N and VAM demonstrated the highest total N%. Possibly Twin N and VAM may have released the nitrogen that it acquired earlier in the season, causing a higher than expected total N%. 15 EXAMPLE 4: Twin N Application To A Cotton Crop. Twin N was applied to Cotton in an effort to reduce nitrogenous fertiliser. Leaf analysis was carried out at flowering to determine the effectiveness of Twin N compared with nitrogenous fertilizer. 20 Trial specifications Crop: Cotton Location: Collarenebri Planting Treatments: T1: Convention cotton, 160 units of nitrogen. 25 T2: Twin N Cotton, (no applied chemical nitrogen) WO 2006/005100 PCT/AU2005/000785 - 19 The planting treatments were not replicated. Twin N (foliar) Twin N (Foliar) was applied post planting in December 2004. This product contained a range of free-living microorganisms that could subsist within plants or in 5 the soil. Twin N (foliar) being a (8x1011 CFU/sachet) was applied at a rate of 1 sachet/Ha, along with fulvic acid at a rate of 1L/ha. Both products were diluted in 1 00L of water/ha and delivered by boomspray. Results From the leaf analysis conducted on 17/01/05, it was determined that 10 percentage of total nitrogen was 3.39 and 3.46 for planting treatments T1 and T2 respectively Conclusion - Twin N, with no additional nitrogenous fertiliser, has a slightly higher total N% than the conventional cotton. 15 EXAMPLE 5: Twin N On Carrot Crop Small non-replicated trials were conducted to determine the effectiveness of Twin N compared with conventional NPK fertiliser treatments. Twin N was applied as a foliar at the three leaf stage. In this instance only one application of Twin N was 20 necessary since visual observations indicated strong vigor during high nitrogen demand and rapid growth. Trial specifications Crop: Carrots Location: "Manzara" Mapleton 25 Planting date: 24/04/04 WO 2006/005100 PCT/AU2005/000785 - 20 Planting Treatments T1: Control (No fertiliser) T2: Twin N (3x1 07 CFU/ml), equivalent to 1 L/Ha X-cell formulation (as described above) was applied at 1 L/ha. 5 T3: NPK (18-18-18), equivalent to 5 bags/Ha Results The weight of carrots were determined at different times being nine weeks after planting (29/06/04), 10 weeks after planting (3/07/04) and 12 weeks after planting 21/07/04), 10 Carrots resulting from planting treatment TI had a mass of 6, 15 and 26 kg per m2 after 9, 10 and 12 weeks respectively. Carrots resulting from planting treatment T2 had a mass of 20.5, 24 and 40 kg per m2 after 9, 10 and 12 weeks respectively. Carrots resulting from planting treatment T3 had a mass of 15, 21 and. 20 kg 15 per m2 after 9, 10 and 12 weeks respectively. Compared with the control T1, carrots treated with T2 (Twin N) and T3 (NPK fertilizer) increased 45% and 16% respectively. Conclusions Results indicate that the Twin N product is beneficial for root crops. Twin N 20 outperformed the NPK fertiliser treatment by 34%. EXAMPLE 6: Twin N On A Bean Crop Small non-replicated plot experiments were conducted to determine the effectiveness of Twin N compared with control and conventional NPK fertiliser WO 2006/005100 PCT/AU2005/000785 -21 treatments. Only one application of Twin N was used since visual observations indicated strong vigor during high nitrogen demand and rapid growth. Trial specifications: Crop: Beans 5 Location: "Manzara" Mapleton Planting date: 18/03/04 Planting Treatments T1: Control T2: Twin N (3x1 07 CFU/ml), equivalent to 1L/Ha. Fulvic acid at 1 L/ha 10 T3: NPK (18-18-18), equivalent to 5 bags/Ha Results First Experiment (18/04/04) All pickings of the bean samples was conducted on the same day and picking was carried out each week for six consecutive weeks on the same day. 15 Bean weight for the control was determined for the first to sixth picking being 2.7, 1.6,10.6,15.2, 13.5 and 21.5 kg per m 2 respectively. The total weight was 64.7 kg per m2 Bean weight for the T2 treatment was determined for the first to sixth picking being 4.1, 18.5, 14.5, 43.3, 25.5 and 42.8 kg per m 2 respectively. The total weight 20 was 148.7 kg per m2. Bean weight for the T3 treatment was determined for the first to sixth picking -being 3, 15.2, 12.6, 22.5, 19.6 and 30.4 kg per m 2 respectively. The total weight was 103.3 kg per M 2 . Carrots treated with T2 and T3 had an increased weight of 57% and 37% over 25 the control.

WO 2006/005100 PCT/AU2005/000785 -22 Second Experiment (19/04/05) Bean weight for the control was determined for the first to seventh picking being 17.5, 4.3, 19, 17.9, 11.6, 4.7 and 1.7 kg per m 2 respectively. The total weight 5 was 76.7 kg per M 2 . Bean weight for the T2 treatment was determined for the first to seventh picking being 5.4, 25.9, 28.9, 27.8, 18.7,18 and 3.4 kg per m 2 respectively. The total weight was 128.1 kg per M 2 . Bean weight for the T3 treatment was determined for the first to seventh 10 picking being 3.6, 14, 22.4,18, 14.8, 15.2 and 2.1 kg per m 2 respectively. The total weight was 90.1 kg per M 2 . Carrots treated with T2 and T3 had an increased weight of 40% and 16% over the control. Conclusions 15 Twin N and NPK fertilizer treatments produced carrots of greater mass than the control treatment. The Twin N treatment was better than the NPK fertilizer treatment in both bean experiments. ADVANTAGES 20 An advantage of the preferred embodiment of the method and system for promoting microbiological nitrogen fixing activity during cultivation of non-leguminous crops is that it provides improved yields through constant monitoring. The method is relatively cheap to implement as no expensive equipment is required and the carbon based media comprises of readily available carbonaceous 25 components.

WO 2006/005100 PCT/AU2005/000785 - 23 The device for determining the concentration of a microbial culture has the advantage in that it can be used in the field, it is relatively simple to operate and does not require power like other measuring devices like the turbidity meter. 5 VARIATIONS It will of course be realised that while the foregoing has been given by way of illustrative example of this invention, all such and other modifications and variations thereto as would be apparent to persons skilled in the art are deemed to fall within the broad scope and ambit of this invention as is herein set forth. 10 Throughout the description and claims this specification the word "comprise" and variations of that word such as "comprises" and "comprising", are not intended to exclude other additives, components, integers or steps.

Claims (8)

1. A system for promoting microbiological nitrogen fixing activity during cultivation of a plant including application of a microbial culture to the plant and or surrounding soil, said 5 microbial culture includes nitrogen fixing strains capable of infecting the plant; application of a carbon based medium to the plant and or surrounding soil for the promotion of microbial growth; periodic monitoring for the presence of endophytic or symbiotic microbes within the plant or surrounding soil or for the presence of nitrate or nitrogen within the 10 plant; reapplication of the carbon based medium when the nitrogen in the plant reaches a predetermined level or the concentration of the endophytic or symbiotic microbes reaches a predetermined level. 15

2. A method for promoting microbiological nitrogen fixing activity during cultivation of a plant including applying a microbial culture to the plant and or surrounding soil, said microbial culture includes nitrogen fixing strains capable of infecting the plant; applying a carbon based medium to the plant and or surrounding soil for the 20 promotion of microbial growth; periodic monitoring for the presence of endophytic or symbiotic microbes within the plant or surrounding soil or for the presence of nitrogen or nitrate or nitrogen within the plant; WO 2006/005100 PCT/AU2005/000785 - 25 reapplying the carbon based medium when the nitrogen or nitrate in the plant reaches a predetermined level or the concentration of the endophytic or symbiotic microbes reaches a predetermined level. 5

3. A system or method as claimed in claims or 2 respectively wherein the plant is non-leguminous and the microbial culture includes free living nitrogen fixing bacteria that can colonize the soil and root zone.

4. A system or method as claimed in any one of the preceding claims wherein 10 the microbial culture includes a mixed culture of diazotrophic bacteria.

5. A system or method as claimed in any one of the preceding claims wherein the microbial culture includes one or more strains of Azorhizobium species. 15

6. A system or method as claimed in any one of the preceding claims wherein the microbial culture includes one or more strains of Azorhizobium caulinodans.

7. A system or method as claimed in any one of the preceding claims wherein the microbial culture includes one or more strains of Azorhizobium caulinodans and 20 one or more strains select from the following group: Azospirillum brasilense, Azospirillum lipoferum, Azotobacter beijerinckii, Azotobacter chroococcum, Azorhizobium caulinodans, Azotobacter vinelandii, Azoarcus indigens, Clostridium pasteuranium, Bacillus megatarium, Bacillus subtiis, Gluconacetobacter diazotrophicus, Burkholderia species, Herbaspirillum species, Klebsiella species, WO 2006/005100 PCT/AU2005/000785 - 26 cyanobacteria, Nostoc species, Anabaena species, Pseudomonas species, Bacillus species, enterobacter species, and Zoogloea species.

8. A system or method as claimed in any one of the preceding claims wherein 5 each strain in the formulation is individually cultured and freeze dried prior to its application.