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    • 专利摘要:
    microbial composition and process for agricultural use. Microbial compositions comprising at least two components are disclosed. The first hyta component is a consortium of microbes derived from fertile soils and commercial sources. The second component comprises at least one of chitin, chitosan, glucosamine and amino acids. The various microbes in hyta are able to fix nitrogen, digest proteins and other biopolymers like chitin and chitosan, provide protection against plant pathogens and supplement the soil microbial flora. Also disclosed are processes wherein the above mentioned mocrobian compositions are used to treat soil, seeds, seedlings and / or plant foliage individually or in combination with chitin, chitosan, glucosamine and / or amino acids.
    公开号:BR112012032040B1
    申请号:R112012032040-8
    申请日:2011-06-15
    公开日:2018-04-17
    发明作者:López-Cervantes Jamie；Reiner Fick Rochin Karl
    申请人:Agrinos AS；
    IPC主号:
    专利说明:

    (54) Title: MICROBIAL COMPOSITIONS, MICROBIAL PROCESS FOR AGRICULTURAL USE AND ACTIVATED HYTA (51) Int.CI .: A01N 63/00; A01N 63/02; A01N 63/04; A01N 37/44; A01N 25/00; A01P 3/00; A01P 7/04 (52) CPC: A01N 63/00, A01N 63/02, A01N 63/04, A01N 37/44, A01N 25/00 (30) Unionist Priority: 06/16/2010 US 61 / 355,447 (73 ) Holder (s): AGRINOS AS (72) Inventor (s): JAMIE LÓPEZ-CERVANTES; KARL REINER FICK ROCHIN
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    MICROBIAL COMPOSITIONS, MICROBIAL PROCESS FOR AGRICULTURAL USE AND ACTIVATED HYTa
    This application claims the benefit in accordance with 35 U.S.C. §119 of provisional application US serial number 61 / 355,447, filed on June 16, 2010.
    FIELD OF THE INVENTION
    Microbial processes and microbial compositions are revealed to increase crop production, increase plant defensive processes, decrease the level of plant pathogens and reduce the amount of fertilizer used.
    BACKGROUND OF THE INVENTION
    Microbes were previously used in agriculture. Examples include those disclosed in US patents 4,952,229; 6,232,270 and 5,266,096.
    Chitin has also been used in agriculture as a protein complex (US patent 4,536,207) or in combination with several microbes (US patents 6,524,998 and 6,060,429).
    Chitosan in combination with other components has been used in agricultural applications. See, for example, US patents 6,649,566; 4,812,159; 6,407,040; 5,374,627 and 5,733,851. It was also used to treat cereal crop seeds. See US patent 4,978,381. US patent 6,524,998 also discloses that chitosan can be used in combination with specific microbes for agricultural use.
    Notwithstanding the above, there is a need to provide improved microbial compositions and processes that improve crop yield and reduce the amount of conventional fungicides and insecticides used in agricultural and horticultural applications.
    SUMMARY OF THE INVENTION
    Microbial compositions comprising at least two components are disclosed. The first component comprises HYTa, which is a consortium of microbes derived from fertile soils and commercial sources. The second component comprises at least one of chitin, chitosan, glucosamine and amino acids. The various microbes in HYTa are able to fix nitrogen, digest proteins and other biopolymers such as chitin and chitosan, providing protection against flat pathogens and supplementing soil microbial flora.
    Processes are also disclosed where the aforementioned microbial compositions or their components are used to treat soil, seeds, seedlings and / or
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    2/33 plant foliage.
    In preferred embodiments, HYTa is activated in an aqueous solution for 24 to 168 hours to allow microbes to grow and reproduce before being used in the process. Incubation conditions influence the general initial properties of HYTa.
    In a preferred embodiment, HYTa is activated in the presence of chitin. Chitin-responsive microbes in HYTa proliferate in this environment. This results in HYTa which has all the properties of HYTa. However, it has an increased capacity against plant pathogens that contain chitin.
    In a preferred embodiment, HYTa is activated in the presence of chitin, chitosan, 10 glucosamine and amino acids. In this modality, after growth, HYTa may contain chitin, chitosan, glucosamine and / or residual amino acids. Under such circumstances, the crop constitutes the revealed microbial composition and can be applied directly to the plant's soil, seed, seedlings or foliage. Alternatively, one or more second components can be added to supplement the second components already in the composition or to alter the components present in the thus formed microbial composition.
    In some embodiments, activated HYTa is combined with one or more second components and applied to soil, seed, seedlings or plant foliage or HYTa and the second component (s) are applied separately. Such second components include chitin, chitosan, glucosamine and amino acids.
    The application of the revealed microbial formulations allows the elimination or significant reduction in the amount of fertilizer, fungicide and insecticide used in agricultural applications. In some modalities, the use of microbial formulations results in a decrease in the amount of greenhouse gas emissions.
    Also disclosed is a treated soil composition comprising soil treated with HYTa.
    Also disclosed is a treated plant comprising a plant treated with HYTa.
    Also disclosed are seeds, seedlings and treated plants comprising seed, seedling or plant treated with HYTa.
    BRIEF DESCRIPTION OF THE DRAWINGS
    Figure 1 is a diagram of the test area involving the growth of durum wheat in Sonora, Mexico where HYTa and HYTb were used.
    Figure 2 is the same diagram as Figure 3 and shows zones that have been
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    3/33 compromised and suffered damage by external factors during the experiment.
    Figure 3 graphically represents the results of treating durum wheat soil and foliage with HYTa and HYTb.
    Figure 4 shows the yield of melons as a function of size for soil 5 and foliage that has not been treated or has been treated with HYTa and HYTb.
    Figure 5 shows the yield of potatoes having diameters greater than 42 mm that were treated with HYTa, HYTb and HYTc compared to untreated potatoes.
    DETAILED DESCRIPTION
    Microbial compositions comprising HYTa and a second component are disclosed. HYTa is a consortium of microbes derived from additional soils and commercial sources. The second component comprises at least one of chitin, chitosan, glucosamine and amino acids. The various microbes in HYTa are able to fix nitrogen, digest proteins and other biopolymers like chitin and chitosan, providing protection against plant pathogens and supplementing the soil microbial flora. Microbial compositions or their components are used to treat soil, seeds, seedlings and / or plant foliage.
    HYTa
    As used here, the term “HYTa” refers to a consortium of microbes derived from fertile soil samples and commercial sources. HYTa was deposited with the
    American Tissue Type Culture (ATCC), Rockville, Maryland, on May 19, 2010 with an assigned deposit designation of PTA-10973.
    Table 1 identifies some of the microbes in HYTa that are believed to be responsible for the beneficial effects seen when used to treat soil and / or foliage.
    Table 1
    Bacteria I. Azotobacter 1. Azotobacter vinlandii II. Clostridium 1. Clostridium pasteurianum 2. Clostridium beijerinckii 3. Clostridium sphenoides 4. Clostridium bifermentans III. Lactobacillus
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    1. Lactobacillus paracasei ss. Paracasei
    2. Lactobacillus acidophillus
    3. Lactobacillus delbrueckii ss. Bulgaricus
    4. Lactobacillus brevis
    IV. Bacilli
    1. Bacillus amyloliquefaciens (Bacillus subtilis ((SILoSil®
    BS))
    2. Bacillus thuringiensis var. kurstakii (Bacillus thuringiensis (HD-1 strains))
    3. Bacillus thuringiensis var. canadensis (Bacillus cereus group)
    4. Bacillus pasteurii (Bacillus cereus group)
    5. Bacillus sphaericus (subgroup I, III, and IV)
    6. Bacillus megaterium (subgroup A)
    V. Acetobacter or Gluconacetobacter
    1. Acetobacter aceti ss. liquefaciens
    2. Acetobacter aceti ss. xylimum
    SAW. Enterococcus
    1. Enterococcus faecium (subgroup A)
    VII. Pediococcus
    1. Pediococcus pentosaceus
    VII. Rhizobium
    1. Rhizobium japonicum
    Fungi
    I. Saccharomyces
    1. Saccharomyces cerevisiae
    II. Penicillium
    1. Penicillium roqueforti
    III. Monascus
    1. Monascus ruber
    IV. Aspergillus
    1. Aspergillus oryzae
    V. Trichoderma
    1. Trichoderma harzianum (TRICHOSIL)
    Plantae
    I. Arthrospiro
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    1. Arthrospira platensis
    II. Ascophyllum
    1. Ascophyllum nodosum
    Other microorganisms contained in HYTa: Nitrobacter, Nitrosomonads, 5 Nitrococcus, Pseudomonas, Micrococcus luteus, Actinomycete, Azotobacter vinelandii, Lactobacillus casei, Trichoderma harzianum, Bacillus licheniformis, Pseudomonas fluorescens.
    Microbes active in HYTa include nitrogen-fixing microorganisms native to the soil. These are Azotobacter vinelandii and Clostridium pasteurianum. Bacillus subtilis provides enzymes to break down plant waste. Bacillus cereus provides additional enzymes to break down plant waste and penicillinase to kill unwanted bacteria. Bacillus megaterium degrades complex sugars after rupture of crop residue. Lactobacillus provides food for microbes in HYTa and controls the pH of the environment. Nitrobacter organisms oxidize ammonia to nitride (NO2) while microbes from
    Nitrosomonas oxidize nitride to nitrate (NO3).
    An important property of HYTa is the fixation of atmospheric nitrogen. The nitrogen-fixing capacity of microbes in HYTa is increased by the assistance of other organisms in HYTa. Nitrogen fixation requires that phosphorus (P), potassium (K) and carbon (C) are available. HYTa contains microbes that are able to break down P, K and C in the soil. In addition, nitrogen-fixing bacteria provide a source of nitrogen for the other microbes in HYTa.
    Nitrogen fixation can occur in a non-symbiotic way by the bacteria Nitrosomonas, Nitrobacter, Azotobacter vinelandii, and Clostridium pasteurinum present in HYTa or in a symbiotic way as occurs in root nodules by means of Rhyzobium bacteria.
    The carbon required by nitrogen-fixing microbes in HYTa is supplied by C decomposers that convert complex organic compounds in soil into simple compounds such as sugars, alcohols and organic acids. C decomposers include many of the microbes identified above.
    Phosphorus is necessary for nitrogen fixing microbes to proliferate and is obtained from the metabolic activity of P decomposers that convert phosphorus immobilized in the soil into a bioavailable phosphorus nutrient. P decompositors in HYTa include Azotobacter, Bacillus subtilis, Pseudomonas fluorescens and Micrococcus luteus.
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    The potassium required by nitrogen fixers is supplied by the decomposing microbes present in HYTa that activate potassium in the soil. Decomposers K in HYTa include Pseudomonas fluorescens.
    Three important microbes in HYTa are Bacillus subtilis (SILoSil® BS) Bacillus 5 thuringiensis strains HD-1 and HD-73 (SILoSil® BT), and Trichoderma harzianum (TRICHOSIL). These bodies are present in ATTC deposit PTA-10973. They were originally obtained from Biotecnologia Agroindustrial S.A. DE C.V., Morelia, Michoacan,
    Mexico.
    Bacillus subtilis ((SILoSil® BS) is a Gram positive bacterium that is mesophilic and grows at an optimum temperature between 25 and 35 ° C. It is aerobic and can grow in anaerobic conditions and uses a wide variety of carbon sources. nitrate reductases, one of which is used for nitrogen assimilation, is capable of secreting amylase, proteases, pululanases, chitinases, xylanases and lipases.
    Bacillus thuringiensis (strains HD-1 and HD-73 (SILoSil® BT)) are facultative anaerobic Gram positive bacteria, in the form of a peritrichous flagella. HD-1 and HD-73 strains synthesize crystals with different geometric shapes of protein and insecticide activity during the spore period. HD-1 and HD-73 strains secrete exoquitanases when in a medium containing chitin and can be used for the degradation of crustacean residues during the production of chitooligosaccharides.
    Trichoderma harzianum (TRICHOSIL) is a saprophytic fungus. It has antibiotic action and biological competition and for this reason it has biological control properties. It produces enzymes that degrade cell walls or a combination of such activities. It produces glucanases, chitinases, lipases and extracellular proteases when it interacts with some pathogenic fungi, such as Fusarium.
    As shown above, the metabolism of each group of bacteria is closely interdependent and lives in a close symbiotic association for proper HYTa performance.
    In addition to carbon, hydrogen, phosphorus, potassium, sulfur and various residual elements, a mixture of special growth factors such as B complex, amino acids
    Free l and ultra-soluble residual elements are important for optimal bacterial growth. Fermentation yeasts are incorporated into HYTa to provide these components. The N2 fixation process requires large amounts of ATP. The amount of naturally present ATP is not sufficient to promote N2 fixation
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    7/33 biological. The yeast fermentation in HYTa compensates for the large energy deficit. During fermentation, organic acids are formed in the respiratory process and together with the phosphorus released by the P decomposers, they form ATP. ATP is used in the biological nitrogen fixation process.
    HYTa contains beneficial enzymes and soil microorganisms that replace those that have been depleted due to excessive use of chemicals that result in decreased crop yields. By increasing the microbial activity in the soil with HYTa, the bacteria causes nutrients and microelements to be absorbed (mineralized) more efficiently and effectively by plants.
    Humus is transformed by some of the microorganisms into HYTa that impregnate both the soil and the plant's radical apparatus. This process provides increased nutrition for the plant. This increases the nutrients and essential elements available in the soil that can be absorbed by plants.
    The use of HYTa individually or in combination with chitin, chitosan, glucosamine and / or amino acids (1) provides nutrients and elements in the soil that increase crop yields by 25 to 55%, (2) reduces greenhouse gas emissions, (3 ) increases the efficiency of mineral fertilizers (3) reduces the use of conventional fungicides and other pesticides, (4) increases the production of plant growth regulators, (5) improves soil structure, crops, and water penetration and retention , (6) cleans chemical residues and (7) shifts the pH of the soil towards neutral pH.
    MICROBIAL COMPOSITIONS
    HYTa can be used, individually or in combination, with one or more components selected from the group of one or more amino acids, chitin, chitosan and / or glucosamine. In some cases, acetyl-D-glucosamine may be included in the microbial composition. The microbial composition includes any and all combinations of the components mentioned above. Particularly preferred combinations include: (1) HYTa and chitin; (2) HYTa and chitosan; (3) HYTa and glucosamine; (4) HYTa and amino acids; (5) HYTa, chitin and amino acids; (6) HYTa, chitin, chitosan and amino acids; (7) HYTa, chitosan, glucosamine and amino acids; (8) HYTa, chitosan and glucosamine and (9) HYTa, chitin, chitosan, glucosamine and amino acids, the latter being particularly preferred.
    As HYTa is grown in the presence of chitin, chitosan and / or amino acids it may contain chitin, chitosan and / or residual amino acids. Under such circumstances, the HYTa culture constitutes the revealed microbial composition and can be applied directly to the soil,
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    8/33 seed, seedlings or plant foliage. Alternatively, one or more of the second components will be added to supplement the second components in the composition or to change its composition.
    As used herein, the term "amino acids" refers to a composition containing 5 two or more amino acids. Amino acids include tryptophan, histidine, threonine, tyrosine, valine, methionine, isoleucine, leucine, phenyl alanine, lysine, aspartic acid, cysteine, glutamic acid, glutamine, serine, glycine, alanine, proline, asparagine and arginine. In preferred embodiments, amino acids are provided by using HYTb (see below).
    As used herein, the term "chitin" refers to a biopolymer consisting predominantly of repeating units of N-acetyl-D-glucosamine bound to beta-1-4-. Chitin is found in the natural environment as a primary structural material in the exoskeleton of animals such as Arthropods, for example, crustaceans, insects, spiders, etc., Molluscs, for example, slugs, squid, etc., Coelentara, for example, organisms such as hyidoids and jellyfish, and Nematodea, as non-segmented worms. Chitin is also found in several fungi including members of the Fusarium genus. Chitin can be extracted from these natural sources by treatment with alkali, or by a process of biodegradation. The molecular weight of chitin varies depending on its source and isolation method. In preferred embodiments, chitin is derived as a solid from the biodegradation of chitin containing Arthropods as described in Bioderpac applications.
    It is preferred that chitin has a diameter of approximately 50 to 75 microns to facilitate its application via spray and drip irrigation systems.
    As used here, the term "chitosan" is a polysaccharide that consists predominantly of D-glucosamine repeat units. Chitosan is obtained by deacetylation of chitin. The degree of deacetylation compared to chitin is preferably greater than 50%, 60%, 70%, 80%, 85%, 90% and 95%. It is preferred that the level of deacetylation is sufficient to make the chitosan soluble in water at acidic pH. The molecular weight of chitosan varies depending on its source and method of isolation. Chitosan includes chitosan oligomers. In preferred embodiments, chitosan is precipitated at pH 9.0 from the aqueous fraction obtained from the biodegradation of chitin containing Arthropods as described in the applications of Bioderpac.
    As used herein, the term "chitosan oligomer" refers to chitosan having one or more D-glucosamine repeat units and, in the case of incomplete chitin deacetylation, one or more N-acetyl-D-glucosamine units. In preferred modalities,
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    9/33 the chitosan oligomers are derived from the aqueous fraction generated in the biodegradation of chitin containing Arthropods as described in the applications of Bioderpac. In some modalities chitosan oligomers are used as the second component of the microbial composition.
    As used here, the term "glucosamine" refers to an amino monosaccharide. In preferred embodiments it is the sugar residue that forms the backbone of the chitin and chitosan biopolymers. Glucosamine is present in the aqueous fraction generated during the biodegradation of chitin containing Arthropods as described in the applications of Bioderpac. Glucosamine induces plants to become chitinase as a defense for pathogens containing chitin.
    HYTb and HYTc
    As used here, the term “HYTb” refers to the aqueous fraction and “HYTc” refers to the solid fraction obtained from the biodegradation of Arthropods as shrimp waste derived from biodegradation or Arthropods containing chitin as described in US patent application serial number 61 / 289,706, filed on 12/23/09 entitled “Biodegradation of crustacean by-products”, US patent application serial number 61 / 299,869, filed on 1/29/10 entitled “Biodegradation process and microbial composition” and patent application US serial number 61 / 355,365, filed on June 16, 2010, entitled “Biodegradtion process and composition” each of which is incorporated by reference here in its entirety.
    In summary, in the arthropod biodegradation process a microbial composition is used to degrade the arthropod or arthropod scrap components. It is a lactic acid fermentation process. The microbial composition contains microbes that produce enzymes that can degrade chitin-containing components from arthropod to chitin, chitosan, N-acetyl glucosamine and glucosamine. It also contains microbes that produce enzymes that can break down proteins and fats to produce amino acids and lipids. A preferred microbial composition for arthropod degradation is referred to as HQE. HQE was deposited with the American Type culture Collection (ATCC) Manassas, VA, USA on April 27, 2010 and received the patent filing designation PTA-10861.
    In a preferred embodiment, the marine arthropod is a crustacean and the preferred crustacean is shrimp. The shrimp by-product comprises cephalothorax and / or shrimp exoskeleton.
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    In the biodegradation process, it is preferred that the fermentation is optional aerobic fermentation. It is also preferred that the fermentation be carried out at a temperature of approximately 30 ° C to 40 ° C. The pH is preferably less than approximately 6, more preferably less than approximately 5.5. However, the pH should be kept above approximately 4.3. Fermentation is carried out for approximately 24 to 96 hours. In some embodiments, fermentation is carried out for approximately 24 to 48 hours and more preferably 24 to 36 hours. These fermentation times are much shorter than the fermentation times of the prior art, typical of 10 to 15 days to obtain substantially the same amount of digestion, although without detectable formation of chitosan and glucosamine.
    The separation of the mixture is preferably by centrifugation. (for example, approximately 920 g). Gravity separation can also be used, but it is not preferred due to the time required to obtain separation.
    The mixture separates into three fractions: solid, aqueous and lipid. The solid fraction 15 comprises chitin and is designated HYTc. The aqueous fraction comprises protein hydrolyzate, amino acids, chitosan and glucosamine and is called HYTb. The lipid fraction comprises sterols, vitamin A and E and carotenoid pigments such as astaxanthin.
    It is preferred that HQE is used in the biodegradation process. In other embodiments, it is preferred that HYTb previously prepared is added to HQE or the fermentation broth. As described above, HYTb contains amino acids, chitosan, glucosamine and trace elements including calcium, magnesium, zinc, copper, iron and manganese. HYTb also contains enzymes such as lactic enzymes, proteases, lipases, chitinases, lactic acid, polypeptides and other carbohydrates. HYTb can also contain microorganisms dormant from a previous biodegradation process. Such microorganisms can become reactivated and, in combination with HQE, contribute to a more robust biodegradation process compared to when HQE is used by itself as otherwise described here.
    More particularly, the process includes the following steps:
    The. Activation of microbial cells in a sugar-based solution to increase their growth and biomass formation.
    B. Grinding of shrimp by-products (cephalothorax and exoskeleton) to make a homogeneous paste.
    ç. Homogeneous mixture of the shrimp by-product paste with at least
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    10% of the activated inoculum.
    d. Adjust pH values to less than 6.0 in the mixture using a citric acid solution to inhibit the growth of microorganisms and promote the development of microbial cells that make up the inoculum.
    and. Fermentation of the mixture in a non-continuous stirred system at temperatures in the range of 30 to 40 ° C for at least 96 hours maintaining the pH at less than 5.0. The pH is monitored periodically. If the pH rises above 5.0, a citric acid buffer is added in an amount to maintain the pH below 5.0.
    f Centrifuging the yeast to separate the three main fractions: chitin, liquid hydrolyzate and pigmented paste.
    g. Rinse the raw chitin and collect the rinse water to recover fine solids or minerals.
    H. Chitin drying and storage.
    i. Drying and storage of the liquid hydrolyzate.
    j. The pigmented paste (lipid fraction) is stored in closed containers for conservation.
    The process and operational fundamentals are better understood with reference to the following detailed description
    Activation of microbial cells
    A microbial composition as disclosed herein is used as an inoculum. The HQE inoculum has a microbial concentration of approximately 2.5 to 3.0% (weight / v). HQE is activated by dilution to 5% in sugar cane solution (3.75% final concentration of sugar cane) and incubated at 37 ° C for 5 days. HYTb (10 ml per liter of culture) is preferably added to provide a source of naturally derived minerals and amino acids. The cell growth of the microorganisms was estimated by optical density measured at 540 nm. Activation is complete at an optical density of approximately 1.7. The concentration of microbes after activation is approximately 1.9 to 3.0% (weight / v).
    Sample preparation
    Samples of shrimp by-products are obtained from shrimp processing plants. Lightly thawed and ground residue (1500 g per batch) is mixed with 99 grams of sugar cane concentration (final concentration 6.6% by weight) and 85.5 ml of
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    HQE activated 5% (v / weight) (optical cell density = 1.7). Then the pH is adjusted to
    5.5 using 2 M citric acid.
    Fermentation control
    The mixture is incubated at 36 ° C with non-continuous shaking for 96 h. during the fermentation process, the pH is monitored using a potentiometer, and the total titratable acidity (TTA,%) was determined by titration with 0.1 N NaOH until a pH of
    8.5 is obtained. TTA is expressed as a percentage of lactic acid.
    Separation conditions
    The fermentation product is a viscous silage that has an intense orange color, due to the presence of astaxanthin. The silage is centrifuged (5 ° C) at 1250 rpm (930 g) for 15 min. to obtain chitin, liquid hydrolysates, and pigment paste. The upper phase (pigment paste) is separated manually. The liquid hydrolysates are separated by decantation, and the sediment that constitutes the crude chitin is washed with distilled water to separate fine solids. The resulting liquid is collected and dried. The crude chitin, liquid hydrolysates and fine solids are dried at 60 ° C. All fractions are stored to protect them from light.
    Other microbial compositions for the production of HYTb and HYTc are shown in table 2 below.
    Table 2
    Culture composition
    Microorganism 1 2 3 4 5 6 7 8 9 10 Bacillus subtilis X X X X X X X X Bacillus cereus X X X X X X Bacillus megaterium X X Azotobacter vinelandii X X X X X X Lactobacillusacidophilus X X X X X X X X Lactobacillus casei X X X X X X Trichoderma harzianum X X X X X X X X Rhizobium japonicum X X X X X X Clostridiumpasteurianum X X X X X X
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    Bacillus licheniformis X X X X X X X X Pseudomonasfluorescens X X X X X Bacillus thuringiensis X X X X X X Streptomyces X X X X X X X Nitrobacter X X X X X Micrococcus X X X X X Proteus vulgaris X X X X X
    These microorganisms are preferably derived from HQE and are referred to as Bacillus subtilis ((SILoSil® BS), Bacillus cereus (Bioderpac, 2008), Bacillus megaterium (Bioderpac, 2008), Azotobacter vinelandii (Bioderpac, 2008), Lactobacillus acidophilus (Bioderpac, 2008 ), Lactobacillus casei (Bioderpac, 2008), Trichoderma harzianum (TRICHOSIL), Rhizobium japonicum (Bioderpac, 2008), Clostridium pasteurianum (Bioderpac, 2008), Bacillus licheniformis (Bioderpac, 2008), Pseudomonas fluorescens, thuringiensis HD-1 and HD-73 (SILoSil® BT), Streptomyces (Bioderpac, 2008), Micrococcus (Bioderpac, 2008), Nitrobacter (Bioderpac, 2008) and Proteus (Bioderpac, 2008). Each of these organisms can be readily isolated from HQE and recombined to form the microbial composition revealed to degrade arthropods to make HYTb and HYTc.
    HYTb contains amino acids (approximately 12% by weight), chitosan (approximately 1.2% by weight), glucosamine (approximately 1% by weight) and trace elements (approximately 6% by weight) including calcium, magnesium, zinc, copper, iron and manganese. It also contains enzymes such as lactic enzymes, proteases, lipases, chitinases, among others, lactic acid, polypeptides and other carbohydrates. The specific gravity of HYTb is typically approximately 1.050 - 1.054. The average amino acid content in HYTb for certain amino acids is shown in table 2.
    Table 3
    Dry powder hydrolysates with an amino acid profile (mg per g dry weight)
    amino acid dry powder hydrolysates aspartic acid 38 glutamic acid 39 Serina 16 Histidine 9
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    amino acid dry powder hydrolysates Glycine 28 Threonine 14 Alanine 36.1 Proline 25.8 Tyrosine 70 Arginine 22.2 Valina 20 Methionine 16.4 Isoleucine 18.3 Tryptophan 3.1 Leucine 23 phenyl alanine 39 Lysine 13 Total 431
    In some embodiments, HYTb may constitute a second component that is combined with HYTa or used separately with a soil correction and / or as a spray of foliage.
    The primary component of HYTc is chitin. It has an average molecular weight of approximately 2300 daltons and constitutes approximately 64% by weight of the composition. Approximately 6% of HYTc contains minerals including calcium, magnesium, zinc, copper, iron and manganese, approximately 24% by weight of protein and 6% of water. It has a specific gravity of approximately 272 kg / m 3 . In some embodiments, HYTc may constitute a second component that is combined with HYTa or used separately as a soil correction and / or as a spray of foliage.
    HYTa is preferably used with HYTb and HYTc in combination or separately as a soil correction or foliage spray.
    The microbes in HYTa require the residual elements calcium, magnesium, sulfur, boron, manganese, zinc, molybdenum, iron, copper, sodium and silicon. These important residual elements can often be obtained from toxic chemical reactions that are not suitable for certified organic products. Therefore, it is preferred that these residual elements are obtained from an organic source such as HYTb and / or HYTc.
    Activation of HYTa
    The aforementioned microbial compositions can be used to treat
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    15/33 soil, seeds, seedlings, and / or plant foliage. However, HYTa is first activated before use.
    In preferred embodiments, HYTa is activated by incubating an inoculum of HYTa in an aqueous solution for 24-168 hours to allow microbes to grow and reproduce before being used in the process of treating soil, seeds, seedlings and / or plant foliage. Incubation conditions influence the general initial properties of HYTa.
    In one embodiment, a HYTa inoculum is diluted with water at a ratio of 1/100 and allowed to incubate at a temperature of approximately 36 ° C at a pH of 6.8 - 7.1 for approximately 24 to approximately 168 hours (7 days). HYTb can be optionally used during this activation. The nitrogen fixing microbes Azotobacter vinelandii and Clostridium pasteurianum proliferate under reduced nitrogen growth conditions. In addition, as the oxygen concentration decreases, Lactobacillus, including Lactobacillus acidophilus and Lactobacillus casei, proliferate. Colony forming units (CFUs) for some of the bacteria in activated HYTa are shown in table 3:
    Table 4
    Azotobactervinelandii Clostridium pasteurianum Bacillus subtilis Bacillus cereus Bacillus megaterium Lactobacillus
    Nitrobacter
    Nitrosomonas
    Total
    101,050,000 Cfu / mL 104,275,000 Cfu / mL 1,100,000 Cfu / mL 25,000 Cfu / mL 10,000 Cfu / mL 500,000 Cfu / mL 5,000 Cfu / mL 2,500 Cfu / mL 206,967,000 Cfu / mL
    The HYTa obtained after this incubation retains the beneficial properties of HYTa but is particularly suitable as a soil correction for treating nitrogen-free soils given the nitrogen fixing capabilities of Azotobacter vinelandii and Clostridium pasteurianum.
    If soil pathogens such as filamentous fungi from the genus Fusarium or nematodes are believed to be present, HYTa can be activated under substantially the same conditions but in the presence of chitin. Chitin stimulates the expansion of chitin-responsive microbes such as Pseudomonas fluorescens,
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    Trichoderma harzianum, Bacillus thuringiensis, Streptomyces sp., Nitrobacter sp., Micrococcus sp., And Bacillus subtilis. HYTa obtained under these conditions has antifungal, fungicidal, anti-nematode, nematodicidal and insecticidal properties to the extent that such pathogens contain chitin. Such microbial compositions can be applied directly to soil or seed, seedlings and / or plant foliage. Such microbial compositions also have the ability to fix nitrogen as in the above mentioned incubation in the absence of chitin.
    In addition to incubating with chitin, HYTa can be activated with chitin and amino acids. A preferred source of chitin is HYTc. When HYTc is used, the protein and minerals in HYTc are also present during activation.
    In addition, HYTa can be activated in the presence of amino acids and chitosan. A preferred source of amino acids and chitosan is HYTb. When HYTb is used glucosamine and the other components of HYTb are also present during activation.
    Optionally, HYTa can be incubated with chitin, amino acids and chitosan.
    A preferred source of chitin is HYTc. A preferred source for amino acids and chitosan is HYTb. When HYTb and HYTc are used the other components in these formulations are also present during activation.
    Use of activated HYTa
    Activated HYTa can be used individually or in combination with other components such as chitin, chitosan, glucosamine and amino acids to treat soil, seed, seedlings or foliage. In some embodiments, combinations of these components can be applied as a mixture. In other modalities, they can be applied separately. In yet other modalities, the components can be applied at different times.
    In one embodiment, activated HYTa can be applied to soil, seeds or seedlings, or used in foliar applications by direct application to foliage. However, when plant pathogens are present, it is preferred that the microbial composition comprises activated HYTa, chitin and / or chitosan. Alternatively, HYTa can be activated in the presence of chitin. Chitosan is known to have bactericidal, fungicidal and antiviral properties, as well as its ability to stimulate plant growth and induce plant resistance to pathogens. In other embodiments, glucosamine is a part of the microbial composition.
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    In a preferred embodiment, HYTa activated individually or in combination with chitin (preferably HYTc) and / or chitin, chitosan and amino acids (preferably HYTb and HYTc) is applied to soil, seeds, seedlings and / or foliage. It is preferred that HYTa is used in combination with chitin, chitosan, glucosamine and amino acids. HYTc is the preferred source of chitin while HYTb is the preferred source of chitosan, glucosamine and amino acids. However, the components of the microbial composition namely HYTa, chitin, chitosan, glucosamine and amino acids can be applied separately or in any combination or subcommunication. They can be applied at the same time or sequentially, in any given order. However, the preferred mode of application is to initially apply everything at the same time. The application of the above components provides for the direct treatment of plant pathogens, the induction of plant pathogen resistance pathways, and the nutrition of HYTa microbes, the native non-pathogenic soil flora and the plant.
    When soil is initially treated with a microbial composition that comprises individually activated HYTa, the microbes present in the composition have an opportunity to populate the soil and change its taxonomic composition. In some situations, initial HYTa colonization provides little or no nutrients for the plant. In such cases, it is important to maintain a nutrient reserve to maintain both the growth of microbes while colonizing the rhizosphere and the growth of plants in the soil. It may be necessary to repeat the application of HYTa, depending on the plant's growth cycle and nutrition regime. In other cases, it may be sufficient to provide additional applications of amino acids, chitin and / or chitosan, for example, HYTb and HYTc, to the previously treated soil.
    When HYTa is used in combination with, for example, HYTb and HYTc, addition nutrients are available for HYTa microbes and plants present in the treated soil.
    Table 5 shows a typical fourteen-week program for the application of HYTa, HYTb and HYTc to drip irrigated crops grown in soil. Values are per hectare. For HYTa and HYTb, the values represent liters per week. For
    HYTc, the values represent kilograms per week.
    Table 5
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    Lts / kg /week W1 W2 W3 W4 W5 W6 W7 W8 W9 W10 Wll W12 W13 W14 HYT-A 3 0 0 1 0 1 0 1 0 1 0 1 0 1 HYT-B 10 5 0 3 2 3 2 3 2 3 2 3 2 3 HYT-C 1 1 1 1
    The pulse in which the microbial composition is injected into the irrigation system must be one in which the microbial composition is able to reach the root system and remain there overnight while the system is turned off. For maximum HYTc performance, it should be applied at the same time as a mixture with HYTa. The 5 protocol must be continued while the plant is still in production. This protocol covers all stages of the plant including germination, root formation, plant growth, flowering, fruit plant, fruit harvesting and new harvesting. This protocol is designed for maximum yield potential that covers nutritional aspects, aspects of biostimulation and protection against diseases such as nematodes and fungi.
    The process can be carried out by contacting soil to form a treated soil. In some cases, the process is repeated. In some cases, plants, seedlings or seeds are already present in the soil before treatment with the microbial composition. In other cases, plants, seedlings or seeds are transplanted into the soil after treatment with the microbial composition.
    In general, before application the number of hectares or acres to be treated is determined. Then, the recommended amount of activated HYTa per hectare or acre is multiplied by the area to be treated and diluted in an amount sufficient to irrigate or spray the soil or crop in the area to be treated. The same procedure can be followed for liquid HYTb. HYTc, being a solid, can be applied directly as a solid or as a suspension in water. HYTc is preferably ground to micron size particles prior to use.
    The process can be carried out with being carried out with infertile soil. Such soils are generally those where at least one of low cation exchange capacity, low water holding capacity, low organic matter content and low levels of available nutrients are present. In general, infertile soil does not support vigorous plant growth and / or produces low crop yields.
    For non-soil systems such as hydroponics, the same protocol applies however
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    Microbial compositions can be used with respect to any plan that includes, but is not limited to, alfalfa, banana, barley, broccoli, carrots, corn, cucumber, garlic, grapes, leek, melon, onion, potato, raspberry, rice , soy, pumpkin, strawberry, sugar cane, tomato and watermelon.
    When applied as a soil correction, the microbial composition containing
    HYTa, chitin, amino acids and chitosan increase crop production and average approximately 25% - 55% compared to the 15 to 25% increase in crop production observed for E2001. From Karl Co. SA de CV, Navojoa, Sonora, Mexico.
    Microbial composites can also result in a decrease in the amount of chitin used. For example, chitin has been used as a soil correction in the prior art. Typically, approximately 600 kg of chitin was used per hectare. However, beneficial effects of such use have not been seen for up to six months. When HYTa was activated in the presence of chitin and then combined with chitin and applied as a soil correction, beneficial effects were observed after seven days with the use of only 4-6 kg of chitin per hectare.
    Although the development is mainly directed to the use of the microbial compositions developed for agricultural applications, such compositions or their components and processes can also be used in horticulture applications to improve the production of foliage and flowers and to decrease the use of conventional insecticides and fungicides.
    When activated HYTa is applied to the soil, seed, seedling or foliage, it forms treated soil, treated seed, treated seedling, treated foliage and treated plants. HYTa is a new microbial composition. Therefore, the soil, seed, seedling, foliage and plants treated with HYTa are also new.
    Treated soil is defined as soil that contains one or more microbes that are unique to HYTa dispersed in treated soil. Such microbes can be detected in the soil treated generically by using a BioChip that detects microbial populations based on DNA. See, for example, US patent publication 2007/0015175, which is incorporated herein by reference. Other methods, such as PCR, which are known to those skilled in the art can also be used. Microbes in HYTa that are particularly preferred are Bacillus subtilis (SILoSil® BS), Bacillus thuringiensis strain HD-1, Bacillus thuringiensis strain HD-73 (SILoSil® BT) and Trichoderma harzianum (TRICHOSIL) each of which can be isolated from the deposit of HYTa or obtained from
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    Biotecnologia Agroindustrial S.A. DE C.V., Morelia, Michoacan, Mexico. Trichoderma harzianum (TRICHOSIL) is more preferred as it is important during HYTa activation as it causes inter-component synergies between the other microbes in HYTa. The identification of one or more of these microorganisms can be further combined with the identification of other microbes in HYTa, if necessary, to confirm the presence of HYTa or that HYTa was present. Each of Bacillus subtilis (SILoSil® BS), Bacillus thuringiensis strains HD-1 and HD-73 (SILoSil® BT) and Trichoderma harzianum (TRICHOSIL) were deposited with the ATCC in_and received the patent filings _, _ and _, respectively.
    Seed, seedlings, foliage and treated plants are similarly defined. In these cases, HYTa microbes are found on the surfaces of seed, seedlings, foliage and treated plants.
    As used herein, the term "consisting essentially of" with respect to 15 HYTa, HYTb and HYTc means any of HYTa, HYTb and / or HYTc individually or in combination without additional microbes.
    Example 1
    The following example compares the growth of Persian cucumber plants using HYTa, HYTb and chitosan compared to a control that has not been treated with
    HYTa, HYTb and chitosan.
    During the development of Persian cucumber seedlings, seeds were incubated for three hours in a mixture of 1 liter of water and 250 grams of HYTc. A bag of peat moss and 250 grams of chitin of 200 micronized mesh (approximately 75 microns in diameter) (HYTc) per bag of peat moss were mixed. The seeds were planted in the peat moss / chitin mixture at 18-24 ° C. the development of the plant after five days after treatment with HYTc was comparable with 9 days of development without treatment.
    Control and treated seedlings were transplanted on 1 hectare of soil in a greenhouse. The control soil and HYTa were treated as shown in table 5.
    Table 6
    HYT-A Control nitrogen fertilizer 150 Kg 280 kg Potash 160 kg 250 Kg
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    Calcium 80 kg 130 Kg phosphor 200 kg 320 Kg Magnesium 20 kg 45 kg residual elements 10 liters 22 liters Fungicides 0 20 liters Insecticides 0 16 liters agricultural soap made fromolive oil and palm oil 10 liters 0
    The soil containing the seedlings treated with HYTa was treated with 2 liters of HYTa and 7 liters of HYTb over time.
    HYTa was diluted in 200 liters of water and activated without the presence of HYTb or
    HYTc.
    In week two, one liter of HYTa and three liters of HYTb were applied to the soil and two liters of HYTb were applied to the foliage of plants treated with HYT.
    There was a significant increase in the yield of cucumbers compared to the control. Control plants produced 3,000 25-pound boxes while HYT-treated plants produced 4,300 boxes. Therefore, this example demonstrates a significant increase in yield using HYT and a decrease in the amount of fertilizer, insecticides, fungicides and other components needed otherwise.
    Example 2
    Septoria leaf and initial rust as well as infection of Roma and beefsteak tomatoes with Phytophthorainfestans can be treated by the protocol shown in table 7. All values are per hectare.
    Table 7
    start Per day Duration Application HYTa 3 liters 0 10 days pulverization HYTa 2 liters 0 10 days systempulverization in HYTc 20 kg 2 kg 10 days pulverization HYTc 500 grams 0 0 systempulverization in HYTb 1 liter 1 liter 10 days pulverization
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    HYTa was diluted in 200 liters of water and activated with HYTc.
    This treatment resulted in the control of these infections.
    Example 3 acres of Roma tomatoes were treated with 4 liters of HYTa, 10 liters of HYTb 5 and 30 pounds of chitin.
    The application protocol was as follows for 10 acres:
    Table 8
    Lts / Lb / week W1 W2 W3 W4 W5 W6 W7 * HYT-A 3 0 0 0 1 0 0 ** HYT-A 2 0 0 0 1 0 0 * HYT-B 6 5 5 0 ** HYT-B 4 2 2 * HYT-C 5 5 ** HYT-C 10 5 5
    * irrigation system: spraying (foliage) ** irrigation system: drip tape
    Values are in liters for HYTa and HYTb and pounds for HYTc. The crop yield was 46 tons of tomatoes per acre compared to 31 tons per acre for the control. This is a 36% increase in yield.
    Example 4
    Root knot nematode Meloidogyne spp. And white fungus disease caused by
    Sclerotiniasclerotiorum have been identified as problematic for the growth of carrots. Figure 2A shows the foliage and carrots obtained from such soil.
    The following protocol was used to treat one hectare. One kg of HYTc was applied to the soil at the time of transplantation. Two weeks after 1 kg of HYTc and 1.5 liters of HYTa were applied. Two weeks after 2 kg of HYTc and 1 liter of HYTb were applied. Thirty days after 1.5 kg of HYTc, 1 liter of HYTb and 1 liter of HYTa was applied.
    The root vesicles caused by the nematode infection were no longer present in the carrots after treatment. The soft cotton rot caused by white fungus was also absent from the carrots after treatment.
    Example 5
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    HYTa, HYTb and HYTc can be used to eradicate and control ROYA (Puccinia dracunculina) in Tarragon (Artemisia dracunculus L.). a total of 6 liters of HYTa, 15 liters of HYTb and 900 grams of HYTc were applied to each hectare.
    The following protocol was used:
    Table 9
    Product Dosage Timecourse in application HYTa 2 liters 5 days pulverization HYTb 5 liters 5 days pulverization HYTc 300 grams 5 days pulverization
    The protocol was repeated twice. This treatment reduced ROYA damage in treated foliage.
    Example 6
    This example reveals a summary of tests done in cooperation with and under the supervision of the Center International of Maize and Wheat Improvement Center (known and referred to here as CIMMYT) http://www.cimmyt.org/.
    This example presents the final harvest data for the different treatments. CIMMYT personnel collected samples according to their scientific methodologies and information.
    These tests were designed to demonstrate the following key benefits of using HYTa alone or in combination with HYTb: (1) the ability to maintain high-performance growth with different fertilizer and mineral regimes, (2) to improve system performance through use of HYTa or HYTa in combination with HYTa, and (3) the ability to restore soil health and increase fertility levels through repeated use of HYT programs.
    The purpose of the test was to determine the effect of crop levels and straw handling in two different soil environments (neighborhood and flood), to investigate the efficiency of different forms, types and doses of mineral fertilizers in combination with Agrinos HYT to make more efficient use of these inputs to increase the profitability of wheat cultivation for the producer.
    Test areas
    These experiments were carried out in an agricultural field associated with the transferee, which has been used widely for the development of
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    This area of experiments is identified by CIMMYT under the coupon code Z 702 module Agirnos-CIMMYT and is in the Irrigation District no. 38, module 4, section 15, irrigation rollers 1049-0 and 1115-0.
    One of the main attributes of HYT ™ products is their ability to improve (rather than degrade) agricultural soils with continuous use. To demonstrate this attribute of the HYT ™ product, the experiments included a test area that was not treated with mineral inputs, E 2001 or any HYT product. The performance of crop plants in this area depends entirely on the state of the soil before planting.
    Other information
    - types of crop: wheat;
    - variety: ATIL (durum wheat);
    - sowing date: December 23 in dry and wet soil in areas 2 and 1 of figure 3. Planting was delayed until January 14, the following year due to a flood caused by an irrigation problem in the adjacent lot.
    - harvest date: May 20-23 (approximately 4 months after planting)
    - size of the test area: 15 hectares
    - mineral fertilization: it was used as the basis for fertilization that is considered to be the best practice of de-mineralizing NPK mineral nutrients generally accepted in the region (BNFP = best nitrogen fertilizer practice).
    Table 10
    Mineral fertilizers and HYT ™ protocols
    Treatment description initial application second application third application treatment 1 areacontrol; without 0 NPK unit, 0 NPK unit,0 liter HYTa or 0 liter HYTa or 0 units of NPK,0 liter of HYTa or treatment 2 fertilizer50% BNFP +HYTa and HYTb HYTb103 units of52 units of HYTbN, 1 liter of HYTa;P, 1 liter of HYTb HYTb1 liter of HYTa;1 liter of HYTb Treatment 3 HYTa + HYTb 1 liter of HYTa1 liter of HYTa 1 liter of HYTa;1 liter of HYTb 1 liter of HYTa;1 liter of HYTb
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    100% BNFP 149 units of N, 61 units of N
    Treatment 4 units of P
    In addition to the main protocols described above, some areas were tested and harvested separately with some additional component, in order to obtain an extra reference point and expand the possibilities for analysis. The designated test was as follows:
    TRT 5: HYT biological treatment plus 100% traditional mineral fertilization program: initial application: 1 liter of HYT + 103-52-0 (NPK), second application 1 liter of HYT + 1 liter of HYT B + 61-0- 0 (NPK), third implementation 1 liter of HYT B. This additional treatment was recommended by CIMMYT to observe the behavior of the traditional program of more complete mineral nitrogen program HYT a + b in the performance of the wheat grain. Only an area of 4 rows was dedicated to this treatment and the information was collected only by a person from CIMMYT.
    A diagram of the test area is shown in figure 1 where “TRT” refers to the treatment identified above.
    External factors in the test area
    Some areas of the test zone have been compromised and have been damaged by external factors. The results of these areas were excluded from the final results of the harvest to allow a reliable comparison. Reference is made to figure 2. These external factors were as follows:
    Highlighted area 1 (zone 1): the variety of wheat used is very susceptible to “Chahuistle”. Due to the proximity of areas 1 and 5 to high voltage power lines, the airplane cannot apply the product in these areas and, consequently, these areas suffered a higher incidence of the pathogen, causing significant loss of performance potential.
    Highlighted areas 1 and 2 (zone 2) suffered flooding due to irrigation problems in the surrounding lots, delaying the planting by 20 days and being affected by the "chauistle".
    Highlighted areas 9 and 10 (zone 3) suffered from irregular irrigation due to the topography of the land that makes erratic crop performance having low and high areas causing non-uniform irrigation that affects the average performance.
    Table 11
    Final total harvest data reported for different treatments
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    Lot tonnesmetricshectare per Lot metric tonsper hectare Area 1 4.1 * Area 9 5.4 / 6.6 * Area 2 4.1 * Area 10 6.2 * Area 3 7.8 Area 11 6.8 Area 4 8.3 Area 12 8.3 Area 5 4 * Area 13 8.3 Area 6 7.1 Area 14 7.7 Area 7 7.4 Area 15 8.2 Area 8 7.9 Area 16 9.0
    * external factors affected this result
    Table 12
    Experiment results
    Performance
    Tons / Ha * Clay* Alluvial * Average* 100% BNFP 7.45 7.40 7.4 (Treatment 4) 50% BNFP plus HYTa and HYTb 7.40 7.20 7.8 (Treatment 2) Control 7.90 8.30 8.2 (treatment 1) HYTa and HYTb only 8.30 6.50 8.7
    (Treatment 3) _
    * results from areas where results affected by external factors are not included Compared to the expected average wheat yield in the region, the repeated historical use E 2001 and HYT ™ contributes to the significant increase in performance by 36% compared to standard fertilization only, see figure 3. This happened without adding any additional elements of NPK or HYT during this agricultural cycle since previous applications of E 2001 and HYT had already recovered the activity and biodiversity of colonies of benign soil microbes creating high levels of organic matter and nutrients available in the soil.
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    The addition of HYTa and HYTb to the plant-soil cycle system continues to improve the soil's ability to supply nutrients to plants, increasing the biological nitrogen fixation capacity. See figure 3.
    Various combinations of standard fertilization regimes, either individually or in combination with HYTa and HYTb, do not appear to improve results compared to the use of HYTa and HYTb. This may be due to the existence of sufficient stored nutrients such as biomass in the soil from previous years.
    When the soil and ecosystem have sufficient nutrients available, through FBN and / or high levels of biomass in the soil, the addition of more fertilizer (NPK) destabilized the biological balance and interfered with the nutrient absorption patterns of the plants, possibly altering the capacity from cultivating to guide your own nutritional program given the resources available in the soil and active biomass in your roots.
    Example 7
    Field experiments were conducted in Pantnagar India under the project entitled ‘Agronomic evaluation of HYT (HYTa, HYTb and HYTc and foliar spray of Suryamin). Details are provided below.
    Culture: wheat Project used: RBD Replication: 3
    Sowing date: 11/19/2010
    Variety: PBW-550
    Gross lot size: 6.0 mx 4.0 m = 24 m 2 Treatments: 12 Treatment details
    T-1: recommended NPK dose
    T-2: application of T-1 + HYTa soil (activated for 72 h) @ 1L at sowing
    T-3: application T-1 + leaf of HYTb + 2L at flower initiation / panicle initiation)
    T-4: application T-1 + HYTc soil @ 2 kg at the time of sowing
    T-5: T-1 + HYTa (activated for 72 hours) @ 1 liter + HYTc @ 2 kg / ha as application of sowing soil
    T-6: T-1 + foliar application of HYTb @ at flower initiation / initiation
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    T-7: T-1 + HYTa (activated for 72 hours) @ 1L + HYTb @ 2L at the time of flower initiation / panicle initiation + HYTc @ 2 kg / ha as application of soil at sowing
    T-8:% dose NPK + HYTa (activated for 72 hours) @ 1L + leaf application of HYTb @ 2L + HYTc @ 2 kg / ha as application of soil at sowing
    T-9: T-1 + application of HYTa soil (activated for 72 hours) @ 2L / ha + leaf application of HYTb @ 5L + HYTc @ 5 kg / ha at sowing
    T-10:% dose NPK + HYTa (activated for 72 hours) @ 2L + foliar application of 10 HYTb @ 5L + HTB-c @ 5kg / ha as application of sowing soil
    T-11: T-1 + 1L / ha of Shriram Suryamin as leaf application in flower initiation T-12: T-1 + 1L / ha of Shriram Suryamin as leaf application each in bud and in flower initiation
    The biological yields, grain and straw are shown in table 13.
    Table 13
    Effect of different HYT organic product on biological yield, grain and straw of wheat culture.
    Treatments Yieldbiological(q / ha) Yieldgrain(q / ha) Yieldstraw(q / ha) T1: Rec. NPK 82.63 32.00 50.63 T2: T1 + HYT-A @ 1.0 l / ha 85.43 33.99 51.50 T3: T1 + HYT-B @ 2.0 l / ha 87.50 35.30 52.20 T4: T1 + HYT-C @ 2.0 kg / ha 76.40 31.33 45.07 T5: T1 + HYT-A + C 87.63 37.90 49.73 T6: T1 + HYT-B + C 84.53 34.80 49.73 T7: T1 + HYT-A + B + C 86.93 35.80 51.93 T 8 : ANPK + HYT-A + B + C 56.90 27.13 29.77 T9: T1 + HYT-A + B + C (dose pluselevated) 88.60 40.27 48.33 T10: ANPK + HYT-A + B + C (more doseelevated) 58.37 28.30 30.07 Tn: T1 + Suryamin (one spray) 82.87 32.93 49.93 T12: Tt + Suryamin (two 83.57 33.17 50.40
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    Treatments Yieldbiological(q / ha) Yieldgrain(q / ha) Yieldstraw(q / ha) sprays) S.In (5%) 4.33 0.92 4.27 CD (5%) 12.68 2.71 12.54
    Table 14 compares the results for grain yield for the various treatments with the HYT components and different combinations.
    Table 14
    Yield CD2.71 standard error0.92 inabovekilo / h grainof 32ectare Control 0 HYTa 1.99 NS * HYTb 3.3 * * HYTc -0.67 NS NS HYTa + c 5.9 * * HYTb + c 2.8 * * HYTa + b + c(1L / 2L / 2Kg) 3.8 * * HYTa + b + c(high dosefrom a, b and c:2L / 5L / 5Kg) 8.27 * *
    NS = Not statistically significant compared to control * = statistically significant compared to control As can be seen, the separate use of HYTa and HYTb improved grain yield by 1.99 and 3.3 kilos per hectare respectively while the use of HYTc individually caused a decrease in yield. When HYTa was combined with
    HYTc yield increase was 5.9 kilos per hectare which is greater than the sum of the results when used separately. The use of HYTb and HYTc resulted in an increase of 2.8 kilos per hectare while the use of HYTa, HYTb and HYTc caused an increase of 3.8 kilos per hectare. The greatest increase in grain yield was observed
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    Example 8
    This example exposes results of pumpkin growth in infertile soil.
    In these experiments, pumpkin seedlings were planted in 5-gallon pots containing infertile "Superstition" sand. The combinations of HYT A, B, and C were applied as shown in table 16. The seedlings were planted on December 21 and the harvest started on January 20 and continued until February 27 of the following year.
    Table 15
    Date treatment fees applied 16 December 200 mL activated HYTA, 10 mL HYT B, and 200 g HYTC perBowl. 23 December 10 mL HYTB per pot. 30 December 5 mL HYTB per vessel. 5 January 5 ml HYTA and 5 ml HYTB per pot. 19 January 10 mL HYTB per pot. 27 January 10 mL HYTA and HYTB per pot. 3 February 10 mL HYTB per pot. 10 February 10 mL HYTA and 10 mL HYTB 17 February 5 mL HYTA applied and 5 mL HYTB in each vessel. 25 February 5 mL HYTB applied to each vessel. 1 March 5 mL HYTA applied and 5 mL HYTB in each vessel.
    The results are shown in table 16.
    Table 16
    treatment Yield(g / vase) Control 0 HYTa 251.6 HYTb 137.9 HYTc 62.6 HYTa + HYTb 472.1 HYTa + HYTc 0 HYTb + HYTc 0
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    treatment Yield(g / vase) HYTa + HYTb + HYTc 62.3 Stat, A ** B ** Ç NS A * B NS B.C ** B * C NS
    NS is not significant at P <0.05.
    *, ** are statistically significant at P <0.05 and P <0.01, respectively.
    As can be seen, there was a substantial increase in yield of zucchini squash when HYTa and HYTb were used separately and when HYTa and HYTb were used in combination.
    Example 9
    The following protocol was used to treat melons.
    Table 16
    PRODU-TO INTERNSHIP> START FLOWERING MATURATION 15 DAYSAFTER READYFORHARVEST APPLICATION Dose (Kg or Lt / Ha) HYT-a ▼ EARTH 3 0.3 0.25 1 0.5 0.25 0.25 HYT-b Foliar 1 1 1 1 1 1 EARTH 3 1 1 3 2 2 2
    The results are shown in table 17 and figure 4.
    Table 17
    WITH HYTa and HYTb TREATMENT ACCUMULATED INFORMATION BY HA SIZE PARTS LBS BOXES PARTS LBS BOXES 9 299 1,289,860 33 7,407 31,958,395 823 12 838 2,859,835 70 20,790 71,007,580 1,733 15 543 1,719,652 36 13,593 43,059,160 906 18 241 604,131 13 6,074 15,266,914 337
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    TOTAL 1,921 6,473 153
    47,864 161,292 3,799
    NO TREATMENT ACCUMULATED INFORMATION PER HA SIZE PARTS LBS BOXES PARTS LBS BOXES 9 181 727,936 20 4,481 18,026,691 498 12 493 1,650,330 41 12,247 41,030,815 1,021 15 463 1,489,587 31 11,568 37,168,321 771 18 176 465,597 10 4,432 11,725,728 246
    TOTAL
    1,313
    4,333
    102
    32,728
    107,952
    2,536
    PERCENTAGE INCREASE
    46.25% 49.41% 49.81%
    BY HA
    Example 10
    Pumpkin was grown according to the following protocol.
    Table 18
    PRODUCT APPLICATION DOSAGE FOR HECTARE 7/3 10/3 03/18 4/4 11/4 4/18 Hyt-a GROUND 2 1 Hyt-b Foliar 1 1 3 1 1 SOLAR 1 1 1 1 1
    The results are shown in table 19.
    Table 19
    SIZE BOXES / HA DIFFERENCEINBOXES / HA DIFFERENCEIN % CONTROL TREATED, X 188 228 40 17.54% XX 63 145 82 56.55% XXX 47 95 48 50.52% BRUCE 29 30 1 3.33%
    Petition 870180001588, of 1/8/2018, p. 39/45
    33/33
    Example 11
    An experiment with HYTa and HYTb was carried out in Norway in a potato crop. The tests with and without HYTa and HYTb were treated with 50 or 100 kg of nitrogen fertilizer / ha. Pesticides were used in normal amounts.
    At the time of the first emergency (June 14), 0.2 liters of HYTa and 0.6 liters of HYTb were applied by decare. After the last application of clay (July 20), 0.2 liters of HYTa, 0.2 liters of HYTb and 50 grams of HYTc were applied by decare. The results are shown in figure 5.
    The use of HYTa and HYTb provided an increase in yield of up to 17% compared to the control. In addition, there was less potato blight in the culture treated with HYTa and HYTb compared to the control.
    Petition 870180001588, of 1/8/2018, p. 40/45
    1/3
    权利要求:
    Claims (3)
    [1]
    1. Microbial composition, characterized by the fact that it comprises HYTa of the ATCC patent filing designation PTA-10973 and at least one of chitin, chitosan, glucosamine and amino acids.
    5 2. Microbial composition according to claim 1, characterized by the fact that it comprises HYTa and chitin.
    3. Microbial composition, according to claim 1, characterized by the fact that it comprises HYTa, chitosan, glucosamine and amino acids.
    4. Microbial composition according to claim 1, characterized by the fact that it comprises HYTa, chitin, chitosan, glucosamine and amino acids.
    5. Microbial composition according to any one of claims 1 to 4, characterized by the fact that chitin is HYTc, wherein said HYTc is the solid fraction obtained from the fermentation of Arthropods containing chitin with a microbial composition comprising HQE of ATCC patent filing designation
    15 PTA-10861.
    6. Microbial composition according to any one of claims 1 to 4, characterized by the fact that chitosan, glucosamine and amino acids are HYTb, in which said HYTb is the solid fraction obtained from the fermentation of Arthropods containing chitin with a microbial composition comprising HQE from the designation of
    20 ATCC patent filing PTA-10861.
    7. Microbial composition according to claim 1, characterized by the fact that it comprises HYTa and at least one of HYTb and HYTc;
    wherein said HYTb is the solid fraction obtained from the fermentation of Arthropods containing chitin with a microbial composition comprising HQE of
    25 ATCC patent filing designation PTA-10861; and where said HYTc is the solid fraction obtained from the fermentation of
    Chitin-containing arthropods with a microbial composition comprising HQE from the ATCC patent filing designation PTA-10861.
    8. Microbial composition according to claim 7, characterized by the fact that it comprises HYTa, HYTb and HYTc.
    9. Process, characterized by the fact that it comprises contacting soil, seed, seedling, or plant foliage with HYTa from the ATCC patent filing designation PTA-10973.
    Petition 870180001588, of 1/8/2018, p. 41/45
    [2]
    2/3
    10. Process, according to claim 9, characterized by the fact that it also comprises contacting soil, seed, seedling, or plant foliage with chitin, chitosan, glucosamine and amino acids.
    11. Process according to claim 9 or 10, characterized by the fact that said chitin is HYTc; wherein said HYTc is the solid fraction obtained from the fermentation of Arthropods containing chitin with a microbial composition comprising HQE from the ATCC patent filing designation PTA-10861.
    12. Process according to claim 9 or 10, characterized by the fact that said chitosan, glucosamine and amino acids are of HYTb, in which said HYTb
    10 is the solid fraction obtained from the fermentation of Arthropods containing chitin with a microbial composition comprising HQE from the ATCC patent filing designation PTA-10861.
    13. Process according to any of claims 9 to 12, characterized by the fact that said HYTa is activated in an aqueous solution for 24 to
    15 168 hours before said contact.
    Process according to any of claims 9 to 12, characterized in that said contact is from said soil to form treated soil.
    15. Process, according to claim 14, characterized by the fact that said method further comprises contacting said treated soil or foliage on said soil with
    20 one or more of HYTa, chitin, or HYTb.
    16. Process, according to claim 9, characterized by the fact that said contact is of said foliage to form treated foliage.
    17. Process, according to claim 16, characterized by the fact that said method further comprises contacting said treated foliage or soil containing a
    25 plant with said foliage treated with one or more of HYTa, chitin, chitosan, glucosamine and amino acids.
    18. Process according to any one of claims 9 to 17, characterized by the fact that said plants, seedlings or seeds are present in said soil before said contact step.
    19. Process according to any of claims 9 to 18, characterized by the fact that before said contact of said treated soil, the plants, seedlings or seeds are transplanted to said treated soil.
    20. Activated HYTa, characterized by the fact that it is done by incubating HYTa from
    Petition 870180001588, of 1/8/2018, p. 42/45
    [3]
    3/3 ATCC patent deposit designation PTA-10973 in the presence of HYTc for 24 to 168 hours, wherein said HYTc is the solid fraction obtained from the fermentation of Arthropods containing chitin with a microbial composition comprising HQE of the deposit designation of ATCC patent PTA-10861.
    21. Process, characterized by the fact that it comprises combining activated HYTa, as defined in claim 20, and at least one of HYTb and HYTc to form a mixture, wherein said HYTb is the solid fraction obtained from the fermentation of Arthropods containing chitin with a microbial composition comprising HQE from the ATCC patent filing designation PTA-10861; and wherein said HYTc is the solid fraction obtained from the fermentation of chitin-containing Arthropods with a microbial composition comprising HQE from the ATCC patent filing designation PTA-10861.
    22. Process, according to claim 21, characterized by the fact that it further comprises applying said mixture to the soil, foliage, seed or seedlings.
    Petition 870180001588, of 1/8/2018, p. 43/45
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    法律状态:
    2017-10-10| B07A| Technical examination (opinion): publication of technical examination (opinion) [chapter 7.1 patent gazette]|
    2017-10-17| B15K| Others concerning applications: alteration of classification|Ipc: A01N 63/00 (1980.01), A01N 63/02 (1980.01), A01N 6 |
    2018-02-14| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|
    2018-04-17| B16A| Patent or certificate of addition of invention granted|
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    US35544710P| true| 2010-06-16|2010-06-16|
    US61/355,447|2010-06-16|
    PCT/EP2011/059936|WO2011157747A2|2010-06-16|2011-06-15|Microbial process and composition for agricultural use|
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