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    • 专利摘要:
    Biostimulant formulation of plant growth and development and resistance induction for the control of diseases caused by phytopathogenic viruses and preparation method. The biostimulant formulation of plant growth and development and resistance inducer for the control of diseases caused by phytopathogenic viruses is composed of extracts and vegetable oils from plant varieties of chihuahuan semi-desert and absolute oils and extracts of aromatic plants, among other components. A method to prepare the biostimulant formulation comprising preparation of the base of the formulation, addition of a premix containing ethanolic extracts of plants, hormones and plant growth regulators and addition of methanolic extract of eucalyptus globulus, addition of a premix containing oils absolutes and addition of acetic acid extract of larrea tridentata and addition of a premix containing hexane extracts and a conditioning agent. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2628278A1
    申请号:ES201631369
    申请日:2016-10-25
    公开日:2017-08-02
    发明作者:Jesús Noel YAÑEZ REYES
    申请人:Greencorp Biorganiks De Mexico SA de CV；
    IPC主号:
    专利说明:

    BIO-STIMULATING FORMULATION OF VEGETABLE GROWTH AND DEVELOPMENT AND RESISTANCE INDUCTOR FOR THE CONTROL OF DISEASES CAUSED BY VIRUS PHYTOOPATHOGENS AND PREPARATION METHOD
    5 Description
    TECHNICAL FIELD OF THE INVENTION
    10 The present invention is in the area of biotechnology and particularly concerns the field of agricultural biotechnology. It refers to the use of plant extracts and other products of natural origin to prepare a biostimulant formulation of plant growth and development and resistance inducer for the control of diseases caused by phytopathogenic viruses.
    BACKGROUND OF THE INVENTION
    lS Plant diseases are the cause of the greatest economic losses in agriculture around the world (Martinelli et al., 2015). Viruses cause many diseases in plants of international importance and are responsible for large losses in terms of production and quality in crops, although it is difficult to calculate those economic losses have been estimated to represent more than 30 billion dollars annually (Sastry and Zitter , 2014).
    twenty Viruses are strictly intracellular pathogens, so their control is complicated and the main measures for their control are linked to the destruction of infected plants or the excessive use of pesticides for the control of organisms that function as vectors of viruses ( Nicaise, 2014). There are practically no antiviral compounds capable of curing viral diseases in plants and current control measures can only mitigate or prevent their occurrence (Gergerich and Dolja, 2006).
    2S The first step required for the management of viral diseases is the identification of the virus. The subsequent management strategy will depend on the way in which a given virus enters the crop, how the virus is transmitted between the plants of the same crop, and how the virus survives in the absence of the crop (Haddidi et al., 1998 ). Some preventive measures include the use of seeds or certified vegetative organs free of viruses, the elimination of possible
    reservoirs of the virus in the surrounding wild vegetation and the modification of practices of
    planting and harvesting. If the virus has a known transmission vector, the control exclusion of
    vector is extremely important, for example: nematodes, insects and fungi vectors that
    they can be controlled with nematicides, insecticides and fungicides, respectively (Gergerich and Dolja,
    S 2006).
    However, an alternative for virus control can be found in the evolution itself
    of plants, which has provided them with resistance mechanisms through interactions
    plant-virus In the last decades, processes have been developed in the identification and
    Understanding the role of phytohormones and other key components in plant response
    10 to biotic stress (Robert-Seilaniantz et al., 2011). Starting from this idea, you can select
    various ingredients of natural origin to integrate an alternative to induce the defense of
    plants to damage by viruses through the activation of systemic resistance mechanisms
    acquired (Delaney al., 1994).
    Traditionally the use of various plant extracts with various applications has been described,
    lS one of them being the development of products based on plant extracts that allow
    favorable development of plants, as well as induction of resistance against various
    phytopathogens For this, several technologies have been described in the state of the art, such as
    describes the international patent application WO 1989009200 A 1, which refers to the use of
    Viscum album extract or white mistletoe as part of a composition for the
    twenty soil conditioning and leaf fertilizer that allows the restoration of soil nutrients and
    the regeneration of the latter. In addition, the use of Viscum extracts has also been reported.
    album in combination with other active compounds or extracts, such is the case of the request for
    U.S. Patent No. 2014/0364309 A1, in which extracts of Aloe Vera and
    seaweed to reduce damage to plants and their fruits caused by insects, parasites and others
    2S phytopathogens, which do not include viruses.
    International patent application WO 2006097700 A 1 refers to individual use or
    combined essential oils of plants such as eucalyptus (Eucalyplus globulus), peppermint
    (Menlha piperita), clove (Syzygium aromalicum) or oregano (Lippia graveolens), in combination
    with vegetable oil and an emulsifying agent to improve plant growth. Likewise
    30 Excerpts from various US patent documents have used extracts from
    Coriandrum sativum with various applications: in the first one, US 8202557 B,
    it has been used to prepare insecticides, while in US patent application 200610194698 A1 reference is made to active phytochemicals to protect plants from weeds, pests and phytopathogens; and although antiviral properties have also been described for Coriandrum sativum extracts, as in US Patent 8529968 82, they have not been linked to viruses
    5 phytopathogens
    On the other hand, in recent years and due to the demand for products that stimulate the growth of plants as well as increase the spectrum of protection against pathogens (viruses, bacteria and fungi) a series of chemical compounds have been developed , as well as a new generation of products called biostimulants, which contain various 10 substances and / or microorganisms that when applied to plants or the rhizosphere improve crop development and responses to biotic stress. Some biostimulants are described in international patent applications WQ 2014020187 A1, WQ 2013030422 A1 and WQ 2012045189 A2, in the first one, biostimulants are made from compounds of mineral or animal origin and algae extracts; in the WQ patent application
    2013030422 A1 waste from beer production is used for the production of biostimulants; while in application WQ 2012045189 A2 the biostimulants are made from chitosan and hydrolyzed from Sacharomyces cerevisae.
    Although a wide use of various plant extracts and essential oils of plants with antiviral activity has been described, few have been directly tested on agricultural crops and 20 specifically with phytopathogenic viruses.
    Some other biostimulant products that are currently on the market are made from glutathione, oligosaccharins and combinations of extracts and oils from plants that provide some effectiveness in the fight against viruses, however none of them describe the combination of extracts and substances which integrate the present invention and which allow to have a synergistic effect to combat DNA and RNA viruses of greater impact on the production of vegetable and fruit crops. Among the main technical advantages of the invention are the following: it prevents and reduces the damage caused by viruses, reduces the number of damaged plants, delays the appearance of symptoms due to virosis, significantly reduces the severity of the damage, reduces the spread of the virus in the plantation, favors the continuity of
    30 growth in plants and ensures greater performance in attack conditions.
    In view of the background in the state of the art, the technical problem solved by the present invention is to provide a formulation based on the use of various plant extracts and essential oils of plants to produce a product with biostimulant action of growth and development of the plants and inducer of resistance to phytopathogenic viruses, which
    5 has the characteristics of being sustainable for its application and safe for the health of vertebrates and invertebrates and of low impact for the environment, which makes the present invention novel, inventive and with a concrete application in the field of agriculture .
    SUMMARY OF THE INVENTION
    An object of the present invention is to provide a formulation based on the use of various plant extracts and essential oils of plants to produce a product with agronomic application. Said vegetable extracts and essential oils include: ethanolic and acetonic extracts from Larrea tridentata, ethanolic extracts from Viscum album, absolute oil from Uppia graveo / ens and hexane extracts from Euphorbia antisyphilitica, Jatropha dioica and Agave americana,
    15 oils of Syzygium aromaticum and Cinnamomum zeylanicum, methanol extract of Eucaliplus globulae, aqueous extracts of Rosmarinus officinalis, Salvia officinalis and Mint piperita, and ethanolic extract of Coriandrum sativum. Additionally, the formulation includes sulfur compounds, amino acids and specific peptides of plant and animal origin, hormones and plant growth regulators, multi-vitamins and polysaccharides.
    Another object of the invention is to provide a formulation that has biostimulant activity of plant growth and development, mainly in plants of the Solanaceae, Cucurbitaceae, Rosaceae, Legumes, Alliaceae, Caricaceae, and Musaceas families, among others of commercial interest.
    Another object of the present invention is to provide an alternative to the prevention and reduction of damage associated with plant diseases caused by phytopathogenic viruses.
    A further object of the present invention is to provide an alternative to induce resistance of plants to diseases caused by phytopathogenic viruses. DNA and RNA type.
    Another object of the present invention includes providing a process for preparing a biostimulant formulation of plant growth and development and resistance inducer for the control of diseases caused by phytopathogenic viruses.
    BRIEF DESCRIPTION OF THE FIGURES
    S Figure 1 shows a process diagram for the preparation of the biostimulant formulation of plant growth and development and resistance inducer for the control of diseases caused by phytopathogenic viruses object of the present invention, which is formed by steps A-G.
    10 Figure 2 shows a graph describing the effect of the formulation object of the present invention on the severity index of the papaya annular virus (PRSV) in Maradol vario papaya plants with formulation treatments at various doses: the line with the marker - represents the dose of 0.50 Uha; the line with the marker -4-represents the dose 0.70 Uha; while the line with the - + - marker represents a dose of 1.00 L / ha. These observations were made with respect to a witness without application.
    lS Figure 3 shows photographs of the aerial parts of Maradol vario papaya plants in the field, infected by the annular papaya virus (PRSV), to compare the severity index before (Day O) and after treatment (Day 28) with the formulation object of the invention at various doses (0.50, 0.70 and 1.00 Uha) with respect to a control without application.
    twenty Figure 4 shows photographs of pepper plants with symptoms of field virosis, before (Day O) and after application of the formulation object of the present invention (Day 8 and Day 18) at doses less than 1.00 Uha.
    2S Figure 5 shows a comparative graph of the average height of tomato plants as a growth parameter for various treatments. T1: positive control; T2: commercial control at a dose of 2 mUL per plant; T3, T4 and T5: treatment with the formulation object of the present invention at doses of 2.50 mUL, 3.75 mLlL and 5.00 mUL per plant, respectively.
    Figure 6 shows a comparative graph of the average number of leaves per tomato plant as a growth parameter for various treatments. T1: positive control; T2: commercial control at a dose of 2 mUL per plant; T3, T 4 and T5: treatment with the formulation object of the present invention at doses of 2.50 mUL, 3.75 mLlL and 5.00 mUL per plant, respectively.
    Figure 7 shows photographs of untreated tomato crop plants with the formulation of the present invention at the beginning (Day O) and after 36 days of field-level evaluation.
    Figure 8 shows photographs of tomato crop plants treated with the formulation of the present invention at the beginning (Day O) and after 36 days of field-level evaluation.
    5 Figure 9 shows photographs of pepper crop plants not treated with the formulation of the present invention at the beginning (Day O) and after 36 days of field-level evaluation.
    Figure 10 shows photographs of pepper crop plants treated with the formulation of the present invention at the beginning (Day O) and after 36 days of field-level evaluation.
    Figure 11 shows the agarose gel electrophoresis images of the detection analysis
    10 of DNA viruses of the genus Begomovirus (Tomato yellow leaf curl virus (TYLCV) and Pepper Huasteco yellow vein virus (PHYVV)) in tomato crop plants at the beginning (Day O) and 36 days after the evaluation.
    Figure 12 shows the agarose gel electrophoresis images of the DNA virus analysis of the genus Begomovirus (Tomato yellow leaf curl virus (TYLCV) and Pepper
    15 Huasteco yellow vein virus (PHYVV)) in pepper crop plants at the beginning (Day O) and 36 days after the evaluation.
    Figure 13 shows the electrophoresis images in agarose gels of the analysis of RNA virus analysis of the genus Torradovirus (ToMarV) in tomato crop plants at the beginning (Day O) and 36 days after the evaluation.
    20 Figure 14 shows the electrophoresis images in agarose gels of the analysis of RNA virus of the genus Torradovirus (ToMarV) in pepper crop plants at the beginning (Day O) and 36 days after the evaluation.
    Detailed description of the invention
    The proposal of the invention concerns the use of extracts and vegetable oils prepared from leaves, roots, stems and bark of plant species from specific regional biotypes of wild and endemic plants of the Chihuahuan semi-seed10, to design a formulation with biostimulant action of the plant growth and inducer of resistance to
    phytopathogenic viruses by having phyto molecules promoting local resistance and Acquired Systemic Resistance (RSA) in plants infected with viruses.
    S The formulation object of the present invention consists of a mixture of multiple extracts of plant species that are not attacked by viruses whereby various properties that impact the final formulation are used, since the active compounds are present in the barks, stems, roots and leaves of these species.
    The biostimulant formulation of plant growth and development and resistance inducer for the control of diseases caused by phytopathogenic viruses, object of the present invention essentially comprises the following components:
    10 a) Vegetable extracts and oils from varieties of plants of the semi-desert Chihuahuan, such as: ethanolic and acetonic extracts of Larrea tridentata, ethanolic extract of Viscum album, absolute oil of Uppia graveo / ens and hexane extracts of Euphorbia antisyphilitica, Jatropha dioica and Agave American
    fifteen b) Absolute oils and aromatic plant extracts, such as yzygium aromaticum and Cinnamomum zeylanicum oils, Eucalyptus globuli methanolic extract, Rosmarinus officinalis aqueous extracts, Salvia officinalis and Peppermint extract, and Coriandrum sativum ethanolic extract.
    twenty c) Sulfur compounds, amino acids and specific peptides of plant and animal origin such as: sulfathiazole, sodium lauryl sulfate, calcium thiosulfates, phenylalanine, soy milk and egg white peptides.
    d) hormones and plant growth regulators such as auxins, gibberellins, salicylic, jasmonates, their precursors, derivatives and salts, among others. acid
    e) Multivitamins and polysaccharides provided by aloe gel.
    2S To obtain the different extracts that conventional described in the state of the art.make uptheinventionbeemploymethods
    The preparation of the biostimulant formulation of plant growth and development and resistance inducer for the control of diseases caused by phytopathogenic viruses consists of adding the components verifying that each one of them is properly dissolved and homogeneous before
    from pursuetotheaggregationof thenextmaterial.Thepreparationbemakesinagitation
    constant and when done in this way does not cause exothermic reactions, nor does it require any
    induction type Due tothephysicochemical nature of some oftheircomponents,is
    necessary prepareseparately some pre-mixesforfavoritsincorporationtothe
    S formulationfinal.Forpreparetheformulationbethey addthecomponentsslowlyfrom
    according to the order described below and which is schematized in Figure 1, it is indicated
    in addition the concentrations of each component of the biostimulant growth formulation
    and plant development and resistance inducer for the control of diseases caused by vi rus
    phytopathogens The process considers the following stages (Figure 1):
    10 A) Preparationfromthebasefromtheformulation, whichiscomposedinitiallybythe
    extracts wateryYthecomponentssolubleinWater.Thecomponentsfromthe
    Base formulation are as follows and are added in sequential order:
    Aqueous extract of Rosmarinus officinalis (0.75 -1.25% v / v)
    Aqueous extract of Peppermint (0.75 -1.25% v / v)
    fifteen Aqueous extract of Salvia officinalis (0.75 -1.25% v / v)
    Soy Milk (1.5 -2.5% v / v)
    Egg White (0.75 -1.25% v / v)
    S.bila gel (0.75 -1.25% v / v)
    Sulfatiazole (0.375 -0.625% w / v)
    twenty Phenylalanine (0.75 -1.25% w / v)
    Calcium thiosulfate (15.0 -25.0% w / v)
    B) Preparation of Premix 1, which contains the components of medium-high polarity:
    extracts plant ethanolic hormones and plant growth regulatorshow
    auxins, gibberellins, salicylic acid, their precursors, derivatives and salts, among others:
    25 Ethanolic former of Larrea tridentata (0.75 -1.25% v / v)
    Ext. Ethanolic from Viscum album (0.75 -1.25% v / v)
    Coriandrum sativum ethanolic ext. (0.75 -1.25% v / v)
    Naphthoxyacetic Acid (0.15 -0.25% w / v)
    6 Benzylaminopurine (0.225 -0.375% w / v)
    30 Salicylic Acid (1.5 -2.5% w / v)
    and) ThePre-mixonebeaddtotheformulationbaseinagitationconstant.Y
    subsequently other components of medium polarity are added, such as:
    Eucalyptus globulus methanolic ext. (0.75 -1.25% v / v)
    O) Preparation of Premix 2, which contains the absolute oils:Syzygium aromaticum oil (0.75 -1.25% v / v)Cinnamomum zeylanicum oil (0.75 -1.25% v / v)
    5 Uppia graveolens oil (0.75 -1.25% v / v) E) Premix 2 is added to the base formulation under constant agitation. Other components of medium polarity are added, such as: Larrea tridentata acetonic extract (0.75 -1.25% v / v) F) Preparation of Premix 3, which contains the extracts of lower polarity (extracts)
    10 hexanes), such as: Euphorbia antisyphilitica hexane ext. (0.75 -1.25% v / v) Jatropha dioica hexane ext. (0.75 -1.25% v / v) Hexagonal bark Agave Americana L (0.75 -1.25 % v / v)
    G) Addition of Premix 3 to the base formulation with constant agitation to obtain 15 of the final formulation. A conditioning agent, such as sodium lauryl sulfate (7.5 -12.5% w / v), is used to perform the formulation.
    Once the biostimulant formulation of plant growth and development and resistance inducer for the control of diseases caused by phytopathogenic viruses is obtained, it is packaged and stored properly for use.
    20 The formulation obtained, with biostimulant activity of plant growth and development and resistance inducer for the control of diseases caused by phytopathogenic viruses, prevents and reduces the damage caused by DNA and RNA viruses with a greater impact on the production of fruit and vegetable crops, reduces the number of damaged plants, delays the appearance of symptoms by virosis, significantly reduces the severity of damage, reduces the spread of the virus in the
    25 plantation, favors the continuity of the growth in the plants and ensures a greater yield in attack conditions. The applications of the formulation object of the invention are proven in various tests carried out for the control of phytopathogenic viruses in different plant species.
    EXAMPLES By way of non-limitation of the present invention, the following examples are described below:
    Example 1. Preparation of the formulation
    To prepare a batch of 1000 L of the biostimulant formulation of growth and development
    5 plant and resistance inducer for the control of diseases caused by phytopathogenic viruses, the components were mixed in purified water according to the previously described process and which is schematized in the diagram of Figure 1, maintaining constant agitation between the addition of One component and another. The components and quantities used specifically for this example are listed below:
    10 Accus extract of Rosmarinus officinalis (9 L) Aqueous extract of Peppermint (7.5 L) Aqueous extract of Salvia officinalis (7.5 L) Soy milk (25 L) Egg white (7.5 L)
    15 S.bila Gel (10 L) Sul! Atiazol (4.5 Kg) Phenylalanine (0.8 Kg) Calcium Thiosulte (150 Kg) Premix 1:
    20 Ethanolic Ext. From Larrea tridentata (9 L) Ethanolic Ext. From Viscum album (8 L) Ethanolic Ext. From Coriandrum sativum (8 L) Naphthoxyacetic Acid (1.5 Kg) 6 Benzylaminopurine (2.5 Kg)
    25 Salicylic Acid (17 Kg) Methanolic Ext. From Eucalyptus globulus (8 L) Premix 2:
    Syzygium aromaticum oil (7.5 L) Cinnamomum zeylanicum oil (7.5 L) 30 Lippia graveolens oil (7.5 L) Larrea tridentata acetonic oil (9 L)
    Premix 3: Hextanic Ext. Of Euphorbia antisyphilitica (8 L) Hextanic Ext. Of Jatropha dioica (8 L) Hexagonal Ext. Of American Agave Bark L (7.5 L)
    5 Sodium lauryl sulfate (85 Kg)
    Once the biostimulant formulation of plant growth and development and resistance inducer for the control of diseases caused by phytopathogenic viruses is obtained, it is packaged and stored properly for use. Based on this formulation, performance tests were carried out against various phytopathogenic viruses that correspond to
    10 following examples.
    Example 2. Antiviral activity of the formulation against PRSV virus in papaya
    The papaya annular spot virus PRSV (Papaya Ringspot Virus) is a virus whose genome is single-stranded RNA. To evaluate the effectiveness of the formulation object of this invention in the control of the PRSV virus, papaya plants cv. Maradol, aged 15, with symptoms of PRSV, who were randomly diagnosed by the ELlSA technique, to corroborate the presence of the virus. Three doses of the formulation were evaluated at 0.50,
    0.75 and 1.00 Uha and an absolute witness (without application). Three successive applications were made at intervals of 7 days between them. The applications were made with a backpack, with a capacity of 15 liters, with a water expense of 500 Uha.
    20 The experimental design was completely randomized with 12 repetitions and the experimental unit was a plant. The planting frame was at a distance between rows of 3.0 m and between plants 2.0 m, with a density of 1,670 plants / ha.
    To evaluate the severity, five evaluations were carried out, the first at the time of the first application and the following at 7-day intervals. On each date the severity of the
    25 disease of each plant, according to the scale proposed by Rivas-Valencia et al., (2003): 1 = Healthy plant. 2 = Symptom onset (yellow spotted, some oil spots poorly defined). 3 = Well defined symptoms, not generalized in the leaves, defined oily spot. 4 = Severe symptoms, generalized in the leaves, concentric rings in fruits.
    30 5 = Very severe symptoms, concentric rings in fruits and foliar leaf reduction.
    6 = Very severe with growth arrest and death of the plant.
    With this nominal severity rating per plant, the population severity index was obtained by applying the following formula:
    5 Where: 18 = severity index; Xki = level of damage at time i; Nki = number of plants with the level of damage at time i, and Nj = total number of plants evaluated.
    10 The severity index values were transformed to a percentage, in order to adjust them and analyze the behavior of the curve. The results were analyzed using the analysis of variance and comparison of Tukey means (0 = 0.05).
    lS The PR8V-P virus control strategy with the use of the formulation object of this invention was successful, since it managed to reduce the final severity of the virus in the plantation by 64% for treatments 1 and 2 (high and intermediate dose, 1.00 and 0.75 Uha, respectively) and in 70% for treatment 3 (low dose 0.50 Uha), while treatment without application (control) increased the severity of the disease by 25% (Figure 2). The severity values of the disease showed significant differences with respect to the control from the second application of the product in any of its doses.
    20 2S As can be seen in Figure 2, at 14 days (after two applications) the remission of symptoms was observed in the treatment of the lowest dose (0.50 Uha), and there was a decrease in the disease in the treatment of 0.75 Uha . From the fourth evaluation (after three applications), remission of symptoms in papaya plants at doses of 1.00 Uha and 0.75 Uha was observed and in the fifth evaluation the remission of symptoms in the three treatments with the dose of the product, without there being significant differences at this time between them. The witness in turn increased its severity index by 25%.
    These results are consistent with what is observed in the images in Figure 3, where 28 days after the treatment the total remission of symptoms occurs if at least three successive applications are used every 7 days between 0.50 and 1 Uha, not happening in the witness.
    Additionally, none of the applied doses of the formulation showed toxicity effect for papaya cultivation during the study.
    Example 3. Activity of the formulation as inducer of resistance to virosis of the pepper at the greenhouse level.
    5 The pepper is one of the horticultural plants that has suffered relentlessly the incidence of viral diseases. In order to demonstrate that the formulation object of this invention can minimize the damage of virosis and induce resistance in the plant, a qualitative study was carried out on pepper plants that show symptoms of virosis. The test consisted in the application of the formulation in an initial dose of 0.70 Uha, followed by a second application
    10 at the same dose 5 days later; Subsequently, a third and fourth application was made at a dose of 0.40 L / ha, but with a spacing of ten days between them. Applications were made with a backpack, with a capacity of 15 liters.
    The effect of the formulation on pepper plants with symptoms of virosis was positive and was observed 8 days after treatment, improving towards day 18 after the first application
    15 (Figure 4). This indicates that the application of the formulation via foliar with doses lower than 1.00 L / ha and with applications every 5 days is effective to induce systemic resistance in plants with symptoms caused by viral agents from the second application. The application of the formulation object of the present invention allows plants to continue their development in a normal manner without showing toxicity effects on the pepper crop.
    20 Example 4. Performance of the tomato yellow leaf virus formulation
    TYLCV.
    The efficacy of the formulation object of the present invention to induce resistance to the spoon virus, or yellow leaf tomato virus TYLCV (Tomato yellow lear curl virus), was evaluated during the vegetative growth phase of a developed tomato crop low
    25 greenhouse in semi-arid climate. The TYCLV virus is a DNA-type virus and is transmitted by a whitefly population called Bemisia tabaci, a species of hemiptera of the Aleyrodidae family.
    The tests were carried out in a multi-tunnel greenhouse of 420 m2, which has passive lateral and overhead ventilation through windows, with opening and closing system
    automated The greenhouse has a sanded soil, formed by a layer of land provided 30 cm thick placed on the original soil of the farm, and covered by a layer of manure about 3 cm thick on which is a layer of 10 cm thick fine sand, as a quilting. For irrigation and fertilization of the crop, 5 drip irrigation facilities are available, with the drip holder branches located on paired lines with a distance of 1.2 m between pairs of paired dripper lines and 0.8 m between the two pairs of lines of adjacent drippers, and with the emitters within the same branch dropper holders located every 50 cm. The drip irrigation system has self-compensating drippers with a unit flow of 3 liters per hour, the dropper. For fertigation programming there is a
    10 irrigation programmer and 5 tanks of concentrated nutrient solution. The planting density used will be 1.5 m-2 plants.
    To perform the test, 5 different treatments were analyzed. In each of them the tests were carried out on 90 different tomato plants and for three repetitions, each consisting of 30 plants per repetition, each repetition of the treatment is in randomized blocks to eliminate
    15 possible experimental errors derived from different infection rates depending on the distribution of climatic conditions inside the greenhouse.
    The evaluation is carried out over a period of four weeks in which each treatment is applied weekly. Two applications were made prior to the inoculation of the plants with the TYCLV virus and two subsequent to it. Below are the
    20 activities carried out in each of the treatments:
    Day O: TransplantDay 7: Entutorado and destalladoDay 9: First application of treatmentsDay 14: DestalladoDay 16: Second application of treatmentsDay 20: InoculationDay 22: Third treatment applicationDay 29: Fourth application of treatmentsDay 35: Sampling growth parameters
    The methodology that was used for the inoculation of the culture is described below: once the culture was established (after 20 days after the transplant) it was inoculated with a population of the pest insect Bemisia tabaci (carrier of the TYCLV virus), for which were introduced inside the greenhouse, next to each of the experimental plots, a
    5 tomato plant infected with the TYCLV virus and an adult population of whitefly ad libitum. In this way, the inoculation of tomato cultivation by the TYCLV virus was encouraged for a period of 5 days, during which time said infected plant material was kept inside the greenhouse. After this period of time, said plant material was removed from the greenhouse.
    10 The treatments that were performed are described below:
    • Treatment 1: Positive control of plants without infection;
    • Treatment 2: Plants with periodic applications with a commercial product widely used in the study area at the dose recommended by the manufacturer (REzist "2 mUL);
    • Treatment 3: Plants with periodic applications of the formulation object of the present invention at the dose of 2.50 mLlL per plant;
    • Treatment 4: Plants with periodic applications of the formulation object of the present invention at the dose of 3.75 mLlL per plant;
    • Treatment 5: Plants with periodic applications of the formulation object of the present invention at the dose of 5.00 mLlL per plant.
    The sampling of growth parameters, after the fourth application of the treatments, consisted of measuring two characteristics: the height of the plant and the number of leaves developed. This measurement was performed on a plot by repetition and treatment, consisting of five plants per plot. In this way it is possible to identify the plants that suffer
    2S growth disorders as a result of having manifested the usual symptoms of the TYCLV virus.
    The results of the growth parameters in relation to the height of the plant are schematized in Figure 5, in which it is possible to observe that from the application of a dose greater than 3.75 thousand per plant of the formulation object of the Present invention (Treatments
    30 4 AND 5) a better development of the height of the tomato plant infected with the virus is obtained
    TYCLV, of the one obtained with commercial control (Treatment 2). Highlighting the dose of 5.00 mUL in which the development of the height of the plant is even greater than in the positive control. Indicating the biostimulant activity of plant growth and development and resistance inducer for the control of diseases caused by phytopathogenic viruses of the formulation.
    S 10 Regarding the results of the growth parameters in terms of the number of leaves developed per plant in each treatment, it was possible to identify that the formulation object of the present invention in any of its doses allows a greater development of leaves per plant that commercial control (Figure 6); and that in the case of an application at a dose of 3.75 mLlL per plant, the formulation has a performance similar to the positive control, this again indicates the activity of the formulation as a biostimulant of plant growth and development and resistance inducer for disease control caused by phytopathogenic viruses.
    Example 5. Activity of the formulation as inducer of resistance to virosis of tomato and pepper at field level.
    lS 20 In order to demonstrate that the formulation object of this invention can minimize the damage of virosis and induce resistance in plants, a study was carried out on tomato and pepper plants at field level that presented symptoms of virosis. The test consisted in the application of the formulation in a dose of 0.5 Uha in soil and subsequently four more foliar applications were made with a spacing of seven days between them. Applications were made with a backpack, with a capacity of 15 liters. As a control, tomato and pepper plants were taken into account without the application of the formulation of the present invention. The percentage of viral incidence was determined by visual observation of the damage caused in tomato and pepper crop plants at field level.
    2S The effect of the formulation on tomato crop plants with viral symptoms was positive and at the beginning of the evaluation a viral incidence of 31% was observed and after 36 days of treatment it decreased to 8.7% where the plants presented a vigorous foliage performance (Figure 8). Tomato crop plants that were not treated with the formulation of the present invention had a lower viral incidence of 7.3% at the beginning of the evaluation, but at 36 days they showed a greater damage to plant yield and foliage (Figure 7) .
    The results of the formulation on the plants of the pepper crop were also positive, where at the beginning of the evaluation a viral incidence of 11% was observed and after 36 days of the treatment it was decreased to 5.7% presenting a greater foliage yield ( Figure 10). It was observed that pepper crop plants that were not treated with the formulation of the
    The present invention showed a tendency similar to those of tomato, because at the beginning of the evaluation they presented a lower viral incidence of 7.2%, but at 36 days they showed a greater damage in the yield and plant foliage (Figure 9).
    This indicates that the application of the formulation in soil and subsequently via foliar with a dose of 0.5 L / ha every 7 days is effective to induce systemic resistance in crop plants
    10 tomatoes and peppers at field level with symptoms caused by viral agents. The application of the formulation object of the present invention allows plants to continue their development in a normal manner without showing toxicity effects on tomato and pepper crop plants at field level.
    Example 6. Molecular detection of DNA virus in tomato and pepper crop plants.
    In order to demonstrate that the formulation object of the present invention can minimize the damage of virosis and induce resistance in plants, a study was conducted to molecularly detect the presence of DNA viruses of the genus Begomovirus in tomato and pepper plants at field level . First the tomato and pepper seeds were washed with ethanol
    20 70% for two minutes and 30% chlorine for 15 minutes, then washed with sterile distilled water and placed in trays with substrate for greenhouse germination.
    After three to four weeks after germination the tomato and pepper plants were inoculated with the DNA virus of the genus Begomovirus (Tomato yellow lear curf virus (TYLCV) and Pepper Huasteco yellow vein virus (PHYW)) by agro infiltration inoculating 2 mL of
    25 DNA The application of the formulation of the present invention was carried out five days after the inoculation of the seeds using a dose of 0.5 L / ha.
    The molecular detection of TYLCV and PHYVV viruses was carried out at the beginning of the application (Day O) and 36 days after the application of the formulation of the present invention. First, DNA extraction was carried out from the apical leaves of each tomato and 30 pepper plant individually based on the 3% CTAB methodology (Zhang el al., 1998).
    Subsequently, a molecular detection was performed by polymerase chain reaction (PCR) in a semiquantitative manner, with the idea of indirectly analyzing the degree of virus replication. The amplified products are shown in images by electrophoresis in 1% agarose gels (Figure 11 and Figure 12).
    S 10 The results obtained in the electrophoresis images in 1% agarose gels showed that the molecular detection of DNA viruses of the genus Begomovirus (Tomato yellow leaf curl virus (TYLCV) and Pepper Huasteco yellow vein virus (PHYVV)) in plants of the Tomato culture was qualitatively less after 36 days of evaluation compared to the initial time of the evaluation (Day O) (Figure 11), so that the formulation of the present invention prevented the progress of viral symptomatology by improving the performance of the Foliage of tomato crop plants at field level.
    lS The electrophoresis images in 1% agarose gels showed that the molecular detection of DNA viruses of the genus Begomovirus (Tomato yellow leaf curl virus (TYLCV) and Pepper Huasteco yellow vein virus (PHYVV)) in pepper plants continues until 36 days of evaluation (Figure 12), however, the formulation of the present invention also succeeded in preventing the progress of viral symptoms by improving the yield of foliage of pepper crop plants at field level.
    Example 7. Molecular detection of pepper virus at field level. of RNAincrop plantsfromtomatoY
    20 2S In order to demonstrate that the formulation object of the present invention can minimize the damage of virosis and induce resistance in plants, a study was conducted to molecularly detect the presence of RNA viruses of the genus Torradovirus (ToMarV) in tomato and pepper plants at field level. First the tomato and pepper seeds were washed with 70% ethanol for two minutes and 30% chlorine for 15 minutes, then washed with sterile distilled water and placed in trays with substrate for greenhouse germination.
    30 After three to four weeks after germination, the tomato and pepper plants were inoculated with the virus of the genus Torradovirus (ToMarV), by agro infiltration inoculating 2 mL of DNA. The application of the formulation of the present invention was carried out five days after the inoculation of the seeds using a dose of 0.5 Uha.
    19
    S The molecular detection of the viruses was carried out at the beginning of the application (Day O) and 36 days after the application of the formulation of the present invention. First, the extraction of RNA from symptomatic leaf tissue of each tomato and pepper plant was carried out individually according to the protocol described by Singh (2002) modified with sodium sulphite. Subsequently, molecular detection was performed by polymerase chain reaction (PCR) with specific primers (ToMarV-FfToMarV-R primers (Verbeek al., 2008) which amplify a 511 bp fragment) for each virus .
    To increase the sensitivity in the detection of ToMarV a nested PCR was performed using
    10 primers pJER-11 23 and pJER-11 24 (Camacho al., 2015), which amplify a 332 bp fragment using the DNA obtained during the first PCR as a temper or template. The nested PCR product was visualized by 1% agarose gel electrophoresis (Figure 13 and Figure 14).
    lS Electrophoresis images in 1% agarose gels showed that simple and nested molecular detection of the RNA virus of the genus TOffadovirus (ToMarV) in tomato plants continues until 36 days of evaluation (Figure 13), however the formulation of The present invention was able to prevent the progress of viral symptoms by improving the yield of foliage of tomato crop plants at field level.
    twenty The results obtained in the electrophoresis images in 1% agarose gels showed that the nested molecular detection of the RNA virus of the genus Torradovirus (ToMarV) in pepper plants was similar at the beginning (Day O) and at 36 days of evaluation ( Figure 14). And the simple molecular detection of the RNA virus of the genus Torradovirus (ToMarV) was qualitatively less than 36 days of evaluation compared to the initial time of the evaluation (Day O) (Figure 14), so the formulation of the present The invention was able to avoid the virus replication rate and the progress of viral symptoms by improving the yield of the foliage of the pepper crop plants at field level.
    25
    权利要求:
    Claims (11)
    [1]
    1. A biostimulant formulation of plant growth and development and resistance inducer for the control of diseases caused by phytopathogenic viruses, characterized in that
    5 includes: a) Extracts and vegetable oils from varieties of plants of the semi-desert Chihuahuense, b) Absolute oils and extracts of aromatic plants, c) Sulfur compounds, amino acids and specific peptides of plant and animal origin,
    10 d) plant growth hormones and regulators, and e) multivitamins and polysaccharides.
    [2]
    2. The biostimulant formulation of plant growth and development and resistance inducer for the control of diseases caused by phytopathogenic viruses according to claim 1,
    15 characterized in that the extracts and vegetable oils from varieties of wild and endemic plants of the semi-desert Chihuahuan are selected from the following group: Larrea tridentata, Viscum album, Lippia graveolens, Euphorbia antisyphilitica, Jatropha dioica and Agave americana.
    The biostimulant formulation of plant growth and development and resistance inducer for the control of diseases caused by phytopathogenic viruses according to claim 1, characterized in that the absolute oils and extracts of aromatic plants are selected from the following group: Syzygium aromaticum, Cinnamomum zeylanicum, Eucalyptus g / obu / os, Rosmarinus officinafis, Salvia officinafis, Peppermint and Coriandrum sativum.
    [4]
    4. The biostimulant formulation of plant growth and development and resistance inducer for the control of diseases caused by phytopathogenic viruses according to claim 1, characterized in that the sulfur compounds, amino acids and peptides of plant and animal origin include: their Ifatiazole, sodium lauryl sulfate, calcium thiosulfates, phenylalanine, peptides
    30 soy milk and egg whites.
    [5]
    5. The biostimulant formulation of plant growth and development and resistance inducer for the control of diseases caused by phytopathogenic viruses according to claim 1, characterized in that the hormones and plant growth regulators are selected from the group: auxins, gibberellins, salicylic acid , jasmonatos, their precursors, derivatives and salts, among others.
    [6]
    6. The biostimulant formulation of plant growth and development and resistance inducer
    5 for the control of diseases caused by phytopathogenic viruses according to claim 1, characterized in that the multivitamins and polysaccharides are provided by aloe gel.
    [7]
    7. The biostimulant formulation of plant growth and development and resistance inducer for
    the control of diseases caused by phytopathogenic viruses according to claims 1 to 10 6, characterized in that it has properties in the regulation of plant development and growth.
    [8]
    8. The biostimulant formulation of plant growth and development and resistance inducer for the control of diseases caused by phytopathogenic viruses according to claims 1 to 6, characterized in that it possesses properties for the biostimulation of defenses and induction of
    15 resistance of plants to phytopathogenic viruses of RNA and DNA type.
    [9]
    9. The biostimulant formulation of plant growth and development and resistance inducer for the control of diseases caused by phytopathogenic viruses according to claims 1 to 8, characterized in that it possesses properties for the biostimulation of cultivated plants of
    20 Solanaceae, Cucurbitaceae, Rosaceae, Legumes, Aliyaceae, Caricaceae and Musaceae families, among others of commercial interest.
    [10]
    10. A method to prepare a biostimulant formulation of plant growth and development and resistance inducer for the control of diseases caused by phytopathogenic viruses,
    25 characterized in that it consists of the following steps: a) Preparation of the base of the formulation, which consists in the sequential addition with constant agitation of aqueous extracts of plants and water soluble components. b) Preparation of Premezcia 1, which contains ethanolic extracts of plants, hormones and plant growth regulators.
    30 c) Addition of Premix 1 and Eucalyptus globulus methanolic extract to the base formulation under constant agitation. d) Preparation of Premix 2, which contains the absolute oils.

    s e) Addition of Premix 2 and Larrea tridentata acetonic extract to the base formulation containing premix 1 under constant agitation. f) Preparation of Premix 3, which contains the hexane extracts. g) Addition of Premix 3 and a conditioning agent to the base formulation containing premixes 1 and 2 under constant stirring.
    10 11. The method for preparing the biostimulant formulation of plant growth and development and resistance inducer for the control of diseases caused by phytopathogenic viruses according to claim 10, characterized in that the aqueous extracts comprise: aqueous extract of Rosmarinus officinalis, aqueous extract of Peppermint mint and aqueous extract of Salvia officinalis.
    fifteen 12. The method for preparing the biostimulant formulation of plant growth and development and resistance inducer for the control of diseases caused by phytopathogenic viruses according to claim 10, characterized in that the soluble components comprise: soy milk, egg white, gel of aloe, sulfathiazole, phenylalanine and calcium thiosulfate.
    twenty 13. The method for preparing the biostimulant formulation of plant growth and development and resistance inducer for the control of diseases caused by phytopathogenic viruses according to claim 10, characterized in that the ethanolic extracts comprise: ethanolic extract of Larrea tridentata, ethanolic extract of Viscum album and ethanolic extract of Coriandrum sativum.
    25 14. The method for preparing the biostimulant formulation of plant growth and development and resistance inducer for the control of diseases caused by phytopathogenic viruses according to claim 10, characterized in that the hormones and plant growth regulators comprise: beta Naphthoxyacetic acid, 6 -benzylaminopurine, salicylic acid, its precursmes, derivatives or salts.
    30 15. The method for preparing the biostimulant formulation of plant growth and development and resistance inducer for the control of diseases caused by phytopathogenic viruses according to claim 10, characterized in that the absolute oils are selected from the
    2. 3
    group: $ yzygium aromaticum oil, Cinnamomum zeylanicum oil and Lippia graveo / ens oil.
    [16]
    16. The method to prepare the biostimulant formulation of plant growth and development and
    5 resistance inducer for the control of diseases caused by phytopathogenic viruses according to claim 10, characterized in that the hexane extracts comprise: hexane extract of Euphorbia antisyphilitica, hexanic extract of Jatropha dioica and hexane extract of Agave americana L.
    17. The method for preparing the biostimulant formulation of plant growth and development and resistance inducer for the control of diseases caused by phytopathogenic viruses according to claim 10, characterized in that the conditioning agent is preferably sodium lauryl sulfate.
    18. The method for preparing the biostimulant formulation of plant growth and development and resistance inducer for the control of diseases caused by phytopathogenic viruses according to claims 10, 11, 13, 15 and 16 characterized in that the plant extracts and Absolute oils are added in a concentration range of 0.75 to 1.25% v / v.
    19. The method for preparing the biostimulant formulation of plant growth and development and resistance inducer for the control of diseases caused by phytopathogenic viruses according to claims 10 and 12 characterized in that the soya milk, the egg white and the gel Aloe Vera are added in a range of 0.75 to 2.5% v / v.
    20. The method for preparing the biostimulant formulation of plant growth and development and resistance inducer for the control of diseases caused by phytopathogenic viruses according to claims 10 and 12 characterized in that sulfathiazole, phenylalanine and calcium thiosulfate are added in a concentration range of 0.075 to 25.0% w / v.
    21. The method for preparing the biostimulation of plant growth and resistance inducer for the control of diseases caused by phytopathogenic viruses according to claims 10 and 14 characterized in that the growth regulators are added in a concentration range of 0.15 at 2.5 p / v.
    [22]
    22. The method for preparing the biostimulant formulation of plant growth and development and resistance inducer for the control of diseases caused by phytopathogenic viruses according to claims 10 and 17 characterized in that the conditioner is added in a
    S concentration range 7.5 -12.5 p / v.
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    同族专利:
    公开号 | 公开日
    ES2628278B2|2017-11-06|
    MX2016001466A|2017-08-01|
    US20170215433A1|2017-08-03|
    US10342237B2|2019-07-09|
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