WO2008077982A1 - Solid product effective for biocontrol of vascular fusariosis of melon, method for preparation thereof and method for use of same - Google Patents

Solid product effective for biocontrol of vascular fusariosis of melon, method for preparation thereof and method for use of same Download PDF

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Publication number
WO2008077982A1
WO2008077982A1 PCT/ES2007/070198 ES2007070198W WO2008077982A1 WO 2008077982 A1 WO2008077982 A1 WO 2008077982A1 ES 2007070198 W ES2007070198 W ES 2007070198W WO 2008077982 A1 WO2008077982 A1 WO 2008077982A1
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melon
biological control
solid product
effective
vascular
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PCT/ES2007/070198
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Spanish (es)
French (fr)
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Noelia Navarro Villamor
Maria Teresa HERNÁNDEZ FERNÁNDEZ
Carlos Javier GARCÍA IZQUIERDO
Agustina Bernal Vicente
Rubén LÓPEZ MONDÉJAR
Ainhoa MARTÍNEZ MEDINA
Ana Isabel ANTÓN GARCÍA
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Consejo Superior De Investigaciones Científicas
Pascual Valero, Jose, Antonio
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Publication of WO2008077982A1 publication Critical patent/WO2008077982A1/en

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/30—Microbial fungi; Substances produced thereby or obtained therefrom
    • A01N63/38—Trichoderma

Definitions

  • the present invention falls within the technical field of conventional and organic farming, and is applicable both in seedlings and in the field. Specifically, it refers to an effective solid product for the biological control of melon vascular fusahosis, its method of obtaining and method of application thereof.
  • Fusariosis is a very common disease, caused by different breeds of Fusarium oxysporum that causes significant crop losses, mainly in areas with high temperatures. Fusariosis is a serious problem in the crop field but above all it is important in the seedbeds, since the cultivation conditions in these favor the development and dispersion of the pathogen due to the production of a large number of plants in a small space and closed.
  • phytopathogens include the use of large amounts of chemical substances such as fungicides, substances that in some cases are classified as carcinogenic and / or toxic and that in many countries are prohibited. These chemicals also, in many cases are ineffective.
  • the biological control of fusariosis through the use of antagonistic microorganisms is one of the most promising alternatives.
  • the most important biological control agents are the fungi belonging to the genus Trichoderma that are capable of controlling a wide range of pathogenic microorganisms of plants of agricultural interest.
  • Five species of Trichoderma (T. hamatum, T. harzianum, T. koningü, T. polysporum and T. viridé) are the most important for biocontrol (Hoitink and Boehm, 1999). However, although they have had relative success, it has not yet been possible to reach a sufficient level in the control of phytopathogens.
  • T. viridae alone (US 6,808,917; 2002) or T hamatum, with other complementary microorganisms such as Flavobacterium balustinum, for the control of R. solani and Pythium ultimun (US 4,900,348; 1987) has also been described. .
  • Trichoderma there are commercial products based on the use of Trichoderma both in liquid formulation (Stimulase (Ecocert France n Q 08- 884, SCPA Agronutrition), Trichomic (AMC Chemical and Trichodex) and Trianum-G (Koppert) as solid in powder form (Tricho-tec Ecocert-SHC n Q VA- 22P-2, Biological control techniques), Trianum-P (Koppert), Trichotem (BCS ⁇ ko-Garantie QUIM_MERI_8244 / 02.04 / 4215-EN, Chemistry Meristem), T.
  • the product object of Ia is solid, the agent shows high levels of survival in the substrate and is applicable both in the seedbed and in the field.
  • An additional advantage of the present invention which is based on the use of a natural product, is that it increases the set of biological remedies to fight against the causative agents of plant diseases, always aimed at decreasing, as far as possible, the indiscriminate use of chemical substances.
  • the present invention provides an effective solid product for the biological control of melon vascular fusahosis, its method of obtaining and method of application thereof.
  • the invention relates to a solid product effective for the biological control of melon vascular fusahosis characterized in that it comprises (i) an antagonistic microorganism of F. oxysporum with biocontrol capacity on it, (ii) A solid support for Ia inoculation of the antagonistic microorganism (iii) and additional factors that improve the biocontrol capacity of the product.
  • the invention relates to a solid product effective for the biological control of melon vascular fusahosis based on the use of T. harzianum as an antagonistic microorganism.
  • the invention relates to a solid product effective for the biological control of melon vascular fusahosis that uses bentonite as solid support.
  • the invention relates to a solid product effective for the biological control of melon vascular fusahosis that incorporates as additional factors CO 3 Ca and chitosan as a pH corrector and inducer of chitinase synthesis, respectively.
  • the invention relates to a process for obtaining a solid product for effective biological control of vascular Ia fusahosis of melon characterized in that its components are incubated at temperatures between 20 to 30 Q C for a time between 1 and 10 days .
  • the invention relates to a method for obtaining an effective solid product for the biological control of melon vascular fusahosis characterized in that its components are mixed, crushed and incubated for 5 days at 25-28 5 C.
  • Another particular aspect of the invention relates to a method for obtaining an effective solid product for the biological control of melon vascular fusahosis characterized in that it comprises the preparation of a solid support such as bentonite, which incorporates as additional factors CO 3 Ca and chitosan as a pH corrector and inducer of chitinase synthesis, respectively, all mixed and dry homogenized.
  • a biologically pure culture of the strain of T. harzianum T78 N Q CECT 20714 is inoculated, and mixed and incubated for 5 days at 28-25 5 C.
  • the resulting product is mixed with 10% peat by use. of a standard mixer used in commercial seedbeds.
  • Another particular aspect of the invention constitutes a method for inducing systemic resistance to diseases caused by phytopathogenic organisms which comprises applying an effective amount of the product object of the invention on the soil or plant to which it is desired to induce systemic resistance to diseases.
  • the invention relates to a process for stimulating the growth of plants which comprises applying an effective amount of the product object of the invention on soil or plant whose growth it is desired to stimulate.
  • Another particular aspect of the invention is the use of the product object of the invention for the biological control of this disease in a seedbed.
  • Another particular aspect of the invention constitutes the use of the product object of the invention for the biological control of vascular fusahosis in the field of culture.
  • the invention relates to the use of
  • Figure 1 Bar graph showing the growth in Petri dishes on a F. oxysporum dual plate assay facing different strains of Trichoderma. In the abscissa axis, the strains of Trichoderma sp. that showed a better result for the interests and / or objectives to be developed (C (Control of Fusarium), TC (commercial strain) and strains T82, T78, T010, T190 and T1) and in the axis of ordinates the final growth at seven days on the Petri dish in centimeters.
  • C Control of Fusarium
  • TC commercial strain
  • strains T82, T78, T010, T190 and T1 strains
  • Figure 2 Growth over peat of Trichoderma sp. according to the suppressivity criterion against F oxysporum (A) and peat survival of strain T78 (B).
  • A the suppressivity criterion against F oxysporum
  • B peat survival of strain T78
  • the different strains studied are represented, (T78; T82, T1, T010; T190 and TC (commercial))
  • the strain of Trichoderma T78 is represented inoculated in peat and peat without inoculation.
  • the number of colony forming units (ufe) x 10 6 per gram of peat analyzed is shown and Figure 2 (B) shows the percentage of CO 2 released in the means, medium.
  • Figure 3 Evolution of F. oxysporum subjected to the different treatments after 15, 37 and 48 days of inoculation respectively.
  • the abscissa axis each of the ways in which the solution of the strain T. harzianum T78 in the peat was inoculated is represented.
  • harzianum T78 in peat after its application in the different supports.
  • the different treatments from 1 to 7 and the control (C) are represented on the abscissa axis.
  • the order number of T. harzianum T78 x 10 6 per gram of peat analyzed at 0, 2, 15, 37 and 48 days is shown on the ordinate axis.
  • Figure 5 Percentage of infection by F. oxysporum (A) and Dry weight (g.) (B) of the melon plants treated with the different supports, after 15, 33 and 48 days of cultivation in seedlings.
  • A Percentage of infection by F. oxysporum
  • B Dry weight of the melon plants treated with the different supports, after 15, 33 and 48 days of cultivation in seedlings.
  • C the control
  • Figure A Percentage of infection by F. oxysporum of a total of 20 plants is shown and in Figure B the grams of dry weight of the melon plants.
  • FIG. 6 Effect of the application of bentonite supplemented with calcium carbonate and chitosan on dry weight (g.) (A) and percentage of infection by F oxysporum of melon plants (B), after 48 days of cultivation in seedlings .
  • T dry weight
  • B percentage of infection by F oxysporum of melon plants
  • Figure 7 Effect of the application on peat of bentonite supplemented with calcium carbonate and chitosan on the strain of T. harzianum T78 (A) and F oxysporum (B) and the percentage of infection by F oxysporum of melon plants (C) after 48 days of growing melon plants in a seedbed.
  • the present invention provides an effective solid product for the biological control of melon vascular fusahosis based on the use of a strain of T. harzianum, pathogen antagonist fungus (F. oxysporum sp. Melonis), on an inert solid matrix.
  • the invention also relates to the method of obtaining the product and its method of application.
  • the invention is based on the use of a natural strain of T. harzianum isolated from organic substrates of similar characteristics to the mobs used in seedbeds, which increases its survival capacity and decreases the frequency of application of the product.
  • bentonite As a solid support, bentonite is used, which has a high moisture retention capacity, allows to fix both reproductive and active vegetative (mycelial) forms of the fungus, at a suitable concentration to facilitate that the antagonist agent begins to act from the moment of application, and maintains high concentrations of this in the substrate.
  • the bentonite acts as a physical protector of the root preventing the entry of pathogens, thus enhancing the biocontrol effect and allows immobilizing enzymes produced by T. harzianum capable of degrading the cell wall of certain pathogens such as F. oxysporum, increasing the effectiveness of the product object of the present invention.
  • the invention uses calcium carbonate to correct the pH of the natural bentonite, up to a pH close to the optimum for the growth of the immobilized T. harzianum strain, which improves its colonization capacity, optimizes its vegetative growth and achieves a greater amount of title in the same volume of support for inoculation.
  • the product object of the present invention incorporates chitosan as a nutritional source for the inoculated strain, a compound that, in some cases, is used by itself in the preventive control of phytopathogens, (Chitomer, BCS ⁇ ko-Garantie QUIM_MERI_8244 / 01.04 / 4161 -EN Meristem chemistry
  • chitosan as a nutritional source for the inoculated strain, a compound that, in some cases, is used by itself in the preventive control of phytopathogens, (Chitomer, BCS ⁇ ko-Garantie QUIM_MERI_8244 / 01.04 / 4161 -EN Meristem chemistry
  • the presence of chitosan in the inoculum induces the activation of the chitinase activity, enzyme responsible for the degradation of chitin present in the cell wall of some pathogenic microorganisms such as F. oxysporum, of so that when a pathogen infection occurs
  • biocontrol capacity of this product is a function of the degree of acetylation, the smaller it is, the greater its biocontrol capacity.
  • its direct use does not offer conclusive results, since, although in certain circumstances it has a preventive effect, in others it has an activating effect of the infection of the pathogen, since it can be used by it as a source of carbon and nutrients.
  • the present invention combines the incorporation of chitosan of low degree of acetylation, calcium carbonate with the strain of T. harzianum, immobilized in an inert solid substrate based on bentonite, which contains fixed both reproductive and active mycelial structures of the fungus.
  • the biocontrol agent was selected and isolated to be used, secondly to study its survival capacity in the usual peat-like organic substrates in the seedbed.
  • the appropriate support was developed to implement its action after inoculation and, once the above aspects were selected, the action of T. harzianum was improved by adding in the support of compounds capable of giving it a greater and faster capacity for action, finally, the final product was validated and the application conditions established.
  • Trichoderma strains Selection and isolation of Trichoderma strains. Evaluation of the biofungicidal capacity against Fusarium oxysporum. Survival study in peat
  • the genus Trichoderma sp was selected, regardless of its species, for the excellent qualities for the fight against various phytopathogenic fungi, their biostimulant and bioregulatory capacity (Samuels,
  • Trichoderma sp strains regardless of their species, were isolated from mobs, green compost and soils from the Spanish southeast, evaluating a total of 11 strains designated T010, T1, T3, T10, T75, T76, T78, T82, T85 , T86 and T190.
  • Trichoderma harzianum was recommended for the preventive control of melon vascular fusahosis (designated commercial T). To perform the evaluation, the strains were subjected to the following tests:
  • strain T78 was selected to allow strain T78 to remain the necessary time in the substrate in contact with the plant and to prevent the pathogen from acting.
  • two liquid supports were analyzed: dissolution of the Trichoderma T78 strain in 2% gum arabic and carboxymethyl cellulose (CMC), two compounds with the ability to confer impregnating effect, a spore suspension of the Ia was also used.
  • strain T78 obtained from petri dish culture; and three solid supports: bentonite, vermiculite and wheat bran, which were prepared by embedding in them a solution of Trichoderma T78. Also superficially inoculated vermiculite was used, that is to say vermiculite previously inoculated with the spore suspension that was incorporated in the upper part of the tray and a spore suspension sprayed during the cultivation.
  • the analysis of the supports was carried out by incorporating them into the peat that was used to germinate melon seeds, on which, once germinated, a spore suspension of the F. oxysporum pathogen was inoculated at the usual concentration of seedling infection, which is around 10 3 -10 5 cfu per gram of peat (Agrios, 1998). After the infection, samples were taken on which the development of the plants, the evolution of the population of the pathogen (Fusarium) and the strain T. harzianum T78 inoculated with the different supports were evaluated. Sampling was carried out throughout the usual period of growing melon in a seedbed (48 days).
  • bentonite After the selection of the strain and the support to be used: bentonite, other factors were incorporated into the product object of the invention that allowed to increase the survival capacity of T. harzianum and its antagonistic effect against pathogenic microorganisms, especially against F oxysporum
  • the pH of the bentonite was corrected by means of the addition of natural calcium carbonate that raises the pH, obtaining a more adequate pH for the development of the strain of T. harzianum T78.
  • chitosan Another factor that improves the biocontrol activity of the product object of the invention is chitosan, which induces an immediate response by T. harzianum, due to the increase in the production of chitinase-like enzymes, enzyme responsible for the degradation of the cell wall of the pathogen.
  • the incorporation of chitosan during the immobilization process of the strain T78 in bentonite avoids that it can be used by the pathogenic microorganism, as occurs in cases where it is used directly, since when the final product is to be inoculated, the chitosan will have already been used by T. harzianum. In this way it is achieved that when T.
  • harzianum is inoculated it can present an ecological "micro-niche" both for the pH conditions and the presence of organic compounds capable of being used, as for the physical protection by the clay, which will improve its survival and will ensure the action for which it has been inoculated.
  • the prototype product was established, it was validated on a commercial scale for which the minimum necessary volume was established, the dosage thereof was optimized in the peat and the application conditions.
  • Trichoderma strains from different sources were isolated: peat, compost, agricultural and forest soils. Some strains previously isolated by the Research group were also studied. A total of 11 strains, designated T010, T1, T3, T10, T75, T76, T78, T82, T85, T86 and T190, were tested. As well as a commercial product containing Trichoderma sp., Designated commercial T. In the product labeling its composition was indicated and its application was recommended for the preventive control of melon fusahosis, among others.
  • the fungicidal capacity of the strains against F. oxysporum was evaluated for which an isolate of F. oxysporum f was used.
  • sp. Melonis race 1, 2 (De Cara et al., 2004J obtained from various seedlings capable of manifesting Ia melon fusariosis, the one that presented a high-very high virulence was chosen, so that the manifestation of the disease was not a limiting factor in the experimentation, this strain was the one that was used in the following examples.
  • the evaluation was carried out through dual culture, which allows to determine the abilities of the different strains of Trichoderma sp. at the time of inhibiting the growth of F. oxysporum.
  • the dual culture trial consists of four centimeter equidistant confrontations of the strains isolated against Fusarium oxysporum, in a petri dish in PDA culture medium, quantifying the growth of the latter with each of the strains (Bell et al., 1982) .
  • the strain T1 despite presenting a large biofungicidal capacity, showed a very low growth capacity in peat; For this reason, it was discarded and the T78 strain was chosen, which presented the greater capacity to inhibit F. oxysporum and the greater capacity to grow on peat.
  • the DNA of this strain was extracted and sequenced, determining that the strain chosen, by sequence homology, belonged to the species of T. harzianum, presenting some variations with respect to those included in the genomic databases, sufficient reason for its registration in Ia Spanish type culture collection under the registration N Q 20714.
  • EXAMPLE N 0 - 2 Selection of bentonite as an inoculation support for T. harzianum. Once the strain of T. harzianum was selected, we proceeded to study the form of inoculation that would be more suitable to facilitate its incorporation into the peat, which would allow plants to provide sufficient protection against phytopathogens.
  • the spore suspension was prepared by culturing the strain in petri dish with PDA for 7 days, after which the spores of the surface of the plates were collected with distilled water, making a total spore count by hematocytometer in Neubauer chamber, this count was around -10 9 cfu August 10 of the strain T78 per milliliter. Next, these spores were immobilized using two types of inoculation supports:
  • T. harzianum T78 Liquid Supports: a solution of T harzianum T78 was used as is, obtained from culture in petri dish and solutions of dissolved spores or in gum arabic or in carboxymethylcellulose (CMC), 2%, in order to confer impregnating effect to the solution of T. harzianum.
  • the gum arabic and CMC were inoculated directly in the seed, given its fixing capacity, acting as cementing substances, while the spore suspension was inoculated in the peat at the time prior to the preparation of the polystyrene trays for Ia Germination and growth of melon seedlings.
  • the number of colony forming units of strain T78 was quantified, using PDA-rose flare per seed, which resulted from 10 4 cfu of strain T78, and that coincides with the degree of inoculation of each of the wells of the polystyrene trays.
  • the amount of inoculum necessary for Ia final amount per gram of peat was in the range of 10 6- 10 7 cfu of the strain T78 was calculated.
  • Solid Supports natural bentonite, vermiculite and wheat bran on which the spore suspension of the strain of T. harzianum T78 was embedded, obtained following the previously proposed method. For the preparation of these supports, a spore suspension of the strain of T. harzianum T78 was incorporated, so that the final spore content per gram of solid support were the same.
  • the inoculums were mixed with the peat so that the amount of inoculum was in the range of 10 6 -10 7 pfu of strain T78 per gram of peat.
  • vermiculite also superficially inoculated vermiculite was used, that is to say vermiculite previously inoculated with the spore suspension that was incorporated in the upper part of the tray and a spore suspension sprayed during the cultivation.
  • the experiment consisted of 8 treatments and 2 controls, each with five replicas, on polystyrene trays used in the germination and growth of seedlings in seedlings, each with 150 melon seedlings.
  • the variety of tendon melon was used, due to its high susceptibility to F oxysporum.
  • the different treatments were subjected to the usual seedbed conditions, with the only difference of the support selected for the inoculation of the strain of T. harzianum T78.
  • the experiment was duplicated, infecting one half with the same strain as in example 1 and the other was kept without infecting to be able to detect the effect of the liquid or solid supports applied in the development of The melon plant without pathogen pressure introduced.
  • the inoculum of the strain T. harzianum T78 was 10 6 -10 7 pfu per gram of peat in all treatments. These concentrations are the most widely used in the literature (Batta, 2004).
  • the different supports inoculated with T. harzianum were mixed with peat. Each treatment was placed in 150-well polystyrene trays each and then sown with melon seeds, coated with vermiculite and moistened. The seeded trays were placed in a commercial germination chamber conditioned for the germination of the melon, at a temperature of 25 QC and 75% humidity, after two days they were taken out of the germination chamber and spread in the seedbed.
  • the trays selected for the treatment with the phytopathogen were inoculated with a suspension of F. oxysporum spores so that the amount in each of the wells was around 10 4 cfu per gram of peat (usual infection dose in seedbed).
  • peat On the organic material (peat), a total of four periodic samples were carried out to know the evolution of the populations of the inoculated microorganisms: strain T. harzianum T78 and F. oxysporum.
  • the samples were subjected to microbiological analysis by the corresponding selective medium (Papavizas et al., 1982 and Komada, 1975, respectively).
  • the size of the leaves, the length and the weight of the stem were analyzed on the collected plant material.
  • the percentage of infection of the melon seedlings was also evaluated by planting a portion of melon stem, with a length of 1 cm previously sterilized, on a general fungal medium (PDA), taking as positive stems those that after five days incubation will show growth of F. oxysporum around it.
  • PDA general fungal medium
  • the treatments in which there was a greater reduction in the population of F oxysporum were the treatments in which the support constituted bentonite (treatment 4) and vermiculite applied superficially to cover the wells of the sowing trays (treatment 7), both inoculated with strain T78 ( Figures 3, 4 and 5).
  • the reduction in the population of F oxysporum was progressive over time, although the one that presented a greater rapidity in the decrease of the same in the peat was the treatment with bentonite, possibly due to its ability to adhere to the roots, thus allowing greater protection over the root system of the plant.
  • the strain T. harzianum T78 showed a 50% decrease in treatments 1 (sporular inoculation), 4 (bentonite) and 7 (superficial vermiculite); Although treatment 1, despite showing a similar behavior regarding the survival of the strain T.
  • Chitinases are enzymes responsible for the degradation of chitin present in the cell wall of some pathogenic microorganisms such as F. oxysporum, part of these enzymes would be immobilized in bentonite, together with the reproductive and mycelial structures of T. harzianum so that when produce an infection by pathogens, the previously generated chitinase activity would help its control.
  • the amount of CO 3 Ca necessary to raise the pH to 7 was calculated first, incorporating different amounts from 1 to 20 grams per 100 grams of bentonite (Table 3). Its incidence was evaluated on the increase in the population of the strain of T. harzianum T78, in peat in seed conditions. Its effect on the germination and growth of melon seedlings was also evaluated as it may be affected by the increase in electrical conductivity with CO 3 Ca. The optimal value found was 5 grams of CO 3 Ca per 100 grams of bentonite , since from this value a decrease in plant development was observed. The incidence of CO 3 Ca on the strain of T. harzianum T78 was positive, producing an increase greater than an order of magnitude (Table 3).
  • the amount of chitosan was determined by adding different amounts from 1 to 20 grams per 100 grams of bentonite (Table 4), the mixture was preinoculated with the strain of T. harzianum T78, as in the previous experiments, with the exception that incubated at 25-28 Q C for 5 days to allow the development of T. harzianum in the support along with the chitosan. Subsequently, a peat test was carried out, inoculating the different mixtures, evaluating their incidence on the increase in the population of the strain of T. harzianum T78 and its effect on the germination and growth of seedlings of cantaloupe.
  • the trays selected for the treatment with the phytopathogen were inoculated with a spore suspension of F. oxysporum so that the amount in each of the wells was 10 3 -10 4 cfu per gram of peat (usual infection dose in seedbed).
  • peat organic material
  • a final analysis was carried out to know the evolution of the populations of the inoculated microorganisms: strain T. harzianum T78 and F. oxysporum (Papavizas et al., 1982 and Komada, 1975, respectively).
  • the leaf size, length and length were analyzed.
  • the percentage of infection of the melon seedlings was also evaluated by planting a portion of the melon stem, with a length of 1 cm previously sterilized on a general fungal medium (PDA), taking as positive stems those who after five days of incubation will show growth of F. oxysporum around it.
  • PDA general fungal medium
  • harzianum T78 is not as prominent, as the decrease in F oxysporum Io, which once again highlights the importance of the proposed combination, which maintains the activity against F oxysporum more effectively, due to the ability to maintain in its structure the chitinases produced during the incubation of T. harzianum with the mixture of bentonite, CO 3 Ca and chitosan.
  • the prototype product was established, it was validated on a commercial scale for which the minimum necessary volume was established, the dosage thereof was optimized in the peat and the application conditions.
  • the 500 kg of bentonite was mixed dry with the 25 kg of CO 3 Ca and 25 kg of chitosan, and once the mixture was homogenized, the 50 liters of the spore suspension was incorporated.
  • this spore solution was dissolved in distilled water in a 1: 10 proportion, so that the final mixture had enough moisture to allow good homogenization, and was sufficiently hydrated to allow the development and growth of the incorporated spores during the 5 days of incubation at 25-28 Q C.
  • the product obtained had a concentration of Trichoderma harzianum T78 of 1, 3 10 8 cfu per gram of bentonite, allowed to dry until 10-15% humidity in order to crush the product and base it, for later incorporation into the peat, obtaining a concentration of Trichoderma harzianum T78 of 0.3 10 8 cfu per gram of bentonite.
  • the inoculum obtained was used in the peat in the proportion of 10% in order to obtain in the substrate T78 of 10 7 ufe of Trichoderma harzianum T78 per gram of peat.
  • a mixture of the inoculum (bentonite + CO 3 Ca + chitosan + Thchoderma) was carried out in the peat by using a characteristic seedbed mixer, for at least 15 minutes to ensure the homogenous distribution of the inoculum in the peat.
  • the treatment of the inoculated substrate was the routine and automated treatment of a commercial seedbed: filling of polystyrene trays automatically with the substrate, following the sowing of melon seeds, humidification, incubation in dark chamber and extension of trays in the seedbed.
  • the melon varieties tested were the one used in the experiment (Tendral), as well as hybrid varieties susceptible to attack by Fusarium oxysporum (Giotto (Ramiro Arnedo)), (Doral and Cyrano (Séminis)).
  • the total volume of trays tested was 1000, each with 150 alveoli, where 500 trays were filled with the inoculated peat-product mixture and the other half with the same peat without the product, the characteristic phytosanitary treatments being carried out to the latter with specific fungicides against fusahosis.
  • Component comprising fungi of the genus Trichoderma useful as a biological control agent and its applications. University of Salamanca; Newbiotechnic, S. A.
  • ES 2219523 (01.12.2004).
  • Component comprising fungi of the genus Trichoderma useful as a biological control agent and its applications. Newbiotechnic, S. A .; University of Salamanca.
  • IL 2103758, (01.10.1997). New isolate from Trichoderma harzianum, T-39, fungicidal compounds containing this isolate and its use against B. cinérea and S. sclerotiorum. Peri development applications (1985) LTD .; Yissum research development company of the Hebrew University of Jerusalem.
  • Trichoderma A review of biology and systematics of the genus. Mycological research, 100 (8), 923-935.

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Abstract

Solid product effective for biocontrol of vascular fusariosis of melon, method for preparation thereof and method for use of same. The invention is part of the technical field of conventional and organic agriculture, is applicable both in fields and nurseries and provides a solid product effective for the biological control of vascular fusariosis of melon based on the use of a strain of T. harzianum, a fungus antagonistic to the pathogen (F. oxysporum sp. melonis), on an inert solid carrier and incorporates additional factors which improve the biocontrol capacity of the product. The invention also relates to the method for preparing the product and the method of use thereof.

Description

TÍTULO TITLE
PRODUCTO SÓLIDO EFICAZ PARA EL CONTROL BIOLÓGICO DE LA FUSARIOSIS VASCULAR DEL MELÓN, SU PROCEDIMIENTO DE OBTENCIÓN Y MÉTODO DE APLICACIÓN DEL MISMO.EFFICIENT SOLID PRODUCT FOR THE BIOLOGICAL CONTROL OF THE VASCULAR FUSARIOSIS OF THE MELON, ITS PROCEDURE OF OBTAINING AND METHOD OF APPLICATION OF THE SAME.
SECTOR DE LA TÉCNICA:SECTOR OF THE TECHNIQUE:
La presente invención se encuadra dentro del campo técnico de Ia agricultura convencional y ecológica, y es aplicable tanto en semillero como en campo. En concreto se refiere a un producto sólido eficaz para el control biológico de fusahosis vascular en melón, su procedimiento de obtención y método de aplicación del mismo.The present invention falls within the technical field of conventional and organic farming, and is applicable both in seedlings and in the field. Specifically, it refers to an effective solid product for the biological control of melon vascular fusahosis, its method of obtaining and method of application thereof.
ESTADO DE LA TÉCNICA:STATE OF THE TECHNIQUE:
La fusahosis vascular del melón es una enfermedad muy común, ocasionada por diferentes razas de Fusarium oxysporum que produce importantes pérdidas en cultivos, principalmente en zonas con temperaturas elevadas. La fusariosis es un problema grave en el campo de cultivo pero sobre todo resulta importante en los semilleros, ya que las condiciones de cultivo en éstos favorecen el desarrollo y dispersión del patógeno debido a Ia producción de un gran número de plantas en un espacio reducido y cerrado.Melon vascular fusahosis is a very common disease, caused by different breeds of Fusarium oxysporum that causes significant crop losses, mainly in areas with high temperatures. Fusariosis is a serious problem in the crop field but above all it is important in the seedbeds, since the cultivation conditions in these favor the development and dispersion of the pathogen due to the production of a large number of plants in a small space and closed.
Los métodos habituales para control de fitopatógenos incluyen el uso de grandes cantidades de sustancias químicas como los fungicidas, sustancias que en algunos casos están clasificadas como cancerígenas y/o tóxicas y que en muchos países están prohibidas. Estas sustancias químicas además, en multitud de ocasiones resultan ineficaces.Common methods for controlling phytopathogens include the use of large amounts of chemical substances such as fungicides, substances that in some cases are classified as carcinogenic and / or toxic and that in many countries are prohibited. These chemicals also, in many cases are ineffective.
Contra Ia fusariosis de melón no hay un control químico efectivo, únicamente es posible Ia desinfección del suelo utilizando productos altamente nocivos tanto para el medioambiente como para Ia salud humana. Por este motivo son muchos los esfuerzos encaminados a investigar métodos alternativos.Against melon fusariosis there is no effective chemical control, only soil disinfection is possible using highly harmful products for both the environment and human health. For this reason, there are many efforts to investigate alternative methods.
El control biológico de Ia fusariosis mediante el empleo de microorganismos antagonistas es una de las alternativas más prometedoras. Entre los agentes de control biológico más importantes se encuentran los hongos pertenecientes al género Trichoderma que son capaces de controlar un amplio abanico de microorganismos patógenos de plantas de interés agrícola. Cinco especies de Trichoderma (T. hamatum, T. harzianum, T. koningü, T. polysporum y T. viridé) son las más importantes para biocontrol (Hoitink y Boehm, 1999). Sin embargo, aunque han tenido un éxito relativo, todavía no ha sido posible alcanzar un nivel suficiente en el control de fitopatógenos. En Ia actualidad existen diferentes métodos de control biológico de fitopatógenos basados en Ia utilización de diversas especies y cepas del hongo Trichoderma. La mayor parte de los métodos relacionados con el control de enfermedades mediante el uso de Trichoderma están basadas en el uso de diferentes especies de este género tales como T. asperellum, T. artroviride, T. inhamatum para el control biológico de fitopatógenos como Rhizoctonia solani (ES 2200602; 2000, y ES 2219523; 2004), T. harzianum por si solo para el control de Botritis. cinérea y de Sclerotinia sclerotiorum (IL2103758; 1997), o en combinación con T viride para el control de Rhizhoctonia solani (ES 2109182; 1995). También se ha descrito el uso de T. viridae por si sólo (US 6,808,917; 2002) o bien T hamatum, con otros microorganismos complementarios como Flavobacterium balustinum, para el control de R. solani y Pythium ultimun (US 4.900.348; 1987). Además, existen productos comerciales basados en el uso de Trichoderma tanto en formulación líquida (Stimulase (Ecocert Francia nQ 08- 884, SCPA Agronutrición), Trichomic (AMC Chemical y Trichodex) y Trianum- G (Koppert) como sólida en forma de polvo (Tricho-tec Ecocert-SHC nQ VA- 22P-2, Técnicas de control biológico), Trianum-P (Koppert), Trichotem (BCS Óko-Garantie QUIM_MERI_8244/02.04/4215-ES, Químicas Meristem), T. harzianum DIB- 32 (Productos Flower). No obstante ninguno de estos productos está descrito de forma específica para el control de Ia fusariosis vascular de melón y en los casos en que se hace mención al control de F. oxysporum es en una gama de microorganismos fitopatógenos.The biological control of fusariosis through the use of antagonistic microorganisms is one of the most promising alternatives. Among the most important biological control agents are the fungi belonging to the genus Trichoderma that are capable of controlling a wide range of pathogenic microorganisms of plants of agricultural interest. Five species of Trichoderma (T. hamatum, T. harzianum, T. koningü, T. polysporum and T. viridé) are the most important for biocontrol (Hoitink and Boehm, 1999). However, although they have had relative success, it has not yet been possible to reach a sufficient level in the control of phytopathogens. At present there are different methods of biological control of phytopathogens based on the use of various species and strains of the Trichoderma fungus. Most of the methods related to disease control through the use of Trichoderma are based on the use of different species of this genus such as T. asperellum, T. artroviride, T. inhamatum for the biological control of phytopathogens such as Rhizoctonia solani (ES 2200602; 2000, and ES 2219523; 2004), T. harzianum alone for the control of Botritis. cinerea and Sclerotinia sclerotiorum (IL2103758; 1997), or in combination with T viride for the control of Rhizhoctonia solani (ES 2109182; 1995). The use of T. viridae alone (US 6,808,917; 2002) or T hamatum, with other complementary microorganisms such as Flavobacterium balustinum, for the control of R. solani and Pythium ultimun (US 4,900,348; 1987) has also been described. . In addition, there are commercial products based on the use of Trichoderma both in liquid formulation (Stimulase (Ecocert France n Q 08- 884, SCPA Agronutrition), Trichomic (AMC Chemical and Trichodex) and Trianum-G (Koppert) as solid in powder form (Tricho-tec Ecocert-SHC n Q VA- 22P-2, Biological control techniques), Trianum-P (Koppert), Trichotem (BCS Óko-Garantie QUIM_MERI_8244 / 02.04 / 4215-EN, Chemistry Meristem), T. harzianum DIB - 32 (Flower Products) However, none of these products is specifically described for the control of melon vascular fusariosis and in cases where the control of F. oxysporum is mentioned, it is in a range of phytopathogenic microorganisms.
La tendencia generalizada del mercado es Ia oferta de productos de amplio espectro, pero hay que tener en cuenta que cada tipo de patógeno tiene un mecanismo de infección distinto, por Io que son necesarios mecanismos de control diferenciados. Se ha propuesto un método específico para el control de Ia fusariosis vascular de melón que está basado en Ia utilización de compost supresivos previamente inoculados con T. harzianum cepa T-34 (ES 188385 A1 ). Sin embargo, el uso de compost supresivos tiene Ia desventaja de que sólo se pueden aplicar a los suelos y no a los sustratos de semilleros ya que pueden inhibir Ia germinación y desarrollo de las plántulas. La patente US0285987; 1985 describe un método de control de fitopatógenos de cuello tales como Pythium sp y Rhizoctonia sp mediante Ia utilización de protoplastos obtenidos de diferentes géneros de Trichoderma, que se embeben en las semillas por si solos o en matrices orgánicas que, una vez germinadas, adquieren resistencia. Su desventaja principal es el bajo contenido final de Trichoderma en el substrato de germinación que es crítico en el control de Ia fusariosis de melón.The general tendency of the market is the offer of broad spectrum products, but it must be taken into account that each type of pathogen has a different infection mechanism, so that differentiated control mechanisms are necessary. A specific method has been proposed for the control of melon vascular fusariosis that is based on the use of suppressive compost previously inoculated with T. harzianum strain T-34 (ES 188385 A1). However, the use of suppressive compost has the disadvantage that they can only be applied to the soil and not to the seedbed substrates since they can inhibit the germination and development of the seedlings. US0285987; 1985 describes a method of control of neck phytopathogens such as Pythium sp and Rhizoctonia sp through the use of protoplasts obtained from different genera of Trichoderma, which are embedded in the seeds by themselves or in organic matrices that, once germinated, acquire resistance . Its main disadvantage is the low final content of Trichoderma in the germination substrate that is critical in the control of melon fusariosis.
La mayor parte de las tecnologías disponibles hasta el momento para el control biológico de fitopatógenos se presentan bajo una formulación líquida, Io que facilita su aplicación pero condiciona su eficacia dada Ia baja capacidad de supervivencia que muestran los microorganismos al pasar de un medio líquido (forma suministrada) a uno sólido (lugar donde debe realizar su acción, bien sea suelo o substrato orgánico). Otra limitación importante de dichas tecnologías es el tiempo de adaptación al nuevo medio y Ia necesidad de encontrar un nicho ecológico del microorganismo inoculado, que limita enormemente su potencial supervivencia en el suelo o substrato orgánico, cuestión que no ha sido resuelta por ninguna de las tecnologías disponibles hasta el momento.Most of the technologies available so far for the biological control of phytopathogens are presented under a liquid formulation, which facilitates their application but determines their effectiveness given the low survival capacity shown by microorganisms when passing from a liquid medium (form supplied) to a solid one (place where it must perform its action, either soil or organic substrate). Another important limitation of these technologies is the time of adaptation to the new medium and the need to find an ecological niche of the inoculated microorganism, which greatly limits its potential survival in the soil or organic substrate, an issue that has not been resolved by any of the technologies available so far.
Por todo ello, y dada Ia problemática específica de Ia fusariosis del melón, es necesario desarrollar productos y procedimientos específicos para el control biológico de Ia fusariosis vascular de melón basados en el uso de microorganismos antagonistas con alta capacidad como agentes biocontrol (Inbar et al., 1994), que sean aplicables tanto en semillero como en campo, capaces de sobrevivir en el sustrato y que Ia inoculación se realice sobre soporte sólido. Este ha sido el objeto de Ia presente invención y su novedad radica en que proporciona un producto específico para el control de Ia fusariosis vascular en melón, con elevada especificidad y alta capacidad como agente biocontrol, hasta ahora inexistente. Además, el producto objeto de Ia presente invención, es sólido, el agente muestra altos niveles de supervivencia en el sustrato y es aplicable tanto en semillero como en campo. Una ventaja adicional de Ia presente invención, que se basa en el uso de un producto natural, es que aumenta el conjunto de remedios biológicos para luchar contra los agentes causantes de enfermedades de plantas, encaminado siempre a disminuir, en Ia medida de Io posible, el uso indiscriminado de sustancias químicas.Therefore, and given the specific problem of melon fusariosis, it is necessary to develop specific products and procedures for the biological control of melon vascular fusariosis based on the use of antagonistic microorganisms with high capacity as biocontrol agents (Inbar et al. , 1994), which are applicable both in the seedbed and in the field, capable of surviving in the substrate and that the inoculation is carried out on solid support. This has been the object of the present invention and its novelty is that it provides a specific product for the control of melon vascular fusariosis, with high specificity and high capacity as a biocontrol agent, hitherto non-existent. In addition, the product object of Ia The present invention is solid, the agent shows high levels of survival in the substrate and is applicable both in the seedbed and in the field. An additional advantage of the present invention, which is based on the use of a natural product, is that it increases the set of biological remedies to fight against the causative agents of plant diseases, always aimed at decreasing, as far as possible, the indiscriminate use of chemical substances.
EXPLICACIÓN DE LA INVENCIÓN La presente invención proporciona un producto sólido eficaz para el control biológico de fusahosis vascular de melón, su procedimiento de obtención y método de aplicación del mismo.EXPLANATION OF THE INVENTION The present invention provides an effective solid product for the biological control of melon vascular fusahosis, its method of obtaining and method of application thereof.
En un aspecto particular Ia invención se refiere a un producto sólido eficaz para el control biológico de Ia fusahosis vascular de melón caracterizado porque comprende (i) un microorganismo antagonista de F. oxysporum con capacidad biocontrol sobre éste, (ii) Un soporte sólido para Ia inoculación del microorganismo antagonista (iii) y factores adicionales que mejorar Ia capacidad biocontrol del producto.In a particular aspect, the invention relates to a solid product effective for the biological control of melon vascular fusahosis characterized in that it comprises (i) an antagonistic microorganism of F. oxysporum with biocontrol capacity on it, (ii) A solid support for Ia inoculation of the antagonistic microorganism (iii) and additional factors that improve the biocontrol capacity of the product.
En otro aspecto particular Ia invención se relaciona con un producto sólido eficaz para el control biológico de Ia fusahosis vascular de melón basado en el uso de T. harzianum como microorganismo antagonista.In another particular aspect, the invention relates to a solid product effective for the biological control of melon vascular fusahosis based on the use of T. harzianum as an antagonistic microorganism.
En otro aspecto particular Ia invención se relaciona con un producto sólido eficaz para el control biológico de Ia fusahosis vascular de melón que utiliza como soporte sólido bentonita. En otro aspecto particular Ia invención se relaciona con un producto sólido eficaz para el control biológico de Ia fusahosis vascular de melón que incorpora como factores adicionales CO3Ca y quitosano como corrector de pH e inductor de Ia síntesis de quitinasas, respectivamente.In another particular aspect, the invention relates to a solid product effective for the biological control of melon vascular fusahosis that uses bentonite as solid support. In another particular aspect, the invention relates to a solid product effective for the biological control of melon vascular fusahosis that incorporates as additional factors CO 3 Ca and chitosan as a pH corrector and inducer of chitinase synthesis, respectively.
En otro aspecto particular Ia invención se relaciona con un procedimiento de obtención de un producto sólido eficaz para el control biológico de Ia fusahosis vascular de melón caracterizado porque sus componentes se incuban a temperaturas entre 20-30QC por un tiempo entre 1 y 10 días. En otro aspecto particular Ia invención se relaciona con un procedimiento para Ia obtención de un producto sólido eficaz para el control biológico de Ia fusahosis vascular de melón caracterizado porque sus componentes se mezclan, trituran e incuban durante 5 días a 25-28 5C. En otro aspecto particular Ia invención se relaciona con un procedimiento para Ia obtención de un producto sólido eficaz para el control biológico de Ia fusahosis vascular de melón caracterizado porque comprende Ia preparación de un soporte sólido como bentonita, que incorpora como factores adicionales CO3Ca y quitosano como corrector de pH e inductor de Ia síntesis de quitinasas, respectivamente, todo ello mezclado y homogeneizado en seco. A dicha mezcla se inocula un cultivo biológicamente puro de Ia cepa de T. harzianum T78 NQ CECT 20714, y se mezcla e incuba durante 5 días a 28-25 5C. El producto resultante se mezcla con turba al 10 % mediante Ie uso de una mezcladora estándar de las utilizadas en los semilleros comerciales. Otro aspecto particular de Ia invención Io constituye un procedimiento para inducir resistencia sistémica a enfermedades causadas por organismos fitopatógenos que comprende aplicar una cantidad eficaz del producto objeto de Ia invención sobre el suelo o Ia planta a Ia que se desea inducir resistencia sistémica a enfermedades. En otro aspecto particular Ia invención se relaciona con un procedimiento para estimular el crecimiento de plantas que comprende aplicar una cantidad eficaz del producto objeto de Ia invención sobre suelo o planta cuyo crecimiento se desea estimular.In another particular aspect the invention relates to a process for obtaining a solid product for effective biological control of vascular Ia fusahosis of melon characterized in that its components are incubated at temperatures between 20 to 30 Q C for a time between 1 and 10 days . In another particular aspect, the invention relates to a method for obtaining an effective solid product for the biological control of melon vascular fusahosis characterized in that its components are mixed, crushed and incubated for 5 days at 25-28 5 C. Another particular aspect of the invention relates to a method for obtaining an effective solid product for the biological control of melon vascular fusahosis characterized in that it comprises the preparation of a solid support such as bentonite, which incorporates as additional factors CO 3 Ca and chitosan as a pH corrector and inducer of chitinase synthesis, respectively, all mixed and dry homogenized. To said mixture a biologically pure culture of the strain of T. harzianum T78 N Q CECT 20714 is inoculated, and mixed and incubated for 5 days at 28-25 5 C. The resulting product is mixed with 10% peat by use. of a standard mixer used in commercial seedbeds. Another particular aspect of the invention constitutes a method for inducing systemic resistance to diseases caused by phytopathogenic organisms which comprises applying an effective amount of the product object of the invention on the soil or plant to which it is desired to induce systemic resistance to diseases. In another particular aspect, the invention relates to a process for stimulating the growth of plants which comprises applying an effective amount of the product object of the invention on soil or plant whose growth it is desired to stimulate.
Otro aspecto particular de Ia invención es Ia utilización del producto objeto de Ia invención para el control biológico de esta enfermedad en semillero.Another particular aspect of the invention is the use of the product object of the invention for the biological control of this disease in a seedbed.
Otro aspecto particular de Ia invención Io constituye Ia utilización del producto objeto de Ia invención para el control biológico de Ia fusahosis vascular en campo de cultivo. En otro aspecto particular Ia invención se relaciona con el uso deAnother particular aspect of the invention constitutes the use of the product object of the invention for the biological control of vascular fusahosis in the field of culture. In another particular aspect, the invention relates to the use of
Trichoderma harzianum, cepa T-78, CECT NQ 20714 para el control biológico de Ia fusahosis vascular. DESCRIPCIÓN DE LAS FIGURASTrichoderma harzianum, strain T-78, CECT N Q 20714 for the biological control of vascular fusahosis. DESCRIPTION OF THE FIGURES
Figura 1 : Gráfico de barras dónde se refleja el crecimiento en placas Petri sobre ensayo en placa dual de F. oxysporum enfrentadas a diferentes cepas de Trichoderma. En el eje de abscisas se representan las cepas de Trichoderma sp. que mostraron un mejor resultado para los intereses y/o objetivos a desarrollar ( C (Control de Fusarium), TC (cepa comercial) y cepas T82, T78, T010, T190 y T1 ) y en el eje de ordenadas el crecimiento final a los siete días sobre Ia placa Petri en centímetros.Figure 1: Bar graph showing the growth in Petri dishes on a F. oxysporum dual plate assay facing different strains of Trichoderma. In the abscissa axis, the strains of Trichoderma sp. that showed a better result for the interests and / or objectives to be developed (C (Control of Fusarium), TC (commercial strain) and strains T82, T78, T010, T190 and T1) and in the axis of ordinates the final growth at seven days on the Petri dish in centimeters.
Figura 2: Crecimiento sobre turba de las cepas de Trichoderma sp. atendiendo al criterio de supresividad frente a F oxysporum (A) y supervivencia en turba de Ia cepa T78 (B). En el eje de abscisas de Ia Figura 2 (A) se representan las diferentes cepas estudiadas, (T78; T82, T1 , T010; T190 y TC (comercial)) y en Ia Figura 2 (B) se representa Ia cepa de Trichoderma T78 inoculada en turba y turba sin inocular. En el eje de ordenadas de Ia Figura 2 (A) se muestra el número de unidades formadoras de colonias (ufe) x 106 por gramo de turba analizado y en Ia Figura 2 (B) se muestra el porcentaje de CO2 desprendido en el medio.Figure 2: Growth over peat of Trichoderma sp. according to the suppressivity criterion against F oxysporum (A) and peat survival of strain T78 (B). In the axis of abscissa of Figure 2 (A) the different strains studied are represented, (T78; T82, T1, T010; T190 and TC (commercial)) and in Figure 2 (B) the strain of Trichoderma T78 is represented inoculated in peat and peat without inoculation. In the ordinate axis of Figure 2 (A) the number of colony forming units (ufe) x 10 6 per gram of peat analyzed is shown and Figure 2 (B) shows the percentage of CO 2 released in the means, medium.
Figura 3: Evolución de F. oxysporum sometido a los diferentes tratamientos pasados 15, 37 y 48 días de su inoculación respectivamente. En el eje de abscisas se representan cada una de las formas en que se inoculó Ia solución de Ia cepa T. harzianum T78 en Ia turba. (C: control sin inocular, 1 : solución de esporas, 2: embebiendo a Ia semilla con una solución de esporas en carboximetilcelulosa, 3: embebiendo a Ia semilla con una solución de esporas en goma arábiga, 4: bentonita previamente inoculada con Ia suspensión de esporas, 5: vermiculita previamente inoculada con Ia suspensión de esporas, 6: incorporación de esporas con una fuente carbonada rica en lignocelulosa, 7: tratamiento similar al tratamiento 5 pero en este caso Ia vermiculita se incorporó en Ia parte superior de las bandeja de germinación y el eje de ordenadas muestra el número de ufe F. oxysporum x 106 por gramo de turba analizado. Figura 4: Evolución de Ia cepa de T. harzianum T78 en turba, tras su aplicación en los diferentes soportes. En el eje de abscisas se representan los diferentes tratamientos del 1 al 7 y el control (C). En el eje de ordenadas se muestra el número de ufe de T. harzianum T78 x 106 por gramo de turba analizado a los 0, 2, 15, 37 y 48 días.Figure 3: Evolution of F. oxysporum subjected to the different treatments after 15, 37 and 48 days of inoculation respectively. In the abscissa axis, each of the ways in which the solution of the strain T. harzianum T78 in the peat was inoculated is represented. (C: control without inoculation, 1: spore solution, 2: embedding the seed with a spore solution in carboxymethyl cellulose, 3: embedding the seed with a spore solution in gum arabic, 4: bentonite previously inoculated with the suspension of spores, 5: vermiculite previously inoculated with the spore suspension, 6: incorporation of spores with a carbon source rich in lignocellulose, 7: treatment similar to treatment 5 but in this case the vermiculite was incorporated in the upper part of the tray Germination and ordinate axis shows the number of CFU F. oxysporum x 10 6 per gram of peat analyzed. Figure 4: Evolution of the strain of T. harzianum T78 in peat, after its application in the different supports. The different treatments from 1 to 7 and the control (C) are represented on the abscissa axis. The order number of T. harzianum T78 x 10 6 per gram of peat analyzed at 0, 2, 15, 37 and 48 days is shown on the ordinate axis.
Figura 5: Porcentaje de infección por F. oxysporum (A) y Peso seco (g.) (B) de las plantas de melón tratados con los distintos soportes, después de 15, 33 y 48 días de cultivo en semillero. En el eje de abscisas se representan los diferentes tratamientos, del 1 al 7 y el control (C). En el eje de ordenadas de Ia Figura A se muestra el porcentaje de infección por F. oxysporum de un total de 20 plantas y en Ia Figura B los gramos de peso seco de las plantas de melón.Figure 5: Percentage of infection by F. oxysporum (A) and Dry weight (g.) (B) of the melon plants treated with the different supports, after 15, 33 and 48 days of cultivation in seedlings. In the axis of abscissa the different treatments are represented, from 1 to 7 and the control (C). In the ordinate axis of Figure A the percentage of infection by F. oxysporum of a total of 20 plants is shown and in Figure B the grams of dry weight of the melon plants.
Figura 6: Efecto de Ia aplicación de bentonita complementada con carbonato calcico y quitosano sobre el peso seco (g.) (A) y porcentaje de infección por F oxysporum de las plantas de melón (B), después de 48 días de cultivo en semillero. En el eje de abscisas se representan los tratamientos T: Turba; Ti:Figure 6: Effect of the application of bentonite supplemented with calcium carbonate and chitosan on dry weight (g.) (A) and percentage of infection by F oxysporum of melon plants (B), after 48 days of cultivation in seedlings . In the axis of abscissa the treatments T are represented: Peat; You:
Turba inoculada con Ia cepa T78 en Bentonita y TiQ: Turba inoculada con Ia cepa T78 en bentonita complementada con CO3Ca y quitosano. En el eje de ordenadas de Ia Figura 6(A) se muestra los gramos de peso seco de las plantas de melón y de Ia Figura 6(B) el porcentaje de infección por F oxysporum de un total de 20 plantas.Peat inoculated with strain T78 in Bentonite TiQ: peat inoculated with strain T78 bentonite supplemented CO 3 Ca and chitosan. In the ordinate axis of Figure 6 (A) the grams of dry weight of the melon plants are shown and of Figure 6 (B) the percentage of infection by F oxysporum of a total of 20 plants.
Figura 7: Efecto de Ia aplicación sobre turba de bentonita complementada con carbonato calcico y quitosano sobre Ia cepa de T. harzianum T78 (A) y F oxysporum (B) y el porcentaje de infección por F oxysporum de las plantas de melón (C) después de 48 días de cultivo de plantas de melón en semillero. En el eje de abscisas se representan los tratamientos, (T: Turba; Ti: Turba inoculada con Ia cepa T78 en Bentonita y TiQ: Turba inoculada con Ia cepa T78 en bentonita complementada con CO3Ca y quitosano), mientras que el eje de ordenadas muestra el número de ufe (escala logarítmica) por gramo de turba analizado de T. harzianum T78 (A), de F oxysporum (B) y en (C) se representa el porcentaje de infección por F oxysporum. DESCRIPCIÓN DETALLADA DE LA INVENCIÓNFigure 7: Effect of the application on peat of bentonite supplemented with calcium carbonate and chitosan on the strain of T. harzianum T78 (A) and F oxysporum (B) and the percentage of infection by F oxysporum of melon plants (C) after 48 days of growing melon plants in a seedbed. In the abscissa axis the treatments are represented, (T: Peat; Ti: Peat inoculated with the strain T78 in Bentonite and TiQ: Peat inoculated with the strain T78 in bentonite supplemented with CO 3 Ca and chitosan), while the axis of ordered shows the number of ufe (logarithmic scale) per gram of analyzed peat of T. harzianum T78 (A), of F oxysporum (B) and in (C) the percentage of infection by F oxysporum is represented. DETAILED DESCRIPTION OF THE INVENTION
La presente invención proporciona un producto sólido eficaz para el control biológico de fusahosis vascular de melón basado en el uso de una cepa de T.harzianum, hongo antagonista del patógeno (F. oxysporum sp. melonis), sobre una matriz sólida inerte. La invención también se refiere al procedimiento de obtención del producto y a su método de aplicación.The present invention provides an effective solid product for the biological control of melon vascular fusahosis based on the use of a strain of T. harzianum, pathogen antagonist fungus (F. oxysporum sp. Melonis), on an inert solid matrix. The invention also relates to the method of obtaining the product and its method of application.
La invención se basa en el uso de una cepa natural de T. harzianum aislada de sustratos orgánicos de similares características a las turbas que se utilizan en semilleros, Io que aumenta su capacidad de supervivencia y disminuye Ia frecuencia de aplicación del producto.The invention is based on the use of a natural strain of T. harzianum isolated from organic substrates of similar characteristics to the mobs used in seedbeds, which increases its survival capacity and decreases the frequency of application of the product.
Como soporte sólido se utiliza bentonita que tiene alta capacidad de retención de humedad, permite fijar tanto formas reproductoras como vegetativas activas (miceliares) del hongo, a una concentración adecuada para facilitar que el agente antagonista empiece a actuar desde el momento de Ia aplicación, y mantiene altas concentraciones de éste en el sustrato. Además, por su elevada capacidad de adherencia, Ia bentonita actúa como protector físico de Ia raíz evitando Ia entrada de patógenos, de este modo potencia el efecto biocontrol y permite inmovilizar enzimas producidas por T. harzianum capaces de degradar Ia pared celular de ciertos patógenos como F. oxysporum, incrementando Ia eficacia del producto objeto de Ia presente invención.As a solid support, bentonite is used, which has a high moisture retention capacity, allows to fix both reproductive and active vegetative (mycelial) forms of the fungus, at a suitable concentration to facilitate that the antagonist agent begins to act from the moment of application, and maintains high concentrations of this in the substrate. In addition, due to its high adhesion capacity, the bentonite acts as a physical protector of the root preventing the entry of pathogens, thus enhancing the biocontrol effect and allows immobilizing enzymes produced by T. harzianum capable of degrading the cell wall of certain pathogens such as F. oxysporum, increasing the effectiveness of the product object of the present invention.
La invención utiliza carbonato calcico para corregir el pH de Ia bentonita natural, hasta un pH próximo al óptimo para el crecimiento de Ia cepa de T. harzianum inmovilizada, Io que mejora su capacidad de colonización, optimiza su crecimiento vegetativo y se consigue mayor cantidad de título en el mismo volumen de soporte para Ia inoculación.The invention uses calcium carbonate to correct the pH of the natural bentonite, up to a pH close to the optimum for the growth of the immobilized T. harzianum strain, which improves its colonization capacity, optimizes its vegetative growth and achieves a greater amount of title in the same volume of support for inoculation.
El producto objeto de Ia presente invención incorpora quitosano como fuente nutritiva para Ia cepa inoculada, compuesto que, en algunos casos, se utiliza por si solo en el control preventivo de fitopatógenos, (Chitomer, BCS Óko-Garantie QUIM_MERI_8244/01.04/4161 -ES, Químicas Meristem. La presencia de quitosano en el inoculo induce Ia activación de Ia actividad quitinasa, enzima responsable de Ia degradación de quitina presente en Ia pared celular de algunos microorganismos patógenos como F. oxysporum, de forma que cuando se produce una infección por patógenos, Ia actividad quitinasa generada previamente evitaría Ia instauración de estos en el suelo. La capacidad de biocontrol de este producto está en función del grado de acetilación, cuanto menor es éste mayor es su capacidad de biocontrol. No obstante su empleo directo no ofrece resultados concluyentes, puesto que, si bien en determinadas circunstancias tiene un efecto preventivo, en otras tiene un efecto activador de Ia infección del patógeno, ya que puede ser utilizado por éste como fuente de carbono y nutrientes.The product object of the present invention incorporates chitosan as a nutritional source for the inoculated strain, a compound that, in some cases, is used by itself in the preventive control of phytopathogens, (Chitomer, BCS Óko-Garantie QUIM_MERI_8244 / 01.04 / 4161 -EN Meristem chemistry The presence of chitosan in the inoculum induces the activation of the chitinase activity, enzyme responsible for the degradation of chitin present in the cell wall of some pathogenic microorganisms such as F. oxysporum, of so that when a pathogen infection occurs, the previously generated chitinase activity would prevent the establishment of these in the soil. The biocontrol capacity of this product is a function of the degree of acetylation, the smaller it is, the greater its biocontrol capacity. However, its direct use does not offer conclusive results, since, although in certain circumstances it has a preventive effect, in others it has an activating effect of the infection of the pathogen, since it can be used by it as a source of carbon and nutrients.
Por tanto, Ia presente invención combina Ia incorporación de quitosano de bajo grado de acetilación, carbonato calcico con Ia cepa de T. harzianum, inmovilizadas en un substrato sólido inerte a base de bentonita, que contiene fijadas estructuras tanto reproductivas como miceliares activas del hongo. Estos componentes se incuban durante un tiempo, previo a su preparación como inoculo, de manera que las densidades eficaces del microorganismo, así como de sus metabolitos (enzimas hidrolíticas), se mantienen durante el periodo de tiempo en el que Ia planta es susceptible de contraer Ia enfermedad. Todo ello permite optimizar el control biológico de cultivos de melón tanto en semillero como en campo, con Ia consecuente obtención de cultivos de calidad que mantienen y mejoran Ia calidad del suelo, con Io que en último término se evolucionará hacia una agricultura respetuosa con el medio ambiente y Ia salud de los seres vivos. Además todos los componentes utilizados para conseguir el formulado final son adecuados para su utilización en agricultura ecológica, por Io que el formulado propuesto permite controlar Ia fusahosis vascular de melón tanto en agricultura convencional como ecológica, mucho más restrictiva en cuanto insumos.Therefore, the present invention combines the incorporation of chitosan of low degree of acetylation, calcium carbonate with the strain of T. harzianum, immobilized in an inert solid substrate based on bentonite, which contains fixed both reproductive and active mycelial structures of the fungus. These components are incubated for a time, prior to their preparation as inoculum, so that the effective densities of the microorganism, as well as its metabolites (hydrolytic enzymes), are maintained during the period of time in which the plant is susceptible to contract The disease All this allows to optimize the biological control of melon crops both in the seedbed and in the field, with the consequent obtaining of quality crops that maintain and improve the quality of the soil, with which ultimately it will evolve towards a respectful agriculture with the environment environment and health of living beings. In addition, all the components used to achieve the final formulation are suitable for use in organic farming, so that the proposed formulation allows to control the melon vascular fusahosis in both conventional and organic farming, much more restrictive in terms of inputs.
Para desarrollar Ia presente invención, en primer lugar se procedió a Ia selección y aislamiento del agente biocontrol a utilizar, en segundo lugar a estudiar su capacidad de supervivencia en los substratos orgánicos tipo turba habituales en semillero. En tercer lugar se procedió a desarrollar el soporte adecuado para implementar su acción tras Ia inoculación y, una vez seleccionados los aspectos anteriores, se procedió a mejorar Ia acción de T. harzianum mediante adición en el soporte de compuestos capaces de darle una mayor y más rápida capacidad de acción, por último, se validó el producto final y se establecieron las condiciones de aplicación.To develop the present invention, in the first place, the biocontrol agent was selected and isolated to be used, secondly to study its survival capacity in the usual peat-like organic substrates in the seedbed. Thirdly, the appropriate support was developed to implement its action after inoculation and, once the above aspects were selected, the action of T. harzianum was improved by adding in the support of compounds capable of giving it a greater and faster capacity for action, finally, the final product was validated and the application conditions established.
Selección y aislamiento de cepas de Trichoderma. Evaluación de Ia capacidad biofungicida frente a Fusarium oxysporum. Estudio de supervivencia en turbaSelection and isolation of Trichoderma strains. Evaluation of the biofungicidal capacity against Fusarium oxysporum. Survival study in peat
Para Ia selección y aislamiento de microorganismos biofungicidas frente al fitopatógeno, se seleccionó el género Trichoderma sp, independientemente de su especie, por las excelentes cualidades para Ia lucha contra diversos hongos fitopatógenos, su capacidad bioestimulante y biorreguladora (Samuels,For the selection and isolation of biofungicidal microorganisms against the phytopathogen, the genus Trichoderma sp was selected, regardless of its species, for the excellent qualities for the fight against various phytopathogenic fungi, their biostimulant and bioregulatory capacity (Samuels,
1996), además de su adaptabilidad, facilidad de manejo y su alta capacidad de colonización (Hoitkin y Boehm, 1999). Una vez seleccionado el conjunto de cepas de Trichoderma, y realizados los ensayos para demostrar el grado de control sobre F. oxysporum, su capacidad de supervivencia y adaptación en los sustratos tipo turba que se utilizan comúnmente en los semilleros, se procedió a elegir aquella que aunase ambas características.1996), in addition to its adaptability, ease of handling and high colonization capacity (Hoitkin and Boehm, 1999). Once the set of Trichoderma strains was selected, and the tests were carried out to demonstrate the degree of control over F. oxysporum, its survivability and adaptation in the peat type substrates that are commonly used in the seedbeds, we proceeded to choose the one that combine both characteristics.
Las cepas de Trichoderma sp, independientemente de su especie, se aislaron a partir de turbas, compost verdes y suelos del sureste español, evaluando un total de 11 cepas designadas T010, T1 , T3, T10, T75, T76, T78, T82, T85, T86 Y T190. También se ensayó un producto comercial que conteníaTrichoderma sp strains, regardless of their species, were isolated from mobs, green compost and soils from the Spanish southeast, evaluating a total of 11 strains designated T010, T1, T3, T10, T75, T76, T78, T82, T85 , T86 and T190. A commercial product containing
Trichoderma harzianum y estaba recomendado para el control preventivo de Ia fusahosis vascular de melón (designado T comercial). Para realizar Ia evaluación las cepas se sometieron a las siguientes pruebas:Trichoderma harzianum and was recommended for the preventive control of melon vascular fusahosis (designated commercial T). To perform the evaluation, the strains were subjected to the following tests:
(i) Evaluación de Ia capacidad biofungicida in vitro de las cepas de Trichoderma frente a F. oxysporum mediante ensayo en placa dual, que consiste en enfrentamientos equidistantes de cuatro centímetros de las cepas aisladas frente a F. oxysporum, en placa petri en medio de cultivo(i) Evaluation of the in vitro biofungicidal capacity of the Trichoderma strains against F. oxysporum by means of a dual plate assay, which consists of equidistant confrontations of four centimeters of the strains isolated against F. oxysporum, in a petri dish in the middle of culture
PDA, cuantificando el crecimiento de este último con cada una de las cepas (Rupela et al., 2003). Estos análisis mostraron que las cepas con mayor capacidad biofungicida eran T78 y Ia T190 (Fig. 1 (A)) pero el crecimiento presentado por las diferentes cepas de Trichoderma sp. frente al patógeno en estudio (Figura 1 (B)) mostró Ia existencia de una cierta influencia negativa de F. oxysporum sobre el desarrollo de Ia cepa T190 hecho que hizo descartarla y seleccionar Ia cepa T1 debido a Ia gran capacidad de crecimiento presentada por esta cepa en presencia de Fusarium oxysporum.PDA, quantifying the growth of the latter with each of the strains (Rupela et al., 2003). These analyzes showed that the strains with the highest biofungicidal capacity were T78 and Ia T190 (Fig. 1 (A)) but the growth presented by the different strains of Trichoderma sp. against the pathogen under study (Figure 1 (B)) showed the existence of a certain negative influence of F. oxysporum on the development of strain T190 fact that made it discard and select the T1 strain due to the large growth capacity presented by this strain in the presence of Fusarium oxysporum.
(ii) Evaluación de Ia capacidad de crecimiento y supervivencia sobre turba, sustrato utilizado en los semilleros durante todo el proceso de germinación y crecimiento de las plántulas de melón. Este análisis se realizó incubando las cepas en turba con las condiciones habituales de germinación y crecimiento aplicadas al cultivo de melón. Los resultados pusieron de manifiesto que Ia cepa T78 presentaba mayor capacidad de crecimiento en este sustrato (Fig. 2 (A)).(ii) Evaluation of the growth and survival capacity on peat, substrate used in the seedlings during the whole germination and growth process of the melon seedlings. This analysis was performed by incubating the peat strains with the usual germination and growth conditions applied to the melon crop. The results showed that strain T78 had greater growth capacity in this substrate (Fig. 2 (A)).
(iii) Evaluación del estado y actividad de Ia cepa T78 en turba: para ello se analizó si su estado era en forma miceliar o activa y/o en forma de esporas, es decir latente. La determinación del estado del microorganismo es importante a Ia hora de evaluar su actividad, ya que es posible que tenga capacidad de supervivencia, pero que no sea activa y, por Io tanto, no desarrolle Ia actividad para Ia que se utiliza. Las esporas son estructuras de resistencia para Ia supervivencia del microorganismo, pero no son activas y es necesario que éstas germinen y den lugar al micelio para que se desarrolle su actividad (Papadivas y Lumsden, 1982; Harman, 2004). Para determinar Ia capacidad de germinación de las esporas de Ia cepa T78 inoculadas sobre turba se midió Ia respiración basal una vez inoculada, comparándola con Ia turba sin inocular con Ia cepa T78, mediante Ia medida del desprendimiento de CO2, en el medio inoculado como medida de su actividad (Pascual et al., 2002). Los resultados obtenidos mostraron que Ia cepa T78 tenía buena capacidad para germinar y desarrollarse en Ia turba.(iii) Evaluation of the state and activity of the T78 strain in peat: for this, it was analyzed if its state was in mycelial or active form and / or in the form of spores, that is to say latent. The determination of the state of the microorganism is important when assessing its activity, since it is possible that it has a survivability, but that it is not active and, therefore, does not develop the activity for which it is used. The spores are resistance structures for the survival of the microorganism, but they are not active and they need to germinate and give rise to the mycelium for their activity to develop (Papadivas and Lumsden, 1982; Harman, 2004). To determine the germination capacity of the spores of the strain T78 inoculated on peat the basal respiration was measured once inoculated, comparing it with the peat without inoculating with the strain T78, by measuring the release of CO 2 , in the inoculated medium as measure of its activity (Pascual et al., 2002). The results obtained showed that strain T78 had a good capacity to germinate and develop in peat.
De todo ello se concluyó que del conjunto de cepas aisladas Ia cepa T78 presentaba una gran capacidad de crecimiento y adaptación en turba, además de presentar una mas que considerable capacidad biofungicida frente a F. oxysporum. El DNA de esta cepa fue extraído y secuenciado determinando que Ia cepa elegida, por homología de secuencia, pertenecía a Ia especie de T. harzianum, presentando algunas variaciones respecto a las incluidas en las bases de datos genómicas, motivo suficiente para su registro en Ia Colección española de cultivos tipo bajo el NQ 20714From all this it was concluded that from the set of isolated strains the strain T78 had a great capacity for growth and adaptation in peat, in addition to presenting a more than considerable biofungicidal capacity against F. oxysporum. The DNA of this strain was extracted and sequenced, determining that the strain chosen, by sequence homology, belonged to the species of T. harzianum, presenting some variations with respect to those included in the Genomic databases, sufficient reason for registration in the Spanish Collection of type crops under N Q 20714
Estudio y desarrollo del soporte adecuado para Ia inoculación de Ia cepa de Trichoderma harzianum T78.Study and development of adequate support for the inoculation of the strain of Trichoderma harzianum T78.
Tras Ia selección de Ia cepa se procedió a Ia selección del soporte de inoculación más apropiado que permitiese a Ia cepa T78 permanecer el tiempo necesario en el sustrato en contacto con Ia planta y que impidiese Ia actuación del patógeno. Para llevar a cabo esta selección se analizaron dos soportes líquidos: disolución de Ia cepa de Trichoderma T78 en goma arábiga y carboximetilcelulosa (CMC) al 2%, dos compuestos con Ia capacidad de conferir efecto impregnante, también se utilizó una suspensión de esporas de Ia cepa T78 obtenida a partir de cultivo en placa petri; y tres soportes sólidos: bentonita, vermiculita y salvado de trigo, que se prepararon embebiendo en ellos una solución de Trichoderma T78. También se utilizó vermiculita inoculada superficialmente, es decir vermiculita previamente inoculada con Ia suspensión de esporas que se incorporó en Ia parte superior de Ia bandeja y una suspensión de esporas pulverizada durante el cultivo.After the selection of the strain, the most appropriate inoculation support was selected to allow strain T78 to remain the necessary time in the substrate in contact with the plant and to prevent the pathogen from acting. To carry out this selection, two liquid supports were analyzed: dissolution of the Trichoderma T78 strain in 2% gum arabic and carboxymethyl cellulose (CMC), two compounds with the ability to confer impregnating effect, a spore suspension of the Ia was also used. strain T78 obtained from petri dish culture; and three solid supports: bentonite, vermiculite and wheat bran, which were prepared by embedding in them a solution of Trichoderma T78. Also superficially inoculated vermiculite was used, that is to say vermiculite previously inoculated with the spore suspension that was incorporated in the upper part of the tray and a spore suspension sprayed during the cultivation.
El análisis de los soportes se realizó incorporándolos a Ia turba que se utilizó para germinar semillas de melón, sobre las que, una vez germinadas, se inoculó una suspensión de esporas del patógeno F. oxysporum a Ia concentración habitual de infección en semillero, que está entorno a 103-105 ufe por gramo de turba (Agrios, 1998). Tras Ia infección se tomaron muestras sobre las que se evaluó el desarrollo de las plantas, Ia evolución de Ia población del patógeno (Fusarium) y de Ia cepa T. harzianum T78 inoculada con los distintos soportes. La toma de muestras se realizó a Io largo de todo el periodo habitual de cultivo de melón en semillero (48 días). Los resultados mostraron que los tratamientos en los que se produjo una mayor reducción de Ia población de Fusarium sp., fueron los tratamientos con bentonita, (tratamiento 4) y con vermiculita superficial (tratamiento 7) inoculada previamente con T. harzianum y aplicada superficialmente para recubrir los pocilios de las bandejas de siembra (Figuras 3, 4 y 5). La reducción en Ia población de F. oxysporum fue progresiva en el tiempo, siendo el tratamiento con bentonita el que presentó una mayor eficacia, posiblemente debido a su capacidad de adherencia a las raíces Io que permite una mayor protección de Trichoderma sobre el sistema radicular de Ia planta. Asimismo, estos dos tratamientos fueron los que mostraron una mejor evolución de Ia cepa de T. harzianum T78 ya que presentaron el número más elevado de ufc/g, destacando el tratamiento con bentonita que mostró mayor concentración de T. harzianum al final del experimento (Fig. 4). También se analizó el efecto de los distintos soportes utilizados sobre el desarrollo de las plantas al final del cultivo en semillero y su efecto sobre el posible riesgo de movimiento de patógeno de semillero a campo al trasplantar plantas sin síntomas, pero portadoras del patógeno, al campo de cultivo. En todos los casos los tratamientos que mostraron menor porcentaje de infección fueron los que incluían bentonita y vermiculita como soporte del inoculo.The analysis of the supports was carried out by incorporating them into the peat that was used to germinate melon seeds, on which, once germinated, a spore suspension of the F. oxysporum pathogen was inoculated at the usual concentration of seedling infection, which is around 10 3 -10 5 cfu per gram of peat (Agrios, 1998). After the infection, samples were taken on which the development of the plants, the evolution of the population of the pathogen (Fusarium) and the strain T. harzianum T78 inoculated with the different supports were evaluated. Sampling was carried out throughout the usual period of growing melon in a seedbed (48 days). The results showed that the treatments in which there was a greater reduction in the population of Fusarium sp., Were the treatments with bentonite, (treatment 4) and with superficial vermiculite (treatment 7) previously inoculated with T. harzianum and applied superficially to cover the wells of the sowing trays (Figures 3, 4 and 5). The reduction in the population of F. oxysporum was progressive over time, being the treatment with bentonite the one that presented greater efficiency, possibly due to its ability to adhere to the roots, which allows greater protection of Trichoderma over the root system of the plant. Also, these two treatments were those that showed a better evolution of the strain of T. harzianum T78 since they presented the highest number of cfu / g, highlighting the treatment with bentonite that showed the highest concentration of T. harzianum at the end of the experiment ( Fig. 4). The effect of the different supports used on the development of the plants at the end of the seedling was also analyzed and their effect on the possible risk of movement of the pathogen from the seedbed to the field when transplanting plants without symptoms, but carrying the pathogen, to the field of cultivation In all cases the treatments that showed the lowest percentage of infection were those that included bentonite and vermiculite as a support for the inoculum.
Por tanto, de los soportes utilizados para inocular Ia cepa T. harzianum T78 en Ia turba, tanto líquidos como sólidos, los más efectivos fueron los sólidos, en particular bentonita y Ia vermiculita aplicada superficialmente, fundamentalmente debido a Ia capacidad de estos de protección de los microorganismos inoculados en el sustrato. Estos tratamientos, además de tener un efecto biocontrol tienen un efecto bioestimulante y biofertilizante sobre las plantas no infectadas por F. oxysporum.Therefore, of the supports used to inoculate the strain T. harzianum T78 in the peat, both liquids and solids, the most effective were the solids, in particular bentonite and the surface-applied vermiculite, mainly due to their ability to protect them. the microorganisms inoculated in the substrate. These treatments, in addition to having a biocontrol effect, have a biostimulant and biofertilizing effect on plants not infected by F. oxysporum.
Incorporación de factores que mejoran Ia capacidad biocontrol de Ia cepa de T. harzianum T78Incorporation of factors that improve the biocontrol capacity of the strain of T. harzianum T78
Tras Ia selección de Ia cepa y el soporte a utilizar: bentonita, se procedió a incorporar otros factores al producto objeto de Ia invención que permitiesen aumentar Ia capacidad de supervivencia de T. harzianum y su efecto antagonista frente a microorganismos patógenos, especialmente frente a F. oxysporum. Para ello se corrigió el pH de Ia bentonita mediante Ia adición de carbonato calcico natural que eleva el pH consiguiendo un pH más adecuado para el desarrollo de Ia cepa de T. harzianum T78.After the selection of the strain and the support to be used: bentonite, other factors were incorporated into the product object of the invention that allowed to increase the survival capacity of T. harzianum and its antagonistic effect against pathogenic microorganisms, especially against F oxysporum For this, the pH of the bentonite was corrected by means of the addition of natural calcium carbonate that raises the pH, obtaining a more adequate pH for the development of the strain of T. harzianum T78.
Otro factor que mejora Ia actividad biocontrol del producto objeto de Ia invención es el quitosano, que induce una respuesta inmediata por parte de T. harzianum, debido al aumento en Ia producción de enzimas tipo quitinasa, enzima responsable de Ia degradación de Ia pared celular del patógeno. La incorporación de quitosano durante el proceso de inmovilización de Ia cepa T78 en bentonita, evita el que éste pueda ser utilizado por el microorganismo patógeno, como ocurre en los casos en que se utiliza directamente, puesto que cuando el producto final vaya a ser inoculado, el quitosano habrá sido ya utilizado por T. harzianum. De este modo se consigue que cuando T. harzianum sea inoculado este pueda presentar un "micro-nicho" ecológico tanto por las condiciones de pH y Ia presencia de compuestos orgánicos capaces de ser utilizados, como por Ia protección física por Ia arcilla, que mejorarán su supervivencia y aseguraran Ia acción para Ia que se ha inoculado.Another factor that improves the biocontrol activity of the product object of the invention is chitosan, which induces an immediate response by T. harzianum, due to the increase in the production of chitinase-like enzymes, enzyme responsible for the degradation of the cell wall of the pathogen. The incorporation of chitosan during the immobilization process of the strain T78 in bentonite, avoids that it can be used by the pathogenic microorganism, as occurs in cases where it is used directly, since when the final product is to be inoculated, the chitosan will have already been used by T. harzianum. In this way it is achieved that when T. harzianum is inoculated it can present an ecological "micro-niche" both for the pH conditions and the presence of organic compounds capable of being used, as for the physical protection by the clay, which will improve its survival and will ensure the action for which it has been inoculated.
Una vez establecido el producto prototipo se procedió a validar el mismo a escala comercial para Io cual se estableció el volumen mínimo necesario, se optimizó Ia dosificación del mismo en Ia turba y las condiciones de aplicación.Once the prototype product was established, it was validated on a commercial scale for which the minimum necessary volume was established, the dosage thereof was optimized in the peat and the application conditions.
Los siguientes ejemplos ilustran Ia invención pero no deben considerarse en sentido limitativo de Ia misma.The following examples illustrate the invention but should not be considered in a limiting sense thereof.
Los experimentos se llevaron a cabo en unos semilleros comerciales, aplicando las condiciones habituales de germinación y crecimiento utilizadas para el cultivo de melón.The experiments were carried out in commercial seedbeds, applying the usual germination and growth conditions used for melon cultivation.
EJ EMPLO NQ1.EXAMPLE N Q 1.
Selección v aislamiento de Ia cepa de T. harzianum T78. Evaluación de su capacidad biofunαicida frente a F. oxysporum y supervivencia en turbaSelection and isolation of the strain of T. harzianum T78. Evaluation of its biofunαicide capacity against F. oxysporum and survival in peat
Para seleccionar Ia cepa de Trichoderma más adecuada se aislaron cepas de diferentes fuentes: turbas, compost, suelos agrícolas y forestales. También se estudiaron algunas cepas aisladas previamente por el grupo de Investigación. Se ensayaron un total de 1 1 cepas, designadas T010, T1 , T3, T10, T75, T76, T78, T82, T85, T86 y T190. Así como un producto comercial que contenía Trichoderma sp., designado T comercial. En el etiquetado del producto se indicaba su composición y se recomendaba su aplicación para el control preventivo de Ia fusahosis de melón, entre otros.To select the most suitable Trichoderma strain, strains from different sources were isolated: peat, compost, agricultural and forest soils. Some strains previously isolated by the Research group were also studied. A total of 11 strains, designated T010, T1, T3, T10, T75, T76, T78, T82, T85, T86 and T190, were tested. As well as a commercial product containing Trichoderma sp., Designated commercial T. In the product labeling its composition was indicated and its application was recommended for the preventive control of melon fusahosis, among others.
Se evaluó Ia capacidad fungicida de las cepas frente a F. oxysporum para Io que se utilizó un aislado de F. oxysporum f. sp. Melonis raza 1 ,2 (De Cara et al., 2004J obtenido de plantones diversos capaces de manifestar Ia fusariosis de melón, se eligió Ia que presentaba una virulencia alta-muy alta, con el fin de que Ia manifestación de Ia enfermedad no fuese factor limitante en Ia experimentación, esta cepa fue Ia que se utilizó en los siguientes ejemplos. La evaluación se realizó mediante cultivo dual, que permite determinar las habilidades de las distintas cepas de Trichoderma sp. a Ia hora de inhibir el crecimiento de F. oxysporum. El ensayo de cultivo dual consiste en enfrentamientos equidistantes de cuatro centímetros de las cepas aisladas frente a Fusarium oxysporum, en placa petri en medio de cultivo PDA, cuantificando el crecimiento de este último con cada una de las cepas (Bell et al., 1982).The fungicidal capacity of the strains against F. oxysporum was evaluated for which an isolate of F. oxysporum f was used. sp. Melonis race 1, 2 (De Cara et al., 2004J obtained from various seedlings capable of manifesting Ia melon fusariosis, the one that presented a high-very high virulence was chosen, so that the manifestation of the disease was not a limiting factor in the experimentation, this strain was the one that was used in the following examples. The evaluation was carried out through dual culture, which allows to determine the abilities of the different strains of Trichoderma sp. at the time of inhibiting the growth of F. oxysporum. The dual culture trial consists of four centimeter equidistant confrontations of the strains isolated against Fusarium oxysporum, in a petri dish in PDA culture medium, quantifying the growth of the latter with each of the strains (Bell et al., 1982) .
Los resultados obtenidos se recogen en Ia Tablai y en Ia Figura 1. Las cepas que presentaron mayor capacidad biofungicida fueron las cepas T1 y T78 (Figura 1 ).The results obtained are shown in the Tablai and in Figure 1. The strains that presented the greatest biofungicidal capacity were strains T1 and T78 (Figure 1).
Tabla 1. Capacidad biofungicida de las diferentes cepas de Trichoderma frente a Fusarium oxysporum (grado de capacidad biofungicida de las cepas de Trichoderma de menor a mayor: -, +/-, +, ++, +++)Table 1. Biofungicidal capacity of the different strains of Trichoderma versus Fusarium oxysporum (degree of biofungicidal capacity of the Trichoderma strains from lowest to highest: -, +/-, +, ++, +++)
Figure imgf000016_0001
Figure imgf000016_0001
A continuación se evaluó Ia capacidad de crecimiento y supervivencia en turba de las cepas, para determinar cuales eran capaces de crecer en turba y cual de ellas lo haría con mayor rapidez y eficacia. Para ello se prepararon varios recipientes con turba y se inocularon con las distintas cepas, pasados 48 días, que es el tiempo de permanencia de las plantas de melón en semillero, desde su germinación hasta su disposición en el campo, se procedió a realizar el recuento de los microorganismos presentes en Ia turba. Los resultados mostraron que Ia cepa T78 presenta mayor capacidad de crecimiento en turba, mientras que las cepas T1 y T comercial presentan Ia menor capacidad de crecer en este sustrato (Figura 2). La cepa T1 , a pesar de presentar una gran capacidad biofungicida, mostró una muy baja capacidad de crecimiento en turba; por este motivo fue descartada y se optó por Ia cepa T78 que presentó Ia mayor capacidad de inhibir a F. oxysporum y Ia mayor capacidad de crecer sobre turba.Next, the capacity for growth and survival in peat of the strains was evaluated, to determine which were capable of growing in peat and which of they would do it more quickly and effectively. For this, several containers were prepared with peat and inoculated with the different strains, after 48 days, which is the time of permanence of the melon plants in the seedbed, from their germination to their disposal in the field, the count was carried out of the microorganisms present in the peat. The results showed that strain T78 has a greater growth capacity in peat, while commercial strains T1 and T have the lowest capacity to grow in this substrate (Figure 2). The strain T1, despite presenting a large biofungicidal capacity, showed a very low growth capacity in peat; For this reason, it was discarded and the T78 strain was chosen, which presented the greater capacity to inhibit F. oxysporum and the greater capacity to grow on peat.
El DNA de esta cepa fue extraído y secuenciado determinando que Ia cepa elegida, por homología de secuencia, pertenecía a Ia especie de T. harzianum, presentando algunas variaciones respecto a las incluidas en las bases de datos genómicas, motivo suficiente para su registro en Ia Colección española de cultivos tipo bajo el NQ de registro 20714.The DNA of this strain was extracted and sequenced, determining that the strain chosen, by sequence homology, belonged to the species of T. harzianum, presenting some variations with respect to those included in the genomic databases, sufficient reason for its registration in Ia Spanish type culture collection under the registration N Q 20714.
Para completar Ia selección de Ia cepa de T. harzianum se determinó su estado y su actividad mediante Ia medida de Ia respiración en el medio inoculado como respuesta a Ia actividad del inoculo. Para ello se prepararon recipientes con turba estéril al 60% de su capacidad de retención hídrica, adicionando una suspensión de esporas de Ia cepa T78 equivalente a 107 ufe por gramo de turba, se incubaron a 28Q C, y se determinó Ia cantidad de CO2 desprendido de forma periódica durante 25 días, el desprendimiento de CO2 es una medida de actividad biológica del microorganismo inoculado (Pascual et al., 2002). Estos análisis mostraron que Ia cepa T78 era capaz de germinar y desarrollarse en Ia turba, con alto nivel de adaptación a ésta, pues después de un corto periodo de tiempo (7 días), presentó el máximo de respiración, disminuyendo su actividad con el tiempo debido a Ia consumición de los nutrientes, llegando a un equilibrio en el resto del tiempo de incubación (Figura 2 (B).To complete the selection of the strain of T. harzianum its state and its activity were determined by means of the measurement of the breath in the inoculated medium in response to the activity of the inoculum. For this purpose, containers with sterile peat at 60% of their water retention capacity were prepared, adding a spore suspension of the strain T78 equivalent to 10 7 cfu per gram of peat, they were incubated at 28 Q C, and the amount of CO 2 released periodically for 25 days, the release of CO 2 is a measure of biological activity of the inoculated microorganism (Pascual et al., 2002). These analyzes showed that the strain T78 was able to germinate and develop in the peat, with a high level of adaptation to it, because after a short period of time (7 days), it presented maximum breathing, decreasing its activity over time due to the consumption of nutrients, reaching a balance in the rest of the incubation time (Figure 2 (B).
EJ EMPLO N0- 2 Selección de bentonita como soporte de inoculación de T. harzianum. Una vez seleccionada Ia cepa de T. harzianum, se procedió a estudiar Ia forma de inoculación que resultara más adecuada para facilitar su incorporación a Ia turba, que permitiera dotar a las plantas de una protección suficiente frente a los fitopatógenos. La suspensión de esporas se preparó mediante cultivo de Ia cepa en placa petri con PDA durante 7 días, tras Io cual se recogieron las esporas de Ia superficie de las placas con agua destilada, realizando un recuento de esporas totales mediante hematocitómetro en cámara Neubauer, este recuento resultó entorno a 108-109 ufe de Ia cepa T78 por mililitro. A continuación, estas esporas se inmovilizaron utilizando dos tipos de soportes de inoculación:EXAMPLE N 0 - 2 Selection of bentonite as an inoculation support for T. harzianum. Once the strain of T. harzianum was selected, we proceeded to study the form of inoculation that would be more suitable to facilitate its incorporation into the peat, which would allow plants to provide sufficient protection against phytopathogens. The spore suspension was prepared by culturing the strain in petri dish with PDA for 7 days, after which the spores of the surface of the plates were collected with distilled water, making a total spore count by hematocytometer in Neubauer chamber, this count was around -10 9 cfu August 10 of the strain T78 per milliliter. Next, these spores were immobilized using two types of inoculation supports:
1 ) Soportes Líquidos: se utilizó una solución de T harzianum T78 tal cual, obtenida a partir de cultivo en placa petri y soluciones de esporas disueltas o bien en goma arábiga y o en carboximetilcelulosa (CMC), al 2%, con el fin de conferir efecto impregnante a Ia solución de T. harzianum. La goma arábiga y Ia CMC se inocularon directamente en Ia semilla, dada su capacidad de fijación, al actuar como sustancias cementantes, mientras que Ia suspensión de esporas se inoculó en Ia turba en el momento anterior a Ia preparación de las bandejas de poliestireno para Ia germinación y crecimiento de las plántulas de melón. Una vez inoculadas las semillas y secadas durante 7 días para estabilizar el inoculo, se cuantificó el número de unidades formadoras de colonias de Ia cepa T78, mediante PDA-rosa bengala por semilla, que resultó de 104 ufe de Ia cepa T78, y que coincide con el grado de inoculación de cada uno de los pocilios de las bandejas de poliestireno. Para el caso de Ia inoculación líquida directa de Ia suspensión de esporas en Ia turba, se calculó Ia cantidad de inoculo necesario para que Ia cantidad final por gramo de turba estuviese en el rango de 106-107 ufe de Ia cepa T78.1) Liquid Supports: a solution of T harzianum T78 was used as is, obtained from culture in petri dish and solutions of dissolved spores or in gum arabic or in carboxymethylcellulose (CMC), 2%, in order to confer impregnating effect to the solution of T. harzianum. The gum arabic and CMC were inoculated directly in the seed, given its fixing capacity, acting as cementing substances, while the spore suspension was inoculated in the peat at the time prior to the preparation of the polystyrene trays for Ia Germination and growth of melon seedlings. Once the seeds were inoculated and dried for 7 days to stabilize the inoculum, the number of colony forming units of strain T78 was quantified, using PDA-rose flare per seed, which resulted from 10 4 cfu of strain T78, and that coincides with the degree of inoculation of each of the wells of the polystyrene trays. In the case of the liquid direct inoculation of spores in the suspension of peat Ia, the amount of inoculum necessary for Ia final amount per gram of peat was in the range of 10 6- 10 7 cfu of the strain T78 was calculated.
2) Soportes Sólidos: bentonita natural, vermiculita y el salvado de trigo sobre los que se embebió Ia suspensión de esporas de Ia cepa de T. harzianum T78, obtenidas siguiendo el método anteriormente propuesto. Para Ia preparación de estos soportes se les incorporó una suspensión de esporas de Ia cepa de T. harzianum T78, de forma que el contenido final de esporas por gramo de soporte sólido fuese igual. Una vez estabilizados en los substratos sólidos, cuya duración aproximada es de 10-15 días, y cuantificado el número de ufe por gramo de substrato mediante PDA-rosa bengala, los inóculos se mezclaron con Ia turba para que Ia cantidad de inoculo estuviese en el rango de 106-107 ufe de Ia cepa T78 por gramo de turba.2) Solid Supports: natural bentonite, vermiculite and wheat bran on which the spore suspension of the strain of T. harzianum T78 was embedded, obtained following the previously proposed method. For the preparation of these supports, a spore suspension of the strain of T. harzianum T78 was incorporated, so that the final spore content per gram of solid support were the same. Once stabilized in the solid substrates, whose approximate duration is 10-15 days, and quantified the number of ufe per gram of substrate by PDA-rose flare, the inoculums were mixed with the peat so that the amount of inoculum was in the range of 10 6 -10 7 pfu of strain T78 per gram of peat.
También se utilizó vermiculita inoculada superficialmente, es decir vermiculita previamente inoculada con Ia suspensión de esporas que se incorporó en Ia parte superior de Ia bandeja y una suspensión de esporas pulverizada durante el cultivo. El experimento constó de 8 tratamientos y 2 controles, cada uno con cinco réplicas, sobre bandejas de poliestireno utilizada en Ia germinación y crecimiento de plántulas en semillero, cada una de ellas con 150 plántulas de melón. Durante los ensayos preliminares se utilizó Ia variedad de melón tendral, por su elevada susceptibilidad a F oxysporum. Una vez desarrollado el protocolo del prototipo, este fue testado sobre otras variedades comerciales para Io que se eligieron aquellas más utilizadas y sensibles al ataque del patógeno según el Vademécum de semillas hortícolas (Portagrano 2003-2004). Los distintos tratamientos se sometieron a las condiciones habituales de semillero, con Ia única diferencia del soporte seleccionado para Ia inoculación de Ia cepa de T. harzianum T78. Con el fin de poder evaluar el efecto biocontrol, se duplicó el experimento, infectando una mitad con Ia misma cepa que en el ejemplo 1 y Ia otra se mantuvo sin infectar para poder detectar el efecto de los soportes líquidos o sólidos aplicados en el desarrollo de Ia planta de melón sin presión de patógeno introducido. El inoculo de Ia cepa T. harzianum T78 fue de 106-107 ufe por gramo de turba en todos los tratamientos. Estas concentraciones son las más ampliamente utilizadas en Ia bibliografía (Batta, 2004). La única excepción fueron los tratamientos con soportes 1 , 2 y 3 (solución de esporas líquida, CMC y Goma Arábiga) debido a su peculiaridad de inoculación, por adhesión a las semillas de melón, por Io que no fue posible alcanzar estos niveles, alcanzando 104 ufe por gramo de turba. En todos los casos una vez preparados los inóculos se procedió a su recuento en PDA-rosa bengala, comprobando que el nivel de ufe era el adecuado para que al inocular sobre Ia turba estuviese en el rango establecido.Also superficially inoculated vermiculite was used, that is to say vermiculite previously inoculated with the spore suspension that was incorporated in the upper part of the tray and a spore suspension sprayed during the cultivation. The experiment consisted of 8 treatments and 2 controls, each with five replicas, on polystyrene trays used in the germination and growth of seedlings in seedlings, each with 150 melon seedlings. During the preliminary tests the variety of tendon melon was used, due to its high susceptibility to F oxysporum. Once the prototype protocol was developed, it was tested on other commercial varieties for which those most used and sensitive to the pathogen attack were chosen according to the Vademecum of horticultural seeds (Portagrano 2003-2004). The different treatments were subjected to the usual seedbed conditions, with the only difference of the support selected for the inoculation of the strain of T. harzianum T78. In order to evaluate the biocontrol effect, the experiment was duplicated, infecting one half with the same strain as in example 1 and the other was kept without infecting to be able to detect the effect of the liquid or solid supports applied in the development of The melon plant without pathogen pressure introduced. The inoculum of the strain T. harzianum T78 was 10 6 -10 7 pfu per gram of peat in all treatments. These concentrations are the most widely used in the literature (Batta, 2004). The only exception was treatments with supports 1, 2 and 3 (solution of liquid spores, CMC and Arabian Gum) due to its peculiarity of inoculation, by adhesion to the melon seeds, so it was not possible to reach these levels, reaching 10 4 cfu per gram of peat. In all cases, once the inoculums were prepared, they were counted in PDA-rose flare, checking that the level of it was adequate so that when inoculating on the peat it was in the established range.
Tabla 2. Nomenclatura establecida para los distintos tratamientosTable 2. Nomenclature established for the different treatments
Figure imgf000020_0001
Figure imgf000020_0001
Una vez preparados los diferentes soportes inoculados con T. harzianum se mezclaron con turba. Cada tratamiento se colocó en bandejas de poliestireno de 150 pocilios cada una y seguidamente fueron sembradas con semillas de melón, recubiertas con vermiculita y humedecidas. Las bandejas sembradas se colocaron en cámara de germinación comercial acondicionada para Ia germinación del melón, a una temperatura de 25Q C y humedad del 75%, pasados dos días se sacaron de Ia cámara de germinación y se extendieron en el semillero.Once prepared the different supports inoculated with T. harzianum were mixed with peat. Each treatment was placed in 150-well polystyrene trays each and then sown with melon seeds, coated with vermiculite and moistened. The seeded trays were placed in a commercial germination chamber conditioned for the germination of the melon, at a temperature of 25 QC and 75% humidity, after two days they were taken out of the germination chamber and spread in the seedbed.
A los 15 días de haber sacado las bandejas de Ia cámara de germinación, y coincidiendo con Ia aparición de Ia segunda hoja verdadera de Ia plántula de melón, las bandejas seleccionadas para el tratamiento con el fitopatógeno (tratamientos C) se inocularon con una suspensión de esporas de F. oxysporum para que Ia cantidad en cada uno de los pocilios, estuviese entorno a 104 ufe por gramo de turba (dosis de infección habitual en semillero). Sobre el material orgánico (turba) se realizaron un total de cuatro muéstreos periódicos para conocer Ia evolución de las poblaciones de los microorganismos inoculados: cepa T. harzianum T78 y F. oxysporum. Las muestras se sometieron a análisis microbiológicos mediante el medio selectivo correspondiente (Papavizas et al., 1982 y Komada, 1975, respectivamente). Sobre el material vegetal recolectado se analizó el tamaño de las hojas, Ia longitud y el peso del tallo. También se evaluó el porcentaje de infección de las plántulas de melón mediante siembra de una porción de tallo de melón, con una longitud de 1 cm previamente esterilizada, sobre un medio general de hongos (PDA), tomándose como tallos positivos aquellos que tras cinco días de incubación presentaran crecimiento de F. oxysporum a su alrededor.15 days after having removed the trays from the germination chamber, and coinciding with the appearance of the second true leaf of the melon seedling, the trays selected for the treatment with the phytopathogen (treatments C) were inoculated with a suspension of F. oxysporum spores so that the amount in each of the wells was around 10 4 cfu per gram of peat (usual infection dose in seedbed). On the organic material (peat), a total of four periodic samples were carried out to know the evolution of the populations of the inoculated microorganisms: strain T. harzianum T78 and F. oxysporum. The samples were subjected to microbiological analysis by the corresponding selective medium (Papavizas et al., 1982 and Komada, 1975, respectively). The size of the leaves, the length and the weight of the stem were analyzed on the collected plant material. The percentage of infection of the melon seedlings was also evaluated by planting a portion of melon stem, with a length of 1 cm previously sterilized, on a general fungal medium (PDA), taking as positive stems those that after five days incubation will show growth of F. oxysporum around it.
Los tratamientos en los que se produjo una mayor reducción de Ia población de F oxysporum, fueron los tratamientos en los que el soporte Io constituía bentonita (tratamiento 4) y vermiculita aplicada superficialmente para recubrir los pocilios de las bandejas de siembra (tratamiento 7), ambos inoculados con Ia cepa T78 (Figuras 3, 4 y 5). La reducción en Ia población de F oxysporum fue progresiva en el tiempo, si bien el que presentó una mayor rapidez en Ia disminución de las mismas en Ia turba fue el tratamiento con bentonita, posiblemente debido a su capacidad de adherencia a las raíces, permitiendo así una mayor protección sobre el sistema radicular de Ia planta.The treatments in which there was a greater reduction in the population of F oxysporum, were the treatments in which the support constituted bentonite (treatment 4) and vermiculite applied superficially to cover the wells of the sowing trays (treatment 7), both inoculated with strain T78 (Figures 3, 4 and 5). The reduction in the population of F oxysporum was progressive over time, although the one that presented a greater rapidity in the decrease of the same in the peat was the treatment with bentonite, possibly due to its ability to adhere to the roots, thus allowing greater protection over the root system of the plant.
En cuanto a Ia evolución de Ia cepa de T. harzianum T78, los tratamientos con bentonita (tratamiento 4) y vermiculita superficial (tratamiento 7) presentaron el número más elevado de ufe, destacando de forma significativa el tratamiento 4 que mostró un mayor número de ufe de Ia cepa T. harzianum T78 al final del estudio (Figura 4). A Io largo del tiempo Ia cepa T. harzianum T78 mostró una disminución del 50 % en los tratamientos 1 (inoculación esporular), 4 (bentonita) y 7 (vermiculita superficial); si bien el tratamiento 1 , a pesar de mostrar un comportamiento similar en cuanto a Ia supervivencia de Ia cepa T. harzianum T78, su efecto sobre Fusarium oxysporum fue menor que en los tratamientos 4 y 7, Io que indica que las esporas no han germinado y por tanto no han podido desarrollar Ia actividad antagónica frente a F oxysporum. Lo que pone de manifiesto Ia importancia del soporte y del nivel de inoculo presente en el efecto sobre F oxysporum. En el resto de tratamientos, Ia cantidad de inoculo existente, tan solo después de dos días, era del 1 % del valor inoculado. Los tratamientos en los que Ia cepa de T. harzianum T78 se embebió en Ia semilla, a pesar de que el descenso observado fue menos pronunciado, dado que no fue posible alcanzar un nivel de inoculo elevado, éste no fue suficiente para el control de F. oxysporum (Fig. 3, 4 y 5). Con el paso del tiempo, se observa que las poblaciones de Ia cepa de T. harzianum T78 capaces de sobrevivir los primeros dos días después de Ia inoculación muestran una mayor capacidad de adaptación, puesto que el descenso observado es menos pronunciado en los distintos tratamientos, destacando Ia capacidad de supervivencia de Ia cepa T. harzianum T78 inmovilizada con bentonita que no presenta disminución alguna. También es de destacar como Ia inoculación en forma esporular (tratamiento 1 ), que en los primeros 15 días se mantenía en niveles similares a los tratamientos 4 y 7, presentó una evolución descendente a partir de los 37 días (Fig. 4), Io que vuelve a poner de manifiesto Ia necesidad de un soporte sólido que permita al microorganismo mantenerse y evitar las pérdidas por lavado durante el riego diario que se aplica a los semilleros. Esto explica que a los 15 días las ufe de F. oxysporum eran mayores en Ia turba tratada con este último tratamiento que en Ia tratada con los tratamientos 4 y 7, debido a que posiblemente T. harzianum estaba latente en forma de espora, sin posibilidad de germinar y por tanto sin capacidad de controlar al patógeno.Regarding the evolution of the strain of T. harzianum T78, the treatments with bentonite (treatment 4) and superficial vermiculite (treatment 7) presented the highest number of ufe, highlighting significantly the treatment 4 that showed a greater number of ufe of the strain T. harzianum T78 at the end of the study (Figure 4). Over time, the strain T. harzianum T78 showed a 50% decrease in treatments 1 (sporular inoculation), 4 (bentonite) and 7 (superficial vermiculite); Although treatment 1, despite showing a similar behavior regarding the survival of the strain T. harzianum T78, its effect on Fusarium oxysporum was lower than in treatments 4 and 7, which indicates that the spores have not germinated and therefore they have not been able to develop the antagonistic activity against F oxysporum. What shows the importance of the support and the level of inoculum present in the effect on F oxysporum. At Other treatments, the amount of inoculum existing, only after two days, was 1% of the inoculated value. The treatments in which the strain of T. harzianum T78 was embedded in the seed, although the observed decrease was less pronounced, since it was not possible to reach a high level of inoculum, this was not sufficient for the control of F oxysporum (Fig. 3, 4 and 5). With the passage of time, it is observed that the populations of the strain of T. harzianum T78 capable of surviving the first two days after inoculation show a greater adaptability, since the observed decrease is less pronounced in the different treatments, highlighting the survival capacity of the strain T. harzianum T78 immobilized with bentonite that has no decrease. It is also noteworthy as the sporular inoculation (treatment 1), which in the first 15 days remained at levels similar to treatments 4 and 7, presented a downward trend after 37 days (Fig. 4), Io which once again shows the need for a solid support that allows the microorganism to maintain itself and avoid washing losses during the daily irrigation applied to the seedbeds. This explains that at 15 days the CFU of F. oxysporum were higher in the peat treated with this last treatment than in the one treated with treatments 4 and 7, because possibly T. harzianum was latent in spore form, without possibility to germinate and therefore unable to control the pathogen.
Del conjunto de plantas que no mostraban síntomas aparentes de afección por F. oxysporum, se tomaron 20 en cada uno de los puntos de muestreo (a Ios15, 33 y 48 días) para evaluar el porcentaje de infección de las plantas que iban a ser transplantadas y el potencial riesgo de movimiento de patógeno de semillero a campo (Figura 6B). De nuevo, los tratamientos con menor porcentaje de infección fueron aquellos en los que el soporte del inoculo Io constituían bentonita, si bien el porcentaje de infección mostró un aumento considerable en el transcurso de Ia última fase del cultivo, Io que sugiere Ia necesidad de complementar Ia cepa de T. harzianum T78 de alguna forma con el fin de que el control de Ia infección pueda mantenerse durante todo el tiempo de cultivo en semillero, evitando así Ia salida de plantas del mismo que, sin presentar síntomas aparentes, sean portadoras del patógeno. Tras los 48 días de Ia periodo habitual de cultivo de las plántulas de melón en semillero, se pesaron 20 plantas de cada uno de los tratamientos, observando que el desarrollo de las plantas a las que se les había aplicado Ia cepa T. harzianum T78, bajo presión de F. oxysporum fue mayor en los tratamientos con bentonita y vermiculita superficial, mientras que el resto presentó un peso similar al control, mostrando todos ellos síntomas de afección además de clorosis, y manchas necróticas en las hojas (Figura 6A).From the set of plants that showed no apparent symptoms of F. oxysporum affection, 20 were taken at each of the sampling points (at Ios15, 33 and 48 days) to assess the percentage of infection of the plants that were to be transplanted and the potential risk of pathogen movement from seedbed to field (Figure 6B). Again, the treatments with the lowest percentage of infection were those in which the inoculum support constituted bentonite, although the percentage of infection showed a considerable increase in the course of the last phase of the culture, which suggests the need to complement The strain of T. harzianum T78 in some way so that the control of the infection can be maintained during all the time of cultivation in a seedbed, thus avoiding the exit of plants from the same that, without presenting apparent symptoms, are carriers of the pathogen . After 48 days of the usual period of cultivation of seedlings of melon in seedlings, 20 plants of each of the treatments were weighed, observing that the development of the plants to which the strain T. harzianum T78 had been applied, under pressure of F. oxysporum was higher in treatments with bentonite and superficial vermiculite, while the rest had a similar weight to the control, all showing symptoms of affection in addition to chlorosis, and necrotic spots on the leaves (Figure 6A).
Las plantas tratadas con Ia cepa T. harzianum T78, sin presión adicional de patógeno, en general, presentaron un mayor peso y por tanto mayor desarrollo que las plantas sin inocular, de nuevo los tratamientos 1 , 4 y 7 fueron los que mostraron los mejores resultados, Io que demuestra que el inoculo además de tener un efecto biocontrol, también presenta efectos biofertilizantes y/o bioestimulantes a tener en cuenta.The plants treated with the strain T. harzianum T78, without additional pathogen pressure, in general, presented a greater weight and therefore greater development than the plants without inoculation, again treatments 1, 4 and 7 were the ones that showed the best Results, which shows that the inoculum, in addition to having a biocontrol effect, also has biofertilizing and / or biostimulant effects to be taken into account.
EJ EMPLO N0- 3.EXAMPLE N 0 - 3.
Mejora de Ia capacidad biocontrol de Ia cepa de T. harzianum T78 en bentonita complementada con carbonato calcico y con quitosano como fuente de inducción de Ia actividadImprovement of the biocontrol capacity of the strain of T. harzianum T78 in bentonite supplemented with calcium carbonate and with chitosan as a source of induction of the activity
Una vez seleccionada Ia bentonita como soporte óptimo para Ia inoculación de Ia cepa de T. harzianum T78, se procedió a mejorar su capacidad biocontrol mediante Ia incorporación de carbonato calcico (CO3Ca) y quitosano de bajo grado de acetilación, incubando Ia cepa de T. harzianum T78 en estas condiciones a 25-28Q C durante 5 días, previo a Ia preparación del producto, al igual que se ha descrito anteriormente. Con este tratamiento se potencia por un lado el crecimiento de Ia cepa de T. harzianum T78, al conseguir un pH próximo a 7, su óptimo de crecimiento, mediante adicción de CO3Ca al 5% y por otro Ia inducción de Ia producción de quitinasas por T. harzianum, con Ia presencia de quitosano. Las quitinasas son enzimas responsables de Ia degradación de quitina presente en Ia pared celular de algunos microorganismos patógenos como F. oxysporum, parte de estas enzimas quedarían inmovilizadas en Ia bentonita, junto a las estructuras reproductivas y miceliares de T. harzianum de forma que cuando se produjera una infección por patógenos, Ia actividad quitinasa generada previamente ayudaría a su control.Once the bentonite was selected as the optimal support for the inoculation of the T. harzianum T78 strain, its biocontrol capacity was improved by incorporating calcium carbonate (CO 3 Ca) and low-grade acetylation chitosan, incubating the strain of T. harzianum T78 under these conditions at 25-28 Q C for 5 days, prior to the preparation of the product, as described above. With this treatment the growth of the strain of T. harzianum T78 is enhanced on the one hand, when achieving a pH close to 7, its optimum growth, by adding 5% CO 3 Ca and on the other the induction of the production of chitinases by T. harzianum, with the presence of chitosan. Chitinases are enzymes responsible for the degradation of chitin present in the cell wall of some pathogenic microorganisms such as F. oxysporum, part of these enzymes would be immobilized in bentonite, together with the reproductive and mycelial structures of T. harzianum so that when produce an infection by pathogens, the previously generated chitinase activity would help its control.
Para ello, en primer lugar se calculó Ia cantidad de CO3Ca necesario para subir el pH hasta 7, incorporando distintas cantidades desde 1 a 20 gramos por cada 100 gramos de bentonita (Tabla 3). Se evaluó su incidencia sobre el aumento en Ia población de Ia cepa de T. harzianum T78, en turba en condiciones de semillero. También se evaluó su efecto sobre Ia germinación y crecimiento de plántulas de melón ya que puede estar afectado por el aumento de Ia conductividad eléctrica con el CO3Ca. El valor óptimo encontrado fue de 5 gramos de CO3Ca por cada 100 gramos de bentonita, puesto que a partir de este valor se observó una disminución del desarrollo vegetal. La incidencia de CO3Ca sobre Ia cepa de T. harzianum T78 fue positiva, produciendo un aumento superior a un orden de magnitud (Tabla 3).To do this, the amount of CO 3 Ca necessary to raise the pH to 7 was calculated first, incorporating different amounts from 1 to 20 grams per 100 grams of bentonite (Table 3). Its incidence was evaluated on the increase in the population of the strain of T. harzianum T78, in peat in seed conditions. Its effect on the germination and growth of melon seedlings was also evaluated as it may be affected by the increase in electrical conductivity with CO 3 Ca. The optimal value found was 5 grams of CO 3 Ca per 100 grams of bentonite , since from this value a decrease in plant development was observed. The incidence of CO 3 Ca on the strain of T. harzianum T78 was positive, producing an increase greater than an order of magnitude (Table 3).
Tabla 3. Determinación de Ia cantidad de carbonato calcico a incorporar.Table 3. Determination of the amount of calcium carbonate to be incorporated.
Figure imgf000024_0001
Figure imgf000024_0001
La cantidad de quitosano se determinó añadiendo distintas cantidades desde 1 a 20 gramos por cada 100 gramos de bentonita (Tabla 4), Ia mezcla se preinoculó con Ia cepa de T. harzianum T78, como en los experimentos anteriores, con Ia salvedad de que se incubó a 25-28Q C durante 5 días para permitir el desarrollo de T. harzianum en el soporte junto con el quitosano. Posteriormente se realizó un ensayo en turba, inoculando las distintas mezclas, evaluando su incidencia sobre el aumento en Ia población de Ia cepa de T. harzianum T78 y su efecto sobre Ia germinación y crecimiento de plántulas de melón. La cantidad máxima de quitosano a inocular sin producir incidencia negativa sobre Ia planta fue de 5 gramos por 100 gramos de bentonita. La incorporación de quitosano produjo un aumento considerable de crecimiento de Ia cepa de T. harzianum T78 (Tabla 4).The amount of chitosan was determined by adding different amounts from 1 to 20 grams per 100 grams of bentonite (Table 4), the mixture was preinoculated with the strain of T. harzianum T78, as in the previous experiments, with the exception that incubated at 25-28 Q C for 5 days to allow the development of T. harzianum in the support along with the chitosan. Subsequently, a peat test was carried out, inoculating the different mixtures, evaluating their incidence on the increase in the population of the strain of T. harzianum T78 and its effect on the germination and growth of seedlings of cantaloupe. The maximum amount of chitosan to be inoculated without producing a negative incidence on the plant was 5 grams per 100 grams of bentonite. The incorporation of chitosan produced a considerable increase in the growth of the strain of T. harzianum T78 (Table 4).
Tabla 4. Determinación de Ia cantidad de quitosano a incorporar.Table 4. Determination of the amount of chitosan to be incorporated.
Figure imgf000025_0001
Figure imgf000025_0001
Una vez establecidos los valores máximos de CO3Ca (5 gramos) y quitosano (5 gramos) se procedió a ensayarlos de forma conjunta bajo presión de F. oxysporum, siguiendo el modelo de trabajo ya descrito anteriormente: una vez preparado el inoculo en bentonita, CO3Ca y quitosano, se mezcló con turba, se rellenaron bandejas de poliestireno de 150 pocilios y se sembraron las semillas de melón. En este caso además de utilizar Ia variedad tendral de melón se utilizaron otras variedades comerciales que se caracterizan por ser mas susceptibles al ataque de F. oxysporum. Una vez sembradas, las bandejas se colocaron en cámara de germinación acondicionada y pasados dos días se sacaron de Ia cámara de germinación y llevaron al semillero donde se extendieron. A los 15 días de haber sacado las bandejas de Ia cámara de germinación, y coincidiendo con Ia aparición de Ia segunda hoja verdadera de Ia plántula de melón, las bandejas seleccionadas para el tratamiento con el fitopatógeno se inocularon con una suspensión de esporas de F. oxysporum para que Ia cantidad en cada uno de los pocilios fuese de 103-104 ufe por gramo de turba (dosis de infección habitual en semillero). Sobre el material orgánico (turba) se realizó un análisis final para conocer Ia evolución de las poblaciones de los microorganismos inoculados: cepa T. harzianum T78 y F. oxysporum (Papavizas et al., 1982 y Komada, 1975, respectivamente). Sobre el material vegetal recolectado se analizaron el tamaño de las hojas, Ia longitud y el peso del tallo. También se evaluó el porcentaje de infección de las plántulas de melón mediante siembra de una porción de tallo de melón, con una longitud de 1 cm previamente esterilizada sobre un medio general de hongos (PDA), tomándose como tallos positivos aquellos que tras cinco días de incubación presentaran crecimiento de F. oxysporum a su alrededor. Los resultados obtenidos mediante Ia inoculación de Ia cepa de T. harzianum T78 en bentonita con CO3Ca y quitosano todo ello incubado previamente a Ia aplicación, demostraron un aumento en el peso seco de las plantas de melón al final del experimento en comparación con Ia turba, significativamente mayor al obtenido con Ia inoculación de Ia cepa de T. harzianum T78 con bentonita únicamente (Figura 6(A)). El porcentaje de tallos infectados en Ia turba tratada con Ia cepa de T. harzianum T78 en bentonita previamente incubada con carbonato calcico y quitosano mostró un considerable descenso (20%) en comparación con Ia tratada con Ia cepa de T. harzianum T78 en bentonita únicamente (50%) (Figura 6(B)), Io que pone de manifiesto Ia mejora en Ia capacidad de controlar el ataque de F. oxysporum. La adición de quitosano y CO3Ca a Ia bentonita muestra una mejora en Ia población de T. harzianum T78, que está íntimamente relacionada con Ia disminución de las poblaciones de F oxysporum en dos ordenes de magnitud de Io conseguido con bentonita únicamente (Figura 7 (A) y (C). Es de destacar que el aumento de Ia cepa de T. harzianum T78 no es tan destacado, como Ia disminución de F oxysporum Io que pone de relieve de nuevo Ia importancia de Ia combinación propuesta, que mantiene Ia actividad frente a F oxysporum de forma mas efectiva, debido a Ia capacidad de mantener en su estructura las quitinasas producidas durante Ia incubación de T. harzianum con Ia mezcla de bentonita, CO3Ca y quitosano.Once the maximum values of CO 3 Ca (5 grams) and chitosan (5 grams) were established, they were tested together under pressure of F. oxysporum, following the work model already described above: once the inoculum was prepared in bentonite , CO 3 Ca and chitosan, mixed with peat, 150-well polystyrene trays were filled and melon seeds sown. In this case, in addition to using the tendon variety of melon, other commercial varieties that are characterized by being more susceptible to the attack of F. oxysporum were used. Once planted, the trays were placed in conditioned germination chamber and after two days they were removed from the germination chamber and taken to the seedbed where they were extended. 15 days after having removed the trays from the germination chamber, and coinciding with the appearance of the second true leaf of the melon seedling, the trays selected for the treatment with the phytopathogen were inoculated with a spore suspension of F. oxysporum so that the amount in each of the wells was 10 3 -10 4 cfu per gram of peat (usual infection dose in seedbed). On the organic material (peat) a final analysis was carried out to know the evolution of the populations of the inoculated microorganisms: strain T. harzianum T78 and F. oxysporum (Papavizas et al., 1982 and Komada, 1975, respectively). On the collected plant material, the leaf size, length and length were analyzed. the weight of the stem. The percentage of infection of the melon seedlings was also evaluated by planting a portion of the melon stem, with a length of 1 cm previously sterilized on a general fungal medium (PDA), taking as positive stems those who after five days of incubation will show growth of F. oxysporum around it. The results obtained by inoculating the strain of T. harzianum T78 in bentonite with CO 3 Ca and chitosan all incubated prior to the application, demonstrated an increase in the dry weight of the melon plants at the end of the experiment in comparison with Ia peat, significantly greater than that obtained with the inoculation of the strain of T. harzianum T78 with bentonite only (Figure 6 (A)). The percentage of infected stems in the peat treated with the strain of T. harzianum T78 in bentonite previously incubated with calcium carbonate and chitosan showed a considerable decrease (20%) compared to that treated with the strain of T. harzianum T78 in bentonite only. (50%) (Figure 6 (B)), which shows the improvement in the ability to control the attack of F. oxysporum. The addition of chitosan and CO 3 Ca to bentonite shows an improvement in the population of T. harzianum T78, which is closely related to the decrease in F oxysporum populations in two orders of magnitude of what was achieved with bentonite only (Figure 7 (A) and (C) It is noteworthy that the increase in the strain of T. harzianum T78 is not as prominent, as the decrease in F oxysporum Io, which once again highlights the importance of the proposed combination, which maintains the activity against F oxysporum more effectively, due to the ability to maintain in its structure the chitinases produced during the incubation of T. harzianum with the mixture of bentonite, CO 3 Ca and chitosan.
EJ EMPLO Na4.EXAMPLE N to 4.
Validación del producto v condiciones de aplicaciónProduct validation and application conditions
Una vez establecido el producto prototipo se procedió a validar el mismo a escala comercial para Io cual se estableció el volumen mínimo necesario, se optimizó Ia dosificación del mismo en Ia turba y las condiciones de aplicación.Once the prototype product was established, it was validated on a commercial scale for which the minimum necessary volume was established, the dosage thereof was optimized in the peat and the application conditions.
Para ello se partió de un volumen equivalente a 500 kg de bentonita comercial, 50 litros de suspensión de esporas de T harzianum T78 (3 109 ufe / mi), 25 kg de CO3Ca y 25 kg de quitosano. Se mezclaron en seco los 500 kg de bentonita con los 25 kg de CO3Ca y 25 kg de quitosano, y una vez homogenizada Ia mezcla, se procedió a incorporar los 50 litros de Ia suspensión de esporas. Con el fin de incorporarla de forma homogénea, esta solución de esporas se disolvió en agua destilada en una proporción 1 :10, de forma que Ia mezcla final tuviera Ia suficiente humedad para permitir una buena homogenización, y estuviera suficientemente hidratada para permitir el desarrollo y crecimiento de las esporas incorporadas durante los 5 días de incubación a 25-28QC. Después de Ia incubación, el producto obtenido, tenía una concentración de Trichoderma harzianum T78 de 1 ,3 108 ufe por gramo de bentonita, se dejó secar hasta un 10-15% de humedad con el fin de poder triturar el producto y embasarlo, para su posterior incorporación en Ia turba, obteniendo una concentración de Trichoderma harzianum T78 de 0,3 108 ufe por gramo de bentonita. El inoculo obtenido se utilizó en Ia turba en Ia proporción del 10% con el fin de obtener en el substrato T78 de 107 ufe de Trichoderma harzianum T78 por gramo de turba. Para ello se realizó una mezcla del inoculo (bentonita+CO3Ca +quitosano+Thchoderma) en Ia turba mediante el uso de una mezcladora característica de semillero, durante al menos 15 minutos para asegurar Ia distribución homogénea del inoculo en Ia turba. A partir de este momento, el tratamiento del substrato inoculado fue el realizado de forma rutinaria y automatizada de un semillero comercial: llenado de bandejas de poliestireno de forma automática con el substrato, siguiendo de Ia siembra de las semillas de melón, humectación, incubación en cámara oscura y extensión de bandejas en el semillero. Las variedades de melón ensayadas fueron Ia utilizada en el experimento (Tendral), así como variedades híbridas susceptibles al ataque de Fusarium oxysporum (Giotto (Ramiro Arnedo)), (Doral y Cyrano (Séminis)).For this, a volume equivalent to 500 kg of commercial bentonite, 50 liters of spore suspension of T harzianum T78 (3 10 9 cfu / mi), 25 kg of CO 3 Ca and 25 kg of chitosan. The 500 kg of bentonite was mixed dry with the 25 kg of CO 3 Ca and 25 kg of chitosan, and once the mixture was homogenized, the 50 liters of the spore suspension was incorporated. In order to incorporate it in a homogeneous way, this spore solution was dissolved in distilled water in a 1: 10 proportion, so that the final mixture had enough moisture to allow good homogenization, and was sufficiently hydrated to allow the development and growth of the incorporated spores during the 5 days of incubation at 25-28 Q C. After incubation, the product obtained had a concentration of Trichoderma harzianum T78 of 1, 3 10 8 cfu per gram of bentonite, allowed to dry until 10-15% humidity in order to crush the product and base it, for later incorporation into the peat, obtaining a concentration of Trichoderma harzianum T78 of 0.3 10 8 cfu per gram of bentonite. The inoculum obtained was used in the peat in the proportion of 10% in order to obtain in the substrate T78 of 10 7 ufe of Trichoderma harzianum T78 per gram of peat. For this, a mixture of the inoculum (bentonite + CO 3 Ca + chitosan + Thchoderma) was carried out in the peat by using a characteristic seedbed mixer, for at least 15 minutes to ensure the homogenous distribution of the inoculum in the peat. From this moment, the treatment of the inoculated substrate was the routine and automated treatment of a commercial seedbed: filling of polystyrene trays automatically with the substrate, following the sowing of melon seeds, humidification, incubation in dark chamber and extension of trays in the seedbed. The melon varieties tested were the one used in the experiment (Tendral), as well as hybrid varieties susceptible to attack by Fusarium oxysporum (Giotto (Ramiro Arnedo)), (Doral and Cyrano (Séminis)).
El volumen total de bandejas ensayadas fue de 1000, cada una de ellas con 150 alvéolos, donde 500 bandejas se llenaron con Ia mezcla turba- producto inoculado y Ia otra mitad con Ia misma turba sin el producto, realizándose a esta última los tratamientos fitosanitarios característicos con fungicidas específicos frente a Ia fusahosis. En este experimento, se buscaron las condiciones más propicias para el desarrollo de Ia fusahosis vascular en melón a nivel de semillero, si bien por el volumen del experimento y Ia necesidad de estudiar Ia eficacia del producto en condiciones de semillero reales, no se infectaron con el patógeno. Los resultados obtenidos (Tabla 5) demostraron Ia eficacia de Ia inoculación en cuanto al porcentaje de infección con patógeno, ufe de Fusarium oxysporum en el substrato, además de demostrar un mayor crecimiento de las plantas no afectadas, debido al efecto bioestimulante y biofertilizante de Ia cepa de Trichoderma utilizada. Todo ello, viene a demostrar que el uso de este inoculo no solo beneficia en cuanto al control de Ia fusahosis de melón sino también a un menor tiempo de estancia de Ia plántula de melón en semillero, con el consiguiente ahorro económico.The total volume of trays tested was 1000, each with 150 alveoli, where 500 trays were filled with the inoculated peat-product mixture and the other half with the same peat without the product, the characteristic phytosanitary treatments being carried out to the latter with specific fungicides against fusahosis. In this experiment, the most favorable conditions for the development of vascular fusahosis in melon at the seedbed were sought, although due to the volume of the experiment and the need to study the efficacy of the product in real seed conditions, they were not infected with the pathogen The results obtained (Table 5) demonstrated the efficacy of the inoculation in terms of the percentage of infection with pathogen, Ufe of Fusarium oxysporum in the substrate, in addition to demonstrating a greater growth of the unaffected plants, due to the biostimulant and biofertilizing effect of Ia Trichoderma strain used. All this shows that the use of this inoculum not only benefits in terms of the control of melon fusahosis but also in a shorter period of stay of the seedling of melon in the seedbed, with the consequent economic savings.
Tabla 5. Porcentaje de infección y peso fresco de plantas no afectadas en el experimentoTable 5. Percentage of infection and fresh weight of unaffected plants in the experiment
Figure imgf000028_0001
Figure imgf000028_0001
BibliografíaBibliography
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Batta, Y.A. (2004). Effect of treatment with Trichoderma harzianum Rifai formulated in invert emulsión on postharvest decay of apple blue mold. International journal of food microbiology, 96(3), 281 -288.Batta, Y.A. (2004). Effect of treatment with Trichoderma harzianum Rifai formulated in invert emulsion on postharvest decay of apple blue mold. International journal of food microbiology, 96 (3), 281-288.
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Claims

REIVINDICACIONES
1. Producto sólido eficaz para el control biológico de Ia fusariosis vascular de melón caracterizado porque comprende los siguientes componentes: (i) Un microorganismo antagonista de F. oxysporum con capacidad biocontrol sobre éste.1. Solid product effective for the biological control of melon vascular fusariosis characterized in that it comprises the following components: (i) An antagonistic microorganism of F. oxysporum with biocontrol capacity over it.
(ii) Un soporte sólido para Ia inoculación del microorganismo antagonista, (iii) Factores adicionales que mejorar Ia capacidad biocontrol del producto. (ii) A solid support for the inoculation of the antagonistic microorganism, (iii) Additional factors that improve the biocontrol capacity of the product.
2. Producto sólido eficaz para el control biológico de Ia fusariosis vascular de melón según reivindicación 1 caracterizado porque como dicho microorganismo antagonista (i) se utiliza una especie del género Trichoderma con capacidad biocontrol sobre F. oxysporum.2. Solid product effective for the biological control of melon vascular fusariosis according to claim 1 characterized in that as said antagonistic microorganism (i) a species of the genus Trichoderma with biocontrol capacity on F. oxysporum is used.
3. Producto sólido eficaz para el control biológico de Ia fusariosis vascular de melón según reivindicación anterior caracterizado porque dicho microorganismo antagonista (i) es una cepa de T. harzianum aislada de turbas, compost verdes o suelos.3. Solid product effective for the biological control of melon vascular fusariosis according to the preceding claim, characterized in that said antagonistic microorganism (i) is a strain of T. harzianum isolated from mobs, green compost or soils.
4. Producto sólido eficaz para el control biológico de Ia fusariosis vascular de melón según reivindicación anterior caracterizado porque como dicho microorganismo antagonista (i) se utiliza Ia cepa T-78 de T. harzianum4. Solid product effective for the biological control of melon vascular fusariosis according to previous claim characterized in that as said antagonistic microorganism (i) the strain T-78 of T. harzianum is used
N0- CECT 20714N 0 - CECT 20714
5. Producto sólido eficaz para el control biológico de Ia fusariosis vascular de melón según reivindicación anterior caracterizado porque dicha cepa de T. harzianum CECT 20714 se inocula para que resulte una concentración final de 106-107 ufe por gramo de turba.5. Solid product effective for the biological control of melon vascular fusariosis according to the preceding claim, characterized in that said strain of T. harzianum CECT 20714 is inoculated so as to result in a final concentration of 10 6 -10 7 pfu per gram of peat.
6. Producto sólido eficaz para el control biológico de Ia fusariosis vascular de melón según reivindicación 1 caracterizado porque como dicho soporte sólido (ii) para Ia inoculación del microorganismo se usa cualquiera de los comprendidos entre diversos tipos de arcillas naturales como Ia bentonita, vermiculita bien mezclada o inoculada superficialmente o el salvado de trigo.6. Solid product effective for the biological control of melon vascular fusariosis according to claim 1 characterized in that as said solid support (ii) for the inoculation of the microorganism any of those comprised between various types of natural clays such as bentonite, vermiculite is used well mixed or superficially inoculated or wheat bran.
7. Producto sólido eficaz para el control biológico de Ia fusariosis vascular de melón según reivindicación anterior caracterizado porque como dicho soporte sólido (ii) para Ia inoculación del microorganismo se utiliza bentonita.7. Solid product effective for the biological control of melon vascular fusariosis according to the preceding claim, characterized in that as said solid support (ii) for the inoculation of the microorganism, bentonite is used.
8. Producto sólido eficaz para el control biológico de Ia fusahosis vascular de melón según reivindicación 1 caracterizado porque como dichos factores adicionales (iii) para mejorar Ia capacidad biocontrol del producto se utilizan correctores de pH, para obtener un pH final en el producto próximo a 7, entre 6 y 8.8. Solid product effective for the biological control of melon vascular fusahosis according to claim 1 characterized in that as said additional factors (iii) to improve the biocontrol capacity of the product, pH correctors are used, to obtain a final pH in the product close to 7, between 6 and 8.
9. Producto sólido eficaz para el control biológico de Ia fusahosis vascular de melón según reivindicación anterior caracterizado porque como dicho factor (iii) corrector de pH se utiliza CO3Ca.9. A solid product for effective biological control of vascular fusahosis melon Ia according claim wherein said factor as (iii) pH corrector CO 3 Ca. used
10. Producto sólido eficaz para el control biológico de Ia fusariosis vascular de melón según reivindicación anterior caracterizado porque como dicho corrector de pH (iii) se utiliza CO3Ca a una concentración entre 1 y 20 gramos por 100 g de bentonita. 10. Solid product effective for the biological control of melon vascular fusariosis according to the preceding claim, characterized in that as said pH corrector (iii) CO 3 Ca is used at a concentration between 1 and 20 grams per 100 g of bentonite.
1 1. Producto sólido eficaz para el control biológico de Ia fusariosis vascular de melón según reivindicación anterior caracterizado porque como dicho corrector de pH (iii) se utiliza CO3Ca a una concentración de 5 gramos por 100 g de bentonita.1 1. Solid product effective for the biological control of melon vascular fusariosis according to the preceding claim, characterized in that as said pH corrector (iii) CO 3 Ca is used at a concentration of 5 grams per 100 g of bentonite.
12. Producto sólido eficaz para el control biológico de Ia fusariosis vascular de melón según reivindicación 1 caracterizado porque como dichos factores adicionales (iii) para mejorar Ia capacidad biocontrol del producto se utilizan inductores de Ia biosíntesis de quitinasas.12. Solid product effective for the biological control of melon vascular fusariosis according to claim 1 characterized in that as said additional factors (iii) to improve the biocontrol capacity of the product inducers of chitinase biosynthesis are used.
13. Producto sólido eficaz para el control biológico de Ia fusariosis vascular de melón según reivindicación anterior caracterizado porque como dicho factor (iii) inductor de Ia síntesis de quitinasas se utiliza quitosano.13. Solid product effective for the biological control of melon vascular fusariosis according to the preceding claim characterized in that said chitosan synthesis factor (iii) inducing chitosanase is used.
14. Producto sólido eficaz para el control biológico de Ia fusariosis vascular de melón según reivindicación anterior caracterizado porque como dicho factor inductor de síntesis de quitinasas (iii) se utiliza quitosano a una concentración entre 1 y 20 g por 100 g de bentonita. 14. Solid product effective for the biological control of melon vascular fusariosis according to the preceding claim, characterized in that as said chitinase synthesis inducing factor (iii) chitosan is used at a concentration between 1 and 20 g per 100 g of bentonite.
15. Producto sólido eficaz para el control biológico de Ia fusariosis vascular de melón según reivindicación anterior caracterizado porque como dicho factor inductor de síntesis de quitinasas (iii) se utiliza quitosano a una concentración de 5 g por 100 gramos de bentonita. 15. Solid product effective for the biological control of melon vascular fusariosis according to the preceding claim, characterized in that as said chitinase synthesis inducing factor (iii) chitosan is used at a concentration of 5 g per 100 grams of bentonite.
16. Producto sólido eficaz para el control biológico de Ia fusariosis vascular de melón según cualquiera de las reivindicaciones 1 a 15 caracterizado porque sus componentes una vez mezclados, se incuban a temperaturas entre 20-30QC por un tiempo entre 1 y 10 días. 16. Solid product effective for the biological control of melon vascular fusariosis according to any of claims 1 to 15, characterized in that its components, once mixed, are incubated at temperatures between 20-30 Q C for a time between 1 and 10 days.
17. Producto sólido eficaz para el control biológico de Ia fusariosis vascular de melón según reivindicación anterior caracterizado porque dicha incubación se realiza a una temperatura entre 25-28 5C durante 5 días.17. Solid product effective for the biological control of melon vascular fusariosis according to the preceding claim, characterized in that said incubation is carried out at a temperature between 25-28 5 C for 5 days.
18. Procedimiento para Ia obtención de un producto sólido eficaz para el control biológico de Ia fusariosis vascular de melón según cualquiera de las reivindicaciones 1 a 17 caracterizado porque comprende Ia preparación de un soporte sólido como bentonita que contiene 5 g de CO3Ca y 5g de quitosano/ 100 g de bentonita, mezclado y homogeneizado en seco, inoculado con 1 mi de un cultivo biológicamente puro de T. harzianum T-78 CECT 20714 a una concentración en torno a 109 ufc/ml, todo ello mezclado y preincubado durante 5 días a 25-28 5C.18. Procedure for obtaining an effective solid product for the biological control of melon vascular fusariosis according to any of claims 1 to 17 characterized in that it comprises the preparation of a solid support such as bentonite containing 5 g of CO 3 Ca and 5g of chitosan / 100 g of bentonite, mixed and dry homogenized, inoculated with 1 ml of a biologically pure culture of T. harzianum T-78 CECT 20714 at a concentration around 10 9 cfu / ml, all mixed and pre-incubated during 5 days at 25-28 5 C.
19. Método de aplicación de un producto sólido eficaz para el control biológico de Ia fusariosis vascular según reivindicación 18, caracterizado porque se mezcla con Ia turba al 10% mediante el uso de una mezcladora estándar de las utilizadas en los semilleros comerciales.19. Method of applying an effective solid product for the biological control of vascular fusariosis according to claim 18, characterized in that it is mixed with 10% peat by using a standard mixer used in commercial seedbeds.
20. Procedimiento para inducir resistencia sistémica a enfermedades causadas por organismos fitopatógenos que comprende aplicar una cantidad eficaz del producto obtenido según cualquiera de las reivindicaciones 1 a 17 o por el procedimiento según reivindicación 18 sobre el suelo o Ia planta a Ia que se desea inducir resistencia sistémica a enfermedades.20. Method for inducing systemic resistance to diseases caused by phytopathogenic organisms comprising applying an effective amount of the product obtained according to any of claims 1 to 17 or by the method according to claim 18 on the soil or plant to which resistance is desired to be induced. Systemic to diseases.
21. Procedimiento para estimular el crecimiento de plantas que comprende aplicar una cantidad eficaz del producto obtenido según cualquiera de las reivindicaciones 1 a 17 o por el procedimiento según reivindicación 18 sobre suelo o planta cuyo crecimiento se desea estimular.21. Method for stimulating the growth of plants comprising applying an effective amount of the product obtained according to any of claims 1 to 17 or by the method according to claim 18 on soil or plant whose growth is to be stimulated.
22. Utilización de un producto sólido eficaz para el control de Ia fusariosis vascular de melón según cualquiera de las reivindicaciones 1 a 17 o por el procedimiento según reivindicación 18 para el control biológico de esta enfermedad en semillero.22. Use of an effective solid product for the control of melon vascular fusariosis according to any of claims 1 to 17 or by the method according to claim 18 for the biological control of this disease in seedbed.
23. Utilización de un producto sólido eficaz para el control de Ia fusariosis vascular de melón según cualquiera de las reivindicaciones 1 a 17 o por el procedimiento según reivindicación 18 para el control biológico de Ia fusariosis vascular en campo de cultivo.23. Use of an effective solid product for the control of melon vascular fusariosis according to any one of claims 1 to 17 or by the method according to claim 18 for the biological control of vascular fusariosis in the culture field.
24. Uso de Trichoderma harzianum, cepa T-78, CECT 20714. para el control biológico de Ia fusariosis vascular. 24. Use of Trichoderma harzianum, strain T-78, CECT 20714. for the biological control of vascular fusariosis.
PCT/ES2007/070198 2006-12-27 2007-12-04 Solid product effective for biocontrol of vascular fusariosis of melon, method for preparation thereof and method for use of same WO2008077982A1 (en)

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ES2360318B2 (en) * 2009-05-08 2012-04-24 Microgaia Biotech, Sl PROCEDURE FOR THE PRODUCTION OF AN ORGANIC SUBSTRATE OF FUNCTIONAL CULTURE, INOCULATED, SUITABLE FOR THE DEVELOPMENT OF SEEDED NUTS, COLES AT THE SEED LEVEL, WITH BIOPESTICIDE, BIO-STIMULATING AND / OR BIOFERTILIZING CAPACITY.
FR3084085B1 (en) * 2018-07-19 2022-12-23 Univ Paris Sud METHOD OF BIOCONTROL OF FUSAARIO wilt in cereals

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