ES2360318B2 - 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. - Google Patents

Abstract

Procedure for the production of a functional organic substrate, inoculated, suitable for the development of horticultural seedlings at the seedbed level, with biopesticide, biostimulant and / or biofertilizer capacity. # Developed of a functional organic substrate and its procedure for obtaining, based in a process of biostabilization of vine pruning remains, characterized in that chitin and the antagonistic microorganism Trichoderma harzianum T-78 are incorporated sequentially and differently, so that the timing and manner of incorporation of the different elements are key to achieve biopesticide, biostimulant and / or biofertilizer functionalities, in an effective, stable and persistent way, in the substrate. # Applied as a substitute for peat, in the cultivation of melon plants at the seedbed level and / or as a functional amendment in applications to the soil, it is characterized by having a high effectiveness in the biological control of fusariosis vascular melon (Fusarium oxysporum sp. Melonis).

Description

Procedure for the production of an organic substrate of functional culture, inoculated, suitable for the development of horticultural seedlings at the seedbed level, with biopesticide, biostimulant and / or biofertilizer capacity.

The present invention provides an organic substrate suitable for seedling development and effective in the biological control of melon vascular fusariosis, its manufacturing process and the methods of application thereof. It refers to a substrate capable of replacing peat with its physical-chemical parameters and with the added value of having an effective, stable and persistent suppressivity against melon vascular fusariosis, in addition to an important biostimulant and biofertilizing effect. It is characterized in that it comprises (i) an antagonistic microorganism (T. harzianum T78) of F. oxysporum with biocontrol capacity over it, (ii) chitin, as an adjuvant; and (iii) a protocol for sequential incorporation of these components, developed in the present invention, during the preparation of the substrate, which induces a synergistic effect of the whole in terms of its biopesticide, biostimulant and biofertilizer functionality.

Sector and state of the art

A substrate is, by definition, any means used to grow plants in a container. The use of substrates in plant production has been experiencing a considerable increase in economic, agronomic and even environmental reasons in recent decades.

The evolution of the techniques and means of cultivation has extended the field of applications of the substrates of important form: ornamental plant, gardening, horticulture, etc., at the same time that the increase of the demand of these produces an increasing sensitivity towards the depletion of non-renewable resources and environmental deterioration. This growing demand for organic substrates causes the use of compost to be plated as an organic substrate, although not all composts can be used as a substrate, because they must meet the minimum requirement of allowing the adequate growth of the plants on it be developed, as well as production management such as irrigation, fertilization, etc.

In Spain, a large amount of pruning waste is produced from the main woody crops. The fate of this type of waste has environmental implications that can be negative, such as burning in the farm itself after being removed from the field; or positive as is the use of physical, physical-chemical, chemical and biological characteristics for use as an organic substrate, alternative to peat, or organic amendment in the soil. For this, this type of material must undergo a composting process that consists in the stabilization of their organic matter and the consequent elimination of phytopathogenic microorganisms that may exist in the plant material used (Costa et al., 1990). In addition, the advantage of handling a plant residue from its origin to obtaining the final product by composting, can allow to increase its beneficial properties, resulting in functional and / or directed composts. In this aspect, depending on the needs of the substrate, the physical characteristics can be addressed; physicochemical, chemical and biological towards different purposes such as control of plant diseases, improve biostimulant and / or biofertilizer capacities.

Most composts have a high content of organic matter, which by natural decomposition allows its use as organic amendments with gradual fertilizing capacity, by decomposing its organic matter and releasing nutrients that can be used by plants. In addition, depending on their origin and composition, they can have a suppressive effect against diseases, although this effect is partial and erratic, being difficult to use for this purpose. Although, in recent times, and given the pressure in the handling of phytosanitary products has received special attention. In addition, composts as such or in combination with mobs have begun to be used as substrates for plant cultivation, although external applications of chemical fertilizers and pesticides are necessary.

Chitin is used by itself in the preventive control of phytopathogens (Chitomer, BCS Öko-Garantie QUIM_MERI_8244 / 01.04 / 4161-EN, Chemistry Meristem). However, the direct use of chitin, which has been used as a fungicidal agent, 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 since it can be used as source of carbon and nutrients by the pathogenic microorganism. In addition, due to the low solubility in water, the application is difficult by irrigation, so for use in the seedbed, it would force an initial mixture with the substrate, which does not have the expected results. Another approach to the management of substrates in a sustainable way is the application just before or during the cultivation of antagonistic microorganisms, in order to prevent the establishment of diseases. Within microorganisms of recognized biocontrol capacity we have different genera such as Trichoderma sp, Bacillus sp, Aspergillus sp, Pseudomonas sp. Some patents are known in which attempts have been made to biologically control different plant diseases through the incorporation of Trichoderma sp. So for example, US. 4,900,348 (1990) describes a method consisting in the production of a suppressor substrate of diseases caused by Rhizoctonia solani and Pythium ultimum that uses a mixture of Trichoderma hamatum strain 382 and Flavobacterium balustinum strain 299. US 4,642,131 (1987), describes a method in which Trichoderma hamatum is used in combination with bacteria. However, none of these methods describe the potential use in the control of melon vascular fusariosis, and more particularly in the process of germination and development of diseases at the seedbed level. On the other hand, there is a patent, ES 2188385 (2004), which consists in the addition of a suspension of conidia of Trichoderma asperellum strain T34 to substrates formulated from mixtures of composts, mobs and other materials, which is effective against Fusarium oxysporum and Rhizoctonia solani in the cultivation of cucumber and tomato respectively. However, the ability of survival and stability of the antagonist, when added to an organic matter already stabilized and with its own micro fl ora already established, is very limited. Nor does it refer to the potential biostimulant and / or biofertilizer effects of the substrate, showing no effect on the effects of its product on melon cultivation at the seedbed level. In another patent, ES200603290, the immobilization of a strain of Trichoderma harzianum T78 is proposed through the use of an inorganic material and other additional factors for the control of vascular fusariosis, being its use comparable to a specific application of a biological control fungicide Substrate preparation as in the previous patent.

Therefore, a substrate such as that proposed in the present invention, conceived from the beginning to be an ecological and nutritional niche of a specific microorganism and developed in a directional way to fulfill said purpose, without prejudice to the fulfillment of the physical-chemical parameters of a standard substrate Considered as a new product.

Description of the invention

The present invention provides a functional organic substrate suitable for seedling production, both at seed and field level, and effective in the biological control of melon vascular fusariosis caused by the pathogen F. oxysporum f. sp. Melonis Its elaboration is based on a process of biostabilization of vine pruning remains, characterized in that chitin and the antagonistic microorganism Trichoderma harzianum T-78 are incorporated sequentially and differentially, so that the timing and manner of incorporation of the different elements are key to achieve biopesticide, biostimulant and / or biofertilizer functionalities, in an efficient, stable and persistent way, in the substrate.

The plant material selected for its elaboration was the rest of the vine pruning, since it turned out to be the most suitable material for the development of the properties that are intended to be enhanced in the new substrate (suppressivity against phytopathogens, biostimulant and biofertilizing activity) due to the adequate establishment of the T. harzianum T-78 microorganism, while presenting physical, chemical, chemical and biological characteristics suitable for the development of horticultural seedlings.

The substrate object of the present invention incorporates chitin as a nutritional source for the inoculated strain, which must be applied at a suitable time during the process of biostabilization of the vine pruning remains, in order to optimize the properties that are intended to enhance in the new substrate (suppresitivity against phytopathogens, biostimulant and biofertilizing activity). In addition, the incorporation of chitin during the composting process not only allows an optimal establishment of T. harzianum T-78, and an increase in the suppressive capacity of the final substrate, but also an increase in biostimulant and biofertilizing activity as a result of partial degradation of the same by T. harzianum T-78, giving rise to metabolites that favor plant growth. In addition, if we start from the base, that in the composting process not all chitin is degraded, the presence of remnants can also fulfill the suppressive effect per se of chitin (Bhaskara Reddy et al., 2000), preventing it from being used by the phytopathogen due to the high establishment of T. harzianum.

The presence of chitin during the biostabilization process of the vine pruning remains, through composting, in addition to contributing to the nitrogen supply, reducing the C / N ratio, favoring the bio-stabilization of the organic matter of the pruning remains of grapevine, induces the activation of the chitinase activity of T. harzianum inoculated, among other microorganisms in the vine pruning residues. Chitinase activity is a key enzyme in the control of pathogenic microorganisms since it is a compound similar to that of the cell wall of some phytopathogenic microorganisms such as F. oxysporum; so that when a pathogen infection occurs, the previously generated chitinase activity would prevent their establishment.

Therefore, the present invention combines the incorporation of low-grade acetylation chitin with the isolate of

T. harzianum, applied sequentially during composting; so that the chitin is applied after the first thermophilic phase, taking advantage of the necessary turning, and the isolated of T. harzianum T-78, is incorporated at the end of the thermophilic phase of the composting process. In this way the suppressive, biostimulant and biofertilizer capacity of the generated organic substrate is optimized, with the consequent obtaining of quality crops, with lower chemical inputs (chemical fertilizers and pesticides), evolving towards an environment-friendly agriculture and the health of living beings. In addition, the components used to achieve said substrate are suitable for use in organic farming, so the proposed substrate allows to control melon vascular fusariosis in both conventional and organic farming, much more restrictive in terms of inputs. In addition, the starting material of this type of substrate: vine pruning remains, would go from being a “waste” to a “resource”, economically recoverable, allowing an adequate environmental exit, avoiding the problem of its elimination, through its indiscriminate burning

Description of the drawings

Fig. 1 is a bar graph of the weight of melon plants expressed in grams, corresponding to the germination and growth of melon seedlings at 30 days (1), at 37 days (2) and at 43 days (3 ) on different organic substrates: T: peat; T-Th: peat inoculated with T. harzianum; SI: compost inoculated with T. harzianum at the beginning of composting; SF: compost inoculated with T. harzianum at the end of composting; S: compost without inoculation; S-Th: compost without inoculation during the composting to which T. harzianum is incorporated before starting the melon cultivation. The results corresponding to treatments not subjected to pathogen pressure are shown with black bars, while the same treatments subjected to pathogen pressure are shown with gray bars. Each bar represents the average of three polystyrene trays, where each one corresponds to 45 plants.

Fig. 2 corresponds to the colony forming units of T. harzianum per gram of the different inoculation treatments of the antagonist microorganism. The treatments tested were compost inoculated with T. harzianum in which the variable factor was the non-incorporation of chitosan (A); incorporation at the beginning of composting (B); incorporation at the end of the thermophilic phase (C); and, incorporation at the end of the maturation phase. Each bar represents the average of CFUs per gram of 5 measurements of each of the different treatments.

Fig. 3., is a bar graph of the weight of melon plants expressed in grams, corresponding to the germination and growth of melon seedlings at 43 days of the following treatments: T: peat; T-Ch: peat in which chitosan is incorporated; C: compost inoculated with T. harzianum without incorporation of chitosan; C-Ch: compost without inoculating with T. harzianum in which chitosan was incorporated; and, C-Ch-Th: compost inoculated with T harzianum in which chitosan was incorporated at the beginning of composting. The results corresponding to treatments not subjected to pathogen pressure are shown with black bars, while the same treatments subjected to pathogen pressure are shown with gray bars. Each bar represents the average of three polystyrene trays, where each one corresponds to 45 plants.

Preferred Embodiment of the Invention

The advantage and differentiation of the product to be patented compared to those existing in the market, is the obtaining of an organic substrate with high suppressive capacity against pathogens, from vine pruning remains through a composting process in which it is introduced Sequential form two components: chitin and the strain of T. harzianum T78, in an adequate and optimized dose of both to achieve the expected effect on the substrate. In the process of obtaining the new substrate with suppressive capacity is key:

1) The use of chitin, both for conferring an added value in terms of reducing the C / N ratio, facilitating the stabilization of the organic matter of the vine pruning remains, such as this being a compound capable of activating the biocontrol activity of T. harzianum T78 and allow further development and implantation of this microorganism in the final compost. As well as providing the organic substrate with a biostimulant power.

2) The use of a specific strain of Trichoderma harzianum that is characterized by its biocontrol capacity against phytopathogenic microorganisms, as well as by its high survival in organic substrates such as plant residues used in the invention.

3) The sequential application of chitin and T. harzianum T78, during the composting process, this being a key aspect in obtaining the final product, since variations of this result in formulations not suitable for use as an organic substrate.

4) Obtaining a final product with a double synergistic effect regarding the suppressive and biostimulant capacity of the organic substrate, due to the application of chitin and T. harzianum T78, a factor that does not occur if either of these two components is used because separated.

The organic substrate obtained by applying the methodology proposed in this Invention may be used for different uses in vegetable crops both in soil, as in its use as a soilless substrate, and in particular as a seedbed substrate, since its use will allow double effect: on the one hand, to solve phytosanitary problems of difficult solution with chemical products, reducing not only the application of fungicides, but of fertilizers due to the suppressive qualities against phytopathogens, biofertilizers and biostimulants described for the new substrate; if not to reduce the residence time of the seedling in the seedbed, given the biostimulant and biofertilizer effect of the new substrate, allowing greater production per unit of time.

In a particular aspect the invention refers to a substrate capable of replacing peat and with the added value of having an effective suppressivity against melon vascular fusariosis, in addition to an important biostimulant and biostimulant effect characterized in that it comprises (i) an antagonistic microorganism (T. harzianum T78) of F. oxysporum with biocontrol capacity over it, (ii) chitin that improves the biocontrol capacity of the product and the development of seedlings; and (iii) the combined effect of the sequential incorporation of chitin and T. harzianum T78 in obtaining the substrate, conferring a synergistic effect of both factors regarding the biostimulant and biofertilizing effect of the resulting organic substrate.

In another particular aspect, the invention relates to an organic substrate suitable for seedling production, both at the seedbed and at the field level.

In another particular aspect the invention relates to an effective organic substrate in the biological control of melon vascular fusariosis based on the use of the isolate of T. harzianum, strain T-78, CECT 20714, as an antagonistic microorganism.

In another particular aspect, the invention relates to an effective organic in the biological control of melon vascular fusariosis which incorporates as an additional chitin factor inducing chitinase synthesis by T. harzianum T-78.

In another particular aspect, the invention relates to a method of obtaining an organic substrate suitable for seedling development and effective in the biological control of melon vascular fusariosis characterized by having developed from pruning remains that are crushed and ground. undergo a process of biostabilization (composting) by sequential incorporation of chitin and T. harzianum T-78, for a period of 5 months.

In another particular aspect the invention relates to an effective organic substrate not only in the suppression of phytopathogens, but the sequential application of chitin and T. harzianum during the process of biostabilization of vine pruning residues, gives rise to a final product with biostimulant and biofertilizer power, allowing the reduction of chemical fertilizers, accelerating the residence time of the seedlings grown on this type of substrate, which directly affects a reduction of costs in the production of seedlings in the seedbed.

Another particular aspect of the invention is the use of the substrate object of the invention for the biological control of phytopathogens in the seedbed for which there is no chemical solution of the same effectiveness as those obtained through the use of the substrate described herein.

Another particular aspect of the invention is the use of the substrate object of the invention for the biological control of vascular fusariosis in a culture field.

The following examples illustrate the invention but should not be construed as limiting it.

Example Nº1

Selection of plant material to be used for compost, more suitable for seedling development and the establishment of the Trichoderma harzianum T-78 isolate

The plant remains used were: pruning of olive, vine and pine, which were compared with Flora Balt peat. All of them were inoculated with the T-78 isolate, assessing its survival. For this, different culture media were prepared on a Petri dish using extracts from the different materials mentioned above. In addition, its survival capacity was validated by an incubation test of the T-78 isolate, in the different organic material plant residues. After a period of eight days, the survival of the isolate was evaluated by counting the CFUs of T. harzianum T-78 in a PDA medium (Potato, Dextrose, Agar) and rose bengal (50 mg / L).

The characterization of the plant remains tested is shown in Table 1, gathering the minimum characteristics to be composted, as they are high in organic matter and average nutrient levels.

TABLE 1

Physicochemical and chemical analysis of selected plant debris, compared with peat

In vitro tests, carried out with the different extracts obtained from the plant remains, showed that the T-78 isolate showed the highest growth in the culture media composed of vine and olive pruning remains, covering the total Petri plate the 3 days of the trial, while the peat and the remains of pine needed 4 and 6 days respectively. The direct inoculation of the T-78 isolate on the different organic materials revealed that the vine pruning remains were those that showed counts of the T-78 isolate significantly higher than the rest of the organic materials used.

Therefore, and according to the selection criteria described above, it was decided to use as a starting material, the remains of pruning of vine, because it was the material that presented the best development of the T-78 isolate, highlighting this over all of them , including peat, reference material for the use of this type of material, a substrate for germination and seedling growth at the seedbed level.

Example Nº2

Obtaining a new organic substrate from vine pruning residues by inoculating T. harzianum T-78 and incorporating chitin during composting

The development of this experiment had a double objective, divided into two distinct phases. In the first one, the timing of the inoculation of T. harzianum T-78 was optimized during the composting process and its response in the plant regarding its suppressive effect against F. oxysporum, as well as its potential biostimulant and / or biofertilizing effect. ; and in the second, the incidence of chitin incorporation in the composting process was evaluated, independently and inoculation of T. harzianum T-78, using the same parameters mentioned above.

Phase 1

Inoculation of T. harzianum T-78 in the composting of vine pruning waste and its use as an organic substrate for seedling growth of melon seedlings

Phase 1a

Optimization of the inoculation of T. harzianum T-78 in composting

Seven piles of vine pruning remains were prepared, which were subjected to a composting process. The inoculation of the T. harzianum T-78 isolate was performed at the beginning of the composting process after the first turning and at the end of the composting phase and beginning of the maturation phase. The so-called S batteries contained non-inoculated sarmiento and were considered as control, the so-called SI were inoculated with the T. harzianum T-78 isolate at the beginning of the composting process and the so-called SF were inoculated at the end of the composting phase (start of the maturation phase).

The piles of vine pruning remains were inoculated with 400 g of a preparation of the T. harzianum T-78 isolate, with a humidity of 45% made with a spore suspension mixture thereof, obtained following the following proportion: 1 milliliter of spore suspension obtained from the scraping of a T. harzianum T78 petri dish culture, equivalent to a spore concentration of 10 (9) CFU / mL, 10 g of bentonite, 20 g of vermiculite and 25 mL of Water.

The temperature records made throughout the composting process, showed that the piles of vine pruning remains inoculated with the T-78 isolate from the beginning of the process, showed a lower temperature than the non-inoculated batteries. On the other hand, the compost piles inoculated at the end of the thermophilic phase in turn recorded a lower temperature record than the non-inoculated ones.

The composts inoculated with the T-78 (SI) isolate presented a greater amount of organic matter at the end of the composting process, compared to the non-inoculated compost. The incorporation of the T-78 isolate at the beginning of the composting phase slowed the decomposition of organic matter during the first composting phases. Composts inoculated with the T-78 isolate at the end of the composting phase (SF), showed again how inoculation of the T-78 isolate caused a slowdown in the decomposition of organic matter. Regardless of the moment of inoculation of the T-78 isolate, the values of organic matter were not significantly different from each other, showing a higher content of organic matter than the non-inoculated compost, denoting the slowdown caused in the mineralization of the organic matter when inoculated the isolated T-78. (Table 2.).

TABLE 2

Total organic matter (calcination) during the composting process of the different sarmiento stacks. (The differences with respect to the control values were significant at P <0.05 according to the multiple test of Tukey HSD, values with the same letter are not significantly different)

(The different letters represent significant differences between the values; no brackets indicate the

statistical significance horizontally and those enclosed in parentheses refer to the

statistical significance in vertical).

The evolution in the physicochemical and nutritional parameters was not affected by the incorporation of the T-78 isolate, giving in general a final product of similar characteristics in all cases (Table 3).

Once the composting process was completed, microbiological analyzes revealed that the composts inoculated in the final phase of the process, SF, had a larger population of the T-78 isolate than those inoculated at the beginning of the process, SI (Table 4.), This shows that the population of the T-78 isolate was affected by the composting process, mainly due to the temperature.

From all this it was concluded that the establishment of the T. harzianum T-78 isolate was greater in the case of inoculation at the end of the thermophilic phase than at the beginning of the composting process, and that the introduction of the T. harzianum T- isolate 78 during composting reflected a reduction in the mineralization of compost organic matter.

TABLE 3

Physicochemical and nutritional analysis of vine pruning residue, as starting material, and composts obtained, inoculated or not with the isolate of T. harzianum T-78. (The differences with respect to the values control were significant at P <0.05 according to the multiple test of Tukey HSD, values with the same letter they are not significantly different)

TABLE 4

Evolution of the population of T. harzianum T-78 isolate throughout the composting process. (The differences with respect to the control values were significant at P <0.05 according to the multiple test of Tukey HSD, values with the same letter are not significantly different)

Phase 1b

Validation of the use of organic substrate obtained for the cultivation of melon plants in seedlings. Evaluation of its suppressive capacity against Fusarium oxysporum f. sp. melonis

The composts obtained were crushed prior to their use as a growth substrate compared to a conventional peat in commercial seed conditions. The cultivation with which it was worked was melon, the treatments being: non-inoculated vine compost (S); compost of sarmiento inoculated at the beginning of the composting process (SI), sarmiento inoculated at the end of the composting process (SF) and compost inoculated at the time of the test (S-T78).

Six polystyrene trays, used in the seedbeds in the process of producing melon seedlings, were prepared for each of the treatments, of which three were used as is and three others were inoculated with F. oxysporum, phytopathogen against which It was intended to evaluate the control capacity of the composts tested. Six trays were also prepared with the usual peat used in the seedbed, which would act as a reference or control during the experiment. The techniques used in the production of melon seedlings were those commonly used by that company, except that on this occasion there was no application of phytosanitary products.

The results obtained showed that a priori, the compost obtained fear characteristics similar to a peat (Table 5.), Despite the nutritional differences mentioned above.

TABLE 5

Physicochemical and chemical characteristics of the materials used as a substrate in the development of Melon seedlings. (The differences with respect to the control values were significant at P <0.05 according to the multiple test of Tukey HSD, values with the same letter no they are significantly different)

In general, the compost inoculated with the T-78 isolate showed a greater seedling weight than the uninoculated compost, in the different samples, these differences being accentuated in the last sampling (Fig. 1), although the weights were in the case of composts smaller than those observed with peat (Fig. 1). This lower growth can be corrected through the pre-enrichment of compost in its production, or with the addition of nutrients during the seedling production process, a fact that was not remedied in order to evaluate the suppressive capacity of the substrates obtained, without interference.

When the plants were subjected to the pressure of F. oxysporum, there was a generalized decrease in weight, registering the greatest weight reductions in the treatments that used peat and compost not inoculated with the T-78 isolate. The use of the composts inoculated with the T-78 isolate showed a greater weight of the melon seedlings grown under the pressure of F. oxysporum. The moment of inoculation of the isolate did not show differences in the weight of the melon plant in the presence of the pathogen in the first samples, although in the last sampling, being significantly higher in the composts inoculated with the T-78 isolate (SI and SF ), compared to inoculated at the time of germination (ST-78; Fig. 1).

The incidence of the pathogen calculated as the percentage of melon plant weight loss in the presence of the pathogen against the same treatment without pathogen pressure, is intended to be a graphical data in which the potential nutritional effect of the treatments is discounted, assessing the incidence of the pathogen. This parameter showed, as the uninoculated organic materials, they presented a high incidence of the pathogen reaching losses of around 50% in the last sampling, time in which the seedlings leave the seedbed for field transplantation (Table 6). Inoculation of the T-78 isolate meant a lower incidence of the pathogen over time, highlighting the lower incidence observed in melon plants grown on compost inoculated at the end of the thermophilic phase, or end of the composting phase itself, with a final incidence of 15%; followed by floods in the compost initially inoculated, with an incidence of 22%; being those treatments in which the inoculation of the isolate was carried out at the time of putting the experiment such as peat and the ST-78 compost, which showed a greater incidence of the set of substrates inoculated with the T-78 isolate (Table 6.).

At this point the importance of the inoculation of the isolate is carried out during composting compared to the inoculation just before cultivation. The causes that can be attributed to this fact is the possibility that the antagonist microorganism has to establish itself in an adequate way in the composting that does not occur if only a physical mixture of the isolate and the substrate is attended. Thus, that an adequate establishment of the antagonist microorganism, as in composting, allows a better protection response against the pathogen under culture conditions.

TABLE 6

Incidence of the pathogen in the growth of melon seedlings, expressed as the percentage of loss Melon plant weight in the presence of the pathogen against the same treatment without pathogen pressure. (The differences with respect to the control values were significant at P <0.05 according to with the multiple test of Tukey HSD, values with the same letter no they are significantly different)

(The letters without parentheses indicate the statistical significance in a horizontal direction and the positions between

parentheses refer to the statistical significance in vertical).

Therefore, the inoculation of T. harzianum T-78, in particular after the thermophilic phase, during composting revealed a lower incidence of the pathogen, compared to those treatments in which the inoculation was performed at the time of preparation of the Materials for germination and growth of melon seedlings at the seedbed level.

Phase 2

Evaluation of the incorporation of chitin during composting on the T. harzianum T-78 isolate and its effect on the suppressivity of the substrate obtained

The incorporation of chitin in the composting process had the objective of: a) promoting the nutritive and biocontrol activity of the T-78 isolate, since this is a characteristic substrate in the metabolism of Trichoderma, b) improving the biocontrol capacity of the substrate, due to to the ability per se of deacetylated chitin to inhibit the growth of phytopathogens and c) promote the nutritive capacity of the substrate, given the nutritional nature of chitin, mainly nitrogen (Table 7).

Phase 2

Optimization of the incorporation of chitin in composting

4 composting piles were prepared, from remnants of vine popo and T-78 isolate, as described in Phase 1, to which was also added deacetylated chitin (chitosan). Chitin was added in different composting processes, at the beginning and end of the thermophilic phases, at the dose of 1%; the batteries were duplicated with and without inoculation of the T-78 isolate, which, if inoculated, was introduced into the battery at the end of the composting phase itself, as just optimized. During the composting process the key parameters of humidity, temperature and aeration were monitored.

The temperature records made throughout the composting process, showed that the incorporation of chitin, regardless of the time of its incorporation caused an increase in temperature, forcing it to perform a turnaround more than in the case of its non-incorporation, independent of Inoculation or not of the isolate. This fact must be attributed to the fact that the incorporation of this compound with a high nutritional component, mainly nitrogen, promoted the mineralization of organic carbon material, due to the decrease in the C / N ratio to optimal values so that greater biodegradation occurred during The composting process.

The evolution in the physical-chemical and nutritional parameters was affected by the incorporation of chitin together with the T-78 isolate, so that at the end of the process a substrate with a higher nutrient content was obtained, mainly of a nitrogen nature (Table. 7.), in addition to a lower content in organic matter (Table 8.), Compared to those treatments in which they were not implemented with chitin. The nutritional content of the substrates was not significantly affected due to the presence or not of the T-78 isolate (Table 7.).

TABLE 7

Physicochemical and chemical characteristics of the materials used as a substrate in the development of Melon seedlings, implemented with chitin at different times in the composting process, inoculated or not with the isolated T78. (The differences with respect to the control values were Significant at P <0.05 according to the Tukey HSD multiple test, values with the same lyrics are not significantly different)

TABLE 8

Total organic matter (calcination) during the composting process of the different batteries in which chitin was incorporated at different key moments of the process, inoculated or not with the isolate of Trichoderma T-78 (The differences with respect to the control values were significant aP <0.05 according to the Tukey HSD multiple test, values with the same letter no they are significantly different)

(Letters without parentheses indicate statistical significance horizontally and those between

parentheses refer to the statistical significance in vertical)

The introduction of chitin affected the evolution of the Trichoderma populations in the compost depending on the moment of their introduction (Fig. 2.), having a positive effect when they were incorporated separately in time with respect to Trichoderma, while if jointly, the observed effect showed a negative effect on Trichoderma, which shows that the population of the T-78 isolate was affected by the incorporation of chitin, due to the increase in temperature observed as a result of the decrease in the C / N ratio to optimal biodegradation values of the organic matter of the global mass obtained (remaining pruning + chitin).

For this reason, among others, only biologically activated composts in which there was a sequential incorporation of chitin and Trichoderma harzianum, as they did not show a negative effect on the Trichoderma populations, were selected as a substrate for the suppressivity tests. the case of the joint incorporation of both components during the composting process. The compost obtained with chitin and not inoculated with Trichoderma harzianum was also used in order to evaluate the potential biostimulant and biofertilizer power of this compound without the need for inoculation of any microorganism.

Phase 2.b

Validation of the use of organic substrate obtained for the cultivation of melon plants in seedlings. Evaluation of its suppressive capacity against Fusarium oxysporum f.sp. melonis

The substrates used for the germination and growth test of melon seedlings in commercial seedlings were: the two new composts incorporating chitin, with and without inoculation of the isolate, the compost inoculated with the isolate, obtained in Example 2, compared with the conventional peat to which both chitin and Trichoderma were incorporated, with the purpose of seeing if there was any difference in the growth of the plant and in the degree of infection of the pathogen with respect to the use of compost.

The suppressivity study was also carried out using the compost that only incorporated chitin without the incorporation of the T-78 isolate, in order to validate the potential effectiveness per se of the chitosan in the potential generation of suppressive substrates, by induction of a natural microbial biomass with biopesticidal capacity.

For the conduct of the experiment, six polystyrene trays were used, used in the seedlings in the process of producing melon seedlings, for each of the treatments, of which three were used as is and three others were inoculated with F. oxysporum, phytopathogen against which it was intended to evaluate the potential fungicidal capacity. Six trays were also prepared with the usual peat used in the seedbed, which would act as a reference or control during the experiment. The techniques used in the production of melon seedlings were those commonly used by that company, except that on this occasion there was no application of phytosanitary products.

In general, the compost that incorporated chitin, presented melon plant weights significantly greater than peat and their respective treatment without the incorporation of chitin (Fig. 3.). In addition, the incorporation of the T-78 isolate, together with chitin, had a positive effect on plant performance, demonstrating a biostimulant and / or biofertilizer effect, resulting from the combination of the ability to produce metabolites with hormonal capacity, as well as a biofertilizing effect, fruit of the mineralization of chitin, releasing nutrients gradually during the growth of the melon seedling. Thus, one of the limitations in the use of vine pruning composts inoculated with Trichoderma, which is the lower growth of the seedlings, despite its high potential biocontrol effect, observed in Phase 1 of the present invention Not only could it be supplied with the incorporation of chitin selectively in the composting process, but also the acceleration of plant growth was achieved in a homogeneous way. The estimation of the residence time in the seedbed was calculated based on the premise of the optimal size of the plants for the transplant, data that was obtained from the average time observed in the growth tests of melon plants with peat. In this way, the use of this new type of substrate would reduce the residence time in the seedbed until its transplant in the field by at least 1/3, going from 40 days (average time of stay with peat) to 27 days (average time of stay with the new substrate implemented with chitin and Trichoderma).

When the plants were subjected to the pressure of F. oxysporum, it was observed that the chitin-based treatment, both in peat and in the compost, without the incorporation of Trichoderma had a negative effect on the development of the melon plants, producing an incidence of the pathogen even greater than peat and compost without the addition of chitin (Fig 3), since chitin can be a source of carbon and nutrients for the pathogenic microorganism. However, the effect that the incorporation of chitin had synergistically to the T-78 isolate, in addition to presenting a nutritive effect, giving rise to larger plants than with peat (Fig. 3.); Significantly reduced the incidence of the pathogen, calculated as the percentage of melon plant weight loss in the presence of the pathogen compared to the same treatment without pathogen pressure (Fig. 3.) Which shows that the presence of chitin and Trichoderma harzianum T-78 in compost of vine pruning remains, is able to prevent the establishment of the pathogen, Fusarium oxysporum, in the substrate and / or preventing the infection of plants, due to a direct antagonistic action against the pathogen, or indirect due to the systemic induction in the plant, which allows to avoid the incidence of the disease.

Therefore, the final result of these studies has been the obtaining of an organic substrate for plant cultivation at the seedbed level, which is not only alternative to peat, but also introduces an important added value in terms of its capacity biostimulant and / or biofertilizer, as well as in the control of a common disease in this crop: melon vascular fusariosis.

The implementation of composting by sequential incorporation of chitin and subsequent inoculation of Trichoderma harzianum T-78 at the end of the composting phase, is revealed as a suitable method for obtaining a suitable substrate for the cultivation of melon seedlings at the level of seedbed, which allows rapid, uniform, homogeneous and adequate growth of melon seedlings and that in conditions of biotic stress has the ability to prevent infection of the plant, allowing to obtain suitable plants for transplantation in the field.

Claims (21)

one.

Procedure for the elaboration of a functional organic substrate, by composting vine pruning remains, characterized by:

1st introduction of chitin, as an adjuvant, during the thermophilic phase of composting. 2nd inoculation of the Trichoderma harzianum microorganism at the end of the thermophilic composting phase.

2.

Process for preparing an organic substrate according to claim 1 characterized in that the strain of the inoculated microorganism is Trichoderma harzianum T78, CECT 20714.

3.

Process for preparing an organic substrate according to claim 1 characterized in that the inoculation of Trichoderma harzianum is carried out by means of a mixture of a spore suspension, bentonite and vermiculite.

Four.

Process for preparing an organic substrate according to claim 1 characterized in that the adjuvant is formed by organisms or parts thereof with high chitin content or from the manufacture of parts thereof.

5.

Process for preparing an organic substrate according to claim 1 characterized in that the adjuvant is chitosan.

6.

Functional organic substrate characterized by being obtained from any of the preceding claims.

7.

Use of an organic substrate made according to claim 6, for the biological control of plant diseases.

8.

Use of an organic substrate made according to claim 6, for the contribution of substances with biostimulant effect.

9. Use of an organic substrate made according to claim 6, for biological control.

10.

Use of an organic substrate made according to claim 6, alone or in combination with another type of organic substrate, for the biological control of vascular diseases in plants.

eleven.

Use of an organic substrate made according to claim 6, alone or in combination with another type of organic substrate, for the control of vascular fusariosis in arable crops.

12.

Use of an organic substrate made according to claim 6 for melon cultivation.

13.

Use of an organic substrate made according to claim 6, for the control of fusariosis in the melon.

14.

Use of an organic substrate made according to claim 6, for the control of fusariosis in melon in soilless crops.

fifteen.

Use of an organic substrate made according to claim 6, for the control of fusariosis in melon in seedlings

16. Use of an organic substrate made according to claim 6, for the cultivation of melon in soil.

17.

Use of an organic substrate made according to claim 6, for the control of fusariosis in melon in soil crops.

18.

Use of a functional organic substrate obtained according to claim 6, alone or in combination with another type of organic substrate, both in the germination, development and growth of plants in soilless culture, as well as their use in the soil, to induce resistance to diseases caused by phytopathogenic organisms.

19.

Use of a functional organic substrate obtained according to claim 6, alone or in combination with another type of organic substrate, both in the germination, development and growth of plants in soilless culture, as well as their use in the soil, for Stimulate plant growth.

twenty.

Use of a method according to claim 1, for obtaining a product with fungicidal, fertilizing or plant growth stimulating capacity.

SPANISH OFFICE OF THE PATENTS AND BRAND Application no .: 200901209 SPAIN Date of submission of the application: 08.05.2009 Priority Date: REPORT ON THE STATE OF THE TECHNIQUE 51 Int. Cl.: See Additional Sheet RELEVANT DOCUMENTS

Category

Documents cited Claims Affected

TO

US 4900348 A (HOITINK HARRY A) 13.02.1990, column 1, lines 36-46; column 4, lines 35-55; column 6, lines 37-46; column 7, lines 40-65. 1-20

TO

BERNAL, A. et al. Biological treatment of melon fusariosis with inoculated compost. Farming. Agricultural Magazine. 2005. Vol. 74, No. 872, pages 188-191. ISSN 0002-1334. 1-20

TO

EN 2311389 A1 (RESEARCH SUPERIOR COUNCIL) 01.02.2009, page 3. 1-20

TO

EN 2188385 A1 (UNIV BARCELONA) 16.06.2003, columns 1-3. 1-20

TO

WO 2007 110686 A2 (COUNCIL SCIENT IND RES et al.) 04.10.2007, page 5, lines 5-10; page 9, lines 1-15; page 13, lines 4-8. 1-7

TO

EP 0485229 A1 (SHELL INT RESEARCH) 13.05.1992, page 6, lines 15-27. 1-10

Category of the documents cited X: of particular relevance Y: of particular relevance combined with other / s of the same category A: reflects the state of the art O: refers to unwritten disclosure P: published between the priority date and the date of priority submission of the application E: previous document, but published after the date of submission of the application

This report has been prepared • for all claims • for claims no:

Date of realization of the report 05.20.2011

Examiner E. Ulloa Calvo Page 1/4

REPORT OF THE STATE OF THE TECHNIQUE Application number: 200901209 CLASSIFICATION OBJECT OF THE APPLICATION A01N63 / 04 (2006.01) C05F17 / 00 (2006.01) C05F11 / 08 (2006.01) C12N1 / 14 (2006.01) C12R1 / 885 (2006.01) Minimum documentation sought (classification system followed by classification symbols) A01N, C05F, C12N, C12R Electronic databases consulted during the search (name of the database and, if possible, search terms used) INVENES, EPODOC, WPI, BIOSIS, XPESP, MEDLINE, NPL, EMBASE, COMPDX, INSPEC State of the Art Report Page 2/4  WRITTEN OPINION Application number: 200901209 Date of Completion of Written Opinion: 05.20.2011 Statement

Novelty (Art. 6.1 LP 11/1986)

Claims Claims 1-20 IF NOT

Inventive activity (Art. 8.1 LP11 / 1986)

Claims Claims 1-20 IF NOT

The application is considered to comply with the industrial application requirement. This requirement was evaluated during the formal and technical examination phase of the application (Article 31.2 Law 11/1986).  Opinion Base.- This opinion has been made on the basis of the patent application as published. State of the Art Report Page 3/4  WRITTEN OPINION Application number: 200901209 1. Documents considered.- The documents belonging to the state of the art taken into consideration for the realization of this opinion are listed below.

Document

Publication or Identification Number publication date

D01

US 4900348 A (HOITINK HARRY A) 13.02.1990

D02

BERNAL, A. et al. Farming. Agricultural Magazine. 2005. Vol. 74, No. 872, pages 188-191. 2005

D03

EN 2311389 A1 (RESEARCH SUPERIOR COUNCIL) 01.02.2009

D04

ES 2188385 A1 (UNIV BARCELONA) 06/16/2003

D05

WO 2007 110686 A2 (COUNCIL SCIENT IND RES et al.) 04.10.2007

D06

EP 0485229 A1 (SHELL INT RESEARCH) 13.05.1992

2. Statement motivated according to articles 29.6 and 29.7 of the Regulations for the execution of Law 11/1986, of March 20, on Patents on novelty and inventive activity; quotes and explanations in support of this statement  NEW AND INVENTIVE ACTIVITY (Art. 6.1 and 8.1 L.P.) The technical object of the application consists of a method of developing a functional substrate by composting vine pruning remains together with chitin and T. harzianum, highlighting the importance of the timing of application of chitin as well as T. harzianum during the composting In this way an optimal substrate for biological control is obtained. Document D01, very close to the application, describes a method of making a functional substrate by composting woody debris with Trichoderma (T. harzianum in some cases), highlighting the importance of applying the microorganism just after the thermophilic peak, at the beginning of the mesophilic phase. It talks about its use for biological control of different pests according to the species used, for example against Fusarium oxysporum. Although, this document does not use chitin at any time during composting, nor part of vine pruning remains as a starting material, aspects that give advantages to the application with respect to D01. Document D02 anticipates the biological treatment of melon fusariosis with compost inoculated with T. harzianum. The inoculation is performed on solid support. In addition, it reflects the need to increase the activity of Trichoderma in the compost for adequate biological control, incorporating chitinase, glucosidase or lipase-like enzymes. Document D03 describes an effective solid product for the biological control of melon vascular fusariosis as well as its method of obtaining. It consists of inoculating T. harzianum T78 CECT 20714 on a solid matrix that carries bentonite, vermiculite and chitosan, and then mixing it with peat. Document D04 anticipates the use of compost from different sources inoculated with Trichoderma for the biological control of Fusarium oxysporum. Document D05 narrates a compound useful for the biological control of pests and that stimulates the growth of plants formed by one or several strains of T. harzianum on a support (compost, for example). It also refers to the use of chitin to increase the population of Trichoderma in the soil. Document D06 refers to a composition based on a mycorrhizal fungus as a plant growth enhancer and, optionally, an antagonistic fungus (T. harzianum) for the biological control of pests and chitin, which is used by the fungus to Use it as a nutrient. The documents closest to the state of the art reflect different aspects of the application, although none of them, taken alone or in combination, make a composting of vine pruning remains by introducing chitin during the thermophilic phase and T. harzianum at the end of the same. The introduction of chitin at that particular time entails an advantage over the non-introduction of it, or the introduction at another time, not only for its suppressor value itself, nor for its contribution of nitrogen to the C / N balance of the compost , but also, and as the application shows in his memory, for favoring the growth of T. harzianum in the compost. The use of vine pruning remains as the most optimal starting material for composting with T. harzianum is also highlighted in the application, an aspect that is not reflected in any previous document. Thus, and in view of the state of the known art, claims 1-20 meet the requirement of novelty and inventive activity. State of the Art Report Page 4/4