BR102014031776A2 - antifungal composition, process for obtaining it and process for fungal control in plants - Google Patents

Abstract

antifungal composition, process for obtaining it and process for fungal control in plants. The present invention provides an improved fungal control means, notably fungi associated with aflatoxin production and / or fungi associated with aflatoxin production in plants, having as a common inventive concept the use of the selected bacterial strain streptomyces sp 80 and combinations thereof. of the same. The present invention also provides a composition comprising said microorganisms in an agriculturally and / or pharmaceutically acceptable carrier. Also disclosed are a process for obtaining said composition and a process for controlling fungi in plants.

Description

Field Description of the Invention Antifungal Composition, Process for Obtaining it and Process for Control of Fungi in Plants Field of the Invention The present invention is in the fields of Microbiology and Agriculture. More specifically, the present invention provides a composition with antifungal activity, a process for obtaining it and a process for controlling fungi in plants.

Background of the Invention The search for fungistatic or fungicidal agents that provide control of plant pathogenic fungi is a current challenge. Specifically, fungicidal agents for controlling aflatoxin levels in plants that have a microorganism as an active component are not common in today's market. Only two approved and commercially available products (Mycostop® and Actinovate®) have as active component for biocontrol a microorganism of the species Streptomyces spp. However, none of these products is recommended for the control of Aspergillus spp., Known genus of aflatoxin-producing fungi. Thus, an important technical problem related to the development of new fungicides in which the active ingredient is a microorganism, such as new strains of actinomycetes, remains unresolved.

It is also known that current fungicidal compositions require low temperature conditions for storage to ensure product stability. Thus, another current technical challenge is also the development of fungicidal formulations that have long stability at room temperature, especially for tropical countries.

Aflatoxins are chemical compounds belonging to the group of mycotoxins (fungal toxins) and are considered the group of mycotoxins of greatest concern from a global health perspective (STREIT et al, 2012). More than 20 types of aflatoxins have been described, the most important being AFB1, AFB2, AFG1, AFG2 and the designations B and G come from their fluorescence color (blue and green respectively) under ultraviolet light and their indices refer to their mobility in a chromatographic plate (ABDIN et al, 2010). Aflatoxin B1 (AFB1) is the most abundant and often referred to as the most potent naturally occurring carcinogen, classified in Group 1 human by the International Cancer Research Agency (IARC) (STREIT et al, 2012). The same authors also point out that the results of high doses of aflatoxin can range from acute aflatoxicosis, causing severe gastrointestinal symptoms and even death, and exposure to aflatoxin can also be associated with rickets in children and immunosuppression. Today, more than five billion people worldwide, or approximately 80% of the world's population, are at risk for developing chronic diseases from exposure to aflatoxins in food. (STREIT et al, 2012) [0005] Aflatoxins have dihydrofuran or tetrahydrofuran substituents fused to a coumarin ring (CALONI; CORTINOVIS, 2011), where group B has a cyclopentanone ring and group G has an unsaturated lactone, as noted in structures I to IV below: The structures indicated above are: aflatoxin Bi (I), aflatoxin B2 (II), aflatoxin G1 (III) and aflatoxin G2 (IV) (HUSSEIN; BRASEL, 2001).

Fungi are responsible for the presence of aflatoxins in the environment. Production is seen in different species and morphological types that differ in competitive capacity, virulence and aflatoxin production (PROBST; COTTY, 2012). Among the fungi of the genus Aspergillus, A. flavus and A. parasiticus species are the most representative among aflatoxin producers, but other species such as A. ochraceoroseus, A. toxicarius, A. arachidicola and A. minisclerotigenes have also been mentioned ( ABDIN et al, 2010).

[0008] Food contamination by aflatoxins is a worldwide food safety issue and various strategies such as chemical, physical and biological control methods are used to manage and investigate aflatoxins in foods (CHOUDHARY; KUMARI, 2010). Among them, biological control seems to be the most promising approach for aflatoxin control. Several bacterial genera, such as Bacillus, Pseudomonas, Ralstonia and Burkholderia spp., Have been shown to inhibit Aspergillus flavus growth under laboratory conditions (REDDY et al, 2010). The same authors also mention that yeast strains were able to significantly inhibit Aspergillus toxin growth and production under laboratory conditions. In most cases, although strains are highly effective in inhibiting fungal and toxin growth under laboratory conditions, there are no good results in the field. One explanation for this is that it is difficult to bring bacterial cells to Aspergillus infection sites (REDDY et al, 2010) because they need a suitable vehicle or formulation.

[0009] The search in patent literature has pointed to some partially relevant documents, described below.

The document by GOMES et al. (Chitinolytic activity of actinomycetes from a cerrado soil and their potential in biocontrol. Letters in Applied Microbiology, 2000, Vol. 30, pp. 146-150) reveals that crude extracts of Streptomyces-specific strains showed inhibitory activity against pathogenic fungi. The present invention differs from said document, among other technical reasons, in that it discloses Streptomyces genus-specific bacterial strains which exhibit inhibitory activity against aflatoxin-producing fungi.

The document by ZUCCHI et al. (Streptomyces sp. ASBV-1 decreases aflatoxin accumulation by Aspergillus parasiticus in peanut grains. J Appl Microbiol., 2008 Dec; v. 105, ed. 6, pp. 2153-60) discloses a specific strain of the genus Streptomyces (Streptomyces sp. ASBV-1) which inhibited the spore viability of aflatoxin-producing fungus A. parasiticus in plants. The present invention differs from said document, among other technical reasons, in that it deals with new bacterial strains of the genus Streptomyces, distinct from that disclosed in the document of ZUCCHI et al., And which showed antifungal activity against aflatoxin-producing fungi.

US 5,403,584 discloses a microorganism of the genus Streptomyces, compositions comprising the microorganism and fungal control method with said compositions. The present invention differs from said document, among other technical reasons, in that the microorganism of the genus Streptomyces described is of a distinct bacterial strain (Streptomyces WYEC 108) from those disclosed in the present invention; furthermore, the inhibition of aflatoxin-producing fungi by said strain is not disclosed in said document.

The document by FRÓES et al. (Selection of Streptomyces Strain Able to Produce Cell Wall Degrading Enzymes and Active against Sclerotinia sclerotiorum. The Journal of Microbiology (2012) Vol. 50, No. 5, pp. 798-806) indicates that one of the streptomyces strains of the bacterium presented antifungal activity, inhibiting the growth of the mycelium form of the fungus Sclerotinia sclerotiorum and the germination of sclerotia. The present invention differs from said document, among other technical reasons in that it discloses the antifungal activity of Streptomyces genus-specific bacterial strains on aflatoxin-producing fungi present in plants and are also different from those disclosed in this invention.

From the literature it appears, no documents were found anticipating or suggesting the teachings of the present invention, so that the solution proposed here, in the eyes of the inventors, has novelty and inventive activity compared to the state of the art.

Thus, in the state of the art there is great difficulty in isolating, characterizing, obtaining and formulating microorganisms with antifungal activity, particularly pathogenic fungi that produce aflatoxins and / or aflatoxins in plants. Also a problem faced in the prior art is the stability and viability of antifungal compositions wherein the active ingredient is an actinomycete microorganism.

SUMMARY OF THE INVENTION The present invention provides in one of its embodiments an improved fungal control means, notably fungi associated with aflatoxin production and / or fungi associated with aflatoxin production in plants, having as common inventive concept the use of microorganism selected from the bacterial strain Streptomyces sp 80.

It is an object of the present invention a composition comprising: an agriculturally and / or pharmaceutically acceptable carrier; and the bacterial strain Streptomyces sp 80.

It is a further object of the present invention a process for preparing an antifungal composition comprising the steps of: a. preparing an agriculturally and / or pharmaceutically acceptable carrier; and b. adding to said carrier the bacterial strain Streptomyces sp 80.

It is also an object of the present invention a plant fungal control process comprising applying an antifungal composition to plants.

[0020] These and other objects of the invention will be immediately appreciated by those skilled in the art and companies having an interest in the segment, and will be described in sufficient detail for their reproduction in the following description.

Brief Description of the Figures Figure 1 shows the graph of the analysis of inoculum stability during storage at room temperature, between 22 and 28 ° C.

[0022] Figure 2 shows a graph of the effect of inoculum on soil fungal count after seven days at 25 ° C, as follows: 1.Control - soil fungal count; 2. Soil inoculated with A. parasiticus (number 10 times higher than the control); 3. Soil inoculated with A. parasiticus (number 100 times higher than the control); 4. Soil inoculated with A. parasiticus (number 1000 times higher than the control); 5. Soil inoculated with A parasiticus (number 1000 times and with the formulation). N = 5. Thin bars represent the maximum and minimum values observed in each test and the gray bars represent the highest and lowest average of each test.

Figure 3 shows the graph with data after in vivo experiment. Average peanut weight, shoot and root dry weight per pot. N = 21 and error bar is the standard deviation. Letters a, b and c show that the means were not different between treatments.

Figure 4 shows the graph with data after in vivo experiment. Average number of pods and flowers per pot.

Figure 5 represents the growth of A. parasiticus in ADM medium after three days of cultivation (A) and the growth of A. parasiticus in BDA medium after 3 days of cultivation (control) (B).

[0026] Figure 6 shows the aflatoxin standard mix chromatogram in the 20μΙ, 30μΙ, 40μΙ and 50μ volumes volumes (top to bottom are: B1, B2. G1 and G2, respectively) and the respective bands. aflatoxins from the agar plug.

Figure 7 depicts the antagonism test (in triplicate) with Streptomyces sp. and A. parasiticus, wherein: A) Strain M7A, B) Strain 70, C) Strain 52, D) Strain 218, E) Strain Q11, F) Strain 80 and G) Control (A. parasiticus fungus).

Figure 8 depicts A. parasiticus antagonism test and inoculum.

Figure 9 represents the inoculum containing Streptomyces sp. 80 (A) and CFU / g inoculum count (10 ~ 7 dilution) (B). -2 [0030] Figure 10 represents the dilution 10 count of A. parasiticus in Dichloran Rose walking stick medium, where: (A) fungi isolated from the original soil; B) Fungi isolated from artificially infested soil 10x the original soil count; C) Fungi isolated from artificially infested soil at 100x the original soil count; D) Fungi isolated from artificially infested soil 1000x the original soil count; E) Fungi isolated from original soil added with 1g of inoculum; F) control.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides in one of its embodiments an improved fungal control means, notably fungi associated with aflatoxin production and / or fungi associated with aflatoxin production in plants, having as common inventive concept the use of of the bacterial strain Streptomyces sp 80.

The composition of the present invention comprises: an agriculturally and / or pharmaceutically acceptable carrier; and the bacterial strain Streptomyces sp 80.

In one embodiment, the composition of the invention provides extended stability and viability at room temperature for long periods, which composition has at least one agriculturally and / or pharmaceutically acceptable carrier of the composition and which comprises emulsion and / or emulsion forming vehicles. suspensions, lyophilizing agents, surfactants, diluting agents, spreading agents and dispersing agents. Diluting agents include, for example, distilled water, sterile water, reverse osmosis treated water, with or without nutrients (carbon source, for example) for the maintenance of strains of microorganisms.

In one embodiment, the agriculturally and / or pharmaceutically acceptable carrier of the composition comprises carriers for forming emulsions and / or suspensions, lyophilizing agents, surfactants, diluting agents, spreading agents and dispersing agents.

In one embodiment, the agriculturally and / or pharmaceutically acceptable carrier of the composition comprises: polysaccharides (such as, for example, amylose and amylopectin), water, amino acids (such as, for example, lysine, threonine, methionine and tryptophan, among others), vegetable oil and / or combinations thereof.

In one embodiment, the agriculturally and / or pharmaceutically acceptable carrier of the composition comprises the combination of: amylose, amylopectin, water, lysine, threonine, methionine, tryptophan and vegetable oil.

In one embodiment, the agriculturally and / or pharmaceutically acceptable carrier of the composition comprises the combination of: amylose (7g to 21g), amylopectin (18g to 48g), water (9g), lysine (1.2g to 3.6g ), threonine (1.2g to 3.6g), methionine (1.2g to 3.6g), tryptophan (up to 1.8g) and vegetable oil.

In one embodiment, the agriculturally and / or pharmaceutically acceptable carrier of the composition is in the form of pellets.

The process of preparing antifungal composition comprises the steps of: preparing an agriculturally and / or pharmaceutically acceptable carrier; and adding to said carrier at least one microorganism selected from the Streptomyces sp 80 bacterial strain.

In one embodiment, the process further comprises: pre-cultivating, in culture medium and controlled conditions, at least one bacterial strain Streptomyces sp 80, followed by adding saline to said medium containing the medium (s). cultivated organism (s); and optionally transferring the culture to a preservative-containing medium such as glycerol. The plant fungal control process comprises the steps of applying the antifungal composition to plants.

In one embodiment, the process comprises applying the composition to plant seeds. The process of the invention provides control of aflatoxin contamination.

Definition of some of the terms used in this patent application Amino acids The term in this patent is to be understood as any amino acid such as aspartic acid (Asp), gutamic acid ( Glu), alanine (Ala), arginine (Arg), asparagine (Asn), cysteine (Cys), phenylalanine (Phe), glycine (Gly), glutamine (Gin), histidine (His), isoleucine (lie), leucine ( Leu), Lysine (Lys), Methionine (Met), Pyrrolysin (Pyl), Proline (Pro), Serine (Ser), Selenocysteine (Sec), Tyrosine (Tyr), Threonine (Thr), Tryptophan (Trp) and Valine ( Go).

[0046] Vegetable Oil [0047] The term in this application is to be understood as any oil of vegetable origin. Non-limiting examples of vegetable oil include soybean oil, canola oil, sunflower oil, castor oil, cottonseed oil, among other vegetable oils which may be used as an agriculturally acceptable vehicle and / or vehicle component. pharmaceutically acceptable.

Polysaccharides The term in this application is to be understood as any type of polysaccharide which may be employed in the formulation of agriculturally and / or pharmaceutically acceptable excipients. Non-limiting examples of polysaccharides include amylose and amylopectin.

Bacterial strain Streptomvces sp 80 or Streptomvces sp. STR80 [0051] The term in this patent application is to be understood as the microorganism (biological material) that is duly deposited in the Brazilian Collection of Microorganisms of Environment and Industry - CBMAI, under the code of collection CBMAI number 1612.

Example 1. Preparation of Composition 1. Materials and methods employed in this embodiment 1.1a. Strain selection as active agent by dual culture antagonism tests Six actinomycete strains were used: Streptomyces sp 80 and Streptomyces sp Q11 and M7A isolated from soil under savannah vegetation in the Central Highlands (GOMES et al. Streptomyces sp 218 isolated from Atlantic Forest forest soil (SEMEDO, 2001), and Streptomyces sp 52 previously isolated from sisal plantation soil in Bahia, Streptomyces sp 70 isolated from Atlantic Forest forest soil (Souza et al, 2006).

First each was inoculated at 2 cm from the edge of a Petri dish in PDA medium and incubated at 28 ° C for three days. Subsequently, an agar plug was added with the fungus grown for seven days. After five days of incubation at 28 ° C, inhibition was calculated by subtracting the distance (mm) of fungus growth (γ0) from Streptomyces strain growth (γ), Δ y = γο - γ, where Δ γ < 5 mm = 0; Δ γ> 5 mm = +; Δ γ> 10 mm = + + and Δ γ> 20 mm = + + + (El-Tarabily et al, 2000). The test was conducted in triplicate (FRÓES et al, 2012).

The strain was selected according to the size of the fungal growth inhibition halo in PDA-containing plate, ie the Streptomyces strain which had the largest inhibition halo after seven days of incubation at 28 ° C was selected. for later steps. 1.1b. Inoculum development and antagonism test using inoculum Strain 80 was inoculated into a petri dish containing YMA medium and incubated at 28 ° C and after five days 0.85% saline was added. With the aid of a loop, the biomass was transferred to cryotube containing 80% glycerol and stored at 4 ° C. A compound containing 99% inert ingredients (starch, water and salts) and 1% active ingredient was added from the bacterial biomass and after seven days of incubation at 28 ° C in the dark, the inoculum was transferred and stored in Falcon tubes. 50 mL.

Inoculum stability was determined according to Souza (2006) as follows: every 30 days, one gram of the inoculum was added to a tube containing 9 mL of 0.85% sterile saline and serial dilutions were made, to the dilution required to obtain a plate count between 30 and 300 colonies. The results obtained were presented in CFU per gram of inoculum. The antagonism test using the inoculum was performed by placing one gram (1 g) of inoculum (6 months preparation) and added 2 cm from the edge of a Petri dish containing PDA medium. At the opposite end 2 cm from the edge an agar plug with the fungus was added and after 10 days of incubation at 28 ° C the inhibition was calculated as described in item 1.1a. of this embodiment. 1.1c. Quantitative and qualitative details of the components of the composition in this embodiment example In relation to the components of the composition, the following are the qualitative and quantitative data of the components of the antifungal composition that was prepared in this example embodiment: Table 1. Components of the Antifungal Composition of this Example Embodiment With respect to Table 1, amylose and amylopectin are polysaccharides and may be substituted for other polysaccharides which may constitute an agriculturally and / or pharmaceutically acceptable carrier. The amino acids lysine, threonine, methionine and tryptophan can also be replaced by amino acids with similar chemical / physical chemical properties, as well as vegetable oil.

Furthermore, it should be noted that quantitative information may vary depending on the formulation to be obtained. In this example embodiment, each component of the composition may vary within a quantitative range but there is also a possibility that larger quantitative variations may occur. For example, instead of 2.4 grams of amino acid for every 100 grams of composition, there should be only 1 gram of amino acid for every 100 grams of composition or 4 grams of amino acid for 100 grams of composition. That is, the amounts disclosed in Table 1 do not limit the quantitative variations of the composition.

Example 2. Fungus Control Process 1.1c. Soil fungal count before and after inoculum application An experiment was performed for soil fungus counting according to HORN et al. (1995) with modifications. The soil was sieved with a 20 mesh sieve and incubated at 80 ° C for drying for two days. Subsequently, 10g soil samples were homogenized with 30 ml of vortex distilled water for 2 minutes. Serial dilutions were made in saline-containing tubes and 0.2 mL of the first three dilutions were plated in 3% NaCl modified Dichloran Rosa Bengal medium incubated for seven days at 25 ° C. Apergillus suggestive colonies were identified directly on the plates according to HORN et al. (1995) and colonies that could not be identified were not counted. After initial count of CFU / g of soil, another four trials were assembled. Samples of 10g of soil were added from fungus spores (spore suspension with known count) at concentrations of 10x, 100x and 1000x of the initial soil fungal count. And in the fourth trial, 10g soil samples containing 1000x the initial fungal count were added with 1g of the inoculum. All tubes were incubated for ten days at 28 ° C in the dark and then serial dilutions were made to count colonies in Petri dishes. 1.1 d. Pilot Experiment and Peanut Growth Method A pilot of the greenhouse experiment was carried out so that the conditions for the experiment could be known in practice. For this purpose, two hundred seeds of the cultivar IAC Tatu-ST were placed to germinate in a conical bottom (3.5cm x 3.5cm) with a capacity of 10 g of soil each. Irrigation was drip and punctual, so each pot received water individually and at the root, and per day each pot received 200 mL of water. The soil used was produced by West Garden, located in São Paulo - SP, and whose packaging presents pest free information. After germination (five days), the seeds were placed in plastic pots no. 04 (4.8 L), kept under natural light in a greenhouse and with irrigation by the automated system. 1.1e. Experiment to evaluate the effect of inoculum on peanut and A. parasiticus growth This experiment was conducted in a greenhouse with automatic irrigation and soil from sugarcane plantation in Ribeirão Preto - SP. Initially, 150 peanut seeds cultivar IAC Tatu-ST were added to three conical-bottom (3.5cm x 3.5cm) seedlings with a capacity of 50 seeds and 10g of soil in each hole. After germination the viable seeds were removed and transferred to plastic pots. Those that emitted radicles within approximately five days were considered viable. The pots with the plants were arranged in a randomized block design. One gram of inoculum was added to the surface of each pot approximately 60 days after seedling emergence. Soil infestation with the fungus was performed with the addition of 10 mL of a spore suspension of A. parasiticus containing 108 spores / mL. Subsequently, the irrigation system was activated to facilitate infiltration of the inoculum and fungus into the soil. The experimental design was: positive control (CP) - untreated seeds - 50 seeds in 50 pots; negative control (CN) - seeds planted in fungus-infested soil - 50 seeds in 50 pots; test (T) - seeds planted in fungus-infested soil and treated with inoculum - 50 seeds in 50 pots. The planting was done in mid-July / 2012 and the harvest was done in mid-November / 2012. The cultivation conditions were the same as those used in the pilot experiment (item 5.5).

Example 3. Aflatoxin Control Process 1.1 f. Qualitative and quantitative analysis of peanut aflatoxins [0063] For the determination of aflatoxins in fresh peanuts by thin layer chromatography, the method referred to by PROBST and COTTY (2012) was used. Each 50g sample was ground in a multiprocessor, then transferred to a 500 mL Erlenmeyer flask with 270 mL methanol and 30 mL 4% aqueous potassium chloride solution. After the mixture was passed through a horizontal shaker at 160 rpm for 1 day, the mixture was filtered on qualitative filter paper, and 150 mL of 30% copper sulfate was added. Again the medium was filtered and passed to a 500 mL separatory funnel with 150 mL chloroform for the liquid-liquid partition. The (lower) chloroform phase was collected and placed in a sample concentrator with a maximum temperature of 55 ° C. The residue obtained after concentration was resuspended with the aid of a Vortex tube shaker with 200 pL chloroform and was used for identification and quantification of aflatoxins by thin layer chromatography.

The sample and the standard solutions were applied, with the aid of (§) a 10 µl microsyringe, on 60 Merck silica gel aluminum chromatograph (without fluorescent indicator, 20 x 20 cm, 02 mm thick) and placed in a glass vial containing chloroformacetone (9: 1). The standard used was Aflatoxin Standard Mix, 5x1 mL, Benzene: Acetonitrile (46300-U Sigma-Aldrich) without dilution. The chromatograms were visualized by Camag TLC Scanner 3 and evaluated using winCATS Planar Chromatography Manager. 1.1g. Statistical data analysis Statistical analyzes were performed using GraphPad InStat 3.06 software (GraphPad Software, San Diego, California, USA). For each treatment (CN, CP and T1) 21 vessels were used. The data were first submitted to the Kolmogorov-Smirnov normality test and subsequently performed the variance analysis (ANOVA) of the main faotr, inoculum. In all tests the significance level was 5%. 2.1 Results obtained in this embodiment 2.1a. Confirmation of fungal species In order to confirm the classification of isolates within the A. flavus species group, growth was performed in ADM medium as described in item 5.1. After three days of incubation in a greenhouse at 25 ° C, there was a change in the pigmentation of the middle back, which turned from yellow to orange, indicating that the isolates may belong to species group A. flavus. In Annex 1 it is possible to compare the pigmentation of culture medium without fungus (B) with triplicates of ADM medium inoculated with fungus (A).

As shown in Annex 2, it is possible through this TLC agar plug technique to check whether the fungus is aflatoxin producer, as in this case. 2.1b. Double Culture Antagonism Testing Petri dish double culture tests revealed that all strains except strain 52 were able to inhibit fungal growth after seven days of incubation, thus reaffirming the potential of this group as agents of biological control. According to Table 2, it is possible to verify that strain 52 did not present an inhibition halo, thus being considered neutral result (0). Strains M7A and 70 showed similar inhibition behavior and halos greater than 1 mm. However strains 80 and Q11 were the ones that most significantly inhibited fungal growth with inhibition halo above 2 mm. Looking at annex 3, it can be seen that there is no pigmentation in the culture media except in (a) where it is possible to see around the bacterial colony an orange color. The control shows how the fungus forms a growth mat in the petri dish in contrast to the poor growth observed in the tests (except in c).

Table 2. Streptomyces strain selection test Strains Streptomyces Antagonism M7Ã TT 70 ++ 52 0 218 ++ Q11 +++ 80 +++ 2.1c. Inoculum Viability, Shelf Life As can be seen from Annex 4, the inoculum test showed that strain 80 was viable, even preserving its ability to inhibit fungal growth even after ten months of storage. Comparing the test with the control, it is noted how fungal growth was inhibited by the inoculum. This result not only demonstrates the efficiency of the bacteria as an active inoculum agent, but also enhances the stability of the inoculum. Annex 5 shows how the inoculum was stored and preserved over ten months at room temperature (A) and it is possible to see the growth of bacteria in petri dishes after dilution of the inoculum in saline solution (B).

Inoculum stability was analyzed by counting CFU for ten months, at 30 day intervals and after storage at room temperature. It can be seen that the inoculum was viable during all storage time (Figure 1). In the first three months, the inoculum remained totally stable and showed a slight decline after four months when it again became stable until the sixth month. In June, the count declines further and stabilizes in the following month. From August to October the bacterial count shows a slight decline and at the end of ten months of storage the inoculum counts above 108 CFU / g of inoculum. 2.1 d. Peanut growth in a greenhouse - pilot experiment [0071] By conducting a pilot experiment, conditions such as time and frequency of plant irrigation, seed germination time, flowering, pod production and ripening and cycle after germination , were later known and used in the greenhouse inoculum test experiment. The planting was carried out in mid-July and harvested in mid-November, confirming the one hundred and twenty-day cycle described in the literature. Thus, it was found that irrigation performed in two periods of the day (morning and afternoon) and for five minutes (200 mL of water per day) provided adequate growth of the plants and kept the soil moisture sufficiently controlled to the point of not being. seen with the naked eye fungal proliferation. The average seed germination time was five days. After five days there was emission of leaflets and between twenty and twenty-five days there was flowering, and from flowering until the formation of the first pods were approximately seven days. 2.1 e. Qualitative analysis of aflatoxins - pilot experiment After harvesting and preparation of peanut seeds according to item 1.1 d., Thin layer chromatographies were performed to identify and quantify aflatoxins in the grains. In annex 2 it is possible to observe the four aflatoxins separated by chromatographic plate and their respective positions, in the order from top to bottom: AFB1, AFB2, AFG1, AFG2. The sample had two bands, which refer to aflatoxins AFB1 and AFG1 of the standard. 2.1f. Soil fungus count before and after inoculum application Figure 2 shows an initial count of 10 CFU / g of soil (point 1), when soil fungi were isolated from Ribeirão Preto - SP. As there is artificial soil infestation with different concentrations of a fungal spore suspension, an increase in the CFU / g soil count increases. At point 2, where there is a count of around 20 CFU / g of soil, the soil was infested with 10x the initial fungal count. At point 3, the count rises to almost 40 CFU / g of soil after artificial infestation with 100x the initial count. At point 4, the count is almost 100 CFU / g of soil, when the artificial infestation was 1000x. At point 5 (when the inoculum was added) the count decreases sharply and becomes very similar to the initial count (1), which represents an 80% decrease compared to point 4. In this test there was addition of the inoculum in the soil. artificially infested with 1000x the initial fungal count. In annex 6, the well-defined colonies suggestive of the Flavi section are noted. This result clearly shows the inoculum's ability to decrease the number of fungi capable of producing aflatoxins in soils. 2.1 g. Biological control experiment with peanut cultivar IAC Tatu-ST and inoculum The results of inoculum antagonism, inoculum stability, soil fungus count and pilot tests indicated that an in vivo biological control experiment would be viable. . Knowing the growing conditions of the peanut plant of the pilot experiment, three trials were assembled as mentioned in item 1.1e.

The average germination time of the seeds was five days and, after seventeen days, there was emission of the leaflets. The flowering period was between twenty and five to thirty days after germination and flowering until pod formation was approximately ten days.

The plants showed good growing conditions throughout the experiment, with the leaves practically intact. Sometimes the leaves showed small black spots, possibly being fungi. This fact did not compromise plant growth or seed production.

The CP plants presented as expected, that is, as a reference of the numbers and weights measured. It was possible to observe (Figure 3) that the peanut weight was similar in the positive control and inoculum plants, and was lower in the plants whose soil was infested with the fungus. The root dry weight was quite similar with a small increase in the fungal plants, while the dry weight of the shoot was virtually unchanged, with a small increase only in the positive control plants. It is also shown (Figure 4) that there was homogeneity in the number of pods per plant in the positive control and in the inoculum plants and a decrease in the number of pods in the plants receiving the fungus. Moreover, it can be seen from Figure 4 that the number of flowers per plant underwent a small increase in the added plants of the inoculum. The results show that the inoculum did not have any negative effect on plant growth and development, as well as on the growth and development of peanut grains, presenting values very similar to those of the positive control. Regarding the quantification of aflatoxins in the grains, it was not possible to obtain a conclusive result, as there were problems related to the thin layer chromatography technique, which made the analysis of the samples impossible. The GraphPad program, through the Kolmogorov-Smirnov test, showed that the data presented a normal distribution and, therefore, a parametric statistical analysis was possible in all data. Through an intergroup comparison, Student's t-test compared the differences in the variables studied between the three groups. Example 4. Composition Stability 2.1 h. Inoculum Viability and Stability The composition of the invention was evaluated for stability. The inoculum remained viable after ten months of preparation, even though it was stored at room temperature (20 ° C -35 ° C). Inocula usually have stability at low temperatures (4 or 5 ° C) as highlighted by YIN et al (2008). Thus, the inoculum produced presents better stability in relation to the products available in the market. The biofungicide T34 Biocontrol (Fargro), whose active agent is the T34 strain of the fungus Trichoderma asperellum, and used against Fusarium wilt, has a storage recommendation at 4 ° C. Rhapsody® ASO ™ QST 713 biofungicide, based on a strain of Bacillus subtilis bacteria, has a storage recommendation at room temperature, but it should be noted that the active agent is lyophilized, contributing to its stability.

Similarly, AflaGuard® is also stored at room temperature, but the durability of the stocked product is only four months. Thus, the composition of the present invention provides greater stability under ambient temperature conditions.

Those skilled in the art will enhance the knowledge presented herein and may reproduce the invention in the embodiments presented and in other embodiments within the scope of the appended claims.

Antifungal composition, Process for obtaining it and Process for fungal control in plants

Claims (12)

An antifungal composition comprising: an agriculturally or pharmaceutically acceptable carrier; and the bacterial strain Streptomyces sp 80. Composition according to Claim 1, characterized in that said microorganism is in the concentration of 0.1% to 10% by mass. Composition according to Claim 1 or 2, characterized in that the agriculturally and / or pharmaceutically acceptable carrier is a combination of: at least one polysaccharide, at least one amino acid, water and vegetable oil. Composition according to Claim 3, characterized in that the agriculturally and / or pharmaceutically acceptable carrier is the combination of: amylose, amylopectin, water, lysine, threonine, methionine, tryptophan and vegetable oil. Composition according to Claim 1, characterized in that the agriculturally and / or pharmaceutically acceptable carrier is selected from the group consisting of: emulsion and / or suspension forming vehicles, lyophilizing agents, surfactants, diluting agents, spreading agents and agents. dispersants. Composition according to Claim 5, characterized in that the agriculturally and / or pharmaceutically acceptable carrier is a diluent agent. A plant fungal control process comprising applying the antifungal composition as defined in any one of claims 1 to 6 on plants. Process according to Claim 7, characterized in that the antifungal composition is applied to plant seeds. Process according to Claim 7 or 8, characterized in that it is for controlling aflatoxin contamination. Process for preparing an antifungal composition comprising the steps of: preparing an agriculturally and / or pharmaceutically acceptable carrier; and adding to said carrier the bacterial strain Streptomyces sp 80. Process according to Claim 10, characterized in that it further comprises pre-culturing, under cultivation medium and controlled conditions, the selected micro-organism of the bacterial strain Streptomyces sp 80, followed by the addition of saline to said medium containing the ) organism (s) cultivated; and optionally transferring the culture to a medium containing a preservative. Process according to Claim 11, characterized in that said preservative is glycerol.