Patent Application
20240373857
The present invention is related to an agricultural composition with bioinsecticide action, containing one or more subspecies of the genre Bacillus thuringiensis and Brevibacillus laterosporus in association with botanical extracts, including the industrial process for obtaining the composition and the use thereof in cultures with agricultural importance with superior performance in the control of insects of the Lepidoptera order, reducing losses caused by these pests, minimizing the use of chemical agricultural pesticides and, therefore, contributing to the agricultural sustainability.
During the past years, the excessive and successive use of insecticide molecules with chemical synthesis with similar action mechanisms and further, with the advent of the genetically modified plants, particularly corn Bt resistant to armyworm such as the Spodoptera frugiperda (fall armyworm), which comprises in their genome genes of the Cry protein of the microorganism Bacillus thuringiensis, have caused undesirable selections in the populations of pest-insects with agricultural importance. The sum of these practices promotes the increase of the resistance of these insect populations to the traditional solutions (pesticides), resulting in the reduction of the effectiveness of the technologies currently used. The final consequence is the increase of the dosages and frequency of application of the chemical pesticides in the crops. In this manner, the adoption of pesticides becomes increasingly frequent and voluminous, going against a more healthy, rational, and sustainable agriculture.
For this reason, added to the concern as to the quality of the foods, studies have been intensified about new control tools and handling of pest-insects (Tavares et al., 2009). Among the alternative practices there is emphasis on the biological control that employs microorganisms, such as fungi and bacteria, whereby this is an important tool for handling the resistance and the conscious use of pesticides.
The bacteria of the genre Bacillus are well-known, particularly B. thuringiensis, which, since 1915, were characterized as having potential insecticide action against insects of the Lepidoptera, Diptera and Coleoptera orders (Habbib and Andrade, 1986; Glare and O'Callaghan, 2000). In 1938 there were developed the first products based on this approach for biological control with the commercialization of this technology in France (Daust, 1990), and, next, they were spread all over the world. The subspecies of B. thuringiensis have entomopathogenic activity, since during the sporulation phase there are produced, among other insecticide toxins, the Cry proteins, being encoded by the cry genes (Glare and O'Callaghan, 2000). The different subspecies of B. thuringiensis present, in their respective genomes, information for encoding distinct types of Cry proteins, whereby these differences are reflected in the specificity and efficiency of the action on insect control. Currently there exist 93 subspecies described (Genbank, 2021). However, the most studied subspecies are, B. thuringienesis subsp. kurstaki; B. thuringienesis subsp. aizawai; B. thuringienesis subsp. israelenses; B. thuringienesis subsp. tenebrionis; B. thuringienesis subsp. galleriae; B. thuringienesis subsp. pakistani; B. thuringienesis subsp. tochigiensis, among others.
Only in Brazil there exist more than 30 registrations with products containing only one subspecies of Bacillus thuringiensis. Since then, no other microbial genre was found that would make up this group with strong bioinsecticide action against the order of the Lepidoptera. Due to the limited variety of action mechanisms, even when referring to biological products, the efficiency in the control of the technology that employs only one controller microorganism is not sufficiently effective in the control of Lepidoptera, whereby the development of new tools is necessary, which make use of different action mechanisms in a single product.
Brevibacillus laterosporus is Gram positive bacteria, aerobic with endospore formation (resistance structures), which are characterized by the ability to produce a parasporal lamelar inclusion in the form of a canoe adjacent the spore. Several studies report the action mechanisms, demonstrating that this species is able to produce enzymes which act as fungicide, antibacterial, insecticides, and mainly used as effective prebiotics (Afrikian, 1989; Orlova et al., 1998; Ghazanchyan et al., 2018). The insecticide action of this species against Coleoptera larvae and velvetbean caterpillar Anticarsia gemmatalis (Oliveira et al., 2004) has already been described. However, these same authors did not find any effect for the fall armyworm Spodoptera frugiperda.
In addition to the selection of new microorganisms for an agricultural composition, the identification and isolation of vegetable compounds having insecticide properties can also be a promising alternative in the development of new products in handling pest insects, generating a new tool relative to the use of synthetic agrochemicals, since there are reports of the increase in resistance of pests in response to these agrochemical substances (Roush and Daly 1990; Raveau et al. 2020).
In a review, carried out by Paredes-Sánchez and collaborators (2021), there were characterized several compounds extracted from plants with insecticide action against the fall armyworm Spodoptera frugiperda, among them being: neem oil (Azadirachta indicae); extract from Atlantic forest fruit tree (Duguetia lanceolata); extract from a weed (Polygonum hydropiperoides); extract from fruit tree such as the papaya (Carica papaya), orange (Citrus sinensis), citronella lemon (C. limonia), lemon (Citrus limon); among others. Another study containing basil essential oil (Ocimum basilicum L.), which active principle the linalool (Silva et al., 2017) presented high insecticide action against Spodoptera frugiperda. Although there are several reports of botanical extracts against caterpillars, there are few reports using the action of the citronella essential oil (Cymbopogon winterianus and Cymbopogon nardus) and tea tree essential oil (Melaleuca alternifólia) with bioinsecticide function, and further, combined with microorganisms in a single product, compatible and effective for agricultural application.
Industrial production, see detailed technical description, of the biological insecticide comprised by Brevibacillus laterosporos, Bacillus thuringiensis subsp. kurstaki and Bacillus thuringiensis subsp. aizawai undergoes a complex process which guarantees the high cellular concentration during the exponential growth phase of bacteria, and, followed by the induction of the spore formation, the parasporal body and the crystals industrially induced by Brevibacillus laterosporos and Bacillus thuringiensis, respectively. This process involves specific parameters such as growth temperature, air volume, agitation, and pressure (related to the rate of oxygen dissolved in the culture medium). Thus, the industrial production with fundamental fermentative parameters for providing the species of Brevibacillus and Bacillus which guarantee the stability of the parasporal body and crystals with high cell viability, enabling the combination with citronella and/or tea tree botanical extracts in a single product for application as an agricultural bioinsecticide composition becomes an important tool for handling and biological control of pest-insects with agronomic interest.
In addition to the control, the diversity of action mechanisms, combined for the first time in an agricultural composition, involving different species of bacteria and botanical extracts will contribute strongly to the reduction of the selection of populations that are resistant to agricultural pests and, consequently, will reduce the need for frequent and voluminous application of chemical pesticides, promoting greater sustainability to the agribusiness.
The new insecticide composition is able to reduce the selection pressure, extending the effects thereof to the longevity of the genetic improvement technologies (conventional or transgenic) allowing the optimization of investments in new materials for agriculture.
The commercial products containing Bacillus thuringiensis undergo processes with energetic and economic costs such as the tangential filtering or drying with spray driers. Said production cost reduces the ability of the biotechnological industry to compete in the market of pesticides with chemical synthesis and low cost.
The current state of the art does not allow the control of insects with Bacillus thuringiensis without additional processes to the steps of fermentation and formulation. The new composition and the industrial process thereof innovate by waiving said steps, increasing competitiveness of the new solutions for sustainable agriculture.
The efficiency of the Brevibacillus sp. as insecticide for the most problematic caterpillar of the tropical agriculture (Spodoptera frugiperda) is not, according to the current state of the art, sufficient to replace pesticides and generate commercial products in industrial scale.
The mixture of vegetable extracts and insecticide microorganisms is not viable in industrial scale as per the current state of the art.
The present invention consists of an agricultural composition that employes microorganisms of the species Brevibacillus laterosporus and Bacillus thuringiensis, wherein the concentration of each microbiological active comprises the range of 1.0×107-1.0×1010 viable spores/mL, associated with botanical extracts, preferably tea tree and citronella essential oils in the range between 0.01-5.0% (v/v), able to potentialize the bioinsecticide effect against pests with agricultural interest in a technical solution that involves the industrial process and use of same. The applicability of the invention is provided, mainly, for application against pests with agricultural interest, whereby it can be used in different cultures and product concentrations, as well as different stages of the phenological stage of plants. The applications are carried out, preferably, via foliar spraying, in which manner the composition is able to reach the targets. Additionally, the applications can vary regarding dosage and frequency (number of applications).
The present invention teaches that, surprisingly, it is possible to develop a biotechnological solution (in industrial scale) containing one or more strains of Brevibacillus laterosporus and Bacillus thuringiensis, associated with botanical extracts that are able to potentialize the bioinsecticide effects against pests with agricultural importance.
Advantageously, the present invention allows obtaining an agricultural composition which can be applied to the field for the resistance of agrochemicals and transgenic cultivars with Bt technology in several cultures with agronomic interest, such as corn, soybean, cotton, among others.
As will be understood by a person skilled in the art, the present invention provides additional parameters for the method of producing an agricultural composition formed by two or more subspecies of Brevibacillus laterosporus and Bacillus thuringiensis fermented in industrial scale, demonstrating the necessary parameters for inducing the formation of endospores and insecticide toxins such as the Cry proteins (crystals) and parasporal bodies in the shape of canoe, such as parameters for pressure, temperature, oxygenation (air volume and agitation) and composition of the culture media, enabling obtaining a biotechnological product with very superior performance to that of the ones currently available.
In a first embodiment, the present invention provides a process for producing an agricultural composition comprising the steps of:
In a surprising manner, the present invention has as its preferred embodiment the potentializing of the bioinsecticide effect in cultures with agronomic interest for high levels of control and industrial viability.
In a surprising manner, the present invention has as its preferred embodiment the potentializing of the bioinsecticide effect with the mixture of microorganisms of Brevibacillus laterosporus and subspecies of Bacillus thuringiensis able to control targets that are difficult to handle, such as the fall armyworm (Spodoptera frugiperda).
For a more complete understanding of the invention, reference must be made now to the embodiments of the invention illustrated in more detail in the attached figures and described by means of the embodiments of the invention.
In a preferred embodiment, according to the present invention, the fermentation step (a)) of the species of Brevibacillus laterosporus and subspecies of Bacillus thuringiensis per batch occurs for approximately 24-168 hours.
In a preferred embodiment, the method of the present invention comprises the sequential expansion (scaling) of the culture of Brevibacillus laterosporus and subspecies of Bacillus thuringiensis for inoculation of the fermentation culture. Preferably, the sequential expansion starts at volumes of 100 L, which serves as inoculum for 1 L. This, in turn, is inoculated in 10 L, which are then inoculated in two flasks in 180 L tanks, are transferred to reactors containing 2.000 L, and which, finally, are transferred to 6.000 L reactors.
In a preferred embodiment, the species of Brevibacillus and subspecies of Bacillus thuringiensis are expanded in flasks of 100 mL by incubation in orbital agitator from 80 rpm to 200 rpm. The incubation time is preferably from 8 hours to 48 hours. Preferably, the species of Brevibacillus and subspecies of Bacillus thuringiensis are cultivated in flasks with about 1 L of culture medium by incubation in orbital agitator at 80 rpm to 200 rpm.
In a preferred embodiment, the air flow of the stainless steel flasks containing 10 L for the cultivation containing the species of Brevibacillus and subspecies of Bacillus thuringiensis is of 0.25 to 1.5 Nm3/h (=0.41-2.5 vvm), and the incubation time if preferably about 8 hours to about 48 hours.
In a preferred embodiment, the incubation temperature for multiplication of the species of Brevibacillus and subspecies of Bacillus thuringiensis according to the present invention is from 22° C. to 38° C.
In a preferred embodiment, the species of Brevibacillus and subspecies of Bacillus thuringiensis are inoculated separately in the scaling process up to 180 L and mixed in the fermentors of 2.000 L as described for the present invention. For this purpose, in a preferred embodiment, after the cultivation of the Brevibacillus and subspecies of Bacillus thuringiensis in 1 L flasks of culture medium they are inoculated in two stainless steel flasks containing 1 L of culture medium each, and after the cultivation being inoculated in two 10 L flasks of culture medium, and then transferred to tanks containing 180 L of specific culture medium for each microorganism, whereby in Table 1 the specific culture medium for the species of Brevibacillus and subspecies of Bacillus thuringiensis; incubated for 24 to 168 hours. The air flow is preferably 1.0 to 15.0 Nm3/h (=0.16-1.25 vvm).
In a preferred embodiment, the step of mixture of the species of Brevibacillus and subspecies of Bacillus thuringiensis is conducted at a temperature from 22° C. to 38° C. The air flow is preferably 1.0 Nm3/h to 2.5 Nm3/h (=0.0085-0.021 vvm). The pressure is preferably of 0.5 to 1.2 kgf/cm3. The agitation is preferably from 40 hz to 45 hz.
Preferably, the concentrations of microbiological actives comprise the range of 1.0×107-1.0×1010 viable spores/mL for each species, and the essential oils employed in the agricultural composition, tea tree and citronella, are in the range between 0.01-5.0% (v/v).
The species of Brevibacillus and subspecies of Bacillus thuringiensis are inoculated separately in flasks containing 100 mL of culture medium as described in Table 1, being incubated in orbital agitator of about 80-200 rpm, at 22-38° C. for approximately 8-48 hours. The next step in the scaling consists in the inoculation of stainless steel flasks containing 1 L of culture medium (Table 1) wherein the species are cultivated separately and incubated for approximately 8-48 hours, with air flow 0.25-1.0 Nm3/h (=4.16-16.67 vvm), at 22-38° C.
After the incubation period, the cultivations of the species of Brevibacillus and subspecies of Bacillus thuringiensis are inoculated in stainless steel flasks containing 10 L of specific culture medium for each microorganism as described in table 1 and incubated for approximately 18-96 hours, with air flow 0.25-1.5 Nm3/h (=0.41-2.5 vvm) and temperature varying from 22 to 38° C.
B. thuringiensis
B. laterosporus
After this period has lapsed, each culture containing two stainless steel flasks with 10 L of the culture medium are inoculated in a tank containing 180 L of the specific culture medium for each microorganism, and incubated for approximately 24-168 hours, with air flow 3.0-10.0 Nm3/h (=0.25-0.83 vvm) and temperature varying between 22-38° C.
After the incubation in tanks of 180 to 300 L, they are then inoculated in three fermentors of 2.000 to 6.000 L containing the specific culture medium for each microorganism. Preferably, the process of sterilization of the culture medium is carried out for approximately 60 to 120 minutes, at a temperature of approximately 121° C. to approximately 130° C. Preferably, the sterilization is carried out at a pressure of approximately 1.0-2.0 Kgf/cm2. After the sterilization and cooling period, the tank containing the Brevibacillus laterosporus, Bacillus thuringiensis subsp. kurstaki and Bacillus thuringiensis subsp. aizawai are then inoculated to the fermentor of 2.000 to 6.000 L and the incubation time is preferably from 4 to 72 hours at a temperature of 22° C.-38° C. The air flow is preferably 1.0 Nm3/h a 2.5 Nm3/h (=0.0085-0.021 vvm). The pressure is preferably 1.0 to 2.0 kgf/cm3. The agitation is preferably from 40 hz to 45 hz.
For the mixture of the species of Brevibacillus and Bacillus thuringiensis in fermentor of 6.000 to 20.000 L, preferably the sterilization process of the fermentor is carried out for approximately 60 to 120 minutes, at a temperature of approximately 121° C. to approximately 130° C. Preferably, the sterilization is carried out at a pressure of approximately 1.0-2.0 Kgf/cm2. After the period of sterilization and cooling, the tank containing Brevibacillus laterosporus, Bacillus thuringiensis subsp. kurstaki and Bacillus thuringiensis subsp. aizawai and 0.01-5.00% of tea tree essential oil or citronella essential oil are then transferred to the fermentor of 6.000 to 20.000 L and the mixture time is preferably of 120 to 180 minutes at a temperature of 22° C. -38° C. The air flow is preferably 1.0 Nm3/h to 2.5 Nm3/h (=0.0085-0.021 vvm). The pressure is preferably from 1.0 to 2.0 kgf/cm3. Agitation is preferably from 40 hz to 45 hz.
Preferably, the product is filled in gallons, package in which the product is stored.
Alternatively, the scaling process of the cultures can be used for larger volumes, however, always respecting the proportion of each microorganism.
Initially, there were carried out laboratory trials to validate the bioinsecticide effect in different concentrations of new species of Brevibacillus lateresporus against fall armyworm (Spodoptera frugiperda) inoculated in artificial diet (
The development of an agricultural composition with two subspecies of Bacillus thuringiensis and a species of Brevibacillus laterosporus was evaluated in the wheat cultivation field searching for the best dosage against the fall armyworm (
Moreover, it was possible to evaluate the potentializing effect of the botanical extract containing 0.01-5% tea tree essential oil or 0.01-5% citronella essential oil against the fall armyworm (
This application is a U.S. National Stage filing of International Patent Application No. PCT/BR2022/050407, filed Oct. 21, 2022, which is incorporated herein by reference in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/BR22/50407 | 10/21/2022 | WO |
