BR102021021042A2 - BIOACTIVE COMPOSITION OF HERMETIA ILLUCENS LARVA EXTRACT WITH APPLICATION FOR THE CONTROL OF COCOA PHYTOPATHOGENS CERATOCYSTIS CACAOFUNESTA, MONILIOPHTHORA PERNICIOSA AND PHYTOPHTHORA PALMIVORE - Google Patents

Abstract

The present invention relates to an extract with fungistatic and fungicidal activity effective in controlling pathogenic agents to the cocoa crop, Ceratocystis cacaofunesta, Moniliophthora perniciosa and Phytophthora palmivora. The composition of said extract comes from larvae of the insect Hermetia illucens (Diptera: Stratiomyidae). These fungi are pathogenic to the cocoa crop in the world, causing, respectively, the diseases Mal do Facão or Ceratocystis Wilt, Witches' Broom, and Black Rot. These diseases are responsible for a decrease in cocoa production, mainly in the southern region of the state of Bahia, in Brazil, aggravated by social, economic and ecological impacts. In the search for new ways to control these pathologies, we evaluated the composition of the H. illucens larvae extract as effective, showing antifungal inhibition activity against some of the phytopathogens that most interfere with cocoa production in the world, as well as having a more sustainable, efficient and economical. The method used for extraction represents a viable option for the commercial scale manufacture of an antifungal product for the control of phytopathogens in the cocoa crop, thus enabling the registration of a product in the plant protection market for the cocoa crop.

Description

BIOACTIVE COMPOSITION OF HERMETIA ILLUCENS LARVA EXTRACT WITH APPLICATION FOR THE CONTROL OF COCOA PHYTOPATHOGENS CERATOCYSTIS CACAOFUNESTA, MONILIOPHTHORA PERNICIOSA AND PHYTOPHTHORA PALMIVORE TECHNICAL FIELD

[001] The present invention concerns the composition of the extract of larvae of the insect Hermetia illucens (Diptera: Stratiomyidae) as a product with effective fungicidal activity in the control of pathogens, Ceratocystis cacaofunesta, Moniliophthora perniciosa and Phytophthora palmivora to the cocoa crop.

STATE OF ART

[002] H. illucens larvae are of great economic importance due to their ability to degrade organic matter from various products, acting as a key point in the cycling of nutrients, in addition to competing directly with other flies for habitat and food, such as the housefly and Fannia canicularis.

[003] In addition, extracts from larvae have potential for the production of proteins, lipids and bioactive substances and, therefore, their antibiotic activity against etiological agents of human pathologies has led to the extensive study of the larvae's immune system, focusing on the search for the identification of the elements responsible for these characteristics of antibiotic action.

[004] Searching the WIPO database, using the words Hermetia illucens in ALLTEXT, it is possible to find 549 patents filed with that term and with a more detailed investigation of them, it is possible to verify that 15 of these have a list of antifungal or antibacterial compounds obtained through of larval extracts of the black soldier fly.

[005] Patent KR1020190024389 with classification A01N 63/00, A23K 10/20, A61K 35/64, A61K 8/98, A61Q 17/00, A61Q 19/00 in the IPC, presents as an invention an antimicrobial composition comprising an extract of H. illucens larva. The composition has excellent antimicrobial activity against various harmful bacteria and resistant bacteria and is useful for controlling various diseases in humans. The patent under registration KR102114996B1 “An antimicrobial composition comprising an extract of an hermetia illucens” comprises an antimicrobial composition from fly larvae extract, against various harmful and resistant bacteria and therefore can be usefully used for various diseases caused by various bacteria.

[006] The patent entitled “Antibacterial composition containing a Hermetia illucens larval extracts or fractions thereof”, with registration KR1020130111854 and classification IPC A61K 35/64 and A61P 31/04, is an antibacterial composition containing an extract of H. illucens or a fraction thereof and is provided to show antibacterial activity against Klebsiella pneumoniae, Neisseria gonorrhoeae or Shigella dysenteriae, preventing or treating pneumonia, gonorrhea or dysentery. Also in the case of treatment against bacteria, the registration WO/2020/234884 entitled “Modified black soldier fly larvae oil with modified lauric acid for treatment against biofilm formation and microorganism growth” aimed at suppressing the development of biofilm and / or growth of microorganisms (for example , from Gram-positive and/or Gram-negative bacteria, fungi and possibly viruses).

[007] Patents under registration KR1020140046193 entitled “Composition comprising extract from larvae of Hermetia illucens for controlling plant pathogenic fungi” and KR20140046193A entitled “Composition comprising the extract from the larvae of hermetia illucens for controlling pathogenic microorganisms in plants” present the application of the extract larvae against plant pathogens. The antifungal and antibacterial activity of H. illucens larvae extract was verified using the radial diffusion method and radial diffusion assay (RDA) on sweet pepper Erwinia carotovora subsp (Pseudomonas marginalis pv. Marginalis, KACC 10466), Xanthomonas campestris pv. vesicatoria (Xanthomonas campestris pv. vesicatoria, KACC 11153), the oomycete Phytophthora infestans (Pseudomonas viridiflava, KACC 10391), pseudomonas (Pseudomonas sp, (KACC 11132), the tobacco wildfire bacterium (Pseudomonas taba. Xanthomonas campestris pv. Vesicatoria ) (Xanthomonas campestris pv. vesicatoria. KACC 12965), Ralstonia solanacearum (Pseudomonas solanacearum - synonym: Ralstonia solanacearum, KACC 10701), Xanthomonas axonopodis. The activity was shown in all bacteria. The techniques used to obtain the larval extract have high costs , in addition to demanding a large amount of time and low income.

[008] Recent studies have demonstrated the existence of more than 50 molecules with effects related to immunity in various insects, such as antimicrobial peptides, which are used by these insects to combat invading pathogens. A compound was extracted and identified from H. illucens larvae that demonstrated in vitro inhibitory effects on the growth and proliferation of Staphylococcus aureus, Methicillin-resistant Staphylococcus aureus, Klebsiella pneumonia, Shigella dysenteriae and plant pathogens.

[009] Diseases such as Machete's Disease (Ceratocystis cacaofunesta Engelbrecht & T.C. Harrington), Witch's Broom (Moniliophthora perniciosa (Stahel) Aime & PhillipsMora) and Black Rot (Phytophthora De Bary) are some of the diseases, considered the main ones, that attack the cocoa plantations in Brazil.

DISCUSSION OF THE PROBLEM OF THE STATE OF THE TECHNIQUE

[010] The cocoa crop is plagued by several diseases of great economic importance. Among them there is the disease Mal do Facão, in which the most efficient and economical control that has been explored is the use of resistant genetic varieties, associated with preventive treatment.

[011] In global terms, because it occurs in all producing countries, the most important disease of them is Black Pond, also known as Phytophthora Rot. Caused by several species of pseudofungi of the genus Phytophthora, this disease was the most important disease of cacao in the southern region of Bahia until the appearance of Broom of Witches in 1989, which lowered its level of importance. The witches' broom disease is caused by the fungus Basidiomycota, Moniliophthora perniciosa, this fungus is one of the most destructive endemic diseases of cacao, causing severe damage and reducing yields and production by up to 100%.

[012] Such lesions can also form on the roots and it is likely that the contact between these structures in different plants is one of the channels for the spread of the disease. The fungus can also penetrate the plant through injuries caused by pruning practices, soil cleaning, harvesting, removal of shoots, or any other activity that may injure the cacao tree, which is the main route of propagation and dissemination of the disease, when use of contaminated tools.

[013] Among the existing strategies for the control of C. cacaofunesta and Phytophthora palmivora, chemical control is the most used, consisting of the application of fungicide, usually based on copper. However, it was observed that in soils where such fungicides are applied, the pH suffers an increase and there is a significant reduction in the levels of essential nutrients: nitrogen, phosphorus and potassium. Also, the isolated use of fungicides increases the cost of production, and is not very efficient in periods of high precipitation. As an alternative to fungicides, the biological control of brown rot has been studied through the use of microorganisms antagonistic to species of the genus Phytophthora that are promising.

[014] The final product obtained through the larvae of H. illucens, has a low cost in the extraction by methanol/ethanol, also in the fractionation procedures due to the techniques and devices used. To obtain the fractions, the time used is short compared to more sophisticated techniques and they increase the yield of the compounds obtained and the efficiency of the fractionation. These factors associated with the efficiency of the extract make it possible to create a commercial product produced on a large industrial scale that can be used through spraying in plantations.

[015] The currently used plant disease control methods, in addition to having low efficiency and causing increased production costs, can have adverse effects on the environment. In this perspective, the need for a viable and more economical alternative for combating phytopathogens is evident.

SUMMARY OF THE INVENTION

[016] The present invention will act in the areas of agronomy, agriculture and phytosanitary in cocoa crops, through the methanolic and ethanolic extracts of Hermetia illucens larvae in the control of the diseases Broom of Witches, Mal do Machete and Black Rot, caused respectively by the fungi Moniliophthora perniciosa and Ceratocystis cacaofunesta and by the pseudofungus Phytophthora palmivora. The use of larvae extract for microbial control can be associated with other methods already used in cocoa plantations, and can be applied not only in Brazil, but in all cultivation centers in the world with the incidence of the studied pathogens.

[017] The growth in the use of biological control is due to the sustainability of the use of natural/biological resources to control an organism harmful to agricultural crops. The compounds present in the extract of H. illucens larvae, in addition to preventing the evolution of Witch's Broom, Mal do Machete and Brown Pond, is cheaper and sustainable, a characteristic increasingly valued by the consumer market

[018] Due to the resistance acquired by pests and pathogens that cause plant diseases to many molecules on the market, as well as the scarcity of new molecules for phytosanitary management, in this way biological products have become an important tool to compose the practices employed integrated management of pests and diseases.

DESCRIPTION OF THE INVENTION

[019] The characterization of the object of this invention is done through figures of the results of research analyzes to obtain the compounds, in such a way that they can be fully extracted and reproduced by appropriate technique, allowing full characterization of the functionality of the claimed object .

[020] From the figures and tables of the analyzes that express the identification and tests of the compounds, the descriptive part of the report is based, through a detailed and consecutive numbering.

[021] In this case we have:

[022] Figure 01. Flowchart of methodological steps to obtain the bioactive extract of black soldier fly larvae, Hermetia illucens L. 1958 (Diptera: Stratiomyidae).

[023] Figure 02. Development of the fungus Moniliophthora perniciosa in CPD and MPG medium and resistance test to ethanolic solution at 30 days after subculture. Thus, to enhance the solubility of the extract, a composition of 15% in ethanol was used to carry out the biological tests.

[024] Figure 03. Moniliophthora perniciosa growth inhibition test against the methanolic extract (extract A) of Hermetia illucens larvae without lyophilization at 30 days after subculture, resulting in the infeasibility of the extract to be used for the following tests

[025] Figure 04. Development of the fungus Moniliophthora perniciosa in CPD culture medium, without ethanol, with 15% ethanol and methanolic extract (extract B) at 30 days after transplanting.

[026] Figure 05. Development of the fungus Moniliophthora perniciosa in CPD culture medium, with ethanol, without ethanol and with methanolic extract (extract C) (respectively) at 30 days after transplanting.

[027] Figure 06. Record of the growth of Phytophthora palmivora in the inhibition test of the extracts, 30 days after subculture in carrot-agar culture medium (CA).

[028] Figure 07. Record of Ceratocystis cacaofunesta growth in the extract inhibition test, 30 days after subculture in potato dextrose agar (PDA) culture medium.

[029] Table 01. Mass of Hermetia illucens larval extracts extracted with solvents of different polarities.

[030] Table 02. Extraction yield of methanolic extracts of Hermetia illucens larvae with and without freeze-drying.

[031] Table 03. Masses of extracts obtained from the extraction of Hermetia illucens associated with different solvents.

[032] H. illucens larvae between the fourth and sixth instars were collected in the insect breeding sector of the Bioenergia e Meio Ambiente (BIOMA) group, in a food substrate composed of chicken feed, orange remains, peel of pineapple and papaya. After collection, the larvae were transferred to the Laboratory of Biological and Semiochemical Control (LaCoBSe), at the State University of Santa Cruz.

[033] They were subjected to the process of triple washing in tap water in order to eliminate impurities. After this process, they were washed in distilled water at room temperature and sterilized by immersion in ethanol for 20 seconds and then spread on a paper towel (room temperature). At the end of these processes, the larvae were weighed, totaling 5 kg of wet mass, and frozen at a temperature of -6ºC.

[034] For the next step of the procedures, two methods were used. In the first, the larvae went to the extraction without any previous drying treatment. In the second method, the larvae were lyophilized for a period of 2 to 4 days, at a temperature of -41º C (Labconcon equipment, Freezone 4.5 model).

[035] The larvae were thawed at room temperature and the extracts were prepared by maceration, using 50 g of dried larvae in 50 mL of solvent. Solvents, used independently and separately, were n-hexane, chloroform, methanol, ethanol and distilled water. After maceration, for an average time of 10 minutes, the mixtures obtained were filtered and evaporated (IKA rotary evaporator, model RV06-ML), under vacuum, at a temperature of 40º C and rotation of 150 rpm. The products of this process were kept under freezing. 15 extracts were obtained, 3 of each solvent used.

[036] To start the microbiological tests, it was necessary to investigate the best medium for the cultivation of the fungus M. perniciosa (biotype C), whose isolates were kindly provided by Professor Sônia Melo from the Laboratory of Fungi Biology at UESC.

[037] The culture media tested were: MPG (Moniliophthora peptone glycerol), medium composed of 2% agar, 2% glycerol and 0.5% peptone; and CPD (Crinipellis peptone dextrose), composed of 2% glucose, 2% peptone and 2% agar added to solid media.

[038] It was also verified the best concentration of ethanol for total miscibility of the extract in the medium, so that there was no influence on the growth of the fungus.

[039] The growth inhibition test consisted of three experiments performed simultaneously independently and in triplicate. The first experiment was the positive control, in which only the culture medium and the fungus were tested. In the second experiment, the solvent control was tested, where the fungus was placed in the same medium plus the solvent, which solubilizes the extract, in the ideal percentage for the normal growth of the fungus. The third experiment consisted of cultivated fungus (pickled) in the culture medium, added with the larval extract solubilized in the percentage identified as ideal.

[040] For the growth of hyphae of M. perniciosa, biotype C, which attacks cacao meristems, was cultivated in sterile petri dishes at 25º C. All tests were evaluated after 21 days.

[041] For the growth of the pathogens C. cacaofunesta and P. palmivora, the culture media PDA (potato dextrose agar) and CA (carrot-agar), respectively, and the Petri dishes used received 8 mL of a mixture of medium, extract and ethanol. Of the total volume, 6.8 mL were composed of culture medium and 1.2 mL of an ethanolic solution of the extract, corresponding to a fraction of 85% and 15%, respectively. The ethanol solution of the extract (ethanol + extract) was produced with the ethanol concentration determined at 15%. Two control tests were also performed: a plate containing only 8 mL of the culture medium and a plate containing 6.8 mL of the medium and 1.2 mL of an aqueous solution of ethanol at a concentration of 15%.

[042] Each experiment was performed in triplicate. The growth zone of the pathogens was checked periodically for 30 days, with an interval of 10 days, in order to observe the inhibitory activity of the extracts.

[043] To verify the best solvent to obtain a bioactive extract, extractions were performed with solvents of different polarities. Two parameters were evaluated, namely: which allows better yield in extracted mass; and which provides the best fungal growth inhibition potential.

[044] Regarding the average amount of mass obtained by independent extractions with different solvents (Table 1), the two best results were the extracts in methanol and in water (without freeze-drying).

[045] After 30 days it was verified that the CPD medium showed the best growth than the MPG medium, and it was also observed that compositions with 10% and 15% ethanol in the medium do not kill the fungus Moniliophthora perniciosa.

[046] The extracts were subjected to a test for inhibiting the growth of M. perniciosa, and it was possible to verify that the extract in methanol inhibits the growth of the fungus more effectively and reproductively.

[047] To continue the other stages of the research, new extractions with methanol were performed, aiming to obtain an additional amount of extract, due to the initial amount being insufficient to carry out the experiments. Thus, new extractions were performed, in which it was possible to observe a significant difference in yield when lyophilization was used or not (Table 2). This procedure sought to verify whether it was possible to obtain a bioactive extract without the need for lyophilization.

[048] To verify the efficiency of the extraction and the existence of the compound of interest in the extract, it was necessary to carry out the fungus growth inhibition test. In the test using extract A, there was an internal contamination in the culture medium. In extracts B and C, the methodology used did not show contamination and it was possible to verify that both significantly inhibited the growth of the fungus M. perniciosa.

[049] Determined the efficiency of extracts B and C in inhibiting the growth of the fungus M. perniciosa, it was possible to infer that to obtain a better yield in the extraction of bioactive compounds, it was necessary to lyophilize the larvae of H. illucens before maceration.

[050] Initial tests with satisfactory results in the laboratory indicate that the methanolic extract of H. illucens larvae has the possibility of application in agriculture. These results allow us to believe in the possibility of generating a commercial product.

[051] To verify the best solvent to obtain a bioactive extract against Ceratocystis cacaofunesta and Phytophthora palmivora, extractions of H. illucens larvae were performed with the solvents: water, methanol, ethanol, chloroform and n-hexane, with three replications for each, as described. Each of the mobile phases and replications generated an extract mass, and the results are shown in Table 3.

[052] The volume of solvent used for the extraction of 50 g of larvae was 50 mL, and the largest masses of extract were produced by water, followed by ethanol, methanol and chloroform. This behavior shows a greater presence of hydrophilic compounds in the composition of the larvae. However, it is possible that the high extract mass associated with the extract with water is due to the lower efficiency of the evaporation step (since water is the solvent with the highest boiling point). Thus, part of the mass of the extract obtained by extraction with water can be attributed to high humidity. Extraction with n-hexane did not produce an appreciable volume of filtrate and, therefore, extract mass, due to the volatilization of the solvent during the development of the methodology, evidencing the difficulty of its use.

[053] The extracts obtained for each of the solvents showed a viscous appearance, and to facilitate their handling, as well as increase their miscibility in the culture media used in the inhibition tests, a dissolution in ethanol was performed. Ethanol was chosen because it is a solvent of intermediate polarity, among those studied, and the extract:ethanol ratio was determined based on the tolerance of the studied microorganisms (P. palmivora and C. cacaofunesta) to alcohol. In order to determine this proportion, an ethanol resistance test was performed.

[054] The methanolic, ethanolic, water and chloroform extracts were diluted with ethanol in the proportion of 85% extract and 15% ethanol, and added to the culture medium in the proportion of 15% diluted extract and 85% medium. Two control tests were carried out: one containing only medium, to verify the viability of the microorganism, and the other containing culture medium and 15% ethanol aqueous solution, to verify the growth zone of the pathogen when exposed to alcohol. In this sense, after periodic observation for 30 days, it was possible to determine the inhibitory activity of the extracts on C. cacaofunesta and P. palmivora.

[055] The controls performed for P. palmivora showed significant growth of this pseudofungus 30 days after subculture in carrot-agar (CA) culture medium, evidencing its viability and its ability to develop in the ethanol concentration used in the tests. Plaques with a positive result for the growth of the pathogen can be identified by the whitish color, resulting from the agglomeration of the white filaments of P. palmivora, since the color of the culture medium is carrot-colored.

[056] In this sense, the development of the microorganism was not observed in the plates containing methanol and ethanol extract, demonstrating its inhibitory effect. In the plates containing water extract, the growth of several different microorganisms was observed, making the test inconclusive for the inhibition of P. palmivora. Finally, the chloroform extract did not produce an inhibitory effect on the microorganism studied.

[057] The control treatments used in the tests for inhibition of C. cacaofunesta showed expressive growth of the fungus, evidencing its viability and its ability to develop in the ethanol concentration used in the tests. Plaques with a positive result for the growth of the pathogen could be identified by the agglomeration of reproductive and infective structures of the fungus, showing a dark color

[058] In this perspective, the growth of the microorganism was not observed in the plates containing the methanol extract and ethanol extract, demonstrating its inhibitory effect. In the plates containing water extract, the growth of several different microorganisms was observed, making the test inconclusive for the inhibition of C. cacaofunesta. Finally, the chloroform extract slowed down the development of the studied microorganism, but did not inhibit it, since the growth halo was smaller than the control.

[059] The test of inhibitory activity of the extracts showed, then, that both pathogens – Ceratocystis cacaofunesta and Phytophthora palmivora – were inhibited/eliminated by the methanol and ethanol extracts. Water and chloroform extracts did not show a conclusive inhibitory effect. This result indicates that the substance(s) that confer(s) antibiotic properties to the extracts have greater affinity with alcohols, among the solvents used. That is, it is likely to have a dipole moment intermediate between water and chloroform. Furthermore, ethanol and methanol have more polar and more non-polar portions in their molecules, making it possible to interact with a variety of low and medium molecular weight molecules.

Claims (3)

Bioactive composition of Hermetia illucens larvae extract with application to control phytopathogens of cacao Ceratocystis cacaofunesta, Moniliophthora perniciosa and Phytophthora palmivora, characterized by its composition with fungistatic and fungicidal action through extraction in methanol and ethanol. Bioactive composition of Hermetia illucens larvae extract with application to control phytopathogens of cocoa Ceratocystis cacaofunesta, Moniliophthora perniciosa and Phytophthora palmivora, according to claim 1, characterized by bioactive fractions extracted by different organic solvents. Bioactive composition of Hermetia illucens larvae extract with application to control the phytopathogens of the cacao tree Ceratocystis cacaofunesta, Moniliophthora perniciosa and Phytophthora palmivora, according to claim 1, characterized in that it is presented in liquid form, either in the form of a suspension or gel, or , in solid form because it is presented in micronized form in powder or granules.

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