Use of volatile organic compounds of parasitic fungi of invertebrates as repellents of the black banana weevil ( cosmopolites sordidus)

Abstract

The present invention relates to the use of the volatile organic compounds of B. bassiana, M. anisopliae and P. chlamydosporia, selected from styrene, benzothiazole, camphor, borneol, 1,3-dimethoxybenzene, 1-octen-3-ol and 3-cyclohepten-1-one, as repellents of Cosmopolites sordidus.

Description

[0001] USE OF VOLATILE ORGANIC COMPOUNDS FROM PARASITIC FUNGI OF INVERTEBRATES AS REPELLENTS OF THE BLACK BANANA WEEVIL (COSMOPOLITES SORDIDUS)

[0002] Field of invention

[0003] The present invention falls within the general field of agrobiotechnology and, in particular, relates to the repellent action of some volatile organic compounds (VOCs) produced by three strains of two entomopathogenic fungi and by one strain of a nematophagous fungus, as repellents of the banana weevil (BW), Cosmopolites sordidus (Germar, 1824) (Coleoptera Curculionidae).

[0004] Prior art

[0005] The banana (Musa sp.) is the most consumed and cultivated fruit in the world (Ferri DV et al. 2012. Genetic variability of Beauveria bassiana and a DNA marker for environmental monitoring of a highly virulent isolate against Cosmopolites sordidus. Iridian Journal of microbiology, 52(4), 569-574). Global production is around 114 million tons per year (FAO, 2018).

[0006] Among the main organisms that affect banana crops, Cosmopolites sordidus is the one that causes the greatest damage and the greatest economic losses in all production areas; that is, it is the key pest of banana plantations. Losses can range from 30% to 90% (Carballo, V. 1998. Mortality of Cosmopolites sordidus with different formulations of Beauveria bassiana. Integrated Pest Management (CATIE) (no. 48) p. 45-48; Musabyimana T. et al. 2001. Effects of neem seed derivatives on behavioral and physiological responses of the Cosmopolites sordidus (Coleoptera: Curculionidae). Journal of economic entomology, 94(2), 449-454; Muñoz-Ruiz, C. 2007. Population fluctuation of the black weevil (Cosmopolites sordidus Germar) of the banana (Musa AAB) in San Carlos, Costa Rica. Revista Tecnología en Marcha, 20(1), 24-41).

[0007] C. sordidus is an insect of the order Coleoptera, belonging to the family Curculionidae and the subfamily Dryophthorinae. The banana weevil is a species native to the Indo-Malayan region (Simmonds NW 1966. Bananas. 2nd ed. Longmans, Green and Co. Ltd., London, pp. 512) that is currently spreading as a pest to all regions where bananas are cultivated, and in Spain, it is present, above all, in the Canary Islands (Carnero HA et al. 2002. Alternative methods for the control of the banana weevil Cosmopolites sordidus Germar, 1824 (Coleoptera: Curculionidae). ICIA activities in Banana Production, p. 75).

[0008] The invasion and spread of the banana planthopper (PNP) is due to its search behavior for food, mates, oviposition sites, and breeding grounds. Furthermore, the PNP has a limited flight capacity and is able to infect the corm, pseudostem, and suckers of banana plants. In this process, the PNP obtains resources that are absolutely essential for its growth and development, and for ensuring the biological fitness of future generations. These efficient search mechanisms of the PNP rely on its antennae, which function as specialized primary chemo- and mechanoreceptors. These precise environmental assessment mechanisms are crucial for ensuring the survival and reproduction of the PNP in its environment.

[0009] The chemoreceptors on the antennae typically detect volatile chemicals. These compounds trigger alerts in the insect about the presence of potential mates, food, suitable egg-laying sites, or dangers to avoid. Therefore, any chemical that could disrupt and / or modify the behavior of the PNP (Polymorphous Nematode) and, in general, its ability to search for a host (Musa sp.) would provide a tool for its sustainable management.

[0010] VOCs are solid or liquid compounds with a carbon base that enter the gaseous phase through vaporization at room temperature (20°C and 0.01 kPa; Pagans E. et al. 2006. Emission of volatile organic compounds from composting of different solid wastes: abatement by biofiltration. Journal of hazardous materials, 757(1-3), 179-186).

[0011] The emission of VOCs plays essential ecological and physiological roles for many organisms, such as fungi, which release a wide spectrum of VOCs (Splivallo R. et al. 2011. Truffle volatiles: from chemical ecology to aroma biosynthesis. New Phytologist, 189(3), 688-699; Kramer R. and Abraham WR 2012. Volatile sesquiterpenes from fungi: what are they good for?. Phytochemistry Reviews, 77(1), 15-37). Fungal VOC emissions belong to different chemical groups, such as monoterpenoids, sesquiterpenes, alcohols, aldehydes, aromatic compounds, esters, furans, hydrocarbons, ketones, or lactones (Campos

[0012] VP et al. 2010. Volatiles produced by interacting microorganisms potentially useful for the control of plant pathogens. Science and Agrotechnology, 34(3), 525-535; Kramer R. and Abraham

[0013] WR 2012. Volatile sesquiterpenes from fimgi: what are they good for? Phytochemistry Reviews, 11(1), 15-37).

[0014] Fungi produce various volatile organic compounds (Crespo R. et al. 2008. Volatile organic compounds released by the entomopathogenic fungi Beauveria bassiana. Microbiological research, 163(2), 148-151; Müller A. et al. 2013. Volatile profiles of fungal chemotyping of species and ecological functions. Fungal Genetics and Biology, 54, 25-33) through metabolic pathways, involved in biological processes of control or communication between microorganisms and their environment. Fungal VOCs are derived from primary and secondary metabolism (Korpi A. et al. 2009. Microbial volatile organic compounds. Critical reviews in Toxicology, 39(2), 139-193), and as they can be propagated through the atmosphere and soil, they are ideal semiochemicals (Morath SU et al. 2012. Fungal volatile organic compounds: a review with emphasis on their biotechnological potential. Fungal Biology Reviews, 26(2-3), 73-83) capable of acting as mediators in interactions between organisms.The production of fungal VOCs is biologically dynamic. The profile of a particular species or strain varies according to the substrate or nutrient, incubation time, temperature, and other environmental parameters (Nilsson A. et al. 2004. Microorganisms and volatile organic compounds in airborne dust from damp residences. Indoor Air, 14(2), 74-82; Fiedler N. et al. 2005. Health effects of a mixture of indoor air volatile organics, their ozone oxidation production, and stress. Environmental health perspectives, 113(11), 1542-1548).

[0015] Currently, there is no integrated control plan for the PNP (Polymorphonuclear Pest), only pest management using pheromone traps for counting and capturing, controlling potential outbreaks, and removing heavily infested plants—an inefficient and expensive method. In this regard, patent ES2600526 describes volatile organic compounds (VOCs) from Beauveria bassiana for use as a specific repellent against Rhynchophorus ferrugineus. The VOCs described in this patent—specifically: methylbenzene, hexamethyl-cyclotetrasiloxanes, 1,4-dimethylbenzene, ethenylbenzene, dodecamethyl-pentasiloxane, benzaldehyde, 5-methylundecane, capric aldehyde, 1,4-bis(1,1-dimethylbenzene), benzothiazole, and 1-decanol—are specific to Rhynchophorus ferrugineus and have no effect on Cosmopolites sordidus.

[0016] The significant impact of the banana weevil (Cosmopolites sordidus) on banana crops is evident, as is the increasing national and European restrictions on the use of synthetic chemical insecticides against these pests. Therefore, there is a need to develop new compounds derived from natural biological control agents to mitigate the damage caused by Cosmopolites sordidus and to eliminate the environmental damage caused by the use of insecticides against this insect.

[0017] Explanation of the invention

[0018] The present invention solves the problems described above, since it provides new VOCs with repellent activity derived from the metabolism of entomopathogenic fungi (Beauveria bassiana, Metarhizium anisopliae) and nematophagous fungi (Pochonia chlamydosporia).

[0019] Thus, in a first aspect, the present invention relates to the use of at least one volatile organic compound from an entomopathogenic and / or nematophagous fungus (hereinafter the volatile organic compound of the present invention), selected from styrene, benzothiazole, camphor, boromol, 1,3-dimethoxybenzene, l-octen-3-ol, 3-cyclohepten-l-one as a repellent of Cosmopolites sordidus.

[0020] In the present invention, "volatile organic compound" refers to a solid and / or liquid carbonaceous chemical substance that appears as an intermediate or final product of the metabolic pathways of the fungi mentioned above.

[0021] In one particular embodiment, the volatile organic compounds of the present invention belong to different chemical families and are produced at different incubation times by the different fungi used.

[0022] In one particular embodiment, the volatile organic compounds of the present invention are selected from:

[0023] Styrene (C8H8) (also referred to in the present invention as compound Cl) belongs to the group of chemical compounds classified as organovinyl compounds. Cl occurs in rice and in Bb1TS11, Pc123, and Ma4TS04.

[0024] Benzothiazole (C7H5NS) (also referred to in the present invention as compound C2), is a heterocyclic aromatic compound identified by the metabolic profile of Bb1TS11.

[0025] Camphor (C 10 H 16O) (also referred to in the present invention as compound C3), is a cyclic ketone also selected by Bb1TS11. This compound, unlike the others, was detected during preliminary tests carried out for the development of the appropriate protocol, with samples of Bb1TS11. Given the known repellent action that this substance has on other insects, such as mosquitoes (Diptera Culicidae) (Nerio LS et al. 2010. Repellent activity of essential oils: a review. Bioresource technology, 101(1), 372-378), it was selected as a repellent candidate, despite not being a major compound.

[0026] Borneol (C 10 H 18 O) (also referred to in the present invention as compound C4), is a terpene identified by the metabolic profile of Bb1TS11.

[0027] 1,3-Dimethoxybenzene (C6H4(OCH3)2) (also referred to in the present invention as compound C5), is an aromatic compound identified by Pc123 and Ma4TS04

[0028] 1-octen-3-ol (CH3(CH2)4CHCH=C2) (also referred to in the present invention as compound C6), is a secondary alcohol selected from the metabolic profiles of Ma4TS04 and Pc123.

[0029] 3-cyclohepten-1-one (C7H10O) (also referred to in the present invention as compound C7), is a cyclic ketone identified by the metabolic profiles of Bb1TS11, Ma4TS04 and Pc123 (Figure 1).

[0030] In a more particular embodiment, the volatile organic compound of the present invention is from an entomopathogenic fungus selected from Beauveria bassiana and Metarhizium anisopliae.

[0031] In another more particular embodiment, the volatile organic compound of the present invention is from the nematophagous fungus Pochonia chlamydosporia.

[0032] In another particular embodiment of the present invention, the volatile organic compound of the present invention is from the entomopathogenic fungus Beauveria bassiana and the volatile organic compound of the present invention is selected from styrene (C8H8), benzothiazole (C7H5NS), camphor (C 10 H 16 O) and borneol.

[0033] In another particular embodiment of the present invention, the volatile organic compound of the present invention is from the entomopathogenic fungus Metarhizium anisopliae and the volatile organic compound of the present invention is selected from among 1,3-dimethoxybenzene (C6H4(OCH3)2), 1-octen-3-ol (CH3(CH2)4CHCH=CH2), 3-cyclohepten-1-one (C7H 10 EITHER).

[0034] In another particular embodiment, the volatile organic compound of the present invention is from the fungus Pochonia chlamydosporia and the volatile organic compound of the present invention is selected from among 1,3-dimethoxybenzene (C6H4(OCH3)2), 1-octen-3-ol (CH3(CH2)4CHCH=CH2), 3-cyclohepten-l-one (C7H 10 O), for use as a repellent against Cosmopolites sordidus.

[0035] In another particular embodiment, the volatile organic compound of the present invention is obtained by chemical synthesis.

[0036] In another particular embodiment, the volatile organic compound of the present invention is in solid form.

[0037] In another particular embodiment, the volatile organic compound of the present invention is in liquid form. In a further particular embodiment, the liquid formulation is impregnated in a matrix.

[0038] In another particular embodiment, the volatile organic compound of the present invention is in gel form.

[0039] In another aspect, the present invention relates to the use of a composition comprising at least one volatile organic compound of the present invention, as a repellent for Cosmopolites sordidus.

[0040] The volatile organic compounds of the present invention have been identified as metabolites of a strain of B. bassiana (Bb1TS11), a strain of Manisopliae (Ma4TS04), and a strain of P. chlamydosporia (Pc123). Bb1TS11 (CECT 21121, NCBI MK 156717) and .U / 4TS04 (CECT 21126; NCBI MK 156715) were isolated from rhizosphere soil samples from banana plantations located in the Canary Islands; therefore, it is assumed that these strains have interacted with the Canary Island population of PNP. Pc123, on the other hand, was isolated from Heterodera avenae eggs in Seville (ATCC No. MYA-4875; CECT No. 20929).

[0041] Brief description of the figures

[0042] Figure 1. Venn diagram representing the VOCs identified by GC / MS-SPME in the samples.

[0043] Figure 2. Gas Chromatography / Mass Spectrometry (GCMS) analysis by SPME for B bassiana 1TS11 after 20 days of incubation.

[0044] Figure 3. Gas Chromatography / Mass Spectrometry (GCMS) analysis by SPME for B bassiana 1TS11 after 30 days of incubation.

[0045] Figure 4. Gas Chromatography / Mass Spectrometry (GCMS) analysis by SPME for M. anisopliae 4TS04 after 10 days of incubation.

[0046] Figure 5. Gas Chromatography / Mass Spectrometry (GCMS) analysis by SPME for P chlamydosporia 123 after 10 days of incubation

[0047] Figure 6. Schematic of the stimuli placed in an olfactometer. E1 represents the individuals that have chosen the attractants; E2 represents all individuals that have come to the repellent of the evaluated candidates or, alternatively, to the absence of stimuli; EC indicates individuals that have chosen not to move.

[0048] Figure 7. Tukey test results in the PCFAs. SL-Ha (Adult PNP in light-free and hungry conditions), SL-SHa (Adult PNP in light-free and non-hungry conditions), L-Ha (Adult PNP in light-free and hungry conditions), L-SHa (Adult PNP in light-free and non-hungry conditions)

[0049] Figure 8. Tukey test results for the PRs, under optimal conditions for their movement (SL-Ha). VOCs: C1 (styrene); C2 (benzothiazole); C3 (camphor); C4 (boneol); C5 (1,3-dimethoxybenzene); C6 (1-octen-3-ol); C7 (3-cyclohepten-1-one). Detailed description of implementation methods

[0050] Insects and banana corm / pseudostem used in bioassays

[0051] Adult PNPs were collected on the Island of Tenerife (Canary Islands, Spain) using pheromone traps baited with ECOSordidina6O (ecobertura ® (ref: 019-FACS60), based on sordidine. The insects were kept in boxes at 28±0.5°C in the dark. The plastic boxes (40x30x21 cm) contained filter paper and a small perforated container with distilled water to maintain humidity levels around 80±5%. Twenty healthy PNP individuals (males and females not distinguished) were randomly selected from the stock population for the two-way olfactometer bioassay experiments.

[0052] Pieces of corm and pseudostem of banana (Musa sp.) were collected from the greenhouses of the University of Alicante, from plants originating in the Canary Islands (CULTESA). 15.6 g of corm / pseudostem were used for the bioassay experiments in a two-way olfactometer.

[0053] Fungus

[0054] Two species of entomopathogenic fungi (Beauveria bassiana and Metarhizium anisopliae) and one species of nematophagous fungus (Pochonia chlamydosporia) have been used.

[0055] We used a strain of B. bassiana Bb1TS11. Bb1TS11 (CECT 21121; NCBI MK156717) isolated from rhizosphere soil samples of banana plantations located in the Canary Islands, specifically Tenerife; therefore, it is assumed that this strain has interacted directly with the Canary Island population of PNP. Regarding M. anisopliae, the strain Ma4TS04 (CECT 21126; NCBI MK156715), like the one mentioned above, was isolated from Canary Island soil. The fungus P. chlamydosporia strain 123, Pc123, was isolated from Heterodera avenae eggs in Seville (ATCC No. MYA-4875; CECT No. 20929). The fungi were maintained in darkness at 4°C on corn flour agar (CMA; BBL Sparks, MD).

[0056] For the use of fungi in GC / MS-SPME analyses, solid formulations of the fungi were prepared from rice substrate (Oryza sativa) according to Güerri-Agulló B. et al. (2011). 250 mL flasks were used, to which 75 g of rice were added as substrate.

[0057] Analysis of volatile organic compounds (VOCs) produced by invertebrate parasitic fungi. The GC / MS (Gas Chromatography-Mass Spectrometry) technique used was Solid Phase Microextraction (SPME). This technique utilizes a fused silica fiber (approximately 1 cm long and 0.110 mm thick) coated with a small amount of extraction phase, placed in a modified syringe commonly called a “holder”.

[0058] Four samples were prepared for VOC analysis, one for each fungal strain, plus a non-inoculated (sterilized) rice sample used as a control. For each sample, 5 g of rice were taken from the culture flasks and placed in a vial (HS, crimb, FB, 20 ml, clr, cert, 100 PK, Agilent Technologies) hermetically sealed with a pressure stopper and a plastic membrane. The samples were incubated individually in a water bath at a fixed temperature of 60°C. For C3, assays were performed up to 100°C.

[0059] The absorption of the released VOCs occurred when the exposed fiber of the holder was placed inside the vial without touching the rice sample for 15 minutes. After this, the holder was inserted into the GC injector where it underwent desorption for four minutes at 150°C in splitless mode. The equipment used was an Agilent 5973 Network mass spectrometer along with an Agilent 6890N gas chromatograph.

[0060] The temperature program used for chromatography was: an initial temperature of 35°C for 5 min, then 150°C at 3°C / min for 1 min, followed by 250°C at 5°C / min until the end of the analysis. The chromatographic analysis lasted 38 minutes. The column used was a J&W Scientific DB624, 30 m long, 0.25 mm ID, and 1.4 mm diameter. The electron impact ionization source was set at 70 eV and 230°C. A single quadrupole detector was used, operating at 150°C. Wiley275 was the library used to identify details of the VOCs.

[0061] The metabolic profiles of the fungi involved in the study were reviewed without the VOCs identified in the control profile, the fiber without sample, and the metabolic profiles of the control substrate, autoclaved rice without fungal inoculum. In addition, from the Total VOCs (T-VOCs) characteristic of each fungus, two categories of VOCs were selected: major VOCs (M-VOCs) and minor VOCs (m-VOCs). M-VOCs are represented by all substances that showed a ≥ 50% match with compounds in the library and a maximum abundance > 100,000 ppm. m-VOCs, on the other hand, are represented by all detected compounds that showed a ≥ 50% match and a maximum abundance between 20,000 ppm and 100,000 ppm. Bb1TS11 presented 49 different T-COVs, characteristic of its metabolic profile (Table 1; Figure 2 and 3).Of these, only three compounds, representing 6.1% of the total, were found to belong to the M-VOCs category; while 12 different compounds were found to belong to the m-VOCs category, representing 24.5% of the total. Among the M-VOCs, 3-cyclohepten-1-one (C7) was found in all six measurements taken during the 60 days of fungal growth; while N-ethylbenzenamine was detected only in the measurements taken 30 and 40 days after substrate inoculation. Finally, 1,3-octadiene was the third M-VOC and was identified only in the measurement taken 50 days after substrate inoculation. Among the m-VOCs, only borneol (C4) was detected at 10, 20, 40, and 50 days after inoculum. The other Bb1TS11 m-COVs are detailed in Table 1. Benzothiazole (C2) was also detected.

[0062] Table 1. VOCs detected in the fungus Bb1TS11 at 10-60 days

[0063] Ma4TS0 m4 exhibited 49 different T-VOCs characteristic of its metabolic profile (Table 2; Figure 4). Of these, only six compounds, representing 12.2% of the total, were found to belong to the M-VOC category; while 11 different compounds, representing 22.4% of the total, were found to belong to the m-VOC category. Among the M-VOCs, 3-cyclohepten-1-one (C7) was found from 10 to 30 days post-inoculation; while l-octen-3-ol (C6) was detected as an M-VOC only in measurements taken 20 and 30 days after substrate inoculation. 4-fluoro-1,4-xylene was detected in measurements taken 20 and 60 days after substrate inoculation. 1,2-Dipentylcyclopropene was also detected in measurements taken 10 and 50 days post-inoculation. 1,3-Dimethoxybenzene (C5) was also detected. The other Ma4TS04 M-COVs are detailed in the table.Among the m-CVs, (+ -)-gymnomitrene was detected at 10, 20, and 40 days post-inoculation. l-octen-3-ol (C6) was also found as an m-CV only at 10 and 40 days post-inoculation. The other m-CVs from Ma4TS04 are detailed in Table 2.

[0064] Table 2. VOCs detected in the fungus Ma4TS04 at 10-60 days

[0065] Pc123 showed 111 different T-VOCs characteristic of its metabolic profile (Table 3; Figure 5). Of these, only 14 compounds, representing 12.6% of the total, were found to belong to the M-VOC category; while 43 different compounds were found to belong to the m-VOC category, representing 38.7% of the total. Of the M-VOCs, 1,3-dimethoxybenzene (C5) was found in all six measurements taken during fungal growth; while l-octen-3-ol (C6) was detected as an M-VOC only in measurements taken 10, 20, 30, and 60 days after substrate inoculation. 3-octanone was identified 20, 40, and 50 days after inoculation. While 3-cyclohepten-1-one (C7) was found only 10 and 20 days after inoculum. Among the m-VOCs, 2-(2-ethoxyethoxy)-ethanol was detected 30 and 40 days after inoculum. Propionin, 2-butanone, and 2-dodecanone were detected 40 and 50 days after inoculum.Also, 16-oxosalutaridine and 3-methylbutanal were detected, but at 50 and 60 days. The other Pc123 m-COVs are detailed in Table 3.

[0066] Table 3. VOCs detected in the fungus Pc123 at 10-60 days.

[0067] Description of the two-way olfactometer or Y-tube

[0068] Two-way olfactometers (Y-tubes) (Rhodes et al. 2012. The role of olfactory cues in the sequential radiation of a gall-boring beetle, Mordellistena convicta. Ecologica Entomology. 37(6): 500-507) were constructed for laboratory bioassays to test the response of adult PNP individuals to stimuli with the aforementioned volatile organic compounds. The olfactometer was constructed entirely from sections of heat-resistant glass tubing, each connected by a 5 mm airtight cone. The glass tubes had an internal diameter of 30 mm, the main body was 200 mm long, and the Y-junction arms were 150 mm long. The angle between each arm and the main body was 75°. The end of each arm was attached to a glass container (60 mm long, 25 mm in diameter) in which the odor chambers (CO) are placed (Bazzocchi GG and Maini S. 2000).The role of volatile semiochimici in the investigation of the Tospite from the parasitoid Diglyphus isaea (Hymenoptera Eulophidae). Olfactometric tests. Bollettino dell'Istituto di Entomologia “G. Grandi” Università di Bologna, 54, 143-154) in which the olfactory stimuli to be analyzed were inserted. The air inside each arm of the olfactometer was kept constant for all experiments with natural airflow. The tubes were covered to prevent the insects from escaping. When changing the substances in the tests, the flasks were washed with ethyl alcohol (Benito Parraga, SL), water, and n-hexane (PanReac AppliChem) to remove all traces of volatiles from previous samples.

[0069] Behavioral bioassays (two-way olfactometer) using the proposed VOCs

[0070] The two-way olfactometer (ODV) was used to evaluate the behavior of the palm weevil (PNP) subjected to different olfactory stimuli (attractants and repellents). Following the studies conducted by J. Jalinas (2016) on the red palm weevil (Rhynchophorus ferrugineus), all tests were performed by placing a single PNP individual in the center of a straight arm. The insects in the test had 10 minutes to move around and choose whether or not to use a stimulus. A total of 10 different bioassays were performed, with 120 PNP individuals in each. Each test consisted of six replicates, each with 20 individuals. The tests were grouped into two different categories: Physio-Environmental Condition Tests (PCFAs) and Repellent Tests (PRs).

[0071] In the first group, called Physio-Environmental Condition Tests (PCFAs), four tests were conducted to assess the attractive activity of the corm / pseudostem. Of the two control points (COs), the natural attractant was placed in one (chosen randomly to the right or left), while in the other, no attractants or repellents were placed, only ambient air. With this olfactory stimulation, two different physio-environmental conditions were tested. In these tests, we aimed to evaluate the impact of an environmental condition; that is, we assessed the influence that the presence (L) or absence (SL) of light has on the movement of the corm / pseudostem. The banana weevil (PNP) is known to have a predominantly nocturnal habit (Gold CS, Pena JE, Karamura EB (2001). Biology and integrated pest management for the banana weevil Cosmopolites sordidus (Germar) (Coleoptera: Curculionidae). Integrated Pest Management Reviews , 6(2), 79-155).

[0072] Furthermore, the movement of the PNP was tested under the physiological condition of starvation. Two subpopulations of PNP were tested: the first consisted of individuals with ad libitum access to food (Musa sp.) (SHa), while the second was composed of individuals with at least one week of starvation (Ha). In this way, it was possible to define the range of physiological and environmental conditions under which C. sordidus exhibits the highest movement rate.

[0073] In the second group, called Repellent Tests (PRs), seven tests were performed with PNP under SL-Ha conditions.

[0074] The tests were conducted with seven potentially repellent compounds (C1-C7), using commercial samples of these compounds (Sigma-Aldrich). In these tests, fresh corms / pseudostems were placed in one of the test chambers, and a dispenser containing either 0.5 ml (C1, C2, C5, C6, and C7) or 0.5 g (C3 and C4) of the pure repellent to be tested was placed in the other. The dispensers were constructed in the laboratory using miracloth sachets (Merck KGaA) (3.5 cm x 2.5 cm) containing 2 g of silica gel 60A (70-200m, Cario Erba). The compound was added directly to the silica.

[0075] The ethological data displayed by the PNPs in the ODV tests were collected in an Excel spreadsheet. As shown in Figure 6, the C. sordidus individuals responded to the test in three different ways: some responded to the El stimuli, which always included the attractants (corm / pseudostem and pheromones). Other individuals responded to the E2 stimulus; in this case, all the tested compounds were presented individually (one compound / trial) or no stimulus was presented at all. Still others preferred not to choose, remaining in what we called the "home" (EC).

[0076] This triple ethological expression was first assessed using ad hoc constructed movement indices to obtain a rapid and explanatory measure of the unique tests performed on ODVs. In addition, the data were tested using the chi-square statistical test with the RStudio statistical software.

[0077] Movement indices arise from the need to have a quick and easily explanatory tool that is also useful for summarizing the ethological situation shown by PNPs in tests.

[0078] It was assumed that a population of PNPs, placed in ODV and the absence of any stimulus, would be evenly distributed across the three possible options while maintaining a relationship. It was also considered that individuals remaining in the EC These are the individuals who did not respond to the attractive stimulus (in the case where no stimulus was placed in E2) or who were rejected by the tested substance (in the case where a repellent candidate was placed in E2). Therefore, three indices were constructed that took into account the relationship between the individuals in EC, E1, and E2 and the total number of individuals evaluated (N). The three indices are as follows: where: mi = number of individuals who have chosen E1, n E2= number of individuals who have chosen E2, n EC = number of individuals remaining in the EC, N = total number of individuals analyzed.

[0079] Finally, to obtain an index that takes into account all these relationships between groups of individuals, the I indices E1 , YO E2 and I EC They have been summarized in a single index: IM (Movement Index): where: 0 < IM> + ¥. The closer the IM is to zero, the more significant the portion of the population that has remained motionless, without reacting or capturing the attractive stimulus.

[0080] The IM was calculated for each of the six trials (N= 20 PNP), generating a data set for each trial. Then, using the statistical software R, ANOVA tests were performed on PCFAs and PRs.

[0081] The physiological-environmental conditions actually interfere with the mobility of the PNP (X 2= 17.952; df = 6; p-value = 0.006352). From the ANOVA analysis performed on the IMs of the individual replicates, there was a significant difference between the conditions (F-value = 3.305; p-value = 0.0413). In the Tukey test (Figure 7), we can see how the conditions of no light (SL) and no hunger (SHa) SL-SHa (IM = 0.53) are those in which the food stimulus attracts the most PNPs. The nocturnal habit of the PNP makes the insect more mobile in the absence of light (SL); in addition, the starvation condition (Ha) further stimulates the PNPs in their search for food and, therefore, gives them greater mobility. The nocturnal behavior of the PNP is evident, as tests conducted under SL conditions, i.e., SL-Ha and SL-SHa (IM= 0.48), show greater mobility of the PNP compared to light (L) conditions, or L-Ha (light-hunger) (IM= 0.36) and L-SHa (IM= 0.22). For these reasons, the SL-Ha condition was used as the experimental condition for the PRs.

[0082] From the analysis of the data relating to the PRs plus the control (SL-Ha), it turned out that there is a significance (X 2 = 34,142; df= 7; p-value = 1.62e -5 Therefore, the observed phenomenon follows a pattern that is not accidental. ANOVA analysis of the IMs revealed statistically significant differences between the tests examined (F-value = 2.798; p-value = 0.0181). Tukey's test (Figure 8) clearly shows that all the tested VOCs have a repellent effect on the PNP, decreasing its mobility compared to the control. Compound C7 exhibits the greatest repellent effect, registering the lowest IM value. There does not appear to be a significant difference between C1 and C6 with respect to the repellency rate. Therefore, according to the IM values, the different VOCs show the following repellency gradient: C7 (IM= 0.11), C5 (IM= 0.18), C2 (IM= 0.26), C1 (IM= 0.28), C4 (IM= 0.28), C3 (IM= 0.45) and C6 (IM= 0.47).

Claims

CLAIMS 1. Use of at least one volatile organic compound from an entomopathogenic and / or nematophagous fungus, selected from styrene, benzothiazole, camphor, borneol, 1,3-dimethoxybenzene, 1-octen-3-ol, 3-cyclohepten-1-one as a repellent of Cosmopolites sordidus.

2. Use of at least one volatile organic compound from an entomopathogenic and / or nematophagous fungus according to claim 1, wherein the entomopathogenic fungus is selected from Beauveria bassiana and Metarhizium anisopliae.

3. Use of at least one volatile organic compound from an entomopathogenic and / or nematophagous fungus according to any of the preceding claims, wherein the nematophagous fungus is Pochonia chlamydosporia.

4. Use of at least one volatile organic compound from an entomopathogenic and / or nematophagous fungus according to any of the preceding claims, wherein the fungus is Beauveria bassiana and the volatile organic compound is selected from styrene (C8H8), benzothiazole (C7H5NS), camphor (C 10 H 16 O) and borneol.

5. Use of at least one volatile organic compound from an entomopathogenic and / or nematophagous fungus according to any of the preceding claims, wherein the fungus is Metarhizium anisopliae and the volatile organic compound is selected from 1,3-dimethoxybenzene (C6H4(OCH3)2), 1-octen-3-ol (CH3(CH2)4CHCH=CH2), 3-cyclohepten-l-one (C7H 10 EITHER).

6. Use of at least one volatile organic compound from an entomopathogenic and / or nematophagous fungus according to any of the preceding claims, wherein the fungus is Pochonia chlamydosporia and the volatile organic compound is selected from 1,3-dimethoxybenzene (C6H4(OCH3)2), 1-octen-3-ol (CH3(CH2)4CHCH=CH2), 3-cyclohepten-l-one (C7H 10 O), for use as a repellent against Cosmopolites sordidus.

7. Use of at least one volatile organic compound according to any of the preceding claims, wherein the volatile organic compound is obtained by chemical synthesis.

8. Use of at least one volatile organic compound according to any of claims 1-7, wherein the volatile organic compound is in solid form.

9. Use of at least one volatile organic compound according to any of claims 1- 7, where the volatile organic compound is in liquid form.

10. Use of at least one volatile organic compound according to claim 9, wherein the liquid formulation is impregnated in a matrix.

11. Use of at least one volatile organic compound according to any of claims 1-7, wherein the volatile organic compound is in gel form.

12. Use of a composition comprising at least one volatile organic compound according to any of claims 1-12, as a repellent for Cosmopolites sordidus.