WO2021058844A1 - Use of volatile organic compounds of parasitic fungi of invertebrates as repellents of the black banana weevil (cosmopolites sordidus) - Google Patents

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

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WO2021058844A1
WO2021058844A1 PCT/ES2020/070563 ES2020070563W WO2021058844A1 WO 2021058844 A1 WO2021058844 A1 WO 2021058844A1 ES 2020070563 W ES2020070563 W ES 2020070563W WO 2021058844 A1 WO2021058844 A1 WO 2021058844A1
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volatile organic
organic compound
fungus
vocs
entomopathogenic
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PCT/ES2020/070563
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Spanish (es)
French (fr)
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Luis Vicente LÓPEZ LLORCA
Federico LÓPEZ MOYA
Ana LOZANO SORIA
Ugo PICCIOTTI
Javier LÓPEZ-CEPERO JIMÉNEZ
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Universidad De Alicante
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Priority to PE2022000487A priority Critical patent/PE20221452A1/en
Publication of WO2021058844A1 publication Critical patent/WO2021058844A1/en
Priority to CONC2022/0005126A priority patent/CO2022005126A2/en

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/30—Microbial fungi; Substances produced thereby or obtained therefrom
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N27/00—Biocides, pest repellants or attractants, or plant growth regulators containing hydrocarbons
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N31/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic oxygen or sulfur compounds
    • A01N31/02—Acyclic compounds
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N31/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic oxygen or sulfur compounds
    • A01N31/06—Oxygen or sulfur directly attached to a cycloaliphatic ring system
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N31/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic oxygen or sulfur compounds
    • A01N31/08—Oxygen or sulfur directly attached to an aromatic ring system
    • A01N31/16—Oxygen or sulfur directly attached to an aromatic ring system with two or more oxygen or sulfur atoms directly attached to the same aromatic ring system
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
    • A01N43/72—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with nitrogen atoms and oxygen or sulfur atoms as ring hetero atoms
    • A01N43/74—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with nitrogen atoms and oxygen or sulfur atoms as ring hetero atoms five-membered rings with one nitrogen atom and either one oxygen atom or one sulfur atom in positions 1,3
    • A01N43/78—1,3-Thiazoles; Hydrogenated 1,3-thiazoles
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention falls within the general field of agrobiotechnology and, in particular, refers to the repellent action of some volatile organic compounds (VOCs) produced by three strains of two entomopathogenic fungi and by a strain of a nematophagous fungus, as repellents of the Banana weevil (PNP), Cosmopolites sordidus (Germar, 1824) (Coleoptera Curculionidae).
  • VOCs volatile organic compounds
  • 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). World production is around 114 million tons per year (FAO, 2018).
  • 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 between 30% and 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 (Coleóptera: Curculionidae).
  • CATIE Integrated Pest Management
  • C. sordidus is an insect of the order Coleoptera, belonging to the Curculionidae family and to the Dryophthorinae subfamily.
  • PNP is a species native to the Indo-Malay region (Simmonds NW 1966. Bananas. 2nd ed. Longmans, Green and Co. Ltd., London, pp. 512) that currently spreads as a pest to all regions where it is grown The banana tree, and in Spain, 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 (Coleóptera: Curculionidae). Activities of the ICIA in Platanera , p. 75).
  • PNP has a low flight capacity and is capable of infecting both the corm of banana trees, such as pseudostem and suckers.
  • the PNP obtains resources that are absolutely essential for its growth and development, and to ensure the biological efficacy of future generations.
  • These efficient PNP search mechanisms are based on its antennas that function as specialized primary chemo- and mechanoreceptor organs. These precise environmental assessment mechanisms are crucial to ensure the survival and reproduction of the NPP in the environment.
  • Antenna chemoreceptors often detect volatile chemicals. These compounds generate alerts in the insect about the presence of possible mates, food, suitable places to deposit their eggs or dangers that they must avoid. Therefore, any chemical product that could interrupt and / or modify the behavior of the PNP and, in general, its capacity to search for the host (Musa sp.) would provide a tool for its sustainable management.
  • VOCs are solid or liquid compounds with a carbon base that enter the gaseous phase by vaporization at room temperature (20 ° C and 0.01 kPa; Pagans E. et al. 2006. Emission of voladle organic compounds from composting of different solid wastes: abatement by biofiltration. Journal of hazardous materials, 757 (1-3), 179-186).
  • VOCs The emission of VOCs performs essential ecological and physiological functions for many organisms, such as fungi, which release a broad spectrum of VOCs (Splivallo R. et al. 2011. Volatile truffle: 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). Emissions of fungal VOCs belong to different chemical groups, such as monoterpenoids, sesquiterpenes, alcohols, aldehydes, aromatics, esters, furans, hydrocarbons, ketones or lactones (Campos
  • Fungi produce various volatile organic compounds (Crespo R. et al. 2008. Volatile organic compounds released by the entomopathogenic fimgus Beauveria bassiana. Microbiological research, 163 (2), 148-151; Müller A. et al. 2013. Volatile profiles of fungi-chemotyping of species and ecological fimctions. 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 derive from primary and secondary metabolism (Korpi A. et al. 2009. Microbial volatile organic compounds.
  • patent ES2600526 describes volatile organic compounds from Beauveria bassiana, for use as a specific repellent for Rhynchophorus ferrugineus.
  • VOCs described in said patent specifically: methylbenzene, hexamethyl-cyclotetrasiloxanes, 1,4-dimethyl benzene, ethenylbenzene, dodecamethyl-pentasiloxane, benzaldehyde, 5-methylundecane, capric aldehyde, 1,4-bis (1,1-dimethyl benzene), benzothiazole and 1-Decanol are specific for Rhyi ichophorus ferrugineus, and have no effect on Cosmopolites sordidus.
  • 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 nematophages (Pochonia clamydosporia).
  • the present invention refers to the use of at least one volatile organic compound of an entomopathogenic and / or nematophagous fungus (hereinafter volatile organic compound of the present invention), selected from styrene, benzothiazole , camphor, bomeol, 1,3-dimethoxybenzene, l-octen-3-ol, 3-cyclohepten-l-one as a repellent for Cosmopolites sordidus.
  • volatile organic compound of the present invention selected from styrene, benzothiazole , camphor, bomeol, 1,3-dimethoxybenzene, l-octen-3-ol, 3-cyclohepten-l-one as a repellent for Cosmopolites sordidus.
  • volatile organic compound refers to a solid and / or liquid carbonaceous chemical that appears as an intermediate or end product of the metabolic pathways of the aforementioned fungi.
  • 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.
  • volatile organic compounds of the present invention are selected from:
  • Styrene (C 8 H 8 ) (also referred to in the present invention as Cl compound), belongs to the group of chemical compounds classified as organic vinyl compounds. Cl it appears in rice and in Bb1TS11, Pc123 and Ma4TS04.
  • Benzothiazole (C 7 H 5 NS) (also referred to in the present invention as compound C2), is a heterocyclic aromatic compound identified by the metabolic profile of Bb1TS11.
  • Camphor (C 10 H 16 O) (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 the 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 el 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 majority compound.
  • 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.
  • 1,3-dimethoxybenzene (C 6 H 4 (OCH 3 ) 2 ) (also referred to in the present invention as compound C5), is an aromatic compound identified by Pc123 and Ma4TS04
  • 3-cyclohepten-1-one (C 7 H1 0 O) (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).
  • the volatile organic compound of the present invention is from an entomopathogenic fungus selected from Beauveria bassiana and Metarhizium anisopliae.
  • the volatile organic compound of the present invention is from the nematophagous fungus Pochonia chlamydosporia.
  • 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 (C 8 H 8 ), benzothiazole (C 7 H 5 NS), camphor (C 10 H 16 O) and borneol.
  • 1,3-dimethoxybenzene C 6 H 4 (OCH 3 ) 2
  • 1-octen-3-ol CH 3
  • 3- cyclohepten-l-one C 7 H 10 O
  • the volatile organic compound of the present invention is obtained by chemical synthesis.
  • the volatile organic compound of the present invention is in solid form.
  • the volatile organic compound of the present invention is in liquid form.
  • the liquid formulation is impregnated in a matrix.
  • the volatile organic compound of the present invention is in gel form.
  • 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.
  • the volatile organic compounds of the present invention have been identified as metabolites of a B. bassiana strain (Bb1TS11), a M anisopliae strain (Ma4TS04) and a P. chlamydosporia strain (Pc123).
  • Bb1TS11 CECT 21121, NCBI MK 156717
  • .U / 4TS04 CECT 21126; NCBI MK 156715
  • Pc123 was isolated from Heterodera avenae eggs, in Seville (ATCC No. MYA-4875; CECT No. 20929).
  • Figure 1 Venn diagram representing the VOCs identified by GC / MS-SPME in the samples.
  • Figure 6 Scheme of the stimuli placed in an olfactometer. He represents the individuals who have chosen the attractants; E2 represents all the individuals who have attended the repellent of the evaluated candidates or, alternatively, the absence of stimuli; EC indicates individuals who have chosen not to move.
  • Figure 7 Results of the Tukey test in the PCFAs.
  • SL-Ha Alults of PNP in conditions without light and with hunger
  • SL-SHa Adults of PNP in conditions without light and without hunger
  • L-Ha Adults of PNP in conditions with light and with hunger
  • L- SHa PNP adults in light conditions and without hunger
  • FIG. 8 Results of the Tukey test in the PRs, in optimal conditions for their movement (SL-Ha).
  • VOCs C1 (styrene); C2 (benzothiazole); C3 (camphor); C4 (bomeol); C5 (1,3-dimethoxybenzene); C6 (1-octen-3-ol); C7 (3-cyclohepten-1-one).
  • PNP adults were collected on the Island of Tenerife (Canary Islands, Spain) using pheromone traps baited with ECOSordidine6O (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 a filter paper and a small perforated container with distilled water, to keep the humidity levels around 80 ⁇ 5%.
  • Twenty healthy PNP individuals were randomly selected from the stock population for two-way olfactometer bioassay experiments.
  • Pieces of corm and banana pseudostem (Musa sp.) Were collected from the greenhouses of the University of Alicante, from plants from the Canary Islands (CULTESA). 15.6 g of corm / pseudostem was used for the two-way olfactometer bioassay experiments.
  • Bb1TS11 CECT 21121; NCBI MK1567157 isolated from soil samples of the rhizosphere of banana plantations located in the Canary Islands, specifically Tenerife; therefore, it is assumed that this strain has directly interacted with the Canarian PNP population.
  • M. anisopliae the Ma4TS04 strain (CECT 21126; NCBI MK156715), like the one mentioned above, has been isolated from Canarian soil.
  • the fungus P. clamydosporia strain 123, Pc123 was isolated from Heterodera avenae eggs, in Seville (ATCC No. MYA-4875; CECT No. 20929). The fungi were kept in the dark at 4 ° C on corn flour agar (CMA; BBL Sparks, MD).
  • solid formulations were prepared from rice substrate (Oryza sativa) according to Güerri-Agulló B. el al. (2011). 250 mL flasks were used to which 75 g of rice were added as a substrate.
  • VOCs volatile organic compounds
  • SPME Solid Phase MicroExtraction
  • the absorption of the released VOCs was produced by placing the fiber of the exposed holder inside the vial without touching the rice sample, for 15 minutes. After this operation, the "holder” was inserted into the GC injector where it was exposed to desorption for four minutes at 150 ° C with the "splitless" mode.
  • the equipment used is an Agilent 5973 Network Mass Spectrometer in conjunction with an Agilent Model 6890N Gas Chromatograph.
  • the temperature program used for the chromatography was: initial temperature of 35 ° C for 5 min and at 3 ° C / min at 150 ° C for 1 min. Subsequently at 5 ° C / min at 250 ° C until the end of the analysis. Chromatographic analysis lasted 38 minutes.
  • the column used is a J&W Scientific 30m, 0.25mm ID, 1.4mm DB624.
  • the ionization source for electronic impact at 70eV and 230 ° C.
  • a simple quadrupole was used as a detector, and its temperature was 150 ° C.
  • Wiley275 is the library used to identify details of VOCs.
  • 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 fungus inoculum.
  • T-VOCs Total VOCs
  • two categories of VOCs were selected: the majority VOCs (M-VOCs) and the minority VOCs (m-VOCs).
  • M-VOCs are represented by all those substances that have presented a coincidence 3 50% with compounds of the library and a maximum abundance> 100000 ppm.
  • the m-VOCs are, on the other hand, represented by all the detected compounds that presented a concordance of 3 50% 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-COVs category; while, it was found that 12 different compounds belonged to the category of m-VOCs and represent 24.5% of the total.
  • M-VOCs 3-cyclohepten-l-one (C7) was found in the six measurements made during the 60 days of fungal growth; while, N-ethyl-benzenamine was detected only in measurements carried out at 30 and 40 days after inoculation of the substrate. Finally, 1,3-octadiene is the third M-VOCs and it was identified only in the measurement carried out 50 days after inoculation of the substrate. Among the m-VOCs, only borneol (C4) was detected at 10, 20, 40 and 50 days from the inoculum. The other m-VOCs of Bb1TS11 are detailed in Table 1. Benzothiazole (C2) was also detected.
  • Ma4TS0 m4 showed 49 different T-COVs 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 category of M-VOCs; while, it was found that 11 different compounds belonged to the category of m-VOCs and represented 22.4% of the total.
  • M-COVs 3-cyclohepten-l-one (C7) was found 10 to 30 days after inoculum; while l-octen-3-ol (C6) was detected as M-VOC only in measurements carried out 20 and 30 days after inoculation of the substrate. 4-fluoro-l, 4-xylene was detected in measurements made 20 and 60 days after inoculation of the substrate.
  • 1,2-dipentyl-cyclopropene was detected in measurements made 10 and 50 days after inoculation.
  • 1,3-Dimethoxybenzene (C5) was also detected.
  • the other M-VOCs of Ma4TS04 are detailed in the table.
  • m-VOCs (+ -) - gymnomitrene was detected 10, 20 and 40 days after the inoculum.
  • l-octen-3-ol (C6) was found as m-COVs only at 10 and 40 days from the inoculum.
  • the other m-VOCs of Ma4TS04 are detailed in Table 2.
  • Pc123 showed 111 different T-COVs 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 category of M-VOCs; while, it was found that 43 different compounds belonged to the category of m-VOCs and represent 38.7% of the total. Of the M-COVs, 1,3-dimethoxybenzene (C5) was found in the six measurements made during the growth of the fungus; while l-octen-3-ol (C6) has been detected as M-COVs only in measurements performed at 10, 20, 30 and 60 days after inoculation of the substrate.
  • C5 1,3-dimethoxybenzene
  • C6 l-octen-3-ol
  • Y tube Two-way olfactometers (“Y” tube) were constructed (Rhodes el al. 2012. The role of olfactory cues in the sequential radiation of a gall-boring beetle, Mordellistena convicta. Ecológica l Entomology. 37 (6): 500- 507.) for laboratory bioassays to check 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 tube each connected by an airtight cone (5 mm). The glass tubes had an internal diameter of 30 mm, the main branch was 200 mm and the arms of the "Y" joint 150 mm in length. The angle between each arm and the main body was 75 °.
  • each arm was attached to a glass container (60 mm in length, 25 mm in diameter) in which the odor chambers (CO) are placed (Bazzocchi GG and Maini S. 2000. Ruolo dei semiochimici volatili nella ricerca delTospite gives part of the parassitoid Diglyphus isaea (Hymenoptera Eulophidae). Prove olfattometriche. Bollettino dell'Istituto di Entomolog ⁇ a "G. Grandi” Universitá di Bologna, 54, 143-154) in which the olfactory stimuli that were analyzed were inserted.
  • the two-way olfactometer was used to evaluate the behavior of PNP subjected to the action of different olfactory stimuli (attractants and repellants).
  • ODV The two-way olfactometer
  • PCFAs Physio-Environmental Conditions Tests
  • the PNP movement was tested with a physiological condition such as hunger.
  • a physiological condition such as hunger.
  • Two subpopulations of PNP were tested: the first consisted of individuals with a food arrangement (Musa sp.) Ad libitum (SHa), while another consisted of individuals with at least one week of starvation (Ha). In this way, it was possible to define the domain of the physio-environmental conditions in which C. sordidus has the highest rate of movement.
  • This triple ethological expression was first evaluated with ad hoc constructed movement indices, to have a quick and explanatory measure of the unique tests performed on ODVs.
  • the data were tested through the chi-square statistical test, with the statistical software Rstudio.
  • Movement indices arise from the need to have a quick and easy explanatory tool and, at the same time, useful to summarize the ethological situation shown by the NPPs in the tests.
  • MI Movement index

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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

USO DE COMPUESTOS ORGÁNICOS VOLÁTILES DE HONGOS PARÁSITOS DE INVERTEBRADOS COMO REPELENTES DEL PICUDO NEGRO DE LA PLATANERA (COSMOPOLITES SORDIDUS) USE OF VOLATILE ORGANIC COMPOUNDS FROM PARASITE FUNGI OF INVERTEBRATES AS REPELLENTS OF THE BLACK PICUDO OF THE PLANTAIN (COSMOPOLITES SORDIDUS)
Campo de la invención Field of the invention
La presente invención se encuadra en el campo general de la agrobiotecnología y, en particular se refiere a la acción repelente de algunos compuestos orgánicos volátiles (COVs) producidos por tres cepas de dos hongos entomopatógenos y por una cepa de un hongo nematófago, como repelentes del picudo negro de la platanera (PNP), Cosmopolites sordidus (Germar, 1824) (Coleóptera Curculionidae). The present invention falls within the general field of agrobiotechnology and, in particular, refers to the repellent action of some volatile organic compounds (VOCs) produced by three strains of two entomopathogenic fungi and by a strain of a nematophagous fungus, as repellents of the Banana weevil (PNP), Cosmopolites sordidus (Germar, 1824) (Coleoptera Curculionidae).
Estado de la técnica anterior Prior state of the art
El plátano ( Musa sp .) es el fruto más consumido y cultivado en el mundo (Ferri D.V. 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). La producción mundial ronda los 114 millones de toneladas al año (FAO, 2018). 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). World production is around 114 million tons per year (FAO, 2018).
Entre los principales organismos que afectan a los cultivos de plataneras, Cosmopolites sordidus es el que causa el mayor daño y las mayores pérdidas económicas en todas las áreas de producción, es decir, es la plaga clave de las plantaciones de plataneras. Las pérdidas pueden oscilar entre el 30% y el 90% (Carballo, V. 1998. Mortalidad de Cosmopolites sordidus con diferentes formulaciones de Beauveria bassiana. Manejo Integrado de Plagas (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 (Coleóptera: Curculionidae). Journal of economic entomology , 94(2), 449-454; Muñoz-Ruiz, C. 2007. Fluctuación poblacional del picudo negro ( Cosmopolites sordidus Germar) del plátano (Musa AAB) en San Carlos, Costa Rica. Revista Tecnología en Marcha, 20(1), 24-41). 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 between 30% and 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 (Coleóptera: 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).
C. sordidus es un insecto del orden Coleóptera, perteneciente a la familia Curculionidae y a la subfamilia Dryophthorinae. El PNP es una especie nativa de la región indo-malaya (Simmonds N.W. 1966. Bananas. 2nd ed. Longmans, Green and Co. Ltd ., London, pp. 512) que actualmente se extiende como plaga a todas las regiones donde se cultiva la platanera, y en España, está presente, sobre todo, en las Islas Canarias (Carnero H.A. etal. 2002. Métodos alternativos para el control del picudo de la platanera Cosmopolites sordidus Germar, 1824 (Coleóptera: Curculionidae). Actividades del ICIA en Platanera , p. 75). C. sordidus is an insect of the order Coleoptera, belonging to the Curculionidae family and to the Dryophthorinae subfamily. PNP is a species native to the Indo-Malay region (Simmonds NW 1966. Bananas. 2nd ed. Longmans, Green and Co. Ltd., London, pp. 512) that currently spreads as a pest to all regions where it is grown The banana tree, and in Spain, 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 (Coleóptera: Curculionidae). Activities of the ICIA in Platanera , p. 75).
La invasión y difusión del PNP se debe a su comportamiento de búsqueda para adquirir alimentos, pareja, oviposición y sitios de reproducción. Por otra parte, el PNP posee una baja capacidad de vuelo y es capaz de infectar tanto al cormo de las plataneras, como pseudotallo e hijuelos. En dicho proceso el PNP consigue recursos que son absolutamente esenciales para su crecimiento y desarrollo y, para asegurar la eficacia biológica de futuras generaciones. Estos eficientes mecanismos de búsqueda del PNP se basan en sus antenas que fúncionan como órganos quimio- y mecanorreceptores primarios especializados. Estos precisos mecanismos de evaluación ambiental son cruciales para asegurar la supervivencia y reproducción del PNP en el medio. The invasion and diffusion of the PNP is due to its search behavior to acquire food, mate, oviposition and breeding sites. On the other hand, PNP has a low flight capacity and is capable of infecting both the corm of banana trees, such as pseudostem and suckers. In this process, the PNP obtains resources that are absolutely essential for its growth and development, and to ensure the biological efficacy of future generations. These efficient PNP search mechanisms are based on its antennas that function as specialized primary chemo- and mechanoreceptor organs. These precise environmental assessment mechanisms are crucial to ensure the survival and reproduction of the NPP in the environment.
Los quimiorreceptores de las antenas suelen detectar productos químicos volátiles. Estos compuestos generan alertas en el insecto sobre la presencia de posibles parejas, alimento, lugares adecuados para depositar sus huevos o peligros que deben evitar. Por lo tanto, cualquier producto químico que pudiese interrumpir y/o modificar el comportamiento del PNP y, en general, su capacidad de búsqueda del huésped ( Musa sp .) proporcionaría una herramienta para su manejo sostenible. Antenna chemoreceptors often detect volatile chemicals. These compounds generate alerts in the insect about the presence of possible mates, food, suitable places to deposit their eggs or dangers that they must avoid. Therefore, any chemical product that could interrupt and / or modify the behavior of the PNP and, in general, its capacity to search for the host (Musa sp.) Would provide a tool for its sustainable management.
Los COVs son compuestos sólidos o líquidos con una base carbonada que entran en fase gaseosa mediante vaporización a temperatura ambiente (20°C y 0,01 kPa; Pagans E. etal. 2006. Emission of voladle organic compounds from composting of different solid wastes: abatement by biofiltration. Journal ofhazardous materials , 757(1-3), 179-186). VOCs are solid or liquid compounds with a carbon base that enter the gaseous phase by vaporization at room temperature (20 ° C and 0.01 kPa; Pagans E. et al. 2006. Emission of voladle organic compounds from composting of different solid wastes: abatement by biofiltration. Journal of hazardous materials, 757 (1-3), 179-186).
La emisión de COVs desempeña fúnciones ecológicas y fisiológicas esenciales para muchos organismos, como los hongos, que liberan un amplio espectro de COVs (Splivallo R. et al. 2011. Truffle volátiles: from Chemical ecology to aroma biosynthesis. New Phytologist , 189(3), 688-699; Kramer R. and Abraham W.R. 2012. Volatile sesquiterpenes from fúngi: what are they good for?. Phytochemistry Reviews, 77(1), 15-37). Las emisiones de COVs füngicos pertenecen a diferentes grupos químicos, como monoterpenoides, sesquiterpenos, alcoholes, aldehidos, compuestos aromáticos, ésteres, furanos, hidrocarburos, cetonas o lactonas (CamposThe emission of VOCs performs essential ecological and physiological functions for many organisms, such as fungi, which release a broad spectrum of VOCs (Splivallo R. et al. 2011. Volatile truffle: 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). Emissions of fungal VOCs belong to different chemical groups, such as monoterpenoids, sesquiterpenes, alcohols, aldehydes, aromatics, esters, furans, hydrocarbons, ketones or lactones (Campos
V.P. et al. 2010. Volátiles produced by interacting microorganisms potentially useful for the control of plant pathogens. Ciencia e Agrotecnologia, 34(3), 525-535; Kramer R. and AbrahamV.P. et al. 2010. Volatile produced by interacting microorganisms potentially useful for the control of plant pathogens. Science and Agrotechnology, 34 (3), 525-535; Kramer R. and Abraham
W.R. 2012. Volatile sesquiterpenes from fimgi: what are they good for? Phytochemistry Reviews , 11(1), 15-37). W.R. 2012. Volatile sesquiterpenes from fimgi: what are they good for? Phytochemistry Reviews, 11 (1), 15-37).
Los hongos producen diversos compuestos orgánicos volátiles (Crespo R. et al. 2008. Volatile organic compounds released by the entomopathogenic fimgus Beauveria bassiana. Microbiological research, 163(2), 148-151; Müller A. et al. 2013. Volatile profiles of fúngi- chemotyping of species and ecological fimctions. Fungal Genetics and Biology, 54, 25-33) a través de vías metabólicas, implicados en procesos biológicos de control o comunicación entre microorganismos y su entorno. Los COVs fungí eos derivan del metabolismo primario y secundario (Korpi A. et al. 2009. Microbial volatile organic compounds. Critical reviews in Toxicology , 39(2), 139-193), y como pueden propagarse a través de la atmósfera y el suelo, son semioquímicos ideales (Morath S.U. et al. 2012. Fungal volatile organic compounds: a review with emphasis on their biotechnological potential. Fungal Biology Reviews, 26(2-3), 73-83) capaces de actuar como mediadores en las interacciones entre organismos. La producción de COVs füngicos es biológicamente dinámica. El perfil de una especie o cepa particular varía según el sustrato o nutriente, el tiempo de incubación, la temperatura y otros parámetros ambientales (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 effeets of a mixture of indoor air volatile organics, their ozone oxidation producís, and stress. Environmental health perspectives, 113(11), 1542-1548). Fungi produce various volatile organic compounds (Crespo R. et al. 2008. Volatile organic compounds released by the entomopathogenic fimgus Beauveria bassiana. Microbiological research, 163 (2), 148-151; Müller A. et al. 2013. Volatile profiles of fungi-chemotyping of species and ecological fimctions. 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 derive from primary and secondary metabolism (Korpi A. et al. 2009. Microbial volatile organic compounds. Critical reviews in Toxicology, 39 (2), 139-193), and how they can spread through the atmosphere and soil, 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 the interactions between organisms. The production of fungal VOCs is biologically dynamic. The profile of a particular species or strain varies according to 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 effeets of a mixture of indoor air volatile organics, their ozone oxidation producís, and stress. Environmental health perspectives, 113 (11), 1542-1548).
Actualmente, no existe ningún plan integrado de control del PNP, únicamente el manejo de la plaga mediante trampas de feromonas para conteo y captura, controlando posibles focos y eliminando las plantas demasiado infectadas, método poco eficiente y caro. En este sentido, la patente ES2600526 describe compuestos orgánicos volátiles de Beauveria bassiana, para su uso como repelente específico de Rhynchophorus ferrugineus. Los COV descritos en dicha patente, concretamente: metilbenceno, hexametil-ciclotetrasiloxanos, 1,4-dimetil benceno, etenilbenceno, dodecametil-pentasiloxano, benzaldehído, 5-metilundecano, aldehido cáprico, 1,4-bis (1,1 -dimetil benceno), benzotiazol y 1-Decanol son específicos para Rhyi ichophorus ferrugineus , y no producen ningún efecto en Cosmopolites sordidus. Currently, there is no integrated PNP control plan, only pest management using pheromone traps for counting and capturing, controlling possible outbreaks and eliminating excessively infected plants, an inefficient and expensive method. In this sense, patent ES2600526 describes volatile organic compounds from Beauveria bassiana, for use as a specific repellent for Rhynchophorus ferrugineus. The VOCs described in said patent, specifically: methylbenzene, hexamethyl-cyclotetrasiloxanes, 1,4-dimethyl benzene, ethenylbenzene, dodecamethyl-pentasiloxane, benzaldehyde, 5-methylundecane, capric aldehyde, 1,4-bis (1,1-dimethyl benzene), benzothiazole and 1-Decanol are specific for Rhyi ichophorus ferrugineus, and have no effect on Cosmopolites sordidus.
Es evidente la gran incidencia que tiene el PNP en los cultivos de platanera y el aumento en las restricciones nacionales y europeas en el uso de insecticidas de síntesis química para estas plagas. Existe pues la necesidad de desarrollar nuevos compuestos derivados de agentes de control biológico naturales para mitigar los daños producidos por Cosmopolites sordidus , y para eliminar los daños provocados en el ambiente cuando se utilizan insecticidas frente a dicho insecto. The great impact that PNP has on banana crops and the increase in national and European restrictions on the use of chemically synthesized insecticides for these pests is evident. There is therefore a need to develop new compounds derived from natural biological control agents to mitigate the damage caused by Cosmopolites sordidus, and to eliminate the damage caused to the environment when insecticides are used against said insect.
Explicación de la invención Explanation of the invention
La presente invención resuelve los problemas descritos anteriormente, ya que proporciona nuevos COVs con actividad repelente derivados del metabolismo de hongos entomopatógenos ( Beauveria bassiana , Metarhizium anisopliae) y nematófagos ( Pochonia clamydosporia).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 nematophages (Pochonia clamydosporia).
Así pues, en un primer aspecto, la presente invención se refiere al uso de al menos un compuesto orgánico volátil de un hongo entomopatógeno y/o nematófago (de aquí en adelante compuesto orgánico volátil de la presente invención), seleccionado de entre estireno, benzotiazol, alcanfor, bomeol, 1,3-dimetoxibenceno, l-octen-3-ol, 3-ciclohepten-l-ona como repelente de Cosmopolites sordidus. Thus, in a first aspect, the present invention refers to the use of at least one volatile organic compound of an entomopathogenic and / or nematophagous fungus (hereinafter volatile organic compound of the present invention), selected from styrene, benzothiazole , camphor, bomeol, 1,3-dimethoxybenzene, l-octen-3-ol, 3-cyclohepten-l-one as a repellent for Cosmopolites sordidus.
En la presente invención por “compuesto orgánico volátil” se refiere a una sustancia química carbonada sólida y/o líquida que aparece como producto intermedio o final de las vías metabólicas de los hongos mencionados anteriormente. In the present invention, "volatile organic compound" refers to a solid and / or liquid carbonaceous chemical that appears as an intermediate or end product of the metabolic pathways of the aforementioned fungi.
En una realización particular, los compuestos orgánicos volátiles de la presente invención pertenecen a diferentes familias químicas y se producen en diferentes tiempos de incubación por los diferentes hongos utilizados. In a 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.
En una realización particular, los compuestos orgánicos volátiles de la presente invención son seleccionados de entre: In a particular embodiment, the volatile organic compounds of the present invention are selected from:
Estireno (C8H8) (también denominado en la presente invención como compuesto Cl), pertenece al grupo de compuestos químicos clasificados como compuestos orgánicos de vinilo. Cl aparece en el arroz y en Bb1TS11, Pc123 y Ma4TS04. Styrene (C 8 H 8 ) (also referred to in the present invention as Cl compound), belongs to the group of chemical compounds classified as organic vinyl compounds. Cl it appears in rice and in Bb1TS11, Pc123 and Ma4TS04.
Benzotiazol (C7H5NS) (también denominado en la presente invención como compuesto C2), es un compuesto aromático heterocíclico identificado por el perfil metabólico de Bb1TS11.Benzothiazole (C 7 H 5 NS) (also referred to in the present invention as compound C2), is a heterocyclic aromatic compound identified by the metabolic profile of Bb1TS11.
Alcanfor (C 10H 16O) (también denominado en la presente invención como compuesto C3), es una cetona cíclica también seleccionada por Bb1TS11. Este compuesto, a diferencia de los otros, se detectó durante las pruebas preliminares realizadas para el desarrollo del protocolo apropiado, con muestras de Bb1TS11. Dada la conocida acción repelente que esta sustancia tiene sobre otros insectos, como mosquitos (Díptera Culicidae) (Nerio L.S. el al. 2010. Repellent activity of essential oils: a review. Bioresource technology, 101(1), 372-378), fue seleccionada como candidato repelente, a pesar de no ser un compuesto mayoritario. Camphor (C 10 H 16 O) (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 the 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 el 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 majority compound.
Borneol (C 10H 18O) (también denominado en la presente invención como compuesto C4), es un terpeno identificado por el perfil metabólico de Bb1TS11. 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.
1,3-dimetoxibenceno (C6H4(OCH3)2) (también denominado en la presente invención como compuesto C5), es un compuesto aromático identificado por Pc123 y Ma4TS04 1,3-dimethoxybenzene (C 6 H 4 (OCH 3 ) 2 ) (also referred to in the present invention as compound C5), is an aromatic compound identified by Pc123 and Ma4TS04
1-octen-3-ol (CH3 (CH2)4CHCH=C 2) (también denominado en la presente invención como compuesto C6), es un alcohol secundario seleccionado de los perfiles metabólicos de Ma4TS04 y Pc123. 1-octen-3-ol (CH 3 (CH 2 ) 4 CHCH = C 2 ) (also referred to in the present invention as compound C6), is a secondary alcohol selected from the metabolic profiles of Ma4TS04 and Pc123.
3-ciclohepten-1-ona (C7H10O) (también denominado en la presente invención como compuesto C7), es una cetona cíclica identificada por los perfiles metabólicos de Bb1TS11, Ma4TS04 y Pc123 (Figura 1). 3-cyclohepten-1-one (C 7 H1 0 O) (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).
En una realización más en particular, el compuesto orgánico volátil de la presente invención, es de un hongo entomopatógeno seleccionado de entre Beauveria bassiana y Metarhizium anisopliae. 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.
En otra realización más en particular, el compuesto orgánico volátil de la presente invención, es del hongo nematófago Pochonia clamydosporia. In another more particular embodiment, the volatile organic compound of the present invention is from the nematophagous fungus Pochonia chlamydosporia.
En otra realización particular de la presente invención, el compuesto orgánico volátil de la presente invención, es del hongo entomopatógeno Beauveria bassiana y el compuesto el compuesto orgánico volátil de la presente invención es seleccionado de entre estireno (C8H8), benzotiazol (C7H5NS), alcanfor (C 10H 16O) y borneol. 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 (C 8 H 8 ), benzothiazole (C 7 H 5 NS), camphor (C 10 H 16 O) and borneol.
En otra realización particular de la presente invención, el compuesto orgánico volátil de la presente invención, es del hongo entomopatógeno Metarhizium anisopliae y el compuesto orgánico volátil de la presente invención, es seleccionado de entre 1,3-dimetoxibenceno (C6H4(OCH3)2), 1-octen-3-ol (CH3(CH2)4CHCH=CH2), 3-ciclohepten-1-ona (C7H10O). 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 1,3-dimethoxybenzene (C 6 H 4 (OCH 3 ) 2 ), 1-octen-3-ol (CH 3 (CH 2 ) 4 CHCH = CH 2 ), 3- cyclohepten-1-one (C 7 H 10 O).
En otra realización particular, el compuesto orgánico volátil de la presente invención, es del hongo Pochonia clamydosporia y el compuesto orgánico volátil de la presente invención, es seleccionado de entre 1,3-dimetoxibenceno (C6H4(OCH3)2), 1-octen-3-ol (CH3(CH2)4CHCH=CH2), 3-ciclohepten-l-ona (C7H10O), para su uso como repelente de Cosmopolites sordidus. In another particular embodiment, the volatile organic compound of the present invention is from the Pochonia clamydosporia fungus and the volatile organic compound of the present invention is selected from 1,3-dimethoxybenzene (C 6 H 4 (OCH 3 ) 2 ), 1-octen-3-ol (CH 3 (CH 2 ) 4 CHCH = CH 2 ), 3- cyclohepten-l-one (C 7 H 10 O), for use as a repellent for Cosmopolites sordidus.
En otra realización particular, el compuesto orgánico volátil de la presente invención, es obtenido por síntesis química. In another particular embodiment, the volatile organic compound of the present invention is obtained by chemical synthesis.
En otra realización particular, el compuesto orgánico volátil de la presente invención, está en forma sólida. In another particular embodiment, the volatile organic compound of the present invention is in solid form.
En otra realización particular, el compuesto orgánico volátil de la presente invención, está en forma líquida. En una realización más en particular, la formulación líquida está impregnada en una matriz. In another particular embodiment, the volatile organic compound of the present invention is in liquid form. In a more particular embodiment, the liquid formulation is impregnated in a matrix.
En otra realización particular, el compuesto orgánico volátil de la presente invención, está en forma gel. In another particular embodiment, the volatile organic compound of the present invention is in gel form.
En otro aspecto, la presente invención se refiere al uso de una composición que comprende al menos un compuesto orgánico volátil de la presente invención, como repelente de Cosmopolites sordidus. 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.
Los compuestos orgánicos volátiles de la presente invención se han identificado como metabolitos de una cepa de B. bassiana (Bb1TS11), una cepa de M anisopliae ( Ma4TS04) y una cepa de P. clamydosporia ( Pc123). Bb1TS11 (CECT 21121, NCBI MK 156717) y .U/4TS04 (CECT 21126; NCBI MK 156715) se aislaron de muestras de suelo de la rizosfera de las plantaciones de platanera ubicadas en Canarias; por lo tanto, se supone que esas cepas han interactuado con la población canaria de PNP. Mientras que Pc123 se aisló de huevos de Heterodera avenae, en Sevilla (ATCC No. MYA-4875; CECT No. 20929). The volatile organic compounds of the present invention have been identified as metabolites of a B. bassiana strain (Bb1TS11), a M anisopliae strain (Ma4TS04) and a P. chlamydosporia strain (Pc123). Bb1TS11 (CECT 21121, NCBI MK 156717) and .U / 4TS04 (CECT 21126; NCBI MK 156715) were isolated from soil samples from the rhizosphere of banana plantations located in the Canary Islands; therefore, it is assumed that these strains have interacted with the Canarian population of PNP. While Pc123 was isolated from Heterodera avenae eggs, in Seville (ATCC No. MYA-4875; CECT No. 20929).
Breve descripción de las figuras Brief description of the figures
Figura 1. Diagrama de Venn que representa los COVs identificados por GC/MS-SPME en las muestras. Figure 1. Venn diagram representing the VOCs identified by GC / MS-SPME in the samples.
Figura 2. Análisis de Cromatografía de Gases/Espectrometría de Masas (GCMS) mediante SPME para B bassiana 1TS11 de 20 días de incubación. Figure 2. Gas Chromatography / Mass Spectrometry (GCMS) analysis using SPME for B bassiana 1TS11 of 20 days of incubation.
Figura 3. Análisis de Cromatografía de Gases/Espectrometría de Masas (GCMS) mediante SPME para B bassiana 1TS11 de 30 días de incubación. Figure 3. Gas Chromatography / Mass Spectrometry (GCMS) analysis using SPME for B bassiana 1TS11 after 30 days of incubation.
Figura 4. Análisis de Cromatografía de Gases/Espectrometría de Masas (GCMS) mediante SPME para M. anisopliae 4TS04 de 10 días de incubación. Figure 4. Gas Chromatography / Mass Spectrometry (GCMS) analysis by SPME for M. anisopliae 4TS04 of 10 days of incubation.
Figura 5. Análisis de Cromatografía de Gases/Espectrometría de Masas (GCMS) mediante SPME para P clamydosporia 123 de 10 días de incubación Figure 5. Gas Chromatography / Mass Spectrometry (GCMS) analysis by SPME for P chlamydosporia 123 of 10 days of incubation
Figura 6. Esquema de los estímulos colocados en un olfactómetro. El representa a los individuos que han elegido los atrayentes; E2 representa a todos los individuos que han acudido al repelente de los candidatos evaluados o, alternativamente, a la ausencia de estímulos; EC indica individuos que han elegido no moverse. Figure 6. Scheme of the stimuli placed in an olfactometer. He represents the individuals who have chosen the attractants; E2 represents all the individuals who have attended the repellent of the evaluated candidates or, alternatively, the absence of stimuli; EC indicates individuals who have chosen not to move.
Figura 7. Resultados de la prueba de Tukey en las PCFAs. SL-Ha (Adultos de PNP en condiciones sin luz y con hambre), SL-SHa (Adultos de PNP en condiciones sin luz y sin hambre), L-Ha (Adultos de PNP en condiciones con luz y con hambre), L-SHa (Adultos de PNP en condiciones con luz y sin hambre) Figure 7. Results of the Tukey test in the PCFAs. SL-Ha (Adults of PNP in conditions without light and with hunger), SL-SHa (Adults of PNP in conditions without light and without hunger), L-Ha (Adults of PNP in conditions with light and with hunger), L- SHa (PNP adults in light conditions and without hunger)
Figura 8. Resultados de la prueba de Tukey en las PRs, en condiciones óptimas para su movimiento (SL-Ha). COVs: C1 (estireno); C2 (benzotiazol); C3 (alcanfor); C4 (bomeol); C5 (1,3-dimetoxibenceno); C6 (1-octen-3-ol); C7 (3-ciclohepten-1-ona). Exposición detallada de modos de realización Figure 8. Results of the Tukey test in the PRs, in optimal conditions for their movement (SL-Ha). VOCs: C1 (styrene); C2 (benzothiazole); C3 (camphor); C4 (bomeol); C5 (1,3-dimethoxybenzene); C6 (1-octen-3-ol); C7 (3-cyclohepten-1-one). Detailed exposition of embodiments
Insectos y cormo/pseudotallo de platanera utilizados en los bioensayos Insects and banana corm / pseudostem used in bioassays
Los adultos de PNP se recogieron en la Isla de Tenerife (Canarias, España) mediante trampas de feromonas cebadas con ECOSordidina6O (ecobertura®, ref: 019-FACS60), basado en sordidina. Los insectos se mantuvieron en cajas a 28±0,5°C en la oscuridad. Las cajas de plástico (40x30x21 cm) contenían un papel de filtro y un pequeño recipiente perforado con agua destilada, para mantener los niveles de humedad en torno al 80±5%. Se seleccionaron al azar 20 individuos (sin distinguir entre machos y hembras) sanos de PNP de la población stock para los experimentos con bioensayos en olfactómetro de dos vías. PNP adults were collected on the Island of Tenerife (Canary Islands, Spain) using pheromone traps baited with ECOSordidine6O (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 a filter paper and a small perforated container with distilled water, to keep the humidity levels around 80 ± 5%. Twenty healthy PNP individuals (without distinguishing between males and females) were randomly selected from the stock population for two-way olfactometer bioassay experiments.
Se recogieron trozos de cormo y pseudotallo de platanera ( Musa sp .) de los invernaderos de la Universidad de Alicante, de plantas procedentes de las Islas Canarias (CULTESA). Se utilizaron 15,6 g de cormo/pseudotallo para los experimentos de bioensayo en olfactómetro de dos vías. Pieces of corm and banana pseudostem (Musa sp.) Were collected from the greenhouses of the University of Alicante, from plants from the Canary Islands (CULTESA). 15.6 g of corm / pseudostem was used for the two-way olfactometer bioassay experiments.
Hongos Mushrooms
Se han utilizado dos especies de hongos entom opatógenos ( Beauveria bassiana y Metarhizium anisopliae) y una especie de hongo nematófago ( Pochonia clamydosporia). Two species of entomopathogenic fungi (Beauveria bassiana and Metarhizium anisopliae) and one species of nematophagous fungus (Pochonia clamydosporia) have been used.
Utilizamos una cepa de B. bassiana Bb1TS11. Bb1TS11 (CECT 21121; NCBI MK156717) aislada de muestras de suelo de la rizosfera de cultivos de plataneras ubicadas en las Islas Canarias, en concreto de Tenerife; por lo tanto, se supone que esta cepa ha interactuado directamente con la población canaria de PNP. En lo que respecta a M. anisopliae , la cepa Ma4TS04 (CECT 21126; NCBI MK156715), como la comentada anteriormente, ha sido aislada de suelo canario. El hongo P. clamydosporia cepa 123, Pc123 se aisló de huevos de Heterodera avenae, en Sevilla (ATCC No. MYA-4875; CECT No. 20929). Los hongos se mantuvieron en oscuridad a 4°C en agar harina de maíz (CMA; BBL Sparks, MD). We used a strain of B. bassiana Bb1TS11. Bb1TS11 (CECT 21121; NCBI MK156717) isolated from soil samples of the rhizosphere of banana plantations located in the Canary Islands, specifically Tenerife; therefore, it is assumed that this strain has directly interacted with the Canarian PNP population. Regarding M. anisopliae, the Ma4TS04 strain (CECT 21126; NCBI MK156715), like the one mentioned above, has been isolated from Canarian soil. The fungus P. clamydosporia strain 123, Pc123 was isolated from Heterodera avenae eggs, in Seville (ATCC No. MYA-4875; CECT No. 20929). The fungi were kept in the dark at 4 ° C on corn flour agar (CMA; BBL Sparks, MD).
Para el uso de los hongos en los análisis de GC/MS-SPME se prepararon formulaciones sólidas de los mismos a partir de sustrato de arroz ( Oryza sativa ) según Güerri-Agulló B. el al. (2011). Se utilizaron matraces de 250 mL a los que se agregaron como sustrato 75 g de arroz. For the use of fungi in GC / MS-SPME analyzes, solid formulations were prepared from rice substrate (Oryza sativa) according to Güerri-Agulló B. el al. (2011). 250 mL flasks were used to which 75 g of rice were added as a substrate.
Análisis de los compuestos orgánicos volátiles (COVs) producidos por los hongos parásitos de invertebrados La técnica GC/MS (Cromatografía de gases-Espectrometría de masas) utilizada fue la MicroExtracción en Fase Sólida (SPME). En ella, se utiliza una fibra de sílice fundida (aproximadamente 1 cm de largo y 0,110 mm) cubierta con una pequeña cantidad de fase de extracción, colocada en una jeringa modificada llamada habitualmente “holder” . Analysis of volatile organic compounds (VOCs) produced by parasitic fungi of invertebrates The GC / MS (Gas Chromatography-Mass Spectrometry) technique used was Solid Phase MicroExtraction (SPME). In it, a fused silica fiber (approximately 1 cm long and 0.110 mm) covered with a small amount of extraction phase is used, placed in a modified syringe commonly called a "holder".
Se prepararon cuatro muestras para analizar los COVs, una para cada cepa de hongos, más una muestra de arroz no inoculada por ningún hongo (esterilizada), utilizada como control. Para cada muestra se tomaron 5 g de arroz de los matraces de cultivo y se colocaron en un vial (HS, crimb, FB, 20 mi, clr, cert, 100 PK, Agilent Technologies) tapado herméticamente con un tapón de presión con una membrana de plástico. Las muestras se incubaron individualmente en un baño termostático, con una temperatura fija de 60°C. Para C3 se realizaron ensayos hasta 100°C.Four samples were prepared to analyze the VOCs, one for each fungal strain, plus a rice sample not inoculated with any fungus (sterilized), 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 capped with a pressure cap with a membrane of plastic. The samples were incubated individually in a thermostatic bath, with a fixed temperature of 60 ° C. For C3 tests were carried out up to 100 ° C.
La absorción de los COVs liberados se produjo al colocar la fibra del “holder” expuesta en el interior del vial sin tocar la muestra de arroz, durante 15 minutos. Después de esta operación, el “holder” se insertó en el inyector de GC donde se expuso a la desorción durante cuatro minutos a 150°C con el modo “splitless”. El equipo utilizado es un espectrómetro de masas Agilent 5973 Network junto con un cromatógrafo de gases, modelo 6890N de Agilent. The absorption of the released VOCs was produced by placing the fiber of the exposed holder inside the vial without touching the rice sample, for 15 minutes. After this operation, the "holder" was inserted into the GC injector where it was exposed to desorption for four minutes at 150 ° C with the "splitless" mode. The equipment used is an Agilent 5973 Network Mass Spectrometer in conjunction with an Agilent Model 6890N Gas Chromatograph.
El programa de temperatura utilizado para la cromatografía fue: temperatura inicial de 35°C durante 5 min y a 3°C/min a 150°C durante 1 min. Posteriormente a 5°C/min a 250°C hasta el final del análisis. El análisis cromatográfico duró 38 minutos. La columna utilizada es un DB624 de J&W Scientific de 30 m, 0,25 mm ID y 1,4 mm. La fuente de ionización para impacto electrónico a 70eV y 230°C. Se utilizó un simple cuadrupolo como detector, y su temperatura fue de 150°C. Wiley275 es la biblioteca utilizada para identificar detalles de los COVs. The temperature program used for the chromatography was: initial temperature of 35 ° C for 5 min and at 3 ° C / min at 150 ° C for 1 min. Subsequently at 5 ° C / min at 250 ° C until the end of the analysis. Chromatographic analysis lasted 38 minutes. The column used is a J&W Scientific 30m, 0.25mm ID, 1.4mm DB624. The ionization source for electronic impact at 70eV and 230 ° C. A simple quadrupole was used as a detector, and its temperature was 150 ° C. Wiley275 is the library used to identify details of VOCs.
Los perfiles metabólicos de los hongos involucrados en el estudio se revisaron sin los COVs identificados en el perfil control, la fibra sin muestra y los perfiles metabólicos del sustrato control, arroz autoclavado sin inoculo de hongo. Además, de los COVs Totales (T-COVs) característicos de cada hongo, se seleccionaron dos categorías de COVs: los COVs mayoritarios (M-COVs) y los COVs minoritarios (m-COV). Los M-COVs están representados por todas aquellas sustancias que han presentado una coincidencia ³ 50% con compuestos de la biblioteca y una abundancia máxima > 100000 ppm. Los m-COVs están, por otra parte, representados por todos los compuestos detectados que presentaron una concordancia de ³ 50% y una abundancia máxima entre 20000 ppm y 100000 ppm. Bb1TS11 presentó 49 T-COVs diferentes, característicos de su perfil metabólico (Tabla 1; Figura 2 y 3). De éstos, solo tres compuestos, que representan el 6,1% del total, se encontraron pertenecientes a la categoría M-COVs; mientras que, se encontró que 12 compuestos diferentes pertenecían a la categoría de m-COVs y representan el 24,5% del total. De los M-COVs, se encontró 3-ciclohepten-l-ona (C7) en las seis mediciones realizadas durante los 60 días de crecimiento de hongos; mientras que, la N-etil-bencenamina se detectó solo en las mediciones realizadas a los 30 y 40 días después de la inoculación del sustrato. Finalmente, el 1,3-octadieno es el tercer M-COVs y se identificó solo en la medición realizada 50 días después de la inoculación del sustrato. Entre los m-COVs solo se detectó borneol (C4) a los 10, 20, 40 y 50 días a partir del inoculo. Los otros m-COVs de Bb1TS11 se detallan en la Tabla 1. También se detectó benzothiazol (C2). 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 fungus inoculum. In addition, from the Total VOCs (T-VOCs) characteristic of each fungus, two categories of VOCs were selected: the majority VOCs (M-VOCs) and the minority VOCs (m-VOCs). The M-VOCs are represented by all those substances that have presented a coincidence ³ 50% with compounds of the library and a maximum abundance> 100000 ppm. The m-VOCs are, on the other hand, represented by all the detected compounds that presented a concordance of ³ 50% 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-COVs category; while, it was found that 12 different compounds belonged to the category of m-VOCs and represent 24.5% of the total. Of the M-VOCs, 3-cyclohepten-l-one (C7) was found in the six measurements made during the 60 days of fungal growth; while, N-ethyl-benzenamine was detected only in measurements carried out at 30 and 40 days after inoculation of the substrate. Finally, 1,3-octadiene is the third M-VOCs and it was identified only in the measurement carried out 50 days after inoculation of the substrate. Among the m-VOCs, only borneol (C4) was detected at 10, 20, 40 and 50 days from the inoculum. The other m-VOCs of Bb1TS11 are detailed in Table 1. Benzothiazole (C2) was also detected.
Tabla 1. COVs detectados en el hongo Bb1TS11 a los 10-60 días
Figure imgf000011_0002
Figure imgf000011_0001
Figure imgf000012_0001
Table 1. VOCs detected in the fungus Bb1TS11 at 10-60 days
Figure imgf000011_0002
Figure imgf000011_0001
Figure imgf000012_0001
Figure imgf000013_0001
Ma4TS0 m4 ostró 49 T-COVs diferentes característicos de su perfil metabólico (Tabla 2; Figura 4). De estos, solo seis compuestos, que representan el 12.2% del total, se encontraron pertenecientes a la categoría de M-COVs; mientras que, se encontró que 11 compuestos diferentes pertenecían a la categoría de m-COVs y representaban el 22.4% del total. De los M- COVs, se encontró 3-ciclohepten-l-ona (C7) de 10 a 30 días después del inoculo; mientras que l-octen-3-ol (C6) se detectó como M-VOC solo en las mediciones realizadas a los 20 y 30 días desde la inoculación del sustrato. El 4-fluoro-l,4-xileno se detectó en las mediciones realizadas los 20 y 60 días de la inoculación del sustrato. También, el 1,2-dipentil-ciclopropeno se detectó en las mediciones realizadas los 10 y 50 días después de la inoculación. También se detectó 1,3-dimetoxibenceno (C5). Los otros M-COVs de Ma4TS04 se detallan en la tabla. Entre los m-COVs, el (+ -)-gymnomitrene se detectó a los 10, 20 y 40 días desde el inoculo. También, se encontró l-octen-3-ol (C6) como m-COVs solo a los 10 y 40 días desde el inoculo. Los otros m-COVs deMa4TS04 se detallan en la Tabla 2.
Figure imgf000013_0001
Ma4TS0 m4 showed 49 different T-COVs 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 category of M-VOCs; while, it was found that 11 different compounds belonged to the category of m-VOCs and represented 22.4% of the total. Of the M-COVs, 3-cyclohepten-l-one (C7) was found 10 to 30 days after inoculum; while l-octen-3-ol (C6) was detected as M-VOC only in measurements carried out 20 and 30 days after inoculation of the substrate. 4-fluoro-l, 4-xylene was detected in measurements made 20 and 60 days after inoculation of the substrate. Also, 1,2-dipentyl-cyclopropene was detected in measurements made 10 and 50 days after inoculation. 1,3-Dimethoxybenzene (C5) was also detected. The other M-VOCs of Ma4TS04 are detailed in the table. Among the m-VOCs, (+ -) - gymnomitrene was detected 10, 20 and 40 days after the inoculum. Also, l-octen-3-ol (C6) was found as m-COVs only at 10 and 40 days from the inoculum. The other m-VOCs of Ma4TS04 are detailed in Table 2.
Tabla 2. COVs detectados en el hongo Ma4TS04 a los 10-60 días
Figure imgf000014_0001
Figure imgf000015_0001
Table 2. VOCs detected in the Ma4TS04 fungus at 10-60 days
Figure imgf000014_0001
Figure imgf000015_0001
Figure imgf000016_0001
Pc123 mostró 111 T-COVs diferentes característicos de su perfil metabólico (Tabla 3; Figura 5). De estos, solo 14 compuestos, que representan el 12.6% del total, se encontraron pertenecientes a la categoría de M-COVs; mientras que, se encontró que 43 compuestos diferentes pertenecían a la categoría de m-COVs y representan el 38,7% del total. De los M- COVs, se encontró 1,3-dimetoxibenceno (C5) en las seis mediciones realizadas durante el crecimiento del hongo; mientras que l-octen-3-ol (C6) ha sido detectado como M-COVs solo en mediciones realizadas a los 10, 20, 30 y 60 días desde la inoculación del sustrato. A los 20, 40 y 50 días de la inoculación, se identificó la 3-octanona; mientras que 3-ciclohepten-l-ona (C7) se encontró solo a los 10 y 20 días desde el inoculo. Entre los m-COVs, se detectó 2-(2- etoxietoxi)-etanol a los 30 y 40 días desde el inoculo. Propionoin, 2-butanona y 2-dodecanona se detectaron a los 40 y 50 días desde el inoculo. También, 16-oxosalutaridina y 3-metil-butanal se detectaron, pero a los 50 y 60 días. Los otros m-COVs dePc123 se detallan en la Tabla 3.
Figure imgf000016_0001
Pc123 showed 111 different T-COVs 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 category of M-VOCs; while, it was found that 43 different compounds belonged to the category of m-VOCs and represent 38.7% of the total. Of the M-COVs, 1,3-dimethoxybenzene (C5) was found in the six measurements made during the growth of the fungus; while l-octen-3-ol (C6) has been detected as M-COVs only in measurements performed at 10, 20, 30 and 60 days after inoculation of the substrate. At 20, 40 and 50 days after inoculation, 3-octanone was identified; while 3-cyclohepten-l-one (C7) was found only 10 and 20 days after the inoculum. Among the m-VOCs, 2- (2-ethoxyethoxy) -ethanol was detected at 30 and 40 days from the inoculum. Propionoin, 2-butanone and 2-dodecanone were detected 40 and 50 days after the inoculum. Also, 16-oxosalutaridine and 3-methyl-butanal were detected, but at 50 and 60 days. The other m-VOCs of Pc123 are detailed in Table 3.
Tabla 3. COVs detectados en el hongo Pc123 a los 10-60 días.
Figure imgf000017_0001
Figure imgf000018_0001
Figure imgf000019_0001
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Table 3. VOCs detected in the fungus Pc123 at 10-60 days.
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Descripción del olfactómetro de dos vías o tubo en “Y” Description of the two-way olfactometer or “Y” tube
Se construyeron olfactómetros de dos vías (tubo en “Y”) (Rhodes el al. 2012. The role of olfactory cues in the sequential radiation of a gall-boring beetle, Mordellistena convicta. Ecológica l Entomology. 37(6): 500-507.) para los bioensayos de laboratorio para comprobar la respuesta de los individuos adultos de PNP a estímulos con los mencionados compuestos orgánicos volátiles. El olfactómetro se construyó en su totalidad a partir de secciones de tubo de vidrio resistente al calor conectadas cada una por un cono hermético (5 mm). Los tubos de vidrio poseían un diámetro interno de 30 mm, la rama principal medía 200 mm y los brazos de la unión en “Y” 150 mm de longitud. El ángulo entre cada brazo y el cuerpo principal fue de 75°. El extremo de cada brazo se unió a un contenedor de vidrio (60 mm de longitud, 25 mm de diámetro) en el que se colocan las cámaras de olor (CO) (Bazzocchi G.G. and Maini S. 2000. Ruolo dei semiochimici volatili nella ricerca delTospite da parte del parassitoide Diglyphus isaea (Hymenoptera Eulophidae). Prove olfattometriche. Bollettino dell'Istituto di Entomología “G. Grandi” Universitá di Bologna, 54 , 143-154) en los cuales se insertaron los estímulos olfativos que se analizaron. El aire en el interior de cada brazo del olfactómetro se mantuvo constante para todos los experimentos con el flujo de aire natural. Los tubos se cubrieron para evitar el escape de los insectos. Al cambiar las sustancias de los ensayos, los frascos se lavaron con alcohol etílico (Benito Parraga, S. L.), agua y con n-hexano (PanReac AppliChem) para eliminar todos los restos de los volátiles de muestras anteriores. Two-way olfactometers (“Y” tube) were constructed (Rhodes el al. 2012. The role of olfactory cues in the sequential radiation of a gall-boring beetle, Mordellistena convicta. Ecológica l Entomology. 37 (6): 500- 507.) for laboratory bioassays to check 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 tube each connected by an airtight cone (5 mm). The glass tubes had an internal diameter of 30 mm, the main branch was 200 mm and the arms of the "Y" joint 150 mm in length. The angle between each arm and the main body was 75 °. The end of each arm was attached to a glass container (60 mm in length, 25 mm in diameter) in which the odor chambers (CO) are placed (Bazzocchi GG and Maini S. 2000. Ruolo dei semiochimici volatili nella ricerca delTospite gives part of the parassitoid Diglyphus isaea (Hymenoptera Eulophidae). Prove olfattometriche. Bollettino dell'Istituto di Entomología "G. Grandi" Universitá di Bologna, 54, 143-154) in which the olfactory stimuli that were 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 escape of insects. When changing the substances of the tests, the bottles were washed with ethyl alcohol (Benito Parraga, S. L.), water and with n-hexane (PanReac AppliChem) to eliminate all the traces of the volatiles from previous samples.
Bioensayos de comportamiento (olfactómetro de dos vías) utilizando los COVs propuestosBehavioral bioassays (two-way olfactometer) using the proposed VOCs
El olfactómetro de dos vías (ODV) se utilizó para evaluar el comportamiento del PNP sometido a la acción de diferentes estímulos olfativos (atrayentes y repelentes). Refiriéndose a los estudios realizados por J. Jalinas (2016) en PRP (Picudo Rojo de las Palmeras, Rhynchophorus ferrugineus), todos se realizaron colocando un individuo de PNP en el centro del brazo recto. Los insectos en prueba tienen 10 minutos para moverse y elegir o no un estímulo. En total se realizaron 10 bioensayos diferentes, con un total de 120 individuos de PNP para cada uno. Cada prueba consta de seis réplicas, cada una con 20 individuos. Los ensayos se agruparon en dos grupos diferentes: Pruebas de Condiciones Fisio- Ambientales (PCFAs) y Pruebas de Repelentes (PRs). The two-way olfactometer (ODV) was used to evaluate the behavior of PNP subjected to the action of different olfactory stimuli (attractants and repellants). Referring to the studies carried out by J. Jalinas (2016) in PRP (Red Palm Weevil, Rhynchophorus ferrugineus), all were carried out by placing a PNP individual in the center of the straight arm. The test insects have 10 minutes to move and choose or not a stimulus. In total, 10 different bioassays were carried out, with a total of 120 PNP individuals for each one. Each test consists of six replicas, each with 20 individuals. The tests were grouped into two different groups: Physio-Environmental Conditions Tests (PCFAs) and Repellent Tests (PRs).
En el primer grupo, llamado Pruebas de Condiciones Fisio- Ambientales (PCFAs), se realizaron cuatro pruebas en las que se probó la actividad atractiva del cormo/pseudotallo. De los dos CO, el atractivo natural se colocó en uno (a la derecha o a la izquierda se eligió al azar) mientras que, en el otro CO, no se colocaron atrayentes ni repelentes, solo aire ambiental. Con esta estimulación olfativa, se probaron dos condiciones fisio-ambientales diferentes. En estas pruebas, quisimos evaluar la incidencia de una condición ambiental, es decir, se evaluó la incidencia que la presencia de luz (L) o su ausencia (SL) tiene en el movimiento del PNP. Se sabe que el PNP tiene un hábito predominantemente nocturno (Gold C.S., Pena J.E., Karamura E.B. (2001). Biology and integrated pest management for the banana weevil Cosmopolites sordidus (Germar) (Coleóptera: Curculionidae). Integrated Pest Management Reviews , 6(2), 79-155). In the first group, called Physio-Environmental Conditions Tests (PCFAs), four tests were carried out in which the attractive activity of the corm / pseudostem was tested. Of the two COs, the natural attraction was placed in one (to the right or left was chosen randomly) while, in the other CO, no attractants or repellants were placed, only ambient air. With this olfactory stimulation, two different physio-environmental conditions were tested. In these tests, we wanted to evaluate the incidence of an environmental condition, that is, the incidence that the presence of light (L) or its absence (SL) has on the movement of the PNP was evaluated. 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).
Además, se probó el movimiento del PNP con una condición fisiológica como es el hambre. Se probaron dos subpoblaciones de PNP: la primera consistió en individuos con un arreglo de comida ( Musa sp.) ad libitum (SHa), mientras que otro estaba compuesto de individuos con al menos una semana de inanición (Ha). De esta manera, fue posible definir el dominio de las condiciones fisio-ambientales en las que C. sordidus tiene la mayor tasa de movimiento.In addition, the PNP movement was tested with a physiological condition such as hunger. Two subpopulations of PNP were tested: the first consisted of individuals with a food arrangement (Musa sp.) Ad libitum (SHa), while another consisted of individuals with at least one week of starvation (Ha). In this way, it was possible to define the domain of the physio-environmental conditions in which C. sordidus has the highest rate of movement.
En el segundo grupo, llamado Pruebas de Repelentes (PRs), se realizaron siete pruebas con PNP en condiciones SL-Ha. In the second group, called Repellent Tests (PRs), seven tests were carried out with PNP under SL-Ha conditions.
Las pruebas se realizaron con siete compuestos potencialmente repelentes (C1-C7), se utilizaron muestras comerciales de dichos compuestos (Sigma-Aldrich). En estas pruebas, en los CO, se colocaron en uno cormo/pseudotallo frescos y en el otro un dispensador de 0,5 mi (Cl, C2, C5, C6 y C7) o 0,5 g (C3 y C4) del repelente puro a ensayar. Los dispensadores utilizados se construyeron en el laboratorio utilizando sobres de miracloth (Merck KGaA) (3,5 cm x 2,5 cm) que contenían 2 g de gel de sílice 60A (70-200m, Cario Erba) en su interior. El volumen de compuesto se añadió directamente a la sílice. The tests were carried out with seven potentially repellent compounds (C1-C7), commercial samples of said compounds (Sigma-Aldrich) were used. In these tests, in the CO, fresh corm / pseudostem were placed in one and in the other a dispenser of 0.5 ml (Cl, C2, C5, C6 and C7) or 0.5 g (C3 and C4) of the repellent pure to rehearse. The dispensers used 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) inside. The volume of compound was added directly to the silica.
Los datos etológicos mostrados por los PNP, en las pruebas de ODV, se recopilaron en una hoja de cálculo de Excel. Como se muestra en la figura 6, los individuos de C. sordidus respondieron a la prueba de tres maneras diferentes: algunos fueron los estímulos El, que siempre incluían los atrayentes (cormo/pseudotallo y feromonas). Otros individuos han recurrido al estímulo E2; en este OC, todos los compuestos ensayados se colocaron individualmente (un compuesto/ensayo) o no se colocó ningún tipo de estímulo. Sin embargo, otros han preferido no elegir, deteniéndose en lo que hemos llamado "casa" (EC). The ethological data shown by the PNPs, in the ODV tests, were compiled in an Excel spreadsheet. As shown in Figure 6, individuals of C. sordidus responded to the test in three different ways: some were the El stimuli, which always included the attractants (corm / pseudostem and pheromones). Other individuals have resorted to the E2 stimulus; in this OC, all the tested compounds were placed individually (one compound / test) or no type of stimulus was placed. However, others have preferred not to choose, stopping at what we have called "home" (EC).
Esta triple expresión etológica se evaluó primero con índices de movimiento construidos ad hoc , para tener una medida rápida y explicativa de las pruebas únicas realizadas en ODVs. Además, los datos fúeron probados a través de la prueba estadística de chi-cuadrado, con el software estadístico Rstudio. This triple ethological expression was first evaluated with ad hoc constructed movement indices, to have a quick and explanatory measure of the unique tests performed on ODVs. In addition, the data were tested through the chi-square statistical test, with the statistical software Rstudio.
Los índices de movimiento nacen de la necesidad de tener una herramienta rápida y fácilmente explicativa y, al mismo tiempo, útil para resumir la situación etológica mostrada por los PNP en las pruebas. Movement indices arise from the need to have a quick and easy explanatory tool and, at the same time, useful to summarize the ethological situation shown by the NPPs in the tests.
Se supuso que una población de PNP, puesta en ODV y la ausencia de algún estímulo se distribuiría de manera uniforme en las tres opciones posibles mientras se mantenía una relación También se consideró que los individuos que permanecen en la EC
Figure imgf000023_0002
son aquellos que no respondieron al estímulo atractivo (en el caso en que no se colocó ningún estímulo en E2) o que fueron rechazados por la sustancia probada (en el caso en que E2 era colocado un candidato repelente). Por lo tanto, se construyeron tres índices que tomaron en consideración la relación entre los individuos en EC, E1 y E2 y el número total de individuos evaluados ( N ). Los tres índices son los siguientes:
Figure imgf000023_0001
donde: mi = número de individuos que han elegido E1, nE2 = número de individuos que han elegido E2, nEC = número de individuos que permanecen en la EC, N = número total de individuos analizados. It was assumed that a population of PNP, put in ODV and the absence of any stimulus would be distributed evenly in the three possible options while maintaining a relationship. It was also considered that the individuals who remain in the CD
Figure imgf000023_0002
are those that did not respond to the attractive stimulus (in the case in which no stimulus was placed in E2) or that were rejected by the tested substance (in the case in which E2 was placed a repellent candidate). Therefore, three indices were constructed that took into account the relationship between individuals in CE, E1 and E2 and the total number of individuals evaluated (N). The three indices are as follows:
Figure imgf000023_0001
where: mi = number of individuals who have chosen E1, n E2 = number of individuals who have chosen E2, n CS = number of individuals who remain in the CS, N = total number of individuals analyzed.
Finalmente, para obtener un índice que tome en consideración todas estas relaciones entre grupos de individuos, los índices IE1 , IE2 e IEC se han resumido en un solo índice: IM (índice de Movimiento):
Figure imgf000024_0001
donde: 0 < IM> + ¥. Cuanto más cerca está de cero el IM, más significativa es la porción de la población que ha permanecido inmóvil, sin reaccionar o capturar el estímulo atractivo. Finally, to obtain an index that takes into account all these relationships between groups of individuals, the I E1 , I E2 and I EC indices have been summarized in a single index: MI (Movement index):
Figure imgf000024_0001
where: 0 <IM> + ¥. The closer the MI is to zero, the more significant is the portion of the population that has remained immobile, without reacting or capturing the attractive stimulus.
El IM se calculó para cada uno de los seis ensayos (N= 20 PNP), generando una serie de datos para cada prueba. Luego, con el software estadístico R, se realizaron pruebas ANOVA en PCFAs, PRs. MI was calculated for each of the six trials (N = 20 PNP), generating a data series for each trial. Then, with the statistical software R, ANOVA tests were performed on PCFAs, PRs.
Las condiciones fisio-ambientales en realidad interfieren con la movilidad de las PNP (X2 = 17.952; df= 6; p-valor = 0.006352). A partir del análisis de ANOVA realizado en los IMs de las réplicas individuales, hubo una diferencia significativa entre las condiciones (F-valor = 3.305; p-valor = 0.0413). En la prueba de Tukey (Figura 7) podemos ver cómo las condiciones de sin luz (SL) y sin hambre (SHa) SL-SHa (IM = 0.53) son aquellas en las que el estímulo alimentario atrae más a los PNPs. El hábito nocturno de el PNP hace que el insecto, en ausencia de luz (SL), sea más móvil; además, la condición de inanición (Ha) estimula aún más los PNPs en la búsqueda de alimentos y, por lo tanto, le da mayor movilidad. La actitud nocturna del PNP es evidente ya que las pruebas realizadas en condiciones de SL, es decir, SL-Ha y SL-SHa (IM= 0,48), muestran una mayor movilidad de los PNP con respecto a las condiciones con luz (L), o L-Ha (luz-hambre) (IM= 0.36) y L-SHa (IM= 0.22). Por estas razones, la condición SL- Ha se usó como condición experimental para las PRs. Physio-environmental conditions actually interfere with PNP mobility (X 2 = 17.952; df = 6; p-value = 0.006352). From the ANOVA analysis performed on the MIs of the individual replicas, 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 (MI = 0.53) are those in which the food stimulus attracts more PNPs. The nocturnal habit of the PNP makes the insect, in the absence of light (SL), more mobile; Furthermore, the starvation condition (Ha) further stimulates the PNPs in the search for food and, therefore, gives them greater mobility. The nocturnal attitude of the PNP is evident since the tests carried out in SL conditions, that is, SL-Ha and SL-SHa (MI = 0.48), show a greater mobility of the PNP with respect to the light conditions ( L), or L-Ha (light-hunger) (MI = 0.36) and L-SHa (MI = 0.22). For these reasons, the SL-Ha condition was used as an experimental condition for the PRs.
Del análisis de los datos relativos a las PRs más el control (SL-Ha), resultó que hay una significación (X2 = 34.142; df= 7; p-valor = 1.62e-5) y que, por lo tanto, el fenómeno observado sigue un patrón que no es accidental. A partir del análisis de ANOVA de los IMs , surgió que hay diferencias estadísticamente significativas entre las pruebas bajo examen (F-valor = 2.798; p-valor = 0.0181). De la prueba de Tukey (Figura 8) está claro que todos los COVs probados tienen una acción repelente hacia el PNP, disminuyendo su movilidad con respecto al control. El compuesto C7 resulta ser el que tiene la mayor acción repelente, registrando el valor de IM más bajo. No parece haber una diferencia significativa entre C1 y C6 con respecto a la tasa de repelencia. Por lo tanto, según los valores de los IMs, los diferentes COVs muestran el siguiente gradiente de repelencia: C7 (IM= 0,11), C5 (IM= 0,18), C2 (IM= 0,26), C1 (IM= 0 ,28), C4 (IM= 0,28), C3 (IM= 0.45) y C6 (IM= 0.47). From the analysis of the data relative 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 ) and that, therefore, the The observed phenomenon follows a pattern that is not accidental. From the ANOVA analysis of the MIs, it emerged that there are statistically significant differences between the tests under examination (F-value = 2.798; p-value = 0.0181). From the Tukey test (Figure 8) it is clear that all VOCs tested They have a repellent action towards the PNP, decreasing its mobility with respect to the control. Compound C7 turns out to be the one with the highest repellent action, registering the lowest MI value. There does not appear to be a significant difference between C1 and C6 with respect to the repellency rate. Therefore, according to the MI values, the different VOCs show the following repellency gradient: C7 (MI = 0.11), C5 (MI = 0.18), C2 (MI = 0.26), C1 ( MI = 0.28), C4 (MI = 0.28), C3 (MI = 0.45) and C6 (MI = 0.47).

Claims

REIVINDICACIONES
1. Uso de al menos un compuesto orgánico volátil de un hongo entomopatógeno y/o nematófago, seleccionado de entre estireno, benzotiazol, alcanfor, borneol, 1,3- dimetoxibenceno, 1-octen-3-ol, 3-ciclohepten-1-ona como repelente de Cosmopolites sordidus. 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- ona as a repellent for Cosmopolites sordidus.
2. Uso de al menos un compuesto orgánico volátil de un hongo entomopatógeno y/o nematófago según la reivindicación 1, donde el hongo entomopatógeno es seleccionado de entr Beauveria bassiana y Metarhizium anisopliae. 2. Use of at least one volatile organic compound of an entomopathogenic and / or nematophagous fungus according to claim 1, wherein the entomopathogenic fungus is selected from Beauveria bassiana and Metarhizium anisopliae.
3. Uso de al menos un compuesto orgánico volátil de un hongo entomopatógeno y/o nematófago según cualquiera de las reivindicaciones anteriores, donde el hongo nematófago es Pochonia clamydosporia. 3. Use of at least one volatile organic compound of an entomopathogenic and / or nematophagous fungus according to any of the preceding claims, wherein the nematophagous fungus is Pochonia chlamydosporia.
4. Uso de al menos un compuesto orgánico volátil de un hongo entomopatógeno y/o nematófago según cualquiera de las reivindicaciones anteriores, donde el hongo es Beauveria bassiana y el compuesto orgánico volátil es seleccionado de entre estireno (C8H8), benzotiazol (C7H5NS), alcanfor (C 10H 16O) y borneol. 4. Use of at least one volatile organic compound of an entomopathogenic and / or nematophagous fungus according to any of the preceding claims, where the fungus is Beauveria bassiana and the volatile organic compound is selected from styrene (C 8 H 8 ), benzothiazole ( C 7 H 5 NS), camphor (C 10 H 16 O) and borneol.
5. Uso de al menos un compuesto orgánico volátil de un hongo entomopatógeno y/o nematófago según cualquiera de las reivindicaciones anteriores, donde el hongo es Metarhizium anisopliae y el compuesto orgánico volátil es seleccionado de entre 1,3-dimetoxibenceno (C6H4(OCH3)2), 1-octen-3-ol (CH3(CH2)4CHCH=CH2), 3-ciclohepten-l-ona (C7H10O). 5. Use of at least one volatile organic compound of an entomopathogenic and / or nematophage fungus according to any of the preceding claims, where the fungus is Metarhizium anisopliae and the volatile organic compound is selected from 1,3-dimethoxybenzene (C 6 H 4 (OCH 3 ) 2 ), 1-octen-3-ol (CH 3 (CH 2 ) 4 CHCH = CH 2 ), 3-cyclohepten-l-one (C 7 H 10 O).
6. Uso de al menos un compuesto orgánico volátil de un hongo entomopatógeno y/o nematófago según cualquiera de las reivindicaciones anteriores, donde el hongo es Pochonia clamydosporia y el compuesto orgánico volátil es seleccionado de entre 1,3-dimetoxibenceno (C6H4(OCH3)2), 1-octen-3-ol (CH3(CH2)4CHCH=CH2), 3-ciclohepten-l-ona (C7H10O), para su uso como repelente de Cosmopolites sordidus. 6. Use of at least one volatile organic compound of an entomopathogenic and / or nematophagous fungus according to any of the preceding claims, where the fungus is Pochonia clamydosporia and the volatile organic compound is selected from 1,3-dimethoxybenzene (C 6 H 4 (OCH 3 ) 2 ), 1-octen-3-ol (CH 3 (CH 2 ) 4 CHCH = CH 2 ), 3-cyclohepten-l-one (C 7 H 10 O), for use as a repellent for Cosmopolites sordidus.
7. Uso de al menos un compuesto orgánico volátil según cualquiera de las reivindicaciones anteriores, donde el compuesto orgánico volátil es obtenido por síntesis química. 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. Uso de al menos un compuesto orgánico volátil según cualquiera de las reivindicaciones 1- 7, donde el compuesto orgánico volátil está en forma sólida. 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. Uso de al menos un compuesto orgánico volátil según cualquiera de las reivindicaciones 1- 7, donde el compuesto orgánico volátil está en forma líquida. 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. Uso de al menos un compuesto orgánico volátil según la reivindicación 9, donde la formulación líquida está impregnada en una matriz. 10. Use of at least one volatile organic compound according to claim 9, wherein the liquid formulation is impregnated in a matrix.
11. Uso de al menos un compuesto orgánico volátil según cualquiera de las reivindicaciones 1- 7, donde el compuesto orgánico volátil está en forma gel. 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. Uso de una composición que comprende al menos un compuesto orgánico volátil según cualquiera de las reivindicaciones 1-12, como repelente de Cosmopolites sordidus. 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.
PCT/ES2020/070563 2019-09-25 2020-09-18 Use of volatile organic compounds of parasitic fungi of invertebrates as repellents of the black banana weevil (cosmopolites sordidus) WO2021058844A1 (en)

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CONC2022/0005126A CO2022005126A2 (en) 2019-09-25 2022-04-25 Repellent composition based on volatile organic compounds of parasitic fungi of invertebrates as repellents of the black banana weevil (Cosmopolites sordidus)

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