ES2815148A1 - USE OF VOLATILE ORGANIC COMPOUNDS FROM PARASITE FUNGI OF INVERTEBRATES AS REPELLENTS OF THE BLACK PICUDO OF THE PLANTAIN (COSMOPOLITES SORDIDUS) (Machine-translation by Google Translate, not legally binding) - Google Patents

Abstract

Use of volatile organic compounds from parasitic fungi of invertebrates as repellants for the banana weevil (Cosmopolites sordidus). The present invention relates to the use of the volatile organic compounds of B. bassiana, M anisopliae and P. chlamydosporia selected from among styrene, benzothiazole, camphor, borneol, 1,3-dimethoxybenzene, 1-octen-3-o1, 3- cyclohepten-1-one, as repellants of Cosmopolites sordidus. (Machine-translation by Google Translate, not legally binding)

Description

DESCRIPTION

USE OF VOLATILE ORGANIC COMPOUNDS FROM PARASITE FUNGI OF INVERTEBRATES AS REPELLENTS OF THE BLACK PICUDO OF THE PLANTAIN ( COSMOPOLITES SORDIDUS)

Field of the invention

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).

Prior state of the art

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. Indian journal of microbiology, 52 (4), 569-574). World production is around 114 million tons per year (FAO, 2018).

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 (Coleoptera: Curculionidae). Journal of economic entomology, 94 (2), 449-454; Muñoz-Ruiz, C. 2007. Population fluctuation of the banana weevil (Cosmopolites sordidus Germar) (Musa AAB) in San Carlos, Costa Rica. Revista Tecnología en Marcha, 20 ( 1), 24-41).

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 andCo. Ltd., London, pp. 512) that currently spreads as a plague to all regions where banana is grown, and in Spain, it is present, above all, in the Canary Islands (Carnero HA et al. 2002. Alternative methods for the control of the banana weevil Cosmopolites sordidus Germar, 1824 (Coleoptera: Curculionidae). ICIA activities in Platanera, p. 75).

The invasion and spread 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.

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 gas phase by vaporization at room temperature (20 ° C and 0.01 kPa; Pagans E. et al. 2006. Emission of volatile organic compounds from composting of different solid wastes : abatement by biofiltration. Journal of hazardous materials, 131 (1-3), 179-186).

The emission of VOCs performs essential ecological and physiological functions for many organisms, such as fungi, which release a wide spectrum of VOCs (Splivallo R. et al.

2011. Truffle volatiles: from chemical ecology to aroma biosynthesis. New Phytologist, 189 (3), 688-699; Kramer R. and Abraham WR 2012. Volatile sesquiterpenes from fungi: what are they good for ?. Phytochemistry Reviews, 11 (1), 15-37). Emissions of fungal VOCs belong to different chemical groups, such as monoterpenoids, sesquiterpenes, alcohols, aldehydes, aromatic compounds, esters, furans, hydrocarbons, ketones or lactones (Campos VP et al. 2010. Volatiles produced by interacting microorganisms potentially useful for the control of plant pathogens. Ciencia e Agrotecnologia, 34 (3), 525-535; Kramer R. and Abraham WR 2012. Volatile sesquiterpenes from fungi: what are they good for? Phytochemistry Reviews, 11 (1), 15-37).

Fungi produce various volatile organic compounds (Crespo R. et al. 2008. Volatile organic compounds released by the entomopathogenic fungus Beauveria bassiana. Microbiological research, 163 (2), 148-151; Müller A. et al. 2013. Volatile profiles of fungichemotyping of species and ecological functions. Fungal Genetics andBiology, 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 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 effects of a mixture of indoor air volatile organics, their ozone oxidation products, and stress. Environmental health perspectives, 113 (11), 1542-1548).

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 Rhynchophorus ferrugineus , and have no effect on Cosmopolites sordidus .

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.

Explanation of the invention

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).

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, borneol, 1,3-dimethoxybenzene, 1-octen-3-ol, 3-cyclohepten-1-one as a repellent for Cosmopolites sordidus.

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.

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.

In a particular embodiment, the volatile organic compounds of the present invention are selected from:

Styrene (CsHs) (also referred to in the present invention as compound C1), belongs to the group of chemical compounds classified as organic vinyl compounds. C1 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 Bb 1TS11.

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 et al. 2010. Repellent activity of essential oils: a review. Bioresource technology, 101 (1), 372-378), it was selected as a repellent candidate, despite not being a 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 BMTS11.

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 (CH ³ (CH ² ) ⁴ CHCH = CH 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-cyclohepten-1-one (C ⁷ H ¹⁰ O) (also referred to in the present invention as compound C7), is a cyclic ketone identified by the metabolic profiles of Bb1TS11, Ma4TS04 and Pc 123 (Figure 1).

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.

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

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 compound the volatile organic compound of the present invention is selected from among styrene (C8H8), benzothiazole (C ⁷ H ⁵ NS), camphor (C ¹⁰ H ¹⁶ O) and borneol.

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 (C6H4 (OCH3) 2), 1-octen-3-ol (CH3 (CH2) 4CHCH = CH2), 3-cyclohepten-1-one (C ⁷ H ¹⁰ O).

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-1-one (C ⁷ H ¹⁰ O), for use as a repellent for Cosmopolites sordidus.

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

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

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.

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

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.

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).

Bb 1TS11 (CECT 21121; NCBI MK156717) and Ma4TS04 (CECT 21126; NCB IMK156715) 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 PNP population. While Pc123 was isolated from Heterodera avenae eggs, in Seville (ATCC No. MYA-4875; CECT No. 20929).

Brief description of the figures

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

Figure 2. Gas Chromatography / Mass Spectrometry (GCMS) analysis using SPME for B. bassiana 1TS11 of 20 days of incubation.

Figure 3. Gas Chromatography / Mass Spectrometry (GCMS) analysis using SPME for B. bassiana 1TS11 of 30 days of incubation.

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

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

Figure 6. Scheme of the stimuli placed in an olfactometer. E1 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 (PNP adults in dark and hungry conditions), SL-SHa (PNP adults in dark and hungry conditions), L-Ha (PNP adults in bright and hungry conditions), L- SHa (PNP adults in light conditions and without hunger)

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 (borneol); C5 (1,3-dimethoxybenzene); C6 (1-octen-3-ol); C7 (3-cyclohepten-1-one).

Detailed exposition of embodiments

Insects and banana corm / pseudostem used in bioassays

PNP adults were collected on the Island of Tenerife (Canary Islands, Spain) using pheromone traps baited with ECOSordidine60 (ecobertura®, ref .: 019-FACS60), based on sordidine. The insects were kept in boxes at 28 ± 0.5 ° C in the dark. Boxes plastic (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.

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.

Mushrooms

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

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).

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

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".

Four samples were prepared to analyze the VOCs, one for each fungal strain, plus one Rice sample not inoculated by 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.

The absorption of the VOCs released 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 30 µm, 0.25 mm ID, 1.4 µm 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. 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> 50% with compounds of the library and a maximum abundance> 100,000 ppm. The m-VOCs are, on the other hand, represented by all the detected compounds that presented a concordance of> 50% and a maximum abundance between 20,000 ppm and 100,000 ppm.

5M TS11 presented 49 different T-VOCs, 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-1-one (C7) was found in the six measurements performed 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 5MTS11 m-VOCs are detailed in Table 1. Benzothiazole (C2) was also detected.

Table 1. VOCs detected in the fungus 5MTS11 at 10-60 days

^{5 -me th oxy -1 -a za -6 -} 2271

3 1.29 NANANANANA

o x a b ic y c lo (3.1.0) h e x a n e 4

6 ¹⁷⁵⁷

^{M e th y ls ila ne 2.10} NANANANANA

290

24 ^{C is -1 -bu ty l-2 -} 13.49 NANA 16304 NANANA me th y lc yc lo p ro pane

25 ^{1 -o cte n e 13.50 N A 16066 N A N A N A N A} 26 ⁴⁵⁰²

^{1, 3 -oc ta d ie ne 15.20 NANANANA} NA 58

27 5008 47571 30613 12058 23341

3-cyclohepten-1-one 15.20 NA _(C7) 6 7 6 ₇ 5

^{M e th y l-1, 4 -d io x id e -} 4847

28 15, 24 NANANANANA

p y ra z in e 7

48 ^{2, 2, 4, 6, 6 -pen ta me th y l-} 21.31 NANA 14184 NANANA hep ta ne

53 ⁴⁰³⁵

^{M e th y lcy c lo hexane 21, 68} NANANANANA

1

54 4, 4 -d im e th y l-2 -pen te ne 21.69 NANA 21528 NANANA 57 ⁴²⁵⁴

^{4 -bu ty l-1 -ox id e -pyr id in e 21, 87} NANANANANA

8

58 ^{d ie th oxyme th y lo c ta decyl} 21.88 NANANANANA 95632

-s ila n e

^{2, 2 -d im e th and lN -} 2814

62 22, 01 NANANANANA

p h e n y lp ro p a n e th i ... 6

63 ^{9 -am in ophenan th re ne 22, 01 NA 27694 NANANANA} 67 ^{d ih yd ro -2 (3 H) -fu ra none 22, 30 NANA 14 115 NANANA} 72 2 -me th y l-2 -bo rn Jan 22 , 71 ²¹¹² NANANANANA

5

73 ^{3 -b ro mo -hep ta ne 22, 71 NANA 13693 NANANA} 78 ^{2 - (2 -e th oxye th oxy) -} 23, 60 NANA 20657 NANANA e th anol

^{2, 2, 4, 4, 6, 8, 8 -} 3894

79 24, 26 NANANANANA hep ta me th and ln onane 3

81 ^{1 -cyano -3 -me th y l-1, 3 -} 24,3 NANANA 25066 NANA cyc lo he xa d ...

82 2, 3 -d im e th y l-1 -pen te ne 24, 30 NANA 254 68 NANANA 87 2, 2, 4 -tr im e th and lp in ta nol ^{24, 77} NANA 16 370 NANANA 32, 99

88 ^{3 -e th and lh ep ta ne 24, 77 NANA 50 802 29098 NANA 1 -me th and l-1 H -1, 2, 4 -} 3361

97 25, 58 NANANANANA tr ia zo le 9

100 ^{2 -me th y l-3, 4 -d ih yd ro -} 25, 70 NANA 18 525 NANANA 2 H -p yra n

^{N -ace ty l-3, 4 -d im e th oxy -} 4744 111 26, 49 NANANANA 38741 pheny lp ro py la m in e 2

113 ¹⁷⁸²

^{B icyc lo o cty l 26, 86 NANANANA} NA 3

115 ^{(-) - la vandu lo l 26, 87 NANA 49139 NANANA} 117 ^{1 -e th and l-1, 3 -d im e th and l-} 26, 87 NANANA 39845 NANA cy c lo hexane

1-1832 124 me th y la lly l (cyc lo oc ta te tr 27, 22 NANANANANA 8 aene) tita n iu m

125 ^{4 -me th y l-1 -decene 27, 41 NANA 12049 NANANA 3, 5, 5 -tr im e th y l-} 2049 128 27, 90 NA 45584 NANANA cyc lo hexene 97

⁷³⁶⁵³ 5 0011 17614 130 ^{N -e th y lb en ze nam in e 27, 90 NANA} NA

0 5 1 141 9627 2055 _(C4) Borneol 29.30 ₃ 24965 NA 31486 ₁ NA 142 ^{2, 3, 3 -tr im e th y l-1, 4 -} 29, 36 NANA 45469 NANANA pen ta d ie ne

143 ²⁸⁷⁸

^{E xo -fe n ch ol 29, 44} NANANANANA

6

2 -c h lo ro -4 - (4 -30, 54

150 ^{me th oxyphenyl) -6 - (4 -} NANA 32 886 NANANA 31.5

n itro p h e n y l) p y r im id in e

154

Benzothiazole 31.00 NA NA 23 152 NA NA NA _(C2)

156 ^{1, 3 -d ie th and l-1, 3 -d im e th and l-} 31.48 NANANANANA 24181

1,3-b i ...

^{1, 3 -d im e th y l-1, 3 -} 3020 157 31.48 NANANANANA d ip heny lu re a 5 ^{(E) -2 - [(N -hydro xy -n -} 2 6, 06

160 NANANA 25065 NANA phenyl) -ami 31.49

^{5 -e th y l-3 -h} 166 ^{yd ro xy -4 -} 33, 21 NANA 20868 NANANA me th y l-2 (5 H) -fu ra none

170 ^{D id e cy l e ste r}

33, 47 NANA 13343 NANANA decaned io ic acid

174 2 -hexy l-1 -o cta nol 34, 71 NANA 14817 NANANA 176 ^{Phyto l 34, 90 NANANA 16500 NANA} 179 ^{H exy l-cy c lo pen te none 34, 98 NANA 19465 NANANA} 2 -me th y l- 4 - (2, 6, 6 - 183 ^{tr im e th y l-1 -cyc lo hexen -} 36, 27 NANA 17549 NANANA 1 -yl) -fo rm a te, (E) -1 -bu te n -1 -ol

189 ^{(+ -) - g y m n o m itre n e 37, 10 N A N A 11441 N A N A 20992}

Ma4TS04 showed 49 different T-VOCs characteristic of its metabolic profile (Table 2; Figure 4). Of these, only six compounds, representing 12.2% of the total, were found to belong to the 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-VOCs, 3-cyclohepten-1-one (C7) was found 10 to 30 days after inoculum; while 1-octen-3-ol (C6) was detected as M-VOC only in the measurements carried out at 20 and 30 days after the inoculation of the substrate. 4-fluoro-1,4-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 m-VOCs, (+ -) - gymnomitrene was detected 10, 20 and 40 days after inoculum. Also, 1-octen-3-ol (C6) was found as m-COVs only 10 and 40 days after inoculum. The other m-VOCs of Ma4TS04 are detailed in Table 2.

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

Ret. Peak Peak Peak Peak Peak Peak Num. Compound Time Height Height Height Height Height (min) 10d 20d 30d 40d 50d 60d 1 Oxirane ^{1.24 -} NA 52843 NANANANA _2.03

1.25 15 459

2 Acetaldehyde NANANANANA

2.45 98

1,1'- 4 bibicyclo (2.2.2) oct 1.32 ^{46298 NANANANANA} yl-4-ca ...

11 Methanamine 3.08 ¹⁷⁰⁶⁸

^NANA NANANA

76

3-methyl-1-

20 ^13.28 NANA 10 542 NANANA

butanol

21 2-chloro-octane 13.45 ^{NANA 11851 NANANA} 26 1.3-octadiene 15.20 ^{NANANA 64942 NANA} 27 3-cyclohepten-1- 24612 10352

15.20 274698 NA NA NA (C7) one 4 ₉

3,5-dimethyl-2- 45 ^20,61 NANANANA 37336 NA propylthiophene

47 1-0-pinene ^{21,24 NANANANANA 41207} Tricholorocyclohex

49 21.40

yl- ^{NANA 17034 NANANA} silane

2,3,3-trimethyl-1- 21.68

52 NA 18503 NANANANA butene 25.58

2,2-dimethyl-N- 62 phenylpropanethi .. 22.01 ^{28023 NANANANANA}

octahydro-2,2,4,4,7,7- 64 ^22.01 NA 23193 Nananana hexamethyl-trans-ih-Indene

68

1 ³⁸⁵³⁷ 14183

_(C6) -octen-3-ol 22.36 26983 _{5 3} 28896 NA NA 3-methoxy- 70 ^22.56 NANANANA 18640 NA benzenamine

1-methyl-4- (1- 76 methylethyl) - 23.18 ^{NANA 28806 NANANA benzene}

2- (2- 78 ethoxyethoxy) - 23.60 ^{NANA 11435 15463 NANA} ethanol

1-cyano-3-methyl- 81 ^24.3 NANANANA 19804 NA 1,3-cyclohexad ...

4- 83 pyridinecarbonitril 24.30 ^{NANANA 29028 NANA} e

6-methyl- (E) -3- 89 24.77 ^{NANANANA 23053 NA} undecene

tert-butyl- 90 isopropyldenecycl 24.77 ^{NANA 14598 NANANA} opropyl-ether

(2-methyl-1- 96 25.57 ^NANA 10315 NANANA propenyl) -benzene

2,2-dimethyl- 99 25.63

propanal ^{NANANANA 18633 NA} 2-amino-3,5- 107 dibromo-6- ^{26,49 NANANANA 23956 NA} methylpyridine

4-hydroxymandelic

109 acid ethyl ester di- 26.49 ^{NANANANANA 17357}

TMS

N-acetyl-3,4-dimethoxy- 111 26.49

phenylpropylamin ^{NANANA 45725 NANA} e

cyclohexane, (1.2- 114 ^26.86 NANANANANA 17450 dimethybutyl ...

1-ethyl-1,3- 117 dimethyl- 26.87 ^{NANANA 43936 32380 NA} cyclohexane

2,6-dimethyl-2- 118 ^26.87 NANA 16844 NANANA trans-6-octadiene

4-ethyl-2,3- 119 ^26.87 NA 27777 NANANANA dimethyl-2-hexene

126 4-octen-3-ol 27.55 ^{NANANANA 86926 NA} 1,2- 127 dipentylcycloprope 27.90 ³⁷²⁰⁴

^{372252 NANANA} NA 8 ne

3,5,5-trimethyl- 53550

128 ^27.90 NANANANANA cyclohexene 7

56405 21279 129 4-fluoro-1,2-xylene 27.90 ^NA NANANA

5 5 N-ethyl- 27609

130 ^27.90 NANANANANA benzenamine 7

136 1,3-dimethoxy- ^28.75 263989 NA NA NA NA NA (C5) benzene

1,5-diisopropyl- 137 2,3-dimethyl- ^{28,77 NANANANANA 20923} cyclohexane

2 (5H) -furanone, 5- 138 ^28.89 NANANA 16709 NANA (2-methyl-2-p ...

1,3,5,7,9-30,54

149 pentaethylcyclope NANA 47968 NANANA 31.49

ntas.

9H- 151 pyrrolo [3 ', 4': 3.4] p and 30.54 ^{NANA 15606 NANANA} rrolo [2 ...

1- (2-furanyl) -3- 152 30.96

butene-1,2-diol ^{NANANA 13738 NANA} 2,4-bis (1,1- 153 dimethylethyl) - 31.00 ^{NA 27688 NANANANA} phenol

159 5-oxoisoboldine 31.48 ^{NANANANA 23875 NA} 4,4-dimethyl-6- 161 benzylamino- 31.50 ^{NANANANANA 17950} dihy ...

176 Phytol 34.90 ^{NANANA 22565 NANA} 2 - [(5- 181 methoxycarbonyl- 35.60 ^{NANA 16521 NANANA} 8-quinoly ...

189 (+ -) - gymnomitrene 37.10 ^{27698 22167 NA 35769 NANA} 1,4- 190 bis (methylene) - 37.10 ^{NANA 37375 NANANA} cyclohexane

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

belonging to the category of M-VOCs; while, it was found that 43 compounds

different belonged to the category of m-VOCs and represented 38.7% of the total. Of the M-

VOCs, 1,3-dimethoxybenzene (C5) was found in the six measurements made during the

fungus growth; while 1-octen-3-ol (C6) has been detected as M-COVs only

in measurements made at 10, 20, 30 and 60 days from the inoculation of the substrate. At 20,

40 and 50 days after inoculation, 3-octanone was identified; while 3-cyclohepten-1-one

(C7) was found only 10 and 20 days after the inoculum. Among the m-VOCs, 2- (2-

ethoxyethoxy) -ethanol at 30 and 40 days from the inoculum. Propionoin, 2-butanone and 2-dodecanone

they were detected 40 and 50 days after the inoculum. Also, 16-oxosalutaridine and 3-methyl-butanal

they were detected, but at 50 and 60 days. The other m-VOCs of Pc123 are detailed in Table 3.

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

Ret. Peak Peak Peak Peak Peak Peak Num. Compound Time Height Height Height Height Height Height (min) 10d 20d 30d 40d 50d 60d

^{1.24 -} 85045

1 O x ira ne 39543 NANANANA

2.03 8

^{1, 25} 16439

2 A ce ta ld ehyde NANANANANA

2.45 45

^{M onoammon iu m sa lt} 71189

5 1.69 NANANANANA ca rb am ic acid 1

6 ¹¹⁹³⁰

^{M e th y ls ila ne 2.10 NA} NANANANA

06

9 ^{1 71522}

^{2 -am in o, 1 -p ro} panol 2.42 NANA NANANA 3

12 2 -bu ta none 6.95 NANANA 86293 48305 NA 13 ^{M e th yl e th yl, 2 -bu ta none 6.99 NANA 19455 NANANA} 14 3 -me th y lb u ta nal 9, 10 NANA 53579 NA 20264 24319 15 ^{H e xa ne 9.41 NANANANANA 18917} 16 ^{2 -me th y lb u ta nal 9.43 NANA 56948 NANANA 6 5274} 12 031

17 D im e th y ld isu lf id e 12.60 NANANANA 4 6

18 ^{3 -hyd ro xy -2 -bu ta none 12.76 NA 3645 0 NANANANA} 19 ^{4 -me th y l-2 -pen ta none 13, 04 NANA 9583 NANANA} 22 3 -me th y lh ep ta ne 13, 45 NANANANA 2 7769 NA

2. 3 ¹⁹⁰⁴⁴

^{3 -me th and l-2 -pen ta none 13.46 NANANA} NANA 2

27 71 470 10274

3-cyclohepten-1-one 15.20 NA NA NA NA _(C7) 4 ₅₃

29 ^{(1 -me th y le th enyl) 15,31} NANA 22 283 NANANA cy c lo p ro penis

30 ^{3 -hyd ro xy -2 -pen ta none 16.41 NANANANA 15454 NA} 31 ^{D ie th y la m in e -D l 16.41 NANANA 19677 NANA} 32 ^{2, 4 -oc ta d ie ne 16, 64 NA 20369 NANANANA} 33 ^{4 -me th and l-3 -hexanone 16.90 NANA 16601 306 11 NANA} 34 ^{N onane 17.17 21416 NANANANANA} 35 ^{3 -me th and l-3 -pen te n -2 -one 17, 18 NANA 12427 NANANA} 36 ^{5 -me th y l-2 -hexanone 17.83 NANANA 6 0436 NANA} 37 a -p in ene 19.40 NANA 18225 NANANA 38 ^{2 -bu to xy -e th anol 19.79 NANANA 25455 NANA} 39 ^{A n is o le 19.80 3629 2 NANANA 16 902 NA} 40 ^{M e th oxyben ze ne 19.82 NANA 18 160 NANANA} 41 P ro p io no in 20, 01 NANANA 38713 23007 NA 42 2, 4 -d im e th y l-3 -hexene 20, 54 NANANA 83 694 NANA 43 ^{3, 4 -d im e th and l-2 -hexene 20.55 NANANANA 31 252 NA}

44 ^{T ra n s-3, 4 -d im e th y l-2 -} 20.55 NANA 83 798 NANANA hexene

46 ^{P -p in e n e 21, 20 N A N A N A N A N A 65861} fifty ^{2 -iso p ro p y l-5 -o x o h e x a n a l 21, 42 N A N A N A N A 434 64 N A}

51 ^{21, 42}

^{6 -me th and l-2 -hep ta none} NANANA 448 53 NANA 25.13

55 ³²¹⁷¹

^{P ro py lc yc lo hexane 21, 69 NANANANANA} 7

56 ^{16 640}

^{D im e th y l-tr isu lf id e 21, 79 NANANANA} NA 62

59 ^{E th eny lp en ta me th y l-} 21.88 NANA 528567 NANANA d is ilo xane

66 2 -me th y lp ro panam id e 22, 14 NA 1 3114 NANANANA 68 30837

1-octen-3-ol 22.36 ¹²³³⁶ 10836

557934 NA NA (C6) _{5 3} 7

12746 15531 10 270

69 3 -o cta none 22, 40 NANANA 5 46 78

71 T rim e th y lp y ra z in e 22, 70 NANANANA 84 154 NA 72 2 -me th y l-2 -bo rn Jan 22, 71 4673 4 NANANANANA 2, 4 -d im e th y lm e th yle ste r- 74 22.95 NA 34740 NANANANA hexane ic acid

1 -me th y l-4 - (1 -me th y le th yl) - 76 23, 18 NANANA 50055 1 6588 NA ben ze ne

77 Iso cyano -ben ze ne 23, 58 NANANANA 23748 NA 78 2 - (2 -e th oxye th oxy) -e th anol 23, 60 NANA 24 002 29 742 NANA 80 1 -ch lo ro -3 -me th y l-2 -bu te ne 24, 30 NANANA 52 096 NANA 1 -cyano -3 -me th y l-1,3 - 81 24, 30 NANANANA 26622 NA cyc lo he xa d ...

84 3, 3 -d im e th y l-1 -pen te ne 24, 31 NANA 26963 NANANA 85 2, 4 -d im e th y lfu ra n 24, 41 NANANANANA 23645

24, 77

87 2, 2, 4 -tr im e th and lp in ta nol NANANANANA 2 0166

32, 99

89 6 -me th and l- (E) -3 -undecene 24, 77 NANA 42811 NANANA 24, 9

91 2 -dode ca none NANANA 50080 74 332 NA 34, 28

92 4 -me th y ln onane 24, 99 NANA 14087 NANANAN - (1 -me th y lh ep ty l) -2 - 93 25.13 NANANANA 19895 NA o cta nam in e

2 -me th and l-2 - (2 -me th and l-2 - 94 25, 47 NANANA 31859 NANA bu te nyl) ...

95 B en ze neme th anol 25, 49 NANANANA 21457 NA 101 4 -T -bu ty lc yc lo hexanone 25, 70 NANANA 3 2817 NANA 6 -me th y l-4 5- 102 25, 70 NANA 17315 NANANA d ¡ hyd ro .a lp ha. [2 H] ...

26, 08 15 264 103 2 -nonanone NANANA 38 059 NA 29, 09 9

3, 3 -d im e th y l-5, 6, 7, 8 - 104 26, 40 NA 21503 NANANANA te tra me th y ...

6, 7 -d im e th oxy -2, 2 -d im e th y l- 105 26, 41 24845 NANANANANA 2 H -1 -ben zo py ra n

1 -a lly l-2, 8 -d im e th o x y -9 H - 15597

106 26, 49 NANANANANA ca rb a z. 3

3 -h y d ro x y m a n d e lic acid

108 26, 49 NA 30385 NANA 87 134 NA e th yle ste rd iT MS

4 -h y d ro x y m a n d e lic acid

109 26, 49 NANANANANA 4 4896 e th yle ste rd iT MS

4 -tr im e th y ls ily l-9, 9 - 110 26, 49 41815 NANANANANA d im e th y l-9 -s ila f lu o re ne

^{(1 -me th and lp ro pyl) -} 10535

116 26, 87 NANANANANA cy c lo o cta ne 9

120 ^{c is -2, 6 -d im e th y l-2, 6 -} 26, 87 20703 NANANANANA oc ta d ie ne

123 ^{E th y l-cy c lo o cta ne 26, 87 NANA 55671 NANANA} 1-13 460 124 me th y la lly l (cy c lo oc ta te tra in 27, 22 NANANANANA 6 e) tita n iu m

127 1,2 -d ip en ty lcy c lo p ro penis 27, 90 NA ¹⁷⁰⁶⁴ NANANANA

4

128 ^{3, 5, 5 -tr im e th y lc yc lo hexene 27, 90 NANA 70395 3 NANANA 12826} 34561 130 ^{N -e th y lb en ze nam in e 27, 90 NANANA} NA

31 9 ^{6 -me th y l-} 6081 2 134 27.93 NANANANANA b ic yc lo [4.1.0] hep ta n -2 -one 5

^{6 7237} 495 08 135 ^{1 -pheny l-2 -p ro panone 28, 22 NANANA} NA 9 3

136 72330 50976 358318 38071 30469 30833 ₍ 1,3-dimethoxy-benzene 28.75 _C5) 98 9 9 70 43 0 139 ^{2 -am in or -4 -me th y lp yr im id in e 29, 14 NA 50671 NANANANA} 1 ^{B ic yc lo [2.2.1] hep ta ne -2 -} 40 29, 14 45 509 NANANANANA ca rb oxy lic acid

145 ^{3 -me th y ls ila cyc lo pen t-3 -} 30, 20 NANA 51365 60183 NA 20685 Jan

146 ^{2 -me th y l-th io phene 30, 21 NANANANA 85 115 NA} 148 ^{(1 H) in do lo [2, 1 -} 30, 54 NANANA 50725 NANA a] iso qu in o lm e ...

2 -c h lo ro -4 - (4 -30, 54

150 ^{me th oxyphenyl) -6 - (4 -} NANA 43086 NANANA 31.5

n itro p h e n y l) p y r im id in e

153 ^{2, 4 -b is (1, 1 -d im e th y le th yl) -} 31.00 3 5545 NANANANANA ph en ol

155 ^{31, 15}

^{2 -undecanone} NANANANA 54235 NA 32, 18

158 ^{16 -oxo sa lu ta rid in e 31, 48 NANANANA 66 214 40033 (E) -2 - [(N -hyd ro xy -n -phenyl) -} 26, 06

160 NANA 17 510 NANANA am i 31.49

^{4 -d im e th y l-6 -b la mi} 161 ^{4, enzyne -} 31.50 NA 19560 NANANANA d ih y ...

162 c is -ch ry sa n th em ic acid 31.80 NANANANA 21985 NA 163 ^{2 -o cta none 32, 19 NANANA 20 918 NANA} 164 3, 5 -d im e th y lo c ta ne 32, 98 NANANA 19633 NANA 165 ^{1 -e th and l-2 -me th and l-} 33, 21 NANANA 22609 NANA cy c lo hexane

^{(2 R, 6 S) -2 -te rt} 167 ^{-bu ty l-6 -e th y l-} 33, 22 NANA 15548 NANANA 6 ...

168 ^{2, 6 -d im e th y lc yc lo hexanone 33, 40 NANA 12143 NANANA} 169 ^{3 -e th y l-3 -me th y lh ep ta ne 33, 44 NANANA 17805 NANA} 171 ^{5 -ace ty l-2 -hyd ra z in o -4 -} 33, 70 NANA 16684 25068 NANA me th y lp and rid in e

172 ^{B is (2, 4 -} 33, 76 NANANANA 16629 NA d im e th y la m in o) pyr im id in e

173 ¹⁰⁷⁷⁴

^{P -e le mene 34, 61 NANANANANA} 6 175 ^{2 -me th oxy -3, 8 -} 34, 86 NANA 16671 NANANA d io xocepha lo ta x -1 -ene

177 D ia my lcy c lo hexanol 34, 97 NANANANA 18490 NA 178 ^{Iso ja sm one 34, 97 NANANA 38429 NANA} 180 ^{8, 8 -d im e th y l-1, 9 -} 35, 54 NANANANANA 19535 d ¡azab ¡ cyclo [5 ...

182 ^{(Z) -3 -Iso p ro py l-3, 6 -d im e th y l-} 36, 27 NANANA 23284 NANA 4, 6 -hep ta d ie n -2 -one

184 C y c lo te tra decane 36, 68 NANANA 29215 NANA ^{B icy c lo [5.2.0] nonane, 4, 8, 8 -} 185 36, 86 NANANANANA 61558 tri ...

186 ^{D odecame th y l-} 36, 88 NA 18438 NANANANA pen ta s ilo xane

^{1 - (2 -tr im e th y ls ilo xy -1, 1 -} 187 36, 89 NANANA 31962 NANA d id eu t ...

3 - (b ro mome th yl) -1, 8 - 188 ^{d im e th oxy -9, 10 -} 36, 90 NANA 21429 NANANA an th ra ce ned io ne

191 ^{2 -tr id ecanone 37, 60 NANANANA 15281 NA} 192 t ra n sP -fa rn e se ne 37, 94 NA 42068 NANANANA 193 ^{P -my rc ene 37, 94 14446 NANANANANA}

Description of the two-way olfactometer or “Y” tube

Two-way olfactometers (“Y” tube) were constructed (Rhodes et al. 2012. The role of olfactory cues in the sequential radiation of a gall-boring beetle, Mordellistena convicta. EcologicalEntomology. 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 (60mm long, 25mm diameter) in which the odor (CO) chambers are placed (Bazzocchi GG and Maini S. 2000.

Ruolo dei semiochimici volatili nella ricerca dell'ospite is part of the parassitoid Diglyphus isaea (Hymenoptera Eulophidae). Prove olfattometriche. Bollettino dell'Istituto di Entomologia “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, SL), water and with n-hexane (PanReac AppliChem) to eliminate all the traces of the volatiles from previous samples.

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

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).

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).

In addition, the PNP movement was tested with a physiological condition such as hunger. I know tested two subpopulations of PNP: 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.

In the second group, called Repellent Tests (PRs), seven tests were carried out with PNP under SL-Ha conditions.

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 (C1, 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 (70-200 ^, Carlo Erba) inside. The volume of compound was added directly to the silica.

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 E1 stimuli, which always included 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).

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.

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.

It was assumed that a PNP population, put on ODV and the absence of any stimulus, would be uniformly distributed in the three possible options while maintaining a 1: 1: 1 relationship ( nE1 = nE2 = n EC). It was also considered that individuals who remain in CD 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:

. _ n E!

E 1 ~ N

. _ n E2

E2 ~ N

. _ n EC

EC = N

where: mi = number of individuals who have chosen E1, m ² = number of individuals who have chosen E2, me = number of individuals who remain in the EC, N = total number of individuals analyzed.

Finally, to obtain an index that takes into account all these relationships between groups of individuals, the Iei, Ie ² and Iec indices have been summarized in a single index: IM (Movement Index):

where: 0 < IM > ro. 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.

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.

Physio-environmental conditions actually interfere with PNP mobility ( x2 = 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.

From the analysis of the data relative to the PRs plus the control (SL-Ha), it turned out that there is a significance ( x2 = 34.142; df = 7; p-value = 1.62e "5) and that, therefore, the phenomenon observed 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 tested VOCs have a repellent action towards 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 seem to be any a significant difference between C1 and C6 with respect to the repellency rate. Therefore, according to the values of the MIs, 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 (12)

1. Use of at least one volatile organic compound of an entomopathogenic and / or nematophagous fungus, selected from among styrene, benzothiazole, camphor, borneol, 1,3-dimethoxybenzene, 1-octen-3-ol, 3-cyclohepten-1- ona as a repellent for Cosmopolites sordidus. 2. Use of at least one volatile organic compound of an entomopathogenic and / or nematophage fungus according to claim 1, wherein the entomopathogenic fungus is selected from Beauveria bassiana and Metarhizium anisopliae. 3. Use of at least one volatile organic compound of an entomopathogenic and / or nematophagous fungus according to any of the preceding claims, wherein the nematophagous fungus is Pochonia chlamydosporia. 4. Use of at least one volatile organic compound of an entomopathogenic and / or nematophagous fungus according to any of the preceding claims, wherein the fungus is Beauveria bassiana and the volatile organic compound is selected from among styrene (CsHs), benzothiazole (C ⁷ H ⁵ NS), camphor (C ¹⁰ H ¹⁶ O) and borneol. 5. 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 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 > CHCH = CH ² ), 3-cyclohepten-1-one (C ⁷ H ¹⁰ O). 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-1-one (C ⁷ H ¹⁰ O), for use as a repellent for Cosmopolites sordidus. 7. Use of at least one volatile organic compound according to any of the preceding claims, wherein the volatile organic compound is obtained by chemical synthesis. 8. Use of at least one volatile organic compound according to any of claims 1 7, wherein the volatile organic compound is in solid form. 9. Use of at least one volatile organic compound according to any of claims 1 7, wherein the volatile organic compound is in liquid form. 10. Use of at least one volatile organic compound according to claim 9, wherein the liquid formulation is impregnated in a matrix. 11. Use of at least one volatile organic compound according to any one of claims 17, wherein the volatile organic compound is in gel form. 12. Use of a composition comprising at least one volatile organic compound according to any of claims 1-12, as a repellent for Cosmopolites sordidus.