Abstract

The study aimed to evaluate the insecticidal activity of extracts of edible mushrooms Pleurotus ostreatus and Lentinula edodes against Rhyssomatus nigerrimus. Methanol:water extracts (70:30) of P. ostreatus and L. edodes were made and evaluated in two in vitro tests (exposure and immersion toxic effect) against R. nigerrimus. Subsequently, the toxicity test of the extracts against Artemia salina was evaluated. These extracts were subjected to colorimetric tests and gas chromatography-mass spectrometry. The results showed a mortality effect against R. nigerrimus of 50% for the P. ostreatus 2 extracts at a concentration of 20% in the immersion test. Likewise, in the toxic effect test, 90% mortality was observed after five days of exposure to a concentration of 10%. On the other hand, for the toxicity test, the extract that showed the values with the highest mortality against A. salina was P. ostreatus, starting with 80% mortality at 100μg/mL. The functional groups present in the extracts were saponins, coumarins, and alkaloids. Furthermore, the presence of more than 7 compounds in the mushroom extracts evaluated is reported. This study demonstrates the insecticidal activity of P. ostreatus and L. edodes fungal extracts and indicates the importance of using different in vitro tests to elucidate the mechanism of action for future studies.

Keywords:
edible fungi; insecticidal activity; Pleurotus; agriculture

Resumo

O estudo teve como objetivo avaliar a atividade inseticida dos extratos dos cogumelos comestíveis Pleurotus ostreatus e Lentinula edodes contra Rhyssomatus nigerrimus. Extratos metanol:água (70:30) de P. ostreatus e L. edodes foram elaborados e avaliados em dois testes in vitro (efeito tóxico de exposição e imersão) contra R. nigerrimus. Posteriormente foi avaliado o teste de toxicidade dos extratos frente à Artemia salina. Esses extratos foram submetidos a testes colorimétricos e cromatografia gasosa-espectrometria de massas. Os resultados mostraram efeito de mortalidade contra R. nigerrimus de 50% para o extrato de P. ostreatus 2 na concentração de 20% no teste de imersão. Da mesma forma, no teste de efeito tóxico, foi observada mortalidade de 90% após 5 dias de exposição à concentração de 10%. Por outro lado, para o teste de toxicidade, o extrato que apresentou os valores com maior mortalidade contra A. salina foi P. ostreatus, iniciando com 80% de mortalidade a 100μg/mL. Os grupos funcionais presentes nos extratos foram saponinas, cumarinas e alcaloides. Além disso, é relatada a presença de mais de 7 compostos nos extratos de cogumelos avaliados. Este estudo demonstra a atividade inseticida dos extratos fúngicos de P. ostreatus e L. edodes e indica a importância da utilização de diferentes testes in vitro para elucidar o mecanismo de ação para estudos futuros.

Palavras-chave:
fungos comestíveis; atividade inseticida; Pleurotus; agricultura

1. Introduction

The biological activity of edible mushroom, especially Pleurotus spp. and their derivatives, have been explored as alternative methods for agricultural pests (Cedillo, 2016CEDILLO, C., 2016. Estudio químico biodirigido del extracto hidroalcoholico del hongo Pleurotus ostreatus con actividad nematicida contra Haemonchus contortus. México: Universidad Politécnica del Estado de Morelos, 65 p. Tesis.; Pineda-Alegría et al., 2017PINEDA-ALEGRÍA, J.A., SÁNCHEZ-VÁZQUEZ, J.E., GONZÁLEZ-CORTÁZAR, M., ZAMILPA, A., LÓPEZ-ARELLANO, M.E., CUEVAS-PADILLA, E.J., MENDOZA-DE-GIVES, P. and AGUILAR-MARCELINO, L., 2017. The edible mushroom Pleurotus djamor produces metabolites with lethal activity against the parasitic nematode Haemonchus contortus. Journal of Medicinal Food, vol. 20, no. 12, pp. 1184-1192. http://dx.doi.org/10.1089/jmf.2017.0031.
http://dx.doi.org/10.1089/jmf.2017.0031... ; Cruz-Arévalo et al., 2020CRUZ-ARÉVALO, J., SÁNCHEZ, J.E., GONZÁLEZ-CORTÁZAR, M., ZAMILPA, A., ANDRADE-GALLEGOS, R.H., MENDOZA-DE-GIVES, P. and AGUILAR-MARCELINO, L., 2020. Chemical Composition of an Anthelmintic Fraction of Pleurotus eryngii against Eggs and Infective Larvae (L3) of Haemonchus contortus. BioMed Research International, vol. 2020, pp. 4138950. http://dx.doi.org/10.1155/2020/4138950.
http://dx.doi.org/10.1155/2020/4138950... ; Comans-Pérez et al., 2021COMANS-PÉREZ, R.J., SÁNCHEZ, J.E., TAWFEEQ AL-ANI, L.K., GONZÁLEZ-CORTÁZAR, M., CASTAÑEDA-RAMÍREZ, G.S., MENDOZA-DE GIVES, P., SÁNCHEZ-GARCÍA, A.D., MILLÁN-OROZCO, J. and AGUILAR-MARCELINO, L., 2021. Biological control of sheep nematode Haemonchus contortus using edible mushrooms. Biological Control, vol. 152, pp. 104420. http://dx.doi.org/10.1016/j.biocontrol.2020.1044200.
http://dx.doi.org/10.1016/j.biocontrol.2... ). In this search, it has been observed that the excessive use of chemical compounds has led to resistance of some pathogenic organisms (nematodes, bacteria and mites), contamination of the environment and the presence of hazardous residues in food for human consumption (RAPAM, 2017RED DE ACCIÓN SOBRE PLAGUICIDAS Y ALTERNATIVAS EN MÉXICO, A. C. – RAPAM, 2017 [viewed 30 June 2023]. Los plaguicidas altamente peligrosos en México. Available from: https://www.rapam.org/wp-content/uploads/2017/09/Libro-Plaguicidas-Final-14-agst-2017sin-portada.pdf
https://www.rapam.org/wp-content/uploads... ; Aguilar-Marcelino et al., 2023AGUILAR-MARCELINO, L., TAWFEEQ-AL-ANI, L.K., WONG-VILLARREAL, A. and SOTELO-LEYVA, C., 2023. Persistence of pesticides residues with chemical food preservatives in fruits and vegetables. In: J. SINGH, A. PANDEY, S. SINGH, V.K. GARG and P.C. RAMAMURTHY, eds. Current developments in biotechnology and bioengineering. Pesticides: human health, environmental impacts and management. USA: Elsevier, pp. 99-118. http://dx.doi.org/10.1016/B978-0-323-91900-5.00007-2.
http://dx.doi.org/10.1016/B978-0-323-919... ). Therefore, the use of these insecticides is being restricted, internationally and nationally through the Stockholm Conventions on Persistent Organic Pollutants and the Rotterdam Convention (SEMARNAT, 2015SECRETARÍA DE MEDIO AMBIENTE Y RECURSOS NATURALES – SEMARNAT, 2015 [viewed 30 June 2023]. Contaminantes orgánicos persistentes. Available from: https://www.gob.mx/semarnat/acciones-y-programas/convenio-de-estocolmo
https://www.gob.mx/semarnat/acciones-y-p... ).

In addition, the search for biological material with insecticidal activity has shown difficulty on the adult stages of insects. Unfortunately, the adult stage is one of the most visible indicators of an infestation in the agricultural crop and where crop damage is sought to be stopped. Although there are reports of insecticidal activity in larval stages, these stages are not as visible to the naked eye in the crop because some occur below the soil of the affected area (Dively et al., 2020DIVELY, G.P., PATTON, T., BARRANCO, L. and KULHANEK, K., 2020. Comparative efficacy of common active ingredients in organic insecticides against difficult to control insect pests. Insects, vol. 11, no. 9, pp. 614. http://dx.doi.org/10.3390/insects11090614. PMid:32911857.
http://dx.doi.org/10.3390/insects1109061... ). For example, the soybean weevil, Rhyssomatus nigerrimus Fahraeus (Coleoptera: Curculionidae), is currently considered the most economically important pest due to its direct and indirect damage to soybean (López-Guillén et al., 2012LÓPEZ-GUILLÉN, G., TERAN-VARGAS, A.P., RUIZ, J.G., LARA, J.S., ROSADO-NETO, G.H., O’BRIEN, C.W., CRUZ-LÓPEZ, L., RODRÍGUEZ-DEL-BOSQUE, L.A. and ALATORRE-ROSAS, R., 2012. First record of Rhyssomatus nigerrimus (Curculionidae: Molytinae: Cleogonini) infestations in soybeans in Mexico. The Florida Entomologist, vol. 95, no. 2, pp. 524-528. http://dx.doi.org/10.1653/024.095.0247.
http://dx.doi.org/10.1653/024.095.0247... ). Currently, R. nigerrimus is controlled by insecticides such as organophosphates, pyrethroids and fipronil (currently restricted in Europe) and aluminium phosphide (RAPAM, 2017RED DE ACCIÓN SOBRE PLAGUICIDAS Y ALTERNATIVAS EN MÉXICO, A. C. – RAPAM, 2017 [viewed 30 June 2023]. Los plaguicidas altamente peligrosos en México. Available from: https://www.rapam.org/wp-content/uploads/2017/09/Libro-Plaguicidas-Final-14-agst-2017sin-portada.pdf
https://www.rapam.org/wp-content/uploads... ; Terán-Vargas and López-Guillén, 2013TERÁN-VARGAS, A.P. and LÓPEZ-GUILLÉN, G., 2013. El picudo de la soya Rhyssomatus nigerrimus Fahraeus 1837 (COLEÓPTERA: CURCULIONIDAE). México: Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias, Folleto Técnico.). However, due to the side effects that chemical insecticides have shown, different alternative control methods are being sought.

A review work was recently carried out where around 150 species and 90 species of edible mushrooms with insecticidal effects (Drosophila melanogaster, Spodoptera littoralis, Tribolium spp., Diatraea magnifactella and Sitophilus zeamais) were reported between 1876 and 2021 (Castañeda-Ramírez et al., 2022aCASTAÑEDA-RAMÍREZ, G.S., AGUILAR-MARCELINO, L. and LÓPEZ-GUILLEN, G., 2022a. Macroscopic and microscopic fungi with insecticidal activity. Chilean Journal of Agricultural Research, vol. 82, no. 2, pp. 348-357. http://dx.doi.org/10.4067/S0718-58392022000200348.
http://dx.doi.org/10.4067/S0718-58392022... ). However, studies have not covered different strains of established fungi and have not been evaluated with the adult stage of R. nigerrimus. For this reason, the present study aimed to evaluate fungal extracts of the genus Pleurotus against R. nigerrimus soybean insects.

2. Material and Methods

The present study was carried out at the Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias (INIFAP), Campo Experimental Rosario Izapa, Tuxtla Chico, Chiapas, Mexico.

2.1. Biological material

The insects were obtained from soybean crops in the municipality of Tapachula, Chiapas, Mexico (located at 14° 56" and 92° 10"). Adults of the weevil, R. nigerrimus, were collected by hand and stored in 1 L plastic jars covered with organdy cloth. The insects were fed with soybean pods in the transfer to the laboratory, and in the laboratory, they were fed with purple sweet potato "Ipomea batatas" (López-Guillén et al., 2016LÓPEZ-GUILLÉN, G., VALDEZ-CARRASCO, J., GÓMEZ-RUIZ, J., MARTÍNEZ-ZARATE, C.J. and CRUZ-LÓPEZ, L., 2016. Sexual dimorphism and ratio of natural populations of Rhyssomatus nigerrimus adults. Southwestern Entomologist, no. 41, pp. 837-844. http://dx.doi.org/10.3958/059.041.0325.
http://dx.doi.org/10.3958/059.041.0325... ).

2.2. Edible mushroom extracts

The basidiomes (1 kg) were obtained from Pleurotus ostreatus mushrooms (nutri-hongos company, Cuernavaca Morelos) and Lentinula edodes (Edible mushroom laboratory of the Colegio de la Frontera Sur - ECOSUR). The basidiomes were dehydrated to a weight of 200 g, then cut into small pieces, respectively. The edible mushroom sample was placed in a methanol:water (70:30) solution and allowed to macerate for 24 h. A second extraction of compounds was carried out on the same material. In the second extraction, a methanol:water solution (70:30) was also used and was identified as the second maceration. The extracts were filtered through a funnel and 15 cm gauze, cotton, and coarse-pore filter paper (#50), and the samples were subsequently concentrated under reduced pressure in a rotary evaporator. Finally, the extracts were dried in a lyophilizer (labconco®) and stored refrigerated at 11 to 4 °C until use. For each species of fungus, two solutions were obtained, called first maceration and second maceration, which were called P. ostreatus 1, P. ostreatus 2, L. edodes 1 and L. edodes 2, respectively.

2.3. In vitro assays against R. nigerrimus

Immersion test: The insects were immersed for 20 seconds in the treatments at a concentration of 2.5, 5, 7.5, 10 and 20% of the four extracts obtained (P. ostreatus 1, P. ostreatus 2, L. edodes 1 and L. edodes 2) and their respective negative control (distilled water) and positive control Fipronil insecticide (0.1%). The insects were exposed to the extract treatments and placed in plastic containers (250 mL), and mortality was observed every day for 15 days (Castañeda-Ramírez et al., 2022bCASTAÑEDA-RAMÍREZ, G.S., LÓPEZ-GUILLÉN, G., AGUILAR-MARCELINO, L., SIU-RIVAS, A. and CRUZ-LÓPEZ, L. 2022b. Insecticidal effect of metabolites identified in edible mushrooms against Rhyssomatus nigerrimus Fahraeus. Brazilian Journal of Biology = Revista Brasileira de Biologia, vol. 84, pp. e264786. https://doi.org/10.1590/1519-6984.264786.
https://doi.org/10.1590/1519-6984.264786... ).

Toxic effect test by exposure: 300 µL of extracts of P. ostreatus 1, P. ostreatus 2, L. edodes 1 and L. edodes 2 at concentrations of 2.5, 5.0, 7.5 and 10% were placed in Petri dishes (9 cm diameter x 1.5 cm height), respectively, sterile water was used as a negative control and firpronil insecticide (0.25%) was used as a positive control. The plates with the treatments were left to dry for 4 h, then 10 adults of R. nigerrimus were placed in each Petri dish. Each treatment was repeated with 10 Petri dishes. Treated insects were observed daily, and the number of insects killed on each day was recorded to calculate mortality (According to the % mortality Equation 1 in the data analysis section) for five days.

2.4. Polyvinylpolypyrrolidone (PVPP) test to rule out polyphenols

Initially, the extracts of the two species of edible mushrooms were prepared in tubes at 20% concentration and the PVPP compound (0.05 g PVPP/mL) was added to the tubes. These concentrations of extract + PVPP were incubated for 3 h (pvpp has the ability to trap or capture polyphenols). After incubating the fungal extracts, the tubes with the samples were centrifuged and the supernatant was transferred to another tube called polyphenol-free extract. The pellet containing the PVPP and the polyphenol mixture was discarded. These tubes with the extracts at concentrations of 20% (treated without polyphenols) were evaluated with the respective in vitro tests and their respective controls (Vargas-Magaña, 2013VARGAS-MAGAÑA, J., 2013. Respuesta fisiológica de los ovinos y sus nematodos gastrointestinales a los taninos. México: Universidad Autónoma de Yucatán, 125 p. Tesis en Doctorado.).

2.5. Artemia Salina test to determine toxicity

The Artemia Salina assay described by McLaughlin (McLaughlin et al., 1998MCLAUGHLIN, J., LINGLING, L. and ROGERS, M.S., 1998. The use of biological assays to evaluate botanicals. Drug Information Journal, vol. 32, no. 2, pp. 513-524. http://dx.doi.org/10.1177/009286159803200223.
http://dx.doi.org/10.1177/00928615980320... ) was used to evaluate the toxicity of fungal extracts on non-target organisms. The relationship between the concentration of the extracts and the effect it causes on a living organism was quantified (Silbergeld, 2023SILBERGELD, E.K., 2023 [viewed 30 June 2023]. Toxicologia. In: INSTITUTO NACIONAL DE SEGURIDAD Y SALUD EN EL TRABAJO, ed. Enciclopedia de salud y seguridad en el trabajo. Available from: https://www.insst.es/documents/94886/161958/Sumario+del+Volumen+I.pdf/18ea3013-6f64-4997-88a1-0aadd719faac?t=1526457520818
https://www.insst.es/documents/94886/161... ). Initially, we sought to hatch the A. salina cysts; for this, a 500 ml flask containing saline water (35 g/L without iodine), the cysts and a constant air flow of 1 to 2.5 L/m was used. to ensure hatching. The different concentrations of each extract of P. ostreatus and L. edodes to be evaluated (10, 100, 300, 600 and 1000 μg/mL) were placed in a 24-well Polystyrene culture plate (CORNING®). Subsequently, 1250μL of saline water was placed with 20 nauplii, to have a final volume of 2.5mL per well. In addition, a control group (lugol) was added and these treatments were left to incubate in artificial light for 24 hours. Subsequently, survivors were counted and median lethal concentration (LC50) and toxicity levels were calculated.

2.6. Preliminary mycochemical tests of edible mushroom extracts

Qualitative phytochemical tests individually processed extracts of P. ostreatus 1, P. ostreatus 2, L. edodes 1 and L. edodes 2. For these tests, different methodologies were used to qualitatively determine possible functional groups of compounds in the samples of the mushroom extracts (Sarker and Nahar, 2012SARKER, S.D. and NAHAR, L., eds., 2012. An introduction to natural products isolation. New York: Humana Press.; Mancilla-Montelongo et al., 2019MANCILLA-MONTELONGO, G., CASTAÑEDA-RAMÍREZ, G.S., BORGES-ARGÁEZ, R., CACERES-FARFAN, M., SANDOVAL-CASTRO, C.A. and TORRES-ACOSTA, J.F.J., 2019. Evaluación fitoquímica preliminar de plantas tropicales con potencial nutracéutico para pequeños rumiantes. Revista Latinoamericana, vol. 47, pp. 71. Suplemento especial.). In 15mL glass test tubes, 5 mg of extract of each fungus plus the reagents of each qualitative biochemical test were added to observe the colouration change after 5 min. In the Liebermann-Burchard reaction, a 5 mg sample of each fungal extract was placed in glass test tubes, to which 1 mL of acetic anhydride, 100 µL of H₂SO₄ and 10 drops (approximately 10µL) of HCL, respectively, were added. The test was considered positive when the solution acquired a purple colouration, suggesting the presence of triterpenes. In the Shinoda reaction, 1 mL of CH₃OH, 2-3 Mg shavings and 2-3 drops (10µL) of HCL were added to 5mg of each extract, respectively. The test was considered positive for the presence of flavonoids when it changed to red colour. For the Molisch test (carbohy drate), 5 mg of each extract was used, adding 1 mL of water, 2 drops of alpha-naphthol (1%) and 7 drops of H₂SO₄ were added. The test was considered positive if the solution with each extract changed colour to violet, which indicated the presence of carbohydrates. In the reaction to identify coumarins, 5 mg of each mushroom extract was used, to which 1 mL of CH₃OH, 1 mL of alcoholic NaOH and 8 drops of HCL, respectively, were added. The test is considered positive when the colouration of the solutions with each fungal extract plus reagents disappears. For the Wagner reaction, 5 mg of each fungal extract was used, to which an iodine chip, KI and 1 mL of distilled water were added; the change of the solution to a brown colouration indicated the presence of alkaloids. Finally, for the case of the saponins test, it was considered positive when the foam was formed by adding 5mg of each mushroom extract and 1 mL of water (Mancilla-Montelongo et al., 2019MANCILLA-MONTELONGO, G., CASTAÑEDA-RAMÍREZ, G.S., BORGES-ARGÁEZ, R., CACERES-FARFAN, M., SANDOVAL-CASTRO, C.A. and TORRES-ACOSTA, J.F.J., 2019. Evaluación fitoquímica preliminar de plantas tropicales con potencial nutracéutico para pequeños rumiantes. Revista Latinoamericana, vol. 47, pp. 71. Suplemento especial.).

2.7. Gas chromatography coupled to mass spectrometry

The lyophiliszed extracts of the fungi were suspended in methanol and subsequently injected into the Gas chromatographic (GC). Gas chromatographic analyses coupled to mass spectrometry of the extracts of each fungal sample were carried out on a Thermo Scientific TRACE gas chromatograph (GC) with an ITQ900 ion trap mass detector (Thermo Electron Corporation, Milan, Italy). The carrier gas was helium, with a 1 mL/min flow rate. The column used was TRACE-5MS (30 m, 0.25 μm film and 0.25 mm internal diameter). The program used to analyze of the samples of each fungal extract was a full sweep, started at a temperature of 50 °C, which was maintained for 1 min; subsequently, a temperature ramp was applied with an increase of 7 °C/min up to 300 °C. The temperature of 300 °C was maintained for five minutes. The interface temperature was 280 °C, and the bulk temperature was 200 °C. Signals of the compounds present were detected and compared with the database by NIST17.L library (Páez-Leon et al., 2022PÁEZ-LEÓN, S.Y., CARRILLO-MORALES, M., GÓMEZ-RODRÍGUEZ, O., LÓPEZ-GUILLÉN, G., CASTAÑEDA-RAMÍREZ, G.S., HERNÁNDEZ-NÚÑEZ, E., WONG-VILLARREAL, A. and AGUILAR-MARCELINO, L., 2022. Nematicidal activity of leaf extract of Moringa oleifera Lam. against Haemonchus contortus and Nacobbus aberrans. Journal of Helminthology, vol. 96, pp. e13. http://dx.doi.org/10.1017/S0022149X22000025. PMid:35195061.
http://dx.doi.org/10.1017/S0022149X22000... ).

2.8. Data analysis

The results obtained from the mortality tests of the different treatments at different hours of exposure were analyszed by means of a generaliszed linear model (GLM) to evaluate the differences for the control. Post hoc, a Tukey's mean comparison test was applied. The analyses were performed with Statgraphics Centurion XV.

The formula used to obtain the insecticide effect was following (Castañeda-Ramirez et al., 2022b):

3. Results

3.1. Mortality of R. nigerrimus adults to extracts of P. ostreatus and L. edodes

The highest percentage of mortality utilizing the immersion test of R. nigerrimus was 52%, with P. ostreatus extract 2 at 20%. The other extracts evaluated did not cause more than 35% mortality. The mortality of R. nigerrimus with the polyphenol-free extract of L. edodes 2 mushroom increased from 35% to 57%; however, with the other extracts, no differences were observed with the PVPP treatment (Table 1).

The extracts of P. ostreatus 1 and P. ostreatus 2, showed more significant insecticidal toxicity test on adults of R. nigerrimus from a concentration of 20% exposure during 5 days, causing more than 98% mortality in adults of R. nigerrimus. The P. ostreatus extract 1 at 2.5% caused 73% mortality of R. nigerrimus adults. The extract of L. edodes 1 showed a mortality of more than 80% of adults of R. nigerrimus from a concentration of 5%, while the extract of L. edodes 2 at all the concentrations evaluated did not show mortality of even 90%. In evaluating the extracts without polyphenols (PVPP), only the extract of L. edodes 2 showed an insecticidal effect, which decreased the percentage of mortality from 86 to 76% (Table 2).

On the other hand, the values of the concentrations that affect 50 or 90% of the treated insects are observed in summary form, being for the case of the EC₅₀ of P. ostreatus 1 (1.74%), P. ostreatus 2 (2.4%) and L. edodes 1 (2.9%) similar according to the confidence intervals (Table 3).

3.2. Artemia Salina testing

Only P. ostreatus extract 1 caused 80% mortality of A. salina at low concentrations (100 μg/mL). The mortality of A. salina was higher than 95% from the concentration of 300 μg/mL of P. ostreatus and L. edodes extracts (Table 4).

The effective concentrations 50 and 90 of the mortality caused by the extracts against A. salina are observed. The extract with the highest activity is P. ostreatus 1 with an EC₅₀ of 22.07 μg/mL, while the other extracts show similar values. For the case of EC₉₀, the P. ostreatus 1 extract stands out more as the one that shows the highest insecticidal activity EC₉₀ 121.68 μg/mL for the other extracts (Table 5).

3.3. Mycochemical evaluation

The presence of flavonoids and carbohydrates was not observed in any of the four extracts evaluated (P. ostreatus 1, P. ostreatus 2, L. edodes 1, L. edodes 2). In the case of the saponins and coumarins test, a slight presence of saponins was observed in three extracts evaluated (P. ostreatus 2, L. edodes 1, L. edodes 2). The presence of alkaloids was only observed in the extracts of L. edodes 1, while the presence of triterpenes was only observed in the extract of L. edodes 2.

3.4. Gas chromatography coupled to mass spectrometry

In the case of P. ostreatus extract, the most abundant compounds were Methyl tris (trimethyl siloxy) silane (51.95%); Cyclotrisiloxane, hexamethyl- (26.3%); Tris (tert-butyl dimethylsilyloxy) arsane (16.01%). On the other hand, for the compounds detected in the extract of L. edodes, Cyclotrisiloxane, hexamethyl- (56.57%) and Tris(tert-butyldimethylsilyloxy) arsane (32.09%) were detected as main compounds. When observing the identified compounds there are 5 compounds present in the two extracts which are: (1) Cyclotrisiloxane, hexamethyl-; 2) Tris(tert-butyldimethylsilyloxy)arsane); 3) Titanium, [(1,2,3,3-.eta.)-2-butenyl](.eta.8-1,3,5,7- cyclooctatetraene)-; 4) 1-Nitro-9,10-dioxo-9,10-dihydro-anthracene-2-carboxylic acid diethylamide; 5) 1,1,1,1,3,5,5,5,5-Heptamethyltrisiloxane (Table 6).

4. Discussion

This study evaluated extracts of edible mushroom, P. ostreatus and L. edodes, against R. nigerrimus. The mortality percentages for the immersion test were low (35%). The highest percentage reached was 52% with the P. ostreatus 2 extract at 20% concentration, which indicates that these extracts, by this mechanism of action, do not show effects more significant than 50%. This is possibly due to the exposure time with the extract, which is only 20 seconds. On the other hand, in the PVPP test, it was possible to observe which extracts showed insecticidal activity, possibly fusing the polyphenols through the immersion test. When the polyphenols were removed from the extracts, only a change was evidenced in the extract of L. edodes 2. The insecticidal activity increased from 35% to 57%, indicating that some polyphenols were blocking the activity of the extract. However, in the case of the other extracts evaluated, their activity was not affected by the polyphenols.

On the other hand, for the second test evaluated "toxicity by exposure", it was possible to observe for the extracts a higher percentage (≥70%) of insecticidal activity in the adults of R. nigerrimus at low concentrations (2.5%). This test, unlike the previous one, shows that prolonged exposure at one concentration affected the adults of R. nigerrimus. Possibly, this is because the exposure time is longer, and some molecules could enter the adults affecting the insects up to mortality of the insect. In addition, since the four extracts evaluated showed activity in the adults, this test could be considered sensitive for detecting biological material with insecticidal activity. In the case of the extracts without polyphenols, the extract that again showed a difference in activity was L. edodes 2, in this case decreasing its insecticidal activity from 86 to 76% mortality, which could indicate that polyphenols have an effect on this extract and its insecticidal activity.

There are few studies on the evaluation of edible mushroom extracts against insect pests; there are no studies specifically against R. nigerrimus, nor are there any studies on the insecticidal activity of the fungus L. edodes. Therefore, this is one of the first works reporting insecticidal activity of L. edodes against R. nigerrimus. On the other hand, reported studies of Pleurotus against insects have shown insecticidal activity. Recently, a study evaluated in vitro extracts of the fungus P. ostreatus for the control of the corn weevil (Sitophilus zeamais Motschulsky) where the tests used were toxicity by contact and fumigation, repellency and antixenosis. Extracts in ethyl acetate and distilled water showed repellent activity and recorded an antixenosis effect (Pino et al., 2019PINO, V., SILVA-AGUAYO, G., FIGUEROA-CARES, I., GERDING-GONZÁLEZ, M., LOYOLA, P., CASTAÑEDA-RAMIREZ, G.S. and AGUILAR-MARCELINO, L., 2019. Eficacia in vitro de extractos del hongo comestible Pleurotus ostreatus kumm para el control de Sitophilus zeamais motschulsky. Chilean Journal of Agricultural & Animal Science, vol. 35, no. 3, pp. 293-303. http://dx.doi.org/10.4067/S0719-38902019005000505.
http://dx.doi.org/10.4067/S0719-38902019... ). This was the first study of Pleurotus spp. Fungi against a member of the Curculionidae family, to which R. nigerrimus belongs, and the results highlight the importance of evaluating the whole extract. These results support the information found on the activity of Pleurotus that could reach more than 90% of insecticidal activity in some tests against some species of the Curculionidae family. In addition, a 2011 study investigated the residual effect of P. ostreatus basidiomonads extracts against Tribolium castaneum adults. Different extracts and fractions of extracts (petroleum ether fraction, methanol-chloroform extract and hot water) were evaluated. The range of results obtained from the fractions (LD₅₀) was from 1.54 mg/cm² to 0.20 mg/cm² after 30 hours of exposure (Rahman et al., 2011RAHMAN, M.F., KARIM, M.R., ALAM, M.J., ISLAM, M.F., HABIB, M.R., UDDIN, M.B. and HOSSAIN, M.T., 2011. Insecticidal effect of oyster mushroom (Pleurotus ostreatus) against Tribolium castaneum (Herbst). Natural Products, vol. 7, no. 4, pp. 187-190.). These results are similar to the insecticidal activity of the contact toxicity test. Further studies and tests are still needed to determine the activity of fungal extracts, as well as to understand the mechanisms of action and to understand better the volatiles that interacts with R. nigerrimus (Espadas-Pinacho et al., 2021ESPADAS-PINACHO, K., LÓPEZ-GUILLÉN, G., GÓMEZ-RUIZA, J. and CRUZ-LÓPEZ, L. 2021. Induced volatiles in the interaction between soybean (Glycine max) and the Mexican soybean weevil (Rhyssomatus nigerrimus). Brazilian Journal of Biology = Revista Brasileira de Biologia, vol. 81, no. 3, pp. 611-620. https://doi.org/10.1590/1519-6984.227271.
https://doi.org/10.1590/1519-6984.227271... ).

The evaluation of A. salina allows us to know the toxicity of the extract being evaluated and the possible effect that the extract has on other populations, such as those in the marine environment or others (Silbergeld, 2023SILBERGELD, E.K., 2023 [viewed 30 June 2023]. Toxicologia. In: INSTITUTO NACIONAL DE SEGURIDAD Y SALUD EN EL TRABAJO, ed. Enciclopedia de salud y seguridad en el trabajo. Available from: https://www.insst.es/documents/94886/161958/Sumario+del+Volumen+I.pdf/18ea3013-6f64-4997-88a1-0aadd719faac?t=1526457520818
https://www.insst.es/documents/94886/161... ). The extracts evaluated in this present investigation have 100% mortality activity against A. salina at lower concentrations (1000 µg/mL) than those evaluated with the insect R. nigerrimus. These differences in concentrations have been shown in studies of insect evaluations against fungi with basidiomata (Castañeda-Ramírez et al., 2022aCASTAÑEDA-RAMÍREZ, G.S., AGUILAR-MARCELINO, L. and LÓPEZ-GUILLEN, G., 2022a. Macroscopic and microscopic fungi with insecticidal activity. Chilean Journal of Agricultural Research, vol. 82, no. 2, pp. 348-357. http://dx.doi.org/10.4067/S0718-58392022000200348.
http://dx.doi.org/10.4067/S0718-58392022... ), where other non-adult stages of insects have been evaluated, and insecticidal effects have been observed at lower concentrations because they are more vulnerable. However, higher concentrations are needed to obtain an insecticidad effect in the adult stage of insects. The A. salina test indicates that the extracts evaluated could show insecticidal activity with other genera of insect pests. However, further studies are needed to confirm the concentrations and the activity that the extracts evaluated in the present work could show.

The compounds reported in Table 6 for the P. ostreatus and L. edodes mushroom extracts have not been reported in other mushrooms of the same genus. However, it is essential to mention that the present work was not a directed study, and the compounds reported are only known to be in the whole extract due to the technique used they are volatiles, but it is not sure that they are the ones that present the activity observed here. There are no reports on the insecticidal activity of the compounds identified in the extracts of P. ostreatus and L. edodes. However, the Methyltris(trimethylsiloxy)silane has shown antibacterial activity (Balachandar et al., 2022BALACHANDAR, R., NAVANEETHAN, R., BIRUNTHA, M., KUMAR, K.K.A., GOVARTHANAN, M. and KARMEGAM, N., 2022. Antibacterial activity of silver nanoparticles phytosynthesized from Glochidion candolleanum leaves. Materials Letters, vol. 311, pp. 131572. http://dx.doi.org/10.1016/j.matlet.2021.131572.
http://dx.doi.org/10.1016/j.matlet.2021.... ). As for the compound identified as Cyclotrisiloxane, hexamethyl- in the extracts of the two edible mushroom species, it has been reported to have antimicrobial activity against Staphylococcus aureus (Priyanka et al. 2015PRIYANKA, C., KUMAR, P., SHIVAKUMAR, P., BANKAR, P. and KARTHIK, L., 2015. In vitro antibacterial activity and gas chromatography-mass spectroscopy analysis of Acacia Karoo and Zizphus mauritiana extracts. Journal of Taibah University for Science : JTUSCI, vol. 9, no. 1, pp. 13-19. http://dx.doi.org/10.1016/j.jtusci.2014.06.007.
http://dx.doi.org/10.1016/j.jtusci.2014.... ; Ismail et al., 2020ISMAIL, G.A., GHEDA, S.F., ABO-SHADY, A.M. and ABDEL-KARIM, O.H., 2020. In vitro potential activity of some seaweeds as antioxidants and inhibitors of diabetic enzymes. Food Science and Technology, vol. 40, no. 3, pp. 681-691. https://doi.org/10.1590/fst.15619.
https://doi.org/10.1590/fst.15619... ). For the case of the other detected compounds, there are still no previous reports of biological activity. More studies are needed to know if these compounds or their mixtures can have insecticidal activity.

On the other hand, the preliminary mycochemical test used helped us to understand the possible groups of compounds present in the evaluated extracts that could not be identified due to the limitations of the mass gas technique used, where complex molecules cannot be detected. The extract of P. ostreatus did not show the presence of any specific group; however, here, it could be that other types of molecules were present in this extract with insecticidal activity. On the other hand, different compounds were detected in the extract of L. edodes, mainly saponins and coumarins. The chemical profiles of these fungi are complex, so it is necessary to perform directed studies to relate the molecules with the reported activity.

5. Conclusion

The extracts of the fungi Pleurotus ostreatus and Lentinula edodes showed an insecticidal effect of more than 80% mortality at a concentration of 20% in the exposure toxicity test against R. nigerrimus in 5 days. Furthermore, the extracts show their effect in toxicity test against A. salina with 100% mortality at 1000 μg/mL concentration.

In addition, the compounds reported by mass gases have shown different biological activity, which could indicate that some of these secondary compounds or their mixtures are responsible for the insecticidal activity. However, there is still a lack of studies to verify which compounds and mixtures may be responsible for the reported insecticidal activity.

Acknowledgements

The project funded this article: "Ecología sensorial del picudo de la soya (Rhyssomatus nigerrimus Fahraeus)", which is funded by SEP-CONACYT Convocatoria de Investigación Científica Básica 2017-2018, project number A1-S-23359. In addition, the present work is part of a CONACYT Postdoctoral fellowship.

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