Evaluación de nematodos entomopatógenos en Agrotis ipsilon (Lepidoptera: Noctuidae) bajo condiciones de laboratorio e invernadero.

El objetivo de este trabajo fue evaluar el potencial de los nematodos entomopatógenos (NEPs) de los géneros Steinernema y Heterorhabditis en el control de Agrotis ipsilon (Hufnagel) (Lepidoptera: Noctuidae). Para esto, se realizaron ensayos de selección de aislamientos, se evaluó la susceptibilidad en diferentes instares del insecto, se analizaron las diferentes concentraciones de NEPs y la producción in vivo de estos aislados, y se realizaron pruebas en invernadero en el Laboratorio de Entomología y control biológico de la Universidade Estadual do Norte do Paraná, Brasil. En los ensayos de selección con nueve aislamientos, los que causan mayor porcentaje de mortalidad en los insectos, se utilizaron diferentes concentraciones (0, 50, 100, 150, 200 Juveniles Infectivos/cm2) sobre diferentes instares del insecto (2do, 3er, 4to, 5to, 6to instar y pupa), en la producción in vivo en larvas de A. ipsilon y en pruebas de patogenicidad en invernadero. En el ensayo de selección, el aislamiento CH3 (Steinernema sp.) causó el 100 % de la mortalidad en A. ipsilon mientras que los aislados GL (Heterorhabditis amazonensis), IBCB-n02 (S. carpocapsae) e IBCB-n05 (H. indica) causaron 90 % de mortalidad. En la prueba de concentraciones, el aislado IBCB-n02 presentó una CL99 de 140 IJs/cm2 y el aislado IBCB-n05, una CL99 de 190 IJs/cm2. Las larvas de 3er y 4to instares fueron más susceptibles. En la prueba in vivo para la producción de larvas de A. ipsilon, no se observó diferencia entre los dos aislamientos. En el ensayo en invernadero, el aislado IBCB-n02 fue el único que difirió del tratamiento de control

Selection of entomopathogenic nematodes and evaluation of their compatibility with cyantraniliprole for the control of Hypothenemus hampei

Attack by the coffee berry borer Hypothenemus hampei causes significant damage to coffee crops because it affects the quality of the coffee fruit during different developmental stages, which results in production losses. Control of the borer is difficult owing to its cryptic behavior and the fact that it spends its entire life cycle inside the coffee berries. This makes it difficult for natural enemies to reach it, as well as for it to come into contact with chemical insecticides. The objective of the present study was to select and evaluate the virulence of entomopathogenic nematodes (EPNs) on the coffee berry borer H. hampei and their compatibility with the insecticide cyantraniliprole under laboratory conditions. Initially, the pathogenicity and virulence of 16 isolates of Steinernema and Heterorhabditis towards coffee berry borer larvae and adults were evaluated. The most virulent isolates to both larvae and adults were determined by topical inoculation tests in coffee fruits (berries) infested by the insect, using a concentration of 100 infective juveniles (IJs)/fruit. The same isolates were also evaluated for viability and infectivity when combined with cyantraniliprole. The isolates S. feltiae (IBCB-n 47) and Heterorhabditis amazonensis (GL) displayed the highest virulence towards adults (54%). For larvae, we observed a high virulence of S. feltiae, Heterorhabditis amazonensis, Heterorhabditis indica, Heterorhabditis sp. (JPM4), Heterorhabditis sp. (NEPET 11), Heterorhabditis sp. (IBCB-n 46), and Heterorhabditis sp. (IBCB-n 44) that promoted 100% mortality. Regarding the topical inoculation test on infested fruits, S. feltiae and Heterorhabditis sp. (IBCB-n 46) were unable to penetrate the fruit through the hole made by the borer, infect, and cause the death of insects. Cyantraniliprole formulation affected the viability of IJs of S. feltiae and Heterorhabditis sp. (IBCB-n 46), mainly after 48 h of exposure

Phenotypic and biochemical characterisation and pathogenicity assessment on Galleria mellonella L. (Lepidoptera: Pyralidae) of symbionts of the entomopathogenic nematode Heterorhabditis amazonensis Andalo et al., 2006

The objective of this study was to describe phenotypically and biochemically the symbiotic bacteria associated with three populations of Heterorhabditis amazonensis Andalo et al., 2006 (isolates: UEL-n 01, UEL-n 07, and UEL-n 08) and evaluate their pathogenicity on Galleria mellonella L. (Lepidoptera: Pyralidae) larvae. Bacteria were isolated by maceration of infective juveniles (IJs) and grown in culture medium (NBTA and MacConkey). The characterization of the bacteria was evaluated by employing motility test and biochemical tests like Gram staining, lipase activity, protease, and lecithinase. The production of antibiotics and bioluminescence was also evaluated. The pathogenicity was evaluated on the last instar larvae of G. mellonella at a concentration of 104 cells/mL. The bacteria from the three entomopathogenic nematodes isolates were positive for all biochemical tests except for lecithinase, and have presented bioluminescence when subjected to ultraviolet light, indicating that they belong to the genus Photorhabdus sp. Both were pathogenic to G. mellonella larvae causing 93.3 to 100.0% mortality

Assessment of entomopathogenic nematodes in Agrotis ipsilon (Lepidoptera: Noctuidae) under laboratory and greenhouse conditions

El objetivo de este trabajo fue evaluar el potencial de los nematodos entomopatógenos (NEPs) de los géneros Steinernema y Heterorhabditis en el control de Agrotis ipsilon (Hufnagel) (Lepidoptera: Noctuidae). Para esto, se realizaron ensayos de selección de aislamientos, se evaluó la susceptibilidad en diferentes instares del insecto, se analizaron las diferentes concentraciones de NEPs y la producción in vivo de estos aislados, y se realizaron pruebas en invernadero en el Laboratorio de Entomología y control biológico de la Universidade Estadual do Norte do Paraná, Brasil. En los ensayos de selección con nueve aislamientos, los que causan mayor porcentaje de mortalidad en los insectos, se utilizaron diferentes concentraciones (0, 50, 100, 150, 200 Juveniles Infectivos/cm2) sobre diferentes instares del insecto (2do, 3er, 4to, 5to, 6to instar y pupa), en la producción in vivo en larvas de A. ipsilon y en pruebas de patogenicidad en invernadero. En el ensayo de selección, el aislamiento CH3 (Steinernema sp.) causó el 100 % de la mortalidad en A. ipsilon mientras que los aislados GL (Heterorhabditis amazonensis), IBCB-n02 (S. carpocapsae) e IBCB-n05 (H. indica) causaron 90 % de mortalidad. En la prueba de concentraciones, el aislado IBCB-n02 presentó una CL99 de 140 IJs/cm2 y el aislado IBCB-n05, una CL99 de 190 IJs/cm2. Las larvas de 3er y 4to instares fueron más susceptibles. En la prueba in vivo para la producción de larvas de A. ipsilon, no se observó diferencia entre los dos aislamientos. En el ensayo en invernadero, el aislado IBCB-n02 fue el único que difirió del tratamiento de control.This work aims to evaluate the potential of Brazilian isolates of entomopathogenic nematodes through the selection of isolates for Agrotis ipsilon (Hufnagel) (Lepidoptera: Noctuidae). In order to analyze the susceptibility at different instars of the insect and different concentrations, to investigate the in vivo production of these isolates, and to perform greenhouse tests in the Laboratory of Entomologia e Controle Microbiano of Universidade Estadual do Norte do Paraná, Brazil. Selection tests were carried out with nine isolates, and those that caused greater percentages of insect mortality were used in concentration tests (0, 50, 100, 150, 200 Infective Juveniles (IJs)/cm2), with different instars of the insect (2nd, 3rd, 4th, 5th, 6th instars and pupa), in the in vivo production of A. ipsilon larvae, and in pathogenicity tests in a greenhouse. In the selection test, the isolate CH3 (Steinernema sp.) caused 100 % of mortality in A. ipsilon, and the isolates GL (Heterorhabditis amazonensis), IBCBn-02 (S. carpocapsae) and IBCB-n05 (H. indica) caused 90 % mortality. In the tests of different concentrations, the isolate IBCB-n02 had CL99 estimated at a concentration of 140 IJs/cm2, and the isolate IBCB-n05 had CL99 estimated at a concentration of 190 IJs/cm2. The most susceptible larval stages were the 3rd and 4th instars. In the in vivo test for production of A. ipsilon larvae, no difference was observed between the two isolates. In the greenhouse test, the isolate IBCB-n02 was the only one that differed from the control treatment

Glycerol as a cryoprotectant agent to the entomopathogenic nematodes Heterorhabditis spp. and Steinernema spp.

A dificuldade de armazenamento e conservação de nematoides entomopatogênicos (NEPs) é um dos principais obstáculos para ampliar seu uso no controle biológico de pragas bem como na manutenção de coleções destes organismos. O objetivo deste estudo foi avaliar a criopreservação como método de armazenamento e conservação de NEPs utilizando o glicerol como crioprotetor. Juvenis infectantes (JIs) das espécies Heterorhabditis amazonensis (RSC 05), H. bacteriophora (HP88), Steinernerma feltiae (Sn) e S. carpocapsae (IBCB-n02) foram submetidos aos seguintes tratamentos: (A) imersão em glicerol em diferentes concentrações (10, 13 e 15%), (B) diferentes tempos de exposição dos isolados ao glicerol (24 e 48 horas) e (C) dois tempos de congelamento em nitrogênio líquido (NL) a –196 ºC (24 e 168 horas). Cada tratamento teve quatro repetições e o delineamento foi inteiramente casualizado em esquema fatorial 3x2x2 (concentrações de glicerol x tempo de exposição ao glicerol x tempo de congelamento). A sobrevivência dos JIs foi avaliada após cada tempo de exposição ao glicerol e tempo de congelamento em NL, e os dados submetidos à análise de variância e as médias comparadas pelo teste de Tukey. S. feltiae e S. carpocapse sobreviveram quando expostos ao glicerol a 10, 13 e 15% por 24 e 48 horas. Após armazenamento em NL por 24 e 168 horas, somente S. feltiae sobreviveu quando exposto ao glicerol por 48 horas nas concentrações de 10, 13 e 15%, com 40,5; 58,2; 57,7% de sobrevivência respectivamente. S. feltiae foi capaz de infectar lagartas de Galleria mellonella após o congelamento, entretanto, para o tempo de 168 horas de congelamento foi observada uma redução de 90% na infectividade.The difficulty of storage and conservation of entomopathogenic nematodes (ENPs) is one of the major obstacles for the expansion of its use in the control of biological pest and in the maintenance of collections of these organisms. The objective of this study was to evaluate cryopreservation as a storage and conservation method for ENPs, using glycerol as cryoprotectant. Infective juveniles (IJs) of the species Heterorhabditis amazonensis (RSC 05), H. bacteriophora (HP88), Steinernema feltiae (Sn) and S. carpocapse (IBCB-n02) were subjected to the following treatments: (A) immersion in glycerol at different concentrations (10, 13 and 15%); (B) different exposure times of the isolates to glycerol (24 and 48 hours); and (C) two freezing times in liquid nitrogen (LN) at –196 ºC (24 and 168 hours). Each treatment was replicated four times, and the design was completely randomized in a factorial 3x2x2 (glycerol concentrations x glycerol exposure time x freezing time in LN). IJs survival was evaluated after each exposure time to glycerol and freezing time in LN. Data were subjected to analysis of variance, and means were compared by the Tukey test. S. feltiae and S. carpocapse survived when exposed to glycerol at 10, 13 and 15% for 24 and 48 hours. After storage in LN for 24 and 168 hours, only S. feltiae survived when exposed to glycerol for 48 hours at concentrations of 10, 13 and 15%, with 40.5; 58.2; and 57.7% survival, respectively. S. feltiae was able to infect Galleria mellonella larvae after freezing. However, for the freezing time of 168 hours, 90% reduction in infectivity was observed