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This work aimed to investigate candidate biological control agents against the cattle tick Rhipicephalus microplus by studying three entomopathogenic nematodes (EPN) isolates from two species, Heterorhabditis bacteriophora (isolates LPP30 and HP88) and Heterorhabditis baujardi (isolate LPP7). The study comprised the pharmacotechnical development of four test formulations, and their biological evaluation, in three stages: 1) selection of the fittest EPN isolate at 16 ± 1 °C after 120 h of incorporation in the formulations; 2) determination of the effects of temperature and incubation time on the survival of the selected isolate in formulations; 3) evaluation of the in vitro efficacy (infectivity) of the selected EPN formulations and their components against R. microplus females after 72 h exposure. H. baujardi LPP7 presented the highest survival rates in 0.1% carboxymethyl cellulose sodium (CMC) formulations (100% at 25 °C, up to 10 days after incorporation). At the highest tested temperature (34.2 °C), 54% of LPP7 survived for up to 72 h in CMC formulations. The infectivity of LPP7-containing formulation against R. microplus was 99%, reducing oviposition, egg production index and larval hatching. Considering the pressing need for safer acaricide products, incorporation of EPNs in pharmaceutical formulations is presented as a promising approach for the biological control of cattle tick infestations, potentially reducing environmental impact and resistance development associated with chemical acaricides.

The present study evaluated, in the laboratory, the virulence of Heterorhabditis bacteriophora LPP30 against engorged females of Rhipicephalus microplus; in addition, we evaluated, in semi-field tests, the effects of four isolates of Heterorhabditis spp. (i.e., H. bacteriophora HP88, Heterorhabditis baujardi LPP7, Heterorhabditis indica LPP1 and H. bacteriophora LPP30) in insect cadaver formulation against the non-parasitic phase of R. microplus. In the first experiment (in vitro), engorged females were exposed, in Petri dish, to H. bacteriophora LPP30 at 75, 150, 300, 600, 1200 and 2400 nematodes/tick (10 ticks per concentration tested). In the second experiment (semi-field), five engorged females and four cadavers of Galleria mellonella infected with nematodes were placed in pots with soil and grown Brachiaria decumbens; the pots were hold outdoor, exposed to natural environment conditions during the tests. In the third experiment (semi-field), 65 days after the cadavers had been placed in the pots for the second experiment, new engorged females (five per pot) were placed in the pots of the groups treated with H. bacteriophora HP88 or H. baujardi LPP7, to assess their persistence in the soil. In the first test, the percent control was higher than 95% in all treatment groups. In the second experiment, at day 22, the mean mortality rate was 78% in the groups treated with H. bacteriophora LPP30 or H. indica LPP1, and reached 100% and 98% when treated with H. bacteriophora HP88 and H. baujardi LPP7, respectively. In this experiment, the egg-laying inhibition index was higher than 90% in the groups treated with H. bacteriophora HP88 (97.2%) or H. baujardi LPP7 (91.9%). In the third experiment with H. bacteriophora HP88 and H. baujardi LPP7, the egg-laying inhibition index was 59.1% and 43.1%, respectively. We concluded that the isolate LPP30 was highly virulent under laboratory conditions, whereas in semi-field tests, HP88 and LPP7 were the most effective isolates. Moreover, HP88 and LPP7 remained active against engorged females of R. microplus in the soil for 65 days after application of EPN-infected cadavers of G. mellonella.

Since the 1980s, research into entomopathogenic nematodes (EPNs) in Latin America has produced many remarkable discoveries. In fact, 16 out of the 117 recognized species of EPNs have been recovered and described in the subcontinent, with many more endemic species and/or strains remaining to be discovered and identified. In addition, from an applied perspective, numerous technological innovations have been accomplished in relation to their implementation in biocontrol. EPNs have been evaluated against over 170 species of agricultural and urban insects, mites, and plant-parasitic nematodes under laboratory and field conditions. While much success has been recorded, many accomplishments remain obscure, due to their publication in non-English journals, thesis dissertations, conference proceedings, and other non-readily available sources. The present review provides a brief history of EPNs in Latin America, including current findings and future perspectives., E. San-Blas, M.G. Rodriguez and P. Morales-Montero were supported by To Fondo de Desarrollo Nacional (FONDEN S.A.), in the form of financial support through “Convenio de Cooperación Integral Cuba-Venezuela”, project “MPPF-FONDEN-CJ-CCATR-XIII-14”. Also, M.G. Rodríguez used the time and facilities granted by the European Union project, “Microbial Uptakes for Sustainable management of major banana pests and diseases” (MUSA, 727624; topic: SFS-11-2016). R. Campos-Herrera’s contribution was supported through an Investigator Program Award (IF/00552/2014) supplied by the Portuguese government. J. Ruiz-Vega thanks the Secretaría de investigación y Posgrado (SIP), Instituto Politécnico Nacional, Mexico for providing resources for the research carried out in Oaxaca from 1998 to 2010.

Porcine pleuropneumonia is an important cause of lowered productivity and economic loss in the pig industry worldwide, associated primarily with Actinobacillus pleuropneumoniae infection. Its colonization and persistence within the upper respiratory tract of affected pigs depends upon interactions between a number of genetically controlled virulence factors, such as pore-forming repeats-in-toxin exoproteins, biofilm formation, and antimicrobial resistance. This study investigated correlations between biofilm-forming capacity, antimicrobial resistance, and virulence of A. pleuropneumoniae obtained from clinical outbreaks of disease, using a Galleria mellonella alternative infection model. Results suggest that virulence is diverse amongst the 21 strains of A. pleuropneumoniae examined and biofilm formation correlated with genetic control of antimicrobial resistance.

The common guava, yellow guava, or lemon guava, Psidium guajava L. (Myrtaceae), is native to the Americas and widely cultivated in more than 50 tropical and subtropical countries. There are many insect pests that limit guava production. Biological control should be considered to avoid extensive use of insecticides. Entomopathogenic nematodes can be very effective against life stages in the soil. Therefore, this review aims to summarize most studies carried out for control of guava pests by entomopathogenic nematodes.

The aim of this study was to evaluate the effect of different concentrations of the entomopathogenic nematodes (EPNs) Heterorhabditis bacteriophora HP88 and Heterorhabditis indica LPP1 on the reproductive biology of partially engorged females of Dermacentor nitens. Four groups were formed, with each group containing 10 females and exposed to concentrations of 0, 75, 300, and 1200 nematodes for each female. This procedure was performed separately for each nematode. The following biological parameters were evaluated: egg mass weight, egg production index, hatching percentage, and percentage of control. H. bacteriophora HP88 at the two highest concentrations (300 and 1200 EPNs/female) caused a reduction (p < 0.05) on the egg mass and egg production index. Was noted a significant reduction (p < 0.05) in the percentage of hatched in all the treated groups. For H. indica LPP1, all treatments resulted in decreased (p < 0.05) values for all the parameters. The percentages of controls obtained at concentrations of 75, 300, and 1200 EPNs/female were 56.3, 89.3, and 98.8 and 77.5, 77.1, and 95.9 for H. bacteriophora HP88 and H. indica LPP1, respectively. Therefore, it is concluded that these nematodes showed pathogenicity toward partially engorged females of D. nitens, thereby negatively affecting the reproductive biology of this tick.

Entomopathogenic nematodes have been evaluated for control of mosquito species for decades. Depending on the nematode and mosquito involved, mortality rates of larvae (L) may reach 100% in vitro. Nonetheless, nematode efficacy at oviposition sites has rarely been assessed. Heterorhabditis indica LPP35 has been shown to kill over 75% of Aedes aegypti L3/L4 in cups and bottles outdoors. To assess its efficacy in indoor oviposition sites, different types/sizes of floor drains and pot saucers, and 65 liter water barrels, were infested with L3/L4 and treated with two doses of infective juveniles (IJs). In floor drains, mortality rates varied from 45 to 82%, with better results in the smallest drains. The adjustable dose of 25 IJs/cm2 of the drain's bottom internal surface gave better results than the fixed dose of 100 IJs/larva. Mortality rates were only 28 to 53% and 0.1 to 1.7% in pot saucers and water barrels, respectively, probably because ridges and grooves that marked the bottom internal surface of these containers hindered the encounter of larvae and IJs. Entomopathogenic nematodes have been evaluated for control of mosquito species for decades. Depending on the nematode and mosquito involved, mortality rates of larvae (L) may reach 100% in vitro. Nonetheless, nematode efficacy at oviposition sites has rarely been assessed. Heterorhabditis indica LPP35 has been shown to kill over 75% of Aedes aegypti L3/L4 in cups and bottles outdoors. To assess its efficacy in indoor oviposition sites, different types/sizes of floor drains and pot saucers, and 65 liter water barrels, were infested with L3/L4 and treated with two doses of infective juveniles (IJs). In floor drains, mortality rates varied from 45 to 82%, with better results in the smallest drains. The adjustable dose of 25 IJs/cm2 of the drain’s bottom internal surface gave better results than the fixed dose of 100 IJs/larva. Mortality rates were only 28 to 53% and 0.1 to 1.7% in pot saucers and water barrels, respectively, probably because ridges and grooves that marked the bottom internal surface of these containers hindered the encounter of larvae and IJs.

Oriental fruit moth Grapholita molesta (Busck, 1916) (Lepidoptera: Tortricidae) is considered a major pest in temperate fruit trees, such as peach and apple. Entomopathogenic nematodes (EPNs) are regarded as viable for pest management control due to their efficiency against tortricid in these trees. The objective of this study was to evaluate the effectiveness of native EPNs from Rio Grande do Sul state against pre-pupae of G. molesta under laboratory and field conditions. In the laboratory, pre-pupae of G. molesta were placed in corrugated cardboard sheets inside glass tubes and exposed to 17 different EPNs strains at concentrations of 6, 12, 24, 48 and 60 IJs/cm2 and maintained at 25 °C, 70 ± 10% RH and photophase of 16 h. Insect mortality was recorded 72 h after inoculation of EPNs. Steinernema rarum RS69 and Heterorhabditis bacteriophora RS33 were the most virulent strains and selected for field application (LC95 of 70.5 and 53.8 IJs/cm2, respectively). Both strains were highly efficient under field conditions when applied in aqueous suspension directed to larvae on peach tree trunk, causing mortality of 94 and 97.0%, respectively.

This study investigated the possible biological, biochemical and histological changes in Bradybaena similaris (Gastropoda: Pulmonata) infected by Heterorhabditis indica (Rhabditida: Heterorhabditidae), strain LPP1. Two groups of 16 snails were formed: the control group (unexposed) and the treated group, which was exposed for three weeks to infective juveniles (J3) of H. indica LPP1. The experiment was conducted in duplicate, using a total of 64 snails. After the exposure period, the snails were dissected to collect the hemolymph and tissues, for evaluation of the physiological changes caused by the infection. The number of eggs laid/snail and the viability of these eggs were also assessed as indicators of the reproductive activity of B. similaris. Intense glycogenolysis was accompanied by a significant reduction (p

The sugarcane borer, Diatraea saccharalis, makes tunnels in the cane stalk, causing weight loss, as well as bud death, impairing germination. The weakened stalks are also more prone to breakage by wind, and in young plants the inner whorl of the leaves can die, resulting in a condition known as “dead heart”. Chemical control is used, but with low efficiency. This work was designed to test biological control of D. saccharalis using entomopathogenic nematodes (NEPs). Two trials in the greenhouse were performed using Heterohabditis baujard LPP7 and Steinernema carpocapse NCAll by foliar application associated with adjuvants. In the first assay, the number of holes per stalk was 3.4 for the control without nematodes and with Joint* Oil; 3.14 for the control without nematodes and with Gotafix®; 2.44 for S. carpocapsae NCAll + Joint* Oil; 2.06 for H. baujardi LPP7 + Gotafix® and also for S. carpocapsae NCAll + Gotafix®; and 1.84 for H. baujardi LPP7 + Joint* Oil. In the second assay, the treatments showed an average of 4.78 holes per stalk for the control without nematodes and with Gotafix®; 4.76 for the control without nematodes and with Joint* Oil; 2.18 for S. carpocapsae NCAll + Joint* Oil and for S. carpocapsae NCAll + Gotafix®; 1.96 for H. baujardi LPP7 + Gotafix®; and 1.90 for H. baujardi LPP7 + Joint* Oil. In the two experiments, the treatments with and without the EPNs differed significantly, but there was no difference between the adjuvants or between the two EPN species. A broca-da-cana, Diatraea saccharalis, causa danos à cana-de-açúcar devido ao seu hábito de formar galerias nos colmos, o que acarreta em perda do peso do produto, redução da sacarose e a seca dos ponteiros. O controle químico é utilizado, porém, com baixa eficiência, pois a larva permanece maior parte do tempo de seu desenvolvimento dentro dos colmos. Este trabalho teve como objetivo testar o controle biológico da D. saccharalis, utilizando-se nematóides entomopatogênicos (NEPs). Para tanto, foram realizados dois ensaios em casa de vegetação utilizando-se Heterohabditis baujardi LPP7 e Steinernema carpocapse NcAll por pulverização foliar, associados a produtos adjuvantes. No primeiro ensaio, o número médio de furos causados pela broca-da-cana, nas testemunhas Joint* Oil e Gotafix® foram de 3,4 e 3,14 respectivamente. Já quanto aos tratamentos, o número de furos por colmo analisado foi de 2,44 para S. carpocapsae NCAll + Joint* Oil; 2,06 para H. baujardi LPP7 + Gotafix®; 2,06 para S. carpocapsae NCAll + Gotafix®; e 1,84 para H. baujardi LPP7 + Joint* Oil. Quando o experimento foi repetido, o número médio de furos nas testemunhas Gotafix® e Joint* Oil, foram respectivamente 4,78 e 4,76. Quanto aos tratamentos S. carpocapsae NCAll + Joint* Oil, S. carpocapsae NCAll + Gotafix®, H. baujardi LPP7 + Gotafix® e H. baujardi LPP7 + Joint* Oil, apresentaram o número médio de 2,18; 2,18; 1,96 e 1,96 furos por colmos avaliados. Assim, ambos nematóides controlaram a broca da cana, contudo não houve diferença significativa entre NEPs e produtos adjuvantes.

The aim of this study was to assess the influence of different exposure times of engorged female the Rhipicephalus microplus to infective juveniles of Heterorhabditis bacteriophora isolate HP88. The engorged females were divided into seven groups (six treatments and one control) of 20 ticks each with statistically similar average weights (p>0.05) and exposed to concentrations of 300 nematodes/tick for periods of 3, 6, 12, 24, 48 and 72h. The following biological parameters were monitored: pre-oviposition period; egg mass weight; hatching percentage; and efficacy of treatment. There was no influence of the exposure time on the pre-oviposition period (p>0.05), while there were significant differences (p

In Brazil, Meloidogyne mayaguensis has become a threat to guava production. Approximately a third of the cultivated area is infested, leading almost inevitably to the decimation of the orchards. Because parasitized trees develop rotten roots as the disease progresses, the possibility that a soil-borne pathogen could be involved was investigated. From several nematode-free or nematode-infested orchards, nearly 2000 root fragments were tested for bacteria and fungi. Positive isolations were obtained from nematode-infested areas only and were predominantly identified as Fusarium sp. In a 5-month microplot experiment, guava seedlings were uninoculated (control) or were inoculated with M. mayaguensis only or with this nematode and 21 days later with one of 11 Fusarium sp. isolates. A Scott–Knot analysis of several vegetative variables and of the extent of root rot allowed the generation of a dissimilarity dendrogram that indicated that four Fusarium sp. isolates were particularly associated with damage to the seedlings. Upon identification of these isolates as Fusarium solani, a 6-month microplot experiment was set up, in which guava seedlings were uninoculated or were inoculated with one of the following: (i) M. mayaguensis only, (ii) four F. solani isolates, separately, (iii) four F. solani isolates separately, combined with physical injury of the roots with a knife, (iv) M. mayaguensis, and 21 days later with four F. solani isolates, separately. No root rot and virtually no effect on all variables were observed in the seedlings inoculated with the fungus isolates, with or without physical injury. Major root rot and a negative effect on all variables were observed in the seedlings inoculated with M. mayaguensis and all four F. solani isolates. This characterizes guava decline as a complex disease caused by the synergistic effect of these organisms, in which parasitism by the nematode predisposes the plants to root decay caused by the fungus.

The Brazilian apple leafroller, Bonagota salubricola (Meyrick, 1937) ( Bonagota cranaodes ) (Lepidoptera: Tortricidae), is one of the main pest problems in apple orchards is Brazil. The purpose of this work was to evaluate B. salubricola larval and pupal susceptibility to different species of entomopathogenic nematodes, isolated in Rio Grande do Sul state in Brazil, under laboratory and field (apple orchard) conditions. Bioassays for isolates selection and determination of lethal concentration were performed in tubes of 1.5 ml (eppendorf), each containing one B. salubricola third instar larvae and filter paper. Field experiments were performed in commercial orchard, with application of 100 infecting juveniles (IJs)/cm 2 for each apple plant previously infected with five B. salubricola larvae covered with plastic trays containing thin cloth. Nematodes H. bacteriophora RS107 and H. bacteriophora RS57 had LC 50 of 13 and 4.5 IJs/larvae (4.3 and 1.5 IJs/cm 2 ), respectively. In the field, H. bacteriophora RS107 and H. bacteriophora RS57, applied with sorbitol as an adjuvant, reached 70.2 and 61.1% larval mortality, respectively. The results showed that both isolates had biological activity against B. salubricola under laboratory and field conditions.

Of the insect pests that attack guava fruits, the guava weevil, Conotrachelus psidii Marshall, 1922 (Coleoptera: Curculionidae), is one of the most important in Brazil. In search of alternatives to chemical pesticides, this study was performed to select fungal isolates of Beauveria bassiana and Metarhizium anisopliae as potential candidates for the control of adult C. psidii. Tests were carried out using three products applied with the entomopathogenic fungi: Tween 80, sunflower oil and Imidacloprid (IMI). A sub-lethal concentration of IMI was determined (100 ppm). The results demonstrated that LPP 19 and LPP 114 were the most effective isolates when used in combination with all of the products. The least virulent isolate, ESALQ 818, when applied in Tween 80 caused only 26.6% mortality, however, this isolate showed significantly improved efficiency when applied together with either sunflower oil or IMI, causing 57.3 and 88.6% mortality, respectively. The efficiency of all the isolates tested here...

In orchards, in cases where the insect pests are concentrated under the canopy, entomopathogenic nematodes can be applied as infected host cadavers. This study evaluated the distribution of Heterorhabditis baujardi Phan, Subbotin, Nguyen & Moens strain LPP7 applied as one or 15 host cadavers per plot in a guava orchard. The plots were located between guava trees, with the host cadavers being placed in the centre. Soil samples were taken at 1, 3, 5, 8 and 10 weeks after cadaver application. On each date, the plots were sampled at 30, 60, 90 and 120 cm from the cadaver application point and at three different depths (0 – 10, 10 – 20 and 20 – 30 cm). The highest average number of recovered infective juveniles was found at 90 cm from the cadaver application point, and at 10 cm depth and on the fifth week after application. There was no significant effect on the number of recovered infective juvenile nematodes, when they were applied as one or 15 cadavers, although the most uniform dispersal was found when 15 ...

Despite massive losses of primary forest, the Amazonian rainforest remains an extremely rich source of biodiversity. In recent years, entomopathogenic nematodes (EPNs) have been isolated from soil in various parts of the world and used successfully as biological control agents against numerous insect pests. Therefore, a sampling in the rainforest of Monte Negro, Rondonia, Brazil was conducted with the aim of discovering new strains and/or species of EPNs for future development as biological control agents. From 156 soil samples taken at nine collecting sites, 19 isolates were obtained, all of them belonging to the genus Heterorhabditis. Four strains were subjected to detailed morphological and molecular evaluation. Based on morphometrics and internal transcribed spacer (ITS) sequence data, the strains LPP1, LPP2 and LPP4 were identified as Heterorhabditis indica, whereas LPP7 was considered Heterorhabditis baujardi. Comparative analysis of the ITS1 sequence of H. indica and H. baujardi isolates showed a polymorphic site for the restriction enzyme Tth 111 that could be used to distinguish the two species. Consequently, strains LPP1, LPP2, LPP3, LPP4, and LPP9 were identified as H. indica, whereas LPP5, LPP7, LPP8 and LPP10 were identified as H. baujardi.

Entomopathogenic nematodes (EPNs) are currently being used as successful biological control agents of soil-dwelling insect pests. Previous field and greenhouse studies demonstrated that application techniques and non-biotic factors (temperature and pressure) have a significant effect on EPNs efficacy. The objective of this study was to evaluate the influence of an irrigation spray application system on the viability, infectivity and host search capability of Heterorhabditis baujardi LPP7 (Nematoda: Heterorhabditidae) infective juveniles (IJ). Two assays were proposed. Their viability was evaluated under the microscope after the IJ passed through the irrigation system. Infectivity on Galleria mellonella larvae, and host search capability, as evidenced by larval mortality, were evaluated in containers (Experiment 1). In the field (Experiment 2), mortality of G. mellonella larvae was evaluated under different nematode concentrations (0, 100,000, 300,000 and 500,000 IJ per tree). No differences were recorded on the viability, infectivity and host search capability of the IJ in Experiment 1. In Experiment 2, differences were recorded among the different concentrations used (p < 0.05), and a higher mortality was observed at the highest nematode concentration (28.3% and 37% in each one of the two experiment repetitions). This irrigation system did not affected adversely the viability, infectivity and host search capability of H. baujardi LPP7. Nematóides entomopatogênicos (NEPs) vêm sendo usados com sucesso como agentes do controle biológico de pragas de solo. Estudos anteriores mostraram que técnicas de aplicação e fatores abióticos (temperatura e pressão) afetam a eficiência dos NEPs em testes de campo e casa-de-vegetação. O objetivo deste trabalho foi avaliar a influência de condições geradas por um sistema de irrigação por microaspersão, na viabilidade, infectividade e na capacidade de busca de hospedeiros nos juvenis infectantes (JI) de Heterorhabditis baujardi LPP7 (Nematoda: Heterorhabditidae). Dois experimentos foram propostos. A viabilidade dos juvenis infectantes (JI) foi avaliada no microscópio imediatamente após sua passagem pelo sistema de irrigação. A infectividade e a capacidade de busca pelo hospedeiro em larvas de Galleria mellonella foram avaliadas em vasos (Experimento 1). A campo (Experimento 2), foi avaliada a mortalidade de larvas de G. mellonella sob diferentes concentrações do nematóide (0, 100.000, 300.000 e 500.000 JI por árvore). A viabilidade, infectividade e a capacidade de busca dos nematóides após a passagem pelo sistema não foi diferente da testemunha. No Experimento 2, houve diferença entre os tratamentos (p < 0.05) e se observou maior mortalidade na maior concentração de nematóides, com mortalidade de 28,3% e 37% em cada uma das duas repetições do experimento. Este sistema de micro-aspersão não afeta a viabilidade, infectividade e capacidade de busca dos JIs de H. baujardi LPP7 até o inseto-alvo.

The guava weevil, Conotrachelus psidii, is a major pest of guava in Brazil causing severe reduction in fruit quality. We assessed its susceptibility to Heterhorhabditis baujardi LPP7 infective juveniles (IJs) in the greenhouse and under field conditions applying the nematodes in cadavers of seventh instar Galleria mellonella larvae. Field persistence of these nematodes in the soil was evaluated through G. mellonella-baiting. Insect cadaver concentrations of 2, 4 and 6 applied in pots in the greenhouse experiment caused significant mortality compared to the control. Significance differences were observed in the field between control and treatments only when six cadavers per 0.25 m2 were applied. Infective juveniles from the cadavers persisted 6 weeks after application in the field, but decreased greatly thereafter. Our work demonstrates that H. baujardi LPP7 IJs emerging from G. mellonella cadavers can be efficacious against guava weevil fourth instar larvae. Also, we demonstrated the long-term pe...

The guava weevil, Conotrachelus psidii, is a major pest of guava in Brazil and causes severe reduction in fruit quality. This weevil is difficult to control with insecticides because adults emerge over a long period, and larvae develop to the fourth-instar inside the fruit and move to the soil for pupation. We assessed the virulence of entomopathogenic nematodes to fourth-instar larvae in soil by comparing their susceptibility to nine species or strains: Heterorhabditis bacteriophora HP88, H. baujardi LPP7, and LPP1, H. indica Hom1, Steinernema carpocapsae All and Mexican, S. feltiae SN, S. glaseri NC, and S. riobrave 355. In petri dish assays with sterile sand at a concentration of 100 infective juveniles (IJs) of a given nematode species/strain, larval mortality ranged from 33.5 to 84.5%, with the heterorhabditids being the most virulent. In sand column assays with H. baujardi LPP7, H. indica Hom1, or S. riobrave 355 at concentrations of 100, 200, and 500 IJs, mortality was greater than the control only for H. baujardi (62.7%) and H. indica (68.3%) at the highest concentration. For H. baujardi LPP7 in a petri dish assay, the time required to kill 50 and 90% of the larvae (LT50 and LT90) for 100 IJs was 6.3 and 9.9 days, whereas the lethal concentration required to kill 50 and 90% of the larvae (LC50 and LC90) over 7 days was 52 and 122.2 IJs. In a greenhouse study with guava trees in 20-L pots, 10 weevil larvae per pot, and concentrations of 500, 1000 or 2000 IJs, H. baujardi LPP7 caused 30 and 58% mortality at the two highest concentrations. These results show that H. baujardi is virulent to fourth-instar larvae and has potential as a biological control agent in IPM programs.

Engorged and partially engorged females of Boophilus microplus were exposed to 600; 3,000; 6,000 and 30,000 infective juveniles of Steinernema carpocapsae Weiser, 1955 Santa Rosa and All strains per dish, under lab conditions. Eggs weight, pre-laying period, laying period, survival period, reproductive efficiency rate, percentage of larval eclosion and lethal concentrations of 50% and 90% (CL50 and CL90) were calculated for engorged females. In the case of partially engorged females, only egg weight, survival period and CL50 and CL90 were calculated. All biological parameters of engorged or partially engorged females were altered by exposition of S. carpocapsae infective juveniles (IJs), Santa Rosa and All strains (pFêmeas ingurgitadas e parcialmente ingurgitadas de B. microplus foram expostas a 600, 3000, 6000 e 30000 juvenis infectivos de Steinernema carpocapsae Weiser, 1955 linhagens Santa Rosa e ALL por placa, sob condições de laboratório. Foram investigados para fêmeas ingurgitadas: peso da massa de ovos, período pré-postura, período de postura, tempo de sobrevivência, índice de eficiência reprodutiva, percentual de aclosão de larvas e concentrações letais 50% e 90% (CL50 e CL90). Para fêmeas parcialmente ingurgitadas somente foram observados peso da massa de ovos, tempo de sobrevivência, CL50 e CL90. Todos os parâmetros biológicos de fêmeas ingurgitadas foram e parcialmente ingurgitadas foram alterados pela exposição a juvenis infectivos de S. carpocapsae linhagens Santa Rosa e ALL (P

Biocontrol success when using entomopathogenic nematodes (EPNs) in the genera Heterorhabditis and Steinernema relies on a variety of factors including components of the application event itself. Successful application encompasses both abiotic and biotic influences (Grewal, 2002; Shapiro-Ilan, Gouge, & Koppenhofer, 2002; Shapiro-Ilan, Gouge, Piggott, & Patterson Fife, 2006; Shapiro-Ilan, Han, & Dolinski, 2012). For example, a diverse array of equipment is available for EPN application including various spray technology and irrigation systems. The specific application equipment that is chosen and parameters associated with EPN distribution can have a direct impact on the level of pest suppression achieved. Additionally, the choice of nematode species, rate of application, and other concurrent management practices are critical to success. In this chapter we review recent literature on EPN application technology, discuss novel innovations, and explore opportunities for future improvement.

Application of entomopathogenic nematodes (EPNs) formulated as insect cadavers has become an alternative to aqueous application for the control of agricultural pests. In this approach, the infected insect host cadaver is applied directly to the target site and pest suppression is achieved by the infective juveniles (IJs) that emerge from the host cadavers. This type of technology could be especially effective for small– and medium–sized growers, with planted areas up to 10 ha, or for use in flower pots and home orchards, etc. The cost of production for cadaver–based formulations is low because it eliminates the need to capture and concentrate infective juveniles (IJs) and reduces storage costs required in production systems that involve aqueous suspension of IJs. Also, the insect cadaver represents a shelter from environmental extremes such as freezing. However, the insect cadaver approach has a number of downsides that demand further study and adaptation. In this chapter we review the development and application of the “cadaver approach” and discuss advantages and disadvantages as well as avenues for future research.

Guava decline, caused by the interaction between the phytonematode Meloidogyne enterolobii and the fungus Fusarium solani, has caused direct and indirect losses to the whole productive chain of guava. Aiming to understand the interaction mechanisms between M. enterolobii and F. solani, this study carried out a bioassay on guava plants with roots in two different treatments: inoculated separatelyor together with the fungus and/or nematode. The nematode parasitism not triggered an systemic effect on the plant become susceptible to root rot caused by the fungus.Therefore, it was concluded that there was a local effect of parasitism by M. enterolobii on the pathogenicity of F. solani in guava roots, making it necessary for the two pathogens to occupy the same space at the same time for occurrence of guava decline. Keywords: complex disease, Fusarium solani, guava root-knot nematode, Meloidogyne enterolobii, Psidium guajava. Cite as Gomes VM, Souza RM, Almeida AM, Dolinski C. Relationships between M. enterolobii and F. solani: spatial and temporal dynamics in the occurrence of guava decline.

Classical hypotheses of the phylogenetic position of Tylenchida within Secernentea are evaluated considering the comparative fine structure of the basal bulb of the pharynx of Basiria gracilis, putatively a representative of the most morphologically conserved members of the order. Unlike other Tylenchida examined in detail, B. gracilis has a full complement of nonglandular cells in the basal bulb including 13 radial muscle cells and six marginal cells. With respect to types and numbers of cells, the basal bulb of B. gracilis most nearly resembles representatives of Cephalobina, differing only by three vs five gland cells. There are more muscle cells in the basal bulb of B. gracilis than in representatives of Rhabditina (ten muscles) and Diplogastrina (six muscles). Considering Teratocephalus as a representative of the outgroup (to establish character polarity), B. gracilis appears to retain most of the conserved features of the basal bulb of Secernentea, with the exception of loss of a heavily cuticularised grinder valve or pump.

This study evaluated the efficacy of four entomopathogenic nematode (EPN) strains in insect cadaver formulations against Rhipicephalus microplus and compared the efficacy of the most virulent EPNs applied in cadavers of Galleria mellonella and Tenebrio molitor. In the first experiment, infected G. mellonela larvae were used as the source of EPNs. Engorged females of R. microplus were placed in pots filled with soil and different numbers of G. mellonella larvae infected with one of four species of nematodes. All treatments with EPNs of the genus Heterorhabditis caused significant reduction (p

Heterorhabditis is a nematode found in the soil that is used as an important biological control agent against various organisms. However, few studies have been performed of its use against snails and the present study is the first to investigate the effect of experimental exposure of Bradybaena similaris to Heterorhabditis indica LPP1. Two groups of 16 snails were formed: the control group (not exposed) and the treatment, which was exposed for three weeks to infective juveniles (J3) of H. indica LPP1. The entire experiment was conducted in duplicate, using a total of 64 snails. After this period, the snails were dissected to collect the hemolymph to evaluate the possible physiological alterations, namely total proteins, uric acid and hemolymph urea, as well as the activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) as a result of the infection. The terrariums were analyzed on alternate days throughout the experiment to count the dead snails. Intense proteolysis was observed in the infected snails. An increase in the level of uric acid and reduction of the hemolymph urea content indicated that the infection by H. indica results in the inversion of the excretion pattern of the host snail. Variations in the aminotransferase activities were also observed, with the infected group presenting significantly higher values (p

O presente trabalho avaliou em laboratorio, a utilizacao de diferentes linhagens de nematoides entomopatogenicos (NEPs) individualmente e combinadas visando ao controle biologico da mosca-do-Mediterrâneo, Ceratitis capitata Wied . (Diptera, Tephritidae). No primeiro bioensaio foram utilizadas oito linhagens individualizadas de NEPs ( Steinernema carpocapsae NCALL, Heterorhabditis bacteriophora HP88, H. baujardi LPP7, H. indica LPP1, H. indica LPP14, H. sp. LPP9, H . sp. LPP17 e H . sp. LPP12) sendo que para cada tratamento foram utilizados 20 tubos de ensaio cada um contendo areia,10 larvas L3 de C. capitata e 100 juvenis infectantes (JIs) diluidos em 1 cm3 de agua destilada. No tratamento controle foi adicionado 1 cm3 de agua destilada. No segundo bioensaio, foram utilizadas cinco larvas de C. capitata e as linhagens de nematoides foram combinadas duas a duas num total de 100 juvenis por repeticao (50 JIs de cada linhagem) Os bioensaios foram conduzidos a 28 oC, 80% UR e 12 de fotoperiodo. A mortalidade media das larvas foi avaliada pelo teste de Tukey a 1%. Individualmente as linhagens H. baujardi LPP7, H. indica LPP14, H. sp. LPP17 e H. sp. LPP12 foram as mais eficientes e causaram mortalidade entre 75 e 98,5%. As combinacoes mais eficientes foram H. indica LPP14 + H. sp. LPP9 e H. sp. LPP17 + H. sp. LPP12 com mortalidade de larvas L3 de 60 e 82%, respectivamente. Conclui-se que tanto separadamente ou em combinacao, algumas linhagens de NEPs podem ser usadas no controle biologico de C. capitata , sendo que quando usadas separadamente, a eficiencia e maior. ABSTRACT The present study investigated under laboratory conditions the use of entomopathogenic nematodes strains separately or in combinations, as biological control agent of Mediterranean fruit fly, Ceratitis capitata Wied. (Diptera, Tephritidae). In the first bioassay, eight strains were used separately ( Steinernema carpocapsae NCALL, Heterorhabditis bacteriophora HP88, H. baujardi LPP7, H. indica LPP1, H. indica LPP14, H. sp. LPP9, H . sp. LPP17 e H . sp. LPP12). For each treatment, 20 test tubes with sand, 10 larvae of C. capitata and 100 infective juveniles (IJs) diluted in 1 cm3 of distilled water were used. In the treatment control only 1 cm3 of distilled water was added. In the second bioassay it was used the same material; however, the number of C. capitata larvae was reduced to five and strains of nematodes combined in pairs, in a total of 100 IJs per replicate (50 individuals of each strain). All treatments were stored in an incubator for 15 days (28 oC, 80% RU and 12 h photoperiod). The average mortality of larvae L3 was evaluated by Tukey test at 1%. The strains H. baujardi LPP7, H. indica LPP14, H. sp. LPP17 and H. sp. LPP12 were the most efficient ones, reaching mortalities range between 75 and 98.5%. In the second experiment, the most effective combinations were H. indica LPP14 + H. sp. LPP9 and H. sp. LPP17 + H. sp. LPP12 with mortality of 60 and 82%, respectively. We concluded that the use of NEPs in the biological control of C. capitata is a feasible alternative either using species separated or in combination, but the first one may reach higher mortality.

Two assays were conducted to assess the influence of infective juveniles (IJs) of Heterorhabditis baujardi LPP7 on the embryogenesis and hatching of Meloidogyne mayaguensis. In the first assay, eggs were incubated in water alone or in the presence of infective juveniles, and completion of embryogenesis was evaluated 14 days later. In the second assay, unhatched second-stage juveniles were incubated in distilled water alone or in the presence of infective juveniles. Cumulative hatching was compared at various time intervals. Embryogenesis was not affected, whereas second-stage juveniles hatching was delayed probably because of the eggs permeability to noxious metabolites released by Photorhabdus luminescens, which is the bacterial symbiont of H. baujardi.

Entomopathogenic nematodes (EPNs) are used for the biological control of soil insect pests worldwide and are generally applied to the soil in aqueous suspension. An alternative application method that could be especially practical and effective under certain conditions is to apply the nematode-killed insect (referred to herein as infected insect cadavers) that are placed on or in the soil and from which the nematodes emerge to seek new hosts. However, physical damage to the insect cadavers during handling and application as well as the potential detrimental impact of various soil biotic and abiotic factors could reduce the efficacy of cadaver applications. Our objective was to test the effectiveness of various protective coverings applied to Galleria mellonella insect cadavers in terms of their potential impact on the emergence and virulence of infective juveniles of the EPN Heterorhabditis baujardi LPP7, and to evaluate whether these coverings influenced cadaver removal by ants (Ectatomma spp.). The protective coveringtreatments included a commercial calcareous powder, a commercial talc powder, and gelatin capsules. The number of emerging infective juveniles (IJs) from insect cadavers formulated with talc powder (9.722 ± 1.382) and gelatin capsules (7.892 ± 1.072) was similar to the control (6.346 ± 1.311), and indicated that these coverings do not interfere with IJ emergence. However, the powdered calcareous covering significantly reduced IJ emergence. High infectivity was observed for IJs that emerged from cadavers in all treatments. Ectatomma spp. ants removed all insect cadavers from the nest entrance to a distance of 20 cm, with the exception of insect cadavers formulated in gelatin capsules, which were not removed. 2009 Elsevier Inc. All rights reserved. Fil: del Valle, Eleodoro Eduardo. Universidad Nacional del Litoral; Argentina Fil: Dolinski, Claudia. Universidade Estadual Do Norte Fluminense Darcy Ribeiro; Brasil Fil: Barreto, Eduardo L.S.. Universidade Estadual Do Norte Fluminense Darcy Ribeiro; Brasil Fil: Souza, R.M.. Universidade Estadual Do Norte Fluminense Darcy Ribeiro; Brasil

Laboratory, greenhouse, and field experiments were performed with the objective of selecting efficient indigenous strains of entomopathogenic nematodes (EPNs) from Rio Grande do Sul (RS) state, Brazil, for controlling the South American fruit fly, Anastrepha fraterculus (Wied.). Laboratory experiments were conducted in 24 well-plates filled with sterile sand and one insect per well. In greenhouse experiments, plastic trays filled with soil collected from the field were used, while in field experiments, holes were made in soil under the edge of peach tree canopies. Among 19 EPN strains tested, Heterorhabditis bacteriophora Poinar RS88 and Steinernema riobrave Cabanillas, Poinar, & Raulston RS59 resulted in higher A. fraterculus larval (pre-pupal) and pupal mortality, with LD(90) of 1630, 457 and 2851, 423 infective juveniles (IJs)/cm(2), respectively. Greenhouse experiments showed no differences in pupal mortality at 250 and 500IJs/cm(2) of either nematode. In the field, H. bacteriophora RS88 and S. riobravae RS59 sprayed individually over natural and artificially infested fruit (250IJs/cm(2)) resulted in A. fraterculus larval mortality of 51.3%, 28.1% and 20%, 24.3%, respectively. There was no significant difference in A. fraterculus pupal mortality sprayed with an aqueous suspension of either nematode; however, when using infected insect cadavers, H. bacteriophora RS88 was more efficient than S. riobrave RS59. Our results showed that H. bacteriophora RS88 was more virulent to insect larvae, with an efficient host search inside the infested fruit and control of pupae in the soil after being applied by aqueous suspension or infected cadavers.

In view of the need to combat the generalized spread of resistance in ticks to commercial acaricides, the objective of this study was to evaluate the action of entomopathogenic nematodes (Steinernema carpocapsae, strains Santa Rosa and ALL) on engorged female Anocentor nitens. Five ticks per Petri dish were exposed to concentrations of 500, 5,000, or 25,000 infective juveniles of S. carpocapsae for 72 h. After transferring the ticks to clean plates, biological parameters were analyzed. Related to strains Santa Rosa, the period of pre-oviposition (p = 0.0001), oviposition (p = 0.041), and the mass weight of eggs (p = 0.005) showed significant differences between the control group and treated group. When the strain ALL was tested, the control and treated groups differed between the periods of pre-oviposition (p = 0.001), oviposition (p = 0.001), and egg mass weight (p = 0.01). The egg mass conversion was less significant in the groups when exposed to strains Santa Rosa (p = 0.002) and ALL (p = 0.001) relative to the control. The efficacy of both entomopathogenic nematode strains used in this study was comparable to other biological control agents, showing their potential against A. nitens in the laboratory.

A multitude of insects and mites attack fruit crops throughout the tropics. The traditional method for controlling most of these pests is the application of chemical pesticides. Growing concern on the negative environmental effects has encouraged the development of alternatives. Inundatively and inoculatively applied microbial control agents (virus, bacteria, fungi, and entomopathogenic nematodes) have been developed as alternative control methods of a wide variety of arthropods including tropical fruit pests. The majority of the research and applications in tropical fruit agroecosystems has been conducted in citrus, banana, coconut, and mango. Successful microbial control initiatives of citrus pests and mites have been reported. Microbial control of arthropod pests of banana includes banana weevil, Cosmopolites sordidus Germar (Coleoptera: Curculionidae) (with EPNs and fungi) among others Oryctes rhinoceros (L.) is one of the most important pests of coconut and one of the most successful uses of non-occluded virus for classical biological control. Key pests of mango that have been controlled with microbial control agents include fruit flies (Diptera: Tephritidae) (with EPNs and fungi), and other pests. Also successful is the microbial control of arthropod pests of guava, papaya and pineapple. The challenge towards a broader application of entomopathogens is the development of successful combinations of entomopathogens, predators, and parasitoids along with other interventions to produce effective and sustainable pest management. Muitos insetos e ácaros atacam fruteiras nos trópicos. O método tradicional para controlar a maioria dessas pragas é a aplicação de inseticidas químicos. A crescente preocupação sobre os efeitos negativos desses produtos vem encorajando o desenvolvimento de alternativas. Agentes do controle biológico aplicados de forma inundativa ou inoculativa têm sido pesquisados como método de controle alternativo para uma variedade de pragas em grande número de culturas, incluindo fruteiras tropicais. A maioria das pesquisas e aplicações em fruteiras tropicais tem sido feita em citros, banana, côco e manga. Especificamente, há vários casos de sucesso em citros para diferentes ácaros e insetos. Em banana, o controle do moleque-da-bananeira, Cosmopolites sordidus Germar (Coleoptera: Curculionidae) com nematóides e fungus também é considerado de sucesso. Oryctes rhinoceros (L.) (Coleoptera: Scarabaeidae) é uma das principais pragas em côco e um dos mais importantes exemplos de controle biológico clássico por vírus não-ocluso. As pragas-chave em manga que têm sido controladas com diferentes agentes de controle microbiano são as moscas-das-frutas (Diptera: Tephritidae), com nematóides e fungos, o gorgulho-da-manga, Sternochetus mangiferae (Fabricius) (Coleoptera: Curculionidae), com fungos, e vários hemípteros com fungo. O controle microbiano de pragas tem sido bem sucedido também em goiaba, mamão e abacaxi. O desafio que se vislumbra para a maior aplicabilidade dessas tecnologias é o desenvolvimento de combinações compatíveis entre entomopatógenos, predadores e parasitóides juntamente com outras técnicas de controle.

Interactions between the nematode Heterorhabditis bacteriophora isolate JPM4 and the fungus Metarhizium anisopliae , isolates LPP45 and LPP39, were studied during dual infections of Diatraea saccharalis . Mortality, production of infective juveniles (IJs) and production of conidia were evaluated. A positive effect was demonstrated for host mortality in duel infections of JPM4 and LPP39, causing 100% mortality with LT 50 and LT 95 values of 1.8 and 2.8 days, respectively. Higher values were seen when using the nematode or fungi individually. However, a combination of JPM4 + LPP39 caused a significant reduction in IJ production. The results show that faster time to death, a moderately virulent fungal isolate could be combined with the nematode, however at the expense of IJ production.

The present study was carried out to evaluate the action of Steinernema glaseri Santa Rosa strain and Heterorhabditis bacteriophora CCA strain as biological control agents of Boophilus microplus. Engorged females ticks were distributed on Petri dishes containing different concentrations of infective juvenile (IJ) nematodes (0, 375, 500, 750, 1,500, 2,500, 5,000 and 25,000). The data showed a reduction of approximately 90% in the eggs laid at a concentration of 5,000 S. glaseri IJs and approximately 80% at a concentration of 1,500 H. bacteriophora IJs. The female mortality increased linearly with the increase in S. glaseri concentrations. However, in the tests with H. bacteriophora this linearity was not observed. The effectiveness of the treatment with both species of entomopathogenic nematodes was compatible with other control methods. The results show the potential of S. glaseri and H. bacteriophora as biological control agents for the control of B. microplus under laboratory conditions.