Role of Synadenium grantii latex proteases in nematicidal activity on Meloidogyne incognita and Panagrellus redivivus / Papel das proteases do látex de Synadenium grantii na atividade nematicida sobre Meloidogyne incognita e Panagrellus redivivus

Abstract Synadenium grantii is a Euphorbiaceae plant commonly found in Brazil, known as Janaúba or Leitosinha, whose latex is traditionally used for several purposes. However, it is not known whether the nematicidal action of this plant latex occurs due to the action of proteases. The present work aims to evaluate the nematicidal activity of proteases from Synadenium grantii latex on Meloidogyne incognita and Panagrellus redivivus. S. grantii latex used in the present study was collected from specimens found in Universidade Federal de Viçosa, Viçosa, Minas Gerais, Brazil. The drained latex was collected in Eppendorf microtubes and immediately stored on ice at 4 °C. After this extraction, the latex was frozen (-20 °C) during 2 hours, thawed at room temperature (25 °C) and centrifuged at 10,000 g at 4 °C for 30 minutes to remove larger particles and concentrate the proteases. After the centrifugation, assays of enzymatic activity were performed in order to know in which of the phases the enzymes were found. S. grantii latex presented protease, but no chitinase activity. The results show that there was a significant difference (p <0.01) between the treated and control groups, with 100% mortality of Meloidogyne incognita and 72% average mortality of Panagrellus redivivus. In addition, it was demonstrated that the nematicidal action occurred due to the action of the proteases, since the control was only differentiated from the treatment by the presence of the enzymes with biological activity.

Resumo Synadenium grantii é uma planta Euphorbiaceae comumente encontrada no Brasil, conhecida como Janaúba ou Leitosinha, e tem seu látex usado tradicionalmente para diferentes propósitos. Entretanto, não se conhece se a atividade nematicida da planta ocorre devido à ação de proteases. O presente trabalho tem como objetivo avaliar a atividade nematicida das proteases do látex de Synadenium grantii sobre Meloidogyne incognita e Panagrellus redivivus. O látex de S. grantii utilizado no presente trabalho foi coletado a partir de espécimes encontradas na Universidade Federal de Viçosa, Viçosa, Minas Gerais, Brazil. O látex foi coletado em microtubos Eppendorf e imediatamente armazenado em gelo a 4 °C. Após esta extração, o látex foi congelado (-20 °C) durante 2 horas, descongelado à temperatura ambiente (25 °C) e centrifugado a 10000 g a 4 °C durante 30 minutos para a remoção de partículas e concentração das proteases. Após a centrifugação, foram realizados ensaios de atividade enzimática para saber em qual das fases as enzimas foram encontradas. O látex de S. grantii apresentou atividade de protease, mas nenhuma atividade de quitinase. Os resultados mostram que houve diferença significativa (p <0,01) entre os grupos tratados e controle, com 100% de mortalidade de Meloidogyne incognita e 72% de mortalidade média de Panagrellus redivivus. Além disso, foi demonstrado que a ação nematicida ocorreu devido à ação das proteases, uma vez que o grupo controle só foi diferenciado do tratamento pela presença das enzimas com atividade biológica.

Potential of entomopathogenic nematodes of the genus Heterorhabditis for the control of Stomoxys calcitrans (Diptera: Muscidae) / Potencial de nematoides entomopatogênicos do gênero Heterorhabditis para o controle de Stomoxys calcitrans (Diptera: Muscidae)

Abstract The present study verified the pathogenic potential of entomopathogenic nematodes (EPNs) of the genus Heterorhabditis (Heterorhabditis bacteriophora, isolate HP88 and Heterorhabditis baujardi isolate LPP7) to immature stages of Stomoxys calcitrans in the laboratory. All EPN concentrations of the H. bacteriophora HP88 strain caused mean larval mortality greater than 90% after four days. Higher concentrations of the H. baujardi LPP7 isolate (≥50 EPNs/larva) eliminated more than 70% of larvae after six days with the concentration 200 EPNs/larva reaching mortality levels of 93.3%. The larval mortality at all concentrations of EPNs (25, 50, 100, 150, and 200 EPNs/larva) for both strains was significant (p<0.05) when compared to the respective control groups. Concentrations of H. bacteriophora HP88 yielded an LC50 of 0.36 EPN/larva and LC90 of 29.1; while H. baujardi LPP7 yielded an LC50 of 39.85 and LC90 of 239.18. H. bacteriophora HP88 provided greater inhibition of the emergence of adults when compared to the response obtained with H. baujardi LPP7. EPNs did not cause considerable mortality when applied directly to pupae. The set of observed results suggests that the EPNs of the genus Heterorhabditis, isolates HP88 and LPP7, are a promising alternative in the control of the stable fly.

Resumo O presente estudo verificou o potencial patogênico de nematoides (NEP) do gênero Heterorhabditis (H. bacteriophora - isolado HP88 e H. baujardi - isolado LPP7) para estágios imaturos de Stomoxys calcitrans em laboratório. Todas as concentrações de NEPs da cepa HP88 causaram mortalidade larval média maior que 90% após quatro dias. Concentrações mais elevadas da cepa LPP7 (≥50 NEPs/larva) eliminaram mais de 70% após seis dias, com a concentração 200 NEPs/larva atingindo níveis de 93,3%. A mortalidade larval em todas as concentrações de NEPs (25, 50, 100, 150, and 200 NEPs/larva) para ambas as cepas foi significativa (p<0,05), quando comparadas aos respectivos grupos controle. Concentrações de H. bacteriophora HP88 obtiveram LC50 de 0,36 NEP/larva e LC90 de 29,1; enquanto H. baujardi LPP7 obteve LC50 de 39,85 e LC90 de 239,18. O isolado H. bacteriophora HP88 propiciou maior inibição da emergência de adultos, quando comparado à resposta obtida com H. baujardi LPP7. NEPs não provocaram mortalidade considerável quando aplicados diretamente sobre pupas. O conjunto de resultados observados indica os nematoides entomopatogênicos do gênero Heterorhabditis (estirpes HP88 e LPP7) como uma alternativa promissora no controle da mosca dos estábulos.

Ecological characterisation of the Colombian entomopathogenic nematode Heterorhabditis sp. SL0708 / Caracterização ecológica do nematoide entomopatogênico colombiano Heterorhabditis sp. SL0708

The entomopathogenic nematode Heterorhabditis sp. SL0708 (Rhabditida: Heterorhabditidae) isolated from soil in Alcalá, Valle del Cauca (Colombia) was characterised ecologically using Galleria mellonella larvae (L) (Pyralidae: Galleriinae) as hosts. The effect of temperature on the viability, infectivity and reproduction, and of moisture on infectivity and storage in liquid were evaluated in infective juveniles (IJs). Significant differences were found in the viability, infectivity and reproduction of the IJs at different temperatures. No nematodes were recovered at 5 °C and 10 °C, and at 35 °C no infectivity was observed. Average daily nematode recovery was best at 25 °C, and survival of the IJs was low in substrates presenting 13% moisture. The optimal storage temperature for Heterorhabditis sp. SL0708 was between 20 °C and 30 °C, keeping its infectivity for up to 8 weeks.

O nematoide entomopatogênico colombiano Heterorhabditis sp. SL0708, isolado do solo de Alcalá, Valle del Cauca (Colombia), foi caracterizado ecologicamente utilizando-se como hospedeiro lagartas de Galleria mellonella (L) (Pyralidae:Galleriinae). O efeito da temperatura na viabilidade, na infecção e na reprodução, e da umidade na infecção e do armazenamento em líquido foi avaliado em juvenis infectantes (JIs). Diferenças significativas na viabilidade, na infecção e na reprodução dos JIs foram observadas nas diferentes temperaturas. Não foram recuperados nematoides a 5 °C e 10 °C, e não se observou infecção a 35 °C. A maior média diária de recuperação de nematoides foi a 25 °C. Por outro lado, a sobrevivência dos JIs foi baixa nos substratos com unidades de 13% e a melhor temperatura de armazenamento para SL0708 foi entre 20 °C e 30 °C, mantendo a infecção até oito semanas.

Influence of soil temperature and moisture on the infectivity of entomopathogenic nematodes (Rhabditida: Heterorhabditidae, Steinernematidae) against larvae of Ceratitis capitata (Wiedemann) (Diptera: Tephritidae)

The Mediterranean fruit fly, Ceratitis capitata (Wiedemann), is considered one of the main pests that affect fruit production in the world. This insect spends part of its life cycle in the soil, making it a target for entomopathogenic nematodes. This work aimed at evaluating the influence of soil temperature and moisture on the infectivity of Heterorhabditis sp. RSC01 and Steinernema carpocapsae ALL to third-instars of C. capitata, and to compare the efficiency of these isolates at five different soil temperatures (19, 22, 25, 28, and 31°C) and three levels of relative soil moisture (100, 75, and 50 percent of field capacity). Ten C. capitata larvae were transferred to plastic jars (12 cm × 6 cm) containing 100 g soil, followed by the application of an aqueous suspension containing 125 infective juveniles (IJ)/cm². In the control treatment, 3 ml of distilled water was applied. Mortality evaluations were made five days later and were confirmed by observations of the characteristic symptoms and cadaver dissection. The infectivity was directly proportional to temperature increase, with maximum percent mortality of 86.7 percent and 80.0 percent for S. carpocapsae and Heterorhabditis sp., respectively, at 31°C. At 25°C, the highest mortality for both species was obtained at 75 percent of field capacity (96.7 percent and 26.7 percent for S. carpocapsae and Heterorhabditis sp., respectively).

Molecular and morphological characterization of heterorhabditid entomopathogenic nematodes from the tropical rainforest in Brazil

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, Rondônia, 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.

Susceptibility of diamond back moth, Plutella xylostella (L) to entomopathogenic nematodes.

Eight entomopathogenic nematode species / strains, Steinernema glaseri (steiner), S. carpocapsae (Weiser), S. feltiae (Filipjev), Steinernema sp. Ecomax strain, Heterorhabditis bacteriophora (Pioner), Heterorhabditis sp. Ecomax strain, two locally isolated strains called as JFC and TFC were tested against the final instar larvae of diamond back moth, Plutella xylostella (L.). All nematodes were found pathogenic. However, H. bacteriophora was adjudged the most pathogenic amongst the test nematodes on the basis of LD50 (9.16 IJS/larva), LT50 (43.26 hr), Lex T50 (3.24 hr) and the propagation potential (average of 271.42 IJS/mg) on the host body weight.

Two new species of nematodes (Rhabditida: Diplogasteridae and Rhabditidae) parasites of Gryllodes Iaplatae (Orthoptera: Gryllidae) in Argentina

Cephalobium magdalensis n.sp. (Rhabditida: Diplogasteridae) found in Magdalena, Buenos Aires, and Cruznema lincolnensis n. sp (Rhabditida: Rhabditidae) found in Lincoln, Buenos Aires, parasitizing the cricket Gryllodes laplatae (Orthoptera: Gryllidae) are described and illustrated. C. magdalensis n. sp. is characterized by having the excretory pore between the pseudobulb and the basal bulb and seven pairs of postanal papillae in the male. C. lincolnensis n.sp. can be distinguished by having meanly two pairs of preanal papillae, one pair of adanal papillae and six pairs of postanal papillae in the male.