Potential of endophytic fungi as biocontrol agents of Duponchelia fovealis (Zeller) (Lepidoptera: Crambidae) / Fungos endofíticos para o biocontrole de Duponchelia fovealis (Zeller) (Lepidoptera: Crambidae)

Abstract This study reports the first assessment of endophytic fungi isolated from strawberry leaves and selection of isolates for the control of Duponchelia fovealis, a new pest of strawberries. A total of 400 strawberry leaves of the cultivar 'Albion' were collected in four commercial farms. Leaves were disinfected, cut in fragments, and placed on Petri dishes containing potato dextrose agar media with tetracycline and incubated for 30 days. Following this time, 517 fungal colonies were isolated, and thirteen genera were identified: Cladosporium, Aspergillus, Nigrospora, Fusarium, Trichoderma, Chaetomium, Alternaria, Paecilomyces, Penicillium, Ulocladium, Bipolaris, Diaporthe, and Phoma. Eight isolates belonging to the genera Aspergillus, Diaporthe, Paecilomyces, and Cladosporium were selected for pathogenicity bioassays against third instar larvae of D. fovealis. Isolates of Paecilomyces induced the highest mortality rates.

Resumo Este trabalho apresenta a primeira contribuição no isolamento de fungos endofíticos de folhas de morangueiro e na seleção de isolados para controle de Duponchelia fovealis, uma nova praga do morangueiro. Foram coletadas 400 folhas da cultivar 'Albion' em quatro lavouras comerciais de morangueiro. As folhas foram desinfetadas, cortadas em fragmentos e depositadas em placas de Petri contendo Ágar Batata Dextrose como meio, em conjunto com tetraciclina e incubados durante 30 dias. Um total de 517 colônias fúngicas e treze gêneros foram isolados: Cladosporium, Aspergillus, Nigrospora, Fusarium, Trichoderma, Chaetomium, Alternaria, Paecilomyces, Penicillium, Ulocladium, Bipolaris, Diaporthe e Phoma. Oito isolados pertencentes aos gêneros Aspergillus, Diaporthe, Paecilomyces e Cladosporium foram selecionados para os bioensaios de virulência contra larvas de 3º instar de D. fovealis. Isolados de Paecilomyces causaram as maiores taxas de mortalidade.

Two new Brazilian isolates of Bacillus thuringiensis toxic to Anticarsia gemmatalis (Lepidoptera: Noctuidae) / Dois novos isolados brasileiros de Bacillus thuringiensis tóxicos para Anticarsia gemmatalis (Lepidoptera: Noctuidae)

Bacillus thuringiensis is a bacterium used for biopesticides production and pest-resistant plants due to the synthesis of protein crystals by cry genes, which are effective in controlling several insect orders such as Lepidoptera. This work aimed at the evaluation and characterisation of two new B. thuringiensis isolates active against A. gemmatalis (Hübner 1818) larvae, which is the soybean major pest. The results showed that Bt117-4 isolate amplified fragments corresponding to cry2 and cry9 genes, and synthesised protein fragments equivalent to 130, 90 and 45 kDa. The Bt3146-4 isolate amplified DNA fragments corresponding to cry9 gene and synthesised protein fragments of 70, 58 and 38 kDa. Transmission electron microscopy revealed the presence of protein crystals in both isolates. CL50 with Cry purified proteins from Bt117-4 and Bt3146-4, corresponded to 0.195 and 0.191 µg larvae-1, respectively. The two B. thuringiensis isolates selected in this study were effective to control velvetbean caterpillar at laboratory conditions. Field tests should be carried on to develop new biopesticides formulation as well for cry genes resource for Anticarsia gemmatalis resistant transgenic plants.

Bacillus thuringiensis é uma bactéria utilizada na produção de biopesticidas e de plantas resistentes às pragas por causa da síntese de cristais proteicos pelos genes cry, os quais são eficazes no controle de diversas ordens de insetos, como os lepidópteros. O presente trabalho objetivou a avaliação e a caracterização de dois novos isolados de B. thuringiensis ativos contra lagartas de A. gemmatalis (Hübner 1818), que é a principal praga da cultura da soja. Os resultados obtidos revelaram que o isolado Bt117-4 amplificou fragmentos correspondentes aos genes cry2 e cry9, sendo que os fragmentos proteicos sintetizados foram equivalentes a 130, 90 e 45 kDa. O isolado Bt3146-4 amplificou fragmentos de DNA que correspondem ao gene cry9 e sintetizou fragmentos proteicos de 70, 58, e 38 kDa. Os dados de microscopia eletrônica de transmissão revelam a presença de cristais proteicos em ambos os isolados. A CL50, com proteínas Cry purificadas de Bt117-4 e Bt3146-4, correspondeu a 0,195 e 0,191 µg lagarta-1, respectivamente. Os dois isolados de B. thuringiensis selecionados neste trabalho mostraram-se eficientes no controle da lagarta-da-soja em laboratório, sendo recomendada sua avaliação a campo para posterior aplicação na formulação de biopesticidas ou como fonte de genes cry para a obtenção de plantas geneticamente modificadas resistentes à Anticarsia gemmatalis.

Plasmid patterns of efficient and inefficient strains of Bacillus thuringiensis against Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae)

Bacillus thuringiensis harbors genes encoding Cry proteins found in chromosomes or plasmids of different sizes (4-150 Mb). Although the smaller plasmids are more abundant in B. thuringiensis, their specific function is unknown. As for the megaplasmids, their main recognized function is to harbor cry genes, although the sequencing of some of these plasmids indicates the occurrence of other important genes. This work used a new protocol for practical and rapid extraction of plasmid DNA in order to characterize the plasmid patterns of Brazilian strains belonging to Embrapa Milho e Sorgo research center B. thuringiensis bank. We tried to further assess the relationship of plasmid patterns with strains belonging to the same serovars and strains causing 100 percent and no mortality to Spodoptera frugiperda (J.E. Smith) larvae. It was possible to characterize 59 strains based on the migration of bands in agarose gel. Strains belonging to the same serovars showed different plasmid sizes (from 1,636 bp to 23,200 bp), with the exception of two strains belonging to serovar galleriae. The strain T09 Bt tolworthi showed a plasmid migration pattern identical to strains belonging to serovar galleriae. Plasmid patterns differed for 46 strains, confirming that this is a useful tool to discriminate specific strains. However, it was not possible to associate the plasmid pattern or the occurrence of particular plasmids with the pathogenicity of a given species towards S. frugiperda larvae.

Seleção de isolados de fungos entomopatogênicos para o controle de Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) e sua compatibilidade com alguns inseticidas usados na cultura do tomateiro / Selection of isolates of entomopathogenic fungi for controlling Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) and their compatibility with insecticides used in tomato crop

The activity of Metarhizium anisopliae and Beauveria bassiana towards eggs and larvae of Tuta absoluta (Meyrick) was evaluated. Our data showed that the isolates were pathogenic to both developmental stages tested and the eggs were more susceptible than the 1st instars. The isolates URPE-6 and URPE-19 of M. anisopliae were more pathogenic to eggs and larvae, respectively. The compatibility of these two isolates with the insecticides chlorfenapyr, spinosad, indoxacarb, abamectin, and neem were evaluated. Spinosad and indoxacarb were compatible with the two M. anisopliae isolates in all tested concentrations. At the average recommended concentration, chlorfenapyr was compatible to URPE-6 and abamectin to UFPE-19. The use of entomopathogenic fungi associated with compatible insecticides may be a useful alternative to control T. absoluta.

Estandarización de un bioensayo y evaluación preliminar de tres formulaciones comerciales de bacillus thuringiensis sobre tuta absoluta (meyrick) (lepidoptera: gelechiidae) / Standardising a bioassay for the preliminary evaluation of threecommercial bacillus thuringiensis formulations against tutaabsoluta (meyrick) (lepidoptera: gelechiidae)

La polilla del tomate (Tuta absoluta Meyrick; Lepidoptera: Gelechiidae) es una de las plagas más devastadorasdel tomate en Colombia y países suramericanos, produciendo pérdidas de hasta el 100% en cultivos sin protección.En 2009, T. absoluta se detectó en España, Portugal y países del mediterráneo, además de Inglaterra,Bulgaria y Alemania. Para su control se utilizan insecticidas químicos que generan resistencia e impactoambiental y de salud. La alternativa de utilizar biopesticidas contra esta plaga es de importancia creciente. Eneste estudio se evaluaron cinco métodos de bioensayo para medir adecuadamente la toxicidad sobre larvasde T. absoluta de tres productos comerciales: Dipel®, XenTary® y Turilav®, formulaciones a base de Bacillusthuringiensis (Bt). El método de Inmersión del folíolo, con el producto Dipel®, causó el 100% de mortalidadde larvas y 96% de supervivencia del testigo; este método presentó diferencias significativas al segundo(F=0,025, p>0,05) y cuarto (F=0,0018, p>0,05) día después de la aplicación (DDA). El método de Aspersiónfoliar por aerógrafo produjo 100% de mortalidad de larvas con Dipel® al segundo DDA (F=7,94x10-10,p> 0,05), y produjo diferencias significativas también al cuarto DDA (F=3,45x10-6, p>0,05). Los métodosFoliolos sumergidos y Medio de cultivo provocaron una alta mortalidad en el control por lo que fueron rechazados.El uso de Dipel®, XenTari® y Turilav® en concentración de 1,25 g/L causó entre 80-100% demortalidad entre el segundo y octavo DDA en tres métodos evaluados válidos (1, 2, 3), además corrobora laactividad biológica de B. thuringiensis sobre este insecto plaga.

The tomato moth (Tuta absoluta Meyrick; Lepidoptera: Gelechiidae) is one of the most devastating tomatopests in Colombia and South-American countries, producing losses of up to 100% in unprotected crops. T.absoluta was detected in Spain, Portugal and Mediterranean countries in 2009, as well as England, Bulgaria andGermany. Chemical insecticides are used for controlling it; however, they produce resistance and an environmentaland human health impact. Finding an alternative to using biopesticides against this pest is becomingincreasingly important. This study evaluated five bioassay methods measuring three commercial products’toxicity on T. absoluta larvae: Dipel, XenTary and Turilav Bacillus thuringiensis (Bt) -based formulations. The leafdipping bioassay method caused 100% larvae mortality with Dipel, the control group having 95% survivalrate. The other products showed significant differences on the 2nd (F=0.025, p>0.05) and 4th (F=0.0018,p>0.05) days after application (DAA). The leaf spray airbrush method produced 100% larvae mortality withDipel on the 2nd DAA, having significant differences from the other products tested on 2nd (DAA F=7.94 x10-10, p>0.05 ), 4th (F=3.45x10-6, p>0.05 ) and 8th (F=1.07x10-5, p>0.05 ) DAA. Submerged leaflet and culturemedium methods caused high mortality in controls and were thus rejected. A variation of the leaflet immersionmethod was standardised. The three commercial products produced high mortality in Lab conditionsregarding T. absolute larvae control at 1.25 g/L concentration, thereby corroborating the biological activity ofB. thuringiensis against this insect pest.

Aucula magnifica (Schaus, 1904) (Lepidoptera: Noctuidae: Agaristinae): morphology of egg and last instar larvae / Aucula magnifica (Schaus, 1904) (Lepidoptera: Noctuidae: Agaristinae): morfologia do ovo e da larva de último ínstar

Aiming to contribute to knowledge of the immature instars of Neotropical Lepidoptera, this study details the morphology of the egg and last instar larvae of Aucula magnifica (Schaus, 1904) (Lepidoptera: Noctuidae: Agaristinae), emphasising the structures of the corium and the chaetotaxy. There is also a report of the occurrence of entomopathogenic action of Nomuraea rileyi (Farlow) Samson fungi on the larva.

Este estudo objetiva contribuir para o conhecimento dos estágios imaturos dos lepidópteros neotropicais. Nele é feito o detalhamento da morfologia das fases de ovo e de larva de Aucula magnifica (Schaus, 1904) (Lepidoptera: Noctuidae: Agaristinae), dando ênfase ao estudo das estruturas do córion e da quetotaxia da larva de último ínstar. Além disso é relatada a ocorrência da ação entomopatogênica do fungo Nomuraea rileyi (Farlow) Samson sobre suas larvas.

Actividades enzimáticas en aislamientos bacterianos de tractos digestivos de larvas y del contenido de pupas de Automeris zugana y Rothschildia lebeau (Lepidoptera: Saturniidae)

Enzymatic activities of bacteria isolated from the digestive tract of caterpillars and the pupal content of Automeris zugana and Rothschildia lebeau (Lepidoptera: Saturniidae). The enzymatic activities of bacteria isolated from the digestive tracts of caterpillars and the pupal contents of Automeris zugana and Rothschildia lebeau was studied. This digestive tract represents an extreme microenvironment due to its high pH and presence of antimicrobial substances secreted by the insect or derived from ingested plant tissue. At the same time, it contains large amounts of nutrient-rich food, for which microbes may compete among themselves and with the caterpillar. There is little information about the microbiota associated with tropical caterpillar guts, although bacteria from different genera have been isolated from gut and pupae samples. The study of the enzymatic activities generated by these organisms constitutes a starting point to understand their metabolic and physiological relationships with their hosts, and to find enzymes that have potential biotechnological applications. In this study we evaluated several enzymatic activities in two collections of bacteria isolated from caterpillar guts and pupae of the tropical lepidopteran species A. zugana and R. lebeau. Bacteria grown under aerobic conditions were tested for an array of enzymes, including gelatinases, caseinases, lipases, esterases, cellulases, xylanases, amylases and chitinases. Both collections displayed similar patterns of enzymatic activity. No isolate showed activity for all enzymatic tests, but as a whole, at least some bacteria in each collection were able to degrade each substrate tested. Isolates with the same taxonomic identification obtained from caterpillar guts and pupae had almost the same enzymatic activities. In both collections, it was possible to group bacterial isolates according to their enzyme activity pattern. In addition to a heterogeneous ensemble of isolates exhibiting two or less enzymatic activities, there were two groups with at least five activities that showed an apparent specialization for the substrates they were able to use. The first consisted exclusively of isolates of the family Enterobacteriaceae, which were positive for lipolytic and chitinolytic activities, but completely lacked amylasic, cellulolytic and xylanolytic activities. The second group, composed mainly of Gram-positive rods, exhibited the opposite pattern: they were positive for amylasic, cellulolytic and xylanolytic activities, lacked chitinolytic activity and had few isolates with lipolytic activity. This work forms the foundation for future research to explore the biotechnological potential of bacterial isolates from caterpillar guts. Rev. Biol. Trop. 55 (2): 401-415. Epub 2007 June, 29.

El tracto digestivo de orugas constituye un microambiente extremo, debido a su elevado pH y presencia de sustancias antimicrobianas secretadas por el insecto o derivadas del tejido vegetal ingerido. Al mismo tiempo, el intestino alberga gran cantidad de alimento, por el cual los microorganismos presentes podrían competir entre sí y con su hospedero. Existe poca información sobre la microbiota asociada con el intestino de orugas tropicales, aunque se ha demostrado la presencia de bacterias de diversos géneros tanto en el intestino como en el interior de pupas. El estudio de las actividades enzimáticas de estos microorganismos constituye un punto de partida en la comprensión de la posible relación metabólica y fisiológica que establecen con sus hospederos, a la vez que permite investigar enzimas con potenciales aplicaciones biotecnológicas. En este trabajo se evaluó la presencia de actividades gelatinolítica, caseinolítica, esterásica, lipolítica, quitinolítica, amilásica, celulolítica y xilanolítica en dos colecciones de aislamientos bacterianos provenientes de tractos digestivos de orugas y de pupas de los lepidópteros Automeris zugana y Rothschildia lebeau. Se utilizaron ensayos bioquímicos tradicionales para detectar enzimas secretadas en condiciones aerobias, en las que ambas colecciones exhibieron un comportamiento enzimático similar. Ningún aislamiento produjo un resultado positivo en todas las pruebas, pero como conjunto ambas colecciones fueron capaces de utilizar todos los sustratos evaluados. Los aislamientos obtenidos de pupas presentaron prácticamente las mismas actividades que sus homólogos provenientes de intestinos. En ambas colecciones fue posible agrupar los aislamientos de acuerdo con su patrón de producción de enzimas. Además de un conjunto heterogéneo de aislamientos poco activos (dos o menos actividades), se destacan dos grupos muy activos (al menos cinco actividades), que manifiestan una aparente especialización en los sustratos que utilizan. El primero de ellos está constituido exclusivamente por miembros de la familia Enterobacteriaceae, los cuales exhibieron un alto porcentaje de positividad en actividades lipolítica y quitinolítica, pero no demostraron la expresión de las actividades amilásica, celulolítica ni xilanolítica. El segundo grupo, formado en su gran mayoría por bacilos Gram-positivos, presenta la situación opuesta: alta positividad en actividades amilásica, celulolítica y xilanolítica, no detección de actividad quitinolítica y pocos aislamientos con actividad lipolítica. Este trabajo pretende ser la base de futuras investigaciones que exploren el potencial biotecnológico de aislamientos bacterianos provenientes del tracto digestivo de orugas.

Bacillus thuringiensis in caterpillars and associated materials collected from protected tropical forests in northwestern Costa Rica

Bacillus thuringiensis (Bt) synthesizes crystalline inclusions that are toxic to caterpillars (Lepidoptera) and other orders of invertebrates. Materials associated with 37 caterpillars from 16 species, collected while feeding on 15 different species of host plants in dry, cloud and rain forests located in the Area de Conservación Guanacaste in northwestern Costa Rica, were examined for the presence of Bt. From a total of 101 derived samples, 25 Bt isolates were cultured: 56% from host plant leaves, 8% from caterpillar guts and 36% from caterpillar fecal pellets. Bt was isolated from at least one sample in 38% of the systems constituted by the food plant, gut and fecal pellets corresponding to a single caterpillar. Four different morphologies of crystalline inclusions were observed, with bipyramidal and irregular crystal morphologies being the most prevalent.

Bacillus thuringiensis (Bt) sintetiza inclusiones cristalinas que resultan tóxicas para algunas larvas de lepidópteros y otros órdenes de invertebrados. Su presencia fue examinada en materiales asociados a 37 orugas de mariposas de 16 especies, las cuales fueron colectadas mientras se alimentaban en 15 especies diferentes de plantas hospederas en bosques secos, nubosos y húmedos localizados dentro del Área de Conservación Guanacaste (ACG) en el noroeste de Costa Rica. A partir de un total de 101 muestras se obtuvo 25 aislamientos de Bt: 56% a partir de material foliar de las plantas hospederas, 8% a partir del contenido intestinal de las larvas y 36% a partir de sus excrementos. Esta bacteria fue cultivada a partir de al menos uno de los 3 diferentes tipos de muestra asociados a una oruga particular (planta hospedera, intestino, excremento) en 38% de los casos posibles. En la colección de aislamientos obtenida se observaron cuatro morfologías de inclusiones cristalinas, siendo aquellas bipiramidales e irregulares las más prevalentes.

Interação de Metarhizium anisopliae (Metsch.) Sorok., Beauveria bassiana (Bals.) Vuill. e o parasitóide Oomyzus sokolowskii (Kurdjumov) (Hymenoptera: Eulophidae) sobre larvas da traça-das-crucíferas, Plutella xylostella (L.) (Lepidoptera: Plutellidae) / Interaction of Metarhizium anisopliae (Metsch.) Sorok., Beauveria bassiana (Bals.) Vuill. and the parasitoid Oomyzus sokolowskii (Kurdjumov) (Hymenoptera: Eulophidae) with larvae of diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae)

Inseticidas químicos são amplamente usados para o controle da traça-das-crucíferas, Plutella xylostella (L.). Esta é considerada a principal praga das crucíferas em todo o mundo e o surgimento de resistência de P. xylostella tem sido freqüentemente relatado. Este trabalho investigou as diferentes interações dos fungos Metarhizium anisopliae (Metsch.) Sorok. e Beauveria bassiana (Bals.) Vuill. com o parasitóide de larva-pupa Oomyzus sokolowskii (Kurdjumov) sobre larvas de P. xylostella. O experimento foi conduzido à temperatura de 26 ± 1°C, UR de 75 ± 5 por cento e fotofase de 12h, empregando-se delineamento inteiramente casualizado, constando de oito tratamentos com seis repetições cada. Foram utilizados os isolados E9 de M. anisopliae e ESALQ 447 de B. bassiana, na concentração de 10(7) conídios ml-1. Os resultados mostram que M. anisopliae e B. bassiana reduziram o parasitismo de P. xylostella por O. sokolowskii . Por outro lado, efeitos aditivos no controle de P. xylostella foram evidenciados nas diferentes combinações dos fungos com o parasitóide, exceto para B. bassiana quando pulverizado 24h antes da exposição a O. sokolowskii. Os fungos M. anisopliae e B. bassiana foram mais eficientes quando aplicados após a exposição das larvas ao parasitóide, e a eficiência de O. sokolowskii foi influenciada negativamente pela presença dos fungos independente da combinação. A associação dos fungos com o parasitóide apresentam potencial para serem testados em campo, objetivando o controle de P. xylostella bem como favorecer economicamente o sistema produtivo das brássicas.

Some aspects of the efficiency of Cotesia sp.[Hymenoptera: Braconidae]in infecting the larose of Tarucus rosacea[Lepidoptera: Lycaenidae]

The study has indicated that Cotesia sp. Parasite is very efficient in infecting the larvae of Traucus rosacea and it infects all the larval stages of the host in the same degree. The highest percentage of parasiting was [76.10 percent] during May-2001. However, the parasite showed no activity during March, August and September of the same year, but it was very active in October [56.25] which is the highest average realized by the parasite in one year. It has been found that these is a clear congruity between the increase in the larvae number and the increase in the host's larvae. The study has also showed that the parasite females do not prefer laying their ages on some of the larvae in particular. No significant difference were noticed in prefer the parasite infecting on host's larvae. There is no effect of the Zizyphus tress on the present of the parasites. The highest percentage of parasiting was [88.88 and 89.74 percent] in April and May in case of the fourth larval stage. The lest percentage of parasiting in the first larval stage was [50.00 and 30.00 percent] in April May respectively

Does a polyphagous caterpillar have the same gut microbiota when feeding on different species of food plants

We used classical culture techniques to explore gut bacteria and changes associated with dietary change in the highly polyphagous, tropical caterpillar Automeris zugana (Saturniidae). Fifty-five third instar wild-caught sibs feeding on Annona purpurea (Annonaceae) in the Area de Conservación Guanacaste (ACG) in northwestern Costa Rica were divided into eight groups. Each of seven groups was reared to the ultimate instar on another species of food plant normally used by A. zugana. Some pupae were also analyzed for the presence of bacteria. Aerobic bacterial cultures were obtained from all 33 caterpillar guts and the eight pupae inventoried. There was no clear pattern in species composition of cultivated bacteria among the eight diets, and each caterpillar on a given food plant carried only a small fraction of the total set of species isolated from the set of caterpillars feeding on that food plant. Taken as a whole, the larvae and pupae contained 22 species of cultivable bacteria in 12 genera. Enterobacter, present in 81.8 of the samples, was the genus most frequently isolated from the caterpillars, followed by Micrococcus and Bacillus. Bacillus thuringiensis was isolated from 30.3 of the dissected caterpillars, but found in caterpillars feeding on only half of the species of food plants.