Entomopathogenic Fungi as Dual Control Agents against Two Phytopathogens and the Lepidopteran Pest Rachiplusia nu in Soybean (Glycine max (L.) Merr).

Soybean (Glycine max) is one of the most important crops worldwide. This crop is prone to diseases caused by the phytopathogenic fungi Macrophomina phaseolina, Fusarium oxysporum, and the lepidopteran pest Rachiplusia nu. Biological control using entomopathogenic fungi is a sustainable alternative to chemical pesticides. In this study, we assessed the antifungal activity of Beauveria bassiana and Metarhizium anisopliae against phytopathogens and the pathogenicity of B. bassiana as an endophyte against R. nu. The antagonic activity of the fungal entomopathogens was evaluated in vitro by dual culture bioassays. The feeding preference of R. nu was evaluated in a "free choice" assay under laboratory conditions. Entomopathogenic fungi inhibited the mycelial growth of the phytopathogens. The best response in all cases was observed when the antagonists were placed in the culture medium two days before the pathogens. B. bassiana was the best antagonist of F. oxysporum, while both antagonists had similar inhibitory effects on M. phaseolina growth. Additionally, B. bassiana, when grown as an endophyte, reduced insects' food preferences and decreased soybean consumption. Findings suggest that entomopathogenic fungi can fulfill multiple roles in the ecosystem. Therefore, the simultaneous expression of both properties should be considered for their application in integrated pest management programs.

Colonzation of Tobacco Plants by Fungal Entomopathogens and the Effect on Consumption over Diabrotica speciosa (Coleoptera: Chrysomelidae).

Entomopathogenic fungi are naturally occurring microorganisms that attack insect pests, making them exceptional allies when developing biocontrol strategies. A particular aspect of the ecology of these fungi is that they interact not only with insects but also with plants, being able to colonize them endophytically without causing symptoms of disease. The objectives of this study were to determine the endophytic capacity of different species of entomopathogenic fungi in tobacco plants by means of foliar spraying, to evaluate the persistence in planta of the entomopathogens and the effect of endophytes on consumption by coleopteran pests. A total of 24 strains were analyzed to test endophytic capacity at 7, 14, 21 and 28 days post inoculation. A significant effect of the strains was found. On days 7, 14 and 21, the strain that showed the highest colonization percentages was B. bassiana LPSc 1215, and at day 28 B. bassiana strains LPSc 1215 and LPSc 1212 exhibited the best endophytic capacity, maintaining elevated colonization percentages. Choice test results indicated that D. speciosa fed indiscriminately on colonized and non-colonized leaves. The results indicate that B. bassiana LPSc 1215 constitutes a strain that would merit further investigation for the purpose of pest management in tobacco cultivation.

ISSR markers to explore entomopathogenic fungi genetic diversity: Implications for biological control of tobacco pests.

Tobacco is one of the major industrial crops cultivated worldwide. Chemical control is the main method employed to reduce damage by insect pests. The use of entomopathogenic fungi represents an alternative to replace insecticides. The search for effective strains in the field constitutes a first step when developing a formulation. The objective of this work was to study genetic differences among isolates of entomopathogenic fungi obtained from tobacco grown soils using ISSR markers. The pathogenicity of the strains towards Helicoverpa gelotopoeon and Diabrotica speciosa was also assessed in order to search for a relationship between virulence and genetic diversity. Nineteen isolates were identified according to morphological features and molecular techniques as Beauveria bassiana (11) and Purpureocillium lilacinum (8). The diversity tree generated by ISSR analysis showed a high diversity among the strains. The pathogenicity towards H. gelotopoeon and D. speciosa was assessed and the logistic models generated showed that B. bassiana isolates LPSc1215 and LPSc1364 were the most pathogenic against both insect pests tested. In the diversity tree, these strains were grouped in a same cluster with a similarity level of approximately 85%, indicating a possible relationship between virulence and the band pattern generated.

Endophytic Effects of Beauveria bassiana on Corn (Zea mays) and Its Herbivore, Rachiplusia nu (Lepidoptera: Noctuidae).

Entomopathogenic fungi are widely recognized as agents of biological control worldwide. Their use in agriculture for the regulation of pest populations is a promising alternative to conventional insecticides. Furthermore, recent studies have shown that entomopathogenic fungi fulfill an additional role in plants as growth promoters. The purpose of this investigation was to assess the growth and yield of corn plants colonized with Beauveria bassiana and its effect on the lepidopteran pest Rachiplusia nu. Effects of the fungus on plant growth, crop yield, and vertical transmission were evaluated in the field. Feeding preferences of R. nu larvae were assessed in the laboratory using a "choice test". Corn plants inoculated with B. bassiana showed an increase in height, number of leaves, grain weight, yield, and percentage of seed germination compared to control plants. Consumption of B. bassiana-colonized corn plants by R. nu larvae was reduced compared to feeding levels observed on non-inoculated plants. This study showed that endophytic B. bassiana can provide multiple benefits to Zea mays and can play an important role in future integrated pest management programs.

Compatibility of chemical insecticides and entomopathogenic fungi for control of soybean defoliating pest, Rachiplusia nu / Compatibilidad de insecticidas químicos y hongos entomopatógenos para el control de Rachiplusia nu, plaga defoliadora de la soja

Rachiplusia nu (Guenée) (Lepidoptera: Noctuidae) is one of the major lepidopteran pests defoliating soybeans (Glycine max Merrill) in Argentina. The combined use of chemical insecticides and entomopathogenic fungi is a promising pest-control option to minimize adverse chemical effects. In this work, we evaluated the interactions between five insecticides-two being considered biorational-and five fungal entomopathogenic strains under laboratory conditions in order to determine the possible usefulness of combinations of these agents against R. nu. The insecticides were tested for compatibility at four doses by in vitro bioassay and for the lethality of R. nu by inoculations at three doses. Fungal strains were applied at 1 x 10(8), 1 x 10(6), and 1 x 10(4) conidia/ml. The combinations of those insecticides with Beauveria bassiana (LPSc 1067, LPSc 1082, LPSc 1098), Metarhizium anisopliae (LPSc 907), and Metarhizium robertsii (LPSc 963) caused higher R. nu-larval mortalities than any of the individual agents alone. We observed significant differences in the in vitro conidial viability, vegetative growth, and conidia production of the five strains of entomopathogenic fungi exposed to different doses of the chemical insecticides. The combination gamma-cyhalothrin-LPSc-1067 caused the highest percent mortality of R. nu larvae, with synergism occurring between the two agents at 50% and 25% of the maximum field doses.

Rachiplusia nu (Guenée) (Lepidoptera: Noctuidae) es una de las principales plagas de lepidópteros defoliadores del cultivo de soja (Glycine max Merrill) en Argentina. El uso combinado de insecticidas químicos y hongos entomopatógenos es una opción de control de plagas prometedora para minimizar los efectos químicos adversos. En este trabajo se evaluaron las interacciones entre 5 insecticidas -2 de ellos considerados biorracionales-- y 5 cepas fúngicas entomopatógenas en condiciones de laboratorio, para determinar la posible utilidad de combinaciones de estos agentes frente a R. nu. Se evaluó la compatibilidad de los insecticidas a 4 dosis mediante bioensayos in vitro y la letalidad de aquellos sobre R. nu mediante inoculaciones a 3 dosis. Las cepas fúngicas se evaluaron a concentraciones de 1 x 10(8), 1 x 10(6) y 1 x 10(4) conidios/ml. Las combinaciones de estos insecticidas con Beauveria bassiana (LPSc1067, LPSc 1082, LPSc 1098), Metarhizium anisopliae (LPSc 907) y Metarhizium robertsii (LPSc 963) causaron una mayor mortalidad de larvas de R. nu que cualquiera de los agentes individuales. Asimismo, se observaron diferencias significativas en la viabilidad de los conidios in vitro, el crecimiento vegetativo y la producción de conidios de las 5 cepas de hongos entomopatógenos expuestos a diferentes dosis de los insecticidas químicos. La combinación gamma-cialotrina-LPSc-1067 causó el mayor porcentaje de mortalidad de larvas de R. nu, con un efecto de sinergismo entre los 2 agentes al 50 y el 25% de las dosis de campo recomendadas por el fabricante.

Compatibility of chemical insecticides and entomopathogenic fungi for control of soybean defoliating pest, Rachiplusia nu.

Rachiplusia nu (Guenée) (Lepidoptera: Noctuidae) is one of the major lepidopteran pests defoliating soybeans (Glycine max Merrill) in Argentina. The combined use of chemical insecticides and entomopathogenic fungi is a promising pest-control option to minimize adverse chemical effects. In this work, we evaluated the interactions between five insecticides-two being considered biorational-and five fungal entomopathogenic strains under laboratory conditions in order to determine the possible usefulness of combinations of these agents against R. nu. The insecticides were tested for compatibility at four doses by in vitro bioassay and for the lethality of R. nu by inoculations at three doses. Fungal strains were applied at 1×108, 1×106, and 1×104conidia/ml. The combinations of those insecticides with Beauveria bassiana (LPSc 1067, LPSc 1082, LPSc 1098), Metarhizium anisopliae (LPSc 907), and Metarhizium robertsii (LPSc 963) caused higher R. nu-larval mortalities than any of the individual agents alone. We observed significant differences in the in vitro conidial viability, vegetative growth, and conidia production of the five strains of entomopathogenic fungi exposed to different doses of the chemical insecticides. The combination gamma-cyhalothrin-LPSc-1067 caused the highest percent mortality of R. nu larvae, with synergism occurring between the two agents at 50% and 25% of the maximum field doses.

Beauveria bassiana (Ascomycota: Hypocreales) Introduced as an Endophyte in Corn Plants and Its Effects on Consumption, Reproductive Capacity, and Food Preference of Dichroplus maculipennis (Orthoptera: Acrididae: Melanoplinae).

In this study, the effects of strain Beauveria bassiana (LPSC 1067) as an endophyte in corn plants on consumption, fecundity, and food preference of Dichroplus maculipennis were examined. We observed that the daily consumption by grasshoppers fed with control plants was almost twice that of those that were fed treated plants. Significant differences in fecundity of grasshoppers that were fed with treated plants compared with those that only fed on control plants were also observed. The number of eggs laid per female fed with control plants was 27.2, while the number of eggs laid per female that were fed during 15 d with treated plants was 17.7. Similar results were observed when the number of embryonated eggs was evaluated. The highest number of embryonated eggs were recorded in those females that only fed on control plants (96%) while fewer embryonated eggs were recorded in grasshoppers fed for 15 d with treated plants only (25%). In relation to food preference the average consumption rate for D. maculipennis females on control corn plants was 303.8 ± 24.5 mg while it was only 25 ± 2.1 mg on plants treated with B. bassiana as an endophyte. In summary, we observed that B. bassiana as a corn plant endophyte negatively affected the daily consumption rate, fecundity and food preference of D. maculipennis.

Endophytic fungi from selected varieties of soybean (Glycine max L. Merr.) and corn (Zea mays L.) grown in an agricultural area of Argentina / Hongos endófitos aislados de cultivares de soja (Glycine max L. Merr) y maíz (Zea mays L.) presentes en áreas agrícolas argentinas

Endophytic fungi are ubiquitous and live within host plants without causing any noticeable symptoms of disease. Little is known about the diversity and function of fungal endophytes in plants, particularly in economically important species. The aim of this study was to determine the identity and diversity of endophytic fungi in leaves, stems and roots of soybean and corn plants and to determine their infection frequencies. Plants were collected in six areas of the provinces of Buenos Aires and Entre Ríos (Argentina) two areas were selected for sampling corn and four for soybean. Leaf, stem and root samples were surface-sterilized, cut into 1 cm² pieces using a sterile scalpel and aseptically transferred to plates containing potato dextrose agar plus antibiotics. The species were identified using both morphological and molecular data. Fungal endophyte colonization in soybean plants was influenced by tissue type and varieties whereas in corn plants only by tissue type. A greater number of endophytes were isolated from stem tissues than from leaves and root tissues in both species of plants. The most frequently isolated species in all soybean cultivars was Fusarium graminearum and the least isolated one was Scopulariopsis brevicaulis. Furthermore, the most frequently isolated species in corn plants was Aspergillus terreus whereas the least isolated one was Aspergillus flavus. These results could be relevant in the search for endophytic fungi isolates that could be of interest in the control of agricultural pests.

Los hongos endófitos son ubicuos y se encuentran en el interior de los tejidos de las plantas de manera asintomática. Se sabe muy poco acerca de la diversidad y la función de estos hongos, particularmente en especies de importancia económica. El objetivo de este trabajo fue determinar la diversidad y la frecuencia de colonización de hongos endófitos en raíces, tallos y hojas de 2 variedades de maíz y de 4 variedades de soja; las muestras se tomaron de 6 áreas diferentes ubicadas en las provincias de Buenos Aires y Entre Ríos (Argentina). Con un bisturí estéril se obtuvieron porciones de 1 cm² de raíz, tallo y hoja, que fueron colocados en placas con agar papa dextrosa más antibiótico. Las especies de hongos fueron identificadas a partir de características morfológicas y moleculares. La colonización de hongos endófitos en soja estuvo influenciada por la variedad y por el tipo de tejido, en tanto que en el maíz solo hubo influencia del tipo de tejido. El mayor número de endófitos se encontró en los tallos de ambas especies. El aislamiento más frecuente en todas las variedades de soja fue Fusarium graminearum y el menos frecuente Scopulariopsis brevicaulis. En ambas variedades de maíz la especie con mayor frecuencia de aislamiento fue Aspergillus terreus y la de menor fue Aspergillus flavus. Estos resultados son relevantes para la búsqueda de especies de hongos endófitos que podrían ser de interés en el control de plagas agrícolas.

Endophytic fungi from selected varieties of soybean (Glycine max L. Merr.) and corn (Zea mays L.) grown in an agricultural area of Argentina.

Endophytic fungi are ubiquitous and live within host plants without causing any noticeable symptoms of disease. Little is known about the diversity and function of fungal endophytes in plants, particularly in economically important species. The aim of this study was to determine the identity and diversity of endophytic fungi in leaves, stems and roots of soybean and corn plants and to determine their infection frequencies. Plants were collected in six areas of the provinces of Buenos Aires and Entre Ríos (Argentina) two areas were selected for sampling corn and four for soybean. Leaf, stem and root samples were surface-sterilized, cut into 1cm(2) pieces using a sterile scalpel and aseptically transferred to plates containing potato dextrose agar plus antibiotics. The species were identified using both morphological and molecular data. Fungal endophyte colonization in soybean plants was influenced by tissue type and varieties whereas in corn plants only by tissue type. A greater number of endophytes were isolated from stem tissues than from leaves and root tissues in both species of plants. The most frequently isolated species in all soybean cultivars was Fusarium graminearum and the least isolated one was Scopulariopsis brevicaulis. Furthermore, the most frequently isolated species in corn plants was Aspergillus terreus whereas the least isolated one was Aspergillus flavus. These results could be relevant in the search for endophytic fungi isolates that could be of interest in the control of agricultural pests.

Incipient loss of flagella in the genus Geolegnia: the emergence of a new clade within Leptolegnia?

The genus Geolegnia represents a poorly documented group of saprolegnialean oomycetes isolated from soils as free-living organisms. Although it is morphologically similar to the facultative parasitic genus Leptolegnia, Geolegnia presents the uncommon property of having lost a flagellate stage in its lifecycle. Based on ITS and large subunit (LSU) rRNA sequence data, we show Geolegnia to be basal to Leptolegnia, and also introduce Geolegnia helicoides sp. nov. Using sequence data of Leptolegnia available in GenBank, supplemented by data derived from culture collections, we show that Geolegnia is nested within Leptolegnia, a genus characterised by its "conventional" biflagellate life cycle. The emergence of Geolegnia is therefore seen as a recent event, and we suggest here an evolutionary context where this loss might have been advantageous. Based on this study, Leptolegnia remains paraphyletic, awaiting the redefinition of genera in this complex.

Pathogenic and enzyme activities of the entomopathogenic fungus Tolypocladium cylindrosporum (Ascomycota: Hypocreales) from Tierra del Fuego, Argentina

Tolypocladium cylindrosporum is an entomopathogenic fungi that has been studied as a biological control agent against insects of several orders. The fungus has been isolated from the soil as well as from insects of the orders Coleoptera, Lepidoptera, Diptera and Hymenoptera. In this study, we analyzed the ability of a strain of T. cylindrosporum, isolated from soil samples taken in Tierra del Fuego, Argentina, to produce hydrolytic enzymes, and to study the relationship of those activities to the fungus pathogenicity against pest aphids. We have made the traditional and molecular characterization of this strain of T. cylindrosporum. The expression of hydrolase activity in the fungal strain was estimated at three incubation temperatures (4ºC, 12ºC and 24ºC), on different agar media supplemented with the following specific substrates: chitin azure, Tween ® 20, casein, and urea for chitinase, lipase, protease, and urease activity, respectively. The hydrolytic-enzyme activity was estimated qualitatively according to the presence of a halo of clarification through hydrolase action, besides was expressed semi-quantitatively as the ratio between the hydrolytic-halo and colony diameters. The pathogenicity of the fungus was tested on adults of the aphid Rhopalosiphum padi at three temperatures of incubation (4ºC, 12ºC and 24ºC). The suspension was adjusted to a concentration of 1x10 7 conidia/ml. In pathogenicity assays at seven days post-inoculation, the fungus caused the mortality of adults of Ropalosiphum padi at different temperatures also showed a broad ability to grow on several agar-culture media, supplemented with different carbon sources at the three incubation temperatures tested. Although, the growth was greater with higher incubation temperatures (with maximum levels at 24°C), the fungus reached similar colony diameters after 15 days of incubation on the medium supplemented with Tween® 20 at the lower two incubation temperatures of 4°C or 12°C. In accordance with the results on colony diameters, the fungus revealed an ability to degrade casein, chitin derivatives, Tween® 20, and urea as evidenced by the appearance of a halo around the fungal colony. Because of its origin and temperature tolerance, this Argentine strain has great potential for use as a biocontrol agent for insect pest control in cold and temperate environments. Rev. Biol. Trop. 60 (2): 833-841. Epub 2012 June 01.

El hongo entomopatógeno Tolypocladium cylindrosporum ha sido estudiado como un agente de control biológico contra insectos de varios órdenes. Esta especie fue aislada del suelo, así como de insectos de los órdenes Coleoptera, Lepidoptera, Diptera e Hymenoptera. En el presente trabajo hemos analizado la capacidad de una cepa de T. cylindrosporum (LPSC Nº1065) aislada del suelo en Tierra del Fuego, Argentina, para producir enzimas hidrolíticas y determinar la relación de esta actividad con la patogenicidad del hongo para combatir la plaga de los áfidos en diferentes temperaturas (4º, 12º y 24ºC). En los ensayos de patogenicidad, siete días posteriores a la inoculación, se registró mortalidad en los adultos del áfido Ropalosiphum padi a diferentes temperaturas y también se demostró una amplia capacidad de crecer en varios medios de cultivos complementados con diferentes fuentes de carbono bajo las tres temperaturas de incubación ensayadas. Debido a su origen y a la tolerancia que tiene a bajas temperaturas esta cepa, presenta un gran potencial para su uso como agente de control biológico para las plagas de insectos de ambientes fríos y templados.

Pathogenic and enzyme activities of the entomopathogenic fungus Tolypocladium cylindrosporum (Ascomycota: Hypocreales) from Tierra del Fuego, Argentina.

Tolypocladium cylindrosporum is an entomopathogenic fungi that has been studied as a biological control agent against insects of several orders. The fungus has been isolated from the soil as well as from insects of the orders Coleoptera, Lepidoptera, Diptera and Hymenoptera. In this study, we analyzed the ability of a strain of T cylindrosporum, isolated from soil samples taken in Tierra del Fuego, Argentina, to produce hydrolytic enzymes, and to study the relationship of those activities to the fungus pathogenicity against pest aphids. We have made the traditional and molecular characterization of this strain of T cylindrosporum. The expression of hydrolase activity in the fungal strain was estimated at three incubation temperatures (4 degreeC, 12 degreeC and 24 degreeC), on different agar media supplemented with the following specific substrates: chitin azure, Tween 20, casein, and urea for chitinase, lipase, protease, and urease activity, respectively. The hydrolytic-enzyme activity was estimated qualitatively according to the presence of a halo of clarification through hydrolase action, besides was expressed semi-quantitatively as the ratio between the hydrolytic-halo and colony diameters. The pathogenicity of the fungus was tested on adults of the aphid Rhopalosiphum padi at three temperatures of incubation (4 degree C, 12 degree C and 24 degree C). The suspension was adjusted to a concentration of 1x10(7) conidia/ml. In pathogenicity assays at seven days post-inoculation, the fungus caused the mortality of adults of Ropalosiphum padi at different temperatures also showed a broad ability to grow on several agar-culture media, supplemented with different carbon sources at the three incubation temperatures tested. Although, the growth was greater with higher incubation temperatures (with maximum levels at 24 degreeC), the fungus reached similar colony diameters after 15 days of incubation on the medium supplemented with Tween 20 at the lower two incubation temperatures of 4 degreeC or 12 degreeC. In accordance with the results on colony diameters, the fungus revealed an ability to degrade casein, chitin derivatives, Tween 20, and urea as evidenced by the appearance of a halo around the fungal colony. Because of its origin and temperature tolerance, this Argentine strain has great potential for use as a biocontrol agent for insect pest control in cold and temperate environments.

Prevalence and distribution of parasites and pathogens of triatominae from Argentina, with emphasis on Triatoma infestans and Triatoma virus TrV.

Chagas' disease is the most important endemic arthropod-zoonosis in Argentina with an estimated 1.6 million people infected with the causative agent Trypanosoma cruzi. Triatoma infestans is the main vector of Chagas' disease in Argentina. A survey for parasites and pathogens of Triatominae was conducted from August 2002 to February 2005. Collections of insects were made in domiciles, peridomiciles, and in the natural habitats of the Triatominae. Insects from these collections were dissected and their organs and tissues examined for flagellates. Frass from these insects was collected and examined for detection of the entomopathogenic virus Triatoma virus (TrV) using AC-ELISA and PCR. Triatominae belonging to four species, T. infestans (n=1646), Triatoma guasayana (n=4), Triatoma platensis (n=1) and Triatoma sordida (n=5) were collected from 62 sites located in 13 provinces of Argentina. Triatoma virus and two protozoan species, Blastocrithidia triatomae and T. cruzi, the etiological agent of Chagas disease, were found infecting Triatominae. The total prevalence of TrV in 1646 T. infestans analyzed by ELISA was 9.66% (159/1646) from 7 to 13 provinces where collections were made. Triatoma virus positive triatomines were found in 17 of 62 populations when examined by AC-ELISA but in 38 of 62 populations when PCR was used for detection. The prevalence of B. triatomae in T. infestans was 0.43% (7/1646), while the prevalence of T. cruzi was 1.3% (21/1646). This is the first study on the diversity, distribution and prevalence of flagellated protozoa and TrV of Triatominae in endemic Chagas' disease regions of Argentina.

[Effect of water quality in mosquito breeding sites on the pathogenicity and infectivity of zoospores from the fungus Leptolegnia chapmanii (Straminipila: Peronosporomycetes)]. / Efecto de la calidad del agua de criaderos de mosquitos (Diptera: Culicidae) sobre la patogenicidad e infectividad de las zoosporas del hongo Leptolegnia chapmanii (Straminipila: Peronosporomycetes).

The fungus Leptolegnia chapmanii is highly pathogenic to mosquito larvae in Argentina. We studied if physical and chemical characteristics of the water from mosquito breeding sites affect pathogenicity, and the infectivity of zoospores of L. chapmanii. Water samples were taken from pools filled by rains, urban ditches with domestic waste water, pools filled by overflow from Río de la Plata, and flower vases from the Cemetery of La Plata city. Sub-samples of water were analyzed for physical and chemical characteristics, while other sub-samples were used for laboratory bioassays. Containers with 150 ml of water samples, 25 Aedes aegypti larvae, and 2.8 x 10(5) zoospores of L. chapmanii, were incubated under controlled environment, and larval mortality was recorded after 48 h. There were highly significant differences among mortalities in water from cemetery vases (70.2%), rain pools water (99.5%), and pools with water from Río de la Plata (95%). There were no significant differences among larval mortalities in water from ditches, rain pools and Río de la Plata pools. Leptolegnia chapmanii was successful as a biological control agent in all kinds of tested water qualities, producing high larval mortality.

Efecto de la calidad del agua de criaderos de mosquitos (Diptera: Culicidae) sobre la patogenicidad e infectividad de las zoosporas del hongo Leptolegnia chapmanii (Straminipila: Peronosporomycetes)

Effect of water quality in mosquito breeding sites on the pathogenicity and infectivity of zoospores from the fungus Leptolegnia chapmanii (Straminipila: Peronosporomycetes). The fungus Leptolegnia chapmanii is highly pathogenic to mosquito larvae in Argentina. We studied if physical and chemical characteristics of the water from mosquito breeding sites affect pathogenicity, and the infectivity of zoospores of L. chapmanii. Water samples were taken from pools filled by rains, urban ditches with domestic waste water, pools filled by overflow from Río de la Plata, and flower vases from the Cemetery of La Plata city. Sub-samples of water were analyzed for physical and chemical characteristics, while other sub-samples were used for laboratory bioassays. Containers with 150 ml of water samples, 25 Aedes aegypti larvae, and 2.8 x 105 zoospores of L. chapmanii, were incubated under controlled environment, and larval mortality was recorded after 48 h. There were highly significant differences among mortalities in water from cemetery vases (70.2%), rain pools water (99.5%), and pools with water from Rio de la Plata (95%). There were no significant differences among larval mortalities in water from ditches, rain pools and Río de la Plata pools. Leptolegnia chapmanii was successful as a biological control agent in all kinds of tested water qualities, producing high larval mortality. Rev. Biol. Trop. 57 (1-2): 371-380. Epub 2009 June 30.

Leptolegnia chapmanii es un potente patógeno de larvas de mosquitos. El objetivo del trabajo fue determinar si las características físico-químicas del agua de criaderos de culícidos de importancia sanitaria a nivel mundial afectan la patogenicidad e infectividad de las zoosporas de L. chapmanii. Se tomaron muestras de cuatro tipos de agua en Argentina: de lluvia, de zanja, del Río de La Plata, y de floreros del cementerio local. A una parte del agua se le realizaron estudios físicos-químicos, y otra parte fue utilizada en ensayos. Se colocó 150 ml de cada muestra en recipientes plásticos, con 25 larvas sanas de Aedes aegypti y 2.8 x 105 zoosporas de L. chapmanii. Fueron incubadas a 25 ºC y 12-12 (L-O). La mortalidad larval fue registrada a las 48 horas. Se utilizaron tres recipientes y un control por tratamiento, con tres repeticiones realizadas en distintos días. Se observaron diferencias altamente significativas entre la mortalidad larval en aguas de floreros del cementerio (70.2%), aguas de lluvias (99.53%), y del río de La Plata (95%). No hubo diferencias significativas entre la mortalidad larval en agua de zanja, lluvia y río. Leptolegnia chapmanii resultó exitoso en los cuatro tipos de aguas evaluados, produciendo elevados niveles de mortalidad larval.

Further research on the production, longevity and infectivity of the zoospores of Leptolegnia chapmanii Seymour (Oomycota: Peronosporomycetes).

The effect of temperature on the production, survival and infectivity of zoospores of an Argentinean isolate of Leptolegnia chapmanii was determined under laboratory conditions. Production of zoospores of L. chapmaniiin vitro and in vivo upon first and fourth instars larvae of the mosquito Aedes aegypti was studied at three different temperatures. Zoospores from infected larvae were infective to mosquito larvae for 51, 12, and 5 consecutive days when maintained at 25, 35, and 10 degrees C, respectively. Maximum zoospore production in infected fourth-instar larvae was 9.6+/-1.4x10(4) zoosp/larva at 48 h at 25 degrees C. The average number of zoospores produced by individual fourth-instar Ae. aegypti larvae infected with L. chapmanii was 3.57+/-0.46x10(5) zoospores during 6 consecutive days at 25 degrees C. Zoospore production in vitro was also affected by temperature with a maximum of zoospores (n=47,666/ml) produced at 25 degrees C. When zoospores produced in vitro were used as inoculum against Ae. aegypti larvae at 25 degrees C, larval mortality was recorded for 5 consecutive weeks. The encystment process for zoospores took 17-20 min; the germination of cysts (excystment) occurred 5 min after exposure in water to mosquito larvae. The minimal time of contact between zoospores and mosquito larvae to develop infection was two minutes. Infection took place by zoospore attachment onto and then penetration through the larval cuticle or by ingestion of cysts as was confirmed by histological studies. Temperature directly affected infectivity and production of zoospores in vivo and in vitro although L. chapmanii zoospores tolerate a wide range of temperatures.

Biotic and abiotic factors affecting Leptolegnia chapmanii infection in Aedes aegypti.

The effects of water volume, container surface area and the density of hosts and fungal zoospores on the infectivity of the oomycete fungus Leptolegnia chapmanii to Aedes aegypti were investigated in the laboratory. Late 3rd or early 4th instars from a laboratory colony were used as hosts in all assays. Fourth instars infected with L. chapmanii for 48 h (6.1 +/- 0.2 x 10(4) zoospores/larva) were used as inoculum. Mortality rates were >90% in containers with 20 or fewer larvae when exposed to 1 infected larva, but they decreased to 37% in containers with 60 larvae. Mortality rates varied from 82% to 92% when healthy 4th instars were exposed to 1 and 2 infected larvae, respectively, whereas 100% mortality was obtained with > or =3 infected larvae. Infection and mortality rates in containers with volumes that varied from 300 to 5,000 ml but that had similar water surface area (397 cm2) varied from 89 to 92%, respectively. When water volume was held constant at 250 ml in containers with variable surface areas (14.5-875 cm(2)), larval mortality varied from 96% to 25%, respectively. The ability of L. chapmanii to infect mosquito larvae is dose dependent and influenced by larval density and surface area.

Isolation of Paecilomyces lilacinus (Thom) Samson (Ascomycota: Hypocreales) from the Chagas disease vector, Triatoma infestans Klug (Hemiptera: Reduviidae) in an endemic area in Argentina.

A survey for entomopathogenic fungi of the Chagas disease vector Triatoma infestans was conducted in two provinces of Argentina from March-December 2003. Field-collected insects that died in the laboratory were individually maintained in moist chamber and incubated at 22 degrees C. Triatominae adults infected with the fungus Paecilomyces lilacinus were found at El Quebracho (27 degrees 34'S-64 degrees 31'W), Santiago del Estero province, Argentina, in December 2003. Paecilomyces lilacinus was cultured and isolated from infected insects in SDAY, PYG and MEA media. Pathogenicity tests were conducted and positive results were recorded. The median survival time (MST) of T. infestans exposed to a P. lilacinus conidial suspension was 12.8 days, and 100% mortality occurred at 30 days post-treatment. This is the first record of natural infection caused by P. lilacinus in T. infestans in the world.

Predation efficiency of indigenous larvivorous fish species on Culex pipiens L. larvae (Diptera: Culicidae) in drainage ditches in Argentina.

Two neotropical freshwater fish species, Cnesterodom decemmaculatus (Poeciliidae) and Jenynsia multidentata (Anablepidae), were collected from human-made ditches, a common habitat of the house mosquito Culex pipiens in La Plata, Argentina. Cnesterodom decemmaculatus was recorded in 62 of the 100 examined ditches, whereas J. multidentata was collected from only 21 ditches sympatrically with C. decemmaculatus. Culex pipiens was the only mosquito species collected, and its larvae and pupae were found in 38 of the 100 ditches. Fish and mosquito larvae and pupae were collected together in only two ditches and were significantly negatively correlated. Siphons of larval Culex and remnants of chironomid larvae, copepods, aquatic mites, and fish were present in the gut contents of two C. decemmaculatus from mosquito-positive ditches, while diatoms and filamentous algae were recorded in every fish dissected. Adult C. decemmaculatus and J. multidentata needed approximately 6.2 h to completely digest one Cx. pipiens 4th instar larva under laboratory conditions. When fish were confined with a density of 60 or fewer Cx. pipiens 4th instar larvae, C. decemmaculatus and J. multidentata adults consumed 100% of them in one day but only 35% and 42%, respectively, when confined with 150 larvae. Eradication of Cx. pipiens from a ditch, where densities had averaged 250 immatures per dip, was achieved 17 days after the introduction of 1,700 C. decemmaculatus.