Natural occurrence of Zoophthora radicans (Entomophthorales: Entomophthoraceae) on Thaumastocoris peregrinus (Heteroptera: Thaumastocoridae), an invasive pest recently found in Brazil

AbstractOccurrence of Zoophthora radicans infecting nymphs and adults of Thaumastocoris peregrinus Carpintero and Dellape, 2006 is reported in Brazil. This is a new record of host for this fungal species and the first fungal pathogen associated with this pest worldwide. Infection of Z. radicans on T. peregrinus populations on commercial Eucalyptus plantation (Eucalyptus spp.) reached up to 100%, and low insect densities were associated with high levels of fungal infection in three out of seven plots. This pathogen seems to be virulent against T. peregrinus and may play an important role in population regulations of this invasive pest through naturally induced epizootics

Carnauba wax enhances the insecticidal activity of entomopathogenic fungi against the blowfly Lucilia sericata (Diptera: Calliphoridae)

Abstract Blowfly, Lucilia sericata (Diptera: Calliphoridae), is a problematic synanthropic insect pest, a vector of microbial pathogens, and the causal agent of secondary myiasis. Fungal biopesticides are considered eco-friendly tools, alternative to synthetic pesticides, for the control of arthropod pests; however, to date, little is known about their bioactivity against blowflies. In this study, we assessed the insecticidal activity of three well-known entomopathogenic fungi, Beauveria bassiana, Beauveria pseudobassiana and Akanthomyces muscarius against L. sericata. In addition, we tested powdered carnauba wax as an electrically charged dust carrier in an attempt to enhance the virulence of fungal spores. Pathogenicity tests on adult flies, by adult immersion in conidial suspension (108 conidia mL−1), showed that the median lethal time (LT50) was 5.3, 5.9, and 6.2 days for B. bassiana, A. muscarius and B. pseudobassiana, respectively. In topical tests, when 108 dry conidia were mixed with or without carnauba wax, the LT50 was 7.7, 10.2, and 14 days without this carrier and 6.9, 8.6, and 13.8 days with it for B. bassiana, B. pseudobassiana and A. muscarius, respectively. Overall, our findings showed that, among the tested fungi, B. bassiana was the most virulent when formulated as a dry powder with carnauba wax, which greatly improved fungal efficacy against the blowfly. We discuss the utility of carnauba wax for electrostatic formulation powder of fungal spores in the integrated management of blowflies as an environmentally sustainable tool to reduce the over-reliance on chemical insecticides and their risk of resistance

Brown dog tick, Rhipicephalus sanguineus sensu lato, infestation of susceptible dog hosts is reduced by slow release of semiochemicals from a less susceptible host

Domestic dog breeds are hosts for the brown dog tick, Rhipicephalus sanguineus sensu lato, but infestation levels vary among breeds. Beagles are less susceptible to tick infestations than English cocker spaniels due to enhanced production of 2-hexanone and benzaldehyde that act as volatile tick repellents. We report the use of prototype slow-release formulations of these compounds to reduce the burden of R. sanguineus s. l. on English cocker spaniel dogs. Twelve dogs were randomly assigned to two groups with six dogs each. The treated group received collars with slow-release formulations of the compounds attached, while the control group received collars with clean formulations attached. Five environmental infestations were performed, with the number of ticks (at all stages) on the dogs being counted twice a day for 45 days. The counts on the number of tick stages found per dog were individually fitted to linear mixed effects models with repeated measures and normal distribution for errors. The mean tick infestation in the treated group was significantly lower than in the control group. For larvae and nymphs, a decrease in tick infestation was observed at the fifth count, and for adults, lower average counts were observed in all counts. The compounds did not interfere with the distribution of the ticks on the body of the dogs, as a similar percentage of ticks was found on the anterior half of the dogs (54.5% for the control group and 56.2% for the treated group). The biological and reproductive parameters of the ticks were not affected by the repellents. This study highlights for the first time the potential use of a novel allomone (repellent)-based formulation for reduction of tick infestation on susceptible dogs

Tolerance to Abiotic Factors of Microsclerotia and Mycelial Pellets From <i>Metarhizium robertsii</i>, and Molecular and Ultrastructural Changes During Microsclerotial Differentiation

Metarhizium species fungi are able to produce resistant structures termed microsclerotia, formed by compacted and melanized threads of hyphae. These propagules are tolerant to desiccation and produce infective conidia, thus they are promising candidates to use in biological control programs. In this study, we investigated the tolerance to both UV-B radiation and heat of microsclerotia of Metarhizium robertsii strain ARSEF 2575. We also adapted the liquid medium and culture conditions to obtain mycelial pellets from the same isolate in order to compare these characteristics between both types of propagules. We followed the peroxisome biogenesis and studied the oxidative stress during differentiation from conidia to microsclerotia by transmission electron microscopy after staining with a peroxidase activity marker, and by the expression pattern of genes potentially involved in these processes. We found that despite their twice smaller size, microsclerotia exhibited higher dry biomass, yield, and conidial productivity than mycelial pellets, both with and without UV-B and heat stresses. From the sixteen genes measured, we found an induction after 96-h differentiation in the oxidative stress marker genes MrcatA, MrcatP, and Mrgpx, the peroxisome biogenesis factors Mrpex5 and Mrpex14/17, and the photoprotection genes Mrlac1, Mrlac2, and Mrlac3. We concluded that an oxidative stress scenario is induced during microsclerotia differentiation in M. robertsii, and confirmed that due to its tolerance to desiccation, heat, and UV-B, this fungal structure could be an excellent candidate for use in biological control of pests under tropical and subtropical climates where heat and UV radiation are detrimental to entomopathogenic fungi survival and persistence.Facultad de Ciencias ExactasInstituto de Investigaciones Bioquímicas de La Plat

Produção por fermentação sólida e líquida e formulação de cepas virulentas dos fungos entomopatogênicos Beauveria bassiana e Isaria fumosorosea contra moscas-brancas

Bemisia tabaci Gennadius (Hemiptera: Aleyrodidae) biotype B is a cosmopolitan, devastating insect pest due to their direct damages and transmission of plant viruses. Entomopathogenic fungi comprise the most diverse group of pathogens regulating arthropod pest populations in agroecosystems. Anamorphic fungal entomopathogens, including Beauveria bassiana, Isaria fumosorosea, and Lecanicillium spp., are among the main biocontrol agents of whitefly populations. Advances in research focusing on virulence, mass production, formulation, and storage stability of fungal propagules are imperative for the development of efficient mycopesticides toward whiteflies and other soft-bodied insects. Therefore, this study placed emphasis on screening for virulent fungal strains, enhancement of efficacy using nonionic surfactants in spray tank-mix, development of liquid culture conditions for rapid production and stabilization processes of single-yeast like cells known as blastospores. Firstly, we selected virulent strains of B. bassiana and I. fumosorosea displaying fastest speed of kill and inciting highest mortality levels of whitefly nymphs and adults along with their ability to produce high numbers of conidia on moistened parboiled rice. Secondly, insecticidal performance was enhanced by combining nonionic surfactants with spore suspensions rendering additive or synergistic effects. These surfactants also allowed reducing the volume application rate without altering fungal bioefficacy. Results from liquid fermentation studies using B. bassiana and I. fumosorosea revealed that appropriate amounts of inexpensive ingredients, such as cottonseed flour and glucose, are suitable for the rapid production of high yields of blastospores (3 days pre-culture and 2-3 days culture). The resultant blastospores of various strains survived well to desiccation and remained viable for more than one year under refrigeration. Moreover, these air-dried blastospores of both fungal species showed higher virulence against whitefly nymphs when compared with solid-substrate produced conidia. Optimized liquid culture production for B. bassiana blastospores was also achieved through the manipulation of oxygen rates and osmotic pressure in the liquid media. Furthermore, these blastospores produced in highly aerated and hyperosmotic liquid medium containing 140 g glucose L-1 were also more virulent to whitefly nymphs than those cells derived from low-osmotic medium amended with 40 g glucose L-1. These optimal conditions were also scaled up in 5-L bioreactor that yielded 1-2 × 1012 viable blastospores L-1 in 6 days at a cost of US$0.19 L-1. These blastospores were formulated with diatomaceous earth for air drying or for spray drying. Formulated blastospores of B. bassiana survived dehydration using both drying methods and showed improved shelf life when stored under vacuumpackaged at 4 °C rather than 28 °C. However, when these blastospores were actively packaged with dual action oxygen-moisture scavenging system, blastospores showed prolonged stability for up to 7 months at 28 °C and still remained virulent to whiteflies. Therefore, this low-cost production and stabilization method for the rapid production of shelf stable, virulent blastospores of B. bassiana and I. fumosorosea may expand the commercial use of mycopesticides for insect control in mainstream agriculture.A mosca-branca, Bemisia tabaci Gennadius (Hemiptera: Aleyrodidae) biótipo B, é uma praga cosmopolita e devastadora devido aos prejuízos oriundos dos seus danos diretos e transmissão de vírus. Fungos entomopatogênicos compreendem um grupo diversificado, que desempenha ação importante na regulação de populações de praga em agroecossistemas. Fungos ascomicetos anamórficos, como Beauveria bassiana, Isaria fumosorosea e Lecanicillium spp., constituem relevantes agentes de biocontrole de moscas-brancas. Avanços na pesquisa focando virulência, produção massal, formulação e estabilização de propágulos fúngicos são fundamentais para o desenvolvimento de micopesticidas eficientes contra moscas-brancas e outros insetos. Desta forma, este trabalho objetivou selecionar isolados fúngicos virulentos à mosca-branca; aumentar a eficácia mediante uso de surfactants não-iônicos em suspensões conidiais; desenvolver meios de cultura para produção rápida e estável por fermentação líquida submersa de células leveduriformes conhecidas por blastosporos. Na primeira etapa, isolados virulentos de B. bassiana e I. fumosorosea foram selecionados pela rápida e elevada atividade inseticida a ninfas e adultos de mosca-branca, bem como alto rendimento de conídios em arroz parboilizado. A adição de surfactantes organosiliconados permitiu a redução do volume de calda aplicado com resultados aditivos ou sinérgicos de controle. Foi ainda verificado que altos rendimentos de blastosporos tanto de B. bassiana como I. fumosorosea foram obtidos em curto tempo de fermentação líquida (3 dias de précultivo e 2-3 dias de cultivo) usando nutrientes de baixo custo, como glucose e farelo de algodão. Esses blastosporos foram tolerantes à dessecação e mantiveram viabilidade por mais de um ano sob refrigeração (4 °C). Os blastosporos foram mais virulentos que conídios aéreos, o que coloca esta estrutura como a mais indicada como ingrediente ativo em bioinseticidas para moscas-brancas. Mediante manipulação nutricional e física do ambiente de fermentação, a produção de blastosporos de B. bassiana foi optimizada mediante aumento da aeração e pressão osmótica do meio líquido. Blastosporos produzidos em meio líquido altamente aerado e hiperosmótico (140 g glucose L-1) mostraram-se mais virulentos à mosca-branca em relação àqueles produzidos em meio hipo-osmótico (40 g glucose L-1). Esse processo foi reproduzido em escala piloto usando biorreator de 5 L resultando numa produção de 1-2 × 1012 blastosporos viáveis L-1 em apenas 3 dias a um custo de US$ 0,19 L-1. Blastosporos de B. bassiana formulados com terra de diatomáceas e secados em fluxo de ar contínuo, ou secados em spray dryer tiveram estabilidade extendida por até 8 meses a 4 °C e superior em relação a 28 °C. Durante empacotamento, o uso de sachês absorventes de oxigênio e umidade prolongou consideravelmente a viabilidade de blastosporos armazenados a 28 °C por até 7 meses sem afetar sua eficiência contra mosca-branca. Em suma, esses resultados demonstram a viabilidade técnica e econômica de produção de blastosporos virulentos de B. bassiana e I. fumosorosea, tolerantes à dessecação e estáveis durante armazenamento. Esta tecnologia é uma nova opção que pode contribuir para expansão comercial de bioinseticidas à base de fungos entomopatogênicos

Microbial control of the potato tuber moth, Phthorimaea operculella (Zeller, 1873) (Lepidoptera: Gelechiidae), using a granulovirus

A traça-da-batata, Phthorimaea operculella (Zeller, 1873) (Lepidoptera: Gelechiidae), é praga-chave da batata (Solanum tuberosum L., Solanaceae), causando sérios prejuízos em países produtores do mundo inteiro, com danos que podem atingir 100% em tubérculos armazenados. O uso de inseticidas químicos para controle de P. operculella geralmente aumenta os custos de produção, gera resíduos tóxicos e causa intoxicações nos produtores. O controle biológico é uma alternativa desejável para ser incorporado no Manejo Integrado de Pragas (MIP). O granulovírus, PhopGV (Baculoviridae), é um importante inimigo natural de P. operculella, causando epizootias frequentes nas populações dessa praga. O presente trabalho investigou o potencial de um isolado nativo de PhopGV sobre a fase larval de P. operculella a diferentes temperaturas de incubação em tubérculo (18, 24 e 30 °C) e na parte aérea de batata. Estudos relacionados à persistência do vírus aplicado em tubérculos e aos efeitos subletais da infecção viral sobre o peso pupal e o sistema imune de lagartas desse inseto foram executados. A suscetibilidade de um hospedeiro alternativo ao vírus, a traça-do-tomateiro Tuta absoluta (Meyrick, 1917) (Lepid.: Gelechiidae), foi avaliada em folhas de tomateiro (Solanum lycopersium L., Solanaceae). Em outros bioensaios, avaliou-se o efeito combinado de dois produtos à base de nim (DalNeem® e NeemAZAl®) com PhopGV sobre a mortalidade larval e rendimento de lagartas infectadas de P. operculella. Uma formulação em pó seco do vírus com talco foi desenvolvida para conferir proteção aos tubérculos. As temperaturas de incubação não afetaram a suscetibilidade de lagartas de P. operculella ao vírus, nem o rendimento de lagartas infectadas e a proporção de lagartas não recuperadas nos bioensaios em tubérculos. Porém, esses parâmetros foram altamente dependentes da concentração viral e 100% de mortalidade larval foi obtida a partir de 1 x 106 OB.mL-1. Apesar de não ter ocorrido diferenças no rendimento de lagartas infectadas entre as temperaturas, o período de incubação até a coleta destas lagartas foi menor a 30 °C. Na parte aérea, lagartas de P. operculella foram menos suscetíveis ao patógeno do que em tubérculos, sugerindo aplicação de diferentes dosagens do vírus para lavoura de batata e tubérculo armazenado. O vírus demonstrou alta persistência durante o armazenamento, mantendo-se viável por até 60 dias com mortalidade > 80%. A transmissão do vírus da lagarta para pupa em baixas concentrações ( 70%) nas concentrações 1 x 106 OB.mL-1 resulted in 100% larval mortality. Although the yield of infected larvae did not vary among the temperatures, the incubation period until the harvest of these larvae was shorter at 30 °C. On potato foliage, PTM larvae were less susceptible to the virus than on tubers, indicating that different virus dosages must be used for applications in field and storage conditions. The pathogen showed high persistence resulting in more than 80% larval mortality 60 days post-treatment. The transmission of the virus from larva to pupa at lower virus inoculum ( 70%) at concentrations < 5 x 106 OB.mL-1 than to nonformulated virus. PhopGV can be produced in vivo on potato tubers at 24-30 °C, and the use of this virus in combination with talc or DalNeem, both at lower concentrations, was very efficient against PTM and must be considered in integrated management programs

Culture media selection for mass production of Isaria fumosorosea and Isaria farinosa

This work investigated the production of the fungi Isaria fumosorosea and Isaria farinosa in biphasic fermentation using agro-industrial products and residues. Combinations of natural liquid substrates, alternative to the complete medium and potato dextrose medium, were evaluated. The best liquid media were sugarcane molasses + rice broth, rice broth + yeast and sugarcane molasses + yeast + rice broth, which resulted in the highest viable propagule concentration. The molasses + rice broth medium was selected for the next phase of the study in which the production of both fungal isolates was evaluated in solid grain substrates. In solid-state fermentation, the best conidia production was achieved with the soybean meal and broken corn for I. farinosa, and whole rice and broken rice for I. fumosorosea. Results demonstrated that the two fungal species could be rapidly produced with higher yield of conidia on agro-industrial resources by using biphasic fermentation techniques.Estudou-se a produção dos fungos Isaria fumosorosea (ESALQ1296) e Isaria farinosa (ESALQ1205) em fermentação líquida e sólida utilizando produtos e resíduos agroindustriais. Inicialmente, avaliaram-se combinações de substratos líquidos naturais e alternativos aos meios completo e batata-dextrose. Os meios líquidos mais promissores foram: melaço + caldo de arroz, caldo de arroz + levedura e melaço + levedura + caldo de arroz, pois resultaram nas maiores produções de propágulos viáveis. O meio melaço + caldo de arroz foi selecionado para etapa seguinte do trabalho, na qual se avaliou a produção dos fungos em grãos. A fermentação sólida foi mais eficiente quando se utilizaram os meios de farelo de soja e milho quirela para I. farinosa,e arroz inteiro e arroz quirela para I. fumosorosea. A fermentação bifásica para os fungos estudados a partir desses produtos agroindustriais selecionados proporcionou elevada produção de conídios em curto intervalo de tempo, podendo ser implantada em escala industrial

Natural occurrence of Zoophthora radicans (Entomophthorales: Entomophthoraceae) on Thaumastocoris peregrinus (Heteroptera: Thaumastocoridae), an invasive pest recently found in Brazil

Occurrence of Zoophthora radicans infecting nymphs and adults of Thaumastocoris peregrinus Carpintero and Dellape, 2006 is reported in Brazil. This is a new record of host for this fungal species and the first fungal pathogen associated with this pest worldwide. Infection of Z. radicans on T. peregrinus populations on commercial Eucalyptus plantation (Eucalyptus spp.) reached up to 100%, and low insect densities were associated with high levels of fungal infection in three out of seven plots. This pathogen seems to be virulent against T. peregrinus and may play an important role in population regulations of this invasive pest through naturally induced epizootics. (c) 2012 Elsevier Inc. All rights reserved