Elza Áurea de Luna Alves Lima

The effects of the fungus Metarhizium anisopliae var. acridum on different stages of Lutzomyia longipalpis (Diptera: Psychodidae)

The control of Visceral Leishmaniasis (VL) vector is often based on the application of chemical residual insecticide. However, this strategy has not been effective. The continuing search for an appropriate vector control may include the use of biological control. This study evaluates the effects of the fungus Metarhizium anisopliae var. acridum on Lutzomyia longipalpis. Five concentrations of the fungus were utilized, 1 x 10(4) to 1 x 10(8) conidia/ml, accompanied by controls. The unhatched eggs, larvae and dead adults previously exposed to fungi were sown to reisolate the fungi and analysis of parameters of growth. The fungus was subsequently identified by PCR and DNA sequencing. M. anisopliae var. acridum reduced egg hatching by 40%. The mortality of infected larvae was significant. The longevity of infected adults was lower than that of negative controls. The effects of fungal infection on the hatching of eggs laid by infected females were also significant. With respect to fungal growth parameters post-infection, only vegetative growth was not significantly higher than that of the fungi before infection. The revalidation of the identification of the reisolated fungus was confirmed post-passage only from adult insects. In terms of larvae mortality and the fecundity of infected females, the results were significant, proving that the main vector species of VL is susceptible to infection by this entomopathogenic fungus in the adult stage.

Biological insect control using Metarhizium anisopliae: morphological, molecular, and ecological aspects

Microbial control of insects is based on the rational use of pathogens to maintain environmentally balanced pest population levels, and Metarhizium anisopliae has been the most studied and most utilized fungal species for that purpose. The natural genetic variability of entomopathogenic fungi is considered one of the principal advantages of microbial insect control. The inter- and intraspecific variability and the genetic diversity and population structures of Metarhizium and other entomopathogenic fungi have been examined using ITS-RFLP, ISSR, and ISSP molecular markers. The persistence of M. anisopliae in the soil and its possible effects on the structures of resident microbial communities must be considered when selecting isolates for biological insect control.

Horizontal transmission and effect of the temperature in pathogenicity of Beauveria bassiana against Diatraea saccharalis (Lepidoptera: Crambidae)

The aim of this work was to evaluate the effect of temperature on the pathogenicity of Beauveria bassiana against Diatraea saccharalis and evaluate the horizontal transmission capacity among the sugarcane borer larvae. The strains were most pathogenic at 26oC, followed by 32oC than at 20oC, with respective mortalities of 100, 50 and 30.3%. The lethal time was also affected by the temperature, with the shortest LT 50 observed at 26oC (2.2 days) and the longest at 20oC (16.2 days). The URM2915 strain appeared most effective in all the experiments as having potential for deployment in biological control programs for the sugarcane borer. Results in horizontal transmission of B. bassiana showed that this procedure could also be adopted as control strategy for the sugarcane borer.

Natural occurrence of the Fusarium solani on Tityus stigmurus (Thorell, 1876) (Scorpiones: Buthidae)

Members of the Fusarium solani species complex are agents of human mycoses, also affecting plants and other animals. Nevertheless, this fungus has not been reported on scorpions. Ten specimens of Tityus stigmurus collected in the field and showing their surface covered by white mycelia were used to assess fungus presence in the animal after its death. Identification of the fungi was based upon the cultural and morphological characteristics. The fungus was isolated from chelicerae and intersegmental regions. Infected individuals had their behaviour modified by reducing feeding and locomotion. None of the infected individuals survived. It is likely that this fungus may have a role in the regulation of field scorpion populations.

Hemocyte characterization of Nasutitermes coxipoensis (Holmgren) (Isoptera: Termitidae) workers and hemocyte evaluation after parasitism by Metarhizium anisopliae

We aimed to characterize the morphology and ultrastructure of hemocytes of Nasutitermes coxipoensis (Holmgren) workers and to quantify the cell types 24 h, 48 h and 72 h after inoculation with Metarhizium anisopliae. Six hemocytes types were identified, plasmatocyte, granulocyte, spherulocyte, prohemocyte, adipohemocyte and eonocytoid Hemocytes did not present any morphological alteration at the several observation periods, but they did have a change in their abundance, as observed for spherulocytes, adipohemocytes and eonocytoids at all intervals, and for plasmatocytes and granulocytes at 48 h after host inoculation. We argue on the possible reasons and implications of the observed changes.

Differentiation of the entomopathogenic fungus Metarhizium flavoviride (Hyphomycetes)

A diferenciacao de um isolado brasileiro de Metarhizium flavoviride (CG 423), candidato a agente de controle biologico de gafanhotos, foi investigada. Conidios semeados em meio de cultura solido (extrato de levedura 1%, agar 2,8%, agua destilada 96,2%) e incubados a 28°C, foram observados durante 26 horas. Para induzir a formacao de apressorios, conidios foram suspensos em meio liquido contendo duas concentracoes de extrato de levedura (0,06 e 1%) e transferidos para placas de Petri plasticas (3,5 cm de diâmetro). A germinacao teve inicio com o aumento do tamanho dos conidios de 5,3 ± 0,6 x 3,1 ± 0,3 µm (0 h de incubacao) para 8,1 ± 0,2 x 6,1 ± 0,2 µm (8 h de incubacao). Os primeiros tubos germinativos comecaram a surgir apos 10 h de incubacao dos conidios, os quais apresentaram acentuada multipolaridade. Vinte e seis horas apos a inoculacao foi observado o inicio da diferenciacao micelial e formacao de anastomoses entre hifas de conidios adjacentes. Apressorios foram formados somente quando conidos foram incubados em meio liquido contendo concentracao minima de nutriente (extrato de levedura 0,0%; peso/volume). Os apressorios formados encontravam-se fortemente aderidos a superficie do fundo plastico da placa de Petri.

MORPHOLOGICAL, CYTOLOGICAL, AND CULTURAL ASPECTS OF CURVULARIA PALLESCENS

Curvularia pallescens Boedijn (Hyphomycetes) is redescribed with the aid of a scanning electron microscope, and the optimal cultural conditions for growing this fungus are discussed. Cytological analysis and nuclear condition, observed through the HCl-Giemsa technique, showed vegetative and reproductive structures (hypha and conidia) formed by uni, bi, tri, and multinucleated segments. Cultures of C. pallescens in Complete medium and in Potato dextrose agar varied on growth, on aspects of the border of the colonies and also on medium pigmentation. The Complete medium and the temperature between 25-28°C were the most indicated for growth of C. pallescens.

Patogenicidade de Metarhizium anisopliae var. anisopliae e Metarhizium anisopliae var. acridum sobre Nasutitermes coxipoensis (Holmgren) (Isoptera: Termitidae)

A acao de Metarhizium anisopliae var. anisopliae e Metarhizium anisopliae var. acridum sobre o cupim de monticulo Nasutitermes coxipoensis (Holmgren) e a producao de conidios sobre os insetos mortos foram estudadas em laboratorio. Doses infectivas foram determinadas atraves do contato direto dos insetos com a cultura fungica apos 12 dias de crescimento e quantificacao dos conidios em câmara de Neubauer. Foram utilizadas as doses 0,5 x 106 e 1,6 x 107 conidos/ml, para M. anisopliae var. anisopliae, e 1,4 x 105 e 1,8 x 106 conidios/ml para M. anisopliae var. acridum, nominadas dose A e dose B, respectivamente. As doses corresponderam a 3 min. e 15 min. de contato direto de 20 cupins com a cultura fungica. A producao de conidios foi avaliada utilizando-se insetos inoculados com o fungo, 10 dias apos a morte. Os bioensaios foram realizados em triplicata. Os percentuais de mortalidade acumulada de N. coxipoensis tratados com M. anisopliae, no terceiro dia apos a inoculacao, foram: controle, 15,7%; dose A 95,7% e dose B 100%; para M. anisopliae var. acridum foram: controle, 13,3%; dose A 66,4 e dose B 88,8%. A media da producao de conidios foi mais elevada para M. anisopliae var. anisopliae do que para M. anisopliae var. acridum. Os resultados mostraram que M. anisopliae var. anisopliae apresentou maior potencial para o controle biologico de N. coxipoensis.

The potential of Isaria spp. as a bioinsecticide for the biological control of Nasutitermes corniger

ABSTRACT The termite Nasutitermes corniger is a serious pest infesting urban areas of Brazil and many other countries. Control largely depends on synthetic pesticides whose indiscriminate use can impact the environment and the health of humans and other animals. Alternative strategies against insect pests, such as biological control by entomopathogenic fungi, could be effective while minimising these deleterious effects. We analysed the actions of the entomopathogenic fungi Isaria farinosa, Isaria fumosorosea, and Isaria javanica against the insect N. corniger. Our results indicated that the fungi examined were pathogenic against N. corniger, with I. farinosa ESALQ1355 being the most efficacious strain, resulting in the death of 95% of the workers (LC50 6.66 × 104 conidia/mL) and 85% of the soldiers (LC50 6.81 × 104 conidia/mL). This is the first report of the pathogenicity of Isaria spp. on N. corniger. These in vitro results suggest that I. farinosa ESALQ1355 demonstrates a significant biological potential for controlling N. corniger.

Pathogenicity of Beauveria bassiana and production of cuticle-degrading enzymes in the presence of Diatraea saccharalis cuticle

The sugarcane borer, Diatraea saccharalis , is one of the worst pests in Brazilian sugarcane crop, causing high levels of financial losses every year. Beauveria bassiana is an entomopathogenic fungus widely used in the biological control of several agricultural pests. The aims of this study were to: (1) evaluate the pathogenicity of B. bassiana strains against D. saccharalis (2) investigate the production of proteases and chitinase by B. bassiana in the presence of the cuticle of sugarcane borer; and, (3) evaluate the relation between the production of enzymes and pathogenicity of the strains. All isolates tested were pathogenic to D. saccharalis and the mortality ranged from 36 to 88%. The production of enzymes was higher in the medium containing cuticle, showing that the process is stimulated by specific components found in the cuticle of the host. Pr1 activity was higher than Pr2 and both were produced at 24 h. The highest production of chitinase was obtained at 96 h of culture for all strains tested. Levels of specific cuticle-degrading enzymes such as proteases correlated positively with specific virulence parameters. B. bassiana URM2915 showed promising features to be used in a biological control program of D. saccharalis . Key words : Biological control, sugarcane, subtilisin-like protease, trypsin-like protease, chitinase.