GENETIC DIVERSIFICATION, SAPROPHYTIC COMPETENCE AND GENETIC ENHANCEMENT OF THE ENTOMOPATHOGENIC FUNGUS METARHIZIUM

Entomopathogenic fungi are being investigated as alternatives to chemical insecticides. This study explored the versatility of the entomopathogenic fungus Metarhizium anisopliae by examining its diversification, saprophytic competence and potential for genetic enhancement. M. anisopliae is a radiating species containing both generalist and specialized lineages with broad and narrow host ranges and as such provides an excellent model system to study the evolution of pathogenesis. Using 18S RNA and protease sequences, I demonstrated that strains can be selected representing evolutionary distances ranging from <1 to 8 MY and their natural molecular variation allows analysis of processes of adaptive change. M. anisopliae is particularly abundant in the rhizosphere. Germination of M. anisopliae strain 2575 was >96% in 1 mg/ml root exudate (RE) and growth in RE resulted in 29 (58%) genes being up-regulated and 21 (42%) down-regulated. The identity of these genes is helping to define the physiological requirements for rhizosphere competence. Hypothetical and orphans proteins (41.4%) were also actively expressed indicating that many previously uncharacterized genes may have functions related to survival at the soil-root interphase. Using the fungus as a delivery vehicle for foreign toxins presents a powerful approach for increasing virulence. M. anisopliae was modified to express a scorpion toxin (AaIT) in insect haemolymph and bioassayed against the coffee berry borer Hypothenemus hampei. AaIT increased mortality up to 56.6%, and reduced the medial lethal concentration (LC 50 ) by 15.7-fold and the average survival time (AST) by 20.1%. The AaIT gene and the M. anisopliae esterase gene (Mest1) were inserted into three strains of Beauveria bassiana (ARSEF 252, 8998 and 9184) with high, medium and low mortality, respectively, against the Colorado potato beetle (CPB) Leptinotarsa decemlineata. Mortality rates were strain- and dose- dependant and increased from 16.1 to 36.7% in single transformants (AaIT or Mest1) and from 7.1 to 33.5% in double transformants (AaIT-Mest1). The AST was reduced up to 33% and the LC50 up to 5.9-fold. Although singly both AaIT and Mest1 increased the killing power of B. bassiana against second instar CPB, combining AaIT and Mest1 together did not produce synergistic effects

Sexual Dimorphism of Pupae and Adults of the Cocoa Pod Borer, Conopomorpha cramerella

This paper describes the main distinguishing characteristics of female and male pupae and adults of cocoa pod borer, Conopomorpha cramerella (Snellen) (Lepidoptera: Gracillariidae). Two pairs of tubercles present on the sterna of segments IX and X of the female pupae are useful in differentiating female from male pupae. The female genital opening is located anterior to the first pair of tubercles and forms a plateau in which the center has a light brown longitudinal depression that indicates the female genital opening. The male genital opening is a conspicuous, brown, longitudinal slit located between the two pairs of tubercles. The sex of the adult moth can be determined by examining the ventrocaudal segments of the abdomen. The last segment of the female abdomen is white, compressed laterally and at the tip, and the hairy anal papillae can be seen. In the male, the ventrocaudal end of the abdomen is black and robust. This information will be useful for laboratory and field diagnosis and while working on sex ratios of this important pest of cocoa

Development of a Multiplex Polymerase Chain Reaction (PCR) Assay for the Potential Detection of Insect Contaminants in Food

Molecular methods can potentially be used to detect insect contaminants of food products. In this study, we used three sets of group-specific primers, two of them targeting the amplification of two regions of the insect’s mitochondrial cytochrome c oxidase subunit I (COI-Fa and COI-Fb) and the other targeting a region of the nuclear protein-coding wingless (wg) gene. Using singleplex and multiplex polymerase chain reaction (PCR), we evaluated the three sets of primers using genomic DNA (gDNA) from 48 insect species including food storage insect pests and known vectors of foodborne pathogens. Seven plant-based food matrices were also evaluated for exclusivity testing. Additionally, we spiked fragments from five insect species in a selected food matrix (whole wheat flour). Singleplex and multiplex PCR amplified single specific bands (401–449 bp), corresponding to the wg gene, from insect species belonging to families Blattidae and Formicidae, and in Plodia interpunctella (Hübner) (Lepidoptera: Pyralidae). The COI-Fa primers amplified specific bands (171–188 bp) in all Dipteran species and the COI-Fb primers amplified a specific band (∼140 bp) in DNA from Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae) and P. interpunctella. However, the presence of specific bands in most Coleopterans was not consistent. No amplicon bands were observed in any of the food matrixes tested and the expected pattern of amplicon bands was seen in multiplex reactions using gDNA from spiked food samples. Our multiplex PCR assay targeted specific groups of insects that commonly contaminate foods without amplifying bands from the food matrixes tested; thus, molecular methods may be suitable for detecting insects or their fragments in foods

Establecimiento del hongo entomopatógeno Beauveria bassiana como endófito en frutos de cacao (Theobroma cacao L.)

The entomopathogenic fungus Beauveria bassiana was re-isolated from surface sterilized pieces of the cocoa pod outer section and peduncle, two and three months after flowers of greenhouse cocoa plants were hand-pollinated and sprayed with a 1x107 mL-1 suspension of B. bassianaconidia. The colonization frequency of B. bassiana on cocoa pods was 0.8% and 1.3% two and three months post-spraying of conidial suspension, respectively. These results indicate that this entomopathogenic fungus can be established as an endophyte on cocoa pods if flowers are sprayed. Other naturally occurring endophytic fungi such as Acremoniumspp., Aspergillus spp., Cladosporium spp. and Penicilliumspp. as well as seven morphospecies of bacteria were also isolated from cocoa pods. These findings suggest that cocoa pods are conducive for endophytic fungal growth of both naturally occurring and inoculated B. bassiana, and could possibly lead to an innovative methodology to manage insect pests in biocontrol programs.El hongo entomopatógeno Beauveria bassiana fue re-aislado de superficies desinfectadas de fragmentos de la parte externa y del pedúnculo del fruto de cacao luego de dos y tres meses de que las flores de plantas criadas en invernadero fueran polinizadas manualmente y asperjadas con una suspensión de 1x107 mL-1 esporas de B. bassiana. La frecuencia de colonización de B. bassiana en el fruto de cacao fue de 0,8% y 1,3% luego de dos y tres meses de la aspersión de las esporas, respectivamente. Estos resultados indican que este hongo entomopatógeno puede ser establecido como hongo endófito de frutos de cacao luego de la aspersión de las flores de caco con esporas del hongo. Durante el estudio, también se aislaron otros hongos endófitos del fruto de cacao como Acremonium spp., Aspergillus spp., Cladosporium spp. y Penicillium spp, y siete morfoespecies de bacterias, demostrando su presencia natural como endófitos de esta planta. Estos resultados sugieren que los frutos de cacao contribuyen al crecimiento tanto de hongos endófitos naturales como del hongo B. bassiana inoculado, y esto podría conllevar al establecimiento de una metodología novedosa para el manejo de insectos perforadores de frutos de cacao en programas de control biológico de insectos plaga.Incluye referencias bibliográfica

ESTABLISHMENT OF THE FUNGAL ENTOMOPATHOGEN Beauveria bassiana AS AN ENDOPHYTE IN COCOA PODS (Theobroma cacao L.) ESTABLECIMIENTO DEL HONGO ENTOMOPATÓGENO Beauveria bassiana COMO ENDÓFITO EN FRUTOS DE CACAO (Theobroma cacao L.)

The entomopathogenic fungus Beauveria bassiana was re-isolated from surface sterilized pieces of the cocoa pod outer section and peduncle, two and three months after flowers of greenhouse cocoa plants were hand-pollinated and sprayed with a 1x10(7) mL-1 suspension of B. bassiana conidia. The colonization frequency of B. bassiana on cocoa pods was 0.8% and 1.3% two and three months post-spraying of conidial suspension, respectively. These results indicate that this entomopathogenic fungus can be established as an endophyte on cocoa pods if flowers are sprayed. Other naturally occurring endophytic fungi such as Acremonium spp., Aspergillus spp., Cladosporium spp. and Penicillium spp. as well as seven morphospecies of bacteria were also isolated from cocoa pods. These findings suggest that cocoa pods are conducive for endophytic fungal growth of both naturally occurring and inoculated B. bassiana, and could possibly lead to an innovative methodology to manage insect pests in biocontrol programs.<br>El hongo entomopatógeno Beauveria bassiana fue re-aislado de superficies desinfectadas de fragmentos de la parte externa y del pedúnculo del fruto de cacao luego de dos y tres meses de que las flores de plantas criadas en invernadero fueran polinizadas manualmente y asperjadas con una suspensión de 1x10(7) mL-1 esporas de B. bassiana. La frecuencia de colonización de B. bassiana en el fruto de cacao fue de 0,8% y 1,3% luego de dos y tres meses de la aspersión de las esporas, respectivamente. Estos resultados indican que este hongo entomopatógeno puede ser establecido como hongo endófito de frutos de cacao luego de la aspersión de las flores de caco con esporas del hongo. Durante el estudio, también se aislaron otros hongos endófitos del fruto de cacao como Acremonium spp., Aspergillus spp., Cladosporium spp. y Penicillium spp, y siete morfoespecies de bacterias, demostrando su presencia natural como endófitos de esta planta. Estos resultados sugieren que los frutos de cacao contribuyen al crecimiento tanto de hongos endófitos naturales como del hongo B. bassiana inoculado, y esto podría conllevar al establecimiento de una metodología novedosa para el manejo de insectos perforadores de frutos de cacao en programas de control biológico de insectos plaga