Isolation, morphological and molecular characterization of Bacillus thuringiensis strains against Hypothenemus hampei Ferrari (Coleoptera: Curculionidae: Scolytinae)

ABSTRACT The coffee berry borer Hypothenemus hampei Ferrari, 1876 (Coleoptera: Curculionidae: Scolytinae) is considered the most serious pest of the coffee crop and is controlled primarily with the use of chemical insecticides. An alternative to this control method is the use of the entomopathogenic bacterium, Bacillus thuringiensis Berliner, 1911. Therefore, the objective of this work was to select strains of B. thuringiensis virulent against H. hampei and characterize them by morphological and molecular methods to identify possible genes for the production of genetically modified plants. To achieve this objective, 34 strains of B. thuringiensis underwent a selective bioassay to evaluate their toxicity to H. hampei first-instar larvae. Among the strains tested, 11 and the standard B. thuringiensis subspecies israelensis (IPS-82) caused mortality above 90%. Then, the median lethal concentration (LC50) was estimated for these strains followed by characterization using morphological, biochemical and molecular methods. The lowest LC50 was obtained for strain BR58, although this concentration did not differ significantly from that of the standard strain IPS-82 or from that of strains BR137, BR80 and BR67. The molecular characterization detected cry4A, cry4B, cry10, cry11 and cyt1 genes in 10 of the most virulent strains (BR58, BR137, BR80, BR81, BR147, BR135, BR146, BR138, BR139, BR140). Strain BR67 differed completely from the others and amplified only the cry3 gene. This strain was more virulent than BR135, BR146, BR138, BR139 and BR140, but it did not differ from BR58, BR137, BR80, BR81 and BR147. The protein profile revealed proteins of 28, 65, 70 and 130 kDa, and the morphological analysis identified spherical crystalline inclusions in all strains. The results showed that the 11 strains studied have potential for use as a gene source for insertion into coffee plants for the control H. hampei, especially the cry3, cry4A, cry4B, cry10, cry11 and cyt1 genes, that were repeated in the most virulent isolates.

Composição química e atividade inseticida do extrato acetônico de Piper alatabaccum Trel & Yuncker (Piperaceae) sobre Hypothenemus hampei Ferrari / Chemical constituents and insecticidal activity of the crude acetonic extract of Piper alatabaccum Trel & Yuncker (Piperaceae) on Hypothenemus hampei Ferrari

O presente trabalho teve como objetivo investigar a composição química e avaliar a atividade inseticida do extrato acetônico das raízes de Piper alatabaccum sobre Hypothenemus hampei. As raízes foram coletadas no campus da Universidade Federal de Rondônia, em Porto Velho-RO. Diluições de 100,0, 50,0, 25,0, 5,0 e 0,5 mg mL-1 foram utilizadas para exposição na superfície contaminada e aplicação tópica. No teste de repelência, utilizou-se as diluições de 25,0 e 100,0 mg mL-1. As avaliações ocorreram durante 48 horas após a exposição ao extrato. O extrato foi eficiente na mortalidade em aplicação tópica e superfície contaminada, não sendo eficiente no efeito de repelência. Estes resultados apontam para o elevado potencial inseticida do extrato de P. alatabaccum no controle de H. hampei. Identificou-se cinco compostos, sendo três amidas, dihydropiplartine piperovatina e piplartina, e duas flavona, 3',4',5,5',7 pentamethoxyflavone e 5,5',7-trimetóxi-3',4'-methylenedioxyflavone.

The purpose of this work was to investigate the chemical composition and to evaluate the insecticidal activity of the acetonic extract of Piper alatabaccum roots on Hypothenemus hampei. Roots of P. alatabaccum were collected at the University of Rondônia in Porto Velho-RO. Dilutions of 100.0, 50.0, 25.0, 5.0 and 0.5 mg mL-1 were used for exposition in the contaminated surface and for topical application. For the repellence test, 25.0 and 100.0 mg mL-1 dilutions were used. The evaluations occurred during 48 hours after exposure to the extract. The extract was efficient in terms of mortality, but no repellence was observed in the bioassays. These results pointed out the high insecticidal potential of the P. alatabaccum extract to control H. hampei. Five compounds, three amids, piperovatine, dihydropiplartine and piplartine, and two flavones, 3',4',5,5',7-pentamethoxyflavone and 5,5',7-trimethoxy-3',4' methylenedioxyflavone were identified.

Alimentary canal and reproductive tract of Hypothenemus hampei (Ferrari) (Coleoptera: Curculionidae, Scolytinae)

The alimentary canal and the reproductive tract of males and females of Hypothenemus hampei Ferrari are described. The alimentary canal of H. hampei showed the crop with several spine-like structures and the midgut with few gastric caeca. We evidenced for the first time that adult females need to feed on coffee in order to produce viable eggs before and during oviposition period. The presence of air bubbles inside the anterior midgut may be due to starvation rather than the response of the environment during flying as previously reported. Two ovaries and the same structures and arrangements common to individuals of the Curculionidae beetles composed the female reproductive system. The male reproductive tract showed a significant difference with respect to other Curculionidae and even other Scolytinae species as it showed a sclerotized aedeagus with several pore-like structures in the terminal portion where sperm is released during mating.

El estudio morfológico del tracto digestivo y el sistema reproductor de machos y hembras de Hypothenemus hampei Ferrari se describe a continuación. El tracto digestivo presenta un arreglo típico descrito para otros insectos. Ahora las diferencias morfológicas se encontraron en el buche del estomodeo, en donde se observaron estructuras espinosas y en el mesenteron se observo un número reducido de ciegos gástricos. Se evidenció por primera vez el comportamiento de la alimentación de las hembras de H. hampei, que requiere aparentemente de la ingesta del endosperma del café para poder producir huevos viables durante la oviposición. La presencia de burbujas de aire al interior del ventrículo anterior se cree que puede ser debido al hambre, más que a la respuesta al medio ambiente durante el vuelo como lo indicaban anteriores observaciones. El sistema reproductor de la hembra presenta dos ovarios y un arreglo estructural común a especímenes de la familia Curculionidae. El sistema reproductor masculino mostró una diferencia significativa con respecto a otros miembros de la familia Curculionidae e incluso a otra especie de la tribu Scolytinae presentando un aedeagus esclerotizado compuesto de varios orificios en la porción terminal, por donde el esperma se expulsa durante el apareamiento.

Seletividade de defensivos agrícolas ao fungo entomopatogênico Beauveria bassiana / Selectivity of insecticides and fungicides to the entomopathogenic fungus Beauveria bassiana

A toxicidade relativa dos inseticidas organofosforados clorpirifós-metílico, dissulfotom, etiom, paratiom-metílico, do organoclorado endosulfam, da mistura do fungicida triadimenol e do inseticida dissulfotom e dos fungicidas inibidores da demetilação de esteróis triadimenol e tebuconazole ao fungo Beauveria bassiana (Balsamo) Vuillemin foi estudada em laboratório. As concentrações dos pesticidas que inibiram 99 por cento do crescimento micelial desse fungo, em miligramas de ingrediente ativo por ml foram: tebuconazole = 0,42; paration-metílico = 1,69; triadimenol = 6,53; triadimenol + dissulfotom = 14,42; clorpirifós-metílico = 31,75; etiom = 7.028,96; endosulfam = 10.326,76 e dissulfotom = 763.959,86. As equações de regressão da inibição do crescimento micelial ( por cento), em função da concentração obtida por ensaio in vitro em meio batata-destrose-ágar (BDA) impregnado com os ingredientes ativos desses compostos, foram usadas para se estimar a seletividade das concentrações prescritas para cafezais, pelos seus respectivos fabricantes. Tebuconazole, paratiom-metílico e clorpirifós-metílico foram altamente tóxicos ao fungo B. bassiana; endosulfam e triadimenol + dissulfotom apresentaram seletividade moderada e triadimenol, etiom e dissulfotom foram seletivos para esse fungo.

The relative toxicity of the insecticides organophosphates methyl-chlorpyrifos, disulfoton, ethion, methyl-parathion, the organochlorinate endosulfan and the mixture of the fungicide triadimenol and the insecticide disulfoton and the sterol demethylation inhibiter fungicide (triadimenol and tebuconazole) to the fungus Beauveria bassiana (Balsamo) Vuillemin was evaluated. Concentrations which inhibited radial mycelium growth (99 percent) of B. bassiana were 0.42; 1.69; 6.53; 14.42; 31.75; 7,028.96; 10,326.76 e 763,959.86 mg i.a/ml of tebuconazole, methyl-parathion, triadimenol, triadimenol + disulfoton, methyl-chlorpyrifos, ethion, endosulfan and disulfoton, respectively. The effects of these products were evaluated based on percentage of inhibition of radial mycelium growth of B. bassiana on potato dextrose-agar impregnated with them, after seven days. Tebuconazole, methyl-parathion and methyl-chlorpyrifos were highly toxic to B. bassiana; endosulfan and triadimenol + disulfoton were moderated toxic; while triadimenol, ethion, and disulfoton were selective to this fungi.

Natural Enemies and Competitors of Hypothenemus hampei (Ferrari) (Coleoptera: Scolytidae) in Colombia / Inimigos Naturais e Competidores da Broca-do-Café Hypothenemus hampei (Ferrari) (Coleoptera: Scolytidae) na Colômbia

O objetivo desta pesquisa foi levantar e registrar os inimigos naturais e competidores da broca-do-café, Hypothenemus hampei (Ferrari), na região cafeeira colombiana. Apesar da recente introdução da broca do café na Colômbia, foram registrados 25 inimigos naturais, incluindo o fungo Beauveria bassiana. Com exceção do fungo, os outros organismos são registros novos de inimigos naturais da broca-do-café, o que demonstra o pouco conhecimento existente sobre esses agentes de controle natural e sobre os competidores que podem contribuir para manter as populações da praga em equilíbrio. O grupo com maior número de inimigos naturais foi o dos predadores, seguido pelos competidores, fungos, bactérias e protozoários. Somente uma vespa parasitóide foi registrada atacando os adultos da broca-do-café.

The objective of this research was to survey and record the natural enemies and competitors of the Coffee Berry Borer (CBB) Hypothenemus hampei (Ferrari) in the Colombian coffee region. Despite the recently introduction of CBB to Colombia, 25 natural enemies were recorded, including Beauveria bassiana. Except for this fungus, the other organisms are new records of natural enemies of CBB. This emphasizes the little knowledge about these natural control agents and competitors that can help to keep the CBB population under control. The predators were the group with higher number of natural enemies followed by the competitors, fungi, bacteria and protozoans. Only one parasitoid wasp was recorded attacking CBB adults.

Ocorrência de Beauveria bassiana (Bals.) Vuill. em broca-do-café (Hypothenemus hampei, ferrari) no estado de Rondônia, Brasil1

In a study conducted to determine natural enemies of coffee berry borer (Hypothenemus hampei, Ferrari) in the state of Rondônia, the occurrence of the fungus Beauveria bassiana (Bals.) Vuill. was identified at Embrapa Rondônia. Coffee fruits with coffee borer containing the fungus were collected in Conilon coffee plantations (Coffea canephora), in Rolim de Moura, Ouro Preto do Oeste and Machadinho do Oeste, state of Rondônia, Brazil, from February to March, 2000. The infection percentage in fruits with coffee berry borer varied from 0.23% to 0.47%, less than reported from other regions of Brazil.

Em trabalho efetuado visando o levantamento de inimigos naturais da broca-do-café (Hypothenemus hampei, Ferrari) no estado de Rondônia, verificou-se a ocorrência do fungo Beauveria bassiana (Bals.) Vuill. conforme identificação da Embrapa Rondônia. Os frutos de café foram coletados em cafezais da cultivar Conilon (Coffea canephora), nos municípios de Rolim de Moura, Ouro Preto do Oeste e Machadinho do Oeste, Rondônia, nos meses de fevereiro a março de 2000. A percentagem média de infecção de B. bassiana em frutos broqueados variou de 0,23% a 0,47%, sendo menor que em outras regiões do Brasil.