Submitted to: Florida Entomologist
Publication Type: Peer reviewed journal
Publication Acceptance Date: 7/10/2006
Publication Date: 12/2/2006
Citation: Weathersbee III, A.A., Lapointe, S.L., Shatters, R.G. 2006. Activity of Bacillus thuringiensis isolates against the root weevil, Diaprepes abbreviatus (Coleoptera: Curculionidae). Florida Entomologist 89:441-448. Interpretive Summary: Bacterial entomopathogens have not been adequately investigated as potential microbial agents for control of the citrus root weevil, Diaprepes abbreviatus. The bacterium, Bacillus thuringiensis is known to be pathogenic to many insect pests but relatively few strains have been found that are active against Coleoptera. Endotoxins produced by B. thuringiensis are activated in the gut of susceptible hosts upon ingestion, and cause feeding inhibition, infection of host tissues, and eventual death. We propose to identify endotoxins that are active against D. abbreviatus. A citrus rootstock genetically-engineered to express one or more of these toxins could offer protection of plant roots from feeding damage caused by D. abbreviatus larva. This approach has been successful in protecting other crop plants from insect pests. Researchers at the U.S. Horticultural Research Laboratory assembled a collection of B. thuringiensis isolates obtained from the Agricultural Research Service Culture Collection, National Center for Agricultural Utilization Research, Peoria, Illinois. The collection contained isolates that were demonstrated to be active against on or more representatives of Coleoptera. Isolates were screened for activity against neonatal larvae of D. abbreviatus. Isolates that were active in screening experiments were included in dose-response experiments to better define their activities. Three bacterial isolates were identified that expressed endotoxins active against D. abbreviatus. The endotoxin genes from which these active toxins are transcribed may be useful in developing a transgenic citrus rootstock that is resistant to D. abbreviatus.
Technical Abstract: A collection of Bacillus thuringiensis isolates plausibly active against coleopteran insects was obtained from the Agricultural Research Service Culture Collection. Each isolate was cultured, spores and delta-endotoxin crystals were pelleted by centrifugation and lyophilized, and the resulting product was incorporated in insect diet for testing against Diaprepes abbreviatus neonates. A bioassay method was developed that utilized small amounts of insect diet and B. thuringiensis spores and '-endotoxin to treat single neonates confined to 0.2 ml clear polymerase chain reaction (PCR) tubes. The method was less expensive in terms of labor and materials as compared to previous methods and reduced control losses due to burrowing and aggressive behaviors of D. abbreviatus larvae confined together. Of 19 B. thuringiensis isolates screened for activity against D. abbreviatus with a discriminating dose of 250 ppm spores and delta-endotoxin on diet, 5 were selected for further evaluation in dose-response experiments. Diaprepes abbreviatus larvae demonstrated a significant dose response to 4 of the 5 isolates tested. The most active isolates were those that expressed CryET33 and CryET34, or Cyt2Ca1 proteins. A wild-type B. thuringiensis strain that expressed Cyt2Ca1 generated the lowest LC50 value (50.7 ug/ml) and steepest slope (1.11) based on log10 probit analysis of the data. These B. thuringiensis delta-endotoxins may have utility in transgenic approaches to citrus rootstock protection from D. abbreviatus.