Pamela Marrone

Specificity and efficacy of purified Bacillus thuringiensis proteins against agronomically important insects.

The host range and relative efficacy of three purified Bacillus thuringiensis insect control proteins were determined against 17 different agronomically important insects representing five orders and one species of mite. The three B. thuringiensis proteins were single gene products from B. thuringiensis ssp. kurstaki HD-1 (CryIA(b)) and HD-73 (CryIA(c)), both lepidopteran-specific proteins, and B. thuringiensis ssp. tenebrionis (CryIIIA), a coleopteran-specific protein. Seven insects showed sensitivity to both B. thuringiensis ssp. kurstaki proteins, whereas only 1 of the 18 insects was sensitive to B. thuringiensis ssp. tenebrionis protein. The level of B. thuringiensis ssp. kurstaki protein required for 50% mortality (LC50) varied by 2000-fold for these 7 insects. A larval growth inhibition assay was developed to determine the amount of B. thuringiensis ssp. kurstaki protein required to inhibit larval growth by 50% (EC50). This extremely sensitive assay enabled detection of B. thuringiensis ssp. kurstaki HD-73 levels as low as 1 ng/ml.

Selection of tobacco budworm for resistance to a genetically engineered Pseudomonas fluorescens containing the δ-endotoxin of Bacillus thuringiensis subsp. kurstaki

Abstract A diet incorporation technique was employed to study the selection response of Heliothis virescens to a genetically engineered strain of Pseudomonas fluorescens . The Pseudomonas expresses a 130-kDa endotoxin protein of Bacillus thuringiensis subsp. kurstaki strain HD-1. Selection was maintained for 14 generations. By generation 3, differences in susceptibility between selected and control lines were significant at 3-fold. Resistance increased to 24-fold by generation 7, and fluctuated between 13 and 20× thereafter. The selected strain was also approximately 4-fold less susceptible to purified HD-1 endotoxin and Dipel. Injection of larvae with engineered or nonengineered Pseudomonas did not result in a significant difference in survival between the two lines, indicating response to bacterial septicemia is a minor part of the resistance mechanism. An unchallenged selected line (F13) maintained its initial level of resistance over two generations, providing evidence for the stability of the trait. The implications of these findings on the development of pest management strategies are discussed.

Phytotoxicity of Sarmentine Isolated from Long Pepper (Piper longum) Fruit

Discovery of novel natural herbicides has become crucial to overcome increasing weed resistance and environmental issues. In this article, we describe the finding that a methanol extract of dry long pepper (Piper longum L.) fruits is phytotoxic to lettuce (Lactuca sativa L.) seedlings. The bioassay-guided fractionation and purification of the crude extract led to isolation of sarmentine (1), a known compound, as the active principle. Phytotoxicity of 1 was examined with a variety of seedlings of field crops and weeds. Results indicated that 1 was a contact herbicide and possessed broad-spectrum herbicidal activity. Moreover, a series of sarmentine analogues were then synthesized to study the structure-activity relationship (SAR). SAR studies suggested that phytotoxicity of sarmentine and its analogues was specific due to chemical structures, i.e., the analogues of the acid moiety of 1 were active, but the amine and its analogues were inactive; the ester analogues and amide analogues with a primary amine of 1 were also inactive. In addition, quantification of 1 from different resources of the dry P. longum fruits using liquid chromatography-mass spectrometry showed a wide variation, ranging from almost zero to 0.57%. This study suggests that 1 has potential as an active lead molecule for synthesized herbicides as well as for bioherbicides derived from natural resources.

Purification and characterization of Bacillus thuringiensis var. tenebrionis insecticidal proteins produced in E. coli

Native and single amino acid variants of the Bacillus thuringiensis var. tenebrionis insecticidal proteins were expressed in Escherichia coli, purified and examined for biological and biochemical properties. A novel, pH dependent, preferential precipitation method was implemented to purify Escherichia coli produced Bacillus thuringiensis var. tenebrionis proteins, which are active against Colorado potato beetle (Leptinotarsa decemlineata) larvae. Cysteine residues of the native Bacillus thuringiensis var. tenebrionis protein were replaced by serine residues by site-directed mutagenesis to investigate the biological and structural importance of the individual cysteine residues. Sulfhydryl determination of the native and amino acid variant Bacillus thuringiensis var. tenebrionis proteins revealed that the native protein contains no disulfide bonds. Modification of the carboxyl terminal cysteine residue (amino acid 540) caused complete inactivation of the protein. Native, truncated and single amino acid variants (other than at amino acid 540) exhibited insecticidal activities comparable to each other and to solubilized crystals from the original strain.

Isolation and Characterization of Burkholderia rinojensis sp. nov., a Non-Burkholderia cepacia Complex Soil Bacterium with Insecticidal and Miticidal Activities

ABSTRACT Isolate A396, a bacterium isolated from a Japanese soil sample demonstrated strong insecticidal and miticidal activities in laboratory bioassays. The isolate was characterized through biochemical methods, fatty acid methyl ester (FAME) analysis, sequencing of 16S rRNA, multilocus sequence typing and analysis, and DNA-DNA hybridization. FAME analysis matched A396 to Burkholderia cenocepacia, but this result was not confirmed by 16S rRNA or DNA-DNA hybridization. 16S rRNA sequencing indicated closest matches with B. glumae and B. plantarii. DNA-DNA hybridization experiments with B. plantarii, B. glumae, B. multivorans, and B. cenocepacia confirmed the low genetic similarity (11.5 to 37.4%) with known members of the genus. PCR-based screening showed that A396 lacks markers associated with members of the B. cepacia complex. Bioassay results indicated two mechanisms of action: through ingestion and contact. The isolate effectively controlled beet armyworms (Spodoptera exigua; BAW) and two-spotted spider mites (Tetranychus urticae; TSSM). In diet overlay bioassays with BAW, 1% to 4% (vol/vol) dilution of the whole-cell broth caused 97% to 100% mortality 4 days postexposure, and leaf disc treatment bioassays attained 75% ± 22% mortality 3 days postexposure. Contact bioassays led to 50% larval mortality, as well as discoloration, stunting, and failure to molt. TSSM mortality reached 93% in treated leaf discs. Activity was maintained in cell-free supernatants and after heat treatment (60°C for 2 h), indicating that a secondary metabolite or excreted thermostable enzyme might be responsible for the activity. Based on these results, we describe the novel species Burkholderia rinojensis, a good candidate for the development of a biocontrol product against insect and mite pests.