Isolation, Purification, and Characterisation of a Phage Tail-Like Bacteriocin from the Insect Pathogenic Bacterium Brevibacillus laterosporus.

The Gram-positive and spore-forming bacterium Brevibacillus laterosporus (Bl) belongs to the Brevibacillus brevis phylogenetic cluster. Isolates of the species have demonstrated pesticidal potency against a wide range of invertebrate pests and plant diseases. Two New Zealand isolates, Bl 1821L and Bl 1951, are under development as biopesticides for control of diamondback moth and other pests. However, due to the often-restricted growth of these endemic isolates, production can be an issue. Based on the previous work, it was hypothesised that the putative phages might be involved. During investigations of the cause of the disrupted growth, electron micrographs of crude lysate of Bl 1821L showed the presence of phages' tail-like structures. A soft agar overlay method with PEG 8000 precipitation was used to differentiate between the antagonistic activity of the putative phage and phage tail-like structures (bacteriocins). Assay tests authenticated the absence of putative phage activity. Using the same method, broad-spectrum antibacterial activity of Bl 1821L lysate against several Gram-positive bacteria was found. SDS-PAGE of sucrose density gradient purified and 10 kD MWCO concentrated lysate showed a prominent protein band of ~48 kD, and transmission electron microscopy revealed the presence of polysheath-like structures. N-terminal sequencing of the ~48 kD protein mapped to a gene with weak predicted amino acid homology to a Bacillus PBSX phage-like element xkdK, the translated product of which shared >90% amino acid similarity to the phage tail-sheath protein of another Bl published genome, LMG15441. Bioinformatic analysis also identified an xkdK homolog in the Bl 1951 genome. However, genome comparison of the region around the xkdK gene between Bl 1821L and Bl 1951 found differences including two glycine rich protein encoding genes which contain imperfect repeats (1700 bp) in Bl 1951, while a putative phage region resides in the analogous Bl 1821L region. Although comparative analysis of the genomic organisation of Bl 1821L and Bl 1951 PBSX-like region with the defective phages PBSX, PBSZ, and PBP 180 of Bacillus subtilis isolates 168 and W23, and Bacillus phage PBP180 revealed low amino acids similarity, the genes encode similar functional proteins in similar arrangements, including phage tail-sheath (XkdK), tail (XkdO), holin (XhlB), and N-acetylmuramoyl-l-alanine (XlyA). AMPA analysis identified a bactericidal stretch of 13 amino acids in the ~48 kD sequenced protein of Bl 1821L. Antagonistic activity of the purified ~48 kD phage tail-like protein in the assays differed remarkably from the crude lysate by causing a decrease of 34.2% in the number of viable cells of Bl 1951, 18 h after treatment as compared to the control. Overall, the identified inducible phage tail-like particle is likely to have implications for the in vitro growth of the insect pathogenic isolate Bl 1821L.

You are what you eat: fungal metabolites and host plant affect the susceptibility of diamondback moth to entomopathogenic fungi.

Background: Beauveria are entomopathogenic fungi of a broad range of arthropod pests. Many strains of Beauveria have been developed and marketed as biopesticides. Beauveria species are well-suited as the active ingredient within biopesticides because of their ease of mass production, ability to kill a wide range of pest species, consistency in different conditions, and safety with respect to human health. However, the efficacy of these biopesticides can be variable under field conditions. Two under-researched areas, which may limit the deployment of Beauveria-based biopesticides, are the type and amount of insecticidal compounds produced by these fungi and the influence of diet on the susceptibility of specific insect pests to these entomopathogens. Methods: To understand and remedy this weakness, we investigated the effect of insect diet and Beauveria-derived toxins on the susceptibility of diamondback moth larvae to Beauveria infection. Two New Zealand-derived fungal isolates, B. pseudobassiana I12 Damo and B. bassiana CTL20, previously identified with high virulence towards diamondback moth larvae, were selected for this study. Larvae of diamondback moth were fed on four different plant diets, based on different types of Brassicaceae, namely broccoli, cabbage, cauliflower, and radish, before their susceptibility to the two isolates of Beauveria was assessed. A second experiment assessed secondary metabolites produced from three genetically diverse isolates of Beauveria for their virulence towards diamondback moth larvae. Results: Diamondback moth larvae fed on broccoli were more susceptible to infection by B. pseudobassiana while larvae fed on radish were more susceptible to infection by B. bassiana. Furthermore, the supernatant from an isolate of B. pseudobassiana resulted in 55% and 65% mortality for half and full-strength culture filtrates, respectively, while the filtrates from two other Beauveria isolates, including a B. bassiana isolate, killed less than 50% of larvae. This study demonstrated different levels of susceptibility of the insects raised on different plant diets and the potential use of metabolites produced by Beauveria isolates in addition to their conidia.

Field Performance of Bt Eggplants (Solanum melongena L.) in the Philippines: Cry1Ac Expression and Control of the Eggplant Fruit and Shoot Borer (Leucinodes orbonalis Guenée).

Plants expressing Cry proteins from the bacterium, Bacillus thuringiensis (Bt), have become a major tactic for controlling insect pests in maize and cotton globally. However, there are few Bt vegetable crops. Eggplant (Solanum melongena) is a popular vegetable grown throughout Asia that is heavily treated with insecticides to control the eggplant fruit and shoot borer, Leucinodes orbonalis (EFSB). Herein we provide the first publicly available data on field performance in Asia of eggplant engineered to produce the Cry1Ac protein. Replicated field trials with five Bt eggplant open-pollinated (OP) lines from transformation event EE-1 and their non-Bt comparators were conducted over three cropping seasons in the Philippines from 2010-2012. Field trials documented levels of Cry1Ac protein expressed in plants and evaluated their efficacy against the primary target pest, EFSB. Cry1Ac concentrations ranged from 0.75-24.7 ppm dry weight with the highest in the terminal leaves (or shoots) and the lowest in the roots. Cry1Ac levels significantly increased from the vegetative to the reproductive stage. Bt eggplant lines demonstrated excellent control of EFSB. Pairwise analysis of means detected highly significant differences between Bt eggplant lines and their non-Bt comparators for all field efficacy parameters tested. Bt eggplant lines demonstrated high levels of control of EFSB shoot damage (98.6-100%) and fruit damage (98.1-99.7%) and reduced EFSB larval infestation (95.8-99.3%) under the most severe pest pressure during trial 2. Moths that emerged from larvae collected from Bt plants in the field and reared in their Bt eggplant hosts did not produce viable eggs or offspring. These results demonstrate that Bt eggplant lines containing Cry1Ac event EE-1 provide outstanding control of EFSB and can dramatically reduce the need for conventional insecticides.