A new biopesticide from a local Bacillus thuringiensis var. tenebrionis (Xd3) against alder leaf beetle (Coleoptera: Chrysomelidae).

Use of chemical pesticides in agriculture harms humans, non-target organisms and environments, and causes increase resistance against chemicals. In order to develop an effective bio-pesticide against coleopterans, particularly against Agelastica alni (Coleoptera: Chrysomelidae) which is one of the serious pests of alder leaf and hazelnut, we tested the insecticidal effect of 21 Bacillus isolates against the larvae and adults of the pest. Bacillus thuringiensis var. tenebrionis-Xd3 (Btt-Xd3) showed the highest insecticidal effect based on screening tests. For toxin protein production and high sporulation of Xd3, the most suitable medium, pH and temperature conditions were determined as nutrient broth medium enriched with salts, pH 7 and 30 °C, respectively. Sporulated Btt-Xd3 in nutrient broth medium enriched with salts transferred to fermentation medium containing soybean flour, glucose and salts. After fermentation, the mixture was dried in a spray dryer, and spore count of the powder product was determined as 1.6 × 1010 c.f.u. g-1. Moisture content, suspensibility and wettability of the formulation were determined as 8.3, 86% and 21 s, respectively. Lethal concentrations (LC50) of formulated Btt-Xd3 were determined as 0.15 × 105 c.f.u. ml-1 for larvae at laboratory conditions. LC50 values were also determined as 0.45 × 106 c.f.u. ml-1 at the field condition on larval stage. Our results showed that a new bio-pesticide developed from B. thuringiensis tenebrionis (Xd3) (Btt-Xd3) may be valuable as a biological control agent for coleopteran pests.

Arthrobacter pityocampae sp. nov., isolated from Thaumetopoea pityocampa (Lep., Thaumetopoeidae).

A bacterium (strain Tp2(T)) was isolated from a caterpillar of the pine processionary moth, Thaumetopoea pityocampa (Den. & Schiff.) (Lepidoptera: Thaumetopoeidae), a destructive pine forest pest. The bacterium is a Gram-stain-positive, red-pigmented coccus, oxidase-negative, nitrate-reducing, non-motile and non-spore-forming. Strain Tp2(T) was subjected to a taxonomic study using polyphasic approach that included morphological and biochemical characterizations, 16S rRNA gene sequence analysis, DNA-DNA hybridization, DNA G+C content analysis, comparative fatty acid profiles, and analyses of quinones and polar lipids. The 16S rRNA gene sequence of strain Tp2(T) revealed that Arthrobacter agilis DSM 20550(T) was the closest known strain (98% 16S rRNA gene sequence similarity). DNA-DNA hybridization of A. agilis DSM 20550(T) and strain Tp2(T) resulted in a DNA-DNA relatedness value of 11.9% (20.2% reciprocal). The DNA base composition of strain Tp2(T) was 69.5 mol%, which is consistent with the other recognized members of Actinobacteria that have a high G+C content in their genome. The polar lipid pattern of strain Tp2(T) consisted of diphosphatidylglycerol (major), phosphatidylglycerol and phosphatidylinositol and unknown glycolipids. The cellular fatty acids were anteiso C15:0 and anteiso C17:0 and the major menaquinone was MK-9(II-H2). The peptidoglycan type was A3α with an L-Lys-L-Thr-L-Ala3 interpeptide bridge. The above-mentioned characterization qualifies strain Tp2(T) as genotypically and phenotypically distinct from closely related species of the genus Arthrobacter with validly published names. Strain Tp2(T) is therefore proposed to represent a novel species of the genus Arthrobacter, described as Arthrobacter pityocampae sp. nov. The type strain is Tp2(T) ( = DSM 21719(T) = NCCB 100254(T)).

Hydrophobin genes of the entomopathogenic fungus, Metarhizium brunneum, are differentially expressed and corresponding mutants are decreased in virulence.

Hydrophobins are small, cysteine-rich, secreted proteins, ubiquitously produced by filamentous fungi that are speculated to function in fungal growth, cell surface properties, and development, although this has been rigorously tested for only a few species. Herein, we report identification of three hydrophobin genes from the entomopathogenic fungus, Metarhizium brunneum, and functional characterization of strains lacking these genes. One gene (HYD1/ssgA) encodes a class I hydrophobin identified previously. Two new genes, HYD3 and HYD2, encode a class I and class II hydrophobin, respectively. To examine function, we deleted all three separately, from the M. brunneum strain KTU-60 genome, using Agrobacterium tumefaciens-mediated transformation. Deletion strains were screened for alterations in developmental phenotypes including growth, sporulation, pigmentation, colony surface properties, and virulence to insects. All deletion strains were reduced in their ability to sporulate and showed alterations in wild-type pigmentation, but all retained wild-type hydrophobicity, except for one individual hyd3 mutant. Complementation with the wild-type HYD3 gene restored hydrophobicity. Each gene, present as a single copy in the genome, showed differential expression patterns dependent on the developmental stage of the fungus. When Spodoptera exigua (beet armyworm) larvae were treated with either conidia or blastospores of each hyd mutant, reductions in virulence and delayed mortality were observed as compared to WT. Together, these results suggest that hydrophobins are differentially expressed and may have distinct, but compensating roles, in conidiation, pigmentation, hydrophobicity, and virulence.

Brevibacterium pityocampae sp. nov., isolated from caterpillars of Thaumetopoea pityocampa (Lepidoptera, Thaumetopoeidae).

This work deals with the taxonomic study of a bacterium, strain Tp12(T), isolated from caterpillars of the pine processionary moth (Thaumetopoea pityocampa Denis & Schiffermüller, 1775; Lepidoptera, Thaumetopoeidae). The isolate was assigned to the genus Brevibacterium on the basis of a polyphasic taxonomic study, including morphological and biochemical characteristics, 16S rRNA gene sequence analysis, fatty acid analysis and DNA G+C content. The highest 16S rRNA gene sequence similarity to this isolate was approximately 96 %, with the type strains of Brevibacterium album and Brevibacterium samyangense. Cellular fatty acids of the isolate are of the branched type, with the major components being anteiso-C(15 : 0) and anteiso-C(17 : 0). The DNA G+C content was 69.8 mol%. Although the strain was related to B. album and B. samyangense according to 16S rRNA gene sequence analysis, it differed from any known species of Brevibacterium. Based on this evidence, the novel species Brevibacterium pityocampae sp. nov. is proposed, with strain Tp12(T) (=DSM 21720(T) =NCCB 100255(T)) as the type strain.