Isolation and identification of bacteria from the invasive pest Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) and evaluation of their biocontrol potential

As an alternative to chemical insecticides, gut bacteria of insects could be used to control insect pests. In this study, bacteria associated with Tuta absoluta, an invasive species that has developed resistance to chemical insecticides, were isolated, and their potential for pest control was investigated. We isolated 13 bacteria from larvae of the pest and identified the isolates on the basis of their morphological, physiological, biochemical, and molecular characteristics as Bacillus thuringiensis (Ta1-8), Staphylococcus petrasii (Ta9), Citrobacter freundii (Ta10), Chishuiella changwenlii (Ta11), Enterococcus casseliflavus (Ta12), and Pseudomonas tremae (Ta13). A laboratory screening test at 109 cfu/ml showed that B. thuringiensis (Bt) isolates caused more than 90% mortality after 3 days. Among the isolates, Bt-Ta1 showed the highest mortality in a short time. The LC50 and LC90 values for Bt-Ta1 were estimated to be 1.2 × 106 and 2 × 109 cfu/ml, respectively. Detailed characterization of Bt-Ta1 revealed that it is one of the serotypes effective on lepidopterans and contains the genes cry1Aa, cry2Aa, and vip3Aa, which encode lepidopteran toxic proteins. Bt-Ta1 isolate has been shown to have the potential to be used in the integrated management of Tuta absoluta. (AU)

Isolation and identification of bacteria from the invasive pest Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) and evaluation of their biocontrol potential.

As an alternative to chemical insecticides, gut bacteria of insects could be used to control insect pests. In this study, bacteria associated with Tuta absoluta, an invasive species that has developed resistance to chemical insecticides, were isolated, and their potential for pest control was investigated. We isolated 13 bacteria from larvae of the pest and identified the isolates on the basis of their morphological, physiological, biochemical, and molecular characteristics as Bacillus thuringiensis (Ta1-8), Staphylococcus petrasii (Ta9), Citrobacter freundii (Ta10), Chishuiella changwenlii (Ta11), Enterococcus casseliflavus (Ta12), and Pseudomonas tremae (Ta13). A laboratory screening test at 109 cfu/ml showed that B. thuringiensis (Bt) isolates caused more than 90% mortality after 3 days. Among the isolates, Bt-Ta1 showed the highest mortality in a short time. The LC50 and LC90 values for Bt-Ta1 were estimated to be 1.2 × 106 and 2 × 109 cfu/ml, respectively. Detailed characterization of Bt-Ta1 revealed that it is one of the serotypes effective on lepidopterans and contains the genes cry1Aa, cry2Aa, and vip3Aa, which encode lepidopteran toxic proteins. Bt-Ta1 isolate has been shown to have the potential to be used in the integrated management of Tuta absoluta.

Microencapsulation of an indigenous isolate of Bacillus thuringiensis by spray drying.

In this study, microencapsulation by spray drying was performed to protect spores and crystals of an indigenous isolate of Bacillus thuringiensis Se13 from environmental stress. The effects of wall material, inlet temperature, and outlet temperature on microencapsulation of Bt-Se13 were investigated using Taguchi's orthogonal array. The most suitable wall material determined as maltodextrin DE10. The optimum inlet and outlet temperatures of spray drier were determined as 160 °C and 70 °C, respectively. The number of viable spores, mean particle size, wetting time, percentage of suspensibility and moisture content of the product produced under optimum conditions were determined as 8.1 × 1011 cfu g-1, 13.462 µm, 25.22 s, 77.66% and 7.29%, respectively. As a result of efficiency studies on Spodoptera exigua in the laboratory conditions, the LC50 was determined as 1.6 × 104 cfu mL-1. Microencapsulated Bt-Se13 based bio-pesticide may be registered for the control of S. exigua and can be tested against other lepidopterans which share the same environment.

Biodiversity and pathogenicity of bacteria associated with the gut microbiota of beet armyworm, Spodoptera exigua Hübner (Lepidoptera: Noctuidae).

In order to find an effective and environmentally friendly biocontrol agent against Spodoptera exigua, we isolated and identified a total of 15 different bacterial species belonging to phyla Firmicutes, Proteobacteria and Bacteroidetes. According to the phenotypic, genotypic and phylogenetic properties, bacterial isolates were identified as Bacillus cereus (Se1), Lysinibacillus macroides (Se2), Pseudomonas geniculata (Se3), Paenibacillus tylopili (Se4), Staphylococcus succinus (Se5), Acinetobacter soli (Se6), Chryseobacterium indologenes (Se7), Bacillus toyonensis (Se8), Serratia marcescens (Se9), Paenibacillus amylolyticus (Se10), Paenibacillus xylanexedens (Se11), Enterobacter ludwigii (Se12), Bacillus thuringiensis (Se13), Bacillus thuringiensis (Se14) and Lysinibacillus fusiformis (Se15). Screening of bacterial isolates for insecticidal potential was conducted at 109 cfu ml-1 bacterial concentration. The highest larvacidal effect was obtained with Bacillus thuringiensis Se13 with 100% mortality. In the dose response experiments performed with this bacterium, the median lethal concentration (LC50) was estimated as 7.5 × 104 cfu ml-1 against 3rd instar larvae of the pest at 10 days post treatment. The median lethal time (LT50) value of 109 cfu ml-1 bacterial concentration was also determined as 1.59 days. Phase-contrast and scanning electron microscope studies exhibited that B. thuringiensis Se13 produced different shape and size crystals (bipyramidal, cubic and spherical). Phylogenetic analysis of cry1 and cry2 gene content of this isolate displayed that B. thuringiensis Se13 had 99% homology with cry1Ac and cry2Aa, respectively. Finding from this study indicated that B. thuringiensis Se13 appears to be a promising microbial control agent for use against S. exigua.

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.