Investigating internal bacteria of Spodoptera littoralis (Boisd.) (Lepidoptera: Noctuidae) larvae and some Bacillus strains as biocontrol agents

Spodoptera littoralis (Boisd.) (Lepidoptera: Noctuidae) is one of the most destructive pests of several vegetables and fruits worldwide. In spite of various control methods, this pest has still continued to cause significant damage. In this study, the culturable bacterial flora of S. littoralis was determined. New isolates from S. littoralis, as well as 12 different Bacillus isolates belong to 5 species that were previously isolated from different pests, were tested on S. littoralis larvae. In total, 9 bacteria were characterized based on their morphological, biochemical, physiological, and molecular characteristics. The bacterial flora of S. littoralis was determined as Flavobacterium sp. (SL1), Klebsiella pneumonia (SL2), Enterobacter sp. (SL3), Enterobacter sp. (SL4), Klebsiella sp. (SL5), Serratia marcescens (SL6), Pseudomonas aeruginosa (SL7), Acinetobacter baumannii (SL8), and Staphylococcus sp. (SL9). The insecticidal activity tests were performed on the third-instar larvae of S. littoralis. SL1 and SL5 from S. littoralis caused the highest mortalities with 67% and 77%, respectively. Among previously isolated Bacillus isolates, Bacillus thuringiensis subsp. kurstaki (MnD) and B. thuringiensis subsp. kurstaki (BnBt) were found to be the most effective, causing 100% mortality within 10 days after treatment. A concentration-response test was conducted with these isolates and it was found that the isolate MnD was more effective than BnBt. Therefore, further bioassay experiments were conducted with the isolate MnD and results were discussed with respect to the biocontrol potential of the bacterial isolates.

Investigating internal bacteria of Spodoptera littoralis (Boisd.) (Lepidoptera: Noctuidae) larvae and some Bacillus strains as biocontrol agents

Spodoptera littoralis (Boisd.) (Lepidoptera: Noctuidae) is one of the most destructive pests of several vegetables and fruits worldwide. In spite of various control methods, this pest has still continued to cause significant damage. In this study, the culturable bacterial flora of S. littoralis was determined. New isolates from S. littoralis, as well as 12 different Bacillus isolates belong to 5 species that were previously isolated from different pests, were tested on S. littoralis larvae. In total, 9 bacteria were characterized based on their morphological, biochemical, physiological, and molecular characteristics. The bacterial flora of S. littoralis was determined as Flavobacterium sp. (SL1), Klebsiella pneumonia (SL2), Enterobacter sp. (SL3), Enterobacter sp. (SL4), Klebsiella sp. (SL5), Serratia marcescens (SL6), Pseudomonas aeruginosa (SL7), Acinetobacter baumannii (SL8), and Staphylococcus sp. (SL9). The insecticidal activity tests were performed on the third-instar larvae of S. littoralis. SL1 and SL5 from S. littoralis caused the highest mortalities with 67% and 77%, respectively. Among previously isolated Bacillus isolates, Bacillus thuringiensis subsp. kurstaki (MnD) and B. thuringiensis subsp. kurstaki (BnBt) were found to be the most effective, causing 100% mortality within 10 days after treatment. A concentration-response test was conducted with these isolates and it was found that the isolate MnD was more effective than BnBt. Therefore, further bioassay experiments were conducted with the isolate MnD and results were discussed with respect to the biocontrol potential of the bacterial isolates.

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  • Lyd6 B. thuringiensis Lymantria dispar (L.) (Lepidoptera: Lymantriidae) Demir et al., 2012
  • Lyd7 B. thuringiensis L. dispar (L.) (Lepidoptera: Lymantriidae) Demir et al., 2012
  • Lyd8 B. thuringiensis L. dispar (L.) (Lepidoptera: Lymantriidae) Demir et al., 2012
  • Ar1 B. circulans Anoplus roboris (Suffr.) (Coleoptera: Curculionidae) Demir et al., 2002
  • Ar4 B. sphaericus A. roboris (Suffr.) (Coleoptera: Curculionidae) Demir et al., 2002
  • MnD B. thuringiensis subsp. kurstaki Malacosoma neustria (L.) (Lepidoptera: Lasiocampidae) Sevim et al., 2012
  • Xd3 B. thuringiensis subsp. tenebrionis Xyleborus dispar (F.) (Coleoptera: Scolytidae) Sezen et al., 2008
  • As3 B. cereus Amphimallon solstitiale (L.) (Coleoptera: Scarabaeidae) Sezen et al., 2005
  • Mm2 B. thuringiensis Melolontha melolontha (L.) (Coleoptera: Scarabaeidae) Sezen et al., 2007
  • Mm5 B. sphaericus M. melolontha (L.) (Coleoptera: Scarabaeidae) Sezen et al., 2007
  • Mm7 B. weihenstephanensis M. melolontha (L.) (Coleoptera: Scarabaeidae) Sezen et al., 2007
  • BnBt B. thuringiensis subsp. kurstaki Balanius nucum (L.) (Coleoptera: Curculionidae) Sezen and Demirbağ, 1999 each replicate were placed on the lettuce in the plastic boxes for 10 days. Three replicates of each treatment group were used, each containing a single bacterial isolate and 10 third-instar larvae.
  • Influence of larval stages and diets on the effect of bioassays More detailed bioassay experiments were conducted with the isolate MnD because of its high virulence compared to the isolate BnBt. For the bioassays on different larval stages, the density of the bacterial cells was adjusted to 1.89 (1.8 × 10 9 cfu/ mL) at OD 600 and 1 mL of bacterial suspension of the isolate MnD was prepared as described above (Moar et al., 1995). After that, lettuce leaves (approximately 10 cm 2 ) were contaminated with the bacterial suspension and individually placed in plastic boxes (30 cm in length and 18 cm in width). Finally, 10 larvae belonging to the first-, second-, third-, and fourth-instar for each replicate were put into these plastic boxes. Three replicates of each treatment group were used, each containing a single concentration of isolate MnD and 10 larvae belonging to different development stages. The mortalities of larvae were checked on day 10. For the effect of different diets on bioassays, the bacterial suspension of isolate MnD was prepared as described above, and freshly collected leaves (approximately 10 cm 2 ) of different diets [chard (Beta vulgaris var. cicla L.), parsley (Petroselinum crispum Mill.), corn (Zea mays L.), bean (Phaseolus vulgaris L.), cabbage (Brassica oleracea var. capitata L.), and lettuce (Lactuca sativa L.)] were contaminated with 1 mL of bacterial suspension. After that, 10 third-instar larvae of the pest for each replicate and the different diets were placed together in each plastic box
  • (30 cm in length and 18 cm in width). Three replicates of each treatment group were used, each containing isolate MnD, different diets, and 10 third-instar larvae. For the control group of all experiments, lettuce leaves were saturated with sterile PBS. All boxes used in all bioassay experiments were kept at 25 °C and 60% relative humidity with a 12:12 photoperiod. After the different diets were eaten completely, fresh untreated leaves were provided for the larvae for the remainder of the 10 days of bioassays. The mortalities of larvae were checked on day 10 after putting the larvae in plastic boxes including contaminated diets with bacterial suspensions. Data analysis of bioassays Mortality data were corrected by Abbott’s formula (Abbott, 1925). The data were subjected to analysis of variance
  • (ANOVA) and subsequently to least significant difference (LSD) multiple comparison tests to compare test isolates with each other and the control group with respect to mortality. S. littoralis and Bacillus isolates were separately evaluated. The effects of different developmental stages of S. littoralis and different diets were also analyzed by ANOVA, followed by LSD multiple comparison post hoc testing with respect to mortality of the pest. Concentrationresponse testing was analyzed by one-way ANOVA. All analyses were performed using SPSS 15.0. GenBank accession numbers of bacterial isolates The GenBank accession numbers for the partial sequence of the 16S rRNA gene sequences for the isolates SL1, SL2, SL3, SL4, SL5, SL6, SL7, SL8, and SL9 are JQ066774, JQ066775, JQ066776, JQ066777, JQ066778, JQ066779, JQ066780, JQ066781, and JQ066782, respectively. Results
  • Isolation and identification of bacteria from Spodoptera littoralis A total of 9 bacteria were isolated from living and dead S. littoralis larvae. Colonies were observed in different colors on nutrient agar. Eight isolates were smooth-round, and only 1 isolate (SL7) was wavy-round. Eight gramnegative bacteria and 1 gram-positive bacterium were determined, and none of them formed spores. Only 1 bacterium (SL9) was isolated from the dead larvae. Other morphological properties of the isolates are given in Table The biochemical characteristics of the bacterial isolates varied depending on the isolate. An API20E test was used for gram-negative bacteria and an API50CH test was used for gram-positive bacteria. Tables 3 and 4 summarize the biochemical properties of the bacterial isolates. Growth of the bacterial isolates at different pH levels, NaCl concentrations, and temperatures also varied depending on the isolate. Although all isolates grew at pH 10, only SL4 and SL8 grew at pH 3. As all isolates grew in 3% NaCl, the increment of the concentration influenced the bacterial growth. SL9 was the only isolate that grew in 15% NaCl concentration. Moreover, none of the isolates could grow at 10 or 50 °C. Other physiological characteristics of the bacterial isolates are given in Table 5. We also sequenced approximately 1.350 bp of the 16S rRNA gene for each isolate to confirm the isolate’s identification. Based on all the identification tests and sequencing analysis, isolates from S. littoralis were identified as Flavobacterium sp. (SL1), Klebsiella pneumonia (SL2), Enterobacter sp. (SL3), Enterobacter sp. (SL4), Klebsiella sp. (SL5), Serratia marcescens (SL6), Pseudomonas aeruginosa (SL7), Acinetobacter baumannii (SL8), and Staphylococcus sp. (SL9) (Table 6). Phylogenetic analysis of the 16S rRNA genes also supports this identification (Figure 1). Virulence of the bacterial isolates Bacterial isolates that were obtained from S. littoralis larvae produced different mortalities in comparison to each other and the control group (F = 36.17; df = 9, 20; P < 0.05). The highest mortalities were obtained from isolates SL1 and SL5 with 67% and 77%, respectively, within 10 Table The morphological characteristics of the bacterial isolates from S. littoralis larvae. Isolates Tests Colony color Shape of colonies Shape of bacteria Gram stain Spore stain Source Growth in NB* SL1 Yellow Smooth-round Bacillus – – Healthy larvae Turbid SL2 Cream Smooth-round Bacillus – – Healthy larvae Turbid SL3 Dark cream Smooth-round Bacillus – – Healthy larvae Turbid SL4 Cream Smooth-round Bacillus – – Healthy larvae Turbid SL5 Cream Smooth-round Bacillus – – Healthy larvae Turbid SL6 Red Smooth-round Bacillus – – Healthy larvae Turbid SL7 Green Wavy- round Bacillus – – Healthy larvae Turbid SL8 Dark cream Smooth-round Coccobacillus – – Healthy larvae Turbid SL9 Yellow Smooth-round Coccus + – Dead larvae Turbid *NB: Nutrient broth. Table The biochemical characteristics of the bacterial isolates from S. littoralis larvae based on conventional and API20E bacterial identification system. Tests Isolates SL1 SL2 SL3 SL4 SL5 SL6 SL7 SL8 SL9 Catalase + + – + – + + + + Oxidase + – – – – – + – + Starch hydrolysis – – – + – + – – – β-Galactosidase + + + + + – – + + Arginine dihydrolase – – + – – + + – – Lysine decarboxylase – + – + + – – – + Ornithine decarboxylase – – + + + – – – – Citrate utilization – + + + + + + – + H 2 S – – – + + – – – – Urease – + + – – – – – + Tryptophan deaminase – – – – + – – – – Indole + – – – + – – + – Acetoin – + + + + – – – + Gelatinase + – – – + + – + – Glucose – + + + + – WP* – + Mannitol – + + + WP – – – + Inositol – + – + WP – – – + Sorbitol – + – + WP – – – + Rhamnose – + + + – – – – + Saccharose – + + + WP – – – + Melibiose – + – + WP + + – + Amygdalin – + + + WP – – – + Arabinose – + + + WP + + – + *WP: Weak positive. days after treatment. The other mortalities ranged from 3% to 57%. Only isolate SL3 caused statistically the same mortality as the control (Figure 2a). The Bacillus species isolated from different pest species caused different mortalities in comparison to the control group (F = 35.96; df = 12, 26; P < 0.05). There was also a significant difference among treatments. Among Bacillus isolates, the highest mortalities were obtained from isolates MnD and BnBt with 100% mortality within 10 days after application. Other mortalities ranged from 30% to 77% (Figure 2b). Statistical differences were found between the MnD and the BnBt with respect to mortality after application of different concentrations (F = 208.79; df = 2, 24; P < 0.05) (Figure 3). The MnD isolate was applied to different development stages of S. littoralis larvae at a concentration of 1.89 × 10 9 cfu/mL. There was a significant difference among the development stages of the pest with regard to mortality (F = 38; df = 4, 10; P < 0.05). It was found that the secondinstar larvae were more resistant than those in the other development stages (Figure 4). There was a significant difference among the chard, parsley, corn, bean, cabbage, and lettuce diets with regard to larval mortality after application of the isolate MnD (F = 07; df = 6, 14; P < 0.05). It was found that S. littoralis larvae were more susceptible to the isolate MnD when lettuce and beans were used in the bioassay (Figure 5). Discussion To date, all bacterial species determined in this study have been isolated from various insect species, except for Acinetobacter baumannii (Inglis et al., 2000; Dugas et Abbott WS (1925). A method of computing the effectiveness of an insecticide. J Econ Entomol 18: 265–7.
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Turkish Journal of Agriculture and Forestry-Cover
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