A growth-based screening method for entomolopathogenic bacteria against Spodoptera frugiperda

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Bioassay data were analyzed using probit analysis with PoloPlus software (LeOra Software 2002, Berkeley, CA, USA). The mortality results obtained from insect bioassays in this paper are values of the adjust mortality, and the calculation formula as follow: adjust mortality (%) = (mortality of treatment － mortality of control) / (1- mortality of control) × 100%. Statistical analyses were also performed using SPSS statistical software v12.0 (SPSS, Inc., Chicago, IL, USA). Mortality, colony diameter and enzyme activity were analyzed using one-way analysis of variance (ANOVA), followed by Tukey’s HSD tests. Pearson correlation analysis was employed to evaluate the relationship between protease, chitinase activity of different bacteria and their toxicity to S. frugiperda. A strong linear correlation was indicated by a Pearson correlation coefficient R of > 0.6, corresponding to an R2 value exceeding 0.36. Table 1, Growth and enzyme activity of different bacteria isolates from field S. frugiperda on milk and chitin medium. Means in the same column followed with the same letter are not significantly different at the 5% level (Tukey’s HSD tests). The data represent the mean of replicates with standard error (SE). Table 2, Insecticidal activity of different bacteria isolates from field S. frugiperda against the pest. The concentration of bacterial suspension for bioassay are 1×1010 cfu/mL. The mortality results obtained from insect bioassays in this paper are values of the adjust mortality. The data represent the mean of replicates with standard error (SE). Means in the same column followed with the same letter are not significantly different at the 5% level (Tukey’s HSD tests). Fig. 2, Correlation analysis between virulence of pathogenic bacteria to S. frugiperda and bacterial growth rate. The mortality results obtained from insect bioassays in this paper are values of the adjust mortality. Table S2, Growth characteristics and toxicity of different bacteria strains isolated in the field. The mortality results obtained from insect bioassays in this paper are values of the adjust mortality. The data represent the mean of replicates with standard error (SE). Data of the genera Serratia and Pediococcus are presented in Table 1 and 2. Fig. S1, Genera level (A) and species level (B) composition of S. frugiperda pathogenic bacteria in different regions of Jiangsu Province.

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1 Determination of growth, extracellular activity of protease and chitinase of bacterial isolates Milk medium (500 mL of sterilized skim-milk mixed with 500 ml of 4% agar solution, heated at 65 ℃ at least 30 min) and chitin medium (0.7 g K2HPO4, 0.3 g KH2PO4, 0.5 g MgSO4·7H2O, 0.01 g Fe2(SO4)3, 1% (w/v) colloid chitin, 20 g agar, 1000 mL distilled water, pH 7.0-7.2) was prepared according to method previously described (Zheng et al., 2011; Xu et al., 2015). 20 mL liquid medium was added to each dish (90mm diameter) and then solidified. 1×1010 cfu/mL pathogenic bacteria were inoculated in plates under aseptic conditions. Each dish was inoculated symmetrically at 3 points, and each isolate was repeatedly inoculated in 3 dishes (The inoculation position on medium in dish has been shown in Fig. 1). After 3 days culture in an incubator (RH > 90%, 28 ℃) for 72h, the 3 colonies on each dish were observed, and the clear transparent circle (hydrolytic circle) around the colony was judged to be an enzyme with corresponding degradation activity. At the same time, the diameter (including hydrolytic circle) of each colony in the plate was measured and recorded, and thus 9 data could be obtained for each bacterial isolates. Quantitative detection of enzyme activity was conducted with the same samples as above, and there was 3 repeats for each bacterial isolate. The protease and chitinase activity of all the bacterial isolates were tested with detection kits from Nanjing JC Detect Biotechnologies Co., Ltd. (Nanjing, China) . Absorbance (OD) was measured at a wavelength of 450 and 540 nm using a spectrophotometer, and the activity of protease and chitinase were calculated using a standard curve. 2 Pathogenicity evaluation of bacterial isolates against S. frugiperda The insecticidal activity of different bacterial isolates against S. frugiperda larvae were tested in order to link virulence and growth characteristics and thus establish new screening method. The bacterial suspension was prepared and inoculated on two LB solid plates respectively, and then cultured in incubator at 28 ℃ for 48 h, after that, the bacteria on the plate were mixed into 10 mL aseptic water and stored at 4 ℃. At the same time, the concentration of the bacterial solution was determined by plate counting method. Bioassay was conducted in 24-well culture plate, 900 μL of artificial food was added to each well of the plate, after solidification of the food, 100 μL 1×1010 cfu/mL bacterial suspension was added to the surface of food in each well, and dried at room temperature, sterilized water treatment were as control. One neonate or second-instar larva of S. frugiperda was then put into every well of a plate, and each treatment repeated 3 times (1 plate as 1 repeat). The mortality were checked at 2, 4, 7, 10 and 13 days later after treatment.

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