Effect of the protein elicitor AMEP412 from Bacillus subtilis artificially fed to adults of the whitefly, Bemisia tabaci (Genn.) (Hemiptera: Aleyrodidae)
- 25 Downloads
In a previous study, we identified a protein elicitor AMEP412 from Bacillus subtilis, which could trigger plant defense response and induce systemic acquired resistance. In the present study, the toxicity of AMEP412 against the whitefly Bemisia tabaci (Genn.) (Hemiptera: Aleyrodidae) was reported. The purified protein samples at different concentrations (1, 5, 10, 20, 40, and 80 μg/ml) caused 17–96% mortality 2 days post-artificial adult feeding, where the median lethal concentration (LC50) was calculated by 15.57 μg/ml. The stability test showed that AMEP412 had a good stability against thermo and natural degradation. The fluorescence localization assay revealed that AMEP412 could be taken into the whitefly adult body and localized in the gut. Based on the feature of this protein, AMEP412 was probably digested by gut proteases and led to the release of hydrophobic fragments in the insect gut. It was deduced that these hydrophobic peptides could insert themselves into the cell membrane and form lytic pores, leading to content leakage and cell lysis, followed by insect death. This study sheds a light on the toxic effect of AMEP412, which not only enriched the function of the protein elicitor but also provided a new choice for the biocontrol of whiteflies.
KeywordsBiocontrol Bacillus subtilis Bemisia tabaci Insecticidal protein Stability
Degrees of freedom
Green fluorescent protein
Lethal concentration to kill 50% population
Yeast malt extract
The whitefly Bemisia tabaci (Genn.) (Hemiptera: Aleyrodidae) is an important insect pest to several crops, including vegetables, cotton, and ornamentals (Byrne and Bellows 1991). It damages crops by feeding on phloem and transmitting plant viruses, leading to tremendous losses in agricultural production and national economies (Reitz 2007; Navas-Castillo et al. 2011). The main strategy of controlling the whitefly was mainly based on chemical insecticides. However, whitefly had developed resistance to those excessively and frequently applied insecticides (Wang et al. 2009; Luo et al. 2010; Houndété et al. 2010; Vassiliou et al. 2011; Kontsedalov et al. 2012). Considering this, exploring insecticides with novel mode of action should be a new focus.
Compared to chemical insecticides, proteins with insecticidal activity acted in different modes, which lead to insect resistance with a relatively low probability. There were many reports about insecticidal proteins for pest control. The most famous example was Cry toxin of Bacillus thuringiensis (Bt), which was developed to Bt-transgenic cotton and effectively controlled the lepidopteran pest larvae. The widespread planting of Bt-transgenic cotton significantly reduced the usage of chemical pesticides. However, none of the Cry toxins was reported effective on whiteflies. In recent years, several researchers focused on screening of insecticidal proteins from plants rarely infested by whiteflies. Das et al. (2009) reported a mannose binding lectin from leaves of Allium sativum that could effectively inhibit whiteflies. Jin et al. (2012) expressed the Pinellia ternata agglutinin in chloroplasts and conferred resistance against whiteflies. Shukla et al. (2016) identified an insecticidal protein (Tma12) from an edible fern and expressed it in transgenic cotton, which showed a high level of resistance to whiteflies. Although these insecticidal proteins showed a good potential for whitefly control, for the low extraction rate or the limit of transgenic plant, there were no matured commercial products in the market.
In a previous study, a protein elicitor (AMEP412) was isolated from Bacillus subtilis, which could interact with plant and induce serials of defense reactions (Shen et al. 2019). The study aimed to determine the toxic effect of AMEP412 and provide a new choice for the biocontrol of whiteflies. The localization of this protein in the insect adults was detected by fluorescent labeling. Moreover, the stability of this protein against thermal and natural degradation treatment was also determined.
Materials and methods
Bacterial strain and growth condition
Bacillus subtilis strain BU412, deposited in China Center for Type Culture Collection (CCTCC M2016142), was used for the production of AMEP412. Yeast malt extract (YME) medium (Schaad et al. 2001) was used for growing the strain.
Preparation of AMEP412 protein
The protein AMEP412 was prepared by serials of purification steps from the supernatant of B. subtilis BU412 culture, following Shen et al. (2019). The culture was centrifuged to obtain the supernatant. The supernatant was filtered through 0.22-μm membrane to remove residues and then applied to AKTA Purifier system (Amersham Biosciences). The purification procedures included anion exchange chromatography and size exclusion chromatography, using Source 15Q 4.6/100 PE column and Superdex 75 10/300 GL column. The fraction of the target protein was collected and adjusted to 1 mg/ml for further determination.
The artificial feeding of whiteflies followed the method described by Upadhyay et al. (2011). Adult whiteflies (1–2 days old) were aspirated from plant leaves into 50-ml specimen tubes. The aqueous artificial diet consisted of 5% yeast extract, and 30% sucrose (Blackburn et al. 2005) was mixed with different concentrations of AMEP412 (1, 5, 10, 20, 40, and 80 μg/ml). The artificial diet without AMEP412 was set as control. Diet (100 μl) was added between two stretched layers of UV-sterilized parafilm on the tube cap, and then, the tube cap was reversed to cover the tube, keeping the diet at inner side. At least 50 whitefly adults were taken in each tube and the experiment was performed three times. The bioassays were carried out for 2 days, and mortality was recorded by counting the dead whitefly adults at the bottom of the tube.
In our previous study, AMEP412 showed a good thermo stability as a protein elicitor. To test its thermo stability as insecticidal protein, AMEP412 was subjected to 95 °C for 15 and 30 min, respectively. Furthermore, the natural degradation of AMEP412 was also determined. The protein was placed in Eppendorf tube at room temperature for 24 and 48 h, respectively. Then, the insecticidal activity of the protein (60 μg/ml) was determined after being exposed to different conditions as described above, with the untreated protein as control. Each treatment was repeated three times.
In vivo localization
As AMEP412 was purified from natural products, green fluorescent protein (GFP) tag was not suitable for labeling the protein. Instead, fluorescein isothiocyanate (FITC) was an ideal tag to label AMEP412. FITC-labeled AMEP412 was prepared, following the previous method (Shen et al. 2019). AMEP412 was incubated by FITC in carbonate buffer (0.05 M, pH 9.0) for 12 h at 4 °C. The FITC and protein mixture was loaded onto a Superdex 75 10/300 GL column for separation according to the different molecular sizes. Subsequently, 60 μg/ml FITC-protein was used as insect diet for feeding bioassays as described above. Dead insects were collected, washed by sterile water, and then monitored by fluorescent microscope (Olympus BX60) with an excitation wavelength of 495 nm for the localization of the tested protein.
The mortality data for whitefly adults in above assays were analyzed by one-way ANOVA and the means were compared by Tukey’s HSD test at α = 5%. The median lethal concentration (LC50) value was calculated by the probit analysis on the SPSS program (version 18).
Results and discussion
The calculated LC50 of AMEP412 against B. tabaci
Lower 95% FL
Upper 95% FL
Slope ± SE
2.10 ± 0.28
AMEP412 showed a good stability against thermo and natural degradation, which was probably attributed to its stable spatial structure. As mentioned in a former study (Shen et al. 2019), AMEP412 formed polymers in aqueous environment, which enhanced its spatial structure against external interferences like thermo and natural degradation. Moreover, the polymerization state could also hide some enzyme digestion sites, leading to a certain resistance to enzymes. This feature will definitely broaden the application areas and improve the insecticidal effects.
AMEP412 localized in whitefly guts
Insecticidal activity of AMEP412 against whitefly showed a good stability against thermo and natural degradation. The fluorescent localization revealed insect guts as its function position. The possible mechanism was discussed based on partial assay result and the feature of AMEP412. In future research, the exact mechanism will be clarified by studying the interaction between AMEP412 and whitefly gut cells. Moreover, the toxicity as insecticidal activity of AMEP412 against other insects will be investigated to fully understand its anti-insect spectrum.
We thank the reviewers whose comments and suggestions helped us to improve this manuscript.
QL was the primary contributor to this study with helpful advice from BZ and YS. KY helped in the data analysis and revised the manuscript. All authors read and approved the final manuscript.
This work was financially supported by Natural Science Foundation of Heilongjiang Province of China (QC2017020), Postdoctoral Science Foundation of Heilongjiang Bayi Agricultural University, Innovation Training Program for College Students in Heilongjiang Province (201910223048) and Heilongjiang Bayi Agricultural University Support Program for San Heng San Zong (ZRCQC201904).
Ethics approval and consent to participate
Consent for publication
The authors declare that they have no competing interests.
- Das S, Banerjee S, Majumder P, Mondal HA, Saha P, Chakraborti D (2009) A mannose binding lectin from leaves of Allium sativum effective against whitefly, and process for its preparation. Indian patent 228783:1-22Google Scholar
- Schaad NW, Jones JB, Chun W (2001) Laboratory guide for the identification of plant pathogenic bacteria. The American Phyto-pathological Society, St. Paul, MN, USAGoogle Scholar
- Shukla AK, Upadhyay SK, Mishra M, Saurabh S, Singh R, Singh H, Thakur N, Rai P, Pandey P, Hans AL, Srivastava S, Rajapure V, Yadav SK, Singh MK, Kumar J, Chandrashekar K, Verma PC, Singh AP, Nair KN, Bhadauria S, Wahajuddin M, Singh S, Sharma S, Omkar URS, Ranade SA, Tuli R, Singh PK (2016) Expression of an insecticidal fern protein in cotton protects against whitefly. Nat Biotechnol 34:1046–1051CrossRefGoogle Scholar
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.