Research Report

Identification of Bacillus thuringiensis Isolates Highly Toxic to Plutella xylostella  

Zhaodong Liang1,2 , Hao Zhong1,2 , Yan Zhou2 , Youzhi Li1
1. College of Life Science and Technology, Guangxi University, Nanning, 530004, P.R. China
2. Hainan Institute of Tropical Agricultural Resources (HITAR), Sanya, 572025, P.R. China
Author    Correspondence author
Bt Research, 2011, Vol. 2, No. 1   doi: 10.5376/bt.2011.02.0001
Received: 12 Jun., 2011    Accepted: 09 Aug., 2011    Published: 18 Aug., 2011
© 2011 BioPublisher Publishing Platform
This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Preferred citation for this article:
Liang et al., 2012, Identification of Bacillus thuringiensis Isolates Highly Toxic to Plutella xylostella, Bt Research, Vol.2, No.1 1-8 (doi: 10.5376/bt.2011.02.0001)

In this research, we employed 31 Bacillus thuringiensis isolates from the soils collected in the Liang Shui Natural Reserve of Heilongjiang province to identifyhighly toxic to Plutella xylostella, an important Lepidopteran pest in agriculture. The results showed that ten of thirty one Bacillus thuringiensis isolates have high toxicity to Plutella xylostella via the bioassay. Microscopic observation for ten seleted isolates was carried out; the results confirmed that these isolates can generate bi-diamond crystal protein which might be the crystalline toxin. The further genotypic analysis by PCR and SDS-PAGE indicated that the genotypes of the selected 10 Bt isolates were consistent with the genotype of cry1 class toxin genes which can generate crystal protein 130 kD in molecular weight. Whilst, the growth curves of the ten isolates exhibited that the bacteria would be in the logarithmic growth phase after culturing for 2 hours to 14 hours, in the stationary phase after culturing 16 hours or 30 hours and in the decline phase after culturing 32 hours. Among them, strains S2685-1 and S2737-3 were further accredited to generate the bi-diamond crystal protein of about 130 kD in molecular weight after being cultured for 22 hours and 20 hours, respectively; toxin expressed by the former stabilizes after 26 hour in culture, whereas toxin expressing levels of the later gradually increases and stabilizes after 22 hours.

The trials of median lethal concentration (LC50) and median lethal time (LT50) of two dominant isolates, S2685-1 and S2737-3, for Diamondback moth (Plutella xylostella) were carried out in this research, and the results showed that the LC50 and LT50 for S2685-1 to diamondback moth were 17.113 ng/mL and 37.343 hours, respectively; while for S2737-3 were 25.782 ng/mL and 36.825 hours, respectively. In contrast, LC50 and LT50 of the reference strain HD-73 to the diamondback moth were 15.414 ng/mL and 35.917 hours. It is obvious that S2685-1 and S2737-3 exhibit strong insecticidal activities in the median lethal concentration and median lethal time against Plutella xylostella. In conclusion, the results of this study that the Bt isolates, S2685-1 and S2737-3, have more prominent insecticidal activities, which might be applied as potential alternative strains in Bt bio-pesticide products as initiate strain instead of standard strain HD-73 strain.

Bacillus thuringiensis; Plutella xylostella; Bt toxin; Bioassay

Bacillus thuringiensis (Bt) is one of the most widely used microbial insecticides and it has toxic activities to many insects. The insecticide principle is that the Bt parasporal crystal protein (ICPs) acts as a toxin via intestinal juice enzyme of the insect, which could lead to insect death (Marco and Porcar, 2012). As Bt are generally considered harmless to the environment and human beings, Bt always attracts scientists’ attention as an ideal insecticide.

Because of the specificity of Bt to insect, Bt bio-pesticides have a narrow insect spectrum and poor efficacy sustainability, especially when Bt is used as the only bio-pesticide product in some areas for a long term, which has made some agricultural pest have insecticide resistance to Bt. It is reported that Plutella xylostella has showed resistance to Bt (Ferre et al., 1991). It is obvious that Using combinations of different Bt strains is an important way in delaying the insect resistance. Therefore, identification of Bacillus thuringiensis strains that are highly toxic to Plutella xylostella becomes a hot spot in the Bt research field.
Bt HD-73 is a well-known strain which is highly toxic to Lepidopteran pests. Adang et al (1985) reported that they have found a gene, cry1Ac encoding a 133 kD insecticidal crystal protein in Kurstaki HD-73. Because the insecticidal crystal protein encodedby cry1Ac is highly toxic to many Lepidopteran pests, Bt HD-73 strain becomes one of the Bt strains that are widely used in bio-pesticide products. Lots of researches about HD-73 were carried out (Wu et al., 2007; Chen et al., 1999). There is no doubt that using Bt strains alone in long-term will lead to insect resistance or gradual accumulation of the resistance. Both searching for alternative strains and using a combination of Bt strains are a promising methods to avoid the resistance or to delay the resistant accumulation. Thus, it is imperative to identify Bacillus thuringiensis strains that are highly toxic to Lepidopteran pests.
In this research, we isolated 31 wild Bt strains from Liang Shui Natural Reserve of Heilongjiang province as test materials. Via morphology observation, the analysis of biological activity, growth curves, SDS-PAGE and genotype as well as the bioassay, we hope to identify the highly toxic Bacillus thuringiensis strains, which can become the alternative strains used in Lepidopteran bio-pesticides.
1 Results and Analysis
1.1 The biological activity assay of Bt strains to Plutella xylostella
We collected protein crude extracts from 31 Bt strains to assay the biological activity against Plutella xylostella. After deleting 21 Bt strains whose 48 hours corrected mortalities are lower than 50%, we obtained 10 Bt strains with high toxicity toward Plutella xylostella, the corrected mortalities of which are between 70%~100%. The serial numbers of these ten strains are S2685-1, S2737-3, S2796-2, S2809-1, S2852-1, S2852-2, S2852-3, S2852-4, S2852-5 and S2853-1, respectively (Table 1). We chose these ten Bt strains as the candidate strains for successive tests; we carried out further study on biological characteristics and ran trials on median lethal concentration (LC50) and median lethal time (LT50). 
Table 1 The corrected mortality of the 10 candidate Bt strains
1.2 Observation of parasporal crystal proteins of 10 candidate Bt strains
Ten candidate Bt strains were activated, cultured in 1/2 LB medium, and shaked at 220 r/min at 30℃. During the sporulation period, we selected a few cells, dyeing with phenol fuchsin and then observed with optical microscope and scanning electron microscope. In this article, we only listed parts of Bt strains’ shape. Observing in optical microscope, all of the S2796-2, S2809-1, S2852-2, and S2852-3 strains generated white spores and red parasporal crystals (Figure 1). After the observation of scanning electron microscope, all of the S2796-2, S2809-1, S2852-2, and S2852-3 strains produced diamond crystal protein.

Figure 1 Micrographs of some Bt strains observed under optical microscope

1.3 Growth curves analysis of 10 candidate Bt strains
Via growth curves analysis of 10 candidate Bt strains, we found that the growth status of these ten candidate Bt strains were similar to standard strain HD-73 in LB culture medium. The lag phase conditions of 10 candidate Bt strains presented was not obvious. Bt strains grew quickly during the first 4~20 h and entered into logarithmic growth phase. They entered stationary phase 20~30 hours after culturing. At this time, the bacterial liquid’s OD600 value was about 5.0. After 32 hours, the strains came into decline phase.

Figure 2 Growth curve of the standard strain HD-73 and 10 Bt strains

1.4 Crystal protein SDS-PAGE analysis of 10 candidate Bt strains
Ten candidate Bt strains were cultured in G-Tris medium, and shaked at 220 r/min at 30℃ for 4 days. Later, the crystal protein of Bt strains was extracted for SDS-PAGE analysis. All of them can produce 130 kD crystal protein (Figure 3).
Figure 3 SDS-PAGE profiles of parasporal crystal proteins of 10 Bt strains during sporulation

1.5 Genotypic analysis of 10 candidate Bt strains
As all 10 candidate Bt strains could express 130 kD crystal protein, we conferred these strains may contain cry1 toxin genes. We chose 2 pairs of universal primers 5Kun2/5Kun2 and 5Kun3/5Kun3 for genotype identification using the PCR reaction. The amplification results were 1.6 kb and 1.45 kb, respectively (Figure 4), which showed that these 10 candidate Bt strains all carried cry1 toxin genes.

Figure 4 The electrophoretic analysis of 10 Bt strains with specific PCR

1.6 Bioassay of median lethal concentration (LC50) and median lethal time (LT50) of 10 candidate Bt strains
We diluted each insecticidal protein solution according to a certain proportion and employed the sterile water and HD-73 protein as the negative control group and positive control group, respectively. And then we counted the Plutella xylostella’s death status after 72 hours. The median lethal concentration (LC50) and median lethal time (LT50) were estimated by SPSS 19.0 software (Table 2; Table 3). The statistics results found that insecticidal activity of S2685-1 and S2737-3 were relatively close to the standard strain HD-73.

Table 2 The toxicity probit analysis of LC50 of 10 Bt strains against Plutella xylostella (Lepidoptera)

Table 3 The toxicity probit analysis of LT50 of 10 Bt strains against susceptible Plutella xylostella (Lepidoptera)

On the other hand, when we analyzed the median lethal concentration (LC50) results, the results displayed that the median lethal concentration (LC50) of the standard strain HD-73 was 15.414 ng/mL, and LC50 of S2685-1 and S2737-3 were close to LC50 of HD-73, whereas the LC50 of S2685-1 and S2737-3 was 17.113 ng/mL and 25.782 ng/mL, respectively. Thus, S2685-1 and S2737-3 have very conspicuous insecticidal toxicity. Further researches about the toxicity need to be carried out.
The analysis of median lethal time (LT50) was also carried out. It was shown that the median lethal time (LT50) of the standard strain HD-73 was 35.917 h. The LT50 of S2685-1 and S2737-3 were 37.343 h and 36.825 h, respectively, which were close to LT50 of HD-73. Thus, it was obvious that S2685-1 and S2737-3 were close to HD-73’s insecticidal time.
1.7 The ICPs change of Bt stains S2685-1 and S2737-3 with the cultured time
We analyzed Bt strains S2685-1 and S2737-3 by SDS-PAGE and observed how their ICPs change with the cultured time (Figure 5).
As shown in Figure 5A, Bt generated the crystal protein about 22 kD in molecular weight after being cultured for 4 hours. The expression levels gradually increased, and it stabilized after 22 hours. There were crystal proteins about 35 kD after 12 hours and the expression levels gradually increased, which was basically stabilized after 18 hours. In the same situation, the expression levels of the crystal protein about 37 kD appeared and was stable at 28 hours and at 48 hours, respectively; meanwhile, the crystal protein about 130 kD was produced and was stable at 22 hours and at 26 hours, respectively. As shown in Figure 5B, Bt generated the crystal protein about 130 kD after 20 hours; the expression levels gradually increased, and was stabilized after 22 hours.
Figure 5 SDS-PAGE profiles of parasporal crystal proteins of Bt strain S2685-1 and S2737-3


2 Discussions
Plutella xylostella is a worldwide pest on vegetables. It is also a common pest in the field in China. Using Bt as a biological control method to Plutella xylostella is safe to human beings, livestock and the environment. Therefore, Bt is very popular among consumers and has wide application prospects (Abdel-Razek et al., 2006).

However, Plutella xylostella becomes less and less sensitive to Bt because of the continuously accumulated resistance of Plutella xylostella and the singleness of the highly toxic strains; besides, the insecticidal activity of some highly toxic strains gradually decreases (Zuo, et al., 2002). The lack of highly toxic strains is still a restrictive factor to extensive utilization of Bt (Tan et al., 2006). Using different types of alternative strains is beneficial for reducing the selection pressure. It is good for delaying the formation of the pest resistance, which will be an effective method to extend the service life of Bt strains and to manage pests resistance.
In this research, we identified that Bt strains S2685-1 and S2737-3 were two wild Bacillus thuringiensis strains that are highly toxic to Lepidopteran pest. We considered that they could be used as alternative strains in pesticides against Lepidopteran pest.
3 Methods and Materials
3.1 Strains and medium
In this experiment, we used 31 wild Bt strains which were isolated from the soil of the Liang Shui Natural Reserve of Heilongjiang province, and the serial numbers are as follows: S2331-1, S2343-2, S2384-1, S2386-4, S2404-3, S2412-2, S2472-3, S2480-1, S2490-1, S2540-1, S2663-2, S2685-1, S2689-1, S2718-3, S2734-1, S2737-3, S2790-2, S2796-2, S2809-1, S2850-1, S2852-1, S2852-2, S2852-3, S2852-4, S2852-5, S2853-1, S2886-2, S2944-3, S2966-1, S2966-2, and S2968-1. LB medium was used to culture Bt strains. G-Tris medium was used for accelerating spore and parasporal crystal generation in Bt strains (Aronson and Thompson, 1971). BP medium was used for analyzing how ICPs change with the cultured times of Bt strains (Xie et al., 2009). Bt strains were cultivated at 30℃ temperature.

3.2 Observation of parasporal crystal by optical microscope
We observed parasporal crystal Bt strains according to Xie et al (2010) and Liu et al (2011)’s method. This method used carbolic acid to dye cells in spore stage of Bt and observed the morphology with an oil-immersion lens of an optical microscope (Nikon YS100).
3.3 Observation of parasporal crystal by Scanning Electron Microscope (SEM)
According to Zhang et al (2010)’s method, we took some Bt strain, generated spore liquid, placed in the EP tubes, repeated washing, suspending, took 2 μL suspension liquid to the clean glass slide, fumigated by osmium acid, fixed up on scanning electron microscope’s (HITACHI S-3400N) object stage, vacuumed, and dyed with heavy metal. Finally, we observed, and photographed.
3.4 Determination of growth curve of Bt strain
The strains were activated and inoculated by Zhang et al (2010)’s method, and shaked with 230 r/min at 30℃, and then 1% bacterial liquid was transferred into LB medium, which was shaked with 230 r/min at 30℃. l mL bacterial liquid was taken into EP tubes every 2 hours. HD-73 was used as the control group and LB medium was used as the blank control. The OD600 nm value was measured, and when the OD value was more than 0.8, we diluted it once.

3.5 The protein extraction of strains and SDS-PAGE analysis
According to Jiang et al (2008)’s method, Bt strains were cultured in 200 mL of G-Tris medium for 96 hours, and then they were centrifuged for 15 mins at 8,000 r/min and 4℃. Then the precipitate was washed with 1 mol/L of NaCl, centrifuged for 10 min at 12000 r/min, and 4℃ again. Discarding the supernatant, the precipitation was dissolved in 30 mL phosphate buffer solution. Then the mixture was treated with ultrasonic (85 w) (model VC-130, Sonics and Materials Inc, USA) for 30 mins, followed by centrifuging for 10 mins at 12000 r/min, and at 4℃ again. Then the precipitate was washed with 1 mol/L of NaCl, centrifuged for 10 mins at12000 r/min and 4℃ for another time. The last precipitate was dissolved in 30 mL  phosphate buffer solution, treated with ultrasonic (85 w) for 30 mins. At last, the precipitate was collected, dissolved in the sterile water, and stored in the -20℃ refrigerator. Procedure of SDS-PAGE followed Liu et al (2010)’s method.

3.6 The determination of parasporal crystal content
The determination of parasporal crystal content followed Bradford (1976)’s method in this study.

3.7 Artificial breeding of Plutella xylostella
The sensitive population of Plutella xylostella was supplied by Hainan Institute of Tropical Agricultural Resources. The artificial breeding of Plutella xylostella was performed by the natural resources. The Plutella xylostella used in this study have been fed for more than 30 generations. The experimental larva age was uniform and larvae were in good growth conditions that reached the bioassay requirement.
3.8 SDS-PAGE analysis on the change ICPs with the time
The strains were activated, transferred into BP medium, cultured and shaked at 230 r/min at 30℃. Some spore-crystal mixtures for SDS-PAGE analysis were harvested every two hours (Xie et al., 2009; Liu et al., 2010).

3.9 Identification of Cry genotype of Bt strains
DNA templates for PCR were prepared following Yu et al (2006)’s method. PCR reaction system contains 2 μL of template DNA, 1 μL of each primer (add dNTP to 250 μmmol/L), 5 μL of 10×PCR buffer,0.5 μL (4 U/μL) of Taq polymerase, and double distilled water to reach a final volume of 50 μL. The PCR was performed with the following procedures: pre-denaturation at 94℃ for 5 min, then followed by 30 cycles of denaturing at 94℃ for 1 min, annealing at 53℃ for 1 min and extension at 72℃ for 3 min, and a final extension at 72℃ for 10 min. At last, PCR products were examined by 1% agarose gel electrophoresis. The primer pairs were K5un2/K3un2 and K5un3/K3un3 which can be used to identify the cry1 gene (Kuo and Chak, 1996).

3.10 Biological activity assay
Based on Crespo et al (2008)’s method, the strains were activated and cultured in 1/2 LB medium at 30℃ shaking at 220 r/min. The supernatant was harvested by centrifugation at 4℃ and 12,000 r/min for 10 minutes. The supernatant was washed by 1 mol/L of NaCl. The precipitation was harvested by centrifugation at 4℃ and 12,000×g for 10 minutes. The distilled water was added into the precipitation to make the protein extracts of strains. The bioassay measurement of Plutella xylostella was preformed (Zhang et al., 2009). Some fresh, uniform sized leaves of cabbage grown in the lab were picked out, washed carefully, and immersed into strains’ protein extracts for about 20 s. Extra attention was given so that each surface of the leaf should be immersed into the protein extract for about 10 s. Then the leaves needed to be dried naturally in bioassay room. Then they were put into Petri dishes. Each dish was placed with 30 Plutella xylostella. Sterile water were used as the blank control. The room temperature would remain (25±1)℃ and 60% relative humidity, under photoperiod of 14 hours light/10 hours dark. 72 hours later, we observed the growth conditions of lavae and calculated the corrected mortality.

3.11 The determination of median lethal concentration (LC50)
We used leaf dipping method to determine the median lethal concentration (LC50) of Plutella xylostella (Xie et al., 2009; Zhang et al., 2009; Crespo et al., 2008). The protein extracts of the strains were diluted into several concentrations. The original concentration was needed to do a series of tests. Once we found that the larval mortality was high with certain concentration, we would set this concentration as the original concentration. And then we diluted the original concentration to five different concentrations. Each concentration was set up with three parallel experiment groups with 30 Plutella xylostella larvae in each group. The LC50 of Plutella xylostella was performed with these six concentrations. Sterile water and HD73 protein was used as negative control group and positive control group, respectively. The number of dead larvae was recorded after 72 hours and the corrected mortality was calculated. The LC50 value was estimated by SPSS software 19.0.

3.12 The determination of median lethal time (LT50)
We referred to Yang et al (2007)’s method to perform the determination of LT50 of Plutella xylostella. The lethal concentration of LC98 was used in this study. The number of dead larvae was recorded every 8 hours. Each treatment was set up with 3 parallel experiment groups. The data was recorded until reaching 72 hours. We also employed sterile water and HD73 protein as negative control group and positive control group, respectively. The corrected mortality in different periods was calculated and then we estimated the LT50 value by SPSS software 19.0.

Authors’ contribution
ZDL conducted all the research work in this paper; YZ and HZ took part in the results analysis and manuscript modification; YZL conceived the experimental design, analyzed the data and modified the manuscript. All authors have read and approved the final manuscript.
This work was initiated by the project of China National Bt Collection Initiative. The authors in this paper appreciated Dr. Fang Xuanjun’s guidance and his valuable suggestions on article correcting as well as Li Zhonggang from Hainan Institute of Tropical Agricultural Resources for providing Plutella xylostella. Part of the research was performed in Prof. Li Youzhi’s laboratory in Guangxi University. All the authors appreciated two anonymous reviewers for their useful critical comments and revising advice to this paper. The mention of trade names or commercial products in this paper is solely for the purpose of providing specific information and does not imply recommendation or endorsement involved in this study.

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