Introduction
Bacillus thuringiensis (Bt) is a gram-positive spore-forming bacterium with entomopathogenic properties. Bt can produce proteinaceous parasporal inclusion bodies during the sporulation phase (
Gill et al., 1992). The crystal proteins of Bt have been found to have a toxic activity toward a target organism. Bt is of special economic importance because its products play a growing role in the biological control of agricultural and forestry pests and insects that are vectors of human and animal diseases. This entomopathogenic property has led to a great interest in finding new Bt isolates, and consequently, growth within-species variation has been found. So far, more than 40000 Bt isolates have been isolated (
Zhang, 2001), and 82 different serovars have been reported (
Lecadet et al., 1999).
Although serotyping remains as a valuable classification of Bt strains, some other methods have been developed to analyze the protein composition of the crystals by polyacrylamide gel electrophoresis as well as the quick and easy characterization of toxin genes by PCR. These methods allow the analysis of a large number of samples, but the bioassay remains as an essential tool to determine the insecticidal activity or a wider spectrum of activity.
The purpose of this study was to characterize a new Bt strain and identify its cry genotype, including crystal protein composition, cry gene content, and the evaluation of their biological activity against a number of insects.
Methods
Bt strains and insects
Bt11 strain was stored in our laboratory, and HD-73 standard strains used in this study was obtained from the Chinese Academy of Agricultural Plant Protection Institute. Larvae of Spodopera exigua and Helicoverpa armigera were obtained from laboratory colonies and reared on the artificial diet.
Bioassays of insecticidal activity
A modified diet overlay assay method (
Zhao et al., 2003;
Guo, 2005) was employed to test the susceptibility of neonates to Bt11. In each cup, 0.1 mL Bt11 solution was applied and distributed over the diet surface. Cups were allowed to air dry for 1.5-2 h before infesting it with the neonates of
Helicoverpa armigera and
Spodoptera exigua. Ten neonates were transferred into each cup. The cups were covered with lids and held at (27±1)°C, (50±2)% RH, and a photoperiod of 16∶8 (L∶D) h for 4 d to determine the mortality or growth inhibition. The POLO program (
LeOra Software, 1997) was used for probit analysis of concentration-mortality response data (
Russell et al., 1977). Mortality was adjusted using Abbott’s formula (
Abbott, 1925) for each probit analysis.
Morphology observation and physiological and biochemical analyses of Bt11
Bt cultures were grown on a 1/2-LB medium (tryptone 0.5%, yeast extract 0.25%, sodium chloride 0.5%, pH7.0) at 30°C for 38 h with agitation until 70%-90% crystal off, with the number of crystal unit volume counted. Observation was made on the morphology of Bt11 strain staining reaction. The method was used to analyze the physiological and biochemical properties, following
Dai and Wang (1997) and
Yu (1990).
Growth characteristics
The wild-type strain Bt11 and the standard strain HD-73 were grown on a 1/2-LB medium at 30°C with agitation, and samples were collected every two hours, respectively. To examine the growth phase, the optical density at 600 nm (OD600) of appropriately diluted culture was measured using a spectrophotometer.
SDS-PAGE analysis
Cell extracts were prepared by resuspending cell pellets in 40 μL sterilized water and 40 μL 2×SDS sample buffer. Samples were loaded on a denaturing gradient (10% to 5%) arylamide gel, and the proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (
Sambrook et al., 1989).
Identification of cry-type genes by PCR-RFLP
The PCR primers in this work were designed following
Kuo and Chak (1996) and
Song et al. (1998). PCR was performed by predenaturing at 94°C for 4 min, followed by 30 cycles at 94°C for 1 min, 52°C for 1 min, and 72°C for 1 min, with the final extension at 72°C for 10 min. The genotype analysis was carried out by the PCR-RFLP following
Kuo and Chak (1996) and
Song et al. (1998).
Results
Insecticidal activity of Bt11 strain
The bioassay results indicated that the Bt11 strain was highly toxic against the neonates of Helicoverpa armigera and Spodoptera exigua (Table 1). The mortality of Helicoverpa armigera and Spodoptera exigua were observed as 100% at the concentration of 5.9×107 spores•mL-1.
Morphological observation
Under the light microscopy (1000×), the parasporal inclusions isolated from Bt11 had a diamondoid, spherical, and squarish morphology, and the spores appeared as oval (Fig. 1).
Physiological and biochemical characteristics
The physiological and biochemical characteristics of the Bt11 strain showed the similarity to those of Bacillus thuringiensis subsp. kurstaki (Table 2).
Growth characteristics of Bt11 strain
Compared with the growth curve of Bt11 strain and HD-73 strain (Fig. 2), the growth characteristics of the wild-type strain Bt11 showed no significant difference with those of the standard strain HD-73. Both of them could be observed in three distinct phases: lag phase after 0-2 h, logarithmic phase after 2-16 h, and stationary phase after 16 h.
SDS-PAGE analysis
SDS-PAGE analysis of the protein composition demonstrates that Bt11 strain contained several proteins ranging from 20 to 130 kDa (Fig. 3). Proteins with molecular masses of 35, 80, and 130 kDa were observed.
Identification of cry-type genes
DNA templates were used for PCR amplification with different universal oligonucleotide primers. The results of RFLP pattern analysis after different digestion of the PCR products are shown in Table 3 and Fig. 4. The sizes of the restriction fragments, the RFLP patterns of the PCR products, and the predicted cry-type genes are listed in Table 3. As a result, we detected cry1, cry1I, cry2, and cry9 genes in Bt11 strain.
Discussion
Insect pests cause a considerable crop loss in agricultural production. According to the FAO statistics, the economic loss that insect pests create every year reaches as high as 14% of the total amount of world agricultural production. Therefore, biological agricultural chemicals that are highly effective, safe, low in poison, low in residue, and low cost are to be developed.
Bacillus thuriniensis has a broad application prospect in the biological control of insect pests. Therefore, seeking for highly effective strains has a vital significance to the production of biological pesticides. The
Bacillus thuriniensis crystal has various shapes including diamondoid, long diamondoid, spherical, circle or ellipse, mosaic shape, amorphous (
Li et al., 1983;
Li et al., 1989), and triangle. In our research, we did not find the triangle parasporal crystal, but other parasporal crystals shapes could be seen obviously. It has been reported that the shapes of parasporal crystals of Bt were responsible for their insecticidal activity (
Li et al., 1983). Therefore, it is significantly vital that we study the formation, shape, and structure of the parasporal crystal of Bt and make further research on the relationship between toxicity and mechanism of Bt.
Wild
Bacillus thuringiensis strain Bt11 was preliminary classified as a subspecies of
Bacillus thuringiensis isolated from the soil samples in China, which further enriched the resources of Chinese Bt, simultaneously providing new resources of Bt strains for the development of new highly effective bioinsecticides. PCR is the most effective and rapid method used to identify the aimed DNA sequence (
Saiki et al., 1988), which has already been used to identify the genes coded for parasporal crystal proteins and forecast their insecticidal activity (
Ceron et al., 1994). In our research, the PCR method was applied to identify
cry-type genes in Bt11strain. The results indicated that the Bt11 strain simultaneously included
cry1Aa,
cry1Ab,
cry1Ia,
cry2Ab, and the
cry9Ea genes. Whether it includes other kinds of
cry genes besides the identified genes needs further researches.
It is reported that Cry1Aa has certain toxicity to the larvae of
Trichoplusia ni, and Cry1Ab has higher toxicity to both
Helicoverpa armigera and Trichoplusia ni larvae. However, both Cry1Aa and Cry1Ab are not so poisonous to
Spodoptera exigua larvae (
Dankocsik et al., 1990;
De Maagd et al., 1996;
Iracheta et al., 2000;
Liao et al., 2002). Cry2Ab has higher toxicity to
Bombyx mori and
Plutella Xylostella. Cry1Ia has higher toxicity to
Bombyx mori and
Plutella Xylostella, but low toxicity to
Spodoptera litura. The biological activity of Cry9Ea has not been reported. The results of our research indicated that the Bt11 strain has high insecticidal toxicity to
Spodoptera exigua,
Helicoverpa armigera, and
Trichoplusia ni larvae, which indicates that the Bt11 strain can harbor many kinds of
cry genes. In the future, we will clone all the
cry genes identified in Bt11 strain to provide beneficial gene resources for transgenetic plants as well as the construction of genetically engineered bacteria.
Higher Education Press and Springer-Verlag Berlin Heidelberg
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