Print version ISSN 1517-8382
Braz. J. Microbiol. vol.32 no.2 São Paulo Apr./June 2001
DETECTION OF CRY1 GENES IN BACILLUS THURINGIENSIS ISOLATES FROM SOUTH OF BRAZIL AND ACTIVITY AGAINST AANTICARSIA GEMMATALIS (LEPIDOPTERA:NOCTUIDAE)
Vera Lucia Bobrowski1*; Giancarlo Pasquali2; Maria Helena Bodanese-Zanettini3; Lidia Mariana Fiuza4
1Departamento de Zoologia e Genética, Instituto de Biologia, Universidade Federal de Pelotas, Pelotas, RS, Brasil. 2Centro de Biotecnologia do Estado do Rio Grande do Sul and 3Departamento de Genética, Instituto de Biociências, Universidade Federal do Rio Grande do Sul, Pelotas, RS, Brasil. 4Departamento de Microbiologia, Universidade do Vale do Rio dos Sinos, Pelotas, RS, Brasil.
Submitted: November 17, 2000; Returned to authors for corrections: February 05, 2001; Approved: May 25, 2001
The bacterium Bacillus thuringiensis (Bt) is characterized by its ability to produce proteic crystalline inclusions during sporulation. Cry1 protein has insecticidal activity and is highly specific to certain insects and not toxic to unrelated insects, plants or vertebrates. In this work, the patogenicity of twelve Bt isolates was tested against Anticarsia gemmatalis, one of the most important insect pests of soybeans. Spore-crystal complex was applied to the surface of artificial diets and the mortality of A. gemmatalis larvae was assessed seven days after each treatment. When compared to a control Bt isolate known by its high toxicity to A. gemmatalis larvae, four novel Bt isolates exhibited even higher toxic activities against the insect, resulting in more than 90% mortality. PCR was used to amplify DNA fragments related to known cry1 genes. Bt strains with high toxicity produced expected PCR products of around 280 bp, whereas non-toxic or low toxic strains did not produce any PCR product or showed amplified fragments of different sizes. Toxic Bt isolates also exhibited an expected protein profile when total protein extracts were evaluated by SDS-PAGE.
Key words: Bacillus thuringiensis, velvetbean caterpillar, Anticarsia gemmatalis, cry1 gene, d-endotoxin
Bacillus thuringiensis (Bt) Berliner is a Gram-positive, aerobic or facultative anaerobic entomopathogenic bacterium found in soil. These bacteria are characterized by their ability to produce crystalline protein inclusions during sporulation that have toxic activity against different invertebrates, especially insects (19,21,25). Bt spores can survive for several years, whereas the stability of the protein crystal is highly variable, lasting from a few days to several months depending on environmental conditions (25). The Bt crystal is composed of proteins called d-endotoxins, cry proteins or insecticidal crystal proteins (ICP) of molecular mass ranging from 25 kDa to 140 kDa (5,9). The morphology, size and number of parasporal inclusions vary among different Bt strains (22).
Many genes encoding toxin proteins have been cloned and sequenced, and are commonly named as cry or cyt genes. Many Bt strains contain multiple cry genes that are usually present on large plasmids (4,14). To date, over 100 cry gene sequences have been described and are classified in 22 families with different subclasses in relation to their amino acid composition (3,8). The toxic protein against lepidopteran, for instance, belong to the cry1, cry2 or cry9 groups (8). Within the cry1 protein group, there are around 10 different subclasses, each subclass has a specific range of activity against different lepidopteran insects (2,13,27).
Despite their classification, cry proteins have similar mechanisms of action. When ingested by insect larvae, the crystal protein is first dissolved in the alkaline midgut and proteolitically converted into smaller toxic polypeptides. These polypeptides bind to specific receptor sites on the apical microvilli of the insect midgut cells. An insertion of the toxin (or a part of it) occurs into the membrane after receptor interaction, forming a pore that causes osmotic swelling of the cell and subsequent lysis, leading to death of the insect (16,20,26).
The use of Bt as a commercial insecticide in agriculture for more than 40 years is based on its high specificity and efficiency to certain insect pests and on its non-toxicity to other unrelated insects, plants and vertebrates. These characteristics point to a clear advantage of Bt formulations over many chemical insecticides, especially when a particular pest is responsible for high losses in agriculture (4,9,15,19). The velvetbean caterpillar cause a lot of damage to soybeans world wide, determining great reductions of leaf areas and, consequently, lowering photosynthesis and productivity (24). Among the Bt strains known to be toxic to A. gemmatalis larvae is Bt kurstaki HD1, which is commercially available. This strain contains at least 5 cry genes including classes cry1 and cry2 (19). Together with strains Bt kurstaki HD73 and Bt thuringiensis 4412, that respectively contain cry1Ac and cry1B genes, HD1 is commonly used as a standard strain when evaluating new Bt isolates (19).
In this work, we selected isolates from south of Brazil with high efficiency against velvetbean caterpillar and confirm cry1 genes presence using PCR techniques and protein spore-crystal analysis.
MATERIALS AND METHODS
New Bt isolates were obtained from soil samples of different districts of the State Rio Grande do Sul (Brazil) for Polanczyk (1999). We used six isolates did not tested against Anticarsia gemmatalis named U68-5, U87-2, U90-1, U96-3, U98-1, U98-4. Strains Bt serovar thuringiensis (IP01), Bt serovar alesti (IP03), Bt serovar fukuokaensis (IP06), Bt serovar kurstaki (HD73), Bt serovar thuringiensis (4412), and Bt serovar kurstaki (HD1-Dipel) were obtained from WHO Collaborating Centre for Entomopathogenic Bacillus, Institut Pasteur (France).
Anticarsia gemmatalis larvae
Adult and larvae A. gemmatalis insects were obtained from the soybean crop plantations of south Brazil and colonies were maintained on artificial diets, prepared as described by Greene et al. (17), at 25 ± 1ºC with a photoperiod of 12:12 (L:D) and 70% relative humidity.
Culture conditions of Bt
Bt strains were grown in glycoside usual medium at 28º ± 1ºC under agitation of 180 rpm until complete autolysis was achieved according to Mahillon and Delcour (23). Spore-crystal complexes were harvested by centrifugation at 5,000 rpm for 20 min at 4ºC. To eliminate extracellular components, including b-exotoxins, known to accumulate in the cell culture supernatant (12), parasporal bodies and cell debris were washed twice in sterile-distilled water by centrifugation (5,000 rpm, 15 min). Pellets were suspended in 8 ml of sterile-distilled water. Cell number was monitored by counting in a Neubauer chamber under phase contrast microscopy (400X) and each suspension was diluted to achieve a title of 3 x 108 spore/ml.
The activity of different Bt isolates were screened among third-instar larvae of A. gemmatalis using a volume of 100 ml of Bt spore/crystal complex (3 x 108 cells/ml) according to Hernandez (18) were applied onto diet surface. In total, 24 larvae were tested for each isolate in this pre-selective assay. In a control group, Bt suspension was replaced by sterile-distilled water. Assays were maintained in a growth chamber at 25ºC ± 1ºC, 70% relative humidity and a photoperiod of 12:12 (L:D). Larval mortality was assessed after 7 days. Mortality of the control larvae reared on the toxin-free diet and under the same conditions was recorded and used to correct the test mortality with Abbot's formula (1).
The protein composition of the spore-crystal complex was analyzed by 10% SDS-PAGE (sodium dodecyl sulfate-polyacrilamide gel eletrophoresis). Gels were stained with Coomasie Blue. The molecular weight of proteins were estimated by comparasion with protein molecular weight standarts (High range, Gibco-BRL).
Cry1 gene related sequence detection by PCR
Bt strains were grown overnight at 30ºC in petri dishes containing solid culture media (3 g/l peptone, 5 g/l beef extract, 0.5 g/l yeast extract, 0.006 g/l MnCl2, 0.08 g/l CaCl2, 0.07 g/l MgCl2, 1.5% agar, on bi-destilled water). The presence of crystals was confirmed under phase contrast microscopy. A loopful of cells from a single colony was transferred to 0.1 ml of sterile bidistilled water in a microfuge tube. The bacterial suspension was frozen at -20ºC for 20 min and directly boiled for 10 min to lyse the cells. The resulting cell lysate was centrifuged briefly at 10,000 rpm and 15 ml of the supernatant was used as DNA sample for PCR. PCR mixtures were prepared using 300 ng of the specific cry1 gene primers: primer 1: 5'-TGTAGAAGAGGAAGTCTATCCA-3' (CJI-1) and primer 2: 5'-TATCGGTTTCTGGGAAGTA-3' (CJI-2) (9), 0.5 U Taq DNA Polymerase (CENBIOT Enzimas®), 0.2 mM dNTP's, 10 mM Tris, 50 mM KCl and 3 mM MgCl2. Amplification was performed in a thermal cycler (Perkin Elmer GeneAmp PCR System 2400) using a single denaturation step (2 min at 95ºC), followed by a 30-cycle program, with each cycle consisting of a denaturation step of 95ºC for 1 min; an anneling step of 52ºC for 1 min, and an extension step of 72ºC for 1 min. A final extension step of 72ºC for 5 min was also included. Ten ml samples from the PCR mixtures were electrophoresed on polyacrylamide gel and stained with ethidium bromide (0.5 ml/ml).
RESULTS AND DISCUSSION
With the objective of isolating new genes coding cry1 proteins lethal to A. gemmatalis larvae, 6 new Bt isolates obtained from soil samples of Rio Grande do Sul (Brazil) were analyzed for their "in vivo" effect and compared to the three known active Bt strains Bt serovar kurstaki HD1 - Dipel, Bt serovar kurstaki HD73 and Bt serovar thuringiensis 4412 from WHO Collaborating Centre for Entomopathogenic Bacillus (Institut Pasteur, France). A series of bioassays were performed by feeding A. gemmatalis larvae with artificial diets containing the Bt spore-crystal complex. As presented in Fig. 1, four (U87-2; U98-1; U98-4 and IP01) out of the 9 Bt isolates tested against A. gemmatalis larvae presented equal or higher mortality than HD1, HD73 and 4412 strains. Although the mortality of A. gemmatalis determined by Bt isolates IP03 and IP06 (respectively 33 % and 51%) were not equivalent to HD1, larvae development was severely reduced when compared to control insects (result not shown). According to others authors, such effect over larval development can consistently reduce plant damage (7,24). Corroborating these observations, diets of larvae treated with IP03 and IP06 were much less consumed than diets given to control larvae (data not shown). Neglectable effects over A. gemmatalis larvae were observed when isolates U68-5, U91-1 and U96-3 were assayed.
Cry1 proteins are known to be active against lepidopteran insects. In order to check for their presence in the new Bt isolates active against A. gemmatalis, total protein extracts were prepared from liquid bacterial cultures and assayed by SDS-PAGE. After Comassie blue staining, isolates IP01, IP06, U87-2 and U98-4 presented a clear band of around 135 kDa, similar to HD1 (Fig. 2). This is the expected weight for cry1 proteins as described by Schenpf et al. (27). Although we could not detect the protein band of 135 kDa with the highly toxic isolate U98-1, all isolates presenting this band demonstrated a mortality of over 90%, suggesting that it may be responsible for the observed activity.
All Bt strains tested in this work were also analyzed for the presence of gene sequences similar to cry1. To do so, synthetic oligonucleotides designed by Cerón et al. (9) were used in PCR. The designed primers are specific to cry1 genes, resulting in the amplification of a DNA fragment of around 280 bp after PCR. Results are illustrated in Fig. 3. Fragments with sizes ranging from 270 bp to 290 bp were observed when DNA from standard strains HD1, HD73 and 4412 (9) and from Bt isolates U87-2, U98-4, IP01 and IP06 were assayed. Isolates IP03 and U98-1 presented similar patterns of DNA amplification with two common DNA bands of approximately 900bp and 200 bp. An extra amplification product of around 1000 bp could also be visualized with the IP03 isolate. No amplification products could be detected after PCR using DNA from isolates U68-5, U91-1 and U96-3. The primers CJI-1 and CJI-2 used in the PCRs help to certify the presence of cry1 genes despite their classification (8). Bt kurstaki HD1 is known to present genes cry1Aa, cry1Ab and cry1Ac; Bt kurstaki HD73 harbours the cry1Ac gene; and the strain 4412 contains the cry1B gene (11,19). The PCR strategy used in this work confirmed the results obtained by Cerón et al. (9) and is a proof for the proposed usefulness of the designed primers. All three standard strains showed single amplification products ranging from 272 bp to 292 bp after PCR with the proposed primers. Although the absence of the 270-290 bp fragment does not prove the inexistence of cry1 genes, our positive PCR results with U87-2, U98-4, IP01 and IP06 strongly indicate that these isolates harbor sequences similar to cry1 genes. Similar results were obtain to strains very effective against Spodoptera frugiperda using these primer, all strain present positive results for cry1 genes (28). Sequencing of the amplified products and cross-hybridizations are underway to confirm this conclusion.
Once again, the amplification of the 270 bp to 290 bp DNA products after PCR may be correlated with the toxicity of the Bt isolates found in our bioassays. With the exception of U98-1, all isolates demonstrating activities superior to 90% against A. gemmatalis larvae clearly presented this PCR product. Absence of this band for isolates U68-5, U91-1 and U96-1 correlates with their absent or low toxic effect against larvae of A. gemmatalis. Bt isolates containing genes type cry1Aa, cry1Ab and cry1Ac independently or in combination were more efficient in bioassays against Spodoptera sp. and Manduca sexta than isolates containing either cry1C or cry1D (10). Since the primers used in the PCRs cannot distinguish among cry1 genes, the presence of different cry1 types in the Bt isolates tested may explain their toxicity differences.
Bt serovar kurstaki HD1 is being used as a standard strain in most of the pathogenicity tests developed over lepidopteran species (6,24,28). Considering the HD1 toxic activity described against lepidopteran insects like Plutella xylostella (28), Spodoptera sp., Diatrea sp., Helicoverpa sp. (6) and Anticarsia sp. (24; this work), the highly active Bt isolates here presented are very promising for new insecticide formulations against A. gemmatalis. More exciting, the results of gene amplification and protein profile here described for the new isolates strongly point them as good sources of genes for the generation of transgenic plants resistant to insect pests.
We thank Dr. Marguerite Lecadet, Institut Pasteur, for providing Bacillus thuringiensis strains, Dr. Rogério F.P. da Silva and his technicians at the Faculdade de Agronomia, UFRGS, for helping with the bioassays. This work was supported by the Secretary of Science and Technology, Estado do Rio Grande do Sul, Brazil. Vera L. Bobrowski acknowledges CAPES for her doctoral fellowship.
Detecção de genes cry1 em isolados de Bacillus thuringiensis do sul do Brasil e sua atividade contra Anticarsia gemmatalis (Lepidoptera:Noctuidae)
A bactéria Bacillus thuringiensis (Bt) é caracterizada pela sua habilidade de produção de corpos paraesporais durante a esporulação. As proteínas cry1 têm atividade inseticida e são altamente específicas para certos insetos e não são tóxicas para outros insetos, plantas e vertebrados. Neste trabalho, a patogenicidade de doze isolados foram testados contra Anticarsia gemmatalis, um dos mais importantes insetos pragas da cultura da soja. Para tanto, foi aplicada sobre a superfície da dieta uma suspensão de esporo-cristal e a mortalidade de lagartas de Anticarsia gemmatalis foi avaliada sete dias após a aplicação. Quando comparado com uma linhagem de Bt controle, conhecido pela sua alta toxicidade para lagartas da soja, quatro novos isolados exibiram atividade tóxica superior, acima de 90% de mortalidade. Foi utilizada a técnica de PCR para amplificar fragmentos de DNA de regiões codificantes de genes cry1. Os isolados de Bt com alta mortalidade produziram produtos de PCR de tamanho esperado, em torno de 280 pb, isolados não tóxicos ou pouco tóxico não produziram qualquer produto de PCR ou mostraram fragmentos amplificados com padrões diferentes do esperado. Os isolados de Bt com alta atividade contra a lagarta da soja mostraram a presença de proteínas com tamanho de aproximadamente 150 kDa, quando o extrato protéico total foi analisado em SDS-PAGE.
Palavras-chave: Bacillus thuringiensis, lagarta da soja, Anticarsia gemmatalis, gene cry1, d-endotoxina
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* Corresponding author. Mailing address: Universidade Federal de Pelotas, IB, Departamento de Zoologia e Genética, Campus Universitário, s/n, Caixa Postal 354. 96010-900, Pelotas, RS, Brasil, Tel: (+5553) 275-7316, Fax: (+5553) 275-7169, E-mail: firstname.lastname@example.org