Molecular detection of nematicidal crystalliferous Bacillus thuringiensis strains of Iran and evaluation of their toxicity on free-living and plant-parasitic nematodes

The characterization of nematode-effective strains and cry genes in the Iranian Bacillus thuringiensis (Bt) collection (70 isolates) is presented. Characterization was based on PCR analysis using 12 specific primers for cry5, cry6, cry12, cry13, cry14, and cry21 genes encoding proteins active against nematodes, crystal morphology, and protein band patterns as well as their nematicidal activity on root-knot nematode (Meloidogyne incognita) and two free-living nematodes (Chiloplacus tenuis and Acrobeloides enoplus). PCR results with primers for these genes showed that 22 isolates (31.5%) contain a minimum of one nematode-active cry gene. Strains containing the cry6 gene were the most abundant and represent 22.8% of the isolates. Bt strains harboring cry14 genes were also abundant (14.2%). cry21 and cry5 genes were less abundant, found in 4.2% and 2.8% of the strains, respectively. In total, six different nematode-active cry gene profiles were detected in this collection. Four isolates did not show the expected PCR product size for cry5, cry6, and cry21 genes; they might contain potentially novel insecticidal crystal protein genes. Twenty-two Bt isolates containing nematode-active cry genes were selected for preliminary bioassays on M. incognita. Based on these bioassays, four isolates were selected for detailed bioassays. Isolates YD5 and KON4 at 2 x 10(8) CFU/mL concentrations showed 77% and 81% toxicity on M. incognita, respectively. The free-living nematodes C. tenuis and A. enoplus were more susceptible and the highest mortality was observed within 48 h of incubation at all of the concentrations tested. Maximum mortality was recorded for isolates SN1 and KON4 at 2 x 10(8) CFU/mL concentrations and resulted in 68% and 77% adults deaths of C. tenuis and 68% and 72% for A. enoplus, respectively. Our results showed that PCR is a useful technique for toxicity prediction of nematicidal Bt isolates.     

RFLP analysis of cry1 and cry2 genes of Bacillus thuringiensis isolates from India

The PCR-RFLP method has been useful for detection of known genes and identification of novel genes. In the present study, degenerate primers were designed from five groups of cry1 genes for PCR-RFLP analysis. Bacillus thuringiensis (Bt) isolates from different regions were evaluated for PCR amplification of various cry1 genes using newly designed primers and cry2 genes using reported primers. PCR analysis showed an abundance of cry1A genes and especially cry1Ac genes in isolates from all regions. RFLP analysis revealed the presence of multiple cry1A genes in isolates from central and southern regions. Unique digestion patterns of cry1A genes were observed in isolates from each region. Few of the isolates represented a digestion pattern of cry1A genes that did match to any of the known cry1A genes. RFLP analysis suggested an abundance of cry2Ab along with a novel cry2 gene in Bt isolates from different regions of India. Sequence analysis of the novel cry2 gene revealed 95% sequence identity to cry2Ab and cry2Ah genes. Phylogenetic analysis revealed that the novel cry2 gene could have diverged earlier than the other cry2 genes. Our results encourage finding of more diverse cry2 genes in Bt isolates. Rarefaction analysis was used to compare cry1A gene diversity in isolates from different soil types. It showed a higher degree of cry1A gene diversity in isolates from central region. In the present study, we propose the use of novel degenerate primers for cry1 genes and the PCR-RFLP method using a single enzyme to distinguish multiple cry1A and cry2 genes as well as identify novel genes.     

5种中国苏云金芽孢杆菌的伴孢 晶体蛋白基因分析

利用聚合酶联反应(PCR)和聚丙烯酰胺凝胶电泳(SDS-PAGE)技术分析了5种中国苏云金杆菌制剂菌株的伴孢晶体蛋白及其基因组成。结果发现,5种菌株均含有cry1Aa和/或c和/或d和/或b基因,只有Bt+Virus菌株含有cry1Ab基因,cry1A基因编码的伴孢晶体蛋白分子量约为130 kD;仅有JS-Bt C菌株含有cry1B基因,其编码的伴孢晶体蛋白分子量约为138 kD;除HB Bt C菌株外,其余4个菌株均含有cry2Aa和/或b基因,这类基因编码分子量为70 kD的伴孢晶体蛋白;所有5个菌株都含有cry1I基因,其编码的伴孢晶体蛋白分子量应为81.2 kD,但实验中未曾检测到cry1I基因的表达;所有的菌株都不含有cry1C和cry1D基因。     

Efficient expression of vip184DeltaP gene under the control of promoters plus Shine-Dalgarno (SD) sequences of cry genes from Bacillus thuringiensis

AIMS: To compare vip184DeltaP gene expression time course and Vip184 protein yield under the control of promoters and Shine-Dalgarno (SD) sequences of vip184, cry3A and cry1A gene from Bacillus thuringiensis respectively. METHODS AND RESULTS: Derived from the shuttle vector pHT3101, recombinant plasmids pHPT3, pHTP3A(Delta)P and pHTP1A(Delta)P were constructed with the native vip184 gene and the vip184(Delta)P gene, either under the control of promoters and SD sequences of cry3A or cry1A genes. When the above plasmids were transformed into an acrystalliferous B. thuringiensis strain Cry(-)B, their expression time course were consistent with those of vip184, cry3A and cry1A gene respectively. The maximum yields of Vip184 protein were increased when under the control of promoters plus SD sequences of cry3A and cry1A gene. CONCLUSIONS: The results showed that both cry3A and cry1A promoter/SD sequence combinations were able to enhance synthesis of Vip184 and change its expression time course. SIGNIFICANCE AND IMPACT OF THE STUDY: Both cry3A and cry1A promoter/SD systems offer a method for improving the expression efficacy of the vip184 gene in B. thuringiensis and it is possible to co-express the vip184 gene and cry genes and accumulate Vip184 in the form of inclusion bodies by these systems in order to construct novel useful B. thuringiensis engineered strains.     

排除法PCR加变性梯度凝胶电泳鉴别未知基因

通过鉴别未知的cry4亚组基因来确定排除法PCR加变性梯度凝脉电泳新方法。方法：应用排除法PCR的组基因和亚组基因引物扩增杀蚊毒素基因,cry4。这些引物是根据已知的cyr4基因的共有或特有DNA片段来设计的。组基因引物扩增产物被用于变性梯度凝胶电泳。平行变性梯度凝胶是由8％的聚丙烯酰胺加上20％到80％的变性剂组成。结果：已知的和未知的cry4来组基因被组基因引物扩增的产物在变性梯度凝胶电泳被分离开。尽管它们之间仅有两个碱基对不同（T在位置224和G在位置394）。应用组基因和亚组基因引物扩增,新发现的基因可被分类到次亚组基因水平。从5个苏云金芽孢杆菌亚菌中发现三个未发表的cry4亚组基因。结论：排除法PCR加变性梯度凝胶电泳是一个高度敏感、特异性和可靠性强的新方法,可用于鉴别各种未知的亚组基因。     

Mining new crystal protein genes from Bacillus thuringiensis on the basis of mixed plasmid-enriched genome sequencing and a computational pipeline

We have designed a high-throughput system for the identification of novel crystal protein genes (cry) from Bacillus thuringiensis strains. The system was developed with two goals: (i) to acquire the mixed plasmid-enriched genomic sequence of B. thuringiensis using next-generation sequencing biotechnology, and (ii) to identify cry genes with a computational pipeline (using BtToxin_scanner). In our pipeline method, we employed three different kinds of well-developed prediction methods, BLAST, hidden Markov model (HMM), and support vector machine (SVM), to predict the presence of Cry toxin genes. The pipeline proved to be fast (average speed, 1.02 Mb/min for proteins and open reading frames [ORFs] and 1.80 Mb/min for nucleotide sequences), sensitive (it detected 40% more protein toxin genes than a keyword extraction method using genomic sequences downloaded from GenBank), and highly specific. Twenty-one strains from our laboratory's collection were selected based on their plasmid pattern and/or crystal morphology. The plasmid-enriched genomic DNA was extracted from these strains and mixed for Illumina sequencing. The sequencing data were de novo assembled, and a total of 113 candidate cry sequences were identified using the computational pipeline. Twenty-seven candidate sequences were selected on the basis of their low level of sequence identity to known cry genes, and eight full-length genes were obtained with PCR. Finally, three new cry-type genes (primary ranks) and five cry holotypes, which were designated cry8Ac1, cry7Ha1, cry21Ca1, cry32Fa1, and cry21Da1 by the B. thuringiensis Toxin Nomenclature Committee, were identified. The system described here is both efficient and cost-effective and can greatly accelerate the discovery of novel cry genes.     

Detection and Identification of cry1I Genes in Bacillus thuringiensis Using Polymerase Chain Reaction and Restriction Fragment Length Polymorphism Analysis

A polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) method for detection and identification of cry1I genes from Bacillus thuringiensis (Bt) was established. Based on the analysis of conserved regions of the cry1I genes, 2 oligonucleotide primers were designed to amplify a 665-bp fragment of the genes. The amplification products were digested with restriction endonuclease HinfI or with RsaI in addition for specific detection of different variants from the known subclasses of cry1I genes. The PCR-RFLP pattern obtained revealed the detection of cry1I genes in 151 of 202 native Bt isolates. Furthermore, cry1I genes were detectable in 10 of 19 standard strains tested. The cry1Ia gene was the most abundant cry1I gene subclass present in 54 of 56 native Bt isolates and in 8 of 10 standard strains. Based on the results obtained, the PCR-RFLP method may be a valuable and reliable tool for specific detection and identification of cry1I genes.     

Distribution and diversity of cry genes in native strains of Bacillus thuringiensis obtained from different ecosystems from Colombia

Colombia is a tropical country located at the north of South America. It is considered to be one of the most important countries in terms of its biodiversity worldwide. One hundred and eight soil samples obtained from agricultural crops and wild ecosystems were evaluated in terms of the presence of Bacillus thuringiensis (Bt) native strains. One hundred and eight different Bt strains were isolated and characterized by the presence of crystal proteins by SDS-PAGE and a multiplex PCR with general and specific primers for cry1 and cry3, cry7, and cry8 gene detection. Most of the Bt strains (73%) reacted with the cry1 general primers; 27.8% of the Bt strains reacted with cry3, cry7, and cry8 general primers and 17.8% of strains did not react with any of these two sets of primers. Thirty different PCR profiles were found in the strains with cry1 genes when they were analyzed with specific primers (cry1A to cry1F). A high frequency of joint occurrence was observed for cry1Aa/cry1Ab, cry1Aa/cry1Ac, cry1Ab/cry1Ac, and cry1C/cry1D genes with a Pearson coefficient of 0.88, 0.74, 0.76, and 0.87, respectively. Other distinctive characteristics were found in the Colombian collection as the presence of 22.2% of native strains which presented, at the same time, lepidopteran and coleopteran active genes. Interesting relations were found as well between the cry gene distribution and the geographical areas sampled. Finally, some strains with moderate to high biopesticide activity against Spodoptera frugiperda (Lepidoptera) and Premnotrypes vorax (Coleoptera) insects were identified, this being important to explore future microbial strategies for the control of these crop pests in the region.     

苏云金芽孢杆菌CrylAb13基因的克隆及表达研究

BtC005是我国自行分离的对多种害虫具有毒杀作用的苏云金芽孢杆菌。经PCR-RFLP系统鉴定,它含有crylAb基因。Southern blot结果显示;PstI酶切C005质粒所得的8.5kb长的DNA片段为crylAb基因的阳性杂交带。以pUCP19为载体,克隆了该片段并证明其含有crylAb基因,对其进行亚克隆和测序,结果表明该基因编码区为3468bp,其编码的蛋白含1155个氨基酸,分子量为130.6kD,等电点为pH4.845。该基因已在GenBank基因库中注册,Accession number为AF254640,并为国际Bt杀虫晶体蛋白基因命名委员会正式命名为crylAb13。将crylAb13基因在Bt无晶体突变株cryB^-中表达,蛋白质电泳结果表明在130kD处有表达带,并证明GryAb对小菜蛾有较高的杀虫活性。     

31株苏云金芽孢杆菌杀虫晶体蛋白基因型鉴定及表达产物研究

利用已建立的苏云金芽孢杆菌cry基因的PCRRFLP鉴定体系,鉴定了31株Bt菌株的cry基因类型,并进行了SDSPAGE分析和杀虫生物活性测定。研究表明：25株含cry1基因,表达蛋白130～150kD;其中16株含有对鞘翅目和鳞翅目害虫皆有活性的cry1I基因,其表达蛋白为81kD;15株同时含有cry1和cry2基因(13株表达蛋白约为60kD);10株含有未知待定基因;6株不含所鉴定的cry基因(其中2株有表达产物)。室内生物测定表明：cry1、cry2基因表达的菌株对鳞翅目害虫具有高杀虫活性,7株对舞毒蛾和膜翅目——杨叶蜂幼虫具有较高杀虫活性;含有cry1Aa\,cry1Ac\,cry2或cry1Ab\,cry1Ac\,cry2基因组合的菌株对棉铃虫幼虫均显示杀虫活性,其中6、12、30号菌株毒力最强。不含上述cry基因的菌株均无杀虫活性。以上结果证明,通过cry基因类型鉴定和表达产物的SDSPAGE分析可以预测菌株的杀虫活性。     

Development of stem borer resistant transgenic parental lines involved in the production of hybrid rice

Stem borer resistant transgenic parental lines, involved in hybrid rice, were produced by Agrobacterium-mediated gene transfer method. Two pSB111 super-binary vectors containing modified cry1Ab/cry1Ac genes driven by maize ubiquitin promoter, and herbicide resistance gene bar driven by cauliflower mosaic virus 35S promoter were, used in this study. Embryogenic calli after co-cultivation with Agrobacterium were selected on the medium containing phosphinothricin. Southern blot analyses of primary transformants revealed the stable integration of bar, cry1Ab and cry1Ac coding sequences into the genomes of three parental lines with a predominant single copy integration and without any rearrangement of T-DNA. T1 progeny plants disclosed a monogenic pattern (3:1) of transgene segregation as confirmed by molecular analyses. Furthermore, the co-segregation of bar and cry genes in T1 progenies suggested that the transgenes are integrated at a single site in the rice genome. In different primary transformants with alien inbuilt resistance, the levels of cry proteins varied between 0.03 and 0.13% of total soluble proteins. These transgenic lines expressing insecticidal proteins afforded substantial resistance against stem borers. This is the first report of its kind dealing with the introduction of Bacillus thuringiensis (Bt) cry genes into the elite parental lines involved in the development of hybrid rice.     

Cloning of cry2Aa and cry2Ab genes from new isolates of Bacillus thuringiensis and their expression in recombinant Bacillus thuringiensis and Escherichia coli strains

Bacillus thuringiensis (Bt) is the major source for transfer of genes to impart insect resistance in transgenic plants. Cry2A proteins of Bt are promising candidates for management of resistance development in insects due to their difference from the currently used Cry1A proteins, in structure and insecticidal mechanism. Two insecticidal crystal protein genes of Bt, viz. cry2Aa and cry2Ab were cloned from new isolates of Bt, 22-4 and 22-11, respectively. Expression of both the genes was studied in an acrystalliferous strain of Bt (4Q7) by fusing the cry2Aa and cry2Ab genes downstream of cry2Aa promoter and orf1 + orf2 sequences. Western blot analysis revealed a low level expression of the cloned cry2Aa and cry2Ab genes in the recombinant Bt strains. High-level expression of cry2Aa and cry2Ab genes was achieved in the recombinant E. coli by cloning the cry2A genes under the control of the T7 promoter.     

Assessment of cry1 gene contents of Bacillus thuringiensis strains by use of DNA microarrays

A single Bacillus thuringiensis strain can harbor numerous different insecticidal crystal protein (cry) genes from 46 known classes or primary ranks. The cry1 primary rank is the best known and contains the highest number of cry genes which currently totals over 130. We have designed an oligonucleotide-based DNA microarray (cryArray) to test the feasibility of using microarrays to identify the cry gene content of B. thuringiensis strains. Specific 50-mer oligonucleotide probes representing the cry1 primary and tertiary ranks were designed based on multiple cry gene sequence alignments. To minimize false-positive results, a consentaneous approach was adopted in which multiple probes against a specific gene must unanimously produce positive hybridization signals to confirm the presence of a particular gene. In order to validate the cryArray, several well-characterized B. thuringiensis strains including isolates from a Mexican strain collection were tested. With few exceptions, our probes performed in agreement with known or PCR-validated results. In one case, hybridization of primary- but not tertiary-ranked cry1I probes indicated the presence of a novel cry1I gene. Amplification and partial sequencing of the cry1I gene in strains IB360 and IB429 revealed the presence of a cry1Ia gene variant. Since a single microarray hybridization can replace hundreds of individual PCRs, DNA microarrays should become an excellent tool for the fast screening of new B. thuringiensis isolates presenting interesting insecticidal activity.     

晶胞粘连苏云金芽胞杆菌C15杀线虫毒力因子发掘与功能鉴定

苏云金芽胞杆菌（Bacillus thuringiensis,Bt）制剂作为一种高效的微生物杀虫剂,在植物病虫害防控领域有着广泛的应用。Bt制剂的主效成分为杀虫晶体和芽胞,其中,杀虫晶体的环境低持久性是Bt农药应用的重要限制因素之一。自然界中存在着一些Bt菌株,其产生的杀虫晶体位于芽胞外壁和芽胞衣之间,这种特殊的表型被称为晶胞粘连（spore-crystal association, SCA）表型。由于芽胞外壁对晶体的保护作用,SCA表型可以提升晶体抵抗不良环境因素的能力,是开发新型Bt生物囊杀虫剂的有效育种策略。本文选取对线虫具有强毒杀能力的 SCA菌株C15作为研究对象。获得了C15菌株的完整基因组序列,包括一个5 637 049 bp的环状染色体和8个不同大小的环形质粒（240 314 bp到3 188 bp）。C15基因编码了5个杀虫蛋白（Cry蛋白）基因：cry21-99、cry21-67、cry21-66、cry21-46和cry-N。在Bt无晶体突变株BMB171中异源表达cry21-99基因,发现其表达产物形成菱形晶体,且对秀丽隐杆线虫（Caenorhabditis elegans）和南方根结线虫（Meloidogyne incognita）均有毒杀活性。同时,还在全基因组范围内预测了Cry毒素以外的杀线虫毒力因子和次生代谢产物。此外,在C15基因组中预测了本团队已报道的苏云金芽胞杆菌幕虫亚种（B. thuringiensis serovar finitimus）菌株YBT-020 SCA表型决定因子的同源基因,缺失后突变体仍然保留稳定的SCA表型,说明C15菌株的SCA表型形成机制与YBT-020不同,该菌株代表了一种新的SCA表型形成机制。本研究为转基因作物防控线虫提供了新的遗传资源,也为研究SCA表型形成机制,开发新型高效Bt制剂提供了新线索。     

Broccoli plants with pyramided cry1Ac and cry1C Bt genes control diamondback moths resistant to Cry1A and Cry1C proteins

This study was undertaken to determine the effects of pyramiding two Bacillus thuringiensis (Bt) genes in the same plant on the production of Bt proteins and the control of diamondback moths (DBM, Plutella xylostella) resistant to one or the other protein. Broccoli lines carrying both cry1Ac and cry1C Bt genes were produced by sexual crosses of cry1Ac- and cry1C-transgenic plants. Plants containing both genes were selected by tests for resistance to kanamycin and hygromycin, and confirmed by PCR analysis for the Bt genes. Both cry1Ac and cry1C mRNAs were detected in the hybrid lines, and Cry1Ac and Cry1C proteins were stably produced at levels comparable to the parental plants. Plants producing both Cry1Ac and Cry1C proteins caused rapid and complete mortality of DBM larvae resistant to Cry1A or Cry1C, and suffered little or no leaf damage. These plants, in combination with the resistant DBM populations available, will allow greenhouse or field studies of resistance management strategies involving gene pyramiding.     

Screening of Bacillus thuringiensis serotypes by polymerase chain reaction (PCR) for insecticidal crystal genes toxic against coffee berry borer

Using PCR,257 isolates of Bacillus thuringiensis(Bt) were screened for cry-type genes. Of 257 isolates/strains, 60 isolates were identified as cry7/8, 10 isolates as cry3 and 36 isolates as cry 1I. One specific strain of B. thuringiensis (sumiyoshiensis; T03B 001) was investigated for the presence of cry7 and cry8 genes. Genes Cry7 and cry8 were first detected in this strain using family primers prior to analysis by exclusion polymerase chain reaction (E-PCR) using specific type primers. E-PCR conducted with the above said primers led to the identification by agarose gel electrophoresis of a remaining 1.5 Kb family band indicating a potentially novel gene. This PCR product, (1.5 Kb), was purified from the gel and cloned in pGEM-T Easy vector. Twenty recombinant colonies bearing 1.5 Kb insert were identified and three randomly selected representatives of the group, clones 7, 8 and 10, were sequenced and compared to all cry7 and cry8 sequences available from Gene Bank. Alignments with available DNA and protein sequences showed that all these clones contained a gene related to cry8Aa1. Analysis using protein sequence alignment showed that the sequence from clone 7 differed from the closest relative, known under the new nomenclature as cry 8Aa1, by 44%. The crystal proteins from B. thuringiensis sumiyoshiensis (T03B 001) was toxic to coffee berry borer larvae.     

Novel cry gene from Paenibacillus lentimorbus strain Semadara inhibits ingestion and promotes insecticidal activity in Anomala cuprea larvae

A positive clone was selected from a library of total cell DNA of Paenibacillus lentimorbus strain Semadara that reacted with an antiserum that was raised against parasporal crystal proteins produced by this strain. The positive clone had a DNA insert containing two whole cry genes (cry43Aa1, cry43Ba1), one partial cry gene (cry43-like), and three smaller genes located upstream. Eight blocks that are conserved in the Cry proteins of Bacillus thuringiensis [Microbiol. Mol. Biol. Rev. 62 (1998) 775] were detected in their deduced amino acid sequences. The Escherichia coli transformant expressing cry43Aa1 caused inhibition of ingestion and 90% mortality in the first stadium larvae of Anomala cuprea. A low concentration of sporangia mixed with the transformant expressing cry43Aa1 easily infected the larvae of A. cuprea. The protein of approximately 150 kDa produced by the transformants expressing the cry genes reacted with antiserum specific for the parasporal crystal proteins. Southern hybridization analysis demonstrated that the cry genes were located on the chromosomal DNA of this strain, which possessed at least four cry genes.     

Identification of novel cry-type genes from Bacillus thuringiensis strains on the basis of restriction fragment length polymorphism of the PCR-amplified DNA.

Two pairs of universal oligonucleotide primers were designed to probe the most conserved regions of all known cryI-type gene sequences so that the amplified PCR fragments of the DNA template from Bacillus thuringiensis strains may contain all possible cryI-type gene sequences. The restriction fragment length polymorphism (RFLP) patterns of the PCR-amplified fragments revealed that 14 distinct cry-type genes have been identified from 20 B. thuringiensis strains. Those cry-type genes included cryIA(a), cryIA(a), cryIA(b), cryIA(b), cryIA(c), cryIB, cryIC, cryIC, cryIC(b), cryID, cryIE, cryIF, cryIF, and cryIII (a dagger at the end of a gene designation indicates a novel cry-type gene determined by restriction mapping or DNA sequences). Among them, the sequences of cryIA(a), cryIA(b), cryIB, cryIC, cryIF, and cryIII were found to be different from the corresponding published cry gene sequences. Interestingly, five cry-type genes [cryIA(a)-, cryIB-, cryIC-, cryIC(b)-, and cryIF-type genes] and seven cry-type genes [cryIA(a)-, cryIA(b)-, cryIB-, cryIC-, cryIC(b)-, cryIF-, and cryIII-type genes] have been detected from B. thuringiensis subsp. morrisoni HD-12 and B. thuringiensis subsp. wuhanensis, respectively. Therefore, the PCR-RFLP typing system is a facile method to detect both known and novel cry genes existing in B. thuringiensis strains.     

苏云金芽孢杆菌分子伴侣串联基因p19-p29的克隆和表达载体的构建

根据已知序列设计一对PCR引物(ORF5S,ORF3N),可从cry2Aa或cry2Ac操纵子中扩增出包含串联分子伴侣基因p19p29的DNA片段,预期大小分别为16kb和20kb。对150株苏云金芽孢杆菌菌株进行PCR检测,从26株中获得了大小为16kb的扩增片段,但未获得20kb的片段。这表明cry2Aa型操纵子p19p29基因存在较广泛,而cry2Ac型较罕见。将来自Y2菌株的16kb片段回收,通过一系列亚克隆,最终构建成一个含有p19p29串联基因的Bt表达载体,为进一步研究p19p29串联基因的功能奠定了基础。     

Contents of cry genes and insecticidal toxicity of Bacillus thuringiensis strains from terrestrial and aquatic habitats

AIMS: Two Bacillus thuringiensis collections from terrestrial and aquatic habitats were compared in order to study the possible interrelationships between habitat and biological characteristics (serovar, cry genes content and toxicity). METHODS AND RESULTS: Bacillus thuringiensis strains were characterized by serology, PCR, and one-dose treatment against the noctuids Helicoverpa armigera and Spodoptera exigua, and the dipteran Tipula oleracea. A total of 12 and 10 different serovars were identified within terrestrial and aquatic strains, respectively. The number of non-toxic strains was greater in aquatic (41.6%) than in terrestrial habitats (5.3%). The genes cry1C, cry1D and cry1E were significantly more frequent in the terrestrial habitat. The cry1B gene was very frequent within thuringiensis strains. CONCLUSIONS: A high diversity was found in terms of serovars present and cry genes content in both collections. The relative frequency of individual cry genes was different in both collections, and a serovar-dependent distribution of the cry1B gene was found. Some strains sharing the same set of cry genes differed in their toxicity, suggesting important differences in gene expression. SIGNIFICANCE AND IMPACT OF THE STUDY: The inter-relationships between serology, cry gene content and toxicity may allow a better understanding of B. thuringiensis ecology.