Journal of Invertebrate Pathology 90 (2005) 91–97

www.elsevier.com/locate/yjipa

Association analysis between serotype, cry gene content, and toxicity to Helicoverpa armigera larvae among Bacillus thuringiensis isolates

native to Spain

Clara Martínez a, Jorge E. Ibarra b,¤, Primitivo Caballero a

a Laboratorio de Entomología Agrícola y Patología de Insectos, Departamento de Producción Agraria, Universidad Pública de Navarra, 31006 Pamplona, Spain

b Departamento de Biotecnología y Bioquímica, CINVESTAV-IPN, Unidad Irapuato, 36500 Irapuato, Gto, Mexico

Received 10 May 2005; accepted 16 May 2005Available online 12 July 2005

Abstract

Serotyping, cry gene content, and toxicity to Helicoverpa armigera were determined for 178 isolates of Bacillus thuringiensis nativeto Spain. A total of 13 diVerent cry1 and cry2 genes were detected when isolates were screened by PCR analysis. Results showed thatcry2 and cry1Ia were the most frequent cry genes in the collection (74 and 57%, respectively); whereas cry1D, cry1Aa, cry1Ab, andcry1C were only moderately abundant (49, 48, 47, and 36%, respectively). The most uncommon cry genes were cry1Ac, cry1E, cry1B,cry1Ib, cry1Ad, cry1F, and cry1G, with frequencies of 24, 14, 13, 8, 5, 5, and 1%, respectively. The distribution of some cry genes wassomewhat associated with particular serovars. For example, genes cry1C and cry1D were especially frequent in the serovar aizawai,while cry1B was very frequent in the serovar thuringiensis. Bioassays against H. armigera larvae showed a wide variation in the insec-ticidal potency, even among strains sharing the same set of cry genes and within the same serotype. 2005 Elsevier Inc. All rights reserved.

Keywords: Bacillus thuringiensis; Serotyping; PCR analysis; cry genes; Helicoverpa armigera

1. Introduction

During the sporulation process, Bacillus thuringiensis(Berliner) forms intracellular crystalline inclusions con-sisting of Cry proteins, that are selectively toxic againsta wide variety of insects, including important pests(Schnepf et al., 1998). Formulations based on B. thurin-giensis have been used as bioinsecticides for almost 50years in agriculture, forestry, and disease vector control(Caballero and Ferré, 2001). More recently, several crygenes have been successfully introduced into plantgenomes, resulting in high levels of protection againstinsect attack (Sanchis, 2000).

* Corresponding author. Fax: +52 462 624 5996.E-mail address: jibarra@ira.cinvestav.mx (J.E. Ibarra).

0022-2011/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.jip.2005.05.003

For decades, worldwide screening followed byisolation and characterization of new B. thuringiensisstrains has taken place to discover strains with novel orparticularly high insecticidal activities (Bel et al., 1997;Bernhard et al., 1997; Bravo et al., 1998; Ibarra et al.,2003; Iriarte et al., 1998). From many B. thuringiensisstrain collections, numerous cry genes have been clonedand sequenced. Cry proteins have been classiWed into 47diVerent groups (from Cry1 to Cry47), with a number ofsubgroups arranged on the basis of the aminoacid sequence homology (Crickmore et al., 1998)(see http://www.biols.susx.ac.uk/home/Neil_Crickmore/Bt/toxins2.html).

Currently, the sub-species classiWcation of B. thur-ingiensis strains is based on their Xagellar (H) antigens(de Barjac, 1981; Lecadet et al., 1999). This system isstill useful although the insecticidal activity rarely

92 C. Martínez et al. / Journal of Invertebrate Pathology 90 (2005) 91–97

corresponds to the serotype. B. thuringiensis strainsfrequently produce crystalline inclusions containingmore than one type of insecticidal protein and varia-tion in toxicity between this inclusions has beenrecently reported between as well as within serovars(Porcar et al., 2000).

Toxicological characterization of B. thuringiensis isbased on bioassays performed on susceptible insect spe-cies, but this is a very time consuming eVort, especiallywhen screening large collections. Prediction of insecti-cidal activity of B. thuringiensis strains by polymerasechain reaction (PCR) product proWles (Carozzi et al.,1991) may help and has been widely exploited for thispurpose. PCR only requires minute amounts of DNAand allows a quick, simultaneous screening of manysamples. This technique has been used to determine thepresence or absence of particular cry genes. It is highlysensitive and fast, and therefore can be easily used on aroutine basis, either to identify cry-type genes (Ben-Dovet al., 1997; Bourque et al., 1993; Ceron et al., 1995; Chaket al., 1994), to study their distribution (Bel et al., 1997;Bravo et al., 1998; Chak et al., 1994; Ferrandis et al.,1999), or to detect new cry genes (Juárez-Pérez et al.,1997; Kalman et al., 1993; Kuo and Chak, 1996). How-ever, normal PCR procedures do not accurately predictthe insecticidal activity of a strain, as other factors, suchas the expression level of the cry genes present, areinvolved in the insecticidal potency of each strain.

Cry1 and Cry2 proteins are known to be highly toxictowards many lepidopteran species, including Heli-coverpa armigera, a major pest of cotton, tobacco, maize,and other crops in many regions of the world. These pro-teins are produced by many of the known B. thuringien-sis strains. Dulmage (1981) demonstrated thatB. thuringiensis strains active against lepidopteran larvaediVer considerably in insecticidal spectra and potency.SpeciWcity and diVerences in the relative potency occurmostly because most of the B. thuringiensis strains carryand express several cry genes (Aronson, 1994). Thus, theinsecticidal activity of a strain is not simply determinedby the properties of a single toxic protein, but by theactivity (MacIntosh et al., 1990), relative proportion, andpotential interactions of the individual Cry proteins(Ceron et al., 1995; Dubois and Dean, 1995) present inits parasporal crystal. For example, larvae of H. armi-gera are particularly susceptible to Cry1Ac while otherproteins, including Cry1Aa, Cry1Ab, Cry2, and Cry1I,have shown diVerent degrees of toxicity toward thisinsect pest (Padidam, 1992). Variation in insecticidalpotency toward H. armigera larvae has been reportedamong B. thuringiensis strains sharing the cry1Ac gene(Padidam, 1992).

In this report, a large collection of B. thuringiensisstrains was characterized by serotyping, cry-gene con-tent, and toxicity against H. armigera, in an attempt tocorrelate these parameters.

2. Materials and methods

2.1. Bacillus thuringiensis strains and culture conditions

The 178 B. thuringiensis strains analyzed in this studywere obtained from the LEAPI collection at the Uni-versidad Pública de Navarra, Pamplona, Spain (Iriarteet al., 1998). The standard strain HD-1 was isolated fromthe commercial product Dipel. Each strain was culturedin 5 ml medium (Stewart et al., 1981) in an orbital shakerset at 250 rpm and 28 °C, until cell lysis. Spores and crys-tals were harvested by centrifugation (9700g, 10 min)and the pellet was washed in NaCl 1 M to avoidany residual endogenous protease activity before re-suspending in 0.5 ml double distilled water. Proteinconcentration of each suspension was quantiWed andconcentrations were adjusted accordingly (see below)and then stored at ¡20 °C until further use.

2.2. Serotyping

Serological identiWcation was performed as describedby de Barjac (1981) using antisera kindly provided bythe WHO Collaborating Center for the Entomopatho-genic Bacillus (Institut Pasteur, Paris, France), to iden-tify the currently recognized Xagellar H-antigens.

2.3. PCR analysis

PCR analysis was used to detect 12 cry1 and one cry2genes. SpeciWc primers for 10 diVerent cry1 genes(Juárez-Pérez et al., 1997; Kalman et al., 1993; Porcaret al., 1999) were used in combination with the cry1 gen-eral primer I(¡) (Juárez-Pérez et al., 1997) to detect:cry1Aa, cry1Ab, cry1Ac, cry1Ad, cry1B, cry1C, cry1D,cry1E, cry1F, and cry1G. For the identiWcation of cry1Iaand cry1Ib two speciWc primer pairs were used (Van Rie,personal communication). The cry2 genes were detectedwith the primer pair cryIIA3–cryIIA5 (Porcar et al.,1999).

DNA templates were obtained from single B. thurin-giensis colonies grown overnight in LB agar, suspendedin 100 �l sterile water and boiled for 10 min. Cell debriswas pelleted by centrifugation. Each reaction contained5 �l DNA template solution (supernatant) and 45 �l ofthe PCR mixture containing 0.25 mM dNTPs, 1 mMMgCl2, 0.6–1 mM each primer, and 1 U of Taq DNApolymerase (Pharmacia). An Eppendorf Mastercyclerthermal cycler was used for all the ampliWcations. PCRprogram included a 2 min step of initial denaturation at95 °C, and a total of 30 ampliWcation cycles, followed byan extra extension step of 10 min at 72 °C. The ampliWca-tion cycles consisted of a denaturation step of 1 min at95 °C; an annealing step of 1 min at 50 °C; and an elon-gation step of 1 min at 72 °C. Each ampliWcation was val-idated with negative (no template DNA) and positive

C. Martínez et al. / Journal of Invertebrate Pathology 90 (2005) 91–97 93

(known template DNA) controls. An aliquot (15 �l) ofeach reaction was analyzed by electrophoresis in 1%agarose, 1£ TAE gel.

For convenience, a scale of four frequency levels wasestablished, considering the frequency of each genewithin the B. thuringiensis collection. Therefore, a crygene found in more than 75% of the strains was regardedas very frequent. Genes present in 50–74% of the strainswere considered frequent; cry genes found in 25–49% ofthe strains were classiWed as moderately frequent; and,Wnally, uncommon cry genes were those found in lessthan 25% of the strains.

2.4. Toxicity bioassays

Insect toxicity tests were carried out on Wrst instarH. armigera larvae which were treated with a single highdose of spore–crystal mixtures as described earlier (Iri-arte et al., 1998). The B. thuringiensis HD-1 strain wasused as a positive control and water served as a negativecontrol. Spore–crystal suspensions of all the isolatestested were adjusted to the protein concentration thatproduced an insect mortality of 70% in the positive con-trol. Lettuce disks of 3 mm in diameter were dipped inspore–crystal suspensions, and then individually placedin a 25-compartment plastic Petri dish, containing a thinlayer of 3% agar as a humidity source. Larvae were indi-vidually transferred into each cell. Three replicates of 25larvae each were conducted for each B. thuringiensisstrain. Larvae were kept at a constant temperature of25 § 2 °C and a photoperiod of 16 h:8h (light:dark), andinsect mortality was recorded after 72 h.

2.5. Statistical analysis

Multiple regression analysis was initially employed toestablish the relationship between the parameters (i.e.,serotypes, toxicity, and cry genes); unfortunately, its use-fulness proved to be very limited. Therefore, cluster anal-ysis was used to group the strains due to a great diversityof combinations among the parameters. Bias towardsthe random distribution of cry genes among serovarswas measured by a �2 goodness-of-Wt test, according tothe observed and expected frequencies. The observedvalues corresponded to the number of B. thuringiensisstrains presenting a given cry gene. Expected values werecalculated assuming a random distribution of the genesamong the serovars analyzed.

3. Results

3.1. Serotyping

Flagellar serotyping of the 178 strains included in thisstudy showed that eight had no Xagella (non-motile) and

two more showed self-agglutination (agglutination inabsence of any antiserum). Throughout this paper, thisgroup of 10 strains will be referred as the group of“unknown serotype.” Among the motile strains, 19diVerent serovars were identiWed: 80 isolates cross-reacted with the aizawai antiserum; 36 with the thuringi-ensis antiserum; 13 isolates were identiWed within theserovar kurstaki; nine within the serovar morrisoni, Wvebelonged to serovar konkukian; four more to serovardarmstadiensis; and three strains agglutinated with eachof the antisera for guiyangiensis, mexicanensis, and sotto.Two isolates cross-reacted with each of the antisera forserovars kenyae and fukuokaensis, and serovars galleriae,entomocidus, kumamotoensis, kyushuensis, nigeriensis,pakistani, silo, and sooncheon were represented only by asingle strain (Fig. 1).

All the strains were arranged in Wve groups: aizawai,kurstaki, morrisoni, thuringiensis; and, for practical pur-poses, one last group (called “communis”) which includesthe remaining 15 uncommon serovars (darmstadiensis,entomocidus, fukuokaensis, galleriae, guiyangiensis,kenyae, konkukian, kumamotoensis, kyushuensis, mexi-canensis, pakistani, nigeriensis, silo, sotto, and sooncheon)along with the “unknown serotype” group.

3.2. IdentiWcation of cry genes

PCR ampliWcation with speciWc primers was used toidentify a total of 12 cry1 and one cry2 genes harbored inthe 178 B. thuringiensis strains included in this work(Fig. 2). Only 26 strains (14.6%) showed no ampliWcationwith the set of primers used.

The frequency of each gene varied greatly among thestrains (Fig. 2). According to the frequency levels estab-lished above, none of the cry genes were very frequent inthe strain collection; cry2 and cry1Ia (74 and 57%,respectively) were considered frequent genes; moderatelyfrequent cry genes were cry1D, cry1Aa, cry1Ab, andcry1C (49, 48, 47, and 36%, respectively); and the lessabundant (uncommon) cry genes were cry1Ac, cry1E,cry1B, cry1Ib, cry1Ad, cry1F, and cry1G (representing24, 14, 13, 8, 5, 5, and 1%, respectively).

Fig. 1. Relative frequencies of the 19 B. thuringiensis serovars found inthe LEAPI strain collection. The communis group includes serovarsdarmstadiensis, entomocidus, fukuokaensis, galleriae, guiyangiensis,kenyae, konkukian, kumamotoensis, kyushuensis, mexicanensis, paki-stani, nigeriensis, silo, sotto, and sooncheon as well as strains ofunknown serovar (see text).

94 C. Martínez et al. / Journal of Invertebrate Pathology 90 (2005) 91–97

3.3. Distribution of cry genes among serovars

The cry gene frequency was also studied within the19 diVerent serovars found in the collection. Fig. 3shows the cry gene frequencies among the isolates ofserovars aizawai, thuringiensis, kurstaki, morrisoni, andthe communis group (80, 36, 13, 9, and 40 strains,respectively).

For the strains of serovar aizawai, genes cry1D andcry 2 were very frequent, while cry1Aa, cry1Ab, cry1C,and cry1Ia were frequent. On the other hand, genescry1Ac, cry1Ad, cry1B, cry1E, cry1G, and cry1Ib wereuncommon. The most frequent cry genes in serovarkurstaki were cry2 (present in all the strains) andcry1Ia; cry1Aa, cry1Ab, and cry1Ac which were fre-quent. Gene cry1D was moderately frequent, andcry1Ad, cry1C, and cry1F were infrequent. In this sero-var, cry1B, cry1E, cry1G, and cry1Ib genes were notdetected. Strains of serovar morrisoni contained fourmoderately frequent genes (cry1Ac, cry1D, cry1Ib, andcry2) and four infrequent genes (cry1Aa, cry1B, cry1C,and cry1E). Genes cry1Ab, cry1Ad, cry1F, cry1G, and

Fig. 2. Frequency of individual cry genes among the 178 B. thuringien-sis strains from the LEAPI collection.

Fig. 3. Frequency of cry1 and cry2 genes of B. thuringiensis strainsfrom the LEAPI collection, within each serovar.

cry1Ia were absent in serovar morrisoni. However, forthe strains of serovar thuringiensis, all cry genes weredetected except for cry1G gene. The highest cry genefrequencies in this serovar were observed in cry1Iaand cry2 genes (very frequent genes), followed bycry1Aa and cry1Ab (frequent genes), cry1Ac andcry1B (moderately frequent genes), and cry1Ad, cry1C,cry1D, cry1E, cry1F, and cry1Ib genes (infrequentgenes). Among the serovars grouped in communis,cry1Ad and cry1G were absent and the remaining crygenes were uncommon, except for cry2, which wasfound in 30% of the strains. More than half (55%) ofthese strains showed no ampliWcation from any pair ofcry primers.

In order to Wnd out if cry genes were randomly dis-tributed among serovars, a series of �2 tests were per-formed to compare the expected and observedfrequencies within the four most common serovars andthe eight most frequent cry genes (cry1Aa, cry1Ab,cry1Ac, cry1B, cry1D, cry1E, cry1Ia, and cry1Ib). Noneof the observed frequencies matched the expected1:1:1:1 random distribution; in all cases �2 values werehighly signiWcant. The largest deviations from a ran-dom distribution were found in the distributions ofcry1B, cry1C, and cry1D (�2 D 25.62, 25.84, 29.15,respectively; df D 3; P < 0.001). This was due to a dis-proportionate abundance of these genes among thestrains of serovars aizawai (cry1C and cry1D) and thur-ingiensis (cry1B).

In order to explore relationships or associationsamong cry genes, serovars, and toxicity, cluster analysesof the strains were performed. Because this analysistakes into account the presence/absence of qualitativevariables, 10 toxicity levels were established, accordingto the mean percent mortality observed in the bioassays.Therefore, a total of 44 variables were analyzed (21 sero-vars, 10 toxicity levels, and 13 cry genes) per strain. Clus-ter analyses performed with all the variables revealedthat most of the strains (129) showed unique combina-tions of the 44 variables.

In regard to the relationship between serovars andspeciWc combinations of cry genes, some common fea-tures were observed in strains of serovars aizawai, thurin-giensis, and kurstaki, some of which form veryhomogeneous groups. The following are the most com-mon combinations found:

(i) all the strains with a gene combination of cry1Ab,cry1D, cry1E, and cry2; and all but one of thestrains showing the combination cry1Aa, cry1Ab,cry1C, cry1D, cry1Ia, and cry2 belong to serovaraizawai.

(ii) all the strains showing a gene combination ofeither cry1Aa, cry1Ac, cry1Ia, and cry2; or cry1Aa,cry1Ab, cry1B, cry1Ia, and cry2 belong to serovarthuringiensis.

C. Martínez et al. / Journal of Invertebrate Pathology 90 (2005) 91–97 95

3.4. Correlation with insect toxicity

The mean mortality of the three replicates againstH. armigera for each strain was estimated. The positivecontrol caused a mortality of about 70%, whereas that ofthe negative control was always 610%. The mean mortal-ity values varied widely, not only between but also withinserovars. Strains within the serovar aizawai producedmortalities from as low as 3% to almost 100%. In otherserovars, the diversity in potency between strains waslower. This variation was not related to cry gene contentin all cases, as some strains sharing the same set of crygenes signiWcantly diVered in their insecticidal potency.For example, strains LO22-4 and LO6-1 both displayedthe genes cry1Aa, cry1Ab, cry1C, cry1D, cry1Ia, and cry2;but the mortalities produced by their spore–crystals sus-pensions were 96 and 24%, respectively. Table 1 shows thelist of strains which displayed the highest insecticidalpotency, where bioassays resulted in >90% mortality.

These strains occurred in the four main serovars, andonly one strain from the communis group (TE21-1, sero-var kenyae). As observed before, the cry gene content ofthese highly toxic strains is variable, ranging from one toseven diVerent cry genes. Interestingly, most of them (16aizawai, 8 thuringiensis, one kurstaki, and one morrisoni)do not harbor the cry1Ac gene which is known to behighly active against H. armigera (Padidam, 1992).

Cluster analyses considering only cry genes and toxic-ity indicated that 55 strains showed unique combinations,suggesting that their distribution among the strains fol-lowed no systematic pattern and that there were nohomogeneous groups. Interestingly, three cry gene combi-nations showed high toxicity: (a) cry1E, cry1Ia, and cry2;(b) cry1Aa, cry1Ab, cry1Ad, cry1C, cry1D, cry1F, andcry2; and (c) cry1D and cry2. Similarly, another genecombination associated with high toxicity (cry1Ac, cry1D,cry1Ib, and cry2) was shared by two morrisoni strains.

Toxicity was the parameter that showed the lowest corre-lation with the other two parameters. No correlation wasobserved between individual cry genes and toxicity; however,most of the strains from which no cry genes were identiWedexhibited low toxicity (36% in average). Among the 15 mosttoxic strains, no common characteristics were found; how-ever, four of them formed a very homogenous group (serovaraizawai with genes cry1Aa, cry1Ab, cry1C, cry1D, cry1Ia,and cry2). Additionally, serovars thuringiensis, kurstaki, andmorrisoni presented a number of toxic strains higher thanthat expected on the basis of their relative frequency.

4. Discussion

Prediction of the insecticidal activity of B. thuringien-sis strains is very important to eYciently assess theentomopathogenic potential of any given strain. This is amajor issue in the screening programs of large

collections, such as those developed during the lastdecades. In this report, we present the results of a com-bined approach to Wnd any possible relationshipbetween the cry gene content, serovar, and toxicity of178 naturally occurring B. thuringiensis strains. For thispurpose, serotyping, PCR identiWcation of 13 cry genes,and bioassays of spore–crystal mixtures against H. armi-gera were carried out, followed by statistical analysis.

The serological results indicated a great diversity ofserovars (19 in total) but they were unevenly distributed

Table 1Content of cry genes in B. thuringiensis strains producing mortalities790% on H. armigera larvae

Serovar and strain cry gene content

aizawaiHU19 1C, 2Z29-9 1DHU8-2 1Aa, 1Ab, 1C, 1D, 1Ia, 2HU10-4 1Aa, 1C, 1Ia, 2CS15-8 1Ab, 1D, 2NA206-1 1Ab, 1D, 1E, 2LO31 1D, 2TE27-1 1Ab, 1C, 1D, 2TE32-8 1Aa, 1Ac, 1D, 1Ia, 2Z6-1 1Ab, 1D, 1E, 2Z21-1 1DZ27-5 1Ab, 1D, 1E, 2LO22-4 1Aa, 1Ab, 1C, 1D, 1Ia, 2NA188-1 1C, 1D, 1Ia, 2NA288-5 1Aa, 1Ab, 1Ib, 2Z8-8 1Aa, 1Ab, 1C, 1D, 1IaZ14-9 1Aa, 1Ab, 1C, 1D, 1Ia, 2

morrisoniZ26 1Ac, 1D, 1Ib, 2Z27-3 1Ac, 1D, 1Ib, 2TE32-2 1E, 1Ia, 2

kurstakiBI29 1Aa, 1Ab, 1C, 1Ia, 2HAV3-1 1Aa, 1Ab, 1Ac, 1Ad, 1F, 1Ia, 2NA166-7 1Ac, 1Ia, 2Z15-1 1Ac, 1Ia, 2LO23-1 1Ia, 2

thuringiensisLO11 1Aa, 1D, 1Ia, 2TE19-8 1Ab, 1Ac, 1Ia, 2Z15-2 1Ac, 1C, 1Ia, 2LO13-2 1Ac, 1Ia, 2LO56-2 1Ac, 1B, 1D, 1Ia, 2LO55-9 1Aa, 1Ac, 1Ia, 2HU33-1 1Aa, 1Ab, 1C, 1Ia, 2LO30 1Aa, 1Ab, 1B, 1Ia, 2NA156-9 1Aa, 1Ab, 1Ia, 2NA180-9 1Aa, 1Ab, 1B, 1IbNA192-1 1D, 1E, 2TE26-2 1Aa, 1Ab, 1B, 1Ia, 2NA190-9 1Aa, 1Ab, 1Ad, 1C, 1D, 1F, 2

kenyaeTE21-1 1Ia

96 C. Martínez et al. / Journal of Invertebrate Pathology 90 (2005) 91–97

among the 178 strains, as one serovar (aizawai) was pres-ent in almost half of the strains (45%). Similarly, a non-random distribution of the identiWed cry genes wasobserved among serovars, as corroborated by a �2 good-ness-of-Wt test. A strong association between cry1C andcry1D in the aizawai strains, and cry1B in the thuringien-sis strains indicated a serovar-dependent distribution ofat least these cry genes. It has been suggested that cry1Cand cry1D are chromosomal genes (Delécluse et al.,1993) and their high frequency in serovar aizawai may berelated to a genetic linkage with Xagellin genes, responsi-ble for the H-antigen. However, the serovar-dependentdistribution of plasmid-borne cry genes is more diYcultto explain, and may be related to some plasmid transferspeciWcity among strains of the same serovar (Baum andGonzalez, 1992). In contrast, despite a lack of direct cor-relation between serovars and insecticidal activities,some serovars are known to contain high numbers ofstrains active against particular insect species (Dulmage,1981; Porcar et al., 2000). Our results are in agreementwith these observations, as this speciWcity may be relatedto the occurrence of certain speciWc genes, such as cry1C,which are more frequent in some serovars, such as aiza-wai (Porcar et al., 2000). In other words, it seems thatserovars will often contain strains that are toxic againstinsects susceptible to the frequently occurring toxins.

Additionally, the PCR identiWcation of cry genes hasemphasized the great diversity of cry gene combinationsamong the strains analyzed. Strains with detectable crygenes (152 strains) revealed from one to as many as 10diVerent genes, detecting a total of 81 diVerent combina-tions. This Wnding is remarkable because, according tothese results, the presence of a particular cry gene is notrelated or associated with any other. This may be due tothe great genetic diversity of B. thuringiensis strains interms of the cry gene content. It also highlights the factthat the cry gene content of a strain has little to do withits taxonomic classiWcation. This Wnding is even moreimportant given that only a small proportion of the largecry gene family was taken into account.

Unexpectedly, some strains sharing the same crygenes signiWcantly diVered in their insecticidal potency.Likewise, some strains producing mortality valueshigher than 90% lacked the most toxic gene knownagainst H. armigera, cry1Ac (Padidam, 1992), or evenlacked any detectable cry gene. An obvious explicationfor these observations is the possible presence of unde-tected cry genes which were responsible for the observedtoxicity. On the other hand, many of these strains con-tained the genes cry1D, cry1E, cry1Ia, and/or cry2, sug-gesting that these genes play a role in the toxicity, eitherindividually or in combination with the associated tox-ins. The wide variety of gene combinations present in thestrains with high insecticidal potencies suggests that sev-eral genes may be implicated in their toxicity. However,the number of possible combinations may be so high

that Wnding the most eVective one would be extremelylaborious; even more so if undetected or novel genes areimplicated. Furthermore, identiWcation was focused oncry genes, but not on expressed Cry proteins, complicat-ing even more the analysis, as some cry genes may becryptic (non-expressed). Therefore, the screening of alarge number of strains against any target pest, usingestablished bioassay techniques, still represents the bestmethod for Wnding highly potent strains.

The main conclusions of the analysis we performedcan be summarized as follows:

(i) The strain collection analyzed here showed a widevariability in terms of serovars present, cry geneoccurrence, and toxicity levels. We were unable toestablish any clear relationships or associationsamong these three parameters.

(ii) Some serovar-dependent distribution of the crygenes was found. The prevalence of toxic strains insome serovars appeared higher than expected.

(iii) Variation in the toxicity levels within and amongserovars, and within and among gene content indi-cates a great diversity in the activity and expressionof the cry genes involved.

(iv) Some unidentiWed toxic factors, possibly due to thepresence of undetected Cry proteins, may beinvolved in the toxicity of some strains.

Acknowledgment

This study received Wnancial support from CICYTProjects BIO97-0935-CO2-02 and AGL2000-0840-CO3-03.

References

Aronson, A.I., 1994. Flexibility in the protoxin composition of Bacillusthuringiensis. FEMS Microbiol. Lett. 117, 21–27.

Baum, J.A., Gonzalez Jr., J.M., 1992. Mode of replication, size and dis-tribution of naturally occurring plasmids in Bacillus thuringiensis.FEMS Microbiol. Lett. 75, 143–148.

Bel, Y., Granero, F., Alberola, T.M., Martínez-Sebastian, M., Ferré,J., 1997. Distribution, frequency and diversity of Bacillus thuringi-ensis in olive tree environments in Spain. Syst. Appl. Microbiol.20, 652–658.

Ben-Dov, E., Zaritsky, A., Dahan, E., Barak, Z., Sinai, R., Manasherob,R., Khamraev, A., Troitskaya, E., Dubitsky, A., Berezina, N., Mar-galith, Y., 1997. Extended screening by PCR for seven cry-groupgenes from Weld-collected strains of Bacillus thuringiensis. Appl.Environ. Microbiol. 63, 4883–4890.

Bernhard, K., Jarrett, P., Meadows, M., Butt, J., Ellis, D.J., Roberts,G.M., Pauli, S., Rodgers, P., Burges, H.D., 1997. Natural isolatesof Bacillus thuringiensis: worldwide distribution, characteriza-tion, and activity against insect pests. J. Invertebr. Pathol. 70,59–68.

Bourque, S.N., Valero, J.R., Mercier, J., Lavoie, M.C., Levesque, R.C.,1993. Multiplex polymerase chain reaction for detection and diVer-

C. Martínez et al. / Journal of Invertebrate Pathology 90 (2005) 91–97 97

entiation of the microbial insecticide Bacillus thuringiensis. Appl.Environ. Microbiol. 59, 523–527.

Bravo, A., Sarabia, S., Lopez, L., Ontiveros, H., Abarca, C., Ortiz, A.,Ortiz, M., Lina, L., Villalobos, F.J., Pena, G., Nunez-Valdez, M.E.,Soberon, M., Quintero, R., 1998. Characterization of cry genes in aMexican Bacillus thuringiensis strain collection. Appl. Environ.Microbiol. 64, 4965–4972.

Caballero, P., Ferré, J., 2001. Bioinsecticidas: fundamentos y aplicacionesde Bacillus thuringiensis en el control integrado de plagas. M.V. Phy-toma-España y Universidad Pública de Navarra Valencia, Spain.

Carozzi, N.B., Kramer, V.C., Warren, G.W., Evola, S., Koziel, M.G.,1991. Prediction of insecticidal activity of Bacillus thuringiensisstrains by polymerase chain reaction product proWles. Appl. Envi-ron. Microbiol. 57, 3057–3061.

Ceron, J., Ortiz, A., Quintero, R., Guereca, L., Bravo, A., 1995. SpeciWcPCR primers directed to identify cryI and cryIII genes within aBacillus thuringiensis strain collection. Appl. Environ. Microbiol.61, 3826–3831.

Chak, K.F., Chao, D.C., Tseng, M.Y., Kao, S.S., Tuan, S.J., Feng, T.,1994. Determination and distribution of cry-type genes of Bacillusthuringiensis isolates from Taiwan. Appl. Environ. Microbiol. 60,2415–2420.

Crickmore, N., Zeigler, D.R., Feitelson, J., Schnepf, E., Van Rie, J.,Lereclus, D., Baum, J., Dean, D.H., 1998. Revision of the nomencla-ture for the Bacillus thuringiensis pesticidal crystal proteins. Micro-biol. Mol. Biol. Rev. 62, 807–813.

de Barjac, H., 1981. IdentiWcation of H-serotypes of Bacillus thuringien-sis. In: Burges, H.D. (Ed.), Microbial Control of Pests and PlantDiseases 1970–1980. Academic Press, London, pp. 35–44.

Delécluse, A., Poncet, S., Klier, A., Rapoport, G.A., 1993. Expression ofcryIVA and cryIVB genes, independently or in a combination, in acrystal-negative strain of Bacillus thuringiensis subsp. israelensis.Appl. Environ. Microbiol. 59, 3922–3927.

Dubois, N.R., Dean, D.H., 1995. Synergism between Cry1A insecticidalcrystal proteins and spores of Bacillus thuringiensis, other bacterialspores, and vegetative cells against Lymantria dispar (Lepidoptera:Lymantriidae) larvae. Environ. Entomol. 24, 1741–1747.

Dulmage, H., 1981. Insecticidal activity of isolates of Bacillus thuringi-ensis and their potential pest control. In: Burges, H.D. (Ed.), Micro-bial Control of Pest and Plant Diseases 1970–1980. Academic Press,New York, pp. 193–222.

Ferrandis, M.D., Juárez-Pérez, V.M., Bel, Y., Ferré, J., 1999. Distribu-tion of cryI, cryII and cryV genes within Bacillus thuringiensisstrains from Spain. Syst. Appl. Microbiol. 28, 440–444.

Ibarra, J.E., Del Rincón, M.C., Ordúz, S., Noriega, D., Benintende, G.,Monnerat, R., Regis, L., de Oliveira, C.M.F., Lanz, H., Rodriguez,M.H., Sánchez, J., Peña, G., Bravo, A., 2003. Diversity of Bacillusthuringiensis strains from Latin America with insecticidal activity

against diVerent mosquito species. Appl. Environ. Microbiol. 69,5269–5274.

Iriarte, J., Bel, Y., Ferrandis, M.D., Andrew, R., Murillo, J., Ferre,J., Caballero, P., 1998. Environmental distribution and diversityof Bacillus thuringiensis in Spain. Syst. Appl. Microbiol. 21, 97–106.

Juárez-Pérez, V.M., Ferrandis, M.D., Frutos, R., 1997. PCR-basedapproach for detection of novel Bacillus thuringiensis cry genes.Appl. Environ. Microbiol. 63, 2997–3002.

Kalman, S., Kiehne, K.L., Libs, J.L., Yamamoto, T., 1993. Cloning of anovel cryIC-type gene from a strain of Bacillus thuringiensis subsp.galleriae. Appl. Environ. Microbiol. 59, 1131–1137.

Kuo, W.S., Chak, K.F., 1996. IdentiWcation of novel cry-type genesfrom Bacillus thuringiensis strains on the basis of restriction frag-ment length polymorphism of the PCR-ampliWed DNA. Appl.Environ. Microbiol. 62, 1369–1377.

Lecadet, M.M., Frachon, E., Dumanoir, V.C., Ripouteau, H.,Hamon, S., Laurent, P., Thiery, I., 1999. Updating the H-antigenclassiWcation of Bacillus thuringiensis. J. Appl. Microbiol. 86,660–672.

MacIntosh, S.C., Stone, T.B., Sims, S.R., Hunst, P.L., Greenplate, J.T.,Marrone, P.G., Perlak, F.J., FischhoV, D.A., Fuchs, R.L., 1990.SpeciWcity and eYcacy of puriWed Bacillus thuringiensis proteinsagainst agronomically important insects. J. Invertebr. Pathol. 56,258–266.

Padidam, M., 1992. The insecticidal crystal protein CryIA(c) fromBacillus thuringiensis is highly toxic for Heliothis armigera. J. Inver-tebr. Pathol. 59, 109–111.

Porcar, M., Iriarte, J., Cosmao Dumanoir, V., Ferrandis, M.D., Leca-det, M., Ferre, J., Caballero, P., 1999. IdentiWcation and character-ization of the new Bacillus thuringiensis serovars pirenaica (serotypeH57) and iberica (serotype H59). J. Appl. Microbiol. 87, 640–648.

Porcar, M., Martínez, C., Caballero, P., 2000. Host range and gene con-tents of Bacillus thuringiensis strains toxic towards Spodopteraexigua. Entomol. Exp. Appl. 97, 339–346.

Sanchis, V., 2000. Biotechnological improvement of Bacillus thuringi-ensis for agricultural control of insect pests: beneWts and ecologi-cal implications. In: Charles, J.F., Delécluse, A., Nielssen-LeRoux,C. (Eds.), Entomopathogenic Bacteria: From Laboratory to FieldApplication. Kluwer Academic Publishers, Dordrecht, pp. 441–459.

Schnepf, E., Crickmore, N., Van Rie, J., Lereclus, D., Baum, J., Feitel-son, J., Zeigler, D.R., Dean, D.H., 1998. Bacillus thuringiensis andits pesticidal crystal proteins. Microbiol. Mol. Biol. Rev. 62, 775–806.

Stewart, G.S., Johnstone, K., Hagelberg, E., Ellar, D.J., 1981. Commit-ment of bacterial spores to germinate. A measure of the triggerreaction. Biochem. J. 198, 101–106.