contents of cry genes and insecticidal toxicity of bacillus thuringiensis strains from terrestrial...

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

C. Martınez and P. CaballeroLaboratorio de Entomologıa Agrıcola y Patologıa de Insectos, Departamento de Produccion Agraria,

Universidad Publica de Navarra, Pamplona, Spain

2001/167: received 3 July 2001, revised 14 September 2001 and accepted 13 November 2001

C. MART INEZ AND P. CABALLERO. 2002.

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

(41Æ6%) than in terrestrial habitats (5Æ3%). 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.

INTRODUCTION

Bacillus thuringiensis is a Gram-positive bacterium which is

being extensively studied because of its ability to synthes-

ize proteinaceous, parasporal crystals (d-endotoxins),

which are very toxic to a wide variety of pests (Schnepf

et al. 1998). This bacterium has been isolated during

numerous studies from natural samples such as soil

(Martin and Travers 1989), insect habitats (Brownbridge

and Margalit 1986), insect larvae (Krieg et al. 1983) and

stored products (Delucca et al. 1982). The characterization

of 32 isolates from foliage samples by Damgaard et al.(1998) showed that 75% of them belonged to serovar

israelensis. In toxicity tests, 84% showed larvicidal activity

against Aedes aegypti, whereas no activity against Pieris

brassicae was detected in any of the isolates. These results

suggest that the occurrence of specific B. thuringiensispopulations correlates with the types of insect feeding on

the foliage and roots. By contrast, Martin and Travers

(1989) analysed 785 B. thuringiensis strains originating

from very diverse habitats and did not find a strong

association between insect environments and high densities

of B. thuringiensis.PCR analysis, bioassays and serological identification of

B. thuringiensis collections demonstrated the large diversity

that can be found among naturally-occurring strains

(Ben-Dov et al. 1997; Bravo et al. 1998; Ferrandis et al.1999). Bacillus thuringiensis strains isolated from different

habitats and geographical locations differ in the occurrence

and diversity of cry genes content (Chak et al. 1994; Bravo

et al. 1998). Moreover, the correlation of the cry genes

profiles with toxicity has been reported to be at least

partially dependent on the serovar (Porcar et al. 2000).

Among these, PCR is the most widely used method for the

first characterization step, but the relative expression of each

Correspondence to: P. Caballero, Laboratorio de Entomologıa Agrıcola y

Patologıa de Insectos, Departamento de Produccion Agraria, Universidad

Publica de Navarra, Campus Arrosadıa, 31006 Pamplona, Spain (e-mail:

[email protected]).

ª 2002 The Society for Applied Microbiology

Journal of Applied Microbiology 2002, 92, 745–752

of the genes, as well as the interactions (either synergistic or

antagonistic) of the crystal proteins, can limit the PCR-

mediated prediction of the toxicity.

In the present study, a combined approach was chosen,

including gene identification, serology and bioassays, in

order to obtain a complete view of the diversity of two

B. thuringiensis collections, and to find out the possible

inter-relationships among serology, toxicity, cry genes

content and habitat. In this work, a total of 62 strains

from two different collections, originating from samples

collected in terrestrial and aquatic habitats in Spain, were

analysed. Insect bioassays were carried out against the

dipteran Tipula oleracea and the Lepidoptera Helicoverpaarmigera and Spodoptera exigua, which are important pests

of economic interest. The frequency of the cry genes in

both collections and within serovars, as well as the toxicity

of the strains and its relation with cry genes contents, are

discussed.

MATERIALS AND METHODS

Bacterial strains

The strains analysed in this work were isolated from a

national screening programme carried out in terrestrial

environments of Spain (Iriarte et al. 1998), and from a

sampling of aquatic environments of the Spanish province

of Navarre (Iriarte et al. 2000). A group of 38 strains

from the former collection and 24 isolates from aquatic

habitats were randomly selected for further analysis by

means of PCR, as well as by serology and insect toxicity.

The B. thuringiensis strains isolated from the commercial

products DipelÒ and XentariÒ were used as positive

controls in bioassays against H. armigera and S. exigua,

respectively, and the strain isolated from B. thuringiensisisraelensis IPS82 was used as control for the dipteran

T. oleracea.

Growth conditions

Bacillus thuringiensis strains were grown in 5 ml sporula-

tion medium (Stewart et al. 1981) at 28°C in a shaker

rotating at 250 rev min–1 for 3 days. The culture was

examined periodically under phase-contrast microscopy

until lysis reached more than 90% of the cells. Then,

NaCl was added to spores and crystals to a final

concentration of 1 mol l–1. The suspension was centri-

fuged for 10 min; the pellet was washed twice with sterile

distilled water and resuspended in 1 ml water. The

optical density (O.D.) of this mixture was measured in

a Spectronic Genesys spectrophotometer (Thermo Spec-

tronic, Rochester, NY, USA) and adjusted to

O.D.595 ¼ 1. Cultures were stored at 4°C until use.

Serological identification

Serotyping of isolates was performed as described by de

Barjac (1981), and the WHO collaborating centre for

entomopathogenic Bacillus (Institut Pasteur, Paris, France)

supplied antisera. Serological identification was carried out

with specific antisera recognizing flagellar antigens H1–H58.

PCR primers

Specific primers were used to identify several cry1 genes, as

well as cry2, cry4A, cry4B, cry10A and cry11A. The

reliability of the oligonucleotides used for the detection of

cry genes was verified by using IPS82 as positive controls,

B. thuringiensis israelensis IPS82 and the B. thuringiensisstrains isolated from the commercial products DipelÒ and

XentariÒ. The cry1 general primer I (–) (Juarez-Perez et al.1997) was used in combination with specific primers in order

to identify the cry genes cry1Aa, cry1Ab, cry1Ac, cry1Ad,

cry1B, cry1C, cry1D, cry1E, cry1F and cry1G. The identi-

fication of cry1Ia and cry1Ib was carried out using a specific

pair of primers for each (Porcar et al. 2000). The cry2 genes

were identified using the primer pair IIA3 and IIA5, and

both cry4A and cry4B genes were amplified using the primer

pair Dip1A and Dip1B (Carozzi et al. 1991). Finally, cry10Aand cry11A were identified using primer pairs 10A3–10A5

and 11A3–11A5 (Porcar et al. 1999).

Preparation of DNA template for PCR

DNA was extracted from overnight cultures on LB agar

plates incubated at 28°C. A single colony was resuspended in

100 ll water and boiled for 10 min. After cooling on ice, 5 ll

of the suspension were transferred to a 0Æ2 ml microcentri-

fuge tube containing 1 U Taq DNA polymerase (Pharmacia

Biotech), 0Æ25 mmol l–1 of each of the four deoxynucleoside

triphosphates, and 1 lmol l–1 of each of the primers, in a total

volume of 50 ll. PCR was performed in a DNA thermal

cycler (Eppendorf Mastercycler, Eppendorf, Hamburg,

Germany) using a single denaturation step (2 min at 95°C),

followed by a 30 reaction cycle consisting of denaturation of

DNA template at 95°C for 1 min, annealing templates and

oligonucleotides primers at 52°C for 1 min, and extension of

PCR products at 72°C for 1 min. An extra extension step was

performed at 72°C for 10 min. Each experiment was done

with a negative (without DNA) and a positive (with a

standard template) control and repeated twice. PCR products

were analysed by 0Æ8% agarose gel electrophoresis.

Insect toxicity assays

Insecticidal toxicity was tested against first instar larvae of

the dipteran T. oleracea obtained from seasonal garden

746 C. MART INEZ AND P. CABALLERO

ª 2002 The Society for Applied Microbiology, Journal of Applied Microbiology, 92, 745–752

populations in Pamplona (Spain). The lepidopterans

H. armigera and S. exigua were obtained from established

cultures in the insectary of this university. Larvae were fed

with a lettuce disc (4 mm in diameter) dipped in a spore-

crystal suspension adjusted to O.D.595 ¼ 1 and mixed with

1 ll of a commercial surfactant. The discs were individually

placed in 25 · 25 cell plastic boxes containing a soft agar

layer of 3% agar for the Lepidoptera and 1Æ5% agar for the

Diptera as humidity source. Each batch included a positive

control of a spore-crystal suspension of B. thuringiensisisraelensis IPS82, against T. oleracea, and the strains isolated

from the commercial products DipelÒ and XentariÒ in

bioassays carried out against H. armigera and S. exigua,

respectively. As negative control, sterile bi-distilled water

with 1 ll of a commercial surfactant was used. Three

replicates of each strain, with 20 larvae each, were

performed. Mortality data were recorded after 48 h. Strains

were considered as active when the mean mortality was

equal or higher than 25%.

Statistical analysis

A v2 analysis was performed to analyse the distribution of

the cry genes between the two collections and among

serovars. The observed values corresponded to the number

of strains bearing a given gene as identified by PCR, and the

expected values to the number of strains with that gene

assuming a random distribution.

RESULTS

Bacterial characterization

Bacterial strains were characterized by serology, PCR and

insecticidal toxicity, as shown in Table 1.

Serovar diversity

In the serological identification, 12 and 10 different serovars

were found within the strains isolated from terrestrial and

aquatic habitats, respectively. Within the terrestrial strains,

11 belonged to serovar aizawai, eight to serovar thuringiensis,eight to morrisoni and three to kurstaki, while the serovars

andalousiensis, entomocidus, mexicanensis, monterrey, novosib-irsk, sotto, tohokuensis and tolworthi were represented by only

one strain. Within aquatic strains, the most frequent serovar

was thuringiensis, with nine of the 24 strains belonging to this

serovar; serovars navarrensis, kurstaki and morrisoni were

represented by two strains each, and serovars aizawai,andalousiensis, nigeriensis, pakistani and sotto, by only one

strain each. One strain (NA323–9) did not react against any

of the antisera and three strains reacted with both pakistaniand amayensis antisera.

Content of cry genes and frequencies

All the strains isolated from terrestrial and aquatic habitats

were characterized by PCR, which allowed determination of

the presence or absence of the specific cry1s, cry2, cry4A,

cry4B, cry10A and cry11A (Table 1 and Fig. 1). The most

frequent cry genes in both habitats were cry1Aa (present in

50% of terrestrial strains and 45Æ8% of aquatic strains),

cry1Ab (50% and 45Æ8%), cry1Ia (52Æ6% and 45Æ8%) and

cry2 (78Æ9% and 54Æ2%). The genes cry1C and cry1D were

very frequent in terrestrial strains (47Æ4% and 60Æ5%,

respectively), whereas the frequency of these two genes was

16Æ6% in strains isolated from aquatic habitats. The genes

cry1Ad, cry1E, cry1F, cry1G and cry1Ib were only detected

in terrestrial strains. In contrast, the cry1B gene was slightly

more frequent in aquatic strains than in terrestrial strains

(33Æ3% and 21%, respectively). Typical dipteran-active

genes such as cry4A, cry4B, cry10A and cry11A were not

found in any of the analysed strains.

A v2 test was applied to analyse the frequency of the

detected cry genes between the two collections. The

frequencies of most of the cry genes were similar in both

collections except for the genes cry1C (v2 ¼ 3Æ91 d.f. ¼ 1),

cry1D (v2 ¼ 6Æ49 d.f. ¼ 1) and cry1E (v2 ¼ 4Æ42, d.f. ¼ 1)

which were significantly more abundant in the terrestrial

collection than in the aquatic ones. However, the cry1Ibfrequency in terrestrial (15Æ8%) and aquatic (0Æ0%) strains

was not found to differ statistically (v2 ¼ 3Æ79, d.f. ¼ 1).

The number of strains bearing cry1B was slightly greater in

aquatic than in terrestrial strains, but this difference was not

statistically different.

Frequency of cry1 and cry2 genesamong serovars

The gene frequency within strains belonging to the three

more common serovars (aizawai, thuringiensis and morrisoni)isolated from terrestrial habitats was also investigated. In

serovar aizawai, cry1C (91%), cry1D (91%), cry1Aa (81Æ8%)

and cry1Ia (72Æ7%) were the most frequent cry1 genes, with

cry1Ac (18Æ2%), cry1Ad and cry1F (9Æ1% each) the least

frequent. Neither cry1B nor cry1G were detected in strains

of this serovar, but cry2 genes were very frequent, with 91%

of the strains bearing this gene. The most frequent cry1genes in serovar thuringiensis were cry1Aa (75%), cry1Ia(75%), cry1Ab (62Æ5%) and cry1B (62Æ5%). Genes cry1Ac,

cry1Ad, cry1E, cry1F, cry1G and cry1Ib were not found in

this serovar. As in the case of the aizawai strains, the cry2gene was found to be very frequent (87Æ5%) in thuringiensisstrains. Finally, all the cry genes analysed in this study were

found within strains of serovar morrisoni. All of the strains

belonging to this serovar contained cry1Ab and cry2, and

87Æ5% of them also contained cry1D. The percentages of

B. THURINGIENSIS STRAINS FROM TERRESTRIAL AND AQUATIC HABITATS 747


Table 1 Content of cry genes and insect toxicity of Bacillus thuringiensis strains from terrestrial and aquatic habitats

Insect toxicity

Strain Serovar cry gene(s) H. a. S. e. T. o.

Terrestrial

NA118* aizawai 1Aa, 1Ab, 1C, 1D, 1Ia, 2 + + )NA145–2* aizawai 1Aa, 1C, 1D, 2 + + )NA142–11* aizawai 1Aa, 1C, 1D, 1E, 1Ia, 2 + + )NA196–5 aizawai 1Aa, 1C, 1D, 1E, 1Ia, 1Ib, 2 + + )NA190–2 aizawai 1Aa, 1Ab, 1Ad, 1C, 1D, 1F, 2 + ) )NA145–1* aizawai 1Aa, 1C, 1D, 1E, 1Ia, 2 ) + )NA195–5* aizawai 1Aa, 1Ab, 1Ac, 1C, 1D, 1Ia, 2 + + )NA148–3* aizawai 1Aa, 1Ac, 1C, 1D, 1E, 1Ia, 2 + + )NA148–7 aizawai 1C, 1D, 1Ia, 1Ib + ) )NA173–3* aizawai 1Aa, 1Ab, 1C, 1D, 1Ia, 2 + + )NA192–6* aizawai 2 + + )NA092 andalousiensis none ) ) )NA193–9* entomocidus 1Aa, 1C, 1D, 1Ia, 2 + + )NA165–2* kurstaki 1Aa, 1Ab, 1Ia, 2 + + )NA166–1* kurstaki 1Ac, 1C, 1D, 1Ia, 2 + + )NA166–3* kurstaki 1Ac, 1C, 1Ia, 2 + + )NA149–3 mexicanensis none + + )NA54 monterrey 1C, 1D ) + )NA196–6 morrisoni 1Ab, 1D, 2 + + )NA300–1* morrisoni 1Aa, 1Ab, 1C, 1D, 2 + + )NA300–2 morrisoni 1Ab, 1Ac, 1B, 1D, 1F, 1Ib, 2 + + )NA300–6 morrisoni 1Ab, 1Ac, 1D, 1F, 1Ib, 2 + + )NA300–8* morrisoni 1Ab, 1Ac, 1D, 1G, 1Ib, 2 + + )NA183–3* morrisoni 1Ab, 1D, 1E, 2 + + )NA178–7 morrisoni 1Ab, 1Ad, 1B, 1E, 1Ia, 2 + ) )NA289–1 morrisoni 1Ab, 1B, 1C, 1D, 2 + ) )NA232–1 novosibirsk 1Aa, 1Ab, 1D, 1E, 1Ib, 2 + ) )NA173–4 sotto 1Ac, 1Ia ) + )NA142–10* thuringiensis 1Aa, 1C, 1D, 1Ia ) + )NA141* thuringiensis 1Aa, 1Ab, 1B, 1Ia, 2 + + )NA150–3* thuringiensis 1Aa, 1Ab, 1B, 1Ia, 2 + + )NA150–6* thuringiensis 1Aa, 1Ab, 1B, 1Ia, 2 + + )NA193–2* thuringiensis 1Aa, 1Ab, 1B, 1C, 1D, 1Ia, 2 + + )NA193–3* thuringiensis 1Aa, 1Ab, 1B, 1Ia, 2 + + )NA169–2 thuringiensis 2 + ) )NA192–5 thuringiensis 2 + + )NA170–2 tohokuensis none ) ) )NA205–3 tolworthi none + ) )

Aquatic

NA335–2 aizawai 1Aa, 1Ab, 1Ac, 1C, 1D, 1Ia, 2 + + )NA317–3 andalouciensis none ) ) )NA326 kurstaki 1Aa, 1Ac, 1Ia, 2 + + )NA340–1 kurstaki 1Aa, 1Ab, 1Ac, 1D, 1Ia, 2 + + )NA317–9 morrisoni 1Ab, 1D, 2 + + )NA332–6 morrisoni none ) ) )NA334–1 navarrensis none ) ) )NA334–3 navarrensis none ) ) )NA322 nigeriensis none ) ) )NA329–1 pakistani none ) ) )NA332–8 PAK-AMA none ) ) )NA332–4 PAK-AMA none + + )



other cry genes in this serovar were: cry1Aa (12Æ5%), cry1Ac(37Æ5%), cry1Ad (12Æ5%), cry1B (37Æ5%), cry1C (25%),

cry1E (25%), cry1F (25%), cry1G (12Æ5%), cry1Ia (12Æ5%)

and cry1Ib (37Æ5%).

The cry gene content of B. thuringiensis serovar thuringi-ensis strains was also compared in both collections. The

identification of cry genes performed by PCR revealed that

the most frequent genetic profile in both collections was

cry1Aa, cry1Ab, cry1B, cry1Ia and cry2.A v2 analysis was performed in order to determine

whether the distribution of the detected cry genes among

serovars corresponded to a random distribution. This was

done with the 27 strains from the terrestrial collection

belonging to serovars aizawai, thuringiensis and morrisoni.One gene, cry1B, was found to lack the aleatory 1:1:1

distribution (v2 ¼ 6Æ34, d.f. ¼ 2), being more frequent in

thuringiensis strains, whereas cry1C (v2 ¼ 5Æ46, d.f. ¼ 2) fit

the random distribution. The rest of the genes did not differ

significantly from an aleatory distribution. The v2 analysis

performed in B. thuringiensis serovar thuringiensis strains

from terrestrial and aquatic collections revealed that the

frequency of cry1Aa, cry1Ab, cry1B, cry1C, cry1D, cry1Iaand cry2 genes did not differ significantly from the random

1:1 distribution.

Insecticidal toxicity

Table 2 shows the insecticidal toxicity of the 38 strains from

terrestrial habitats and the 24 strains from aquatic habitats.

Within the terrestrial strains, 65Æ8% were toxic against the

two Lepidoptera tested and only 5Æ3% were not toxic against

either of them. Ten strains (26Æ3%) were toxic against one

species and no strains were found to be toxic against the

Diptera (T. oleracea) larvae. In the aquatic collection, 41Æ6%

of strains were non-toxic, 29Æ2% were toxic against all three

species, 25% were active against both lepidoptera species,

Table 1 (Continued)

Insect toxicity

Strain Serovar cry gene(s) H. a. S. e. T. o.

NA334–7 PAK-AMA none ) ) )NA338–2 sotto none ) ) )NA334–5 thuringiensis 2 ) ) )NA319–5 thuringiensis 1Aa, 1Ab, 1B, 1C, 1Ia, 2 + + +

NA319–6 thuringiensis 1Aa, 1Ab, 1B, 1Ia, 2 + + +


NA321–3 thuringiensis 1Aa, 1Ab, 1B, 1C, 1Ia, 2 + + +


NA332–9 thuringiensis 1Aa, 1Ab, 1B, 1Ia, 2 + + )NA335–4 thuringiensis 1Aa, 1Ab, 1B, 1C, 1Ia, 2 + + +

NA335–5 thuringiensis 1Aa, 1Ab, 1B, 1D, 1Ia, 2 + + +

NA323–9 unknown none + ) )

H.a., Helicoverpa armigera; S.e., Spodoptera exigua; T.o., Tipula oleracea.

+ , Mortality ‡ 25%; –, mortality < 25%.

*The cry gene content of these strains has been previously reported (Porcar et al. 2000).

No cry genes were identified with the primer pairs used in the PCR as described in Materials and Methods.

Fig. 1 Frequency of cry1 and cry2 genes in

Bacillus thuringiensis strains from terrestrial

and aquatic habitats. (h), Terrestrial collec-

tion; (j), aquatic collection



and one strain was only toxic against H. armigera. The

relationship between toxicity and gene contents was inves-

tigated. The number of terrestrial strains containing both

cry1 and cry2 was 27 (71%), four of them being toxic to

H. armigera, one toxic to S. exigua and 22 toxic to both.

Seven strains contained either cry1 or cry2 genes, all of them

being active against either H. armigera or S. exigua or both.

Four of the 38 strains did not contain any of the cry genes

tested, one of these strains being toxic to both lepidoptera

species and another, toxic only to H. armigera. Twelve

(50%) of the strains isolated from aquatic habitats contained

cry1 and cry2 genes. All of these strains exhibited dual

toxicity to H. armigera and S. exigua and seven of them were

additionally toxic to T. oleracea.

The number of strains lacking all the genes tested was 11

and only two strains were toxic, one of them being active

against both lepidopteran species and the other against

H. armigera. The crystal protein composition of the strains

serovar thuringiensis was previously analysed by SDS-PAGE

gel electrophoresis (Iriarte et al. 1998, 2000), and it was

shown that cry2 was only expressed in the seven strains of

aquatic habitats that were toxic against T. oleracea.

DISCUSSION

The characterization (serological identification, PCR-based

determination of cry genes contents and insect toxicity) of

two B. thuringiensis collections, originating from terrestrial

and aquatic environments, is reported. A great serological

diversity was found in both habitats. In the terrestrial

habitat, three main serovars (aizawai, thuringiensis and

morrisoni) and nine other serovars with a lower number of

strains were found. In the aquatic habitat, the most

abundant serovar was thuringiensis, with nine strains; eight

other serovars were represented by only one or two strains.

The serological diversity found in this work is in accordance

with Damgaard (2000), who also reported frequency vari-

ation of the serovars found in soil. However, in both

habitats, kurstaki and israelensis serovars were not found in

their expected frequency. In fact, kurstaki is known to be a

very frequent serovar among soil-originating strains (Damg-

aard 2000) although here, only three isolates belonging to

this serovar were found. Also, israelensis is known to be one

of most abundant serovars in mosquito breeding habitats,

such as stagnant ponds (Damgaard, 2000), but no strains of

this serovar were found in the present study. Serovar

israelensis must be very rare or even absent in the surveyed

area, since no strains of this serovar were found in the wide

B. thuringiensis sampling programme performed in Spain

(Iriarte et al. 1998).The determination of cry genes by PCR revealed a high

genetic diversity in both habitats. It was found that the

relative proportion of some genes was very high, whereas for

some other genes, this proportion was very low in the

B. thuringiensis from both habitats. The relative proportion

of cry1Aa, cry1Ab, cry1Ia and cry2 was very high in both

habitats, while the proportion of cry1Ad, cry1E, cry1F,

cry1G and cry1Ib was very low in the terrestrial habitat and

zero in the aquatic habitat. In accordance with the present

results, previous reports have also found that collections of

strains from different environments may differ in their crygenes content. For example, Bravo et al. (1998) found that

the distribution of cry1A, cry1B, cry1C and cry1D was more

frequent than cry1E and cry1F, and Chak et al. (1994)

reported that cry1A, cry1C and cry1D were more abundant

in B. thuringiensis from Taiwan. The distribution of three

Table 2 Insecticidal activity of Bacillus thuringiensis from aquatic and terrestrial habitats

Insecticidal activityà

Gene combination* Habitat n H. a. S. e. H. a + S. e. H. a + S. e + T. o. Non-toxic

cry1 + cry2 Terrestrial 27 4 1 22 0 0

Aquatic 12 0 0 5 7 0

cry1 Terrestrial 4 1 3 0 0 0

Aquatic 0 0 0 0 0 0

cry2 Terrestrial 3 1 0 2 0 0

Aquatic 1 0 0 0 0 1

None Terrestrial 4 1 0 1 0 2

Aquatic 11 1 0 1 0 9

Total Terrestrial 38 7 4 25 0 2

Aquatic 24 1 0 6 7 10

*Gene combinations given correspond to cry1 and cry2 genes shown in Table 1. The gene combination ‘none’ corresponds to strains which lacked

amplification in all PCR performed.

Data given correspond to number of strains.

àStrains were considered as toxic when mortality was ‡ 25%.



genes, cry1C, cry1D and cry1E, was found to be statistically

different in both collections, being more common within the

terrestrial strains. A possible explanation for this might be

the high frequency of these genes in serovars aizawai and

morrisoni, which, although very abundant in the terrestrial

habitat, are very low in the aquatic habitat (see below).

Additionally, the occurrence of cry genes within the three

more abundant serovars in the group of terrestrial strains

was analysed, and the results suggest a non-random

distribution of the cry1B gene among the different serovars.

This gene was very frequent in B. thuringiensis serovar

thuringiensis (62Æ5%) while it was not detected in B. thurin-giensis serovar aizawai. The non-random cry genes–serovar

distribution had been previously reported by Porcar et al.(2000), who found that cry1B was more frequent in serovar

thuringiensis, and by Ferrandis et al. (1999), who found a

similar result for the cry1C gene in strains belonging to

serovar aizawai.It has been proposed that cry1C and cry1D are located on

the bacterial chromosome (Lereclus et al. 1993) and that

they might be genetically linked, at least in some B. thurin-giensis strains (Ferrandis et al. 1999). The present study

suggests this linkage between cry1C and cry1D genes in

strains from serovar aizawai but not in morrisoni strains,

where the frequency of cry1D was greater than cry1C.

Studying the relationship between content of cry genes and

toxicity in both collections, it was found that all the strains

containing cry1 genes alone or combined with cry2 genes were

active on either H. armigera or S. exigua. These results were

expected since the protein products of cry1 and cry2 are

known to be toxic in lepidopteran species (Schnepf et al.1998). It was also found that some strains sharing the same set

of cry genes differed in their toxicity. This result might be

explained by a variation in the level of gene expression, which

can strongly influence the insect toxicity of the bacterial

strain. This is in accordance with the low expression level, if

expressed at all, reported for some cry genes (Lereclus et al.1993; Masson et al. 1998). Insect toxicity studies revealed

that most of the strains were toxic against at least one

lepidopteran. Several strains also exhibited biological activity

against the dipteran T. oleracea. Seven from a total of 17

strains of serovar thuringiensis also exhibited toxicity against

T. oleracea larvae, and all of them were isolated from the

aquatic habitat. Sixteen of these strains carried cry2 genes,

which are known to be dipteran active, but a typical band

in the SDS-PAGE gel corresponding to Cry2 was only

observed in the seven toxic aquatic strains. The dipteran

insecticidal activity of these strains was attributed to the

expression of the cry2 gene, since other dipteran-active genes,

such as cry4A, cry4B, cry10A or cry11A, were not present

in any of the strains analysed. The lack of toxicity against

T. oleracea larvae of the other B. thuringiensis strains in this

study bearing the cry2 gene was attributed to the low or

non-expression of this gene, as has been previously reported

by Lereclus et al. (1993).

It was found that two of 38 (5Æ3%) strains of the terrestrial

habitat and nine of 24 (37Æ5%) of the aquatic habitat lacked

all the cry genes tested by PCR, and these strains were not

active against any of the tested insect species. Other reports,

using different criteria for activity and different target

insects, have also found non-toxic strains, even at high rates

(Ohba and Aizawa 1986; Bernhard et al. 1997), and it is not

clear why an entomopathogen carries genes for the produc-

tion of a non-toxic crystal. However, since these strains

present the parasporal body characteristic of B. thuringiensis,other cry genes coding for proteins but lacking activity

against the species bioassayed may be present and expressed.

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