Distribution of cryl, cryll and cryV Genes within Bacillus thuringiensis Isolates from Spain

System. Appl. Microbiol. 22,179-185 (1999) © Urban & Fischer Verlag _htt-,p_:/_/www __ .u_rb_a_nf_is_ch_e_r.d_e-'./jo_u_rn_a_ls_/s_am _______________

SYSTEfv\41lC AND

APPUED MICROBIOlOGY

Distribution of cry/, cryl/ and cryV Genes within Bacillus thuringiensis Isolates from Spain MARIA D. FERRANDIS 1, VICTOR M. JUAREZ-PEREZ 2 , ROGER FRUTOS 2 , YOLANDA BELl, and JUAN FERREl IDepartament de Genetica, Universitat de Valencia, Burjassot (Valencia), Spain 2BIOTROP-IGEPAM, CIRAD, Montpellier Cedex 1, France Received: February 12, 1999

Summary Using a PCR-based approach, a collection of 223 isolates of Bacillus thuringiensis from Spain was screened for the presence of cry genes belonging to three families. Genes from the cryI, cryII and cryV families were found in 54%, 42%, and 66% of the isolates, respectively. Only 23% of the isolates did not show the presence of any of the genes tested. Frequencies of these genes were compared in isolates from soil samples and from samples of cereal stores and mills, being this higher in the latter. Specific primers were used to detect cryIA(a}, cryIA(b}, cryIA(c}, cryIA(d}, cryIA(e}, cryIB, cryIC, cryID, cryIE, cryIF and cryIG genes. Within the cryI family, the most frequent gene was cryIA(c} (62%), followed by cryIA (a) , cryID, cryIC and cryIA(b} (49, 43, 35, and 34%, respectively). A high frequency of joint occurrence was observed for cryIC and cryID; the latter was present in 93% of the isolates containing cryIC. A random sample of 97 isolates was tested for toxicity against the insect pests Plutella xylostella and Spodoptera exigua. Among the isolates showing toxicity, the most common gene combination was cryIA-cryIC-cryID-cryII-cry V. Although in most cases toxicity could be related to gene content, in some others toxicity was unexpected according to the results obtained by PCR. We found no apparent relationship between gene content in our isolates and the serovar to which they belong. Key words: Bacillus thuringiensis - cry genes - PCR

Introduction The first systematic classification of Bt insecticidal proteins was based on host specificity and sequence homology. Five major classes were distinguished: CryI, Lepidoptera specific; CryU, Lepidoptera and Diptera specific; CryIII, Coleoptera specific; Cry IV, Diptera specific, and Cyt, Diptera specific and cytolytic to a variety of invertebrate and vertebrate cells (HOFTE and WHITELEY, 1989). A new class of Coleoptera and Lepidoptera specific crystal proteins was named CryV (TAILOR et aI., 1992). Inconsistencies in this nomenclature system due to the rapidly increasing number of Cry proteins discovered and the finding of different insecticidal spectra within Cry proteins with high homology, lead to the recent proposal of a new systematic nomenclature based solely on amino acid sequence identity (CRICKMORE et aI., 1998). Screening programs carried out all over the world yielded thousands of Bt isolates, which are maintained in public and private institutions. One of the most important aspects in a Bt collection is its potential to contain

isolates with novel pesticidal activities or increased pesticidal potency. Although the ultimate proof of the toxicity of a given isolate is bioassay testing, methodologies based on PCR have been developed to help the screen process. PCR analysis is not only used to determine the identity of cry genes (genes coding for Cry proteins) in an isolate (CAROZZI et aI., 1991; BOURQUE et aI., 1993; CER6N et al., 1994; CER6N et al., 1995), but also to detect the presence of novel cry genes (KALMAN et al., 1993; Kuo and CHAK, 1996; JUAREZ-PEREZ et aI., 1997). In an effort to characterise a collection of 223 Bt isolates, we have looked for the presence of cryl, cryll and cry V genes and their distribution in isolates from different sources (soil, cereal stores and mills). Gene frequency, as well as the frequency of pair-wise combinations, has been determined for 11 genes belonging to the cryl family. In addition, toxicity tests were performed in 97 isolates against two lepidopteran species, Plutella xylostella and Spodoptera exigua, to determine the relationship between the gene content and the toxicity spectrum. 0723-2020/99/22/02-179 $ 12.00/0

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Material and Methods Bacterial isolates: The Bt isolates used in the present study are part of a collection obtained from an extensive Bt screening program carried out in Spain (lRIARTE et aI., 1998) and were isolated following conventional methods (OHBA and AIZAWA, 1986). From a total of 149 samples collected from soil from agricultural or non cultivated fields, and dust or debris from cereal stores and mills in the eastern and southern of Spain, 3038 Bacillus-like colonies were observed by phase contrast microscopy, from these, 658 isolates were classified as Bt on the basis of the presence of parasporal inclusions. All isolates were subjected to SDS-PAGE (LAEMMLI, 1970) of the crystal proteins to select, within a same sample, those isolates that presented different patterns of protein bands, to obtain the maximum variability and reduce the number of sibling strains, selecting a total of 223 isolates. DNA extraction and PCR analysis: Total DNA was extracted and purified following the method described by DELfCLUSE et al. (1991) with minor modifications: 3 M potassium acetate buffer (pH 5.5) was used to precipitate proteins, and the phenol extraction step was avoided. PCR mixtures were prepared with 1 ]ll of the DNA template in a final volume of 50 Jll as described previously (JUAREZ-PEREZ et aI., 1997; GLEAVE et aI., 1993). Amplifications were carried out in a Perkin-Elmer Cetus thermal cycler following conditions described by .JUAREZ-PEREZ et al. (1997) for cryI and cryIl, and by GLEAVE et al. (1993) for cry V. PCR products were analysed by 1 % agarose gel electrophoresis in 90 mM Tris-borate/2 mM EDTA (pH 7.5-7.8) for 1 hour at 100 V and visualized staining with ethidium bromide. PCR primers: Genes belonging to the cryJ and cryIl families were detected using primers designed to recognise conserved regions for these families and are heretofore referred to as "family primers". The sequence for cryll family primers were designed by multiple alignment of cryll genes, using the Megalign program of DNAStar software package. Two high homology sequences were selected, corresponding to the primers II( +) (5'-AACTCCATCGTTATTTGTAG-3') and II(-) (5'-TAAAGAAAGTGGGGAGT CT T-3') (MASSON et aI., 1998). Primers for cryI (JUAREZ-PEREZ et aI., 1997) and cryV genes (GLEAVE et aI., 1993) have been described previously. The size of expected bands are: 1.5 kb for the cryI family, 1.6 kb for the cryIl family, and 1.2 kb for the cry V family. Identification of genes within the cryl family was carried out using the 1(-) family primer along with a specific primer for either cryIA, cryIB, cryIC, cryID, cry IE, crylF, cryIG, cryIA(a), cryIA(b), cryIA(c), or cryJA(d) (JUAREZ-PEREZ et aI., 1997). For cryIA(e), 1(-) was used in combination with the specific primer (5'-CTCTACTTTTTATAGAAACC-3') (.JUAREZ-PEREZ, 1998) designed from a highly variable region of the gene sequence. An isolate was considered to contain a determined gene only when the amplification product was of the expected size.

Statistical analyses: Frequencies of a given gene in isolates from different sources were compared using the contingency test (SNEDECOR, 1962). Differences were considered significant if the contingency chi-square value was >0.05. Insect toxicity assays: Insecticidal activity against Plutella xylostella and Spodoptera exigua was tested in third instar larvae and neonate larvae, respectively. The assays were performed using spore/crystal mixtures by surface contamination of an artificial diet as described by IRIARTE et al. (1998). Mortality was monitored after 2 days in P. xylostella and after 4 days in S. exigua. As positive controls in the bioassays, spore/crystal mixture prepared from spores from the commercial product DIPEL (Abbot, with Bt kurstaki as the active ingredient) was used for P. xylostella, and from XENTARI (Sandoz, containing Bt aizawai as the active ingredient) for S. exigua. Serological identification: H-serotyping was carried out with the 58 antisera belonging to the serovars described at the moment, according to the method described by DE BARJAC (1981) with minor modifications (IRIARTE et aI., 1998).

Results Identification of genes from the cryI, cryIJ, and cryV families Family primers were used to analyse 223 Bt isolates for the presence of genes from the cryI, cryIl, and cryV families (Table 1). Of these, 52 isolates (23 %) did not show the presence of genes from these families; 121 isolates (54%) contained cryI genes, (42%) contained cryIl genes and 148 (66%) contained cryV genes. Differences in gene frequency were found depending on the source of the sample. The proportion of isolates without any gene of the tested families was significantly higher within isolates from soil samples (42% vs. 18% in isolates from stores-mills samples). The frequency of cry I, cry II, and cry V genes was significantly lower in isolates from soil samples (29%, 21 %, and 52%, respectively) than from stores and mills samples (62%,49%, and 71 %, respectively). However, considering just the subgroup of isolates with cry genes, the frequency of cry V was not significantly different in isolates from the two sources of samples (90% in isolates from soil vs. 86% in isolates from stores and mills), whereas the frequency of the other type of genes was significantly lower in isolates from soil (50% for cry I and 37% for cryIl) than in isolates from stores and mills (75% for cryI and 59% for cryIl).

Table 1. Distribution of genes from the cryJ, cryIl and cryV families within Bt isolates from different sources. Source

Total isolates

No. of isolates without any of the tested cry genes

No. of isolates with cry genes cryJ

cryIl

cryV

Cereal stores and mills Soil

171 52

30 (18%) 22 (42%)

106 (62%) 15 (29%)

83 (49%) 11(21%)

121 (71%) 27 (52%)

Total

223

52 (23%)

121 (54%)

94 (42%)

148 (66%)

Distribution of cryI, cryIJ and cry V genes

Identification of genes within the cryI family Isolates giving a positive reaction with cryI family primers were tested with specific primers for 11 genes of this family (Table 2). All the isolates reacting positively with the cryI family primers contained at least one gene of the cryIA group. Regarding the frequency of the individual genes, the most frequent was eryIA(c), which appeared in 62 % of the isolates. Other genes found at high frequency were cryIA(a), cryID, cryIC, and cryIA(b), which appeared in 34-49% of the isolates; cryIB appeared at a moderate frequency (17% of the isolates) and the rest of genes appeared at a low or very low frequency (0.8-7.4%). Regarding the source of the isolates, no significant difference in frequencies were found for any given gene according to the contingency test.

Pair-wise combinations between cryI genes Of the 121 isolates containing genes of the cryI family, 98 contained combinations of two or more cryI genes. In the few isolates containing a single gene, this was either cryIA(a), cryIA(b), or cryIA(c). Table 3 shows the frequency of pair-wise combinations between commonly occurring cryI genes (cryIA(a), cryIA(b), cryIA(c), cryIB, cryIC and cryID, referred to as "genes tested", which appeared in at least 17% of the isolates) and each of the cryI genes tested (referred to as "associated genes"). Two genes clearly stood out against the others because of their high frequency of joint occurrence: eryIC and cryID. The latter was present in 93% of the isolates containing

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cryIC, and the former in 72% of the isolates containing cryID. Among the other genes, cryIA(a) was found in 58-89% of the isolates containing either cryIA(b), eryIA(c), cryIB, cryIC, or cryID, and cryIA(c) in 63-74% of the isolates containing either cryIA(a), cryIA(b), cryIB, cryIC, or cryID. Other combinations found in a frequency higher than 0.5 were cryIC and cryID in isolates containing cryIA(a), and cryID in isolates containing cryIA(b). Insecticidal activity of the isolates A random sample of 97 isolates was tested against the two lepidopterans P. xylostella and S. exigua. Among them, 19 isolates were found to be highly toxic for at least one of the two species (Table 4). The highest number of highly toxic isolates was found within the group containing the cryIA-cryIC-cryID-cryll-cryV gene combination. Other highly toxic isolates were found to contain the cryIA-cryll-cry V, cryIA-cryIB-cryll-cry V, cryIAeryIC-cryID-cry V and cryIA-cryIB-cryIC-cryID-cryllcry V gene combinations. Interestingly, two highly toxic isolates were found within the group of isolates without any of the cryI genes tested, though they both contained a cryV gene and one of them a cryll gene as well.

Serological identification Some of the isolates containing different cryI gene combinations were classified on the basis of their flagel-

Table 2. Distribution of genes from the cry! family within Bacillus thuringiensis isolates from different sources. Source

Cereal stores and mills Soil

Total isolates with cryI genes

Isolates containing a determined cryI gene (percentage)!

106

52 (49)

36 (34)

31 (61)

15

7 (47)

5 (33)

10 (67)

cryIA(a) cry/Alb) cry/A (c) cryIA(d) cryIA(e) cryIB 1 (1)

6 (6)

cryIC

cryID

cryIE cryIF

15 (14) 38 (36) 48 (45) 9 (8) 5 (33)

4 (27)

4 (27)

1 (1)

cryIG 2 (2)

-

------------------------------------------------------ -------------------------------------------

Total 1

121

59 (49)

41 (34)

75 (62)

1 (1)

6 (5)

20 (17) 42 (35) 52 (43) 9 (7)

1 (1)

2 (2)

Percentage are referred to the number of times a determined gene appears within the isolates from a particular source.

Table 3. Frequency of pair-wise combinations between cryI genes. The only combinations considered were those between commonly occurring cryI genes (referred to as "genes tested", which appeared in at least 17% of the isolates) and each of the cryI genes tested (referred to as "associated genes"). Gene tested cryIA(a) cryIA(b) cryIA(c) cryIB cryIC cryID

Frequency of associated gene None

cryIA(a) cryIA(b) cryIA(c) cryIA(d) cryIA(e) cryIB

0.05 0.02 0.25 0 0 0

0.65 0.58 0.89 0.88 0.70

0.45 0.36 0.32 0.38 0.49

0.73 0.63 0.63 0.74 0.64

0 0.02 0.01 0 0.02 0.02

0.06 0.15 0.04 0 0 0.02

0.25 0.17 0.16 0.17 0.15

cryIC

cryID

cryIE cryIF cryIG

0.60 0.44 0.41 0.37

0.58 0.63 0.45 0.42 0.93

0.05 0.22 0.04 0.05 0.02 0.15

0.72

0 0.02 0.01 0 0 0.02

0 0 0.02 0 0 0

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Table 4. Toxicity against Plutella xylostella and Spodoptera exigua in a sample of isolates with different combinations of cryI genes. No. of isolates tested

Gene combination

7

No cryI, cryII, or cryV gene cry V cryII, cryV cryIA cryIA, cryIJ cryIA, cryV cryIA, cryIJ, cryV cryIA, cryIB, cryIJ, cryV cryIA, cryIC, cryIJ, cryV cryIA, cryID cryIA, cryID, cryII, cry V cryIA, cryIE, cryV cryIA, cryIG, cryII, cryV cryIA, cryIC, cryID cryIA, cryIC, cryID, cryV cryIA, cryIC, cryID, cryII, cryV cryIA, cryID, cryIE, cryIJ, cryV cryIA, cryIB, cryIC, cryID, cryIJ, cryV cryIA, cryIC, cryID, cryIp, cryII, cryV

No. of highly toxic isolates a

P. xylostella

S. exigua

1

36 6 5 1

1

1 9 5 1

1

5

1 1 2 2

2 3 12

4

3 4

1 1

1 1

Strains were considered highly toxic when mortality was as similar or higher than in the positive control. Spore/crystal mixtures prepared from DIPEL or from XENTARI were used as positive controls for P. xylostella and S. exigua, respectively.

a

Table 5. Serological classification of isolates with different cry! gene combinations. Gene combination

n

serovar

No cryI gene cryIA cryIA, cryIB cryIA, cryIC cryIA, cryID cryIA, cryIE cryIA, cryIG cryIA, cryIC, cryID cryIA, cryID, cryIE cryIA, cryIB, cryIC, cryID cryIA, cryIC, cryID, cryIF

7 9 2 2 2 2 2 9 2 2

dendrolimus, kenyae, fukuokaensis, leesis, londrina, mexicanensis ostriniae, thuringiensis, mexicanensis, alesti, kurstaki, tohokuensis thuringiensis, kurstaki aizawai, sumiyoshiensis morrisoni fukuokaensis galleriae kurstaki, medellin, aizawai, thuringiensis, darmstadiensis, thompsoni ostriniae kenyae aizawai

1

lar antigen (Table 5). Isolates containing the same gene combination were found to belong to different serovars. Conversely, a same serovar was assigned to isolates with different gene combinations. It is interesting to note that the cryIF and cryIG genes were detected in isolates belonging to the same serovars in which they were originally described (aizawai and galleriae, respectively) CHAMBERS et al., 1991; SMULEVITCH et al., 1991).

Discussion Genes from the cryI, cryll and cryV families were found widely distributed within our isolates. Comparing isolates from soil with those from cereal stores and mills, the former presented a lower frequency of genes from the

above families and a higher percentage of isolates without any of the tested genes. Several studies on the occurrence of cry genes in Bt collections have rendered different results. In the present study, cryI genes were detected in 121 out of 223 isolates (54.2 % ). In a similar study of distribution of cry-type genes carried out in Taiwan (CHAK et al., 1994) cryI genes were found in 221 out of 225 isolates (98.2%), and in a study of Bt isolates from Israel, Kazakhstan and Uzbekistan, the occurrence was in 72 out of 215 isolates (33.5%) (BEN-Dov et al., 1997). Using an enzyme-linked immunosorbent assay, cryI-type proteins were detected in 47 out of 241 isolates (19.5%) in a Bt collection of Mexican isolates (CER6N et al., 1994). Studies on the frequency of cryll genes are more restricted: cryll genes were found in 61 out of 215 isolates (28.4%) in samples from Israel, Kazakhstan and

Distribution of cryI, cryll and cryV genes Uzbekistan (BEN-Doy et al., 1997), whereas in 94 out of 223 isolates (42.2%) in the present work. The high variability found in the distribution of cryl and cryll genes among different studies may reflect changes in frequency depending of the habitat and the geographical area where the samples were collected. To our knowledge, there are no previous studies on environmental distribution of cry V genes, however, they have been found in 12 out of 24 isolates (50%) from the Bacillus Genetic Stock Center (the Ohio State University, Columbus, Ohio, U.S.A.) (SHIN et al., 1995) and in 7 out of 21 different serotypes (30%) (GLEAVE et al., 1993). Our results indicate that cry V genes are commonly distributed among Bt isolates (in 148 out of 223 isolates, or 66.4%, in the present study). Even though the frequency of cryI genes was lower in samples from soil, within the group of isolates containing cryl genes, the frequency for any individual cryl gene was similar in isolates from the two sources. The cryl gene variability in our collection is higher than that found in other similar studies (CER6 N et al., 1994; CHAK et al., 1994; BEN-Doy et al., 1997). Although at low frequencies, we could detect the cryIA(d), cryIA(e), cryIB, crylE, cryIF and cryIG genes. Among these genes, CER6N et a1. (1995), using primers for cryIB, cryIE, cryIF, and cryIG, could not find cryIE, cryIP nor cryIG in a collection of 181 isolates; in the other two studies (CHAK et al., 1994; BEN-Doy et al., 1997), using primers for cryIB, cryIE, cryIF, neither of these genes were detected in collections of 225 and 215 isolates. In contrast with other studies (BEN-Doy et al., 1997; CHAK et al., 1994; CER6N et al., 1994) all our isolates containing cryI genes harboured at least one gene of the cryiA group. The most frequent situation is to find combinations of more than one cryI gene, although cryIA(a), cryIA(b} and cryIA(c} could be found alone. Table 3 shows that cryIC and cryID genes appear frequently together: cryIC was practically found only when crylD was present, whereas cryID could be found at a moderate frequency without cryIG. These results have also been found by other authors (CHAK et al., 1994; BEN-Doy et aI., 1997) and suggest that there is a strong genetic linkage between these two genes. A Bt aizawai strain was shown to contain cryIC and cryiD separated 3 kb apart in the same replicon (SANCHIS et al., 1988 ). The fact that the frequency of isolates carrying crylD but not cryIC is much higher than those carrying cryIC without cryiD might be a consequence of some evolutionary event as, for example, one in which cryIC would have become deleted, or one in which cryIC would have been negatively selected for after a recombination process that separated both genes. Our results also show that cryIB and cryIC appear commonly in combination with cryIA(a}. Although results can vary depending on the type of bioassay and the source of the purified crystal protein, it is widely accepted that P. xylostella is highl y susceptible to CryIA's and CryIB toxins, somewhat less susceptible to CryIC and CryIF, not susceptible to CryIE, and that there are contradictory results regarding CryID (FERRE et al., 1991; GRANERO et al., 1996; TABASHNIK et aI. , 1994; TANG et al., 1992). It is also accepted that S. exigua is

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susceptible to CryIC and CryID, little or not susceptible to CryIA's, CryIB and CryIF, and that there are contradictory results with CryIE (CHILCOIT and WIGLEY, 1993; DE MAAGD et aI., 1996; CHAMBERS et aI., 1991; VISSER et al., 1990; MASSON et al., 1992; MACINTOSH et al., 1990). If PCR screening was to be applied to find isolates toxic to these two target insects, isolates with the cryIA and cryIB genes would have been selected as potentially toxic to P. xylostella, and isolates with the cryIC and cryID genes as potentially toxic to S. exigua. In the present work, among the isolates that showed high toxicity against S. exigua, 8 harboured cryIC and cryID genes, but 5 only had cryIA genes and one had no gene of the cryI family. For P. xylostella, 5 isolates harboured cryIA genes, but one had no cryI gene. Therefore, screening based on the gene content would have missed a considerable number of highly toxic isolates and, thus, the opportunity to discover novel genes coding for toxic proteins. However, for screening purposes, PCR approaches will give a fast answer on the presence/absence of cry genes but PCR does not tell if the genes are expressed or not. A cry gene, detected by PCR, can be interrupted, mutated, or under control of a defective promoter. This means that a gene can be detected but the corresponding protein may not be present or present at reduced levels, therefore, contributing nothing or minimally to the toxicity. PCR is useful when there is a very high number of isolates and the goal is to select toxic ones no matter missing a certain percentage of them or when finding novel genes is secondary. Bioassay is always the ultimate test to assess the toxicity of an isolate. It is well known that the classification based on flagellar antigens does not reflect the pathotype of a strain (DE BARJAC, 1990). Since strain toxicity is a consequence of the cry gene content, it was not surprising to find no apparent relationship between serovar and the gene content in our isolates. It is interesting to note that the only serotyped isolate containing cryIF was classified as serovar aizawai, since the isolate from which the gene was first discovered belonged to this same serovar. Similarly, the two serotyped isolates containing cryIG were classified as serovar galleriae, also the same serovar of the isolate from which this gene was first discovered . Therefore, for at least these two cases it seems that there is a relationship between the type of gene and the serovar. We do not know whether this relationship might reflect a more recent evolutionary origin of these genes, and thus that not enough time would have elapsed as to allow them to become more randomly distributed among serovars by natural conjugation. Acknowledgements We acknowledge PATRICIA LOUISOR for her assistance in PCR screening. This research was supported by the Spanish Comisi6n Interministerial de Ciencia y Tecnologfa (CICYT), and by contract with INDUSTRIAS AFRASA, S. A., within the Plan Tecnologico de la Comunidad Valenciana (IMPIVA). M. D. F. was supported by a grant from the Ministerio de Educacion y Ciencia and V. J. P. by a grant from CONACyT (Mexico D. E, Mexico).

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M. D. FERRAND IS et al.

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Corresponding author: Dr. JUAN FERRE, Departament de Genetica, Universitat de Valencia, Dr. Moliner, 50, 46100 Burjassot, Valencia, Spain. Phone: (3496) 386-4506; Fax: (3496) 398-3029 e-mail: [email protected]