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Telomere fingerprinting for assessing chromosome number, isolate typing and recombination in the entomopathogen Beauveria bassiana
Mycol. Res. 107 (5): 572–580 (May 2003). f The British Mycological Society
572
DOI: 10.1017/S0953756203007573 Printed in the United Kingdom.
Telomere fingerprinting for assessing chromosome number, isolate typing and recombination in the entomopathogen Beauveria bassiana
J. PADMAVATHI, K. UMA DEVI*, C. Uma Maheswara RAO and N. Nageswara Rao REDDY Department of Botany, Andhra University, Visakhapatnam, 530 003, AP, India. E-mail : [email protected] Received 22 June 2002; accepted 18 January 2003.
Beauveria bassiana is a popular biocontrol agent used as ‘ green’ pesticide in crop insect pest management. Chromosome number has been variously reported as five, six, seven and eight in this species. The range of chromosome number and the minimum chromosome number in this economically important fungus were assessed through telomere fingerprint analysis of a sample of 17 isolates from different and similar hosts and distant and same geographic origin. Genomic DNA digested with EcoRI, which has no cutting site in the telomere repeat sequence arrays was probed with a radioisotope-labelled (5k-TTAGGG-3k)8 oligonucleotide. The probe-hybridised regions appeared as discrete bands – each representing a telomere. The number of bands in each lane was counted and halved to arrive at the chromosome number of that isolate. The chromosome number varied from 5 to 10 in the different isolates. The telomere probe hybridised bands were also scored for presence or absence in a 0–1 matrix and a dendrogram based on similarities between the isolates was constructed using the NTSYS-pc ver. 2.02i software. The isolates showed very little similarity ; the overall similarity was 14 %. Only two isolates which were of diverse host and geographic origin showed 100% similarity. Isolates from the same epizootic that showed 43 % similarity in their telomere fingerprints had 96 % similarity in their RAPD (Random amplified polymorphic DNA) fingerprints with 10 primers. The genetic distances computed from any one DNA fingerprinting method thus do not reflect the true genetic similarities of the isolates. The frequency distribution pattern of the pair-wise similarities computed from telomere fingerprints hinted at the occurrence of recombination in this fungus. Telomere fingerprinting proved very useful in typing isolates since each of them was found to have a unique fingerprint. Isolates with the same chromosome number neither showed a distinct morphology or virulence character nor a close similarity in telomere or RAPD fingerprints to merit their subgrouping into a taxonomically relevant or practically useful unit.
INTRODUCTION Beauveria bassiana is an ubiquitous, mitosporic fungus with a very wide and diverse host range of nearly 750 insect species (Inglis, Magalhaes & Inglis 2001). It has the longest history as an experimental mycoinsecticide (Leathers, Gupta & Alexander 1993), and is reported to have been widely used and to be one of the seven registered mycopesticides now on the market (Butt, Jackson & Magan 2001). Knowledge of the genomic organization of this fungus is important when strain improvement programmes are undertaken. Karyotype analysis is one approach to the characterization of entomopathogenic fungal isolates because, a high level of chromosomal polymorphism (in number and size) is known to exist in these organisms (Kistler & Miao 1992, Zolan 1995, Bidochka 2001). The karyotype of an isolate has been found to be a stable genetic marker in * Corresponding author.
many fungal species (Zolan 1995) including B. bassiana (Couteaudier & Viaud 1997). In mitosporic fungi, observation of chromosomes under a microscope is difficult because mitotic material is not suitable for slide preparations for such analysis (Zolan 1995). Usually, pulsed field gel electrophoresis method involving clamped homogenous electrical field (CHEF) termed ‘electrophoretic karyotyping ’ is employed for karyotype studies in such fungi (Zolan 1995, Bidochka 2001). In B. bassiana isolates, the chromosome number estimated from telomere fingerprints corresponded to the number estimated through the CHEF technique (Viaud et al. 1996). Telomere fingerprints were used also in other entomopathogenic fungi, notably Nomuraea rileyi and Metarhizium anisopliae, to assess the chromosome number (Boucias et al. 2000, Inglis et al. 1999). Telomere fingerprinting is reported to substitute for inadequately resolved chromosomes in electrophoretic karyotyping in estimation of chromosome numbers (Zolan 1995). B. bassiana has been reported to be
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Table 1. Details of isolates of Beauveria bassiana used in the telomere fingerprint analysis. Isolatea
Source insect
Order of host
Geographical origin
Chromosome no.
BB2 ARSEF 3120 ARSEF 739 ARSEF 326 ITCC 913 ARSEF 1512 ARSEF 1149 ARSEF 1316 ARSEF 1169 ITCC 4521 ARSEF 2860 ARSEF 1314 ARSEF 1166 ITCC 1253 ARSEF 3286 NRRL 20699 NRRL 3108
Spodoptera litura Senecio sp. Diabrotica paranoense Chilo plejadellus Unknown Spodoptera littoralis Helicoverpa armigera Helicoverpa virescens Sitona lineatus Diatraea saccharalis Schizaphis graminum Helicoverpa virescens Helicoverpa armigera Musca domestica Spodoptera littoralis Unknown Ostrinia nubilalis
Lepidoptera Homoptera Lepidoptera Lepidoptera Unknown Lepidoptera Lepidoptera Lepidoptera Coleoptera Lepidoptera Homoptera Lepidoptera Lepidoptera Diptera Lepidoptera Unknown Lepidoptera
Bangalore, S. India Yvelines, France CNPAF, Brazil Queensland, Australia Netherlands La Minie`re, France Cordoba, Spain La Minie`re, France Sennecille, France Karnal, N. India Idaho, USA La Minie`re, France Cordoba, Spain Mumbai, India Montpellier, France Illinois, USA Unknown
10 8 7 7 7 6 6 6 6 6 6 6 5 5 5 5 5
a
ARSEF, USDA-ARS collection of Entomopathogenic Fungi (ARSEF, Ithaca, New York); NRRL, Peoria, Illinois; ITCC, Indian Type Culture Collection, IARI, New Delhi. BB2 was isolated from a local field and is being accessioned.
haploid (St Leger et al. 1992). The chromosome number in this species has been variously reported to be five, six, seven and eight (Paccola-Meirelles & Azevedo 1991, Shimizu, Higashiyama & Matusumoto 1993, Pfeiffer & Khachatourians 1993, Viaud et al. 1996). Few isolates have been examined. In all filamentous fungi whose telomeres have been investigated, a 6 bp repeat sequence of 5k-TTAGGG-3k has been found (Levis et al. 1997). The Bal 31 exonuclease sensitivity of genomic regions (bands) hybridising with the telomere specific probe (TTAGGG)n was tested in B. bassiana (Viaud et al. 1996). The results confirmed the presence of this repeat in the telomeres. No telomere probe hybridised band was found that was insensitive to Bal 31 nuclease digestion (Viaud et al. 1996) indicating that telomere repeat sequences are not present in the intercalary regions of the chromosomes in B. bassiana. When a restriction enzyme with no recognition sequence in the telomeres constituted by a 5k-TTAGGG3k repeat array is used to digest the genomic DNA, among the fragments, those containing mostly telomeric DNA are generated. The size of the telomeres is reported to vary between chromosomes and between chromosome arms of a chromosome, in the chromosome complement of an organism (Zakian 1989). If the size differences are discrete and non-overlapping, the number of telomeres would equal the number of hybridising bands in the autoradiograms. In B. bassiana and other entomopathogenic fungi, the telomere probehybridised regions appeared as discrete bands (Viaud et al. 1996, Couteaudier & Viaud 1997, Inglis et al. 1999, Boucias et al. 2000). The range of variation in chromosome number in B. bassiana is studied using a larger sample size of 17 isolates through telomere fingerprinting. Telomere fingerprinting is a less sophisticated technique than CHEF ; besides, it provides a means of simultaneously typing individual isolates and assessing the level of genetic polymorphism in the species.
MATERIALS AND METHODS Isolates Beauveria bassiana isolates procured from the ARSEF, NRRL and ITCC culture collections, and from our local fields, were analysed (Table 1). The sample composition was chosen to represent both isolates of diverse host and geographic origin as well as isolates from similar hosts collected from the same region (Table 1). Telomere fingerprinting The fungal isolates were grown in liquid Sabouraud dextrose medium and DNA was isolated from mycelium as described by Uma Devi et al. (2001). The DNA (8–10 mg) was digested to completion with EcoRI (Genei, Bangalore) following manufacturers instructions. Eight isolates were also digested with HindIII (Bangalore Genei). The standard protocol for RFLP (Restriction fragment length polymorphism) as described in Sambrook, Fritsch & Maniatis (1989) was followed. A 1 kb ladder (Life Technologies, Alameda, CA) and l HindIII and wr174 Hae III digests (Finnzymes, Finland) were used as DNA size markers. An oligonucleotide constituted by telomere repeat sequence – (5k-TTAGGG-3k)8 (Genmed Synthesis, San Francisco) was 5k-end-labelled with P32 using T4 polynucleotide kinase to serve as the probe. Analysis of autoradiograms The autoradiograms were marked to indicate the position of the DNA size markers and photographed under white light with a Kodak DC 120 digital camera. The number of bands in each lane was counted, and the intensity of each band was noted. Sizes for individual bands were assigned using Kodak 1D software. The chromosome number was estimated by halving the total
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(a)
(b)
Fig. 1. Autoradiograms showing telomeric fingerprints of isolates of Beauveria bassiana : (a) and (b) are fingerprints of genomic DNA digested with EcoRI and HindIII respectively. The size (in kb) of DNA fragments are shown (1 kb ladder, Life technologies) on the left and l HindIII+wr174 HaeIII (Finnzymes) on the right.
number of bands since each chromosome has two telomeres. When the number of bands was odd, it was rounded up to the next even number (Viaud et al. 1996, Boucias et al. 2000) since, one of the bands in such cases, may represent the overlapping of two similar sized telomeres. The correlation of differences in chromosome number among the B. bassiana isolates to their morphological traits for which they had been characterized (Padmavathi 2002) was examined. The bands in all lanes were scored manually as a binary matrix (1=present, 0=absent). The data was analysed using NTSYS-pc ver. 2.02i software. A similarity matrix was generated using the SIMQUAL program and Jaccard’s similarity coefficient (Sneath & Sokal 1973). A dendrogram displaying the similarities between the isolates was generated using the SAHN program by UPGMA (Sneath & Sokal 1973). The genetic distances between the Beauveria bassiana isolates was
visualized in a 3D-picture plotted along the first 3 principal coordinate axes through principle coordinate analysis (PCA). The distribution of the distances was graphically plotted to assess the reproductive pattern in the worldwide population of B. bassiana. A comparison of the phenetic groups based on telomere fingerprints of these B. bassiana isolates with those observed in the analysis of RAPD fingerprints generated with ten primers (Padmavathi 2002) was made.
RESULTS The regions that hybridised with the telomere sequence appeared as discrete bands. In autoradiograms of EcoRI cut DNA, 9–19 bands were observed (Fig. 1a, Table 2) while the HindIII cut DNA showed 9–25 bands
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Table 2. Number and size range of DNA fragments that hybridized with telomere specific probe (TTAGGG)n in the autoradiograms and the estimated chromosome number in the isolates of Beauveria bassiana. EcoRI Bands HindIII Bands Band sizes (in kb) Isolate
No.
Size (Kb) range
Unique
Rare
Commona
No.
Size (Kb) range
Chromosomes no.b
BB2
19
23.5–0.6
5.6, 2.5
23.5, 13, 4.6
25
13.0–0.57
10
ARSEF 3120
15
23.0–0.56
ARSEF 739
14
23.0–1.6
ARSEF 326
13
24.0–0.74
ITCC 913
13
20.0–0.56
ARSEF 1512
12
23.0–1.0
ARSEF 1149
12
23.0–1.0
ARSEF 1316
12
20.0–0.6
ARSEF 1169
12
23.0–0.6
ITCC 4521
11
14.0–1.7
ARSEF 2860
11
14.0–1.0
ARSEF 1314
11
20.0–0.6
ARSEF 1166
10
23.0–1.0
ITCC 1253
10
8.3–1.0
ARSEF 3286 NRRL 20699 NRRL 3108
10 9 9
23.5–0.39 23.5–2.0 20.0–1.2
23, 20, 16, 11.9, 9.3, 8.5, 7.9, 3.9, 3.6, 3.4, 2.7, 1.3, 1.2, 0.6 23, 16, 9.3, 8.9, 7.9, 6.5, 6.3, 5.5, 4.5, 4.3, 3.9, 3.3, 3, 1.9, 0.56 23, 16, 9.3, 8.5, 8.3, 5.7, 3.9, 3.6, 3.5, 3.2, 2.7, 1.9, 1.6 20, 16, 11.9, 8.9, 7.9, 6.3, 4.3, 3.6, 2.6, 2.4, 1.9 20, 14, 11.9, 9.3, 8.9, 8.5, 8.3, 6.5, 5.7, 4.3, 3.6, 2.7, 0.56 23, 20, 16, 11.1, 8.5, 8.3, 6.3, 4.3, 3.4, 2.4, 1.9, 1 23, 16, 11.1, 8.5, 8.3, 6.3, 4.3, 3.4, 2.4, 1.9, 1 20, 16, 9.3, 8.9, 7.9, 6.5, 5.7, 4.5, 3.2, 2.6, 1.9, 0.6 23, 20, 16, 14, 11.9, 9.3, 8.5, 7.9, 6.3, 4.5, 3.9, 0.6 14, 9.3, 8.9, 8.3, 6.5, 5.7, 4.5, 3.5, 3.2, 2.4 14, 11.9, 9.3, 8.5, 5.7, 5.5, 4.3, 3, 2.6, 2, 1 20, 16, 11.9, 8.9, 6.5, 5.7, 2.7, 1.9, 1, 0.6 23, 20, 16, 11.1, 8.5, 8.3, 6.3, 4.3, 3.4, 2.4, 1.9, 1 8.3, 7.9, 6.5, 6.3, 4.3, 3.6, 3.3, 2.6, 1.3, 1 20, 11.1, 8.5, 7.9, 5.5, 3.4, 1.9 16, 14, 11.1, 8.3, 6.3, 4.3, 3.4, 2 20, 16, 14, 7.9, 5.5, 4.5, 3.9, 1.6, 1.2
a b
13 24, 0.74
21
1.7
4.6
4.6 0.39
23.5, 21 23.5, 21
–
8
11
9.0–0.39
7
7
5.5–0.56
7
–
7
–
6
–
6
–
6
12
6.1–0.51
– 19
6 6
12.0–0.56
6
–
6
–
5
–
5
16 9 10
10.8–0.8 6.0–0.55 6.1–0.6
5 5 5
Present in more than three isolates. Estimated from number of EcoRI fragments.
(Fig. 1b, Table 2). The telomere sequences of only eight of the 17 fungal isolates analysed were cut with HindIII. The size range of the bands in EcoRI blots was 24 kb to 0.39 kb while in HindIII blots, it was 13 kb to 0.39 kb (Figs 1a–b, Table 2). Thus bands that hybridised with the telomere sequence probe were smaller in size in HindIII digests than in EcoRI digests. In the HindIII digests, the bands were more intense than in the EcoRI blots (Fig. 1b). The much smaller sized and more intensely dark telomere probe-hybridised bands in HindIII autoradiogram compared to the EcoRI autoradiogram, hints at the presence of recognition sequence of this enzyme in the telomere regions. The chromosome number as estimated from the number of bands in the EcoRI autoradiogram ranged from 5–10 in the different isolates (Table 2). The isolates with similar chromosome number were not found to have a distinct morphology (Table 3) or any relation to the original host or geographic region (Table 1). The cluster analysis of the telomere banding pattern observed in EcoRI generated fragments revealed a very high degree of variability among the isolates (Fig. 2).
Mean similarity values for pair-wise comparisons ranged from 0.14 to 1.00. There was just 14 % overall similarity among the isolates. A cophenetic correlation value of c=0.89 was obtained. It indicated that the UPGMA cluster analysis was statistically significant. Only two isolates ARSEF 1166 and ARSEF 1512 showed 100 % similarity (Fig. 2). They are of different host and geographic origin, the former being from France and the later from Spain (Table 1). These two isolates showed 85 % similarity with ARSEF 1149, which is from the same place in Spain as ARSEF 1166 collected two days later in the same epizootic (Table 1, Fig. 2). On the other hand, isolates ARSEF 1314 and ARSEF 1316 collected in the same place from the same insect species at the same time (Table 1) showed only 43 % similarity (Fig. 2) in telomere fingerprints. The wide variation among the isolates is evident from the dispersed distribution of the Beauveria bassiana isolates on the 3D-scatter plot generated from PCA (Fig. 3). The mean and variance of the pair-wise distances in the similarity matrix are 3.56 and 1.82 respectively. The graphical representation of frequency distribution
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Table 3. Morphological characteristics and phenetic group based on RAPD analysis of the isolates of Beauveria bassiana with different chromosome number (as determined from telomere fingerprinting). Range of pH Isolate
Chr. No.
Colony morphologya
Growth rateb
BB2 ARSEF 3120 ARSEF 739 ARSEF 326 ITCC 913 ARSEF 1512 ARSEF 1149 ARSEF 1316 ARSEF 1169 ITCC 4521 ARSEF 2860 ARSEF 1314 ARSEF 1166 ITCC 1253 ARSEF 3286 NRRL 20699 NRRL 3108
10 8 7 7 7 6 6 6 6 6 6 6 5 5 5 5 5
Dusty Fluffy Chalky Fluffy Dusty Dusty Dusty Chalky Fluffy Matty Chalky Chalky Dusty Chalky Fluffy Matty Dusty
High Medium Medium High High High High Medium Medium High Medium Medium High Medium Low High High
Conidia cmx2 c
Germ. Speedd
Tolerance
Optimum
RAPD phenetic groupe
Medium High Low High Medium Medium Medium Low High Low Low Low Medium Low High Low Medium
Medium Slow Fast Medium Slow Fast Fast Slow Slow Fast Fast Fast Medium Slow Fast Slow Slow
4–14 5–13 5–14 4–13 4–14 4–14 4–14 5–14 5–13 4–13 5–14 5–14 4–14 5–14 5–13 4–13 4–14
6–12 5–13 5–13 9–13 5–13 5–8 5–8 5–13 5–13 5–13 5–13 9–13 5–8 5–13 6–13 5–13 9–10
1Aixa 3 1B Iy 1A Iy 1B iyI aii a 1B Ix bI 1B Ix aib 1B Ix aii 1A Ixb 1B IIx 1B Iiy b 1B Ix aia 1B Ix bI 1B Ix bii 1A IIx 3 1A IIy
a
Gross colony morphology on solid Sabouraud dextrose medium (MacLeod 1954). As measured from dry weight of biomass accumulated in 10 d cultures in liquid Sabouraud dextrose medium at 25¡1 x – High o400; Medium o300 & low f300 mg. c Conidia produced per cm2 on 10 d cultures on solid Sabouraud dextrose medium overlaid with cellophane film at 25¡1 x (High c4.5r108 ; Medium c2.5r109 ; Low c1.5r10 9 ; Very low c1.5r10 6). d Time taken for y100% conidial germination on solid Sabouraud dextrose medium at 25¡1 x (Fast=12 h; Medium=14 h and Slow=20 h). e From Padmavathi (2002). b
of the distance values (Fig. 4) indicates the prevalence of recombination in these isolates (Taylor et al. 1999). Isolates ARSEF 1166 and ARSEF 1512 that showed 100 % similarity in their telomere fingerprints (Fig. 2), also had a close (87 %) resemblance in their RAPD profiles generated from ten primers (Fig. 5). The other isolate, ARSEF 1149 which showed 85 % similarity in its telomere fingerprint to these two isolates (Fig. 2), however, had a very different RAPD profile and shared only 54 % similarity with them in the RAPD profiles (Padmavathi 2002). ARSEF 1149 had very similar RAPD pattern as ARSEF 1314 and ARSEF 1316 (Fig. 5) and clustered with them at 94% similarity (Padmavathi 2002). The isolates ARSEF 1314 and ARSEF 1316 are from the same epizootic (Table 1) collected on the same day, but in their telomere fingerprints they shared only 43 % similarity between them and 18 % similarity with ARSEF 1149, the isolate which they very closely resembled in their RAPD fingerprints (Fig. 5).
DISCUSSION Different workers have reported the chromosome number in Beauveria bassiana variously as five (PaccolaMeirelles & Azevedo 1991), at least six (Shimizu et al. 1993), eight (Pfeiffer & Khachatourians 1993) and seven or eight (Viaud et al. 1996). However, different isolates were analysed by each group. The differences in the reported chromosome numbers indicate the existence
of chromosomal number polymorphism in this species. In our investigation, chromosome numbers of five, six, seven, eight and ten were observed among the different isolates. Isolate-BB2, that had ten chromosomes, has two unique bands and a few rare bands (Table 2) ; The higher chromosome number in this isolate may be due to the presence of an accessory chromosome, a B chromosome. Fungi are reported to harbour B chromosomes (Kistler & Miao 1992). The minimum chromosome number in B. bassiana as evidenced from our work and previous reports, is five. The chromosome number above the minimum five, may not be considered as aneuploidy arising from parasexual cycle as hypothesised by Couteaudier et al. (1996). If the extra chromosomes were the chromosomes yet to be lost subsequent to a parasexual cycle, they would soon be lost resulting in a change in the telomere fingerprint profile of the isolates. The telomere fingerprints in B. bassiana are however reported to be quite stable, remaining unchanged through subcultures in the laboratory for over 3 years and even after passing an in vivo cycle through an insect (Couteaudier & Viaud 1997). Fungal isolates with different chromosome numbers have been referred to as different chromosomal ‘races ’ (Bidochka 2001). The B. bassiana isolates used in this study have been characterized both morphologically and for their virulence to several insect spp. belonging to different taxonomic orders (Padmavathi 2002). The isolates with similar chromosome number were not found to have a distinct morphology ; the host range and virulence characteristics of the genetically distinct
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577 ARSEF-1166 ARSEF-1512 ARSEF-1149 ARSEF-3286 ARSEF-3120 ARSEF-326 ITCC-1253 NRRL-3108 ARSEF-1169 BB2 ARSEF-739 ARSEF-1314 ARSEF-1316 ITCC-4521 ITCC-913 ARSEF-2860 NRRL-20699
0.14
0.35
0.57 Coefficient
0.78
1.00
Fig. 2. Dendrogram constructed based on RFLP polymorphism observed in the isolates of Beauveria bassiana when hybridised with telomere repeat sequence (TTAGGG)8. Relationships were derived from pairwise genetic distance estimates between the 17 isolates. Cluster analysis was performed using the UPGMA method. Except for isolates ARSEF 1166 and ARSEF 1512, which showed 100% similarity, the isolates showed no close similarity. z ITCC-1253 ITCC-4521 ARSEF-2860 ITCC-913 ARSEF-1314
NRRL-20699 ARSEF-326
ARSEF-1316
ARSEF-739 ARSEF-1149
ARSEF-3120 ARSEF-3286
ARSEF-1512 & ARSEF-1166*
BB2 NRRL-3108 ARSEF-169
Y
X
Fig. 3. Distribution of Beauveria bassiana isolates along first three principal coordinate axes based on telomeric fingerprints. (The first axis (x) explained 276% the second ( y) 53 % and the third (z) 933% of the variation. * Isolates ARSEF-1512 and ARSEF-1166 showed identical telomere fingerprints.
isolates in B. bassiana, were not found to be as well defined as in plant pathogenic fungi (Padmavathi 2002). Classifying isolates with similar chromosome number, as different varieties, special forms, or races, based on
morphology or host range/virulence is thus not justified. In B. bassiana, isolates with closely similar telomere fingerprints have been reported to belong to the same vegetative compatibility group (VCG) which
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Frequency
60 56 52 48 44
Mean 3.56 Variance 1.82
40 36 32 28 24 20 16 12 8 4 0
Mean
– 0.75 – 0.25 0.25 0.75
1.25 1.75 2.25 2.75 3.25 3.75 4.25 4.75 5.25
5.75
Distance
Fig. 4. Distribution of genetic distances obtained from pairwise comparison of telomere fingerprints in 17 isolates of Beauveria bassiana. The distribution pattern and the value of variance testify the prevalence of recombination in the worldwide population of B. bassiana.
can intercross but are reproductively isolated from isolates with a different telomere fingerprint (Couteaudier & Viaud 1997). In the present study, the B. bassiana isolates with similar chromosome number were found to have very different telomere fingerprints. Since isolates with the same chromosome number can have very different telomere fingerprints, chromosome number cannot be used to determine the VCG of a particular isolate. In Fusarium spp., the RAPD patterns were reported to be generally VCG specific (Crowhurst et al. 1991, Bentley, Pegg & Dale 1995) but this does not appear to be true for B. bassiana. Our results demonstrate that RAPD analysis, using ten primers (Padmavathi 2002), of isolates with the same chromosome number do not have similar RAPD profiles. Further, a good correlation between groups determined by RAPD and electrophoretic karyotyping has been reported to exist in Fusarium oxysporum f.sp. cubense meriting the recognition of isolates with different chromosome numbers as ‘races ’ with reproductive compatibility among isolates within the race (same VCG) and incompatibility between races (Bentley, Pegg & Dale 1995). Reproductive isolation is the basic premise on which a taxonomically permissible subgroup, a species if the ‘species complex ’ classification of B. bassiana (Inglis et al. 2001) is accepted, can be made. Chromosome number differences as a sole basis for such subgrouping of the B. bassiana isolates is not justified. The significance of the difference in chromosome numbers among isolates of B. bassiana thus remains enigmatic. From a comparison of the telomere fingerprints generated in the EcoRI and HindIII autoradiograms, it is clear that some of the telomeres have a HindIII site. A similar conclusion can be drawn from the results presented in B. bassiana by Viaud et al. (1996). Satellite DNA is reported to have internal heterogeneity
of the repeated units due to mutations (Weising et al. 1995). The frequency distribution of the similarity values computed from the telomere fingerprints of B. bassiana isolates, revealed the occurrence of recombination in this fungus which has been described as mitosporic. Recombination through parasexual cycle has been demonstrated in this fungus in the laboratory (PaccolaMeirelles & Azevedo 1991) but, its occurrence in natural conditions is yet to be shown. However, a teleomorph, Cordyceps bassiana, has recently reported for B. bassiana (Li et al. 2001). The telomeric fingerprint analysis points to the existence of recombination, whether it is through a sexual or a parasexual mode. In our telomere fingerprint analysis, except for two isolates, all the remaining 15 B. bassiana isolates could be unambiguously differentiated by the banding pattern typical to each of them. Telomere fingerprints have been reported to be stable (Couteaudier & Viaud 1997). Thus telomere DNA fingerprinting is a good tool for molecular typing of individuals. Typing of isolates is required for proprietary reasons when isolates are registered for use as biopesticides (Butt et al. 2001). Isolates from the same epizootic (ARSEF 1314 and 1316), which showed only 43 % similarity in their telomere fingerprints, showed very similar RAPD patterns with ten primers and clustered with 96 % similarity. These two isolates from the same epizootic thus represent a single genotype, with differences in the telomere fingerprints having been generated recently. Entomopathogenic fungal populations causing natural epizootics, have been demonstrated through RAPD and telomere fingerprinting to be constituted by genetically diverse isolates (e.g. Castrillo & Brooks 1998, Boucias et al. 2000). Though the cause for this diversity has not yet been investigated, it is likely that it can arise either due to diversification of a single genotype into several
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(a)
579
(b)
(c)
Fig. 5. RAPD patterns of Beauveria bassiana isolates collected from epizootics in the fields of France and Spain obtained with: (a) RC 12 (5k-GGGCAATGA-3k) (b) RC 30 (5k-ACCGCTGTG-3k) ; and (c) RC 20 (5k-ACCCGGACA) primers. Isolates ARSEF 1314 and ARSEF 1316 from France and ARSEF-1149 from Spain which showed very similar RAPD profiles had very different telomere fingerprints. Isolates ARSEF 1166 and ARSEF 1512 with identical telomere fingerprints showed similar RAPD fingerprints with these primers.
forms, through accumulations of changes in the genome, or, due to recombination between genetically different individuals, either by sexual or parasexual means. Minisatellite sequences, which constitute the telomeres are recognized as hotspots of recombination (Chakravarti, Elbein & Permutt 1986, Steinmetz, Uematsu & Fischer 1987) and fragile sites (Oliva, Sutherland & Richards 2000). Two of the B. bassiana isolates in the present investigation, ARSEF 1149 and ARSEF 1314 were among the isolates analysed for allozyme polymorphism by St Leger et al. (1992). They shared a close similarity in their allozyme profiles and clustered in one
group (St Leger et al. 1992). They also share similar RAPD patterns (Padmavathi 2002) but, they have very dissimilar telomere fingerprints. Thus, the genetic distances computed based on any one molecular typing method (telomere fingerprint, RAPD etc.) is not a real indicator of genetic similarity/dissimilarity between isolates. The existence of isolates with similar DNA fingerprints in different geographic areas like ARSEF 1166 and ARSEF 1512 in the present study may not indicate that the same genotype is distributed in several geographical regions. Instead, it is likely that similar changes in the minisatellite or simple sequence or other
Telomere fingerprinting of Beauveria bassiana DNA sequences may have occurred independently in different genotypes. ACKNOWLEDGEMENTS J.P. and N. N.R.R. are thankful to UGC and CSIR (respectively) New Delhi, for their research fellowships. K. U.D. and C. U.M. are thankful to the DST, Government of India, New Delhi for financial support through research grant no. SP/So/A-10/97. We also thank: Richard A. Humber (USDA-ARS Collection of Entomopathogenic Fungal Cultures, Ithaca, NY); Kerry O’Donnell (Microbial Properties Research, NRRL, Peoria, ILL); and the Indian type culture collection (IARI, New Delhi) for providing isolates.
REFERENCES Bentley, S., Pegg, K. G. & Dale, J. L. (1995) Genetic variation among a world wide collection of isolates of Fusarium oxysporium f. sp. cubense analysed by RAPD-PCR fingerprinting. Mycological Research 99: 1378–1384. Bidochka, M. J. (2001) Monitoring the fate of biocontrol fungi – fungal biological control agents: progress, problems and potential. In Fungi as Biocontrol Agents (T. M. Butt, C. Jackson & N. Magan, eds): 193–198. CAB International, Wallingford. Boucias, D. G., Tigano, M. S., Sosa-Gomez, D. R., Glare, T. R. & Inglis, P. W. (2000) Genotypic properties of the entomopathogenic fungus Nomuraea rileyi. Biological Control 19: 124–138. Butt, M. T., Jackson, C. & Magan, N. (2001) Introduction – fungal biological control agents: progress, problems and potential. In Fungi as Biocontrol Agent (T. M. Butt, C. Jackson & N. Magan, eds): 1–8. CAB International, Wallingford. Castrillo, L. A. & Brooks, W. M. (1998) Differentiation of Beauveria bassiana isolates from the darkling beetle, Alphitobius diaperinus, using isozyme and RAPD analysis. Journal of Invertebrate pathology 72: 190–196. Chakravarti, A., Elbein, S. C. & Permutt, M. A. (1986) Evidence for increased recombination near the human insulin gene. Proceedings of National Academy of Sciences, USA 83: 1045–1049. Couteaudier, Y. & Viaud, M. (1997) New insights into population structure of Beauveria bassiana with regard to vegetative compatibility groups and telomeric restriction fragment length polymorphisms. FEMS Microbiology Ecology 22: 175–182. Couteaudier, Y., Viaud, M. & Riba, G. (1996) Genetic nature, stability and improved virulence of hybrids from protoplast fusion in Beauveria. Microbiology and Ecology 32: 1–10. Crowhurst, R. N., Hawthorne, B. T., Rikkerink, E. H. A. & Templeton, M. D. (1991) Differentiation of Fusarium solani f. sp. cucurbitae races 1 and 2 by random amplification of polymorphic DNA. Current Genetics 20: 391–396. Inglis, W. P., Magalhaes, B. P. & Inglis, C. V. (1999) Genetic variability in Metarhizium flavoviride revealed by telomeric fingerprinting. FEMS Microbiology Letters 179: 49–52. Inglis, D. G., Goettel, S. M., Butt, M. T. & Strasser, H. (2001) Use of hyphomycetous fungi for managing insect pests. In Fungi as Biocontrol Agents (T. M. Butt, C. Jackson & N. Magan, eds): 23–69. CAB International, Wallingford. Kistler, H. C. & Miao, V. P. W. (1992) New modes of genetic change in filamentous fungi. Annual Review of Phytopathology 30: 131–152.
580 Leathers, T. D., Gupta, S. C. & Alexander, N. J. (1993) Mycopesticides: status, challenges and potential. Journal of Industrial Microbiology 12 : 69–75. Levis, C., Giraud, T., Duterytre, M., Foritni, D. & Brygoo, Y. (1997) Telomeric DNA of Botrytis cinera: a useful tool for strain identification. FEMS Microbiology Letters 157: 267–272. Li, Z. Z., Li, C. R., Huang, B. & Fan, M. Z. (2001) Discovery and demonstration of the teleomorph of Beauveria bassiana (Bals.) Vuill., an important entomogenous fungus. Chinese Science Bulletin 46: 751–753. MacLeod, D. M. (1954) Investigations on the genera Beauveria Vuill. and Tritirachium Limber. Canadian Journal of Botany 32: 818–890. Oliva, H., Sutherland, G. R. & Richards, R. I. (2000) Fragile sites and minisatellite repeat instability. Molecular Genetics and Metabolism 70: 99–105. Paccola-Meirelles, L. D. & Azevedo, J. L. (1991) Parasexuality in Beauveria bassiana. Journal of Invertebrate Pathology 57: 172–176. Padmavathi, J. (2002) A study of morphology, host specificity and genetic structure through DNA fingerprinting of the insect pathogenic asexual fungal species Beauveria bassiana (Bals.) Vuillemin – a potential biopesticide. PhD thesis, Andhra University, Visakhapatnam. Pfeiffer, T. A. & Khachatourians, G. G. (1993) Electrophoretic karyotype of the entomopathogenic deuteromycete Beauveria bassiana. Journal of Invertebrate Pathology 61: 231–235. St Leger, R. J., Allee, L. L., May, B., Staples, R. C. & Roberts, D. W. (1992) World-wide distribution of genetic variation among isolates of Beauveria spp. Mycological Research 96: 1007–1015. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989) Molecular Cloning: a laboratory manual. 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Shimizu, S., Higashiyama, R. & Matusumoto, T. (1993) Chromosome length polymorphism in Beauveria bassiana. Journal of Sericulture Science Japan 62: 45–49. Sneath, P. A. & Sokal, R. R. (1973) Numerical Taxonomy. W. H. Freeman, San Franscisco. Steinmetz, M., Uematsu, Y. & Fischer, L. K. (1987) Hotspots of homologous recombination in mammalian genomes. Trends in Genetics 3 : 7–10. Taylor, W. J., Geiser, D. M., Burt, A. & Koufopanou, V. (1999) The evolutionary biology and population genetics underlying fungal strain typing. Clinical Microbiological Reviews 1999: 126–146. Uma Devi, K., Padmavathi, J., Sharma, H. C. & Seetharama, N. (2001) Laboratory evaluation of the virulence of Beauveria bassiana isolates to the sorghum shoot borer Chilo partellus Swinhoe (Lepidoptera : Pyralidae) and their characterization by RAPDPCR. World Journal of Microbiology and Biotechnology 17: 131–137. Viaud, M., Couteaudier, Y., Levis, C. & Riba, G. (1996) Genomic organisation of Beauveria bassiana: electrophoretic karyotyping, gene mapping and telomeric fingerprinting. Fungal Genetics and Biology 20: 175–183. Weising, K., Nybom, H., Wolff, K. & Mayer, W. (1995) DNA Fingerprinting in Plants and Fungi. CRC Press, Boca Raton. Zakian, V. A. (1989) Structure and function of telomeres. Annual Reviews of Genetics 23: 579–604. Zolan, M. E. (1995) Chromosome-length polymorphism in fungi. Microbiological Reviews 59: 686–698.
Corresponding Editor: D. J. Bond
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DOI: 10.1017/S0953756203007573 Printed in the United Kingdom.
Telomere fingerprinting for assessing chromosome number, isolate typing and recombination in the entomopathogen Beauveria bassiana
J. PADMAVATHI, K. UMA DEVI*, C. Uma Maheswara RAO and N. Nageswara Rao REDDY Department of Botany, Andhra University, Visakhapatnam, 530 003, AP, India. E-mail : [email protected] Received 22 June 2002; accepted 18 January 2003.
Beauveria bassiana is a popular biocontrol agent used as ‘ green’ pesticide in crop insect pest management. Chromosome number has been variously reported as five, six, seven and eight in this species. The range of chromosome number and the minimum chromosome number in this economically important fungus were assessed through telomere fingerprint analysis of a sample of 17 isolates from different and similar hosts and distant and same geographic origin. Genomic DNA digested with EcoRI, which has no cutting site in the telomere repeat sequence arrays was probed with a radioisotope-labelled (5k-TTAGGG-3k)8 oligonucleotide. The probe-hybridised regions appeared as discrete bands – each representing a telomere. The number of bands in each lane was counted and halved to arrive at the chromosome number of that isolate. The chromosome number varied from 5 to 10 in the different isolates. The telomere probe hybridised bands were also scored for presence or absence in a 0–1 matrix and a dendrogram based on similarities between the isolates was constructed using the NTSYS-pc ver. 2.02i software. The isolates showed very little similarity ; the overall similarity was 14 %. Only two isolates which were of diverse host and geographic origin showed 100% similarity. Isolates from the same epizootic that showed 43 % similarity in their telomere fingerprints had 96 % similarity in their RAPD (Random amplified polymorphic DNA) fingerprints with 10 primers. The genetic distances computed from any one DNA fingerprinting method thus do not reflect the true genetic similarities of the isolates. The frequency distribution pattern of the pair-wise similarities computed from telomere fingerprints hinted at the occurrence of recombination in this fungus. Telomere fingerprinting proved very useful in typing isolates since each of them was found to have a unique fingerprint. Isolates with the same chromosome number neither showed a distinct morphology or virulence character nor a close similarity in telomere or RAPD fingerprints to merit their subgrouping into a taxonomically relevant or practically useful unit.
INTRODUCTION Beauveria bassiana is an ubiquitous, mitosporic fungus with a very wide and diverse host range of nearly 750 insect species (Inglis, Magalhaes & Inglis 2001). It has the longest history as an experimental mycoinsecticide (Leathers, Gupta & Alexander 1993), and is reported to have been widely used and to be one of the seven registered mycopesticides now on the market (Butt, Jackson & Magan 2001). Knowledge of the genomic organization of this fungus is important when strain improvement programmes are undertaken. Karyotype analysis is one approach to the characterization of entomopathogenic fungal isolates because, a high level of chromosomal polymorphism (in number and size) is known to exist in these organisms (Kistler & Miao 1992, Zolan 1995, Bidochka 2001). The karyotype of an isolate has been found to be a stable genetic marker in * Corresponding author.
many fungal species (Zolan 1995) including B. bassiana (Couteaudier & Viaud 1997). In mitosporic fungi, observation of chromosomes under a microscope is difficult because mitotic material is not suitable for slide preparations for such analysis (Zolan 1995). Usually, pulsed field gel electrophoresis method involving clamped homogenous electrical field (CHEF) termed ‘electrophoretic karyotyping ’ is employed for karyotype studies in such fungi (Zolan 1995, Bidochka 2001). In B. bassiana isolates, the chromosome number estimated from telomere fingerprints corresponded to the number estimated through the CHEF technique (Viaud et al. 1996). Telomere fingerprints were used also in other entomopathogenic fungi, notably Nomuraea rileyi and Metarhizium anisopliae, to assess the chromosome number (Boucias et al. 2000, Inglis et al. 1999). Telomere fingerprinting is reported to substitute for inadequately resolved chromosomes in electrophoretic karyotyping in estimation of chromosome numbers (Zolan 1995). B. bassiana has been reported to be
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Table 1. Details of isolates of Beauveria bassiana used in the telomere fingerprint analysis. Isolatea
Source insect
Order of host
Geographical origin
Chromosome no.
BB2 ARSEF 3120 ARSEF 739 ARSEF 326 ITCC 913 ARSEF 1512 ARSEF 1149 ARSEF 1316 ARSEF 1169 ITCC 4521 ARSEF 2860 ARSEF 1314 ARSEF 1166 ITCC 1253 ARSEF 3286 NRRL 20699 NRRL 3108
Spodoptera litura Senecio sp. Diabrotica paranoense Chilo plejadellus Unknown Spodoptera littoralis Helicoverpa armigera Helicoverpa virescens Sitona lineatus Diatraea saccharalis Schizaphis graminum Helicoverpa virescens Helicoverpa armigera Musca domestica Spodoptera littoralis Unknown Ostrinia nubilalis
Lepidoptera Homoptera Lepidoptera Lepidoptera Unknown Lepidoptera Lepidoptera Lepidoptera Coleoptera Lepidoptera Homoptera Lepidoptera Lepidoptera Diptera Lepidoptera Unknown Lepidoptera
Bangalore, S. India Yvelines, France CNPAF, Brazil Queensland, Australia Netherlands La Minie`re, France Cordoba, Spain La Minie`re, France Sennecille, France Karnal, N. India Idaho, USA La Minie`re, France Cordoba, Spain Mumbai, India Montpellier, France Illinois, USA Unknown
10 8 7 7 7 6 6 6 6 6 6 6 5 5 5 5 5
a
ARSEF, USDA-ARS collection of Entomopathogenic Fungi (ARSEF, Ithaca, New York); NRRL, Peoria, Illinois; ITCC, Indian Type Culture Collection, IARI, New Delhi. BB2 was isolated from a local field and is being accessioned.
haploid (St Leger et al. 1992). The chromosome number in this species has been variously reported to be five, six, seven and eight (Paccola-Meirelles & Azevedo 1991, Shimizu, Higashiyama & Matusumoto 1993, Pfeiffer & Khachatourians 1993, Viaud et al. 1996). Few isolates have been examined. In all filamentous fungi whose telomeres have been investigated, a 6 bp repeat sequence of 5k-TTAGGG-3k has been found (Levis et al. 1997). The Bal 31 exonuclease sensitivity of genomic regions (bands) hybridising with the telomere specific probe (TTAGGG)n was tested in B. bassiana (Viaud et al. 1996). The results confirmed the presence of this repeat in the telomeres. No telomere probe hybridised band was found that was insensitive to Bal 31 nuclease digestion (Viaud et al. 1996) indicating that telomere repeat sequences are not present in the intercalary regions of the chromosomes in B. bassiana. When a restriction enzyme with no recognition sequence in the telomeres constituted by a 5k-TTAGGG3k repeat array is used to digest the genomic DNA, among the fragments, those containing mostly telomeric DNA are generated. The size of the telomeres is reported to vary between chromosomes and between chromosome arms of a chromosome, in the chromosome complement of an organism (Zakian 1989). If the size differences are discrete and non-overlapping, the number of telomeres would equal the number of hybridising bands in the autoradiograms. In B. bassiana and other entomopathogenic fungi, the telomere probehybridised regions appeared as discrete bands (Viaud et al. 1996, Couteaudier & Viaud 1997, Inglis et al. 1999, Boucias et al. 2000). The range of variation in chromosome number in B. bassiana is studied using a larger sample size of 17 isolates through telomere fingerprinting. Telomere fingerprinting is a less sophisticated technique than CHEF ; besides, it provides a means of simultaneously typing individual isolates and assessing the level of genetic polymorphism in the species.
MATERIALS AND METHODS Isolates Beauveria bassiana isolates procured from the ARSEF, NRRL and ITCC culture collections, and from our local fields, were analysed (Table 1). The sample composition was chosen to represent both isolates of diverse host and geographic origin as well as isolates from similar hosts collected from the same region (Table 1). Telomere fingerprinting The fungal isolates were grown in liquid Sabouraud dextrose medium and DNA was isolated from mycelium as described by Uma Devi et al. (2001). The DNA (8–10 mg) was digested to completion with EcoRI (Genei, Bangalore) following manufacturers instructions. Eight isolates were also digested with HindIII (Bangalore Genei). The standard protocol for RFLP (Restriction fragment length polymorphism) as described in Sambrook, Fritsch & Maniatis (1989) was followed. A 1 kb ladder (Life Technologies, Alameda, CA) and l HindIII and wr174 Hae III digests (Finnzymes, Finland) were used as DNA size markers. An oligonucleotide constituted by telomere repeat sequence – (5k-TTAGGG-3k)8 (Genmed Synthesis, San Francisco) was 5k-end-labelled with P32 using T4 polynucleotide kinase to serve as the probe. Analysis of autoradiograms The autoradiograms were marked to indicate the position of the DNA size markers and photographed under white light with a Kodak DC 120 digital camera. The number of bands in each lane was counted, and the intensity of each band was noted. Sizes for individual bands were assigned using Kodak 1D software. The chromosome number was estimated by halving the total
Telomere fingerprinting of Beauveria bassiana
574
(a)
(b)
Fig. 1. Autoradiograms showing telomeric fingerprints of isolates of Beauveria bassiana : (a) and (b) are fingerprints of genomic DNA digested with EcoRI and HindIII respectively. The size (in kb) of DNA fragments are shown (1 kb ladder, Life technologies) on the left and l HindIII+wr174 HaeIII (Finnzymes) on the right.
number of bands since each chromosome has two telomeres. When the number of bands was odd, it was rounded up to the next even number (Viaud et al. 1996, Boucias et al. 2000) since, one of the bands in such cases, may represent the overlapping of two similar sized telomeres. The correlation of differences in chromosome number among the B. bassiana isolates to their morphological traits for which they had been characterized (Padmavathi 2002) was examined. The bands in all lanes were scored manually as a binary matrix (1=present, 0=absent). The data was analysed using NTSYS-pc ver. 2.02i software. A similarity matrix was generated using the SIMQUAL program and Jaccard’s similarity coefficient (Sneath & Sokal 1973). A dendrogram displaying the similarities between the isolates was generated using the SAHN program by UPGMA (Sneath & Sokal 1973). The genetic distances between the Beauveria bassiana isolates was
visualized in a 3D-picture plotted along the first 3 principal coordinate axes through principle coordinate analysis (PCA). The distribution of the distances was graphically plotted to assess the reproductive pattern in the worldwide population of B. bassiana. A comparison of the phenetic groups based on telomere fingerprints of these B. bassiana isolates with those observed in the analysis of RAPD fingerprints generated with ten primers (Padmavathi 2002) was made.
RESULTS The regions that hybridised with the telomere sequence appeared as discrete bands. In autoradiograms of EcoRI cut DNA, 9–19 bands were observed (Fig. 1a, Table 2) while the HindIII cut DNA showed 9–25 bands
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Table 2. Number and size range of DNA fragments that hybridized with telomere specific probe (TTAGGG)n in the autoradiograms and the estimated chromosome number in the isolates of Beauveria bassiana. EcoRI Bands HindIII Bands Band sizes (in kb) Isolate
No.
Size (Kb) range
Unique
Rare
Commona
No.
Size (Kb) range
Chromosomes no.b
BB2
19
23.5–0.6
5.6, 2.5
23.5, 13, 4.6
25
13.0–0.57
10
ARSEF 3120
15
23.0–0.56
ARSEF 739
14
23.0–1.6
ARSEF 326
13
24.0–0.74
ITCC 913
13
20.0–0.56
ARSEF 1512
12
23.0–1.0
ARSEF 1149
12
23.0–1.0
ARSEF 1316
12
20.0–0.6
ARSEF 1169
12
23.0–0.6
ITCC 4521
11
14.0–1.7
ARSEF 2860
11
14.0–1.0
ARSEF 1314
11
20.0–0.6
ARSEF 1166
10
23.0–1.0
ITCC 1253
10
8.3–1.0
ARSEF 3286 NRRL 20699 NRRL 3108
10 9 9
23.5–0.39 23.5–2.0 20.0–1.2
23, 20, 16, 11.9, 9.3, 8.5, 7.9, 3.9, 3.6, 3.4, 2.7, 1.3, 1.2, 0.6 23, 16, 9.3, 8.9, 7.9, 6.5, 6.3, 5.5, 4.5, 4.3, 3.9, 3.3, 3, 1.9, 0.56 23, 16, 9.3, 8.5, 8.3, 5.7, 3.9, 3.6, 3.5, 3.2, 2.7, 1.9, 1.6 20, 16, 11.9, 8.9, 7.9, 6.3, 4.3, 3.6, 2.6, 2.4, 1.9 20, 14, 11.9, 9.3, 8.9, 8.5, 8.3, 6.5, 5.7, 4.3, 3.6, 2.7, 0.56 23, 20, 16, 11.1, 8.5, 8.3, 6.3, 4.3, 3.4, 2.4, 1.9, 1 23, 16, 11.1, 8.5, 8.3, 6.3, 4.3, 3.4, 2.4, 1.9, 1 20, 16, 9.3, 8.9, 7.9, 6.5, 5.7, 4.5, 3.2, 2.6, 1.9, 0.6 23, 20, 16, 14, 11.9, 9.3, 8.5, 7.9, 6.3, 4.5, 3.9, 0.6 14, 9.3, 8.9, 8.3, 6.5, 5.7, 4.5, 3.5, 3.2, 2.4 14, 11.9, 9.3, 8.5, 5.7, 5.5, 4.3, 3, 2.6, 2, 1 20, 16, 11.9, 8.9, 6.5, 5.7, 2.7, 1.9, 1, 0.6 23, 20, 16, 11.1, 8.5, 8.3, 6.3, 4.3, 3.4, 2.4, 1.9, 1 8.3, 7.9, 6.5, 6.3, 4.3, 3.6, 3.3, 2.6, 1.3, 1 20, 11.1, 8.5, 7.9, 5.5, 3.4, 1.9 16, 14, 11.1, 8.3, 6.3, 4.3, 3.4, 2 20, 16, 14, 7.9, 5.5, 4.5, 3.9, 1.6, 1.2
a b
13 24, 0.74
21
1.7
4.6
4.6 0.39
23.5, 21 23.5, 21
–
8
11
9.0–0.39
7
7
5.5–0.56
7
–
7
–
6
–
6
–
6
12
6.1–0.51
– 19
6 6
12.0–0.56
6
–
6
–
5
–
5
16 9 10
10.8–0.8 6.0–0.55 6.1–0.6
5 5 5
Present in more than three isolates. Estimated from number of EcoRI fragments.
(Fig. 1b, Table 2). The telomere sequences of only eight of the 17 fungal isolates analysed were cut with HindIII. The size range of the bands in EcoRI blots was 24 kb to 0.39 kb while in HindIII blots, it was 13 kb to 0.39 kb (Figs 1a–b, Table 2). Thus bands that hybridised with the telomere sequence probe were smaller in size in HindIII digests than in EcoRI digests. In the HindIII digests, the bands were more intense than in the EcoRI blots (Fig. 1b). The much smaller sized and more intensely dark telomere probe-hybridised bands in HindIII autoradiogram compared to the EcoRI autoradiogram, hints at the presence of recognition sequence of this enzyme in the telomere regions. The chromosome number as estimated from the number of bands in the EcoRI autoradiogram ranged from 5–10 in the different isolates (Table 2). The isolates with similar chromosome number were not found to have a distinct morphology (Table 3) or any relation to the original host or geographic region (Table 1). The cluster analysis of the telomere banding pattern observed in EcoRI generated fragments revealed a very high degree of variability among the isolates (Fig. 2).
Mean similarity values for pair-wise comparisons ranged from 0.14 to 1.00. There was just 14 % overall similarity among the isolates. A cophenetic correlation value of c=0.89 was obtained. It indicated that the UPGMA cluster analysis was statistically significant. Only two isolates ARSEF 1166 and ARSEF 1512 showed 100 % similarity (Fig. 2). They are of different host and geographic origin, the former being from France and the later from Spain (Table 1). These two isolates showed 85 % similarity with ARSEF 1149, which is from the same place in Spain as ARSEF 1166 collected two days later in the same epizootic (Table 1, Fig. 2). On the other hand, isolates ARSEF 1314 and ARSEF 1316 collected in the same place from the same insect species at the same time (Table 1) showed only 43 % similarity (Fig. 2) in telomere fingerprints. The wide variation among the isolates is evident from the dispersed distribution of the Beauveria bassiana isolates on the 3D-scatter plot generated from PCA (Fig. 3). The mean and variance of the pair-wise distances in the similarity matrix are 3.56 and 1.82 respectively. The graphical representation of frequency distribution
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576
Table 3. Morphological characteristics and phenetic group based on RAPD analysis of the isolates of Beauveria bassiana with different chromosome number (as determined from telomere fingerprinting). Range of pH Isolate
Chr. No.
Colony morphologya
Growth rateb
BB2 ARSEF 3120 ARSEF 739 ARSEF 326 ITCC 913 ARSEF 1512 ARSEF 1149 ARSEF 1316 ARSEF 1169 ITCC 4521 ARSEF 2860 ARSEF 1314 ARSEF 1166 ITCC 1253 ARSEF 3286 NRRL 20699 NRRL 3108
10 8 7 7 7 6 6 6 6 6 6 6 5 5 5 5 5
Dusty Fluffy Chalky Fluffy Dusty Dusty Dusty Chalky Fluffy Matty Chalky Chalky Dusty Chalky Fluffy Matty Dusty
High Medium Medium High High High High Medium Medium High Medium Medium High Medium Low High High
Conidia cmx2 c
Germ. Speedd
Tolerance
Optimum
RAPD phenetic groupe
Medium High Low High Medium Medium Medium Low High Low Low Low Medium Low High Low Medium
Medium Slow Fast Medium Slow Fast Fast Slow Slow Fast Fast Fast Medium Slow Fast Slow Slow
4–14 5–13 5–14 4–13 4–14 4–14 4–14 5–14 5–13 4–13 5–14 5–14 4–14 5–14 5–13 4–13 4–14
6–12 5–13 5–13 9–13 5–13 5–8 5–8 5–13 5–13 5–13 5–13 9–13 5–8 5–13 6–13 5–13 9–10
1Aixa 3 1B Iy 1A Iy 1B iyI aii a 1B Ix bI 1B Ix aib 1B Ix aii 1A Ixb 1B IIx 1B Iiy b 1B Ix aia 1B Ix bI 1B Ix bii 1A IIx 3 1A IIy
a
Gross colony morphology on solid Sabouraud dextrose medium (MacLeod 1954). As measured from dry weight of biomass accumulated in 10 d cultures in liquid Sabouraud dextrose medium at 25¡1 x – High o400; Medium o300 & low f300 mg. c Conidia produced per cm2 on 10 d cultures on solid Sabouraud dextrose medium overlaid with cellophane film at 25¡1 x (High c4.5r108 ; Medium c2.5r109 ; Low c1.5r10 9 ; Very low c1.5r10 6). d Time taken for y100% conidial germination on solid Sabouraud dextrose medium at 25¡1 x (Fast=12 h; Medium=14 h and Slow=20 h). e From Padmavathi (2002). b
of the distance values (Fig. 4) indicates the prevalence of recombination in these isolates (Taylor et al. 1999). Isolates ARSEF 1166 and ARSEF 1512 that showed 100 % similarity in their telomere fingerprints (Fig. 2), also had a close (87 %) resemblance in their RAPD profiles generated from ten primers (Fig. 5). The other isolate, ARSEF 1149 which showed 85 % similarity in its telomere fingerprint to these two isolates (Fig. 2), however, had a very different RAPD profile and shared only 54 % similarity with them in the RAPD profiles (Padmavathi 2002). ARSEF 1149 had very similar RAPD pattern as ARSEF 1314 and ARSEF 1316 (Fig. 5) and clustered with them at 94% similarity (Padmavathi 2002). The isolates ARSEF 1314 and ARSEF 1316 are from the same epizootic (Table 1) collected on the same day, but in their telomere fingerprints they shared only 43 % similarity between them and 18 % similarity with ARSEF 1149, the isolate which they very closely resembled in their RAPD fingerprints (Fig. 5).
DISCUSSION Different workers have reported the chromosome number in Beauveria bassiana variously as five (PaccolaMeirelles & Azevedo 1991), at least six (Shimizu et al. 1993), eight (Pfeiffer & Khachatourians 1993) and seven or eight (Viaud et al. 1996). However, different isolates were analysed by each group. The differences in the reported chromosome numbers indicate the existence
of chromosomal number polymorphism in this species. In our investigation, chromosome numbers of five, six, seven, eight and ten were observed among the different isolates. Isolate-BB2, that had ten chromosomes, has two unique bands and a few rare bands (Table 2) ; The higher chromosome number in this isolate may be due to the presence of an accessory chromosome, a B chromosome. Fungi are reported to harbour B chromosomes (Kistler & Miao 1992). The minimum chromosome number in B. bassiana as evidenced from our work and previous reports, is five. The chromosome number above the minimum five, may not be considered as aneuploidy arising from parasexual cycle as hypothesised by Couteaudier et al. (1996). If the extra chromosomes were the chromosomes yet to be lost subsequent to a parasexual cycle, they would soon be lost resulting in a change in the telomere fingerprint profile of the isolates. The telomere fingerprints in B. bassiana are however reported to be quite stable, remaining unchanged through subcultures in the laboratory for over 3 years and even after passing an in vivo cycle through an insect (Couteaudier & Viaud 1997). Fungal isolates with different chromosome numbers have been referred to as different chromosomal ‘races ’ (Bidochka 2001). The B. bassiana isolates used in this study have been characterized both morphologically and for their virulence to several insect spp. belonging to different taxonomic orders (Padmavathi 2002). The isolates with similar chromosome number were not found to have a distinct morphology ; the host range and virulence characteristics of the genetically distinct
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577 ARSEF-1166 ARSEF-1512 ARSEF-1149 ARSEF-3286 ARSEF-3120 ARSEF-326 ITCC-1253 NRRL-3108 ARSEF-1169 BB2 ARSEF-739 ARSEF-1314 ARSEF-1316 ITCC-4521 ITCC-913 ARSEF-2860 NRRL-20699
0.14
0.35
0.57 Coefficient
0.78
1.00
Fig. 2. Dendrogram constructed based on RFLP polymorphism observed in the isolates of Beauveria bassiana when hybridised with telomere repeat sequence (TTAGGG)8. Relationships were derived from pairwise genetic distance estimates between the 17 isolates. Cluster analysis was performed using the UPGMA method. Except for isolates ARSEF 1166 and ARSEF 1512, which showed 100% similarity, the isolates showed no close similarity. z ITCC-1253 ITCC-4521 ARSEF-2860 ITCC-913 ARSEF-1314
NRRL-20699 ARSEF-326
ARSEF-1316
ARSEF-739 ARSEF-1149
ARSEF-3120 ARSEF-3286
ARSEF-1512 & ARSEF-1166*
BB2 NRRL-3108 ARSEF-169
Y
X
Fig. 3. Distribution of Beauveria bassiana isolates along first three principal coordinate axes based on telomeric fingerprints. (The first axis (x) explained 276% the second ( y) 53 % and the third (z) 933% of the variation. * Isolates ARSEF-1512 and ARSEF-1166 showed identical telomere fingerprints.
isolates in B. bassiana, were not found to be as well defined as in plant pathogenic fungi (Padmavathi 2002). Classifying isolates with similar chromosome number, as different varieties, special forms, or races, based on
morphology or host range/virulence is thus not justified. In B. bassiana, isolates with closely similar telomere fingerprints have been reported to belong to the same vegetative compatibility group (VCG) which
Telomere fingerprinting of Beauveria bassiana
578
Frequency
60 56 52 48 44
Mean 3.56 Variance 1.82
40 36 32 28 24 20 16 12 8 4 0
Mean
– 0.75 – 0.25 0.25 0.75
1.25 1.75 2.25 2.75 3.25 3.75 4.25 4.75 5.25
5.75
Distance
Fig. 4. Distribution of genetic distances obtained from pairwise comparison of telomere fingerprints in 17 isolates of Beauveria bassiana. The distribution pattern and the value of variance testify the prevalence of recombination in the worldwide population of B. bassiana.
can intercross but are reproductively isolated from isolates with a different telomere fingerprint (Couteaudier & Viaud 1997). In the present study, the B. bassiana isolates with similar chromosome number were found to have very different telomere fingerprints. Since isolates with the same chromosome number can have very different telomere fingerprints, chromosome number cannot be used to determine the VCG of a particular isolate. In Fusarium spp., the RAPD patterns were reported to be generally VCG specific (Crowhurst et al. 1991, Bentley, Pegg & Dale 1995) but this does not appear to be true for B. bassiana. Our results demonstrate that RAPD analysis, using ten primers (Padmavathi 2002), of isolates with the same chromosome number do not have similar RAPD profiles. Further, a good correlation between groups determined by RAPD and electrophoretic karyotyping has been reported to exist in Fusarium oxysporum f.sp. cubense meriting the recognition of isolates with different chromosome numbers as ‘races ’ with reproductive compatibility among isolates within the race (same VCG) and incompatibility between races (Bentley, Pegg & Dale 1995). Reproductive isolation is the basic premise on which a taxonomically permissible subgroup, a species if the ‘species complex ’ classification of B. bassiana (Inglis et al. 2001) is accepted, can be made. Chromosome number differences as a sole basis for such subgrouping of the B. bassiana isolates is not justified. The significance of the difference in chromosome numbers among isolates of B. bassiana thus remains enigmatic. From a comparison of the telomere fingerprints generated in the EcoRI and HindIII autoradiograms, it is clear that some of the telomeres have a HindIII site. A similar conclusion can be drawn from the results presented in B. bassiana by Viaud et al. (1996). Satellite DNA is reported to have internal heterogeneity
of the repeated units due to mutations (Weising et al. 1995). The frequency distribution of the similarity values computed from the telomere fingerprints of B. bassiana isolates, revealed the occurrence of recombination in this fungus which has been described as mitosporic. Recombination through parasexual cycle has been demonstrated in this fungus in the laboratory (PaccolaMeirelles & Azevedo 1991) but, its occurrence in natural conditions is yet to be shown. However, a teleomorph, Cordyceps bassiana, has recently reported for B. bassiana (Li et al. 2001). The telomeric fingerprint analysis points to the existence of recombination, whether it is through a sexual or a parasexual mode. In our telomere fingerprint analysis, except for two isolates, all the remaining 15 B. bassiana isolates could be unambiguously differentiated by the banding pattern typical to each of them. Telomere fingerprints have been reported to be stable (Couteaudier & Viaud 1997). Thus telomere DNA fingerprinting is a good tool for molecular typing of individuals. Typing of isolates is required for proprietary reasons when isolates are registered for use as biopesticides (Butt et al. 2001). Isolates from the same epizootic (ARSEF 1314 and 1316), which showed only 43 % similarity in their telomere fingerprints, showed very similar RAPD patterns with ten primers and clustered with 96 % similarity. These two isolates from the same epizootic thus represent a single genotype, with differences in the telomere fingerprints having been generated recently. Entomopathogenic fungal populations causing natural epizootics, have been demonstrated through RAPD and telomere fingerprinting to be constituted by genetically diverse isolates (e.g. Castrillo & Brooks 1998, Boucias et al. 2000). Though the cause for this diversity has not yet been investigated, it is likely that it can arise either due to diversification of a single genotype into several
J. Padmavathi and others
(a)
579
(b)
(c)
Fig. 5. RAPD patterns of Beauveria bassiana isolates collected from epizootics in the fields of France and Spain obtained with: (a) RC 12 (5k-GGGCAATGA-3k) (b) RC 30 (5k-ACCGCTGTG-3k) ; and (c) RC 20 (5k-ACCCGGACA) primers. Isolates ARSEF 1314 and ARSEF 1316 from France and ARSEF-1149 from Spain which showed very similar RAPD profiles had very different telomere fingerprints. Isolates ARSEF 1166 and ARSEF 1512 with identical telomere fingerprints showed similar RAPD fingerprints with these primers.
forms, through accumulations of changes in the genome, or, due to recombination between genetically different individuals, either by sexual or parasexual means. Minisatellite sequences, which constitute the telomeres are recognized as hotspots of recombination (Chakravarti, Elbein & Permutt 1986, Steinmetz, Uematsu & Fischer 1987) and fragile sites (Oliva, Sutherland & Richards 2000). Two of the B. bassiana isolates in the present investigation, ARSEF 1149 and ARSEF 1314 were among the isolates analysed for allozyme polymorphism by St Leger et al. (1992). They shared a close similarity in their allozyme profiles and clustered in one
group (St Leger et al. 1992). They also share similar RAPD patterns (Padmavathi 2002) but, they have very dissimilar telomere fingerprints. Thus, the genetic distances computed based on any one molecular typing method (telomere fingerprint, RAPD etc.) is not a real indicator of genetic similarity/dissimilarity between isolates. The existence of isolates with similar DNA fingerprints in different geographic areas like ARSEF 1166 and ARSEF 1512 in the present study may not indicate that the same genotype is distributed in several geographical regions. Instead, it is likely that similar changes in the minisatellite or simple sequence or other
Telomere fingerprinting of Beauveria bassiana DNA sequences may have occurred independently in different genotypes. ACKNOWLEDGEMENTS J.P. and N. N.R.R. are thankful to UGC and CSIR (respectively) New Delhi, for their research fellowships. K. U.D. and C. U.M. are thankful to the DST, Government of India, New Delhi for financial support through research grant no. SP/So/A-10/97. We also thank: Richard A. Humber (USDA-ARS Collection of Entomopathogenic Fungal Cultures, Ithaca, NY); Kerry O’Donnell (Microbial Properties Research, NRRL, Peoria, ILL); and the Indian type culture collection (IARI, New Delhi) for providing isolates.
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Corresponding Editor: D. J. Bond