Variation in the electrophoretic karyotype of Brazilian strains of Metarhizium anisopliae

Variation in the electrophoretic karyotype of Brazilian strains of Metarhizium anisopliae

Maria Cléria Valadares-Inglis¹ and John F. Peberdy ²

¹ Centro Nacional de Pesquisa de Recursos Genéticos e Biotecnologia (CENARGEN/EMBRAPA), Caixa Postal 02372, 70849-070 Brasília, DF, Brasil. Tel: [phone]. E.mail: [e-mail].

Send correspondence to M.C.V.I.

² Department of Life Science, University of Nottingham, Nottingham NG7 2RD, UK.

ABSTRACT

Pulsed-field gel electrophoresis (PFGE) was used to separate chromosome-sized DNA molecules of four strains of

Metarhizium anisopliae from Brazil.

Metarhizium anisopliae isolates from Japan have been reported as possessing seven chromosomes. Variation was observed among the Brazilian strains and the chromosomal DNA was resolved into eight bands for strain CG46. Densitometric analysis of PFGE gels suggested that the other three Brazilian strains also possess eight chromosomes, with two chromosomes migrating as doublets under the electrophoretic conditions used. The genome size was estimated as varying between 23.39 to 31.88 Mb, not including possible doublet chromosomes.

INTRODUCTION

Pulsed-field gel electrophoresis (PFGE) is an effective method for separation of chromosome-sized DNA. This technique, along with a refinement known as countour-clamped electric field (CHEF) gel electrophoresis, has been successfully applied to resolve the genome of several organisms into defined chromosomal bands. Electrophoretic karyotyping has been used in many yeast and fungi such as

Saccharomyces cerevisiae (Schwartz and Cantor, 1984; Carle and Olson, 1985),

Schizosaccharomyces pombe (Vollrath and Davies, 1987; Smith

et al., 1987),

Neurospora crassa (Orbach

et al., 1988),

Aspergillus nidulans (Brody and Carbon, 1989),

Ustilago hordei (McCluskey

et al., 1990),

Aspergillus niger (Debets

et al., 1990),

Septoria tritici (MacDonald and Martinez, 1991),

Paecilomyces fumosoroseus (Shimizu

et al., 1991),

Coprinus cinereus (Pukkila and Casselton, 1991),

Metarhizium anisopliae (Shimizu

et al., 1992),

Trichoderma reesei (Carter

et al., 1992),

Podospora anserina (Javerzat

et al., 1993),

Agaricus bisporus (Lodder

et al., 1993),

Fusarium spp (Migheli

et al., 1993) and

Fusarium solani (Nazareth and Bruschi, 1994).

The entomopathogenic fungus Metarhizium anisopliae is recognised as having significant potential as a mycopesticide. M. anisopliae var. anisopliae chromosomal DNA has previously been separated into seven bands, and the total genome size of different isolates from Japan is estimated to be in the range of 29.6 to 32.1 Mb (Shimizu et al., 1992). However M. anisopliae is known to demonstrate high genetic variation in relation to geographical source, and in the present study electrophoretic karyotyping has been used to study the differences between four Brazilians isolates.

MATERIAL AND METHODS

Strains

All four strains of

Metarhizium anisopliae var.

anisopliae used were obtained from the CENARGEN/ EMBRAPA-Brazil collection. The strains were originally isolated from

Deois sp. (Homoptera: Cercopidae).

Protoplast isolation

Spores of the strains, produced on MYG medium (2 g/l malt extract, 2 g/l yeast extract, 20 g/l glucose, and 20 g/l agar), were harvested and inoculated into liquid ACM (6 g/l NaNO

₃, 0.52 g/l MgSO

₄.7H

₂O, 0.52 g/l KCl, 1.52 g/l KH

₂PO

₄

.7H

₂O, traces of FeSO

₄ 7H

₂O and ZnSO

₄ 7H

₂O, 10 g/l glucose, 1.5 g/l casein hydrolysate, 2.0 g/l peptone, 2.0 g/l yeast extract, pH 6.5) and incubated at 28°C, 180 rpm for 48 h. Mycelium was collected by filtration and washed twice in distilled water. Protoplasts were obtained by digestion of the mycelium with Novozym 234 (Novo Nordisk) and cellulase CP (Sturge Enzymes Ltd.) prepared in 0.7 M KCl in phosphate buffer (2 mM, pH 5.8). After 2-h digestion, protoplasts were recovered by filtration in 30-mm mesh filters, centrifuged at 2000

g and washed twice with 0.7 M KCl.

Preparation of chromosome-sized DNA and electrophoresis conditions

Suspension containing approximately 5 x 10

⁸ protoplasts ml

^-1 was heated to 42°C and mixed with an equal volume of molten 1.4% (w/v) Sea Plaque agarose (FMC). The mixture was poured into a plug mould (Bio-Rad) and held on ice for 10 min. The agarose plugs were immersed in NDS buffer (1% SDS, 0.5 M EDTA, 10 mM Tris-HCl, pH 8.5) at 50°C for 24 h. The plugs were then washed three times over 8 h with 50 mM EDTA and stored at 4°C. PFGE was performed using CHEF-DRII apparatus (Bio-Rad). The DNA-agarose plugs were inserted into the wells of a 0.5% chromosomal grade agarose gel (Bio-Rad) and sealed with 0.7% Sea Plaque agarose. Gels were electrophoresed at 4°C in 0.5x TBE buffer at 60 V with linearly ramped initial and final pulse lengths of 3000 s and 1300 s, respectively, for 167 h.

Gel analysis

After staining in ethidium bromide for 15 min, the gel was destained in several changes of electrophoresis buffer over 4 h and photographed under UV illumination. Analysis was performed by using the Gel Imaging and Analysis System software package from QuantiGel Corporation (Kendrick Laboratories). RESULTS AND DISCUSSION Intact chromosomes from four different strains of

M. anisopliae were isolated by CHEF gel electrophoresis (

Figure 1). Variation was observed between strains in terms of chromosome size and number. Strains CG57, CG42 and CG32 showed seven chromosomes and strain CG46 clearly demonstrated eight chromosomes, which was confirmed by densitometric analysis of the gels (

Figure 2). However, chromosomes migrating as doublets could be an explanation for the high densitometric intensity of the first chromosomal bands of strains CG32 and CG42, and the second band of strain CG57.

Figure 1
- CHEF gel analysis of chromosomal DNAs from M. anisopliae strains. (1) Strain CG57, (2) strain CG46, (3) strain CG32, and (4) strain CG42. Chromosomes of S. cerevisiae and S. pombe were used as size markers as indicated.

Figure 2
- Densitometric analysis of banding intensity of chromosomal DNA of M. anisopliae. (A) Strain CG57, (B) strain CG32, (C) strain CG46, and (D) strain CG42, resolved by CHEF gel electrophoresis. (X = element number and Y = film density).

The size of the genome was estimated by comparison of the peak areas corresponding to chromosomes and standard chromosomal markers of S. cerevisiae and S. pombe, and varied from 23.39 to 31.88 Mb (Table I). Different genome and chromosome sizes were obtained by Shimizu et al. (1992), who resolved seven chromosomes and genome sizes, varying between 29.6 and 32.3 Mb from strains isolated in Japan. Differences observed in chromosome number and length in strains belonging to the same species are not necessarily surprising, as such variability has previously been observed in other fungal species, such as Fusarium oxysporum (Migheli et al., 1993) and Ustilago hordei (McCluskey et al., 1990). Variation in the M. anisopliae genome sizes between strains isolated from different geographical areas should be expected, since genetic variation is widely observed using other techniques such as RAPD-PCR (Fegan et al., 1993; Curran et al., 1994). Also, variation in phenotypic characteristics such as colony morphology, spore size or the ability to kill insects (Luna, 1985; Samuels et al., 1989) is widely observed in M. anisopliae strains.

Table I
- Size estimated (Mb) of chromosome-sized molecules of Metarhizium anisopliae isolates as determined by CHEF analysis. (*) Possible doublet choromosomes.

Band No.	Strain
	CG57	CG46	CG32	CG42
1	5.87	6.02	5.97*	5.75*
2	4.98*	5.60	5.16	4.65
3	4.01	5.23	4.19	3.85
4	2.67	4.49	2.53	2.69
5	2.27	3.65	2.36	2.47
6	2.10	2.65	2.28	2.27
7	1.49	2.23	1.44	2.09
8	-	2.01	-	-
Total	23.39	31.88	23.93	23.77

M. anisopliae. Future work will allow the physical determination of chromosome linkages using cloned genes, several of which are already available for

M. anisopliae, such as chitinase (Valadares-Inglis

et al., 1997), protease (St. Leger

et al., 1992a), starvation stress gene (St. Leger

et al., 1992b) and some other heterologous genes. ACKNOWLEDGMENTS We acknowledge financial support from CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico/RHAE and EMBRAPA/ CENARGEN. We thank Mr. B.V. Case for help with photographic work and Dr. P.W. Inglis for corrections of this manuscript. RESUMO A eletroforese de campo pulsado foi usada para separar cromossomos de quatro isolados de

Metarhizium anisopliae procedentes do Brasil. Isolados de

M. anisopliae procedentes do Japão foram descritos como possuindo sete cromossomos. Variações em relação aos isolados brasileiros foram observadas, tendo sido claramente detectadas oito bandas no isolado CG46. A análise densitométrica da intensidade dos cromossomos sugere que, sob as condições utilizadas, os outros isolados brasileiros também apresentam oito cromossomos, com dois cromossomos migrando conjuntamente, como duplos. O tamanho do genoma foi estimado como variando entre 23.39 e 31.88 Mb, não incluindo os cromossomos possivelmente resolvidos como duplos. REFERENCES

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(Received March 3, 1997)

Publication Dates

• Publication in this collection
  06 Jan 1999
• Date of issue
  Mar 1998

History

• Received
  03 Mar 1997