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Print version ISSN 1517-8382
Braz. J. Microbiol. vol.32 no.2 São Paulo Apr./June 2001
DNA POLYMORPHISM AND TOTAL PROTEIN IN MUTANTS OF METARHIZIUM ANISOPLIAE VAR. ANISOPLIAE (METSCH.) SOROKIN STRAIN E9
Laurineide Lopes de Carvalho Freire; Ana Bolena Lima da Costa; Larissa Brandão Góes; Neiva Tinti de Oliveira*
Departamento de Micologia, Centro de Ciências Biológicas,Universidade Federal de Pernambuco, Recife, PE, Brasil
Submitted: August 01, 2000; Returned to authors for corrections: November 10, 2000; Approved: May 25, 2001
Five mutants (MaE10, MaE27, MaE24, MaE41 e MaE49) of Metarhizium anisopliae wild strain E9 were analysed for DNA profile through the RAPD technique and for changes in total protein content by spectrophotometry, polyacrylamide gel electrophoresis and densitometry. The pattern of RAPD markers showed genetic polymorphism among the strains: out of twenty primers seven were selected, producing 113 bands. Forty seven bands were present in all strains (41.6% of monomorphic bands) and 66 showed polymorphism (58.4%). The mean coefficient of similarity among all strains was 0.75 (75%). The total protein content varied, staining in the interval of 6.0-8.0 µg/µl. The electrophoresis analysis, through zymogram and protein fraction profiles by densitometry, allowed the observation of seven bands for the wild strain E9 and five bands for the mutants MaE10, MaE27, MaE34, MaE41 and MaE49, evidence of variations in µg% among protein fractions. The RAPD technique was very sensitive to detect genetic differences between the wild type and the mutants obtained through gamma radiation. The total protein analysis also showed changes in quantity and pattern of bands after electrophoresis in the mutants compared to the wild type.
Key words: Metarhizium anisopliae, mutants, RAPD, total protein
Among the entomopathogenic fungi, Metarhizium anisopliae presents a wide spectrum of pathogenicity and infectivity reaching different species of pest insects. In Brazil this fungus is used with success for the control of Mahanarva posticata Stal on sugar-cane and against Deois flavopicta Stal. and Zulia entreriana Berg on pastures (2,12). In entomopathogenic fungi genetic manipulation allows the alteration of factors such sporulation, dispersion and tolerance to stress in the spores, with the purpose of increasing their efficiency in biological control (29). Knowledge of recombination mechanisms and variability in M. anisopliae made it possible to accomplish improved techniques for obtaining strains with expressive characteristics of pathogenicity and infectivity important for biological control (5,17,19,20). Using gamma radiation Oliveira et al. (19) obtained mutants of the strain E9 of M. anisopliae var. anisopliae with altered germination and suggested the possibility of its ultilization in genetic crossings for incorporation of desirable characteristics.
The RAPD (Random Amplified Polymorphic DNA) technique described by Williams et al. (28) and Welsh and Mc Clelland (27) has been used in studies of genetic variability, taxonomy and ecology of several fungi (1,3,6,9,21). The RAPD markers are important tools for evaluation of the variability among different isolates of a species, the degree of genetic relationship among isolates, and may also be useful to help distinguish isolates of nonpathogenic and pathogenic fungi (8,13,16,31,32). Fegan et al. (10) observed a great genetic variability in isolates of M. anisopliae var. anisopliae using this technique. Using RAPD in 13 isolates of M. anisopliae var. anisopliae Fungaro et al. (11) verified a great genetic diversity among them. There was less genetic variability among the isolates from insects, suggesting a certain degree of host specificity. Schlick et al. (26) analyzed strains of Trichoderma harzianum (Rifai) and mutants of a wild isolate induced by gamma radiation through RAPD, and verified that it was possible to differentiate all mutants by at least one primer. The authors emphasised the great importance of the possibility of discriminating these mutants of the original strain for a single technique of genomic fingerprint for use in protection of patents of fungal strains.
The electrophoresis profile of proteins in fungi and techniques for direct observation of DNA have been of great usefulness in taxonomical, phylogenetical and ecological studies (14,18,22,30).
We report the differentiation of five mutants originated from the wild strain E9 of M. anisopliae var. anisopliae obtained by gamma radiation, using the RAPD technique and protein electrophoresis.
MATERIALS AND METHODS
E9 strain of M. anisopliae var. anisopliae and five mutants (MaE34, MaE49, MaE41, MaE10 and MaE27) obtained by gamma radiation (19) were used.
Erlenmeyer flasks with 100 ml of Complete Media (4,23) were inoculated, separately, with 108 conidia/ml of the wild strain E9 and mutants MaE27, MaE34, MaE41, MaE49 and MaE10, and incubated under agitation at 28ºC for 96 hs. The mycelium was recovered by vacuum filtration, washed with sterilized water, and stored at 12ºC.
Extraction and amplification of DNA
The total genomic DNA of the samples was extracted from mycelium as described by Raeder and Broda (24). Twenty primers of the kit OPW were tested and selected for amplification with the DNA of the wild strain E9. Kit OPW, Taq DNA polymerase and the dNTPs for RAPD were supplied by Operon Technologies, CA, USA. Amplification reactions were carried out in a total volume of 25 µl containing approximately 20-25 ng of template DNA, primer 0.4 µmol l-1 and MgCl2 3.0 mmol l-1. The reaction control consisted of all components, except the genomic DNA. The amplification process was accomplished in a thermal cycler (MJ Research), programmed to accomplish an initial denaturation of 5 minutes at 92ºC, 40 cycles of 1 minute at 92ºC (denaturation), 1 minute and 30 seconds at 39ºC (annealing), 2 minutes at 72ºC (extension) and finally 5 minutes in 72ºC for a final extension, during 40 cycles. Samples of 22 µl of the amplification products were assayed by electrophoresis in 1.4% agarose gels at 3 V/cm of distance among the electrodes, running with Tris-Borate-EDTA (TBE) buffer. Gels were stained with ethidium bromide, and photographed under U.V. light.
The NTSYS.PC (Numerical Taxonomy System Applied Biostatistics, Setauket, New York) computer program was used for data analysis. The data (band presence or absence) were introduced in the form of a binary matrix and a pairwise similarity matrix was constructed using the JACCARD coefficient (25). The values were obtained by the double number of shared bands between two patterns divided by the sum of all the bands in the same pattern (value 1 indicates identical patterns for two individuals and value 0 indicates completely different patterns). The UPGMA (Unweighted pair-group method with arithmetical averages) grouping of the values was generated using the NTSYS program.
Analysis of the total protein extracts
Humid masses of mycelium were triturated in a mortar with protein extraction buffer (15), centrifuged at 10,000 rpm for 5 minutes and the supernatants stored at 12ºC. The amount of protein present in the extracts was measured spectrophotometrically at 595 nm according to Bradford (7). All reactions were accomplished in triplicate. Aliquots of the extracts were submitted to polyacrylamide gel electrophoresis using a vertical run and a discontinuous system of buffers (22). Each sample was prepared by mixing 100 µl of the sample with 10 µl of bromophenol blue solution (0.025 g bromophenol blue + 10 ml Tris-HCl buffer, pH 6.7). The run buffer was Tris-glycine, pH 8.3. The running was started at 15 mA, corrected to 20 mA after the bromophenol blue front passed through the stacking gel. A bovine serum albumin solution was used as standard (0.001g of bovine serum albumin in 5ml NaCl 0.15M solution). After running, the gel was fixed in 20% glacial acetic acid + methanol (1:1, v/v) for 30 minutes and stained with a fresh solution of Coomassie Brilliant Blue R 250 + 10% glacial acetic acid. An aqueous mixture of 40% glacial acetic acid and 2.5% glycerol was added at a proportion of 1:20. Minimum staining time was of 8 hours. The gel was destained in a 20% methanol + 5% acetic acid + 2.5% glycerol solution. After drying the gel was photographed and submitted to the densitometer (DS-35 of CELM).
RESULTS AND DISCUSSION
Analysis of the RAPD profiles
The RAPD profiles obtained with the selected primers (OPW01, OPW02, OPW03, OPW04, OPW09, OPW12, OPW13) are shown in Figs. 1 and 2. The seven primers provided a total of 113 bands, with an average of 16 bands per primer. 41.6% were monomorphic, because they were present in all samples, while 58.4% were polymorphic, because at least one sample did not present the band. A similarity matrix and a dendrogram (Fig. 2) using the UPGMA method were constructed. All the mutants of the wild strain E9 presented a ramification point around 0.75, equivalent to 75% similarity, which corresponds to a moderate variation degree. The MaE34 and MaE49 mutants presented the highest similarity index (about 87.5%). These mutants presented the same physiological characteristics such as mycelial growth in complete media and minimum media, percentage of germination, beginning of conidia germination, and colony morphology, as reported by Oliveira et al. (19). The MaE10 mutant presented a ramification point around 0.81 (81.2% of similarity); the wild strain 0.78 (78% of similarity) and the MaE27 and MaE41 mutants 0.71 and 0.62, corresponding to 71% and 62% of similarity, respectively.
The RAPD molecular markers allowed verification of DNA polymorphism among the wild strain E9 and the mutants obtained by gamma radiation. This confirms the capability of the RAPD technique to distinguish mutants obtained by gamma radiation, as observed by Schlick et al. (26). These authors used the RAPD technique to detect mutants of Trichoderma harzianum obtained by gamma radiation, observing an evident DNA polymorphism among the wild strain and the mutants this was interpreted as an indication of the occurrence of deletions and inserts, detected by the molecular markers of RAPD.
Total protein analysis
The concentration of total protein in the mycelium extracts of the wild strain E9 of M. anisopliae var. anisopliae and its mutants varied from 6.0 to 8.0 µg/µl (Table 1). The zymogram obtained after electrophoresis of the total proteins is shown in Fig. 3. The wild strain E9 presented seven bands (considered as 100% for a comparative analysis), and the mutants five bands, resulting therefore in 71.4% of monomorphic bands and 28.6% of polimorphic bands. Quantitative analysis of the fractions of total proteins obtained by densitometry are shown Fig. 4. As shown in Table 1, the wild strain E9 presented seven fractions of proteins (A, B, C, D, E, F and G) in the electrophoretic profile. Variation in the amount of proteins in fractions was observed. All samples presented the fractions A, C, E, F, G. Fractions B and D were not observed in mutants. The amount of protein in fraction A varied from 1.44 to 2.5 µg%, and the MaE10 mutant presented the highest percentage. For fraction C the amount varied from 1.7 to 2.63 µg%. The percentage of protein in fraction E varied from 0.37 to 1.12 µg%; MaE49 presented the highest percentage. The MaE34 mutant presented the highest percentage of protein for the fraction F and MaE10 the lowest. For the fraction G the MaE41 mutant presented the lowest while the MaE34 showed the highest percentage of protein.
The RAPD technique was sensitive enough to detect genetic polymorphism among mutants of the wild strain E9 of M. anisopliae var. anisopliae obtained by gamma radiation. The average similarity index revealed a moderate degree of variation. The zymogram and protein electrophoretical profiles also indicated genetic variation, revealing the presence of seven proteins bands in the wild strain and five in the mutants.
The authors are indebted to CNPq and CAPES for financial support
Polimorfismo de DNA e proteína total em mutantes da linhagem E9 de Metarhizium anisopliae var. anisopliae (Metsch.) Sorokin
Foram analisados cinco mutantes MaE (MaE10, MaE27, MaE34, MaE41 e MaE49) da linhagem selvagem E9 de Metarhizium anisopliae var. anisopliae quanto ao perfil de DNA pela técnica de RAPD e também quanto ao conteúdo de proteína total por espectrometria e eletroforese em gel de poliacrilamida e densitometria. O padrão de marcadores de RAPD evidenciou polimorfismo nas amostras; dos 20 primers testados foram selecionados 7 que geraram 113 bandas. Deste total, 47 estavam presentes em todas as amostras (41.6% de bandas monomórficas) e 66 mostraram polimorfismo (58.4%). O coeficiente médio de similaridade foi de 75%. O conteúdo de proteína total variou de 6 a 8 µg/µl. O zimograma e perfís das frações de proteínas obtidos por densitometria revelaram 7 bandas para a linhagem selvagem E9 e 5 bandas para os mutantes com variações nos percentuais em µg% entre as frações de proteínas. A técnica de RAPD mostrou-se bastante sensível para detectar diferenças entre a linhagem selvagem e os mutantes obtidos por radiação gama. A análise de proteína total também evidenciou mudanças ocorridas na quantidade e no padrão de bandas nos mutantes em relação à linhagem selvagem.
Palavras-chave: Metarhizium anisopliae, mutantes, RAPD, proteína total
1. Abbasi, P.A.; Miller, S.A.; Meulia,T.; Hoitink, H.A.J.; Kim, J.M. Precise detection and tracing of Trichoderma hamatum 382 in compost-amended potting mixes by using moleculars markers. Appl. Environ. Microbiol., 65(12):5421-5426. 1999. [ Links ]
2. Alves, S.B. Fungos Entomopatogênicos. In: ALVES, S.B., ed Controle microbiano de Insetos. Editora ESALQ-USP, Piracicaba, SP, Brasil. p.289-381, 1998. [ Links ]
3. Azevedo, A.C.S. Variabilidade genética entre isolados de B. bassiana obtidos de Schizoterium palens, Castria licus e Deois flavopicta Dissertação (Monografia). Universidade Estadual de Londrina, Centro de Ciências Biológicas, Departamento de Biologia Geral. Londrina, 1996. [ Links ]
4. Azevedo, J.L.; Costa, S.O.P. Exercícios práticos de Genética. Orgs. São Paulo, Ed. Nacional, ED. USP, 288p. 1973. [ Links ]
5. Bagalhi, E.; Valadares, M.C.C.; Azevedo, J.L. Parameiosis in the entomopathogenic fungus Metarhizium anisopliae (Metsch.) Sorokin. Rev. Bras. Gen, 14(2):261-271, 1991. [ Links ]
6. Bidochka, M.J.; Mc Donald, M.A.; ST-Leger, R.J.; Roberts, D.W. Differentiation of species and strains of entomopathogenic fungi by Randon Amplified Polymorphic DNA (RAPD). Curr. Gen., 25; 107-113, 1994. [ Links ]
7. Bradford, M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye bynding. Anal. Biochem., 72:248-254, 1976. [ Links ]
8. Chew, J.S.K.; Strongman, D.B.; Mackay, R.M. Comparisons of twenty isolates of the entomopathogen Paecilomyces farinosus by analysis of RAPD markers. Mycol. Res., 102(10):1254-1258, 1998. [ Links ]
9. Crowhurst, R.N.; King, F.Y.; Hawthorne, B.T.; Sanderson, F.R.; Choi-Pheng, Y., RAPD characterization of Fusarium oxysporum associated with wilt of angsana (Pterocarpus indicus) in Singapore. Mycol. Res., 99(1):14-18, 1995. [ Links ]
10. Fegan, M.; Manners, J.M.; Maclean, D.J.; Irwim, J.A.G.; Samuels, K.D.Z.; Holdom, D.G.; Li, D.P. Randon amplified polymorphic DNA markers reveal a high degree of genetic diversity in the entomopathogenic fungus Metarhizium anisopliae var anisopliae. J. Gen. Microbiol., 139:2075-2081, 1993. [ Links ]
11. Fungaro, M.H.P.; Vieira, M.L.C.; Pizzirani-Kleiner, A.A.; Azevedo, J.L. Diversity among soil and insect isolates of Metarhizium anisopliae var. anisopliae detected by RAPD. Lett. Appl. Microbiol., 22:389-392, 1996. [ Links ]
12. Garcia, A.S.; Messias, C.L.; Souza, H.M.L.; Piedrabuena, A.E. Patogenicidade de Metarhizium anisopliae var. anisopliae em Ceratitis capitata (Wied.) (Diptera; Tephritidae). Rev. Bras. Entomol., 28:421-424, 1984. [ Links ]
13. Gomez, I.; Chet, I.; Herreraestrela, A. Genetic diversity and vegetative compatibility among Trichoderma harzianum isolates. Mol. Gen. Genetics, 256:127-135, 1997. [ Links ]
14. Gorinstein, S.; Paccola-Meirelles, L.D.; Belo, V.A.; Azevedo, J.L. Characterization of Beauveria bassiana, Metarhizium anisoplie and Aspergillus nidulans through eletrophoretic patterns of their protein fractions. J. Ferm. Bioeng., 82(2):89-92, 1996. [ Links ]
15. Hanson, L.C.; Wells, K. Characterization of tree Tremella species by isozymes analysis. Mycologia, 83(4):446-454, 1991. [ Links ]
16. Manulis, S.; Kogan, N.; Reuven, M.; Ben-Yephet, Y. Use of the RAPD Tecnique for identification of Fusarium oxysporum f. sp. dianthi from carnation. Phytopathology, 84(1):98-101, 1993. [ Links ]
17. Messias, C.L.; Azevedo, J.L. Parassexuality in the deuteromycete Metarhizium anisopliae. Trans. Brit. Mycol. Soc., 75:473-477, 1980. [ Links ]
18. Oliveira, N.T.; Messias, C.L. Variability of strains of Metarhizium anisopliae var. anisopliae (Metsch.) Sorokin in terms of protein content and spore germination. Rev. Microbiol., 27:268-272, 1996. [ Links ]
19. Oliveira, M.G.; Oliveira, N.T.; Luna-Alves Lima, E.A. Novos biotipos de Metarhizium anisopliae var. anisopliae (Metsch.) Sorokin com germinação alterada de conídios, obtidos pela exposição à radiação gama radiação gama. Arq. Biol. Tecnol., 40(3):586-595, 1997. [ Links ]
20. Oliveira, M.G.; Oliveira, N.T.; Luna-Alves Lima, E.A. Characterization of new biotypes of P157 strain of Metarhizium anisopliae var. anisopliae, got by treatment with gamma radiation. Bol. Micol. 12(1-2):41-48, 1997. [ Links ]
21. Paavanem-Huhtala, S.; Avikaiiven, H.; Yli-Matila, T. Development of strain specific primer for a strain of Gliocladium catenulatum used in biological control. Eur. J. Plant Pathol., 106(2):187-198, 2000. [ Links ]
22. Paccola-Meirelles, L.D.; Valarini. M.J.; Azevedo, J.L.; Alfenas, A.C. Manual de Técnicas Eletroforéticas em Microorganismos. Piracicaba: FEALQ, 54p, 1988. [ Links ]
23. Pontecorvo, G.; Roper, J.A.; Hemons, J.M.; Mac Donald, K.D.; Button, A.W.J. The genetics of Aspergillus nidulans. Adv. Gen., 5:141-238, 1953. [ Links ]
24. Raeder, U.; Broda, P. Rapid preparation of DNA from filamentous fungi. Lett. Appl. Microbiol., 1: 16-20, 1985. [ Links ]
25. Sneath, P.H.A.; Sokal, R.R. Numerical Taxonomy: The Principles and Practice of Numerical Classification. San Francisco: Freeman, 1973, 573p. [ Links ]
26. Schlick, A.; Kuhls, K.; Meyer, W.; Lieck Fel Dt, E.; Borner, T.; Messner, K. Fingerprinting reveals gamma-ray induced mutations in fungal DNA: implications for identification of patent strains of Trichoderma harzianum. Curr. Gen., 26:74-78, 1994. [ Links ]
27. Welsh, J.; Mc Clelland, M. Fingerprinting genomes using PCR with arbitrary primers. Nucleic Acid Res., 18:7213-7218, 1990. [ Links ]
28. Williams, J.G.K.; Kubelik, A.R.; Livak, K.J.; Rafalski, J.A.; Tingey, S.V. DNA polymorphisms amplified by arbitrary primers. Nucleic Acid Res., 18(22):6531-6535, 1990. [ Links ]
29. Wilson, M.; Lindow, S.E. Release of Recombinant Microorganismos. Ann. Rev. Microbiol., 47:913-944, 1993. [ Links ]
30. Zervakis, G.; Sourdis, J.; Balis, C. Genetic variability and systematics of eleven Pleurotus species based on isozyme analysis. Mycol. Res., 98(3):329-341, 1994. [ Links ]
31. Zimand, G.; Valinsky, L.; Elad, Y.; Chet, I.; Manulis, S. Use of the RAPD procedure for the identification of Trichoderma strains. Mycol. Res., 98(5):531-534, 1994. [ Links ]
32. Zinno, T.D.; Longrée, H.; Maraite, H. Diversity of Pyrenophora tritici-repentis isolates from warm wheat growing areas: pathogenicity, toxin production and RAPD analysis. In: Helminthosporium blights of wheat: spot blotch and tan spot. Duveiller et al. (eds.), Mexico, D.F.: CIMMYT, pp.302-311, 1998. [ Links ]
* Corresponding author. Mailing address: Universidade Federal de Pernambuco, Centro de Ciências Biológicas-CCB, Departamento de Micologia, Av. Prof. Nelson Chaves S/N - Cidade Universitária, 50670-901, Recife, PE, Brasil. Telefax: (+5581) 271-8482 / 271-8483. E-mail: firstname.lastname@example.org