Isolation and molecular characterization of endophytic bacteria associated with the culture of forage cactus (Opuntia spp.)

M. C. C. P. de Lyra; D. C. Santos; C. Mondragon-Jacobo; M.L.R.B. da Silva; A. C. E.S Mergulhão; E. Martínez-Romero

doi:10.7324/JABB.2013.1103

Research Article | Volume: 1, Issue: 1, May - June, 2013

Isolation and molecular characterization of endophytic bacteria associated with the culture of forage cactus (Opuntia spp.)

M. C. C. P. de Lyra, D. C. Santos, C. Mondragon-Jacobo, M.L.R.B. da Silva, A. C. E.S Mergulhão , E. Martínez-Romero

Open Access

Published: Jun 28, 2013

DOI: 10.7324/JABB.2013.1103

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Abstract

In Brazil, the forage cactus, has great economic interest because such plants are the nutritional base for livestock from semiarid regions. However little is known of endophytic diazotrophs in this culture. This work is aimed at characterizing molecularly in different semi-solid media using 22 isolates from seven cultivars of Opuntia:IPA 90-92; Marmillon Fodder (1317); F8 (438); F3 Rojo Vigor; Mexico vegetable (1294); Chile Fruit (1371); COPENA - V1 – isolated in different semi-solid media specific. The plant was inoculated with 100 µL of plant crude extract in a semi-solid medium free of nitrogen. The isolates were grown in DYGS medium with malic acid (NFB, JNFb, LGI and LG), and sucrose in DYGS medium for the isolates of LGI-P medium. The Wizard Genomic DNA Purification Kit (Promega) was employed for the extraction of DNA. The use of four ISSR markers (UBC 808, UBC 809, UBC 810 and UBC 812), and the ARDRA technique consisting of five restriction enzymes (DdeI, AvaI, HaeIII, HinfI and MspI), followed by amplification of the 16S rDNA region (primers fD1 and rD1). This research points to great diversity of bacteria living in association with cactus and it will be necessary to undertake more detailed studies with molecular markers to obtain a conclusive characterization of the genetic diversity of plants with these endophytes, which could be achieved working with ribosomal genes and subsequent sequencing of these microorganisms.

Keyword: Cactaceae Nopalea 16S rDNA ARDRA ISSR

Copyright: Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike license.

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1. Bazzicalupo, M, Okon, Y. Associative and endophytic symbiosis. In: F Pedrosa, Hungria M, Yates, MG and Newton, WE (Eds.). Nitrogen fixation: from molecules to crop productivity. Dordrecht, Kluwer Academic Publishers. 2000, p.409-410.
2. Boddey RM, Oliveira OC, Urquiaga S, Reis V, Olivares F, Baldani VLD, Döbereiner J. Biological nitrogen fixation associated with sugarcane and rice: contribution and prospects for improvement. Plant and Soil. 1995; 174: 195-209. https://doi.org/10.1007/BF00032247
3. Cheang AJR, Cerón IDT, Bringas YF, Badía MMR, Casta-eda JM, Pérez MH. Caracterización fisiológica de la comunidad microbiana endófita de la ca-a de azúcar. Revista Colombiana de Biotecnología. 2005; 1: 66-75.
4. Döbereiner J. Biological nitrogen fixation in the tropics: social and economic contributions. Soil Biology & Biochemistry. 1997; 29: 771-774. https://doi.org/10.1016/S0038-0717(96)00226-X
5. Dobereiner J, Baldani VLD, Baldani JI. Como isolar e identificar bacterias diazotróficas de plantas não-leguminosas. – Brasilia:EMBRAPA-SPI: Itaguai, RJ: EMBRAPA-CNPAB. 60p. 1995.
6. Fang DQ, Roose ML. Identification of closely related citrus cultivars with inter-simple sequence repeat markers. Theoretical and Applied Genetics. 1997; 95 : 408-417. https://doi.org/10.1007/s001220050577
7. Govindarajan M, Kwon SW. Isolation, molecular characterization and growth-promoting activities of endophytic sugarcane diazotroph Klebsiellasp. GR9. World Journal Microbiology Biotechnology. 2007; 23: 997-1006. https://doi.org/10.1007/s11274-006-9326-y
8. Gupta M, Chyi YS, Romeo-Severson J, Owen JL. Amplification of DNA markers from evolutionarily diverse genomes using single primers of simple-sequence repeats. Theoretical and Applied Genetics, 1994; 89:998-1006. https://doi.org/10.1007/BF00224530
9. James EK. Nitrogen fixation in endophytic and associative symbiosis. Field Crops Research. 2000; 65: 197-209. https://doi.org/10.1016/S0378-4290(99)00087-8
10. Melloni R, Nóbrega RSA, Moreira FMS, Siqueira JO. Densidade e diversidade fenotípica de bactérias diazotróficas endofíticas em solos de mineração de bauxita em reabilitação (1). Revista Brasileira Ciência do Solo. 2004; 28:85-93. https://doi.org/10.1590/S0100-06832004000100009
11. Pimienta-Barrios E, Mu-oz-Urias A. Domestication of Opuntias and cultivated varieties. In: Barbera, G., P. Inglese and B.E. Pimienta (Eds.), Agroecology, cultivation and uses of cactus pear, 1994. p 58-63.
12. Reis Junior FB, Mendes IC, Texeira KRS, Reis VM. Uso de ferramentas moleculares em estudos da diversidade de microrganismos do solo Planaltina, DF: Embrapa Cerrados, 33p. (Documentos / Embrapa Cerrados, ISSN 1517-5111; 51). 2002.
13. Sanchez de la Hoz MP, Davalia, JA, Loarce Y, Ferrer E. Simple sequence repeat primers used in polymerase chain reaction amplification to study genetic diversity in barley. Genome. 1996; 39:112 – 117 https://doi.org/10.1139/g96-015
14. Sofalian O, Chaparzadeh N, Javanmard A, Hejazi MS. Study the genetic diversity of wheat landraces from northwest of Iran based on ISSR molecular markers. International Journal of Agriculture e Biologia. 2008; 10: 465–468.
15. Weisburg WG, Barns SM, Pelletier DA, Lane DJ. 16S DNA ribosomal amplification for phylogenetic study. Journal of Bacteriology 1991; 173: 697-703. https://doi.org/10.1128/jb.173.2.697-703.1991
16. Zietkiewicz E, Rafalski A, Labuda D. Genome fingerprinting by simple sequence repeat (SSR)-anchored polymorphism chain reaction amplification. Genomics. 1994; 20:176-183. https://doi.org/10.1006/geno.1994.1151

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1. Bazzicalupo, M, Okon, Y. Associative and endophytic symbiosis. In: F Pedrosa, Hungria M, Yates, MG and Newton, WE (Eds.). Nitrogen fixation: from molecules to crop productivity. Dordrecht, Kluwer Academic Publishers. 2000, p.409-410.

2. Boddey RM, Oliveira OC, Urquiaga S, Reis V, Olivares F, Baldani VLD, Döbereiner J. Biological nitrogen fixation associated with sugarcane and rice: contribution and prospects for improvement. Plant and Soil. 1995; 174: 195-209. https://doi.org/10.1007/BF00032247

3. Cheang AJR, Cerón IDT, Bringas YF, Badía MMR, Casta-eda JM, Pérez MH. Caracterización fisiológica de la comunidad microbiana endófita de la ca-a de azúcar. Revista Colombiana de Biotecnología. 2005; 1: 66-75.

4. Döbereiner J. Biological nitrogen fixation in the tropics: social and economic contributions. Soil Biology & Biochemistry. 1997; 29: 771-774. https://doi.org/10.1016/S0038-0717(96)00226-X

5. Dobereiner J, Baldani VLD, Baldani JI. Como isolar e identificar bacterias diazotróficas de plantas não-leguminosas. – Brasilia:EMBRAPA-SPI: Itaguai, RJ: EMBRAPA-CNPAB. 60p. 1995.

6. Fang DQ, Roose ML. Identification of closely related citrus cultivars with inter-simple sequence repeat markers. Theoretical and Applied Genetics. 1997; 95 : 408-417. https://doi.org/10.1007/s001220050577

7. Govindarajan M, Kwon SW. Isolation, molecular characterization and growth-promoting activities of endophytic sugarcane diazotroph Klebsiellasp. GR9. World Journal Microbiology Biotechnology. 2007; 23: 997-1006. https://doi.org/10.1007/s11274-006-9326-y

8. Gupta M, Chyi YS, Romeo-Severson J, Owen JL. Amplification of DNA markers from evolutionarily diverse genomes using single primers of simple-sequence repeats. Theoretical and Applied Genetics, 1994; 89:998-1006. https://doi.org/10.1007/BF00224530

9. James EK. Nitrogen fixation in endophytic and associative symbiosis. Field Crops Research. 2000; 65: 197-209. https://doi.org/10.1016/S0378-4290(99)00087-8

10. Melloni R, Nóbrega RSA, Moreira FMS, Siqueira JO. Densidade e diversidade fenotípica de bactérias diazotróficas endofíticas em solos de mineração de bauxita em reabilitação (1). Revista Brasileira Ciência do Solo. 2004; 28:85-93. https://doi.org/10.1590/S0100-06832004000100009

11. Pimienta-Barrios E, Mu-oz-Urias A. Domestication of Opuntias and cultivated varieties. In: Barbera, G., P. Inglese and B.E. Pimienta (Eds.), Agroecology, cultivation and uses of cactus pear, 1994. p 58-63.

12. Reis Junior FB, Mendes IC, Texeira KRS, Reis VM. Uso de ferramentas moleculares em estudos da diversidade de microrganismos do solo Planaltina, DF: Embrapa Cerrados, 33p. (Documentos / Embrapa Cerrados, ISSN 1517-5111; 51). 2002.

13. Sanchez de la Hoz MP, Davalia, JA, Loarce Y, Ferrer E. Simple sequence repeat primers used in polymerase chain reaction amplification to study genetic diversity in barley. Genome. 1996; 39:112 – 117 https://doi.org/10.1139/g96-015

14. Sofalian O, Chaparzadeh N, Javanmard A, Hejazi MS. Study the genetic diversity of wheat landraces from northwest of Iran based on ISSR molecular markers. International Journal of Agriculture e Biologia. 2008; 10: 465–468.

15. Weisburg WG, Barns SM, Pelletier DA, Lane DJ. 16S DNA ribosomal amplification for phylogenetic study. Journal of Bacteriology 1991; 173: 697-703. https://doi.org/10.1128/jb.173.2.697-703.1991

16. Zietkiewicz E, Rafalski A, Labuda D. Genome fingerprinting by simple sequence repeat (SSR)-anchored polymorphism chain reaction amplification. Genomics. 1994; 20:176-183. https://doi.org/10.1006/geno.1994.1151

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