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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 Mergulhao and E. Martinez-Romero
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.
Keywords: Cactaceae, Nopalea, 16S rDNA, ARDRA, ISSR