Tobacco as many other plants has its own microbiota. There are very few studies determining the evolution of this microbiota during tobacco storage, which may affect the quality of tobacco. Polymerase chain reaction (PCR) combined with denaturing gradient gel electrophoresis (DGGE) were used to determine changes in the microbiota of tobacco during the aging of eleven different tobacco grades stored at three different locations for twelve months. The microbial fraction of these tobacco grades was extracted, and the bacterial 16S and the fungal 18S ribosomal RNA gene (rDNA) sequences were PCR amplified before being segregated by DGGE. The bacterial complexity of the tobacco grades was represented by DGGE migrating banding profiles that varied between 20 and 30 bands. Some variations in the banding profiles were observed between the tobacco grades, but overall no substantial changes occurred in the bacterial population of the different grades during their storage at different locations. Most of the fungal DGGE profiles were identical and had only one dominating band related to the genus Aspergillus. Bacterial and fungal isolates were also derived from the microbial fractions of the tobacco, and part of their respective 16S and 18S rDNA sequences were determined. Bacterial isolates belonged to Bacillales and gamma Protobacteria. Fungal isolates belonged to the genus Aspergillus. Our results showed that the bacterial and fungal biota of tobacco are relatively stable throughout 12 months storage time.
If the inline PDF is not rendering correctly, you can download the PDF file here.
1. Zhao M. B. Wang F. Li L. Qiu F. Li S. Wang and J. Cui: Analysis of bacterial communities on aging flue-cured tobacco leaves by 16S rDNA PCR–DGGE technology; Appl. Microbiol. Biotechnol. 73 (2007) 1435–1440.
2. Muyzer G. E. C. De Waal and A. G. Uitterlinden: Pro-filing of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction amplified genes coding for 16S rRNA; Appl. Environ. Microbiol. 59 (1993) 695–700.
3. Di Giacomo M. M. Paolino D. Silvestro G. Vigliotta F. Imperi P. Visca P. Alifano and D. Parente: Microbial community structure and dynamics of dark fire-cured tobacco fermentation; Appl. Environ. Microbiol. 73 (2007) 825–837.
4. Welty R. E. and G. B. Lucas: Fungi isolated from flue-cured tobacco at time of sale and after storage; Appl. Microbiol. 17 (1969) 360–365.
5. Welty R. E. and L. A. Nelson: Growth of Aspergillus repens in flue-cured tobacco; Appl. Microbiol. 21 (1971) 854–859.
6. Tso T. C.: Seed to Smoke; in: Tobacco: Production Chemistry and Technology edited by D. Layten Davis and M. T. Nielsen Blackwell Science Inc. Malden MA. 1999 pp. 1–31.
7. CORESTA: Determination of reducing substances in tobacco by continous flow analysis; Method No37 (1994).
8. Borneman J. and R. J. Hartin: PCR primers that amplify fungal rRNA genes from environmental samples; Appl. Environ. Microbiol. 66 (2000) 4356–4360.
9. Qiu L. M. Zhao X. Yue W. Qi and W. Zhang: Iso-lation and identification of the microflora on tobacco leaves during the natural fermentation of flue-cured tobacco; Tob. Sci. Technol. 3 (2000) 14–17.
10. Lane D. J.: 16S/23S rRNA sequencing; in: Nucleic Acid Techniques in Bacterial Systematics edited by E. Stackebrandt and M. Goodfellow John Wiley & Sons Chichester U. K. 1991 pp. 115–175
11. Edwards U. T. Rogall H. Blöcker M. Emde and E. C. Böttger: Isolation and direct complete nucleotide determination of entire genes: characterization of a gene coding for 16S ribosomal RNA; Nucleic Acids Res 17 (1989) 7843–7853.
12. Brown P. B. and G. V. Wolfe: Protist genetic diversity in the acidic hydrothermal environments of Lassen volcanic national park USA; J. Eukaryotic Microbiol. 53 (2006) 420–431.