Submitted to: Applied Microbiology and Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/2/2005
Publication Date: 7/1/2006
Citation: Stevenson, D., Muck, R.E., Shinners, K., Weimer, P.J. 2006. Use of real time polymerase chain reaction to determine population profiles of individual species of lactic acid bacteria in alfalfa silage and stored corn stover. Applied Microbiology and Biotechnology. 71(3):329-338. Interpretive Summary: We commonly preserve crops for cattle by a fermentation process known as ensiling. This fermentation is done by lactic acid bacteria eating sugars and producing lactic acid and other products that help preserve the crop. A crop that has been fermented this way is called silage. Some lactic acid bacteria are better than others in preserving the nutritional value of the crop and so farmers may add commercial strains of lactic acid bacteria to the crop to guarantee a good fermentation. Many times it would be useful to know which bacterial species were the most active in preserving the crop. However, the techniques to do that have been very time-consuming, expensive and not particularly sensitive. We developed a method based on amplifying the bacterial DNA for each species that allowed us to measure the presence of seven important lactic acid bacterial species in silages. This method allows us to quantify not only the major species that are active in silage but also the minor players. The method is also much more rapid and inexpensive than older methods. We expect that this new method will be useful now for scientists trying to understand what happens during ensiling and how it may affect the nutritional value of the crop. In the future, this method may be useful in diagnosing why a farmer's silage is not liked by his or her cows.
Technical Abstract: Real-time polymerase chain reaction (RT-PCR) was used to quantify seven species of lactic acid bacteria (LAB) in alfalfa silage prepared at laboratory scale in the presence or absence of four commercial inoculants, and corn stover harvested at different moisture levels and stored for ~9 months under a variety of field conditions without inoculum addition. Species-specific PCR primers were designed based on known recA gene sequences for five species, and from sequences determined from two other species following cloning of the gene into E. coli. For alfalfa silage, species corresponding to those in commercial inoculants displayed dramatic reductions in relative population sizes within 24 h of inoculation. Lactobacillus brevis was the most aboundant LAB (12 to 32 % of total bacterial DNA) in all of the alfalfa silages by 96 h. Modest populations (up to 10%) of L. plantarum were observed in inoculated silage, and small populations (0.1 to 1%) of L. buchneri and Lactococcus lactis were observed in all silages, while L. pentosus and Enterococcus faecium were near or below detection limits. Corn stover generally displayed higher populations of L. plantarum and L. brevis and lower populations of L. buchneri and L. pentosus, while Enterococcus faecium and Lactococus lactis were near or below the minimum detectable limits of the RT-PCR assay (~0.001% of total DNA). The data illustrate the utility of RT-PCR for quantifying individual species of LAB in conserved forages prepared under a wide variety of conditions.