Localization of ruminal cellulolytic bacteria on plant fibrous materials as determined by fluorescence in situ hybridization and real-time polymerase chain reaction Running title: FISH detection of ruminal cellulolytic bacteria

25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 To visualize and localize specific bacteria associated with plant materials, a new fluorescence in situ hybridization (FISH) protocol was established. By using this protocol, we successfully minimized the autofluorescence of orchard grass hay and detected rumen bacteria attached to the hay under a fluorescence microscope. Real-time polymerase chain reaction assays were also employed to quantitatively monitor the representative fibrolytic species Fibrobacter succinogenes and Ruminococcus flavefaciens, and also total bacteria attached to the hay. F. succinogenes was found firmly attached to not only the cut edges but also undamaged inner surfaces of the hay. Cells of phylogenetic group 1 of F. succinogenes were detected on many stem and leaf sheath fragments of the hay, even on fragments on which few other bacteria were seen. Cells of phylogenetic group 2 of F. succinogenes were often detected on hay fragments coexisting with many other bacteria. On the basis of 16S rDNA copy number analysis, the numbers of bacteria attached to the leaf sheaths were higher than those attached to the stems (P < 0.05). In addition, R. flavefaciens had a greater tendency than F. succinogenes to be found on the leaf sheath (P < 0.01) with formation of many pits. F. succinogenes, particularly phylogenetic group 1, is suggested to possibly play an important role in fiber digestion, because it is clearly detectable by FISH and is the bacterium with the largest population size in the less easily degradable hay stem.