Cellulolytic bacterial biodiversity in long-term manure experimental sites

Abstract

To study the Influence of long-term application of manure on cellulolytic bacterial diversity, 54 efficient cellulolytic bacterial cultures were isolated from two long-term manure experimental field sites of Berlin-Dahlem (established in 1923) and BadLauchstadt (established in 1902). The sequence divergence of highly conserved region of 16S rDNA was exploited by restriction analysis of PCR-amplified 16S rDNA using restriction enzyme ScrFI to assay the evolutionary relatedness of isolates. Restriction analysis identified 10 genetically diverse pattern groups comprising five groups each of bacterial and actinomycetal domain. Irrespective of manuring, a dominant pattern group (H2) was identified, containing 31.48% of total isolates. On the other hand, two site-specific pattern groups highly specific for the brown soil (H2, 9.25% of total isolates) and for black soil (J2, 11.11% of total isolates), respectively were identified. In general, the composition of cellulolytic isolates in two sites displayed differences with respect manure application and soil properties. Manure strongly influenced the abundance of cellulolytic bacterial diversity in brown soil. The terminal restriction fragment length polymorphism (T-RFLP) data revealed a distinct relationship of total bacterial diversity with long-term manure application. This influence is more prominent in nutrient poor brown soil. Based on 16S rDNA sequence analysis, isolates of the dominant as well as the specific pattern groups could be assigned to the genus Streptomyces comprising species of diverse phylogenetic affiliation. Furthermore, sequencing of 16S rDNA of isolates of five bacterial pattern groups revealed a high phylogenetic diversity among these isolates, including Streptococcus, Paenibacillus, Bacillus, Bacillus megaterium and Bacillus pumilus. Key words: Cellulolytic bacteria, 16SrDNA gene, restriction analysis, restriction fragment length polymorphism (T-RFLP), terminal restriction fragment length polymorphism (T-RFLP), phylogenetic diversity.