Specific detection and real-time PCR quantification of potentially mycophagous bacteria belonging to the genus Collimonas in different soil ecosystems

Abstract

The bacterial genus Collimonas has the remarkable characteristic that it grows at the expense of living fungal hyphae under laboratory conditions. Here, we report the first field inventory of the occurrence and abundance of Collimonas in soils (n = 45) with naturally different fungal densities, which was performed in order to test the null hypothesis that there is a relationship between the presence of Collimonas and fungal biomass. Estimates of fungal densities were based on ergosterol measurements. Each soil was also characterized in terms of its physical and chemical properties and vegetation and management types. Culturable Collimonas was identified in plate-spread soil samples by its ability to clear colloidal chitin, in combination with a Collimonas-specific restriction fragment length polymorphism analysis of 16S rRNA PCR amplified from individual colonies. Using this approach, we found culturable collimonads only in (semi)natural grasslands. A real-time PCR assay for the specific quantification of Collimonas 16S rRNA in total soil DNA was developed. Collimonas was detectable in 80% of the soil samples, with densities up to 10(5) cells g(-1) (dry weight) soil. The numbers of Collimonas cells per gram of soil were consistently lowest in fungus-poor arable soils and, surprisingly, also in fungus-rich organic layers of forest soils. When all soils were included, no significant correlation was observed between the number of Collimonas cells and ergosterol-based soil fungal biomass. Based on this result, we rejected our null hypothesis, and possible explanations for this were addressed.

FIG. 1.

Alignment of partial 16S rRNA sequences from previously described collimonads and closely related species. The numbers correspond to the numbers in the 16S rRNA sequence of C. fungivorans Ter331 (accession no. AJ310395). Dashes indicate nucleotides identical to those in the Ter331 sequence. The locations of primers Eddy3for and Eddy3rev, as well as the dual-labeled probe Sophie, are indicated. The boxed nucleotides indicate the BstBI restriction site unique to collimonads. The accession numbers for the 16S rRNA sequences of strains Ter166, Ter300, Ter228, Ter14, Ter165, Ter299, Ter90, Ter94, Ter227, Ter291, DSM 9628^T, DSM 1522^T, DSM 6445^T, ATCC 19308^T, and DSM 13128^T are AY281140, AY281145, AY281148, AY281135, AY281139, AY281144, AY281136, AY281138, AJ496445, AY281143, Y08845, Y08846, Y10146, AB021424, and AJ238358, respectively.

FIG. 2.

(a and b) Average ergosterol contents (mg kg⁻¹ [dry weight] soil) in the upper layer (0 to 10 cm) of the mineral soils at arable, forest, and (semi)natural grassland sites (a) and in the organic and mineral layers at five forest sites (b). (c and d) Average log-transformed numbers of Collimonas genome equivalents per gram (dry weight) of soil in the upper layer (0 to 10 cm) of the mineral soils at arable, forest, and (semi)natural grassland sites (c) and in organic and mineral layers at five forest sites (d). Different letters indicate significant differences at the 0.05% level (as determined by a modified Tukey's honestly significant difference test).

FIG. 3.

RFLP analysis of BstBI-digested 16S rRNA genes amplified almost to completion from J. agaricidamnosum DSM 9628^T (lane A), J. lividum DSM 1522^T (lane B), H. rubrisubalbicans ATCC 19308^T (lane C), H. seropedicae DSM 6445^T (lane D), H. frisingense DSM 13128^T (lane E), C. fungivorans Ter331 (lane F), Collimonas sp. from soil sample 19 (lanes G and H), Collimonas sp. from soil sample 26 (lane I), Collimonas sp. from soil sample 15 (lane J), Collimonas sp. from soil sample 26 (lane K), isolate 142 from soil sample 16 (= Flavobacterium sp.) (lane L), isolate 134 from soil sample 27 (= Burkholderia sp.) (lane M), isolate 186 from soil sample 36 (= Rhodanobacter sp.) (lane N), and isolate 88 from soil sample 9 (= Pedobacter sp.) (lane O). The lanes indicated by an asterisk contained a 1-kb marker. The strains in lanes A to E were obtained from DSMZ (Braunschweig, Germany). The strains in lanes G to O were chitinolytic isolates from our inventory study. Identification of the strains in lanes G to O (see above) was based on sequence analysis of the 16S rRNA gene fragment amplified by primers pA and 1492r (15).

FIG. 4.

Standard curve for the Collimonas-specific dual-labeled probe PCR assays. This curve was created using known amounts of DNA ranging from 3.7 × 10¹ to 3.7 × 10⁶ genome equivalents of C. fungivorans Ter331 genomic DNA per reaction mixture. The values shown are representative results derived from multiple independent assays (n = 5).