Table 4.

PCR detection of P. putida mt-2 cells andB. globigii spores in soils that differ in humic acid and background DNA concentrations

Soil typeAmt (μg) of soil DNAaAmt (μg) of humic acidsbOrganismVisible amplicon with P. putida CFU or B. globigii spores seeded per g of soilc
1071061051041030
N. Mex. soil0.18 (0.006)49 (10) P. putida ++NT++++++----
B. globigii ++++++++++----
Ohio soil21.3 (5.6)908 (440) P. putida ++NT++++--------
B. globigii ++++++++--------
Ariz. soil13.3 (2.6)2,200 (762) P. putida ++NT++++--------
B. globigii ++++++++--------
Ariz. cinders14.6 (5.4)1,933 (614) P. putida ++NT++------------
B. globigii ++++++------------
  • a Per gram (wet weight) of soil. Values are means of three to four replicate extractions, and numbers in parentheses are standard deviations. DNA was quantified by a PicoGreen spectrophotometric assay as described in Materials and Methods.

  • b Per gram (wet weight) of soil. Values are means of three to four replicate extractions, and numbers in parentheses are standard deviations. Humic acids were quantified by UV spectroscopy as described in Materials and Methods. Values presented are the amounts of humic material present in crude extracts prior to removal by Sephadex G200 spin microcolumns.

  • c P. putida cells were seeded at 4 × 107, 4 × 105, 4 × 104, and 4 × 103 CFU per g of soil, andB. globigii endospores were seeded at 2.5 × 107, 2.5 × 106, 2.5 × 105, 2.5 × 104, and 2.5 × 103 spores per g of soil. Results are the presence (++) or absence (----) of a visible amplicon, detected by ethidium bromide staining 1/10 of the PCR mixture in an agarose gel. All reactions were positive for amplification of general bacterial and fungal rDNA fragments using the P3MOD-PC5B and NS1-NS2 primer pairs, respectively, at template concentrations of 100 pg and 1 ng (data not shown), indicating that they were sufficiently free of inhibitors to support PCR. NT, not tested.