Table 1.
Overview of nematode diversity at genus level (microscopic analysis) in the topsoil (depth 0–25 cm) of the former arable field and the adjacent pristine beech forest.
Figure 1.
Precipitation and temperature in relation to total nematode densities in open (field) and closed (forest) canopies.
Weekly averages of daily temperature (red) and total rainfall over 21 days before sampling (blue) as measured by the Royal Dutch Meteorological Institute (KNMI) are shown above. At the bottom, average nematode densities per 100 ml of soil from a since 25 years abandoned arable field (open canopy, yellow bars) and adjacent pristine beech forest (close canopy, green bars) are given. Sites sampled in 2009 at regular intervals between March 17 (week 1) and December 18 (week 39).
Table 2.
Molecular overview of the nematode families and genera monitored in our study.
Figure 2.
Temporal patterns of bacterivorous, omnivorous and predatory nematode families.
We determined DNA-based variation in the nematode densities per 100 ml soil (note differences in y-axes) of representatives from seven bacterivorous families: Teratocephalidae, Prismatolaimidae, Cephalobidae, Plectidae (i.e., all Plectidae excl. Anaplectus and ‘Anaplectus’, both in a dashed gray box), Alaimidae, Metateratocephalidae, Monhysteridae; the omnivorous family Dorylaimidae (D3 region sensu [65]); and the predatory families Mononchidae (M3, [65]) and Mylonchulidae. Sampling weeks as x-axes (constant scales); samples from the field are represented by orange triangles and samples from the forest by green diamonds. Trends are given as two-period moving averages: the averaged 2nd and 3rd data points are portrayed by the 1st data point and so forth.
Figure 3.
Temporal patterns of fungivorous nematode families.
Seasonal variation in densities for fungivores in the field (A) and in the forest (B). Please note that the y-axis scales differ. Aphelenchidae (Aphe, blue), Aphelenchoididae (Acho, red), and two genera belonging to Diphtherophoridae –viz. Tylolaimophorus (Tylo, green) and Diphtherophora (Diph, yellow)– show different patterns over the seasons between open and close canopies. As these taxa represent all observed fungivores, a partial Mantel analysis performed in a matrix describing the community structure in the field (open canopies, matrix Y) and in the forest (close canopies, matrix X) using the squared Euclidean distance was performed using the total entries and the same set of entities. A positive association between the matrices is indicated over the seasons by observed Z greater than average Z from randomized runs (P = 0.0297).
Figure 4.
Development and testing of a nematode family-specific primer combination.
Here we use the Metateratocephalidae (one bacterivorous family harboring the Metateratocephalus and Euteratocephalus genera) as an example of primer development. (A) All primers were designed to have optimal annealing temperature (Ta) of 63°C, with Ct values varying at temperatures above and below the target Ta. (B) Specificity test of a Metateratocephalidae primer combination with plasmid DNAs from three target species, SSU rDNA fragments from 11 potential false positives (as selected by ARB [39]) and a negative water control. Clade numbers are according to Van Megen et al. [23]. In the quantitative PCR graph the gap between the target and the non-target signal (ΔCt) is shown. (C) Pictures of the head region of a representative of both genera. (D) The relationship between Ct values and numbers of nematodes for quantification of densities. A linear relationship between Ct values and numbers of nematodes till 1/1,000 part of a single nematode is shown (equivalent to a single nematode cell harboring ∼50 copies of the ribosomal DNA cistron). Hand-picked individuals of Metateratocephalus (purple circles) and Euteratocephalus (blue squares) were used to quantify the Metateratocephalidae-specific primers.
Figure 5.
Quantitative coverage of the DNA-based tool using environmental samples.
Logarithm of the total of individuals as detected by optical microscopy (x-axis) plotted against the logarithm of the total of individuals as estimated by quantitative PCR (y-axis). The correlations of quantitative PCR with classical analyses seem to be accurate, with no Studentized residuals higher than | 2 |. The solid line shows the trend of all data and the two dashed lines show the boundaries of one-order-of-magnitude precision. The dotted line represents an equal amount of nematodes for both methods. Such a coverage is expected to be lower than 100% as obligate plant parasites were not included, although the fungivorous Aphelenchidae and Aphelenchoididae may harbor facultative plant parasites as shown in Table 2. Given that taxa like Rhabditidae, Qudsianematidae or Nordiidae appear to be both poly- and paraphyletic [23], [65], no rDNA-based detection assay on family level could be developed for those nematodes.