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R. CAMPOS-HERRERA ET AL.
endoparasitic fungi are obligate parasites of nematodes and some other microscopic
metazoans (Kerry & Jaffee, 1997).
Because the predation rate on EPNs cannot be reliably inferred from the
abundance of many NF species in soil, more direct methods of assessing predation
are needed. Duncan et al. (2007) developed an assay to enumerate NF species
recovered directly from nematodes rather than soil, which better reflects levels of
predatory rather than saprophytic behavior. Circumscribed soil cylinders were
defined and isolated in situ by pounding PVC tubes to a depth of 20 cm in soil
beneath the citrus tree canopy. These relatively undisturbed soil cylinders were
baited with large numbers of EPNs and then recovered from the field after 3 days.
The nematodes extracted from the soil cylinders were placed on water agar to allow
growth of NF from nematode cadavers. This method effectively recovered predators
and parasites of nematodes, which were invariably all killed within 5 days.
However it was necessary to add fresh EPNs to the agar plates after 5 days to induce
the formation of fungal fruiting bodies for species identification and the estimation
of population abundance based on numbers of EPNs killed. The long period of time
during which the fungi competed with one another on the water agar likely skewed
the abundance estimates in favor of species best adapted to this artificial habitat.
A more reliable estimate of NF predation rates in soil requires the identification
of infected nematodes immediately following extraction from soil. Indeed, direct
quantification of target populations rather than estimation from bioassays would
facilitate understanding the roles in food webs of many organisms that are currently
poorly understood. For example, possible effects of Paenibacillus on EPN
prevalence can be inferred in bioassays by the degree of spore encumbrance of EPN
IJs emerging from sentinel insects (Duncan et al., 2007). However, spore
encumbrance increases with the length of time that IJs are in the vicinity of bacteriainfected
cadavers in these assays, and the detection of bacteria using sentinel insects
depends on EPN abundance in soil. Therefore, methods to directly measure the
abundance of these bacteria in soil or on nematodes extracted from soil are needed
to accurately assess the degree to which EPNs and Paenibacillus interact at different
times or in different habitats.
Real-time PCR (or quantitative PCR, qPCR) provides an efficient method of
quantifying soilborne organisms such as bacteria and fungi using molecular probes
(Atkins, Clark, Pande, Hirsch, & Kerry, 2005; Klob, Knief, Stubner, & Conrad,
2003). The abundance of S. kraussei and S. affine in fields and meadows was
recently compared using qPCR (Torr, Spiridonov, Heritage, & Wilson, 2007). The
methods most commonly employed involve the use of fluorescent products that link
to double stranded DNA causing increased fluorescence (e.g., SYBR Green®) or the
design of specific fluorescent probes (e.g., TaqMan® or hydrolysis probes). In both
cases, species-specific primers designed for the target taxon are used. Both systems
can function with a high degree of species specificity but, due to the use of a probe
that adds an additional level of specificity to the primers, hydolysis probes are
generally reported to be more reliable in this regard (Holeva et al., 2006; Leal,
Green, Allen, Humble, & Rott, 2007). In both systems, the amount of fluorescence
increases during PCR cycling. The quantification cycle (Cq) (also called threshold
cycle or Ct) is that at which product amplification enters an exponential phase.