[Edited_by_A._Ciancio,_C.N.R.,_Bari,_Italy_and_K.(Bookos.org)

IPM THROUGH ENTOMOPARASITES
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Once the primers/probes have been validated, sampling and extraction methods
can be selected that exploit the advantages and limitations of qPCR. Many protocols
for sampling fungi and bacteria in soil extract DNA directly from soil samples that
can be as small as a few grams or less. Smith and Jaffee (2009) suggest using a
combination of culturing and molecular detection of trapping fungi because primers
specific for a major group of trapping fungi (Orbiliales) yielded clones representing
just 3 of 8 species that were isolated through culturing, but revealed the presence of
18 species that had not been detected through culturing. It is unclear whether uneven
distribution of the species in the small samples (12.5 cm 3 soil) affected the results.
In order to optimize sampling and extraction methods, additional studies comparing
both species-specific primers and culturing are needed, and they should involve
DNA that has been extracted and concentrated from a range of soil sample sizes.
MacMillan et al. (2006) accurately quantified numbers of slug-parasitic
nematodes by extracting DNA directly from 10 g soil samples but were not able to
do so from 1 g soil samples. However, the samples were artificially infested with
nematodes and, in contrast to bacteria or fungal propagules, it is unlikely that the
relatively low numbers of nematodes that occur naturally in soil can be reliably
detected from such small samples. Studies and diagnostic services that use qPCR to
quantify metazoans such as nematodes in soil routinely employ standard methods of
sampling and extraction and, therefore, are able to rely on conventional sampling
programs designed to optimize cost and accuracy (Hollaway, Ophel-Keller, Taylor,
Burns, & McKay, 2004; Stirling et al., 2004; Ophel-Keller, McKay, Hartley,
Herdina, & Curran, 2008; Donn, Griffiths, Nielson, & Daniell, 2008). Such
programs employ a variety of sampling methods that address the highly aggregated
spatial patterns of nematodes, usually by collecting large numbers of samples that
are pooled, mixed and subsampled for nematode extraction (Been & Schomaker,
2006).
Subsamples of 500–1000 cm 3 are routinely processed by methods such as
sucrose centrifugation to recover nematodes (Jenkins, 1964). There is no reason that
such large subsamples cannot be used for molecular diagnostics although a number
of studies report the use of smaller subsamples (Table 2), perhaps to reduce the
recovery of soil chemicals that can interfere with DNA extraction and PCR reactions
(see below). If the objective is estimating predation rates by measuring only those
NF intimately associated with nematodes, the nematodes can be further separated
from the small residue of soil remaining after sucrose centrifugation by additional
centrifugation in a magnesium sulphate density gradient (Duncan et al., 2007).
Preliminary experiments (Duncan et al., unpublished data) revealed less fungal DNA
and fewer nematodes recovered following the two-step extraction procedure
compared to just sucrose centrifugation; and there was no evidence of significant NF
DNA in the soil residue. This observation should be confirmed using a variety of
soils and NF targets because use of one rather than two extractions reduces time and
cost, and the recovery of more nematodes increases the detection efficiency of the
system.
A variety of methods have been used to extract nematode DNA from soil or
from nematodes recovered from soil (Table 2). Kits involve higher costs than
standard laboratory protocols to purify DNA but are far simpler and faster to use if