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Development of a soil DNA extraction and quantitative PCR method for detecting two
Cylindrocarpon species in soil
INTRODUCTION
C.M. Probst{ XE "Probst, C.M." }, M.V. Jaspers, E.E. Jones and H.J. Ridgway
Faculty of Agriculture and Life Sciences, Lincoln University, PO Box 84, Lincoln 8150, New Zealand
Cylindrocarpon black foot disease has been identified worldwide
as a common cause of vine death in nurseries and in young
vineyards (1), especially in sites converted from orchards or
replanted from grapes. This indicates that the existing soil
conditions may have contributed to the disease, although very
little information is available regarding the survival of the
pathogens in soil. In New Zealand, the three Cylindrocarpon
species equally responsible for black foot disease on grapevines
are C. destructans, C. macrodidymum and C. liriodendri (1).
Recently, quantitative PCR (qPCR) has begun to supersede soil
dilution plating as a method for precise determination of soil
inoculum levels (2). In this research program, qPCR was
optimised to test large soil samples for presence of two
Cylindrocarpon species.
MATERIALS AND METHODS
Fungal isolates. The Lincoln University Culture Collection
provided isolates of the three Cylindrocarpon species, which had
been obtained from symptomatic New Zealand grapevines.
Single spore colonies of 10 randomly selected isolates per
species were grown on potato dextrose agar (PDA) at 20°C in the
dark.
RESULTS
The primer pairs were specific for each of the two species and no
cross reactivity was observed. They produced a 300 bp and 197
bp product for C. macrodidymum and C. liriodendri, respectively.
They were also suitable for a range of genetically diverse
isolates. Each of the primers was able to detect as little as 30 pg
DNA in a standard PCR and 3 pg DNA in a nested PCR. The qPCR
could detect pure DNA at the same level as the nested PCR. For
spore suspensions, the qPCR was able to detect as little as 100
spores in soil. The time course experiment showed that for each
species, less than half of the nuclei remained 1 week after
infesting the soil with conidia. However, at this time, the DNA
could still be visualised on agarose gels of the qPCR products
(Fig 1). After 2, 3 and 6 weeks, the DNA could not be detected.
M ng Weeks
30 3 0.3 0.03 0.003 0 0 1 2
Session 1B—Disease surveys
Soil Infestation. A mixed conidium suspension (10 6 conidia /mL)
was obtained for each species using three isolates. They were
used to infest 5 L pots of soil, three replicates per species, with a
final concentration of 10 5 conidia per gram of soil. Controls were
treated with a similar quantity of water. The 5 L pots were sunk
into, and level with, the ground in the Lincoln University
Vineyard. Two 15 g soil samples were taken from each pot for
DNA extraction at 0, 1, 2, 3 and 6 weeks after set up. All three
species were inoculated into each pot to ensure detection
specificity
DNA extraction. For pure culture extraction, a small plug of
mycelium was transferred from a 5 d old PDA culture to potato
dextrose broth (PDB) and incubated for 7 d at room
temperature, in 12 h light/ dark. The mycelium was harvested
and stored at ‐80°C until DNA extraction using a PureGene DNA
extraction kit (Qiagen). Spectrophotometry was used to quantify
the genomic DNA. DNA was also extracted from 10 6 conidia of
each isolate in a similar way.
For DNA extraction from soil samples, 10 g of soil was placed in a
250 mL bottle with 45 mL water containing 0.01% agar. The
bottles were shaken for 10 min and left to stand for 10 min. The
supernatant was put into two 15 mL tubes, and centrifuged at
4000 x g for 15 min. The pellets were combined and centrifuged
again, with the final pellet being used for DNA extraction using a
PowerSoil kit (MO BIO Laboratories Inc.). Genomic DNA quality
was assessed by visualisation on a 1% agarose gel.
PCR amplification. Species specific primers for the β− tubulin
region of C. macrodidymum and C. liriodendri, donated by Dr
Lizel Mostert (University of Stellenbosch, South Africa), were
used in qPCR with SYBR green chemistry and a Rox internal
standard on an ABI Prism 7700 sequence detector. After the
qPCR, the products were separated by electrophoresis on a 1.5%
agarose gel and visualised under UV light.
Figure 1. 1.5% agarose gel of products amplified during qPCR with the
primers specific for C. liriodendri.
DISCUSSION
The results show that the species specific primers used in a qPCR
system could detect as little as 3 pg of pure DNA, which is
equivalent to 30 Cylindrocarpon macroconidia. When spores
were recovered from soil a sensitivity of 100 spores was
achieved. Following soil inoculation, DNA of fewer than 50% of
the conidium nuclei in the soil was detected after 1 week.
Additional unpublished data has indicated that in the soil
environment, the 4‐celled conidia were often converted to 2
chlamydospores (uninucleate). It is possible that the >50%
decrease observed is a reflection of that conversion, rather than
the death of conidia. Research is continuing to investigate the
population dynamics of these Cylindrocarpon species in soil.
Future research will include the development of C. destructans
specific primers.
ACKNOWLEDGEMENTS
We gratefully acknowledge Lizel Mostert for providing the
primers sequences and Winegrowers New Zealand and TECNZ
for funding this project.
REFERENCES
1. Halleen, F., Fourie, P.H. and Crous P.W. (2006). A review of black
foot disease of grapevine. Phytopathologia Mediterranea 45: S55‐
S67.
2. Kernaghan, G., Reeleder, R.D. and Hoke, S.M.T. (2007).
Quantification of Cylindrocarpon destructans f. sp. Panacis in soils
by real‐time PCR. Plant Pathology 56: 508–516.
APPS 2009 | PLANT HEALTH MANAGEMENT: AN INTEGRATED APPROACH 25