prog detail - International Mycological Association

S40PS1 - 0484
The utility and limitations of positive and negative controls for PCR detection of quarantine pathogens.
M Glen 1, AC Alfenas 2, EAV Zauza 2, SRH Langrell 1
1 CSIRO ensis FBP, Hobart, Australia, 2 Federal University of Viçosa, Viçosa, Brazil
Species-specific PCR test methods are increasingly being developed for the detection of plant pathogens of
quarantine importance. The possibility of false negatives, and their significance for bio-security measures, is an ongoing
concern with the application of this technology, particularly as PCR is commonly prone to failure due to enzyme
inhibition or operator error. Incorporation of Internal Amplification Controls (IACs) can increase confidence in negative
test results by demonstrating the success of each PCR reaction, but IACs alone may be insufficient to prevent false
negative results. Here we describe the development of IACs for PCR detection of Puccinia psidii, a pathogen with
potential devastating consequences for Australian biodiversity if introduced, and discuss the need for additional
controls.
IAC plasmids were constructed by cloning PCR products generated using ‘hybrid’ primers. The 5’ end of each ‘hybrid’
primer comprised a P. psidii-specific primer with an additional 3’ portion complementary to an appropriate region of
plasmid pUC18.
A 1200bp fragment was successfully amplified from recombinant plasmids p04L1-6 and p04L2-4 using the P. psidii
nested PCR diagnostic primers P1/P6 and P2/P4. PCR product was detectable from an IAC template concentration
of 0.1 fg/Ìl, but the addition of plant or fungal DNA necessitated an increase in IAC concentration to 1 fg/Ìl. A high
concentration of P. psidii template DNA competitively inhibited the amplification of the larger product from the IAC.
The inclusion of an IAC can increase confidence in negative test results, but may also be misleading. In addition to
the inclusion of IAC, DNA amplification quality should be demonstrated by amplification with non-specific primers as
degradation of DNA before or after extraction or accidental loss of DNA during the purification process will give a false
negative that is not detected by the addition of an extraneous IAC. The advantages of exogenous rather than
endogenous IACs for P. psidii is also discussed. In addition to positive controls, the requirements for appropriate
negative controls are discussed.
Perhaps this could be widened to include reasons for the inhibition, as t5his is what we are continually trying to avoid
with PCR for such purposes. Standardisation of DNBA extraction and post extraction clean up is an issue. Rember, more
and more folk are accepting that a test method is more than just the primers, but also a complimentary, relevant DNA
extraction procedure (and to that end a sampling approach….).
S40PS2 - 0634
Dissemination of aerial and soilborne Phytophthoras by human vectors
J F Webber, J Rose
Forest Research, Farnham, United Kingdom
Two new invasive Phytophthora pathogens, Phytophthora kernoviae and P. ramorum, have recently established in the
UK. They are most prevalent in the south west of England in woodland areas where they cause intense episodes of
dieback on wild rhododendrons (Rhododendron ponticum), but also cause lethal stem cankers on a range of
broadleaf trees. As both these Phytophthoras are aerial pathogens their deciduous sporangia, produced on foliage
of infected rhododendron and other foliar hosts, are dispersed by wind and rain splash on a local basis. However,
patterns of disease spread suggest that vertebrate vectors may also aid the spread of these pathogens over longer
distances. Infected rhododendron leaves are quickly shed and incorporated into the dense litter layer and people
and animals frequently walk through these contaminated areas. To assess the likelihood of walkers picking up
infested soil or litter on their feet, a study was set up to analyse how frequently Phytophthora could be isolated from
the soil or litter attached to people’s boots, particularly those walking in known P. kernoviae/P. ramorum woodlands
and gardens. The study started in July 2004 and has continued through 2005 and 2006. As the aim of the study was
to determine (1) which species and (2) how frequently viable Phytophthora inoculum was moved by human vectors,
so baiting methods were employed for isolation and detection. Several different species of Phytophthora have been
isolated, and more than 30% of samples collected from walker’s boots were contaminated with Phytophthora. The
most commonly occurring species was P. citricola, but 10-15% of the samples contained either P. ramorum or P.
kernoviae. Other Phytophthoras found include P. ilicis (another aerial Phytophthora), and also P. cambivora, P.
cryptogea, P. gonapodyides and a single finding of P. hibernalis. It has yet to be established if the amount of
Phytophthora inoculum carried on boots can initiate a new infection focus in an area remote from the source of
Phytophthora inoculum, but it is clear from this study that human vectors could provide significant pathways for
disease spread for quarantine pathogens such as P. ramorum and P. kernoviae as well as other aerial and soilborne
Phytophthoras.
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