Folie 1

1
Development of specific SCAR Primers to detect
the active ingredients of an antagonistic plant
protection product against fire blight
Loncaric I.1, Moosbeckhofer R.1, Antlinger B.2,
Oberlerchner J.T.1,Ertl C. 3, Donat C.3 1
Austrian Agency for Health and Food Safety,
Institute for Apiculture, Spargelfeldstraße 191,
1226 Vienna 2 University of Natural Resources and
Applied Life Sciences, Konrad Lorenz Str. 20,
A-3430 Tulln 3 bio-ferm Biotechnolgische
Entwicklung und Produktion GmbH Konrad Lorenz
Strasse 20, 3430 Tulln, Austria Contact
igor.loncaric_at_ages.at christina.donat_at_bio-ferm.com
Introduction Aureobasidium pullulans (Ap) is a
dematiaceous fungus distributed widely throughout
environment. Two environmental strains of Ap CF10
and CF40, active ingredients of Blossom Protect,
showed antagonistic potential against Erwinia
amylovora (Ea), causative agent of fire blight.
By using these two strains of Ap as to defend the
blossoms of pome fruits against fire blight we
were facing the problem to recover the strains
against the natural occurring background of
autochton Ap, yeasts and other fungi by using the
common cultivation techniques. Similar as for
chemical plant protection products there is also
for BCAs the need to detect them during
application trials and to monitor the persistence
and behaviour of the micro organisms in use
during toxicological and environmental studies.
Therefore, the aim of this study to develop a
specific identification method for these two
strains of Ap.
Development of a specific detection method for
strains CF10 and CF40
Principle Application of RAPD- and
duplex-RAPD-PCR with several RAPD primers and
Duplex combinations to more than 200 Ap strains
to find a specific DNA fragment for CF10 and CF40
strains
Results RAPD- and duplex-RAPD 5 specific
fragments after RAPD-PCR 5 specific fragments
after duplex-RAPD-PCR Several SCAR primers sets
per DNA fragment were developed One specific
primer set/fragment. Tm was modified for single
tube multiplex-PCR and was specific if assessed
against all fungal strains. The limit of
detection was 50 pg
Sequencing of the DNA fragment to design SCAR
primers specific to strains CF10 and CF40
Optimisation of PCR conditions to obtain
stringency as well as to obtain single tube
diagnostic PCR for both strains
Specificity and sensitivity tests of SCAR primers
under multiplex-PCR conditions
Fig. 2 Results Marker (100bp MBI Fermentas),
CF40, CF10, CF40, Mix
Implementation of new developed multiplex-PCR

• Application of Blossom Protect with the help of
  honeybees
• Detection of Ap strains CF10 and CF40 dispersed
  by honeybees (A. mellifera ) in two greenhouse
  trials tests
• Use of honeybees as vectors for fire blight
  antagonists in field experiments
• After dispersal of Ap strains CF10 and CF40 by
  honey bees in the 1st test 8 and in 2nd 9 out of
  10 collected flowers gave positive results
  without any unspecific PCR reaction

• Persistence and Behaviour of Blossom Protect in
  the Environment
• Detection of Ap strains CF10 and CF40 against
  background level of other environmental
  microorganisms
• Pathenogenicity
• verify the identity of isolates gained by
  investigation of the organs (brain, lung,
  intestinal,..) of rats during several toxicity
  studies.

Conclusion New multiplex-PCR provides a valuable
tool for rapid, specific and sensitive
identification of strains Ap CF10 and CF40,
active ingredients of Blossom Protect. The
primers were successfully applied to identify the
microbial plant protection product during
degradation experiments in soil and water as well
as for checking the identity of micro organisms
isolated during pathogenicity studies with rats.
for Material and Methods as well as Results look
at our Poster Use of honeybees (Apis mellifera)
as vectors for fire blight antagonists in field
experiments