sdu faculty of forestry journal special edition 2009 - Orman Fakültesi

SDÜ Faculty of Forestry Journal
A newer and more aggressive form was since 1970´s distinguished into two
races - an Eurasian (EAN), probably originated in Moldavia and the Ukraine, and a
North American race isolate (NAN). In 1991, the more aggressive form was
described by Brasier as a new species O. novo-ulmi Bras (Brasier 1991). Ten years
later, Brasier et al. (2001) designated races EAN and NAN as subspecies of O.
novo-ulmi ssp. novo-ulmi and O. novo-ulmi ssp. americana. Specific group of DED
pathogens are hybrids of O. novo-ulmi subspecies.
O. novo-ulmi, its subspecies and their hybrids were for the first time recorded
in the Czech Republic by Dvořák et al. (2006, 2007) . The aim of present study is
a complex characterisation of strains, which are actually spreading in the area of
the Czech Republic.
2. MATERIALS AND METHODS
2.1. Isolation of the pathogen: Coordinates of position and altitude of every
infected elm with inner DED symptoms was achieved by GPS. Samples of twigs
were cut from different parts of drying crowns and cultivated on cycloheximide 3%
MEA medium. Each strain was deposited into the culture collection of MUAF
Brno.
2.2. Molecular-biological characterisation: DNA was obtained from pure
MEA cultures by PowerSoil DNA Kit (Mo Bio). Identification of species and
subspecies was performed by the mean of Polymerase Chain Reaction (PCR) and
Restriction Fragment Length Polymorphism (RFLP) using two gene regions. The
former is a cerato-ulmin (cu) gene region, described in more detail in Pipe et al.
(1997) and the latter gene region is called col1 and encodes colony type (Konrad et
al., 2002). Amplified fragments of these two gene regions were restricted by
endonucleases, Hph I used for cu gene region and Bfa I for col1 region (Konrad et
al., 2002). RFLP fragments were visualized on 3% agarose gel and evaluated.
Sequencing was provided in a few cases and submitted to Gen Bank.
2.3. Production of cerato-ulmin: Amount of cerato-ulmin produced by liquid
cultures of 15 isolates was determined by chromatography (Scala et al., 1994).
The tested group of isolates was composed from 4 cultures of ssp. novo-ulmi, 2
cultures of ssp. americana and 9 strains of genetic hybrids of precedent strains. In
this tested group, well known isolates H328 ssp. novo-ulmi and 182E ssp.
americana were used as reference strains. Liquid cultures of dilutions 1:1, 1:2, 1:4
and 1:8 were stirred to obtain turbid milky solutions. The turbidity of the samples
at the various dilutions was immediately assayed for optical density at 400 nm.
Results of the dilution with the most variable values represent the best
measurement for comparing the cu-production of the strains.
2.4. Mating type, vegetative compatibility and fertility tests: General
characterisation of 13 isolates has been performed according to Brasier (1981). A
special medium – Elm Sapwood Agar was used. Occurrence of elm sapwood in the
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