pathogen of Norway spruce [8]. C. laricicola, also found
across Eurasia and Japan, is restricted to larch and associated
with the bark beetle Ips cembrae.
C. adiposa is distantly related and morphologically distinct
from the above-described species. However, we include
it in this study since it has been isolated from
wood chips and sawn lumber in North America [9].
Species of Ceratocystis can be di⁄cult to di¡erentiate.
For example, C. coerulescens, C. resinifera, and C. ru¢penni
overlap in host and geographical range. Although some
morphological di¡erences exist, such as the diameter of
perithecial bases and ascospore length, there is considerable
overlap in these characters and they can require
months to develop in culture. The polymerase chain reaction-
restriction fragment length polymorphism (PCRRFLP)
method has proved useful for the rapid and sensitive
detection and di¡erentiation of many plant pathogens.
Ribosomal DNA (rDNA) is a common target sequence
and has been used extensively for the di¡erentiation of
sapstain species [10,11]. However, these methods have
been unable to separate closely related Ceratocystis species
due to low sequence heterogeneity [12]. The L-tubulin gene
has also been successfully used for PCR-RFLP based differentiation
of species of Babesia and Theileria [13] and
distinguishing between groups of Leishmania [14]. Tubulin
proteins have a relatively high degree of conservation at
the amino acid and nucleotide level, but a relatively high
degree of variability in the intronic sequences.
In this paper we describe a rapid and reliable PCRRFLP
identi¢cation method to di¡erentiate species of Ceratocystis
using a fragment of the L-tubulin gene sequence,
which had higher sequence variability than rDNA. This
method was used to con¢rm the identity and to di¡erentiate
Ceratocystis sapstain fungi, which our lab routinely
isolates from stained logs and lumber in Canada.