Mycologia, 97(3), 2005, pp. 612–620.

q 2005 by The Mycological Society of America, Lawrence, KS 66044-8897

Molecular and genetic analyses of geographic variation in isolates of

Phoma macrostoma used for biological weed control

Lecong Zhou al 2001, Tebeest et al 1992, Teng and Yang 1993,

K.L. Bailey 1 Templeton 1992, Wapshere 1974). One aspect of the
C.Y. Chen biosafety assessment is to determine the genetic var-
Mario Keri iability of the biocontrol strain in relation to other
Saskatoon Research Centre, Agriculture and Agri-Food similar isolates and species that may exist in the in-
Canada, 107 Science Place, Saskatoon, Saskatchewan, tended area of release. For the purposes of register-
Canada S7N 0X2
ing a microbial pest control product in Canada, the
Pest Management Regulatory Agency has designated
five ecozones across the country that are based on
Abstract: Molecular and genetic approaches were
large, ecologically distinctive areas resulting from
used to evaluate the genetic relatedness among iso-
generalized criteria involving landform, soil, water,
lates of the fungus Phoma macrostoma Montagne orig-
climate, flora, fauna and human factors (FIG. 1). To
inating from Canada and Europe and to other spe-
avoid or minimize the risk of introducing new alleles
cies in the genus Phoma. Distinct differences were
into an area through asexual or sexual interactions,
observed in genetic variation among nine species of
it should be known that the biocontrol strain is sim-
the genus Phoma. Randomly amplified polymorphic
ilar genetically to the local populations before re-
DNA (RAPD) revealed the presence of intraspecific
leasing it across ecozones (Hintz et al 2001, Slatkin
genetic variation among the isolates of P. macrostoma,
1987, Taylor et al 1995, Templeton et al 1979).
with the isolates being used for biological weed con-
trol being distributed in a distinct phylogenetic clus- Various broadleaf weed pests including Canada
ter. Additional variation within the biocontrol isolate thistle (Cirsium arvense L. [Scop.]), dandelion (Ta-
cluster in P. macrostoma was revealed by pulsed field raxacum officinale Weber ex F.H. Wigg.), chickweed
gel electrophoresis (PFGE), which showed that bio- (Stellaria media [L.] Vill.) and scentless chamomile
control isolates generated two different chromosom- (Matricaria perforata Mérat) are economically impor-
al profiles, however the profiles did not relate to their tant in western Canada (Van Aker et al 2000). Ten
Canadian ecozone origin. Mating studies showed that isolates of Phoma macrostoma Montagne recently were
biocontrol isolates of P. macrostoma from Canada did discovered to have bioherbicidal effects on these
not produce sexual reproductive structures and were weeds (Bailey and Derby 2001). Application of these
incapable of crossing. These studies also confirmed isolates to soil resulted in chlorosis and seedling
that no obvious differentiation exists among the bio- death of several broadleaf weed species but had no
control isolates of P. macrostoma from Canadian Eco- effect on monocotyledonous plants. The 10 isolates
zones 3 and 4. originated in two Canadian ecozones and their bio-
Key words: biocontrol, genetic variation, mating, logical and morphological characteristics appeared
PFGE, Phoma macrostoma, RAPD similar, but the genetic variation occurring in them
was unknown.
Randomly amplified polymorphic DNA (RAPD) is
an effective molecular fingerprinting tool that iden-
INTRODUCTION
tifies genetic variation at the species level and higher
The application of micro-organisms for biological analyses (Arisan-Atac et al 1995, Chang et al 1996,
pest control is an approach that has gained accep- Gosselin et al 1996, Hintz et al 2001, McDermott et
tance as a useful tool for integrated pest management al 1994, Nicholson and Rezanoor 1994, Zimand et al
programs. However applications using large quanti- 1994). Pulsed field gel electrophoresis (PFGE) also
ties of specific micro-organisms to a defined area of has been used widely and proved to be a reliable tool
release need to be studied to ensure environmental in studies of genetic variation in Phoma lingam Tode
safety (Charudattan 1988, Cook et al 1996, Hintz et ex Fr. (sexual state Leptosphaeria maculans [Desm.]
Ces & de Not.) (Chen et al 1996; Howlett 1997; Koch
Accepted for publication 23 Feb 2005.
1 Corresponding author. Phone: 11 (306) 956 7260; Fax: 11 (306) et al 1991; Lim and Howlett 1994; Morales et al 1993;
956 7247; E-mail: BaileyK@agr.gc.ca Moreno-Rico et al 2002; Plummer and Howlett 1993,

612
 ZHOU ET AL: GENETIC VARIATION IN PHOMA MACROSTOMA 613

DNA extraction.—Mycelial tufts (100 mg) were scraped from

the surface of 14 d old colonies growing on V-8 juice agar,
weighed and transferred into an 1.5 mL microcentrifuge
tube. The sample was ground into fine powder with liquid
nitrogen and a plastic minipestle before being used imme-
diately for DNA extraction with the DNeasyt Plant Mini Kit
(QIAGEN) according to the supplier’s protocols.

RAPD analyses.—The primers used were 10-mer oligonu-

cleotides purchased from the Biotechnology Laboratory
(University of British Columbia, Vancouver, British Colum-
bia) (TABLE II). DNA amplification was performed in 25
mL reaction mixtures, each containing 20 ng template
DNA, 1 unit AmpliTaq Gold Polymerase (5U/mL, Applied
Biosystems), 1/10 volume (2.5 mL) of GeneAmpt 103PCR
Buffer II, 2.0 mM MgCl2, 0.2 mM primer, and 100 mM of
each dNTPs (MBI Fermentas). The PCR program was ini-
tiated at 94 C for 10 min, followed by 45 cycles of 94 C 1
min, 35 C 1 min, 72 C 2 min, at 72 C 10 min and held at
10 C. All amplification reactions were performed in an Al-
pha Unity Block Assembly for PTC DNA Enginey Systems
(MJ Research Inc.). Amplification products were resolved
by electrophoresis in 1.5% agarose gels. Each amplified
DNA fragment was scored as either present or absent for
each isolate. To check reproducibility of amplicons, the am-
plification of the DNA templates was repeated twice; there
were no problems with reproducibility.
FIG. 1. Five microbial pesticide ecozones of Canada.
(Source: Pest Management Regulatory Agency, 2001). PFGE analysis.—The chromosomal DNA of the biocontrol
isolates of P. macrostoma from Ecozones 3 and 4, with one
isolate of P. medicaginis and three isolates of P. herbarum,
1995). More conventional studies on genetic varia- was compared by pulsed field gel electrophoresis (PFGE)
analysis. Single-spore cultures of the isolates were main-
tion within a species may be evaluated by observing
tained by subculture on 10% V8-juice agar and incubated
mating between fungal isolates (Cozijnsen et al 2000,
at room temperature (20–24 C) with a 16 h photoperiod
Mengistu et al 1995, Moreno-Rico et al 2002). These at 100–150 mE/m2/s. Pycnidiospores of 14 d old cultures
molecular tools and mating studies presumably were harvested by scraping the surface with 0.05% Tween
would be useful to determine the genetic variability 80 and filtering the spore suspension through two layers of
in the biocontrol isolates of P. macrostoma. Miracloth. Chromosome inserts were prepared as described
The objectives of the present study were to evalu- by Plummer and Howlett (1993). Electrokaryotyping was
ate the genetic variation occurring among isolates P. performed on a contour-clamped homogeneous electric
macrostoma with different geographic origins and to field (CHEF) DR II system (Bio-Rad, Mississauga, Ontario)
show the genetic relatedness of P. macrostoma with with 0.53 TBE buffer at 11 C according to Chen and Sé-
other species of Phoma. guin-Swartz (1999), with minor modifications: The initial
switch time was 600 s and final switch time was 600 s at 100
V for 72 h, followed by initial 400 s and final 400 s at 100
MATERIALS AND METHODS V for 46 h. Gels were stained in 0.1 mg/mL ethidium bro-
mide solution and photographed. The chromosomal DNA
Fungal strains and growth conditions.—Ten biocontrol iso- bands were recorded for each isolate as present or absent.
lates of P. macrostoma and 18 reference isolates comprising
nine Phoma spp. were used. The biocontrol isolates came Genetic mating.—Five biocontrol isolates of P. macrostoma
from Canada thistle plants found in Canadian Ecozone 3 (94- from Ecozones 3 and 4 were crossed among themselves and
44B, 85-24B, 95-268B, 89-25A2, 94-359A, 97-12B and 97-15B2) to tester isolates of P. lingam with different mating types
and Ecozone 4 (95-54A1, 94-26 and 94-134). They are on (Mat1 or Mat2). Although no sexual state for P. macros-
deposit with the International Depositary Authority of Can- toma has been reported, it was important to confirm that
ada, Winnipeg, Manitoba. The other isolates originated in crossing could not occur among the biocontrol isolates.
North America and Europe. The host plant and geographic Phoma lingam was used because it has known mating types,
origins of all the fungal isolates are listed (TABLE I). All fun- can reproduce sexually under laboratory conditions, is an
gal isolates were derived from single-spore cultures and important plant pathogen of canola (Brassica napus and B.
grown on V-8 juice agar at room temperature (20–24 C) un- rapa) in Ecozones 3 and 4 and might have some genetic
der a 16 h photoperiod at 100–150 mE/m2/s. relatedness to P. macrostoma. All fungal isolates were cul-
614 MYCOLOGIA

TABLE I. Phoma isolates used in the studies

Species Isolate Host of origin Place of origina Sourceb

Phoma macrostoma 85-24B Cirsium arvense (L.) Scop. Saskatchewan, Canada [3] SRC
P. macrostoma 89-25A2 Cirsium arvense Saskatchewan, Canada [3] SRC
P. macrostoma 94-26 Cirsium arvense Ontario, Canada [4] SRC
P. macrostoma 94-44B Cirsium arvense Saskatchewan, Canada [3] SRC
P. macrostoma 94-134 Cirsium arvense New Brunswick, Canada [4] SRC
P. macrostoma 94-359A Cirsium arvense Saskatchewan, Canada [3] SRC
P. macrostoma 95-54A1 Cirsium arvense Nova Scotia, Canada [4] SRC
P. macrostoma 95-268B Cirsium arvense Saskatchewan, Canada [3] SRC
P. macrostoma 97-12B Cirsium arvense Alberta, Canada [3] SRC
P. macrostoma 97-15B2 Cirsium arvense Alberta, Canada [3] SRC
P. macrostoma var. incolorata CBS 839.84 Hordeum vulgare L. Monheim, Germany CBS
P. macrostoma var. macrostoma CBS 154.83 Philadelphus coronaries L. Baarn, Netherlands CBS
P. macrostoma var. macrostoma CBS 482.95 Larix decidua Mill. München, Germany CBS
P. macrostoma var. macrostoma CBS 488.94 Forsythia sp. Baarn, Netherlands CBS
P. macrostoma var. macrostoma CBS 837.84 Triticum aestivum L. Monheim, Germany CBS
P. dennisii var. dennisii CBS 135.96 Solidago canadensis L. Ontario, Canada [4] CBS
P. lingam Leroy Brassica napus L. Saskatchewan, Canada [3] SRC
P. lingam Peace-3 Brassica napus British Columbia, Canada [1] SRC
P. lingam Pl 86-12 Brassica napus Manitoba, Canada [3] SRC
P. lingam Pl 89-19 Brassica sp. Unknown, Australia SRC
P. lingam Pl 89-21 Brassica napus Mt Barker, Australia SRC
P. herbarum Al Taraxacum officinale Ontario, Canada [4] G. Boland
Webber ex F.H. Wigg.
P. herbarum AIV Taraxacum officinale Ontario, Canada [4] G. Boland
P. herbarum G/5/2 Taraxacum officinale Ontario, Canada [4] G. Boland
P. chrysanthemicola 90-64 Ambrosia artemisifolia L. Ontario, Canada [4] SRC
P. chrysanthemicola 91-27I Ambrosia artemisifolia Ontario, Canada [4] SRC
P. exigua 92-180-1 Cirsium arvense Manitoba, Canada [3] SRC
P. medicaginis 94-335A1 Medicago lupulina L. Saskatchewan, Canada [3] SRC
P. nebulosa 92-74 Cirsium arvense Saskatchewan, Canada [3] SRC
P. pomorum 91-177 Ambrosia artemisifolia Iowa, USA SRC
a
The number in parentheses refers to the Canadian ecozone.
SRC 5 Saskatoon Research Centre, Agriculture and Agri-Food Canada, Saskatoon, Canada; CBS 5 Centraalbureau voor
b

Schimmelcultures, Utrecht, The Netherlands.

tured on 2% V-8 juice agar medium for 14 d. On a fresh isolate. A sterile wooden toothpick was placed between the
agar plate, a 10 mm mycelial plug from each isolate in the two isolates at the junction where they would meet and
mating pair combination was placed ca. 7.5 cm apart. The merge (Mengistu et al 1993, 1995) and incubated at 10 C
cultures were incubated at 16 h photoperiod under cool with the same light conditions 4–7 wk. The toothpick and
white fluorescent light (100–150 mE/m2/s) at 22 C for 3 d the culture media were examined at 5, 6 and 7 wk for ma-
or until it was possible to determine the growth rate of each ture pseudothecia under a binocular microscope at 403

TABLE II. The 10-mer oligonucleotide primers selected for RAPD analyses

Primer No. Sequence GC % Source

308 59AGCGGCTAGG39 70 University of British Columbia
350 59TGACGCGCTC39 70 University of British Columbia
356 59GCGGCCCTCT39 80 University of British Columbia
357 59AGGCCAAATG39 50 University of British Columbia
382 59ATACACCAGC39 50 University of British Columbia
734 59GGAGAGGGAG39 70 University of British Columbia
736 59GAGGGAGGAG39 70 University of British Columbia
740 59GGAGGGAGGA39 70 University of British Columbia
 ZHOU ET AL: GENETIC VARIATION IN PHOMA MACROSTOMA 615

magnification. Pseudothecia that formed on toothpicks or herbarum were genetically distinct from the biocon-
culture medium in the plates were extracted and crushed trol isolates (FIG. 2B). The primers did not detect
on a glass slide in a drop of water. The slide was examined genetic variation among P. lingam Leroy, P. nebulosa
for asci containing ascospores with a light microscope at 92-74, P. pomorum 91-177, P. chrysanthemicola 90-64,
40–1003 magnification. The presence or absence of ma-
P. exigua 92-180-1 and P. medicaginis 94-335A1. These
ture pseudothecia, asci and ascospores was recorded.
latter species were more distantly related to the bio-
Phylogenetic data analyses.—The presence (1) or absence control isolates.
(0) of DNA bands in agarose gel were converted as 0–1
matrix. The data were analyzed by the phylogenetic soft- Genetic variation in Phoma isolates as revealed by
ware package TREECONt for Windows (Version 1.3b, Van PFGE.—Twenty-seven polymorphic chromosomal
de Peer and de Wachter 1994). The evolutionary distance DNA bands were generated with the CHEF analysis.
estimation was performed according to Nei and Li (1979). The chromosomal profiles of P. medicaginis and P.
An unweighted pair group cluster method with arithmetic herbarum were different from those of P. macrostoma
averages (UPGMA; Benzécri 1973) was used to infer tree (FIG. 3A) but exhibited some similarity to P. macros-
topology. Bootstrap analyses were included in the distance toma. However the biocontrol isolates of P. macrosto-
estimation and tree topology to place confidence intervals ma separated into two different categories of chro-
on phylogenies (Efron and Gong 1983, Felsenstein 1985,
mosomal profiles (Types I and II). Type I included
Sworfford et al 1996).
isolates 94-44B, 85-24B, 94-26, 95-268B and 95-54A1,
while the other five biocontrol isolates belonged to
RESULTS Type II (FIG. 3B). Similar to that revealed by RAPD
analysis, isolates from Ecozone 3 (94-44B, 85-24B, 95-
Genetic variation in Phoma isolates as revealed by
268B, 89-25A2, 94-359A, 97-12B and 97-15B2) and
RAPD.—A total of 96 polymorphic DNA bands were
Ecozone 4 (95-54A1, 94-26 and 94-134) were distrib-
produced by amplification with eight random prim-
uted randomly in these two chromosome profile
ers, and a phylogenetic tree was generated from the
types. The phylogenetic tree also showed that the iso-
data showing only the results from two primers (FIG.
lates of P. herbarum separated into two chromosome
2A, B). With primers 734 and 736, banding patterns
profiles (FIG. 3B).
were detected for all isolates of P. macrostoma, P. lin-
gam and one isolate of P. chrysanthemicola. Other Genetic variation in Phoma isolates as revealed by cross-
primers (not shown) detected P. exigua, P. medicagin- ing studies.—The crossing pair combinations and for-
is, P. nebulosa or P. pomorum, but not P. macrostoma, mation of asexual or sexual reproductive structures
which was the species of prime interest. Both inter- are presented (TABLE III). Only mycelial growth and
and intraspecific genetic variation in the Phoma spe- asexual sporulation from pycnidia were observed
cies and isolates was observed for all primers. when all P. macrostoma isolates were crossed with
All 10 biocontrol isolates of P. macrostoma were re- themselves, among each other or with the different
lated closely and clustered within the group desig- mating types of P. lingam. Pseudothecia containing
nated as ‘‘P. macrostoma Biocontrol Isolates’’ (FIG. ascospores were formed only by crossing different
2B). The isolates were somewhat variable from each mating type isolates of P. lingam.
other, to a limited extent within this group. For ex-
ample 97-15B2 had a banding pattern slightly differ-
DISCUSSION
ent than the other biocontrol isolates using Primer
734 (FIG. 2A). However the biocontrol isolates did Genetic variation in the population of a biocontrol
not form distinct subgroups based on their geograph- fungus must be assessed to understand the affect of
ic origins because the isolates from Ecozone 3 (94- a specific strain on the natural population after the
44B, 85-24B, 95-268B, 89-25A2, 94-359A and 97-12B) release (Hintz et al 2001). Concerning the release of
and Ecozone 4 (95-54A1, 94-26 and 94-134) were dis- a bioherbicide across ecozones, the biocontrol isolate
tributed within the same cluster. should be genetically similar to local populations to
The RAPD fragment patterns from the biocontrol reduce the potential unexpected affects, such as in-
isolates were different when compared to the other troducing new alleles that could alter host range or
isolates of P. macrostoma var. macrostoma, P. macros- pathogen growth and survival. Various approaches
toma var. incolorata and the other isolates of various have been adopted for such assessment. Recent re-
Phoma species (FIG. 2A, B). The isolates of P. macros- ports on the mycoherbicide Chondrostereum purpu-
toma var. macrostoma originating from Larix, Forsyth- reum (Pers. : Fr.) Pouzar by biochemical and molec-
ia, Philadelphus and Triticum were more similar to P. ular marker analyses showed no host or geographic
dennisii from Solidago than to the biocontrol isolates specialization in this fungus and that the introduc-
from Canada thistle (FIG. 2B). Also the isolates of P. tion of rare pathogenicity alleles from isolates used
616 MYCOLOGIA
 ZHOU ET AL: GENETIC VARIATION IN PHOMA MACROSTOMA 617

as biocontrol agents was unlikely (Shamoun and Wall into a local population. Genetic variation in fungal
1996; Ramesfield et al 1996, 1999; Gosselin et al populations is common as illustrated by P. lingam
1999; Hintz et al 2001). In our present study molec- ( Johnson and Lewis 1990, McGee and Petrie 1978,
ular and genetic tools were used to investigate the Petrie 1969, Hassan et al 1991, Kuusk et al 2002).
genetic variation of a potential broadleaf biocontrol Our RAPD study also showed that the genetic dis-
fungus, P. macrostoma. tances among three P. lingam isolates were signifi-
RAPD has been used widely as an effective molec- cantly different based on the confidence intervals
ular tool to evaluate genetic variance at or below the used for bootstrap analyses. This demonstrated that
species level. In our RAPD analyses different species these P. lingam populations were genetically diverse
and isolates of the genus Phoma produced different and movement of these populations likely would in-
DNA profiles on the gels denoting significant inter- troduce new alleles into an area. With P. macrostoma
and intraspecific genetic variation. The biocontrol there was obvious genetic variation among isolates
isolates of P. macrostoma were most similar to each from different continents, although the isolates from
other but uniquely different than other isolates with- the two Canadian ecozones were relatively similar.
in the same species (P. macrostoma var. macrostoma PFGE also was effective in distinguishing among
and P. macrostoma var. incolorata) and quite different different species and isolates according to chromo-
from other Phoma species, including P. lingam, P. her- somal profiles. The phylogenetic analysis indicated
barum, P. denisii var. denisii, P. medicaginis, P. nebu- that P. macrostoma may be more closely related to P.
losa, P. pomorum, P. chrysanthemicola and P. exigua. A herbarum than other Phoma spp. The present study
related study on molecular monitoring of the biocon- showed two distinctly different chromosomal profiles
trol isolates of P. macrostoma in plant and soil envi- within the species P. macrostoma. However it is inter-
ronments also supported our findings. In that study esting to note that the 10 biocontrol isolates of P.
a pair of PCR primers designed from the genomic macrostoma orginating from two different ecozones
DNA of the biocontrol isolates was found to be spe- were distributed into the two chromosomal sub-
cific to only the biocontrol isolates and no bands groups. At this time no other biological traits (such
were generated from other common soil fungi such as host range or growth characteristics) have been
as Cochliobolus, Epicoccum, Fusarium, Penicillium, associated with these two subgroups (personal obser-
Pythium, Sclerotinia and Septoria (Zhou et al 2004). vation based on unpublished data).
The intraspecific variation occurring in P. macrostoma Studies of mating types of fungal isolates also may
may be due to either geographic origin (Europe vs. be useful for subspecies classification and evaluation
Canada) or a unique trait (i.e. bioherbicidal activity) of genetic variation. In the case of P. macrostoma it
that evolved with a host-pathogen interaction. Al- was demonstrated that the biocontrol isolates did not
though some variation occurred within the 10 bio- produce a sexual state in mating studies among
control isolates, their distribution in the phylogenetic themselves or when paired with known mating types
tree generated from the RAPD data were not based from another Phoma species possessing the capability
on geographic origin. This suggested that the P. ma- of sexual reproduction. Other researchers also have
crostoma biological control isolates, which were iso- noted that P. macrostoma has no known teleomorph
lated from Ecozone 3 or Ecozone 4, are genetically (Boerema et al 2004). The use of a crossing strategy
similar, so the use of those isolates across these two was used to demonstrate to regulators that Canadian
ecozones has little risk of introducing novel alleles isolates of P. macrostoma were unable to reproduce

←
FIG. 2. A. RAPD analyses of genetic variations among Phoma isolates with two 10-mer oligonucleotide primers (734 on
top and 736 on bottom). Lanes 1 and 31, GeneRulery 1kb DNA Ladder (MBI Fermentas); Lanes 2–11, Phoma macrostoma
isolates 85-24B, 89-25A2, 94-26, 94-44B, 94-134, 94-359A, 95-54A1, 95-268B, 97-12B, and 97-15B2; Lane 12, P. dennisii var.
dennisii CBS135.96; Lanes 13–16, P. macrostoma var. macrostoma isolates CBS154.83, CBS482.95, CBS488.94 and CBS837.84;
Lane 17, P. macrostoma var. incolorata CBS839.84; Lanes 18–20, P. lingam isolates Leroy, Peace-3 and Pl86-12; Lanes 21–
23, P. herbarum isolates AI, AIV and G/5/2; Lanes 24–25, P. chrysanthemicola 90-64 and 91-27I; Lanes 26–29, P. exigua 92-
180-1, P. medicaginis 94-335A1, P. nebulosa 92-74, and P. pomorum 91-177; Lane 30, Blank. B. Phylogenetic relationship
among the biocontrol isolates of 10 Phoma macrostoma and 18 reference Phoma isolates generated by TREECONt for
Windows with RAPD data. The biocontrol isolates were clustered together and genetically distant from the reference
isolates. Within the cluster of biocontrol isolates, the isolates originating from two Canadian ecozones were scattered
randomly. The evolutionary distance scale was placed on the top of the figure and a bootstrap value was presented on
each node of the tree.
618 MYCOLOGIA

FIG. 3. A. Electrokaryotypes of P. macrostoma and several other Phoma isolates separated by pulsed field gel electro-
phoresis. Lane M corresponds to Saccaromyces cerevisiae marker; Lanes 1–10 to the P. macrostoma isolates 85-24B, 89-25A2,
94-26, 94-44B, 94-134, 94-359A, 95-54A1, 95-268B, 97-12B, and 97-15B2, respectively; Lane 11 to P. medicaginis 94-335 A1;
and Lanes 12–14 to P. herbarum isolates AI, AIV and G5/5/2, respectively. B. Phylogenetic relationship among several
Phoma isolates revealed by CHEF profiles. Two distinct subgroups were clustered within which limited variation was found.
Biocontrol agents 94-44B, 85-24B, 94-26, 95-268B and 95-54A1 were clustered in subgroup I (Type I profile), while isolates
89-25A2, 94-134, 94-359A, 97-12B, and 97-15B2 in subgroup II (Type II profile). The evolutionary distance scale (placed
on the top of the figure) and bootstrap values (presented on nodes of the tree) were calculated with TREECONt for
Windows software.

sexually thus reducing the risk of developing new al- not consider the occurrence of other mechanisms of
leles through recombination and to demonstrate to genetic exchange such as mutation or the parasexual
plant breeders that there was no exchange of genetic cycle. This would require the use of a visible marker
material with an economically important plant path- strategy, such as deploying antibiotic resistance in
ogen, P. lingam. However the crossing approach did one isolate and the green fluorescent protein marker
 ZHOU ET AL: GENETIC VARIATION IN PHOMA MACROSTOMA 619

TABLE III. Presence (1) or absence (0) of pseudothecia and ascospores after crossing biocontrol isolates of Phoma macro-
stoma (PM) among themselves and with P. lingam (PL) tester isolates with different mating types (Mat1 or Mat2)

PL 186-12
Phoma species and isolate PM 94-44B PM 85-24B PM 95-54A1 PM 94-26 PM 97-15B2 PM 94-134 (Mat2)
P. macrostoma 94-44B 0 0 0 0 0 0 0
P. macrostoma 85-24B 0 0 0 0 0 0 0
P. macrostoma 95-54A1 0 0 0 0 0 0 0
P. macrostoma 94-26 0 0 0 0 0 0 0
P. macrostoma 97-15B2 0 0 0 0 0 0 0
P. lingam PL189-21 (Mat1) 0 0 0 0 0 0 1
P. lingam PL189-19 (Mat1) 0 0 0 0 0 0 1
P. lingam PL186-12 (Mat2) 0 0 0 0 0 0 0

in another isolate, and then subculturing the inter- specific and infra-specific taxa in culture. Wallingford,
mingled colonies to determine which clones now UK: CABI Publishing.
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In conclusion P. macrostoma may be distinguished plified polymorphic DNA markers for the detection of
genetic variation in Phytophthora cinnamomi in Taiwan.
genetically from other Phoma spp. Ecological zones
Bot Bull Acad Sin 37:165–171.
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biocontrol isolates of P. macrostoma, as determined digenous fungal pathogens. In: Burge MN, ed. Fungi
by molecular and genetic approaches. Therefore any in biological control systems. New York: Manchester
P. macrostoma weed biocontrol isolates originating University Press. 269 p.
from Ecozone 3 and Ecozone 4 could be considered Chen CY, Séguin-Swartz G. 1999. Reaction of wild crucifers
by the regulators for release across these ecozones as to Leptosphaeria maculans, the causal agent of blackleg
a biopesticide with low risk of gene flow from asexual of crucifers. Can J Plant Pathol 21:361–367.
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ACKNOWLEDGMENTS Pathol 102:349–352.
Cook RJ, Bruckart WL, Coulson JR, Goettel MS, Humber
The authors are grateful to AAFC-SRC staff members YB RA, Lumsden RD, Maddox JV, McManus ML, Moore
Fu, A Hannoufa, SR Rimmer, J Derby, J Nettleton and R L, Meyer SF, Quimby PC Jr, Stack JP, Vaughn JL. 1996.
Underwood for their kind assistance concerning research Safety of microorganisms intended for pest and plant
facilities, fungal cultures or figure preparation. We also disease control: a framework for scientific evaluation.
thank Dr Greg Boland, University of Guelph, for providing Biol Control 7:333–351.
cultures of Phoma herbarum. This research was supported Cozijnsen AJ, Popa KM, Purwantara A, Rolls BD, Howlett
by a grant from Saskatchewan Agriculture Development BJ. 2000. Genome analysis of the plant pathogenic as-
Fund through the ADF Project 20010062-33AD. A visiting comycete Leptosphaeria maculans; mapping mating
fellowship in a Canadian government laboratory from the type and host-specificity loci. Mol Plant Pathol 1:293–
Natural Sciences and Engineering Research Council of Can- 302.
ada to Dr Lecong Zhou is acknowledged. Efron B, Gong G. 1983. A leisurely look at the bootstrap,
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