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Received: 27 June 2016 Accepted: 10 October 2016 DOI: 10.1111/efp.12323
ORIGINAL ARTICLE
Current status of the Dutch elm disease pathogen populations affecting Ulmus minor in Greece G. T. Tziros | Z. G. Nakopoulou | C. Perlerou | S. Diamandis Forest Research Institute, Hellenic Agricultural Organization, Vassilika, Thessaloniki, Greece Correspondence George T. Tziros, Forest Research Institute, Hellenic Agricultural Organization, Vassilika, Thessaloniki, 57006, Greece. Email:
[email protected] Editor: A. Lehtijärvi
Summary One hundred and eight Ophiostoma ulmi sensu lato isolates were collected from field elm trees with symptoms in 14 Prefectures of Greece. The purpose of this study was to assign Greek isolates to species and afterwards subspecies of the DED fungi and to analyse the genetic variability within the Greek populations of these pathogens. Isolates were compared with six reference strains belonging to O. ulmi and the two subspecies of O. novo-ulmi. The structure of the population has been analysed by means of morpho-physiological features (growth rates, colony morphology and fertility responses) and by DNA sequencing and PCR-RFLP amplification of the cerato- ulmin (cu) and the colony type (col1) gene regions. According to fertility tests, both subspecies of O. novo-ulmi were detected in Greece, but none of the isolates collected was identified as O. ulmi. O. novo-ulmi ssp. novo-ulmi occurred more frequently than ssp. americana (73 and 35 isolates, respectively) and their ranges overlapped. All isolates that behaved as ssp. novo-ulmi in the fertility tests had the cu, as well as the col1 profile of ssp. novo-ulmi. Surprisingly, all isolates that behaved as O. novo-ulmi ssp. americana in the fertility test had the cu, as well as the col1 (with one exception) profile of O. novo-ulmi ssp. novo-ulmi. A possible explanation for this inconsistency could be the occurrence of hybridization between the two subspecies in Greece.
1 | INTRODUCTION
O. novo-ulmi ssp. novo-ulmi and ssp. americana is suspected to occur (Brasier, 2001).
Dutch elm disease (DED) is one of the most destructive diseases in
Three elm species occur in Greece: Ulmus minor (Field elm), the
the Northern Hemisphere (Brasier, 1991), resulting in the death of the
most common species, found in plains and lowland all over the country
majority of mature elms (Brasier & Buck, 2002). Two destructive pan-
(e.g. along country roads and river banks), U. glabra (Wych elm) present
demics of the disease have occurred across both Europe and North
in mountainous and moist habitats, and U. laevis (European white elm)
America over the last 100 years, each caused by a different species
found only in riparian ecosystems.
of Ophiostoma. The first pandemic was caused by the less aggres-
The first record of Dutch elm disease in Greece, probably caused
sive Ophiostoma ulmi and the second one by the more aggressive
by Ophiostoma ulmi, was reported on Ulmus campestris in 1962 (Gibbs,
Ophiostoma novo-ulmi (Brasier, 1990; Brasier & Buck, 2002), which is
1981). In 1968, the disease was recorded in some Prefectures of
separated into two distinct subspecies, ssp. novo-ulmi and ssp. amer-
Macedonia, northern Greece (Thessaloniki, Serres and Kilkis), in
icana (Brasier & Kirk, 2001), formerly known as the Eurasian (EAN)
Thessaly and in Thrace, central and eastern Greece, respectively
and the North American (NAN) races, respectively (Brasier, 1979).
(Pantidou, 1973). Since then, it has spread all over the country elimi-
The ranges of the two subspecies presently overlap in several parts
nating elm trees growing in natural ecosystems as well as those used
of Europe (Brasier, Buck, Paoletti, Crawford, & Kirk, 2004; Brasier &
for landscaping. Prior to this study, almost nothing was known about
Kirk, 2001, 2010). In those parts of Europe where the ranges of the
the DED pathogens in Greece. The only previous reports were those
subspecies are presently overlapping, free hybridization between
of Brasier and Kirk (2001, 2010) and Brasier et al. (2004) who reported
Forest Pathology. 2017;47:e12323. wileyonlinelibrary.com/journal/efp https://doi.org/10.1111/efp.12323
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that only the subspecies O. novo-ulmi ssp. novo-ulmi occurred in Greece, and Santini, Montaghi, Vendramin, and Capretti (2005) identified two Greek isolates as O. novo-ulmi ssp. novo-ulmi. Several studies on the diversity of DED fungal populations were
et al
2 | MATERIALS AND METHODS 2.1 | Study area and fungal isolation
conducted previously, and the isolates were examined using vegeta-
The survey was carried out in three successive years (from 2008 to
tive compatibility reactions (Brasier, 1988; Brasier & Kirk, 1990), DNA
2010) in localities, which were included in 14 Prefectures, and sam-
sequence analysis and restriction fragment length polymorphisms
pling was based on the map distribution of Ulmus minor trees pre-
(RFLP) of nuclear and mitochondrial DNA (Bates, Buck, & Brasier,
sented in Boratynski, Browicz, and Zielinski (1992). Knowledge of the
1993a, 1993b; Diaz et al., 2009; Dvorak, Jankovsky, & Krajnakova,
distribution of this species in Greece was obtained during a previous
2009; Dvorak, Tomsovsky, Jankovsky, & Novotny, 2007; Hintz, Jeng,
European research project on the conservation of elm genetic re-
Hubbes, & Horgen, 1991; Jeng, Duchesne, Sabourln, & Hubbes,
sources in Europe (Diamandis & Perlerou, 2005; Solla et al., 2005).
1991; Kirisits & Konrad, 2004; Konrad, Kirisits, Riegler, Halmschlager,
Two additional Prefectures (Trikala and Evia) were included in the sur-
& Stauffer, 2002), direct amplification of minisatellite-region DNA
vey, but no diseased trees were detected (Figure 1).
(Santini et al., 2005) and polymerase chain reaction of random am-
To assess the variability of the Greek DED fungal population, 108
plified polymorphic DNA (PCR-RAPD) (Hoegger, Binz, & Heininger,
Ophiostoma ulmi sensu lato isolates were obtained from symptomatic
1996; Pipe, Buck, & Brasier, 1995; Solla, Dacasa, Nasmith, Hubbes,
trees growing at least 1 km apart. Each isolate was derived from dis-
& Gil, 2008).
eased elm twigs with live bark collected from an individual diseased
The aim of this study was to investigate the current distribution
elm tree, using standard isolation methods described by Brasier
of species and subspecies causing Dutch elm disease in Greece. For
(1981). Slivers of xylem showing dark streaks characteristic of infec-
this purpose, Greek isolates were examined for their growth rates,
tion were removed with a scalpel, plated on a selective medium con-
colony morphology and fertility responses and using molecular
taining cycloheximide, penicillin and streptomycin (Brasier, 1981) and
methods. For the latter, the cerato-ulmin (cu) (Bowden, Hintz, Jeng,
incubated at 20°C. Single spore isolates were prepared from all Greek
Hubbes, & Horgen, 1994; Jeng, Hintz, Bowden, Horgen, & Hubbes,
isolates (Table 1) and cultured at 20°C on Malt Extract Agar (MEA,
1996; Pipe, Buck, & Brasier, 1997) and the colony type (col1) (Pereira
Oxoid). Short-term stock cultures were maintained on MEA at 20°C
et al., 2000) gene regions were amplified and restricted by HphI and
and subcultured at 2-week intervals, while long-term stock cultures
BfaI endonucleases.
were maintained on MEA slopes at −20°C.
F I G U R E 1 Geographical distribution of Ophiostoma novo-ulmi subspecies in Greek surveyed Prefectures based on phenotypic characteristics. Capital letters stand for surveyed Prefectures in Table 1. Open circles, subsp. novo-ulmi; closed circles, Prefectures where subsp. novo-ulmi and americana overlapped; closed squares, Prefectures of Trikala (TRK) and Evia (EVI) included in the survey but without any Ophiostoma isolates obtained
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T A B L E 1 Origin of Greek isolates of Ophiostoma spp. and results of mating type, fertility test and PCR-RFLP of cerato-ulmin (cu) and colony type (col1) gene region Isolate codea
Prefecture
Coordinates
Year of isolation
THE1
Thessaloniki
40°32′N022°59′E
2008
Growthb at 20°C
33°C
Fertility testc
2.86
0.16
O.n.a.
Mating type 1
Subspecies cu
col1
O.n.n.
O.n.n.
LRS1
Larissa
39°53′N022°40′E
2008
3.00
0.06
O.n.n.
2
O.n.n.
O.n.n.
LRS3
Larissa
39°52′N022°42′E
2008
2.96
0.03
O.n.n.
2
O.n.n.
O.n.n.
LRS4
Larissa
39°52′N022°42′E
2008
2.56
0.22
O.n.n.
2
O.n.n.
O.n.n.
LRS5
Larissa
39°52′N022°42′E
2008
3.14
0.00
O.n.n.
2
O.n.n.
O.n.n.
LRS6
Larissa
39°56′N022°39′E
2008
3.28
0.00
O.n.n.
2
O.n.n.
O.n.n.
LRS7
Larissa
39°56′N022°40′E
2008
2.92
0.03
O.n.n.
2
O.n.n.
O.n.n.
LRS8
Larissa
39°56′N022°41′E
2008
2.30
0.08
O.n.n.
2
O.n.n.
O.n.n.
LRS9
Larissa
39°56′N022°41′E
2008
3.14
0.08
O.n.n.
2
O.n.n.
O.n.n.
LRS10
Larissa
39°57′N022°41′E
2008
3.14
0.03
O.n.n.
2
O.n.n.
O.n.n.
KOZ1
Kozani
40°17′N021°25′E
2008
3.26
0.00
O.n.a.
2
O.n.n.
O.n.n.
KOZ2
Kozani
40°18′N021°25′E
2008
3.22
0.15
O.n.n.
2
O.n.n.
O.n.n.
KOZ3
Kozani
40°18′N021°23′E
2008
2.90
0.14
O.n.n.
2
O.n.n.
O.n.n.
KOZ4
Kozani
40°18′N021°21′E
2008
3.06
0.00
O.n.a.
1
O.n.n.
O.n.n.
KOZ5
Kozani
40°18′N021°21′E
2008
4.62
0.00
O.n.n.
2
O.n.n.
O.n.n.
KOZ6
Kozani
40°17′N021°21′E
2008
3.88
0.15
O.n.n.
2
O.n.n.
O.n.n.
KOZ7
Kozani
40°17′N021°20′E
2008
3.00
0.00
O.n.n.
2
O.n.n.
O.n.n.
KOZ8
Kozani
40°16′N021°19′E
2008
2.58
0.14
O.n.n.
2
O.n.n.
O.n.n.
THE6
Thessaloniki
40°43′N022°37′E
2008
2.56
0.25
O.n.n.
2
O.n.n.
O.n.n.
PEL4
Pella
40°47′N021°52′E
2008
5.08
0.10
O.n.a.
1
O.n.n.
O.n.n.
SER2
Serres
41°10′N023°09′E
2008
2.74
0.23
O.n.a.
1
O.n.n.
O.n.n.
SER3
Serres
41°11′N023°05′E
2008
2.58
0.03
O.n.n.
2
O.n.n.
O.n.n.
HMA4
Imathia
45°35′N022°17′E
2008
3.52
0.03
O.n.n.
2
O.n.n.
O.n.n.
HMA5
Imathia
40°35′N022°17′E
2008
2.78
0.19
O.n.a.
2
O.n.n.
O.n.n.
HMA6
Imathia
40°38′N022°29′E
2008
3.34
0.20
O.n.a.
1
O.n.n.
O.n.n.
HMA7
Imathia
40°38′N022°30′E
2008
2.74
0.00
O.n.n.
2
O.n.n.
O.n.n.
THE7
Thessaloniki
40°49′Ν023°29′Ε
2008
3.66
0.25
O.n.n.
2
O.n.n.
O.n.n.
THE8
Thessaloniki
40°45′N023°32′E
2008
3.02
0.00
O.n.n.
2
O.n.n.
O.n.n.
KIL5
Kilkis
40°59′N022°56′E
2008
3.38
0.08
O.n.n.
2
O.n.n.
O.n.n.
KIL9
Kilkis
41°04′N022°30′E
2008
2.18
0.04
O.n.n.
2
O.n.n.
O.n.n.
PIR1
Pieria
40°09′N022°32′E
2009
2.24
0.03
O.n.a.
1
O.n.n.
O.n.n.
PIR2
Pieria
40°10′N022°29′E
2009
3.26
0.10
O.n.n.
2
O.n.n.
O.n.n.
PIR3
Pieria
40°10′N022°29′E
2009
3.46
0.05
O.n.n.
2
O.n.n.
O.n.n.
PIR4
Pieria
40°14′N022°24′E
2009
3.10
0.09
O.n.a.
1
O.n.n.
O.n.n.
PIR5
Pieria
40°15′N022°24′E
2009
3.12
0.17
O.n.a.
1
O.n.n.
O.n.n.
PIR6
Pieria
40°15′N022°21′E
2009
2.92
0.21
O.n.n.
2
O.n.n.
O.n.n.
PIR7
Pieria
40°15′N022°24′E
2009
2.20
0.20
O.n.n.
2
O.n.n.
O.n.n.
THE10
Thessaloniki
40°38′N022°42′E
2009
2.58
0.19
O.n.n.
2
O.n.n.
O.n.n.
THE11
Thessaloniki
40°38′N022°42′E
2009
2.30
0.23
O.n.n.
2
O.n.n.
O.n.n.
THE13
Thessaloniki
40°39′N022°42′E
2009
2.36
0.18
O.n.a.
1
O.n.n.
O.n.n.
THE14
Thessaloniki
40°35′N022°39′E
2009
3.00
0.38
O.n.a.
1
O.n.n.
O.n.n.
HMA8
Imathia
40°34′N022°30′E
2009
1.98
0.00
O.n.n.
2
O.n.n.
O.n.n.
HMA9
Imathia
40°35′N022°30′E
2009
2.06
0.08
O.n.n.
2
O.n.n.
O.n.n.
HMA10
Imathia
40°33′N022°31′E
2009
2.26
0.10
O.n.n.
2
O.n.n.
O.n.n. (Continues)
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T A B L E 1 (Continued) Isolate codea
Prefecture
Coordinates
Year of isolation
Growthb at 20°C
33°C
Fertility testc
Mating type
Subspecies cu
col1
PEL5
Pella
40°53′N022°04′E
2009
2.30
0.30
O.n.a.
1
O.n.n.
O.n.n.
PEL6
Pella
40°57′N022°04′E
2009
3.16
0.16
O.n.a.
2
O.n.n.
O.n.n.
PEL7
Pella
41°01′N022°04′E
2009
2.06
0.45
O.n.n.
2
O.n.n.
O.n.n.
PEL8
Pella
41°02′N022°07′E
2009
1.98
0.13
O.n.n.
2
O.n.n.
O.n.n.
PEL9
Pella
41°02′N022°08′E
2009
3.92
0.03
O.n.a.
1
O.n.n.
O.n.n.
PEL10
Pella
41°03′N022°08′E
2009
3.00
0.19
O.n.a.
1
O.n.n.
O.n.n.
PEL11
Pella
41°03′N022°10′E
2009
3.02
0.15
O.n.n.
2
O.n.n.
O.n.n.
PEL12
Pella
41°02′N022°10′E
2009
3.26
0.15
O.n.a.
2
O.n.n.
O.n.n.
PEL13
Pella
41°02′N022°11′E
2009
3.12
0.16
O.n.a.
2
O.n.n.
O.n.n.
THE15
Thessaloniki
40°43′N022°38′E
2009
2.18
0.15
O.n.a.
2
O.n.n.
O.n.n.
THE16
Thessaloniki
40°46′N022°37′E
2009
2.02
0.21
O.n.n.
1
O.n.n.
O.n.n.
THE17
Thessaloniki
40°47′N022°37′E
2009
2.20
0.13
O.n.n.
2
O.n.n.
O.n.n.
THE18
Thessaloniki
40°47′N022°38′E
2009
3.32
0.16
O.n.n.
2
O.n.n.
O.n.n.
THE19
Thessaloniki
40°40′N022°37′E
2009
3.28
0.19
O.n.n.
2
O.n.n.
O.n.n.
THE20
Thessaloniki
40°48′N022°35′E
2009
2.76
0.56
O.n.a.
1
O.n.n.
O.n.n.
THE21
Thessaloniki
40°48′N022°34′E
2009
3.14
0.20
O.n.a.
1
O.n.n.
O.n.n.
PIR8
Pieria
40°30′N022°34′E
2009
3.20
0.08
O.n.n.
2
O.n.n.
O.n.n.
PIR9
Pieria
40°30′N022°34′E
2009
2.76
0.25
O.n.n.
2
O.n.n.
O.n.n.
PIR10
Pieria
40°31′N022°34′E
2009
4.28
0.15
O.n.n.
2
O.n.n.
O.n.n.
LRS11
Larissa
39°33′N022°17′E
2009
2.70
0.15
O.n.n.
2
O.n.n.
O.n.n.
KAR2
Kardirsa
39°26′N022°04′E
2009
2.46
0.17
O.n.n.
2
O.n.n.
O.n.n.
XAN1
Xanthi
40°25′N023°30′E
2009
2.50
0.13
O.n.n.
2
O.n.n.
O.n.n.
XAN3
Xanthi
41°05′N024°50′E
2009
3.02
0.29
O.n.n.
2
O.n.n.
O.n.n.
XAN5
Xanthi
40°53′N024°48′E
2009
2.58
0.17
O.n.n.
2
O.n.n.
O.n.n.
XAN6
Xanthi
40°54′N024°48′E
2009
3.70
0.01
O.n.n.
2
O.n.n.
O.n.n.
XAN7
Xanthi
41°06′N024°53′E
2009
3.24
0.16
O.n.a.
1
O.n.n.
O.n.n.
KAV1
Kavala
40°55′N024°38′E
2009
2.62
0.16
O.n.n.
2
O.n.n.
O.n.n.
KAV3
Kavala
40°53′N024°41′E
2009
3.00
0.16
O.n.n.
2
O.n.n.
O.n.n.
KAV4
Kavala
40°53′N024°42′E
2009
3.02
0.16
O.n.n.
2
O.n.n.
O.n.n.
KAV5
Kavala
40°51′N024°11′E
2009
3.32
0.14
O.n.n.
2
O.n.n.
O.n.n.
ROD1
Rodopi
41°07′N025°13′E
2010
2.10
0.00
O.n.n.
2
O.n.n.
O.n.n.
ROD3
Rodopi
41°07′N025°16′E
2010
3.16
0.08
O.n.n.
2
O.n.n.
O.n.n.
ROD4
Rodopi
41°08′N025°26′E
2010
2.82
0.09
O.n.n.
2
O.n.n.
O.n.n.
ROD5
Rodopi
41°10′N025°26′E
2010
3.20
0.10
O.n.a.
1
O.n.n.
O.n.n.
ROD6
Rodopi
41°10′N025°28′E
2010
2.74
0.03
O.n.n.
2
O.n.n.
O.n.n.
ROD7
Rodopi
41°03′N025°28′E
2010
3.66
0.06
O.n.n.
2
O.n.n.
O.n.n.
ROD9
Rodopi
41°00′N025°28′E
2010
3.80
0.00
O.n.n.
2
O.n.n.
O.n.n.
ROD10
Rodopi
40°59′N025°26′E
2010
3.38
0.09
O.n.a.
2
O.n.n.
O.n.n.
ROD11
Rodopi
41°00′N025°25′E
2010
3.44
0.05
O.n.n.
2
O.n.n.
O.n.n.
PEL14
Pella
40°46′N021°55′E
2010
3.48
0.00
O.n.a.
1
O.n.n.
O.n.n.
PEL15
Pella
40°45′N021°51′E
2010
3.60
0.01
O.n.n.
2
O.n.n.
O.n.n.
PEL16
Pella
40°43′N021°49′E
2010
4.00
0.00
O.n.n.
2
O.n.n.
O.n.n.
FLO1
Florina
40°41′N021°47′E
2010
3.60
0.00
O.n.n.
2
O.n.n.
O.n.n.
FLO2
Florina
40°40′N021°46′E
2010
3.66
0.00
O.n.n.
2
O.n.n.
O.n.n.
FLO3
Florina
40°39′N021°42′E
2010
4.16
0.01
O.n.n.
2
O.n.n.
O.n.n. (Continues)
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T A B L E 1 (Continued) Isolate codea
Prefecture
Coordinates
Year of isolation
Growthb at 20°C
33°C
Fertility testc
Mating type
Subspecies cu
col1
FLO4
Florina
40°46′N021°23′E
2010
4.30
0.00
O.n.n.
2
O.n.n.
O.n.n.
FLO5
Florina
40°46′N021°22′E
2010
3.34
0.00
O.n.n.
2
O.n.n.
O.n.n.
FLO6
Florina
40°45′N021°25′E
2010
3.08
0.00
O.n.a.
1
O.n.n.
O.n.n.
FLO7
Florina
40°44′N021°26′E
2010
2.92
0.03
O.n.n.
2
O.n.n.
O.n.n.
FLO8
Florina
40°43′N021°27′E
2010
3.32
0.00
O.n.n.
2
O.n.n.
O.n.n.
FLO9
Florina
40°41′N021°28′E
2010
3.58
0.07
O.n.a.
2
O.n.n.
O.n.n.
FLO10
Florina
40°40′N021°40′E
2010
2.94
0.00
O.n.a.
2
O.n.n.
O.n.n.
FLO11
Florina
40°39′N021°41′E
2010
3.46
0.05
O.n.n.
2
O.n.n.
O.n.n.
FLO12
Florina
40°36′N021°41′E
2010
3.88
0.00
O.n.n.
2
O.n.n.
O.n.n.
FLO13
Florina
40°34′N021°41′E
2010
3.56
0.00
O.n.a.
2
O.n.n.
O.n.n.
FLO14
Florina
40°36′N021°41′E
2010
4.18
0.01
O.n.a.
1
O.n.n.
O.n.n.
THE22
Thessaloniki
40°35′N023°46′E
2010
3.04
0.24
O.n.a.
1
O.n.n.
O.n.a.
KOZ9
Kozani
40°17′N021°26′E
2010
2.38
0.14
O.n.a.
1
O.n.n.
O.n.n.
KOZ10
Kozani
40°18′N021°24′E
2010
2.80
0.00
O.n.a.
1
O.n.n.
O.n.n.
GRE1
Grevena
40°05′N021°26′E
2010
3.06
0.16
O.n.a.
1
O.n.n.
O.n.n.
GRE2
Grevena
40°04′N021°25′E
2010
2.80
0.15
O.n.n.
2
O.n.n.
O.n.n.
GRE3
Grevena
40°05′N021°23′E
2010
2.78
0.00
O.n.n.
2
O.n.n.
O.n.n.
GRE4
Grevena
40°04′N021°26′E
2010
2.18
0.05
O.n.a.
1
O.n.n.
O.n.n.
GRE5
Grevena
40°03′N021°23′E
2010
2.74
0.20
O.n.n.
2
O.n.n.
O.n.n.
a
Isolates are presented in chronological order of sampling. Growth rate in mm/day on Oxoid malt extract agar. c O.n.n., O. novo-ulmi ssp. novo-ulmi; O.n.a., novo-ulmi ssp. americana. b
Subspecies novo-ulmi tester strains AST-20 and AST-27 (one and
2.2 | Colony morphology, growth rate, mating type, species and subspecies determination of isolates
two mating types, respectively), as well as ssp. americana strains
Determination of growth rates at 20 and 33°C, colony morphology
used in the mating type tests and as recipients and donors in the fer-
and mating type of isolates was performed as described by Brasier
tility tests. Additionally, O. ulmi isolates R21 and P32 (one and two
(1981). Malt Extract Agar (MEA) and Elm Sapwood Agar (ESA) were
mating types, respectively) were used in this study.
MM2/1 and RDT-38 (one and two mating types, respectively), were
prepared as described in Brasier (1981). For MEA, 16.5 g of Malt
Ophiostoma ulmi and O. novo-ulmi are bipolar heterothallic, with
Extract Agar (Oxoid) and 5 g of agar (Oxoid) were suspended in 500 ml
mating types MAT-1 and MAT-2 (Paoletti, Buck, & Brasier, 2005), pre-
of distilled water. ESA medium was prepared using 50 g of peeled and
viously known as MAT-A and MAT-B, respectively. To test for the mat-
milled sapwood from elm twigs up to 0.5 cm in diameter, 15 g of agar
ing type of an isolate, an ESA plate was inoculated with the unknown
and 500 ml of distilled water.
isolate and with one and two mating type tester strains. On each plate,
For strain identification, 2-mm inoculum plugs of each isolate were
the inocula were equidistant from each other. Plates were incubated
inoculated centrally on Petri dishes (diameter 9 cm) containing ca. 20 ml
for 7 days in the dark at 20°C and then were placed on the labora-
of MEA, and the experiment was repeated twice. After 10 days of incu-
tory bench at room temperature in diffuse light for a further period
bation at 20°C in the dark, two colony diameters at right angles at the
of 2 weeks. The mating type test was performed three times for each
reverse side of the plates were measured. The colony diameters were
isolate. Perithecia were formed at the boundary between the MAT-2
remeasured after an additional incubation of 5 days, and the mean ra-
tester strain and the unknown isolate if the latter was of mating type
dial growth rate (mm/day) was calculated over the final 5-day period.
1, and at the boundary between the MAT-1 tester strain and the un-
Plates were then placed on the laboratory bench at room temperature
known isolate if it belonged to mating type 2.
in diffuse light for a further period of 10 days and were examined for
Subspecies determination was performed using fertility tests
colony morphology. A second growth rate at 33°C was carried out to
(Brasier, 1981) based on the fact that O. novo-ulmi ssp. novo-ulmi shows
support the differentiation into aggressive and non-aggressive strains.
a partial reproductive barrier against O. novo-ulmi ssp. americana.
In contrast with the first test at 20°C, in this second test, a period of
Therefore, in a novo-ulmi (female) x americana (male) pairing, ca. 90%
10 days intermediated between the first and the second growth rate
fewer perithecia are produced than in a reciprocal cross (Brasier,
measurements.
1979). To identify the subspecies of an O. novo-ulmi MAT-2 isolate,
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two ESA plates were inoculated with the MAT-1 tester strains (one
at 72°C for 5 min. Electrophoresis on a 1% agarose gel (Sigma), pre-
with AST-20 and the other with MM2/1) to act as recipients. After
pared in 2×TBE electrophoresis buffer, stained with ethidium bromide
14 days of incubation in the dark at 20°C and 7 days in diffuse light
and visualized with a UV transilluminator (Syngene) was carried out to
at room temperature, conidia from the surface of the MAT-2 isolates
confirm the success of amplification. A 100-bp DNA ladder (Invitrogen)
(unknown and donors) were scraped and applied to a 2-cm2 patch on
was used as a size marker. PCR products were purified with PureLink™
the surface of each recipient strain. In the same way, conidia of the
PCR Purification Kit (Invitrogen) and custom-sequenced (Cemia,
MAT-2 tester strains (AST-27 and RDT-38), which were used as do-
Greece) without cloning (Gyllensten & Allen, 1993). The sequences
nors, were applied to the recipient strains. After 10 days of incubation
were initially visualized by ChromasLite® (Technelysium Pty Ltd.) and
in diffuse light at room temperature, recipient strain AST-20 differ-
visually aligned. All DNA sequences were determined in both directions
entiated between the O. novo-ulmi ssp. novo-ulmi and ssp. americana
assigning isolates to O. ulmi and O. novo-ulmi species of the DED fungi.
donors. Fertilization with conidia of O. novo-ulmi ssp. americana gave
Phylogenetic analysis was conducted using MEGA version 5.05, and
10–30 times fewer perithecia in comparison with the fertilization with
the phylogenetic tree was constructed from the concatenated cu and
O. novo-ulmi ssp. novo-ulmi, which gave abundant perithecia. Recipient
col1 loci using maximum-likelihood algorithm (100 bootstrap replicates)
tester strain MM2/1 did not differentiate O. novo-ulmi ssp. novo-ulmi
and neighbor-joining algorithm (550 bootstrap replicates). Distances
and ssp. americana donors, as scored perithecia were abundant, and
were calculated using Kimura-2p in both phylogenetic inferences.
thus served as a control. In the same way, to identify the subspecies of
The amplified cu gene fragments of all O. novo-ulmi isolates were
an O. novo-ulmi MAT-1 isolate, tester strains AST-27 and RDT-38 were
cleaved by restriction endonuclease HphI (New England Biolabs) and
used as recipients and tester strains AST-20 and MM2/1 as donors.
by the restriction enzyme BfaI (New England Biolabs) for the col1 gene
The unknown MAT-1 isolates themselves acted as recipients.
to distinguish the two subspecies among all studied isolates. PCR- RFLP fragments were visualized on 2% agarose gels described before, and a 100-bp DNA ladder (Invitrogen) was used as a size standard. The
2.3 | DNA extraction
evaluation was performed according to Pipe et al. (1997) and Konrad
Petri dishes (diameter 9 cm) containing ca. 20 ml MEA, overlain
et al. (2002).
with sterilized cellophane sheet (Gel drying frames, Sigma-Aldrich), were inoculated with 4-mm-diameter mycelial plugs and incubated for 7–10 days in the dark at 20°C. The mycelium was scraped from each plate, lyophilized and ground to a fine powder. Genomic DNA was extracted from this material using the DNeasy Plant Mini Kit (Qiagen) following the manufacturer’s instructions. DNA quality and
3 | RESULTS 3.1 | Colony morphology, growth rate, mating type, species and subspecies determination of isolates
concentration were evaluated on 0.8% agarose gel in the presence of
All the isolates used in this study were assigned to Ophiostoma novo-
λDNA/Hind III Fragments Ladder (Invitrogen).
ulmi based on their growth rates (Table 1) and colony morphology. O. novo-ulmi colonies formed aerial mycelium, which gave them a characteristic fibrous-striate appearance, a petaloid pattern and mod-
2.4 | cu and col1 amplification, sequencing and PCR-RFLP
erate to strong diurnal zonation. None of the Greek isolates showed an irregular culture aspect,
The species and subspecies were distinguished by polymerase chain
which is typical for d-infected strains, and has been reported for strains
reaction (PCR) and restriction fragment length polymorphism (RFLP)
infected with deleterious fungal viruses (d-factors) (Brasier, 1983).
of two gene regions: the cerato-ulmin (cu) gene region (Bowden et al.,
For further characterization, the isolates were tested for mating
1994; Jeng et al., 1996; Pipe et al., 1997) and the col1 gene that en-
types and were assigned to subspecies (Table 1). Fertility and subspe-
codes colony type (Pereira et al., 2000). The cu gene region was am-
cies test made it possible to distinguish 73 isolates of the subspecies
plified with the primers CU1 5′-GGGCAGCTTACCAGAGTGAAC-3′
O. novo-ulmi ssp. novo-ulmi of which one isolate belonged to MAT-1
and
et al.,
and 72 isolates belonged to MAT-2. The remaining 35 isolates were
1997). A 482-bp fragment of the col1 gene was amplified with
CU2
5′-GCGTTATGATGTAGCGGTGGC-3′
(Pipe
determined as O. novo-ulmi ssp. americana of which 25 isolates were
the F-primer 5′-GCAGTTGTTGACATGTATG-3′ and the R-primer
MAT-1 and 10 isolates were MAT-2 (Table 1).
5′-TGCTTGACGTAGATCTCG-3′ designed by Konrad et al. (2002). The PCR volume of each sample consisted of 2, 5 μl 10 × amplification buffer (Promega), 2 mM MgCl2, 200 μM of each dNTP, 10 μM of each primer, 2 units of Taq DNA polymerase (Promega) and 18 ng of
3.2 | cu and col1 amplification, sequencing and PCR-RFLP
genomic DNA in a final reaction volume of 25 μl. PCR was performed
The results of cu and col1 PCR-RFLP for Greek isolates are summarized
with a thermocycler (MyCycler, Bio-Rad) with the following amplifica-
in Table 1. The results of the fertility test did not correspond with their
tion conditions: initial denaturation step at 94°C for 4 min, followed by
cu and col1 profile for all studied isolates. Seventy-three isolates de-
30 cycles of 94°C for 15 s, annealing at 68°C for 1 min (for cu) and at
fined as ssp. novo-ulmi in the fertility test had cu and col1 profile cor-
58°C (for col1) and denaturation at 72°C for 2 min with final extension
responding absolutely to fertility test. On the contrary, 34 isolates that
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et al
behaved as ssp. americana in the fertility test had cu and col1 profile of
is in agreement with the reported decline of this species in other
ssp. novo-ulmi. Isolate THE22 behaved as ssp. americana in the fertility
European countries (e.g. Hoegger et al., 1996; Santini et al., 2005;
test but showed the cu profile of ssp. novo-ulmi and the col1 profile of
Solla et al., 2008) following the emergence of the more aggressive
ssp. americana. Subspecies novo-ulmi and ssp. americana tester strains
and competitive O. novo-ulmi. Gremmen, Heybroek, and De Kam
included in this study had the corresponding cu and col1 RFLP profiles.
(1976) and Brasier (1983) suppose that in Europe, O. novo-ulmi had
DNA sequences containing both cu and col1 gene regions of all
replaced O. ulmi by the end of the 1970s. It is believed that O. ulmi has
Greek isolates used in this study were aligned constructing a con-
disappeared, which is characteristic from areas where O. novo-ulmi
catenated phylogenetic tree (Figure 2). The concatenated nucleotide
has already been present for a relatively long time (Brasier, 1991).
sequences of the reference isolates R21, P32 (O. ulmi), AST-20 (O. no-
Furthermore, the interaction between O. ulmi as a “resident” species
vo-ulmi ssp. novo-ulmi) and MM2/1 and RDT-38 (O. novo-ulmi ssp.
and O. novo-ulmi as an “invader” has led to the rapid decline and virtual
americana) were used. Cu and col1 sequences of O. himal-ulmi isolate
extinction of O. ulmi throughout most of its known range and its re-
HP50 retrieved from Genbank (KJ540068 for cu and KJ540073 for
placement by O. novo-ulmi (Brasier, 1986; Brasier & Kirk, 2010).
col1) were used to root the tree. To access the reliability of the tree ob-
About 24% of the isolates belonged to MAT-1 mating type, while
tained, bootstrap analysis consisting of 500 replicates was performed
76% of the isolates were of MAT-2 mating type. The proportion of
using the CLUSTAL X2.0 program. The concatenated sequences of 105
MAT-2 isolates varies from 70 to 100%, with a mean value of about
isolates were identical to the one of O. novo-ulmi ssp. novo-ulmi tes-
85% (Brasier, 1988; Brasier & Kirk, 1990). In Switzerland, Italy and
ter isolate AST-20 and are represented by FLO4 haplotype (Figure 2).
Spain, for example, the proportions of MAT-2 were reported to be 62,
In contrast, the cu and col1 gene sequence of isolates PIR4 and PIR5
66 and 95%, respectively (Hoegger et al., 1996; Santini et al., 2005;
presents a significant genetic distance compared with those of isolate
Solla et al., 2008). The lower presence of MAT-1 isolates is suspected
AST-20 and FLO4 resembling two new Greek haplotypes. On the other
to be caused by their slightly lower level of aggressiveness and slower
hand, an isolate THE22 correlates well with reference isolates MM2/1
growth rate, leading to selective disadvantage in their competition
and RDT-38 and has also a reliable genetic distance represents a fourth
with MAT-2 isolates (Brasier & Gibbs, 1975; Brasier & Kirk, 2001).
haplotype. Bootstrapping analysis with 108 samples showed that the
Results of the fertility tests showed that 73 isolates belonged to
concatenated cu and col1 gene regions of the isolates of the two spe-
subspecies novo-ulmi and 35 to ssp. americana. The two subspecies
cies formed two distinct groups in 100% of the trees, totally distinct
of O. novo-ulmi overlap in the majority of the Prefectures surveyed in
from the O. himal-ulmi group represented by the isolate HP50.
this study, often in close proximity one to another (Figure 2). Greek isolates examined in previous studies (Brasier & Kirk, 2001; Brasier
4 | DISCUSSION
et al., 2004; Brasier & Kirk, 2010; Santini et al. 2005) were assigned only to subspecies novo-ulmi. It was suspected that the two subspecies of O. novo-ulmi most
In this study, a set of 108 isolates of Ophiostoma ulmi sensu lato from
likely hybridize in Greece due to the overlap in their distribution in the
Greece were evaluated and assigned to species and subspecies. No
country. To verify this possibility, DNA sequencing and PCR-RFLP of
isolate collected in this survey was identified as O. ulmi. This finding
nuclear genes that are polymorphic and consistently distinguish the
F I G U R E 2 Neighbor-joining (NJ) and maximum-likelihood (ML) phylogenetic tree of concatenated cu and col1 gene nucleotide sequences of Greek isolates with Ophiostoma himal-ulmi isolate HP50 as an out-group. Isolate FLO4 represents 105 studied isolates. The numbers above nodes stand for the statistical support; first number indicates NJ, and the second one, ML bootstrap support values
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et al
two subspecies of O. novo-ulmi were followed. The 108 Greek isolates
indicating that hybrids had already emerged by that time (Konrad
that had previously been designated to ssp. novo-ulmi or ssp. ameri-
et al., 2002). In addition, in Czech Republic, 6–9 isolates were iden-
cana on the basis of their fertility response (Table 1) were examined
tified as intraspecific hybrids between the two subspecies amplify-
by the PCR-RFLP method of the cu gene. In 73 isolates, the fertil-
ing the same gene regions as mentioned above (Dvorak et al., 2007,
ity test and RFLP of cu gene gave identical results. However, in 35
2009). All these authors proposed hybridization between the two
isolates, the fertility response and cu RFLP profile differed, indicating
subspecies to explain conflicting results in fertility test and molecular
that they likely correspond to O. novo-ulmi x ssp. americana hybrids.
methods.
To find further evidence for hybridization between the subspecies of
These results show that the fertility test commonly used to differen-
O. novo-ulmi, all 108 Greek isolates were investigated by RFLP of the
tiate the two subspecies is no longer sufficient for definite designation
col1 gene. Although all 73 isolates identified as ssp. novo-ulmi in the
of isolates of O. novo-ulmi, as ssp. novo-ulmi x americana hybrids can
fertility test had col1 profiles of the same subspecies, 34 of 35 isolates
show fertility responses resembling those of either subspecies (Brasier,
defined as ssp. americana in the fertility test had col1 profiles of ssp.
1986). Surprisingly, the Greek isolates designated as O. novo-ulmi ssp.
novo-ulmi. Most likely, these isolates could be subspecies hybrids, as
americana using fertility tests had cu and col1 profiles (with one ex-
they gave conflicting results in the fertility tests and the cu and col1
ception for col1 gene amplification—isolate THE22) of ssp. novo-ulmi.
RFLP profiles. The hybrid isolates were detected in the majority of the
With only two genes regions amplified, it is likely that some of the iso-
localities surveyed in the country and also were present in the same
lates behaved as O. novo-ulmi ssp. americana in the fertility tests were
Prefecture.
carrying undetected amounts of DNA of ssp. novo-ulmi. Brasier and
The relationship among the analysed isolates is presented in a
Kirk (2010) ended up in such a conclusion examining 10 distinguishing
concatenated phylogenetic tree (Figure 2). The clusters were de-
RAPD and phenotypic markers in total of two European populations,
signed as O. ulmi, O. novo-ulmi ssp. novo-ulmi and O. novo-ulmi ssp.
which consisted ca. 70–80% of hybrids.
americana in the presence of reference strains. The species O. ulmi
Most likely, Greece forms part of a hybrid zone between
and O. novo-ulmi were separated into two major clusters, and the lat-
O. novo-ulmi ssp. novo-ulmi and ssp. americana. This was first sus-
ter species was further divided into its two subspecies. Four haplo-
pected because of the overlap of the subspecies in Greece and has
types (including the putative hybrids) were identified within the 108
subsequently been proven through the identification of ssp. novo-ulmi
Greek isolates analysed. The low number of types found in this study
x ssp. americana hybrids in several parts of the country. A large num-
is probably indicatory of the post-epidemic situation in Greece. This
ber of Greek isolates (35 of 108) were identified as hybrids between
assumption together with the 35 cases of inconsistency, between the
ssp. novo-ulmi and ssp. americana based on fertility response and RFLP
fertility tests and the two amplified gene regions, could be correlated
of the cerato-ulmin gene and colony type gene. As a consequence,
to a recent invasion. That means that perhaps, ssp. americana has not
subspecies hybrids may become the dominant forms of O. novo-ulmi
reached the country yet or the specific subspecies has been intro-
in Greece, as has previously been suggested (Brasier, 2001; Brasier &
duced very recently.
Kirk, 2010) for other parts of Europe.
Several studies have confirmed that hybrids are emerging in var-
This is the first study dealing with the genetic diversity of DED
ious parts of Europe. Evidence of hybridization between the two
pathogens in Greece. According to Konrad et al. (2002), PCR-RFLP
subspecies has already been obtained from physiological features
provides a reliable and rapid diagnostic technique to identify ssp.
(Brasier, 1986) and RAPD polymorphisms (Brasier et al., 2004). Based
novo-ulmi and ssp. americana isolates. However, 35 Greek isolates
on isozyme analysis, Jeng, Bernier, and Brasier (1988) suggested that a
showed contrasting results between fertility tests and PCR-RFLP
French isolate could be a subspecies hybrid and Hoegger et al. (1996)
profiles. Hybridization between the two subspecies is commonly pro-
proposed that a Swiss isolate giving conflicting results in fertility
posed to explain conflicting results in fertility test and molecular meth-
tests and RAPD analysis is a subspecies hybrid. A molecular analysis
ods (e.g. in Hoegger et al., 1996; Jeng et al., 1996; Konrad et al., 2002;
using RAPDs of genomic DNA, rDNA RFLPs and cerato-ulmin gene
Kirisits & Konrad, 2004 and Dvorak et al., 2009). Many hybrids could
sequences revealed that three isolates were interspecific hybrids
not be detected using only PCR-RFLP of cu and col1, and it will thus
(Brasier, Kirk, Pipe, & Buck, 1998). Santini et al. (2005) identified sev-
be necessary to include other nuclear genes from different chromo-
eral Italian isolates as subspecies hybrids from a combination of their
somes (Bowden et al., 1994; Pereira et al., 2000), as well as mitochon-
PCR profile with the M13 primer and their fertility response, while
drial genes or anonymous markers such as AFLP and RAPD, to obtain
Solla et al. (2008) identified two isolates from Spain as subspecies hy-
reliable results on the occurrence and frequency of hybrids (Konrad
brids based on RAPD profiles and fertility responses. Furthermore,
et al., 2002).
seven isolates from Austria were identified as hybrids between ssp. novo-ulmi and ssp. americana, based on fertility response and RFLP amplification of the cerato-ulmin (cu) and colony type (col1) gene
AC KNOW L ED G EM ENTS
regions (Kirisits & Konrad, 2004; Konrad et al., 2002). Konrad et al.
The authors gratefully acknowledge Clive M. Brasier and Susan A.
(2002) found also that an isolate (P150, Brasier & Kirk, 2000) that
Kirk (Forest Research Station, Alice Holt Lodge, Farnham, Surrey,
was included in their study as a tester isolate showed a hybrid pat-
U.K.) for providing reference strains of the DED fungi, and Vassilis
tern. Interestingly enough, that strain was collected in Poland in 1980
Christopoulos (Forest Research Institute, Thessaloniki, Greece)
TZIROS
et al
for his assistance with sampling from diseased elm trees. We also thank Dr. Dimitrios N. Avtzis for his helpful comments and suggestions.
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