Eur J Plant Pathol (2007) 119:421–428
DOI 10.1007/s10658-007-9178-9
FULL RESEARCH PAPER
Characteristics of a Plasmopara angustiterminalis isolate
from Xanthium strumarium
Hedvig Komjáti Æ Ilona Walcz Æ Ferenc Virányi Æ Reinhard Zipper Æ
Marco Thines Æ Otmar Spring
Received: 18 December 2006 / Accepted: 21 May 2007 / Published online: 3 July 2007
KNPV 2007
Abstract Leaves of Xanthium strumarium infected
with downy mildew were collected in the vicinity of a
sunflower field in southern Hungary in 2003. Based
on phenotypic characteristics of sporangiophores,
sporangia and oospores as well as host preference
the pathogen was classified as Plasmopara angustiterminalis. Additional phenotypic characters were
investigated such as the size of sporangia, the number
of zoospores per sporangium and the time-course of
their release. Infection studies revealed infectivity of
the P. angustiterminalis isolate to both X. strumarium
and Helianthus annuus. Inoculation of the sunflower
inbred line, HA-335 with resistance to all known P.
halstedii pathotypes, resulted in profuse sporulation
on cotyledons and formation of oospores in the bases
of hypocotyls. Infections of sunflower differential
lines often led to damping-off. Molecular genetic
analysis using simple sequence repeat primers and
nuclear rDNA sequences revealed clear differences to
H. Komjáti F. Virányi (&)
Department of Plant Protection, Szent István University,
Godollo H-2103, Hungary
e-mail: viranyi.ferenc@mkk.szie.hu
I. Walcz
Forage Crops Research Institute, University of Kaposvár,
Iregszemcse-Bicserd H-7095, Hungary
R. Zipper M. Thines O. Spring
Institute of Botany, University of Hohenheim, 70593
Stuttgart, Germany
Plasmopara halstedii, the downy mildew pathogen of
sunflower.
Keywords Downy mildew Helianthus annuus
Host specificity Internal transcribed spacer
Peronosporaceae Plasmopara halstedii
Introduction
Novotelnova (1962) had separated Plasmopara angustiterminalis, the downy mildew pathogenic to
Xanthium strumarium (common cocklebur) from
Plasmopara halstedii. The latter has long been
considered as a single species complex with a broad
host range showing infectivity to > 80 genera of the
Asteroideae and Cichorioideae subfamilies of the
Asteraceae (Leppik 1966). The first revision on this
species complex by Savulescu (1941) was based on
detailed investigation of the morphology. It was
followed by Novotelnova (1962, 1963, 1966) who,
on the basis of morphology and host preference,
separated seven new species from the P. halstedii
complex. The name P. angustiterminalis f. sp.
angustiterminalis was introduced for the Plasmopara
isolates from Xanthium sp. (Novotelnova 1962), while
isolates from Heliantheae were named as P. helianthi
with further divisions as f. sp. helianthi, perennis, and
patens. Accordingly, the name P. halstedii remained
exclusive for the isolates infective on Eupatorieae, on
which this pathogen was first described. However, this
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concept has not been accepted generally by the
scientific community because (i) host specialisation
within the Plasmopara species complex could not be
reproduced (Virányi 1992), (ii) spore morphology
seemed to be a highly variable feature inappropriate
for species deliniation (Sackston 1981) and (iii) it was
argued that the new classification did not adhere to the
rules of the International Code of Botanical Nomenclature (Constantinescu cited in Gulya et al. 1997).
The separation of P. angustiterminalis had been
based mainly on differences in sporangiophore morphology and morphometric characters of zoosporangia and oospores, but host specificity was also an
important feature (Novotelnova 1966). However,
little more has yet been published on the taxonomy
of P. angustiterminalis, except for some molecular
phylogenetic analysis which grouped the taxon basal
to P. halstedii s.l. on Flaveria and Helianthus species
(Spring et al. 2003; Voglmayr et al. 2004).
Downy mildew isolates from X. strumarium plants
collected in Hungary in the early 1980s were found to
infect cultivated sunflower, and their morphological
characters were stated to be similar to P. halstedii
species (Viranyi 1984). A more recent recollection
from downy mildewed cocklebur plants in the south
of Hungary allowed us to establish a viable culture of
this obligate biotrophic pathogen for initiating more
detailed investigations into the taxonomy and pathogenicity of P. angustiterminalis. Intensive comparisons of phenotypic and genotypic characters with
features of P. halstedii isolates from cultivated
sunflower were conducted in order to clarify the
distinctiveness or conspecific status of the two taxa.
Moreover, infection studies on sunflower genotypes
with defined resistance genes should give insights
into the epidemiological potential of P. angustiterminalis to affect sunflower cultivation in areas where
X. strumarium occurs.
Materials and methods
Isolation of the pathogen and procedure for
obtaining single sporangium lines
The Plasmopara isolate used in this study was
collected from X. strumarium in southern Hungary
in the vicinity of a commercial sunflower field near
Bicsérd in 2003 (sample Pa676) and again from the
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Eur J Plant Pathol (2007) 119:421–428
same site in 2004 (voucher Pa671 HUH) and 2006
(BX06, SZIUH). Sporangia from sample Pa676 were
rinsed off from diseased leaves and used to inoculate
seedlings of a generally susceptible sunflower cv.
GK-70 (voucher Pa676GK70, HUH) with the whole
seedling inoculation (WSI) technique described by
Cohen and Sackston (1973).
From the first spore yield, single sporangial strains
were obtained by applying a modified technique of
Spring et al. (1997). Freshly produced sporangia were
picked up from the surface of water agar by a
micromanipulator and each was transferred to the
radicle of a germling of the sunflower line HA-335.
The germlings were then placed in 25 multiwell
microplates with 0.5 ml water per well. After 16 h
incubation at 16C the germlings were planted in
sterilized soil and grown in a climate chamber (16C,
80% RH, 14 h photoperiod) for two weeks prior to
inducing sporulation of the downy mildew pathogen.
Microscopic observation
Fresh sporangia were spread on 1% water agar plates
and incubated at 16C in the dark in order to observe
zoospore release and the counting of the number of
zoospores per sporangium. Furthermore, sporangial
shape was observed and the average length and width
determined by measuring 100 fresh zoosporangia in
water under a light microscope (Laborlux, Leitz,
Wetzlar, Germany) at 400· magnification.
Re-infection experiments
Fruits of X. strumarium collected from the same
location where the Xanthium downy mildew isolates
obtained were used for re-infection experiments. Seeds
were germinated after stratification and inoculated by
using the WSI method (Cohen and Sackston 1973).
Alternatively, the apical-bud was inoculated with a
sporangial suspension (approximately 5000 zoosporangia/bud) on 2 week-old X. strumarium plants, which
were grown in a climate chamber (16C, 80% RH, 14 h
photoperiod). To induce sporulation, plants were kept
in a humid chamber (100% RH) for 1 day at 16C.
Virulence tests
Virulence phenotype determination was carried out
using the internationally accepted sunflower differ-
Eur J Plant Pathol (2007) 119:421–428
ential test lines (Tourvieille et al. 2000) by soil
drench inoculation. Two day-old seedlings (10 per
test line) of each line were planted in pots filled with
a commercial soil mixture and 5000 sporangia/
seedling were applied on the soil around the roots.
Experiments were repeated twice with 10 seedlings
each time. Plants were grown for 2 weeks in a growth
chamber under controlled conditions and sporulation
induced as described above.
Fatty acid composition analysis
The fatty acid (FA) extraction followed by gas
chromatography analysis of approximately 2 mg of
sporangia and sporangiophores of the isolate Pa676
propagated on sunflower differential lines RHA-274
(Pa676–274) and HA-335 (Pa676–335) was done
according to Spring and Haas (2002). Average values
of FA composition of P. halstedii from different
geographic origins propagated on sunflower were
cited from Spring and Haas (2002).
Metalaxyl sensitivity test
Fifteen seeds of the sunflower line HA-335 were
allowed to germinate for 2 days on filter-paper soaked
with 1 ppm metalaxyl (Ciba-Geigy, Switzerland)
solution. After planting, 5000 sporangia of Pa676 per
seedling were applied by soil drenching. The pots
were watered once with 50 ml 1 ppm metalaxyl
solution after planting. Controls were made by
inoculating 15 plants with the pathogen in the
absence of metalaxyl. Two weeks later sporulation
was induced as described earlier.
Molecular characterisation
Total nucleic acid was extracted from approximately
15 mg of sporangia and sporangiophores using Genomic DNA purification KIT (Fermentas, Germany).
PCR was conducted in an Eppendorf Mastercycler
(Eppendorf, Germany). SSR primed PCRs were performed using (CAC) 4 RC, (GTG) 5 , (GT) 7 , and
(GAG)4RC simple sequence repeat primers, applying
the programme: 94C for 5 min denaturation followed
by 30 cycles of 94C for 1 min, 50C for 1 min, 72C
for 2 min, ending with a final 10 min elongation at
72C. Each reaction contained 30 ng of template, 1 unit
Taq-polymerase (Fermentas, Germany), 1x Buffer,
423
1.5 mM MgCl2, 100 mM dNTPs, and 1 mM primer.
Amplification products of the Xanthium downy mildew isolate Pa676 were compared to samples of four
P. halstedii isolates having the representative virulence
phenotypes 700, 703, 710, or 730, respectively, and
another isolate of P. halstedii with host preference to
Helianthus x. laetiflorus infective on sunflower (isolate
characterised in Spring et al. 2003). Fragments were
scored as present or absent. The generated binary data
were analysed by the Treeconw programme (Van de
Peer and De Wachter 1994) using neighbour-joining
(Saitou and Nei 1987) and UPGMA analyses (Sokal
and Michener 1958), and robustness was evaluated by
bootstrapping (Felsenstein 1985).
The variable D1/D2/D3 domain of the 50 end of the
nuLSU rDNA was amplified and sequenced according to Riethmüller et al. (2002). The oomycete
specific primer pairs LR0R (5 0 GTACCCGCTGAACGGAAGC) and LR6-O (50 CGCCAGACGAGCTTACC) were used for amplification. Sequencing
was initiated with the universal primers NL1
(5 0 GCATATCAATAAGCGGAGGAAAAG) and
NL4 (50 GGTCCGTGTTTCAAGACGG) from O’
Donnell (1993). Sequence was compared to P. angustiterminalis nrLSU sequence data from GenBank
(accession number AY 178535), using the SeqMan
programme (DNAStar, Lasergene, Madison, USA).
Internal transcribed spacer (ITS) regions were
amplified according to Thines et al. (2005). The
product was cloned into pGEM-Teasy T/A cloning
vector (Promega, Bio-Science, Hungary). For
sequencing, additional primers were designed: XIF1
(50 ATCGTAACATGACTTCCGGTG), XIF2 (50
ATTGGTGAACCGTAGTTATAG) and XIR1
(50 GAAGAAAATGTATGATATGTCG). XIF1 primer was used for direct sequencing on the cloned
product while products obtained by primer pairs XIF2
and XIR1 were cloned as previously prior to
sequencing. Sequences were assembled manually
with SeqMan and sequence analysis was conducted
by MegAlign software from a DNAStar programme
package (Lasergene, Wisconsin, USA). ITS sequences were compared to the corresponding data
from GenBank Plasmopara halstedii DQ665670, P.
geranii DQ131916, P. obducens DQ665671, P.
viticola DQ665668, Plasmoverna pygmaea
DQ665671 and Phytophthora infestans AF228084.
ITS sequences were aligned using MAFFT 5.8
(Katoh et al. 2002) by FFT-NSi option. The dendo-
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gram was created by applying NJ (Saitou and Nei
1987) of 610 conserved sites by the Jukes-Cantor
substitution model (Jukes and Cantor 1969). The
support for the internal nodes of the tree was assessed
with bootstrap analysis (Felsenstein 1985) using 1000
replicates. The dendogram was visualised by TreeViewX 0.5.0. (Page 1996).
Eur J Plant Pathol (2007) 119:421–428
HA-335 sunflower seedlings showed hyphae and
haustoria of the pathogen in both the cortical and pith
parenchyma of the hypocotyls. Cell necrosis also
developed in these tissues. Oospores were detected in
the roots of the 12 week-old infected sunflower plants
(Fig. 1c).
Single sporangium infections
Results
Microscopic observations
The sporangiophores of the isolate Pa676 showed
monopodial branching (Fig. 1a). Sporangial shape
varied from round, through ovate to elongated or
peanut shaped, measuring 25–50 · 20–30 (average
41.1 ± 7.8 · 27.0 ± 4.6) mm (Fig. 1b). The incubation
time prior to zoospore release was 30–45 min; the
number of released zoospores from one single
sporangium varied between 4 and 13, and most
frequently six zoospores were observed (average
6.29 ± 2.0). Cross sections of 8 day-old infected
The single-spore technique to inoculate sunflower
cotyledons and/or true leaves in order to prepare
genetically homogenous lines from the field isolates
was unsuccessful. On the other hand, single sporangial strains were successfully prepared applying
selected sporangia to the roots of whole seedlings.
As a result, 2 weeks after single spore infection, weak
sporulation was visible on the cotyledons. Infection
rate using single sporangia as inoculum reached 8%.
Host specificity
Inoculation of the sunflower cv. GK-70 with fresh
sporangia of the Plasmopara isolate collected from X.
Fig. 1 (A) Monopodial sporangiophore of Plasmopara angustiterminalis from Xanthium strumarium. (bar: 35 mm), (B) Diverse
shape and size of sporangia of P. angustiterminalis (bar: 25 mm). (C) Oospores produced by P. angustiterminalis in sunflower roots
(bar: 40 mm). (D) Sunflower line HA-335 infected by P. angustiterminalis symptoms showing stunting, destroyed primary root,
secondary root development, and sporulation on cotyledons. (E) WSI-infected Xanthium strumarium seedling showing sparse
sporulation on cotyledons. (F) Secondary local infection of X. strumarium by P. angustiterminalis with chlorosis, necrosis and leaf
deformation. (G) Systemic spread of infection by P. angustiterminalis, revealed by the development of chlorosis on a X. strumarium
leaf
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Eur J Plant Pathol (2007) 119:421–428
425
strumarium by WSI method resulted in sporulation
on the cotyledons. However, the infection remained
cotyledon-limited (CLI), i.e. the fungus did not
colonise the epicotyl, and the typical systemic
symptom of leaf chlorosis was not observed. Nevertheless, infected plants showed profuse sporulation on
cotyledons, hypocotyls and roots. They became
stunted and often showed necrosis on the soil-covered
parts of the hypocotyl, and roots (Fig. 1d). Infected
plants survived for months, but heavy infection often
caused damping-off of the sunflower seedlings.
Re-infection of X. strumarium with sporangia
obtained after cultivation of the pathogen on sunflower resulted in symptom development. Cotyledons
were partially chlorotic and sporulation occurred
(Fig. 1e). These chlorotic areas later became brown
due to necrosis. Under laboratory conditions, no
systemic growth of the fungus was observed into the
epicotyl of inoculated seedlings. However, inoculation through the apical bud of Xanthium seedlings
resulted in some cases of local chlorosis on the true
leaves (Fig. 1f) that eventually turned necrotic, but
occasionally systemic spread of the pathogen into the
leaves could be observed (Fig. 1g). In general, the
disease remained restricted to the infected leaf
segment.
Virulence character
The virulence phenotype of the single sporangial
strain Pa676-SSL, designated as CLI-type 717, was
quite similar to that observed for the parental isolate
(Pa676) (Table 1), except for two sunflower lines
PM-17 and 803-1 where Pa676-SSL caused sparse
sporulation on cotyledons. Infections remained cotyledon-limited (CLI-type) or caused damping-off.
Systemic symptoms such as leaf chlorosis and/or
stunting were not observed for any of the sunflower
genotypes.
Metalaxyl sensitivity
Inoculated control plants not treated with metalaxyl
showed strong sporulation on their cotyledons but
metalaxyl-treated plants all remained without symptoms. In addition, subsequent microscopic observations of the cross-sections of the hypocotyls from
metalaxyl-treated HA-335 sunflowers failed to show
any fungal structure.
Fatty acid composition
FA patterns of the sporangia of the Plasmopara
isolate Pa676 grown on the sunflower lines RHA-274
and HA-335, and those from P. halstedii are given in
Table 2. Samples of Pa676 propagated on sunflower
had a significantly higher C18:0 content, whereas the
relative ratio in C18:1, 20:1 and 22:1 was lower than
in P. halstedii isolates. Eicosapentaenoic acid
(C20:5) dominated the pattern in both pathogens.
Molecular characterisation
PCR analysis using simple sequence repeat primers
highlighted clear differences between the Plasmopara isolate Pa676 from Xanthium and P. halstedii.
Screening of Pa676, Pa676-SSL and five different
pathotypes of P. halstedii with the primers
(CAC)4RC, (GTG)5, (GT)7 and (GAG)4RC yielded
a total of 92 amplification products. Of these, 80 were
polymorphic for Pa676. Calculation of similarity
based on the binary data (presence or absence of
amplification products) clearly separated the Pa676
samples (here assigned as P. angustiterminalis) from
the P. halstedii group (Fig. 2).
Sequence of the partial nuLSU of rDNA from
Pa676 was analysed and deposited in the NCBI
database (DQ457006). The Xanthium downy mildew
isolate Pa676 cultivated on sunflower and P. angust-
Table 1 Virulence phenotye of a Xanthium field isolate of Plasmopara angustiterminalis (Pa676) and its single sporangial strain
(Pa676-SSL) on sunflower differential test lines based on the percentage of cotyledon-limited (CLI) type of infection
Fungal isolate/line
Sunflower differential line
HA-304
Pa676
Pa676-SSL
a
RHA-265
RHA-274
DM-2
PM-17
803-1
HAR-4
HAR-5
HA-335
80
80
100
100
0
10
90
100
80
100
66
100
100
30a
62a
100
100
100
Only sparse sporulation observed
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Eur J Plant Pathol (2007) 119:421–428
Table 2 Fatty acid composition (ratio of total peak area) assayed by GC analysis of sporangia and sporangiophores of P. angustiterminalis propagated on sunflower lines RHA-274 (Pa676-274) and HA-335 (Pa676-335), respectively
Fatty acid
P. angustiterminalis
C14:0a
C16:0
C18:0
C18:1
C18:2
C20:1
C20:5
C22:0
C22:1
Pa676–274
0.9 b
13.9 b
9.8 c
1.6 b
11.0 b
tr c
20.3 b
–
1.6 b
Pa676–335
2.0 a
19.9 b
3.5 b
2.2 b
23.2 a
0.8 b
34.5 a
1.2 b
2.2 c
P. halstedii average and
SD
1.9 a
(0.6)
17.4 a
(2.3)
0.9 a
(0.5)
11.0 a
(3.2)
22.0 a
(2.3)
1.9 a
(0.4)
30.9 a
(3.9)
0.8 a
(0.1)
3.9 a
(0.8)
Comparative values for P. halstedii from different geographic origin propagated on sunflower were cited from Spring and Haas
(2002). Figures with the same letters did not differ at P = 0.05
a
Number of carbon atoms: number of double bonds
tr = trace amounts of <0.1% of total peak area
with P. halstedii, indicating that these species might
constitute a monophyletic group.
Discussion
Fig. 2 Dendogram created on SSR-primed PCR patterns of P.
angustiterminalis and P. halstedii s.l. with representative
pathotypes (indicated in brackets) or host preference. Isolates
were all propagated on H. annuus. Distances (relative
similarity) were calculated on binary data by Neighbourjoining method and UPGMA analysis using Treeconw.
Numbers on branches indicate bootstrap values
iterminalis from an Austrian population of X. strumarium (AY178535) (Spring et al. 2003) shared
100% identity regarding partial nrLSU sequence, thus
unambiguously identifying Pa676 as P. angustiterminalis.
ITS amplification resulted in a 3225 base length
product which is deposited in GenBank (accession
number DQ993167). Sequence analysis revealed that
ITS1 (base:1–218) had 92.7% similarity to P.
halstedii. The more conserved 5.8S region (bases
219–374) shared 100% identity, and the next 76 bases
(bases 375–450) of ITS-2 region showed 92.1%
similarity between the two species. The extraordinary
length of ITS2 with 2815 bases in total (bases 375–
3190) was due to a large insertion containing 14
repeated elements (RE), which are homologous to the
RE described for P. halstedii and other downy
mildew species by Thines (2007). Phylogenetic trees
estimated by Minimum Evolution (Rzhetsky and Nei
1993) using Jukes-Cantor distances on 610 bases of
the conserved parts of ITS sequences of Plasmopara
species (Fig. 3) grouped P. angustiterminalis together
123
Light microscopy revealed differences in the Xanthium
downy mildew isolate compared to P. halstedii from
sunflower in several aspects: (i) sporangial morphology, (ii) time of incubation required for zoospore
release and (iii) the number of released zoospores from
one single sporangium. Sporangial size given by
Novotelnova (12–27 · 9–18 mm) for P. angustiterminalis (Novotelnova 1966) was relatively small when
compared with our isolate. This may be due to the
strong variation we observed even within the same
isolate in the course of several rounds of propagation.
The time-course of zoosporangial germination took
<1 h for the Xanthium downy mildew isolate, compared to the usual 2–3 h for P. halstedii from sunflower
(Virányi and Oros 1991; Spring et al. 1998). As an
average, six zoospores were released per sporangium,
less than the average of 20 zoospores observed under
similar conditions for a P. halstedii isolate.
Spring and Thines (2004) claimed that besides
classical morphometric measurements and hostbound criteria there was a necessity for considering
new characters for the classification of oomycete
species. Accordingly, fatty acid analysis indicated
differences between P. angustiterminalis and P.
halstedii particularly in the ratio of stearic acid
(C18:0) and oleic acid (C18:1). Both pathogens were
rich in eicosapentaenoic acid (C20:5), a characteristic
compound of P. halstedii (Spring and Haas 2002)
underlining the relatedness of both taxa. However,
Eur J Plant Pathol (2007) 119:421–428
427
Fig. 3 Minimum Evolution model using JC distances on 610 bp of ITS1-5.8S-ITS2 sequences of Plasmopara species. Phytophthora
infestans served as outgroup. Numbers above the branches indicate bootstrap values (1000 replicates)
molecular phylogenetic analyses based on simple
sequence repeats and ITS-sequence indicated clear
differences in the genetic background of the two
species. The ITS-2 region of P. angustiterminalis
Pa676 contained a large insertion sharing sequence
similarity with previously identified repeated elements (Thines 2007). The total ITS length (3190 bp)
exceeded the known size found so far within the
Plasmopara genus by >20% (Spring et al. 2006;
Voglmayr et al. 2006). For the phylogenetic analysis
only conserved regions were used in the model to
exclude the influences of in/dels (Mitchell and
Zuccaro 2006). The resulting tree topology was
similar to what was observed during nuLSU analysis
by Spring et al. (2003). Analysis of nuLSU obtained
for P. angustiterminalis Pa676 was different from that
of P. halstedii (Spring et al. 2003) and it was identical
to the previous GenBank nuLSU sequence of P.
angustiterminalis. The latter sequence had been
established from a sample collected in Austria indicating the presence of this pathogen in other regions.
The host–pathogen interaction found with our P.
angustiterminalis isolate on sunflower differential
lines was characterised by the appearance of CLI-type
infections. In this type of infection the plant prevents
the pathogen from advancing into the epicotyl, but the
pathogen stays alive (Heller et al. 1997). Notably, the
generally susceptible sunflower line HA-304 (with no
known resistance gene) responded with profuse sporulation of the fungus on the hypocotyl and cotyledons.
On the other hand, the sunflower line HA-335, known
to carry effective resistance genes against all known
virulence phenotypes of P. halstedii, also permitted
CLI-type infections. It is noteworthy that despite the
restriction of the mycelium to cotyledons and lower
plant parts, the pathogen remained alive over several
months and was able to complete its sexual life-cycle
within its host (see Fig. 1).
Although not yet isolated from naturally infected
sunflower, our studies with zoosporangia of P.
angustiterminalis clearly indicated that cultivated
sunflower is a potential host plant for this downy
mildew pathogen. Its natural host, X. strumarium, is
common in Hungary and eastern parts of Austria, and
our isolate was obtained from a population in close
vicinity of sunflower fields. If advantageous natural
conditions allow sporangia or oospores of P. angustiterminalis to infect cultivated sunflower, the capability to produce oospores in CLI-type infections may
help to generate recombinant offspring for soilborne
infection in the next season. Moreover, it cannot be
excluded that co-infection with P. halstedii could
lead to genetic exchange through hybridisation. The
resistance of the cultivars currently used in sunflower
cultivation will not be sufficient to prevent infection.
However, our fungicide tests showed that phenylamides are still effective in controlling P. angustiterminalis.
Acknowledgements This work was kindly supported by a
DAAD grant A/03/30179 for H. Komjáti.
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