DIVISION OF PLANT MEDICINE

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DIVISION OF PLANT MEDICINE
RESEARCH ACTIVITIES
Department of Virology
Transmissibility of Wheat dwarf virus (WDV)
strains by leafhopper vectors (MZe
0002700603)
The transmissibility of WDV strains (wheat and
barley) by leafhopper (Psammotettix alienus)
vectors was compared in one- and three-day
acquisition feeding period (AFP) with the aim to
select optimal AFP. The leafhopper nymphs
(individually) were allowed to feed in a six-day
inoculation feeding period (IFP) on spring wheat
(wheat strain) and spring barley (barley strain).
The percentage of transmissibility was evaluated by
symptom manifestation and by DAS-ELISA four
weeks after inoculation. The transmissibility in oneday AFP was 73% in wheat strain and 45% in barley
strain, in three-day AFP 53.6% in wheat strain and
60.7% in barley strain. Hence, our results indicate
higher transmissibility of the wheat strain of WDV
in a shorter AFP. (Širlová L.)
Molecular characterisation of grapevine
viruses of the genus Foveavirus (MZe
0002700603)
Samples of dormant canes were taken from vines
grown at Karlštejn Research Station of Viticulture.
Total RNA was isolated from the phloem tissue
scraped from these canes. Degenerate primers
were used in RT-PCR procedure. The obtained
fragments were cloned into a pGEM-T Easy plasmid
(Promega) and sequenced. The 412 nucleotides
long sequence of a polymerase gene of Rupestris
stem pitting-associated virus (RSPaV) is now
available in the GenBank under acc. no. AY731821.
(Komínek P., Bryxiová M.)
Molecular and immunoenzymatic methods
for the detection of grapevine viruses from
the genus Vitivirus (MZe 0002700603)
Samples of dormant canes were collected from
three geographic regions of grapevine cultivation
in the Czech Republic. These samples were tested
by DAS-ELISA for the presence of Grapevine virus A
– GVA. Out of the total of 62 tested grapevines,
9 were found to be infected with GVA. Several
isolates of GVA were obtained from cuttings taken
from positive vines. (Komínek P.)
Reliable detection of European stone-fruit
yellows (ESFY) (MZe 0002700603)
Two hundred sixty-four ESFY symptomless apricot
trees of different apricot cultivars were tested by PCR
for the presence of ESFY. ESFY was detected in
39 samples out of 116 tested using a nested PCR. In
another experiment 52 apricot trees were tested by
nested PCR and ESFY was detected in 7 trees. Forty
trees of apricot cultivars Maďarská, Velkopavlovická,
Veecot, Bergeron, Karola (only 1 tree) and Leskora
(only 1 tree) with ESFY positive reaction and 26 trees
of apricots (Velkopavlovická-25, Veecot-1) with ESFY
negative reaction in nested PCR were verified using a
biological test on peach GF-305 (according to the
EPPO recommendation). Three seedlings of peach
GF-305 were inoculated by chip-budding using buds
from one tested apricot tree. Thirty-seven out of
40 apricot trees with detected ESFY by PCR did not
show any reaction on GF-305 (no ESFY symptoms
were observed). The results of testing of the three
remaining apricot trees were not unambiguous. Out
of the 3 tested plants one GF-305 indicator died after
inoculation with 1 tree of cv. Maďarská and 1 tree of
cv. Bergeron, and 2 GF-305 plants out of the 3 tested
ones died after inoculation with apricot tree cv.
Leskora. On the other hand, 25 out of 26 apricot trees
with PCR negative reaction to ESFY did not show any
reaction on GF-305. One GF-305 plant out of the
3 tested ones died after inoculation with one tree of
cv. Velkopavlovická. The results of biological testing
indicate that only 1 apricot tree of cv. Leskora seems
to be ESFY infected, however the dying of GF-305
trees is not associated with typical ESFY symptoms.
The results of molecular testing (nested PCR) were
completely different from the biological test. Further
experiments will be necessary to explain this
discrepancy between the results of molecular and
biological detection of ESFY in apricots. (Polák J.,
Bryxiová M., Salava J., Svoboda J.)
The occurrence of Wheat dwarf virus (WDV)
strains in cereals and grasses in the Czech
Republic (MZe 0002700603)
The screening of WDV incidence was carried out
in selected areas of Central, Western, Northern and
Eastern Bohemia and Southern Moravia. Thirty-six
samples of winter wheat and 19 samples of winter
barley suspicious of WDV infection were collected
and tested by DAS-ELISA. WDV was detected in
28 samples of winter wheat and 16 samples of
winter barley. Fifteen samples of winter wheat and
13 samples of winter barley infected by WDV (ELISA
positive) were verified by PCR using strain specific
primers. Wheat strain of WDV was found in all tested
wheat samples and only in three tested barley
samples (fields in the localities Žebrák, Ejpovice,
Kuchař). Barley strain of WDV was detected in
10 barley samples. Fourteen couch-grass samples
with dwarfing and reddening symptoms were
collected from the vicinity of winter wheat fields
(Velvary and Praha-Zličín) with previously detected
WDV. The virus was not detected by DAS-ELISA in
the above couch-grass samples. (Širlová L.)
The occurrence of pome fruit tree viruses in
apple and pear nurseries (MZe 0002700603)
An RT-PCR assay was developed for detection of
ACLSV (Apple chlorotic leaf spot virus) and ApMV
(Apple mosaic virus). Total RNA was isolated using
RNeasy mini kit (Qiagen). Both viruses were
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successfully detected throughout the year using
different plant tissues (leaves and dormant buds)
by RT-PCR as described by Menzel et al. (2002). The
same samples were tested by DAS-ELISA. The results
show that DAS-ELISA is reliable only during spring
but later in the season it gave erratic results. Hence
we recommended to verify negative-ELISA results
by RT-PCR. (Hassan M., Svoboda J.)
Apple stem pitting virus (ASPV) was detected in
70 accessions of apple and pear trees. Samples were
obtained from the germplasm at RBIP, Holovousy.
The RT-PCR and primers were used as described by
Menzel et al. (2002). The infection rate of ASPV was
high, up to 90% infection. One-tube Multiplex-RTPCR protocol for simultaneous detection of four
apple viruses (ASGV, ASPV, ApMV and ACLSV) with
RNA internal was optimised. The validation of the
above protocol by ELISA and woody indexing
techniques is under way. (Hasan M., Polák J.)
Research on the distribution of economically
important viruses of fruit trees and
grapevine (MZe 0002700603)
One hundred fifty apricot varieties and 25
rootstocks from the germplasm of FH MUAF in
Lednice were tested for the presence of five
economically important viruses: Plum pox virus,
(PPV), Apple chlorotic leaf spot virus (ACLSV),
Prunus necrotic ring spot virus (PNRSV), Plum
dwarf virus (PDV) and Apple mosaic virus (ApMV).
Total RNA was isolated from leaves according to
Astruc et al. (1996). These viruses were detected by
molecular hybridisation conducted in IBMCP-UPV,
Valencia, Spain. (Hassan M., Čiháková K.)
A screening of the sanitary status of grapevine in
the Czech Republic was done by sampling dormant
canes from grapevines grown in three areas of
Southern Moravia. In total 62 samples were tested by
DAS-ELISA for the presence of 15 viruses: Grapevine
fanleaf virus – GFLV, Arabis mosaic virus – ArMV,
Strawberry latent ringspot virus – SLRSV, Tomato
black ring virus – TBRV, Grapevine virus A – GVA,
Grapevine virus B – GVB, Grapevine fleck virus –
GFkV, two serotypes of Raspberry ringspot virus –
RpRSV-g and RpRSV-ch and a complex of grapevine
leafroll viruses: Grapevine leafroll-associated virus
1, 2, 3, 5, 6, 7 – GLRaV-1, 2, 3, 5, 6, 7.
15 grapevines were found to be positive for
GLRaV-1, 9 for GVA, 8 for GFkV, 1 for GLRaV-2, 1 for
GLRaV-5, 5 for GLRaV-7, 2 for GLRaV-3, 2 for GFLV,
3 for ArMV, 2 for GVB, 2 for TBRV and 2 for SLRSV.
The presence of GLRaV-6, RpRSV-g and RpRSV-ch
was not proved. (Komínek P.)
The occurrence and spread of Plum pox virus
– PPV (PPV-M and PPV-D) strains, Prune
dwarf virus – PDV and Prunus necrotic ring
spot virus –PNRSV in natural sources
(MZe 0002700603)
The occurrence of PPV and two Ilarviruses PDV
and PNRSV was tested (by ELISA) in natural hosts of
these viruses including myrobalan, blackthorn
(Prunus spinosa), plum (P. domestica) and Prunus
avionum throughout the growing session (from
April to July) in the fruit-growing areas of Bohemia
and Moravia. For the identification of PPV and its
strains polyclonal and monoclonal antiserum
(commercial kits) was used. The presence of PPV
was not proved in the above-mentioned trees. A
total 23 trees of sweet cherry and sour cherry was
tested for the presence of PDV and PNRSV. In
addition Apple chlorotic leaf spot virus – ACLSV and
Cherry leaf roll virus – CLRV were also tested. PDV
was detected in two sweet cherry trees in Prostějov
(Olšany). The other viruses were not detected in
the Moravian region. ACLSV was not detected in
sweet cherry trees either in Central or Western
Bohemia. PNRSV was detected in a single sour
cherry tree from Starý Plzenec and an old sweet
cherry tree from Šťáhlavy in Western Bohemia.
CLRV was detected in a single sweet cherry tree
from Nezbavětice. The presence of PDV was not
proved in the tested sweet cherry and sour cherry
trees from Western Bohemia. (Polák J., Pívalová J.)
The identification and distribution of the
nematode genera Xiphinema and Longidorus
– vectors of Nepoviruses (MZe 0002700603)
A survey of the incidence of nematode genera
Xiphinema and Longidorus was conducted in soil
samples of the rhizosphere in fruit tree orchards
and vineyards from 37 localities of Southern
Moravia. The nematode genus Xiphinema was
identified in 24 localities including X. pachtaicum
(in 3 localities), X. simile (in 2 localities) and X.
vuittenezi (in all 24 localities). The genus
Longidorus was not detected in any of the abovementioned localities. A permanent slide of the
isolated
nematodes
was
prepared
for
morphological and morphometrical tests. The
identification of X. vuittenezi was verified by PCR.
A duplex-PCR was used for the detection of X.
vuittenezi and X. diversicaudatum. (Kumari S.)
The occurrence and distribution of Zucchini
yellow mosaic virus (ZYMV), Water melon
mosaic virus (WMV-2) and Cucumber mosaic
virus (CMV) in cucurbit vegetables
(MZe 0002700603)
During the vegetation period in 2004 leaf samples
with symptoms were collected from 10 localities in
Central Moravia and fruit samples from 5 localities in
Bohemia. A total of 98 samples was collected and
tested by DAS-ELISA for the presence of ZYMV,
WMV-2 and CMV. ZYMV was detected in 53 plants,
WMV-2 in 23 plants and CMV in 7 plants. These
viruses were found to be distributed equally in
samples collected in Moravia as well as in Bohemia.
The viruses were detected in samples (s) collected in
Central Moravia: 27 s ZYMV, 11 s CMV and 3 s WMV2 and in Bohemia: 26 s ZYMV, 12 s CMV and 4 s
WMV-2. The presence of the viruses was verified by
mechanical inoculation in indicator hosts and by
electron microscopy. (Svoboda J., Jokeš M.)
Evaluation of the source and status of
resistance to Plum pox virus in apricot and
peach cultivars (MZe 0002700603)
Three-year results of PPV resistance test in apricot
and peach conducted from 2002 to 2004 were
summarized (in the first two years 2002 & 2003
research was conducted under previous project
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MZe 01-01-03). Preliminary results showed the
apricot cultivar Harlayne as immune, Betinka as
resistant, and Velkopavlovická and Karola as
susceptible to PPV infection. Different PPV isolates
(both PPV belonging to serogroups PPV-M and PPVD) including PPV-D (original isolate from Dideron),
PPV-D (isolated from P. insititia L., CR), PPV-M
(isolated from apricot, CR), PPV-recombinant (PPVM x PPV-D, isolated from plum, CR) were used for
the resistance test. The leaves of cultivar ‘Harlayne’
remained symptomless during three years after the
inoculation of different strains of PPV. The fruits
and stones of cv. ‘Harlayne’ were also symptomless
(evaluation in 2004). PPV was not detected in leaf
samples of the plants of cv. ‘Harlayne’ either by
ELISA or by RT-PCR. Similarly, leaves and fruits of
cv. Betinka remained symptomless and virus-free
(detected by ELISA) within 3 years since PPV
inoculation. However, in 2004 a single plant out of
3 plants (cv. Betinka) inoculated with PPV-M
showed diffuse mosaic ‘Sharka’ symptoms. Similar
symptoms were also observed in fruits of this plant
and the presence of PPV was proved by ELISA.
(Polák J., Komínek P., Pívalová J.)
The resistance test was also conducted in cultivars
and interspecies hybrid including Barier (Prunus
davidiana x P. persica), Cadaman (P. davidiana x P.
persica), Pumiselect (P. pumila), MRS (P. cerasifera x
P. spinosa), NBS 540-73 (P. cerasifera x P. holoserica
x P. domestica), GF 677 (P. amygdalopersica) and
Fire (P. amygdalopersica) by grafting onto peach
infected by PPV. The Sharka symptoms were
evaluated in leaves. The relative concentration of the
virus was estimated by ELISA and the presence of
PPV was verified by immunosorbent electron
microscopy (ISEM). The cultivars and the hybrid
without Sharka symptoms were proved virus-free by
ELISA. These results were confirmed by RT-PCR
again. (Polák J., Pívalová J., Komínek P., Jokeš M.)
presence of twelve economically important viruses.
Only a few vines were found to be infected
with Grapevine virus A and Grapevine leafrollassociated virus 5. (Jandurová O., Komínek P.)
The investigation into possible occurrence of
new viruses of cereals and grasses (QD 1301)
A survey of the occurrence of cereal viruses was
conducted in different winter barley fields of the
CR that included Chomutov (5 fields), Rokycany
(4 fields), Plzeň (3 fields) and Benešov (1 field).
Barley yellow mosaic virus (BaYMV) and Barley
mild mosaic virus (BaMMV) were not detected in
the above field samples by DAS-ELISA (using
polyclonal antibodies) and leaf dip preparation of
electron microscopy (EM).
A filamentous virus was isolated from the tall oatgrass (Arhenatherrum elatius) displaying mosaic-like
symptoms (the plant containing a complex of
isometric and filamentous virus particles). The
filamentous virus was separated through passage by
mechanical inoculations to oat plants. A similar
filamentous virus was also found in yellow oat-grass
(Trisetum flavescens) showing mosaic symptoms. The
virus expressed an eyespot mosaic symptom in oat
plants (Fig.). The leaf dip preparation of EM showed
the virus particle length of 700 nm. The identity of
both filamentous viruses with the family Potyviridae
was confirmed by EM observation of a potyvirus-like
pinwheel inclusion in cytoplasm (in ultra-thin
section) and by RT-PCR using universal primers
(PV1/PV2). The DAS-ELISA with specific antibodies of
other cereal viruses of Potyviridae such as ONMV,
SCMV, MSMV and WSMV reacted negatively when the
above-mentioned filamentous virus infected oat plants
were used. (Širlová L., Hassan M., Jokeš M.)
Identification of resistance of Prunus
domestica cv. Jojo to Plum pox virus strains
(MZe 0002700603)
The trees of Prunus domestica L., cv. ‘Jojo’, were
graft-inoculated by 3 PPV strains: PPV-M, PPV-D and
PPV recombinant (PPV-M x PPV-D). Different
reactions to PPV infection were observed. One year
after inoculation with PPV-M and PPV-recombinant
a strong hypersensitive reaction was recorded, but
not all inoculated trees died. The virus was
transmitted from the cv. Jojo scion to rootstocks (St.
Julien). A systemic Sharka symptom was observed
in rootstocks where the Jojo scion died. The virus
was detected only in the leaf samples of rootstocks,
but not in the Jojo scion. In the tress of cv.
Jojo inoculated with PPV-D strain a partial
hypersensitive reaction was recorded. (Polák J.,
Pívalová J., Jokeš M.)
Sanitation of grapevine propagation material
(MZe 0002700603)
Maintenance breeding of the newly recognized
grapevine cultivar Auxerois started at Karlštejn
Research Station of Viticulture. The sanitary status
of propagation material of this cultivar was
evaluated by DAS-ELISA testing of vines for the
Filamentous virus naturally infecting yellow
oat-grass – eyespot mosaic symptoms on oat leaf
(on the right), healthy oat leaf (on the left)
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Determination of Wheat dwarf virus (WDV)
strains by PCR (QD 1301)
PCR assay was optimised for the identification of
barley and wheat strains of WDV. An expeditious
procedure was used for DNA extraction from
different WDV hosts (Poaceae) and leafhopper
(Psammotettix alienus) vectors. This procedure
consists in homogenisation of plants or leafhoppers
in liquid nitrogen, dilution in 1% Tris buffer, boiling
in the water bath (5 min.) and cooling on ice.
Before PCR the samples were centrifuged and the
supernatant was diluted 1:100 in distilled water
(plants) or without dilution (leafhoppers). The
procedure is equally suitable for DNA preparation
as a classical procedure described elsewhere
(Bendahmane et al., 1995). Different primers were
designed for the detection of WDV (HWDA/HVDU,
WD1A/WD1R, WD1A/WD2R, WD2A/WD2R and
WD3A/WD3R) and differentiation of WDV barley
strains (HWDA/HWDJ) and wheat strains
(WD4A/WD4R) for PCR. The PCR with the primer
pair WD1A/WD1R and WD1A/WD2R followed by an
RFLP assay with RSaI was also used for the specific
determination of WDV strains. (Širlová L.)
The incidence of PPV strains in natural hosts
(QC 1301)
The investigations of the occurrence of PPV in
natural hosts myrobalan and blackthorn (Prunus
spinosa) continued like in the previous year. More
than 150 samples were collected from different parts
of the Czech Republic (Louny, Budějovice, Vlašim,
Karlovary, Chomutov and Olomouc). PPV was
detected by DAS-ELISA (using polyclonal antibodies)
in 29 myrobalan and 3 blackthorn trees. The PPV
particles were found in these samples in leaf dip
preparations of electron microscopy. ELISA test with
monoclonal antibodies against PPV-M showed the
presence of this strain only in 5 myrobalan trees.
These results suggest that PPV in myrobalan and
blackthorn trees is either PPV-D strain or PPVrecombinant. (Polák J., Pívalová J., Jokeš M.)
A method for the detection of a recombinant
strain of Plum pox virus (PPV-Rec) (QC1301)
A Czech PPV isolate from plum was characterised
on a RNA level and typed as a newly recognized
recombinant strain of the virus (PPV-Rec), which
is probably a natural recombinant between
two common PPV strains PPV-M and PPV-D.
The recombination breakpoint is located in the
C-terminal part of the NIb gene of viral RNA. A
partial sequence of the Czech isolate of PPV from
plum encompassing the end of NIb and the
beginning of CP genes of the virus was obtained
(GenBank accession number AY324843). Similarly,
PPV isolate PPV-Horoměřice was proved as a
recombinant and sequences of different parts of
the genome of this isolate are also available in
GenBank under acc. no. AY324884 (from end of
NIb to beginning of CP gene), AY553377 (from end
of HC to beginning of P3 genes) and AY553373
(certain portion of HC gene). The genes spanning
NIb-CP of isolate PPV-Nectagrand, which belongs
to PPV-M strain, were also sequenced (GenBank
acc. no. AY324839).
A method of RT-PCR amplification with
subsequent restriction analysis was developed to
determine PPV strains including recombinant ones.
(Komínek P.)
Assessment of the level of resistance to Wheat
dwarf virus infection in selected species of
the genus Aegilops and winter wheat varieties
(QD 1350)
The level of resistance to early infection with
wheat strain of WDV was evaluated in nine selected
species of the genus Aegilops (A. columnaris, A.
cylindrica, A. geniculata, A. kobscleyi, A lorenbii, A.
neglecta, A. tauschii, A. triuncinalis, A. umbelluata)
and 27 winter wheat varieties (Apache, Banquet,
Batis, Bill, Clever, Complet, Contra, Corsaire,
Drifter, Hana, Ilona, Ludwig, Mladka, Nela, Niagara,
Record, Rheia, Rialto, Semper, Sepstra, Solara,
Sulamit, Svitava, Tower, Trend, Vlasta and Winsdor).
The evaluation was conducted (in a 2-year period)
according to symptom intensity, plant height
reduction, dry weight of shoot biomass, heading
reduction and grain yield losses (only in winter
wheat). All tested materials were highly sensitive to
WDV infection except A. tauschii. The sensitivity
was manifested by strong dwarfing (height
reduction in Aegilops from 39.91 to 67.91%, winter
wheat varieties from 45.55 to 95.97%), deformation
of younger leaves, colour changes, decrease in
shoot biomass weight (weight reduction in
Aegilops from 54.91 to 85.69%, winter wheat
varieties from 59.45 to 97.98%), heading failure
(heading reduction in winter wheat varieties from
9.01 to 100%), premature mortality and grain yield
reduction in winter wheat varieties from 87.3 to
100%. The summarisation of our results indicates A.
tauschii as moderately resistant and all other tested
species of Aegilops and winter wheat varieties as
susceptible (moderately susceptible, susceptible
and highly susceptible) to WDV. (Širlová L.)
Purification of Zucchini yellow mosaic virus
ZYMV (QD 1357)
The purification of a ZYMV isolate was carried
out and experimental animals were immunised in
order to prepare specific antibodies. The obtained
results were used to develop the “Methodology for
the purification of ZYMV intended for the
immunisation of experimental animals and the
preparation of specific antibodies against viral
strains isolated in the Czech Republic”. (Svoboda J., Polák J.)
Methodology for the protection of vegetables
from the family Cucurbitaceae against ZYMV
(QD 1357)
Winter-hardy weed species growing in the
vicinity of plots where species from the gourd
family were grown in the previous year were
collected in the spring season and analysed for
the presence of ZYMV by ELISA. ZYMV was
detected in the following three plant species:
Stellaria media (L.) Vill., Trifolium repens (L.) and
Tripleurospermum maritimum (L.) Sch. Bip. The
research on ZYMV epidemiology included the
testing to reveal whether the virus could be
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transferred by seeds of infected cucumbers,
zucchinis and gourds, contaminated soil, plant
residues, virus-infected plant sap and aphids. The
results obtained in this project served for the
development of the “Methodology for the
protection of vegetables from the gourd family
against ZYMV”. (Svoboda J., Polák J.)
Search for new invasive viruses infecting
vegetables from the family Cucurbitaceae in
the Czech Republic (QD 1357)
Samples of leaves from vegetables from the gourd
family collected at selected sites in southern
Moravia and Bohemia during the growing season in
2004 were tested for the presence of invasive
viruses such as PRSV, ZYFV, SqMV, MNSV, TSWV
and CGMMV, and to determine the distribution of
ZYMV, CMV and WMV-II using ELISA. It was found
out that ZYMV, CMV, and WMV-II occurred in
Southern Moravia and Central Bohemia (the Polabí
region). No other viruses were detected. (Svoboda J., Polák J.)
Propagation of Myrobalan latent ringspot
virus (MLRSV) and its purification (QC 1359)
MLRSV was propagated in Chenopodium quinoa
Willd. by mechanical transmission onto plants at the
6th leaf stage with 0.007M phosphate buffer (pH
8.6) containing 0.01M veronal, 0.01M cystein
hydrochloride and 0.007M EDTA. The virus was
purified by clarification with n-octanol and
differential centrifugation as previously modified for
SLRSV purification (Polák, 1991). This approach was
complemented by ultra-centrifugation on a sucrose
gradient and the virus fraction was collected. The
fraction was then ultra-centrifuged and the pellet
was re-suspended in 0.02M phosphate buffer
(pH 8.0). The suspension was dialysed in a
physiological solution. The virus suspension
prepared in this way was used for rabbit
immunisation. Intravenous and intramuscular
(adding complete and incomplete Freund adjuvant)
injections were applied. (Polák J., Chaloupková M.)
Mechanical transmission of MLRSV from
trees of Ishtara and Prunus persica cv. GF 305
to a herbaceous indicator; investigation of
the transmission of TBRV from C. quinoa to a
woody indicator; determination of SLRSV and
CLRV in stone-fruit trees by ELISA (QC 1359)
A reliable method of Myrobalan latent ringspot
virus (MLRSV) transmission was verified. MLRSV
was mechanically inoculated from Ishtara to a
herbaceous indicator, Chenopodium quinoa. The
inoculation buffer containing antioxidants proved
to be necessary for a successful transmission. The
Nepoviruses like Strawberry latent ringspot virus
(SLRSV) and Cherry leaf spot virus (CLRV) were
determined in stone-fruit trees by ELISA and
mechanical transmission to herbaceous indicators
(C. quinoa, C. amaranticolor). The transmission of
a Slovak isolate of Tomato black ring virus (TBRV)
to C. quinoa was unsuccessful. However, two Polish
isolates (L10/Cuc) of TBRV were successfully
transmitted to herbaceous hosts. The transmission
of TBRV to a woody indicator, Prunus persica cv.
GF 305, failed on our part. (Polák J., Chaloupková M., Jokeš M.)
Reliable procedure for the detection of Apple
chlorotic leafspot virus in apple cultivars and
rootstocks (QC 1359)
We summarised the results of Apple chlorotic
leafspot virus (ACLSV) detection in 124 cultivars
and 43 rootstocks of pear trees by DAS-ELISA (the
same procedure as it was used for ACLSV detection
in apple). ACLSV was detected in a few pear
cultivars but not in any of the tested rootstocks. The
results showed that the incidence of ACLSV in pear
was less frequent than in apple (previously detected
almost 100% in apple). (Polák J., Svoboda J.)
Reliable procedure for the detection of Apple
mosaic virus in apple cultivars and rootstocks
(QC 1359)
A total of 80 apple trees from different cultivars
(10 trees of each cv.) were tested for Apple mosaic
virus (ApMV) using DAS-ELISA. ApMV was detected
in all 10 trees of Kidd’s Orange and Starkrimson,
2 trees of MacIntosh, 3 trees of Spartan and a single
tree of Stark Earliest. The virus was not detected in
any of the tested trees of cvs. Melrose, Vista Bela
and Idared. The relative virus concentration was
evaluated according to the virus titre detected in
5 apple trees from 2 cultivars. Different apple
tissues from different periods of the year were
tested that included bark, buds, flowers and leaves.
Young leaves from buds (beginning of flowering, in
April) showed the best tissues for reliable detection
as they contained high virus concentrations.
(Polák J., Svoboda J.)
Symptom evaluation of Czech plum isolate of
PPV in transgenic plum Prunus domestica
(QD 1360)
The leaves of transgenic plum trees inoculated
with PPV (by grafting) were examined for symptom
evaluation in one-month intervals since May 2004.
The first ‘Sharka’ symptom, a diffuse mosaic,
appeared in June 2004 in 8 trees out of 10 tested.
The highest symptom severity in leaves was
recorded in June and July while it remained low for
the rest of the vegetation period. The symptom
severity was rapidly reduced in the transgenic part
of trees. No symptoms were observed in noninoculated transgenic trees. (Polák J., Pívalová J.)
The identification of Czech plum isolate of
PPV in transgenic plum Prunus domestica by
ELISA, ISEM and RT-PCR (QD 1360)
The presence of PPV-M in transgenic plum was
proved by DAS-ELISA in leaf samples of 8 trees out of
10 tested. The virus was not detected by ELISA in a
single tree where no PPV symptom was observed.
Two trees out of 11 planted were dead and the
presence of the virus of these trees was verified by RTPCR. The presence of PPV in transgenic plum leaves
was verified by immunosorbent electron microscopy
(ISEM) and 750 nm virus particles were detected.
(Polák J., Pívalová J., Komínek P., Jokeš M.)
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Evaluation of PPV, PDV and ACLSV symptoms
in leaves of transgenic plum (C-5), Prunus
domestica graft, inoculated with virus
combinations PPV-M+ACLSV, PPV-M+PDV and
PPV-M+PDV +ACLSV (QD 1360)
‘Sharka’ symptoms caused by PPV were observed
in all virus combinations in June. In the
combination
PPV-M+PDV
symptoms
were
observed in the transgenic part of all 10 tested
trees. No PDV symptoms were recorded in these
trees. In the combination PPV-M+PDV symptoms
were observed in the transgenic part of all 11 tested
trees. No ACLSV symptoms were recorded in these
trees. In the combination PPV-M+ACLSV+PDV
symptoms were also observed in the transgenic
part of all 11 tested trees. No ACLSV or PDV
symptoms were recorded in these trees. Strong PPV
symptoms were observed in non-transgenic
branches of the tested trees. (Polák J., Pívalová J.)
combination PPV-M+ACLSV+PDV. ACLSV was
detected in all tested trees; however PDV was
detected only in non-transgenic branches of the trees.
The PPV detection was verified by ISEM and RT-PCR
again. (Polák J., Komínek P., Pívalová J., Jokeš M.)
Assessment of the sanitary status of basic
materials of apple, pear, cherry, apricot and
peach cultivars for recovering (1B44051)
An RT-PCR assay was used for the detection of
ACLSV, ApMV, ASGV and ASPV in apple and pear
samples from RBIP Holovousy as described by
Menzel et al. (2002). ASPV was detected in apple
cultivars Idared and Šampion Red and pear
cultivars Alexander Lucas and Max Red Bartlet.
ACLSV was detected in apple cultivars Idared and
Šampion Red, and ASGV in Šampion Red. The
presence of ApMV and CLRV was not detected in
cherry cultivars Kordia and Karešova by ELISA. A
PCR based test was conducted for the detection of
phytoplasms using universal primers (P1/Tint) as
described by Smart et al. (1996) in apple, pear,
sweet cherry, apricot and peach trees. The
phytoplasms were not detected in any of the tested
trees. (Bryxiová M., Hassan M.)
PPV-infected buds of plum, apricot and peach
cultivars grafted onto virus-free rootstocks
and testing for the presence of PPV and
others pathogens (1B44051)
Four plum cultivars including Blue free, Švestka
domácí, Anna Späth and Hanita (from RBIP
Holovousy) were grafted onto virus-free rootstocks
St. Julien (169 trees in total).
Peach cultivars Sentry (16 trees) and Fairhaven
apricot cultivars Karola (12 trees, infected by PPV-M
and PPV-D) and Velkopavlovická and plum cultivar
Hanita were grafted onto virus-free rootstocks
(Maxma, Puebla and Barier). Plum cultivars Hanita,
Blue free, Švestka domácí were tested by ELISA for the
presence of PPV, PNRSV, PDV, ACLSV and CLRV. Trees
of apricot and peach were tested for the presence of
ApMV, ACLSV, PNRSV and PDV. In all tested samples
only PPV was detected. (Polák J., Chaloupková M.,
Blanco G., Bryxiová M., Hassan M.)
Diffuse rings, symptoms of PPV in leaves of P. domestica,
clone 5 infected with PPV-M+PDV+ACLSV
Identification of viruses in transgenic plum
(C-5), Prunus domestica graft, inoculated
with virus combinations PPV-M+ACLSV, PPVM+PDV and PPV-M+PDV+ACLSV (QD1360)
The presence of PPV-M was proved by ELISA in
leaf samples of all 10 tested transgenic plums
inoculated with PPV-M+PDV and verified by RTPCR. The PPV particles were confirmed by
immunosorbent electron microscopy (ISEM). PDV
was not detected in transgenic scions of the trees
by ELISA; however, the virus was detected in the
leaves of non-transgenic branches.
PPV-M and ACLSV were detected in all 11 tested
transgenic trees inoculated with PPV-M+ACLSV. The
PPV detection was verified by ISEM and RT-PCR.
The presence of PPV-M was proved again in 10
transgenic trees out of 11 tested by ELISA in the
Thermotherapy of plum, apricot and peach
cultivars infected by PPV (1B44051)
Thermotherapy was conducted in a growth
chamber with selected temperature, day 39.5° C and
night 37.5° C. Four trees of plum cultivars Švestka
domácí and Hanita were exposed to these conditions
during 21 and 24 days. In the case of one tree (Hanita)
the thermotherapy was interrupted because of
wilting. Another tree shed leaves after therapy, but
both trees were regenerated in a greenhouse. The
trees of cv. Švestka domácí survived better in the
same conditions. The apricot cv. Karola and peach cv.
Sentry were kept at gradually increasing
temperatures from 25° C to 38° C (duration of two
weeks). After thermotherapy growing tips were
collected and cultivated in an in vitro culture. All
samples were tested by ELISA for the presence of PPV
after thermotherapy and the virus was not detected.
However, the PPV test will be repeated in the spring
2005. (Polák J., Chaloupková M., Blanco G.)
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Establishment of in vitro cultures of plum,
apricot and peach trees infected by PPV
(1B44051)
During the vegetation period shoots were collected
from the plum cultivars Švestka domácí, Hanita, Blue
free, apricot cv. Karola and peach cv. Sentry, infected
by PPV. In vitro cultures were isolated from bud
segments and apical meristem tissues of source
plants. The plant material was disinfected with
different reagents. To overcome the infection of
endogenous bacteria the explants was disinfected in
a flow-box through infusion in ethanol and the
antibiotics (Streptomycin sulphate, 10mg/l) were
added to the medium. The medium MS (Murashige &
Skoog, 1962) and WPM (Lloyd & McCown, 1981) with
modifications of concentrations of vitamins,
phytohormones and sucrose were used. A new
laboratory of in vitro cultures and climatic chamber
were established. (Chaloupková M., Blanco G.)
Reaction to Barley yellow dwarf virus (BYDV)
infection in varieties and breeding lines of
(winter and spring) barley and wheat
(QE 1107; QE 1311)
Selected genotypes (153 winter barley, 95 winter
wheat, 67 spring barley and 62 spring wheat) were
tested in small-plot trials that involved planting and
infection of spring genotypes by viruliferous
aphids (Rhopalosiphum padi) and evaluation of
resistance of winter (infected last year) and spring
genotypes to BYDV-PAV. The reaction of the tested
genotypes to BYDV-PAV infection was evaluated
by symptom intensity (10-point scale; 0 –
symptomless, 9 – dead plant) in the flowering stage
of the non-infected variant. The harvest analysis
was aimed at yield reduction, number of grains per
ear, weight of grains per ear and thousand grain
weight in winter wheat varieties and breeding
lines. Susceptibility index was calculated. The
tested materials were attributed different levels of
susceptibility or resistance and the results were
given to users. The experiments were conducted in
cooperation with Division of Genetics and Plant
Breeding of RICP (Chrpová J., Šíp V.) and SELGEN
(P. Mařík, O. Veškrna). (Širlová L.)
Characterisation of closteroviruses
associated with grapevine leafroll disease in
the Czech Republic (GA ČR 522/01/D131)
Selected grapevine plants from Karlštejn Research
Station of Viticulture were tested for the presence of
Grapevine leafroll-associated virus 1 (GLRaV-1). The
virus was detected using DAS-ELISA, RT-PCR and
molecular hybridisation with non-radioactive RNA
probes. Both molecular methods were based on
detection of the GLRaV-1 heat-shock protein 70
(HSP70) gene and showed a higher sensitivity in the
detection of GLRaV-1 compared to DAS-ELISA. RNA
probes are considered more suitable for the GLRaV1 detection as their application can overcome
potential minor sequence variability, which may
cause the detection by RT-PCR to be less reliable,
especially when the variability occurs in the genome
region targeted by RT-PCR primers. Based on
additional DAS-ELISA, mixed infections of GLRaV-1
with other viruses (Grapevine leafroll-associated
virus 3, Grapevine virus A and Grapevine fleck virus)
were recorded frequently. (Komínek P.)
Comparison of relative Barley yellow dwarf
virus (BYDV-PAV) concentration in DH-lines
of spring barley, Igri/Atlas Yd2+ and Yd2genotypes (GA ČR 521/03/0137)
The relative virus concentration (RVC) of BYDVPAV was evaluated in resistant (gene Yd2+) and
susceptible genotypes (gene Yd2-) of spring barley
Igri/Atlas DH-lines. The experiment was conducted
(in 3 trails) in a growth chamber. The plants were
inoculated in the 1-2 leaf stage. The root samples
were collected in 0.5, 1, 3, 6, 9, 12, 15, 18, 21, 24, 27,
30, 33, 36, 39, 42 and 45 days after inoculation and
subsequently stored at –20° C. RVC was estimated by
TAS-ELISA. Significant differences between Yd2+ and
Yd2- were proved from 6 to 18 days after inoculation.
The average absorbance value was higher in
susceptible lines compared to resistant lines. The
highest difference in RVC between Yd2+ and Yd2genotypes was found at 9th day after inoculation.
The virus was detected earlier in susceptible lines,
which suggests a higher virus replication. (Širlová L.,
Chrpová J., Šíp V., Vojáčková M.)
Collection of plant viruses and homologous
antibodies (E-97/01-3160-0200)
The plant viruses (and virus strains) including
Apple stem pitting virus (2 isolates), Beet western
yellows virus, Potato leaf roll virus, Turnip mosaic
virus, Plum pox virus (4 isolates), Wheat dwarf
virus (wheat and barley strains), Brome mosaic
virus, Barley yellow dwarf virus and Wheat streak
mosaic virus were maintained on indicator hosts.
Reactivation of antigens dehydrated under calcium
chloride was accomplished with twelve viruses
that included Apple chlorotic leaf spot virus, Apple
stem grooving virus, Arabis mosaic virus, Bean
common mosaic virus, Cherry leaf roll virus,
Cucumber mosaic virus, Hop mosaic virus, Lettuce
mosaic virus, Myrobalan latent ring spot virus,
Strawberry latent ring spot virus, Turnip yellow
mosaic virus, Watermelon mosaic virus-2 and
Zucchini yellow mosaic virus. In 2004 the
collection was extended with Pepper mild mottle
virus. The collection consists of 28 plant viruses
Capsicum annuum, cv. Stalagnit F1, infected by Pepper mild
mottle virus
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48
and homologous antibodies in total. (Červená Z.,
Chaloupková M., Komínek P., Kumari S.,
Pívalová J., Polák J., Svoboda J., Širlová L.)
Reference laboratory for diagnostics of
selected quarantine plant viruses
The activities of reference laboratory in 2004
were focused on diagnosis of Plum pox virus. A
total of 36 plum samples was tested by ELISA. PPV
was not detected in all tested samples. PPV test was
also conducted in nursery plum materials for the
company Vino Blatel, a. s. PPV was detected in 11
samples (green bark) out of 34 tested samples
originating from Germany. The Sharka symptoms
were observed in most trees of cultivars (cv.)
Stanley and a few trees of cv. Čačanská lepotica and
Č. najbolaja. The leaf samples were tested for PPV
by ELISA. PPV was detected in 19 samples out of 20
tested in cv. Stanley and in all 3 samples of cv.
Čačanská lepotica and Č. Najbolaja. Proposed
eradication of infected nursery materials was one
of the basic results for Vino Blatel, a.s. (Polák J.)
Molecular Plant Pathogen Diagnostics
Laboratory
Molecular characterisation of Apple stem
pitting virus isolates from the Czech Republic
(QC 0048)
We carried out an analysis of coat protein (CP) gene
of 3 Czech isolates of Apple stem pitting virus (ASPV).
The isolates were collected in 3 different Bohemian
apple orchards and were denoted as CzAp9, CzAp2
and CzAp3. All of them were asymptomatic on their
hosts. The nucleotide sequence identity of nine
sequence variants and isolates including the Czech
ASPV isolates CzAp2 and CzAp3 and foreign ASPV
isolates GerD, BraAY and PolST132 ranged from
70.3% to 99%. The deduced amino acid number of
coat protein varied from 365 to 385 in the three
isolates and the six Czech variants. Their identities
ranged from 76.6 to 99.7% (Fig. 3). Nucleotide
distances were determined and estimated between
five variants obtained from the CzAp9 isolate. We
found considerable nucleotide distances between the
100
90
95
95
100
0.02
● CzAp36
● CzAp318
PolST132
BraAY572458
GerD21829
GerPVYV21828
PolST181
● CzAp39
▲ CzAp214
100
▲ CzAp211
99
63 ▲ CzAp29
Phylogenetic tree for the amino acid sequence of coat protein
in isolates and sequence variants of ASPV. Czech isolates
characterised in this study are denoted as ▲ (CzAp2) and ●
(CzAp3)
five variants. The nucleotide distance between
CzAp91 and CzAp94 was 0.1858, and between
CzAp95 and CzAp94 it was 0.1799. Only between the
variants CzAp92 and CzAp93 was there no significant
genetic distance. These data indicate that the variants
CzAp91 and CzAp94 might originate from a mixed
infection of two ASPV isolates with diverse sequence
variants. (Hassan M., Kundu J. K., Salava J.)
Analysis of Synchytrium endobioticum
ribosomal DNA sequences (QC 0048)
A region of about 1850 bp of ribosomal DNA was
sequenced from six pathotypes of Synchytrium
endobioticum from the Czech Republic and a
pathotype from Germany. This region consists of
about 1300 bp at the 3’end of the 18S rRNA gene, the
5.8S rRNA gene and two internal transcribed spacers
(ITS). The partial sequence of 18S rRNA gene
showed the greatest degree of homology to those of
Neocallimastix sp., Rhizophlyctis rosea and
Catenomyces sp., which confirms the classification
of S. endobioticum as a member of the phylum
Chytridiomycota. In the pathotypes the sequences
were found to be identical over the region studied.
The DNA sequences of both ITS may be used to
design primers for specific detection and
determination of S. endobioticum. (Salava J.)
Inheritance of resistance to Plum pox virus in
apricot (MZe 0002700603)
In order to determine the inheritance of resistance
to Plum pox virus (PPV) in apricot three crosses
between resistant and susceptible cultivars and
selections were performed. The B1 seedlings were
inoculated with the PPV-M strain by an infected bud.
PPV infection was evaluated over 4 consecutive
growth periods through visual symptoms, ELISA and
in some cases by reverse transcriptase PCR assays. Chisquare analysis of each B1 progeny was performed to
determine if the segregation ratio differed from the
expected ratio. PPV resistance segregated in three
apricot B1 progenies at a 1:7 (resistant:susceptible)
ratio, indicating that resistance was controlled by three
independent complementary genes. This knowledge
will help in planning apricot breeding programs.
(Salava J., Polák J., Pívalová J., Komínek P.)
Department of Bacteriology
Characterisation of plant pathogenic bacteria
associated with premature decline of apricot
trees in the Czech Republic (MZe 0002700603)
Sixty-two isolates of fluorescent pseudomonads
were isolated from infected apricot trees in tree
localities in southern Moravia. Out of the 54
fluorescent pseudomonads isolated, 94% were
identified as Pseudomonas syringae pv. syringae van
Hall (Pss) and 6% as Pseudomonas fluorescens
(Trevisan) Migula 1895 (Pf). None of the fluorescent
isolates belonged to Pseudomonas syringae pv.
morsprunorum (Wormald 1893) Young, Dye &
Wikie 1978 (Pmp). Out of the 25 isolates that
expressed ice nucleation activity, 88% were
classified to Pss and 12% to Pf. In a pathogenicity test
on immature cherry fruits, all Pss isolates were
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49
pathogenic and all Pf isolates were non-pathogenic.
Pss isolates showed slight differences in biochemical
characteristics (e.g. in utilisation of L-histidine,
psicose and D,L-carnitine and in gelatine
liquefaction) as well as in pathogenicity (according
to the size of necrosis in immature sweet cherry
trees) and ice nucleation activities. Some Pss isolates
nucleated ice at a temperature relatively as high as
–1° C, another isolates nucleated at a temperature
near to –5° C. (Krejzar V., Krejzarová R., Kůdela V.)
Possibility to detect non-fluid strains of
Clavibacter michiganensis subsp. sepedonicus
in potato tuber macerates (QC 0048)
Bacterial ring rot disease of potato, caused by
Clavibacter michiganensis subsp. sepedonicus
(Cms), is a major concern of producers of seed
potatoes in all countries of the world. Current
methods of detection of this pathogen primarily
rely upon the use of serological procedures
including an indirect fluorescent antibody staining
assay (IFAS) and enzyme-linked immunosorbent
assay (ELISA). Accurate identification of Cms is
especially difficult when population levels are
below 10,000 CFU per ml of plant tissue or millilitre
of plant sap. A further complication arises when
potato samples contain other bacteria that exhibit
cross-reactivity in IFAS and ELISA. Moreover, a
certain non-fluid variant of Cms that may occur in
various natural populations exhibits antigenic
variation and escapes serological detection.
In our tests, potato tubers from samples free of Cms
were used. The internal tissue was removed from
tubers and homogenized. Cms strains were added to
the homogenates to obtain final concentrations 104,
105, 106 and 107 CFU/ml. Bacterial extractions were
prepared from homogenates. Out of the 29 Cms
tested, 22 strains belonged to fluid type, 3 to
intermediate type and 4 to non-fluid type.
Commercial monoclonal antibody Agdia (USA)
against Cms was used for determination. Suitability of
the monoclonal antibody for detecting various types
of Cms strains was evaluated according to percentage
of positive reactions. Using 22 fluid Cms strains, the
percentages of positive reactions in potato tuber
homogenates at concentrations 104, 105, 106 and 107
CFU/ml were 95, 91, 86 and 86, respectively. On the
other hand, using 7 intermediate and non-fluid Cms
strains, the percentages of positive reactions in
potato tuber homogenates at concentrations 104, 105,
106 and 107 CFU/ml were 33-50, 33-50, 0-25, 0-50,
respectively. It is interesting that the percentage of
positive reactions at a respective Cms concentration
in phosphate buffered saline was lower in
comparison with Cms concentration in potato
extracts. (Krejzarová R., Krejzar V., Bryxiová M.)
Use of PCR for detection of Clavibacter
michiganensis subs. insidiosus in lucerne
seeds (QC 0048)
Bacterial
wilt,
caused
by
Clavibacter
michiganensis subs. insidiosus (Cmi), is a serious
disease of lucerne. The pathogen is of North
American origin and has spread to many other
countries. Cmi is an EPPO A2 quarantine pest, i.e. it
is the quarantine pest present in some areas within
the region. Seed transmission is virtually the only
way of introducing the pathogen to previously free
areas. A reliable and rapid method is needed in
practice to check seed lots for infection.
The objective of this study was to determine
whether a polymerase chain reaction (PCR) based
assay could be used to detect Cmi in lucerne seeds.
Samples of 0.2 g from a seed lot were prepared,
ground in a mortar and suspended with 5 ml culture
medium. One millilitre of sterile water (as a negative
control) and 1 ml of Cmi suspension at
concentrations of 108, 107, 106, 105, 104, 103 and 102 cfu
were added to each 5 ml seed macerate. The
macerates were shaken at room temperature for
24 h. Total genomic DNA from macerates was
isolated using NucleoSpin kit (Macherey-Nagel).
DNA primers were designed and used to detect Cmi
in isolated DNA. Cmi was detected reliably in seed
macerates which contained from 108 to 102 cfu. The
detection of Cmi in lucerne seed was improved
using the PCR method. (Bryxiová M., Kůdela V.)
Evaluation of resistance to fire blight in
Czech pear cultivars (QD 1053)
Newly bred Czech pear cultivars (14 cultivars)
were tested for resistance to fire blight. Tests were
carried out at Slany research station of RICP. Tested
cultivars were grown in technical isolation.
Artificial inoculations were carried out in the
period of strong extension growth. Inoculations
were done by decapitation of shoot tips with
scissors dipped in the bacterial suspension
(concentration 106 cfu). The bacterial suspension
was composed of 5 virulent strains of Erwinia
amylowora. The virulence of the pathogen was
verified by testing on forced shoots of Pyrus
ussuriensis. The level of resistance was determined
from the ratio (in %) of bacterial lesions to total
length of shoots (in cm) after 40 days. We used the
adjusted USDA scale for evaluation. The tests
proved high resistance to fire blight in cultivars
‘Bohemica’ (higher than in ‘Alexander Lucas’) and
‘Jana’. This resistance was comparable to newly
bred US-625-63-4. On the other hand, cultivars
‘Isolda’, ‘Vonka’, ‘Beta’, ‘Decora’ and ‘Petra’ showed
strong symptoms of infection. Growth and yield
characteristics of three cultivars (‘Red Bartlett’,
‘Conference’, ‘Alexander Lucas’) on rootstocks
resistant to fire blight were evaluated in an
experimental orchard at RBIP Holovousy. The
following rootstocks were tested: OHxF 69, 87, 230
and 333. Based on the present results, OHxF 87
rootstock appears to be the best for all three
cultivars concerning growth and yield. (Paprštein
F., Korba J., Kosina J., Šillerová J.)
Identification and characteristics of bacteria
accompanying populations of Clavibacter
michiganensis subsp. sepedonicus in potato
tuber and eggplant samples (QC 0048)
Clavibacter michiganensis subsp. sepedonicus
(Spieckermann & Kotthoff) Davis et al. (Cms),
causing ring rot of potato, is a quarantine organism.
Detection and diagnosis of Cms are carried out in
accordance with the requirements of EC Directive
No. 93/85/EEC based on indirect fluorescent
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50
antibody staining (IFAS) and a bioassay with eggplant
as an indicator plant. Further diagnostic methods
including immunochemical, biochemical and
molecular tests are frequently used to definitively
identify Cms. Epiphytic and endophytic bacteria
associating with Cms in potato samples could
complicate its detection. Extracts from potato tubers,
extracts from eggplants inoculated with extracts
from potato tubers and extracts from eggplants
inoculated with pure Cms cultures were screened. A
total of 170 non-Cms strains of bacteria were found at
populations of 103 – 105 cfu/ml per one tuber potato
or eggplant sample, ca. 2/3 gram-negative and ca. 1/3
gram-positive ones. The ratio of bacteria crossreacting with monoclonal antibodies against Cms
(Agdia, USA) in immunochemical methods was about
3%, both in DAS-ELISA and IFAS. In contrast, PCR test
proved to be very specific because no amplification
products were observed with the non-Cms strains.
Using the Microbial Identification System BIOLOG,
ca. 70% recovered bacterial strains were identified.
Among the gram-negative bacteria, Pseudomonas
fluorescens, Pantoea dispersa and Aureobacterium
saperdae were found the most frequently, while
among the gram-positive strains these were
Staphylococcus epidermidis, Staphylococcus pasteuri,
Curtobacterium albidum, Clavibacter michiganensis
and Bacillus sp. The potential plant-pathogenic
bacteria Agrobacterium tumefaciens, Pseudomonas
syringae and Xanthomonas vesicatoria were also
recovered. (Kokošková B., Klenová H.)
Reliability of diagnostic techniques for
detection of Clavibacter michiganensis
subsp. sepedonicus under specific conditions
(QC 0048)
Clavibacter michiganensis subsp. sepedonicus
(Cms), the causal agent of bacterial ring rot of potato,
was detected in potato tissue extracts and pure
bacterial cultures by means of the polymerase chain
reaction (PCR), indirect fluorescent antibody
staining (IFAS) and double antibody sandwich
enzyme-linked immunosorbent assay (DAS-ELISA).
Monoclonal antibodies of the company Agdia (USA)
were used for IFAS and DAS-ELISA and the primers
according to Mills et al. (1997) synthesised by the
company Biotech (CR) were used for PCR. The
identification of all tested Cms strains previously
confirmed a ring rot bacterium in the eggplant tests.
In assays of pure cultures, Cms was determined to
amount to 104 – 103 CFU/ml in PCR and IFAS reactions
and to 105 – 104 CFU/ml in DAS-ELISA reaction. In
assays of potato extracts kept at –20° C the presence
of Cms was detected only in 92% of samples. The
most sensitive IFAS determined Cms in extracts to
dilution 1:1000, by PCR to dilution 1:10 and by DASELISA to dilution 1:100. After fluctuations from
freezing to above-zero temperatures (–20 to +25° C),
the efficiency of Cms detection in potato tuber
samples decreased on average by 51% and 56% in
IFAS and PCR respectively, and even by 86% in DASELISA. Pastrik and Rainey (1999) primers seemed to
be of better quality than Mills et al. (1997) primers. In
all experiments, 10% of false positives were recorded
in DAS-ELISA while only 3% in PCR and IFAS.
(Kokošková B.)
Relationship between the virulence of strains
of Clavibacter michiganensis subsp.
sepedonicus and the intensity of metabolic
activity of several sugars based on Microbial
Identification System BIOLOG
Clavibacter michiganensis subsp. sepedonicus
(Spieckermann & Kotthoff) Davis et al (Cms)
causing ring rot of potato is subject to strict
quarantine in several European countries,
including the Czech Republic. The proof of the ring
rot bacterium in potato tuber samples is primarily
based on a bioassay using eggplants (cv. Black
Beauty). Differences in the virulence of Cms strains
are evident in the eggplant bioassay. Cms strains
with fluidal and non-fluidal colony morphology
were tested using the eggplant bioassay and the
Microbial
Identification
System
BIOLOG.
Monomers including galactose, glucose, mannose,
and ribose are known to be highly involved in the
production of extracellular polysaccharides (EPS).
A higher metabolic activity of these particular
monomers may be associated with increased
virulence of specific Cms strains. We found out that
a majority of Cms strains that were highly virulent
in the eggplant bioassay also showed a higher
metabolic activity of sugars EPS than less virulent
Cms strains. However, the correlation was not
universal for all tested strains. (Kokošková B.)
Bacteria associated fire blight symptoms
could complicate detection of Erwinia
amylovora (MZe 0002700603)
A total of 20 putative Erwinia amylovora-like
(Ea-like) isolates originating from 11 samples of
host plants with symptoms of fire blight were
analysed. Commercial polyclonal antibodies
(Loewe Co., Germany and Adgen Co., Scotland)
were used in immunochemical tests. Fourteen
strains reacted negatively in all tests. Six strains
reacted positively with a polyclonal antibody for
PTA-ELISA (plate-trapped antigen – enzyme linked
immunosorbent assay) at a concentration
corresponding to A620 = 0.1 while the results were
negative at A620 readings of 0.01 and 0.001. Five
strains reacted positively with a polyclonal
antibody for an indirect IF (immunofluorescent)
test at all tested concentrations (A620 = 0.1, 0.01 and
0.001). Three of these strains were positive in a PCR
test with primers AMSbL and AMSbR designed for
detection of E. amylovora. In hypersensitivity tests
in tobacco, all putative Ea-like isolates were
negative while the known reference strain of E.
amylovora exhibited a typical HR reaction. After
modification of PCR protocol, three putative Ealike isolates reacted as negatives. Our PCR test was
optimised by finding an optimal annealing
temperature and time for primers. Using the
microbial identification system BIOLOG the three
strains were identified as Pantoea dispersa (one
strain) and Pantoea agglomerans (two strains). The
identified strains were white variants of the species
P. dispersa and P. agglomerans that occur less
frequently than yellow variants. Since there were
positive reactions in our immunochemical tests,
these strains could cause false positives in a routine
screening of plant samples. (Kokošková B., Mráz I.)
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Collection of plant pathogenic bacteria and
antibodies
The collection of plant pathogenic bacteria and
antibodies contains about 1 467 items out of which
approximately 90% belong to plant pathogenic
bacteria and 10% to saprophytic ones. Most bacterial
strains belong to the genera Agrobacterium
(=Rhizobium), Erwinia, Clavibacter, Curtobacterium,
Pantoea, Pseudomonas, Ralstonia, Xanthomonas
and Xylella. Bacterial strains are kept in microbanks
at –70° C and/or as lyophylisates. In 2004 the
collection was enlarged, mostly by accessions of
strains of Clavibacter michiganensis subsp.
insidiosus, C. m. subsp. michiganensis, Pseudomonas
syringae pv. syringae and others and of reference
strains of some plant pathogenic bacteria.
The collection also comprises about 90
polyclonal antibodies from our production and
41 commercial antibodies (Agdia, Adgen, Löewe)
for detection of plant pathogenic bacteria. (Krejzarová R., Pánková I., Krejzar V.)
Reference laboratory for diagnostics and
monitoring of plant pathogenic quarantine
bacteria
For the needs of State Phytosanitary Authority
(SPA) and other subjects, diagnostic analyses of plant
samples suspicious of the presence of quarantine
and other important bacteria were conducted. For
the Central Institute for Supervising and Testing
in Agriculture (CISTA) Bratislava, Slovakia,
bacteriological analyses of plant samples suspicious
of the infection with Erwinia amylovora (Ea), the
causal agent of fire blight, were carried out. In the
framework of an optimised procedure for detection
of Ea, some diagnostic methods were compared.
Using the microbial identification system BIOLOG,
Ea isolates were identified very reliably as fire blight
bacterium. In immunochemical tests (ELISA, IFA), a
high percentage of false positives occurred. The
antibodies of Adgen and Loewe Companies that
were used were not of the first-rate quality. A
polyclonal antibody for determination of Ea was
released for SPA in the CR and for CISTA in Slovakia.
Reference strains of the ring rot and brown rot
pathogens used as standards in diagnostic tests were
released for the needs of SPA. As SPA required, the
ring tests focused on diagnostics of quarantine
bacteria of potato by IFA techniques in accordance
with EU directives were organised. As usual, the staff
of Dept. of Bacteriology gave lectures and practicals
for undergraduate and postgraduate students, plant
pathologists from research institutes and SPA.
(Kokošková B.)
Department of Mycology
The occurrence of cereal fungal diseases
(MZe 000270603)
Natural infestation of the fungi – the genus
Fusarium on cereals (wheat and barley) was very
weak in 2004. Like in previous years Fusarium
graminearum was the most frequent species. We
did not find out any new sources of resistance. The
samples of brown leaf spotting of wheat were
1
2
3
Symptoms of wheat leaf spot diseases caused by Pyrenophora
tritici-repentis (1), Phaeosphaeria nodorum (2), Mycosphaerella
graminicola (3)
obtained in 2004 from 12 districts and 15 localities
of the Czech Republic. Pyrenophora tritici-repentis
was present in 40% of samples of cereals,
Phaeoseptoria nodorum was isolated from 29% of
samples, Mycosphaerella graminicola from 13%
and Cochliobolus sativus from 9% of samples. From
these collections we prepared 38 monosporic
isolates of Pyrenophora tritici-repentis which are
deposited in our collection of fungi for subsequent
testing. (Šárová J., Hýsek J., Brožová J., Sychrová E.)
Study of the aetiology of charcoal disease of
sunflower (MZe 000270603)
We found out diseased plants after flowering and
the most frequently at the beginning of ripening.
The occurrence of this disease was searched in all
areas of sunflower cultivation in the Czech
Republic. Diseased plants had smaller flowers and
ill-developed seeds. This disease can be divided into
3 groups: a) bad development of root system,
b) infestation of plants with the fungus
Macrophomina phaseolina – charcoal disease of
sunflower, c) another non-specific cause. The
highest incidence of charcoal disease of sunflower
caused by Macrophomina phaseolina was
influenced by extremely hot and dry weather at the
end of summer and at the beginning of autumn.
The incidence of the disease was high not only in
the Louny area like in previous years but also in the
south of Moravia. Inoculation tests in the field
showed large differences in the resistance of
sunflower hybrids. The results must be confirmed
in subsequent trials. (Šárová J., Veverka K.,
Kudlíková I.)
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52
Control of the occurrence of pathogenic,
potentially pathogenic and saprotrophic
fungi on fruit tree species and on small fruit
shrubs (MZe 000270603)
In the last year we studied the mycobiota (fruit
tree species) of leaves, trunks and roots and the
mycobiota of pear branches. We found out
different composition on the above-mentioned
organs. Dominant species were Alternaria
alternata and Aureobasidium pullulans. These
dominant species belong to the organisms which
occurred in plant parts above the ground. The
occurrence of the genera Seimatosporium and
Geniculosporium was very interesting. In the
tissues of pear branches we found out 7 species of
microscopic fungi – the species Seimatosporium
cf. pestalotioides occurred most frequently. At
present we are studying the mycoflora of
grapevine. Endophytic fungi were isolated from
44% of segments. Microscopic fungi Alternaria
alternata, Aureobasidium pullulans, Phoma spp.,
Cladosporium cladosporoides and C. herbarum
prevailed in all tissues. (Novotný D.)
Coryneum sp. – dematiaceous conidia isolated from apple
branches
Epicoccum nigrum – conidia isolated from an apple leaf
Study of the resistance of selected
agricultural crops to economically
important fungal species in the year 2004
(MZe 000270603)
By the method of RAPD (Random Amplified
Polymorphic DNA) we continually analysed a
number of pathotypes of oat rust in selected
economically important crops (cereals originating
from various regions of Europe and Middle East –
Belorussia, Czech Republic, Israel, Austria, Serbia
and Monte Negro, Sweden). The comparison of P.
coronata f.sp. avenae and P. graminis f.sp. avenae
showed molecular differences. We proposed that
for the evaluation of differences between particular
pathotypes of oat rust on the molecular level it will
be possible to establish the tests with other
accidental primers or to use other molecular
methods (AFLP). For the genetic study of selected
donors of resistance to oat rust (Pc 50-2, Pc 50-4,
Avena sterilis VIR 343-1, Avena sterilis VIR 343-2)
we already multiplied the generations F0 and F1 for
the study of resistance. (Klenová H.)
Foundation of the collection of studied
pathogens (species of the genus
Macrophomina, Phytophthora, Verticillium,
Colletotrichum and others) isolated from
host plants and their characterisation,
preparation of antigens and antibodies to
detect these pathogens (MZe 000270603)
The temperature growth optimum of Macrophomina phaseolina strains obtained from various
localities was evaluated. Most strains had their
growth optimum at 30° C, some strains showed the
optimum at 35° C. The hypothesis of temperature
adaptation of the strains coming from colder
localities was not proved. To prepare antigens we
selected three strains with rich production of mycelia
and microsclerotia at 30° C. After eight days of
cultivation on a liquid medium, mycelia mass was
harvested and antigens were extracted in the form of
protein fraction. Antigens were used for
immunisation of laboratory rabbits.
The titre of the obtained sera prepared against
Macrophomina phaseolina amounted to 1:6 400 and
1:12 800 IgG. From the sera we isolated protein A by
precipitation with ammonium sulphate and by
subsequent immunoaffinity chromatography. The
specificity of antibodies was examined for possible
cross-reactions against other soil-borne fungal
pathogens of sunflower (F. oxysporum, F. solani,
Verticillium albo-atrum and Sclerotinia sclerotiorum).
We found out that after purification IgG sensitivity
decreased substantially (titre 1:3 200) and we
observed strong cross-reactions against Verticillium
albo-atrum (for the antigen concentration 1µg/ml).
(Šárová J., Kudlíková I., Chalupníková J., Kutíková M.)
Collection of isolates of the pathogen
Plasmodiophora brassicae, antigen
preparation and immunisation of
experimental animals (MZe 000270603)
As the first step in the preparation of specific
polyclonal antibodies for detection of Plasmodiophora brassicae we obtained purified resting
spores of the pathogen. Resting spores were
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53
isolated from root galls of Chinese cabbage cv.
Granaat by centrifugation on saccharose gradients.
A
suspension
(6x107/ml)
was
used
for
immunisation of laboratory rabbits. IgGs were
isolated from sera by precipitation with ammonium
sulphate
and
subsequent
immunoaffinity
chromatography on protein A. The specificity of
antibodies was examined for possible crossreactions against other soil-born fungal pathogens
of Brassicacae vegetables (Alternaria sp. div.,
Botrytis
cinerea,
Fusarium
oxysporum,
Verticillium albo-atrum). We did not observe any
significant cross-reactions against the mentioned
pathogens. (Kudlíková I., Chalupníková J.)
Collection of isolates of the pathogenic
genera Phytophthora, Verticillium and
Colletotrichum, preparation of antigens and
immunisation of experimental animals
(MZe 000270603)
In 2004 we focused our research on the
preparation of antigens from economically
important pathogens of apple trees and strawberry.
We used isolates obtained in the last years
(Phytophthora cinnamomi) and isolates received
from internal and international collections of
micromycetes
(Colletotrichum
acutatum,
Phytophthora cactorum, Verticillium albo-atrum).
Pathogenicity of these selected isolates was verified
on susceptible cultivars of young apple trees and
strawberry. Antigens in the form of protein fractions
were extracted from mycelial mass after cultivation
in a stationary culture on liquid media. Purified
protein antigens were used as immunogens.
Laboratory rabbits (Chinchila grande) were
immunised in three-week intervals by four or five
subcutaneous injections of immunisation dose with
increasing content of the mite proteins (50, 100, 100,
250 and 500 µg of proteins per dose). (Kudlíková I.,
Novotný D., Chalupníková J.)
Assessment of the influence of
biopreparations on the soil mycoflora with
special regard to phytopathogenic fungi and
the ways of management (MZe 000270603)
We observed a qualitative and quantitative
increase in the species of soil mycoflora and on the
surface of spring barley plants. After the spraying
of spring barley (the varieties Akcent and Tolar)
with biopreparations no differences either in
the spectrum of soil micromycetes or in
phytopathogenic species of the genus Fusarium
appeared. The influence on soil microorganisms was
totally negative. The occurrence of phytopathogenic
fungi
from
the
genera
Drechslera
and
Rhynchosporium caused spotting and stripe on the
surface of spring barley leaves; there was a difference
of about 15% (cultivation test) in comparison with
untreated control. When the biopreparations
(Supresivit, Polyversum, Ibefungin) were used as
seed treatment in spring barley, after two months
since the application we found out that this process
led to the lowering of the fungi of the genus
Fusarium and the prevailing saprotrophic genera
Penicillium and Acremonium. Lower occurrence of
pathogenic fungi led to a reduction in leaf fungal
diseases (septorioses, helminthosporioses) – about
15% in comparison with the control.
When the biopreparations were used in a mixture
with mineral fertilizer, the lowering of the fungi of
the genus Fusarium was lower than in the case of
the application of biopreparations as seed
treatment. The occurrence of phytopathogenic
fungi (Septoria, Drechslera, Rhynchosporium) on
the leaves was about 15% lower in comparison with
the control. (Hýsek J., Brožová J., Vach M.)
The influence of biopreparations on
decreasing the infestation with fungal
pathogens (MZe 000270603)
When the biopreparations (Polyversum, Ibefungin
and Supresivit) were used in a mixture with mineral
fertilizer (ammonium saltpetre with limestone ASL)
for winter wheat (cultivar EBI) and after
conventional ploughing, a high lowering of the genus
Fusarium (the number of colonies decreased
approximately by about 20-30%) occurred especially
before the use of Supresivit. The occurrence of
phytopathogenic fungi of the genera Drechslera and
Septoria on leaves (leaf spot) was about 15% lower in
comparison with untreated control. In the variants
with ploughed-in straw a higher influence on the
lowering of the genus Fusarium was found out after
the application of biopreparations (Supresivit and
Polyversum) with mineral fertilizers ASL. A higher
influence on the genus Fusarium (the lowering
about 20% and more) was found out before the
application of the biopreparations like seed
treatment especially before the use of Supresivit.
The spectrum of phytopathogenic species of the
genera Fusarium and Drechslera was limited.
The yield increased consequently about 3-5%. The
biopreparations influenced the balance of the
populations of phytopathogenic and saprotrophic
fungi in the soil, their influence on the seed was
very weak because the application was distant
from the yield. (Brožová J., Hýsek J., Sychrová E.,
Vach M.)
The influence of biopreparations on winter wheat yield before
conventional ploughing in 2004
LAV = ASL
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Hop plants – left control, right diseased plant, cultivar Sládek,
infected with Verticillium albo-atrum
Phytophthora spp.) on the branches of apples.
Dominant species on healthy branches were
Pleurophoma
cava,
Alternaria
alternata,
Aureobasidium pullulans, Seimatosporium cf.
pestalotioides, Phomopsis cf. mali, coelomycete sp.
1 and Coniothyrium cf. olivaceum. On the
branches with necrotic spots five fungal species
were
observed:
Phaeoacremonium
similar
anamorph and basidiomycete sp. 1 were isolated
most frequently. We prepared the antigens in the
form of total protein extract from mycelial matter
of P. malicicornis and P. alba. Antigens were
prepared from international collections. From
selected strains of Phytophthora cinnamoni, P.
cambivora with verified pathogenicity (on young
apples cv. Spartan) antigens and first antisera were
prepared. Antigens and antisera were also prepared
from the species Pseudomonas syringae.
(Novotný D., Kudlíková I., Šárová J.)
The effect of the quarantine fungus
Verticillium albo-atrum on different varieties
of Czech hop (1G 46060)
Hop varieties and new breeding lines cultivated
from meristem cultures (Bor, Sládek, Premiant, OK
31, OK 72 and OK 114) were artificially infected in a
quarantine glasshouse with the quarantine fungus
Verticillium albo-atrum (German strain) on the
roots at a concentration of 1 million conidia per 1 ml.
The infection had only very weak effects on these
varieties. Some necroses appeared on the roots. The
fungus occurred on the roots and on the root collar
only in the varieties Bor and Sládek. The
biopreparation Supresivit suppressed the given
fungus sufficiently in the soil. Among the chemical
compounds Imazalil was the most effective for the
fungus suppression in the soil. (Hýsek J., Brožová J.)
Decrease in qualitative and quantitative
losses after treatment and storage of food
grains in an agricultural enterprise.
(Study of the mycoflora on grains after
different methods of treatment) (QD 1201)
The evaluation of the spectra of microscopic
fungi on the seeds of different plants (pea, maize,
barley, wheat) was carried out before storage under
different conditions. Protective atmosphere with
carbon oxide appeared to be very efficient and it
depressed toxicogenic fungi of the genus
Fusarium also in wet conditions. It seems that this
method might be very useful for the protection of
seeds in storage conditions. (Hýsek J., Brožová J.)
The ecology, identification and diagnostics of
selected fungal pathogens of fruits from the
aspect of requirements of the European
Union (QF 4074)
The occurrence and ecology of the fungi
Glomerella cingulata, Pezizula malicorticis, P. alba
and Phytophthora spp. were examined on the
branches of apples and pears in the Czech Republic
before the optimisation of immunodiagnostic
methods for detection of introduced fungi. We
investigated the populations of healthy and
diseased (with necrotic spots) branches of apples
and the frequency of introduced fungi (excluding
Phomopsis sp. from the sample of apple branches
Pleurophoma cava – colonies isolated from an apple branch
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55
Detection methods of Phytophthora species
on apple trees (QC 1359)
In 2004 development of detection and
determination methods of the genus Phytophthora
in host plants continued. We carried out artificial
inoculation of five apple cultivars (Spartan, Golden
Delicious, MacIntosh, Idared, Zvonkové) by three
isolates of the genus Phytophthora. Tissue samples
were collected two months after inoculation. To
prove the presence of the pathogens in plant tissue
we used immunochemical methods (PTA-ELISA,
dot-blot on nitrocellulose membrane). Although
samples of bark showed only weak symptoms,
immunochemical tests were positive for all used
Phytophthora isolates. The use of sentinel baiting
plants (Fragaria vesca cv. Alpina, seedlings of apple
cv. Spartan) for the detection of pathogens was
assessed. For the detection of P. cactorum, Fragaria
vesca was evaluated as the most suitable and
seedlings of apple cv. Spartan were also satisfactory.
For the detection of P. cryptogea seedlings cv.
Spartan were effective. But for reliable detections
of these pathogens of the genus Phytophthora,
the application of two different methods is recommended. (Kudlíková I., Krátká J., Dunaiová J.)
Diagnostics of phytopathogenic fungi
causing economically important and
quarantine plant diseases (QC 1301)
Assessment of potato leaf resistance to
Phytophthora infestans on the basis of the
quantification of pathogen proteins in tissue was
carried out. Leaf discs (2.5 cm in diameter) of ten
potato cultivars (Vera, Cordoba, Lenka, Adéla,
Impala, Filea, Liseta, Arnika, Rosella, and Désirée)
were cut and inoculated on the abaxial side by
conidia suspension (1x104 CFU) of three strains of
Phytophthora infestans. Discs were placed on the
surface of sterile distilled water in Petri dishes and
cultivated at 17° C. Leaf discs were removed for
evaluation 2 to 7 days after inoculation.
The prepared polyclonal anti-P. infestans IgG K57
was applied for the pathogen detection in artificially
inoculated leaves. PTA-ELISA was used to prove the
presence of the pathogen and to quantify its proteins
in plant tissue. The level of resistance was
determined for each cultivar on the basis of the
calculated total amount of pathogen proteins in all
tested leaves and of the number of leaves assessed as
positive. Cultivars Arnika, Rosella and Désirée
appeared as the most susceptible according to both
criteria. Likewise, cultivar Lenka was assessed as the
most resistant according to both criteria. On the
basis of the amount of pathogen proteins, a
continuous range of leaf resistance in the tested
cultivars was observed from the most resistant Lenka
(5.3 µg of proteins/ml) to the most susceptible
Arnika (38.8 µg of proteins/ml). (Kudlíková I.,
Krátká J., Chalupníková J., Kutíková M.)
Application of immunochemical methods
to detect Plasmodiophora brassicae in soil
(QD 1357)
In 2004 the effect of antagonistic plants on the
amount of resting spores of Plasmodiophora
brassicae was proved in artificially infested soil.
Symptoms on an apple tree cv. Gold Spur after inoculation
with P. cinnamoni
We used the following species: buckwheat
(Fagopyrum esculentum cv. Prego), mint (Mentha
piperita cv. Variegata), thyme (Thymus vulgaris cv.
Compactus), and English caper (Tropaeolum
majus). These antagonistic plants were grown in
the artificially inoculated soil with different levels
of infestation. After three months soil samples were
collected and evaluated by immunochemical and
biological tests. Indirect immunofluorescence did
not reveal any significant differences in the number
of resting spores between control and artificially
inoculated soil samples. On the basis of a biological
test (assessed number and size of root galls of
Leaf disc of potato cv. Adéla seven days after inoculation with
P. infestans
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Brassica pekinensis) we observed a moderate
decrease of disease variability for the experimental
variant: infestation 1x105 resting spores/g of soil,
antagonistic plant mint. For the other antagonistic
plants we did not find out any significant
differences in disease variability in Brassica
pekinensis. (Kudlíková I., Kutíková M., Dunaiová J.)
Complex methodology to monitor and detect
pest infestation of stored cereals by means of
physico-chemical, immunochemical and
molecular methods (QF 4071)
The aim of our work for the year 2004 was to
prepare antigens from Acarus siro and Tribolium
castaneum and subsequently to raise specific
polyclonal antibodies for the detection of these
stored product pests. After surface sterilisation,
10 grams of the mites and larvae were homogenised
in an extraction buffer. The homogenates were
centrifuged and proteins from the supernatant were
precipitated by a saturated solution of ammonium
sulphate. Purified protein antigens isolated from
Acarus siro and Tribolium castaneum were used as
immunogens. Laboratory rabbits (Chinchila grande)
were immunised in three-week intervals by four or
five subcutaneous injections of immunisation dose
with increasing content of proteins. IgGs were
isolated by precipitation with ammonium sulphate
and subsequent immunoaffinity chromatography on
protein A. The specificity and sensitivity of the
obtained antibodies were evaluated by PTA-ELISA on
microtitration plates and dot-blot. Antibodies against
A. siro showed the detection threshold lower than
0.1 µg of A. siro proteins/ml. We did not observe any
significant cross-reactivity with other stored product
mites (Aleuroglyphus ovatus, Caloglyphus sp.,
Tyrophagus putrescentiae) and insect species
(Tribolium castaneum, Sitophilus granarius and
Ephestia kuehniella) frequently found in Czech grain
and flour stores. The in-vitro detection threshold for
two anti-T. castaneum was determined as 0.05 µg and
0.1 µg of T. castaneum proteins/ml. No crossreactions against other stored product insects and
mites were observed. (Kudlíková I., Chalupníková J.)
Development and utilisation of effective
methods for selection of genetic resources
and production of initial breeding materials
of Brassica vegetables with desired traits
(QD 1356)
In 2004 we applied the prepared polyclonal
antibodies against resting spores of Plasmodiophora
brassicae to probe artificially and naturally infected
soil by indirect immunofluorescence. Soil samples
(10 g) were processed by sieving through two sieves
(with meshes of 1mm and 45µm) and settling on a
2M saccharose layer. The pellet remaining on the
saccharose layer was evaluated by an indirect
immunofluorescence technique (fluorochrome
FITC). Selected anti-P. brassicae quantified the
number of resting spores in soil at a concentration
104 and 105 with sufficient precision (about 70%). We
obtained a linear model estimating P. brassicae-K38
that showed a high specificity to cross-reactions
against other soil-borne fungi up to 10% that are
considered being negligible. The described method
enabled to determine the dependence of the number
of recovered resting spores on the number of resting
spores originally added into soil. The linear regression
model was significant (F = 160.95, p < 0.001) and
explained about 81% of total variability. For naturally
infected soil samples we obtained a wide range of the
infestation level from 2.6x102 to 1.3x105 resting
spores/g of soil. (Kudlíková I., Dunaiová J.)
Screening of digestive enzymes of mites as
novel candidates for protein microarrays
(COST P1OC853.003)
In the first year of the project we focused on the
following aims: screening of digestive enzymes
(amylase, lysozyme, cysteine and serine proteinase)
in whole-body homogenates and faeces of tested
mite species by biochemical methods; optimising of
immunochemical methods based on ELISA system to
detect antigens (digestive enzymes) of Acarus siro in
its excrements and infested grain kernels. Amylase,
cysteine and serine proteinase were detected
in excrements and whole-body homogenates of
all model species (Acarus siro, Tyrophagus
putrescentiae, Lepidoglyphus destructor and
Dermatophagoides pteronyssinus) at different rates.
Prepared polyclonal antibodies were used to detect
and quantify digestive enzymes in A. siro excrements
by PTA-ELISA. The presence of digestive enzymes
was assessed in a range of purified excrements of
0.5-10µg/ml of an extraction buffer. The used
technique reached the detection limit with the value
1-2µg of excrements/ml. We obtained a linear model
estimating the dependence of absorbance on the
weight of excrements: abs = 0.03 x weight (µg/ml) +
0.06. Detection of digestive enzymes was carried out
in artificially infested grain kernel samples (with the
known amount of excrements). PTA-ELISA enabled
to detect excrements from 1µg of excrements/25g
of kernels. Reliable detection limit had the value
6.8µg of excrements/100g of kernels. (Kudlíková I.,
Hubert J., Chalupníková J.)
Diagnostics and detection of phytopathogenic
fungi
In 2004 the analyses of 300 samples of spring and
winter barley were carried out to detect the
occurrence of the fungus Ramularia collo-cygni.
The method of the creation of colour compounds
after the placing of the last leaves on water agar and
after artificial shining was used. But this fungus was
not detected this year contrary to the year 2003. It
was probably due to a long-term dry weather.
(Hýsek J., Brožová J.)
At the State Phytosanitary Authority’s request an
analysis of wilted Dianthus plants of different
cultivars was carried out. After cultivation in wet
chambers we isolated filamentous micromycetes
on common isolation media. The putative causal
pathogen Phialophora cinerescens was not found
in any of the analysed samples. We isolated and
determined several species of the genus Fusarium
that are considered as saprotrophic or facultative
pathogens. From three samples we isolated a dark
filamentous fungus forming the setous coeloma
that was determined as Colletotrium cf. dematium.
(Novotný D., Kudlíková I.)
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Reference laboratory for diagnostics and
monitoring of phytopathogenic quarantine
fungi
At a grower’s request we analysed strawberry plants
on the suspicion of infestation by the quarantine
pathogen Phytophthora fragariae. Samples were
probed by immunochemical (ELISA) and biological
tests. By means of ELISA we proved the presence of
the genus Phytophthora in plant tissues. On the basis
of a biological test we excluded the presence of the
quarantine pathogen P. fragariae in all samples.
According to morphological traits of oospores,
frequently found in roots of sensitive baiting plants,
the pathogen was determined as P. cactorum.
(Hýsek J., Kudlíková I., Chalupníková J.)
Department of Entomology
Spiders (Araneae) in the biological and
integrated pest management of apple in the
Czech Republic (MZe 0002700603)
Biological and integrated systems were compared
with respect to the control of the major pest, codling
moth (Cydia pomonella) in apple orchards. The aim
of the study was to assess the effect of these two
systems on arboreal spiders. The biological system
was based on the use of biological preparations,
whereas in the integrated system selective pesticides
were employed. The control plot had no pesticide
treatment. The abundance of spiders was similar on
all studied plots during three years of study. Diversity
was higher on the biological plot than on the control
and the integrated plots, suggesting that the
response of spiders to management was guild
specific. Four spider families dominated on all plots:
Araneidae (orb weavers), Theridiidae (space-web
weavers), and Philodromidae and Thomisidae
(ambushers). While Araneidae and Thomisidae
were similarly abundant on all plots, the density of
Theridiidae and Philodromidae differed. On the
integrated plot there were significantly more
theridiid spiders, whereas on the control plot
philodromid spiders were significantly more
abundant. On the biological plot, the two families
were similarly abundant. These differences were
attributed to different age of trees, prey spectrum,
susceptibility of the two families to applied
chemicals, and intraguild predation of theridiids by
philodromids. (Pekár S., Kocourek F.)
Estimation of seasonal changes in population
feeding activity of the oribatid mite Galumna
elimata (Acari: Oribatida) (MZe 0002700603)
The abundance of oribatids in natural biotopes in
the temperate zone is characterised by two peaks,
in spring and in autumn. We tested whether these
abundance peaks were accompanied by high
feeding activity of Galumna elimata population.
The defecation was used as an indirect estimation
of the feeding activity. The model of seasonal
changes in the population defecation activity
was constructed. Temperature dependence of
defecation measured previously in laboratory and
Galumna abundance data from 4 meadow plots in
Central Bohemia were used for the model. The
mites were sampled monthly from 1992 to 1993.
The monthly defecation per Galumna population
on each plot was adjusted to mean monthly
temperatures and to mean monthly abundance.
The estimated maximal population faecal pellet
production was not correlated with the abundance
peaks on three out of four plots. The model showed
that mites defecated particularly from May to
September. In the autumn, when abundance
was high, negligible defecation was found. The
model of seasonal changes indicates that oribatid
defecation may influence the activity of
microorganisms mainly in late spring and in
summer. (Hubert J., Pekár S.)
Spreading of invasive molluscan species
(MZe 0002700603)
In the last years, invasive spreading of alien
molluscan species caused great damage to several
crops. Therefore we started to study factors
determining the invasions because molluscs are, at
the first sight, poorly adapted for long distance
dispersal. Among several invasive molluscan
species we selected Cepaea nemoralis, whose
dispersal is slow and well documented. In the
territory of the Czech Republic, this species
originating from western Europe was recorded for
the first time in the mid-19th century. Since then its
spreading was slow until the 1950s, when the
species occurred in northern Bohemia and
simultaneously also in southern Moravia. The
species territory was extended particularly since
the late 1980s and the vigorous spread has
continued until now. Over the period of the last
20 years C. nemoralis spread to about one quarter
of the territory. New records were established
particularly in central Bohemia and northern
Moravia. The spreading of snails is mostly
facilitated by human activities, particularly railway
traffic and distribution of potted garden plants.
Studying the change in the rate of dispersal is
important for predicting invasive behaviour of
molluscan species of high economic importance.
(Honěk A., Martinková Z.)
Evaluation of the effect of different
herbivores on regulation of weeds
(MZe 0002700603)
Effects of phytophagous entomofauna on the
injury of different weeds were studied. Positive
results indicating the effect of flea beetles on
reduction of leaf apparatus and production of
overground biomass of weeds were obtained.
Average number of leaves per plant was lower by
30% than in control variant. On average, 20% of the
leaf apparatus of plants was injured by herbivores
(J. Štolcová). Summary of biology and pest status of
Gastrophysa viridula L. was published. Data on
temperature dependence of Gastrophysa viridula
development, feed consumption and synergistic
effect of Ustilago rumicis on Gastrophysa viridula
were summarized. Data on spreading of this
initially mountain species in lowlands, utilisation
of this species in protection against weeds and
its control in cultivated crops were published.
(Honěk A., Martinková Z.)
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Development of the ground-beetle
parasitoids Brachinus explodens and B.
crepitans (Coleoptera: Carabidae): effect of
temperature (MZe 0002700603)
Establishing the thermal requirements of insects
is useful for understanding when they are present
during a season and predicting the period of
their maximum abundance. Here the thermal
requirements for the development of all stages and
certain phases within larval instars of Brachinus
explodens Duftschmid and B. crepitans (Linnaeus)
were established at three constant temperatures
between 17.7 and 27.4° C. The lower development
threshold (LDT) for eggs is 9.4° C for B. explodens
and 7.2° C for B. crepitans, respectively; the sums of
effective temperatures (SET) are 154.4 and 180.7
degree-days, respectively. LDT for the total
postembryonic development (except the searching
phase) is 12.3° C in B. explodens and 10.5° C in B.
crepitans, respectively, and SET are 209.2 and 289.5
degree-days, respectively. Thermal constants for the
searching phase of the first instar larva were not
calculated because its duration is independent of
temperature. (Saska P., Honěk A.)
Development of the beetle parasitoids
Brachinus explodens and B. crepitans
(Coleoptera: Carabidae) (MZe 0002700603)
Earlier authors demonstrated that wetland species
of the genus Brachinus (Coleoptera: Carabidae) are
pupal ectoparasitoids of water beetles of the
families Dytiscidae, Gyrinidae and Hydrophilidae.
Here we demonstrate that the dryland species,
Brachinus explodens and B. crepitans, develop on
pupae of ground beetles of the genus Amara
(Carabidae). As Brachinus and Amara species often
occur together at high densities in crops, the rearing
of both Brachinus species was examined in the
laboratory. Females laid eggs in the soil and the
first instar larva searched for a host. After finding
an Amara pupa a Brachinus larva bit it and then
fed on the haemolymph exuding from the wound.
The second instar larva continued feeding in this
way. The third instar larva attached the Amara
pupa to its dorsum and ate it completely within
ca. 2 days. Feeding was followed by a resting
phase and moulting to pupa and adult. The total
postembryonic development was completed
within 20.0 days in B. explodens and 24.0 days in
B. crepitans in 24.7° C. (Saska P., Honěk A.)
Flight pattern of Archips podana (Lep.:
Tortricidae) based on data from pheromone
traps (MZe 0002700603)
In 1993 – 2003 the flight activity of Archips podana
was investigated by pheromone traps placed in four
apple orchards in Central and Eastern Bohemia. The
cumulative catches of A. podana males were plotted
against the time of the catch expressed as sum of
degree-days (DD) above 10° C and approximated by
Richards’ function. The common parameters of
Richards’ function could be found for the
overwintering generation of A. podana from all
localities. The reasonable predicting of the
beginning, the peak and the end of the flight activity
of the overwintering generation of A. podana
population in Central and Eastern Bohemia was
ascertained by help of DD. It was found that A.
podana is usually bivoltine in the Czech Republic,
exceptionally univoltine in cold years or cold
localities. It was impossible to construct the flight
pattern of the first summer generation of A. podana
because the course of flight of this generation in
dependence on DD differed significantly in particular years and localities. (Stará J., Kocourek F.)
Evaluation of Psylla pyri resistance to
insecticides (MZe 0002700603)
The efficacy of different insecticides in the
control of adults and nymphae of P. pyri from
localities Litoměřice, Doksany and Slaný was
evaluated in biological tests. Resistance of P. pyri to
preparation Nomolt was proved in the tests.
Simultaneously, decreased efficacy of preparations
Dimilin and Zolone on P. pyri populations resistant
to Nomolt was proved when applied on both adults
and nymphae (Dimilin) or only on nymphae
(Zolone). The highest efficacy on P. pyri adults was
found when Zolone was applied. Preparation
Cascade was efficient from 85% when applied at an
increased concentration 0.25%. The efficacy of
other tested preparations (Calypso, Vaztac,
Vertimec) applied at registered concentrations on
adults of P. pyri was not sufficient, not even when
they were applied at concentrations twice or three
times higher than registered (Dimilin, Nomolt).
The highest efficacy on P. pyri nymphae was found
when Calypso (100%) and Vertimec (92%) were
applied. On the basis of the results, preparations
Calypso, Vertimec and Cascade were recommended
for antiresistant strategies against P. pyri. (Kocourek F., Stará J.)
Systemic applications of neem in the control
of Cameraria ohridella, a pest of horse
chestnut (Aesculus hippocastanum)
(MZe 0002700603)
Systemic injection of neem (azadirachtin) was
tested in the control of horse chestnut leafminer,
Cameraria
ohridella
Deschka
&
Dimic
(Lepidoptera: Gracillariidae), a pest of horse
chestnut trees (Aesculus hippocastanum L.:
Hippocastanaceae). The 0.15 and 0.25 g a.i.
concentrations resulted in 100% reduction of pupae
in all generations; the 0.08 g a.i. concentration was
Larva of Cameraria ohridella
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Aesculus hippocastanum branch in August after azadirachtin
injection treatment
Aesculus hippocastanum branch in August without
azadirachtin treatment (control)
effective against the horse chestnut leafminer for at
least 23 weeks and led to 70-80% reduction of pupae.
Treated trees did not lose their leaves before winter.
Neem application via injection is cheap, with no
environmental hazards, and thus this treatment is
recommended for the control of C. ohridella in
horse chestnut trees. (Pavela R.)
Potential insecticidal activity of extracts from
some species of medicinal plants on larvae of
Spodoptera littoralis (MZe 0002700603)
Insecticidal activity of methanol extracts from
18 species of medicinal plants was tested on the
3rd instar larvae of the Egyptian cottonworm
(Spodoptera littoralis). All extracts were toxic
to larvae. Extracts from Ocimum basilicum,
Origanum majorana, Picea excelsa and Salvia
officinalis were highly toxic. Extracts from
Melilotus officinalis, Pinus silvestris, Taraxacum
officinalis and Achillea ptarmica were moderately
toxic. The relative growth rate, food consumption
and conversion efficiency of the digested food were
calculated. Clear correlations were found between
weight increase, quantity of ingested food, and the
quantity of excrements produced during the whole
assay period. These results indicate an antifeedant
property of the tested extracts (Pavela R.).
Utilisation of the “attract and kill” method
against codling moth (Cydia pomonella)
(QD 1048)
The “attract and kill” method based on the
combination of attractant and insecticide was tested
in Prague – Ruzyně in an experimental apple
orchard. The preparation LastCallTM CM (permethrin
6% + codlemone 0.16%) produced by IPM
Technologies was tested on a 0.34 ha plot. The
efficacy of the tested preparation LastCallTM CM was
determined by the evaluation of the number of
codling moth larvae in paper belt traps and by the
evaluation of fruit injury caused by codling moth
larvae on treated and untreated plots. The number
of codling moth larvae in paper belt traps was 8.2
per tree on the untreated plot and 2.4 larvae per tree
on the plot treated with LastCallTM CM. The fruit
injury at harvest reached 17.5% on the untreated plot
compared to 0.8% on the plot treated with
LastCallTM CM. The efficiency of LastCallTM according
to fruit injury at harvest was 95.4% in comparison
with the untreated plot. (Stará J., Kocourek F.)
Biology and phenology of Cacopsylla pruni
and detection of ESFY phytoplasma in
Cacopsylla pruni adults (QD 1048)
Psyllids of Cacopsylla sp. were collected in 2004
in Lednice, Bulhary, Velké Bílovice and Valtice. The
population density of Cacopsylla adults in these
localities was assessed and the individuals of
Cacopsylla were determined into the species. The
individuals of C. pruni and C. melanoneura were
separated for the further analysis of ESFY
phytoplasma. In total, 156 adults of C. pruni
collected in 2004 were used for the ESFY analysis.
The incidence of ESFY phytoplasma was detected
in samples from all localities. In 2004 the incidence
of ESFY phytoplasma in C. pruni adults ranged from
5% to 26% in all localities. In Lednice locality, the
proportion of ESFY positive C. pruni individuals
increased from 10% positive individuals collected
in March to 26% positive individuals collected in
April. (Kocourek F., Navrátil M., Lauterer P.)
Thermal requirements for development and
resource partitioning in aphidophagous
guilds (QD 1350)
The duration of the increase, peak and decline in
abundance of the immature stages of cereal aphids is
ephemeral each year. These temporary resources are
exploited by a sequence of aphidophagous insect
predators. The temporal sequence in the appearance
of the immature stages of coccinellids and syrphids in
the sycamore and cereal aphid systems is defined. In
spring and early summer the immature stages of
syrphids consistently appeared before those of
coccinellids. These temporal patterns in the attack
sequence are associated with a difference in the
lower developmental thresholds (LDT) of these two
groups of predators. The LDT of syrphids (4° C)
enables them to be active at lower temperatures and
to develop faster between 10° and 27° C than
coccinellids, whose LDT is 10° C. As a consequence,
when temperatures are low but increasing, syrphids
can appear before coccinellids. Thus, the niche
shift between syrphids and coccinellids is possibly
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a consequence of a phylogenetic constraint rather
than a response to competition and/or intraguild
predation. (Dixon A. F. G., Jarošík V., Honěk A.)
Monitoring of thrips on cabbage by
combination of yellow water traps and sum
of effective temperatures (QD 1357)
In 2004 alike in previous years the flight activity of
Thrips tabaci and its natural enemies, thrips of the
family Aeolothrips, was monitored by yellow water
traps in cabbage. On the basis of data from 2002 –
2004, DDs were defined for the maximum of flight
activity of particular generations, which allows a
short-term prognosis of flight activity of particular
generations of Thrips tabaci. The critical period for
chemical control applied against thrips on cabbage
is at SET from 300 to 800° C. The initiation of control
is signalled at the beginning of cabbage head
formation. An optimal term of treatment is in the
period of maximum incidence of adults, before egg
laying. (Kocourek F., Karasová T.)
Monitoring of flight activity of Agrotis
segetum, Mamestra brassicae and Lacanobia
oleracea by light traps and temperature
models (QD 1357)
In 2004 alike in 2002 – 2003 the flight activity of
Agrotis segetum, Mamestra brassicae and Lacanobia
oleracea was monitored in five localities by light
traps and pheromone traps. Both these methods
were efficient for Agrotis segetum. For Mamestra
brassicae and Lacanobia oleracea the used
pheromones were not efficient enough. The flight
patterns of the overwintering generations of all
three species differed in DD between localities as
well as between years. Consequently, it was
impossible to construct the flight patterns of the
overwintering generations of these species. For
Mamestra brassicae it is possible to use DD 1 190° C
for the prediction of flight activity of summer
generation. The timing of the control of incidence in
the field and optimisation of dates of treatment are
possible according to data from light traps. Increased
catches of individuals in particular generations of
Agrotis segetum, Mamestra brassicae and Lacanobia
oleracea signal the gradation of the species in the
surroundings. (Kocourek F., Holý K., Vošta M.)
Evaluation of efficacy of a Trichogramma –
– based biopreparation in the control of pests
in vegetables (QD 1357)
Field experiments with the evaluation of biological
efficacy of a preparation based on Trichogramma
(Trichoplus) in the control of pests in vegetables
continued in 2004. In a two-year experiment good
efficacy of a new formulation of the preparation
Trichoplus was proved against Mamestra brassicae
in cabbage in comparison with the efficacy of
synthetic insecticides. Good efficacy of Trichoplus
was also proved against Lacanobia oleracea in
glasshouse tomatoes. The timing of treatment with
Trichoplus is based on the monitoring of flight
activity by light traps and control of incidence of
eggs in the field. The developed method of vegetable
protection by the preparation Trichoplus involves
the method of application, timing of treatment
including the combination of Trichoplus with other
means of control such as preparations based on
Bacillus thuringiensis and botanical insecticides.
(Kocourek F., Holý K.)
Host range and population growth of stem
and bulb nematode (Ditylenchus dipsaci)
populations isolated from garlic and chicory
(QD 1357)
Pot experiments aimed at the study of host range
and population growth of two Ditylenchus dipsaci
(Stem and Bulb Nematode) populations on five
vegetable crops continued in 2004. Inoculation was
performed by placing a droplet of 1.5% CMC
suspension containing nematodes among the
primary leaves. Normal population growth occurred
in the variants of original host plants, i.e. on salad
chicory inoculated with chicory population and on
garlic and onion inoculated with garlic population.
Survival of the particular nematodes on garlic and
spinach inoculated with chicory population and on
chicory inoculated with garlic population was
registered. Nematodes of the chicory population did
not survive on onion and leek, the same result was
obtained in the garlic population inoculated to
spinach and leek. The influence of nematode
infection on dry weight of the green parts of plants
was not indicated. The symptoms of the infections
were registered on chicory inoculated with chicory
population. In substrate the nematodes were
recorded only in the variants with normal
population growth. (Douda O.)
Effect of azadirachtin applied systemically
through roots of plants on mortality,
development and fecundity of the cabbage
aphid (Brevicoryne brassicae) (QD 1357)
The effect of low concentrations of azadirachtin
applied systemically through root tissues of rape
(Brassica napus subsp. napus) plants on mortality,
development period, longevity and fecundity of
the cabbage aphid (Brevicoryne brassicae L.)
(Sternorrhyncha: Aphididae) was studied. The
mortality of cabbage aphid nymphs, particularly
during ecdysis, increased significantly with
increasing concentrations. Azadirachtin had no
effect on the length of the development period
of the respective immature stages of cabbage
aphids. The average longevity of the cabbage
aphids decreased with increasing azadirachtin
concentration and time spent by feeding. The
fecundity of aphids decreased after the application
of azadirachtin. (Pavela R., Bárnet M., Kocourek F.)
Transgenic Bt-maize and parasitic wasp
Trichogramma – two compatible strategies in
the control of Ostrinia nubilalis in maize
(QD 1360)
In 2004 alike in two previous years the efficacy of
Bt-maize in the regulation of the Ostrinia nubilalis
population in comparison with biological control by
Trichogramma wasp was studied in two localities.
Bt-maize MON 810, its isoline Monumental and local
hybrid Raissa were evaluated. Trichogramma wasp
was applied in the preparation Trichocarp. Before
harvest plant injuries in the untreated variant were
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from 0.75 to 2.5 per plant in 2002, from 0.22 to 1.4 in
2003 and from 0.68 to 1.55 in 2004. Biological
efficacy of Bt-maize was 100% in 2002 – 2004,
biological efficacy of Trichogramma wasp ranged
from 35 to 69% in 2002 – 2003 and from 12 to 53%
in 2004. An increase in grain yield ranged in Bt-maize
variant from 21 to 27% in 2002, from 7 to 15% in
2003 and from 0 to 4.6% in 2004. A yield increase in
variants treated with Trichogramma wasp ranged
from 0.6 to 17%. A mathematical model of population
dynamics of Ostrinia nubilalis on strips treated with
Trichogramma wasp in comparison with untreated
strips was constructed. The model indicates that
Trichogramma wasp can be used in antiresistant
strategies against Ostrinia nubilalis. Strips of
sensitive non-transgenic maize can be used as refuges
that moderate or prevent the spreading of individuals
resistant to Bt-toxin. (Kocourek F., Říha K.)
The species spectrum of toxicogenic
micromycetes and incidence of mycotoxins
in maize grain at different levels of
infestation by Ostrinia nubilalis (QD 1360)
In 2004 alike in two previous years toxicogenic
micromycetes were detected from maize cobs
originating from the experiments with Bt-maize
MON 810 and its isoline in variants treated with
Trichogramma wasp and variants without
treatment carried out in two localities. 15 species
from the family Fusarium and 9 species from the
family Penicillium were diagnosed in total. The
spectrum of micromycete species was similar in
Bt-maize and non-transgenic maize. The incidence
of Fusarium in maize cobs according to symptoms
correlated with the injury of maize cobs caused by
Ostrinia nubilalis larvae. No injury caused by
larvae of Ostrinia nubilalis was found in Bt-maize.
Frequency of occurrence of several Fusarium
species was markedly lower in Bt-maize variant
after mechanical injury than in non-transgenic
maize after injury caused by Ostrinia nubilalis
larvae. The frequency of occurrence of F.
proliferatum, F. sporotrichioides, F. verticillioides,
F. oxysporum and F. subglutinans was lower in
Bt-maize by 86%, 62%, 78%, 36% and 32%,
respectively. In 2002 and 2004 the concentration of
mycotoxins in grain (DON, 2EA, FUM and T-2
toxin) was markedly lower in Bt-maize than in nontransgenic maize and in 2003 it was below the
detection limit in both Bt-maize and non-transgenic
maize. (Slezáková J., Remešová J., Kocourek F., Říha K.)
Evaluation of the Bt-maize effect on diversity
and abundance of soil arthropods,
microorganisms and biomass decomposition
(QD 1360)
The fauna of soil arthropods was monitored by
earth formalin pitfall traps in two localities in 2002 –
2004 on Bt-maize MON 810 and its non-transgenic
isoline in the variants treated with Trichogramma
wasp and without treatment. During three years of
growing of the same hybrid in the same field, no
significant differences were found in pathway
diversity and abundance of spiders and carabids in
both localities. Bt-maize had no influence on
communities of soil arthropods. Effects of Bt-maize
on post-harvest residue decomposition, soil
microflora and soil fauna were studied. No
significant effects of Bt-maize on studied parameters
were recorded. In laboratory fresh post-harvest
residues or post-harvest residues exposed in soil for
60 days from the above field experiment were used
to study the effect of Bt-maize on the population
growth of Enchytraeus crypticus (Oligochaeta:
Enchytraedae). Significant (about 30%) reductions
of E. crypticus population growth on fresh Bt-maize
litter in comparison with non Bt-maize were
observed. This was not observed in litter exposed in
soil for 60 days. Bt-maize may have a deleterious
effect on decomposers in the laboratory, but these
effects were minor and restricted to the initial stages
of decomposition. (Kocourek F., Pekár S., Řezáč
M., Saska P., Frouz J., Elhotová D., Souzková M.)
Evaluation of the resistance of insect pests
of agricultural crops to insecticides
(GA ČR 522/04/P181)
Laboratory tests were conducted to determine
discriminating concentrations of insecticides, LC50
value and LC90 value when applied on the eggs, L1
larvae and L5 larvae of Cydia pomonella. The
discriminating concentration of 0.005% and 0.34%
and LC50 values of 0.0015% and 0.115% were
determined when the preparation Zolone was
applied on L1 and L5 larvae, respectively. For Dimilin,
the discriminating concentration of 0.001% was
determined for application on L1 larvae. The
resistance of C. pomonella population from Velké
Bílovice locality to insect growth inhibitors has
not been detected yet. However, resistance to
organophosphates was detected in a part of this C.
pomonella population. A great part of four tested
populations of L. decemlineata was resistant to
pyrethroids, organophosphates and their composite
preparations. The greatest part of individuals in
L. decemlineata populations was resistant to
pyrethroids (from 70% to 95%). The proportion of
individuals resistant to organophosphates and
composite preparation Nurelle D was consistent in
different populations (from 40% to 85%). The
resistance of L. decemlineata to neonicotinoids was
not detected. However, fast selection of resistant
individuals was proved after repeated applications of
neonicotinoids. (Stará J.)
Carabid larvae as predators of weed seeds:
granivory in larvae of Amara eurynota
(Coleoptera: Carabidae) (GA ČR 521/03/0171)
The food requirements of larvae of Amara
eurynota (Panzer) were investigated in the
laboratory and a hypothesis that they are
granivorous was tested. Insect diet (Tenebrio
molitor larvae), three seed diets (seeds of Artemisia
vulgaris, Tripleurospermum inodorum and Urtica
dioica) or a mixed diet (T. molitor + A. vulgaris)
were used as food. Seeds are essential for successful
completion of development in larvae of A. eurynota
because all those fed the pure insect diet died
before pupation. Differences in suitability were
observed between pure seed diets. Larvae fed seeds
of A. vulgaris had the lowest mortality and fastest
development on the seed diets. Those fed seeds
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of T. inodorum had low mortality, but the
development was prolonged in the third instar. In
contrast, development of larvae reared on seeds of
U. dioica was the slowest of the tested diets and
could not be completed as all individuals died
before pupation. When insects received the seed
diet of A. vulgaris (mixed diet), the duration of
development shortened, but mortality remained
the same when compared to the seed diet of T.
inodorum. It was concluded from the results that
larvae of A. eurynota are granivorous. The mixed
diet and seed diets of A. vulgaris and T. inodorum
were suitable and insect diet and seeds of U. dioica
were unsuitable diets in this experiment. (Saska P.)
Study of biological control of ant pests using
ant-eating spiders (COST – OC 842.40)
A laboratory experiment was performed to find
evidence for the group hunting of Zodarion spiders.
Foraging behaviour of spiderlings of the second
instar was studied in two ant species Tapinoma and
Messor. Groups of 3 and 6 spiders had better capture
success than a single individual. The paralysis time of
Messor ants shortened as the number of spiders in
the group increased while that of Tapinoma ants
was similar for all groups. On Tapinoma ants 1 or 2
spiders were feeding at the same time while on
Messor ants even all six individuals could feed
simultaneously. The group of 6 spiders fed
significantly shorter on Tapinoma ants than the
group of 3 spiders. In spiders feeding on Messor the
individual gain was on average 1.5-times higher than
that when feeding on Tapinoma. The gain was not
shared equally between the group members: the
catchers gained 2.1-times more than the intruders
did. The catchers experienced a higher capture risk
than the intruders. The obtained results show that
Zodarion spiders exhibit solitary foraging behaviour
when hunting small ants, but co-operative behaviour
when hunting large ants. The observed foraging
behaviour seems to be an example of mutual
parasitism, which may be considered on a long-term
scale as co-operation. This is the first evidence of cooperation in a solitary non web-building spider.
(Pekár S., Hrušková M.)
Collection of animal pests of agricultural
crops and their natural enemies
The collection of pests of agricultural crops and
their natural enemies involves live colonies and
prepared insects. Laboratory colonies contain 21
species of insects and nematodes in 27 strains, which
are used for experiments in the framework of
existing projects as well as for the preparation
of new projects and for elaboration of diploma and
dissertation theses of students of Czech Agricultural
University and Charles University. Currently, the
collection of prepared insects contains ca. 100 000
exemplars of more than 15 000 species from the
orders Coleoptera, Dermaptera, Diptera, Heteroptera,
Hymenoptera, Lepidoptera, Thysanoptera. The
collection is gradually rearranged and completed to
improve its accessibility to daily handling. It serves as
a comparative material for diagnostics and
determination of species from field experiments.
(Saska P.)
Reference laboratory for detection and
determination of quarantine pests
The laboratory is authorised to carry out reference
diagnostics and to elaborate procedures for
determination of 42 taxa of quarantine organisms.
The work station also serves as a training centre of
specialists from regional laboratories of the State
Phytosanitary Authority. In 2004, adults of
Helicoverpa armigera were diagnosed from light
traps. Further, populations of Leptinotarsa
decemlineata resistant to pyrethroids and
organophosphates and a population of Adoxophyes
orana resistant to organophosphates were
diagnosed. It was found that the population of
Adoxophyes orana in outbreak was resistant to
organophosphates (Zolone) and highly tolerant to
pyrethroids and neonicotinoids (Mospilan). It is
probably an imported population from Western
Europe which spreads from localities in Eastern
Bohemia to other apple growing areas. (Kocourek F.)
Department of Stored-Product Pest
Control
Bean flour suppresses population
development of stored-product pest mite
Acarus siro (COST – 1P04OC842.20)
It is well known that pea flour is rich in insecticidal
proteins that protect beans against graminivores.
Recently the combination of stored grain and pea
flour was found to protect grain against storedproduct beetles. Except pea, the common bean
Phaseolus vulgaris contains a high amount of
insecticidal proteins (arcelins, inhibitors, etc.).
Therefore we tested the application of bean flour
(the concentration from 0.01 to 2%) into stored grain
to protect it from the development of a storedproduct mite (Acarus siro). The pest population
growth was suppressed on bean flour enriched grain
in comparison with control grain, but the extinction
of pests was not observed. (Hubert J.)
A novel strategy of combined biological and
digestive enzyme inhibitor control of storage
pest arthropods (COST 842,20)
We investigated the effect of a selective α-amylase
inhibitor on dynamics of the pest-predator trophic
system. The parthenogenetic adult females of
Cheyletus malaccenis were reared on Acarus siro,
which fed either on the control diet or on the diet
containing the inhibitor. The inhibitor significantly
suppressed the population of Acarus siro in the
experiments without predator, and the efficiency
was higher than in the experiments conducted
with the predator in the absence of the inhibitor.
The high efficiency of suppression by the
combined biological control is associated with
low or no adverse effect of the selective inhibitor
on C. malaccensis. The applied concentrations of
α-amylase inhibitor in the trophic system were
set low, and the transfer through the trophic
chain further decreased its concentration. This
conclusion is supported by the absence of
a negative effect on the oviposition period and
longevity of C. malaccensis females. We observed
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a negative side-effect on the predator’s fecundity,
however, we suppose that this is an indirect result
of the inhibitor in the trophic system. The
suppressive effect of α-amylase inhibitor is based
on nutrient deficiency of A. siro, and the
consumption of such a prey resulted in lower
fecundity of C. malaccensis females. (Hubert J.)
Polyclonal antibodies for detection of the flour
mite Acarus siro (Acari: Acaridida) (QC 4071)
Acaroid mites are serious pests of stored products
and households. They produce allergens and
transmit mycotoxin-producing fungi. The infestation
of stored food by mites strongly decreases food
safety and endangers public health. There is an
urgent need for rapid detection methods such as
ELISA. Therefore the aim of our work was to prepare
and characterise antibodies for detection of the flour
mite (Acarus siro). Two polyclonal antibodies (antiA.siroK53 and anti-A.siroK54) were prepared
against an antigen in the form of protein fraction
from whole body homogenates of Acarus siro.
Specificity and sensitivity of the prepared antibodies
were evaluated. We did not observe any crossreactivity with stored beetles, moths, other mite
species, micro-fungi, rearing diet of mites (yeast and
wheat diet) and extracts from wheat kernels. Crossreactivity to Acarus gracilis was found out. This
phenomenon is discussed in relation to the
evolution position of this species. The obtained
antibodies (anti-A.siroK53 and anti-A.siroK54)
enabled to detect and quantify the stored-product
mite Acarus siro and its faeces in the tested samples.
The potential of the obtained antibodies indicates
their possible application to rapid detection of food
contamination by stored-product pests. (Hubert J.,
Kudlíková I., Stejskal V.)
Field evidence and symptoms of pasta
infestation by Sitophilus oryzae in the Czech
Republic (MZe 0002700603)
Heavy infestation of pasta by the internally feeding
pest Sitophilus oryzae (Coleoptera: Curculionidae)
was recorded for the first time under field
conditions in the ČR. Symptoms of hidden
infestation (egg, larva, pupa, adult) and comparison
of optical methods for their detection were
described for the needs of farmers, plant health
and phytoquarantine inspectors. (Stejskal V.,
Kučerová Z., Lukáš J.)
Arthropod infestation in samples of stored
seeds in the Czech Republic (MZe 0002700603)
Twenty-one types of seed samples (mainly vegetable
and grass seed) were analysed in laboratory and 60%
arthropod infestation (14 Acarina, 5 Psocoptera species)
was found. The seeds of beet, grass, onion, radish and
lettuce were most sensitive to infestation. Acarus siro
was a dominant mite pest from all aspects (frequency,
abundance and seed diversity infestation), followed by
Tyrophagus putrescentiae, Tarsonemus granarius and
Lepidoglyphus destructor. Cheyletus eruditus was a
dominant predatory mite. Lepinotus patruelis was the
most frequent psocid pest. (Kučerová Z., Horák P.)
Pesticidal activity of bean α-amylase
inhibitors a-AI-1 (MZe 0002700603)
The α-amylase inhibitor a-AI-1 belongs to the family of
related defence proteins in seeds of common bean
Phaseolus vulgaris and other species of the genus
Phaseolus. This family also includes homologous
phytohaemagglutinins and arcelins the genes of which
evolved by duplication and divergence of a single
ancestral gene. In spite of the considerable sequence
homology, the mode of action of these proteins in
protection of seeds is different. Phytohaemagglutinins
and arcelins are toxic to insects due to their binding to
midgut epithelial cells. The bean α-amylase inhibitors
were reported to interact with digestive α-amylases, but
show high specificity. In vitro screening indicates that
a-AI-1 has no inhibitory activity for α-amylases of Acarus
siro and Ephestia kuehniella, but blocks α-amylases of
Tribolium castaneum. The inhibitory specificity of a-AI-1
determined in vitro was compared with the selectivity
of its activity in vivo. The a-AI-1 was incorporated in the
feeding diet of model species, and the effect of the
ingested a-AI-1 was monitored by biological parameters.
The obtained results showed a significant suppression
of Tribolium castaneum development, contrary to
insignificant changes in the development of Ephestia
kuehniella and Acarus siro. (Hubert J.)
Reference laboratory for detection and
determination of stored-product pests
Over 294 samples of various commodities from
agricultural and food processing companies were
examined for detection of stored-product arthropods.
About 1 330 determinations of 33 pest species were
made. Species belong to the following taxonomic
groups: Acarina (9), Psocoptera (7), Coleoptera (16)
and Lepidoptera (1). (Kučerová Z.)
STAFF PUBLICATIONS
Abernathy D., Zhebentyayeva T., Abbott G. A.,
Vilanová S., Badenes L. M., Salava J., Polák J., Krška
B., Damsteegt V. J.: Molecular genetic mapping of
the Plum pox virus resistance genes in apricot. Acta
Horticulturae, 657, 2004, pp. 283-288.
Bryxiová M., Korba J., Kůdela V.: A survey on
possible occurrence of quarantine bacteria Xylella
fastidiosa in Bohemian and Moravian landscape.
Acta fytotechnica et zootechnica, 7, 2004, Special
number, pp. 41-44. Internet publication on URL:
http://www.fem.uniag.sk/
Dvořák L., Honěk A.: The spreading of the Brown
Lipped Snail, Cepaea nemoralis, in the Czech
Republic. Časopis Národniho muzea, Řada
přírodovědecká, 174, 2004, pp. 97-103.
Fialová R., Navrátil M., Válová P., Lauterer, P.,
Kocourek F., Poncarová-Voráčková Z.:
Epidemiology of European stone fruit yellows
phytoplasma in the Czech Republic. Acta
Horticulturae, 657, 2004, pp. 483-487.
Glasa M., Palkovics L., Komínek P., Labonne G.,
Pittnerová S., Kůdela O., Candresse T., Šubr Z.:
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Geographically and temporally distant natural
recombinant isolates of Plum pox virus (PPV) are
genetically very similar and form a unique PPV
subgroup. Journal of General Virology, 85, 2004,
pp. 2671-2681.
Honěk A.: Holland J.M. (ed): The Agroecology of
Carabid beetles. (Recenze). European Journal of
Entomology, 101, 2004, p. 36.
Honěk A., Martinková Z.: Host plant age and
population development of a cereal aphid, Metopolophium dirhodum (Hemiptera: Aphididae). Bulletin
of Entomological Research, 94, 2004, pp. 19-26.
Honěk A., Martinková Z.: The effect of
environmentally induced variation of host-plant
vigour on abundance of cereal aphids. In: Simon
J.C., Dedryver C.A., Rispe C., Hulle M. (eds.): Aphids
in a new millenium. Institute National de la
Recherche Agronomique, Paris, 2004, pp. 319-324.
Honěk A., Martinková Z.: Seed consumption by
ground beetles. In: Gasquez J. (ed.): 12eme Colloque
International sur la Biologie des Mauvaises Herbes,
Dijon - France. AFPP-INRA-ENESAD, Dijon, 2004, pp.
251-256.
Honěk A., Martinková Z.: Seed predation in
dandelion, Taraxacum officinale. In:
Gasque J. (ed.): 12eme Colloque International sur
la Biologie des Mauvaises Herbes, Dijon - France.
AFPP-INRA-ENESAD, Dijon, 2004, pp. 433-438.
Hubert J., Šustr V., Pekár S.: Estimation of seasonal
changes of population feeding activity of the
oribatid mite Galumna elimata (Acari: Oribatida).
Acarologia, 44, 2004, pp. 253-260.
Hýsek J., Vach M., Brožová J.: Biological
protection of spring barley against fungal diseases.
Acta fytotechnica et zootechnica (Nitra, Slovakia),
vol. 7, 2004, Special number, pp. 101-103. Internet
publication on URL: http://www.fem.uniag.sk/
Chodová D., Mikulka J., Jokeš M.: Changes in
kochia (Kochia scoparia L.) ploidy in relation to
resistance to acetolactate synthase inhibitors. Acta
fytotechnica et zootechnica, 7, 2004, Special
number, pp. 104-106. Internet publication on URL:
http://www.fem.uniag.sk/
Chodová D., Mikulka J., Kočová M., Salava J.:
Origin, mechanism and molecular basis of weed
resistance to herbicides. Plant Protection Science,
40, 2004, pp. 151-158.
Chodová D., Salava J.: Chromosome number,
ploidy level and the molecular basis for the atrazine
resistance in Czech kochia (Kochia scoparia)
biotypes. Herbologia, 5, 2004, pp. 73-84.
Chodová D., Salava J.: Herbicid rezistentní
plevele–následek rozsáhlého používání přípravků.
In: Otázky biologické bezpečnosti GMO a
mezinárodní závazky ČR, Praha, 2004, pp. 69-75.
Chodová D., Kočová M., Holá D.: Weed resistance
in relation to GMO growing. Acta fytotechnica et
zootechnica, 7, 2004, Special number, pp. 101-103.
Internet publication on URL:
http://www.fem.uniag.sk/
Chodová D., Salava J.: The evolution and present
state of weed resistance to herbicides in the Czech
Republic. Herbologia, 5, 2004, pp. 11-21.
Chrpová J., Šíp V., Sýkorová S., Sychrová E.,
Matějová E.: Beitrag zur Problematik der
Ährenfusariosen bei Getreide, Journal of Applied
Botany and Food Quality, 78, 2004, pp. 153-156.
Jarošík V., Kratochvíl L., Honěk A., Dixon . F. G.:
A general rule for the dependence of developmental
rate on temperature in ectothermic animals.
Biology Letters, Proceedings of the Royal Society
of London B (Supplementum), 2004,
DOI 10.1098/rsbl.2003.0145.
Kokošková B., Jeřábková R.: The use of nutrient
semi-selective media for detection and
determination of Clavibacter michiganensis subsp.
sepedonicus. Proceedings of the XVI. Slovak and
Czech Plant Protection Conference, 2003, Nitra.
Acta fytotechnica et zootechnica, 7, 2004, Special
number, pp. 141-143. Internet publication on URL:
http://www.fem.uniag.sk/
Kokošková B., Klenová H.: The study of
saprophytic bacteria in potato tuber samples with
the confirmed occurence of Clavibacter
michiganensis subsp. sepedonicus. Proceedings of
the XVI. Slovak and Czech Plant Protection
Conference, 2003, Nitra. Acta fytotechnica et
zootechnica, 7, 2004, Special number, pp.138-140.
Internet publication on URL: http://www.fem.uniag.sk/
Komínek P., Bryxiová M., Glasa M.: Partial
molecular characterization of a Czech isolate of
Grapevine leafroll-associated virus 3. Journal of
Phytopathology, 152, 2004, pp. 427-431.
Korba J., Kůdela V.: Evaluation of the fire blight
susceptibility of pear genotypes following
inoculation. Proceedings of the XVI. Slovak and
Czech Plant Protection Conference, 2003, Nitra. Acta
fytotechnica et zootechnica, 7, 2004, Special
number, 2004: pp.144-146. Internet publication on
URL: http://www.fem.uniag.sk/
Krejzar V., Jeřábková R., Kůdela V.: Suitability of
biolog system for differentiating fluorescent
pseudomonads associated with premature dying of
apricot trees. Acta fytotechnica et zootechnica, 7,
2004, Special number, pp.147-149. Internet
publication on URL: http://www.fem.uniag.sk/
Kubátová A., Kolařík M., Prášil K., Novotný D.: Bark
beetles and their galleries: well-known niches for
little known fungi on the example of Geosmithia.
Czech Mycol., 56, 2004, (1-2), pp. 1-18.
Kůdela V.: Plant Health Care Terminology – 21.
Post-harvest plant pathology. Plant Protect. Sci., 40,
2004, (3), pp. I-XVII.
Kůdela V.: Plant Health Care Terminology – 22.
Certification of phatogen-free planting material.
Plant Protect. Sci., 40, 2004, (4), pp. I-XI.
Kůdela V., Korba J., Bryxiová M., Krejzar V.:
The possible association of xylem-inhabiting nonfastidious bacteria in leaf scorch and twig dieback in
some shade and forest trees. Proceedings of the XVI.
Slovak and Czech Plant Protection Conference,
2003, Nitra. Acta fytotechnica et zootechnica, 7,
2004, Special number, pp.153-155. Internet
publication on URL: http://www.fem.uniag.sk/
Kumari S. : The occurrence of Xiphinema vuittenezi,
X. pachtaicum and Longidorus leptocephalus
(Nematoda: Dorylaimida) in the Central Czech
Republic. Helminthologia, 41, 2004, (2), pp. 103-108.
Kumari S., Kundu J. K., Polák J.: Určování
nematoda Xiphinema vuittenezi pomocí
polymerázové řetězové reakce. Plant Protect. Sci.,
40, 2004, (1), pp. 1-4.
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Kundu J. K., Ryšánek P.: Detection of Beet yellows
virus by RT-PCR and IC-RT-PCR in Tetragonia
expansa and Beta vulgaris. Acta Virologica, 48,
2004, pp. 177-182.
Martinková Z., Honěk A.: Gastrophysa viridula
(Coleoptera: Chrysomelidae) and biocontrol of
Rumex - a review. Plant Soil and Environment, 50,
2004, pp. 1-9.
Martinková Z., Honěk A.: Seed afterripening in
Echinochloa crus-galli (barnyardgras). Zeitschrift
für Pflanzenkrankheiten und Pflanzenschutz
(Sonderheft), 19, 2004, pp. 197-200.
Novák I., Honěk A.: Plant health care terminology.
18. Animal - food relationship. Plant protection
Science, 40, 2004, (Supplement), pp. 1-15.
Navrátil M., Fialová R., Kocourek F., Lauterer P.,
Válová P., Šafářová D., Poncarová-Voráčková Z.:
Problems of European stone fruit yellows
phytoplasma in the Czech. In: Acta fytotechnica et
zootechnica, 7, 2004, Special number, pp. 217-219.
Internet publication on URL:
http://www.fem.uniag.sk/
Novotný D.: Contribution to the knowledge tree
tissue cultures associated mycobiota. Acta
fytotechnica et zootechnica, (Nitra, Slovakia), 7,
2004, Special number, pp. 227-230. Internet
publication on URL: http://www.fem.uniag.sk/
Novotný D., Šrůtka P.: Ophiostoma stenoceras and
O. grandicarpum (Ophiostomatales), first records
in the Czech Republic. Czech Mycol., 56, 2004, (1-2),
pp. 19-32.
Pavela R., Bárnet M., Kocourek F.: Effect of
azadirachtin applied systemically through roots of
plants on the mortality, development and fecundity
of the cabbage aphid (Brevicoryne brassicae).
Phytoparasitica, 32, 2004, pp. 286-294.
Pavela R., Chermenskaya T.: Potential insecticidal
activity of extracts from 18 species of medical plants
on larvae of Spodoptera littoralis. Plant Protection
Science, 40, 2004, pp. 145-150.
Pavela R.: Growth inhibitory effects of extracts
from Tagetes erecta on larvae Spodoptera littoralis.
Acta fytotechnica et zootechnica, 7, 2004, pp. 237239. Internet publication on URL:
http://www.fem.uniag.sk/
Pavela R.: Insecticidal activity of certain medicinal
plants. Fitoterapia, 75, 2004, pp. 745-749.
Pekár S.: Poor display repertoire, tolerance and
kleptobiosis: results of specialization in an ant-eating
spider (Araneae, Zodariidae). Journal of Insect
Behavior, 17, 2004a, pp. 555-568.
Pekár S., Kocourek F.: Spiders (Araneae) in the
biological and integrated pest management of apple
in the Czech Republic. Journal of Applied
Entomology, 128, 2004, pp. 561-566.
Pekár S., Žďárková E.: A model of the biological
control of Acarus siro by Cheyletus eruditus (Acari:
Acaridae, Cheyletidae) on grain. Journal of Pest
Science, 77, 2004, pp. 1-10.
Polák J.: To the distribution of PPV-M strain in selected
orchards of apricots and peaches in the Czech
Republic. Hort. Sci. (Prague) 31, 2004, (2), pp. 44-46.
Polák J.: Variability in susceptibility to Plum pox virus
in natural woody hosts, myrobalan and blackthorn.
Acta Horticulturae, 657, 2004, pp. 261-264.
Polák J.,Chaloupková M., Jokeš M.: Biological
and serological procedures to detect three
nepoviruses in fruit trees. Plant Protect. Sci. 40,
2004, pp. 121-127.
Salava J.: Analysis of ribosomal DNA sequences of
Synchytrium endobioticum (Schilberzsky) Percival.
Acta fytotechnica et zootechnica, 7, 2004, Special
number, pp. 260-262. Internet publication on URL:
http://www.fem.uniag.sk/
Salava J., Abernathy D., Krška B., Polák J., Abbott A.:
Mapping of resistance genes to Plum pox virus. In:
Proceedings of International Symposium "Advances
in Molecular Biology: Methods for Genotype
Identification, Plant Breeding and Product Control",
Prague, 2004, pp. 39-42.
Salava J., Chodová D., Mikulka J.: Molecular basis
of acetolactate synthase-inhibitor resistance in
Czech biotypes of kochia. Zeitschrift für
Pflanzenkrankheiten und Pflanzenschuzt, 19, 2004,
pp. 915-919.
Salava J., Chodová D., Nováková K.: The emergence
of an atrazine resistant black nightshade (Solanum
nigrum) biotype and molecular basis of the resistance.
Plant Protection Science, 40, 2004, pp. 94-100.
Salava J., Chodová D., Mikulka J.: Molecular
study on acetolactate synthase gene of two Czech
biotypes of kochia (Kochia scoparia L.). Acta
fytotechnica et zootechnica, 7, 2004, Special
number, pp. 263-265. Internet publication on URL:
http://www.fem.uniag.sk/
Saska P., Honěk A.: Development of the beetle
parasitoids, Brachinus explodens and B. crepitans
(Coleoptera: Carabidae). Journal of Zoology,
London, 262, 2004, pp. 29-36.
Stará J., Kocourek F.: Flight pattern of Archips
podana (Lep.: Tortricidae) based on data from
pheromone traps. Plant Protection Science, 40,
2004, pp. 75-81.
Stará J., Kocourek F.: Utilization of the „attract and
kill“ method against codling moth (Cydia pomonella
L., Lep.: Tortricidae). Acta fytotechnica and
zootechnica, 7, 2004, Special number, pp. 301-304.
Internet publication on URL: http://www.fem.uniag.sk/
Svoboda J., Polák J.: Preliminary evaluation of squash
cultivars for resistance to a Czech isolate of zucchini
yellow mosaic virus. In: Proceedings of Cucurbitaceae
2004, the 8th EUCARPIA Meeting on Cucurbit
Genetics and Breeding "Progress in Cucurbit
Genetics and Breeding Research", July 12-17, 2004,
Olomouc, Czech Republic. 2004, pp. 231-235.
Svoboda J.: Ecology of Zucchini Yellow Mosaic
Virus (ZYMV) and contribution to its epidemiology
in the Czech Republic. Acta fytotechnica et
zootechnica, 7, 2004, Special number, pp. 307-310.
Internet publication on URL: http://www.fem.uniag.sk/
Ali S., Šárová J., Wakulinski W.: Pyrenophora triticirepentis races in the Czech Republic and Poland.
Phytopathology, 94, 2004, p. S3.
Širlová L., Vacke J., Jokeš M.: Characteristics of a
Potyvirus associated with a mosaic disease of yellow
oat-grass. Plant Protect. Sci., 40, 2004, pp. 37- 41.
Širlová L.: Virus zakrslosti pšenice a virus žluté
zakrslosti ječmene – významní patogeni obilnin v
ČR (charakteristika, symptomy, diagnostika,
odolnost odrůd). Sborník ze semináře VÚRV
Choroby rostlin, s důrazem na obilniny, jejich
symptomy a rezistence, molekulární a biochemická
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charakteristika interakcí hostitel - patogen,
4.11.2004 Praha-Ruzyně. 2004, pp. 33-37.
Širlová L.: Study on the transmissibility of Barely
yellow dwarf virus, PAV strain by the russian wheat
aphid (Diuraphis noxia). Acta fytotechnica et
zootechnica, 7, 2004, Special number,. pp. 279-281.
Internet publication on URL: http://www.fem.uniag.sk/
Veverka K., Ptáčníková V.: Ausbildung zum
Pflanzenarzt: Beruf und gesselschaftlicher Status.
Phytomedizin, 34, 2004, pp. 74-78.
STAFF OF THE DIVISION
Doc. Ing. Jaroslav Polák, DrSc. (Head of Division)
Blanka Klapová (Personal Secretary)
Department of Virology
Ing. Gabriel Blanco Argollo (by 30. 9. 2004)
Marie Bouzková
Ing. Zuzana Červená
Miloslava Ducháčová
František Dvořák (from 15. 4. 2004 to 31. 10. 2004)
Klára Gajdůšková (from 15. 4. 2004 to 31. 12. 2004)
Ing. Michala Chaloupková
Milan Jokeš
Ing. Petr Komínek, Ph.D.
Jitka Pívalová
Zdenka Pojezdná (by 30. 4. 2004)
Ing. Jiří Svoboda
Ing. Lenka Širlová
Vendula Špelinová (since 15. 3. 2004)
Ing. Shesh Kumari, Ph.D. (nematology)
Markéta Hrabovská (nematology, since 1. 9. 2004)
Department of Mycology
RNDr. Josef Hýsek, CSc. (Head of Dept.)
Ing. Jana Brožová, Ph.D.
Mgr. Julie Chalupníková
Evžena Davidová
Jitka Dunaiová
Zora Hauová
Ing. Hana Klenová
RNDr. Jiřina Krátká, DrSc.
Mgr. Iva Kudlíková
Miroslava Kutíková
Anna Macáková (since 16. 8. 2004)
Zuzana Macháčková
RNDr. David Novotný, Ph.D.
RNDr. Eliška Sychrová
Mgr. Jana Šárová, Ph.D.
Doc. Ing. Josef Šebesta, DrSc.
Eva Šíchová
Lenka Urbánková
Prof. Ing. Karel Veverka, DrSc.
Department of Entomology
Molecular Plant Pathogen Diagnostics Laboratory
Dr. Ing. Jaroslav Salava (Head of Laboratory)
Ing. Dita Bohdanecká
Ing. Marcela Bryxiová
Mgr. Katarína Čiháková
Mohamed A. A. Hassan, MSc.
RNDr. Daniela Chodová, CSc.
Lucie Sýkorová (by 23.7.2004)
Department of Bacteriology
Ing. Václav Krejzar (Head of Dept.)
Jitka Chytráčková
Ing. Blanka Kokošková, CSc.
Ing. Radka Krejzarová (Jeřábková)
Prof. Ing. Václav Kůdela, DrSc.
Anna Machová
Ing. Iveta Pánková, Ph.D.
Doc. RNDr. Ing. František Kocourek, CSc. (Head of
Dept.)
Ing. Martin Bárnet
Ing. Ondřej Douda
RNDr. Alois Honěk, CSc.
Ing. Kamil Holý
Ludmila Kreslová
Irena Kubečková
Bc. Roman Pavela
Doc. Stano Pekár, Ph.D.
Ing. Karel Říha
Mgr. Pavel Saska, Ph.D.
Lenka Slámová
Ing. Jitka Stará, Ph.D.
Doc. RNDr. Josef Šedivý, DrSc.
RNDr. Jindra Štolcová
Zdeňka Víznerová
Department of Stored-Product Pest Control
Slaný Research Station
Ing. Josef Korba (Head of Station)
Johana Branská (by 31.3.2004)
Olga Hojnicová (since 1.4.2004)
Pharm. Dr. Jana Šilerová
Ing. Václav Stejskal, Ph.D (Head of Dept.)
Radek Aulický
Pavel Horák
Mgr. Jan Hubert
Ing. Zuzana Kučerová
Ing. Jan Lukáš, Ph.D.
Bc. Marta Nesvorná
Šárka Tučková