DIVISION OF PLANT MEDICINE

36
DIVISION OF PLANT MEDICINE
RESEARCH ACTIVITIES
Department of Virology
Molecular and biological methods of Wheat
dwarf virus (WDV) strain differentiation
(MZe ČR 0002700603)
During a survey conducted in 2004, winter barley
plants were found to be infected by WDV using
universal polyclonal antibodies in DAS-ELISA. Three
out of these plants were found to contain a WDV
wheat-specific form as proved by PCR-specific
primers. One of those non-specific wheat isolates
(Cz-Wbar3), and wheat specific isolate (CzWhRu5), both isolates were cloned and the
complete sequences were determined.
The genome size of (CzWbar3) and (Cz-WhRu5)
(2 749 nucleotides) was exactly the same as that of
WDV-[Enk1] and WDV-[SE], but one nucleotide was
shorter than that of WDV-[FR] and WDV-[CZ]. Both
isolates (Cz-WhRu5) and (CzWbar3) contained four
genes (MP, CP, repA, rep) as well as the LIR and SIR.
Analyses including these and previously published
sequences showed that the wheat-infecting isolates
of WDV detected in barley or wheat displayed less
than 3% of divergence.
The closer relationship of Czech isolates
(CzWbar3) and (Cz-WhRu5) to wheat-infecting
Hungarian isolates than to other French or Swedish
isolates was supported by phylogenetic analyses.
Analysis of barley-infecting isolates seems to be
necessary to complete the information needed
about this virus in the CR, and it will be conducted
2006. (Širlová L., Hassan M., Bouzková M.)
Molecular detection and analysis
of biodiversity of grapevine viruses from
the genus Foveavirus (MZe ČR 0002700603)
A Czech isolate of Rupestris stem pittingassociated virus-1 (RSPaV-1) obtained from
Karlštejn Research Station of Viticulture was
studied on a molecular level. Twelve primer pairs
were designed spanning 3′ half of the virus
genome. Fragments obtained after RT-PCR with
these primers were cloned into a plasmid vector
and commercially sequenced. 45 partial sequences
were obtained in this way. These sequences were
analysed and found to belong to different isolates
occurring in a natural mixture in one grapevine.
Such a high variability did not allow to complete
one sequence of the virus by joining its different
parts. (Bryxiová M., Komínek P.)
Detection of viruses of the genus Nepovirus
in fruit trees by immunochemical,
electron-microscopic and molecular methods
(MZe ČR 0002700603)
Tomato black ring virus (TBRV) was maintained on
indicator plants of Chenopodium quinoa Willd. by mechanical inoculation using 0.01 M veronal – 0.007 M
phosphate buffer pH 7.8 during the year 2005. The
virus from infected indicator plants was transmitted
mechanically by veronal-phosphate, nicotine and
caffeine buffer onto young plants (six leaves, four
weeks old) of peach (Prunus persica, cv. GF 305).
Inoculated plants of peach were kept in a greenhouse
and tested for the presence of TBRV by ELISA and by
electron microscope after two weeks periodically.
The presence of the virus was not proved by ELISA,
no symptoms were observed, and no particles of
viruses in GF 305 were detected. Trials for the
transmission of TBRV by its vector Longidorus
elongatus were not successful either. (Chaloupková
M., Polák J., Kumari S., Ducháčová M.)
Purification of MLRSV, immunization
of rabbits and antiserum preparation
(MZe ČR 0002700603)
Myrobalan latent ringspot virus (MLRSV) was
purified from infected indicator plants of Chenopodium quinoa Willd. For purification 100 g of
systematically infected leaves were used. The virus
was purified by homogenizing leaves with noctanol and by differential centrifugation in an
ultracentrifuge and finally by centrifugation on
a saccharose gradient. Rabbits were immunized by
combination of intravenous and intramuscular
injections of antigen, emulsified with complete and
non-complete Freund’s adjuvant. Antibody titre was
assessed by a drop precipitation method. High titre
(1:512) of the virus was achieved. Final titre of the
acquired antiserum was 1:1024 and the serum was
kept at 4°C. (Polák J., Chaloupková M., Ducháčová M.)
Molecular and immunoenzymatic detection
of grapevine viruses from the genus Vitivirus
(MZe ČR 0002700603)
Isolates of Grapevine virus A (GVA) were
analysed on the RNA level. A selected part of its
genome was amplified by RT-PCR and sequenced.
Partial sequences of two isolates coming from
Research Station of Viticulture were obtained. They
cover movement protein and coat protein genes.
The first sequence is 1 523 nucleotides long; the
second is 1 522 nucleotides long. In the second
sequence a deletion of one nucleotide was found,
creating a stop codon in the middle of the open
reading frame for movement protein. (Komínek
P., Bryxiová M.)
Research on a reliable diagnostic method
for ESFY (MZe ČR 0002700603)
Research on a reliable diagnostic method for
ESFY continued in 2005. Thirty-eight experimental
trees of apricot cvs. Velkopavlovická, Bocuria,
Liscia, Goldrich and Bergeron were grown in the
orchard. In 2004, eight trees showed symptoms of
ESFY (yellowing and curling of leaves, branches
without leaves and reduction of leaf size). These
trees were evaluated and tested during 2005.
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Two trees with symptoms of ESFY died in 2004.
The other six trees with symptoms of ESFY died in
June 2005. Symptoms of ESFY were also observed
on another four trees in June 2005. In total 40 trees
were tested. In the first PCR 10 trees were positive;
in the second PCR 15 trees were positive. Three
trees showed symptoms of ESFY. One plant (sample
no. 24) with symptoms of ESFY was negative in
PCR. Buds from a scion of a single tree were grafted
on woody indicator plants GF 305 (three
replications were grafted on each tree). Positive
results were achieved after transmission from eight
trees (two or all three indicator plants died); all
these trees were without symptoms of ESFY. The
results of molecular test do not match the results of
biological test or the visual evaluation of symptoms.
These experiments will be continued in 2006.
(Polák J., Bryxiová M., Salava J., Svoboda J.)
Development of a pentaplex RT-PCR method
for simultaneous detection of four pome fruit
viruses (MZe ČR 0002700603)
A pentaplex reverse-transcription polymerase
chain reaction (Pentaplex RT-PCR) in a single tube
was developed for the simultaneous detection of
these pome fruit viruses: Apple stem pitting virus
(ASPV), Apple stem grooving virus (ASGV), Apple
chlorotic leaf spot virus (ACLSV) and Apple mosaic
virus (ApMV). This is the first report of the
simultaneous detection of all four viruses and
host mRNA as an internal specific control.
Pentaplex RT-PCR was applied successfully
throughout the year, using different plant organs
(leaves or dormant buds). The sensitivity of
detection by monoplex and pentaplex RT-PCR
assays was comparable. Different combinations of
mixed infections of viruses were identified in
samples of infected apple and pear trees from
different geographical regions. The developed
pentaplex RT-PCR assay was sensitive, simple,
rapid, and reliable for the simultaneous detection
of four viruses in extracts of leaves or dormant
buds. (Hassan M., Polák J.)
Survey of the occurrence of viruses
in the germplasm of apples and pears
(MZe ČR 0002700603)
A total of sixty accessions covering native and
foreign cultivars and rootstocks were collected and
tested for the presence of four viruses by pentaplex
RT-PCR. All samples were also tested by ELISA for
the presence of ACLSV, ASGV and ApMV.
Regardless of the cultivars, different combinations
of mixed infections of viruses were identified in
samples. All positive samples detected by ELISA
were confirmed by pentaplex RT-PCR. However,
RT-PCR revealed more infected trees than detected
by ELISA. Regardless of the species, ACLSV and
ASPV were the most prevailing viruses in apple and
pear, the two viruses were detected in almost all
mixed infections. Only apple accessions were
found to be infected by ApMV and ASGV, while
none of pear samples was infected by any of them.
For further confirmation of the health condition
of these trees testing of samples will be repeated in
spring 2006. (Hassan M., Polák J.)
Survey of the occurrence of pome fruit tree
viruses in nurseries (MZe ČR 0002700603)
A survey of the occurrence of Apple stem pitting
virus (ASPV), Apple stem grooving virus (ASGV),
Apple chlorotic leaf spot virus (ACLSV) and Apple
mosaic virus (ApMV) was carried out in nurseries
on different sources of apples and pears (e.g.
Holovousy, Mcely, Litoměřice and others). Each
virus was detected by ELISA (ASGV, ACLSV and
ApMV) and by RT-PCR (ASPV, ASGV and ACLSV). In
total 77 leaf samples of apples and pears were
tested by ELISA. Out of these 77 leaf samples, 17
were positive for ACLSV and 18 for ASGV. ApMV
was not detected in any of the tested plants. Ten
cultivars of apple and pear were tested by RT-PCR,
in total 55 plants (5 trees of each cultivar). ASPV
was detected in 25 plants, ASGV in 2 plants only
and ACLSV in 17 plants. ASPV and ACLSV were
detected in all five tested plants in some cultivars.
(Kundu J. K., Svoboda J.)
Survey for the presence of grapevine viruses
(MZe ČR 0002700603)
A screening of the health status of grapevine in the
Czech Republic was done by sampling dormant
canes from grapevines grown in a grapevineproduction area in the South Moravian Region.
Samples were taken from the localities Boršice
u Buchlovice, Bzenec, Louka u Ostrohu, Mutěnice
and Uherské Hradiště. In total 50 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 leafrollassociated virus 1, 2, 3, 5, 6, 7 – GLRaV-1, 2, 3, 5, 6, 7.
16 grapevines were found to be positive for
GLRaV-1, 10 for GFkV, 8 for GVA, 1 for GLRaV-5 and
1 for GLRaV-3. The other viruses: GLRaV-2, GLRaV6, GLRaV-7, GFLV, ArMV, GVB, TBRV, SLRSV,
RpRSV-g and RpRSV-ch were not detected.
(Komínek P.)
Analysis of variability of Grapevine leafrollassociated virus 1 (MZe ČR 0002700603)
Variability of a 540-nucleotide-long part of HSP70
protein gene of Grapevine leafroll-associated virus
1 (GLRaV-1) was evaluated. Several grapevines
infected with GLRaV-1 were selected; the analyzed
region was amplified by RT-PCR and sequenced.
The results showed a grouping of the sequences
into two groups tentatively called A and E. Genetic
divergence between group A and group E reached
13.9%. Restriction enzymes distinguishing group
A and E were found. SalI site is present only in type
A isolates, whereas BsiWI cleaves group E only.
These enzymes were used for a large-scale testing of
GLRaV-1 infected grapevine samples from the
Czech Republic and Turkey. Based on our tests,
GLRaV-1 isolates of both group A and E occur in
these countries. A mixed infection of group A and
E was also found in a single grapevine. (Komínek
P., Bryxiová M.)
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Occurrence and distribution of natural
sources of Plum pox virus strains PPV-M,
PPV-D, Prune dwarf virus (PDV) and Prunus
necrotic ringspot virus (PNRSV)
(MZe ČR 0002700603)
In 2005, research on the distribution of natural
sources of five stone fruit viruses continued in
different localities of fruit growing areas of
Bohemia and Moravia. During the vegetation
season from April to July samples of leaves of main
potential natural hosts (myrobalan, blackthorn,
plum and cherry) were collected and tested by
ELISA. The occurrence of single virus and complex
infections of PPV strains, PDV, PNRSV, ACLSV and
ApMV was detected by ELISA. Leaf samples were
taken from trees with suspicion of infection
symptoms on leaves, especially with symptoms of
diffuse spots and rings, oak mosaic, vein clearing
and interveinal mosaic. The presence of viruses was
also detected in tree leaves which had no visual
symptoms of the disease. In total 129 trees were
tested in the areas of Kladno, Polabí, Příbram,
Stříbro, Domažlice, Hradec Králové, Rychnov,
Jeseníky, Osoblažsko, Bruntál, Šumperk, Sedlčany,
Kokořín and Česká Lípa. Plum pox virus was
identified in 44 natural sources, PDV on eleven
trees; PPV-M and PPV-C strains were not detected.
PNRSV was detected on twenty trees. ACLSV and
ApMV in stone fruits were not detected in natural
sources of infection. Complex infection by two
viruses was found only in four cases and by three
viruses in one case. The first results showed
dominant distribution of Plum pox virus in
myrobalan and plum, much smaller distribution on
blackthorns and negligible significance of stone
fruits as natural sources of ACLSV and ApMV.
(Polák J., Pívalová J.)
In 37 leaf samples from naturally growing trees
and bushes of myrobalan, plum and blackthorn (in
which the presence of PPV was proved by
polyclonal antibodies), the presence of PPV-M was
detected by strain specific monoclonal antibodies
(Agritest, Italy). The presence of PPV-M, PPV-D or
PPV-Rec was established by RT-PCR in two different
regions of virus genome and further by restriction
enzyme digestion of PCR products. Using
monoclonal antibodies the presence of PPV-M was
not detected in 21 myrobalan trees, 13 plum trees
and 3 blackthorn bushes. RT-PCR and RFLP showed
the presence of PPV-D strain in 36 trees. In one
myrobalan tree (Příbram locality) complex
infection of PPV-D and PPV-M, or PPV-Rec
was detected. These results confirmed previously
detected and published sporadic occurrence
of PPV-M in nature and also sporadic occurrence of
PPV-Rec. (Polák J., Komínek P., Pívalová J.)
Growth tips and apical meristems were isolated
under microscope from plum (cvs. Prunus
domestica, Hanita, Bluefree), apricot (cv. Carola)
and peach (cvs. Galla, Fireprince) trees infected by
Plum pox virus (PPV). Samples (0.5-2 mm) were
fixed, dehydrated and poured by a polymerization
process in resin Spurr-kit. Contrasted ultra-thin
60nm slices were examined under electron
microscope 208 S Philips on a grid. At the same
time samples from in vitro cultures after
thermotherapy were examined and possible
occurrence of particles or inclusions of PPV was
determined. In total eight samples from diseased
plants and seven samples from in vitro cultures and
meristems after thermotherapy were prepared and
examined. The presence of virus particles and
inclusions was not detected in any sample from
diseased plants, not even in recovered tissue and in
vitro plant cultures and species of plum, apricot
and peach trees. (Chaloupková M., Ducháčová
M., Polák J.)
Identification and distribution of nematodes
of the genus Longidorus and Xiphinema
in the Czech Republic (MZe ČR 0002700603)
The distribution and occurrence of longidorid
nematodes in Southern and Western Bohemia were
studied from April to August, 2005. Thirty-five fruit
orchards were surveyed for the presence of
longidorid nematodes. Soil samples were taken at
a depth of 0-90 cm with a soil auger. Nematodes
were recovered from the soil by a sieving and
decanting technique, heat killed, fixed in TAF,
processed in a slow glycerin process and mounted
in anhydrous glycerin on slides. Photomicrographs
were recorded with a digital camera connected to
a computer and measurements were made with the
aid of imaging software (Olympus DP-soft). X.
diversicaudatum was identified from three
orchards and X. vuittenezi also from three
orchards. The genus Longidorus was detected in
ten orchards.
Morphological and morphometrical identification of L. elongatus was verified by a polymerase
chain reaction. Total genomic DNA was extracted
from a single nematode. A fragment of
approximately 847 bp was amplified with speciesspecific reverse primer (5' TTA TCG TAC GTA TTC
CCA GTT CT 3') and universal forward primer (5'
TTC ATT ACG TCC CTG CCC TTT GT 3') for all
studied individuals. (Kumari S., Chaloupková
M., Jokeš M., Polák J.)
Occurrence and distribution of Zucchini
Yellow Mosaic Virus (ZYMV), Watermelon
Mosaic Virus 2 (WMV-2) and Cucumber
Mosaic Virus (CMV) in vegetables from
the gourd family (MZe ČR 0002700603)
Samples of leaves from vegetables belonging to
the gourd family that showed symptoms of viral
disease were collected at five sites in Northern
Moravia (the area of Karviná, Opava, Bruntál) and
at four sites in Southern Bohemia (the area of Písek,
Tábor, České Budějovice) during the growing
season in 2005. A total of 107 collected plant
samples were examined for the presence of ZYMV,
WMV-2, and CMV using the DAS-ELISA method.
ZYMV was found in 2 positive plants (Bruntál)
while CMV was detected in 19 plants and WMV-2
was not detected in any sample. Positive findings
were confirmed by mechanical transmission onto
indicator zucchini plants and by using an electron
microscopy. (Svoboda J., Jokeš M.)
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Sources of resistance and assessment of
apricot and peach immunity to PPV
(MZe ČR 0002700603)
The apricot cultivars Harlayne and Betinka
(resistant to PPV), and Velkopavlovická and Karola
(susceptible to PPV) were inoculated by six strains
and isolates of PPV (three different strains of PPVM, two strains of PPV-D and PPV-Rec). The
occurrence and intensity of virus symptoms on
leaves and fruits were evaluated. The presence of
PPV was tested on leaves of apricot cultivars
Harlayne and Betinka by RT-PCR. Medium severe
symptoms of PPV on leaves of susceptible cultivars
Velkopavlovická and Karola, severe symptoms,
diffuse spots and deformation of fruits on cv. Carola
and medium severe spots and rings on fruits of cv.
Velkopavlovická were observed. Leaves and fruits
of cv. Harlayne, inoculated by different PPV strains,
were without symptoms whereas the virus was not
detected either by ELISA or by RT-PCR. Cv. Harlayne
is immune to different strains of PPV. The results of
evaluation of cv. Betinka were the same as in 2004.
Cv. Betinka is immune to four PPV strains from the
group PPV-D, PPV-Rec and PPV-M, but only
resistant to original PPV-M strain, when two trees
were without symptoms and it was not possible to
detect the virus by ELISA and RT-PCR, while
sporadic leaf symptoms appeared on the third tree
(mild diffuse spots and rings) and mild diffuse
spots on several fruits and stones. The presence of
the virus was proved by ELISA and RT-PCR. (Polák
J., Komínek P., Pívalová J.)
The source of resistance to Plum pox virus (PPV)
on peach-tree (Prunus persica) has not been
located until now. Prunus davidiana has shown
high resistance to PPV. This variety can be
crossbred with Prunus persica, but the results of
such crossbreeding did not bring any satisfactory
results. This is the reason why interspecific hybrids
of the genus Prunus were tested as candidate
sources of resistance to PPV. The hybrids Barier,
Cadaman, Pumiselect, MRS, NBS 540-73, GF 677 and
Fire were grafted on peach trees in spring 2003
before these trees were inoculated by PPV using
infected buds. In the years 2003, 2004 and 2005
symptoms of PPV were evaluated. The presence of
PPV in leaves of evaluated trees of Prunus hybrids
was determined by ELISA in May 2004. Relative
concentration of PPV in leaves of tested varieties
was determined by ELISA in 2004 and 2005.
Preliminary results of three-year evaluation of PPV
symptoms and two-year evaluation by ELISA
showed that NBS 540-73 hybrid (P. cerasifera x P.
holoserica x P. domestica) was susceptible to PPV,
Barier hybrid (Prunus davidiana x P. persica) was
medium-susceptible, Pumiselect hybrid (P. pumila)
was tolerant to PPV, MRS (P. cerasifera x P. spinosa)
medium-resistant, Fire (P. amygdalus x P. persica)
resistant to PPV, Cadaman hybrid (P. davidiana x P.
persica) resistant to highly resistant and GF 677
hybrid (P. amygdalus x P. persica) immune to PPV.
The hybrids Cadaman and GF-677 can be suitable
sources of resistance to PPV for crossbreeding with
peach tree. Fire is the third possible candidate.
Evaluation of these candidate hybrids will continue
in 2006. RT-PCR will also be used for the detection
of PPV in tested plants. (Polák J., Pívalová J.,
Kundu J. K., Jokeš M.)
Genetic mapping of Plum pox virus
resistance in apricot (MZe ČR 0002700603)
An integrated genetic linkage map for apricot
(Prunus armeniaca L.) was obtained using B1
progeny of ‘LE-3246’ x ‘Vestar’. A total of 316
molecular markers (290 AFLPs, 26 SSRs) and
resistance to PPV were assigned to 8 linkage groups
covering 574 cM of the apricot genome. The
average distance between adjacent loci is 2.5 cM.
The map was anchored to the Prunus reference
map through published SSR markers. The PPVres1
locus conditioning resistance to PPV was mapped
in linkage group 1. Several AFLP loci tightly linked
to the PPVres1 locus were identified. Four of them
were converted into codominant SSR markers. SSR
markers linked to resistance to PPV could allow the
plant breeders to use marker assisted selection to
screen cultivars and segregate progenies in apricot
and potentially in other Prunus species. (Salava J.,
Polák J., Pívalová J., Komínek P.)
Evaluation of resistance of Prunus domestica
cv. Jojo to PPV and its strains
(MZe ČR 0002700603)
Sixteen trees of Prunus domestica cv. Jojo were
evaluated. Four trees were used as controls. In
2003, four trees were inoculated by grafting PPVRec strain, four trees by PPV-D and four trees by
PPV-M. Symptom evaluation and tests for the
presence of single strains in infected plants by
ELISA and RT-PCR were carried out. Control plants
showed normal growth, they were without
symptoms and the presence of the virus was not
detected. The whole trunks of grafted trees
infected with PPV-Rec died in 2005, only scions of
St. Julien rootstock survived with severe symptoms
of PPV on leaves. The presence of the virus was
proved by ELISA in all four rootstocks. The results
of RT-PCR detection for PPV-Rec, PPV-M, and PPVD with specific primers were positive in rootstocks
of tree no. 1 and 4 with all three primers. Negative
reaction in the rootstock of tree no. 2 with all three
primers and positive reaction with primers PPVM and PPV-Rec in St. Julien rootstock of tree no. 3
were found out. The results of RT-PCR are
inconsistent and do not match with PPV symptoms
and results of ELISA in leaf samples from all four
rootstocks. Therefore, it would be necessary to
repeat the experiments in 2006. Out of the four
trees infected with PPV-D only tree no. 1 died,
where scions of St. Julien rootstock started to show
shoot growth with symptoms of PPV and the virus
was confirmed by ELISA; tree no. 2 and 3 showed
normal growth without symptoms and ELISA was
also negative. In tree no. 4 the whole crown died,
shoots on the rootstock grew with symptoms of
PPV on leaves. The virus was detected by ELISA.
However, later shoots (without symptoms on
leaves) started to grow from the bottom part of the
stem of Jojo variety. PPV-D was detected on tree no.
1 and 3 (rootstock) by RT-PCR and strain specific
primers, however also on growing shoots of Jojo
40
variety in tree no. 3. This is an indication of
important results; therefore the evaluation will
continue in 2006. All four trees of cv. Jojo infected
with PPV-M died. On all young shoots severe
symptoms of PPV were observed and the virus was
detected by ELISA. By RT-PCR and strain specific
primers PPV-M was proved in tree no. 2, 3 and 4, the
test in tree no. 1 was negative. The results of RTPCR with specific primers for PPV-Rec and
PPV-D were negative for all four trees. (Polák J.,
Kundu J. K., Pívalová J.)
Evaluation of the health status of a newly
recognized grapevine cultivar Auxerois
(MZe ČR 0002700603)
Maintenance breeding of newly recognized
grapevine cultivar Auxerois started at Karlštejn
Research Station of Viticulture. The health status of
propagation material of this cultivar was evaluated
by DAS-ELISA, testing vines for the presence of
twelve economically important viruses. 28 grapevines were found to be free of the viruses, eight
grapevines were found to be infected with
Grapevine leafroll-associated virus 5 and four
grapevines were infected with Arabis mosaic virus.
Virus-infected grapevines were maintained in vitro
and sanitised by thermotherapy at 35° C for 40 days.
After this process they will be tested for the presence
of viruses again. (Jandurová O., Komínek P.)
Molecular characterization of potyvirus
occurring in yellow oatgrass plants
(MZe ČR 0002700603)
Yellow oatgrass (Trisetum flavescens) plants with
mild mosaic symptoms were observed in 24
locations in the Czech Republic. Electron
microscope observations of symptomatic plants
revealed the presence of filamentous particles and
inclusion bodies characteristic of the family
Potyviridae. The virus was readily mechanically
transmitted to its original host plus a narrow host
range of monocotyledons. Results were negative in
ELISA using antisera specific for WSMV, ONMV,
SCMV and MDMV. RT-PCR tests with family
Potyviridae-specific primers were used to detect and
amplify the 3’ terminal of the viral RNA genome,
which covered the coding region of the C-terminal
part of the nuclear inclusion b (NIb), coat protein
(CP) gene, and 3’ untranslated region (UTR).
The 3’ region of the genome was cloned,
sequenced and compared with other Potyviridae
species. Pair-wise sequence comparisons of the
nucleotide and amino acid of CPs showed that this
virus was more closely related to viruses from the
genus Tritimovirus than to other genera within
Potyviridae.
Phylogenetic analyses of the coat protein cistron
and flanking genomic regions suggest the presence
of a distinct viral species of the genus Tritimovirus,
tentatively named Yellow oatgrass mosaic virus
(YOgMV) in Trisetum flavescens. (Hassan M.,
Širlová L., Bouzková M.)
Detection of point mutations associated
with organophosphate resistance in
Colorado potato beetle (MZe ČR 0002700603)
The enzyme acetyl cholinesterase (AChE) is
a target site for organophosphate insecticides. The
resistance to organophosphate insecticides in the
Colorado potato beetle (Leptinotarsa decemlineata
(Say)) is associated with a point mutation in
location 980 that results in a serine to glycine
change (S291G) in the resistant AChE gene. We
developed a simple molecular assay based on
restriction fragment length polymorphism (RFLP)
of PCR products to detect the mutation. Out of 20
individuals originating from potato fields all over
the Czech Republic, 8 were characterized as
homozygous resistant, 10 as heterozygous resistant,
and 2 as homozygous susceptible ones. These
results were verified by the bi-directional
polymerase chain reaction (Bi-PASA PCR) method
and sequencing of PCR products. The RFLP-PCR
assay is expected to implement a distinct resistance
monitoring for resistance management and control
of Colorado potato beetle field populations in the
Czech Republic. (Bohdanecká D., Salava J.)
Thermotherapy in vivo of plum, apricot
and peach trees infected with PPV (1B44051)
The thermotherapy of a plum tree of Prunus
domestica L., peach tree of cv. Sentry and apricot
trees of cvs. Puebla and Carola was carried out in
2005. Different levels of temperature were applied.
After the therapy trees were tested for the presence
of PPV by ELISA. The presence of PPV was proved
only in the peach tree of cv. Sentry. (Chaloupková
M., Polák J., Hauptmanová A.)
Preparation of primary sources of apricot
and peach trees infected with PPV (1B44051)
Rootstock cvs. Maxma, Barier, and Puebla were
repeatedly budded with peach (cvs. Redhaven,
Galla) and apricot (cv. Velkopavlovická) in 2005.
The presence of PPV in leaves of shoots grown from
infected buds was tested by ELISA. Additional
primary sources were obtained from the SEVAFLORA Company in Valtice in autumn 2005:
apricots (Velkopavlovická, Leskora), peaches
(Earliglo, Redhaven), and plums (Švestka domácí,
Čačanská lepotica). New primary sources will be
infected with PPV by grafting and budding in
spring 2006. (Svoboda J., Chaloupková M.,
Hassan M., Hauptmanová A., Polák J.)
41
Chemotherapy of in vitro cultures of plum, apricot, and peach trees by Ribavirine (1B44051)
Chemotherapy of plum cvs. Švestka domácí and
Hanita, and of peach cv. Sentry began in September
2005 using Ribavirine. MS medium (Murashige and
Skoog, 1962) with different concentrations of Ribavirine (0.1, 0.2, 0.5, 1.0 mg/l of medium) was used.
Cultures regenerated the best in a medium with
0.2 mg of Ribavirine per litre. Cultures of cv. Švestka domácí regenerated well using different
concentration of Ribavirine. Plants of cv. Hanita were
tested for the presence of PPV (ELISA kit mfd by
Loewe Company) with negative results. 50% of plants
of cv. Sentry survived in a medium with the Ribavirine concentration of 0.1 mg/l, the concentration
0.2 mg/l was better for the regeneration of in vitro
cultures. The chemotherapy will continue in 2006.
(Hauptmanová A., Chaloupková M., Polák J.)
Evaluation of the effect of thermotherapy
of self-rooted grapevine plants using ELISA
and RT-PCR (1B44051)
Self-rooted plants of selected grapevine clones
were sanitised by thermotherapy at 36° C for 6
weeks. After this time they were tested by ELISA
and RT-PCR for the presence of viruses which were
objects of sanitation.
Clone MT 23/37 T3 42 – two plants were tested
after thermotherapy, one plant was positive in RTPCR for Grapevine leafroll-associated virus 1
(GLRaV-1), the other plant was negative.
Clones MT 26/19 T4 22 and MT 33/16 T3 16 – all
tested plants were positive in RT-PCR for GLRaV-1.
However they were all negative in ELISA showing
this method as less sensitive and less suitable for
GLRaV-1 testing.
Clone MT 26/19 T4 43 – three plants were tested
after thermotherapy, they were all positive in RTPCR for GLRaV-1. Moreover, one of them was
positive and two other plants were negative for
Grapevine fanleaf virus (GFLV), which was present
in this clone before thermotherapy.
The same partial sanitation from GFLV, but not
from GLRaV-1, was achieved in clones MT 26/19 T3
60, MT 33/16 T3 7 and PM 30/40 T3 26.
Before thermotherapy clone PM 11/48 T3 2 was
positive for GLRaV-1 and Grapevine fleck virus
(GFkV). After thermotherapy, four plants were
tested, they were all positive for both viruses, none of
them was sanitised. (Jandurová O., Komínek P.)
Evaluation of symptoms in leaves of transgenic
plum Prunus domestica L., clone C-5, inoculated
by grafting of PPV-M strain, and with combinations of the viruses PPV-M + ACLSV, PPV-M
+ PDV and PPV-M + ACLSV + PDV (1B53054)
Leaves of trees of transgenic plum Prunus
domestica L., clone C-5, inoculated with
combinations of PPV-M + ACLSV, PPV-M + PDV, and
PPV-M + ACLSV + PDV by grafting were examined
monthly during the vegetation period 2005. PPV
symptoms appeared in leaves at the beginning of
June as mild to severe diffuse spots. The intensity of
symptoms was severe till July, afterwards it was
getting milder. No symptoms of ACLSV and PDV
were observed during the whole vegetation period.
Diffuse spots caused by PPV appeared in leaves of
infected trees. Medium severe diffuse spots were
observed in leaves of trees inoculated with PPV-M +
ACLSV + PDV. (Polák J., Pívalová J.)
Detection of PPV-M in trees of transgenic
Prunus domestica L., clone C-5, inoculated
with M strain, and with combinations of PPVM + ACLSV, PPV-M + PDV, and PPV-M + ACLSV
+ PDV by ELISA, ISEM and RT-PCR (1B53054)
PPV-M was detected by ELISA in all nine trees
inoculated with PPV-M. The titre of PPV-M protein
varied from 1:128 to 1: 2048 and was not in
correlation with the intensity of evaluated
symptoms. PPV-M was detected by ELISA in all trees
inoculated with PPV-M + ACLSV, in nine trees out of
ten inoculated with PPV-M + PDV, and in all eleven
trees inoculated with PPV-M + ACLSV + PDV. The
relative concentration of PPV-M protein in leaves of
trees inoculated with the particular combinations of
viruses was relatively high, and the combinations
PPV-M + ACLSV and PPV-M + PDV were comparable
with trees infected only with PPV-M, while in the
combination PPV-M + ACLSV + PDV the
concentration of PPV-M was a little higher. The
presence of PPV-M, ACLSV, and PDV was not proved
in healthy control trees of transgenic plum C-5, and
no symptoms of viral infection were observed.
The presence of PPV-M particles was proved by
ISEM in leaves of infected trees.
PPV-M was detected in all trees in combination
PPV-M + ACSLV, in ten trees out of eleven in
combination PPV-M + PDV (in agreement with the
results of ELISA), and only in six trees out of eleven
in combination PPV-M + ACLSV + PDV while by
ELISA in all eleven trees.
ACLSV was detected by one-step RT-PCR almost in
all trees infected with PPV-M + ACLSV and PPV-M +
ACLSV + PDV. Similar results were obtained in the
case of PDV detection by one-step RT-PCR in trees
inoculated with both virus combinations, PPV-M +
PDV, PPV-M + PDV + ACLSV. PDV was proved
almost in all trees. (Polák J., Pívalová J., Kundu J.
K., Komínek P.)
Molecular hybridization detection of
grapevine viruses (QG50083)
The work was aimed at the cloning of appropriate
genome segments of selected grapevine viruses:
Grapevine virus A (GVA), Grapevine virus B (GVB)
and Grapevine fleck virus (GFkV). Primers were
designed to amplify these segments using RT-PCR: in
the case of GVA designed primers amplify a 933nucleotide-long fragment encompassing the whole
gene for coat protein. Primers for GVB amplify a 151nucleotide-long fragment covering 3′ end of coat
protein gene and 5′ end of RNA binding protein gene.
Primers designed by the team of Prof. Martelli
(University of Bari, Italy) were used for
amplification of GFkV genome fragment 383
nucleotides long from the region of replicase gene.
Fragments of viral genome were successfully
amplified by RT-PCR and cloned into pGEM-T Easy
plasmid (Promega). Plasmids were commercially
sequenced to verify the specificity of obtained
fragments. (Komínek P., Bryxiová M.)
42
Establishment of small field plot experiments
to evaluate the infection of cereals by Wheat
dwarf virus (QG50081)
The source of infection, a wheat isolate of Wheat
dwarf virus (WDV), was maintained and
reproduced in the greenhouse. The vector
Psammotettix alienus Dahlb. was collected from
the surrounding fields of RICP and Slaný. The
vector was located in the greenhouse and on small
field plots isolated with screens. Twenty-eight small
field plots (1 m2) were prepared. Nine cultivars of
Aegilops (A. columnaris, A. cyclindrica, A. geniculata, A. kobscley, A. lorenbii, A. neglecta, A.
tauschii, A. triuncinalis, A. umbellate) and five
cultivars of winter wheat (Clarus, Rapsodia, Herold,
Hana, Ilona) were sown (100 seeds per plot) in two
variants (infected plants and healthy controls).
Plots which were supposed to be inoculated by
WDV were isolated with screens after seed
emergence. The acquisition period for the
leafhopper was 3 days and duration of inoculation
feeding was 14 days on each plot. One hundred
leafhoppers were located on each plot. After the
acquisition period, numbers of leafhoppers were
tested by ELISA for the presence of WDV. Due to
the shortage of leafhoppers only 5 cultivars of
wheat were inoculated. Ten samples were collected
from each cultivar after inoculation and tested for
the presence of WDV. Serological test (ELISA)
showed the presence of WDV, but titres were low
in all samples (except one). Next year in spring we
will continue to observe the development of the
disease and to evaluate reactions of particular
wheat cultivars to WDV infection. (Kundu J. K.,
Bouzková M.)
Evaluation of the level of field resistance
to BYDV in selected sources of resistance
of winter barley (IG 57060)
The maintenance of BYDV-PAV isolate ‘Blatno 85’
and vector Rhopalosiphum padi was carried out in
the Department of Virology as a part of the project.
In collaboration with the Department of Breeding
Methods (J. Chrpová, V. Šíp) tests of resistance to
BYDV-PAV were conducted in different cultivars
and breeding lines of winter barley. The field tests
were conducted on small plots. Viruliferous aphids
R. padi were used for the transmission of BYDVPAV. The reproduction and infection of R. padi
were carried out in the Department of Virology. In
2005 in the RICP collection of material there were
of 41 samples from CRI Fiurenzuola d’Arda (Italy),
20 samples from IER Aschersleben (Germany), and
other 28 samples were from different foreign
countries.
Further,
double
haploid
lines
IGRI/ATLAS68 from the material of RICP were also
inoculated. Evaluation of symptoms was performed
according to Schaller and Qualset (1980). Overall,
cultivars from the Italian collection were
susceptible. Resistance to BYDV-PAV was also tested
in 20 samples from Aschersleben. A good level of
field resistance was recorded in 13 lines. A high
level of resistance in general is conferred in
samples by resistance carrying gene Yd2. (Kundu
J. K., Bouzková M.)
The study on resistance of Triticum aestivum
to BYDV (QG50076)
Barley yellow dwarf virus (BYDV), PAV isolate
‘Blatno 85’, and the vector aphid Rhopalosiphum
padi were maintained. Winter and spring wheat
cultivars and breeding lines were tested for their
resistance to BYDV-PAV (in collaboration with the
Department of Breeding Methods and Selgen Ltd.).
A field test was performed on small plots where
plants were inoculated with the virus by
viruliferous aphids (R. padi). In total 133 winter
wheat (in Ruzyne and Stupice) and 108 spring
wheat (in Ruzyne) cultivars and breeding lines
were tested. The virus infection was proved in
randomly collected samples by ELISA. (Kundu J.
K., Bouzková M.)
Co-ordination of Research on Genetic
Resistance to Plant Pathogenic Viruses, and
their Vectors, in European Crops (ResistVir)
(FOOD-CT-2005-006961, FP6 EC, priority 5,
Food Quality and Safety)
We attended and actively participated in
discussion at the Kick-off Meeting in Rome, Italy in
February 2005 and at the 6th Month Meeting in
Ankara, Turkey in July 2005. We were involved in
work on Package 3 “Mechanisms and sources of
resistance”. We elaborated a report on economically significant viruses and virus diseases of
agricultural crops in the Czech Republic. We
completed an address list of Biotechnology and
Plant Breeding Roof Federations and Companies in
the Czech Republic. (Polák J., Salava J.)
Collection of plant viruses and homologous
antibodies
Fourteen plant viruses and strains were
maintained and regularly inoculated to new
indicator plants (apple and pear strains of Apple
stem pitting virus, Beet western yellows virus,
Potato leaf roll virus, Turnip mosaic virus, plum,
peach and apricot strains of Plum pox virus, wheat
and barley strains of Wheat dwarf virus, Brome
mosaic virus, Barley yellow dwarf virus and Wheat
streak mosaic virus). Nineteen viruses and isolates
(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,
Pepper mild mottle virus, Strawberry latent ring
spot virus, Tomato aspermy virus, Tomato black
ring virus, Turnip yellow mosaic virus,
Watermelon mosaic virus 2 and Zucchini yellow
mosaic virus – 3 isolates) were reactivated on
indicator plants and dehydrated under calcium
chloride. In 2005 Potato potyvirus Y – pepper
isolate extended the collection. (Bouzková M.,
Červená Z., Hauptmanová A., Chaloupková M.,
Komínek P., Kumari S., Pívalová J., Polák J.,
Svoboda J., Širlová L.)
Reference laboratory for diagnostics
of selected plant viruses
The activities of reference laboratory were aimed
at testing the presence of quarantine Plum pox
43
virus (PPV). Molecular analysis of PPV strains in 41
samples from a survey showed the presence of PPVD strain only. PPV-M strain was detected in an
apricot orchard near Hrušky, Břeclav district. The
presence of PPV-Rec strain was not recorded. The
presence of PPV in RT-PCR positive samples was
confirmed by DAS-ELISA using polyclonal antibodies.
A survey was done on grapevines grown in
Southern Moravia for the presence of quarantine
nepoviruses Tomato ringspot virus and Tobacco
ringspot virus. In 48 tested plants no quarantine
nepoviruses were found. (Polák J., Komínek P.)
Report on expert activities
The main objectives in the area of diagnostics and
detection of cereal viruses were to test samples for
the presence of Wheat dwarf virus (WDV) and
Barley yellow dwarf virus (BYDV). Tests were
conducted mainly for the State Phytosanitary
Service. Fifteen samples of wheat and barley were
tested for the presence of WDV in the area of
Kladno and Slaný. Wheat samples were from the
following localities: Lukov, Zlonice, KnovízPopelka,
Knovíz
Švermov,
Slatina,
Bílek,
Slatina-Žlaby, Voříšek-Vyšínek, Hořešovice, Černoc),
and barley samples were from these localities:
Bílek, Bilichov, Hořešovice, Zbrašín). The presence
of WDV was proved in three samples. Eight samples
of wheat were also tested for the presence of WDV
and BYDV-PAV in the area of Morava. The presence
of WDV was recorded in five samples and BYDVPAV in one sample. (Bouzková M., Kundu J. K.)
Thirty-five trees of plums, apricots and myrobalans
were tested using ELISA for the presence of Plum
pox virus, Prunus necrotic ringspot virus, Prune
dwarf virus, Apple chlorotic leafspot virus and Apple
mosaic virus. Three trees of myrobalan were found
to be positive for Plum pox virus and one plum tree
was found to be infected by Prune dwarf virus.
(Polák J., Pívalová J.)
A grapevine plant of cv. Portugais Bleu was
suspected to be infected by a virus. The plant was
tested by ELISA for the presence of grapevine
viruses present in the Czech Republic: Grapevine
fanleaf virus – GFLV, Arabis mosaic virus – ArMV,
Grapevine virus A – GVA, Grapevine virus B – GVB,
Grapevine fleck virus – GFkV and a complex of
grapevine leafroll viruses: Grapevine leafrollassociated virus 1, 2, 3, 5, 6, 7 – GLRaV-1, 2, 3, 5, 6,
7. The tests did not prove that the symptoms were
caused by a virus, a possible explanation is damage
by herbicides. (Komínek P.)
Department of Bacteriology
The first report on Pseudomonas savastanoi
pv. nerii on oleander in the Czech Republic
(MZe ČR 0002700603)
The bacterium Pseudomonas savastanoi pv. nerii
was identified as the causal agent of
parenchymatous galls on leaves of potted oleander
plants grown in Brno, Moravia, in 2004. The plants
originated from cuttings made from firm shoots of
a supposedly asymptomatic plant grown in and
introduced from the Mediterranean region. The
Biolog GN microplate system was used to identify
the isolated bacterial strains. Successful inoculation
of Nerium oleander seedlings proved the
pathogenicity of the isolates. This is the first record
of P. savastanoi pv. nerii in the Czech Republic.
(Kůdela V., Krejzar V., Korba J.)
Destruction of chlorophyll in emerging
seedlings of spring barley associated with
environmental stresses (MZe ČR 0002700603)
An unusual disorder occurred on spring barley
seedlings in Southern and Central Bohemia in April
2005. Affected seedlings showed conspicuous
bleached or straw-coloured areas on tips of the first
leaves. There were very sharp, distinct, horizontal
boundaries between the green basal and
discoloured tip parts of the leaves. It was
remarkable that these boundaries were at the same
position on all affected first leaves of seedlings
from the same field. Affected seedlings were
randomly distributed in the fields, and their
incidence ranged from about 20 to 70% according
to the location. Based on meteorological data it can
be concluded that the colour abnormality in
emerging seedlings was associated with specific
environmental stresses, being effective in a certain
chronological order on sensitive plant tissues
during the period from 6 to 8 days after seeding.
These stresses include frosty mornings followed by
clear days with high solar radiation with a high UV
Index. (Kůdela V., Krejzarová R., Krejzar V.)
Occurrence of fruit doubles in the 2004
season associated with heat and drought
stress in the previous year (MZe ČR
0002700603)
A higher incidence of fruit doubles than the
normal one occurred in the Czech Republic in the
2004 season. Fruit doubles or twinning were most
frequently observed among plum fruits sold on
town markets, in mirabelle in small gardens in
Prague and its environs, and in apple, James Grieve
variety, on experimental plots of the Research
Institute of Crop Production in Prague-Ruzyně. In
other cases, only the terminal fruits were fully
developed, usually in one apple spur, while basal
fruits were retarded. Based on meteorological data
and literature reports, it can be concluded that the
higher incidence of fruit doubles in the CR in 2004
is correlated with heat and drought stress in 2003.
The period between August 3 and August 15, 2003,
was the time when differentiating and developing
flower buds might have been stressed and injured.
In this period, there were high temperatures (30 to
38.9°C) each day and trees suffered from water
stress. (Kůdela V., Krejzar V.)
Evaluation of epiphytic bacteria for potential
control of fire blight pathogen
(MZe ČR 0002700603)
In the Czech Republic, fire blight of rosaceous
plants, caused by Erwinia amylovora (Ea), was
first observed in 1986. Nineteen years later, in
2005, the disease occurs in all fruit-growing areas.
The use of antibiotics against the fire blight
44
bacterium is not allowed for field sprays in the CR,
similarly like in many other European countries.
The use of copper compounds is restricted because
of their phytotoxicity. Therefore, biological control
would be a welcome alternative to streptomycin
and other antibiotics. The purpose of our
investigation was to isolate microorganisms from
epiphytic microflora that might be antagonistic
against Ea. Among the isolated epiphytic
microorganisms, a total of 10 isolates of Gramvariable,
endospore-forming
bacteria
(GVS
bacteria) was obtained from blight-infected host
plants (Malus domestica, Pyrus communis and
Cotoneaster monogyna). The screening of their
antagonistic behaviour against Ea included agar
plate tests and pear fruit tests. Antagonistic
effectiveness of 10 isolates of GNS bacteria was
compared with the effectiveness of copper
hydroxide
(Koside
2005,
0.3%),
copper
oxychloride (Kupricol, 0.6%) and streptomycin
(500 ppm). In agar plate tests and pear fruit tests,
three strains of GNS bacteria (K 3Y, K 11, L1)
inhibited Ea comparably with copper compounds
and one strain (K 11) inhibited Ea comparably with
streptomycin. Although these GNS bacteria were
found promising in agar plate tests and pear fruit
tests, they have to be tested further in greenhouse
assays and under field conditions. (Korba J.,
Šillerová J.)
Reliability of diagnostic techniques
for Clavibacter michiganensis subsp.
michiganensis, the causal agent of bacterial
cancer of tomato (MZe ČR 0002700603)
Clavibacter
michiganensis
subsp.
michiganensis (Smith) Davis et al. (Cmm) is
a quarantine bacterium causing serious losses of
both glasshouse and field tomato crops in areas
where tomato is planted. Sensitivity and
specificity of polyclonal antibodies (Adgen Co.,
Scotland) to Cmm were verified using immunochemical techniques PTA-ELISA and IFA. Our
primers (Cmm 1F and Cmm 1R) designed for
detection of Cmm were used in PCR. Plant
pathogenic and saprophytic bacteria, potentially
associated with Cmm in plant and seed samples of
tomato infected by the causal organism, were
screened. Altogether 15 reference strains of
different bacterial species were tested in both
immunochemical methods and 8 of them in PCR
after the isolation of bacterial DNA. By means of
IFA, reference strains of Cmm were reliably
determined up to a concentration 104 cfu/ml. In
IFA tests, strong cross-reactions to the used
polyclonal antibody were found in coryneform
bacteria of C. m. subsp. insidiosus, C. m. subsp.
sepedonicus and C. m. subsp. nebraskensis. Slight
cross-reactions occurred in saprophytic bacteria
Pantoea dispersa, P. agglomerans and Rahnella
aquatilis and also in pectinolytic erwinias
Erwinia chrysanthemi and E. carotovora subsp.
carotovora. By means of PTA-ELISA, reference
strains of Cmm were reliably determined up to
a concentration 105 cfu/ml. In PTA-ELISA, crossreactions to the used polyclonal antibody
occurred in C. m. subsp. insidiosus, Curtobac-
terium flaccumfaciens, E. c. subsp. atroseptica
and E. chrysanthemi. PCR showed much higher
specificity than the used immunochemical
techniques. A strong positive reaction was shown
to the target organism, while only very slight
false positives occurred in C. flaccumfaciens
and Ralstonia solanacearum. (Kokošková B.,
Mráz I.)
Comparison of immunochemical and
molecular techniques for reliable detection
of fire blight bacterium (MZe ČR 0002700603)
Erwinia amylovora (Burrill) Winslow et al.
(Ea), causing fire blight, is a harmful quarantine
organism. Fire blight is one of the most serious
diseases affecting fruit-bearing and ornamental
plants of the family Rosaceae that has spread in
many countries all over the world. Diagnosis of
the pathogen is based on IFA and bioassay on
immature pear fruit, and also other diagnostic
techniques are recommended to use for diagnosis
of the fire blight organism. PTA-ELISA and IFA,
working with polyclonal antibodies of Adgen Co.
(Scotland), were used for the testing of 81 isolates
of Ea and 38 symptomatic plant samples.
Sensitivity of PTA-ELISA reached the value 106-5
CFU/ml, but IFA and PCR were ten times more
sensitive, i.e. 105-4 CFU/ml. IFA was not only more
sensitive but also more specific than PTA-ELISA,
because 11 cross-reactions were recorded using
IFA in spite of 16 ones in PTA-ELISA. IFA appeared
as very sensitive, particularly for the screening of
plant tissue homogenates. At a dilution 1:100 and
1:000, Ea was detected using IFA in 100% and 89%
of plant samples, respectively. In PTA-ELISA, Ea
was reliably detected only up to a dilution 1:100.
In PCR tests, our primers (Ea f72 and Ea r560)
designed for the detection of Ea were used. PCR
was much more specific than PTA-ELISA and IFA
because no false positives were observed.
(Kokošková B., Mráz I., Hýblová J.)
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 a research station of RICP in Slany.
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 pathogen was
verified by testing on forced shoots of Pyrus
ussuriensis. The level of resistance was determined
from the percentage proportion of bacterial lesions
in the total length of shoots (in cm) after 40 days.
We used an adjusted USDA scale for evaluations.
The tests proved high resistance to fire blight in the
cultivar ‘Bohemica’ (higher than in ‘Alexander
Lucas’) and ‘Jana’. This resistance was comparable
to that of newly bred US-625-63-4. On the other
hand, cultivars ‘Isolda’, ‘Vonka’, ‘Beta’, ‘Decora’ and
‘Petra’ showed strong symptoms of infection.
45
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 in 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 from the aspect of growth and yield.
(Paprštein F., Kosina J., Korba J., Šillerová J.)
Collection of plant pathogenic bacteria
and antibodies
The collection of plant pathogenic bacteria and
antibodies contains about 930 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 maintained in microbanks at –70° C and /or as
lyophylisates. In 2005 the collection was enlarged,
mostly by accessions of strains of Streptomyces
aureofaciens, S. albidoflavus, S. europaeiscabiei, S.
intermedius, S. ipomoeae, S. reticuliscabiei, S.
scabiei, S. stelliscabiei and Xylophilus ampelinus
and others and of reference strains of some plant
pathogenic bacteria.
The collection also comprises about 54
polyclonal antibodies from our production and 21
commercial antibodies (Agdia, Adgen, LÖEWE) for
detection
of
plant
pathogenic
bacteria.
(Krejzarová R., Pánková I.)
Reference laboratory for diagnostics
and monitoring of plant pathogenic
quarantine bacteria
For the needs of the State Phytosanitary Authority
(SPA) and other organisations, diagnostic analyses
of plant samples suspicious of the presence of
quarantine and other important bacterial
pathogens
were
conducted.
Pseudomonas
syringae pv. syringae was confirmed in samples
of pear and apricot plants from Southern Moravia.
In samples of tomato plants from glasshouses
in Southern Moravia, the strong infection by
Clavibacter michiganensis subsp. michiganensis,
the causal agent of bacterial canker of tomato, was
confirmed, and Pseudomonas corrugata and
Pseudomonas syringae pv. tomato were also
recovered. In samples of geranium plants from
a glasshouse area in North-Western Bohemia,
the presence of Xanthomonas hortorum pv.
pelargonii, the causal agent of geranium blight, was
confirmed. Reference strains of Clavibacter
michiganensis subsp. sepedonicus and Ralstonia
solanacearum used as standards in immunochemical, molecular and biological tests were
released for diagnostic laboratories of SPA in
Olomouc and Research Institute of Potatoes in
Havlíčkův Brod. As usual, the staff of Dept. of
Bacteriology gave lectures and practicals for
undergraduate and postgraduate students, and
plant pathologists from research institutes and SPA.
(Kokošková B., Bryxiová M.)
Effectivity of essential oils against
pectinolytic erwinias and pseudomonads
Soft rot bacteria infect many species of plants.
They might cause high economic losses on
vegetables and ornamental flowers planted in
glasshouses and fields because symptomatic plants
are refused on the market. Chemical compounds
have been used against soft rot bacteria, however
resistance to them occurring in bacterial
populations decreases their effectivity, and
therefore new chemicals should be available on the
market. In our experiment, about 30 essential oils
obtained from various plants were tested if they
inhibited pectinolytic erwinias and pseudomonads.
The antimicrobial activity tests were conducted in
vitro. The inhibitory zones were measured on agar
plates where extracts were dropped. Streptomycin
and erythromycin were used as controls.
Pseudomonas marginalis pv. marginalis – a weak
inhibitory activity, but higher than in streptomycin,
was found in essential oils from Citrus aurantifolia,
Lavandula latifolia, Melaleuca quinquenervia, M.
spicata, Melissa officinalis, Origanum majorana,
Pelargonium roseum, P. graveolens, Rosmarinus
officinalis and Thymus mastichina (zones 6 – 10
mm); medium inhibitory effectivity was proved in
essential oils from Ocimum basilicum, Tagetes
bipinnata and Tsuga canadensis (zones 11 – 15
mm); essential oils from Origanum compactum, O.
vulgare and Thymus vulgaris (zones 16 – 20 mm)
showed a strong inhibitory activity. Erwinia
chrysanthemi – a weak inhibitory activity, but
higher than in erythromycin, was found in essential
oils from Eugenia caryophyllata, Lavandula
angustifolia, L. latifolia, Melaleuca quinquenervia,
Melissa officinalis, Mentha pulegium, Origanum
majorana, Pelargonium graveolens, P. roseum,
Rosmarinus officinalis, Salvia officinalis, Thuja
occidentalis and Thymus mastichina (zones 6 – 10
mm); medium inhibitory effectivity was proved in
essential oils from Artemisia absinthium and
Ocimum basilicum (zones 11 – 15 mm); essential
oils from Origanum compactum, O. vulgare and
Thymus vulgaris (zones 16 – 20 mm) showed
a strong inhibitory activity. (Kokošková B., Pavela R.)
Effectivity of essential oils against
Xanthomonas hortorum pv. pelargonii, the
causal agent of bacterial blight on geraniums
Xanthomonas hortorum pv. pelargonii (Brown)
Vauterin et al. (Xhp) is an important plant
pathogenic bacterium causing wilting, leaf spot and
stem rot of plants resulting in serious losses in areas
where geraniums (Pelargonium spp.) have been
planted. The pathogen is transmitted by seed,
disseminated by infected nursery plants and
cuttings where it latently persists. The choice of
chemicals affective against Xhp on the market is not
sufficient. In our experiment, about 30 essential
oils obtained from various plants were tested for
antimicrobial effectivity against Xhp. The screening
was conducted in laboratory conditions on agar
plates, where averages of inhibitory zones were
measured. Streptomycin in concentrations of 0.02
– 0.2% was used as control (zones 2.5 – 7.0 mm).
46
Weak bactericidal effectivity against Xhp was found
in essential oils from Lavandula latifolia,
Melaleuca quinquenervia, Mentha spicata, Tagetes
bipinnata and Zingiber officinale (zones 7 – 10
mm), medium effectivity was found in essential oils
from Citrus aurantifolia, Eugenia caryophyllata,
Lavandula angustifolia, Melissa officinalis,
Mentha arvensis, M. citrata, M. pulegium, Nepeta
cataria, Origanum majorana, Rosmarinus
officinalis, Thuja occidentalis and Thymus
mastichina (zones 11 – 20 mm); strong inhibitory
effectivity was proved in essential oils from
Artemisia absinthium, Ocimum basilicum and
Salvia officinalis (zones 21 – 30 mm); the strongest
bactericidal effectivity was determined in essential
oils from Origanum compactum and Thymus
vulgaris (zones 31 – 50 mm). (Kokošková B.,
Pavela R.)
Study of Pseudomonas sp. bacteria related
to larvae of some species of the family
Chloropidae
The investigation of Pseudomonas sp. related to
larvae of selected species of the family Chloropidae
harmful on cereals and grasses focused on
Pseudomonas syringae was finished. Out of 80
recovered bacterial strains from the phyllosphere
of plants, about 2/3 were identified using a Biolog
Bacteria method. Fluorescent pseudomonads such
as Pseudomonas fluorescens, P. syringae, P. putida
and P. viridiflava were found the most frequently.
Patovars of P. syringae were not reliably identified
because the Biolog Bacteria method does not make
it possible. P. syringae pv. syringae strains were
also confirmed by serological and HR tests on
tobacco. Different strains of gram-negative and
gram-positive bacteria were isolated from digestive
tracts of larvae and tissues of galls formed by larvae.
Among the isolates from plants, six ice nucleation
strains belonging to the species Pseudomonas
syringae were found. (Kokošková B., Horváthová
M., Zámečník J.)
The occurrence of wheat leaf spot pathogens in samples collected
in the Czech Republic in 2005
Department of Mycology
Occurrence of cereal fungal diseases
(the species of the genera Fusarium,
Pyrenophora, Mycosphaerella, Phaeoseptoria,
Pseudocercosporella, Gaeumannomyces,
Plasmopara) in some agricultural crops
(cereals, sunflower, fruit-trees)
Research on the occurrence of cereal pathogenic
fungi (MZe ČR 000270603)
The occurrence of pathogenic fungi was
influenced by the dynamics of meteorological
conditions. In relatively warm weather conditions
in January the fungus Mycosphaerella graminis
was widespread in the form of ascospores
practically on all stands. The fungus was in infested
leaves in the latent phase and after snowfall at the
end of March many dark fruit-bodies were
observable on the leaves. The following spread of
the pathogen was limited by dry spring. The fungus
was widespread at the end of May and at the
beginning of June. The spots were found out on
almost all stands of wheat (namely winter wheat),
but the losses of yield were very low. The glume
blotch (Phaeoseptoria nodorum) occurred on all
stands only to a very small extent. The spread was
also influenced by dry weather conditions. This
fungus was recorded in all regions of the Czech
Republic at the end of ripening. Higher occurrence
was local without greater influence on grain
production. The powdery mildew (Blumeria
graminis) occurred less than in the previous year
and it was only a very weak infection in the ears.
The rust occurrence was also very weak. The
occurrence of the genus Fusarium was mainly
influenced by the forecrop. The highest influence
was found out after maize for grain, and also after
clover plants and cereals (except oat). The
infection was not visible on the leaves and the ears,
the highest spread was before ear ripening. The
occurrence was supported to the highest extent by
the forecrop namely maize for grain, clover plants
and cereals (except oat). The
occurrence on leaves was
only very weak, the conditions
during flowering were not
favourable for the infection
spread. The fungus was widespread during ear ripening.
Especially at the end of wheat
ripening we observed ear
fusarioses in many wheat
localities (untreated with suitable fungicide). The intensity of
the infestation depended on
local conditions (forecrop and
dynamics of weather conditions). Fusarium graminearum
prevailed in 2005. The eyespot
and sharp eyespot, and the takeall (the pathogens Pseudocercosporella
herpotrichoides,
Rhizoctonia sp. and Gaeumannomyces graminis) occurred only locally (the dependence
on the forecrop and humidity).
47
Higher infestation was local and limited to the parts
of the parcels. The cereal infestation was weaker in
2005 than in the previous year 2004. The
occurrence was influenced by spring drought and
dry weather conditions at the time of flowering and
at the beginning of grain development in the ears.
The conditions during ripening in most areas in the
Czech Republic were not favourable for pathogen
spreading. We obtained the samples of tan spot of
wheat from 5 districts (Jičín, Pelhřimov, Svitavy,
Třebíč, Znojmo) and from 7 localities of the Czech
Republic. The isolation and determination of the
species spectrum of phytopathogenic fungi were
done by the method of humid chamber and
microscopically. We examined 17 samples by these
methods. The highest occurrence was found in
Mycosphaerella graminicola (59% of samples),
Phaeoseptoria nodorum (53%), Pyrenophora
tritici-repentis (41%) and Ascochyta sp. (12%)
(Graph). We prepared 9 monosporic isolates of
Pyrenophora tritici-repentis which are deposited in
our fungal collection.
Natural infestation of the fungi – the genus
Fusarium on cereals (wheat and barley) was very
weak in the year 2005. Fusarium graminearum
was the most frequently prevailing species like in
the previous year. We did not find out any new
sources of resistance. The samples of brown leaf
spotting of wheat were obtained in 2005 from 12
districts and 15 localities of the Czech Republic.
Pyrenophora tritici-repentis was in 40% of the
samples of cereals, Phaeoseptoria nodorum was
isolated from 29% of samples, Mycosphaerella
graminicola from 13% and Cochliobolus sativus
from 9% of samples. From this collection we
prepared 38 monosporic isolates of Pyrenophora
tritici-repentis which are deposited in our
collection of fungi for subsequent testing. (ŠárováPalicová J., Hýsek J., Sychrová E.)
Study of aetiology of charcoal disease
of sunflower (MZe ČR 000270603)
The fungus Macrophomina phaseolina (Tassi)
Goid. is one of the infection sources of charcoal
disease of sunflower. This warm-requiring
polyphagous fungus did not occur in this country
previously. Since 1999 we have observed its
occurrence. The occurrence on other crops was
not observed. Macrophomina phaseolina was
mostly found out in the area with less rainfall in the
districts Žatec, Louny and Kolín. Scarce findings
were in Southern Moravia. The highest occurrence
was in 2004 and also in Moravia. The comparison of
weather dynamics in the Czech Republic and in
Southern Europe showed that in the years 19992004 summer temperatures were very extreme.
The temperatures reached the average of the
regions of Southern Europe where the disease is
normally present. In comparison with previous
years the dynamics of weather conditions was very
different in 2005. In the time that has the main
influence on the disease dynamics on leaves and
stalks the weather was colder and damper. The
plant infestation of leaves and stalks was higher
than in previous years. (Šárová-Palicová J.,
Veverka K., Kudlíková I.)
The occurrence and the intensity of the pathogen
Macrophomina phaseolina in studied stands in the Czech
Republic in 2005
Locality
Stand
No.
Infestation
with M.
phaseolina
Prague–West Středokluky
1
–
Louny
Postoloprty
2
–
Postoloprty
3
–
area
place
Bítozeves
4
–
Vidovle
5
–
Seménkovice
6
–
Pavlov
7
–
Pavlov
8
–
Jeneč
(Pavlov direction)
Český Brod
9
–
10
–
Rostoklaty
11
–
Č. Brod
12
–
Přišimasy
13
–
Vitice
14
–
Tuklaty
Vitice
15
–
Lipany
16
–
Southern
Brno – Pisárky
17
–
Moravia
Chrlice
18
–
Blučina
19
–
Slavkov
20
–
Hustopeče
21
–
Velké Pavlovice
22
–
Rakvice
23
+++
Rakvice
24
–
Velké Bílovice
25
–
Velké Bílovice
26
–
Podivín
27
–
Ostrožská Lhota
28
–
Ostrožská Lhota
29
–
+++ highest occurrence
–
no occurrence
The control of the occurrence of pathogenic,
potentially pathogenic and saprotrophic
fungi on fruit tree species and on small fruit
shrubs (MZe ČR 000270603)
This year we investigated the qualitative and
quantitative composition of leaf mycoflora on
apple trees from two localities: one locality was
Bítouchov near Semily (Northern Bohemia) and the
other locality was Praha-Ruzyně (Central Bohemia).
The samples were taken on three dates (in May, July
and October). We took the samples in both
localities (10 leaves from 5 trees). The leaves were
divided into leafstalks and leaf blades. We
investigated 300 leaves in total. The samples were
taken into polyethylene bags. The leaves were
washed with running water and superficially
48
sterilized (70% ethanol for 30 s, sodium
hypochlorite (NaClO) with 1% Cl for 1 min, 70%
ethanol for 15 s) and rinsed in sterile water. They
were cut into segments 3-5 x 3-5 x 1 mm in size
which were put in 5 samples on 2% malt extract
agar in Petri dishes. The incubation was done at 1820° C and alternation of artificial and black-light
lighting. After 4 weeks the cultures were isolated
and determined.
In the Bítouchov locality we isolated and
determined 13 species of the fungi. A part of the
isolates did not form any sexual or asexual
structures necessary for correct determination. The
most frequent species were: Alternaria alternata,
Aureobasidium pullulans, Cladosporium herbarum and Spilocea sp. from Venturia inequalis.
A list of the fungal species occurring in the Bítouchov locality
Alternaria alternata
arthroconidial hyphomycete
Aureobasidium pullulans
Cladosporium cladosporoides
Cladosporium herbarum
Nodulisporium sp.
Phoma sp.
Pleurophoma cava
Seimatosporium sp.
Spilacea sp. from
Venturia inaequalis
A list of the fungi occurring in leaves
Alternaria alternata
basidiomycete sp. 1
basidiomycete sp. 2
arthroconidial hyphomycete
Botrytis cinerea
Cladosporium cladosporoides
Cladosporium sphaerospermum
Cladosporium macrocarpum
Fusarium sp.
In the Praha – Ruzyně locality we isolated and
determined 15 fungal species. A part of the isolated
fungi did not form any sexual or asexual structures
for the determination of the fungal genus and they
are not mentioned. The most frequent species were
Alternaria alternata, Aureobasidium pullulans
and Cladosporium herbarum.
A list of the fungal species occurring in the Praha-Ruzynû
locality
Aureobasidium pullulans
basidiomycete sp. 1
Botrytis cinerea
Cladosporium cladosporoides
Cladosporium herbarum
Epicoccum nigrum
Geniculosporium sp.
in two localities in the Czech Republic that were
chosen deliberately according to their different
climate: Research Station for Viticulture in
Karlštejn and in Nosislav (south of Brno) in
Southern Moravia.
The endophytes of bark, xylem and periderm,
nodes and internodes, leafstalks, leaf blades and
venation were isolated from 80 branches from two
localities. The fungi were determined from more
than 4 500 segments from the Vrše vineyard in
Karlštejn and from the vineyard in Nosislav. We
determined 13 fungal species, and 14 fungal species
in the branches. The fungi were found out in more
than 40% of the segments. In all tissues these
microscopic fungi predominated: Alternaria
alternata, Aureobasidium pullulans, Phoma spp.,
Cladosporium cladosporoides and Cladosporium
herbarum.
Nodulisporium sp.
Phoma sp. 1
Phoma sp. 2
Phomopsis sp.
Pleurophoma cava
Sordaria sp.
pilacea sp. from
Venturia inaequalis
This year the mycoflora of trunks and roots from
infected young pears from the Slaný locality (the
station of RICP) was studied. The samples were
examined in the same way as the samples from
apple trees. We found out 4 fungal species in the
roots:
Cylindrocarpon
sp.,
Fusarium
sp.,
Gliocladium roseum and Phomopsis sp. The
prevailing genera were Cylindrocarpon sp. and
Fusarium sp., which occurred in more than 50% of
the samples. In trunks 4 fungal species were
detected: Alternaria alternata, Phomopsis sp.,
Microphaeropsis sp. and Fusarium sp.
We also continued our research to determine the
mycoflora in one-year branches of grapevine,
which began last year. The research was conducted
Epicoccum nigrum
Fusarium sp.
Hormonema
dematiodes
Penicillium sp.
Phoma spp.
A list of the fungi occurring in one-year branches:
Alternaria alternata
Aureobasidium pullulans
Botrytis cinerea
Diplodia sp.
Cladosporium cladosporoides
Cladosporium herbarum
Cladosporium sp
Epicoccum nigrum
Fusarium sp.
Hormonema
dematiodes
Penicillium sp.
cf. Phialophora
Phoma spp.
Seimatosporium sp. 2
(Novotný D.)
Occurrence of pathogenic and potentially
pathogenic and saprophytic fungi in fruit
trees and grapevine (MZe ČR 000270603)
The mycobiota of apple-tree leaves and pear-tree
stems and roots was investigated. The samples from
apple trees were taken in 2 localities of the Czech
Republic. Thirteen species were detected in the
leaves of apple trees. Alternaria alternata,
Aureobasidium pullulans, Cladosporium herbarum and Spilocea sp., Venturia inequalis were
dominant species of leaves. The samples from pear
trees were taken in one locality. Four species were
isolated from roots while Cylindrocarpon sp. and
Fusarium sp. occurred most frequently. Four
species were recorded also in stems. Alternaria
alternata and Fusarium sp. were dominant species.
The occurrence of fungi in 1-year-old branches and
leaves of grapevine from two studied localities was
investigated. Fungi were isolated from more than
40% of the segments of leaves and branches. So far
fourteen species were detected in the leaves and
branches of investigated grapevine. Alternaria
49
alternata, Phoma spp., Aureobasidium pullulans,
Cladosporium cladosporoides and Cladosporium
herbarum were dominant species of the
investigated parts of Vitis vinifera. (Novotný D.)
Puccinia graminis genotypes formed an individual
group and they were clearly distinguished from
Puccinia coronata f. sp. avenae genotypes.
(Klenová H., Šebesta J.)
Study of the resistance of selected
agricultural crops to economically important
fungal species in 2004 (MZE ČR 000270603)
Pathologic and molecular methods of detection
of resistance of the genus Avena to Puccinia
coronata f. sp. avenae. The inheritance of oat
crown rust resistance was investigated in the line
Pc 50-4 (Avena sterilis L.). Hybridological F2 and F3
analyses performed in field and glasshouse
conditions showed that in the Pc 50-4 line the
resistance to recently identified pathotypes of oat
crown rust was conditioned by one major gene. It
was shown that the Pc 50-4 line did not contain any
other independent resistance gene(s) effective in
the control of the used pathotypes. In total 71
isolates of Puccinia coronata f. sp. avenae were
used to define the current efficiency of the gene Pc
50-4. Analyses were performed in the juvenile stage
of the plant in glasshouse conditions. The
efficiency of Pc 50-4 is very high, the current
valuation is 0.9296.
Resistance efficiency of selected Pc genes was
evaluated. The Pc genes were divided into four
groups according to the value of resistance
efficiency. The first group with low efficiency
containing the genes Pc 40, Pc 45, and the cv. Azur
had the value of efficiency 0–0.399. The second
group with medium efficiency comprising the
genes Pc 38, Pc 46, Pc 51, Pc 54, Pc 56, Pc 64, Pc 67
and the accessions of Avena sterilis, VIR 343-1 and
VIR 343-2 had the value of efficiency 0.400–0.799.
The third group with high efficiency having the
value 0.800–0.899 contained Pc 60, Pc 61, Pc 96
and the cv. Vok. The last group with very high
efficiency had the value 0.900–1.00. This group
consisted of Pc 39, Pc 48, Pc 50-2, Pc 50-4, Pc 52, Pc
54-1, Pc 55, Pc 58, Pc 59, Pc 62, Pc 68 and Pc 94.
Molecular characterization of 17 different
Puccinia coronata f. sp. avenae pathotypes by
means of RAPD (Random Amplified Polymorphic
DNA) method was finished. Two different
pathotypes of Puccinia graminis were the positive
control. Forty-six random primers from the
Operon technologies (Inc. Alameda, Ca. USA) were
used in the analyses. The banding pattern of 45
primers was polymorphic; only 1 primer provided
a monomorphic pattern. No primer alone (out of
the 46 studied) could differentiate all the Puccinia
coronata f. sp. avenae genotypes. The level of
polymorphism was different with different primers
between different genotypes. A similarity matrix
for Nei and Li’s coefficient of 17 oat crown rust
genotypes was created. Coefficients of similarity
varied from 0.69444 to 0.90291. A dendrogram of
19 rust genotypes (seventeen Puccinia coronata f.
sp. avenae genotypes and two Puccinia graminis
genotypes) developed from RAPD data using the
unweighted pair group of arithmetic means
(UPGMA) was constructed. Four associated groups
and three detached isolates were identified from
Puccinia coronata f. sp. avenae genotypes.
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
The pathogenicity of Macrophomina
phaseolina isolates, the preparation
of antibodies and their characteristics
(MZe ČR 000270603)
During the vegetation period we monitored
sunflower stands and took samples of plants
suspicious of the infestation by M. phaseolina. The
occurrence of this pathogen was proved in one
stand. The isolation of the pathogen was not
successful. We confirmed the pathogenicity of
some collection strains. The strains were cultivated
on a liquid nutrient medium and from mycelial
mass. We prepared the following antigens:
Antigens of Macrophomina phaseolina
No.
Collection strain No.
Type of antigen
A329b
78
extracellular proteins
A333
85
total extract
A334
85
intracellular proteins
A394a
72
intracellular proteins
A394b
72
total extract
A395a
76
intracellular proteins
A395b
76
total extract
A396a
78
intracellular proteins
A396b
78
total extract
A397a
95
intracellular proteins
A397b
95
total extract
These antigens were used as immunogens for the
preparation of polyclonal antibodies and for the
characterisation of prepared polyclonal antibodies.
(Šárová-Palicová J., Veverka K., Kudlíková I.)
The antigen preparation for the diagnostics
of Plasmodiophora brassicae (MZe ČR
000270603)
We continued the preparation of the antigen for
antisera. We obtained two isolates of the pathogen
from the Velenka locality (kohlrabi, cabbage). The
antigen was prepared in the form of a suspension
of persistent spores from plant tumours on
Chinese cabbage. Persistent spores were separated
from plant cells in three steps by means of
centrifugation based on the saccharose gradient
(step 1 and 2: 0.5 – 2.5 M saccharose, step 3: 0.6 –
1.0 M saccharose). The prepared antigen (the
concentration of about 3x105 cfu/ml) was used
for the immunisation of rabbits. (Kudlíková I.,
Krátká J., Chalupníková J.)
50
The enlargement of the collection
of pathogenic species Phytophthora,
Verticillium and Colletotrichum, the
preparation of antigens (MZe ČR 000270603)
This year (2005) we isolated the following strains
of important phytopathogenic fungi in some
localities. The most important species was
Colletotrichum acutatum on strawberries and
cherries from the area of the CR. This species was
evaluated in three localities and two areas of the
CR. This fungus was determined on the basis of
micromorphological, macromorphological, physiological and molecular-genetic signs. We also
isolated a strain of the genus Colletotrichum from
bean (Lupinus polyphylus) and its determination
on the species level is prepared. The cultivation and
the preparation of antigens of some strains of
Colletotrichum acutatum isolated from strawberries were carried out. (Kudlíková I., Novotný D.,
Krátká J., Chalupníková J.)
The purification of polyclonal antibodies
and hybridomas prepared for the antibody
production for detection of the pathogen
Macrophomina phaseolina (MZe ČR
000270603)
The antigen in the form of intracellular proteins
isolated from the pathogen mycelium was used
for the immunisation of laboratory rabbits by a
standard immunisation scheme (4-5 immunisation
subcutaneous doses in a three-week interval,
protein content in the immunisation dose was 100
– 250 µg). We isolated IgG from the serum of
immunised animals by precipitation with
ammonium sulphate and by means of affinity
chromatography (A-Sepharose). We purified two
monoclonal antibodies (anti-M. phaseolina K-65
and anti-M. phaseolina K 66). Their specificity and
sensitivity were tested by means of PTA-ELISA. The
antibody anti-M. phaseolina K-65 showed higher
sensitivity. Its detection limit was evaluated to be
70 ηg of proteins of M. phaseolina.ml-1 at a working
concentration of IgG 1 µg.ml-1.
Cross reaction of antibody anti-M. phaseolina K65 (%)
Antigen
Macrophomina phaseolina
Isolate
72
Type
Concentration
of antigen 1 µg/ml 10 µg/ml
total extract
11
26
Macrophomina phaseolina
76
total extract
13
12
Macrophomina phaseolina
78
total extract
15
57
Macrophomina phaseolina
95
total extract
37
68
Macrophomina phaseolina
78
total extract
32
74
Macrophomina phaseolina
85
immunogen
100
100
Pythium ultimum
total extract
4
9
Sclerotinia sclerotiorum
total extract
0
7
Botrytis cinerea
total extract
1
3
Polyclonal antibody anti-M. phaseolina K65 was
evaluated as sufficiently susceptible and highly
specific, and it was used for subsequent tests of the
pathogen in the host’s tissues. The immunisation of
laboratory mice was done with antigens A334 and
A335. We evaluated the titre of antibodies in the
serum during the immunisation cycle. Because
none of the mice reached the titre 1:25 000 or
higher, we decided to give up the preparation of
hybridomas. For successful productive hybridomas
we have to prepare an immunogenic antigen.
(Kudlíková I., Krátká J., Chalupníková J.)
Botrytis cinerea – conidiophor with conidia
The purification and characterization of
polyclonal antibodies for the determination
of Plasmopara brassicae (MZe ČR 000270603)
We purified IgG from the serum of an immunized
rabbit by the method of affinity purification on the
protein A. The cross-reactions against the following
soil fungal pathogens were evaluated: Fusarium
solani, Botrytis cinerea, Sclerotinia sclerotiorum,
Trichoderma harzianum, Pythium ultimum,
Phytophthora cactorum and Phytophthora cryptogea. We found out cross-reactions against Trichoderma
harzianum,
Botrytis
cinerea
and
Sclerotinia sclerotiorum. The susceptibility of the
prepared antibody was evaluated by means of PTAELISA as 5 µg of hydrolyzed proteins of persistent
spores.ml-1. (Kudlíková I., Krátká J., Chalupníková J.)
Evaluation of the effects of biopreparations
on the soil mycoflora in relation to
phytopathogenic fungi and ways of
management
The effects of biopreparations on a decrease
in fungal diseases of cereals (MZe ČR
000270603)
In 2005 a small-parcel experiment was
established with spring barley (cultivars Akcent
and Tolar). All treated variants were fertilized with
the same dose of ammonium sulphate 30 kg N/ha
and in the phase of the 3rd leaf 30 kg N/ha was
added. The seed of the cultivar Akcent was not
chemically treated contrary to the cultivar Tolar,
whose seed was treated with Vitavax. We
established the control variant without fertilizing
and without any treatment, and the variant
fertilized only with the dose of ammonium sulphate
51
The yield of winter wheat after different ways of soil cultivation in 2005
but later not treated. The cultivar Akcent had
a lower yield as a result of no seed treatment in
comparison with the cultivar Tolar. The best results
were obtained for the seed treatment with
Supresivit and for the combination of ammonium
sulphate with Supresivit. The genus Fusarium was
observed on treated variants to a smaller extent, no
occurrence of eyespot and take-all was recorded.
The occurrence of pathogenic species of the genus
Fusarium in the soil was very limited (F. culmorum,
F. graminearum). The figure shows 3 variants of
winter wheat and their yields. (Hýsek J., Vach M.)
The influence of the year (2005) on
the spectrum of soil and plant pathogens
and their influence on crop yields in
connection with the way of cultivation
(MZe ČR 000270603)
The analysis of the spectrum of soil pathogen
species was carried out after different ways of
winter wheat cultivation. We observed a lower
number of soil fungi of the genus Fusarium and a
lower number of its toxicogenic species (F.
culmorum, F. graminearum) in the conventional
way of cultivation. The method with shallowploughed straw resulted in a higher level of the
infestation with Helminthosporium and increased
the population of this fungus by about 20% and
also the population of the genus Fusarium in the
soil (more than 10%) and the occurrence of
toxicogenic species F. culmorum and F.
graminearum. The results in the previous year
were different because the number of plants
infested by Helminthosporium was lower.
(Hýsek J., Sychrová E.)
The study of harmfulness of pathogenic
and potentially pathogenic fungi in some
crops in relation to the quality of agricultural
products (MZe ČR 000270603)
We investigated the infestation of winter wheat
by phytopathogenic fungi, namely by the genus
Fusarium, and its influence on seed quality. Among
physical parameters we examined the humidity
(%), and we also studied the falling number,
nitrogen matters (%), content of aleurone, gluten
index and sedimentation test. The falling number
for winter wheat is the time in seconds for the fall
of a stirrer to a limited distance in gelatinized
suspension (minimal value for winter wheat is 250
sec). The values are influenced by the locality.
Sedimentation test indicates the sedimentation
value which is influenced by a higher content of
aleurone and its higher swelling ability – the cause
of lower sedimentation. According to the
assumptions the fertilized variants had a higher
content of nitrogen matters and also aleurone in
comparison with unfertilized variants. If we
compare the values obtained in variants with
conservation tillage with conventional variants and
with regard to the level of nitrogen fertilization, the
results were very variable on average of the
particular years. Gluten index was defined as the
portion of wet aleurone in the total amount of
washed aleurone which remained on the surface of
the sedimentation sieve after centrifugation.
Optimal values from the aspect of suitability for
bread-making were 60-90. In the experiments with
winter wheat EBI these values were in this
limitation amount. A higher trend was observed on
soil protecting variants. (Hýsek J., Sychrová E.)
52
The effect of the quarantine fungus
Verticillium albo-atrum on different varieties
of Czech hop (1G 46060)
The artificial infection of different varieties of
Czech hop was done, both on young plants
cultivated from explant cultures and on older
plants. The plants were infected with different
strains of the quarantine fungus Verticillium alboatrum obtained from Slovenia, England and
Netherlands. We prepared a spore suspension (the
concentration of 106 conidia/ml) and applied it to
cut roots. Adult plants died within 2 months and
young plants after one month. Tracheomycosis of
plants was on the leaves in the form of mosaic and
the plants withered and also died. A lower
infestation was found in the cultivars (new lines)
OK-71 and OK-72. We reisolated the quarantine
fungus which caused this disease. Koch’s
postulates were confirmed. Fungicidal preparations (Ridomil Gold 42,5 WP, Aliette 80 WP,
Cuproxat SC, Aliette Bordeaux Cursate K and
Kuprikol 50) did not control this fungus in the
concentrations of 0.05% and 0.1%. Biological
preparations Supresivit and Ibefungin also had
only very weak effects, the biopreparation
Polyversum had no effect. (Hýsek J.)
Ecology, identification and diagnostics
of selected phytopathogenic fungi of fruit
trees with special respect to the EU
requirements (QF 4074)
The ecology and occurrence of Pezicula
malicorticis, P. alba, Glomerella cingulata and
Phytophthora spp. in apple and pear trees in the
Czech Republic were investigated. Immunodiagnostic methods for detection of the abovementioned fungal species were developed and
optimized. The composition of the fungal
community of healthy branches and frequency of
occurrence of the above-mentioned fungal species
(excluding Phytophthora spp.) in apple-tree
branches were investigated in two study sites. Until
now Glomerella cingulata, Pezicula malicorticis, P.
alba have not been recorded in apple- or pear-tree
branches but different species of Cryptosporiopsis
Sordaria fimicola from the apple’s leaf
(anamorphic state of Pezicula) were isolated. More
than twenty fungal species were detected in
healthy apple-tree branches. Pleurophoma cava,
Alternaria alternata, Aureobasidium pullulans,
Seimatosporium cf. pestalotioides, Phomopsis cf.
mali, Coelomycete sp. 1 and Coniothyrium cf.
olivaceum were dominant fungi in healthy
branches. Antigens from Pezicula malicorticis and
P. alba were prepared in the form of total and
protein extracts. Antigens and antiserums were
prepared from Phytophthora cinnamomi, P.
cambivora and P. syringae. (Novotný D.,
Kudlíková I., Šárová-Palicová J.)
Species, ecology and distribution
of ophiostomatoid fungi in the Czech
Republic (GA ČR 206/05/P279)
Diversity of ophiostomatoid fungi in the Czech
Republic was investigated. The fungi were isolated
Colony growing from the apple’s leaf
Ophiostoma bicolor isolated from Ips typographus
53
from the body of bark beetles, branches and stems
of various trees species. So far 10 species of
ophiostomatoid fungi been have detected.
Ceratocystis polonica, Ophiostoma bicolor, O.
grandicarpum, O. piceae, O. quercus, O. novo-ulmi,
O. minor, O. minus, O. stenoceras, Graphium
fimbriisporum were isolated. Approximately
another five species were isolated, but they were
not identified on the species level. Some strains of
the isolated and determined fungi were deposited
in the Collection of Phytopathogenic Fungi of RICP
or Culture Collection of Fungi of the Department of
Botany at Faculty of Science of Charles University.
(Novotný D.)
Screening of digestive enzymes of mites
as novel candidates for protein microarrays
(COST P1OC853.003)
In the second year of the project we focused on
the following partial aims: screening of digestive
enzymes (amylase, lysozyme, cysteine and serine
proteinase) in whole-body homogenates and faeces
of tested mite species by biochemical methods;
detailed study of the presence of lysozyme in
whole-body homogenates and faeces, and study of
persistence of amylase and α and β glucosidases in
Acarus siro spent growth medium.
The presence of lysozyme was shown by in vitro
tests in the whole spectra of studied stored-product
mites. The highest activity was found in medium
acid pH (4-5).
The activity of amylase and α and β glucosidases
was observed in the spent growth medium after
6 months of cultivation in control conditions
(25° C, 85% RH). After this period the activity
of amylase slightly decreased by 35% in
comparison with fresh control, while the activity
of α and β glucosidases had a rising tendency.
(Hubert J., Kudlíková I.)
Immunochemical methods for detection
of food contamination by stored-product
pests (1B53040)
Tyrophagus putrescentiae is a widely distributed species inhabiting agricultural soil, nests of
birds and rodents, stored food and house dust.
This species is of medical importance. The latest
detection techniques of this species are based on
classical methods such as extraction in TulgrenBerllese funnels mainly, but there is an urgent
need for rapid detection techniques. Therefore
we developed polyclonal antibodies (Pabs) for
the detection of T. putrescentiae. Two Pabs (antiTyr-putK60 and anti-Tyr-putK61) were prepared
against the antigen in the form of protein fraction
from whole-body homogenates of the mite.
Specificity and sensitivity of the purified
Pabs were evaluated by PTA-ELISA in the range
of working concentration of Pabs from 0.01 to
1 µg/ml. Both Pabs have the identical detection
limit 50 ηg of antigen proteins/ml (approx. 3
individuals) at a working concentration of 1 µg of
IgG/ml. The Pabs showed strong cross-reactivities
to mites of the family Acaridae, Carpoglyphidae
and Glyciphagidae. No cross-reaction was found
out for the family Pyroglyphidae, stored-product
insects, micro-fungi, rearing diet of mites (yeast
and wheat diet) and extracts from wheat kernels.
The obtained antibodies enabled to detect (i) the
mites, (ii) their faeces and (iii) mite remnants in
the gut of their predators. (Kudlíková I., Hubert J., Chalupníková J.)
Complex methodology to monitor and detect
pest infestation of stored cereals by means
of physicochemical, immunochemical,
and molecular methods (QF 4071)
The red flour beetle, Tribolium castaneum
(Coleoptera: Tenebrionidae), is an important pest
of stored grain that has had a long association
with human stored food. This species is known as
an allergen producer and in addition it produces
quinol substances with a carcinogenic potential.
Therefore there is an urgent need to improve the
detection and monitoring of this species.
Immunochemical techniques based on polyclonal
antibodies (Pabs) seem to be perspective for the
detection of stored-product pests.
We prepared polyclonal antibodies (Pabs) antiTriCasK51 and anti-TriCasK52 against the antigen
in the form of protein fraction from whole-body
homogenates of Tribolium castaneum larvae.
Specificity and sensitivity of the purified Pabs
were evaluated by PTA-ELISA in the range of
working concentration of Pabs from 0.1 to 1µg of
IgG/ml. Both Pabs had a similar detection limit –
50 ηg of antigen proteins/ml at a working
concentration of 1µg of IgG/ml. The Pabs showed
no cross-reactivities to stored-product mites,
moths, micro-fungi, and extracts from wheat
kernels. Cross reactivity to other Tribolium
species (T. destructor, T. confusum) was significant.
The obtained antibodies enable to detect adults,
larvae and eggs as well as the faeces with different
sensitivity. (Kudlíková I., Hubert J., Krátká J.,
Chalupníková J.)
Collection of phytopathogenic fungi
and homologous antibodies
Two hundred and ten strains of phytopathogenic and potentially pathogenic fungi
and 18 polyclonal antibodies are deposited in
the collection of phytopathogenic fungi. The
majority of deposited strains belong to Deuteromycetes (approx. 175), Oomycetes (11 strains),
Heterobasidiomycetes (18 strains), Ascomycetes
(6 strains) and Zygomycetes (1 strain). In the
collection there are also antibodies against
Phytophthora spp., Plasmopara spp., Fusarium
spp., Pythium sp. and Colletotrichum spp. All
strains were isolated from plants and often from
organs and tissues with symptoms of infestation.
The main function of the collection is the holding
of cultures of phytopathogenic and potentially
phytopathogenic fungal species and the antibodies against them. Collection cultures are
prepared for the use by scientists from other
institutes, universities and from diagnostic and
industrial laboratories. (Novotný D., ŠárováPalicová J., Kudlíková I.)
54
Department of Entomology
The complex of pupal parasitoids
of Coccinella septempunctata
(MZe ČR 0002700603)
The abundance of C. septempunctata has
dramatically decreased since the mid-1990s.
Increased parasitisation was also hypothesized
among the causes of this change. Pupa, the stage
particularly vulnerable to parasitism, is attacked by
several Hymenoptera and Diptera species. The
occurrence of these parasitoids was recorded in
Prague, in maize stands grown for three years
at the same site. The abundance of parasitoids
varied between years. It was high in 2002,
when Phalacrotophora fasciata (Fallén) (Diptera,
Phoridae) was a dominant parasitoid and in
2004, when Tetrastichus coccinellae Kurdjumov
(Hymenoptera, Eulophidae) was a dominant
species. Other species, Homalotylus spp.
(Hymenoptera, Encyrtidae) and Dinocampus
coccinellae (Schrank) (Hymenoptera, Braconidae)
were scarce. In 2003 the abundance of parasitoids
was low. The proportions of parasitised pupae in
samples collected from other crops (sugar beet,
cereals) were similar to samples collected from
maize. Since parasitoid development was longer
than that of coccinellid pupae, the proportion of
parasitised pupae was apparently higher at the end
of the period of C. septempunctata pupation than
at its beginning. In all years, the total proportion of
parasitised pupae was low and could hardly affect
the C. septempunctata abundance. (Honěk A.,
Martinková Z.)
Development of the ground-beetle
parasitoids Brachinus explodens and B.
crepitans (Coleoptera: Carabidae): effect
of temperature (MZe ČR 0002700603)
To establish the thermal requirements of insects
is useful for understanding when they are present
during a season and for 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; the sums of effective
temperatures (SET) are 154.4 and 180.7 daydegrees, 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, and SET are 209.2 and 289.5 day-degrees,
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.)
Horizontal and vertical distribution
of spiders (Araneae) in sunflowers
(MZe ČR 0002700603)
Sunflowers are an increasingly important crop
plant in the Czech Republic. The spider fauna of
this crop has not been investigated yet. The aim of
this study was to monitor the spider fauna of
sunflowers and to study the seasonal change in the
spatial and vertical distribution of this fauna. For
this purpose a small experimental area was used
where spiders were visually checked on each single
leave of 50 sunflower plants at monthly intervals
from spring to autumn. The density of spiders
increased during the season reaching a maximum
seven spiders/plant in autumn shortly before
harvest. The spatial distribution changed
accordingly, being random in spring and early
summer and normal or aggregated toward late
summer. Two spider species, Neottiura bimaculata
and Theridion impressum (Theridiidae), dominated (96% of all individuals) throughout the season.
These two species exhibited a different microhabitat preference: N. bimaculata individuals were
found particularly on the lower sunflower leaves, T.
impressum preferred higher leaves. The density of
the spiders (per leaf) was independent of the
density of two dominant pest species, aphids and
leafhoppers. (Pekár S.)
Morphometric characterization of the
Ditylenchus dipsaci population isolated from
garlic (MZe ČR 0002700603)
Activity was included in problem solution
because the light microscopy diagnostic should
anticipate the use of molecular diagnostic methods.
The population isolated from garlic in 2004 in the
area of the village Blatnice pod Svatým Antonínkem
was used for characterization. The characterization
was done by measuring the basic body proportions
of the nematodes and calculating measurement
codes. The following values were estimated for
males: body length from 1 182 to 1 525 µm with
average value 1 347; value “a” 39 – 49, average 45;
value “b” 6.7 – 7.8, average 7.2; value "c" 14 – 18,
average 15; value “c” 4 – 7, average 5; value “MB” 28
– 42, average 32; stylet length10 – 12, average 11;
greatest body diameter 28 – 35 µm, average 31;
body width at the anus level 13 – 21 µm, average 18;
pharynx length 164 – 218 µm, average 190; tail 80 –
101 µm, average 91; spicule 20 – 29 µm, average 25.
The following values were estimated for females:
body length from 1 249 to 1 488 µm with average
value 1 347; value “a” 35 – 48, average 40; value “b”
6.7 – 8.5, average 7.4; value “c” 14 –17, average 15;
value “c” 5 – 6, average 5; value “MB” 26 – 36,
average 30; value “V” 80 – 83, average 81; value “V”
86 – 89, average 87; value “VA/T” 1.5 – 2.3, average
1.8; stylet length 10 – 12 µm, average 11; greatest
body diameter 27 – 40 µm, average 34; body width
at the anus level 16 – 19 µm, average 17; pharynx
length 164 – 199 µm, average 182; tail length 80 –
99 µm, average 90. The measurement of a similar
species Ditylenchus destructor was performed for
comparison. (Douda O.)
Evaluation of biological efficacy
of Adoxophyes orana granulovirus in the
control of Adoxophyes orana populations
by PCR detection (MZe ČR 0002700603)
The presence of Adoxophyes orana granulovirus
(AdorGV) in larvae of the summer fruit tortrix
(Adoxophyes orana) after treatment with AdorGV-
55
based preparation Capex® 2 against A. orana was
tested in three localities in the Czech Republic in
2003-2005. A PCR assay was developed for the
detection of AdorGV in A. orana larvae, which
could be used to identify the frequency of AdorGV
in the insect populations infected with AdorGV as
well as in natural populations. The AdorGV was
detected in larvae of A. orana from all three
localities treated with Capex® 2. The portion of the
overwintering larvae positive for AdorGV differed
according to locality and after the treatment it
ranged from 5% to 80%. The AdorGV was detected
from 10% to 80% of larvae from localities where
AdorGV was disseminated by special pheromone
traps. No AdorGV was detected in larvae collected
in Holovousy without virus treatment. The results
suggest a strong persistence of AdorGV in surviving
larvae after direct treatment with AdorGV causing a
high mortality of larvae in subsequent generations.
The population density was reduced by AdorGV
below the damage threshold during two years after
the virus treatment. (Stará J., Bohdanecká D.,
Kocourek F., Kundu J. K.)
Biological efficacy of three control strategies
used against Ostrinia nubialis and their effect
on the fauna of epigeic arthropods
(MZe ČR 0002700603)
The effect of three control strategies (1.
transgenic insect-resistant maize (Bt maize); 2.
biological control by Trichogramma wasps on an
isogenic maize hybrid or local hybrid; 3.
conventional control, i.e. the use of isogenic maize
hybrid (or local hybrid) with only a deep ploughing
of straw) used against Ostrinia nubialis on the
abundance and diversity of epigeic communities of
spiders, harvestmen, carabid and staphylinid
beetles in maize was evaluated. Epigeic arthropods
were collected by means of pitfall traps. In total, 9
498 individuals of carabid beetles (46 species), 2
608 individuals of spiders (60 species), and 575
individuals of harvestmen (5 species) were
collected at the study sites during three years of
research. The annual abundance and diversity of
arachnids were similar for all three control
strategies. The overall abundance of spiders
decreased over three years. There was not any
difference in the family and guild (hunters versus
web-builders) composition between strategies but
we observed a displacement of linyphiid spiders by
lycosids on all plots over time. Thus the three
control strategies had a similar effect on the epigeic
arthropods. We failed to find any adverse effect of
Bt maize on the abundance and diversity of epigeic
arthropods. Biological efficacy of Bt maize on the
regulation of O. nubilalis was 100% in 2002-2004,
biological efficacy of Trichogramma wasp ranged
from 35 to 69%. (Řezáč M., Pekár S., Kocourek
F., Saska P., Říha K., Stará J.)
Evaluation of Cacopsylla pyri resistance
to insecticides (MZe ČR 0002700603)
Resistance of C. pyri populations originating
from an orchard intensively treated with: Homoly
15 SC (teflubenzuron), Dimilin 48 SC (diflubenzuron), Zolone 35 EC (phosalone) and Trebon
30 EC (etofenprox) was proved in laboratory tests.
The population of C. pyri from an untreated locality
was susceptible to all tested insecticides. The
highest efficacy in the control of C. pyri nymphs
was found in tests in net slives when Calypso 480
SC (thiacloprid) and Vertimec 1.8 EC (abamectin)
were applied. The efficacy of the tested insecticides
against C. pyri adults in a tarsal test was sufficient
except for Cascade 5 EC (flufenoxuron) and Zolone
35 EC, when the C. pyri population from the
chemically treated orchard was tested. The efficacy
of Sanmite 20 WP (pyridaben) and Calypso 480 SC
in the control of new-hatched nymphs of C. pyri
showing multiple resistance in field experiments in
two different localities was excellent. On the basis
of the results, Sanmite 20 WP, Calypso 480 SC and
Cascade 5 EC were recommended for antiresistant
strategy against C. pyri. Sanmite 20 WP was
recommended for the registration in the Czech
Republic against C. pyri in pear orchards.
(Kocourek F., Stará J.)
Insecticidal activity of some essential oils
against Spodoptera littoralis larvae
(MZe ČR 0002700603)
Thirty-four essential oils were tested for their
insecticidal activity (fumigation or topical
application) against Spodoptera littoralis larvae.
Twenty essential oils applied by fumigation were
highly toxic to the third instar of S. littoralis larvae.
Two essential oils from Nepeta cataria and Thuja
occidentalis were highly toxic, with LC50 ≤ 10.0
ml/m3. Five essential oils from Salvia sclarea,
Thymus mastichina, Origanum majorana,
Pogostemon cablin and Mentha pulegium were
toxic with LC50 between 10.1 and 20.0 l ml/m3.
Twenty-three essential oils were highly toxic to the
third instar of S. littoralis larvae after topical
application. Eight essential oils from Mentha
citrata, N. cataria, S. sclarea, O. vulgare, O.
compactum, Melissa officinalis, T. mastichina, and
Lavandula angustifolia were highly toxic with
LD50 ≤ 0.05 µl/larva. (Pavela R.)
New control technologies against pests based
on azadirachtin (MZe ČR 0002700603)
The effect of low concentrations of azadirachtin
A (AzaA) applied systemically through root tissues
of tomato plants (Lycopersicon lycopersicum L.) cv.
Vilma on the development and population size of
greenhouse whitefly (Trialeurodes vaporariorum
Westwood; Hemiptera: Aleyrodidae) was studied.
The tested concentrations of AzaA were 5 and
0.5 ppm. The effectiveness increased, depending
on the dosage and number of applications. If at
least two applications at 5 ppm or 3 applications at
0.5 ppm were made, the number of each stage was
reduced from 70 to 80%. Such a significant
decrease in the T. vaporariorum population can be
sufficient for pest regulation in tomato plants
grown in hydroponics and may serve as a model
basis for other crops and ornamentals growing
hydroponically. (Pavela R.)
56
Factors affecting egg load in Coccinella
septempunctata (QG50081)
Egg load measured as instantaneous potential
fecundity (number of eggs available for oviposition
within the next 24 h) is determined by several
factors. The effects of female age, body size and
nutrition were studied in C. septempunctata.
Females collected in different crop or wild plant
stands on several dates were allowed to lay eggs for
24 h under conditions that forced them to deposit
all available eggs. The number and mass of eggs and
the residual mass of the females were determined.
The number of deposited eggs was significantly
positively correlated with the female mass. In cereal
stands the slope of fecundity vs. female body mass
regression was not affected by female age (within
15 days of oviposition period) or prey availability
(abundance of aphids). However, the slope of
fecundity vs. mass regression was significantly
lower in a sample of females collected on host plant
stands populated by a rejected prey (Uroleucon
spp.). The body mass vs. fecundity relationship is
thus significantly affected by food quality. The
proportion of females not laying eggs increased
after feeding the rejected prey and with female age.
(Honěk A., Martinková Z.)
The production of high-quality and safe
cereal products using different control
strategies of maize and store products
(1B 53043)
Development of the method of monitoring
of European corn borer (ECB) resistance to
Bt-toxin and biological efficacy of different
control strategies in maize against ECB
A laboratory bioassay for the evaluation of ECB
(Ostrinia nubilalis) resistance to Bt-toxin was
developed. The bioassay is based on the evaluation
of mortality of ECB larvae reared on a semisynthetic diet with defined content of lyophilized
biomass of Bt-maize. In 2005, 2 field experiments
with 4 control strategies against ECB were
established: untreated control, chemical control
(Mospilan, Integro), biological control (Trichogramma) and Bt-maize MON 810. The biological
efficacy of Bt-maize against ECB reached 100%, the
biological efficacy of Integro was about 90%. The
efficacy of Mospilan and biological control were
insufficient in both experimental localities in 2005.
An important increase in maize yield was observed
in “Bt-maize” and “Integro” variants. The incidence
of toxicogenic micromycetes and content of
mycotoxins in grain were evaluated in all the
experimental variants of maize control. Spring
barley and winter wheat stands were established
on plots where maize with different control
strategies was grown in 2004. (Kocourek F., Stará
J., Říha K.)
Toxicogenic micromycetes and their
mycotoxins in grains of transgenic Bt-maize
hybrid and nontransgenic hybrids (1B 53043)
The efficiency of Bt-maize hybrid in comparison
with biological control by the introduction of
Trichogramma wasp and untreated control hybrid
was proved in two localities in the Czech Republic
(Praha-Ruzyně and Ivanovice na Hané). In total, 15
taxa of the genus Fusarium and 9 taxa of the genus
Penicillium were identified. The occurrence of
toxicogenic species in Bt-maize and nontransgenic
hybrids was similar in dependence on different
damage of plants, caused by larvae of European
corn borer (ECB). Frequencies of Fusarium species
in Bt-maize with mechanical damage were significantly lower in comparison with nontransgenic
hybrids. The frequencies of Fusarium species were
lower in Bt-maize by 35.6% in the species Fusarium
oxysporum, by 86.1% in F. proliferatum, by 61.6% in
F. sporotrichioides, by 32.4% in F. subglutinans and
by 77.6% in the species F. verticillioides. Changes in
the presence of micromycetes of the genus
Fusarium in ears were explained by approximately
13-48% changes in the occurrence of ECB in
Ivanovice na Hané locality and approximately 18.468% in Praha-Ruzyně locality. The relationship
between Bt-maize, occurrence of toxicogenic
species and concentration of four selected mycotoxins was demonstrated by the lower occurrence
of mycotoxins in Bt-maize grain. (Slezáková L.,
Remešová J., Kocourek F., Říha K.)
Causes of long-term changes in the
abundance of seven-spot ladybird, Coccinella
septempunctata (GA ČR 522/05/0765)
Since 1978, the abundance of Coccinella
septempunctata L. has been recorded at a
hibernation site in the western part of the Czech
Republic. Between years the abundances varied by
two orders of magnitude and a long-term trend of a
decrease in C. septempunctata abundance has been
observed since the early 1990s. This recession was
correlated with the decreasing abundance of its
dominant prey, cereal aphids. The acreage of small
grain cereals and other crops convenient for the
breeding of C. septempunctata populations also
decreased. The change in the abundance of cereal
aphids may be promoted by a dramatic decrease in
fertilizer input after 1990. Decreasing fertilizer
doses changed the quality of small grain cereal
crops reflected by lower yields. (Honěk A.,
Martinková Z.)
Contrary food requirements of the larvae of
two Curtonotus (Coleoptera: Carabidae:
Amara) species (GA ČR 521/03/0171)
The larvae of carabids that are granivorous as
adults can be granivorous, omnivorous or
carnivorous. The differences in larval food
preferences of Amara aulica and A. convexiuscula,
two closely related species of the subgenus
Curtonotus, were studied. The survival and
duration of development of non-diapausing first
and second instar larvae were compared. The third
instar larvae were not studied because they go into
diapause prior to pupation. The larvae were fed
diets consisting of insect larvae (Tenebrio molitor),
seeds of Artemisia vulgaris or Tripleurospermum
inodorum or Urtica dioica or Cirsium arvense, and
a mixed diet consisting of insect larvae and seeds.
Larvae of A. aulica required seeds for successful
development, whereas those of A. convexiuscula
developed on both the insect diet and seeds
57
of Artemisia. The difference in larval food
requirements facilitates the co-existence of these
closely related species, which frequently share the
same habitat. (Saska P.)
Seed predation by carabids in organic wheat
fields (GA ČR 521/03/0171)
In a study carried out in 2004 in organic winter
wheat fields in the Netherlands we focused on
post-dispersal seed predation by carabid beetles
(Coleoptera: Carabidae). The aim was to
investigate the temporal and spatial variation in
seed predation. Two preferred (Capsella bursapastoris and Stellaria media), two non-preferred
(Veronica persica and Lamium amplexicaule)
types of seeds and one seed of unknown
preference (Poa annua) were used. Seeds were
glued on cards and exposed weekly, from early
April to the end of July at a different distance from
the field margin (0-50 m). The study was replicated
in two organic fields. The number of removed
seeds greatly varied between species. C. bursapastoris and P. annua were eaten to the largest
extent. Seed consumption also varied in time, and
was significant only in some of the experimental
weeks. Seed predation was highest close to the
field margin, probably because the activity density
of ground beetles was higher at the field margin
than in the field interior. This work was done in
cooperation
with
Wageningen
University,
Wageningen, the Netherlands. (Saska P.)
The evaluation of Cydia pomonella
and Leptinotarsa decemlineata resistance
to insecticides (GA ČR 522/04/P181)
Discriminating concentration and LC50 value
were determined for Dimilin 48 SC, Nomolt 15 SC,
Calypso 480 SC and Insegar 25 WP when applied to
the eggs, L1 larvae or L5 larvae of C. pomonella from
a laboratory colony. Resistance to Zolone 35 EC,
Nomolt 15 SC and Dimilin 48 SC was detected in a
part of the C. pomonella population from Velké
Bílovice when tested on L5 overwintering larvae.
Resistance to Dimilin 48 SC and Nomolt 15 SC was
found in 20% and 30-80% of the tested larvae,
respectively. Resistance to Insegar 25 WP was
observed in 31.2% of the tested C. pomonella
population from Velké Bílovice when tested on the
eggs.
Decreased efficacy of pyrethroids (Decis EW 50)
and organophosphates (Sumithion Super) was
proved against L. decemlineata from three
localities, no resistance of L. decemlineata was
found to neonicotinoids (Mospilan 20 SP). High
efficacy of Mospilan 20 SP on larvae of L.
decemlineata was found after its application to
plants in contrast to insufficient efficacy after the
contact application to larvae. High efficacy of
Spintor and BAS 320 I was found against L4 larvae
from
Lednice
and
Svitavy,
resistant
to
organophosphates and pyrethroids. (Stará J., Naďová K., Kocourek F.)
The antifeedant effect of extracts from
Leuzea carthamoides (Willd.) DC. on
Leptinotarsa decemlineata Say (1P05ME764)
The inhibitory effect of a methanol extract from
Leuzea carthamoides (Willd.) DC. crude seeds on
Colorado potato beetle (Leptinotarsa decemlineata Say) larval and adult feeding was studied.
The antifeedant activity was detected both at
a lower 0.2% (w/v) concentration and at a higher
4.0% (w/v) concentration of the crude seed
extract. In our tests the crude extract showed the
relatively long-lasting antifeedant effect that
declined in dependence on time and concentration. (Pavela R.)
Department of Stored-Product Pest
Control
Fungi associated with rodent faeces in stored
grain environment in the Czech Republic
(MZe ČR 0002700603)
Fungi cause deterioration of stored crops and
may contaminate them with mycotoxins and
allergens. Although fungi-rodent associations are
known from nature, there is little data from the
environment of stored products. Therefore, the
goal of this work was to identify the fungi
connected with the occurrence of mouse faeces in
stored grain in the Czech Republic. Mouse
populations were sampled in three grain stores and
their faeces were subjected to mycological analysis.
We isolated 11 genera (the highest species diversity
was found in Penicillium /13 species/, Aspergillus
/5 species/ and Mucor /5 species/) and 35 species
of fungi (the most frequent were: Eurotium repem,
Microascus brevicaulis, Aspergillus niger, Mucor
mucedo, Penicillium aurantiogriseum, and
Thamnidium elegans). The toxicogenic and
allergenic potential of isolated fungi was evaluated.
(Stejskal V., Hubert J.)
Outdoor occurrence of stored-product pests
(Coleoptera) in the vicinity of a grain store
(MZe ČR 0002700603)
The species composition, frequency and
abundance of insect pests (Coleoptera) occurring
outside a grain store were explored. Wheat bait
traps were used for pest monitoring; they were
changed every month from April to September. For
the first time the outdoor occurrence of primary
and secondary stored-product pests was documented in the Czech Republic. Seven species of
Coleoptera were found outside the grain store. Pest
abundance and frequency were in high correlation.
The primary pest Sitophilus granarius was
dominant. (Kučerová Z., Aulický R., Stejskal V.)
Arthropod pests and their natural enemies
in stored crops in northern Namibia
(MZe ČR 0002700603)
A survey of stored-product pests, their natural
enemies, grain storage and pest management
approaches in northern Namibia was conducted.
The survey revealed five types of crop commodities
(pearl millet, sorghum, cowpea, maize, feed
mixture) stored in four types of grain stores
(baskets, bag stags-flat stores, concrete bin, and
metal containers). Neither synthetic pesticides nor
58
plastic sheeting was recorded. The only protectant
used is a wooden-ash natural pesticide. Ten insect
pest species were found affiliated to the orders of
Coleoptera (Attagenus fasciatus, Callosobruchus
subinnotatus, Cryptolestes ferrugineus, Oryzaephilus surinamensis, Rhyzopertha dominica and
Tribolium castaneum), Lepidoptera (Corcyra
cephalonica, Sitotroga cerealella), Psocoptera
(Liposcelis paeta) and Blattodea (Blattella
germanica). Out of them, seven species were
recognized as new stored-product pests for
Namibia. Although listed for the South African
region, no storage pest-mites (Acari) and Sitophilus
spp., Prostephanus sp. or Trogoderma sp. beetles
were traced. We found four new species of natural
enemies of storage pests recruiting from insects
(Habrobracon hebetor, Cephalonomia wattersoni,
Brachymeria sp.) and mites (Blattisocius tarsalis).
The occurrence of natural enemies indicates a
potential for the pest bio-control in Namibian grain
stores. (Stejskal V.)
Trogoderma longisetosum and Trogoderma
variabile (Coleoptera, Dermestidae) as two
new stored-product pests for the Czech
Republic (MZe ČR 0002700603)
A revision of material from the stored-product
pest collection of the Research Institute of Crop
Production, Prague, was carried out. While so far
five species of Trogoderma (all synanthropic ones)
have been known from the Czech Republic, the
revision revealed two new species and two new
stored-product pests for the Czech Republic: T.
longisetosum and T. variabile. A key to determine
T. longisetosum and T. glabrum is given. Faunistic
records of the former quarantine species T.
granarium are listed. (Stejskal V., Kučerová Z.,
Háva J.)
In vitro and in vivo inhibition of α-amylases
of the stored-product mite Acarus siro
(MZe ČR 0002700063)
Stored-product mites are the most abundant and
frequent group of pests living on stored food
products in Europe. They endanger public health
since they produce allergens and transmit
mycotoxin-producing fungi. Novel acaricidal
compounds with inhibitory effects on the digestive
enzymes of arthropods are a safe alternative to the
traditional neurotoxic pesticides used for the
control of stored-product pests. In this work, we
explored the properties of acarbose, the low
molecular weight inhibitor of α-amylases (AI), as
a novel acaricide candidate for the protection of
stored products from infestation by Acarus siro
(Acari: Acaridae). In vitro analysis revealed that AI
efficiently blocked the enzymatic activity of
digestive amylases of A. siro, and decreased the
physiological capacity of the mite’s gut in utilizing
a starch component of grain flour. In vivo
experiments showed that AI suppressed the
population growth of A. siro. The mites were kept
for three weeks on an experimental diet enriched
with AI in a concentration range of 0.005 to 0.25%.
Population growth of A. siro was negatively
correlated with the content of AI in the treated diet
with a half population dose of 0.125%. The
suppressive effect of AIs on stored-product mites is
discussed in the context of their potential
application in GMO crops. (Hubert J., Kudlíková
I., Stejskal V.)
Biological control of stored-product pest
mites using the predatory mite Cheyletus
eruditus (Cost 1P04OC842.10)
The effects of various temperatures and initial
densities on the dynamics of predator (Cheyletus
eruditus) and prey (Acarus siro) interactions under
laboratory conditions were studied. The temperature played a key role in this type of interaction: in
the majority of the initial populations the control of
predator to prey densities was successful at 25°C.
This, however, was not the case at 15°C. The
relations between population densities of pest
mites (Acari: Acaridida) and Cheyletus spp.
predatory mites (Acari: Cheyletidae) were
investigated in 147 grain stores in the Czech
Republic. More than 1.000.000 individuals of pest
mites and 40.000 individuals of predatory mites
were extracted. We found that 29% of samples did
not contain any mites, 41% contained only pest
mites, 4% only predatory mites, and in 26% both
groups occurred simultaneously. The most
abundant predatory mites were C. eruditus (79%)
followed by “minor” species: C. aversor (10 %), C.
trouessarti (9 %) and C. malaccensis (2 %). There
was a significantly positive correlation between the
occurrence and population density of the
predatory and pest mites, except for C.
malaccensis. The flight performance of females of
the thelytokous and arrhenotokous strain of
Venturia canescens was investigated during a shortterm mission. The age of wasps influenced the total
distance flown, the total time in flight, the distance
traversed in the longest single flight and the time
invested in it. The influence of the mode of
reproduction on the mean flight distance and time
was shown in a complex interaction with other
variables. The flight parameter most clearly affected
by the mode of reproduction was the mean flight
velocity. Arrhenotokous wasps were faster than
their thelytokous counterparts of the same size. The
distance traversed in the longest single flight and
the time invested on it as well as the mean distance
and mean time flown were also affected by the
mode of reproduction but only in interaction with
other variables. The total number of flights was
influenced by the age and size of wasps but not by
the mode of reproduction. (Lukáš J.)
Amylase inhibitor is without any adverse
effect on the parasitoid Venturia canescens
(GA ČR 522/04/1286)
The amylase inhibitors (AI) are suitable
candidates for transgenic plants to increase their
resistance to insect pests. A detail study of
suppressive effects of AI on target and non-target
insects is necessary before their incorporation into
GMO plants. Therefore we tested the effect of a
selected AI inhibitor (acarbose) on the pest flour
moth (Ephestia kuehniella) and its parasitoid wasp
(Venturia canescens) in laboratory experiments.
59
Various concentrations of AI were incorporated
into a diet for E. kuehniella larvae and their
mortality was observed to find sublethal doses of
AI.
The
larvae
influenced
by
sublethal
concentrations of AI (0.001 and 0.0001%) were
parasitized by the wasp. The morphological
parameters of wasps treated with AI and of the
control were compared. The AI in the range from
0.1 to 0.01% caused 100% mortality of Ephestia
kuehniella larvae, the concentration AI 0.001%
suppressed an increase in larval weight and
prolonged the developmental period. We did not
observe any suppressive effect of AI on Venturia
canescens; but we found differences in morphological parameters between wasps treated on (i)
control larvae and on larvae fed on 0.001% AI and
(ii) 0.0001% AI. The measured parameters of wasps
influenced by AI 0.0001% were significantly higher
in most cases (i.e. weight, hind tibia length
and wing size). These results demonstrate
a possibility of the combination of biocontrol and
AI that would provide higher pest control
efficiency than these methods used individually.
(Hubert J.)
Biological control of Oryzaephilus
surinamensis (Coleoptera: Cucujidae)
using Cephalonomia tarsalis (Hymenoptera:
Bethylidae) (GA ČR 522/04/P169)
The following experiments on Cephalonomia
tarsalis were conducted in 2005: temperature and
time dependent survival and fecundity of C.
tarsalis, temperature dependent functional
response and susceptibility of C. tarsalis adults to
deltamethrin. Handling time (Th) was inversely
proportional to temperature and ranged from 0.167
at 21°C to 0.024 at 30°C. Instantaneous search rate
(a) also changed with temperature. It was lowest at
30°C and highest at 27°C. The predicted maximum
number of paralyzed larvae in 1 day (1/Th) was
highest at 30°C (41.7 larvae/day) and 27°C (21.3
larvae/day). But the value of Th at 30°C was on the
level of significance (p=0.051). The temperaturemediated functional response equation explained
77% of the variance in the paralyzation rate. The
preoviposition period was nonlinearly temperature
dependent. Females that oviposited first did so at
30°C after 1-2 days. At 21°C females started to
oviposit at the age of 2-8 days. An extended
preoviposition period of 1-6 days of age was
recorded at 33°C. Adult longevity was temperature
and sex dependent. Adults lived longer at a lower
temperature and females lived longer than males.
The overall mean longevity of males was 4-8 days at
temperatures ranging between 21 and 33°C. At
21°C males lived for up to 17 days. Mean longevity
of females varied between 43 days at 30°C and 82
days at 21°C. An individual female lived more than
90 days at 21°C. The oviposition period was
negatively linearly temperature dependent.
Females oviposited on average for 54 days at 21°C
but for 33 days at 30°C. Egg production was
nonlinearly temperature dependent. Up to 198
eggs were laid by an individual female during its
lifetime at 27°C. Maximal mean fecundity of 110
eggs per female was recorded at 27°C but only 32
eggs per female (mean) were laid at 21°C. Males and
females of C. tarsalis were unequally sensitive to
deltamethrin (F=6.1, df=1.13, p<0.05); males were
more sensitive than females. (Lukáš J.)
Secretory cells of the digestive tract
of Tribolium castaneum larvae
(MŠMT 1P05ME 758)
To understand the effect of an insecticide on the
midgut of Tribolium castaneum larvae we explored
and described the structure of secretory cells using
optical and fluorescent microscopy. The larvae were
(i) dissected and the gut was removed and observed
by staining with calcofluor or methylene blue; (ii)
fixed in Bouin-Duboaque-Brazil fixation fluid,
embedded in paraffin and sectioned. The sections
were stained in Masson’s trichrome stain, alciane
blue and peroxide-acid Schiff’s reagents. The
digestive tract composes of 3 parts: stomodaeum
(foregut), mesodaeum (midgut) and proctodaeum
(hindgut). The mesodaeum is quite a simple tube,
without any caeca or diverticula. In the middle
part of mesodauem there are cylindrical cells.
The short middle zone secrets no enzymes.
A multi-layer peritrophic membrane is present. The
secretory cells are localized into (i) anterior
mesodaeum and (ii) posterior mesodaeum. The
anterior part of mesodaeum contains tall and
columnar cells and the membrane on the luminal
side forms microvilli. Apical extrusions at the apical
parts of cells release the lumen into the gut. The cells
of posterior mesodaeum create multicellular crypts
and apical extrusions can also be observed here. The
cells in both the anterior and posterior mesodaeum
contain large nuclei and are strongly vacuolized
indicating a high secretory activity. (Hubert J.)
Morphology of Dorypteryx domestica
(Psocoptera) nymphs (MŠMT 1P05ME733)
Dorypteryx domestica is an expansive psocid
often occurring in synanthropic localities. So far it
has been possible to identify only adults and eggs,
since the description of nymphs has not been
available. The morphological study deals with
overall appearance and size measurements of
particular instars. Five developmental instars are
distinguished and described. Morphological
characters include mainly the number of flagellar
segments, the number of ommatidia in compound
eyes, and the level of wing development.
Morphological details are illustrated with scanning
electron microscope micrographs and macro
photos. (Kučerová Z.)
Diagnostic laboratory for detection
and determination of stored-product pests
(350/5309)
Over 30 samples of various agricultural and food
commodities were examined for detection of
stored-product arthropods. About 250 determinations of 17 pest species were done. The species
belong to the following taxonomic groups: Acarina
(4), Psocoptera (6), Coleoptera (5), Lepidoptera
(2). (Kučerová Z.)
60
Cultures and collections of stored-product
mites, insects and microscopic fungi
(350/1373)
Over 70 species of insects, 12 species of Acarina
and 3 species of microscopic fungi (altogether 108
tribes) are kept in culture. The collection com-
prises more than 10 000 items of taxonomically
classified mites and 30 000 items of insects.
Cultures and collections of stored-product pests
serve for the purposes of research, education,
training and diagnostics. (Aulický R.)
STAFF OF THE DIVISION
Doc. Ing. Jaroslav Polák, DrSc. (Head of Division)
Ing. Josef Drozen (Scientific Secretary)
Blanka Klapová (Personal Secretary)
DEPARTMENT OF VIROLOGY
Doc. Ing. Jaroslav Polák, DrSc. (Head of Dept.)
Marie Bouzková
Ing. Zuzana Červená
Markéta Dubayová (since 1.12.2005)
Miloslava Ducháčová
Bc. Lenka Frýdlová (since 15.11.2005)
Bc. Alena Hauptmanová (since 1.7.2005)
Ing. Michala Chaloupková (by 31.7.2005)
Milan Jokeš
Maxim Kalačňuk (by 31.5.2005)
Ing. Petr Komínek, Ph.D.
Ing. Jiban K. Kundu, Ph.D.
Jan Novák (from 15.6.2005 to 15.11.2005)
Jitka Pívalová
Ing. Jiří Svoboda
Ing. Lenka Širlová
Vendula Špelinová
MOLECULAR PLANT PATHOGEN
DIAGNOSTICS LABORATORY
Dr. Ing. Jaroslav Salava (Head of Laboratory)
Ing. Dita Bohdanecká
Ing. Marcela Bryxiová
Mgr. Katarína Čiháková
Markéta Hrabovská (nematology)
Mohamed A. A. Hassan, MSc.
RNDr. Daniela Chodová, CSc.
Ing. Shesh Kumari, Ph.D. (nematology)
DEPARTMENT OF MYCOLOGY
RNDr. Josef Hýsek, CSc. (Head of Dept.)
Mgr. Julie Chalupníková
Evžena Davidová
Jitka Dunaiová
Ing. Hana Klenová
RNDr. Jiřina Krátká, DrSc.
Mgr. Iva Kudlíková
Miroslava Kutíková
Anna Macáková
RNDr. David Novotný, Ph.D.
Iveta Slaninová (since 1.8.2005)
RNDr. Eliška Sychrová
Mgr. Jana Šárová – Palicová, Ph.D.
Doc. Ing. Josef Šebesta, DrSc.
Eva Šíchová
Ing. Lada Urbanová (since 1.10.2005)
Lenka Urbánková
Prof. Ing. Karel Veverka, DrSc.
DEPARTMENT OF ENTOMOLOGY
Doc. RNDr. Ing. František Kocourek, CSc. (Head of
Dept.)
Ing. Ondřej Douda
RNDr. Alois Honěk, CSc.
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.
RNDr. Jindra Štolcová
Zdeňka Víznerová
DEPARTMENT OF BACTERIOLOGY
Ing. Václav Krejzar, Ph.D. (Head of Dept.)
Prof. Ing. Václav Kůdela, DrSc.
Ing. Iveta Pánková, Ph.D.
Ing. Radka Krejzarová
Jitka Chytráčková
Ing. Blanka Kokošková, CSc.
Slaný Research Station
Ing. Josef Korba (Head of Station)
Olga Hojnicová
Pharm. Dr. Jana Šillerová
DEPARTMENT OF STORED-PRODUCT
PEST CONTROL
Ing. Václav Stejskal, Ph.D. (Head of Dept.)
Ing. Radek Aulický
Pavel Horák
Mgr. Jan Hubert, Ph.D.
Ing. Zuzana Kučerová
Ing. Jan Lukáš, Ph.D.
Bc. Marta Nesvorná
Šárka Tučková