RNDr. Barbora Mieslerov

1
Case study Interaction Solanum spp. Oidium
neolycopersici
RNDr. Barbora Mieslerová, Ph.D. Katedra
botaniky Prírodovedecká fakulta Univerzita
Palackého Olomouc
2
There are only two ways to live your life. One
is as though nothing is a miracle. The other is
as though everything is a miracle.
Albert Einstein
3
Solanum

• Solanum spp. is a large and diverse genus of
  annual and perennial plants.
• They grow as forbs, vines, subshrubs, shrubs, and
  small trees, and often have attractive fruit and
  flowers.
• Many formerly independent genera like
  Lycopersicon (the tomatoes) or Cyphomandra are
  included in Solanum as subgenera or sections
  today.
• Thus, the genus nowadays contains roughly
  1,500-2,000 species.
• Several species are cultivated, including three
  globally important food crops
• Tomato, S. lycopersicum

Potato, S. tuberosum
Eggplant, S. melongena
4
Solanum (Lycopersicon) spp.
Variability of fruits and flowers of r. Solanum
sect. Lycopersicon, sect. Juglandifolia a sect.
Lycopersicoides (Peralta et al., 2008).
5
Taxonomy of genus Solanum earlier taxonomy

• According to the former concept of Rick (1979
  1995) there were discriminated two large
  species-complexes within genus Lycopersicon,
  namely Esculentum-complex and Peruvianum-complex.
  
• Esculentum-complex encompassed 7 species L.
  esculentum (newly Solanum lycopersicum), L.
  cheesmanii (S. cheesmaniae), L. chmielewskii (S.
  chmielewskii), L. hirsutum (S. habrochaites), L.
  parviflorum (S. neorickii), L. pennellii (S.
  pennellii) and L. pimpinellifolium (S.
  pimpinellifolium).
• In Peruvianum-complex were placed two species L.
  chilense (S. chilense) and L. peruvianum (S.
  peruvianum).

6
Crossability polygon of Solanum (Lycopersicon)
species (Lindhout et al., 1994)
Esculentum-complex
PENN
ESC
HIRS
CHEES
PIM
PARV CHMIE
Strong barrier of interspecific hybridization
PER
CHIL
PERHU
Peruvianum-complex
7
Lycopersicon esculentum var. cerasiforme (Solanum
lycopersicum)
L. pimpinellifolium (S. pimpinellifolium )
8
Lycopersicon hirsutum f. glabratum (Solanum
habrochaites)
L. pennellii (S. pennellii)
9
http//digi.azz.cz/Book001/images/Solanum_peruvian
um_A327.jpg
L. chmielewskii (S. chmielewskii)
L. peruvianum (S. peruvianum)
10
Taxonomy of genus Solanum recent taxonomy

• Recently, it is widely accepted that tomato and
  its wild relatives belong to the genus Solanum
  subgen. Potatoe (G. Don) DArcy, sect.
  Lycopersicon (Mill.) Wettst., subsect.
  Lycopersicon (e.g. Child, 1990 Spooner et al.,
  2005 Ji and Scott, 2007 Peralta et al., 2008)
• Child (1990) also propounded representatives of
  Solanum sect. Lycopersicoides Child (including S.
  lycopersicoides and S. sitiens), and sect.
  Juglandifolium (Rydb.) Child (included S.
  juglandifolium and S. ochranthum) as the closest
  relatives of subsect. Lycopersicon.
• Peralta et al. (2008) recently distinguished 13
  species belonging to Solanum sect. Lycopersicon
  and four closely related species (S.
  juglandifolium, S. lycopersicoides, S. ochranthum
  and S. sitiens).

11
Comparison of earlier (Rick, 1979) and recent
classification (Peralta et al., 2008) of genus
Solanum sect. Lycopersicon (according to
Grandillo et al., 2011)
12
Tomato powdery mildew (Oidium neolycopersici)

• Tomato powdery mildew (Oidium neolycopersici)
  belongs to the order Erysiphales (powdery
  mildews) and it is arelatively new disease
  occurring predominantly on glasshouses tomato
  crops throughout Europe and New World

13
Distribution of Oidium neolycopersici

• Information is given on the geographical
  distribution in
• EUROPE (Bulgaria, Czech Republic, Denmark,
  France, Germany, Greece, Hungary, Italy (mainland
  Italy), Netherlands, Poland, Spain, Switzerland,
  UK (England)),
• ASIA (Bhutan, China (Hong Kong), India (Jammu
  and Kashmir, Karnataka, Uttar Pradesh), Japan,
  Malaysia, Nepal, Taiwan, Thailand),
• AFRICA (Tanzania),
• NORTH AMERICA (Canada (Alberta, British
  Columbia, Ontario, Quebec), USA (California,
  Connecticut, Florida, Maryland, New Jersey, New
  York)),
• CENTRAL AMERICA AND CARIBBEAN (Guadeloupe,
  Jamaica),
• SOUTH AMERICA (Argentina, Venezuela).

14
Distribution of Oidium neolycopersici
http//agro.biodiver.se/2007/04/whats-so-special-a
bout-oidium-neolycopersici/
15
The map of the first records of Oidium
neolycopersici occurrence in Europe
Lebeda, A., Mieslerová, B. Plant Prot. Sci. 36
(4)156-162, 2000.
16
Symptoms of disease

• The first symptoms of the disease start to occur
  in EARLY SUMMER, seldom in late spring.
• On the UPPER, seldom on the lower LEAF SURFACES
  white pustules of powdery mildew appear.
• YOUNGER LEAVES are mostly WITHOUT SYMPTOMS.
• The SMALL CIRCULAR INITIAL PUSTULES, 3-10 mm
  diam., enlarge quickly and can COVER THE WHOLE
  LEAF SURFACE within a few days.
• In highly suscpetible tomato cultivars, the
  STEMS AND PETIOLES are also affected
• Infected plant parts GROW SLOWLY, which is
  followed by CHLOROSIS of the colonized tissue,
  DEFOLIATION AND DRYING of the plant.
• NO SYMPTOMS are recorded on tomato FRUIT.

17
Symptoms of tomato powdery mildew (O.
neolycopersici) infection on susceptible S.
lycopersicum. (A) The initial symptoms of powdery
mildew. (B) Intensive disease infestation. (C)
Necrosis after intensive disease development.
Photo B. Mieslerová
18
Tomato powdery mildew (Oidium neolycopersici).
(A) Conidiophores. (B) Conidia. (C) Germinating
conidium. (D) Dense mycelial coat with
conidiophores on leaf of susceptible tomato.
Photo R. Novotný (A, B) and B. Mieslerová (C, D)
19
Chemical protection - registered preparations
against tomato powdery mildew in the Czech
Republic
Preparation Effective compound
BIOAN Lecitihin, Albumin, Milk Cassein
KUMULUS WG Sulphur
ORTIVA Azoxystrobin
SCORE 250 EC Difenoconazole
TALENT Myclobutanil
TOPAS 100 EC Penconazole
20
Morphological characterization and possible
taxonomic position

• The exact taxonomic determination of Oidium
  neolycopersici is difficult
• Till now the TELEOMORPH STAGE was NOT FOUND. The
  attempt to initiate formation of cleistothecia
  under laboratory conditions failed

• Jones et al. (2000) on the basis of the complex
  study including light microscopy, SEM analysis
  and ITS sequence analysis this species assign to
   ERYSIPHE SECT. ERYSIPHE, and found that is very
  close relative (nearly identical) to Erysiphe
  aquilegiae var. ranunculi and clearly distinguish
  from Golovinomyces orontii and G. cichoracearum.
• Kiss et al. (2001) identified earlier described
  powdery mildew on tomatoes from AUSTRALIA
  (OIDIUM LYCOPERSICI) as a species different from
  tomato powdery mildew widespread in EUROPE,
  AFRICA, NORTH AND SOUTH AMERICA AND ASIA (OIDIUM
  NEOLYCOPERSICI).

21
Parsimony tree of the phylogenetic analysis of
ITS4 -5,8S- ITS 5 regions.
Jones et al.. Can. J. Bot.781361-1366, 2000.
22
Kiss et al. Mycol. Res. 105 684-697, 2001
O. neolycopersici isolate Pseudoidium type
O. lycopersici isolate from South Australia
Euoidium type
23
Taxonomical position
Phylogenetic analysis of the internal transcribed
spacer (ITS) region of the ribosomal RNA gene for
12 Pseudoidium anamorphs (according to Kiss et
al., 2001)
24
Morphological comparative study

• Trying to solve the problem of taxonomical
  position of O. neolycopersici, comparative
  morphological studies of 14 isolates of powdery
  mildew 10 of O. neolycopersici (OL),
• 1 Golovinomyces cichoracearum (GC)
• 1 - Golovinomyces orontii (GO)
• 1 Sphaerotheca fusca (SF)
• 1 Erysiphe aquilegiae var. ranunculi (EAR)
• using light and Scanning electron microscopy
• Our COMPARATIVE MORPHOLOGICAL STUDY revealed
  DIFFERENCE of Oidium neolycopersici from
  Golovinomyces cichoracearum, G. orontii and
  Sphaerotheca fusca and close SIMILARITY to
  Erysiphe aquilegiae var. ranunculi

Mieslerová, B., Lebeda, A., Kennedy, R., Novotný,
R. Acta Phytopathol. Entomol. Hungar., 37 (1-3)
57-74, 2002.
25
Dendrogram constructed on morphological data
showing similarity between isolates of O.
neolycopersici (OL), Erysiphe aquilegiae var.
ranunculi (EAR), G. cichoracearum (GC), G.
orontii (GO) and Sphaerotheca fusca (SF).
Mieslerová, B., Lebeda, A., Kennedy, R., Novotný,
R. Acta Phytopathol. Entomol. Hungar., 37 (1-3)
57-74, 2002.
26
SEM photographs of selected powdery mildews
Sphaerotheca fusca
Oidium neolycopersici
Mieslerová, B., Lebeda, A., Kennedy, R., Novotný,
R. Acta Phytopathol. Entomol. Hungar., 37 (1-3)
57-74, 2002.
Golovinomyces cichoracearum
27
BIOLOGY OF THE PATHOGEN (Oidium neolycopersici)

• The influence of environmental conditions on
  development of tomato powdery mildews has been
  reported by various authors (e.g. (Fletcher et
  al., 1988 Hannig, 1996 Whipps and Budge, 2000
  Jacob et al. 2008 Mieslerová and Lebeda, 2010).
  ).
• The EFFECT OF TEMPERATURE and LIGHT CONDITIONS
  (spectral quality, intensity and photoperiod) on
  germination, development and conidiation of
  tomato powdery mildew (Oidium neolycopersici) on
  the highly susceptible tomato cv. Amateur were
  studied. CONIDIA GERMINATED across the whole
  range of tested temperatures (10 35C) however,
  at the end-point temperatures, germination was
  strongly limited.
• Suitable conditions for O. neolycopersici
  development were narrower than for germination.
  At temperatures slightly lower than optimum
  (2025C), MYCELIAL DEVELOPMENT and time of
  appearance of the first conidiophores was
  delayed. CONIDIATION occurred within the range of
  1525C, however was most intense between
  2025C.
• Basic conditions important for development and
  conidia formation of O. neolycopersici have also
  been studied (Fletcher et al., 1988 Hanning,
  1996 Whipps and Budge 2000 Jacob et al., 2008)
  with similar results concerning temperature
  conditions. As for RELATIVE HUMIDITY, the highest
  percentage of infections was found on tomatoes
  growing at 60-80 R.H.

28
Mieslerová, B., Lebeda, A. J. Phytopathol. 112
(2010)
Mean length of the conidial germ tubes of Oidium
neolycopersici in various temperature conditions

29
Light conditions

• Pathogen development was also markedly
  influenced by the LIGHT CONDITIONS. At each light
  regime, the percentage of CONIDIA GERMINATION was
  relatively HIGH, and after 48 hpi ranged 7895
• Light intensity significantly influenced
  pathogen development. Conidiation and mycelium
  development was greatest at light intensities of
  approximately 5562 umol /m2 per second.
• At LOWER INTENSITIES, pathogen DEVELOPMENT WAS
  DELAYED, and in the dark, conidiation was
  completely inhibited.
• The results regarding the effect of LIGHT
  SPECTRUM are more complicated. Pathogen
  development was MORE RAPID UNDER RED, blue and
  green plastic foil, that under white light.
  However, CONIDIATION was PROFUSE after 8 dpi
  under ALL COLOUR foils.
• A dark period of 24 h after inoculation had no
  stimulatory effect on later mycelium development,
  however complete dark for 8 days reduced mycelium
  development and no sporulation occurred.
• Very interesting results were obtaineed when
  only inoculated LEAF was COVERED WITH ALUMINIUM
  FOIL while whole plant was placed in photoperiod
  12h/12h. - intensive mycelium development and
  slight subsequent sporulation on covered leaf was
  recorded.

30
Mieslerová, B., Lebeda, A. J. Phytopathol. 112
(2010)
Mean length of the conidial germ tubes of Oidium
neolycopersici in various light conditions
31
Host range of O. neolycopersici

• O. neolycopersici is NOT ABLE TO INFECT
  economicaly important species from the families
  Brassicaceae (Brassica oleracea var. botrytis
  Brassica oleracea var. capitata), Compositae
  (Asteraceae), Leguminosae (Phaseolus lunatus,
  Pisum sativum) and Poaceae (Zea mays, Triticum
  aestivum) (Arredondo et al., 1996 Whipps et al.,
  1998).
• On the other hand, some SUSCEPTIBLE SPECIES WERE
  FOUND in the families Apocynaceae, Campanulaceae,
  Crassulaceae, Cistaceae, Linaceae, Malvaceae,
  Papaveraceae, Pedialiaceae, Scrophulariaceae,
  Valerianaceae a Violaceae (Whipps et al., 1998).
• We tested in host-range studies 70 species of 20
  genera of Solanaceae and 7 species of
  Cucurbitaceae. The most interesting findings were
  the results concerning the family Solanaceae
  there were confirmed the completely resistant
  genotypes, moderatelly resistant genotypes (e.g.
  Ancistus spp., Atropa sp., Browalia sp., most of
  the representatives of Capsicum spp., Hyoscyamus,
  some Solanum)
• On the end of this spectrum are susceptible
  genotypes of genera Datura sp., Nicotiana sp.,
  Petunia sp., Schizanthus sp., and Solanum
  capsicoides, S. jamaicense, S. laciniatum, S.
  lycopersicoides, S. melongena, S. sysimbriifolium
  (Lebeda and Mieslerová, 1999)

32
Records on ability of different Oidium
neolycopersici isolates to infect cucumber,
tobacco and eggplant -
susceptible - - resistant nd - not
determined



Lebeda, A., Mieslerová, B. Plant Prot. Sci. 36
(4)156-162, 2000.
Lebeda, A., Mieslerová, B. Acta Phytopathologica
and Entomologica Hungarica, 34 (1-2), 13-25, 1999.
33
Wild Solanum and Lycopersicon germplasm as
sources of resistance

• Extensive screening of tomato cultivars,
  foregoing the study of wild relatives of tomato
  (Solanum spp.), showed that in assortments of
  TOMATO CULTIVARS (SOLANUM LYCOPERSICUM) available
  till the end of 20th century, DIDNT EXIST ANY
  EFFECTIVE SOURCES OF RESISTANCE to O.
  neolycopersici. Therefore the effort of breeders
  and phytopathologist turned out to wild relatives
  of tomato.
• Generally, among the most important SOURCES OF
  RESISTANCE in earlier genus Lycopersicon
  (recently Solanum) can be considered some
  genotypes of S. habrochaites (L. hirsutum), S.
  parviflorum (L. parviflorum), S. peruvianum (L.
  peruvianum) and S. pennellii (L. pennellii)
  (Lindhout et al., 1994a Ignatova et al., 1997
  Milotay a Dormanns-Simon, 1997 Ciccarese et al.,
  1998 Mieslerová et al., 2000 Matsuda et al.,
  2005).
• On the other hand within species S. lycopersicon
  (L. esculentum) and S. pimpinellifolium (L.
  pimpinellifolium), which are the closest
  relatives of cultivated tomatoes, there were
  found only few resistant genotypes (Georgiev a
  Angelov, 1993 Kumar et al., 1995 Ciccarese et
  al., 1998 Mieslerová et al., 2000) and most of
  the closest relatives are highly susceptible to
  infection of powdery mildew.

34
Succesive clustering of Lycopersicon spp. based
on inoculation experiments with Oidium
neolycopersici (C-1) (154 Lycopersicon spp.
accessions)
Mieslerová, B., Lebeda, A., Chetelat, R.T.
Journal of Phytopathology 148, 303-311, 2000.
35
Intraspecific pathogenic variability within
Oidium neolycopersici

• Differences in host range experiments postulate
  existence of DIFFERENT PATHOTYPES (formae
  speciales) of O. neolycopersici
• The COMPARISON OF PATHOGENICITY of four O.
  neolycopersici isolates originating from the
  CZECH REPUBLIC, GERMANY, THE NETHERLANDS AND
  ENGLAND on Lycopersicon spp. genotypes revealed
  variability on level of race specialization. The
  English isolate of O. neolycopersici considerably
  differs from others higher of susceptible
  responses (according inoculation experiments on
  35 accessions of wild Lycopersicon species).
• The PRELIMINARY DIFFERENTIAL SET OF LYCOPERSICON
  spp. genotypes was proposed.
• Existence of three races was proposed.

36
Comparison of O. neolycopersici isolates
originating from the Czech Republic (C1/96),
Germany (G/97), the Netherlands (W1/97) and
England (E/98) based on inoculation tests with 35
Lycopersicon spp. accessions
Lebeda, A., Mieslerová, B. J. Plant. Dis. Prot.
109 (2) 129-141, 2002.
37
The list of Lycopersicon spp. accessions
recommended as a base for preliminary
differential set and postulated pathogen races
Lebeda, A., Mieslerová, B. J. Plant. Dis. Prot.
109 (2) 129-141, 2002.
Reaction pattern R - resistant ( max ID
between 0-30) M - moderately
resistant/susceptible ( max ID between 30-60)
S - susceptible ( max ID between 60-100)
38
Intraspecific variability within Oidium
neolycopersici

• In the Netherland Huang et al. (2001) studied O.
  neolycopersici variability by AFLP analysis of
  four Dutch isolates. They revelaed at least two
  different patterns related to two types of O.
  neolycopersici isolates.
• Study of intraspecific variability of Oidium
  neolycopersici isolates originating from various
  countries of Europe, North America and Japan
  showed that ITS SEQUENCES were identical for all
  10 isolates of O. neolycopersici, however AFLP
  ANALYSIS discovered high diversity of all
  isolates and they were represented by different
  genotypes (Jankovicz et al., 2008).
• Probably may exist UNKNOWN MANNER OF SEXUAL
  RECOMBINATION or other genetic mechanisms, who is
  responsible for such broad genetic variability of
  O. neolycopersici. Nevertheless, until now was
  not found any clear relationship betweeen
  virulence and AFLP patterns of studied of O.
  neolycopersici isolates.

In the research of this subject is the most
difficult problem separate study of intraspecific
variation by molecular genetic methods and study
of virulence variation.
39
Infection cycle of O. neolycopersici

• Some detailed studies of infection cycle of O.
  neolycopersici on tomato and wild Solanum spp.
  were realized (Huang et al., 1998 Jones et al.,
  2000 Lebeda and Mieslerová, 2000 Lebeda et al.,
  2002 Mieslerová et al., 2004).

3-6 hpi germination started 3-24 hpi
deposits of extracellular matrix (ECM) 8-
hpi primary short germ tube, ending in a
primary appressorium, from which a primary
haustorium Till 24 hpi secondary appressorium,
secondary haustorium Till 72 hpi third and
fourth germ tubes 89-120 hpi the first
conidiophores
Huang et al., 1998 Jones et al., 2000 Lebeda
and Mieslerová, 2000 Lebeda et al., 2002
Mieslerová et al., 2004
40
168 hpi
http//beta-media.padil.gov.au/species/136595/2723
-large.jpg
Schematic representation of Oidium neolycopersici
development at 8, 24 and 72 hpi on leaf discs of
susceptible genotype Solanum lycopersicum cv.
Amateur. (according to Mieslerová and Lebeda,
2010)
41
Comparison of Oidium neolycopersici germination
on Lycopersicon spp. accessions in various
intervals after inoculation
Mieslerova, B., Lebeda, A., Kennedy, R. Ann.
appl. Biol. 144 237-248, 2004.
42
Comparison of Oidium neolycopersici development
on Lycopersicon spp. accessions (72 hpi)
Mieslerova, B., Lebeda, A., Kennedy, R. Ann.
appl. Biol. 144 237-248, 2004.
43
Resistance mechanisms of Lycopersicon spp. to O.
neolycopersici

• Both Huang et al. (1998) and Mieslerová et al.
  (2004) reported that in resistant Solanum (sect.
  Lycopersicon) accessions, many epidermal cells,
  in which a primary haustorium was formed, became
  necrotic, indicating a HYPERSENSITIVE RESPONSE
  (HR). Another resistance MECHANISM NOT BASED ON
  HYPERSENSITIVITY was revealed in L. hirsutum
  (LA 1347) (Mieslerová et al., 2004)
• Huang et al. (1998), who recorded papillae
  beneath some appressoria at very low frequencies
  in all accessions including the susceptible
  control. Haustoria were present in at least 50
  of the cells where papilla was induced.
  Therefore, papilla formation seems NOT TO BE AN
  EFFECTIVE OR A COMMON MECHANISM OF SOLANUM SPP.
  RESISTANCE TO O. NEOLYCOPERSICI.
• The phenomenon of CALLOSE DEPOSITION in the sites
  of pathogen penetration was described in
  pathosystems with powdery mildew. Experiments
  realized by Li et al. (2007) found that
  accumulation of callose are related with the
  resistance given by genes Ol-1 and Ol-4, what is
  manifested by hypersensitive response and also
  linked with the resistance based on recessive
  gene ol-2, which is connected with papillae
  formation.
• In our experiments no changes in the deposition
  of LIGNIN were observed in diseased or healthy
  plants of wild Solanum spp. during the first 120
  hpi (Tománková et al., 2006).

44
Mieslerova, B., Lebeda, A., Kennedy, R. Ann.
appl. Biol. 144 237-248, 2004.
Hypersensitive response of tomato leaf tissue
after infection of powdery mildew (Oidium
neolycopersici)
45
Papilae formation after initial infection of
tomato leaf tissue of powdery mildew (Oidium
neolycopersici)
Mieslerova, B., Lebeda, A., Kennedy, R. Ann.
appl. Biol. 144 237-248, 2004.
46
Resistance mechanisms of Lycopersicon spp. to O.
neolycopersici

• The existence of ADULT PLANT RESISTANCE in tomato
  line OR 4061 was confirmed. Rapid development and
  profuse sporulation of O. neolycopersici was
  observed on juvenile plants (6-8 w), however this
  was in contrast to the slow development and
  sporadic sporulation observed on 4 month old
  plants.
• The phenomenon of FIELD RESISTANCE is only very
  little known in interaction between wild Solanum
  spp. and tomato and O. neolycopersici. Glasshouse
  infection experiment with ten Solanum accessions
  (Mieslerová and Lebeda, unpubl. results) showed
  significant differences in the disease progress
  during the growing period (ca 4 month) and the
  level of field resistance to O. neolycopersici.
• In the end of experiment (110th day after
  inoculation of spread plants) susceptible tomato
  cv. Amateur was heavily infested. However, some
  other accessions (S. pennellii /LA 2560/, S.
  peruvianum /LA 445/, tomato line OR 4061) did not
  exceed 20 of the maximum infection degree (ID)
  and expressed slower rate of diseases
  development, i.e. high level of field resistance.
  

47
Field resistance in the interaction between wild
Solanum spp. and tomato powdery mildew
Solanum spp. accession SmaxID ABC (leaf
disc experiments) S. lycopersicum cv. Amateur
100 5918.75 S. lycopersicum OR 4061 12.5
1328.00 S. lycopersicum OR 960008 50 2685.00 S.
chmielewskii LA 2663 36.66 0 S. habrochaites
LA 1347 28.33 0 S. habrochaites LA 1738 3.33
0 S. habrochaites f. glabratum LA 2120
3.33 0 S. neorickii LA 1322 0 c 0 S.
pennellii LA 2560 14.44 1440.00 S. peruvianum
LA 445 63.33 1493.75
48
Physiology and biochemistry of host-pathogen
interaction

• One of the first responses of host cells after
  beginning of the interaction between plant and
  pathogen is the increased PRODUCTION OF REACTIVE
  OXYGEN SPECIES (ROS).
• PEROXIDASES (POXS) represent one of the important
  groups of enzymes, which participate in the
  metabolism of ROS in plants
• Reactive ROS are apparently involved in the
  INDUCTION OF HYPERSENSITIVE RESPONSE and they
  function also as SIGNAL MOLECULES in the
  programmed cell death (Lamb and Dixon, 1997
  Hückelhoven and Kogel, 2003).
• NITRIC OXIDE (NO), the ubiquitous intra- and
  extracellular messenger, has a wide spectrum of
  regulatory functions in plant growth, ontogenesis
  and responses to various stress stimuli. The key
  role of NO AS A SIGNAL MOLECULE and in defense
  processes of plants was documented

49
Production of ROS in the interaction between
Lycopersicon spp. and Oidium neolycopersici

• Defence reactions occurring in tissue of three
  Lycopersicon spp. were investigated during the
  first 120 hpi. Changes in accumulation of
  HYDROGEN PEROXIDE and enzymes involved in its
  metabolism (CATALASE, PEROXIDASES, SUPEROXIDE
  DISMUTASE) were monitored.
• A hypersensitive reaction was detected after 48
  hpi in both resistant tomato accessions.
• High production of SUPEROXIDE ANION was observed
  mainly in infected leaves of highly susceptible
  Lycopersicon esculentum cv. Amateur during the
  first hours post inoculation (hpi).
• The production of HYDROGEN PEROXIDE as well as
  an INCREASE OF PEROXIDASE (POX) activity were
  detected mainly in RESISTANT ACCESSIONS at 412
  hpi and at the second phase (20-48 hpi).
• INCREASED SOLUBLE POX AND CATALASE ACTIVITY in
  leaf extracts of resistant accessions L.
  chmielewskii (LA 2663) and L. hirsutum (LA 2128)
  (20 hpi) CORRELATED with the of NECROTIC CELLS
  in infection sites.
• The correlation between production of reactive
  oxygen species (ROS) and activity of enzymes
  participating in their metabolism and
  hypersensitive response was evident during plant
  defence response.

50
Time course of hydrogen peroxide concentration in
leaf tissues of Lycopersicon spp. accessions
after inoculation by O. neolycopersici. -
infected, ? - control plants.
Tománková, K., Luhová, L., Petrivalský, M., Pec,
P., Lebeda, A. Physiol. Mol. Plant. Pathol. 68
2232, 2006.
Mlícková, K., Luhová, L., Lebeda, A., Mieslerová,
B., Pec, P. Plant Physiol. Biochem. 42 753-761,
2004.
51
Time course of peroxidase activity in leaves of
Lycopersicon spp. accessions after inoculation by
O. neolycopersici
Tománková, K., Luhová, L., Petrivalský, M., Pec,
P., Lebeda, A. Physiol. Mol. Plant. Pathol. 68
2232, 2006.
Mlícková, K., Luhová, L., Lebeda, A., Mieslerová,
B., Pec, P. Plant Physiol. Biochem. 42 753-761,
2004.
52
Time course of catalase activity in leaves of
Lycopersicon spp. accessions after inoculation by
O. neolycopersici
Tománková, K., Luhová, L., Petrivalský, M., Pec,
P., Lebeda, A. Physiol. Mol. Plant. Pathol. 68
2232, 2006.
Mlícková, K., Luhová, L., Lebeda, A., Mieslerová,
B., Pec, P. Plant Physiol. Biochem. 42 753-761,
2004.
53
Tománková, K., Luhová, L., Petrivalský, M., Pec,
P., Lebeda, A. Physiol. Mol. Plant. Pathol. 68
2232, 2006.
Mlícková, K., Luhová, L., Lebeda, A., Mieslerová,
B., Pec, P. Plant Physiol. Biochem. 42 753-761,
2004.
54
Local and systemic production of nitric oxide in
tomato responses to powdery mildew infection

• NO production was determined in PLANT LEAF
  EXTRACTS of L. esculentum cv. Amateur
  (susceptible), L. chmielewskii (moderately
  resistant) and L. hirsutum f. glabratum (highly
  resistant) by the oxyhaemoglobin method during
  216 h post-inoculation.
• In SUSCEPTIBLE GENOTYPE, elevated NO production
  was observed only during the EARLY INTERVAL
  following inoculation, at 4-8 hpi.
• A specific, TWO-PHASE INCREASE IN NO PRODUCTION
  was observed in the extracts of infected leaves
  of MODERATELY AND HIGHLY RESISTANT genotypes.
  Second phase started from 96 hpi and lasted up to
  end of the studied interval at 216 hpi.
• Moreover, transmission of a SYSTEMIC RESPONSE
  THROUGHOUT THE PLANT was observed as an increase
  in NO production within tissues of uninoculated
  leaves.
• In resistant tomato genotypes, increased NO
  production was LOCALIZED IN INFECTED TISSUES by
  confocal laser scanning microscopy using the
  fluorescent probe 4-amino-5- methylamino-2',7'-dif
  luorofluorescein diacetate.

55
Piterková, J., Petrivalský, M., Luhová, L.,
Mieslerová, B., Sedlárová, M., Lebeda, A. Mol.
Plant Pathol. 10 501-513, 2009.
Localization of nitric oxide (NO) at later stages
of Oidium neolycopersici pathogenesis (168 hpi)
on Lycopersicon chmielewskii (LA 2663) - Confocal
fluorescence
staining with DAF-FM DA (4-amino-5-(N-methylamino)
-2,7-difluorofluorescein diacetate)
56
Piterková, J., Petrivalský, M., Luhová, L.,
Mieslerová, B., Sedlárová, M., Lebeda, A. Mol.
Plant Pathol. 10 501-513, 2009.
Increase of NO production in infected compared to
control non-infected plants 4, 8 and 216 hpi in
the leaves under (brown column) and above (green
column) inoculated (red column) leaves of L.
esculentum cv. Amateur (susceptible genotype), L.
hirsutum f. glabratum (LA 2128) (highly
resistant) and L. chmielewskii (LA 2663)
(moderately resistant).
57
Changes in photosynthesis of Lycopersicon spp.
plants induced by tomato powdery mildew
infection in combination with heat shock
pre-treatment

• Effect of POWDERY MILDEW Oidium neolycopersici
  ON PHOTOSYNTHESIS in tomato leaves was
  investigated DURING 9 DAYS after inoculation
  using CO2 exchange measurement and chlorophyll
  fluorescence imaging.
• In both MODERATELY RESISTANT (Lycopersicon
  chmielewskii) and SUSCEPTIBLE (Lycopersicon
  esculentum cv. Amateur) genotypes the infection
  caused only minimal impairment of photosynthesis.
• Because in many host-pathogen interactions,
  PLANT RESISTANCE and/or susceptibility is
  INFLUENCED BY TEMPERATURE, we studied effect of
  short heat stimulus (40,5C 2 h) on pathogen
  development and changes of photosynthesis.
• When the plants were PRE-TREATED BY HEAT SHOCK
  (40.5 C, 2 H) before inoculation, RESISTANCE
  RESPONSE OF L. chmielewskii was NOT AFFECTED,
  whereas in L. esculentum CHLOROSES/NECROSES
  DEVELOPED and rate of CO2 assimilation and
  maximal quantum yield of photosystem II
  photochemistry (FV/FM) decreased in infected
  leaves.
• The HS-pretreatment did not change significantly
  the resistance in L. chmielewskii and increase
  susceptibility in L. esculentum.

58
Photographs (A-D) of representative healthy and
powdery mildew infected leaflets of the
SUSCEPTIBLE TOMATO (L. esculentum) with
(HS-treated) or without (non-treated) heat shock
pre-treatment the image of MAXIMAL QUANTUM YIELD
OF PHOTOSYSTEM II PHOTOCHEMISTRY (FV/FM E-H) and
steady-state value of NON-PHOTOCHEMICAL
FLUORESCENCE QUENCHING (NPQ I-L) in the same
leaflets (9dpi).
Prokopová, J.,Mieslerová, B., Hlavácková, V.,
Hlavinka, J., Lebeda, A., Nauš, J., Špundová, M.
. Physiol. Mol. Plant Pathol. 2010 (in print).
59
Genetic basis of resistance

• Only few experiments tried to study the genetic
  background of resistance to O. neolycopersici in
  wild Lycopersicon spp..
• The resistance in the pathosystem Lycopericon
  spp. - O. neolycopersici is conferred by
  monogenic genes (Bai et al., 2005 Huang et l.,
  2000 Li et al., 2007).
• DOMINANT RESISTANCE GENES (Ol -1, Ol- 3, Ol -4,
  Ol -5, Ol- 6) confer race-specific resistance by
  hampering the fungal growth via Hypersensitive
  response of the host rpidermal cells, whereas the
  RECESSIVE GENE ol-2 confers reistance via papilla
  formation. POLYGENIC RESISTANCE locus linked
  on Chr 6- L. hirsutum PI247087.

Resistance gene Origin Author
Ol -1 L. hirsutum G1.1560 Huang et al., 2000
ol-2 L. esculentum var. cerasiforme Ciccarese et al., 1998
Ol- 3 L. hirsutum G1. 1290 Huang et al., 2000
Ol -4 L. peruvianum LA2172 Bai et al., 2004
Ol -5 L. hirsutum PI247087 Bai et al., 2005
Ol- 6 ABLs Bai et al., 2005
Ol-QTLs 1-3 L. parviflorum G1.1601 Bai et al., 2003
60
Rozvoj oboru rostlinolékarství na katedre
botaniky PrF UP

• Pedagogická cást
• Stávající výuka predmetu Základy fytopatologie
  bude rozšírena výukou predmetu
• Fytopatologie pro pokrocilé (výuka od školního
  roku 2013/2014)
• Fytopatologická exkurze (výuka od školního roku
  2012/2013) spolupráce s MZLU
• Výstavy pro verejnost napr. v botanické zahrade
  UP
• Prednášky pro verejnost

61
Rozvoj oboru rostlinolékarství na katedre
botaniky PrF UP

• Vedecká cást
• Vedení bakalárských a diplomových prací popr.
  SOC
• Studium vnitrodruhové patogenní variability
  obligátních biotrofních parazitu rostlin
  (klasický fytopatologický prístup) výhledove
  doplnit o molekulární metody- zatím se darí pouze
  u nekterých patogenu
• Studium mechanismu rezistence hostitelu vuci
  biotrofním parazitum použití nových metod
  detekce napr. hypersenzitivní reakce spolupráce
  s katedrou biochemie (produkce enzymu
  podílejících se obranných reakcích) téma
  rozšírit o studium stresem podmínené zmeny
  rezistence/náchylnosti.
• Studium biologie biotrofních patogenu. Soustredit
  se na problematiku prezimování a reinfekce na
  jare
• Prohloubit studium výskytu biotrofních parazitu
  na okrasných rostlinách