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RNDr. Barbora Mieslerová, Ph.D. Katedra botaniky Přírodovědecká fakulta Univerzita Palackého Olomouc Case study: Interaction Solanum spp. – Oidium neolycopersici.

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Presentation on theme: "RNDr. Barbora Mieslerová, Ph.D. Katedra botaniky Přírodovědecká fakulta Univerzita Palackého Olomouc Case study: Interaction Solanum spp. – Oidium neolycopersici."— Presentation transcript:

1 RNDr. Barbora Mieslerová, Ph.D. Katedra botaniky Přírodovědecká fakulta Univerzita Palackého Olomouc Case study: Interaction Solanum spp. – Oidium neolycopersici

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. tuberosumEggplant, 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 CHEES Strong barrier of interspecific hybridization ESC PIM PENN PARV CHMIE HIRS CHIL PER PERHU Esculentum-complex Peruvianum-complex Crossability polygon of Solanum (Lycopersicon) species (Lindhout et al., 1994)

7 Lycopersicon esculentum var. cerasiforme (Solanum lycopersicum) L. pimpinellifolium (S. pimpinellifolium )

8 Lycopersicon hirsutum f. glabratum (Solanum habrochaites) L. pennellii (S. pennellii)

9 L. peruvianum (S. peruvianum) L. chmielewskii (S. chmielewskii) pg

10 Recently, it is widely accepted that tomato and its wild relatives belong to the genus Solanum subgen. Potatoe (G. Don) D´Arcy, 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). Taxonomy of genus Solanum – recent taxonomy

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

15 The map of the first records of Oidium neolycopersici occurrence in Europe Lebeda, A., Mieslerová, B. Plant Prot. Sci. 36 (4): , 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 PreparationEffective compound BIOANLecitihin, Albumin, Milk Cassein KUMULUS WGSulphur ORTIVAAzoxystrobin SCORE 250 ECDifenoconazole TALENTMyclobutanil TOPAS 100 ECPenconazole

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.78: , 2000.

22 O. neolycopersici isolate Pseudoidium type O. lycopersici isolate from South Australia – Euoidium type Kiss et al. Mycol. Res. 105: , 2001

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  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, Morphological comparative study

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 Golovinomyces cichoracearum Sphaerotheca fusca Oidium neolycopersici Mieslerová, B., Lebeda, A., Kennedy, R., Novotný, R. Acta Phytopathol. Entomol. Hungar., 37 (1-3): 57-74, 2002.

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– 35°C); 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 (20–25°C), MYCELIAL DEVELOPMENT and time of appearance of the first conidiophores was delayed. CONIDIATION occurred within the range of 15–25°C, however was most intense between 20–25°C.  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 Mean length of the conidial germ tubes of Oidium neolycopersici in various temperature conditions Mieslerová, B., Lebeda, A. J. Phytopathol. 1–12 (2010)

29  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 78–95%  Light intensity significantly influenced pathogen development. Conidiation and mycelium development was greatest at light intensities of approximately 55–62 umol ⁄m 2 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. Light conditions

30 Mean length of the conidial germ tubes of Oidium neolycopersici in various light conditions Mieslerová, B., Lebeda, A. J. Phytopathol. 1–12 (2010)

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. Acta Phytopathologica and Entomologica Hungarica, 34 (1-2), 13-25, Lebeda, A., Mieslerová, B.: Plant Prot. Sci. 36 (4): , 2000.

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, DIDN´T 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, , 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) , 2002.

37 The list of Lycopersicon spp. accessions recommended as a base for preliminary differential set and postulated pathogen races Reaction pattern: R - resistant (% max ID between 0-30) M - moderately resistant/susceptible (% max ID between 30-60) S - susceptible (% max ID between ) Lebeda, A., Mieslerová, B. J. Plant. Dis. Prot. 109 (2) , 2002.

38  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. Intraspecific variability within Oidium neolycopersici 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- hpiprimary short germ tube, ending in a primary appressorium, from which a primary haustorium Till 24 hpisecondary appressorium, secondary haustorium Till 72 hpi third and fourth germ tubes hpithe first conidiophores Huang et al., 1998; Jones et al., 2000; Lebeda and Mieslerová, 2000; Lebeda et al., 2002; Mieslerová et al., 2004

40 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) 168 hpi large.jpg

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: , 2004.

42 Comparison of Oidium neolycopersici development on Lycopersicon spp. accessions (72 hpi) Mieslerova, B., Lebeda, A., Kennedy, R.: Ann. appl. Biol. 144: , 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 Hypersensitive response of tomato leaf tissue after infection of powdery mildew (Oidium neolycopersici) Mieslerova, B., Lebeda, A., Kennedy, R.: Ann. appl. Biol. 144: , 2004.

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: , 2004.

46 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 (110 th 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. Resistance mechanisms of Lycopersicon spp. to O. neolycopersici

47 Solanum spp. accessionΣ%maxID ABC (leaf disc experiments) S. lycopersicum cv. Amateur S. lycopersicum OR S. lycopersicum OR S. chmielewskii LA S. habrochaites LA S. habrochaites LA S. habrochaites f. glabratum LA S. neorickii LA c 0 S. pennellii LA S. peruvianum LA Field resistance in the interaction between wild Solanum spp. and tomato powdery mildew

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 4–12 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., Petřivalský, M., Peč, P., Lebeda, A. Physiol. Mol. Plant. Pathol. 68: 22–32, Mlíčková, K., Luhová, L., Lebeda, A., Mieslerová, B., Peč, P. Plant Physiol. Biochem. 42: , 2004.

51 Time course of peroxidase activity in leaves of Lycopersicon spp. accessions after inoculation by O. neolycopersici Tománková, K., Luhová, L., Petřivalský, M., Peč, P., Lebeda, A. Physiol. Mol. Plant. Pathol. 68: 22–32, Mlíčková, K., Luhová, L., Lebeda, A., Mieslerová, B., Peč, P. Plant Physiol. Biochem. 42: , 2004.

52 Time course of catalase activity in leaves of Lycopersicon spp. accessions after inoculation by O. neolycopersici Tománková, K., Luhová, L., Petřivalský, M., Peč, P., Lebeda, A. Physiol. Mol. Plant. Pathol. 68: 22–32, Mlíčková, K., Luhová, L., Lebeda, A., Mieslerová, B., Peč, P. Plant Physiol. Biochem. 42: , 2004.

53 Tománková, K., Luhová, L., Petřivalský, M., Peč, P., Lebeda, A. Physiol. Mol. Plant. Pathol. 68: 22–32, Mlíčková, K., Luhová, L., Lebeda, A., Mieslerová, B., Peč, P. Plant Physiol. Biochem. 42: , 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′-difluorofluorescein diacetate.

55 Localization of nitric oxide (NO) at later stages of Oidium neolycopersici pathogenesis (168 hpi) on Lycopersicon chmielewskii (LA 2663) - Confocal fluorescence Piterková, J., Petřivalský, M., Luhová, L., Mieslerová, B., Sedlářová, M., Lebeda, A. Mol. Plant Pathol. 10: , staining with DAF-FM DA (4-amino-5-(N-methylamino)-2`,7`-difluorofluorescein diacetate)

56 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). Piterková, J., Petřivalský, M., Luhová, L., Mieslerová, B., Sedlářová, M., Lebeda, A. Mol. Plant Pathol. 10: , 2009.

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 CO 2 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 (40,5°C 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 CO 2 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., Hlaváčková, V., Hlavinka, J., Lebeda, A., Nauš, J., Špundová, M.. Physiol. Mol. Plant Pathol (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 PI Resistance geneOriginAuthor Ol -1L. hirsutum G1.1560Huang et al., 2000 ol-2L. esculentum var. cerasiforme Ciccarese et al., 1998 Ol- 3L. hirsutum G Huang et al., 2000 Ol -4L. peruvianum LA2172Bai et al., 2004 Ol -5L. hirsutum PI247087Bai et al., 2005 Ol- 6ABLsBai et al., 2005 Ol-QTLs 1-3L. parviflorum G Bai et al., 2003

60 Rozvoj oboru rostlinolékařství na katedře botaniky PřF UP Pedagogická část: Stávající výuka předmětů Základy fytopatologie bude rozšířena výukou předmětů: Fytopatologie pro pokročilé (výuka od školního roku 2013/2014) Fytopatologická exkurze (výuka od školního roku 2012/2013) – spolupráce s MZLU Výstavy pro veřejnost např. v botanické zahradě UP Přednášky pro veřejnost

61 Rozvoj oboru rostlinolékařství na katedře botaniky PřF UP Vědecká část : Vedení bakalářských a diplomových prací; popř. SOČ Studium vnitrodruhové patogenní variability obligátních biotrofních parazitů rostlin (klasický fytopatologický přístup) – výhledově doplnit o molekulární metody- zatím se daří pouze u některých patogenů Studium mechanismů rezistence hostitelů vůči biotrofním parazitům – použití nových metod detekce např. hypersenzitivní reakce; spolupráce s katedrou biochemie (produkce enzymů podílejících se obranných reakcích); téma rozšířit o studium stresem podmíněné změny rezistence/náchylnosti. Studium biologie biotrofních patogenů. Soustředit se na problematiku přezimování a reinfekce na jaře Prohloubit studium výskytu biotrofních parazitů na okrasných rostlinách


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