RNDr. Barbora Mieslerová, Ph.D. Katedra botaniky Přírodovědecká fakulta Univerzita Palackého

RNDr. Barbora Mieslerov, Ph.D.Katedra botanikyProdovdeck fakultaUniverzita PalackhoOlomoucCase study: Interaction Solanum spp. Oidium neolycopersici

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

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. lycopersicumPotato,S. tuberosumEggplant,S. melongena

Solanum (Lycopersicon) spp.

Variability of fruits and flowers of r. Solanum sect. Lycopersicon, sect. Juglandifolia a sect. Lycopersicoides (Peralta et al., 2008).

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).

CHEESStrong barrier of interspecific hybridization ESCPIMPENNPARVCHMIEHIRSCHILPERPERHUEsculentum-complexPeruvianum-complexCrossability polygon of Solanum (Lycopersicon) species (Lindhout et al., 1994)

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

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

L. peruvianum (S. peruvianum)L. chmielewskii (S. chmielewskii)http://digi.azz.cz/Book001/images/Solanum_peruvianum_A327.jpg

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). Taxonomy of genus Solanum recent taxonomy

Comparison of earlier (Rick, 1979) and recent classification (Peralta et al., 2008) of genus Solanum sect. Lycopersicon (according to Grandillo et al., 2011)

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

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).

http://agro.biodiver.se/2007/04/whats-so-special-about-oidium-neolycopersici/ Distribution of Oidium neolycopersici

The map of the first records of Oidium neolycopersici occurrence in EuropeLebeda, A., Mieslerov, B. Plant Prot. Sci. 36 (4):156-162, 2000.

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.

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. Mieslerov

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. Novotn (A, B) and B. Mieslerov (C, D)

Chemical protection - registered preparations against tomato powdery mildew in the Czech Republic

Preparation Effective compound BIOANLecitihin, Albumin, Milk CasseinKUMULUS WGSulphurORTIVAAzoxystrobin SCORE 250 EC DifenoconazoleTALENT Myclobutanil TOPAS 100 ECPenconazole

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).

Parsimony tree of the phylogenetic analysis of ITS4 -5,8S- ITS 5 regions. Jones et al.. Can. J. Bot.78:1361-1366, 2000.

O. neolycopersici isolate Pseudoidium type O. lycopersici isolate from South Australia Euoidium type Kiss et al. Mycol. Res. 105: 684-697, 2001

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)

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

Mieslerov, B., Lebeda, A., Kennedy, R., Novotn, R. Acta Phytopathol. Entomol. Hungar., 37 (1-3): 57-74, 2002. Morphological comparative study

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).

Mieslerov, B., Lebeda, A., Kennedy, R., Novotn, R. Acta Phytopathol. Entomol. Hungar., 37 (1-3): 57-74, 2002.

Dissimilarity

OL C-1

OL W-1

OL G-3

OL RZ-1

OL W-2

OL G-2

OL G-4

OL G-5

OL CKV

OLC1CS

OL E-1

EAR5

GO

GC

SF13

0.00

0.50

1.00

1.50

2.00

SEM photographs of selected powdery mildews Golovinomyces cichoracearum Sphaerotheca fuscaOidium neolycopersici Mieslerov, B., Lebeda, A., Kennedy, R., Novotn, R. Acta Phytopathol. Entomol. Hungar., 37 (1-3): 57-74, 2002.

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; Mieslerov 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.

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

Graf1

12.214.7514.9316.7214.3214.98

21.3518.261926.2622.720

20.911928.1645.5121.8322.87

20.9437.85108.2200.3121.8123.99

T10

T15

T20

T25

T30

T35

Hours post inoculation

Mean length of the germ tube (um)

List1

6hpi11 hpi24 hpi48 hpi

T1012.221.3520.9120.94

T1514.7518.261937.85

T2014.931928.16108.2

T2516.7226.2645.51200.31

T3014.3222.721.8321.81

T3514.982022.8723.99

T102.447.938.615.24

T155.256.366.5124.24

T205.388.6315.1557.69

T256.1914.9731.994.31

T3011.5214.1617.4515.69

T354.213.6615.718.16

List1

000000

000000

000000

000000

T10

T15

T20

T25

T30

T35



List2

List3

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. Light conditions

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

Graf2

10.614.4412.2714.6211.0710.889.0511.67

20.7420.6220.6921.3920.2118.5722.0917.64

22.2428.9831.4130.525.2120.9521.422.32

73.892.7101.9675.2460.7537.6748.0330.02

A white light

B blue light

C red light

D green light

E reduced light intensity

F reduced light intensity

G reduced light intensity

H dark



List1

6hpi11 hpi24 hpi48 hpi

T1012.221.3520.9120.94

T1514.7518.2619.7137.85

T2014.931928.16108.2

T2516.7226.2645.51200.31

T3014.3222.721.8321.81

T3514.9820.7422.8723.99

T102.447.938.615.24

T155.256.366.5124.24

T205.388.6315.1557.69

T256.1914.9731.994.31

T3011.5214.1617.4515.69

T354.213.6615.718.16

List1

000000

000000

000000

000000

T10

T15

T20

T25

T30

T35



List2

T1020.945.24

T1537.8524.24

T20108.257.69

T25200.3194.31

T3021.8115.69

T3523.998.16

List2

05.245.24

024.2424.24

057.6957.69

094.3194.31

015.6915.69

08.168.16

Experimental temperatures


List3

6 hpi11 hpi24 hpi48 hpi

A white light10.6020.7422.2473.8

B blue light14.4420.6228.9892.7

C red light12.2720.6931.41101.96

D green light14.6221.3930.575.24

E reduced light intensity11.0720.2125.2160.75

F reduced light intensity10.8818.5720.9537.67

G reduced light intensity9.0522.0921.448.03

H dark11.6717.6422.3230.02

List3

00000000

00000000

00000000

00000000

A white light

B blue light

C red light

D green light

E reduced light intensity

F reduced light intensity

G reduced light intensity

H dark



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 Mieslerov, 1999)

Records on ability of different Oidium neolycopersici isolates to infect cucumber, tobacco and eggplant

+ - susceptible- - resistant nd - not determined Lebeda, A., Mieslerov, B. Acta Phytopathologica and Entomologica Hungarica, 34 (1-2), 13-25, 1999.Lebeda, A., Mieslerov, B.: Plant Prot. Sci. 36 (4):156-162, 2000.

Plant species

Cucumis

Nicotiana

Solanum

Origin

Report

sativus

tabacum

melongena

CS

Lebeda, Mieslerov

(1999)

+

-

-

HU

Kiss (1996)

-

-

nd

SW

Corbaz (1993)

+

+

nd

NL

Huang et al. (1998)

-

+

+

UK

Fletcher et al. (1988)

-

+

+

UK

Whipps et al. (1998)

+

+

+

RUS

Ignatova et al. (1997)

+

+

nd

Wild Solanum and Lycopersicon germplasm as sources of resistanceExtensive 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; Mieslerov 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; Mieslerov et al., 2000) and most of the closest relatives are highly susceptible to infection of powdery mildew.

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

L. esculentum

L. pimpinellifolium

L. esc. cerasiforme

L. chmielewskii

L. peruvianum

L. parviflorum

L. pennellii

L. hirs. glabratum

L. hirsutum

L. chilense

L. cheesmanii

Dissimilarity

0,00

1,00

2,00

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.

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., Mieslerov, B. J. Plant. Dis. Prot. 109 (2) 129-141, 2002.

E/98

W1/97

G/97

C1/96

Dissimilarity

0.00

0.50

1.00

1.50

2.00

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 60-100)

Lebeda, A., Mieslerov, B. J. Plant. Dis. Prot. 109 (2) 129-141, 2002.

Lycopersicon spp. Accession

O. neolycopersici isolate / race/ response

W1/97

C1/96

G/97

E/98

OL1

OL2

OL2

OL3

L. esculentum

cv. Amateur

S

S

S

S

L. hirsutum

LA 94

R

S

S

M

L. hirsutum

LA 1738

R

R

R

S

L. hirsutum

LA 1731

R

R

R

M

L. hirsutum f. glabratum

LA 2128

R

R

R

R

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 neolycopersiciIn the research of this subject is the most difficult problem separate study of intraspecific variation by molecular genetic methods and study of virulence variation.

Infection cycle of O. neolycopersiciSome 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 Mieslerov, 2000; Lebeda et al., 2002; Mieslerov et al., 2004). 3-6 hpi germination started3-24 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 haustoriumTill 72 hpi third and fourth germ tubes 89-120 hpithe first conidiophoresHuang et al., 1998; Jones et al., 2000; Lebeda and Mieslerov, 2000; Lebeda et al., 2002; Mieslerov et al., 2004

Schematic representation of Oidium neolycopersici development at 8, 24 and 72 hpi on leaf discs of susceptible genotype Solanum lycopersicum cv. Amateur. (according to Mieslerov and Lebeda, 2010) 168 hpihttp://beta-media.padil.gov.au/species/136595/2723-large.jpg

Comparison of Oidium neolycopersici germination on Lycopersicon spp. accessions in various intervals after inoculationMieslerova, B., Lebeda, A., Kennedy, R.: Ann. appl. Biol. 144: 237-248, 2004.

Graf1

0.50.840.91

0.720.860.83

0.710.90.81

0.280.820.79

0.090.590.68

0.070.670.7

0.030.530.61

0.070.740.88

0.140.710.81

0.350.890.93

6 h

24 h

48 h

% germination

List1

6 h24 h48 h

L. esc. Amateur50%84%0.91

L. esc. OR 40610.720.860.83

L. esc. OR 9600080.710.90.81

L. chmie LA 26630.280.820.79

L. hir. LA 13470.090.590.68

L. hir. LA 17380.070.670.7

L. hir. glab. LA 21280.030.530.61

L. parv. LA 13220.070.740.88

L. penn. LA 25600.140.710.81

L. per. LA 4450.350.890.93

List1

000

000

000

000

000

000

000

000

000

000

6 h

24 h

48 h

% germination

List2

List3

Comparison of Oidium neolycopersici development on Lycopersicon spp. accessions (72 hpi)Mieslerova, B., Lebeda, A., Kennedy, R.: Ann. appl. Biol. 144: 237-248, 2004.

Graf1

00558

0151570

0713553

0919.371.7

2111155

1103106

5108110

01041817

01107120

031580

01223139

0918115

Conidia with the first non-appressorial germ tube

Conidia with the first appressorial germ tube

Conidia with the second germ tube

Conidia with the third germ tube

List1

72L. esc. AmateurL. esc. OR 4061 JL. esc. OR 4061AL. esc. OR 960008 JL. esc. OR 96 0008 AL. chmiel. LA 2663L. hirs. LA 1347L. hirs. LA 1738L. hirs. f. glabr. LA 2128L. parv. LA 1322L. penn. LA 2560L. peruv. LA 445

Conidia with the first non-appressorial germ tube000021500000

Conidia with the first appressorial germ tube0157191111031081041103129

Conidia with the second germ tube5153519.31510111871152318

Conidia with the third germ tube58705371.7560172080139115

48ControlL. esculentum OR4061 JL. esculentum OR 4061AL. esculentum OR 960008 JL. esculentum OR 96 0008 AL. chmielewskii LA 2663L. hirsutum LA 1347L. hirsutum LA 1738L. hirs. f. glabratum LA 2128L. parviflorum LA 1322L. pennellii LA 2560L. peruvianum LA 445

Conidia with primary non-appressorial germ tube00101.8043.813.8000

Conidia with primary appressorial germ tube8565.73.671.61091026375.911122.7

Conidia with secondary germ tube1345.725.230.316.5912248.6263333.8

Conidia with terciary germ tube7849.48.166.110329.61.7506263.5


Conidia with primary non-appressorial germ tube14.111.252.52.521617.833.95113.4

Conidia with primary appressorial germ tube39.369.889.272.595979359.364.4855830.5

Conidia with secondary germ tube46.518.95.8252.5211222.91.7295066.1

Conidia with terciary germ tube000000000000


Conidia with primary non-appressorial germ tube6969.27463.554.611110388.686.8879591.1

Conidia with primary appressorial germ tube3130.82636.545.49811.413.21298.9

Conidia with secondary germ tube000000000000

Conidia with terciary germ tube000000000000

List1

00558

0151570

0713553

0919.371.7

2111155

1103106

5108110

01041817

01107120

031580

01223139

0918115

Conidia with the first non-appressorial germ tube

Conidia with the first appressorial germ tube

Conidia with the second germ tube

Conidia with the third germ tube

List2

081378

0545.749.4

165.725.28.1

03.630.366.1

1.871.616.510

010993

4102122

3.863249.6

13.875.98.61.7

0112650

0123362

02.733.863.5

Conidia with primary non-appressorial germ tube

Conidia with primary appressorial germ tube

Conidia with secondary germ tube

Conidia with terciary germ tube

List3

0000

0000

0000

0000

0000

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Resistance mechanisms of Lycopersicon spp. to O. neolycopersici Both Huang et al. (1998) and Mieslerov 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) (Mieslerov 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 (Tomnkov et al., 2006).

Hypersensitive response of tomato leaf tissue after infection of powdery mildew (Oidium neolycopersici) Mieslerova, B., Lebeda, A., Kennedy, R.: Ann. appl. Biol. 144: 237-248, 2004.

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.

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 (Mieslerov 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.

Resistance mechanisms of Lycopersicon spp. to O. neolycopersici

Solanum spp. accession%maxID ABC(leaf disc experiments)

S. lycopersicum cv. Amateur 1005918.75S. lycopersicum OR 406112.5 1328.00S. lycopersicum OR 960008502685.00S. chmielewskii LA 266336.660S. habrochaites LA 134728.33 0S. habrochaites LA 17383.33 0S. habrochaites f. glabratum LA 2120 3.330S. neorickii LA 1322 0 c 0S. pennellii LA 256014.44 1440.00S. peruvianum LA 44563.33 1493.75

Field resistance in the interaction between wild Solanum spp. and tomato powdery mildew

Physiology and biochemistry of host-pathogen interactionOne 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; Hckelhoven 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

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.

Time course of hydrogen peroxide concentration in leaf tissues of Lycopersicon spp. accessions after inoculation by O. neolycopersici. - infected, - control plants.Tomnkov, K., Luhov, L., Petivalsk, M., Pe, P., Lebeda, A. Physiol. Mol. Plant. Pathol. 68: 2232, 2006. Mlkov, K., Luhov, L., Lebeda, A., Mieslerov, B., Pe, P. Plant Physiol. Biochem. 42: 753-761, 2004.

Time course of peroxidase activity in leaves of Lycopersicon spp. accessions after inoculation by O. neolycopersiciTomnkov, K., Luhov, L., Petivalsk, M., Pe, P., Lebeda, A. Physiol. Mol. Plant. Pathol. 68: 2232, 2006. Mlkov, K., Luhov, L., Lebeda, A., Mieslerov, B., Pe, P. Plant Physiol. Biochem. 42: 753-761, 2004.

Graf2

37.9138.08

32.0835

56.3134.35

49.127.87

53.4740.67

7044.56

77.143.91

74.6744

63.7661.73

49.7457.73

68.8646.01

69.0348.44

86.4854.23

94.0751.85

175.3269.35

199.0745.7

76.99100.73

71.5588.7

86.9893.14

79.5975.58

104.7493.07

164.47101.62

222.9690.66

328.68117.17

L. esculentum cv. Amateur

L. chmielewskii(LA 2663)

L. hirsutum f. glabratum (LA 2128)

Infected plants

Healthy plants

Time (hpi)

Peroxidase activity (nkat/ml)

Catalasa

Infected plantsHealthy plants

635.2642.86

1219.428.02

2417.8929.35

3618.0534.43

5027.6736.11

7249.663.16

1207073.72

16864.1139.47

610.89.4

1281.2

245.41.2

3639.1516.2

5021.8710.8

7236.452.6

120560.210.8

168641.21.2

639.521.3

1217.435.5

2435.537.5

3624.713.2

5028.116.6

7267.531.4

12069.242.2

168221.444.3

Catalasa

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00


L. chmielewskii (LA 2663)


Infected plants

Healthy plants

Catalase activity (nkat/ml)

Peroxidase


637.9138.08

1232.0835

2456.3134.35

3649.127.87

5053.4740.67

727044.56

12077.143.91

16874.6744

663.7661.73

1249.7457.73

2468.8646.01

3669.0348.44

5086.4854.23

7294.0751.85

120175.3269.35

168199.0745.7

676.99100.73

1271.5588.7

2486.9893.14

3679.5975.58

50104.7493.07

72164.47101.62

120222.9690.66

168328.68117.17

Peroxidase

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00




Infected plants

Healthy plants

Time (hpi)


List3

MBD0007A85B.xls

Graf7

91025.44

794542.22

615062.85

Germination (48 hpi)

Hypersensitive reactions (72 hpi)

Peroxidase reaction mkat/ml (72 hpi)

Germination, HR (%)


Peroxidasa

Infected plantsHealthy plantsAmateur

637.9138.08

1232.0835

2456.3134.35

3649.127.87

4853.4740.67

727044.56

12077.143.91

16874.6744

663.7661.73

1249.7457.52

2468.8646.01

3669.0348.44Chmielewski

4886.4854.23

7294.0751.85

120175.3269.35

168199.0745.7

676.99100.73

1271.5588.7

2486.9893.14

3679.5975.58

48104.7493.07

72164.47101.62Hirsutum

120222.9690.66

168328.68117.17

Peroxidasa

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00


L. chmielewskii LA 2663

L. hirsutum f. glabratum LA 2128

Infected plants

Healthy plants


Catalasa


635.2642.86

1219.428.02

2417.8929.35

3618.0534.43

5027.6736.11

7249.663.16

1207073.72

16864.1139.47

610.89.4

1281.2

245.41.2


5021.8710.8

7236.452.6

120560.210.8

168641.21.2

639.521.3

1217.435.5

2435.537.5

3624.713.2

4828.116.6

7267.531.4Hirsutum

12069.242.2

168221.444.3

Catalasa

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00




Infected plants

Healthy plants


List2

Germination (48 hpi)Hypersensitive reactions (72 hpi)Peroxidase reaction mkat/ml (72 hpi)

L. esculentum cv. Amateur91025.44

L. chmielewskii LA 2663794542.22

L. hirsutum f. glabratum LA 2128615062.85

List2

000

000

000

&A

Page &P




Germination, HR (%)


List3

Time course of catalase activity in leaves of Lycopersicon spp. accessions after inoculation by O. neolycopersiciTomnkov, K., Luhov, L., Petivalsk, M., Pe, P., Lebeda, A. Physiol. Mol. Plant. Pathol. 68: 2232, 2006. Mlkov, K., Luhov, L., Lebeda, A., Mieslerov, B., Pe, P. Plant Physiol. Biochem. 42: 753-761, 2004.

Graf1

35.2642.86

19.428.02

17.8929.35

18.0534.43

27.6736.11

49.663.16

7073.72

64.1139.47

10.89.4

81.2

5.41.2

39.1516.2

21.8710.8

36.452.6

560.210.8

641.21.2

39.521.3

17.435.5

35.537.5

24.713.2

28.116.6

67.531.4

69.242.2

221.444.3




Infected plants

Healthy plants


Catalasa


635.2642.86

1219.428.02

2417.8929.35

3618.0534.43

5027.6736.11

7249.663.16

1207073.72

16864.1139.47

610.89.4

1281.2

245.41.2

3639.1516.2

5021.8710.8

7236.452.6

120560.210.8

168641.21.2

639.521.3

1217.435.5

2435.537.5

3624.713.2

5028.116.6

7267.531.4

12069.242.2

168221.444.3

Catalasa

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00




Infected plants

Healthy plants


Peroxidase


637.9138.08

1232.0835

2456.3134.35

3649.127.87

5053.4740.67

727044.56

12077.143.91

16874.6744

663.7661.73

1249.7457.73

2468.8646.01

3669.0348.44

5086.4854.23

7294.0751.85

120175.3269.35

168199.0745.7

676.99100.73

1271.5588.7

2486.9893.14

3679.5975.58

50104.7493.07

72164.47101.62

120222.9690.66

168328.68117.17

Peroxidase

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00




Infected plants

Healthy plants

Time (hpi)


List3

MBD0007A85B.xls

Graf7

91025.44

794542.22

615062.85




Germination, HR (%)


Peroxidasa


637.9138.08

1232.0835

2456.3134.35

3649.127.87

4853.4740.67

727044.56

12077.143.91

16874.6744

663.7661.73

1249.7457.52

2468.8646.01


4886.4854.23

7294.0751.85

120175.3269.35

168199.0745.7

676.99100.73

1271.5588.7

2486.9893.14

3679.5975.58

48104.7493.07

72164.47101.62Hirsutum

120222.9690.66

168328.68117.17

Peroxidasa

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00




Infected plants

Healthy plants


Catalasa


635.2642.86

1219.428.02

2417.8929.35

3618.0534.43

5027.6736.11

7249.663.16

1207073.72

16864.1139.47

610.89.4

1281.2

245.41.2


5021.8710.8

7236.452.6

120560.210.8

168641.21.2

639.521.3

1217.435.5

2435.537.5

3624.713.2

4828.116.6

7267.531.4Hirsutum

12069.242.2

168221.444.3

Catalasa

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00




Infected plants

Healthy plants


List2

Germination (48 hpi)Hypersensitive reactions (72 hpi)Peroxidase reaction mkat/ml (72 hpi)

L. esculentum cv. Amateur91025.44

L. chmielewskii LA 2663794542.22

L. hirsutum f. glabratum LA 2128615062.85

List2

000

000

000

&A

Page &P




Germination, HR (%)


List3

Tomnkov, K., Luhov, L., Petivalsk, M., Pe, P., Lebeda, A. Physiol. Mol. Plant. Pathol. 68: 2232, 2006. Mlkov, K., Luhov, L., Lebeda, A., Mieslerov, B., Pe, P. Plant Physiol. Biochem. 42: 753-761, 2004.

Graf12

025.44096.5

4542.2233.8530.4

5062.8536.134.9

Hypersensitive reaction (72 hpi)

Changes of peroxidase activity nkat/ml (72 hpi)

Changes of catalase activity nkat/ml (72 hpi)

Mean length of germ tube (um) 48 hpi

HR (%); length of germ tube (um)

Changes of enzyme activity (nkat/ml)

Catalasa


635.2642.86

1219.428.02

2417.8929.35

3618.0534.43

5027.6736.11

7249.663.16

1207073.72

16864.1139.47

610.89.4

1281.2

245.41.2

3639.1516.2

5021.8710.8

7236.452.6

120560.210.8

168641.21.2

639.521.3

1217.435.5

2435.537.5

3624.713.2

5028.116.6

7267.531.4

12069.242.2

168221.444.3

Catalasa




Infected plants

Healthy plants


Peroxidase


637.9138.08

1232.0835

2456.3134.35

3649.127.87

5053.4740.67

727044.56

12077.143.91

16874.6744

663.7661.73

1249.7457.73

2468.8646.01

3669.0348.44

5086.4854.23

7294.0751.85

120175.3269.35

168199.0745.7

676.99100.73

1271.5588.7

2486.9893.14

3679.5975.58

50104.7493.07

72164.47101.62

120222.9690.66

168328.68117.17

Peroxidase




Infected plants

Healthy plants

Time (hpi)


List3

Germination (48 hpi)Hypersensitive reaction (72 hpi)Changes of peroxidase activity nkat/ml (72 hpi)Changes of catalase activity nkat/ml (72 hpi)Mean length of germ tube (um) 48 hpi

L. esculentum91025.44096.5

L. chmielewskii794542.2233.8530.4

L. hirsutum615062.8536.134.9




List3

&A

Page &P


Hypersensitive reaction (72 hpi)

Changes of peroxidase activity nkat/ml (72 hpi)

Changes of catalase activity nkat/ml (72 hpi)

Germination, HR (%)

Changes of enzyme activity (nkat/ml)

Local and systemic production of nitric oxide in tomatoresponses 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.

Localization of nitric oxide (NO) at later stages of Oidium neolycopersici pathogenesis (168 hpi) on Lycopersicon chmielewskii (LA 2663) - Confocal fluorescencePiterkov, J., Petivalsk, M., Luhov, L., Mieslerov, B., Sedlov, M., Lebeda, A. Mol. Plant Pathol. 10: 501-513, 2009.staining with DAF-FM DA (4-amino-5-(N-methylamino)-2`,7`-difluorofluorescein diacetate)

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).Piterkov, J., Petivalsk, M., Luhov, L., Mieslerov, B., Sedlov, M., Lebeda, A. Mol. Plant Pathol. 10: 501-513, 2009.

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.

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). Prokopov, J.,Mieslerov, B., Hlavkov, V., Hlavinka, J., Lebeda, A., Nau, J., pundov, M. . Physiol. Mol. Plant Pathol. 2010 (in print).

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 -1L. hirsutum G1.1560Huang et al., 2000ol-2L. esculentum var. cerasiforme Ciccarese et al., 1998Ol- 3L. hirsutum G1. 1290Huang et al., 2000Ol -4L. peruvianum LA2172Bai et al., 2004Ol -5L. hirsutum PI247087Bai et al., 2005Ol- 6ABLs Bai et al., 2005Ol-QTLs 1-3L. parviflorum G1.1601 Bai et al., 2003

Rozvoj oboru rostlinolkastv na katede botaniky PF UPPedagogick st:Stvajc vuka pedmt Zklady fytopatologie bude rozena vukou pedmt:Fytopatologie pro pokroil (vuka od kolnho roku 2013/2014)Fytopatologick exkurze (vuka od kolnho roku 2012/2013) spoluprce s MZLUVstavy pro veejnost nap. v botanick zahrad UPPednky pro veejnost

Rozvoj oboru rostlinolkastv na katede botaniky PF UPVdeck st :Veden bakalskch a diplomovch prac; pop. SOStudium vnitrodruhov patogenn variability obligtnch biotrofnch parazit rostlin (klasick fytopatologick pstup) vhledov doplnit o molekulrn metody- zatm se da pouze u nkterch patogen Studium mechanism rezistence hostitel vi biotrofnm parazitm pouit novch metod detekce nap. hypersenzitivn reakce; spoluprce s katedrou biochemie (produkce enzym podlejcch se obrannch reakcch); tma rozit o studium stresem podmnn zmny rezistence/nchylnosti.Studium biologie biotrofnch patogen. Soustedit se na problematiku pezimovn a reinfekce na jae Prohloubit studium vskytu biotrofnch parazit na okrasnch rostlinch

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Documents

RNDr. Barbora Mieslerová, Ph.D. Katedra botaniky Přírodovědecká fakulta Univerzita Palackého