Patterns of virulence variation in the interaction between Lactuca spp. and lettuce powdery mildew (Golovinomyces cichoracearum)

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Patterns of virulence variation in the interaction between Lactuca spp. and lettuce powdery mildew (Golovinomyces cichoracearum) 
I. PETRZELOV 
P. KORBELOVA,
E. CESNEKOV
A. LEBEDA*, B. MIESLEROVA, A, A 
University in Olomouc, Faculty of Science, Department of Botany, Slechtitel  Palacky u 11, 783 71 Olomouc-Holice, Czech Republic

article info

abstract

Article history:

Golovinomyces cichoracearum sensu stricto (lettuce powdery mildew) is a common pathogen of

Received 14 December 2011

the family Asteraceae (Compositae), mainly of species of the tribe Lactuceae. However,

Revision received 6 March 2012

information on the natural distribution and virulence variation of G. cichoracearum on wild

Accepted 30 March 2012

Lactuca spp. is rather limited. In the Czech Republic, samples of G. cichoracearum were

Available online 14 May 2012

collected in wild populations of prickly lettuce (Lactuca serriola) during 2005e2008. These

Corresponding editor:

isolates of G. cichoracearum were screened on Lactuca species (L. sativa, L. serriola, L. sat-

Jeff Powell

iva
L. serriola, L. saligna and L. virosa) by using a leaf-disc assay. The main goals were to assess

Keywords:

virulence

variation

in

natural

populations

of

this

pathogen,

determine

host-pathogen reaction patterns and search for the race-specificity in interactions between

Lettuce powdery mildew

Lactuca species and isolates of G. cichoracearum. On the basis of results from the inoculation

Lactuca serriola

studies, a Lactuca spp. differential set was developed for characterization of virulence

Lactuca sativa

variation of this pathogen. The experimental work was divided into four sequential steps.

Leaf-disc assay

In 2005, mainly L. serriola accessions were used for the virulence study. In 2006, the set was

Prickly lettuce

enlarged with genotypes of L. sativa, and L. sativa
L. serriola hybrids. In 2007, genotypes of

Partial resistance

L. sativa and L. serriola previously showing clear differential responses to G. cichoracearum

Race-specific interactions

isolates were selected and the set was supplemented with some accessions of L. saligna and

Resistance screening

L. virosa. Finally, in 2008, the number of differentials was settled on 13 accessions of Lactuca spp. (six cultivars of L. sativa; one L. sativa
L. serriola hybrid; two accessions of L. serriola, L. saligna and L. virosa) that showed broad variation in host-pathogen reaction patterns. Overall, the inoculation studies confirmed the existence of many pathogen races, and demonstrated that the interaction between Lactuca spp. and G. cichoracearum is race-specific. In the future, more experimental studies on the pathogenic variability of G. cichoracearum s. str., as well as application of the genetic and molecular genetic approaches, is essential to understand this host-pathogen interaction in detail. These data may be important for applications in crop breeding and improvement. ª 2012 Elsevier Ltd and The British Mycological Society. All rights reserved.

Introduction The majority of records of powdery mildew occurrence on Lactuca spp. in Europe are Golovinomyces cichoracearum.

However, in Southern France occurrence of a different species of powdery mildew (probably a new species belonging to the genus Erysiphe) was recorded on Lactuca viminea subsp. chondrilliflora (Lebeda et al. 2002a). Furthermore, occurrence of

* Corresponding author. Tel.: þ420 585634800; fax: þ420 585634824. E-mail address: [email protected] (A. Lebeda). 1754-5048/$ e see front matter ª 2012 Elsevier Ltd and The British Mycological Society. All rights reserved. doi:10.1016/j.funeco.2012.03.005

Interaction between Lactuca spp. and lettuce powdery mildew

powdery mildew caused by Podosphaera fusca (syn. Sphaerotheca fusca) has also been reported on Lactuca spp. (Shin et al. 2006). G. cichoracearum is an obligate biotrophic parasitic fungus, causing lettuce powdery mildew disease in Lactuca spp., which has a world-wide distribution. The symptoms of this disease are most abundant from early summer to early autumn when leaves of the host plants can be completely covered with a white, flour-like growth of the fungus (Lebeda
2011). During the summer, G. cichoracearum more & Mieslerova commonly reproduces asexually (formation of conidiophores with conidia macroscopically visible as a white powder) while sexual reproduction (formation of chasmothecia with ascii) occurs mostly at the end of the growing season (Lebeda & Buczkowski 1986). Detailed information on the taxonomy, morphology, biology and ecology of lettuce powdery mildew
(G. cichoracearum) is given elsewhere (Lebeda & Mieslerova 2011). Among plant growers and breeders, the name Golovinomyces (formerly Erysiphe) cichoracearum is often used for powdery mildew occurring on a wide range of host plants, including members of the Asteraceae (Compositae), Cucurbitaceae, Nicotiana L. spp. and Arabidopsis, and others (e.g., Cunnington et al. 2010; Kenneth & Palti 1984; Lebeda
2011; Shin 2000). However, by some other & Mieslerova authors (e.g., Braun 1987, 1995; Matsuda & Takamatsu 2003; Nagy & Kiss 2006) the name has been recently assigned only to the races parasitizing representatives of the family Asteraceae (Compositae). A high number of host species of G. cichoracearum sensu stricto belong to the tribe Lactuceae (e.g., Matsuda & Takamatsu 2003). The genus Lactuca is taxonomically classified in the family Asteraceae (Compositae), subfamily Cichorioideae, tribe Lactuceae, and subtribe Lactucinae (Lebeda et al. 2004, 2007). The generic concept was previously well elaborated (at least for
kova
(1977), and more the European species of Lactuca) by Fera recently by Lebeda & Astley (1999) and Lebeda et al. (2007) including also the complex of Lactuca spp., originating from other continents (Lebeda et al. 2004). In their classification, the genus Lactuca is divided into seven sections and two (African and North American) geographical groups. Within the genus, L. serriola is the most frequent weedy species in Europe
kova
1977; Lebeda et al. 2001a) with significant expansion (Fera to new areas, especially within the last 50 years (Hooftman et al. 2006; D’Andrea et al. 2009). Its spread is closely connected to human activities as L. serriola is a ruderal, pioneer plant which readily occupies newly arisen habitats or disturbed soils, and disseminates easily through the production of huge numbers of achenes capable of long-distance
kova
1977; Hooftman et al. 2006; Novotna
et al. dispersal (Fera 2011). Most attention has focused on monitoring the occurrence of powdery mildew (G. cichoracearum s. str.) on cultivated lettuce. Reports are available from the USA, Israel, France, Greece, Switzerland, former USSR, UK, Germany and former
2011). In comparison, Czechoslovakia (Lebeda & Mieslerova there is only limited information on the geographic distribution of lettuce powdery mildew on wild Lactuca species (e.g., Blumer 1967; Dixon 1978; Crute & Burns 1983; Lebeda 1985a; Braun 1995; Paulech 1995; Lebeda et al. 2001a, 2008; Lebeda et al. 2011). In Europe, the natural hosts of G.

671

cichoracearum include several Lactuca spp. (L. perennis, L. quercina, L. saligna, L. sativa, L. serriola, L. sibirica, L. viminea and L. virosa) (Braun 1995; Lebeda 1985a, 1994; Lebeda et al. 2001a;
et al. 2001). However, it can be most frequently alova Dolez found on L. serriola (Lebeda 1985a, b, c, 1994; Lebeda et al.
et al. 2007b), often accompanying its 2001a, 2008; Mieslerova other common pathogen, which is Bremia lactucae an oomy
et al. 2007b). Both pathocete (Lebeda et al. 2008; Mieslerova gens can co-occur in one L. serriola population (even on leaves of the same plants) and either of them may dominate. Nevertheless, they can be easily distinguished by different
et al. 2007b; macroscopical symptoms on leaves (Mieslerova Lebeda et al. 2008). Until now, most of the studies dealing with Lactuca spp. resistance to powdery mildew have been based on limited screenings with one local isolate of G. cichoracearum. Such assessments have been primarily conducted on lettuce (L. sativa), for example, in the USA (Schnathorst & Bardin 1958; Matheron & Porchas 2003), Czech Republic (Lebeda 1985c), United Kingdom (Knight et al. 1986) and India (Husain & Akram 1996). Most of the lettuce cultivars studied were susceptible, while only a few were resistant to the pathogen isolates used. Only a limited number of previous studies have evaluated wild Lactuca species for resistance to lettuce powdery mildew (Lebeda 1985a, b, 1994; Lebeda & Buczkowski
et al. 2007a). Results of screening more than 1986; Mieslerova one hundred accessions of various Lactuca species (namely L. aculeata, L. dentata, L. perennis, L. saligna, L. serriola, L. tatarica, L. tenerrima, L. viminea and L. virosa), under natural infection, revealed high variability in resistance to G. cichoracearum (Lebeda 1985a, b, 1994; Lebeda & Buczkowski 1986). However, until now a differential set of common Lactuca host spp. has not been available for differentiation of G. cichoracearum
2011). Also, the virulence and races (Lebeda & Mieslerova patterns of virulence variation of this pathogen have not been studied in detail yet. The main aims of the present study were: (1) to assess virulence variation in natural populations of G. cichoracearum; (2) to determine host-pathogen reaction patterns (race-specificity) in interactions between Lactuca species and isolates of G. cichoracearum s. str. originating from L. serriola; and (3) on the basis of results from the inoculation studies, to develop a preliminary Lactuca spp. differential set (a set of host genotypes that react in a way that distinguishes among race-specific isolates of a pathogen) for characterization of virulence variation of this pathogen.

Materials and methods Collection of G. cichoracearum samples, isolation of pure cultures and their maintenance During 2005e2008 natural populations of L. serriola (prickly lettuce), occurring in the Czech Republic (mostly in Moravia and Eastern Bohemia), were surveyed (Fig 1) for occurrence of its two most common pathogens, B. lactucae (Lebeda et al. 2008)
et al. and G. cichoracearum s. str. (Lebeda et al. 2008; Mieslerova 2007b). In individual localities with powdery mildew disease present, one or two infected leaves were collected from

672

A. Lebeda et al.

Fig 1 e Areas and localities (dots) in the Czech Republic surveyed for the natural distribution of Golovinomyces cichoracearum sensu stricto in Lactuca serriola and L. sativa populations during years 2005e2008.

a randomly selected L. serriola plant. Leaf samples were transported to the laboratory in plastic boxes lined with moistened cellulose cotton-wool for subsequent isolation of pure cultures of the pathogen. Individual isolates were obtained by transfer of conidia (by dusting or tapping), from a single sporulating colony of powdery mildew present on a leaf sample, onto a healthy leaf of an 8e10 week old seedling of a susceptible L. serriola genotype (LSE/57/15). Inoculated plants separated by transparent PVC-foil covers were transferred into the growth chamber and there maintained at a day/night temperature of 20/18  C, under a 12 hr photoperiod and a light intensity of 100 mmol m2 s1. Under these conditions, white mycelium and conidiophores of G. cichoracearum developed on susceptible plants during 7e9 d of incubation. Isolates were maintained for ca 14e18 d on leaves of L. serriola plants and then re-inoculation on healthy leaves of new plants was necessary. The number of G. cichoracearum isolates obtained from L. serriola and studied in the years 2005e2008 is shown in

Table 1. In addition, one isolate (24/07) from 2007, originating from L. sativa (cv. RH04-1104-4, Salinas, CA, USA), was included in the experiments for comparison.

Plant material Seeds of Lactuca spp. accessions used for the study originated from the seed collection maintained at the Department of Botany (Laboratory of Plant Pathology), Faculty of Science,
University in Olomouc (Czech Republic). The compoPalacky sition of the Lactuca spp. differential set was adjusted several times during the project, mainly according to the experimental infection data obtained in preceding years. In 2005, the preliminary differential set comprised 15 Lactuca spp. accessions (i.e., one cultivar of L. sativa, two L. sativa
L. serriola hybrids and twelve accessions of L. serriola) (Table 2). In 2006, it included 22 Lactuca spp. accessions (sixteen cultivars of L. sativa (including the one from the 2005 set), one L. sativa
L. serriola hybrid and five accessions of L. serriola

Table 1 e Number of Golovinomyces cichoracearum isolates collected in the Czech Republic in the years 2005e2008 and used for development of the Lactuca spp. differential set. Year of collection 2005 2006 2007 2008

Number of GC isolates used for the screening

Total number of Lactuca spp. accessions tested

Total number of the studied host-pathogen interactionsb

7 20 23 (þ1a) 16

15 22 11 13

103 389 262 196

a isolate originating from L. sativa (from the USA). b data are not available for all interactions between Lactuca spp. accessions and GC isolates because we were not able to keep the respective isolates alive until the test had finished.

Interaction between Lactuca spp. and lettuce powdery mildew

673

Table 2 e Responses of Lactuca spp. accessions/cultivars to isolates of Golovinomyces cichoracearum collected in 2005. Lactuca spp. (accession/cultivar)

L. serriola (LSE/57/15) L. serriola (PI 273617) L. serriola (LSE/18) L. serriola (PIVT 1309) L. serriola (CGN 05153/PIVT 1544) L. serriola (CGN 491178) L. serriola (CGN 491229) L. serriola
L. sativa (CS-RL) L. serriola (CGN 14255) L. serriola (CGN 14256) L. serriola (CGN 14270) L. serriola (CGN 14280) L. serriola (LS-102) Hilde
L. serriola (H
B) L. sativa Cobham Green Total value of ‘% max ID’* G. cichoracearum isolates F value1.11 df 6 Prob. level0.3650

Isolate of G. cichoracearum/ Lactuca spp. reaction pattern 1/05

2/05

3/05

4/05

10/05

13/05

17/05

S S S S S S S S S S S S S S S 93.7

S S S S S S S S S S S S S 92.5

S MR S S S S S S S S S S S S S 89.4

S R S S S S S S S S S S S R MR 79.7

S S S S S S S S S S S S S MR S 90.3

S MR S S S S S S S S S S S R S 86.0

S R S S S S S S S S S S S S S 81.2

Total value of ‘% max ID’ * 100c** 48.5a 88.7bc 89.5c 98.0c 95.9c 89.1bc 89.5c 97.7c 90.3c 89.8bc 96.0c 97.4c 64.3ab 77.7bc Lactuca spp. 5.93 14 0.000000

R ¼ resistance (‘% max ID’  30); MR ¼ moderate resistance/susceptibility (30 < ‘% max ID’  60); S ¼ susceptibility (60 < ‘% max ID’  100); - ¼ data not available. * Bonferroni (With Control) Multiple-Comparison Test. **Different letters indicate significant differences (P  0.05) among Lactuca spp. accessions/GC isolates.

(both included in the 2005 set)) (Table 3). In 2007, 11 Lactuca spp. accessions (five cultivars of L. sativa (all included in the 2006 set), two accessions of L. serriola (included in the 2005 and 2006 set), two of L. saligna and two of L. virosa (neither previously tested)) were used (Table 4). Finally, in 2008, the number of differentials for further tests settled on 13 accessions (six cultivars of L. sativa, one L. sativa
L. serriola hybrid, two accessions of L. serriola, two of L. saligna and two of L. virosa) (Table 5), and this set was used for determination of G. cichoracearum pathogenic variability in subsequent years.

Preparation of plants for tests Lactuca spp. seeds were sown into plastic pots (7 cm in diam eter) filled with moistened agroperlite (Perlit Ltd, Senov u

Noveho Jicına, Czech Republic). Pots were placed in a glasshouse under a day/night temperature of 22/20  C and light conditions equivalent to ambient conditions with artificial lighting 12 hr, if needed. Individual seedlings with fully developed cotyledons were transplanted into 7-cm plastic pots containing a mixture of garden soil and peat (2:1, v:v) and then grown in a glasshouse at 20e22  C. At the age of 8e10 weeks plants were used for inoculation experiments. Leaf discs (12 mm in diameter) were cut out of true leaves of plants with a cork borer and those were then placed in Petri dishes (9 cm in diameter) with their abaxial side down on moistened cellulose cotton-wool covered with a sheet of filter paper. For each combination of a Lactuca spp. accession and a G. cichoracearum isolate the tests were repeated with 3 plants. In total, 8e10 leaf discs were tested (mostly 3 discs from one plant, i.e., in total 9 leaf discs). However, the total number of discs

taken from one plant depended on a leaf blade size. L. serriola
(LSE/57/15) served as a highly susceptible control (Mieslerova et al. 2007a).

Inoculation and incubation As a source of inoculum for each test, heavily infected leaves (from approximately 80 % covered with sporulating (ca 8e10 d old) mycelium of an individual isolate of G. cichoracearum) of the susceptible L. serriola (LSE/57/15) were used. The upper side of each leaf disc was inoculated with conidia from the sporulating mycelium by surface contact (by tapping). After inoculation, Petri dishes were placed into the growth chamber and incubated under the same conditions as used for previous multiplication of the powdery mildew isolates.

Assessment of sporulation intensity The infection degree (ID) of individual leaf discs was evaluated macroscopically 14 d after inoculation by using a 0e3 scale
et al. 2007a): 0 ¼ leaf disc without any symptoms of (Mieslerova powdery mildew development; 1 ¼ separate small individual colonies of powdery mildew visible on a leaf disc; 2  50 % of the leaf disc area covered with well-developed and sporulating mycelium; 3  50 and 100 % of the leaf disc area covered with well-developed and sporulating mycelium. However, for final evaluation of results, it was necessary to get one value for each Lactuca accession/pathogen isolate interaction. Therefore, for each pairwise inoculation of a Lactuca spp. accession with a G. cichoracearum isolate, the percentage of the maximum potential infection degree (‘% max ID’,

674

Table 3 e Responses of Lactuca spp. accessions/cultivars to isolates of Golovinomyces cichoracearum collected in 2006. Lactuca spp. (accession/cultivar) L. sativa Argeles L. sativa British Hilde L. sativa Capitan L. sativa Cobham Green L. sativa Colorado L. sativa Dandie L. sativa Discovery
L. sativa Lednicky L. sativa Line 4/57/D L. sativa Ninja L. sativa Pennlake L. sativa Sabine L. sativa UCDM10 L. sativa UCDM14 L. sativa UCDM2 L. sativa Valmaine L. serriola (LS-102) L. serriola (LSE/18) L. serriola (PI 273617) L. serriola (PIVT 1309) Hilde
L. serriola (H
B) L. serriola (LSE/57/15) Total value of ‘% max ID’* G. cichoracearum isolates F value1.72 df 19 Prob. level 0.03205

Isolate of G. cichoracearum/Lactuca spp. reaction pattern 3/06 4/06 5/06 6/06 7/06 8/06 9/06 10/06 14/06 15/06 17/06 18/06 19/06 20/06 21/06 22/06 23/06 24/06 31/06 32/06 S MR MR R S S S S S MR S R S S S S S 69.0

S S S S S S S S S S S S S MR R MR S R S S S 74.0

S S MR S MR S S MR S S S MR MR S S S S S 78.9

MR S R S R S S MR S S R S S S MR S S S 69.1

S S S S MR S S MR S S S S S S S S S 78.4

S S S S MR S S S S S S S S S S S S S S S S 89.5

S S S S MR S S S S S R S S R MR S R S S S 73.9

S S S S R S S S S S S S S S S S S S R S S S 83.5

S S S S MR S S S S S S S S S S S S S S S S 80.6

S S S S MR S S MR MR S S S S S S S S S R S S S 78.9

R S S S S S S S S S S S S S S MR S S S S S S 86.4

S R MR R S S MR S S S S S S MR S 69.5

S S S S R S S S S MR S S S S S R S S S 80.1

S S S S R S S MR MR R S MR S S S MR S S R MR S S 67.9

S S S S R S S MR S S MR MR S MR S S R S MR S 66.1

S S S S MR S S MR MR S S MR S S S S S R S MR S 72.3

S MR S S S S S S S S S S S MR R S S S 78.2

S S S S R S MR S S S S S S S S R S S S 77.0

S S S MR S S S S S S S S S S S S 82.1

MR S S S S S S S S S S S S S S S S S S 85.4

Total value of ‘% max ID’* 85.3cdef** 83.1bcdef 80.5bcde 86.7cdef 33.8a 94.3ef 83.9bcdef 68.9bc 83.4bcdef 86.6cdef 86.4cdef 64.9b 83.1bcdef 85.3bcdef 73.9bcd 72.2bcd 95.9def 91.7def 36.2a 90.0def 80.2bcde 100f Lactuca spp. 12.28 21 0.000000

R ¼ resistance (‘% max ID’  30); MR ¼ moderate resistance/susceptibility (30 < ‘% max ID’  60); S ¼ susceptibility (60 < ‘% max ID’  100); - ¼ data not available. *Bonferroni (With Control) Multiple-Comparison Test. **Different letters indicate significant differences (P  0.05) among Lactuca spp. accessions/GC isolates.

A. Lebeda et al.

L. serriola (LSE/57/15) L. serriola (PI 273617) L. sativa Capitan L. sativa Colorado L. sativa Ninja L. sativa Sabine L. sativa UCDM2 L. saligna (09-H58-1013) L. saligna (09-H58-1010) L. virosa (LVIR/50) L. virosa (09-H58-998) Total value of ‘% max ID’* G. cichoracearum isolates F value1.89 df22 Prob. level 0.010

Isolate of G. cichoracearum/Lactuca spp. reaction pattern

1/07

Total value of ‘% 2/07 3/07 4/07 5/07 6/07 7/07 8/07 9/07 10/07 11/07 12/07 13/07 14/07 15/07 16/07 17/07 18/07 19/07 20/07 21/07 22/07 23/07 24/07 max ID’*

S

S

S

S

S

S

S

S

S

S

S

S

S

S

S

S

S

S

S

S

S

S

S

S

100c**

MR

S

R

S

S

S

S

S

S

R

MR

MR

MR

S

MR

S

R

S

R

MR

R

R

S

S

53.4a

S

S

S

MR

S

MR

S

S

S

S

S

S

S

S

S

S

S

S

S

S

S

S

S

S

86.2bc

R

S

R

MR

S

S

R

S

S

MR

R

R

S

S

R

S

MR

R

R

MR

R

R

S

S

45.9a

S

S

S

S

S

S

S

S

S

S

S

S

S

S

S

S

S

S

S

S

S

S

S

S

98.5c

MR

S

S

S

S

S

S

S

S

S

S

S

S

S

S

S

MR

MR

MR

S

R

MR

S

S

76.9b

S

S

S

S

S

S

S

S

S

S

S

S

S

S

S

S

MR

S

S

S

S

S

S

S

89.3bc

R

S

R

MR

S

S

R

MR

MR

R

R

-

-

R

R

S

R

R

R

S

R

MR

S

S

36.2a

S

S

S

S

MR

S

S

S

MR

S

S

S

MR

S

MR

S

S

S

S

R

S

S

S

S

76.5b

R

MR

R

MR

MR

MR

MR

MR

S

MR

R

R

S

S

MR

MR

MR

R

MR

S

R

MR

MR

S

42.6a

S

S

S

S

S

S

S

S

MR

R

MR

S

S

MR

S

S

S

S

S

S

S

MR

S

S

85.8bc

Interaction between Lactuca spp. and lettuce powdery mildew

Table 4 e Responses of Lactuca spp. accessions/cultivars to isolates of Golovinomyces cichoracearum collected in 2007. Lactuca spp. (accession/ cultivar)

61.7ab** 87.4ab 60.9ab 73.9ab 79.0ab 85.5ab 72.9ab 83.2ab 78.1ab 61.2ab 62.7ab 76.1ab 81.1ab 74.3ab 62.6ab 89.3ab 58.0ab 65.2ab 67.1ab 70.7ab 52.8a 57.9ab 89.8ab 94.6b Lactuca spp. 29.12 10 0.000

R ¼ resistance (‘% max ID’  30); MR ¼ moderate resistance/susceptibility (30 < ‘% max ID’  60); S ¼ susceptibility (60 < ‘% max ID’  100); - ¼ data not available. * Bonferroni (With Control) Multiple-Comparison Test. **Different letters indicate significant differences (P  0.05) among Lactuca spp. accessions/GC isolates.

675

676

Table 5 e Responses of Lactuca spp. accessions/cultivars to isolates of Golovinomyces cichoracearum collected in 2008. Lactuca spp. (accession/cultivar)

L. serriola (LSE/57/15) L. serriola (PI 273617) Hilde
L. serriola (H
B) L. sativa Capitan L. sativa Colorado L. sativa Argeles L. sativa Sabine L. sativa UCDM2 L. sativa Cobham Green L. saligna (09-H58-1013) L. saligna (09-H58-1010) L. virosa (LVIR/50) L. virosa (09-H58-998) Total value of ‘% max ID’* G. cichoracearum isolates F value1.63 df 15 Prob. level 0.070

Isolate of G. cichoracearum/Lactuca spp. reaction pattern 1/08

2/08

3/08

4/08

5/08

6/08

7/08

8/08

9/08

10/08

11/08

12/08

13/08

14/08

15/08

16/08

S MR S S S S S S MR MR MR 72.6ab**

S MR S S S S S S S R S R S 79.1ab

S R S S MR S S S R S R MR 65.5ab

S R S S R S S S S R S R MR 68.5ab

S R S S MR S S S S R MR MR 70.8ab

S R S MR S S MR MR MR S 65.5ab

S MR MR S S S S S S MR S R S 74.4ab

S MR S S S S S S S S S R S 88.8ab

S MR MR MR MR S S S S R S R MR 64.5ab

S R R MR S S R R MR 53.3a

S MR S S R S S S R S S MR 74.9ab

S S S S S S S S S S S MR MR 86.6ab

S MR S S S S S S S MR S S S 79.9ab

S S S S S S S S S S S S S 95.8b

S MR S S R S S S S S S MR MR 74.7ab

S MR S S MR S S S S R S MR S 71.0ab

Total value of ‘% max ID’* 100c** 41.7a 86.1c 92.4c 56.1ab 95.2c 85.6c 92.5c 95.1c 44.8ab 89.8c 42.8ab 61.5b Lactuca spp. 22.21 12 0.000

R ¼ resistance (‘% max ID  30); MR ¼ moderate resistance/susceptibility (30 < ‘% max ID’  60); S ¼ susceptibility (60 < ‘% max ID’  100); - ¼ data not available. * Bonferroni (With Control) Multiple-Comparison Test. ** Different letters indicate significant differences (P  0.05) among Lactuca spp. accessions/GC isolates.

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Interaction between Lactuca spp. and lettuce powdery mildew

i.e., percentage of the overall evaluated leaf discs area (sum of 8e10 leaf discs) of an accession infected) was calculated from data recorded 14 d after inoculation, according to the formula of Townsend & Heuberger (1943): P ðn$vÞ$100 % max ID ¼ x$N where: % max ID ¼ percentage of maximum infection degree; n ¼ number of plants in each assessed category (infection degree); v ¼ infection degree; x ¼ highest degree of infection scale; N ¼ total number of assessed plants. Based on the values of ‘% max ID’ interactions were categorized qualitatively: R ¼ resistant (‘% max ID’  30), MR ¼ moderately resistant/susceptible (30 < ‘% max ID’  60) and S ¼ susceptible (60 < ‘% max ID’  100).

Statistical analysis NCSS 97 Programme (NCSS 97, Kaysville, Utah) was used for statistical analyses. The mean ‘% max ID’ calculated for each pairwise inoculation, using individual Lactuca spp. accessions and G. cichoracearum isolates, were compared using one-way Analysis of Variance (ANOVA). Differences between interactions were tested with the Bonferroni (With Control) MultipleComparison Test.

Results Virulence of G. cichoracearum sensu stricto (hereafter referred to as ‘GC’) isolates (obtained from natural populations of Lactuca serriola between years 2005 and 2008) to Lactuca species was tested in a leaf-disc assay, to assess virulence variation among isolates as well as the resistance of different Lactuca species to this pathogen. During the screening experiments there was no substantial interplant variation inside individual Lactuca spp. accessions used (Tables 2e5). However, to determine the pathogen virulence variation, it was first necessary to develop a differential set consisting of suitable Lactuca spp. accessions showing different resistance reaction patterns. The primary criterion for selection of suitable Lactuca species and accessions was detection of race-specific variation in host reaction to the collection of GC isolates. Because of the absence of knowledge in this area, the differential set was refined sequentially over four consecutive years. In year 2005, a preliminary Lactuca spp. differential set comprising mainly L. serriola (12 accessions) (Table 2) was screened for variation in responses to 7 isolates of GC originating from naturally infected L. serriola. The results revealed high susceptibility of most accessions of L. serriola and the L. serriola
L. sativa (CS-RL) hybrid (total ‘% max ID’ to all isolates used ¼ 88.7e100) (Table 2). Only one accession of L. serriola (PI 273617) and the L. sativa Hilde
L. serriola hybrid showed some resistant responses to GC isolates. In view of these results it was evident that the used accessions of L. serriola could not be considered as important sources of race-specific resistance against lettuce powdery mildew originated from naturally infected plants of this species and we therefore assessed patterns of resistance variation in other Lactuca species.

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In 2006, 16 cultivars of L. sativa, whose assortment was inspired by the differential set used for virulence analysis of B. lactucae (lettuce downy mildew) isolates (Lebeda &
2010), along with five further accessions of L. serelova Petrz riola and the L. sativa
L. serriola (H
B) hybrid were screened with 20 isolates of GC collected that year (Table 3). A differentiated reaction with some resistances was recorded only for one L. serriola (PI 273617) with total ‘% max ID’ ¼ 36.2 and a few L. sativa cultivars, namely Colorado (‘% max ID’ ¼ 33.8), Sabine (64.9), UCDM2 (73.9), Capitan (80.5), British Hilde (83.1) and Argeles (85.3) (Table 3). In 2007, tests were conducted with the cultivars / accessions of L. sativa and L. serriola showing the most differentiated responses in the two previous years and some accessions of L. saligna and L. virosa (Table 4). The set was tested for resistance to a total of 11 isolates of GC (Fig 1, Table 1). Both newly included Lactuca species showed highly differentiated reaction patterns to GC. The highest proportion of resistant responses was recorded for the following accessions: L. saligna (09-H581013), total ‘% max ID’ ¼ 36.2; L. virosa (LVIR/50), 42.6; L. sativa (cv. Colorado), 45.9; and L. serriola (PI 273617), 53.4 (Table 4). In 2008, the number of differentials was settled on 13 accessions of Lactuca spp., including L. serriola, L. sativa, L. saligna, L. virosa and a L. sativa
L. serriola hybrid (Table 5). This set was used for GC virulence studies in all subsequent years (Lebeda et al., unpublished results). The set was screened in 2008 for resistance to 16 GC isolates and, like the previous year, the highest proportion of resistant responses to the used GC isolates was recorded for L. saligna (09-H58-1013) and L. virosa (LVIR/50), and additionally also for L. serriola (PI 273617) (Table 5). The results of screening confirmed the relevance of selecting those Lactuca species and accessions as suitable ones for the differentiation of GC races. To summarize, the results of tests showed clear variation in virulence among GC isolates. Complex virulence to all Lactuca spp. accessions was the most frequent outcome of the studied interactions (Tables 2e5). Further, resistance of the tested Lactuca species (L. sativa, L. serriola, L. saligna and L. virosa) to GC is race-specific. Frequency of individual response categories recorded for particular Lactuca species and accessions after inoculation by GC isolates are shown in Figs 2and 3, respectively. The highest frequency of resistant and moderately resistant responses was recorded for interactions of GC with L. saligna and L. virosa, while accessions of L. serriola were found to be the most susceptible (Fig 2). None of the Lactuca spp. accessions expressed resistance to all GC isolates used.

Discussion For decades crop pathosystems have commanded most of the attention of plant breeders and pathologists, while interactions between populations or communities of naturally growing plants and their pathogens remained nearly unexplored. The most important wild plant pathosystems so far studied were reviewed recently (e.g., Laine et al. 2011). It is well known that while crop pathosystems (due to the high genetic uniformity of the hosts) are mostly characterized by an epidemic occurrence of pathogens which may lead to

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Fig 2 e Frequency (%) of response categories by Lactuca species after inoculation by Golovinomyces cichoracearum isolates originating from L. serriola and collected in the period 2005e2008.

a complete crop damage, relationships between host and pathogen in natural plant pathosystems are highly diverse, allowing sustainability of interacting taxa in natural ecosystems (Laine 2004). Furthermore, we must realize that weed plant species are important alternative hosts for pathogens also attacking crop plants. Hence, detailed studies of wild plant pathosystems have practical importance as they could potentially reveal a lot of information applicable in sustainable agriculture (Burdon & Thrall 2008), involving resistance
& breeding and practical plant disease management (Lenne Wood 1991). Until now, only a few wild plant-powdery mildew pathosystems have been studied in detail. Some papers concerning variation in host resistance and virulence of the interacting pathogen species have been published (e.g. for the pathosystems Senecio vulgaris-G. cichoracearum var. fischeri (formerly Erysiphe fischeri): Clarke & Akhkha 2002; Bevan et al. 1993a, b, c; Harry & Clarke 1986, 1987), and family Poaceae-Blumeria graminis (formerly Erysiphe graminis) (e.g., Gustafsson & Claesson 1988; Wahl et al. 1978). Extensive metapopulation studies, focused mainly on spatial variation in populations of both

A. Lebeda et al.

interacting species, have been conducted in the pathosystem Plantago lanceolata-Podosphaera plantaginis (e.g., Laine 2004, 2005; Soubeyrand et al. 2009). Our current paper reports on results of the first extensive study evaluating variation in the wild plant pathosystem Lactuca spp.-G. cichoracearum sensu stricto (abbrev. GC) and further contributes to recent knowledge about this
2011). host-pathogen system (Lebeda & Mieslerova Our results provide clear evidence of race-specific interactions between Lactuca species and GC originating from populations of L. serriola (prickly lettuce) growing as weeds in the Czech Republic. The same preliminary conclusion was made in our previous papers (Lebeda 1985a, b, 1994). However, for the species L. serriola, there was little variation in responses to various GC isolates found among the studied accessions. Most responses were categorized as susceptible, which shows very close adaptation of GC isolates to their original host, L. serriola. Similar results (i.e., prevalence of susceptibility) were previously reported for responses of L. serriola accessions to isolates of another common pathogen of that species, i.e., B. lactucae (lettuce downy mildew) (Lebeda et al. 2008). The only exception among accessions of L. serriola tested to GC, which showed a highly differentiated pattern of responses to various isolates tested during 2005e2008, was PI 273617 (Tables 2e5). However, no data are available on resistance variation in the particular L. serriola populations from which the tested GC isolates originate. Moreover, we have no possibility to compare our results with the results concerning GC originating from L. serriola from any other country, because of unavailability of suitable pathogen isolates. Therefore, it is difficult to draw general conclusions about possible specialization of the local GC isolates (races) to local populations of L. serriola. On the contrary, L. sativa as well as accessions of L. saligna and L. virosa gave more variation in race-specific patterns after inoculation with GC isolates, which was especially true for some cultivars and/or accessions (Tables 3e5). However, also for these species, susceptibility or moderate susceptibility were the most frequently recorded responses to GC isolates from naturally growing L. serriola. In contrast, L. sativa accessions mostly expressed resistance to the lettuce downy mildew isolates from L. serriola plants (Lebeda et al.

Fig 3 e Frequency (%) of response categories by Lactuca spp. accessions after inoculation by Golovinomyces cichoracearum isolates originating from L. serriola and collected in the period 2005e2008. Legend: L. ser. [ Lactuca serriola, L. sat. [ Lactuca sativa, L. vir. [ Lactuca virosa, L. sal. [ Lactuca saligna.

Interaction between Lactuca spp. and lettuce powdery mildew

2008). To summarize, the highest numbers of resistant responses and the broadest variation of race-specificity to the range of GC isolates used in inoculation trials were recorded for four Lactuca spp. accessions: (L. virosa (LVIR/50), L. saligna (09-H58-1013), L. sativa (cv. Colorado) and L. serriola (PI 273617)). These can therefore be considered as the most valuable sources of resistance to G. cichoracearum s. str., however, they did not display resistance to all GC isolates used. These conclusions accord with results from earlier field/glasshouse observations (Lebeda 1985a, b, c, 1994; Lebeda & Buczkowski 1986). The current study included one GC isolate (24/07) originating from L. sativa. This isolate showed the strongest pathogenicity to the set of Lactuca spp. accessions tested in 2007 (‘% max ID’ ¼ 94.6) (Table 4). The low level of variation in its virulence could be explained partially as a result of the species composition of the differential set used in that year (5 of 11 accessions tested were L. sativa) and a close specialization to the original host species L. sativa. However, it was highly virulent also on accessions of other Lactuca species (Table 4). This confirms previously published results regarding differences between GC isolates originating from L. sativa and/or L. serriola plants (Schnathorst et al. 1958), and showing existence of different races of GC. The main problem in breeding for crop resistance to diseases is the identification and introduction of resistance genes that can provide efficient and durable resistance (e.g., Pink 2002). Each plant species has a combination of different resistance mechanisms that provide protection against pathogens (e.g., Gustafsson & Claesson 1988; Lebeda et al. 2002b; Lindhout 2002; Mauch-Mani 2002; Niks & Rubiales 2002). Race-specificity is a general phenomenon of plantepathogen interactions (Crute & Pink 1996). However, since its discovery by Flor (1955), resistance based on a genefor-gene relationship between hosts and pathogens (also called ‘race-specific’, ‘single-gene’ or ‘monogenic’ resistance) has become the most frequent type of resistance used in cultivated species. Resistance of a plant cultivar under genefor-gene control lies in the utilization of one or more single dominant R-genes (Crute & Pink 1996; Pink 2002). Thus, resistance is dominant to susceptibility. However, at the same time the pathogen must have corresponding dominant avirulence gene(s) so that the result of a host-pathogen interaction could be the resistance of the host. The main disadvantage of monogenic resistance is the fact that most plant pathogens are capable of rapidly evolving new virulent strains. The emergence of such strains which are able to overcome genes in previously resistant plant cultivars is a direct response to R gene introduction. Such situations have been well described e.g., from the interaction between lettuce and its oomycete pathogen B. lactucae causing lettuce downy mildew (e.g., Lebeda & Zinkernagel 2003; van der Arend et al., 2006). Therefore, permanent control of a disease requires continual supply of new resistance genes (Michelmore et al. 2009). Successful breeding of a given plant species for major gene resistance is thus conditional on characterization of genetic diversity for host resistance. Pathogen isolates originating from wild species can help significantly to reveal new sources of resistance, both in cultivated and wild plants
& Lebeda 2011). elova (Lebeda et al. 2008; Petrz

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For example, with regard to powdery mildews the genefor-gene relationship is well explored e.g., in interactions between Senecio vulgaris and G. cichoracearum var. fischeri (previously Erysiphe fischeri) (Bevan et al. 1993a, b, c; Harry & Clarke 1987). In Lactuca species, numerous mechanisms of resistance against B. lactucae (e.g., Lebeda et al. 2001b, 2002b) have been described. In contrast, there is little information available about resistance mechanisms operating in Lactuca species, or specific genes conferring resistance to the lettuce powdery mildew pathogen. Variability of interactions recorded between Lactuca spp. and isolates of G. cichoracearum clearly indicates the existence of race-specific resistance in this pathosystem. Resistance controlled by a gene-for-gene interaction is often associated with a hypersensitive response, in which infected cells and some neighboring ones die due to the numerous biochemical reactions (Mansfield et al. 1997). During our experiments we often observed dark spots on leaves (in sites of pathogen penetration) similar to the leaf spots commonly occurring as a result of the hypersensitive response. However, in our study spot formation was often followed by further pathogen development including sporulation. Nevertheless, in our preliminary microscopic study on the resistance mechanisms operating in this 
2008) we postulated that hypersenpathosystem (Cesnekov a sitive responses do not need to be completely effective to prevent powdery mildew pathogen development. Our results indicate that not only isolate-specific complete resistance (expressed by a total absence of pathogen development depending on its race) but probably also isolate-specific partial resistance (expressed by reduced development of mycelium and limited sporulation of some pathogen races) play roles in protecting Lactuca spp. plants against G. cichoracearum. Partially expressed resistance in relation to the powdery mildew pathogen has been previously described also in Senecio vulgaris, and it is supposed that this resistance is controlled by incompletely dominant (or recessive) resistance genes (Bevan et al. 1993b, c; Harry & Clarke 1987), and in Linum spp. e Melampsora spp. host-pathogen system (Antonovics et al. 2011). Limited availability of durable sources of resistance to diseases in cultivated plants has stimulated interest among breeders in the potential to find new sources in wild crop relatives (e.g., Lebeda et al. 2009; Jeuken & Lindhout 2002, 2004; Michelmore et al. 2009). Thus, considerable progress has been made with regard to identification of sources of resistance within wild Lactuca species, and also in lettuce breeding for resistance to diseases and pests (summarized in Lebeda et al. 2002b, 2007, 2009). However, practical exploitation of resistance in the wild hosts in lettuce breeding depends largely on crossability of those species with L. sativa. Regarding this issue, Lactuca species are categorized into three gene pools (Lebeda et al. 2007, 2009). Generally, the primary gene pool is represented by numerous cultivars and landraces of L. sativa together with their wild ancestor, L. serriola (Lebeda et al. 2009). The wild ‘serriola-like’ species from South-Western Asia (i.e., L. aculeata, L. altaica, L. azerbaijanica, L. georgica and L. scarioloides) and the South African species L. dregeana (none of them tested in our experiments) all display similar levels of cross-compatibility and inter-fertility with cultivated lettuce, and also belong to the primary gene pool. On the other hand, species such as L. saligna and L. virosa are more difficult to

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cross with L. sativa and are included in the secondary and tertiary gene pools, respectively. According to recent studies (e.g., Jeuken & Lindhout 2002, 2004; Lebeda et al. 2007, 2009), from the above mentioned species only L. saligna and L. virosa are considered as the most important potential sources of efficient and durable resistance. These species displayed quite variable responses to isolates of G. cichoracearum (Figs 2and 3) which makes them potential candidates for exploitation in lettuce breeding programmes. In comparison, L. serriola, which has been commonly used as a source of race-specific resistance to the lettuce downy mildew (e.g., Lebeda et al. 2002b, 2007), showed high susceptibility and only limited variability in responses to the powdery mildew isolates originating from L. serriola. Similar results were obtained for cultivated L. sativa. Therefore, we do not recommend using L. serriola in breeding for resistance to powdery mildew. In the future, more research on the pathogenic variability of G. cichoracearum s. str. is needed. Ideally, this would include a larger series of isolates originating from L. sativa and related species (and originating also from other countries), and their testing on the stable Lactuca spp. differential set as proposed in this study. Moreover, genetic (e.g., detection of QTLs) and molecular genetic (polymorphism analysis by using e.g., AFLP) studies would be essential to understand more detail of this host-pathogen interaction. All these data are important, not only for better understanding of the evolutionary ecology of the Lactuca spp.-G. cichoracearum interaction, but also have practical application in the development of disease resistant lettuce cultivars.

Acknowledgements Critical reading and valuable remarks by Dr. P.H. Thrall (CSIRO Plant Industry, Canberra, Australia) are gratefully acknowledged. This research was supported by grant: MSM 6198959215. Some isolates used in this study are maintained under National Programme of Genepool Conservation of Microorganisms and Small Animals of Economic Importance, funded by Ministry of Agriculture of the Czech Republic.

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