Characterization of New Highly Virulent German Isolates of Bremia lactucae and Efficiency of Resistance in Wild Lactuca spp. Germplasm

Four German isolates (FS1, SR2, SAW1 and DEG2) of Bremia lactucae originating from lettuce cultivars with R‐factors R18 and Dm6 + R36 were used for detailed characterization of virulence factors (v‐factors) and for the study of the resistance efficiency in wild Lactuca spp. germplasm. The highest complexity of v‐phenotype was recognized in isolate DEG2, which overcomes resistance in cv. Mariska (R18) and line CS‐RL (L. serriola × L. sativa, R18 + ?), until now known as resistant to all known races of B. lactucae in Europe. However, some sparse sporulation also occurred on cv. Titan (Dm6 + R36). The isolates SR2 and SAW1 overcome the resistance based on the gene R36, but are avirulent to R18. The v‐phenotype of SR2 is highly complex with the most important v‐factors being present except for v14 and v18. The isolate FS1 is the first in Germany originating from a cultivar with R18 (cv. Samourai). The search for efficient sources of resistance in 64 accessions of 11 wild Lactuca spp. and primitive forms of L. sativa showed broad variation in accession–isolate interactions. Expression of race‐specific resistance in wild Lactuca spp. (L. serriola, L. viminea, L. virosa) was recorded frequently. Lactuca indica and L. saligna could be considered as the most efficient sources of resistance against isolates FS1, SR2 and SAW1. The isolate DEG2 showed the highest level of virulence. On seedlings of L. saligna, which is considered as a most important source of resistance against B. lactucae, there was frequently recorded limited sporulation, however this response cannot be considered as a susceptible. Except for some L. saligna accessions (CGN 05310 and CGN 05315), the resistance to all studied isolates was only observed in one accession of L. serriola (PI 253467).     

Resistance to Bremia lactucae in natural populations of Lactuca saligna from some Middle Eastern countries and France

The results of the first detailed screening of a resistance to Bremia lactucae in naturally growing populations of Lactuca saligna are presented here. In total, 146 accessions from 25 populations of L. saligna originating in Israel (N = 136), France (N = 8), Jordan (N = 1) and Turkey (N = 1) were tested at seedling stage for their resistance to 10 highly virulent isolates (races) of B. lactucae from Lactuca sativa (DEG2, Bl:5, Bl:15, Bl:16, Bl:17, Bl:18, Bl:21, Bl:22, Bl:24 and Bl:25). Our study strongly supports the suggestion that L. saligna is indeed generally highly resistant to B. lactucae. However, our results provide evidence that at least at a seedling stage L. saligna may not be a non‐host plant for B. lactucae, as was hypothesised for approximately the last 30 years. Some accessions expressed a differential (i.e. race‐specific) response, which accords with other recently published data for this Lactuca species. Furthermore, some geographical differences in race‐specific resistance were observed, too. Tests performed at an adult‐plant stage, however, did not prove race‐specificity of the respective accessions. To summarise, what is behind the race‐specific character of the responses observed at a seedling stage is still uncertain, as is its comparability with the race‐specific resistance of some other Lactuca species such as L. sativa or L. serriola. The presence of plant stage‐dependent resistance, governed by a combined effect of different quantitative trait loci in young and adult plants of L. saligna, is discussed.     

Host specificity and virulence variation in populations of lettuce powdery mildew pathogen (Golovinomyces cichoracearum s. str.) from prickly lettuce (Lactuca serriola)

Fifty-four isolates of Golovinomyces cichoracearum (GC) sensu stricto were collected during the period 2008–2010 from natural populations of prickly lettuce (Lactuca serriola) growing in the Czech Republic. Population variation in virulence was assessed with a previously developed race differential set, comprising 13 accessions of four Lactuca species (L. serriola, L. sativa, L. saligna, L. virosa, and L. sativa ‘Hilde’ × L. serriola hybrid). GC isolates differed in their host specificity and virulence; most were able to infect most Lactuca spp. accessions (i.e., complete compatibility), but some showed only moderate virulence (i.e., incomplete compatibility). ‘Avirulent’ reactions were the least common, and these were most frequently observed on L. virosa (LVIR/50) and L. saligna (09-H58-1013). Furthermore, a temporal shift in virulence variability was recorded for the Czech GC populations. Generally, virulence of GC to the Lactuca spp. accessions increased during the study period. Across individual years, the most variable were reactions of GC to L. serriola accession (PI 273617), L. sativa cv. ‘Colorado’, L. saligna (09-H58-1013) and L. virosa (LVIR/50). A broad spectrum of virulence (v)-phenotypes was determined among the 54 isolates; however, differences in the reaction patterns of individual isolates were mostly small and related to differentiation between moderately and completely compatible interactions. However, most of the v-phenotypes characterized were unique to a given population in one year. Altogether, 16 v-phenotypes were identified among 16 isolates tested in 2008, 10 v-phenotypes were determined among 10 isolates tested in 2009, and 24 v-phenotypes among 28 isolates tested in 2010; i.e., different v-phenotypes were identified in different localities.     

Occurrence of Erysiphe cichoracearum Perithecia on Wild Lactuca Species

Perithecia of Erysiphe cichoracearum (powdery mildew) were found on several, but not all, accessions of Lactuca serriola, L. saligna, L. aculeata and L. serriola × L. sativa hybrid. Their occurrence varied between Lactuca species and different accessions of the same species. Perithecia were globular, had a mean diameter of 125.5 μm and contained an average 13–14 asci. Asci had mean dimensions of 55.0 × 26.1 μm and each contained two ascospores which measured an average 17.4 × 10.6 μm. There were however significant differences in the dimensions of spores and other structures obtained from different Lactuca accessions.     

Molecular diversity at the major cluster of disease resistance genes in cultivated and wild Lactuca spp.

Diversity was analyzed in wild and cultivated Lactuca germplasm using molecular markers derived from resistance genes of the NBS-LRR type. Three molecular markers, one microsatellite marker and two SCAR markers that amplified LRR-encoding regions, were developed from sequences of resistance gene homologs at the main resistance gene cluster in lettuce. Variation for these markers were assessed in germplasm including accessions of cultivated lettuce, Lactuca sativa L. and three wild Lactuca spp., L. serriola L., L. saligna and L. virosa L. Diversity was also studied within and between natural populations of L. serriola from Israel and California; the former is close to the center of diversity for Lactuca spp. while the latter is an area of more recent colonization. Large numbers of haplotypes were detected indicating the presence of numerous resistance genes in wild species. The diversity in haplotypes provided evidence for gene duplication and unequal crossing-over during the evolution of this cluster of resistance genes. However, there was no evidence for duplications and deletions within the LRR-encoding regions studied. The three markers were highly correlated with resistance phenotypes in L. sativa. They were able to discriminate between accessions that had previously been shown to be resistant to all known isolates of Bremia lactucae. Therefore, these markers will be highly informative for the establishment of core collections and marker-aided selection. A hierarchical analysis of the population structure of L. serriola showed that countries, as well as locations, were significantly differentiated. These differences may reflect local founder effects and/or divergent selection. Received: 7 March 1999 / Accepted: 25 March 1999     

Crossing experiments of lettuce cultivars and species (Lactuca sect.Lactuca,Compositae)

The degree of relationships withinLactuca sativa and three wild relativesL. serriola, L. saligna, andL. virosa was studied by observing the performance, vigour and fertility of the F 1 hybrids obtained from crosses made in and between the four species. The crosses ofL. saligna ×L. virosa and the reciprocal crosses produced no hybrids.L. saligna andL. virosa are the least related of the four species.L. sativa ×L. serriola and the reciprocal crosses were successful and produced fertile hybrids These two species are genetically very closely related.L. saligna is known to produce, as a female parent, hybrids withL. sativa andL. serriola. Now the reciprocal cross was successful for the first time, so the unability to obtain hybrids in the past was based on the choice of accessions and not caused by unilateral incompatibility.L. virosa ×L. sativa and the reciprocal combination produced hybrids. The combinationL. serriola ×L. virosa produced hybrids with very limited fertility. In contrast to earlier reports (sterile hybrids) one combination of the reciprocal cross too produced hybrids with very limited fertility.—Some of theL. saligna ×L. sativa (and reciprocal) hybrids were found to look strikingly likeL. serriola. This adds evidence for the descent ofL. serriola andL. sativa:L. saligna also made part of the ancestral complex of the cultivated lettuce.     

Chromosome banding patterns in Lettuce species (Lactuca sect.Lactuca,Compositae)

Chromosome banding patterns obtained with C- and N- banding, and AgNO₃ staining were studied in somatic metaphase complements of fourLactuca species.L. sativa andL. serriola have almost identical chromosome morphology, andL. saligna differs only slightly from them, butL. virosa is quite distinct from the other species. A gross comparison of the banded karyotypes suggests a closer relationship ofL. saligna toL. sativa/serriola than toL. virosa. Our data agree with the results of previous crossing experiments in these species but conflict partly with recent RFLP data which indicate a closer phenetic relationship ofL. saligna toL. virosa than toL. sativa/serriola. Such a discrepancy may be explained assuming that domestication ofL. sativa/serriola resulted in an increased selection pressure on unique DNA sequences as demonstrated by the RFLP data. Differential evolution of specific heterochromatin classes (and presumably of highly repetitive DNA classes), as revealed by chromosome banding techniques was not linked to domestication. Thus the disparity in conclusions about relationship (in terms of genetic similarity) as based on the different experimental approaches reflects a non-parallel evolution of highly repetitive vs. unique DNA classes.     

Numerical morphological analysis of Lettuce cultivars and species (Lactuca sect.Lactuca,Asteraceae)

L. sativa L. cultivars were compared morphologically with accessions ofL. saligna L.,L. serriola L., andL. virosa L. in order to define the infra- and interspecific variation. Multivariate analysis (principal component analysis) distinguished four groups corresponding with the four species. Infraspecific variation (e.g., colour, leaf shape) withinL. sativa is the largest, because breeding has caused a large variation in vegetative features; infraspecific variation withinL. serriola is also large. Three plants of one accession were placed betweenL. sativa andL. serriola because they showed morphological characteristics of both species. They are still classified asL. sativa. The morphological differences betweenL. sativa andL. serriola are too large to consider these two as one species. The descent of the cultivar groups and the four species is described.     

An Association Between High Peroxidase Activity in Lettuce (Lactuca sativa) and Field Resistance to Downy Mildew (Bremia lactucae)

The association between variation for pre-infection peroxidase activity and levels of field resistance-susceptibility to downy mildew (Bremia lactucae) was investigated in lettuce (Lactuca sativa) cultivars, accessions of L. serriola (prickly lettuce), segregating F₂ populations and selected F₃ families from a cross between field resistant and susceptible lettuce cultivars. A trend was apparent in this series of experiments indicating that one component of field resistance could be related to a high level of peroxidase activity prior to infection. The data suggest that in breeding programmes there could be merit in imposing primary selection for high peroxidase activity prior to field selection for resistance.     

A Lactuca universal hybridizer, and its use in creation of fertile interspecific somatic hybrids

A Lactuca sativa cv. Ardente line heterozygous for a gene encoding resistance to kanamycin, a positive and dominant trait, was crossed with cv. Girelle, which is heterozygous for a recessive albinism marker. The resulting seeds yielded 25% albino seedlings, of which 50% were also resistant to kanamycin. Such plantlets (KR, a) grown in vitro were used for preparation of universal hybridizer protoplasts, since green buds that can develop on kanamycin containing-medium should result from fusion with any wild-type protoplast. To test the practicability of this selection scheme, we fused L. sativa KR, a protoplasts with protoplasts derived from various wild Lactuca as well as various other related species. Protoplast-derived cell colonies were selected for resistance to kanamycin at the regeneration stage. Green buds were regenerated after fusion with protoplasts of L. tatarica and of L. perennis. So far, 9 interspecific hybrid plants have been characterized morphologically. In addition, random amplified polymorphic DNA (RAPD) analysis with selected primers confirmed that these plants are indeed interspecific hybrids. Some plants are female-fertile and production of backcross progenies with L. sativa is in progress. Since many desirable traits such as resistances to viruses, bacteria and fungi (Bremia lactucae) have been characterized in wild Lactuca species, the use of somatic hybridization in breeding programmes now appears a practical possibility.     

Field water relations of a compass plant, Lactuca serriola L.

Abstract Field water relations of Lactuca serriola serriola and L. serriola integrifolia were examined. Leaf conductance to water vapour was high early in the morning and declined rapidly during the midday hours. Leaf water potentials decreased to their minima early in the morning and remained low all day. Afternoon recovery of leaf conductance occurred occasionally. Leaf conductance was shown to have a linear response to vapour concentration difference. No differences were seen between L. serriola serriola and L. serriola integrifolia. The pattern of diurnal gas-exchange activity appeared to be complemented by the pattern of intercepted solar irradiance which results from the compass plant leaf orientation observed in L. serriola.     

Non-random leaf orientation in Lactuca serriola L.

Abstract Leaf shapes and leaf orientation of Lactuca serriola serriola and serriola integrifolia were studied. Leaf shapes in L. serriola serriola differed greatly from those of L.serriola integrifolia, but leaf surface areas were similar. In exposed habitats, leaf orientation of cauline leaves of both forms was non-random, with leaves almost vertical and tending to orient with their lamina normal to the east and west. In the shade, cauline leaves oriented randomly. An experiment demonstrated that the orientation of leaves did not change significantly once they were fully expanded. The leaf orientation in L. serriola affected the diurnal distribution of solar irradiance intercepted by a leaf. Peak solar radiation fluxes are incident on the rosette leaves at midday, but on the cauline leaves the peak solar radiation flux occurs early in the morning and again late in the afternoon. The significance of this unusual leaf orientation is discussed in relation to water loss and carbon gain.     

PCR-based fingerprinting using AFLPs as a tool for studying genetic relationships in Lactuca spp.

AFLP markers were evaluated for determining the phylogenetic relationships Lactuca spp. Genetic distances based on AFLP data were estimated for 44 morphologically diverse lines of cultivated L. sativa and 13 accessions of the wild species L. serriola, L. saligna, L. virosa, L. perennis, and L. indica. The same genotypes were analyzed as in a previous study that had utilized RFLP markers. The phenetic tree based on AFLP data was consistent with known taxonomic relationships and similar to a tree developed with RFLP data. The genetic distance matrices derived from AFLP and RFLP data were compared using least squares regression analysis and, for the cultivar data, by principal component analysis. There was also a positive linear relationship between distance estimates based on AFLP data and kinship coefficients calculated from pedigree data. AFLPs represent reliable PCR-based markers for studies of genetic relationships at a variety of taxonomic levels.     

Distribution,Disease Level and Virulence Variation of Bremia lactucae on Lactuca sativa in the Czech Republic in the Period 1999–2011

We report the distribution and disease level of downy mildew on lettuce (Lactuca sativa) and virulence variation in populations of its causal agent (Bremia lactucae) in the Czech Republic during the period 1999–2011. Disease incidence was not high; among a total of 92 different localities surveyed, 43.4% of them were infected by lettuce downy mildew at least once during the whole period. However, among individual years, differences were found in disease incidence that ranged from 4.8% (2009) to 66.7% (2004). A total of 128 isolates of B. lactucae collected from infected leaf samples in 35 different localities during the surveying period were included in the virulence analysis. Virulence was examined on a set of 19 differential genotypes of Lactuca sativa and Lactuca serriola (EU‐A test set). Isolates exhibited quite a broad variation in virulence to individual Lactuca differential genotypes. Eighteen of 19 virulence factors (v‐factors) tested were present in the samples. The most frequently detected factors were v1–4, v5/8, v6, v7, v10–14, v16, v36 and v38; factor v17 was not found. The most pronounced temporal shift was recorded for factors v36 and v38 whose frequency increased during the studied period. V‐factors 15, 17, 18 and 37 were present in low frequencies in a pathogen population, and their corresponding gene (Dm15) or resistance factors (R17, R18 and R37) may have the best potential for resistance breeding in the Czech Republic. Broad diversity of v‐phenotypes (63 different ones) was identified during the study period. The numbers of v‐factors per v‐phenotype (resp. isolate) varied within a range of 5–15. Within the 128 analysed isolates, only 9 v‐phenotypes were recorded repeatedly (three or more times). Possible reasons of recorded virulence variation are discussed.     

Occurrence of Bremia lactucae in Natural Populations of Lactuca serriola

In the period 1996–2001 the natural occurrence of Bremia lactucae (lettuce downy mildew) on Asteraceae plants was studied in the Czech Republic. Lactuca serriola (prickly lettuce) is the most common naturally growing host species of B. lactucae. Infection of plants was recorded during the whole vegetation season with the first occurrence in April and last in November. Bremia lactucae was found on host plants in all developmental stages. High percentages of naturally infected populations of L. serriola were recorded. Host plants exhibited broad variation in phenotypic expression of disease symptoms and degree of infection, however, the intensity of infection was rather low in the majority of populations. Geographic distribution of B. lactucae was studied in the two main parts of Czech Republic, central and southern Moravia, and eastern, northern and central Bohemia. Bremia lactucae was recorded in all these areas. Nevertheless, in the warmest parts of the Czech Republic (southern Moravia) only sporadic occurrence of the pathogen was recorded. Bremia lactucae infection on L. serriola and disease severity was judged also in relation to the type of habitat, and the size and density of host plant populations. However, no substantial differences among various habitats were found; only host plants growing in urban areas were frequently free of infection and the degree of infection was very low. Nevertheless, these plants were commonly infected with powdery mildew (Golovinomyces cichoracearum), which is most aggressive pathogen of this type of habitat.     

Effect of plant–soil feedbacks on the growth and competition of <Emphasis Type="Italic">Lactuca</Emphasis> species

Plant species generate specific soil communities that feedback on plant growth and competition. These feedbacks have been implicated in plant community composition and dispersion. We used Lactuca sativa and its wild progenitor Lactuca serriola to test the hypotheses that separate Lactuca species generate unique soil communities and that these soil communities differentially influence host, and neighboring, plant growth and competition. We grew each Lactuca in competition with the other, in sterile and non-sterile soils. We then examined the growth of each Lactuca species in sterile, non-sterile, and preconditioned soil. Finally, we used TRFLP techniques to explore whether the two Lactuca species generate significantly different bacterial communities in their rhizosphere soils. L. sativa proved to be the stronger competitor of the two species. However, sterilization increased the competitive effect of L. serriola background competitors. The growth experiment showed a significant effect on plant species, soil treatment, and the interaction of the two. Preconditioning soil caused reduced growth in both Lactuca species. Only L. serriola showed significantly increased growth in sterile soils. Our TRFLP analysis showed that the L. sativa soil community was significantly less diverse and that soil preconditioning had the largest impact on the community composition. These results show that Lactuca serriola’s rhizosphere communities generate a stronger negative feedback for plant growth than do the communities associated with L. sativa. Our study suggests that selection for plants that are able to grow in dense monoculture may have released Lactuca from species-specific negative soil feedbacks. This has important implications for both agriculture and the evolution of invasive plant species.     

Geographical distribution of wildLactuca species (Asteraceae, Lactuceae)

A broad survey of available world literature showed that at least 98 wildLactuca spp. (Asteraceae) have been described taxonomically. The distribution of the genusLactuca worldwide includes 17 species in Europe, 51 in Asia, 43 in Africa, and 12 in the Americas (mostly the North American subcontinent). Species originating in Asia, Africa, and the Americas form ca. 83% of knownLactuca spp. richness; however, they are very poorly documented from the viewpoint of taxonomic relationships, ecogeography, and variability. The phytogeography ofLactuca spp. regarding their distribution on different continents and in relation to the structure of the lettuce gene pool is discussed. A more detailed analysis of geographical distribution and habitats is given for some species (L. serriola, L. saligna, L. virosa, L. perennis, L. quercina, L. tatarica), which represent the primary, secondary, and tertiary gene pools of cultivated lettuce (L. sativa). Original and synanthropic distributions ofLactuca spp. and their occurrence in natural and secondary habitats are discussed, along with the representation of wildLactuca spp. in world gene-bank collections. Global biodiversity ofLactuca spp. and their representation in germplasm collections are poorly documented. Future studies of taxonomy, phytogeography, ecology, phylogenetic relationships, and genetic diversity are needed for a more complete understanding of this genus and taxonomically related genera.     

Recognition of lettuce downy mildew effector BLR38 in Lactuca serriola LS102 requires two unlinked loci

Plant-pathogenic oomycetes secrete effector proteins to suppress host immune responses. Resistance proteins may recognize effectors and activate immunity, which is often associated with a hypersensitive response (HR). Transient expression of effectors in plant germplasm and screening for HR has proven to be a powerful tool in the identification of new resistance genes. In this study, 14 effectors from the lettuce downy mildew Bremia lactucae race Bl:24 were screened for HR induction in over 150 lettuce accessions. Three effectors—BLN06, BLR38 and BLR40—were recognized in specific lettuce lines. The recognition of effector BLR38 in Lactuca serriola LS102 did not co-segregate with resistance against race Bl:24, but was linked to resistance against multiple other B. lactucae races. Two unlinked loci are both required for effector recognition and are located near known major resistance clusters. Gene dosage affects the intensity of the BLR38-triggered HR, but is of minor importance for disease resistance.     

Virulence of Bremia lactucae in Sweden and race-specific resistance of lettuce cultivars to Swedish isolates of the fungus

Virulence surveys of Swedish Bremia lactucae populations confirmed that the virulence factors vl to v12 were present in high or very high frequencies. Virulence associated with recently defined new resistance genes was also present. Laboratory tests of lettuce cultivars and Lactuca accessions using different Bremia isolates and field tests with natural inoculum showed that previously undetected virulence factors were present. Due to a lack of highly effective genes for specific resistance and the frequent sexual recombination of virulence genes it is suggested that any future breeding programmes concentrate on non-specific resistance.