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.     

Linkage Between Virulence Genes, Compatibility Types and Sexual Recombination in the Swedish Population of Bremia lactucae

The host pathogen interaction between Lactuca sativa and Bremia lactucae fits a gene-for-gene model well. Twelve resistance genes of the host are matched by twelve genes for virulence in the pathogen. The evolution of the parasite involves drastic changes in virulence frequencies, and a great diversity in virulence even on a sub-poipulation level. Bremia is a heterothallic, obligate parasite, in which presence of two mating types is needed for sexual reproduction. Sexual recombination probably occurs frequently, indicated by simultaneous occurrence of mating types in commercial lettuce crops, zygote formation, and sufficiently high oospore germination. The pattern of variation agrees well with that of a diploid, out- crossing organism with frequent sexual recombination. Unexpected high frequencies of some of the unnecessary v-genes are probably due to genetic linkage with another "necessary" v-gene.     

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.     

New virulence gene combinations in British isolates of Bremia lactucae

Isolates of Bremia lactucae made in 1974 from new Dutch-bred lettuce cultivars carrying the R gene combinations 3, 4, 7 or 3, 4, 7, 8 proved to have the V gene combination 1, 3, 4, 5, 6, 7, 8 predominant. An isolate obtained from cv. Larganda (R2, 7) was found to overcome all ten R genes identified in lettuce cultivars to date and could be designated V 1, 2, 3, 4, 5, 6, 7, 8, 9, 10. The virulence combinations 3, 7, and 2, 3, 7 have not previously been reported in British B. lactucae collections although the former has been found in Holland and both occur in Sweden. The practical implications of these findings are discussed.     

Pathogenic variation and sexual reproduction in Swedish populations of Bremia lactucae

Summary The host-pathogen interaction between lettuce (Lactuca sativa) and downy mildew (Bremia lactucae) is mainly differential and the resistance so far utilized in the host is vertical. As in many other obligate parasites, the introduction of cultivars with new vertical resistance has exerted a strong selection pressure on the pathogen resulting in significant changes in virulence frequencies and in the establishment of races with new combinations of virulence. Genetic diversity in pathogen populations may arise through mutation and gene flow, and new virulence genotypes may then be established through parasexuality and sexual recombination. In Swedish populations of Bremia lactucae, the pattern of variation in the parasite agrees well with that which might be expected in a diploid, outcrossing organism with frequent sexual reproduction. This is supported by: two or more isolates, different in virulence and mating type, may occur together on the same lettuce leaf; zygotes (oospores) are formed in all populations investigated and the frequency varies from 22% to 98%; oospores germinate rather frequently under suitable conditions. To breed for resistance in dynamic host-pathogen systems such as this one is difficult and the program should preferably be based on race-non-specific resistance.     

Histochemical Detection and Role of Phenolic Compounds in the Defense Response of Lactuca spp. to Lettuce Downy Mildew (Bremia lactucae)

The occurrence of phenolic compounds (PC) in the defence reaction of Lactuca spp. was detected by histochemical methods. Four staining methods, including three for light and one for fluorescence microscopy, were used for imaging the location of PC in cells of genotypes with different resistance mechanisms after infection by Bremia lactucae , race NL16. The results showed that the major role of phenolic compounds in studied Lactuca spp. is connected with their overexpression and localized accumulation during a hypersensitive response (HR). In incompatible interactions a slight accumulation of phenols near the cell wall of infected cells was detected. The negative reaction to staining with aniline sulphate verified the absence of lignin creation. In both compatible and incompatible interactions structural modifications in the host cells occurred as a callose deposition. Frequently, these deposits were widespread in susceptible genotypes. Intensive and rapid accumulation of autofluorescent phenolics was linked with the onset of HR, the main cytological feature of resistance to lettuce downy mildew in Lactuca spp.     

Studies on a further race (W. 4) of Bremia lactucae Regel

A further race (W. 4) of Bremia lactucae was identified in a sporangial collection from an infected field crop of the cultivar Avondefiance by tests on detached seedlings and leaf disks of various diagnostic lettuce cultivars. Race W. 4 appeared to be unstable, with a similar host range to race W. 3 , but differed from it in its inability to infect the crisp-lettuce cultivars Grand Rapids, Calmar, Caravan, Francisco and Great Lakes.
Races W. 1, W. 2 and W. 3 were also found in tests starting with the sporangial collection from Avondefiance. Although W. 3 could have been present in small quantities in this original collection, it was considered that W. 1 and W. 2 arose spontaneously from W. 4 during subculturing.
A means of eliminating W. 2 from mixtures of W. 1 and W. 2 is described.
A laboratory experiment indicated that when competing in a crop of Avon-defiance or Cobham Green, race W. 2 might predominate over race W. 3.     

Studies on a method of carry-over of downy mildew (Bremia lactucae) of lettuce

Summary Bremia lactucae produced oospores in the decayed stem tissue of infected lettuce plants. These oospores caused cotyledon and first leaf infections ofBremia lactucae when added to the rooting medium of lettuce seedlings grown under sterile conditions.     

The virulence characteristics of Bremia lactucae populations present in Sweden from 1971 to 1976

One hundred and four Swedish isolates of Bremia lactucae were tested between 1971 and 1976 for their virulence phenotypes. Eighty of these isolates showed patterns of virulence previously recorded in other countries while the remainder conformed to seven previously unreported virulence factor combinations. The simplest resistance factor combination that could give resistance to all isolates collected was R2 plus Rl 1. This combination now exists in some recently bred lettuce cultivars. Most of the possible virulence factor combinations occurred in the population at the expected frequency. However, several combinations, which had not been directly selected (by the growing of cultivars carrying the comparable resistance factor combinations) were present at higher or lower than expected frequencies.     

Lactuca saligna,a non-host for lettuce downy mildew ( Bremia lactucae), harbors a new race-specific Dm gene and three QTLs for resistance

Lactuca sativa (lettuce) is susceptible to Bremia lactucae (downy mildew). In cultivated and wild Lactuca species, Dm genes have been identified that confer race-specific resistance. However, these genes were soon rendered ineffective by adaptation of the pathogen. Lactuca saligna (wild lettuce) is resistant to all downy mildew races and can be considered as a non-host. Therefore, L. saligna might be an alternative source for a more-durable resistance to downy mildew in lettuce. In order to analyze this resistance, we have developed an F(2) population based on a resistant L. saligna x susceptible L. sativa cross. This F(2) population was fingerprinted with AFLP markers and tested for resistance to two Bremia races NL14 and NL16. The F(2) population showed a wide and continuous range of resistance levels from completely resistant to completely susceptible. By comparison of disease tests, we observed a quantitative resistance against both Bremia races as well as a race-specific resistance to Bremia race NL16 and not to NL14. QTL mapping revealed a qualitative gene ( R39) involved in the race-specific resistance and three QTLs ( RBQ1, RBQ2 and RBQ3) involved in the quantitative resistance. The qualitative gene R39 is a dominant gene that gives nearly complete resistance to race NL16 in L. saligna CGN 5271 and therefore it showed features similar to Dm genes. The three QTLs explained 51% of the quantitative resistance against NL14, which indicated that probably only the major QTLs have been detected in this F(2) population. The perspectives for breeding for durable resistance are discussed.     

Gonidial morphology and the specialization of Bremia lactucae Regel (Peronosporaceae) on hosts in the family Compositae

SKIDMORE, D. I. & INGRAM, D. S., 1985. Conidial morphology and the specialization of Bremia lactucae Regel (Peronosporaceae) on hosts in the family Compositae . Conidia of Bremia lactucae were collected from Lactuca saliva, Sonchus oleraceus, S. asper, Senecio vulgaris, S. bicolor subsp. cineraria, Cirsium arvense, Centaurea nigra and Picris hieracioides , from a wide distribution in the British Isles. There were no consistent differences between isolates taken from the different hosts in the lengths, breadths or length: breadth ratios of the conidia, the lengths of the conidiophores or the numbers of sterigmata per conidiophorc branch. Therefore, as there are no significant morphological differences between the isolates of Bremia lactucae from these hosts, the pathogen is classified upon host specialization. A review is presented of cross-infection studies in which isolates of B. lactucae had been inoculated on to a range of species of Compositae. On the basis of these investigations it is recommended that the term 'formae speciales' is used to denote host specialization of isolates within the species Bremia lactucae .     

The genetics of race specific resistance in lettuce (Lactuca sativa) to downy mildew (Bremia lactucae)

Data are presented on the segregation of resistance to five British races and two Dutch races of Bremia lactucae in the F₂ progenies of crosses involving seven resistant and several susceptible lettuce cultivars. These data and also those previously published by other workers are considered in relation to the systematic model proposed by Crute & Johnson (1976) to explain the genetics of race specific resistance to B. lactucae in lettuce. It is shown that, with minor modifications, the model accommodates almost all of the previously published data and correctly predicts the new data, except for one set which cannot at present be interpreted. It is concluded that genetic evidence exists for the presence, among various cultivars of lettuce, of at least four and possibly five different dominant resistance genes of major effect designated Dm₂, Dm₃, Dm₄, Dm₆ and Dm₈; and of a pair of dominant genes with complementary effect designated Dm7/1 and Dm7/2. The resistance conferred by these genes is specified in relation to five British races, five Dutch, three Israeli and one United States race of the fungus. Resistance genotypes are proposed for cultivars Avoncrisp, Avondefiance, Calmar, Great Lakes 659, Kares, Meikoningen, Mildura, Proeftuins Blackpool, Solito, Valverde, Ventura and the USDA line PI 164937.     

Localisation and metabolism of reactive oxygen species during Bremia lactucae pathogenesis in Lactuca sativa and wild Lactuca spp.

A plant's physiology is modified simultaneously with Oomycete pathogen penetration, starting with release and accumulation of reactive oxygen species (ROS). Localisation of superoxide, hydrogen peroxide, peroxidase and variation in their activity, and the isoenzyme profile of antioxidant enzymes peroxidase (1.11.1.7), catalase (EC 1.11.1.6), superoxide dismutase (EC 1.15.1.1) were studied in six genotypes of four Lactuca spp. (L. sativa, L. serriola, L. saligna and L. virosa) challenged with Bremia lactucae (race NL16). These factors were related to the differential expression of resistance during the course of 96h after inoculation (hai). Accumulation of hydrogen peroxide in infected cells together with enhanced activity of H(2)O(2)-scavenging enzymes in leaf extracts characterised resistant Lactuca spp. genotypes 6-12hai, and peaked at 48-96hai with expression of a hypersensitive reaction. Substantial changes of guaiacol peroxidase activity were detected only in the cytosolic enzyme; activities of the membrane-bound and the ion-bound enzymes were insignificant in the interactions of host genotypes and pathogen isolate examined. The most significant modifications of ROS metabolism were found in resistant L. virosa (NVRS 10.001 602), a genotype responding to pathogen ingress by a rapid and extensive hypersensitive reaction. Formation of the superoxide anion was not detected in either susceptible or resistant plants, and there was also no increase of superoxide dismutase activity or changes in its isozyme profile. The significance of precise balancing the intracellular level of hydrogen peroxide for variability of phenotypic expression of responses to B. lactucae infection in Lactuca spp. is discussed.     

Incidence of Virulence Factors and Antibiotic Resistance among Enterococci Isolated from Food

The incidence of virulence factors among 48 Enterococcus faecium and 47 Enterococcus faecalis strains from foods and their antibiotic susceptibility were investigated. No strain was resistant to all antibiotics, and for some strains, multiple resistances were observed. Of E. faecium strains, 10.4% were positive for one or more virulence determinants, compared to 78.7% of E. faecalis strains. Strains exhibiting virulence traits were not necessarily positive for all traits; thus, the incidence of virulence factors may be considered to be strain specific.     

From Grazing Resistance to Pathogenesis: The Coincidental Evolution of Virulence Factors

To many pathogenic bacteria, human hosts are an evolutionary dead end. This begs the question what evolutionary forces have shaped their virulence traits. Why are these bacteria so virulent? The coincidental evolution hypothesis suggests that such virulence factors result from adaptation to other ecological niches. In particular, virulence traits in bacteria might result from selective pressure exerted by protozoan predator. Thus, grazing resistance may be an evolutionarily exaptation for bacterial pathogenicity. This hypothesis was tested by subjecting a well characterized collection of 31 Escherichia coli strains (human commensal or extra-intestinal pathogenic) to grazing by the social haploid amoeba Dictyostelium discoideum. We then assessed how resistance to grazing correlates with some bacterial traits, such as the presence of virulence genes. Whatever the relative population size (bacteria/amoeba) for a non-pathogenic bacteria strain, D. discoideum was able to phagocytise, digest and grow. In contrast, a pathogenic bacterium strain killed D. discoideum above a certain bacteria/amoeba population size. A plating assay was then carried out using the E. coli collection faced to the grazing of D. discoideum. E. coli strains carrying virulence genes such as iroN, irp2, fyuA involved in iron uptake, belonging to the B2 phylogenetic group and being virulent in a mouse model of septicaemia were resistant to the grazing from D. discoideum. Experimental proof of the key role of the irp gene in the grazing resistance was evidenced with a mutant strain lacking this gene. Such determinant of virulence may well be originally selected and (or) further maintained for their role in natural habitat: resistance to digestion by free-living protozoa, rather than for virulence per se.     

Linkage analysis of genes for resistance to downy mildew (Bremia lactucae) in lettuce (Lactuca sativa)

Summary The genetics of specific resistance was studied in F₂ populations which segregated for either one or two resistance genes. The resistance factors 1, 11 and 14 which had not previously been characterized genetically segregated as single dominant genes (Dm). Resistance was determined by three linkage groups; R 1/14, 2, 3, and 6 in the first, R 5/8, and 10 in the second and R 4, 7 and 11 in the third. Cultivars of lettuce commonly used in the differential series to detect virulence to R3 and R10, were demonstrated to carry two tightly linked resistance genes. Implications of this linkage arrangement to the manipulation and characterization of these resistance genes are discussed.     

Variation in seedling susceptibility to Bremia lactucae (lettuce downy mildew) in the absence of effective major resistance genes

In the absence of effective major genes the importance of interactions between cultivar genotype, isolate genotype and environment was investigated in experiments using seedlings of eight lettuce cultivars inoculated with three isolates of Bremia lactucae and grown under a number of different environmental conditions. The outcome of the cultivar-isolate association was measured using four criteria and the data were examined by analysis of variance and correlation. The relative susceptibility of cultivars was generally independent of environment and there was no evidence that isolates were adapted to particular cultivars. Although significant cultivar X isolate interactions were found in individual experiments they were not consistent between experiments, even where these were conducted under apparently identical conditions. Variation resulted from either cultivar X environment or isolate X environment interaction with the environment always the dominant variable.