Bremia Lactucae Research Articles

Identification of New Sources of Resistance to Downy Mildew in Lactuca Spp.

Abstract More than 300 accessions of Lactuca spp. recently collected in the Mediterranean region and California were screened for resistance to downy mildew (Bremia lactucae Regel). Accessions were evaluated in the seedling stage with two isolates of B. lactucae, which together have virulence to all known resistance factors. Four accessions demonstrated complete resistance to both isolates, indicating that new resistance was present. This resistance was confirmed using further isolates of B. lactucae. None of these lines, however, was resistant to all the isolates used.

Zur Auswirkung von Resistenzreaktionen auf Pflanzenwachstum und Ertrag in inkompatiblen Wirt‐Parasit‐Systemen

Abstract Effects of defense reactions on plant growth and yield in incompatible host‐parasite‐systems Investigations in incompatible host‐parasite‐systems (barley –Erysiphe graminis and lettuce –Bremia lactucae) were carried out to prove, whether the induction of defense reactions leads to measurable effects on the yield performance of plants. Permanent or frequent inoculations with avirulent races decreased yield of barley and lettuce remarkably, expressed in reduced fresh weight and drym, atter. Less grains were produced and their contents of carbohydrates and amino acids were diminished significantly. In young plants a reduction in sugar content was evident already after a single inoculation. In barley as in lettuce this effect was shown, not only for inoculated, but also for plant parts not present at the time of inoculation. There, in addition especially aromatic amino acids increased, while glutamate, aspartate and their amides diminished. The consequences of a permanent maintenance of postinfectional resistance mechanisms either of a hypersensitive or non‐hypersensitive reaction type for growth and yield performance are discussed with regard to an increased consumption of energy and carbohydrates.

The identification and characteristics of a mutation in Bremia lactucae resulting in large lipid inclusions in the conidiosporangia

During an investigation involving the production of sexual recombinants of Bremia lactucae, isolates were identified which contained atypical large spherical inclusions in their conidiosporangia. These were visible under the light microscope. Since these inclusions stained red with Sudan III and disappeared as the conidia germinated, they were assumed to be reserves of lipid. Storage lipid in normal spores is distributed as numerous small droplets at the perimeter of the spore near the plasmalemma, whereas in the mutant, lipid occurred as large droplets within the spore. There was no significant difference between normal and mutant spores in their total lipid content or their hydrocarbon or fatty acid profiles. Mutant spores germinated as readily as normal spores and there was no apparent effect of the mutation on pathogenic competence. The character appeared to be inherited as a single recessive gene. The use of this marker for investigations on the relative fitness of isolates under different environmental conditions is discussed.

An assessment of genetic variability in somacloned lettuce plants (Lactuca sativa) and their offspring

SUMMARYPlants were regenerated from callus derived from cotyledons and first true‐leaves of the lettuce cultivars Salad Bowl, Lobjoits Cos and Pennlake. Sexual progeny of these regenerants were assessed under glasshouse and field conditions for variation including reaction to lettuce mosaic virus (LMV) and downy mildew (Bremia lactucae). All three cultivars exhibited somaclonal variation. Mutations detected at the seedling stage included reduced vigour, albinism and changes in chlorophyll content, with most being recessive. Variation for leaf shape and vigour was detected in mature plants. One line exhibited increased yield and chlorophyll content together with early flowering. Enhanced and reduced susceptibility to both LMV and B. lactucae were observed. Reduced susceptibility to B. lactucae was indicated by an extended latent period following inoculation in two lines. Reduced susceptibility to LMV in glasshouse trials could not be confirmed in the field although one such line exhibited an improved yield and a second line segregated 1:1 in glasshouse tests for plants which were obviously infected and those without symptoms. All variable lines were diploid.

Gene-dosage effects on the relationship between Bremia lactucae (downy mildew) and Lactuca sativa (lettuce): the relevance to a mechanistic understanding of host—parasite specificity

The expectations for the reaction phenotypes of four models proposed to explain the genetic regulation of cultivar-isolate specificity in gene-for-gene relationships were different when gene-dosage effects (incomplete dominance) were taken into account. To test these hypotheses, F 2 individuals from two crosses involving the segregation of gene R6 for resistance in Lactuca sativa (lettuce) to Bremia lactucae (downy mildew) were inoculated with isolates homozygous ( AA or aa ) or heterozygous ( Aa ) at the matching avirulence locus. Assessment of pathogen development revealed evidence for gene-dosage dependence in the expression of both resistance and avirulence. However, these effects were only observed in incompatible genotype combinations and the data only satisfactorily fit the hypothesis that specificity is regulated by the interaction between specific alleles for resistance ( R ) and avirulence ( A ). The implications of gene-dosage dependence to a mechanistic understanding of specificity are discussed.

Specificity of Interactions Between Wild Lactuca spp. and Bremia lactucae Isolates from Lactuca serriola

AbstractSpecificity of interactions between eight Lactuca species and 8 Bremia lactucae isolates was studied in seedlings and adult plants of 36 Lactuca accessions plus one L. serriola × L. sativa hybrid. Pathogenicity of the isolates and/or plant susceptibility was expressed by sporulation intensity.A highly compatible relationship was observed in all of L. serriola accession/isolate interactions tested. A differential reaction was found in numerous cases testifying to physiological specialization of the pathogen in a wild pathosystem. Nonspecific nondifferential compatibility (quantitative resistance) can be expected in L. serriola PI 281876. Age dependent resistance (seedlings versus adult plants, and vice versa) and heterogeneity of reactions were also recorded. The comparisonof B. lactucae isolates from L. sativa and L. serriola has shown a significant shift of pathogenicity in favour of L. serriola accessions. A high level of resistance was found in the L. serriola × L. sativa hybrid.The existence of basic incompatibility can be expected in L. saligna and L. virosa as well as in such taxonomically remote species as L. viminea, L. squarrosa and L. biennis. Other taxonomically remote species, i.e. L. dentata and L. alpina, exhibited a compatible reaction, although the reaction of the former was differential. A high level of sporulation was recorded in all accession/isolate interactions of L. alpina.

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

SUMMARYVirulence 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.

Effect of Aliette on Bremia lactucae on lettuce

Aliette is a systemic fungicide effective against diseases caused by lower fungi but there is some question whether it acts directly on pathogens or indirectly via host defence. Downy mildew development on lettuce cotyledons was markedly decreased by application of 100–500 μg Aliette, active ingredient (a.i.), ml −1 to roots. Sporangial germination on glass and cotyledons was prevented by direct application of 10 and 25 μg Aliette ml −1 respectively. Germ-tube growth and penetration into cotyledons were markedly decreased when 25 μg Aliette ml −1 was applied directly 2 h after sporangial inoculation. The size of secondary vesicles and growth of intercellular hyphae in cotyledons were markedly affected when 250 μg Aliette ml −1 was applied 4 h after sporangial inoculation. Hyphal growth was seen to be affected 24 h later; a low incidence of necrosis of host cells was observed after a further 24 h. The findings gave reason to believe that Aliette acted directly on the pathogen in this system.

Untersuchungen zur Anfälligkeit und Resistenz von Kopfsalat (Lactuca sativa) gegen falschen Mehltau (Bremia lactucae) III. Peroxydase‐, peroxydatische Katalase‐und Polyphenoloxydase‐Aktivitäten

Abstract Investigations on the susceptibility of head lettuce (Lactuca sativa) to downy mildew (Bremia lactucae) III. Activities of peroxidase, catalase and polyphenoloxidase Host cell walls in contact with intercellular hyphae of Bremia lactucae stain electron positively in susceptible and incompletely resistant varieties of lettuce after appropriate electron microscopy preparation for peroxidase activity. The outer membranes of the mitochondria of the parasite also stained darkly in susceptible varieties whereas in incompletely resistant plants Bremia innermost mitochondrial membranes and host cell mitochondria were darkly stained. This latter observation suggests increased respiration and could be explained as a resistance reaction. Catalase activity was observed in the microbodies of susceptible, in incompletely resistant and healthy varieties. There were no differences in stain intensity in the three kinds of varieties suggesting that catalase activity is not involved in resistance reactions.Polyphenoloxidase activity was infrequently observed on the host cell wall in susceptible and healthy plants, whereas strong activity in incompletely resistant varieties was observed in vesicles in the haustorial sheath. These vesicles were not surrounded by unit membranes and therefore could not have originated from the unit membranes of the extrahaustorial matrix or from the host plasmalemma. They may have been derived from the host protoplast and involved in inactivation of parasite produced toxins thereby contributing to resistance.

Untersuchungen zum Einflufß von Aluminiumfosetyl auf den pflanzlichen Phenolstoffwechsel in den Pathogen‐Wirt‐Beziehungen Phytophtbora fragariae – Erdbeere und Bretmia lactucae ‐ Salat

Abstract Investigations on the influence of aluminium ethyl phosphite on the plant phenolic metabolism in the pathogen‐host‐interactions Phytophthora fragariae ‐ strawberry and Bremia lactucae ‐ lettuce Aluminium ethyl phosphite exhibited pronounced preventive and very good curative activities. In strawberries, 4000 ppm of the compound affected an eminent protection against Phytophthora fragariae after root and leaf applications; the earlier the fungicide treatment was carried out, the more distinct was the control effect. Similar relations were established in the pathogen‐host‐interaction Bremia lactucae, in which 5000 ppm of the fungicide proved to be highly active following pre‐ and postinfectional applications.In both parasite‐host‐interactions, an enrichment of phenolics in consequence of a de‐novo‐synthesis in diseased plant tissue was observed only very late and exclusively, when fungicide and parasite had come together; inoculation on one hand or fungicide treatment on the other resulted in no remarkable effect.With great certainty, however, the augmentation of phenolics was not the cause, but rather the consequence of fungicidal efficiency which resulted in killing of the fungus and partial destruction of host tissue after direct influence of aluminium ethyl phosphite on the parasite itself. A clear proof for a main and causal evidence of aluminium ethyl phosphite‐induced phenolics for protection of strawberries against Phytophthora fragariae and of lettuce against Bremia lactucae could not be furnished.

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.

Further characterization of field resistance in lettuce to Bremia lactucae (downy mildew)

The development of five Bremia lactucae isolates on the field‐resistant crisphead lettuce cultivar Iceberg was compared under field conditions with that on the more susceptible crisphead cultivar Ithaca. With each of five isolates, the epidemic developed more rapidly on Ithaca than Iceberg; lesions on Ithaca were more numerous, larger and sporulated more profusely than on Iceberg. There was no evidence for adaptation of isolates to Iceberg, which provides some additional evidence that the resistance of this cultivar could be non‐differential (horizontal). There was, however, evidence of non‐specific differences between isolates in their quantitative virulence.

Pathogenic variation and sexual reproduction in Swedish populations of Bremia lactucae

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.

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

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.

The nature of virulence in isolates of Bremia lactucae from South Devon

Nine isolates of Bremia lactucae from South Devon showed the presence of virulence factors against all 11 resistance factors available in lettuce, but no insensitivity to metalaxyl. The virulence phenotype of a single–sporangium isolate of the fungus changed from VI, 3, 4 and 7 to 1, 2, 3, 4, 6 and 7 after it was maintained on Lobjoit Green Cos (RO) for 10 months.

Untersuchungen zur Anfälligkeit und Resistenz von Kopfsalat (Lactuca sativa) gegen falschen Mehltau (Bremia lactucae): II. Licht‐ und elektronenmikroskopische Untersuchungen zur Morphologie des Wirt‐Parasit‐Verhältnisses

Abstract Investigations on the susceptibility and resistance of head lettuce (Lactuca sativa) to downy mildew (Bremia lactucae) II. Light and electron microscopic examinations of the host‐parasite interface Infected leaves of lettuce varieties susceptible and incompletely resistant to Bremia lactucae were observed by light and electron microscopy. Primary infection structures in the epidermal cells as well as intercellular hyphae with the adjacent haustoria could be seen by differential interference contrast microscopy. The haustoria in host cells of susceptible varieties collapsed before degeneration of the invaded host cell. On the contrary, host cells of incompletely resistant varieties died before the haustoria in these cells showed any sign of degeneration. Electron microscopic investigations confirmed the observations with light microscopy. In incompletely resistant varieties, an electron transparent sheath enveloped the haustorium. In the sheath fragments of membranes are localized. These membrane particles as seen by using the goniometer in electron microscopic work were flat faced. The sheath material consists of transformed host cell wall material and involves fragments of the host plasmalemma as well as fragments of the unit membrane separating the sheath from extrahaustorial matrix. The sheath has an important role as a special filter to prevent the passage of nutrients from the host cell into the haustorium. Thus the incomplete resistance is based not only on an impeded penetration of the parasite into the epidermal cells and their hypersensitive reactions in case of a successful penetration but also on hypersensitivity of mesophyll cells which does not necessarily lead to death of the parasite but does impede the absorption of nutrients.

Comparative ultrastructure of lettuce cotyledons infected with Botrytis cinerea and Bremia lactucae and the histochemical localization of acid phosphatase

The ultrastructure of the compatible interactions between lettuce cotyledons and Botrytis cinerea and Bremia lactucae were compared. Infection by B. cinerea was achieved, following direct penetration through the cuticle, by growth of intramural and intracellular hyphae and was associated with rapid and widespread destruction of host tissue. Using a modified Gomori lead-salt precipitation method acid phosphatase activity was localized in the modified epidermal cell wall surrounding the penetration site of B. cinerea , at various vesicular locations in infected plant cells and dispersed throughout the disorganized cytoplasm of cells at an advanced stage of necrosis. Infection by B. lactucae caused no apparent adverse reaction in the cytoplasm of infected plant cells and acid phosphatase activity was largely confined to those sites, such as the dictyosomes, which were typical of healthy tissue. However, intense enzyme activity was localized within the walls of intruding haustoria and with the collar of apposition material deposited by the host around the necks of these fungus structures. Acid phosphatase was also a conspicuous feature of the apposition material deposited by lettuce in response to infection by B. cinerea .

Inheritance of virulence in Bremia lactucae to match several resistance factors in lettuce

A 1∶1 mixture of spores of the physiologic races NL5 and NL6 ofBremia lactucae was sprayed on leaf discs of the lettuce cultivar Olof. The resulting oospores were allowed to germinate on seedlings after a two-month ripening period. Monospore lines of 49 of the 79 recovered isolates were tested on cotyledons and leaf discs of a differential series for virulence to 11 resistance factors. All monospore lines were also tested for the mating type factor. A backcross of a progeny line, line H, with NL5 was made and 41 isolates tested on the differential series.

The genetic control and expression of specificity in Bremia lactucae (lettuce downy mildew)

The inheritance of specific virulence in Bremia lactucae was studied in crosses involving 12 heterothallic isolates of the fungus. In one cross, virulence to eight of the II specific resistance factors examined segregated in the F1 generation. Although there were exceptions, most of the data were consistent with the hypothesis that pathogenicity was controlled by independent single loci with avirulence dominant to virulence. Linkage between loci determining virulence on R2 and R11 was confirmed. Loci controlling virulence on R5 and R8 also appeared to be linked but these may be identical R‐factors. Contrary to a previous suggestion, the locus determining virulence to R10 was independent of that for R5/R8. The expression of virulence to three R‐factors (Rl, R4 and R5/R8) was influenced by independent second loci. The presence of a dominant allele at the second locus inhibited avirulence. The expression of avirulence on R6 seemed to be influenced by modifier genes and environment in some isolates. Although the cultivars Mildura, Bourguignonne, Sucrine and Captain were originally thought to contain a single resistance factor, these data suggest that Sucrine carries R5/R8 in addition to R10 whilst Mildura may carry Rl in addition to R3. The stock of Bourguignonne appeared to be a mixture of resistance genotypes. The data add additional support to the suggestion that Capitan (Rll) may carry two resistance factors.

Integration of environmental and fungicidal control of Bremia lactucae in a glasshouse lettuce crop

The effect of combinations of night ventilation regimes and fungicide treatments on levels of downy mildew ( Bremia lactucae) was investigated in a November-sown glasshouse lettuce crop. Environmental regimes were allocated in a randomized-block design to glasshouse compartments in which fungicide treatments were applied to sub-plots from which sub-sub-plots were harvested on each of four dates. The benefit of night ventilation was strikingly shown. It was almost as effective as the systemic fungicide metalaxyl which gave complete control of B. lactucae for 20 weeks after its incorporation in the peat blocks. A ‘heat-purging’ treatment increased the incidence of downy mildew, while other fungicidal regimes failed to control late infection. In the period close to cutting, when fungicide usage is not permitted, night ventilation may be the only effective means of controlling the disease.