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.     

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.     

Host‐induced gene silencing inhibits the biotrophic pathogen causing downy mildew of lettuce

Host‐induced gene silencing (HIGS) is an RNA interference‐based approach in which small interfering RNAs (siRNAs) are produced in the host plant and subsequently move into the pathogen to silence pathogen genes. As a proof‐of‐concept, we generated stable transgenic lettuce plants expressing siRNAs targeting potentially vital genes of Bremia lactucae, a biotrophic oomycete that causes downy mildew, the most important disease of lettuce worldwide. Transgenic plants, expressing inverted repeats of fragments of either the Highly Abundant Message #34 (HAM34) or Cellulose Synthase (CES1) genes of B. lactucae, specifically suppressed expression of these genes, resulting in greatly reduced growth and inhibition of sporulation of B. lactucae. This demonstrates that HIGS can provide effective control of B. lactucae in lettuce; such control does not rely on ephemeral resistance conferred by major resistance genes and therefore offers new opportunities for durable control of diverse diseases in numerous crops.     

Identification of <Emphasis Type="Italic">Aspergillus</Emphasis> section <Emphasis Type="Italic">Flavi</Emphasis> in maize in northeastern China

Bremia lactucae Regel (Chromista, Peronosporaceae) is an economically destructive pathogen, which causes downy mildew disease on lettuce (Lactuca sativa L.) worldwide. The ribosomal internal transcribed spacer (ITS) of Bremia lactucae isolates was analyzed for the first time. The ITS region of lettuce downy mildew was observed to have a size of 2458 bp; thereby, having one of the longest ITS sizes recorded to date. The majority of the extremely large sized ITS2 length of 2086 was attributed to the additional presences of nine repetitive elements with lengths of 179–194 bp, which between them shared the low homology of 48–69%. Comparison of the ITS2 sequences with the B. lactucae isolates from other host plants showed that isolates present on Lactuca sativa were distinct from those on L. indica var. laciniata, as well as Hemistepta and Youngia. We suggest the high degree of sequence heterogeneity exhibited in the ITS2 region of B. lactucae may warrant the specific detection and diagnosis of this destructive pathogen or its division into several distinct species.     

The genetic relationship between races of Bremiae lactucae and cultivars of Lactuca sativa

The reactions of lettuce cultivars to physiologic races of Bremia lactucae are interpreted in terms of a gene-for-gene relationship between pathogen and host. The hypothesis takes into account the parentage of cultivars and the origins of their resistance, the characteristics of the resistance reactions and data available from detailed genetical analysis of various race/cultivar combinations. Cultivars are classified with respect to ten postulated resistance genes and B. lactucae races are defined by the virulence genes present. The practical significance of these studies is discussed in relation to both future lettuce breeding programmes and to the choice of cultivars available to counteract any given local race situation.     

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

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

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.     

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.     

First Record of Bremia lactucae Infecting Sonchus oleraceus and Sonchus asper in Brazil and its Infectivity to Lettuce

Bremia lactucae is recorded for the first time causing downy mildew on common sowthistle (Sonchus oleraceus) and spiny sowthistle (Sonchus asper) in Brazil. The disease and etiological agent are described. Pathogencity of sporangia obtained from S. oleraceus was tested on 12 species belonging to the Asteraceae, already recorded in the literature as hosts of B. lactucae, and four commonly cultivated varieties of lettuce. All four cultivars of lettuce, S. oleraceus and S. asper showed symptoms of the disease and sporulation of the pathogen 5 days after inoculation. It has been observed that the disease occurs on Sonchus spp. throughout the year in Viçosa (state of Minas Gerais), being more common on these two hosts than on lettuce. Both weeds are very common invaders of vegetable growing areas in Brazil. This indicates that those two hosts for the fungus may be important inoculum reservoirs for disease occurring in lettuce, highlighting the need for control of these weeds as part of the procedures aimed at controlling this disease. These results are also in agreement with the views that B. lactucae is split into host‐specific infra‐specific taxa. Lettuce and sowthistles are regarded as closely related, belonging to the same subfamily and tribe in the Asteraceae (Subfamily Cichorioideae: Tribe Lactuceae).     

Evidence for a race-specific resistance factor in some lettuce (Lactuca sativa L.) cultivars previously considered to be universally susceptible to Bremia lactucae regel

Summary Previously undetected race-specific resistance to Bremia lactucae (downy mildew) was located in many lettuce cultivars hitherto considered to be universally susceptible to this disease. This resistance factor(s) may also be widely distributed in other cultivars known to carry combinations of already recognised factors R1 to R11. Specific virulence to match this resistance is almost invariably present in pathogen collections. This situation may be either a relic of the evolutionary history of the B. lactucae — L. sativa asssociation or may reflect a rare mutation in B. lactucae for avirulence on all but a few specialised L. sativa genotypes.     

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.     

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.     

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.     

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

Plants 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.     

Laboratory and field tests of zineb and other fungicides against downy mildew of lettuce (Bremia lactucae Regel)

In laboratory tests on young lettuce plants, the protection provided by sprays of zineb wettable powder against artificial inoculations with Bremia lactucae was less than that obtained with tank-mix zineb, and with several other dithiocarbamates and related fungicides. Experiments on autumn lettuce in the field also showed that tank-mix zineb was more effective than the wettable powder. On winter lettuce in frames, however, zineb wettable powder gave control similar to or better than that given by any of the other materials tested. Infection in most of these outdoor experiments was not heavy. None of the fungicides gave very high control or markedly increased the number of hearted or marketable plants. When zineb wettable powder was sprayed on autumn lettuce at the rate of 2 lb./acre it gave similar disease reductions whether applied in low, medium or high volumes of water.     

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

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.     

The influence of seedling age and development on the infection of lettuce by Bremia lactucae

Laboratory methods are described for the culture and observation of Bremia lactucae on lettuce. Mycelial growth of the fungus within the cotyledons of the cultivar Cheshunt Early Giant progressed to a minimum density at which sporulation occurred, and then continued to a maximum density coinciding with peak sporulation. The timing and intensity of sporulation was influenced by the concentration of the inoculum. Seedlings often became systemically infected and this, together with intensity of sporulation, was a good indicator of fungal growth and hence host susceptibility. The severity of disease development on four susceptible lettuce cultivars (Cheshunt Early Giant, Paris White, Trocadero Improved and Crackling Ice) decreased with age and development of the seedlings at inoculation. The reasons for this are discussed.     

Further work on the genetics of race specific resistance in lettuce (Lactuca sativa) to downy mildew (Bremia lactucae)

Data are presented on the segregation of resistance to four races of Bremia lactucae in the F2 progenies of crosses involving 15 resistant and various susceptible lettuce cultivars. Most of these data and those recently published by other workers fit the systematic model for the genetics of race specific resistance to B. lactucae proposed by Crute & Johnson(1976). Seven different dominant resistance genes of major effect were found. There was also evidence of a pair of dominant genes with complementary effect, one of which may sometimes be effective on its own. Two of the genes may be linked and another may have two different alleles for resistance at the same locus. The resistance conferred by these genes is specified in relation to two British, two Dutch and four French races of the fungus. Resistance genotypes are proposed for 16 cultivars.     

Application of Cluster Analysis for Establishment of Genetic Similarity in Gene-For-Gene Host-Parasite Relationships

Genetic similarity and/or dissimilarity among virulence phenotypes of phytopathogenic organisms and generally in gene-for-gene relationships can be exactly expressed by cluster analysis. The present paper deals with the application of a definite hierarchical agglomerative clustering method based on μ-connected clusters as a means for describing genetic structure of virulence in a set of 48 physiological races (isolates) of Bremia lactucae (lettuce downy mildew). A dendrogram and a minimums, panning tree were used to distinguish between virulence phenotypes of B. lactucae. Further application of the methods of numerical taxonomy to phytopathology is briefly discussed.