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.     

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.     

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.     

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.     

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.     

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.     

Frequency and geographical distribution of specific virulence factors in Bremia lactucae populations in England from 1973 to 1975

Samples of lettuces infected with Bremia lactucae were collected from seven areas of England during 1973-5. These were tested under controlled environmental conditions on a range of differentially resistant cultivars to determine the frequency of virulence (V) factors in the pathogen population. In most cases the results could be accommodated by the relationship advanced by Crate & Johnson (1976). A virulence phenotype was determined for each isolate and individual virulences within isolates were identified as present at high or low frequencies. The most common V factors were V3, V4, V6 and V8 which reflected the resistance (R) factor complement of lettuce cultivars currently in commercial production. There were few gross differences in the geographical distribution of virulence factors between the seven areas. In the West Central region, however, which encompasses the Evesham and Lancashire production areas, results indicated that the use of cultivars containing resistance (R) factors 2 and 8 respectively would have provided protection from the disease during 1974. There were differences in virulence combinations at the holding or locality level within each large area, which could be exploited by growers providing testing was done in their particular locality. Universally susceptible cultivars and those with only limited resistance factors were often found to be infected with B. lactucae populations which contained large numbers of V-factors. It might have been expected that these pathogen populations would have been restricted to cultivars with large numbers of R-factors. This finding conflicts with the suggestion that pathogen populations with complex virulence patterns are ‘unfit’ as compared with those with more simple virulence components.     

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.     

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.     

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.     

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.     

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).     

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.     

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.     

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.     

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.     

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.     

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.