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.     

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.     

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.     

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.     

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.     

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.     

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.     

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).     

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.     

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).     

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.     

Distribution,Host Range and Disease Severity of Pseudoperonospora cubensis on Cucurbits in the Czech Republic

Cucurbit downy mildew, caused by Pseudoperonospora cubensis, is a major cucumber disease in the Czech Republic. Disease prevalence, host range and disease severity were evaluated from 2001 to 2009. The geographical distribution of P. cubensis was assessed on ca 80–100 locations per year in two main regions of the Czech Republic (central and southern Moravia, and eastern, northern and central Bohemia). Infection by P. cubensis was observed primarily on cucumber (Cucumis sativus) but only on the leaves. During the study, disease prevalence ranged from 66 to 100%. The majority of C. sativus crops were heavily infected at the end of the growing season (second half of August). Generally, P. cubensis was present at high or very high disease severity. The loss of foliage results in the reduction in the quality and quantity of marketable yield of fruit. Pseudoperonospora cubensis was widespread across the whole area of the Czech Republic studied. Very rarely, infection was recorded in muskmelon (Cucumis melo) and Cucurbita moschata. Of other pathogens, the most frequently recorded was the cucurbit powdery mildew (Golovinomyces cichoracearum and Podosphaera xanthii).     

Morphological variability of achenes of some European populations of Lactuca serriola L.

This study focuses on the morphological variability of Lactuca serriola achenes in relation to eco-geographic features. Fifty L. serriola populations from four European countries, Czech Republic, Germany, the Netherlands and United Kingdom, were studied. Five morphological characters of the achenes - length and width of achene, length/width index, length of beak, and number of ribs - were evaluated. Significant differences exist in achene morphology of two leaf forms of L. serriola, forma serriola and forma integrifolia. Achenes of f. serriola are shorter, thinner, shorter beaked, lower length/width index, and higher number of ribs compared to f. integrifolia. There was significant variation in the measured characters. Statistical analysis indicated that achene length and width increased along an east-west transect from 2.95 to 3.35 mm and 0.93 to 1.00 mm, respectively. Mean beak length had a similar trend with the exception of German achenes. They had shorter beaks than achenes originating from the Czech Republic with 4.38 and Germany with 4.33 mm. The same trend was evident for L/W index from Czech with a ratio of 3.21 and Germany with 3.14. The number of ribs increased from east to the west in continental Europe, whereas the lowest number of ribs was recorded in achenes collected in Czech with 10.89 and the UK with 10.59. Achene morphology was significantly correlated with three eco-geographic features; longitude, latitude, soil texture of the habitats. The other eco-geographic factors, altitude and population size, did not significantly correlate with the studied characters of L. serriola achenes.     

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.     

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.     

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

Specificity 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.     

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.     

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.