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.     

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

Callus cultures derived from isogenic lines of the tomato cultivars Moneymaker and Craigella, resistant or susceptible to F. oxysporum f. sp. lycopersici, were inoculated with Fusarium oxysporum f. sp. lycopersici race 1. Fungal growth was restricted on callus derived from resistant plants, after inoculation with a conidial suspension, whereas callus derived from susceptible plants was totally overgrown by the fungus within 7 days. The concentration of the phytoalexin rishitin was significantly higher in the callus culture derived from a resistant tomato line compared with the callus culture from a susceptible line, 2 and 3 days after inoculation with mycelium. The results of the experiments were compared with experiments with whole plants. Rishitin production as well as growth of the fungus was comparable with responses in plant-fungus interaction. Therefore callus culture may be useful in studying the interaction between tomato plants and race 1 of F. oxysporum f. sp. lycopersici.     

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

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

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

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

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

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

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.     

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.     

Interspecific somatic hybridization between lettuce (Lactuca sativa) and wild species L. virosa

Somatic hybrids between cultivated lettuce (Lactuca sativa) and a wild species L. virosa were produced by protoplast electrofusion. Hybrid selection was based on inactivation of L. sativa with 20mM iodoacetamide for 15 min, and the inability of L. virosa protoplasts to divide in the culture conditions used. Protoplasts were cultured in agarose beads in a revised MS media. In all 71 calli were formed and 21 of them differentiated shoots on LS medium containing 0.1mg/l NAA and 0.2mg/l BA. Most regenerated plants exhibited intermediate morphology. These plants were confirmed as hybrids by isoenzyme analysis. The majority of somatic hybrids had 2n=4x=36 chromosomes, and had more vigorous growth than either parent. Hybrids had normal flower morphology, but all were sterile.     

Mitochondrial metabolism of reactive oxygen species

Oxidative stress is considered a major contributor to etiology of both normal senescence and severe pathologies with serious public health implications. Mitochondria generate reactive oxygen species (ROS) that are thought to augment intracellular oxidative stress. Mitochondria possess at least nine known sites that are capable of generating superoxide anion, a progenitor ROS. Mitochondria also possess numerous ROS defense systems that are much less studied. Studies of the last three decades shed light on many important mechanistic details of mitochondrial ROS production, but the bigger picture remains obscure. This review summarizes the current knowledge about major components involved in mitochondrial ROS metabolism and factors that regulate ROS generation and removal. An integrative, systemic approach is applied to analysis of mitochondrial ROS metabolism, which is now dissected into mitochondrial ROS production, mitochondrial ROS removal, and mitochondrial ROS emission. It is suggested that mitochondria augment intracellular oxidative stress due primarily to failure of their ROS removal systems, whereas the role of mitochondrial ROS emission is yet to be determined and a net increase in mitochondrial ROS production in situ remains to be demonstrated.Translated from Biokhimiya, Vol. 70, No. 2, 2005, pp. 246–264.Original Russian Text Copyright © 2005 by Andreyev, Kushnareva, Starkov.This revised version was published online in April 2005 with corrections to the post codes.     

Mitochondrial metabolism of reactive oxygen species

For a long time mitochondria have mainly been considered for their role in the aerobic energy production in eukaryotic cells, being the sites of the oxidative phosphorylation, which couples the electron transfer from respiratory substrates to oxygen with the ATP synthesis. Subsequently, it was showed that electron transfer along mitochondrial respiratory chain also leads to the formation of radicals and other reactive oxygen species, commonly indicated as ROS. The finding that such species are able to damage cellular components, suggested mitochondrial involvement in degenerative processes underlying several diseases and aging.More recently, a new role for mitochondria, as a system able to supply protection against cellular oxidative damage, is emerging. Experimental evidence indicates that the systems, evolved to protect mitochondria against endogenously produced ROS, can also scavenge ROS produced by other cellular sources. It is possible that this action, particularly relevant in physio-pathological conditions leading to increased cellular ROS production, is more effective in tissues provided with abundant mitochondrial population. Moreover, the mitochondrial dysfunction, resulting from ROS-induced inactivation of important mitochondrial components, can be attenuated by the cell purification from old ROS-overproducing mitochondria, which are characterized by high susceptibility to oxidative damage. Such an elimination is likely due to two sequential processes, named mitoptosis and mitophagy, which are usually believed to be induced by enhanced mitochondrial ROS generation. However, they could also be elicited by great amounts of ROS produced by other cellular sources and diffusing into mitochondria, leading to the elimination of the old dysfunctional mitochondrial subpopulation.     

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.     

Biodiversity of Lactuca aculeata germplasm assessed by SSR and AFLP markers,and resistance variation to Bremia lactucae

In total, seventy two Lactuca aculeata and three Lactuca serriola samples originating from natural populations of these species in Turkey, Jordan, and Israel were analysed by eight microsatellite and 287 amplified fragment length polymorphism (AFLP) markers. Neighbor–Network and Bayesian clustering were used for visualisation of the differences among the analysed L. aculeata and L. serriola samples, and to confirm hybrid origin (L. aculeata × L. serriola) of three samples (343-8A, 343-8B, 54/07) previously indicated by their morphological traits. Molecular data reflect the geographical origin, i.e., the clustering of samples according to their country of origin. Samples from neighbouring parts of Jordan and Israel expressed similar genetic characteristics, indicating the possibility of migration or artificial introduction of plant material. Forty-one L. aculeata samples were screened for their response to five Bremia lactucae races (Bl: 17, Bl: 18, Bl: 24, Bl: 27, and Bl: 28). Susceptible reactions of L. aculeata prevailed. L. aculeata samples were most frequently susceptible to races Bl: 18, Bl: 24, Bl: 27, Bl: 28; and least susceptible to Bl: 17. No highly efficient source of resistance was detected; however, race-specific reaction patterns were frequently recorded, indicating the possible presence of some race-specific resistance factors/genes in the studied samples of L. aculeata. Conservation and exploitation of this material in lettuce breeding is discussed.     

The behaviour of Bretnia lactucae on cultivars of Lactuca sativa and on other composites

The spores of an isolate of Bremia lactucae obtained from lettuce germinated equally well on Cellophane and on the surfaces of all plant species inoculated. The fungus penetrated the epidermal cells of both host and non-host plants but extensively invaded only certain lettuce cultivars. Many of the species and cultivars inoculated showed no macroscopic symptoms but exhibited a hypersensitive response to invasion that was limited to a few cells and only visible microscopically. However, the hypersensitive response was not always so limited and in some species, fungal development and associated host cell necrosis was more extensive and visible macroscopically. In such instances, limited sporulation occasionally occurred. On some lettuce cultivars symptoms developed more slowly than on fully susceptible ones and less seedlings became diseased. This was due to the heterogeneity of the inoculum with respect to its ability to overcome certain race specific resistance genes. Such cultivars reacted hypersensitively to the majority, but not all, of the fungal propagules applied and the observed effect was thus due to a lower effective inoculum concentration. Without due care, both these types of ‘intermediate’ reaction could easily be misinterpreted as race non-specific resistance particularly under field conditions. These observations emphasized the problems of categorizing cultivars and species rigidly as resistant or susceptible when inoculum could be heterogenous or when resistance reactions allowed some macroscopic symptom development.     

Cell death and reactive oxygen species metabolism during accelerated ageing of soybean axes

In this paper, Glycine max L. seeds under accelerated ageing condition (40°C and 100% relative humidity) were used as experimental material to study the relationships between seed viability and cell death, production and scavenging of reactive oxygen species (ROS) during accelerated ageing. Water content of seeds gradually increased, while the final germination percentage, germination rate of seeds and fresh weight of seedlings produced decreased with increasing accelerated ageing time. The accelerated ageing time (T ₅₀) when final seed germination decreased to 50% was about 10.5 days. During the period of accelerated ageing, the viability of root cells was lost gradually as manifested by the increase in staining with Evans blue. The respiration rate of seeds, ^·O₂ production rate, and H₂O₂ content of axes increased, peaked at the 10 days of accelerated ageing, and then decreased. Activities of superoxide dismutase, ascorbate peroxidase, catalase, and glutathione reductase of axes decreased; and malondialdehyde contents of axes markedly increased. A sceme to explain relationships between seed vigor, cell death, and production and scavenging of ROS during accelerated ageing was suggested. This text was submitted by the authors in English.     

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.     

Root architecture in cultivated and wild lettuce (Lactuca spp.)

Root architecture is described for intact root systems of cultivated (Lactuca sativa L.) and wild (L. serriola L.) lettuce, grown for 5 to 6 weeks in greenhouse pot and cylinder experiments in coarse-textured soil. L. sativa cv. Salinas and a sclinas line of L. serriola attained the same biomass at 4 to 6 weeks after planting. Root biomass allocation was also similar, but root architecture differed. In the top zone along the tap root (0 to 5 cm), Salinas tended to produce more laterals, a greater total root length, and more external links (segments that originate at a branch point and end in a meristem) than wild lettuce. In the 5 to 55cm zone of the tap root, these measures were greater in the wild species. These patterns of root structure were generally corroborated by a second cylinder study with a different pair of L. sativa and L. serriola. Regressions of root structural traits were made against total root dry weight as a means to compare root architecture independently of the size of the root system. Regressions demonstrated that production of root segments differed between the two species; for example the slope for the regression of summed external link length in the top 0 to 5 cm with total root dry weight was significantly higher for Salinas, indicating that the rate of construction in the top 0 to 5cm was greater for cultivated than for wild lettuce. Yet, from 5 cm depth to the tap root tip, the rate of construction of external link length was greater in L. serriola. For many of these types of regression, r² and mean slope ± SE suggested that more variation occurred in cultivated than wild lettuce, yet genetic heterogeneity was probably low within the studied taxa. Inadvertent selection may have occurred in the breeding of cultivated lettuce varieties for increased root growth in the surface zone where water and fertilizer are applied, and for greater plasticity in construction of root segments, which might maximize the efficiency of exploitation of soil moisture and nutrients.     

Microsatellite fingerprinting in lettuce (Lactuca sativa L.) and wild relatives

Southern hybridisation with a single microsatellite probe, (TCT)₁₀, sufficed to discriminate between a representative set of cultivars/accessions of lettuce, Lactuca sativa L., and its wild relatives L. serriola, L. saligna and L. virosa. Variability within cultivars was tested in a relatively modern cultivar (Hector), where no variation was found, and in an older and morphologically more variable cultivar (Madrilene), where heterogeneity was observed in the TCT fingerprint. (TCT)₁₀ fingerprinting should be useful for variety identification and homogeneity testing in lettuce. Received: 25 July 1997 / Revision received: 5 August 1997 / Accepted: 30 August 1997     

Generation of reactive oxygen species during mouse hepatic microsomal metabolism of cannabidiol and cannabidiol hydroxy-quinone

We investigated whether cannabidiol (CBD) and cannabidiol hydroxy-quinone (CBDHQ) generate reactive oxygen species (ROS) during metabolism with mouse hepatic microsomes. CBD and CBDHQ (91.5 microM) significantly suppressed lipid peroxidation in the mouse hepatic microsomes. CBDHQ also significantly decreased NADH-cytochrome b5 reductase (fp1) activity by 25% of the control activity in the hepatic microsomes, and tended to increase NADPH-cytochrome c (P450) reductase (fp2) activity. CBDHQ also significantly inhibited superoxide dismutase and catalase activities in mouse hepatic 105,000 xg supernatant. Moreover, CBDHQ significantly increased glutathione reductase activity and significantly inhibited NAD(P)H-quinone reductase activity. CBD exhibited similar effects on these enzymes, except that cannabinoid significantly inhibited glutathione reductase activity in mouse hepatic 105,000 xg supernatant. These results suggest that CBDHQ is easily converted to the semiquinone form rather than the hydroquinone form. It was also suggested that CBDHQ and CBD were capable of generating ROS as superoxide anion radicals during their metabolism with mouse hepatic microsomes or with purified fp2 by electron spin resonance spin trapping methods with 5,5-dimethyl-1-pyrroline-N-oxide. The present results suggest that CBDHQ formed during hepatic microsomal metabolism of CBD is capable of generating ROS and inducing cell toxicity.     

Effects of reactive oxygen species on eicosanoid metabolism in human endothelial cells.

The influence of reactive oxygen species (H2O2 was used as model substance) on the formation and release of PGI2 and TXA2 by cultured human endothelial cells was analyzed. In the presence of H2O2 concentrations which did not induce a general cell damage (analyzed by estimation of the cellular concentration of energy rich phosphates and extent of lipid peroxidation), the formation of both eicosanoids exhibited a sigmoidal shape with respect to time. Increasing H2O2 concentration shortened the half time of PGI2 and TXA2 production. The maximum rates of PGI2 and TXA2 formation were separated by a delay of the TXA2 production. The ratio of PGI2 and TXA2 formation was 100 to 1 at the time of maximum PGI2 formation and 1-2 to 1 at the time of maximum TXA2 formation. This effect of reactive oxygen species could contribute to the reduction of the protective function of the endothelium in hemostasis and vascular tone. Using antioxidants, the modulating function of reactive oxygen species on the eicosanoid metabolism in endothelial cells was verified.     

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.