The incidence and distribution of viruses infecting lettuce, cultivated Brassica and associated natural vegetation in Spain

A virus survey was conducted during the spring and autumn of 2001 and 2002 to determine the presence, prevalence and distribution in Spain of the viruses that are most commonly found infecting lettuce and Brassica worldwide. Crop plants showing virus symptoms from the principal lettuce and Brassica-growing regions of Spain, and some samples of the annual and perennial flora nearby, were tested by enzyme-linked immunosorbent assays using specific commercial antibodies against the following viruses: Alfalfa mosaic virus (AMV), Broad bean wilt virus 1 (BBWV-1), Beet western yellows virus (BWYV), Cauliflower mosaic virus (CaMV), Cucumber mosaic virus (CMV), Lettuce mosaic virus (LMV), Pea seed-borne mosaic virus (PSbMV), Turnip mosaic virus (TuMV) and Tomato spotted wilt virus (TSWV). Samples were also tested with a Potyvirus genus antibody. Virus incidence was much lower in spring than in autumn, especially in 2001. In spring 2002, CMV and LMV were the most prevalent viruses in lettuce, while CaMV was the most important virus present in Brassica crops grown in Navarra, followed by CMV and BWYV. In the autumn, the spectrum of viruses was different; potyviruses were widespread in lettuce grown in Madrid, but TSWV and BWYV were predominant in the Murcia region. The prevalent Potyvirus detected in lettuce fields was LMV, but none of the samples collected were positive for PSbMV or TuMV. In Brassica crops, TSWV was the most abundant in autumn-sown crops, especially in the Navarra region. All of the viruses present in lettuce and Brassica were also frequently detected in their associated natural vegetation at the same time, suggesting that they probably play an important role as virus reservoirs. Sonchus spp. were particularly common and were frequently infected with CMV, LMV and BWYV. Another common species, Chenopodium album, was often infected with TSWV and BWYV. Multiple infections were common, especially in non-crop plants, and the most common combination was BWYV and TSWV. The role of weeds in the epidemiology of viruses that infect lettuce and Brassica crops in Spain is discussed.     

Effects of long-term storage on phytochrome-mediated germination in lettuce seeds

The effects of long-term seed storage on the physiological properties of phytochrome-mediated germination including water uptake, the temperature and light flunnce dependencies of germination and dark germination were studied. The fluenceresponse relationships of the brief irradiation with monochromatic red (660 nm, 7.5 W m⁻²) and far-red (750 nm, 6.6 W m⁻²) light at various times after sowing were also studied. The samples used consisted of three lots of seeds ofLactuca sativa L. cv. MSU-16, which had been harvested in 1976, 1979 and 1985 and stored dry for 9, 6 and 0 years, respectively, in darkness at 23±2 C until the experiments were carried out in July–August, 1985. Seeds with the longer storage periods showed the higher ability to germinate in both continuous darkness and continuous white fluorescent light at 20–30 C. In the seeds stored for 6 or 9 years, red light irradiation for 20 sec given at 15 min or more after sowing at 25 C induced as high a percent germination (85–95%) as those under continuous white fluorescent light. In the freshly harvested seeds, however, germination under continuous white fluorescent light (46%) was considerably lower than the germination induced by the red pulse (97%). Germination of the seeds decreased when the intervals between sowing and a far-red irradiation for 20 sec increased up to 100 min (or 30 min in the freshly harvested seeds). The far-red pulse given later than 100 min (or 6 hr in the freshly harvested seeds) after sowing resulted in an increased germination up to the dark-germination levels with increasing intervals between sowing and the pulse irradiation. Before or at 3 min after sowing, the seeds stored for 6 or 9 years were responsive to the far-red pulse although they were not or hardly responsive to the red pulse, while the freshly harvested seeds were responsive to both the far-red and the red pulses. These data indicate that normal functions of phytochrome completely survived in the dry seeds during storage at 25 C for as long as 6 or 9 years and that these functions are restored into full operation by means of imbibition. The differences in the dependence of germination on the time and fluence of a single pulse of red or far-red light seems to be related to the smaller water content throughout the imbibition in the seeds with the longer storage periods. The greater ability to germinate in the dark indicates the greater amounts of P_FR or the greater responsivity to P_FR, in the seeds with the longer storage periods.     

Differences in the nature of thermodormancy and far-red dormancy in lettuce seeds

Lettuce seeds cv. Noran germinate at 23°C in light as well as in darkness. However dormancy can be induced either by a long exposure (24 h) to far-red radiation or by an exposure of 48–72 h to a temperature of 37°C. The difference in response of these two types of dormant seeds to conditions inducing germination indicate that in both types P_fr is inactivated, but that a dark process required for immediate action of P_fr does not proceed at 37°C as it does during far-red radiation.     

Secondary dormancy (skotodormancy) in seeds of lettuce (Lactuca sativa cv. Grand Rapids) and its release by light, gibberellic acid and benzyladenine

Lettuce seeds (Lactuca sativa L. cv. Grand Rapids) imbibed in darkness at supra-optimal temperatures (23 ± 1°C) develop a secondary dormancy, termed skotodormancy. The seeds first lose their ability to be promoted to germinate by gibberellic acid, and then lose their ability to be promoted by red light. A combination of red light and gibberellic acid will break skotodormancy for longer than either alone, but red light and benzyladenine together are much more effective. Desiccation of skotodormant seeds does not diminish their dormancy. Embryos dissected from skotodormant seeds will germinate, and are as capable of radicle expansion in the osmoticum polyethylene glycol as are newly-imbibed seeds. Hence skotodormancy is a whole seed dormancy and does not reside within the embryo as an inherent block to germination processes, but as an inability to respond to the stimulation of red light or to hormone.     

Fluorescence polarization and spin-label studies of the fluidity of stromal and granal chloroplast membranes

The lipid fluidity of thylakoid membrane regions separated by Yeda press and sonication methods has been investigated using diphenylhexatriene fluorescence polarization measurements and rotational correlation times derived from the ESR spectra of the spin-labels 5-doxyldecane and 12-doxylstearate. According to both techniques, stromal lamellae vesicles with essentially only Photosystem I activity were more fluid than the granal membranes. The differences in lipid fluidity between the two fractions were interpreted in terms of the ratio of the amounts of protein compared to lipid in the membranes. Stromal lamellae fractions contained lower protein/lipid ratios compared with the granal membranes.     

Development of reliable PCR-based markers linked to downy mildew resistance genes in lettuce

Summary Sequence characterized amplified regions (SCARs) were derived from eight random amplified polymorphic DNA (RAPD) markers linked to disease resistance genes in lettuce. SCARs are PCR-based markers that represent single, genetically defined loci that are identified by PCR amplification of genomic DNA with pairs of specific oligonucleotide primers; they may contain high-copy, dispersed genomic sequences within the amplified region. Amplified RAPD products were cloned and sequenced. The sequence was used to design 24-mer oligonucleotide primers for each end. All pairs of SCAR primers resulted in the amplification of single major bands the same size as the RAPD fragment cloned. Polymorphism was either retained as the presence or absence of amplification of the band or appeared as length polymorphisms that converted dominant RAPD loci into codominant SCAR markers. This study provided information on the molecular basis of RAPD markers. The amplified fragment contained no obvious repeated sequences beyond the primer sequence. Five out of eight pairs of SCAR primers amplified an alternate allele from both parents of the mapping population; therefore, the original RAPD polymorphism was likely due to mismatch at the primer sites.     

Molecular analysis of irradiation-induced and spontaneous deletion mutants at a disease resistance locus inLactuca sativa

The major cluster of disease resistance genes in lettuce (Lactuca sativa) contains at least nine downy mildew resistance genes (Dm) spanning a genetic distance of 20 cM and a physical distance of at least 6 Mb. Nine molecular markers that were genetically tightly linked toDm3 were used to analyze nine independent deletion mutants and construct a map of the region surroundingDm3. This analysis identified a linear order of deletion breakpoints and markers along the chromosome. There was no evidence for chromosomal rearrangements associated with the deletions. The region is not highly recombinagenic and the deletion breakpoints provided greater genetic resolution than meiotic recombinants. The region contains a mixture of high- and low-copy-number sequences; no single-copy sequences were detected. Three markers hybridized to low-copy-number families of sequences that are duplicated predominantly close toDm3. This was not true for sequences related to the triose-phosphate isomerase gene; these had been shown previously to be linked toDm3, as well as to two independent clusters ofDm genes, and elsewhere in the genome. Two spontaneous mutants ofDm3 were identified; several markers flankingDm3 are absent in one of these two mutants. The stability of theDm3 region was also studied by analyzing the genotypes of diverse related cultivars. The 1.5 Mb region surroundingDm3 has remained stable through many generations of breeding with and without selection forDm3 activity.     

Conditioning as a factor in parasitoid host plant preference

The relative effects of larval and adult conditioning on Diglyphus isaea host plant preference and subsequent parasitism were investigated. Parasitoid larvae were reared on Chromatomyia syngenesiae on lettuce, chrysanthemum or in isolation, and female parasitoids were exposed to hosts on either lettuce or chrysanthemum. Hosts on lettuce and chrysanthemum were then presented to assess parasitoid preference. Larval and adult D. isaea conditioning only slightly affected the females selection of host plant type on which the host larvae were located. The major effect of conditioning was a reduction in the number of eggs laid after larvae and adults were conditioned on chrysanthemum and particularly when both stages were conditioned on this host plant.     

Biocontrol of Slugs in Protected Lettuce Using the Rhabditid Nematode Phasmarhabditis hermaphrodita

In two experiments, the rhabditid nematode Phasmarhabditis hermaphrodita, a parasite of slugs, was cultured in vitro and applied as a drench to soil at four dose rates (3 108, 1 109, 3 109 and 1 1010 ha-1) 1 or 4 days before planting lettuce seedlings in a polythene tunnel. The effects of the four nematode doses on slug damage during the first 3 weeks after planting and on the numbers of slugs found within and below lettuce plants at harvest were measured. Results were then compared with untreated plots and with plots where methiocarb pellets were applied at the recommended field rate. In the first experiment, methiocarb pellets significantly reduced the percentage of plants damaged by slugs, but the nematode did not. In the second experiment, methiocarb pellets and the second highest dose of nematodes significantly reduced the percentage of plants damaged by slugs. The different effect of the nematode in the two experiments may have resulted from differences in the timing of nematode application and/or differences in the pattern of slug damage between experiments. At the end of the first experiment, the highest two doses of nematodes and methiocarb pellets had significantly reduced the number of slugs found within lettuce plants at harvest and on the soil surface below the plants. At the end of the second experiment, analysis of variance showed no significant effects of any treatment on slug numbers or biomass, but regression analysis showed significant negative relationships between nematode dose and total slug numbers, numbers of Arion ater agg. and biomass of Deroceras reticulatum. In both experiments, increasing nematode dose significantly reduced the numbers of slugs found contaminating the harvested lettuce. At the end of the second experiment, the mean weight of individuals of A. ater agg. increased with rising nematode dose.     

An integrated interspecific AFLP map of lettuce (Lactuca) based on two L. sativa × L. saligna F2 populations

AFLP markers were obtained with 12 EcoRI/ MseI primer combinations on two independent F₂ populations of Lactuca sativa ×Lactuca saligna. The polymorphism rates of the AFLP products between the two different L. saligna lines was 39%, between the two different L. sativa cultivars 13% and between the L. sativa and L. saligna parents on average 81%. In both F₂ populations segregation distortion was found, but only Chromosome 5 showed skewness that was similar for both populations. Two independent genetic maps of the two F₂ populations were constructed that could be integrated due to the high similarity in marker order and map distances of 124 markers common to both populations. The integrated map consisted of 476 AFLP markers and 12 SSRs on nine linkage groups spanning 854 cM. The AFLP markers on the integrated map were randomly distributed with an average spacing between markers of 1.8 cM and a maximal distance of 16 cM. Furthermore, the AFLP markers did not show severe clustering. This AFLP map provides good opportunities for use in QTL mapping and marker-assisted selection. Received: 13 July 2000 / Accepted: 19 January 2001