Cytopathology of Lettuce Mosaic Virus-infected Lettuce Seeds and Seedlings

The ultrastructure of lettuce mosaic potyvirus (LMV)-infected lettuce seeds and seedlings was studied by transmission electron microscopy. Conventional thin-section electron microscopy and immunogold cytochemistry were both successfully employed to study the location of LMV in embryonic and non-embryonic seed parts. LMV particle aggregates and cytoplasmic “pinwheel” inclusions characteristic of potyviruses were observed throughout the embryonic tissues (radicle, hypocotyl and cotyledon) of infected lettuce seeds and seedlings, and also in the non-embryonic endosperm layer. LMV particles, but not inclusions, were also located in the non-embryonic pericarp layer.     

Cytopathology of Mature Ovaries from Lettuce Plants Infected by Lettuce Mosaic Potyvirus

The location of lettuce mosaic potyvirus (LMV) in mature ovaries of lettuce plants ( Lactuca sativa ) was studied by conventional thin-section electron microscopy and immunogold cytochemistry. Flexuous filamentous particles and inclusion bodies characteristic of LMV infection were observed in the integumentary tapetum, integument and ovary wall of the mature ovary but not in the embryo sac.     

Temporal and spatial spread of Lettuce mosaic virus in lettuce crops in central Spain: factors involved in Lettuce mosaic virus epidemics

Lettuce mosaic virus (LMV) is transmitted by aphid vectors in a nonpersistent manner as well as by seeds. The virus causes severe disease outbreaks in commercial lettuce crops in several regions of Spain. The temporal and spatial patterns of spread of LMV were studied in autumn 2002 in the central region of Spain. Symptomatic lettuce (var. Cazorla) plant samples were collected weekly, first at the seedling stage from the greenhouse nursery and later outdoors after transplantation. The exact position of symptomatic plants sampled in the field was recorded and then material was tested by enzyme‐linked immunosorbent assay to assess virus infection. Cumulative spatial data for infected plants at different growth stages were analysed using spatial analysis by distance indices. For temporal analysis, the monomolecular, Gompertz, logistic and exponential models were evaluated for goodness of fit to the entire set of disease progress data obtained. The results indicated that the disease progress curve of LMV epidemics in the selected area is best described by a Gompertz model and that the epidemic follows a polycyclic disease progression. Our data suggest that secondary cycle of spread occurs when noncolonising aphid species land on the primary infected plants (probably coming from infected seed) and move to adjacent plants before leaving the crop. The role of weeds growing close to lettuce fields as potential inoculum sources of virus and the aphid species most likely involved in the transmission of LMV were also identified.     

The Response of Lettuce Cultivars against Two Lettuce mosaic virus isolates,One Able to Systemically Infect the Resistant Cultivar Salinas 88

The response of seven lettuce cultivars to two geographically different Lettuce mosaic virus (LMV) isolates (LMV‐A, LMV‐T) was statistically evaluated based on infection rate, virus accumulation and symptom severity in different time trials. LMV‐A is characterized by the ability to systemically infect cv. Salinas 88 (mo1²‐carrying resistant cultivar), and inducing mild mosaic symptoms. Among lettuce cultivars, Varamin (a native cultivar) similar to cv. Salinas showed the most susceptibility to both LMV isolates, whereas another native cultivar, Varesh, was tolerant to the virus with minimal viral accumulation and symptom scores, significantly different from other cultivars at P < 0.05. LMV‐A systemically infects all susceptible lettuce cultivars more rapidly and at a higher rate than LMV‐T. This isolate accumulated in lettuce cultivars at a significantly higher level, determined by semiquantitative ELISA and induced more severe symptoms than LMV‐T isolate at 21 dpi. This is the first evidence for a LMV isolate with ability to systemically infect mo1²‐carrying resistant cultivar of lettuce from Iran. In this study, accumulation level of LMV showed statistically meaningful positive correlation with symptom severity on lettuce plants. Based on the results, three evaluated parameters differed considerably by lettuce cultivar and virus isolate.     

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.     

Lettuce mosaic virus: from pathogen diversity to host interactors

TAXONOMY: Lettuce mosaic virus (LMV) belongs to the genus Potyvirus (type species Potato virus Y) in the family Potyviridae. PHYSICAL PROPERTIES: The virion is filamentous, flexuous with a length of 750 nm and a width of 15 nm. The particles are made of a genomic RNA of 10 080 nucleotides, covalently linked to a viral-encoded protein (the VPg) at the 5' end and with a 3' poly A tail, and encapsidated in a single type of capsid protein. The molecular weight of the capsid protein subunit has been estimated electrophoretically to be 34 kDa and estimated from the amino acid sequence to be 31 kDa. GENOME ORGANIZATION: The genome is expressed as a polyprotein of 3255 amino-acid residues, processed by three virus-specific proteinases into ten mature proteins. HOSTS: LMV has a worldwide distribution and a relatively broad host range among several families. Weeds and ornamentals can act as local reservoirs for lettuce crops. In particular, many species within the family Asteraceae are susceptible to LMV, including cultivated and ornamental species such as common (Lactuca sativa), prickly (L. serriola) or wild (L. virosa) lettuce, endive/escarole (Cichorium endiva), safflower (Carthamus tinctorius), starthistle (Centaurea solstitialis), Cape daisy (Osteospermum spp.) and gazania (Gazania rigens). In addition, several species within the families Brassicaceae, Cucurbitaceae, Fabaceae, Solanaceae and Chenopodiaceae are natural or experimental hosts of LMV. Genetic control of resistance to LMV: The only resistance genes currently used to protect lettuce crops worldwide are the recessive genes mo1(1) and mo1(2) corresponding to mutant alleles of the gene encoding the translation initiation factor eIF4E in lettuce. It is believed that at least one intact copy of eIF4E must be present to ensure virus accumulation. TRANSMISSION: LMV is transmitted in a non-persistent manner by a high number of aphid species. Myzus persicae and Macrosiphum euphorbiae are particularly active in disseminating this virus in the fields. LMV is also seedborne in lettuce. The effectiveness of LMV transmission depends on the cultivar and the age of the seed carrier at the inoculation time. SYMPTOMS: The characteristic symptoms on susceptible lettuce cultivars are dwarfism, mosaic, distortion and yellowing of the leaves with sometimes a much reduced heart of lettuce (failure to form heads). The differences in virus strains, cultivars and the physiological stage of the host at the moment of the attack cause different symptom severity: from a very slight discoloration of the veins to severe necrosis leading to the death of the plant.     

Ultrastructural Impact of Tobacco Rattle Virus on Tobacco and Pepper Ovary and Anther Tissues

The one‐third of plant viruses are seed transmitted, and this has significant economic consequences. Tobacco rattle virus (TRV), belonging to the genus Tobravirus and family Virgaviridae, has one of the widest host range of any known plant viruses. TRV infects vegetative organ and effects seed and pollen development that results in a decrease in crop yield. The mechanisms by which Tobravirus is transmissible to seeds are still poorly understood. The presence of the virus in pollen grains and inside ovaries is linked with seed transmission and can have effects on virus particles' transport during the pollination and fertilization process. This paper focuses on the significant impact of TRV on pepper and tobacco anthers and ultrastructure changes in ovaries. The presence of two types of TRV particles in ovary wall parenchyma and vascular tissues as well as in placenta cells was demonstrated via ultrastructural analysis. For the first time, the regular inclusion of virus particles was reported in both ovule integuments and nucellus parenchyma cells. Immunolocalization of TRV capsid proteins indicated the deposition of TRV CP epitope in ovary vascular bundles and in placenta cells. Moreover, the presence of virus particles was demonstrated inside pepper seeds in endothelium and integument parenchyma layers as well as on the embryo cell wall. Virus particles were found not only on the surface of pollen grains but also inside pepper pollen protoplasts in mature anthers. Also, this is the first time where TRV particles are reported in both differentiated endothecium cells and the remaining tapetum cells. Moreover, the detection of TRV capsid protein epitope in tobacco and pepper vascular anther tissues as well as in tapetum and endothecium cells was correlated with TRV distribution in infected anthers. Demonstrated analyses indicated that pollen grains and ovaries with ovules as well as could be a natural source of TRV transmission.     

Effects of Abiotic and Biotic Stresses on the Internalization and Dissemination of Human Norovirus Surrogates in Growing Romaine Lettuce

Human norovirus (NoV) is the major causative agent of fresh-produce-related outbreaks of gastroenteritis; however, the ecology and persistence of human NoV in produce systems are poorly understood. In this study, the effects of abiotic and biotic stresses on the internalization and dissemination of two human NoV surrogates (murine norovirus 1 [MNV-1] and Tulane virus [TV]) in romaine lettuce were determined. To induce abiotic stress, romaine lettuce was grown under drought and flood conditions that mimic extreme weather events, followed by inoculation of soil with MNV-1 or TV. Independently, lettuce plants were infected with lettuce mosaic virus (LMV) to induce biotic stress, followed by inoculation with TV. Plants were grown for 14 days, and viral titers in harvested tissues were determined by plaque assays. It was found that drought stress significantly decreased the rates of both MNV-1 and TV internalization and dissemination. In contrast, neither flood stress nor biotic stress significantly impacted viral internalization or dissemination. Additionally, the rates of TV internalization and dissemination in soil-grown lettuce were significantly higher than those for MNV-1. Collectively, these results demonstrated that (i) human NoV surrogates can be internalized via roots and disseminated to shoots and leaves of romaine lettuce grown in soil, (ii) abiotic stress (drought) but not biotic stress (LMV infection) affects the rates of viral internalization and dissemination, and (iii) the type of virus affects the efficiency of internalization and dissemination. This study also highlights the need to develop effective measures to eliminate internalized viruses in fresh produce.     

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.     

Occurrence and distribution of lettuce mosaic disease in Tehran province from Iran

Lettuce mosaic virus (LMV) Cucumber mosaic virus (CMV) and Tomato spotted wilt virus (TSWV) were identified in fields of Tehran province. In this study 452 leaf samples were collected from the fields throughout the Tehran province during 2002 and 2003. Distribution of Lettuce mosaic virus (LMV), Cucumber mosaic virus (CMV), Tomato spotted wilt virus (TSWV)and Arabis mosaic virus (ArMV) was determined with DAS-ELISA. Percentage of single Infection to LMV. CMV or TSWV was 20.58, 15.93 and 9.96% respectively. Also 15.28% of samples were co- infected with LMV+CMV, 8.19% with LMV+TSMV and 7.74% with CMV+TSWV. 4.65% of samples were Infected to all of these three viruses. LMV was found in 48.69%, CMV in 43.59% and TSWV in 30.54% of samples totally. Therefore LMV is major dominant agent of lettuce mosaic disease in Tehran province. This is the first report of occurrence of TSWV on lettuce in Iran and first report of CMV and LMV in Tehran province.     

Transmission Studies with Cucumber Green Mottle Mosaic Virus

Cucumber green mottle mosaic virus may spread in bottlegourd under field conditions through soil contaminated with infected plant debris followed by contact. No seed transmission was noticed in bottlegourd (Lagenaria siceraria) or vegetable marrow (Cucurbita pepo) although pollen grains and cotyledons from infected bottlegourd flowers or seeds, respectively, contained negliginle amounts of virus. Cucumber leaf beerles (Raphidopalpa fevicollis) are probable vectors since their regurgitated fluid and excretes contained infective virus particles. No vector fungi were found in the soil around infected bottlegourd plants.     

Interveinal Yellowing Caused by Tomato infectious chlorosis virus in Lettuce and Escarole in Southern Italy

In 2005 and 2006, a severe disease of lettuce and escarole, characterized by interveinal yellowing of the leaves, was observed in the Calabria region (Southern Italy). Samples collected were positive in RT‐PCR assay when specific primers for Crinivirus were used. The identity of the virus was determined by using Tomato infectious chlorosis virus (TICV)‐specific primers and probe. An amplicon of the same size (501 bp) as that from TICV‐infected tomato controls, corresponding to the partial Hsp70 viral gene, was obtained from 36 of 40 lettuce samples and from 19 of 24 escarole samples tested in 2005. The sequences obtained had 99–100% nucleotide sequence identity with other TICV sequences. In an extensive survey carried out in 2006 with TICV‐specific probe, 92% of symptomatic lettuce and 89% of escarole samples were infected with TICV. Adults of Trialeurodes vaporariorum, harvested from symptomatic lettuce and escarole plants, transmitted the virus to healthy plants of the same species. This is the first evidence of a disease caused by TICV in lettuce in Italy and the first record of TICV infection in escarole.     

The eukaryotic translation initiation factor 4E controls lettuce susceptibility to the Potyvirus Lettuce mosaic virus

The eIF4E and eIF(iso)4E cDNAs from several genotypes of lettuce (Lactuca sativa) that are susceptible, tolerant, or resistant to infection by Lettuce mosaic virus (LMV; genus Potyvirus) were cloned and sequenced. Although Ls-eIF(iso)4E was monomorphic in sequence, three types of Ls-eIF4E differed by point sequence variations, and a short in-frame deletion in one of them. The amino acid variations specific to Ls-eIF4E(1) and Ls-eIF4E(2) were predicted to be located near the cap recognition pocket in a homology-based tridimensional protein model. In 19 lettuce genotypes, including two near-isogenic pairs, there was a strict correlation between these three allelic types and the presence or absence of the recessive LMV resistance genes mo1(1) and mo1(2). Ls-eIF4E(1) and mo1(1) cosegregated in the progeny of two separate crosses between susceptible genotypes and an mo1(1) genotype. Finally, transient ectopic expression of Ls-eIF4E restored systemic accumulation of a green fluorescent protein-tagged LMV in LMV-resistant mo1(2) plants and a recombinant LMV expressing Ls-eIF4E degrees from its genome, but not Ls-eIF4E(1) or Ls-eIF(iso)4E, accumulated and produced symptoms in mo1(1) or mo1(2) genotypes. Therefore, sequence correlation, tight genetic linkage, and functional complementation strongly suggest that eIF4E plays a role in the LMV cycle in lettuce and that mo1(1) and mo1(2) are alleles coding for forms of eIF4E unable or less effective to fulfill this role. More generally, the isoforms of eIF4E appear to be host factors involved in the cycle of potyviruses in plants, probably through a general mechanism yet to be clarified.     

The effect of virus infection on morphology and protein components of pollen grains

Morphology of pollen grains collected from healthy and virus infected plants ofChenopodium quinoa L.,Chenopodium album L. andNicotiana tabacum L. cv. Samsun was investigated using scanning electron microscopy (SEM). Pollen grains from tobacco plans infected with tobacco mosaic virus (TMV) were smaller, with rounded shape and conspicuous deformation of aperture unlike oval and smooth pollen grains from healthy plants. No morphological alterations were observed inC. quinoa andC. album plants infected with TMV and cucumber mosaic virus (CMV). Polyacrylamide gel electrophoresis of pollen proteins revealed substantial quantitative and qualitative differences in protein components of pollen grains collected from healthy and virus infected plants     

Male-sterile mutation alters Zea m 1 (β-expansin 1) accumulation in a maize mutant

gaMS-2 is a gametophytic male-sterile mutant of maize, with sterile pollen grains developmentally blocked at the binucleate stage. To characterise differentially expressed proteins in gaMS-2 pollen, we compared protein profiles of anthers and mature pollen from heterozygous GaMS-2/gaMS-2 plants and wild type (wt) plants by two-dimensional electrophoresis (2-DE). A basic protein present at a greatly reduced level in GaMS-2/gaMS-2 anthers was subsequently identified by tandem mass spectrometry as Zea m 1 (a glycoprotein of 31 kDa), the major group-1 allergen of maize pollen and a member of the -expansin 1 family. Moreover, we show that Zea m 1 can be deglycosylated by peptide N-glycosidase F. After deglycosylation, four major isoforms—Zea m 1a (more acetic), Zea m 1b, Zea m1c and Zea m 1d (more basic)—can be discriminated in wt anther in 2-DE immunoblots probed with a monoclonal antibody against the group-1 pollen allergen, whereas all the isoforms, especially Zea m 1a, exist at reduced levels in GaMS-2/gaMS-2 anthers. Furthermore, the reduced Zea m 1 accumulation in the mutant appears to occur in immature pollen but not in anther sporophytic tissues. Finally, we separated sterile pollen grains (at the mononucleate stage) from fertile ones using 42% Percoll solution, and found that Zea m 1 is barely detectable in sterile pollen grains. Together, our results indicate that a reduced Zea m 1 level is associated with the sterile phenotype of gaMS-2.W. Wang and M. Scali contributed equally to this study     

马铃薯Y病毒组病毒高产量提取方法的建立

本文报道了高产量提取芜菁花叶病毒（ＴｕＭＶ）、莴苣花叶病毒（ＬＭＶ）、芋花叶病毒（ＤＭＶ）和大豆花叶病毒（ＳＭＶ）的提取方法。本方法通过使用高盐浓度的磷酸盐缓冲液以及在缓冲液中加入氯化镁和脲,并用ＴｒｉｔｏｎＸ－１００作为澄清剂,替代常规使用的氯仿和正丁醇,成功的提取到了大量病毒粒子,上述四种病毒提取的得率分别是ＴｕＭＶ为１７３．３ｍｇ／ｋｇ病叶,ＬＭＶ为９６ｍｇ／ｋｇ病叶,ＳＭＶ为１９９．２ｍｇ／ｋｇ病叶,ＤＭＶ为１７６．６ｍｇ／ｋｇ病叶。     

Precocious pollen germination in Arabidopsis plants with altered callose deposition during microsporogenesis

Pollination is essential for seed reproduction and for exchanges of genetic information between individual plants. In angiosperms, mature pollen grains released from dehisced anthers are transferred to the stigma where they become hydrated and begin to germinate. Pollen grains of wild-type Arabidopsis thaliana do not germinate inside the anther under normal growth conditions. We report two Arabidopsis lines that produced pollen grains able to in situ precociously germinate inside the anther. One of them was a callose synthase 9 (cs9) knockout mutant with a T-DNA insertion in the Callose Synthase 9 gene (CalS9). Male gametophytes carrying a cs9 mutant allele were defective and no homozygous progeny could be produced. Heterozygous mutant plants (cs9/+) produced approximately 50% defective pollen grains with an altered male germ unit (MGU) and aberrant callose deposition in bicellular pollen. Bicellular pollen grains germinated precociously inside the anther. Another line, a transgenic plant expressing callose synthase 5 (CalS5) under the CaMV 35S promoter, also contained abnormal callose deposition during microsporogenesis and displaced MGUs in pollen grains. We also observed that precocious pollen germination could be induced in wild-type plants by incubation with medium containing sucrose and calcium ion and by wounding in the anther. These results demonstrate that precocious pollen germination in Arabidopsis could be triggered by a genetic alteration and a physiological condition.     

Properties of cherry ring mottle, a distinctive strain of prune dwarf virus

A virus was transmitted from sweet cherry trees with cherry ring mottle disease to cucurbitaceous plants with the aid of liquid nitrogen, caffeine or polyethylene glycol, which were more effective than sodium diethyldithiocarbamate, polyvinylpyrrolidone and other materials used in sap-transmission studies. The virus was transmitted from dormant buds, petals, young leaves, anthers and pollen. Of 172 herbaceous species and varieties tested, twenty-five (thirteen spp.) became infected with virus. Ribes nigrum and peach seedlings were also infected. Of the systems produced in woody plants, those in Italian Prune resembled the symptoms caused by prune dwarf virus, but those in other Prunus spp. did not. In cucumber extracts the thermal inactivation point was between 40 and 44 d?C; dilution end-point was 1/16 to 1/32 and longevity in vitro 8–16 h. Formaldehyde (4%) fixed the particles and preserved their shape for electron microscopy; they were spherical, with a mean diameter of 24 nm. The virus reacted with prune dwarf virus antiserum but differed in several ways from other isolates     

Confocal Microscopic Observations on Actin Filament Distribution in Lily Pollen Protoplasts

Actin filaments (F-actin) were localized in the isolated pollen protoplasts of lily using TRITC-phalloidin probe and confocal microscopy. Two kinds of pollen protoplasts were examined: one from pollen grains of non-dehiscent anthers(referred to as ‘nearly mature’ pollen); and the other from pollen grains of just dehiscent anthers(referred to as ‘just mature’ pollen). In the cytoplasm of the pollen protoplasts of the ‘nearly mature’ pollen there was a very well organized actin network made up of thick actin bundles. Two types of bundle connections were seen in the network; namely ‘branch’ connections and 'junction' connections. The ‘branch’ connection (or branching points) was formed due to branching or merging of bundies. The ‘junction’ connection (or 'junction' point) had two or more bundles associated with it. Some of the ‘junction’ points might be actin filament organization: centres. The generative cell in iht pollen protoplasts of the ‘nearly mature’ pollen also contained an actin network. But this network was structurally quite loose and the pundles made up the network were short and thick. In the cytoplasm of the pollen protoplasts of the ‘just mature’ pollen the actin net work was more densely packed. The bundles made up the network were also thinner. The actin network in the generative cell was, however, less densely packed. If the pollen protoplasts from both the ‘nearly mature’ and the 'just mature' pollen grains were transferred from a B5 medium into a Brewbaker and Kwack medium supplemented with sucrose, protoplasts rapidly (i.e. within 2 to 3 hours) developed vacuoles and transvacuolar strand. In these va cuolated protoplasts the vegetative nucleus andthe generative cell became tightly surrounded by a new actin network. In the transvacuolar strands there were numerous actin bundles. The “ends” of some of these bundles appeared to be tightly attached to the protoplast membrane indicating that some kind of structures might be present in the protoplast membrane for actin filament attachment.