Identification and Partial Characterisation of <Emphasis Type="Italic">Lettuce big-vein associated virus</Emphasis> and <Emphasis Type="Italic">Mirafiori lettuce big-vein virus</Emphasis> in Common Weeds Found Amongst Spanish Lettuce Crops and their Role in Lettuce Big-vein Disease Transmission

The potential role of 10 frequently occurring weed species found amongst Spanish lettuce crops as host plants for the two viruses associated with the lettuce big-vein disease, Lettuce big-vein associated virus (LBVaV) and Mirafiori lettuce big-vein virus (MLBVV), was studied. The results showed that both viruses can infect naturally growing Sonchus oleraceus (common sowthistle) plants, the unique susceptible species detected among the analysed weeds. The sequences of the coat protein (CP) genes of the LBVaV and MLBVV isolates recovered from S. oleraceus plants were determined. Phylogenetic studies revealed a very close relationship between the CP sequences from these weed isolates and those from Spanish lettuce. Moreover, we showed that S. oleraceus can act as a source of lettuce infection by means of Olpidium brassicae, the vector fungus of both viruses.     

Die Breitadrigkeit des Salats — ein Komplex aus zwei Viren

Since many years lettuce big-vein disease (LBVD) occurring in lettuce (Lactuca sativa) and endivia (Cichorium endivia) is a well known disease. It is widely spread all over the world and can cause important economical losses. For more than 20 years the lettuce big-vein virus (LBVV) was thought to be the causal agent. New results indicate that another virus, named “Mirafiori lettuce big-vein ophiovirus” (MLBVV), is responsible for the typical symptoms. Mostly both viruses are detected together in diseased plants. But also the presence of only one virus can be observed as shown in a sample which was sent to the Bavarian State Research Center for Agriculture (LfL), Freising, in the spring of 2005. In further investigations at the German Federal Authority and Federal Research Centre (BBA), Braunschweig, only the MLBVV was found. Like any other virus disease LBVD cannot be controlled directly. Therefore prevention is of utmost importance. If LBVD is already present integrated disease management strategies combining cultural and phytosanitary measures as well as growing tolerant lettuce cultivars are the only way to minimize economical damage.     

Acidic soil conditions suppress zoospore release from zoosporangia in <Emphasis Type="Italic">Olpidium virulentus</Emphasis>

Lettuce big-vein disease, caused by Mirafiori lettuce big-vein virus and Lettuce big-vein associated virus, is suppressed when the pH of field soil becomes acidic. Therefore, we evaluated the effect of soil pH on the activities of Olpidium virulentus, the vector of the viruses. We found that acidic soil, pH less than 6.0, significantly reduced O. virulentus infection of the root and influenced the detection rate of zoospores released in the surrounding water. We concluded that acidic soil suppresses zoospore release from zoosporangia.     

Occurrence of Mirafiori Lettuce Virus and Lettuce Big-vein Virus in Relation to Development of Big-vein Symptoms in Lettuce Crops

Big-vein disease (BV) of lettuce has been attributed to infection by Lettuce big-vein virus (LBVV), vectored by the soil fungus Olpidium brassicae. The finding of a second soil-borne virus in lettuce, Mirafiori lettuce virus (MiLV), led to a re-investigation of the role of LBVV in big-vein disease, with evidence emerging that both MiLV and LBVV are vectored by O. brassicae, and that MiLV, not LBVV, is the cause of BV (Lot et al. (2002), Phytopathology 92: 288–293). The two viruses have coat proteins of similar size but have different morphologies and are serologically unrelated. We tested individual lettuce plants in BV-prone fields and protected crops in France and Italy for the presence of the two viruses, using DAS-ELISA and antisera specific for each virus. Both MiLV and LBVV were found at high incidence, often together but sometimes separately. Symptoms were frequently found to be associated with MiLV alone or both viruses, but rarely LBVV alone. However, no absolute correlation emerged, because sometimes MiLV was present in the absence of symptoms, and vice versa. To clarify the situation, individual lettuce plants were examined over a period of time in two further surveys. In surveys of protected crops in France, plants with big-vein were always ELISA-positive for MiLV, or else symptomless plants positive for MiLV were later seen to develop big-vein symptoms. Presence or absence of LBVV appeared to have no effect on symptom development. In surveys of open fields in Italy, all combinations were found: presence of both viruses, apparent absence of both viruses, or presence of each one alone, in plants that developed BV. At the end of the observation period, nearly all plants had BV and contained both viruses.     

Tobacco stunt and lettuce big-vein viruses: biology,economic impact and control1

Two soil-borne virus diseases, tobacco stunt and lettuce big-vein, have many unique features in common, such as highly sensitive temperature dependency for disease development, in vivo acquisition of virus by the fungus vector Olpidium brassicae, long-term persistence of virus in the fungal resting spores, extreme instability of virus in vitro, dsRNA and coat protein unique for rod-shaped viruses, and others. Recent changes in agricultural practices have increased the incidence of these old diseases, in particular lettuce big-vein, causing serious damage to industry. Frequent incidence of the latter has been reported from intensively managed glasshouses and from the use of soil-free environments, where plants were grown with circulating liquid nutrients on certain support matrices. Chemical control of O. brassicae, particularly at the zoospore stage, appears feasible by continuous application of certain fungicides and/or non-ionic liquid surfactants that are effective in killing virus-transmitting zoospores. In the long term, breeding of cultivars resistant to the fungus or to both the fungus and virus, and biological control of the vector with other rhizosphere-inhabiting microorganisms may be possible.     

Further evidence of Mirafiori lettuce big-vein virus but not of Lettuce big-vein associated virus with big-vein disease in lettuce

Mirafiori lettuce big-vein virus (MLBVV) and Lettuce big-vein associated virus (LBVaV) are found in association with big-vein disease of lettuce. Discrimination between the two viruses is critical for elucidating the etiology of big-vein disease. Using specific antibodies to MLBVV and LBVaV for western blotting and exploiting differences between MLBVV and LBVaV in host reaction of cucumber and temperature dependence in lettuce, we separated the two viruses by transfering each virus from doubly infected lettuce plants to cucumber or lettuce plants. A virus-free fungal isolate was allowed to acquire the two viruses individually or together. To confirm the separation, zoospores from MLBVV-, LBVaV-, and dually infected lettuce plants were used for serial inoculations of lettuce seedlings 12 successive times. Lettuce seedlings were infected at each transfer either with MLBVV alone, LBVaV alone, or both viruses together, depending on the virus carried by the vector. Lettuce seedlings infected with MLBVV alone developed the big-vein symptoms, while those infected with LBVaV alone developed no symptoms. In field surveys, MLBVV was consistently detected in lettuce plants from big-vein-affected fields, whereas LBVaV was detected in lettuce plants not only from big-vein-affected fields but also from big-vein-free fields. LBVaV occurred widely at high rates in winter-spring lettuce-growing regions irrespective of the presence of MLBVV and, hence, of the presence of the big-vein disease.     

The use of methyl bromide and carbendazim for the control of lettuce big-vein disease

Lettuce seedlings raised in peat blocks placed on contaminated soil subsequently developed lettuce big-vein disease symptoms when grown in pots of sterilized compost. Incorporation of 0.01 g carbendazim per 4.3 cm³ peat block reduced the number of plants with disease symptoms but did not prevent root infection by Olpidium brassicae , the vector of the big-vein agent. Similar results were obtained when seedlings propagated in the absence of big-vein disease were grown in pots of contaminated soil but carbendazim was less effective when the treated blocks were planted in contaminated field plots. Methyl bromide applied at 500, 750 or 1000 kg/ha temporarily controlled the disease but re-contamination occurred and was complete after three consecutive crops.
Bromide residues in lettuce heads reached 9240 μg/g dry tissue in the first crop but fell to a maximum 772 μg/g by the third crop. Bromide residues in soil fell to natural levels over this period.     

Evidence for Lettuce big‐vein associated virus as the causal agent of a syndrome of necrotic rings and spots in lettuce

Lettuce big‐vein associated virus (LBVaV, genus Varicosavirus) was shown to be responsible for characteristic necrotic symptoms observed in combination with big‐vein symptoms in lettuce breeding lines when tested for their susceptibility to lettuce big‐vein disease (BVD) using viruliferous Olpidium virulentus spores in a nutrient film technique (NFT) system. Lettuce plants showing BVD are generally infected by two viruses: Mirafiori lettuce big‐vein virus (MiLBVV, genus Ophiovirus) and LBVaV. New mechanical inoculation methods were developed to separate the two viruses from each other and to transfer both viruses to indicator plants and lettuce. After mechanical inoculation onto lettuce plants MiLBVV induced vein‐band chlorosis, which is the characteristic symptom of BVD. LBVaV caused a syndrome of necrotic spots and rings which was also observed earlier in lettuce plants inoculated in the NFT system, resembling symptoms described for lettuce ring necrosis disease (RND). This observation is in contrast with the idea that LBVaV only causes latent infections in lettuce. De novo next‐generation sequencing demonstrated that LBVaV was the only pathogen present in a mechanically inoculated lettuce plant with symptoms, providing evidence that LBVaV was the causal agent of the observed necrotic syndrome and thus fulfilling Koch’s postulates for this virus. The necrotic syndrome caused by LBVaV in lettuce is referred to as LBVaV‐associated necrosis (LAN).     

Transmission by Olpidium brassicae of Mirafiori lettuce virus and Lettuce big-vein virus, and Their Roles in Lettuce Big-Vein Etiology

ABSTRACT Big-vein disease occurs on lettuce worldwide in temperate conditions; the causal agent has been presumed to be Lettuce big-vein virus (LBVV), genus Varicosavirus, vectored by the soilborne fungus Olpidium brassicae. Recently, the role of LBVV in the etiology of big-vein disease has been questioned because a second soilborne virus, Mirafiori lettuce virus (MiLV), genus Ophiovirus, has been found frequently in big-vein-affected lettuce. LBVV and MiLV, detectable and distinguishable by enzyme-linked immunosorbent assay using specific antisera, were tested for their ability to be transmitted from lettuce to lettuce by mechanical inoculation of sap extracts, or by zoospores of O. brassicae, and to cause big-vein disease. Both viruses were mechanically transmissible from lettuce to herbaceous hosts and to lettuce, but very erratically. LBVV was transmitted by O. brassicae but lettuce infected with only this virus never showed symptoms. MiLV was transmitted in the same manner, and lettuce infected with this virus alone consistently developed big-vein symptoms regardless of the presence or absence of LBVV. With repeated mechanical transmission, isolates of both viruses appeared to lose the ability to be vectored, and MiLV appeared to lose the ability to cause big-vein symptoms. The recovery of MiLV (Mendocino isolate, from Cali-fornia) from stored O. brassicae resting spores puts the earliest directly demonstrable existence of MiLV at 1990.     

Lettuce ring necrosis,caused by a chytrid-borne agent distinct from lettuce big-vein ‘virus’

Ring necrosis is a serious disease of lettuce (Lactuca sativa) with often coalescing necrotic rings and ring-like patterns on middle leaves of plants or groups of plants in glasshouses during winter. Affected leaves may decay and plants rapidly become unmarketable. The disease was shown to be soil-borne and transmitted by the zoospores ofOlpidium brassicae. Symptoms in lettuce do not appear before seven weeks after inoculation via the soil. Additives to the inoculum and chilling of source leaves, inoculum buffer and utensils enabled mechanical transmission of a pathogenic agent toChenopodium quinoa, C. amaranticolor, Nicotiana benthamiana, N. clevelandii, N. hesperis, andN. occidentalis but not to lettuce. TheChenopodium spp. reacted with local lesions, infection was symptomless inN. clevelandii and mostly so inN. benthamiana, butN. hesperis andN. occidentalis reacted with leaf spotting and plant stunting. With zoospores of an originally pathogen-free fungus culture further cultivated on the roots of cuttings from sap-inoculated plants ofN. clevelandii andN. occidentalis, the agent could be transferred back to lettuce and the symptoms of ring necrosis be reproduced. The agent biologically resembles those of lettuce big-vein (LBV) and freesia leaf necrosis and the tobacco stunt virus. In lettuce it often occurs together with LBV virus but differs in longer incubation period, type of symptoms and symptom appearance only during winter. It could be separated from a mixture with LBV virus by serial transfer always selecting plants without LBV symptoms. So far cultural hygiene, including soil disinfection addressing the vector, is the main means of control.     

Investigation of soilborne mosaic virus diseases transmitted by <Emphasis Type="Italic">Polymyxa graminis</Emphasis> in cereal production areas of the Anatolian part of Turkey

Polymyxa graminis is the vector of several important viruses, including Soilborne cereal mosaic virus, Wheat spindle streak mosaic virus, Barley yellow mosaic virus and Barley mild mosaic virus, of winter cereals worldwide. Surveys were carried out to detect these viruses and their vector P. graminis in 300 soil samples from the main wheat and barley production areas of the Anatolian part of Turkey collected in May 2002, June 2004 and May 2005. For these surveys, various susceptible wheat and barley cultivars were pot grown in the collected soil samples in a greenhouse and then analysed using ELISA and RT-PCR to detect the presence of different virus species. In addition, a combination of light microscopy following roots staining with acid fuchsin and PCR was used for detection of P. graminis. All soil samples analysed were found to be free of these soilborne viruses and their vector.     

Molecular analysis and virus transmission tests place Olpidium virulentus, a vector of Mirafiori lettuce big-vein virus and tobacco stunt virus, as a distinct species rather than a strain of Olpidium brassicae

The rDNA-ITS sequences of ten single-sporangium isolates of Olpidium virulentus (a noncrucifer strain of Olpidium brassicae), which transmits Mirafiori lettuce big-vein virus (MLBVV) and tobacco stunt virus (TStV), were compared with those of six single-sporangium isolates of O. brassicae. The sequence similarity within isolates of O. virulentus or O. brassicae was almost identical (98.5%–100.0%), but was low between the two species (79.7%–81.8%). In a phylogenetic analysis of the rDNA-ITS region, O. virulentus and O. brassicae fell into two distinct clusters, indicating that O. virulentus, a vector of MLBVV and TStV, is a distinct species rather than a strain of O. brassicae.     

Quantification of Mirafiori lettuce big‐vein virus and its vector,Olpidium virulentus,from soil using real‐time PCR

Big vein disease of lettuce (Lactuca sativa) is an economically important disease transmitted through soil by Olpidium virulentus, and has occurred in most production areas worldwide. The disease is assumed to be caused by Mirafiori lettuce big‐vein virus (MiLBVV). To understand the dynamics of the virus and its vector, MiLBVV and O. virulentus were directly detected in soil. DNA and RNA were extracted from 5 g soil using a bead beating method, followed by purification using adsorption to a column. Detection and quantification were performed using real‐time PCR and a TaqMan probe that was prepared based on the CP region of MiLBVV and the rDNA‐ITS region of O. virulentus, respectively. Furthermore, using a visual assessment of the incidence rate of big vein disease on lettuce in agricultural fields, the C_t values of MiLBVV and O. virulentus from soil were also determined using real‐time PCR. The results showed that MiLBVV concentrations in the soil were high in the field, as also determined by a visual assessment of the incidence rate of big vein disease on lettuce. However, the amount of O. virulentus in soil was not directly correlated with the incidence of MiLBVV. From these results, it is suggested that the risk of lettuce crops developing big vein disease can be estimated using an index of the amount of MiLBVV in the soil.     

Viruses and virus-like pathogens transmitted by zoosporic fungi1

The viruses and virus-like pathogens transmitted by zoosporic fungi are reviewed. The nine furoviruses (and possible members of the group), with labile rod-shaped particles, have nearly all been shown to be transmitted by plasmodiophoromycete vectors. As they have been reviewed extensively elsewhere, they are covered only briefly; important examples are beet necrotic yellow vein furovirus and potato mop-top furovirus. Five viruses with filamentous particles, tentatively recognized as poty viruses, are transmitted by Polymyxa graminis. Within this group, wheat yellow mosaic virus should be considered to include wheat spindle streak mosaic virus, while the M and NM forms of barley yellow mosaic virus, the best known members of the group, should probably be regarded as distinct viruses. Chytrids (especially Olpidium brassicae) transmit a variety of viruses in different groups (e.g. tobacco necrosis necrovirus, lettuce big-vein virus, melon necrotic spot carmovirus, red clover necrotic mosaic dianthovirus). Finally, several diseases caused by uncharacterized pathogens appear to be transmitted by O. brassicae: freesia leaf necrosis, lettuce ring necrosis, pepper yellow vein, watercress chlorotic leaf spot.     

Preparation and characterization of polyclonal antibody against resting spores of Olpidium virulentus, fungal vector of lettuce big-vein disease

Research on Olpidium virulentus, the vector of the serious lettuce big-vein disease, is difficult because its resting spores persist in soil and retain virus for years and the fungus is not culturable. We developed protocols to obtain purified, viable resting spores from infected roots using an enzymatic treatment and two-step density gradient centrifugation and to generate a polyclonal antibody against these spores. The antibody was species-specific in a direct immunostaining assay and in western blots produced two specific bands (ca. 30.5, 29.0 kDa) against resting spores. The detection limit for resting spores in an indirect ELISA was 200 spores/100 μL.     

Molecular and biological characterization of two potyviruses infecting lettuce in southeastern France

Several potyviruses affect lettuce (Lactuca sativa) and chicory (Cichorium spp.) crops worldwide and are important constraints for production because of the direct losses that they induce and/or because of their seed transmission. Here, the molecular and biological properties are described of two potyviruses that were recently isolated from lettuce plants showing mosaic or strong necrotic symptoms in an experimental field in southeastern France. The first potyvirus belongs to the species Endive necrotic mosaic virus and is present in a large number of wild plant species, especially Tragopogon pratensis. It is unable to infect lettuce cultivars with a resistance to Turnip mosaic virus that is present in many European cultivars and probably conferred by the Tu gene. The second potyvirus belongs to the tentative species lettuce Italian necrotic virus and was not observed in wild plants. It infected all tested lettuce cultivars. Wild accessions of Lactuca serriola, Lactuca saligna, Lactuca virosa and Lactuca perennis were identified as resistant to one or the other potyvirus and could be used for resistance breeding in lettuce. No resistance against these two potyviruses was observed in the tested Cichorium endivia cultivars. In contrast, all tested Cichorium intybus cultivars or accessions were resistant.     

Effects of temperature increase on the epidemiology of three major vector-borne viruses

The epidemiologies of Maize streak virus (MSV), Maize stripe virus (MSpV), and Maize mosaic virus (MMV) were compared in La Réunion over a three year-period. Disease incidence caused by each virus was assessed, and the leaf and planthopper vector populations (Cicadulina mbila and Peregrinus maidis) were estimated in weekly sowings of the temperate, virus-susceptible maize hybrid INRA 508 and of the composite resistant cv. IRAT 297. MSV caused the most prevalent disease and MMV the least, with lower incidences in cv. IRAT 297 than in INRA 508. For each plant–virus–vector combination, (a) disease incidence was positively correlated to vector abundance, often with 1 month of time lag; (b) annual periodicity of disease incidence and of vector numbers was consistent with highest autocorrelations and a time lag of 12 months, (c) vector numbers and disease incidence were closely associated with temperature fluctuations, both remaining relatively constant below 24°C and increasing rapidly above this threshold temperature. By contrast, relationships with rainfall and relative humidity (RH) were less consistent. Overall, 63 to 80% of the variance of disease incidence was explained through stepwise regression with vector number, temperature, and sometimes also rainfall or RH. The simple epidemiological model proposed underlines the close link between increased temperature and possible (re-) emergence of these three diseases in a maize cropping area.