Cucumber-mosaic-virus-associated RNA-5: XII. — Symptom-modulating effect is codetermined by the helper virus satellite replication support function

In tomato, the disease-modulating effects of a cucumber mosaic virus (CMV) satellite isolate from Belgium, here designated T-CARNA-5 (CARNA-5 = CMV-associated RNA-5), were found to be different depending on the supporting helper virus strain. With two CMV strains, T-CARNA-5 induced lethal necrosis, but with a third strain from Ixora spp. (CMV-Ix), aggravated stunting was observed. However, the primary structure of the T-CARNA-5 contained within virus isolated from tobacco or tomato infected with each of these three CMV strains, conformed to the conserved sequence profile of CARNA-5 isolates which are necrogenic in tomato. Dilution end-point bioassay of T-CARNA-5 established a direct cause-effect relationship between it and tomato necrosis or stunting, depending on the helper virus. Total nucleic acid extracts taken at different times from tomato plants infected with the above CMV strains and T- or S-CARNA-5 (used as non-necrogenic control) showed viral RNA, ssCARNA-5 and dsCARNA-5 to be present in significant amounts, but in sometimes dissimilar proportions depending on the combination; except in CMV-Ix/S-CARNA-5 infection where neither ss-nor dsCARNA-5 was found.The experiments established that CARNA-5 biological expression studies in CMV-infected tomato have to take into account the helper virus satellite replication support function, which may be a primary codeterminant of quantitative or qualitative differences in the symptom modulation observed.     

Grafting to manage infections of top stunting and necrogenic strains of cucumber mosaic virus in tomato

Cucumber mosaic virus (CMV) lists among the most important etiological agents of tomato diseases. Some isolates of CMV function as helper virus for replication, encapsidation and transmission of satellite RNAs (satRNA), which may exacerbate symptoms induced by CMV in certain hosts. Outbreaks of CMV strains supporting hypervirulent variants of satRNAs are recurrent in tomato with devastating effects on crop production and efficient control measures are still unavailable. In this study, we examined the dynamics of infection of the CMV strains tomato top stunting (TTS) and 77 supporting replication of satRNA variants that codetermine top stunting (TTS‐satRNA) and necrotic (77‐satRNA) phenotypes in two tomato cultivars denoted Solanum lycopersicum Manduria (Sl‐Ma) and S. lycopersicum UC82 (Sl‐UC). Sl‐Ma but not Sl‐UC recovered from disease symptoms induced by CMV‐TTS while both the cultivars succumbed to the infection of CMV‐77 and its necrogenic satRNA. Ability to recover of the Sl‐Ma plants was transmitted by grafting to the susceptible genotype Sl‐UC. More interestingly, recovery was observed also against the challenge inoculation of CMV plus 77‐satRNA in plants grafted on Sl‐Ma and in self‐grafted plants of both the Sl‐Ma and Sl‐UC cultivars. Analysis of small RNAs and genes of the defence plant response based on RNA interference (RNAi) suggested that RNAi is involved in the recovery of Sl‐Ma against CMV with hypervirulent satRNAs and in scions grafted on this rootstock. The response of Sl‐Ma to the inoculation of CMV‐77 plus 77‐satRNA was compared with that of the transgenic tomato line S. lycopersicum transgenic line UCTC5.9.2 that expresses constitutively the benign variant of the satRNA denoted Tfn‐satRNA. Comparative analysis suggested that the response may operate via similar mechanisms, which involve RNAi, the graft and the presence of the satRNA.     

Studies on plant growth-regulating substances LV. The Plant growth-promoting properties of some β-aryl-propionic acids

Severe diseases of pepper (Capsicum annuum), tomato (Lycopersicon esculentum), eggplant (Solanum melongena) and tomato eggplant (Solanum integrifolium) in West Africa were induced by pepper veinal mottle virus (PVMV). Five selected virus isolates were serologically similar and readily transmissible by aphids in the non-persistent manner, but they differed in host range and/or symptoms induced in some susceptible species. One isolate from eggplant failed to infect pepper, Chenopodium quinoa and C. amaranticolor, and induced only local infections in tomato. An isolate from tomato failed to infect eggplant, and an isolate from tomato eggplant induced severe stunting in Physalis floridana. The type strain, like the isolate from tomato, failed to infect Nicotiana tabacum systemically, but each caused severe systemic leaf and stem necrosis in tomato. None of the isolates infected S. melongena cv. Long Purple, suggesting that PVMV might be controlled in this and perhaps other crop species by the use of immune or tolerant cultivars. All five isolates were serologically related to potato virus Y and some to six of 12 other potyviruses.     

Vertical Transmission Selects for Reduced Virulence in a Plant Virus and for Increased Resistance in the Host

For the last three decades, evolutionary biologists have sought to understand which factors modulate the evolution of parasite virulence. Although theory has identified several of these modulators, their effect has seldom been analysed experimentally. We investigated the role of two such major factors—the mode of transmission, and host adaptation in response to parasite evolution—in the evolution of virulence of the plant virus Cucumber mosaic virus (CMV) in its natural host Arabidopsis thaliana. To do so, we serially passaged three CMV strains under strict vertical and strict horizontal transmission, alternating both modes of transmission. We quantified seed (vertical) transmission rate, virus accumulation, effect on plant growth and virulence of evolved and non-evolved viruses in the original plants and in plants derived after five passages of vertical transmission. Our results indicated that vertical passaging led to adaptation of the virus to greater vertical transmission, which was associated with reductions of virus accumulation and virulence. On the other hand, horizontal serial passages did not significantly modify virus accumulation and virulence. The observed increases in CMV seed transmission, and reductions in virus accumulation and virulence in vertically passaged viruses were due also to reciprocal host adaptation during vertical passages, which additionally reduced virulence and multiplication of vertically passaged viruses. This result is consistent with plant-virus co-evolution. Host adaptation to vertically passaged viruses was traded-off against reduced resistance to the non-evolved viruses. Thus, we provide evidence of the key role that the interplay between mode of transmission and host-parasite co-evolution has in determining the evolution of virulence.     

Virus–vector Relationships,Host Range,Detection and Sequence Comparison of Chilli leaf curl virus Associated with an Epidemic of Leaf Curl Disease of Chilli in Jodhpur,India

An epidemic of chilli leaf curl disease was recorded in 2004 in Jodhpur, a major chilli‐growing area in Rajasthan, India. Several isolates were efficiently transmitted by the whitefly (Bemisia tabaci), all of which induced severe leaf curl symptoms in chilli. A single whitefly was capable of transmitting the virus, and eight or more whiteflies per plant resulted in 100% transmission. The minimum acquisition access period (AAP) and inoculation access period (IAP) were 180 and 60 min, respectively. The virus persisted in whiteflies for up to 5 days postacquisition. Of 25 species tested, the virus infected only five (Capsicum annuum, Carica papaya, Solanum lycopersicum, Nicotiana tabacum and N. benthamiana). The virus was identified as Chilli leaf curl virus (ChiLCV), which shared the closest sequence identity (96.1%) with an isolate of ChiLCV from potato in Pakistan and showed sequence diversity up to 12.3% among the ChiLCV isolates reported from India and Pakistan. A betasatellite was identified, which resembled most closely (97.3%) that of Tomato leaf curl Bangladesh betasatellite previously reported from chilli and tomato leaf curl in India. The betasatellite was very different from that reported from chilli leaf curl in Pakistan, indicating that different betasatellites are associated with chilli leaf curl in India and Pakistan. We describe here for the first time the virus–vector relationships and host range of ChiLCV.     

Evolution of Virulence in Emerging Epidemics

Theory predicts that selection for pathogen virulence and horizontal transmission is highest at the onset of an epidemic but decreases thereafter, as the epidemic depletes the pool of susceptible hosts. We tested this prediction by tracking the competition between the latent bacteriophage λ and its virulent mutant λcI857 throughout experimental epidemics taking place in continuous cultures of Escherichia coli. As expected, the virulent λcI857 is strongly favored in the early stage of the epidemic, but loses competition with the latent virus as prevalence increases. We show that the observed transient selection for virulence and horizontal transmission can be fully explained within the framework of evolutionary epidemiology theory. This experimental validation of our predictions is a key step towards a predictive theory for the evolution of virulence in emerging infectious diseases.     

Transmission Comparisons of Cucumber Mosaic Virus Subgroup I and II Isolates by Different Aphid Species

To investigate the transmission differences between Cucumber mosaic virus (CMV) subgroup isolates, we carried out a comparative study with five aphid species Myzus persicae, Aphis gossypii, Lipaphis erysimi, Aphis craccivora and Megoura viciae in laboratory and field experiments to evaluate spread of CMV Subgroup I NX and subgroup II AG isolates in tobacco. Both NX and AG varied in transmission efficiency by the five aphids, and our transmission results revealed important differences in transmission efficiency of two isolates by Myzus persicae and Aphis gossypii. In contrast, significant transmission differences were not detected with Lipaphis erysimi, Aphis craccivora or Megoura viciae. Interestingly, the overall transmission efficiencies of the two different subgroup strains were almost equal when field transmissions were tested with mixed populations of the five aphid species. Our results together with our previously reported experiments on competition of CMV subgroup isolates in tobacco suggest that variations in aphid vector populations contribute substantially to the epidemic potential of CMV subgroup isolates.     

Identification of a 334-ribonucleotide viral satellite as principal aetiological agent in a tomato necrosis epidemic

Cucumber mosaic virus (CMV), a widespread and economically important virus of vegetable crops, often contains a satellite RNA, here designated CARNA-5 (for CMV-associated RNA 5). Viral satellites are small nucleic acids that are sequence-unrelated to, but replicatively dependent upon, the viral genome. They essentially are molecular parasites of their helper viruses, and thereby frequently modulate viral symptom expression. Some isolates of CARNA-5 change normally moderate CMV symptoms in tomato into a lethal disease named tomato necrosis; others ameliorate CMV symptoms in tomato and other important crop plants. Here we report on the identification and molecular characterization of a 334-nucleotide necrogenic CARNA-5 isolated from tomato fields in southern Italy, where a massive outbreak of lethal necrosis occurred in the summer of 1988. This is the first time that direct evidence is given for the involvement of a viral satellite in a crop disease of epidemic scale. The possible molecular interrelationships between plant, virus, satellite and other factors that influence the satellite-induced symptom modulation underlying such a catastrophe are discussed.     

Virulence and community dynamics of fungal species with vertical and horizontal transmission on a plant with multiple infections

The virulence evolution of multiple infections of parasites from the same species has been modeled widely in evolution theory. However, experimental studies on this topic remain scarce, particularly regarding multiple infections by different parasite species. Here, we characterized the virulence and community dynamics of fungal pathogens on the invasive plant Ageratina adenophora to verify the predictions made by the model. We observed that A. adenophora was highly susceptible to diverse foliar pathogens with mixed vertical and horizontal transmission within leaf spots. The transmission mode mainly determined the pathogen community structure at the leaf spot level. Over time, the pathogen community within a leaf spot showed decreased Shannon diversity; moreover, the vertically transmitted pathogens exhibited decreased virulence to the host A. adenophora, but the horizontally transmitted pathogens exhibited increased virulence to the host. Our results demonstrate that the predictions of classical models for the virulence evolution of multiple infections are still valid in a complex realistic environment and highlight the impact of transmission mode on disease epidemics of foliar fungal pathogens. We also propose that seedborne fungi play an important role in structuring the foliar pathogen community from multiple infections within a leaf spot.     

First Report of a Croton yellow vein mosaic virus (CYVMV) Associated with Tomato Leaf Curl Disease in India

Leaf curl disease symptoms were observed in tomato crop grown in a tomato field at Matera district of Bahraich, India, in March 2013 with an 85% disease incidence. The infected plants exhibited leaf curl symptoms accompanied with puckering, vein swelling and stunting of the whole plant. PCR carried out with begomovirus coat protein gene and DNA beta‐specific primer sets resulted in positive amplification of ~775 bp and 1.35 kbp, respectively, with all symptom‐bearing plant samples. BLASTn and phylogenetic analyses of CP gene sequences showed highest and close relationship with Croton yellow vein mosaic virus (CYVMV) isolates, while the phylogenetic study of betasatellite sequence showed distinct relationships with other begomovirus associated betasatellites reported from India and abroad. This is a first report of a CYVMV associated with tomato leaf curl disease in India.     

Severe outbreaks of tomato yellow leaf curl Sardinia virus in Calabria, Southern Italy

During the winter 2003--2004 a serious disease was observed in protected tomato crops in Castrovillari, Reggio Calabria province, Southern Italy. Symptoms consisted in marginal leaf yellowing, leaf curling, plant stunting, flower abortion. The disease was detected in a group of greenhouses (about 10ha) where several tomato cultivars were grown hydroponically. The highest incidence of infection (60-100%) was observed in tomatoes grafted on Beaufort DRS tomato rootstock. Since the symptoms were similar to those described for Tomato yellow leaf curl Sardinia virus (TYLCSV) and Tomato yellow leaf curl virus (TYLCV), detection assays for these viruses were used. In DAS-ELISA positive results were obtained with a abroad-spectrums reagent combination (distributed by Bioreba AG) detecting TYLCV, TYLCSV, and other begomoviruses. When DNA probes were used in tissue print assays, positive reactions were obtained for TYLCSV, but not for TYLCV. The two probes consisted of digoxigenin-labelled DNAs representing the coat protein gene of either TYLCSV or TYLCV. Attempts to isolate the viral agent by mechanical inoculation failed, except in few cases where Potato virus Y and Tobacco mosaic virus were identified following transmission from symptomatic plants to herbaceous indicatorpplants. By contrast, grafting onto tomato seedlings always successfully transmitted the disease. In the Castrovillari area TYLCSV was not reported before. The rootstocks that nurseries used for grafting were obtained from Sicily, where the disease is endemic and both TYLCSV and TYLCV are widespread. Probably the grafted plantlets represented the primary source of infection from which subsequent diffusion by way of the vector Bemisia tabaci followed. In fact the vector had previously been detected in both the glasshouse-grown and open field tomato crops in Calabria region. TYLCV was previously reported in a different area of Calabria in 1991, but apparently it was an occasional outbreak, and B. tabaci was not detected. Since in the Castrovillari area surveyed in the present study tomato is grown throughtout the year in protected crops, the whitefly vector of the virus is present, and some natural hosts of the virus are found, it is feared that TYLCSV may become endemic, as already happened in Sicily, Sardinia, and Spain several years ago. In Spain and Sicily TYLCV, together with TYLCSV, was reported as the causal agent of very severe tomato crop losses. Therefore the danger exists that also TYLCV will reach this area, furthermore complicating the management of tomato crops.     

番茄曲叶病及其血清学和PCR测定

我国曾报道的番茄病毒病有多种,其中最常见的是黄瓜花叶病毒(CMV)和烟草花叶病毒(TMV)引起的花叶病。柯冲等(1964)在大陆首次报道烟粉虱(Bemisia tabaci)传播的番茄病毒病——番茄黄顶病,此病在50～60年代曾在广州市郊流行,造成大面积减产。Green等(1984)报道台湾发生番茄黄曲叶病,此病与日本的番茄黄矮病(Tomato yellow dwarf)相似,并且与烟草曲叶病毒(TLCV)有血清学关系。印度、委内瑞拉等国也曾报道发生由烟粉虱传播的番茄曲叶病和番茄黄曲叶病。1991和1992年秋,在广西南宁市郊发现一种症状表现为植株矮缩,叶片向上向内卷曲,叶背面产生耳状或杯状增生物,对光看有时可见叶脉呈墨绿色,不结果或少结果的番茄病害。1992年秋广西农业科学院的番茄试验地发病率高达6.8％,对当地秋番茄生产构成了威胁。作者对病害症状、传播、血清学反应及PCR分析等方面与烟草曲叶病毒进行了比较研究,证实了该病的病原与烟草曲叶病毒有很高的同源性。现将研究结果简报如下。     

Estimation of the effective number of founders that initiate an infection after aphid transmission of a multipartite plant virus

The fecundity of RNA viruses can be very high. Thus, it is often assumed that viruses have large populations, and RNA virus evolution has been mostly explained using purely deterministic models. However, population bottlenecks during the virus life cycle could result in effective population numbers being much smaller than reported censuses, and random genetic drift could be important in virus evolution. A step at which population bottlenecks may be severe is host-to-host transmission. We report here an estimate of the size of the population that starts a new infection when Cucumber mosaic virus (CMV) is transmitted by the aphid Aphis gossypii, based on the segregation of two CMV genotypes in plants infected by aphids that acquired the virus from plants infected by both genotypes. Results show very small effective numbers of founders, between one and two, both in experiments in which the three-partite genome of CMV was aphid transmitted and in experiments in which a fourth RNA, CMV satellite RNA, was also transmitted. These numbers are very similar to those published for Potato virus Y, which has a monopartite genome and is transmitted by aphids according to a different mechanism than CMV. Thus, the number of genomic segments seems not to be a major determinant of the effective number of founders. Also, our results suggest that the occurrence of severe bottlenecks during horizontal transmission is general for viruses nonpersistently transmitted by aphids, indicating that random genetic drift should be considered when modeling virus evolution.     

Molecular Variability in the Replicase Gene of Viruses Causing Tomato Leaf Curl Disease in India

Tomato Leaf Curl Virus (ToLCV)-infected samples collected from four different ecological zones of India: Northern zone, NewDelhi (IARI) [ToLCNDV-IARI]; Central zone, Jabalpur [ToLCV-Jb], Raipur [ToLCV-Rp] and Southern zone, Dharwad [ToLCV-Dh] were maintained by whitefly transmission into tomato cv Pusa Early Dwarf. Replicase gene coding for replication initiator protein (Rep) was amplified by PCR using gene specific primers, yielding an amplicon of 1086 bp for all the isolates. Sequence analysis of the rep amplicons of all the Indian isolates of ToLCV causing the disease clearly shows the presence oftwo subgroups irrespective of their geographical evolution. Isolates belonging to subgroup I comprising of tomato leaf curl virus-New Delhi (ToLCV-NDe) having bipartite genome are conserved within themselves and showed 94–95% nucleotide sequence homology. Second subgroup comprising of tomato leaf curl virus-Bangalore (ToLCBV) having monopartite genome and conserved among themselves, but showed only 73-75% homology with the subgroup I. In phylogenetic tree analysis, ToLCV-NDe appeared in a different cluster than ToLCV-Ban. All the four isolates used in this study fall in ToLCV-NDe group based on their replicase gene sequences. The specificity determinant located at N terminal suggests ToLCV-NDe and ToLCV-Rp as mild isolates.     

The reactions of swede (Brassica napus) to infection by turnip mosaic virus

Turnip mosiac virus (TuMV) and cucumber mosaic virus (CMV) were the only viruses commonly isolated from naturally diseased swedes (Brassica napus) showing leaf mosaic and leaf and root necrosis. Only TuMV caused these symptoms when re-inoculated to swede. TuMV-infected plants showed a severe loss in leaf (55%) and root (63%) fresh weight after 140 days. Systemic leaf symptoms in infected swede plants varied greatly, but were predominantly necrotic (N) or mosaic (M). Plants were classified into one of seven reaction classes ranging from slight or severe necrosis, and mosaics with and without slight veinal necrosis. Swede cultivars differed markedly in their reaction to TuMV and contained different proportions of N- or M-reacting plants. The reactions of progeny of four resistant cv. Bangholm plants were separately inherited; progeny of two plants reacting either symptomlessly or necrotically and those of the other two plants developing mosaic symptoms only. Five isolates of TuMV from swede crops in different regions caused similar reactions but differed in virulence in the progeny of a self-pollinated resistant swede plant.