Spread and interaction of Pepino mosaic virus (PepMV) and Pythium aphanidermatum in a closed nutrient solution recirculation system: effects on tomato growth and yield

Pepino mosaic virus (PepMV) was shown to be efficiently transmitted between tomato plants grown in a closed recirculating hydroponic system. PepMV was detected in all plant parts after transmission via contaminated nutrient solution using ELISA, immunocapture RT‐PCR, RT‐PCR, electron microscopy, and by inoculation to indicator plants. Detection of PepMV in nutrient solution was only possible after concentration by ultracentrifugation followed by RT‐PCR. Roots tested positive for PepMV 1–3 weeks after inoculation, and subsequently a rapid spread from the roots into the young leaves and developing fruits was found within 1 week. PepMV was only occasionally detected in the older leaves. None of the infected plants showed any symptoms on fruits, leaves or other organs. Pre‐infection of roots of tomato cv. Hildares with Pythium aphanidermatum significantly delayed PepMV root infections. When mechanically inoculated with PepMV at the 2–4 leaf stage, yield loss was observed in all plants. However, only plants of cv. Castle Rock recorded significant yield losses when infected via contaminated nutrient solution. Yield losses induced by infection with PepMV and/or P. aphanidermatum ranged from 0·4 up to 40% depending on experimental conditions.     

Molecular variability of Spanish and Hungarian isolates of Tomato torrado virus

The population structure and genetic variation of Tomato torrado virus (ToTV) were estimated from 19 Spanish isolates collected from 2001 to 2009 in different tomato‐production areas by analyses of the partial nucleotide sequences of five regions of the virus genome: the protease cofactor (Pro‐Co) and the RNA‐dependent RNA polymerase (RdRp) in RNA1, and the movement protein (MP) and two subunits of the coat protein (CP; viz. Vp35 and Vp23) in RNA2. Three Hungarian isolates of the virus were also included in the analyses. All the ToTV isolates clustered together in the phylogenetic analysis of the nucleotide sequences of the different regions. However, some genetic diversity was observed in the case of the two CP subunits among the Gran Canaria isolates and the remaining ToTV‐isolates analysed, which grouped together. A high similarity was observed among all the isolates and the two published ToTV isolates: the ToTV type isolate (PRI‐ToTV0301) and the Polish isolate Wal03. The most variable encoding regions studied were those on RNA2. In general, no correlation was found between genetic diversity and collection date. Studying the genetic distances between pairs of sequences, the ratio between nonsynonymous (amino‐acid‐replacing) and synonymous (silent mutational) substitutions was low, indicating a strong negative selective pressure in the studied regions. Nine negatively selected sites (distributed in Pro‐Co, MP, Vp23 and Vp35) and just one positively selected one (in Pro‐Co) were found for all the genome regions studied.     

Susceptibility of different plant species and tomato cultivars to two isolates of <Emphasis Type="Italic">Pepino mosaic virus</Emphasis>

As Pepino mosaic virus has become a pathogen of major importance in worldwide tomato production, information is needed on possible differences between the sensitivity of cultivars towards infection. Furthermore, it is important what hosts other than Solanaceae may be virus reservoirs and are, therefore, threats for tomato cultivation. Two PepMV isolates (PepMV-Sav, E397, a European tomato isolate and PV-0554, a Peruvian pepino isolate) differing in their origin and virulence were used for several experiments to investigate these issues. The response to mechanical inoculation with PepMV was studied using 25 tomato cultivars, seven indicator plant species, and nine other possible horticultural host plants. Symptom development after infection with PepMV was monitored and the virus was detected by DAS-ELISA and IC-RT-PCR. Garlic and broad bean were shown to be additional hosts of PepMV depending on the virus isolate. Nicotiana benthamiana seems to be the most sensitive indicator among all tested indicator plants developing symptoms. Both PepMV isolates infected all tested tomato cultivars. Development of disease symptoms depended on the cultivar and the virus isolate but symptoms were not visible in all cases. None of the cultivars showed tolerance against the two isolates but two responded with a lower susceptibility at an absorbance level of 0.2 (healthy control 0.09). It was observed that some cultivars grown hydroponically showed also lower losses in biomass and yield. Data indicated a correlation between absorbance level in DAS-ELISA and reduction in total tomato growth.     

Distribution and accumulation of cassava brown streak viruses within infected cassava (Manihot esculenta) plants

Cassava brown streak disease (CBSD), caused by Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV), ranks among the top seven biological threats to global food security. The disease poses a significant threat to cassava production in East and Central Africa (ECA). In Uganda, overall CBSD incidence increased by c. 20% since it re‐emerged in 2004, and the disease persistently reduces cassava yields and storage root qualities. The spread of CBSD has been studied spatially in fields in different agroecologies. However, within‐host distribution and accumulation of CBSV and UCBSV in naturally infected cassava plants is unknown. Therefore, within‐host CBSV and UCBSV distribution was studied to correlate CBSD symptoms with virus titre in organs of infected cassava. Leaf, stem and storage root samples, with and without symptoms, were collected from 10 genotypes of field‐grown cassava. Presence of CBSV and UCBSV was detected by RT‐PCR and virus levels determined by qRT‐PCR. CBSV was present in 100% of CBSD samples with symptoms, with 45·3% positive for presence of both CBSV and UCBSV. Tolerant cassava genotypes were infected with CBSV alone and accumulated higher titre in roots than in aerial organs. Susceptible genotypes were co‐infected with CBSV and UCBSV and exhibited variation in virus titre in each organ. Across genotypes, virus titre was lowest in the youngest leaves and highest in mature non‐senescing leaves. This information provides insight into the relationship between CBSV, UCBSV and their cassava host, and is valuable for CBSD resistance breeding, epidemiology studies and CBSD control.     

Tomato chlorosis virus in pepper: prevalence in commercial crops in southeastern Spain and symptomatology under experimental conditions

Tomato chlorosis virus (ToCV), a member of the genus Crinivirus (family Closteroviridae), has been present in Spain since at least 1997, causing annual epidemics of yellowing in protected tomato crops. In 1999, sweet pepper plants exhibiting stunting and symptoms of interveinal yellowing and mild upward curling in the leaves, were found to be infected with ToCV in a greenhouse heavily infested with the whitefly Bemisia tabaci in the province of Almería, southeastern Spain. This study investigated the prevalence of ToCV in tomato and pepper crops in the major growing areas of southeastern Spain (Murcia, Almería and Málaga provinces) over a 3‐year period. In addition, an experimental system was developed for ToCV inoculation using B. tabaci as a vector, which allowed analysis of susceptibility of different pepper cultivars to the virus. The disease syndrome and yield losses induced by ToCV in pepper were also studied under experimental conditions, confirming severe yield reduction in infected plants.     

Arabidopsis thaliana,an experimental host for tomato yellow leaf curl disease‐associated begomoviruses by agroinoculation and whitefly transmission

Tomato yellow leaf curl disease is one of the most devastating viral diseases affecting tomato crops worldwide. This disease is caused by several begomoviruses (genus Begomovirus, family Geminiviridae), such as Tomato yellow leaf curl virus (TYLCV), that are transmitted in nature by the whitefly vector Bemisia tabaci. An efficient control of this vector‐transmitted disease requires a thorough knowledge of the plant–virus–vector triple interaction. The possibility of using Arabidopsis thaliana as an experimental host would provide the opportunity to use a wide variety of genetic resources and tools to understand interactions that are not feasible in agronomically important hosts. In this study, it is demonstrated that isolates of two strains (Israel, IL and Mild, Mld) of TYLCV can replicate and systemically infect A. thaliana ecotype Columbia plants either by Agrobacterium tumefaciens‐mediated inoculation or through the natural vector Bemisia tabaci. The virus can also be acquired from A. thaliana‐infected plants by B. tabaci and transmitted to either A. thaliana or tomato plants. Therefore, A. thaliana is a suitable host for TYLCV–insect vector–plant host interaction studies. Interestingly, an isolate of the Spain (ES) strain of a related begomovirus, Tomato yellow leaf curl Sardinia virus (TYLCSV‐ES), is unable to infect this ecotype of A. thaliana efficiently. Using infectious chimeric viral clones between TYLCV‐Mld and TYLCSV‐ES, candidate viral factors involved in an efficient infection of A. thaliana were identified.     

新疆番茄病毒病检测及南方番茄病毒全基因组序列测定与传播特性分析

为了解新疆番茄上病毒病的发生情况,利用一步法RT-PCR技术检测了南北疆番茄上南方番茄病毒(Southern tomato virus,STV)、黄瓜花叶病毒(Cucumber mosaic virus,CMV)、番茄花叶病毒(Tomato mosaic virus,ToMV)以及马铃薯Y病毒(Potato virus Y,PVY)的感染情况,并利用分段克隆的方法进行全基因组测序,通过RT-PCR方法检测健康植株与携带病毒植株杂交育种的F1代植株带毒率以分析STV的种子传播特性。结果显示,新疆番茄上CMV、STV、ToMV和PVY在北疆的检出率分别为52%、37%、27%和14%;在南疆的检出率分别为79%、60%、69%和0;且以STV、CMV及ToMV的复合侵染为主。从我国加工番茄上首次获得了长3 437 nt的STV SHZ-1核苷酸序列,序列比对分析发现其与已报道STV只有1~9个核苷酸的变异,且序列变异与地域无相关性。分析杂交F1代加工番茄植株上STV的传播特性,发现其除可由种子传播外,也可通过花粉传播。表明STV是侵染新疆番茄的主要病毒之一,且该病毒可通过种子或杂交育种途径进行传播。     

Transmission of the Pepino mosaic virus by whitefly

Two experiments were conducted to investigate the transmission of the Pepino mosaic virus (PepMV) by the greenhouse whitefly (Trialeurodes vaporariorum) from tomato to tomato. In the 1:1 system (in which a single virus-contaminated plant was placed next to a healthy plant in a cage containing 469 whiteflies on average) the virus was transmitted to three out of 10 plants. In the 1:4 system (in which a virus-contaminated plant was surrounded by four healthy plants in a cage with 601 whiteflies on average) the virus was transmitted to five out of 32 plants. In order to investigate the mechanism involved in the transmission, the insect bodies were washed to determine the external presence of viral particles. The results showed that the number of PepMV particles carried on whitefly bodies was low, with an average occurrence of 1.33 on the 55 whiteflies tested after the insects were in contact with infected plants for 5 days. This low occurrence was confirmed by observation under microscope, which showed an absence of PepMV-contaminated tomato sap on the insect bodies, suggesting that PepMV transmission by whiteflies could occur when they feed on the plant.     

Biological characterization of Tomato leaf curl New Delhi virus from Spain

Tomato leaf curl New Delhi virus (ToLCNDV; family Geminiviridae, genus Begomovirus) is an emerging virus in horticulture crops in Asia, and has recently been introduced in Spain, Tunisia and Italy. No betasatellite DNA was detected in infected tomato and zucchini squash samples from Spain, and agroinoculated viral DNA‐A and DNA‐B were sufficient to reproduce symptoms in plants of both crop species. Infected tomato and zucchini squash plants also served as inoculum sources for efficient transmission either mechanically or using Bemisia tabaci whiteflies. Cucumber, melon, watermelon, zucchini squash, tomato, eggplant and pepper, but not common bean, were readily infected using viruliferous whiteflies and expressed symptoms 8–15 days post‐inoculation. New full‐length sequences from zucchini squash and tomato indicated a high genetic homogeneity (>99% sequence identity) in the ToLCNDV populations in Spain, pointing to a single recent introduction event.     

Detection,characterization and host range studies of <Emphasis Type="Italic">Pepino mosaic virus</Emphasis> in Cyprus

Pepino mosaic virus (PepMV, Genus Potexvirus, Family Flexiviridae) is a mechanically transmitted viral disease that has emerged as a significant problem of greenhouse tomato crops in Europe and around the world. Although previous studies in Cyprus suggested that the virus was not present on the island, in 2009 tomato fruits from two major tomato production areas exhibited symptoms of yellow mosaic and discolouration, similar to those induced by PepMV. Consequently, an extensive survey was conducted in all tomato producing areas of the country to identify the incidence and prevalence of PepMV in protected and open field tomato crops. Analysis of 3500 leaf samples from tomato plants and weeds with DAS-ELISA and real-time RT-PCR showed that PepMV was present in all tomato growing areas of the island. The virus was detected in both protected and open field tomato plants, as well as in 20 weed species in the families of Amaranthaceae, Chenopodiaceae, Compositae, Convolvulaceae, Malvaceae, Plantaginaceae and Solanaceae. All Cypriot isolates assayed belonged to the CH2 genotype. Biological assays with two Cypriot isolates showed that they could infect cultivated and weed species including Vigna unguiculata, Solanum melongena, Nicotiana tabacum, Malva parviflora, Sonchus oleraceus, Solanum nigrum, Convolvulus arvensis, Chrysanthemum segetum and Calendula arvensis. To our knowledge, this is the first study to report Chrysanthemum segetum and Calendula arvensis as hosts of PepMV.     

Temporal distribution and pathogenicity of the predominant tomato‐infecting begomoviruses in Taiwan

Between 1998 and 2009, the four tomato‐infecting begomovirus species detected in Taiwan were Ageratum yellow vein Hualien virus (AYVHuV), Tomato leaf curl Taiwan virus (ToLCTWV), Tomato yellow leaf curl Thailand virus (TYLCTHV) and a newly defined species Tomato leaf curl Hsinchu virus (ToLCHsV). AYVHuV was detected occasionally in 2003 and ToLCHsV only in 2000–2001, whilst ToLCTWV was detected throughout the period. TYLCTHV was first detected in 2005. Between 1998 and 2005, >99% of the begomovirus‐positive samples were infected with ToLCTWV. In 2007 in western Taiwan, 16% of the positive samples were infected with ToLCTWV, 35% with TYLCTHV and 49% with mixed infection (ToLCTWV/TYLCTHV). In contrast, in eastern Taiwan the proportions were 84% ToLCTWV, 2% TYLCTHV and 14% mixed infection. However, throughout Taiwan in 2008–2009, most positive samples were either identified as TYLCTHV (51%) or mixed infection (ToLCTWV/TYLCTHV; 41%), and only 8% were ToLCTWV. This shows a clear trend of shifting from ToLCTWV to TYLCTHV and mixed infection over a short time period in Taiwan. Sequence analyses indicated that tomato‐infecting AYVHuV, an apparent recombinant between ToLCTWV and AYVHuV from Ageratum, represents a new strain Hsinchu. TYLCTHV Taiwan isolates were highly similar to each other, whereas ToLCTWV isolates had greater diversity and were classified into three strains which had one country‐wide and two local distributions. ToLCTWV and TYLCTHV were confirmed as monopartite and bipartite begomoviruses, respectively, by agroinfection followed by transmission with Bemisia tabaci biotype B. In addition, TYLCTHV was found to be mechanically transmissible together with viral DNA‐B.     

Viruses affecting tomato crops in Serbia

In a two-year survey (2011–2012), 3220 samples were collected and analyzed in order to determine the presence and distribution of viruses in tomato crops at 56 localities of 18 districts in Serbia. Out of 12 viruses tested, Cucumber mosaic virus (CMV), Potato virus Y (PVY), Alfalfa mosaic virus (AMV), Tomato spotted wilt virus (TSWV), Tomato mosaic virus (ToMV) and Tobacco mosaic virus (TMV) were detected in 42.1, 40, 11, 8.6, 2.3 and 1.3% of the total tested samples, respectively. The results revealed that CMV was prevalent in 2011 and PVY in 2012. CMV and PVY, apart from being predominant, were also the most widespread viruses. In general, single infections were the most frequent type of infection. Additionally, the most common mixed infections were double infections and the most prevalent combination was CMV and PVY. In 2011, the incidence of diseases and the percentage of all infection types were significantly higher than in 2012. Furthermore, in 2011, regardless of total single infections being prevalent compared to mixed infections, two prevailing viruses were commonly detected in mixed infections. The additional molecular testing of ELISA-negative samples using virus specific primers did not reveal the presence of Pepino mosaic virus (PepMV), Tomato yellow leaf curl virus (TYLC), Tomato infections chlorosis virus (TICV) and Tomato chlorosis virus (ToCV).     

Genetic characterization of <Emphasis Type="Italic">Pepino mosaic virus</Emphasis> isolates from Belgian greenhouse tomatoes reveals genetic recombination

Over a period of a few years, Pepino mosaic virus (PepMV) has become one of the most important viral diseases in tomato production worldwide. Infection by PepMV can cause a broad range of symptoms on tomato plants, often leading to significant financial losses. At present, five PepMV genotypes (EU, LP, CH2, US1 and US2) have been described, three of which (EU, LP and US2) have been reported in Europe. Thus far, no correlation has been found between different PepMV genotypes and the symptoms expressed in infected plants. In this paper, the genetic diversity of the PepMV population in Belgian greenhouses is studied and related to symptom development in tomato crops. A novel assay based on restriction fragment length polymorphism (RFLP) was developed to discriminate between the different PepMV genotypes. Both RFLP and sequence analysis revealed the occurrence of two genotypes, the EU genotype and the CH2 genotype, within tomato production in Belgium. Whereas no differences were observed in symptom expression between plants infected by one of the two genotypes, co-infection with both genotypes resulted in more severe PepMV symptoms. Furthermore, our study revealed that PepMV recombinants frequently occur in mixed infections under natural conditions. This may possibly result in the generation of viral variants with increased aggressiveness.     

Occurrence and distribution of viruses infecting tomato and pepper in Alibori in northern Benin

In surveys conducted in 2011 and 2012 to identify the viruses causing diseases on pepper and tomato in the department of Alibori in northern Benin, 451 samples of pepper and tomato were analyzed by ELISA using 11 specific antibodies. The highest virus incidence among the surveyed districts was recorded on pepper in Malanville (56.18%), followed by Karimama (39.32%). The most frequently found viruses were Pepper veinal mottle virus (PVMV), Cucumber mosaic virus (CMV), and Potato virus Y-necrotic (PVY-n), accounting respectively for 22.39%, 21.73% and 15.96% of the collected samples. Tomato yellow leaf curl virus (TYLCV) was detected in only 2.43% of the samples, whereas Alfalfa mosaic virus (AMV), Chilli veinal mottle virus (ChiVMV), Tomato mosaic virus (ToMV) and Tomato spotted wilt virus (TSWV) were not detected in any of the samples tested. Double and triple infections involving different virus combinations were found, respectively, in 14.86% and 4% of the samples. Five plant species (Euphorbia hirta Linnaeus, Moringa oleifera Lam, Leucas martinicencis (Jacquin) R. Brown, Combretum micranthum G. Don, Abelmoschus esculentus L. Moench.) out of 30 samples belonging to 13 botanical families, collected within or nearby tomato and pepper fields, were found infected with PVMV, PVY-n, and CMV. Control measures to reduce the impact of viruses on pepper and tomato production are discussed. This is the first report of viruses infecting pepper and tomato in Benin.     

Methods of controlling Tomato yellow leaf curl virus (TYLCV) and its vector Bemisia tabaci in the Maltese Islands*

Bemisia tabaci and Tomato yellow leaf curl virus (TYLCV) were first observed in Malta in the early 1990s and caused serious damage to glasshouse and outdoor tomato crops. Chemical, physical and biological control methods have been developed, but the effective method has been the use of virus‐tolerant cultivars.     

Lack of synergistic effects in tomato plants co-infected with tomato severe rugose virus and tomato chlorosis virus

The begomovirus Tomato severe rugose virus (ToSRV) and the crinivirus Tomato chlorosis virus (ToCV), in single and co-infections, are very common in tomato crops in Brazil. Both viruses are transmitted by the whitefly Bemisia tabaciMEAM1 (biotype B). The objective of this study was to analyse the interaction between ToSRV and ToCV in tomato plants of cultivars Santa Clara and Kada. Plants at 15, 30 and 45 days after emergence were inoculated with 30 viruliferous B. tabaci per plant. The following treatments were compared: plants inoculated with ToSRV, ToCV, ToSRV + ToCV, and healthy (control). The interaction between these viruses was analysed by measuring the virus titre by qPCR and the fresh and dry weights of the aerial parts of the tomato plants. Based on two independent assays, no significant effects for co-infection of ToSRV and ToCV on virus titres and plant development were observed compared to single infections. The dry weight of tomato plants of both cultivars infected with ToSRV, ToCV, or co-infected did not differ significantly. However, the dry weight of Santa Clara tomato plants infected with ToSRV, ToCV and ToSRV + ToCV showed mean reductions of 21.5%, 25.5% and 32%, respectively, compared to healthy plants, and mean reductions for Kada were 31.7%, 37.5% and 38%, respectively.     

Genetic variation and evolutionary analysis of Pepino mosaic virus in Sicily: insights into the dispersion and epidemiology

Pepino mosaic virus (PepMV) is a highly infectious potexvirus that causes a severe disease in tomato (Solanum lycopersicum) crops worldwide. In Sicily, the first outbreak was detected in a single greenhouse in 2005 and it was promptly eradicated. However, in 2008, a large number of greenhouses were simultaneously affected, and it was impossible to eradicate or control the virus. This study addressed the dispersion and the genetic diversity of PepMV isolates obtained from the outbreak in Sicily, in comparison with worldwide PepMV isolates, to gain insight into the factors determining the evolution and epidemiology of the virus. A total of 1800 samples from plants with and without symptoms were collected in the Sicilian provinces of Agrigento, Caltanissetta, Palermo, Ragusa, Siracusa and Trapani. Three isolates collected at different times were biologically characterized. The incidence of the virus increased rapidly from 13% in 2011 to 63% in 2013, and phylogenetic analysis showed that all Sicilian isolates of PepMV belonged to the CH2 strain, one of the six strains previously described. Nucleotide diversity of the Sicilian isolates was low, thus suggesting rapid spread and genetic stability.     

Occurrence of a new recombinant begomovirus species infecting tomato in the Al‐Batinah region of Oman

Whitefly‐transmitted begomoviruses are the most important limiting factor for tomato cultivation in Oman, particularly in the Al‐Batinah region, the major agricultural area of the country. Commercial farms in the Al‐Batinah region were surveyed during January–March 2013. Samples of tomato showing leaf curl disease symptoms typical of begomoviruses were collected and analysed. Full‐length sequences of five clones were shown to have relatively low percentage identity values to known begomoviruses, with the highest (88·6%) to isolates of Tomato leaf curl Oman virus (ToLCOMV), a begomovirus previously reported in Oman, indicating that these represent a newly identified species, for which the name Tomato leaf curl Barka virus (ToLCBrV) is proposed. Four isolates of ToLCBrV were found associated with Tomato leaf curl betasatellite (ToLCB). The five isolates of ToLCBrV characterized in this study were shown to be recombinants, with ToLCOMV as the major parent, and a fragment of Croton yellow vein virus (CrYVV) spanning the 3′ half of the replication‐associated protein. The significance of these findings is discussed.