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.     

Phytophthora species causing crown and root rot of tomato in southern Italy*

Phytophthora capsici, Phytophthora cryptogea and Phytophthora nicotianae were isolated from tomato plants with symptoms of crown and root rot in plastic‐house crops in Sicilia and Calabria (southern Italy). The species were identified primarily on the basis of morphological and cultural characteristics. The identification was confirmed using molecular methods, polyacrylamide gel electrophoresis (PAGE) of mycelial proteins and polymorphism of DNA sequences amplified by polymerase chain reaction using random primers (RAPD‐PCR). P. capsici caused significant losses in tomato crops that had succeeded capsicum crops. P. cryptogea was found to be the most frequent species causing basal stem rot of tomato, a disease of increasing importance in commercial tomato crops in plastic houses in Sicilia. P. nicotianae was common in plastic houses where poor drainage resulted in standing water.     

Genetic resistance derived from Solanum pimpinellifolium AAU2019 is monogenic recessive to tomato leaf curl virus

Tomato leaf curl disease is a severe threat to tomato production. Yield losses are generally high in the absence of effective management strategies. The disease is caused by tomato leaf curl virus (ToLCV) and is transmitted by a whitefly vector that is challenging to control. Resistance to ToLCV is absent from most cultivated tomato gene pools, although the use of resistant cultivars would provide a better control option than minimizing the vector population. Unfortunately, resistance sources based on field screening break down when virus pressure is severe. Our previous screening and virus testing of 40 tomato genotypes led to the identification of a highly resistant genotype, Solanum pimpinellifolium AAU2019, as a new source of resistance. In this study, we investigated the inheritance and genetics of resistance to ToLCV in the cross of Pusa Ruby × S. pimpinellifolium AAU2019 in F₂ and BC₁P_s populations, revealing a monogenic recessive gene (best-fit ratios of resistance: susceptible to be 1:3 and 0:1) responsible for ToLCV resistance in S. pimpinellifolium AAU2019. Hence, S. pimpinellifolium AAU2019 could be considered as a potential donor parent in breeding programmes to develop tomato cultivars with resistance to ToLCV.     

Tomato infectious chlorosis virus — a new clostero-like virus transmitted byTrialeurodes vaporariorum

A previously undescribed virus disease of tomato, other crops and weed hosts was found in California. Affected tomato plants exhibited interveinal yellowing, necrosis and severe yield losses. Leaf dips and purified preparations contained closterovirus-like long flexuous, filamentous particles approximately 12×850–900 nm. The virus, designated as tomato infectious chlorosis virus (TICV), is transmitted in a semipersistent manner by the greenhouse whitefly,Trialeurodes vaporariorum. The host range of the virus is moderate (26 species in 8 plant families) but includes some important crops and ornamental species including tomato, (Lycopersicon esculentum), tomatillo (Physalis ixocarpa), potato (Solanum tuberosum), artichoke (Cynara scolymus), lettuce (Lactuca sativa) and petunia (Petunia hybrida). The virus has been found in a number of different locations in California and has a number of potential vehicles of movement including greenhouse grown ornamentals, tomato transplants, artichoke cuttings and potato seed. The virus has the potential to spread to other growing regions with resident populations of the greenhouse whitefly. The host range, particle size, insect transmission, and serology clearly distinguish TICV from previously described viruses.     

Screening tomato genotypes for resistance and tolerance to Tomato chlorosis virus

Tomato chlorosis virus (ToCV) is an emerging crinivirus in Brazil that causes an economically important disease in tomato (Solanum lycopersicum) and other solanaceous species. ToCV is transmitted predominantly by the whitefly Bemisia tabaci Middle East‐Asia Minor 1 (MEAM1, formerly biotype B), in a semipersistent manner. As all cultivated tomato varieties and hybrids are susceptible to this crinivirus, the main alternatives for the control of the disease are the use of healthy seedlings for transplanting and the chemical control of the insect vector. The objective of this work was to evaluate the responses of tomato genotypes to infection with this crinivirus and their tolerance to the disease in order to support the development of other alternatives for disease control. Resistance to infection was evaluated by ToCV inoculation with viruliferous B. tabaciMEAM1 followed by virus detection by RT‐PCR and RT‐qPCR. To measure tolerance to the disease, plant development and fruit yield of ToCV‐infected and healthy plants were compared. Among 56 genotypes, only the lineage IAC‐CN‐RT (S. lycopersicum ‘Angela Gigante’ × S. peruvianum ‘LA 444‐1’) was highly resistant to infection with ToCV. Tolerance to the disease over two trials with different genotypes showed variable results. The effect of ToCV on plant development varied from 2.9% to 71.9% reduction, while yield loss varied from 0.2% to 51.8%. The highly ToCV‐resistant lineage IAC‐CN‐RT, which is also resistant to a Spanish isolate of ToCV, might be useful for tomato breeding programmes.     

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.     

Detection and molecular characterization of viruses associated with tomato yellow leaf curl disease in cucurbit crops in Jordan

In this study, Tomato yellow leaf curl Sardinia virus (TYLCSV) and the strains Israel and Mild of Tomato yellow leaf curl virus (TYLCV-IL, TYLCV-Mld) were detected for the first time in four cucurbit crops in Jordan by nested polymerase chain reaction (nPCR). These viruses cause the tomato yellow leaf curl disease (TYLCD) in tomato. Cucumber, squash, melon and watermelon plants inoculated with TYLCV-IL[JO:Cuc], TYLCV-Mld, TYLCSV-IT[IT:Sar:88] and the Jordanian isolate of TYLCV (TYLCV-JV) did not show disease symptoms. However, virus-specific fragments were detected in uppermost leaves of symptomless plants by nPCR. A whitefly transmission test showed that Bemisia tabaci could transmit TYLCV-Mld from cucumber into tomato and jimsonweed plants. However, all infected tomato plants remained symptomless. In addition, results of semi-quantitative PCR (sqPCR) analysis showed that the relative amount of TYLCV-Mld DNA acquired by B. tabaci from cucumber plants was less than that acquired from tomato plants.     

Pyramiding Ty‐2 and Ty‐3 genes for resistance to monopartite and bipartite tomato leaf curl viruses of India

Breeding resistance to whitefly‐transmitted begomoviruses is an important goal of tomato breeding programmes worldwide. So far, resistance to begomoviruses in tomato has been achieved using wild species, and at least five resistance genes (Ty genes) have been studied. The present study was undertaken to combine Ty‐2 and Ty‐3 and to determine the effect of pyramiding on infection of tomato by three diverse begomovirus species. The diagnostic ability of the markers linked to Ty genes was assessed and marker‐assisted selection was used to develop pyramided tomato lines from the crosses between Ty stocks. Five stable pyramided tomato lines that differ in fruit morphology and yield potential were developed. The horticultural performance of pyramided lines in field trials showed that the yield and horticultural traits are well maintained in the plants. The response of these lines was assessed using agroinoculation and field tests in a disease hotspot. The pyramided lines and Ty‐3‐carrying lines exhibited a high level of resistance to the monopartite and two bipartite begomoviruses tested. The pyramided tomato lines developed in this study could be important genetic resources for sustainable tomato production in areas affected by tomato leaf curl virus disease. The combined results of disease resistance tests also showed that Ty‐3 is critical for achieving broad‐spectrum resistance. The limitations of relying on a single gene and the importance of pyramiding are discussed in the light of available evidence on frequent recombination in begomoviruses.     

Farmers' perceptions and practices for managing tomato leaf curl virus disease in southern India

A survey of 174 tomato farmers in five districts of Karnataka State, South India, was conducted between March and July 1998 with the objectives of assessing farmers' knowledge and perceptions of tomato leaf curl virus disease (ToLCVD) and examining their current control practices. The findings showed that farmers were familiar with ToLCVD and regarded it as a serious constraint upon production, particularly in the hot season. However, only 2% of farmers were aware that tomato leaf curl virus (ToLCV) was transmitted by a whitefly vector, Bemisia tabaci . A similarly small number of farmers knew that ToLCVD was caused by a virus, whilst 86% of farmers believed that it was caused by high temperatures. Approximately 90% of farmers relied primarily on pesticides for control of ToLCVD. Few ToLCV-resistant varieties were available but one such variety, Avinash II, was grown by 13% of farmers in the hot season when incidence of the disease is highest. Farmers visiting experimental trials at a farmer field day, showed a strong interest in new advanced breeding tomato lines with resistance to ToLCV. Factors affecting the adoption of ToLCV-resistant varieties by tomato farmers are discussed.     

Panel of real‐time PCRs for the multiplexed detection of two tomato‐infecting begomoviruses and their cognate whitefly vector species

A new approach for the simultaneous identification of the viruses and vectors responsible for tomato yellow leaf curl disease (TYLCD) epidemics is presented. A panel of quantitative multiplexed real‐time PCR assays was developed for the sensitive and reliable detection of Tomato yellow leaf curl virus‐Israel (TYLCV‐IL), Tomato leaf curl virus (ToLCV), Bemisia tabaci Middle East Asia Minor 1 species (MEAM1, B biotype) and B. tabaci Mediterranean species (MED, Q biotype) from either plant or whitefly samples. For quality‐assurance purposes, two internal control assays were included in the assay panel for the co‐amplification of solanaceous plant DNA or B. tabaci DNA. All assays were shown to be specific and reproducible. The multiplexed assays were able to reliably detect as few as 10 plasmid copies of TYLCV‐IL, 100 plasmid copies of ToLCV, 500 fg B. tabaci MEAM1 and 300 fg B. tabaci MED DNA. Evaluated methods for routine testing of field‐collected whiteflies are presented, including protocols for processing B. tabaci captured on yellow sticky traps and for bulking of multiple B. tabaci individuals prior to DNA extraction. This work assembles all of the essential features of a validated and quality‐assured diagnostic method for the identification and discrimination of tomato‐infecting begomovirus and B. tabaci vector species in Australia. This flexible panel of assays will facilitate improved quarantine, biosecurity and disease‐management programmes both in Australia and worldwide.     

Ultrastructural aspects of tomato leaves infected by Tomato torrado virus (ToTV) and co‐infected by other viruses

Optical and electron microscopy studies were carried out to investigate the cytopathology induced in tomato leaves infected by Tomato torrado virus (ToTV), a new picorna‐like virus associated with the ‘Torrado’ disease. Infected leaves, showing typical Torrado disease symptoms were surveyed in commercial greenhouses in the main tomato production areas of Spain. The effect of the co‐infection of ToTV with other viruses which commonly infect tomato crops was also studied. Ultra‐thin sections of ToTV‐infected tomato leaves did not show a strong cellular alteration. However, crystalline arrays of isometric virus‐like particles (VLPs) of 20–30 nm in the inclusion bodies were observed in phloem parenchyma cells of the infected tissues. Tissues co‐infected by ToTV and either Tomato chlorosis virus (ToCV) or Pepino mosaic virus (PepMV) presented more severe cellular alterations. The most deleterious consequences for tomato cells were found in triple infections of ToTV, PepMV and Tomato spotted wilt virus (TSWV), where characteristic cell wall overgrowth was distinguishable, together with a large amount of necrotic cells.     

Evaluation of Datura stramonium and Nicandra physaloides as reservoirs of tomato severe rugose virus and whiteflies

Tomato severe rugose virus (ToSRV) is the most important begomovirus transmitted and spread by the whitefly Bemisia tabaci in tomato crops in Brazil. Cultural practices are being adopted, along with insecticides, for controlling this virus. However, little is known about the importance of weeds in the pathosystem, which can contribute to the failure of these practices. This work aimed to evaluate the role of Datura stramonium and Nicandra physaloides as alternative hosts of ToSRV and verify the viral influence on the biological performance of Bemisia tabaci Middle East-Asia Minor 1 (MEAM1) and Mediterranean (MED) cryptic species. N. physaloides was a better alternative host for ToSRV when combined with MEAM1 whiteflies, while D. stramonium was mostly a good host for whitefly reproduction. Viral infection improved MEAM1 performance on both host plants but affected MED performance negatively. These data suggest that both weeds can be of some importance for the pathosystem, and their control should be included in management programmes.     

Comparison of Resistance to Tomato Leaf Curl Virus (India) and Tomato Yellow Leaf Curl Virus (Israel) among Lycopersicon Wild Species, Breeding Lines and Hybrids

The objective of this study was to screen wild and domesticated tomatoes for resistance to Tomato yellow leaf curl virus, Israel (TYLCV-Is) and Tomato leaf curl virus from Bangalore isolate 4, India (ToLCV-[Ban4]) to find sources of resistance to both viruses. A total of 34 tomato genotypes resistant/tolerant to TYLCV-Is were screened for resistance to ToLCV-[Ban4] under glasshouse and field conditions at the University of Agricultural Sciences, Bangalore, India. Resistance was assessed by criteria like disease incidence, symptom severity and squash-blot hybridization. All the tomato genotypes inoculated with ToLCV-[Ban4] by the whitefly vector Bemisia tabaci (Gennadius) produced disease symptoms. In some plants of the lines 902 and 910, however, the virus was not detected by hybridization. The tomato genotypes susceptible to ToLCV-[Ban4] by whitefly-mediated inoculation were also found susceptible to the virus under field conditions. However, there were substantial differences between genotypes in disease incidence, spread, symptom severity and crop yield. Despite early disease incidence, many genotypes produced substantially higher yields than the local hybrid, Avinash-2. Sixteen tomato genotypes from India resistant/tolerant to ToLCV-[Ban4] were also tested for TYLCV-Is resistance at the Hebrew University of Jerusalem, Rehovot, Israel. Accessions of wild species, Lycopersicon hirsutum LA 1777 and PI 390659 were the best sources of resistance to both viruses. Lines 902 and 910, which were, resistant to TYLCV-Is were only tolerant to ToLCV-[Ban4] and accession Lycopersicon peruvianum CMV Sel. INRA, resistant to ToLCV-[Ban4], was only tolerant to TYLCV-Is. Implications of using the resistant lines in breeding programme is discussed.     

A selective barrier in the midgut epithelial cell membrane of the nonvector whitefly <Emphasis Type="Italic">Trialeurodes vaporariorum</Emphasis> to <Emphasis Type="Italic">Tomato yellow leaf curl virus</Emphasis> uptake

We studied the presence of a potential transmission barrier that blocks Tomato yellow leaf curl virus in the nonvector greenhouse whitefly, Trialeurodes vaporariorum. Because T. vaporariorum can ingest and retain the virus after acquisition feeding on an infected plant, comparable to the vector whitefly Bemisia tabaci, circumstance evidence suggested that a transmission barrier presents at location(s) where the virus moves from the digestive tract lumen to the hemolymph. To provide direct evidence for the site of a transmission barrier in the nonvector insect, we compared the accumulation levels and localization of the virus between the two species of whiteflies. Quantitative real-time and conventional PCR analysis showed that accumulation of the virus during acquisition feeding and retention after a short acquisition period were indistinguishable between the two species, but the circulation of the virus within the whiteflies differed significantly between the species. In an immunofluorescence analysis using an antibody specific to the coat protein of the virus, the virus was restricted to the luminal surface of the midgut epithelial cells and did not enter their cytoplasm or that of the salivary glands in T. vaporariorum. In contrast, the virus was localized within the cytoplasm of the midgut epithelial cells and in the paired salivary glands of B. tabaci adults. This direct evidence shows that a selective transmission barrier at the luminal membrane surface of midgut epithelial cells in the nonvector whitefly blocks entrance of the virus into the midgut epithelial cells, resulting in incompetence as a vector of the virus.     

Detection and typing of tomato yellow leaf curl viruses

The denomination Tomato yellow leaf curl virus (TYLCV) comprises several viruses that cause severe damage to tomato crops in warm and temperate regions worldwide. TYLCV viruses are widespread in the Mediterranean Basin, in which two species have been reported: Tomato yellow leaf curl Sardinia virus (TYLCSV) and Tomato yellow leaf curl virus (TYLCV, previously TYLCV-Is). The availability of methods convenient for the diagnosis of these viruses is essential. We have investigated several alternatives for reliable detection and differentiation of TYLCSV and TYLCV. Triple-antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA) proved to be very useful for large-scale diagnosis in field situations, but lacked discriminating capacity and sensitivity in the stages of infection in which low virus titre is present. The DNA-based methods are suited to laboratory operations and plant disease clinics, where accuracy of detection and discrimination of viruses is required. Polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) was the most reliable method to discriminate between TYLCSV and TYLCV, but is not suited to high sample turnover. For large-scale testing, tissue print hybridization assay provides a reliable and sensitive alternative to PCR.     

Tomato yellow leaf curl virus: host range and virus-vector relationships

The Jordanian isolate of tomato yellow leaf curl virus has a narrow host range restricted to a few solanaceous plants. Severe symptoms developed on tomatoes and Datura stramonium , whereas Nicotiana glutinosa and N. tabacum cvs Samsun and Havana 423 were infected without showing symptoms. The whitefly Bemisa tabaci is an efficient vector; a single whitefly was able to transmit the virus. The minimum acquisition and inoculation feeding periods were 60 and 30 min, respectively, and the latent period was 20–24 h. The virus was retained by Bemisia tabaci for 11 days. The host range and virus vector relationships of the Jordanian isolate are reported for the first time.     

Diversity of European begomoviruses: identification of a new disease complex*

The diversity of whitefly‐transmitted begomoviruses in Europe is low, most being exotic, introduced species. The only agriculturally important viruses are two species causing tomato yellow leaf curl. These viruses are believed to have originated in the Middle East but have since spread right across the Mediterranean region. Two ornamentals (Abutilon and Lonicera japonica) were introduced into Europe from the New World and the Far East, respectively, for the striking symptoms induced by the viruses which infect them. The virus infecting honeysuckle (Honeysuckle yellow vein mosaic virus) has been shown to be part of newly identified cluster of begomoviruses which require an additional component, a satellite molecule termed DNA β, to induce symptoms in their host plants. A further begomovirus, Ipomoea yellow vein virus, which infects the weed Ipomoea indica, is present in the Mediterranean region. The precise origin and relationship of this virus to other begomoviruses is unclear.     

The virus-vector relationship of the tomato leafcurl virus (TLCV) and its vector,Bemisia tabaci gennadius (Hemiptera: aleyrodidae)

Basic studies carried out in India showed that the incubation period of TLCV in plants varied from 8 days in August to 90 days in winter. The acquisition threshold for the whitefly,Bemisia tabaci Gen., was 31 min; it resulted in 3% transmission. An acquisition access of 24 h for a female whitefly on a TLCV source resulted in 30% transmission. A minimum feeding period of 32 min was required by a viruliferous whitefly to cause infection on tomato test plants; this gave 4% transmission. With inoculation access of 24 h on tomato test plants, the transmission rose to 24%. Starving the vector for 1 h pre-acquisition or 1 h pre-inoculation resulted in higher levels of transmission of TLCV: 36 and 40%, respectively, compared with 20% for non-starved whiteflies. Extending the fasting period beyond 1 h resulted in a reduced transmission level. The whiteflies could acquire the virus from the cotyledonary leaves of an infected tomato plant, with a resultant 28% transmission; but infection did not occur when the whiteflies had an inoculation access to such leaves. Higher transmission rates were obtained when the younger leaves on tomato plants were used for acquisition and inoculation. Transmission was 8 and 38% when five whiteflies per plant were allowed 24 h of acquisition access to 11- and 2-month-old virus sources, respectively. After an acquisition access of 24 h to a TLCV source, male and female whiteflies retained their infectivity for 5 and 53 days, respectively. Nymphs can acquire and transmit the virus. When ten whiteflies of each sex were given 24 h of acquisition and of inoculation access, the subsequent transmission rate of males and females was 56 and 86%, respectively. This virus is not transovarially transmitted. Whitefly colonies raised on brinjal were more efficient (70 and 84% transmission in two experiments) than those raised on chilli, cotton, cowpea, tobacco or tomato.     

Spread of Tomato yellow leaf curl virus in Sicily: Partial Displacement of Another Geminivirus Originally Present

The geminivirus Tomato yellow leaf curl virus (TYLCV) was reported for the first time in Italy in 2002. We have followed its spread in Sicily, where Tomato yellow leaf curl Sardinia virus (TYLCSV), another tomato-infecting geminivirus, is endemic and has been causing severe crop losses since 1989. The presence of the two viruses was monitored in the main tomato growing area, the Ragusa province, analyzing samples with yellow leaf curling symptoms. At first (spring–summer 2002) both viruses were always found in mixed infections, but in 2003 and 2004 18–35% of plants were found infected by TYLCV alone and 8–28% by TYLCSV alone, with 41–69% carrying both viruses. TYLCV has spread quickly in the area, demonstrating, as in other parts of the world, its high virulence and invasiveness; however it has not, so far, completely displaced TYLCSV. An infectious clone of TYLCV from Sicily (TYLCV-IT) was sequenced. The nucleotide sequence was 97% identical to other TYLCV strains of the ‘severe’ type, found in many countries worldwide.     

媒介虫口密度、植株苗龄及温度对B型烟粉虱传播台湾番茄曲叶病毒的影响

台湾番茄曲叶病毒(Tomato leafcurl Taiwan virus,ToLCTWV)近年来在我国部分番茄种植区流行成灾,对番茄生产造成了严重损失。作者研究了B型烟粉虱对ToLCTWV的获取、保持,并测定了虫口密度、温度及植株苗龄对其传播该病毒的影响。结果表明,B型烟粉虱在感染ToLCTWV的番茄植株上取食0.5h即可在3.3%个体内检测到ToLCTWV DNA,取食48h后,带毒率达100%;ToLCTWV DNA可在B型烟粉虱体内终生存留。B型烟粉虱是ToLCTWV的高效媒介,每株1头带毒烟粉虱取食48h,植株发病率达50%～60%;每株5头取食,发病率达到或接近100%。介体虫口密度及植株苗龄是决定苗期发病程度的重要因子,虫口密度增加,病情指数上升;苗龄增大,病情指数下降。温度对发病率有显著影响,但对病情指数影响不显著。此外,温度与苗龄的互作、虫口密度与苗龄的互作以及三因子之间的互作都对病情指数有显著影响。这些结果提示,培育无病壮苗是防治台湾番茄曲叶病毒病的关键。