First Report of Tomato Yellow Leaf Curl Virus-Israel Species Infecting Tomato, Pepper and Bean in Tunisia

Tomato yellow leaf curl virus disease (TYLCVD) has been observed in Tunisia for more than 20 years. Until year 2004, only the Tomato yellow leaf curl Sardinia virus‐Sicily (TYLCSV‐[Sic]) was detected in tomato, pepper and bean crops. In the Sahel region, some tomato samples showing severe TYLCVD symptoms were collected from greenhouses in 2004 and 2005. Typing of these isolates revealed for the first time the presence of the TYLCV Israel in Tunisia. This result was confirmed by using several sets of specific primers and by sequencing. This species has also been detected on pepper and bean collected from fields in the same region. The sequencing of a tomato and a bean isolate showed that they both share more than 97% of sequence identity with the TYLCV from Dominican Republic ( AF024715 ). The TYLCV has been found in single and mixed infection with the TYLCSV‐[Sic].     

Occurrence of Six Begomoviruses Infecting Tomato Fields in Venezuela and Genetic Characterization of Potato Yellow Mosaic Virus Isolates

Begomoviruses are one of the major pathogens in tomato crops worldwide. In Venezuela, six begomovirus species have been described infecting tomato: Potato yellow mosaic virus (PYMV), Euphorbia mosaic Venezuela virus (EuMVV), Merremia mosaic virus (MeMV), Tomato chlorotic leaf distortion virus (ToCLDV), Tomato yellow margin leaf curl virus (TYMLCV) and Tomato yellow leaf curl virus (TYLCV). In this study, the occurrence of these viruses was analysed by PCR in 338 tomato plants exhibiting virus‐like symptoms. Sixty‐three per cent of the plants were positive at least to one of the begomoviruses tested. PYMV and TYLCV were the most frequent viruses showing 39.6 and 23.7% occurrence, respectively. Phylogenetic analyses revealed two groups of PYMV isolates from several Caribbean Basin countries. The first group clustered isolates from several countries, including Venezuela, and the second group clustered only Colombian isolates. Due to the high prevalence of PYMV and TYLCV in Venezuela, it is suggested that the surveillance and control strategies currently applied in the country should be focused on these two begomoviruses.     

Rapid Detection and Identification of Six Tomato yellow leaf curl virus Isolates from Different Regions Using Polymerase Chain Reaction and Restriction Enzyme Analysis

The combinational analysis of polymerase chain reaction and restriction enzyme analysis (PCR‐RE) to distinguish six Tomato yellow leaf curl virus (TYLCV) isolates from five countries was developed. Tomato yellow leaf curl virus has spread from the Middle East to Western Europe, Central America and Eastern Asia, and occurs on infected crops such as tomatoes, peppers, cucurbits and beans. Tomato yellow leaf curl virus isolates from Jordan (TYLCV‐Mld[Jo:Cuc] and TYLCV‐IL[Jo:Cuc]), Israel (TYLCV‐IL[IL:Reo:86]), Spain (TYLCV‐Mld[ES72/97]), USA (TYLCV‐IL[US:F10:04]) and Korea (TYLCV‐KR) were collected, and the sequences of the six isolates were analysed to distinguish them by PCR‐RE combination analysis. Oligonucleotide primers for the six TYLCV isolates were designed to amplify approximately 740 base pairs including the intergenic region (IR) and parts of V1 and V2 ORF. Unique restriction enzyme sites were analysed to identify isolate‐specific restriction enzyme sites on the PCR products of each isolate. Three enzymes (DdeI, FauI and BssSI) were selected by in silico analysis, and then, the PCR products following the serial digestion of each restriction enzyme were separated by agarose gel electrophoresis to distinguish the TYLCV isolates. Taken together, the PCR‐RE combination analysis by serial digestion with three restriction enzymes could be a useful method for distinguishing the six isolates.     

Cytopathology of Tomato torrado virus Infection in Tomato and Nicotiana benthamiana

Electron microscopy studies were carried out to investigate the cytopathological changes induced in tomato leaves by Tomato torrado virus (ToTV) that infects tomato plants worldwide causing severe necrotic symptoms. Plants infected with one of the Polish isolates of ToTV were used for cytopathological research. The results revealed severe cellular alterations, especially in Solanum lycopersicum. Moreover, it was shown that crystalline aggregates of virions occurred not only within the phloem cells as it has been previously reported.     

Begomoviruses Associated with Leaf Curl Disease of Tomato in Java,Indonesia*

We report that several begomoviruses are associated with tomato leaf curl disease in Java, Indonesia. Tomato plants with leaf curl symptoms were collected from Bandung (west Java), Purwokerto (central Java), Magelang (central Java) and Malang (east Java) of Indonesia, the major tomato‐growing areas of the country. Viruses were detected using the polymerase chain reaction (PCR), with universal primers for the genus Begomovirus. PCR‐amplified fragments were cloned and sequenced. Based on sequence comparisons and phylogenetic analyses, the viruses were divided into three groups. With respect to amino acid (aa) identities of the N‐terminal halves of the coat proteins compared in this study, group I was most closely related to Ageratum yellow vein virus (AYVV) (97%), Ageratum yellow vein China virus‐[Hn2] (AYVCNV‐[Hn2]) (96%) and Ageratum yellow vein virus‐[Taiwan] (AYVV‐[Tai]) (95%), and ageratum‐infecting begomovirus from Java (99%). Group II had high sequence identity with a tentative species of tomato leaf curl Java virus (ToLCJAV) (96% aa) for the CP. Group III was most closely related to a proposed species of Pepper yellow leaf curl Indonesia virus (PepYLCIDV) (90% aa identity) by its partial CP sequence.     

Begomoviruses Associated with Yellow Leaf Curl Disease of Tomato in Iran*

The occurrence of Tomato yellow leaf curl virus (TYLCV; genus Begomovirus, family Geminiviridae) in the major tomato‐growing areas of Iran was determined using TAS‐ELISA and PCR. The nucleotide sequences of the coat protein (CP) gene and intergenic region (IR) of eight Iranian isolates were determined. CP nucleotide identities among the Iranian isolates were 96–98%, and showed 94–96% identity with TYLCV‐IR [IR:Ira:98] and TYLCV‐IL [IL:Reo:86]. However, they showed low identity (68–69%) with ToLCIRV‐[IR:Ira]. Sequence analyses of IR indicated that seven Iranian isolates had sequence identity of 93–100% with each other, and 76% identity with the Jiroft isolate; identities of 75–79% with TYLCV‐IR[IR:Ira:98] were observed in every case, and 59–62% identity with ToLCIRV‐[IR:Ira]. The IR nucleotide sequences of Iranian isolates showed 92–93% identity with TYLCV‐IL[IL:Reo:86], except the Jiroft isolate (75%). The CP and IR sequence analyses suggested that eight Iranian TYLCV isolates probably differ from ToLCIRV‐[IR:Ira]. Based on IR sequence comparisons and phylogenetic analyses, the Iranian isolates were divided into two groups. The first major group (A), consists of seven virus isolates, was most closely related to TYLCV‐IL[IL:Reo:86], and relatively divergent from TYLCV‐IR [IR:Ira:98] and ToLCIRV‐[IR:Ira]. However, the Jiroft isolate from group B did not show high similarity with TYLCV‐IR[IR:Ira:98], ToLCIRV‐[IR:Ira], and TYLCV‐IL[IL:Reo:86], suggesting that the isolate may be a divergent variant. The differences are in a range that suggests different strains or species from TYLCV‐IR[IR:Ira:98] and ToLCIRV‐[IR:Ira] are probably associated with tomato yellow leaf curl disease in Iran.     

Identification of Mungbean yellow mosaic Indian virus Associated with Tomato Leaf Curl Betasatellite Infecting Phaseolus vulgaris in Oman

Kidney bean (Phaseolus vulgaris) plants exhibiting foliar yellow mosaic symptoms and some leaf crumpling were identified in the Al Batinah region of Oman. Rolling circle amplification and polymerase chain reaction identified a bipartite begomovirus (family Geminiviridae) and a betasatellite in association with the symptomatic plants. Analysis of full‐length sequences showed the virus to be Mungbean yellow mosaic Indian virus (MYMIV) and the betasatellite Tomato leaf curl betasatellite (ToLCB). This is the first identification of a legume‐adapted begomovirus in Oman and the first identification of MYMIV in association with the betasatellite ToLCB. The isolate of MYMIV from Oman shows the greatest levels of sequence identity to isolates occurring in South Asia and South‐East Asia, suggesting that the virus has only recently been introduced. The significance of these findings is discussed.     

Identification of a distinct strain of Cotton leaf curl Gezira virus infecting tomato in Oman

Tomato (Solanum lycopersicum) plants exhibiting yellowing, curling and stunting symptoms were identified in fields of the Tawoos Agricultural Systems, in Al‐Batinah in Oman. Cloning and sequencing of restriction endonuclease digested rolling circle amplified viral DNA identified a cotton begomovirus (family Geminiviridae) associated with the symptomatic tomato plants. Detailed analysis of complete sequences showed the virus to be a previously unknown strain of Cotton leaf curl Gezira virus (CLCuGeV) in association with the betasatellite Tomato leaf curl betasatellite (ToLCB). The new CLCuGeV strain, for which the name “Al Batinah” strain is suggested, has the greatest levels of sequence identity (91.9%) to an isolate of CLCuGeV recently reported from the neighbouring United Arab Emirates. Additionally, CLCuGeV‐Al Batinah was shown to have a recombinant origin with sequences donated by an African cassava mosaic virus‐like parent. This is the first identification of this Malvaceae‐adapted begomovirus in tomato. Although ToLCB is common in Oman, being one of only two betasatellites identified there so far, this is the first identification of this betasatellite with CLCuGeV. The significance of these findings is discussed.     

Low Genetic Diversity Suggests a Single Introduction and Recent Spread of Tomato chlorosis virus in Brazil

By comparing the partial nucleotide sequences of the heat shock protein HSP70 homologue gene, we assessed the genetic diversity of Brazilian tomato isolates of Tomato chlorosis virus (ToCV), as well as their relationship with other ToCV isolates found worldwide. The Brazilian ToCV isolates shared 99.9–100% nucleotide identity, which indicates low genetic diversity. Brazilian ToCV isolates showed a closer evolutionary relationship to those from Mediterranean countries. Based on these results, the origin of Brazilian ToCV isolates and the possible number of introductions of the virus into Brazil are discussed.     

Identification and Molecular Characterization of a New Geminivirus Betasatellite Infecting Malvastrum coromandelianum in China

The complete nucleotide sequence of a satellite molecule associated with Malvastrum leaf curl Guangdong virus (MLCuGdV) infecting M. coromandelianum plants exhibiting leaf curl symptoms in a suburb of Guangzhou, Guangdong Province of China, is described and analysed. The molecule has typical features of betasatellites, containing a single ORF in the complementary‐sense strand, an A‐rich region, the satellite‐conserved region and a stem–loop structure. Compared with the geminivirus betasatellites in GenBank database, this molecule shows the highest nucleotide sequence identity of 71.9% with Tomato leaf curl Philippine betasatellite isolate Laguna1 (ToLCPB, AB307732). Phylogenetic analysis indicates that it is more related to isolate Laguna 1 and Laguna 2 of ToLCPB. According to the proposed species demarcation threshold of betasatellites (78% nucleotide identity), it is a novel betasatellite species, for which we propose the name Malvastrum leaf curl Guangdong betasatellite (MLCuGdB).     

Natural Spread of Plant Viruses by Birds

Observations made in Mali strongly suggest that Rice yellow mottle virus (RYMV) is spread by weaverbirds (Quelea quelea) below and around baobab trees (Adansonia digitata) in which they nest. Rice leaves in bird nests appeared to be infected. In Spain, an infection of Southern bean mosaic virus (SBMV) in string (climbing) beans (Phaseolus vulgaris) was apparently introduced and spread by sparrows (Passer domesticus) judging from the damage caused on flowers and bean pods. Damaged leaves and pods on SBMV‐infected plants were also found in a screenhouse visited by sparrows and bulbuls (Pycnonotus barbatus) in Morocco. These observations showed that both viruses could be spread by birds when either collecting infected leaves for nesting or feeding on infected plants.     

Different effects of exogenous jasmonic acid on preference and performance of viruliferous Bemisia tabaci B and Q

The sweetpotato whitefly, Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae), B and Q biotypes have caused severe losses to crops and vegetables through virus transmission. Our previous studies showed that Q is more efficient than B in acquisition and transmission of tomato yellow leaf curl virus (TYLCV) and viruliferous Q is better equipped than B in counterattacking jasmonic acid (JA)‐related plant defense. To understand how plant‐mediated defensive responses involving JA affect insect vectors within a tritrophic framework of plants, insects, and viruses, we examined the effects of exogenous JA on preference and performance of non‐viruliferous and viruliferous B and Q on tomato plants (Solanum lycopersicum L., Solanaceae). Our results demonstrated a significantly lower fecundity, shorter longevity, shorter developmental time, and lower survival rate of whiteflies on JA‐treated than on control plants. In addition, viruliferous Q performed significantly better than B in fecundity, longevity, developmental time, and survival rate. When given a choice between JA‐treated and control tomato plants, viruliferous Q was not repelled to JA‐treated plants when the JA concentration was 0.01 and 0.1 mm , whereas others all preferred the untreated control plants. Exogenous JA increased the concentration and the composition of plant volatiles, such as α‐terpinene and β‐ocimene, which deterred whiteflies in a Y‐tube bioassay. It is worth noting that Q has a mutualistic relationship with TYLCV to counteract the host defenses. A better understanding of tritrophic interactions between plants, insects, and viruses will facilitate the development of sustainable management of this invasive global pest.     

Recombination Among Begomoviruses on Malvaceous Plants Leads to the Evolution of Okra Enation Leaf Curl Virus in Pakistan

Whitefly transmitted begomoviruses (family Geminiviridae) are the major reason for significant yield losses of dicotyledonous crops in tropics and subtropics. Okra (Abelmoschus esculentus) is one of the important vegetable crops, and leaf curl disease caused by geminiviruses is the most important limiting factor for its production in Pakistan. Here, we report a new species of okra‐infecting begomovirus in south‐eastern region of Pakistan and the name Okra enation leaf curl virus (OELCuV) complex is proposed. This okra enation leaf curl disease complex (OELCuD) in Pakistan is found to be associated with Ageratum conyzoides symptomless alphasatellite (AConSLA). All efforts to clone the betasatellite were unsuccessful. Comprehensive sequence analyses suggest that intermalvaceous recombination between okra and cotton‐infecting begomoviruses resulted in the evolution of the new species. Surprisingly, Bhendi yellow vein mosaic virus (BYVMV) which has not been reported previously from Pakistan is the major parent while Cotton leaf curl Multan virus (CLCuMV) acts as a distant parent of the virus. Comparative recombination analysis also reveals that okra‐infecting begomoviruses from south and north‐western India is causing OELCuD in the Pakistan by recombining with CLCuMV at the Rep (1964–1513 nts). Recombination is common among geminiviruses and recombining of BYVMV and CLCuMV resulted in a new species: OELCuV. To the best of our knowledge, this evolution of a new species of okra‐infecting begomovirus is the first report of intermalvaceous recombination where Rep acts as the target region.     

Biological and Molecular Characterization of Alfalfa Mosaic Virus Infecting Trumpet Creeper (Campsis radicans) in Iran

Samples of trumpet creeper (Campsis radicans) leaves showing mottling and mosaic were collected from plants growing in a private garden in Tehran province, Iran, in 2012. Symptomatic leaf samples were tested for Alfalfa mosaic virus (AMV), Cucumber mosaic virus (CMV) and Peanut stunt virus (PSV) infection in enzyme‐linked immunosorbent assay (ELISA), using specific antibodies. None of the samples were positive for CMV and PSV; however, all reacted positively with that of AMV antiserum. In biological assay, systemic infection was found on Datura stramonium, Nicotiana tabacum cvs., White Burley, and Xanthi, 21 days postinoculation (DPI), while necrotic local lesions were obtained following inoculation of Phaseolus vulgaris and Vigna unguiculata within three to four DPI. Using a pair of primers specific for AMV, a DNA fragment of 880 bp was RT‐PCR‐amplified. Analysis of the sequences revealed the presence of 657 nucleotides of AMV complete coat protein (CP) gene (translating 218 amino acid residues). Phylogenetic analysis using neighbour‐joining (NJ) method clustered AMV isolates into two main types and the IRN‐Tru (GenBank Accession No. JX865593 ) isolate fell into type I. Pairwise nucleotide distances also confirmed two main types with the highest and lowest similarities for type I and II, respectively. The association of AMV with mosaic disease of C. radicans represents the first record from the world.     

First Report of Cucumber mosaic virus Infecting Chinese Mallow in China

The virus in naturally infected, stunted Chinese mallow plants and mosaic leaves was identified as Cucumber mosaic virus (CMV). Six symptomatic plants and one symptomless plant were collected in Chongqing, China. DAS‐ELISA suggested CMV was likely associated with the diseased Chinese mallow. Double‐stranded RNA was extracted from the samples, analysed by RT‐PCR, and the coding sequences of their coat proteins (CPs) were sequenced. The results further confirmed CMV was the pathogen causing Chinese mallow stunted, mosaic disease. The isolate was named CMV‐DXC. The full sequence of CMV‐DXC CP was determined, and it had the highest nucleotide identity (99.4%) of those of CMV‐lily, CMV‐WSJ and CMV‐Hnt, respectively. Phylogenetic analysis shows that CMV‐DXC belongs to CMV subgroup II. To our knowledge, this is the first report of CMV infecting Chinese mallow in China.     

Temporal Dynamics of Tomato Severe Rugose Virus and Bemisia tabaci in Tomato Fields in São Paulo,Brazil

Temporal progress of a begomovirus disease in tomato fields and the abundance of its whitefly vector, Bemisia tabaci biotype B, were evaluated during three consecutive tomato plantings in the municipality of Sumaré, state of São Paulo, Brazil, in 2006 and 2007. The incidence of symptomatic plants and the number of adult whiteflies were weekly monitored on experimental plots randomly chosen in tomato commercial fields. Tomato severe rugose virus (ToSRV) was identified as the causal agent of the disease, and its relationships with other Brazilian begomoviruses was confirmed by partial and complete nucleotide sequencing of the viral genome. The disease temporal progress was analysed by fitting different models to disease incidence. The monomolecular model showed the best fit, which is consistent with a predominant role of primary spread in the epidemiology of ToSRV. A higher number of adult whiteflies were observed at the borders of the plots, also suggesting primary spread of ToSRV from external sources of inoculum, which might be represented by weeds and volunteer tomato‐infected plants. In Brazil, since 2004, there is a legislative measure that mandates, for some regions of processing tomato plantings, a 2‐month crop‐free period during the year. Based on our results, we suggest the extension of this measure to all tomato‐producing regions, including fresh market tomato. We also suggest that growers emphasize the elimination of old plants from harvested fields that can serve as virus reservoirs several weeks prior to new plantings and weeds nearby the fields to limit the primary spread of ToSRV.     

Begomovirus and Associated Satellite Components Infecting Cluster Bean (Cyamopsis tetragonoloba) in Pakistan

Cluster bean (Cyamopsis tetragonoloba) is a legume that is grown widely on the Indian subcontinent. Leaf curl symptoms of cluster bean plants collected in the Punjab, Pakistan, were shown to be associated with the begomovirus Papaya leaf curl virus; the first time this virus has been identified infecting cluster bean in Pakistan. The virus was shown to be associated with Tomato leaf curl betasatellite. Additionally, some cluster bean plants were shown to also harbour Cotton leaf curl Multan alphasatellite. The significance of these findings is discussed.     

Marker Gene Overexpression in Flowers Treated with Resistance Inducers does not Correlate with Protection Against Flower‐Infecting Fungi in Tomato and Blueberry

Flowers can serve as infection courts for specialized and unspecialized plant pathogens, but little is known about the ability of floral tissues to undergo induced resistance (IR) responses against these pathogens. We studied the expression of IR marker genes in tomato and blueberry flowers treated with the inducers methyl jasmonate (MeJA), benzothiadiazole‐S‐methyl ester (BTH) and 2,6‐dichloroisonicotinic acid (INA). In tomato, spray application of MeJA and BTH (but not INA) to entire plants (leaves, stems and flowers) resulted in a significant (P < 0.05) overexpression of Pin2 (5.2‐fold) and PR‐4 (5.6‐fold) in pistil tissues, respectively. A statistically similar expression was obtained in pistils when flowers were protected from direct spray, indicating a systemic response. In blueberry, where information about IR marker genes is limited, PR‐3 and PR‐4 orthologs were first identified and characterized using in silico and wet‐laboratory techniques. In subsequent induction experiments, INA and BTH induced overexpression of PR‐4 in blueberry pistils by 3.2‐ and 1.8‐fold, respectively, when entire plants were treated. In blueberry flowers protected from spray applications, all chemicals applied to vegetative tissues led to significant overexpression of PR‐4 (MeJA: 1.4‐fold, BTH: 2.9‐fold and INA: 1.6‐fold), with BTH also inducing PR‐3 (1.7‐fold). The effect of these responses in protecting flowers was studied by inoculating treated tomato flowers with the necrotroph Botrytis cinerea and blueberry flowers with the hemi‐biotroph Monilinia vaccinii‐corymbosi. In both pathosystems, no significant disease suppression associated with resistance inducer application was observed under the conditions studied. Thus, although IR marker genes were shown to be inducible in floral tissue, the magnitude of this response was insufficient to suppress pathogen ingress.