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A major resistance gene of the melon accession PI414723 to zucchini yellow mosaic virus (ZYMV) is located at the Zym-1 locus on chromosome 2, but the underlying defense mechanisms are poorly understood. The physiological responses and expression of selected genes at Zym-1 were assessed in PI414723 and in the susceptible genotype Védrantais. Viral titers and the expression of genes related to systemic acquired resistance (SAR) were evaluated in inoculated (Inoc) and non-inoculated (Non-Inoc) portions of the cotyledons at 3, 7 and 10 days after inoculation (dai) and in apical leaves at 10 dai. ZYMV was detected in both portions of the cotyledons but not in the apical leaves of PI414723 plants. Also, ZYMV was recovered in a susceptible zucchini only from Inoc portions at 3 dai. By contrast, in Védrantais ZYMV was detected and recovered from all tissues at high concentrations. Starchy local lesions and accumulation of transcripts of the SAR marker genes PR1 and PR4 were also detected in the resistant genotype. Plus, transcripts of one candidate resistance gene analog previously located at Zym-1 and of two melon homologs of restricted tobacco etch virus movement 2 (RTM2) genes located close to Zym-1 , accumulated only in PI414723. It is proposed that resistance results from the combined action of the R gene, involved in restricting ZYMV replication after a supposed recognition event and of the RTM genes which impact viral systemic movement to distal apical tissues.

Adenium obesum plants showing virus-like symptoms were collected in several regions of Brazil. Mottling symptoms like those observed in symptomatic plants in the field were reproduced in mechanically inoculated A. obesum plants. This potexvirus was named "desert rose mottle virus" (DRMoV), and its genome sequence was first determined by high-throughput sequencing and then confirmed by Sanger sequencing. The complete genome of DRMoV is 6,781 nt in length, excluding the poly(A) tail, and five ORFs were predicted in order from 5' to 3': Rep-TGB1-TGB2-TGB3-CP. Phylogenetic analysis based on Rep amino acid sequences showed different clustering among potexviruses. These data suggest that RDMoV is a new member of the genus Potexvirus, and the binomial name "Potexvirus adenii" is proposed for its species., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Plumeria pudica , known as bridal bouquet, exhibiting characteristic symptoms of orthotospovirus infection were found in different localities in Brazil. Symptoms were restricted to leaves of the middle and lower thirds of a few branches of each plant. Electron microscopy, molecular analyses, and complete genome sequencing identified the orthotospovirus as groundnut ringspot virus (GRSV),member of the species Orthotospovirus arachianuli . The virus was poorly transmitted mechanically to P. pudica . Reverse transcription polymerase chain reaction (RT-PCR) and reverse transcription quantitative polymerase chain reaction (RT-qPCR) analyses performed using total RNA extracted from leaf blades, primary veins, petioles, and regions of petiole insertion on branches indicated the presence of GRSV, predominantly in the symptomatic leaf blades. Symptomatic branches propagate vegetatively, often resulting in plants expressing GRSV symptoms. In contrast, vegetative propagation of the asymptomatic branches of infected plants predominantly generates plants without GRSV symptoms. The resistance of P. pudica plants to GRSV infection, restricted systemic viral movement, and expression of symptoms in infected plants suggest that this orthotospovirus does not threaten this ornamental plant., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Favara, Oliveira, Ferro, Kraide, Carmo, Bello, Ribeiro-Junior, Krause-Sakate, Kitajima and Rezende.)

Tomato severe rugose virus (ToSRV) is one of Brazil's main begomoviruses infecting tomato ( Solanum lycopersicum ). Recent studies indicate that soybean ( Glycine max ) crops harboring the whitefly Bemisia tabaci Middle East-Asia Minor 1 (MEAM1) may have epidemiological significance by acting as an asymptomatic amplifier host for the virus. In this study, we gathered experimental greenhouse and field evidence of the role of soybean in the epidemiology of the disease caused by ToSRV. Tomato and Nicandra physalodes , known as good sources of inoculum of this begomovirus, were used as references. The infection rates of soybean, tomato, and N. physalodes with ToSRV in greenhouse no-choice transmission tests with B. tabaci MEAM1 were 50, 71.4, and 64.2%, respectively. The transmission efficiencies of ToSRV to tomato when B. tabaci MEAM1 acquired the virus in ToSRV-infected soybean, tomato, and N. physalodes were 43, 33, and 20%, respectively. Leaves of ToSRV-infected soybean, tomato, and N. physalodes used as sources of inoculum had similar virus titers. In the host preference assay, viruliferous whiteflies preferred to land on tomato rather than soybean and N. physalodes , whereas aviruliferous whiteflies landed indistinctly on these plants. Under experimental field conditions, the transmission efficiency of ToSRV to tomato was higher when tomato was used as a source of inoculum, followed by N. physalodes and soybean. Considering that soybean is extensively cultivated in several Brazilian states that also grow tomato, it can serve as an efficient asymptomatic source of inoculum and support the recent hypothesis that it can also play, under certain conditions, a relevant role as an amplifier host in the epidemiology of the disease caused by ToSRV.

Zinnia sp. is a genus belonging to Asteraceae family, originated in Mexico and adapted to a warm-hot climate (Hemmati and Mehrnoosh, 2017). Several types of zinnias with different flower color and forms are cultivated in Brazil (Min et al., 2020 and Souza Jr. et al., 2020). Characteristic symptoms of infection caused by orthotospovirus, including chlorotic spots and concentric rings on the leaves, were observed in two plants of Zinnia sp. of a florist located in the city of Piracicaba, State of São Paulo, Brazil. Orthotospovirus-like particles were observed by transmission electron microscope in leaf extracts from both plants, stained negatively with 1% uranyl acetate. By analyzing ultrathin sections of infected leaf tissues, particles of 80-100 nm in diameter were found in the lumen of the endoplasmic reticulum and nucleocapsid aggregates in the cytoplasm. Total RNA extracted separately from the leaves of both samples, using the Purelink Viral DNA / RNA kit (Thermo Fisher Scientific), was used to detect the virus by reverse transcription polymerase chain reaction (RT-PCR), using the universal primers for orthotospovirus BR60, complementary to the 3' end of the non-translated region of the S RNA (position 1 to 15 nt), and BR65, matching the nucleocapsid gene (N) (position 433 to 453 nt), generating and amplicon of 453 nt (Eiras et al., 2001). Amplicons of the expected size were obtained for the two samples. An amplicon was purified with the Wizard SV Gel and PCR Clean-Up System kit (Promega) and sequenced in both directions at Macrogen Inc (South Korea). The nucleotide sequence (GenBank MW629018) showed 99.29-99.76% identity with nucleotide sequences of the orthotospovirus groundnut ringspot virus (GRSV) isolates (GenBank MH686229 and KY400110). Leaf extracts from symptomatic plants were also analyzed by plate-trapped antigen-enzyme-linked immunosorbent assay (PTA-ELISA), using polyclonal antiserum produced against the GRSV nucleocapsid protein (Esquivel et al., 2019). The absorbance values obtained for the extracts of the two symptomatic plants of Zinnia sp. (1.3 and 1.7) were twice as high as the value obtained for the healthy plant extract (0.5). Leaf extract of symptomatic Zinnia sp. was inoculated mechanically onto leaves of healthy plants of Zinnia sp., Capsicum annuum cv. Dara, Cucumis sativus, Cucurbita pepo cv. Caserta, Chenopodium amaranticolor, Datura stramonium, Nicotiana tabacum cv. Turkish and Solanum lycopersicum cv. Compack. At 5 days post inoculation (dpi), inoculated leaves of D. stramonium reacted with local lesions, and at 9 dpi, newly developed leaves of inoculated S. lycopersicum plants showed necrotic spot and concentric ring symptoms, whereas C. annuum exhibited concentric rings at 10 dpi. Inoculated zinnia plants showed systemic chlorotic spot and concentric ring symptoms at 20 dpi, indistinguishable from those observed under natural infection. The other inoculated plant species were not symptomatic, nor the virus was detected. PTA-ELISA and RT-PCR confirmed infection with GRSV in symptomatic plants. The amplicons generated by RT-PCR of total RNA extracted from an experimentally infected plant of C. annuum and D. stramonium, and two plants of Zinnia sp. were sent for nucleotide sequencing. The obtained nucleotide sequences (MW629019, MW629020, MW629021, MW629022) shares 100% identity with the nucleotide sequence corresponding to the original GRSV isolate (MW629018) identified in Zinnia sp. This is the first report of the natural occurrence of GRSV in Zinnia sp. in Brazil. Studies on incidence and damage are needed to recommend alternatives for management.

Tomato chlorosis virus (ToCV; genus Crinivirus , family Closteroviridae ) was identified in tomato crops in São Paulo State, Brazil, in 2006. Management strategies to control external sources of inoculum are necessary, because chemical control of the whitefly vector Bemisia tabaci Middle East-Asia Minor 1 (MEAM1) has not efficiently prevented virus infections and no commercial tomato varieties or hybrids are resistant to this crinivirus. We first evaluated the natural infection rate of some known wild and cultivated ToCV-susceptible hosts and their attractiveness for B. tabaci MEAM1 oviposition. Physalis angulata was the most susceptible to natural infection in all six exposures in 2018 and 2019. No plants of Capsicum annuum 'Dahra' or Chenopodium album became infected. Solanum melongena 'Napoli' had only two infected plants of 60 exposed. Capsicum annuum and Chenopodium album were the least preferred, and Nicotiana tabacum and S. melongena were the most preferred for whitefly oviposition. In addition, from 2016 to 2019, we surveyed different tomato crops and the surrounding vegetation to identify ToCV in weeds and cultivated plants in the region of Sumaré, São Paulo State. Only S. americanum , vila vila ( S. sisymbriifolium ), and Chenopodium album were found naturally infected, with incidences of 18, 20, and 1.4%, respectively. Finally, we estimated the ToCV titer (U.S. and Brazilian isolates ToCV-FL and ToCV-SP, respectively) by quantitative reverse transcription PCR in different ToCV-susceptible host plants and evaluated the relationship between virus acquisition and transmission by B. tabaci MEAM1. The results clearly showed significant differences in ToCV concentrations in the tissues of ToCV-susceptible host plants, which appeared to be influenced by the virus isolate. The concentration of the virus in plant tissues, in turn, directly influenced the ToCV- B. tabaci MEAM1 relationship and subsequent transmission to tomato plants. To minimize or prevent damage from tomato yellowing disease through management of external sources of ToCV, it is necessary to correctly identify potentially important ToCV-susceptible hosts in the vicinity of new plantings.

Tradescantia spathacea (family Commelinaceae) is cultivated worldwide as an ornamental (Golczyk et al., 2013) and as medicinal plant (Tan et al., 2020). In 2019, 90 of ~180 plants of T. spathacea, grown in two beds of 4 m2 and exhibiting leaf mosaic were found in an experimental area at ESALQ/USP (Piracicaba municipality, São Paulo state, Brazil). Potyvirus-like flexuous filamentous particles were observed by transmission electron microscopy in foliar extracts of two symptomatic plants stained with 1% uranyl acetate. Total RNA was extracted using the Purelink viral RNA/DNA kit (Thermo Fisher Scientific) from leaves of two symptomatic plants and separately subjected to a reverse transcription polymerase chain reaction (RT-PCR). The potyviruses degenerate pairs of primers CIFor/CIRev (Ha et al. 2008), which amplifies a fragment corresponding to part of the cylindrical inclusion protein gene, and WCIEN/PV1 (Maciel et al. 2011), which amplifies a fragment containing part of the capsid protein gene and the 3' untranslated region, were used. The expected amplicons (~700bp) were obtained from both total RNA extracts. Two amplicons from one sample were purified using the Wizard SV Gel and PCR Clean-Up System kit (Promega) and directly sequenced in both directions at Macrogen Inc (Seoul, South Korea). The obtained nucleotide sequences (GenBank MW430005 and MW503934) shared 95.32% and 97.79% nucleotide identity, respectively, with the corresponding sequences of the Brazilian isolate of the potyvirus costus stripe mosaic virus (CoSMV, MK286375) (Alexandre et al. 2020). Extract from an infected plant of T. spathacea was mechanically inoculated in 10 healthy plants of T. spathacea and two plants each of the following species: Capsicum annuum, Chenopodium amaranticolor, Commelina benghalensis, Datura stramonium, Gomphrena globosa, Nicandra physaloides, Nicotiana tabacum cvs. Turkish and Samsun, Solanum lycopersicum, T. palida, and T. zebrina. All T. spathacea plants exhibited mosaic and severe leaf malformation. C. benghalensis plants developed mild mosaic, whereas infected T. zebrina plants were asymptomatic. The plants of other species were not infected. RT-PCR with specific CoSMV primers CoSMVHC-F and CoSMVHC-R (Alexandre et al. 2020) confirmed the infection. Nucleotide sequences of amplicons obtained from experimentally inoculated T. spathacea and T. zebrina (MW430007 and MW430008) shared 94.56% and 94.94% identity with the corresponding sequence of a Brazilian CoSMV isolate (MK286375). None of eight virus-free plants of T. spathacea inoculated with CoSMV using Aphis craccivora exhibited symptoms, nor was CoSMV detected by RT-PCR. Lack of CoSMV transmission by A. solanella, Myzus persicae, and Uroleucon sonchi was previously reported (Alexandre et al. 2020). T. spathacea plants are commonly propagated vegetatively, and by seeds. Virus-free seeds, if available, can provide an efficient and easy way to obtain healthy plants. Only three viruses were reported in plants of the genus Tradescantia: Commelina mosaic virus, tradescantia mild mosaic virus, and a not fully characterized potyvirus (Baker and Zettler, 1988; Ciuffo et al., 2006; Kitajima 2020). CoSMV was recently reported infecting Costus spiralis and C. comosus (Alexandre et al. 2020). As far as we know, this is the first report of CoSMV infecting T. spathacea plants.

Strongylodon macrobotrys, commonly known as the jade vine, emerald vine, or turquoise jade vine, is a species of Fabaceae native to the Philippines. The plants have blue-green color inflorescences, which makinge them one of the most admired ornamental plants in Brazil (Muniz et al. 2015). In addition, the plants contain compounds with anticancer properties (Ragasa et al. (2014) isolated compounds from S. macrobotrys with anticancer properties. In March 2019, an adult jade plant, grown under the trellis system in an experimental area at the campus of the University of São Paulo (USP), Piracicaba, state of São Paulo, was found showing mosaic symptoms typical of a virus infection. Preliminary examination of negatively stained leaf extracts by transmission electron microscopy detected elongated, flexuous particles similar tolike thoseat of a potyviruses. Further observations of thin sections of symptomatic leaf tissues revealed the presence of cylindrical inclusions, as well as bundles of thin, elongated, and filamentous particles, typical of potyvirus infection in epidermal, parenchymalparenchymal, and vascular regions, as well as bundles of thin, elongated and filamentous particles. Subsequent molecular and biological assays confirmed the presence of a potyvirusTo identify the species of the virus, .Presence of a potyvirus was confirmed by subsequent molecular and biological assays. Ttotal RNA was extracted from a pool of symptomatic leaves from the plant using the Purelink viral RNA/DNA kit (Thermo Fisher Scientific), and analyzed by one- step RT-PCR using potyviruses universal primers PV1/SP6 and WCIEN-sense (Mackenzie et al. 1998; Maciel et al. 2011), which amplify a 750-bp fragment. Total RNA extracted from an asymptomatic jade vine, obtained from a florist shop, was used as a negative controlincluded in the assay. PCR products at the expected size (~750-bp) were observed in the symptomatic plant but not in the asymptomatic plant. BLASTn analysis of the Nnucleotide sequence of the amplicon obtained only from total RNA of the symptomatic plant (GenBank accession no. MN970030) showed that it shares 90.82% to 97.859% identity with corresponding nucleotide sequences of the Korean isolate WS162 of soybean mosaic virus (SMV) deposited at the GenBank (, accession no. FJ640973, FJ640956, D88616). Extracts from symptomatic leaves of the jade plant wereas mechanically inoculated onto leaves of healthy plants of jade vine, Jack bean (Canavalia ensiformis), soybean cv. NA 5909 (Glycine max), cowpea (Vigna unguiculata), and passion fruit (Passiflora edulis f. flavicarpa). One plant of jade plant and four plants of each other species were inoculated , and infection was assessed based and monitored for symptom expression on symptom expression, and RT-PCR. The jade vine and Jack bean plants were infected by SMV, showingdeveloped mild mosaic symptoms approximately 60 and 15 days after inoculation, respectively , whereas the plants of other species were absent of any visible symptoms . To confirm the potyvirus identity, the jade vine samples were also tested by cConventional RT-PCR with SMV-specific primers pairs CP-F-SMV/CP-R-SMV (Jaramillo Mesa et al., 2018) and SMV-CPf/SMV-CPr (Wang and Ghabrial, 2002), thawhicht amplify fragments of 1000 990-bp and 469-bp90, respectively, nucleotides offrom the CP geneome region of SMV was performed, respectively. Amplicons of expected sizes were obtained from the total RNA of the leaves of field-infected and the mechanically inoculated plant of jade plantsvine as well as the Jack bean plants, but not from the asymptomatic jade plantvine and plants of other species the negative control. The viral nucleotide sequences obtained with the above pairs of primersBLASTn analysis of nucleotide sequences of the amplicons showed that they share 96.81% and 97.63% identity, respectively, with the same Korean SMV isolate WS162. These results demonstrate that… the field-symptomatic jade vine was infected with SMV, which is naturally transmitted by aphids speciess in a non-persistent manner and via soybean infected seeds (Hajimorad et al. 2018)( ). The virus appears to have has a restricted narrow natural host range., Aapart from soybean, and to date, it has only been reported the natural infection has been documented only in soybean, Lagenaria siceraria, Passiflora spp., Pinellia ternata, Senna occidentalis, and Vigna angularis (Almeida et al., 2002; Chakraborty et al. 2016; Hajimorad et al. 2018). To our knowledge, this is the first report of SMV in S. macrobotrys in the world. Further surveys are necessary to determine the incidence of the virus in ornamental jade plants vines and its importance as virus reservoirs for commercial soybean crops.

The complete nucleotide sequence of a new member of the family Potyviridae, which we propose to name "Arachis virus Y" (ArVY), is reported from forage peanut plants (Arachis pintoi) exhibiting virus-like symptoms. The ArVY positive-sense RNA genome is 9,213 nucleotides long and encodes a polyprotein with 2,947 amino acids that is predicted to be cleaved into 10 mature proteins. The complete single open reading frame (ORF) of ArVY shares 47% and 34% nucleotide and amino acid sequence identity, respectively, with the closest related virus, soybean yellow shoot virus. Electron microscopic analysis revealed elongated viral particles typical of those found in plant cells infected with potyviruses.

Centella asiatica is a perennial, herbaceous creeper plant that belongs to the family Apiaceae. It has been known since prehistoric times and has been used for therapeutic and cosmetic purposes (James and Dubery 2009; Gohil et al. 2010), and is easily propagated vegetatively. In 2018, plants of C. asiatica exhibiting foliar symptoms of mosaic and malformation were found in the botanical garden of the Plantarum Institute (Nova Odessa municipality, São Paulo state - 22°46'45.8"S 47°18'47.5"W) and in an experimental area at ESALQ/USP (Piracicaba municipality, São Paulo state - 22°42'26.0"S 47°37'48.6"W). In both locations the plants were grown in beds of approximately 4 m2 and all of them were symptomatic. Initially, leaf extract from symptomatic C. asiatica plants was examined by transmission electron microscopy (TEM) after being negatively stained with 1% uranyl acetate. Potyvirus-like flexuous filamentous particles were observed in leaf samples from both locations. TEM of thin sections of symptomatic leaf tissues revealed the presence of cylindrical inclusions, characteristic of infection by potyviruses, in the cytoplasm of epidermal, parenchymal, and vascular cells. Total RNA was extracted from symptomatic leaves collected in the Plantarum Institute (3 samples), and at Escola Superior de Agricultura Luiz de Queiroz (1 sample) using the Purelink viral RNA/DNA kit (Thermo Fisher Scientific, Waltham, USA). Reverse Transcription -Polymerase Chain Reaction (RT-PCR) was performed using the degenerate primers CIFor (5'-GGIVVIGTIGGIWSIGGIAARTCIAC-3') and CIRev (5'-ACICCRTTYTCDATDATRTTIGTIGC-3'), which amplify a fragment of approximately 700 bp within the the cylindrical inclusion protein gene of potyviruses (Ha et al. 2008). Amplicons of the expected size were obtained for all four samples analysed. One amplicon per location was purified using the Wizard® SV Gel and PCR Clean-Up System kit (Promega), and directly sequenced in both directions at Macrogen Inc (Seoul, South Korea). The nucleotide sequences obtained from the symptomatic C. asiatica plants collected in the Plantarum Institute (GenBank Acc. No. MT668627), and at ESALQ/USP (GenBank Acc. No. MT668626) showed 97.1% and 96.2% identity, respectively, with the nucleotide sequence of a Brazilian isolate of bidens mosaic virus (BiMV), family Potyviridae, genus Potyvirus (GenBank Acc. No. KF649336). To confirm the infection of C. asiatica plants with BiMV, the previously extracted RNAs were analyzed by RT-PCR using the specific primers 8331 (5'-CGTGGGGCTATCCTGAATTG-3') and 9046 (5'-CCACATCAGAGAAGTGTGCC-3'), which amplify a fragment of 715 bp corresponding to the BiMV coat protein gene (Suzuki et al. 2009). The expected size amplicons were obtained for all four samples of symptomatic plants of C. asiatica. The nucleotide sequences of two amplicons (GenBank Acc. Nos. MT668628, and MT668629), representing plants from each location, showed 94.6% to 95.6% identities with corresponding nucleotide sequences of the coat protein gene of BiMV from Brazil (GenBank Acc. Nos. KF649336, AY960150, and AY960151). A leaf extract of a symptomatic C. asiatica plant was mechanically inoculated to healthy plants of Apium graveolens, Bidens pilosa, C. asiatica, Chenopodium amaranticolor, C. quinoa, Coriander sativum, Nicotiana benthamiana, N. tabacum and Petroselinum crispum. C. asiatica became systemically infected, reproducing the original symptoms of leaf mosaic and malformation. N. benthamiana was infected and developed severe mosaic symptoms, whereas C. amaranticolor and C. quinoa reacted only with necrotic and chlorotic local lesions, respectively. Other assayed plants were not infected. Potyvirus-like particles were observed by TEM in the infected plants and BiMV infection was confirmed by RT-PCR. Transmission assays of the BiMV isolate by aphids Myzus persicae and Aphys gossypii to healthy C. asiatica plants were also performed. Virus-free aphids of the two species, reared on Capsicum annuum and Gossypium hirsutum respectively, were fasted for 30 min and then placed, separately, on symptomatic leaves of C. asiatica for an acquisition access period (AAP) of 10 min. After that, groups of six insects were transferred, separately, to four healthy C. asiatica plants for an inoculation access period (IAP) of 24 h. After inoculation the insects were killed manually. Approximately 30 days later, one plant inoculated with each species of aphid exhibited symptoms and infection was confirmed by RT-PCR and nucleotide sequencing of the amplicons. BiMV was absent in control, non-inoculated plants in both mechacial and aphid transmission assays. Infection of spontaneously growing C. asiatica plants by potyvirus, determined by TEM, was previously reported in Curitiba and Colombo, state of Paraná, Brazil by Lima Neto and Souza (1981), but the virus was not fully characterized and identified. In addition to BiMV, plants of C. asiatica are also suscptible to infection with cucumber mosaic virus (CMV), as reported by Cardin and Moury (2010) in Madagascar. This is the first identification of BiMV naturally infecting C. asiatica. Additional works on effects of BiMV infection of C. asiatica on commercial production and pharmaceutical properties are required.

Senna rizzinii is a flowering shrub found mainly in the northeast region of Brazil. Here, we report the coding-complete genome sequence, particle morphology, mode of transmission, and the indicator host responses of an isolate of the putative allexivirus cassia mild mosaic virus (CaMMV) found in S. rizzinii. The virus was transmitted mechanically to Chenopodium amaranticolor, C. quinoa, Gomphrena globosa, which showed local lesions, and S. rizzinii, and S. occidentalis, which were infected systemically. It was also efficiently transmitted to S. rizzinii by grafting. Seed transmission was not observed. The near-complete genome sequence of the virus is 7829 nucleotides in length, containing six open reading frames (ORF), like other allexiviruses.

Current control of tomato golden mosaic disease, caused in Brazil predominantly by tomato severe rugose virus (ToSRV), is dependent on both, planting resistant/tolerant hybrids and intensive insecticide sprays (two to three per week) for controlling Bemisia tabaci , the vector of ToSRV. Resistant hybrids only confer moderate resistance to infection by ToSRV and some tolerance to the disease. Insecticide sprays, although widely used, have failed in most tomato production areas in Brazil, as they are unable to reduce primary spread, i.e., infection caused by the influx of viruliferous whiteflies coming from external sources of inoculum. Severe epidemics are recurrently observed in some tomato fields in several Brazilian regions, which prompted us to postulate the existence in the agroecosystem, in some places and time, of amplifier hosts that provide the necessary force of infection for epidemics to occur, even in the absence of secondary spread in the target crop. Amplifier hosts are ideally asymptomatic, occur in high density near the target crop, and support growth of both virus and vector. Soybean and common bean are potential amplifier hosts for begomovirus in tomato crops. Our results support the hypothesis that soybean plants may play an important role as an amplifier host of ToSRV for tomato crops in the field, although this does not seem to be a frequent phenomenon. Successful amplification will depend on several factors, including the soybean cultivar, the soybean stage of development at the moment of infection, the ToSRV isolate, and the perfect synchrony between the beginning of a soybean field and the end of a ToSRV-infected crop, and, later, between the senescence of the ToSRV-infected soybean plants and the new tomato crop. The concept of amplifier hosts has been widely used in ecology of zoonoses but, to our knowledge, has never been used in botanical epidemiology., (Copyright © 2020 Bergamin Filho, Macedo, Favara, Bampi, Oliveira and Rezende.)

Physalis peruviana is a perennial solanaceous plant that has recently been established as a commercial crop in Brazil. This work reports the near-complete genome sequence, particle morphology, and plant host responses to a putative new sobemovirus, named "physalis rugose mosaic virus". The virus, characterized by isometric particles of ca. 30 nm in diameter, causes foliar symptoms of mosaic, malformation and blistering, accompanied by stunting. The near-complete genome sequence comprises 4175 nucleotides and contains five open reading frames that are similar to those of other sobemoviruses. In addition to P. peruviana, the new virus systemically infected Capsicum annuum, Nicotiana tabacum and Solanum lycopersicum by mechanical inoculation. Thus, this virus may cause disease in these crops in the field.

Insect-borne plant viruses usually alter the interactions between host plant and insect vector in ways conducive to their transmission ('host manipulation hypothesis'). Most studies have tested this hypothesis with persistently and non-persistently transmitted viruses, while few have examined semi-persistently transmitted viruses. The crinivirus Tomato chlorosis virus (ToCV) is semi-persistently transmitted virus by whiteflies, and has been recently reported infecting potato plants in Brazil, where Bemisia tabaci Middle East Asia Minor 1 (MEAM1) is a competent vector. We investigated how ToCV infection modifies the interaction between potato plants and B. tabaci in ways that increase the likelihood of ToCV transmission, in two clones, one susceptible ('Agata') and the other moderately resistant (Bach-4) to B. tabaci. Whiteflies alighted and laid more eggs on ToCV-infected plants than mock-inoculated plants of Bach-4. When non-viruliferous whiteflies were released on ToCV-infected plants near mock-inoculated plants, adults moved more intensely towards non-infected plants than in the reverse condition for both clones. Feeding on ToCV-infected plants reduced egg-incubation period in both clones, but the egg-adult cycle was similar for whiteflies fed on ToCV-infected and mock-inoculated plants. Our results demonstrated that ToCV infection in potato plants alters B. tabaci behaviour and development in distinct ways depending on the host clone, with potential implications for ToCV spread.

The crinivirus Tomato chlorosis virus (ToCV) is often found infecting tomato crops in Brazil, with variable incidence, but associated with prevalence of its primary vector, Bemisia tabaci MEAM1. ToCV control is difficult because there are no resistant commercial tomato varieties or hybrids available and chemical spray for control of the whitefly vector has not been effective. The present study evaluated the partial host range of a Brazilian isolate of ToCV and the preference of B. tabaci MEAM1 for oviposition on those species identified as susceptible to the virus. Subsequently, transmission tests were performed using plants of each ToCV host species as sources of inoculum to elucidate the epidemiological importance of nontomato sources of inoculum for infection of tomato. Among 80 species experimentally inoculated, 25 were susceptible, including 6 previously not known to be hosts ( Jaltomata procumbens , Physalis pruinosa, Solanum aculeatissimum, S. viarum , Beta vulgaris var. cicla , and Chenopodium quinoa ). Preference of whitefly for oviposition and infection by ToCV under free-choice transmission tests varied among the susceptible species. When ToCV-infected tomato, eggplant, and C. quinoa were used separately as sources of inoculum for virus transmission to tomato plants, mean percentages of infected plants were 76.6, 3, and 0%, respectively. Average oviposition of Bemisia tabaci on these three hosts were 2.7, 10.6, and 0.0 eggs/cm 2 , respectively. Additional studies will be necessary to evaluate the importance of ToCV host plants under field conditions and their efficiency as sources of inoculum for virus acquisition and transmission to tomato crops.

Tomato severe rugose virus (ToSRV) and Tomato chlorosis virus (ToCV) are among the major viruses that affect tomato (Solanum lycopersicum) development and yield in Brazil. ToSRV and ToCV are transmitted in a persistent circulative and semipersistent manner, respectively, by the whitefly Bemisia tabaci Middle East-Asia Minor 1, considered the main vector of these viruses. In this study, the kinetics of systemic invasion and the latent and incubation periods of ToSRV and ToCV were evaluated in singly and doubly infected tomato plants. Both viruses moved systemically into tomato plants as early as 1 day after inoculation. The mean ToCV latent periods in single infections and co-infections with ToSRV were 13 and 11 days, respectively, while incubation periods in single and co-infections were, on average, 30 and 31 days, respectively. For ToSRV, the mean latent period was 7 days in single infections and 6 days in co-infections with ToCV. Incubation periods were, on average, 18 and 17 days in single and co-infections, respectively. Because latent periods for both viruses were shorter than their respective incubation periods, field-infected tomato plants may act as sources of inocula soon after infection and before onset of symptoms.

Erigeron sp. plants showing symptoms of witches' broom and stunting were found near orchards of passion fruit in São Paulo state, Brazil. These symptoms were indicative of infection by phytoplasmas. Thus, the aim of this study was to detect and identify possible phytoplasmas associated with diseased plants. Total DNA was extracted from symptomatic and asymptomatic plants and used in nested PCR conducted with the primer pairs P1/Tint and R16F2n/16R2. Amplification of genomic fragments of 1.2 kb from the 16S rRNA gene confirmed the presence of phytoplasma in all symptomatic samples. The sequence identity scores between the 16S rRNA gene of the phytoplasma strain identified in the current study and those of previously reported 'Candidatus Phytoplasma fraxini'-related strains ranged from 98% to 99% indicating the phytoplasma to be a strain affiliated with 'Candidatus Phytoplasma fraxini'. The results from a phylogenetic analysis and virtual RFLP analysis of the 16S rRNA gene sequence with 17 restriction enzymes revealed that the phytoplasma strain belongs to the ash yellows phytoplasma group (16SrVII); the similarity coefficient of RFLP patterns further suggested that the phytoplasma represents a novel subgroup, designated 16SrVII-D. The representative of this new subgroup was named EboWB phytoplasma (Erigeron bonariensis Witches' Broom).

Beet necrotic yellow vein virus (BNYVV) is an economically important pathogen of sugar beet (Beta vulgaris var. saccharifera) in several European, and Asian countries and in the United States (3). The virus is transmitted by the soil-inhabiting plasmodiophorid Polymyxa betae and causes the rhizomania disease of sugar beet. In November 2012, plants of B. vulgaris subsp. vulgaris cv. Boro (red table beet) exhibiting mainly severe characteristic root symptom of rhizomania were found in a commercial field located in the municipality of São José do Rio Pardo, State of São Paulo, Brazil. No characteristic virus-inducing foliar symptom was observed on diseased plants. The incidence of diseased plants was around 70% in the two visited crops. As the hairy root symptom is indicative of infection by BNYVV, the present study aimed to detect and identify this virus associated with the diseased plants. Preliminary leaf dip analysis by transmission electron microscopy revealed the presence of very few benyvirus-like particles. Total RNA was extracted from roots of three symptomatic plants and one asymptomatic plant according to Toth et al. (3). One-step reverse-transcription-polymerase chain reaction (RT-PCR) was performed as described by Morris et al. (2) with primers that amplify part of the coat protein gene at RNA2. The initial assumption that the hairy root symptom was associated with BNYVV infection was confirmed by the amplification of a fragment of ~500 bp from all three symptomatic samples. No amplicon was obtained from the asymptomatic control plant. Amplicons were directly sequenced, and the consensus nucleotide and deduced amino acid sequences showed 100% identity. The nucleotide sequence for one amplicon (Accession No. KM433683) was compared with other sequences deposited in GenBank. The nucleotide (468 nt) and deduced amino acid (156 aa) sequences shared 93 to 100 and 97 to 99% identity, respectively with the corresponding nucleotide and amino acid sequences for other isolates of type A of BNYVV. The virus was transmitted to three of 10 red table beet plants inoculated with contaminated soil, and infection was confirmed by nested RT-PCR, as described by Morris et al. (1), and nucleotide sequencing. This is the first report on the occurrence of BNYVV in Brazil, which certainly will affect the yield of red table beet in the producing region. Therefore, mapping of the occurrence of BNYVV in red table beet-producing areas in Brazil for containment of the spread of the virus is urgent. In the meantime, precautions should be taken to control the movement of contaminated soil and beet roots, carrots, or any vegetable grown on infested land that might introduce the virus to still virus-free regions. References: (1) J. Morris et al. J. Virol. Methods 95:163, 2001. (2) D. D. Sutic et al. Handbook of Plant Virus Diseases. CRC Press, Boca Raton, Florida, 1999. (3) I. K. Toth et al. Methods for the Detection and Quantification of Erwinia carotovora subsp. atroseptica (Pectobacterium carotovorum subsb. atrosepticum) on Potatoes: A Laboratory Manual. Scottish Crop Research Institute, Dundee, Scotland, 2002.

Mandevilla (Apocynaceae) is an ornamental tropical vine popular for its bright and attractive flowers. During 2012 to 2013, 12 Mandevilla sp. samples from Minnesota and Florida nurseries were submitted for analysis at the University of Minnesota Plant Disease Clinic. Plants showed mosaic symptoms, leaf deformation, premature leaf senescence, and vine dieback. Filamentous virus particles with modal lengths 700 to 900 nm were observed by transmission electron microscopy (TEM) in partially purified preparations from symptomatic leaves. Partially purified virions were obtained using 30% sucrose cushion centrifuged at 109,000 g max for 2 h at 10°C (5). No other virus particles were observed in these samples, nor were any observed in non-symptomatic samples. One sample was submitted as potted plant (Mandevilla 'Sunmandeho' Sun Parasol Giant White) and was kept under greenhouse conditions for subsequent analyses. Total RNA (Qiagen) was extracted from this sample, and Potyvirus was detected using the universal primers Poty S (5'-GGN AAY AAY AGY GGN CAR CC-3') and PV1 (5'-20(T)V-3') (1) by reverse transcription (RT)-PCR (3). The amplified product was the expected ~1.7-kb, corresponding to the partial nuclear inclusion body gene, the coat protein (CP) gene, and the 3' end untranslated region. The RT-PCR amplicon was cloned (NEB) and sequenced, and the 1,720-bp consensus sequence was deposited in GenBank (Accession No. KM243928). NCBI BLAST analysis at the nucleotide level revealed highest identity (83%) with an isolate of Catharanthus mosaic virus (CatMV) from Brazil (Accession No. DQ365928). Pairwise analysis of the predicted 256 amino acid CP revealed 91% identity with the CatMV Brazilian isolate (ABI94824) and 68% or less identity with other potyviruses. Two potyviruses are usually considered the same species if their CP amino acid sequences are greater than 80% identical (2). Serological analysis of the infected sample Mandevilla 'Sunmandeho' Sun Parasol Giant White using a CatMV specific antiserum (4) resulted in positive indirect ELISA reactions. CatMV has been previously reported in periwinkle (Catharanthus roseus) in Brazil (4). Based on the analyses by TEM, RT-PCR, nucleotide and amino acid sequence identities, and serological reactivity, we identify this virus as a U.S. Mandevilla isolate of CatMV. To our knowledge, this is the first report of Catharanthus mosaic virus both in the United States and in Mandevilla. References: (1) J. Chen et al. Arch Virol. 146:757, 2001. (2) A. Gibbs and K. Ohshima. Ann. Rev. Phytopathol. 48:205, 2010. (3) R. L. Jordan et al. Acta Hortic. 901:159, 2011. (4) S. C. Maciell et al. Sci. Agric. Piracicaba, Brazil. 68:687, 2011. (5) D. Mollov et al. Arch Virol. 158:1917, 2013.

The antidote vines or nhandirobas (Fevillea trilobata L. [Cucurbitaceae]) are dioecious plant species native to the South American Neotropics (1). Genetic materials of these species are now being domesticated and evaluated as potential crops for seed-oil extraction aiming to produce biodiesel fuel (2). Plants of F. trilobata (Accession No. CNPH-001) were cultivated from seeds under open field conditions during the years 2008 through 2011 in Brasília-DF, Brazil. Approximately 200 plants exhibiting mosaic symptoms and severe leaf malformation (with typical bubble-like patches) were found in all fields every year. Apical mosaic was slightly more severe in female than in male plants. Electron microscopy examination of negatively stained extracts of symptomatic leaf tissue showed the presence of filamentous particles about 700 to 800 nm long. Analysis of ultra-thin sections of the same tissues revealed the presence of lamellar inclusions typical of a potyvirus infection. No aphid colonies were observed on field-grown F. trilobata plants. The virus was mechanically transmitted to healthy Cucurbita pepo cv. Caserta and Luffa cylindrica, causing systemic mosaic. Sap from these infected plants reacted in PTA-ELISA with polyclonal antiserum against Zucchini yellow mosaic virus (ZYMV), but not with antisera against Papaya ringspot virus - type W (PRSV-W), Cucumber mosaic virus (CMV), and Zucchini lethal chlorosis virus (ZLCV). Total RNA extracted from experimentally infected C. pepo was analyzed by RT-PCR using specific pairs of primers for the coat protein gene of ZYMV (3). A cDNA fragment of approximately 1,186 bp was amplified and the nucleotide sequence obtained by direct sequencing. Comparisons of the nucleotide (837 nt) and deduced amino acid (279 aa) sequences of the coat protein genomic segment (GenBank Accession No. JX502677) revealed 93 to 98% and 97 to 98% identity, respectively, with the corresponding nucleotide and amino acid sequences of a group of ZYMV isolates from distinct hosts (AY188994, AY279000, and NC_003224). The infection by ZYMV might cause fruit yield losses to F. trilobata. In addition, the infected F. trilobata crops might work as a reservoir of ZYMV providing inoculum to other cucurbit hosts since it has been managed as a semi-perennial crop. To our knowledge, this is the first report of the genus Fevillea as a natural host of ZYMV. References: (1) M. Nee et al. Syst. Bot. 34:704, 2009. (2) E. G. Shay. Biomass Bioenergy 4:227, 1993. (3) K. G. Thomson et al. J. Virol. Meth. 55:83, 1995.

In Brazil, serious epidemics of begomovirus diseases have been successively reported since the mid-90s, among them those caused by Tomato yellow spot virus (ToYSV) (1). In July 2009 and October 2010, high incidences (40 to 60%) of plants of the weed Leonurus sibiricus (Lamiaceae) exhibiting symptoms of yellow leaf mosaic were found near soybean (Glycine max) crops within the municipalities of Marechal Cândido Rondon and Tapejara, in the states of Paraná and Rio Grande do Sul. Leaves from 21 symptomatic and seven asymptomatic L. sibiricus plants were collected from both localities and tested for the presence of begomovirus. Total DNA was extracted from each sample using Dneasy Plant Mini Kit (Qiagen) and submitted to PCR using begomovirus universal oligonucleotides PAL1v1978/PAR1c496 (3). One fragment of approximately 1,300 bp comprising the 5'-region of the replication-associated protein (Rep) gene, the entire intergenic region (IR), and the 5'-region of the coat protein (CP) gene was amplified from all symptomatic, but not from asymptomatic samples. Amplified fragments corresponding to all isolates were directly sequenced and nucleotide sequence comparisons indicated 98 to 99% nucleotide identity among themselves, and 93 to 94% identity with the corresponding nucleotide sequences for the DNA-A of the begomovirus ToYSV (GenBank Accession No. DQ336350). To confirm these results, the full genome of ToYSV Mc-7 isolated from Marechal Cândido Rondon was cloned and completely sequenced by primer walking (Macrogen Seoul, Korea). The DNA-A of ToYSV Mc-7 (JX513952) was 2,592 nt long and shared 92 and 91% identity with isolates of ToYSV from Argentina (FJ538207) and Brazil (DQ336350), respectively. The DNA-B of ToYSV Mc-7 (JX513952) was 2,568 nt long and shared 91% identity with DNA-B of a Brazilian isolate of ToYSV (DQ336351). The ToYSV Mc-7 isolate is a new strain named Tomato yellow spot virus (Brazil:Marechal Candido Rondon 7:Leonurus:2009) [ToYSV-(BR:MCR7:Le:09)]. To demonstrate pathogenicity, virus-free adults of Bemisia tabaci biotype B were confined on symptomatic L. sibiricus plants for a 48-h acquisition period. The whiteflies were then transferred to healthy L. sibiricus, bean (Phaseolus vulgaris), soybean, and tomato (Solanum lycopersicum) plants. L. sibiricus plants showed the original symptoms on the leaves (five symptomatic plants, seven inoculated plants), whereas bean (3/7), soybean (4/10), and tomato plants (5/10) exhibited mild yellow leaf mosaic. The infection in these symptomatic plants was confirmed by PCR with oligonucleotides PAL1v1978/PAR1c496 (3) and subsequent direct nucleotide sequencing of the 5'-region of the CP gene, which confirmed the identity of the transmitted virus as ToYSV. ToYSV was first reported infecting tomato plants in Minas Gerais state, Brazil (1). Recently, ToYSV was found infecting bean and soybean plants in northwestern Argentina (2). Because L. sibiricus is a weed widely distributed throughout Brazil, and the ToYSV vector B. tabaci is also common, this weed may become a potential source of inoculun of ToYSV to bean, soybean, and tomato crops. To our knowledge, this is the first report of L. sibiricus as a natural host of ToYSV. References: (1) R. F. Calegario et al. Pesq. Agropec. Bras. 42:1335, 2007. (2) P. E. Rodríguez-Pardina et al. Ann. Appl. Biol. 158:69, 2011. (3) M. R. Rojas et al. Plant Dis. 77:340, 1993.

Potato plants (Solanum tuberosum cv. Ágata) exhibiting symptoms of leaf roll and interveinal chlorosis, especially on older leaves, were found in a commercial crop in the County of Cristalina, State of Goiás, Brazil in June 2011. The crop was severely infested by whitefly Bemisia tabaci biotype B. Four potato tubers from symptomatic plants were indexed for the presence of the following viruses: Tomato chlorosis virus (ToCV), Potato leaf roll virus (PLRV), Tomato severe rugose virus (ToSRV), and Potato virus Y (PVY). Total RNA was extracted separately from each tuber and used for reverse transcription (RT)-PCR using the HS-11/HS-12 primer pair, which amplifies a fragment of 587 bp from the highly conserved region of the heat shock protein (HSP-70) homolog gene reported for ToCV. The RT-PCR product was subsequently tested by nested-PCR for detection of ToCV with specific primers ToC-5/ToC-6 (2). Amplicons of 463 bp, amplified from total RNA separately extracted from three tubers, were purified and directly sequenced. Comparisons among the three consensus sequences of 448 bp (GenBank Accession Nos. JQ288896, JQ288897, and JQ288898) revealed respectively, 98, 100, and 100% identity with the reported sequence of a tomato isolate of ToCV from Brazil (GenBank Accession No. EU868927) (1). For ToSRV detection, total DNA was extracted from two tubers and a fragment of approximately 820 bp was amplified by PCR with specific primers (3). PLRV and PVY were indexed in two and three tubers, respectively, by double-antibody sandwich-ELISA (SASA, Edinburg, Scotland). Virus-free B. tabaci biotype B were separately transferred to potato and tomato leaves infected with ToCV for an acquisition access period of 24 h. Groups of 30 viruliferous whitefly were transferred to four, young, sprout-grown potato plants cv. Ágata (two plants per virus isolate) for 24-h inoculation access period. After 37 days of inoculation, one plant inoculated with the potato and tomato isolates of ToCV, respectively exhibited symptoms of leaf roll and interveinal chlorosis on order leaves, which were similar to that induced by PLRV. Experimental infection of potato plants with ToCV, which induced leaf roll symptoms resembling PLRV infection, was reported in the United States by Wisler et al. (4). The potato isolate of ToCV was also transmitted by B. tabaci to one of two inoculated tomato plants. The presence of ToCV in all inoculated plants was detected by nested-RT-PCR as described above. To our knowledge, this is the first report on detection of ToCV in field potato plants in the world. Considering that ToCV occurs in innumerous countries around the world, it is transmitted by a cosmopolitan insect, and it induces symptoms similar to PLRV, this finding triggers an alert to field dependent seed-potato multiplication, virus inspector, and certification system. References: (1) J. C. Barbosa et al. Plant Dis. 92:1709, 2008. (2) C. I. Dovas et al. Plant Dis. 86:1345, 2002. (3) F. R. Fernandes et al. Trop. Plant Pathol. 35:43, 2010. (4) G. C. Wisler et al. Plant Dis. 82:270, 1998.

We report on the production and evaluation of passionflower transgenic lines for resistance to Cowpea aphid borne mosaic virus (CABMV). Genetic transformation was done using Agrobacterium tumefaciens and transgene integration was confirmed by Southern blot analyses, resulting in nine transgenic lines for 'IAC 275' and three for 'IAC 277'. Transgenic lines were clonally propagated and evaluated for resistance to CABMV. After the third inoculation, under higher inoculum pressure, only propagated plants of the transgenic line T16 remained asymptomatic, indicating a high resistance to infection with CABMV. This transgenic line was self-pollinated and the R 1 generation was evaluated together with the R 1 generation of another resistant transgenic line (T2) identified previously. Plants were inoculated with CABMV by means of viruliferous Myzus nicotianae. All 524 T2R 1 plants became infected, whereas 13 of 279 T16R 1 remained asymptomatic after four successive inoculations. A T16R 2 generation was obtained and plants were inoculated with CABMV mechanically or by aphids. After successive inoculations, 118 of 258 plants were symptomless, suggesting that the resistance to CABMV was maintained in the plant genome as the homozygous condition was achieved. Five selected resistant T16R 2 plants which contained the capsid protein gene are being crossed for further analyses.

Sesame (Sesamum indicum L.) is cultivated mainly in the central region of the Departamento de San Pedro in Paraguay from October to February and the seed are exported to Asia. The crop is grown on 100,000 ha annually and Escoba blanca is the most common cultivar. The crop plays an important socioeconomical role since it is cultivated mostly by small growers. A disease characterized by yellowing and curling down leaves and shortening of the internodes has been observed in almost all sesame-growing areas. It is referred to locally as "ka'are" because the affected sesame plant resembles Chenopodium ambrosioides L. This disease occurred occasionally and was of marginal importance prior to 2005, but during the last five growing seasons the disease incidence has increased substantially, with some growers losing the entire crop. To determine the causal agent, symptomatic leaf samples were collected from five commercial fields near Colonia San Pedro and Choré, Departamento San Pedro in December 2009. Preliminary transmission electron microscopy (TEM; Zeiss EM900) of extracts from symptomatic leaves revealed the presence of elongated flexible particles resembling a potyvirus. Mechanical transmission assays resulted in chlorotic local lesions on C. quinoa and C. amaranticolor, mosaic on Vigna unguiculata and Nicotiana benthamiana, and symptoms on sesame that are similar to those observed in the field. The disease could also be reproduced in sesame by aphid (Myzus persicae) transmission in a nonpersistent manner. TEM examination of leaf sections of these naturally or experimentally infected plants showed the presence of the type I cylindrical inclusions and masses of filamentous particles. Leaf extracts of naturally or experimentally infected sesame and test plants were positive for Cowpea aphid-borne mosaic virus (CABMV) on the basis of plate-trapped antigen (PTA)-ELISA. CABMV as the causal agent of "ka'are" disease of sesame in Paraguay was further confirmed by analyzing part of the nucleotide sequence of CABMV coat protein and 3' nontranslated region that were obtained directly from reverse transcription-PCR product amplified with PV1-antisense primer (5'-gatttaggtgacactatagt 17 -3') and WCIEN-sense primer (5'-atggtttggtgyatygaraat-3') (1,2). Comparisons of the 676-bp nucleotide sequence of two sesame virus isolates (GenBank Accession Nos. HQ336402 and HQ336403) revealed 92% identity with the corresponding nucleotide sequence of CABMV available in the GenBank (Accession No. AF348210). Thus, all the assays indicated that the "ka'are" disease of sesame in Paraguay is caused by an isolate of CABMV. Several cowpea fields, nearby sesame diseased crops, also contained plants exhibiting mosaic symptoms. Transmission assays, electron microscopy, PTA-ELISA, and nucleotide sequence analysis indicated that they were also infected by CABMV and may play an important role in the epidemiology of this disease on sesame. CABMV isolates from passion fruit and cowpea from Brazil were mechanically transmitted to sesame but induced milder symptoms. CABMV-infected sesame was described in the United States (3), but to our knowledge, this is the first report of a severe disease on sesame caused by this virus in Paraguay. References: (1) A. Gibbs and A. Mackenzie. J. Virol. Methods 63:9, 1997. (2) L. D. C. Mota et al. Plant Pathol. 53:368, 2004. (3) H. R. Pappu et al. Arch. Virol. 142:1919, 1997.

Sida is a genus of flowering herbs in the family Malvaceae, which includes several species that are weeds in Brazil. Plants of a Sida sp. exhibiting symptoms characterized by stunting, chlorosis, small leaves, and witches'-broom, indicative of infection by phytoplasmas, were found in a field previously cultivated with tomato, located in the region of Campinas, State of São Paulo, in December 2008. To demonstrate the presence of phytoplasmas in diseased tissues, DNA was extracted from shoots and leaves from three symptomatic and eight asymptomatic plants. Nested PCR was performed using primers P1/Tint followed by primer pair R16F2n/R16R2 (1). DNA fragments of 1.2 kb, corresponding to 16S rDNA, were amplified only for DNA from two symptomatic samples. Phytoplasma identification was initially carried out by restriction fragment length polymorphism (RFLP) analysis through digesting the PCR products with the restriction enzymes AluI, HhaI, HaeIII, HpaII, MseI, and RsaI. The two phytoplasma isolates found to be infecting a Sida sp. showed identical RFLP patterns, which were indistinguishable from the phytoplasma previously reported in association with hibiscus (Hibiscus rosa-sinensis) witches'-broom in Brazil (2). Nucleotide sequence alignment revealed that 16S rDNA of both phytoplasma isolates found in a Sida sp. (GenBank Accession No. HQ230579) shared 99.9% sequence similarity with 16S rDNA from hibiscus witches'-broom phytoplasma (HibWB) (GenBank Accession No. AF147708). HibWB is the representative of the 16SrXV group and it was proposed as a putative species nominated "Candidatus Phytoplasma brasiliense" (2). The disease is frequently observed in hibiscus plants used as ornamentals in the states of São Paulo (4) and Rio de Janeiro (2). "Ca. Phytoplasma brasiliense" has only been reported in Brazil to be infecting hibiscus (2,4) and periwinkle (Catharanthus roseus) (3). The presence of a phytoplasma belonging to group 16SrXV in a Sida sp. expands its natural host range. The role of this weed as a potential source of inoculum for crops should be investigated. References: (1) I. M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998. (2) H. G. Montano et al. Int. J. Syst. Evol. Microbiol. 51:1109, 2001. (3) H. G. Montano et al. Plant Dis. 85:1209, 2001. (4) E. G. Silva et al. Summa Phytopathol. 35:234, 2009.

Watercress (Nasturtium officinale L.), a member of the family Brassicaceae, is consumed mainly as salad. Medicinal properties have also been attributed to this species. In Brazil, watercress is grown mainly by very small farmers. The crop is primarily seed propagated and growers can harvest several times per year in an established planting. Very few diseases have been reported in this crop worldwide. In Brazil, watercress infection by Cauliflower mosaic virus (CaMV) (3), Cucumber mosaic virus (CMV) (1), and an unidentified potyvirus (2) were previously reported. In January 2009, 80% of watercress plants, cv. Gigante Redondo, exhibiting severe mosaic, leaf size reduction, and plant stunting were observed in a crop in Marechal Floriano Municipality, State of Espírito Santo, Brazil. Preliminary leaf dip analysis by transmission electron microscopy revealed the presence of potyvirus-like particles. Sap from five infected plants reacted in plate-trapped antigen (PTA)-ELISA with polyclonal antiserum against Turnip mosaic virus (TuMV), but not with antiserum against CMV. Both antisera were produced in the Plant Virology Laboratory, ESALQ/USP. Mechanically inoculated watercress plants developed similar systemic mosaic symptoms. The virus was also transmitted to Nicotiana benthamiana, which exhibited severe mosaic and stunting. The presence of TuMV on these inoculated plants was confirmed by PTA-ELISA and reverse transcription (RT)-PCR. Total RNA extracted from infected and healthy watercress and infected N. benthamiana was analyzed by RT-PCR using specific pairs of primers flanking the coat protein gene of TuMV. Degenerated anti-sense (5'-t/caacccctt/gaacgcca/cagt/ca-3') and sense (5'-gcaggtgaa/gacg/acttgat/ca/gc-3') primers were designed after analysis to an alignment of the nucleotide sequences for five isolates of TuMV available in the GenBank (Accession Nos. NC_002509, D10927, EU680574, AB362513, and D88614). One fragment of 838 bp was amplified from samples in the infected plants, but not in the healthy controls. Two amplicons were purified and directly sequenced in both directions. Comparisons of the 731-bp consensus nucleotide sequence (Accession No. HM008961) to several other isolates of TuMV revealed 94 to 95% identity in the coat protein region. To our knowledge, this is the first report of TuMV in watercress in Brazil. Management of the disease should include propagation by seeds instead of vegetative parts of the plants and rouging of diseased plants to prevent mechanical transmission during successive harvestings. References: (1) A. J. Boari et al. Fitopatol. Bras. 25:438, 2000. (2) A. J. Boari et al. Fitopatol. Bras. 27:S200, 2002. (3) M. L. R. Z. C. Lima et al. Fitopatol. Bras. 9:403, 1984.

Trichosanthes cucumerina L., known as snake gourd, is a cucurbitaceous plant that is probably native to and originally domesticated in India. It is cultivated in humid subtropical and tropical countries of Australia, Latin America, and Africa (2). Plants of this species exhibiting symptoms of mosaic and leaf malformation were found during November 2008 near an experimental field of the Departamento de Fitopatologia e Nematologia, Universidade de São Paulo, Piracicaba, State of São Paulo, Brazil. Electron microscopy examination of negatively stained extract of infected tissue showed the presence of filamentous potyvirus-like particles. Sap from these infected plants reacted in plate-trapped antigen (PTA)-ELISA with the antiserum against Papaya ringspot virus-type W (PRSV-W) or Zucchini yellow mosaic virus (ZYMV), but not with the antiserum against Cucumber mosaic virus (CMV) or Zucchini lethal chlorosis virus (ZLCV). PRSV-W and ZYMV were simultaneously transmitted by mechanical inoculation to four plants of Cucurbita pepo cv. Caserta and one plant of T. cucumerina, causing mosaic. In addition, PRSV-W and ZYMV isolates from our virus collection separately infected one plant of T. cucumerina after mechanical inoculation. Infections were confirmed by PTA-ELISA. Total RNA extracted from infected and healthy T. cucumerina was analyzed by reverse transcription (RT)-PCR using a primer pair specific to the coat protein (CP) gene of PRSV-W (4) or ZYMV (3). Fragments of 864 bp and 1,045 bp were amplified with each pair of primers, respectively. Nucleotide sequences directly obtained from purified PCR products were used for further identification of these potyviruses. The nucleotide and deduced amino acid sequences of part of the CP gene (792 nt) of PRSV-W (GenBank Accession No. GU586789) shared 99 and 98% identity, respectively, with that of the Brazilian isolate PRSV-W-C (GenBank Accession No. 4152). The nucleotide and deduced amino acid sequences of the entire CP gene (837 nt) of ZYMV (GenBank Accession No. 6790) shared 91 to 98% and 94 to 100% identity, respectively, with innumerous isolates of ZYMV deposited in the GenBank (e.g., Accession Nos. AB004640, D13914, AB004641, and AJ420019). Natural infection of T. cucumerina by PRSV-W was reported in Nepal (1). To our knowledge, this is the first report of T. cucumerina infected by PRSV-W and ZYMV in Brazil. References: (1) G. Dahal et al. Ann. Appl. Biol. 130:491, 1997. (2) R. W. Robinson and D. S. Decker-Walters. Cucurbits. CAB International, Wallingford, UK. 1997. (3) K. G. Thomson et al. J. Virol. Methods 55:83, 1995. (4) M. G. S. D. Vechia. Fitopatol. Bras. 28:678, 2003.

In June of 2009, sweet pepper (Capsicum annuum cvs. Elisa and Prador) plants exhibiting interveinal chlorosis, some necrosis, and mild upward leaf curling on the intermediate leaves were found in three protected crops in the municipality of São Miguel Arcanjo, São Paulo state, Brazil. Incidence of symptomatic plants varied from 70 to 100%. Abundant whitefly adults were seen in all crops. Initially, total DNA was separately extracted from seven symptomatic plants and submitted to a PCR reaction using the universal primer pairs PAL1v1978/PAR1c496 and PBL1v2040/PCRc1 for begomovirus (3). The results were negative. The same samples were also analyzed for infection with Tomato infectious chlorosis virus (TICV) and Tomato chlorosis virus (ToCV) (genus Crinivirus, family Closteroviridae). Total RNA was extracted separately from leaves of each symptomatic plant and used for one-step reverse transcription (RT)-PCR using the HS-11/HS-12 primer pair, which amplifies a fragment of 587 bp from the highly conserved region of the heat shock protein (HSP-70) homolog gene reported for TICV and ToCV (1). The RT-PCR product was subsequently tested by nested-PCR for single detection of TICV and ToCV using primer pairs TIC-3/TIC-4 and ToC-5/ToC-6, respectively (1). Only one fragment of approximately 463 bp was amplified from the five plants with the primer pair specific for ToCV. No amplification was obtained with the primers specific for TICV. Four purified amplicons of 463 bp were directly sequenced in both directions. Sequence comparisons of the 419-bp consensus sequence, encompassing nucleotides 750 and 1,167 of the HSP-70 homolog gene, revealed 98% identity with the reported sequences of tomato infecting isolates of ToCV from Brazil (GenBank Accession No. EU868927) and the United States (GenBank Accession No. AY903448). Virus-free adults of Bemisia tabaci biotype B were confined on symptomatic pepper leaves for a 48-h acquisition access period. Twenty adults were transferred to one plant of sweet pepper cv. Magda for a 24-h inoculation access period. The sweet pepper plant exhibited the original symptoms on the leaves 67 days after inoculation under greenhouse conditions. Infection by ToCV was confirmed by RT-PCR. The susceptibility of sweet pepper plants to ToCV was previously reported in Spain (2), whereas in the United States, this species was experimentally found as nonhost for this virus (4). Further studies are needed to better understand the variable susceptibility of sweet pepper to ToCV. References: (1) C. I. Dovas et al. Plant Dis. 86:1345, 2002. (2) G. Lozano et al. Plant Dis. 88:224, 2004. (3) M. R. Rojas et al. Plant Dis. 77:340, 1993. (4) W. M. Wintermantel et al. Plant Dis. 90:814, 2006.

During 2006 and 2007 in the region of Sumaré, state of São Paulo, Brazil, surveys were done on tomato (Solanum lycopersicum L.) virus diseases in three open field-grown crops. The data revealed low incidence (0.25 to 3.42%) of randomly distributed plants exhibiting interveinal chlorosis and some necrosis on the basal leaves. Symptoms were only observed on old fruit-bearing plants. Preliminary analysis of thin sections of symptomatic leaves from one plant by transmission electron microscopy revealed the presence of aggregates of thin, flexible, and elongated particles in some phloem vessels, suggesting infection with a member of the genus Crinivirus, family Closteroviridae. Total RNA was extracted separately from leaves of 10 symptomatic plants and used for one-step reverse transcription (RT)-PCR using the HS-11/HS-12 primer pair, which amplifies a fragment of 587 bp from the highly conserved region of the heat shock protein (HSP-70) homolog gene reported for Tomato infectious chlorosis virus (TICV) and Tomato chlorosis virus (ToCV) (1). The RT-PCR product was subsequently tested by nested-PCR for single detection of TICV and ToCV using primer pairs TIC-3/TIC-4 and ToC-5/ToC-6, respectively (1). Only one fragment of approximately 463 bp was amplified from 7 of the 10 plants with the primer pair specific for ToCV. No amplification was obtained with the primers specific for TICV. Two amplicons of 463 bp were purified and directly sequenced in both directions. Sequence comparisons of the 463-bp consensus sequence (GenBank Accession No. EU868927) revealed 99% identity with the reported sequence of ToCV from the United States (GenBank Accession No. AY903448) (3). Virus-free adults of Bemisia tabaci biotype B confined on symptomatic tomato leaves for a 24-h acquisition access period were able to transmit the virus to healthy tomato plants, which reproduced the original symptoms on the bottom leaves 65 days after inoculation under greenhouse conditions. Infection from transmission was confirmed by RT-PCR using the HS-11/HS-12 primer pair. In addition to B. tabaci biotype B, the greenhouse whitefly, Trialeurodes vaporariorum, has also been reported as a vector of ToCV, although it is less efficient than the B. tabaci biotype B in transmission of this virus (4). T. vaporariorum, which was previously considered limited to greenhouses, was recently reported in tomato and green bean (Phaseolus vulgaris L.) crops under field conditions in São Paulo State (2). Therefore, it might also contribute to the spread of ToCV in tomato crops in São Paulo. To our knowledge, this is the first report of ToCV in Brazil and South America. References: (1) C. I. Dovas et al. Plant Dis.86:1345, 2002. (2) A. L. Lourenção et al. Neotrop. Entomol. 37:89, 2008. (3) W. M. Wintermantel et al. Arch. Virol. 15:2287, 2005. (4) W. M. Wintermantel and G. C. Wisler. Plant Dis. 90:814, 2006.

We report the use of the coat protein (CP) gene from Passion fruit woodiness virus (PWV) to produce resistant transgenic plants of yellow passion fruit. A full-length CP gene from a severe PWV isolate from the state of São Paulo, Brazil (PWV-SP) was cloned into pCAMBIA 2300 binary vector, which was further introduced into Agrobacterium tumefaciens strain EHA 105. Leaf disks were used as explants for transformation assays, e.g., 2,700 and 2,730 disks excised from plants from the Brazilian cultivars IAC-275 and IAC-277, respectively. In vitro selection was performed in kanamycin. After transferring to the elongation medium, 119 and 109 plantlets of IAC-275 and IAC-277, respectively, were recovered. Integration of the PWV CP gene was confirmed in seven of eight plants evaluated by Southern blot analysis, showing different numbers of insertional events for the CP gene. Three transgenic plants (T3, T4, and T7) expressed the expected transcript, but the 32 kDa PWV CP was detected by Western blot in only two plants (T3 and T4). The results of three successive mechanical inoculations against the transgenic plants using three PWV isolates showed that the primary transformant T2 of IAC-277 was immune to all isolates.

Stuffing cucumber (Cyclanthera pedata var. edulis Schrad.) is native to the Americas, where it often occurs as an escape. The species is monoecious, with small flowers and large and deeply palmately lobed leaves. Fruits are puffy, partially hollow, and measure 5 × 15 cm long. The plant has soft spines, a tapered neck, and black seeds. The species has long been cultivated in Asia, where fruits are eaten raw as a substitute for cucumber or cooked (1). One plant showing intense mosaic, without leaf malformation, was found near a squash crop (Cucurbita moschata) in Anhembi County, State of São Paulo. Electron microscopic examination of a negatively stained sap preparation from leaves of this plant showed that it contained numerous flexuous rod-shaped particles, approximately 700 × 760 nm long, similar to those of potyviruses. Extracts from symptomatic leaves were rub-inoculated to zucchini squash (Cucurbita pepo), Carica papaya, Chenopodium amaranticolor, C. quinoa, Gomphrena globosa, and Phaseolus vulgaris cv. Black Turtle 2. Only zucchini squash was infected and developed severe mosaic with intense leaf malformation. Extracts from field infected stuffing cucumber and experimentally inoculated zucchini squash were tested by plate-trapped antigen enzyme linked immunosorbent assay (PTA-ELISA) with antisera against the following potyviruses: Papaya ringspot virus-type W (PRSV-W), Zucchini yellow mosaic virus (ZYMV), and Watermelon mosaic virus-2 (WMV-2). Samples were also tested with antisera against a tospovirus (Zucchini lethal chlorosis virus) and a cucumovirus (Cucumber mosaic virus). Both samples were positive in PTA-ELISA only with PRSV-W antiserum. This is the first report of C. pedata var. edulis as a natural host for PRSV-W in Brazil. References: (1) R. W. Robinson and D. S. Decker-Walters. 1997. Cucurbits. CAB International, UK.

Cucurbits species grown in 38 of 40 agricultural regions in the state of São Paulo, Brazil, were surveyed for the relative incidence of Cucumber mosaic virus (CMV), Papaya ringspot virus-type W (PRSV-W), Watermelon mosaic virus-2 (WMV-2), Zucchini lethal chlorosis virus(ZLCV), and Zucchini yellow mosaic virus (ZYMV) during May 1997 and June 1999. Samples from 621 plants, representing eight cultivated species, six wild species, and one commercial hybrid (Cucurbita moschata × C. maxima), were analyzed by plate trapped antigen enzyme-linked immunosorbent assay (PTA-ELISA). PRSV-W and ZYMV were the most frequently found viruses, accounting for 49.1 and 24.8%, respectively, of 605 samples tested. ZLCV, CMV, and WMV-2 were detected in 7.8, 6.0, and 4.5% of 612, 497, and 423 samples tested, respectively. Double infection was found in 97 samples, and triple infection was found in 10 samples. Quadruple infection was detected in one C. pepo sample. Plants that were symptomatic but negative by PTA-ELISA might be due to abiotic agents, infection by virus for which antiserum was not available, such as Squash mosaic virus, or infection with an as yet uncharacterized virus.

Cayaponia tibiricae Cogn. (CT) is a wild Cucurbitaceae species found in secondary forests in the State of São Paulo, Brazil. The species has indefinite growth and bears oblong dark green fruits, 15 to 20 mm long (1,2). CT plants showing yellow mosaic symptoms were found in Atibaia County. Extracts from symptomatic plants were rub inoculated to zucchini squash (Cucurbita pepo L.) and Chenopodium amaranticolor Coste & Reyn. Zucchini squash plants developed severe yellow mosaic with intense leaf malformation, while C. amaranticolor showed necrotic local lesions. Extracts from naturally infected CT, zucchini squash, and C. amaranticolor were tested by plate trapped antigen-enzyme-linked immunosorbent assay (PTA-ELISA) with antisera against papaya ringspot virus type W (PRSV-W), zucchini yellow mosaic virus (ZYMV), zucchini lethal chlorosis virus (ZLCV), watermelon mosaic virus 2 (WMV-2), and cucumber mosaic virus (CMV). All samples were positive in PTA-ELISA only with ZYMV antiserum. Also, in Western blot (immunoblot) assay, ZYMV antiserum labeled a protein of approximately 36 kDa. Electron microscopic examination of ultrathin sections from infected CT tissue revealed the presence of pinwheel inclusions typical of potyvirus (type 1) infection in the cytoplasm of the cell. CT seedlings were susceptible to mechanical inoculation with the ZYMV isolated from this species. This is the first report of CT as a natural host for ZYMV in Brazil. References: (1) A. Cogniaux. Flora Brasiliensis 6:1, 1878. (2) M. Pio Corrêa. 1926. Diccionário das plantas úteis do Brasil e das exóticas cultivadas. Vol 1. Ministério da Agricultura, Rio de Janeiro, Brazil.

Two mild strains of papaya ringspot virus-type W (PRSV-W) were tested under greenhouse and field conditions to study the potential of cross-protection for control of zucchini squash mosaic. Protected plants of zucchini squash cultivars Caserta and Clarinda were challenged with three severe strains from different geographic regions of the country in tests carried out in the greenhouse. Challenge inoculations were done mechanically 10 and 20 days after the protective inoculation. The mild strains did not have a visible negative effect on the development of the plants and offered effective protection against the severe strains. Field tests of protected Caserta plants were carried out in Piracicaba County, São Paulo. Comparative evaluation based on the symptoms and development of protected and unprotected plants of zucchini squash showed that both mild strains effectively protected the plants against the effects of the severe strain present in the field. Yield of marketable fruits harvested from protected plants was only 10% less than that of the healthy plants in one field trial. Compared with the yield from plants infected with the severe strains, protected plants showed an increase of 511 and 633% in the number and weight of marketable fruits, respectively, in a test in 1994. In a second experiment in 1995, an increase of 327% in the number and 344% in the weight of marketable fruits was recorded. These studies demonstrate the effectiveness of cross-protection for the control of the mosaic disease caused by PRSV-W in zucchini squash and offer growers a method for large-scale application of this technology.