Biological and molecular characterization of a new potexvirus isolated from Adenium obesum.

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.

Infection of groundnut ringspot virus in Plumeria pudica characterized by irregular virus distribution and intermittent expression of symptoms.

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.

Characterization of Soybean, Tomato, and Nicandra physalodes as Sources of Inoculum of Tomato Severe Rugose Virus to Tomato Crops.

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.

First report of groundnut ringspot virus infecting Zinnia sp. in Brazil.

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.

First report of costus stripe mosaic virus infecting Tradescantia spathacea plants in Brazil.

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: new host of soybean mosaic virus in Brazil.

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.

Natural Infection Rate of Known Tomato chlorosis virus-Susceptible Hosts and the Influence of the Host Plant on the Virus Relationship With Bemisia tabaci MEAM1.

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.

First report of bidens mosaic virus infecting Centella asiatica in Brazil.

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.

Arachis virus Y, a new potyvirid from Brazilian forage peanut (Arachis pintoi).

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.

Near-complete genome sequence and biological properties of an allexivirus found in Senna rizzinii in Brazil.

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.

Ocurrence of pepper yellow mosaic virus and cucumber mosaic virus on Capsicum chinense in the state of Amazonas, Brazil / Ocorrência do pepper yellow mosaic virus e cucumber mosaic virus em Capsicum chinense no estado do Amazonas, Brasil

The habanero chilli pepper, Capsicum chinense is an important crop in the Amazon Basin, mainly grown by small-scale producers. Capsicum chinense plants in an experimental field in the northern Brazilian state of Amazonas were found exhibiting characteristic symptoms of viral infection. Leaf sap from symptomatic plants examined under a transmission electron microscope revealed the presence of elongated flexuous particles and isometric particles. Using molecular assays, the viruses were identified as pepper yellow mosaic virus (PepYMV) and cucumber mosaic virus (CMV). Aphids, identified as Aphis gossypii, were found colonizing the C. chinense plants in the field and may be the vector for both PepYMV and CMV. We report the first occurrence of these viruses infecting C. chinense in the state of Amazonas.

A pimenta-de-cheiro, Capsicum chinense é uma cultura importante na Bacia Amazônica, cultivada principalmente por pequenos produtores. Plantas de C. chinense em um campo experimental localizado no norte do estado brasileiro do Amazonas, foram encontradas apresentando sintomas característicos de infecção viral. Extratos de amostras de folhas sintomáticas examinados ao microscópio eletrônico de transmissão revelaram a presença de partículas alongadas e flexuosas e de partículas isométricas. Análises moleculares permitiram identificar a presença do pepper yellow mosaic virus (PepYMV) e do cucumber mosaic virus (CMV). Pulgões, identificados como Aphis gossypii foram encontrados colonizando pimenteiras-de-cheiro neste campo experimental e podem representar o provável vetor de PepYMV e CMV. Este trabalho relata a primeira ocorrência desses vírus infectando C. chinense no estado do Amazonas.

Ocurrence of pepper yellow mosaic virus and cucumber mosaic virus on Capsicum chinense in the state of Amazonas, Brazil

The habanero chilli pepper, Capsicum chinense is an important crop in the Amazon Basin, mainly grown by small-scale producers. Capsicum chinense plants in an experimental field in the northern Brazilian state of Amazonas were found exhibiting characteristic symptoms of viral infection. Leaf sap from symptomatic plants examined under a transmission electron microscope revealed the presence of elongated flexuous particles and isometric particles. Using molecular assays, the viruses were identified as pepper yellow mosaic virus (PepYMV) and cucumber mosaic virus (CMV). Aphids, identified as Aphis gossypii, were found colonizing the C. chinense plants in the field and may be the vector for both PepYMV and CMV. We report the first occurrence of these viruses infecting C. chinense in the state of Amazonas. (AU)

Molecular and biological characterization of a putative new sobemovirus infecting Physalis peruviana.

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.

Kinetics of Systemic Invasion and Latent and Incubation Periods of Tomato severe rugose virus and Tomato chlorosis virus in Single and Co-Infections in Tomato Plants.

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.

A highly divergent isolate of tomato blistering mosaic virus from Solanum violaefolium.

The complete genome of a tymovirus infecting Solanum violaefolium was sequenced. The genome comprised 6284 nt, with a 5'-UTR of 137 nt and a comparatively longer 3'-UTR of 121 nt. Sequence analysis confirmed three ORFs encoding a movement protein, a polyprotein, and a coat protein (CP). The isolate was considered to be the Tomato blistering mosaic virus (ToBMV) based on a CP amino acid sequence identity of 95.3 %. The nucleotide sequence of the complete genome of the S. violaefolium isolate, however, differed markedly from the other two reported ToBMV isolates, with identities of 76.6 and 76.3 %, below one of the demarcation criteria of the genus Tymovirus (overall genome identity of 80 %). No recombination signals were detected in the genome of this isolate. The high identity of the CP amino acid sequence and similar host responses suggest that the S. violaefolium isolate belongs to the same species as the Tomato blistering mosaic virus. The sequence analysis of this ToBMV isolate thus suggests that the demarcation criterion of 80 % overall genome sequence identity in the genus Tymovirus may require revision.

A novel subgroup 16SrVII-D phytoplasma identified in association with Erigeron witches' broom.

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).

Diversity and localization of bacterial endosymbionts from whitefly species collected in Brazil.

Whiteflies (Hemiptera: Aleyrodidae) are sap-sucking insect pests, and some cause serious damage in agricultural crops by direct feeding and by transmitting plant viruses. Whiteflies maintain close associations with bacterial endosymbionts that can significantly influence their biology. All whitefly species harbor a primary endosymbiont, and a diverse array of secondary endosymbionts. In this study, we surveyed 34 whitefly populations collected from the states of Sao Paulo, Bahia, Minas Gerais and Parana in Brazil, for species identification and for infection with secondary endosymbionts. Sequencing the mitochondrial Cytochrome Oxidase I gene revealed the existence of five whitefly species: The sweetpotato whitefly Bemisia tabaci B biotype (recently termed Middle East-Asia Minor 1 or MEAM1), the greenhouse whitefly Trialeurodes vaporariorum, B. tabaci A biotype (recently termed New World 2 or NW2) collected only from Euphorbia, the Acacia whitefly Tetraleurodes acaciae and Bemisia tuberculata both were detected only on cassava. Sequencing rRNA genes showed that Hamiltonella and Rickettsia were highly prevalent in all MEAM1 populations, while Cardinium was close to fixation in only three populations. Surprisingly, some MEAM1 individuals and one NW2 population were infected with Fritschea. Arsenopnohus was the only endosymbiont detected in T. vaporariorum. In T. acaciae and B. tuberculata populations collected from cassava, Wolbachia was fixed in B. tuberculata and was highly prevalent in T. acaciae. Interestingly, while B. tuberculata was additionally infected with Arsenophonus, T. acaciae was infected with Cardinium and Fritschea. Fluorescence in situ hybridization analysis on representative individuals showed that Hamiltonella, Arsenopnohus and Fritschea were localized inside the bacteriome, Cardinium and Wolbachia exhibited dual localization patterns inside and outside the bacteriome, and Rickettsia showed strict localization outside the bacteriome. This study is the first survey of whitely populations collected in Brazil, and provides further insights into the complexity of infection with secondary endosymionts in whiteflies.

Genome analysis of a severe and a mild isolate of Papaya ringspot virus-type W found in Brazil.

Papaya ringspot virus-type W (PRSV-W) is one of the most economically threatening viruses of cucurbits in Brazil. Premunization is one of the most effective PRSV control measures currently applied in squash and zucchini crops. PRSV-W-1, a mild and premunizing strain of PRSV has been successfully used to protect cucurbits against both the severe PRSV-W-C strain and other Brazilian PRSVs. To aid in understanding the mechanism by which PRSV-W-1 premunization operates, the complete genome sequences of PRSV-W-1 and PRSV-W-C were determined. PRSV-W-1 had a genome size of 10,332 nucleotides, whereas indels within the coat protein encoding gene meant that the genome size of PRSV-W-C was six nucleotides shorter than that of the mild strain. The genomes of the two strains shared 94.63% nucleotide sequence identity, with the 5' UTR and P1 being the most variable regions, and the coat protein and 3' UTR being the most conserved. Rigorous recombination analysis revealed that neither PRSV-W-1 nor PRSV-W-C was obviously recombinant, there was significant evidence that many other fully sequenced PRSV genomes were recombinant.