RESUMO -Objetivou-se com este trabalho avaliar o efeito da adubação nitrogenada em cobertura na presença e na ausência de bactérias do gênero Azospirillum brasilense em milho safrinha. O experimento foi realizado na safrinha de 2015, em um solo classificado como Latossolo Vermelho Amarelo distrófico, no município de Alta Floresta-MT.O delineamento experimental utilizado foi em blocos ao acaso, em arranjo fatorial 4 x 2, com quatro repetições. Os tratamentos consistiram de ausência e presença de Azospirillum e quatro doses de N em cobertura (0, 35, 70, 105 kg ha -1 ). Foram avaliados a altura de plantas, a altura de inserção da espiga, o comprimento de entrenó, o diâmetro do colmo, o comprimento da espiga, o diâmetro da espiga, a massa de sabugo, o diâmetro do sabugo, o número de grãos por fileira e o número de fileiras por espiga, a massa de 100 grãos e a produtividade de grãos. Na associação de Azospirillum com N, houve incremento linear no diâmetro do colmo e no número de grãos por fileira. As doses de N em cobertura apresentaram ajuste quadrático, em que a produtividade aumentou em 15% com a dose de 69,33 kg ha USE OF Azospirillum brasilense TO INCREASE THE EFFICIENCY OF NITROGEN FERTILIZATION ON CORNABSTRACT -This study aims to evaluate the effect of nitrogen fertilization in top dress with and without the Azospirillum brasilense bacteria with interim-harvest of corn. The experiment was completed during 2015's interim harvest on a soil classified as Distrophic Red Yellow Latosol in Alta Floresta, MT. The experiment was conducted using a 4x2 factorial arrangement in random blocks, with four repetitions. The treatments were both with and without the Azospirilllum and four doses of top dress Nitrogen (0, 35, 70, 105 kg ha -1 ). Were evaluated the plant's height, the ear of the corn insertion height, the length of the internode, the diameter of the stalks, the length of the ear, the diameter of the ear, the corncob mass, the corncob diameter, the number of kernels per row and the rows per corncob, the 100 kernels weight and the kernels productivity. In the association between Azospirillum and nitrogen, there was linear increase at the diameter of the stalks and the numbers of kernels per row. The dosage of top dress nitrogen promoted an increase of 15% at the production with 69.33 kg ha -1 of nitrogen in topdress.
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.
Physalis is an herbaceous species native to the Andes region. Currently, it is cultivated in various Brazilian states due to the economic interest of growers for this new fruit.Physalis plants grown in the field showed symptoms of shoot proliferation, leaf malformation, and chlorosis. Since these symptoms are commonly induced by phytoplasmas, this study investigated to confirm the presence of these prokaryotes in symptomatic plants. After DNA extraction from symptomatic and asymptomatic plants, phytoplasmas were found in all affected plants through the nested PCR. Examination by transmission electron microscopy (TEM) using appropriately prepared segments of leaf veins allowed the visualization of typical pleomorphic cells of phytoplasmas in the phloem of symptomatic plants. The computer-simulated RFLP patterns and the phylogenetic analysis allowed identifying the detected phytoplasmas as a 'Candidatus Phytoplasma fraxini'-related strain belonging to the 16SrVII-B subgroup. Moreover, physalis was identified as an additional host species for phytoplasmas in the 16SrVII group, expanding the current knowledge on the host range of phytoplasmas in this group.
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.
This contribution describes biological and molecular features of an isolate of the chrysanthemum stem necrosis orthotospovirus (CSNV) found naturally infecting sweet pepper plants (Capsicum annuum L.). High-throughput sequencing resulted in three contig sequences of 2948, 4829 and 8959 nucleotides for CSNV-Ca small, medium and large RNAs, respectively. They shared 98.4%, 99.3% and 99.1% nucleotide identities, respectively, with the corresponding nucleotide sequences of a CSNV isolate from Slovenia. The virus was mechanically transmitted to sweet pepper and some additional test plants, and RT-PCR confirmed the infection with specific primers for CSNV. Additionally, transmission electron microscopic analysis of ultrathin sections prepared from symptomatic sweet pepper fruit tissue showed orthotospoviruses-like particles in elements of the endoplasmic reticulum. Studies are needed to evaluate the occurrence of CSNV infecting sweet pepper in commercial crops, to assess yield damage and develop recommendations for management.
Tomato severe rugose virus (ToSRV) is one of Brazil’s main begomoviruses infecting tomatoes (Solanum lycopersicum). Recent studies indicate that soybean (Glycine max) crops harboring the whitefly Bemisia tabaci 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 soybeans 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 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 tomatoes, 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.
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.
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