Microbiota of Attine Ants' Gardens: Visualizing a Microbial Landscape by Scanning Electron Microscopy

In macroscale ecosystems, such as rainforests or coral reefs, the spatial localization of organisms is the basis of our understanding of community ...

University of São Paulo (USP)

A betabaculovirus (DisaGV) was isolated from Diatraea saccharalis (Lepidoptera: Crambidae), one of the most important insect pests of the sugarcane and other monocot cultures in Brazil.

A betabaculovirus encoding a gp64 homolog.

Citrus leprosis virus C (CiLV-C) causes a severe disease affecting citrus orchards in the Western hemisphere. This study reveals the molecular variability of the virus by analyzing four genomic regions (p29, p15, MP and RNA2-intergenic region) distributed over its two RNAs. Nucleotide diversity (π) values were relatively low but statistically different over the analyzed genes and subpopulations, indicating their distinct evolutionary history. Values of πp29 and πMP were higher than those of πp15 and πRNA2-IR, whereas πMP was increased due to novel discovered isolates phylogenetically clustered in a divergent clade that we called SJP. Isolate BR_SP_SJP_01 RNA1 and RNA2 sequences, clade SJP, showed an identity of 85.6% and 88.4%, respectively, with those corresponding to CiLV-C, the type member of the genus Cilevirus, and its RNA2 5'-proximal region was revealed as a minor donor in a putative inter-clade recombination event. In addition to citrus, BR_SP_SJP_01 naturally infects the weed Commelina benghalensis and is efficiently transmitted by Brevipalpus yothersi mites. Our data demonstrated that negative selection was the major force operating in the evaluated viral coding regions and defined amino acids putatively relevant for the biological function of cilevirus proteins. This work provides molecular tools and sets up a framework for further epidemiological studies.

Phylogenetic and Molecular Variability Studies Reveal a New Genetic Clade of Citrus leprosis virus C.

Citrus leprosis has been one of the most destructive diseases of citrus in the Americas. In the last decade important progress has been achieved such as the complete genome sequencing of its main causal agent, Citrus leprosis virus C (CiLV-C), belonging to a new genus Cilevirus. It is transmitted by Brevipalpus yothersi Baker (Acari: Tenuipalpidae), and is characterized by the localized symptoms it induces on the leaves, fruits and stems. It occurs in the American continents from Mexico to Argentina. The virus was until recently considered restricted to Citrus spp. However, it was found naturally infecting other plants species as Swinglea glutinosa Merrill and Commelina benghalensis L., and has been experimentally transmitted by B. yothersi to a large number of plant species. Despite these advances little is known about the virus-vector relationship that is a key to understanding the epidemiology of the disease. Some components of the CiLV-C/B. yothersi relationship were determined using the common bean (Phaseolus vulgaris L. cv. 'IAC Una') as a test plant. They included: (a) the virus acquisition access period was 4 h; (b) the virus inoculation access period was 2 h; (c) the latent period between acquisition and inoculation was 7 h; (d) the period of retention of the virus by a single viruliferous mite was at least 12 days; (d) the percentage of viruliferous individuals from mite colonies on infected tissues ranged from 25 to 60%. The experiments confirmed previous data that all developmental stages of B. yothersi (larva, protonymph and deutonymph, adult female and male) were able to transmit CiLV-C and that transovarial transmission of the virus did not occur. CiLV-C can be acquired from lesions on leaves, fruits and stems by B. yothersi. Based on the distribution of lesions produced by single viruliferous B. yothersi on bean leaves, it is concluded that they tend to feed in restricted areas, usually near the veins. The short latent and transmission periods during the larval stage suggest that the CiLV-C/B. yothersi relationship is of the persistent circulative type.

Virus-vector relationship in the Citrus leprosis pathosystem.

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.

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

Endive (Cichorium endivia L.) is a very important cash crop for small farmers in Brazil. During inspections conducted in the summer season of 2019-2020, leaf samples of C. endivia 'La Spezia' seedlings exhibiting typical symptoms of orthotospoviruses infection (viz. concentric chlorotic spots and apical leaf deformation; ≈ 10%) were collected in commercial greenhouses in Brasília-DF, Central Brazil. Leaves of one healthy and three symptomatic plants were initially evaluated via double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) with polyclonal antibodies (produced at CNPH) raised against the nucleoprotein of the three major orthotospoviruses: tomato spotted wilt orthotospovirus (TSWV), groundnut ringspot orthotospovirus (GRSV) and tomato chlorotic spot orthotospovirus (TCSV). Strong serological reactions were observed only against GRSV antibodies exclusively in extracts from symptomatic samples. In order to confirm the causal agent of those symptoms, total RNA was extracted (Trizol®; Sigma) from infected leaf samples and used in a two-step reverse transcriptase polymerase chain reaction (RT-PCR) approach. Synthesis of the cDNA was carried out with the J13 primer (5'-CCC GGA TCC AGA GCA AT-3') (Cortez et al., 2001) followed by PCR assays with the primer pair BR60 (5'-AGA GCA ATC GTG TCA-3`) and BR65 (5'-ATC AAG CCT TCT GAA AGT CAT-3') (Eiras et al., 2001). This primer set amplifies a fragment of 453 bp including the untranslated region at the 3' terminus of the small RNA and the protein N-coding gene of at least five orthotospoviruses: TSWV, GRSV, TCSV, chrysanthemum stem necrosis orthotospovirus (CSNV) and zucchini lethal chlorosis orthotospovirus (ZLCV) (Eiras et al., 2001). The obtained amplicons (≈ 432 bp) were subsequently subjected to Sanger dideoxy nucleotide sequencing at CNPH. BLASTn analysis showed >99% identity with a wide array of GRSV isolates available in the GenBank. The nucleotide sequence of Tospo #1 (MT215222) and Tospo #3 (MT215224) isolates displayed 100% identity between them, whereas the Tospo #2 (MT215223) isolate displayed one non-synonymous point mutation in the 3' untranslated region in comparison with the former two isolates. Three plants of C. endivia, Capsicum annuum L. cv. Ikeda, tomato (Solanum lycopersicum L.) cv. Santa Clara and its isoline 'LAM-147' (with the Sw-5 resistance gene), Nicotiana rustica L., Lactuca sativa L. ('Vanda' and 'PI-342444') and Gomphrena globosa L. were mechanically inoculated individually with each GRSV isolate in order to confirm their pathogenicity. Chlorotic lesions and mosaic were observed seven days after inoculation of all plant materials, except the tomato inbred line 'LAM-147', which has the Sw-5 gene that confers broad-spectrum resistance to all Brazilian orthotospoviruses (Boiteux and Giordano, 1993). The GRSV infection was confirmed via DAS-ELISA and RT-PCR 15 days after inoculation, using the same set of antibodies and the primer pair BR60 / BR65. Transmission electron microscopy of ultrathin sections from symptomatic leaf tissues, both from field-infected and experimentally inoculated endive revealed the presence of typical orthotospovirus particles, within endoplasmic reticulum cisternae. Natural infection of endive by TSWV has been reported in Greece (Chatzivassiliou et al., 2000) and by TCSV in São Paulo State, Brazil and in Florida, USA (Subramanya Sastry et al., 2019). To our knowledge, it is the first report of GRSV naturally infecting this Asteraceae species in Brazil. Confirmation of GRSV infection of C. endivia plants is a relevant piece of information aiming to design effective disease management strategies. References: Boiteux, L.S. and Giordano, L. B. 1993. Euphytica 71: 151. Eiras, M. et al. 2001. Fitopatol. Bras. 26: 170. Chatzivassiliou, E.K. et al. 2000 Ann. Appl. Biol. 137: 127. Cortez, I., et al. 2001. Arch. Virol. 146: 265. Subramanya Sastry, K., et al. 2019. Encyclopedia of plant viruses and viroids. Springer, New Delhi. https://doi.org/10.1007/978-81-322-3912-3.

First Report of Cichorium endivia (Asteraceae) as a Natural Host of Groundnut ringspot orthotospovirus 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.

Strongylodon macrobotrys: new host of soybean mosaic virus 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.

Elliot Watanabe Kitajima

Plant Pathology and Nematology Department,
Escola Superior de Agricultura Luiz de Queiroz,
Plant Pathology and Nematology Department, Escola Superior de Agricultura Luiz de Queiroz

University of São Paulo (USP)

Microbiota of Attine Ants' Gardens: Visualizing a Microbial Landscape by Scanning Electron Microscopy

Elliot Watanabe Kitajima

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Plant Pathology and Nematology Department,Escola Superior de Agricultura Luiz de Queiroz,Plant Pathology and Nematology Department, Escola Superior de Agricultura Luiz de Queiroz

University of São Paulo (USP)

Microbiota of Attine Ants' Gardens: Visualizing a Microbial Landscape by Scanning Electron Microscopy

Plant Pathology and Nematology Department,
Escola Superior de Agricultura Luiz de Queiroz,
Plant Pathology and Nematology Department, Escola Superior de Agricultura Luiz de Queiroz