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Since 2007, a new disease in broccoli (Brassica oleracea var. italica Plenck) has been observed in the São Paulo state, Brazil. The characteristic symptoms of the disease are plant stunting, inflorescence malformation, reddening of the leaves and phloem necrosis. Nested polymerase chain reaction with P1/Tint and F2n/R2 primer pairs revealed the presence of phytoplasmas in diseased broccoli plants. Restriction fragment length polymorphism and phylogenetic analysis of the 16S rDNA gene showed that phytoplasmas belonging to 16SrI, III and XIII groups were associated with the plants. To the best of our knowledge, this is the first report of phytoplasmas in this Brassica species in Brazil, as well the first time phytoplasmas of 16SrIII and XIII groups have been associated with broccoli plants.

Symptoms resembling giant calyx, a graft-transmissible disease, were observed on 1-5 % of eggplant (aubergine; Solanum melongena L.) plants in production fields in Sao Paulo state, Brazil. Phytoplasmas were detected in 12 of 12 samples from symptomatic plants that were analysed by a nested PCR assay employing 16S rRNA gene primers R16mF2/R16mR1 followed by R16F2n/ R16R2. RFLP analysis of the resulting rRNA gene products (1.2 kb) indicated that all plants contained similar phytoplasmas, each closely resembling strains previously classified as members of RFLP group 16SrIII (X-disease group). Virtual RFLP and phylogenetic analyses of sequences derived from PCR products identified phytoplasmas infecting eggplant crops grown in Piracicaba as a lineage of the subgroup 16SrIII-J, whereas phytoplasmas detected in plants grown in Braganç a Paulista were tentatively classified as members of a novel subgroup 16SrIII-U. These findings confirm eggplant as a new host of group 16SrIII-J phytoplasmas and extend the known diversity of strains belonging to this group in Brazil. INTRODUCTIONEggplant (aubergine; Solanum melongena L.) is among the most important edible crop species grown in Brazil. Sao Paulo state is a leader in the production of this crop, where it is currently cultivated over 1037 ha, with a total annual yield of 47 549 tonnes (figures for 2009). A disease of low incidence affecting approximately 1-5 % of plants was recently observed in fields of eggplant grown in Bragança Paulista and Piracicaba. Symptoms of affected plants included foliar chlorosis, shoot proliferation, shortened internodes, stunting, enlarged calyces, small flowers and fruit of reduced size. Collectively, these symptoms closely resembled those described previously for giant calyx, a disease of eggplant first reported to occur in Brazil during the 1990s (Boiteaux et al., 1994). The latter disease was suspected to have a phytoplasma aetiology based on consistent observation by electron microscopy of phytoplasma cells in the phloem sieve elements of diseased plants. A subsequent investigation of DNA extracts derived from diseased eggplants in Sao Paulo state and Brasília-DF by PCR-RFLP analysis concluded that phytoplasmas associated with eggplant giant calyx were most similar to strains previously classified as members of 16Sr RFLP group 16SrIII, subgroup B (16SrIII-B; Barros et al., 1998).Eggplant diseases associated with phytoplasmas have been reported in other countries. In Japan, a phytoplasma affiliated to group 16SrI was characterized molecularly from eggplant with dwarfing symptoms (Okuda et al., 1997;Lee et al., 1998). A disease called little leaf was reported in Bangladesh and molecular identification indicated that the phytoplasma belonged to group 16SrVI (Siddique et al., 2001). The study also demonstrated the phytoplasma to be indistinguishable from brinjal little leaf (BLL) phytoplasma characterized previously in India (Lee et al., 1998; Seemüller et al., 1998). Recently, the BLL strain was classified as a member of group 16SrVI, subg...

No Brasil, ainda há poucas pesquisas sobre os microrganismos associados às sementes florestais. O objetivo desse trabalho foi detectar fungos associados às sementes de seis espécies florestais nativas: açoita-cavalo (Luehea divaricata), araçá amarelo (Psidium cattleyanum), corticeira (Erythrina falcata), rabode-bugio (Lonchocarpus sp.), vassoura-vermelha (Dodonea viscosa) e paujacaré (Piptadenia gonoacantha ABSTRACTIn Brazil, there is still scarce research on microorganisms associated with forest seeds. The aim of this study was to detect fungi associated with seeds of six native forest species: "açoita-cavalo" (Luehea divaricata), "araçá amarelo" (Psidium cattleyanum), "corticeira" (Erythrina falcata), "rabo-de-bugio" (Lonchocarpus sp.), "vassoura-vermelha" (Dodonea viscosa) and "pau-jacaré" (Piptadenia gonoacantha). Eight genera of fungi were identified as potentially Carmo, A.L.M.; Mazaratto, E.J.; Eckstein, B.; Santos, A.F. Association of Fungi with Seeds of Native Forest Species. Summa Phytopathologica, v.43, n.3, p.246-247, 2017. pathogenic: Cladosporyum sp., Fusarium sp., Pestalotiopsis sp., Phomopsis sp., Coletotrichum sp., Alternaria sp., Phoma sp. and Botrytis sp., and saprophytic fungi: Aspergillus sp., Penicillium sp., Rhizopus sp., Epicoccum sp., Torula sp., Trichoderma sp., Gliocladium sp., Chaetomium sp. and Trichothecium sp. Results evidence the diversity of fungi associated with native forest seeds, including several potentially phytopathogenic fungi.Keywords: Seed pathology; sanitary quality; native species.Espécies nativas possuem grande relevância ecológica, devido ao potencial em reflorestamento e recomposição de áreas degradadas. No entanto, muitas espécies com destaque pela qualidade da madeira, uso medicinal, ornamental e industrial, acabam sendo dizimadas pelo desmatamento e cultivos agrícolas (12). Algumas espécies florestais como açoita-cavalo (Luehea divaricata Mart.) araçá amarelo (Psidium cattleyanum Sabine L.), corticeira (Erythrina falcata Benth.), rabode-bugio (Lonchocarpus sp. Mart. ex Benth.), vassoura-vermelha (Dodonea viscosa Jacq.) e pau-jacaré (Piptadenia gonoacantha [Mart.] J.F.Macbr.) apresentam diferentes finalidades comerciais, no entanto, poucas informações estão disponíveis quanto à qualidade sanitária de sementes dessas espécies (3). Além disso, as sementes florestais nativas têm uma oferta reduzida que depende da espécie, do ano e do indivíduo (10), dificultando a execução de trabalhos de pesquisa nesta área.A demanda por sementes de espécies florestais nativas tem aumentado nos últimos anos, principalmente para atender a lei nº 12.651 (2) que dispõe sobre a proteção da vegetação nativa no Brasil, as quais englobam também as áreas de preservação permanente e reserva legal. Com o aumento do intercâmbio de sementes entre regiões, aumenta-se também o risco de transporte de fitopatógenos associados às sementes, representando uma forma de disseminação de patógenos para novas áreas (3). Em algumas espécies nativas, alguns autores já mostraram a associação de fungos (...

Recently, disease in broccoli plants (Brassica oleracea var. italica) was associated with three distinct phytoplasmas in Brazil. The disease named broccoli stunt (BS) has caused significant economic losses in São Paulo State. Group 16SrIII phytoplasmas is the most common group that have been associated with BS. The BS disease is still poorly understood, and the lack of information about the vectors further impairs its management. In this study, leafhoppers belonging to 18 different species were collected from weeds thriving near broccoli fields that were affected by group 16SrIII phytoplasmas. Specific primers revealed the presence of group 16SrIII phytoplasmas in five leafhoppers: Empoasca spp., Agallia albidula Uhler, Agalliana sticticollis (Stål), Planicephalus flavicosta (Stål), and Atanus nitidus (Linnavuori). The identity of the phytoplasmas was confirmed through DNA sequencing analysis. The leafhoppers were infected by phytoplasmas of the 16SrIII group (proposed "Candidatus Phytoplasma pruni" species) and are phylogenetically related to the broccoli stunt phytoplasma (BSP) strains detected in the study area and, thus, are considered potential vectors of group 16SrIII phytoplasmas to broccoli plants.

Periwinkle (Catharanthus roseus), a tropical perennial plant, was found to be infected by a phytoplasma. Plants exhibiting virescence, phyllody and variegation symptoms were collected in the states of Minas Gerais and São Paulo, Brazil. The phytoplasma was transmitted by grafting from an infected periwinkle plant to healthy plants and by dodder to a citrus plant. Phytoplasma isolates from periwinkle plants from Brazil had the 16S rDNA gene sequenced and were classified in the 16SrIX group, subgroup A, belonging to the 'Candidatus P. phoenicium' species.

A Deus, por estar sempre do meu lado e me guiar para caminhos seguros. Ao meu amor Júlio, pela dedicação, carinho, por me dar forças para ir sempre mais longe do que poderia imaginar DEDICO Aos meus pais Adalberto e Marlene Por todo amor, e pelo esforço que fizeram a fim de me oferecerem o melhor A minha irmã Angélica e minha sobrinha Maria Eduarda Por todo amor, e alegria que trazem para minha vida OFEREÇO 4 À amiga Eliane Gonçalves da Silva, por toda ajuda no laboratório, e, principalmente pela amizade, incentivo e conselhos; 6 À amiga Raquel de Cássia Neroni, que aos poucos conquistou minha admiração, confiança e por me oferecer uma amizade sincera que sempre me faz tão bem; Aos amigos que fiz durante o período de pós-graduação Ana Querales, por toda a atenção e força que me deram durante este período; Aos amigos de longa data Adriano L. Spanhol, Andréa Ruver, Idiana Marina Dalastra e Luciana Iurkiv, pela amizade que distancia nenhuma reduz; Aos amigos e colegas do Laborátorio Thais, e demais colegas do Departamento por tudo que me ensinaram e pelos bons momentos; Aos professores e funcionários do Departamento de Fitopatologia e Nematologia da ESALQ, que muito me ensinaram e sempre foram atenciosos quando necessitei de ajuda; A todos que de alguma forma contribuíram para a minha formação. Obrigada! 7 SUMÁRIO RESUMO . ABSTRACT Broccolo stunt: identification of phytoplasmas, potential insect vectors and alternative hosts and epidemiology of the diseaseBroccoli (Brassica oleraceae var. italica) is one of the most important vegetables in Brazil, whose trading volume in CEAGESP is approximately 13 000 tons per year. Recently, a new disease has caused significant losses in this crop cultivated in the largest producing region of the São Paulo State. The characteristic symptoms of the disease are expressed by plant stunting and necrosis of phloem vessels. Because these symptoms indicate the presence of phytoplasmas in cabbage and cauliflower crops, grown in the same geographical region, it was suspected that the same pathogens could be associated with the affected broccoli plants. Therefore, the total DNA from symptomatic plants of broccoli was analyzed by PCR with specific primers for the 16S rDNA of phytoplasmas. Through the techniques of RFLP and nucleotide sequencing of the same genomic region, the phytoplasmas were identified as belonging to the groups 16SrI, 16SrIII and 16SrXIII. Through RFLP analysis, phytoplasmas were also identified in several species of weeds and leafhoppers in the family Cicadellidae collected in adjacent areas of broccoli fields. Phytoplasmas belonging of the 16SrIII group were identified in the weeds belonging to the species Agetarum conyzoides, Crotalaria lanceolata, Lepidium virginicum, Nicandra physalodes, Paulicourea marcgravii, Ricinus communis, Sida rhombifolia, Sonchus oleraceae, Bidens pilosa, Erigeron bonariensis, Emilia sonchifolia, Leonorus sibiricus, while phytoplasmas of the 16SrVII group were found in the last four mentioned species. In respect to insects, phytoplasmas were ...

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.