Olive trees exhibiting slow development, yellowing, and high intensity of shoot proliferation with small leaves were observed in commercial plantings, in the municipality of Extrema, Minas Gerais (MG) state in 2015. The incidence of symptomatic plants was about 70% and diseased trees presented yield reduction. Here we report the association of symptomatic olive trees with a phytoplasma and describe its molecular identification. Symptomatic plants (38 trees) were sampled in three growing areas located in the same municipality. The samples consisted of bunch of leaves and young shoots. The total DNA was extracted using DNeasy® Plant Mini Kit (Qiagen, Hilden, Germany). Phytoplasma detection was conducted by nested PCR with primers P1/16S-SR (Lee et al. 2004) followed by R16F2n/R16R2 (Gundersen and Lee 1996). PCR assays generated amplicons (~1.2 kb) from 28 trees out of 38 symptomatic plants, confirming the association of phytoplasma with diseased plants. The disease was named olive witches’ broom. The genomic fragments amplified by nested PCR were cloned into Escherichia coli DH5α and sequenced. The sequence representative of the olive phytoplasma was designated OWB-Br01 (Olive Wiches' Broom-Brazil 01) and deposited in GenBank under accession number MH141985. This sequence shared 99% sequence identity with phytoplasmas affiliated with 16SrVII group. According to the iPhyClassifier online tool (Zhao et al. 2009) the olive witches’-broom phytoplasma was classified as a variant of subgroup 16SrVII-B with a pattern similarity coefficient of 0.99. The phylogenetic tree showed that OWB-Br01 phytoplasma emerges from the same branch of the reference phytoplasma of the 16SrVII-B subgroup (Erigeron witches᾽-broom phytoplasma - GenBank AY034608), indicating that the olive tree phytoplasma is a member of the 16SrVII-B subgroup. The pathogenicity test was performed with 28 healthy plants (cultivar Arbequina) grown in pots, which were grafted by simple english forklift with scions obtained from olive plants (Arbequina) six years old, naturally infected by the phytoplasma. The initial symptoms were observed four months after grafting and at eight months 22 grafted plants exhibited slow growth, yellowing, and small leaves as those naturally observed in the fields. Molecular characterization allowed identify the phytoplasma as a member of the 16SrVII-B subgroup. In Brazil, representatives of the 16SrVII group were previously reported in association with diverse botanical species. Thus, a strain of 16SrVII-C subgroup was identified in sunn hemp (Flôres et al. 2013); the reference phytoplasma of 16SrVII-D subgroup was found in erigeron plants (Flôres et al. 2015); and the representative of 16SrVII-F was detected in the wild species Vernonia brasiliana. (Fugita et al. 2017). Specifically regarding subgroup 16SrVII-B, the reference phytoplasma of this subgroup was described from erigeron and periwinkle (Barros et al. 2002), while other members of this subgroup were reported in cauliflower (Pereira et al. 2016a) and ming aralia (Pereira et al. 2016b). The disease here studied is a threat since olive planting is in large expansion in Brazil. A potential control option could be use of propagative material from sources free of the pathogen. Based on our findings, olive tree represents a new host for subgroup 16SrVII-B phytoplasma, which is different from 16Sr groups previously reported as associated with olive witches’ broom in other countries.

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.

Acerola bushes were observed showing symptoms of shoot proliferation, generalized stunting, yellowing and decline. Since these symptoms are typically induced by phytoplasmas, this survey was carried out with the aim of detecting, identifying and classifying the supposed phytoplasma present in symptomatic bushes. Total DNA was extracted from symptomatic and asymptomatic samples and used in nested PCR conducted by the primer pairs R16mF2/mR1 followed by R16F2n/R2. Amplifications of expected genomic fragments of 1.2 kb revealed the presence of phytoplasma in 73 % of the symptomatic samples. Molecular analyses, using computer-simulated RFLP patterns, similarity coefficient calculation and phylogenetic analysis allowed for classifying the bacterium as a 'Candidatus Phytoplasma pruni' -related strain (subgroup 16SrIII-F). The phytoplasma induced the same symptoms in healthy acerola plants inoculated by grafting and showed molecular identity with the strain identified in naturally infected bushes. Although various strains belonging to distinct subgroups within the 16SrIII group have been previously identified in Brazil, this is the first report of the presence of a representative of the 16SrIII-F subgroup in the Brazilian agroecosystem. Considering that phytoplasmas can be systemically distributed throughout the plant and acerola plants are vegetatively propagated, it is recommended that propagation material be obtained from mother plants free of the pathogen.

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Efeito do Acibenzolar-S-metílico e filtrado da suspensão de células de Streptomyces scabies na indução de resistência de plantas de milho ao enfezamento vermelho causado por fitoplasma Cláudia Alves de Almeida Tese apresentada para obtenção do título de Doutora em Ciências. Área de concentração: Fitopatologia Piracicaba 2020 1. Zea mays 2. Mollicutes 3. Dalbulus maidis 4. Alterações bioquímicas 5. Controle alternativo I. Título