Olive tree represents a new host of a subgroup 16SrVII-B phytoplasma associated with witches' broom disease in Brazil.

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 witchesá¾½-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.

Delineation of a novel subgroup 16SrXIII-J phytoplasma, a 'Candidatus Phytoplasma hispanicum'-related strain, based on computer-simulated RFLP and phylogenetic analysis.

Symptoms of fruit phyllody and slow growth, which are suggestive of phytoplasma infection, were observed in strawberry plants cultivated in commercial fields. In order to provide evidence of association of phytoplasma with affected plants, assays for detecting and identifying were performed through computer-simulated restriction fragment length polymorphism (RFLP) and phylogenetic analysis. Total DNA was extracted from symptomatic and asymptomatic samples and used as template in nested PCR primed by the primers P1/Tint followed by R16F2n/16R2. Amplified DNA fragments of 1.2 kb from the 16S rRNA gene revealed the presence of phytoplasma in all symptomatic samples. Molecular detection was confirmed by electron transmission microscopy, which evidenced pleomorphic bodies in the phloem vessels. Nucleotide sequence representative of the strawberry phytoplasma shared 97.2 to 99 % similarity with phytoplasmas currently classified as members of the distinct subgroups within the 16SrXIII group. Similarity coefficient (F) values ranged from 0.70 to 0.92, indicating that strawberry phytoplasma delineates a new strain in addition to 'Candidatus Phytoplasma hispanicum'-related strains. The evolutionary tree displayed that this strain emerges as a new branch in relation to those previously described. The novel strain, designated SFP (strawberry fruit phyllody) phytoplasma represents the new 16SrXIII-J subgroup and its sequence, denominated SFP-Br02, was deposited in the GenBank database (EU719108). These findings contribute for the knowledge of the genetic diversity existing among members of the group 16SrXIII and establishes strawberry as an additional host of representatives of this group in Brazil.

Molecular characterization of a phytoplasma affiliated with the 16SrVII group representative of the novel 16SrVII-F subgroup.

Vernonia brasiliana is a wild perennial shrub frequently found in pasture areas. Plants of this species have been observed displaying typical symptoms induced by phytoplasmas, which were characterized by shoot proliferation, deformed leaves and leaf chlorosis. The present study confirmed the presence of phytoplasmas in association with affected plants. Sequencing of the 16S rRNA gene, computer-simulated RFLP analysis and phylogenetic analysis revealed that one of the phytoplasmas identified was representative of novel subgroup. The sequence identity scores between the novel strain and those of previously described 'CandidatusPhytoplasma fraxini'-related strains was 99 %, while similarity coefficient values were lower than 0.97. These findings provide support to delineate the phytoplasma found in vernonia plants as a reference phytoplasma for a novel subgroup designated 16SrVII-F. This representative of the novel subgroup was denominated VbSP phytoplasma (Vernonia brasiliana Shoot Proliferation; GenBank KX342018). The results of the present study revealed V. brasiliana to be a host of phytoplasmas, evidenced a novel phytoplasma associated with phytoplasmal disease in Brazil and extended the knowledge of the genetic diversity existing within the 16SrVII group.

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).

Identification by computer-simulated RFLP of phytoplasmas associated with eggplant giant calyx representative of two subgroups, a lineage of 16SrIII-J and the new subgroup 16SrIII-U.

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 Bragança 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.