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New Disease Reports (2017) 35, 28. [http://dx.doi.org/10.5197/j.2044-0588.2017.035.028]
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First report of a phytoplasma associated with witches' broom symptoms in Waltheria indica in Brazil

M.S. Alves, G.M. Ribeiro, A.N. Souza and C.M. Carvalho*

*claudine.carvalho@ufv.br

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Received: 13 Feb 2017; Published: 18 May 2017

Waltheria indica (Malvaceae) is a short-lived shrub, native to the neotropics but with a pantropical distribution, which is frequently used for medicinal purposes (Zongo et al., 2013). In January 2016, W. indica plants located in a coastal area of Vila Velha (state of Espirito Santo, Brazil, S20°31'42.51'', W40°22'24.1356'') were found exhibiting symptoms of witches' broom and yellowing (Fig. 1).

Leaf samples were collected from branches of three symptom-bearing and two symptomless W. indica plants. Total DNA was extracted from each sample and subjected to nested polymerase chain reaction (nPCR) amplification of the phytoplasma 16S rRNA gene with primers P1/P7 (Deng & Hiruki, 1991) and R16F2n/R2 (Gundersen & Lee, 1996). Amplicons of the expected size (1.2 kb) were obtained from all plants showing symptoms but not from symptomless plants. The amplicons were purified using an Illustra GFX PCR DNA and Gel Band Purification Kit (GE Healthcare, UK), cloned using the pGEM®-T Easy Vector System I (Promega, USA) and sequenced by Macrogen Inc. (Korea). BLAST searches confirmed that the consensus 16S rDNA sequence of the Waltheria indica witches' broom phytoplasma (WIWB) (GenBank Accession No. KX691443) shared 99.4% sequence identity with those of 'Candidatus Phytoplasma asteris' (16SrI group) species (e.g. HM067755, KP864664, AF222066). The WIWB phytoplasma was preliminary classified as a member of the 16SrI-S subgroup, with a similarity coefficient of 0.99 based on RFLP analysis when compared to 16SrI phytoplasmas using iPhyClassifier (Zhao et al., 2013). Phylogenetic analysis using the neighbour-joining method in MEGA7 software (www.megasoftware.net/) supported the sequencing and iPhyClassifier results (Fig. 2).

The occurrence of phytoplasmas in W. indica has been previously reported in Brazil and Australia (Kitajima & Costa, 1971; Schneider et al., 1999), associated with phytoplasmas of group 16SrII 'Ca. P. aurantifolia'. To our knowledge, this is the first report of a member of 16SrI phytoplasma associated with W. indica plants in Brazil, which may pose a threat to other nearby plant species.

Figure1+
Figure 1: Symptoms of witches' broom and yellowing observed in Waltheria indica in Vila Velha, Brazil. 
Figure 1: Symptoms of witches' broom and yellowing observed in Waltheria indica in Vila Velha, Brazil. 
Figure2+
Figure 2: Phylogenetic tree based on 16S rDNA sequence of Waltheria indica witches' broom (WIWB) phytoplasma and those from other 16Sr phytoplasma groups. Acholeplasma palmae was used as outgroup. Phytoplasma group and GenBank accessions are indicated for each phytoplasma. Numbers above branches indicate the bootstrapping confidence values performed 1,000 times. Bars indicate 0.01 substitutions per nucleotide position. Ca. P: 'Candidatus Phytoplasma species'. SBS: Sorghum bunchy shoot phytoplasma strain; MSCY: Mauritius sugar cane yellows phytoplasma strains D3T1 & D3T2; PBP: Poinsettia branch-inducing phytoplasma strain; ICP: Italian clover phyllody phytoplasma strain; MYP: Milkweed yellows phytoplasma strain; VACWB: Vaccinium witches' broom phytoplasma strain; EPWB: Echinacea purpurea witches's broom phytoplasma strain; PnWB: Peanut witches's broom phytoplasma strain; SPLLV4: Sweet potato little leaf phytoplasma strain V4; ROL: Rice orange leaf phytoplasma strain; AYWB: Aster yellows witches's broom phytoplasma strain; WBD: Wheat blue dwarf phytoplasma strain; OYM: Onion yellows phytoplasma strain M; MBS: Maize bushy stunt phytoplasma strain; WTWB: Weeping tea tree witches' broom phytoplasma strain; BVGY: Buckland Valley grapevine yellows phytoplasma strain; HDP: Havana derbid phytoplasma strain.
Figure 2: Phylogenetic tree based on 16S rDNA sequence of Waltheria indica witches' broom (WIWB) phytoplasma and those from other 16Sr phytoplasma groups. Acholeplasma palmae was used as outgroup. Phytoplasma group and GenBank accessions are indicated for each phytoplasma. Numbers above branches indicate the bootstrapping confidence values performed 1,000 times. Bars indicate 0.01 substitutions per nucleotide position. Ca. P: 'Candidatus Phytoplasma species'. SBS: Sorghum bunchy shoot phytoplasma strain; MSCY: Mauritius sugar cane yellows phytoplasma strains D3T1 & D3T2; PBP: Poinsettia branch-inducing phytoplasma strain; ICP: Italian clover phyllody phytoplasma strain; MYP: Milkweed yellows phytoplasma strain; VACWB: Vaccinium witches' broom phytoplasma strain; EPWB: Echinacea purpurea witches's broom phytoplasma strain; PnWB: Peanut witches's broom phytoplasma strain; SPLLV4: Sweet potato little leaf phytoplasma strain V4; ROL: Rice orange leaf phytoplasma strain; AYWB: Aster yellows witches's broom phytoplasma strain; WBD: Wheat blue dwarf phytoplasma strain; OYM: Onion yellows phytoplasma strain M; MBS: Maize bushy stunt phytoplasma strain; WTWB: Weeping tea tree witches' broom phytoplasma strain; BVGY: Buckland Valley grapevine yellows phytoplasma strain; HDP: Havana derbid phytoplasma strain.

Acknowledgements

The authors would like to thank Vale S.A. for financial support. CMC is in receipt of a Conselho Nacional de Desenvolvimento Técnico e Científico (CNPq) productivity grant.

References

  1. Deng S, Hiruki C, 1991. Amplification of 16S rRNA genes from culturable and nonculturable mollicutes. Journal of Microbiological Methods 14, 53-61. [http://dx.doi.org/10.1016/0167-7012(91)90007-D]
  2. Gundersen DE, Lee IM, 1996. Ultrasensitive detection of phytoplasmas by nested-PCR assays using two universal primer sets. Phytopathologia Mediterranea 35, 144-151.
  3. Kitajima EW, Costa AS, 1971. Corpúsculos do tipo micoplasma associados a diversas moléstias de plantas, do grupo amarelo, no estado de São Paulo. Ciência e Cultura (São Paulo) 23, 285-291.
  4. Schneider B, Padovan A, De La Rue S, Eichner R, Davis R, Bernuetz A, Gibb K,1999. Detection and differentiation of phytoplasmas in Australia: an update. Australian Journal of Agricultural Research 50, 333-342. [http://dx.doi.org/10.1071/A98106]
  5. Zhao Y, Wei W, Lee I-M, Shao J, Suo X, Davis RE, 2013. The iPhyClassifier, an interactive online tool for phytoplasma classification and taxonomic assignment. Methods in Molecular Biology 938, 329-338. [http://dx.doi.org/10.1007/978-1-62703-089-2_28]
  6. Zongo F, Ribuot C, Boumendjel A, Guissou I, 2013. Botany, traditional uses, phytochemistry and pharmacology of Waltheria indica L. (syn. Waltheria americana): a review. Journal of Ethnopharmacology 148, 14-26. [http://dx.doi.org/10.1016/j.jep.2013.03.080]
To cite this report: Alves MS, Ribeiro GM, Souza AN, Carvalho CM, 2017. First report of a phytoplasma associated with witches' broom symptoms in Waltheria indica in Brazil. New Disease Reports 35, 28. [http://dx.doi.org/10.5197/j.2044-0588.2017.035.028]

©2017 The Authors