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of Phytopathology

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  • Phytopathological note

Detection and molecular characterization of a 16SrII group phytoplasma associated with ‘witches broom’ disease in cactus (Opuntia sp.)

By Candelario Ortega Acosta, Reyna Isabel Rojas Martínez*, Daniel L. Ochoa Martínez, Manuel Silva Valenzuela

* Corresponding Author. Email: / Institution: Colegio de Postgraduados

Accepted: 19/February/2024 – Published: 06/March/2024 – DOI: https://doi.org/10.18781/R.MEX.FIT.2310-2

Abstract Background/Objective. Phytoplasmas are obligate plant pathogens that exhibit strong specificity with their hosts. Typical symptoms induced by these pathogens include stunted growth and general decline, among others, and they rarely lead to plant death. The aim of this research was to determine the phytoplasma associated with the ‘witch’s broom’ symptom in an ornamental cactus (Opuntia sp.).

Materials and Methods. Four samples of ornamental cacti exhibiting ‘witch’s broom’ symptoms were collected from four commercial nurseries in Texcoco, State of Mexico. DNA extraction was performed on the samples, followed by PCR using specific primers for phytoplasmas (P1/P7 and R16F2n/R16R2). Phytoplasma determination was carried out through PCR, in vitro RFLP, sequencing, and phylogenetic analysis.

Results. According to the various analyses conducted, it was determined that the phytoplasma associated with the ornamental cactus belongs to the subgroup 16SrII-C.

Conclusion. Based on the obtained results, it is established that a phytoplasma from the 16SrII-C subgroup is associated with the ‘witch’s broom’ symptom in the ornamental cactus (Opuntia sp.).

Keywords: deformation, uncultivable pathogens, in vitro RFLP

Figure 1. A) Amplifications of 16S rDNA of phytoplasmas obtained using primers R16F2n/R16R2. Lane M; Molecular marker 100 pb, lane +; DNA from Dimorphotheca sinuata infected with “Candidatus Phytoplasma asteris” (16SrI-B), lane -; Negative control, PCR without a template, lane 1-4; samples of cactus (<em>Opuntia</em> sp.) with “witches’ broom” syndrome, from nurseries located in Texcoco, State of Mexico; B-C) “Witches’ broom” symptoms in an ornamental cactus.
Figure 1. A) Amplifications of 16S rDNA of phytoplasmas obtained using primers R16F2n/R16R2. Lane M; Molecular marker 100 pb, lane +; DNA from Dimorphotheca sinuata infected with “Candidatus Phytoplasma asteris” (16SrI-B), lane -; Negative control, PCR without a template, lane 1-4; samples of cactus (Opuntia sp.) with “witches’ broom” syndrome, from nurseries located in Texcoco, State of Mexico; B-C) “Witches’ broom” symptoms in an ornamental cactus.
Figure 2. RFLP analysis of the 16S rDNA of phytoplasmas amplified using primers R16F2n/R16R2 and digested with five restriction enzymes: EcoRI, HaeIII, KpnI, MseI and RsaI M: molecular marker 100 pb (Promega, USA); A) Positive control ‘Candidatus Phytoplasma asteris’ (I-B); B) Symptomatic sample of cactus from this study (Accession number: 0N413680); C) Restriction patterns <em>in silico</em>, generated from the sequences of gene 16S rDNA of the Cactus witches’-broom phytoplasma 16SrII-C (Accession number: AJ293216.2) of the reconnaissance sites for 17 restriction enzymes.
Figure 2. RFLP analysis of the 16S rDNA of phytoplasmas amplified using primers R16F2n/R16R2 and digested with five restriction enzymes: EcoRI, HaeIII, KpnI, MseI and RsaI M: molecular marker 100 pb (Promega, USA); A) Positive control ‘Candidatus Phytoplasma asteris’ (I-B); B) Symptomatic sample of cactus from this study (Accession number: 0N413680); C) Restriction patterns in silico, generated from the sequences of gene 16S rDNA of the Cactus witches’-broom phytoplasma 16SrII-C (Accession number: AJ293216.2) of the reconnaissance sites for 17 restriction enzymes.
Table 1. Phylogenetic tree created using the neighbor-joining method, with sequences of the 16S rDNA deposited in the GeneBank, showing the relationship between the phytoplasmas for groups 16SrI and 16SrII with the phytoplasma that induced “witches’ broom” in cactus (<em>Opuntia</em> sp.) (Accession number: 0N413680.1). The bar indicates the number of substitutions per nucleotides
Table 1. Phylogenetic tree created using the neighbor-joining method, with sequences of the 16S rDNA deposited in the GeneBank, showing the relationship between the phytoplasmas for groups 16SrI and 16SrII with the phytoplasma that induced “witches’ broom” in cactus (Opuntia sp.) (Accession number: 0N413680.1). The bar indicates the number of substitutions per nucleotides