Presence of a Phytoplasma Associated with Witches’-Broom Disease in Ugni molinae Turcz. and Gaultheria phillyreifolia (Pers.) Sleumer Determined by DAPI, PCR, and DNA Sequencing

Abstract

Murta (Ugni molinae Turcz.) and common chaura (Gaultheria phillyreifolia (Pers.) Sleumer) are native species of Chile. Plants of both species have shown over-branching like witches' broom. The causal agents of these symptoms in many plants are phytoplasma. To verify the presence of these microorganisms, DAPI (4',6-diamidino-2-phenylindole) staining analysis and polymerase chain reaction (PCR) were performed in symptomatic and asymptomatic plants. Positive PCR samples were sequenced to identify the pathogens involved. In individuals of both species with witches' broom symptoms, DAPI staining showed fluorescent bodies in the phloem tissues, but not in asymptomatic plants. Verification by nested-PCR, phytoplasmatic DNA was amplified from diseased murta and chaura, but not in apparently healthy plants. Sequencing of amplified products allowed locating phytoplasma within the ash yellows group (16SrVII) and related to Candidatus phytoplasma fraxini. This is the first report of phytoplasma in Chilean native species. Considering the diversity of plant species infected by the ash yellows group suggests that G. phillyreifolia and U. molinae could be a phytoplasma reservoir for other economically important agricultural crops.

Identification of a phytoplasma associated with witches’ broom symptoms in calafate (Berberis microphylla G. Forst.)

“…The 16SrXIII group is present in region of South America affecting various crops such as potatoes (Solanum tuberosum L.), broccoli (Brassica oleracea L.), strawberry (Fragaria ×ananassa Duchesne ex Rozier) and papaya (Carica papaya L.), among others (Santos-Cervantes et al, 2010;Eckstein et al, 2013;Melo et al, 2013;Fernández et al, 2015). Results shown in this study indicate that the group 16SrXIII-F also affects Berberidaceae family, and therefore is added to the number of reports that indicate the presence of phytoplasmas in diverse crops from Chile such as natives plants (Arismendi et al, 2010a) and fruit trees (Gajardo et al, 2009;González et al, 2011). To our knowledge, this is the first report of a phytoplasma affecting Calafate plants in Chile.…”

Discovery of mutations in Chenopodium quinoa Willd through EMS mutagenesis and mutation screening using pre-selection phenotypic data and next-generation sequencing

“…When the M2 plants developed true leaves, two individual tissue samples were taken, frozen in liquid nitrogen and stored at −70°C for DNA isolation. DNA extraction was performed using the cetyl trimethylammonium bromide protocol as described in Arismendi et al (2010). The quantity and quality of DNA was determined using a Qubit fluorometer (Life Technologies Corporation, Carlsbad, CA, USA).…”

Caracterización molecular del gen psbA en Chenopodium quinoa Willd

“…La extracción de ADN se realizó usando un protocolo CTAB modificado (Arismendi, Andrade, Riegel, & Carrillo, 2010) en el Laboratorio de Biología Molecular de la Facultad de Ciencias Agrarias. El ADN aislado se cuantificó midiendo la absorbancia a 260 nm, y se diluyó con agua destilada a una concentración aproximada de 20 ng ul -1 y se almacenó a -20 ºC para análisis futuros.…”