First Report of 16SrIV Palm Lethal Yellowing Group Phytoplasma (‘Candidatus Phytoplasma palmae’) in Palmilla de Taco (Brahea brandegeei) and Palma Colorada (Washingtonia robusta) in the State of Baja California Sur, Mexico

The Mexican state of Baja California Sur (BCS) is part of Peninsular Range Province (PRP), the arid mountainous region from Southern California to the southern tip of the Baja California Peninsula. This is the northwestern limit in the geographic range of the palms family (Arecaceae) in the Americas (Munnich et al. 2011). Brahea brandegeei (Bb) and Washingtonia robusta (Wr) are among the native ornamental fan palms of PRP most widely cultivated worldwide. In BCS, Wr grows largely throughout oases at the base of mountains and in urban areas, whereas Bb favors high-elevation oases and is rarely found in urban zones (Leon de la Luz et al. 2014). In July 2014, the symptoms of lethal yellowing (LY) diseases were observed in two Wr and one Bb palms in a private garden in El Centenario,10 km north of La Paz. Bb manifested bunchy-top-like symptoms, with midcanopy yellowing and chlorosis, necrotic tips of lateral leaves, and streaks in rachis and necrosis in some inflorescences. Wr palms had necrosis at the lower leaves and partial necrotic inflorescences. Samples from palm leaves, rachises, and inflorescences were collected and analyzed by scanning electron microscopy (SEM) and molecular techniques. Using SEM, phytoplasma cells were detected in phloem tissue of tested samples, with variable distribution in different specimens, and with average sizes 400 to 1,800 nm. Total DNA from eight collected samples of leaves and inflorescences from two palms was extracted (Tapia-Tussell et al. 2005), and nested polymerase chain reaction (PCR) was performed using P1/P7 and R16F2n/R16R2 primer pairs (Lee et al. 1998). Amplicons of ∼1.2 kb were obtained from all samples. Nested PCR products from three samples, two from Bb (2a and 3a) and one from Wr (Kb), were cloned into pGEM-T-easy vector (Promega, Madison, WI) and sequenced (University of California, Davis, CA, http://dnaseq.ucdavis.edu/). Analysis in NCBI BLASTn database of sequences Kb and 3a showed 99.28% (1,234/1,243 bp) and 99.35% (1,228/1,236 bp) similarity with 14 accessions from 16SrIV group, and the nearest matching sequence was MG234701, whereas sequence 2a displayed 99.36% identity (1,235/1,243 bp) to 35 GenBank accessions from 16SrIV group, such as MK421966. The analyzed three sequences, KX982666 (Kb), KX982667 (2a), and KX982668 (3a), were deposited in GenBank database. The use of iPhyClassifier tool (https://plantpathology.ba.ars.usda.gov/cgi-bin/resource/iphyclassifier.cgi) confirmed their 99% similarity with ‘Candidatus Phytoplasma palmae’ reference strain (accession no. U18747). Virtual restriction fragment length polymorphism (RFLP) analysis (Zhao et al. 2009) of sequence 2a (KX982667) identified the phytoplasma as a variant of 16SrIV-D, based on 0.98 similarity coefficient (F) to virtual pattern from the reference strain 16SrIV-D (AF237615). Analysis of virtual RFLP of sequences 3a (KX982668) and Kb (KX982666) indicates the most similarity with the reference pattern of 16SrIV-A (AF498307), with F ≤ 0.97 (0.95 for 3a and 0.96 for Kb). Further research is required to prove if these two phytoplasmas represent new subgroups within the 16SrIV group. To our knowledge, this is the first report of 16SrIV palm LY phytoplasma in two native palm species in the Baja California Peninsula; worldwide, this is the first report of 16SrIV-D ‘Ca. P. palmae’ in a new species, Brahea brabdegeei. Research is in progress to investigate the presence of phytoplasmas in Bb and Wr in the mountainous region of BCS, to establish the possible source of LY phytoplasma in their native ecosystem.