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Molecular characterization of palm species, phytoplasma and associated Auchenorrhyncha occurrence in Puerto Rico
Palm crops worldwide are experiencing adverse effects in crop yields and survivorship from exposure to phytopathogens, including phytoplasmas (Ntushello et al., 2013). Phytoplasmas are plant-pathogenic bacteria that are associated with over 1000 plant diseases. (Duan et al., 2013; Ntushello et al., 2013). The 16SrIV group of phytoplasmas is associated with lethal yellowing diseases that affect at least 30 species of palms worldwide. Available information for Puerto Rico indicates the existence of 16SrIV phytoplasma group in some palm species, including the Royal palm Roystonea borinquena O. F. Cook, the Fishtail palm Caryota mitis Lour, the Carpentaria palm Carpentaria acuminata (H. Wendl. & Drude) Becc., and the Coconut palm Cocos nucifera L. The visual symptoms of the diseases include leaf discoloration (e.g., yellowing, bronzing), flower malformation, premature nuts drop, and death of the plant. This work aimed to identify and characterize palm species, phytoplasma, and associated Auchenorrhyncha occurrence in Puerto Rico. For this study, we collected samples from over 1,027 palms between July 2015 to May 2017 around the island of Puerto Rico. The samples comprise 40 palm species representing species native to the island and introduced ornamental ones. Sixty percent of the collected palms presented symptoms associated with phytoplasma diseases, mainly with premature leave yellowing and decay. We used the coding ribulose-bisphosphate carboxylase gene (rbcL) and maturase K gene (matK) to determine their power to distinguish between species in the palm family Arecaceae in Puerto Rico. Our study found that rbcL and matK place the species with 100% accuracy at the family and subfamily levels. However, additional markers may be needed to identify species within the Arecaceae. To amplify the phytoplasma DNA, we carried out direct and nested PCRs to a sub-sample of 192 palms from the initial sample (n=1,027). Forty-three percent of the samples tested showed amplification using specific primers in the nested PCR. Of these, only 1.5% of the samples were positive for phytoplasma 16SrIV-D associated with lethal bronzing disease of palms. The three confirmed sequences of phytoplasma were from positive samples: two from Dypsis lutescens and one Aiphanes minima, both collected at the UPR Botanical Garden. Phloem feeder insects are the vectors of phytoplasmas. The suborder Auchenorrhyncha contains phloem-feeder insects that are listed as vectors of phytoplasmas. We surveyed Auchenorrhyncha insects associated with palms in three localities of different altitudes between August 2016 to October 2018: the UPR Botanical Garden in San Juan, Punta Santiago Natural Reserve in Humacao, and El Verde Field Station (El Yunque) in Rio Grande. Thirty-three species of planthoppers and leafhoppers were sweep-collected from palms and associated weeds. Genetic analyses evaluated the mitochondrial gene region for cytochrome c oxidase subunit I (COI) to support morphological identification. A subsample of (n=120) insects was screened for hytoplasma by direct and nested PCR assays. We found seven samples that tested positive for phytoplasma. Three samples belonged to Hortensia similis (Walker) and four to Xyphon reticulatum (Signoret), all collected from C. nucifera in Humacao. To our knowledge, this is the first report of these Cicadellidae species associated with phytoplasma in Puerto Rico. The early detection and characterization of phytoplasma-related diseases and the identification of vector species can help reduce the spread of this organism and avoid the negative impact on crop yields in palm species of agricultural importance....