First Report of a 16SrI (Aster Yellows) Group Phytoplasma on Garlic (<i>Allium sativum</i>) in the United States

During the growing season of 2012, 35 garlic plant samples were submitted to the University of Minnesota Plant Disease Clinic for disease diagnosis. Samples originated from multiple counties throughout Minnesota as well as Iowa, Wisconsin, and South Dakota. Symptoms first appeared at the time plants were starting to produce scapes. Symptoms included leaf discoloration that varied from yellow to purple, plant stunting, and leaf tip necrosis. In severe cases, the plants wilted and died. Bulbs of affected plants ranged from being soft and small to almost normal-looking. Symptoms were similar to those associated with phytoplasma infection in other plants. Total genomic DNA was extracted from 30 symptomatic samples and five asymptomatic leaf samples using a Qiagen DNeasy Plant Mini Kit (Qiagen, Germantown, MD) according to the manufacturer's instructions, and used with the universal phytoplasma primers P1/P7 in a direct PCR assay, and with P1/AYint in a nested PCR assay (2) to yield amplicons of 1.8 and 1.6 kb, respectively. Asymptomatic plants did not produce amplicons. Garlic cultivars displaying a range of symptoms tested positive for the presence of phytoplasma. These cultivars included: Susanville, Middle Eastern, Music, Ajo Rojo, Spanish Roja, Inchelium Red, Silver White, Asian Tempest, Chesnok Red, and Purple Glazer. The P1/P7 PCR products of 1,830 bp were purified using the PureLink PCR Purification kit (Life Technologies, Carlsbad, CA), and cloned in a pGem T-Easy vector system (Promega, Madison, WI). Sequences from a clone from each of Wisconsin, Iowa, and Minnesota were deposited in GenBank under the accession numbers KC000005, KC000006, and KC000007, respectively. A BLASTn similarity search revealed that the Wisconsin and Iowa isolates shared 99% homology to the sequences of 16SrI-A group phytoplasmas, aster yellows phytoplasma (AY389827), and aconitum proliferation phytoplasma (AF510323). The Minnesota isolate had 99% sequence homology to a 16SrI-B group phytoplasma, mulberry yellow dwarf phytoplasma (GQ249410). Also, the iPhyClassifier 16Sr group/subgroup classification based on similarity (3) analyses showed that the Wisconsin and Iowa phytoplasma isolates had 16S rDNA sequences in the 16SrI-A group with similarity coefficients of 0.97 and 1.00, respectively, to aster yellows witches'-broom phytoplasma AYWB (NC_007716). The same analysis revealed that the Minnesota phytoplasma isolate 16S rDNA sequence grouped with the 16SrI-B group onion yellows phytoplasma (NC_005303) with a similarity coefficient of 1.0. A phylogenic tree was deduced by the neighbor joining algorithm, clustering together the Iowa, Minnesota, and Wisconsin isolate sequences with a 16SrI group phytoplasma. Aster yellows phytoplasma has been reported in North America, but only in Canada (1). This is the first documented occurrence of 16SrI aster yellows group phytoplasma in garlic in the United States. The spring of 2012 was unusually warm, and high leafhopper pressure was observed throughout the Midwest; above average numbers of many ornamental crops and small grains were infected with phytoplasma. These events may have contributed to the phytoplasma infection in garlic. References: (1) A. H. Khadhair et al. Microbiol. Res. 157:161, 2002. (2) C. D. Smart et al. Appl. Env. Microbiol. 62:2988, 1996. (3) Y. Zhao et al. Int. J. Syst. Evol. Microbiol. 59:2582, 2009.

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