Rehman, M.

From 2006 to 2009, all commercial potato fields in Azacualpa F.M. Honduras were heavily infested with the potato psyllid Bactericera cockerelli (Sulc.). Plants exhibited interveinal chlorosis, vein-greening, downward curling, stunting, above ground tuber formation, and brownish flecks in some tubers. Disease incidence ranged from 50 to 95%. Leaf samples and psyllids were collected from seven fields in two potato-growing regions of Honduras. Total DNA was purified from the leaves of 30 symptomatic and three asymptomatic plants. DNA was extracted from 20 adult and 10 immature (4th to 5th instar) psyllids according to Frohlich et al (1). PCR primers, PSY680F 5′-GTTCGGAATAACTGGGCGTA-3′ and PSY1R 5′-CCCATAAGGGCCATGAGGACT-3′, were used to amplify a 680-bp fragment of the 16S rDNA for the recently described “Candidatus Liberibacter physallaurous” (2) and “Ca. L. solanacearum” (3). PSY1R/PSY680F primer design was based on the association of a previously undescribed liberibacter with vein-greening symptoms in greenhouse tomato plants in Arizona from 2006 to 2007 (GenBank Accession No. GQ926918) that lead to the hypothesis that a similar bacterium could be associated with symptomatic potato plants in Honduras. PCR amplification, cloning, and sequencing of the resultant 16S rDNA amplicons indicated that 17 of 30 potato plants, 8 of 20 adult and 7 of 10 third to fourth instar psyllids, respectively, were positive for liberibacter based on 99 to 100% shared nucleotide sequence (nt) identity with the analogous sequence from liberibacter (EU812558 [2]). To substantiate these results, a second molecular marker was targeted using the 1611F and 480R primers (~980 bp) that amplify the 16S-23S-ITSrDNA of liberibacter (2) for selected liberibacter-positive samples (above). Amplicons of the expected size were obtained from 12 of 17 potato and 7 of 10 immature psyllids. No PCR product of the expected size was obtained from asymptomatic potato samples or the PCR negative (water) control. The resultant PCR amplicons were cloned and 12 to 15 clones per amplicon were sequenced. The sequences were aligned and the percentage pair wise nt identity was calculated by Clustal W revealing that the 16S rDNA and 16S-23S-ITS sequences, respectively, shared 99 to 100% nt identity with each other. BLAST analysis against the NCBI database indicated that the 16S rRNA sequences from potato plants (GQ926922) and immature psyllids (GQ926923), and the 16S-ITS-23S sequence from potato plants (GQ926924) and immature psyllids (GQ926925), shared 98.5 to 100% nt identity with ‘Ca. Liberibacter’ reported from potato (EU812556; [2,4]) and tomato (EU812558, EU812559, EU935005; [2,3]). Evidence for the widespread presence of liberibacter and the potato psyllid in potato fields in Honduras, together with foliar and tuber symptoms that are reminiscent of those recently described in potato plants in the United States affected with ‘zebra chip’ disease (4), suggest that a similar or identical disease of the potato also occurs in Honduras. This emergent disease poses a serious threat to potato production in Honduras and elsewhere in Central America. References: (1) D. R. Frohlich et al., Mol. Ecol. 8:1683, 1999. (2) A. K. Hansen et al. Appl. Environ. Microbiol. 78:5862, 2008. (3) L. W. Liefting et al. Plant Dis. 93:208, 2009. (4) J. E. Munyaneza et al. J. Econ. Entomol. 100:656, 2007.

During the winter of 2006–2007, plants in commercial tomato greenhouses (GH-1 and GH-2; total 320 acres [129.5 ha]) in Arizona were infested with the potato psyllid Bactericera cockerelli (Sulc) and more than 60% and ~20% of the plants, respectively, exhibited leaf curling, chlorosis, and shortened internodes. In addition, some plants in GH-1 developed an unusual ‘vein-greening’ phenotype. Nucleic acids were isolated from 10 symptomatic and three asymptomatic plants from each greenhouse. PCR primers designed to amplify a phytoplasma-like 16S rDNA (850 bp) yielded the expected size product from GH-1 samples, whereas samples from GH-2 and the asymptomatic samples from both greenhouses did not. Several 16S rDNA PCR products (3 of 60) when cloned and sequenced, surprisingly shared 97% homology with ‘Candidatus Liberibacter asiaticus’ (GenBank No. GQ926917). PCR primers PSY680F 5′-GTTCGGAATAACTGGGCGTA-3′ and PSY1R 5′-CCCATAAGGGCCATGAGGACT-3′, based on the resultant 16S rDNA sequences, were used to amplify a 680-bp fragment from plant DNA extracts and psyllid lysates (1). A robust PCR product (~680 bp) was obtained from 10 of 10 GH-1 plant extracts (GQ926918) and from a GH-1-derived psyllid colony (28 of 35 adults) (GQ926919) and the tomato plants on which they were reared. In contrast, no 680-bp product was obtained from GH-1 asymptomatic plants (0 of 3), GH-2 plants (0 of 10 symptomatic; 0 of 3 asymptomatic), GH-2-derived psyllid colonies (0 of 35 adults), or psyllid colony tomato plants (data not shown). At least three 680-bp amplicons for each sample type were cloned and 8 to 10 inserts were sequenced for each. BLAST analysis revealed that all 680-bp sequences shared 99 to 100% nt identity with the analogous 16SrDNA from “Ca. Liberibacter psyllaurous” (2) and synonym “Ca. L. solanacearum” (3). A second molecular marker was obtained with the 1611F and 480R primers (2) to amplify the 16SrDNA-23S-ITS (980 bp) from >3 plant extracts and psyllid lysates that tested positive for liberibacter. Clustal W alignment of the 16S-23S-ITS sequences from GH-1 original tomato plants and psyllid colony plants (GQ926920) and psyllids (GQ926921) indicated they were 100% identical to each other and BLAST analysis indicated 99 to 100% shared identity with “Ca. L. psyllaurous” (EU812558) (synonym “Ca. L. solanacearum”). Transmission electron microscopy examination of GH-1 and GH-2 psyllids revealed rod and pleomorphic-shaped bacteria (0.5 to 2.0+ μm) at the brain-salivary gland interface in psyllids from the GH-1 liberibacter-positive colony. No such bacteria were observed in GH-2 liberibacter-negative psyllids. These results support an etiological role of a new liberibacter spp. in the development of the ‘vein-greening’ symptom phenotype. In contrast, the GH-2 ‘yellows’ phenotype is reminiscent of ‘psyllid toxicity’ in tomato colonized by B. cockerelli (4). To our knowledge, this is the first report of distinct psyllid-associated diseases in greenhouse tomato in Arizona, one associated with a new ‘Ca. Liberibacter’ spp., manifest as ‘vein-greening’ disease, and the other associated with psyllid feeding, in which liberibacter is undetectable in plants and psyllids, and is manifest as the ‘tomato psyllid yellows’ disease. References: (1) D. R. Frohlich et al. Mol. Ecol. 8:1683, 1999. (2) A. K. Hansen et al. Appl. Environ. Microbiol. 74:5862, 2008. (3) L. W. Liefting et al. Plant Dis. 93:208, 2009. (4) H. J. Pack. Utah Agric. Exp. Stn. Bull. 209, 1929.