Plant Science

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.

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.

The specificity and sensitivity of polymerase chain reaction (PCR) primers developed for ‘Candidatus Liberibacter solanacearum’ and ‘Candidatus Liberibacter psyllaurous’ were evaluated in conventional and real-time PCR assays. All PCR primers were specific for ‘Ca. L. psyllaurous’ and ‘Ca. L. solanacearum’ insomuch as they did not detect other prokaryotic plant pathogens that affect potato except for the putative pathogens associated with psyllid-yellows and haywire. Conventional PCR assays were capable of detecting 0.19 to 1.56 ng of total DNA per reaction, and real-time PCR was found capable of detecting 1.56 to 6.25 ng of total DNA per reaction, depending on the specific PCR primer set used. ‘Ca. Liberibacter’ species associated with zebra complex disease (ZC) was confirmed in plants affected by this disease throughout Texas from 2005 to 2008, in seed tubers produced in Wyoming in 2007, and in Colorado, Kansas, Nebraska, and Mexico in 2008. A multiplex PCR assay using ‘Ca. L. solanacearum’–specific primers and primers specific for the β-tubulin DNA regions from potato was developed, providing possible utility of the multiplex assay for ‘Ca. Liberibacter’ detection in different solanaceous plant species. Preliminary studies suggest silverleaf nightshade (Solanum elaeagnifolium), wolfberry (Lycium barbarum), black nightshade (S. ptychanthum), and jalapeno pepper (Capsicum annuum) as additional solanaceous hosts for the ZC-associated bacterium. The ‘Ca. Liberibacter’ species detected in all samples divided into two clusters sharing similarity of 99.8% in their partial 16S rRNA gene sequences and 99.3% in their partial intergenic spacer region (ISR)-23S rRNA gene sequences. Genetic variation in the 16S rDNA region consistently matched that of the ISR-23S rDNA region. In this partial 16S-ISR-23S rDNA region, there was a total of eight single nucleotide polymorphisms among ‘Ca. L. psyllaurous’ and ‘Ca. L. solanacearum’ “strains” investigated in this study. ‘Ca. L. solanacearum’ and ‘Ca. L. psyllaurous’ were shown to be very closely related bacteria, if not the same, by successful amplification using a combination of forward primer of ‘Ca. L. solanacearum’ and reverse primer of ‘Ca. L. psyllaurous’ in ZC-affected potato samples. This finding clarifies the current taxonomic status of ‘Ca. L. solanacearum’ and ‘Ca. L. psyllaurous’. The detection of ‘Ca. L. solanacearum’ from haywire-symptomatic potato samples demonstrates that this bacterium might also be associated with this disease.

Bell pepper (Capsicum annuum) plants exhibiting symptoms that resembled those of potato psyllid (Bactericera cockerelli Sulc) damage and “Candidatus Liberibacter solanacearum” infection (2) were observed in a pepper field in La Cruz de Elota, Sinaloa, México in March 2009, with an infection rate of 1.5%. Plants exhibited chlorotic or pale green apical growth and leaf cupping, sharp tapering of the leaf apex, shortened internodes, and an overall stunting (2). Total DNA was extracted from the top whole leaf tissue of nine symptomatic and five asymptomatic pepper plants with cetyltrimethylammoniumbromide (CTAB) buffer (3,4). Seven and eight of the nine selected symptomatic pepper plants yielded the expected 1,168-bp 16S rDNA and the expected 669-bp rplJ/rplL ribosomal protein gene amplicons with the “Ca. L. solanacearum” specific OA2/OI2c and CL514F/CL514R primer pairs, respectively, indicating the presence of liberibacter (2,4). Nucleic acid from asymptomatic pepper plants yielded no products with these primers. Three amplicons generated from symptomatic pepper plants with each primer pair were cloned into pCRII-TOPO plasmid vectors (Invitrogen, Carlsbad, CA) and three clones of each amplicon were sequenced in both directions (ACGT, Inc., Wheeling, IL). BLAST analysis of the 16S rDNA consensus sequence (GenBank Accession No. FJ957896) showed 100% identity to 16S rDNA sequences of “Ca. L. solanacearum” amplified from Solanum betaceum (EU935004) and S. lycopersicum (EU834130) from New Zealand (2), and “Ca. L. psyllaurous” from potato psyllids (EU812559) (1). The ribosomal protein gene consensus sequence (GenBank Accession No. FJ957894) was 100% identical to the analogous rplJ and rplL “Ca. L. solanacearum” ribosomal protein gene sequence amplified from S. lycopersicum (EU834131) from New Zealand (2) and to ‘Ca. Liberibacter’ sp. sequence amplified from zebra chip-infected potato tubers from Lancaster, CA (FJ498803). To our knowledge, this is the first report of “Ca. L. solanacearum” associated with bell pepper in México. “Ca. L. solanacearum” was first reported in tomato and pepper plants in 2008 in New Zealand, where it has resulted in plant decline and significant yield loss, resulting in millions of dollars in losses to the commercial glasshouse tomato and pepper industry (2). Zebra chip, a new and emerging potato disease associated with ‘Ca. Liberibacter’ sp., was first identified in México in 1994, where it has caused significant economic damage, often leading to abandonment of entire potato fields (3,4). References: (1) A. K. Hansen et al. Appl. Environ. Microbiol. 74:5862, 2008. (2) L. W. Liefting et al. Plant Dis. 93:208, 2009. (3) J. E. Munyaneza et al. J. Econ. Entomol. 100:656, 2007. (4) J. E. Munyaneza et al. Plant Dis. 93:552, 2009.

Tomato (Solanum lycopersicum) plants exhibiting symptoms resembling those of permanent yellowing disease (known in Mexico as “permanente del tomate”) that is commonly associated with phytoplasmas (1) were observed in tomato fields in Sinaloa, México in March 2009. Plant symptoms also resembled those caused by “Candidatus Liberibacter solanacearum” infection (2). Affected plants showed an overall chlorosis, severe stunting, leaf cupping, purple discoloration of veins, excessive branching of axillary shoots, and leaf scorching (1,2). Symptom incidence ranged from 18 to 40%. To investigate whether liberibacter is associated with permanent yellowing disease of tomato in México, eight symptomatic and five asymptomatic tomato plants were collected from two fields in La Cruz de Elota and Culiacán, Sinaloa. Total DNA was extracted from the top whole leaf tissue of symptomatic and asymptomatic plants with cetyltrimethylammoniumbromide (CTAB) buffer (3,4). DNA samples were tested by PCR using primer pairs OA2/OI2c and CL514F/CL514R, which amplify a sequence from the 16S rDNA and rplJ and rplL ribosomal protein genes, respectively, of “Ca. L. solanacearum” (2,4). The DNA samples were also tested for phytoplasmas with nested PCR using universal primer pairs P1/P7 and fU5/rU3 (3). DNA from five and four symptomatic plants yielded the expected 1,168-bp 16S rDNA and 669-bp rplJ/rplL amplicons, respectively, indicating the presence of liberibacter. Extracts from asymptomatic plants yielded no products with these primers. Amplicons generated from three symptomatic plants with each primer pair were cloned into pCRII-TOPO plasmid vectors (Invitrogen, Carlsbad, CA) and three clones of each of these amplicons were subsequently sequenced in both directions (ACGT, Inc., Wheeling, IL). BLAST analysis of the 16S rDNA consensus sequence (GenBank Accession No. FJ957897) showed 100% identity to 16S rDNA sequences of “Ca. L. solanacearum” amplified from S. betaceum (EU935004) and S. lycopersicum (EU834130) from New Zealand (2), and “Ca. L. psyllaurous” from potato psyllids (EU812559). The rplJ/rplL consensus sequence (GenBank Accession No. FJ957895) was 100% identical to the analogous rplJ and rplL “Ca. L. solanacearum” ribosomal protein gene sequence amplified from S. lycopersicum (EU834131) from New Zealand (2) and ‘Ca. Liberibacter’ sp. sequence amplified from zebra chip-infected potatoes from Lancaster, CA (FJ498803). No phytoplasmas were detected in the symptomatic tomato plants. To our knowledge, this is the first report of “Ca. L. solanacearum” associated with tomatoes in México. In 2008, this bacterium was detected in glasshouse tomatoes in New Zealand and caused millions of dollars in losses to the commercial glasshouse tomato industry (2). References: (1) R. L. Holguín-Peña et al. Plant Dis. 91:328, 2007. (2) L. W. Liefting et al. Plant Dis. 93:208, 2009. (3) J. E. Munyaneza et al. J. Econ. Entomol. 100:656, 2007. (4) J. E. Munyaneza et al. Plant Dis. 93:552, 2009.

Greenhouse tomato growers from Fort Lupton, CO contacted the USDA-ARS-USHRL in 2002 regarding plant symptoms resembling “psyllid yellows” associated with Bactericera cockerelli (Sulc) infestations that initially begin as retarded growth, erectness of new growth, chlorosis, and purpling of leaves followed by widespread chlorosis and production of many small, poor-quality fruit (1). Symptoms appeared ≈6 weeks after psyllids were observed and were generally restricted to the top half of the plant. Leaf cuttings from beefsteak tomatoes cv. Quest were immediately placed in RNAlater (Applied Biosystems, Austin, TX). Samples from symptomatic and asymptomatic plants were collected in September and December of 2002. At each date, leaves were sampled from multiple plants and placed in separate RNAlater bottles. September samples exhibited initial “psyllid yellows” symptoms and December samples exhibited severe symptoms. Samples remained at 4°C in RNAlater for 6 years until recent findings suggested that a new species of bacteria, named either “Candidatus Liberibacter psyllaurous” (2) or “Ca. L. solanacearum” (3), may be the causal agent of “psyllid yellows”. The Qiagen (Valencia, CA) DNeasy Plant Kit and recommended protocols were used for four separate DNA isolations from each of the four tomato samples that had previously remained unopened. Five PCR primer pairs designed to amplify three distinct genetic regions within the “Ca L. psyllaurous” rrn operon (16S rRNA, 16S-23S rRNA intergenic region, and 23S rRNA) were used and one primer pair specific to the tomato DNA (18S rRNA gene) that successfully amplified from all samples was used as a positive control. Bacterial primers included one pair designed specifically for 16S rRNA sequences of ‘Ca. L. asiaticus’, ‘americanus’, and ‘africanus’ species (USHRL-CL1) and four sets, Lp-1 through Lp-4, previously described (2) that amplify nonoverlapping regions of the 16S-23S rRNA operon. The USHRL-CL1 primers (USHRL-CL1f: 5′-CTTACCAGCCCTTGACATGTATAGGA-3′, and USHRL-CL1r: 5′-TCCCTATAAAGTACCCAACATCTAGGTAAA-3′) amplify a 195-bp fragment from bp 895 to 1,089 of the ‘Ca. Liberibacter’ sp. 16S rRNA Genbank Accession No. L22532. Only samples from severe symptomatic plants collected in December 2002 yielded amplicons that were purified and sequenced (Genbank: USHRL-CL1, FJ871062; Lp-1, FJ871058; Lp-2, FJ871059; Lp-3, FJ871060; Lp-4, FJ871061). For each bacterial primer pair, the fragment amplified was highly homologous (98 to 100% identity) to “Ca. L. psyllaurous” rRNA gene/intergenic space sequences. The 16S rRNA coding region was identical to two GenBank ‘Ca. Liberibacter’ sp. entries: EU921627 and EU921626 from B. cockerelli samples collected in Dalhart, TX and zebra chip potato samples from Garden City, KS, respectively; however, the whole 2,500 bp amplified and sequenced from our sample contained 11 to 14 polymorphisms when compared to nine “Ca. L. psyllaurous” sequences. Our results clearly indicate that “Ca. L. psyllaurous” isolates were associated with tomato “psyllid yellows” symptoms in Colorado as early as 2002 and significant sequence variation exists within the 16S/23S rRNA intergenic region and 23S rRNA coding region to allow analysis of genetic diversity among “Ca. L. psyllaurous” isolates. References: (1) L. B. Daniels. Ph.D. diss. University of Minnesota, St. Paul, 1954. (2) A. K. Hansen et al. Appl. Environ. Microbiol. 74:5862, 2008. (3) L. W. Liefting et al. Plant Dis. 93:208, 2009.