Plant Science

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.

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.

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.

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.

In January of 2009, potato plants (Solanum tuberosum) from a commercial crop in the Waikato Region, New Zealand were observed to have symptoms of upward rolling and purpling of the leaves. The symptoms appeared similar to those of “zebra chip”, a disorder of potato recently found to be associated with ‘Candidatus Liberibacter solanacearum’ in New Zealand and the United States (4). Total DNA from the leaf midveins and tubers from one of the symptomatic plants was separately extracted with an InviMag Plant DNA Mini Kit (Invitek GmbH, Berlin, Germany) and a KingFisher mL workstation (Thermo Scientific, Waltham, MA). DNA extracted from leaf midveins and tubers tested negative for ‘Ca. L. solanacearum’ by nested-PCR using primer pair OA2/OI2c (4) followed by Lib16S01F/Lib16S01R (5′-TTCTACGGGATAACGCACGG-3′ and 5′-CGTCAGTATCAGGCCAGTGAG-3′), which amplifies a 580-bp region of the 16S rRNA gene. However, DNA extracted from the tuber tissue tested positive for phytoplasma by TaqMan real-time PCR (3). No phytoplasma was detected in the DNA extracted from leaf tissue. The 16S rRNA gene, 16S-23S rRNA intergenic spacer region, and part of the 23S rRNA gene of the phytoplasma were amplified with primers P1/P7 (1). The PCR product was cloned into the pCR 4-TOPO vector (Invitrogen, Carlsbad, CA) and sequenced (GenBank Accession No. FJ943262). BLAST analysis showed 100% identity to ‘Ca. Phytoplasma australiense’ (16SrXII, Stolbur group). A fragment of approximately 850-bp of the Tuf gene was also amplified (2) and sequenced directly (GenBank Accession No. FJ943263). BLAST analysis showed 100% identity to Tuf gene variant IX of ‘Ca. P. australiense’ (2). An additional 14 plants showing similar leaf symptoms and also production of aerial tubers were collected from seven different potato fields from the Auckland and Waikato regions. Total DNA from the leaf midveins, stem, and tubers were separately extracted from each of the plants. The samples were tested for phytoplasma by nested-PCR using primer pair R16F2/R16R2, followed by NGF/NGR (1), and tested for ‘Ca. L. solanacearum’ by nested-PCR as described above. Seven plants tested positive only for phytoplasma, three tested positive for only ‘Ca. L. solanacearum’, and four plants tested positive for both pathogens. The pathogens were most commonly detected in samples extracted from the stem with 9 and 5 of the 14 samples testing positive for phytoplasma and liberibacter, respectively. Six of each of the leaf and tuber samples tested positive for phytoplasma. Liberibacter was detected in one of the leaf samples and in four of the tuber samples. ‘Ca. P. australiense’ has only been reported from New Zealand and Australia. The only other known hosts of ‘Ca. P. australiense’ in New Zealand are strawberry and native plants belonging to the genera Cordyline, Coprosma, and Phormium (2). In Australia, ‘Ca. P. australiense’ is associated with Australian grapevine yellows and Papaya dieback (2). To our knowledge, this is the first report of ‘Ca. P. australiense’ infecting potato as well as the first report of phytoplasma and ‘Ca. L. solanacearum’ mixed infections in potato. References: (1) M. T. Andersen et al. Plant Pathol. 47:188, 1998. (2) M. T. Andersen et al. Phytopathology 96:838, 2006. (3) N. M. Christensen et al. Mol. Plant Microbe Interact. 17:1175, 2004. (4) L. W. Liefting et al. Plant Dis. 93:208, 2009.

Plantas de hibisco com superbrotamento e definhamento seguido de morte têm sido observadas nos municípios de São Paulo, Campinas e Piracicaba. Como os sintomas são sugestivos daqueles induzidos por fitoplasmas, o presente trabalho buscou identificar o possível fitoplasma associado com a doença. Assim, 14 plantas sintomáticas de hibisco foram coletadas em Piracicaba (SP) e submetidas ao PCR duplo com os primers P1/Tint-R16F2n/R2 e ao exame em microscópio eletrônico de transmissão. A identificação foi realizada por análise de RFLP com as enzimas de restrição BfaI, DraI, HaeIII, HhaI, HpaII, MboI, MseI, RsaI e TaqI. Testes de transmissão foram conduzidos com enxertia de ramos e uso de Cuscuta subinclusa. Os resultados de nested-PCR revelaram a presença consistente de fitoplasmas em todas as plantas sintomáticas e foram confirmados pela observação de corpúsculos pleomórficos no floema, através da microscopia eletrônica. A análise de RFLP mostrou que o fitoplasma encontrado em hibisco pertence ao grupo 16SrXV, o mesmo grupo do Candidatus Phytoplasma brasiliense. O fitoplasma foi transmitido de planta doente para sadia, tanto pela enxertia como pela C. subinclusa, demonstrando ser o agente do superbrotamento do hibisco.