Plant Science

In North America, elm yellows, aster yellows (AY), and X-disease phytoplasmas have been detected in American grapevines (1), and recently, Bois noir was detected in Canadian vineyards from British Columbia (BC) and Ontario (ON) (2). Typical symptoms of grapevine yellows (GY) include leaf rolling and chlorosis, uneven or total lack of lignification of canes, flower abortion or berry withering, and stunting. In 2006 and 2007, independent surveys were conducted by the Canadian Food Inspection Agency (CFIA) and Agriculture and Agri-Food Canada (AAFC) to detect phytoplasmas in Canadian vineyards containing different cultivars in BC, ON, Québec (QC), Nova Scotia, New Brunswick, and Prince Edward Island. The CFIA collected and tested 651 fresh leaf samples from recently imported grapevines and older grapevines in the same or neighboring blocks displaying symptoms typical of those associated with disease caused by phytoplasmas. Many vineyards were surveyed only once. AAFC collected and tested 3,485 samples from symptomatic and asymptomatic grapevines from established vineyards in ON, BC, and QC. The same vineyards were sampled in ON and BC both years; QC vineyards were only sampled in 2007. AAFC-collected leaf samples were freeze dried and stored at –20°C before processing. CFIA samples were tested by a modified real-time PCR assay and TaqMan probe targeting the 16S ribosomal RNA gene that detects a wide range of known phytoplasmas (2). Positive samples were confirmed by conventional PCR using the phytoplasma-specific primers P1/P7 (3) and the resulting ~1,800-bp fragment was cloned and sequenced as previously described (2). DNA extracted by AAFC was amplified by nested PCR technology with universal phytoplasma specific primer pairs P1/P6 and R16R2/R16F2 (3) and the resulting 1,200-bp fragment was cloned and sequenced. Two plants, one located in ON in 2006 and the other in BC in 2007, were found to be infected with an AY-like phytoplasma by the CFIA. The phytoplasmas detected in both infected plants had a 99.9% nt sequence identity with AY phytoplasma sequences from GenBank (Accession Nos. AF222063 and AY665676, respectively), with the BC isolate also showing 100% identity to a strain of AY, ash witches'-broom phytoplasma (GenBank Accession No. AY566302). AAFC detected phytoplasma DNA in both years in a total of 17 symptomatic plants and 21 asymptomatic plants from different vine varieties in ON, BC, and QC. Positive samples were found to have a 99.0% nt sequence identity to AY subgroup 16SrI-A (GenBank Accession No. AY180956). Sequences were exchanged for confirmation of phytoplasma identity and were deposited in Genbank under Accession Nos. FJ659844 and FJ824597. Phytoplasma strains were identified for all plants in which phytoplasmas were detected. Results show that AY is present in vineyards in the provinces of ON, BC, and QC. To our knowledge, this is the first report of AY being detected in grapevines in Canada. References: (1) E. Boudon-Padieu. Bull. O I V, 79:299, 2003. (2) M. Rott et al. Plant Dis. 91:1682, 2007. (3) E. Tanne et al. Phytopathology 91:741, 2001.

A new disease of potatoes, tentatively named zebra chip (ZC) because of the intermittent dark and light symptom pattern in affected tubers which is enhanced by frying, was first found in Mexico in 1994 and in the southwestern United States in 2000. The disease can cause severe economic losses in all market classes of potatoes. The cause of ZC has been elusive, and only recently has been associated with ‘Candidatus Liberibacter’ sp. Field samples of potato plants were collected from several locations in the United States, Mexico, and Guatemala to determine transmission to potato and tomato by grafting of ZC-infected scions and psyllid feeding. The disease was successfully transmitted, through up to three generations, by sequential top- and side-grafting ZC-infection scions to several potato cultivars and to tomato. The disease was also successfully transmitted to potato and tomato plants in greenhouse experiments by potato psyllids collected from potato plants naturally affected with ZC. Transmission electron microscopic observation of ZC-affected tissues revealed the presence of bacteria-like organisms (BLOs) in the phloem of potato and tomato plants inoculated by grafting and psyllid feeding. The BLOs were morphologically similar in appearance to BLOs associated with other plant diseases. Polymerase chain reaction (PCR) amplified 16S rDNA sequences from samples representing different geographic areas, including the United States, Mexico, and Guatemala, were almost identical to the 16S rDNA of ‘Ca. L. solanacearum’ previously reported from solanaceous plants in New Zealand and the United States. Two subclades were identified that differed in two single base-pair substitutions. New specific primers along with an innovative rapid PCR were developed. This test allows the detection of the bacteria in less than 90 min. These data confirm the association of ‘Ca. L. solanacearum’ with potatoes affected by ZC in the United States, Mexico, and Guatemala.

In August 2008, unusual symptoms were observed in Mexican lime trees (Citrus aurantifolia (Christm.) Swing) in the municipality of Luperón, province of Puerto Plata on the north coast of the Dominican Republic. Symptoms observed in young and old trees included blotchy mottle on leaves, healthy-appearing larger branches with smaller side branches that displayed chlorotic leaves, abscised and lopsided fruit, and branch dieback, all symptoms similar to those of citrus huanglongbing associated with ‘Candidatus Liberibacter’ spp. (1). Symptoms were observed in an area of ~100 ha surrounding Luperón, where Mexican lime trees were grown as seedlings and no commercial plantings of other citrus were present. Symptomatic leaves were collected from 16 trees in September 2008, and DNA was extracted from petioles and midveins with a DNeasy kit (Qiagen, Gaithersburg, MD) or with chloroform/isoamyl alcohol (24:1). Real-time PCR with the16S rDNA primer/probe set specific to ‘Ca. Liberibacter asiaticus’ and performed as described (2) gave Ct values comparable with the positive control for five samples. Conventional PCR with the forward (5′-tcgagcgcgtatgcaatacg-3′) and reverse (5′-ctacctttttctacgggataacgc-3′) primers used in real-time PCR (2) amplified a 75-bp product from these five samples. Eleven to twelve clones were sequenced from each sample and BLAST analysis of a consensus sequence for each sample (GenBank Accession Nos. FJ489643–FJ489647) showed 98% (e.g., EU921622, EU921618) to 100% identity (e.g., FJ236554, FJ263702) with 16S rDNA sequences of ‘Ca. Liberibacter asiaticus’. A larger 912-bp portion of the 16S rDNA gene was amplified from each sample by conventional PCR with sense (5′-gagcctaccaaggctacgat-3′) and antisense (5′-gcgttatcccgtagaaaaaggtag-3′) primers designed from the sequence of ‘Ca. Liberibacter asiaticus’ strain LJZ-4730 (GenBank Accession No. FJ263700). Nine to twelve clones were sequenced for each sample and BLAST analysis of a consensus sequence for each sample (GenBank Accession Nos. FJ811891–FJ811895) showed 100% identity to ‘Ca. Liberibacter asiaticus’ (DQ471900, DQ4719010) from Florida and Brazil, respectively. To confirm this identification, 693 bp of the outer membrane protein gene (omp) were amplified from each of the five positive samples by conventional PCR using sense (5′-gtgattctgagggtgagcg-3′) and antisense (5′-cgaactcactgagaactgatc-3′) primers designed from nucleotides 15 to 33 and 687 to 707, respectively, of the omp gene from ‘Ca. Liberibacter asiaticus’ strain MZ (GenBank Accession No. EF580135). The consensus sequences from 12 clones from each sample (GenBank Accession Nos. FJ489638–FJ489642) showed 99% (e.g., FJ236564, AY842429) to 100% (e.g., FJ236566, EF580135) nucleotide identity and the predicted translation product showed 100% amino acid identity (e.g., AAX47433, AAX47431) with the omp gene from ‘Ca. Liberibacter asiaticus’. These results confirm the presence of and to our knowledge, this is the first report of ‘Ca. Liberibacter asiaticus’ in the Dominican Republic. Huanglongbing is a destructive disease of citrus (1) and its spread is expected to adversely affect citrus production in the Dominican Republic with subsequent negative effects predicted for the employment of thousands of people. References: (1) J. M. Bové, J. Plant Pathol. 88:7, 2006. (2) W. Li et al. J. Microbiol. Methods 66:104, 2006.

A disease that severely affects processing potatoes (Solanum tuberosum L.), termed zebra chip (ZC), has been identified in several locations in the United States (Texas, Nebraska, Colorado, Kansas, New Mexico, Arizona, and Nevada), Mexico, and Central America (4). The disease name comes from the rapid oxidative darkening of freshly cut tubers and the dark stripes and blotches that occur in chips processed from infected tubers. Recently, the disorder has been associated with a new ‘Candidatus Liberibacter’ species in New Zealand (3). Also, a bacterium designated ‘Candidatus Liberibacter psyllaurous’ has been identified recently in potato plants with “psyllid yellows” symptoms that resemble foliar symptoms of ZC (2). In the fall of 2008, 10 tubers were received at the Prosser laboratory from a commercial potato grower and five had symptoms characteristic of ZC. The tubers, cv. Dakota Pearl, were grown near Lancaster in southern California. The tubers showed rapid oxidation upon slicing and the sunken stolon attachment characteristic of ZC (4). Nucleic acid was extracted from symptomatic tubers (1) and tested by PCR for ‘Ca. Liberibacter’ species with primer pairs OA2/OI2c (5′-GCGCTTATTTTTAATAGGAGCGGCA-3′ and 5′-GCCTCGCGACTTCGCAACCCAT-3′) and CL514F/R (5′-CTCTAAGATTTCGGTTGGTT-3′ and 5′-TATATCTATCGTTGCACCAG-3′), which amplify from the 16S rDNA and rplJ and rplL ribosomal protein genes, respectively (3). Four of the five tubers with distinct ZC symptoms yielded the expected amplicons with both primer pairs. Two tubers with mild internal discoloration yielded correctly sized amplicons but in lesser amounts than from the severely affected tubers. Nucleic acid from healthy potato tubers yielded no product with these primers. One clone of the 1,168-bp OA2/OI2c amplicon and two clones of the 669-bp CL514F/R amplicon from a strongly positive sample were sequenced in both directions (ACGT, Inc., Wheeling, IL). BLAST alignments of the consensus sequences of the OA2/OI2c and CL514F/R amplicons (GenBank Accessions Nos. FJ498802 and FJ498803, respectively) revealed 100% identity with analogous ‘Ca. Liberibacter’ sequences reported from ZC-symptomatic potatoes in New Zealand (GenBank Accession Nos. EU849020 and EU919514). The OA2/OI2c amplicon was also identical to a sequence of ‘Ca. Liberibacter psyllaurous’ (GenBank Accession No. EU812559) from psyllid yellows-affected potatoes in the United States (2) and also showed a 99% identity with sequences from a ‘Ca. Liberibacter’ species reported in ZC tubers from Kansas (GenBank Accession No. EU921626). Potato crops with symptoms of ZC have been observed previously in California (4), but this is the first identification of ‘Ca. Liberibacter psyllaurous’ from diseased potatoes grown in California. Since ZC was first reported in the mid- to late-1990s, information from potato growers and processors suggests that ZC is becoming more important. The disease has caused millions of dollars in losses, particularly in south Texas (4). The identification of ‘Ca. Liberibacter psyllaurous’ in California provides additional evidence that the disease is increasing in importance in other potato-growing regions. References: (1) J. M. Crosslin et al. Plant Dis. 90:663, 2006. (2) A. K. Hansen et al. Appl. Environ. Microbiol. 74:5862, 2008. (3) L. W. Liefting et al. Plant Dis. 92:1474, 2008. (4) J. E. Munyaneza et al. Subtrop. Plant Sci. 59:30, 2007.

Bamboos are ecologically and economically valuable plants. Young shoots of almost all species are edible, either raw or cooked, and are major components of Asian cuisine while culms are used for furniture or handicrafts as well as fuel wood. Symptoms indicative of phytoplasma disease were observed on Sasa fortunei (van Houtte) Fiori during a survey in Yangling, Shaanxi Province, China during 2007. Symptoms included internode shortening, a mosaic pattern on leaves of diminished size, stunted growth, and death of entire plants. Total nucleic acids were extracted from leaf veins of 10 diseased plants and 6 symptomless plants with a modified cetyltrimethylammoniumbromide (CTAB) method (1). Phytoplasma infection of plants was demonstrated by a nested PCR assay employing primer pair R16mF2/R16mR1 followed by R16F2n/R16R2 (2), which generated a 16S rDNA product of approximately 1.2 kb from all symptomatic plants only. Restriction fragment length polymorphism analysis by digestion of nested PCR products with restriction enzymes AluI and MseI indicated that S. fortunei plants contained group 16SrI (aster yellows), subgroup B phytoplasmas. Sequencing and phylogenetic analysis of phytoplasma 16S rDNA from S. fortunei (GenBank Accession No. FJ501958) revealed this strain to be very similar (99.7 and 99.6%, respectively) to phytoplasmas previously associated with Henon bamboo (Phyllostachys nigra) witches'-broom (GenBank Accession No. AB242433) (4) and sasa (S. borealis) witches'-broom (GenBank Accession No. AB293421) in Korea but less so (98.6%) to bamboo (Phyllostachys spp.) witches'-broom (GenBank Accession No. AY635145) disease in China (3). To our knowledge, this is the first report of a ‘Ca. Phytoplasma asteris’-related strain infecting S. fortunei in China. References: (1) E. Angelini et al. Vitis 40:79, 2001. (2) D. E. Gundersen and I.-M. Lee. Phytopathol. Mediterr. 35:144, 1996. (3) C. Hong et al. Plant Prot. 31:39, 2005. (4) H. Jung et al. J. Gen. Plant Pathol. 72:261, 2006.

Zebra Chip (ZC), an emerging disease of potato (Solanum tuberosum L.) first documented in potato fields around Saltillo in México in 1994, has been identified in the southwestern United States, México, and Central America and is causing losses of millions of dollars to the potato industry (4). Recently, this damaging potato disease was also documented in New Zealand (3). This disease is characterized by a striped pattern of necrosis in tubers produced on infected plants, and fried chips processed from these infected tubers are commercially unacceptable (4). Recent studies conducted in the United States and New Zealand have associated ZC with a new species of ‘Candidatus Liberibacter’ vectored by the potato psyllid, Bactericera cockerelli Sulc (1,3,4). A bacterium designated ‘Candidatus Liberibacter psyllaurous’ has recently been identified in potato plants with “psyllid yellows” symptoms that resemble those of ZC (2). To investigate whether liberibacter is associated with ZC in México, 11 potato (cv. Atlantic) tuber samples exhibiting strong ZC symptoms and six asymptomatic tubers were collected from a ZC-affected commercial potato field near Saltillo City, Coahuila, México in September 2008 and tested for this bacterium by PCR. Total DNA was extracted from symptomatic and asymptomatic tubers with cetyltrimethylammoniumbromide (CTAB) buffer (4). DNA samples were tested by PCR using primer pair OA2/OI2c (5′-GCGCTTATTTTTAATAGGAGCGGCA-3′ and 5′-GCCTCGCGACTTCGCAACCCAT-3′, respectively) specific for 16S rDNA and primer pair CL514F/R (5′-CTCTAAGATTTCGGTTGGTT-3′ and 5′-TATATCTATCGTTGCACCAG-3′, respectively) designed from ribosomal protein genes (3). Seven of eleven (63.7%) ZC-symptomatic tubers and one of six (16.7%) asymptomatic potatoes yielded the expected 1,168-bp 16S rDNA and 669-bp CL514F/R amplicons, indicating the presence of liberibacter. Amplicons generated from symptomatic tubers were cloned into pCR2.1-Topo plasmid vectors (Invitrogen, Carlsbad, CA) and one clone of each amplicon was sequenced in both directions (ACGT, Inc., Wheeling, IL). BLAST analysis of the ZC OA2/OI2c sequence (GenBank Accession No. FJ498806) showed 100% identity to liberibacter 16S rDNA sequences amplified from potato psyllids from Dalhart, TX and potato tubers from Garden City, KS (GenBank Accession Nos. EU921627 and EU921626, respectively). The ZC CL514F/R sequence (GenBank Accession No. FJ498807) was 98% identical to analogous rplJ and rplL liberibacter ribosomal protein gene sequences amplified from several solanaceous plants in New Zealand (GenBank Accession Nos. EU834131 and EU935005). The OA2/OI2c sequence was also identical to the 16S rDNA sequence (Genbank Accession No. EU812559) of ‘Ca. Liberibacter psyllaurous’ (2). To our knowledge, this is the first report of ‘Ca. Liberibacter psyllaurous’ associated with ZC-affected potatoes in México. References: (1) J. A. Abad et al. Plant Dis. 93:108, 2009. (2) A. K. Hansen et al. Appl. Environ. Microbiol. 74:5862, 2008. (3) L. W. Liefting et al. Plant Dis. 92:1474, 2008. (4) J. E. Munyaneza et al. J. Econ. Entomol. 100:656, 2007.

In São Paulo State, Brazil, ‘Candidatus Liberibacter americanus’ and ‘Candidatus Liberibacter asiaticus’ are associated with huanglongbing (HLB). Affected municipalities occur mainly in the central and southern regions, where the annual number of hours above 30°C is two to five times lower than that in the extreme northern and western regions. The influence of temperature on sweet orange trees infected with ‘Ca. L. asiaticus’ or ‘Ca. L. americanus’ was studied in temperature-controlled growth chambers. Symptom progression on new shoots of naturally infected and experimentally graft-inoculated symptomatic sweet orange trees was assessed. Mottled leaves developed on all infected trees at 22 to 24°C, but not on any ‘Ca. L. americanus’–infected trees at 27 to 32°C. Quantitative, real time-PCR was used to determine the liberibacter titers in the trees. After 90 days, ‘Ca. L. asiaticus’–infected trees had high titers at 32 and 35°C, but not at 38°C, while ‘Ca. L. americanus’–infected trees had high titers at 24°C, but at 32°C the titers were very low or the liberibacters could not be detected. Thus, the multiplication of ‘Ca. L. asiaticus’ is not yet affected at 35°C, while a temperature of 32°C is detrimental to ‘Ca. L. americanus’. Thus, ‘Ca. L. americanus’ is less heat tolerant than ‘Ca. L. asiaticus’. The uneven distribution of these two liberibacters in São Paulo State might be in relation with these results.

In Brazil ‘Candidatus Liberibacter asiaticus’ and ‘Ca. L. americanus’ cause huanglongbing (also known as greening), the most destructive citrus disease. A shift in pathogen prevalence was observed over time, with a disproportional increase in ‘Ca. L. asiaticus’ occurrence. Graft transmission experiments were used for a comparative study of both species using budsticks from symptomatic branches of field-affected trees as inoculum. The plants were inoculated with ‘Ca. L. asiaticus’ or ‘Ca. L. americanus’ alone, or simultaneously with both species. Symptom manifestation and conventional and quantitative real-time polymerase chain reaction were used for plant evaluations. ‘Ca. L. americanus’ was detected mainly in symptomatic plants and ‘Ca. L. asiaticus’ was detected in symptomatic plants as well as in infected plants prior to symptom manifestation. Transmission percentages varied from 54.7 to 88.0% for ‘Ca. L. asiaticus’ and 10.0 to 45.2% for ‘Ca. L. americanus’ in two experiments. In co-inoculated plants, 12.9% contained ‘Ca. L. americanus’ only, 40.3% contained ‘Ca. L. asiaticus’ only, and 19.3% contained both species. Average bacterial titers for ‘Ca. L. asiaticus’ and ‘Ca. L. americanus’, in log cells per gram of leaf midrib, were 6.42 and 4.87 for the experimental plants and 6.67 and 5.74 for the field trees used as the source of inoculum. The higher bacterial populations of the ‘Ca. L. asiaticus’-infected plants provided an explanation for the disproportional increase in field prevalence of this species over time, based on the greater likelihood for pathogen transmission by the insect vector.