Plant Disease

Huanglongbing (HLB), also known as citrus greening, is one of the most destructive citrus diseases worldwide and is seen as a major threat to the multimillion dollar citrus industry in California. The vector of the two bacterial species associated with this disease, Candidatus Liberibacter asiaticus and Ca. L. americanus, is the Asian citrus psyllid (ACP), Diaphorina citri (4). ACP was detected in California in August of 2008 and has since been detected in nine counties in southern California. As part of a long term survey and testing program for the ACP carrying the HLB associated bacteria, groups of ACP nymphs and adults were submitted to the Jerry Dimitman Citrus Research Board/Citrus Pest and Disease Prevention Program Laboratory in Riverside, CA. In March 2012, DNA extracted using the Qiagen MagAttract 96 DNA plant kit (QIAGEN Inc., 27220 Turnberry Lane, Suite 200, Valencia, CA 91355) from a group of three ACP adults tested positive for Ca. L. asiaticus with the real-time PCR assay developed by Li et al. (4). ACP adults were collected from a residential citrus tree located in the Hacienda Heights area of Los Angeles County, California. The approximately 1.8 meter tall lemon tree had 23 graft unions, primarily of lemon (Citrus × meyeri) and pomelo (Citrus maxima) varieties. The tree was unthrifty, with yellow shoots and chlorotic leaves. Symptoms on the lemon and pomelo leaves included asymmetrical blotchy mottling, yellowing, and corking of the leaf veins, with the blotchy mottle more prominent in the pomelo leaves. Pomelo leaves appeared crinkled along the thickened veins. Lemon leaves had yellow veins and a few had islands of green tissue completely surrounded by yellow tissue. The entire tree was removed, cut into sections, bagged, and transported to the CDFA Plant Pest Diagnostics Lab for analysis. Two hundred milligrams of petiole and midrib tissue from leaves apical to each graft union was collected, and DNA from each sample was extracted using the Qiagen DNeasy plant mini kit. DNA extracted from both lemon and pomelo leaves tested positive for Ca. L. asiaticus using real-time PCR (4). A 1,160-bp fragment of the 16S ribosomal RNA gene was amplified from the insect and plant DNA extracts using conventional PCR with primers Ol1 and OI2c (2). A 703-bp fragment of the β-operon gene was amplified from the insect and plant extracts with primers A2 and J5 (1). The 16S rDNA fragments from the insect and plant respectively (GenBank Accession Nos. JX430434 and JX455745) and the β-operon fragments (JX430435 and JX455746) showed 100% identity with the corresponding regions of Ca. L. asiaticus (CP001677) strain psy 62. Our 16S rDNA sequence showed 98% identity with Ca. L. africanus (EU921620), 97% identity with Ca. L. solanacearum (HM246509), and 96% with Ca. L. americanus (FJ036892). In response to the detection of HLB, a 241 km2 quarantine area around the detection site was established. Surveys for ACP and symptomatic host plants within the HLB quarantine area are ongoing. To date, there have been no additional positive detections. In the United States, HLB was first detected in Florida in 2005 (4) and in Texas in January of 2012 (3). To our knowledge, this is the first confirmed report of Ca. L. asiaticus associated with HLB in California. References: (1) A. Hocquellet et al. Mol. Cell. Probes 13:373, 1999. (2) S. Jagoueix et al. Mol. Cell. Probes 10:43, 1996. (3) M. Kunta et al. Phytopathology 102:S4.66, 2012. (4) W. Li et al. J. Microbiol. Methods 66:104, 2006.

While performing a routine field survey on 2-year-old canes of Rubus fruticosus (cv. Evergreen Thornless) in the region of Plovdiv (central southern Bulgaria), severe stunting of single or grouped plants (3 to 4 in a row) was found in late August of 2009. It was noteworthy that the leaves of these plants were curved upwards and stayed green until the end of the season. The bushy aspect of the diseased plants led to the assumption of a phytoplasma origin; therefore, specific PCR and sequence based identification methods were applied on leaves, petioles, and stems from three infected Rubus plants grown in different rows of the field (midsummer, nine samples in total) and the same number of asymptomatic samples. Partial amplification of the 16S ribosomal RNA gene with generic phytoplasma primer pairs P1/P7 and fu5/ru3 (3), followed by a nested PCR specific for all members of the Phytoplasma stolbur subgroup by means of the stol11 primers (1), and an RFLP analysis of the tuf gene (elongation factor Tu) fragment produced with PCR primers tufAY/r tufAY (3), were used for the identification and characterization of the pathogen. All target amplicons were also sequenced by Macrogen (Seoul, South Korea) following gel purification (Nucleospoin Plant II, Macher-Nagel). Identical sequences were obtained from each of the P1/P7-derived amplicons (100% homology between samples) and a consensus 1,142 bp sequence was delineated and submitted to NCBI GenBank with accession no. JF293091. It had the highest similarity (99 to 100%) to sequences of ‘Bois noir’ phytoplasma (e.g. HQ589193; Candidatus Phytoplasma solani, position 29 to 1,171). The fu5/ru3 amplicons produced sequences that showed 99.5% homology to the Ca. Phytoplasma solani strains of a southern Russian and Romanian phytoplasma survey on different hosts (potato, tomato, Convolvulus) (GenBank Accession No. HM449999 to HM4450002). The stolbur specific primers also produced an amplicon in all samples and again the consensus sequence was identified (100% homology between the samples) and deposited in GenBank (JN561701). RFLP analysis of the tuf gene with the enzymes HindIII, HinfI, HpaII, and TaqI (Fermentas) produced the same profile types for the different samples and clearly allocated the phytoplasma in the tuf type-b (VKII), according to (2). This type is commonly reported as associated with bindweed (Convolvulus arvensis). Additionally, the sequenced tufAY fragment also confirmed a 100% correspondence with the submitted Tu elongation factor fragments of Ca. Phytoplasma solani strains in GenBank. No phytoplasma was detected in symptomless blackberry plants that were sampled from the same plot. In the molecular identification tests, a stolbur phytoplasma control (potato isolate), a Rubus stunt (EY subgroup, 16SrV) and an apple proliferation phytoplasma (AP subgroup, 16SrX) were used as controls. Based on the symptoms and the laboratory results, we concluded that the Rubus plants were infected by Ca. Phytoplasma solani, a species belonging to the stolbur subgroup (16SrXII-A). To our knowledge, this is the first report of Ca. Phytoplasma solani on Rubus fruticosus in Bulgaria. The disease is not likely to be an isolated case in the future because of the pathogen's spread on other hosts and the expected increase in blackberry fields. References: (1) X. Daire et al. Eur. J. Plant Pathol. 103:507, 1997. (2) M. Langer and M. Maixner. Vitis 43:191, 2004. (3) K.-H. Lorenz et al. Phytopathology 85:771, 1995.

Phytoplasmas of the 16SrVII group in ornamental Fraxinus uhdei trees (1) growing in different cities of the Colombian Andes have been reported (2). In surveys made in Bogotá during March and May 2011, symptoms suggestive of phytoplasma infection were observed in ornamental woody species: Croton spp. (Euphorbiaceae), Pittosporum undulatum (Pittosporaceae) and Populus nigra (Salicaceae) trees, growing close to infected F. uhdei (Oleaceae). Symptoms included witches' broom, yellowing, dieback, epicormic sprouts, tufted foliage, abnormal elongation or shortening of internodes, and deliquescent branching leading to dramatic changes in crown architecture. P. undulatum and F. uhdei are introduced species representing the second and third most abundant trees in the city. P. nigra is an introduced species and Croton spp. is an Andean genus. In order to screen for the presence of phytoplasmas in Croton spp., P. undulatum, and P. nigra, four individuals of each species and two F. uhdei trees were sampled. For DNA extraction, 1 g of vascular tissue from young stems was used. Samples were tested by nested PCR with primers P1A/P7A (4) followed by R16F2n/R16R2 (3). The frequency of phytoplasma detection varied among species; P. undulatum and Croton spp. had three positives each, while P. nigra had one positive. Both F. uhdei were positive. Sequences from the amplicons (three reads) were aligned. BLAST analysis of 16S rDNA sequences from the four species tested had 99.2 to 99.7% similarity to 16SrI group sequences. Phylogenetic analysis further confirmed this relationship. Virtual sequence analysis using the iPhyclassifier tool ( http://plantpathology.ba.ars.usda.gov/cgi-bin/resource/iphyclassifier.cgi ) showed that the sequence derived from P. undulatum (JQ730861) produced an identical RFLP pattern to group 16SrI-B (reference sequence NC_005303). RFLP similarity coefficients of the phytoplasmas from F. uhdei, Croton spp., and P. nigra (JQ730859, JQ730859 and JQ730861) were less than 0.97, suggesting the presence of a new subgroup within group 16SrI. The vectors of phytoplasmas are unknown in the region. Phytoplasma hosts previously reported in Colombia are: Solanum quitoense (16SrIII), Manihot esculenta (16SrIII), Liquidambar styraciflua (16SrI and 16SrVII), Elaeis guineensis (16SrI and 16SrIII), Coffea arabica (16SrIII), Cordia alliodora (16SrIII), Solanum tuberosum (16SrV and 16SrXII), and Zea mays (16SrI). To our knowledge, this is the first report of Croton spp. and P. undulatum as phytoplasma hosts. Phytoplasmas of group 16SrI are known to infect more than 100 species of different families worldwide. Detection of this group in several tree species and the observation of similar symptoms in other trees species raises concerns about a possible epidemic affecting plants in the Andean region. Implications are at several levels: i) epidemiological, with infected trees representing a potential inoculum source for other ornamental plants or crops growing in the agricultural surrounding areas; ii) economic, since eventually it will be necessary to replace diseased plants; and iii) environmental, because of the negative impact on the services provided by trees and green areas. References: (1) J. J. Filgueira et al. Plant Pathology 53:520, 2004. (2) L. Franco-Lara et al. Fitopatología Colombiana 29:32, 2005. (3) D. E Gundersen et al. Phytopathol. Mediterr. 35:144, 1996. (4) I-M. Lee et al. Int. J. Syst. Evol. Microbiol. 54:1037, 2004.

In the summer of 2008, symptoms of leaf curling with yellow, bronze, and purple discoloration, twisting of petioles, stunting of shoots and tap roots, and proliferation of secondary roots were observed in 18 commercial carrot (Daucus carota L.) fields (~62 ha) severely infested with psyllids (mainly Bactericera sp.) from 52 fields (297 ha) located in Alicante and Albacete provinces of Spain. Incidence of symptomatic plants was variable among fields. Similar symptoms were observed in 2009, 2010, and 2011. Symptoms resembled those associated with phytoplasma, spiroplasma, or the bacterium ‘Candidatus Liberibacter solanacearum’ infections in carrot (1–4). Aster yellows and stolbur phytoplasmas and Spiroplasma citri have previously been reported from carrot in mainland Spain but liberibacter infection has not been documented in this region (1). Studies were conducted to determine if ‘Ca. L. solanacearum’ was associated with the symptoms. Petiole samples of symptomatic carrot plants were collected in 2009 (25 from 9 fields in Alicante and Albacete provinces) and early 2010 (21 from 8 fields in Alicante, Albacete, and Valencia provinces) from symptomatic fields where incidence ranged from 50 to 90%. In addition, one sample collected in 2008 in Alicante was included in the assay. Also, samples were collected from five asymptomatic carrot plants. Total DNA was extracted from 0.5 g of petiole tissue of each sample with the CTAB extraction buffer method (3,4). DNA extractions were analyzed by PCR assay using primer pairs OA2/OI2c and CL514F/R to amplify a portion of 16S rDNA and rplJ/rplL ribosomal protein genes, respectively, of ‘Ca. L. solanacearum’ (3,4). DNA samples were also tested for phytoplasmas and S. citri by nested-PCR assays using primer pairs P1/P7 followed by R16F2n/R16R2n and ScR16F1/ScR16R1 followed by ScR16F1A/ScR16R2, respectively (2). A 1,168-bp fragment of 16S rDNA was detected in DNA extracted from 1, 12, and 12 symptomatic samples collected in 2008, 2009, and 2010, respectively, suggesting the presence of ‘Ca. L. solanacearum’ in the carrot samples. A 669-bp rplJ/rplL fragment also was amplified from DNA of the same samples. Liberibacter was not detected in asymptomatic plants. Eight and two samples were infected with S. citri and aster yellows phytoplasmas, respectively. Three samples were infected with S. citri and ‘Ca. L. solanacearum’ and one sample was infected with all three pathogens. Three amplicons obtained from the PCR assays with both primer pairs from carrot samples collected in 2009 and 2010 were sequenced directly. BLAST analysis of the 16S rDNA sequences (GenBank Nos. HQ454302, HQ454303, and HQ454304) showed 99% nucleotide identity to those of ‘Ca. L. solanacearum’ amplified from carrot in Finland (GU373049). The rplJ/rplL nucleotide sequences (HQ454305, HQ454306, and HQ454307) were 97% identical to sequences of the analogous rplJ/rplL ‘Ca. L. solanacearum’ ribosomal protein gene from carrot in Finland (GU373051). To our knowledge, this is the first report of ‘Ca. L. solanacearum’ in carrot in mainland Spain and also the first evidence of mixed infections of S. citri, ‘Ca. L. solanacearum’, and phytoplasmas in carrot. References: (1) M. C. Cebrián et al. Plant Dis. 94:1264, 2010. (2) I.-M. Lee et al. Plant Dis. 90:989, 2006. (3) J. E. Munyaneza et al. J. Econ. Entomol. 103:1060, 2010. (4) J. E. Munyaneza et al. Plant Dis. 94:639, 2010.

In 2009 and 2010, commercial carrot (Daucus carota L.) fields located in Tenerife (Canary Islands, Spain) showed symptoms of curling, yellow, bronze, and purple discoloration of leaves, stunting of shoots and tap roots, and proliferation of secondary roots. A large population of the psyllid Bactericera trigonica was noted in those fields. Similar symptoms were reported previously in carrot-production areas of the Canary Islands and mainland Spain that were associated with stolbur and aster yellows (1997 and 1998) (2) and Spiroplasma citri and phytoplasmas (2009 and 2010) (1). These symptoms were also reported in southern Finland in 2008 and associated with ‘Candidatus Liberibacter solanacerum’ (4). Studies were conducted to investigate whether these pathogens and the psyllid B. trigonica were associated with the observed symptoms in carrot in Tenerife. A total of 18 petiole samples of symptomatic carrots were collected (13 samples in 2009 and 5 samples 2010). Five asymptomatic plants were also sampled. Three samples of psyllids (five individuals grouped) collected from one affected field in 2010 were also included in the assay. Total DNA was extracted with the DNeasy Plant Mini Kit (Qiagen, Valencia, CA), and analyzed by nested-PCR assays using primer pairs P1/P7 and R16F2n/R16R2n for phytoplasmas and ScR16F1/ScR16R1 followed by ScR16F1A/ScR16R2 for S. citri detection as described previously (3). PCR was performed using primer pairs OA2/OI2c and CL514F/R to amplify a portion of 16S rDNA and rplJ/rplL ribosomal protein genes, respectively, for ‘Ca. L. solanacearum’ (4). S. citri and phytoplasmas were not detected in any of the studied samples. However, a 1,168-bp 16S rDNA fragment and a 669-bp rplJ/rplL fragment were amplified from DNA from 16 symptomatic carrot samples and three psyllid grouped samples using specific primers for ‘Ca. L. solanacearum’. No DNA was amplified from the asymptomatic samples. These results indicate the presence of ‘Ca. L. solanacearum’ in the affected carrot and psyllid samples collected in Tenerife (Canary Islands). Four and one PCR products obtained from DNA of carrot and psyllid samples, respectively, with both primer pairs were sequenced. BLAST analysis of the 16S rDNA sequences obtained from infected carrots (GenBank Accession Nos. HQ454312, HQ454313, HQ454314, and HQ454315) and psyllids (HQ454316) showed 99% identity to those of ‘Ca. L. solanacearum’ amplified from carrot in Finland (GU373049) and B. cockerelli (EU812557). The rplJ/rplL nucleotide sequences obtained from infected carrots (Accession Nos. HQ454317, HQ454318, HQ454319, and HQ454320) and psyllid (HQ454321) were 98% identical to the analogous rplJ/rplL ‘Ca.L. solanacearum’ ribosomal protein gene from carrot (GU373051) in Finland and tomato (EU834131) from New Zealand. To our knowledge, this is the first report of ‘Ca. L. solanacearum’ associated with psyllid-affected carrots in the Canary Islands (Tenerife, Spain) and also the first report of this plant pathogen associated with B. trigonica. References: (1) M. C. Cebrián et al. Plant Dis. 94:1264, 2010. (2) M. I. Font et al. Bol. San. Veg. Plagas 25:405, 1999. (3) I.-M. Lee et al. Plant Dis. 90:989, 2006. (4) J. E. Munyaneza et al. Plant Dis. 94:639, 2010.

In September 2011, potato (Solanum tuberosum L.) tubers graded in a packing facility in south-central Idaho were observed with internal discolorations suggestive of zebra chip disease (ZC). Symptoms were observed in 1 to 2% of tubers of cv. Russet Norkotah and included brown spots and streaks especially in and near the vascular tissue. Some tubers also showed a dark and sunken stolon attachment typical of ZC (1). Initially, tissue samples were taken from seven symptomatic tubers and tested by PCR for “Candidatus Liberibacter solanacearum”, the bacterium associated with ZC. Primers specific for the 16S rDNA (primers CLipoF [4] and OI2c [3]) and the outer membrane protein (OMB 1482f and 2086r) (2) were used. Six of these samples were positive for the bacterium. The amplified 16S rDNA and OMB products from two symptomatic tubers of cv. Russet Norkotah were cloned and three clones of each were sequenced. The 16S sequences (1,071 bp; GenBank Accession Nos. JN848755 and JN848756) from the two tubers varied by one nucleotide and had 99 to 100% sequence identity to numerous “Ca. L. solanacearum” sequences in GenBank (e.g., Accession Nos. HM246509, FJ957897, and EU935004). Sequences of the two OMB clones (605 bp; GenBank Accession Nos. JN848757 and JN848758) had 97% sequence identity to the two “Ca. L. solanacearum” OMB sequences in GenBank (Accession Nos. CP002371 and FJ914617). Six of eight additional symptomatic field-collected cv. Russet Norkotah tubers corresponding to tubers collected in the packing facility were also positive for “Ca. L. solanacearum” by PCR. Additional severely symptomatic tubers of cvs. Russet Burbank, Yukon Gold, and raw cut French fries of Ranger Russet produced in south-central Idaho were subsequently tested by PCR and were found to be positive for “Ca. L. solanacearum” as well. On the basis of the symptoms, specific PCR amplification with two distinct primer pairs and DNA sequence analysis, zebra chip disease caused by “Ca. L. solanacearum” was determined to be present in Idaho. This disease has caused significant economic damage to potatoes in many regions, including Texas, Mexico, Central America, and New Zealand (1). Idaho is the largest potato-producing state in the United States, with over 150,000 ha planted this year, and therefore, ZC potentially poses a significant risk to agriculture in this state. References: (1) J. M. Crosslin et al. Online publication. doi:10.1094/PHP-2010-0317-01-RV, Plant Health Progress, 2010. (2) J. M. Crosslin et al. Southwest. Entomol. 36:125, 2011. (3) S. Jagoueix et al. Mol. Cell. Probes 10:43, 1996. (4) G. A. Secor. Plant Dis. 93:574, 2009.

Carrot (Daucus carota) plants with symptoms resembling those associated with the carrot psyllid Trioza apicalis and the bacterium “Candidatus Liberibacter solanacearum” (1–4) were observed in 70 to 80% of commercial fields and experimental plots in southeastern Norway from late July to mid-September of 2011; all cultivars grown were affected with approximately 10 to 100% symptomatic plants per field. T. apicalis, a pest of carrot in northern and central Europe, including Norway, can cause as much as 100% crop loss and is associated with “Ca. L. solanacearum” (1–4). Symptoms on affected plants include leaf curling, yellow and purple discoloration of leaves, stunted growth of shoots and roots, and proliferation of secondary roots. Carrot plant samples were collected from five T. apicalis-infested fields in Ostfold, Vestfold, Oppland, and Hedmark counties. Total DNA was extracted from petiole and root tissues of 54 plants, including 27 symptomatic plants and 27 asymptomatic plants from four cultivars (Namdal, Panther, Romance, and Yukon) with the cetyltrimethylammonium bromide (CTAB) buffer extraction method (2,3). DNA samples were tested by PCR assay using primer pairs OA2/OI2c and CL514F/R to amplify a portion of 16S rDNA and rplJ/rplL ribosomal protein genes, respectively, of “Ca. L. solanacearum” (2,3). A 1,168-bp 16S rDNA fragment was detected in the DNA from 22 (81.5%) symptomatic plants and a 668-bp rplJ/rplL fragment was amplified from the DNA of 26 (96.3%) symptomatic and 5 (18.5%) asymptomatic plants, indicating the presence of liberibacter. No liberibacter was detected in the asymptomatic carrot plants with the primer pair OA2/OI2c. Amplicons from the DNA of four carrot root samples with each primer pair were cloned (pCR2.1-TOPO; Invitrogen, Carlsbad, CA) and three clones of each of the eight amplicons were sequenced (MCLAB, San Francisco, CA). BLAST analysis of the 16S rDNA consensus sequence from the carrot root tissues (GenBank Accession No. JN863097) showed 100% identity to those of “Ca. L. solanacearum” previously amplified from carrot (GU373048 and GU373049) and T. apicalis (GU477254 and GU477255) in Finland (2,3). The rplJ/rplL consensus sequence from the carrots (GenBank Accession No. JN863098) was 99% identical to the sequences of rplJ/rplL “Ca. L. solanacearum” ribosomal protein gene from carrots in Finland (GU373050 and GU373051). To our knowledge, this is the first report of “Ca. L. solanacearum” associated with carrot in Norway. This bacterial species has caused millions of dollars in losses to potato and several other solanaceous crops in North and Central America and New Zealand (1). This plant pathogen has also been reported from carrots and T. apicalis in Finland, where it has caused significant economic damage to carrot crops (2–4). References: (1) J. E. Munyaneza. Southwest. Entomol. 35:471, 2010. (2) J. E. Munyaneza et al. Plant Dis. 94:639, 2010. (3) J. E. Munyaneza et al. J. Econ. Entomol. 103:1060, 2010. (4) A. Nissinen et al. Entomol. Exp. Appl. 125:277, 2007.

Carrot (Daucus carota) plants with symptoms resembling those associated with the carrot psyllid Trioza apicalis and the bacterium “Candidatus Liberibacter solanacearum” (1–4) were observed in 70% of commercial fields in southern Sweden in August 2011, with approximately 1 to 45% symptomatic plants per field. T. apicalis, a pest of carrot in northern and central Europe, including Sweden, can cause as much as 100% crop loss and is associated with “Ca. L. solanacearum” (1–4). Symptoms on affected plants include leaf curling, yellow and purple discoloration of leaves, stunted growth of shoots and roots, and proliferation of secondary roots (3). Carrot plant and psyllid samples were collected from fields in the province of Halland. Total DNA was extracted from petiole and root tissues of 33 symptomatic and 16 asymptomatic plants (cvs. Nevis and Florida), with the cetyltrimethylammonium bromide (CTAB) buffer extraction method (2,3). DNA was also extracted from 155 psyllids (3). DNA samples were tested by PCR using primer pairs OA2/OI2c (5′'-GCGCTTATTTTTAATAGGAGCGGCA-3′/5′-GCCTCGCGACTTCGCAACCCAT-3′) and CL514F/R (5′-CTCTAAGATTTCGGTTGGTT-3′/5′-TATATCTATCGTTGCACCAG-3′), to amplify a portion of 16S rDNA and rplJ/rplL ribosomal protein genes, respectively, of “Ca. L. solanacearum” (2,3). A 1,168-bp 16S rDNA fragment was detected in the DNA from all 33 symptomatic and two asymptomatic plants, and a 668-bp rplJ/rplL fragment was amplified from the DNA of all 33 symptomatic and four asymptomatic plants, indicating the presence of liberibacter. DNA from 23 and 49 psyllid samples yielded similar amplicons with OA2/OI2c and CL514F/R primer pairs, respectively. Amplicons from the DNA of four carrot roots and three T. apicalis with each primer pair were cloned (pCR2.1-TOPO; Invitrogen, Carlsbad, CA) and three clones of each of the 14 amplicons were sequenced (MCLAB, San Francisco, CA). BLAST analysis of the 16S rDNA consensus sequences from carrot (GenBank Accession No. JN863095) and T. apicalis (GenBank Accession No. NJ863096) showed 100% identity to those of “Ca. L. solanacearum” previously amplified from carrot (GU373048 and GU373049) and T. apicalis (GU477254 and GU477255) from Finland (2,3). The rplJ/rplL consensus sequences from carrot (GenBank Accession No. JN863093) and T. apicalis (GenBank Accession No. JN863094) were 99% identical to the sequences of rplJ/rplL “Ca. L. solanacearum” ribosomal protein gene from carrots in Finland (GU373050 and GU373051). To our knowledge, this is the first report of “Ca. L. solanacearum” associated with carrot and T. apicalis in Sweden. The disease associated with this bacterium caused millions of dollars in losses to potato and several other solanaceous crops in North and Central America and New Zealand (1). This plant pathogen is also associated with significant economic damage to carrot crops observed in Finland (2,3). References: (1) J. E. Munyaneza. Southwest. Entomol. 35:471, 2010. (2) J. E. Munyaneza et al. Plant Dis. 94:639, 2010. (3) J. E. Munyaneza et al. J. Econ. Entomol. 103:1060, 2010. (4) A. Nissinen et al. Entomol. Exp. Appl. 125:277, 2007.

In August of 2011, potato (Solanum tuberosum) tubers grown in the lower Columbia Basin of southern Washington State and northern Oregon were observed with internal discolorations suggestive of zebra chip disease (ZC). Symptoms included brown spots, streaks, and stripes in and near the vascular tissue, typical of ZC (1). Symptoms were observed in cvs. Alturas, Russet Norkotah, Pike, Ranger Russet, Umatilla Russet, and Russet Burbank. Foliar symptoms on plants that produced symptomatic tubers included purple discoloration in upper leaves, leaf rolling, axial bud elongation, chlorosis, leaf scorch, and wilt. Tissue was taken from two symptomatic tubers each of cvs. Alturas and Russet Norkotah, three tubers of cv. Umatilla Russet, and one tuber of cv. Pike. These tubers were tested by PCR for “Candidatus Liberibacter solanacearum”, an unculturable alphaproteobacterium associated with ZC (1,4). Primers specific for the 16S rDNA were CLipoF (4) and OI2c (3), and primers OMB 1482f and 2086r were specific for the outer membrane protein (2). All of these samples, except one Umatilla tuber, were positive for the bacterium. The 16S rDNA and OMB amplicons from one symptomatic tuber each of Alturas (from Washington) and Pike (from Oregon) were cloned and three clones of each were sequenced. BLAST analysis of the consensus sequences confirmed “Ca. L. solanacearum”. The 16S sequences (1,071 bp) from the two tubers were identical and showed 99 to 100% identity to a number of 16S rDNA sequences of “Ca. L. solanaceaum” in GenBank (e.g., Accession Nos. HM246509 and FJ957897). The 16S rDNA sequences were deposited in GenBank as Accession Nos. JN848751 and JN848753. Consensus sequences of the two OMB clones (605 bp; deposited in GenBank as Accession Nos. JN848752 and JN848754) were identical and showed 97% identity to the two “Ca. L. solanacearum” OMB sequences in GenBank (Accession Nos. CP002371 and FJ914617). Potato psyllids (Bactericera cockerelli Sulc), the vector of “Ca. L. solanacearum”, were present in ZC-affected fields in Oregon and Washington and the bacterium was confirmed by PCR in 5 to 10% of 128 adult psyllids collected from two fields. On the basis of foliar and tuber symptoms, specific PCR amplification with two primer pairs, sequence analyses, and the presence of Liberibacter-infected potato psyllids, ZC and “Ca. L. solanacearum” are present in potatoes in Oregon and Washington State. Washington and Oregon together grow ~80,000 ha of potatoes. ZC has caused significant economic damage to potatoes in Texas, Mexico, Central America, and New Zealand (1). Therefore, ZC may pose a risk to agriculture in Oregon, Washington, and neighboring states. However, the potential for development of widespread and serious disease will depend upon the arrival time and number of infective potato psyllids entering the region. References: (1) J. M. Crosslin et al. Online publication. doi:10.1094/PHP-2010-0317-01-RV, Plant Health Progress, 2010. (2) J. M. Crosslin et al. Southwest. Entomol. 36:125, 2011. (3) S. Jagoueix et al. Mol. Cell. Probes 10:43, 1996. (4) G. A. Secor. Plant Dis. 93:574, 2009.

Temperature has been shown to have a significant effect on development of liberibacter species associated with citrus Huanglongbing disease. ‘Candidatus Liberibacter africanus’ and ‘Ca. L. americanus’ are both heat sensitive, whereas ‘Ca. L. asiaticus’ is heat tolerant. The recently described ‘Ca. L. solanacearum’ is associated with zebra chip (ZC), a newly emerging and economically important disease of potato worldwide. This psyllid-transmitted liberibacter species severely affects several other solanaceous crops and carrot. Experiments were conducted to evaluate effects of temperature on development of ‘Ca. L. solanacearum’ and ZC disease. Potato plants were inoculated with ‘Ca. L. solanacearum’ by briefly exposing them to liberibacter-infective potato psyllids at various temperatures under laboratory conditions. Following insect exposure, the plants were maintained at selected temperature regimes in growth chambers, monitored for ZC symptom development, and later tested for liberibacter by polymerase chain reaction to confirm infection. Results indicated that temperatures below 17°C appear to slow development of ‘Ca. L. solanacearum’ and ZC symptoms, whereas temperatures above 32°C are detrimental to this liberibacter. Compared to Huanglongbing liberibacters, ‘Ca. L. solanacearum’ appears heat sensitive. The sensitivity of this bacterium and its insect vector to temperature may partially explain incidence, severity, and distribution of ZC in affected regions.