Plant Disease

In this study, two polyclonal antibodies were produced against the Omp protein of ‘Candidatus Liberibacter asiaticus’. First, omp genes were sequenced to exhibit 99.9% identity among 137 isolates collected from different geographical origins. Then, two peptides containing the hydrophobic polypeptide-transport-associated (POTRA) domain and β-barrel domain, respectively, were identified on Omp protein. After that, these two peptides were overexpressed in Escherichia coli and purified by affinity chromatography to immunize the white rabbits. Finally, the antiserum was purified by affinity chromatography. The two Omp antibodies gave positive results (0.454 to 0.633, 1:1,600 dilution) in enzyme-linked immunosorbent assay against ‘Ca. L. asiaticus’-infected samples collected from different geographical origins but revealed negative results against other pathogen-infected, nutrient-deficient and healthy samples. The antibody against the POTRA domain of Omp protein could detect ‘Ca. L. asiaticus’ in 45.7% of the symptomatic samples compared with a 56.2% detection rate with a polymerase chain reaction assay. These new antibodies will provide a very useful supplement to the current approaches to ‘Ca. L. asiaticus’ detection and also provide powerful research tools for tracking distribution of this pathogen in vivo.

In April of 2012, tobacco (Nicotiana tabacum) plants with symptoms resembling those caused by viral infection were observed in commercial fields in several departments in Nicaragua, including Esteli and Nueva Segovia. Heavy infestations of the psyllid Bactericera cockerelli, a major insect pest of potato and other solanaceous crops and vector of the bacterium “Candidatus Liberibacter solanacearum” (Lso) (2,3), were observed in the affected fields. All cultivars grown were affected and 5 to 100% of plants in each field were symptomatic. Symptoms on affected plants included apical leaf curling and stunting, overall chlorosis and plant stunting, young plant deformation with cabbage-like leaves, wilting, internal vascular necrosis of stems and leaf petioles, and overall poor leaf quality. Plant samples were collected from a total of three psyllid-infested fields in the municipalities of Esteli, Condega, and Jalapa (one field/municipality). The plant samples were first tested for viruses, including Potato virus Y, Tobacco mosaic virus, Cucumber mosaic virus, and Impatiens necrotic spot virus, using Immunostrips (Agdia, Elkhart, IN) and no virus was detected. Total DNA was extracted from leaf tissues of a total of 22 plants, including 17 symptomatic plants and five asymptomatic plants from two cultivars (Corojo and Habano) with the cetyltrimethylammonium bromide (CTAB) buffer extraction method (2,4). The DNA samples were tested by PCR using specific primer pairs OA2/OI2c and OMB 1482f/2086r, to amplify a portion of 16S rDNA and the outer membrane protein (OMB) genes, respectively, of Lso (2). 16 rDNA and OMB gene-derived fragments of 1,168 and 605 bp, respectively, were amplified from the DNA of 13 of 17 (76.5%) symptomatic plants, indicating the presence of Lso. No Lso was detected in the five asymptomatic plants. DNA amplicons of three plant samples (one plant/field) with each primer pair were cloned and two to four clones of each of the six amplicons were sequenced. BLASTn analysis of the 16S rDNA consensus sequences was the same for all three locations (GenBank Accession Nos. KC768323, KC768324, and KC768325) and showed 100% identity to numerous 16 rDNA sequences of Lso in GenBank, including accessions HM245242, JF811596, and JX559779. Similarly, identical OMB consensus sequences were observed in all three locations (KC768331 and KC768332 for Jalapa and Condega, respectively) that are 97 to 100% identical to a number of Lso sequences in GenBank (e.g., CP002371, FJ914617, JN848754, and JN848752). A second OMB sequence was isolated from the Esteli sample (KC768333) that was 98% identical with the consensus sequences from this and other sites and 100% identical to an OMB sequence from Lso in GenBank (JN848754). To our knowledge, this is the first report of Lso associated with tobacco. Tobacco is an important crop in many parts of the world, including Central and South America. This bacterium has also caused millions of dollars in losses to potato and several other solanaceous crops in the Americas and New Zealand (3). In addition, this plant pathogen has been reported as serious pest of carrot in Europe, where it is associated with the psyllids Trioza apicalis and B. trigonica (1,4). References: (1) A. Alfaro-Fernandez et al. Plant Dis. 96:581, 2012. (2) J. M. Crosslin. Southwest. Entomol. 36:125, 2011. (3) J. E. Munyaneza. Am. J. Pot. Res. 89:329, 2012. (4) J. E. Munyaneza et al. J. Econ. Entomol. 103:1060, 2010.

In April of 2012, tomato plants (Solanum lycopersicum) grown near the town of Yuroconte in the municipality of La Palma, Chalatenango, El Salvador, were observed with symptoms resembling those of “Candidatus Liberibacter solanacearum” infection. The symptoms included overall chlorosis, severe stunting, leaf cupping, excessive branching of axillary shoots, and leaf purpling and scorching (1,2,3). Disease incidence in several fields in the area ranged from 40 to 60%. Heavy infestations of the potato/tomato psyllid, Bactericera cockerelli, were observed in the affected fields and this insect has been shown to transmit “Ca. L. solanacearum” to tomato and other solanaceous species (1,2,3). Leaf samples and psyllids were collected from one of the fields and total DNA was purified from the leaves of 8 and 10 symptomatic and asymptomatic plants, respectively (2,3). DNA was also extracted from the psyllids and the samples were tested by PCR for species confirmation. PCR oligonucleotide primers specific for both 16S rDNA (OA2 and OI2c) and a gene for a surface antigen for the outer membrane protein (OMB) (OMB 1482f and 2086r) of “Ca. L. solanacearum” were used to confirm the presence of the bacterium in infected tomatoes (1). Four of the eight symptomatic tomatoes (50%) tested positive for “Ca. L. solanacearum” using both primer pairs and all asymptomatic plants were negative for the bacterium. The collected psyllids were first identified through a morphological key, then verified using species-specific PCR primers (CO1 F3 and CO1 meltR) that generated a 94-bp fragment that was consistent with DNA from B. cockerelli (4). Amplicons generated with DNA from two plant samples with each primer pair were cloned and four clones of each of the four amplicons were sequenced. BLASTn analysis of the 16S rDNA consensus sequences from the clones (1,168 bp; deposited in GenBank as Accession Nos. KC768318 and KC768319) showed 100% identity to “Ca. L. solanacearum” sequences in GenBank (HM246509 and HM245242, respectively). Two OMB consensus sequences were 98% identical (deposited in GenBank as KC768326 and KC768327) and both sequences were 97 to 100% identical to a number of “Ca. L. solanacearum” sequences in GenBank (e.g., CP002371, FJ914617, JN848754, and JN848752). To our knowledge, this is the first report of “Ca. L. solanacearum” associated with tomato in El Salvador and the first formal report of the bacterium in the country. This bacterium has caused millions of dollars in losses to the tomato industry in New Zealand, Mexico and the United States (2,3). Tomatoes are an economically important commodity in Central America and are severely damaged by “Ca. L. solanacearum” infection. The confirmation of “Ca. L. solanacearum” infections in El Salvador alerts the agricultural sector to the presence of this serious pathogen. References: (1) J. M. Crosslin. Southwest. Entomol. 36:125, 2011. (2) L. W. Liefting et al. Plant Dis. 93:208, 2009. (3) J. E. Munyaneza et al. Plant Dis. 93:1076, 2009. (4) K. D. Swisher et al. Environ. Entomol. 41:1019, 2012.

Huanglongbing (HLB) is a devastating citrus disease. It is associated with a phloem-restricted bacterium, ‘Candidatus Liberibacter asiaticus’, and primarily transmitted by Asian citrus psyllid in Florida. Because Liberibacter cannot be cultured, early diagnosis of HLB relies on DNA-based polymerase chain reaction (PCR), including real-time quantitative (q)PCR. Although estimating genomes from live bacteria (GLB) is critical for HLB research, PCR does not distinguish between live and dead cells and, thus, does not estimate GLB in hosts. Propidium monoazide (PMA), a novel DNA-binding dye, has been successfully used on many bacterial pathogens to effectively remove DNA from dead cells but there is no report of its use on uncultured bacteria. In this study, PMA-qPCR protocols were first optimized to work with plant and psyllid samples, respectively. Both TissueLyser treatment and plant tissue were demonstrated to have an insignificant impact on the GLB detected by PMA-qPCR. Finally, a standard curve for GLB determination was successfully established between PMA-qPCR results and microscopic counts and then applied in two studies with different greenhouse plant samples. This rapid qPCR method provides a more accurate way to determine GLB in HLB hosts which, in turn, should benefit disease epidemiology studies and serve as a crucial component in HLB management.

In November 2010, approximately 2% of the chrysanthemum (Chrysanthemum morifolium) cv. Paniz plants showed numerous small leaves in the top and stunting in a field collection of the National Research Center of Ornamental Plants in Mahallat, Iran. Next to these plants, some plants of the same collection showed leaves with a reddish and/or chlorotic discoloration around the veins. The observed symptoms were believed to represent infection by a phytoplasma and/or a viroid. Two plants with each type of the symptoms were individually analyzed. Using a total RNA extract from diseased leaves, RT-PCR with primer pairs targeting all known pospiviroids, including Chrysanthemum stunt viroid (CSVd) (3), were negative. Purified DNA was examined for the highly conserved phytoplasma 16S rRNA gene by nested-PCR using the universal primer sets P1/P7 and R16F2n/R16R2 (2). Fragments of 1.2 kb, obtained only from the plants with the small leaves and stunting, were sequenced and one of these sequences, which were identical, was deposited in GenBank (Accession No. KC176800). BLAST analysis of the chrysanthemum phytoplasma sequence exhibited 99% identity to Candidatus Phytoplasma phoenicium (Ca. P. phoenicium) species of the 16SrIX group. Subsequently, in silico RFLP analysis of the nested PCR product with the pDRAW32 program using AluI and TaqI restriction sites used for 16SrIX subgroups A, B, C, D, and E indicated that the 16SrIX chrysanthemum isolate belonged to subgroup D (1). Recently, based on GenBank sequences, several strains of Ca. P. phoenicium have been isolated and identified from diverse host species like Lactuca serriola, L. sativa, Solanum lycopersicon, Sonchus sp. [16SrIX-E], Carthamus tinctorius, and Prunus amygdalus [16SrIX-B] (4) in Iran. The vector species transmitting Ca. P. phoenicium to C. morifolium still needs to be identified. The leafhopper Neoaliturus fenestratus may be a potential vector as it is an often encountered efficient transmitter vector of 16SrIX group phytoplasmas in Iran (2). Next to the susceptibility of chrysanthemum to members of aster yellows, stolbur, and Ca. P. aurantifolia phytoplasma groups, this is, to our knowledge, the first report of a 16SrIX group member infecting chrysanthemum. The detection of this phytoplasma in chrysanthemum can form a new threat to this crop and other ornamentals in the Mahallat flower production region. References: (1) R. E. Davis et al. New Dis. Rep. 20:35, 2010. (2) M. Salehi et al. Plant Pathol. 56:669, 2007. (3) J. Th. J. Verhoeven et al. Eur. J. Plant Pathol. 110:823, 2004. (4) M. G. Zamharir. Afr. J. Microbiol. Res. 5:6013, 2011.

In September 2011, potato (Solanum tuberosum) tubers grown in Nicaragua outside of Estelí and Jinotega were observed with internal discoloration suggestive of zebra chip (ZC); and the plants showed foliar symptoms of chlorosis, leaf scorching, wilting, vascular discoloration, swollen nodes, twisted stems, and aerial tubers (3). Disease incidence ranged from 50 to 95% in eight fields ranging from 5 to 12 ha in the Estelí and Jinotega regions of Nicaragua. Leaf samples and psyllids were collected from two fields, and total DNA was purified from the leaves of 17 symptomatic and 10 asymptomatic plants. DNA was also extracted from 20 individual potato psyllids. Primers specific for 16S rDNA (OA2 and OI2c) and the surface antigen gene (OMB 1482f and 2086r) of Candidatus Liberibacter solanacearum (CLs) were used to confirm the presence of the pathogen in infected potatoes and insects (2). All symptomatic potato leaf samples tested positive for the presence of CLs using both primers, and no asymptomatic plants had positive results. Seven insects tested positive for the presence of CLs. 16S rDNA sequences obtained for all positive samples (1,071 bp) were identical and showed 99 to 100% identity to a number of rDNA sequences of CLs in GenBank (Accession Nos. HM246509 and FJ957897). 16S rDNA sequences from two CLs-infected plants, one from Estelí, Nicaragua (JX559779) and one from Jinotega, Nicaragua (JK559780), were deposited in GenBank. Identity of insects was done using a morphological key, and then verified as Bactericera cockerelli using a real-time PCR assay with melt temperature analysis of the cytochrome c oxidase 1 gene, as described by Chapman et al. (1). Sequencing of the amplified DNA yielded an approximately 63-bp read, with 100% homology to reference sequences of B. cockerelli (AY971886) and those obtained from psyllids collected in McAllen, TX, in 2010. B. cockerelli samples were collected from both locations. Similar to previous reports of ZC in new locations, foliar and tuber symptoms associated with ZC were observed in all eight fields in these two Nicaraguan potato-growing regions, specific PCR amplification with two primer pairs was completed, 16S rDNA sequence analyses showed 100% similarity to reference sequences of CLs, and the presence of potato psyllids which tested positive for the presence of CLs provide evidence that ZC is now present in Nicaragua. Potatoes rank in the top 20 commodities produced in Nicaragua, resulting in >$4.5M annual revenue. Because CLs has caused significant economic damage to potatoes in the United States, Mexico, Guatemala, and Honduras, this finding has significance for potato production in Central America. References: (1) R. I. Chapman et al. Southwest. Entomol. 37:475, 2012. (2) J. M. Crosslin. Southwest. Entomol. 36:125, 2011. (3) L. W. Liefting et al. Internat. J. Syst. Evol. Microbiol. 59:2274, 2009.

Cauliflower stunt, caused by a phytoplasma of the group 16SrIII-J, was reported in the beginning of 2012 and has occurred with high incidences of infected plants (up to 90%) in crops located in the state of São Paulo in the southeast region of Brazil (3). Diseased plants exhibit general stunting, malformation of inflorescence, reddening leaves, and vessel necrosis (3). Further investigations with plants displaying identical symptoms collected in Nova Bassano, state of Rio Grande do Sul, Brazilian south region, have revealed the presence of a phytoplasma distinct from 16SrIII-J subgroup. Four symptomatic plus four asymptomatic samples were assayed from a field, and the presence of phytoplasma was evidenced by nested PCR assays performed with primers P1/Tint followed by R16F2n/16R2 in three affected plants, which amplified genomic fragments of 1.2 kb from the 16S rRNA gene. No amplification occurred in non-affected samples. Nested PCR products analyzed by conventional RFLP (2) using the enzymes AluI, RsaI, KpnI, HpaII, MseI, HhaI, MboI, and BstUI pointed to the presence of a phytoplasma belonging to group 16SrXV-A in all three phytoplasma-positive samples. Virtual RFLP analysis based on restriction patterns, derived from in silico digestion with 17 endonucleases (4), confirmed the previous results obtained from those samples by conventional RFLP. The 16S rDNA sequences of this phytoplasma identified in cauliflower (GenBank Accession No. JN818845) shared 99% sequence similarity with the reference phytoplasma for subgroup 16SrXV-A (Hibiscus witches'-broom phytoplasma, AF147708), designated ‘Candidatus Phytoplasma brasiliense.’ Analysis of putative restriction sites showed excellent identity between the phytoplasma studied here and the reference phytoplasma. In addition, the arrangement of branches of a phylogenetic tree constructed with phytoplasmas representing diverse 16Sr groups and subgroups supported that the phytoplasma found in cauliflower is closed related to the representative of the subgroup 16SrXV-A. Association of distinct phytoplasmas with the same kind of disease is not rare and the present pathosystem constitutes a new example. Members of this subgroup have been described almost exclusively in Brazil and previously reported in Sida sp., periwinkle, and hibiscus (1). In some European countries, as well as in the United States and Canada, phytoplasmas belonging to group 16SrI has been associated with this type of disease, which has been reported for various species of the genus Brassica, as published in previous works (3). However, a representative of the group 16SrVI was described in infected plants in Iran (3). Although the 16SrIII-J phytoplasma is currently the most important agent of cauliflower stunt in Brazil, and members of 16SrI are prevalent in other countries, this study revealed that a 16Sr XV-A phytoplasma may be also associated with this important disease of brassicas. Besides, the findings here reported expand the natural host range, including cauliflower as new host for phytoplasmas affiliated with 16SrXV-A. References: (1) B. Eckstein et al. Plant Dis. 95:363, 2009. (2) I. M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998. (3) M. C. C. Rappussi et al. Eur. J. Plant. Pathol. 133:829, 2012. (4) Wei et al. Int. J. Syst. Evol. Microbiol. 57:1855, 2007.

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.