Plant Science

The foxtail palm (Wodyetia bifurcata), an Australian native species, is an adaptable and fast-growing landscape tree. The foxtail palm is most commonly used in landscaping in Malaysia. Coconut yellow decline (CYD) is the major disease of coconut associated with 16SrXIV phytoplasma group in Malaysia (1). Symptoms consistent with CYD, such as severe chlorosis, stunting, general decline, and death were observed in foxtail palms from the state of Selangor in Malaysia, indicating putative phytoplasma infection. Symptomatic trees loses their green and vivid appearance as a decorative and landscape ornament. To determine the presence of phytoplasma, samples were collected from the fronds of 12 symptomatic and four asymptomatic palms in September 2012, and total DNA was extracted using the CTAB method (3). Phytoplasma DNA was detected in eight symptomatic palms using nested PCR with universal phytoplasma 16S rDNA primer pairs, P1/P7 followed by R16F2n/R16R2 (2). Amplicons (1.2 kb in length) were generated from symptomatic foxtail palms but not from symptomless plants. Phytoplasma 16S rDNAs were cloned using a TOPO TA cloning kit (Invitrogen). Several white colonies from rDNA PCR products amplified from one sample with R16F2n/R16R2 were sequenced. Phytoplasma 16S rDNA gene sequences from single symptomatic foxtail palms showed 99% homology with a phytoplasma that causes Bermuda grass white leaf (AF248961) and coconut yellow decline (EU636906), which are both members of the 16SrXIV ‘Candidatus Phytoplasma cynodontis’ group. The sequences also showed 99% sequence identity with the onion yellows phytoplasma, OY-M strain, (NR074811), from the ‘Candidatus Phytoplasma asteris’ 16SrI-B subgroup. Sequences were deposited in the NCBI GenBank database (Accession Nos. KC751560 and KC751561). Restriction fragment length polymorphism (RFLP) analysis was done on nested PCR products produced with the primer pair R16F2n/R16R2. Amplified products were digested separately with AluI, HhaI, RsaI, and EcoRI restriction enzymes based on manufacturer's specifications. RFLP analysis of 16S rRNA gene sequences from symptomatic plants revealed two distinct profiles belonging to groups 16SrXIV and 16SrI with majority of the 16SrXIV group. RFLP results independently corroborated the findings from DNA sequencing. Additional virtual patterns were obtained by iPhyclassifier software (4). Actual and virtual patterns yielded identical profiles, similar to the reference patterns for the 16SrXIV-A and 16SrI-B subgroups. Both the sequence and RFLP results indicated that symptoms in infected foxtail palms were associated with two distinct phytoplasma species in Malaysia. These phytoplasmas, which are members of two different taxonomic groups, were found in symptomatic palms. Our results revealed that popular evergreen foxtail palms are susceptible to and severely affected by phytoplasma. To our knowledge, this is the first report of a mixed infection of a single host, Wodyetia bifurcata, by two different phytoplasma species, Candidatus Phytoplasma cynodontis and Candidatus Phytoplasma asteris, in Malaysia. References: (1) N. Nejat et al. Plant Pathol. 58:1152, 2009. (2) N. Nejat et al. Plant Pathol. J. 9:101, 2010. (3) Y. P. Zhang et al. J. Virol. Meth. 71:45, 1998. (4) Y. Zhao et al. Int. J. Syst. Evol. Microbiol. 59:2582, 2009.

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 L.) plants with symptoms resembling those associated with viral infection were observed in commercial fields in the Department of El-Paraíso, Honduras. Symptoms on affected plants included apical leaf curling and stunting, overall chlorosis and plant stunting, young plant deformation with cabbage-like leaves, wilting, and internal vascular necrosis of stems and leaf petioles. All cultivars grown were affected, with disease incidence ranging from 5 to 80% of symptomatic plants per field. The fields were also heavily infested with the psyllid Bactericera cockerelli. This psyllid is a serious pest of solanaceous crops in the United States, Mexico, Central America, and New Zealand and has been shown to transmit the bacterium “Candidatus Liberibacter solanacearum” to potato, tomato, and other solanaceous species (2,3). Tobacco (cv. Habano criollo) plant samples were collected from one field in the municipality of Trojes. Initial testing of the plant samples for viruses, including Tobacco mosaic virus, Impatiens necrotic spot virus, Cucumber mosaic virus, and Potato virus Y, using Immunostrips (Agdia, Elkhart, IN) were negative. Total DNA was then extracted from leaf tissues of a total of 13 plants, including eight symptomatic plants and five asymptomatic plants with the cetyltrimethylammonium bromide (CTAB) buffer extraction method (2,4). The DNA samples were tested by PCR using specific PCR primer pairs OA2/OI2c and OMB 1482f/2086r, to amplify a portion of 16S rDNA and the outer membrane protein (OMB) gene of “Ca. L. solanacearum,” respectively (2). All eight (100%) symptomatic plant samples were positive for “Ca. L. solanacearum” with both sets of primer pairs. “Ca. L. solanacearum” was not detected in the asymptomatic plants. The 16S rDNA and OMB gene amplicons of two plant samples each were cloned and four clones of each of the four amplicons were sequenced. BLASTn analysis of the consensus sequences confirmed “Ca. L. solanaeacrum” in the tobacco samples. The 16S rDNA consensus sequences (1,168 bp) of all amplicons were identical and showed 100% identity with several 16S rDNA sequences of “Ca. L. solanacearum” in GenBank (e.g., Accession Nos. HM245242, JF811596, and JX559779). The consensus sequence of the OMB amplicon (605 bp) showed 97 to 100% homology with a number of “Ca. L. solanacearum” OMB sequences in GenBank, including Accession Nos. CP002371, FJ914617, JN848754 and JN848752. The tobacco-associated consensus 16S rDNA and OMB sequences from this study were deposited in GenBank as Accession Nos. KC768320 and KC768328, respectively. To our knowledge, this is the first report of “Ca. L. solanacearum” associated with tobacco in Honduras, where this cash crop is economically important. This bacterium has also caused millions of dollars in losses to potato, tomato, and several other solanaceous crops in North and Central America and New Zealand, particularly in regions where B. cockerelli is present (3). Furthermore, “Ca. L. solanacearum” has caused significant economic damage to carrot crops in Europe, where it is transmitted by the psyllids Trioza apicalis in northern Europe (4) and B. trigonica in the Mediterranean region (1). 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.

Based on an earlier survey of putative psyllid vectors of apple proliferation (AP), carried out in 2009 and 2010, Cacopsylla picta (Förster) populations infected with ‘Candidatus Phytoplasma mali’ were detected in at least two commercial apple (Malus domestica Borkh.) orchards in southern Finland (1). To establish the presence of ‘Ca. P. mali’ in apple trees, a survey was conducted in 17 commercial apple orchards in August 2012. Phytosanitary inspectors tracked the source of the ‘Ca. P. mali’ by collecting 33 leaf samples from trees showing probable symptoms. Typical symptoms, including elongated stipules and witches' broom, were rare. Total DNA was extracted from leaves using a DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) and screened for ‘Ca. P. mali’ with real-time PCR (2) and the commercial Apple Proliferation Group – complete PCR reaction kit (Loewe Biochemica GmbH, Sauerlach, Germany). Two samples tested positive and results were confirmed with TaqMan PCR and conventional PCR assays and DNA sequencing in the Food and Environment Research Agency (Fera), in the United Kingdom. One positive sample was taken from an orchard in Lohja, southern Finland, where high ‘Ca. P. mali’ incidence in overwintered C. picta was observed in 2010 (1). ‘Ca. P. mali’ was found in a >40-year-old ‘Red Melba’ tree with witches' broom but without elongated stipule symptoms. The other positive sample was collected from an orchard in the Aland Islands, where the infected ‘Lobo’ tree showed symptoms of elongated stipules. This orchard was not monitored for AP vectors. No small fruit symptoms were noted by inspectors or growers in either of the orchards. The positive samples were further analyzed for subtypes using PCR/RFLP and primers AP13/AP10 (3). The amplicons (776 bp) were sequenced and digested with HincII and BspHI (New England BioLabs Inc., Ipswich, MA) following manufacturer's instructions. Both samples proved to be apple proliferation subtypes AT-1 on the basis of RFLP and the sequenced 776-bp region. Sequences of the 776-bp amplicon of the Lohja and Aland isolates showed 100% and 99% identity, respectively, with sequences of apple proliferation isolates (accession nos. L22217.1 and CU469464.1) in GenBank. Both suspected psyllid vectors of ‘Ca. P. mali’ C. picta and C. melanoneura (Förster) occur in Finland, but their distribution, abundance, and transmission specificity is inadequately documented. The next step to evaluate the risk of spread of apple proliferation in commercial orchards is an extensive survey of the occurrence of Cacopsylla species infected with ‘Ca. P. mali’. References: (1) A. Lemmetty et al. B. Insectol. 64:257, 2011. (2) P. Nikolić et al. Mol. Cell. Probes. 24:303, 2010. (3) W. Jarausch et al. Mol. Cell. Probes 14:17, 2000.

Tomato (Lycopersicum esculentum) crops grown in several departments of Honduras and heavily infested with the psyllid Bactericera cockerelli were observed in April of 2012 with plant symptoms suggestive of “Candidatus Liberibacter solanacearum” infection. B. cockerelli is a serious pest of potato, tomato, and other solanaceous plants and a vector of “Ca. L. solanacearum” (1,2,3,4). The symptoms included overall chlorosis, severe stunting, leaf cupping, excessive branching of axillary shoots, and leaf purpling and scorching (2,3). Disease incidence ranged from 5 to 50% symptomatic plants per field. Tomato (cv. Pony) plant samples were collected from two psyllid-infested commercial fields in the municipalities of Danli and Comayagua in the departments of El-Paraiso and Comayagua, respectively. Total DNA was extracted from leaf tissues of 50 and 20 symptomatic and asymptomatic plants, respectively, with the cetyltrimethylammonium bromide (CTAB) buffer extraction method (1,3). The DNA samples were tested for “Ca. L. solanacearum” by PCR with primer pairs specific for 16S rDNA (OA2 and OI2c) and the outer membrane protein gene (OMB 1482f and 2086r) of the bacterium (1,2). Ten (20%) of the 50 symptomatic tomato samples were positive for “Ca. L. solanacearum” using both primer pairs and the remaining samples were negative for the bacterium with both primer sets. None of the 20 asymptomatic plants tested positive for “Ca. L. solanacearum”. Amplicons from DNA of two plant samples (one plant/municipality) 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 (deposited in GenBank as Accession Nos. KC768321 and KC768322) were identical for both locations and showed 99 to 100% identity to several “Ca. L. solanacearum” sequences in GenBank (e.g., JN848753, JN84856, and HM246509). The OMB consensus sequences from the two tomato plants (deposited in GenBank as KC768329 and KC768330) were 100% identical to OMB sequences of Lso in GenBank (CP002371 and JN48754, respectively). To our knowledge, this is the first report of “Ca. Liberibacter solanacearum” associated with tomato crops in Honduras. This bacterium has caused millions of dollars in losses to the tomato industry in the United States, Mexico, and New Zealand (2,3,4). Serious damages to tomato crops due to “Ca. L. solanacearum” could expand throughout Central America, especially in those countries where B. cockerelli occurs. 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) J. E. Munyaneza. Am. J. Pot. Res. 89:329, 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 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.