Progress 02/05/99 to 09/30/03
Outputs
Spread of coconut lethal yellowing (LY) disease continues among coconut palm populations along the Atlantic coast of Central America. In addition to southern Mexico, Belize and Honduras, presence of LY was most recently confirmed in Guatemala. LY-type diseases were also identified for the first time affecting coconut production in the states of Guerrero and Oaxaca on the Pacific coast of Mexico and were also responsible for a fatal decline of mature, ornamental Canary Island date palms in Corpus Christi, Texas. Characterization by phylogenetic analysis of 16S rRNA gene sequences PCR-amplified from phytoplasmas determined that the associated strains detected in dying palms in southwestern Mexico differed from those strains infecting Texas Phoenix palms. Collectively, strains at both locations were very similar to, albeit distinct from, the LY agent and were classified subsequently as new members of the coconut lethal yellows (16SrIV) phytoplasma group. These findings
provide important insights into strain variation among LY group phytoplasmas whose plant host range is limited to palms (Arecaceae) and the palm like aborescent monocots Pandanus utilis (Pandanceae) and Carludovica palmata (Cyclanthaceae) only. RFLP analysis of PCR-amplified rRNA operon sequences used as a basis for comparing phytoplasmas revealed differences among strains associated with LY disease of coconut in Florida, Mexico Honduras and Jamaica and was attributed to rRNA interopeon sequence heterogenity. Strains examined in Florida, Mexico and Honduras all possessed this attribute whereas Jamaica strains uniformly lacked this attibute. However, this distinguishing genetic characteristic could not be linked to unexpected widespread losses to LY of resistant Malayan dwarf and hybrid MayPan coconuts in Jamaica during the last three years as similar mortality of these cultivars has also occurred in southern Florida. An in situ PCR investigation clearly revealed phytoplasma DNA in
embryos from some fruit aborted from LY-symptomatic coconut palms thereby raising the possibility of seed transmission of LY in this palm species. However, one important limitation to phytoplasma detection by DNA-based methods such as in situ PCR is that none of these methods are capable of selectively discriminating viable from nonviable phytoplasma. Future progress toward clearly resolving the fate of phytoplasma-positive seeds will require study of a more amenable herbaceous plant pathosystem rather than an intractable woody perennial host such as coconut. Thirty nine monoclonal antibodies (Mabs) were raised against sonicated (LY) phytoplasma cells extracted from coconut tissues and purified by discontinuous Percoll density gradient centrifugation. Of these, Mabs most useful for distinguishing LY-diseased from healthy coconut extracts were identified by indirect ELISA assay of coconut trunk phloem samples. Development of a lateral flow test for rapid field diagnosis of LY disease
of palms is in progress in partnership with Pocket Diagnostics, a private sector company based in the UK.
Impacts
Development of a sero-diagnostic assasy for rapid, practical field diagnosis of coconut lethal yellowing (LY) disease in conjunction with a detailed understanding of plant host range and genetic variation among strains of the uncultivable phytopathogenic mollicute that causes LY will facilitate new strategies for disease contol and assist those charged with long term coconut germplasm improvement and dissemination.
Publications
- Cordova, I., Jones, P., Harrison, N. A. and Orpopeza, C. 2003. In situ PCR detection of phytoplasma DNA in embryos from coconut palms with lethal yellowing disease. Mol. Plant Pathol. 4: 99-108.
- Harrison, N.A., Boa, E. and Carpio, M. L. 2003. Characterization of phytoplasmas detected in Chinaberry trees with symptoms of leaf yellowing and decline in Bolivia. Plant Pathol. 52: 147-157.
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Progress 10/01/01 to 10/01/02
Outputs
Nineteen HindIII restriction fragments of lethal yellowing (LY) phytoplasma DNA cloned in pUC19 and Escherichia coli DH5-alpha cells were chosen for sequencing based on their hybridization to probe DNA derived from LY-diseased coconut palm and lack of hybridization to DNA from healthy coconut palm. Partial or full-length sequences from clones ranging from 0.6 to 5.5 kb in size were subjected to database searches for homologous nucleotide and protein sequences on the National Center for Biotechnology Information (NCBI) network server using the BLAST program. Prediction of open reading frames (ORFs) in sequence data was obtained using ORF Finder. Collectively, cloned fragments shared little or no homology with eukaryote DNA sequences. Among sequences, partial or complete ORFs sharing significant homology with the following prokaryote genes: gyrA and gyrB; dnaK; dTMP kinase; DNA-directed RNA polymerase beta-subunit; secA; peptide release factor 2 (RF2);
cation-transporting ATPase, DNA-dependent RNA helicase, tRNA synthetases class I, as well as 50S and 30S ribosomal protein genes, were identified. As predicted, these putative genes share greatest similarity with those of gram-positive bacterial species such as Bacillus subtilis, B. halodurans and Clostridium perfringens, or with cultivable mollicutes.
Impacts
Genome sequence information will help to define strain diversity and current geographic range of the noncultivable lethal yellowing (LY) phytoplasma that is associated with a fast-spreading, fatal disease of coconut and other palms in the Americas. It is expected that this information will benefit those responsible for coconut improvement, coconut rehabilitation programs or international transfer of coconut genetic resources.
Publications
- Ebbert, M. A., Jeffers, D. P., Harrison, N. A. and Nault, L. R. 2001. Lack of specificity in the interaction between two maize stunting pathogens and field collected Dalbulus leafhoppers. Entomologia Experimentalis et Applicata 101: 49-57.
- Harrison, N. A., Womack, M., and Carpio, M. L. 2002. Detection and characterization of a lethal yellowing (16SrIV) group phytoplasma in Canary Island date palms affected by lethal decline disease in Texas. Plant Disease. 86: 676-681.
- Harrison, N. A., Oropeza, C., Cordova, I., and Carpio, M. L. 2002. First report of group 16SrIV phytoplasmas infecting coconut palms with leaf yellowing symptoms on the Pacific coast of Mexico. BSPP New Disease Reports Vol. 5. http://www.bspp.org.uk/ndr/
- Harrison, N. A., Myrie, W., Jones, P., Carpio, M. L., Castillo, M., Doyle, M. M., and Oropeza, C. 2002.16SrRNA interoperon sequence heterogeneity distinguishes strain populations of palm lethal yellowing phytoplasma in the Caribbean region. Annals of Applied Biology 141: 183-193.
- Broschat, T., Harrison, N. A., and Donselman, H. 2002. Losses to lethal yellowing cast doubt on coconut cultivar resistance. Palms 46: 185-189.
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Progress 10/01/00 to 10/01/01
Outputs
Polymerase chain reaction (PCR) assays employing phytoplasma universal rRNA primer pair P1/P7 were used to detect phytoplasmas in 10 of 11 declining Canary Island date (Phoenix canariensis) palms displaying symptoms similar to coconut lethal yellowing disease in coastal southern Texas. AluI endonuclease digests and sequencing of P1/P7 products revealed that nontarget Bacillus-megaterium-related rDNA sequences of similar size were co-amplified along with phytoplasma rDNA from 10 palms. Phytoplasma rDNA products were selectively obtained from all 11 symptomatic palms when initial P1/P7 products were reamplified by PCR employing nested 16S rRNA primer pair LY16Sf/LY16Sr. Restriction site analysis of nested products revealed that palm-infecting phytoplasmas were uniform and most similar to strains composing the coconut lethal yellowing phytoplasma (16SrIV) group. Analysis of 16SrDNA sequences determined the Texas Phoenix palm decline (TPD) phytoplasma to be
phylogenetically closest to the Carludovica palmata leaf yellowing (CPY) phytoplasma. rDNA profiles of strains TPD and CPY obtained with AluI were co-identical and distinct from other known 16SrIV group phytoplasmas. On this basis, both strains were classified as members of a new subgroup, 16SrIV-D. The majority (>80%) of all mature, apparently symptomless Virginia creeper (Parthenocissus quinquefolia) plants randomly sampled on a 100 acre site in subtropical southeastern Florida harbored phytoplasmas following their detection in plants by polymerase chain reaction (PCR) assays employing phytoplasma-specific rRNA primer pairs. Detected phytoplasmas characterized by restriction site and sequence analysis of 16S rDNA amplification products were uniform and identified as members of the elm yellows (16SrV) phytoplasma group. Phylogenetic classification based on 16S rDNA sequences indicated Virginia creeper-inhabiting phytoplasmas to be most similar to European alder yellows (ALY), an
established 16SrV-C subgroup phytoplasma
Impacts
Specific etiological information will help define the current geographic range of lethal yellowing (LY), a fast-spreading, fatal disease of palms that poses the most importnat threat to coconut production worldwide. It is expected that this information will benefit those charged with coconut improvement and rehabilitation programs or international exchange of coconut genetic resources.
Publications
- Harrison, N. A., Griffiths, H. M., Carpio, M. L. and Richardson, P. A. 2001. Detection and characterization of an elm yellows (16SrV) group phytoplasma infecting Virginia creeper plants in southern Florida. Plant Disease 85: 1055-1062.
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Progress 10/01/99 to 09/30/00
Outputs
Recent increased incidence of coconut lethal yellowing (LY) disease amongst resistant Malayan dwarf ecotype and hybrid Maypan coconut palms in Jamaica could not be attributed to any demonstrable change in the causal LY phytoplasma. Variability amongst strains of the LY phytoplasma was assessed by HinfI restriction endonuclease digestion of a portion (1.8 kb) of the rRNA operon selectively amplified from DNAs of diseased palms by the polymerase chain reaction (PCR) employing phytoplasma-specific rRNA primer pair P1/P7. Resulting rDNA RFLP fragment patterns were uniform for strains infecting all 30 diseased coconut palms that were examined. Patterns also distinguished Jamaican LY phytoplasmas from strains associated with LY in Florida and other western Caribbean localities, as has been previously noted. Phytoplasma infestation of embryos recovered from aborted seed of LY-diseased coconut palms was shown definitively by in situ PCR methodology. Amplifications
incorporating DIG-11-dUTP and phytoplasma-specific rRNA primer pair P1/P7 were performed on serial sections of excised embryos harvested from fruit of healthy and diseased coconut palms. Following application of anti-DIG-AP conjugate and chromogenic substrate, discrete accumulations of DIG-11-dUTP were detected in several embryos from diseased palms only. Staining appeared to be confined to a region of the embryo containing the plumule (embryonic shoot). In situ PCR analysis employing LY-phytoplasma-specific nonribosomal primer pair LYF1/R1 was repeated on tissues from all positively testing embryos. Assay results mirrored those obtained previously by P1/P7-primed PCR. Similarly, accumulations of DIG-11-dUTP were confined to phloem sieve tubes in sections of immature leaf bases from an LY-diseased coconut palm whereas no staining of comparable tissues from a healthy coconut palm was apparent. These findings suggests that seed transmission of the coconut LY phytoplasma may be possible.
In vitro germination of coconut embryos to obtain plantlets to be analyzed for phytoplasma infection is presently being attempted.
Impacts
Specific etiological information will help to define the current geographic range of lethal yellowing (LY), a fast-spreading, fatal disease of palms that poses the most important threat to coconut production worldwide. It is expected that this information will benefit those charged with coconut improvement and rehabilitation programs or international transfer of coconut genetic resources.
Publications
- Cordova, I., Oropeza, C., Almeyda, H. and Harrison, N. A. 2000. First report of a phytoplasma-associated leaf-yellowing syndrome of Palma Jipi plants in southern Mexico. Plant Disease 84: 807.
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Progress 10/01/98 to 09/30/99
Outputs
Coconut palm-associated isolates of the palm lethal yellowing (LY) phytoplasma from six locations within the Caribbean region were compared by RFLP analysis of a 1.8 kb rDNA product amplified from palm DNA samples by PCR employing phytoplasma-specific primers P1 and P7. RFLP patterns generated by separate digestion of P1/P7 products with each of 14 endonucleases revealed no differences between the majority of LY phytoplasma isolates from these locations. However, HinfI restriction fragment patterns for Jamaican isolates (type I pattern) differentiated these isolates from all remaining LY isolates collected in Belize, Florida, Honduras and southeastern Mexico (type II). Both type patterns were represented among isolates from Dominican Republic. Cloning, RFLP and sequence analysis of P1/P7-primed rDNA amplimers showed that more complex type II restriction patterns were a product of sequence heterogeneity between two rRNA operons present within LY phytoplasma isolates.
These data provide the first evidence of variation among isolates although the biological significance of such variation remains to be determined. The association of phytoplasmas with coconut palms displaying foliar yellowing symptoms in LY disease-free southwestern states of Guerrero and Oaxaca, Mexico was confirmed by P1/P7-primed PCR. RFLP analysis of P1/P7 products revealed patterns distinct from those of the LY phytoplasma thereby alleviating concerns that LY disease had spread to these states, the central region of coconut production in Mexico. Comparison of rDNA RFLP patterns with those of other known phytoplasmas identified these newly detected isolates as similar, or identical, to coconut lethal decline (LDY), a phytoplasma of unknown epidemiological significance first reported from the state of Tabasco, southeastern Mexico. According to recently proposed classification schemes, the LDY phytoplasma is phylogenetically related to LY and an additional member of 16S rDNA RFLP
group, 16SrIV (coconut lethal yellows group). Among six putative LDY phytoplasma isolates that were examined in detail, two identical variants were detected that did not conform precisely to the LDY type strain based on their respective AluI RFLP patterns. A subsequent analysis of cloned P1/P7 products revealed the presence of two rRNA operons in each variant that differed by a single AluI restriction site whereas rDNA sequences cloned from the LDY type strain lacked this singular dissimilarity.
Impacts
Specific etiological information will help to define the current geographic range of lethal yellowing (LY), a fast-spreading, fatal disease of palms that poses the most important threat to coconut production worldwide. It is expected that this information will benefit those charged with coconut improvement and rehabilitation programs or international transfer of coconut genetic resources.
Publications
- No publications reported this period
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