Progress 09/01/09 to 08/31/12
Outputs
OUTPUTS: Following the first outbreak of pineapple heart rot disease on the island of Oahu, repeated surveys were conducted on the islands of Oahu and Maui. Bacteria were isolated from infected pineapples, irrigation channels, and reservoirs. All Hawaiian pineapple strains were identified using biochemical and molecular methods. Strains were further characterized genetically to determine relationships to reference strains from other hosts and diseased pineapple in Malaysia. Genetic relationships were evaluated using multilocus sequence analysis and DNA-DNA hybridization data. Genetic markers of the Hawaiian pineapple strains were compared to strains isolated from infected plants imported from Costa Rica, Honduras, and the Philippines to determine the possible origin of the exotic pathogen. Rapid field tests were developed to identify the pathogen directly from field samples. Two Dickeya-specific monoclonal antibodies that reacted specifically with the pineapple pathogen were generated and an immunodiagnostic assay was developed to identify new isolates from field samples. Later, a genomic method based on sequence analysis of the dnaA gene was developed. Both methods clearly identified the pineapple pathogen and distinguished it from nonpathogenic Dickeya and/or Erwinia strains from plants, soil and water. Meetings were held with pineapple producers, Hawaii Department of Agriculture regulatory personnel, Plant Quarantine, and APHIS personnel. As a service to the Hawaii State Department of Agriculture we surveyed pineapple fields on two islands to determine the extent and spread of the disease. As products, two hybridoma cell lines were generated that produce monoclonal antibodies specific for the pineapple heart rot pathogen. Another product was development of a database for dnaA sequence information. Scientists may post a sequence of an unknown and determine relatedness to previously sequenced Dickeya strains, thus resolving their identity. Students who assisted with sample collection, conducting experiments, and data analysis were mentored during the year and three students graduated with a M.S. degrees. Research results were presented at the annual CTAHR Student Research Symposium and at annual meetings of the American Phytopathological Society, August, 2010. Three M.S. level students have graduated based on research work accomplished in this program. Two have gone on to graduate work at the Ph.D. level and one is preparing for a Doctorate Degree in Plant Medicine at the University of Florida. Results were disseminated at public presentations of MS Thesis research at two college-wide thesis defense seminars (Glori Marrero and Van Luu) and one departmental seminar (Gabriel Peckham). Updates on pineapple heart disease were presented to Pineapple Industry representatives in meetings and via the Pineapple Newsletter. Discussions with regulatory officials Hawaii Department of Agriculture, USDA-APHIS occurred at regular intervals. Publications that advanced knowledge of the distribution and spread of pineapple heart rot disease were distributed. PARTICIPANTS: The PI was Anne Alvarez, Plant Pathologist. Wendy Kaneshiro was a technical assistant who was involved in all sample collection and processing along with a second technical assistant, Asoka de Silva. Both did all the initial bacteriological and pathogenicity tests and initial genetic analysis using rep-PCR. Glorimar Marrero was a graduate student who genetic characterized all the strains using multilocus sequence analysis. Kevin Schneider developed a dna marker for rapid characterization of Dickeya strains. Dr. Gabriel Peckham was a post-doctoral researcher who generated hybridoma cell lines in the laboratory of Dr. John Berestecky, an instructor at Kapiolani Community College. Van Luu was a MS graduate student who assisted with the development of a rapid immunodiagnostic test for identifying Dickeya in field samples. Dr. Paul Patek was a colleague from the Department of Microbiology who gave assistance and provided funds for one of the graduate students, van Luu. Gernot Presting is a colleague in the Department of Molecular Biology and Bioengineering who provided guidance of two students, Glori Marrero and Kevin Schneider with bioinformatic approaches. Partner Organizations who collaborated in the project: the Hawaii Department of Agriculture brought samples for analysis and gave guidance on regulatory issues. APHIS personnel were involved in the initial inspections of fields and set regulations that required further surveys. Directors from the two local pineapple companies provided trucks, transportation, and personnel for field surveys. The companies also provided healthy pineapple shoots for pathogenicity testing. All collaborators and agencies called frequently for updates on the spread of the disease in Hawaii and followed the epidemiological information provided by the genetic analysis of pathogenic bacterial strains. . Collaborators and contacts Lyle Wong, HDOA, consulted frequently on the status of the surveys. We also had frequent calls from other HDOA (Amy Takahashi) and APHIS personnel (Yolisa Ishibashi, Dorothy Alantoga) and pineapple industry representatives. Glenn Taniguchi, staff member in the Department of Plant and Environmental Protection Sciences, gave frequent guidance on the status of the epidemic from a field perspective. He is now helping us find collaborators outside the United States who may be able to provide bacterial strains from other pineapple heart rot epidemics worldwide. Training or professional development: Students Glori Marrero and Van Luu were trained in disease recognition, isolation and characterization of bacterial pathogens. Dr. Gabriel Peckham, the postdoctoral researcher associated with the project received excellent training from Dr. John Berestecky in generation of hybridoma cell lines, selection strategies, and maintenance of cell lines. He Xu was a MS student in Tropical Plant Pathology who assisted with the collections and identification of bacteria and finished his MS degree requirements during this project. TARGET AUDIENCES: Pineapple producers, field managers, and agricultural laborers were all involved in this project. Most of the pineapple workers are from the Philippines and South Asia. As a result of this project, we corresponded with colleagues in the Philippines, Costa Rica, and Malasia who discussed implications of latent infections, regulation, and detection. Some colleagues were able to provide strains for genetic comparisons. The results of the project affected these communities because the pineapple industry in Hawaii is a major source of labor and income to these groups. Efforts to deliver science-based knowledge to workers and plantation managers included formal talks at the plantation office houses as well as informal talks with workers and managers as we did the field surveys in plantation trucks. Two talks were given at college-sponsored workshops. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Biology and etiology of the pineapple heart rot disease was significantly improved during this project. The bacterial pathogen from Hawaii, originally called Erwinia chrysanthemi, was thoroughly characterized by biochemical tests, molecular assays and DNA-DNA hybridization data and now is classified as Dickeya zeae. Pineapple strains are unique, however, and they were placed into a separate subgroup from D. zeae strains previously found in water, soil and ornamentals. Hawaiian pineapple strains further formed two clearly separate groups, each with distinct DNA fingerprints and different reaction patterns with Dickeya-specific monoclonal antibodies. Strains associated with pattern A were initially less virulent than strains associated with patterns B and C, and the A strains were not encountered in later surveys. The A, B, and C types were originally associated only with the planting stocks received from Central America. In subsequent years, C types were also found in drainage ditches bordering affected fields and in a reservoir used for irrigation. New strains (D and E types) later appeared in pineapple fields and irrigation water. Dickeya zeae was identified in an outbreak of bacterial stalk rot of corn (maize) on Oahu, and the corn strains were genetically similar to the C-type pineapple strains. Two pineapple strains from Malaysia and four strains from the Philippines were similar to C-type strains found in Hawaii. For the first time it is now possible to identify the pineapple pathogen with a few genetic markers and to determine its closest relatives in a world-wide collection of Dickeya strains. Our specific monoclonal antibody will distinguish the pineapple pathogen from contaminants and other Dickeya species. New knowledge generated about disease spread from infected sites in Hawaii has impacted plant quarantine regulations as well as industry practices. No pineapple heart rot disease was found in Maui following four extensive surveys of entire plantations. As a result, commercial plantations on Maui were declared disease-free. Movement of planting stocks from Oahu to Maui was prohibited and the disease outbreak was contained on Oahu. Results will eventually impact on the pineapple industries in the Philippines where the disease is prevalent and measures to control the disease are enhanced by the ability to rapidly identify and destroy the major sources of infected materials. The methods and products generated in this study will enable scientists in pineapple growing countries to answer epidemiological questions. The discovery that Dickeya strains genetically similar to the pineapple pathogen was found on ornamentals, corn and taro on Oahu could have major impact on taro distribution to other islands and could impact the future expansion of the corn seed industry in Hawaii. The impact of pineapple heart rot disease on local pineapple production was immense because of the regulatory issues that followed the first discovery of the disease in Hawaii.
Publications
- Marrero, G., Schneider, K.L., Jenkins, D.M., and Alvarez, A.M. 2012. Phylogeny and classification of Dickeya based on multilocus sequence analysis. International Journal of Systematic Evolutionary Microbiology (accepted).
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Progress 09/01/10 to 08/31/11
Outputs
OUTPUTS: Surveys of pineapple fields for the heart rot pathogen were continued in 2010 and 2011. Additional bacterial strains were isolated, characterized with bacteriological tests. Strains were genetically characterized using multi-locus sequence analysis and the positions of representative strains in a phylogenetic tree were compared with reference strains of six Dickeya sp. Pathogenicity tests were undertaken at different temperatures in both laboratory and greenhouse environments. Students who assisted with sample collection, conducting experiments, and data analysis were mentored during the year and three students graduated with a M.S. degrees. Events: research results were presented at the CTAHR Student Research Symposium, April, 2011, and at the annual meetings of the American Phytopathological Society, August, 2011. Services included consulting with pineapple plantation managers and supervisors, correspondence with pineapple researchers and industry representatives in the Philippines, Australia, Costa Rica. Products include development of a publically available database for phytopathogenic bacteria (the RIF database) which contains all strains that were amplified with specific RIF primers. Other products include a) phylogenetic trees showing the relationships between reference strains of Dickeya and the pineapple strains; b) a large collection of Dickeya pineapple and other reference strains; c) improved methods of genetic analysis such as multilocus sequence analysis and phylotyping based on dnaA. The genetic analysis of this genus has been a significant improvement over rep-PCR. Three M.S. level students have graduated based on research work accomplished in this program. Two have gone on to graduate work at the Ph.D. level and one is preparing for a Doctorate Degree in Plant Medicine at the University of Florida. Dissemination: An update on pineapple heart disease was presented in a newsletters to Pineapple Industry. Discussions with regulatory officials Hawaii Department of Agriculture, USDA-APHIS. Papers that advanced knowledge of the distribution and spread of pineapple heart rot disease were distributed as pdf files by internet to intended audiences. Topics were followed up with e-mail correspondence with researchers in Australia, Philippines, Costa Rica. PARTICIPANTS: Individuals who worked on the project: The PI was Anne Alvarez, Plant Pathologist. Wendy Kaneshiro was a technical assistant who was involved in all sample collection and processing along with a second technical assistant, Asoka de Silva. Both did all the initial bacteriological and pathogenicity tests and initial genetic analysis using rep-PCR. Dr. Diane Sether assisted with epidemiological studies. Glorimar Marrero was a graduate student who genetic characterized all the strains using multilocus sequence analysis. Kevin Schneider developed a dna marker for rapid characterization of Dickeya strains. Dr. Gabriel Peckham was a post-doctoral researcher who generated hybridoma cell lines in the laboratory of Dr. John Berestecky, an instructor at Kapiolani Community College. Van Luu was a MS graduate student who assisted with the development of a rapid immunodiagnostic test for identifying Dickeya in field samples. Dr. Paul Patek was a colleague from the Department of Microbiology who gave assistance and provided funds for one of the graduate students, van Luu. Gernot Presting is a colleague in the Department of Molecular Biology and Bioengineering who provided guidance of two students, Glori Marrero and Kevin Schneider with bioinformatic approaches. An additional MS student, He Xu, assisted with field collections and isolation. He completed the MS degree and proceeded to the Doctor of Plant Medicine program at the University of Florida. TARGET AUDIENCES: Pineapple producers, field managers, and agricultural laborers were all involved in this project. Most of the pineapple workers are from the Philippines and South Asia. The results of the project affected these communities because the pineapple industry in Hawaii is a major source of labor and income to these groups. PROJECT MODIFICATIONS: We have expanded our surveys for the pineapple heart rot pathogen to corn, ornamentals and taro, since these were local hosts that harbored strains genetically similar to the strains originally found on pineapple.
Impacts
Now that we have attributed the pineapple disease to a new species and have genetic markers to classify strains collected over a period of seven years, it is possible to trace the spread of the disease from sites of initial infection and to other possible hosts in Hawaii. The pineapple heart rot pathogen, originally called Erwinia chrysanthemi, was thoroughly characterized and identified as a new species of Dickeya based on phylogenetic analysis and biochemical tests. For the first time it is now possible to identify the pineapple pathogen with a few genetic markers and to determine its closest relatives in a world-wide collection of strains. Our specific monoclonal antibody will distinguish the pineapple pathogen from contaminants and other Dickeya species in field setting.In Hawaii, the new knowledge generated about disease spread from infected sites has impacted plant quarantine regulations as well as industry practices. Based on results of a thorough disease survey on Maui and Oahu, commercial plantations on Maui were declared disease-free. Dickeya strains with a genotype identical to Oahu strains was detected in irrigation channels but no disease was observed in commercial plantings. On this basis, movement of planting stocks from Oahu to Maui was prohibited and the disease outbreak has been restricted to Oahu. The results will eventually have impact on the pineapple industries in the Philippines where the disease is prevalent and measures to control the disease are enhanced by the ability to rapidly identify and destroy the major sources of infected materials. The methods we have generated will enable scientists in pineapple growing countries to answer epidemiological questions. Dickeya sp. with the same genetic profile as the pineapple pathogen was isolated from ornamentals, taro and corn on Oahu. This discovery could have major impact on taro distribution to other islands and could impact the future expansion of the corn and taro industries in Hawaii.
Publications
- Marrero, G., and Alvarez, A. M. 2011. Hawaiian strains of Erwinia chrysanthemi (Dickeya sp.) associated with pineapple heart rot disease. Pineapple News. 18:57-59.
- Marrero, G., Schneider, K., Alvarez, A. 2008. Relationships of bacterial strains causing heart rot of pineapple to Dickeya species based on 16S-23S intergenic spacer and dnaA sequences. Phytopathology 98:S98
- Marrero, G., Schneider, K.L., Alvarez, A.M. 2009. Relationships between Dickeya species and heart rot of pineapple based on sequence comparison of dnaJ, gyrB, dnaA, and recN genes. Phytopathology 99:S80
- Schneider, K.L., Marrero, G., Alvarez, A.M., and Presting, G.G. 2011. Rapid identification of plant pathogenic bacteria below the species level using a computationally derived DNA marker. PLoS One 6:e18496. doi: 10.1371/journal.pone.0018496 (on-line publication).
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Progress 09/01/09 to 08/31/10
Outputs
OUTPUTS: Bacterial strains were collected from infected pineapples from repeated surveys on the islands of Oahu and Maui. Phenotypic and genetic traits of pineapple strains were compared with strains from other hosts and with reference strains isolated from diseased pineapple in Malaysia. Genetic traits were compared with reference strains from well-characterized Dickeya species to determine their genotypic relationships. We are now comparing genetic traits of the Hawaiian pineapple strains to strains isolated from infected plants imported from Costa Rica, Honduras, and the Philippines to determine the possible origin of the exotic pathogen in Hawaii; ii) We have generated two Dickeya-specific monoclonal antibodies that react specifically with the pineapple pathogen. The pathogens fell into two serological groups, which also were identified using data obtained with DNA-fingerprinting analysis of the same strains. A rapid immunodiagnostic assay was developed to identify new isolates from field samples and a genomic method based on sequence analysis of the dnaA gene was developed. Both methods clearly identify the pineapple pathogen. Meetings with pineapple producers, Hawaii Department of Agriculture regulatory personnel, Plant Quarantine, and APHIS personnel. As a service to the State of Hawaii we surveyed pineapple fields on two islands to determine the extent and spread of the disease. As products, two hybridoma cell lines were generated that produce monoclonal antibodies specific for the pineapple heart rot pathogen. Results were disseminated at public presentations of MS Thesis research at two college-wide thesis defense seminars (Glori Marrero and Van Luu) and one departmental seminar (Gabriel Peckham). PARTICIPANTS: Individuals who worked on the project: The PI was Anne Alvarez, Plant Pathologist. Wendy Kaneshiro was a technical assistant who was involved in all sample collection and processing along with a second technical assistant, Asoka de Silva. Both did all the initial bacteriological and pathogenicity tests and initial genetic analysis using rep-PCR. Glorimar Marrero was a graduate student who genetic characterized all the strains using multilocus sequence analysis. Kevin Schneider developed a dna marker for rapid characterization of Dickeya strains. Dr. Gabriel Peckham was a post-doctoral researcher who generated hybridoma cell lines in the laboratory of Dr. John Berestecky, an instructor at Kapiolani Community College. Van Luu was a MS graduate student who assisted with the development of a rapid immunodiagnostic test for identifying Dickeya in field samples. Dr. Paul Patek was a colleague from the Department of Microbiology who gave assistance and provided funds for one of the graduate students, van Luu. Gernot Presting is a colleague in the Department of Molecular Biology and Bioengineering who provided guidance of two students, Glori Marrero and Kevin Schneider with bioinformatic approaches. Partner Organizations who collaborated in the project: the Hawaii Department of Agriculture brought samples for analysis and gave guidance on regulatory issues. APHIS personnel were involved in the initial inspections of fields and set regulations that required further surveys. Directors from the two local pineapple companies provided trucks, transportation, and personnel for field surveys. The companies also provided healthy pineapple shoots for pathogenicity testing. All collaborators and agencies called frequently for updates on the spread of the disease in Hawaii and followed the epidemiological information provided by the genetic analysis of pathogenic bacterial strains. . Collaborators and contacts Lyle Wong, HDOA, consulted frequently on the status of the surveys. We also had frequent calls from other HDOA (Amy Takahashi) and APHIS personnel (Yolisa Ishibashi, Dorothy Alantoga) and pineapple industry representatives. Glenn Taniguchi, staff member in the Department of Plant and Environmental Protection Sciences, gave frequent guidance on the status of the epidemic from a field perspective. He is now helping us find collaborators outside the United States who may be able to provide bacterial strains from other pineapple heart rot epidemics worldwide. Training or professional development: Students Glori Marrero and Van Luu were trained in disease recognition, isolation and characterization of bacterial pathogens. Dr. Gabriel Peckham, the postdoctoral researcher associated with the project received excellent training from Dr. John Berestecky in generation of hybridoma cell lines, selection strategies, and maintenance of cell lines. TARGET AUDIENCES: Target audiences: Pineapple producers, field managers, and agricultural laborers were all involved in this project. Most of the pineapple workers are from the Philippines and South Asia. The results of the project affected these communities because the pineapple industry in Hawaii is a major source of labor and income to these groups. Efforts to deliver science-based knowledge to workers and plantation managers included formal talks at the plantation office houses as well as informal talks with workers and managers as we did the field surveys in plantation trucks. Two talks were given at college-sponsored workshops. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Biology of the pineapple heart rot disease was significantly improved during this project: Unexpectedly, the pineapple pathogen in Hawaii formed two clearly separate groups, each with distinct DNA fingerprints and different reactivities with Dickeya-specific monoclonal antibodies. Strains associated with pattern A were initially less virulent than strains associated with patterns B and C and the A strains were not encountered in later surveys. Initially, A, B, and C types were associated only with the planting stocks received from Central America. Later, C types were also found in drainage ditches bordering affected fields and in a reservoir used for irrigation. Later, new strains (D and E types) appeared in pineapple fields and irrigation water. No pineapple heart rot disease was found in Maui following four extensive surveys of entire plantations. A multi-locus sequence analysis performed on all pineapple heart rot strains as well as reference strains from six Dickeya species revealed that the bacteria associated with the heart rot disease in Hawaii are unique and should be placed into a new species of Dickeya. Changing conditions for industry resulted in changes in industry approach to the disease. The impact of pineapple heart rot disease on local pineapple production was immense because of the regulatory issues that followed the first discovery of the disease in Hawaii. The disease outbreak resulted in immediate state and federal restrictions on movement of planting materials and fruits. As a result of our island-wide surveys on the islands of Oahu and Maui, the state regulatory officer made decisions on further interisland exchange of planting materials between these two islands.
Publications
- Peckham, GD, Kaneshiro WS, Luu V, Berestecky JM, and Alvarez AM. 2010. Specificity of monoclonal antibodies to strains of Dickeya sp.that cause bacterial heart rot of pineapple. Hybridoma 29:383-389.
- Marrero G, Kaneshiro-Sueno W, de Silva AS and Alvarez AM. 2010. Insights into the introduction of bacterial heart rot of pineapple to Hawaiian plantations on the basis of molecular and biochemical analyses. Phytopathology 100:S78.
- Peckham, G.D., Marrero, G., Kaneshiro, W.S., Luu, V.P., Berestecky, J.M., and Alvarez, A.M. 2009. Generation of monoclonal antibodies based on phylogenetic relationships of Dickeya sp. associated with pineapple heart rot disease. Phytopathology 99:S101.
- Schneider, K.L., Marrero, G., Alvarez, A., and G.G. Presting. 2009. Comparative analysis of whole bacterial genomes and derivation of RIF, a DNA identification marker for bacterial phytopathogens. Phytopathology 99:S115.
- Marrero, G. 2010. Bacterial Heart Rot of Pineapple and the genus Dickeya. M.S. Thesis, University of Hawaii, 86 pp.
- Luu, V. P. 2010. Development of a non-enzyme based capillary dipstick assay for detection and purification of environmental pathogens. M.S. Thesis, University of Hawaii, 95 pp.
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