Progress 09/15/06 to 09/14/09
Outputs
OUTPUTS: Activities: Research on detection with immunodiagnostic and molecular techniques were implemented in greenhouse and field studies on pathogen detection and survival. Potting media and soil studies were set up in greenhouses on Oahu as well as Hilo. Effects of treatments with essential oils at different concentrations (0.04, 0.07 and 0.14 % v/v) on R. solanacearum and other bacterial strains were evaluated with epifluorescence microscopy and micro-Raman spectroscopy. R. solanacearum cells treated with the plant essential oils were stained using syto9 (green) and propidium iodide (red) and observed using epifluorescence microscopy. Our group also built a greenhouse in Hilo for current and future studies on bacterial wilt of ginger. Experimental plots were set up by graduate students and our collaborators in Hilo. Events: Bi-monthly meetings with the Hawaii Ginger Group; two grower workshops, and presentation of videos designed for grower groups. Services: We assisted local growers making contact with who proposed alternative marketing strategies to local growers and demonstrated value-added ginger products. Products: Our project supplied pathogen-free ginger pieces for field production. Planting materials not used by the research group were made available to local ginger growers. Dissemination: Outreach to reach intended audiences: Findings from our studies have been made in videos, in oral presentations at meetings attended by growers and ginger product industry representatives. Methodology for detection of the pathogen and disease management was demonstrated online at the University of Hawaii/CTAHR website. The videos and short research papers posted there serve as useful resource material for ginger farmers, academic personnel and students. The results from our studies also were presented at the APS meeting in Portland, 2009. PARTICIPANTS: Current and potential partners and collaborators: Current collaborators are those who worked on the project. The follow-up project will include Bernie Kratky, CTAHR and Michael Shintaku (CAFRM), and Susan Miyasaka, Agronomist, CTAHR, University of Hawaii. Partner Organizations: CTAHR, University of Hawaii Manoa and CTAHR, Hilo; College of Agriculture, Forestry and Natural Resource Management (CAFNRM) in Hilo, USDA-ARS-Pacific Basin Agricultural Research Center (PBARC) Hilo, and the Komohana Extension Training and professional development: Two students coming from an educational background in horticulture were trained to accurately identify R. solanacearum using PCR and a newly devised LAMP reaction for specific strain identification. A Ph.D. student, Mathews Paret, has now graduated and is furthering his career in plant pathology at the University of Florida. Individuals who worked on the project: The PD was Anne Alvarez, Professor and Plant Pathologist, from the College of Tropical Agriculture and Human Resources (CTAHR). Collaborators and assistants were Bernie Kratky, CTAHR, Hilo, Kate Nishijima and Lisa Keith, USDA-ARS-PBARC, Mike Shintaku, College of Agriculture and Forestry Resource Management (CAFNRM), University of Hawaii, Hilo; Kent Fleming, Extension Specialist and Dwight Sato, Extension Agent, Komohana Extension Office; Roxana Cabos, a graduate student located in Hilo, and Mathews Paret, a graduate student located on Oahu. They were assisted by Asoka de Silva, technician and Richard Criley, Horticulturalist from the Department of Tropical Plant and Soil Sciences (TPSS) and Daniel Jenkins, Department of Molecular Biosciences and Bioengineering (MBBE) department; Shiv Sharma, from the Hawaii Institute of Geophysics and Planetology helped guide Mathews Paret in studies with laser Raman spectroscopy. TARGET AUDIENCES: Target audiences: Ginger growers, wholesale buyers for ginger products, shippers, restaurant owners, and tourist industry. Training Efforts: Students and technicians were trained in molecular detection and identification methods. Findings from our studies have been made in videos on detection of the pathogen and disease management which are available online at our college website. This serves as a useful resource material for numerous ginger farmers, academic personnel and students. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Change in knowledge: Bacterial wilt disease was known to be caused by Ralstonia solanacearum (Rs) but little was known and/or reported about race 4, which specifically affects ginger and its relatives. Our group provided new information on survival of the pathogen Hawaiian soils and epidemiology of the disease in the field. Survival of R. solanacearum in symptomless ginger rhizomes was a major cause of spread to the next generation of plants. The pathogen was also detected from drainage water from potting medium and soil containing infected ginger plants. Survival on root surfaces of weeds and symptomless solanaceous hosts was another source of inoculum in ginger fields. One R. solanacearum strain, which was being used for biological control of Kahili ginger in Hawaiian forest reserves was proven to be highly pathogenic to both edible and ornamental ginger species in Hawaii. This finding was significant because our results contrasted with two previously published reports, which stated that the biocontrol strains of Rs did not affect ornamental gingers and hence were of no risk to local agriculture. Proof that the biocontrol strain had an extended host range was a significant finding that changed biological control practices in Hawaii, once the new information was published. Bioindicator hosts identified and tested in this project now provides a more sensitive method of detecting low numbers of R. solanacearum in large field plots, enabling evaluation of large soil samples (1 Kg) whereas previously only small sample sizes (a few microliters) could be evaluated by molecular methods including PCR. Biocontrol: Essential oils of palmarosa, lemongrass and eucalyptus showed promise as biofumigants for control of R. solanacearum. In our study, epifluorescence microscopy confirmed the bactericidal and bacteriostatic properties of the treated cells and micro-Raman spectroscopy gave new information on the changes in the cell at the molecular level. Change in actions: Pathogen-free ginger seed pieces (32 grams each) were generated for production of seed stocks for planting new production fields. The virulent strain of R. solanacearum previously used for biological control of kahili ginger in national and state forest reserves is no longer applied in the forests and foresters are now evaluating other methods which pose less risk to the environment. Changing conditions for industry: Hydroponic methods of obtaining large quantities of high quality ginger for planting stocks were demonstrated to growers and there was a large surplus from the demonstration plots enabling us to provide pathogen-free ginger planting materials to growers. This demonstration was significant in encouraging ginger growers to adopt similar growing methods for their own planting stocks in the future. Results of our project led to a follow-up proposal submitted to the Western SARE grants program for field control of bacterial wilt. The ginger research-extension working group will continue to undertake further field research followed by plot demonstrations and grower workshops to help reestablish a sound ginger industry in Hawaii.
Publications
- Journal articles; Paret ML, de Silva, A. Criley, RA and Alvarez, AM. 2008. Bioindicators for Ralstonia solanacearum race 4: plants in the Zingiberaceae and Costaceae families. Australasian Plant Pathology 38:6-12.
- Paret ML, de Silva, A. and Alvarez, AM. 2008. Detection of Ralstonia solanacearum with an immunostrip assay; its specificity and sensitivity. Indian Phytopath. 61 (4) 518-522.
- Dissertation: Paret, ML. 2009. Management of bacterial wilt of ginger (Zingiber officinale R.) caused by Ralstonia solanacearum with plant essential oils. Ph.D. Dissertation, University of Hawaii, Honolulu, HI.
- Abstract: Paret ML, Green L, Sharma, SK, Alvarez AM. 2009. Biochemical characterization of effects of plant essential oils on Ralstonia solanacearum by laser Raman spectroscopy. Phytopathology 99:S99
- Extension productions (videos: Alvarez AM, Paret ML, Kratky BA, Shintaku M, Cabos R, Bernabe C. 2009. Ginger Production and Disease Management Strategies. Tropical and Sub-Tropical Agricultural Research project. ed. Kadooka C. (http://mm.ctahr.hawaii.edu/t-star/Ginger2.wmv)
- Alvarez AM, Paret ML, deSilva AS, Kubota R, Peckham G. 2008. Saving ginger root in Hawaii. Tropical and Sub-Tropical Agricultural Research project. ed. Miyahara (http://www.ctahr.hawaii.edu/t-star/TSTARHilitePage.htm)
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Progress 09/15/07 to 09/14/08
Outputs
OUTPUTS: Serological and DNA-based assays were compared and optimized for detecting low populations of the bacterial wilt pathogen, Ralstonia solanacearum (Rs), in drainage water from infested soils. Survival of Rs in drainage water was determined by viable plate counts on a semiselective medium, an immunostrip assay, ELISA and a polymerase chain reaction (PCR) assay using primers from the fliC gene. The most sensitive and reliable tests were then used to determine the effect of biofumigants (palmarosa, lemongrass, and eucalyptus oil) on survival of Rs in potting soils. Bactericidal and bacteriostatic properties of biofumigants were evaluated first in the laboratory using disk, well- and vapor-diffusion assays. Structural characteristics of Rs cells treated with essential oils were determined using scanning and transmission electron microscopy. Pot studies to determine efficacy of essential oils were carried out at the UH-Hilo agricultural farm. For pot studies, second generation tissue-cultured plants were multiplied in bag culture prior to greenhouse testing. Treatments were compared as average yield per treatment, average number of tillers per treatment, average height of tillers per treatment. Survival of Rs in potting soils was evaluated with immunostrips and inoculum potential was evaluated with bioindicator hosts. For the latter purpose, tissue-cultured ginger planting stocks were generated from pathogen-free stocks and compared to other bioindicator hosts in the Zingiberaceae and Costaceae families. Results were disseminated at regular meetings of the ginger research-extension group in Hilo and presented at the annual meeting of the American Phytopathological Society in St. Paul, Minnesota, at the Annual meeting of the American Horticultural Society in Orlando, Florida, and in publications and abstracts in professional journals. PARTICIPANTS: Researchers and extension personnel at University of Hawaii, campuses at Manoa and Hilo: Anne Alvarez, Bernie Kratky, Kent Fleming, Dwight Sato and Mike Shintaku. UH Manoa graduate student: Mathews Paret. ARS-Pacific Basin Agricultural Research Center, Hilo: Kate Nishijima, and Lisa Keith. Independent entrepreneur/edible ginger grower at Hilo: Roxana Cabos TARGET AUDIENCES: Growers and of edible and ornamental ginger in Hawaii and other tropical environments. Producers of ginger-based commercial products. Horticulturalists, plant pathologists and extension personnel. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The comparison between serological and DNA-based assays for detection of Rs in drainage water showed that an immunostrip assay based on an Rs-specific anti-EPS monoclonal antibody (MAb Ps1a), was the most sensitive of the three assays, giving positive results as low as log 2 cfu/mL. The ELISA (also using MAb Ps1a) showed positive results for Rs populations greater than log 5 cfu/mL. PCR (using fliC primer) detected Rs populations above log 3 cfu/mL; however, it failed to detect Rs in many samples containing high populations (log 4 - 6) probably due to PCR inhibitors in environmental samples. Since the immunostrip assay and culturing methods were most sensitive and consistent, they were used to evaluate the effects of palmarosa, lemongrass and eucalyptus oils (1,400, 700, and 400 ppm v/v) on survival of Rs in pot studies. Among the three potential biofumigants tested for bactericidal and bacteriostatic properties in lab assays, lemongrass oil was the most effective bactericide based on pot and culture amendment studies. Bactericidal effects included cell wall rupture and formation of cellular debris as observed in scanning and transmission electron microscopy. Eucalyptus oil had only bacteriostatic properties as treated cells were distorted but were viable when re-cultured. All tested essential oils produced clear zones of inhibition in culture plates by disk, well and vapor diffusion assays. Palmarosa and lemongrass oils significantly reduced Rs populations in culture amendment assays. Palmarosa and lemongrass oils at 1,400 and 700 ppm (v/v) completely eradicated the pathogen in pot studies, and the plants remained disease-free until the end of the experiment (180 days). At the lowest concentration (400 ppm) results were variable. All the plants in the four separate controls wilted during the period of the experiment but no plants in pots treated with plant essential oils showed any wilting. Data on the average number of tillers per treatment, average height of tillers per treatment, average number of leaves per tiller per treatment and average yield per treatment recorded at 60, 90, 120, 150 days after planting indicated no significant differences between essential oil treated pots and Control- No Rs. Thus, the essential oils did not reduce plant growth or yield of edible ginger. The repeated greenhouse studies using bio-indicator hosts to assess viable pathogen populations confirmed last year's results and again showed that bioindicator hosts are sensitive at lower threshold levels than immunodiagnostic or DNA-based assays. The initial pot studies at UH Manoa have now been confirmed in larger scale greenhouse tests in Hilo. We have again demonstrated that two plant essential oils are effective bio-fumigants for the bacterial wilt pathogen and warrant further testing in larger field studies.
Publications
- Paret, M.L., Kubota, R. Jenkins, D.M., Alvarez, A.M. 2008. Detection of Ralstonia solanacearum race 4, in field samples using a combination of serological and molecular assays. Phytopathology 98:S120.
- Paret, M. L., Kratky, B., Alvarez, A.M. 2008. Bio-fumigation with plant essential oils for bacterial wilt of edible ginger (Zingiber officinale Rosc.) caused by Ralstonia solanacearum race 4. HortScience 43:1225.
- Paret, M.L; De Silva, A. S; and Alvarez, A.M. 2008. Bio-indicators for Ralstonia solanacearum race 4: Plants in the Zingiberaceae and Costaceae families. Australasian Plant Pathology. (in press).
- Paret, M.L; De Silva, A. S; Criley, R.A and Alvarez, A.M. 2008. Ralstonia solanacearum race 4: risk assessment for edible ginger and floriculture ginger industries in Hawaii. HortTechnology 18: 90-96.
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Progress 09/15/06 to 09/14/07
Outputs
OUTPUTS: Studies on bacterial wilt of edible ginger caused by Ralstonia solanacearum were continued. This year we used previously developed methods to quantify the survival of the bacterial pathogen in ginger field soil and the commercially-used potting medium. Survival of the pathogen in soil and potting mix was first studied in micro-experiments conducted at the Pope greenhouse facility at the University of Hawaii at Manoa. Based on findings two larger replicated experiments were conducted at our recently constructed greenhouse facility at the agricultural farm at the University of Hawaii at Hilo. The latter experiments included tests using palmarosa oil and lemongrass oil as potential bio-fumigants to eradicate the pathogen from infested soils. The effectiveness of biofumigation for pathogen kill was then compared with solarization. Bio-indicator plants in the ginger family were also used to detect low numbers of the pathogen in field soil. Soil survival studies were performed
during the fallow season for ginger cultivation in Hawaii, a four-month period from November to February. The ability of pathogen to survive in ginger field soil during this period was used as an indicator of its capacity to re-infest the subsequent crop. Pathogen-free ginger, which was initially established in tissue culture, was then increased in bag culture over the past year. The suitability of three ginger species as bio-indicator hosts for detecting low populations of Ralstonia solanacearum race 4 was compared to immunodiagnostic and DNA-based detection methods. Results from pathogen survival studies and bio-indicator hosts have been disseminated at a local symposium and at the annual meetings of the American Phytopathological Society. Production of pathogen-free ginger from tissue-culture followed by bag-culture increase was demonstrated to growers. A meeting was organized by the extension horticulturalist and county agent for growers and producers/and retailers of ginger
products. The use of micro-sized, bio-indicator plants in the Zingiberaceae family for pathogen detection has been demonstrated and will later be used to evaluate soils collected directly from ginger production fields.
PARTICIPANTS: Participants: Researchers and extension personnel at University of Hawaii, campuses at Manoa and Hilo: Anne Alvarez, Bernie Kratky, Kent Fleming, Dwight Sato, and Mike Shintaku. ARS-Pacific Basin Agricultural Research Center, Hilo: Francis Zee, Kate Nishijima, and Lisa Keith. UH Manoa Student: Mathews Paret
TARGET AUDIENCES: Target audiences: Growers and of edible and ornamental ginger in Hawaii and other tropical environments. Producers of ginger-based commercial products. Researchers and extension personnel.
PROJECT MODIFICATIONS: Project modifications: The project was extended beyond the initial deadline in order to produce sufficient amounts of pathogen-free ginger all the way from tissue culture through a two-year clean bag culture method. The ginger rhizomes were multiplied in an isolated greenhouse in pathogen-free soil to produce sufficient numbers of clean plants for the bacterial survival and disease management studies. The interdisciplinary group of ginger researchers also constructed another isolated glasshouse for controlled studies, using personnel from all four participating institutions (UH Manoa, UH-Hilo, USDA-ARS, and the State of Hawaii). Logistical problems inherent in inter-island and inter-institutional projects delayed completion of the greenhouse, but once accomplished, the new site has made a significant impact on the reproducibility of the greenhouse studies.
Impacts
Ralstonia solanacearum survived in ginger field soil for more than four months after inoculation of ginger field soil and potting medium with or without edible ginger. The pathogen survived for 120 days in soil and potting medium that contained no visible plant debris. This long survival period underscores the risk of introducing the pathogen into an ecosystem following ginger cultivation. Tissue-cultured ginger, micro-sized red ginger and micro-sized spiral ginger plantlets wilted within 9 - 39 days after planting into pre-infested potting medium. The rate of symptom production depended on the initial level of the bacterial inoculum. Most significant is that specific bio-indicator plants attracted low populations of the specific pathogen from large volumes of potting medium. The plant then acts as a trap which increases the pathogen population to levels detectable by previously-developed immunodetection and DNA-based assays. Tissue-cultured ginger enhanced the
pathogen population to its highest value at log 9 colony forming units (cfu) per gram while micro-sized red ginger and micro-sized spiral ginger enhanced the population to approximately log 7 cfu. All three bio-indicator plants tested are now used to monitor pathogen populations in field soil, but eventually, the tests will be limited to a single bio-indicator host. Bio-fumigation of the potting medium with palmarosa oil and lemongrass oil at 700 ppm (v/v) and 400 ppm (v/v) successfully eliminated Ralstonia solanacearum from potting medium. Pathogen-free ginger rhizomes planted into the bio-fumigated medium remained healthy during the subsequent six-month growing period. This suggests that a pre-plant fumigation of ginger fields with previous history of bacterial wilt may become part of an effective disease management strategy. Two significant challenges were met over the past year; one was the repeated use of bio-indicator hosts to better assess viable pathogen populations, which
could not be accurately estimated with immunodiagnostic or DNA-based assays; the second achievement was a demonstration of the effectiveness of two plant essential oils for use as bio-fumigants. These plant products can now be further evaluated as sustainable treatments in a reduced-pesticide disease management program for bacterial wilt.
Publications
- Paret, M.L; De Silva, A. S; and Alvarez, A.M. 2007. Detection of Ralstonia solanacearum race 4, with micro-sized, bio-indicator plants in the Zingiberaceae family. Phytopathology 97: S89
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