Progress 11/01/10 to 09/30/14
Outputs
Target Audience: Anthurium producers in Hawaii county, Philippines, West Indies, Trinidad, Netherlands, and India. Hawaii Floriculture and Nursery Association. Researchers in department of Molecular Biosciences (MBBE) and Bioengineering, University of Hawaii, who worked on senescence genes and programmed cell death in anthurium in the effort to extend shelf life. The similarities between pathogen-induced programmed cell death and stress or age induced PCD were examined by graduate students. Members of APS Press, a division of the American Phytopathological Association invited us to submit a chapter on Xanthomonas axonopodis pv. dieffenbachiae in the book, "Virulence Mechanisms of Plant Pathogenic Bacteria". A bioinformatics professor in the MBBE department used our type strain of the pathogen for sequencing in the effort to determine its relationship to other Xanthomonas axonopodis pathovars. State of Hawaii Department of Agriculture, which sent representatives to visit our laboratories, attend our workshops and conferences, and invited six members of our research team to submit a proposal for state funding (but later failed to fund because all the funds were needed for the Agricultural Research Service needs on the Island of Hawaii). Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Two undergraduate biology students from the University of Hawaii profited from the training provided in development of assessment methods, statistical comparisons and data evaluations. These students took directed research credits in my laboratory. A technician associated with the project expanded his knowledge of translating analog to digital systems with the assistance of an astronomer who used simple high resolution camera systems for repeated measurements of light emissions. Another student used this opportunity to search for and compare additional software needed to translate leaf areas colonized by bacteria into a standardized disease tolerance index. A post-doctoral researcher working with the Hawaii Agricultural Research Center (HARC) came to our laboratory to learn the techniques used to trace bacterial colonization of plant tissues. She used our evaluation system to explore the possibility of using the green fluorescent protein gene reporter system as an alternative. She profited from the experience but was not able to match quantity of light emitted from our previously-developed Lux reporter strain. New opportunities for young researchers in the anthurium industry were provided. One student from Hilo was assisted by a distance learning opportunity and took an entire course in Bacterial Plant Interactions from the P.I. using the Google Hangouts platform. A postdoctoral researcher who had previously specialized in nematology assisted with plant evaluations and statistical analysis of all our laboratory, greenhouse, and field studies. He was provided intensive training in plant bacteriology for which he had no previous experience. How have the results been disseminated to communities of interest? Results have been disseminated periodically via meetings/workshops with collaborators at the Hawaii Agriculture Research Center (HARC). At one such workshop collaborating researchers examined the third set of experiments involving inoculation of mature plants. They helped evaluate infection using a visual assessment key, evaluation of autophotographs, and increase of leaf colonization rates over a 12 week period. New, more accurate ratings of commercially available "resistant" cultivars were established in the workshop. New findings were transmitted directly to growers through the Hawaii County Extension Agent, Kelvin Sewake. During the course of this project he organized several symposia, the details of which are in previous reports. The University of Hawaii's College of Tropical Agriculture and Human Resources (CTAHR) and the USDA Risk Management Agency (RMA) sponsored the 2012 Hawaii Floriculture Risk Management School -Floriculture Symposium and Farm Tours on July 24 -25, in Hilo, Hawaii. In 2013 and 2014 the conferences were also co-sponsored by the Hawaii Floriculture and Nursery Association (HFNA). Each year Dr. Tessie Amore provided updates on the UH Anthurium and Dendrobium Breeding Program. At the last conference growers had immediate concerns about nematode damage and impending restrictions on use of the available and commonly used nematicides. As a result of their urgent concerns, our research group responded by forming a new team that included nematologists who would address grower concerns for nematode control in a follow-up project, that continues the goal of reducing bacterial blight. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
We developed, assessed, and optimized a new and rapid for screening anthurium plants for resistance to bacterial blight caused by Xanthomonas axonopodis pv. dieffenbachiae. Based on our tests, none of the cultivars were immune to disease. As a result, a tolerance index was developed based on a 10-point scale that correlated observable symptoms with internal tissue colonization by bacteria. A reference strain of the pathogen which had been bioengineered to fluoresce when actively multiplying in plant tissues was used for this purpose. A high resolution camera captured sufficient photons from the leaf when bacteria were actively colonizing tissues. Area colonized by the pathogen was compared to total leaf area of each cultivar to develop the quantitative rating system for blight tolerance. Comparisons between bioluminescence emission using the Vibrio lux gene cassette (our system) and light captured from the pathogen bioengineered with the green-fluorescent protein were made. The Lux gene produced far more light and was more appropriate for disease tolerance evaluations. Growth of anthurium microplants (at stage IV when plantlets are normally transplanted into community pots) was accelerated by combination of beneficial bacteria and specific nutrients. When microplants transition from axenic culture to community pots they are initially sterile. Supplementation with naturally-occuring bacteria accelerated the growth rate and plants were hardier after three-months growth in the community pots than plants which received no supplements. Further supplementation with specific amino acids increased survival of beneficial bacteria and protected microplants from infection by Xanthomonas (Xad). Microplant cultivars tolerant to blight were selected for further multiplication and advanced testing. This project was integrated with a separate project 868H, which enabled researchers to deliver and grow new cultivars on the Agricultural Research Station on the Island of Hawaii. On field days, cultivars were observed and rated by growers for subsequent planting into commercial fields.
Publications
- Type:
Other
Status:
Other
Year Published:
2014
Citation:
Toves, P. and Alvarez, A. 2014. Amino acid additives enhance biocontrol of anthurium blight on microplants in shadehouses. Unpublished report to anthurium growers, Hilo Hawaii.
- Type:
Other
Status:
Other
Year Published:
2014
Citation:
Alvarez, A., de Silva, A. and Ayin, C. 2014. A comparison of analog and digital imaging evaluation methods for assessing tissue colonized by Xanthomonas axonopodis pv. dieffenbachiae
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Progress 10/01/12 to 09/30/13
Outputs
Target Audience: The target audience consisted of anthurium growers on the Islands of Hawaii, Maui, Kauai and Oahu along with extension agents, University researchers, industry representatives, and researchers in the ARS-PBARC laboratories in Hilo Hawaii. These audiences were reached individually and in two conferences (described later in the report). Extension agents on Oahu and the island of Hawaii were instrumental in organizing and carrying out both conferences.Individuals who worked on the project from University of Hawaii: Asoka de Silva and Tomie Vowell, technicians, PEPS Department; Kelvin Sewake, Extension Agent, Plant and Environmental Protection Sciences (PEPS); Teresita Amore, plant breeder, and Richard Criley, ornamentals researcher, Tropical Plant and Soil Science Department (TPSS); Juan Castillo, statistician; (PEPS and TPSS part time hire) John Tonry, Astronomy Department; Maureen Fitch, Xiaoling (Linda) He, research scientists, Hawaii Agricultural Research Center. Changes/Problems: In 2011 we reported that the use of the green-fluorescent protein (GFP) method of evaluating resistance was unsatisfactory for evaluation of large plants, but we continued to compare the LUX with the GFP method for several more months. Results from 2012 confirmed our earlier conclusions; thus, we abandoned the GFP approach and continued only with disease resistance evaluations based on plant colonization trials using the LUX reporter gene in the bacterial pathogen. We compared analog vs. digital methods in the attempt to save project costs by using a digital camera to register light rather than a sensitive x-ray film. The digital method showed promise, but the number of photons emitted from the plants over a series of repeated autophotographs was insufficient, giving a weak image in comparison to the original film-based method. For the final year of the studies we relied only on autophotography, which statistically gave us the best results. What opportunities for training and professional development has the project provided? This year we have provided new opportunities for young researchers in the anthurium industry. One is a nematologist who is assisting with statistical analysis of all our laboratory, greenhouse, and field studies and will later assist with the development of the tissue-cultured, clean-microplant program to be introduced to the ornamental industry in Hawaii. We propose to provide rooted microplants as pathogen free planting stocks three months after deflasking and establishing plants under controlled environmental conditions. How have the results been disseminated to communities of interest? Kelvin Sewake, County Extension Agent with UH Manoa CTAHR and Team LIFE member organized a symposium for 103 participants in June, 2013. This was a follow up to last year's conference when the University of Hawaii's College of Tropical Agriculture and Human Resources (CTAHR) and the USDA Risk Management Agency (RMA) sponsored the 2012 Hawaii Floriculture Risk Management School –Floriculture Symposium and Farm Tours on July 24 –25, in Hilo, Hawaii. CTAHR’s County Extension Agent and collaborator on our project, Mr. Kelvin Sewake, organized both events. In 2012 the conference was also co-sponsored by the Hawaii Floriculture and Nursery Association (HFNA). Day 1 took place at the University of Hawai’i at Hilo’s Campus Dining Room and included 16 presentations mostly from CTAHR researchers, but also included Dr. Maureen Fitch from the Hawaii Agriculture Research Center speaking onAnthurium Transformation for Crop Improvementand Dr. Robert Hollingsworth from USDA-PBARC speaking on new insect pests. Mr. Eric Tanouye, President of HFNA, provided the welcome address stating the need to work together to reverse the trend of declining industry value. Dr. Tessie Amore provided updates on theUH Anthurium and Dendrobium Breeding Program; Mr. Kelvin Sewake spoke onChemical Controls for Burrowing Nematodes on Anthuriums and Foliar Nematodes on Oncidiums; and Dr. Paul Singleton addressed the issue ofWater and Nutrient Management to Improve Productivity in Our Flower Industries. The program also included research updates and useful information on cultural practices, disease and insect pest management, and postharvest management practices. What do you plan to do during the next reporting period to accomplish the goals? This year we have focused on statistical analysis of laboratory, greenhouse and field experiments. Next year our group will publish all research results in refereed journals, news-letters, book chapters and extension publications. We will continue to rate new anthurium hybrids for resistance to blight and generate a universal rating scale that can be used to improve planting design (blocking and spacing plants). The outcome will be protection of highly susceptible cultivars from infection by inter-planting with resistant A. antioquiense cultivars.
Impacts
What was accomplished under these goals?
Traditional anthurium cultivars and new hybrids were evaluated for susceptibility/resistance to bacterial blight following inoculation with Xanthomonas axonopodis pv. dieffenbachiae. Flowering plant evaluations were based on successive measurements of leaf areas colonized by the pathogen over a 12-week period following inoculation. The experiment was repeated three times with six replicates per cultivar per treatment. Microplants recently deflasked from tissue culture were evaluated four weeks after inoculation using leaf blot analysis of detached leaves. Analog and digital imaging methods were used to calculate disease severity indices and rate the selected cultivars. An X-ray film assessment method was compared to a potentially more cost effective way to monitor bacterial colonization of anthurium cultivars. Experiments were designed to obtain images of luminance from diseased leaves that would match the intensity of light absorbed onto the X-ray film. Sensitivity (ISO), exposure times, aperture settings, and positions of the camera were manipulated to get a desirable depth of field and software was developed to calculate the number of photons emitted in the infected leaves. Four of nine new hybrid cultivars showed greater resistance than Kalapana (resistant control) but none of the cultivars were more susceptible than Marian Seefurth.
Publications
- Type:
Book Chapters
Status:
Accepted
Year Published:
2014
Citation:
Alvarez, A.M. Vowell, T.S. and Toves, P.J. 2013. Anthurium blight caused by Xanthomonas axonopodis pv. dieffenbachiae. Chapter In: "Virulence Mechanisms of Plant Pathogenic Bacteria" N.Wang, J.Jones, G.Sundin; F. White, S. Hogenhout, C.Roper, L. De La Fuente, JH Ham, eds. APS Press, St. Paul, MN (In press)
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Alvarez, A.M., Toves, P.J. and Vowell, T.S. 2013. Biological control of bacterial blight of anthurium caused by Xanthomonas axonopodis pv. dieffenbachiae Phytopathology, S148-P.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Alvarez, A.M., de Silva, A.S., and Amore, T.D. 2013. Development of a blight susceptibility index for anthurium cultivars evaluated in a resistance breeding program Phytopathology, S264-P
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2013
Citation:
Amore, T.D. 2013. Anthurium and Dendrobium Breeding at the University of Hawaii. 2013 Hawaii Floriculture Risk Management School Symposium. Hilo, Hawaii. June 26, 2013.
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Progress 10/01/11 to 09/30/12
Outputs
OUTPUTS: Anthurium cultivars were evaluated for resistance and susceptibility to bacterial blight following inoculation with the pathogen Xanthomonas axonopodis pv. dieffenbachiae (Xad). Whole plant evaluations were based on successive measurements of leaf areas colonized by the pathogen over a 12-week period following inoculation. Large plant experiments were repeated three times with six replicates per cultivar per treatment. Small plants (recently removed from tissue culture) were evaluated four weeks after inoculation using leaf blot analysis of detached leaves. Analog and digital imaging methods were used to calculate disease severity indices and rate resistance and susceptibility of the selected cultivars. Original plant materials were propagated to increase the supply of reference materials and about 80 plants of each of the 4 Dutch cultivars are currently maintained at the Hawaii Agriculture Research center (HARC) facilities. In addition, we have Kalapana, New Pahoa Red, and Marian Seefurth for reference comparisons. The X-ray film assessment method was compared to a potentially more efficient and cost effective way of evaluating bacterial colonization of anthurium cultivars. Experiments were designed to obtain images of luminance of diseased leaves that would match the intensity of light absorbed on to the X-ray film. Sensitivity (ISO), exposure times, aperture settings, and positions of the camera were manipulated to get a desirable depth of field. Software was developed to calculate the number of photons emitted in the infected leaves. PARTICIPANTS: Individuals who worked on the project from University of Hawaii: Asoka de Silva and Tomie Vowell, technicians, PEPS Department; Kelvin Sewake, Extension Agent, PEPS Department; Teresita Amore, plant breeder, and Richard Criley, ornamentals researcher, Tropical Plant and Soil Science Department; John Tonry, Astronomy Department; Maureen Fitch, Xiaoling (Linda) He, research scientists, Hawaii Agricultural Research Center. TARGET AUDIENCES: The people directly affected by our research are anthurium growers in Hawaii and worldwide. national plant pathologists, plant breeders and horticulturalists iplant breeders in the Netherlands and the West Indies; Quantitative evaluations of plant resistance will assist growers in selecting suitable cultivars, growth under suitable conditions, and in distribution of cultivars and in placement in production fields. TARGET AUDIENCES: Target audiences are anthurium growers, researchers in tropical ornamentals, extension personnel, and plant breeders. We made presentations at the Hawaii Floral Industry Conference in July, 2012, attended by grower groups, industry personnel, and researchers from the Hawaii Agricultural Research Center, the USDA-ARS Pacific Agricultural Research Center (PBARC, and ornamental growers throughout the state. The extension agent on our project, Kelvin Sewake, organized a field tour following the conference which was well attended by both researchers and growers. PROJECT MODIFICATIONS: Last year we reported that the use of the green-fluorescent protein (GFP) method of evaluating resistance was unsatisfactory for evaluation of large plants, but we continued to compare the LUX with the GFP method for several more months. This year's results confirmed our earlier conclusions and we have now abandoned the GFP approach and continued only with disease resistance evaluations based on plant colonization trials using the LUX reporter gene in the pathogen. We compared analog vs. digital methods in the attempt to save project costs by using a digital camera to register light rather than a sensitive x-ray film. The digital method shows promise, but the number of photons emitted from the plants over a series of repeated photos is insufficient, giving a weak image in comparison to the older film-based method.
Impacts
None of the commercial hybrid cultivars showed adequate resistance to Xad, although these cultivars were characterized as `tolerant or resistant' by commercial companies prior to evaluation. The lux reporter clearly demonstrated that plant leaves and vascular tissues were fully colonized prior to symptom development. Within 10 weeks following inoculation, most of the plants were dead. Severity indices for each cultivar showed delayed colonization of leaf tissues over the 3 to 12 week period compared to the most susceptible cultivar, Marion Seefurth. Based on the mean severity index at week 7, the cultivars were ranked from lowest (most resistant) to highest (most susceptible): Acropolis (13.10); Laguna (22.15); Senator (22.71); Fantasia (27.45); Marian Seefurth (44.33). The small plants comparisons of nine anthurium cultivars (Acropolis, A697, UH711, UH711xA582-I-92, UH711xA582-I-94, Uh1349, Uh1469, Flamingo and Monohara) showed the range of resistance ratings using the detached leaf assay four weeks after inoculation. Disease incidence (percentage of leaves infected) was as follows: A697 (10.87%); Acropolis (11.29%); UH1349 (12.12%); Flamingo (17.24%); Kalapana (18.26%); UH711 (28.95%); UH1469 (30.77%); UH711x(A582) I-94 (39.62%); Manohara (41.67%) UH711x(A582) I-92 (54.55%)and Marian Seefurth (55.81%). Four cultivars showed greater resistance than the standard Kalapana (resistant control) but none of the cultivars were more susceptible than Marian Seefurth (susceptible control). None of the variables applied to the digital camera helped resulted in an image that matched the intensity of the X-ray image. Increasing the ISO values higher than 1600 (6400 is the maximum ISO value for the camera) produced `white noise' that interfered with the luminance from the pathogen. Increasing the exposure time gave similar results and a grainy image. Image intensity was increased by taking a series of repetitive images of infected leaves. Twenty-second exposures were repeated 20 times without changing the camera or leaf positions. Resolution was increased by superimposing the 20 digital files and computing the total light emitted. By taking 100 to 1000 repeated images the intensity of digitally recorded images matched the luminance recorded in the X-ray film (following 8 hour exposures to infected leaves). Given the setting and the resources available to us, the digital method is not practical. However, if repeated imaging can be automated the images will be suitable for the evaluation of disease incidence among the cultivars. Conclusions: cameras using charge-coupled device (CCD) technology are available and more sensitive than the camera used in this study. However, CCD cameras are too expensive and beyond the capacity of this project. Despite our attempt to adapt an affordable moderately priced camera for pathogen colonization measurements, we still conclude that the X-ray imaging is still the best method for this type of study if the investigator has to operate within a tight budget. Nevertheless it is important make an effort to switch to a digital method in the world where the analog methods are slowly disappearing including emulsion film stocks.
Publications
- No publications reported this period
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Progress 10/01/10 to 09/30/11
Outputs
OUTPUTS: Five hundred plants (two partially resistant and two susceptible cultivars) sent to us by a Dutch company were grown at HARC facilities. Half of the plants were dedicated to assays with the lux strain and half to future studies to be carried out with a genetically modified Xad strain that carries the green fluorescent protein (gfp). In initial experiments with four flowering anthurium cultivars, we used a transgenic strain of the bacterial pathogen, Xanthomonas (Xad), which contains the lux gene cassette and produces light as the bacterium colonizes leaves and stems of anthurium plants. A disease severity index was calculated as the percentage of leaf area colonized by the pathogen at intervals over a period of twelve weeks. The percentage tissue colonized is calculated and transformed to a severity index. In the first two experiments with flowering plants of four Dutch cultivars and one Hawaiian cultivar, one cultivar (Acropolis) was clearly more resistant than the other three, so this cultivar was used to make crosses between resistant Dutch and susceptible Hawaiian germplasm. Progeny were selected and propagated in vitro for further studies. Sufficient numbers of plantlets have been produced to start leaf-blot analysis on the progeny of the Acropolis x Hawaiian cultivar crosses. In order to reduce the cost of evaluating resistance with expensive x-ray film, we tested a high-end digital camera (Canon EOS 5D Mark II with high-speed 50 mm f/1.4 lens) which was able to capture sufficient levels of bioluminescence to separate diseased and healthy leaves. Each leaf was photographed in the light and later in the dark. The two images were analyzed using an algorithm developed by Dr. John Tonry (UH Dept of Astronomy), who assisted by developing software that converts the image to pixels, analyzes the image and assigns a luminence score. Meanwhile, three new Hawaiian varieties were produced by T. Amore: a) 658 Kuuipo was released and published in the Hilo Hearld Tribune, February 2011; b)UH 1651 was published as a new cultivar release in CTAHR series, 2011, and c) UH 1679 Iiwi. These cultivars will be evaluated by the optimized lux protocol (using camera) and assigned a resistance rating over the next year of the project. To disseminate results, we provided information to the extension agent who is liaison with the anthurium industry and the UH research team. Information on UH anthurium cultivars was disseminated to industry growers at the 2011 Hawaii Export Nursery Association's MIDPAC Conference and Trade Show and at various educational meetings. An anthurium conference/field day is planned to update growers on new UH anthurium cultivars and selections. The agent is developing a DVD to graphically illustrate complex concepts and techniques of breeding anthuriums as well as current scientific techniques used to evaluate anthuriums for Xad resistance. PARTICIPANTS: Individuals who worked on the project from University of Hawaii: Asoka de Silva, technician, PEPS Department Tomie Vowell, technician, PEPS Department Kelvin Sewake, Extension Agent, PEPS Department Teresita Amore, plant breeder, TPSS Department Richard Criley, ornamentals researcher, TPSS Department John Tonry, Astronomy Department Hawaii Agricultural Research Center: Maureen Fitch, Xiaoling (Linda) He TARGET AUDIENCES: The people directly affected by our research are anthurium researchers in temperate and tropical regions; plant breeders in the Netherlands and the West Indies; Anthurium growers in Hawaii and worldwide. Quantitative evaluations of plant resistance will assist growers in selecting suitable cultivars, growth under suitable conditions, and in distribution of cultivars and in placement in production fields. PROJECT MODIFICATIONS: Based on early studies, the gfp method of evaluating resistance has been unsuitable for our purposes whereas the lux method has already shown reproducible results. We will continue to compare the lux with the gfp method for several more months, but if the gfp methodology is still unsuitable after several more evaluations, we plan to abandon this approach and complete the evaluations with the lux method alone.
Impacts
Two Dutch cultivars, previously characterized as resistant, were fully colonized by the bacterial blight pathogen (Xad) in as little as nine weeks after inoculation as demonstrated by the lux method of evaluation. This represents a major change in thinking about resistance with respect to Anthurium andreanum cultivars and this information has caused researchers to develop better quantitative methods to characterize and quantify resistance. In our studies so far, the lux method using the transgenic Xad-lux strain has been more efficient than the gfp method which uses a transgenic strain that carries the green fluorescent protein. Nevertheless, the lux procedure is expensive for large-scale screening. Based on promising results from initial assays, we have now purchased a digital camera as part of the project and will calibrate the digital system by comparing a series of infected leaves with both the digital and the x-ray film- based methods. A technician has been trained by the software developer and can now make direct comparisons between the X-ray film and digital methods. Once the digital data are validated with repeated studies, we should be able to discontinue use of the older and more expensive x-ray method of leaf evaluation.
Publications
- No publications reported this period
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