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ARS Home » Pacific West Area » Hilo, Hawaii » Daniel K. Inouye U.S. Pacific Basin Agricultural Research Center » Tropical Plant Genetic Resources and Disease Research » Research » Research Project #424839

Research Project: Molecular Resources for the Improvement of Tropical Ornamental and Fruit Crops

Location: Tropical Plant Genetic Resources and Disease Research

2018 Annual Report

1a. Objectives (from AD-416):
Objective 1: Develop and assess transgenic plants to control plant growth and development, disease resistance, and shelf life. [NP 301, C1, PS 1A and NP 301, C3, PS, 3A] Sub-objective 1a: Screen and select transgenic and/or non-transgenic anthuriums for resistance/tolerance to bacterial blight. Sub-objective 1b: Develop and evaluate an improved screening method for determining resistance and tolerance to burrowing nematodes in anthurium. Sub-objective 1c: Develop and screen transgenic anthurium lines with improved gene constructs for resistance/tolerance to bacterial blight and burrowing nematodes. Sub-objective 1d: Develop management options to control flowering of pineapple under Hawaii conditions. Sub-objective 1e: Obtain PRSV-resistant Hawaiian papaya that have reduced amount of fruit blemishes through recurrent selection. Objective 2: Identify genes or genetic elements useful for improving horticulturally and commercially important traits in floral crops. [NP 301, C3, PS 3A] Sub-objective 2a: Identification of pathways and molecular components for floral color improvement of anthurium. Objective 3: Improve horticultural characteristics and cultivation practices of subtropical and tropical crops. [NP 305, C1, PS 1B1] Sub-objective 3a: Develop improved practices for coffee production to mitigate the damage of coffee berry borer (CBB). Sub-objective 3b: Develop improved practices for coffee production that will serve as an industry model. Sub-objective 3c: Develop improved practices for sustainable, tropical/subtropical, diversified crop production that will serve as an industry model for zero waste.

1b. Approach (from AD-416):
1) Use genetic engineering to develop anthurium that are resistant or tolerant to bacterial blight and nematode pests. Vegetatively propagate the best lines and initiate a large screening trial of the few selected bacterial tolerant lines in a cinder bed trial to simulate commercial conditions. Evaluate the impact of plant resistance or tolerance on the nematode (reproduction) and the impact of the nematode on the plant (flower yield, plant growth). Develop new screening methods for long term yield evaluations. Develop transformation protocols in house (PBARC) and use new antimicrobial peptides for bacterial resistance and a combination of cystatin and plant proteinase inhibitors for nematode resistance; optimize for expression in monocots/anthuriums. 2) Use genetic engineering to develop pineapple that are resistant to natural flowering. Develop a robust transformation system and a realistic and effective screening regime for natural flowering. 3) Develop color enhanced cultivars of anthurium through the identification of key pigment pathways and subsequent genetic engineering for transferring the traits. Develop more comprehensive molecular biological and biochemical tools to fill the information gap required for supporting improvement of other commercially important traits and a cooperative resource for cultivar development with the University of Hawaii and industry growers. 4) Mitigate the impact of coffee berry borer through effective use of a biological control agent (Beauveria bassiana). Determine viability and persistence of Beauveria spores on coffee cherries, measure Beauveria infection rates in infested coffee berries and monitor infestation levels from field counts of CBB-infested and uninfested coffee berries. 5) Mitigate the impact of coffee berry borer by using plant hormones to synchronize coffee flowering. Define a method to time flowering and “schedule” harvesting for efficient distribution of labor for hand harvested areas and optimize flowering to one or two major flowering times for mechanically harvested areas. 6) Develop virus resistant Hawaiian solo papaya with blemish-free fruit through recurrent selection. Evaluate fruit and either backcross or self lines to increase freckle free phenotype; select fruits with the lowest amount of freckles, desirable fruit shape, high total soluble sugars and good fruit quality. Determine shipping longevity of freckle free fruit.

3. Progress Report:
This is the final report for this project, which has been replaced by 2040-21000-017-00D, "Genetic Improvement and Sustainable Production Systems for Sub-tropical and Tropical Crops in the Pacific Basin". For additional information, see the new project report. Over the course of the project, progress has been made on all objectives of this project. Objective 1: Anthuriums that were transformed to be bacterial blight resistant were screened and screening methods were improved. Directly inoculating the transgenic anthurium plants on a greenhouse bench rather than incubating them in a humid chamber allowed us to screen twice as many plants at one time. In addition, a modified bench inoculation method was developed to improve the rate and progression of infection. Within four weeks all test plants become infected and showed systemic infection similar to plants that were incubated for 24 hours in a humid chamber. This inoculation method provided a more efficient way to inoculate many plants and reduced the length of the trials. Anthurium lines that showed increased tolerance to bacterial blight in initial pot tests were multiplied for greenhouse and field testing. To assess the potential for transgenic plants to manage disease and help to make Hawaii-grown anthuriums more marketable and competitive in the global market, cinder bed trials of promising transformed lines of anthurium that showed tolerance to Xanthomonas axonopodis pv. dieffenbachia in pot/bench trials were conducted. No transformed line was able to survive disease pressure; promising lines from earlier pot trials did not maintain their tolerance under field conditions. New constructs have been transformed into anthuriums with monocot promoters for increased expression, the presence of the transgene confirmed, and transformed plants are able to suppress the bacterial growth and survive longer than control plants in in vitro assays. Potted plant and field bioassays were conducted to evaluate anthurium plants transformed for resistance to burrowing nematode, Radopholus similis. Nematode reproduction and plant health were assessed by inoculating burrowing nematode on transformed potted plants. Lines displaying resistance or tolerance to nematode infestation were vegetatively propagated and assayed in cinder beds simulating grower conditions. Flower size and yield were measured in addition to root vigor and nematode populations. Lines showing resistance or tolerance in previous studies did not hold up in heavily infested field conditions. Future emphasis will focus on transformation by improving existing constructs with root-specific promoters and developing new Ribonucleic acid interference (RNAi) constructs targeting nematode effector genes. Naturally differentiated flowering of pineapples results in smaller pineapples overall and unmarketable pineapple fruits scattered throughout the season. One potential method to control this is to reduce the stress induced plant hormone, ethylene, produced by Aminocyclopropane-1-carboxylic acid (ACC) synthase. "Smooth Cayenne” pineapple plants transformed for reduced expression of ACC synthase from cooperators in Australia are being evaluated for flowering under controlled conditions. The same constructs are being transformed into low acid pineapple varieties. To improve marketability of Hawaiian papayas, scientists are breeding and selecting for papayas with a blemish-free skin, papaya ringspot virus resistance and good fruit flavor. Three generations of papaya have been bred and selected for these important horticultural traits and are in the process of being propagated by tissue culture and seed production. Objective 2: Anthurium amnicola was utilized as a model plant for gene sequencing and mining. A. amnicola is a parental species of unique, purple-colored anthurium cut flower and potted plant hybrids. Expressed genes in four organs of ornamental and horticultural importance in anthurium: the leaf, spathe, spadix and roots were determined by RNA sequencing in A. amnicola. Genes for putative transcription factors to induce expression of anthocyanin, the main pigment of the floral stem as well putative genes involved in the enzymatic biosynthesis of this pigment were identified from among expressed sequences. A method to test putative anthocyanin biosynthesis pathway genes in bacteria (Escherichia coli) was employed to support characterization of enzymatic function of plant anthocyanin genes that was more rapid and facile compared to plant-based methods. This line of research led to the first report to produce peonidin, an anthocyanin of pharmaceutical and commercial importance in bacteria. Sequencing of RNA from different A. amnicola organs identified genes that are expressed in an organ-specific manner. This information serves as a resource for potential organ-specific promoters that would be valuable for regulating expression of important ornamental traits such as floral color in the spathe and spadix, organs of the floral stem that are specific only to Anthurium and other members of the aroid family. Tissue specific promoters will also be useful in directing expression of pathogenesis related (anti-microbial, anti-fungal, nemacidal) transgenes at the most relevant entry point in anthurium and related plants. To support sequencing of promoter DNA regions and genomic DNA studies in A. amnicola and related germplasm, methods to isolate high molecular weight DNAs were initiated. Collection of Anthurium species and heritage cultivars were initiated to insure future availability of germplasm for molecular studies, as well as for breeding research and growers. Objective 3: ARS scientists in Hilo, Hawaii, have implemented improved horticultural practices to reduce sanitation for coffee berry borer (CBB) control, optimized Beauveria biocontrol sprays, and provided added value for farm waste products. CBB was discovered in the Kona coffee growing region of Hawaii in 2010, on Oahu in December 2014 and on Maui in January 2017. Current recommended control methods include monitoring for CBB through traps or field surveys, field sanitation to remove CBB reservoirs between seasons and biopesticide applications of the entomopathogenic strain Beauveria bassiana GHA. Plant growth regulators have been used to effectively promote coffee flowering events to reduce the amount of coffee at the end of the coffee growing season. To further optimize these results, both plant growth regulator sprays and horticultural practices such as pruning will be used to further synchronize coffee harvests and optimize timing for biopesticide applications of Beauveria bassiana. Effects of Beauveria spray regimes and management practices on CBB infestation were documented on cooperator farms located at different microclimates throughout the Kona coffee region. Results from these locations showed excellent control of CBB, to levels beneath 5% infestation. The monitoring and spray regimes are recommended parts of the Area-wide CBB IPM efforts. A zero-waste approach makes agriculture more profitable and addresses food and energy security issues in Hawaii. Research optimized and increased production of algae oil and animal feed ingredients to production scale utilizing the heterotrophic algae, Auxenochlorella protothecoides (formally Chlorella protothecoides) and papaya as a feedstock. A 40-gallon Steam-In-Place Bioreactor was installed at the ARS Daniel K. Inouye U.S. Pacific Basin Agricultural Research Center (DKI-PBARC) in Hilo, Hawaii, and results from lab scale were successfully replicated. Demonstration phase production was initiated as a collaboration between DKI-PBARC, the Hawaii Department of Agriculture, Agribusiness Development Corporation (HDOA-ADC) and Big Island Biodiesel. The scale-up of the demo phase resulted in multiple, successful 150-gallon, 7 day runs of A. protothecoides and papaya in 270-gallon reactors. Results were comparable to experiments conducted at DKI-PBARC.

4. Accomplishments
1. Optimizing commercial Beauveria sprays for coffee berry borer control. Coffee berry borer (CBB) is the most devastating insect pest in coffee production and Beauveria bassiana is the most important natural enemy of CBB. ARS scientists in Hilo, Hawaii, studied the effect of altitude, temperature, rainfall, frequency and timing of spray application, in combination with other control methods on Beauveria GHA control of CBB infestation. Evaluation of the results over multiple seasons since 2013 discovered that combining well-timed B. bassiana GHA spray applications with strip picking was found to be the most effective treatment tested. Results from this work have been utilized as best management practices by University of Hawaii Extension. This work not only contributes to the overall understanding of the occurrence and outbreaks of CBB in relation to seasonal, geographic and biological variables, as well as cultural practices used on production farms, but is also being utilized to strengthen prediction models.

2. Two species of Ceratocystis cause Rapid Ohia Death. Two new species of fungi have been identified as the puzzling killer of hundreds of thousands of Hawaii’s most ecologically and culturally significant ohia trees. An ARS scientist from Hilo, Hawaii, and colleagues from the University of Pretoria, University of Hawaii, and Iowa State University, used a combination of the phylogenetic, morphological and biological species concepts, as well as pathogenicity tests and microsatellite analyses, to characterize isolates collected from diseased ohia trees across Hawaii Island. Besides fully characterizing these two important fungal species, the ARS scientist consulted with an Hawaiian cultural expert at the Edith Kanakaole Foundation to help select the names Ceratocystis huliohia (disruptor of ohia) and C. lukuohia (destroyer of ohia). This marks the first-time Hawaiian names have been given to plant pathogens.

Review Publications
Suzuki, J.Y., Horrell, J.R., Imamura, J.L., Amore, T.D., Matsumoto Brower, T.K. 2017. Floral developmental timing in the ornamental progenitor species Anthurium amnicola Dressler. Aroideana. 40:40-48.

Aristizabal, L.F., Johnson, M., Shriner, S., Hollingsworth, R.G., Manoukis, N., Myers, R.Y., Bayman, P., Arthurs, S.P. 2017. Integrated pest management of coffee berry borer in Hawaii and Puerto Rico: Current status and prospects. Insects. 8(4):123.

Asner, G., Martin, R., Keith, L.M., Heller, W., Hughes, M., Vaughn, N., Hughes, F., Balzotti, C. 2018. A spectral mapping signature for the Rapid Ohia Death (ROD) pathogen in Hawaiian forests. Remote Sensing. 10(3):404-417. doi:10.3390/rs10030404.

Barnes, I., Fourie, A., Wingfield, M., Harrington, T., Mcnew, D., Sugiyama, L.S., Luiz, B., Heller, W., Keith, L.M. 2018. New Ceratocystis species associated with rapid death of Metrosideros polymorpha in Hawai'i. Persoonia: Molecular Phylogeny and Evolution of Fungi. 40:154-181.

Myers, R.Y., Follett, P.A., Mello, C.L., Snook, K.A. 2018. Effects of irradiation on the reproduction of Rotylenchulus reniformis. Nematology. 20:265-269.

Gutierrez-Coarite, R., Heller, W., Wright, M., Keith, L.M., Sugiyama, L.S., Mollinedo, J., Chun, S. 2018. Entomopathogenic fungi as mortality factors of macadamia felted coccid, eriococcus ironsidei (hemiptera:eriococcidae) in Hawai'i. Hawaiian Entomological Society Proceedings. 50:9-16.

Myers, R.Y., Mello, C.L., Keith, L.M. 2017. Evaluation of edible ginger and turmeric cultivars for root-knot nematode resistance. Nematropica. 47:99-105.

Suzuki, J.Y., Geib, S.M., Carlsen, M.M., Henriquez, C.L., Amore, T.D., Sim, S., Matsumoto Brower, T.K., Keith, L.M., Myers, R.Y. 2017. Development of chloroplast single nucleotide polymorphisms (SNPs) as a tool towards interspecies typing of Anthurium germplasm. Acta Horticulturae. 1167:257-270.

Amore, T.D., Matsumoto Brower, T.K., Suzuki, J.Y., Toves, P.J., Lichty, J.S. 2017. Rebuilding Hawaii’s Anthurium germplasm collection for cultivar and species preservation, breeding, and biotechnological research. Acta Horticulturae. 1185:117-122.