Location: Tropical Plant Genetic Resources and Disease Research
2019 Annual Report
Objectives
Objective 1: Develop papayas with superior disease resistance and value added traits.
Subobjective 1A: Generate papaya lines with wide-spectrum resistance to papaya ringspot virus.
Subobjective 1B: Breed new papaya cultivars with superior disease resistance, quality, flavor, and value added products.
Objective 2: Devise integrated horticultural management practices and enhanced germplasm to improve plant health, yield, quality, and product value of coffee.
Subobjective 2A: Evaluate coffee rootstocks to identify genotypes with resistance or tolerance to root-knot nematodes under field conditions.
Subobjective 2B: Evaluate horticultural practices to identify those that yield optimal vegetative growth, flowering, fruiting, and quality of coffee.
Subobjective 2C: Combine the preceding knowledge into an integrated package of genetic and horticultural management solutions to optimize coffee production in Hawai'i.
Objective 3: Genetic improvement of disease and pest resistances and ornamental traits in anthuriums.
Subobjective 3A: Identify and assess the efficacy of selected transgenes for controlling burrowing nematode, Radopholus similus in anthurium.
Subobjective 3B: Identify metabolic pathways and molecular components governing novel flower color traits in anthurium.
Subobjective 3C: Generate and assess anthurium plants with transgenes for enhanced resistance to plant-parasitic nematodes and bacterial diseases, and novel flower colors.
Approach
Objective 1: Focus on improving for disease resistance and improved appearance of papaya through both molecular and conventional breeding.
Subobjective 1A: Utilize Clustered Regularly Short Palindromic Repeats (CRISPR)/Cas9 or alternatively Cas9/ subgenomic RNA (sgRNA) directed mutation of an endogenous papaya gene eIF4E or eIF(iso)4E gene to confer broad viral resistance resulting in commercial papaya cultivars with wide-spectrum resistance to papaya ringspot virus (PRSV) and related viruses using a combination of transformation and crossing.
Subobjective 1B: Develop commercially acceptable papaya cultivars with PRSV resistance, blemish free skin, and improved flavor using conventional breeding and selection.
Objective 2: Develop coffee management practices to facilitate coffee harvest and improve resistance to pests such as nematodes and CBB.
Subobjective 2A: Use grafting techniques to test if yields of Arabica coffee will be higher when grafted on Coffea canephora 'Nemaya' rootstock in nematode (Meloidogyne konaensis) infested fields when compared to un-grafted trees.
Subobjective 2B: Focus on a combination of pruning, fertilization, and applications of plant growth regulators to synchronize coffee flowering and subsequent fruit development to concentrate harvests and reduce the amount of immature berries at the end of the growing season.
Subobjective 2C: Use the on farm site surveys to evaluate commercial strain of Beauveria bassiana GHA to help to mitigate damage caused by the coffee berry borer in commercial coffee farms.
Objective 3: Utilize transient expression and functional analysis of genes to identify key effectors of nematode resistance and pigment production and use transformation to generate bacterial and nematode resistant anthurium varieties.
Subobjective 3A: Use transient expression of genes involved with nematode resistance to identify potential transcripts for control of the burrowing nematode, Radopholus similis, and improve transformation efficiency for anthuriums.
Subobjective 3B: Use molecular techniques for functional analysis of regulatory genes or biosynthetic genes for pigment pathways and identification of organ-specific promoters to prove that genes identified by sequence homology will function in anthurium color pathways similar to those reported in model systems.
Subobjective 3C: Use molecular transformation to generate and assess anthurium plants with transgenes for resistance to plant-parasitic nematodes and bacterial diseases.
Progress Report
Research continued in Objective 1 on developing papayas with superior disease resistance and value-added traits.
To increase the success of Sub-objective 1A, a collaboration was established with a plant pathology lab with extensive experience in Cas9/sgRNA design and host resistance gene targeting. It was determined that additional analyses to identify the optimal eiF4E isoform target(s) in papaya would increase likelihood of host resistance to papaya ringspot virus (PRSV). Although the refocus of the design portion of the project delayed the transformation experiments, an improved working protocol for harvesting and disinfesting embryos from ‘Kapoho’ papaya fruits for future plant transformations has been established.
In support of Sub-objective 1B, selected lines of `Sun Up’ and N08-75 hybrids are being grown in the cooperator field along with selected lines of `Kapoho’. Planting was delayed due to the loss of the seedlings during the government furlough. Due to the lava flow on Hawaii island in 2018, much of the land used for commercial production was lost and relocated to other areas. This resulted in a lack of hybrid ‘Rainbow’ seeds. To increase seed production, ARS researchers are providing technical expertise to the papaya industry and the University of Hawaii at Manoa for seed production, cleaning and storage.
Research continued under Objective 2 on integrated horticultural management practices and enhanced germplasm to improve plant health, yield, quality, and product value of coffee.
In support of Sub-objective 2A, research continued on the evaluation of coffee rootstocks to identify genotypes with resistance or tolerance to root-knot nematodes. Coffee yields, plant health, and nematode populations were used to select potential nematode tolerant lines. Seeds and vegetative cuttings were collected and propagated from these promising lines. In addition to identifying new lines, an improved technique was developed to rapidly and more accurately assess root-knot nematode populations in coffee fields. To evaluate future cultivars for nematode resistance, select Ethiopian accessions with nematode resistance imported from Centro Agronómico Tropical de Investigación y Enseñanza (CATIE), Costa Rica by collaborators at Hawaii Agriculture Research Center, have been transferred to ARS researchers in Hilo, Hawaii, for further evaluation.
Under Sub-objective 2B, coffee trees in Kunia, Oahu, Kona and Kau, Hawaii, were either stump, stumped with nurse, hedged or umbrella pruned. Coffee was harvested throughout the season to determine yields and coffee berry borer (CBB) infestation rates. Bean size and quality are also being evaluated to determine the effect of pruning over multiple seasons.
In Sub-objective 2C, this is the first year of a project to find the efficacy of the commercial mycoinsecticide BotaniGard (Beauveria bassiana GHA) in combination with three pruning techniques (hedged, stumped and umbrella) of coffee trees, to reduce CBB infestations at two Kau coffee farms. Persistence and efficacy of Beauveria was determined for the inside, middle and outside of the tree canopy and data collection commenced in March. Initial results suggest that pruning style may affect distribution of Beauveria applications. Hedging and umbrella pruning may allow for better coverage throughout the entire canopy, but additional work is necessary to determine if current trends show significant differences from stumping.
Research continued in Objective 3 on genetic improvement of disease and pest resistances and ornamental traits in anthurium.
Under Sub-objective 3A, research identifying potential gene targets for controlling burrowing nematode in anthurium is ongoing. The transcriptome of the burrowing nematode was sequenced and assembled. Six potential targets related to locomotion and feeding were identified. To expedite the prioritization of these genes for transformation into anthuriums, carrot hairy roots will be used as a model system. The protocol for culturing and transformation of carrot hairy roots was optimized.
In previous work under Sub-objective 3B, potential organ-specific genes, as well as genes for key enzymes and regulatory genes involved in controlling the biosynthesis of the important floral pigment anthocyanin, were identified by next generation sequencing of expressed genes in anthurium. Progress has been made in development of tools for optimizing the technique of transient gene expression to further enable analysis of identified ornamental trait gene functions in this non-model plant. This includes improvements in cell imaging techniques and the construction of new reporter genes for use in anthurium and other monocots. Research continued on the identification and functional testing of organ-specific promoters for use as a tool to control color and other traits in different organs including structures unique to the floral stem of anthurium.
Under Sub-objective 3C, stable transformants of anthurium plants expressing anti-bacterial genes or reporter genes have been evaluated in the greenhouse. High ß-glucuronidase (GUS) expression has been observed throughout all tissues, including the leaves, stems and roots of the anthurium plants. Preliminary results show that transformed potted plants survived multiple inoculations with of the bacterial blight pathogen, Xanthomonas axonopodis pv. diffienbachia (Xad). In order to determine if bacterial blight populations have evolved over time, Xad populations were collected and morphologically and molecularly compared to the original Xad isolate collected in 2004, which has been used for all previous bacterial blight resistance screening. Fourteen new isolates were collected from multiple anthurium varieties containing typical bacterial blight symptoms from three farms on the east side of Hawaii Island. While there are slight differences in mucoidy on agar plates, eight gene sequences are identical for the 2018 and 2004 isolates. Pathogenicity tests are ongoing.
Accomplishments
Review Publications
Wang, D., Hamin, I., Borth, W.B., Melzer, M.J., Suzuki, J.Y., Wall, M.M., Matsumoto Brower, T.K., Sun, G.F., Hu, J.S. 2018. First report of Apple of Peru (Nicandra physalodes) infected with pepper mottle virus in Hawai'i. Plant Disease. 103(1):169. https://doi.org/10.1094/PDIS-06-18-1061-PDN.
Hamim, I., Borth, W.B., Melzer, M.J., Suzuki, J.Y., Wall, M.M., Hu, J.S. 2019. Occurrence of tomato leaf curl Bangladesh virus and associated subviral DNA molecules in papaya in Bangladesh: molecular detection and characterization. Archives of Virology. 164(6):1661-1665. https://doi.org/10.1007/s00705-019-04235-8.
Sipes B., Myers R. 2018. Plant parasitic nematodes in Hawaiian agriculture. In: Subbotin, S., Chitambar, J., editors. Plant Parasitic Nematodes in Sustainable Agriculture of North America. Sustainability in Plant and Crop Protection. Cham, Switzerland: Springer Nature. p. 193-209. https://doi.org/10.1007/978-3-319-99585-4_7.
Wang, D., Boluk, G., Quinto, E., Hamin, I., Borth, W., Melzer, M., Green, J., Suzuki, J.Y., Wall, M.M., Matsumoto Brower, T.K., Sun, G., Hu, J. 2019. First report of zucchini tigre mosaic virus infecting bitter melon (momordica charantia) in Hawaii. Plant Disease. 103(5). https://doi.org/10.1094/PDIS-08-18-1391-PDN.
