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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 #434947

Research Project: Genetic Improvement and Sustainable Production Systems for Sub-tropical and Tropical Crops in the Pacific Basin

Location: Tropical Plant Genetic Resources and Disease Research

2021 Annual Report

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.

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
Progress was made this year on most objectives and sub-objectives which fall under National Program 301, Crop Genetic Improvement, Problem Statement 1B: New crops, new varieties, and enhanced germplasm with superior traits. This project contributes to the National Program Anticipated Products/Accomplishments by providing: 1) crop plants with resistance or tolerance to diseases and pests, 2) crop plants with superior product quality for consumers and producers, and 3) new crops and current crops with new traits for new uses. In support of Sub-objective 1A, research is being conducted on Clustered Regularly Short Palindromic Repeats (CRISPR)/Cas9 or Cas9/subgenomic RNA (sgRNA) directed mutation of the endogenous papaya gene eIF4E or eIF(iso)4E gene for the development of broad viral resistance in commercial papaya cultivars. Protocols for improving the delivery of transgenes into plant cells and tissue, a bottleneck for efficient application of gene editing, are currently being developed in papaya. Improved gene delivery methods aim to support the greater utility of CRISPR technology for agronomic trait improvement in papaya and wider adoption in other crop plants. In support of Sub-objective 1B, the final selection of the freckle-free parental lines of papaya has been completed and will be crossed with the Kapoho parental line. These plants will produce seeds that will produce yellow-fleshed papaya for Hawaii’s export market and will be resistant to the Hawaiian strain of Papaya Ringspot Virus (PRSV). Selections for the larger Mexican papaya that are resistant to the Hawaiian strain of PRSV are complete. We will be working with our Cooperative Research and Development Agreement (CRADA) partners to explore a plant variety protection and release of this new cultivar. We continue to assist the University of Hawaii and the Hawaii papaya farmers with generating Rainbow papaya seeds and assisted researchers at the University of Florida in completing their research project that compared the growth, yield, and fruit characteristics of different papaya cultivars in Florida and Hawaii. In support of Sub-objective 2A, research on evaluating nematode tolerant and resistant coffee genotypes is ongoing. A greenhouse bioassay to evaluate nematode-resistant Ethiopian accessions and coffee leaf rust-resistant varieties against local isolates of the coffee root-knot nematode was completed. A greenhouse and field trial to test the efficacy of chemical and biological nematicides against coffee root-knot nematode in nematode susceptible and tolerant genotypes is ongoing. In support of Sub-objective 2B, research continued to evaluate pruned tree growth and impact of coffee leaf rust and coffee berry borer (CBB) on tree health and harvest quality. Determination of the economic impact of plant growth regulators on flowering of coffee was postponed due to the expiration of the Special Local Needs Label for the plant growth regulators. We are working with the Hawaii Department of Agriculture to obtain an exemption from the crop destruct requirement. For Sub-objective 2C, research has been published on the persistence of the Beauveria bassiana (Bb) strain GHA and correlations between observed persistence and environmental conditions to control Coffee Berry Borer. Briefly, the recommendations include: 1) Integrated Pest Management guidelines provided by University of Hawaii should be followed to the greatest extent possible. Early-season control practices are most critical in controlling initial female infecting the coffee berries, 2) Sanitation is the single most effective control practice, and reduces the need for Beauveria bassiana applications, 3) All feral coffee trees in unmanaged areas bordering fields should be removed or at least subject to strict sanitation, 4) Bb strain GHA-based biopesticide products are highly temperature sensitive, storage at temperatures less than 27 degrees C (naturally cool storage spaces at high elevation, or air-conditioned spaces at low elevations), 5) Beauveria sprays should be applied during late afternoon–evening, with reduced solar radiation and when no rainfall is forecast, 6) In areas with high CBB present, spraying after monitoring is suggested, however if a calendar schedule is necessary, a maximum interval of three weeks is suggested, and ) After CBB is established in a substantial portion of the coffee berries especially in August/September frequent harvest or pre-harvest strip picking is the most effective control practice. In support of Sub-objective 3A, research to identify gene targets for control of burrowing nematode in anthurium continued. Constructs targeting nematode effector genes and genes responsible for locomotion were developed and transformed in carrot hairy root cultures for evaluation. Six constructs were screened against burrowing nematode in laboratory bioassays with four of the constructs effectively reducing nematode populations. The most promising lines supported 400x less nematodes than untransformed roots after two months. Additional novel effector genes were discovered from the burrowing nematode transcriptome and confirmed through in situ hybridization. In support of Sub-objective 3B, further progress is being made in optimization of a novel, nonbacterial-mediated gene delivery system for functional analysis of ornamental trait genes in different floral tissue targets including development of protocols for improved imaging and detection of transient transgene expression. In support of Sub-objective 3C, the developed gene delivery and transient transgene expression system is used to evaluate floral trait genes from anthurium and other species for transformation and production of anthurium with novel ornamental traits. Stable transformants for bacterial blight resistance are being multiplied for potted and simulated field assays in cinder beds. These lines are being challenged with Xanthomonas axonopodis pv. diffienbachia (Xad), through cuts with razors dipped in bacterial suspensions for potted assays and sprays with bacterial suspensions for the bed assays.

1. An integrated management response to coffee berry borer in Hawaii. The coffee berry borer (CBB), known as the most devastating insect pest to affect coffee worldwide, has greatly impacted Hawaii’s coffee industry (valued at $100M per year). Since large-scale applications of Beauveria bassiana (Bb) strain GHA had never been used in Hawaii prior to the introduction of the CBB, recommendations for more accurate use for CBB integrated pest management (IPM) were required. ARS researchers at Hilo, Hawaii, and Ithaca, New York, compared timing and frequency of various BotaniGard® application rates applied in grower managed fields, assessed their impact on CBB, and adjusted techniques as necessary for Hawaii conditions resulting in improved recommendations using various control treatments. This research provides coffee growers valuable tools for CBB management, including the use of Bb as a key control component of an IPM strategy, and is being used in the Hawaiian Islands to improve the production and quality of coffee by reducing CBB to manageable levels.

Review Publications
Myers, R.Y., Bushe, B., Mello, C.L., Lichity, J., Hara, A., Wang, K., Sipes, B.S. 2020. Yield increases in burrowing nematode-infested anthurium with fluopyram and trifloxystrobin applications. HortTechnology. 30(5):603-607.
Laurora, A., Bingham, J., Poojary, M.M., Wall, M.M., Ho, K. 2021. Varietal differences in carotenoid composition and their bioaccessibility from papaya cultivars in Hawaii. Journal of Food Composition and Analysis. 101. Article 103984.
Castrillo, L.A., Wraight, S.P., Galaini-Wraight, S., Matsumoto Brower, T.K., Howes, R.L., Keith, L.M. 2020. Genetic diversity among naturally-occurring strains of Beauveria bassiana associated with the introduced coffee berry borer, Hypothenemus hampei, (Coleoptera: Curculionidae) on Hawai'i Island. Journal of Invertebrate Pathology. 175.
Puig, A. S., Keith, L. M., Matsumoto, T. K., Gutierrez, O. A., & Marelli, J. P. (2021). Virulence tests of Neofusicoccum parvum, Lasiodiplodia theobromae, and Phytophthora palmivora on Theobroma cacao. European Journal of Plant Pathology. 159:851–862.
Puig, A.S., Quintanilla, W.E., Matsumoto Brower, T.K., Keith, L.M., Gutierrez, O.A., Marelli, J. 2021. Phytophthora palmivora causing disease on theobroma cacao in hawaii. Phytopathology. 11(5). Article 396.
Waisen, P., Wang, K., Uyeda, J., Myers, R.Y. 2021. Effects of fluopyram and azadirachtin integration with sunn hemp on nematode communities in zucchini, tomato and sweet potato in Hawaii. Journal of Nematology. 53:1-15.