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

2020 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
Progress was made this year on all objectives and sub-objectives under Objective 1, Develop papayas with superior disease resistance and value added traits. In support of Sub-objective 1A, research is being developed to 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. Progress is being made on efficient delivery and identification of transformed papaya lines through the optimized tissue culture method developed previously. In support of Sub-objective 1B, backcrosses of the freckle free papaya with Papaya Ringspot Virus (PRSV) resistance are being grown in the field and by tissue culture. Social distancing during the pandemic has not allowed for simulations to be shipped, however, these lines continue to be maintained and additional crosses were made to allow future experiments to be conducted. In addition to these projects, we are actively involved in assisting the University of Hawaii and the Hawaii papaya farmers with generating Rainbow papaya seeds due to an increase in demand following the lava flow and shortfall in hybrid seed production. We are also actively assisting the University of Florida with a research project that compared the growth, yield and fruit characteristics of different papaya cultivars grown in Florida and Hawaii. In support of Sub-objective 2A, research continued to evaluate nematode tolerant and resistant coffee genotypes. A greenhouse bioassay was initiated to evaluate nematode resistant Ethiopian accessions against local isolates of coffee root-knot nematode. A greenhouse and field trial were established to test the efficacy of chemical and biological nematicides against coffee root-knot nematode in susceptible and tolerant plants. In support of Sub-objective 2B, research continued to evaluate pruned tree growth and prune second set of coffee trees. Total harvest yield on select trees in Kona have been collected in addition to damage caused by Coffee Berry Borer (CBB) with applications of Beauveria. Determination of the economic impact of plant growth regulators on flowering of coffee was postponed due to quarantine but plans have been made to continue this research next season. For Sub-objective 2C, research analysis continued in comparing treated and untreated field samples to determine the persistence of Beauveria: bassiana strain GHA and correlations between observed persistence and environmental conditions. In support of Sub-objective 3A, research is ongoing to identify gene targets for control of burrowing nematode in anthurium. Constructs targeting nematode locomotion and feeding were identified through transcriptome analysis and comparison with other nematode species. Candidate genes have been transformed in carrot hairy root cultures to determine the best genes to transform into anthurium. The burrowing nematode transcriptome continues to be mined with novel effector genes located in the esophageal glands and genital primordia being discovered and confirmed through in situ hybridization. In support of Sub-objective 3B, substantial progress was made toward characterizing anthurium flower color trait-related genes, regulatory genes and promoter functions through adoption and optimization of a novel, nonbacterial-mediated gene delivery system. This technique will improve the efficiency and reliability of transient gene expression analysis in intact floral tissue. Use of fluorescent protein reporter genes developed in this work with strong promoter activity in anthurium tissue was key in detecting and optimizing the gene delivery method. In support of Sub-objective 3C, transformations of anthuriums with antibacterial peptide constructs and multiplication of lines continue for screening in the greenhouse. In addition, in vitro assays for bacterial resistance has been conducted with transformed lines showing different intensities of bacterial blight symptoms and death. Fourteen recently collected isolates of the bacterial blight pathogen, Xanthomonas axonopodis pv. diffienbachia (Xad), were tested for pathogenicity and % disease severity on Anthurium andraeanum. A revised disease rating scale (0 to 9; symptoms include necrotic spots, systemic infection, % leaf drop and death) was developed. Final ratings 60 days post-inoculation ranged from systemic infection (avg. rating of 5.3) to 100% leaf drop and death (avg. rating of 9), showing that all recently collected isolates are as virulent as the original Xad isolate collected in 2004. To prepare for the screening of transformed commercial anthurium cultivars with antibacterial genes, a raised anthurium bed was constructed.

Accomplishments
1. Safety permit from China for Hawaii’s genetically modified (GM) papaya. The genetically modified (GM) Rainbow papaya is resistant to the papaya ringspot virus (PRSV) and comprises 85% of Hawaii’s production. Until recently, this GM papaya had been approved in Canada and Japan, but not in China. Market access in China would boost the economic strength of the industry. ARS researchers in Hilo, Hawaii, led a team of University and industry partners to petition the China Ministry of Agriculture for safety approval to import Hawaii’s GM papaya into China. The safety certificate was issued in December 2019. Therefore, the Rainbow papaya is the first GM fresh commodity to be deregulated in China. Completion of U.S.-China bilateral trade and quarantine agreements is the next critical step to enable shipment of Rainbow papaya to China.

2. Identification of critical control point for papaya shipping averts Hawaii fruit spoilage issue. The largest supplier of Hawaii grown papaya ships ~10 million pounds a year to the U.S. Mainland. Because Hawaii Island no longer has wide body airfreight service, the supplier has moved almost exclusively to ocean freight and reusable plastic containers (RPC) for shipping. This past year the supplier started seeing an increase in post-harvest problems and losses of upwards of 30% due to spoiled fruit reaching the mainland (total container value of $50K). ARS researchers in Hilo, Hawaii, identified the fungal pathogens causing the problem and determined the source of cross-contamination. A critical control point was identified, and recommendations were made to minimize disease incidence and severity levels incurred during ocean freight shipments. Improved standard operating procedures were adopted and losses returned to traditionally acceptable levels.

Review Publications
Wang, X., Larrea-Sarmiento, A., Borth, W.B., Barone, R., Olmedo-Velarde, A., Melzer, M.J., Suzuki, J.Y., Wall, M.M., Hu, J.S. 2020. First report of Basella alba naturally infected with basella rugose mosaic virus in Hawai'i. Plant Disease. 104(8):2296. https://doi.org/10.1094/PDIS-06-19-1266-PDN.
Larrea-Sarmiento, A., Wang, X., Borth, W.B., Barone, R., Olmedo-Verlarde, A., Melzer, M.J., Sugano, J., Galanti, R., Suzuki, J.Y., Wall, M.M., Hu, J.S. 2019. First report of bean common mosaic virus infecting flowering ginger (Alpinia purpurata) in Hawai'i. Plant Disease. 104(2):63. https://doi.org/10.1094/PDIS-06-19-1264-PDN.
Myers, R.Y., Sylva, C.D., Mello, C.L., Snook, K.A. 2020. Reduced emergence of cylas formicarius elegantulus(Coleoptera:curculionidae) from sweet potato roots by heterorhabditis indica. Journal of Economic Entomology. 113(3):1129-1133. https://doi.org/10.1093/jee/toaa054.
Larrea-Sarmiento, A., Olmedo-Velarde, A., Green, J.C., Al Rwahnih, M., Wang, X., Li, Y., Wu, W., Zhang, J., Matsumoto Brower, T.K., Suzuki, J.Y., Wall, M.M., Melzer, M.J., Hu, J.S. 2020. Identification and complete genomic sequence of a novel sadwavirus discovered in pineapple (Ananas comosus). Archives of Virology. 165:1245-1248. https://doi.org/10.1007/s00705-020-04592-9.
Myers, R.Y., Kawabata, A., Nakamoto, S., Cho, A. 2020. Grafted coffee increases yield and survivability. HortTechnology. 30(3):428-432. https://doi.org/10.21273/HORTTECH04550-20.
Wang, D., Ocenar, J.R., Hamim, I., Borth, W.B., Fukada, M.T., Melzer, M.J., Suzuki, J.Y., Wall, M.M., Matsumoto Brower, T.K., Sun, G., Ko, M., Hu, J.S. 2018. First report of bean yellow mosaic virus infecting nasturtium (tropical majus) in Hawaii. Plant Disease. 103(1):168. https://doi.org/10.1094/PDIS-06-18-1082-PDN.
Zhang, M., Yang, X., Wei, Y., Wall, M.M., Songsak, T., Wongwiwatthananukit, S., Chang, L. 2019. Bioactive sesquiterpene lactones isolated from the whole plants of Vernonia cinerea. Journal of Natural Products. 82(8):2124-2131. https://doi.org/10.1021/acs.jnatprod.8b01078.
Hamim, I., Al Rwahnih, M., Borth, W.B., Suzuki, J.Y., Melzer, M.J., Wall, M.M., Green, J.C., Hu, J.S. 2019. Papaya ringspot virus isolates from papaya in Bangladesh: detection, characterization, and distribution. Plant Disease. 103(11):2920-2924. https://doi.org/10.1094/PDIS-12-18-2186-RE.
Xu, Y., Xu, H., Wall, M.M., Yang, J. 2020. Roles of transcription factor SQUAMOSA promoter binding protein-like gene family in papaya (Carica papaya) development and ripening. Genomics. 112(4):2734-2747. https://doi.org/10.1016/j.ygeno.2020.03.009.