Location: Tropical Plant Genetic Resources and Disease Research
2022 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.
Sub-objective 2D: Evaluate selected coffee genetic resources and experimental mapping and breeding populations for resistance and susceptibility to different genetic types of coffee leaf rust (CLR); apply integrated genomic and quantitative genetic approaches to the evaluation results to ascertain the quantitative and functional genetic bases for coffee host-plant resistance to CLR; and armed with the preceding knowledge, devise and apply optimal genetic enhancement and breeding designs and strategies to breed superior new coffee varieties with resistance to CLR, profitable yields, and superior cupping quality.
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
In support of Sub-objective 1A, research is continuing on Clustered Regularly Short Palindromic Repeats (CRISPR)/Cas9 or Cas9/sub-genomic ribonucleic acid (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 isolation of high molecular weight (HMW) DNA are being optimized to enable single molecule long read, whole genome sequencing, gene editing, characterization of cultivar-specific alleles for disease-resistance and other agronomic traits in different genetic backgrounds of papaya and other tropical crops. Testing of newly constructed reporter genes is continuing with the aim of improving transformation and transient gene expression of CRISPR and other gene components through sensitive monitoring of gene delivery in recalcitrant cultivars or species. In support of Sub-objective 1B, we are hybridizing the freckle-free parental lines of papaya with select lines of Kapoho and testing the progeny of this cross in the field for comparison with the industry standard, Rainbow papaya. In addition, we are increasing the seeds of the larger Mexican papaya plants that are resistant to the Hawaiian strain of papaya ringspot virus (PRSV). We are also assisting the University of Hawaii and the Hawaii papaya farmers with generating Rainbow papaya seeds.
In support of Sub-objective 2A, the evaluation of coffee genotypes for resistance or tolerance to Kona coffee root-knot nematode is ongoing. Nematode resistance bioassays are being conducted on semi-wild Ethiopian Arabica accessions and genotypes resistant to coffee leaf rust (CLR). Nematode populations, plant growth, and root health of infected plants are being compared to genotypes with known nematode tolerance or resistance. The results will inform growers if new CLR-resistant genotypes should be grafted to nematode-tolerant rootstocks before planting. A greenhouse bioassay evaluating chemical and biological nematicides against Kona coffee root-knot nematode has been completed and a corresponding field trial is ongoing. The nematicide fluopyram reduced nematode populations and increased plant vigor, although it is not yet labeled for coffee in Hawaii.
For Sub-objective 2B, pruned coffee plants are being monitored. We are awaiting Hawaii Department of Agriculture approval to have the plant growth regulator treatment be exempt from crop destruct. Due to the recent discovery of Coffee Leaf Rust in Hawaii, we are also researching different fungicide applications to determine the best control of this disease and effects it may have on coffee production.
In support of Sub-objective 3A, research to identify gene targets for control of burrowing nematode continued. Nematode resistance bioassays were repeated for six constructs to evaluate reproduction of burrowing nematode on transformed carrot hairy root lines. These constructs target movement genes and effector genes derived from the esophageal glands in the nematode. Lines containing the three most promising constructs resulted in nematode reproductive factors below 1.0 signifying high levels of resistance. In addition, other promising novel effector genes discovered in the burrowing nematode transcriptome were transiently expressed in tobacco using a potato virus X (PVX)-based vector for gene validation.
In support of Sub-objective 3B, research is continuing for improvement of gene delivery systems to enable direct functional characterization of candidate ornamental trait genes in floral tissue targets. Further improvement of gene delivery and gene function analyses are being made for efficient transformation and production of anthurium with novel color, new ornamental traits and other horticulturally beneficial traits.
In support of Sub-objective 3C, greenhouse trials are on-going for evaluating bacterial tolerance/resistance in anthuriums. Promising lines are being placed back into tissue culture to be clonally propagated and multiplied for retesting.
Accomplishments
Review Publications
Heureux, A.M., Matsumoto Brower, T.K., Keith, L.M. 2022. Toward a zero-waste model: Potential for microorganism growth on agricultural waste products in Hawaii. Algal Research. 62. Article 102640. https://doi.org/10.1016/j.algal.2022.102640.
Wang, X., Larrea-Sarmiento, A., Olmedo-Velarde, A., Borth, W., Suzuki, J.Y., Wall, M.M., Melzer, M., Hu, J. 2022. Complete genome organization and characterization of Hippeastrum latent virus. Virus Genes. 58:367-371. https://doi.org/10.1007/s11262-022-01901-z.
Larrea-Sarmiento, A., Olmedo-Velarde, A., Wang, X., Borth, W., Matsumoto Brower, T.K., Suzuki, J.Y., Wall, M.M., Melzer, M., Hu, J.S. 2021. A novel ampelovirus associated with mealybug wilt of pineapple (Ananas comosus). Virus Genes. 57:464-468. https://doi.org/10.1007/s11262-021-01852-x.
Wang, X., Olmedo-Velarde, A., Larrea-Sarmiento, A., Simon, A.E., Kong, A., Borth, W., Suzuki, J.Y., Wall, M.M., Hu, J.S., Melzer, M. 2021. Genome characterization of fig umbra-like virus. Virus Genes. 57:566-570. https://doi.org/10.1007/s11262-021-01867-4.
