Location: Tropical Plant Genetic Resources and Disease Research
2023 Annual Report
Objectives
Objective 1: Develop papayas with superior disease resistance and value added traits.
Sub-objective 1A: Generate papaya lines with wide-spectrum resistance to papaya ringspot virus.
Sub-objective 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.
Sub-objective 2A: Evaluate coffee rootstocks to identify genotypes with resistance or tolerance to root-knot nematodes under field conditions.
Sub-objective 2B: Evaluate horticultural practices to identify those that yield optimal vegetative growth, flowering, fruiting, and quality of coffee.
Sub-objective 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.
Sub-objective 2E: Conduct microbial genomic and bioinformatics research to elucidate the content and genetic structure of beneficial and pathogenic soil microbiome communities associated with the rhizosphere of macadamia trees and coffee plants, as a means of controlling pathogens occupying soils and assembling soil microbial communities that maximize macadamia tree and coffee plant health and productivity.
Objective 3: Genetic improvement of disease and pest resistances and ornamental traits in anthuriums.
Sub-objective 3A: Identify and assess the efficacy of selected transgenes for controlling burrowing nematode, Radopholus similus in anthurium.
Sub-objective 3B: Identify metabolic pathways and molecular components governing novel flower color traits in anthurium.
Sub-objective 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.
Sub-objective 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.
Sub-objective 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.
Sub-objective 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.
Sub-objective 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.
Sub-objective 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.
Sub-objective 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.
Sub-objective 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.
Sub-objective 3C: Use molecular transformation to generate and assess anthurium plants with transgenes for resistance to plant-parasitic nematodes and bacterial diseases.
Progress Report
This is the final report for project 2040-21000-017-000D, titled "Genetic Improvement and Sustainable Production Systems for Sub-tropical and Tropical Crops in the Pacific Basin", which terminated on 5/30/2023. It has been replaced by new project 2040-21000-019-000D, titled, “Crop Health and Genetic Improvement of Sub-Tropical and Tropical Crops in the Pacific”. For additional information about the new research objectives and sub-objectives, please review the new project report.
Substantial results were realized over the five years of this project. The goal of Objective 1 was to develop papayas with superior disease resistance and value-added traits. Scientists bred for improved papaya with a blemish-free appearance and papaya ringspot virus resistance. In support of Sub-objective 1A, ARS researchers in Hilo, Hawai'i conducted to generate papaya lines with wide-spectrum resistance to papaya ringspot virus (PRSV). 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 the likelihood of host resistance to PRSV. Isolation of high molecular weight (HMW) DNA has been optimized and long read, > 50 X coverage whole genome sequencing in papaya achieved to identify cultivar-specific alleles for virus and disease-resistance traits and off-target editing sites. These protocols are being applied for sequencing novel cultivars and recalcitrant tropical crops. An efficient, non-destructive method for monitoring gene expression using visible reporter gene(s) is being used to optimize transformation and transient gene expression to increase the efficiency of CRISPR for disease resistance and transgene-based horticultural trait improvement. In collaboration with university partners, new plant viruses continue to be identified, and diagnostics optimized for the detection and management of viral diseases of papaya and other economically important tropical crops. In support of Sub-objective 1B, selected lines of `Sun Up’ and N08-75 hybrids were backcrossed to the N08-75 parent. Offspring were selected for PRSV resistance conferred by the transgene in the ‘Sun Up’ parent. The selected lines with blemish-free appearance and PRSV resistance were crossed with ‘Kapoho’ to develop material with yellow-fleshed papaya for Hawaii’s export market. In addition, selections for the larger Mexican papaya resistant to the Hawaiian strain of PRSV are complete. A plant variety protection and release of this new cultivar are being explored with our CRADA partner. In addition, ARS researchers provided technical expertise to the University of Hawaii at Manoa to generate ‘Rainbow’ papaya seeds for the Hawai'i papaya industry.
The goal of Objective 2 is to devise integrated horticultural management practices and enhanced germplasm to improve plant health, yield, quality, and product value of coffee. In support of Sub-objective 2A, ARS researchers in Hilo, Hawai'i evaluated 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. The field trial evaluated the tolerance of six coffee varieties used as rootstocks to Meloidogyne konaensis, the Kona coffee root-knot nematode. Plant health and yield were evaluated and compared to non-grafted Arabica. Four varieties of the Liberica species and one Robusta variety had high yields and optimal plant health in contrast to low yields and high mortality in non-grafted trees. In addition to identifying new lines, an improved technique was developed to assess root-knot nematode populations rapidly and more accurately in coffee fields. A greenhouse bioassay was conducted to evaluate the nematode resistance and tolerance of wild Ethiopian accessions and coffee leaf rust (CLR)-resistant varieties. The Ethiopian accessions demonstrated high levels of root-knot nematode resistance compared to the CLR-resistant varieties Obata and Tupi-HI, which were highly susceptible and intolerant to the nematode. A greenhouse bioassay was conducted to evaluate six chemical and biological nematicides on coffee for their efficacy in controlling M. konaensis. Plants treated with fluopyram had the highest growth rates and lowest nematode populations. A coffee field trial evaluating spirotetramat, Burkholderia, and compost resulted in the compost-treated trees having the highest yields for two seasons. In support of Sub-objective 2B, research was conducted on coffee trees in Kunia, Oahu, Kona, and Kau, Hawaii. Coffee trees were either stumped, stumped with a nurse, hedged, or umbrella pruned. Umbrella-pruned trees had the highest yields and Kona-style trees had the highest coffee berry borer (CBB) infestation. The determination of the economic impact of plant growth regulators on the flowering of coffee was postponed due to the expiration of the Special Local Needs Label for the plant growth regulators. ARS researchers are working with researchers at the Hawai'i Department of Agriculture to obtain an exemption from crop destruct requirements. Due to the discovery of CLR in Hawaii, fungicide tests were conducted and the translaminar fungicide Priaxor Xemium effectively controlled this disease. In support of Sub-objective 2C, research has been published on the persistence of Beauveria bassiana (Bb) GHA and correlations between observed persistence and environmental conditions to control CBB. Briefly, the recommendations include: 1) Integrated pest management (IPM) guidelines provided by the University of Hawaii should be followed to the greatest extent possible. Early-season control practices are most critical in controlling initial female CBB infecting the coffee berries. 2) Sanitation is the single most effective control practice and reduces the need for Bb 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 < 27 degrees C. 5) Beauveria sprays should be applied during the late afternoon–evening, with reduced solar radiation and 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 3 weeks is suggested. 7) After CBB is established in a substantial portion of the coffee berries, frequent harvests or pre-harvest strip picking is the most effective control practice.
The goal of Objective 3 is the genetic improvement of disease and pest resistance and ornamental traits in anthuriums. In support of Sub-objective 3A, ARS researchers have identified gene targets to control burrowing nematode in anthurium. The transcriptome of the burrowing nematode was sequenced and assembled. The transcriptome continues to be mined with novel effector genes in the esophageal glands and genital primordia being discovered and confirmed through in situ hybridization. Six constructs targeting effector and movement genes of burrowing nematodes were evaluated in a model carrot hairy root system. Three constructs prevented nematode reproduction with reproductive factors lower than 1.0 when evaluated with in-vitro assays. The most promising lines supported 400x fewer nematodes than untransformed roots after two months. The transcriptome also revealed novel effector genes from the esophageal glands of the nematode. These genes were validated using a PVX-based vector in tobacco. In support of Sub-objective 3B, research was conducted to develop tools to optimize transient gene expression to analyze gene functions, including improving cell imaging techniques and constructing new reporter genes for use in anthuriums and other monocots. A new method was developed to characterize anthurium flower color trait-related genes, regulatory genes, and promoter functions through optimization of a novel, nonbacterial-mediated gene delivery system. The 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, research was conducted on the stable transformants of anthurium plants. High transgene expression has been observed throughout all tissues, including the leaves, stems, and roots of the greenhouse grown anthurium plants. Preliminary results show that transformed potted plants survived multiple inoculations with the bacterial blight pathogen, Xanthomonas axonopodis pv. diffienbachia (Xad). At least two promising anthurium lines have been re-initiated into tissue culture from the previously inoculated greenhouse studies. These plants are being grown and multiplied for additional testing. 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. Fourteen new isolates were collected from multiple anthurium varieties containing typical bacterial blight symptoms from three farms on the east side of Hawai'i Island. While there are slight differences in mucoidy on agar plates, eight gene sequences are identical for the 2018 and 2004 isolates. The 14 Xad isolates were tested for pathogenicity and % disease severity on anthurium plants. 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 isolates collected in 2004.
Accomplishments
1. Grafting coffee leaf rust resistant cultivars onto nematode-tolerant rootstock. Root-knot nematodes are a devastating pest in coffee production, causing reduced yield and plant vigor. With the discovery of Coffee Leaf Rust (CLR) in Hawaii, growers are interested in growing new CLR-resistant coffee cultivars; however, the susceptibility of these cultivars is unknown. ARS researchers at Hilo, Hawaii, have identified that CLR-resistant cultivars Obata and Tupi-HI are susceptible to root-knot nematodes and should be grafted onto nematode tolerant rootstocks. This research will enable Hawaii coffee growers to produce plants tolerant to both CLR and nematodes.
Review Publications
Kawabata, A., Myers, R.Y., Miyahira, M., Yamauchi, N., Nakamoto, S.T. 2023. Field efficacy of spinetoram for the management of coffee berry borer (Hypothenemus hampei). Insects. 14(3). Article 287. https://doi.org/10.3390/insects14030287.
Liu, F., O'Donnell, T., Park, E., Kovacs, S., Nakamura, K., Dave, A., Luo, Y., Sun, R., Wall, M.M., Wongwiwatthananukit, S., Silva, D., Williams, P., Pezzuto, J., Chang, L. 2023. Anti-inflammatory quinoline alkaloids from the roots of Waltheria indica. Journal of Natural Products. 86(2):276-289. https://doi.org/10.1021/acs.jnatprod.2c00861.
Budhathoki, S., Sipes, B.S., Shikano, I., Myers, R.Y., Manandhar, R., Wang, K. 2022. Integrating trap crops and entomopathogenic nematode foliar sprays to manage diamondback moth and imported cabbage worm. Horticulturae. 8(11). Article 1073. https://doi.org/10.3390/horticulturae8111073.
Ramirez-Camejo, L.A., Keith, L.M., Matsumoto Brower, T.K., Sugiyama, L.S., Fukada, M., Brann, M., Moffitt, A., Liu, J., Aime, M.C. 2022. Coffee leaf rust (Hemileia vastatrix) from the recent invasion into Hawaii shares a genotypic relationship with Latin American populations. The Journal of Fungi. 8(2). Article 189. https://doi.org/10.3390/jof8020189.
Myers, R.Y., Mello, C.L., Nagai, C., Sipes, B.S., Matsumoto Brower, T.K. 2023. Evaluation of Coffea arabica cultivars for resistance to Meloidogyne konaensis. Agriculture. 13(6). Article 1168. https://doi.org/10.3390/agriculture13061168.
