Location: Tropical Plant Genetic Resources and Disease Research
2024 Annual Report
Objectives
Hawaii is one of the most geographically isolated areas in the world. Although this geographic isolation has been beneficial in the past, increased global transportation and commerce have provided a pathway for introducing invasive pests and pathogens. This isolation also results in higher transportation costs for importing essential farm supplies such as fertilizer, pesticides, and other supplies and exporting goods to the continental U.S. and global market. In sub-tropical and tropical growing climates, plant diseases flourish in warm, humid environments. Understanding disease development is the first step to effective control methods, either through chemical control or enhanced techniques for breeding and deploying resistant crops. Objective 1 of this plan aims to develop methods for early detection, mitigation, and management of diseases in economically important crops in Hawaii. It focuses on addressing a potentially destructive disease in coffee.
Objective 1: Generate new knowledge of disease etiology and the epidemiology of emerging invasive species for tropical crops.Sub-objective 1.A: Identify and characterize emerging pathogens attacking important sub-tropical and tropical crops including cacao, mango, and avocado. Sub-objective 1.B: Identify and characterize emerging Colletotrichum spp. attacking coffee.
Objective 2 of this proposal addresses the need to develop and screen new coffee varieties for disease resistance, productivity, and cupping quality for long-term sustainable production. In the meantime, cultural control methods and pesticides must be investigated for short-term solutions to combat these pests and pathogens.
Objective 2: Develop or characterize new genotypes, sustainable management strategies, or combined approaches to control pests and diseases for tropical crops. Sub-objective 2.A: Develop disease management strategies for coffee leaf rust control. Sub-objective 2.B: Evaluate coffee varieties for resistance to coffee leaf rust, root-knot nematode, horticultural characteristics, and cupping quality from World Coffee Research (WCR), Hawaii Agriculture Research Center (HARC), and other coffee sources.
Objective 3 utilizes comparative transcriptome profiling of nematode infected roots to identify genes in coffee that trigger resistance to root-knot nematode. Furthermore, objective 4 implements the use of these genes for nematode resistance in bananas and anthuriums.
Objective 3: Increase genomic information for important tropical crops to accelerate gene discovery and germplasm improvement. Sub-objective 3.A: Identify genes that are associated with resistance to root-knot nematodes in coffee. Sub-objective 3.B: Identify genes for product quality traits in tropical crops.
Objective 4: Improve disease resistance and value-added traits in tropical crops using conventional breeding and biotechnology. Sub-objective 4.A: Utilize RNAi technology targeting novel effector genes to control burrowing nematode, Radopholus similis. Sub-objective 4.B: Develop papaya lines with universal resistance to PRSV.
Approach
Novel and sustainable strategies for controlling pathogens and pests are needed to decrease production costs while maintaining high product quality. In order to receive premium prices, these specialty products must maintain high quality both in appearance and taste. The goals of this research are to 1) develop methods for early detection and characterization of plant diseases; 2) develop sustainable management strategies for coffee leaf rust and new rust-resistant varieties; 3) develop genomic tools for nematode resistance, novel color, and prevention of browning; and 4) develop new genotypes for resistance to nematodes and papayas resistant to Papaya ringspot virus. The specific goals and approaches for each objective are noted below.
Objective 1: Develop a system for early and accurate identification of emerging plant pathogens as a crucial first step to limiting the spread, reducing their economic impact, and preventing future disease outbreaks. Sub-objective 1A: Develop a system for early and accurate identification of emerging plant pathogens as a crucial first step to limiting the spread, reducing their economic impact, and preventing future disease outbreaks. Sub-objective 1B: The species of Colletotrichum infecting coffee in Hawaii can be genetically classified and sources of resistance can be identified among coffee accessions.
Objective 2: Develop or characterize new genotypes, sustainable management strategies, or combined approaches to control pests and diseases for tropical crops. Sub-objective 2A: Determine which fungicides are most effective in controlling CLR and optimize the strategy and timing for rotation of products so that maximum control can be achieved and fungicide resistance and copper toxicity can be avoided.. Sub-objective 2B: Evaluate coffee varieties for resistance to coffee leaf rust, root-knot nematode, horticultural characteristics, and cupping quality from World Coffee Research (WCR), Hawaii Agriculture Research Center (HARC), and other coffee sources.
Objective 3: Increase genomic information for important tropical crops to accelerate gene discovery and germplasm improvement. Sub-objective 3A: Identify resistance genes against M. konaensis from nematode-resistant coffee cultivars and develop molecular markers to increase the efficiency of nematode resistance screening for coffee breeding programs. Sub-objective 3B: Develop a reference genome and perform RNA sequencing to provide information on metabolic genes involved in quality traits such as color and browning for cultivar improvement in anthurium.
Objective 4: Improve disease resistance and value-added traits in tropical crops using conventional breeding and biotechnology. Sub-objective 4A: Burrowing nematode reproduction will be suppressed on roots expressing dsRNA from nematode effector genes. Sub-objective 4B: Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 or Cas9/ sub genomic RNA (sgRNA) directed mutation of an endogenous papaya gene eIF4E or eIF(iso)4E gene will confer broad viral resistance and result in commercial papaya cultivars with universal resistance to PRSV and related viruses.
Progress Report
This report documents the progress for project, “Crop Health and Genetic Improvement of Sub-Tropical and Tropical Crops in the Pacific”. The approval of this project occurred after the annual report deadline for FY23, milestones for years one and two were combined into the following report.
In support of Sub-objective 1A, ARS scientists in Hilo, Hawaii, continued their research efforts to isolate and identify emerging pathogens. Many macadamia trees across Hawaii Island are exhibiting increased symptoms of slow decline, including loss of vigor, leaf chlorosis, defoliation, canopy thinning, and death of the branch tips. Isolations from branch and trunk samples collected throughout Hawaii Island have yielded 37 non-Phytophthora isolates. Isolates were identified by morphology and molecular analysis. Pathogenicity studies have begun with 10 isolates and two-year-old macadamia nut seedlings. Currently four potential pathogens are causing a range of slow decline symptoms. Numerous commercial fungicides were tested in vitro for antimicrobial activity against growth of three species of Phytophthora (cinnamomi, heveae, and tropicalis) known to cause disease in macadamia, all of which are present in Hawaii, and two of which are responsible for macadamia tree losses on Hawaii Island due to Macadamia Quick Decline. Results suggest that commercially available products of varying active ingredients may be effective at combating Phytophthora species infecting and killing macadamia nut trees. Screening for Phytophthora-resistant macadamia germplasm using seedling bioassays is ongoing. Seventeen commercial and noncommercial varieties have been testing for resistance to four Phytophthora species, including P. cinnamomi, P. tropicalis, P. heveae, and P. palmivora. Results indicate that tolerance or resistance is present in the macadamia collection. In addition, surveys were conducted, and samples were collected to identify emerging pathogens. In the past year, disease samples (including fruit, leaves, stems, and trunks) from cacao, coffee, macadamia, mango, soursop, cherimoya, Monstera, and Red Ginger were collected or received. A total of 71 isolates were recovered and identified using both morphology and molecular analysis. Pathogenicity trials are being conducted with 20 isolates from cacao, coffee, and mango. Many are suspect pathogens that are emerging or re-emerging. Cacao mild mosaic virus (CaMMV) surveys were conducted at the germplasm repository greenhouses and fields, and at multiple farms across Hawaii Island and Oahu. More than 900 cacao samples were tested, and results suggest that CaMMV is widespread in Hawaii.
For Sub-objective 1B, Colletotrichum isolation and identification is ongoing. In the past year, over 20 Colletotrichum isolates have been isolated from diseased leaves, stems, and fruits of cacao, coffee, mango, macadamia, soursop, and Red Ginger. Isolates were identified by morphology and molecular analysis. Pathogenicity studies have been conducted with five coffee and two mango isolates.
Under Sub-objective 2A, a field trial was conducted at a coffee farm in Holualoa from June to December 2023. Eleven products were applied to trees at varying timings throughout the trial period, and coffee leaf rust (CLR) incidence data was collected. Results provide new management strategies for coffee leaf rust control. In-vitro testing has been used interchangeably with in-planta testing in growth chambers in the laboratory as the first attempt at screening new products against CLR. Three environmentally friendly, biobased products were applied to potted coffee plants which were subsequently inoculated with Hemileia vastatrix. Incidence and severity data was recorded weekly for eight weeks. Results for two of the products show promise to protect coffee seedlings against CLR infection. In a separate experiment, four fungal isolates recovered from a CLR mycoparasite survey were applied to potted coffee plants. Studies on potential biocontrol of CLR are ongoing.
In support of Sub-objective 2B, hundreds of F2 Catimor X Hawaiian commercial varieties from the Hawaii Agricultural Research Center (HARC) have been screened for CLR resistance using optimized leaf disc assays. Results suggest greater than 60% of the crosses show tolerance or resistance to CLR. Testing is ongoing. World Coffee Research (WCR) varieties are currently being propagated for testing and leaf disc assays will be utilized for screening when sufficient leaf material is available.
In addition, five coffee varieties from HARC were screened for coffee root-knot nematode resistance and tolerance. Two CLR-resistant varieties were found to be intolerant and susceptible to the nematode and grafting with nematode tolerant rootstock is recommended. Three wild accessions, susceptible to CLR, were found to be resistant to root-knot nematode and good candidates for the coffee breeding program. A greenhouse bioassay is ongoing to screen ten Catimor crosses from the University of Hawaii, Tropical Agriculture and Human Resources (UH CTAHR). A survey for root-knot nematodes in coffee plantings was conducted on Oahu and Hawaii Island. Species-specific primers were developed for the coffee root-knot nematode for more efficient molecular identification of samples. New Coffee Leaf Rust resistant cultivars have been successfully imported and are currently being propagated under an USDA Animal and Plan Health Inspection Service (APHIS) Controlled Import Permit. These lines include coffee plants from the WCR Innovea Arabica Breeding Network and five commercial cultivars through an agreement with San Francisco Bay Coffee. In addition, ARS scientists continue to propagate a local Coffee Leaf Rust resistant cultivar and have sent this to HARC for mass multiplication, as well as lines from the WCR International Multi-Location Variety Trial.
In support of Sub-objective 3A, coffee varieties were initiated into tissue culture using somatic embryogenesis. These plantlets are currently being acclimated in the greenhouse and grown out for comparative transcriptome analysis. Somatic embryos of a nematode tolerant variety were transferred to HARC for further multiplication and distribution to growers for use as a rootstock for new CLR-resistant varieties. In support of Sub-objective 3B, progress was made in improving quality and amount of high molecular weight (HMW) DNA from limited tissue to enable long read sequencing of the anthurium genome for the discovery of genes to improve ornamental, horticultural traits. DNA isolation protocols have been applied for routine sequencing of other recalcitrant tropical crops, including papaya, and will be useful where germplasm tissue is limited. In addition, multiple isolation methods have been tested to identify one that yields intact RNA to support full-length RNA sequencing in anthurium for information on genes important for ornamental or quality traits such as color or browning.
Under Sub-objective 4A, disease-free anthurium and banana are being propagated in tissue culture for transformation with nematode resistance genes.
In support of Sub-objective 4B, research is continuing for Clustered Regularly Short Palindromic Repeats (CRISPR)/Cas9 or Cas9/subgenomic RNA (sgRNA)-directed editing of the papaya gene eIF4E or eIF(iso)4E gene for development of broad viral resistance in commercial papaya cultivars. Chromosome level whole genome sequencing and assembly in papaya has been optimized to improve gene editing design and detection of cultivar-specific alleles for virus and disease-resistance. Application of non-destructive methods for monitoring transient gene expression with visible reporter gene(s), including the Ruby gene for beet color, is supporting gene delivery optimization for transformation and gene editing for plant disease resistance and horticultural trait improvement in papaya and anthurium. Improved gene delivery systems will advance understanding of function of new genes and trait development in other specialty and recalcitrant crops.
Accomplishments
1. First ever potential coffee leaf rust hyperparasite discovered in Hawaii. Coffee leaf rust (CLR, Hemileia vastatrix), is an aggressive, economically devastating pathogen of coffee plants worldwide. Coffee leaf rust was discovered in Hawaii in 2020 and quickly spread throughout the state. Conventional fungicides have played a major role in the suppression of CLR, but due to a recent shift toward eco-friendly farming practices, additional novel, effective, and sustainable strategies for CLR control are needed. To meet this need, ARS researchers in Hilo, Hawaii, conducted the first ever survey of fungi associated with CLR lesions on Hawaii Island. Potential fungal antagonists were isolated and identified and several of the genera are known antagonists of CLR or other plant pathogens. This critical first step may lead to new technologies to help safeguard Hawaii’s coffee industry against H. vastatrix, protect its economic viability, and preserve its cultural heritage.
2. A new bacterium killing the popular ornamental plant, Monstera, discovered in Hawaii. Monstera, a tropical plant with unique leaf holes often referred to as the Swiss cheese plant, is a popular ornamental house plant that is also sold as cut foliage in Hawaii. At a plant nursery in Hilo, all variegated plants in four-inch pots were showing signs of infecttion, with something causing large brown, necrotic spots, making them unmarketable. ARS researchers in Hilo, Hawaii, put on their sleuthing hats to determine what was causing this problem using microbiology, molecular techniques, and pathogenicity studies. The plant pathogenic bacterium, Pseudomonas cichorii, was determined to be causing the disease, and once identified, the ARS researchers were able to provide recommendations to the industry to avoid further spread and protect the marketability of this ornamental plant prized for its leaves.
Review Publications
Han, C., Raksat, A., Atanu, M., Chang, L., Wall, M.M., Chang, L. 2023. Investigation of antimicrobial, antioxidant, and cytotoxic activities of Boesenbergia rotunda rhizome extract. Journal of Current Science and Technology. 14(1). Article 20. https://doi.org/10.59796/jcst.V14N1.2024.20.
Raksat, A., Atanu, M., Mendez, S., de la Zerda, R., Sun, R., Cheenpracha, S., Wall, M.M., Simmons, C.J., Williams, P.G., Tan, G.T., Wongwiwatthananukit, S., Chang, L. 2024. Bioassay-guided isolation and identification of cytotoxic compounds from Melaleuca quinquenervia fruits. ACS Omega. 9(16):17691-18687. https://doi.org/10.1021/acsomega.4c00769.
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.
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.
Budhathoki, S., Sipes, B.S., Shikano, I., Myers, R.Y., Manandhar, R., Wang, K. 2022. Integrating trap cropping and entomopathogenic nematode foliar sprays to manage diamondback moth and imported cabbage worm. Horticulturae. 8(11). Article 1073. https://doi.org/10.3390/horticulturae8111073.
Luiz, B.C., Sugiyama, L.S., Brill, E., Keith, L.M. 2024. Survey of potential fungal antagonists of coffee leaf rust (Hemileia vastatrix) on Coffea arabica in Hawai‘i, USA. Brazilian Journal of Microbiology. https://doi.org/10.1007/s42770-024-01304-2.