Recipient Organization
UNIV OF IDAHO
875 PERIMETER DRIVE
MOSCOW,ID 83844-9803
Performing Department
(N/A)
Non Technical Summary
Global crop yields are estimated to decline about 20-30% per year because of plant pests and pathogens, threatening worldwide food security. Plant-parasitic nematodes (PPNs) represent particularly pernicious pathogens responsible for causing crop losses exceeding $100 billion annually. Farmers employ diverse strategies to mitigate nematode damage. These approaches include crop rotation, soil sterilization using plastic coverings, and the cultivation of nematode-resistant crop varieties, all of which contribute to managing the issue. However, the most potent and efficient solution remains the application of pesticides to treat fields. For decades, synthetic nematicides have been pivotal in managing PPNs, but legitimate concerns regarding their environmental toxicity and human safety have led to justified bans on most used nematicides. For example, Oxamyl, a systemic compound with basipetal translocation properties, stands as the sole commercially available product utilized for foliar treatment. However, its use as a liquid formulation is restricted in many countries due to concerns related to toxicity. This has resulted in a glaring scarcity of effective agents for PPN control. Out of the 20 key nematicides utilized during the twentieth century, only 4 presently hold approval for use in the European Union, with a mere 3 available in the USA without constraints. While these regulatory actions are warranted, they have left farmers with limited options and no viable means of controlling numerous PPNs. In this proposed study, economically important potato cyst nematode (PCN) Globodera pallidais selected as our target control to manage crop health and maximize yields. G. pallidais a globally regulated pest of potato (Solanum tuberosum) and is of great economic importance in many countries throughout the world. It is a quarantine pest in the state of Idaho where it was detected in 2006. G. pallidais a highly specialized obligate endoparasitic nematode that requires a living potato plant as a host to complete its life cycle. In highly infested fields, the nematode can reduce tuber yields up to 80% and is spread mainly through the movement of soil, tubers, or farm equipment. Significantly, not only does PCN cause major yield losses in potato industry, PCN-infested and surrounding fields are regulated by USDA-APHIS and ISDA. Control of soil-borne pests and diseases has traditionally been accomplished by soil fumigation. The most widely used compound has been volatile methyl bromide which is toxic and has been phased out as a class 1 ozone depleting substance under the Montreal Protocol (https://www.epa.gov/ods-phaseout/methyl-bromide). Infested fields are now being fumigated with the less effective soil fumigant, Telone. Hence, there is an urgent need to develop new classes of nematicides with novel bioactivity, that are effective whether applied to soil or directly to crops, while remaining environmentally benign and specifically designed to target the intended pests.Nematodes generate a distinctive, evolutionarily conserved group of pheromones known as ascarosides. These signaling molecules play pivotal roles in orchestrating nematode's developmental processes and facilitate communication both within nematode's own community and with other organisms. Ascarosides are the derivatives of dideoxy sugar ascarylose, undergoing combinations with diverse lipophilic side chains derived from fatty acids and other moieties originating from primary metabolism. It has been reportedthat ascaroside Ascr#18 acting as a pathogen-associated molecular pattern (PAMP) triggers plantdefense mechanism including activation of (a) mitogen-activated protein kinases, (b) salicylic acid- and jasmonic acid-mediated signaling pathways, and (c) defense gene expression. Moreover, the plant innate immunity elicited by Ascr#18 offers comprehensive protection against a broad spectrum of pathogens. Ascaroside pheromones also play a significant role in the chemotaxis behavior of nematodes, particularly in their response to environmental cues and interactions with other organisms. Different ascarosides can have both attractant and repellent effects on nematodes, depending on their concentration and the specific ascaroside involved. Some ascarosides attract nematodes towards favorable conditions, such as areas rich in food sources, while others can repel them from unfavorable environments or deter them from potential threats.Conventional pesticides often enter the environment through processes such as leaching, volatilization, and rainwater runoff, leading to a notable reduction in their effectiveness. This loss of pesticides has significant adverse effects on ecosystems and poses health hazards to humans. Therefore, the development of innovative technologies to mitigate pesticide losses, improve utilization efficiency, and reduce associated pollution is of paramount importance. In recent years, a range of slow-release pesticides has emerged, utilizing technologies such as microcapsules, micro/nano composites, and organosilicone. While these innovations have managed to reduce losses and enhance pesticide utilization efficiency to some degree, precise control of their release to meet crop requirements and significantly boost overall utilization efficiency has proven challenging. This limitation has hindered their widespread adoption. Consequently, the development of controlled-release pesticides has become a focal point in both agricultural and environmental fields.Historically, conventional synthetic hydrogels typically made from acrylic and polyacrylamide materials were utilized in agriculture. Nevertheless, their lack of biodegradability raised concerns about potential pollution. Additionally, the gradual breakdown of synthetic polymers in agricultural applications could negatively impact soil fertility. Given the growing emphasis on environmental issues, biodegradable hydrogels have gained preference over their synthetic counterparts in certain situations. Biodegradable hydrogels offer advantages due to their renewability, biocompatibility, and non-toxic properties, making them an appealing choice for agricultural purposes such as moisture retentionand pest control.Poly-γ-glutamic acid (γ-PGA), initially identified in Bacillus anthracis, is a naturally occurring biopolymer composed of glutamic acid monomers linked by amide bonds between γ-carboxylic acid groups. γ-PGA has garnered considerable interest for its versatile applications in the medical, agricultural, and food sectors, owing to its unique attributes of biocompatibility, biodegradability, and water solubility.γ-PGA hydrogels feature a three-dimensional network structure capable of absorbing substantial amounts of water and expanding in volume without dissolution. Consequently, they hold promise as delivery systems, not only for controlled pesticide release but also for water conservation in agricultural settings, such as mitigating water loss through surface evaporation and facilitating rehydration via irrigation or rainfall. For this proposed study, we intend to develop light-responsive γ-PGA based hydrogels for controlled nematicide delivery.
Animal Health Component
30%
Research Effort Categories
Basic
40%
Applied
30%
Developmental
30%
Goals / Objectives
The long-term goal of this proposed project is to provide agriculture in the USA with environmentally friendly approaches for better water management, pest and disease control, crop protection, and agricultural production. The goal will be achieved through (1) light-responsive poly-γ-glutamic acid (γ-PGA) tethered with nematode pheromone (ascaroside) for controlled nematicide delivery, and (2) study of the efficacy of ascaroside-tagged photocleavable γ-PGA in planta under UV-light exposure with resistance to nematode infection. Accomplishment of this goal would greatly contribute to the rural economy, the welfare of farmers, and our society in general.Objectives: Control of pests and pathogenic diseases has traditionally relied on pesticide/fungicide sprays. These tools not only threaten the human health and ecosystem but also generate uncontrollable multidrug-resistant pathogenic strains. Thus, alternative approaches to manage pests in a sustainable and eco-friendly manner are needed to maintain high crop production. Using natural compounds instead of synthetic compounds for pest and disease control is much desirable because they are environmentally friendly. Together with previous findings [1,2], our preliminary results suggest that one of the ascarosides (i.e., Ascr#18) has great potential for use as a pesticide against potato cyst nematode infection on potato. Since Idaho leads the United States in potato production and produces nearly 1/3 of all U.S.potatoes (Idaho State Department of Agriculture - https://agri.idaho.gov/main/about/about-idaho-agriculture/idaho-crops), harnessing Ascr#18 for pest control is pertinent approach for Idaho's and Pacific Northwest's potato industry. However, ascarosides, including Ascr#18, are glycosides of the dideoxysugar ascarylose with a lipophilic side chain and only moderately stable and undergoes degradation over time. It is hypothesized that the activity of Ascr#18 can be extended if it is tethered with light-responsive biopolymer and slow control release of Ascr#18 is achievable by photo-cleavage under UV light exposure. With control delivery of such an approach, we could achieve greater effectiveness of much small dose in a prolonged releasing time frame without negative environmental consequences.For this seed grant proposal, our team will initiate a proof-of-concept investigation of the anti-nematode effectiveness of ascaroside-tagged photocleavable γ-PGA via foliar spray under UV-light irradiation. The successful outcome of this proposed study will lay the framework for PD and Co-PD to pursue future AFRI grants. To accomplish the goals set by this seed grant project, specific studies will be conducted with the following objectives.Objective 1: Fabrication of light responsive Ascr#18-functionalized γ-PGA for controlled nematicide deliveryObjective 2: Efficacy of ascaroside-tagged photocleavable γ-PGA in planta under UV-light exposure with resistance to nematode infection
Project Methods
Methods for Objective 1Design and synthesis of ?-PGA functionalized with photocleavable linker As shown in Scheme 1, γ-PGA (1, cat#G1049, Sigma-Aldrich) will first react with 3-azido-1-propylamine (2, cat #762016, Sigma-Aldrich) which is a building block used to modify carbonyl groups in the presence of activators such as 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC). The introduction of azide functionality into γ-PGA through stable amide bond leads to γ-PGA-azide (3 in Scheme 1) which will be used for subsequent ligation to terminal alkynes via Cu-catalyzed azide alkyne 1,3-dipolar cycloaddition (CuAAC, a.k.a. click reaction). In this task, the influence of the molar ratio of the reagents, the pH of the medium, and the reaction time on the degree of substitution (represented k in Scheme 1) of the product gamma-PGA-azide (3) will be studied. Photocleavable linker 1-(5-methoxy-2-nitro-4-prop-2-ynyloxyphenyl)ethyl N-succinimidyl carbonate (4) used for the click reaction will be synthesized according to the reported protocol [35]. In brief, 1-(5-methoxy-2-nitro-4-prop-2-ynyloxyphenyl)ethanol (cat#B23571117, BenchChem) will be added in dry acetonitrile in the presence of trimethylamine, N,N'-disuccinimidyl carbonate. The reaction will be protected from light and stirred under nitrogen atmosphere at room temperature for 5 h. The reaction mixture will then be concentrated under reduced pressure. The product (4) will be extracted with chloroform. Then, γ-PGA-azide will react with the synthesized photocleavable linker via CuAAC to obtain gamma-PGA functionalized with photocleavable linker (5). All of the products (3, 4, and 5) will be characterized by FT-IR and 1H NMR spectroscopy. We expect the azido group introduced into gamma-PGA will be confirmed by the characteristic band of the stretching vibrations of the azido group at 2,100 cm-1 in the FT-IR spectrum of azido derivative 3. Additionally, the 1H NMR spectra of products 3 and 5 will be similar, which confirms the intact of the polymer structure of γ-PGA.Light-responsive control release of Ascr#18 In order to have Ascr#18 (cat#HY-N8393, MCE) ligated to γ-PGA functionalized with photocleavable linker (5), ethylenediamine (NH2CH2CH2NH2, cat#E26266, Sigma-Aldrich), with short chain length ensuing minimal steric effect and virtually no hydrophobic interaction, will be employed for amino-alkylation of Ascr#18 by forming amide bond via EDC carbodiimide-mediated coupling (product 6 in Scheme 2). Then, γ-PGA functionalized with photocleavable linker (5) reacts with the amine (NH2) endowed on Ascr#18 via the succinimidyl ester to form Ascr#18-tagged photo-responsive γ-PGA (7) with the release of N-hydroxysuccinimide. Once product 7 is exposed to UV irradiation wavelength = 350 nm), Ascr#18 will be released via the photo-cleavage reaction of the nitrobenzyl group in the photocleavable linker with the release of CO2.Chemotaxis assay Given that in vitro treatment with Ascr#18 can attract nematode J2 worms, we will follow the published protocols to set up the chemotaxis plates to determine the efficiency of Ascr#18 release upon UV irradiation. Chemotaxis plates will be prepared by pouring 8 mL of a 2% agar solution into 6-cm Petri dishes. Before introducing the G. pallida J2 worms, 10 µL of a buffer solution, containing varying amounts of Ascr#18-tagged photoresponsive γ-PGA or a buffer solution alone, will be dispensed on opposite sides of the plate. Approximately 100 J2 worms will be positioned in the central region of the plates. Subsequently, the plates will undergo UV light irradiation at a wavelength of 350 nm for different durations, followed by incubation in a 25°C environment for 4 hours. Afterwards, worm counts on both sides of the plates will be conducted, excluding those remaining within the central 0.5-cm-wide strip. The chemotactic index will be calculated as (A - B) / (A + B), where A and B represent the numbers of nematodes on the side with the test solution and the side with the mock solution, respectively. To serve as a positive control, an experiment will be carried out in which the Ascr#18-tagged photo-responsive γ-PGA solution will be substituted with an Ascr#18-containing solution. Three of most promising Ascr#18-tagged photo-responsive γ-PGAs obtained from this task will be used for the Objective 2 studies.Methods for Objective 2Following the task of Objective 1, given that Ascr#18 can be used as a pesticide for disease control, particularly dealing with potato cyst nematode in Idaho, we will determine the efficacy of Ascr#18-tagged photocleavable γ-PGA in plants and evaluate resistance to potato cyst nematode conferred by application of Ascr#18-tagged photocleavable γ-PGA under UV-light irradiation, which will be accomplished by examining 1) the activation of immune signaling in potato cells triggered by Ascr#18-tagged photocleavable γ-PGA; and 2) the resistance to G. pallida in potato upon foliar application of Ascr#18-tagged photocleavable γ-PGA under the exposure of UV-light.Determine the immune signaling by Ascr#18-tagged photocleavable γ-PGA In order to determine the effectiveness of Ascr#18-tagged photocleavable γ-PGA in plant cells. We will examine whether Ascr#18-tagged photocleavable γ-PGA can activate both local immune signaling in the inoculated area and systemic immune signaling in the distant area. To this end, potato leaves will be sprayed with Ascr#18-tagged photocleavable γ-PGA or un-coupled Ascr#18 with and without UV-light irradiation. The local induction of immune signaling will be determined by examining the expression of three immune marker genes (FRK1, GSTF6, and PR1) in potato leaves at 6 and 12 hours after spraying, whereas the systemic activation of immune signaling will be determined by the expression of immune marker genes in potato roots at 24 hours after foliar spraying. Gene expression levels will be determined by quantitative real-time PCR assay using gene specific primers, which will reflect the capacity of Ascr#18-tagged photocleavable γ-PGA exposed to UV-light to trigger both local and system immune signaling in comparison with un-coupled Ascr#18.Evaluate Ascr#18-tagged photocleavable γ-PGA triggered resistance to nematode (G. pallida) under UV-light exposure To verify the resistance to PCN G. pallida triggered by foliar application of Ascr#18-tagged photocleavable γ-PGA under UV-light exposure, relatively younger 4-week-old potato plants will be sprayed with Ascr#18-tagged photocleavable γ-PGA or uncoupled Ascr#18. 48 hours after foliar application, potato plants will be inoculated with G. pallida cysts (10 cysts per plant) in the root zone. To evaluate infection rate, half of tested plants will be subjected for an acid fuchsin assay to score all nematode life stages six weeks post-inoculation. To evaluate reproductive success, fully formed cysts will be recovered from the dry soil of remaining plants using an elutriator ten weeks post-inoculation. We will determine the number of cysts formed per plant, number of eggs per cyst, egg viability (Meldola blue staining), and calculate the nematode reproduction factor [Rf = (final egg population density)/(initial egg population density)]. This comprehensive assessment will allow us to evaluate the efficacy of Ascr#18-tagged photocleavable γ-PGA triggered resistance to G. pallida in comparison with the un-coupled Ascr#18.