Recipient Organization
UNIV OF IDAHO
875 PERIMETER DRIVE
MOSCOW,ID 83844-9803
Performing Department
(N/A)
Non Technical Summary
To meet the dietary needs of an estimated 9.5-10 billion people by 2050, it is critical to sustain the efficient production of nutritionally dense staple foods such as potatoes. Potato is the third most important food crop worldwide, but a leading crop for pesticide usage due to the prevalence of invasive pests such as potato cyst nematodes (PCN), Globodera pallida and G. rostochiensis. Currently to control PCN producers rely on synthetic pesticides that are not always effective, costly, and can inflict harm on non-targeted beneficial nematode species.To address this issue, we propose a novel approach for control of PCN by capitalizing on specific host-pest relationship of PCN that relies on chemical signaling for plant host location and infection. For example, other plants from Solanum family such as Solanum sisymbriifolium can also promote hatch of and be infected by PCN; however, they do not allow nematode reproduction. The proposed project will test the combination of mustard derived 4-hydroxybenzyl alcohol as a nematode eggshell permeability enhancer and nematocidal compounds from Solanum sisymbriifolium to develop a "smart nematicide" system that will selectively target PCN while sustaining beneficial species. Quantifying and characterizing of nematicides will be done using chromatography and mass spectrometry techniques. In vitro and in planta nematode bioassays will be used for the system efficiency evaluation.Our research project aligns with the A1112 Pests and Beneficial Species in Agricultural Production Systems program goal to develop innovative biologically-based strategies to manage pests and falls under following program objectives: 1) abiotic factors affecting the abundance or spread of agriculturally-important plant pests and 2) behavioral attributes of pests and communication systems relevant to pest management.
Animal Health Component
100%
Research Effort Categories
Basic
(N/A)
Applied
100%
Developmental
(N/A)
Goals / Objectives
While we are focusing on the potato cyst nematodeG. pallidaas a model pest of agricultural and quarantine significance, the proposed work is fundamental for advancing our understanding of nematode-plant interaction and ways to control them in the field. Traditional nematode control methods such as use of fumigants are not specific, generally toxic to the environment, and can result in unanticipated crop contamination. We are proposing to develop a selective nematode control by capitalizing on natural mechanism of nematode-host plant interactions. We are proposing to develop a "smart chemical" system that is based on combining a specific hatch promoter with a natural nematicidal compounds, a concept that can be extended to other plant-parasitic nematodes.Solanum species are known to synthesize secondary metabolites with antimicrobial, nematicidal and other toxicological properties. In fact, crude plant extracts from S. sisymbriifolium can inhibit hatch of G. pallida.3 Specifically, our study showed significant inhibition of G. pallida hatching by the S. sisymbriifolium root and leaves extracts even in the presence of potato root diffusate containing hatching stimulants. This effect can partially be attributed to glycoalkaloids - compounds that are naturally present in the solanaceous family. Specifically, when four pure glycoalkaloids (solamargine, solasodine, solamarine, and solasonine) were used, G. pallida hatch rate decreased up to six-fold but only in the presence of potato root diffusates.3 This suggests that the hatching factor present in potato root diffusate may increase eggshell permeability which may be important for diffusion of the large glycoalkaloid molecule into the egg. Thus, enhancement of PCN egg permeability may be required for nematicidal action of these highly toxic compounds.The development of novel approaches for control ofG. pallidais highly needed to keep potato production profitable and sustainable while preserving soil and environment health. One of such approaches is to capitalize on specific host-pest relationship ofG. pallidathat relies on chemical signaling for activation of hatch, and host location and infection. By utilizing the chemistry of these natural cues, targeted, efficient, and selective "smart nematicides" system for PCN control can be developed. Availability of such "smart nematicides" will advance the sustainability of U.S. agriculture by helping to solve a critical constraint on production of a major food crop, reducing reliance on environmentally damaging chemical fumigation, and providing biologically based control methods for cyst and other nematodes that burden crop production. The implication of "smart nematicides" will also potentially improve the profitability and economic viability of farm operations and related economic sectors.Our aim is to develop a biologically based nematicidal material for control of G. pallida, a quarantine pest of the staple food, potato. The goal of this project is to develop a targeted "smart nematicide" system by exploring the chemistry of S. sisymbriifolium, a highly effective trap crop for G. pallida that is also resistant to root knot and lesion nematodes. We plan to study the efficacy of Solanaceous nematocidal compounds on PCN life cycle and pursue optimizing nematocidal potential by pairing with HBA, a compound that we have demonstrated to enhance hatch of PCN. Specific objectives are:Objective 1: Quantify the ability of S. sisymbriifolium fractionated extracts or pure glycoalkaloid compounds to disrupt population development through inhibition of hatch, parasitism, and reproduction of G. pallida.Objective 2: Identify potential nematocidal compounds from S. sisymbriifolium by target screening and activity-guided separation.Objective 3: Evaluate changes in expression of oxidative stress enzyme activity in G. pallida in response to pure glycoalkaloids and fractionated extracts from S. sisymbriifolium to understand the possible nematocidal mechanism of these bioactive compounds.Objective 4: Optimize the targeted action of S. sisymbriifolium nematocidal compounds by activating nematode eggs with HBA.
Project Methods
Objective 1. Quantify the ability of S. sisymbriifolium fractionated extracts or pure glycoalkaloid compounds to disrupt population development through inhibition of hatch, parasitism, and reproduction of G. pallidaPlant material preparation, glycoalkaloids extraction and fractionation. Solanum sisymbriifolium plants will be grown under greenhouse condition for 6 weeks. Freeze-dried leaf or root samples will be exhaustively extracted with methanol using Omni Prep homogenizer. A methanolic extract will be reconstituted in water and fractionated using liquid-liquid sequential extraction using hexane, dichloromethane, ethyl acetate, and n-butanol to selectively collect glycoalkaloids and other bioactive compounds based on their chemical properties. Each fraction will be analysed by Agilent 6230 HPLC Time-of-Flight mass spectrometer (TOF MS) and evaluated for biological activity toward G. pallida. Reverse phase porous and HPLC chromatography separation columns will be used to accommodate the polarity range of chemicals. Fractions with largest biological potential will be subjected to metabolomic analysis. Identification will be based on analytical standards or software packages that integrate chemical databases with mass spectrometry cheminformatics.In vitro PCN hatching inhibition and viability assays. Standard in vitro hatching and viability assays will be performed by incubating cysts with plant fractions or pure glycoalkaloids at different concentrations (200 ug/ml & 100ug/ml) with PRD and PRD containing HBA for 7 or 14 days. Treated cysts will be transferred to 96 well plates containing PRD or sterile water and incubated at 20?. Hatched juveniles (J2) will be counted after 14 days and percentage hatching will be calculated as: (total number of J2)/ (total egg remained in the cysts) × 100. Viability assessments will be made with the fluorescent acridine orange stain. Treatments will be replicated, and experiments repeated. Data will be analysed through Glimmix procedure of SAS.Deleterious effect of toxins on PCN. The impact of bioactive S. sisymbriifolium fractions and pure glycoalkaloids on PCN infection and parasitism, will be conducted through in planta bioassays by using previously published protocols. Briefly, the PCN cysts after incubation with bioactive compounds will be transferred onto potato and maintained in the greenhouse. Infection and life stage development will be assessed by the acid fuchsin method. Reproduction efficiency will be evaluated by extracting cysts 16 weeks post planting into soil infested with treated cysts. All treatments will be replicated, and experiments will be repeated. Treatment significance will be analysed by using the Glimmix procedure in SAS.Objective 2: Identify potential nematocidal compounds from S. sisymbriifolium by target screening and activity-guided separation.Glycoalkaloids purification and fractionation from S. sisymbriifolium. Glycoalkaloids with high biological activity will be purified from extracts using column chromatography, silica gel or RP-18 gel. Briefly, preparative chromatography will be carried out using Agilent 1100 Prep HPLC in reversed-phase Luna® (5 µm, 100 Å, 250×10 mm) column (Phenomenex, Torrance, CA) at the flow rate of 3.0?mL/min. Detection will be monitored by UV/vis detector. Collected fractions will be separated on semi-preparative Agilent 1100 HPLC DAD/RID system and then analysed by HPLC TOF MS. Identification of S. sisymbriifolium bioactive fractions. One and two-dimensional (1D and 2D) nuclear magnetic resonance (NMR) will determine molecular structure and stereochemistry. Bruker AVANCE III HD 500 MHz NMR equipped with a broadband SMART probe and with a PRODIGY broadband cold probe will acquire 1D and 2D NMR spectra over 200 - 800 Da range. Coupling constants in hertz, and chemical shifts in δ (parts per million, ppm) will be provided with tetramethyl silane as an internal standard. Structural elucidation of chemical group will be done by TOF MS.4 Structural characteristics will be assessed through ionization and identified based on published fragmentation patterns for glycoalkaloids.Objective 3: Evaluate changes in expression of oxidative stress enzyme activity in G. pallida in response to pure glycoalkaloids and fractionated extracts from S. sisymbriifolium to understand the possible nematocidal mechanism of these bioactive compounds.Extraction, purification, and quantification of protein from PCN. Protein extraction and lysis buffer systems (G Bioscience) will be used for the total protein extraction from eggs/juveniles. Proteins will be quantified by using the bicinchoninic acid method; concentrations will be estimated by the protein standard, Bovine Serum Albumin. All treatments will be replicated, and experiments repeated, and the data will be analysed using Softmax Pro software.Enzyme activity assays for PCN detoxification enzymes. To study mechanisms of nematicidal activity, S. sisymbriifolium fractions treated PCN eggs and juveniles will be analysed for the following enzymatic activities: catalase, peroxidase, glutathione peroxidase, and superoxide dismutase.Objective 4: Optimize the targeted action of S. sisymbriifolium nematocidal compounds by activating nematode eggs with HBA. Hatching assays of nematode cysts treated with glycoalkaloids and isolated glycoalkaloid fractions combined with 4-HBA will be conducted as described in the Objective 1. A range of concentrations of both HBA and glycoalkaloids will be used to identify the optimum combination. To study the changes in enzyme activity, protein extraction and quantification form PCN eggs will be achieved by the methods described in Objective 1. Enzyme activity will be analysed by the methods described in Objective 1.