Project : USDA ARS

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Research Project: Molecular Systematics, Identification, Biology, and Management of Crop-Parasitic Nematodes

Location: Mycology and Nematology Genetic Diversity and Biology Laboratory

2019 Annual Report

Objectives
Objective 1: Identify and characterize nematode proteins and lipids that inhibit the metabolic pathways critical to fundamental processes in plant-parasitic nematodes. Sub-objective 1A: Discover and characterize endogenous nematode agonists or antagonists, including bioactive peptides, which affect metabolism and behavior. Sub-objective 1B: Characterize protease and other enzyme profiles in plant-parasitic nematodes, and determine their roles and modes of action in regulating development and hatching. Sub-objective 1C. Identify lipids and steroids with likely regulatory roles in plant-parasitic nematodes and design methods to disrupt this regulation. Objective 2: Improve the effectiveness of nematode management based on amendments and beneficial organisms, and by identifying and characterizing plant, microbe or nematode products antagonistic to nematodes. Sub-objective 2A: Evaluate cover crops, soil amendments and microbes for suppressing plant-parasitic nematode populations on agricultural crops. Sub-objective 2B: Determine effects of natural products from plants, microbes, and nematodes on plant-parasitic nematodes, and evaluate selected nematode-antagonistic products as potential nematotoxins. Objective 3: Assay and analyze the variability in DNA genetic markers across nematode taxa to ascertain those markers optimal for identifying priority crop parasitic nematodes and for refining their phylogenies. [NP303, C1, PS1] Objective 4: Apply microscopic and DNA genetic marker methods to a diversity of crop parasitic nematode taxa and specimens to identify unknown specimens and to improve diagnostic approaches. [NP303, C1, PS1]

Approach
1. Discovering and characterizing internal plant parasitic nematode regulatory molecules, including peptides, proteases, inhibitors, and nematode-unique lipids and steroids, that act as effectors of behavior, development, hatching and metabolism, will provide novel targets for disruption of nematode life processes. Analyzing external signals, including plant chemicals, and their interactions with internal nematode pathways, will expand the number of molecular targets for nematode control. 2. Evaluating cover crops, soil amendments, microbes, and other beneficial agents, and improving strategies for their application, will lead to enhanced plant-parasitic nematode suppression and improved plant yields. Assessing the biological effects of selected beneficial organisms, and the analysis of natural products from plant, microbial and nematode origins, will reveal novel nematotoxins and other suppressive agents for plant-parasitic nematode control and agricultural crop protection. 3. Expanding ribosomal and nuclear gene sequences to more nematode species beyond the relatively few in GenBank will produce more informative family trees demonstrating the distribution of plant parasitism for improved pathology prediction. 4. Advanced microscopy techniques and appropriately selected and designed DNA genetic markers will reveal more detailed features and increase consistency of nematode description for improved diagnosis of nematodes that may damage agriculturally important plants.

Progress Report
This report is for Project 8042-22000-300-00D “Molecular Systematics, Identification, Biology, and Management of Crop-Parasitic Nematodes.” Progress was made under Objectives 2, 3, and 4. No progress was recorded under Objective 1 due to the retirement of the two scientists associated with this objective. The work in this project falls under National Program 303, C1, PS1; C2, PS2A; and C3, PS3B. Regarding Objective 2, research was conducted with scientists from the Universities of Maryland and Hawaii with soil samples processed from field trials designed to investigate effects of cover crops, living mulches and varying tillage practices on nematode populations in the soil. In other research, selected crop plant lines were investigated to determine susceptibility to nematodes and production of nematode-antagonistic compounds. Grass lines associated with fungal endophytes were also studied for host status to root-knot nematodes, and assays conducted to determine correlation of plant chemistry with activity against nematodes. Several separate studies determined nematotoxicity of natural products tested as extracts and/or solutions. These investigations are important for enhancing or developing methods for managing plant-parasitic nematodes and improving crop yields. As part of Objective 3, multiple molecular markers were developed and used for parasitic and other nematodes, thereby improving identification and construction of comprehensive phylogenetic trees. New primers were adapted to more reliably generate 18S ribosomal DNA markers for nematodes previously difficult to amplify. Primers were also produced to generate a much longer ribosomal DNA sequence spanning three regions for more accurate and rapid molecular diagnoses. In collaboration with scientists from University of Alexandria, Egypt, the first studies on resistance of selected plant species and cultivars to the root-knot nematodes Meloidogyne spp. was determined. For Objective 3B nematodes were enumerated, imaged, described and DNA sequences generated from long-term Farming System Plots in Beltsville, Maryland. For Objectives 3 and 4, three new species of plant-parasitic nematodes were identified and/or described using microscopic techniques to detail anatomical features. Two new lesions, one new stunt parasitizing soybean in North Dakota, three new country records of cyst nematodes (Heterodera medicaginis, Punctodera stonei, Heterodera pratensis) on alfalfa fields in Kansas, Montana, and on golf courses from Southern Oregon and Michigan, respectively, were discovered. In addition, three new records of quarantine important nematodes, one root-knot (Meloidogyne enterolobii), one lesion (Pratylenchus fallax) and one cyst nematode (Vittatidera zeaphila) parasitizing guava, soybean and corn in South Carolina, Wisconsin and Indiana, respectively, were recorded. Bursaphelenchus antoniae found in the U.S. on white pine in Massachusetts, was characterized. Confirmation that October 2018 inoculation of Litylenchus crenatae parasitizing American beech trees and involved in Beech Leaf Disease in an Ohio greenhouse was the same nematode reisolated in May 2019 was made, providing evidence that Beech Leaf Disease may be caused by a nematode. As part of the service component of Objective 4, ARS scientists provided nematode identifications and expertise during the last year for over 430 samples submitted by various customers for research, regulation and control purposes. The USDA Nematode Collection, one of the largest in existence, was curated, managed and expanded with the addition of 414 valuable slides and vials, bringing the total number to 50,069. Twenty-five slides were loaned to scientists, and a computerized internet-accessible database was updated for the collection with thousands of sample records on hosts, occurrence and distribution. Over 200 specimen records were entered in the database for a total of 50,115 current records. During FY 2019 accurate nematode identifications and related expertise were provided for 282 urgent plant and soil samples intercepted by Animal Plant Health Inspection Service (APHIS) at ports-of-entry or during domestic surveys. Domestic surveys included national surveys for the presence of potato pale cyst nematodes and surveys in New York and Idaho to delineate the distribution of golden and pale cyst nematodes and the viability of cysts following soil fumigation. Nematodes identified from plant materials intercepted at ports of entry included a variety of cyst, root-knot, lesion, dagger, foliar, bulb and stem, pin, spiral, lance, and ring nematodes. Related expertise provided to APHIS included information about the distribution and pathogenicity of many of these nematodes as well as potentially invasive nematode species, information on morphological and molecular protocols useful for identification, and information about which nematode species pose threats to agriculture. The results of these identifications were used by APHIS for taking appropriate regulatory actions beneficial to growers and the public. These taxonomic reports and resources are used by research scientists, extension agencies and regulatory action agencies involved in nematode research and control.

Accomplishments
1. Vetiver grass shoots suppress plant-parasitic nematodes. Plant-associated nematodes (roundworms) harm billions of dollars’ worth of crop plants around the world each year. ARS scientists in Beltsville, Maryland, and collaborators in Thailand discovered that amending soil with shoots of vetiver cv. Sierra suppressed root-knot nematode populations on cucumber roots. Vetiver is an economically important bunch grass that is widely used in tropical areas for oil production, reduction of soil erosion, and to conserve water, improve soil and reduce weeds. This work demonstrated that planting vetiver and/or amending soil with vetiver plant parts could reduce numbers of nematodes that attack commercially important plants. This work will assist growers as part of a broad strategy for suppression of plant-parasitic nematodes on vegetable crops.

2. Nematodes (roundworms) cause beech leaf disease (BLD) symptoms. Beech leaf disease causes severely deformed leaves and tree mortality to American and European beech and has been spreading in Ohio, Pennsylvania, New York, and Ontario, Canada since 2012. ARS researchers in Beltsville, Maryland, and USDA Forest Service, the Holden Arboretum and Canadian collaborators confirmed that nematodes could cause symptoms associated with beech leaf disease in North America. This research is being used by scientists, extension agents, growers, forest managers and quarantine officials to understand the cause, manage the disease where it currently occurs and prevent its spread into new areas.

3. Camellia tea seed cake and tree peony extracts suppress plant-parasitic nematodes. Plant-associated nematodes (roundworms) harm billions of dollars’ worth of crop plants around the world each year. ARS scientists in Beltsville, Maryland, and Chinese collaborators discovered that extracts from tea seed cake and tree peony stems suppressed egg hatch and killed juveniles of soybean cyst and root-knot nematodes. Tea seed cake applied as a soil amendment reduced soybean cyst nematode populations in greenhouse trials. These studies provide knowledge about the nematicidal activity of these natural byproducts. This work will assist growers and plant disease management professionals as part of a broad strategy for suppression of plant-parasitic nematodes on vegetable crops.

4. New DNA primers improve DNA identification of nematodes. Identification and distinction of plant-parasitic or plant beneficial nematodes can be difficult due to limited morphological differences. Molecular tools called primers for identifying nematodes with their DNA sequences are used like a fingerprint but are sometimes inadequate for certain nematode species. ARS scientists in Beltsville, Maryland, developed new DNA primers to improve the identification of plant-associated nematodes. This work will result in accurately and reliably identified nematodes associated with globally traded commodities and will be used by agricultural researchers, plant health professionals, and plant disease diagnosticians in the U.S. and other countries.

U.S. DEPARTMENT OF AGRICULTURE

Mycology and Nematology Genetic Diversity and Biology Laboratory: Beltsville, MD