Project : USDA ARS

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Research Project: Plant-associated Nematode Management and Systematics and USDA Nematode Collection Curation

Location: Mycology and Nematology Genetic Diversity and Biology Laboratory

2023 Annual Report

Objectives
Objective 1. Improve molecular methodologies for the identification and classification of nematodes to better predict and respond to nematode outbreaks in food and forage crops. (NP303, C1, PS1B) Objective 2. Curate and expand the USDA Nematode Collection, including enhancing the availability of web-based research information to support accurate morphological nematode identification. (Service-oriented; NP303, C1, PS1B) Objective 3: Identify and determine the efficacy of management approaches, natural products, and beneficial organisms for managing plant-parasitic nematodes. (NP303, C3, PS3B). Sub-objective 3A: Evaluate cover crops, soil amendments, and beneficial microbes for suppressing plant-parasitic nematode populations on agricultural crops. Sub-objective 3B: Determine effects of natural products on plant-parasitic nematodes, and evaluate selected products as potential nematotoxins.

Approach
Plant-parasitic nematodes pose a serious threat to crop productivity in the U.S., causing extensive reductions in yield and quality. This project comprises a multifaceted approach to identifying nematodes of food and forage crops and controlling the damage they cause. Accurate nematode identifications will be achieved through (1) analysis of acquired morphological and molecular data; (2) molecular phylogenetic studies aimed at clarifying or establishing evolutionary relationships and species boundaries; and (3) combining the above with biogeographic, pathogenicity, and host range data to achieve integrated phylogenetic schemes to improve risk assessments and tracking of emerging nematode outbreaks. The service component activities within this project will (4) provide authoritative identification of plant-associated nematodes to USDA-ARS, USDA-APHIS, and other state and federal researchers and action agencies; and (5) provide reference specimens from the USDA Nematode Collection to scientists around the world. The USDA Nematode Collection and database will be expanded and modernized through (6) digital documentation of slides, drawings, and associated specimen information. Moreover, (7) we will attempt recovery of DNA from formalin-fixed specimens to liberate a previously untapped source of molecular information from key collection specimens. Novel molecular targets for nematode control, including genes involved in key nematode life processes or parasitism will be identified via (8) genomic and transcriptomic methods and next-generation sequencing. New bio-based methods to control plant-parasitic nematodes and improved strategies for their application will be identified through (9) evaluation of cover crops, soil amendments, microbes, and other beneficial agents; or through (10) isolation and analysis of natural products from plant, microbial and nematode origins to identify nematotoxic compounds or other agents with nematode-suppressive activity.

Progress Report
Progress was made on all three objectives, which fall under NP303. Under Objective C1 PS1B and C3, PS3B. For progress under Objective 1, ARS researchers in Beltsville, Maryland, provided nematode identifications and expertise for over 412 samples submitted by various customers for research, regulation, and control purposes. Detailed morphological and molecular descriptions were generated for several nematodes representing new state or country records obtained from field surveys or international trade interceptions, including: the barley root-knot nematode Meloidogyne naasi from golf course turfgrass in Idaho and from an unknown host in Pennsylvania, a Boleodorus species isolated from industrial hemp in Maryland, burrowing nematode Radopholus bridgei on the aquarium ornamental Anubias barteri from Indonesia, and cyst nematode Heterodera ustinovi from a golf course in New York. New insights into the phylogenetic relationships among several cyst nematode isolates from Greece were demonstrated through graphical representation of DNA marker variability. These studies provide critical anatomical and molecular information necessary for accurate species identifications and support management and regulatory measures. Significant advances on molecular characterization of the beech leaf disease nematode, Litylenchus crenatae mccannii includes the development of microsatellite markers to further elucidate its geographical origin, likely number of introductions, and pathways of dispersion in North America. Esophageal gland cell transcriptome libraries were generated for the first time for L. crenatae mccannii and a detailed microscopic assessment of the cellular basis of beech leaf disease provides new insights into the mechanism of infection. Improved understanding of the nematode’s life history and disease progression will be used by scientists, extension agents, forest managers and quarantine officials to eventually inform methods to effectively mitigate the spread of this disease. In regard to Objective 2, accurate nematode identifications and related expertise were provided for 40 urgent plant and soil samples intercepted by APHIS at ports-of-entry or during domestic surveys. National surveys for the presence of potato pale cyst nematodes and surveys in New York and Idaho 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, foliar, dagger, stunt, ring, spiral, rice root, several free living, nematodes of both Rhabditid and Dorylaimid groups as well as burrowing nematodes. Related expertise provided to APHIS included information about the distribution and pathogenicity of many of these nematodes and potentially invasive nematode species, 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 implantation of regulatory actions beneficial to growers and the public. Taxonomic reports and resources are used by research scientists, extension agencies and regulatory action agencies involved in nematode research and control. The USDA Nematode Collection was expanded by 657 additional slides and 111 vials, bringing the specimen total to 68,079. Thirteen samples (about 150 specimens) were given to scientists for molecular analysis. An internet-accessible database was updated with thousands of sample records on hosts, occurrence, and distribution. Over 534 specimen records were entered in the database for a total of 56,490 current records. Under Objective 3, ARS scientists conducted research on the identification and characterization of genes from root-knot nematodes (Meloidogyne javanica and M. incognita) that encode a class of peptide effectors with high sequence similarity to those found in both bacterial and plants. Synthetic versions of similar peptides stimulate root growth in Arabidopsis, and silencing of the corresponding genes decreased their expression and reduced root galling and egg production, suggesting that these peptides serve as nematode virulence factors. These investigations are important for enhancing or developing methods for managing economically important root-knot nematodes. Other research is focused on identification and functional characterization of parasitism-related genes of the Columbia root-knot nematode Meloidogyne chitwoodi from potato fields in Washington State, and the burrowing nematode Radopholus similis from banana fields in Hawaii. Progress was made on generation of new transcriptomic data for different populations of the lesion nematode, Pratylenchus neglectus, causing yield reduction of barley, lentil, and wheat in the state of Montana. These results are significant because they provide new molecular data for different nematode species/populations parasitizing different agriculture crops in the U.S., as well as new nematode target genes to be tested for the control of these specific nematodes. Advances were also made in the study of existing strawberry cultivars and breeding selections to identify lines with resistance to the northern root-knot nematode, an important pathogen of strawberry that can reduce yields via feeding on the plant roots. This research may yield markers and genotypes associated with nematode resistance will be used for breeding nematode resistant strawberry. New research being developed under Objective 3, includes the study of beneficial nematodes as soil health indicators in urban agricultural systems that may experience different stresses from those occurring in conventional rural farming systems. A survey of biological indicator nematodes is planned to assess the soil health status of urban agricultural sites in the DC-Maryland-Virginia metropolitan area. Another line of research with an urban agriculture focus includes the evaluation of organic mulches on beneficial nematode abundance and diversity in ethnic hibiscus and jute leaf crop systems. These studies will be of interest to small growers of ethnic crops in culturally diverse per-urban and urban environments and will be used to inform future research and provide general information on soil health to urban gardeners. Another planned study will investigate the impacts of organic agricultural practices on bacterial feeding soil organisms, which help to cycle nutrients in the soil by converting nutrients to forms available for plant uptake and use. Results from the study will be used to provide recommendations to organic producers for increasing bacterivore nematode abundance in soil. Another study will evaluate the impact of natural organic products with lower leaching potential on beneficial nematodes within a managed turfgrass environment, with an emphasis on nematode community structure and abundance. This research may help turfgrass managers make informed decisions on long-term fertilizer use and the impacts on nematodes which contribute to soil health. Other work is being developed to investigate anaerobic soil disinfestation (ASD) carbon sources that suppress plant-parasitic nematode numbers. ASD is a non-chemical management practice that employs different carbon sources to reduce plant-parasitic nematode numbers prior to planting certain crops. Research to determine optimal combinations of carbon source and oxygen ASD will inform subsequent research projects as well as enhance ASD recommendations for plant-parasitic nematode management for growers in the southeastern U.S.

Accomplishments
1. Nematode responsible for beech leaf disease now found in Michigan. The beech leaf disease (BLD) nematode (Litylenchus crenatae mccanii) is a newly emergent nematode species that causes severely deformed leaves and buds and mortality in seedlings and mature beech trees (Fagus spp.) in North America. Beech leaf disease has been spreading steadily from Ohio to 10 additional states since 2012. ARS scientists from Beltsville, Maryland, and colleagues from Michigan State University used morphological features and DNA markers to identify this nematode for the first time in the state of Michigan. This discovery provides vital information about the expanding reach of this pest to scientists, growers, action agencies, and extension agencies involved with controlling its spread.

2. DNA information recovered from old nematodes provides new insights. The United States Department of Agriculture Nematode Collection (USDANC) is currently one of the largest nematode collections in the world. For years, it has not been possible to recover intact DNA from collection specimens that are typically preserved in a DNA-damaging fixative called formalin. In this study, successful DNA extraction was achieved from formalin-fixed nematodes, allowing comparison of current isolates with decades-old collection specimens to confirm the identity of a stunt nematode called Sauertylenchus maximus from Arlington, Virginia. This discovery is significant because it represents the first report of this nematode from Virginia and the first report of successful DNA extraction from fixed nematode specimens deposited in the USDANC. This research opens more opportunities for ARS scientists to re-examine material from the collection to shed new light on current nematode discoveries.

3. Genetic markers for resistance to a highly virulent root-knot nematode identified in watermelon. The guava root-knot nematode is a highly virulent and damaging pest of many crops that has emerged as a serious threat to vegetable production, particularly on watermelon in the southeastern United States. ARS scientists in Beltsville, Maryland, and Charleston, South Carolina, with colleagues from Clemson University, evaluated watermelon breeding lines to identify genetic regions in watermelon that may contribute to disease resistance. These findings advance understanding of nematode resistance in watermelon and potentially other crops. These markers and breeding lines will be useful to plant breeders to develop resistant watermelons that would provide a pesticide alternative to control this aggressive nematode species.

4. A new species of virus-transmitting needle nematode was found in Maryland. Needle nematodes of the genus Longidorus are a major group of plant pathogens with a wide distribution and host range, and they sometimes are vectors of plant pathogenic viruses. A team of ARS scientists from Beltsville, Maryland, and a colleague from California described a new species of needle nematode that was discovered in two diverse environments: one on tall fescue grass growing along the bank of the Patuxent River in Maryland, and the other found near a walnut tree in California. Anatomical and DNA traits described for the new species are essential for future identifications of this needle nematode by scientists, diagnosticians, action, and extension agencies involved in nematode research and control.

5. Microbes vs nematodes: Insights into biocontrol through antagonistic organisms to control Root-knot nematodes. Chemical nematicides are a common approach to combat and suppress parasitic nematodes that damage crop plants, but many of these compounds are being phased out due to concerns about toxicity and environmental effects. An ARS scientist from Beltsville, Maryland, and collaborators from the Aligarh Muslim University, Aligarh, India, worked together to assemble comprehensive, current information on natural alternatives to chemical controls for nematodes. This work describes the ways in which these biocontrol agents, which include bacteria, fungi, their metabolites, or natural products can be exploited to control nematodes, either by directly killing them or by preventing them from multiplying in host plants. Scientists, diagnosticians, growers, action agencies, and extension agencies involved in nematode research and control will use this reference work as a guide for managing plant-parasitic nematodes.

6. Elucidating potential of fly ash in root-knot nematode (M. incognita) suppression on okra. One alternative to chemical control of root-knot nematodes is the application of soil amendments that either stimulate plant growth or inhibit nematode reproduction. An ARS scientist from Beltsville, Maryland, in cooperation with collaborators from Aligarh Muslim University, Aligarh, India, determined that okra grown in pots with a soil amendment called fly ash showed improved soil health, growth, yield, and antioxidant properties, with fewer harmful effects of root-knot nematode compared to other treatments. The success of fly ash application for control of nematodes is important for scientists, growers, and extension agencies involved in nematode research and control.

U.S. DEPARTMENT OF AGRICULTURE

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