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

2024 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
The work in this project falls under National Program 303, C1 PS1B and C3, PS3B. For progress under Objective 1, ARS researchers in Beltsville, Maryland, provided nematode identifications and expertise for over 387 samples submitted by various customers for research, regulation, and control purposes. Detailed descriptions were generated for several nematodes representing new species, new state or country records obtained from field surveys or international trade interceptions, including a new fungal feeding nematode (Laimaphelenchus) from beech trees in Pennsylvania. Integrated taxonomic approaches involving morphological, morphometric, and molecular phylogenetic analysis were employed to describe a new cyst nematode (Punctodera) from corn in North Carolina. Other identifications included the barley root-knot nematode, Meloidogyne naasi, from naturally infected fowl manna grass in Pennsylvania, lesion nematode, Pratylenchus pseudocoffae, on dahlia and stubby-root nematode, Paratrichodorus renifer, from blueberry in Pennsylvania, and pine wood nematode, Bursaphelenchus xylophilus, on Pinus sylvestris from Idaho. These studies provide critical anatomical and molecular information necessary for accurate species identifications and support management and regulatory measures. Progress in Objective 1 was also achieved in several studies examining DNA marker variability among nematode populations. Phylogenetic relationships among several cyst nematode isolates from several crops in Greece were revealed through graphical statistical parsimony methods that highlight population diversity and global distribution. This method was also used to characterize a sting nematode (Belonolaimus longicaudatus) isolate encountered in Maryland for the first time in soil from an athletic field. This finding further expands the known distribution of this important species that not only can cause substantial visual decline in turfgrass but also has a wide host range. In another study, an ARS researcher in Beltsville, Maryland, and a collaborator from the Connecticut Agricultural Experiment Station applied microsatellite markers to study the population genetic structure of the beech leaf disease (BLD) nematode, Litylenchus crenatae subps. mccannii (Lcm), across North America and Japan. In related research, ARS scientists developed a cost-effective, rapid visual detection approach to diagnose beech leaf disease and associated nematodes within beech buds. Related progress on Lcm under Objective 3 included new research with Pennsylvania State University to understand the contribution of abiotic (rain and wind) and biotic vectors (insects) potentially contributing to the local transmission of Lcm in natural forest systems. These tools are needed to effectively manage BLD. Advances were made on research under Objective 1 that involved genome sequencing of nematodes. These include a collaboration between ARS labs in Beltsville, Maryland, and Corvallis, Oregon, and Oregon State University to sequence and assemble the genome of the most economically important nematode of hops, the hop cyst nematode. This genome will allow development of novel molecular diagnostic markers for this species and provides an essential resource to investigate insights into the basic biology of cyst nematodes. Additionally, this genome is the basis for an ongoing population genetics study to understand the global movement and introduction of hop cyst nematode to the Pacific Northwest. While sequencing hop cyst nematode, the endosymbiont bacteria Wolbachia was detected, and assembly of this genome is ongoing. This research provides genome data for studies to understand the differences between Wolbachia strains from different plant-parasitic nematodes and how Wolbachia can be manipulated to manage plant-parasitic nematodes (new research in Objective 3). In a collaboration that included ARS researchers from Beltsville, Maryland, Corvallis, Oregon, and Hilo, Hawaii, and the Universidad de Costa Rica, whole genome sequencing was achieved for 18 global populations of the burrowing nematode. These genomes will serve as a source of new diagnostic markers to track host range and distribution of this nematode, which is an important pest of banana, citrus, ginger, and anthurium. For Objective 2, accurate nematode identifications and related expertise were provided for 274 urgent plant and soil samples intercepted by APHIS at ports-of-entry or during domestic surveys. National surveys for the presence of pale potato 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, lance, ring, spiral, and 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 in support 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 616 additional slides and 4 vials, bringing the specimen total to 68,896. USDA Nematode Collection specimens numbering 135 were loaned to scientists for research purposes. Fifteen samples (about 300 specimens) were provided to scientists for molecular analysis. An internet-accessible database hosted by the National Agricultural Library Azure Cloud was developed for the thousands of sample records available including information on hosts, occurrence, and distribution. Over 181 specimen records were entered in the database for a total of 56,671 current records. Under Objective 3, ARS scientists made progress on the identification and characterization of effector genes from the lesion nematode, Pratylenchus fallax. Through in-depth transcriptome analyses of this nematode, dozens of candidate effector genes have been identified and validated in the nematode esophageal glands by in situ hybridization. To functionally characterize these effectors, different assays have been performed using Potato Virus X (PVX)-based transient expression and subcellular localization assays in planta. This functional characterization will enable a more in-depth knowledge of this plant-nematode interaction, to better understand the function of these effectors in nematode parasitism. In parallel, an ARS scientist from Beltsville, Maryland, with collaborators from the University of Coimbra, Portugal, generated novel transcriptomic data from potato cultivars exhibiting varying degrees of susceptibility to infection by the root lesion nematode Pratylenchus penetrans. Furthermore, progress on Objectives 1 and 3 involved identification and functional characterization of effector genes from the Columbia root-knot nematode, Meloidogyne chitwoodi, from potato fields in Washington State (in collaboration with Washington State University, Pullman, Washington). Additionally, the most comprehensive effector repertoire of the sugar beet cyst nematode, Heterodera schachtii, was identified (in collaboration with the University of Cambridge, United Kingdom). These comprehensive catalogues of effector genes from economically significant plant-parasitic nematodes serve as a foundational resource for understanding nematode parasitism and inform the development of strategies for crop protection. New research under Objective 3 addresses the emerging opportunity to tailor crop production to meet the specific food preferences of historically underserved populations near urban metropolitan areas in the mid-Atlantic region. A better understanding of how ground covers impact nematode communities is important for sustainable production practices in ethnic crops such as jute leaf. ARS scientists in Beltsville, Maryland, with colleagues from the University of the District of Columbia, investigated the effect of four ground covers and three jute leaf cultivars on soil nematode communities. Early findings showed that research plots with wood chips plus compost or straw plus compost had high numbers of beneficial nematodes compared to compost-only ground covers, but these treatments also had higher numbers of plant-parasitic nematodes nematodes. Progress is also being made on using nanotechnology to develop novel nematode controls from plant-derived compounds. Cowpea plants, an important food and forage crop, are susceptible to damage from root-knot nematodes as well as certain species of fungi. Chemicals originally found in walnuts that were made in the laboratory were found to significantly improve plant growth. ARS scientists from Beltsville, Maryland, are collaborating with university scientists from Aligarh, India, and Saudi Arabia to harness walnut-based zinc oxide nanoparticles as a sustainable approach to combat the disease complex of Meloidogyne arenaria and the fungus Macrophomina phaseolina in cowpea. This research will create novel delivery systems to control nematodes in place of traditional chemical controls. Another study is examining the effect of endophytic microbes on forage grass resistance to nematodes. Tall fescue used as livestock forage is vulnerable to damage by plant-parasitic nematode feeding. ARS researchers from Beltsville, Maryland, and Lexington, Kentucky, along with collaborators at the University of Kentucky, are investigating resistance to Pralylenchus nematodes that is conferred on tall fescue by the action of fungal endophytes.

Accomplishments
1. Insights into the cellular mechanism underlying beech leaf disease. The beech leaf disease (BLD) nematode (Litylenchus crenatae mccanii) is a rapidly spreading nematode species that poses a severe threat to American beech (Fagus spp.), a tree species valued as a host and food source for a wide range of birds, bears, and other wildlife. The nematode causes severely deformed leaves and buds and mortality in seedlings and mature beech trees alike. USDA ARS scientists from Beltsville, Maryland, used light and scanning electron microscopy to reveal extensive cellular changes in nematode-infected buds and leaf tissues, which effectively captured the sequential, temporal cellular events associated with the development of beech leaf disease, and simultaneously provided an in-depth, mechanistic overview of this disease. This approach not only provided insight into the development of the nematode feeding site but also revealed several critical time periods for future molecular and physiological studies of BLD. This research is being utilized by scientists, extension agents, forest managers, and quarantine officials to establish effective measures to mitigate the spread of this nematode and control the disease through by targeting vulnerable points in its life cycle.

2. Novel method for identification of guava root-knot nematode. The guava root-knot nematode Meloidogyne enterolobii is a highly aggressive species that causes significant damage in a wide variety of crops worldwide. This nematode was first detected in South Carolina in 2018, where it poses a significant threat to the sweet potato industry, the most valuable vegetable commodity in the state, valued at $164 million in 2022. In order to facilitate rapid and accurate screening for this nematode, ARS researchers from Beltsville, Maryland, and Charleston, South Carolina, along with university researchers from Clemson University, developed a new methodology for identifying the guava root-knot nematode from large batches of sweet potatoes by extracting total DNA from sweet potato skins and using molecular markers to detect this nematode. This new identification methodology will provide regulatory agencies with efficient, rapid testing for guava root-knot nematode in sweet potato storage roots at export and will help growers to reduce the spread of this nematode by ensuring that seed potato stocks are clean prior to planting.

3. Root-knot nematodes produce functional mimics of plant defense peptides. One way that pests and pathogens cause disease is to overcome host plant immunity through mimicry of normal plant defense mechanisms. Scientists from ARS in Beltsville, Maryland, along with colleagues from University of California, Davis, report the identification of genes from root-knot nematodes (Meloidogyne spp.) predicted to encode PSY-like peptides (MigPSYs) with high sequence similarity to normal host plant immunity activators called PSYs as well as the known bacterial mimic peptide RaxX. Downregulation of MigPSY gene expression reduces root galling and egg production, suggesting that the MigPSYs serve as nematode virulence factors. Together, these results indicate that nematodes and bacteria hijack plant developmental signaling pathways to facilitate parasitism. This discovery will allow the development of new biotechnology tools for plant pathologists and other scientists to mitigate the impact of root-knot nematodes in agriculture systems.

4. Molecular diversity of nematodes from golf courses and athletic fields. The turfgrass industry in the United States has an estimated value of $105 billion (2018), and yet information on the presence and impact of nematodes on turfgrass in Maryland is essentially non-existent. To address this knowledge gap, total of 28 golf courses and 10 athletic fields were surveyed by ARS scientists from Beltsville, Maryland, and a University of Maryland scientist, revealing the prevalence and abundance of 13 plant-parasitic nematode taxa in the region, with some showing symptoms or present above typical threshold levels. Among the nematodes identified, the sting nematode, Belonolaimus longicaudatus, was encountered for the first time in Maryland. In another study with Pennsylvania State University, the barley root-knot nematode Meloidogyne hapla was identified for the first time from golf course turfgrass in Idaho. Genetic diversity studies of these nematodes revealed their relationship to other populations of the same species. This research will be useful to scientists, extension agents, and turf industry partners interested limiting the impact of nematodes on the aesthetics and functionality of turfgrass in athletic fields and golf courses.

5. New Wolbachia genomes shed light on nematode-endosymbiont dynamics. While manipulating symbiotic bacteria within disease-causing parasites shows promise as a biocontrol strategy, the extent to which Wolbachia is present as an endosymbiont of plant-parasitic nematode species is relatively unknown. ARS researchers from Beltsville, Maryland, and Corvallis, Oregon, and Oregon State University, used whole genome sequencing of 52 populations of 12 species of plant-parasitic nematodes to discover Wolbachia in new nematode hosts. These Wolbachia genomes will serve as a valuable genomic resource for nematology researchers to understand how this bacterium interacts with its nematode host and to what extent this interaction can be manipulated for plant-parasitic nematode management.

6. Identification and reproduction of dagger nematode from potato. Dagger nematode (Xiphinema) species are of importance to agriculture as plant parasites, causing damage to host plants due to direct feeding on root cells, while also acting as vectors of plant viruses. In this study, an ARS scientist from Beltsville, Maryland, in cooperation with North Dakota State University, identified the dagger nematode Xiphinema americanum by both molecular and morphological means and found that nematode reproduction was higher on yellow potato cultivars than white and Russet cultivars. This research will facilitate future identifications of dagger nematode and may improve management decisions based on potato cultivar and will be used by scientists, diagnosticians, growers, action agencies, and extension agencies involved in dagger nematode research and control.

7. Plant-based nitrogen fertilizers leach less and reduce plant-parasitic nematodes in turfgrass production systems. Synthetic fertilizers commonly used for turfgrass are prone to leaching which can contaminate groundwater within residential communities. An ARS researcher in Beltsville, Maryland, in collaboration with scientists from University of Maryland completed a long-term study of three slow-release fertilizers that are less prone to leaching for effects on nutrient content in soil, turfgrass quality, disease development, and nematode community structure. The plant-based fertilizer plots had more beneficial omnivore-predator nematodes and fewer plant-parasitic lance nematodes than the biosolid fertilizer plots. These findings are relevant to turfgrass producers, extension specialists, and researchers looking to manage nematode communities in turfgrass using sustainable practices.

U.S. DEPARTMENT OF AGRICULTURE

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