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

Location: Mycology and Nematology Genetic Diversity and Biology Laboratory

2022 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] Objective 5: Identify and describe invasive and emerging plant-parasitic nematodes from forage and biomass legumes, turf, grasses, and associated rotation crops - including species of quarantine significance - to enable specific and timely management of these pests. [NP303, C1, PS1] Objective 6: Improve molecular methodologies for the identification and classification of nematodes from forage legumes and grasses in order to better predict and respond to nematode outbreaks and to improve detection and diagnostic methodologies to distinguish closely related or morphologically similar species. [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 is the final report for project 8042-22000-300-000D, “Molecular Systematics, Identification, Biology, and Management of Crop-Parasitic Nematodes,” which combined projects: 8042-22000-275-000D, “Improved Classification and Taxonomic Methods for Agriculturally Important Nematodes,” 8042-22000-283-000D, “Plant-Parasitic Nematode Management: Amendments, Beneficial Organisms, and Nematode Antagonistic Molecules,” and 8042-22000-309-000D, “Improved Identification and Diagnostics of Plant Nematodes for Sustainable Grass and Forage Production Systems.” This work falls under National Program 303, C1, PS1; C2, PS2A; and C3, PS3B. The following is a summary of progress over the life of the project. Advances under Objective 1 included research on essential components of nematode biology to discover novel agents for nematode control. Progress included discovery and characterization of protease inhibitors known to disrupt plant-parasitic nematode metabolism and reproduction. No further progress was recorded due to the retirements of two participating scientists associated with this objective; new lines of investigation were initiated by the scientist hired in early FY22. Progress (relating to Objectives 1 and 3) include a reference transcriptome generated for the first time for root lesion nematode Pratylenchus fallax, from soybean in Wisconsin. This study provides a large DNA sequence dataset for developing new molecular markers useful for species identification. Progress also included the identification of parasitism genes from P. fallax, the sugar beet cyst nematode Heterodera schachtii, and the Columbia root-knot nematode Meloidogyne chitwoodi from potato fields in Washington State. These results are significant because they can provide new nematode target genes to be tested for the control of these specific nematodes. Under Objective 2, microbes were studied as potential biological control agents and as sources of compounds active against nematodes. Research was also conducted to determine nematotoxicity of natural products. These investigations are important for enhancing or developing methods for managing plant-parasitic nematodes. The nematotoxicity or inhibitory properties of several plants or plant products was demonstrated, including vetiver, amylose inclusion complexes of vegetable oil derivatives (patent application 0128.18), a natural compound and its analogs (provisional patent 63/123,615 - DN. 76.20), and of camelia tea seed cake. Materials from roots of corn, cucumber, mustard, pepper, soybean, and nematode-antagonistic marigold caused immobility and paralysis of soybean cyst and root-knot nematodes. Soybean root extracts also contained factors causing abnormal behaviors in infective juveniles. Other research assessed suppression of phytoparasitic nematodes in forage grasses with and without fungal endophytes. Together this research demonstrated the effectiveness and limitations of various cover crops and natural compounds for nematode control. Additional progress was contributed by a second new scientist hired in FY22. ARS scientists from Beltsville, Maryland, and Charleston, South Carolina, evaluated watermelon breeding lines to identify genetic regions that may contribute to resistance to guava root-knot nematode. These findings will assist plant breeders in the development of resistant watermelons, requiring less pesticide for nematode control. Flood irrigation was also studied as a nematode management tool. An ARS scientist from Beltsville, Maryland, with colleagues from Gainesville, Florida, counted nematodes from flooded soil and overhead irrigated soil in a greenhouse experiment. Statistically fewer plant-feeding nematodes were found in flood irrigated soil than overhead-irrigated soil. These studies will be used by researchers and growers to confirm if flood irrigation reduces nematode abundance in large-scale agricultural fields. Under Objectives 3 and 4 (relating to nematode identifications), ARS researchers in Beltsville, Maryland, and collaborators from the Ohio Department of Agriculture identified a new nematode, Litylenchus crenatae mccannii from beech trees affected by Beech Leaf Disease (BLD), sparking a multidisciplinary, multinational effort to determine the cause of this disease decimating trees in several states (ARS Tellus 2019 Cover story). Subsequently, ARS scientists from Beltsville, Maryland, and colleagues from Ontario, Canada, and Rhode Island identified molecular markers from L. crenatae mcannii to facilitate tracking of this subspecies, believed to be an invasive species from Asia to which North American native trees have little to no resistance. This research is being used by scientists, extension agents, growers, forest managers and quarantine officials to determine the cause of BLD and eventually propose methods to manage this disease. Under Objective 3, the highly virulent root-knot nematode, Meloidogyne enterolobii was identified for the first time from sweet potato in South Carolina. In another study, ARS scientists from Beltsville, Maryland and Tifton, Georgia, with colleagues from Brazil, identified this species for the first time from cotton in Brazil, and showed M. enterolobii was able to reproduce successfully on seven genotypes of cotton, several of which contained resistance to other root-knot species. This research will aid growers and regulators in limiting the damage to many crops cause by this highly aggressive nematode. Other progress included descriptions of several plant-parasitic and fungal-feeding nematodes using microscopic techniques to detail anatomical features, and molecular techniques for generating DNA sequences and phylogenetic analyses. These included one new stunt nematode and two new lesion nematodes parasitizing soybean in North Dakota, and a ring nematode (Mesocriconema curvatum) from corn in North Dakota. ARS scientists from Beltsville, Maryland, worked with Egyptian colleagues to identify for the first time the highly damaging golden potato cyst nematode, Globodera rostochiensis, from Egypt. A new species of tylenchid nematode was characterized from corn in South Carolina. New insights into the phylogenetic relationships among cyst nematodes from Greece was demonstrated through graphical representation of DNA marker variability. ARS scientists from Beltsville, Maryland, and colleagues from California Department of Food & Agriculture identified a new species of pin nematode, Paratylenchus beltsvilleiensis, from soil around a pine tree (Pinus virginiana) using morphological and molecular approaches. These studies provide critical anatomical and molecular information necessary for accurate species identifications and support management and regulatory measures. As part of the service component of Objective 4, during FY 2022, accurate nematode identifications and related expertise were provided for 236 urgent plant and soil samples intercepted by the Animal Plant Health Inspection Service (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, and burrowing 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 implentation of regulatory actions beneficial to growers and the public. Axonomic reports and resources are used by research scientists, extension agencies and regulatory action agencies involved in nematode research and control. In addition, nematode identifications and expertise were provided for over 348 samples submitted by various customers for research, regulation, and control purposes. The USDA Nematode Collection was expanded by 224 additional slides and vials, bringing the specimen total to 66,568. Twenty-six slides and 15 samples (about 210 specimens) were loaned to scientists or sent for molecular analysis. An internet-accessible database was updated with thousands of sample records on hosts, occurrence, and distribution. Over 331 specimen records were entered in the database for a total of 55,905 current records. Progress under intersecting Objectives 3-6 also focused on identification of nematodes from forage legumes and grasses. This included identification of alfalfa cyst nematode, Heterodera medicaginis, from alfalfa fields in Kansas, Montana, Idaho, and Utah, representing the first records of this species in North America. This species poses a significant emerging threat to alfalfa, which is grown in all 50 states and is the third most valuable field crop. Other notable identifications on grasses included a stunt nematode Sauertylenchus maximus, isolated from the roots of tall fescue turf grass in Virginia. This research demonstrated for the first-time recovery of DNA sequences from 22-year old fixed material from the USDA Nematode Collection. Cyst nematodes from the same soil sampling, Cactodera torreyanae and Punctodera stonei, were described, and a new country record of cyst nematode (Heterodera pratensis) on golf courses from Michigan was reported. This research provides critical anatomical descriptions and molecular markers needed to identify important nematodes of forage and grasses.

Accomplishments
1. Genetic characterization of nematode isolated from diseased beech leaves. Beech leaf disease (BLD), which causes severely deformed leaves and buds and mortality in seedlings and mature trees, has been spreading in Ohio, Pennsylvania, and New York, since 2012. ARS researchers in Beltsville, Maryland, identified this nematode in Virginia. A team of ARS researchers then generated the first reference transcriptome from this population, revealing a set of nematode parasitism genes. This information is necessary for determining the molecular mechanism between the nematode, the tree, and BLD and to distinguish L. crenatae mcannii from closely related species. This research will be used by scientists, growers, action agencies, and extension agencies to effectively monitor and control the spread of this nematode.

2. Meloidogyne hispanica newly discovered on corn from South Carolina. Corn is the number one crop grown in the United States and a staple crop worldwide. Several nematode species are important pests of corn, including the root-knot nematodes. ARS scientist from Beltsville, Maryland, Clemson University in South Carolina, and California Department of Food & Agriculture used anatomical features and DNA markers to identify for the first time in the United States the Seville root-knot nematode, Meloidogyne hispanica, from corn in South Carolina. This discovery provides new morphological and molecular data that can be used by scientists, growers, action agencies, and extension agencies necessary to monitor and control the spread of this newly discovered nematode.

3. Detection and identification of 139 new virus species associated with nematodes and other soil-inhabiting organisms. In this study, a team of ARS scientists from Beltsville, Maryland, and colleagues from California Department of Food and Agriculture and Virginia Tech University performed extensive meta-transcriptomic analyses of soil nematode samples collected in different regions of the US. This study demonstrated for the first time the existence of a totally unknown large diversity of viruses associated with nematodes and other soil-inhabiting communities. This study will be of interest to a wide range of plant pathologists, plant breeders, soil ecologists and regulatory officials.

4. Susceptibility of potato cultivars to lesion nematode. The root lesion nematode Pratylenchus penetrans is considered one the most important species of this genus due to its cosmopolitan distribution and wide host range, comprising more than 400 plant species. This lesion nematode is an important plant-parasitic nematode of potato, causing damage to roots and tubers, and as part of the disease complex Potato Early Dying. In this study, a team of ARS scientists from Beltsville, Maryland, and colleagues from the University of Coimbra in Portugal assessed the susceptibility of ten commercial potato cultivars to P. penetrans. This study is significant because provides new information regarding the different levels of susceptibility of the different cultivars against this nematode. This research will be used by scientists and growers to mitigate and better manage the impact of this nematode in potato cultivars.

5. New stubby-root nematode identified from Maryland. A team of ARS scientists from Beltsville, Maryland, and researchers from California Department of Food & Agriculture and Ghent University, Belgium, identified Trichodorus marylandi, a new species of plant parasitic nematode from a group known to carry nepoviruses of plants. Methods were developed to distinguish the new species from closely related stubby-root nematodes based upon DNA markers and anatomical features. This discovery is significant because information obtained for this new species will facilitate future identifications and further research and management of this emerging pest.

Review Publications
Decraemer, W., Kantor, M.R., Handoo, Z.A., Subbotin, S. 2021. Description of Trichodorus maryland in. sp. (Nematoda: Trichodoridae) from Maryland, USA. Nematology. https://doi.org/10.1163/15685411-bja10132.
Skantar, A.M., Handoo, Z.A., Subbotin, S.A., Kantor, M., Vieira, P., Agudelo, P., Hult, M.N., Rogers, S.T. 2021. Molecular and morphological evidence for the first report of Seville root-knot nematode, Meloidogyne hispanica (Nematoda: Meloidogynidae) in the USA and North America. Journal of Nematology. https://doi.org/10.21307/jofnem-2021-098.
Handoo, Z.A., Skantar, A.M., Subbotin, S., Kantor, M.R., Hult, M.N., Grabowski, M. 2021. Molecular and morphological characterization of a first report of Cactodera torreyanae Cid del Prado Vera & Subbotin, 2014 (Nematoda: Heteroderidae) from Minnesota, the United States of America. Journal of Nematology. https://doi.org/10.21307/jofnem-2021-093.
Haroon, S., Handoo, Z.A., Kantor, M.R., Skantar, A.M., Hult, M. 2021. Molecular and morphological characterization of Globodera rostochiensis (Wollenweber, 1923) Skarbilovich, 1959 from Egypt. Notulae Scientia Biologicae. 13(4):11083. https://doi.org/10.15835/nsb13411083.
Figueiredo, J., Reis Vieira, P.C., Abrantes, I., Esteves, I. 2022. Commercial potato cultivars exhibit distinct susceptibility to the root lesion nematode Pratylenchus penetrans. Horticulturae. 8:244. https://doi.org/10.3390/horticulturae8030244.
Ibrahim, I.K., Handoo, Z.A., Zeyadah, S.A., Kantor, M., Kawanna, M.I. 2022. Pathogenicity and control of Meloidogyne spp. on some spinach, Swiss chard, and table beet plant cultivars. Notulae Scientia Biologicae. 14(1). https://doi.org/10.15835/nsb14111175.
Nemchinov, L.G., Irish, B.M., Grinstead, S.C., Shao, J.Y., Vieira, P. 2022. Diversity of the virome associated with alfalfa (Medicago sativa L.) in the U.S. Pacific Northwest. Scientific Reports. 12:8726. https://doi.org/10.1038/s41598-022-12802-4.
Vieira, P., Subbotin, S.A., Alkharouf, N., Eisenback, J.D., Nemchinov, L.G. 2022. Advancing virus discovery in nematodes and other soil-inhabiting organisms. Virus Evolution. 8(1):1–8. https://doi.org/10.1093/ve/veac019.
Morphological and molecular characterization of Paratylenchus beltsvilleiensis n.sp.(Tylenchida: Paratylenchidae) from the rhizosphere of pine tree (Pinus virginiana Mill) in Maryland, USA. Journal of Nematology. 53(1). https://doi.org/10.21307/jofnem-2021-079.