Research Project: Biological Control in Integrated Weed Management of Invasive Weeds from Europe, Asia, and Africa

Location: European Biological Control Laboratory

2025 Annual Report

Objectives
Objective 1: Explore for natural enemies, primarily arthropods, of invasive weeds identified as high priority targets by the ARS Office of National Programs, performing collections, importations and exportations in compliance with local and international regulations. High priority pests include Sahara mustard, French broom, annual grasses, stinkwort, tree of heaven and swallow-worts. Objective 2: Perform taxonomic, population genetic, and phylogeographic studies of target weeds and biological control agents to better understand the origin of invasive populations, arthropod-plant relationships, and clarify species. • Sub-objective 2A Phylogeography of Sahara mustard, stinkwort, ventenata. • Sub-objective 2B Genetic characterization of potential biocontrol agents. Objective 3: Identify the biological, physical and chemical parameters that affect the efficacy, specificity, and safety of biological control agents, and those underlying the interactions between target weeds, biological control agents, and the environment. • Sub-objective 3A Assess the potential host specificity on selected plant species, focusing on host use of candidate agents for tree of heaven, stinkwort, swallow-worts, medusahead and Sahara mustard. • Sub-objective 3B Investigate foraging behavior and impact of biological control agents with French broom weevil and French broom psyllid. • Sub-objective 3C Investigate safety procedures of biological control agents, including French broom arthropod candidates and other potential biological control agents. • Sub-objective 3D Compare the impact of rhizospheric bacteria and fungi from native and invasive ranges on the fitness of the annual grass weed ventenata.

Approach
The goal of this current 5-year plan is to conduct a customer-driven research program that will enhance our ability to manage invasive weeds in the U.S. Successful alien weeds usually lack natural enemies, which control them in their native ranges. The European Biological Control Laboratory research team and associated cooperators will accomplish this by using a combination of hypothesis and goal-driven research toward the discovery and development of exotic natural enemies for classical (=importation) biological control of alien weeds in the U.S. The long-term research plan will lead to environmentally safe and sustainable management of weeds that threaten U.S. agriculture and natural ecosystems over large areas. The process usually involves three phases that can be conducted in parallel: 1) exploration to discover and characterize candidate arthropod biological control agents and their target host plants in full compliance with current regulations in host countries regarding exportation of live organisms and benefit-sharing, 2) morphological and genetic characterization of the field collected arthropod material and related natural enemies, and 3) evaluation of the host specificity and effectiveness of the best selected candidate biological control agents. Candidates that are found to be safe and effective are then proposed for release in the U.S., and must undergo a rigorous review and permitting process involving state and federal agencies. Research on Sahara mustard, French broom, annual grasses, tree of heaven and swallow-worts will continue, and additional weeds will be targeted in response to stakeholder demand and available resources. EBCL plays a key role providing research and prospective agents to federal and state cooperators necessary for the successful control of target weeds. The research proposed in classical biological control is highly cost effective, and critical to achieving ecologically rational, sustainable management of some of the most important invasive weeds in the United States.

Progress Report
Under Objective 1, for Sahara mustard, ARS scientists from France conducted field exploration to Egypt and Israel in early 2023. About ten species of arthropods were collected from leaf, stem and root material. All material was identified (See Sub-objective 2B). The field impact of each of the enemy collected was not significant, but particular interest was devoted to a curculionid beetle that was found on the Sahara mustard root system. That feeding niche could reveal a close specificity to the target weed. Unfortunately, it was not possible to travel back to Egypt since 2023 because a military conflict. For Stinkwort, several field surveys were conducted from 2022 – 2025 in the Mediterranean basin including, Cyprus, Crete, France, Spain, and Portugal. One major natural enemy, Condica viscosa (Lepidoptera: Tortricidae), was collected from foliage with strong defoliation observed impacts in some locations (Cyprus, Crete). All the other moths collected from stinkwort were well known as polyphagous, after a morphological identification by an Italian taxonomist (e.g. Heliothis peltigera and Helicoverpa armigera) that was confirmed by genetic screening (see Sub-objective 2A). Under Sub-objective 2A, with Sahara mustard (Brassica tournefortii), genetic comparisons using a genotyping-by-sequencing approach were conducted on 52 populations in the invaded United States range, alongside 16 populations in the native range, which includes Egypt, France, Israel, Italy, Morocco, Turkey, and Qatar. Egypt, along with potentially connected groups in Morocco and France, appears to be the probable source of the largest invasive populations in California, while Australia is thought to be the origin of the recently introduced invasive population found near Nipomo, California. This finding has helped in streamlining the search for natural enemies of Sahara mustard in its native range (See Objective 1). The genetic analysis, carried at European Biological Control Laboratory (EBCL) with stinkwort (Dittrichia graveolens), using 20 natural populations along the invasion path in California by using the population genetic markers developed from 2020 onwards showed that the level of genetic diversity of stinkwort is very low in California, represented by only two genotypes which are differently distributed between the North and South of the state. The genetic analysis of the native range, with samples from 32 populations across six European countries; Spain, France, Italy, Greece, Cyprus, and Portugal was carried out in 2024. The genotypes found in California have not yet been identified in the Mediterranean Basin in the native range. For the grass ventenata (Ventenata dubia), the genetic data analysis of native populations in Eurasia still needs to be completed by our partner laboratory at Boise State University, Boise, Idaho. A previous genetic analysis of the invasive populations of ventenata was published in 2020. Under Sub-objective 2B, DNA barcoding was conducted by ARS scientists at EBCL France to identify natural enemies of stinkwort using samples collected in Cyprus, resulting in the identification of one foliage feeder pyralid moth, two foliage-feeding moths, three scentless plant bugs, along with one plume moth. All these natural enemies are deemed non-specific to stinkwort, unlike the moth Condica viscosa, suggesting it has the greatest potential for biocontrol. Genetic analyses confirmed that C. viscosa populations from Cyprus in 2021 and 2023 belong to a single species, hence mitigating the risk of unintentional introductions of another species in quarantine testing. A comparable DNA barcoding approach was applied to natural enemies associated with Sahara mustard collected from Israel and Egypt in 2023, resulting in identification of two leaf mining flies, a root weevil and the stem miner. All except the root weevil are considered nonspecific to Sahara mustard, so it is unlikely that they will be retained as prospective biocontrol agents for release in California. For Tree of heaven (“ToH”, Ailanthus altissima), the eriophyid mite collected in Spain and Portugal in 2024 was confirmed through DNA barcoding phylogenetic approaches to be Aculus taihangensis, the most promising candidate biocontrol agent of ToH. The molecular and phylogenetic studies helped to determine the species status of this organism in all European countries (three publications). The DNA barcoding method was utilized for the natural enemies linked to ToH congeners collected from Vietnam in 2024, leading to the identification of one leaf-feeding psyllid from the Psylloidea superfamily and one leaf-feeding sawfly that has still to be identified. For medusahead (Taeniatherum caput-medusae), the Eurytomidae wasp (Tetramesa amica) is currently the most promising biocontrol agent for this target. DNA barcoding in 2024 verified its morphological identification together with another wasp, Eurythoma amicophaga, which is found associated with T. amica on medusahead in Greece. The DNA barcoding approach led to the development of a diagnostic method which offers a reasonably low-cost basis for an extensive monitoring of these two wasps in the field. For Sub-objective 3A targeting Sahara mustard, the weevil Rhytideres plicatus was collected in Egypt by ARS scientists from France. It has potential but could not be fully evaluated due to travel restrictions: Host range testing with this potential native biocontrol candidate was blocked due to the impossibility of collecting more fresh material. Regarding Stinkwort, ARS scientists in France evaluated the moth Condica viscosa with more than 26 selected non target species in the Asteraceae (cultivated, United States native, ornamental species), two species outside the target led to a complete larval development to adult. However, both species are nonnative to the United States as they belong to the sub-tribe Inulinae which is absent from North America. None of the cultivated species tested (sunflower, tagetes) were used by the moth. For ToH, a field experiment was conducted in France in 2024-25 for measuring the impact of the mite Aculus taihangensis on field seedlings of ToH after a controlled inoculation. In 2021, the mite was shown as extremely host specific to ToH. The current field impact study in France showed a 50 to 94% biomass reduction for inoculated ToH seedlings vs controls. Also included in Sub-objective 3A, over the course of the project (2021-23), the foraging behavior and the ecological risk of attack on non-target plants of the French broom psyllid (FBP) was investigated by ARS scientists in France. During year 1 and year 2, two field experiments that included French broom, and five species of non-target plants showed limited dispersal ability of the psyllid and limited risk of establishment on non-target plants. In parallel, behavioral investigations in an olfactometer with FBP adults showed no attraction toward the volatile organic compounds (VOCs) of non-target plants, confirming field results. However, psyllids failed to show attraction toward French broom VOCs, whether in a vegetative or blooming state. During 2023, an electrophysiology study was also conducted with FBP, exposing the antennae of FBP adults to VOCs of host plants as well as blends of 40 common plant VOCs in an electroantennography (EAG) setup. Despite testing an extensive number of insects, no antennal responses to VOCs were observed. Interestingly, the antennae of French broom psyllids were extremely sensitive to vibrations but did not respond to VOCs. Thus results suggests that 1) the risks of attraction toward non-host plants and of establishment on non-host plants of FBP is minimal; and 2) FBP may not rely on olfaction to locate its host plants in the field. In Sub-objective 3B, no chemical analyses could be conducted, because of persistent technical difficulties at the chemical ecology platform of our partner. This was an unexpected issue as preliminary experiments were conducted in a proper manner. In Sub-objective 3C, the sanitary status of two biocontrol agents of French broom, Lepidapion argentatum and Arytinnis hakani, was assessed by ARS scientists in France. The two reared colonies were found free of detectable plant and insect pathogens. For stinkwort, Coleosporium inulae, a rust fungus, was collected in Cyprus, Portugal, and France, while a Sporisorium species (smut fungus) was collected on cogongrass (Imperata cylindrica) in Spain. Additionally, cherry leaf roll virus was detected in symptomatic ToH samples collected in Greece, which were also infested with a mite species currently under evaluation as a biological control agent. Since mites are known vectors of some viruses, those collected from the same location are currently being tested for the presence of the virus. However, transmission via mites is considered unlikely, as cherry leaf roll virus is typically pollen-borne. Under Sub-objective 3D, from 2022 to 2024 soil and seed samples of Ventenata dubia were collected from both native (Slovakia), and invasive (United States) ranges by ARS scientists from France. Preliminary tests established optimal growing conditions, and a transplant experiment began in October 2024 using combinations of seed and soil from native and invasive origins to study their impact on plant growth and seed production. At the end of the experiment in 2025, seed production, weight, and viability were assessed to evaluate the effects of soil and seed origins from both the native and invasive ranges of Ventenata. Slovak seeds consistently produced more and heavier seeds than United States seeds regardless of soil origin. While seed weight of United States seeds did not differ significantly between soils, both were lower than Slovak seeds grown in either soil. These results suggest that seed and soil origin influence reproductive success but challenge the assumption that invasive-range genotypes are better adapted to local conditions.

Accomplishments
1. The good mite against the bad and the ugly. Across the United States, Tree-of-heaven directly impacts agroecosystems by outcompeting surrounding native plants for water and sunlight and damages agriculture and farmers as a host of the spotted lanternfly. An invisible, host-specific mite from China, which is widespread in Europe and recently recorded from the United States, impacts Tree of heaven by its predation. An ARS team in France has completed field experiments in biological control, demonstrating that the mite reduced seedlings’ growth by 50 to 94% after controlled releases in early Spring. The highest effect on young plants was seen in shady areas. By reducing the tree-of-heaven densities, the mite, if applied in the United States, could indirectly impact spotted lanternfly populations perhaps as a cascade effect. Therefore, this mite could help control both an alien plant and an insect invader. Mass rearing the mite and releasing it is easy, and both technologies can be transferred to stakeholders, reducing the use of the labor-intensive mechanical control. The outcome of the research on this mite will not only positively help management of Tree of heaven in urban, natural and agricultural areas but also support reducing the spread of the spotted lanternfly invader that seriously threats the United States grape industry economy.

Review Publications
Marsch, D., Deines, L., Rausch, J.L., Tindon, Y., Sforza, R., Melton, A.E., Novak, S.J. 2025. Reconstructing the introduction history of the invasive grass Taeniatherum caput-medusae subsp. asperum in the western United States: low within-population genetic diversity does not preclude invasion. American Journal of Botany. e70001. https://doi.org/10.1002/ajb2.70001.
Hinkle, M., Sforza, R., Smith, J., Serpe, M., Novak, S. 2025. Disentangling Taxonomic Complexity in the Native Range: Morphological and Genetic Differentiation Among Subspecies of Taeniatherum caput-medusae. Agronomy Journal. 15:454. https://doi.org/10.3390/agronomy15020454.
Sforza, R., Breugnot, D., Tannieres, M. 2025. New European distribution of Coleosporium inulae (Coleosporiaceae) on Dittrichia graveolens (Asteraceae) and considerations for weed biological control. Botanica. 31(1):39-47. https://doi.org/10.35513/Botlit.2025.1.4.
Lesieur, V., Thomann, T., Jourdan, M., Kashefi, J., Bon, M. 2025. Fly in the ointment: Host specificity challenges for Botanophila turcica, a candidate agent for the biological control of saffron thistle in Australia. Insects. 16(4):357. https://doi.org/10.3390/insects16040357.