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

Location: European Biological Control Laboratory

2021 Annual Report

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.

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
3a. Because of COVID-19 pandemics, all foreign exploration surveys but one to Greece were cancelled. Only a limited number of domestic travels within France were authorized and led to the discovery of a minute arthropod belonging to the Eriophyidae Family, Aculus mosoniensis on the target alien tree, tree of heaven (Objective 1, Explore for natural enemies, primarily arthropods, in the native range of prioritized invasive weed targets, such as Sahara mustard, French broom, annual grasses, tree of heaven, and other weeds). Eriophyid mites are generally host specific and could be an important tool for biological control (One article submitted). This mite was found attacking tree of heaven in a recreation park near Paris. In September, additional populations from the same mite species were discovered in four more locations in southeastern France. This is the first time that the mite has been discovered in France. In addition, two microlepidoptera and one Lepidoptera were collected in France from Stinkwort (Dittrichia graveolens), an invasive weed in California. In Greece, a stem gall maker was also discovered that needs to be recollected for emergence and identification. 3b. In order to pinpoint the genetic origin of a very fast spreading weed in California, Sahara mustard, (Brassica tournefortii), seeds from 28 populations in nine countries, including France, Qatar, Israel, Turkey, Italy, Jordan, Morocco, Australia, and Egypt, were collected (Objective 2, Sub-objective 2A). Seeds were sown in quarantine and their genomic DNAs were extracted, quantified and shipped to the U.S. cooperators. Genetic analysis, using SNPs to be performed by cooperating a scientist working for U.S. Geological Survey, Reston, CO, is aimed at pinpointing the origin of this weed in the U.S. Collections of stinkwort, Dittrichia graveolens, were made from seven countries, including South Africa, Sicily, Spain, Cyprus, Crete, France and Greece, for a total of 14 populations. Enriched DNA libraries were prepared from these materials in order to develop microsatellites markers that are still missing although critical to conduct future phylogeography studies. A total of close to 1.5 million reads were obtained, allowing the design of 116 microsatellites markers. The study on the genetic analysis of the 23 European populations of ventenata (Ventenata dubia), and performed by Boise State University, ID, USA, was suspended due to the COVID-19 restrictions at BSU. 3c. Prior to release a biocontrol agent in the U.S., a detailed and accurate genetic characterization is highly needed. Under Objective 2, Sub-objective 2B, (Genetic characterization of potential biocontrol agents), this characterization was performed on an eriophyid mite Aculus mosoniensis collected from tree of heaven in three countries, including France, Greece, and Bulgaria, for a total of seven populations. Genetic barcoding confirmed the genetic similarity of these populations with the Italian reference population of which the potential biological control agent is currently under evaluation (1 article submitted). Because of the genetic similarities this will allow host specificity testing of in field sites in France. Genetic characterization was performed on lepidopterans reared from stinkwort (Dittrichia graveolens) in Crete. The barcoding analysis identified these natural enemies as two different species belonging to two different families, Epischnia illotella (Pyralidae family) and to Cochylidia heydeniana (Tortricidae family) with 100% and 99.54% confidence, respectively. This is the first report of the association of these two lepidopteran species with stinkwort. 3d. Evaluating non-target impacts of a weed biocontrol candidate is mandatory for every petition submitted in the U.S. Under Objective 3, 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), adults of a chrysomelid beetle, Chrysochus asclepiadeus, a prospective agent of swallow-worts (Vincetoxicum spp.), were evaluated on non-target species in an open field test in the city of Lyon (central France). Insect colonies were maintained at EBCL. Two target species, Vincetoxicum rossicum and V. hirundinaria, and one non-target species, Asclepias syriaca (the host plant of the Monarch butterfly) were tested in open circular field gardens where chrysomelid adults were released centrally. As a preliminary, but promising result, both target weeds were infested and damaged by adults and larvae of the beetle. No beetles (adults and larvae) were found on the non-target species. This is very promising for the future of this chrysomelid beetle as a biological control agent. Due to the pandemics, no laboratory arthropod colonies were maintained on medusahead, ventenata and tree of heaven. 3e. Maximizing the number of biocontrol agents is a recognized approach for a successful biocontrol program (Objective 3, Sub-Objective 3B, Investigate foraging behavior and impact of biological control agents with French broom weevil and French broom psyllid). To control French broom (Genista monspessulana), two insects are currently under consideration. The first is a seed-feeding weevil (Lepidapion argentatum) that was collected every two weeks in southern France (except during Pandemic lockdowns). Field studies were conducted to understand the weevil’s life cycle and impact. A single generation was identified with two population peaks in the summer. The second is a sapsucker insect, Arytinnis hakani, the French broom psyllid A highly successful rearing of the psyllid has been developed in outdoor cages under field conditions, producing >25,000 psyllids in early 2021. This mass rearing method is extremely effective producing large numbers of high-quality insects and in case of permission to release in U.S., could be slightly modified to be used to mass-produce this biological control agent candidate. As such, a subgroup of the psyllid colony was released in an outdoor experiment to verify if the biocontrol candidate attacks various species of lupines: Lupinus longifolius, L. chamissonis, L. formosus, L. arboreus, and L. albifrons, compared to French broom. These lupines are native to California, and their anticipated evaluation in France will help to verify whether the psyllid is a safe candidate for submitting a petition to release. The design of the experiment is innovative with two circles designed at 5 and 10 meters from the central release point. The experiment is still in progress. In addition, psyllid adults were being tested for their olfactory preferences in a four-arm olfactometer under laboratory conditions. This approach could be further used for a push-pull strategy if specific odors are recognized by the psyllid. Psyllids were given the choice between their host plant, French broom, and three blank odor sources. The host plant was presented to the psyllids either at the vegetative or the blooming stage, and either infested with psyllids or not. Results indicated that while large numbers of the psyllids made a choice in olfactometers but choices were highly variable.

1. Barcoding for minute biocontrol organisms. Developing a DNA barcoding protocol for very small insects and mites that improve the efficacy and sensitivity for detection will be highly beneficial to ARS laboratories. Current techniques require DNA extraction, a process that is not only time consuming and costly but also subject to DNA loss. In addition, pooling multiple insects and mites for extraction can lead to a failure in detecting different species when mixed during the sampling. To improve this technique for small insect identification, EBCL scientists modified the classical PCR conditions that are commonly used for the genetic analysis of individual eriophyid mites as biological control agents. This protocol proved successful to amplify the barcode region in an individual mite that is being considered as a biological control agent of tree of heaven, a highly invasive tree in eastern U.S. This protocol greatly reduces the chance to amplify different DNAs from a mixed sample that occurs when tiny eriophyid mites are pooled together because in past protocols they did not provide enough genetic material for analysis. [304, C1, PS1A, 2021]

Review Publications
Desurmont, G., Bon, M., Kerdellant, E., Guermache, F., Pfingstl, T., Tixier, M. 2020. An integrative approach combining molecular analyses and experiments to investigate predation of insect eggs by a mite. Ecosphere. 11(3).
Gaskin, J.F., Endriss, S.B., Fettig, C., Hufbauer, R., Norton, A., Sforza, R. 2021. One genotype dominates a facultatively outcrossing plant invasion. Biological Invasions.
Marini, F., Vidovic, B., Lonis, S., Wibawa, M.I., De Lillo, E., Kashefi, J., Cristofaro, M., Smith, L. 2020. Comparison of the performance of an eriophyid mite, Aceria salsolae, on nontarget plants in the laboratory and in the field. Biological Control. 152. Article 104455.
Pervukhina-Smith, I., Sforza, R., Cristofaro, M., Smith, J.F., Novak, S.J. 2020. Genetic analysis of invasive populations of Ventenata dubia (Poaceae): an assessment of propagule pressure and pattern of range expansion in the USA. Biological Invasions. 22:3575–3592.