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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

2022 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. Under Objective 1, several foreign exploration surveys were cancelled during the summer 2021, as most borders were closed. Few islands in the Mediterranean basin were safely surveyed by EBCL staff (Crete, Malta, Cyprus, Sicily). A series of potential BC candidates were found on stinkwort, Sahara mustard and medusahead. For stinkwort (Dittrichia graveolens), collections in Greece, Cyprus and southern France led to the discovery of several foliage feeder moths (Lepidoptera). A total of seven Lepidoptera and 2 Hymenoptera were field collected . Lepidoptera were identified as: Condica viscosa, first time reported from stinkwort and apparently is a very active defoliator; Heliothis peltigera and Autographa gamma, feed on multiple plant species; Epischia illotella already reported from Crete on two different host plants including stinkwort; Cochydia heydeniana, Helicoverpa armigera and Gymnoscelis rufifasciata also known as feeding on multiple plant species. All the above species, except Condica viscosa, are not suitable as a agents. The Hymenoptera species are being identified. Surveys in southern France and Spain on Sahara mustard (Brassica tournefortii) showed no suitable natural enemies for classical biological control. As per medusahead (Taeniatherum caput-medusae), two new locations with the medusahead gall maker (Tetramesa amica) were found in eastern Greece. 3b. Under Sub-objective 2A, a genetic analysis of Sahara mustard was conducted by cooperators at the U.S. Geological Survey Center, UT. Genetic analysis using next generation sequencing of Sahara mustard representing 24 sites across 9 countries mainly in the Mediterranean region was conducted by including the dataset obtained from previous work on the species invaded range (52 sites across CA and AZ). In total, raw DNA sequences of 1,009 individual samples have been processed using a supercomputer cluster to identify polymorphic genetic markers. Analyses confirmed the establishment of three distinct populations in the U.S., likely originating from multiple introductions, and the wide distribution throughout CA and AZ of one genotype. Preliminary spatial interpolation showed that Morocco and Jordan are likely the original native sources of Sahara mustard. Under Sub-Objective 2A stinkwort were collected from 6 countries, including France, Spain, Italy, Greece, Cyprus, and South Africa totaling 25 populations and in CA by our ARS cooperator in Albany for 12 populations. EBCL is aiming to pinpoint the origin of this weed in the U.S. Twenty markers were developed and tested on 60 individuals from four populations in South Africa, Cyprus and France. Nine were found diverse enough to allow their application at a larger scale for pinpointing the origin of this weed in the US. 3c. Under Sub-Objective 2B, the assemblage of natural enemies associated with stinkwort was surveyed, to ascertain its diversity. A DNA barcoding approach was applied to identify natural enemies in particular the juvenile stage (nymphs) which classical morphology is no help. We identified five moths (out of seven) belonging to four insect families (see above Obj. 1). Most moths, except Condica viscosa, were nonspecific to stinkwort in the literature, so it is unlikely that they be prospective biocontrol agents in CA. Additionally under Sub-Objective 2B, the same DNA barcoding approach was applied to identify the assemblage of natural enemies (at the nymphal stage) associated with Sahara mustard collected in Italy and Israel. Barcoding analysis revealed the prevalence of weevils, belonging to the beetle genus Ceutorhynchus in both countries. Under Sub-Objective 2B, the taxonomy of the mite Aculus mosoniensis associated with tree-of-heaven (Ailanthus altissima) in Europe needs clarification, owing to the potential this mite species as a biological control agent in the U.S. An integrative taxonomic approach was conducted in cooperation with taxonomists in Italy and Serbia. The phylogenetic analysis between Aculus mosoniensis collected in European countries and the Chinese Aculops taihangensis coupled with morphological examination allowed the synonymization of Aculus mosoniensis with Aculops taihangensis. In addition, this same species of eriophyid mite is occurring on tree-of-heaven in many European countries. Laboratory and field experiments are on-going with this mite by EBCL and the European groups involved in host range testing of this candidate agent. 3d. Under Objective 3, Sub-Objective 3A, adults of a leaf 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, swallow-worts (Vincetoxicum rossicum and V. hirundinaria), and two non-target species, e.g. milkweeds (Aclepias syriaca and A. tuberosa) were tested in field gardens where adult leaf beetles were released. As a preliminary, but promising result, both target weeds were infested and damaged by adults and nymphs of the beetle. No beetles were found on milkweeds. This is promising for the future of this leaf beetle as a biological control agent, and preliminary assessments to draft a petition to release in the U.S. and Canada is ongoing. 3e. Under Objective 3, Sub-Objective 3B, as in 2021, the foraging behavior and impact of biological control agents with the sapsucker French broom psyllid, (Arytinnis hakani) to control French broom (Genista monspessulana) continued. EBCL maintains a successful rearing of the psyllid in outdoor cages under field conditions, producing >25,000 psyllids in early 2022. This rearing method, if permission to release in U.S. occurs, could be modified to mass-produce this candidate for release. As such, a subgroup of the psyllid colony was released 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 evaluation in France will elucidate and the the feeding specificity of this psyllid. In addition, under Sub-Objective 3B, the foraging preferences of the French broom psyllid were investigated both in a device (olfactometer) that traps odors emitted by the plants (investigating the role of chemical cues on foraging preferences) and in a wind tunnel setting (investigating the role of chemical and visual cues on foraging preferences). In both settings, vegetative and flowering French broom plants were used. The results did not show any significant attraction to host plants. In the wind tunnel, psyllids were not found to fly upwind toward their host plant, even when the plant is placed at a short distance (40 cm). These results suggest that the dispersal behavior of the psyllid is not driven by visual or chemical cues. Under Objective 3, Sub-Objective 3C, specimens of the French broom weevil (Lepidapion argentatum), stem galls and infested seed-pods were collected (Genista monspessulana) in France in June 2022. Isolation of microbes from adults, larvae developing in seedpods or stem galls are ongoing. The presence of microbes negatively impacting plants and insects will be investigated using microbiology and molecular tools to identify the microbes. In addition, a metagenomic analysis was conducted to study the bacterial diversity associated with the psyllid Arytinnis hakani , and the psyllid spartiophilia, a BCA of Scotch broom Cytisus scoparius in New Zealand (NZ) and Australia and accidentally introduced into California. No phytopathogenic bacteria were detected the rearing of A. hakani, and A. spartiophilia from the U.S. However, it revealed the presence of A. spartiophilia and C. scoparius from the UK of Candidatus liberibacter sp., whose members of this genus include bacterial pathogenic agent of important disease on citrus and potatoes and vectored by a psyllid. As the presence of a bacterium (Candidatus liberibacter europaeus) in Scotch broom and the psyllid A. spartiophila was reported in NZ in 2013, a retrospective risk assessment was conducted to investigate the pathway by which the bacterium arrived in New Zealand. Using molecular tools, the bacterium was detected in Scotch broom and psyllids from the UK and NZ but not in ornamental brooms and other host plants of interest in NZ. The NZ surveys also show that the absence of the bacterium coincides with broom psyllids absence. In addition, the partial DNA sequences of this bacterium in Scotch broom and broom psyllids from the UK and NZ are identical. All the results strongly support the hypothesis of the accidental introduction of the bacterium into NZ via the release of the biocontrol agent originated from the U.K.. It remains uncertain whether the bacterium is symptomless in Scotch broom, or inducing symptoms like several other bacteria (Candidatus Liberibacter spp.). Nevertheless, molecular characterisation of insect microbiomes could become an essential procedure for pre-release safety screening of biocontrol agents that are potential vectors of plant pathogens. Under Objective 3, Sub-Objective 3D, ten kilograms of soil samples from France where the African wiregrass (Ventenata dubia; Triticeae) occurs, have been collected in May 2022. Preliminary tests are ongoing at the EBCL quarantine and EBCL property. Seeds and soil from the U.S where the dominant genotype occurs will be collected by co-operators and sent shortly to EBCL. Seeds and soil from Slovakia where the same genotype occurs will be soon collected. The aim of these collections is to set up a transplant experiment with various soil and seed origins of the African wiregrass.

1. First surveys for enemies against stinkwort. Several Mediterranean islands in the native range were surveyed in 2021 for natural enemies of stinkwort. The assemblage of prospective biocontrol agents associated with stinkwort was collected. Prior to these surveys, nothing was known about its diversity. A DNA barcoding approach, together with a morphological approach, has been applied to help identify accurately these natural enemies in particular at the juvenile stage (nymphs) of which classical morphology examination cannot help. This approach allowed us to identify a total of five moths (out of seven) belonging to four insect families. Most moths, except one, Condica viscosa, are feeding on a number of different plant species. As a result, Condica viscosa, collected from Cyprus is the most promising candidate agent to date.

Review Publications
Kashefi, J., Guermache, F., Cristofaro, M., Bon, M. 2021. Occurence of Aculus mosoniensis (Ripka & Érsek) comb. nov. (Acari; Prostigmata; Eriophyoidea) on tree of heaven (Ailanthus altissima Mill.) is expanding across Europe. First record in France confirmed by Barcoding. Phytoparasitica.
De Lillo, E., Marini, F., Cristofaro, M., Valenzano, D., Petanovic, R., Vidovic, B., Cvrkovic, T., Bon, M. 2022. Integrative taxonomy of Aculus mosoniensis (Acari: Eriophyidae), a potential biological control agent for tree-of-heaven (Ailanthus altissima). Insects. 13(5), 489.
Fowler, S.V., Lange, C., Beard, S., Cheeseman, D.F., Houliston, G.J., Paynter, Q., Peterson, P., Pitman, A., Smith, L., Tannieres, M., Thompson, S., Winks, C. 2021. Accidental introduction of Candidatus Liberibacter europaeus into New Zealand via a weed biocontrol agent from the UK. Biological Control. 160(2021) 104697.
Gaskin, J.F., Goolsby, J., Bon, M., Calatayud, P., Cristofaro, M. 2022. Identifying the geographic origins of invasive Megathyrsus maximus in the United States using molecular data. Invasive Plant Science and Management. 15(2):67-71.
Kerdellant, E., Thomann, T., Sheppard, A., Sforza, R.F. 2021. Host specificity and preliminary impact of Lepidapion argentatum (Coleoptera, Brentidae), a biocontrol candidate for French broom (Genisata monspessulana, Fabaceae). Insects. 12(8), 691.
Marini, F., Profeta, E., Vidovic, B., Petanovic, R., De Lillo, E., Weyl, P., Hinz, H., Moffat, C., Bon, M., Cvrkovic, T., Kashefi, J., Sforza, R., Cristofaro, M. 2021. Field assessment of the host range of Aculus mosoniensis (Acari: Eriophyidae), a biological control agent of the Tree of Heaven (Ailanthus altissima). Insects. 12:637.
Cuny, M.A., La Forgia, D., Desurmont, G., Glauser, G., Benrey, B. 2022. Top-down cascading effects of seed-feeding insects and their parasitoids on plant responses and early season herbivores. Functional Ecology. 00, 1-13.