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

Location: European Biological Control Laboratory

2023 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
Objective 1. Permits for collecting and exporting living material were also obtained prior to traveling to Cyprus and Egypt. For stinkwort (Dittrichia graveolens), collections in Cyprus led to the discovery of several foliage feeder moths (Lepidoptera). A total of two Lepidoptera were field collected from stinkwort. Condica viscosa (Lep.: Noctuidae) and Heliothis peltigera (Lep.: Noctuidae). Condica viscosa represents the most promising candidate to date. For Sahara mustard, exploration in Egypt led to the discovery of several foliage feeder insects: a leaf miner Phytomyza horticola (Dip., Agromyzidae), and two weevil species, Rhytideres plicatus, Ceutorhynchus pallidactylus. The most promising species is R. plicatus, the reseda weevil. From swallow-worts, the host specific chrysomelid beetle, Chrysochus asclepiadeus was field collected prior shipping to the U.S. Objective 1. Sahara Mustard (Brassica tournefortii). Since the search in Europe for natural enemies of the weed did not result in identification of promising insects, a trip to Israel to explore for the natural enemies of the weed was undertaken in February 2023. The following insects were reared from the collected material during this trip and molecular identification was performed based on identity of the sequence of a barcoding gene to known sequence data present in Genbank: 1. Barcoding results for collected species 1: Best sequence match is to Genus Ocnogyna (closest species is Ocnogyna clathrata (Lepidoptera; Erebidae) found in Israel. The sequences are 99.33% identical indicating that they are likely the same species. 2. Barcoding results for collected species 2: Best sequence match is to Evergestis isatidalis for which barcoding sequence data is provided from a voucher sample labeled Lep24 (lepidoptera; Crambidae) . The sequences are 100% identical. 3. Barcoding results for collected species 3: Best sequence match is to Ceutorhynchus pallidactylus (Coleoptera; Curculionidae: cabbage stem weevil). The sequences are 99.39% identical indicating that they are likely the same species. 4. Barcoding results for collected species 4. Best sequence match is to Triaspis species (hym:Braconidae). The sequences are 99.28% identical indicating that they are likely the same species. 5. Barcoding results for collected species 5. Best sequence match is to Plutella xylostella (Lepidoptera; Plutellidae: Diamondback moth) which is an important pest of Cole crops in California, and this makes Sahara mustard as an invasive weed a potential pool of infestation for Diamondback moth in California. Further collections are needed since none of the above seems to be a highly host specific candidate for biological control of Sahara mustard. Objective 2A. A very first genetic analysis of individuals of stinkwort representing the invaded range (10 sites across CA) and three countries in the Mediterranean region in the native range was conducted by using population genetic markers developed at EBCL. In total, 141 individual samples representing mainly the invaded range have been processed to identify 13 polymorphic microsatellite markers. Genetic analysis evidenced the establishment of two distinct populations in CA, which are differently distributed between the North and the South of CA. Preliminary spatial interpolation already discarded Cyprus as an original native source of stinkwort in CA. Additionally, under Sub-Objective 2A the genetic analysis of Sahara mustard in the native range (8 countries mainly in the Mediterranean region) and the invaded range across CA, TX, UT and AZ was finalized by our cooperator at the US Geological Survey Center, UT. Across all invasive samples, analyses generally pointed to East Cairo, Egypt as a major source of invaded populations. Objective 2B. The survey of the assemblage of natural enemies associated with stinkwort collected from Crete and Cyprus in the native range under Objective 1 was done as little is known about its diversity. A DNA barcoding 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 two foliage feeder moths, Heliothis peltigera and Helicoverpa armigera (Lepidoptera: Noctuidae) also found last year in Cyprus and France, one aphid, Uroleucon inulae (Hemiptera: Aphididae), one scentless plant bug Stictopleurus sp. (Hemiptera: Rhopalidae) and one seed bug, Nysius graminicola (Hemiptera: Lygaeidae). All are given as nonspecific to stinkwort in the literature, so it is unlikely that they will be retained as prospective biocontrol agents in CA. Additionally under Sub-Objective 2B, the taxonomy of two tiny mites associated with tree-of-heaven (Ailanthus altissima) and found beyond the native range of this invasive, namely Aculus taihangensis present in Europe and Aculops alilanthi (Acari: Eriophyididae) in the U.S. calls for clarification as both mites seem to have the potential to be considered as biological control agents of Tree of Heaven. In collaboration with taxonomists of eriophyid mites in Serbia and our cooperator at BBCA in Italy, molecular phylogeny between A. alilanthi collected in four states in the U.S. and Ac. taihangensis collected in fourteen European countries coupled with morphological examination strongly suggested treating Ac. taihangensis as a junior synonym of A. alilanthi. Consequently, outcomes of ongoing laboratory and field experiments with Ac. taihangensis by EBCL and the European groups involved in host range testing will be compared to similar tests conducted on A. alilanthi in the U.S., enabling an effective identification of cues critical to the effectiveness and specificity of these biological control agents. Objective 3A. For swallow-worts, fecundity and longevity of females Chrysochus asclepiadeus, the principal BC agent, were studied in controlled conditions and showed that 25% of the the total tested female (n=40) lived over 70d, with a maximum lifespan at 100d. In controlled conditions, 97% of egg clusters are hidden (in rolled leaves, under cardboard), which is consistent with the field behaviour with eggs laid in the ground. Mean fecundity is of 246eggs/female. For three of heaven, the eriophyid mite, Aculus taihangensis was massively reared at EBCL, and augmentatively released in seven experimental sites (+3 controls) in France. An integrated approach of controlling tree of heaven by using mechanical method (debarking) and augmentative biocontrol was pursued. The purpose of field releases was to verify establishment and spread in natural conditions. Additional tests on wind dispersal were conducting (on-going). Host range testing with five U.S. non-target plant species was also conducted. Objective 3A. Research on French broom conducted in 2023 was a direct development of the results collected in 2021 and 2022 at EBCL and investigated the electrophysiology of the French bromm psyllid Arytinnis hakani, a promising natural enemy and potential biocontrol agent of French broom. In 2021 and 2022, research results showed limited dispersal ability of the psyllid on target and non-target plants under field conditions and no preferences for plant odors in olfactometer tests. These results led to the hypothesis that this species does not rely on olfaction to locate its host plants in the field. In order to test this hypothesis, the research project conducted in 2023 directly investigated the responses of French broom psyllid antennae to plant odors in an Electroantennography (EAG) and a gas chromatography-electroantennographic detection (GC-EAD) setting at CEFE Montpellier. Objective 3A. Antennae of males and females of French broom psyllids originating from the rearing maintained at EBCL were exposed to odors of French broom at the vegetative and blooming stage as well as to 40 volatile organic compounds commonly found in plant volatile blends. Despite testing an extensive number of insects, no responses to any volatile organic compound were observed. Interestingly, the antennae of French broom psyllids were extremely sensitive to vibrations but did not show any response to volatile organic compounds. These results support the hypothesis that the French broom psyllid does not rely on olfaction to locate its host plants in the field and has important implications for the release of this insect as a biocontrol agent in the U.S. Objective 3C. sampling seedpods, galls and adults from a native population in southern France was performed to check their sanitary status to make sure they are free from pathogens. Using microbial isolation and molecular methods, eight worldwide distributed fungal genera were identified and no insect pathogens. Isolated fungi belong to fungal groups generally described as saprophytic fungi or opportunistic plant pathogens. These results, in addition to the double ability of L. argentatum to develop in stems and pods which increases its potential value as a biocontrol agent against French broom, indicate that L. argentatum could be an efficient and safe candidate for the control of the invasive French Broom, Objective 3D. Preliminary tests have been conducted with soil from France. Plants were grown in two different sizes of pot and in greenhouse or growth chamber to identify the best conditions to grow Ventenata dubia. Seeds were obtained after six months under all the conditions, but plant mortality were higher in small pots and in the greenhouse. The biggest pots and growth chamber conditions were thus selected to conduct the transplant experiment. Seeds and soil from Slovakia have been collected and seeds and soil from the U.S. will be collected this summer to start the transplant experiment in September 2023.

Accomplishments
1. Development of genetic markers that show the diversity of invasive Stinkwort plants in the U.S. Stinkwort is a plant species in the sunflower family, native to southern Europe, North Africa, and western Asia. It has become naturalized in California and can kill grazing animals and can cause allergic reactions in people who come in contact with the sticky resin. From 1984 to 2012, stinkwort has expanded at an exponential rate, invading 36 of the 58 California counties. A very first genetic analysis of individuals of stinkwort representing the invaded range (10 sites across CA) and three countries in the Mediterranean region in the native range was conducted using population genetic markers developed by ARS researchers in Montpellier, France. Genetic analysis demonstrated the establishment of two distinct populations in CA, which are differently distributed between the North and the South of CA. This information can now be used to identify where to look in the native range for biological control agents that would provide the best control of this invasive and damaging weed.

Review Publications
Bon, M., Goolsby, J., Mercadier, G., Guermache, F., Kashefi, J., Cristofaro, M., Vacek, A., Kirk, A. 2023. The so-called ambrosia of the Arundo leaf miner, Lasioptera donacis, harbors a diverse endophytic fungal assemblage. Diversity. 15(4). Article 571. https://doi.org/10.3390/d15040571.
Lesieur, V., Sforza, R.F., Sheppard, A., Shaw, R. 2023. Prioritising environmental invasive weeds of European concern for classical biological control: A reanalysis. Weed Research. 1-14. https://doi.org/10.1111/wre.12582.
Hoelmer, K.A., Sforza, R., Cristofaro, M. 2023. Accessing biological control genetic resources: the United States perspective. BioControl. 68: 269–280. https://doi.org/10.1007/s10526-023-10179-5.