Research Project: Biological Control of Invasive Weeds from Eurasia and Africa

Location: EUROPEAN BIOLOGICAL CONTROL LABORATORY

2020 Annual Report

Objectives
Objective 1: Explore for natural enemies, including arthropods and microorganisms, 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 hoary cress, swallow-worts, thistle species, giant reed, tree of heaven, French broom, and medusahead grass. Objective 2: Perform taxonomic, population genetic, and phylogeographic studies as necessary to support the successful development of biological control agents for invasive weeds. Objective 3: Identify the biological and physical parameters that affect the efficacy and safety of potential agents, including climatic conditions, host specificity, effective rearing conditions, and how interactions that occur between a target weed (e.g. hoary cress) and multiple natural enemies (e.g., a root gall weevil and a root pathogen) enhance or inhibit biocontrol.

Approach
The goal of the proposed research is to improve management of several important, invasive alien weeds of the contiguous United States. The Bureau of Land Management estimates that over 4,600 acres of western rangelands are lost to the spread of invasive weeds each day. Successful invaders usually lack natural enemies, which control them in their native ranges. Classical biological control is a highly cost-effective, sustainable, environmentally-sound method of controlling exotic weeds over large areas. This method requires research aimed at understanding the basic biology and ecology of target weeds and their interactions with the environment and natural enemies. Genetic characterization of target weeds in their native and adventive ranges can identify regions of origin of invasive populations and help to understand the invasion process. Foreign exploration in the region of origin provides candidate biological control agents such as insects and mites for evaluation. Host specificity and potential effectiveness of candidates will be evaluated in field and laboratory studies. Experiments will also examine the evolutionary, ecological, and physiological aspects of plant-insect interactions relevant to weed biological control. Research on yellow starthistle, hoary cress, giant reed, medusahead, and French broom 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 here is critical to achieving ecologically rational, sustainable management of some of the most important invasive weeds in the United States.

Progress Report
Under Objective 1, since 2019, a colony of an eriophyid mite ACULOPS MOSONIENSIS collected from tree of heaven (AILANTHUS ALTISSIMA) in Italy, is maintained at EBCL. Seeds of non-target plant species were received from the U.S., as to be tested for host specificity with the potential arthropod agent. Adults of a seed feeder weevil (LEPIDAPION ARGENTATUM), a prospective agent of French broom (GENISTA MONSPESSULANA), were collected throughout the season in southern France. Field studies were conducted to understand its life cycle, and its impact, and revealed one single generation. Adults of a chrysomelid beetle, CHRYSOCHUS ASCLEPIADEUS, a prospective agent of swallow-worts (VINCETOXICUM SPP.), were collected in the French Jura for setting up an open field test in the city of Lyon. Two target species, VINCETOXICUM ROSSICUM and V. HIRUNDINARIA, and one non-target species, ASCELPIAS SYRIACA (the host plant of the Monarch butterfly) were tested in circled open field gardens where 20 couples were released per garden. Beetles' feeding activity and presence on selected plants were recorded. Exploration for eriophyid mites on VENTENATA DUBIA has revealed no presence from three populations in France and Germany. Under Sub-objective 2A, a genetic analysis of the invasive grass, VENTENATA DUBIA was conducted by our cooperator at Boise State University, ID. Genetic analysis of 51 invasive populations (1636 individuals) of VENTENATA DUBIA, coupled with historical records, suggests moderate propagule pressure from multiple introductions, followed by local or regional range expansion. The most common genotype revealed was widely distributed throughout the Pacific Northwest and was found in ca. 60% of the populations analyzed. Despite multiple introductions, invasive populations exhibit low levels of genetic admixture, low levels of genetic diversity within populations and high genetic differentiation among populations. The apparent reduced evolutionary potential of most VENTENATA DUBIA populations did not preclude the initial establishment and rapid spread of this species across its new range in the western US (1 article submitted). Collections of spurges (EUPHORBIA ESULA, E. PSEUDO-VIRGATA, E. CYPARISSIAS) were made from 13 countries, including France, Spain, Italy, Greece, Belgium, Bulgaria, Hungary, Slovakia, Slovenia, Switzerland, Kazakhstan, Armenia, and Czech Republic, for a total of 40 populations. Genetic analysis, using microsatellites, to be performed by cooperating ARS scientists in Sidney, MT, is aimed to pinpoint the origin of spurges invasive in the US. Additionally, under Sub-Objective 2A, the stem-feeding shoot fly (CRYPTONEVRA NIGRITARSIS) is one of the dipteran species infesting ARUNDO DONAX shoots in the south of France. Although it is considered as a prospective biocontrol agent of ARUNDO DONAX in the USA, very little is known of its biology and ecology, casting doubts on the impact and specificity of this agent. A DNA barcoding diagnosis tool has been purposely developed in order to assess in the shoots the prevalence of CRYPTONEVRA NIGRITARSIS among the dipteran larvae and pupae of which morphology examination cannot help identifying species. This method allowed us to separate 341 larvae and pupae sampled in three sites in South France in three groups: individuals belonging to the species CRYPTONEVRA NIGRITARSIS, individuals belonging to the CHLOROPIDAE (excluding CRYPTONEVRA NIGRITARSIS), and individuals belonging to another dipteran family. Genetic analyses revealed that the overall prevalence of CRYPTONEVRA NIGRITARSIS among the dipteran larvae and pupae found in ARUNDO DONAX shoots was 54.3% that CRYPTONEVRA NIGRITARSIS larvae were found every month of the year, and pupae were found to be the main overwintering stage. Overall, these results shed light on some important aspects on the biology, ecology, and impact of this potential agent of ARUNDO DONAX. Under Sub-Objective 2B, the root gall weevil (CEUTORHYNCHUS ASSIMILIS) associated with hoary cress is made at least by a specialist lineage strictly associated with hoary cress (LEPIDIUM DRABA) and a generalist lineage, which develops on several Brassicaceae. Genetic analysis of two progenies resulting from the interbreeding assays between these two lineages evidenced that a cross contamination occurred between the cages where parental populations were maintained. So at this point we cannot be conclude whether the two lineages interbreed or not, and if they belong to two different species or one species although previous molecular analyses rather suggested that these two lineages belong to the same species. Under Sub-Objective 2B, a common garden experiment was conducted by our American cooperator in Thessaloniki in Greece to test the host plant specificity of the prospective biological control agent LARINUS FILIFORMIS for yellow starthistle (CENTAUREA SOLSTITIALIS). The garden contained 10 replicates of 14 different kinds of plants and it is located at the American Farm School, in Thessaloniki, in Greece. The adult insects were released in May and mature flower heads sampled about every 2 weeks during the summer. The flower heads were dissected to record insect damage and 94 immature weevils were preserved for DNA analysis to determine which species they are. The barcode analysis revealed that LARINUS FILIFORMIS was only found on yellow starthistle confirming the specificity of the agent. Under Sub-Objective 3A, a field experiment with four conditions (hoary cress [LEPIDIUM DRABA] alone, hoary cress with the root gall weevil (CEUTORHYNCHUS ASSIMILIS), hoary cress with the soilborne pathogenic fungus RHIZOCTONIA SOLANI which is present in the USA, and all three organisms) was conducted at EBCL to measure possible interactions among the three organisms. Data were recently re-analyzed. Plant mortality rate tended to be higher in the weevil plus pathogen condition (21.8%) than in the weevil alone condition (13.8%), probably because the insect damage provided entry points for RHIZOCTONIA to attack the plant. Although we know now that the root gall weevil will not be retained as a biocontrol agent, this research opens the door for future research where such synergistic relationship between a root gall weevil and a soilborne pathogen will increasing the likelihood of a higher impact on the target. Under Sub-Objective 3B, a summer open field experiment to measure the herbivory and the oviposition behavior of CHRYSOCHUS ASCLEPIADEUS, a prospective agent of swallow-worts (VINCETOXICUM SPP.), was conducted in Lyon (France). Five circular gardens with potted plants of VINCETOXICUM HIRUNDINARIA, V. ROSSICUM, and ASCLEPIAS SYRIACA were tested. A total of 100 couples were released. No feeding damage was observed outside the genus VINCETOXICUM (30 plants attacked/30 plants exposed). A second, open field test with potted VINCETOXICUM HIRUNDINARIA and ASCLEPIAS SYRIACA was set up in the Jura (France). Plants were randomly displayed in a pasture where CHRYSOCHUS ASCLEPIADEUS naturally lives. In November 2019, checking of potted plants for presence of underground larvae of C. ASCLEPIADEUS, we counted tens from both VINCETOXICUM SPP. and only four from ASCLEPIAS SYRIACA. In May 2020, we recorded the emergence of tens of C. ASCLEPIADEUS from both VINCETOXICUM SPP, and only one adult emerged from one ASCLEPIAS SYRIACA plant. This reveals that C. ASCLEPIADEUS can overwinter on roots of ASCLEPIAS SYRIACA, but this is not enough to discard the beetle for future prospection in biocontrol. Based on our data, CHRYSOCHUS ASCLEPIADEUS is still sought to be petitioned for release. Sentinel GENISTA MONSPESSULANA plants were exposed from April to October in Southern France. We determined the seasonal activity and natural density of the French broom weevil (LEPIDAPION ARGENTATUM), inducing both gall formation, and seedhead damage on the target weed. A peak population from galls was observed on mid June, and a second peak occurred in late July from seedheads. In addition, EBCL has developed microbiological research on the assessment of the sanitary status of biocontrol agents under evaluation at EBCL which may bear microbial pathogens and could vector and spread disease in the introduced environment. A metagenomic analysis was conducted to study the bacterial diversity associated to the psyllid ARYTINNIS HAKANI under evaluation at EBCL as BCA against French broom GENISTA MONSPESSULANA, and the psyllid ARYTAINILLA SPARTIOPHILA, a biocontrol agent of Scotch broom CYTISUS SCOPARIUS in New Zealand and Australia and accidentally introduced in California. It revealed the presence in A. SPARTIOPHILA and C. SCOPARIUS from the UK of a CANDIDATUS LIBERIBACTER sp., whose members of this genus include bacterial pathogenic agent of important disease on citrus and potatoes and vectored by a psyllid. This C. LIBERIBACTER sp. from the UK is currently being identified more precisely as C. LIBERIBACTER EUROPAEUS (Leu) has been reported in Scotch broom and A. SPARTIOPHILA in 2013 in NZ and could support a scenario of the accidental co-introduction of Leu and A. SPARTIOPHILA from the UK to NZ for the biological control of Scotch broom.

Accomplishments
1. New understanding of host preference and herbivory impact of a potential biocontrol agent of swallow-worts. Invasive swallow-worts are composed of two species of VINCETOXICUM that invade the north-eastern USA and Canada. Biocontrol agents of swallow-worts were needed for release that were host-specific, safe for the environment, and did not attack the Monarch butterfly host-plant, the common milkweed, ASCLEPIAS SYRIACA. Data showed zero attack on milkweeds (0/20 plants exposed), meaning no foliage feeding by the beetle, and substantial defoliating on the weed target (30/30 plants exposed). However, the winter observation of larvae on the root system revealed the presence of hundreds of larvae on all the VINCETOXICUM plants, and four larvae on ASCLEPIAS SYRIACA. In May 2020, only one larva of the four observed emerged as an adult. This result was encouraging as 1) only one larva emerged, and 2) no feeding activity was observed on foliage of ASCLEPIAS SYRIACA in the course of the summer. Additional tests are required in 2021. Controlling swallow-worts in the U.S. and Canada will increase native biodiversity understory in natural parks and reserves, and recreational areas.

2. Rearing protocol developed for flies infesting shoots of ARUNDO DONAX. This developed protocol used cut A. DONAX shoots placed in a layer of water agar and proved successful to rear CRYPTONEVRA NIGRITARSIS, a prospective biological control agent of A. DONAX. This protocol greatly reduces the amount of time and efforts needed to grow A. DONAX shoots and improves rearing productivity of a potential biocontrol agent.

Review Publications
Escobar, Y., Guermache, F., Bon, M., Kerdellant, E., Petoux, L., Desurmont, G. 2020. Biology and ecology of Cryptonevra nigritarsis, a potential biological control agent against the giant reed Arundo donax. Biological Control. 147:104287. https://doi.org/10.1016/j.biocontrol.2020.104287.
Milbrath, L.R., Dolgovskaya, M., Volkovitsh, M., Sforza, R.F., Biazzo, J. 2019. Photoperiodic response of Abrostola asclepiadis (Lepidoptera: Noctuidae), a candidate biological control agent for swallow-worts (Vincetoxicum, Apocynaceae). Great Lakes Entomologist. 52(2).
Smith, L., Cristofaro, M., Bon, M., De Biase, A., Petanovic, R., Vidovic, B. 2018. The importance of cryptic species and subspecific populations in classic biological control of weeds: a North American perspective. Biocontrol. 3(417-425). doi.org/10.1007/s10526-017-9859-z.
Tannieres, M., Manaargadoo-Catin, L., Shaw, R. 2020. First report of Candidatus Liberibacter europaeus in the United Kingdom. New Disease Reports. 41:3. https://doi.org/10.5197/j.2044-0588.2020.041.003.