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

Location: EUROPEAN BIOLOGICAL CONTROL LABORATORY

2018 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
Progress was made on two objectives and their subobjectives, all of which fall under National Program 304, Crop Protection and Quarantine, Component 2: develop innovative control methods and integrated pest management (IPM) strategies for weeds, regarding Problem Statement 2A3: Systems approach to environmentally sound weed management in cropping systems and Problem Statement 2B3: Systems approach to environmentally sound weed management in natural ecosystems. Under Objective 1, ARS researchers at the European Biological Control Laboratory (EBCL), Montpellier, France, made significant progress in exploration for prospective biological control agents of several target weeds. The weevil (LEPIDAPION ARGENTATUM), a prospective biological control agent of French broom (GENISTA MONSPESSULANA), was collected in two localities in Sicily (Italy), which is the source population of the original morphological identification of this species in the 19th century, to verify the identity of the insects we are studying. In order to study the microbial diversity surrounding roots of the annual grass ventenata (VENTENATA DUBIA), soil was sampled in 2017 (3 replicates + 3 controls) in France, Bulgaria, and Slovakia to compare the diversity of the rhizoplan, rhizosphere, seed endophytes, and control soil. In 2018, a first analysis was done on two soils from France, to study the similarity of bacterial and fungal communities using 16S and ITS sequencing. Preliminary analysis suggests that the bacterial communities in the rhizoplan are under strong influence of plant exudates. Indeed, plant invasions often involve a shift in the associated microbial communities. The seed head weevil LARINUS FILIFORMIS, a prospective biological control agent of yellow starthistle (CENTAUREA SOLSTITIALIS), was collected by collaborators in Bulgaria. A life history study was conducted in quarantine and the weevil was multiplied to send to cooperators in California for host specificity studies. The eriophyid mite ACERIA SALSOLAE was collected in Greece, and a colony was established at EBCL. The mite was also released in a field garden experiment being conducted by collaborators in Italy to measure its host plant specificity. The seed-feeding moth, GYMNANCYLA CANELLA was collected in France and is being multiplied to send to cooperators in California for host specificity studies. The eriophyid mite, ACERIA DRABAE, was collected in Greece and sent to a collaborator in Montana in preparation for release to help control hoary cress (LEPIDIUM DRABA). Studies were conducted in collaboration with an ARS scientist in Edinburg, TX to establish a nematode-free colony of the arundo leaf miner (LASIOPTERA DONACIS) for release in the USA to help control giant reed (ARUNDO DONAX). Under Objective 2a, ARS researchers at EBCL made significant progress identifying the geographic origins of target weeds. In order to direct where to look for prospective biological control agents, surveys were conducted to collect samples of several target weeds for molecular genetic analysis. Collections of seeds of the invasive grass, VENTENATA DUBIA, were made in western Europe including 8 populations from 3 different countries, France, Spain and Portugal including 2 countries, Spain and Portugal, where VENTENATA DUBIA is considered extremely rare or nearly extinct. Genetic analysis conducted by our cooperator at the University of California, Irvine using genome-wide single nucleotide polymorphisms of populations of Sahara mustard (BRASSICA TOURNEFORTII) collected in 2016-2017 across Western Europe, Northern Africa and the Middle East by EBCL and Biotechnology and Biological Control Agency (BBCA) scientists identified as many as 7 distinct populations in Sahara mustard’s native range and matched one site in Morocco and two sites in Jordan populations as probable origins for the invasive weed. New populations of medusahead (TAENIATHERUM CAPUT-MEDUSAE), were collected in 3 Eurasian countries (Sicily, Armenia and Iran) by a visiting scientist and BBCA. Seeds were germinated at EBCL, and DNA was extracted and sent to ARS cooperator at Reno for genetic analysis using genotype by sequencing (GBS). New populations of cheatgrass (ANISANTHA TECTORUM), and red brome (ANISANTHA RUBENS) were collected by a visiting ARS scientist and BBCA cooperators in 6 Eurasian countries (Bulgaria, Greece, Iran, Armenia, Morocco and Kazakhstan) and one African country (Kenya). Genetic analysis using GBS revealed three main lineages of cheatgrass that have been introduced into the USA, with the suspected center of origin in central Asia. The results also support the close phylogenetic relationship between ANISANTHA TECTORUM and ANISANTHA RUBENS. Under Objective 2b, the genetic analysis using mitochondrial DNA of the root gall weevil (CEUTORHYNCHUS ASSIMILIS) associated with the invasive weed hoary cress (LEPIDIUM DRABA) revealed three distinct evolutionary lineages, one of which, the specialist lineage was only found on hoary cress, and occurred in a restricted area, ranging from northern Spain to northern Italy. However, this specialist lineage is going to be abandoned as a biocontrol agent because it was able to develop to adult on three federally listed threatened and endangered (T&E) species (STREPTANTHUS) as recently demonstrated by CABI in collaboration with EBCL. Under Objective 2C, a population of yellow starthistle (CENTAUREA SOLSTITIALIS) sampled in the native range in Sicily was evaluated by a botanist in Spain, and it appears to be a new species unknown to science. Genetic analysis of the stem-feeding shootfly (CRYPTONEVRA sp.), a prospective biocontrol agent of the giant reed (ARUNDO DONAX) in USA, reared from phragmites and arundo is ongoing to determine if there is a genetic lineage that is specific to giant reed. A field study was conducted to investigate the biology and ecology of CRYPTONEVRA sp. in the south of France and determine its impact on natural populations of Arundo. The arundo leaf miner (LASIOPTERA DONACIS) is a permitted biological control agent of giant reed; however, it is infested by a nematode (TRIPIUS GYRALOURA), which is not permitted for release. We developed a PCR-based method to detect and identify the nematode. We used genetic sequencing of French and Sicilian populations of the French broom weevil (LEPIDAPION ARGENTATUM), a prospective biological control agent of French broom (GENISTA MONSPESSULANA), to confirm that populations sampled from different parts of the plant (pods, galls) belong to one single species. Results evidenced that Sicilian populations were genetically different from French populations, though all belong to the same species, LEPIDAPION ARGENTATUM. Under Objective 3a, a field experiment to measure possible interactions among the three organisms (hoary cress [LEPIDIUM DRABA], the hoary cress gall weevil [CEUTORHYNCHUS ASSIMILIS], and the soilborne pathogenic fungus [RHIZOCTONIA SOLANI]) was conducted at EBCL. Real-time PCR used to quantify the natural and introduced populations of RHIZOCTONIA SOLANI in the metagenomic DNA extracted from the soil of the different conditions showed that Rhizoctonia populations did not vary significantly. Soil bacterial communities analyzed by sequencing the 16S rRNA gene showed a non-significant modification of their structure and the diversity in presence of the weevil and/or RHIZOCTONIA SOLANI. Host plant specificity of the French broom weevil (LEPIDAPION ARGENTATUM), a prospective biological control agent of French broom (GENISTA MONSPESSULANA) progressed. Seven nontarget lupine species endemic to California (LUPINUS FORMOSUS, L. CHAMISSONNIS, L. PERENNIS, L. ALBIFRONS, L. MICROCARPUS, L. LONGIFOLIUS, L. POLYPHYLUS) plus French broom (GENISTA MONSPESSULANA) controls were evaluated in no-choice host specificity tests. Feeding damage was observed on L. LONGIFOLIUS, L. PERENNIS, and on the control plants. From all plant species tested, only French broom developed a total of 26 galls among the 8 tested plants. Under Objective 3b, Sentinel GENISTA MONSPESSULANA plants were exposed from April to October (6 plants every two weeks) in two wild fields in Southern France to determine the seasonal activity and natural density of insect galling. Overwintering pupae of the leaf-feeding moth, ABROSTOLA ASCLEPIADIS, a prospective biological control agent of swallow-wort (VINCETOXICUM SPP.), provided adults in May. About 200 eggs were obtained in early June that provided a total of 72 pupae in late June. We still want to determine if the moth can produce more than one generation during the summer period.

Accomplishments
1. Assessment of safety of root gall weevil (CEUTORHYNCHUS ASSIMILIS) as a potential biological control agent for the noxious weed, Hoary Cress. Hoary Cress (LEPIDIUM DRABA) has spread throughout the western and northeastern U.S., and is now declared a noxious weed in 14 US states and three Canadian provinces. Host-range tests and molecular genetic studies conducted at the European Biological Control Laboratory in Montpellier, France indicated that a population of the root gall weevil (CEUTORHYNCHUS ASSIMILIS) in southern France is only found on L. draba. Experiments conducted by collaborators at CABI in Switzerland showed a preference by this French weevil for L. draba, especially under choice and open-field conditions, but also evidenced that the weevil was able to develop to adult on 39 species, including three threatened and endangered (T&E) species, and attacked one of the T&E species under multiple-choice conditions. Collectively these experiments lead to the conclusion that this weevil is probably not safe to use for biological control and should be abandoned.

Review Publications
Kellenberger, R., Desurmont, G., Schlueter, P., Shiestl, F. 2018. Trans-generational inheritance of herbivory-induced phenotypic changes in Brassica rapa. Scientific Reports. 8(1), article 3536. 10.1038/s41598-018-21880-2.
Madeline, M., Goolsby, J., Vacek, A.T., Kirk, A., Moran, P.J., Cortes, E., Cristofaro, M., Bownes, A., Mastoras, A., Kashefi, J. 2018. Densities of the Arundo Wasp, Tetramesa Romana (Hymenoptera: Eurytomidae) across its native range in Mediterranean Europe and introduced ranges in North America and Africa. Biocontrol Science and Technology. 28(8):772-785.
Shaw, R., Ellison, C., Marchante, E., Pratt, C., Schaffner, U., Sforza, R. 2017. Weed biocontrol in the EU: from serendipity to strategy. Biocontrol. 63:333. doi.org/10.1007/s10526-017-9844-6.