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- Widmer, Timothy
- Moore, Ryan
- 2021
- Crop Protection & Quarantine (304)

Research Project: Biological Control of Weeds Endemic to Southeast Asia and Australia that are Invasive in the United States **Location:
                            Office of International Research Engagement and Cooperation** 2021 Annual Report **Objectives**
                                

Discover and develop biological control agents for invasive terrestrial and aquatic weeds, with emphasis on those invasive to Florida. **Approach**
                                

Travel within Australasia, SE Asia, and China to a range of habitats to identify, collect and evaluate potential biocontrol agents of target invasive weeds in consultation with the ARS PI. Current targets will include the climbing fern, Lygodium microphyllum, downy rose myrtle, Rhodomyrtus tomentosa, Melaleuca and hydrilla, Hydrilla verticillata. Promising candidate agents will be shipped to U.S. cooperators for further study and development at U.S. quarantine facilities. **Progress Report**
                                

Objective 1. New biological control agents were evaluated in Australia for invasive weeds targeted for management in the United States. 

Earleaf acacia, Acacia auriculiformis (Fabaceae) is invasive in Florida, and evaluation of potential biocontrol agents of focused on the gall forming wasp, Trichilogaster sp. Preliminary host testing is complete and indicates specificity to its host.  Plant impact assessments have been completed for saplings and being initiated for small seedlings. Seasonality surveys were initiated in North Queensland where the wasp is native, and new sites were located west of Cairns in Chillagoe. Laboratory evaluation of the leaf feeding Chrysomelidae beetle, Calomela intermerata compared the performance of Queensland and Northern Territory populations of this species on the Florida introduced genotype of earleaf acacia (origin Northern Territory). The beetles (Northern Territory) held on live plants matching its host genotype had superior performance when comparing feeding damage, size and rate of development of immatures. Surveys for new agents of earleaf acacia and monitoring of herbivore populations in the native range were conducted in the Northern Territory, the region from which the U.S. introductions are thought to have originated. Molecular diagnostics of C. intermerata collected from new remote sites in the Tiwi Islands and Arnhem Land in the Northern Territory clustered with specimens from other sites in the same region. Local indigenous rangers were re-engaged in remote areas to assist with field surveys through access and insect/plant collections.
Old World climbing fern, Lygodium microphyllum (Lygodiaceae) is invasive in Florida and surveys were conducted in new and unexplored areas of its Australian native range at Lawn Hill (Boodjamulla) National Park in remote northwestern Queensland. Survey sites were located for future surveys, and insect herbivore damage was recorded. Sites in North Queensland, primarily near Cairns and Bamaga, were surveyed and defoliating Callopistria and Musotiminae moths were collected to establish cultures in Brisbane. The performance of these moth species will be evaluated on L. microphyllum genotypes sourced from the native range in Queensland and from the invasive range in Florida. Performance of the Callopistria will be compared to results of a similar/same species collected in Hong Kong and being tested at the USDA ARS Invasive Plant Research Laboratory in Fort Lauderdale. Damaged stems sections containing Siamusotima stem borers were also collected from Bamaga for development of laboratory rearing techniques. A site has been prepared and plant stocks generated to initiate a L. microphyllum common garden plot where native and U.S. invasive genotypes of this fern will be grown and monitor to evaluate the evolution of increased competitive ability theory. 
Mosquito fern, Azolla pinnata (Salviniaceae) is a floating aquatic plant that is invasive in Florida. The false scale, Dysmicoccus sp., that damages mosquito fern, was collected across a broad geographic range of Australia and identified by an expert taxonomist who determined it to be an undescribed species whose close relatives have floating ferns as hosts. Rearing techniques have been successfully developed for this candidate agent and preliminary host range testing indicates that Mosquito fern is its primary host. Colonies of the foliage-feeding weevil Bagous clarenciensis have been established for each of its field hosts, A. pinnata and A. filiculoides, and multigeneration studies are underway to contrast the performance of B. clarenciensis from each field host on both Australian native Azolla species. This year, field surveys were extended to new sites in southern Australia in Victoria and South Australia. 
Our exploratory research of aquatic plant targets focused within Australia. Surveys in Asia for collaborative research with the US Army Corps of Engineers, Engineer Research and Development Center, were limited to our Republic of Korea and Chinese collaborators exploring for biological control agents and conducting demography studies of the invasive floating hearts, Nymphoides peltata (Menyanthaceae). Nymphoides peltata is invasive across many US waterways. In demography studies, plant densities, reproduction, plant size, and plant chemistry (including Nitrogen/Phosphorus/Potassium and leaf toughness) are monitored at select sites. A current list of herbivores collected thus far on N. peltata in China and the Republic of Korea contains 19 insect species for further evaluation. Those prioritised for further evaluation include three Bagous weevils (Coleoptera: Curculionidae), two of which severely damaged the flowers (adults) and fruits (larvae), and a leaf-mining Hydrellia sp. fly (Diptera: Ephydridae).
Monoecious and dioecious forms of the submerged aquatic weed hydrilla, Hydrilla verticillata (Hydrocharitaceae) are invasive in the U.S.  Exploration in the native range for biological control agents of hydrilla was conducted in temperate regions of Australia and sites have been identified which are climatically similar to regions of the northern U.S. where hydrilla, particularly monoecious hydrilla, is invasive and where the existing biological control agents are not effective. Sites were located along the Murray River both along the border between the states of New South Wales and Victoria near the cooler Alpine region of Australia, and in the same river in South Australia.  Hydrilla across Australia and its associated herbivores, more specifically the leaf-mining Hydrellia flies, have also been surveyed to determine the biogeography of plant reproductive biotypes and the phylogenies of hydrilla/and associated Hydrellia flies. The data combined with laboratory evaluations will determine if unknown and cold tolerant Hydrellia genotypes exist which could be evaluated for use in the U.S. to control hydrilla. Testing was completed and results published on a Korean Hydrellia species, but this fly was rejected as a biocontrol agent due to its development on the related aquatic plant species, Elodea canadensis, native to North America, and the pondweed, Potamogeton crispus. A new introduction of hydrilla has recently been discovered in the Connecticut River. Given our previous extensive surveys of hydrilla in China and Korea and genetic characterization of plant samples we were able to rapidly determine the likely origin of the US introduction and report on the herbivores collected that have been collected from this genotype. The sites in China and the Republic of Korea matching the Connecticut River introduction were published, and the paper detailed the associated herbivores and potential biological control agents, including leaf-mining flies, Bagous weevils and Chironomidae midges.
Downy rose myrtle, Rhodomyrtus tomentosa (Myrtaceae) is an invasive weed in Florida.  Exploratory research has ceased on this weed, but data obtained on the stem-boring moth, Casmara subagronoma (Lepidoptera: Oecophoridae) was published summarizing its biological control potential. If the project is restarted, this insect could be prioritised immediately and developed as a biological control agent.
No insects being evaluated as biological control agents of plant targets were requested or required from stateside USDA ARS collaborators for shipment to quarantine facilities in the U.S. 
Objective 2. Progress was made to develop an iterative approach to explore for biological control agents in their native and introduced ranges using a decision process based on Next Generation Sequencing of weed targets and their natural enemies.  Engagement of intuitions with genomics expertise was required given this capacity is not available within ABCL. A subcontract was negotiated with the University of Queensland, School of Biosciences, and collaboration has been initiated. Acacia auriculiformis will be the model weed used for this research, though all weed targets will be utilized in this approach. Currently Lygodium microphyllum DNA is being extracted from our field samples from the native and invasive ranges for genotyping by sequencing to investigate mite resistance post release in Florida. Collaborative efforts are shared across three laboratories, the USDA ARS Australian Biological Control Laboratory, the USDA ARS Invasive Plant Research Laboratory and the University of Queensland.
Objective 3.  Investigation of hydrilla datasets accumulated since the mid-1980s identified gaps in the geographic surveys for biological control agents of hydrilla, Hydrilla verticillata in Australia. There is potential to explore for cold tolerant herbivores in temperate regions that could be suitable for use in the northern U.S. where hydrilla is problematic and where existing agents don’t overwinter. These surveys have bene initiated.    Interrogation of historic hydrilla collection data and field preserved specimens also identified that there is potential to evaluate the biogeography of hydrilla reproductive biotypes and the genetic phylogenies of hydrilla and associated leaf-mining Hydrellia flies. **Accomplishments**
                                

1. Overseas genetic surveys accelerate biological control development for a new Hydrilla verticillata introduction in the USA. The submerged aquatic weed hydrilla, Hydrilla verticillata is invasive in the U.S. A new introduction of hydrilla was recently discovered in the Connecticut River. Existing biocontrol agents are ineffective in the cooler regions of the U.S. like New England. Given our previous extensive surveys of hydrilla in Asia and genetic characterization of collected plant samples, we were able to rapidly determine that the likely origin of this new introduction was from sites in China or the Republic of Korea. From our surveys we could immediately report on the natural enemies and potential biological control agents adapted to this hydrilla genotype, which included leaf-mining flies, weevils, and midges. 2. New biological control agent specific against the weed earleaf Acacia. Biological control agents are urgently required for earleaf Acacia, an Australian native tree invasive in Florida. Without control this invasive tree could rapidly expand, reducing biodiversity by invading protected areas such as the Everglades National Park. A damaging gall-forming wasp was prioritized for evaluation as a biological control agent as closely related species have successfully controlled Acacia species in Africa. After successful colonization, preliminary no-choice host range testing has been completed and determined that this wasp is highly specific. This potential biological control agent can be sent to U.S. quarantine when required for final testing. 3. Laboratory populations established of insect biological control agents to evaluate as biological control agents against aquatic mosquito fern. Mosquito fern is a floating aquatic plant that is invasive in waterways in Florida. This plant has dramatically expanded its range in the state despite immediate efforts by land managers to eradicate it using herbicides because of its production of dense mats that impede irrigation and recreational activities. Natural controls are needed for aquatic environments where it is invasive. Two insects, a false scale and weevil, that kill mosquito fern were found in Australia, which is the native range for this invasive weed.  Populations were successfully established in the laboratory, which is important so that there are enough of them to do further host range tests and other studies.  These insects will also be tested on the American native floating fern.  If successful, these will be the first two insect biological control agents against mosquito fern.


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