M.L. 2013 Minnesota Aquatic Invasive Species Research Center Subproject Abstract For the Period Ending June 30, 2019 SUBPROJECT TITLE: MAISRC Subproject 2: Metagenomic Approaches to Develop Biological Control Strategies for Aquatic Invasive Species - Phase II: Development of Potential Microbiological Control Agents for Aquatic Invasive Species SUBPROJECT MANAGER: Michael J. Sadowsky AFFILIATION: University of Minnesota – Minnesota Aquatic Invasive Species Research Center MAILING ADDRESS: 140 Gortner Lab, 1479 Gortner Avenue CITY/STATE/ZIP: St. Paul, MN 55108 PHONE: (612) 624-2706 E-MAIL: sadowsky@umn.edu WEBSITE: http://www.maisrc.umn.edu/ FUNDING SOURCE: Environment and Natural Resources Trust Fund (ENRTF) LEGAL CITATION: M.L. 2013, Chp. 52, Sec. 2, Subd. 06a SUBPROJECT BUDGET AMOUNT: $303,217 AMOUNT SPENT: $286,610 AMOUNT REMAINING: $16,607 Sound bite of Subproject Outcomes and Results This project evaluated the potential for harnessing natural microbes for use as biocontrol agents against Eurasian watermilfoil and zebra mussels. Several microorganisms were isolated that could be pathogenic to zebra mussels, but none met safety requirements for testing. EWM is associated with elevated concentrations of E. coli and human pathogens. Overall Subproject Outcomes and Results: Aquatic invasive species (AIS), including Eurasian watermilfoil (EWM) and zebra mussels (ZMs) pose a serious threat to the health and function of aquatic ecosystems. Traditional approaches for AIS management, including use of chemicals and manual removal, have been ineffective. This requires development of new management and eradication strategies, such as the use of (micro)biological control agents. Some microorganisms have evolved to live in close association with aquatic organisms and such relationships could be exploited to develop microbe-mediated AIS management strategies. As the first step towards the identification of potential biocontrol strategies, microbial communities associated with ‘healthy’ AIS were compared with that of ‘diseased’ AIS or to native species. Since no natural diseased mussels were available, we opted to develop an experimental model system, which allowed for the application of different intensities of stress – heat (17, 25, 33℃) and salinity (1.5, 13.5 ppt), to promote the proliferation of opportunistic pathogens. High-throughput DNA sequencing of 414 samples (providing 32 million DNA reads) resulted in the identification of several potentially ‘pathogenic’ microbial groups that were strongly associated with ZM mortality. These included Aeromonas, Chryseobacterium, Flavobacterium, Acidaminobacter, Clostridiaceae 1 sp., Rhodobacteraceae sp., Acinetobacter, Shewanella, and Clostridium sensu stricto 13. For the identification of EWM-specific microbiota, high-throughput DNA sequencing was performed on 315 samples (46 million reads) derived from leaf and root compartments of EWM and six native macrophyte species. This resulted in the identification of taxa that were significantly enriched in EWM leaves and roots compared to native plants. Though several AIS-associated microorganisms were isolated that could be pathogenic to invasive mussels (e.g. Aeromonas) - none of them met our safety requirements for further testing. Future studies must isolate and evaluate the efficacy of ‘host-specific and pathogenic’ biocontrol candidates that will only infect invasive mussel species.

Subproject Results Use and Dissemination

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Our research findings were disseminated via oral and poster presentations at the following (international/ national/ local) conferences: 61st International Association for Great Lakes Research conference (Toronto, Canada), UNC Water Microbiology Conference 2019 (Chapel Hill, NC), 20th International Conference on Aquatic Invasive Species (Fort Lauderdale, FL), 5th Upper Midwest Invasive Species Conference (Rochester, MN), 119th General Meeting of the American Society for Microbiology (San Francisco, CA), and the AIS Research Management Showcase in 2017 & 2018 (St. Paul, MN). Two papers were published in the journals ‘FEMS Microbiology Ecology’ and ‘Science of the Total Environment’ during this project period. One manuscript is currently undergoing peer-review and two additional manuscripts are under preparation. All sequencing data generated in this project will be publicly available (via submission to NCBI Genbank) and all publications will list accession numbers to link to short read archive of all samples. Thus far, all sequence data mentioned in current publications is directly linked to a publicly available web site for download.

Environment and Natural Resources Trust Fund (ENRTF) M.L. 2013 Minnesota Aquatic Invasive Species Research Center Sub-Project Work Plan Final Report

Date of Report: August 8, 2019

Final Report

Date of Work Plan Approval: May 23, 2017

Sub-Project Completion Date: June 30, 2019

Project Completion Date: June 30, 2019

SUB-PROJECT TITLE: MAISRC Sub-Project 2: Metagenomic Approaches to Develop Biological Control Strategies for Aquatic Invasive Species - Phase II: Development of Potential Microbiological Control Agents for Aquatic Invasive Species Sub-Project Manager: Michael J. Sadowsky

Organization: University of Minnesota – Minnesota Aquatic Invasive Species Research Center

Mailing Address: 140 Gortner Lab, 1479 Gortner Avenue

City/State/Zip Code: St. Paul, MN 55108

Telephone Number: (612) 624-2706

Email Address: sadowsky@umn.edu

Web Address: http://www.maisrc.umn.edu/ Location: Minnesota Statewide

Total ENRTF Sub-Project Budget: Sub-Project Budget: $303,217 Amount Spent: $286,610 Balance: $16,607

Legal Citation: M.L. 2013, Chp. 52, Sec. 2, Subd. 06a Appropriation Language: $4,350,000 the first year and $4,350,000 the second year are from the trust fund to the Board of Regents of the University of Minnesota to develop and support an aquatic invasive species (AIS) research center at the University of Minnesota that will develop new techniques to control aquatic invasive species including Asian carp, zebra mussels, and plant species. This appropriation is available until June 30, 2019, by which time the project must be completed and final products delivered.

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I. SUB-PROJECT TITLE: Metagenomic Approaches to Develop Biological Control Strategies for Aquatic Invasive Species - Phase II: Development of Potential Microbiological Control Agents for Aquatic Invasive Species II. SUB-PROJECT STATEMENT: Aquatic invasive species (AIS), including Eurasian watermilfoil (EWM) and zebra/quagga mussels (ZM/QM), pose a serious threat to the health, structure, and function of aquatic ecosystems. Traditional approaches for AIS control, including the use of chemicals and manual removal, have been mostly ineffective. This problem requires the use of innovative management and eradication tools, such as (micro)biological control strategies. Some microorganisms have evolved to live in close association with aquatic organisms, and these interactions may be commensal, symbiotic, or pathogenic in nature. Such relationships could potentially be exploited to develop microbe-mediated AIS management strategies. During the first phase of this project (years 1 & 2), we used high-throughput sequencing approaches to characterize the total microbial community (bacterial and fungal) structure associated with ZM/QM and EWM, in Minnesota waterways across time and space. This has provided a distributional map of microbes specifically associated with AIS and these will be key for the development of microbiological control strategies for AIS. The work proposed in Phase II (years 3 & 4) will build upon the results obtained in Phase I. Specific objectives in Phase II are to: (1) identify and isolate microbes that are potentially pathogenic to AIS, and, (2) evaluate the specificity and effectiveness of potential biocontrol agents in laboratory microcosms. The following activities will be performed to accomplish these objectives: (1) AIS sample collection and processing, (2) isolation and characterization of potential pathogens, (3) challenge/infectivity experiments. The proposed work is about 40% basic, 55% applied research, and 5% outreach in nature. These studies will put Minnesota at the forefront of this important area of AIS research. Project outcomes will provide important information for conservation practices of native aquatic species and management of natural resources in Minnesota. III. SUB-PROJECT STATUS UPDATES: Sub-Project Status as of January 31, 2018: Work began on this project in earnest. Field sampling commenced in July and ended in October. Native plants (seven different species), EWM, water and sediment were collected from the same nine lakes (Josephine, Vadnais, White Bear, Phalen, Cedar, Minnetonka, Bush, Lower Prior, and Nokomis) that were extensively sampled in 2016. Meta data were also measured at each site. DNA was extracted from all samples (n=315) and were sequenced at the University of Minnesota Genomics Center. A few native mussels have also been collected with the help from collaborators at SAFL. Sub-Project Status as of July 31, 2018 Significant progress has been made in this project sampling for the 2018 field season commenced in June. Sequence analyses have been completed for all the samples (which included invasive and native macrophytes) procured during the 2017 field season and the results look promising. Targeted cultivation of select microbes has begun based on information obtained from Phase 1. The experimental setup for zebra mussel stress experiments have been completed, which are currently underway. A junior researcher (Jonathan Bertram) was hired on April 16 and replaced Hannah Dunn. Updated information has been provided in column A on the attached budget. No amendments are necessary to accommodate these changes. Sub-Project Status as of January 31, 2019: We have made significant progress since our last project update. In particular, several stress experiments were performed on ~2,500 zebra mussels that were collected during the 2018 field sampling season. This was done to develop a disease model for zebra mussles to test the affect of potential biocontrol microbes. Several aquaria were maintained under controlled conditions, and the effect of temperature and salinity on zebra mussel

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survival was examined. Work is currently underway to elucidate changes within microbial communities associated with these invasive mussels under stressed conditions. We have requested three amendments as part of this project status update: (1) creation of an additional sub-category within ‘Equipment/Tools/Supplies’, (2) transfer funds from “Personnel (Wages and Benefits)” to “Professional /Technical Services and Contracts” and “Equipment/ tools/ Supplies” categories, (3) and transfer of funds from “Services – Lab and/ or Medical: Research and Analysis lab” sub-category to the “Services – Lab and/ or Medical: UMGC”. Amendment Request January 31, 2019 As explained in the project status of Activity, we did not have access to any natural zebra mussel die-off events, necessitating a change in experiemental plans. Due to these changes, we anticipate increased expenses within the following sub-categories: (1) Services – Lab and/ or Medical: UMGC, (2) Rental: MAISRC AIS holding facility, and (3) Supplies – Lab and/or Field. We request to amend our budget as follows: Amendment 1 We request that $12,500 be transferred from Personnel (Wages and Benefits) in Activity 2 and split between Professional/Technical Services and Contracts and Equipment/Tools/Supplies. Surplus funds within Personnel exist due to a five month gap (November 2017 – April 2018) between the departure and subsequent hiring of a new junior researcher.

• $5,500 will be used to pay for additional expenses within Rental: MAISRC AIS holding facility in Activity 3. We will need to rent space at this facility for at least two additional 12-week periods during our zebra mussel and milfoil microcosm trail experiments.

• $5,500 will be used to purchase additional supplies [i.e. DNA extraction kits ($3,000), PCR reagents ($1,500) and disposable plastic ware ($1,000)] in Supplies – Lab and/or Field for Activity 1.

• $1,500 will be used to purchase new pipettes in Supplies – Lab and/or Field for Activity 3, to replace those that were damaged (and beyond repair) during the course of this project.

Amendment 2 We request that $3,000 from Services – Lab and/or Medical: Research Analytical Lab (Activity 1) be transferred to Services – Lab and/ or Medical: University of Minnesota Genomics Center (Activity 2). Given our current studies, we do not anticipate that all the funds previously allocated to “Services- Research Analytical Labs” to be used. Once transferred to the new category, these funds will be used to pay for an additional sequencing runs. Amendments Approved by LCCMR: 02/26/2019 Overall Sub-Project Outcomes and Results: Aquatic invasive species (AIS), including Eurasian watermilfoil (EWM) and zebra mussels (ZMs) pose a serious threat to the health and function of aquatic ecosystems. Traditional approaches for AIS management, including use of chemicals and manual removal, have been ineffective. This requires development of new management and eradication strategies, such as the use of (micro)biological control agents. Some microorganisms have evolved to live in close association with aquatic organisms and such relationships could be exploited to develop microbe-mediated AIS management strategies. As the first step towards the identification of potential biocontrol strategies, microbial communities associated with ‘healthy’ AIS were compared with that of ‘diseased’ AIS or to native species. Since no natural diseased mussels were available, we opted to develop an experimental model system, which allowed for the application of different intensities of stress – heat (17, 25, 33℃) and salinity (1.5, 13.5 ppt), to promote the proliferation of opportunistic pathogens. High-throughput DNA sequencing of 414 samples (providing 32 million DNA reads) resulted in the identification of several potentially ‘pathogenic’ microbial groups that were strongly associated with ZM mortality. These included Aeromonas, Chryseobacterium, Flavobacterium, Acidaminobacter, Clostridiaceae 1 sp., Rhodobacteraceae sp., Acinetobacter,

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Shewanella, and Clostridium sensu stricto 13. For the identification of EWM-specific microbiota, high-throughput DNA sequencing was performed on 315 samples (46 million reads) derived from leaf and root compartments of EWM and six native macrophyte species. This resulted in the identification of taxa that were significantly enriched in EWM leaves and roots compared to native plants. Though several AIS-associated microorganisms were isolated that could be pathogenic to invasive mussels (e.g. Aeromonas) - none of them met our safety requirements for further testing. Future studies must isolate and evaluate the efficacy of ‘host-specific and pathogenic’ biocontrol candidates that will only infect invasive mussel species. IV. SUB-PROJECT ACTIVITIES AND OUTCOMES: ACTIVITY 1: Sample collection and processing Description: Sampling efforts in Phase II will complement those performed in Phase I, which primarily focused on characterizing the spatio-temporal variation of microbial communities (bacterial and fungal) associated with apparently “healthy” ZM/QM and EWM. The proposed work will mostly focus on collecting samples from diseased/stressed AIS (and healthy AIS, if applicable), and taxonomically related native species (macrophytes and bivalves) during the summer and fall over a 2-year period. Sampling sites will be selected based, in part, on consultation with the Minnesota Department of Natural Resources (MN DNR) and MAISRC faculty. Invasive mussels (~50 per sampling site) will be collected from water bodies with reported mortality events, and native bivalves will be sampled in collaboration with the Center for Aquatic Mollusk Programs (Lake City, MN), Minnesota Zoo (Apple Valley, MN) and Dr. Jessica Kozarek (St. Anthony Falls Lab, UMN). Site selection will also be done in consultation with our collaborator, Dr. Michael McCartney (UMN) and the MN DNR. Mussels will be placed in 2L bottles containing fresh aerated water from the sampling site, and brought to the lab for processing. Mussels (and DNA) collected in this project will be shared with an ongoing MAISRC Sub-Project by project manager Dr. Nick Phelps, entitled “Developing eradication tools for invasive species. Phase II: Virus discovery and evaluation for use as potential biocontrol agents” New EWM sampling sites will be selected in consultation with our collaborator, Dr. Ray Newman (UMN) and MN DNR. Diseased EWM tissues (including lesions) will be identified, confirmed via microscopy, and rated via a disease-factor index. In addition, EWM tissues affected by the milfoil-weevil (Euhrychiopsis lecontei) will be collected. Samples will also be collected from lakes within the Midwest region (e.g., Lake Auburn, Cenaiko Lake, Otter Lake, and Lake Minnetonka) with documented reports of EWM decline. In addition, native, phylogenetically related macrophytes, and water and sediment samples will be collected and analyzed from each sampling location. This will aid us in determining taxonomic specificity of the microbiota prior to testing pathogenicity, efficacy and host specificity. We will also record environmental parameters (e.g., temperature, dissolved oxygen, and pH) at each site when sampled. All field samples will be processed within 24 h of collection. Mussel samples will be processed as whole tissue and as specific dissected organs, whereas EWM (and other macrophytes) tissue samples will be processed to characterize both epiphytic and endophytic microbes. The majority of subsamples will be stored at -20˚C for DNA extraction, while others will be preserved in glycerol at -80˚C for future cultivation-based experiments. Water samples will be filtered onto 5um and 0.22um filters to concentrate cells, and the filtrate will be submitted to the Research Analytical Labs (UMN) for nutrient analysis. DNA will be extracted from biomass samples and filters using the DNeasy PowerSoil Kit, as per the manufacturer’s instructions.

Summary Budget Information for Activity 1: ENRTF Budget: $67,000 Amount Spent: $61,688 Balance: $5,312

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Activity Completion Date: Outcome Completion Date 1. Collect and process 150 native mussel and macrophyte samples December 2018 2. Collect and process 100 samples from ZM/QM mortality events December 2018 3. Collect and process 150 diseased and weevil-infected EWM December 2018

Activity Status as of January 31, 2018: Samples (EWM, non-targeted native plants, water, and sediment) were collected from nine lakes around the Twin Cities Metro Area (Josephine, Vadnais, White Bear, Phalen, Cedar, Minnetonka, Bush, Lower Prior, and Nokomis). Sampling was performed at all sites twice (in July and September). All sampling sites were the same as those selected in phase 1, with the exception of lake Holland which was not accessible this year due to construction. In addition to EWM, the following non-target plants were also collected: Pondweed (broad-leaf, large-leaf, curly-leaf), Wild Celery, Canada waterweed, Lillypads, and Coontail. Physical site data (temperature, dissolved O2, pH and conductivity) were recorded for each sampling event. All samples were processed in the lab within 24 hours of collection. DNA was extracted from all samples. A few native mussels (threeridge, white heelsplitter, and plain pocketbook) have been collected in collaboration with Dr. Jessica Kozarek at the St. Anthony Falls Laboratory, Minneapolis. Activity Status as of July 31, 2018: An experimental setup was designed, constructed, and has been completed to conduct stress experiments on zebra mussels, which are being maintained at MAISRC’s holding facility (UMN). Zebra mussels were collected from Lake Minnetonka for preliminary survival experiments. The effect of environmental variables (e.g., water temperature, pH, dissolved oxygen, and salinity) on the structure of zebra mussel-associated microbiota will be evaluated during summer and fall. This data will be used for targeted microbe experiments. Activity Status as of January 31, 2019: Since we did not have access to any natural zebra mussel die-off events, we focused our effort to evaluate whether changes in environmental conditions (e.g. temperature and salinity stress) resulted in the enrichment of potential pathogens in zebra mussels. Prior to the larger stress experiment, a pilot scale experiment (1 month) was conducted to study the physiology, behavior, and adaptability of zebra mussels in a laboratory aquarium environment. A pilot aquarium was built with appropriate conditions such as pH, light, aeration, and anchoring points, following which, over 400 zebra mussels were collected from Lake Minnetonka and monitored for morbidity or mortality. For the final experiment (2.5 months), ~2500 individual zebra mussels were collected from Gull Lake. Nine aquarium tanks were set up with custom-designed riggings and airflow. All zebra mussels were first acclimatized to lab conditions and then increased until reaching a setpoint. Specifically, the following conditions were evaluated: (a) high temperature and low salinity, (b) high temperature and high salinity, (c) low temperature and high salinity, (d) high temperature and low salinity, (e) medium temperature and low salinity, and (f) low temperature and low salinity. Each tank was monitored daily for zebra mussel mortality and water quality (e.g. ammonia concentrations, dissolved oxygen, pH). Throughout the experiment, zebra mussels were extracted at pre-determined timepoints in order to get a snapshot of the microbial community at that time point. Dead zebra mussels were also collected and processed, where applicable. Glycerol stocks were also prepared for each sampling point, which would allow for targeted isolation in the future. Our results indicated that increases in temperature and salinity contributed towards higher zebra mussel mortality. Final Report Summary: Zebra mussels: Zebra mussel (ZM) die-off events were not reported during the course of this project. To overcome this problem, we developed a model system to determine the effect of multiple environmental stressors (i.e. heat and salinity)

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on ZM survival and impact on its associated microbial communities. This led us to perform several experiments outlined in Activity 3, as part of Activity 1. It was hypothesized that these stressors would weaken the immunity of ZMs and result in the enrichment of opportunistic microbial pathogens. Prior to the larger stress experiment, a month-long pilot scale experiment was conducted on > 400 ZMs (collected from Lake Minnetonka) to establish ideal baseline conditions (e.g. pH, light, aeration, water replacement rate, feeding frequency) for ZM survival in a laboratory aquarium environment. For the final experiment, roughly 2,500 ZMs were collected from Gull Lake and transported to MAISRC’s BSL-2 containment lab. All ZMs were first acclimatized to baseline conditions, and stressors were added and gradually increased until reaching a setpoint. The following stressors were applied: fluctuating high temperature + low salinity [Tank A], high temperature + high salinity [Tank B], low temperature + high salinity [Tank C], high temperature + low salinity [Tank D], medium temperature + low salinity [Tank E], and low temperature + low salinity [Tank F: Control]. Each tank was monitored daily for ZM mortality and water quality (e.g., ammonia concentrations, dissolved oxygen, pH) throughout the course of this experiment (~ 2.5 months). Live ZMs were collected and processed at pre-determined time-points, as well as dead ZMs - where applicable. All processed ZM samples [n=384] stored at -20℃ for DNA extraction and preserved in glycerol at -80℃ for future microbial isolation experiments. Pooled ZM tissue samples, collected from 30 different time-points during this experiment, were shared with an ongoing MAISRC sub-Project “Developing eradication tools for invasive species. Phase II: Virus discovery and evaluation for use as potential biocontrol agents” for shotgun sequencing. Results from our tank experiments indicated that acute heat was the primary driver of ZM mortality, though salinity increased the likelihood of mortality as well [Fig. 1]. In particular, mortalities began increasing drastically when water temperatures reached 30℃ in Tanks A, B, and D. In contrast, no mortality was observed in the control Tank F (maintained at 17±1°C) during both acclimation and experimental phases. This established a rigorous model to use for future infection studies. Eurasian watermilfoil: During the first phase of this project, epiphytic bacterial communities associated with Eurasian watermilfoil (EWM) was compared with those in surrounding water and sediment, collected from 10 lakes in Minnesota, once a month for six consecutive months. Building on our results obtained from Phase 1, we sought to evaluate whether EWM (leaves and roots) harbored bacterial communities that are distinct from those associated with native aquatic plants. This information would help us shortlist specific microbes for isolation and evaluation of pathogenicity and host-specificity (Activity 3). Samples (EWM and native plants, water, and sediment) were collected from nine lakes around the Twin Cities Metro Area (Josephine, Vadnais, White Bear, Phalen, Cedar, Minnetonka, Bush, Lower Prior, and Nokomis) during summer and fall seasons [Table 1]. These sites were similar to those selected in phase 1 with the exception of Lake Holland which was not accessible due to construction. In addition to EWM, approximately 1-3 different native plant species were collected from each site, which included Pondweed (broad-leaf, large-leaf), Wild Celery, Canada waterweed, Lillypads, and Coontail. Physical site data (temperature, dissolved O2, and pH) were recorded for each sampling event [Table 1]. Diseased- and weevil-infested EWM tissues could not be identified during these field sampling trips. A total of 315 samples (leaves:, roots, sediment, water) were processed in the lab within 24 hours of collection. All processed samples were split and stored at -20℃ for DNA extraction and preserved in glycerol (EWM only) at -80℃ for future microbial isolation experiments. ACTIVITY 2: Identification and cultivation of potential candidates for microbiological control Description: Metagenomic characterization of ZM/QM and EWM-associated microbiota (i.e., comparing results from Phase I [non-diseased] and Phase II [diseased]) will help identify indigenous microorganisms that may be specifically associated with these AIS, in a symbiotic, commensal, or pathogenic manner. Candidate microbes will be selected based on the following criteria: (1) close and exclusive association with the target AIS, (2) predominance in relation to the diseased species, and (3) presence of desired traits such as lytic enzyme production, and (4) phylogenetic relatedness to previously identified pathogens. Several potential pathogenic microbes were previously identified in Phase I.

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Microbes that display some of the above characteristics will be isolated using multiple cultivation strategies, and extensive laboratory screening will be conducted for the desired traits and potential interactions. We propose the following experiments to accomplish this activity: (a) High-throughput DNA sequencing (HTS; amplicon- and shotgun-based) will be done based on samples collected in Activity 1 to decipher bacterial and fungal communities specifically associated with diseased AIS and native species. HTS will be performed using Illumina HiSeq and MiSeq platforms at the University of Minnesota Genomics Center. Spatio-temporal HTS data from Phase I (‘healthy’ AIS) and Phase II (‘diseased’ AIS and native species) will be compared to identify potential candidates for targeted cultivation. Resources of the Minnesota Supercomputing Institute will be utilized for bioinformatics analysis and data storage. (b) Targeted cultivation will be performed on fresh and frozen (glycerol stocks) AIS biomass samples collected as part of Phase I and Phase II (Activity 1). Microbes will be isolated on a variety of permissive, semi-selective, and selective microbiological growth media. Candidates for enrichment culturing will also be selected based on HTS results and as per criteria described above. Biomass samples from lab-based EWM decay experiments (Phase I) will be used to enrich for microorganisms involved in cellulase and pectinase production. In addition, other potential pathogens will also be cultivated and tested. Novel, potentially useful, microbes, if identified during these experiments, will undergo extensive phenotypic and biochemical characterization.

Summary Budget Information for Activity 2: ENRTF Budget: $146,000 Amount Spent: $145,078 Balance: $922

Activity Completion Date: Outcome Completion Date 1. Submit 1,200 DNA samples for high-throughput sequencing December 2018 2. Complete bioinformatics and statistical analyses for 1,200 samples December 2018 3. Complete targeted cultivation of at least 10 potential AIS-specific pathogens June 2019

Activity Status as of January 31, 2018: DNA that was extracted from EWM, non-targeted native plants, sediment and water samples were submitted for high-throughput sequencing, ahead of the proposed timeline. A total of 315 DNA samples were successfully sequenced at the University of Minnesota Genomics Center. The sequence data will be analyzed within the next few months. Activity Status as of July 31, 2018: High-throughput DNA sequencing, targeting the 16S rRNA gene, was completed for all samples (n=315) collected during the 2017 field season. The DNA samples included those derived from invasive- (Eurasian watermilfoil) and native macrophytes (Wild celery, Canada waterweed, Curly-leaf pondweed, Broad-leaf pondweed, and Coontail), water, and sediment. Illumina output files from all sequencing runs were combined (9 million reads) and analyzed using software packages such as QIIME. In addition, metadata such as water temperature, dissolved oxygen, and pH were included in the analysis. Preliminary results indicated that the microbial community structure was significantly different for each matrix (i.e., macrophytes, water and sediment). We found that a large proportion of epibacterial communities were shared between these macrophyte species. Microbial communities associated with root (rhizoid) compartments were more similar to those found in sediment, compared to those within the leaf (phylloid). Efforts are underway to identify taxa that are enriched or specific to Eurasian watermilfoil compared to other native plant species. In addition, our initial phase I studies suggested that several microbial taxa (including Acinetobacter, Aeromonas, Pseudomonas, Citrobacter, Enterobacter, Pantoea, Herbaspirillum, Klebsiella, Serratia, Plesiomonas, Bacillus, Exiguobacterium, Erwinia, and Comamonas) inhabited EWM. Based on this, we have commenced targeted cultivation of select microbes, such as Aeromonas, to test if it may be useful in biocontrol efforts. Activity Status as of January 31, 2019:

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We are currently extracting DNA from ~450 biomass samples derived from the zebra mussel stress experiment. These samples will be shortly submitted for high-throughput DNA sequencing to the University of Minnesota Genomics Center. Once we receive the data, our effort will be directed towards identifying zebra mussel-associated microbes that are enriched during stressed environmental conditions such as temperature and salinity. Our data will also be complemented with shotgun sequencing data on 30 pooled samples, generated by Dr. Nick Phelps’s research group. Final Report Summary: Zebra mussels: DNA was extracted from samples derived from the stress experiment, which included whole-tissue homogenates from 384 ZMs, 27 water samples and 3 algal feed samples. All DNA samples (n=414) were submitted to the University of Minnesota Genomics Center for high-throughput amplicon sequencing of the bacterial 16S rRNA gene. Fourteen samples were resequenced to meet the minimum threshold for target number of reads per sample. Overall, a total of 32 million reads were generated with an average sequencing depth of 76,585 reads per sample. Illumina output files from three MiSeq runs were combined and analyzed using the software package QIIME. Beta diversity analysis using PCoA plots indicated that dead ZMs (from Tanks A-E) clustered separately from live ZM from the control tank [Fig. 2]. Alpha diversity analyses showed significantly lower diversity and evenness in dead ZMs compared to live ZMs [Fig. 3]. Different statistical methods such as canonical correspondence analysis [Fig. 4a], redundancy analysis [Fig. 4b], and linear discriminant analysis [Fig. 5a] indicated a strong association between the presence of a few microbial groups and ZM mortality [Fig. 5a, 5b]. These included: Aeromonas, Chryseobacterium, Flavobacterium, Acidaminobacter, Clostridiaceae 1 sp., Rhodobacteraceae sp., Acinetobacter, Shewanella, and Clostridium sensu stricto 13 [Fig. 5a]. In particular, the genus Aeromonas was highly enriched in dead ZMs. Quantitative PCR analysis targeting the cytotoxic enterotoxin (aerolysin) gene confirmed the significant increase in pathogenic Aeromonas species in dead ZMs [Fig. 6]. We were also able to successful isolate 93 Aeromonas strains from glycerol stocks prepared from tissues, which was confirmed via Sanger sequencing [Fig. 7]. Efforts are currently underway to isolate other bacteria (non-Aeromonas) that were significantly associated with ZM mortality [Fig. 5a]. Results from this study suggest that stressed conditions such as high temperature and salinity can induce ZM die-offs, which could be linked to the increase in opportunistic pathogens. We are hesitant to use an Aeromonas strain as a biocontrol agent until the bacterium can be shown to be safe to non-target humans and fish. Eurasian watermilfoil: DNA was extracted from samples included those derived from leafs and roots of invasive and native macrophytes (n=207), water (n=54) and sediment (n=54). A total of 315 DNA samples were submitted to the University of Minnesota Genomics Center for high-throughput amplicon sequencing of the bacterial 16S rRNA gene and the fungal ITS2 region. A total of 27 million reads (2 MiSeq runs) and 19 million reads (1 MiSeq run) were obtained for bacterial 16S rRNA gene- and the fungal ITS2 region-based sequencing, respectively. Illumina output files were combined for each target region and analyzed using the software package QIIME. In contrast to the bacterial 16S rRNA gene, the fungal ITS2 region was not able to provide an in-depth taxonomic resolution of fungal communities (data not shown) with majority of the sequences unclassified at phylum-level. Beta diversity analysis using PCoA plots indicated clustering of samples by compartment (i.e. phylloid, rhizoid, water and sediment) [Fig. 8]. Microbial communities associated with roots (rhizoid) compartments were more similar to those found in sediment, compared to those within leaves (phylloid). However, a large proportion of bacterial communities were shared between different macrophyte species, including EWM [Fig. 10]. Nonetheless, linear discriminant analysis identified a few taxa that were strongly associated with each compartment [roots/leaves] of EWM compared to native plants [Fig. 11]. Though efforts are currently underway, we are yet to isolate bacterial species that are exclusively associated with EWM. Similar to what was done with mussels, stressed/ diseased models might be needed to identify host-specific (micro)biocontrol agents.

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ACTIVITY 3: Evaluate the specificity and effectiveness of isolated candidates for microbiological control Description: Initially 10 candidate microbes (bacteria and fungi) will be used for challenge experiments against both native species and target AIS. This number will be increased if necessary. To test for host-specificity, mortality tests will be performed on each target AIS and two native species, which will be selected based on their phylogenetic relatedness to the target AIS. The pathogenic potential (i.e., effectiveness) of each isolated microbe will be assessed in lab-scale microcosms in terms of growth stage and environmental conditions. Freshly collected samples will be maintained in aerated glass tanks at the MAISRC facility, containing lake water, at either 10 or 20˚C under a 12h light/dark photoperiod cycle. The microbial isolates will be inoculated into tanks to test for pathogenicity. Each treatment “infection assay” will be performed in triplicate, with negative controls. Microcosm variables such as treatment duration (up to 2 weeks), temperature (10˚C vs 20˚C), inoculum concentrations (from to 105-108 cells/ml), with or without herbicide/molluscicides will be examined. Microbes that show specific pathogenic behavior to AIS will need to satisfy Koch’s postulates, i.e., be re-isolated and shown to again cause disease. To be considered for future studies, the shortlisted microbiological control agent must display pathogenic behavior only against the target AIS and not against native species.

Summary Budget Information for Activity 3: ENRTF Budget: $72,000 Amount Spent: $66,670 Balance: $5,330

Activity Completion Date: Outcome Completion Date 1. Test the specificity of at least 10 isolated microbes on select macrophytes and mussels in microcosms

June 2019

2. Test the effectiveness of at least 10 isolated microbes on ZM/QM and EWM in microcosms

June 2019

Activity Status as of January 31, 2018: Progress on this activity will begin after the 2018 field sampling season. Activity Status as of July 31, 2018: Progress on this activity will begin after the 2018 field sampling season. Activity Status as of January 31, 2019: Progress on this activity will begin in Feburary 2019. Final Report Summary: Zebra mussels: Due to the change in experimental approach, it was necessary to perform some of the experiments proposed in Activity 3 during Activity 1 period, such as using microcosm-based stress experiments to identify opportunistic pathogens. As described in Activity 2, we were able to identify several taxa that were strongly associated with ZM mortality: Aeromonas, Chryseobacterium, Flavobacterium, Acidaminobacter, Clostridiaceae 1 sp., Rhodobacteraceae sp., Acinetobacter, Shewanella, and Clostridium sensu stricto 13 [Fig. 5a]. Amongst these ten microbial groups, we have only been able to isolate 93 strains within the genus Aeromonas [Fig. 7]. However, several species within Aeromonas are well-known fish and human pathogens and as a result, did not meet our criteria for further testing. Efforts are currently underway to isolate other bacterial species listed in Fig. 5a that were strongly associated with ZM mortality but also meet our safety and specificity criteria for further evaluation.

10

Eurasian watermilfoil: Activity 3 was intiated by growing EWM (in 15 microcosms) under controlled conditions in plant growth chambers for 7 months. However, we could not pursue this activity further as we were not successful in isolating bacterial species (other than Aeromonas) that were strongly associated with EWM [Fig. 11] and not other macrophytes. V. DISSEMINATION: Description: This project has high potential to engage the public regarding use of state-of-the art research, application of this work to real problems in our state, and discussions about the implications of policy decisions on our natural resources. Project data and research findings will be disseminated via public outreach programs done in collaboration with the Minnesota Aquatic Invasive Species Research Center, presentations to State agencies, scientific conferences presentations, and peer-reviewed publications. We believe that one of the best approaches to protect native species in Minnesota is to engage the public through outreach programs done in collaboration with the MAISRC. We will work with MAISRC and the BioTechnology Institute (BTI) outreach personnel to publicize our findings, which includes the development of a website resource, magazine articles, press releases and tweets. Results from this project will also be disseminated via more traditional routes, such as reports made to the LCCMR, presentations at local, national and international conferences, and in peer-reviewed scientific publications. Status as of January 31, 2018: Dr. Michael Sadowsky gave two talks titled ‘No guts, no glory! The inside story about using biocontrol for invasive plants and mussels at the AIS Research and Management Showcase held at the University of Minnesota, St. Paul on September 13, 2017. Dr. Prince Mathai gave a poster presentation titled ‘Microbial Communities Associated with Aquatic Invasive Species Using High-Throughput Sequencing Approaches’ at the 20th International Conference on Aquatic Invasive Species at Fort Lauderdale on October 23, 2017. Michael Sadowsky will be making a presentation to USGS and DNR later this year. We are currently working on several manuscripts with data from phase I of this project for publication in peer-reviewed journals. Status as of July 31, 2018: Our manuscript titled ‘Spatial and Temporal Characterization of Epiphytic Microbial Communities Associated with Eurasian Watermilfoil: A Highly Invasive Macrophyte in North America’ is in the last round of revisions for publication in the journal ‘FEMS Microbiology Ecology’. This version will be resubmitted on 7/20/18. Dr. Prince Mathai will give a talk titled ‘Unraveling the Microorganisms Associated with Eurasian Watermilfoil: Potential Candidates for Biological Control?’ at the 2018 Upper Midwest Invasive Species Conference, which will be held from October 15-18, 2018 at Rochester (MN). Michael Sadowsky also gave a talk at the IAGLR conference (June 21, 2018, Toronto, Canada) entitled: ‘Use of DNA Sequence Technology to Determine Sources and Sinks of Microbes in Waterways’. Status as of January 31, 2019: Dr. Prince Mathai gave a talk titled ‘Unraveling the Microorganisms Associated with Eurasian Watermilfoil: Potential Candidates for Biological Control?’ at the 2018 Upper Midwest Invasive Species Conference, which was held from October 15-18, 2018 at Rochester, MN. Two papers were published during this period:

1. Mathai PP, Dunn HM, Magnone P, Brown CM, Chun CL, Sadowsky MJ (2018). Spatial and temporal characterization of epiphytic microbial communities associated with Eurasian watermilfoil: a highly invasive macrophyte in North America. FEMS Microbiol Ecol. 94:1-9.

11

2. Mathai PP, Dunn HM, Magnone P, Brown CM, Zhang Q, Chun CL, Ishii S, Sadowsky MJ (2019). Association between submerged aquatic vegetation and elevated levels of Escherichia coli and potential bacterial pathogens in freshwater lakes. Sci Total Environ. 657:319-324.

In addition, two manuscripts are currently under preparation. Final Report Summary: Presentations: Dr. Michael Sadowsky gave two oral presentations at the ‘AIS Research Management Showcase’ which is held at the University of Minnesota (St. Paul, MN) each year to update the public on research findings and progress:

1. ‘No guts, no glory! The inside story about using biocontrol for invasive plants and mussels’ on September 13, 2017.

2. “Mussel poo holds the answer for biocontrol” on September 12, 2018.

Dr. Michael Sadowsky also presented project results at two international/ national conferences: 1. ‘Use of DNA sequence technology to determine sources and sinks of microbes in waterways’ at the 61st

International Association for Great Lakes Research conference at Toronto, Canada on June 21, 2018. 2. ‘Relationship Between Microbiota and Aquatic Vegetation Affecting Health’ at the UNC Water

Microbiology Conference 2019 at Chapel Hill (NC) on May 14, 2019. Dr. Prince Mathai presented project results at three international/ national conferences:

1. ‘Microbial communities associated with aquatic invasive Species Using High-Throughput Sequencing Approaches’ at the 20th International Conference on Aquatic Invasive Species at Fort Lauderdale (FL) on October 23, 2017.

2. ‘Unraveling the Microorganisms Associated with Eurasian Watermilfoil: Potential Candidates for Biological Control?’ at the 5th Upper Midwest Invasive Species Conference at Rochester (MN) on October 17, 2018.

3. ‘Spatial and Temporal heterogeneity of microbial communities associated with invasive dreissenid mussels in freshwater lakes’ at the 119th General Meeting of the American Society for Microbiology at San Francisco (CA) on June 22, 2019.

Publications: Two papers were published during this period:

1. Mathai PP, Dunn HM, Magnone P, Brown CM, Chun CL, Sadowsky MJ (2018). Spatial and temporal characterization of epiphytic microbial communities associated with Eurasian watermilfoil: a highly invasive macrophyte in North America. FEMS Microbiol Ecol. 94:1-9.

2. Mathai PP, Dunn HM, Magnone P, Brown CM, Zhang Q, Chun CL, Ishii S, Sadowsky MJ (2019). Association between submerged aquatic vegetation and elevated levels of Escherichia coli and potential bacterial pathogens in freshwater lakes. Sci Total Environ. 657:319-324.

A manuscript titled “Water and sediment act as reservoirs for microbial taxa associated with tissues of invasive dreissenid mussels” is currently under peer-review in the journal “Science of the Total Environment” – submitted in July 2019. In addition, two manuscripts are currently under preparation for rapid submsission to peer-reveiewed journals. Data deposition: All sequencing data, including meta-data, generated in this project has been, and will be, submitted to Genbank (https://www.ncbi.nlm.nih.gov/genbank/) and all publications will list accession numbers to link to short read

12

archive of all samples. Thus far, all sequence data mentioned in current publications is directly linked to a publicly available web site at www.ncbi.nlm.nih.gov/genbank for sequence download. VI. SUB-PROJECT BUDGET SUMMARY: A. Preliminary ENRTF Budget Overview: *See Attached Budget Explanation of Use of Classified Staff: N/A Explanation of Capital Expenditures Greater Than $5,000: N/A Number of Full-time Equivalents (FTE) Directly Funded with this ENRTF Appropriation: 2 Number of Full-time Equivalents (FTE) Estimated to Be Funded through Contracts with this ENRTF Appropriation: N/A B. Other Funds:

Source of Funds $ Amount Proposed

$ Amount Spent Use of Other Funds

Non-state $ $ State $ $

TOTAL OTHER FUNDS: $ $ VII. SUB-PROJECT STRATEGY: A. Sub-Project Team/Partners: The project will be carried out under the direction of Drs. Sadowsky (PI) and Mathai (Co-PI). This work will be done in consultation with the Minnesota Department of Natural Resources and MAISRC faculty. In addition, we will also collaborate with the Center for Aquatic Mollusk Programs (Lake City, MN), Minnesota Zoo (Apple Valley, MN), Dr. Jessica Kozarek (St. Anthony Falls Lab, UMN), and the Minnehaha Creek Watershed District. B. Sub-Project Impact and Long-term Strategy: Results from this work may allow us to develop efficient, specific, and cost-effective microbiological control agents for AIS management. This will allow us to protect Minnesota’s valuable water resources. Project outcome will provide insight for the protection of native species, maintenance of aquatic biodiversity, and management of natural resources in Minnesota. Federal and state agencies such as the US Environmental Protection Agency, US Geological Survey, US Army Corps of Engineers, US Fish and Wildlife, watershed districts, lake associations, and MN-DNR will be interested in the results from this study. Future long-term funding for these studies will be obtained from the MN DNR, USGS, the National Science Foundation, and other funding agencies, and foundations. C. Spending History:

Funding Source M.L. 2008 or

FY09

M.L. 2009 or

FY10

M.L. 2010 or

FY11

M.L. 2011 or

FY12-13

M.L. 2013 or

FY14 ML 2013 ENRTF (MAISRC) Subproject #2, Phase I

$303,217

13

VIII. ACQUISITION/RESTORATION LIST: N/A IX. VISUAL ELEMENT or MAP(S): N/A X. ACQUISITION/RESTORATION REQUIREMENTS WORKSHEET: N/A XI. RESEARCH PROPOSAL: Attached XII. REPORTING REQUIREMENTS: Periodic work plan status update reports will be submitted no later than January 31, 2018, July 31, 2018, and January 31, 2019. A final report and associated products will be submitted within two months of the anticipated sub-project completion of June 30, 2019.

14

APPENDIX Figure 1: Tank experiments showing the effect of water temperature and salinity on ZM survival

15

Figure 2: PCoA plot displaying variability within microbial communities associated with dead ZMs (from experimental Tanks A-E) and live ZMs (from control Tank F)

Figure 3: Alpha diversity indices comparing microbial communities associated with dead ZMs (from experimental Tanks A-E) and live ZMs (from control Tank F)

-0.4

-0.3

-0.2

-0.1

0

0.1

0.2

0.3

0.4

0.5

-0.6 -0.5 -0.4 -0.3 -0.2 -0.1 0 0.1 0.2 0.3 0.4 0.5

F2 (8

.76%

)

F1 (23.97%)

Dead ZMs

Live ZMs

16

Figure 4a: Canonical correspondence analysis

Figure 4b: Redundancy analysis

17

Figure 5a: LEfSe analysis showing taxa that were significantly in dead ZMs (from experimental Tanks A-E) compared to live ZMs (from control Tank F)

• Only taxa with ≥3.5 LDA cut-off score are shown

Figure 5b: Relative abundance of taxa that were significantly in dead ZMs (from experimental Tanks A-E) compared to live ZMs (from control Tank F)

**** = <0.0001, *** = 0.0001, ** = 0.001

18

Figure 6: Quantification of total bacteria and a Aeromonas-specific virulence gene in dead ZMs (from experimental Tanks A-E) and live ZMs (from control Tank F)

• 16S rRNA gene was used to quantify total bacteria • Cytotoxic enterotoxin gene was used to quantify pathogenic Aeromonas species

Figure 7: Taxonomic classification of strains isolated from tissues of dead ZMs based on full-length 16S rRNA gene profiling

19

Figure 8: PCoA plot displaying spatial and temporal variability within microbial communities associated with leaves- and roots of invasive (Eurasian watermilfoil) and native (Wild celery) plants, sediment, and water

EWM = Eurasian watermilfoil; WC = Wild celery

Figure 9: Alpha diversity indices comparing comparing microbial communities associated with leaves- and roots of invasive (Eurasian watermilfoil) and native (Wild celery) plants, sediment, and water

EWM = Eurasian watermilfoil; WC = Wild celery

-0.4

-0.2

0

0.2

0.4

0.6

-0.3 -0.1 0.1 0.3 0.5 0.7

F2 (1

4.3%

)

F1 (20.1%)

Water

Sediment

EWM (Leaves)

WC (Leaves)

WC (Roots)

EWM (Roots)

20

Figure 10: Taxonomic classification of microbial communities associated with leaves- and roots of invasive (Eurasian watermilfoil) and native (Wild celery) plants, sediment, and water A) Phylum-level

B) Order-level

EWM = Eurasian watermilfoil; WC = Wild celery

21

Figure 11: List of taxa enriched in leaves and roots of Eurasian watermilfoil, compared to Wild celery

• Only taxa with ≥3.0 LDA cut-off score are shown

22

Table 1: Sampling locations and water parameters

Lake Name GPS coordinates Temperature (℃) Dissolved oxygen pH

Summer Fall Summer Fall Summer Fall

1 Josephine 45.033533, -93.149375 26.1 18.5 8.6 10.2 8.97 8.60

2 Vadnais 45.051643, -93.094741 26.8 19.3 9.0 9.5 9.26 8.64

3 White Bear 45.076200, -93.016177 27.1 19.5 6.8 9.1 9.25 8.60

4 Phalen 44.994196, -93.059331 25.0 20.3 7.7 9.8 8.79 8.73

6 Cedar 44.956391, -93.325737 25.4 18.7 8.1 7.1 8.96 8.55

7 Minnetonka 44.935675, -93.526984 25.3 18.5 6.7 7.8 8.65 8.49

8 Bush 44.831242, -93.381547 26.5 18.9 10.0 8.7 9.23 8.07

9 Lower Prior 44.739517, -93.407006 28.0 20.1 8.9 9.1 9.07 8.51

1

23456789

1011

12

1314

18

2223

25

26

27

28

A B D E F H I J L M N P Q R T UEnvironment and Natural Resources Trust FundM.L. 2013 Sub-Project Budget of M.L. 2013-06a: Aquatic Invasive Species Research Center

Project Title: Aquatic Invasive Species Research Center Sub-Project 2: Metagenomic Approaches to Develop Biological Strategies to Control Aquatic Invasive Species: Phase II: Development of Potential Microbiological Control Agents for Aquatic Invasive SpeciesLegal Citation: M.L. 2013, Chp. 52, Sec. 2, Subd. 06aProject Manager: Michael J. SadowskyOrganization: University of Minnesota - Minnesota Aquatic Invasive Species Research CenterSub-Project Budget: $303,217Sub-Project Phase 2 Length and Completion Date: 1 year, 11 months; June 30, 2019Project Length and Completion Date: 6 Years, June 30, 2019Date of Report: August 8, 2019

ENVIRONMENT AND NATURAL RESOURCES TRUST FUND BUDGETBUDGET ITEM

Activity 1 Budget

Amount Spent

Activity 1Balance

Activity 2 Budget

Amount Spent

Activity 2Balance

Activity 3 Budget

Amount Spent

Activity 3Balance

Activity 4 Budget

Amount Spent

Activity 4Balance

Personnel (Wages and Benefits) - Total $44,500 $44,500 $0 $94,000 $94,000 $0 $52,000 $52,000 $0 $7,570 $7,570 $0 $198,070 $198,070 $0Prince Mathai - Research Associate: $96,979 salary, $32,681 benefits (33.5% fringe rate); 1.0 FTE for 23 monthsJonathan Bertram - Junior Researcher (Civil Service): $63,501 salary, $17,399 (27.2% fringe rate); 1.0 FTE for 23 monthsProfessional/Technical Services and Contracts - Total $2,000 $417 $1,583 $33,500 $33,078 $422 $13,500 $8,170 $5,330 $5,500 $668 $4,832 $54,500 $42,333 $12,168Services – Office and General operation: (shipping, software etc.) $1,000 $417 $583 $0.00 $0.00 $0 $0 $0 $0 $0 $0 $0 $1,000 $417 $583

Services – Lab and/ or Medical: University of Minnesota Genomics Center: High-throughput sequencing: 8 MiSeq runs ($2,930*8=$23,440) + 2 HiSeq runs ($5,900*2=$11,640). Total: ~1,200 samples

$0 $0 $0 $33,000 $33,000 $0 $5,000 $2,385 $2,615 $0 $0 $0 $38,000 $35,385 $2,615

Services – Lab and/ or Medical: Minnesota Supercomputing Institute. Data storage at MSI

$0 $0 $0 $500 $78 $422 $0 $0 $0 $0 $0 $0 $500 $78 $422

Services – Lab and/ or Medical: Research Analytical Lab (UMN) – Nutrient analysis: $55 per sample. Total: 70 samples

$1,000 $0 $1,000 $0 $0 $0 $0 $0 $0 $0 $0 $0 $1,000 $0 $1,000

Activity 4: Dissemination

TOTALBALANCE

Activity 1: Sampling and processing Activity 2: Identification and cultivation of potential candidates for microbiological

control

Activity 3: Evaluate the specificity and effectiveness of isolated candidates for

microbiological control

TOTAL BUDGET

TOTALSPENT

12

13

A B D E F H I J L M N P Q R T U

ENVIRONMENT AND NATURAL RESOURCES TRUST FUND BUDGETBUDGET ITEM

Activity 1 Budget

Amount Spent

Activity 1Balance

Activity 2 Budget

Amount Spent

Activity 2Balance

Activity 3 Budget

Amount Spent

Activity 3Balance

Activity 4 Budget

Amount Spent

Activity 4Balance

Activity 4: Dissemination

TOTALBALANCE

Activity 1: Sampling and processing Activity 2: Identification and cultivation of potential candidates for microbiological

control

Activity 3: Evaluate the specificity and effectiveness of isolated candidates for

microbiological control

TOTAL BUDGET

TOTALSPENT

29313234

353642

44

4551

Professional Services and Contracts: Publication costs for dissemination. $500-$2000 per manuscript. Total: 3-4 manuscripts

$0 $0 $0 $0 $0 $0 $0 $0 $0 $5,500 $668 $4,832 $5,500 $668 $4,832

Rental: MAISRC AIS holding facility $0 $0 $0 $0 $0 $0 $8,500 $5,785 $2,715 $0 $0 $0 $8,500 $5,785 $2,715Equipment/Tools/Supplies - Total $12,500 $12,500 $0 $18,500 $18,000 $500 $6,500 $6,500 $0 $0 $0 $0 $37,500 $37,000 $500Supplies – Office & Gen Operations $0 $0 $0 $500 $0 $500 $0 $0 $0 $0 $0 $0 $500 $0 $500Supplies – Lab and/ or Field: Field sampling ($2,500), sample processing ($6,000), DNA extraction/enrichment kits ($5,000), PCR/qPCR reagents ($4,000), culture media/ingredients ($7,000), chemicals ($2,500), disposable plasticware ($3,000).

$12,500 $12,500 $0 $18,000 $18,000 $0 $5,000 $5,000 $0 $0 $0 $0 $35,500 $35,500 $0

Equipment - Non-Capital Lab and/or Field: pipetters ($1,500)] $0 $0 $0 $0 $0 $0 $1,500 $1,500 $0 $0 $0 $0 $1,500 $1,500 $0Travel - Total $8,000 $4,271 $3,729 $0 $0 $0 $0 $0 $0 $5,147 $4,937 $210 $13,147 $9,208 $3,939Travel – MN: In-state sampling – Transportation: UMN car rental ($45/day) + mileage ($0.17/mile): 24 field trips per year (~2000 miles) X 2 years = $2500. Lodging and meals during field trips: 2 people X 5

$6,000 $2,271 $3,729 $0 $0 $0 $0 $0 $0 $500 $290 $210 $6,500 $2,561 $3,939

Travel – Domestic: Near-state (WI) sampling: Transportation: UMN car rental ($45/day) + mileage ($0.17/mile): 4 field trips per year (~1000 miles) X 2 years = $750. Lodging and meals during field trips:

$2,000 $2,000 $0 $0 $0 $0 $0 $0 $0 $4,647 $4,647 $0 $6,647 $6,647 $0

COLUMN TOTAL $67,000 $61,688 $5,312 $146,000 $145,078 $922 $72,000 $66,670 $5,330 $18,217 $13,175 $5,042 $303,217 $286,610 $16,607