Quarterly Progress Report: Third Quarter FY 08

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EPA Demonstration Project on Hydrilla and Hygrophila in the Upper Kissimmee Chain of Lakes; EPA Grant #X796433105-1 (Hydrilla / Hygrophila Demonstration Projects - Osceola County, FL)

Project description and Goals:
This project is a combination of studies that will result in improved water quality and natural habitat in Osceola County lakes. Hydrilla and Hygrophila are exotic invasive weeds that are dominating the lakes aquatic ecosystem, shading out natural vegetation, reducing oxygen level, increasing the amount of sediments, and impacting flood control in lakes and canals. The project objectives are to evaluate the effectiveness of Experimental Use Permit herbicides, biological controls and application methods of herbicides in the treatment and control of Hydrilla and Hygrophila using small-scale fieldwork, large-scale field demonstrations and public outreach on the progress and findings.

Participants: Hilda Hatzell, EPA Project Officer, Region 4
Eleanor Foerste, Project Manager, Osceola County

Reporting period: Third Quarter, EPA Fiscal Year 2008 April 1-June 30, 2008

Project activities during reporting period:

What work was accomplished for this reporting quarter?
- The semi-annual QA/QC Report for Element 3 Task 1 was completed by Lane Tredway.

Element 1

Treated:

Stewart Rd pond with FL EUP number FL08-EUP-01 at a rate of 50ppb.
Granada Blvd pond was treated with FL EUP number FL08-EUP-02 at a rate of 400ppb
Oak Tree Pond was treated with FL EUP number FL08-EUP-02 at a rate of 400ppb.

Continued monitoring transects in Lake Jackson following Galleon treatment in November and December 2007 to record effects on native plant communities and Hydrilla control.

Element 2

Monitored:

Three April 2008, large-scale Aquathol applications on Lake Tohopekaliga.
- October 2007 shallow water treatments conducted in the north end of Lake Tohopekaliga. To date, we are still observing good Hydrilla control in these areas at approximately 8 months post-treatment.
- December 2007 Aquathol treatment on the north end of Lake Hatchineha. June survey indicated that hydrilla recovery was very limited.
- March 2008 Rhodamine dye dispersion from the S-61 structure into Lake Cypress.

The longevity of the shallow water treatments in the north end of Lake Toho, as well as the longevity of the treatment in the north end of Lake Hatchineha would suggest that larger scale treatments conducted in the late fall can provide good efficacy well into the next growing season.

Element 3 Task 1

Resolved issue of loss of virulence in Mt fungus through re-isolation
Conducted greenhouse aquarium study of liquid and dry formulated Mt material
Initiated field testing in Osceola County Corrections pond May 2008 with liquid and dry formulated Mt material.
Results of the field trial support laboratory results indicating that active liquid cultures have more virulence than dry product on a per unit production basis.

Element 3 Task 2

Collected Hygophila samples from 5 Florida counties and India for DNA analysis.
Findings suggest almost no genetic variation in the Florida Hygrophila.

Element 3 Task 3

Reared 17 species of chironomids in the lab in Burundi from Hydrilla.
In Uganda, 35 species if chironomids were reared from Hydrilla.

Element 4

Planning and preparation for July 1, 2008 Hydrilla Field Day.
Planning and preparation for teacher training with CAIP staff for July 28-31, 2008.
Advertised and recruited to fill Extension Agent Aquatics position vacancy.
Continued to update website http://plants.ifas.ufl.edu/osceola
Development of template for public information displays.

Is the project work on schedule?

This quarter? Yes
For the project?

Element 3 Task 2 Though efforts are being made, it is likely that establishing sub-contracts with Indian collaborators (PDBC) could result in some delays in accomplishing project goals and objectives due to complex sub-contracting regulations at the University of Florida.

If the project is not on schedule, what is proposed for a revised schedule?

Element 3 Task 1 Virulence issues have resulted in delays in decision to scale-up and test at larger field scales. However, a significant new production process is under investigation, and a commercial scale-up of production is now targeted for fall 2008.

Does the new schedule require a time extension? A time extension is not necessary at this time.
Element 3 Task 1 Per work plan, there is still a window for operating under revised schedule without any formal time extension. However, as we finish FY per recent discussion with Osceola grant coordinator Eleanor Foerste, we will discuss status of project to confirm appropriate timing for remainder of grant.
Is there a change in the Grantee’s Authorized Representative or any of the key contacts?
- Dr. Tina Bond, Extension Agent Aquatics and Element 4 Coordinator resigned and accepted another position with Osceola County. The position has been re-advertised and candidates screened. Candidates will be interviewed in early August, 2008.

Project spending for reporting period:
$30,004.95 was expended this quarter; with total project costs at 06/30/2008 totaling $1,094,916.47. Project balance is $2,881,000 – 1,094,916.47 = $ 1,786,083.53.

Reporting period: Third Quarter, EPA Fiscal Year 2008 April 1-June 30, 2008

Person Reporting:      Sarah T. Berger, Senior Biologist
                                    Dr. William T. Haller, University of Florida

Element: Element 1: Development and Testing of New Aquatic Herbicides

Project activities during reporting period:

What work was accomplished for this reporting quarter?

The following activities were undertaken from April 1-June 30, 2008 in Osceola County, Florida:

1. The storm-water pond located at the corner of Stewart Rd and Pleasant Hill Rd was treated April 30, 2008 with FL EUP number FL08-EUP-01 at a rate of 50ppb. Plant heights were measured on a bi-weekly basis and this continued through the current quarter. The trend is shown below.

Figure 1. Plant Height vs. Time

2. The storm-water pond located at the corner of Granada Blvd and Pleasant Hill Rd was treated on June 9, 2008 with FL EUP number FL08-EUP-02 at a rate of 400ppb Plant heights were measured on a bi-weekly basis and this continued through the current quarter. The data is shown below.

Table 1. Granada Pond Data

3. The storm-water pond located across from Pleasant Hill Estates on the east side of Pleasant Hill Rd (referred to as “Oak Tree Pond”) was treated on June 9, 2008 with FL EUP number FL08-EUP-02 at a rate of 400ppb. Plant heights were measured on a bi-weekly basis and this continued through the current quarter. The data is shown below.

Table 2. Oak Tree Pond Data

4. Lake Jackson, located in Osceola County, was treated with Galleon in November and December 2007 by the South Florida Water Management District. A 6-9 ppb concentration was kept through 100 days. Plant transects were established in the lake to determine impact of the treatment on hydrilla efficacy and selectivity. The transect data is listed below as percentage of points that each plant was present.

Table 3: Lake Jackson transect data January 2008 vs. May 2008

Reporting period: Third Quarter, EPA Fiscal Year 2008 April 1-June 30, 2008

Person Reporting: Michael D. Netherland, US Army Engineer Research and Development Center, UF-IFAS Center for Aquatic and Invasive Plants, Gainesville, FL

Element: Element 2 Evaluation of Currently Registered Herbicides for Control of Hydrilla

Project activities during reporting period:

What work was accomplished for this reporting quarter?

Summary of Ongoing Work:
Within Element 2 the demonstration objectives include evaluation of registered herbicides for hydrilla treatments in shallow waters, evaluation of the timing of herbicide application on efficacy and longevity (winter vs. spring vs. summer vs. fall), the potential for novel application techniques, and the use of herbicide combinations to enhance herbicide efficacy.

In conjunction with the Florida Department of Environmental Protection (FDEP) and South Florida Water Management District (SFWMD) we monitored large-scale Aquathol applications conducted on Lake Tohopekaliga beginning on April 21, 2008. Treatments included a 1300 acre treatment block in the south end of the lake, a 2400 acre treatment block in the center of the lake, and a 420 acre treatment block in Goblit’s Cove (Figure 1). There were also 3 research sites established near the large treatment block in the south end of the lake to allow us to compare Aquathol at 2.0 and 3.0 mg/L vs. Reward at 0.37 mg/L + Aquathol at 0.5 mg/L (Figure 1). We established 30 sampling stations both within and outside of the treated areas and collected water samples for endothall residues and took water quality readings starting the day of treatment. By the May 6th sample date, there was no detection of endothall residues either within or outside of the plots. Hydrilla began to collapse and uproot within 1 to 3 weeks of treatment and the treatment areas as well as immediate adjacent sites were largely free of rooted hydrilla during our June 19th assessment. During this June survey, small mats of hundreds of floating root crowns (stems are leafless and the roots are missing) were observed in various areas of the lake. We continue to monitor the efficacy in the treatment blocks and adjacent areas.

We also continue to monitor the efficacy of the October 2007 shallow water treatments conducted in the north end of Lake Tohopekaliga. To date, we are still observing good hydrilla control in these areas at ~8 months post-treatment. While some hydrilla recovery is noted within the aquatic grasses, the treated area remains largely free of hydrilla. It remains difficult to ascertain efficacy differences between the plots that received Aquathol only, and the plots that received a combination of Aquathol and Reward. This late fall treatment has provided both selective hydrilla control and good longevity to date. We will continue to evaluate the recovery of hydrilla in this area.

We also continue to monitor the 450-acre Aquathol treatment that was applied via helicopter on December 11th, 2007 on the north end of Lake Hatchineha. This treatment was conducted in deeper water (2 m average depth) compared to fall treatment in Lake Tohopekaliga. The initial efficacy of this treatment was very good and an early June survey indicated that hydrilla recovery was very limited.

Following a March 31, 2008 Rhodamine dye injection at the S-61 structure, we monitored dye dispersion in Lake Cypress over an 8 day period. Dye was injected into the canal at 8 ppb for a 24-hour period and we monitored the downstream movement of residues into and throughout Lake Cypress. The dye concentrations were quantified (to 0.2 ppb) via the use of a field fluorometer. Results from this work suggest that the large surface-matted hydrilla bed in the southwest corner of Lake Cypress was a significant initial barrier to dye penetration. A drip application of the contact herbicide Aquathol would not be advisable when the plants are in a surface canopy condition. Further trials are planned when the plants are not in a surface canopy condition. Dye trials for the canal linking East Lake Tohopekaliga to Goblit’s cove are being planned to determine the best method for controlling hydrilla.

What Milestones have been met for your Element?

During this quarter we focused on characterizing the ability to utilize a drip application for flowing water. We evaluated residues and efficacy following the use of herbicide combinations and we monitored the efficacy of large-scale applications that utilized different use rates. All of these projects include monitoring of long-term efficacy, selectivity, and potential recovery of the hydrilla, so each project can last for several quarters.

What problems, if any, were encountered? There were no specific problems during this phase of the work.

If a problem was encountered, what action was taken to correct it? No significant problems were encountered.
Is the project work on schedule?
- This quarter? - Work is on schedule for this quarter
- For the project? – Work is on schedule for the project.
Are there any significant findings this quarter?

The longevity of the shallow water treatments in the north end of Lake Toho, as well as the longevity of the treatment in the north end of Lake Hatchineha would suggest that larger scale treatments conducted in the late fall can provide good efficacy well into the next growing season. There is significant discussion at this point in time regarding the implementation of several large-scale hydrilla control efforts for the late fall of 2008.

What is the future direction of the work?

We will continue to monitor large-scale operational treatments as these projects have yielded valuable insight. Due to concerns associated with plant management and the endangered snail kite, we are looking to further evaluate the selectivity of endothall on the submersed native plant Vallisneria. While there is strong anecdotal evidence that Aquathol treatments are not active against Vallisneria, we feel that further development of empirical data will support efforts to control hydrilla in areas where the two species co-exist.

Is there a change in the key contacts for this element? There is no change in key contacts for this element.

Figure 2. April 2008 Aquathol treatment plots on Lake Tohopekaliga, Fl.

Reporting period: Third Quarter, EPA Fiscal Year 2008 April 1 – June 30, 2008

Person Reporting: Mark A. Heilman

Element: Element 3 Task 1 Demonstration of Hydrilla Control in Osceola County, Florida using Mycoleptodiscus terrestris (Mt), a new contact bioherbicide, for Aquatic Plant management

Project activities during reporting period:

What work was accomplished for this reporting quarter?

1. Laboratory Evaluations of Mt Formulations and Methods

In the winter of 2008, project team detected an apparent decrease in virulence in Mt cultures produced for testing. Through various re-isolation efforts, this issue was resolved in the early spring and quickly evaluated through bioassays on hydrilla in early April (see April 9 study below). Results confirmed good rate response for three different strains.

Figure 3: Bioassy of 3 strains of Mt, April 9, 2008

With re-isolated materials, plans were made to initiate studies examining the field performance of a pure Mt liquid culture produced in the USDA pilot fermentors compared to dried Mt product from the same pilot source. To support this field trial per work plan protocols, a greenhouse aquarium study was started in early May by US Army ERDC (Dr. Shearer) to corroborate mid-May field testing in Osceola County of liquid and dry formulated Mt material. The results below show good virulence for dry and both liquid culture batches. Since it requires roughly 80 liters of fermentation to produce approximately 3.5 kg of dry Mt material, a 20 mg/L dry test rate should be equal in virulence to a pure liquid culture rate of 0.46 ml/L without any loss of activity from drying. Since the 20 mg/L dry rate produced the same level of injury as a 0.04 ml/L rate of liquid in this aquarium study, the results suggest that drying Mt cultures results in a 10X loss of potentially infective propagules. Based on this result and some previous evaluations of dry versus liquid cultures, the field portion of this May study would attempt to confirm this difference in activity.

Figure 4: Aquaria testing of dry formulation vs. liquid culture of Mt
(Study conducted by Dr. Judy Shearer, ERDC, Vicksburg, MS, May 7, 2008)

In parallel collaborative research supported by the FDEP Bureau of Invasive Plant Management, USDA has further refined temperature requirements for Mt strain being utilized for hydrilla control demonstration studies. Graphs below document that Mt growth in culture, germination of dormant dry microsclerotia, and Mt spore (conidia) production are all optimal in discrete temperature window of 22-30C. These results confirm that 1) summer conditions with elevated temperatures in topped-out hydrilla mats will limit Mt bioherbicide performance, and 2) late fall – early spring should be optimal timeframes for Mt bioherbicide use in Osceola County based on known seasonal water temperature fluctuations in central Florida.

Figure 5: Biomass accumulation of Mt at various temperatures

Figure 6: Germination of Mt mycrosclerotia at various temperatures

Figure 7: Conidia production of Mt at various temperatures

2. Collaborative Research with NC State

Collaborative studies on physiological impacts of Mt infection of hydrilla are now scheduled to begin in July with recent Mt re-isolates and new graduate student of Dr. Rob Richardson.

3. Pilot-scale commercial production

This scale-up decision was delayed due to the virulence question and missing the optimal spring window for Mt treatments in Osceola County. Discussions between SePRO and USDA have early fall target for production with final determination of a commercial partner during the upcoming quarter.

4. Outdoor testing of best formulations

Due to seasonal conditions, the spring field evaluation was supported with confirmation study conducted in greenhouse at ERDC in Vicksburg, MS as described above.

5. Small-scale field demonstrations in Osceola County

On May 15, applications of liquid Mt bioherbicide culture and dried formulation were conducted in a pond at the Osceola County Corrections Facility off Simpson Road. Dry material was applied to a 108 square-foot plot, while liquid materials were applied to 4000 square foot plots. As shown below, the results of one-month post-treatment harvest indicate that Mt applications resulted in approximately a 2X reduction in hydrilla growth versus untreated plot. Also below are temperature profiles during the month period post-treatment recorded by dataloggers placed 1 foot below the water surface and 1 foot above the bottom within pond control plot (approximate water depth 6 feet). Average pond surface temperature was approximately 28C with significant daily variation and slow increase during the period. Bottom water temperature was a more constant 25 – 26C. Both ranges suggest that water temperature should have been relatively optimal for Mt performance with exception of daily peaks that likely for short periods exceeded 30C at the immediate water surface in dense hydrilla mats.

Figure 8: Treatment photos, May 15, 2008 Clockwise from upper left: 1) Starting application to 0.1 ml/L liquid plot (right shoreline), 2) and 3) application of liquid Mt product, 4) Untreated plot (left shore)

Figure 9: One month assessment, June 16, 2008 Clockwise from upper left: 1) Plants off surface in 0.2 ml/L Mt liquid plot (note: water level was up ~6 inches from day of treatment, 2) floating hydrilla fragments damaged by bioherbicide, 3) Defoliation of hydrilla noted in 0.2 ml/L Mt liquid plot, 4) undefoliated hydrilla in untreated control plot.

Figure 10: Hydrilla field trial comparing liquid culture vs. dry formulation, May 15, 2008

Figure 11: Surface and bottom water temperature at Corrections field plot, May 14-June 16,

Relative to greenhouse mesocosm study, results of this field trial confirm that there is a major difference in performance of bioherbicide under laboratory versus field conditions. In mesocosm study, much greater hydrilla control was achieved at lower Mt use rates. Possible reasons for difference in laboratory response include softer, fresher tissue for lab plants and water column aeration in laboratory providing for greater mixing and more Mt contact points with hydrilla. The difference between field and lab response indicates that lab mesocosm studies, while appropriate to confirm basic virulence of bioherbicide, are not well-correlated with field results in terms of specific effective use rate.

Results of the field trial also support laboratory results indicating that active liquid cultures have more virulence than dry product on a per unit production basis. In this trial, the 161 mg/L test rate of dry product produced the same level of hydrilla growth inhibition as 0.1 and 0.2 ml/L test rates of liquid culture. In the absence of activity loss from the drying process, the equivalent theoretical rate of liquid culture needed to produce a 161 mg/L rate of dry product is 3.2 ml liquid culture/L. When compared to actual liquid rates of 0.1-0.2 ml/L used for the field trial, the difference in activity between dry and liquid material on equivalent basis is 16-32X. This outcome indicates that increasing yield of viable dry propagules or further stabilizing a dewatered live culture are potential routes for bioherbicide improvement.

While not a commercially acceptable result in terms of control of existing biomass, the field study does document a potential use pattern for Mt bioherbicide. If cost effective, early season applications of bioherbicide to subsurface hydrilla could be a viable use pattern if management goal is hydrilla suppression versus complete control.

Publications

“Laboratory and Field Demonstration of weed control properties of dry formulations of Mycoleptodiscus terrestris, a potential fungal bioherbicide for control of Hydrilla verticillata”
Florida Department of Environmental Protection Contract #PL114 - 1st Annual Report, 2007-2008, Co-PIs, Mark A. Jackson1 and Mark A. Heilman2
1 USDA, Agriculture Research Service, National Center for Agricultural Utilization Research, Crop Bioprotection Research Unit, Peoria, IL, 61604
2 SePRO Corporation, SePRO Research and Technology Campus, 16013 Watson Seed Farm Rd., Whitakers, NC 27891

What Milestones have been met for your Element?

An additional field demonstration study of Mt bioherbicide was conducted in Osceola County along with supporting laboratory mesocosm work.
On a side note, the project field day activities on July 1 were supported by SePRO staff. Thanks to Eleanor and the Osceola team for their efforts at this event.

Were there any significant problems encountered?

Virulence issue observed in Q2 carried over to the start of Q3. Based on 2007 work and continued investigation of bioherbicide environmental limits, summer temperatures will prevent further pond testing in Osceola County until mid fall.

If so, what measures were taken to correct the problem? Mt strains were re-isolated and tested to confirm proper virulence.
Is the project work on schedule?
- This quarter? For this quarter, the field and mesocosm activities described in the Q2 report were accomplished.

For the project?

We are behind schedule in terms of decision to scale-up and test at larger field scales. However, a significant new production process is under investigation, and a commercial scale-up of production is now targeted for fall 2008.

If the project is not on schedule, what is proposed for a revised schedule?

Per comments above, schedule calls for resumption of significant field testing in the fall.

Does the new schedule require a time extension?

Per work plan, there is still a window for operating under revised schedule without any formal time extension. However, as we finish FY per recent discussion with Osceola grant coordinator Eleanor Foerste, we will discuss status of project to confirm appropriate timing for remainder of grant.

Are there any significant findings this quarter? Documentation of liquid Mt versus dry Mt effects.
What is the future direction of the work?

SePRO will be discussing with Osceola grant coordinator a short-term shift in study focus for field studies in the summer. In collaboration with other grant PIs, we believe that there is an opportunity to help support additional chemical herbicide research under other elements as our field staff waits for more favorable fall conditions for further Mt field efforts. Limited contact chemical herbicide treatments will also be used to encourage fresh hydrilla growth for later season bioherbicide demonstration studies.

Is there a change in the key contacts for this element? No.

Reporting period: Third Quarter, EPA Fiscal Year 2008
May 1, 2008- July 31, 2008

Person Reporting: James P. Cuda

Element: Element 3, Task 2: Foreign Exploration for Natural Enemies of Hygrophila

Project activities during reporting period:

Domestic survey to identify insects associated with Hygrophila.
Presentations and publications.

What Milestones have been met for your Element?

Activity 1 Milestone:
Foreign exploration for natural enemies on Hygrophila spp.:
Searching for natural enemies was only a small part of our initial surveys in 2007. However, during the course of surveys to locate hygrophila populations we observed some insect feeding damage on the leaves of the plant (Fig. 1), but we were unable to collect or identify the natural enemies at the time.

Fig 12: Insect feeding damage on hygrophila,

Fig 12: Insect feeding damage on hygrophila,
West Bengal, India.

Fig 13: H. auriculata leaves showing insect damage

While in India surveying for natural enemies of hygrophila in 2007, we located another species of the same genus, Hygrophila auriculata (Schum). While collecting the samples, we found insect damage and disease symptoms affecting the plant (Fig 2). Although we were unable to specifically identify the natural enemies impacting H. auriculata, our findings clearly showed the potential for obtaining insects and pathogens from this congener of hygrophila. It is noteworthy that there is a published report of larvae of an agromyzid fly boring into the stems of H. auriculata (Sankaran and Rao 1972, Sankaran 1990). Additional surveys in 2008 will focus on this and other natural enemies.

Activity 2 Milestone:
Domestic survey to identify insects associated with hygrophila
No activity in this quarter to report

Activity 3 Milestone:
Genetic analysis of Hygrophila polysperma using microsatellite primers

Collection of plant samples for DNA extraction. In total, 66 samples of hygrophila were collected from 5 Florida counties, namely Dixie, Columbia, Hillsborough, Osceola, and Pinellas (Fig 3). FLDEP personnel John Rodgers, Joe Hinkle and Ed Harris collected samples for us from Hillsborough, Columbia and Osceola Counties, respectively. With the help of Mr. Marcos J. De Jesus, Texas Parks & Wildlife Dept, Inland Fisheries Management, we were able to obtain samples of hygrophila from the San Marcos River in Texas as well. We also collected 24 hygrophila samples from India for DNA analysis.

Fig 14: Hygrophila sampling locations in the USA for DNA extraction

Development of Microsatellite primers. In total, 12 microsatellite primers were developed initially to genotype hygrophila. On further screening for amplification, 6 primers (Table 1) were found consistently to amplify in hygrophila DNA. The results of our initial analysis are shown in Fig. 4. It is evident that in all cases the primers amplified at almost identical loci (see arrows ). This finding suggests almost no genetic variation in the Florida hygrophila. We are currently undertaking extensive analyses using all 6 primers to amplify all DNA samples collected, including those from India.

Fig 15: Hygrophila fragment analysis results showing scored alleles. In all cases, the primer amplified two identical loci (see arrows), which indicates no intra-population genetic variation in Florida populations of hygrophila.

Table 4: Sequences of 6 microsatellite primers found consistent in amplifying hygrophila DNA

What problems, if any, were encountered? None
If a problem was encountered, what action was taken to correct it?
Is the project work on schedule?
This quarter? Yes
For the project? Though efforts are being made, it is likely that establishing sub-contracts with Indian collaborators (PDBC) could result in some delays in accomplishing project goals and objectives due to complex sub-contracting regulations at the University of Florida
If the project is not on schedule, what is proposed for a revised schedule? There is no need to revise the schedule at this point.
Does the new schedule require a time extension? A time extension is not anticipated at this time.
Is there a change in the key contacts for this element? No.

Reporting period: Third Quarter, EPA Fiscal Year 2008 April 1 – June 30, 2008

Person Reporting: Willam A. Overholt

Element: Element 3 Task 3 Collaborative effort to search for natural enemies of hydrilla in East Africa (Project Ongoing)

Project activities during reporting period:

Surveys in Burundi and Uganda indicate that chironomid midges comprise the great majority of insects associated with Hydrilla. In total, 21,817 chironomids, representing 17 species have been reared from collections made in Lake Tanganyika, Burundi, with Dicrotendipes fusconotatus accounting for 84% of individuals. The putative Hydrilla herbivores, Polypedilum dewulfi and Polypedilum wittei, were reared in relatively large number from two locations near Bujumbura in Lake Tanganyika, and efforts to colonize these insects have been initiated. In Uganda, a total of 1886 chironomids representing 35 species were reared from Hydrilla, 20 of which were collected in Lake Bisina, the most frequently sampled lake. Dicrotendipes septemmaculatus, a close relative of D. fusconotatus, accounted for 27% of specimens. Neither of the two target Polypedilum spp. was commonly reared in Uganda. Berlese funnels set up in Burundi recovered chironomids, but only one curculionid larva. One pyralid larva was collected from Hydrilla in Lake Tanganyika on a leaf whose margin had been partially chewed. No insects were reared from over 100 Hydrilla tubers collected at Cercle Nautique, Lake Tanganyika.

A plant diversity survey in Lake Bisina, Uganda found 10 plant species, with 9 submersed macrophytes and Nymphea nuchalli. Najas horrida was the most widespread and abundant species. Hydrilla was found at about one-half of sampled sites and was the fourth most abundant plant. Hydrilla was typically found growing in communities with 3-4 other plants species.

DNA has been extracted from a total of 488 Hydrilla samples. Seventeen polymorphic Hydrilla microsatellite loci have also been isolated and dye labeled. We expect at least 10 of these loci will provide easily interpretable genotypes. We successfully amplified a portion of the pds gene using nuclear DNA as judged by its perfect sequence alignment to the published cDNA sequence of the pds gene. We are currently testing this region’s utility as a marker for resistance using fluridone resistant strains of Hydrilla.

What Milestones have been met for your Element?

Checklists of insects associated with hydrilla in Burundi and Uganda have been prepared (see tables 1, 2, 3 below). This information will soon be posted on the web.

Were there any significant problems encountered?

No major problems, albeit we have not been able to unequivocally associate plant damage with any of the insects associated with hydrilla.

If so, what measures were taken to correct the problem?

We plan to 1) continue plant dissections to examine for plant damage and herbivores and 2) a major effect will be placed on rearing two species of chironomids (Polypedilum dewulfi and P. wittei) which are thought to be plant feeders.

Is the project work on schedule?
- This quarter? Yes
- For the project? Yes
Are there any significant findings this quarter?

We have identified areas in Lake Tanganyika near Bujumbura where the two putative hervivores ((Polypedilum dewulfi and P. wittei) are relatively more abundant than at other sampling sites. Earlier, our major problem was that these two species were not commonly recovered – now that we have areas where they are more abundant, it will give us a greater opportunity for laboratory colonization.

What is the future direction of the work?

Colonization of the two Polypedilum spp.
Test a hypothesis that herbivory by fish may be a factor in regulating hydrilla in Africa.

Is there a change in the key contacts for this element? No

Reporting period: Third Quarter, EPA Fiscal Year 2008, April 1- June, 30, 2008

Person Reporting: Karen Brown
Eleanor Foerste

Element: Element 4 Demonstration and Outreach

Project activities during reporting period:

What work was accomplished for this reporting quarter?

Completed Quarterly and Budget Reports for Second Quarter 2007/08.
Planning and preparation of displays for July 1, 2008 Hydrilla Field Day
Planning and preparation for teacher training with CAIP staff for July 28-31, 2008.
Advertise and recruit to fill Extension Agent Aquatics position vacancy.
Development of template for public information displays.
Continued to update website http://plants.ifas.ufl.edu/osceola
Attended Florida Exotic Pest Plant Council Annual Meeting (April 21-24, 2008) and gave presentation
Participated in Florida Lakes Management Society annual meeting (June 2-5, 2008) and gave presentation.

What Milestones have been met for this Element?

Task 1, Year 2 - Demonstration field days and media events to keep stakeholders informed on the progress of hydrilla and hygrophila management work.

Year 1 - Kick off Event – complete.
Year 2 – Field demonstration day – a Great Outdoors Days event (organized by Tina Bond) was held October 27th-28th, 2007 in Kissimmee.

A display featuring aquatic plant management methods and Hydrilla and Hygrophila information was set up and staffed by Amy Richard and Rob Horsburgh of the UF / IFAS Center for Aquatic and Invasive Plants. Full color pamphlets about hydrilla and hygrophila and the Demonstration project were distributed at the event. They also are available at the Osceola County Extension office and other venues.
A set of business card sized “web cards” (2” x 3.5”) for both hydrilla and hygrophila were produced and printed for handing out at public venues. The cards feature images of the plants and include the Demonstration Project web address for easy reference. The cards were given to everyone who visited the CAIP/UF-IFAS and the Osceola County booths at the Great Outdoors Days in late October 2007.

Task 2, Year 2 – An interactive website will keep citizens/agencies informed of the various stages of research and allow citizens to communicate directly with researchers and county officials with questions and/or concerns.

The website continues to be developed and expanded as new material is made available. According to Google Analytics, the website had 417 visits and 588 page-views during this quarter.
The following new web pages were created during this quarter:

Task 3, Year 2 - Database development.

Annotated citations to the scientific literature pertaining to hygrophila and hydrilla management in Osceola County and Kissimmee water bodies are listed in the demonstration project website under “Research Publications from the APIRS Database.” New citations will be posted as they are received.
Other citations from the APIRS collection that could provide additional information to citizens of Osceola County will be posted as they are located and evaluated.
Printed copies of publications are available from the CAIP Information office or as downloadable PDF files.

Task 4, Year 2 – Develop portable Aquatic Plant Management Displays/ Kiosks that can be used for local events such as the County Fair, Earth Day, Great Outdoors Days, scientific conferences and at various public areas such as boat ramps, parks, libraries and county offices.

Year 1 - Three introductory displays (retractable banner stands) (Oct 06 – Dec 06) – complete.
Year 2 - Second set of displays (Dec 07). The large format public information displays used in recent public events are currently under review for ideas on developing future materials. Continuation of this phase of the project is currently on hold pending the hiring of a replacement extension faculty. In the meantime, four templates (dimensions: 24” X 36”) have been created. One of the panels features six of the worst invasive plant species currently being managed in Osceola County (including hydrilla and hygrophila) along with information on how the public can help. Slight alterations of text and images will be needed for final production, based on edits/suggestions from the new extension faculty.

Task 5, Year 2 - Development of interactive science curricula for Osceola County teachers: 4 PPT lessons, 2 web-based activities, hands-on lab/field activities & modules tailored for Osceola County for UE, MS, HS. (June 08)

CAIP staff made contact with a number of science teachers in Osceola County and provided them with materials and lessons to test in their classroom. A number of activities have been adapted for testing in the upcoming July teacher workshop slated for July 28-31 (i.e., the hydrilla game and the Aquatic Plant ID activity).

Task 6, Year 2 - Development of additional demonstration and outreach strategies.

Additional strategies to demonstrate and educate stakeholders about hydrilla and hygrophila management are being considered:

Video plant identification modules on hydrilla and hygrophila have been placed on the Osceola website for use by the general public and by field personnel.
Full color informational brochures about hydrilla and hygrophila and the Demonstration project were designed, printed and distributed at the Great Outdoors Days event in late October, 2007. They also are available at the Osceola County Extension office and other venues.
A set of “web cards” for both hydrilla and hygrophila have been produced and printed for handing out at public venues. The business card sized cards feature images of the plants and include the Demonstration Project web address for easy reference.
A pilot project will be presented to teachers at the upcoming workshop (July 28-31), at which time teachers will be trained and encouraged to use new media technologies to inspire students to communicate/raise awareness about invasive plants. Using these techniques, students can create PSAs, virtual posters, and other cool graphic communications. The finished products can then be posted and viewed from the Osceola County hydrilla project website, as well as the CAIP education website, and be used to generate further interest and excitement among teachers, students, and parents in Osceola County.

Task 7 - One paper and one poster per year to share research progress and management technology, to be published on EDIS and presented at meetings.

Element 4 Presentations:
Bond, Tina M. Project Update. Florida Exotic Pest Plant Council Annual Meeting
(April 21-24, 2008).
Foerste, Eleanor C. Project Update, presented at Florida Lakes Management Society Annual Meeting (June 2-5, 2008).

Element 3 Task 2 Publications:
Cuda J. P., R. Charudattan, M. J. Grodowitz, R. M. Newman, J. F. Shearer, M. L. Tamayo and B. Villegas. 2008. Recent advances in biological control of submersed aquatic weeds. J. Aquatic Plant Manage 46: 15-32.

Mukherjee A, J. P., Cuda, W. A., Overholt, & C. Ellison. 2008. Biological Control of Hygrophila polysperma: Searching for Natural Enemies in India-First Trip Report. Aquatics. 30(1): 20-22.

What problems, if any, were encountered? None.
- If a problem was encountered, what action was taken to correct it?
Is the project work on schedule?

This quarter? Yes.
For the project? Yes.

Is there a change in the key contacts for this element?
- Dr. Tina Bond, Extension Agent Aquatics and Element 4 Coordinator resigned and accepted another position with Osceola County. The position has been re-advertised and candidates screened. Candidates will be interviewed in early August, 2008. Eleanor Foerste, Project Manager is providing leadership for the Hydrilla Field Day and assisting with the teacher workshop as these activities were under time deadlines that required action this quarter.