IDENTIFYING RESERVOIRS AND INSECT VECTORS OF PINE WILT DISEASE IN THE FRONT RANGE

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: MCINTIRE-STENNIS
• Reporting Frequency: Annual
• Accession No.: 1016206
• Project Start Date: Jul 1, 2018
• Project End Date: Jun 30, 2020
• Program Code: [(N/A)]- (N/A)

Recipient Organization
COLORADO STATE UNIVERSITY
(N/A)
FORT COLLINS,CO 80523

Performing Department
Forest and Rangeland Stewardship

Non Technical Summary
In this project CSU scientists will determine the rate of infection of an emerging disease complex (pine wilt nematode) that threatens forest health in Colorado and elsewhere. The project will focus on assessing forests in the Front Range region of Colorado, and evaluating several species of insect for their potential to vector this disease. CSU scientistswill also construct simple behavioral models that can be used to estimate timing of disease exposure from insect vectors. To achieve these goals, researchers will employ basic forestry and field ecology measurements, as well as genomic techniques and statistical modelling procedures. Ultimately, these approaches are intended to increase knowledge about a potentially harmful forest disease and provide new knowledge to practitioners that can be used to develop management strategies.

Animal Health Component

50%

Research Effort Categories

Basic

50%

Applied

50%

Developmental

0%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
212	0612	1130	50%
212	0612	1160	50%

Knowledge Area
212 - Pathogens and Nematodes Affecting Plants;

Subject Of Investigation
0612 - Conifer forests of the West;

Field Of Science
1130 - Entomology and acarology; 1160 - Pathology;

Keywords

forest health

tree diseases

insect vectors

disease model

Goals / Objectives
The major goals of this project are to:(1) Assess the incidence of pine wilt nematode disease in forests of the Front Range region of Colorodo;(2) Determine the primary insect vector of the disease and vector density; and(3) Produce a flight phenology model to describe potential windows of disease exposure based on vector behaviors.

Project Methods
Objective 1: Determine incidence of PWN in Front Range forests50 1-ha fixed area plots will be established in stands containing P. ponderosa and/or P. contorta on public lands across five counties in the Front Range, including Larimer, Boulder, Jefferson, Teller, and El Paso counties. Ten plots will be distributed latitudinally within each county, and plot corners will be marked using rebar and GPS coordinates. During each project field season, trees on each fixed area plot will be censused for symptoms consistent with PWN.Trees displaying disease symptoms consistent with PWN, including slowed current years' growth, needle discoloration, crown dieback, and branch death. Five 3 cm × 10 cm cores will be extracted from the branches and/or bole of symptomatic trees. In the lab, samples will be cut into 3 cm3 pieces. Testing for PWN will include both extraction of nematodes from wood and direct molecular testing on wood. For each wood sample, 50g will be placed into a Baermann funnel to release nematodes from wood. Extracted nematodes will be identified using both morphological charactersor using a species-specific molecular assay. This molecular assay is a Loop-mediated isothermal amplification (LAMP) procedure that requires no thermal cycling due to its complex primer design. To utilize this assay, total genomic DNA will be extracted from nematodes using the DNeasy Blood and Tissue Mini kit (Qiagen, Germantown, MD) following manufacturers specifications. Lastly, 5g of each wood sample will also be tested directly using the species-specific LAMP assay to determine presence of PWN.Analytical approach: Simple parametric tests (two-sample Student's t-test) will be used to compare the mean frequency of infection between both potential host species (P. ponderosa and P. contorta). We will use ANOVA methods to compare the response of mean proportion of infected trees due to the fixed effect of 'county'-allowing for conclusions about which counties support the greatest incidence of PWN.Objective 2: Characterize the primary insect vectors of PWN in the Front Range and evaluate vector abundanceInsect vectors (Monochamus spp.) will be surveyed using a targeted trapping approach to assess vector flight timing and abundance in the 2018 and 2019 field seasons.On half of the plots (N=25, five trapping sites in each county, randomly chosen from forest survey plots), black crossvane traps will bedeployedand baited with a lure containing the sex pheromone monochamol (2-undecyloxy-1-ethanol) and a proprietary blend of host volatiles. Baited traps will be collected weekly from May-October. Typically, one lure per trap is sufficient to retain attractancy throughout the field season, and traps will be placed at a height of 1.5 m between the collection cup and the ground surface.Trap contents will be sorted and identified by project staff. Degree-day accumulation (base 10°C) will be recorded throughout the sampling period each year at trapping sites using data loggers.Monochamus spp. captured in flight traps will be assayed for the presence of PWN. Briefly, insects will be macerated in 2 ml of sterile distilled water. Released nematodes will be both morphologically and molecularly identified. The species specific molecular assay, LAMP, will be used to verify the presence of PWN . Using an inverted glass microscope slide, the number of nematodes in 1ml will be used to extrapolate the total number of nematodes (i.e., PWN/beetle) carried by each insect.Analytical approach: Parametric tests (two-sample Student's t-test) will be used to compare mean PWN/beetle between the two potential vector species (M. clamator and M. scutellatus), as well as any other wood-boring insects that are consistently identified as associated with PWN. ANOVA methods will also be used to examine the fixed effect of 'county' on the response of mean abundance of captured insect vectors.Objective 3: Develop a phenology model to determine the potential window of exposure to PWNInsect phenology models will bedeveloped from trapping studiesto predict the temporal window of exposure of plant populations to insect pests. Cumulative trap captures of vector insects will be modeled as a function of multiple predictor variables including ordinal day (i.e., day-of-year), thermal accumulations (degree-days base 10°C), and thermal thresholds (number of days ≥10°C) using a two-parameter logistic model. Models based on each predictor variable (day-of-year, thermal accumulation, or thermal threshold) will be compared using Akaike's information criterion (AIC), and the model minimizing AIC will be selected for further interpretation.These models can be used to predict the cumulative proportion (percent) of the vector population that has initiated directed flight in response to attractive odor sources.By modeling beetle flight in this way practitioners can easily determine probable initiation and terminus of the flight period as well as the relative proportion of the population which has initiated host-finding or mate-finding behavior. Host-finding and mate-finding are coincident with primary and secondary transmission of PWN, thus, these models can be used to predict probable periods of tree exposure to PWN vectors. Flight phenology models will be produced for both M. scutellatus and M. clamator. An additional flight phenology model will be constructed based on the pooled number of Monochamus beetles (i.e., incorporating both species) that are infective at each weekly collection period.Analytical approach: Analysis will be addressed using a model comparison approach, employing a metric of model parsimony (AIC) to evaluate logistic models constructed using different predictor variables. A 'best' model will be identified in this way for both M. scutellatus and M. clamator.

Progress 07/01/18 to 06/30/20

Outputs
Target Audience:The target audience for the project includes scientists, university extension specialists, and the general public. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?We have disseminated project results to regional managers in several workshops and meetings during 2019 and 2020. These workshops were led by the project graduate student, providing them with an opportunity to develop public speaking skills. The project graduate student also attended the national conference of the Entomological Society of America in 2019 (St. Louis, MO). How have the results been disseminated to communities of interest?We have given multiple professional seminars and workshops, as mentioned above. In addition, we have published an extension publication on the disease and its regional patterns, and a peer-reviewed publication is currently in production with an open-access journal (Ecological Solutions and Evidence). What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? (1) We have completed our survey of disease incidence and determined that 3.6% of tested trees are positive for pine wilt nematode disease in the Front Range region. Many of these hosts are outwardly asymptomatic and do not express disease symptoms until environmental stress occurs. We alsoidentified four epicenters within the region that should be targeted for mitigation efforts. In addition, by surveying insect vectors of the disease we also found that 4.2% of sampled sawyer beetles (out of a total sample size of >5000 beetles). (2) We have determined that both Monochamus clamator and M. scutellatus are abundant in the region and both insects are vectors of pine wilt nematode disease. We report the first association between M. clamator and pine wilt disease in the United States, and in the Front Range region M. clamator was ~5 times more abundant than M. scutellatus, indicating that it is the most likely the 'primary' vector of the disease regionally. (3) We have developed flight phenology models for both species and determined that timing of flight activity is similar for both species, but flight timing can vary from year-to-year. Flight activity typically initiates by early August, peaks in late August/early September, and flight activity ends by early October. We have also determined that the flight activity of infected beetles tends to occur earlier in the growing season at identified disease epicenters, with likely dispersal of infected individuals to proximal locations. We have also determined using flight trap methods that beetle populations are not supported within urban forest landscapes, and that natural forest stands in the wildland urban interface are the primary reservoir for insects vectoring the disease. By late in the growing season, infected vectors emigrate into urban areas, causing disease transmission in susceptible landscape tree species.

Publications

• Type: Journal Articles Status: Accepted Year Published: 2021 Citation: Atkins DH, Davis TS, Stewart JE. 2021. Probability of occurrence and phenology of pine wilt disease transmission by insect vectors in the Rocky Mountains. Ecological Solutions and Evidence. In press.
• Type: Other Status: Published Year Published: 2020 Citation: Atkins, D.H., Davis, T.S., Stewart, J.E. Pine wilt disease in the Front Range. Colorado State University Extension. Fact Sheet 2.915. Gardening Series: Diseases.

Progress 10/01/18 to 09/30/19

Outputs
Target Audience:Target audiences include private homeowners, municipalities, and natural resource management practitioners. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? During 2019 the project graduate student has attended two international conferences and presented research findings to scientists and peers. During 2019 the project graduate student attended two stakeholder meetings/extension workshops and has trained practitioners in the identification and management of PWN disease. The project graduate student has also completed the requirements necessary to receive a professional certificate in applied statistics, in addition to their graduate degree. How have the results been disseminated to communities of interest? As above, project results have been presented to both peer and stakeholder audiences through attendance at professional conferences and stakeholder meetings. An extension publication is currently under review to describe project results in a short format for interpretation by the public. The project graduate student and PI's have interacted extensively with practitioners in the region to describe project results in one-on-one meetings. What do you plan to do during the next reporting period to accomplish the goals? One thesis will be published by the project graduate student during the spring of 2020. At least one peer-reviewed publication will be submitted to an international journal (Journal of Applied Ecology)to describe project results. If time and funding allow, flight phenology model results will be developed into an online calculator tool for access by practitioners.

Impacts
What was accomplished under these goals? (1) Assess the incidence of pine wilt nematode disease in forests of the Front Range region of Colorado All insect and tissue samples have been tested for PWN and this objective is now complete. (2) Determine the primary insect vector of the disease and vector density All primary data have been analyzed and this objective is now complete. We have also developed models based on landscape factors that predict the prevalence of the disease in the region. (3) Produce a flight phenology model to describe potential windows of disease exposure based on vector behaviors. Flight phenology models are developed for both species and this objective is now complete. We are now coupling the models to climate data to analyze how temperature and deviation from winter normals are likely to impact windows of disease exposure.

Publications

• Type: Other Status: Under Review Year Published: 2020 Citation: Atkins, D.H., Davis, T.S., Stewart, J.E. Pine wilt disease in the Front Range of Colorado. Colorado State University Extension. Topic: 6.30 Insect: Trees and Shrubs. Fact Sheet # 2019-0817.

Progress 07/01/18 to 09/30/18

Outputs
Target Audience:Target audiences include private homeowners, municipalities, and natural resource management practitioners. Changes/Problems:The project was initially intended to focus on both ponderosa pine and lodgepole pine, but has since been narrowed to focus only on ponderosa pine. The rationale for this was that the sampling efforts may have been stretched too thin to be convincing if two pine species were considered simulatneously, and ponderosa pine is generally more prevalent in the region. What opportunities for training and professional development has the project provided?Training opportunities: A graduate student working on the project has recieved considerable training in (a) the deployment of pheromone traps for sampling pest insect populations, (b) using forestry techniques to establish and sample field sites, and (c) laboratory techniques for extraction and identification of nematodes using standard molecular methods. The student has also taken coursework in analytical statistics and has gained direct experience in experimental research design. Professional development: The student has also participated in a mentoring program that has given them the opportunity to direct the work of undergraduate technicians. The student has also assisted the PI's in the development of small grants to continue funding the research, and will attend at least two professional conferences in the next calender year. How have the results been disseminated to communities of interest?Results will be orally disseminated at professional conferences by the PI's and the project graduate student in early 2019. Both extension and peer-reviewed publications are projected to result from the research near the end of 2019. What do you plan to do during the next reporting period to accomplish the goals?An additional year of collections will be made in 2019 to sample insect vector species at the 30 established sites. In addition, several new sites will be added and sampled as well. All insect and pine tissue samples will be analyzed using molecular methods to test for presence of PWN in 2019.

Impacts
What was accomplished under these goals? Pinewood nematode (PWN, Bursaphelenchus xylophilus) was first discovered in the Rocky Mountain region in 2006 in Larimer and Weld counties (CO) in Scots pine (Pinus sylvestris), a widely planted urban landscape tree. Since then, the disease has been reported in Austrian pine (P. nigra) and P. sylvestris from urban landscapes in the Front Range and western Colorado. In 2016, the first report of PWN infecting P. ponderosa was made near Carter Lake (CO) in native forest ecosystems. Subsequent samplings identified P. ponderosa at an additional five locations along the Front Range in wildland-urban interface forests infected with PWN. This has prompted a need to develop a more detailed survey of incidence and severity of PWN infection in native pines. It is currently unknown if native pines harbor endemic PWN populations that may act as a reservoir for transmission of the disease to urban forests and susceptible landscape trees or if PWN is moving into wildlands from infected urban trees.However, the disease is a major agent of tree mortality and economic damage worldwide, and could have considerable consequences for forest landscapesof the Front Range. Our project will impact a change in knowledge regarding the frequency of PWN disease in Colorado, and help to determine which native insect species are vectors of the disease. This will allow managers and homeowners to make decisions regarding treatment of PWN in susceptible host trees, and determine optimal timing of potential treatments and risk of infection. Project Goals and progress towards each goal are listed below: (1) Assess the incidence of pine wilt nematode disease in forests of the Front Range region of Colorado Activities: A total of 30 trapping sites have been established across multiple counties in wildland-urban interface forests ofColorado. All pine trees at insect trapping sites have been sampled for PWN. Data collected:Approximately 1000 tissue samples have been collected from across the Front Range region for processing and testing using molecular methods. Nematode extractions have been made from all collected tissues. Summary statistics: Nematodes were found to be present in every pine tissue sample collected thus far. However, identification of nematode species is currently underway. Key outcomes: Approximately 1000 pine tissue samples have resulted in the collection of large amounts of nematodes from all sample trees. Continued analysis of nematode samples using morphometric and molecular techniques will allow us to determine disease frequency in native pines of the Front Range. (2) Determine the primary insect vector of the disease and vector density Activities: Insects vectors were sampled at study sites (30 sites, mentioned above) from July - October in 2018. Samples were collected using pheromone-baited traps and collections were made at weekly intervals. Data collected: Insect specimens have been collected using pheromone traps across the Front Range region, and consist of two main species. Both Monochamus scutellatus and M. clamator were captured, as expected. Summary statistics: Approximately 2600 specimens were collected during the trapping season. It was determined thatM. scutellatuscomprised approximately 60% of the sample and M. clamatorcomprised the remaining 30%. Key outcomes: Continued work in the next calender year will utilize PCR methods to directly sample PWN from vectors, and this will allow us to determine which species is able to vector PWN. We will also be able to determine the relative frequency of vector insects that are infected with the disease. This will effect a change in knowledge regarding the primary insect vector of PWN in the Front Range region. (3) Produce a flight phenology model to describe potential windows of disease exposure based on vector behaviors Activities: Insect trapping listed under (2) above constitutes the activity needed to deduce flight timing of vector species. Data collected:2600 vector insect specimens were collected at weekly intervals, and abundances of each focal species will be analyzed using standard statistical procedures. Summary statistics:The majority of the flight period of both species occurred in August and Septemer, indicating that exposure to PWN disease may be most likely in early fall months. Key outcomes:Although extensive trap capture data has already been collected and can be used to characterize vector flight timing,flight timings can vary between years considerably. Accordingly, an additional collection of insect specimens will occur in 2019, and characterize the flight period from May - October. This will effect a change in knowledge regarding critical windows of high vector densities, and allow us to determine vector abundances from spring through fall.

Publications