2016 Inoculation of whitebark pine seedlings with native ectomycorrhizal fungi: field restoration project on a burn

General Project Information

Project Title:

Inoculation of whitebark pine seedlings with native ectomycorrhizal fungi: field restoration project on a burn

Project Dates: 2016

Year project implementation started: 2015

How many more years is this project expected to continue, if any? 2017

Project Contact: (Please provide complete information for primary contact(s), e.g., name, position, phone number, email, agency name, unit/sub-unit)

Dr. Cathy L. Cripps, Plant Sciences & Plant Pathology Dept., Montana State University, Bozeman, MT 59717, 406-994-5226, ccripps@montana.edu

Location (Land management agency or ownership and name of geographic area(s) where project was implemented. This information should be specific enough to identify a general project location on a map but not specific enough to compromise the project.)

Burn that resulted from the Eureka Fire, in the GYGT, Gravelly Range, SW, MT

Cooperators: (List cooperating agencies and sub-units, other companies/organizations, and individuals as needed.)


Project Funding

Funding Sources (amount FHP/amount other incl. in-kind)

Forest Health Protection funding                   $ 7,500

Other funding                                                 $ 5,787

Total                                                               $12,787*


*This is funding we received in 2015. We had additional previous funding to use in addition to this current annual budget. We received an extension for this research until 2017.

Did Whitebark Restoration funding get used or obligated? (If not, please explain.)

All of the remaining funds for the budget above and the previous funds are designated (primarily for graduate student summer salary and travel to sites for monitoring). We did receive an extension to use remaining funds.


Project Details

Scope and/or Size of Project or Treated Area: (Include a short description of the project or treatment area if helpful in understanding the scope of the project.)

Planting is being done by the USDA Forest Service for restoration purposes.

Site 1: 120 acres were planted in whitebark pine in 2015; of these, 10 acres are currently in our study and were monitored in 2016.

Site 2: 10 acres were added in 2016 and will be monitored in 2017.

Number of Acres or Other Units Treated, Monitored, or Surveyed:

About 120 acres in 2015; 123 acres planted in 2016.

Specific location of project or treated area(s): (If desired, add more specific project location information here, e.g., UTMs, Lat-long, specific landmark. Otherwise, indicate if more information is available by request.)

Montana, Gravelly Range, Beaverhead- Deerlodge Forest; Site 1: 9200 ft. elevation and site 2: 8500 ft.

Eureka Burn, 44.854 N, 111.86 W

Objective(s) (from original request):

Original objective: Assess how inoculation/colonization with native ectomycorrhizal suilloid fungi affects survival (and other parameters) of whitebark pine seedlings planted in spring on the Eureka burn in comparison to uncolonized seedlings.

The burned area in the GYGT is a result of the 2013 Eureka Fire; it is in the Gravelly Range in the Beaverhead-Deerlodge Forest. This was a severe burn and is devoid of living trees (standing dead only) with a sparse grass understory. The area was previously in a mixed forest type that included whitebark pine. According to Mahalovich (pers. comm.), the fire resulted in the loss of a significantly unique whitebark pine genetic source near this area known as the West Fork genetically unique whitebark pine population. We expect that significant areas of this burn are likely to be devoid of appropriate ectomycorrhizal fungi due to the intensity of the fire and lack of living trees.

Our original goal was to inoculate seedlings with native ectomycorrhizal fungi (spore slurry) in the nursery before planting and to monitor the survival of seedlings compared to uninoculated controls after at least one year, and hopefully longer. In addition, we would monitor microsite and other abiotic parameters. Due to a misinterpretation of when it would be possible to inoculate seedlings for spring planting, the original objective is pushed back 1 year. However, we did manage to initiate an additional project using already colonized seedlings.


The project is revised as follows:

Objective 1: (year 1) Assess how ‘natural colonization’ (no inoculation)’ with native ectomycorrhizal fungi affects the survival (and other parameters) of whitebark pine seedlings planted on the Eureka burn in spring 2015.

We were not able inoculate seedlings planted in spring 2015 with native mycorrhizal fungi, since they were frozen at this time. However, we previously noted that many whitebark pine seedlings coming out of the Coeur D’Alene nursery recently have been well colonized by ectomycorrhizal fungi. DNA analysis revealed that these are native suilloid fungi of unknown origin. Therefore, at planting in spring 2015, 400 seedlings well-colonized with these fungi, and 400 largely devoid of these fungi were separated into two batches, tagged, and planted on the Eureka burn. GPS coordinates were taken for each seedling and microsite and other relevant data recorded. Seedlings were monitored for survival in Fall (Oct. 15, 2015) and were monitored again in 2016 (at least).

Objective 2: (year 2) Assess how ‘inoculation with native ectomycorrhizal fungi’ affects the survival (and other parameters) of whitebark pine seedlings planted in spring on the Eureka burn (in 2016). [Note: This is our original objective which was pushed back 1 year.]

Spore slurries (inoculum) made from native suilloid fungi (EB 105-15) from whitebark pine forests were used to inoculate 400 whitebark pine seedlings on Oct. 22, 2015 at the Coeur D’Alene nursery, and 800 seedlings (400 controls) were tagged. Mycorrhizal colonization was allowed to occur over the next few weeks before they were frozen at the end of the year. At planting in spring 2016, GPS coordinates were taken for these seedlings, data was recorded, and we will, subsequently, monitor them in 2017 (at least).

Objective 3: Assess how ‘natural colonization’ of seedlings in the greenhouse with suilloid fungi (of unknown origin) affects seedling parameters and nitrogen content of needles. [This objective was added for a small sample of leftover seedlings.]

Over half of two batches of whitebark pine seedlings we received from the Coeur D’Alene nursery (2014, 2015) that were already colonized while at that nursery were assessed by our own DNA analysis and found to be colonized with native suilloid fungi. The origin of these native fungi is unknown*. The ‘naturally colonized’ seedlings were obviously larger, which was an unexpected response given the slow growing nature of this tree species and because colonization with mycorrhizal fungi can cause an initial carbon drain to seedlings. Therefore, the biomass and nitrogen content of seedlings were assessed to determine if mycorrhizal colonization was related to increased growth and nitrogen content of seedlings. Nitrogen isotope analysis was used to directly determine if additional N in seedlings was in fact a result of the presence of mycorrhizal fungi.

*The origin of this inoculum could possibly be from spores of Suillus species fruiting near the nursery (in the cone production orchard of western white pine). We did observe these species of Suillus fruiting when we visited the nursery. See article in Nov/Dec 2015 issue of Nutcracker Notes (Cripps & Jenkins 2015).

Planting: (Please answer the following questions if the project includes plantings or cone collections.)

Number of seedlings planted (List by location if applicable):

We are currently monitoring 800 of 36,000 seedlings planted in 2015. We will monitor a 2nd set of 800 seedlings planted in spring 2016.

Was the seed source screened for resistance? (If other, explain.)

Seedlings came from local trees in areas heavily impacted by the rust; I do not believe they were actually tested (CrockettLake11; WB03030092 and Gravellys11;14CO18-2016W.M.)

Were Plus trees used? Unknown


Objective 1, site 1: The 800 seedlings planted in spring 2015, were tagged and have GPS coordinates, and GIS was used for mapping.

First monitoring: Initial survival after 3 months was high, as expected, at 98% overall for all seedlings. Of these 90% had a high health rating of 4/5. Differences between colonized /not were not expected after just 3 months. It was noted that some seedlings downhill of stumps used as microhabitat were flooded where rain water pooled. Analysis of microhabitat types is also part of the study as it relates to survival and is ongoing.

Second monitoring in 2016: Seedling survival was still high overall after one year (93.5%). Only a few seedlings died in each category. A majority (80.5%) still had the highest health rating of 4 overall, but this was lower than the first monitoring. A higher number of mycorrhizal colonized seedlings (91%) had the highest health rating of 4 in comparison to 83% for uncolonized seedlings. Less than 1% were counted as dead in the ‘0’ category.


Health data for whitebark pine seedlings excluding those that were buried or pulled/eaten by animals. Highest health rating = 4 and lowest = 0.

Health rating Number colonized seedlings Number uncolonized seedlings % colonized seedlings % uncolonized seedlings
4 327 271 91% 83%
3 25 39 7% 12%
2 4 9 1% 3%
1 0 3 0% 1%
0 3 4 1% 1%


Objective 2, site 2: Slurry for seedlings was prepared and 400 seedlings were inoculated and tagged on Oct. 22, 2015. Seedlings were planted in spring 2016 on the Eureka burn and will be monitored in fall 2017 (at least) as funding allows. Inoculum was Suillus sibiricus EB105-15.

Objective 3: Greenhouse experiment. Results for a preliminary sample of N = 6 is given below. Results for the whole sample set of N = 140 is currently being analyzed for a thesis, and results so far tend to mirror that for the small sample. Carbon isotope analysis is underway.

Seedlings in the uncolonized group were less than 1% colonized and seedlings in the colonized group were 10 – 30% colonized with ectomycorrhizae (suillioid type). On average, the colonized seedlings had 68% greater biomass and 66% higher total foliar nitrogen content. Wilcoxon Rank-Sum tests showed that colonized seedlings had a significantly higher biomass (p = 0.015) and a significantly higher foliar nitrogen content (p = .0086, with one outlier excluded).

DNA Analysis of Mycorrhizae: A majority of the ectomycorrhizae appeared to be of the Suillus ‘hand-type morphology; a neglible number were of the thelephoroid or Cenococcum types. Molecular analysis picked up Suillus discolor in four blast matches, Suillus sibiricus in two, confirming that these native species were present in the nursery seedlings. These two are the native species that we normally use in inoculation.


Project Status (Is the project complete? If not, what remains to be accomplished and when?

For site 1, the planted seedlings were monitored in 2015 and 2016. Results are being analyzed statistically (Marlee Jenkins M.Sc Thesis). We hope to continue this monitoring with another student in the future.

For site 2, all seedlings were planted and received GPS coordinates and tags. This site will also be monitored by a new graduate student in 2017 given there is additional funding.

Will outcome meet objectives?

Yes, it should meet the original objective 1 (one year late) given that all goes smoothly now. The additional objectives will also be met.


Project Follow-Up

Are there plans for monitoring or follow-up? (If not, please explain.)

Yes, we will continue to monitor as long as possible depending on funding.

Changes Needed or Problems Encountered:

We did encounter a delay (see objectives), but this time was used to expand the study to examine not only the effect of purposeful mycorrhizal inoculation on the survival of whitebark pine seedlings, but also the effect of suilloid colonization occurring without inoculation in the Idaho nursery for survival of seedlings planted on the burn.

In lieu of the delay, we officially extended the End Date to complete the project in 2017.

Sharing Results/Products/Outcomes: (Please include pertinent photos and links to any relevant reports or publications)

Products currently in progress:

Cripps, C.L. and M. Jenkins. 2015. An unexpected growth response in whitebark pine seedlings colonized with ectomycorrhizal fungi in the greenhouse. Nutcracker Notes, Nov/Dec issue. (published)

Jenkins, Marlee. M.Sc. Thesis, Montana State University. Expected May 2017.

We expect one or two papers to come out of this thesis as well.


We thank:

Anton Brennick and Bruce Schuelke USDA Forest Service for their help with this project.