2008 Mycorrhizal Inoculation with Native Fungi

Project: Mycorrhizal Inoculation (of whitebark pine seedlings) with Native Fungi

Attachments: MSU_Cripps_2008_Report

MSU_Cripps_2008_ Whitebark pine inoculation presentation

MSU_Cripps_2008_ Paper Phase 1

Agency/forest or park/ district  N/A

Project coordinator:  Cathy Cripps

Contact:  Cathy Cripps, Plant Sciences & Plant Pathology Dept. Montana State University, Bozeman, MT 59717-3150. Tele: 406-994-5226, FAX: 406-994-7600, email: ccripps@montana.edu

Cooperators

Melissa Jenkins, USDA FS, Targhee National Forest who will provide seeds for the project and the USDA FS Nursery in Coeur d’Alene Idaho which may donate labor. Alternatively, we will pay for seedlings with funds from this proposal, and then budget would need to be adjusted.

Source of funding

FHP: $7,800

Supplemental funding: $4,000 from Dr. Gene Ford time (in kind), $600 from Coeur D’Alene Nursery (seedlings).

Dates of restoration efforts

All 2008

Objectives

The main goal of this project is to develop methods for inoculation of whitebark pine seedlings with native ectomycorrhizal fungi under nursery conditions. Specific objectives are to:

1. Evaluate native fungi collected from whitebark pine forests for their potential as inoculum.
2. Compare inoculation methods for efficiency of mycorrhizal colonization.
3. Determine optimum seedling age for inoculation.
4. Evaluate influence of various fertilization regimes on mycorrhizal colonization.

Acres/ha treated  N/A

Methods

Seedlings will be grown at the USDA FS nursery in Couer d’Alene ID. The experiment will take place in the Montana State University’s Plant Growth Center (PGC), a facility designed for greenhouse projects and adjacent to the mycology lab. Fungal isolates are from native whitebark pine forests in the GYE.

We currently have around 15 species of fungi in culture which will be tested for their rate of growth in Petri dishes (mean colony diameter) and in 1:9 peat:vermiculate (with added MMN agar media) in test tubes (Mahony 2005). Two to four species of fungi will be selected for further testing based on growth characteristics (100 seedlings for each species). For each species, two inoculation methods will be compared using 50 seedlings each (Fig. 1):

1. “soil inoculum” (mycelium grown in peat:vermiculate) added to seedlings.
2. “filter paper wrap” (mycelial growth on the paper wrapped around seedlings).

For each method, seedlings will be inoculated at 3-4 different ages (12 seedlings x 4 ages = 48). Each group of 12 will be again divided into those that will receive typical levels of nursery fertilizer and those that will receive none (fertilization typically decreases mycorrhizal colonization). The age of seedling at inoculation may be critical to successful mycorrhizal colonization as well (Rincon et al. 2005). Ideally seedlings would be stagger-started at the nursery with 50 each at 0 time, 2 weeks, 8 weeks and 16 weeks later. If this is not possible we hope to obtain very young seedlings and to stagger inoculate them. Inoculation could start as early as 2, 4, 8 and 16 weeks depending on the age of seedlings we initially receive. Seedlings could be older. Seedlings would be maintained in the PGC greenhouse for 4 to 6 months (checked at 4 months for colonization and left longer if necessary).

Due to the nature of the tree species, we do not expect differences in seedling growth at the nursery stage, however seedling height and stem diameters would be measured at the beginning and end of the experiment. Each seedling would then be assessed for mycorrhizal colonization at the end of the experiment. This would be done according to Brundrett (1996), and seedling roots would be soaked overnight in water to gently remove soil. The total number of mycorrhizal root tips would be counted using a dissecting scope and compared to the total number of root tips for % colonization (alternatively a grid method of assessment would be used). Our lab is experienced in delineating the morphotypes of ectomycorrhizal fungi and if necessary can use molecular methods to confirm results are from native fungi. Comparisons of the % mycorrhizal colonization for all treatments will be analyzed statistically using multivariate analysis to determine effects of: fungal species, inoculation method, age of inoculation and fertilization regime.  We will inoculate any extra seedlings using spores (as liquid slurry) from dried/fresh sporocarps when/if they become available. This would give preliminary data for another inoculation method.

Planting? If so, source of seedlings? Resistance?  No

Outcome

The main goal of this project was to initiate development of methods for inoculation of whitebark pine seedlings with native ectomycorrhizal fungi under nursery conditions.We have made significant progress in capturing and storing native fungi from whitebark pine forests in the GYE for this project (a difficult task) and screening them for potential as inoculum for whitebark pine seedlings. This is an important step since commercial inoculum has the potential to upset sensitive whitebark pine systems and should not be used. Successful mycorrhization occurred in the greenhouse with certain fungi and for particular methods. Therefore, this research was effective in initiating this avenue of research. Next would be trials to refine methods for consistent and reliable mycorrhizal colonization on a larger scale.

Monitoring since completion of the project

Subsequent monitoring does not apply to this greenhouse project which was destructively measured and analyzed.

Dates– N/A

Plans for future monitoring? –N/A

Will outcome meet goals?

Yes, the outcomes did meet the goals of the project (see review summary in the report)

Future actions/follow up?

Future trials were based on results of these experiments (see subsequent reports and summary below).

Miscellaneous comments

A summary of the research is included. The paper is attached separately.