2013 Inoculation of WBP Seedlings with Native Ectomychorrhizal Fungi
Project: Inoculation of WBP Seedlings with Native Ectomycorrhizal Fungi
Attachment: MSU_Cripps_2013_ Lonergan Paper
Agency/Forest or Park/District: MSU Plant Growth Center, Bozeman, Montana
Project coordinator: Dr. Cathy L. Cripps
Contact: Dr. Cathy L. Cripps, Plant Sciences & Plant Pathology Dept., Montana State University, Bozeman, MT 59717, 406-994-5226, ccripps@montana.edu
Cooperators
Dr. Bill Hoch, horticulture specialist, assistant professor, Montana State University.
Dr. John Schwandt, USDA FS Forest Health Protection, Coeur D’Alene, Idaho
Source of funding /amount
FHP: $6,500
Supplemental funding: $3,600 from MSU, Cripps in kind salary and greenhouse rental from other projects contributes to rent.
Dates of restoration efforts
2013-2014
Objectives
1. Examine additional fertilizer regimes conducive to mycorrhizal colonization (by native fungi) of whitebark pine seedlings in the greenhouse.
2. Examine how different container types affect mycorrhization and seedling root development in the greenhouse and root exploration on out-planting.
3. Briefly examine if root preparation (snipping) enhances root exploration on out-planting.
4. Examine the viability of spore slurries of native ectomycorrhizal fungi specific to whitebark pine to determine shelf life of the inoculum.
5. Provide slurries of native ectomycorrhizal fungi for various projects and help in monitoring.
Acres/ha treated N/A
Methods
Objective 1: Seedlings grown from seed (with warm stratification) and (left-over) seedlings from the Forest Nursery in Idaho will be inoculated with slurries of native ectomycorrhizal fungi in the greenhouse at MSU. After vernalization, fertilizer (with a form of N accessible only to the fungus) will be applied to some seedlings and not to controls. Assessment will determine if this fertilizer type is conducive to mycorrhizal colonization, so that both inoculation and fertilization can be applied.
Objective 2&3: Small experiments using ‘extra seedlings’ will determine how root systems form in various containers. Seedlings will be transplanted into various pot types and root-trainer pots and inoculated with native ectomycorrhizal fungi. Root formation will be assessed visually for shape and quantitatively for biomass and volume. Then seedlings will be put in a glass-sided viewers to observe how roots spread out into native soils. For objective 3, seedlings grown in cone-tainers will also be planted in glass-sided viewers and some will first be ‘snipped’ at the base to remove compacted roots. These will also be visually assessed for effective root expansion into the soil.
Objective 4: Spore slurries of various suilloid fungi of different ages and from fresh and dried sporocarps will be tested for viability using various techniques (Trypan blue, dapi-staining and epi-fluorescence). The proportion of viable spores will be assessed using a hemocytometer to count viable versus dead spores. All methods have implicit ‘assumptions’ that may/may not be true for suilloid spores and methods will be compared for general trends that can be easily used in applied research.
Objective 5: Fruiting bodies of ectomycorrhizal fungi will continue to be collected each fall. Slurries will be ‘stock-piled’ for use in our research and will also be available for other restoration purposes.
Planting? If so, source of seedlings? Resistance?
No, this is currently a greenhouse study; seedlings it will not be outplanted at the end of the experiment. We are using a variety of ‘extra’ seedlings from lots obtained from the Idaho nursery [TOMBEAL09, WB03030092, SURPRISE 10, FREEZOUT 10, LITTLEJOE 10, BIGMTN 11]. We assume that these have been screened or are being screened for rust resistance. We would check on this if they are eventually to be out-planted.
Outcome
The project is not complete yet. Although we have completed most of the objectives, we still have one experiment going in the greenhouse with the older seedlings. We still need to analyze results from some experiments. We still hope to be able to out-plant our inoculated seedlings in a test plot. Except for this last objective, we should be able to complete everything by the final report date in May.
1. In the 1st experiment we addressed nitrogen levels in fertilization: We learned that a low nitrogen fertilizer is amenable to mycorrhizal colonization, although colonization is still lower than on un-inoculated control seedlings.
2. In the 2nd experiment which is completed but not yet completely analyzed, we addressed the use of different types of phosphorus in fertilization: a low P:N ratio, a low N with phytic acid, a balanced N:P ration, and low N with rock phosphate. Colonization of the older seedlings (used of necessity because they were all we had at the time) will not yield usable results. Colonization of young seedlings (from seeds we stratified and germinated) revealed again that a low N fertilizer was conducive to mycorrhizal colonization, but colonization was low with low N + phytic acid and also with a balanced N:P regime. However, of course, seedling height and biomass were increased with all fertilizer types. Interestingly, pH appeared to remain lower (around 5.5) with fertilization and climbed to 6.0 without fertilization. We do know our water supply has a high pH. Graphs are at the end of this document, the experiment is not yet completely analyzed.
3. The 3rd experiment is ongoing, and is examining inoculation of older seedlings, with the goal of monitoring how fast ectomycorrhizal colonization occurs in the greenhouse so we know how long the process takes just before out-planting. NOT ANALYZED YET
4. We learned that long containers (3.8 x 21 cm) were more conducive to mycorrhizal colonization than short containers (3.8 x 14 cm), but intermediate length containers should be tested.
5. We learned that ectomycorrhizal spore slurries could be made from dried sporocarps, but that there is a lag time for colonization compared to slurry made from fresh sporocarps, and also vernalization might be necessary for the spores in the ‘dried slurry’ to germinate.
6. We learned that the method of ‘dripping’ spores on to roots with a pipette and the method of injecting spores into the soil with an injection device were equally effective but the latter is much more time efficient and is less likely to spread disease.
7. We tested to see if roots snipped lengthwise or crosswise would spread out into soil more rapidly. But results from the small experiment were inconclusive. Roots in all treatments did spread out into the soil, including those of the control. This may/may not result from sufficient watering and warmth in the greenhouse. It has been our experience that roots of out-planted seedlings stay bound in the pot for a substantial length of time as observed at the Yellowstone Club planting site.
Monitoring since completion of the project N/A
Dates
Plans for future monitoring?
Will outcome meet goals?
Yes, except perhaps for the last one concerning out-planting which we will not be able to do. Also data may be limited for the spore viability tests which are also ongoing.
Future actions/follow up?
Yes, we do plan to continue this avenue of research, and in fact results from this research were used to determine the method of inoculation for whitebark pine seedlings that were planted in Waterton Lakes National Park and Glacier National Park. Seedlings in WLNP were monitored for 2 years and information will be available in a recently accepted paper.
Miscellaneous comments
APPENDIX
Results:
1. In the 1st experiment we addressed nitrogen levels in fertilization: We learned that a low nitrogen fertilizer is amenable to mycorrhizal colonization, although colonization is still lower than on un-inoculated control seedlings.
2. In the 2nd experiment which is completed but not yet analyzed, we addressed the use of different types of phosphorus in fertilization: a low P:N ratio, a low N with phytic acid, a balanced N:P ration, and low N with rock phosphate. Colonization of the older seedlings (used of necessity because they were all we had at the time) will not yield usable results. Colonization of young seedlings (from seeds we stratified and germinated) revealed again that a low N fertilizer was conducive to mycorrhizal colonization, but colonization was low with low N + phytic acid and also with a balanced N:P regime. However, of course, seedling height and biomass were increased with all fertilizer types. Interestingly, pH appeared to remain lower (around 5.5) with fertilization and climbed to 6.0 without fertilization. We do know our water supply has a high pH.
3. The 3rd experiment is ongoing, and is examining inoculation of older seedlings, with the goal of monitoring how fast ectomycorrhizal colonization occurs in the greenhouse so we know how long the process takes just before out-planting.
4. We learned that long containers (3.8 x 21 cm) were more conducive to mycorrhizal colonization than short containers (3.8 x 14 cm), but intermediate length containers should be tested.
5. We learned that ectomycorrhizal spore slurries could be made from dried sporocarps, but that there is a lag time for colonization compared to slurry made from fresh sporocarps, and also vernalization might be necessary for the spores in the ‘dried slurry’ to germinate.
6. We learned that the method of ‘dripping’ spores on to roots with a pipette and the method of injecting spores into the soil with an injection device were equally effective but the latter is much more time efficient and is less likely to spread disease.
7. We tested to see if roots snipped lengthwise or crosswise would spread out into soil more rapidly. But results from the small experiment were inconclusive. Roots in all treatments did spread out into the soil, including those of the control. This may/may not result from sufficient watering and warmth in the greenhouse. It has been our experience that roots of out-planted seedlings stay bound in the pot for a substantial length of time as observed at the Yellowstone Club planting site.
PAPER ATTACHED SEPARATELY
Research from the WPEF was followed up by a big study in the International Peace Park funded by Parks Canada and Glacier National Park. Although this research was not funded by WPEF, we include the paper here since it was made possible by the WPEF grants for the preliminary research i.e. determining the fungi to used, the inoculation method, the soil substrate, and the fertilization method.
Lonergan, E., Cripps, CL and C Smith (accepted). Influence of site conditions, shelter objects and ectomycorrhizal inoculation on the early survival of whitebark pine seedlings planted in Waterton Lakes National Park. Forest Science (slated for 2014).
Inoculated seedlings increased survival 17-25% for seedlings planted in/near beargrass on unburned sites in WLNP.