Inoculation of whitebark pine with native mycorrhizal fungiA strategy for increasing out-planting success?

Cathy L. Cripps & Paul TrustyDepartment of Plant Sciences and Plant Pathology, Montana State University, Bozeman, MT

1. MYCORRHIZAE 2. DISCOVERY 3. MONITORING 4. APPLICATION-awaiting results5. EUROPEAN METHOD 6. BIOASSAY 7. FINAL THOUGHTS

USDA FS 2008 Whitebark Pine Restoration Project

Note: this presentation has been abbreviated for the Progress Report

Background info

Current project

Slides removed include:

Threats to whitebark pine - Schwandt, J. 2006. Whitebark pine in peril. USDA FS R1-06-28.

Restoration of whitebark pine - Tomback, Arno & Keane 2001. Whitebark pine communities: Ecology and Restoration

• development of seed germination methods (Burr et al. 2001)

• nursery production of whitebark pine seedlings (Burr et al. 2001)

• selection of rust resistant strains (Mahalovich et al 2004)

• research on seedling diseases (Dumroese 2008)

• planting over 200,000 nursery seedlings (Izlar 2008)

• use of burned sites (openings, reduced competition, for out-plantings) (Keane et al 2000)

There was no information on the native mycorrhizal fungi with whitebark pine and the potential for inoculation of nursery seedlings prior to our reseach

What are Mycorrhizae?

No fruiting body, just sporesMushrooms, “truffles”, etc

Glomeromycota: AM FungiPrimarily Basidiomycota

Fungi: 150 speciesFungi: 6000 species

Forbs, grasses, some woody plantsMost trees, some shrubs

ARBUSCULAR MYCORRHIZAEECTOMYCORRHIZAE

2 MAIN TYPES

A mutualistic relationship between certain fungi and plant roots

“beneficial to both”

Benefits to fungus

get sugars from the plant

Potential benefits to plant host

enhanced phosphorus uptake

improved access to nitrogen

protection from

drought

soil pathogens/grazers

heavy metals

Mycorrhizal fungi can also

aggregate soil

provide links to other plantsImproved health & survival

Cripps, CL 2002. Mycorrhiza. In: Pscheidt & Ocamb, Pacfic NW Plant Disease Management Handbook. Brundrett et al. 2007.

ectomycorrhizae

Mycelium in soil

Fruiting body

PINES CANNOT SURVIVE IN NATURE WITHOUT APPROPRIATE MYCORRHIZAL FUNGIWebsite: Whitebark Pine Ecosystem Foundation, Ecology, Mycorrhizae http://www.whitebarkfound.org/

ARE MYCORRHIZAL FUNGI IMPORTANT IN WHITEBARK PINE SYSTEMS?GOALS OF OUR RESEARCH PROGRAM

“MYCORRHIZAL FUNGI OF WHITEBARK PINE”

1. DISCOVERY (Who are they?)GREATER YELLOWSTONE ECOSYSTEM

WATERTON LAKES NATIONAL PARK

GLACIER NATIONAL PARK

2. MONITORING (Where are they?)DUNRAVEN PASS, YNP

FRIDLEY BURN, GALLATIN NATIONAL FOREST, MT

3. APPLICATION (Are they useful in restoration?)INOCULATION OF NURSERY SEEDLINGS

Cathy L Cripps, PhD, Montana State University

Paul Trusty, graduate student, MSU

Kate Mohatt, graduate student, MSU & USDA Forest Service, Girdwood Alaska

Ben Johnson, undergraduate, MSU

Don Bachman, freelance field assistant

Cooperators: John Schwandt, USDA FS Forest Health Protection, Coeur D’AleneMary Hecktner, YNP Resource Manager, Yellowstone National Park Dan Reinhart, Resource specialist, YNP & Kay Izlar, U of MBob Keane, USFS Fire Ecologist, Missoula Fire Office Julie Shea, USFS Fire Officer Gallatin National Forest, MTStan Cook, USFS Silviculturist, Gallatin National Forest, MTCyndi Smith, Parks Canada Ecologist, Waterton Lakes National ParkJoyce Lapp, Park Service Silviculturist, Glacier National ParkKay Izlar, graduate student, University of Montana

Seedling planted on Dunraven Pass, YNP

1. DISCOVERYof the native mycorrhizal fungi with white bark pine

Mohatt, Cripps & Lavin (in ed) Ectomycorrhizal fungi of whitebark pine (a tree in peril) revealed by sporocarps and molecular analysis of mycorrhizae from treeline forests in the Greater Yellowstone Ecosystem. Can. J. Bot. 86(1): 14-25. Cripps & Mohatt 2005. Preliminary results on the Ectomycorrhizal Fungi of Whitebark Pine Forests. Nutcracker Notes 7: 9-11.

BASIDIOMYCOTAAMANITACEAE Amanita "alpina"HYGROPHORACEAEHygrophorus gliocyclusHygrophorus marzuolus (Fr.)Hygrophorus olivaceoalbusHygrophorus subalpinusTRICHOLOMATACEAELeucopaxillus paradoxisTricholoma moseriCORTINARIACEAECortinarius clandestinusCortinarius duracinusCortinarius “flavobasalis”Cortinarius “flavoroseus”Cortinarius aff. fulminoidesCortinarius subolivescensCortinarius sp.Dermocybe crocea (Schff.) Mos.Inocybe sp. RUSSULALESLactarius deliciosusRussula cf tortulosa /queletiiRussula sp. 2, Russula sp. 3

BOLETALESBoletus edulisChroogomphus sp. nov. Rhizopogon cf milleriRhizopogon cf evadensRhizopogon spp. Suillus subalpinusSuillus sibiricusSuillus tomentosus var. discolorSuillus sp.THELEPHORALESPseudotomentella nigraTomentelloid type 1Tomentelloid type 2Thelephora sp.ASCOMYCOTACenococcum geophilumWilcoxina remhii & mikolaeOTHERAmphenima byssoidesPiloderma sp.

40+ species of ECM fungi confirmed with whitebark pine by fruiting bodies or ectomycorrhizae on roots

Unculturable, shared with other conifers

OLDER TREES

Unculturable, some may be specific for pines or shared with other conifers

MOSTLY OLDER TREES

SUILLOIDS – mostly specific for pines, 5-needle pines & stone pines

SEEDLINGS, YOUNGER TREES, & OLDER TREES

Waterton Lakes National ParkGlacier National Park

Yellowstone National Park

New World District, Gravelly Mountains, Sacajawea Peak, Big Sky Ski Area, Golden Trout Lakes, Dunraven Pass, Waterton Park, Glacier Park, Gallatin County (Fridley Burn), Yellowstone National Park

GENERALISTS – with many hosts

SEEDLINGS, YOUNGER TREES, & OLDER TREES

Suillus sibericus – stone pinesSuillus subalpinus – 5-needle pine/stone pines

Cripps photos

Moser 2004. In Cripps: Fungi in Forest Ecosystems, NYBG Press. (fungi with 5-needle pines in Alps, Altai, Rocky Mts)

Fruiting bodies of Suilloids

Rhizopogon milleri & R. evadens(pine/5-needle/stone pine)

UNDERGROUND “POGIES”

These fungi are eaten by squirrels, deer, elk & bears which spread the spores

Czares & Trappe 1994. Spore dispersal of ectomycorrhizal fungi by mammal mycophagy. Mycologia 86NPS PHOTO

Ectomycorrhizae of Whitebark pine on Roots

E-strain fungus Suillus species

PseudotomentellaRhizopogon species Suilloid type

Unknown type

Cripps photos: native fungi from Greater Yellowstone Ecosystem, Waterton Lakes-Glacier International Peace Park

2. MONITORINGthe mycorrhizal status of planted white bark pine seedlings & how might restoration strategies affect their presence?

Project 1: Whitebark Pine Restoration, Dunraven Pass Yellowstone National Park

Project 2: Post-fire Restoration Treatments on Fridley Burn, Gallatin NF, MT: seedlings planted in burn adjacent to unburned mature whitebark pine forest

• 96% of root tips were mycorrhizal after 5 years (some nursery fungi persisted)• Suilloids were present on planted seedlings in the burn at low levels • A lower diversity of mycorrhizal fungi existed on the burn• A “shift” in mycorrhizal taxa after the burn on planted & natural seedlings• Functional significance of shift is unknown at this point

Project 3: Waterton Lakes National Park, occurrence of mycorrhizae in microhabitats

“ideal” restoration conditions for fungal colonization

• a higher diversity of ECM fungi on seedlings on nurse trees• lowest on seedlings in Beargrass

• Mycorrhizal colonization only on healthy (not on compromised) seedlings• No colonization of seedlings on south side of pass, only north side (mature forests) • Colonization levels (mostly suilloids) were very low (< 5% of roots) after 1 year

Cripps, CL, Smith, C, Lapp, J. and T. Carolin 2008. Assessment of Ectomycorrhizal Fungi with Whitebark Pine: Waterton-Glacier International Peace Park. Nutcracker Notes, 14: 12-14.

3. APPLICATIONInoculation of whitebark pine with native mycorrhizal fungi:A beneficial strategy for increasing seedling survival on out-planting?

Spore slurry

Mycelium slurry

Root to root inoculation

Soil inoculum

or from roots

Liquid inoculum

Soil inoculum

MSU WHITEBARK PINE CULTURES

Ground fruiting bodies

Spore slurries

Landis et al. 1990. The Container Tree Nursery Manual Vol. 5: Nursery Pests and Mycorrhizae. USDA FS Agriculture Handbook 674.

+SP. albicaulis?grizzly scat YellowstoneRhizopogon spWashS?P. albicaulissporocarpNew WorldHypogeous 2NW Hyp 2S?P. albicaulissporocarpNew WorldHypogeous 1NW hyp 1

+SP. albicaulissporocarpNew WorldThaxterogasterCLC 2282 +SP. albicaulissporocarpNew WorldR. olivaceofusca 4,5 CLC 2281a +SP. albicaulissporocarpsYellowstoneR. sp. (yellow) (R3)CLC 2280b +SP. albicaulissporocarpYellowstoneR. cf molligleba(R2)CLC 2280a +SP. albicaulissporocarpYellowstoneR. cf evadens(Rz 1)CLC 2279 +SP. albicaulissporocarpBeartoothsR. subpurpureusCLC 2277 +SP. albicaulissporocarpBeartoothsS. sibericusCLC 2275 ++M +coniferrootsEastern USCenococcumVT

MP. albicaulissporocarpYellowstoneS. subalpinusCLC 2247c

M -Mixed conifersporocarpYellowstoneS. cf. brevipesCLC 2246 MP. albicaulissporocarpNew WorldS. sibericus (thin)CLC 2245b

++M +P. albicaulissporocarpYellowstoneS. sibericus (thick)CLC 2245a ++M+P. albicaulissporocarpNew WorldS. variegatusCLC 2244 ++ M+P. albicaulissporocarpNew WorldS. subalpinusCLC 2241 ++M+P. flexilissporocarpYellowstoneR. subbadiusCLC 2294 ++M+P. albi & cont.sporocarpYellowstoneSuillus sp. (veil)CLC 2199

MP. albicaulisSoil, nativeFridley BurnR. sp (Ben) UB 7MP. flexilisrootsGlacier ParkR. sp. *GDP 1MP. flexilissporocarpWaterton ParkR. cf salebrosusHyp 1M -P. contortasporocarpWaterton ParkR. cf rubescensRhiz 1wM -P. albicaulissporocarpNew WorldTricholoma moseriWO 81.1MP. albicaulissporocarpNew WorldR. spCLC 2036

++M P. albicaulissporocarpNew WorldRhizopogon subpurCLC 2035

Seedlings inoculated

Soil & liquid

Myceliumor slurry

hostIsolated fromSource locationSpeciesNo.

SCREENING ISOLATES OF NATIVE ECTOMYCORRHIZAL FUNGI FROM WHITEBARK PINE

INOCULATION OF PINE SEEDLINGS FOR EVALUATION TRIALS

ADDING SPORE SLURRY with pippette

ADDING SOIL INOCULUM

Steinfeld, Amaranthus and Cazares 2003. Survival of Ponderosa pine seedlings outplanted with Rhizopogon mycorrhizae inoculated with spores at the nursery. Journal of Arboriculture 29(4): 197-208.

Rincon, Paride and Pera 2005. Effects of ectomycorrhizal inoculation and the types of substrate for mycorrhization, growth and nutrition of containerized Pinus pinea seedlings produced in a commercial nursery. Ann. For. Sci. 62: 817-822.

500 rust resistant WHITEBARK PINE SEEDLINGS/SEEDS Forest Nursery Coeur d’Alene ID

PLANT GROWTH CENTER, MSU

Mahony, C 2004. Effects of native ectomycorrhizal fungi on aspen seedlings in greenhouse studies: inoculation methods, fertilizer regimes, and plant uptake of selected elements in smelter-impacted soils. MSU, M.S. Thesis (Cripps Lab).

• rust resistant seedlings styrofoam blocks conetainersage of seedlings at inoculation

• species/isolates of fungiscreeningspore slurries (fresh)spore slurries (aged)mycelium liquidsoil inoculum+/- fertilizer

• inoculation in the nurserybefore/after transplanting to larger pots

• transplant potting media types peat sawdust soil types suppressive soils?

Awaiting results!

SEEDS PLANTED DIRECTLY INTO CONETAINERS

EXPERIMENTAL DESIGN --variables

Rincon A, Paride, Ja and J Pera 2005. Effects of ectomycorrhizal inoculation and the types of substrate for mycorrhization, growth and nutrition of containerized Pinus pinea seedlings produced in a commercial nursery. Ann. For. Sci. 62: 817-822.

Visit to Federal Institute Nursery near Innsbruck, Austria, 2008

Bachman photo

Pinus cembraEuropean stone pines

HISTORY of INOCULATION

• MEINHARD MOSER (1960’s) inoculates stone pines (Pinus cembra)

• increased survival of stone pines seedling from 20% to 80 % at high elevations

• biodegradable pots help in transfer of ectomycorrhizal fungi

• nursery maintained Moser’s fungi since that time (over 50 years!)

• Moser’s great-great-grand students use molecular techniques to identify the mycorrhizal fungi on nursry roots and discover the original species!

Dr Weisleitner, head forester European stone pines in pots

BIODEGRADABLE POTS WITH ROOTS GROWING OUTHEAVILY MYCORRHIZAL ROOT SYSTEM!

Bachman photo

HOLDER FOR BIODEGRADABLE CONTAINERSBIODEGRADABLE POTS BREAKING DOWN

Cripps photos

HIGHLY SUCCESSFUL MYCORRHIZAL COLONIZATION ON POTTED PINES!

Bachman photo

Colonization of Pinus cembra (European stone pine) by native Suilloid fungi

Molecular (DNA) techniques have identified these fungi as native suilloid fungi specific for stone pines!

Rhizopogon & Suillus species

Coralloid ectomycorrhizae: important in N acquisition

The bio-containers cleared US Customs & arrived safely from Germany

Our new horticulture post doc from Germany Dr. Eva Grimme will join the lab in November.

Our goal is to adapt the European system to the USA system for growing whitebark pines

Inoculation of seedlings with native ECM can increase seedling survival and fitness (Smith 1998)

Inoculation of European stone pines increased survival from 20% to 80% (Moser 1960’s)Inoculation is necessary on sites where mycorrhizal fungi are lacking (Wiensczyk et al 2002, J. Ecosystem & Management 2: 1-20).

Brundrett et al. 1996. Working with Mycorrhizas in Agriculture. CSIRO.

3. APPLICATIONFLOW CHART FOR DETERMINING WHEN INOCULATION IS NECESSARY

HOW CAN WE OPTIMIZE THE SYSTEM?

DO PLANTED PINES HAVE THE “RIGHT STUFF”ON THEIR ROOTS FOR SURVIVAL?

1. MONITORING TO DETERMINE WHERE FUNGI ARE LACKING ON SEEDLINGS

2. BIOASSAY TO PRE-DETERMINE THE PRESENCE OF NATIVE SUILLOID FUNGI IN THE SOIL --before large out-plantings

• Bioassay 1: native and replaced soils along Dunraven Pass

--suilloid fungi were found in native & replaced soil --nursery fungi (E strain) were found to persist, but not prevent suilloids--few other mycorrhizal fungi were discovered

BIOASSAY (in development stage):

--gather soil from test site --plant seedlings/seeds of whitebark pine in native soil in pots --incubate under greenhouse conditions --assay seedling roots to determine fungi present in soil

• Bioassay 2: burned and undburned soil (Fridley Burn, Gallatin National Forest)

--suilloid fungi found in burned and unburned soils --results don’t reflect the diversity of fungi present (suilloid bias)

Bioassays can determine if appropriate suilloids are present!

FUTURE PLANS

--adapt the European system of mycorrhizal inoculation to that of the current USA system for production of whitebark pine seedlings

SOME FINAL THOUGHTS

COMMERICAL MYCORRHIZAL INOCULUM SHOULD NOT BE USED IN SENSITIVE WHITEBARK PINE SYSTEMS

--they do not contain the appropriate species for whitebark pine --they may promote other competitive tree species--they may contain EXOTIC fungi (especially for National Parks) --local mammals may depend on particular species of fungi for food (odors)

NURSERY FUNGI SUCH AS E-STRAIN AND THELEPHORA WERE FOUND IN LOW NUMBERS ON NURSERY WHITEBARK PINE SEEDLINGS

--presently they are not considered a problem in nursery seedlings (more research needed)

We would like to thank the USDA Forest Service & panel for the Whitebark Pine 2008 Restoration Projects that provided funding for this project. Special thanks goes to Dr John Schwandt (Forest Health Protection) and Dr. Kent Eggleston and crew at the Coeur d’Alene Nursery) for their help with this research.

Cripps photo