MONITORING MYCORRHIZAL FUNGI ON PLANTED WHITEBARK PINES IN THE GYEDO PLANTED PINES HAVE THE “RIGHT STUFF” ON THEIR ROOTS FOR SURVIVAL?

GOALS OF OUR RESEARCH PROGRAM

“MYCORRHIZAL FUNGI OF WHITEBARK PINE”

1. DISCOVERY - ongoing

2. MONITORING – today’s topic

WHAT ARE MYCORRHIZAL FUNGI?

DUNRAVEN PASS, YNP

FRIDLEY BURN, GALLATIN NATIONAL FOREST, MT

3. APPLICATION - the future

Cathy L Cripps, PhD, Montana State University

Paul Trusty, graduate student, MSU

Kate Mohatt, graduate student, MSU

Ben Johnson, undergraduate, MSU

Don Bachman, freelance field assistant

Cooperators: Mary 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 Park

Seedling planted along Dunraven Pass

•White bark pine blister rust

•Mountain pine beetle

•Fire exclusion

•Climate change

THREATS

WHITE BARK PINE FORESTS ARE SERIOUSLY DECLINING

Tomback, Arno & Keane 2001

Significant restoration efforts have been ongoing for the last 15 years, yet no one has addressed mycorrhizal fungi and whitebark pine!

WHAT ARE MYCORRHIZAE?ECTOMYCORRHIZAE ARBUSCULAR MYCORRHIZAE

Most trees, some shrubs Forbs, grasses, some woody plants

Fungi: 6000 species Fungi: 150 species

Primarily Basidiomycota Glomeromycota: AM Fungi

Mushrooms, “truffles” No fruiting body, just spores

2 MAIN TYPES

A mutualistic relationship between certain fungi and plant roots

“beneficial to both”

mycelium ectomycorrhizae

Fruiting bodies

spores Benefits to fungus

get sugars from the plant i.e. Food!

Potential benefits to plant

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 plants

Brundrett et al. 2007 website

ARE MYCORRHIZAL FUNGI IMPORTANT IN WHITEBARK PINE SYSTEMS?

• PINES CANNOT SURVIVE IN NATURE WITHOUT MYCORRHIZAL FUNGI

• PINES ASSOCIATE WITH ONLY A SUBSET OF THE 6,000 SPECIES OF ECM FUNGI

• SOME ECM FUNGI ARE SPECIFIC FOR: PINES, 5-NEEDLE PINES, OR EVEN STONE PINES

• SOME ECM FUNGI ASSOCIATE WITH MANY TREE SPECIES (GENERALISTS)

• TYPICALLY ONE TREE HOSTS MANY SPECIES OF ECM FUNGI

ECM FUNGI ARE NOT ALL THE SAME, EACH PROVIDES UNIQUE BENEFITS TO THE TREE

ECM FUNGI ARE CRUCIAL TO ESTABLISHMENT, SURVIVAL, SUSTAINABILITY OF WHITEBARK PINE!

Cripps 2001 Encyclopedia of Plant Pathology; Smith and Read 1998. Mycorrhizal Symbiosis

YES! But which ones?

HOW DO WE STUDY ECTOMYCORRHIZAL FUNGI?

1) Identification of ectomycorrhizal fruiting bodies in pure whitebark pine stands (& DNA analysis)

2) Sampling ectomycorrhizae on roots (DNA analysis of ITS region)

Matching DNA to that of sporocarps or Genbank Library for identification

Fruiting bodies Ectomycorrhizae on roots

ITS-1

DNA

Amanita alpina Courtesy CSIRO

TO IDENTIFY FUNGI ON ROOTS & CONFIRM ASSOCIATION

Gardes & Bruns 1993. ITS primers with enhanced specificity for basidiomycetes-application for the identification of mycorrhizae and rusts. Mol. Ecol. 2: 113-118.

1. DISCOVERYWHICH MYCORRHIZAL FUNGI ASSOCIATE WITH WHITE BARK PINE IN THE GREATER YELLOWSTONE ECOSYSTEM & THE SURROUNDING REGION?

New World District, Gravelly Mountains, Sacajawea Peak, Big Sky Ski Area, Golden Trout Lakes, Dunraven Pass, Waterton Park, Glacier Park

Cripps & Mohatt 2005 (Nutcracker Notes), Mohatt 2006 (MSU Thesis)

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. (coming soon!)

BASIDIOMYCOTAAMANITACEAE Amanita "alpina" HYGROPHORACEAEHygrophorus gliocyclus Hygrophorus marzuolus (Fr.)Hygrophorus olivaceoalbus Hygrophorus subalpinus TRICHOLOMATACEAELeucopaxillus paradoxis Tricholoma moseri CORTINARIACEAECortinarius clandestinus Cortinarius duracinus Cortinarius “flavobasalis” Cortinarius “flavoroseus”Cortinarius aff. fulminoidesCortinarius subolivescens Cortinarius sp.Dermocybe crocea (Schff.) Mos.Inocybe sp. RUSSULALESLactarius deliciosusRussula cf tortulosa /queletiiRussula sp. 2, Russula sp. 3

BOLETALESBoletus edulis Chroogomphus sp. nov. Rhizopogon cf milleriRhizopogon cf evadensRhizopogon spp. Suillus subalpinusSuillus sibiricus Suillus tomentosus var. discolorSuillus sp.PHALLALES - GOMPHALESHysterangium separabile THELEPHORALESTomentelloid type 1Tomentelloid type 2

ASCOMYCOTACenococcum geophilum

32 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

Generalist, important in dry conditions for relations

SEEDLINGS UNDER CANOPY & OLDER TREES

Waterton Lakes National ParkGlacier National Park

Yellowstone National Park

Suilloids–show host specificity

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

Chroogomphus sp nov (new species)- whitebark pine

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

Cripps photos

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

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

UNDERGROUND MYCORRHIZAL FUNGI COMMON IN THE SYSTEM

pinepine

pine-spruce-fir Peaco photo, YNP

Rhizopogon

Gautieria

“POGIES”

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

Ashkannejhad & Horton 2006. Ectomycorrhizal ecology on coastal dunes: interactions involving Pinus contorta, Suilloid fungi and deer. New Phytol. 169:345-354.

Mattson et al. 2002. Consumption of sporocarps by Yellowstone grizzly bears. Ursus 13:95-103. Cripps photos

Spore dispersal by deer

“POGIES”

DUNRAVEN PASS, YNP

2. MONITORINGProject 1: Whitebark Pine Restoration, Dunraven Pass Yellowstone National Park: Monitoring the mycorrhizal status of planted whitebark pine seedlings.

Greater Yellowstone Coordinating Committee & Rocky Mountain Research Station funding. Cooperators: Mary Hecktner Resource manager YNP, Dan Reinhart YNP, and Kay Izlar, U of M.

UMT E UMT N elevation aspect slope vegetation burned

1 543253 4959160 8900 ft W 300 50% -

2 543225 4959232 8885 ft flat flat 5%, disturbed, bare -

3 543239 4959241 8880 ft W 400 20%, rocky, duff, bare mix -

4 543130 4957295 8583 ft SE 250 50%, grasses, rocky, stable -

5 543172 4957556 8600 ft ESE 150 30%, disturbed, pioneer -

6 543884 4961573 8897 ft NNW 200 30%, brome, lupine, rocky +

7 543940 4961640 8844 ft WNW 300 10%, unstable, bare rocks +

8 543467 4962733 8667 ft S 5-250 25%, bare soil, small rocks -

9 544060 4961780 8880 ft W+E? 0-100 10-90%, native grass/bare -

11 543322 4960009 8873 ft NW 5-20% 10%, some overstory -

• Sept. 11-13 2006 whitebark pine seedlings planted along Dunraven Pass

• Sept. 11-13 2006 Kay Izlar (U of M) set up 10 plots along the pass

• Sept 14: We selected 10 of nursery seedlings from this batch to check roots for nursery fungi

• June 2006 Izlar measured % survival on 10 plots

• June 2007 We sampled roots of seedlings near/on each plot (after 9 months)

10 sites where whitebark pine seedlings were planted & monitored (Izlar data)

WHAT IS THE MYCORRHIZAL CONDITION OF NURSERY SEEDLINGS BEFORE OUT- PLANTING?

Thelephora sp – typical in nurseries E-strain fungus – typical in nurseries

FUNGI ON NURSERY SEEDLINGS? BENEFIT OR CONCERN?

•98% non-mycorrhizal roots for sample of 10 nursery seedlings from Coeur D'Alene nursery

• 2% nursery fungi on roots : Thelephora & E-strain

• exotic non-native fungi (benefit or concern?)

• do they persist in soil after out-planting and spread?

• evidence of fungal root pathogens

Root hairs = non-mycorrhizal

OBJECTIVES: To determine

•if planted seedlings are effectively colonized by native fungi [ON-SITE MONITORING]

•inoculum potential of replaced soil [GREENHOUSE BIOASSAY]

•if imported nursery fungi persist on planted seedlings [MOLECULAR TOOLS]

Cripps photos

sites Healthy seedlings

Compromised seedlings

% Survival

Habitat notes Vegetation

1. Top Pass – 8900’, W, 300 15 0 87% slope behind visitor area to north 50%

2. Top Pass – 8885’, flat 32 0 96% disturbed area adjacent to parking lot 5%, disturbed

3. Top Pass – 8880’, W, 400 8 0 81% slope behind visitor area to south 20%, rocky

4. S Pass – 8583’, SE, 250 0 0 84% disturbed area adjacent to parking lot 50%, grasses

5. S Pass – 8600’, ESE, 150 0 0 58%open slope adjacent to distant conifer forest 30%, disturbed

6. N Pass – 8897’, NNW, 200, burned 670 0 43% burned in 1988, previously mixed conifer 30%, brome

7. N Pass –8844’, WNW, 300, burned 136 0 100% burned in 1988, previously mixed conifer 10%, rocky

8. N Pass – 8667’, S, 5-250 0 0 30% windy, exposed, no trees 25%, rocky

9. N Pass – 8880’, W+E, 0-100 117 69 94% Unburned below mature whitebark pine 10-90% grass

11. N Pass – 8873’, NW 5-200 14 0 44% unburned whitebark pine, spruce-fir 10%, overstory

Total 992 69

Average/tree 99.2 6.9

Number of mycorrhizal root tips per seedling sampled on each siteIzlar data Izlar notes

• No (little) mycorrhizal colonization on compromised seedlings

• Native fungi were colonizing roots on sites 1, 6, 7, 9 and 11 (50% of sites). Suilloids

• No colonization of sites 4, 5, 8 (S & SE aspect).

• Only nursery fungi were present on sites 2 and 3.

Number of viable mycorrhizal root tips for healthy seedlings by depth

sites 0-4 cm 4-8 cm 8-12 cm 12-16 cm % mycorrhizal

1. Top of Pass 0 0 15 0 < 1%

2. Top of Pass 0 0 5 27 < 1%

3. Top of Pass 0 8 0 0 < 1%

4. S of Pass 0 0 0 0 0%

5. S of Pass 0 0 0 0 0%

6. N of Pass 65 312 245 48 < 30%

7. N of Pass 76 12 28 20 < 5%

8. N of Pass 0 0 0 0 0%

9. N of Pass 0 82 15 10 < 5%

11. N of Pass 0 14 0 0 < 1%

Total 141 428 308 105 44

Average 14.1 42.8 30.8 10.5 4.4%

• Most seedlings were not well-colonized after 9 months

• A majority of mycorrhizae were 4-12 cm deep.

Percent mycorrhizal colonization of root tips and depth of ectomycorrhizae on cone-tainerized roots systems

Mycorrhizal colonization of cone-tainerized roots

Cripps photos

Kay Izlar’s “star performer”: seedling #1, site 6 Collected below a mature whitebark pine

NATIVE ECTOMYCORRHIZAE ON WHITEBARK PINE SEEDLINGS PLANTED ALONG DUNRAVEN PASS (sampled 9 months after planting)

Suilloid type Rhizopogon

Cenococcum & sclerotia Piloderma

A DIVERSITY OF NATIVE SUILLOIDS WERE PRESENT, ALONG WITH OTHER NATIVE ECTOMYCORRHIZAL FUNGI (Rhizopogon species, Suillus species).

Molecular identifications by Paul Trusty

Cripps photos

METHOD: SOIL BIOASSAY IN GREENHOUSE

T1: REPLACED SOIL, UNSTERILIZED

T2: NATIVE WBP FOREST SOIL, UNSTERILIZED

T3: REPLACED SOIL, STERILIZED (CONTROL)

T4: NATIVE SOIL, STERILIZED (CONTROL)

Develop of a method to test various soils for presence of appropriate fungi---------

before out-planting

ARE MYCORRHIZAL FUNGI FOUND IN REPLACED SOIL ON DUNRAVEN PASS AND AVAILABLE TO SEEDLINGS?

Nursery fungi?

Native fungi?

T1. Replaced soil N=10

T2. native soil N=10

T3. Sterilized replaced soil (control) N=10

T4. sterilized native soil (control) N=10

% seedling survival 100% 100% 80% 90%

% seedlings with nursery fungi

60% 80%?* 10-40%? 50%?

% of seedlings with native fungi

60% 50% 10%† 0%

3-6 species of native fungi

2-5 species of native fungi

E-strain E-strain

Mycorrhizal colonization of 2-year-old nursery seedlings planted in various soils from Dunraven Pass (YNP) after 1 year in greenhouse conditions

Native fungi: all Suilloid fungi (Rhizopogon subbadius, R. sp. 1, and Suillus aff. borealis, Suillus sp., Amphinema sp.). Nursery fungi: Wilcoxina (E-strain) confirmed on some, not confirmed molecularly on all)

† incomplete sterilization

• Results are not correlated with individual sites (soil samples were mixed)

• Suilloid fungi present

• Nursery fungi may have persisted on seedlings in both soils

Molecular ID by P Trusty

• Soil bioassay revealed native mycorrhizal fungi appropriate for colonization of whitebark pine in replaced & native soil on Dunraven Pass.

CONCLUSIONS

RECOMMENDATIONS

• Colonization by native mycorrhizal fungi initiated on 50% of sites after 9 months.

• Most of the colonization N side of Pass

• No colonization (1 exception) on compromised seedlings.

• Colonization levels low on most seedlings after 9 month (one notable exception).

• Suilloid fungi are present in soil of at least 50% of sites, also in replaced soil on Pass.

• Second monitoring for % survival & mycorrhizal colonization (2009).

• Planting seedlings S of pass (with controls)

- with native soil from whitebark pine forests (2008/2009)

- inoculated in greenhouse with native fungi from Yellowstone Park

-Greenhouse (2008), field (2009)

- test retroactive methods for inoculation (2008/9)

• a commercial inoculum of exotic mycorrhizal fungi should NOT be used in sensitive whitebark pine forests!

Assessment of mycorrhizal colonization of rust resistant white bark pine seedlings planted in Post-fire Restoration Treatments on a severe burn (Fridley Burn, Gallatin NF, MT) as a measure of sustainability.

2. MONITORING

Rocky Mountain Research Station Funding. Cooperator Bob Keane, Fire Ecologist, Missoula, MT. Julie Shea, USFS Fire Officer Gallatin NF, Stan Cook, USFS Silviculturist, Gallatin NF, MT

NASA EARTH OBSERVATORY AUG 19, 2001

Fire started Aug 19 2001

Cause Lightening

Acres 26,373

Severe: High fuel loads, 40 mph winds, below ave. precip, low humidity, in whitebark pine

Pacific Biodiversity Institute

Paul Trusty graduate student MSU

• In 2002, rust-resistant seedlings from the Coeur D’Alene nursery were planted on burn

OBJECTIVES: To determine

•if planted seedlings are effectively colonized on Fridley burn [ON-SITE MONITORING]

•inoculum potential of soil from a severe burn [GREENHOUSE BIOASSAY]

•if imported nursery fungi persist on planted seedlings [MOLECULAR TOOLS]

•Compare mycorrhizal fungi on seedlings:

2. planted rust resistant seedlings

3. naturally regenerating seedlings1. Naturally regenerating seedlings

Cripps photos

METHODS: Monitoring by comparison 2006

3 transects in burn and 3 in adjacent unburned whitebark pine forest, 2X samplings

Minimally destructive sampling: root samples removed from seedlings in situ

N=60 T1 = burned, planted seedlings N=60 T2 = unburned adjacent forest, naturally regenerating seedlings N=24 T3 = burned, naturally regenerating seedlings

Cripps photo

RESEARCH PROJECT OF PAUL TRUSTY, MSU GRADUATE STUDENT

NViable/Nonviable= % Mycorrhizal

Total # Species

Mean # Species/ Tree

1 Burned, Planted 60 60/36 =96% 10 1.883

2 Unburned, Natural 60 32/67 = 98% 8 2.25*

3 Burned, Natural 24 33/55 = 88% 7 1.625

1+2: p-value=0.02742,

1+3: p-value=0.27540

2+3: p-value=0.00182

PRELIMINARY RESULTS:

• 88-98% of root tips were mycorrhizal for all treatments

• Range of 1-5 “morphotypes” of fungi per seedling

• More “morphotypes” per seedling for unburned forest vs those on burns (planted/natural)

PAUL TRUSTY DATA & PHOTOS 2006

Amphinema - burn

Cortinarius - unburned

Byssocorticium - burn

Mycorrhizal colonization of whitebark pine seedlings 5 years after burn

Pseudotomentella- burn

Importance value = relative frequency & relative abundance

Relative Importance of Ectomycorrhizal types by “treatment”

Species shift: 1. Mycorrhizal fungi on burn different from those in unburned forests.

2. Natural & planted seedlings on burn share fungal taxa.

3. Suilloid fungi coming into burned area after 5 years.

3. Unclear at this point if nursery fungi persist after 5 years.

4. Functional significance of shift is unknown at this point. PAUL TRUSTY DATA 2007

A “shift” in mycorrhizal taxa after the burn

unburnedburned

PAUL TRUSTY DATA 2007

Correspondence analysis

• each symbol represents 1 seedling & mycorrhizal fungi on that seedling

• Unburned seedlings are clearly separate

• natural & planted seedlings on burn overlap

CONDITIONS WERE OPTIMIZED FOR MYCORRHIZAL COLONIZATION IN PLANTINGS OF RUST RESISTENT SEEDLINGS ON THE FRIDLEY BURN

• rust resistant seedlings were planted 1 year after burn.

• the planting area was in close proximity to mature unburned whitebark pine trees as a source of mycorrhizal inoculum.

• burned area was previously in whitebark pine, “sporebanks” of suilloid fungi may persist in the soil.

• mammals that have potential to disperse suilloid fungi and “inoculate” seedlings are present

• moisture conditions were sufficient

Naturally regenerating seedlings

Cone-tainerized root systems on planted seedlings after 5 years

GREENHOUSE BIOASSY:

Picked up Suilloids & E-strain on burned & unburned soil

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

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

3. APPLICATIONFLOW CHART FOR DETERMINING WHEN INOCULATION IS NECESSARY

Cripps, CL  2003. Native mycorrhizal fungi with aspen on smelter-impacted sites in the Northern Rocky Mountains: occurrence and potential use in reclamation. Amercian Society of Mined Land Reclamation, Lexington, KY. Pgs. 193-208.

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

HOW CAN WE OPTIMIZE THE SYSTEM?

FUTURE DIRECTIONS

• Continue to determine the mycorrhizal fungi important to whitebark pine

currently in Yellowstone, Glacier & Waterton Parks

• Monitor whitebark pine seedling planted under a variety of conditions

primarily in areas where inoculum in not likely to be available & seedlings compromised.

need suggestions on where to monitor

• Capture & screen native mycorrhizal fungi for use as inoculation

we now have about 15 species in culture

• Developing methods for inoculation of seedlings

We need cooperators & access to young whitebark seedlings, a few weeks or months old

• Test to see if inoculation of seedlings can be retroactive.

THANKS TO ALL THOSE WHO HAVE HELPED WITH OUR WHTEBARK PROJECTS:

Mary Hecktner, YNP Resource Manager, Yellowstone National Park; Dan Reinhart, YNP & Kay Izlar, U of M; Bob Keane, USFS Fire Ecologist, Missoula Fire Office; Julie Shea, USFS Fire Officer Gallatin NF, MT; Stan Cook, USFS Silviculturist, Gallatin NF, MT; Cyndi Smith, Parks Canada Ecologist, Waterton Lakes National Park; Joyce Lapp, Park Service Silviculturist, Glacier National Park; Tara Carolin, Glacier National Park.

THE END…….

OR JUST THE BEGINNING?