Transcript Slide 1

MONITORING MYCORRHIZAL FUNGI ON
PLANTED WHITEBARK PINES IN THE GYE
DO PLANTED PINES HAVE THE “RIGHT STUFF” ON THEIR ROOTS FOR SURVIVAL?
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 M
Bob Keane, USFS Fire Ecologist, Missoula Fire Office
Julie Shea, USFS Fire Officer Gallatin National Forest, MT
Stan Cook, USFS Silviculturist, Gallatin National Forest, MT
Cyndi Smith, Parks Canada Ecologist, Waterton Lakes National Park
Joyce Lapp, Park Service Silviculturist, Glacier National Park
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
Seedling planted along Dunraven Pass
WHITE BARK PINE FORESTS ARE SERIOUSLY DECLINING
THREATS
•White bark pine blister rust
•Mountain pine beetle
•Fire exclusion
•Climate change
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?
A mutualistic relationship between
certain fungi and plant roots
“beneficial to both”
spores
2 MAIN TYPES
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
Benefits to fungus
get sugars from the plant i.e. Food!
Fruiting bodies
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
mycelium
Brundrett et al. 2007 website
ectomycorrhizae
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
YES! But which ones?
ECM FUNGI ARE CRUCIAL TO ESTABLISHMENT, SURVIVAL, SUSTAINABILITY OF WHITEBARK PINE!
Cripps 2001 Encyclopedia of Plant Pathology; Smith and Read 1998. Mycorrhizal Symbiosis
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
ITS-1
DNA
Amanita alpina
Fruiting bodies
Courtesy CSIRO
Ectomycorrhizae on roots
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. DISCOVERY
WHICH 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
32 species of ECM fungi confirmed with whitebark
pine by fruiting bodies or ectomycorrhizae on roots
BASIDIOMYCOTA
AMANITACEAE
Amanita "alpina"
HYGROPHORACEAE
Hygrophorus gliocyclus
Hygrophorus marzuolus (Fr.)
Hygrophorus olivaceoalbus
Hygrophorus subalpinus
TRICHOLOMATACEAE
Leucopaxillus paradoxis
Tricholoma moseri
CORTINARIACEAE
Cortinarius clandestinus
Cortinarius duracinus
Cortinarius “flavobasalis”
Cortinarius “flavoroseus”
Cortinarius aff. fulminoides
Cortinarius subolivescens
Cortinarius sp.
Dermocybe crocea (Schff.) Mos.
Inocybe sp.
RUSSULALES
Lactarius deliciosus
Russula cf tortulosa /queletii
Russula sp. 2, Russula sp. 3
Waterton Lakes National Park
Glacier National
Park
Unculturable, shared
with other conifers
OLDER TREES
Unculturable, some
may be specific for
pines or shared with
other conifers
MOSTLY OLDER
TREES
Yellowstone
National Park
BOLETALES
Boletus edulis
Chroogomphus sp. nov.
Rhizopogon cf milleri
Rhizopogon cf evadens
Rhizopogon spp.
Suillus subalpinus
Suillus sibiricus
Suillus tomentosus var. discolor
Suillus sp.
PHALLALES - GOMPHALES
Hysterangium separabile
THELEPHORALES
Tomentelloid type 1
Tomentelloid type 2
ASCOMYCOTA
Cenococcum geophilum
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
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!)
Cripps photos
Suilloids–show host specificity
Moser 2004. In Cripps: Fungi in Forest Ecosystems, NYBG
Press. (fungi with 5-needle pines in Alps, Altai, Rocky Mts)
Suillus subalpinus – 5-needle pine/stone pines
Chroogomphus sp nov (new species)whitebark pine
Suillus sibericus – stone pines
Rhizopogon milleri & R. evadens
(pine/5-needle/stone pine)
UNDERGROUND MYCORRHIZAL FUNGI COMMON IN THE SYSTEM
Rhizopogon
pine
“POGIES”
pine
These fungi are eaten by squirrels, deer,
elk & bears which spread the spores
Gautieria
pine-spruce-fir
Peaco photo, YNP
Czares & Trappe 1994. Spore dispersal of ectomycorrhizal fungi by mammal mycophagy. Mycologia 86
Spore dispersal by deer
DUNRAVEN PASS, YNP
“POGIES”
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
2. MONITORING
Project 1: Whitebark Pine Restoration, Dunraven Pass Yellowstone National
Park: Monitoring the mycorrhizal status of planted whitebark pine seedlings.
• 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)
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
-
Greater Yellowstone Coordinating Committee & Rocky Mountain Research Station funding. Cooperators: Mary
Hecktner Resource manager YNP, Dan Reinhart YNP, and Kay Izlar, U of M.
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]
WHAT IS THE MYCORRHIZAL CONDITION OF NURSERY SEEDLINGS BEFORE OUT- PLANTING?
•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
FUNGI ON NURSERY SEEDLINGS? BENEFIT OR CONCERN?
Thelephora sp – typical in nurseries
Root hairs = non-mycorrhizal
E-strain fungus – typical in nurseries
Cripps photos
Number of mycorrhizal root tips per seedling sampled on each site
Izlar data
sites
Healthy
seedlings
Compromised
seedlings
1. Top Pass – 8900’, W, 300
15
2. Top Pass – 8885’, flat
Izlar notes
%
Survival
Habitat notes
Vegetation
0
87%
slope behind visitor area to north
50%
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
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
8. N Pass – 8667’, S, 5-250
• No (little) mycorrhizal colonization on compromised seedlings
• Native fungi were colonizing roots on sites 1, 6, 7, 9 and 11 (50% of sites).
• No colonization of sites 4, 5, 8 (S & SE aspect).
• Only nursery fungi were present on sites 2 and 3.
Suilloids
Percent mycorrhizal colonization of root tips and depth of ectomycorrhizae on
cone-tainerized roots systems
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.
Kay Izlar’s “star performer”: seedling #1, site 6
Collected below a mature whitebark pine
Mycorrhizal
colonization of
cone-tainerized
roots
Cripps photos
NATIVE ECTOMYCORRHIZAE ON WHITEBARK PINE SEEDLINGS
PLANTED ALONG DUNRAVEN PASS (sampled 9 months after planting)
Suilloid type
Cenococcum & sclerotia
Rhizopogon
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
ARE MYCORRHIZAL FUNGI FOUND IN REPLACED SOIL ON DUNRAVEN PASS
AND AVAILABLE TO SEEDLINGS?
METHOD: SOIL BIOASSAY IN GREENHOUSE
T1: REPLACED SOIL, UNSTERILIZED
T2: NATIVE WBP FOREST SOIL, UNSTERILIZED
T3: REPLACED SOIL, STERILIZED (CONTROL)
Native fungi?
Nursery fungi?
T4: NATIVE SOIL, STERILIZED (CONTROL)
Develop of a method to test
various soils for presence of
appropriate fungi--------before out-planting
Mycorrhizal colonization of 2-year-old nursery seedlings planted in various soils from
Dunraven Pass (YNP) after 1 year in greenhouse conditions
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
† incomplete sterilization
Molecular ID by P Trusty
• Soil bioassay revealed native mycorrhizal fungi appropriate for colonization
of whitebark pine in replaced & native soil on Dunraven Pass.
• Results are not correlated with individual sites (soil samples were mixed)
• Suilloid fungi present
• Nursery fungi may have persisted on seedlings in both soils
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)
CONCLUSIONS
• 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.
RECOMMENDATIONS
• 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!
2. MONITORING
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.
Paul Trusty
graduate student
MSU
Fire started
Aug 19 2001
Cause
Lightening
Acres
26,373
Pacific Biodiversity Institute
Severe: High fuel loads, 40
mph winds, below ave. precip,
low humidity, in whitebark pine
NASA EARTH OBSERVATORY AUG 19, 2001
• In 2002, rust-resistant seedlings from the Coeur D’Alene nursery were planted on burn
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
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
1. Naturally regenerating seedlings
3. 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
RESEARCH PROJECT OF PAUL TRUSTY, MSU GRADUATE STUDENT
Cripps photo
PRELIMINARY RESULTS:
Mycorrhizal colonization of whitebark pine seedlings 5 years after burn
N
Viable/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
Cortinarius - unburned
1+2: p-value=0.02742,
1+3: p-value=0.27540
2+3: p-value=0.00182
•
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
Pseudotomentella- burn
Byssocorticium - burn
Relative Importance of Ectomycorrhizal types by “treatment”
A “shift” in mycorrhizal taxa after the burn
Natural Burned
Planted Burned
.
1.20
Importance Value
1.40
1.00
Natural Unburned
burned
unburned
0.80
0.60
0.40
0.20
0.00
Thelephoroid
group
Wilcoxina rehmii Pseudotomentella
nigra
Amphinema
Suilloid group
byssiodes
Piloderma
Cenococcum
byssinum
geophilum
Cortinarius sp.
Ectomycorrhizal Taxa
Importance value = relative frequency & relative abundance
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
Correspondence
analysis
• each symbol represents 1 seedling
& mycorrhizal fungi on that seedling
• Unburned seedlings are clearly
separate
• natural & planted seedlings on burn
overlap
PAUL TRUSTY DATA 2007
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
Cone-tainerized root systems on
planted seedlings after 5 years
Naturally regenerating
seedlings
GREENHOUSE BIOASSY:
Picked up Suilloids & E-strain
on burned & unburned soil
3. APPLICATION
FLOW CHART FOR DETERMINING WHEN INOCULATION IS NECESSARY
HOW CAN WE OPTIMIZE THE SYSTEM?
Inoculation of seedlings with native ECM can
increase seedling survival and fitness (Smith 1998)
Cripps, CL 2003. Native mycorrhizal fungi with aspen on smelterimpacted sites in the Northern Rocky Mountains: occurrence and
potential use in reclamation. Amercian Society of Mined Land
Reclamation, Lexington, KY. Pgs. 193-208.
Brundrett et al. 1996. Working with Mycorrhizas in Agriculture. CSIRO.
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 smelterimpacted soils. M.S. Thesis (Cripps),
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?