SURVEY FOR THE PRESENCE OF
PHYTOPHTHORA CINNAMOMI ON
RECLAIMED MINED LANDS IN OHIO
CHOSEN FOR RESTORATION OF THE
AMERICAN CHESTNUT
Shiv Hiremath, Kirsten Lehtoma,
and Jenise M. Bauman
Burnham 1988
*7/8
*15/16
* Advanced 15/16
Chestnut in Ohio Coal Mine Reclamation:
• Fast growth rate
• Compliment native range
• Provides a venue for assessment
Early Reports: C. dentata mortality
Phytophthora species are causal
agents of “ink disease”
Its presence in the Appalachian
region, has been noticed on
chestnut seedlings (Rhoades et al.,
2003, 2009; James 2012).
Backcrossed chestnuts are not
breed for resistance to chestnut ink
disease.
Jeffers 2009
History of Chestnut Ink Disease
Prior to chestnut blight, several
Phytophthoras were introduced to the
U.S. (1700-1800s, Jeffers et al. 2009).
Castanea spp. reported dying of
unknown causes as early as 1824
(Crandall et al. 1945)
Not reported until 1932 (Milburn and
Gravatt 1932)
Found in all throughout N. America
including 28 States of the U.S.
Crandall et al. 1945
Phytophthora: “Plant Destroyer”
• Crown Rot
• Fruit Rot of Pumpkin
• Rhododendron
Dieback (shoot rot)
• Bleeding cankers
• Citrus rot
• Port-Orchard-Cedar
Root Rot
Phytophthora cactorum
Phytophthora capsici
Phytophthora citricola
Phytophthora palmivora
Phytophthora parasitica
Phytophthora lateralis
Infamous Phytophthoras…
P. infestans
P. ramorum
Phytophthora cinnamomi
• Symptoms:
- Rot of fine feeder roots
- Wilts, stem cankers, collar
rot
- decline in yield and
decreased fruit size
• Host plants: 3000 species
Examples: Eucalyptus, Avocados, Pineapples, Rhododendron,
Beech, Walnut, Oak, Chestnut
Phytophthora cinnamomi Rands
• Taxonomic Class:
Oomycetes
• Stramenopiles =
flagella hairs
• Geographic Origin:
Southeast Asia
Southern Africa
• First Described:
Portugal in 1838
• Global Distribution
Hardham 2005
Infective Structures
• Sporangia
• Zoospores
• Oospores
Bar = 25m
Disease Cycle
Infection Mechanism
Zoospore finds host
Encysts
Direct Penetration
Range of Phytophthora in N. America
• A 2004-05 survey of soils
associated with oak species for
the occurrence of Phytophthora
• Soils sampled from bases of
healthy and declining oak trees
• P. cinnamomi was the most
frequently isolated species
(69.4%) of the Phytophthoras
• The absence of P. cinnamomi
above the 40ºN latitude
Balci et al. 2007
Range of P. cinnamomi in Ohio
• Survey of Phytophthora
species in white oak
decline in southern Ohio
• The most common
species was P. cinnamomi
• P. cinnamomi was more
commonly isolated with
wet soils
• The population densities
of P. cinnamomi were
significantly with
declining oaks
Balci et al. 2010
Study Objective:
• Survey reclaimed mine sites in
southern Ohio for presence of
P. cinnamomi
• Compared sites that were
reclaimed under 5 years to
those reclaimed 25 years prior
• Compared two Phytophthora
sampling techniques: direct
plating and leaf baiting
Study Sites
YEAR
LOCATION
SITES
METHOD
2008
Greendale, Ohio, Ora Anderson Park
Ironton District, WNF
8
6
Direct plating
Direct plating
2009
The Wilds, International Road
The Wilds, Lake Trail
The Wilds, Site 2
Nelsonville, Ohio
4
4
4
5
Direct plating
Direct plating
Direct plating
Direct plating
2011
The Wilds, Site 1
The Wilds, Forest Site C07
New Straitsville, OH
7
2
10
Leaf baiting
Leaf baiting
Leaf baiting
Ironton District, WNF
Nelsonville, Ohio
The Wilds, International Road
The Wilds, Lake Trail
Methods: Direct Plating
Soil was collected at a depth of 4-5 inches
Ten grams of soil were diluted in 100 ml
dH2O and plated onto PAR(PH)-V8
(Johnson and Curl 1972)
Plates were incubated in the dark at 22 oC
and examined under a dissecting
microscope for the presence hyphae
Subcultures were made on the same
selective medium to obtain a pure culture.
The fungus was allowed to grow for 3
weeks and harvested
Methods: Leaf Baiting
250 g of soil from each site was
mixed with 1.25 L deionized water
American chestnut leaves were
floated on the surface of the water
After 3-5 days, necrotic areas were
examined for sporangia
These areas were plated onto
PAR(PH)-V8 plates as described
above to obtain a pure culture
(Balci et al. 2007)
http://www.forestryimages.org/browse/detail.cfm?imgnum=5408716
Methods: Molecular Identification
• Final identification of the fungus was made by sequencing the
internal transcribed spacer region of the ribosomal DNA
• PCR reactions were analyzed on a 0.7% agarose gel
• Bands produced from the primer pair were isolated from the gel
using the Geneclean (MP Biochemicals)
• Sequences were analyzed in the GENBANK using BLAST
Primer
Yph1F
Yph2R
Ycin3F
Ycin4R
Sequence
5’CGACCATKGGTGTGGACTTT3’
5’ACGTTCTCMCAGGCGTATCT3’
5’GTCCTATTCGCCTGTTGGAA3’
5’GGTTTTCTCTACATAACCATCCTATAA3’
Phytophthora genus-specific(470bp)
P. cinnamomi –specific(243bp)
Methods: Resulting Gels
P. cinnamomi –specific
Phytophthora genus-specific
1.0
0.6
0.2
Pos 2
Pos 1
OA-6-2
OA-6-1
OA-1
Neg
Ycin3F + Ycin4R
Pos 2
Pos 1
OA-6-2
OA-6-1
OA-1
Kbp M
Neg
Yph1F + Yph2R
Results: Wayne National Forest
Year Location
P. cinnamomi
Phytophthora
2008 Ora Anderson Park
Ironton District
0%
0%
0%
0%
2009 Nelsonville, Ohio
0%
0%
2011 New Straitsville, OH
0%
10 %
P. cinnamomi was not detected. We found another species, P.
citricola. In addition to Phytophthora, we also detected another
plant pathogen belonging to the genus Pythium at one of the
sites (Ironton District).
Results: The Wilds
Year Location
P. cinnamomi
Phytophthora
2009 The Wilds, Grassland
The Wilds, Lake Trail
The Wilds, Site 2
0%
0%
0%
0%
0%
0%
2011 The Wilds, Site 1
The Wilds, Forest
0%
0%
0%
50 %
We also found P. citricola in the forested site. This indicates
that at least some of the Phytophthora species are present in
these reclaimed lands. However, their presence was not
common or widespread.
Summary
Northern tip of the distribution of P.
cinnamomi and the pathogen may have not
be able to survive the freezing conditions.
Since these were reclaimed lands, fresh top
soil layer would have been added and
pathogen may not have had time to relocate
to these soils.
It is not known how conducive the harsh soil
conditions (low nutrients, pH extremes,
toxic metals) are for the establishment of the
pathogen.
If this pathogen cannot survive in the mined
lands, they would make highly suitable
locations for the American chestnut
restoration (Barton, et al, 2010).
Future Studies:
Continue to evaluate hybrid seedlings for resistance to P.
cinnamomi.
Identify backcrossed lines with high levels of resistance
Difficulty is that resistance appears to be incompletely
dominant and regulated by more than one gene (Jeffers et al.
2010).
Pre-screening for soils that do not harbor P. cinnamomi.
Proper site selection of areas that have proper drainage
Continue experimental plantings that identify methods that
improve establishment and long-term survival of chestnut.