Phytophthora Root Rot on FirResearch Findings from North Carolina and BeyondJohn FramptonProfessor & Christmas Tree Geneticist
Department of Forestry & Natural Resources
North Carolina State University
CTFANY2018 Winter ConventionSyracuseJanuary 19th
Presentation Outline
ØBackground Information
ØScreening for resistance to Phytophthora
cinnamomi
ØOther Phytophthora species
ØStrategies
Presentation Outline
ØBackground Information
ØScreening for resistance to Phytophthora
cinnamomi
ØOther Phytophthora species
ØStrategies
2nd ranked state in U.S.5-6 million trees harvested annually$100+ million wholesale value realized annually 2,500+ Christmas tree growers98%+ of the harvested Christmas trees are Fraser fir produced in the western mountainous portion of the state80%+ of the Fraser fir production is wholesale and shipped country-wide
North Carolina’s Christmas Tree Industry
Fraser fir (Abies fraseri)-------------------------------------------------------------------------------------Virginia pine (Pinus virginiana)Eastern white pine (Pinus strobus)Eastern redcedar (Juniperus virginiana)Leyland cypress (x Cupressocyparis leylandii 'Leighton Green')Arizona cypress (Cupressus arizonica var. glabra 'Carolina Sapphire' & 'Clemson Greenspire')Atlantic white cedar (Chamaecyparis thyoides)Various spruces (Picea spp.)
North Carolina’s Christmas Tree IndustrySpecies List
Phytophthorav Name is Greek for “plant destroyer”v Genus of water mold (Oomycetes) v About 100 species (and counting)v Can multiply exponentially v Many species have mating strains designated as
A1 and A2v Several spore stagesv Zoospores infect conifer roots and have two
unlike flagella to sense and swim toward host
Electron micrograph of Phytophthora cinnamomi zoospores encysting. (Giles Hardy)
Zoospore infection of host plant. (Jim Deacon)
Sporangium releasing zoospores. (Oregon State University)
Brief History of Phytophthora cinnamomiin the Southeast U.S.
Early 19th
Centuryv Pc believed to be introduced on exotic flora imported through southern ports vReports of American chestnut and chinkapinmortality from lower elevations due to a root disease
1930 1st definitive report of Pc in the U.S. on three rhododendron species
1932 Pc identified as cause of �ink disease� on American chestnuts
1940s Littleleaf emerged as important disease on shortleaf pine
1963 1st Report of Pc on Fraser fir in North Carolina
1976-1977 Fraser fir survey – 10% of sites infested NC Christmas tree industry < 1 million trees
1997-1998 Fraser fir survey – 9% of sites infested NC Christmas tree industry 6-7 million trees
Crandall, B.S., G.F. Gravatt and M.M. Ryan. 1945. Phytopathology 35:162-180.
Impact on Fraser Fir
v Primarily caused by P. cinnamomiv Results in $6-9 million in revenue losses annuallyv Control with Subdue feasible in nursery bedsv Site selection & clean planting stock are our only
preventative measures for the fieldv No resistance found in Fraser fir although other
fir species have some resistance
Presentation Outline
ØBackground Information
ØScreening for resistance to Phytophthora
cinnamomi
ØOther Phytophthora species
ØStrategies
Resistance Screening in Fraser Fir
Complete mortality in two greenhouse
inoculations studies:
• 100 open-pollinated families from Fraser fir
seed orchard from Roan Mountain (1999)
Resistance Screening in Fraser Fir
Complete mortality in two greenhouse inoculations
studies:
• 100 open-pollinated families from Fraser fir seed
orchard from Roan Mountain (1999)
• 99 open-pollinated families from all six of
provenances of Fraser fir (2003)
Resistance Screening in Fraser Fir
v Little, if any resistance in Fraser fir
v Other fir species may offer resistance
Variation in Resistance to P. cinnamomiamong Abies Species
Objectives:
vRank the relative resistance of fir speciesvIdentify potential sources of fir resistance
The Genus AbiesThe True Firs
v39-55 Species – 2nd largest genus in Pinaceae
vTemperate and frigid regions
vSea-level to over 5,000 m elevation
vNorthern Hemisphere - 14°N to 67 ° N latitude
Worldwide distribution of the genus Abies (Farjon 1990)
Methods
vGrew seedlings in greenhouse for two or three years
v Inoculated with rice grains colonized with P. cinnamomi
vPlaced into an outdoor lath house and recorded
mortality every two weeks for a total of 16 weeks
v32 Abies species, 50 seed sources
v6,629 seedlings total
Time from Inoculation (wks)
0 2 4 6 8 10 12
Mor
talit
y (p
ropo
rtion
)
0.0
0.2
0.4
0.6
0.8
1.0
MeanA. bornmuelleriana A. firma A. fraseri A. pindrow
SpeciesMortality(%)
A. firma (momi) 11.3A. pindrow (pindrow, Himalayan) 30.0A. bornmuelleriana (Turkish) 61.3A. cilicica (Cilcican, Toros) 63.2A. siberica (Siberian) 68.8A. borisii-regis (King Boris) 75.0A. nordmanniana (Nordmann) 77.0A. cephalonica (Greek) 82.5A. chensiensis (Chinese) 82.5A. equi-trojani (Trojan) 84.2
A. concolor (concolor, white) 84.4
Species Mortality (%)A. holophylla 88.8A. guatamalensis 90.2A. fabri 90.6A. alba 91.3A. ernestii 91.9A. fargesii 92.5A. delavayi 95.6A. bracteata 96.9A. procera 97.4A. numidica 97.5A. sachalinensis 97.5A. grandis 98.4A. balsamea 98.6A. lasiocarpa 98.7A. amabilis 99.2A. georgei 99.4A. koreana 99.4A. veitchii 99.4A. fraseri 100.0A. magnifica 100.0A. nephrolepis 100.0
Variation among Abies Species
Conclusions
v Most fir species are highly susceptible to P. cinnamomi
v Some resistance exits in the Mediterranean and central Asia regions
v Momi and West Himalayan (pindrow) fir appear to be relatively resistant
vTechnique may overlook some types of resistanceØYoung treesØSevere disease conditions
§ Roots confined§ High inoculum load§ Continuously wet medium
vSome mortality may not be due to Phytophthora
vLimited sampling of provenances within species
Caveats
• 1st Momi fir
– Poor Christmas tree quality
– Breaks bud about one month earlier than Fraser fir
• 2nd Pindrow fir
– No little known about this species
– Very long needles
– Does not produce many buds on branches
• 3rd Turkish fir
– Closely related to Nordmann fir
– Used as a Christmas tree in Europe & North America
Three Most Resistant Species
SpeciesMortality(%)
A. firma (momi) 11.3A. pindrow (pindrow, Himalayan) 30.0A. bornmuelleriana (Turkish) 61.3A. cilicica (Cilcican, Toros) 63.2A. siberica (Siberian) 68.8A. borisii-regis (King Boris) 75.0A. nordmanniana (Nordmann) 77.0A. cephalonica (Greek) 82.5A. chensiensis (Chinese) 82.5A. equi-trojani (Trojan) 84.2
A. concolor (concolor, white) 84.4
Species Mortality (%)A. holophylla 88.8A. guatamalensis 90.2A. fabri 90.6A. alba 91.3A. ernestii 91.9A. fargesii 92.5A. delavayi 95.6A. bracteata 96.9A. procera 97.4A. numidica 97.5A. sachalinensis 97.5A. grandis 98.4A. balsamea 98.6A. lasiocarpa 98.7A. amabilis 99.2A. georgei 99.4A. koreana 99.4A. veitchii 99.4A. fraseri 100.0A. magnifica 100.0A. nephrolepis 100.0
Turkish & Trojan FirWithin Species VariationJohn Frampton and Fikret Isik
2005 Cone Collection Trip
Trojan Fir(Abies equi-trojani)1 Kazdagi2 Can
Turkish Fir(Abies bornmulleriana)3 Uludag4 Akyazi5 Bolu6 Safranbolu
6 Provenances20 Trees/Provenance
----- -----------------------120 Trees Total
Black Sea
2
13
4 5 6
Turkish Fir Seeds
Trojan Fir(Abies equi-trojani)1 Kazdagi2 Can
Turkish Fir(Abies bornmulleriana)3 Uludag4 Akyazi5 Bolu6 Safranbolu
6 Provenances20 Trees/Provenance
----- -----------------------120 Trees Total
Black Sea
254.5%
158.2%
350.8%
433.3%
524.1% 6
23.2%
Geographic Pattern of Resistance
v Although P. cinnamomi is not believed to be native to
Turkey, other Phytophthora species are present (Balci and
Halmschlager 2003)
v Current or past contact with Phytophthora or other
Oomycete species
v Adaptation to environmental factors such as rainfall, soil
temperature, and/or soil characteristics (e.g., texture)
model (generalized estimation equations) was fit to alldata of the seedlings inoculated from both species to testthe fixed effects (species and provenance) over time. We
modeled the probability of mortality (π) while accountingfor random replication and family effects and repeatedmeasure:
ηijklmn ¼ log p= 1" pð Þ½ & ¼ μ þ Ri þ Sj þ PðSÞkðjÞ þ R ( Sij þ R ( PðSÞikðjÞ þ F SPð ÞlðkjÞþ R ( F SPð ÞilðjkÞ þ b1Wn þ b2W ( Wn þ b3 W ( Rð Þin þ b4 W ( W ( Rð Þin þ b5 W ( Sð Þjnþ b6 W ( W ( Sð Þjn þ b7 W ( PðSÞð ÞljðkÞn þ b8 W ( W ( PðSÞð ÞkðjÞn þ b9 W ( F PSð Þð Þl jkð Þnþ b10 W ( W ( F PSð Þð Þl kjð Þn þ T SPFð Þijklm þ eijklmn
ð1Þ
where,
ηijklmn is the link function [g(μ)] of mortalityof the nth time of the mth seedling,jth species, kth provenance, lth family,and in the ith replication
log [π/(1−π)] is the logit value or log of odds ofmortality
π is the probability of moralityμ is the conditional meanRi is the ith replication effect, i04
0
10
20
30
40
50
60
0
20
40
60
80
100
2 4 6 8 10 12 14 162 4 6 8 10 12 14 16
00
10
20
30
40
50
60
70
80
90
100
42 6 8 10 12 14 16
Mor
talit
y (%
)
Mor
talit
y (%
)M
orta
lity
(%)
Weeks after inoculationWeeks after inoculation
Weeks after inoculation
Akyazı
Uluda
Kazdagı
Karabük
Çan
Bolu
a b
c
Fig. 2 Mortality curves of fir seedlings over a 16-week period followinginoculation with P. cinnamomi. a Mortality of four fir species (Fraser,Turkish, Trojan, andmomi). bMortality of two Trojan fir (Kazda ı, Çan)and four Turkish fir (Uludağ, Akyazı, Bolu, Karabük) provenances. c
Mortality of 34 families of Trojan fir (solid blue lines), 71 families ofTurkish fir (dashed black lines), and the overall mean (thick red line). Theaverage family size was about 45 seedlings
Tree Genetics & Genomes
Author's personal copy
Family Means for Mortality
Heritability Estimates16 Weeks after Inoculation
ConclusionsvUseful levels of resistance exist in Turkish and Trojan
fir
vTurkish fir has a higher frequency of resistance than
Trojan fir
vThe frequency of resistance increase from west to
east in the Trojan-Turkish fir range
vResistance is under strong genetic control
Genetic Basis of Phytophthora Resistance in Trojan fir
Research of PhD graduate student, Will KohlwayFunded by USDA Specialty Crops Grant
2 Approaches
Genotyping by Sequenceing (GBS)Genomic markers to select for resistant planting stockDoes not address biological cause for resistance
RNA-seq“Snap-shot” of cellular response against PhytophthoraStudy gene expression patternsIdentifies genes important to root rot resistance
Presentation Outline
ØBackground Information
ØScreening for resistance to Phytophthora
cinnamomi
ØOther Phytophthora species
ØStrategies
Phytophthora Species Survey
Research of PhD graduate student, Martin Pettersson
Funded by:Gunnar and Lillian Nicholson Graduate Fellowship and Faculty Exchange FundNCSU Christmas Tree Genetics ProgramNIFA USDA SCRI
Rationale
Growers have contracted out-of-state nurseries
to produce their planting stock
This may have led to introduction of new
Phytophthora species into the region
Katie McKeever (PhD, Washington State University)
- isolated P. cryptogea (taxon kelmania) and P. pini
3%
15%
15%
44%
23%
ME MI PA OR WA
Survey Christmas Tree Growers(n=89 grower, 123 farms, 13 counties)
54% of growers surveyed use out-of state planting stockAll large growers (>100 acres) surveyed use out-of-state planting stock78 % of acreage of surveyed growers planted with out-of-state planting stockMost transplants originate from the Pacific Northwest
Origin of Out-of-State Transplants
0102030405060708090
100
1970
1974
1978
1982
1986
1990
1994
1998
2002
2006
2010
Acr
eage
pla
nted
in O
ut-o
f-St
ate
Pla
ntin
g St
ock
(%)
103 sites
309 diseased trees32 healthy controls
682 petri dishes
Results80% isolation success per site54% isolation success per tree
6 species identified
P. cinnamomi
P. cryptogea (P. sp. kelmania)
P. pini (P. citricola complex)
P. europaea
P. citrophthora
P. sansomeana
71%
23%
2% 2% 1% 1%
P. cinnamomi
P. cryptogea
P. citrophthora
P. europaea
P. pini
P. sansomeana
Results
Summary
In this survey we found more Phytophthora species in Fraser fir Christmas tree plantations in the Southern Appalachians compared to previous published surveys.
While P. cinnamomi remains the predominant species (71%), P. cryptogea appears to have become an important pathogen (23%) contributing to losses to the Christmas tree industry in the Southern Appalachian Mountains.
Summary
P. citrophthora, P. europaea, P. pini and P. sansomeanahave not been reported in previously published Fraser fir surveys conducted in the region.
P. citrophthora that has only been found once before on an Abies species – Martin proved pathogenecity on Fraser fir (Koch’s Postulates)
New York Christmas Trees
Katie McKeever & Gary
Chastagner WSU 2016
v P. cryptogea (taxon kelmania)
v P. pulirivora
v P. pini
v P catorum (CT)
Brian Eshenaur & Shawn
Kenaley – Cornell 2012
v P. cryptogea/drechsleri
v P catorum
v P. citricola
Research of MS graduate student, Will KohlwayFunded by NCDA & CS Specialty Crops Block Grant
Objective: to determine differences in aggressiveness of specific genetic isolates of Phytophthora on various fir species.
Interactions between Fir and Phytophthora
Genetic Interactions between Fir and Phytophthora
Species Isolate Mating Type Host Plant2322 A1 Camilla2325 A2 Shore Juniper2327 A2 Cedar
23ss04 A2 Fraser fir23ss11 A2 Fraser firC161 A2 FirC198 A2 Fir
P. cinnamomi
P. cryptogea
4 Fir SpeciesFraser firMomi firTrojan firTurkish Fir
7 Phytophthora Isolates
Difference in Agressivenes of Phytophthora Species
Presentation Outline
ØBackground Information
ØScreening for resistance to Phytophthora
cinnamomi
ØOther Phytophthora species
ØStrategies
StrategiesShort-term
vGraft Fraser fir onto momi fir (A. firma) rootstock for
deployment on infested sites
vPlant out resistant provenances (Bolu) of Turkish fir
StrategiesIntermediate
v Establish Turkish/Trojan fir seed orchard using survivors from
past inoculation trials
v Study genetic control of resistance and develop DNA markers to
select resistant trees from field trials (four in NC)
Long-term
v Produce genetically transformed resistant Fraser fir
v Breed for resistance within Turkish and other resistant fir species
v Develop resistant interspecific hybrids and backcrosses with
Fraser fir
Planting Season
Careful site selectionü No past disease problemsü Well-drained, low clay contentü Minimize compaction during site prep
Don’t plant Fraser fir on sites known to have Phytophthora ü Fraser fir grafted onto resistant fir ü Resistant firü Spruceü White pine
Planting Season
Carefully inspect all planting stockShoot symptomsü Off-colorü Low vigor / little growthü Short needles
Root symptomsü Brown or black lesionsü Dead roots
Do not plant suspicious stockHave suspicious stock tested for PhytophthoraReport positive results to Cooperative Extension Service
Phytophthora ImmunoStrips
25 = $145, $5.80 / test
Phytophthora ImmunoStrips
agdia.com