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

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