Alien tree species and m

icrofungaldiversity

JarkkoHantula

The Finnish Forest Research Institute

Cry

phonec

tria

para

sitica

-an introduced disaster

•100 years ago one tree out of four was

american

chestnut in certain areas of

eastern USA.

•Trade with Japanese chestnuts started in

1870´s, and by the early 20th century

Cry

phonec

tria

para

sitica

was

introduced several tim

es in

contaminated seedlings.

•In 1913 the fungus was transferred from

China to NA in order to confirm

that it

was the cause of symptoms. It escaped

to nature.

•The fungus is genetically highly

polymorphic in NA due to several

introductions

•American chestnut became endangered

in 1950´s

•C. para

sitica

was introduced to Europe

in 1938

•In Europe viruses have restricted the

disease; and are also used by m

an for

control.

•In NA viruses have been alm

ost

useless, probably because of more wide

genetic diversity of the pathogen.

•Neither has breeding for resistance

provided a solution

Other international catastrophs

•Dutch elm

disease (Ophiostomasp.) in Europe and North America

•White pine blister rust (Cro

nartium

ribicola) in Europe and North America

•Phytophth

ora

cinnamomiin Australia

•Siroco

ccusclavigig

enti-jugla

ndace

aru

min USA

•Phytophto

raaln

iin Europe

•Phytophth

ora

ramoru

min North America

The history of introduced tree pathogens in Finland

•Ento

leuca

mammata

was probably

introduced from North-A

merica to

Europe several centuries ago

•White pine blister rust destroyed

strobus-plantations in the early 20th

century

•Neo

fabre

apopuliwas probably

introduced with american

aspen in

1950´s.

•Dutch elm

disease came in but was

removed by quick actions in 1960´s

•Uninucleate

Rhizoctonia

was observed

in nurseries in 1990´s. It probably cam

e

from Norw

ay.

•Phytophth

ora

cactoru

mwas observed

on birch in nurseries in 1990´s. The

origin of the disease is unknown, and its

local origin has not been completely

ruled out.

The lessons learned from introduced pathogens

•We know

–that the number of introduced diseases is increasing despite efforts to restrict their

dispersal

–that introduced diseases have often turned out to be real catastrophs

–that the more genetic variation in introduced pathogens exist, the more difficult

they are to control

–that m

icrofungimay be non-pathogenic or only m

ild pathogens on one host, but

turn out to be serious diseases on another one -especially in a new

environment

–that m

icrofungimay m

igrate in nonsymptomaticplant material

•We do not know

–how often m

icrofungiare succesfullyco-introduced with their plant hosts

–how often m

icrofunginonsymptomaticon an introduced host causes disease on the

naturally occurring plants in the new

environment

–how often introduced m

icrofungihybridize with their relatives in the new

environment

–how often new

pathogenic capabilities appear through a hybridization of

microfungi

How often are m

icrofungisuccesfullycointroducedwith their

plant hosts -could we learn something from the needle

endophytes?

•E

ndophyte

sare

fungi, that occur in

pla

nts

without causin

g a

ny s

ym

pto

ms

•U

p to 1

00 e

ndophytic

mic

rofu

ngihave b

een reported to inhabit n

eedle

s o

f

Norw

ay s

pru

ce

•In

theory

–endophyte

sof in

troduced s

pecie

s a

re a

ble

to c

o-m

igra

te e

asily

with their

hosts

, as n

o a

ctions a

re taken to s

top their m

igra

tion in p

lant trade

–in

the introduced a

rea, th

e resourc

e p

rovid

ed b

y their just in

troduced h

ost

pla

nt is

low

in the n

ew

environm

ent

–lo

cal endophyte

sm

ay m

ove to the introduced p

lant and c

om

pete

with c

o-

introduced e

ndophyte

s

•In

the c

ase o

f Rhabdoclineparkeri

it w

as o

bserv

ed that in

isola

ted s

tands the

endophyte

div

ers

ity w

as low

er th

an in larg

e p

opula

tions

•B

ased o

n a

vaila

ble

info

rmation it is

im

possib

le to d

educe w

heth

er th

e c

o-

mig

rationalcapacity o

f needle

endophyte

sis

restric

ted o

r not

•In

tern

al fu

ngi of needle

(m

ostly e

ndophyte

s) do p

rovid

e a

well

defined a

nd

div

erg

ent m

odel syste

m to a

naly

se the e

ffic

iency o

f th

e c

o-introductional

capability

of m

icro

fungiin

genera

l

Endophytesof Siberian larch (Larixsibirica)

JarkkoHantula, MinnaKauhanen, EevaVainio, GudidurG. Eyjolfsdottir, & PekkaNiemelä

•S

iberian larc

h is a

n introduced s

pecie

s in F

inla

nd

•It h

as b

een introduced to F

inla

nd s

evera

l tim

es d

uring the last 300 y

ears

•It w

as introduced to Icela

nd d

uring the 2

0th

centu

ry

•Four cla

sses o

f la

rch s

tands w

ere

sam

ple

d

–N

ative s

tand in A

rchangel, R

ussia

–O

ld introduced s

tands in F

inla

nd

•R

aiv

ola

, P

unkaharju

and K

itee

–N

ew

introduced s

tands in F

inla

nd

•R

uots

inkylä

and S

olb

öle

–N

ew

introduced s

tand in Icela

nd, surrounded b

y a

sea

•A

ltogeth

er 986 m

ycelia

lculture

s w

ere

isola

ted fro

m s

urface s

teriliz

ed n

eedle

s

•The isola

tes w

ere

cla

ssifie

d to O

pera

tional Taxonom

ic U

nits (O

TU

) usin

g 1

8S

rDN

Apro

filin

g a

nd w

hen n

ecessary

als

o o

ther m

ole

cula

r m

eth

ods

–at le

ast m

ost of th

e O

TU

sshould

be e

quiv

ale

nt to

bio

logic

al specie

s

Denaturing Gradient Gel Electrophoresis (D

GGE)

•DGGE is a mutation detection system

based on two principles

•The melting point of DNA double helix in a denaturing gradient depends on its

nucleotide sequence

•The migration rates of dsD

NA, partially m

elted DNA and ssD

NAare different in

acrylamid

gel electrophoresis

FungalidentificationbyDGGE profiling

•In DGGE profilingonePCR-productis

producedfrom

allspeciesin the sample

–accordingto the specificityof the

primers

Sam

plesof differentfungalspecies

from

pure cultures(V

ainio &

Hantula 2000)

Sam

plesfrom

decayingwood(V

ainio

& Hantula 2000)

18S rDNAprofiling of needle endophytesof Siberian larch

•Partially amplified 18S rDNA-fragments were separated in

DGGE

•It has previously been shown that a combination of the tw

o

primer pairs used in this study separate different species

from each others efficiently (Vainio &

Hantula 2000;

MycolRes

104, 927)

•The amplification products from m

ost m

orphologically

different isolates were separated in DGGE

–in some cases isolates with slightly different

morphologies migrated identically, and were grouped

to a single OTU

•The amplification products from m

ost m

orphologically

similar isolates migrated identically in DGGE

–additional analyses based on ITS were conducted on

these exceptional cases

The occurrence of OTUsin different stands

•Two of the endophytesin Archangel

and Finland were found also in

Iceland

•All endophytescommon in the

Archangel area occurred also in

Finland

•The most common OTU 21-24 was

not present in Solböleor Iceland

•There were several OTUsin

introduced areas that did not occur in

Archangel

–potential examples of host

changes from other plants

(possibly European larch) to

Siberian larch

0 %

10 %

20 %

30 %

40 %

50 %

60 %

70 %

80 %

90 %

100 %

AR

KI

PU

RU

SIC

Diversity of Siberian larch endophytesin different

populations

•The lowest diversities were observed in Iceland and in Archangel

–both based on the number of OTU´s and Shannon-W

eaver index

•There were no systematic differences in diversities according tothe stand age in Finland

Shannon-W

eaver in

dex

0

0.51

1.52

2.5

Arc

hangel

Raiv

ola

Kite

ePunka

harju

Ruots

inky

läS

olb

öle

Icela

nd

num

ber of O

TU

´s

05

10

15

20

25

30

35

Arc

hangel

Raivola

Kitee

Punkaharju

Ruots

inkylä

Solb

öle

Icela

nd

Taxonomic considerations

•Comparison of 18S rDNAsequences of

endophytesto those in GenBankdid not lead to

species identifications

•A phylogeny was determined for some

interesting OTUsand closely m

atching

GenBanksequences

•Some of the endophyteswere more or less

closely related to some other species

OTU G7 ~ H

ypoxy

lonsu

bmonticu

losu

m

OTU G6 ~ G9 ~ P

ara

phaeo

sphaer

ia

quadrise

ptata

and Sep

torianodoru

m

OTU G10 ~ L

euco

stomaper

soonii

OTU G11 ~ D

isco

sphaer

inafagi

OTU G17 ~ C

occ

odiniu

mbartsc

hii

•All relatives of endophytesbelonged to

ascomycetousgroups including pathogens

Interpreting the data: three groups of endophytes

•Only two endophyteshad co-m

igrated to Iceland

–suggests that only a small number of endophyteswould be co-introduced with their

host plants

•group I: man-m

ediated invaders

•hypothesis I: low frequency

•All species common in the natural area were found in introduced stands in Finland

–suggests that the endophytesnaturally follow their host plants to new

sites provided

that the distance from natural area is not too long

•group II: followers

•hypothesis II: commonly observed within the natural spore dispersal area

•Several endophyticspecies were found in introduced stands but not in the natural stand

–might suggest that new

species could inhabit the introduced plant in the new

area

•group III: host extenders

•hypothesis III: relatively common

Could further inform

ation on the history of endophytesbe

obtained based on their population structures?

•a man-m

ediated invader endophyte

–should be found in Iceland

–the degree of genetic differentiation should be high at least in

Iceland

–the degree of genetic variation should be low in Iceland

–in Finland the degree of genetic differentiation among populations depends on the

gene flow over short distances

•an endophyte that naturally followed the host to new

areas

–should be found only in Archangel and Finland

–the degree of genetic differentiation should be low

•a generalist endophyte that expanded its host range from another

plant species

–should be found in all locations where its original host occurs

–the degree of genetic differentiation should be extremely high between Iceland and

other populations (norm

al spore dispersal) or

–there should be no genetic differentiation at all (exceptional long distance spore

dispersal)

The population structure of OTU G21-24

•24 and 20 alleeleswere observed among the two hypervariableloci in

a sequence analysis of 59 isolates from three populations

•No differentiation was observed between the three stands (F

STwas

0.008)

•No clustering of isolates from single was observed in dendrogram

analysis of alleles ( �

Archangel, �Raivola, �Ruotsinkylä)

•Neither were there considerable differences in gene diversities

between the populations

•Thus, no evidence for genetic bottlenecks was observed

Location

Locus 1

Locus 2

Com

bin

ed

num

ber

of

allele

s

gene

div

ers

ity

Fst

num

ber

of

allele

s

gene

div

ers

ity

Fst

gene

div

ers

ity

Fst

Ark

angel

80.7

95

11

0.8

68

0.8

32

Raiv

ola

12

0.9

21

10

0.8

63

0.8

92

Ruots

inkylä

12

0.9

24

50.6

49

0.7

87

Com

ple

tedata

24

0.0

13

20

0.0

03

0.0

08

Interpreting the data: population structure and history of the

most common endophyte (G21-24)

•Not observed in Iceland or Solböle

•Elsew

here populations extrem

ely variable, but not genetically differentiated

–suggests efficient gene flow between the populations

•Observations in relatively good accordance with G21-24 being a fungus that

migrated to Finland after the larch stands had been established

•Could also be a host expander, provided that the original host does not exist in

Iceland

The population structure of OTU G2-5

•Five populations were analysed using

sequences in one specific m

arker

–Archangel, Kitee, Punkaharju, Solböle

and Iceland

•Only four alleles were detected

•Only six isolates were available from the

Archangel population

–no variation occurred among them

•The population were genetically

differentiated as Gstvalues were high

–among all populations 0.254

–among all -Archangel 0.192

–among all -Iceland 0.295

–among Finnish populations 0.206

Distribution of alleles in different

populations

0 %

20 %

40 %

60 %

80 %

100 %

Arc

hangel

Kite

e

Punka

harju

Solb

öle

Icela

nd

Interpreting the data: population structure and history of the

secondmostcommon endophyte (G2-5)

•Common or relatively common in all locations

•Populations genetically differentiated

–the degree of differentiation roughly equal among all populations

–suggests lack of gene flow between populations i.e. low m

igration

potential

•Observations in relatively good accordance with G2-5 being a m

an-m

ediated

invader that was co-introduced with its host to Iceland (and probably also to

Finland)

•However...

–one of the isolates in Iceland had a specific allele

–the degree of genetic variation was equal to other populations

The alder rust story

•Jarkko Hantula

•Tim

oKurkela

•Stephen Hendry

•TakehiroYam

aguchi

•HalvorSolheim

•Adrian Bolay

•Thomas Cech

•Erhard Halmschlager

•Marja-LeenaSantanen

Photograph: ErkkiOksanen

Occurrence of a new

alder rust epidem

ic in Finland

•A new

alder rust was observed in Estonia, Lithuania, and Latvia in 1996

•In Finland, Poland, Austria and Germany in 1997

•In Norw

ay and Hungary in 2001

•In Switzerland in 2002

•More recently also in Slovakia

•Based on urediniospore

morphology it was different from the alder rust

previously described in Europe, and shown by Roll-H

ansen and Roll-H

ansen

to be closely related to the birch rust, M

elampso

ridiu

mbetulinum

•We collected isolates from m

any countries in Europe, Scotland (where alder

rust has continuously occurred) and Japan

Variation in the sequence of the ITS-region of the ribosomal

gene cluster

1 1 1 1 1 1 1 1 1 1 1 2 2 3 3 4 4 4 4 4 5 5 5

2 2 3 4 7 0 1 1 2 3 3 3 3 4 8 9 4 6 0 9 0 0 2 9 9 0 0 2

5 8 5 0 8 9 0 1 7 3 4 6 7 7 5 0 0 7 9 5 3 7 8 0 1 0 6 7

AUT2 C T G A A - - T T - - T T A A A A A T T C C A C A G A T

CH2 . . . . . - - - . - - . - . . . . - . . . . . . . . . .

FIN1 . . . . . - - . . - - . . . . . G . . . T . T . . . . .

FIN4 . . . . . - - . . - - . - . . . . . . . . . . . . . . .

JAP1 A . . . . - - . . - - . - . . . . . . . . T . . . . . .

JAP2 . . . . . - - . . - - - - . . T . . C . . . . . . . . .

NOR1 . . . . . - - . . - - . - . . . . . . . . . . . . . . .

SAL6 . - A - G C - . A A T . A C - . . . . G T . T T G A G .

SKG2 . - . - G C - . A A T . A C - . . . . G T . T T G A G C

SKG6 . - . - G C - . A A T . A C - . . . . G T . T T G A G .

SKOU3 . - . - G C C . A A T . A C - . . . . G T . T T G A G .

M._betulinum . - . - G - - . A A T . A C - . . . . G T . T T G A G .

Clustering analysis of the ITS sequences

•In Neighborjoining analysis

two groups of sequences do

occur

–Cluster 1 is composed of

sequences from new

epidemic isolates and

Japanese isolates

–Cluster 2 is composed of

sequences from Scotland

and birch rust

•Therefore the new

alder rust

must be an invader from East

Asia, and at least in Finland has

taken advantage of the

increasing number of larch trees

in the country

CH

1

JA

P1

FIN

3

FIN

1

FIN

4

JA

P2

CH

2

NO

R1

NO

R6

AU

T2

NO

R2

SK

OU

3

M. betu

linum

SK

G6

SK

G2

SA

L6

55

22

28

99

113

0.0

02

Phytophth

ora

sp. on Rhododendron in Finland

Arja Lilja, Anna Rytkönen, Mirkka Kokkola, Päivi Parikka & Jarkko Hantula

•Phytophth

ora

ramoru

mhas

beenfound

since

2004 in imported

Rhododendrons, and

also

in FinnishRhododendron seedlings

•In analysesof theseseedlingsalso

another

and relativelypoorlyknownPhytophth

ora

inflata

was

observed

–inoculationexperim

entswere

conducted

in co-operationbetween

Finnish Forest Research Institute ,

MTT AgrifoodResearch Finland and

Finnish Food Safety Authority Evira

–allNorw

ayspruce

seedlingstested

got

killedin greenhouse

conditions

•Thereis no evidence

for the dispersalor

pathogenicsymptomsassociated

withthese

Phytophth

orasin the Finnishnature

PhotographbyDr. Arja Lilja

General implications for Finnish forests

•In Finland four dominant tree species occur

•Due to the restricted number of tree species, the

forests in Finland would suffer considerably if an

extremely serious pathogen, equalling to

Cry

phonec

tria

para

sitica, would invade in the

country

–possible threats could be for example

Cro

nartiu

mquer

quum/P

erid

ermiu

mhark

nes

sii,

Inonotu

sto

men

tosu

sand P

hellinusweirii.

–the risk for introduction from North America

seems to be relatively low but not negligible due

to the relatively inefficient co-introduction,

whereas pathogens from Asia cause a high risk

•a recent example is the appearance of

Melampso

ridiu

mhiratsuka

numfrom East

Asia to Finland (and other Europe)

Per

ider

miu

mhark

nes

siion pine

PhotographProf. Tim

o Kurkela

General implications for Finnish forests

•The second type of risk is caused by the

endophytesthemselves

–the capability of endophytesto act as

pathogens on new

host species

•Phytophth

ora

inflata?

–the capability of microfungito gain

new

pathogenic capabilities via

hybridization

•Fortunately, man-m

ediated introductants

seem to be rare.

•However, the question is -are we doing

enough to avoid disastrous introduced

pathogens in future?

Photograph: Erkki Oksanen

Acknowledgments

•Majand TorNesslingfoundation

•Academ

y of Finland