BIOLOGY ECOLOGY GENETICSof FOREST FUNGI

• Lesson 1 of BEG Spring 2011

• Taught by Dr. Matteo Garbelotto– Matteog@berkeley.edu– Office: 338 Hilgard Hall

– Office hours:

WEDNESDAYS at 3 pm, THURSDAYS at 1 pm

Course info

• One two hour lecture per week• One quiz every week on previous lecture and

reading assignments• NO syllabus, but….• Some readings will be emailed to students• Web site: www.matteolab.org

» Link to UCB course» POWERPOINT LECTURES, assigned readings» Posted on Fridays

Course info

• One final quiz (3 questions out of 9)• One short Powerpoint presentation• Possibility to improve grade by writing paper

(5 pages) on same topic as oral presentation• One computer lab: use of software for genetic

analyses• Participation in SOD blitz mandatory

GRADING

• 25% class participation

• 25% weekly quizzes

• 25% final quiz

• 25% oral presentation

COURSE PROGRAM-1

• What is an emergent disease ?• What do plant diseases exactly do?• How do plant and pathogens interact?• What are the evolutionary consequences of

emergent diseases?• What causes plant diseases• How can we use genetic information to identify

a pathogen? DNA, chromosomes and loci

COURSE PROGRAM-2

• DNA used to identify a species • DNA used to identify an individual within a

species• Following an individual pathogen to understand

infection process • Using genes to tag individuals; how far does

an individual go?• Identify populations and migration among

populations

COURSE PROGRAM-3

• Identify source of a pathogen and phylogeography

• Determine whether a population is introduced or native

• Rapid evolutionary changes linked to introductions

• Epigenetics of disease

EMERGENT FOREST EMERGENT FOREST DISEASES: ARE THEY A DISEASES: ARE THEY A

THREAT TO NATIVE THREAT TO NATIVE ECOSYSTEMS?ECOSYSTEMS?

Matteo Garbelotto

U.C.Berkeley

““Native diseases”:Native diseases”:

• Indigenous microbesIndigenous microbes

• Plant ecosystems have co-evolved to resistancePlant ecosystems have co-evolved to resistance

• Native diseases are an important component of Native diseases are an important component of forest ecosystemsforest ecosystems

““Roles of native diseases”:Roles of native diseases”:

• Thin natural populations of treesThin natural populations of trees

• Optimal allocation of resourcesOptimal allocation of resources

• Selection for a genetically diverse host populationSelection for a genetically diverse host population

• Maintain tree rangesMaintain tree ranges

• Succession: nutrient cyclingSuccession: nutrient cycling

““Emergent diseases”:Emergent diseases”:diseases on the risediseases on the rise

• New introduced, exotic, organismsNew introduced, exotic, organisms

• Good gone bad: e.g. climate change or Good gone bad: e.g. climate change or human activities trigger excessive human activities trigger excessive pathogenicity of native organismpathogenicity of native organism

DISEASE TRIANGLEDISEASE TRIANGLE

PathogenPathogenHostHost

EnvironmentEnvironment

DISEASE TRIANGLEDISEASE TRIANGLE

PathogenPathogenHostHost

EnvironmentEnvironment

HIGH DISEASE

PathogenPathogen

Does it need a wound to infect a host?Does it need a wound to infect a host?

Can it survive in the environment without a host?Can it survive in the environment without a host?soil, watersoil, wateron alternate hoston alternate host

How does it move around?How does it move around?airborne/waterborneairborne/waterborneanimal vectorsanimal vectorshumanshumans

HostHost

Must be physically present with pathogenMust be physically present with pathogen

Must be physically compatible with pathogenMust be physically compatible with pathogen

Must provide window of opportunity for infection Must provide window of opportunity for infection

Genetic resistance?Genetic resistance?often lack of resistance often lack of resistance if disease is introducedif disease is introduced

What type of resistance?What type of resistance?simple= one genesimple= one genecomplex=several genescomplex=several genes 0

102030405060708090

100

SimpleComplex

EnvironmentEnvironment

ClimaticClimatic

As modified per human managementAs modified per human management

Species compositionSpecies compositionPlant densityPlant density

““Emergent diseases”:Emergent diseases”:1: 1: hosthost

• New host-pathogen combinations:New host-pathogen combinations:

exotic hostsexotic hosts

hosts planted off sitehosts planted off site

Cypress canker by Cypress canker by Seiridium Seiridium cardinalecardinale

• Pathogen was first described in California in the 20s. Later it was described in Italy where it started a serous epidemic of Italian cypress

• Belief that pathogen is native to California: is that true and why is it then causing a significant disease in our state?

Conidia of Conidia of Seiridium Seiridium cardinalecardinale observed by observed by

optical microscope optical microscope and SEMand SEM

Use of molecular genetics to Use of molecular genetics to resolve issue of origin of resolve issue of origin of

pathogenpathogen

• Used a technique similar to the one used in human forensics

• Native populations should comprise many different individuals genetically

• Introduced populations should be genetically simpler because of bottleneck related to introduction events

RESULTS: CA vs. EuropeRESULTS: CA vs. Europe

o California population diverse genetically= native to the state

o European population show no diversity=introduced

Symptoms caused by Symptoms caused by Seiridium cardinaleSeiridium cardinale on on Cupressus macrocarpaCupressus macrocarpa (above) and (above) and xxCupressocyparisCupressocyparis leylandiileylandii (right) (right)

Fig. 3

Why a disease in CA?Why a disease in CA?

• If pathogen is native to California, why is it causing such a serious disease?

• We observed that disease incidence is variable with:– cypress species, – location,

Range of susceptibilityRange of susceptibility

• Leyland cypress, Italian, monterey are listed as most susceptible

• Arizona and McKnob are regarded as more resistant

Range of susceptibilityRange of susceptibility

• 90% of Leyland are heavily infected

• 10% of monterey

• LEYLAND CYPRESS IS AN ORNAMENTAL CROSS, NOT NATIVE

Range of susceptibilityRange of susceptibility

• Monterey is more susceptible in inland areas where it is NOT NATIVE: we believe that colder temperatures cause more wounds that lead to infection

CONCLUSIONSCONCLUSIONS

• Cypress canker is a serious disease in Europe because pathogen was introduced

• Cypress canker is a serious disease in California because hosts were introduced either through planting off range (Monterey cypress) or because host is artificial creation (Leyland cypress); extinction of LEYLAND is most likely

““Emergent diseases”:Emergent diseases”:2: 2: environmental changesenvironmental changes

• Forestry and intensive forest use:Forestry and intensive forest use:

timber productiontimber production

tree felling and creation of stumpstree felling and creation of stumps

fire exclusion and increase in densityfire exclusion and increase in density

oversimplified forest compositionoversimplified forest composition

changes in forest compositionchanges in forest composition

changes in forest structure changes in forest structure

HeterobasidionHeterobasidion root disease root disease

• Heterobasidion (a bracket or shelf mushroom) infects trees through wounds and stumps, then it spreads through the roots to neighboring trees

• With tree felling,stumps and wounds are created, suddenly exponentially increasing infection levels

Heterobasidion shelf fruit-body

Use of molecular geneticsUse of molecular genetics:

• Differentiate Heterobasidion on fir/sequoias (H. occidentalis) from that on pine/junipers (H.irregularis)

• Show that airborne meiospores are responsible for most infection of Heterobasidion

• Show that in pines most infections start on stumps and that in true firs most infections on wounds

True firs

Pines

Each spore is a genetically different individual:

In pines we found the same genetic individual in stumps andadjacent trees indicating direct contagion between the two

In true firs and true firs/sequoias we find same individual in adjacent standing trees indicating infection not linkedto stumps but to wounds on standing trees

CONCLUSIONS:CONCLUSIONS:

• Logging activities increase Heterobasidion infection because of stump creation in pines and because of wounding in true firs sequoias

• We have shown that in pine stumps H. irregularis and H. occidentalis can both be present and create a new hybrid entity

• We have shown that in the past these hybridization events have lead to sharing of genes among these two species (Horizontal gene transfers)

ArmillariaArmillaria root diseases root diseases

• Armillaria, the honey mushroom, normally infects the roots of trees. It can be a saprobe and a pathogen and is common amongst oaks

• If woodland composition shifts to pine/oak, pines become the target of attacks and gaps in canopy enlarge over time. Stress (e.g. flooding) exacerbates susceptibility

Clusters of Clusters of ArmillariaArmillaria

How Does it Infect?How Does it Infect?

SOURCE: http://www.forestpathology.org/dis_arm.html

Two means of dispersal to other trees:

1.Mycelium can grow through direct root contacts and grafts with uninfected trees.

2.Rhizomorphs can grow through soil to contact uninfected trees.

DEAD OAK

OAK or PINE

What are Rhizomorphs?What are Rhizomorphs?• …“conglomerations of

differentiated parallel

hyphae with a protective

melanized black rind

on the outside.”

• Rhizomorphs are able to transport food and nutrients long distances which allows the fungus to grow through nutrient poor areas located between large food sources such as stumps.

SOURCE: http://www.nifg.org.uk/armillaria.htm

SOURCE: http://botit.botany.wisc.edu/toms_fungi/apr2002.html

Humongous FungusHumongous Fungus

It’s One of U-HAUL’s “Bizarre Roadside Attractions”

http://botit.botany.wisc.edu/toms_fungi/apr2002.html

CONCLUSIONSCONCLUSIONS

Human activities shifting from oak woodlands to mixed oak-pine lead

to large mortality gaps in pines around oaks if honey mushroom is

present

CHANGING SPECIES COMPOSITION LEADS TO

SEVERE DISEASE

Many gaps with very little regeneration and have not closed in

Change in gap area 1972-1999

YearArea in gaps

(m2) Percent in gaps

1972 6125 3.5

1999 53,981 31

““Emergent diseases”:Emergent diseases”:3: 3: exotic pathogensexotic pathogens

• 99% of times human responsible for their 99% of times human responsible for their introduction introduction

Like the conquistadores brought Like the conquistadores brought diseases that were lethal to diseases that were lethal to those who had never been those who had never been

exposed to them, so do exotic exposed to them, so do exotic diseases cause true devastation diseases cause true devastation in plant communities because of in plant communities because of

lack of coevolution between lack of coevolution between hosts and microbeshosts and microbes

California invaded: 1849 A.D.California invaded: 1849 A.D.

New hybrid root pathogenNew hybrid root pathogen1990s1990s

White pine blister rustWhite pine blister rust1930s1930s

Port Orford Cedar Root DiseasePort Orford Cedar Root Disease1950s1950s

Pitch canker diseasePitch canker disease1980s1980s

Dutch Elm DiseaseDutch Elm Disease1960s1960s

Sudden Oak DeathSudden Oak Death1990s1990s

Oak root cankerOak root canker20002000

Manzanita/madrone Manzanita/madrone die-backdie-back

Canker-stain ofCanker-stain ofSycamores 1980’sSycamores 1980’s

How can people transport How can people transport pathogenspathogens

• By transporting plants and plant parts– Crops, and seeds– Raw food– Ornamental plantsUntreated lumberSoilInsects vectoring fungiMilitary activity

The Irish Potato FamineThe Irish Potato Famine

• From 1845 to 1850• Phytophthora

infestans• Resulted in the

death of 750,000 • Emigration of over 2

million, mainly to the United States.

Is it exotic?Is it exotic?

• Our studies have indicated that California population is extremely simplified, basically two strains reproducing clonally as expected of an introduced organism

• Many hosts appear to have no resistance at all

• Limited geographic distribution

Where does it come from?Where does it come from?

• It is unknown where pathogen originally comes from, but previous studies have shown that California forest population is derived from a relatively genetically diversified US nursery population, indicating ornamental nurseries were the most likely avenue for pathogen introduction

Where was it introduced?Where was it introduced?

• First reports mid 90’s• Pathogen identified in

2000• By then, the pathogen

was widespread• CLUES: severity of

symptoms and anedoctal stories

Positive isolationP. ramorum

We found We found same same genotypes in genotypes in nurseries and nurseries and forests proving forests proving origin of wild origin of wild outbreakoutbreak

nurseries

Introduction phase

1- Escape of pathogen fromInfected nursery plants at twolocations: Mount Tamalpais(Marin County), and Scott’sValley (Santa Cruz County)

2- Nurseries and two siteshave identical strain composition, but distancebetween sites is impossiblefor natural spread of organism

What favors invasion of exotic What favors invasion of exotic fungi ?fungi ?

– Density of host increases severity of disease

– Corridors linking natural habitats

– Synchronicity between host susceptibility and pathogen life cycle

– Ecological and environmental conditions

Bay/OakBay/Oak association association

Bleeding cankerBleeding canker

Canker margin in phloemCanker margin in phloem

Bay Coast Live Oak (no sporulation)

SporangiaSporangia

Synchrony pathogen-host

Susceptibility of oaksSusceptibility of oaks(lesion size)(lesion size)

0

2 0

4 0

6 0

8 0

0 6 1 2 1 8 2 4 3 0 3 6 4 2 4 8 5 4

T i m e ( h )

Average lesion (mm_)

0

1 0

2 0

3 0

4 0

5 0

1 5 1 7 1 9 2 1 2 3 2 5 2 7 2 9

T e m p e r a t u r e ( ° C )

Lesion area (mm

2

)

Wetness > 12 h

Temp >19 C

Bay Laurel / Tanoak SOD Spore Survey

Date

Temp (C)

Rain (mm)

How to control emergent How to control emergent exotic diseasesexotic diseases

• PREVENT THEIR INTRODUCTION• LIMIT THE HUMAN-SPREAD OF

PATHOGENS (infected plants, plant parts, dirty tools)

• EMPLOY HOST RESISTANCE• CHEMICAL AND OTHER MITIGATION

STRATEGIES

Forest pathogens can never be Forest pathogens can never be eradicatederadicated

PREVENT: DiagnosePREVENT: Diagnose

Symptoms relatively generic, veryvariable, and pathogen not always culturable

LAB CULTURESDNA TESTS

AgriFos and AgriFos and PentraBark PentraBark

Topical Topical ApplicationApplication

++

0

2

4

6

8

10

12

14

Azomite Positive Control Agrifos

Agrifos vs. Azomite Treatments(efficacy 1 - 24 months)

Can

ker

Siz

e (m

m)

aa aa

bb

Why emphasis on molecular analyses?

• As a way to identify and quantify microbes in the environment

• As a way to understand microbial biology: how do microbes reproduce and infect hosts

• As a way to determine epidemiology: follow the movement of a strain

Why emphasis on molecular analyses?

• As a way to determine potential for spread: use genes as markers for individuals

• As a way to determine whether population of microbes is exotic or native

• As a way to identify source of a pathogen and migration patterns

Why emphasis on molecular analyses?

• As a way to determine the size of the gene pool of a pathogen, Important to scale management options

• As a way to determine rapid evolutionary changes linked to an introduction

• As a way to determine epigenetic effects