The FUN-TASTICThe FUN-TASTIC

world ofworld of

FUN-GIFUN-GI

ESPM 107ESPM 107

Matteo Matteo GarbelottoGarbelotto

Photo: Amanita muscaria in Picea sp. root.

WHAT ARE FUNGI ?WHAT ARE FUNGI ?

• Individuals belonging to the kingdom fungi

• They are actually more closely related to animals than

plants (cell wall contains chitin, do not photosynthesize)

• The feed by extrogenous digestion (food base digested

outside their cells and then simple elements absorbed )

• Indefinite growth: size can range from unicellular

organisms such as yeasts to the largest beings in the

world

Largest living organisms in theLargest living organisms in the

world are fungiworld are fungi

Aerial view of a single fungalAerial view of a single fungal

individual growing in the soil andindividual growing in the soil and

killing treeskilling trees

Tens of acres occupied by a single individual, older thanTens of acres occupied by a single individual, older than

5000 years!!!! (Northern5000 years!!!! (Northern Michigan)Michigan)

Thanks to their web-like indefiniteThanks to their web-like indefinite

growth in soil and plant substratesgrowth in soil and plant substrates

and their way of digesting nutrientsand their way of digesting nutrients

fungi play a critical role in recyclingfungi play a critical role in recycling

nutrients which can then benutrients which can then be

reutilized by plantsreutilized by plants

•Fungi like this one will actually decay

the woody matter and physically free

space for new generations of trees,

besides recycling the nutrients

The The weblike weblike structure of fungi, usuallystructure of fungi, usually

immersed in the soil or in plant matter isimmersed in the soil or in plant matter is

involved in an essential symbiosis thatinvolved in an essential symbiosis that

greatly enhances the ability of plants togreatly enhances the ability of plants to growgrow

•piant

•fungus

The visible part of root tips ofThe visible part of root tips of

most trees is actually a mantlemost trees is actually a mantle

of fungal of fungal hyphae hyphae fused with thefused with the

plant tissueplant tissue

What is the deal of this

mutualism?

• Fungus absorbs nutrients for plants

• Plant gives fungus carbohydrates it produces via

photosynthesis

There are thousands of There are thousands of mycorrhzial mycorrhzial fungalfungal

species, and only at times do they produce thespecies, and only at times do they produce the

classical fruit body (e.g.mushrooms) aboveclassical fruit body (e.g.mushrooms) above

groundground

•In absence of fruit body: how can we identify them?

Diversity of fungi, but all have ideal structure for plantinfection:– hypha/cord/rhizomorph/infection peg/appressorium

– Sexual vs. asexual reproduction: can do both

– Do not photosinthesize

– Chitin in cell wall

– Exogenous digestion

– Indefinite growth

– Phenotypic plasticity and pleomorphisms

Septa

Pores

Pores

CELLS

Fungi… again!

•• ASCOASCOMYCETES

•• BASIDIOBASIDIOMYCETES

• OOMYCETES (fungus-like, water molds)

ASCOASCOMYCETES

•• Yeasts (fermentation,Yeasts (fermentation, human mycoses)human mycoses)

•• Truffles, morelsTruffles, morels

•• Penicillia Penicillia (penicillin), (penicillin), Fusaria Fusaria (potent(potent

toxins, damping off of seedlings), moldstoxins, damping off of seedlings), molds

Ascus is the sack in which the

spores are contained

Asci can be placed on a disk

(apothecium), many apothecia

can be together in a fruitbody

Morel fruitbody

Asci can be carried inside a

flask (perithecium)

Nectria

BASIDIOBASIDIOMYCETES

•• Mushrooms. Mushrooms. mycorrhizalmycorrhizal

•• Wood decay organismsWood decay organisms

• Rusts, Smuts

• Yeasts and damping off

Toadstools and huitacochle are

both basidiomycetes

Basidium means “club”, it

carries the basidiospores

(dispersion propagules) naked

Mycorrhizae are Fungi => absorptive(heterotrophic) => they need hosts =Plants.

Principal mycorrhizal groups:Ascomycetes, Basidiomycetes andZygomycetes.

Mycorrhizae: definition

• Mycorrhiza = in Greek “fungus-root” (Frank, 1885).

• Mycorrhizas are multifaceted associations comprising diverse

morphological, functional and evolutionary categories.

• Mycorrhizas are associations between fungal hyphae and organs of higher

plants concerned with absorption of substances from the soil.

“A symbiotic association essential for one or both partners,

between a fungus (specialized for life in soils and plants) and a

root (or other substrate-contacting organ) of a living plant, that

is primarily responsible for nutrient transfer. Mycorrhizas

occur in a specialized plant organ where intimate contact

results from synchronized plant-fungus development”.

• Most mycorrhizae mutualistic (= beneficial association relationship

between a plant root and a fungus), but not all. Full spectrum of variation

in plant-fungus association.

• There appears to be a continuum of plant responses to mycorrhizal

colonization ranging from positive to neutral or negative.

• In this way mycorrhizal fungi have a wide diversity of roles and can also

function as endophytes, necrotrophs and antagonists of non-host plants,

with roles that can vary during the life of associations. Possible phases:

» Free-living.

» Endophytic.

» Balanced mycorrhizas.

» Exploitative mycorrhizas

» Antagonistic.

» Necrotrophic.

Description and Ecology (1)

Description and ecology (2)

• Most mycorrhizas can be described as balanced mutualistic

associations in which the fungus and plant exchange commodities

required for the growth and survival of both partners.

• The presence of mycorrhiza can increase the uptake of plant nutrients,

specially immobile ions such as P, Zn and Cu, but also mobile ions

such as S, Ca, K, Fe, Mg, Mn, Cl, Br, and N.

• They can increase plant resistance to drought, through improved

absorbance area.

• Increase tolerance to adverse conditions: heavy metals, salinity, pH,…

• The harsher the environment for plants, the more important the role of

mycorrhizae.

• The role of mycorrhizae can also change when the host plant is

stressed, e.g.: Tricholoma matsutake.

• E.g.: high levels of P in soil can inhibit spore germination in some VAM

species.

• Many other benefits: growth hormones, protection from pathogens,…

• Many species in the same plant species, some mycorrhizae shared by

several species, even between woody and herbaceous.

• There can be mycelial connections between neighbor plants (hyphal

networks).

• The majority of mycorrhizal associations provide substantial benefits to

both plants and fungi, and most plants (>90%) need them for their

development. Plants vs. mycorrhizae:

– Obligately mycorrhizal.

– Facultatively mycorrhizal.

– Nonmycorrhizal.

Description and ecology (3)

• ECM species belong to asco-, basidio-, and zygomycetes.

• ECM associations are characterized by the presence of the Hartig Net.

(=labyrinthine hyphae between root cells) and the mantle:

Hartig Net: hyphal envelope around active cortical cells. Primary zone

nutrient transfer fungus-plant.

Mantle: mat of fungal symbiont envoloping around ECM roots, either

smooth or with radiating hyphae (mycelial strands). Increase

absorption area.

• Most ECM are considered obligate simbionts incapable of independent life

without plants.

• ECM infections are initiated from spores, sclerotia (“mycelia =

underground spores”) , hyphae and/or roots of trees.

• Most common among forest and ornamental tree species, in families such

as: Pinaceae, Salicaceae , Betulaceae, Rosaceae, Leguminosae, Ericaceae,

and Juglandaceae.

ECM (Ectomycorrhizas) (1)

• ECM species can range from one host to many.

• Many ECM fruit bodies are edible mushrooms.

• Two basic morphological categories:

• Angiosperms (epidermal).

• Gimnosperms (cortical).

ECM (Ectomycorrhizas) (2)

Amanita caesarea

Different ECM in Angiosperms

and Gimnosperms

(epidermal and cortical).

Diagram of

typical ECM

A.muscaria in P. strobus

Mantle of ECM in P. tremuloidesAstraeus pteridis in E. maculata

A. muscaria in P. radiata

Hartig net in P. tremuloides root.

Suillus subluteus with P.

resinosa

• Vesicular arbuscular mycorrhizas (VAM) (= endomycorrhizas, endotrophic

mycorrhizas)

• Characterized by intracellular root penetration of root cortical cells:

Arbuscules: branch haustoria within root cortex cells; nutrient

exchange between the two symbionts.

Vesicules: hyphal swellings formed within the cells of plant roots (not

always present), to accumulate storage products .

• Produced by Glomalean fungi: Class Glomeromycota (=Zygomicota,

Glomales).

• Some plants can have both VAM and ECM.

• VAM are the most widespread and common root-fungus associations.

• All VAM are obligate simbionts.

• Structures in roots: hyphae, arbuscules, vesicules.

• Structure in soils: hyphae spores

VAM (Vesicular arbuscular mycorrhizas) (1)

VAM (2)

• About 80% plant species present VAM.

• However, apparently little diversity: few genera and few dozenspecies.

Arbuscule Glomus mosseae

VAM in Asarum canadense root

Importance of mycorrhizae in forestry and thefood industry (1)

Forestry and Range Management:

• Spatial distribution of mycorrhizae complex => relation with

establishment of ectomycorrhizal vegetation.

• Reforestations: inoculations. 2 billion tree seedlings grown annually in

USA => need of mycorrhizae (enhance survival).

• Mycorrhizae in herbaceous communities: increase importance in

restoration with herbaceous communities. Much research still needed.

• Relations herbaceous vs. woody species mycorrhizae.

• Consequences to forest and range management: environmental

conditions vs. mushrooms (ECM, basically).

• Particularly important in restoration of arid and semiarid ecosystems

• (=> Mediterranean areas, among others).

Food industry

• There are about 2500 sp of edible mushrooms. It includes about 200 edible

ECM, among them the most expensive and sought: T. melanosporum, T.

magnatum, Tricholoma matsutake, Boletus edulis, Cantharellus cibarius

and Amanita caesarea.

• Estimated annual value of mushrooms: 7.5x109 U$.

• Most of the ECM mushrooms are now supplied from forest harvests.

Others

• Recreational collecting.

• Importance of commercial harvesting for many regional economies.

• VAM as biofertilizer:

• Increase crops with lower levels of nutrients.

• Reduction in agrochemical.

• Bioethical aspects.

• Mycorrhiza in control of soil borne pathogens: interactions mycorrhiza-

pathogen enhance breeding resistance.

Importance of mycorrhizae in forestry and thefood industry (2)

Genetic variation and evolution in fungi

Fungi possess many features that make them ideal for genetic research:

• Most fungi can be grown in pure culture: avoid environmental variability.

• High grow rates, very large number of progeny from each cross, short life

cycle, short genome size and low chromosome number. E.g. 23

chromosomes in humans against 7-20 in Eumycota => 3000 millions of bp

in nuclear DNA in humans vs. 19-47x106 bp in Eumycota.

• Fungi alternate difference n, 2n and/or n+n life patterns = more complex

life cycle, more variety of genetic approaches Haploid life cycles, many

genes expressed then => easy to detect mutant alleles compared with

diploid organisms.

Molecular techniques have been used to accurately identify EMs and their

isolates, detect infections, and monitor the development of infections in

soils.

• In EM among other markers PCR-RFLP (ITS, nuclear rRNA, mRNA) and

RAPD (in disuse). Isozymes to determine species.

• In VAM SSCP and RFLP, mainly (also RAPD).

However, microorganisms populations genetics have been delayed from

animals and plants pop gen, much information is now generation.

Three essential concepts:

1. Individual: careful with mycelial fungi (like mycorrhizae). Take into

account genetic identity and physical continuity, e.g., a colony from a

single spore = one individual.

2. Population (genetics): individuals which, as a result of proximity and

genetic similarity, readily interbreed and exchange genes.

3. Species: theoretical and practical difficulties in defining and

delimiting species => use of genospecies (limits of genetic

exchange).

Population genetics in fungi

Life cycles, mating systems and geneticexchange

Life cycles and the sexual process:

2 Cell (n) fusion (plasmogamy) 1 Dykarion (n+n)

Nuclear fusion (karyogamy) (2n) Meiosis (n)

Mating systems = breeding systems = sel-sterile = heterothallic: prevention of

mating between genetically identical cells. There are several kinds of

mating systems (bifactorial, two loci involved: AB) => increase variability.

Genetic exchange: if genetic differences between homologous chromosomes,

two ways of genetic recombination:

i. Chromosomes from both partners in the preceeding sexual fusion.

ii. Crossing-over = homologous chromosomes break and exchange genetic

material.

=> Increase variability.

(See Figure 5.4. page 214. Carlile and Watkinson, 1994).

Life cycle ofAscomycetes

Life cycle ofBasidiomycetes

Life cycle ofZygomycetes

• Traditional approach: morphology.

• Current approach: molecular biology. Importance of molecular

biology and population genetics on mycorrhizae and on fungi

markets.

• Future: classification trees based on genetics.

• “Mycorrhizas are three way-way interactions of plants, fungi,

and soils, so we must expect environmental and edaphic

factors to affect their structure and function”.

Mycorrhizas and molecular biology

Need of genetic characterization

• Soils that support vegetation are also inhabited by different communities

of fungi, with different and some unknown connections.

• In a forest, each single tree support sa complex community of

mycorrhizal species, that change with the development of the plant.

As an example I discuss Quercus garryana from Valentine et al. (2004):

• Virtually all root tips of Quercus garryana are ectomycorrhizal.

• Identification of species using morphological characteristics and RFLP.

Sample size: 20 trees, 160 samples in southern Oregon.

• Samples from the 10-20 cm of soil and from sporocarps above ground

(haploid).

• 39 ECM morphotypes: number reduced when RFLP.

• DNA analyses usually shows that morphotypes with slight variations in

morphology are genetically distinct. But not always.

• In other species and studies up to 80 morphotypes.

The mycorrhizal community (1)

• Usually many species occurring few times (79% in only one soil core).

• The most abundant Cenococcum geophilum (75% of soil cores): easily

recognizable from its characteristic radiate mantle morphology. Does this fact

partly affect its abundance in most studies?.

• Decrease in mycorrhizal diversity as a function of distance to the tree,

tending towards zero (consequences to management).

• ITS-RFLP patterns from ECM growing below ground did not match those

of sporocarps growing above ground: saprotrophes, fruiting

heterogeneity,… => Generally poor correlation between sporocarps and

ECM status or abundance.

Multiple species of mycorrhizas coexist within cm on a single fine root.

“Island effect”: isolated trees have limited access to ectomycorrhizal inocula

and therefore attain low ectomycorrhizal diversity.

The mycorrhizal community (2)

TheMycorrhizalcommunity

(3)

Morphotypes!!!

Tanoak mycorrhizal communities

Douglas-fir: large mycorrhizal tips,

Tanoak: extremely small

Materials and methods: DNA is

electrically charged and if you put into an electric

field and in a gel matrix it will move through the

gel, following the current

Electrophoresis

The smaller the DNAfragment, the faster (thefurther) it will move

Gel placed in EthidiumBromide, fluorescesunder UV light

Conclusions

• EM taxa shared between two species, but notshown the same individual network is shared

• Lithocarpus may benefit, either if carbon isobtained from Douglas-fir, or at least cost ofmycorrhization is decreased for tanoaks

• More diversity in Douglas-fir probably explainedby older age

• A whole group of EM taxa more abundant ontanoaks: PREFERENTIAL ON TANOAKS?

• Shared EM are common, but great spatialheterogeneity

Study Area

Whitethorn

18S 5.8S 28S

ITS1 ITS2

Lactarius sp.

ITS Alignments

Hydnellumcaeruleum

Cenococcum

Ascom

ycete 37

Phialophora 1

Phialophora 2

Ascom

ycete 6

Lactarius 1

Basidiom

ycete 26

Basidiom

ycete 10

Ram

aria fennica var. violaceibrunnea

Ram

aria formosa

Cortinariod sp. 2

Piloderm

a 3

Basidiom

ycete 12

Cortinariod sp. 5

Melanogaster tuberiform

is

Russula 6

Russula 8

Tricholom

a 3

Ascom

ycete 11

Ascom

ycete 12

Ascom

ycete 2

Phialocephala fortinii

Ascom

ycete 29

Basidiom

ycete 14

Basidiom

ycete 23

Basidiom

ycete 32

Basidiom

ycete 4

Cortinariod sp. 1

Fungal 3

Piloderm

a 1

Russula 1

Russula em

etica

Russula 4

Russula 7

Sebacina 1

Tricholom

a 1

Tricholom

a 5

Alpolva trappei

Am

anita 1

Am

anita gemm

ata

Ascom

ycete 1

Ascom

ycete 10

Ascom

ycete 13

Ascom

ycete 14

Ascom

ycete 15

Ascom

ycete 16

Ascom

ycete 17

Ascom

ycete 18

Ascom

ycete 19

Ascom

ycete 20

Ascom

ycete 21

Ascom

ycete 22

Mortierella verticillata

Ascom

ycete 24

Ascom

ycete 25

Ascom

ycete 26

Ascom

ycete 28

Ascom

ycete 3

Ascom

ycete 30

Ascom

ycete 31

Neonectria radicola var. radicola

Ascom

ycete 33

Ascom

ycete 34

Ascom

ycete 35

Ascom

ycete 36

Ascom

ycete 4

Ascom

ycete 5

Ascom

ycete 7

Ascom

ycete 8

Ascom

ycete 9

Basidiom

ycete 1

Hydnellum

caeruleum

Basidiom

ycete 13

Basidiom

ycete 17

Basidiom

ycete 18

Basidiom

ycete 19

Basidiom

ycete 2

Entolom

a alpicola

Basidiom

ycete 21

Inocybe flocculosa

Basidiom

ycete 24

Basidiom

ycete 25

Basidiom

ycete 27

Basidiom

ycete 28

Basidiom

ycete 29

Basidiom

ycete 3

Basidiom

ycete 30

Basidiom

ycete 31

Basidiom

ycete 5

Basidiom

ycete 6

Basidiom

ycete 7

Basidiom

ycete 8

Basidiom

ycete 9

Boletus cf. edulis

Cortinariod sp. 3

Cortinariod sp. 4

Fungal 1

Fungal 2

Hygrophoroid sp. 1

Hym

enoscyphus 1

Lactarius 2

Lactarius 3

Lactarius 4

Lyophyllum 1

Phialophora 3

Piloderm

a 2

Piloderm

a 4

Russula 2

Russula 9

Sebacina 2

Thelephoroid 1

Thelephoroid 2

Thelephoroid 3

Thelephoroid 4

Thelephoroid 5

Tom

entella 1

Tom

entella 2

Tom

entella 3

Tom

entella 4

Tom

entella 5

Tom

entella 6

Tom

entella 7

Tom

entella 8

Tricholom

a 2

Tricholom

a 4

Tricholom

a

Tricholo

Trich

T

taxon sampled

Basidiomycete treeTricholomatoid

Cortinarioid

Amanitoid

Boletoid

RussuloidAtheliod

Thelephoroid

Gomphoid

Sebacina

Ramaria formosa

Tricholoma matsutake

Cenococcum geophilum

Sebacina

Tricholoma

Russuloid

6 SPECIES

3 SPECIES

6 SPECIES

11 SPECIES

In Moorea…

• Unknown number of VAM

• One species of EM (in Guyana 75)

– On exotic Pinus caribaea

– DNA places it as close relative of nurserygenotype from Europe

? Two characteristics are associated with EMin tropical and subtropical regions:monodominance and invasiveness (ability tofare well independent of soil) ?

Ectomycorrhizal symbionts

Photo: Todd Osmundson

Rhizopogon sp.

Found only with the introduced Caribbean Pine (Pinus caribaea),and possibly with She-oak (a.k.a., Australian Ironwood; Casaurinaequisetifolia).

In Ecuador…

• Pinus radiata introduced at high elevation

• Trees dwarfed, but mushroom productionastonishingly high

• Only five mycorrhizal species introduced (hencelack of competition)

• Mycorrhizae may be able to use carbon sourcefrom rich volcanic carbon layer where grassesgrow---->long term impact on site productivity!

In Guyana…• Legumes, mostly Cesalpionidae

• No effect of EM on litter decomposition

• Clear effect of Ca

In South-East Asia

• Family Dipterocarpaceae is EM

• Tropical soils poor in minerals (especially if primarycover is taken out)

• EM increase survival and growth of seedlings in poorsoils (P, K, Mg, and CA)

• Significant host-specificity reported: possibly related topattern of distribution of trees (e.g. in Guyana, Costa-Rica, and Mexico EM associated with monodominance)

DISEASE!!

• Symptoms vs. signs; e.g. chlorosis vs.

fruit-body

• The disease triangle

host-pathogen-environment

• Susceptibility of individuals or of portionsof individuals

• Genetic variability

• Basic compatibility (susceptibility) betweenhost and pathogen

• Ability to withstand physiologicalalterations

Cankers byCankers by P. ramorum P. ramorum at 3 monthsat 3 monthsfrom time of inoculation on two coastfrom time of inoculation on two coastlive oakslive oaks

host-pathogen-environment

• Basic compatibility with host (virulence)

• Ability to maintain diversity: sex vs. no sex

• Size of genetic pool

• Agressiveness (pathogenicity) towardshosts

• Ability to survive without host

Chlamydospores of Chlamydospores of P. ramorumP. ramorum

Pr75Pr75 Qa Qa MontereyMonterey Pr87 Am Marin Pr87 Am Marin Pr86b Am Marin Pr86b Am Marin Pr86a Am Marin Pr86a Am Marin Pr84 Soil Marin Pr84 Soil Marin Pr82 Vo Marin Pr82 Vo Marin Pr80 Vo Marin Pr80 Vo Marin Pr72 Pr72 Rh Rh Alameda Alameda Pr65 Pr65 Qp Qp Santa Cruz Santa Cruz Pr58 Vo Marin Pr58 Vo Marin Pr50 Pr50 Qa Qa Sonoma Sonoma Pr201b Pr201b Rh Rh Santa Cruz Santa Cruz Pr201a Pr201a Rh Rh Santa Cruz Santa Cruz Pr47b Pr47b Qa Qa Sonoma Sonoma Pr47a Pr47a Qa Qa Sonoma Sonoma Pr35 Pr35 Qa Qa Sonoma Sonoma Pr28 Ld Sonoma Pr28 Ld Sonoma Pr24 Pr24 Qa Qa Sonoma Sonoma Pr22 Pr22 Qa Qa Sonoma Sonoma Pr20 Pr20 Qa Qa Sonoma Sonoma Pr19 Pr19 Qa Qa Napa Napa Pr16 Pr16 Qa Qa Santa Cruz Santa Cruz Pr13 Pr13 Qa Qa Santa Cruz Santa Cruz Pr11b Pr11b Qa Qa Monterey Monterey Pr11a Pr11a Qa Qa Monterey Monterey Pr10 Ld Monterey Pr10 Ld Monterey Pr08 Pr08 Qa Qa Napa Napa Pr06 Pr06 Qa Qa Marin Marin Pr05 Ld Marin Pr05 Ld Marin Pr04 Pr04 Qk Qk Marin Marin Pr03 Ld Marin Pr03 Ld Marin Pr88 Pr88 Uc Uc Sonoma Sonoma Pr89 Pr89 Uc Uc Sonoma Sonoma Pr90 Pr90 Qa Qa Marin Marin Pr91 Pr91 Uc Uc Sonoma Sonoma Pr97 Pr97 Qa Qa Napa Napa Pr102 Pr102 Qa Qa Marin Marin Pr103 Ld Marin Pr103 Ld Marin Pr104 Ld Marin Pr104 Ld Marin Pr107 Pr107 Uc Uc Sonoma Sonoma Pr110 Pr110 Uc Uc Marin Marin Pr112 Pr112 Uc Uc Marin Marin Pr113 Pr113 Uc Uc Marin Marin Pr114 Pr114 Uc Uc Marin Marin Pr115 Pr115 Uc Uc Marin Marin Pr116 Pr116 Uc Uc Marin Marin Pr136 Pr136 Uc Uc Marin Marin Pr156 Ld Oregon Pr156 Ld Oregon Pr157 Ld Oregon Pr157 Ld Oregon Pr158 Ld Oregon Pr158 Ld Oregon PrJL3.1 Ss Sonoma PrJL3.1 Ss Sonoma PrSDC21.6 Ss Sonoma PrSDC21.6 Ss Sonoma

Pr36 Pr36 Qa Qa Sonoma Sonoma Pr27 Pr27 Qa Qa Marin Marin

Pr57 Ld Santa Clara Pr57 Ld Santa Clara Pr70 Vo Marin Pr70 Vo Marin Pr159 Ld Oregon Pr159 Ld Oregon

Pr52a Pr52a Rh Rh Santa Cruz Santa Cruz Pr52b Pr52b Rh Rh Santa Cruz Santa Cruz PrCoen Rh PrCoen Rh Santa Cruz Santa Cruz

PrJL3.5.3 Ss Sonoma PrJL3.5.3 Ss Sonoma Pr106 Pr106 Uc Uc Sonoma Sonoma

Pr71 Pr71 Qa Qa Sonoma Sonoma Pr01 Pr01 Qa Qa Marin Marin

PrE9/95 PrE9/95 Rh Rh Germany Germany PrE16/99 PrE16/99 Vb Vb Germany Germany

PrE12/98 PrE12/98 Rh Rh Germany Germany PrE104 Water Germany PrE104 Water Germany

PrE69082 PrE69082 Rh Rh Germany Germany PrE9/3 Water Germany PrE9/3 Water Germany

PrE14/98-a PrE14/98-a Rh Rh Germany Germany Pl33 Cl Del Norte

Pl16 Soil Josephine Pl27 Tb Del Norte0.1

Clone groupClone group

European groupEuropean group

P. P. lateralislateralis

((outgroupoutgroup))

100100

9696

9393

8989

6767

host-pathogen-environment

• Temperatures

• Shading

• Relative humidity

• Free standing water

• pH and any potentially predisposingfactors

• Nutrient status

Co

lon

y d

iam

ete

r (m

m)

at

13 d

ay

sC

olo

ny

dia

me

ter

(mm

) a

t 13 d

ay

s

Presence of free water

Between 6 and 12 hours requiredBetween 6 and 12 hours requiredfor infection of bay leavesfor infection of bay leaves

++

Temperature at 19 CTemperature at 19 C

COMMON GARDEN EXPERIMENTS NECESSARY

DISEASE: plant microbe

interaction• Basic compatibility need to be present

• Chemotaxis, thighmotropy

• Avirulence in pathogen matched by resistancein host according to the gene for gene model(vertical resistance)

• Pathogenicity factors such as toxins andenzymes important in the infection process

• Multi-locus , additive, horizontal resistanceconferred by multiple loci: harder to harness inbreeding programs but more durable

Gene for gene

• Resistance in host is dominant

• Virulence is recessive

ar aR

Ar AR

Because there are costs involved in havingresistance and in being virulent, bothtypes of alleles are maintained. This leadsto maintenance of genetic diversity of host.

Host specificity in tropical

ecosystems• Obvious signs of ongoing co-evolution

(pathogen more aggressive on plants that arerelated) indicate host specialization important intropics

• Gradient from biotrophic (high hostspecialization) to necrotrophic (low hostspecialization)

• If low diversity in host: more host specialization

• In limiting conditions (ability to grow in anoxia)then generalism expected

Some pathogen roles in

natural plant communities

• Selection of individuals best suited for the site

• Maintenance of genetic diversity and stability in host

plant populations

• Establishment or maintenance of host geographic

ranges

• Natural succession

• Regulation of stand density, structure, and

composition

Human activities affecting

disease incidence in forests

• Introduction of exotic pathogens

• Planting trees in inappropriate sites

• Simplifying genetic make-up of host

• Changing stand density, age structure,

composition, fire frequency

• Wound creation

• Pollution, etc.

Effects of fire exclusion

Effects of diseases on host

mortality, growth and

reproduction

• Young plants killed before reaching

reproductive age

• Affect reproductive output

• Directly affect flowers and fruits

Categories of wild plant

diseases• Seed decay

• Seedling diseases

• Foliage diseases

• Systemic infections

• Parasitic plants

• Cankers, wilts , and diebacks

• Root and butt rots

• Floral diseases

Seed diseases

• Up to 88% mortality in tropical Uganda

• More significant when seed production is

episodic

Stress cone crop BS on DF

Seedling diseases

• Specific diseases, but also diseases of adulttrees can affect seedlings

• Pythium, Phytophthora, Rhizoctonia, Fusariumare the three most important ones

• Pre- vs. post-emergence

• Impact: up to 65% mortality in black cherry.These diseases build up in litter

• Shady and moist environment is very conduciveto these diseases

Foliar diseases

• In general they reduce photosynthetic ability by reducing

leaf area. At times this reduction is actually beneficial

• Problem is accentuated in the case of small plants and in

the case other health issues are superimposed

• Often, e.g. with anthracnose,needle cast and rust

diseases leaves are point of entry for twig and branch

infection with permanent damage inflicted

A foliar pathogen employed as a

biological control of Myconia• Myconia extremely invasive plant from South

and Central America (ERH/EA)

• Fungal pathogen Colletotrichumgleosporoidoedes can be a foliar pathogen

• Released in Tahiti in early 2000s

• Pros: it seems to spread naturally even betweenislands

• Cons: it does not seem very effective especiallyin lower areas and/or where host populations aresmaller

Photo:

Donald E. Gardner, University of Hawaii

Better understanding why

Colletotrichum is not

effective• Are plants responding differently to pathogen

because they are genetically different: test withcommon garden experiment and artificialinoculation

• Are environmental conditions driving infectionsuccess: test with field inoculations at differentelevation

• Survey and check for relationship betweendensity and infection levels

Systemic infections

• Viral?

• Phytoplasmas

• Peronospora and smuts can lead to over

50% mortality

• Endophytism: usually considered

beneficial

Grass endophytes• Clavicipetaceae and grasses, e.g. tall fescue

• Mutualism: antiherbivory, protection fromdrought, increased productivity

• Classic example of coevolutionary development:Epichloe infects “flowers” of sexuallyreproducing fescue, Neotyphodium is verticallytransmitted in species whose sexualreproductive ability has been aborted

Endophytes: from mutualism

to parasitism

• In wheat: pathogenic fungi and phylloplane fungi bothpresents endophytically

• In Europe and Puerto Rico: leaves of healthy woodyplants harbor wood decay fungi (asociation with veins?)

• In coniferous forests: some wood decay fungi endophyticand their growth triggered by increased oxygen let in bywounds or self pruning, e.g. Echinodontium tinctorium

Tropical leaves: “hotspots of

diversity”• In a transect from the arctic to Panama:

– Increased incidence

– Increased diversity

– Broader host range

• Endophytes include many undescribed species. 20+ species perhost. Would increase at least 4 times the estimate of fungi in theworld from, 1.5 (based on 6 taxa per plant in the UK) to 6 M. Manyspecies not found anywhere else!!

• Horizontal transfer, favored by leaf wetness, but timing and hostpreference are still unclear

• Because of the large cryptic diversity of soil andplant-associated fungi, an important component of the2008 fungal sampling was the development ofprotocols for obtaining fungal DNA from substratesincluding soil, leaves, and wood, and the constructionof clone libraries from which DNA can be sequencedand analyzed to assess phyletic diversity. Culturing ofleaf and wood substrates was conducted in order toprovide collections for taxonomic identification.Sampling of airborne spores was conducted in order tocapture additional fungal species not associated withthe focal plant species.

Environmental sampling: overview

Leaf library 1 (3 host species):• High PCR/sequencing success rate from colony picks

• Tags obtained through search on 79 of 95 T3/T7-primed clones• 44 unique top BLAST hits

• (BLAST-based) “species” shared between 3 hosts =4; 2 hosts=9

Preliminary Results (III): clone libraries

Testing for timing of infection

and host preference

• Monitor leaves from flushing to maturity of two sympatricspecies (bag some leaves as control)

• Isolate endophytic fungi from both after surfacesterilization to avoid contaminants

• Subculture isolates on media amended with leaves fromeither host and see if there is an effect

Do endophytes of Miconia

protect it from Colletotrichum

• Theobroma cacao more resistant to infection byPhytophthora when leaves are endophytically colonized

• If it is possible to obtain Miconia without endophytes acomparative inoculation could be performed

Floral diseases

• Pollinator vectored smut on silene offers an example of

well known dynamic interaction in which pathogen drives

genetic variability of hosts and is affected by

environmental condition

• Puccinia monoica produces pseudoflowers that mimic

real flowers. Effects: reduction in seed production,

reduction in pollinators visits

Cankers, wilts, and die-backs

• Includes extremely aggressive, often easy

to import tree diseases: pine pitch canker,

Dutch elm disease, Chestnut blight, White

pine blister rust

• Lethal in most cases, generally narrow

host range with the exception of Sudden

Oak Death

Root diseases

• Extremely common, probably representthe most economically damaging type ofdiseases

• Effects: tree mortality (direct and indirect),cull, effect on forest structure, effect oncomposition, stand density, growth rate

• Heterobasidion, Armillaria, Phellinus weirii,Phytophthora cinnamomi

Wood-decay fungi

• The most important group of macrofungi (both basidio-

and ascomycetes) on Moorea

• Considered essential element for nutrient cycling, soil

formation and carbon budget in ecosystem

• Important element for successful regeneration

• Important in creating physical space for new growth in

understory

DEADWOODOLOGY:the ecology of dead wood

• White rotters (attack

lignin)

• Large substrates

(trunk)

• Host specific

• Endophytic stage

• Early colonizers

• Pathogenic

• Persistent fruitbodies

• Brown rotters (only

attack cellulose)

• Small substrates

(twigs,small branch)

• Generalist

• Active decayers only

• Late colonizers

• Saprobic

• Ephemeral fruitbodies

Species richness of decay fungi

• Amount of coarse woody debris (CWD):estimated that 90% reduction in CWD, 50%species extinction

• Size of CWD positively correlated to absolutenumbers (not per unit of volume though): space& ecology (presence of heartwood, etc.)

• Size of habitat not as important as age of habitat

Wood decay in tropics

• Richness of spp. correlates with better decomposition:Cyrilla racemiflora in Puerto Rico

• CWD not larger in tropics: why? Faster decay rates,more effective and/or rich decay communities?

• Number of macrofungi correlates with number of trees

• How does this all fit with suggested– Less host specificity of fungi in tropics

– Less diversity of fungi in tropics (based on macrofungi)

Testing for host specificity

• Survey mushrooms: focus on common ones, and oneones growing on relatively intact substrates

• Based on observations, make prediction of host specificvs. generalist decayer

• Grow in media amended with host tissue andunameneded

• Show enhanced growth of host-specific fungus on host-amended medium

Sampling: transects + sporocarp

collecting

• Vast majority consists of wood decay species,particularly Polyporaceae s.l., Marasmiaceae s.l.,Mycenaceae

Preliminary Results (I): sporocarp

taxonomy

Wood saprotrophs and pathogens

Photos: Todd Osmundson

Most commonly observed in Inocarpus fagifer (Tahitian Chestnut) -Hibiscus tiliaceus forests; occasionally on coconut palm (Cocosnucifera) and in cloud forests.

LEFT: Ganoderma sp. (lucidumgroup), a white rot fungus

ABOVE RIGHT: Auricularia cf. polytricha, the “wood ear” or “tree ear” fungus. Aclose relative of this rubbery-gelatinous mushroom is commonly found, thinlysliced, in Chinese soups.

RIGHT: Schizophyllum commune, the“split-gill fungus,” is reported nearlyworldwide.

LEFT: Pleurotus sp., a relative of the oyster mushroom.

Wood saprotrophs and pathogens

(continued)

Photos: Todd Osmundson

ABOVE: Polypores (Polyporus and relatives); diverse and difficult to identify. A varietyof morphological forms (shelflike, crustlike, stalked, etc.) are found on Moorea.

LEFT: Lentinus sajor-caju, reported from tropicalhabitats in many parts of the world.

ABOVE RIGHT: A variety of small, white mushroomscan be found on decaying wood; Campanella sp. is justone example.

Litter saprotrophs

Photos: Todd Osmundson

Decomposers of dead plant leaves and herbaceousstems

Phallus sp., a netted stinkhorn.

Agaricus sp., a relative of the portobellomushroom (though the species pictured here isprobably mildly toxic).

Seed saprotrophsAt present, only reported from seeds of Tahitian Chestnut(Inocarpus fagifer)

LEFT: Melanotus? sp., a fungus that appears to be restrictedto I. fagifer seeds.

RIGHT: Psathyrella sp. Similar-looking fungi are found on theground in Moorea; DNA sequence data are therefore beingexamined to determine whether the taxon found on decaying I.fagifer seeds is distinct.

Photos: Todd Osmundson

Dung saprotrophs

Photos: Todd Osmundson

Coprinus sp. onhorse dung

Anellaria sp.on horse dung

Additionally, Psilocybesp. is reported by localsto be found on cattledung, but has not beencollected to-date byBiocode mycologists

Found on dung of introduced mammalian herbivores.

POPULATION DYNAMICS

Species interactions and diversity

Density-dependence

• Most diseases show positive density dependence

• Negative dependence likely to be linked to limited

inoculum: e.g. vectors limited

• If pathogen is host-specific overall density may not be

best parameter, but density of susceptible host/race

• In some cases opposite may be true especially if

alternate hosts are taken into account

Counterweights to numerical

effects

• Compensatory response of survival can

exceed negative effect of pathogen

• “carry over” effects?

– NEGATIVE: progeny of infected individuals

less fit;

– POSITIVE; progeny more resistant (shown

with herbivory)

Disease and competition

• Competition normally is conducive to

increased rates of disease: limited

resources weaken hosts, contagion is

easier

• Pathogens can actually cryptically drive

competition, by disproportionally affecting

one species and favoring another

Diseases and succession

• Soil feedbacks; normally it’s negative.

Plants growing in their own soil repeatedly

have higher mortality rate. This is the

main reason for agricultural rotations and

in natural systems ensures a trajectory

towards maintaining diversity

• Phellinus weirii takes out Douglas fir and

hemlock leaving room for alder

Janzen-Connol

• Regeneration near parents more at riak of becoming

infected by disease because of proximity to mother

(Botryosphaeria, Phytophthora spp.). Maintains spatial

heterogeneity in tropical forests

• Effects are difficult to measure if there is little host

diversity, not enough host-specificity on the pathogen

side, and if periodic disturbances play an important role

in the life of the ecosystem

The red queen hypothesis

• Coevolutionary arm race

• Dependent on:

– Generation time has a direct effect on rates of evolutionary

change

– Genetic variability available

– Rates of outcrossing (Hardy-weinberg equilibrium)

– Metapopulation structure

• Rapid generation time of pathogens. Reticulated

evolution very likely. Pathogens will be selected for

INCREASED virulence

• In the short/medium term with long lived trees a

pathogen is likely to increase its virulence

• In long term, selection pressure should result in

widespread resistance among the host

Island Biogeography: a fungal

perspective• In the 1920’s widespread was the belief

that viable fungal spores were everywhereand that they would germinate whereverconditions were favorable

• This is still a strong belief for microbialecologists

• Fungi are microbes, but in many respectstheir ecology and evolution is explained byecology and evolution of their hosts

Genetic structure

• Seems to be highest for host specific fungi

and lowest for generalist species.

Wood saprobes (large metapopulations)

Generalist pathogens (need habitat)

Mycorrhizal fungi (need habitat)

Host specific pathogens (genetic structure mimicsclosely host or is more restricted than hostdepending on biology)

Effects shaping current structure

• Age of isolation

• Distance from neighbor

• Size of population

• Dispersal ability

• Specificity of substrate

• Lack of habitat

• Presence of geographicalbarrier

• Number and size offounding events

•Sp

ore

loa

d

•distance

Results• Simplified genetic structure of populations

– Low genotypic diversity

– Loss of polymorphisms

– Higher number of fixed and rare alleles

• Simplified communities– Lower number of species

• Highly structured populations– Lack of migration where habitat not present

– Inability to overcome barriers (mountains)

• Speciation

Fungal examples 1: broad

metapopulations• Generalist late phase wood decayer

Schyzophyllum communis & Lentinus eodesspp. complex

• Migration over large areas, not planetary, butthousands of km

• Dispersal hypothesized: airborne in strong windsfor first and infected wood for second

Fungal examples 2: barriers to gene flow• Tricholoma matsutake is genetically structured between

valleys in Hunnan, but not within valleys

Fungal examples 3: lack of habitat• Coriolopsis caperata grows exclusively on white

mangroves. When mangroves are missing for more than50 km we see strong structure as a result of lack ofmigration. If population is small, loss of polymorphismswas detected. Mangrove stands are like islands

Distances =PhiST between pairs of

populations. Above diagonal is the Probability

Random distance > Observed distance (1000

iterations).

Coco Solo Bocas David

Coco Solo 0.000 0.000 0.000

Bocas 0.2083 0.000 0.000

David 0.1109 0.2533 0.000

•Fungus from mangrove

not the same as from other

hosts: mangrove stands

can be regarded as islands

Coalescent analyses identified split

between Caribbean and Pacific

populations. Unidirectional migration from

Caribbean followed by lack of gene flow:

protosecies?

Fungal examples 4: effect of lack of

habitat (lack of host) on mycorrhizal

community:Islands of trees which survived Mount Vision Fire

log(S) = 0.53 + 0.20 Log(Area)

Log(m2)

0 1 2 3 4 5 6

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

R? = 0.74

Power Law Species Area Relationship for EM Fungi

p = 0.0003

Log S

pecie

s

•Trend for distance of patch from forest but not as strong as size