Fungi
• Eukaryotic, filamentous, branched,
spore-producing microorganisms
• Lack chlorophyll
• Cell walls (chitin, cellulose, or both)
Biological Roles of Fungi
•
•
•
•
Decomposers
Pathogens
Allergens
Producers of enzymes, new drugs
(Secondary metabolites)
• Fermentation, biotechnology
• Foods for humans and animals directly
and indirectly
Secondary metabolites
Toxins
Antibiotics
Hormones
Others
Via polyketide pathways,
non-ribosomal peptide pathways
Gene cluster
horizontal transfer
Hybrid
Fungal Toxins:
• Phytotoxins or pathotoxins (vs. phytoalexins)
• Mycotoxins (aflatoxins, fumonisins, trichothecines, ergot alkaloids)
• Not all plant pathogenic fungi cause disease by means of toxins
• Host-Selective Toxins (HSTs)―Cochliobolus [Anamorph: Bipolaris
(Helminthosporium)] and Alternaria (>20 HSTs)
• Non Host-Selective Toxins (most)
• Associate with part or all of the symptoms (toxin alone) in susceptible
hosts
• Exotoxins vs. endotoxins
• Vary between and within the species
Characteristics:
• Low M.W. except ToxA (PrtA) produced by Pyrenophora tritici-repentis
• Secondary metabolites―not need for normal growth, reproduction, or
survival
• Diverse biochemistry―cyclic nonribosomally synthesized peptides,
sphingolides, polyketides, terpenoids, saccharides, and others
• Mobile―symptoms occur at regions with no sign of fungal colonization
(some distance from the infection sites )
• Active at low conc. (ca.10 pM-1 μM)
• Non-enzymatic, non-hormonal substances
• Antimetabolites (physiological and biochemical)
• Often disturb the permeability of cell membrane, inactivate enzymes
• Electrolyte leakage (dying cells)
• Mitochondria (energy and respiration), chloroplasts
• Do not affect structural integrity
• Induce chlorosis and necrosis when injected
Possible ecological roles:
• Phytotoxicity
• Contributing to pathogenicity/virulence (debilitating plant cells, altering plant
gene expression, preventing host defense response)
• Contributing to pathogen fitness― promoting nutrient leakage, altering plant
metabolism
How does one determine if a toxin has a role in disease?
• Isolation of the toxin from diseased plants
• Reproduction of part or all of symptoms when toxin is applied to plants
• Host range, correlation of virulence/pathogenicity with sensitivity to toxin
• Correlation of virulence/pathogenicity with ability of pathogen to produce
toxin
• Production at a key point in pathogenesis
• Genetic and molecular analyses
Morphology
Vegetative body:
mycelium (pl. mycelia)
Hypha (pl. hyphae)
0.5-100 µm
Coenocytic: no septae in hyphae with multiple nuclei
Anastomosis (Hyphal fusion)
Hypo-virulent viruses
Cryphonectria parasitica: Chestnut blight
Vegetative compatibility or incompatibility
Multiple genetic loci
Self-anastomosis- pathogenicity
Alternaria brassicicola
(Craven et al., 2008 Eukaryot Cell)
Kingdom Fungi made up of multicellular
eukaryotes. Fungi are actually more closely
related to Kingdom Animalia than to Kingdom
Plantae.
Four phylums:
•
•
•
•
Chytridiomycota (fossil fungus)
Zygomycota
Ascomycota (the sac fungi)
Basidiomycota (the club fungimushrooms)
• Imperfect fungi: Lack of sexual stage or not
known
• Perfect fungi: asexual and sexual stages
Phylum Chytridiomycota (pot fungi),
lack true mycelium, fossil fungi
flagellated zoospores
gametangia
gametothallus
Gamete
Rhizoid
Gamete: A reproduction
cell, male of female
Sporangium (spore)
Classified as true fungi
based on ITS.
Phylum Zygomycota
Zygotes or zygospores
(2N)
Phylum Ascomycota (the sac fungi)
>75% plant pathogens
Phylum Basidiomycota
• (the club fungi-mushrooms)
Fungal Species
• >100,000 species, most are
saprophytic
• ~50 species, pathogens of
humans/animals (0.5%)
• ~8,000 species, plant pathogens
(8%)
• Over 65% of plant diseases are
caused by fungi
Type of Fungal Pathogens
• Heterotrophs: lack photosynthesis
• Obligate parasites (Biotrophs):
organisms can’t live or multiply without
their “hosts”. Parasites increase their
fitness by exploiting hosts for food,
habitat and dispersal. ---Parasitism
• Non-obligate parasites: opportunists,
necrotrophs (cause the death of host
tissues and live on dead substrate)
Reproduction
• Spores (reproductive bodies, one or
multiple cells)
• Asexual spores: derived from mycelium
or specialized cells
• Sexual spores: Karyogamy (the fusion
of nuclei of two cells) and meiosis
Type of spores: Asexual Spores
• Sporangium (Sac): with or without
flagella
Zoospores
Type of spores: Asexual Spores
• Conidiophores and conidia (Conidium)
Simple, unbranched conidiophores:
•Some species form conidia on single,
unbranched hyphae (conidiophores).
•e.g. Geotrichum candidum
Branched conidiophores: Example of a
branching pattern of conidiophores bearing
clusters of conidia at their tips.
•e.g. Trichoderma spp.
Trichoderma
Coremium(a):
•Conidiophores are aggregated together
to form a vertical stalk-like coremium.
•At the top of the coremium the
conidiophores branch and conidia
develop at the tips of the branches.
•e.g. Penicillium spp.
Acervulus(i):
•A flat, open pad of short conidiophores
growing side-by-side.
•The conidiophores develop from the
underlying mass of somatic hyphae.
•e.g. Colletotrichum species.
Pycnidium(a):
•A flask-shaped structure with
conidiophores developing from cells of
the pycnidial wall.
•e.g. Phoma species.
Sporodochium(a):
•A cushion-shaped mass of short
conidiophores.
•e.g. Epicoccum species.
Fusarium
Chlamydospores (Thicken cell wall)
Homothallic
Heterothallic
Fungal Sexual Cycle: perfect fungi
Sexual Spores
• Zygospores
Zygote:
Fusion of two
similar gametes
Zygote: Fusion of two unequal gametes and
producing Oospores
Ascospores: produced within the ascus(i)
Perithecium (ascocarp)
Asci
Ascospores
Basidiospores: produced on
basidium (mushrooms)
Alternate Hosts
Autoecious fungus:
complete its entire life cycle on the same
host.
Alternate host
Spermatia in spermogonium
(Rust)
Aeciospores in aecium (rust)
Uredospores in uredium (Rust)
Teliospores in telium (Rust)
Haploid (N)
Dikaryotic (N+N)
Haploid (N)
Dikaryotic (N+N)
Diploid (2N)
Hermaphroditic (Isogamous) Fungi:
Male and female gametes are
produced on the same mycelium.
Homothallic
Heterothallic
Ecology and Spread
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•
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Entirely on their host plants (Biotrophs)
Partially on host plants, soil or plant debris
Parasite and saprophyte (two life styles)
On dead tissues (Necrotrophs)
Parasites vs. mutualistic or symbionts
Endophytes
Ecology and Spread
• Propagule or inoculum: spores,
mycelium, zoospores, sclerotia
• Environmental conditions (light,
moisture, wind-blown rain, air-borne,
soil-borne, rain splash, insects,
animals)
Germination, Infection
(penetration), and colonization
Penetration
• Direct penetration
• Through natural openings
• Wounds
Figure 1. Diagram showing infection of barley by the fungal
pathogen Erysiphe graminis f. sp. hordei, an obligate parasite that
infects barley leaves using a specialized cell called an appressorium
which penetrates the leaf cuticle.
The fungus is an extremely efficient parasite of the living plant, and
produces a specialized feeding structure, the haustorium, that allows it to
subsist in leaf epidermal cells.
cv. “Chivery” 48 hpi
cv. “Groupzest” 72 hpi
a
pp
a
pp
cn
ds
gt
ds
cn
pp
pp
a
ds
pp
a
cn
gt
cn
pp
On fruit surface
Below appressorium
a
In cuticle layer
On fruit surface
Colletotrichum acutatum on Capsicum spp.
(Liao et al., 2012 Plant Pathol)
ds
pp
ds
Below appressorium In cuticle layer
More on Ascomycota
(the sac fungi)
• Naked asci
• Apothecium
• Perithecium
• Cleistothecium
Naked Asci
Apothecium
Perithecium
Cleistothecium
Concepts of diseases:
disruption of normal function
(physiology, differentiation, development, genetics); Consequence of
diseases: weakening, disturbing, consuming, killing
The types of disorder—depending on the types of cells and tissues
affected.
Root (root rot)-- water and nutrition uptake
Xylem vessels (vascular wilts)-- translocation
Foliage (leaf spots, blights etc) -- photosynthesis
Cortex (canker) -- downward translocation
Flowers (blight) -- reproduction
Fruit (fruit rots) –
Hyperplasia (overgrowth due to increased cell division) and
hypertrophy (overgrowth due to cell enlargement)
Most plants are healthy and diseases are exceptional.
Race: genetic variability within a pathogen
(restricted to certain varieties of a host species)
Strain (an isolate, a race): the descendants of a
single isolation in culture
Forma specialis (f. sp.): a pathogen species only
infects a certain host genus or species
Pathogenicity: the capability of a pathogen to
cause diseases
Virulence: the degree of pathogenicity
Symptom: the external and internal reactions or
alternations of a plant as a result of disease. (disease
expression, structural, functional, and physiological
disorders, local vs. systemic)
Sign: the pathogen or its parts or products seen on a
diseased plant.
Latent infection: the state for a pathogen to infect a host
plant but does not show any symptoms.
Human and plant diseases: Larger acreages and density,
new cultivation, uniform crops (monoculture), increased
fertilization, varieties, irrigation, pesticides, and
mechanization; breeding, nursery stock, frequent travel,
Monocyclic:
Polycyclic:
Polyetic:
Epiphytic growthQuiescent (latent)
Infections-
Disease cycles---life cycle of pathogen
Inoculation:
• Inoculum—any part of the pathogen that can initiate
infection such as spores, mycelium, sclerotia etc.
• Propagule---one unit of inoculum.
• Primary inoculum: inoculum that survives the winter or
summer and causes the original infections (primary
infection).
• Secondary inoculum: inoculum produced from primary
infections
• Sources of inoculum: plant debris, soil, seeds,
transplants, tubers, or other propagative organs,
alternate or weed hosts.
• Means for dispersal: wind, water (rain, tropical storm),
tools, insect vectors, animals, and humans.
2). Penetration: germination, attachment, recognition, and penetration.
--Direct penetration (appressorium-mechanical force, penetration peg) (enzymes,
toxins)
--Through natural openings: stromata, hydathodes, nectarthodes, and lenticels
--Through wounds
(Penetration does not always lead to infection.)
3). Establishment of infection--- on susceptible hosts
4). Colonization (invasion) —intracellular or intercellular mycelium, hausteria,
and stylet (local vs. systemic)
5). Growth and reproduction of the pathogen
6). Dissemination of pathogen — direct contact, air, water, insects, mites,
nematodes, and other vectors, human
Control of Plant Diseases
Principles:
• to reduce initial pathogen populations;
• to create environment conditions which do
not favor pathogen growth;
• shall tie up with economic and environmental
concerns; an integral component;
• understand the diseases;
• requires frequent adjustment,
• Control methods that eradicate or reduce the
pathogen inoculum—crop rotation, host
eradication, sanitation, creating conditions
unfavorable to the pathogen, appropriate
cultural practices, fungicides
Control of Plant Diseases
• Protection—regulatory control
measures, cultural control methods,
biological control, chemical and
physical methods
• Exclusion—quarantines, inspections
• Eradication ?
• Use of pathogen-free propagating
materials
Control of Plant Diseases
• Biological control— suppressive soil, cross
protection (mycoparasitism, competition for
space or food, antibiotics or toxin)
•
• Use of resistant varieties—
•
• Induced resistance—
• Cross protection—
• Genetic modification—
Grass Endophytes
Corn Smart-Edible fungus
Mexican truffle
Edible smart fungus
Ustilago esculenta in
aquatic perennial grass, Zizania latifolia
Fun Guy:
This fungus will trap you if you are a nematode.
Nematode-trapping fungi
sticky knobs, constricting loops, or hyphal matrices
Parasitism
Biocontrols?
Postbloom fruit drop (PFD)
Lime anthracnose (KLA)
Colletotrichum acutatum
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•
KLA strain
Lime anthracnose (flowers,
fruits, leaves and shoots)
PFD strain
Postbloom fruit drop on
sweet orange (flowers)
PFD isolate: Blossom blight (Anthracnose), and postbloom
fruit drop
KLA isolate: Anthracnose, blossom blight, and postbloom
fruit drop
AZ-C
AZ-A
PFD:
Young fruit drop caused by C.
acutatum [abscission occurs between
the calyx and the fruit (AZ-C)]
Persistent calyces
(buttons)
Natural fruit drop [abscission
occurs between the shoot and
the peduncle (AZ-A)]
Mechanisms of natural tree fruit drops:
‡
‡
‡
‡
‡
Degree of fertilization
Disturbance of embryogenesis
Adverse environmental conditions
Nutrition competition
Hormone regulation
Single lesion can result in fruit drop and button formation
Formation of persistent calyces (Buttons)
Leaves surrounding infected flowers become distorted
(twisted laminae and enlarged veins)
Hypothesis: Plant hormones or growth regulators are
involved in postbloom fruit drop
To investigate the possible causes of young fruit drop-specifically focus
on hormone imbalance
--Differential gene expressions
--Analyses of hormone contents
--Assessment of fruit retention after applying hormone inhibitors
• Ethylene
• Indole-3-acetic acid (IAA)
• Abscisic acid (ABA)
• Jasmonic acid (JA)
• 12-oxo-phytodienoic acid (PDA)
• Salicylic acid (SA)
(Lahey et al., 2005 MPMI)
Acervulus with
conidia
Scab infested fruit, twigs & leaves
Life cycle of Elsinoë sp.
Young fruit, twigs
& leaves
Rain-splashed
Hyaline
conidia
Airborne
Spindle-shaped
conidia
Chung, KR, 2011
Mol. Plant Pathol
H
O
O
OCH3
H
H
OCH3
OCH3
CHOHCH3
CHOHCH3
H
H
O
O
O
CH2
Elsinochrome D
H
H
O
O
OCH3
H
O
O
OCH3
O
OCH3
H
H
OCH3
H
O
OCH3
H
R1
OCH3
R2
OCH3
H
H
Tautomerization
H
H
Elsinochrome A: R1 = R2 = -CO-CH3
Elsinochrome B: R1 = -CO-CH3, R2 = -CHOH-CH3
Elsinochrome C: R1 = R2 = -CHOH-CH3
O
O
H
R1
R2
OCH3
Liao & Chung, 2008
New Phytologist
MPMI
Rf
0.69
0.48
0.23
0.12
4
6
9
12
15
18 (days)
Elsinochrome Toxin Biosynthetic Gene Mini-Cluster
EfHP1
2
EfPKS1:
TSF1:
OXR1:
RDT1:
ECT1:
PRF1:
EfHP1–4:
RDT1
3
TSF1 OXR1 EfPKS1 PRF1 ECT1EfHP4
Involved in condensation, chain elongation and cyclization
Transcription regulator
Involved in oxidation and hydroxylation
Involved in oxidation and hydroxylation
Involved in ESC exportation
Unknown function
Hypothetical proteins
Chung & Liao, 2008
Microbiology
Alternaria brown spot on fruit, twigs & leaves
Host-Selective Toxins (HSTs): Pathogenicity factor
Tangerine pathotype: ACT toxin (A. citri-tangerine)
Toxicity: Disruption of plasma membrane (Kohmoto et al., 1993, Phytopath.)
Rough lemon pathotype: ACRL toxin
Toxicity: Mitochondria functions (uncoupling of oxidative
phosphorylation, membrane potential, inhibition of malate
oxidation)
(Akimitsu et al., 1989, Plant Physiol.)
9,10-epoxy-8-hydroxy9-methyl-decatrienoic acid
Dihydropyrone ring
Accumulation of H2O2 (DAB staining)
50
H2O2 (μM)
mock
Lin et al., 2011
Curr Microbiol
WT
40
30
mock
20
10
A. alternata
0
1
bright light
Lipid peroxidation
2
3
fluorescent light (2 dpi)
A. alternata
Malondialdehyde
(mM)
3
mock
2
Cell death
1
mock
0
1
2
3
Time (dpi)
4
4
(dpi)
Lin et al., 2009 MPMI
Yang et al., 2010 PMPP
Fungal cell
Nucleus
AAAAAAA
N
C
toxin
Fungal detoxification systems
H2O2
Cell death & necrosis
H2O2
Oxidase
O2
H2O2
O2·NADPH oxidase
Citrus Cell
Reactive Oxygen Species (ROS)
Point inoculation
WT
WT
Mock
(4 dpi)
Mock
D1
D1
D2
WT
Mock
D1
D2
D2
Cp1
Cp1
Cp2
Cp1
Cp2
Defective at
penetration &
colonization
Spray inoculation (4 dpi)
WT
D1
Cp1
Mock
Cp2
Stress
Development
2O2·
2O2
NoxA
NoxR
H2O2
H2O2
Other
stimuli
membrane
NoxB
RAC
Unknown
component
SOD
GDP
GTP
H2O2 as a signal
Nucleus
AaAP1
AaHOG1
AaNoxA
Transcriptional activation
Conidiation
Oxidative stress response
Pathogenicity
Yang & Chung, 2012 MPP
Chen et al., 2012 FGB