Slides from my presentation at the Czech Greenkeepers School of Education at Lednice, Czech Republic, 4th December 2019

4. How did I get into turfgrass?

9. Stress responses in plants
Abiotic and Biotic
Pathogens and the infection
process
Plant defence mechanisms
Constitutive v Induced
responses
Todays Talk
Response induction – Elicitors
First line of induced response -
Hypersensitive Response
Antimicrobial compounds –
Phytoalexins
Long term response – Systemic
Acquired Resistance
Defence activators

10. Turfgrass responses to stress

11. A wound response occurs when a leaf is chewed
or injured….or cut as with a mower
• Can lead to rapid production of proteinase
inhibitors throughout the plant
• Defence pathways are initiated
• Signaling pathway involved
• Jasmonic acid
• Salicylic acid

13. • Changes in various morphological and physiological elements
• Inhibition of photosynthesis and respiration processes
• Reductions in chlorophyll concentrations
• Cell membrane stability
• Leaf water content
• Antioxidant enzyme activity and increased generation of reactive
oxygen species (ROS).

14. Responses to Abiotic stress are similar to Biotic
challenges

15. Turfgrass – Abiotic/Biotic interactions

17. Responses to Abiotic stress are similar to Biotic
challenges

18. Disease - the malfunctioning
of host cells and tissues
that results from their
continuous irritation by a
pathogenic agent and leads
to the development of
symptoms (Agrios, 1988).
Problems caused by
a pathogen on
amenity turfgrasses
Visual quality
Playing quality
Concentrate today on biotic challenges and responses

19. Plants are a rich source of nutrients for many organisms
including bacteria, fungi, protoctista, insects, and
vertebrates…

21. Fungi and bacteria can also be beneficial to plants
• Mycorrhizal fungi
• Nitrogen-fixing bacteria like Rhizobium
• Plant growth-promoting rhizobia
• Bacteria provide substances that support plant
growth
• Can also limit the growth of pathogenic soil bacteria

22. Some Cool-season pathogens

23. Fungicides withdrawn in last 5
years:
• Carbendazim
• Chlorothalonil
• Iprodione
• Propiconazole
So why are defence responses important?
Legislative restrictions!!
Available Fungicides:
• Azoxystrobin
• Pyraclostrobin
• Difenoconazole
• Fludioxonil
• Trifloxystrobin
• Tebuconazole
• Fluopyram

25. Two forms-
Basal rot
Foliar blight
Basel rot -
affects crowns, stem bases, and roots.
Mostly affects Poa annua
Youngest leaf red - black rot at base - stunted roots

26. Foliar blight-
Mostly mid-summer
leaves and shoots discolour, similar
appearance to drought stress attacks the
leaves and stems

27. Indicates a problem with turf
Reduce stress
Help turfgrass to respond with their
inbuilt defence mechanisms

31. Host – All cool season turfgrass species
Conditions
Persistent humidity, moist surface, high N + pH

32. Microdochium active on turfgrass

33. Using pot samples
and infected greens

42. • Hyphae/conidia in the soil/thatch are the main
source of inoculum
• Environmental conditions allow infection to
commence
• Mycelium grows from the base of the plant
• Infection by means of stomatal
penetration/appressoria formation
• Infects the plant extracting nutrients
• Then emerges from plant producing conidia which
are the means of propagation and dispersal
• Infected plants respond with chemical defences
Host
recognition

44. Constitutive v Induced Defenses

45. Protection from a pathogen’s initial invasion is achieved via
passive defences, such as physical and/or chemical barriers

46. • The thickness of the cuticle, the presence of a secondary cell wall, and
the size of the stomatal pores can all affect the success with which a
pathogen invades a host.
• Some plants also have leaves which have a vertical orientation. This
prevents the formation of moisture films on the leaf surface, inhibiting
infection by pathogens that are reliant on water for motility
• Some plants have very thick cell walls and/or cuticles, and bark which
can all provide a better barrier to infection.
Constitutive Defenses

47. Silica for disease suppression

48. Constitutive defences - Phytoanticipins
Phytoanticipins – Low
molecular weight compounds
which are present in plants
prior to infection
They act as antimicrobial
compounds
Important to note - they are
present in plants before
challenge by microorganisms

49. Constitutive defences

51. Induced defences
• Elicitors
• Hypersensitive response
• Programmed Cell Death
• ROS
• Pathogenesis Related Proteins
• Phenolics
• Phytoalexins
• Systemic Acquired Resistance
• Induced Systemic Resistance
• Salicylic Acid

53. Inducible defences

54. Do plants have an immune system?
Plants do not have an immune system comparable to
animals…
… but they have developed an array of structural, chemical
and protein-based defences designed to detect invading
organisms and stop them before they are able to cause
extensive damage.

55. Recognising an attack
Plants are not passive – they are able to respond rapidly to pathogen
attacks.
• Receptors in the cells respond to molecules from the pathogens, or to
chemicals produced when the plant cell wall is attacked - Elicitors
• This stimulates the release of signalling molecules that switch on genes
in the nucleus.
• This in turn triggers cellular responses:
o Producing defensive chemicals
o Sending alarm signals to unaffected cells to trigger their defences
o Physically strengthening the cell walls

63. Recognition of the pathogen leads to
hypersensitive response (HR)
• Leads to a very rapid cell death around
the site of attack
• Also to longer term, whole plant
resistance
• Reactive Oxygen Species (ROS) generated
• Hydrogen peroxide and nitric oxide key compounds
• Seals off the wounded tissue to prevent the pathogen or pest from moving into
rest of the plant
• Leads to signal cascade of chemical events resulting in localised host cell death
- Programmed Cell Death (PCD)

64. Sequence of events leading to the hypersensitive reaction in plants
infected by incompatible pathogens

65. Programmed Cell Death - PCD

67. H2O2 (hydrogen peroxide)
H2O2 plays multiple roles:
• Induces defense-related genes
• Induces apoptosis (PCD)
• Causes cross-linking of cell wall
proteins (more resistant to wall-
degrading enzymes)
• May directly kill pathogens

72. Also these defence compounds
require available resources from
the plants
No free lunches!

74. Hypersensitive response leads onto:
• Cell wall reinforcement
• Stimulation of secondary metabolite pathways
• Synthesis of Thionins
• Synthesis of Pathogenesis Related Proteins
Antimicrobials – phytoalexins, phenolic compounds

76. Secondary metabolites which contain a hydroxyl functional group on an aromatic ring –
(Cyclic (ring-shaped) structures have a ring of bonds that gives increased stability compared to
other geometric arrangements with the same set of atoms)
• Some are water soluble only in organic solvents
• Some are water soluble carboxylic acids and glycosides
• Some are insoluble polymer
Many serve as defence compounds against pathogens
Plants produce a variety of compounds that contain one or more phenol groups -
called phenolics - Thousands of phenolics occur in plants

77. Occurrence of phenolics

78. Important enzymes

79. Turfgrass response in infection - phenolics

81. Phytoalexins

82. Phytoalexins are synthesized
and accumulated in plants after
exposure to microorganisms
Phytoanticipins are also low
molecular weight and
antimicrobial compounds but
these are preformed which are
present in plants before
challenge by microorganisms
Low molecular mass antimicrobial
metabolites synthesized de novo
from primary metabolites in
response to infection
Structurally diverse group of
metabolites with the isoflavonoids
can be an example
The isoflavonoids phytolaexins
are synthesised from the
flavonoids branch of the
phenylpropanoid pathways

83. PRODUCTION OF PHYTOALEXINS
• Production of phytoalexins may be stimulated by elicitors.
• High molecular weight substances found in the cell wall such as
glucans, glycoprotein, Chitin
• Gases such as ethylene (C2H4)
• In susceptible plants, a pathogen may prevent the formation of
phytoalexins, by the action of suppressors produced by the pathogen
• The suppressor also can be a glucan, a glycoprotein, or a toxin
produced by the pathogen

84. Phytoalexins produced in plants act as
toxins to the attacking organism.
They may puncture the cell wall.
Delay maturation, disrupt metabolism or
prevent reproduction of the pathogen.
Their importance in plant defence is
indicated by an increase in
susceptibility to infection in their
absence.
They show defence reaction only in
living cells.
They can't be inherited.

87. Systemic v Induced resistance

90. Long term defences – Systemic resistance

92. Systemic Acquired Resistance
• Systemic acquired resistance causes systemic
expression of defense genes and is a long-lasting
response
• Salicylic acid is synthesized around the infection site and
is the signal that triggers systemic acquired resistance
• When a plant survives the infection of a pathogen at one
site it can develop increased resistance to subsequent
attacks.
• Although plants don’t have “immune systems” they have
signaling mechanisms that can act in this way.

93. • Accumulates in both local and
systemic tissues
• Removal of SA prevents induction
of SAR
• Analogs: INA or BTH
The SA-dependent defense pathway can be
activated by treatment of plants with
chemical inducers such as benzo-
thiadiazole-carbothioic acid-S-methyl ester
(acibenzolar-S-methyl, ASM or BTH, Bion)

96. Resistance to infection overview

97. Resistance to infection overview

98. • Silica
• Civitas
• Chitin
• Salicylic acid
• Harpin
• Phosphite
Defence activators
Treatments to control
diseases by priming
the expression of plant
defences

99. Silica for disease suppression

103. Chitin
Chitin is a long-chain polymer derivative of glucose. It is a primary
component of the exoskeletons of crustaceans and insects……but also
the cell walls of fungi
In recent years chitin-containing products have been used agriculture
A number of modes of action have been proposed for how chitin and its
derivatives can improve crop yield, be toxic to plant pests and pathogens,
stimulate the growth and activity of beneficial microbes.
And importantly …..induce plant defences

107. Form of Phosphorus (P) a major nutrient of plant growth
Taken up as Phosphate - Phosphoric acid (H3PO4)
Phosphite - Phosphorous acid (H3PO3)
Phosphite not metabolised
in plants

108. • Phosphite derived from Phosphorous acid – (H3PO3) pH 2.2 - has to be
modified with an alkali salt to usable pH
• First used in 1980’s - Rhone- Poulenc fosetyl Al (aluminum tris (O-ethyl
phosphonate))
• Potassium hydroxide (KOH) - Forms Potassium dihydrogen phosphite
(KH2PO3) or dipotassium hydrogen phosphite (K2HPO3)
Potassium phosphite
Ammonium phosphite, Magnesium phosphites and Calcium phosphites

109. Anthracnose
Microdochium majus in cereals
No research into
Phosphite and Microdochium nivale
Oomycete pathogens
not fungi
Suppresses plant pathogens
Pythium and Phytophthora

110. Four year mean values of percent M. nivale incidence on P. annua, A. canina canina
and A. stolonifera trial plots.

111. • Inhibits mycelial growth and conidial germination
• Disrupts hyphal morphology
• Causes release of stress metabolites
In the plant –
• Slows the growth of the pathogen
• Allows for faster recognition of the pathogen by
the plant
Primes the plant defences prior to infection

112. • Measured using reagent and spectroscopy
• Important response to stress and pathogen challenge
• Visualised using fluorescent microscopy
• Hydrogen peroxide
• Phenolic compounds

113. • Hypersensitive response
• Generation of ROS
• Hydrogen peroxide key
component

114. 0.00
5.00
10.00
15.00
20.00
25.00
30.00
35.00
0 hpi 1 hpi 6 hpi 12 hpi 24 hpi 48 hpi 72 hpi
μmolH2O2/gfw
P. annua
0.00
5.00
10.00
15.00
20.00
25.00
30.00
35.00
0 hpi 1 hpi 6 hpi 12 hpi 24 hpi 48 hpi 72 hpi
μmolH2O2/gfw
A. stolonifera

116. H2O2 concentrations in M.
nivale infected greenhouse
turfgrass tissues
H2O2 concentrations in
• Control
• Phosphate (Pi)
• Phosphite (Phi)
treated tissues turfgrass
10 days post inoculation

119. M. nivale infected P.annua leaf, viewed under UV
Fluorescence
Blue hyphae are visible with H2O2 fluorescencing at stomatal infection
sites.

120. Accumulations of H2O2 in
response to M. nivale
infection in turfgrass leaves
A: M. nivale hyphae entering stoma
blue stain shows H2O2
accumulation.
B: Infected stoma showing H2O2
accumulation.
C: P. annua leaf showing H2O2 in
response to infection
D: Infected A. stolonifera leaf
showing autofluorescence of
phenolic compounds (light
yellow).

121. Induced Systemic Resistance (ISR)
Systemic Acquired Resistance (SAR)
• Pathogenesis related proteins
• Phytoalexins
• Salicylic acid
All polyphenols

122. Total phenolic compounds
Total phenolic compounds as GAE mg/g dry weight
Comparing levels between infected and non-infected turfgrass from trial plots
0.00
0.50
1.00
1.50
2.00
2.50
3.00
Control Infected Control Infected Control Infected
2012 2013 2014
GAEmg/g
P. annua
0.00
0.50
1.00
1.50
2.00
2.50
3.00
Control Infected Control Infected Control Infected
2012 2013 2014
GAEmg/g
A. stolonifera

123. Total phenolic compounds
Total phenolic compounds as GAE mg/g dry weight
Comparing levels between infected and non-infected greenhouse turfgrass
0.00
0.50
1.00
1.50
2.00
2.50
3.00
Control Infected Control Infected Control Infected
2012 2013 2014
GAEmg/g
P. annua
0.00
0.50
1.00
1.50
2.00
2.50
3.00
Control Infected Control Infected Control Infected
2012 2013 2014
GAEmg/g
A. stolonifera

125. TPC as GAE mg/g dw, in
turfgrass tissues sampled
from greenhouse plants
Total phenolic compounds
in turfgrass following six,
monthly applications of
• Control
• Phosphate
• Phosphite

126. TPC in M. nivale infected
P. annua over 10 dpi in
greenhouse turfgrasses
Total phenolic compounds
in infected P. annua over
10 dpi following treatment
with
• Control
• Phosphate (Pi)
• Phosphite (Phi)

128. • Phenolic compounds and H2O2 are a component of initial defence
responses
• Phosphite treatment led to enhanced responses in regard to total phenolic
compound accumulation
• Results of H2O2 extractions indicated that phosphite treatment did not
influence H2O2 responses
• Fluorescence microscopy determined that phosphite treatment did
enhance this response

129. Phosphite suppressing M. nivale
incidence by enhancing the
plants natural defences.
Inclusion of
• Calcium
• Silica
• Salicylic acid
Working synergistically to
reduce disease incidence.

130. • Phosphite induced and
enhanced defence
responses
• Calcium, Manganese and
Zinc all key to plant
defence responses
• Salicylic acid
stimulating Systemic
Acquired Resistance
• Silica strengthening cell
walls

131. Research supporting disease suppression from defence
enhancement

132. Research supporting disease suppression from defence
enhancement

133. Research supporting disease suppression from defence
enhancement

134. Abiotic and Biotic stress
responses similar
First line of defence
involves physical barriers
eg waxes, cutin
Constitutive defences are in
place prior to infection
A secondary arsenal of
inducible defence
processes are then
produced
Summary!
Recognition of pathogen vital
for successful response -
elicitors
Rapid induction of short term
defences then can occur
Hypersensitive response first
induced response
Generation of Reactive
Oxygen Species (ROS),
Programmed Cell Death (PCD
Induced Systemic Resistance (ISR)
System Acquired Resistance (SAR)
Salicylic acid
Phytoalexins
Phenolic compounds
Defence activation!

135. Priming and enhancement of plant
defences
Defence activators
• Silica
• Civitas
• Chitin
• Salicylic acid
• Harpin
• Phosphite

136. In conclusion:
Plant responses to stress either abiotic or biotic involves complex
interactions and signalling
Regarding pathogen challenge defences are in place prior to infection
or induced upon recognition of the attack
Priming and/or enhancement of these responses can be a key to
disease suppression and control