The role of phosphite in turfgrass management quebec

The Role of Phosphite in
Turfgrass Management

Greenkeeper since 1980’s
Course manager 1993

BSc in Turfgrass science – Myerscough college
Final year research project-
The Effects of Phosphite on the Growth and Disease
Susceptibility of Agrostis stolonifera

Centre for Research in Biosciences
PhD in Plant Pathology
Suppression of Microdochium nivale by
Phosphite in Cool-season Turfgrass
In vitro studies comparing activities of
antimicrobial photodynamic therapy and
electrochemically activated solutions

Does phosphite suppress Microdochium nivale
infection?
Means of suppression?
What other effects might phosphite have on turfgrass?
Field trials
Laboratory studies

That’s enough about me………
what about you guys?

Todays workshop
The journey through ten years of research
Talking about the methodology used and the
results we obtained
Hopefully we can have plenty of interaction

Microdochium infection process from inoculum source through infection,
reproduction and dissemination also how turfgrass respond defensively
Phosphite:
• Description, history and usage
• Field trials re disease suppression
• In vitro lab work showing the effect on M. nivale
• The uptake and fate of foliar applied phosphite
• Does phosphite stimulate defences?
• Effects on turfgrass growth and quality

Three components of this research

mold
Uk and Ireland it’s the most common pathogen in turfgrass
Its also the most economically important winter disease of
turfgrass in the northern and alpine regions
Of the US and Canada
Ascomycete fungus - Fusarium patch or Pink
snow-mould
What is Microdochium nivale?

Scope for alternative means of disease control
Phosphite is one possible method
Reliance on fungicides-
Expensive
Inhibition of beneficial organisms
Legislative controls
Microdochium active on turfgrass

Infection process
Fluorescence microscopy and stains
Using pot samples
and infected greens

Sampled from infected golf greens
and trial areas at Royal Curragh

• Analysis of the thatch
layers and the upper 5 cm
from rootzones of the golf
greens and trial plots
showed constant levels of
hyphal inoculum
• This was observed at all
times through the year
• M. nivale conidia were also
observed, but to a much
lesser extent than hyphae

• Soil samples taken
from the upper 5 cm of
a golf green
• Viewed following
fluorescence staining,
using a combination of
UV and bright
microscopy
• M. nivale hyphae can
be seen fluorescing
and growing through
the soil particles.

• Hyphae next to the
root and root hairs
• M. nivale isolates were originally
identified on the basis of colony
characteristics, conidial morphology
and on re-infection symptoms
• These identifications were later
confirmed following DNA extractions
in TrisEDTA buffer and testing by
polymerase chain reaction (PCR),
using primers, EFniv-F/EF-Mic-R
(Glynn et al., 2005)
Some photos from
Żur et al. (2011), Physiological and
Molecular Plant Pathology 76(3-4) 189-
196.
Dubas et al. (2010). Acta Physiologiae
Plantarum 33 (2) 529-537

• When
environmental
conditions were
right, hyphae
emerged from
the soil/plant
interface
• Hyphae then
grew up the
sheaths and
crowns rapidly
covering them in
a mass of
mycelium

M. nivale hyphal growth on infected turfgrass leaves
following emergence from the soil/thatch interface
• Hyphae on the
surface of leaf cells

Hyphae enters plant via
stomata
• Hyphae on leaf surface.
Red autofluorescence of
chlorophyll
• Stomata apparatus
penetration by hyphae

• Hyphae in leaf
epidermis cells

• Hyphae protruding
thorough the stomata
apparatus on the
surface of epidermis

L.E. Jewell and T. Hsiang, 2012, School of Environmental Sciences,
University of Guelph, Guelph, 10 Ontario, N1G 2W1, Canada.

• Assessment of numerous infection
incidences in both the field and in
greenhouses determined that hyphae
are the main source of M. nivale
inoculum and that infection was by
means of stomatal penetration
• Conidia produced via sporodochia
following infection are the means of
propagation and dispersal
Host recognition

• Measured using reagent and spectroscopy
• Important response to stress and pathogen challenge
• Visualised using fluorescent microscopy
Turfgrass defence responses
Phenolic compounds
Hydrogen peroxide

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
2012 2013 2014
GAEmg/g
A. stolonifera

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
2012 2013 2014
GAEmg/g
P. annua
0.00
0.50
1.00
1.50
2.00
2.50
3.00
2012 2013 2014
GAEmg/g
A. stolonifera

Phenolic content in turfgrass
over 10 days
following inoculation with
Microdochium nivale

Hydrogen peroxide accumulation 0 to 72 hpi
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

Hydrogen peroxide concentrations as μmol H2O2/g fw
Sampled from infected turfgrass over 10 days post inoculation
0.00
5.00
10.00
15.00
20.00
25.00
30.00
35.00
0 dpi 2 dpi 4 dpi 6 dpi 8 dpi 10 dpi
μmolH2O2/gfw
P. annua
Hydrogen peroxide accumulation 0 to 10 dpi
0.00
5.00
10.00
15.00
20.00
25.00
30.00
35.00
μmolH2O2/gfw
A. stolonifera

D
Accumulations of H2O2 and
TPC in response to M. nivale
infection in turfgrass leaves.
A: M. nivale hyphae entering
stoma, (arrow) with TMB
fluorescence indicating H2O2
accumulation.
B: view of infected stoma
showing H2O2 accumulation.
C: P. annua leaf following TMB
staining showing H2O2
synthesis in response to
infection (red
autofluorescence of
chlorophyll).
D: Infected A. stolonifera leaf
showing autofluorescence of
phenolic compounds (light
yellow).
TMB - 3,5,3′,5′-tetramethylbenzidine

Defence activators
Phosphite
Can these defences be stimulated or enhanced?
Treatments to control diseases by priming the
expression of plant defences

What is Phosphite?
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

• 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 Hydroxide, Magnesium phosphites and Calcium phosphites
Phosphite use

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

Joshua Cook; Peter Landschoot, Ph.D.; and Max Schlossberg, Ph.D.

In vitro mycelial growth rate of Microdochium majus measured on
PDA amended with potassium phosphite solution – Hofgaard et al 2010

BSc Study at Myerscough College
Dr. Andy Owen
‘The Effect of Phosphite Treatments on the Growth and Disease
Susceptibility of Agrostis stolonifera L.’

Conclusions of the Myerscough Study

Trials ran from September to
March each year
2010 to 2014
Phosphite applied alone or in
combinations with fungicides and
biostimulants
Agrostis canina canina
Agrostis stolonifera
Poa annua
Disease incidence assessed
monthly
Turf quality was also assessed
Royal Curragh
field trials

Figure 3-2 Monthly disease incidence, P. annua, January 2011 (year 1). Treatment effect on percent M.
nivale incidence on trial plots (n=5), of P. annua, during the month of greatest disease incidence in year 1
of the trial, January 2011.

Figure 3-3 Monthly disease incidence, A. canina, December 2010 (year 1). Treatment effect on percent
M. nivale incidence on trial plots greatest disease incidence in year 1 of the trial, December 2010.

Figure 3-7 Monthly disease incidence, A. stolonifera, November 2011 (Year 2). Treatment effect on percent M.
nivale incidence on trial plots (n=5), of A. stolonifera during the month of greatest disease incidence in year 2 of the
trial, November 2011.

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

We assessed the effect of phosphite treatment on turfgrass quality
Turfgrass quality P. annua, A. canina and A. stolonifera, September 2011 to March 2012

Turfgrass quality mean values over four years 2010 to 2014

Agrostis canina plots – January 2012

Poa annua plots – January 2011

Poa annua plot 5 – Phosphite

Agrostis stolonifera plot 7 – Phosphite

Agrostis stolonifera plot 5 – Control

Agrostis canina plot 9 – Phosphite

Agrostis canina canina plots – January 2011
CONTROL PHOSPHITE

Poa annua plots – October 2011
CONTROL PHOSPHITE

Phosphite treated plants – January 2008

Control plants – January 2008

Sequential applications of phosphite significantly reduced
Microdochium nivale incidence
The addition of phosphite to fungicides significantly
enhanced disease suppression
Significant improvement in turfgrass quality

Means of suppression
• Inhibits pathogen
Direct
• Stimulates plants defences
Indirect
Combination of both

In Vitro Study - Assess the effect phosphite has on the mycelial growth of
Microdochium nivale
Microdochium propagated
from infected turfgrass
Grown on and used for in vitro
study
To assess inhibition of
mycelial growth

Figure 2-5 Percent inhibition of M. nivale mycelial growth on H3PO3, KH2PO3, H3PO4, KH2PO4 and
KOH amended PDA.

Mycelial Growth on Amended PDA - 4 days p.i.

Hyphal morphology
Unamended 75µg/ml Phosphite

Experiment 2, the fungicidal or fungistatic properties of Phosphite

Effects of phosphite on conidial germination

In vitro conclusions
• 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
• Quicker response to infection
So what else is going on?
What happens when phosphite is applied
to turfgrass?

High
Performance
Ion
Chromatography

Phosphite uptake following foliar application – February 2011
Figure 4-3 Accumulation of Phi in A. stolonifera leaf
and root tissues, 96 hours p.a. in February
2011.
Figure 4-4 Accumulation of Phi in P. annua leaf and
root tissues, 96 hours p.a. in February 2011.

Phosphite uptake following foliar application – February 2011
Figure 4-5 Accumulation of Phi in A. stolonifera
leaf and root tissues, 6 weeks p.a. in February
2011.
Figure 4-6 Accumulation of Phi in P. annua
leaf and root tissues, 6 weeks p.a. in
February 2011.

Phosphite uptake following foliar application – July 2011
Figure 4-7 Accumulation of Phi in A. stolonifera leaf
and root tissues, 96 hours p.a. in July 2012.
Figure 4-8 Accumulation of Phi in P.annua leaf
and root tissues, 96 hours p.a. in July 2012.

Phosphite uptake following foliar application – July 2011
Figure 4-9 Accumulation of Phi in A.
stolonifera leaf and root tissues, 6 weeks
p.a. in July 2012.
Figure 4-10 Accumulation of Phi in P.
annua leaf and root tissues, 6 weeks p.a.
in July 2012.

HPIC Results – Phosphate
Figure 4-11 Phosphate amounts in leaf and root tissues of A. stolonifera.

HPIC Results – Phosphate
Figure 4-12 Pi amounts in leaf and root tissues of P. annua.

To assess the effect on turfgrass tissues and rootzones of sequential phosphite
applications to A. stolonifera and P. annua at monthly intervals, from July 2012 to
July 2014.
Take up and accumulation phosphite following long term
sequential applications
Treatments comprised of phosphite and phosphate applied at 0.35 g/m2 PO33- and
PO43- respectively
Phosphate was applied to assess the effect on long term soil P status
compared to the phosphite treatment.

Figure 4-14 phosphite accumulations
in P. annua leaf and root tissues
between July 2012 and July 2014.
Figure 4-13 phosphite accumulations
in A. stolonifera leaf and root tissues
between July 2012 and July 2014.

P levels in phosphite treated
rootzones increased
significantly from 37 to 51 ppm,
t(5) = 20.147, p < 0.01.
P levels in phosphate treated
rootzones increased
significantly from 37 ppm to 40
ppm, t(5) = 3.354, p = 0.02.
Phosphite treated rootzones
displayed significantly greater
P levels than in the phosphate
treated samples, t(5) = 14.094,
p < 0.01.
Analyses of rootzones

Quick summary
In vitro studies determined:
• Phosphite suppressed M. nivale mycelial growth
• Caused disruption of hyphal morphology
• inhibited conidial germination
Field trials determined:
• Phosphite significantly reduced M. nivale infection
• enhanced the efficacy of turfgrass fungicides
• HPIC found:
• Phosphite is rapidly taken up and translocated by turfgrass
Microdochium infection:
• Looked at infection process
• Phenolic compounds and H2O2 are key components of turfgrass defences
Can phosphite enhance or stimulate
defence responses???

Can phosphite enhance or stimulate defence responses?

0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
control Phosphate Phosphite
GAEmg/gdw
P.annua
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
GAEmg/gdw
A. stolonifera
TPC as GAE mg/g dw, in
turfgrass tissues sampled
from field trial plots
in turfgrass tissues
sampled from field trial
plots following six, monthly
applications of SDW
(control), phosphate and
phosphite Analysis carried
out 48 hpa.

TPC as GAE mg/g dw, in
turfgrass tissues sampled
from greenhouse plants
in turfgrass tissues
sampled from greenhouse
plants following six,
monthly applications of
SDW (control), phosphate
and phosphite Analysis
carried out 48 hpa.
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
GAEmg/gDW
P.annua
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
GAEmg/gDW
A. stolonifera

0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
0 dpi 1 dpi 2 dpi 3 dpi 4 dpi 5 dpi 6 dpi 7 dpi 8 dpi 9 dpi 10 dpi
GAEmg/gdw
P. annua
Control Pi Phi (6 apps)
TPC as GAE mg/g dw, in M.
nivale infected P. annua
over 10 dpi in greenhouse
turfgrasses
Total phenolic compounds in
M. nivale infected P. annua
over 10 dpi following
treatment with SDW (control),
phosphate and phosphite (6
apps)

0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
0 dpi 1 dpi 2 dpi 3 dpi 4 dpi 5 dpi 6 dpi 7 dpi 8 dpi 9 dpi 10 dpi
GAEmg/gdw
A. stolonifera
Control Pi Phi (6 apps)
TPC as GAE mg/g dw, in M.
nivale infected A. stolonifera
over 10 dpi in greenhouse
turfgrasses
Total phenolic compounds in M.
nivale infected A. stolonifera
over 10 dpi following treatment
with SDW (control), phosphate
and phosphite (6 apps)

0.00
5.00
10.00
15.00
20.00
25.00
30.00
35.00
μmolH2O2/gfw
P. annua
Control Pi Phi ( 6 apps)
0.00
5.00
10.00
15.00
20.00
25.00
30.00
35.00
μmolH2O2/gfw
A. stolonifera
Control Pi Phi ( 6 apps)
H2O2 concentrations in M.
nivale infected greenhouse
turfgrass tissues
H2O2 concentrations in SDW
(control), phosphate and
phosphite treated tissues of M.
nivale infected P. annua and A.
stolonifera greenhouse plants
over 10 days post inoculation

M. nivale infected P.annua leaf, viewed under UV
Fluorescence
Blue hyphae are visible with H2O2 fluorescencing
at stomatal infection sites.
TMB stained leaf tissues showing H2O2
fluorescence at infection sites
Fluorescence microscopy images of infected tissues

Accumulations of H2O2 and
TPC 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).

• 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

Microdochium nivale suppression
• inhibited conidial germination
Microdochium suppression:
• Phosphite in the plant slows the infection process
• Phosphite pre-treatment primes the plants defences
• Phosphite treated plants respond in a more timely manner than untreated

Phosphite as a nutrient or biostimulant?
Can Phosphite provide P nutrition?
Effects on turfgrass growth
What else happens when phosphite is applied to turfgrass?
We set up an experiment to assess the properties of
phosphite as a source of P nutrition for turfgrass growing
in different soil P conditions and to determine treatment
effect on turfgrass growth and development

Lolium perenne and Poa annua
P sufficient and deficient rootzones
Three treatments
• Phosphate -KH2PO4
• Phosphite - KH2PO3
• Control - KCl
Assessed
• Shoot growth
• Crowns
• Root growth
Phosphite - effects on turfgrass growth

Figure 4-19 Treatment effect on the growth
L. perenne in a P deficient rootzone.
Figure 4-20 Treatment effect on the
growth P. annua in a P deficient rootzone

Figure 4-23 Treatment effect on P levels of
L. perenne growing in a P sufficient rootzone
Figure 4-24 Treatment effect on P levels of
P. annua growing in a P sufficient rootzone

Control Phosphate Phosphite
• In P deficient
rootzones foliar-
applied phosphite
does not supply a
usable form of P and
represses deficiency
responses
• In P sufficient
rootzones foliar-
applied phosphite
increased biomass in
all plants, but also
led to a reduction in
root to shoot ratios
L. Perenne in P deficient rootzone

Summary!
Microdochium nivale infection:
• Hyphae are the main source of inoculum
• Infection is by means of stomatal penetration
• Conidia are the means of propagation and dispersal
• Generation of phenolic compounds and H2O2 are key
components of turfgrass defence

Summary!
• Enhanced the efficacy of turfgrass fungicides
• Led to improved turfgrass quality

Summary!
What are the means of suppression?
• Inhibited conidial germination.
Defence responses:
• Phosphite primes the plant prior to infection
• Enhances synthesis of phenolics and H2O2 as turfgrass
defence responses

Summary!
Means of suppression
• HPIC analyses shows phosphite is rapidly taken up
and translocated and does not convert to PO4 in the
plant
• Loss in the leaf is more rapid during high growth
periods
• Maintaining 3000 ppm and above will suppress disease
development

Summary!
Phosphite effect on turfgrass growth
• Phosphite is rapidly taken up and translocated but
does not supply a usable form of Phosphorus
• In P deficient rootzones plant deficiency
responses were repressed
• In P sufficient rootzones phosphite increased
biomass and improved turf quality
• Long-term phosphite treatment can lead to
cumulative increases in meristematic tissues and
can cause increases in soil P levels

In conclusion:
The last ten years work has shown that phosphite, when applied sequentially,
as a component of a balanced nutrient programme, will suppress M. nivale
incidence, increase the efficacy of turfgrass fungicides and lead to an
enhancement of turfgrass quality.
Phosphite is recommended as a valuable addition to turfgrass management
practices, as part of a foliar applied nutrient program, which will result in
reduced incidence of M. nivale infection and improved turfgrass quality.

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The role of phosphite in turfgrass management quebec

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