Centre for Research in Biosciences

Suppression of Microdochium nivale by Phosphite in amenity Turfgrasses

John Dempsey BSc(Hons)Centre for Research in Biosciences, Bristol, UK

Centre for Research in Biosciences

PhD Research objectives -

Does phosphite reduce Microdochium nivale infection?

Means of reduction?

Two components of this research

Microdochium nivale Phosphite

Most common pathogen in cool-season turfgrass

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 organismsLegislative controls

Microdochium active on turfgrass

Phosphite?

Form of Phosphorous (P) a major nutrient of plant growth

Taken up as Phosphate - Phosphoric acid (H3PO4)

Phosphite - phosphorous acid (H3PO3)

Phosphite not metabolised in plants

Suppresses phytopathogens

Pythium and Phytophthora

Anthracnose

Microdochium majus in cereals

No research intoPhosphite and Microdochium nivale

Oomycete pathogens

Field trials Laboratory studies

Agrostis canina canina

Agrostis stolonifera

Poa annua

Curragh golf course –field trials

120 2 x 2 m plots

Range of phosphite treatments and assessments

Published Trials –

Phosphite

(PO33- 0.37g/m-2)

Phosphite

+Biostimulant

Iprodione

(Fungicide)

Iprodione +Phosphite

NPK Control

Untreated Control

Trials running since Sept 2010

Treatments applied bi-weekly

Five replications

Disease incidence assessed monthly

Percent disease incidence

Phosphite

Phosphite+Biostimulant

Fungicide Fungicide+Phosphite

NPK Control Control

Agrostis canina canina plots – January 2012

Agrostis canina canina plots – January 2012

Poa annua plots – January 2011

Poa annua plot 5 – Iprodione + Phosphite

Poa annua plot 6 – Phosphite

Poa annua plot 16 - Control

Agrostis stolonifera plot 5 – Control

Agrostis stolonifera plot 7 – Phosphite

Agrostis canina plot 3 - Control

Agrostis canina plot 9 – Phosphite

Field trial conclusions

• Sequential applications of phosphite significantly reduced Microdochium nivale incidence

• The addition of phosphite to iprodione significantly enhanced suppression of Microdochium nivale

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 ofmycelial growth

Amended growth media

Amended PDA

Range of phosphite and phosphate

From 0.5 μg/ml to 1000 μg/ml

Compared with unamended controls

Control + 4 days

Phosphate - 100 μg/ml + 4 days

Phosphite - 100 μg/ml + 4 days

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

Hyphal morphology

Unamended 75µg/ml Phosphite

Hyphal morphology

Unamended 75µg/ml Phosphite

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

• Measure assimilation rate• Track translocation• Determine accumulation amounts• Assess the fate

Targets

What happens when phosphite is applied to turfgrass?

• Treat turfgrass• Collect samples• Six week period• Analyse using HPIC

Methods

0h 1h 6h 12h 24h 48h 1wk 2wk 4wk 6wk0

1000

2000

3000

4000

5000

6000

0

639

3193

3876

4205

4889

3334

2561

715

393

0 55 111 120

376

116 126

492338 265

Phosphite accumulation in Agrostis stolonifera

Leaf phosphite Root phosphite

Time post-application

ppm

0h 1h 6h 12h 24h 48h 1wk 2wk 4wk 6wk0

2000

4000

6000

8000

10000

12000

Phosphate accumulation in Agrostis stolonifera

Leaf phosphate Root phosphate Leaf phosphate -Control Root phosphate -ControlTime post-application

ppm

Long term effects of phosphite applications

• Phosphite, phosphate and control areas

• Application at 3 week intervals

• Commenced June 2011

• Assessment of PO33- and PO4

3- in leaf, crowns and roots

• Assessments of soils P levels

Long term results – 4 weeks post application

Leaf Crown Root0

200

400

600

800

1000

1200

1400

1600

1104

1493

183

802

1055

156

6 months 4 wks pa 12 months 4 wks pa

ppm

1250 ppm in first study

Samples taken – January and July 2012

715 ppm in first study

338 ppm in first study

HPIC Conclusions

• Phosphite is rapidly assimilated by turfgrass

• Translocates throughout the plant

• Accumulates in the leaf tissues

• 3-4 week application period maintains levels within the leaf

• Long term applications show metabolic rate effects accumulation period in tissue

• Slight increase in meristematic areas• Effect on soil P amounts yet to be calculated

Does Phosphite enhance the defence responses in infected turfgrass?

Need to understand the M. nivale infection process and turfgrass responses

Defence related compounds-

• Hydrogen peroxide• Nitric oxide• Phenols• Phytoalexins• Salicylic acid

Infection process

Fluorescent microscopy and stains

Using pot samples and infected greens

• Inoculum in the soil –conidia, mycelium

• Infection first in the crown and sheath area

• Moves to the leaf and enters plant through stomata

• The plant recognises the pathogen, this leads in induction of defence responses

Hydrogen peroxide

• Direct measurement Titanium oxysulphate and spectroscopy

• Histological stains using fluorescent microscopy TMB (tetramethylbenzidine)

DAB (diaminobenzidine)

• Confocal microscopy dichlorofluorescein diacetate - H2-DCFDA

TMB staining DAB staining

Hydrogen peroxide detection in Triticale seedlings inoculated with M. nivale, stained with DAB. The brown colour around penetration sites indicates H2O2 generation (Dubas et al., 2010)

Phenolic compounds

• Measure using reagent and spectroscopy

A. Autofluorescence of phenolic compounds (yellow) in leaf close to the hyphae (blue)

B. Callose (light-green) in leaf cells after aniline blue staining(Zur et al., 2011)

• Another important response to pathogen challenge

• Visualise using fluorescent microscopy

Systemic Acquired Resistance

Salicylic acid – signal molecule for SAR

HPLC – compare untreated to phosphite treated turfgrass

• Tracked Microdochium infection process

Results to date

• Rapidly assimilated, translocated by turfgrass

• Significant reduction in Microdochium nivale incidence

• In combination with fungicide enhanced disease suppression

• Inhibits mycelial growth and conidial germination

• Disrupts hyphal morphology

Centre for Research in Biosciences

Further Research

Fields trials are continuing

In vitro HPIC analyses

Defence processes – ROSNO2

Phenolic compounds and phytoalexins

Systemic Acquired Resistance -

Measure salicylic acid

Infection process in turfgrass

Follow updates on Twitter - @J_J_Dempsey

Thanks for listening

Any questions?