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
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Thanks for listening
Any questions?