suppression of microdochium nivale by phosphite in amenity turfgrasses
DESCRIPTION
Suppression of Microdochium nivale by Phosphite in amenity Turfgrasses Presentation for the HLS annual research forum at Uwe, BristolTRANSCRIPT
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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
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Centre for Research in Biosciences
PhD Research objectives -
Does phosphite reduce Microdochium nivale infection?
Means of reduction?
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Two components of this research
Microdochium nivale Phosphite
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Most common pathogen in cool-season turfgrass
Ascomycete fungus - Fusarium patch or Pink snow-mould
What is Microdochium nivale?
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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
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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
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Suppresses phytopathogens
Pythium and Phytophthora
Anthracnose
Microdochium majus in cereals
No research intoPhosphite and Microdochium nivale
Oomycete pathogens
Field trials Laboratory studies
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Agrostis canina canina
Agrostis stolonifera
Poa annua
Curragh golf course –field trials
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120 2 x 2 m plots
Range of phosphite treatments and assessments
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Published Trials –
Phosphite
(PO33- 0.37g/m-2)
Phosphite
+Biostimulant
Iprodione
(Fungicide)
Iprodione +Phosphite
NPK Control
Untreated Control
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Trials running since Sept 2010
Treatments applied bi-weekly
Five replications
Disease incidence assessed monthly
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Percent disease incidence
Phosphite
Phosphite+Biostimulant
Fungicide Fungicide+Phosphite
NPK Control Control
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Agrostis canina canina plots – January 2012
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Agrostis canina canina plots – January 2012
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Poa annua plots – January 2011
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Poa annua plot 5 – Iprodione + Phosphite
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Poa annua plot 6 – Phosphite
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Poa annua plot 16 - Control
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Agrostis stolonifera plot 5 – Control
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Agrostis stolonifera plot 7 – Phosphite
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Agrostis canina plot 3 - Control
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Agrostis canina plot 9 – Phosphite
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Field trial conclusions
• Sequential applications of phosphite significantly reduced Microdochium nivale incidence
• The addition of phosphite to iprodione significantly enhanced suppression of Microdochium nivale
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Means of suppression
• Inhibits pathogen
Direct
• Stimulates plants defences
Indirect
Combination of both
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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
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Amended growth media
Amended PDA
Range of phosphite and phosphate
From 0.5 μg/ml to 1000 μg/ml
Compared with unamended controls
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Control + 4 days
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Phosphate - 100 μg/ml + 4 days
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Phosphite - 100 μg/ml + 4 days
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Mycelial Growth on Amended PDA -4 days p.i.
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Hyphal morphology
Unamended 75µg/ml Phosphite
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Hyphal morphology
Unamended 75µg/ml Phosphite
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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
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• Measure assimilation rate• Track translocation• Determine accumulation amounts• Assess the fate
Targets
What happens when phosphite is applied to turfgrass?
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• Treat turfgrass• Collect samples• Six week period• Analyse using HPIC
Methods
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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
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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
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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
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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
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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
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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
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Infection process
Fluorescent microscopy and stains
Using pot samples and infected greens
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• 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
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Hydrogen peroxide
• Direct measurement Titanium oxysulphate and spectroscopy
• Histological stains using fluorescent microscopy TMB (tetramethylbenzidine)
DAB (diaminobenzidine)
• Confocal microscopy dichlorofluorescein diacetate - H2-DCFDA
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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)
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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
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Systemic Acquired Resistance
Salicylic acid – signal molecule for SAR
HPLC – compare untreated to phosphite treated turfgrass
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• 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
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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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Follow updates on Twitter - @J_J_Dempsey
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Thanks for listening
Any questions?