Suppression of Fusarium patch by Phosphite in cool season turfgrasses

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1 Centre for Research in Biosciences Suppression of Fusarium patch by Phosphite in cool season turfgrasses John Dempsey BSc(Hons) Centre for Research in Biosciences, Bristol, UK

2 Greenkeeper since mid 1980 s Course manager at Curragh Golf Course since 1993 BSc in Turfgrass science Myerscough college, UK Currently carrying out postgraduate research for a PhD in turfgrass pathology The mechanism by which phosphite reduces susceptibility to Microdochium nivale

3 Todays talk- Phosphite - what is it, its history and current usage Does phosphite suppress Microdochium nivale in turfgrasses? By what means does suppression occur? Microdochium infection process Turfgrass responses

4 Two components of this research Microdochium nivale Phosphite

5 What is Microdochium nivale? Ascomycete fungus - Fusarium patch or Pink snow-mould Most common pathogen in cool-season turfgrass

6 Microdochium active on Poa annua

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

8 Phosphite? Form of Phosphorous (P) a major nutrient of plant growth Taken up as Phosphate - Phosphoric acid (H 3 PO 4 ) Phosphite - Phosphorous acid (H 3 PO 3 )

9 Phosphite use Phosphite derived from Phosphorous acid (H 3 PO 3 ) ph has to be modified with an alkali salt to usable ph First used in 1980 s - Rhone- Poulenc fosetyl Al (aluminum tris (Oethyl phosphonate)) Potassium hydroxide (KOH) - Forms Potassium dihydrogen phosphite (KH 2 PO 3 ) or dipotassium hydrogen phosphite (K 2 HPO 3 ) Potassium phosphite Ammonium Hydroxide, Magnesium phosphites and Calcium phosphites

10 Phosphite not metabolised in plants Phosphite should not be applied to plants in sub-optimal phosphate conditions Phosphite only converts to phosphate by means of soil organisms Bacteria can metabolise Phosphite to Phosphate - Escherichia coli, Pseudomonas stutzeri, Alcaligenes faecalis and Xanthobacter flavus Half-life for oxidation in soil is approximately weeks (Adams and Conrad 1953).

11 Suppresses phytopathogens Pythium and Phytophthora Oomycete pathogens Anthracnose Microdochium in cereals Improved turf quality No research into Phosphite and Microdochium nivale

12 Performance of Phosphonate Fertilizers and Fungicides on Pythium Blight Development on a Perennial Ryegrass Golf Course Fairway Horvath, B.J. and D.S. McCall, Virginia Tech University

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

15 BSc Study at Myerscough Dr. Andy Owen Establish Agrostis stolonifera swards Apply a range of phosphite treatments Assess treated and untreated controls Growth Turf quality Disease occurrence

16 Methodology Agrostis stolonifera established in Greenhouses Trial plots

17 Treatments Phosphite 0.35g/m g/m g/m 2 Phosphate 0.35g/m g/m g/m 2 Control Nil Applied every 3 weeks - Replicated 6 times

18 Assessments - Growth Shoots Crowns Collected dried and weighed

19 Results - shoot Growth

20 Results shoot and crowns

21 Control Phosphite December 2008

22 Assessments Turf Quality Turf qualitygreenhouse and trial plots Visually assessed 2 weekly intervals Scale of- 1 10

23 Results Turf quality

24 Turf quality Trial plots

25 Assessments Disease Occurrence Disease assessments- Greenhouse and trial plots Visually assessed 2 weekly intervals Rated on percentage of disease occurrence

26 Results Greenhouse

27 Results Trial plots

28 Control Phosphite January 2009

29 Phosphate Phosphite November 2008

30 Control Phosphite December 2008

31 Phosphite treated plants January 2009

32 Control plants January 2009

33 Conclusions of the Myerscough Study Not metabolised Phosphite treatment- Improved turfgrass quality Reduced disease

34 European Turfgrass Society 2 nd Conference Angers, France 2010

35 Centre for Research in Biosciences PhD Research objectives - Phosphite reduce Microdochium? Means of reduction? Field trials Laboratory studies

36 Curragh golf course field trials Three turfgrass species Agrostis canina canina Agrostis stolonifera Poa annua Range of phosphite treatments and assessments

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38 Published Trials Griggs and Turfcare PK Plus 20L ha -1 (PO g/m -2 ) PK Plus 20L ha -1 +Ultraplex 10L ha -1 Chipco 20L ha -1 Iprodione 20L ha -1 Chipco 20L ha -1 Trials running since Sept 2010 Treatments applied bi-weekly Disease incidence assessed monthly +PK Plus 20L ha -1 NPK Control (3:7:18 20l ha -1 ) Untreated Control

39 Disease Assessments Poa Plots

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41 Poa annua plots - mean disease incidence over two years (n=5), letters indicate significant differences at p< 0.01

42 Agrostis stolonifera plots mean disease incidence over one year (n=5), bars represent standard error, letters indicate significant differences at p< 0.01

43 Agrostis canina canina plots

44 Agrostis canina canina plots mean disease incidence over two years (n=5), bars represent standard error, letters indicate significant differences at p< 0.01

45 Mean disease incidence over two years (n=5), bars represent standard error, letters indicate significant differences at p< 0.01 PK Plus +Ultraplex Chipco Chipco +PK Plus NPK Control Control

46 Assessment of Turf quality 2010 to 2012

47 Turf quality average over two years P. annua A. canina canina a a a a a a b 5.00 b 4.00 b b b b PK Plus Pk Plus + Ultraplex Chipco Green Chipco Green + PK Plus NPK control Control

48 Agrostis canina canina plots January 2012

49 Agrostis canina canina plots January 2012

50 Poa annua plots January 2011

51 Poa annua plots October 2011 CONTROL PHOSPHITE

52 Poa annua plot 5 Phosphite

53 Poa annua plot 16 - Control

54 Agrostis stolonifera plot 7 Phosphite

55 Agrostis stolonifera plot 5 Control

56 Agrostis canina plot 9 Phosphite

57 Agrostis canina plot 3 - Control

58 Field trial conclusions Sequential applications of phosphite significantly reduced Microdochium nivale incidence The addition of phosphite to iprodione significantly enhanced suppression of Microdochium nivale Significant improvement in turfgrass quality

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

60 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

61 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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64 Control + 4 days

65 Phosphate μg/ml + 4 days

66 Phosphite μg/ml + 4 days

67 Mycelial Growth on Amended PDA Phosphite 100µg/ml Phosphate 100µg/ml Control

68 Hyphal morphology Unamended 75µg/ml Phosphite

69 Hyphal morphology Unamended 75µg/ml Phosphite

70 Fungicide or Fungistat? Does phosphite kill the pathogen? Or just inhibits the growth? Immersed the mycelium in a range of phosphite and phosphate concentrations for 10 days Extracted, washed and placed them on fresh PDA Observed the effects on re-growth

71 Radial growth -mm Mycelial growth after immersion for 10 days M. nivale mycelial growth - four days post inoculation Control 50µM 100µM 500µM 1mM 5mM Control PO4 PO3

72 In vitro conclusions Inhibits mycelial growth and conidial germination Disrupts hyphal morphology In the plant Slows the growth of the pathogen Causes release of stress metabolites Allows for increased time for the plant to initiate defence responses

73 What happens when phosphite is applied to turfgrass? Targets Measure assimilation rate Track translocation Determine accumulation amounts Assess the fate Methods Treat turfgrass Collect samples Six week period Analyse using HPIC

74 ppm Phosphite accumulation in Agrostis stolonifera h 1h 6h 12h 24h 48h 1wk 2wk 4wk 6wk Time post-application Leaf phosphite Root phosphite

75 HPIC Results Phosphate ppm Phosphate accumulation in Agrostis stolonifera h 1h 6h 12h 24h 48h 1wk 2wk 4wk 6wk Time post-application Leaf phosphate Root phosphate Leaf phosphate -Control Root phosphate -Control

76 HPIC Results Phosphite +Phosphate

77 Long term results 4 weeks post application ppm ppm in first study ppm in first study ppm in first study Leaf Crown Root 6 months 4 wks pa 12 months 4 wks pa Samples taken January and July 2012

78 Long term results 8 weeks post application ppm Leaf Crown Root 6 months 8 wks pa 12 months 8 wks pa Samples taken January and July 2012

79 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

80 Does Phosphite enhance the defence responses in infected turfgrass? Need to understand the infection process and turfgrass responses Using pot samples and infected greens

81 Fluorescent microscopy and stains

82 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 to induction of defence responses

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84 Hydrogen peroxide Nitric oxide Phenols Phytoalexins Salicylic acid

85 Defence related compounds Histological stains using fluorescent microscopy TMB - tetramethylbenzidine DAB - diaminobenzidine Direct measurement Titanium oxysulphate and spectroscopy Confocal microscopy H 2 -DCFDA - dichlorofluorescein diacetate DAF2-DA - diaminofluorescein diacetate

86 TMB staining DAB staining Hydrogen peroxide detection in Triticale seedlings inoculated with M. nivale, stained with DAB. The brown colour around penetration sites indicates H 2 O 2 generation (Dubas et al., 2010)

87 Phenolic compounds Another important response to pathogen challenge Measure using reagent and spectroscopy Visualise using fluorescent microscopy 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)

88 Systemic Acquired Resistance Salicylic acid signal molecule for SAR HPLC compare untreated to phosphite treated turfgrass

89 Results to date Significant reduction in Microdochium nivale incidence In combination with fungicide enhanced disease suppression Rapidly assimilated, translocated by turfgrass Inhibits mycelial growth and conidial germination Disrupts hyphal morphology Tracked Microdochium infection process

90 Further Research Centre for Research in Biosciences Fields trials are continuing In vitro studies HPIC analyses Infection process in turfgrass Defence processes Hydrogen peroxide Nitric oxide Phenolic compounds SAR- Salicylic acid synthesis

91 Summary Alternative means to suppress disease are required Phosphite application significantly reduces Microdochium nivale Phosphite is rapidly assimilated and translocated by turfgrass Phosphite has a direct inhibitory effect on mycelial growth Tracked Microdochium infection process defence responses Enhance Inducible defence mechanisms - ROS Systemic Acquired resistance -phytoalexins and salicylic acid

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93 Thanks for listening Any questions?