Abstract on the research into the assimilation and accumulation of phosphite in A. stolonifera and the in vitro effect phosphite has on the mycelial growth of M. nivale

1. Assimilation of Phosphite by Agrostis stolonifera L. and its In Vitro Effect on
Microdochium nivale
J.J. Dempsey, I.D. Wilson, P.T.N Spencer-Phillips, and D.L. Arnold
Centre for Research in Biosciences, University of the West of England, Bristol, BS16 1QY.
Keywords: Agrostis stolonifera, High Performance Ion Chromatography, in vitro, mycelium.
Introduction
Microdochium nivale (Fr.) Samuels and Hallett, (teleomorph Monographella nivalis (Schafnitt) is
a major pathogen of cool-season turfgrasses (Vargas, 2005).The search for new or improved means to
reduce susceptibility to M. nivale is an ongoing target for turfgrass research. One possible means is the
use of phosphite (PO33-, Phi) which is derived from phosphorous acid (H3PO3) and commonly modified
with potassium hydroxide (KOH), forming KH2PO3 or K2HPO3 (potassium phosphite)(Ouimette and
Coffey, 1988). In turfgrass management these form the active substances in numerous products currently
marketed as either fungicides or fertilisers and used as a component of an integrated approach to disease
management (Landschoot and Cook, 2005). Phi has proven efficacy in reducing susceptibility to
oomycete pathogens in numerous plant species. While there are data showing similar success in
controlling ascomycete pathogens (Hofgaard et al., 2010) and reports of Phi mediated reduction of M.
nivale in turfgrass (Dempsey and Owen, 2010), studies into Phi uptake in turfgrass and its possible direct
inhibition of M. nivale are lacking. The objectives of this study therefore, were to determine the mode of
assimilation of Phi in A. stolonifera L. and to assess the inhibitory effects Phi may have on the in vitro
mycelial growth of M.nivale.
Materials and Methods
The assimilation rate, translocation and accumulation of Phi and phosphate (PO43-, Pi) in A.
stolonifera L. were determined using High Performance Ion Chromatography (HPIC). A potassium
phosphite solution was prepared by adjusting a 1M solution of phosphorous acid (H3PO3) to a pH of 6.4,
by titrating with 10 M potassium hydroxide (KOH). This was used to apply a foliar treatment to A.
stolonifera, at a rate of 0.35 g PO33-/ m-2. Leaf, crown and root tissues from treated and control plants
were harvested 1, 6, 12, 24, 48 hours and 1, 2, 3, 4, 5, and 6 weeks post application (p.a.). The dried and
ground tissues were extracted into 10 ml of deionised water and injected via a 0.47 micron filter into a
Dionex HPIC system, using 9 mM sodium carbonate eluent. Results shown are means (n=4) of Phi and Pi
as parts per million (ppm) of dry tissue.
In vitro inhibition of M. nivale mycelial growth was determined by amending PDA (19 g/l) with
Phi and Pi. Amendments ranged from 0.5 to 1000 μg/ml-1 (n=6). Mycelial radial growth measurements
taken 96 hours post inoculation (p.i.)
with M. nivale were used to calculate
percent relative growth (PRG) on Phi
amended PDA compared to
unamended and Pi amended PDA. The
effective concentration that reduced
mycelial growth by 50% (EC50) was
determined by probit transforming the
PRG and regressing against the Log10
of amendments.
Data were subject to analysis
of variance (ANOVA) and significant
treatment differences were separated
by Tukey Least Significant Difference
(LSD) Test at p< 0.01 (SPSS Statistics
19.0).
Figure 1 Phi and Pi accumulations (ppm) in A. stolonifera (n=4) six
weeks p.a. with 0.35 g PO33-/ m-2

2. Results and discussion
Figure 1 shows Phi and Pi
accumulations in A. stolonifera tissues over
6 weeks p.a. Phi accumulations in leaf
tissues 48 h p.a. were 4889 ppm, with 3193
ppm (65% of maximum accumulation) at 6
h p.a, proving rapid assimilation. Amounts
declined to 2561 ppm at 2 weeks p.a.,
indicating a 3-4 week application cycle
would maintain in planta levels consistently
between 2000 and 4000 ppm. Root
accumulations peaked 2 weeks p.a. at 492
ppm, less than leaf amounts but confirming
that Phi is symplastically mobile. Analysis
of the crowns determined 1250 and 484
ppm at 4 and 6 weeks p.a. Pi amounts in
both treated and control tissues were not
significantly different, indicating that Phi
was not metabolised in planta to Pi.
In vitro analysis determined that PDA Figure 2 Inhibition of in vitro mycelial growth of M. nivale on Phi
and Pi amended PDA. Data are mean of two experiments
amended with Phi concentrations of (n=6), bars are standard mean error.
100μg/ml and above fully inhibited M. nivale
mycelial growth, with an EC50 value of 38μg/ml. As can be seen in Figure 2, Pi amended PDA caused no
inhibition. Microscopic analysis of hyphal morphology showed distinct irregularities in M. nivale
growing on Phi amended PDA (Figure 3). While on Pi amended PDA mycelial growth was normal.
Conclusions A B
Results have shown that Phi is rapidly
assimilated and translocated by turfgrass; and
that sequential applications applied on a 3
week cycle would maintain leaf tissue
amounts of approximately 3000 ppm.
In vitro research determined that Phi
has a direct inhibitory effect on mycelial
growth of M. nivale, with total growth
inhibition at amounts of 100μg/ml and above Figure 3 Effect of Phi on hyphal morphology of M. nivale (A)
with an EC50 value of 38μg/ml. Normally developed mycelium grown on unamended PDA.
Further research on treated turfgrass (B) Short-branched and swollen hyphae grown on PDA
is evaluating secondary metabolic processes amended with 50μg/ml PO33-.
to determine the role of Phi in activating
inducible defence mechanisms and stimulation of Systemic Acquired Resistance.
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