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Biochemical effects of phosphite on the phytopathogenicity of Phytophthora cinnamomi Rands

Stasikowski, Patricia (2012) Biochemical effects of phosphite on the phytopathogenicity of Phytophthora cinnamomi Rands. PhD thesis, Murdoch University.

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Phosphite, a chemical analogue of orthophosphate, controls disease symptoms and spread of Oomycete plant pathogens, particularly those caused by Phythophthora spp. Phosphite can be applied to horticultural and native plant species as a foliar spray or trunk injection and results in in planta phosphite concentrations of between 25 - 425 μg g-1 dry weight (equivalent to 0.3 – 6.0 mM). However, despite its extensive use it is not known why phosphite is biostatic towards oomycetes, although several mechanisms have been proposed.

This thesis aims to devise and test a biochemical model of phosphite action that could account for the observed effects of phosphite on the interaction between Phytophthora cinnamomi and a susceptible plant host. However, prior to this it was necessary to devise a test to assess the concentration of phosphite in planta and to establish that phosphite needs to be present at the plant /pathogen interface in order to have an effect. A silver nitrate staining method was developed and its ability to detect phosphite was assessed in a variety of native Australian and horticultural plants. The method demonstrated that phosphite concentrations of between 1 and 3 mM were present in the tips of the roots of lupins that had been foliar sprayed with 0.5 % phosphite (equivalent to 62 mM) and that in most instances, these concentrations were sufficient to completely control the development of disease symptoms.

As phosphite is chemically similar to orthophosphate its presence in a cell is likely to interfere with many aspects phosphate metabolism in both plant and pathogen. In order to discern the mechanism of action of phosphite it was important to separate the antipathogenic effects from the general/ pleotropic effects. A bioassay was devised whereby the roots of lupin seedlings were inoculated with filter paper discs that had been colonised with P. cinnamomi isolate MP94-48 and then treated with phosphite or other chemicals that would be expected to reduce its pathogenicity. The extent of lesion development and the root growth below the point of inoculation were the two parameters by which the effect of the chemicals on pathogenicity was assessed.

Increasing either the concentration of orthophosphate (0 – 100 mM) or phosphite (0 – 10 mM) in the growth medium of P. cinnamomi colonised discs reduced lesion development on inoculated lupin seedling roots. Orthophosphate concentrations of between 3 – 10 mM, in combination with 1 mM phosphite did not reduce the extent of lesion development. In contrast, plants inoculated with discs treated with concentrations of orthophosphate above 10 mM together with 3 and 10 mM phosphite, lesions were reduced when compared to plants inoculated with discs treated with phosphite alone.

The inhibition of phosphatase activity in P. cinnamomi is often proposed to be a primary effect of phosphite. Treatment of P. cinnamomi colonized discs with the phosphatase inhibitors okadaic acid, sodium fluoride, and a mixture of inhibitors containing sodium vanadate, sodium molybdate, sodium tartrate and imidazole, neither decreased nor increased the development of lesions, and no change in the degree of phosphorylation of cytosolic proteins could be detected by Pro-Q Diamond phosphoproteins staining. The addition of the kinase inhibitor staurosporine (0.1 - 1 mM) reduced lesion development on lupins and this effect was augmented slightly, but significantly, by the addition of phosphite (3 mM). It was not possible to draw any conclusions from the results of experiments testing the effect of addition of exogenous cAMP or the phosphatase inhibitor phenyl arsine oxide to colonised discs on the ability of P. cinnamomi to produce lesions in lupins. These results suggest that phosphorylation reactions and cascades may not be the primary control mechanism in either initiation or inhibition of phytopathogenesis. However, the addition of glucose (30 mM) increased pathogenicity and the development of lesions.

As evidence exists that abscisic acid (ABA) increases the susceptibility of plants to infection by Phytophthora spp. and that ABA signaling involves phospholipase D (PLD) the effect of inhibitors on this signaling pathway were tested on the ability of P. cinnamomi to produce lesions. Primary, 2o and 3o butyl alcohol, as well as the guanine nucleotide exchange factor (GEF) inhibitor brefeldin A, and ABA itself were added to cultures of P. cinnamomi. The application of either 1o, 2o or 3o butyl alcohol to P. cinnamomi colonised discs had no effect on lesion development, which would be expected were the generation of phosphatidic acid per se was vital to pathogenicity. However, Brefeldrin A (10 - 250 μM) had a highly significant and concentration dependent effect on the development of lesion on lupins. These results suggested that a member of the Ras-superfamily (such as an ADP ribosylation factor) is likely to be involved in the development of lesions and that the exchange of GDP for GTP on this protein is required for pathogenesis. The results from the bioassays of addition of exogenous ABA to P. cinnamomi colonised discs were ambiguous and additional experimentation is needed to elucidate the role of ABA in the phytopathogenesis of P. cinnamomi.

Calcium ion signatures and cytosolic gradients are known to be important components of many signal transduction pathways and increased soil calcium can limit the development of Phytophthora disease. The effect of external calcium ion concentration and the calcium channel blockers ruthenium red (RR), lanthanum chloride (La3+) and the calcium ion chelator EGTA on the development of lesions was investigated. The results of the bioassays indicated that external calcium ion concentration, RR and La3+ (100 μM) reduced lesion development significantly as did EGTA (1 mM) and that this reduction was further enhanced in the presence of phosphite.

The combined role of phosphite and external calcium ion concentration was further investigated in a glasshouse pathogenicity trial using P. cinnamomi and the Australian plant Banksia leptophilia in a factorial nested pot design with foliar phosphite and soil calcium sulphate concentration as independent variables. The results one year post-inoculation confirmed that when foliar phosphite (0.1% - 0.3%) was used in conjunction with soil supplementation with calcium sulphate (3 – 30 mM) disease symptoms and lesion development were significantly reduced and general plant health was improved.

The combined results of these experiments suggested not only a role for calcium ion concentration and signaling in pathogenicity but, together with the 35-fold increase in PPi concentration, imply that inhibition of the calcium dependent ATPase responsible for regulating cytosolic Ca 2+ concentration may be the cause of the antipathogenic effect of phosphite in P. cinnamomi. Calcium dependent ATPases are known to be involved in the gravitropic response of roots as well as the polar growth of pollen tubes (i.e. presence of the Ca2+ channel blocker La3+ results in inhibition of the gravitropic and polar response). Preliminary results of the effect of phosphite on the gravitropism of lupin seedling roots indicate that phosphite does inhibit the gravitropic response, suggesting that there is a causal link between the mechanism of action of phosphite and calcium-dependent ATPases.

Item Type: Thesis (PhD)
Murdoch Affiliation(s): School of Biological Sciences and Biotechnology
Supervisor(s): Hardy, Giles, McComb, Jen and O'Brien, Phllip
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