Philip O'Brien

Phosphorus is one of the most critical macronutrients for plants and taken up from the soil in the form of phosphate (H2PO4-, Pi) by specific transporters. It is frequently a growth limiting factor due to low availability in the soil. Hence plants have developed adaptations to Pi starvation that include the alteration of root architecture and the secretion of organic acids to enhance Pi uptake capacity. Pi depleted plants also increase the expression of genes involved in Pi acquisition, e.g. Pi transporters and purple acid phosphatases. Phosphite (H2PO3-, Phi) is the reduced form of Pi and taken up by plants through phosphate transporters. Although metabolically inert, Phi is able to suppress some Pi-starvation responses which exacerbates Pi depletion leading to an inhibition of plant growth. In addition, Phi induces plant defence responses and effectively inhibits colonisation by oomycete pathogens (e.g. Phytophthora spp.). Although phosphite is the only reliable measure to control these pathogens, its mode of action remains unclear. To better understand the effects of Phi on plant growth and induced pathogen resistance we are analysing the genetic basis of Phi sensitivity in Arabidopsis thaliana. We have investigated the phenotypic responses of 18 Arabidopsis accessions and also EMS mutagenized lines to Phi treatment. This has led to the identification of a major QTL and also a mutant line with increased phosphite tolerance. Gene expression studies also reveal differences in the response of two contrasting accessions. The findings will improve our knowledge of the mode of phosphite action in plant defence responses, and may have implications for the understanding of Pi signalling or metabolism.

Phosphorus is one of the most critical macronutrients for plants and taken up from the soil in the form of phosphate (H2PO4-, Pi) by specific transporters. It is frequently a growth limiting factor due to low availability in the soil. Hence plants have developed strategies to adjust to Pi starvation with adaptations including the alteration of root architecture or the secretion of organic acids to raise Pi uptake capacity. Pi depleted plants also increase the expression of genes involved in Pi acquisition, e.g. Pi transporters and purple acid phosphatases. Phosphite (H2PO3-, Phi) is the reduced form of Pi and taken up by plants through phosphate transporters. Although metabolically inert, Phi is able to suppress Pi starvation responses which exacerbates Pi depletion leading to an inhibition of plant growth. In addition, Phi induces plant defence responses and effectively inhibits colonisation by oomycete pathogens (e.g. Phytophthora spp.), which have devastating effects in horticultural and native ecosystems. Although phosphite is the only reliable measure to control these pathogens its mode of action remains unclear. To better understand the effects of Phi on plant growth and induced pathogen resistance we are analysing the genetic basis of Phi sensitivity in Arabidopsis thaliana. We have investigated the phenotypic responses of 18 Arabidopsis accessions and EMS mutagenized lines to Phi treatment. This has led to the identification of a major QTL and also a mutant line with increased phosphite tolerance. Findings from this research will improve our knowledge of the mode of phosphite action in plant defence responses, and may have implications for the understanding of Pi signalling or metabolism.

Foliar application of phosphite, a systemic fungicide, to Phytophthora cinnamomi infected plants results in the control of disease symptoms and a reduction in the spread and impact of the pathogenic in native plant communities. Calcium ions have also been shown to affect the interaction between Phytophthora species and their plant hosts and to reduce the impact and spread of disease caused by soil‐borne Phytophthora species. Calcium may enhance plant defence mechanisms or interfere with sporangial production, zoospore release and encystment on plant roots. Phosphite has been shown to have similar effects. The addition of calcium salts to soil inhibits the infection of plants by P. cinnamomi, and there is a correlation between the incidence in dieback disease caused by P. cinnamomi in natural ecosystems and the distribution of calcareous soil. This study used a susceptible Australian native plant species Banksia leptophilia, to investigate whether the disease control of P. cinnamomi by phosphite could be augmented by soil supplementation with calcium sulphate. The results showed that the effects of applying both calcium and phosphite were synergistic, and that the addition of calcium sulphate to the soil augmented and significantly prolonged the effect of foliar phosphite application. A mechanism involving the disruption of intracellular calcium signatures caused by phosphite induced accumulation of pyrophosphate in the cytosol of P. cinnamomi is discussed.

Phosphite (phosphonate) is widely applied to plant communities to control the spread and impact of Phytophthora species in natural and peri‐urban woodland and forest ecosystems. Determining (1) if phosphite applications have been successfully taken up in planta, (2) how phosphite is distributed around plants across seasons, and (3) when plants need to be retreated to maintain effective pathogen control is problematic due to the time and costs associated with current methods. This paper describes a direct chemical method of rapidly and effectively estimating the concentration of phosphite in plant material using a silver nitrate reagent. Glass fiber filter papers (Whatman GF/B) are saturated with acidified silver nitrate (1 M) and dried for 2 hours at 600C. 20 uL of a PVPP treated aqueous plant extract is then adsorbed on to the filter paper and incubated in the dark at room temperature for 1 hour. The presence of phosphite in the extract reduces the silver ions to elemental silver resulting in a grey‐black precipitate that is clearly visible. The method was successfully tested on the roots and leaves of a range of exotic and Australian native plants species from different families and genera which had been treated with 0.3% phosphite. The method is rapid, sensitive and inexpensive, and can detect phosphite at concentrations of 1 mM in 20 ul of aqueous extract from 100 mg of fresh plant material, equivalent to 82 ug g‐1 fresh weight, or 20 nmol phosphite per sample. The concentrations detected by the silver nitrate method equated well with the more expensive and less rapid HPLC method that we used to confirm the accuracy of the assay.

Phosphorus is one of the most critical macronutrients for plants and is taken up from the soil in the form of phosphate (H2PO4 −, Pi). It is frequently not readily available to plants as it is often found in low concentrations and bound to other soil components. Hence plants have developed strategies to adjust to Pi starvation with adaptations such as alterations of primary root length, number of lateral roots or secretion of organic acids to raise Pi uptake capacity. In addition, Pi depleted plants increase the expression of genes involved in Pi acquisition, e.g. Pi transporters and purple acid phosphatases. Phosphite (H2PO3 −, Phi) is the more reduced form of Pi and taken up by plants through phosphate transporters. Although metabolically inert, Phi is able to suppress Pi starvation responses, which results in an exacerbated Pi depletion leading to an inhibition of plant growth. In addition, Phi accumulation can lead to toxicity, likely through interference with Pi dependent reactions or remobilization. Phi is also used as an inducer of plant defense and biostat against plant pathogens, especially oomycetes (e.g. Phytophthora spp.), which have devastating effects in horticultural and native ecosystems. The only reliable measure to control these pathogens is Phi, but its mode of action is yet to be elucidated. To gain a better understanding on the effects of Phi on plant growth and defense we have started to investigate the natural genetic variation of Phi sensitivity in Arabidopsis thaliana. Findings from this research will improve our knowledge of the mode of action of Phi on plant defense responses, and might also have implications for the understanding of Pi signaling or metabolism. Investigations into the genetic background of Phi dependent adaptation have been made by observing the phenotypic responses of 18 different Arabidopsis accessions. We will present first results obtained by screening for root phenotypes on media containing varying ratios of Pi and Phi.

Phosphite (H2PO3 -, Phi) is an analog of phosphate (H3PO4 -, Pi) which is not metabolized by plants and therefore accumulates in the tissue. Because of their close steric resemblance Phi is able to mimic Pi, thus impeding Pi sensing and signalling mechanisms. In Pi limited plants Phi inhibits phosphate starvation responses, i.e. adaptations aimed at coping with limited Pi supply and increasing Pi uptake capacity. Thus, Phi has constrictive effects on plant growth under low P supply. Phi is also commercially marketed as a biostat against oomycete pathogens (e.g. Phytophthora spp.) for agricultural application and even used on an ecosystems scale. Phi directly inhibits pathogen growth through interference with phosphate-dependent metabolism and/or phosphate signaling which is paralleled in plants grown on high phosphite concentrations. In addition, Phi activates plant defense responses, e.g. by SA-dependent induction of defense gene expression or increased callose deposition, by a yet unknown mode of action. We have started to characterize the impact of prolonged phosphite treatment by analyses of growth responses, root development, gene expression and metabolic adjustments. We show that at low levels Phi has indirect impact on plant growth by inhibiting phosphate uptake whereas at elevated tissue contents Phi directly impairs growth. Phi is not able to suppress the induction of lateral roots under P limiting conditions and the up-regulation of a set of phosphate starvation induced genes. Furthermore, phosphite induces specific changes in the abundance of several metabolites, especially amino acids, which may have relevance for the understanding of phosphite induced resistance.

Phytophthora cinnamomi is known to survive more than 50 years on impacted sites in the Eucalyptus marginata forest. One of the most severely impacted landscapes within this area are the ‘black gravel’ sites and persistence of the pathogen has made these areas extremely difficult to rehabilitate. Previous research has shown that P. cinnamomi is a poor competitive saprophyte so it was postulated that complete removal of the vegetation will kill the pathogen. Eradication experiments on black gravel sites investigated the length of time P. cinnamomi can survive in the soil without living plant tissue. Results encourage the view that the pathogen can be eliminated from infested sites as recoveries decreased significantly two years after removal of living plants. Annual and herbaceous perennials play an unexpectedly important role in the disease cycle and must be eliminated if eradication is to be successful.

Many species of the Phytophthora genus are pathogenic to a broad range of plants. Recent outbreaks of pathogenic Phytophthora sp. In the United States and European Union have been linked to internationally-traded nursery plants. A targeted survey was conducted at two wholesale plant nurseries in Western Australia over the course of one year, to identify Phytophthora sp. present in plant stock being sold to retail nurseries. One nursery was accredited with the Nursery and Garden Industry Australia (NGIA) whilst the other was not. Multiple species of Phytophthora were detected at the non-accredited nursery, but none at the accredited nursery. Species included P. cinnamomi and P. multivora. The results suggest that the best management practices suggested by the NGIA are important for maintaining the disease free status of plants sold through nurseries, and preventing the spread of plant pathogens.

Phosphite (H2PO3 ‐) is a phosphate analog widely used to protect plants from oomycete pathogens such as Phytophthora and Pythium. Phytophthora species are prominent pathogens in agriculture, e.g. Phytophthora infestans causing potato late blight. Phytophthora cinnamomi has devastating effects on native ecosystems with over 4000 plant species at risk in Western Australia alone. Phosphite is a well‐known protectant of plants and exhibits a complex mode of action. I It directly inhibits the pathogen’s growth by interference with its phosphate‐dependent metabolism. At the same time it also inhibits the plant’s phosphate starvation response, e.g. the up‐regulation of high‐affinity phosphate transporters, and thus has constrictive effects on plant growth under low phosphate supply. In addition to these direct effects, phosphite also induces the plant’s defence responses with increased expression of defence genes. However, the underlying mechanism of this indirect effect is not understood. We have started to characterise the impact of phosphite on plant defence responses by analyses of transgenic plants, metabolic pathways and the natural genetic variation in the plant Arabidopsis thaliana.