Jen McComb

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.

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.

In this study we evaluated the role of plant defence pathways in resistance to Phytophthora cinnamomi by testing the susceptibility of mutants of Arabidopsis thaliana impaired in various defence pathways to P. cinnamomi infection. Susceptibility to infection was assessed by measuring the number of callose papillae, production of hydrogen peroxide, and measurements of pathogen biomass using quantitative PCR. Mutants impaired in the salicylic acid, jasmonic acid, ethylene, and phytoalexin camalexin pathways did not show increased susceptibility to P. cinnamomi compared to their wild type background Col‐0. However, the aba2 mutant deficient in abscisic acid (ABA) signalling displayed a very much higher level of susceptibility than the wild type parent, Col‐0. The results show that resistance of A. thaliana to P. cinnamomi is mediated by the ABA signalling pathway.

A common approach for measuring the susceptibility of a plant to infection by a pathogen is to measure the pathogen biomass in planta using quantitative PCR. However, the processes such as the production of phenolics, or degradation of cellular DNA and cell collapse associated with necrosis process in plant tissue can lead to variation in DNA extraction efficiency, inhibition of PCR, or overestimation of pathogen biomass thereby affecting accurate measurement of pathogen biomass. Our approach to this is to add a control DNA (plasmid) to the plant tissue before the DNA extraction. By measuring the amount of plasmid DNA recovered in the DNA extract, and by normalising the amount of pathogen DNA to plasmid DNA we can allow for differences in DNA extraction efficiency and avoid overestimation of pathogen biomass. In both lupin–Phytophthora cinnamomi and Arabidopsis–Phytophthora cinnamomi pathosystems, cell collapse and tissue necrosis were observed and led to overestimation of pathogen biomass when normalised to host plant DNA. In lupin we observed up to 17 fold overestimation of pathogen biomass compared to three fold overestimation found in infected A. thaliana 72 h after inoculation with P. cinnamomi. Our results suggested that by increasing the degree of necrosis due to necrotrophic infection, the level of overestimation of pathogen biomass increases if normalised based on host DNA. We demonstrated the capability and robustness of this developed technique in two different plant‐pathogen interactions with various levels of resistance. This method can also be adapted in quantification of pathogen biomass in other pathosystems.

Many species of Phytophthora are significant pathogens of plants of agricultural, forestry and native plant systems throughout the world. Of these, Phytophthora cinnamomi is of particular significance due to its broad host range and worldwide distribution. CPSM’s successful bid to have the P. cinnamomi genome sequenced through the Joint Genome Institute’s Community Sequencing Program gives many Phytophthora researchers the opportunity to collaborate and unlock some of the central conundrums of this pathogen including, but not limited to its extraordinarily wide host range, response to control measures and diagnostics. The proposed approach and expected outcomes, along with opportunities for collaboration will be presented and discussed.

Over the years several hypotheses have been put forward to account for the “fungistatic” action of phosphite against Phytophthora, but biochemical detail is scarce. The complexity of the interaction between host and pathogen is such that no single aspect of phosphite chemistry is able to explain all the various effects of phosphite that have been observed. For instance, why does the temporal efficacy of phosphite vary up to ten-fold between horticultural and native plants? And how can this observation be reconciled with the fact that pathogen growth in vitro is not inhibited by the concentration of phosphite that gives disease control in planta? This talk will outline a biochemical model that explains how and why phosphite has the effects on Phytophthora that it has. However, when considering models of phosphite action it should be noted that “Essentially, all models are wrong, but some are useful” (George Box), and within this context “usefulness” can be defined as increased disease control and better management of Phytophthora dieback. The model suggested that the efficacy of phosphite in controlling disease caused by Phytophthora may be enhanced by maintaining high levels of extracellular calcium. Results from a glasshouse trial confirmed this, and showed that although phosphite and calcium are not lethal to the pathogen, combined treatments of soil calcium supplementation and foliar phosphite application were synergistic in controlling infection caused by Phytophthora cinnamomi in Banksia leptophilia. Rigorous biochemical testing is needed to confirm that inhibition of the calcium-dependent ATPase by phosphite does in fact cause the synergistic effect. However, in the absence of evidence to the contrary, the suggested model of phosphite action has proved useful.