Bruce R. Grant

THE COMPLEX ACTION OF PHOSPHONATES AS ANTIFUNGAL AGENTS

C O N T E S T S

The Fungicide Phosphonate Disrupts the Phosphate-Starvation Response in Brassica nigra Seedlings

The development of Brassica nigra seedlings over 20 d of growth was disrupted by the fungicide phosphonate (Phi) in a manner inversely correlated with nutritional inorganic phosphate (Pi) levels. The growth of Pi-sufficient (1.25 mM Pi) seedlings was suppressed when 10, but not 5, mM Phi was added to the nutrient medium. In contrast, the fresh weights and root:shoot ratios of Pi-limited (0.15 mM) seedlings were significantly reduced at 1.5 mM Phi, and they progressively declined to about 40% of control values as medium Phi concentration was increased to 10 mM. Intracellular Pi levels generally decreased in Phi-treated seedlings, and Phi accumulated in leaves and roots to levels up to 6- and 16-fold that of Pi in Pi-sufficient and Pi-limited plants, respectively. Extractable activities of the Pi-starvation-inducible enzymes phosphoenolpyruvate phosphatase and inorganic pyrophosphate-dependent phosphofructokinase were unaltered in Pi-sufficient seedlings grown on 5 or 10 mM Phi. However, when Pi-limited seedlings were grown on 1.5 to 10 mM Phi (a) the induction of phosphoenolpyruvate phosphatase and inorganic pyrophosphate-dependent phosphofructokinase activities by Pi limitation was reduced by 40 to 90%, whereas (b) soluble protein concentrations and the activities of the ATP-dependent phosphofructokinase and pyruvate kinase were unaffacted. It is concluded that Phi specifically interrupts processes involved in regulation of the Pi-starvation response in B. nigra.

Disruption of the phosphate-starvation response of oilseed rape suspension cells by the fungicide phosphonate

Abstract. The influence of the anti-fungal agent phosphonate (Phi) on the response of oilseed rape (Brassica napus L. cv. Jet Neuf ) cell suspensions to inorganic phosphate (Pi) starvation was examined. Subculture of the cells for 7 d in the absence of Pi increased acid phosphatase (APase; EC 3.1.3.2) and pyrophosphate (PPi)-dependent phosphofructokinase (PFP; EC 2.7.1.90) activities by 4.5- and 2.8-fold, respectively, and led to a 19-fold increase in Vmax and a 14-fold decrease in Km (Pi) for Pi uptake. Addition of 2 mM Phi to the nutrient media caused dramatic reductions in the growth and Pi content of the Pi-starved, but not Pi-sufficient cells, and largely abolished the Pi-starvation-dependent induction of PFP, APase, and the high-affinity plasmalemma Pi translocator. Immunoblotting indicated the cells contain three APase isoforms that are synthesized de novo following Pi stress, and that Phi treatment represses this process. Phosphonate treatment of Pi-starved cells significantly altered the relative extent of in-vivo 32P-labelling of polypeptides having Mrs of 66, 55, 45 and 40 kDa. However, Phi had no effect on the total adenylate pool of Pi-starved cells which was about 32% lower than that of Pi-sufficient cells by day 7. Soluble protein levels, and activities of pyruvate kinase (EC 2.7.1.40) and ATP-dependent phosphofructokinase (EC 2.7.1.11) were unaffected by Pi starvation and/or Phi treatment. The effects of Phi on the growth, and APase and PFP activities of Pi-starved B. napus seedlings were similar to those observed in the suspension cells. The results are consistent with the hypothesis that a primary site of Phi action in higher plants is at the level of the signal transduction chain by which plants perceive and respond to Pi stress at the molecular level.

The Effect of Cations on Zoospores of the Fungus Phytophthora cinnamomi

Summary: Swimming zoospores of the fungus Phytophthora cinnamomi exposed to a range of ions were much more sensitive to cations than anions. One cation, Ca2+, induced zoospores to encyst and subsequently germinate, but most cations induced encystment only and were toxic at higher concentrations. In general, the sensitivity of zoospores to a cation increased with its charge density. At 0.3 μm, La3+reduced the viability of a zoospore population to 50%, while Fe3+(20 μm) and Mn2+(50 μm) induced encystment with only a slight decrease in viability. The other divalent and monovalent cations tested (Mg2+, Ca2+, Ba2+, Li+, Na+, K+, Cs+, NH4 +) were effective in inducing encystment or reducing viability at higher concentrations. Each ion showed a distinctive concentration–response curve. Only F−and CH3COO−among the anions tested (Cl−, NO3 −, F−, H2PO4 2-, SO4 2-, CH3COO−) had any effect on zoospores, and at 20 mM (pH 6.0) they reduced viability. The cysts (cystospores) of the fungus were generally less sensitive to cations than were swimming zoospores, and only Cs+and K+(50 mM) reduced viability to the same extent in each population. Both zoospores and cysts of this fungus had a broad tolerance to pH and temperature, but cysts were more resistant to low temperatures than were motile zoospores.

Role of RNA and protein synthesis in stimulated germination of zoospores of the pathogenic fungusPhytophthora palmivora

Abstract The role of RNA and protein synthesis in differentiating spores of the fungus Phytophthora palmivora was investigated using inhibitors, precursor assimilation, and in vitro translation of directly extracted RNA. Distinct differences in the capacity to take up small molecules, including exogenous precursors of protein and RNA synthesis, were detected between the different stages of differentiation. Zoospores and cysts of this fungus were impermeable to amino acids, glucose, and inorganic phosphate. Uptake of these metabolites was observed only after emergence of the germ tube. By contrast, inhibitors of protein and RNA synthesis penetrated these cells. In the presence of either cycloheximide or actinomycin D, the cells remained as cysts and did not germinate. Although encystment was not dependent on de novo transcription or translation, an increase in the pool of polyadenylated RNA was detected in the cells during this stage. This polyadenylated RNA showed a rapid and sustained increase in template activity, as measured by cell-free translation assays. The increase commenced very early in the cyst stage, immediately following pectin stimulation, and was attributed to the transcription of new mRNA. This transcription during encystment, which was also shown to be inhibited by actinomycin D, appears to be essential for germination in this species of fungus.

The mechanism of phosphonic (phosphorous) acid action in Phytophthora

Phosphonates exhibit a complex mode of action in their control of fungi. Using Phytophthora palmivora as a model, we have shown that phosphonate anion can act directly on the fungus, reducing growth, and that growth reduction is associated with a rapid reduction in the total pool of adenylate. At lower concentrations, phosphonate alters fungal metabolism without reduction in growth rate. Under these conditions there is a reduction in the amount of macromolecular materials which can be washed from the surface of the mycelia grown on an agar plate. We suggest that these materials contain suppressors, which normally act to delay the expression of the host defence systems. Therefore, a reduction in their synthesis induced by phosphonate could alter the host response and change the host reaction from compatible to incompatible. Superimposed on these effects is that of orthophosphate, which competes for the same carrier as phosphonate and determines the rate at which phosphonate is absorbed into the fungus.

The effects of sub-toxic levels of phosphonate on the metabolism and potential virulence factors of Phytophthora palmivora

Abstract Potassium phosphonate (phosphite) altered the metabolism of P. palmivora in liquid cultures even under conditions where growth of the fungus was not restricted. However, under these conditions, the composition of the lipid and cell wall material was altered, as were the standing concentrations of intermediary metabolites (e.g. amino acids). The compound dehydroabietic acid, a diterpenoid, was detected as part of a neutral/cationic macromolecule in young Phytophthora mycelium. In the presence of phosphonate, this compound was absent. These changes in fungal metabolism and in the composition of the cell wall complex suggest a mechanism by which phosphonate may reduce the virulence of the pathogen in the host plant and consequently expose it to the natural host defences at an earlier stage of the invasion processes. Under the experimental conditions used, the fungus was able to reduce intracellular levels of phosphonate to levels below detection during the exponential growth stage. As phosphonate concentrations within the fungus fell, the number of metabolic changes observed in the fungus were also reduced.

Calcium efflux associated with encystment of Phytophthora palmivora zoospores

Zoospores of the fungus Phytophthora palmivora, pre-labeled with 45Ca, excreted up to 30% of their total 45Ca when stimulated to encyst. Excretion was essentially completed within 90 sec of the application of the stimulus. Encystment of the population was completed within 5 min. Four different stimuli were used: pectin addition (420 micrograms ml-1), Sr2+ addition (5 mM), cyclic AMP addition (6.7 mM) and mechanical agitation. The kinetics and amount of Ca excretion were essentially the same in each case. The calcium ionophore A23187 increased the rate of 45Ca uptake by motile zoospores, incubated in 100 microM CaCl2, but did not induce encystment under these conditions. The ionophore did not induce 45Ca efflux from pre-labeled zoospores. Incubation in EGTA and in K+ failed to induce either encystment or 45Ca excretion. We conclude that rapid excretion of a significant proportion of the zoospore calcium is linked to the early stage of stimulus-induced encystment, and that this comes from an intracellularly located, non-cytoplasmic source, such as the peripheral vesicles, but that changes in cellular Ca2+ are not necessarily the single controlling factor in the induction of encystment.

31P NMR studies on the effect of phosphite on Phytophthora palmivora

31P NMR spectra were obtained from perchloric acid (PCA) and KOH extracts of Phytophthora palmivora mycelium. Signals indicating the presence of large amounts of short-chain polyphosphate were observed in the spectra of PCA extracts of mycelia grown under both low (0.1 mM) and high (10 mM) phosphate conditions. The mean chain length of polyphosphate was calculated from the relative areas of signals arising from terminal and internal P nuclei in the polyphosphate chain. The small amount of polyphosphate evident in the KOH extract had an average chain length similar to PCA-soluble polyphosphate. 32P tracer studies indicated that phosphorus in the PCA fraction accounted for between 50 and 60% of total phosphorus, the bulk of the remainder being divided between the lipid and KOH extracts. The presence of the fungicide phosphorous acid markedly reduced the average chain length of acid-soluble polyphosphate. This reduction occurred both under low-phosphate conditions, in which treatment with phosphorous acid retards growth, and under high-phosphate conditions, in which no significant growth retardation is observed. Treatment with phosphorous acid perturbed phosphorus distribution and lipid composition under low-phosphate conditions.

Calcium control of differentiation in Phytophthora palmivora

A method has been developed for the preparation of zoospores from Phytophthora palmivora which allows the ionic composition of the suspension medium to be closely controlled. Sub-micromolar concentrations of calcium ions have been shown to play a key role in maintaining the zoospore state and in the transition to the cyst stage. Restriction of free Ca2+ to between 0.2 and 1 μM resulted in zoospores which could be maintained for several hours before they finally encysted and germinated. When exposed to citrus-pectin, or 3 mM SrCl2, or to vigorous shaking, these zoospores underwent rapid synchronous encystment. At free Ca2+ concentrations below 0.1 μM, zoospores lysed slowly. If exposed to inducers of encystment before lysis had occurred, the zoospores failed to respond to pectin or to vigorous shaking. However, they did differentiate in response to SrCl2 addition. Provided the free Ca2+ was maintained between 0.02 and 0.2 μM, zoospores survived gentle centrifugation, a procedure which previously had resulted in encystment.