T. YAMAKAWA, Department of Plant Resources, Faculty of Agriculture, Plant Nutrition Laboratory, Kyushu University, Fukuoka 812-8581, Japan. Email: firstname.lastname@example.org
Phosphite (; Pi) uptake in cell suspension culture, information on how Phi affects the Pi uptake of intact plants remains to be determined. The present study was conducted to investigate the effect of Phi on Pi absorption of intact komatsuna plants (Brassica rapa var. peruviridis cv. Ajisai) in hydroponic culture. Phosphite markedly decreased Pi absorption of the intact komatsuna plants under both low (0.05 mmol L−1) and high (0.5 mmol L−1) Pi supply, although the growth (both shoots and roots) and water uptake of the high Pi-supplied plants was not affected by Phi. The inhibiting effect of Phi was small at 0.2 mmol L−1, but became large at 2 mmol L−1. Using relatively large seedlings (28 days old) to better assess the influence of Phi on Pi absorption early in the treatment, the results indicated that there was an immediate decrease in Pi absorption within the first 2-day period of Phi treatment when the water absorption of the plants was not affected. Taken together, the results suggested that there was a strong inhibiting effect of Phi on Pi uptake of intact komatsuna plants and this effect is exerted most likely by competition between Phi and Pi at uptake level. We speculate that the application of Phi to plant roots in an environment that is unfavorable for Phi-to-Pi conversion (e.g. hydroponic culture) may need to increase the amount of required Pi fertilization of plants to compensate for the reduction in Pi uptake by Phi. Further research is needed to confirm our results.
Phosphite (; Pi) is the active agent in various fungicides, and has been used widely in agriculture to control many important plant diseases caused by Oomycetes pathogens, particularly by Phytophthora spp. (Fenn and Coffey 1984; Forster et al. 1998; Grant et al. 1992; Guest and Grant 1991; Guest et al. 1995; Jackson et al. 2000; Jee et al. 2002). It has long been assumed that Phi has a selective effect on the Pi metabolism of Oomycete and that the host plants remain unaffected. However, a number of recent studies have demonstrated that Phi influences not only fungal metabolism, but also the growth and adaptation of plants to phosphorus (P) limitation (Abel et al. 2002; Carswell et al. 1996, 1997; Thao et al. 2008, 2009; Ticconi et al. 2001; Varadarajan et al. 2002). Several studies using cell suspension culture have indicated that Phi inhibits Pi uptake in Brassica cells (Carswell et al. 1997) and tobacco cells (Danova-Alt et al. 2008). However, information on how Phi affects the Pi uptake of intact plants remains to be determined. The effect of Phi on plant growth is strongly dependent on the Pi nutrient status of the plants and plants insufficiently fertilized with Pi are at high risk of the deleterious effects of Phi (Thao et al. 2009). As Phi products are intensively used in agriculture and Pi fertilization of plants needs to be managed to maintain good crops without causing environmental problems, information on how Phi affects the Pi uptake of intact plants merits further study to ensure good Pi management of crops and to minimize any undesired effects from the use of Phi.
The present study was conducted to investigate the effect of Phi on the uptake of Pi by intact plants grown under different Pi supplies. Komatsuna plants (Brassica rapa var. peruviridis cv. Ajisai) were used because this plant grows rapidly in hydroponic culture.
Materials and methods
Two uptake experiments (Experiment 1 and Experiment 2) were conducted in a 25°C-controlled room under natural light conditions at Kyushu University, Japan, to investigate the effect of Phi on Pi absorption by plants. Komatsuna seeds obtained from Tohoku Seed Company (Tohoku Seed Co. Ltd., Tochigi, Japan) were germinated and grown in vermiculite in the same room. The seedlings were watered daily and received half-strength Hoagland solution twice per week. To guarantee uniformity in the seedlings and to reduce the shock to the plants during transfer to the treatment solutions, after growing in vermiculite for 16 days, the seedlings were transplanted into one-eighth-strength Hoagland solution (pH 7.0) in 40-L hydroponic culture containers for 6 days of pre-treatment before being used in Experiment 1. For Experiment 2, the seedlings were grown for another 6 days of pre-treatment in the same container containing one-fourth-strength Hoagland solution (pH 7.0).
In both experiments, deionized water was used to prepare the treatment solutions.
Experiment 1 had a total of six treatments, a combination of two Pi concentrations (0.05 mmol L−1 [low level] and 0.5 mmol L−1 [high level]) and three Phi concentrations (0, 0.2 and 2 mmol L−1), arranged in a completely randomized design with three pots per treatment as three replicates. The Pi and Phi used were H3PO4 and H3PO3, respectively. Uniform seedlings, 22 days old, were selected, carefully washed, dried using absorbent papers and then transferred to 3-L pots (three plants per pot) containing 3 kg of one-fourth-strength Hoagland solution (pH 7.0), with P modified according to the treatments. The solutions were renewed every 2 days for a total of five times (the five 2-day absorption periods are referred to as day 1–2, day 3–4, day 5–6, day 7–8 and day 9–10 of the treatments) to measure the absorption of water, Pi and Phi in each period. The weights of the fresh (before absorption) and remaining (after absorption) solutions in the pots were carefully recorded for every 2-day absorption period. Any solution attached to the roots (a part of the remaining solution) was measured at each solution change by blotting the roots of each pot carefully in absorbent papers and then recording the increase in weight of the absorbent papers. The rate of water absorption was calculated based on the decrease in the solution at each period and this rate was measured for all five periods. The Pi and Phi absorption rates were calculated based on the initial amount and the remaining amount of each anion after each absorption period. There were three additional non-plant pots that were used in the same way to assess any changes in water, Pi and Phi without plants and these pots were used as blanks for calculation. As differences in nutrient absorption by the plants were difficult to detect during the early stages of the treatments, because the plants were small, the rates of Pi and Phi absorption were measured only during the last 2-day periods (i.e. day 5–6, day 7–8 and day 9–10 after treatment).
The plants were harvested after 10 days of treatment for dry weight (DW) and total P analysis.
To be able to better detect the effect of Phi on Pi absorption in plants in the early stages of the treatments, this experiment was conducted using middle-aged seedlings (28 days old) because these plants would require a more nutrients. The same 3-L pots with three komatsuna plants per pot were used. The experiment had a total of three treatments, that is, three different Phi levels (0, 0.2 and 2 mmol L−1), and Pi was supplied equally at a concentration of 0.1 mmol L−1. Uniform seedlings were selected, carefully washed and transferred into pots containing 3 kg of one-fourth-strength Hoagland solution (pH 7.0) with P was modified as the treatments. During the 6 days of the experiment, the solutions were renewed three times (renewed every two days) (the three absorption periods are referred to as day 1–2, day 3–4 and day 5–6 after treatment). The water, Pi and Phi absorption rates were determined as described for Experiment 1 for all periods.
Analysis of the samples
After harvesting, the plant samples from Experiment 1 were washed first in running tap water and then in deionized water. Dry weights of the shoots and roots of the plants were obtained after drying the samples for 3 days at 70°C.
Determination of total P in the plant tissue
Dried, ground plant samples from Experiment 1 were digested using the H2SO4–H2O2 Kjeldahl digestion method, which was designed for nitrate-containing samples (Ohyama et al. 1991), before measurement of P using the colorimetric procedure of Murphy and Riley (1962).
Determination of Pi and Phi in the nutrient solutions
Phosphate and Phi in the solution samples were simultaneously determined by ion chromatography as described by Thao et al. (2008).
Treatment effects were assessed by ANOVA using IRRISTAT for Windows version 4.0 (Biometric Unit, International Rice Research Institute, Laguna, Philippines). Mean separation was carried out using least significant difference (LSD) at P = 0.05 whenever a significant ANOVA (P < 0.05) result was found.
Phosphite did not affect the water uptake of the high-Pi-supplied plants, but it significantly decreased the water uptake of the low-Pi-supplied plants from day 5–6 after treatment (Fig. 1). In addition, after this time, the low-Pi-supplied plants wilted easily during the daytime (Fig. 2). At day 9–10 after treatment, the water uptake of the low-Pi-supplied plants was down by 38% in the 0.2 mmol L−1 Phi treatment and by 51% in the 2 mmol L−1 Phi treatment (Fig. 1).
Figure 3 shows that Pi absorption of komatsuna plants decreased under Phi treatment (P < 0.01) with both low and high Pi supply. Under high Pi supply, the decrease was significant only in the 2 mmol L−1 Phi treatment and was not affected by time of treatment. However, under low Pi supply, the decrease was significant at both Phi levels and became more profound with treatment time. For example, in the 2 mmol L−1 Phi treatment, Pi absorption of high-Pi-supplied plants was down by 27–33% and not affected by treatment duration, whereas for low-Pi-supplied plants, absorption was reduced by 43% at day 5–6 after treatment and by 79% at day 9–10 after treatment (Fig. 3).
Figure 4 shows that Phi was taken up by the plants under both low and high Pi supplies. The rate of Phi uptake in the high-Pi-supplied plants was significantly higher than that in the low-Pi-supplied plants.
Plant growth and P accumulation in plants
The results in Fig. 5 show that over 10 days of treatment, Phi did not affect root and shoot growth in the high-Pi-supplied komatsuna plants, but it decreased substantially both root and shoot growth in the low-Pi-supplied plants. Plants grown under a low Pi supply easily wilted under sunny conditions from day 6 of the Phi treatment (Fig. 2). The concentration of P in the plant tissues increased substantially under both low and high Pi supplies (Fig. 5c,d), confirming that plants absorbed Phi under both conditions.
The results detailed in Fig. 6 show that the addition of Phi at both levels had little effect on water uptake of the plants early in the treatment (from day 1 to day 4); the water uptake significantly decreased only at day 5–6 in the 2 mmol L−1 Phi treatment. However, the rate of Pi absorption was strongly decreased by Phi right from the start, that is, from day 1–2 after treatment (Fig. 7). This decrease was small at 0.2 mmol L−1 Phi, but large at 2 mmol L−1 Phi. Averaged over the three periods, the reduction in Pi absorption by 2 mmol L−1 Phi was approximately 26%.
There was a large reduction in Pi uptake in intact komatsuna plants by Phi from day 5–6 of the treatment under both low and high Pi supplies (Fig. 3). No effect of Phi on water uptake (Fig. 1) or plant growth (both shoots and roots) (Fig. 5a,b) was found in the high-Pi-treated plants throughout the experiment. The results suggested that there was an inhibiting effect of Phi on Pi absorption in intact komatsuna plants, regardless of whether or not plant growth (shoots and roots) was affected by Phi. Results from Experiment 2 (Fig. 6) indicate that there was an immediate decrease in Pi absorption in plants right from the first 2-day period of Phi treatment when the water uptake of the plants was not affected. Taken together, the results confirm the inhibiting effect of Phi on Pi absorption in intact plants at an uptake level. Our results support previous research using cell suspension culture that indicated that Phi inhibits the Pi influx of tobacco cells in a competitive manner (Danova-Alt et al. 2008) and that Phi inhibits the Pi uptake in Brassica cells, altering both Km (the substrate concentration that gives half Vmax) and Vmax (the maximum enzyme velocity) (Carswell et al. 1997). The effects of Phi on Pi absorption and transport have been more extensively investigated in fungi. Research conducted on different Phytophthora sp. provides evidence that Pi and Phi anions share common transport systems and that they are competitive inhibitors for each other’s transport (Barchietto et al. 1989; Griffith et al. 1989). Examination of the Phi effect on two yeast pho mutants revealed that Phi potentially targets PHO84, a plasmalemma high-affinity Pi transporter (McDonald et al. 2001b). Based on the competitive effect of Phi on Pi uptake in tobacco cells and the intracellular accumulation of Phi, it is assumed that Phi enters the plant cells via a high-affinity Pi transport system (Danova-Alt et al. 2008).
A number of studies have clearly shown severe interference of Phi with plant adaptations to Pi limitation; these studies have also shown that this interference suppresses the typical molecular and developmental responses of plants to P deficiency (Abel et al. 2002; Carswell et al. 1996, 1997; Ticconi et al. 2001; Varadarajan et al. 2002). Phosphite intensifies the effects of P deficiency by tricking Pi-deprived plant cells into sensing that they are Pi sufficient when in fact their cellular Pi content is extremely low (McDonald et al. 2001a). A deleterious effect of Phi was not evident in Pi-sufficient plants, but plants fertilized with sufficient Pi for approximately 80–90% of their maximum growth may still be at risk of the effect (Thao et al. 2008, 2009). The results of the present study further indicate that the harmful effect of Phi on the plants can also be attributed to its direct inhibition of Pi absorption by intact plants. It is possible to speculate that the application of Phi to plant roots in an environment that is unfavorable for Phi-to-Pi oxidation (e.g. hydroponic culture) may need to increase the amount of required Pi fertilization of plants to compensate for the reduction in Pi uptake by Phi under these conditions and therefore can avoid the undesirable effects of Phi on plants. Although Phi can be converted to Pi in soils by microorganisms, this process is very slow, the approximate half-life for Phi oxidation to Pi in soil is approximately 12–16 weeks (Adams and Conrad 1953). In addition, although some microbes are capable of oxidizing Phi, they preferentially use Pi over Phi as a source of P. The inoculation test of soil bacteria in a mixed culture of Phi and Pi conducted by Adams and Conrad (1953) revealed that the Phi was not used by the soil bacteria until all Pi in the culture had been depleted and thus all traces of Pi would have been scavenged by the microbes before Phi oxidation occurred. Therefore, an inhibiting effect of Phi on Pi uptake of plants may need to be considered, particularly in the early time of cropping when Phi-to-Pi conversion is negligible. Further research is required to better understand this process.
The reduction in Pi uptake in the low-Pi-supplied plants became more severe with time in the Phi treatment, whereas for the high-Pi-supplied plants the reduction remained unchanged (Fig. 3). Water uptake of the low-Pi-supplied plants was reduced from day 5–6 after Phi treatment (Fig. 1), together with the appearance of root damage (Fig. 2b) and the plants easily wilted under sunny conditions (Fig. 2a). Root and shoot growth of these plants was significantly reduced after 10 days of Phi treatment (Fig. 5b). The results imply that the root growth and root function of low-Pi-supplied plants could be inhibited by Phi as early as 5 or 6 days after treatment. This effect contributed to the further reduction in Pi uptake with time in the Phi treatment.
It was expected that the high-Pi-supplied plants would uptake less Phi than the low-Pi-supplied plants because of competition between both anions at the uptake level. However, the results in Fig. 4 did not show this; that is, Phi uptake in the high-Pi-supplied plants was significantly higher than that in the low-Pi-supplied plants. This result may be explained by the inhibiting effect of Phi on root growth and root function (Figs 2, 5b) in the low-Pi-supplied plants, leading to a decrease in nutrient uptake, including Phi.
The present study demonstrated the strong inhibiting effect of Phi on Pi absorption in intact komatsuna plants under both low and high Pi supply, regardless of whether or not plant growth was affected by Phi, and this effect is likely to have resulted from competition between both anions at the uptake level. Under low Pi supply, prolonged Phi treatment further decreased the Pi absorption rate as a result of the inhibiting effect of Phi on root growth and hence root function under this condition. We speculate that the amount of required Pi fertilization in plants may need to be increased if Phi products are applied to the roots under conditions that are unfavorable for Phi-to-Pi oxidation (e.g. hydroponic culture). Further research is needed to confirm our results.