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Influence of nutrient solution concentration and a root pathogen (Pythium aphanidermatum) on tomato root growth and morphology
Scientia Horticulturae 97 (2003) 109–120
Influence of nutrient solution concentration and a root pathogen (Pythium aphanidermatum) on tomato root growth and morphology Dietmar Schwarz*, Rita Grosch Institute for Vegetable and Ornamental Crops, Theodor-Echtermeyer-Weg 1, D-14979 Grossbeeren, Germany Accepted 17 July 2002
Abstract Young roots, especially those in the root hair zone, are most important for nutrient uptake. However, these roots are also sensitive to high nutrient solution concentration and root pathogens, major problems in soilless culture systems. Supply of nutrient solution at a constant electrical conductivity (EC) can increase the EC in the root environment up to 10 dS m1. Pythiaceae are among the most important root pathogens in soilless systems. Therefore, investigations were carried out to quantify and compare root damage by high nutrient solution EC and by Pythium aphanidermatum (Edson) Fitzp. Three tomato plants at two-leaf-stage (Lycopersicon esculentum [Mill] L. cv. Counter) were transferred to a 2 l container filled with aerated nutrient solution. They grew in climate chambers for 14 days (16 h light 600 mmol m2 s1, 30/25 8C day/night). In one experiment, a basic nutrient solution of 1.5 dS m1 was supplemented with either NaCl or macronutrients to give an EC of 1.5, 5 or 9 dS m1. In another experiment, plants were inoculated with P. aphanidermatum (0, 102, or 104 oospores ml1 solution). All treatments were replicated five and four times, respectively. On days 3, 7, 10, and 13 after treatment one plant was sampled from each container. Fresh and dry mass of shoots and roots, total root length, number of adventitious roots and of all tap root laterals decreased with increasing nutrient solution EC. Dry matter content of roots and tap root diameter were not influenced while shoot dry matter content increased with increasing EC. NaCl enhanced root fresh matter, tap root diameter, total root length and number of adventitious roots compared with the macronutrient treatment. On the contrary shoot dry matter content and number of all laterals were reduced by NaCl. Three days after inoculation with P. aphanidermatum the number and length of roots were reduced significantly by the treatment 104 oospores ml1. P. aphanidermatum effects were greatest on young and thin roots, particularly those of the second and third laterals. Reduction increased with time and * Corresponding author. Tel.: þ49-33701-78206; fax: þ49-33701-55391. E-mail address: [email protected] (D. Schwarz).
0304-4238/02/$ – see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 2 3 8 ( 0 2 ) 0 0 1 4 3 - 7
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increasing number of inoculated oospores. Root diameters were greatest in the treatment with 104 oospores ml1. Results confirm that attempts to extrapolate results obtained on average root systems to single root formation could be misleading. Furthermore, a tomato plant can compensate to some extent its water uptake of a root system reduced by nutrition or pathogen effects. # 2002 Elsevier Science B.V. All rights reserved. Keywords: EC; Growth rate; Lateral roots; Nutrient solution; Osmotic stress; Pythium aphanidermatum; Root length; Root diameter; Tap root; Water uptake
1. Introduction Young roots, particularly those in the root hair zone, are most important for nutrient uptake. However, these roots are also most sensitive to high nutrient solution concentration and root pathogens, the main problems in soilless culture systems nowadays. Supply of nutrient solution at a constant electrical conductivity (EC) as common in horticultural practice can increase the EC in the root environment up to 10 dS m1 (Van Noordwijk and Raats, 1980). High salt concentrations result in low water potentials and thus reduce root elongation for many crops (Kafkafi, 1996). For radish, root elongation was reduced by 4% per 0.1 MPa (Hassan and Overstreet, 1952) while maize root length was reduced by 54% at about 100 mmol l1 NaCl (0.4 MPa) compared to the control without NaCl (Dalton et al., 1997). Furthermore, root biomass could be negatively affected by cell growth restriction. For tomato at EC above 4–6 dS m1 plants have a significantly reduced water uptake (Cuartero and Fernandez-Munoz, 1999). The decrease in water uptake is strongly and linearly correlated to EC (Dalton et al., 1997). Decreased water uptake decreases dry matter allocation to shoots more than to roots (Cruz and Cuartero, 1990). As a consequence transpiration coefficient and shoot/root ratio decrease with increasing EC. In nutrient solution systems an enrichment of ions, such as sodium chloride and sulphates, is typical. An enrichment imposes additional stress to that of the osmotic stress, which might lead to ionic imbalances or reduction in the uptake of some ions and thus to further growth depressions or even cell death (Kafkafi, 1996; Cuartero and Fernandez-Munoz, 1999). Cramer et al. (1995) reported that supplying more than 70 mmol l1 NaCl inhibited nitrate uptake and reduced photosynthesis. On the other hand supply of Ca2þ might compensate for the negative effects of Naþ (Yermiyahu et al., 1997). Pythiaceae are among the most important root pathogens in soilless systems causing root rot in tomato and cucumber (Jenkins and Averre, 1983). Pythium aphanidermatum (Edson) Fitzp. is an oomycete with high pathogenic potential under soilless culture conditions, that has a broad host range (Mitchell and Deacon, 1986). The fungus reproduces asexually and is dispersed by zoospores, which respond to chemo attractants, such as amino acids, produced by the host. The zoospores swim to and accumulate on the root surface, where they encyst. Zoospore cysts germinate rapidly and the germ tubes from the cysts penetrate root tissue. Root infection by Pythium spp. may reduce yield of plants through decays of root tissue. On the other hand, yield losses can also occur in the absence of any obvious symptoms of root necrosis (Favrin et al., 1988). Reasons for such asymptomatic yield loss
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might be slight infections, a plant physiology that influences the behaviour of the fungi, or the activity of specific Pythium spp., such as Pythium group F (Moulin et al., 1994). With or without root symptoms, Pythium spp. reduces water uptake and may cause leaves or shoots to wilt during warm temperatures (Couteaudier and Lemanceau, 1989), effects that are similar to those of osmotic stress. Relatively little research has studied the nuances in characteristics that exist among the various constituents of the root system (Waisel and Eshel, 1992). Metabolically active roots of older tomato are mostly laterals growing from several branching levels. Nutritional and hormonal conditions affect pericycle cells presumably activating specific root forming genes (Barlow and Adams, 1988). Different gene expressions determine the uptake capability of tap and lateral roots. Waisel and Eshel (1992) reported that the metabolic nutrient uptake system in tap roots dominates that of the non-metabolic uptake system. Depending on cultivar, under salinity treatments tomato developed numerous small lateral feeder roots (Zobel, 1975). Pythium spp. effects on root formation are not described in the literature. Similar influences of osmotic stress and P. aphanidermatum inoculation on water uptake and plant growth reduction led us to the question whether their effects on tomato roots are comparable. Therefore, experiments were carried out to quantify and compare their effects on (i) total root system morphology, (ii) root formation, and (iii) productivity of the root system with respect to water uptake and dry matter production.
2. Materials and methods Tomato seeds were (Lycopersicon esculentum [Mill] L. cv. Counter) germinated in coarse sand and kept moist until the seedlings reached the two-leaf-stage. Then three tomatoes were transferred to a 2 l container filled with aerated nutrient solution and covered with a plastic foil to prevent algae growth and evaporation. Plants were secured in a foam disc (10 mm thick) flooding on the surface of the solution. They grew in climate chambers for 14 days at relative humidity of 70/90% (day/night) and photosynthetic active radiation of 600 mmol m2 s1 for 14 h per day. The stem above the tap root was wrapped up to a height of 15 mm with flexible foam to prevent growth of adventitious roots. To determine the effect of nutrient solution composition and concentration a first experiment was carried out at day/night temperature of 25/20 8C (13–26 June 1997). The basic nutrient solution of 1.5 dS m1 (T1.5, control) contained in mmol l1: NH4 1, NO3 10.8, K 6.5, Ca 3.8, SO4 1.5, P 1.2, and in mM l1: Fe 15; Mn 10; B 20; Mo 0.5 (De Kreij et al., 1997). To give an EC of 5 and 9 dS m1 the basic solution was supplemented either with 38 mmol l1 NaCl or 81 mmol l1 NaCl (N5, N9) or with macronutrients (T5, T9). The pH was adjusted daily to 5.6. Every treatment included five replications with 75 total plants in a randomised design. To determine the effect of P. aphanidermatum (Edson) Fitzp. a second experiment was conducted at day/night temperature of 30/25 8C using a basic nutrient solution at EC 2.5 dS m1 and pH 5.6 (11–24 July 1997). In the first experiment treatments started with seedlings transfer to the containers, while in the second experiment treatments started 3 days after seedling transfer. Plants were inoculated with 0, 102, or 104 oospores ml1
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solution (O0, O2, O4). Every treatment included four replications with 36 total plants in a randomised design. On days 4, 7, and 13 after treatment (DAT) during the first experiment, and on days 3, 7 and 10 during the second experiment, one plant was removed from the container and analysed. Total water uptake during the preceding period (Wup, ml per plant), total fresh and dry mass of shoots (Dsp, g per plant) and roots (Drp, g per plant) were measured. To determine the treatment effect on the whole root system total root number (Nrp, no. per plant) was counted and total length (Lrp, m per plant) was estimated using the line intersect method of Tennant (1975). Tap root and adventitious roots were excluded but measured separately with a ruler. Root diameters were measured on 10 roots randomly sampled from the second and third laterals (every 10th line root intersection until 10 readings were obtained) and mean root diameter was calculated (2Rr, mm). From these measurements we calculated specific root 1 2 3 length (Lrw ¼ Lrp D1 rp , m g ), root surface area (Arp ¼ Lrp pRr 2 10 , m per plant) 1 1 and root frequency number per length (Nrl ¼ Nrp Lrp , no. m ). First, second, and third laterals of the tap root and any adventitious roots that grew under the foam were counted to test treatment influences on root formation and initiation. Relative root reduction was calculated for first to third laterals as ratio to the respective control. Tap root diameter was measured 10 mm away from shoot and 10 mm before where the root ended. From these data, we calculated total plant growth rate (GRR, g m2 per day) and water uptake rate (WUR, ml m2 per day) both related to root surface area: ðDsp i þ Drp i Þ ðDsp i1 þ Drp i1 Þ ; 0:5ðti ti1 ÞðArp i þ Arp i1 Þ Wup i WUR ¼ 0:5ðti ti1 ÞðArp i þ Arp i1 Þ
GRR ¼
where t is DAT, and i the index for DAT. Data were subjected to analysis of variance procedures with treatment EC and inoculum density as factors. Means at the different measurement dates were separated by Tukey’s test procedure at P ¼ 0:05. Significant differences are presented by different letters in the table but omitted in the figures for clarity. Instead the figures show only standard deviation bars. For statistical procedures Statistica for Windows (1998) software was used.
3. Results 3.1. Plant growth analysis At 4 DAT dry matter content but not dry mass of shoots and roots was already enhanced by treatments T5 and T9 (data not shown). Three days later, treatments diminished shoot growth significantly and further 6 days root mass was also reduced significantly (Fig. 1A and B). Effects of P. aphanidermatum were significant at 3 DAT for root fresh and dry mass and from 7 DAT afterwards for shoot and root fresh and dry masses (Fig. 1A and B). O2 resulted in a 71% reduction of shoot growth and was not much stronger compared to the influence of N9 with 64%. Root decrease was smaller than shoot decrease with 63% but significantly stronger compared to a 40% reduction reached by N9. Dry matter content of roots was
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Fig. 1. Shoot (A) and root (B) dry mass of tomato grown in 2 l containers. Left: treated with nutrient solution concentration of EC 1.5 (T1.5), 5, and 9 dS m1 made by either increasing the macronutrient concentration (T5, T9) or supply of NaCl (N5, N9). Right: treated with 0 (O0), 102 (O2) and 104 (O4) oospores ml1 of P. aphanidermatum.
reduced significantly at O4 (Table 2). Shoot dry matter content increased with increasing EC but not for NaCl treatments. 3.2. Morphology of the root system Total root number, length (Fig. 2), and surface area of the whole root system increased with time for all treatments (data not shown). Only O4 showed no further increase or even a decrease on the last measurement in most of these characteristics compared to results 7
Fig. 2. Total root length of tomato grown in 2 l containers. Left: treated with nutrient solution concentration of EC 1.5 (T1.5), 5, and 9 dS m1 made by either increasing the macronutrient concentration (T5, T9) or supply of NaCl (N5, N9). Right: treated with 0 (O0), 102 (O2) and 104 (O4) oospores ml1 of P. aphanidermatum.
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Table 1 Root characteristics of tomato for treatments EC 1.5 (T1.5), 5, or 9 dS m1 made by supply of either macronutrients (T5, T9) or NaCl (N5, N9)a Characteristic 1
Dry matter content, root (g kg ) Dry matter content, shoot (g kg1) Root surface area (m2 per plant) Tap root length (mm) Diameter, beginning of tap root (mm) Diameter, end of tap root (mm) Diameter, root branches (mm) Root number, branch 1 (no. per plant) Root number, branch 2 (no. per plant) Root number, branch 3 (no. per plant) Adventitious roots (no. per plant) a
T1.5
T5
T9
N5
N9
30.2 a 71.8 c 0.0723 a 44.6 a 5026 a 1077 b 279 a 36.4 a 1906 a 1032 a 20.6 a
31.1 a 77.9 b 0.0594 b 33.0 ab 4318 a 1313 b 309 a 35.8 a 1659 a 797 a 14.4 b
30.2 a 86.4 a 0.0482 b 36.8 ab 4041 a 1221 b 307 a 36.6 a 1285 b 659 a 13.4 b
28.7 a 62.7 d 0.0742 a 34.0 ab 4656 a 1856 a 278 a 32.8 ab 845 bc 718 a 18.0 a
26.2 a 61.7 d 0.0561 b 30.6 b 4359 a 1436 ab 270 a 26.8 b 531 c 309 b 8.4 c
Different letters depict significant differences at P < 0:05.
DAT. As depicted in Fig. 2 for root length all of these characteristics were affected by EC and inoculum density. Reduction of root length and surface area was significant for the EC treatments 13 DAT but for P. aphanidermatum already 3 DAT (Tables 1 and 2; Fig. 2). Total root number was reduced 4 DAT for T9 where 89 and N9 where 82 roots were counted compared to 147 roots at T1.5. The root length per dry mass changed with treatments for both experiments already at the first measurement date (Fig. 3A). While NaCl treatments resulted in an increased specific root length it was reduced with increasing inoculum density of P. aphanidermatum. The number of roots per length was affected in an almost opposite way (Fig. 3B). It decreased for the NaCl treatments but increased for the treatments with oospores of P. aphaniderTable 2 Root characteristics of tomato inoculated with 0 (O0), 102 (O2), or 104 (O4) oospores ml1 of P. aphanidermatuma Characteristic
O0 1
Dry matter content, root (g kg ) Dry matter content, shoot (g kg1) Root surface area (m2 per plant) Tap root length (mm) Diameter, beginning of tap root (mm) Diameter, end of tap root (mm) Diameter, root branches (mm) Root number, branch 1 (no. per plant) Root number, branch 2 (no. per plant) Root number, branch 3 (no. per plant) Adventitious roots (no. per plant) a
33.0 a
O2 b
22.7 bb
c
c
c
0.0721 a 39.0 b 5115 a 987 a 264 b 37.5 b 2680 a 1322 a
0.0218 b 33.8 c 3949 b 897 a 265 b 35.2 b 869 b 774 b
0.0069 c 53.5 a 3744 b 308 b 378 a 70.2 a 178 c 29.8 c
c
c
c
Different letters depict significant differences at P < 0:05. Determined 7 days after the treatment was started. c No measurements. b
32.2 a
O4 b
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Fig. 3. Specific root length per dry mass (Lrw) (A) and number of roots per length (B) for tomato. Left: treated with nutrient solution concentration of EC 1.5 (T1.5), 5, and 9 dS m1 made by either increasing the macronutrient concentration (T5, T9) or supply of NaCl (N5, N9). Right: treated with 0 (O0), 102 (O2) and 104 (O4) oospores ml1 of P. aphanidermatum.
matum. However, the lower inoculum density O2 increased the root numbers per length more than the highest density O4 (data are not shown). Mean root diameter of all laterals varied between 264 and 309 mm for all treatments, except O4. Here, it increased significantly to 378 mm (Table 2). 3.3. Effects on root formation Adventitious roots were counted only during the EC experiment (Table 1). The number increased linearly until 7 DAT and was then diminished with increasing EC, most at N9 with 59% (data not shown). Three levels of branches from the main root were determined. Compared with the control more roots grew 10 DAT with P. aphanidermatum within the first laterals, at O4 almost 100%, and within the second laterals 7 DAT at N5 60% (Fig. 4). Number within the second and third laterals was reduced 13 DAT (Fig. 4). The strongest reduction by EC treatments occurred within the second laterals at N9, while the strongest reduction by P. aphanidermatum treatments occurred within the second and third laterals at O4. Compared with the second laterals number of third laterals was less diminished. Tap root length amounted to 68 mm as a maximum at T9 4 DAT and 30 mm as a minimum at O0 7 DAT (data not shown). It was influenced by treatments. While N9 and O2 reduced it O4 increased the length compared to the controls (Table 1). The diameter at the beginning of the tap root was reduced by P. aphanidermatum treatments 7 and 10 DAT
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Fig. 4. Relative root reduction (dimensionless) of first to third laterals of tomato tap roots related to the respective control. Upper figures: treated with nutrient solution concentration of EC 1.5 (T1.5), 5, and 9 dS m1 made by either increasing the macronutrient concentration (T5, T9) or supply of NaCl (N5, N9). Lower figures: treated with 0 (O0), 102 (O2) and 104 (O4) oospores ml1 of P. aphanidermatum.
Fig. 5. Growth rate of total tomato plant dry mass (GRR) (A) and water uptake rate (WUR) (B) per root surface area. Left: treated with nutrient solution concentration of EC 1.5 (T1.5), 5, and 9 dS m1 made by either increasing the macronutrient concentration (T5, T9) or supply of NaCl (N5, N9). Right: treated with 0 (O0), 102 (O2) and 104 (O4) oospores ml1 of P. aphanidermatum.
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(Table 2). It was reduced as well close to the end of the tap root by O4 to 79% but increased by N5 at 72%. 3.4. Growth and water uptake related to root growth Total plant growth rate and water uptake were related to 1 m1 m root surface area to characterise treatment influence on root productivity. GRR decreased with time independent of the treatments (Fig. 5A). The same root surface was connected to a lower growth rate for NaCl treatments compared with balanced macronutrient treatments, 7 and 13 DAT. Three DAT at O4 GRR increased immediately but afterwards it decreased more severely than the control and O2. It was negative on 10 DAT (Fig. 5B). Here, the mass produced was smaller than that destroyed. WUR decreased with time as well but it was not affected by EC treatments. On the other hand, it increased with increasing inoculum density caused by a larger reduction in root than in shoot growth. Roots were able to double WUR at O4 compared to the control. Thus they compensated the smaller surface by an increased water uptake rate.
4. Discussion The reduction of plant growth characteristics in general and of root growth in particular with increasing nutrient solution EC and with P. aphanidermatum inoculation agree with the results of several authors (e.g. Kafkafi, 1996; Wulff et al., 1998). The EC effect is mainly an osmotic stress and was significant already at EC 5 dS m1 which is the threshold value of 4–6 dS m1 mentioned by Cuartero and Fernandez-Munoz (1999). The more severe reduction of shoot mass by the NaCl treatments compared with balanced macronutrient treatments indicates an additional ionic effect (Kafkafi, 1996). The opposite effect by NaCl treatments was observed for total root length (Fig. 2). Several reasons are possible for the reduced root length: cell growth restriction because of low water potential of external medium, interference of the ions or the toxicity of accumulated ions (Cuartero and Fernandez-Munoz, 1999). Root length reduction was related to a larger specific root length for the NaCl treatments compared to the macronutrient treatments (Fig. 3). Normally, increased specific root length is accompanied by decreased average root diameters (Schwarz et al., 1995). Mean root diameters of NaCl treatments were >30 mm smaller than those of the macronutrient treatments (Table 1). Changes in the root structure (Pessarakli and Tucker, 1988) or less available assimilates for the roots, e.g. by reduced photosynthesis (Cramer et al., 1995), could have caused the reduction. A lower dry matter (Table 1) confirmed changes in root structure. It is possible that in the NaCl treatments alleviation of osmotic stress might have increased the production of osmotically active substances, such as proline (Sudhakar et al., 1993) or myo-inositol (Sacher and Staples, 1985). P. aphanidermatum reduced root length before it affected shoot and root mass. This was also observed in previous investigations at low inoculum densities of 10 oospores ml1 without measurable effects on plant growth (Grosch and Schwarz, 1998). The early reduction of root length is connected to a strong reduction in specific root length (Fig. 3A).
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A larger diameter measured for O4 can partly explain the decrease in specific root length. It is possible that the pathogen started cell destruction and dissolution after penetrating the root exodermis (Wulff et al., 1998). A significantly lower dry matter content for O4 as well as a reduction of the number of the younger and thinner roots indicate this process (Table 2). Although EC and P. aphanidermatum immediately affected roots, shoots were more affected after a few days (Figs. 1–3 and 5A) as has been reported by others (Cruz and Cuartero, 1990; Dalton and Poss, 1990; Plant et al., 1991). The authors explained these findings by changes in dry matter allocation in favour of the roots caused by lowered root indole-3-acetic acid (IAA) concentration or root to shoot signalling by reduced water uptake and thus increase in abscisic acid (ABA) in the xylem. Treatments affected the levels of laterals differently (Fig. 4). Gulmon and Turner (1978) showed that nutrient deficiency resulted in fewer tomato roots and reduced length of the second and third branches independent of the cultivars. Barlow and Adams (1988) cultured tomato root axes and lateral roots for 8 days in media containing three different concentrations (0.5, 1.5, 2%) of sucrose. The medium sucrose concentration resulted in a significantly larger number of laterals and increased total length. Both, nutrient deficiency as well as nutrient surplus seem to decrease root initiation. The reduced number of second and third laterals (Fig. 4) could be explained by an overall reduced root length. Therefore, the root frequency per length was only diminished for the NaCl treatments 13 DAT (N5, N9) but not for increasing macro nutrient concentration (Fig. 3B). In contrast, P. aphanidermatum inoculation increased the root frequency per length. A similar development has been described by Zobel (1975) typically for salinity effects inducing the development of numerous small lateral feeder roots. The conflicting findings might be due to different growing media and cultivars used. The roots in experiments presented grew directly in solution and not in soil as in the experiments by Zobel (1975). The same author also mentioned that some cultivars investigated did not show a strong development of these feeder roots. That roots appear later with salt (Abrisqueta et al. (1991) in Cuartero and Fernandez-Munoz, 1999) was confirmed particularly for the third laterals. Four DAT no roots were found at N9 and also the number for the other treatments were much lower compared to the control at EC 1.5 dS m1. Muday and Haworth (1994) showed that IAA transport decreased or prevented formation of lateral roots. This was not found for first laterals except N9 in tomato experiments. IAA concentrations sufficient to inhibit growth of second and third laterals may have stimulated growth of first laterals as it was the case for treatment O4. Here, also stimulation could have occurred by IAA production by the pathogen itself. P. aphanidermatum is able to influence root growth negatively by producing IAA (Wulff et al., 1998). A lack of production may result in low IAA concentrations and thus in a positive or at least not root growth reducing effect (Schroth et al., 1984). It is also possible that different effects on root formation are caused by specific root forming genes presumably activated in the pericycle cells (Barlow and Adams, 1988) or by the activity of other hormones, such as ethylene, cytokinins, and gibberellins (Cruz and Cuartero, 1990). All of these hormones affect root initiation and branching and are affected by the nutritional status of the plant (Barlow and Adams, 1988; Waisel and Eshel, 1992) or by microorganism and pathogens (Donaldson and Deacon, 1993). In the experiment presented the pathogen colonisation of the different laterals were not quantified. To explain plant and
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particular single root and pathogen interactions it is necessary to investigate population dynamics of the pathogen, e.g. by serological methods. From our results it can be assumed that pathogen density was higher on second and third laterals than on the first laterals. In addition to the hormonal effect a direct damage in form of cell destruction and dissolution might be responsible although in the O2 treatment no symptoms were visible. It can be summarised that general treatment effects of EC and Pythium resulted in similar patterns but they had different effects on root formation. Therefore, attempts to extrapolate results obtained on average root systems and their characteristics to single root formation could be misleading (Waisel and Eshel, 1992). The reduction of the root surface area was compensated by increased water uptake rates per unit root particularly after treatment start while the effect disappeared later (Fig. 5A and B). The increased water uptake of roots confirms the functional equilibrium between root and shoot growth (De Willigen and Van Noordwijk, 1987). The increase in water uptake per root surface or length was longer lasting after inoculation with P. aphanidermatum. Infected roots as well as EC affected roots react probably as a sink for photosynthetically produced carbon (Whipps, 1986).
Acknowledgements The Ministries of Agriculture of the Federal Republic of Germany and the States of Brandenburg and Thueringen supported this study. Keith T. Ingram (Griffin Experiment Station, University of Georgia) is gratefully acknowledged for his critical comments. References Barlow, P.W., Adams, J.S., 1988. The position and growth of lateral roots on cultured root axes of tomato, Lycopersicon esculentum (Solanaceae). Plant Syst. Evol. 158, 141–154. Couteaudier, Y., Lemanceau, P., 1989. Culture hors-sol et maladies parasitaires. P.H.M.-Revue Horticole 301, 9–18. Cramer, M.D., Schierholt, A., Wang, Y.Z., Lips, S.H., 1995. The influence of salinity on the utilization of root anaplerotic carbon and nitrogen metabolism in tomato seedlings. J. Exp. Bot. 46 (291), 1569–1577. Cruz, V., Cuartero, J., 1990. Effects of salinity at several developmental stages of six genotypes of tomato (Lycopersicon spp.). In: Cuartero, J., Gomez-Guillamon, M.L., Fernandez-Munoz, R. (Eds.), Proceedings of the XI Eucarpia Meeting on Tomato Genetics and Breeding, Eucarpia Tomato 90, Malaga, Spain, pp. 81–86. Cuartero, J., Fernandez-Munoz, R., 1999. Tomato and salinity. Sci. Hort. 78, 83–125. Dalton, F.N., Poss, J.A., 1990. Water transport and salt loading: a unified concept of plant response to salinity. Acta Hort. 278, 187–194. Dalton, F.N., Maggio, A., Piccinni, G., 1997. Effect of root temperature on plant response functions for tomato: comparison of static and dynamic salinity stress indices. Plant and Soil 192, 307–319. De Kreij, C., Voogt. W., Van den Bos, A.L., Baas, R., 1997. Voedingsoplossingen voor de teelt van tomaat in gesloten teeltsystemen. Brochure VG Tomaat, PBG Naaldwijk, The Netherlands, 22 pp. De Willigen, P., Van Noordwijk, M., 1987. Plant production and nutrient use efficiency. Thesis. Agricultural University of Wageningen, 282 pp. Donaldson, S.P., Deacon, J.W., 1993. Differential encystment of zoospores of Pythium species by saccharides in relation to establishment on roots. Physiol. Mol. Plant Pathol. 42, 177–184. Favrin, R.J., Rahe, J.E., Mauza, B., 1988. Pythium spp. associated with crown rot of cucumbers in British Columbia greenhouses. Plant Dis. 72, 683–687.
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Grosch, R., Schwarz, D., 1998. Auswirkungen eines Pathogenbefalles durch Phytium aphanidermatum auf die Wurzelmorphologie von Tomate. In: Merbach, W. (Ed.), Pflanzenerna¨ hrung, Wurzelleistung, Exsudation. 8. ¨ kophysiologie des Wurzelraumes 1997, B.G. Teubner Verlagsgesellschaft Borkheider Seminar zur O Stuttgart, Leipzig, pp. 65–72. Gulmon, S.L., Turner, N.C., 1978. Differences in root and shoot development of tomato (Lycopersicon esculentum L.) varieties across contrasting soil environments. Plant and Soil 19, 127–136. Hassan, M.N., Overstreet, R., 1952. Elongation of seedlings as a biological test of alkali soils. 1. Effects of ions on elongation. Soil Sci. 73, 315–326. Jenkins, S.F., Averre, C.W., 1983. Root diseases of vegetables in hydroponic culture systems in North Carolina greenhouses. Plant Dis. 67, 968–970. Kafkafi, U., 1996. Root growth under stress-salinity. In: Waisel, Y., Eshel, A., Kafkafi, U. (Eds.), Plant Roots— The Hidden Half, 2nd ed. Marcel Dekker, New York, pp. 375–391. Mitchell, R.T., Deacon, J.W., 1986. Differential (host specific) accumulation of zoospores of Pythium on roots of graminaceous and non-graminaceous plants. New Phytol. 102, 113–122. Moulin, F., Lemanceau, P., Alabouvette, C., 1994. Pathogenicity of Pythium species on cucumber in peat-sand, rockwool and hydroponics. Eur. J. Plant Pathol. 100, 3–17. Muday, G.K., Haworth, P., 1994. Tomato root growth, gravitropism, and lateral development: correlation with auxin transport. Plant Physiol. Biochem. 32, 193–203. Pessarakli, M., Tucker, T.C., 1988. Dry matter yield and N-15 uptake by tomatoes under sodium-chloride stress. Soil Sci. Am. J. 52, 698–700. Plant, A.L., Cohen, A., Moses, M.S., Bray, E.A., 1991. Nucleotide sequence and spatial expression pattern of a drought- and abscisic acid-induced gene of tomato. Plant Physiol. 97, 900–906. Sacher, R.F., Staples, R.C., 1985. Inosito and sugars in adaptation of tomat to salt. Plant Physiol. 77, 206–210. Schroth, M.N., Loper, J.E., Hildebrand, D.C., 1984. Bacteria as biological control agents for plant disease. In: Klug, M.J., Reddy, C.A. (Eds.), Current Perspectives in Microbial Ecology. American Society of Microbiology, Washington, DC, pp. 362–369. Schwarz, D., Heinen, M., Van Noordwijk, M., 1995. Rooting characteristics of lettuce grown in irrigated sand beds. Plant and Soil 176, 205–217. Statistica for Windows, 1998. Reference Manual. StatSoft Inc., Tulsa, OK, USA, 3781 pp. Sudhakar, C., Reddy, P.S., Veeranjaneyulu, K., 1993. Effect of salt stress on the enzymes of proline synthesis and oxidation in greengram (Phaseolous aureus Roxb.) seedlings. J. Plant Physiol. 141, 621–623. Tennant, D., 1975. A test of a modified line intersect method of estimating root length. J. Ecol. 63, 995–1001. Van Noordwijk, M., Raats, P.A.C., 1980. Drip and drainage systems for rockwool cultures in relation to accumulation and leaching of salts. In: Proceedings of the Fifth International Congress on Soilless Culture, Wageningen, 1980, pp. 279–287. Waisel, Y., Eshel, A., 1992. Differences in ion uptake among roots of various types. J. Plant Nutr. 15, 945–958. Whipps, J.M., 1986. Carbon economy. In: Curl, E.A., Truelove, B. (Eds.), The Rhizosphere. Springer, Berlin. Wulff, E.G., Pham, A.T.H., Cherif, M., Rey, P., Tirilly, Y., Hockenhull, J., 1998. Inoculation of cucumber roots with zoospores of mycoparasitic and plant pathogenic Pythium species: differential zoospore accumulation, colonization ability and plant growth response. Eur. J. Plant Pathol. 104, 69–76. Yermiyahu, U., Shlomo, N., Ben-Hayyim, G., Kafkafi, U., Kinraide, T.B., 1997. Root elongation in saline solution related to calcium binding to root cell plasma membranes. Plant and Soil 191, 67–76. Zobel, R.W., 1975. The genetics of root development. In: Torrey, J.G. Clarkson, D.T. (Eds.), The Development and Function of Roots. Academic Press, New York, pp. 261–275.
Influence of nutrient solution concentration and a root pathogen (Pythium aphanidermatum) on tomato root growth and morphology Dietmar Schwarz*, Rita Grosch Institute for Vegetable and Ornamental Crops, Theodor-Echtermeyer-Weg 1, D-14979 Grossbeeren, Germany Accepted 17 July 2002
Abstract Young roots, especially those in the root hair zone, are most important for nutrient uptake. However, these roots are also sensitive to high nutrient solution concentration and root pathogens, major problems in soilless culture systems. Supply of nutrient solution at a constant electrical conductivity (EC) can increase the EC in the root environment up to 10 dS m1. Pythiaceae are among the most important root pathogens in soilless systems. Therefore, investigations were carried out to quantify and compare root damage by high nutrient solution EC and by Pythium aphanidermatum (Edson) Fitzp. Three tomato plants at two-leaf-stage (Lycopersicon esculentum [Mill] L. cv. Counter) were transferred to a 2 l container filled with aerated nutrient solution. They grew in climate chambers for 14 days (16 h light 600 mmol m2 s1, 30/25 8C day/night). In one experiment, a basic nutrient solution of 1.5 dS m1 was supplemented with either NaCl or macronutrients to give an EC of 1.5, 5 or 9 dS m1. In another experiment, plants were inoculated with P. aphanidermatum (0, 102, or 104 oospores ml1 solution). All treatments were replicated five and four times, respectively. On days 3, 7, 10, and 13 after treatment one plant was sampled from each container. Fresh and dry mass of shoots and roots, total root length, number of adventitious roots and of all tap root laterals decreased with increasing nutrient solution EC. Dry matter content of roots and tap root diameter were not influenced while shoot dry matter content increased with increasing EC. NaCl enhanced root fresh matter, tap root diameter, total root length and number of adventitious roots compared with the macronutrient treatment. On the contrary shoot dry matter content and number of all laterals were reduced by NaCl. Three days after inoculation with P. aphanidermatum the number and length of roots were reduced significantly by the treatment 104 oospores ml1. P. aphanidermatum effects were greatest on young and thin roots, particularly those of the second and third laterals. Reduction increased with time and * Corresponding author. Tel.: þ49-33701-78206; fax: þ49-33701-55391. E-mail address: [email protected] (D. Schwarz).
0304-4238/02/$ – see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 2 3 8 ( 0 2 ) 0 0 1 4 3 - 7
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increasing number of inoculated oospores. Root diameters were greatest in the treatment with 104 oospores ml1. Results confirm that attempts to extrapolate results obtained on average root systems to single root formation could be misleading. Furthermore, a tomato plant can compensate to some extent its water uptake of a root system reduced by nutrition or pathogen effects. # 2002 Elsevier Science B.V. All rights reserved. Keywords: EC; Growth rate; Lateral roots; Nutrient solution; Osmotic stress; Pythium aphanidermatum; Root length; Root diameter; Tap root; Water uptake
1. Introduction Young roots, particularly those in the root hair zone, are most important for nutrient uptake. However, these roots are also most sensitive to high nutrient solution concentration and root pathogens, the main problems in soilless culture systems nowadays. Supply of nutrient solution at a constant electrical conductivity (EC) as common in horticultural practice can increase the EC in the root environment up to 10 dS m1 (Van Noordwijk and Raats, 1980). High salt concentrations result in low water potentials and thus reduce root elongation for many crops (Kafkafi, 1996). For radish, root elongation was reduced by 4% per 0.1 MPa (Hassan and Overstreet, 1952) while maize root length was reduced by 54% at about 100 mmol l1 NaCl (0.4 MPa) compared to the control without NaCl (Dalton et al., 1997). Furthermore, root biomass could be negatively affected by cell growth restriction. For tomato at EC above 4–6 dS m1 plants have a significantly reduced water uptake (Cuartero and Fernandez-Munoz, 1999). The decrease in water uptake is strongly and linearly correlated to EC (Dalton et al., 1997). Decreased water uptake decreases dry matter allocation to shoots more than to roots (Cruz and Cuartero, 1990). As a consequence transpiration coefficient and shoot/root ratio decrease with increasing EC. In nutrient solution systems an enrichment of ions, such as sodium chloride and sulphates, is typical. An enrichment imposes additional stress to that of the osmotic stress, which might lead to ionic imbalances or reduction in the uptake of some ions and thus to further growth depressions or even cell death (Kafkafi, 1996; Cuartero and Fernandez-Munoz, 1999). Cramer et al. (1995) reported that supplying more than 70 mmol l1 NaCl inhibited nitrate uptake and reduced photosynthesis. On the other hand supply of Ca2þ might compensate for the negative effects of Naþ (Yermiyahu et al., 1997). Pythiaceae are among the most important root pathogens in soilless systems causing root rot in tomato and cucumber (Jenkins and Averre, 1983). Pythium aphanidermatum (Edson) Fitzp. is an oomycete with high pathogenic potential under soilless culture conditions, that has a broad host range (Mitchell and Deacon, 1986). The fungus reproduces asexually and is dispersed by zoospores, which respond to chemo attractants, such as amino acids, produced by the host. The zoospores swim to and accumulate on the root surface, where they encyst. Zoospore cysts germinate rapidly and the germ tubes from the cysts penetrate root tissue. Root infection by Pythium spp. may reduce yield of plants through decays of root tissue. On the other hand, yield losses can also occur in the absence of any obvious symptoms of root necrosis (Favrin et al., 1988). Reasons for such asymptomatic yield loss
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might be slight infections, a plant physiology that influences the behaviour of the fungi, or the activity of specific Pythium spp., such as Pythium group F (Moulin et al., 1994). With or without root symptoms, Pythium spp. reduces water uptake and may cause leaves or shoots to wilt during warm temperatures (Couteaudier and Lemanceau, 1989), effects that are similar to those of osmotic stress. Relatively little research has studied the nuances in characteristics that exist among the various constituents of the root system (Waisel and Eshel, 1992). Metabolically active roots of older tomato are mostly laterals growing from several branching levels. Nutritional and hormonal conditions affect pericycle cells presumably activating specific root forming genes (Barlow and Adams, 1988). Different gene expressions determine the uptake capability of tap and lateral roots. Waisel and Eshel (1992) reported that the metabolic nutrient uptake system in tap roots dominates that of the non-metabolic uptake system. Depending on cultivar, under salinity treatments tomato developed numerous small lateral feeder roots (Zobel, 1975). Pythium spp. effects on root formation are not described in the literature. Similar influences of osmotic stress and P. aphanidermatum inoculation on water uptake and plant growth reduction led us to the question whether their effects on tomato roots are comparable. Therefore, experiments were carried out to quantify and compare their effects on (i) total root system morphology, (ii) root formation, and (iii) productivity of the root system with respect to water uptake and dry matter production.
2. Materials and methods Tomato seeds were (Lycopersicon esculentum [Mill] L. cv. Counter) germinated in coarse sand and kept moist until the seedlings reached the two-leaf-stage. Then three tomatoes were transferred to a 2 l container filled with aerated nutrient solution and covered with a plastic foil to prevent algae growth and evaporation. Plants were secured in a foam disc (10 mm thick) flooding on the surface of the solution. They grew in climate chambers for 14 days at relative humidity of 70/90% (day/night) and photosynthetic active radiation of 600 mmol m2 s1 for 14 h per day. The stem above the tap root was wrapped up to a height of 15 mm with flexible foam to prevent growth of adventitious roots. To determine the effect of nutrient solution composition and concentration a first experiment was carried out at day/night temperature of 25/20 8C (13–26 June 1997). The basic nutrient solution of 1.5 dS m1 (T1.5, control) contained in mmol l1: NH4 1, NO3 10.8, K 6.5, Ca 3.8, SO4 1.5, P 1.2, and in mM l1: Fe 15; Mn 10; B 20; Mo 0.5 (De Kreij et al., 1997). To give an EC of 5 and 9 dS m1 the basic solution was supplemented either with 38 mmol l1 NaCl or 81 mmol l1 NaCl (N5, N9) or with macronutrients (T5, T9). The pH was adjusted daily to 5.6. Every treatment included five replications with 75 total plants in a randomised design. To determine the effect of P. aphanidermatum (Edson) Fitzp. a second experiment was conducted at day/night temperature of 30/25 8C using a basic nutrient solution at EC 2.5 dS m1 and pH 5.6 (11–24 July 1997). In the first experiment treatments started with seedlings transfer to the containers, while in the second experiment treatments started 3 days after seedling transfer. Plants were inoculated with 0, 102, or 104 oospores ml1
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solution (O0, O2, O4). Every treatment included four replications with 36 total plants in a randomised design. On days 4, 7, and 13 after treatment (DAT) during the first experiment, and on days 3, 7 and 10 during the second experiment, one plant was removed from the container and analysed. Total water uptake during the preceding period (Wup, ml per plant), total fresh and dry mass of shoots (Dsp, g per plant) and roots (Drp, g per plant) were measured. To determine the treatment effect on the whole root system total root number (Nrp, no. per plant) was counted and total length (Lrp, m per plant) was estimated using the line intersect method of Tennant (1975). Tap root and adventitious roots were excluded but measured separately with a ruler. Root diameters were measured on 10 roots randomly sampled from the second and third laterals (every 10th line root intersection until 10 readings were obtained) and mean root diameter was calculated (2Rr, mm). From these measurements we calculated specific root 1 2 3 length (Lrw ¼ Lrp D1 rp , m g ), root surface area (Arp ¼ Lrp pRr 2 10 , m per plant) 1 1 and root frequency number per length (Nrl ¼ Nrp Lrp , no. m ). First, second, and third laterals of the tap root and any adventitious roots that grew under the foam were counted to test treatment influences on root formation and initiation. Relative root reduction was calculated for first to third laterals as ratio to the respective control. Tap root diameter was measured 10 mm away from shoot and 10 mm before where the root ended. From these data, we calculated total plant growth rate (GRR, g m2 per day) and water uptake rate (WUR, ml m2 per day) both related to root surface area: ðDsp i þ Drp i Þ ðDsp i1 þ Drp i1 Þ ; 0:5ðti ti1 ÞðArp i þ Arp i1 Þ Wup i WUR ¼ 0:5ðti ti1 ÞðArp i þ Arp i1 Þ
GRR ¼
where t is DAT, and i the index for DAT. Data were subjected to analysis of variance procedures with treatment EC and inoculum density as factors. Means at the different measurement dates were separated by Tukey’s test procedure at P ¼ 0:05. Significant differences are presented by different letters in the table but omitted in the figures for clarity. Instead the figures show only standard deviation bars. For statistical procedures Statistica for Windows (1998) software was used.
3. Results 3.1. Plant growth analysis At 4 DAT dry matter content but not dry mass of shoots and roots was already enhanced by treatments T5 and T9 (data not shown). Three days later, treatments diminished shoot growth significantly and further 6 days root mass was also reduced significantly (Fig. 1A and B). Effects of P. aphanidermatum were significant at 3 DAT for root fresh and dry mass and from 7 DAT afterwards for shoot and root fresh and dry masses (Fig. 1A and B). O2 resulted in a 71% reduction of shoot growth and was not much stronger compared to the influence of N9 with 64%. Root decrease was smaller than shoot decrease with 63% but significantly stronger compared to a 40% reduction reached by N9. Dry matter content of roots was
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Fig. 1. Shoot (A) and root (B) dry mass of tomato grown in 2 l containers. Left: treated with nutrient solution concentration of EC 1.5 (T1.5), 5, and 9 dS m1 made by either increasing the macronutrient concentration (T5, T9) or supply of NaCl (N5, N9). Right: treated with 0 (O0), 102 (O2) and 104 (O4) oospores ml1 of P. aphanidermatum.
reduced significantly at O4 (Table 2). Shoot dry matter content increased with increasing EC but not for NaCl treatments. 3.2. Morphology of the root system Total root number, length (Fig. 2), and surface area of the whole root system increased with time for all treatments (data not shown). Only O4 showed no further increase or even a decrease on the last measurement in most of these characteristics compared to results 7
Fig. 2. Total root length of tomato grown in 2 l containers. Left: treated with nutrient solution concentration of EC 1.5 (T1.5), 5, and 9 dS m1 made by either increasing the macronutrient concentration (T5, T9) or supply of NaCl (N5, N9). Right: treated with 0 (O0), 102 (O2) and 104 (O4) oospores ml1 of P. aphanidermatum.
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Table 1 Root characteristics of tomato for treatments EC 1.5 (T1.5), 5, or 9 dS m1 made by supply of either macronutrients (T5, T9) or NaCl (N5, N9)a Characteristic 1
Dry matter content, root (g kg ) Dry matter content, shoot (g kg1) Root surface area (m2 per plant) Tap root length (mm) Diameter, beginning of tap root (mm) Diameter, end of tap root (mm) Diameter, root branches (mm) Root number, branch 1 (no. per plant) Root number, branch 2 (no. per plant) Root number, branch 3 (no. per plant) Adventitious roots (no. per plant) a
T1.5
T5
T9
N5
N9
30.2 a 71.8 c 0.0723 a 44.6 a 5026 a 1077 b 279 a 36.4 a 1906 a 1032 a 20.6 a
31.1 a 77.9 b 0.0594 b 33.0 ab 4318 a 1313 b 309 a 35.8 a 1659 a 797 a 14.4 b
30.2 a 86.4 a 0.0482 b 36.8 ab 4041 a 1221 b 307 a 36.6 a 1285 b 659 a 13.4 b
28.7 a 62.7 d 0.0742 a 34.0 ab 4656 a 1856 a 278 a 32.8 ab 845 bc 718 a 18.0 a
26.2 a 61.7 d 0.0561 b 30.6 b 4359 a 1436 ab 270 a 26.8 b 531 c 309 b 8.4 c
Different letters depict significant differences at P < 0:05.
DAT. As depicted in Fig. 2 for root length all of these characteristics were affected by EC and inoculum density. Reduction of root length and surface area was significant for the EC treatments 13 DAT but for P. aphanidermatum already 3 DAT (Tables 1 and 2; Fig. 2). Total root number was reduced 4 DAT for T9 where 89 and N9 where 82 roots were counted compared to 147 roots at T1.5. The root length per dry mass changed with treatments for both experiments already at the first measurement date (Fig. 3A). While NaCl treatments resulted in an increased specific root length it was reduced with increasing inoculum density of P. aphanidermatum. The number of roots per length was affected in an almost opposite way (Fig. 3B). It decreased for the NaCl treatments but increased for the treatments with oospores of P. aphaniderTable 2 Root characteristics of tomato inoculated with 0 (O0), 102 (O2), or 104 (O4) oospores ml1 of P. aphanidermatuma Characteristic
O0 1
Dry matter content, root (g kg ) Dry matter content, shoot (g kg1) Root surface area (m2 per plant) Tap root length (mm) Diameter, beginning of tap root (mm) Diameter, end of tap root (mm) Diameter, root branches (mm) Root number, branch 1 (no. per plant) Root number, branch 2 (no. per plant) Root number, branch 3 (no. per plant) Adventitious roots (no. per plant) a
33.0 a
O2 b
22.7 bb
c
c
c
0.0721 a 39.0 b 5115 a 987 a 264 b 37.5 b 2680 a 1322 a
0.0218 b 33.8 c 3949 b 897 a 265 b 35.2 b 869 b 774 b
0.0069 c 53.5 a 3744 b 308 b 378 a 70.2 a 178 c 29.8 c
c
c
c
Different letters depict significant differences at P < 0:05. Determined 7 days after the treatment was started. c No measurements. b
32.2 a
O4 b
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Fig. 3. Specific root length per dry mass (Lrw) (A) and number of roots per length (B) for tomato. Left: treated with nutrient solution concentration of EC 1.5 (T1.5), 5, and 9 dS m1 made by either increasing the macronutrient concentration (T5, T9) or supply of NaCl (N5, N9). Right: treated with 0 (O0), 102 (O2) and 104 (O4) oospores ml1 of P. aphanidermatum.
matum. However, the lower inoculum density O2 increased the root numbers per length more than the highest density O4 (data are not shown). Mean root diameter of all laterals varied between 264 and 309 mm for all treatments, except O4. Here, it increased significantly to 378 mm (Table 2). 3.3. Effects on root formation Adventitious roots were counted only during the EC experiment (Table 1). The number increased linearly until 7 DAT and was then diminished with increasing EC, most at N9 with 59% (data not shown). Three levels of branches from the main root were determined. Compared with the control more roots grew 10 DAT with P. aphanidermatum within the first laterals, at O4 almost 100%, and within the second laterals 7 DAT at N5 60% (Fig. 4). Number within the second and third laterals was reduced 13 DAT (Fig. 4). The strongest reduction by EC treatments occurred within the second laterals at N9, while the strongest reduction by P. aphanidermatum treatments occurred within the second and third laterals at O4. Compared with the second laterals number of third laterals was less diminished. Tap root length amounted to 68 mm as a maximum at T9 4 DAT and 30 mm as a minimum at O0 7 DAT (data not shown). It was influenced by treatments. While N9 and O2 reduced it O4 increased the length compared to the controls (Table 1). The diameter at the beginning of the tap root was reduced by P. aphanidermatum treatments 7 and 10 DAT
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Fig. 4. Relative root reduction (dimensionless) of first to third laterals of tomato tap roots related to the respective control. Upper figures: treated with nutrient solution concentration of EC 1.5 (T1.5), 5, and 9 dS m1 made by either increasing the macronutrient concentration (T5, T9) or supply of NaCl (N5, N9). Lower figures: treated with 0 (O0), 102 (O2) and 104 (O4) oospores ml1 of P. aphanidermatum.
Fig. 5. Growth rate of total tomato plant dry mass (GRR) (A) and water uptake rate (WUR) (B) per root surface area. Left: treated with nutrient solution concentration of EC 1.5 (T1.5), 5, and 9 dS m1 made by either increasing the macronutrient concentration (T5, T9) or supply of NaCl (N5, N9). Right: treated with 0 (O0), 102 (O2) and 104 (O4) oospores ml1 of P. aphanidermatum.
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(Table 2). It was reduced as well close to the end of the tap root by O4 to 79% but increased by N5 at 72%. 3.4. Growth and water uptake related to root growth Total plant growth rate and water uptake were related to 1 m1 m root surface area to characterise treatment influence on root productivity. GRR decreased with time independent of the treatments (Fig. 5A). The same root surface was connected to a lower growth rate for NaCl treatments compared with balanced macronutrient treatments, 7 and 13 DAT. Three DAT at O4 GRR increased immediately but afterwards it decreased more severely than the control and O2. It was negative on 10 DAT (Fig. 5B). Here, the mass produced was smaller than that destroyed. WUR decreased with time as well but it was not affected by EC treatments. On the other hand, it increased with increasing inoculum density caused by a larger reduction in root than in shoot growth. Roots were able to double WUR at O4 compared to the control. Thus they compensated the smaller surface by an increased water uptake rate.
4. Discussion The reduction of plant growth characteristics in general and of root growth in particular with increasing nutrient solution EC and with P. aphanidermatum inoculation agree with the results of several authors (e.g. Kafkafi, 1996; Wulff et al., 1998). The EC effect is mainly an osmotic stress and was significant already at EC 5 dS m1 which is the threshold value of 4–6 dS m1 mentioned by Cuartero and Fernandez-Munoz (1999). The more severe reduction of shoot mass by the NaCl treatments compared with balanced macronutrient treatments indicates an additional ionic effect (Kafkafi, 1996). The opposite effect by NaCl treatments was observed for total root length (Fig. 2). Several reasons are possible for the reduced root length: cell growth restriction because of low water potential of external medium, interference of the ions or the toxicity of accumulated ions (Cuartero and Fernandez-Munoz, 1999). Root length reduction was related to a larger specific root length for the NaCl treatments compared to the macronutrient treatments (Fig. 3). Normally, increased specific root length is accompanied by decreased average root diameters (Schwarz et al., 1995). Mean root diameters of NaCl treatments were >30 mm smaller than those of the macronutrient treatments (Table 1). Changes in the root structure (Pessarakli and Tucker, 1988) or less available assimilates for the roots, e.g. by reduced photosynthesis (Cramer et al., 1995), could have caused the reduction. A lower dry matter (Table 1) confirmed changes in root structure. It is possible that in the NaCl treatments alleviation of osmotic stress might have increased the production of osmotically active substances, such as proline (Sudhakar et al., 1993) or myo-inositol (Sacher and Staples, 1985). P. aphanidermatum reduced root length before it affected shoot and root mass. This was also observed in previous investigations at low inoculum densities of 10 oospores ml1 without measurable effects on plant growth (Grosch and Schwarz, 1998). The early reduction of root length is connected to a strong reduction in specific root length (Fig. 3A).
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A larger diameter measured for O4 can partly explain the decrease in specific root length. It is possible that the pathogen started cell destruction and dissolution after penetrating the root exodermis (Wulff et al., 1998). A significantly lower dry matter content for O4 as well as a reduction of the number of the younger and thinner roots indicate this process (Table 2). Although EC and P. aphanidermatum immediately affected roots, shoots were more affected after a few days (Figs. 1–3 and 5A) as has been reported by others (Cruz and Cuartero, 1990; Dalton and Poss, 1990; Plant et al., 1991). The authors explained these findings by changes in dry matter allocation in favour of the roots caused by lowered root indole-3-acetic acid (IAA) concentration or root to shoot signalling by reduced water uptake and thus increase in abscisic acid (ABA) in the xylem. Treatments affected the levels of laterals differently (Fig. 4). Gulmon and Turner (1978) showed that nutrient deficiency resulted in fewer tomato roots and reduced length of the second and third branches independent of the cultivars. Barlow and Adams (1988) cultured tomato root axes and lateral roots for 8 days in media containing three different concentrations (0.5, 1.5, 2%) of sucrose. The medium sucrose concentration resulted in a significantly larger number of laterals and increased total length. Both, nutrient deficiency as well as nutrient surplus seem to decrease root initiation. The reduced number of second and third laterals (Fig. 4) could be explained by an overall reduced root length. Therefore, the root frequency per length was only diminished for the NaCl treatments 13 DAT (N5, N9) but not for increasing macro nutrient concentration (Fig. 3B). In contrast, P. aphanidermatum inoculation increased the root frequency per length. A similar development has been described by Zobel (1975) typically for salinity effects inducing the development of numerous small lateral feeder roots. The conflicting findings might be due to different growing media and cultivars used. The roots in experiments presented grew directly in solution and not in soil as in the experiments by Zobel (1975). The same author also mentioned that some cultivars investigated did not show a strong development of these feeder roots. That roots appear later with salt (Abrisqueta et al. (1991) in Cuartero and Fernandez-Munoz, 1999) was confirmed particularly for the third laterals. Four DAT no roots were found at N9 and also the number for the other treatments were much lower compared to the control at EC 1.5 dS m1. Muday and Haworth (1994) showed that IAA transport decreased or prevented formation of lateral roots. This was not found for first laterals except N9 in tomato experiments. IAA concentrations sufficient to inhibit growth of second and third laterals may have stimulated growth of first laterals as it was the case for treatment O4. Here, also stimulation could have occurred by IAA production by the pathogen itself. P. aphanidermatum is able to influence root growth negatively by producing IAA (Wulff et al., 1998). A lack of production may result in low IAA concentrations and thus in a positive or at least not root growth reducing effect (Schroth et al., 1984). It is also possible that different effects on root formation are caused by specific root forming genes presumably activated in the pericycle cells (Barlow and Adams, 1988) or by the activity of other hormones, such as ethylene, cytokinins, and gibberellins (Cruz and Cuartero, 1990). All of these hormones affect root initiation and branching and are affected by the nutritional status of the plant (Barlow and Adams, 1988; Waisel and Eshel, 1992) or by microorganism and pathogens (Donaldson and Deacon, 1993). In the experiment presented the pathogen colonisation of the different laterals were not quantified. To explain plant and
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particular single root and pathogen interactions it is necessary to investigate population dynamics of the pathogen, e.g. by serological methods. From our results it can be assumed that pathogen density was higher on second and third laterals than on the first laterals. In addition to the hormonal effect a direct damage in form of cell destruction and dissolution might be responsible although in the O2 treatment no symptoms were visible. It can be summarised that general treatment effects of EC and Pythium resulted in similar patterns but they had different effects on root formation. Therefore, attempts to extrapolate results obtained on average root systems and their characteristics to single root formation could be misleading (Waisel and Eshel, 1992). The reduction of the root surface area was compensated by increased water uptake rates per unit root particularly after treatment start while the effect disappeared later (Fig. 5A and B). The increased water uptake of roots confirms the functional equilibrium between root and shoot growth (De Willigen and Van Noordwijk, 1987). The increase in water uptake per root surface or length was longer lasting after inoculation with P. aphanidermatum. Infected roots as well as EC affected roots react probably as a sink for photosynthetically produced carbon (Whipps, 1986).
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