HIGH LEVEL OF COPPER APPLICATION TO SOIL AND LEAVES REDUCE THE GROWTH AND YIELD OF TOMATO PLANTS

Copper-containing fertilizers, fungicides and bactericides are extensively used in greenhouses in Turkey. Informations on effects of these applications to plants are scarce. The aim of the present study was to investigate effects of Cu application to a calcareous soil and to leaves on the yield and growth of tomato plants. Cu was first applied to soil as CuSO4.5H2O in three different levels (0, 1000, and 2000 mg Cu kg) and then to leaves in three different frequencies (no application, biweekly and weekly) using two cupric fungicides (Cu oxychloride, and Cu salts of fatty and rosin acids) in pot experiments carried out in a computer-controlled greenhouse. Total yield, fruit number, dry root weight and plant height decreased with increasing Cu application to soil. Increasing levels of Cu applied to soil and leaves resulted in decreasing final fruit number, dry root weight and plant height in 4, 5 and 6 weeks. Combined applications of Cu to soil and leaves could be more deleterious to plants than when Cu is applied only to soil or leaves.


INTRODUCTION
Copper contents of the majority of plant species varies between 20 and 30 mg kg -1 dry weight.The critical copper deficiency level in vegetative plant parts is generally 3 to 5 mg kg -1 dry weight (Robson & Reuther, 1981); in young grain plants it was reported to be 1.5 mg kg -1 dry weight (Robson et al., 1984).
Copper is an essential element for various metabolic processes.Because it is required only in trace amounts, Cu becomes toxic at high concentrations (Delas, 1963;Alva & Chen, 1995).In non-tolerant plants, inhibition of root elongation and damage of root cell membranes are the immediate responses to high Cu levels (Wainwright & Woolhouse, 1977).Changes in root morphology, such as inhibited elongation and enhanced lateral root formation (Savage et al., 1981), might be related to the sharp decrease in Indol Acetic Acid oxidase activity in roots exposed to high Cu concentrations (Coombs et al., 1976).Zheng et al. (2004) reported that excessive copper reduced plant root length, root dry weight, total dry weight, root to shoot ratio, leaf area and specific leaf area in three ornamental crops (Dendronthema × grandiflorum L. 'Fina', Rosa × hybrida L. 'Laulinger', Pelargonium × hortorum L. 'Evening Glow') grown in solution culture.Copper-containing fertilizers, fungicides and bactericides have been used extensively in the greenhouses in Antalya, Turkey.Kaplan (1999) reported that circa 8% of soils in Antalya contained DTPA-extractable Cu greater than the critical toxicity level (20 mg kg -1 ), and the Cu concentration in leaf samples was very high as a result of the intensive use of foliar applied Cu-containing chemicals.
Application of copper containing fertilizers, pesticides and fungicides to leaf or soil has increased gradually over the years in Mediterranean regions where soils are calcareous with neutral or alkaline pH.It is known that Cu solubility decreases as soil pH increases.Therefore, it is thought that levels of bio-available Cu in the Mediterranean region are low.There are no previous reports on the effects of high levels of Cucontaining fungicides on yield and growth of tomato plants in the Mediterranean region.The effects of Cu toxicity on yield and growth of tomato plants, under high levels of Cu applications were evaluated in this study.
Two separate experiments were carried out, each using different cupric fungicide: Cu oxychloride or copper salts of fatty and rosin acids.The former contains 25% Cu oxychloride and is sold as a powder.The latter is a liquid fungicide containing 58% copper salts of fatty and rosin acids (CAS # 61789-22-8), equivalent to 51.4 mg L -1 metallic Cu.

Experimental Design
Twenty kg of air-dried soil were passed through a 4 mm mesh sieve and mixed with 5 kg of a 75% turf: 25% perlyte mixture, and distributed in 25-L pots, fertilized with mono ammonium phosphate and potassium sulphate (36 kg N ha -1 , 80 kg P ha -1 and 112 kg K ha -1 ).Copper was applied to soil at three different rates [0 (Cu1), 1000 (Cu2) and 2000 mg kg -1 (Cu3)] as CuSO 4 .5H 2 O.One seedling of tomato (Lycopersicon esculentum (L.) Mill.Cv.F144) was planted per pot.Fungicides were applied at three different frequencies [control, no application (L1), biweekly (L2) and weekly (L3)].The treatments were set up based on Kaplan (1999).Trials were set up in a completely randomized factorial design with nine treatments: three levels of Cu application to soil and three frequencies of fungicide application to leaves, in all possible combinations, (n = 4).

Processes During and at the End of the Experiment Period
Pots were incubated for two weeks after addition of copper and before planting.Copper application to leaves started at four weeks after planting.All pots were fertilized once a week with mono ammonium phosphate, potassium nitrate, ammonium nitrate, and magnesium sulfate.Total amounts of nutrients provided to each pot were: 195 kg N ha -1 , 62 kg P ha -1 , 177 kg K ha -1 , and 16 kg Mg ha -1 .Pots also received 3.0 kg Fe ha -1 , 3.0 kg Mn ha -1 , 1.13 kg Zn ha -1 , 0.38 kg B ha -1 and 0.08 kg Mo ha -1 .
Plant height was measured weekly from the 4 th week after planting.Fruit numbers per plant were recorded and harvested fruits were weighed.At the end of the experiment, plant roots were washed to detach soil particles, dried in a forced-air oven (65 o C; 72 h), and weighed.

Statistical Analysis
Statistical analysis was carried out using the MSTAT-C software.Means were compared by analysis of variance (ANOVA) and the LSD test (α = 0.05).A factorial analysis was used to determine interaction effects of copper application to soil and leaves on yield and growth of tomato plants.

Experiment I (Cu Oxy Chloride Fungicide)
Copper application to soil affected total yield, fruit number, dry root weight, and plant height (P < 0.01).The greatest total yield, fruit number, and dry root weight were obtained when no copper was applied to soil (Cu1); performance traits decreased from treatment Cu1 to Cu3 (Table 1).
Increasing the level of Cu application to soil resulted in decreased plant height.The greatest plant heights during the 11 weeks were observed when no copper was applied.On average, Cu application to soil resulted in 39% and 50% reductions in plant height in Cu2 and Cu3, respectively, in comparison with treatment Cu1.
Increasing levels of Cu application to leaves affected dry root weight and plant height after the 5 th week.The smallest dry root weight was observed in L3.Similarly, after the 5 th week, the smallest plant heights were registered for L3 (Table 2).Cu application to leaves did not affect plant height during the first four weeks.After the 5 th week, however, plant height decreased as a result of increasing levels of Cu application.The interaction between Cu application to soil and leaves was significant for fruit number and plant height in the 4 th , 5 th and 6 th week (Table 3).In treatment L1, in which no copper was applied to leaves, Cu application to soil did not change fruit number per plant, whereas in the treatments L2 and L3, fruit number decreased with increasing level of Cu application to soil.While the smallest fruit number in treatment L2 (11 fruits, 69.4% decrease in comparison to the control) was recorded for treatment Cu3, the smallest fruit number in treatment L3 was obtained recorded for both treatments Cu2 and Cu3 (64.2% and 75.5% decrease, respectively) (Table 3).Plant height in the 4 th , 5 th and 6 th weeks decreased from Cu1 to Cu2 when no fungicide was applied or when it was applied biweekly, while the weekly application of fungicide led to a further decrease in plant height from Cu2 to Cu3 (Table 3).

Experiment II (Copper Salts of Fatty and Rosin Acids Containing Fungicide)
Total yield, fruit number, dry root weight and plant height were affected by the level of Cu application to soil (P < 0.01).The greatest total yield, fruit number and dry root weight were obtained when no copper was applied (Table 1).As compared with treatment Cu1; total yield, fruit number and dry root weight decreased in the treatment Cu2 (33.5%, 35.0% and 60.3%, respectively) and Cu3 (63.9%, 55.0% and 74.5%, respectively).The greatest plant heights dur-ing the experiment were also obtained when no copper was applied to soil (Table 1).On average, increasing the level of Cu application to soil resulted in a 40.9% and 50.4% reduction in plant height in treatments Cu2 and Cu3, respectively, as compared to treatment Cu1.
Cu application to leaves resulted in a decrease in dry root weight.The greatest dry root weight was obtained in treatment L1 (Table 2).The interaction between Cu application to soil and leaves was significant only for dry root weight (P < 0.01).Copper application to soil and leaves led to a sharper decrease in dry root weight than when copper was only applied to soil (Table 3).

DISCUSSION
Successful tomato production is ordinarily associated with healthy vegetative top and root growth throughout the growing season.High levels of Cu application to soil and leaves seriously disrupted normal plant growth.There was an increasing reduction total yield, fruit number, dry root weight and plant height with increasing levels of Cu application to soil and leaves.Copper is a transition metal that participates in redox reactions.When in excess, Cu causes over-production of oxy radicals, which is believed to be its primary toxic effect in plant cells.Furthermore, Cu-induced cell disturbances have consequences on main physiological processes, and impair growth   (Marschner, 1986).Application of high levels of Cu usually inhibits root growth before affecting shoot production.However, this does not necessarily mean that roots are more sensitive to high copper concentrations, but probably, derives from the fact that roots are in an environment where copper is in excess (Lexmond & Vorm, 1981).
Copper application to soil decreased total yield, fruit number, dry root weight and plant height in both experiments (Table 1).Karataglis & Babalonas (1985) reported that plant height, shoot and root biomass, flower and fruit production decreased with increasing Cu concentration.Hunter (1981) reported that root growth was almost completely inhibited by Cu treatment in maize.Alva et al. (2000) reported 20% reduction in root weight for 62 and 271 mg kg -1 Cu in the roots of citrus seedlings grown in soils with pH 5.7 and 6.5, respectively.Mazhoudi et al. (1997) reported a decline in the growth rate of tomato plants after addition of 50 μM Cu to a nutrient medium.Lidon & Henriques (1992) reported that Cu toxicity, as expressed by reduced root length, appeared to be a direct result of the accumulation of excess Cu in tissues.Similarly, Rhoads et al. (1992) found that plant dry weight decreased with increasing Cu level in 'Florida 502' oats.Lombardini & Sebastiani (2005) registered that Prunus cerasifera plantlets grown in vitro had smaller growth rate (for both fresh and dry weight) at 100 mμM of copper.
Cu application to leaves affected dry weight of roots in both experiments.Plant height after the 5 th week was also affected by foliar application of the Cu oxy chloride fungicide, but not by the copper salts of fatty and rosin acids fungicide (Table 2).Reasons for these differences are unknown.However, there are other reports of excessive Cu +2 depressing root (Hill et al., 2000;Lidon & Henriques, 1992;Ouzounidou, 1994) and shoot growth in other plants (Lin et al., 2003;Maksymiec & Baszynski, 1996).
While the interaction between Cu application to soil and leaves was found to be significant for fruit number and plant height in the 4 th , 5 th and 6 th week, when Cu oxychloride-containing fungicide was used, it was significant only for dry root weight when copper salts of fatty and rosin acids-containing fungicide was used (Table 3).Copper application to soil and leaves resulted in a sharper decrease in dry root weight, fruit number, and plant height in the 4 th , 5 th and 6 th week than when copper was only applied to soil.High levels of Cu application to soil and leaves to control plant diseases, can negatively affect yield and growth of tomato plants.The combined application of Cu to soil and leaves could be more deleterious to plants than when Cu is applied only to soil or leaves.

Table 1 -
The Effects of Cu Application to Soil on Total Yield, Fruit Number, Dry Root Weight and Plant Height.

Table 2 -
The Effects of Cu Application to Leaves on Total Yield, Fruit Number, Dry Root Weight and Plant Height.Means in the same row followed by the same letter are not significantly different (LSD; P > 0.05).Fungicide I: Cu oxychloride Fungicide.Fungicide II: Copper salts of fatty and rosin acids Fungicide.

Table 3 -
Significant Interactions Between Cu Application to Soil and Leaves.Means in the same column followed by the same letter are not significantly different at (LSD; P > 0.05).Fungicide I: Cu oxychloride containing Fungicide.Fungicide II: Copper salts of fatty and rosin acids containing Fungicide.