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© by PSP Volume 24 – No 1b. 2015
Fresenius Environmental Bulletin
THE EFFECTS OF THE INSECTICIDE PYRIPROXYFEN ON GERMINATION, DEVELOPMENT AND GROWTH RESPONSES OF MAIZE SEEDLINGS Yasemin Coskun*, Semra Kilic and Ragbet Ezgi Duran Suleyman Demirel University, Faculty of Arts and Sciences, Department of Biology, Isparta, Turkey
ABSTRACT
1. INTRODUCTION
Phytotoxicity is defined as the detrimental effects of chemical products used for pest eliminating or growth regulating purposes on some morphological, anatomical and physiological processes of plants. We examined the effects of pyriproxyfen, which is used to protect crops against whitefly, to determine whether it adversely affects maize plants. For this purpose, maize (Zea mays L. saccharata Sturt.) seeds were treated with several pyriproxyfen concentrations (0.0, 0.1, 0.2, 0.4, and 0.6 ppm) for 72 h under controlled conditions. The results showed that, while low concentrations of the insecticide had little effect on the growth of maize seedlings, increasing the concentration gradually had harmful effects on the growth. The highest insecticide concentration led to a remarkable decrease in all the growth parameters of maize seedlings. It was found that maize growth was significantly inhibited by increasing the concentration of pyriproxyfen, which adversely affected seed germination and seedling growth. Stomata index decreased in both adaxial and abaxial surfaces of leaves treated with increasing concentrations compared with the control group. The adaxial surfaces of maize leaves always had fewer stomata than the abaxial surfaces. As insecticide concentration increased, photosynthetic pigment contents decreased except for anthocyanin, but the proline accumulation increased as an adaptation to toxic treatment. This work suggests that excessive and uncontrolled usage of pesticide pyriproxyfen results in phytotoxic effects by inducing some morphological, anatomical, physiological and metabolic processes. In addition, as chemical compounds accumulate, pyriproxyfen is thought to adversely affect all living beings feeding on plants that are exposed to chemicals, and also negatively affects the environmental factors such as, soil, air and water. KEYWORDS: Insecticide, photosynthetic pyriproxyfen, maize, stomatal index
* Corresponding author
pigment,
proline,
Pesticides are chemical substances that are used to eliminate or remove pests which damage agricultural products during production, consumption and storage of nutrients, causing product loss [1-3]. Chemical control of harmful organisms is being strictly managed. Pesticides are applied to crops throughout the world, but extensive application in agricultural areas, horticulture and home gardens leads to environmental pollution, especially in soil, water and air. Pesticide residue, left after treatment, may penetrate into plant tissues, and can cause various deformations in their structures [4-7]. More importantly, the involvement of pesticide into the food chain may have adverse effects on human and other species [8], and especially the accumulation of pesticide residues is known to have potential cancerogenic effects [9]. Insecticides have become more widespread as a new way to harvest more crops. As a result, living things have been exposed to a wide variety of insecticides lately. Pyriproxyfen [4-phenoxyphenyl (RS)-2-(2-pyridyloxy) propyl ether] is an influential insecticide used to protect crops against whitefly [10]. It is a pyridine-based pesticide which easily dissolves in water, and harms both animals and plants in aquatic systems [11]. Thus, it is readily translocated in plant tissue. Pyriproxyfen prevents larvae from developing into maturity (inhibits insect maturation process from cell division to elongation) and thus their replication is inhibited [12, 13], but its activity against non-target organisms such as plants has not been tested. However, it is known that the presence of certain functional groups (COOR, -CO.NH2, -NH2, -NHR, -NR3, -0H,) in the chemical structure of the pesticides further facilitate adsorption of the soil humus [14]. Pesticide residues dissolved in water reduces nutrient uptake from soil [15, 16] and depolarizes the plasma membrane of the root cells [17]. This can cause abnormalities in different growth parameters [1821]. Therefore, it is necessary to consider the possible hazardous effects of these insecticides used extensively in agriculture on ecological balance. In the present work, the adverse effects of different concentrations of pyriproxyfen have been evaluated on growth parameters (morphological, anatomical and physiological) of maize seedlings. In this way, ecotoxicological
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© by PSP Volume 24 – No 1b. 2015
Fresenius Environmental Bulletin
profiles of pyriproxyfen can be determined in relation to the structural modification of pyriproxyfen.
2. MATERIALS AND METHODS 2.1 Plant material and chemicals
Uniform-sized seeds (n=25) of a commercial variety of maize (Zea mays L. saccharata Sturt.) were used as the test plant. The Pyriproxyfen was obtained from Bayer. Other chemicals were purchased from Sigma (USA) and Fluka AG (Buchs, Switzerland). 2.2 Germination test
Seeds of maize were surface sterilized with 0.5% sodium hypochlorite for 10 min, followed by extensive washing in sterile distilled water. Treatment concentrations were prepared using control (distilled water) and 0.1, 0.2, 0.4, and 0.6 ppm of original insecticide solution. The application doses were prepared as recommended by the manufacturing company on the label. Uniform-sized seeds were placed into a clean 50 ml glass beaker and were pretreated with the pyriproxyfen concentrations for 72 hours. At the end of 72 h, seeds of maize were sown in 12 cm petri dishes lined with two layers of filter paper (Whatman 1) moistened with 10 ml of the distilled water, and maintained in an incubator at 25°C for 7 days. The experiment was replicated three times at each concentration of Pyriproxyfen. Seeds were considered to be germinated with the emergence of the radicle. At the end of the 7th day, the radicle and coleoptile lengths of seedlings were measured with a millimetric ruler and germination percentages were calculated according to Khan and Ungar [22]. 2.3 Anatomical parameters
The seedlings of each application were placed in 4-L pots with perlite containing Hoagland’s nutrient solution. Plants were cultivated in a climate chamber with controlled conditions (photoperiod 12-h, temperature 25±2°C, relative humidity 60±5%, light intensity 160 μmol m-2 s-1) for 45 days. Seedling growth was provided with the nutrient solution added on a regular basis. Epidermal tissue was stripped from the adaxial and abaxial surfaces of leaf lamina pieces to determine leaf stomatal density, expressed as the number of stomata per unit leaf area, mounted on a glass slide, immediately covered with a cover slip, and then lightly pressured with fine-point tweezers. For each independent measurement, numbers of stomata (s) and epidermal cells (e) for each film strip were counted from both the adaxial and abaxial surfaces. The leaf stomatal index was calculated using the formula [s / (e + s)]×100, as defined by Meidner and Mansfield [23]. Stomatal size was defined as width and length in micrometres. 2.4 Physiological parameters tested
The proline content was determined using to the method of Bates et al. [24]. Proline was extracted from leaf
samples of 100 mg FW with 2 ml of 40% methanol. 1 ml extract was mixed with 1 ml of a mixture of glacial acetic acid and orthophosphoric acid (6 M) (3: 2, v/v) and 25 mg ninhydrin. After 1 h incubation at 100 °C, the tubes were cooled and 5 ml toluene was added. The absorbance of the upper phase was spectrophotometrically determined at 528 nm and total proline amount was calculated with the help of standard curve and expressed as µg proline g-1 FW. For Chlorophyll a, chlorophyll b, total chlorophyll, carotenoids and anthocyanin measurement, the tissue samples (1.0 cm2 in most cases) were ground in 2 ml cold acetone/Tris buffer solution (80:20 vol:vol, pH = 7.8), centrifuged to remove particulates, and the supernatant was diluted to a final volume of 6 ml with additional acetone/Tris buffer. The absorbance of the extract solutions was measured with the spectrophotometer (Jenway 6300) using an external cuvette holder [25]. 2.5 Statistical analysis
The statistical analysis of variance (ANOVA) was performed on all of the experimental data reported in the present paper and statistical significance (P < 0.05) of means of at least two independent assays with three replicates was judged by the Duncan’s New Multiple Range Test using SPSS software 13.0. All data were expressed as means ± S.D.
3. RESULTS 3.1 Seed germination and seedling growth responses
There was a clear propensity that the growth of plant decreased with increasing dosage level of pyriproxyfen (Table 1). Statistical analysis showed that the germination and seedling growth of the seeds treated with pyriproxyfen was rather different from the control group (P
