Interaction effects of selected pesticides on soil enzymes.

The laboratory studies were conducted to resolute the effects of imidacloprid (insecticide) and triadimefon (fungicide) singly and in combination on enzymatic activities of soil microorganisms in tomato cultivated soils at different concentrations of 0.2, 0.5 and 0.7 kg/ha. The rate of amylase activity was stimulated by the application of pesticides at field rate. High dosage decreased the activity of amylase. Decline in the activity of cellulase was observed at all concentrations than control. Imidacloprid had an improved activity of cellulase at 0.5 μg/g than tridimefon and combination. At higher concentration (0.7 μg/g), the combination of insecticide and fungicide showed an antagonistic interaction toward cellulase. After 24 h, maximum inhibition was observed in invertase enzyme rate at all examined dosages. After 48 h, the activity was revived to some extent and imidacloprid showed enhanced activity at 0.5 μg/g (field rate). However at 0.7 μg/g, imidacloprid has a noticeable effect on the invertase. The pesticide application in single and in combination (0.2-0.7 μg/g soil) triggered the dehydrogenase activity. At field rate triadimefon significantly quickened the activity.

INTRODUCTION

Modern agriculture depends upon a wide variety of synthetically produced chemicals including insecticides, fungicides, herbicides and other pesticides.[1] When a synthetic pesticide is released into the environment, about 0.1% is reaching the target organism, while the remaining 0.99% of interferes local metabolism or enzymatic activities, and also affects human health by entering into the food chain, which has raised considerable public concern.[2] Soil is a living dynamic system containing many free enzymes, immobilized extracellular enzymes and enzymes within microbial cells. Soil enzymes are the soil quality indicators which play an important role in organic matter decomposition and nutrient cycling.[34] Thus, it is required to estimate soil biological responses to the pesticides in terms of soil enzyme activities.

Tomato (Lycopersicum esculentum) is a major vegetable crop in Madanapalle, Chittoor district of Rayalaseema region, Andhra Pradesh, India. It is grown in abundance in the district with an average of 35,000 acres producing 3-4 lakh million tons per annum and extensively used in fruit processing industries. Pesticides like imidacloprid and triadimefon are commonly used for pest control in tomato crop nowadays. Imidacloprid is a systemic nicotinic compound with a potent insecticidal activity against a wide range of pests of vegetable crops.[5] and triadimefon is a systemic triazole foliar fungicide with a good fungicidal activity (Extension Technology Network, Cornell university). Despite of their potent role in pest control, there is no information available on the interaction effects of imidacloprid and triadimefon in tomato cultivated fields of Madanapalle.

Cellulose, the most abundant organic compound in the biosphere comprising almost 50% of the biomass synthesized by photosynthetic fixation of CO2. Cellulases catalyse the degradation of cellulose and polysaccharide buildup of β-1, four linked glucose units.[6] Amylase hydrolyses α bond of polysaccharides or starch in soil yielding glucose and maltose.[7] Invertase is an enzyme that catalyses the hydrolysis of sucrose to fructose and glucose.[8] Dehydrogenases, as respiratory chain enzymes, play a major role in the energy production of organisms, which are responsible for the decomposition and conversion of organic matter.[9]

It is the farmer's practice in Madanapalle to use pesticides in combination to prevent the number of pests of tomato at the same time, which will enable sustainable management of pests. The rampant use of these pesticides exhibits a detrimental effect on non-target forms, which are beneficial to the agricultural fields. The present study will provide information on whether the selected pesticides are beneficial or harmful to the soil microbial activities. Inspite of the increased use of pesticides imidacloprid and triadimefon in the agricultural sector of Madanapalle, Chittor District of Andhra Pradesh, which occupied 19% of India's total tomato production, no research was performed in the direction of interaction of pesticides on soil microbial activities in this area. The present study has been aimed to determine the effects of pesticides on amylase, cellulase, invertase and dehydrogenase enzymes in tomato cultivated fields of Madanapalle.

MATERIALS AND METHODS

Soil

Deep red loamy soil from tomato cultivated field at Madanapalle which is a semi-arid zone of Andhra Pradesh, India, was collected to a depth of 12 cm from the four corner parts and the center of the field. The samples were pooled, sieved through the 2 mm mesh, and brought to the laboratory in polyethylene bags and kept in the refrigerator at 5-6°C to maintain the biological activity of the soil microbes. The same was done during the entire studies. The soil physico chemical properties were studied and represented in Table 1.

Table 1

Soil physico-chemical properties

Pesticides

Two pesticides, imidacloprid (insecticide) and triadimefon (fungicide) were selected for the present investigation in view of their usage in Madanapalle tomato cultivation for the control of insect pests and fungi. Imidacloprid and triadimefon were purchased from Saraswathi agrochemicals, Jammu and Kashmir and Bayer Crop sciences, Himatnagar, India. Imidacloprid: The chemical formula of imidacloprid is C9H10ClN5O2 and the international union of pure and applied chemistry (IUPAC) name is (E)-1-(6-chloro-3-pyridylmethyl)-N-nitroimidazolidin-2-ylideneamine. It is also known as nicotinamide and a common insecticide. The chemical formula of triadimefon (Triazole) is C14H16ClN3O2 and the IUPAC name of it is 1-(4-chlorophenoxy)-3, 3-dimethyl-1-(1H-1, 2, 4-triazol-1-yl) butan-2-one. It is a common fungicide.

Amylase activity

A total of 5 g soil samples in test tubes (15 mm × 150 mm) were incubated with selected pesticides singly and in combination. Duplicate soil samples were withdrawn after 10 days of incubation at room temperature (28 ± 4°C) to determine the amylase activity. The method employed for determining amylase activity is the method adapted by Tu.[10]

Assay of soil amylase

Soil samples were transferred to 100 ml erlenmeyer flasks and 1 ml of toluene was added. After 15 min, 6 ml of 0.2 M acetate-phosphate buffer (pH - 5.5) containing 2% of starch was added to the soil samples and the flasks were stoppered and held for 24 and 72 h at 30°C. Soil extracts were passed through Whatmann No. 1 filter paper and glucose content in the filtrate was assayed.

Invertase activity

The soil treatments are maintained at 60% of water holding capacity (WHC) and incubated at 28 ± 4°C. After 10 days, soil samples in duplicates were withdrawn for the assay of invertase activity. The method employed for determining invertase is developed by Cole.[11]

Assay of soil invertase

Soil samples were transferred to 100 ml erlenmeyer flasks and 1 ml of toluene was added. After 15 min, 6 ml of 0.2 M acetate phosphate buffer containing 18 Mm sucrose was added to the soil samples. The flasks were closed and held for 24 h and 48 h at 30°C. Soil extracts in sterile distilled water were passed through Whatmann No. 1 filter paper and glucose in the filtrate was assayed (Nelson, 1944). Suitable aliquots of filtrate were taken in test tubes and 1 ml alkaline copper reagent was added and covered with marbles, placed in boiling water bath for 20 min. The tubes were cooled under running tap water and then 1 ml of arsenomolybdate reagent was added. The final volume was made up to 5 ml with distilled water and bluish green color developed was read at 500 nm in a spectronic 20D spectrometer. The amount of glucose was calculated by referring to a calibration curve.

Cellulase activity

A total of 5 g portions of soil placed in 15 mm × 150 mm test tubes were amended with imidacloprid and triadimefon singly and in combination. All the treatments including untreated controls were maintained at 60% WHC and incubated in the laboratory at 28 ± 4°C. Moisture levels were restored to their initial levels during incubation. Duplicate samples of soils were withdrawn after 10 days of incubation for determining cellulase activity following the method of Pancholy and Rice.[12]

Assay of soil cellulase

The soil samples were transferred into 100 ml of erlenmeyer flasks and 0.5 ml of toluene was added. The contents in the flasks were mixed thoroughly and after 15 min 10 ml of acetone buffer at PH 5.9 was added followed by 10 ml of 1% of carboxy methyl cellulose. The flasks were then incubated for 24 h at 30°C. At the end of this period, 50 ml of distilled water was added. The suspension was filtered through Whatmann No. 1 filter paper and the volume of the contents was made up to 100 ml with distilled water. The reducing sugar content in the filtrate was determined by the method of Nelson-Somagi (1944).

Suitable aliquots of filtrate were taken in test tubes and 1 ml of alkaline copper reagent was added, covered with marbles and placed in boiling water bath for 20 min. The tubes were then cooled under running tap water and then 1 ml of arsenomolybdate reagent was added. The final volume in tubes was made up to 5 ml with distilled water and bluish green color was read at 500 nm in a spectronic 20D spectrophotometer. The amount of glucose was calculated by referring to a calibration curve.

Dehydrogenase activity

To determine the dehydrogenase activity, 5 g portions of soil were incubated in test tubes with selected pesticides. Soil samples were withdrawn after 10 days of incubation to determine the dehydrogenase activity by the method of Casida et al.[13] adapted by Adam and Duncan (2001).

Assay of dehydrogenase

Assay of dehydrogenase activity was based on the reduction of 2, 3, 5-tri phenyl tetrazolium chloride (TTC). Soil samples were treated with 0.1 g of CaCO3 and 1 ml of 0.18M aqueous TTC and incubated for 24 h at 30°C. The tri phenyl formazen formed was extracted in methanol from the reaction mixture and assayed at 485 nm in a spectronic 20D spectrophotometer.

Statistical analysis

Statistical analysis was performed by using Duncan's multiple range test.

RESULTS AND DISCUSSION

After 24 h of incubation with starch, the pesticide treatment individually and in combination at 0.5 μg/g (field rate) showed an increment in the amylase activity than that of control. The combination of pesticides at field rate augmented the rate of enzyme activity. The higher dosage drastically reduced the activity. After 72 h, there is continuous enhancement in the activity at field rate. However, the activity was restored to some extent at remaining application rates. The insecticide endosulfan and quinalphos are at normal residue to an elevated level (0-100) ppm which is equivalent to field application rates of 0-10 kg/ha was studied. Amylase enzyme activity was declined significantly after the application of pesticides at higher concentrations (7.5-10 kg/h), amylase activity showed individual increments of 53-171, 45-139 and 34-192, 69-183% of increase at 24 and 72 h of black paddy soil. The activity was decreased gradually on the prolonged period of incubation up to 30 and 40 days. Over all higher concentrations were toxic or innocuous to amylase activity.[14] This supported our observations. Triadimefon reduced the rate of enzyme activity at high dosage. The effect of two triazole compounds triadimefon and hexaconazole on carbohydrate metabolism was studied at the treatments 50 mg/L and 10 mg/L respectively. Both the triazoles resulted in a marginal increase in starch content and decreased the sugar contents. The α and β amylase activities were reduced under triadimefon and hexaconazole treatment.[15] Data pertaining to amylase activity was represented in Figures 1 and 2.

Figure 1

Effect of pesticides on amylase activity after 10 days of incubation. Bars marked by the same letter are not significantly different (P < 0.05) from each other according to Duncan's multiple range test

Figure 2

Effect of pesticides on amylase activity after 10 days of incubation. Bars marked by the same letter are not significantly different (P < 0.05) from each other according to Duncan's multiple range test

We observed that there is a suppressed activity in the cellulase enzyme with individual and combined treatments of pesticides at low field rate (0.2 μg/g), at field rate (0.5 μg/g) and high field rate (0.7 μg/g) compared to control. However, imidacloprid had an improved activity of cellulase enzyme. At high field rate, the combination of insecticides and fungicide showed an antagonistic interaction toward the activity, i.e., the percentage of glucose released from cellulose was only 43 compared with control. Contradictory to the present results, Mohiddin et al.[16] reported that the activity of cellulase in terms of glucose released from cellulose was more pronounced at 0.5 kg/ha soil, under the influence of two insecticides imidacloprid and acephate. However at higher concentrations of 7.5 and 10 kg/ha, both the insecticides were either stimulatory or innocuous to cellulase activity. Cellulase activity in soil treated with two fungicides, brominal and selecron was inhibited at field application rate and fivefold field rates after most incubation periods.[17] According to Moharram et al.,[18] Kocide and Rodomil Plus (systemic fungicides) were incorporated in the liquid culture medium specified for enzyme production; there was a significant reduction in cellulase production particularly at higher doses (200-400 ppm). Exceptions were observed with lower doses (50 and 100 ppm). The data pertaining to cellulase was represented in Figure 3.

Figure 3

Effect of pesticides on cellulase activity after 10 days of incubation. Bars marked by the same letter are not significantly different (P < 0.05) from each other according to Duncan's multiple range test

After 24 h of incubation with sucrose, we observed a maximum decrease in invertase activity in both individual and combined treatments pesticides at all dosages. Mixture of pesticides at a lower dose showed much decline in activity. The depressed activity might be due to the toxicity of the pesticide to soil microbes. At the prolonged incubation with sucrose (72 h), the activity was revived to extent than before. Imidacloprid showed an intensified activity of the invertase enzyme as that in control at 0.5 μg/g. But at higher dose, there was a marked effect of imidacloprid on the activity. Soils were treated with napropamide insecticide at 0, 2, 10, 20, 40 and 80 mg/kg soil and sampled at intervals of 1, 3, 7, 14, and 56 days. Application of napropamide at 2-80 mg/kg soil had an inhibitory effect on invertase activity. The depressed enzyme might be due to the toxicity of pesticides to soil microbes.[19] The activity of invertase in the rhizosphere of potato plants was determined under field conditions with the application of pyrethrins and neemix-4E. The effects were neither drastic nor prolonged enough to be considered deleterious to the invertase activity which is important to soil fertility.[20] These studies correlated with our observations. Data of invertase activity represented in Figures 4 and 5.

Figure 4

Effect of pesticides on invertase activity after 10 days of incubation. Bars marked by the same letter are not significantly different (P < 0.05) from each other according to Duncan's multiple range test

Figure 5

Effect of pesticides on invertase activity after 10 days of incubation. Bars marked by the same letter are not significantly different (P < 0.05) from each other according to Duncan's multiple range test

The insecticide (imidacloprid) and fungicide (triadimefon) applied to soil stimulated the dehydrogenase activity at all concentration (0.2 μg/g, 0.5 μg/g and 0.7 μg/g. Triadimefon showed its supremacy with a significant raise of dehydrogenase activity at field rate. In contrast to this, the insecticide diazinon dosage at three different dosages of 7, 35 and 700 mg/kg soil were studied in sandy soils on dehydrogenase activity. The activity decreased in the soil treated with the higher dosage of insecticide.[21] The impact of fungicides benomyl, kitazine, mancozeb and tridimorph on dehydrogenase activity was studied at 0.37 kg/ha, 0.98 L/ha, 2.0 kg/ha and 0.62 L/ha respectively. The enzyme activity was disrupted with the application. However, stimulation in dehydrogenase activity was observed with benomyl and tridimorph treated soils.[22] Data pertaining to dehydrogenase represented in Figure 6.

Figure 6

Effect of pesticides on dehydrogenase activity after 10 days. Bars marked by the same letter are not significantly different (P < 0.05) from each other according to Duncan's multiple range test

CONCLUSIONS

The effect of pesticides generally decreased with the increase in incubation period. Increase in the concentration of pesticides decreased the rate of enzyme activities. The rate of amylase was restored after 72 h. The higher concentration was toxic or innocuous to the amylase activity. Imidacloprid increased the cellulase activity much. However, the combination at high dosage was detrimental towards cellulase activity. There was a deleterious effect of pesticide on invertase enzyme earlier, but later the activity was restored. Imidacloprid had a great effect in the raise of invertase activity rate. Dehydrogenase activity was stimulated with individual and mixture of pesticide concentrations at all levels. Triadimefon ameliorated the dehydrogenase activity. It was concluded that the enzyme activities were not harmed at the recommended field rates. Future work is to clarify whether the changes in enzymatic activities is due to the effect of the pesticides rather than the heterogenecity of soil itself.