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Agrochemicals influencing nitrogenase, biomass of N2-fixing cyanobacteria and yield of rice in wetland cultivation
Biocatalysis and Agricultural Biotechnology 9 (2017) 28–34
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Agrochemicals influencing nitrogenase, biomass of N2-fixing cyanobacteria and yield of rice in wetland cultivation Nalinaxya Prasad Dasha, Ajay Kumarb, Manish Singh Kaushikb, Gerard Abrahamc, ⁎ Pawan Kumar Singhb, a b c
Directorate of Vocational Education, Shikhya Soudha Unit-V, Bhubaneshwar, India Centre for Advance Studies in Botany, Banaras Hindu University, Varanasi 221005, India Centre for Conservation and Utilization of Blue Green Algae, Indian Agricultural Research Institute, New Delhi 110012, India
A R T I C L E I N F O
A BS T RAC T
Keywords: Cyanobacteria Nitrogenase Biomass N-yield Agrochemicals Rice crop
Cyanobacteria maintain soil fertility by performing N2-fixation and act as a key biocatalyst in nitrogen cycle. Chemical N-fertilizers and pesticides as agrochemicals are intensively being used in rice farming to boost rice production, this work deals with the first hand information on their influence on native N2-fixing cyanobacteria, which play an important role in maintaining soil health. A field study was conducted for three consecutive seasons in water logged rice field to observe the influence of agrochemicals, urea, benthiocarb and carbofuran in isolation and in combinations on biomass, acetylene reduction activity (ARA) and N-yield of native cyanobacteria as well as, on growth and yield of rice. The ARA and N-yield followed almost same trend. It is discernible that both urea and benthiocarb had deleterious effects whereas, carbofuran was promoting effects on cyanobacterial growth, ARA and N-yield. The combination of all the three above agrochemicals was found inhibitory, but inhibition was comparatively less than that of urea or benthiocarb in isolation or urea plus benthiocarb treatments. It is concluded that the combination of agrochemicals was toxic, in comparison to the control, but was better than application of urea N or benthiocarb alone or with their combinations. It was recorded that along with rice straw and gain yields, panicle numbers were the maximum at the combination with treatments of benthiocarb+carbofuran. Adverse effects of used agrochemicals on cyanobacteria in wetland rice cultivation could be avoided by a prudent use of chemical N-fertilizers and pesticide(s) in combination.
1. Introduction Rice is widely cultivated in wetlands having high temperature, low soil organic carbon and high humidity. Such environmental conditions are favorable for cyanobacteria mainly due to their ability to fix atmospheric nitrogen and carbon. Cyanobacteria are considered as key biocatalysts in the N2 cycle (Vitousek et al., 2002; Latysheva et al., 2012). Cyanobacteria were reported as first agent to fix atmospheric nitrogen in flooded rice soils (Singh, 1961) and maintenance of natural soil fertility in such ecosystem was attributed mainly due to these organisms (De, 1939; Singh, 1961). Before introduction of chemical fertilizers, rice has been cultivated year after year without losing soil fertility which was considered mainly due to cyanobacteria and thus role of these organisms in N- economy of rice fields has been widely advocated (Singh, 1961; Venkataraman, 1972; Roger and Kulasooriya, 1980; Swarnalakshmi et al., 2006). With the introduction of high yielding rice varieties, agrochemicals are invariably being used to
⁎
obtain high yields. Thus, simultaneous or sequential application of agrochemicals such as herbicides, insecticides and nitrogenous fertilizers is indispensible in modern rice farming. But these chemicals affect other plants, animals and microorganisms which prevail in such habitat (Chen et al., 2007; Aktar et al., 2009; Casida, 2009; Geisseler and Scow, 2014). When two or more chemicals are applied together as a mixture or one after another during a cropping season, they interact and cause synergistic, additive or antagonistic responses (Tammes, 1964; Akobundu et al., 1975; Magnusson et al., 2010; Padhy and Rath, 2015). Therefore, information on the interactions of these chemicals or their degradation products with rice field ecosystem is very important. It is particularly relevant in case of cyanobacteria, which are the dominating flora of the tropical rice fields and are known to play important role in building soil organic carbon and nitrogen in the flooded rice ecosystem (Venkataraman, 1972; Singh, 1978; Singh and Bisyoi, 1989; Whitton, 2000; Nayak et al., 2004; Swarnalakshmi et al., 2006). Effects of pesticides on cyanobacteria in laboratory cultures
Corresponding author. E-mail address: [email protected] (P.K. Singh).
http://dx.doi.org/10.1016/j.bcab.2016.11.001 Received 7 September 2016; Received in revised form 22 October 2016; Accepted 2 November 2016 Available online 08 November 2016 1878-8181/ © 2016 Published by Elsevier Ltd.
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and graminaceous weeds was applied in one dose at the time of puddling whereas insecticide carbofuran (1 kg a.i. ha−1) a systematic and contact poison acts as miticide was applied in two equal splits at 25 and 45 DAT. The P fertilizer (20 kg P ha−1) was applied in 3 equal split doses at a weekly interval after transplantation (Bisyoi and Singh, 1988a).
were reported earlier (Singh, 1973; Das and Singh, 1978; Tiwari et al., 2001; Chen et al., 2007; Padhy and Rath, 2015). Recently, Das et al. (2015) reported from pot experiment conducted with planted rice that herbicide (butachlor) application adversely affected native and inoculated cyanobacteria whereas insecticide (metacid) application was favorable to them. The information on the interaction of different agrochemicals with cyanobacteria in paddy fields is inadequate. Therefore, in present investigation, an experiment was performed to analyse the impact of commonly used agrochemicals (i.e. herbicide, insecticide and Nfertilizer) in isolation and in combination for three consecutive seasons on native cyanobacterial biomass, nitrogenase activity in terms of acetylene reduction assay (ARA), cyanobacterial N-yield as well as growth and yield of rice.
2.5. Growth of native cyanobacteria in experimental field The rice fields of CRRI farm harbour N2-fixing cyanobacteria abundantly and the dominating species mostly belonged to genera Aulosira, Aphanothece, Gloeotrichia, Anabaena and Nostoc. To promote their growth superphosphate at a rate of 20 kg P ha−1 was applied in three equal splits at 7 days interval (Bisyoi and Singh, 1988a).
2. Materials and methods 2.6. Collection and processing of cyanobacteria samples and measurement of their growth
2.1. Experimental design and treatments A field experiment was carried out for three consecutive seasons (two dry seasons and one wet season) with native cyanobacteria in plots of 5 m×2 m size using urea (CH4N2O), Benthiocarb (trade name Saturn) (S-(4-Chlorobenzyl) N, Ndiethylthiolcarbamate; C12H16CINOS) and carbofuran (trade name furadan) (2,2-dimethyl3H-1-benzofuran-7-yl) N-methylcarbamate; C12H15NO3) as N fertilizer, herbicide and insecticide, respectively. The experiment had a completely randomized block design with three replications and had the following treatments: T1 control (no urea, benthiocarb, carbofuran); T2-Urea (60 kg N ha−1); T3-benthiocarb (1 kg active ingredient (a.i.) ha−1); T4-carbofuran (1 kg a.i. ha−1); T5-T2+T3; T6-T2+T4; T7T3+T4; T8-T2+T3+T4. The treatments T5, T6, T7 and T8 had the same doses as used in T2, T3 and T4. The soil of experimental plots was classified as coastal alluvium sandy clay loam (Aeric Endoaquept) (Soil Survey Staff, 2010) with 0.68% organic C, 0.07% total N (C: N ratio 9.7), 11 µg g−1 available P (Olson), 59.9% P-fixing capacity and pH 6.4. A high yielding rice variety (Oryza sativa L.) IR-36 of 120 days duration was used in the experiment. Other experimental details and methodologies adopted in the experiment are given below.
Ten cyanobacterial samples were randomly collected from each plot with a metallic quadrate (25 cm×25 cm×25 cm) having both ends open (Singh, 1961). The cyanobacterial biomass inside the quadrate was collected by passing the flood water through two layers of cheese cloth. The samples were then washed several times with water to remove the contaminating materials and blotted with the help of blotting paper to remove excess water and utmost care was taken to remove green algae and other contaminants. However, in such field studies, total contaminants cannot be removed and dominating biomass has to be taken into consideration for further study. The fresh weight of the cyanobacterial biomass was recorded immediately after collection and 20 g fresh material was dried in an oven at 80 °C for 24 h for determination of dry weight. From this, the total dry weight was computed and the dry weight of the sample was expressed in kg ha−1 (Bisyoi and Singh, 1988a, 1988b).
2.7. Measurement of nitrogenase activity (acetylene reduction activity; ARA)
2.2. Season and weather conditions
The nitrogenase activity (ARA) was measured following the method of Reddy and Roger (1988). Ten soil-water core samples were randomly collected within each plot with the help of glass tubes of 1.8 cm diameter and 10 cm length. The flood water along with top 0.5 cm of the soil core suspension was adjusted with the distilled water to make 254 mL, a volume that equals to 10 times (in cm2) of the soil surface cored by 10 samples, giving a dilution of 10−1 on the surface basis. The suspension was then stirred at 400 rpm for 30 min to disrupt the cyanobacterial clumps and serially diluted to 10−3 dilution. 5 mL of the diluted sample was incubated in the field with 10% acetylene (v/v) for 1 h. After incubation, 0.5 mL of the gas mixture was injected into the gas chromatograph (AIMIL-5500 series, Nucon Engineers, New Delhi). The amount of ethylene produced was expressed in nmole C2H4 cm−2 h x 10−3 (Schollhorn and Burris, 1966; Stewart et al., 1968).
The experiment was carried out at the farm of Central Rice Research Institute, Cuttack, India. It is situated on the bank of river Mahanadi in Orissa state (Eastern India) at latitude of 25.5°N, longitude of 86°E, altitude of 23.48 m above the mean sea level and is 80 km in the west from the Bay of Bengal in the main rice growing tract of India. During the period of experimentation, average annual rainfall was 1553.2 mm. Mean maximum temperature was 35.3–36.5 °C during May and June, whereas the mean minimum temperature was 13.6–15.1 °C during January and December and relative humidity was 86–97% during July-September months. 2.3. Field preparation and transplanting The fields were ploughed, cross ploughed, puddled, levelled and thick bounds were raised to avoid mixing of treatments. The healthy and sprouted rice seeds were sown in seed beds and 25–30 days old healthy seedlings were transplanted at the rate of 2 seedlings hill−1 with a spacing of 15×10 cm.
2.8. Measurement of cyanobacterial N-yield The total N content (%) of cyanobacteria was estimated by the micro-kjeldahl method (Jackson, 1973). 100 mg of dried cyanobacterial samples were taken in duplicate in 100 mL kjeldahl flasks to which a pinch of digestion mixture (K2SO4, CuSO4 and SiO2 in a 100:10:1 ratio) and 2 mL of concentrated H2SO4 (36N) were added. The flasks were heated in the digestion chamber for 2–3 h and the digested samples were used for determination of N content. The N-yield was calculated by multiplying the N content with the dry weight and expressed in kg N ha−1.
2.4. Biocides applications The half of the N fertilizer (urea at the rate of 60 kg N ha−1) was applied as a basal dose during puddling and a quarter each at 25 and 45 days after transplanting (DAT). The pre-emergence herbicide benthiocarb (1 kg a.i. ha−1) having intergenic selectivity between rice plants 29
Biocatalysis and Agricultural Biotechnology 9 (2017) 28–34
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2.9. Rice post harvest observation 2.9.1. Panicle number, grain and straw yield The rice plants in 1 m2 area were harvested at their maturity and the numbers of panicles were counted. The rice crop from an area of 7.57 m2 from each plot was harvested, threshed and the grain weight was recorded. The moisture content of grains was measured in an OSAW Universal moisture meter and the grain yield was expressed at 14% moisture. The straw samples were completely sun dried before weighing. 2.10. Statistical analysis The data were analyzed statistically with one-way analysis of variance using software SPSS.16 advance version, where means for each treatment at different days after transplanting (DAT) were compared with control using Tukey HSD post hoc analysis. 3. Results
Fig. 1. Influence of chemical-N fertilizer and pesticides on growth (kg dry weight ha−1) of rice field's native cyanobacteria. Data are the presented as mean ± SD (n=3) of growth measured at different rice crop stages from both dry and wet season. Multivariate ANOVA was performed for repeated measures of treatments and their interactions with different parameters. Level of significance: *p < 0.05; **p < 0.01; ***p < 0.001.
3.1. Growth of cyanobacteria as influenced by agrochemicals in rice field The growth of the native cyanobacteria in terms of dry weight (kg ha−1) was found maximum at 60 days after transplanting (DAT) and minimum at 30 DAT. The cyanobacterial growth in wet season (WS) was comparatively lower than that in the dry season (DS) (Table 1; Fig. 1). Urea applied at rate of 60 kg N ha−1 was observed to be highly inhibitory for growth of cyanobacteria. It reduced their growth at 60 DAT by 55% and 62% over the control. The herbicide benthiocarb applied at rate of 1.0 kg a.i. ha−1 decreased the growth at 30, 60, 90 DAT by 16%, 8%, and 19% respectively during DS and 22%, 11% and 23% respectively at 30, 60, 90 DAT in WS. The insecticide carbofuran applied at rate of 1.0 kg a.i. ha−1 was observed to be growth stimulatory on all sampling dates and in both the seasons. The significant stimulatory effect of this insecticide in terms of percentage increase in growth over control was from 16% to 30% in both seasons. Urea and benthiocarb interaction was significant during both dry and wet seasons. A combination of urea and benthiocarb showed lower growth as compared to treatment without urea and benthiocarb (Table 1) and decrease in growth over control at 60 DAT was 38% and 42% during dry and wet season respectively. Their combination had produced significantly lower cyanobacterial biomass in comparison to treatment receiving only herbicide whereas it was statistically similar with treatment receiving only nitrogen. The interaction between the urea and carbofuran had significant effect on the cyanobacterial growth during WS and DS. The decrease in cyanobacterial growth due to use of
urea plus carbofuran was 12–22% over the control during wet and dry season respectively. Thus, combination of these two chemicals showed less cyanobacterial growth than the treatment receiving only carbofuran whereas it was superior to treatment receiving only urea. The combined application of benthiocarb plus carbofuran significantly increased growth of cyanobacteria in both the seasons. Cyanobacterial growth was significantly higher in treatment receiving only herbicide and lower in the treatment receiving only insecticide. All the three factors (urea+benthiocarb+carbofuran) significantly decreased the growth at all the three 30, 60 and 90 DAT in both the seasons. The combination of these three factors recorded significantly higher cyanobacterial growth than the treatments receiving urea and urea plus benthiocarb whereas it recorded lower growth than treatments receiving carbofuran, benthiocarb, benthiocarb plus carbofuran and urea and carbofuran. 3.2. Nitrogenase activity (ARA) of cyanobacteria in rice field as influenced by agrochemicals Nitrogenase activity (ARA) in wet season was comparatively lower than the dry season and it almost followed trend of the growth (Table 2; Fig. 2). The ARA was found lowest in all the treatments and seasons at 30 DAT and was highest at 60 DAT. The minimum and maximum ARA
Table 1 Influence of chemical-N fertilizer and pesticides on growth (kg dry weight ha−1) of rice field's native cyanobacteria. Data presented as mean ± SD (n=3). Treatments
Control( no N-fertilizer, herbicide and insecticide) Urea-N Benthiocarb Urea+ Benthiocarb Carbofuran Benthiocarb+ Carbofuran Urea N+ Carbofuran Urea-N+ Carbofuran +Benthiocarb
Dry season (kg dry wt ha−1)
Wet season (kg dry wt ha−1)
30 DAT
60 DAT
90 DAT
30 DAT
60 DAT
90 DAT
94.4 ± 0.21
209.4 ± 0.89
98 ± 0.37
67.1 ± 0.11
181.1 ± 0.21
76.11 ± 0.31
43.2 ± 0.20 79 ± 0.40 61 ± 0.91 115 ± 0.3 103 ± 0.65 73.3 ± 0.21 67 ± 0.11
(-54%) (−16%) (−35%) (+22%) (+9%) (−22%) (−29%)
93.3 ± 0.13 193 ± 0.45 130 ± 0.63 244 ± 0.30 217.5 ± 0.17 163 ± 0.35 149 ± 0.59
(−55%) (−8%) (−38%) (+16%) (+4%) (−22%) (−29%)
49 ± 0.41 79 ± 0.37 62 ± 0.72 124 ± 0.50 102 ± 0.37 79.3 ± 0.79 73 ± 0.97
(−50%) (−19%) (−37%) (+26.5%) (+4%) (−19%) (−25.5%)
27.4 ± 0.30 52.35 ± 0.29 43 ± 0.16 79.5 ± 0.31 83.41 ± 0.25 53.2 ± 0.42 47.2 ± 0.23
(−59%) (−22%) (−36%) (+18%) (+24%) (−20%) (−30%)
67.1 ± 0.19 161.4 ± 0.31 103.3 ± 0.30 215.2 ± 0.98 202 ± 0.63 169 ± 1.01 128.6 ± 0.41
DAT-Days after transplanting N-Fertilizer- Urea (60 kg N ha−1), Herbicide- benthiocarb (1.0 kg a.i. ha−1), Insecticide- carbofuran (1.0 kg a.i. ha−1) The data given in bracket “()” showed the percentage (%) increase or decrease in growth after treatments with respect to control in each column.
30
(−62%) (−11%) (−42%) (+19%) (+11%) (−12%) (−30%)
36 ± 0.45 58.11 ± 0.21 46.14 ± 0.20 99.2 ± 0.28 84.45 ± 0.81 59.34 ± 0.20 55.11 ± 0.22
(−53%) (−23%) (−39%) (+30%) (+10%) (−22%) (−27%)
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Table 2 Influence of chemical-N fertilizer and pesticides on acetylene reduction activity or ARA (n moles C2H4 cm−2 h−1 ×10−3) of rice field's native cyanobacteria. Data presented as mean ± SD (n=3). Dry season (n moles C2H4 cm−2 h−1 ×10−3)
Wet Season (n moles C2H4 cm−2 h−1 ×10−3)
Treatments
30 DAT
60 DAT
90 DAT
30 DAT
60 DAT
80 DAT
Control ( no Nfertilizer, herbicide and insecticide) Urea-N Benthiocarb Urea + Benthiocarb Carbofuran Benthiocarb+ Carbofuran Urea N + Carbofuran Urea-N + Carbofuran +Benthiocarb
147 ± 0.10
355 ± 0.90
251 ± 0.45
119.50 ± 0.50
291.30 ± 0.15
05.70 ± 0.30
353 ± 0.15 120 ± 0.67 48 ± 0.20 215 ± 0.79 161 ± 0.50
(−76%) (−18%) (−67%) (+46%) (+9.7%)
90 ± 0.71 307.5 ± 0.25 121 ± 0.9 552.5 ± 1.21 395 ± 0.67
(−75%) (−13%) (−66%) (−55%) (+11%)
72.5 ± 0.3 217 ± 0.13 93 ± 0.43 377 ± 0.39 289 ± 0.45
(−71%) (−13%) (−63%) (+50%) (+15%)
25.70 ± 0.09 87.40 ± 0.69 32.10 ± 0.17 160.70 ± 0.60 128.50 ± 0.70
(−79%) (−27%) (−73%) (+34%) (+7%)
66.60 ± 0.19 224.90 ± 0.51 79.50 ± 0.71 415.30 ± 0.22 321.40 ± 0.40
(−77%) (−23%) (−73%) (+43%) (+10%)
52.30 ± 0.45 161.40 ± 0.28 59.60 ± 0.30 295 ± 0.20 231.40 ± 0.15
(−74%) (−22%) (−71%) (+4%) (+12%)
58.45 ± 0 0.21 54 ± 0.67
(−60%) (−63%)
146.5 ± 0.49 132 ± 0.88
(−59%) (−63%)
120 ± 0.53 106 ± 0.35
(−52%) (−58%)
42.40 ± 0.69 45.00 ± 0.49
(−64.5%) (−62.3%)
102.60 ± 0.38 95.10 ± 0.71
(−65%) (−67%)
81.40 ± 0.89 79.60 ± 0.42
(−61%) (−61%)
DAT-Days after transplanting N-Fertilizer- Urea (60 kg N ha−1), Herbicide- benthiocarb (1.0 kg a.i. ha−1), Insecticide- carbofuran (1.0 kg a.i. ha−1) The data given in bracket “()” showed the percentage (%) increase or decrease in growth after treatments with respect to control in each column.
reduced activity significantly by 13–27% over control, although this herbicide was observed to be less deleterious as compared to urea. The insecticide carbofuran was found to be stimulatory to ARA and it significantly enhanced activity by 43–55% over control. As both urea and benthiocarb were inhibitory to cyanobacterial ARA, their combined application showed a drastic reduction in ARA. Application of the urea and benthiocarb together recorded significantly lower ARA than herbicide treatment but it was similar with nitrogen treatment. When the carbofuran was applied along with urea, inhibitory effect of latter was lowered and the ARA values were significantly higher than that of urea treatment. Application of benthiocarb and carbofuran in combination significantly increased ARA over the control without benthiocarb and carbofuran during both the season. The percentage of increase was in between 7–15% during both the seasons. The combined use of benthiocarb and carbofuran showed significantly lower ARA than application of the carbofuran but recorded higher ARA than application of benthiocarb suggesting reduction of inhibitory effect of benthiocarb when it was applied along with carbofuran. Three factors (urea, benthiocarb and carbofuran) interaction had significant effect on ARA in both the seasons.
Fig. 2. Influence of chemical-N fertilizer and pesticides on ARA (n moles C2H4 cm−2 h×10−3) of rice field's native cyanobacteria. Data are the presented as mean ± SD (n=3) of ARA measured at different rice crop stages from both dry and wet season. Multivariate ANOVA was performed for repeated measures of treatments and their interactions with different parameters. Level of significance: *p < 0.05; **p < 0.01; ***p < 0.001.
3.3. Agrochemicals affecting nitrogen accumulation by cyanobacteria The nitrogen accumulation (N-yield) by cyanobacteria was measured at 30, 60 and 90 days after transplanting (DAT) in both dry and wet seasons and N-yield (kg N ha−1) was found to be minimum at 30 DAT and maximum at 60 DAT irrespective of treatments and seasons (Table 3; Fig. 3). The N-yield of different treatments showed a similar trend to that of ARA. Urea (60 kg N ha−1) significantly decreased N-
was 25.7 and 552.9 n moles C2H4 cm2 h−1 ×10−3. Urea (60 kg N ha−1) was found to be highly inhibitory to ARA of the cyanobacteria and the extent of reduction over the control during different seasons was more than 65%. The herbicide benthiocarb (1 kg a.i. ha−1) showed a deleterious effect on cyanobacterial ARA, and
Table 3 Influence of chemical-N fertilizer and pesticides on N-yield (kg N ha−1) of rice field's native cyanobacteria. Data presented as mean ± SD (n=3). Dry season (kg N ha−1) Treatments Control( no N-fertilizer, herbicide and insecticide) Urea-N Benthiocarb Urea + Benthiocarb Carbofuran Benthiocarb+ Carbofuran Urea + Carbofuran
30 DAT 2.9 ± 0.30 1.43 ± 0.03 2.38 ± 0.20 2 ± 0.10 3.89 ± 0.09 3.44 ± 0.04 2.34 ± 0.34
Wet season (kg N ha−1) 60 DAT 6.6 ± 0.10
(−50%) (−17%) (−31%) (+34%) (+19%) (−19%)
2.97 ± 0.02 6.2 ± 0.05 4.28 ± 0.28 7.66 ± 0.33 7.09 ± 0.09 5.52 ± 0.12
90 DAT 3.5 ± 0.25 (−55%) (−6%) (−35%) (+16%) (+7%) (−16%)
1.7 ± 0.30 2.98 ± 0.69 2.2 ± 0.11 4.33 ± 0.22 3.74 ± 0.14 2.75 ± 0.25
30 DAT 0.74 ± 0.02 (−50%) (−15%) (−37%) (+28%) (+7%) (−21%)
0.49 ± 0.09 0.81 ± 0.29 0.52 ± 0.30 1.48 ± 0.47 1.01 ± 0.09 0.76 ± 0.03
60 DAT 2.14 ± 0.14 (−33%) (+9%) (−30%) (+100%) (+36%) (+3%)
1.16 ± 0.13 2.02 ± 0.02 1.81 ± 0.29 3.65 ± 0.13 2.68 ± 0.27 2.45 ± 0.15
DAT-Days after transplanting N-Fertilizer- Urea (60 kg N ha−1), Herbicide- benthiocarb (1.0 kg a.i. ha−1), Insecticide- carbofuran (1.0 kg a.i. ha−1). The data given in bracket “()” showed the percentage (%) increase or decrease in growth after treatments with respect to control in each column.
31
90 DAT 1.20 ± 0.10 (−46%) (−7%) (−15%) (+70%) (+25%) (+14%)
0.66 ± 0.33 1.04 ± 0.11 1.05 ± 0.17 1.71 ± 0.04 1.46 ± 0.70 0.98 ± 0.39
(−45%) (−13%) (−13%) (+42.5%) (+22%) (−18%)
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+carbofuran whereas minimum increase of 3% was observed with benthiocarb. The combined treatments showed maximum yields in comparison to their separate applications except urea. Separate application of urea, benthiocarb and carbofuran enhanced the yields by 9%, 3% and 6% respectively whereas treatments of urea+benthiocarb, urea+carbofuran, benthiocarbcarbofuran, urea+benthiocarb+carbofuran enhanced yield by 7%, 13%, 7% and 10% respectively. It is revealed from the data that all the treatments enhanced the straw yields in comparison to the control except herbicide. Application of urea was also found promotary to straw yield in combinations or separate applications (Table 4). 3.4.3. Panicle number The number of panicles m2 also followed almost same trend as that of grain and straw yields. Urea significantly increased the number of panicles m2 by 9% over the control. The benthiocarb did not have a significant effect on the number of panicles, whereas carbofuran application enhanced the number of panicles by 8%. The treatments urea+benthiocarb, urea+carbofuran, benthiocarb+carbofuran, urea +benthiocarb+carbofuran agrochemicals enhanced panicles by 7%, 9%, 5.5% and 7% respectively in comparison to control (Table 4).
Fig. 3. Influence of chemical-N fertilizer and pesticides on N- yield (kg N ha−1) of rice field's native cyanobacteria. Data are the presented as mean ± SD (n=3) of N-yield measured at different rice crop stages from both dry and wet season. Multivariate ANOVA was performed for repeated measures of treatments and their interactions with different parameters. Level of significance: *p < 0.05; **p < 0.01; ***p < 0.001.
4. Discussion
yield of rice field's native cyanobacteria during both the seasons. The percentage of reduction over control without urea at 60 DAT was 55% and 46% during dry and wet season respectively. The carbofuran (1 kg a.i. ha−1) significantly enhanced N-yield over untreated control. The interaction of urea+benthiocarb was observed to have a significant effect on cyanobacterial N-yield. The combined application of urea and benthiocarb decreased N-yield by 31%, 35% and 37% at 30, 60 and 90 DAT over the control in dry season whereas 30%, 15% and 13% at 30, 60, 90 DAT in wet season respectively. This combination recorded significantly lower N-yield than the benthiocarb treatment but it was superior to the application of urea alone, during both dry and wet seasons. The interaction of the urea+carbofuran was significant at 30 DAT during dry season and at all the three DAT during wet season. The retarding effect of the urea fertilizer on N-yield was lowered when it was applied along with insecticide. The urea+carbofuran combination showed significantly increased N-yield over urea treatment but recorded lower N-yield than the control. A combination of benthiocarb +carbofuran increased the N-yield at 30, 60 and 90 DAT by 10%, 7% and 7% over the control during dry season respectively. The N-yield in this treatment was significantly lower than that in the carbofuran treatment but higher than that of benthiocarb treatment.
Among different food crops, rice ranked second to wheat in terms of area harvested, but in terms of importance as a food crop rice provides more calories than any other cereal crop (De Datta, 1981). The sustainability of agriculture and ecosystems is considered crucial and therefore increasing demand of improving yields using extensive agrochemicals and other modern technologies should also ensure protection of environmental integrity and biodiversity conservation. Since agrochemicals are extensively being used in rice cultivation (Das et al., 2015), it is necessary to study impact of these agrochemicals on non-target organism like cyanobacteria, which is a key biocatalyst of nitrogen cycle, increase soil fertility and rice yield by fixing nitrogen. Cyanobacteria are known to thrive well in tropical flooded rice fields and therefore, due care needs to be taken while using the agrochemicals in rice cultivation for protecting these beneficial organisms in rice ecosystem and enabling to sustain soil fertility on long term basis. Advantages to cyanobacteria in this environment are due to two major metabolic activities i.e. N2-fixation and photosynthesis, due to which they grow well under poor nitrogen and low organic-carbon containing soils provided other environmental and soil conditions are favorable. Roger (1996) described various components of flooded water rice ecosystem, their properties, and activities highlighting importance of photosynthetic aquatic biomass and effects on N dynamics in this system. In the current investigation, in depth study for three consecutive seasons was undertaken using same high yielding rice variety in a traditional rice growing area of India to find out effects of nitrogenous fertilizer (urea), herbicide (benthiocarb) and insecticide (carbofuran) commonly used in rice cultivation as per rice cultivation practices at different stages of crop. During the course of investigation, very interesting observations were recorded. Most of the earlier studies either conducted in laboratories using cyanobacterial cultures or randomly samples taken from rice fields using single input parameter. The deleterious effects of combined N sources on cyanobacteria have been reported (Singh, 1978, 1985; Watanabe and Cholitkul, 1979; Roger and Kulasooriya, 1980; Singh and Bisyoi, 1989; Swarnalakshmi et al., 2006). Among different nitrogenous fertilizers, urea is widely used in rice cultivation. The cyanobacteria occurring in the flooded rice ecosystem takes up urea in cells and degrade into ammonium using enzyme urease. The ammonium is taken up by cells as N source. However, urea is found to be inhibitory to cyanobacteria in this study which might be due to its indirect effects (Roger, 1996). Urea is reported to reduce soil pH (Geisseler and Scow, 2014) whereas cyanobacteria prefer to grow well in neutral to alkaline pH. Thus,
3.4. Crop parameters response as affected by agrochemicals 3.4.1. Grain yield The impact of application of chemical-N fertilizer on the grain yield was found significantly maximum in case of urea+carbofuran (35%), whereas minimum in case of herbicide benthiocarb (4%). The yield was found maximum where urea+benthiocarb+carbofuran were applied in combinations in comparison to singly applications. Applications of urea, benthiocarb and carbofuran separately enhanced the yield by 20%, 4% and 11% respectively whereas in case of treatments urea +benthiocarb, urea+carbofuran, benthiocarb+carbofuran, urea +benthiocarb+carbofuran enhanced grain yield by 17%, 35%, 13% and 27% respectively. The results stated that all treatments enhance the grain yield significantly except benthiocarb. Application of urea applied alone or in different combinations was found to enhance grain yield (Table 4). 3.4.2. Straw yield The straw yield followed almost same trend as that of grain yield. Maximum increase in yield (13%) was found in the treatment of urea 32
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Table 4 Crop parameters (grain yield, straw yield and panicles number) of rice variety IR-36 as influenced by chemical-N fertilizer and pesticides. Data presented as mean ± SD (n=3). Treatments
Grain yield (t ha−1)
Control ( no N-fertilizer, herbicide and insecticide) Urea-N Benthiocarb Urea + Benthiocarb Carbofuran Benthiocarb+Carbofuran Urea+Carbofuran Urea+Carbofuran+Benthiocarb
2.78 ± 0.03 3.34 ± 0.30 2.90 ± 0.45 3.27 ± 0.09 3.10 ± 0.05 3.16 ± 0.16 3.76 ± 0.30 3.53 ± 0.25
Straw yield (t ha−1)
(+20%) (+4%) (+17%) (+11%) (+13%) (+35%) (+27%)
2.39 ± 0.09 2.62 ± 0.02 2.47 ± 0.13 2.56 ± 0.14 2.53 ± 0.53 2.56 ± 0.41 2.71 ± 0.89 2.63 ± 0.71
Panicle numbers (m−2)
(+9%) (+3%) (+7%) (+6%) (+7%) (+13%) (+10%)
327 ± 0.90 358 ± 0.40 334 ± 0.67 351 ± 0.21 353 ± 0.75 345 ± 0.89 358 ± 1.15 350 ± 0.99
(+9%) (+2%) (+7%) (+8%) (+5.5%) (+9%) (+7%)
N-Fertilizer- Urea (60 kg N ha−1), Herbicide- benthiocarb (1.0 kg a.i. ha−1), Insecticide- carbofuran (1.0 kg a.i. ha−1). The data given in bracket “()” showed the percentage (%) increase or decrease in growth after treatments with respect to control in each column.
The cyanobacterial N-yield also followed similar trend as that of growth and N2-fixation. The biological nitrogen fixation by photodependent cyanobacteria in the photic zone is favoured in flooded soilrice ecosystem. The N balance study conducted in the presence and absence of light, rice and flooding reported significant N gain due to photo-dependent BNF (Singh and Singh, 1987). A visible growth of native cyanobacteria generally corresponds to less than 10 kg N ha−1 (Roger, 1996). In the present study N-yield is observed to be less than 10 kg N ha−1 in most of the treatments. Although under flooded rice field conditions, N-yield determined from biomass measurement is likely to be underestimated because turnover of cyanobacterial biomass is not taken into account (Roger, 1996). The total N estimated and effects of agrochemicals in different treatments followed the observations which were discussed earlier on growth of cyanobacteria. The combinations of urea plus benthiocarb, benthiocarb plus carbofuran, carbofuran plus urea and urea plus benthiocarb plus carbofuran along with rice in field trials have provided first hand informations. Informations on such systematic study in rice ecosystem with reference to cyanobacteria were not available earlier. Antagonistic effect of the benthiocarb and carbofuran on the growth, N2 -fixation and N-yield of cyanobacteria was observed. A similar antagonistic relationship between benthiocarb and carbofuran was also reported in cotton (Arle, 1968). Effect of three factors interaction (urea, herbicide and insecticide) was mostly regulated by the urea-N, probably because the cyanobacteria are more susceptible to combined-N whether applied alone or in combination with the pesticides. Regarding rice crop response, the studies showed that application of urea alone or in combination with other agrochemicals improved the grain and straw yields as reported earlier (De Datta, 1981). Besides, benthiocarb and carbofuran when applied together also increased the grain yield significantly. One of the notable points in this investigation was that urea plus carbofuran treatment produced higher grain yield than the urea plus benthiocarb treatment which implies that the total increase in rice yield could not be attributed to urea alone. Since urea plus carbofuran treatment also recorded a higher cyanobacterial growth than the urea plus benthiocarb treatment, the increased rice yield in former treatment could also be partly due to cyanobacterial response. The control of grazers (Raghu and Mac Rae, 1967) by use of the carbofuran might have led to production of higher biomass and accumulation of more nitrogen in cyanobacteria which ultimately increased the yield of rice. The lower grain yield in the urea plus benthiocarb treatment might be due to retarding effect of benthiocarb on the growth and N2-fixation of rice field's cyanobacteria (Singh and Singh, 1983). This result may also be applicable to other paddy ecosystems for cyanobacteria and other microorganisms.
decline in pH might have affected the growth of these organisms in rice ecosystem. To overcome the adverse effects of surface applied urea, attempts were made in past to use slow release N fertilizers, deep placement of urea super granules and methods of N fertilizers applications in rice ecosystem on free living and symbiotic cyanobacteria. It was inferred from these studies that inhibitory effects of surface applied urea could be reduced by deep placing or incorporating it into the soil (Manna and Singh, 1988c, 1990; Roger, 1996). The inhibition of cyanobacterial growth by herbicide benthiocarb in this study is supported by earlier observations based mostly from laboratory studies (Zarger and Dar, 1990; Singh, 1974; Das and Singh, 1976; Zaitseva, 1979; Higazy and Fayez, 1989). It was interesting to note that pesticide carbofuran which used to be considered as a potential insecticide for insect control in rice fields, promotes growth of native cyanobacteria. Kar and Singh (1978) reported similar growth promoting effects of carbofuran in a laboratory study on cyanobacteria. Also they studied effects of nutritional factors, pH, light intensity on carbofuran and cyanobacteria interaction (Kar and Singh, 1978, 1979). Cyanobacteria are likely to metabolise or biodegrade carbofuran and metabolic products could be used as growth and N2-fixation promotary as reported by Kar and Singh (1978). Besides pests of cyanobacteria are likely to be affected adversely in the fields favouring cyanobacteria. The combination of carbofuran and benthiocarb promoted above activities but was less than carbofuran alone. Therefore, positive response in the combined treatment is likely to be due to carbofuran. The carbofuran is also reported as mutagenic in our earlier study (Kar and Singh, 1979). Das et al. (2015) reported interaction of pesticides and cyanobacteria in pot studies where herbicide butachlor decreased their growth but insecticide metacid application was found to be growth promotary. The N2- fixation activity of microorganisms in general is reported to be inhibited by chemical N sources, where free living cyanobacteria seems to be more susceptible to inhibition than heterotrophs. The inhibitory effect of surface urea application is confirmed by acetylene reduction activity on photo-dependent biological N2-fixation (BNF). Although it was not the complete inhibition but decrease during the growth cycle was because of N uptake by plants and also due to various losses. Based on extrapolated data from nitrogenase activity (ARA) measurement, the visible growth of cyanobacteria may contribute up to 30 kg N ha−1 crop−1 in control plots without addition of N fertilizer. But the growth reduced to one-fourth with broadcast of urea and almost half when it was deep placed (Roger, 1996). Cyanobacterial nitrogenase activity is expressed in the absence of combined N source. The ammonium negatively affects nitrogenase synthesis besides its effects on heterocyst differentiation (Flores and Herrero, 1994). Therefore, ammonium produced in cells after uptake of urea likely to inhibit ARA in this study. Thus, nitrogenase activity (ARA) might be getting affected directly and also due to indirect effect on growth of cyanobacteria. The pesticides have initial depressive effect on N2-fixing activity followed by increase or decrease in activity. The insecticides generally have less effect except some on N2-fixing activity of cyanobacteria (Roger and Kulasooriya, 1980).
5. Conclusion The cyanobacteria are known to play an important role in maintaining soil fertility in flooded rice ecosystem. With the demand of intensive rice cultivation to obtain high yields, high doses of chemical 33
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Kar, S., Singh, P.K., 1978. Toxicity of carbofuran to blue-green alga Nostoc muscorum. Environ. Contam. Toxicol. 20, 707–714. Kar, S., Singh, P.K., 1979. Effect of nutrients on the toxicity of pesticides, carbofuran and hexachlorocyclohexane to blue-green alga Nostoc muscorum. Z. All. Mikrobiol. 19, 467–472. Latysheva, N., Junker, V.L., Palmer, W.J., Codd, G.A., Barker, D., 2012. The evolution of nitrogen fixation in cyanobacteria. Bioinformatics 2, 603–606. Magnusson, M., Heimann, K., Quayle, P., Negri, A.P., 2010. Additive toxicity of herbicide mixtures and comparative sensitivity of tropical benthic microalgae. Mar. Poll. Bull 60, 1978–1987. Manna, A.B., Singh, P.K., 1990. Effect of nitrogenous fertilizer application methods on growth Aacetylene reduction activity and Azolla Pinnata and yield of rice. Fert. Res 28, 25–30. Manna, A.B., Singh, P.K., 1988c. Effect of different nitrogen sources on growth, Acetylene reduction activity and Azolla Pinnata and yield of rice. Plant Soil 107, 165–171. Nayak, S., Prasanna, R., Pabby, A., Dominic, T.K., Singh, P.K., 2004. Effect of urea, bluegreen algae and Azolla on nitrogen fixation and chlorophyll accumulation in soil under rice. Biol. Fert. Soil 40, 67–72. Padhy, R.N., Rath, S., 2015. Probit analysis of carbamates-pesticide-toxicity at soil-water interface to N2-fixing cyanobacterium. Rice Sci. 22, 89–98. Raghu, K., Mac Rae, I.C., 1967. The effect of the gamma isomer of benzene hexachloride upon the microflora of submerged rice soils. Can. J. Microbiol. 13, 173–180. Reddy, P.M., Roger, P.A., 1988. Dynamics of algal populations and acetylene-reducing activity in five rice soils inoculated with blue- green algae. Biol. Fert. Soil 6, 14–21. Roger, P.A., Kulasooriya, S.A., 1980. Blue green algae and rice. International Rice Res. Ins., Los Banos Laguna, Philippines. Roger P.A, Biology and management of the flood water ecosystem in rice fields. Int. Rice Res. Ins., Los Banos, Laguna, Philippines 1996 250. Schollhorn, R., Burris, R.H., 1966. Study of intermediates in nitrogen fixation. Fed. Am. Soc. Exp. Biol. 25, 7–10. Singh, P.K., Bisyoi, R.N., 1989. Blue green algae in rice fields. Phykos 28, 181–195. Singh, A.L., Singh, P.K., 1987. Nitrogen fixation and balance studies of rice soil. Biol. Fert. Soil 4, 15–19. Singh, P.K., Singh, A.L., 1983. Comparative studies on Azolla and blue-green algae biofertilization to rice crop. In: Proceedings Appl. Phycol. Cong., Kanpur, India, p. 334-349. Singh, P.K., 1973. Effect of pesticides on the blue-green algae. Arch. Microbiol. 89, 317–320. Singh, P.K., 1974. Algicidal effect of 2, 4-dichlorophenoxyacetic acid on blue-green alga. Cylindrospermum sp. Arch. Microbiol. 97, 69–72. Singh, P.K., 1978. Effect of ammonia on growth and nitrogen fixation of blue green algae in rice fields (In:). Advances in cyanophyta Research, BHU, Varanasi, India, 29–32. Singh, P.K., 1985. Nitrogen fixation by blue green algae in paddy fields (In:). Rice Research in India, ICAR, New Delhi, 344–362. Singh, R.N., 1961. Role of blue green algae in nitrogen economy of Indian agriculture. ICAR, New Delhi. Soil Survey Staff, 2010. In: Keys to Soil Taxonomy, Eleventh Eds., UDSA-Natural Resources. Stewart, W.D.P., Fitzgerld, G.P., Burris, R.H., 1968. Acetylene reduction by nitrogen fixing blue green algae. Arch. Microbiol. 62, 336–348. Swarnalakshmi, K., Dhar, D.W., Singh, P.K., 2006. Blue green algae a potential biofertilizer for sustainable rice cultivation. Proceedings Natl. Acad. Sci., India 72, pp.135-143. Tammes, P.M.L., 1964. Isoboles, a graphic representation in pesticides. Neth. J. Plant Pathol. 70, 73–80. Tiwari, D.N., Prassana, R., Yadav, A.K., Dhar, D.W., Singh, P.K., 2001. Growth potential and biocide tolerance of non heterocystous filamentous cyanobacterial isolates from rice fields of Uttar Pradesh, India. Biol. Fert. Soil 34, 291–295. Venkataraman, G.S., 1972. Algal biofertilizer and rice cultivation. Today and Tomorrows Print and Publication Faridabad, India. P.M. Vitousek K. Cassman C. Cleveland T. Crews C.B. Field N.B. Grimm R.W. Howarth R. Marino L. Martinelli E.B. Rastetter J.I. Sprent Towards an ecological understanding of biological nitrogen fixation 2002. Watanabe, I., Cholitkul, W., 1979. Field studies on nitrogen fixation in paddy soils In: Nitrogen and phosphorous fertilizer. Soil Sci. Plant Nut 26, 301–307. Whitton, B.A., 2000. Soils and Rice fields. In: Whitton, B.A., Potts, M. (Eds.), The Ecology of cyanobacteria: Their Diversity in Time and Space. Kluwer Academic Publishers, 233–255. Zaitseva, I.I., 1979. The influence of soil herbicides on the nitrogen fixing activity of bluegreen algae. Byulleten vsesoyuznogo, Nauchno-issledovatel Skogo, Inst. selskokhozyaistvennoi Mikrobiol. 32, 60–62. Zarger, M.Y., Dar, G.H., 1990. Effect of benthiocarb and butachlor on growth and nitrogen by cyanobacteria. Bull. Environ. Contam. Toxicol. 45, 232–241.
N-fertilizers are being used. Herbicides and insecticides have also become essential schedule in rice cultivation. Hence, it is essential to have reliable informations from field studies on the effects of these chemicals on the beneficial microorganisms. The experiment was conducted using a moderate dose of urea-N and recommended levels of herbicide benthiocarb and insecticide carbofuran in planted waterlogged rice field to observe their influence on native cyanobacterial biomass, N2-fixation, N-yield and also on rice crop production. It was observed that urea-N and benthiocarb showed deleterious effects whereas carbofuran was promotary to cyanobacterial biomass and N2-fixation. Rice crop response studies revealed that urea alone or in combination with benthiocarb and carbofuran or in combined use of these biocides improved grain and straw yields. It is concluded from this study that combination of all agrochemicals was toxic when compared to the control but better than urea-N or benthiocarb alone or with their combinations. Hence, it is suggested to take utmost care to avoid adverse effects of these agrochemicals on beneficial organisms in rice ecosystem. Acknowledgements Authors would like to thank Director, Central Rice Research Institute, Cuttack for providing experimental facilities and also thankful to Head, Department of Botany, B.H.U., Varanasi and Indian National Science Academy, New Delhi, India. References Aktar, M.W., Sengupta, D., Chowdhury, A., 2009. Impact of pesticide use in agriculture: their benefits and hazards. Interdiscip. Toxicol. 2, 1–12. Akobundu, I.O., Sweet, R.D., Duke, W.B., 1975. A method of calculating herbicide combinations and determining herbicide synergism. Weed Sci. 23, 20–25. Arle, H.F., 1968. Trifluralin-sysematic insecticide interactions on seedling cotton. Weed Sci. 16, 430–432. Bisyoi, R.N., Singh, P.K., 1988a. Effect of phosphorus fertilization on blue-green algal inoculum production and nitrogen yield under field conditions. Biol. Fert. Soil 5, 338–343. Bisyoi, R.N., Singh, P.K., 1988b. Effect of seasonal changes on cyanobacterial production and nitrogen-yield. Microb. Ecol. 16, 149–154. Casida, J.E., 2009. Pest toxicology: the primary mechanisms of pesticide action. Chem. Res. Toxicol. 22, 609–619. Chen, Z., Juneau, P., Baosheng, Q., 2007. Effects of three pesticides on the growth, photosynthesis and photoinhibition of the edible cyanobacteria Ge-Xian-Mi (Nostoc). Aqua. Toxicol. 81, 256–265. Das, B., Singh, P.K., 1978. Pesticides (hexachlorocytohexane) inhibition of growth and nitrogen fixation in blue-green algae Anabaena perisraciforskii and Anabaena aphanizomenoides. Z. Fur. Allg. Mikrobiol. 18, 161–167. Das, B., Singh, P.K., 1976. Effect of 2, 4- dichlorophenoxy acetic acid on growth and nitrogen fixation of blue-green alga Anabaenopsis raciborskii. Arch. Environ. Contam. Toxicol. 5, 437–445. Das, N.P., Kumar, A., Singh, P.K., 2015. Cyanobacteria, pesticides and rice interaction. Biodivers. Conserv. 24, 995–1005. De Datta, S.K., 1981. Principles and practices of rice production. John Wiley and Sons, New York, 618. De, P.K., 1939. The role of blue green algae in nitrogen fixation in rice fields. Proc. R. Soc. Lond. B Biol. Sci. 127, 121–139. Flores, E., Herrero, A., 1994. Assimilatory nitrogen metabolism and its regulation. In: Bryant, D.A. (Ed.), The molecular biology of cyanobacteria. Kluwer Academic Publication, The Netherlands, 487–517. Geisseler, D., Scow, K.M., 2014. Long term effects of mineral fertilizers on soil microorganisms-A review. Soil Biol. Biochem. 75, 54–63. Higazy, A., Fayez, M., 1989. Effect of some herbicides on growth and diazotrophy of cyanobacteria isolated from Egyptian rice fields. Ann. Agri. Sci. Cairo 34 (2), 765–779. Jackson, M.L., 1973. Chemical analysis. Prentice Hall of India (P) Ltd, New Delhi.
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Agrochemicals influencing nitrogenase, biomass of N2-fixing cyanobacteria and yield of rice in wetland cultivation Nalinaxya Prasad Dasha, Ajay Kumarb, Manish Singh Kaushikb, Gerard Abrahamc, ⁎ Pawan Kumar Singhb, a b c
Directorate of Vocational Education, Shikhya Soudha Unit-V, Bhubaneshwar, India Centre for Advance Studies in Botany, Banaras Hindu University, Varanasi 221005, India Centre for Conservation and Utilization of Blue Green Algae, Indian Agricultural Research Institute, New Delhi 110012, India
A R T I C L E I N F O
A BS T RAC T
Keywords: Cyanobacteria Nitrogenase Biomass N-yield Agrochemicals Rice crop
Cyanobacteria maintain soil fertility by performing N2-fixation and act as a key biocatalyst in nitrogen cycle. Chemical N-fertilizers and pesticides as agrochemicals are intensively being used in rice farming to boost rice production, this work deals with the first hand information on their influence on native N2-fixing cyanobacteria, which play an important role in maintaining soil health. A field study was conducted for three consecutive seasons in water logged rice field to observe the influence of agrochemicals, urea, benthiocarb and carbofuran in isolation and in combinations on biomass, acetylene reduction activity (ARA) and N-yield of native cyanobacteria as well as, on growth and yield of rice. The ARA and N-yield followed almost same trend. It is discernible that both urea and benthiocarb had deleterious effects whereas, carbofuran was promoting effects on cyanobacterial growth, ARA and N-yield. The combination of all the three above agrochemicals was found inhibitory, but inhibition was comparatively less than that of urea or benthiocarb in isolation or urea plus benthiocarb treatments. It is concluded that the combination of agrochemicals was toxic, in comparison to the control, but was better than application of urea N or benthiocarb alone or with their combinations. It was recorded that along with rice straw and gain yields, panicle numbers were the maximum at the combination with treatments of benthiocarb+carbofuran. Adverse effects of used agrochemicals on cyanobacteria in wetland rice cultivation could be avoided by a prudent use of chemical N-fertilizers and pesticide(s) in combination.
1. Introduction Rice is widely cultivated in wetlands having high temperature, low soil organic carbon and high humidity. Such environmental conditions are favorable for cyanobacteria mainly due to their ability to fix atmospheric nitrogen and carbon. Cyanobacteria are considered as key biocatalysts in the N2 cycle (Vitousek et al., 2002; Latysheva et al., 2012). Cyanobacteria were reported as first agent to fix atmospheric nitrogen in flooded rice soils (Singh, 1961) and maintenance of natural soil fertility in such ecosystem was attributed mainly due to these organisms (De, 1939; Singh, 1961). Before introduction of chemical fertilizers, rice has been cultivated year after year without losing soil fertility which was considered mainly due to cyanobacteria and thus role of these organisms in N- economy of rice fields has been widely advocated (Singh, 1961; Venkataraman, 1972; Roger and Kulasooriya, 1980; Swarnalakshmi et al., 2006). With the introduction of high yielding rice varieties, agrochemicals are invariably being used to
⁎
obtain high yields. Thus, simultaneous or sequential application of agrochemicals such as herbicides, insecticides and nitrogenous fertilizers is indispensible in modern rice farming. But these chemicals affect other plants, animals and microorganisms which prevail in such habitat (Chen et al., 2007; Aktar et al., 2009; Casida, 2009; Geisseler and Scow, 2014). When two or more chemicals are applied together as a mixture or one after another during a cropping season, they interact and cause synergistic, additive or antagonistic responses (Tammes, 1964; Akobundu et al., 1975; Magnusson et al., 2010; Padhy and Rath, 2015). Therefore, information on the interactions of these chemicals or their degradation products with rice field ecosystem is very important. It is particularly relevant in case of cyanobacteria, which are the dominating flora of the tropical rice fields and are known to play important role in building soil organic carbon and nitrogen in the flooded rice ecosystem (Venkataraman, 1972; Singh, 1978; Singh and Bisyoi, 1989; Whitton, 2000; Nayak et al., 2004; Swarnalakshmi et al., 2006). Effects of pesticides on cyanobacteria in laboratory cultures
Corresponding author. E-mail address: [email protected] (P.K. Singh).
http://dx.doi.org/10.1016/j.bcab.2016.11.001 Received 7 September 2016; Received in revised form 22 October 2016; Accepted 2 November 2016 Available online 08 November 2016 1878-8181/ © 2016 Published by Elsevier Ltd.
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and graminaceous weeds was applied in one dose at the time of puddling whereas insecticide carbofuran (1 kg a.i. ha−1) a systematic and contact poison acts as miticide was applied in two equal splits at 25 and 45 DAT. The P fertilizer (20 kg P ha−1) was applied in 3 equal split doses at a weekly interval after transplantation (Bisyoi and Singh, 1988a).
were reported earlier (Singh, 1973; Das and Singh, 1978; Tiwari et al., 2001; Chen et al., 2007; Padhy and Rath, 2015). Recently, Das et al. (2015) reported from pot experiment conducted with planted rice that herbicide (butachlor) application adversely affected native and inoculated cyanobacteria whereas insecticide (metacid) application was favorable to them. The information on the interaction of different agrochemicals with cyanobacteria in paddy fields is inadequate. Therefore, in present investigation, an experiment was performed to analyse the impact of commonly used agrochemicals (i.e. herbicide, insecticide and Nfertilizer) in isolation and in combination for three consecutive seasons on native cyanobacterial biomass, nitrogenase activity in terms of acetylene reduction assay (ARA), cyanobacterial N-yield as well as growth and yield of rice.
2.5. Growth of native cyanobacteria in experimental field The rice fields of CRRI farm harbour N2-fixing cyanobacteria abundantly and the dominating species mostly belonged to genera Aulosira, Aphanothece, Gloeotrichia, Anabaena and Nostoc. To promote their growth superphosphate at a rate of 20 kg P ha−1 was applied in three equal splits at 7 days interval (Bisyoi and Singh, 1988a).
2. Materials and methods 2.6. Collection and processing of cyanobacteria samples and measurement of their growth
2.1. Experimental design and treatments A field experiment was carried out for three consecutive seasons (two dry seasons and one wet season) with native cyanobacteria in plots of 5 m×2 m size using urea (CH4N2O), Benthiocarb (trade name Saturn) (S-(4-Chlorobenzyl) N, Ndiethylthiolcarbamate; C12H16CINOS) and carbofuran (trade name furadan) (2,2-dimethyl3H-1-benzofuran-7-yl) N-methylcarbamate; C12H15NO3) as N fertilizer, herbicide and insecticide, respectively. The experiment had a completely randomized block design with three replications and had the following treatments: T1 control (no urea, benthiocarb, carbofuran); T2-Urea (60 kg N ha−1); T3-benthiocarb (1 kg active ingredient (a.i.) ha−1); T4-carbofuran (1 kg a.i. ha−1); T5-T2+T3; T6-T2+T4; T7T3+T4; T8-T2+T3+T4. The treatments T5, T6, T7 and T8 had the same doses as used in T2, T3 and T4. The soil of experimental plots was classified as coastal alluvium sandy clay loam (Aeric Endoaquept) (Soil Survey Staff, 2010) with 0.68% organic C, 0.07% total N (C: N ratio 9.7), 11 µg g−1 available P (Olson), 59.9% P-fixing capacity and pH 6.4. A high yielding rice variety (Oryza sativa L.) IR-36 of 120 days duration was used in the experiment. Other experimental details and methodologies adopted in the experiment are given below.
Ten cyanobacterial samples were randomly collected from each plot with a metallic quadrate (25 cm×25 cm×25 cm) having both ends open (Singh, 1961). The cyanobacterial biomass inside the quadrate was collected by passing the flood water through two layers of cheese cloth. The samples were then washed several times with water to remove the contaminating materials and blotted with the help of blotting paper to remove excess water and utmost care was taken to remove green algae and other contaminants. However, in such field studies, total contaminants cannot be removed and dominating biomass has to be taken into consideration for further study. The fresh weight of the cyanobacterial biomass was recorded immediately after collection and 20 g fresh material was dried in an oven at 80 °C for 24 h for determination of dry weight. From this, the total dry weight was computed and the dry weight of the sample was expressed in kg ha−1 (Bisyoi and Singh, 1988a, 1988b).
2.7. Measurement of nitrogenase activity (acetylene reduction activity; ARA)
2.2. Season and weather conditions
The nitrogenase activity (ARA) was measured following the method of Reddy and Roger (1988). Ten soil-water core samples were randomly collected within each plot with the help of glass tubes of 1.8 cm diameter and 10 cm length. The flood water along with top 0.5 cm of the soil core suspension was adjusted with the distilled water to make 254 mL, a volume that equals to 10 times (in cm2) of the soil surface cored by 10 samples, giving a dilution of 10−1 on the surface basis. The suspension was then stirred at 400 rpm for 30 min to disrupt the cyanobacterial clumps and serially diluted to 10−3 dilution. 5 mL of the diluted sample was incubated in the field with 10% acetylene (v/v) for 1 h. After incubation, 0.5 mL of the gas mixture was injected into the gas chromatograph (AIMIL-5500 series, Nucon Engineers, New Delhi). The amount of ethylene produced was expressed in nmole C2H4 cm−2 h x 10−3 (Schollhorn and Burris, 1966; Stewart et al., 1968).
The experiment was carried out at the farm of Central Rice Research Institute, Cuttack, India. It is situated on the bank of river Mahanadi in Orissa state (Eastern India) at latitude of 25.5°N, longitude of 86°E, altitude of 23.48 m above the mean sea level and is 80 km in the west from the Bay of Bengal in the main rice growing tract of India. During the period of experimentation, average annual rainfall was 1553.2 mm. Mean maximum temperature was 35.3–36.5 °C during May and June, whereas the mean minimum temperature was 13.6–15.1 °C during January and December and relative humidity was 86–97% during July-September months. 2.3. Field preparation and transplanting The fields were ploughed, cross ploughed, puddled, levelled and thick bounds were raised to avoid mixing of treatments. The healthy and sprouted rice seeds were sown in seed beds and 25–30 days old healthy seedlings were transplanted at the rate of 2 seedlings hill−1 with a spacing of 15×10 cm.
2.8. Measurement of cyanobacterial N-yield The total N content (%) of cyanobacteria was estimated by the micro-kjeldahl method (Jackson, 1973). 100 mg of dried cyanobacterial samples were taken in duplicate in 100 mL kjeldahl flasks to which a pinch of digestion mixture (K2SO4, CuSO4 and SiO2 in a 100:10:1 ratio) and 2 mL of concentrated H2SO4 (36N) were added. The flasks were heated in the digestion chamber for 2–3 h and the digested samples were used for determination of N content. The N-yield was calculated by multiplying the N content with the dry weight and expressed in kg N ha−1.
2.4. Biocides applications The half of the N fertilizer (urea at the rate of 60 kg N ha−1) was applied as a basal dose during puddling and a quarter each at 25 and 45 days after transplanting (DAT). The pre-emergence herbicide benthiocarb (1 kg a.i. ha−1) having intergenic selectivity between rice plants 29
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2.9. Rice post harvest observation 2.9.1. Panicle number, grain and straw yield The rice plants in 1 m2 area were harvested at their maturity and the numbers of panicles were counted. The rice crop from an area of 7.57 m2 from each plot was harvested, threshed and the grain weight was recorded. The moisture content of grains was measured in an OSAW Universal moisture meter and the grain yield was expressed at 14% moisture. The straw samples were completely sun dried before weighing. 2.10. Statistical analysis The data were analyzed statistically with one-way analysis of variance using software SPSS.16 advance version, where means for each treatment at different days after transplanting (DAT) were compared with control using Tukey HSD post hoc analysis. 3. Results
Fig. 1. Influence of chemical-N fertilizer and pesticides on growth (kg dry weight ha−1) of rice field's native cyanobacteria. Data are the presented as mean ± SD (n=3) of growth measured at different rice crop stages from both dry and wet season. Multivariate ANOVA was performed for repeated measures of treatments and their interactions with different parameters. Level of significance: *p < 0.05; **p < 0.01; ***p < 0.001.
3.1. Growth of cyanobacteria as influenced by agrochemicals in rice field The growth of the native cyanobacteria in terms of dry weight (kg ha−1) was found maximum at 60 days after transplanting (DAT) and minimum at 30 DAT. The cyanobacterial growth in wet season (WS) was comparatively lower than that in the dry season (DS) (Table 1; Fig. 1). Urea applied at rate of 60 kg N ha−1 was observed to be highly inhibitory for growth of cyanobacteria. It reduced their growth at 60 DAT by 55% and 62% over the control. The herbicide benthiocarb applied at rate of 1.0 kg a.i. ha−1 decreased the growth at 30, 60, 90 DAT by 16%, 8%, and 19% respectively during DS and 22%, 11% and 23% respectively at 30, 60, 90 DAT in WS. The insecticide carbofuran applied at rate of 1.0 kg a.i. ha−1 was observed to be growth stimulatory on all sampling dates and in both the seasons. The significant stimulatory effect of this insecticide in terms of percentage increase in growth over control was from 16% to 30% in both seasons. Urea and benthiocarb interaction was significant during both dry and wet seasons. A combination of urea and benthiocarb showed lower growth as compared to treatment without urea and benthiocarb (Table 1) and decrease in growth over control at 60 DAT was 38% and 42% during dry and wet season respectively. Their combination had produced significantly lower cyanobacterial biomass in comparison to treatment receiving only herbicide whereas it was statistically similar with treatment receiving only nitrogen. The interaction between the urea and carbofuran had significant effect on the cyanobacterial growth during WS and DS. The decrease in cyanobacterial growth due to use of
urea plus carbofuran was 12–22% over the control during wet and dry season respectively. Thus, combination of these two chemicals showed less cyanobacterial growth than the treatment receiving only carbofuran whereas it was superior to treatment receiving only urea. The combined application of benthiocarb plus carbofuran significantly increased growth of cyanobacteria in both the seasons. Cyanobacterial growth was significantly higher in treatment receiving only herbicide and lower in the treatment receiving only insecticide. All the three factors (urea+benthiocarb+carbofuran) significantly decreased the growth at all the three 30, 60 and 90 DAT in both the seasons. The combination of these three factors recorded significantly higher cyanobacterial growth than the treatments receiving urea and urea plus benthiocarb whereas it recorded lower growth than treatments receiving carbofuran, benthiocarb, benthiocarb plus carbofuran and urea and carbofuran. 3.2. Nitrogenase activity (ARA) of cyanobacteria in rice field as influenced by agrochemicals Nitrogenase activity (ARA) in wet season was comparatively lower than the dry season and it almost followed trend of the growth (Table 2; Fig. 2). The ARA was found lowest in all the treatments and seasons at 30 DAT and was highest at 60 DAT. The minimum and maximum ARA
Table 1 Influence of chemical-N fertilizer and pesticides on growth (kg dry weight ha−1) of rice field's native cyanobacteria. Data presented as mean ± SD (n=3). Treatments
Control( no N-fertilizer, herbicide and insecticide) Urea-N Benthiocarb Urea+ Benthiocarb Carbofuran Benthiocarb+ Carbofuran Urea N+ Carbofuran Urea-N+ Carbofuran +Benthiocarb
Dry season (kg dry wt ha−1)
Wet season (kg dry wt ha−1)
30 DAT
60 DAT
90 DAT
30 DAT
60 DAT
90 DAT
94.4 ± 0.21
209.4 ± 0.89
98 ± 0.37
67.1 ± 0.11
181.1 ± 0.21
76.11 ± 0.31
43.2 ± 0.20 79 ± 0.40 61 ± 0.91 115 ± 0.3 103 ± 0.65 73.3 ± 0.21 67 ± 0.11
(-54%) (−16%) (−35%) (+22%) (+9%) (−22%) (−29%)
93.3 ± 0.13 193 ± 0.45 130 ± 0.63 244 ± 0.30 217.5 ± 0.17 163 ± 0.35 149 ± 0.59
(−55%) (−8%) (−38%) (+16%) (+4%) (−22%) (−29%)
49 ± 0.41 79 ± 0.37 62 ± 0.72 124 ± 0.50 102 ± 0.37 79.3 ± 0.79 73 ± 0.97
(−50%) (−19%) (−37%) (+26.5%) (+4%) (−19%) (−25.5%)
27.4 ± 0.30 52.35 ± 0.29 43 ± 0.16 79.5 ± 0.31 83.41 ± 0.25 53.2 ± 0.42 47.2 ± 0.23
(−59%) (−22%) (−36%) (+18%) (+24%) (−20%) (−30%)
67.1 ± 0.19 161.4 ± 0.31 103.3 ± 0.30 215.2 ± 0.98 202 ± 0.63 169 ± 1.01 128.6 ± 0.41
DAT-Days after transplanting N-Fertilizer- Urea (60 kg N ha−1), Herbicide- benthiocarb (1.0 kg a.i. ha−1), Insecticide- carbofuran (1.0 kg a.i. ha−1) The data given in bracket “()” showed the percentage (%) increase or decrease in growth after treatments with respect to control in each column.
30
(−62%) (−11%) (−42%) (+19%) (+11%) (−12%) (−30%)
36 ± 0.45 58.11 ± 0.21 46.14 ± 0.20 99.2 ± 0.28 84.45 ± 0.81 59.34 ± 0.20 55.11 ± 0.22
(−53%) (−23%) (−39%) (+30%) (+10%) (−22%) (−27%)
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Table 2 Influence of chemical-N fertilizer and pesticides on acetylene reduction activity or ARA (n moles C2H4 cm−2 h−1 ×10−3) of rice field's native cyanobacteria. Data presented as mean ± SD (n=3). Dry season (n moles C2H4 cm−2 h−1 ×10−3)
Wet Season (n moles C2H4 cm−2 h−1 ×10−3)
Treatments
30 DAT
60 DAT
90 DAT
30 DAT
60 DAT
80 DAT
Control ( no Nfertilizer, herbicide and insecticide) Urea-N Benthiocarb Urea + Benthiocarb Carbofuran Benthiocarb+ Carbofuran Urea N + Carbofuran Urea-N + Carbofuran +Benthiocarb
147 ± 0.10
355 ± 0.90
251 ± 0.45
119.50 ± 0.50
291.30 ± 0.15
05.70 ± 0.30
353 ± 0.15 120 ± 0.67 48 ± 0.20 215 ± 0.79 161 ± 0.50
(−76%) (−18%) (−67%) (+46%) (+9.7%)
90 ± 0.71 307.5 ± 0.25 121 ± 0.9 552.5 ± 1.21 395 ± 0.67
(−75%) (−13%) (−66%) (−55%) (+11%)
72.5 ± 0.3 217 ± 0.13 93 ± 0.43 377 ± 0.39 289 ± 0.45
(−71%) (−13%) (−63%) (+50%) (+15%)
25.70 ± 0.09 87.40 ± 0.69 32.10 ± 0.17 160.70 ± 0.60 128.50 ± 0.70
(−79%) (−27%) (−73%) (+34%) (+7%)
66.60 ± 0.19 224.90 ± 0.51 79.50 ± 0.71 415.30 ± 0.22 321.40 ± 0.40
(−77%) (−23%) (−73%) (+43%) (+10%)
52.30 ± 0.45 161.40 ± 0.28 59.60 ± 0.30 295 ± 0.20 231.40 ± 0.15
(−74%) (−22%) (−71%) (+4%) (+12%)
58.45 ± 0 0.21 54 ± 0.67
(−60%) (−63%)
146.5 ± 0.49 132 ± 0.88
(−59%) (−63%)
120 ± 0.53 106 ± 0.35
(−52%) (−58%)
42.40 ± 0.69 45.00 ± 0.49
(−64.5%) (−62.3%)
102.60 ± 0.38 95.10 ± 0.71
(−65%) (−67%)
81.40 ± 0.89 79.60 ± 0.42
(−61%) (−61%)
DAT-Days after transplanting N-Fertilizer- Urea (60 kg N ha−1), Herbicide- benthiocarb (1.0 kg a.i. ha−1), Insecticide- carbofuran (1.0 kg a.i. ha−1) The data given in bracket “()” showed the percentage (%) increase or decrease in growth after treatments with respect to control in each column.
reduced activity significantly by 13–27% over control, although this herbicide was observed to be less deleterious as compared to urea. The insecticide carbofuran was found to be stimulatory to ARA and it significantly enhanced activity by 43–55% over control. As both urea and benthiocarb were inhibitory to cyanobacterial ARA, their combined application showed a drastic reduction in ARA. Application of the urea and benthiocarb together recorded significantly lower ARA than herbicide treatment but it was similar with nitrogen treatment. When the carbofuran was applied along with urea, inhibitory effect of latter was lowered and the ARA values were significantly higher than that of urea treatment. Application of benthiocarb and carbofuran in combination significantly increased ARA over the control without benthiocarb and carbofuran during both the season. The percentage of increase was in between 7–15% during both the seasons. The combined use of benthiocarb and carbofuran showed significantly lower ARA than application of the carbofuran but recorded higher ARA than application of benthiocarb suggesting reduction of inhibitory effect of benthiocarb when it was applied along with carbofuran. Three factors (urea, benthiocarb and carbofuran) interaction had significant effect on ARA in both the seasons.
Fig. 2. Influence of chemical-N fertilizer and pesticides on ARA (n moles C2H4 cm−2 h×10−3) of rice field's native cyanobacteria. Data are the presented as mean ± SD (n=3) of ARA measured at different rice crop stages from both dry and wet season. Multivariate ANOVA was performed for repeated measures of treatments and their interactions with different parameters. Level of significance: *p < 0.05; **p < 0.01; ***p < 0.001.
3.3. Agrochemicals affecting nitrogen accumulation by cyanobacteria The nitrogen accumulation (N-yield) by cyanobacteria was measured at 30, 60 and 90 days after transplanting (DAT) in both dry and wet seasons and N-yield (kg N ha−1) was found to be minimum at 30 DAT and maximum at 60 DAT irrespective of treatments and seasons (Table 3; Fig. 3). The N-yield of different treatments showed a similar trend to that of ARA. Urea (60 kg N ha−1) significantly decreased N-
was 25.7 and 552.9 n moles C2H4 cm2 h−1 ×10−3. Urea (60 kg N ha−1) was found to be highly inhibitory to ARA of the cyanobacteria and the extent of reduction over the control during different seasons was more than 65%. The herbicide benthiocarb (1 kg a.i. ha−1) showed a deleterious effect on cyanobacterial ARA, and
Table 3 Influence of chemical-N fertilizer and pesticides on N-yield (kg N ha−1) of rice field's native cyanobacteria. Data presented as mean ± SD (n=3). Dry season (kg N ha−1) Treatments Control( no N-fertilizer, herbicide and insecticide) Urea-N Benthiocarb Urea + Benthiocarb Carbofuran Benthiocarb+ Carbofuran Urea + Carbofuran
30 DAT 2.9 ± 0.30 1.43 ± 0.03 2.38 ± 0.20 2 ± 0.10 3.89 ± 0.09 3.44 ± 0.04 2.34 ± 0.34
Wet season (kg N ha−1) 60 DAT 6.6 ± 0.10
(−50%) (−17%) (−31%) (+34%) (+19%) (−19%)
2.97 ± 0.02 6.2 ± 0.05 4.28 ± 0.28 7.66 ± 0.33 7.09 ± 0.09 5.52 ± 0.12
90 DAT 3.5 ± 0.25 (−55%) (−6%) (−35%) (+16%) (+7%) (−16%)
1.7 ± 0.30 2.98 ± 0.69 2.2 ± 0.11 4.33 ± 0.22 3.74 ± 0.14 2.75 ± 0.25
30 DAT 0.74 ± 0.02 (−50%) (−15%) (−37%) (+28%) (+7%) (−21%)
0.49 ± 0.09 0.81 ± 0.29 0.52 ± 0.30 1.48 ± 0.47 1.01 ± 0.09 0.76 ± 0.03
60 DAT 2.14 ± 0.14 (−33%) (+9%) (−30%) (+100%) (+36%) (+3%)
1.16 ± 0.13 2.02 ± 0.02 1.81 ± 0.29 3.65 ± 0.13 2.68 ± 0.27 2.45 ± 0.15
DAT-Days after transplanting N-Fertilizer- Urea (60 kg N ha−1), Herbicide- benthiocarb (1.0 kg a.i. ha−1), Insecticide- carbofuran (1.0 kg a.i. ha−1). The data given in bracket “()” showed the percentage (%) increase or decrease in growth after treatments with respect to control in each column.
31
90 DAT 1.20 ± 0.10 (−46%) (−7%) (−15%) (+70%) (+25%) (+14%)
0.66 ± 0.33 1.04 ± 0.11 1.05 ± 0.17 1.71 ± 0.04 1.46 ± 0.70 0.98 ± 0.39
(−45%) (−13%) (−13%) (+42.5%) (+22%) (−18%)
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+carbofuran whereas minimum increase of 3% was observed with benthiocarb. The combined treatments showed maximum yields in comparison to their separate applications except urea. Separate application of urea, benthiocarb and carbofuran enhanced the yields by 9%, 3% and 6% respectively whereas treatments of urea+benthiocarb, urea+carbofuran, benthiocarbcarbofuran, urea+benthiocarb+carbofuran enhanced yield by 7%, 13%, 7% and 10% respectively. It is revealed from the data that all the treatments enhanced the straw yields in comparison to the control except herbicide. Application of urea was also found promotary to straw yield in combinations or separate applications (Table 4). 3.4.3. Panicle number The number of panicles m2 also followed almost same trend as that of grain and straw yields. Urea significantly increased the number of panicles m2 by 9% over the control. The benthiocarb did not have a significant effect on the number of panicles, whereas carbofuran application enhanced the number of panicles by 8%. The treatments urea+benthiocarb, urea+carbofuran, benthiocarb+carbofuran, urea +benthiocarb+carbofuran agrochemicals enhanced panicles by 7%, 9%, 5.5% and 7% respectively in comparison to control (Table 4).
Fig. 3. Influence of chemical-N fertilizer and pesticides on N- yield (kg N ha−1) of rice field's native cyanobacteria. Data are the presented as mean ± SD (n=3) of N-yield measured at different rice crop stages from both dry and wet season. Multivariate ANOVA was performed for repeated measures of treatments and their interactions with different parameters. Level of significance: *p < 0.05; **p < 0.01; ***p < 0.001.
4. Discussion
yield of rice field's native cyanobacteria during both the seasons. The percentage of reduction over control without urea at 60 DAT was 55% and 46% during dry and wet season respectively. The carbofuran (1 kg a.i. ha−1) significantly enhanced N-yield over untreated control. The interaction of urea+benthiocarb was observed to have a significant effect on cyanobacterial N-yield. The combined application of urea and benthiocarb decreased N-yield by 31%, 35% and 37% at 30, 60 and 90 DAT over the control in dry season whereas 30%, 15% and 13% at 30, 60, 90 DAT in wet season respectively. This combination recorded significantly lower N-yield than the benthiocarb treatment but it was superior to the application of urea alone, during both dry and wet seasons. The interaction of the urea+carbofuran was significant at 30 DAT during dry season and at all the three DAT during wet season. The retarding effect of the urea fertilizer on N-yield was lowered when it was applied along with insecticide. The urea+carbofuran combination showed significantly increased N-yield over urea treatment but recorded lower N-yield than the control. A combination of benthiocarb +carbofuran increased the N-yield at 30, 60 and 90 DAT by 10%, 7% and 7% over the control during dry season respectively. The N-yield in this treatment was significantly lower than that in the carbofuran treatment but higher than that of benthiocarb treatment.
Among different food crops, rice ranked second to wheat in terms of area harvested, but in terms of importance as a food crop rice provides more calories than any other cereal crop (De Datta, 1981). The sustainability of agriculture and ecosystems is considered crucial and therefore increasing demand of improving yields using extensive agrochemicals and other modern technologies should also ensure protection of environmental integrity and biodiversity conservation. Since agrochemicals are extensively being used in rice cultivation (Das et al., 2015), it is necessary to study impact of these agrochemicals on non-target organism like cyanobacteria, which is a key biocatalyst of nitrogen cycle, increase soil fertility and rice yield by fixing nitrogen. Cyanobacteria are known to thrive well in tropical flooded rice fields and therefore, due care needs to be taken while using the agrochemicals in rice cultivation for protecting these beneficial organisms in rice ecosystem and enabling to sustain soil fertility on long term basis. Advantages to cyanobacteria in this environment are due to two major metabolic activities i.e. N2-fixation and photosynthesis, due to which they grow well under poor nitrogen and low organic-carbon containing soils provided other environmental and soil conditions are favorable. Roger (1996) described various components of flooded water rice ecosystem, their properties, and activities highlighting importance of photosynthetic aquatic biomass and effects on N dynamics in this system. In the current investigation, in depth study for three consecutive seasons was undertaken using same high yielding rice variety in a traditional rice growing area of India to find out effects of nitrogenous fertilizer (urea), herbicide (benthiocarb) and insecticide (carbofuran) commonly used in rice cultivation as per rice cultivation practices at different stages of crop. During the course of investigation, very interesting observations were recorded. Most of the earlier studies either conducted in laboratories using cyanobacterial cultures or randomly samples taken from rice fields using single input parameter. The deleterious effects of combined N sources on cyanobacteria have been reported (Singh, 1978, 1985; Watanabe and Cholitkul, 1979; Roger and Kulasooriya, 1980; Singh and Bisyoi, 1989; Swarnalakshmi et al., 2006). Among different nitrogenous fertilizers, urea is widely used in rice cultivation. The cyanobacteria occurring in the flooded rice ecosystem takes up urea in cells and degrade into ammonium using enzyme urease. The ammonium is taken up by cells as N source. However, urea is found to be inhibitory to cyanobacteria in this study which might be due to its indirect effects (Roger, 1996). Urea is reported to reduce soil pH (Geisseler and Scow, 2014) whereas cyanobacteria prefer to grow well in neutral to alkaline pH. Thus,
3.4. Crop parameters response as affected by agrochemicals 3.4.1. Grain yield The impact of application of chemical-N fertilizer on the grain yield was found significantly maximum in case of urea+carbofuran (35%), whereas minimum in case of herbicide benthiocarb (4%). The yield was found maximum where urea+benthiocarb+carbofuran were applied in combinations in comparison to singly applications. Applications of urea, benthiocarb and carbofuran separately enhanced the yield by 20%, 4% and 11% respectively whereas in case of treatments urea +benthiocarb, urea+carbofuran, benthiocarb+carbofuran, urea +benthiocarb+carbofuran enhanced grain yield by 17%, 35%, 13% and 27% respectively. The results stated that all treatments enhance the grain yield significantly except benthiocarb. Application of urea applied alone or in different combinations was found to enhance grain yield (Table 4). 3.4.2. Straw yield The straw yield followed almost same trend as that of grain yield. Maximum increase in yield (13%) was found in the treatment of urea 32
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Table 4 Crop parameters (grain yield, straw yield and panicles number) of rice variety IR-36 as influenced by chemical-N fertilizer and pesticides. Data presented as mean ± SD (n=3). Treatments
Grain yield (t ha−1)
Control ( no N-fertilizer, herbicide and insecticide) Urea-N Benthiocarb Urea + Benthiocarb Carbofuran Benthiocarb+Carbofuran Urea+Carbofuran Urea+Carbofuran+Benthiocarb
2.78 ± 0.03 3.34 ± 0.30 2.90 ± 0.45 3.27 ± 0.09 3.10 ± 0.05 3.16 ± 0.16 3.76 ± 0.30 3.53 ± 0.25
Straw yield (t ha−1)
(+20%) (+4%) (+17%) (+11%) (+13%) (+35%) (+27%)
2.39 ± 0.09 2.62 ± 0.02 2.47 ± 0.13 2.56 ± 0.14 2.53 ± 0.53 2.56 ± 0.41 2.71 ± 0.89 2.63 ± 0.71
Panicle numbers (m−2)
(+9%) (+3%) (+7%) (+6%) (+7%) (+13%) (+10%)
327 ± 0.90 358 ± 0.40 334 ± 0.67 351 ± 0.21 353 ± 0.75 345 ± 0.89 358 ± 1.15 350 ± 0.99
(+9%) (+2%) (+7%) (+8%) (+5.5%) (+9%) (+7%)
N-Fertilizer- Urea (60 kg N ha−1), Herbicide- benthiocarb (1.0 kg a.i. ha−1), Insecticide- carbofuran (1.0 kg a.i. ha−1). The data given in bracket “()” showed the percentage (%) increase or decrease in growth after treatments with respect to control in each column.
The cyanobacterial N-yield also followed similar trend as that of growth and N2-fixation. The biological nitrogen fixation by photodependent cyanobacteria in the photic zone is favoured in flooded soilrice ecosystem. The N balance study conducted in the presence and absence of light, rice and flooding reported significant N gain due to photo-dependent BNF (Singh and Singh, 1987). A visible growth of native cyanobacteria generally corresponds to less than 10 kg N ha−1 (Roger, 1996). In the present study N-yield is observed to be less than 10 kg N ha−1 in most of the treatments. Although under flooded rice field conditions, N-yield determined from biomass measurement is likely to be underestimated because turnover of cyanobacterial biomass is not taken into account (Roger, 1996). The total N estimated and effects of agrochemicals in different treatments followed the observations which were discussed earlier on growth of cyanobacteria. The combinations of urea plus benthiocarb, benthiocarb plus carbofuran, carbofuran plus urea and urea plus benthiocarb plus carbofuran along with rice in field trials have provided first hand informations. Informations on such systematic study in rice ecosystem with reference to cyanobacteria were not available earlier. Antagonistic effect of the benthiocarb and carbofuran on the growth, N2 -fixation and N-yield of cyanobacteria was observed. A similar antagonistic relationship between benthiocarb and carbofuran was also reported in cotton (Arle, 1968). Effect of three factors interaction (urea, herbicide and insecticide) was mostly regulated by the urea-N, probably because the cyanobacteria are more susceptible to combined-N whether applied alone or in combination with the pesticides. Regarding rice crop response, the studies showed that application of urea alone or in combination with other agrochemicals improved the grain and straw yields as reported earlier (De Datta, 1981). Besides, benthiocarb and carbofuran when applied together also increased the grain yield significantly. One of the notable points in this investigation was that urea plus carbofuran treatment produced higher grain yield than the urea plus benthiocarb treatment which implies that the total increase in rice yield could not be attributed to urea alone. Since urea plus carbofuran treatment also recorded a higher cyanobacterial growth than the urea plus benthiocarb treatment, the increased rice yield in former treatment could also be partly due to cyanobacterial response. The control of grazers (Raghu and Mac Rae, 1967) by use of the carbofuran might have led to production of higher biomass and accumulation of more nitrogen in cyanobacteria which ultimately increased the yield of rice. The lower grain yield in the urea plus benthiocarb treatment might be due to retarding effect of benthiocarb on the growth and N2-fixation of rice field's cyanobacteria (Singh and Singh, 1983). This result may also be applicable to other paddy ecosystems for cyanobacteria and other microorganisms.
decline in pH might have affected the growth of these organisms in rice ecosystem. To overcome the adverse effects of surface applied urea, attempts were made in past to use slow release N fertilizers, deep placement of urea super granules and methods of N fertilizers applications in rice ecosystem on free living and symbiotic cyanobacteria. It was inferred from these studies that inhibitory effects of surface applied urea could be reduced by deep placing or incorporating it into the soil (Manna and Singh, 1988c, 1990; Roger, 1996). The inhibition of cyanobacterial growth by herbicide benthiocarb in this study is supported by earlier observations based mostly from laboratory studies (Zarger and Dar, 1990; Singh, 1974; Das and Singh, 1976; Zaitseva, 1979; Higazy and Fayez, 1989). It was interesting to note that pesticide carbofuran which used to be considered as a potential insecticide for insect control in rice fields, promotes growth of native cyanobacteria. Kar and Singh (1978) reported similar growth promoting effects of carbofuran in a laboratory study on cyanobacteria. Also they studied effects of nutritional factors, pH, light intensity on carbofuran and cyanobacteria interaction (Kar and Singh, 1978, 1979). Cyanobacteria are likely to metabolise or biodegrade carbofuran and metabolic products could be used as growth and N2-fixation promotary as reported by Kar and Singh (1978). Besides pests of cyanobacteria are likely to be affected adversely in the fields favouring cyanobacteria. The combination of carbofuran and benthiocarb promoted above activities but was less than carbofuran alone. Therefore, positive response in the combined treatment is likely to be due to carbofuran. The carbofuran is also reported as mutagenic in our earlier study (Kar and Singh, 1979). Das et al. (2015) reported interaction of pesticides and cyanobacteria in pot studies where herbicide butachlor decreased their growth but insecticide metacid application was found to be growth promotary. The N2- fixation activity of microorganisms in general is reported to be inhibited by chemical N sources, where free living cyanobacteria seems to be more susceptible to inhibition than heterotrophs. The inhibitory effect of surface urea application is confirmed by acetylene reduction activity on photo-dependent biological N2-fixation (BNF). Although it was not the complete inhibition but decrease during the growth cycle was because of N uptake by plants and also due to various losses. Based on extrapolated data from nitrogenase activity (ARA) measurement, the visible growth of cyanobacteria may contribute up to 30 kg N ha−1 crop−1 in control plots without addition of N fertilizer. But the growth reduced to one-fourth with broadcast of urea and almost half when it was deep placed (Roger, 1996). Cyanobacterial nitrogenase activity is expressed in the absence of combined N source. The ammonium negatively affects nitrogenase synthesis besides its effects on heterocyst differentiation (Flores and Herrero, 1994). Therefore, ammonium produced in cells after uptake of urea likely to inhibit ARA in this study. Thus, nitrogenase activity (ARA) might be getting affected directly and also due to indirect effect on growth of cyanobacteria. The pesticides have initial depressive effect on N2-fixing activity followed by increase or decrease in activity. The insecticides generally have less effect except some on N2-fixing activity of cyanobacteria (Roger and Kulasooriya, 1980).
5. Conclusion The cyanobacteria are known to play an important role in maintaining soil fertility in flooded rice ecosystem. With the demand of intensive rice cultivation to obtain high yields, high doses of chemical 33
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N-fertilizers are being used. Herbicides and insecticides have also become essential schedule in rice cultivation. Hence, it is essential to have reliable informations from field studies on the effects of these chemicals on the beneficial microorganisms. The experiment was conducted using a moderate dose of urea-N and recommended levels of herbicide benthiocarb and insecticide carbofuran in planted waterlogged rice field to observe their influence on native cyanobacterial biomass, N2-fixation, N-yield and also on rice crop production. It was observed that urea-N and benthiocarb showed deleterious effects whereas carbofuran was promotary to cyanobacterial biomass and N2-fixation. Rice crop response studies revealed that urea alone or in combination with benthiocarb and carbofuran or in combined use of these biocides improved grain and straw yields. It is concluded from this study that combination of all agrochemicals was toxic when compared to the control but better than urea-N or benthiocarb alone or with their combinations. Hence, it is suggested to take utmost care to avoid adverse effects of these agrochemicals on beneficial organisms in rice ecosystem. Acknowledgements Authors would like to thank Director, Central Rice Research Institute, Cuttack for providing experimental facilities and also thankful to Head, Department of Botany, B.H.U., Varanasi and Indian National Science Academy, New Delhi, India. References Aktar, M.W., Sengupta, D., Chowdhury, A., 2009. Impact of pesticide use in agriculture: their benefits and hazards. Interdiscip. Toxicol. 2, 1–12. Akobundu, I.O., Sweet, R.D., Duke, W.B., 1975. A method of calculating herbicide combinations and determining herbicide synergism. Weed Sci. 23, 20–25. Arle, H.F., 1968. Trifluralin-sysematic insecticide interactions on seedling cotton. Weed Sci. 16, 430–432. Bisyoi, R.N., Singh, P.K., 1988a. Effect of phosphorus fertilization on blue-green algal inoculum production and nitrogen yield under field conditions. Biol. Fert. Soil 5, 338–343. Bisyoi, R.N., Singh, P.K., 1988b. Effect of seasonal changes on cyanobacterial production and nitrogen-yield. Microb. Ecol. 16, 149–154. Casida, J.E., 2009. Pest toxicology: the primary mechanisms of pesticide action. Chem. Res. Toxicol. 22, 609–619. Chen, Z., Juneau, P., Baosheng, Q., 2007. Effects of three pesticides on the growth, photosynthesis and photoinhibition of the edible cyanobacteria Ge-Xian-Mi (Nostoc). Aqua. Toxicol. 81, 256–265. Das, B., Singh, P.K., 1978. Pesticides (hexachlorocytohexane) inhibition of growth and nitrogen fixation in blue-green algae Anabaena perisraciforskii and Anabaena aphanizomenoides. Z. Fur. Allg. Mikrobiol. 18, 161–167. Das, B., Singh, P.K., 1976. Effect of 2, 4- dichlorophenoxy acetic acid on growth and nitrogen fixation of blue-green alga Anabaenopsis raciborskii. Arch. Environ. Contam. Toxicol. 5, 437–445. Das, N.P., Kumar, A., Singh, P.K., 2015. Cyanobacteria, pesticides and rice interaction. Biodivers. Conserv. 24, 995–1005. De Datta, S.K., 1981. Principles and practices of rice production. John Wiley and Sons, New York, 618. De, P.K., 1939. The role of blue green algae in nitrogen fixation in rice fields. Proc. R. Soc. Lond. B Biol. Sci. 127, 121–139. Flores, E., Herrero, A., 1994. Assimilatory nitrogen metabolism and its regulation. In: Bryant, D.A. (Ed.), The molecular biology of cyanobacteria. Kluwer Academic Publication, The Netherlands, 487–517. Geisseler, D., Scow, K.M., 2014. Long term effects of mineral fertilizers on soil microorganisms-A review. Soil Biol. Biochem. 75, 54–63. Higazy, A., Fayez, M., 1989. Effect of some herbicides on growth and diazotrophy of cyanobacteria isolated from Egyptian rice fields. Ann. Agri. Sci. Cairo 34 (2), 765–779. Jackson, M.L., 1973. Chemical analysis. Prentice Hall of India (P) Ltd, New Delhi.
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