effects of transgenic bt cotton on soil fertility and biology under field conditions in subtropical...

Effects of transgenic Bt cotton on soil fertility and biologyunder field conditions in subtropical inceptisol

Raman Jeet Singh & I. P. S. Ahlawat &Surender Singh

Received: 6 August 2011 /Accepted: 3 February 2012 /Published online: 21 February 2012# Springer Science+Business Media B.V. 2012

Abstract Although there is large-scale adoption of Btcotton by the farmers because of immediate financialgain, there is concern that Bt crops release Bt toxinsinto the soil environment which reduces soil chemicaland biological activities. However, the majorities ofsuch studies were mainly performed under pot experi-ments, relatively little research has examined the directand indirect effects of associated cover crop of peanutwith fertilization by combined application of organicand inorganic sources of nitrogen under field condi-tions. We compared soil chemical and biologicalparameters of Bt cotton with pure crop of peanut toarrive on a valid conclusion. Significantly higher de-hydrogenase enzyme activity and KMnO4-N contentof soil were observed in Bt cotton with cover crop ofpeanut over pure Bt cotton followed by pure peanut at

all the crop growth stages. However, higher microbialpopulation was maintained by pure peanut over inter-cropped Bt cotton, but these differences were relatedto the presence of high amount of KMnO4-N contentof soil. By growing cover crop of peanut between Btcotton rows, bacteria, fungi, and actinomycetes popu-lation increased by 60%, 14%, and 10%, respectively,over Bt cotton alone. Bt cotton fertilized by combinedapplication of urea and farm yard manure (FYM)maintained higher dehydrogenase enzyme activity,KMnO4-N content of soil and microbial populationover urea alone. Significant positive correlations wereobserved for dry matter accumulation, dehydrogenaseenzyme activity, KMnO4-N content, and microbialpopulation of soil of Bt cotton, which indicates noharmful effects of Bt cotton on soil biological param-eters and associated cover crop. Our results suggestthat inclusion of cover crop of peanut and FYM in Btcotton enhanced soil chemical and biological parame-ters which can mask any negative effect of the Bt toxinon microbial activity and thus on enzymatic activities.

Keywords Bt cotton . Dehydrogenase . FYM .

Microbial activity . Nitrogen . Peanut . Soil

Introduction

India made its long-awaited entry into commercialagricultural biotechnology in March 2002 with the

Environ Monit Assess (2013) 185:485–495DOI 10.1007/s10661-012-2569-1

R. J. Singh (*) : I. P. S. AhlawatDivision of Agronomy,Indian Agricultural Research Institute (IARI),New Delhi 110-012, Indiae-mail: [email protected]

R. J. SinghDivision of Soil Science and Agronomy, Central Soil andWater Conservation Research and Training Institute,Dehradun, Uttarakhand 248-195, India

S. SinghDivision of Microbiology,Indian Agricultural Research Institute (IARI),New Delhi 110-012, India

approval of three Bt cotton hybrids for commercialcultivation. Bt cotton has occupied 66% of the 9.4million hectares of the total cotton area in India in2007 (Venugopalan et al. 2009). In 2007, Bt cottonoccupied globally 15 million hectares which comprised43% of the total cotton area of 35 million hectares innine countries namely USA, Mexico, China, Argentina,South Africa, Colombia, India, and Brazil (ISAAA2008). Although there is large-scale adoption of Btcotton by the farmers because of immediate financialgain, there is concern that transgenic Bt crops release Bttoxins into the environment which affect associated andsucceeding crops due to reduction in soil chemical andbiological activities (O’Callaghan et al. 2005; Sarkar etal. 2008). This toxin is produced in every major part ofBt cotton plants like leaves, stem, and root (Dong and Li2007). During crop growth, soil micro-organisms comeinto direct contact with transgenic Cry endotoxin and itis released from Bt crops in root exudates and fromdecomposing tissues (Motavalli et al. 2004). While Btoccurs naturally in soil, growth of transgenic Bt cropscauses a large increase in the amount of Cry endotoxinpresent in agricultural systems, e.g., roughly 0.25 gha−1

produced naturally (calculated from approximately1,000 Bt spores g−1 soil) vs. 650 gha−1 in case of Btcotton crop, excluding grain (Blackwood and Buyer2004). Thus the transgenic plants, either through theproducts of introduced genes and modified rhizospherechemistry or through altered crop residue quality, havethe potential to significantly change the essential eco-system functions such as soil enzymatic activity, nutri-ent mineralization, microbial population, and plantgrowth (Dunfield and Germida 2004; Motavalli et al.2004; O’Callaghan et al. 2005). Although some researchhas examined the environmental impacts of the trans-genic crops (O’Callaghan et al. 2005; Sarkar et al.2008), relatively less research effort has examined theeffects of these crops on associated legume crop orlegume effect on these transgenic crops. Among all theapproaches of increasing the agricultural productivity,intercropping is one of the highly promising possibilitieswhich involves growing of two or more crops simulta-neously with distinct row arrangement for complemen-tary use of natural resources and enhancing productivity(Mandal and Mahapatra 1990). Cotton is sown at widerrow spacing (90–120 cm) hence provides sufficientspace for cultivation of short duration intercrop likepeanut (Singh et al. 2009). Intercropped legumes benefit

the associated cotton crop by either transferring a part offixed N2 or sparing effect because of their less N re-quirement (Subba Rao et al. 2001; Lupwayi andKennedy 2007). This practice stabilizes the productivitybesides enhancing the total returns (Blaise et al. 2005).Among the agronomic packages of Bt cotton, nitrogen(N) management is the most important factor decidingthe crop performance and maintenance of soil fertility,which is important in sustaining cotton productivity andprofitability (Karlen et al. 1998). Since N is costly inputand Bt cotton reduces N mineralization activity overnon-Bt cotton (O’Callaghan et al. 2005; Sarkar et al.2008), so efficient N management through optimumsynergistic combination of inorganic and organic sour-ces of N is essential for higher productivity, sustainabil-ity, and input use efficiency of Bt cotton. Up to now,much work has been done regarding the effects oforganic and inorganic amendments on soil biologicalactivity and/or enzymatic activity of Bt crops (Tu et al.2006; Bastida et al. 2008). However, the majorities ofsuch studies were mainly performed under laboratoryincubation conditions where short-term effects weresimulated only (Crecchio et al. 2001; Feng andSimpson 2009). Information is still scant on soil biolog-ical activities of Bt crops as influenced by application oforganic and inorganic amendments, especially seasonalsoil biological activities under field conditions in Indiaand global level. There is also relatively less informationis available on the interactive effects of cover crops andorganic amendments on soil biological activity under Btcotton field experimental conditions. Such informationplay a crucial role in maintaining balanced seasonalnutrient supply and higher soil productivity for Bt cot-ton. More importantly, there is a paucity of informationon the effects of intercropping and integrated N man-agement practices on enzymatic activity, N availability,and microbial population in soil of Bt cotton itself. Asadequate nutrient supply and efficient management arevery important to harness and sustain the potential yieldof Bt cotton, obviously there is a need to improvethe soil biological activities in the soil of Bt cotton. Inthis backdrop, a field experiment was undertakenwith the hypothesis that whether the peanut inter-cropping and substitution of N requirement of Btcotton with farm yard manure (FYM) nullifies the ad-verse effects of Bt cotton on soil biological activitiesunder kharif-irrigated subtropical situation in thenorthern zone of India.

486 Environ Monit Assess (2013) 185:485–495

Materials and methods

Experimental site

This field experiment was conducted in the kharifseason (warmer rainy season, June–November) of theyears 2006 and 2007 at the Indian AgriculturalResearch Institute, New Delhi, situated at 28° 35′Nlatitude and 77° 12′E longitude at an altitude of about228.61 m above mean sea level. It has a semi-arid andsubtropical climate with hot dry summers and severecold winters. The total precipitation during the studyperiod was 505.8 mm during 2006 and 457.0 mmduring 2007. Mean monthly relative humidity in2006 and 2007 ranged from 47.1% to 95.0%, and31.0% to 95.0%, respectively, during the period ofexperimentation. A composite representative soil sam-ple was collected from the experimental field prior toexperimentation and analyzed (Table 1). It belongs tothe hyperthermic family of typic haplustept. Watertable remained below 3.5 m deep from ground surfaceduring crop growth period.

Treatments

There were eight treatments comprising combinationof two cropping systems (C1, sole cotton; C2, cotton+

peanut) and four fertility levels [F0, control (0 N); F1

100% recommended dose of nitrogen (RDN) of cottonthrough urea; F2, 75% RDN of cotton through urea+25% N through FYM; and F3, 50% RDN of cottonthrough urea+50% N through FYM] to cotton werelaid out in a randomized block design with threereplications. In cotton, 150 kg N ha−1 was used asRDN. Peanut is another popular legume crop ofcotton-growing zones of India and the world; so tomake a comparison of legume vs. Bt cotton crop, apure stand of peanut crop was maintained during bothyears along with main experiment on cotton. Field wasinitially plowed twice in May after the harvest ofpreviously grown uniform crop of wheat and the grossplots of 18.0×6.0 m were marked. Conventionallyprepared FYM from cattle dung mixed with left-overcrop residues and well composted in a pit for over6 months was used. This well-decomposed FYM wasuniformly incorporated into the soil 7 days beforesowing as per treatments. FYM on dry weight basiscontained 178–5.0–2.0–5.0 gkg−1 organic C–N–P–K,respectively. Nonetheless, application of differentquantities of FYM resulted into variable amounts ofnutrient addition and their release pattern might havecaused changes in soil properties, hence crop growthand soil activities. Half N and full dose of P wereapplied at the time of sowing. Remaining N was top-dressed in the form of urea at square initiation stage ofcotton at 60 days after swing (DAS) along with secondirrigation. A uniform dose of 26 kg P ha−1 throughsingle superphosphate was applied at sowing to all thetreatments. Cotton “RCH-134 Bt” (180 days) wassown by dibbling with 120×60 cm geometry on 17June in 2006 and on 2 June in 2007. Five rows of non-Bt isogenic lines at the border as refugia crop werealso planted. In intercropped cotton, three rows ofpeanut “Punjab No. 1 (110 days)” were planted simul-taneously in between two cotton rows with 30×10 cmgeometry (additive series) without any extra doses offertilizers. In sole peanut, a uniform basal dose of20 kg N+26.2 kg P ha−1 was applied. One day afterthe sowing of both crops, a pre-emergence weedicide“pendimethalin” was applied in all the treatments. Inthe second cropping cycle (2007), the experiment wasrepeated in the different location with same layout.Sole and intercropped peanut was harvested in lastweek of September in both of years while cotton washarvested manually in two pickings in second and first

Table 1 Initial characteristics of the experimental soil from 0 to30 cm depth (air dry weight basis)

Parameters Value

Sand (%) 66.8

Silt (%) 14.3

Clay (%) 18.9

Textural class Sandy loam

Bulk density (Mg m−3) 1.52

Field capacity (%) 17.48

Permanent wilting point (%) 4.32

Organic carbon (mg kg−1 soil) 490

KMnO4-N (mg kg-1 soil) 96.4

Olsen’s P (mg kg−1 soil) 5.9

NH4OAc–K (mg kg−1 soil) 122.7

pH (1:2.5, soil/water) 7.8

ECe (dSm−1 at 25°C) 0.32

CEC (Cmol kg−1) 10.3

Dehydrogenase activity (μg TPF g soil−1 day−1) 238.6

Environ Monit Assess (2013) 185:485–495 487

fortnight of November, respectively in both the years.The entire above ground biomass of cotton and peanutwas removed at harvest. There was no incidence ofbollworms during study period in Bt cotton.

Sampling and analysis of soil and plants

Destructive soil samples (0–30 cm) were collected at0, 60, 90, 120 DAS, and at harvesting (180 DAS) ofcotton. At day 0 (just before FYM mixing), the soilsample was taken immediately. Second sampling(60 DAS) was carried out just before second topdressing of urea. Soils in between two cotton rows(0.15–0.20 kg) were collected to represent soil sample.At all the crop growth stages, five randomly selectedcotton plants from each plot were removed and kept inpaper bags for sun drying. Sun-dried samples weretransferred into a thermostatic drying oven and weredried at 65°C (36–48 h) to obtain a constant dry weight.Dehydrogenase activity was determined by usingthe method of Casida et al. (1964). The KMnO4-Nwas estimated by alkaline KMnO4 method sug-gested by Subbiah and Asija (1956). The rhizo-sphere soil samples collected at midstage of cotton andpeanut (90 DAS) were analyzed for population ofbacteria, fungi, and actinomycetes. The soil adher-ing to the roots was carefully collected and usedfor enumeration of total bacteria, fungi, and acti-nomycetes by standard serial dilution plate counttechnique using soil extract agar (Bunt and Rovira1955) for bacteria count, Martin’s Rose Bengalagar for fungi (Martin 1950) and Kusters ager foractinomycetes (Kuster and Williams 1964). Thenumber of effective root nodules at 90 DAS ofpeanut was counted by pinching with the help offingers, and those containing pink fluid were con-sidered effective in terms of biological N2 fixation(Idnani and Singh 2008).

Statistical analysis

The data collected on different parameters were sub-jected to appropriate statistical analysis following theprocedure described by Cochran and Cox (1957).Significant difference between means was testedthrough “F” test and the critical difference was workedout where variance ratio was found significant fortreatment effect. The treatment effects were tested at5% probability level for their significance. The data

pertaining to cotton and peanut were analyzed in ran-domized block design. The correlation coefficient wascalculated between different parameters by using themethod given by Snedecor and Cochran (1968). Thelevels of significance (P<0.01 and P<0.05) are basedon Pearson’s coefficients.

Results

Dry matter accumulation of cotton

In general, the rate of dry matter (DM) accumulation ofcotton was relatively slow up to 60 days (square initia-tion stage). Thereafter, a rapid increase in DM accumu-lation was noticed up to 120 days. Cropping system didnot significantly influence the DM accumulation at allthe crop growth stages but fertility levels did (Table 2).All the N treatments being at par with each otherrecorded higher DM over control at all the crop growthstages except at 90 DAS where substitution of 25%RDN of cotton through FYM being on par with 100%RDN of cotton through urea accumulated significantlyhigher DM over 50% RDN substitution through FYMand control. At all the crop growth stages, substitution of25% RDN of cotton through FYM recorded the highestcrop growth rate over other N treatments whereas con-trol (0 N) recorded the least crop growth rate.

Table 2 Effect of cropping systems and fertility levels on drymatter accumulation (gram per plant) of Bt cotton (mean data of2 years)

Treatment 60 DAS 90 DAS 120 DAS At harvest

Cropping systems

Sole cotton 66.5 148.5 373.0 495.5

Cotton+peanut 79.0 167.0 406.0 559.5

±SEM 4.00 5.24 19.28 18.55

CD (P00.05) NS NS NS NS

N dose (kg ha−1) and source (% urea–N–% FYM–N)

Control (0–0) 57.0 109.5 237.5 372.0

150 (100–0) 79.5 174.0 441.5 579.0

150 (75–25) 83.0 190.0 471.5 619.0

150 (50–50) 69.5 157.5 406.5 540.5

±SEM 5.65 7.41 27.26 26.23

CD (P00.05) 17.15 22.47 82.69 79.56

CD critical difference


Dehydrogenase activity of soil

The dehydrogenase activity of soil of both cotton andpeanut crops, in general, decreased from initial valuesup to the harvest but more decrease in dehydrogenaseenzyme was observed with peanut crop (Table 3).However, between 120 DAS and harvest of cottonand peanut, there was a sharp increase in dehydroge-nase activity of soil. Dehydrogenase activity of soilwas only increased in FYM containing treatments at60 DAS of cotton. Cropping system as well as fertilitylevels significantly affected dehydrogenase activity ofsoil at all the crop growth stages. Higher dehydroge-nase activity of soil was noticed with cotton intercrop-ped with peanut over sole cotton, at all the cropgrowth stages. Sole peanut maintained less dehydro-genase activity of soil than sole or intercropped cottonat all the crop growth stages. Higher dehydrogenaseactivity of soil was observed in 50% RDN of cottonsubstitution through FYM at all the crop growthstages, except at 60 DAS where 25% RDN of cottonsubstitution through FYM recorded higher activityover other fertility levels. The lowest dehydrogenaseactivity of soil was noticed in control (0 N) at all thecrop growth stages.

Interaction of cropping systems and fertility levelson dehydrogenase activity of soil at cotton harvest wasfound to be significant (Table 4). Cotton intercropped

with peanut fertilized by substitution of 50% RDN ofcotton through FYM maintained significantly higherdehydrogenase activity of soil over sole cotton.However, in both cropping systems substitution of50% RDN of cotton through FYM recorded signifi-cantly the highest dehydrogenase activity over otherfertility levels. Both the cropping systems maintainedmoreover similar dehydrogenase activity of soil atcontrol (0 N) and substitution of 25% RDN of cottonthrough FYM.

KMnO4-N content of soil

The soil KMnO4-N content was increased at all the cropgrowth stages of both crops in all the treatments incomparison to initial values except in control (0 N) at60, 120 DAS, and at harvest of cotton (Table 5).Cropping systems significantly influenced the KMnO4-N content of soil at all the crop growth stages except at60 DAS. After 60 DAS, cotton+peanut system main-tained significantly higher KMnO4 soil N over sole cot-ton. In between 120 DAS to harvest, soil KMnO4-Ndeclined in both cropping systems but this magnitudewas less in intercropped cotton (6%) over sole cotton(21%). Sole peanut maintained less KMnO4-N content ofsoil over cotton intercropped with peanut in contrast tosole cotton at all the crop growth stages. Fertility levelssignificantly influenced the KMnO4 soil N at all the cropgrowth stages. All the N treatments being at par with eachother maintained significantly higher KMnO4-N contentof soil over control at all the crop growth stages except at90 DAS where substitution of 25% RDN of cottonthrough FYM and 100% RDN of cotton through urea

Table 3 Effect of cropping systems and fertility levels ondehydrogenase activity of soil (microgram TPF per gram of soilper day) of Bt cotton and peanut (mean data of 2 years)


Cropping systems

Sole cotton 214.2 112.4 50.1 137.5

Cotton+peanut 227.6 123.5 59.3 162.7

±SEM 2.46 2.64 1.21 2.29

CD (P00.05) 7.41 7.91 3.64 6.87

Sole peanut 187.7 94.4 49.9 105.4


Control (0–0) 148.2 64.6 17.9 83.6

150 (100–0) 206.4 106.6 38.0 119.9

150 (75–25) 277.8 141.2 69.6 159.6

150 (50–50) 251.1 159.4 93.2 237.3

±SEM 3.49 3.73 1.72 3.24

CD (P00.05) 10.47 11.19 5.15 9.71


Table 4 Interaction effect of cropping system and fertility levelon dehydrogenase activity of soil (microgram TPF per gram ofsoil per day) at harvest of Bt cotton (mean data of 2 years)

N dose (kg ha−1) and source(% urea–N–% FYM–N)

Cropping system

Sole cotton Cotton+peanut

Control (0–0) 86.4 90.7

150 (100–0) 103.6 123.3

150 (75–25) 150.6 159.6

150 (50–50) 209.4 237.3

±SEM 4.58

CD (P00.05) 13.73



were equally effective. At harvest of cotton, 100% RDNthrough urea and control (0 N) were also maintainedsimilar KMnO4-N content of soil. Control (0 N) had thelowest KMnO4 soil N at all the crop growth stages.

Microbial population and effective root nodules

Among three experimental crops, sole peanut had thehighest microbial population at midstage of the crop(Table 6). Cotton intercropped with peanut maintained18%, 42%, and 45% less bacteria, fungi, and actino-mycetes population, respectively, over sole peanut. Byinclusion of peanut between cotton rows, bacteria

population increased by 60%, fungi 14%, and actino-mycetes 10% over pure stand of cotton. The number ofeffective root nodules per plant of intercropped peanut at90 DAS was 30% less than pure stand of peanut.Among 3 N management practices, cotton fertilized by50% RDN substitution by FYM maintained the highestmicrobial population which had 54% more bacteria,40% fungi, and 35% actinomycetes populations than100% RDN of cotton through urea. Control (0 N) hadthe lowest microbial population among all the fertilitylevels; however, this treatment maintained the highestnumber of effective root nodules of intercropped peanutbetween cotton rows. Cotton fertilized by 100% RDNthrough urea, maintained the lowest number of effectiveroot nodules of intercropped peanut.

Correlation matrix for crop productivity and soilproperties

DM accumulation of Bt cotton as a function of soilchemical and biological activities at various cropgrowth stages was positively correlated. The correla-tion relationship derived from mean data of two grow-ing seasons (Table 7) showed that DM accumulationwas significantly correlated with dehydrogenase activ-ity of soil at 60 DAS, after that a linear decrease wasobserved up to the harvest of cotton. Similarly, DMaccumulation was also significantly positively corre-lated with soil KMnO4-N content and microbial pop-ulation except bacteria population at 90 DAS. At90 DAS of cotton, DM accumulation and dehydroge-nase activity of soil were highly positively correlatedwith actinomycetes population than other microbialpopulation. Dehydrogenase activity and KMnO4-N

Table 5 Effect of cropping systems and fertility levels onKMnO4-N content (milligram per kilogram per soil) of soil ofBt cotton and peanut (mean data of 2 years)


Cropping systems

Sole cotton 121.3 133.1 134.4 110.8

Cotton+peanut 132.9 166.3 160.9 151.4

±SEM 10.90 5.65 7.68 10.99

CD (P00.05) NS 17.21 23.5 33.03

Sole Peanut 115.5 137.7 144.0 134.3


Control (0–0) 91.8 112.0 87.5 81.7

150 (100–0) 126.7 140.0 140.4 119.5

150 (75–25) 141.8 142.2 141.9 136.5

150 (50–50) 148.4 171.1 172.0 152.4

±SEM 15.42 7.99 10.87 15.58

CD (P00.05) 46.22 23.95 32.53 46.71


Table 6 Effect of croppingsystems and fertility levels onsoil microbial population of Btcotton and peanut and number ofeffective root nodules of peanutat mid crop stage (90 DAS;mean data of 2 years)

Treatment Bacteria Fungi Actinomycetes Number of effectiveroot nodule plant-1(×103) (×103) (×103)

Cropping systems

Sole cotton 135.6 36.7 98.9 –

Cotton+peanut 217.0 41.8 109.2 15.7

Sole peanut 265.7 72.0 197.0 22.5


Control (0–0) 101.2 31.7 71.6 18.5

150 (100–0) 128.2 34.6 99.5 11.8

150 (75–25) 145.4 42.5 110.7 15.0

150 (50–50) 197.5 48.3 134.2 17.2

Initial 42.6 17.2 33.0 –


content of soil was also highly positively correlated at60 DAS of cotton. Soil KMnO4-N content at 90 DASwas highly positively correlated with bacteria popula-tion than other microbial population. At this stage,bacteria population was more closely associated withfungi than actinomycetes population.

Discussion

Dry matter accumulation

Cotton intercropped with peanut did not significantlyaffect the DM accumulation over sole cotton. This mightbe due to noncompetitive environment between maincrop and intercrop in respect of available growth resour-ces such as solar radiation, moisture, and nutrients ow-ing to different growth habits of companion crops insystem (Sharma and Behera 2009; Blaise et al. 2005).The cotton crop fertilized by substitution of 25% RDNthrough FYM being at par with 100% RDN throughurea had the highest DM accumulation at all the cropgrowth stages. This might be due to availability of more

KMnO4-N in soil and uptake by plant parts with thesetreatments. Higher plant growth parameters with com-bined application of organic (FYM) and inorganic (urea)sources of N might be due to extended period of avail-ability of nutrients from combined source compared tourea alone. This might have increased photosyntheticactivities of plant which ultimately helped to realizedgreater DM accumulation (Das et al. 2006; Srinivasuluet al. 2004).

Dehydrogenase activity and KMnO4-N content of soil

Soil enzyme such as dehydrogenase was chosen as anindex of microbial activity because it refers to a group ofmostly intercellular enzymes which catalyzes the oxida-tion of soil organic matter and influenced by agronomicpractices (Forster et al. 1993). Dehydrogenase is anenzyme that involved in microbial oxido-reductase me-tabolism. Activity of this enzyme significantly correlateswith soil biomass carbon. Thus, dehydrogenase activityis indicated to be a good indicator of soil microbialactivity (Garcia et al. 1994). Dehydrogenase enzymeactivity increased with leguminous intercrop (peanut)

Table 7 Pearson’s correlation (r) matrix for plant parameters and soil biological properties during the growth period of Bt cotton

Properties Dry matteraccumulation

Dehydrogenase activity Soil KMnO4-N content Microbial population

a b c d a b c d a b c d B F A

Dry matteraccumulation

a 0.74** 0.72**

b 0.72** 0.57* 0.43 0.52* 0.64**

c 0.64** 0.77**

d 0.52* 0.78**

Dehydrogenaseactivity

a 0.94**

b 0.85** 0.72** 0.95** 0.98**

c 0.87**

d 0.86**

Soil KMnO4-Ncontent

a

b 0.95** 0.88** 0.91**

c

d

Microbialpopulation

B 0.82** 0.78**

F 0.95**

A

a 60 DAS, b 90 DAS, c 120 DAS, d at harvest, B bacteria, F fungi, A Actinomycetes

***Significant correlation at P<0.05 (n014)

**Significant correlation at P<0.01 (n014)


because of enhanced microbial activity and enzyme syn-thesis and accumulation due to increased carbon turn-over and nutrient availability (Dinesh et al. 2004; Meleroet al. 2007). Similarly, Martínez et al. (2007) alsoreported enhanced microbial and biochemical propertiesof soil by addition of peanut crop in cotton system atGeorgia, USA. The least values of dehydrogenase activ-ity of soil was observed in our field study at similarlocation or different location in comparison to pot studyof Bt cotton and non Bt cotton (Sarkar et al. 2008; Chenet al. 2011). On the one hand, the test soil in our studyhad lower organic carbon, less silt, and higher pH thanstudies on Bt cottons in pot experiments, and thesefactors might cause less dehydrogenase enzyme to accu-mulate in soil and on the other hand, the planting densityin our field experiment was lower than that in the potexperiment condition which might not contribute togather exogenous enzymes from Bt cottons and decreasetheir effects on soil microbial and enzyme activities. Incontrast to several studies which reported deleterious(O’Callaghan et al. 2005; Sarkar et al. 2008) or noapparent effect (Shen et al. 2006; Devare et al. 2004;Icoz and Stotzky 2008) in soil biological activities bygrowing Bt crops, we found that the dehydrogenaseactivity of Bt cotton soil either in pure stand or inintercropping with peanut was higher in comparison topure stand of peanut at all the crop growth stages. Thisfinding is in close agreement with the findings of Sun etal. (2006) who reported stimulated microbial and en-zyme activities of Bt cotton soil over non-Bt cotton soil.This effect can mask any negative effect of the Bt toxinon microbial activity and thus on enzyme activities.

Higher dehydrogenase activity of soil was observedin FYM containing treatments at all the crop growthstages over 100% RDN through urea alone (Sainju etal. 2008; Ramesh et al. 2006). This can be attributedmainly due to the large number of variable, dead andlivingmicroorganisms, and the large quantities of readilyutilizable energy sources introduced by organic manure(Ladd et al. 1994). Increased microbial activity in soils,as indicated by the results of soil enzymes, might also beresponsible for increased nutrient availability in FYMcontaining treatments (Gaofei et al. 2010). Substitutionof 50% RDN of cotton through FYM had the highestKMnO4 soil N at all the growth stages of cotton. Thismight be due to multidimensional role of FYM rangingfrom building up of organic matter, improving soilaggregation, soil permeability, and related physicalproperties to long-lasting supply of several macro and

micronutrients besides improving the cation exchangecapacity of soil (Ramesh et al. 2006; Sarkar et al. 2008).Increase in available Nmight be due to the direct additionof N through the FYM to the available pool of the soil. Itcould also be attributed to the greater multiplication ofsoil microbes which could convert organically bound Nto mineralized form (Ge et al. 2010; Hu et al. 2011).Mineral N in various soil fractions strongly affected claymineral type andmicrobes after the combined applicationof organic materials and synthetic N fertilizer (Qiu et al.2012; Diacono and Montemurro 2010).

Microbial population, effective root nodules,and correlation studies

Intercropped peanut between Bt cotton rows enhancedsoil microbial population over pure stand of Bt cotton.Dinesh et al. (2004) and Martínez et al. (2007) alsoreported enhanced microbial population of soil byaddition of peanut crop and rye grass (Zablotowiczet al. 2007) in cotton-based cropping systems due toincreased carbon turnover and nutrient availability.Changes of microbial population or the small propor-tion of active microbial biomass during the conversionperiod could explain the lack of correlation betweenbacteria population and DM accumulation at mid stage(90 DAS) of cotton. Cotton fertilized by 50% RDNsubstitution by FYM maintained the highest microbialpopulation than inorganic fertilization alone becauseorganic matter addition leads to a rapid increase of soilcarbon evaluation and increases the availability ofvarious enzymes and ultimately microbial population.FYM addition provided a stable supply of carbon andenergy for microorganisms and thus increased themicrobial population over inorganic fertilizer alone. Geet al. 2010) and Hu et al. (2011) reported significantlydecreased microbial activity of soil by long-term appli-cation of inorganic fertilizers alone in China. They rec-ommended combined use of organic manure withinorganic fertilizers to maintain soil fertility and micro-bial activities. There were also significant correlationsbetween soil microbial population, Soil KMnO4-N con-tent and dehydrogenase enzyme activity of soil (Ge et al.2010). This indicates that productivity of Bt cottongrown in subtropical inceptisols was enhanced and con-trolled by these soil parameters. Katkar et al. (2011) alsoreported similar correlation between soil KMnO4-Ncontent and dehydrogenase activity of soil with cropyield of sorghum and wheat in the vertisols of India.


The number of effective root nodules of intercroppedpeanut at 90 DAS was 30% less than pure stand ofpeanut because of enhanced N content of soil due tostimulated microbial activity of soil due to addition ofcotton tissues (Sun et al. 2006). Control (0 N) main-tained the highest number of effective root nodules ofintercropped peanut between cotton rows over N treat-ments. This might be due to reason that up to 60 DAS,cotton crop of N treatments did not offer N competitionto intercropped peanut because of less N requirement ofcotton (sparing effect) and high N requirement of inter-cropped peanut up to 40 DAS (no biological N2 fixa-tion) but in control (0 N) this mutual association couldnot established due to less availability of KMnO4-N(Subba Rao et al. 2001). At later stages, intercroppedpeanut in control did not faced solar radiation and Ncompetitions due to less shading effect from poor cottoncrop and high biological N2 fixation by peanut due toless KMnO4-N availability in soil (Lupwayi andKennedy 2007). Less number of effective root nodulesunder intercropping system over a pure stand of peanutis an indication that there was less atmospheric nitrogenfixation in the crop mixture (Maingi et al. 2001). Earlierstudies show a complementary use of N sources innonlegume–legume intercrops where the legume isforced to rely on nitrogen fixation because the nonlegumeis more competitive for soil inorganic N (Hauggaard-Nielsen et al. 2001).

Conclusion

The results of this field study indicate that Bt cottonhad no adverse effects on dehydrogeanse enzyme ac-tivity of soil but some adverse effects on soil microbialpopulation and KMnO4-N was observed when com-pared with pure crop of peanut. These adverse effectswere more pronounced when Bt cotton was fertilizedby urea alone. These effects can be nullified by in-volving peanut as an intercrop between wide inter rowspaces of Bt cotton and substitution of 25–50% ofrecommended dose of nitrogen through organic sour-ces like FYM for sustainable crop production on frag-mented and small holdings of Bt cotton belt areas oftropical countries like India.

Acknowledgments The authors greatly acknowledge the In-dian Agricultural Research Institute, New Delhi for providingfinancial assistance to conduct this study.

References

Bastida, F., Zsolnay, A., Hernández, T., & García, C. (2008).Past, present and future of soil quality indices: a biologicalperspective. Geoderma, 147, 159–171.

Blackwood, C. B., & Buyer, J. S. (2004). Soil microbial com-munities associated with Bt and non-Bt corn in three soils.Journal of Environment Quality, 33, 832–836.

Blaise, D., Bonde, A. N., & Chaudhary, R. S. (2005). Nutrientuptake and balance of cotton+pigeonpea strip intercrop-ping on rainfed vertisols of Central India. Nutrient Cyclingin Agroecosystem, 73(2–3), 135–145.

Bunt, J. S., & Rovira, A. D. (1955). Microbiological studies ofsub-antarctic soils. Journal of Soil Science, 6, 119–122.

Casida, L. E., Klein, D. P., & Santora, T. (1964). Solid dehy-drogenase activity. Soil Science, 93, 371–376.

Chen, Z. H., Chen, L. J., Zhang, Y. L., & Wu, Z. J. (2011).Microbial properties, enzyme activities and the persistenceof exogenous proteins in soil under consecutive cultivationof transgenic cottons (Gossypium hirsutum L.). Plant, Soiland Environment, 57(2), 67–74.

Cochran, W. G., & Cox, G. M. (1957). Experimental designs(2nd ed.). Wiley: New York.

Crecchio, C., Curci, M., Miinni, R., Ricciuti, P., & Ruggier, P.(2001). Short term effects of municipal solid wastes com-post amendments on soil carbon and nitrogen content,some enzyme activities and genetic diversity. Biology andFertility of Soils, 34, 311–318.

Das, A., Prasad, M., Gautam, R. C., & Shivay, Y. S. (2006).Productivity of cotton as influenced by organic and inor-ganic sources of nitrogen. Indian Journal of AgriculturalSciences, 76(6), 354–357.

Devare, M. H., Jones, C. M., & Thies, J. E. (2004). Effect ofCry3Bb transgenic corn and tefluthrin on the soil microbialcommunity: biomass, activity, and diversity. Journal ofEnvironment Quality, 33, 837–843.

Diacono, M., & Montemurro, F. (2010). Long-term effects oforganic amendments on soil fertility. A review. Agronomyfor Sustainable Development, 30, 401–422.

Dinesh, R., Suryanarayana, M. A., Ghosha, S., & Sheeja, T. E.(2004). Long-term influence of leguminous cover crops onthe biochemical property of a sandy clay loam soils. Soil &Tillage Research, 77, 69–77.

Dong, H. Z., & Li, W. J. (2007). Variability of endotoxinexpression in Bt transgenic cotton. Journal of Agronomy& Crop Sciences, 193, 21–29.

Dunfield, K. E., & Germida, J. J. (2004). Impact of geneticallymodified crops on soil- and plant-associated microbial com-munities. Journal of Environment Quality, 33, 806–815.

Feng, X. J., & Simpson, M. J. (2009). Temperature and substratecontrols on microbial phospholipid fatty acid compositionduring incubation of grassland soils contrasting in organicmatter quality. Soil Biology and Biochemistry, 41, 804–812.

Forster, J. C., Zech, W., & Wiirdinger, E. (1993). Comparison ofchemical and microbiological methods for the characterizationof the maturity of composts from contrasting sources. Biologyand Fertility of Soils, 16, 433–439.

Gaofei, G., Zhaojun, L., Fenliang, F., Guixin, C., Zhenan, H., &Yongchao, L. (2010). Soil biological activity and their


seasonal variations in response to long-term application oforganic and inorganic fertilizers. Plant and Soil, 326, 31–44.

Garcia, C., Hernandez, T., Costa, F., & Ceccanti, B. (1994).Biochemical parameters in soil regenerated by addition oforganic waste. Waste Management & Research, 12, 456–466.

Ge, G., Li, Z., Fan, F., Chu, G., Hou, Z., & Liang, Y. (2010).Soil biological activity and their seasonal variations inresponse to long-term application of organic and inorganicfertilizers. Plant and Soil, 326, 31–44.

Hauggaard-Nielsen, H., Ambus, P., & Jansen, E. S. (2001).Interspecific competition, N use and interference withweeds in pea-barley intercropping. Field Crops Research,70, 101–109.

Hu, J., Lin, X., Wang, J., Dai, J., Chen, R., Zhang, J., &Wong, M.H. (2011). Microbial functional diversity, metabolic quotient,and invertase activity of a sandy loam soil as affected bylong-term application of organic amendment and mineralfertilizer. Journal of Soils Sediments, 11, 271–280.

Icoz, I., & Stotzky, G. (2008). Fate and effects of insect-resistantBt crops in soil ecosystems. Soil Biology and Biochemistry,40, 559–586.

Idnani, L. K., & Singh, R. J. (2008). Effect of irrigation regimes,planting and irrigation methods and arbuscular mycorrhi-zae on productivity, nutrient uptake, economics and watereconomization of summer greengram (Vigna radiata var.radiata). Indian Journal of Agricultural Sciences, 78(1),53–57.

ISAAA. (2008). International Service for the Acquisition ofAgri-Biotech Applications (ISAAA). http://www.isaaa.org

Karlen, D. L., Kramer, L. A., & Logsdon, S. D. (1998). Field-scale nitrogen balance associated with long-term continu-ous corn production. Agronomy Journal, 90, 644–650.

Katkar, R. N., Sonune, B. A., & Kadu, P. R. (2011). Long-termeffect of fertilization on soil chemical and biological char-acteristics and productivity under sorghum (Sorghum bi-color)–wheat (Triticum aestivum) system in Vertisol.Indian Journal of Agricultural Sciences, 81(8), 734–739.

Kuster, E., & Williams, S. T. (1964). Selection of media forisolation of Streptomyces. Nature, 202, 926–929.

Ladd, J. N., Amato, M., & Zhou, L. K. (1994). Differentialeffects of rotation, plant residues and nitrogen fertilizeron microbial biomass and organic matter in Australianalfisol. Soil Biology and Biochemistry, 26, 821–831.

Lupwayi, N. Z., & Kennedy, A. C. (2007). Grain legumesin northern plains: impacts on selected biological pro-cesses. Agronomy Journal, 99, 1700–1709. doi:10.2134/agronj2006.0313s.

Maingi, M. J., Shisanya, A. C., Gitonga, M. N., & Hornetz, B.(2001). Nitrogen fixation by common bean (Phaseolusvulgaris L.) in pure and mixed stands in semi arid Southeast Kenya. European Journal of Agronomy, 14, 1–12.

Mandal, B. K., & Mahapatra, S. K. (1990). Barley, lentil andflax yield under different intercropping systems. AgronomyJournal, 82, 1066–1068.

Martin, J. P. (1950). Use of acid rose Bengal and streptomycin inplate method for estimating soil fungi. Soil Science, 9,215–232.

Martínez, V. A., Rowland, D., Sorensen, R. B., & Yeater, K. M.(2007). Microbial community structure and functionality

under peanut-based cropping systems in a sandy soil. Bi-ology and Fertility of Soils, 44(5), 681–692.

Melero, S., Madejon, E., Ruiz, J. C., & Herencia, J. F. (2007).Chemical and biochemical properties of a clay soil underdryland agriculture system as affected by organic fertiliza-tion. European Journal of Agronomy, 26, 327–334.

Motavalli, P. P., Kremer, R. J., Fang, M., & Means, N. E. (2004).Impact of genetically modified crops and their manage-ment on soil microbially mediated plant nutrient transfor-mations. Journal of Environment Quality, 33, 816–824.

O’Callaghan, M., Glare, T. R., Burgess, E. P. J., & Malone, L.A. (2005). Effects of plants genetically modified for insectresistance on nontarget organisms. Annual Review of En-tomology, 50, 271–292.

Qiu, S. J., Peng, P. Q., Li, L., He, P., Liu, Q., Wu, J. S., Christie,P., & Ju, X. T. (2012). Effects of applied urea and straw onvarious nitrogen fractions in two Chinese paddy soils withdiffering clay mineralogy. Biology and Fertility of Soils,48, 161–172.

Ramesh, P., Singh, M., Panwar, N. R., Singh, A. B., & Ramana,S. (2006). Response of pigeonpea varieties to organicmanures and their influence on fertility and enzyme activ-ity of soil. Indian Journal of Agricultural Sciences, 76(4),252–254.

Sainju, U. M., Senwo, Z. N., Nyakatawa, E. Z., Tazisong, I. A.,& Reddy, K. C. (2008). Tillage, cropping systems, andnitrogen fertilizer source effects on soil carbon sequestra-tion and fractions. Journal of Environment Quality, 37,880–888.

Sarkar, B., Patra, A. K., & Purakayastha, T. J. (2008). Trans-genic Bt-cotton affects enzyme activity and nutrient avail-ability in a sub-tropical inceptisol. Journal of Agronomy &Crop Sciences, 194, 289–296.

Sharma, A. R., & Behera, U. K. (2009). Recycling of legumeresidues for nitrogen economy and higher productivity inmaize-wheat cropping system. Nutrient Cycling in Agro-ecosystem, 83, 197–210.

Shen, R. F., Cai, H., & Gong, W. H. (2006). Transgenic Btcotton has no apparent effect on enzymatic activities orfunctional diversity of microbial communities in rhizo-sphere soil. Plant and Soil, 285, 149–159.

Singh, R. J., Ahalawat, I. P. S., & Gangaiah, B. (2009). Directand residual effects of nitrogen requirement in Bt cotton–wheat cropping system. Indian Journal of Agronomy, 54(4), 401–408.

Snedecor, G. W., & Cochran, W. G. (1968). Statistical methods.New Delhi: IBH Publishing.

Srinivasulu, K., Hema, K., & Krishna, R. (2004) Integratednutrient management in enhancing and sustaining cotton(Gossypium hirsutum L.) productivity. National Symp. onChanging World Order-Cotton research, development andpolicy in context. ANGRAU, Hyderabad, Aug. 10–12,2004.

Subba Rao, G.V., Kumar Rao, J.V.D.K., Kumar, J., Johansen,C., Deb, U. K., & Ahmed, I. (2001). Spatial distributionand quantification of rice fallow in South Asia—potentialfor legumes. ICRISAT, Hyderabad; NRSA, Hyderabad andDFID, UK, p 315

Subbiah, B. V., & Asija, G. L. (1956). A rapid procedure forestimation of available nitrogen in soils. Current Science,25(8), 259–260.


http://www.isaaa.org

http://dx.doi.org/10.2134/agronj2006.0313s

http://dx.doi.org/10.2134/agronj2006.0313s

Sun, C. X., Chen, L. J., Wu, Z. J., Zhou, L. K., & Shimizu, H.(2006). Soil persistence of Bacillus thuringiensis (Bt) toxinfrom transgenic Bt cotton tissues and its effect on soil enzymeactivities. Biology and Fertility of Soils, 43(5), 617–620.

Tu, C., Rustaino, J. B., & Hu, S. (2006). Soil microbial biomassand activity in organic tomato farming systems: effects oforganic inputs and straw mulching. Soil Biology and Bio-chemistry, 38, 247–255.

Venugopalan, M. V., Sankaranarayanan, K., Blaise, D., Nalayini,P., Prahraj, C. S., & Gangaiah, B. (2009). Bt cotton in Indiaand its agronomic requirements. Indian Journal of Agrono-my, 54(4), 343–360.

Zablotowicz, R. M., Locke, M. A., & Gaston, L. A. (2007).Tillage and cover effects on soil microbial properties andfluometuron degradation. Biology and Fertility of Soils, 44(1), 27–35.


effects of transgenic bt cotton on soil fertility and biology under field conditions in subtropical...

Documents