PLANT MICROBE INTERACTIONS

Bacterial Community Structure in the Rhizosphereof a Cry1Ac Bt -Brinjal Crop and Comparisonto Its Non-transgenic Counterpart in the Tropical Soil

Amit Kishore Singh &Govind Kumar Rai &Major Singh &

Suresh Kumar Dubey

Received: 18 December 2012 /Accepted: 30 August 2013 /Published online: 18 September 2013# Springer Science+Business Media New York 2013

Abstract To elucidate whether the transgenic crop altersthe rhizospheric bacterial community structure, a 2-yearstudy was performed with Cry1Ac gene-inserted brinjalcrop (Bt ) and their near isogenic non-transformed trait(non-Bt ). The event of Bt crop (VRBT-8) was screenedusing an insect bioassay and enzyme-linked immunosor-bent assay. Soil moisture, NH4

+-N, NO3−-N, and PO4

−-Plevel had non-significant variation. Quantitative polymer-ase chain reaction revealed that abundance of bacterial 16SrRNA gene copies were lower in soils associated with Btbrinjal. Microbial biomass carbon (MBC) showed slightreduction in Bt brinjal soils. Higher MBC values in thenon-Bt crop soil may be attributed to increased root activityand availability of readily metabolizable carbon com-pounds. The restriction fragment length polymorphism ofPCR-amplified rRNA gene fragments detected 13 differentbacterial groups with the exclusive presence of β-Proteobacteria, Chloroflexus, Planctomycetes, and Fuso-bacteria in non-Bt , and Cyanobacteria and Bacteroidetesin Bt soils, respectively, reflecting minor changes in thecommunity structure. Despite the detection of Cry1Acprotein in the rhizospheric soil, the overall impact ofCry1Ac expressing Bt brinjal was less compared to thatdue to seasonal changes.

Introduction

Brinjal (Solanum melongena L.; eggplant) is an importantsolanaceous vegetable crop in South Asia, particularly India,Bangladesh, Nepal and Sri Lanka. This region accounts foralmost 50 % of the global land area under brinjal cultivation,amounting to 1.85million ha producing 32million metric tons(MTs) [21]. Brinjal (average annual production 12.6 millionMTs, cultivation area 0.69 million ha) ranks second to tomato(average annual production 18.6 million MTs, cultivation area0.91 million ha), in India [39]. During 2007–2008, 34 millionkilograms of brinjal (worth US$0.38 million) was exportedmainly to the UK, the Netherlands, South Arabia, and theMiddle East [19].

Brinjal is highly susceptible to insects and pests, and spe-cially, fruits are highly prone to damagemainly by Leucinodesorbonalis , the fruit and shoot borer (FSB) insect. The FSBlarvae lower marketable fruit yield (60–70 %) [13]. Currently,control measures of FSB mainly rely on the extensive appli-cation of chemical pesticides. Farmers usually spray insecti-cides twice a week with 15–40 sprays or more in one season,depending upon the extent of infestation; as a consequence,pesticides continue to accumulate in fruits [13]. Pesticideconsumption may cause severe health hazards to humans,such as mild cognitive dysfunction and hormonal imbalancesleading to infertility, breast pain, menstrual disturbances, ad-renal gland exhaustions, and early menopause [58]. Bt spray(mixture of cells, spores, and parasporal crystals of subspeciesof Bacillus thuringiensis ) as insecticide is not very effectivedue to higher cost, rapid environmental inactivation, and poorcrop coverage [7]. Genetically engineered crops could providean alternative to pesticides used as they have the potential forhigh productivity and thereby help address the food securityproblem especially for small-scale agriculture in developingcountries [4].

Electronic supplementary material The online version of this article(doi:10.1007/s00248-013-0287-z) contains supplementary material,which is available to authorized users.

A. K. Singh : S. K. Dubey (*)Department of Botany, Banaras Hindu University, Varanasi 221005,Indiae-mail: skdubey@bhu.ac.in

G. K. Rai :M. SinghIndian Institute of Vegetable Research, Varanasi 221305, India

Microb Ecol (2013) 66:927–939DOI 10.1007/s00248-013-0287-z

Transgenic crops, in many cases, enhance crop productiv-ity by minimizing the crop damage through pest-resistantvarieties. The number of countries growing transgenic cropsincreased from 6 in 1996 to 29 in 2011, and the trend is likelyto continue [30]. Risks of transgenic crop include transgeneflow in the indigenous plants or organisms through pollentransfer [16], plant invasiveness [15], development of resistantpests and pathogens [15], and loss of biodiversity [9]. Moststudies on GMOs focus on above-ground processes and hu-man health risks. GMOs also alter soil microbes and theprocess they mediate [29, 49]. Since these processes contrib-ute to the fertility of the soil, it is interesting to understand theplant–microbe interaction. The influence of transgenic cropson soil microbial communities varies with gene insert, plant,and environment [42].

Rhizosphere is considered the hotspot for the soil microbialdiversity and activity, and the associated microbial communi-ties may vary in species composition depending on the planttype and age, among others [43, 46]. Root exudates vary withthe plant development stages and help structure the rhizo-sphere microbial community [31]. Genetic modifications ofthe plants can change the biochemical nature and also thequality of root exudates that, in turn, may affect the develop-ment of rhizospheric microbial community. Many plants (e.g.,corn, cotton, potato, canola, and rice) are now geneticallyengineered to express the B. thuringiensis -derived Cry pro-teins. Cry proteins confer resistance to agriculturally impor-tant pests, when taken up and transformed to the active δ-endotoxin in the midgut of the target organism. Truncatedforms of gene-encoding toxins are genetically engineered intothe plants and express active larvicidal proteins in tissues, thusmaking the plant resistant to various insects and pests [47].There is a possibility of soil exposure of Cry proteins via theroot exudates or through plant tissues and decomposed plantlitter [28, 48]. These proteins remain active in soil, whereadsorption to clay particles and humic acids protects againstmicrobial degradation [14], and could affect microbial com-munities and the processes they mediate.

In a previous study, Pal et al. [40] developed seven inde-pendent transgenic Bt brinjal events of cultivar “Kashi Taru(IVBL-9)” using the insect resistance conferring Cry1Acgene. In the present follow-up, we select an efficient Bt brinjaland assess changes in the rhizosphere bacterial communitystructure (diversity and density) following cultivation of Btbrinjal.

Materials and Methods

Experimental Site and Plant Material

Field trial experiments were performed during two consecu-tive years (2010 and 2011) at the agricultural farm of the

Indian Institute of Vegetable Research, Varanasi, India (25°08′N latitude, 83° 03′ E longitude, 90 m of elevation from sealevel, and average temperature of a maximum of 33 °C andminimum of 20 °C). The site has been used for intensivevegetable production. The soil (WHC 39.9 %) is pale brownsilty loam, Inceptisol with pH 6.7, organic C (0.73 %), total N(0.09 %) [57].

Seven independent transgenic events (VRBT-1, VRBT-2,VRBT-4, VRBT-6, VRBT-7, VRBT-8, and VRBT-9) ofbrinjal (S. melongena L. var. Kashi Taru) were developedthrough inserting the Cry1Ac gene, a strategy applied toprotect it against a variety of lepidopteron insect pests. Theexpression of this gene is controlled by the Cauliflower mo-saic virus 35S promoter and octopine synthase gene. Forkanamycin resistance, the transgenic plants incorporated theneomycin phosphotransferase II gene (npt II) controlled bynopaline synthase promoter [40].

Such Bt brinjal transgenic events were grown in random-ized blocks with three replications during June 2010. Seeds ofthe most efficient transgenic event (VRBT-8) and its non-transgenic counterpart (IVBL-9) were grown in pots(29 cm×31.5 cm×19.5 cm) with sufficient amount of fullydecomposed farm yard manure (FYM) mixed with soil duringthe last week of August. Healthy and disease-free seedlings(with three to four leaves) were transplanted in the insect proofcontainment net house facility with spacing maintained as60×60 cm from plant to plant and row to row. Transplantingwas done in complete randomized block design in six plots,each at 12 m2 (three each for transgenic and non-transgenic,respectively). Recommended brinjal cultivation practice wasadopted for soils before transplantation by adding 25–30 tonnes/ha FYM along with NPK application (100–120 kgN, 75–85 kg P and 45–50 kg K) [11]. Irrigation was doneevery 10–15 days to optimize the yield.

Plant, Soil Sampling, and Their Analyses

Leaf, shoot, fruit, and root were collected at the maturationstage for the screening of the efficient Bt brinjal event byusing the insect bioassay as well as the enzyme-linked immu-nosorbent assay (ELISA) test. After screening, the soil sam-ples (in triplicate) were collected at branching, flowering, andmaturation including pre-plantation and postharvest stage ofthe crop during the two consecutive years (2010–2011). Threeplants per plot of non-Bt and Bt were gently removed, and therhizospheric soils were collected by gentle shaking of roots todislodge the adhering small soil clumps. Sticky soils attachedto the root system were carefully removed using forceps andmixed evenly to form a composite soil sample. Soil samples atpre-vegetation and post-harvest stage were collected from 0 to10 cm of depth using a 5-cm diameter soil corer [57]. Toremove the plant material, all the soil samples were passedthrough a 2-mm sieve and stored (3–5 days) at 4 °C to slow

928 A.K. Singh et al.

down the microbial activity for consecutive analyses. Soilmoisture was determined gravimetrically, and available nutri-ents like ammonium-N (NH4

+-N), nitrate-N (NO3−-N), and

phosphate-P (PO4−-P) were determined as per the standard

method [3].Microbial biomass C (MBC)was estimated by thechloroform fumigation–extraction method [56].

Insect Bioassay

Insect bioassay was conducted to assess the efficacy of thegene in the seven transgenic events. One laboratory-rearedthird-instar pre-weighed FSB larva (L. orbonalis) was releasedonto 3-mm-thick aseptic slices of detached fruits of seven Btbrinjal events and those of the non-transgenic one (IVBL-9)along with a susceptible check (Punjab Sadabahar). One pre-weighed larva was released onto one slice, and each slice wasplaced in a separate Petri dish (90 mm×15 mm diam) contain-ing water-soaked Whatman paper. The larvae were allowed tofeed in the laboratory (28±2 °C and 70±5%RH) for 6, 10, 14,18, and 22 days; the fruit slices were replaced after every5 days. Feeding of the larva was monitored regularly forweight gain and mortality at 6, 10, 14, 18, and 22 days ofinterval. Three replications were carried out for each Bt brinjalevent as well as feeding durations. Larval mortality (in per-centage) was assessed at end of the feeding durations [40].

ELISATest

Cry1Ac protein expression in the different plant parts of theseven transgenic brinjal events along with the non-Bt cultivarwas quantified through quantitative ELISA analysis [40]. TheELISA was conducted using DesiGen® Quan-T Cry1Ac/Cry1Ab ELISA Kit (Mahyco, Jalna, India) coated with theCry1Ac/Ab-1 monoclonal antibodies against Cry1Ac, follow-ing the manufacturer's instructions. Fresh leaf, shoot, fruit, orroot samples (20 mg) from the transgenic events along withthe non-transformed plant were ground in 100 μl of extractionbuffer, and the homogenate was trypsinized (37 °C, 30 min).Anti-Cry1Ac/Ab-2 antibody was added to the wells, followedby the trypsinized sample homogenate, and incubated (37 °C,1.5 h). The plates were washed twice, incubated at 37 °C(45 min) with AP-conjugated Cry1Ac/Ab-3 antibody, andagain washed twice. Finally, 1 mg ml−1 para -nitrophenolphosphate substrate was added following dark incubation(30 min), and the absorbance was measured at 450 nm usingan ELISA plate reader (Thermo Corp., Helsinki, Finland).

The rhizospheric soils of the transgenic event (VRBT-8)and the non-Bt cultivar (IVBL-9) were subjected to ELISA forquantification of the Cry1Ac protein during different growthstages of the Bt and non-Bt brinjal plants. To extract theCry1Ac protein from the soil samples, a buffer solution asdescribed by Palm et al. [41] was used. The buffer contained10 mM ascorbic acid, 50 mM sodium borate (pH 10.5),

0.75 M potassium chloride, and 0.075 % Tween 20. Samples(500mg) were mixed with the extraction buffer in the 1:2 ratio(w/v), and homogenized (10–15 s; 11,000×g ) using a FastPrep® (MP Biol, USA). Soil suspension was incubated(37 °C, 45 min) and then centrifuged (5–6 min, 16,000×g ).Supernatants were filtered (0.25-μm syringe filters;Millipore), and 100 μl of the supernatant was loaded ontothe ELISA plate wells coated with Cry1Ac/Ab-1 monoclonalantibodies against Cry1Ac. The rest of the steps that followedwere as per the manufacturer's instruction, as discussed earlier.The concentration of Cry1Ac protein was estimated by refer-ring to a seven-point standard curve of purified Cry1Ac stan-dard provided with kit and included in each microplate.

Soil DNA Extraction

Total genomic DNA (in triplicate) was extracted from 0.5 g ofrhizosphere soil using Fast DNA® Spin Kit (MP Biol, USA)combined with Fast DNA prep bead beater according to themanufacturer's protocol. The gDNAwas eluted in 50 μl DESand stored (−20 °C) for subsequent analysis. The concentra-tion and purity levels were determined using Nanodrop Spec-trophotometer (ND 1000; NanoDrop Technologies Inc., Wil-mington, DE, USA).

Real-Time Quantitative PCR and Quantification of TotalBacterial 16S rRNA Gene Copy Number

Real-time PCR amplification was performed (qPCR, ABI750; Applied Biosystems®, CA, USA) to quantify the abun-dance of total bacterial 16S rRNA gene copy number usinguniversal primers (Eub338F 5′-ACT CCT ACG GGA GGCAGC AG-3′) and (Eub518 R 5′-ATT ACC GCG GCT GCTGG′-3) [20]. The PCR reaction was carried out in a final 20-μlvolume containing 10× SYBR Green PCR Master Mix(Fermentas, USA). The reaction mixture (20 μl) comprisedof 7.5 μl master mix, 10 pmol each of primer, and 40 ngtemplate. The PCR condition was as follows: initial denatur-ation at 95 °C (15 min), followed by 40 cycles of 95 °C(1 min), annealing at 53 °C (30 s), and extension at 72 °C(1 min) [20]. Plasmid DNA containing the respective targetgene (16S rRNA) sequence, used as the standard DNA inqPCR, was obtained by PCR cloning using the universalbacterial primer set [20]. The standard curve revealed a slopeof −3.143 corresponding to an efficiency of 108.06 % and R2

of 0.999, similar to those reported by others [26, 59].

PCR Amplification, Cloning, Restriction Fragment LengthPolymorphism, and Phylogenetic Analysis of 16S rRNAGene

Total bacterial 16S rRNA gene in the extracted DNA of non-Bt- and Bt-planted soils was amplified (in triplicate) using

Bacterial Community Structure and Transgenic Brinjal Crop 929

bacterial universal primers, 27F (5′-AGAGTTTGATCMTGGCTCAG-3′) and 1492R (5′-GGYTACCTTGTTACGACTT-3′) [33]. The PCR was carried out using thermal cycler(Bio-Rad, CA, USA) under the following conditions: (94 °C,5 min; 30 cycles of denaturation at 94 °C (1 min), annealing at60 °C (1 min and 30 s), and extension at 72 °C (1 min) and72 °C (5 min). The reaction mixture (25 μl) contained 2.5 μlof 10× buffer (Bangalore Genei, Bangalore, India), 0.5 μl of40 mM dNTPs (Fermentas, USA), 1.25 μl each of 10 μMforward and reverse primers (Sigma), 2.5 U Taq DNA poly-merase (Bangalore Genei), and a 1-μl template (40 ng). Therest of the volume was maintained by nuclease-free water. Theamplified PCR products were electrophoresed on agarose gel(1.5 %). The PCR products of 1,500 bp were pooled andpurified using the QIAprep Spin MiniPrep Kit.

The purified PCR products (in triplicate) were pooled andligated into the p-GEM®T Easy Vector (Promega, USA) asper the manufacturer's protocol and transformed into theEscherichia coli DH5α competent cells by heat shock. Thescreening of blue and white colonies was performed on am-picillin plates (100 μg ml−1) supplemented with X-gal(0.5 mM) and IPTG. Positive insert verification was donemanually by plasmid isolation and desired insert size of pos-itive clones checked through M13 colony PCR [57]. The 16SrRNA clone library of each sampling stage was analyzed bydigesting the positive M13 PCR products (20 μl) using 1 Ueach of tetra cutterMsp I andHaeIII restriction enzymes (NewEngland Biolabs, Beverly, MA, USA) as described by Ahmadet al. [2]. A total of 283 16S rRNA gene-positive clones ofnon-Bt and 275 clones of Bt brinjal-planted soils weresubjected to restriction fragment length polymorphism(RFLP) analysis. The zero–one matrices were prepared onthe basis of RFLP pattern and operational taxonomic units(OTUs) grouped for each soil sample in the form of dendro-gram by the CLUSTALW program using the NTSYS version2.1 software. More than one representative of each OTU wassequenced.

The sequencing of the amplified clone was performed onboth the strands in Applied Biosystems 3130 Genetic An-alyzer (Applied Biosystems) using the Big Dye TerminatorKit (Version 3.1). Clones were finally checked for chimericartifacts by using CHECK-CHIMERA of the RibosomalDatabase Project, and the chimeric sequences werediscarded. The 16S rRNA sequences obtained were initiallyrecognized and aligned against the known sequences in theGenBank database by using the BLAST program of theNational Center for Biotechnology Information (NCBI,http://www.ncbi.nlm.nih.gov/) search. The 16S rRNAclones obtained from the non-Bt - and Bt -plantedrhizospheric soils with >90 % similarity with the NCBIdatabase were adopted for phylogenetic analysis usingMEGA software version 4.0 [54]. Further details regardingthe sequence analysis are given by Shukla et al. [52].

Statistical Analysis

The complete randomized design with three replicates wasused. Analysis of variance (ANOVA) was performed to de-termine the effect of treatments (non-Bt and Bt) at differentstages. All parameters were compared for significant changesby using Tukey's highest significant difference test (HSD)with SPSS 16.0. The results reported are at p= 0.05.

Nucleotide Sequence Accession Numbers

The nucleotide partial sequences of the 16S rRNA gene fromthe representative OTUs were deposited in NCBI under thefollowing accession numbers: JN573624–JN573650,JN637767–JN637778, HQ663864, HQ663865, HQ663859,HQ663854, HQ663861, HQ663875, and HQ663868,JQ346700, and JQ346701.

Results

Insect Bioassay

There were two groups of responses to Bt. Several constructskilled almost all the larvae, and some doubled the mortalitybut were not as effective. Expression of Cry1Ac increasedinsect mortality from 44% in controls to 60–99% inBt brinjal(Table 1). One cultivar, VRBT-8, showed the highest signifi-cant mortality rates and killed up to 99 % of the larvae in22 days.

Quantification of Cry 1Ac Protein in the Transgenic Events

The Cry1Ac protein was detected in all plant parts of thetransgenic events, and not in non-Bt plants. The amount ofCry1Ac in all the seven transgenic events ranged from 69 to97 ng g−1 in leaf, from 50 to 68 ng g−1 in shoot, from 59 to80 ng g−1 in fruit, and from 27 to 42 ng g−1 in the root(Table 2). In VRBT-8, the level of Cry1Ac was the highestover all the plant parts tested with the following sequence: leaf(97 ng g−1) > fruit (80 ng g−1) > shoot (68 ng g−1) > root(43 ng g−1). FSB mortality correlated positively (R2=0.65;P=0.012) with Cry 1Ac protein content.

Soil Parameters

Inorganic nutrients and soil moisture content did not differsignificantly between the Bt and non-Bt brinjal rhizosphericsoil (Table 3); however, soil parameters varied significantlybetween sampling stages (Table 4).

930 A.K. Singh et al.

Bacterial Population Size

A significant difference (P <0.05) in the bacterial populationsize was observed between the Bt and non-Bt brinjal rhizo-sphere soil. The 16S rRNA gene copy number was higher innon-Bt brinjal soils compared to Bt brinjal during the year2010. The same trend was also observed in the following year(Fig. 1). Among the stages, 16S rRNA gene copy numberswere significantly higher at the flowering stage followed bymaturation, branching, post-harvest, and pre-vegetation stagein both years (Fig. 1). ANOVA indicated significant differ-ences due to year and treatment (Table 4).

Quantitative Analysis of Cry1Ac Protein from Rhizosphereof VRBT-8

In the rhizosphere soils, the Cry1Ac protein level was verylow relative to protein (Cry1Ac) detected in plant tissue.Interestingly, this was observed at several stages in 2010, witha maximum at flowering stage. During the year 2011, theflowering stage also exhibited a similar trend (Table 5).

Soil Microbial Biomass Carbon

Microbial biomass was significantly lower in samples gener-ated from Bt brinjal plots than in the parallel controls (Fig. 2).

The flowering stage had the highest MBC value in non-Bt andBt brinjal compared to other sampling stages. ANOVA indi-cated significant differences due to treatment and stages;however, the effect of year was not significant (Table 4). Apositive correlation was found between bacterial populationsize and MBC content (R2=0.69; P=0.006) and Bt (R2=0.72; P=0.002) for non-Bt -planted soils.

Phylogenetic Analysis of Bacterial 16S rRNA GeneSequences from Non-Bt and Bt Brinjal Soil

The 46 OTUs defined by RFLP showed their closest phylo-genetic resemblance with 13 different bacterial groups:Firmicutes, uncultured bacteria, δ-Proteobacteria, α-Proteobacteria, Fusobacteria, Actinobacteria, Chloroflexus,Planctomycetes, Cyanobacteria, Acidobacteria, β-Proteobacteria, γ-Proteobacteria, and Bacteroidetes (Fig. 3).

Firmicutes are clustered with seven OTUs in which TBR6II, AMB2 III, AMB1 I, and AMB3 IVwere detected under Btbrinjal soil during branching, flowering, pre-vegetation, andmaturation stage, while EML9 I, EML11 V, and EML10 IVwere detected in the non-Bt brinjal-planted soil during pre-vegetation, post-harvest, and maturation stage. TBR6 II,AMB2 III, and EML9 I showed >90 % resemblance withPaenibacillus , Bacillus algicola , and Cohnella sp., respec-tively, while AMB1I, EML11 V, EML10 IV, AMB3 IV, and

Table 2 Concentration ofCry1Ac protein (in nanogram pergram fwt) in different parts of thenon-Bt and Bt plants

Data represent the mean values±SE of three different plants (n =3). Differences between meanswere compared by Tukey's HSDtest (P<0.05); mean values hav-ing the same letter are not signif-icantly different

ND not detected

Plant Leaf Shoot Fruit Root

Non-Bt ND ND ND ND

VRBT-1 82.84±1.23 bc 60.50±3.02 ab 72.32±3.99 ab 34.71±1.55 a

VRBT-2 74.16±2.16 bc 53.19±3.42 bc 75.69±1.48 ab 41.15±3.33 a

VRBT-4 80.01±0.83 bc 65.46±2.32 a 77.92±4.0 ab 37.45±3.68 a

VRBT-6 69.68±2.37 c 50.42±0.28 c 67.39±3.88 ab 27.20±3.84 a

VRBT-7 84.86±4.40 ab 53.28±3.65 bc 70.61±2.77 a 31.63±3.52 a

VRBT-8 97.58±2.45 a 68.10±3.17 a 79.92±4.09 a 42.68±3.83 a

VRBT-9 81.23±5.14 bc 50.40±0.42 c 59.11±4.71 b 38.85±2.15 a

HSD0.05 8.65 7.55 9.29 8.72

Table 1 Mortality (in percent-age) of fruit and shoot borer neo-nate larvae on fruit slices of dif-ferent Cry1Ac-inserted transgenicbrinjal events

Data represent the mean values±SE of three different experi-ments. Differences betweenmeans were compared byTukey's HSD test (P <0.05).Common lowercase letters in-dicate that values are not sig-nificantly different

Plants 6 days 10 days 14 days 18 days 22 days

Non-Bt 19.6±0.5 c 20.0±0.8 e 20.4±1.0 d 19.3±0.5 d 43.6±1.2 c

VRBT-1 60.2±1.2 b 82.8±1.0 b 80.0±0.7 c 84.5±0.4 b 86.4±1.2 b

VRBT-2 60.6±0.9 b 60.7±0.5 d 81.0±1.3 c 81.0±1.6 b 84.6±1.4 b

VRBT-4 60.0±0.1 b 78.2±0.4 c 82.7±0.5 c 96.4±1.4 a 97.5±1.1 a

VRBT-6 80.1±0.6 a 80.0±0.9 bc 80.6±1.4 c 83.2±1.6 b 98.0±0.8 a

VRBT-7 79.8±1.0 a 83.2±0.7 b 90.6±0.6 b 92.2±1.2 a 93.3±0.3 a

VRBT-8 83.5±1.6 a 89.8±0.1 a 98.9±1.0 a 98.2±1.1 a 99.0±0.4 a

VRBT-9 80.5±0.5 a 80.0±0.8 bc 78.0±0.9 c 71.2±1.2 c 80.4±0.7 b

HSD0.05 3.73 3.92 4.38 5.29 4.34

Bacterial Community Structure and Transgenic Brinjal Crop 931

AMB 4 V were clustered with Bacillus sp. in a separate clade.OTUs EML8 IVand AMB7 IV were detected only during thematuration stage of non-Bt and Bt brinjal-planted soils, re-spectively, having >94 % similarity with the uncultured bac-teria (Supplementary Tables S1 and S2).

α-Proteobacteria dominated all stages sampled of non-Bt-and Bt-planted soils. OTUs of pre-vegetation stages such as

EML 4 I and PV2Ib were 95 % affiliated with Rhizobiumtropici , while EML1 II and EML3Vof non-Bt and AMB17Vof Bt brinjal-planted soil were grouped separately. Again, inthe same α-Proteobacterial group, EML2 III and EML5 IVofnon-Bt and AMB16 IV were grouped with the unculturedBeijerinckiaceae (98 % similarity). AMB15 III was affiliated(98 %) to Devosia sp., while AMB14 II was grouped with R.

Table 3 Variation in soil mois-ture (in percentage), NH4

+-N (inmicrogram per gram), NO3

−-N (inmicrogram per gram), and PO4

−-P(in microgram per gram) contentsin non-Bt and Bt brinjal-plantedsoils. Stage 1, pre-vegetation; 2,branching; 3, flowering; 4, matu-ration; and 5, post-harvest

Letters a, b, c, and d, where com-mon, indicate that mean valuesare not significantly different (P <0.05 by Tukey's HSD test)

Stages Crop Year Soil moisture NH4+-N NO3

−-N PO4−-P

1 Non-Bt 2010 15.4±1.08 a 05.29±0.13 a 03.41±0.56 a 10.61±0.33 a

2011 14.52±0.62 a 05.49±0.73 a 03.27±0.34 a 10.06±0.44 a

Bt 2010 15.4±1.08 a 05.29±0.14 a 03.41±0.57 a 10.61±0.34 a

2011 14.52±0.63 a 05.49±0.74 a 03.27±0.34 a 10.06±0.44 a

2 Non-Bt 2010 17.81±0.89 b 09.03±0.21b 05.61±0.38 b 09.25±0.20 b

2011 16.61±0.51b 09.79±0.11b 05.73±0.42 b 09.80±0.47 b

Bt 2010 17.92±0.69 b 09.87±0.41b 05.65±0.23 b 09.02±0.15 b

2011 17.06±0.62 b 08.96±0.21 b 05.36±0.53 b 09.18±0.10 b

3 Non-Bt 2010 18.36±0.23c 10.77±0.29 c 06.0±0.18c 06.55±0.51 c

2011 18.59±0.36 c 10.8±0.24 c 06.20±0.18 c 06.12±0.42 c

Bt 2010 18.55±0.81 c 11.06±0.14 c 06.26±0.28 c 06.01±0.31 c

2011 19.05±0.40 c 10.59±0.33 c 06.38±0.32 c 06.22±0.30 c

4 Non-Bt 2010 16.42±0.62 b 08.40±0.45 b 07.50±0.26 d 04.28±0.28 d

2011 17.47±0.43 b 08.08±0.18 b 07.20±0.31d 04.16±0.19 d

Bt 2010 17.08±0.25 b 09.50±0.28 b 07.44±0.29 d 04.41±0.27 d

2011 17.23±0.27 c 09.20±0.43 b 07.37±0.37 d 04.10±0.17 d

5 Non-Bt 2010 14.62±0.36 a 05.27±0.23 a 06.51±0.44 c 03.82±0.21d

2011 14.38±0.79 a 05.07±0.17 a 06.63±0.41 c 04.17±0.11 d

Bt 2010 13.99±0.30 a 05.16±0.44 a 06.17±0.61 c 04.04±0.19 d

2011 14.05±0.39 a 05.39±0.43 a 06.26±0.14 c 03.92±0.19 d

Table 4 Results of multivariate analysis of variance for observed parameters. Treatment: non-Bt and Bt

Factors Year Statistical parameters Observed parameters

Soil moisture NH4+-N NO3

−-N PO4−-P MBC Bacterial population

Treatment 2010 F1, 20 0.02 5.89 0.07 0.99 12.03 5.18

P 0.87 0.25 0.78 0.33 0.01 0.03

2011 F1, 20 0.62 0.08 0.12 0.16 13.52 13.34

P 0.43 0.77 0.73 0.68 0.01 0.02

Stage 2010 F4, 20 16.74 131.00 36.55 64.24 275.12 4.46

P 0.00 0.00 0.00 0.00 0.01 0.01

2011 F4, 20 29.26 72.88 36.65 28.45 278.09 14.09

P 0.00 0.00 0.00 0.00 0.01 0.01

Year Pooled F1, 40 0.10 0.07 0.04 3.89 0.31 14.14

P 0.74 0.78 0.95 0.05 0.58 0.01

Treatment Pooled F1, 40 0.36 2.97 0.19 0.87 25.55 17.43

P 0.55 0.09 0.66 0.35 0.01 0.01

Stage Pooled F4, 40 38.65 189.5 72.95 83.16 55.13 16.61

P 0.00 0.00 0.00 0.00 0.01 0.01

932 A.K. Singh et al.

tropici in a separate clade at flowering and branching stages,respectively. Within δ-Proteobacteria, only one OTU (EML13III) was detected during the flowering stage in case of non-Btbrinjal, while AMB8 II and AMB9Vwere detected during thebranching and maturation stages (relative abundance, 35 and19 %) and showed >93 % relatedness with Stigmatellaaurantiaca (Figs. 3 and 4). EML6 II belonging to γ-Proteobacteria was present only in rhizosphere soils during

the branching stage of non-Bt brinjal (relative abundance,8 %) and was 94 % related to Azotobacter chroococcum .AMB12 II andAMB13 III were recovered from Bt brinjal-planted soils during the branching and flowering stages (rel-ative abundance 32 and 4 %, respectively) and >92 % simi-larity with gamma Proteobacteria (Fig. 4).

Acidobacteria-related OTUs recovered mostly from Btbrinjal soil were detected during branching (EML16 II), mat-uration (EML18 IV), flowering (EML17 III), and post-harveststage (EML19V), while single OTU (AMB11 III) was foundduring the flowering stage, and PV3 I, during the pre-vegetation stage. These OTUs were similar (>94 %) touncultured Acidobacteria.

OTUs TBR4 II, AMB6 IV, and AMB5 III were foundin the Bt brinjal-planted soils, while NBR1 II, NBR6 IV,and NBR 10 III were common during branching, matu-ration, and flowering stages of non-Bt brinjal-plantedsoils. AMB5 III, NBR1 II, NBR6 IV, and NBR 10 IIIshowed resemblance (>90 %) with the unculturedAcidomicrobiales and Nocardioides, while other OTUs(TBR4 II and AMB6 IV) attained separate positionswithin the Actinobacterial group (Fig. 3).

Groups showed similarity to β-Proteobacteria, Plan-ctomycetes, Chloroflexus, and Fusobacteria exclusively during

Fig. 1 Variation in bacterialpopulation size in non-Bt and Btbrinjal crop-planted soil atdifferent crop growth stages.Different letters denotesignificant difference (P <0.05)estimated by Tukey's HSD, andthe bar indicates extent ofvariation from the mean

Table 5 Cry 1Ac protein content (in nanogram per gram fwt) at differentplant stages of Bt brinjal (VRBT-8)

Stages 2010 2011

Non-Bt ND ND

Pre-vegetation ND ND

Branching 0.31±0.10 ND

Flowering 0.69±0.14a 0.61±0.12a

Maturation 0.30±0.11 ND

Postharvest ND ND

HSD0.05 0.30 ND

ND not detecteda Significant changes from maturation stage

Bacterial Community Structure and Transgenic Brinjal Crop 933

the flowering stage exclusively in non-Bt brinjalrhizospheric soil (Fig. 3). OTUs like EML7 III (94 %),EML15 III (94 %), and EML14 III (90 %) were similarto Thiobacillus , uncultured Planctomycetes, and uncul-tured Fusobacteria, respectively. However, in Chloro-flexus affiliating OTUs, EML20 IV (87 %) was detectedonly during the maturation stage with a relative abun-dance of 9 %. Similarly, exclusive OTUs generated fromBt brinjal-planted rhizospheric soil showed similarity toCyanobacteria and Bacteroidetes (Fig. 3). AMB10 II wassimilar (99 %) to the uncultured Cyanobacteria and wasdetected only during the branching stage (relative abundance,4 %). However, Bacteroidetes related to both the OTUs suchas AMB18 IV (92 %) and AMB19 IV (98 %) detected duringmaturation stage (25 % relative abundance) were similar touncultured Bacteroidetes (Fig. 4).

Discussion

Selection of the Efficient Event for Cry1Ac Activity

The data on insect bioassay and ELISA are in agreement withthe findings of Pal et al. [40] who reported significantly higher

percentage of FSB larval mortality and adverse effects onlarval growth for larvae fed on the transformed brinjal shootand fruit tissues. The transformation of brinjal was ascertainedby the toxic effects of Cry1Ac protein [22] that could have ledto significant changes in larval periodic weight gain andmortality.

Soil Moisture Content and Available Soil Nutrients

In the present study, variations in the soil moisture andavailable soil nutrients (NH4

+-N, NO3−-N, and PO4

−-P)were more influenced by crop development stage than thecrop type (non-Bt vs. Bt-brinjal). Moisture content differedbetween crop developmental stages. Soil moisture influ-ences soil processes dramatically [36], but soil processrates such as decomposition and soil respiration were notmeasured in this study. Similarly, significant variation inthe levels of available nutrients among the plant develop-mental stages could be attributed to plant–microbe compe-tition for soil nutrient uptake [18]. Transgenic plants altersoil nutrient transformations [37] and decrease inorganicnitrogen availability [45], but such changes were not ob-served in this study.

Fig. 2 Variation in MBC content(in microgram per gram) in non-Bt and Bt brinjal rhizosphericsoil. Different letters denotesignificant difference (P <0.05)estimated by Tukey's HSD testover error bar

934 A.K. Singh et al.

Fig. 3 Phylogenetic analysis of bacterial 16S rRNA sequences andrelated species by neighbor-joining method obtained from the non-Bt(NBt) and Bt brinjal crop-planted soil across the different crop growthsincluding pre-vegetation (PV) and post-harvest. Stages are denoted byroman letters I (pre-vegetation), II (branching), III (flowering), IV

(maturation), and V (post-harvest). Boot strap values above 50 % areindicated at the nodes. The scale bars represent 0.05 substitutions persite. GenBank accession numbers are indicated for each sequence clone inparenthesis

Bacterial Community Structure and Transgenic Brinjal Crop 935

Cry1Ac Protein in the Rhizosphere of VRBT-8

High Cry1Ac protein detected at the flowering stage mayreflect high root activity at this stage [27] and the associatedleakage from border cells [32]. Cry proteins enter soils throughroot exudates [28, 48], and Bt toxin in the rhizosphere peaks atthe advanced crop stage [44]. The low percentage of clay in thesoils studied herein [57] could explain the relatively low pro-tein concentrations, as clay particles protect proteins frommicrobial degradation. Cry proteins degrade rapidly in soilswith low clay content [6, 28]. The present observations agreewith those of Head et al. [25] and Muchaonyerwa et al. [38]that the Cry proteins in transgenic plants are short-lived in thesoil.

Bacterial Population Size

A significant variation in the bacterial population sizebetween the rhizosphere soil of non-Bt and Bt crops wasalso observed. This change is consistent with the previousreports that insertion of Bt gene into transgenic plants mayalter either the amount or character of one or more rootexudates vital to microorganisms in rhizosphere, therebyaffecting the bacterial population [17]. In addition, cropgrowth stage also affected the bacterial population. Themaximum 16S rRNA gene copy number as detected inthe flowering stage in non-Bt and Bt brinjal clearly

suggests enrichment of the bacterial population duringthe flowering stage owing to the optimum nutrient avail-ability. The results are consistent with the findings ofothers [53] and also strongly support the findings ofGschwendtner et al. [23] wherein the influence of plantdevelopmental stage on the 16S rRNA gene abundancewas more pronounced, although the trends are in contrastto the present report. This is because they reported thattotal bacterial 16S rRNA gene copy number decreased withthe increasing plant age in field trials. The present studyindicates that Bt brinjal might exert a little negative impacton the bacterial population.

Molecular Characterization of Rhizospheric BacterialCommunity

The exclusive groups detected in non-Bt (β-Proteobacteria,Fusobacteria, Planctomycetes, and Chloroflexus) and Bt(Bacteroidetes and Cyanobacteria) brinjal-planted soil mightbe due to Cry1Ac gene-mediated genetic modifications inbrinjal. These exclusive groups were confined to eitherflowering or maturation stages and were low in the relativeabundance compared to the dominant groups (α-Proteobacteria, Firmicutes, Acidobacteria, and Actinobacteria).This observation leads one to assume that genetic modificationcould unintentionally change the plant characteristics (root exu-dates) in several ways [24] that might selectively enrich the

Fig. 4 Relative abundance ofbacterial 16S rRNA clones acrossthe different crop growth stages innon-Bt and Bt brinjal crop-planted soils at different samplingstages

936 A.K. Singh et al.

bacterial community distinct from the bacterial community as-sociated with the non-Bt-planted soils. Brusetti et al. [10] re-ported that exudates from transgenic plants selected particulargroups of bacteria. Insertion of genes can change exudate qual-ity, which will select for different bacteria [50].

Changes in the microbial community in the rhizosphere ofsoils associated with transgenic brinjal were less than thevariations between crop stages; however, the effects wereminor compared to the shifts caused by the crop developmen-tal stages. Our result are in agreement with those of Barriusoet al. [5] who observed nomajor variation in the rhizobacterialcommunity associated with the non-Bt and Bt maize during a4-year study. Similarly, Miethling-Graff et al. [35] found nosignificant difference between the rhizospheric bacterial com-munity structure of the Bt maize and other cultivars over thethree consecutive years of the study. Rasche et al. [42] report-ed on minor or transient effect of transgenic potatoes (express-ing T4 lysozyme) on the rhizosphere-associated bacterialcommunity compared to variations induced by soil type, plantgenotype, plant age, and pathogen exposure. Despite thelimitation of the methods [1], significant changes were detect-ed. The changes may bemore profound than reported [50, 51].

Microbial Biomass Carbon

Lower MBC in treatments could reflect the changes in rootactivity, level of root exudates, and the availability of readilymetabolizable carbon compounds [34]. Reduced bacterialpopulation in Bt brinjal could also reduce the MBC. Thesetrends are consistent with the findings of Tarafdar et al. [55]and Chen et al. [12] regarding the significant reductions ofMBC under Bt cotton compared to that of non-Bt cotton.High MBC during the flowering stage is consistent withprevious studies [51] and reflects changes in root exudatesand moisture [8]. Our observations that such changes weregreater than those associated with GMOs agree with those of aprevious report [18].

Concluding Remarks

MBC and bacterial population were significantly reducedunder Bt brinjal compared to non-Bt brinjal soil. Cry Acprotein and several OTUs were detected exclusively in therhizosphere associated with transgenic plants. The exclusivepresence of β-Proteobacteria, Chloroflexus, Planctomycetes,and Fusobacteria in non-Bt , and Cyanobacteria andBacteroidetes in Bt crop-planted soils indicated minorchanges in the community structure. The effect of geneticallymodified brinjal on the bacterial community structure is lesscompared to seasonal variations.

Acknowledgments This research work was supported by Indian Insti-tute of Vegetable Research, India. One of the authors (AKS) is grateful tothe Council of Scientific and Industrial Research, NewDelhi, for financialassistance in the form of JRF and SRF.We are also grateful to anonymousreviewers whose constructive comments improved the manuscript.

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