Rhizospheric fungal community structure of a Bt brinjal and a near isogenic variety

ORIGINAL ARTICLE

Rhizospheric fungal community structure of a Bt brinjal anda near isogenic varietyA.K. Singh1, M. Singh2 and S.K. Dubey1

1 Department of Botany, Banaras Hindu University, Varanasi, India

2 Indian Institute of Vegetable Research, Varanasi, India

Keywords

Bt brinjal, Cry1Ac gene, fungal communities,

ITS-rRNA-RFLP, rhizosphere.

Correspondence

Suresh Kumar Dubey, Department of Botany,

Banaras Hindu University, Varanasi 221005,

India.

E-mails: [email protected];

[email protected]

2014/0697: received 2 April 2014, revised 17

May 2014 and accepted 17 May 2014

doi:10.1111/jam.12549

Abstract

Aims: The objective of this study was to investigate the influence of Cry1Ac

gene expressing brinjal (VRBT-8) on the rhizospheric fungal community

structure.

Methods and Results: qPCR indicated variations in the fungal ITS rRNA copy

numbers of non-Bt (1�43–4�43) 9 109 g�1 dws and Bt (1�43–3�32) 9 109 g�1

dws plots. Phylogenetic analysis of ITS rRNA clones indicated fungal-related

group majority of being Ascomycota compared to that of Basidiomycota and

Zygomycota in non-Bt- and Bt-planted soils. Sordariomycetes was the dominant

class detected in all the stages.

Conclusions: Despite the variations in the population size and the distribution

pattern observed across the non-Bt and Bt brinjal, plant-growth-dependent

variability was more prominent compared with genetic modification.

Therefore, this study concludes that genetic modification of brinjal crop has

minor effect on the fungal community.

Significance and Impact of the Study: Brinjal, the important solanaceous crop,

is also prone to attack by many insect pests, especially by Leucinoides orbonalis,

resulting in significant losses in the crop yield. However, the reports on the

effect of transgenic crops and the associated microbial community are

inconsistent. The present communication takes into account for the first time

the possible interactions between Bt brinjal and the associated fungal

community; the latter playing a significant role in maintaining soil fertility. As

this study is limited to the structural diversity of fungal community, additional

information regarding the functional diversity of the group seems imperative

before recommending the commercialization of GM crops.

Introduction

With the development and use of transgenic technology,

the possible effects of transgenic plants on the environ-

ment have become issues of public concern (Saxena et al.

2002). The Bt gene originating from Bacillus thuringiensis

is widely exploited for endowing plant resistance against

pests (Benedict and Ring 2004). Truncated forms of the

genes that code for Bt toxin protein have been genetically

engineered into plants. It is reported that transgenes (like

Cry proteins) expressed by the Bt crops enter the soil

through root exudates and bind rapidly onto a surface

active components (clay minerals, humic acids and on

the complexes of M-humic acids-Al hydroxypolymers) in

soil and become less accessible for microbial degradation

but retain insecticidal activity (Crecchio and Stotzky

1998; Young and Stotzky 1999; Vettorri et al. 2003). As a

result, these proteins could accumulate in soil and may

influence the biological and chemical processes in the soil

as well as the microbial community and composition.

Numerous field studies were carried out to investigate the

persistence of Cry proteins in rhizospheric soil (Icoz and

Stotzky 2008; Wang et al. 2009). The effect of the Cry

proteins on soil microbial communities and soil-mediated

processes may vary depending on the type of plant and

gene insert (Icoz et al. 2008). A summary of the available

Journal of Applied Microbiology 117, 750--765 © 2014 The Society for Applied Microbiology750

Journal of Applied Microbiology ISSN 1364-5072

published information on the impact of Cry1Ac express-

ing transgenic crops on soil ecosystem is given in Table

S1. It is clear from this summary that earlier workers

have studied the effect of genetically modified corn, cot-

ton, potato, cabbage and rice on microbial community

and soil fertility. Most of these studies are concerned only

with the effect on the culturable fungal population den-

sity as determined by plate counts (Donegan et al. 1995,

1996; Dutta et al. 2012; Tarafdar et al. 2012; Velmou-

rougane and Sahu 2013). Tan et al. (2010) and Wei et al.

(2012) studied the effect of Bt corn and Bt rice on fungal

diversity, respectively. They observed no or little effect in

their respective studies. Shen et al. (2006), Sun et al.

(2007), Sarkar et al. (2008), Chen et al. (2011), Tarafdar

et al. (2012) and Chen et al. (2012) have studied the

effect of Cry1Ac protein on enzyme activities in soil, but

here, again the results are inconsistent. Reports of MAH-

YCO (www.envfor.nic.in/divisions/csurv/geac/bt_brinjal.

html) focused only on the effect of Bt brinjal on cultur-

able fungal populations. Singh et al. (2013b,c) have

recently assessed the variation in actinomycetes and bac-

terial community in soil planted with Cry1Ac expressing

Bt brinjal. However, no effort has been made to assess

the effect of Bt brinjal on fungal community structure

(diversity and density). Therefore, analysis of fungal com-

munity structure in relation to Bt brinjal is warranted.

This study seems relevant as fungi are considered a

potential soil fertility indicator along with actinomycetes

and bacteria.

The rhizosphere is considered the most active micro-

habitat in the field for microbial activity. Soil microbes

quickly respond to changes in root exudate quantity and

chemical composition and are believed to be plant spe-

cies, cultivar and growth stage specific (Saxena et al.

1999; Rengel 2002; Marcial-Gomes et al. 2003). Soil

micro-organisms are one of the main sources of soil

enzymes (Nannipieri et al. 1983). Like bacteria, fungi are

ubiquitous micro-organisms, which play an important

role in the soil ecosystem as major decomposers of

organic matter and release nutrients via nutrient cycling

that stimulate plant growth in tropical soil (Lodge 1995).

Some fungi are well known to cause a range of plant

diseases, some that devastate the entire agricultural crop

(Jarosz and Davelos 1995; Thorn 1997). Other fungi pos-

sess antagonistic properties towards plant pathogens.

The global area under brinjal cultivation is expected to

be 1�72 million hectare with total production of brinjal

fruit of about 43 million MTs (FAO 2012). India

accounts for about 12�6 million MTs of production with

a cultivation area of 0�69 million hectares (NHB 2011).

Brinjal cultivars are susceptible to a variety of stress con-

ditions, which significantly limit productivity. The most

important biotic stress is the damage caused by the

Leucinoides orbonalis commonly known as Brinjal Shoot

and Fruit Borer (BSFB) forcing farmers to use higher

doses of some insecticides, for example indoxacarb,

imidacloprid, cypermethrin, which is a matter of serious

health concern. Therefore, to reduce the dependency on

insecticides, transgenic technology as an alternative tech-

nique is being adopted. Insecticidal crystal proteins

(ICPs) derived from B. thuringiensis (Bt) have proved to

be most economical in limiting the use of insecticides

(Icoz and Stotzky 2008).

Soil microbiological and biochemical properties have

often been proposed as sensitive indicators (microbial

population, microbial biomass and soil enzymes) of

anthropogenic effects on soil ecosystems (Dick and Taba-

tabai 1993; Kandeler et al. 1999). Soil enzymes play an

important role in catalysing microbial-mediated soil pro-

cesses, such as organic matter decomposition and nutri-

ent cycling (Nannipieri et al. 1990). Dehydrogenase

(DHA) and fluorescein diacetate (FDA) activities indicate

energy transfer and thus microbial viability. Urease, acid

phosphomonoesterase and invertase indicate nutrient

transformation in soil (Guan et al. 1986; Green et al.

2006). Bt toxin released from transgenic crops might

compete with soil enzymes for binding sites of soil parti-

cles and may influence the soil microbial activities that

are useful for soil health. No information is available

about the effects of Cry1Ac conferred transgenic brinjal

on soil enzymes.

Therefore, the objective of this study was to investigate

the effect of Cry1Ac gene expressing Bt brinjal on struc-

ture of fungal communities and selected soil enzymes

under field conditions.

Materials and methods

Sampling site description and plant material

Field experiments were performed in the agricultural

farm of Indian Institute of Vegetable Research (I.I.V.R.),

Varanasi, India (25°080N latitude, 83°030E longitude,

90 m elevation from sea level, average temperature maxi-

mum 33°C and minimum 20°C). Previously, the site had

been used for intensive vegetable productions such as

tomato, cauliflower, cabbage, okra, chilli, bean, pumpkin,

bottle gourd, radish, carrot, but has never been exposed

to any transgenic crop cultivation prior to this study

which occurred during the consecutive years of 2010 and

2011. The soil was classified as inceptisol with 39�9%water-holding capacity (WHC), pale brown silty loam

(sand 30%, silt 70%, clay 2%), pH 6�7, organic C 0�73%and total N 0�09% (Vishwakarma et al. 2010).

Ten-day-old seedlings of the VRBT-8 transgenic line

were selected as the efficient event validated both by

Journal of Applied Microbiology 117, 750--765 © 2014 The Society for Applied Microbiology 751

A.K. Singh et al. Fungal community structure and Bt brinjal

insect bioassay and ELISA (Singh et al. 2013c). The trans-

genic line VRBT-8 was developed by inserting Cry1Ac

gene, which is a strategy applied to brinjal to protect it

against a variety of lepidopteran insect pests. Expression

of this gene is controlled by the Cauliflower mosaic virus

(CAMV) 35S promoter and octopine synthase gene

(OCS). To select the kanamycin resistance, the transgenic

plants were incorporated with the neomycin phospho-

transferase II gene (npt II) controlled by nopaline syn-

thase (NOS) promoter (Pal et al. 2009).

Experimental design and soil sampling

The field experiment was conducted with Bt brinjal

(VRBT-8) and its near isogenic nontransgenic brinjal

(IVBL-9). Plants were grown in six plots of randomised

blocks design each of 12 m2 (three replications each for

transgenic VRBT-8 and nontransgenic, respectively)

under containment conditions as per biosafety regulations

approved by biosafety committee, IIVR, Varanasi. Rec-

ommended brinjal cultivation practice was adopted in

which soil was prepared before transplantation by adding

25–30 tonnes/ha farmyard manure (FYM) along with

NPK application (100–120 kg N, 75–85 kg P and

45–50 kg K per hectare) (Chadha 2001). Irrigation was

carried out at the interval of every 10–15 days to main-

tain the optimum moisture condition.

Soil samples (in triplicate) were collected at branching,

flowering and maturation including prevegetation and

postharvest stages of the crop during the two consecutive

years of 2010 and 2011. Three plants from plots of non-

Bt and Bt were gently removed, and the rhizospheric soils

from (about 20 cm rooting depth) were collected by

shaking the roots to dislodge small clumps of soil adher-

ing to the roots. The remaining soil attached to root sys-

tem was considered as rhizosphere soil. Soil adhering to

the root system was carefully removed using forceps and

mixed evenly to form a composite soil sample. Soil sam-

ples at prevegetation and postharvest stage were collected

from 0–20 cm depth using a 5-cm-diameter soil corer

(Vishwakarma et al. 2010). To remove plant material, all

the soil samples were passed through a 2-mm sieve and

stored at 4°C for consecutive analyses.

Detection of Cry1Ac protein in the soil

The rhizospheric soils of the transgenic line (VRBT-8)

and the non-Bt cultivar (IVBL-9) were subjected to

ELISA for quantification of the Cry1Ac protein during

different growth stages of the Bt and non-Bt brinjal

plants. A buffer solution as described by Palm et al.

(1994) was used to extract the Cry1Ac protein from the

soil samples. Soil samples (500 mg) were mixed with the

extraction buffer in the 1 : 2 ratio (w/v) and homoge-

nized (10–15 s; 11 000 g) using a Fast Prep� (MP Biol,

Irvine, CA). The soil suspension was incubated (37°C,45 min.) and then centrifuged (5–6 min, 16 000 g). Su-

pernatants were filtered (0�25-l filter syringe; Millipore

GmbH, Eschborn, Germany), and 100 ll of the superna-

tant was loaded onto the ELISA plate wells coated with

Cry1Ac/Ab-1 monoclonal antibodies against Cry1Ac. Rest

of the steps followed were as per the manufacturer’s

instruction, as discussed by (Pal et al. 2009). Concentra-

tion of Cry1Ac protein was determined by referring to a

seven-point standard curve of purified Cry1Ac standard

provided with kit and included in each microplate.

Enzymatic assay

Urease, dehydrogenase and acid phosphomonoesterase

activities in soil were determined as described by Tabata-

bai (1994). Invertase was analysed according to Guan

et al. (1986), and FDA hydrolysis as per the protocol of

Adam and Duncan (2001). For DHA activity, 5 g field

moist soil sample (triplicate) was hydrated to field capac-

ity in a test tube, mixed with a solution of 2, 3, 5-triphe-

nyltetrazolium chloride (TCC) 3% and dark incubated

(37°C). The soil mixture was then washed with methanol.

The intensity of the resulting pink-coloured formazon

was measured at 485 nm. Urease activity was assessed

using urea as the substrate. The ammonia released was

determined at 578 nm. Acid phosphomonoesterase activ-

ity assay used p-nitrophenyl phosphate as the substrate,

and CaCl2 (0�5 mol l�1) and NaOH (0�5 mol l�1) added

to precipitate humic molecules and the extract (p-nitro-

phenol) measured at 410 nm.

Soil invertase activity determination used sucrose solu-

tion as the substrate. The amount of reactant glucose fol-

lowing incubation was determined. For FDA assay, the

soil sample (2 g) was mixed with 20 ml of 60 mmol l�1

phosphate and incubated (60 min, 30°C). Following

incubation, 20 ml of 2 : 1 chloroform/methanol was

added to stop the reaction. Centrifugation (5000 g,

5 min) settled the soil, and the aqueous (buffer) and the

organic (chloroform/methanol) phases (5 ml) were fil-

tered and the intensity read at wavelength 490 nm. The

experimental conditions for all the enzymatic assays are

given in Table S2. Enzymatic activities were quantified

colorimetrically (in triplicate).

Soil DNA extraction

Total genomic DNA (in triplicate from each sampling

stages) was extracted from 0�5 g rhizosphere soil using

Fast DNA� spin kit (MP Biol) combined with Fast DNA

prep bead beater according to manufacturer’s protocol.


Fungal community structure and Bt brinjal A.K. Singh et al.

The genomic DNA was eluted in 50 ll DNA elution

solution (DES) and stored (�20°C) for subsequent analy-sis. The concentration and purity levels were determined

using nanodrop spectrophotometer (ND 1000; Nano

Drop Technologies, Inc., Wilmington, DE).

Real-time quantitative PCR and quantification of total

fungal ITS rRNA gene copy number

Real time Quantitative PCR (qPCR) amplification was per-

formed to quantify the abundance of total fungal internal

transcribed spacer (ITS) rRNA gene copy number using

universal primer 5�8 s F (50-CGC TGC GTT CTT CAT

CG0-3) suggested by Vilgalys and Hester (1990) and ITS1f

R 50-TCC GTA GGT GAA CCT GCG G0-3 (Fierer et al.

2005). The amplifications were carried out in a final vol-

ume of 25 ll containing 109 SYBR Green PCR master mix

(Fermentas, St. Leon-Rot, Germany). The reaction mixture

(25 ll) comprised of 7�5 ll master mix, 10 pmol each of

primer and 50 ng DNA template. The PCR conditions

were, initial denaturation 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.) (Fierer et al. (2005). The ampli-

fied products (300 bp) of the fungal ITS rRNA gene were

purified using the PCR purification kit (QIAprep spin

MiniPrep kit; Qiagen, Hilden, Germany), ligated into

pGEM�-T easy vector (Promega, Madison, WI) and

cloned into Escherichia coli DH5 a stain.For the quantification of total fungal ITS rRNA copy

number, a standard was prepared from recombinant E. coli

plasmid containing the targeted gene. Ten-fold serial dilu-

tions (10�1 to 10�9) were used to construct the calibration

curve. The threshold cycle (Ct), that is, the cycle at which

the fluorescence in the sample increased above a predefined

threshold, was determined. Standard curve was generated

by linear regression obtained between threshold cycle (Ct)

and copy number of target gene using ABI PRISM 7900 SDS

2.2.2 software (Applied Biosystems, Weiterstadt, Ger-

many) and used for estimation of gene abundance in

unknown DNA samples. The standard curve revealed a

slope of–3�425 corresponding to an efficiency of 95�8% and

R2 of 0�992, similar to those reported in other studies

(Zhang and Fang (2006); Hussain et al. 2011).

PCR amplification, cloning, RFLP and phylogenetic

analysis of fungal ITS rRNA

Total fungal-specific ITS rRNA from the extracted DNA of

different sampling stages was amplified (in triplicate for all

sampling stages) using primers sets, ITS 1F (50-CTT GGT

CAT TTA GAG GAA GTA A-30) and universal ITS4 R (50-TCC TCC GCT TAT TGA TAT GC-30) (Lord et al. 2002;

Fierer et al. 2005) because fungal ITS sequences generally

provide greater taxonomic resolution than sequences gen-

erated from coding regions (18S rRNA) (White et al.

(1990); Anderson et al. 2003; Klamer and Hedlund 2004).

PCR amplification as described by Klamer and Hedlund

(2004) was followed using thermal cycler (My CyclerTM

Thermal Cycler, Bio-Rad Laboratories, Hercules, CA)

under the following conditions: 5 min of initial denatur-

ation at 95 ⁰C followed by 35 cycles of 0�5-min denaturation

at 94 ⁰C, 2 min of annealing at 52 ⁰C and 3-min extension

at 72 ⁰C. Final extension was 5 min at 72°C. Reaction mix-

ture (25 ll) contained 2�5 ll of 109 buffer (Bangalore Ge-

nei, Bangalore, India), 0�5 ll of 40 mmol l�1 dNTPs

(Fermentas), 10 pmol each of forward and reverse primers

(Sigma, St. Louis, MO), 2�5 U Taq DNA polymerase (Ban-

galore Genei) and 1 ll template (40 ng). Rest of the vol-

ume was maintained by nuclease-free water. The amplified

PCR products were electrophoresed on agarose gel (1�5%).

The PCR products of 600 bp approx. were pooled and

purified using the Qiagen Mini Prep kit (Qiagen).

The purified PCR products were ligated into the

p-GEM�T Easy vector (Promega) as per manufacturer’s

protocol and transformed into the E. coli DH5a competent

cells by heat shock. The screening of blue and white colo-

nies was performed on ampicillin plates (100 lg ml�1)

supplemented with X-gal (0�5 mmol l�1) and IPTG. Posi-

tive insert verification was carried out manually by plasmid

isolation, and desired insert size of positive clones checked

through M13 colony PCR (Vishwakarma et al. 2010).

The ITS rRNA clone library was constructed by digest-

ing the positive M13 PCR products (20 ll) using 1U

each of tetra cutter Hinf I and Hae III restriction enzymes

(New England Biolabs, Beverly, MA) as described by

Chen and Cairney (2002) and Curlevski et al. (2010). A

total of 273 ITS rRNA gene-positive clones of non-Bt and

272 clones of Bt-brinjal-planted soils were subjected for

restriction fragment length polymorphism (RFLP) analy-

sis. The zero–one matrices were prepared on the basis of

RFLP pattern, and operational taxonomic units (OTUs)

were grouped for each soil sample in the form of dendro-

gram by the CLUSTAL W program using the NTSYS ver. 2.1

software. More than one representative of each OTU was

selected for sequencing.

Phylogenetic analysis of ITS rRNA genes

The sequencing of the amplified clones were performed

on both the strands in ABI PRISM� 3100 Genetic Analyzer

(ABI, Applied Biosystems, Foster City, CA) using the Big

Dye Terminator Kit (ver. 3.1; Cycle Sequencing Kit

(Applied Biosystems, Rotkreuz, Switzerland)). Electroph-

orograms were edited using the Chromas freeware (ver.

2.01; Chromas lite Technelysium Pvt. Ltd., South Bris-

bane, Australia). Clones were finally checked for chimeric



artefacts using CHECK-CHIMERA of the Ribosomal

Database Project, and the chimeric sequences were dis-

carded. The ITS rRNA sequences obtained were initially

recognized and aligned against the known sequences in

the GenBank database using the BLAST program of the

National Centre for Biotechnology Information (NCBI,

http://www.ncbi.nlm.nih.gov/). The ITS rRNA clones

obtained from the non-Bt- and Bt-planted rhizospheric

soils with >90% similarity with the NCBI database were

adopted for phylogenetic analysis. All the sequences were

initially aligned using CLUSTAL W (Saitou and Nei

1987) available in MEGA4 with opening and at the gap

penalty of 10. The phylogenetic relatedness was estimated

using the neighbour-joining method (Tamura et al.

2004). The evolutionary distance was computed using the

maximum composite likelihood method (Felsenstein

1985). All positions containing gaps and missing data

were eliminated from the data set (complete deletion

option). One thousand bootstrap replications were per-

formed to place the confidence estimates on the major

groups resolved in the tree. The bootstrap consensus tree

inferred from 1000 replicates represents the evolutionary

history of the sequences analysed (Tamura et al. 2007).

Branches corresponding to partitions reproduced in

<50% were collapsed. The phylogenetic analysis was car-

ried out using MEGA software ver. 4.0. (Tamura et al.

2007).

Statistical analysis

All data were examined for homogeneity of variance and

were found to have normal distributions. Multivariate

analysis of variance (MANOVA) was performed to deter-

mine the treatment effects (non-Bt and Bt) on selected

soil enzymes and fungal community structure at different

crop growth stages. All the parameters were compared for

significant changes using Tukey’s test (HSD), (SPSS 16.0;

SPSS Inc., Chicago, IL). All results reported are at P = 0�05.

Nucleotide sequence accession numbers

The nucleotide partial sequences of the ITS rRNA clones

derived from this study have been deposited to the NCBI

database under accession numbers: JQ989288–JQ989345.

Results

Cry1Ac protein content and soil enzymes activity in the

non-Bt- and Bt-brinjal-planted soils

Cry1Ac toxin content was assessed using ELISA in the

rhizospheric soil of Bt brinjal and compared with the non-

Bt one at different sampling stages. Cry1Ac concentration

varied across the sampling stages in the Bt-brinjal-planted

soils, and the protein level was 0�31, 0�69 and 0�30 ng g�1

at branching, flowering and maturation stages of crop,

respectively, in 2010, while in 2011, this protein was

detected only at flowering stage (0�61 ng g�1). The non-Bt

planted soils did not contain Cry1Ac protein.

Significant reductions in DHA and FDA activities were

found in the Bt brinjal soils compared with non-Bt soils

(P < 0�05) in 2010. Similar trends occurred in the follow-

ing year (Fig. 1a,b). Significant variation in DHA and

FDA activities was also observed among the crop growth

stages. Enzyme activity increased initially according to the

availability of the substrates and reached its highest rate

at flowering stages and decreased thereafter, indicating

the role of plant growth in governing the microbial activ-

ities. Soil enzymes such as invertase, urease, acid phos-

phomonoesterase were not significant different between

rhizospheric soils of non-Bt and Bt variety (Fig. 1c–e).However, similarly to FDA and dehydrogenase, significant

differences in the amount of enzymes were detected

between plant growth stages.

Fungal population size

Real time Quantitative PCR-based results indicated a sig-

nificantly higher fungal population size in the rhizosphere

of non-Bt brinjal compared with Bt brinjal in both years

(P < 0�05) (Fig. 2). Irrespective of crops (Bt or non-Bt),

ITS copy number was also varied significantly among the

crop stages and followed the order–flowering > matura-

tion > branching > postharvest and > prevegetation stages

(Fig. 2). Mean values at all stages were significantly dif-

ferent from each other except for pre- and postvegetation

stages. ANOVA indicated significant differences attributable

to the year and the crop (Table 1).

Fungal community analysis

PCR-RFLP fingerprinting analysis revealed a total of fifty-

six OTUs generated from the non-Bt- and Bt-planted soil

that comprised of Ascomycota, Basidiomycota and Zygo-

mycota as the major groups (Fig. 3). Of 56 OTUs, 3

OTUs (EMF 71, EMF20 I and EMF2 I) were detected

during the prevegetation stage, while the other OTUs

were detected at different stages (branching, flowering,

maturation and postharvest stages) of non-Bt and Bt

brinjal crops (Tables 2 and 3). Ascomycota was the dom-

inant group with most of the ITS rRNA clones generated

from non-Bt- and Bt-planted soils clustering within the

class Sordariomycetes (Fig. 3). Apodus oryzae- and Chaeto-

mium-related clones had higher relative abundance (>30and >25%, respectively) that were detected in both non-

Bt- and Bt-brinjal-planted rhizospheric soils. Apodus



oryzae-related OTUs from non-Bt- and Bt-brinjal-planted

soils were detected in branching, flowering, stages

including prevegetation (EMF7 I PV) and postharvest

stages (EMF5 V). However, Chaetomium associated OTUs

(EMF56 III, EMF 57 IV and EMF58 V from non-Bt

brinjal soils and EMF6 III, EMF11 IV and EMF17 V from

Bt brinjal rhizospheric soils) were detected during the last

three consecutive plant stages, that is, flowering, matura-

tion and postharvest. OTUs like EMF45 III from non-Bt

brinjal and EMF 23 III and EMF24 III from Bt-brinjal-

planted soils were detected only during flowering stage

having 10% relative abundance and showed 98% affilia-

tion with Penicillium glabrum. Verticillium-related OTUs

(EMF35 IV and EMF 37V from non-Bt and EMF 13 IV

and EMFV from Bt-planted rhizospheric soils), detected

in maturation and postharvest stages of the non-Bt

and Bt crops, had more than 3% relative abundance

(Fig. 4).

Interestingly, within Ascomycota, some OTUs affiliated

to Pseudallescheria, Podospora, Pyronemataceae, Gelasi-

nospora and Neurospora including some uncultured

Pezizomycotina, Hymenoscyphus and Lasiosphaeriaceae

were detected exclusively in non-Bt-brinjal-planted soil

(Fig. 3). OTUs like, EMF44 II, EMF32 III, EMF33 IV

and EMF41 V, detected during the branching, flowering,

maturation and postharvest stages, had 16, 5, 6 and 12%

relative abundance at the corresponding stages and were

95% similar to soil inhabiting Gelasinospora bonaerensis

(Table 2). Three respective representative OTUs, EMF54

II, EMF28 IV and EMF40 V, detected during branching,

maturation and postharvest stages and each having more

than 5% relative abundance, showed 86% similarity with

Pseudallescheria boydii. EMF47 II and EMF42 III were

99% similar to Pyronemataceae detected during the

branching and flowering stages having 8 and 5% relative

abundance, respectively. In addition to postharvest stage,

uncultured Hymenoscyphus-related OTUs (EMF29 III and

EMF39 IV) were also detected in flowering stage along

with uncultured Pezizomycotina- and Lasiosphaeriaceae-

related respective OTUs–EMF29 III (100%), EMF50 III

(92%) and EMF51 III (>90%) with relative abundance of

7 and 10%, respectively (Fig. 4).

160

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(a) (b)

Figure 1 (a, b) Microbial activity in non-Bt- and Bt-brinjal-planted soils in terms of dehydrogenase and FDA activities. Letters a, b, c denote signif-

icant variation among stages of non-Bt and letter x, y in case of Bt brinjal. Letters sharing the same letter are not significantly different between

the treatment and the sampling stages by Tukey’s HSD at (P < 0�05). Bars indicate extent of variation from the mean. (c–e) Enzymatic activity

involved in C, N, P nutrient cycling in non-Bt- and Bt-brinjal-planted soil. Different letters denote significant difference (P < 0�05) estimated by Tu-

key’s HSD, and bars indicate extent of variation from the mean. ( ) Non-Bt and ( ) Bt.



Likewise, Bt brinjal rhizospheric soils also harboured

some exclusive OTUs that were affiliated to Zopfiella-,

Melanopsamma- and Gibberella-related taxa (Fig. 3).

EMF3 II, EMF9 III and EMF10 IV were detected during

branching, flowering and maturation stages, respectively,

with relative abundance of 30, 20 and 30% at the

70 0·5

0·4

0·3

0·2

0·1

0

0·5

0·4

Ure

ase

(µg

NH

4 + g

–1 s

oil)

0·3

0·2

0·1

0

60

50a a

a aa

2010 2010

20112011

a

b

2010

b

a aa a

b b

c c

aa a

a

b ba aa a a a

b bb b c

c

d d

d db b

40

30

20

10

0

70

Inve

rtas

e (µ

g gl

ucos

e g–

1 so

il)

60

50

40

30

20

10

0

500

300

200

100

0

Aci

d ph

osph

omon

oest

eras

e (m

g p-

nitr

ophe

nol k

g–1

soil)

400

a a

b b

c cd d

a a

a a

b b

c cd d

a a

2011500

300

200

100

0

400

Stages StagesPre

-veg

etat

ion

Branc

hing

Flower

ing

Mat

urat

ion

Post-h

arve

st

Stages

Pre-v

eget

ation

Branc

hing

Flower

ing

Mat

urat

ion

Post-h

arve

st

Pre-v

eget

ation

Branc

hing

Flower

ing

Mat

urat

ion

Post-h

arve

st

(c) (d)

(e)

Figure 1 (Continued)



corresponding stages and were 99% similar to Zopfiella

latipes. EMF15 III detected only during the flowering

stage with relative abundance of 15% and showed 100%

similarity with Melanopsamma pomiformis. Similarly, Gib-

berella-related OTUs, EMF4 III and EMF22 V (96%),

were detected during flowering and maturation stages

with 5% relative abundance. However, some of the

sequences (EMF21 II and EMF8 III), found during

branching and flowering stages each having 10% relative

abundance, did not resemble with any of the taxa and

showed 99% affinity with the ascomycete (Table 3).

Unlike Ascomycota, other OTUs with relative abun-

dance >30% from both the fields were assigned under

Zygomycota and showed more than 95% affinity only

with Mortierella sp (Tables 2 and 3). OTUs like EMF2 I

PV were detected during prevegetation. EMF34 II,

EMF38 III, EMF30 IV and EMF31 V from non-Bt and

EMF1 II, EMF26 III, EMF25 IV and EMF27 V from Bt

brinjal rhizosphere were detected throughout the vegeta-

tion and postharvest stage. However, OTUs under Basidi-

omycota were related to Mycena and Cortinarius taxa.

Both, EMF20 I and EMF59 IV, detected during prevege-

tation and maturation stages of non-Bt brinjal and

EMF20 I detected only during prevegetation stage of

Bt-brinjal-planted rhizospheric soil, were 99% similar to

Mycena sp. having relative abundance >8% (Fig. 4). Simi-

larly, EMF49 V and EMF9 V, respectively, from the rhi-

zospheric soil of non-Bt- and Bt-brinjal-planted soils

were detected only during postharvest stage with 13 and

10% relative abundance and were 99% similar to Cortina-

rius (Tables 2 and 3).

Discussion

Cry1Ac protein content and soil enzymes activity in the

non-Bt- and Bt-brinjal-planted soils

The relatively high amount of Cry1Ac protein at flowering

stage compared with other stages reflects high root activity

during flowering as reported earlier (Icoz et al. 2008). The

low percentage of clay (2%) in the soils used for the pres-

ent study could explain the relatively low protein concen-

trations, as clay particles protect proteins from microbial

degradation. Baumgarte and Tebbe (2005) suggested Cry

proteins rapidly degrade in soils. Chen et al. (2011)

5

4

a a

b

x

c

y

d

zb

x

Pre-v

eget

ation

Branc

hing

Flower

ing

Mat

urat

ion

Post-h

arve

st

aaa

a

b

x

c

yd

z

2010

2011

3

2

1

0

5

4

3

2

1

0

Gen

e co

pies

(×

109

) g–

1 dw

s

Stages

Figure 2 Variations in fungal population size in non-Bt- and Bt-brin-

jal-planted soil during 2010 and 2011. Different letters denote signifi-

cant difference (P < 0�05) estimated by Tukey’s HSD, and bars denote

the extent of variation from the mean. ( ) Non-Bt and ( ) Bt.

Table 1 Results of multivariate analysis of variance for observed parameters

Parameters

2010 2011 Pooled

Stages Treatment Stages Treatment Year Stages Treatment

F4,20 P F1,20 P F4,20 P F1,20 P F1,40 P F4,40 P F1,40 P

Dehydrogenase 163�10 0�001 5�81 0�026 148�11 0�040 9�13 0�007 0�27 0�602 310�33 0�002 14�81 0�000FDA 42�18 0�030 29�45 0�010 50�33 0�030 44�15 0�000 0�73 0�398 90�90 0�020 71�47 0�000Urease 59�61 0�020 0�18 0�676 46�71 0�010 0�00 1�000 5�00 0�031 103�25 0�030 0�07 0�790Invertase 69�42 0�010 3�83 0�064 37�02 0�020 0�30 0�587 1�95 0�169 91�13 0�010 2�24 0�142Acid

phospho-monoesterase

349�77 0�020 0�14 0�70 203�77 0�010 0�77 0�391 0�06 0�808 517�49 0�040 0�86 0�358

Fungal population 5�31 0�004 7�05 0�015 29�39 0�020 13�11 0�002 14�94 0�000 22�06 0�001 17�02 0�020



reported varied levels of Cry1Ac protein from the rhizo-

spheric soil of transgenic cotton after three and 4 years of

growth revealing the persistence of Cry protein in trans-

genic cotton rhizosphere soil. On the basis of literature

reviewed, the persistence of Bt toxins in soil, as well as

their microbial degradation, is a function of soil type,

environmental conditions, source of the proteins and

the particular Cry protein studied (Clark et al. 2005).

EMF7 I (JQ989294)PVEMF10 IV (JQ989297)Bt

EMF9 III (JQ989296)BtEMF53 V (JQ989339)NBtEMF36 II (JQ989322)NBt

EMF55 III (JQ989341)NBt

EMF5 V (JQ989292)Bt

EMF51 IV (JQ989337)NBtEMF46 II (JQ989332)NBt

EMF41 V (JQ989327)NBt

EMF52 IV (JQ989338)NBt

EMF33 IV (JQ989319)NBtEMF44 II (JQ989330)NBt

EMF56 III (JQ989342)NBtEMF6 III (JQ989293)BtEMF11 IV (JQ989298)BtEMF17 V (JQ989304)Bt

EMF57 IV (JQ989343)NBtEMF58 V (JQ989344)NBt

EMF4 III (JQ989291)BtEMF22 V (JQ989308)Bt

EMF15 III (JQ989302)Bt

EMF21 II (JQ989307)BtEMF8 III (JQ989295)Bt

EMF13 IV (JQ989300)BtEMF37 V (JQ989323)NBtEMF35 IV (JQ989321)NBtEMF14 V (JQ989301)Bt

EMF29 III (JQ989315)NBtEMF39 V (JQ989325)NBt

EMF23 III (JQ989309)BtEMF24 III (JQ989310)BtEMF45 III (JQ989331)NBt

EMF42 III (JQ989328)NBtEMF47 II (JQ989333)NBt

EMF59 IV (JQ989345)NBt

EMF20 I (JQ989306)PV

EMF49 V (JQ989335)NBtEMF19 V (JQ989305)Bt

EMF34 II (JQ989320)NBt

EMF2 I (JQ989289)PV

EMF31 V (JQ989317)NBt

EMF25 IV (JQ989311)BtEMF30 IV (JQ989316)NBtEMF38 III (JQ989324)NBtEMF1 II (JQ989288)BtEMF26 III (JQ989312)Bt

0·02

81

6463

9956

9999

97

69

9999

99

99

99

99

99

99

99

99

99

9963

69

9562

94

66

66

82

88

98

60

63

69

99

7954

62

76

99

EMF27 V (JQ989313)BtZ

ygo

myc

ota

Bas

idio

myc

ota

Asc

om

yco

ta


EMF28 IV (JQ989314)NBtEMF54 II (JQ989340)NBtEMF40 V (JQ989326)NBt



EMF48 III NBt

Apodus oryzae (AY681200)Zopfiella latipes (AY999129)

Podospora didyma (AY999127)

Neurospora sp. (JN207303)

Gelasinospora endodonta (AY681191)

Chaetomium (AJ279466)

Pseudallescheria boydii (GU566237)

Melanospora pomiformis (AF081478)

Verticillium (EF641856)

Uncultured Hymenoscyphus (AB456655)

Uncultured Pezizomycotina (AY394904)

Penicillium asperosporum (AF033412)

Pyronemataceae sp. (EF372405)

Mycena murina (AF335444)

Cortinarius pilatii (GQ159836)

Mortierella humilis (JN943013)

Uncultured fungus (FN397316)

Mortierella alpina (AJ271630)

Uncultured Lasiosphaeriaceae (EU754956)

EMF3 II (JQ989290)Bt

Leotiomycetes

Sordariomycetes

Eurotiomycetes

Pezizomycetes

Figure 3 Phylogenetic analysis of fungal-specific ITS rRNA sequences and related species by neighbour-joining method obtained from the non-Bt

and Bt brinjal crop growth stages including prevegetation and postharvest stages. Stages were mentioned in roman letters; Stages-I (prevegeta-

tion), II (branching), III (flowering), IV (maturation) and V (postharvest). Boot strap values above the 50% are indicated at the nodes. The scale

bars represent 0�02 substitutions per site. Accession numbers are indicated for each sequenced clone (bold letter) in parentheses.



Therefore, differences in soil characteristics, environ-

mental conditions and their interaction with transgenic

lines are likely to determine the fate of Cry proteins under

natural conditions (Icoz and Stotzky 2008).

Dehydrogenase is present in all viable micro-organisms

(Dick 1994) and is considered to reflect the microbial

activity of the soil. FDA hydrolysis has also been sug-

gested to be a measure of overall microbial activity (Guan

et al. 1986; Masto et al. 2006). Significant reduction in

soil dehydrogenase and FDA observed in the rhizospheric

soils of Bt-brinjal-planted field indicates that a fraction of

microbes including fungi might be inhibited and does

not contribute to the metabolic activities of the soil

(Nannipieri et al. 2003). A significant positive correlation

was found between the fungal population and dehydroge-

nase (R2 = 0�83, P = 0�03 for non-Bt; R2 = 0�68,P = 0�002 for Bt brinjal soil) and FDA activities

(R2 = 0�79, P = 0�006 for non-Bt; R2 = 0�71 P = 0�04 for

Bt brinjal soil). Chen et al. (2012) detected varied Cry1Ac

protein content (4�5–26�7 ng g�1 soil) causing significant

reduction in the DHA and FDA activities in soil planted

with Bt cotton. Yang et al. (2012) reported that Bt toxin

levels (56�14 ng g�1) peaked at the flowering stage of Bt

cotton and promoted DHA activity in rhizospheric soil as

compared to soil with non-Bt cotton. Differences in soil

dehydrogenase activity are an indication of variation in

labile organic carbon (Singh et al. 2013a). This is sup-

ported by our previous observation (Singh et al. 2013b),

where the organic carbon content in the soil of Bt brinjal

was significantly lower than in the non-Bt brinjal soil. On

the other hand, no significant changes in the dehydroge-

nase activity were also found in the study conducted

with transgenic cotton (Baumgarte and Tebbe 2005; Liu

et al. 2008), transgenic rice (Fliebbach et al. 2011) and

Table 2 Nucleotide sequence BLAST results of fungal-specific ITS rRNA OTUs from non-Bt brinjal soil

OTUs Close NCBI BLAST match Taxonomic affinity Similarity (%) Habitat

EMF7 I PV Apodus oryzae Ascomycota 99 Environmental sample

EMF36 II A. oryzae Ascomycota 99 Environmental sample

EMF53 V A. oryzae Ascomycota 99 Environmental sample

EMF56 III Chaetomium Ascomycota 99 Common reed

EMF57 IV Chaetomium Ascomycota 100 Common reed

EMF58 V Chaetomium Ascomycota 100 Common reed

EMF55 III Uncultured Lasiosphaeriaceae Ascomycota 99 Oil seed rape field

EMF46 II Podospora sp. R198 Ascomycota 93 Silver wounded birch

EMF48 III Podospora sp. R199 Ascomycota 92 Silver wounded birch

EMF51 IV Podospora sp. R200 Ascomycota 92 Silver wounded birch

EMF43 III Neurospora sp. P24E1 Ascomycota 99 Semiarid grassland

EMF52 IV Neurospora sp. P24E2 Ascomycota 99 Semiarid grassland

EMF44 II Gelasinospora bonaerensis Ascomycota 95 Soil

EMF32 III G. bonaerensis Ascomycota 95 Soil

EMF33 IV G. bonaerensis Ascomycota 95 Soil

EMF41 V G. bonaerensis Ascomycota 95 Soil

EMF35 IV Verticillium biguttatum Ascomycota 92 Environmental sample

EMF37 V V. biguttatum Ascomycota 92 Environmental sample

EMF54 II Pseudallescheria boydii Ascomycota 86 Environmental sample

EMF28 IV P. boydii Ascomycota 85 Environmental sample

EMF40 V P. boydii Ascomycota 86 Environmental sample

EMF29 III Uncultured Hymenoscyphus Ascomycota 100 Environmental sample

EMF39 V Uncultured Hymenoscyphus Ascomycota 100 Environmental sample

EMF45 III Penicillium glabrum Ascomycota 98 Wood

EMF50 III Uncultured Pezizomycotina Ascomycota 99 Environmental sample

EMF47 II Pyronemataceae Ascomycota 99 Orchids

EMF42 III Pyronemataceae Ascomycota 99 Orchids

EMF20 I PV Uncultured Mycena Basidiomycota 99 Rhizosphere soil

EMF59 IV Uncultured Mycena Basidiomycota 99 Rhizosphere soil

EMF49 V Cortinarius pilatii Basidiomycota 99 Root

EMF34 II Uncultured fungus Zygomycota 98 Rice field soil

EMF2 I PV Uncultured Mortierellales Zygomycota 97 Tomato rhizosphere

EMF38 III Mortierella alpine Zygomycota 99 Soil

EMF30 IV M. alpine Zygomycota 99 Soil

EMF31 V M. alpine Zygomycota 98 Soil



transgenic corn (Rasche et al. 2006). Moreover, in the

present study, all the soil enzyme activities in relation to

crop stages peaked at the flowering stage and decreased at

the later stages which are consistent with other studies

(Lu et al. 2002; Yang et al. 2012). The reduced enzyme

activities at later growth stages of brinjal crop are due to

less organic inputs by the roots into the soil. This might

be due to the fact that Bt plants have more intact roots

and do not disperse organic matter to attract fungi. Soil

dehydrogenase, urease and phosphatase activities could be

used as indicators for the effect of toxin on soil microbio-

logical activity (Jepson et al. 1994). In contrast to dehy-

drogenase and FDA, no significant changes were observed

in the invertase, urease or acid phosphomonoesterase

activity between the non-Bt- and Bt-planted rhizospheric

soils. This result is similar to earlier findings with trans-

genic cotton (Shen et al. 2006; Sun et al. 2007). However,

some researchers have also reported significant effects due

to transgenes (Wei et al. 2006; Miethling-Graff et al.

2010). This contradiction may be attributed to insufficient

levels of transgenic proteins and soil characteristics affect-

ing the persistency of the transgene proteins (Icoz and

Stotzky 2008; Gschwendtner et al. 2010).

Variation in fungal population size

Variation in the fungal population between Bt and non-

Bt brinjal (Fig. 2) is probably due to changes in available

100%

80%

60%

40%

20%

0%Non-Bt Bt Non-Bt Bt Non-Bt Bt Non-Bt Bt Non-Bt

Post-harvestMaturationFlowering

Stages

BranchingPre-vegetation

Bt

Rea

ltive

abu

ndan

ce o

f fun

gal

spec

ific

ITS

rR

NA

clo

nes

in %

Figure 4 Relative proportion of fungal-

specific ITS rRNA clones across the different

sampling stages in non-Bt- and Bt-brinjal-

planted soil. ( ) Gibberella; ( ) Cortinarius;

( ) Neurospora; ( ) Uncultured

Hymenoscyphus; ( ) Gelasinospora; ( )

Pseudallescheria; ( ) Melanopsamma; ( )

Uncultured Mycena; ( ) Penicillium glabrum;

( ) Uncultured Pezizomycotina; ( )

Podospora; ( ) Apodus oryzae; ( ) Zopfiella;

( ) Verticillium; ( ) Uncultured

Lasiosphaeriaceae; ( ) Chaetomium; ( )

Pyronemataceae and ( ) Mortierella.

Table 3 Nucleotide sequence BLAST results of fungal-specific ITS rRNA OTUs from Bt brinjal soil

OTUs Close NCBI BLAST match Taxonomic affinity Similarity (%) Habitat

EMF10 IV Zopfiella latipes Ascomycota 99 Environmental sample

EMF9 III Z. latipes Ascomycota 98 Environmental sample

EMF5V Apodus oryzae Ascomycota 99 Environmental sample

EMF3 II A. oryzae Ascomycota 99 Environmental sample

EMF6 III Chaetomium Ascomycota 100 Common reed

EMF11 IV Chaetomium Ascomycota 100 Common reed

EMF17 V Chaetomium Ascomycota 99 Common reed

EMF4 III Gibberella Ascomycota 96 Mangrove endophytic fungus

EMF22 V Gibberella Ascomycota 96 Mangrove endophytic fungus

EMF15 III Melanopsamma pomiformis Ascomycota 100 Environmental sample

EMF8 III Ascomycete sp. Ascomycota 99 Monument surface

EMF21 II Ascomycete sp. Ascomycota 99 Monument surface

EMF13 IV Verticillium biguttatum Ascomycota 92 Environmental sample

EMF14 V V. biguttatum Ascomycota 92 Environmental sample

EMF23 III Penicillium glabrum Ascomycota 98 Wood

EMF24 III P. glabrum Ascomycota 98 Wood

EMF19 V Cortinarius pilatii Basidiomycota 99 Root

EMF 27 V Uncultured Mortierellales Zygomycota 90 Tomato rhizosphere

EMF26 III Mortierella alpine Zygomycota 99 Soil

EMF1 II M. alpine Zygomycota 100 Soil

EMF25 IV M. alpine Zygomycota 99 Soil



nutrients and microbial biomass C in both the treatments

as reported earlier (Singh et al. 2013b,c). Miethling-Graff

et al. (2010) suggested that alterations in quality and

composition of root exudates from genetically modified

plants result in a differently structured community. Other

studies also revealed significant differences in the fungal

population due to cultivation of transgenic cotton

(Hannula et al. 2012). These studies suggest that genetic

manipulation of plants can change plant characteristics

including root exudate quality and composition in addi-

tion to Bt toxin production.

Among the crop growth stages, maximal ITS rRNA

copy number detection during flowering stages could be

due to the availability of nutrients such as NH4+-N, NO�

3

-N, PO�4 - P as reported earlier (Singh et al. 2013b,c).

According to Bais et al. (2006), plant rhizodeposition

results in increased microbial populations including the

fungal community. The results are consistent with the

finding of Hannula et al. (2012) and Gschwendtner et al.

(2010) wherein the influence of the plant developmental

stage on the ITS rRNA gene abundance was more pro-

nounced in transgenic potato, although the trends are in

contrast to the present report finding a decrease in total

fungal ITS rRNA gene copy number with increasing plant

age in a field trial. The fungal population dynamics dis-

cussed in the present study indicated the sensitivity of

real-time PCR over the conventional plate count method

(Hannula et al. 2012). Thus, our results suggest that

Cry1Ac gene expressing Bt brinjal exerts a small but sig-

nificant impact on the fungal population size.

Effects of Bt brinjal and its near isogenic line on fungal

diversity

In present study, the majority of the fungal sequences

recovered belonged to Ascomycota which corroborates

other studies conducted in agricultural ecosystems (Nakas

et al. 1987; Klaubauf et al. 2010). Surprisingly, within the

Ascomycota, OTUs differences at the class level were more

prominent between non-Bt and Bt rhizospheric soils. The

classes most abundantly represented by these clones were

Sordariomycetes and Pezizomycetes supporting the finding

of Lee et al. (2011). All of the ascomycetes identified are

common residents of agricultural soils. The presence of

exclusive taxa, such as Podospora, Neurospora, Gilasinos-

pora, Pseudallescheria and Pyronemataceae including

uncultured Lasiosphaeriaceae, Hymenoscyphus and Pezizo-

mycotina in non-Bt and Z. latipes, Gibberella and M. pomi-

formis in Bt-planted soil, suggests specificity of the plant

for particular groups which is similar to the findings of

Hussain et al. (2011) who reported new OTUs of Ascomy-

cota (Sordariomycetes, Pezizomycetes and Leotiomycetes) in

soil with hybrid rice cultivar compared to soil with conven-

tional rice cultivar. Genetic transformation of plants could

possibly lead to changes in plant characteristics including

root exudates (Weinert et al. 2009). As a consequence,

rhizospheric microbial communities respond quickly to

changes in root exudate quality and chemical composition

that are believed to depend on natural factors such as plant

species, cultivar and growth stages (Saxena et al. 1999;

Rengel 2002). The apparent changes in Ascomycota in the

present study indicate that cultivation of Bt brinjal favours

this particular group of fungi (Ascomycota) with the possi-

ble suppression of other groups.

OTUs assigned to Basidiomycota and Zygomycota were

few and are similar to Mycena and Mortierella related sp.,

respectively. This refers to the dominancy of Ascomycota

over these groups which is in accordance with earlier

studies on the agroecosystems, conducted with rice

(Rengel et al. 1998) and potato (Broeckling et al. 2008;

Nemergut et al. 2008). However, temporal shift within

the fungal communities was observed with the highest

OTUs detected in flowering stages, indicating the

dynamic nature of rhizospheric fungal community influ-

enced by the plant growth stages. It has been reported

that flowering stage may be favourable for microbial pro-

liferation due to active release of root exudates (Broec-

kling et al. 2008; Wang et al. 2009). Changes in organic

C across the sampling stage (Singh et al. 2013b) probably

leads to alterations in the fungal community that was

similar at prevegetation stage compared with subsequent

sampling stages (Fig. 4). Furthermore, in our study, the

presence of different OTUs during the maturation stage

was surprising and could be due to substrate availability

through root exudates in order to sustain biological pro-

cesses such as decomposition (Broeckling et al. 2008).

Liu et al. (2008) reported no significant differences in

the fungal community composition based on DGGE and

T-RFLP polymorphism fingerprints in the rhizospheric

soil of parental non-Bt and Bt rice cultivars during crop

development. Wei et al. (2012) observed little effect of

Cry1Ac protein on the dominant fungal community in

the transgenic rice rhizosphere. The specificity of the pri-

mer set (ITS1F-ITS4) used in present study favours the

detection of Ascomycota compared with Zygomycota

(Chen and Cairney 2002), so this could be one reason for

estimating minor changes under the influence of Bt brin-

jal. As the community analysis was only carried out for

one year and in view of the limitations and biases of the

PCR-based methods, it is difficult to assess the influence

of transgenic brinjal on fungal community. From the

abundance of data available, it is clear that the effect of

transgenic plants strongly depends on the particular

plant, transgene and conditions considered. Based on the

above finding, we can hypothesize that the impact of

genetic modification was detected only in the Ascomycota



but was transient and minor. However, the temporal

changes in the fungal community observed during the

brinjal cultivation period were more affected by crop

growth stages.

In conclusion, the present study indicates that popula-

tion size and diversity of fungal community varied signif-

icantly between transgenic and nontransgenic brinjal

crop. Diverse groups in the fungal community were

detected with the dominance of Ascomycota between the

non-Bt and Bt crops. Significant differences in the dehy-

drogenase and FDA activity were also observed between

the respective crops. Therefore, it can be inferred that

crop growth was the overriding factor that masks the

effect of Cry1Ac gene expressing Bt brinjal. The knowl-

edge obtained through the present study is helpful in

understanding the variability of fungal community struc-

ture associated with the rhizosphere of Bt brinjal. In the

context of genetically modified crops for field applica-

tions, it is imperative for further research to explore the

impact of such applications on the structural and func-

tional diversity of both soil bacteria and fungi.

Acknowledgements

This research work was supported by Indian Institute of

Vegetable Research, India. One of the authors (AKS) is

grateful to Council of Scientific and Industrial Research,

New Delhi, for financial assistance in the form of JRF

and SRF. The insights provided by the Editor, and anon-

ymous reviewers helped us to improve the ms in present

form.

Conflict of Interest

No conflict of interest declared.

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Supporting Information

Additional Supporting Information may be found in the

online version of this article:

Table S1 Summary of published available work on the

impact of Cry 1Ac gene expressing transgenic crops on

fungal communities in soil

Table S2 Enzyme activities assayed in non-Bt and Bt

soil



Documents

Rhizospheric fungal community structure of a Bt brinjal and a near isogenic variety