Phytoparasitica

https://doi.org/10.1007/s12600-019-00738-x

Widespread infestation of pink bollworm, Pectinophora

gossypiella (Saunders) (Lepidoptera: Gelechidae) on Bt
cotton in Central India: a new threat and concerns
for cotton production
Babasaheb B. Fand & V. S. Nagrare & S. P. Gawande & D. T. Nagrale &
B. V. Naikwadi & Vrushali Deshmukh & Nandini Gokte-Narkhedkar &
V. N. Waghmare

Received: 9 August 2018 / Accepted: 17 May 2019

# Springer Nature B.V. 2019

Abstract The pink bollworm Pectinophora gossypiella
(Saundars) (Lepidoptera: Gelechidae) has recently
emerged as a serious menace on Bt cotton in India.
Extensive roving surveys were conducted in 83 locations
spread across 16 major cotton growing districts of
Maharashtra State, India to assess the level of pink boll-
worm infestation in Bt cotton. The dynamics and severity
of pink bollworm damage during the different stages of
boll development (90–130 days of crop age) were
assessed based on random sampling of green bolls,
opened bolls and picked bolls per plant and number of
locules damaged per boll. Survey results revealed a wide-
spread infestation of pink bollworm on Bt cotton across
Highlights
• Widespread infestation of pink bollworm in Bt cotton ranging
between 40 – 95 % accounting for estimated yield losses to the
tune of 20 – 30 % have been reported from 16 major cotton
growing districts of Maharashtra, a leading cotton producing
state of Central India.
• The intensification of boll infestation and locular damage with
the advancement of the crop season was observed.
• The pink bollworm has resumed its pestilence against Bt cotton,
which has serious ecological and economic implications for
country’s cotton production.
• The integrated measures for restoring susceptibility in pink
bollworm and ensuring viability of Bt cotton technology have
been suggested to mitigate its potential threat to cotton
production.
the surveyed sites in a range of 40–95% which accounts
for anticipated yield losses between 20 and 30%. A
typical pattern of progressive increase in the level of pink
bollworm infestation and intensification of locular dam-
age with the advancement of the crop season was ob-
served. The pink bollworm hitherto was supposed to be
under control since the introduction of Bt cotton in India,
now it appears to be widespread, especially on Bt cotton
throughout the cotton growing areas of Maharashtra State
which shares approximately 36% and 25% of the
country’s cotton area and production, respectively. This
article discusses the implications of serious outbreak of
pink bollworm infestations in Bt cotton and also suggest
measures necessary to mitigate its potential threat to
Indian cotton production. The modulation of manage-
ment practices aimed at manipulation of cotton cropping
pattern being followed in the country in general and
Maharashtra in particular could strengthen the manage-
ment of pink bollworm populations effectively. We dis-
cuss the practicality of several management options
aimed at achieving this goal.
Keywords Bt cotton . India . Maharashtra . Pink
bollworm . Resistance development . Widespread
infestation . Yield losses

B. B. Fand (*) : V. S. Nagrare : S. P. Gawande : Introduction

D. T. Nagrale : B. V. Naikwadi : V. Deshmukh :
N. Gokte-Narkhedkar : V. N. Waghmare
The pink bollworm Pectinophora gossypiella (Saunders)
ICAR-Central Institute for Cotton Research, Nagpur, Maharashtra
440 010, India (Lepidoptera: Gelechidae) has recently emerged as a
e-mail: babasahebfand@gmail.com serious concern for cotton production in India. The insect

has its origin from Indo-Pak region (Saunders 1843) and
is widely distributed throughout tropical America, Africa,
Asia, Australia, Egypt, USA and Mexico, wherever cot-
ton is grown. The pink bollworm, hereafter referred as
PBW, is a stenophagous insect species which has co-
evolved with Malvaceous food plants like cotton, okra,
deccan hemp and roselle (CABI 2017). The destructive
larvae of PBW feeds on flowers, buds, bolls and seeds of
cotton plant, causing rosetting and malformation of
flowers, premature opening and heavy shedding of
infested bolls, reduction in fiber length and poor quality
of lint due to staining (Singh et al. 1988). Pink bollworm
was a key pest of cotton in India accounting for yield
losses to the level of 20–90%, prior to the use of broad
spectrum insecticides and introduction of transgenic cot-
ton (Patil 2003). Synthetic pyrethroids introduced in
India during 1980’s played an instrumental role in sup-
pressing this notorious and difficult to manage pest of
cotton. However, intensive use of chemical insecticides
caused widespread ecological disruption leading to exac-
erbation of bollworm and secondary pest problems in
cotton ecosystem (Kranthi et al. 2002; Kranthi and
Russell 2009; ICAC 2010). Subsequently, development
of genetically engineered transgenic cotton carrying
genes encoding delta-endotoxin proteins from entomo-
pathogenic soil bacterium Bacillus thuringiensis had
opened new avenues for managing dreaded bollworms
in cotton and since then Indian cotton ecosystem has
witnessed phenomenal changes in its pest status. Single
gene (Cry 1Ac) and dual gene (Cry 1Ac + Cry 2Ab) Bt
cotton hybrids were commercially introduced in India
during 2002 and 2006, respectively (Choudhary and
Gaur 2010), targeting the dreaded bollworm complex
which included American bollworm Helicoverpa
armigera (Hubner), spotted bollworm Earias vitella
(Fabricious) in addition to PBW. Besides bollworms, Bt
cotton offers protection against other minor Lepidopteran
insect pests like semiloopers, hairy caterpillars and leaf
eating caterpillars. Initially, the Bt cotton technology
performed well and offered promising control of boll-
worm complex until 2009. As such, the PBW infestations
remained at very low levels in the first decade of release
of Bt cotton. Even after 15 years of continuous cultivation
of Bt cotton in India, negligible resistance was observed
in H. armigera and E. vitella. However, after a hiatus of
nearly two decades, PBW has suddenly emerged as a
major pest problem in central and southern cotton grow-
ing belt of India where the pest have been reported
feeding and surviving on single gene (Bollgard I) and
Phytoparasitica
dual gene (Bollgard II) Bt cotton. The infestations of
PBW have been reported in Bt cotton fields of Gujarat,
Maharashtra, Karnataka, Andhra Pradesh and Telangana
states (Monsanto 2010; Kranthi 2015; Durga Prasad et al.
2016; Naik et al. 2017), causing yield losses of 5–10%
after second picking (Kranthi 2015). ICAR-Central
Institute for Cotton Research (ICAR-CICR), Nagpur
has anticipated yield losses ranging between 10 and
30% in Maharashtra State alone due to PBW infestation
in Bt cotton during cotton season of 2017.
The reasons for BWhy pinkies have come back again
to trouble the cotton?^ may be manifold, but certainly it
has put forth several unanswered questions related to
resuming of its pestilence. Prima facie, it appears that
certain factors like i. Complete evasion of refuse strate-
gy by cotton farmers, ii. Widespread cultivation of long
duration Bt cotton hybrids with different flowering and
fruiting windows, and iii. Extension of normal crop
season through ratooning or providing supplementary
irrigation and fertilizer applications may perhaps have
exerted selection pressure high enough for accelerating
the development of Bt resistance in PBW (Kranthi
2015). Whatsoever could be the reasons, the re-entry
of PBW, that too especially on Bt cotton in India has
come as a big blow to the cotton stakeholders (technol-
ogy developers, farmers, researchers, seed companies,
policy makers, etc.). The main reason for this is that Bt
cotton, the country’s first and sole commercially ap-
proved genetically engineered technology (Choudhary
and Gaur 2010), has lost the battle against PBW.
India is the largest producer of cotton in the world,
accounting for 26% of the world cotton production. In
2016–17, the area under cotton cultivation in India was
10.8 million ha and the production was 34.5 million bales
(CCI STAT 2017). Infestations of the devastating pest,
PBW on wide areas across the major cotton growing
states of India have posed serious threat to cotton farming
in India, the World’s No. 1 producer of cotton. There has
been a lot of hue and cry through print and electronic
mass media (Aryal 2016; The Hindu 2017), and various
farmers’ and scientific forums on the issue of outbreak of
PBW infestations on Bt cotton in India in general and
Maharashtra in particular. In these contexts, lot of con-
troversy has already been generated in the country on Bt
cotton technology right from its introduction. Few studies
in India reported development of resistance in PBW
against Cry1Ac, a Bt toxin produced by first generation
transgenic cotton i.e. Bollgard (Dennehy et al. 2010;
Dhurua and Gujar 2011; Mohan et al., 2016). Yet there
Phytoparasitica
is no published evidence from the country based on
detailed scientific investigations on the field occurrence
and development of resistance in PBW against second
generation transgenic cotton called Bollgard II, which
currently occupies more than 93.14% of the total area
of cotton cultivated in the country (DCD 2017). In this
framework, we aimed at documenting widespread field
level infestation of PBW in Bt cotton from Maharashtra
State, India, which ranks first in the country in terms of
both area (3.81 m ha) and production (8.9 m bales) of
cotton (CCI STAT 2017). Roving surveys of 83 villages
from 16 major cotton growing districts of the State were
made to assess the level of field infestation of PBW in
cotton based on randomly sampled green bolls and dam-
aged open bolls. The present study is aimed at alerting the
wider stakeholder community of cotton production
across the globe, on probable threat of PBW to the
world’s cotton farming and to undertake timely steps
necessary towards its mitigation.
Materials and methods
Sampling plan for survey and assessment of PBW
infestation
Roving field surveys were conducted in Bt cotton fields
in intensive cotton growing areas of Khandesh (03
districts viz., Dhule, Jalgaon, Nandurbar), Vidarbha
(08 districts viz., Akola, Amravati, Buldana,
Chandrapur, Nagpur, Wardha, Washim, Yavatmal) and
Marathwada (05 districts viz., Beed, Latur, Nanded,
Osmanabad, Parbhani) regions of Maharashtra State of
India during September – December, 2017 for assessing
the infestation of PBW. A total of 83 locations covering
15 major cotton growing districts of the State (Fig. 1)
were surveyed at peak of PBW infestation stage [90–
130 days after sowing, (DAS)] coinciding with boll
maturity to boll bursting stages representing PBW larval
population at pre–picking and picking stages, respec-
tively. A random survey of the road side villages having
large area under cotton was done. Around 5–8 km dis-
tance was maintained between two villages and in each
village at least two cotton fields were selected for survey
(Tanwar et al. 2011; Fand et al. 2017). The sampling
procedure accounted for the various parameters like
green bolls, opened bolls and picked bolls per plant
and number of locules damaged for assessing the dy-
namics and severity of PBW damage during the
different stages of boll development. For assessment of
the green boll damage due to PBW, destructive sam-
pling was done of about 40–50 green bolls collected
randomly from a unit area of one acre at each location.
The care was taken while sampling green bolls, that a
single boll was sampled from each plant so as to collect
more representative sample of the population. The num-
bers of infested bolls recorded and converted to percent
infestation (Durga Prasad et al. 2016; Naik et al. 2017).
Additionally, at each survey location in eight districts,
the damage to open bolls due to PBW infestation was
recorded individually from 25 to 30 randomly selected
cotton plants from one acre area and expressed as per-
centage of open boll damage. For detailed analysis of
potential damage due to PBW infestation in cotton, the
number of total and infested picked bolls, open bolls and
green bolls per plant were recorded and the proportion
of bolls with varying degree of locular damage were
estimated for three locations surveyed in Akola district
as an example. Majority of the places that are far away
were surveyed once, whereas the nearby places in two
districts viz. Akola and Yavatmal were visited more than
once so as to get a clear picture of dynamics of PBW
infestation with the advancement of crop season
(Nagrare et al. 2009).
Confirmation of identity of PBW
The identity of PBW was confirmed based on visual
morphological characters and DNA barcoding by am-
plifying cytochrome oxidase gene (COI) of mitochon-
drial DNA of adult moths reared from sampled larvae.
About 3–4 larvae from each of the 83 surveyed locations
were individually observed under stereo zoom micro-
scope (Leica S8 APO) for their morphological charac-
ters (e.g. shape, size, colour, etc) in accordance with
Saunders (1843) and USDA (1948). Based on the sim-
ilarities of morphological characters and nature of dam-
age caused to green bolls (and also to opened bolls in the
field), the sampled larvae were pooled into three groups
representing each of the cotton growing regions of
Maharashtra State i.e. Khandesh, Vidarbha and
Marathwada. These pooled larvae were reared till the
emergence of adult moths. Since, majority of the PBW
larvae recovered from field collected green boll samples
were in third or fourth instar of the larval stage, they
were reared until adulthood on the sampled green bolls.
Individual PBW larvae were secured inside the dissect-
ed open bolls by tying their opened locules with rubber

Fig. 1 The map of Maharashtra State of India showing locations
of survey sites where PBW infestations occurred in Bt cotton. The
figures in the parentheses are serial numbers of sites surveyed in
each district. Khandesh Region: Nandurbar (1–4), Dhule (5–7),
Jalgaon (8–14); Vidarbha Region: Buldana (15–18), Akola (19–
bands. The locular sutures and cut ends of the stalks of
the bolls were sealed with molten paraffin wax to avoid
moisture loss and decay due to secondary infection of
fungus or bacteria. The stalks were wrapped with a
cotton wick and were immersed individually in eppendorf
tubes (2 ml capacity) filled with water. This helped to
keep the bolls fresh till the larvae enter pupal stage. Bolls
were then kept inside the plastic jars (45 × 20 cm size) @
10 bolls per jar; the mouth of jar was covered with a white
muslin cloth and secured with a rubber band. The jars
were maintained in the laboratory at 27 ± 1 °C tempera-
ture and 65 ± 5% relative humidity. Newly emerged adult
moths were provided with 10% honey solution to ensure
longevity. About 10 pupae and 10 adult moths from
pooled samples of each region were examined for stan-
dard morphometric characters (e.g. shape, size, colour and
scale pattern of adult wings, etc). Additionally, six adult
moths of PBW, two from each of the three cotton growing
regions of Maharashtra were sent for identification to the
Insect Identification Service of the Division of
Entomology, ICAR-Indian Agricultural Research
Institute (ICAR-IARI), New Delhi.
Phytoparasitica
25), Washim (26–27), Amravati (28–31), Nagpur (32–41),
Chandrapur (42–50),Wardha (51–56), Yavatmal (57–64);
Marathwada Region: Nanded (65–69), Parbhani (70–73), Latur
(74–76), Beed (77–81), Osmanabad (82–83)
As this was the case of resurgence of PBW in Bt
cotton, the identity of sampled insects was further con-
firmed by molecular characterization using DNA
barcoding technique. For extraction of DNA, a total of
four newly emerged adult moths were chosen from
pooled samples of all the three cotton growing regions
viz., Khandesh (01 moth), Vidarbha (02 moths one from
Western Vidarbha and another one from Central
Vidarbha), and Marathwada (01 moth). The genomic
DNA (gDNA) was extracted from the thorax of sampled
adult moths by using the DNeasy Plant Mini Kit
(QIAGEN) with slight modification. For amplification
of the standard barcoding region of Cytochrome oxidase
I (COI) gene of mtDNA, we followed the procedure as
described by Folmer et al. (1994). The PCR reaction
volume mixture of 25 μl consisted of: 10 pmol of each
forward (5′- TTGATTTTTTGGTCATCCAGAAGT-3′)
and reverse (5’-TCCAATGCACTAATCTGCCA
TATTA -3′) COI primers, 2.5 μl of 10X Taq buffer with
25 mM MgCl 2 (Genaxy), 10 mM dNTP mixture
(Promega) and 1 Unit (U) of Taq DNA polymerase
(Genaxy) including 1.5 μl of template DNA. PCR was
Phytoparasitica
set using standard protocol (Sridhar et al. 2016;
Choudhary et al. 2017). The annealing temperature of
51 °C for 45 s was used. The PCR amplification prod-
ucts were confirmed on 1.5% agarose gel, stained with
ethidium bromide (10 μg/ml) and visualized in a
BioRad gel documentation system. Ten μl of PCR prod-
ucts were purified using Exo-SAP IT clean up solution
(Affymetrix). To resolve uncertainty in nucleotide se-
quence data, the purified PCR products having ampli-
fied mitochondrial COI regions were processed to dou-
ble pass. The sequencing was outsourced from M/S
Eurofins Genomics India Pvt. Ltd., Bengaluru, India.
The DNA sequences obtained were analyzed using
Sequence Scanner software 1.0 (Applied Biosystems)
program. Sequence alignment program MEGA6
(Tamura et al. 2013) was used for alignment of the
nucleotide sequences. BLASTn search against the nu-
cleotide sequence database in GenBank was carried out
for determining the identity of obtained sequences. The
annotated 4 unique sequences were deposited in NCBI
GenBank repository (Table 1).
Results
Confirmation of identity of PBW
Both the morphological and molecular identification
revealed that all the samples collected from cotton fields
from a total of 83 number of surveyed sites comprised of
single species i.e. pink bollworm, Pectinophora
gossypiella (Saunders) Lepidoptera: Gelechidae). The
morphological identification of larvae, pupae and
adults was based on the descriptions of Saunders
(1843) and USDA (1948). All the examined larvae were
deep pink colored with head capsule dark brown, and
were found inside the infested bolls causing damage to
the cotton seeds and lint. The newly formed pupae were
light brown colored which later turned to dark brown.
The adult moths emerged from all the collections were
light brown colored with undefined blackish spots on
the fore wings and silvery grey colored hind wings. The
adult moths were identified as Pectinophora gossypiella
based on morphological examinations by Dr. P.
Shashank, a taxonomist from Insect identification
service of the Division of Entomology, ICAR-
IARI, New Delhi (Voucher specimen no. RRS
No. 1885–1890/17, deposited at National Pusa
Collection of ICAR-IARI).
The standard insect DNA barcoding using the uni-
versal COI primers (Choudhary et al. 2017) employed in
present study successfully amplified the target fragment
( 800 bp) of mitochondrial COI gene of PBW. Sequence
comparisons using BLASTn search of all the four sam-
ples with those already deposited in NCBI-GenBank
showed 99–100% similarity (Table 1). There was no
variation among nucleotide sequences of four samples
analyzed from three different cotton growing regions of
the state. All the four sequences of PBW were deposited
in NCBI-GenBank as representatives of three major
cotton growing regions of Maharashtra viz., Khandesh
(01), Vidarbha (02) and Marathwada (01) (Table 1).
Thus, it was confirmed that the pest which was earlier
known to be susceptible to the second generation Bt
cotton i.e. Bollgard II, now appears to have developed
resistance against it, causing widespread damage across
the cotton growing areas of Maharashtra State. This has
put forth a major challenge to the cotton production of
the country.
Status of PBW infestation in Maharashtra state
The results of roving surveys conducted during
September – December, 2017 in cotton growing areas
of Khandesh, Vidarbha and Marathwada regions of
Maharashtra State indicated a progressive increase in
green boll infestation by PBW from low – moderate (<
30%) during pre-boll bursting stage (90–100 DAS) to
severe (> 65%) during boll bursting to subsequent pick-
ing stages (>120 DAS) of the cotton crop. The mean
PBW infestation at 90–100 days of cotton crop
(September 28 – October 10, 2017) in five cotton grow-
ing districts of Vidarbha region ranged between 18.33 ±
8.33% (Buldana district) and 30.00 ± 5.00% (Amravati
district) (Fig. 2). The pest was found attacking flowers
and developing green bolls of Bt cotton crop with ex-
tensive damage mainly to the green bolls (Fig. 3).
Subsequent survey of 13 districts during November
01 – December 15, 2017 revealed that in majority of the
places, the mean green boll infestation exceeded 65%
during the late reproductive growth stage of the cotton
crop that coincided with the boll bursting and subse-
quent pickings (> 120 DAS), except for Wardha and
Nagpur districts where the mean green boll infestation
was found well below 45%. The highest green boll
infestation was observed in Jalgaon district of
Khandesh region (94.99 ± 3.17%) followed by Latur,
Nanded, Parbhani and Osmanabad districts of

Table 1 Molecular identification of representative samples of Pink bollworm
Sample No. Source of Collection BLASTn similarity (%)
1. Marathwada region 99
2. Western Vidarbha region 100
3. Khandesh Region 100
4. Central Vidarbha region 100
Marathwada region, wherein the infestation levels in
randomly sampled green bolls were found consistently
well above 90% (Fig. 4). The progressive increase in
green boll infestation between pre-boll bursting and boll
bursting to subsequent picking stages of the cotton crop
was clearly visualized from the case of Akola and
Yavatmal districts of Vidarbha region which were sur-
veyed at both pre-boll opening and boll opening stages
of the cotton crop (Fig. 5).
The data on mean open boll damage expressed as the
ratio of number of damaged open bolls and total number
of open bolls per plant was reasonably correlated with
the high levels of green boll infestation in respective
districts (Fig. 6). More than 60% of the opened bolls
were found damaged by PBW in all the surveyed dis-
tricts; except for Nagpur district (35.32 ± 4.72%). The
highest open boll damage was recorded in Nanded
district of Marathwada region (71.28 ± 5.16%).
Sampling of cotton plants (> 120 DAS) bearing simul-
taneously all the three staggered stages of boll develop-
ment i.e. picked bolls with only dried bracts/ locules left
out at the bottom, opened bolls with lint in the middle
and green unopened bolls at the top portion indicated a
typical pattern of progressive increase in the level of
PBW infestation as the crop season proceeds (Fig. 7).
Fig. 2 Mean percent infestation of PBW (based on randomly
sampled green bolls) at pre-boll bursting stage in five districts of
Vidarbha region of Maharashtra State. The sample size (n) is 30
green bolls per acre at each survey location and minimum of four
locations surveyed in each district
Phytoparasitica
NCBI-GenBank accession number Insect identified
MG738712 Pectinophora gossypiella
MG738713 Pectinophora gossypiella
MG738714 Pectinophora gossypiella
MG738715 Pectinophora gossypiella
The detailed analysis of locular damage by PBW at this
stage of the cotton crop indicated severe intensity of
PBW infestation in open bolls as revealed from the
highest proportion of bolls with all the four locules
damaged (45.56 ± 5.38%) (Figs. 8 and 9).
Discussion
Status of PBW infestation and resistance to Bt cotton
We report, based on extensive field surveys, the wide-
spread infestation of PBW in second generation Bt
cotton, i.e. Bollgard II in Maharashtra, which is a lead-
ing cotton producing state of India. Astonishingly, the
pest had been recovered from all the 83 sampled loca-
tions spread across the 16 major cotton growing districts
Fig. 3 PBW damage to the flowers and green bolls of Bt cotton.
Rosetting of infested flower (a), Infested flower showing feeding
damage and PBW larva (inside the red circle) (b), green boll
showing larval exit hole (c), opened green boll with extensive
damage due to larval feeding (d)
Phytoparasitica
Fig. 4 Mean percent infestation of PBW (based on randomly
sampled green bolls) at boll bursting to first picking stage in major
cotton growing districts of Maharashtra State. The sample size (n)
of the State. The average infestation at State level based
on the damage to green bolls and open bolls was found
to be 77.75 ± 3.81% and 60.99 ± 5.88%, respectively.
The progressive increase in PBW infestation with
intensification of locular damage over the crop
season was noticed.
Occurrence and extensive damage of PBW on Bt
cotton in India in general and Maharashtra State in
particular has posed a serious concern to the entire
country’s cotton farming. In this context, the present
report thus assumes its own significance because of
the fact that the pest has resumed its pestilence state
against Bt cotton, a novel genetically engineered tech-
nology intended for combating the bollworm complex
on cotton. Being a pest species native to India, PBW had
already established and adapted to the wider climatic
conditions of the country, wherever cotton is grown.
Fig. 5 Increase in mean per cent infestation of PBW (based on
randomly sampled green bolls) from pre-boll bursting to first
picking stage in Akola and Yavatmal districts of Vidarbha region
of Maharashtra State. The sample size (n) is 40 green bolls per acre
at each survey location and minimum of four locations surveyed in
each district
is 40 green bolls per acre at each survey location and minimum of
four locations surveyed in each district
Moreover, pest like PBW establishes and perpetuates
more easily compared to other insect pests because of
its hidden biology/cryptic habitat and, protection from
insecticidal applications and natural enemies. Thus,
PBW comes under the category of ‘difficult to manage
pests’. In this context, it would not be surprising if the
pest causes havoc in Indian Bt cotton in the absence of
timely action.
There exists very few published reports documenting
PBW resistance to first generation Bt cotton i.e.
Bollgard carrying single gene Cry 1Ac in India. The
diet incorporation bioassay studies carried out by
Dhurua and Gujar (2011) on a field population of
PBW collected from Gujarat State during cotton season
of 2008 revealed the development of resistance to Bt
toxin Cry 1Ac. The mutations in a gene encoding a
cadherin protein that binds Bt toxin Cry1Ac have been
reported to be associated with field-evolved resistance
of PBW to Cry1Ac produced by transgenic cotton in
India (Fabrick et al. 2014). Although enough attention
had already been paid towards the issue of comeback
and successful feeding and survival of PBW on dual
gene Bt cotton i.e. Bollgard II in India through popular
articles (Kranthi 2015), technical bulletins (Durga
Prasad et al. 2016; Naik et al. 2017) and or print and
electronic media (Aryal 2016; The Hindu 2017), so far,
there are no published systematic research reports from
the country documenting widespread infestation of and
resistance development in PBW against second genera-
tion Bt cotton (Bollgard II). One cannot neglect the very
possibility that the pest that had broken out seriously in
one part of the country may also pose severe threat to the

Fig. 6 Mean percent infestation of PBW (based on randomly
sampled plants with opened bolls) at boll bursting to first picking
stage in major cotton growing districts of Maharashtra State. The
other parts in due course of time causing havoc.
Therefore, we feel this is the right time to alert the
stakeholders of cotton production on the seriousness of
the issue and to devise strategies and policies appropri-
ate for the effective and eco-friendly management of this
serious pest of cotton. In this perspective we presented,
through extensive surveying and repetitive sampling at
Fig. 7 Relative proportion of picked, open and green bolls (total
and damaged) at boll burst to first picking stage in Akola district of
Maharashtra State. The sample size is 25 plants with open bolls per
Phytoparasitica
sample size (n) is >25 cotton plants with opened bolls per acre at
each survey location and minimum of four locations surveyed in
each district
various growth stages of cotton crop, a detailed picture
of PBW infestation levels in various cotton grow-
ing areas of Maharashtra. The knowledge generat-
ed in present study will be crucial in getting
deeper insights into the dimensions of PBW infes-
tations that may help in assessing the potential yield
losses in cotton crop.
acre at each survey location and total of three locations were
surveyed in Akola District
Phytoparasitica

Fig. 8 Mean proportion of

damaged open bolls per plant
with varying degree of locule
damage due to PBW infestation at
boll burst to first picking stage in
Akola district of Maharashtra
State. The sample size is 25 plants
with open bolls per acre at each
survey location and total of three
locations were surveyed in Akola
District

What factors triggered resumption of the pestilence
in PBW?
In depth analysis of the phenomena underlying resur-
gence of PBW in Indian Bt cotton pinpoints on some of
the hardcore facts about the cotton farming practices
being adopted in the country. The authors are of opinion
that, non-compliance of refuge strategy comes to the
forefront of all the reasons therein. A refuge, an indis-
pensable component of Bt cotton technology had been
proposed as the most effective insecticide resistance
management (IRM) strategy that was supposed to be
crucial in delaying the process of Bt resistance develop-
ment in target insects (Head and Greenplate, 2012;
Kranthi et al. 2017). As per the guidelines of Genetic
Engineering Appraisal Committee (GEAC), Ministry of
Environment, Forest and Climate Change (Government
of India), it has been made mandatory that at least 20%
of the total area under Bt cotton hybrids must be sown
with non-Bt cotton hybrids having same duration,
flowering and fruiting windows (GEAC Guidelines for
refuge in Bt Cotton 2011; Kranthi et al. 2017) as refuge
crop. However, refuge strategy did not get enough pop-
ularity among the Indian cotton growers. The fear

Fig. 9 Typical pattern of

progressive increase in locular
damage by PBW based on
random sampling of opened bolls.
Boll with single locule damaged
(a), boll with two locules
damaged (b), boll with three
locules damaged (c) and boll with
all the four locules damaged (d)

psychosis among the cotton growers was that cultivating
non-Bt cotton over 20% of the area would accelerate the
bollworm problem, thus warranting frequent insecticide
sprays and perhaps lower the yields (Kranthi 2012).
Since most of the Bt cotton growers in India are small
scale farmers, implementation of the refuge strategy had
neither been encouraging nor profitable to them on
account of reduced margin of profit due to sparing of a
valuable portion of already scarce land for non-
productive purpose such as refuge planting
(Sadashivappa and Qaim 2009). In a nutshell, Bt cotton
is being extensively cultivated in India for more than
one and half a decade but with near-absence of refuge.
Presently, Bt cotton occupies >93% of the total area
under cotton in the country (DCD 2017). Thus, large
acreages of Bt cotton with non-enforcement of refuge
strategies has placed the Bt technology at greatest risk of
failure and appears to be the prime cause for high
selection pressure and eventual development of Bt re-
sistance in PBW.
Another, yet important dimension that adds to the
intensification of Bt resistance development in PBW, is
the growing of long duration cotton hybrids and exten-
sion of cotton season in late winters that supports con-
tinuous multiplication of the pest. The pink bollworm
appears mostly from flowering stage onwards and in-
tensity of its infestation goes on increasing with subse-
quent boll formation until maturity. Traditionally, PBW
is a late season pest of cotton with more prominence on
long duration crop grown for more than 180 days. This
has also been clearly shown by our survey results that
the PBW infestation increased progressively from pre-
boll opening to post boll opening phases of cotton crop
with intensification of locular damage. In ideal condi-
tions, timely termination of crop by the end of
December forces the larvae of PBW to enter into hiber-
nation in the absence of food. However, extension of
cotton crop beyond normal season prevents the larvae of
PBW from entering into hibernation, enabling it to
continue multiplication on the newly produced host
resources i.e. flowers and bolls (Kranthi 2012, 2015).
The crop extension, sometimes even up to late winters
(March–April) through supplementary irrigation and
fertilizer applications in view of harnessing additional
yields has been observed as a common practice among
cotton farmers of the surveyed villages. Further, sowing
of next season’s crop is taken up from April end to mid
of May (pre-monsoon sowing) in areas having protected
irrigation facilities. These situations of round the year
Phytoparasitica
availability of cotton crop might have accelerated the
process of resistance development in PBW against Bt
cotton due to increased selection pressure from
prolonged exposure to Bt toxins.
Bt cotton with possibly low expression levels of Cry
toxins could be the another yet important factor hasten-
ing Bt resistance development in PBW due to exposure
to sub-lethal dose of toxins. Certain factors like high
temperature, drought, and crop age have been found to
be affecting transgene expression in Bt cotton resulting
in reduced production of Bt toxins leading to enhanced
susceptibility of the cotton to bollworms (Kaiser 1996;
Hilder and Boulter 1999; Kranthi et al. 2005). The
important point to be noted here is that the Bt toxin
expression in transgenic cotton decreases with the in-
crease in the age of plant. PBW which comes to play its
role late in the season might be getting exposed to sub-
lethal dose of Bt toxins since the beginning of Bt cotton
era and this may be the reason for resumption of its
resistance to Bt cotton earlier than the other two species
of bollworms.
Globally PBW is an oligophagous pest, reported
damaging other than cotton, few Malvaceous plants like
okra, deccan hemp and roselle. However, it is supposed
to be monophagous under Indian conditions where large
acreage of land is put under the cultivation of cotton
crop for longer part of the year. The extensive monocul-
tures of Bt cotton being practiced since past one and half
decade (presently occupying >93% of the total cotton
area in the country) must have exerted continuous high
selection pressure on this monophagous insect pest,
triggering the process of resistance development.
Moreover being indigenous pest species, rapid combi-
nations of alleles having multiple adaptive mechanisms
for resistance to Bt toxins could have accelerated the
evolution of Bt resistant populations in PBW relative to
other polyphagous bollworms (Ojha et al. 2014).
The way forward
The lessons learnt from the past experiences entails that
the only feasible option to sustain the durability of Bt
cotton technology is to adopt the integrated measures for
PBW management that helps to restore the susceptibility
in PBW population. In this context, we propose certain
measures for achieving this uphill task. Provision of non-
Bt cotton as refuge still figures at the top of all the
measures of PBW management. The refuge planting will
help in moderating the high selection pressure and thus
Phytoparasitica
restoring susceptible genes in monophagous bollworm
pest like PBW. However, prior experience with the adop-
tion of planting structured refuge of non-Bt cotton seeds
that are supplied separately in a small bag along with the
main bag of Bt cotton seeds has not been encouraging.
Instead of providing separate bags of non-Bt cotton
seeds, provision of a single bag carrying an appropriate
mixture of Bt and non-Bt seeds could be the promising
option for ensuring high compliance on refuge planting,
the concept has witnessed remarkable success with Bt
corn in USA (Onstad et al. 2011). Hence, revised policy
is being proposed here for promoting implementation of
refuge strategy, which includes: i. provision of a single
seed bag carrying mixture of Bt seeds (90–95%) and non-
Bt seeds (5–10%) (According to the latest notification of
The Gazette of India 2016), ii. giving incentives to the
farmers those who comply with the stringent require-
ments of refuge planting, and iii. Making refuge planting
as a mandatory requirement for receiving compensation
from Government agencies in case of eventual damage
and yield loss due to PBW. Another, yet feasible way to
lessen the PBW menace in cotton is to avoid pre-
monsoon sowing of crop in the months of April–May
in irrigated pockets. An early sown irrigated crop sup-
ports the initial establishment and perpetuation of proge-
ny of PBW moths emerged from hibernating residual
population which further spreads onto the rainfed cotton
crop planted during June–July. If early planting of the
cotton crop is avoided, the PBW moths from hibernating
populations emerge only to die for the need for appropri-
ate host stage to survive. This is called ‘suicidal emer-
gence’ which substantially reduces the PBW inoculum
load on coming season’s cotton crop. Therefore a new
PBW management policy should focus on: i. creating
awareness among farmers through mass campaign re-
garding non-practicing of early season cotton planting,
and ii. enforcement of legislative control over the seed
sector, the biggest private agri-based enterprise in the
country for regulating the seed market so as to make seed
availability only for the normal planting season of cotton
crop. The promising options for successfully bringing
down the PBW populations below the economical dam-
aging levels include: i. monitoring the PBW infestation
through sex pheromone traps (carrying female sex pher-
omone ‘Gossyplure’), ii. inundative releases of egg par-
asitoid Trichogramma bactrie @ 60,000 adult wasps/ ha
three times at 15 days interval commencing from
flowering stage of the cotton crop and iii. Need based
application of insecticides having ovicidal action such as
chlorpyriphos, quinalphos and thiodicarb. The larvae of
PBW hibernating in seeds of harvested cotton stored in
bulk at places such as godowns, market yards and gin-
ning mills serve as primary source for built up of pest
inoculum that infests the new season’s crop. Therefore it
is essential to create awareness among the owners of
cotton gowdowns, market yards and ginning mills re-
garding installation of pheromone traps in the vicinity for
mass trapping and killing of post-season emerging moths.
Thus, an effective PBW management strategy has to be
viewed as collaborative efforts from all the stakeholders
of cotton industry. Further, the research efforts should be
focused on delineation of geographical hotspots with
varying levels of Bt resistance and strain variation in
PBW populations which will enable to devise and under-
take region specific management strategies.
Acknowledgements This work was supported by grants from the
‘Crop Pest Surveillance and Advisory Project^ (CROPSAP), spon-
sored by Agriculture Department, Government of Maharashtra
(India). The authors gratefully acknowledge the Head, Division of
Crop Protection and the Director of ICAR-CICR, Nagpur for insti-
tutional support in execution of the present work. The authors are
also thankful to Dr. P. Shashank, Scientist (Entomology) from
Division of Entomology, ICAR-Indian Agricultural Research
Institute, New Delhi for his valuable insect identification service.
Author contribution BBF, VSN, NGN and VNW conceived
and designed the research plan. BBF, VSN, SPG, DTN, BVN and
VD conducted the surveys and collected data on PBW infestation.
BVN and VD reared the insect in laboratory for identification of all
the life stages. SPG and DTN carried out DNA barcoding for
molecular identification of PBW. BVN and VD compiled the data.
BBF and VSN analyzed the data and drafted the manuscript. All
authors edited the manuscript.
Compliance with ethical standards
Conflict of interest Authors of this manuscript declare that there
exists no any conflict of interest with respect to the content of this
manuscript. All the sources of information accessed have been
duly acknowledged.
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