Impact Analysis of Genetically Modified ( Bt ) Cotton Genotypes on Economically Important Natural Enemies under Field Conditions

Field studies were conducted at Hisar during Kharif, 2009-2010 and 2010-2011 under natural and unsprayed condition. RCH 134 Bt, HS 6 (non Bt), H 1117 and Ganganagar Ageti (non Bt) were selected for the studies. The experiment was laid down in Randomized Block Design replicated thrice having plant spacing 67.5 × 60 cm. Among the bollworms, Erias spp. exhibited positive correlation with rainfall, minimum temperature and relative humidity morning hours significantly at 5 percent level whereas Helicoverpa and Pectinophora displayed positive relations only with evening hours relative humidity significantly while Spodoptera were significantly positive correlated with relative humidity of morning and evening hours. Bollworm complex was negatively correlated with all the weather parameters employed to study although being non-significant in case of rainfall, maximum and minimum of temperature, morning and evening hours of relative humidity. Among the sucking pests, leafhoppers, whitefly, thrips and aphids population showed significantly negative correlation with weather parameters. The comparison of natural bio-agents in cotton hybrids under study revealed that overall mean population of natural enemies were higher in Bt cotton hybrid as compared to non-Bt. These results confirm that use of genetically modified (Bt) cotton in lieu of conventional genotypes could positively impact non-target and beneficial insect species by preserving their host populations.


Introduction
Cotton is one of the major commercial crops and backbone of textile industry in India which provides employment to vast majority of population directly or indirectly.It provides livelihood of 60 million people depending on cotton cultivation, processing trade and textiles.Textile industry contributes 4% of GDP, 14% of total industrial product, 20% of total work force, 17% of country's exports earning and employment to 30 million people [1].It is also one of the major sources that accounts for more than 30% of exports, making it India's largest net foreign exchange industry to the tune of $10 -12 billion annually from exports of cotton yarn, thread, fabrics, apparel and made-ups [2].India produces around 11 percent of the world's cotton from 20 percent of the area.In India, cotton area was 11.7 million ha with productivity of 496.4 kg lint/ha and total production of 35.6 million bales [3].
The pest spectrum of cotton is quite complex and as many as 1326 species of insects have been recorded in the world [4].Cotton has the most fragile ecosystem amongst the field crops where approximately 162 insectpests damage the crop in India [5].Among the vast array of insect pests, major insect-pests which cause economic losses in cotton are bollworm complex includes American bollworm (Helicoverpa armigera), pink bollworm (Pectionphora gossypiella) and spotted bollworm (Earias spp.) while sucking pests are white fly, Bemisia tabaci (Gennadius); leafhopper, Amrasca biguttula biguttula (Ishida); mealy bug, Pnenacoccus solenopsis Tinsley; thrips, Thrips tabaci Lindeman; and aphid, Apis gossypii Glover [6].Since the introduction of Bt cotton in agriculture in 1987 in USA by Monsanto, Delta and Pine companies [7] scenario has been changed as Bt replaced the conventional cotton and provided growers with a new tool for managing bollworms in cotton.In this context, amazingly, Bt cotton is one of the few technologies that have the safest bio-safety profile.It comes as an alternative to the previously used hazardous concoction of insecticides mixtures.Insecticides used on cotton were known to have ravaged ecology, disrupted the environment, played havoc with human and animal health, were toxic to honey bees, insect-parasitoid and predators.Bt cotton removed that to a great extent [8].Criticism also focused on the bio-safety issues starting from adverse impact on soil microbes, pet animals and reported presence of Bt in human blood and placenta.Most of these reports were characterized by methodology errors and clearly lacked scientific credence in establishing a clear, cause and effect relationship [9].Moreover, it's a common myth that Bt cotton has adverse effect on the population of economically important arthropods particularly natural enemies, which we have tried to study in these investigations.As bio-control agents considered to be an integral part of Integrated Pest Management (IPM) strategies, many workers have made assiduous study to nullify the assumptions of adverse effect on insects particularly of beneficial species [10] observed low incidence of plant damage, low density of lepidopteran and homopteran pests with relatively high densities of Chrysoperla spp in Bt. cotton hybrids as compared to non-Bt hybrids.Similarly, higher populations of spiders, predatory bugs (Geocoris spp.), green lace wing (Chrysopa spp.) and coccinellids (Coccinella spp.) were observed [11] [12] in Bt hybrids than non-Bt hybrids of cotton.An increase of 25 percent population of natural enemies in Bt cotton fields was observed [13] than non-Bt fields.Keeping in the mind regarding the assumption of adverse effect on living organism particularly of natural enemies of insect-pests in cotton, in present studies the attempt has been made to study the population dynamics and correlation of important insect-pests along with natural enemies on Bt as well as non-Bt cotton hybrids.

Materials and Methods
The experiments were conducted at the Cotton Research Farm, Department of Plant Breeding and Genetics, CCS Haryana Agricultural University, Hisar during 2009-2010 and 2010-2011 under natural conditions.Hisar city also known as city of steel and traditional cotton growing area, geographically located in Indo-Gangetic plains of North-West India at 215.2 meter (705 ft MSL) coordinates at 29˚10'0"N, 75˚43'0"E on map, receives average precipitation 490.6 mm annually with average summer and winter temperature 32.5˚C and 17.6˚C, respectively.RCH 134 Bt, HS 6, H 1117 (non-Bt) and Ganganagar Ageti (non-Bt) were selected for the study.RCH 134 Bt is popularly grown Bt hybrid, H 1117 and HS 6 are highly susceptible to bollworm complex and Ganganagar Ageti highly susceptible for sucking pests and moderately susceptible to bollworms.Seeds of RCH 134 Bt is of Rasi Seed Company, HS 6 and H 1117 procured from Haryana Agricultural University, Hisar and Ganganagar Ageti made available by Regional Research Station, Swami Keshwanand Rajasthan Agricultural University, Sri Ganganagar.

Research Farm-layout plan
The experiment was laid down in Randomized Block Design (RBD) replicated thrice having plant spacing 67.5 × 60 cm in a plot of 50 × 20 square meter.All agronomical practices were followed as per the recommendations of "Package of Practices of Kharif Crops" except plant protection measures [14].Fertilizers namely Nitrogen, Phosphorous, Potassium and Zinc sulphate were applied at the rate of 70, 24, 24 and 2.100 kg in the field of cotton crop during the crop season.Of the total fertilizers, one third of nitrogen, total of Phosphorous, Potassium and Zinc Sulphate were applied at the time of sowing followed by one third of nitrogen at the time of square formation and remaining one third at the time of flowering.First irrigation was provided to crop after 30 to 40 days after sowing, subsequently field irrigated at the interval of 15 -20 days.Irrigation of cotton field synchronized especially with flowering and boll formations during the crop season.Standard procedures were adopted to record the observations on population of economically important insect-pests damaging cotton crop namely bollworms included American, Helicoverpa armigera; pink, Pectinophora gossypiella, spotted, Earias insulana, E. vittella (Fabricius) and Spodoptera litura.The observations on the population of bollworms were recorded at weekly intervals during period of study.The observation on bollworms infestation in green fruiting bodies (buds, flowers, squares and bolls) was started from August for both the years of study.Population and damage in open bolls and loculi was taken at the time of maturity.The sample size and method employed for sampling the population of bollworms was as under.

Intact Fruiting Bodies
Observations on the incidence of bollworm complex on the intact fruiting bodies (square, flower, bolls) were recorded at weekly interval from randomly selected five plants from each plot.The infestation was calculated by following formula: No. of fruiting bodies infested Percent bollworm infestation 100 Total number of fruiting bodies = × Pink Bollworm; Pectinophora gossypiella (Saund.)For pink bollworm infestation, 50 unopened green bolls were plucked from each plot randomly at 80, 110 and 140 days after sowing.

Open Bolls
Boll infestation was recorded in open bolls on boll and locule basis from randomly selected 5 plants/treatment.

Boll Basis
Total number of open bolls from randomly selected 5 plants were counted from each plot and examined critically for bollworm infestation.The per cent bollworm infestation in the bolls was calculated by following formula:

( )
No. of bolls infested Percent boll infestation Boll basis 100 Total number of bolls = ×

Locule Basis
To work out the bollworm infestation on locule basis, the total number of open bolls of randomly selected 5 plants was counted.All the loculi of these bolls were examined carefully for bollworms infestation.Presence of hole in the septa of the locule was considered as infected.The per cent infestation was calculated with the following formula: ( ) No. of infested loculi Percent boll infestation locule basis 100 Total number of loculi = × Simultaneously, to record the bollworm complex on pheromones (Make PCI, India ltd) trap catches basis, trap catches were installed in the field at the rate of four trap per plot.Pheromones capsules were replaced at ten days interval.Observations were recorded daily and then total numbers of catches per day were cumulated to weekly pheromones trap catch of an individual bollworm species.

Sucking Insect-Pests
The sucking pests included white fly, Bemisia tabaci; leafhopper, Amrasca biguttula biguttula; mealy bug, Pnenacoccus solenopsis; thrips, Thrips tabaci; aphid, Apis gossypii and mirid bugs of different genera.The observations on sucking pests were recorded at weekly intervals, starting from 24 th standard week to the end of 42 nd standard week.The observations of whitefly, leafhopper, aphids and thrips were recorded from randomly selected five plants on the lower surface of 3 randomly selected leaves (one each from upper, middle and lower plant canopy) per plot at weekly intervals.Mirid bugs were recorded from 100 squares (20 top squares/plant from 5 plant).

Natural Enemies
The natural enemies viz., lady bird beetle, Coccinella septumpuncata; green lace wing, Chrysoperla carnea; spiders, yellow wasps etc. were recorded at weekly intervals on randomly selected 5 plants in each variety and standard method i.e., beat basket method (dimension of basket-diameter 30 cm, ht.37.5 cm) to record the natural enemies was used for sampling.Total trap catch was observed per day of a particular bollworm and then counted cumulatively per standard week.

Statistical Analysis
The data collected during studies in the above experiments were got computed for analysis of variance [15].In case of sucking pest population the data were analyzed for variance by adopting n + 1 transformation.Significant differences were set at P ≤ 0.05 and P ≤ 0.01 to workout correlations coefficient in between weather parameters, insect-pests population and natural enemies counts for the statistical analysis of the data [15].

Results
The results of studies conducted at during Kharif 2009-2010 and 2010-2011 revealed that the sucking pest's population started their build up from 24 th standard week and continued till 42 nd standard week in Bt as well as non-Bt hybrids.On the other hand, presence of bollworms in the field recorded in-between 29 th to 31 st of standard week.

Sucking Insect-Pests
In non-Bt cotton hybrids (Ganganagar Ageti and HS 6) leaf hopper population 6.5 per three leaves was maximum in 33 rd standard meteorological week (SMW), while whitefly population was maximum 5.55 per three leaves 26 th SMW in the crop season 2010-2011 (Figure 1).Leaf hoppers 4.9 per three leaves in 30 th and whitefly 7.44 per three leaves in 25 th standard week recorded maximum in Bt cotton hybrid RCH 134 (Figure 2).
The population of mirid bugs was observed only in non-Bt hybrids while in Bt Hybrid (RCH 134 Bt) their population was almost negligible (Figure 2).Population of thrips was observed as maximum in the 29 th SMW whereas, mirid bug population was recorded maximum in the 40 th , SMW.However the presence of aphids in cotton field was recorded 42 nd SMW in late growth stages of crop (Figure 1 and Figure 2).

Bollworm Complex
It has been observed that the pheromones trap catches of all the bollworms were first noticed in 32 nd SMW except pink bollworm.The trap catch of Erias spp.increased up to 40 th week then decreased.In case of trap catches, Helicoverpa armigera peaked in 40 th SMW and then decreased and again increased in 44 th SMW.There were gradual increase in catches of Spodoptera up to 40 th SMW and afterwards a decline was observed and again there was a spurt of Spodoptera population in 44 th SMW.Pectinophora gossypiella catches were noticed in 38 th SMW and peak was in 40 th SMW (Figure 3

Population Dynamics of Insect Pests during 2009-2010 Sucking Insect Pests and Bollworm Complex
During the cotton crop season 2009-10, it was quite conspicuous that leafhopper and whitefly populations remained below the economic threshold in both varieties H 1117 (Figure 4) and RCH 134 Bt (Figure 5).However    there was a gradual increase followed by a decrease in the population of these two sucking pests.
During the crop season 2009-2010, maximum leafhopper population was observed in 35 th SMW, while whitefly population buildup was maximum in 40 th SMW in H-1117.Bollworm complex remained above five per cent level during crop season from 38 th till the end of the cotton season registering highest in 40 th SMW.Leaf roller was recorded during 34 th to 43 rd standard week and marked their peak in 38 th SMW (Figure 4).
In RCH 134Bt maximum population of sucking pests was observed in 35 th SMW, while bollworm complex remained below economic threshold from 36 th SMW till the end of fruiting satges.Leaf roller remained confined in ends of crop season from 35 th to 42 nd SMW showing a peak in 38 th SMW 1.95 per plant (Figure 5).

Population Dynamics of Natural Enemies 2010-2011 3.3.1. Spiders
The population of spiders was present throughout the crop season on all three hybrids.Ganganagar Ageti harboured the maximum number of spiders which was higher than that on other two hybrids.However in case of HS 6, spiders were recorded continuously throughout the season whereas the spiders could not be recorded on Ganganagar Ageti during 33 SMW and on RCH 134 Bt during 31 and 42 SMW.Ganganagar Ageti was observed supporting maximum numbers of spiders (0.23) followed by 0.19 in RCH 134 Bt and 0.18 in HS 6 per plant.

Coccinellid
Number of Coccinellid was in HS 6 and Ganganagar Ageti whereas it was 0.01 in RCH 134 Bt.Population of Coccinellids on hybrids was lower as compared to other two natural enemies.

Others
The natural enemies like dragonflies, damselflies, yellow wasp etc. was also recorded on the test cultivars.Their population was i.e., maximum 0.53 and 0.33 per plant on HS 6 and RCH 134 Bt.During 37 th SMW with mean population of 0.15 and 0.13 per plant, respectively.On Ganganagar Ageti it was maximum (0.63 per plant) in 25 th SMW with an average of 0.19 per plant.2).Maximum spiders were observed at 29 th standard week in RCH 134 Bt while it was 33 rd week in H 1117.

Chrysoperlla
Mean number of Chrysoperla (0.17/plant) was higher in RCH 134 Bt as compared to (0.10/plant) in H 1117. Maximum population (0.34/plant) was recorded in RCH 134 Bt in 37 th SMW while in H 1117 maximum population (0.25/plant) was observed in 33 rd SMW.

Coccinellid
Mean population of Coccinellids in both the hybrids seems to be comparable with each other.Maximum numbers, 0.15 and 0.40/plant were observed in 37 th and 34 th SMW in RCH 134 Bt and H 1117, respectively.

Others
As regards the population of other natural enemies like dragonflies, damselflies, yellow wasp, etc. in test hybrids, H 1117 harboured maximum number (0.49 per plant) and the peak reached in 34 th SMW having mean of 0.18 per plant, whereas, in case of RCH 134 Bt maximum number (0.43 per plant) was recorded in 29 th SMW having average number (0.24 per plant) of these natural enemies.

Correlation between Natural Enemies and Insect-Pests of Cotton 2010-2011
During 2010-2011 crop season (Table 3)-Correlation worked out between natural enemies and insect-pests of cotton, it was quite interesting to note that bollworms have exhibited negative association with the natural biocontrol agents i.   correlation coefficient.While others bio-agents shown preference r = −0.380and −0.451 in HS6 and GA, respectively.Whitefly shown the positive correlation with natural enemies particularly worth mention of Chrysoperla (r = 0.340) in GA, Coccinellids in HS6 (r = 0.646) and GA (r = 0.479).In HS 6 others # bio-agent were having positive association (r = 0.288).In case of thrips, it were learned that only spiders (r = 0.500) in HS 6, Chrysoperla (r = 0.417) in GA and Coccinellids (r = 0.423) in RCH 134 Bt shown positive association.In mirid bugs Spiders (r = 0.409), Chrysoperla (r = 0.350) and other # bio agents (r = 0.387) in HS 6 were positively correlated, while aphid shown positive association (r = 0384) with Chrysoperla in HS 6.

Correlation between Natural Enemies and Insect-Pests of Cotton 2009-2010
During 2009-2010 crop season (Table 4), natural enemies and insect-pest correlation shown that most of the bio-control agents were of preference to sucking pest at most.Spiders (r = 0.663, 0.752) and Chrysoperla (r = 0.554, 0.809) preferred leafhoppers in Bt and non-Bt cotton hybrid.The same trend were apparent in case of whitefly in RCH 134 Bt and H 1117 during the crop season for predation of spiders (r = 0.399, 0.740) and Chrysoperla (r = 0.345, 0.743).Only spiders (r = 0.304) preferred bollworm in RCH 134 Bt.

Correlation of Abiotic Factor and Insect-Pests of Cotton (Pooled)
Table 5 exhibited that Erias spp.positively correlated with rainfall (r = 0.516), minimum (r = 0.583) and maximum (r = 0.363) of temperature, relative humidity (RH) of morning (r = 0.578) and evening (r = 0.277) hours, significantly.Helicoverpa was positively related to RH of morning (r = 0.384) and evening (r = 0.572) hours.In pink bollworm, rainfall (r = −0.422)and minimum (r = −0.370)temperature were found negatively associated, while RH of evening (r = 0.690) hours was positive correlated.In Spodoptera, maximum (r = 0.381) temperature, RH of morning (r = 0.655) and evening (0.827) hours were found positive correlated.Overall bollworm complex was negative correlated with all the parameters studied, although minimum (r = −0.386)temperature and RH of  morning (r = −0.320)hours are of worth mention.Leafhoppers, whitefly and thrips were of negative association significantly in all weather parameters.In case of aphid, all abiotic factors were positively correlated significantly except RH of evening hours.Mirid bug was positive correlated with rainfall (r = 0.295), minimum (r = 0.320) and maximum (r = 0.365) of temperature and RH of morning (r = 0.357) hours.

Correlation of Abiotic Factor and Natural Enemies of Insect-Pests of Cotton (2010-2011)
Correlations Table 6 revealed that in HS 6 cotton hybrid, spiders were found negative associated with minimum (r = −0.424)temperature and RH of evening (r = −0.476)hours.Chrysoperla was negatively correlated in maximum (r = −0.265)and minimum (r = −0.369)temperature and RH of evening (r = −0.268)while, it was positive associated with RH of morning (r = 0.345) hours.In others category, which included dragonflies, damselflies, yellow wasp etc. were negative correlated with minimum (r = −0.366)temperature and RH of evening (r = −0.410).

Correlation of Abiotic Factor and Natural Enemies of Insect-Pests of Cotton (2009-2010)
Chrysoperla was negative correlated with rainfall (r = −326), while Coccinellid was positive in RH of evening (r = 0.265) hours in non-Bt cotton hybrid (Table 7).
In Bt hybrid RCH 134, Chrysoperla was negative with the rainfall (r = −0.279),while in RH of morning (r = 0.269) hours was positively associated.Coccinellids positive associated with minimum (r = 0.301) temperature and RH of evening (r = 0.323) hours.

Discussion
The correlations between the populations of insect pests of cotton hybrids and their natural enemies were worked out are presented in Tables 3-5.Different meteorological parameters were correlated with insect pests invading cotton crop are presented in Tables 5-7.
Among the bollworms, Erias spp.exhibited significantly positive correlation with rainfall, minimum temperature and Morning RH whereas Helicoverpa and Pectinophora populations were positively related only with Evening RH and Spodoptera was significantly positive correlated with morning and evening RH.The overall population of bollworm complex was negatively correlated with all the weather parameters used in the study although it was statistically non-significant.In case of sucking pests, leafhoppers, whitefly, thrips and aphids inverse  [16] and lady bird beetle showed significantly positive correlation with sunshine hours and significantly negative correlation with minimum temperature and relative humidity [17].
The comparison of natural bio-agents in cotton hybrids under study (Table 8 & Table 9) revealed that overall mean population of natural enemies were higher in Bt cotton hybrid as compared to non-Bt.The present scenario corroborates the findings of [13] report that 25 percent increase in natural enemies of pests in Bt cotton fields and observed higher number of natural enemies such as wasps (Vespoidea), aphids consuming flies (Syrphus spp.) and ladybirds (Hippodamia spp.) than in non-Bt fields [12].Natural enemies, viz.spiders, predatory bugs (Geocoris spp.), green lace wing (Chrysoperla spp.) and Coccinellids (Coccinella spp.) were significantly higher in Bt hybrids than non-Bt hybrids [11].

Conclusion
In conclusion, the present results confirm that use of Bt cotton in lieu of conventional could positively impact non-target and beneficial organisms by preserving their host populations [18]- [20].
(a) and Figure 3(b)).Bollworms infestation throughout the fruiting season remained above economic threshold (5 per cent infestation in shed fruiting bodies) from mid August till crop maturity.

Table 1 .
Population of natural enemies during the crop season in cotton hybrids, 2010-2011.

Table 2 .
Population of natural enemies during the crop season in cotton hybrids, 2009-2010.

Table 3 .
Correlation coefficient (=r) of natural enemies and insect-pests on cotton hybrid during crop season 2010-2011.

Table 4 .
Correlation coefficient (=r) of natural enemies and insect-pests on cotton hybrid during crop season 2009-2010.

Table 6 .
Correlation coefficient (=r) of abiotic factors and natural enemies population on cotton during crop season 2010-2011.

Correlation coefficient (=r) for natural enemies population HS 6 Ganganagar Ageti RCH 134 Bt Spiders Chrysoperla Coccinelids Others # Spiders Chrysoperla Coccinelids Others # Spiders Chrysoperla Coccinelids Others #
with all the weather parameters exception case of aphids being non-significant with evening RH.Mirid bugs population did not show any correlation with the weather parameters studied.Correlations for the natural enemies during crop season 2010-2011 in relation to abiotic factors were non-significant with most of the parameters in hybrids HS 6, Ganaganagar Ageti and RCH 134 Bt except negative correlations between spiders and evening RH, Coccinellid & Chrysoperla with morning RH.Coccinellids had significantly positive correlation with maximum temperature only in hybrid Ganganagar Ageti.During the crop season 2009-2010, none of the weather parameters was found to have any correlation in case of H 1117 and RCH 134 Bt.Negative correlation of green lace wing with all abiotic factors relationship

Table 8 .
Population of natural enemies in Bt and non-Bt genotypes of cotton, 2009-2010.

Table 9 .
Population of natural enemies in Bt and non-Bt genotypes of cotton, 2010-2011.