Efficiency of cotton bollworm(Helicoverpa armigera Hübner)control of different Bt cotton varieties in North China

LÜ Limin,LUOJunyu,ZHANGShuai,YUQianlin,MALigang,LIUXiaofeng,WANGChunyi,MAXiaoyan,MAYan*and CUIJinjie*

Abstract Background:The cotton bollworm(Helicoverpa armigera)isone of cotton’smost destructive insect pestsin termsof yield and quality.Since 1997,China hasgrown commercially available transgenic Bacillusthuringiensis(Bt)cotton.We aimed to investigate the variation in resistance of transgenic Bt cotton varietiesto cotton bollworm in North China.Methods:Populationsof cotton bollworm were monitored from 2008 to 2015 in environmentswhere Bt cotton was planted adjacent to other non-Bt crops.The study included 197 Bt cotton varietiesplanted in 42 counties/locationsin three provinces(Hebei,Shandong and Henan)of North China,which were evaluated through field investigations,bioassays,and enzyme-linked immunosorbent assays(ELISA).Results:The average number of cotton bollwormsnever exceeded the action threshold(10 larvae per 100 cotton plants),however,their number reached 19.55 per 100 cotton plantsin 2011.The ratiosof damaged plantsto total Bt cotton stem-tips,buds,and bolls was low except in 2010,when the ratios reached 1.82%,2.09%,and 10.63%,respectively.The resultsof bioassay showed that the corrected mortality were higher at the second generation cotton bollworm stage than the third and fourth germination stages.Totally,Bt protein content declined sharply at the seedling stage from 2008 to 2015.Conclusions:Thisstudy indicated that almost all Bt cotton varietieswere capable to effectively control the populations of cotton bollworm in North China.

Keywords:Upland cotton,North China,Bt cotton varieties,Cotton bollworm,Efficiency evaluation

Background

The cotton bollworm,Helicoverpa armigera Hübner,is one of cotton’s most destructive insect pests.In the 1990s,both cotton yield and quality were severely affected by cotton bollworm outbreaks in major cotton production provinces of China.Since 1997,China has grown commercially available transgenic Bacillus thuringiensis(Bt)cotton.Since its introduction,Bt cotton has been produced on a large scale in three provinces of Hebei,Shandong,and Henan in China(Liu et al.2010;Wu and Guo 2005;Wu et al.2008).

Transgenic Bt cotton has been grown for 19 years in China.Over time,and as the plantation areas for Bt cotton have expanded,cotton bollworms have become increasingly resistant to Bt insecticidal proteins,prompting researchers to conduct investigations(Liu et al.2010;Zhang et al.2011).Bt-resistant cotton bollworm strains have been reported to habe 2 893.3-fold higher resisitance than susceptible strains when fed Bt toxins in the laboratory(Liang et al.2008).

The mechanism of bollworm resistance has been investigated in the laboratory,with particular focus on the Bt insecticidal protein(Gunning et al.2005;Zhang et al.2009).Field-evolved resistance of cotton bollworm to Bt crops has been documented in several countries(Dhurua and Gujar 2011;Van Rensburg 2007;Zhang et al.2011).In field-selected populations,recessive cadherin alleles accounted for 75%–84%of the resistance alleles detected;most resistance alleles occur as heterozygotes,and 59%–94% of the resistant individuals carried at least one recessive resistance allele(Zhang et al.2012).Currently,resistance management tactics use large refuges and pyramiding approaches to maintain low inherent resistance of cotton bollworm to Bt toxins.Bt cotton production is also integrated with other pest management tactics.These are promising approaches and useful techniques for managing bollworm resistance in Bt cotton.Some refuge crops such as corn,soybean,and peanut have been screened and assessed(Carriere et al.2016;Jin et al.2015;Lu et al.2013;Wu et al.2002).The experimental model used in this study of planting Bt cotton in large areas with non-Bt crops present(e.g.,watermelon,peanut,corn,pepper,and sorghum)has been extensively used in North China(Fig.1a to e)for the past eight years.

However,investigations on variations in field resistance to bollworms in different Bt cotton production varieties over an extended period are limited.The area under Bt cotton production in the three provinces has decreased in favor of other major crops in the last 8 years (Additional file 1:Table S3,Additional file 2:Figure S1).Changes in Bt insecticidal protein concentration of the Bt cotton varieties planted during this time have been monitored using enzyme-linked immunosorbent assays(ELISA).The effects on cotton bollworms of Bt insecticidal protein samples collected from 197 Bt cotton varieties were determined using bioassays.

The major aims of this study were to investigate variations in the field resistance to cotton bollworms of 197 Bt cotton varieties(Additional file 1:Table S1–1)in three major cultivation provinces(Hebei,Shandong,and Henan;Additional file 1:Table S2)for an extended period(8 years)and to evaluate the efficiency of different Bt cotton varieties in controlling cotton bollworm in North China.

Methods

Experimental Bt cotton varieties,locations,and planting patterns

A total of 197 transgenic Bt cotton varieties were monitored for Bt-induced mortality of cotton bollworm and the ability of Bt cotton to control cotton bollworm in the field from 2008 to 2015(Additional file 1:Table S1–1).These Bt cotton varieties included 113 unique varieties in different locations and years(Additional file 1:Table S1–2).These experimental Bt cotton varieties were supplied by the county and planted 42 locations(Additional file 1:Table S2)in Henan,Hebei,and Shandong provinces in North China.

The same Bt cotton varieties were planted at the East experimental farm(36°5′34.8”N,114°31′47.19″E)of the Institute of Cotton Research of Chinese Academy of Agricultural Sciences every year,and the buds,leaves,and bolls of these varieties were collected and used in the bioassays and Bt protein analyses from 2008 to 2015.

The area planted with Bt cotton is continually decreasing(Additional file 1:Table S3,Additional file 2:Figure S1).The experimental planting patterns consisted of Bt cotton situated adjacent to corn,watermelon,pepper,sorghum,and peanut(Fig.1a to e).

Field investigations

Three field investigations to determine the number of Bt-induced live cotton bollworm larvae were conducted in the period June,July and August each year from 2008 to 2015.A total of four generations of cotton bollworm emerged during May and August each year in North China,generally in a cycle of one generation per month.The first generation of cotton bollworm fed on wheat during May 10 to June 10.Upon maturity and harvest of wheat,cotton bollworms transferred to the cotton field to lay eggs after June 10,which was named as the second generation of cotton bollworms.Regarding the life history of cotton bollworm,we conducted field investigations during the peak period of cotton bollworm larvae proliferation,from the second to fourth generation larval stage of cotton bollworm during the third 10-day period every month.

We investigated three sites forming a triangle for each Bt cotton variety.And twenty plants were selected per site.We recorded the number of cotton bollworm larvae,damaged tip-stems,buds,and bolls,and healthy buds and bolls.

Bioassay of cotton bollworms

Newly-hatched cotton bollworm larvae of the first generation were used in the bioassays.The populations of larvae were all Bt-susceptible and were maintained separately at(25±1) °C and 60%–70%relative humidity(RH),with a photoperiod of 16 h∶8 h(L∶D).

The leaf-feeding bioassay method was performed based on the national standard(Ministry of Agriculture of China,2013b).Briefly,five cotton bollwormsof the first generation were fed with cotton leaves,with 20 leaves per variety with three replicates.The newly-hatched first-generation larvae were used in the bioassays that were conducted from the mid-June to August each year.We selected 20 healthy cotton leaves from each variety,which were placed in a plastic bioassay box(4-cm diameter×4-cm high),onto which five newly-hatched larvae were gently placed.After that,bioassay boxes were placed in a culture room(26–28 °C,RH=60%).The CK(CCRI 49)variety was used as the non-Bt cotton control.After five days,the number of dead larvae wasrecorded.

Fig.1 The planting pattern of Bt cotton with other cropsin the different locations(a to e).a The intercropping of Bt cotton and watermelon.b Bt cotton adjacent peanuts.c Bt cotton adjacent corn.d Bt cotton adjacent peppers.e Bt cotton adjacent sorghum

Bt protein content analysis using ELISA

Each of the Bt cotton varieties was planted in three experimental plots as three replicates.Bt protein concentrations in all of Bt cotton varieties planted on the East experimental farm of the Institute of Cotton Research of CAAS were determined by ELISA according to standardized methods(Ministry of Agriculture of China,2013a)using QualiPlate™Kit for Cry1Ab/Cry1Ac(Envirologix Inc.,500 Riverside Industrial Parkway,Portland,Maine,United States)each year.Twenty leaves from all experimental Bt cotton varieties were collected,and then rapidly immersed in liquid nitrogen.All samples were transported back to the laboratory.The 20 fresh cotton leaves of each variety were ground,and then 0.4 g of each powdered sample was used to extract Bt protein,following the instructions provided in the QualiPlate™kit for Cry1Ab/Cry1Ac.

Data analysis

The data were analyzed using Microsoft Excel 2007 and DPS7.0.Standard errors of data were calculated using the STDEV and SQRT functions.The following equations were used in the calculations(Püntener,1981):

Results

Monitoring of cotton bollworms in the field

Cotton bollworm larvae on Bt cotton varieties were investigated from 2008 to 2015.The results showed that the number of cotton bollworm larvae peaked in 2011,the only year in which the average number of cotton bollworm larvae per 100 plants exceeded the action threshold of 10 larvae at the second cotton bollworm generation stage.In the other years,the average number of cotton bollworm larvae of all varieties per 100 plants remained below the action threshold(Fig.2a)for all stages.The average number of cotton bollworms remained below the action threshold at the third and fourth cotton bollworm generation stages in seven of the 8 study years(Fig.2b and c).Standard deviations were calculated for the samples of cotton bollworm larvae collected from all investigated cotton varieties(Fig.2a to c).

The study results also revealed the ratio and the number of varieties exceeding the action threshold as well as the bollworm mortality induced by Bt protein in each cotton bollworm generation from 2008 to 2015(Table 1).Our results indicated that,in 2011,9 of the 22 varieties exceeded the action threshold at the second cotton bollworm generation stage.Similarly,in 2009,5 of 29 varietiesexceeded the action threshold at the second and third cotton bollworm generation stages.Whereas during other years,the number of the varieties reached the action threshold was less than 2011 and 2009 at the second and third generations.The results showed there were differences in the cotton bollworm resistanceability among different Bt cotton varieties.

Fig.2 The changes in the number of cotton bollwormsand the ratios of damaged cotton stem-tips,buds,and bolls.a to c Changes in the number of cotton bollworms found at the second,third and fourth cotton bollworm generation stages from 2008 to 2015;d to f The ratios of damaged cotton stem-tips,buds,and bolls from 2008 to 2015.The error barsare SEs

Table 1 The percentages and number exceeding the action threshold for each cotton bollworm generation from 2008 to 2015

Ratio of damaged stem-tips,buds,and bolls

The ratio of damaged tip-stems is an essential evaluation index of Bt cotton resistance to cotton bollwormsat the second cotton bollworm generation.The highest ratio of damaged tip-stems was 1.82%,whereas the average ratio ranged from 0.00 to 0.95%(Fig.2d).The ratio of damaged buds and bolls is an essential evaluation index for Bt cotton resistance at the third and fourth cotton bollworm generation(Fig.2e and f).The results indicate that the highest ratios of damaged buds and bolls were 2.09%and 10.63%respectively,at the third and fourth cotton bollworm generation in 2010.

Bioassay analysis

The corrected bollworm mortalities of the tested Bt cotton varieties were recorded for each cotton bollworm generation.The corrected Bt-induced mortality at the second cotton bollworm generation stage was higher than at the third and fourth cotton bollworm generationsin most years from 2008 to 2015.The corrected mortality at the second cotton bollworm generation stage initially increased,then subsequently decreased.Moreover,the average corrected mortality of all varieties exceeded 90%in 2010 and 2011,which was the highest resistance level(Fig.3a).However,the resistancelevel hasdeclined in thepast few years.

The overall resistance level decreased at the third and fourth cotton bollworm generations from 2008 to 2015(Fig.3b and c).Bt-induced mortality ranged from 19.13%to 48.35%in the third cotton bollworm generation from 2008 to 2015 and from 11.86%to 84.46%in the fourth cotton bollworm generation(Fig.3c).

Changes in Bt protein content

Bt protein content at the seedling,bud and boll stages from 2008 to 2015 was assessed using ELISA.Bt protein content significantly differed at variousdevelopmental stages.At the seedling stage,the Bt protein content decreased from 2008 to 2015(Fig.3d),from 692.88 ng·g−1to 300.51 ng·g−1.Similarly,Bt protein content in the bud-stage leaves decreased from 371.24 ng·g−1to 158.89 ng·g−1from 2008 to 2015,and that of boll-stage leaves decreased from 435.56 ng·g−1to 100.51 ng·g−1(Fig.3eand f).

Discussion

The results of this study indicate that Bt cotton varieties currently under production are capable of controlling cotton bollworms,although the field resultsshowed that different varieties have varying levels of bollworm resistance.Field monitoring shows that most of the main cotton varieties are effective in controlling the cotton bollworm population.The average number of cotton bollworms exceeded the action threshold in only 1 year during the 8-year study period and generally remained below the action threshold in most of the Bt cotton varieties(Table1,Fig.2a).

In 2011,we investigated infestation of cotton bollworms during the second-generation stage at Nanpi County in Hebei.At this location,there was a variety of weeds growing around the cotton field.These weeds provided abundant hosts for the bollworm population(Rajapakse and Walter,2007).It was speculated that this might be one reason for more cotton bollworms at Nanpi in this year.In 2010,there was a high ratio of damaged cotton buds and bolls at the fourth generation stage.The main reason was cotton boll rot,caused by above-average rainfall in July and August of thisyear.

Fig.3 The changesin corrected mortality and Bt protein content.a to c The changesin corrected mortality at the second,third,and fourth cotton bollworm generationsfrom 2008 to 2015;d to f the changesin Bt protein content at the seedling,bud,and bollstagesfrom 2008to 2015.The error barsare SEs

The bioassay results of this study demonstrated that Bt-induced bollworm mortality decreased in the second cotton bollworm generation,which is one of the most destructive generations for cotton.This implies that the cotton bollworms can evolve resistance to Bt cotton;resistant insects have been produced on Bt cotton both in the field and in the laboratory(Liang et al.2008;Tabashnik et al.2003;Zhang et al.2011).In addition,in field-selected populations,most resistance alleles occur as heterozygotes,and 59%–94%of resistant individuals carried at least one recessive resistance allele,even though the resistance level to Bt cotton was low(Zhang et al.2012).

These reports show that the mechanism of resistance to cotton bollworm is complex.However,the change in mortality ratio or resistance may be the result of reduced Bt insecticidal protein concentration in Bt cotton.The ELISA resultsshowed that the Bt cotton Cry1Ac contents in seedlings significantly decreased from 2008 to 2015.The reduced Cry1Ac contents in Bt cotton were correlated with changes in bollworm mortality in the field.This result shows that the bollwormsdevelop resistance to current cotton varieties;therefore,new cotton varieties with multi-resistance to bollworm should be developed to delay the evolution of bollworm resistance to Bt cotton in the field(Baker and Tann,2014;Ye et al.2015).Consequently,the pyramid strategy for delaying the evolution of pest resistance to Bt crops is apparently risky(Brevault et al.2013;Carriere et al.2015).The use of natural refuge crops is a good strategy to delay cotton bollworm resistance(Baker and Tann 2014;Ye et al.2015).

The results of our bioassay and ELISA showed some years were unusual in terms of corrected mortality and Bt protein.This might be due to significant differences in temperature and rainfall in 2009 and 2014.Results of previous studies have shown that the efficacy of Bt cotton plants is affected by environmental factors such as changes in light intensity,water and nitrogen availability or insect and wind damage.Differing environmental conditions can regulate the transcript levels of cry1Ac,and specifically modulate Bt gene expression(Trtikova et al.2015;Wan et al.2005).

The results of the present study reveal that the most practical strategy to control the bollworm population is to plant different Bt cotton varieties.Bt cotton varieties from different breeding units play an important role in restraining cotton bollworm damage.Employing refuge crops that are designed to increase the dominance or magnitude of fitness is particularly useful in delaying pest resistance(Gassmann et al.2009).Adjacent non-Bt crops that function as bollworm host plants may provide sufficient natural refuges to delay the evolution of bollworm resistance to Bt cotton(Carriere et al.2016;Jin et al.2015).The resultsof this8-year study show that planting Bt cotton with any other non-Bt crops is a strategy that may have contributed to the control of the bollworm population.

Conclusions

This study evaluated 197 Bt cotton varieties(113 unique varieties)planted in 42 counties/locationsof threeprovinces in northern China.Field investigations,bioassays,and ELISA indicated that different Bt cotton varieties were inherently equipped to control thedamageincurred by cotton bollworms,and almost all Bt cotton varieties effectively controlled the populations of cotton bollworm during the yearsinvestigated.

Additional files

Additional file 1:Table S1–1.Bt cotton varietiesmonitored from 2008 to 2015.Table S1–2.Single Bt cotton varietiesafter repeat planted varieties were deleted from 2008 to 2015.Table S2.The study sitesthat were investigated from 2008 to 2015.Table S3.Plantation areafor cotton and other cropsthat were assessed from 2008 to 2015.(XLSX 31 kb)

Additional file 2:Figure S1.The percentage of planted Bt cotton area monitored from 2008 to 2015.(TIF149 kb)

Acknowledgments

We thank Cotton Technological Guidance Station of Shandong Province,Cash Crops Technological Guidance Station of Hebei Province and Cash Crops Extension Station of Henan Province in China.

Funding

This research was supported by the project of China Agriculture Research System(CARS-15-20).

Availability of data and materials

Data sharing not applicable to this article as no datasetswere generated or analyzed during the current study.

Authors’contributions

Cui JJand Ma Yconceived and designed the experiments.LüLM and Luo JYcontributed equally to this work.LüLM,Luo JY,Zhang S,Yu QL,Ma LG,Liu XF,Wang CYand Ma XYanalysed the data and prepared the figures and tables.LüLMwrote the manuscript.All of the authors reviewed the manuscript.All authors read and approved the final manuscript.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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Author details

1Institute of Cotton Research,Chinese Academy of Agricultural Sciences(CAAS),State Key Laboratory of Cotton Biology,Anyang 455000,Henan,China.2Cotton Technological Guidance Station of Shandong Province,Jinan 250000,China.3Cash Crops Technological Guidance Station of Hebei Province,Shijiazhuang 050000,China.4Cash Crops Extension Station of Henan Province,Zhengzhou 450000,China.

Received:30 March 2018 Accepted:28 May 2018