- Open Access
Efficiency of cotton bollworm (Helicoverpa armigera Hübner) control of different Bt cotton varieties in North China
- LÜ Limin †1,
- LUO Junyu †1,
- ZHANG Shuai 1,
- YU Qianlin 2,
- MA Ligang 3,
- LIU Xiaofeng 4,
- WANG Chunyi 1,
- MA Xiaoyan 1,
- MA Yan 1Email author and
- CUI Jinjie 1Email author
© The Author(s) 2018
Received: 30 March 2018
Accepted: 28 May 2018
Published: 29 June 2018
The cotton bollworm (Helicoverpa armigera) is one of cotton’s most destructive insect pests in terms of yield and quality. Since 1997, China has grown commercially available transgenic Bacillus thuringiensis (Bt) cotton. We aimed to investigate the variation in resistance of transgenic Bt cotton varieties to cotton bollworm in North China.
Populations of cotton bollworm were monitored from 2008 to 2015 in environments where Bt cotton was planted adjacent to other non-Bt crops. The study included 197 Bt cotton varieties planted in 42 counties/locations in three provinces (Hebei, Shandong and Henan) of North China, which were evaluated through field investigations, bioassays, and enzyme-linked immunosorbent assays (ELISA).
The average number of cotton bollworms never exceeded the action threshold (10 larvae per 100 cotton plants), however, their number reached 19.55 per 100 cotton plants in 2011. The ratios of damaged plants to 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 results of 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.
This study indicated that almost all Bt cotton varieties were capable to effectively control the populations of cotton bollworm in North China.
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 have 28,93.3-fold higher resistance than susceptible strains when fed Bt toxins in the laboratory (Liang et al. 2008).
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.
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).
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 bollworms of 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 was recorded.
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.
Monitoring of cotton bollworms in the field
The percentages and number exceeding the action threshold for each cotton bollworm generation from 2008 to 2015
Number of varieties
2nd generation (> 10 larvae)a
3rd generation (> 13 larvae)b
4th generation (> 13 larvae)c
Ratiosd per generation/%
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 bollworms at 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.
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 various developmental stages. At the seedling stage, the Bt protein content decreased from 2008 to 2015 (Fig. 3d), from 692.88 ng·g− 1 to 300.51 ng·g− 1. Similarly, Bt protein content in the bud-stage leaves decreased from 371.24 ng·g− 1 to 158.89 ng·g− 1 from 2008 to 2015, and that of boll-stage leaves decreased from 435.56 ng·g− 1 to 100.51 ng·g− 1 (Fig. 3e and f).
The results of this study indicate that Bt cotton varieties currently under production are capable of controlling cotton bollworms, although the field results showed 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 (Table 1, 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 this year.
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 results showed 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 bollworms develop 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 results of this 8-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.
This study evaluated 197 Bt cotton varieties (113 unique varieties) planted in 42 counties/locations of three provinces in northern China. Field investigations, bioassays, and ELISA indicated that different Bt cotton varieties were inherently equipped to control the damage incurred by cotton bollworms, and almost all Bt cotton varieties effectively controlled the populations of cotton bollworm during the years investigated.
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.
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 datasets were generated or analyzed during the current study.
Cui JJ and Ma Y conceived and designed the experiments. Lü LM and Luo JY contributed equally to this work. Lü LM, Luo JY, Zhang S, Yu QL, Ma LG, Liu XF, Wang CY and Ma XY analysed the data and prepared the figures and tables. Lü LM wrote the manuscript. All of the authors reviewed the manuscript. All authors read and approved the final manuscript.
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- Baker GH, Tann CR. Refuge crop performance as part of the Bt resistance management strategy for Helicoverpa spp. (Lepidoptera: Noctuidae) in Australian cotton production systems. Aust J Entomol. 2014;53(2):240–7. https://doi.org/10.1111/aen.12072.View ArticleGoogle Scholar
- Brevault T, Heuberger S, Zhang M, Ellers-Kirk C, Ni XZ, Masson L, et al. Potential shortfall of pyramided transgenic cotton for insect resistance management. Proc Natl Acad Sci U S A. 2013;110(15):5806–11. https://doi.org/10.1073/pnas.1216719110. View ArticlePubMedPubMed CentralGoogle Scholar
- Carriere Y, Crickmore N, Tabashnik BE. Optimizing pyramided transgenic Bt crops for sustainable pest management. Nat Biotechnol. 2015;33(2):161–8. https://doi.org/10.1038/nbt.3099.View ArticlePubMedGoogle Scholar
- Carriere Y, Fabrick JA, Tabashnik BE. Can pyramids and seed mixtures delay resistance to Bt crops? Trends Biotechnol. 2016;34(4):291–302. https://doi.org/10.1016/j.tibtech.2015.12.011.View ArticlePubMedGoogle Scholar
- Dhurua S, Gujar GT. Field-evolved resistance to Bt toxin Cry1Ac in the pink bollworm, Pectinophora gossypiella (Saunders) (Lepidoptera: Gelechiidae), from India. Pest Manag Sci. 2011;67(8):898–903. https://doi.org/10.1002/ps.2127.View ArticlePubMedGoogle Scholar
- Gassmann AJ, Carriere Y, Tabashnik BE. Fitness costs of insect resistance to Bacillus thuringiensis. Annu Rev Entomol. 2009;54:147–63. https://doi.org/10.1146/annurev.ento.54.110807.090518.View ArticlePubMedGoogle Scholar
- Gunning RV, Dang HT, Kemp FC, Nicholson IC, Moores GD. New resistance mechanism in Helicoverpa armigera threatens transgenic crops expressing Bacillus thuringiensis Cry1Ac toxin. Appl Environ Microbiol. 2005;71(5):2558–63. https://doi.org/10.1128/AEM.71.5.2558-2563.2005.View ArticlePubMedPubMed CentralGoogle Scholar
- Jin L, Zhang HN, Lu YH, Yang YH, Wu KM, Tabashnik BE, et al. Large-scale test of the natural refuge strategy for delaying insect resistance to transgenic Bt crops. Nat Biotechnol. 2015;33(2):169–74. https://doi.org/10.1038/nbt.3100.View ArticlePubMedGoogle Scholar
- Liang GM, Wu KM, Yu HK, Li KK, Feng X, Guo YY. Changes of inheritance mode and fitness in Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae) along with its resistance evolution to Cry1Ac toxin. J Invertebr Pathol. 2008;97(2):142–9. https://doi.org/10.1016/j.jip.2007.09.007.View ArticlePubMedGoogle Scholar
- Liu FY, Xu ZP, Zhu YC, Huang FN, Wang YH, Li HL, et al. Evidence of field-evolved resistance to Cry1Ac-expressing Bt cotton in Helicoverpa armigera (Lepidoptera: Noctuidae) in northern China. Pest Manag Sci. 2010;66(2):155–61. https://doi.org/10.1002/ps.1849.PubMedGoogle Scholar
- Lu ZZ, Zalucki MP, Perkins LE, Wang DY, Wu LL. Towards a resistance management strategy for Helicoverpa armigera in Bt-cotton in northwestern China: an assessment of potential refuge crops. J Pest Sci. 2013;86(4):695–703. https://doi.org/10.1007/s10340-013-0517-7.View ArticleGoogle Scholar
- Ministry of Agriculture of China. Detection of genetically modified plants and derived products-technical specification for quantitative detection of exogenous proteins in Bt transgenic cotton: No. of 1485 announcement of Ministry of Agriculture, 14–2010. Beijing: Ministry of Agriculture, China; 2013a.Google Scholar
- Ministry of Agriculture of China. Evaluation of environmental impact of genetically modified plants and it’s derived products - insect-resistant cotton part 1: evaluation of insect pest resistance: No. of 1943 announcement of Ministry of Agriculture-3-2013. Beijing: Ministry of Agriculture, China; 2013b.Google Scholar
- Püntener W. Manual for field trials in plant protection. 2nd ed. Basle: CibaGeiji Limited; 1981.Google Scholar
- Rajapakse CNK, Walter GH. Polyphagy and primary host plants: oviposition preference versus larval performance in the lepidopteran pest Helicoverpa armigera. Arthropod Plant Interact. 2007;1(1):17–26. https://doi.org/10.1007/s11829-007-9003-6.View ArticleGoogle Scholar
- Tabashnik BE, Carriere Y, Dennehy TJ, Morin S, Sisterson MS, Roush RT, et al. Insect resistance to transgenic Bt crops: lessons from the laboratory and field. J Econ Entomol. 2003;96(4):1031–8. https://doi.org/10.1093/jee/96.4.1031.View ArticlePubMedGoogle Scholar
- Trtikova M, Wikmark OG, Zemp N, Widmer A, Hilbeck A. Transgene expression and Bt protein content in transgenic Bt maize (MON810) under optimal and stressful environmental conditions. PLoS One. 2015;10(4):e0123011. https://doi.org/10.1371/journal.pone.0123011.View ArticlePubMedPubMed CentralGoogle Scholar
- Van Rensburg JBJ. First report of field resistance by the stem borer, Busseola fusca (fuller) to Bt-transgenic maize. S Afr J Plant Soil. 2007;24(3):147–51. https://doi.org/10.1080/02571862.2007.10634798.View ArticleGoogle Scholar
- Wan P, Zhang Y, Wu K, Huang M. Seasonal expression profiles of insecticidal protein and control efficacy against Helicoverpa armigera for Bt cotton in the Yangtze River valley of China. J Econ Entomol. 2005;98(1):195–201.View ArticlePubMedGoogle Scholar
- Wu KM, Guo YY. The evolution of cotton pest management practices in China. Annu Rev Entomol. 2005;50:31–52. https://doi.org/10.1146/annurev.ento.50.071803.130349.View ArticlePubMedGoogle Scholar
- Wu KM, Guo YY, Gao SS. Evaluation of the natural refuge function for Helicoverpa armigera (Lepidoptera : Noctuidae) within Bacillus thuringiensis transgenic cotton growing areas in North China. J Econ Entomol. 2002;95(4):832–7. https://doi.org/10.1603/0022-0493-95.4.832.View ArticlePubMedGoogle Scholar
- Wu KM, Lu YH, Feng HQ, Jiang YY, Zhao JZ. Suppression of cotton bollworm in multiple crops in China in areas with Bt toxin-containing cotton. Sci. 2008;321(5896):1676–8. https://doi.org/10.1126/science.1160550.View ArticleGoogle Scholar
- Ye LF, Fu X, Ouyang F, Xie BY, Ge F. Determining the major Bt refuge crops for cotton bollworm in North China. Insect Sci. 2015;22(6):829–39. https://doi.org/10.1111/1744-7917.12171.View ArticlePubMedGoogle Scholar
- Zhang HN, Tian W, Zhao J, Jin L, Yang J, Liu CH, et al. Diverse genetic basis of field-evolved resistance to Bt cotton in cotton bollworm from China. Proc Natl Acad Sci U S A. 2012;109(26):10275–80. https://doi.org/10.1073/pnas.1200156109. View ArticlePubMedPubMed CentralGoogle Scholar
- Zhang HN, Yin W, Zhao J, Jin L, Yang YH, Wu SW, et al. Early warning of cotton bollworm resistance associated with intensive planting of Bt cotton in China. PLoS One. 2011;6(8) https://doi.org/10.1371/journal.pone.0022874.
- Zhang SP, Cheng HM, Gao YL, Wang GR, Liang GM, Wu KM. Mutation of an aminopeptidase N gene is associated with Helicoverpa armigera resistance to bacillus thuringiensis Cry1Ac toxin. Insect Biochem Mol Biol. 2009;39(7):421–9. https://doi.org/10.1016/j.ibmb.2009.04.003.View ArticlePubMedGoogle Scholar