July 26, 2021
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Bt-Resistance in Cotton Pink Bollworms: Was it Inevitable from the Start? Dr. Hadi Husain Khan1, Dr. Mohd. Monobrullah2, Sirumoni Phukon3, Nitumoni Mahanta4, Jishnu Pratim Mudoi5, Suraj Baruah6 & Jabir Ahmed7 1Research Associate, ICAR-DRMR-APART, Dhubri -783324 (Assam), India.

Bt-Resistance in Cotton Pink Bollworms: Was it Inevitable from the Start?
Dr. Hadi Husain Khan1, Dr. Mohd. Monobrullah2, Sirumoni Phukon3, Nitumoni Mahanta4, Jishnu Pratim Mudoi5, Suraj Baruah6 & Jabir Ahmed7
1Research Associate, ICAR-DRMR-APART, Dhubri -783324 (Assam), India.
2Principal Scientist, Division of Crop Research, ICAR-RCER, Patna- 800014 (Bihar), India.
3ADO, Simlitola, Office of the District Agricultural Officer, Dhubri – 783324 (Assam), India.
4ADO, Alomganj, Office of the District Agricultural Officer, Dhubri – 783324 (Assam), India.
5ADO, Madhusoulmari, Office of the District Agricultural Officer, Dhubri – 783324 (Assam), India.
6ADO, Barundanga, Office of the District Agricultural Officer, Dhubri – 783324 (Assam), India.
7ADO, Halakura, Office of the District Agricultural Officer, Dhubri – 783324 (Assam), India.

India is considered as second largest producer of cotton in the world after China. Bollworms (particularly pink bollworm: Pectinophora gossypiella) are the major pests, feeding on fibres and seeds, causing serious yield reductions and economic losses in cotton. Though its origin is still not clear to us, pink bollworm infestation in cotton was first reported from India during 1842 (Tabashnik and Carrière, 2019). Pink bollworms have very narrow ranges of plant hosts other than cotton. Generally, in a newly grown cotton field, bollworm larvae of first generations feed upon flower-buds (locules) but their subsequent generations feed on entire contents of cotton bolls. Breakthrough happened in 1995 when United States approved the field commercialization of Bt-cotton, followed by China (1997) and India (March, 2002). Genetically engineered cotton, inserted with insecticidal proteins from soil-borne, gram positive, Bacillus thuringiensis (Bt) bacterium, was developed to kill targeted herbivorous insect-pests and to reduce insecticidal sprays. The crystalline (Cry) toxins from Bt must be passed to larval midgut (alkaline pH) to pose toxicity.
Based on the insecticidal activity and sequences in amino acid, Cry toxins are grouped into five classes:
1. Cry1 (effective against Lepidoptera).
2. Cry2 (effective against Lepidoptera and Diptera).
3. Cry3 (effective against Coleoptera).
4. Cry4 (effective against Diptera).
5. Cry5 (effective against Lepidoptera and Coleoptera)

In India, Bt-cotton (Bollgard) was developed by Maharashtra Hybrid Seed Company Limited (Mahyco) in a joint venture with Monsanto (US) using Cry1Ac. The Bollgard-II was developed in 2006, combining both Cry1Ac and Cry2Ab genes. Currently, Bollgard-II occupies more than 90% Gossypium hirsutum-cotton areas in India (Naik et. al., 2020). With the extension of Bt-cotton areas, ‘refuge’ (non-Bt) cotton areas started to decline and the Bt-resistance in pink bollworms began to develop (Naik et. al., 2020). The pink bollworm shows Cry-toxin resistance through disruption of binding sites between Cry-toxin and midgut protein receptors (Wan et. al., 2017).

First Reports of Bt-Resistance:
There were many predictions of Bt-resistance in bollworms but the laboratory DNA-screening of field populations in Arizona first confirmed that Bt-resistant alleles (r1, r2, and r3) of cadherin (BtR) protein were inherited recessively to pink bollworms against Cry1Ac toxin (Morin et. al., 2003). However, this resistance was found rare in cotton growing areas of Arizona, California, and Texas during 2001-2005 (Tabashnik et. al., 2006). In India, first pink bollworm resistances against Bt-toxins (Cry1Ac and Cry2Ab) were reported from Amreli district of Gujrat, followed by Sriganganagar in Rajasthan, Surat in Gujrat, and Dharwad in Karnataka (Dhurua and Gujar, 2011). Now a day, reports from all cotton growing areas of north, central, and south India confirmed the population extension of Bt-resistant pink bollworms (Naik et. al., 2020). In China, first report of pink bollworm resistance against Cry1Ac was detected through dietary bio-assay in laboratory during 2008 but unlike India, failures of Bt-cotton at field levels were not seen (Tabashnik et. al., 2012).

Types of Bt-Resistances by Pink Bollworm:
Tabashnik and Carrière, 2019 categorized pink bollworm resistances against Bt-cotton as followed:

1. No decrease in susceptibility: Statistically non-significant decrease in pink bollworm population susceptibility against Bt-cotton (e.g., Bt-resistance of pink bollworm in US).

2. Practical resistance: It is field-evolved resistance against Bt-cotton, reducing its efficacy to control pink bollworm (e.g., Bt-resistance of pink bollworm in India).

3. Early warning of resistance: It includes monitoring all field-evolved resistance cases in a particular region (country or collection of different cotton growing regions under study) where at least one population of pink bollworm have to be resistant, but the others must stay susceptible (e.g., Bt-resistance of pink bollworm in China).

Strategies Adopted in Major Cotton Growing Countries:
Factors that delayed the Bt-resistances of pink bollworms in major commercial cotton producing countries (China, India, and US) include maintaining random abundance of refuges (non-Bt cotton), Bt-resistance associated fitness costs, incomplete resistance for some period of time, and recessive inheritances (Tabashnik and Carrière, 2019).

1. Partially sterile pink bollworm moths (irradiated) were released in huge number into Bt-cotton fields of Arizona, US. This huge number of sterile moths mated with rare resistant pink bollworm moths from Btcotton fields and only few larvae, aroused from those reproductions, became heterozygous recessive resistant. Thus, their survival upon to Bt-cotton became negligible. Even, these larvae cannot become fertile moths without exposure to Bt-toxins. As a result, Bt-cotton (carrying both Cry1Ac and Cry2Ab) acreage increased near 100%; while decrement in refuge cottons (non-Bt) was observed from more than 25% to less than 5% in 2010. Based on extensive field monitoring from 2010 to 2018, pink bollworm was declared eradicated by United States Secretary of Agriculture in the year of 2018 (Tabashnik and Carrière, 2019).
2. China adopted a seed mixture strategy to delay Bt-resistance in pink bollworms. This novel mixture was performed by crossing Bt-cotton with non-Bt cotton and planting F2 generations in field. Crossing between non-Bt cotton and Bt-cotton produces F1 hemizygotes (carrying Bt-toxin). Self-pollination of these F1 hemizygotes generates F2 seeds which contain Bt-hemizygotes (50%) and Bt-homozygotes (25%), producing Bt-toxin (Cry1Ac), and non-Bt cottons (25% homozygotes) which don’t have Bt-toxin. This resulted in 25% randomly dispersed non-Bt cottons (refuges) in Bt-cotton fields. These hybrids also yielded higher than their parents due to heterosis and this fact was implemented by seed companies in China. When the non-Bt cotton areas (ha) got increased to 67% during 2011-2015, 96% decrease in pink bollworm population density was observed (Wan et. al., 2017).
3. In 2002, Genetic Engineering Approval Committee (GEAC) of India approved Bollgard to be grown with refuge (non-Bt) cottons in 20% Bt-cotton sown areas. In fact, Indian seed companies supplied seeds of nonBt cotton (120 g) along with Bt-cotton packets, carrying 450g of Bt-seeds but due to lack of proper knowledge about refuge cottons and need for maximizing yield, Indian farmers stayed away from planting non-Bt cotton seeds (Mohan, 2017). As an impact, pink bollworm developed practical resistance and unlike US and China, it is already too late for India to delay the resistance through ‘refuge strategy’. Thus, experts are recommending Integrated Pest Management (IPM) to achieve pest suppression in long run.

Some IPM strategies to reduce pink bollworm infestation in cotton fields include:
a. Pheromones trapping and disruption of mating.
b. Insecticidal spray based on threshold levels.
c. Use of natural enemies and bio-pesticides of microbial origin.
d. Use of chemical repellents.
e. Avoiding mono-cropping and extended cotton growing season.
f. Planting early duration cotton hybrids.
g. Avoiding rationing of cotton.
h. Destruction of infested crop residues.

In contrast to US and China, sparse areas for refuge non-Bt cottons in India allowed Bt-resistant pink bollworms to proliferate rapidly. During 2014, mean Bt-resistance ratio for pink bollworms were found to be 78-times higher for Cry2Ab and 310-times higher in case of Cry1Ac which together accounted nearly 52% destruction of Bt-cotton bolls in India (Naik et. al., 2020). The PgCad1 gene mutation encrypts Cry1Acbinding cadherin protein, responsible for Cry1Ac resistance; while, mutation of PgABCA2 gene encodes ATP-binding cassette (transporter protein), responsible for pink bollworm resistance against Cry2Ab toxin (Morin et. al., 2003; Tabashnik and Carrière, 2019). In India, the surge in resistant pink bollworms renders the Bt-cotton production unfavourable and inefficient. Experts are not suggesting Indian seed companies to introduce multiple toxins producing Bt-cottons as it might help these pests to evolve their resistance in upcoming years. Thus, return to IPM is the only option left for India to control pest resistance and sustain cotton production.

1. Dhurua S. and Gujar G.T., (2011). Field‐evolved resistance to Bt toxin Cry1Ac in the pink bollworm, Pectinophora gossypiella (Saunders) (Lepidoptera: Gelechiidae), from India. Pest Management Science. 67:898-903.
2. Mohan K.S., (2017). An area-wide approach to pink bollworm management on Bt cotton in India–a dire necessity with community participation. Current Science. 112:2016-2020.
3. Morin S., Biggs R.W., Sisterson M.S., Shriver L., Ellers-Kirk C., Higginson D., Holley D., Gahan L.J., Heckel D.G., Carrière Y. and Dennehy T.J., (2003). Three cadherin alleles associated with resistance to Bacillus thuringiensis in pink bollworm. Proceedings of the National Academy of Sciences. 100:5004-5009.
4. Naik V.C.B., Pusadkar P.P., Waghmare S.T., Raghavendra K.P., Kranthi S., Kumbhare S., Nagrare V.S., Kumar R., Prabhulinga T., Gokte-Narkhedkar N. and Waghmare V.N., (2020). Evidence for population expansion of cotton pink bollworm Pectinophora gossypiella (Saunders) (Lepidoptera: Gelechiidae) in India. Scientific Reports. 10:1-11.
5. Tabashnik B.E. and Carrière Y., (2019). Global patterns of resistance to Bt crops highlighting pink bollworm in the United States, China, and India. Journal of Economic Entomology. 112:2513-2523.
6. Tabashnik B.E., Fabrick J.A., Henderson S., Biggs R.W., Yafuso C.M., Nyboer M.E., Manhardt N.M., Coughlin L.A., Sollome J., Carrière Y. and Dennehy T.J., (2006). DNA screening reveals pink bollworm resistance to Bt cotton remains rare after a decade of exposure. Journal of Economic Entomology. 99:1525-1530.
7. Tabashnik B.E., Wu K. and Wu Y., (2012). Early detection of field-evolved resistance to Bt cotton in China: Cotton bollworm and pink bollworm. Journal of Invertebrate Pathology. 110:301-306.
8. Wan P., Xu D., Cong S., Jiang Y., Huang Y., Wang J., Wu H., Wang L., Wu K., Carrière Y. and Mathias A., (2017). Hybridizing transgenic Bt cotton with non-Bt cotton counters resistance in pink bollworm. Proceedings of the National Academy of Sciences. 114:5413-5418.

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