Nitrogen spraying affects seed Bt toxin concentration and yield in Bt cotton

ZHANG Xiang,ZHOU Ming-yuan,LI Ya-bing,LIU Zhen-yu,CHEN Yuan,CHEN De-hua

1 Jiangsu Key Laboratory of Crop Genetics and Physiology/Co-Innovation Center for Modern Production Technology of Grain Crops,Yangzhou University,Yangzhou 225009,P.R.China

2 Cotton Research Institute,Chinese Academy of Agricultural Sciences,Anyang 455000,P.R.China

Abstract Cotton bolls exhibit the lowest insecticidal efficacy among all organs of Bt cotton,which would ultimately affect the yield formation.The objective of this study was to investigate the effects of different urea concentrations on the seed Bt protein contents,seed cotton yield and the corresponding protein metabolism mechanism.The experiments were conducted during 2017–2018 cotton growing seasons.Two cultivars,Sikang 3 (hybrid,SK3) and Sikang 1 (conventional,SK1),were treated with six urea concentrations and their seed Bt protein contents were compared during boll formation period.The urea spray concentration had a significant effect on the seed Bt toxin content and seed cotton yield.Spraying of either 5 or 6%urea led to higher insecticidal protein contents and higher seed cotton yield for both cultivars.Moreover,the highest amino acid and soluble protein contents,as well as GPT and GOT activities,and lower protease and peptidase activities were observed at the 5 to 6% urea levels.Significant positive correlations between the seed Bt toxin and amino acid contents,and between the seed Bt toxin content and GPT activities were detected.The lower boll worm number and hazard boll rate were also observed with the 5 to 6% urea treatments,which may be the reason why nitrogen spraying increased the seed cotton yield.Therefore,our results suggested that the seed Bt toxin content and insect resistance were impacted markedly by external nitrogen application,and 5 to 6% urea had the greatest effect on insect resistance.

Keywords:Bt cotton,external nitrogen application,urea concentration,Bt toxin,protein metabolism

1.Introduction

Unstable insecticidal characteristics in Bt cotton have been observed worldwide (Cui and Xia 1999;Stone 2011),often characterized by high insect resistance before flowering,but markedly reduced insecticidal protein expression during the boll formation period (Greenplateet al.2000;Adamczyk and Meredith 2004;Zhang and Wen 2011),and the leaves typically exhibit higher insecticidal ability than reproductive organs (Shenget al.2010;Stone 2011).Also,adverse environmental conditions affected the insect resistance(Benedictet al.1993;Wanget al.2009;Chenet al.2012a,b).Switching off the Bt gene,dilution of Bt protein in the organs and inactivation of Bt protein due to the binding of Bt toxin with tannin all contribute to the insecticidal protein concentration reduction (Xia and Guo 2004;Yuanet al.2012).

The Bt insect resistance is related to Bt protein concentration,and synthesis of Bt protein is associated with nitrogen metabolism.Therefore,nitrogen metabolism might play an important role in insect resistance.Some studies have confirmed that nitrogen fertilizer in soil increased Bt protein content and insecticidal ability (Oosterhuis and Brown 2000;Coviella 2002;Yanget al.2005;Wanget al.2012).Other investigations further indicated that nitrogen metabolic physiology has close relationships with Bt protein expression in Bt cotton (Chenet al.2005,2013),nitrogen metabolic strength impacts the Bt protein concentration of cotton organs (Chenet al.2004;Yuanet al.2012),and nitrogen deficit reduces Bt protein contents in Bt cotton(Chenet al.2018,2019).However,most studies focused mainly on the effect of soil nitrogen application on leaf Bt protein expression in cotton.The reproductive organs of cotton are the primary target of boll worm,and the most severe damage from boll worm infestation usually happens at the bolling stage.Thus,increasing of the Bt toxin content,especially for reproductive organs,becomes the main goal in Bt cotton production.Nitrogen spraying is highly efficient and the method providing the easiest absorption.Therefore,we hypothesized that external application of nitrogen would affect the cotton seed Bt protein concentration in Bt cotton,and that the alteration was related to nitrogen metabolism.

The objectives of this research were:(i) to determine the effect of external nitrogen application on seed Bt toxin content;and (ii) to study the nitrogen metabolic mechanism of the change in seed Bt protein content under different nitrogen spraying concentrations.

2.Materials and methods

2.1.Plant materials and experimental design

Field experiments were carried out at Yangzhou University Farm,Jiangsu Province,China (32°30´N,119°25´E) in 2017–2018.Sikang 1 (conventional,SK1) and Sikang 3(hybrid,SK3),which are two widely grown Bt cotton cultivars in China,were used in this study with the planting densities of 27 000 (SK3) and 37 500 (SK1) plants ha–1.Seeds were sown on 15 April (2017) and 16 April (2018) in a plastic cover Lilliputian greenhouse.Seedlings were transplanted to the field on 13 May (2017) and 15 May (2018).The soil(sandy loam texture (Typical fluvaquents,Entisols (U.S.taxonomy))) contained 21.6 and 22.3 g kg–1organic matter,108.3,110.2,24.2 and 25.3,82.1,81.3 mg kg–1available N-P-K in 2017 and 2018,respectively.Cultivation practices,including applications of fertilizers and insecticides,chemical plant growth retardant DPC (1,1-dimethyl piperidinium chloride) spray,and irrigation,were carried out following local recommendations.

Before planting,K (100 kg ha–1as KCl) and P (300 kg ha–1as single superphosphate) were applied.At early flowering,K (100 kg ha–1as KCl) and P (300 kg ha–1as single superphosphate) were top-dressed.N (urea) was applied before transplanting (20%),at early flowering (25%) and at peak flowering (55%).The nitrogen fertilization dose in the experiments is 300 kg ha–1.

The experiment was arranged with split plot designs.The main plot treatment was cultivars (SK1 and SK3),and the subplot treatment consisted of six urea spraying concentrations,0,2,4,6,8,and 10% in 2017,and 0,1,3,5,7,and 9% in 2018.The solutions of the treatments were sprayed on the cotton plants at the peak flowering stage.Three replications were used in the field.Each plot consisted of 5-m lengths of rows,with rows spaced 0.83 m apart.

2.2.Preparation of plant materials

SamplingFive bolls were harvested from the first position of the 4th to 6th fruiting branches in each plot,and seeds of the same position from the boll were picked for measurements of Bt protein and the chemicals of nitrogen metabolism.

The Cry1Ac protein contentImmunological analysis ELISA was used to determine the Cry1Ac content in the seed extracts as described by Chenet al.(1997).The seeds (0.2 g FW) were used to determine the toxin protein content.

Free amino acid and soluble protein contentBased on Yemmet al.(1955),analysis of the total free amino acid content was measured by the ninhydrin assay.The Coomassie blue dye-binding assay of Bradford (1976) was used for total soluble protein content determination.

Glutamic-pyruvic transaminase (GPT) and glutamate oxaloacetate transaminase (GOT) activitiesThe seeds(0.2 g FW) were homogenized in 0.05 mmol L–1Tris-HCl,pH 7.2 buffer.The supernatant was collected after centrifugation at 26 100×g for 10 min at 4°C.For GOT activity assay,0.2 mL of the supernatant was added to a mixture containing 0.5 mL of 0.8 mol L–1alanine in 0.1 mol L–1Tris-HCl (pH 7.5),0.1 mL of 2 mmol L–1pyriodoxal phosphate solution and 0.2 mL of 0.1 mol L–12-oxoglutarate solution.The reaction mixture was incubated at 37°C for 10 min followed by addition of 0.1 mL of a 0.2-mol L–1trichloroacetic acid solution to stop the reaction.The color intensity was read at 520 nm.The GPT activity assay was similar to the GOT assay.In the GPT assay,0.5 mL of a 0.1-mol L–1buffered aspartate solution was used in the reaction mixture instead of 0.5 mL of 0.8-mol L–1alanine in 0.1 mol L–1Tris-HCl (pH 7.5) (Tonhazyet al.1950).

Protease and peptidase activitiesThe seeds (0.8 g FW)were homogenized at 4°C in 1 mL of β-mercaptoethanol extraction buffer (a mixture of ethylene glycol,sucrose and phenyl methyl sulfonyl fluoride at pH 6.8).The supernatant was collected to estimate the square protease.Protease activity was determined spectrophotometrically at 400 nm using azocasein as a substrate (Vanceet al.1979) and expressed as mg protein g–1flower FW h–1.The seed samples (0.5 g FW) were homogenized at 4°C in 8 mL of Tris-HCl extraction buffer (a mixture of 4 mmol L–1DTT,4 mmol L–1EDTA,1% PVP,pH 7.5).The supernatant(0.4 mL) was collected by centrifugation at 15 000×g for 30 min at 4°C,added to a reaction mixture (0.4 mL acetate buffer (pH 4.8),1% bovine hemoglobin compounded with 0.2 mL acetate buffer (pH 4.8)) and incubated at 38°C for 60 min.A total of 1 mL of a 10% trichloroacetic acid solution was added to stop the reaction.The supernatant collected by centrifugation (4 000×g for 5 min) was used for amino acid content analysis by the ninhydrin assay (Yemmet al.1955),and peptidase activity was expressed as µmol amino acid g–1flower FW h–1.

Investigation of the number of boll worms and hazard rates of the abscission bollIn the 2018 insect harm experiment,the number of boll worms per 10 plants and hazard rates of the abscission bolls by boll worm were surveyed for every plot.

Seed cotton yieldFor each plot,all open bolls were manually harvested once on 31 October,and weighed for seed cotton yield.

2.3.Statistical analysis

Analyses of quadratic regression for relationships were conducted with PROC ANOVA in SAS 6.Multiple comparisons were evaluated by Duncan’s multiple range tests atP≤0.05.The data were summarized and presented by year.

3.Results

3.1.Effect of spraying different urea concentrations on cotton seed Bt toxin content and insect resistance

Significant declines in cotton seed Bt protein content were observed with boll development,lower toxin contents at 20 days after flowering (DAF20) and DAF25 were detected relative to that at DAF15.The trends of the seed Bt protein content variation with increased urea concentration were similar at the three growth stages for both studied cultivars(Figs.1 and 2).In 2017,the toxin content increased from 2 to 6% urea,and declined from 6 to 10%.In 2018,the toxin content increased with the increasing urea spraying concentration at first,but then decreased when the concentration exceeded 5% urea.The greatest cotton seed Bt toxin contents were observed at 6% (2017) and 5% urea (2018).In comparison to the control,the largest values represented increases by 92.9 and 60.0% (2017),and 57.8 and 54.8% (2018) at DAF15 in SK1 and SK3,respectively and the differences were significant in both years.Similar results were found at DAF20 and DAF25,but the extents of the increments were reduced.Although SK3(hybrid) had higher seed Bt protein content,the conventional cultivar (SK1) had a higher extent of increment with urea spraying.The results showed that an optimum urea spraying concentration would significantly increase cotton seed Bt protein content,especially for the conventional cultivar.

The investigation of field boll worm hazard (Table 1)further confirmed the results in Figs.1 and 2.The lowest boll worm number per 10 plants and percent of damaged squares were detected at 6% urea (2017) and 5% urea(2018).These findings showed that spraying 5–6% urea could increase insect resistance in Bt cotton.

3.2.Effect of spraying different urea concentrations on seed cotton yield

Significant differences of seed cotton yield were observed both in 2017 and 2018 (Fig.3).The trends of the seed cotton yield variations with increased urea concentration were similar for SK1 and SK3.The highest seed cotton yields were observed at 6% (2017) and 5% urea (2018),and the yields were more than 4 442.9 kg ha–1,respectively.Compared with the 0% treatment,the seed cotton yields for spraying 6% urea respectively increased by 28.3 and 32.0%for SK1 and SK3 in 2017.Similarly,the yield for spraying 5% urea respectively increased by 24.5 and 25.4% for SK1 and SK3 in 2018.

The above results (Figs.1–3;Table 1) showed that spraying urea increased insect resistance and control of cotton bollworms,and this may be the reason why nitrogen spraying increased the seed cotton yield.Spraying of 5 to 6% urea produced high yield and high insect resistance at the same time for both cultivars.

3.3.Effect of spraying different urea concentrations on protein metabolism of cotton seed

Fig.1 The seed insecticidal protein contents of the cotton seed under different urea concentrations in 2017 at 15 days after flowering (DAF) (A),20 DAF (B) and 25 DAF (C).SK1 and SK3,Sikang 1 and Sikang 3,respectively.The relationship between the seed Bt toxin content and urea spraying concentration(%) can be quantified by a parabolic equation at each boll developmental stage,where y stands for the seed toxin content,and x represents urea spraying concentration.Bars mean SD(n=3).

Soluble protein and amino acids contentsIncreases in the seed soluble protein contents were observed with boll development (Table 2),with the highest soluble protein content detected at DAF25.The trend of soluble protein content under different urea spraying concentrations was similar to that of the Bt protein content,with the highest soluble protein content observed at 6% in 2017 and 5%in 2018.In comparison to the control,the highest values were increased by 107.8 and 125.0% (2017),and by 91.5 and 95.1% (2018) at DAF 15 for cultivars SK1 and SK3,respectively.Similar results were found at DAF20 and DAF25.Hybrid cultivar SK3 had both higher seed soluble protein content and extent of increment than conventional cultivar SK1 with the different urea spraying concentrations.

Fig.2 The seed insecticidal proteins contents of the cotton seed under different urea concentrations in 2018 at 15 days after flowering (DAF) (A),20 DAF (B) and 25 DAF (C).SK1 and SK3,Sikang 1 and Sikang 3,respectively.The relationship between the seed Bt toxin content and urea spraying concentration (%) can be quantified by a parabolic equation at each boll developmental stage,where y stands for the seed toxin content,and x represents urea spraying concentration.Bars mean SD (n=3).

Similar to the results of the seed soluble protein content,the seed amino acid content increased with boll growth seasons (Table 3).The amino acid content was bolstered from 2 to 6% urea,but then reduced from 6 to 10% urea in 2017,and it was enhanced from 1 to 5% urea,but then declined from 5 to 9% urea in 2018.The highest seed amino acid contents were observed at 6% (2017) and 5% urea(2018).In comparison to the control,the highest values were enhanced by 40.7 and 20.2% (2017),and by 41.7 and 31.1%(2018) at DAF15 for SK1 and SK3,respectively.Similar results were found at both DAF20 and DAF25,but the extents of increases were bolstered.Hybrid cultivar SK3 had a higher seed amino content than the conventional cultivar.The correlation analysis showed significant linear positive correlations between Bt protein and amino acid contents(Fig.4),with the correlation coefficients of 0.938**,0.666*and 0.891**for SK1 (2017);and 0.666*,0.912**and 0.813**for SK3 (2018) at DAF15,DAF20 and DAF25,respectively.

Table 1 Insecticidal efficacy at the peak boll period under different urea concentrations in 2017 and 2018

Table 2 The cotton seed soluble protein contents at three boll developmental stages under different urea concentrations in 2017 and 2018 (mg g–1)

Fig.3 The seed cotton yields under different urea concentrations in 2017 and 2018.SK1 and SK3,Sikang 1 and Sikang 3,respectively.Differences among treatments labeled with the same letters are not statistically significant at P=0.05.Bars mean SE (n=3).

GPT and GOT activitiesReduced seed GPT activity was observed with growth seasons,and GPT activity declinedfrom DAF15 to DAF25.The seed GPT activity was enhanced at first,but then reduced with increasing urea spraying concentrations (Table 4).The greatest seed GPT activities were detected at 6% (2017) and 7% (2018) urea,respectively.In comparison to the control,the largest values were increased by 58.4 and 30.5% (2017),and by 61.3 and 57.7% (2018) at DAF15 for SK1 and SK3,respectively.Similar results were found at both DAF20 and DAF25,and the extents of the increments declined.The conventional cultivar SK1 had a greater increment than SK3 at optimum urea concentrations.There were significant linear positive correlations between Bt protein content and GPT activity(Fig.5),the correlation coefficients were 0.968**,0.669*and 0.610*(2017),and 0.697*,0.692*and 0.701*(2018) at DAF15,DAF 20 and DAF 25,respectively.

Table 3 The cotton seed amino acid contents at three boll developmental stages under different urea concentrations in 2017 and 2018 (μg g–1)

Fig.4 The correlations between the seed Bt toxin and amino acid contents of two Bt cotton cultivars in 2017 and 2018 cotton growing seasons.A shows the fitted linear equations at days after flowering (DAF)15,DAF20 and DAF25,in 2017.B shows the fitted linear equations at DAF15,DAF20 and DAF25,in 2018.

Similar results were observed for the seed GOT activity.However,the greatest seed GOT activities were detected at 6% (2017) and 5% (2018) urea spraying concentrations(Table 5).In comparison to the control,the greatest values were increased by 28.1 and 26.1% (2017),and by 12.7 and 10.5% (2018) at DAF15 for SK1 and SK3,respectively.The conventional cultivar (SK1) had greater increments than the hybrid cultivar.

Protease and peptidase activitiesThe seed protease activity increased from DAF15 to DAF25,and decreased with increasing urea concentration (Table 6).The higher the urea concentration,the lower the protease activity,with the lowest seed protease activities detected at 10% (2017)and 9% (2018) urea.In comparison to the corresponding controls,the lowest values were decreased by 28.8 and 28.5% (2017),and by 34.6 and 28.9% (2018) at DAF15 for SK1 and SK3,respectively;similar results were also found at DAF20 and DAF25.

Similar to the results of the protease activity,the seed peptidase activity also increased with boll growth,and declined with enhanced urea concentration (Table 7).The highest peptidase activity appeared in the control,and the lowest values were found at 10% (2017) and 9% (2018)urea.Compared to the corresponding controls,the 10%urea treatment was reduced by 47.0 and 30.8% (2017),while the 9% urea treatment was decreased by 39.1 and 41.6% (2018) at DAF15 for SK1 and SK3,respectively.Similar results were also observed at both DAF20 and DAF25.

Table 4 The cotton seed glutamic-pyruvic transaminase (GPT) activities at three boll developmental stages under different urea concentrations in 2017 and 2018 (µmol g–1 h–1)

Fig.5 The correlations between the seed Bt toxin content and glutamic-pyruvic transaminase (GPT) activity of two Bt cotton cultivars in 2017 and 2018 cotton growing seasons.A shows the fitted linear equations at days after flowering (DAF)15,DAF20 and DAF25 in 2017.B shows the fitted linear equations at DAF15,DAF20 and DAF25 in 2018.

4.Discussion

4.1.An optimum rate of urea spraying could increase cotton seed Bt toxin content and seed cotton yield markedly

The improvement ofCry1Acprotein expression in Bt cotton through agronomic practices such as high doses of N fertilizer in soil were reported previously.The concentrations of δ-endotoxin in plant tissues were significantly correlated with the plant tissue concentrations of total soluble protein and overall nitrogen (Bruns and Abel 2003;Donget al.2007).The soil nitrogen and plant growth regulator (PGR)applications also influenced the insecticidal efficacy for boll worms (Zhou and Wu 2000;Rochester 2006;Chenet al.2021).Pettigrew and Adamczyk (2006) reported that 112 kg N ha–1applied preplant plus 56 kg N ha–1applied sidedress as a urea-ammonium nitrate solution produced a 14%greater leafCry1Acconcentration than other treatments(112 kg N ha–1anhydrous;112 kg N ha–1liquid;56 kg N ha–1applied preplant plus 56 kg N ha–1applied sidedress as a urea-ammonium nitrate solution).Liet al.(2014) found that a 50% increment of nitrogen application at early flower stage enhanced leaf Bt toxin content by 132.4%.In our present experiment,external urea spraying increased the seed Bt protein concentration,the extent of the seed Bt protein content enhancement depended on the urea concentration,the greatest increments were detected at 5 to 6% urea,and the seed Bt protein content was increased by 54.8 to 92.9%.These results were further confirmed with a field boll worm hazard investigation (Table 1),with the lowest boll worm numbers per 10 plants and percent of damaged squares detected at 6% (2017) and 5% (2018)urea treatments during both years.Therefore,exogenous

nitrogen application on Bt cotton could bolster insect resistance of cotton seeds markedly.At the harvesting stage,the seed cotton yields for the treatments of spraying with 5–6% urea were the highest.The results suggest that the increments of both Bt protein and seed cotton yield can be achieved by external nitrogen spraying application,which is the simplest method to rapidly enhance insect resistance in Bt cotton due to the quick absorbance and easy application characteristics of external spraying.Moreover,this method is the fastest way to increase insecticidal ability when a breakout of the boll worm is predicted,and ultimately achieve higher yield.

Table 5 The cotton seed glutamate oxaloacetate transaminase (GOT) activity at three boll developmental stages under different urea concentrations during 2017 to 2018 experiments (µmol g–1 h–1)

Table 6 The cotton seed protease activity at three boll developmental stages under different urea concentrations in 2017 and 2018 (mg g–1 h–1)

Table 7 The cotton seed peptidase activity at three boll developmental stages under different urea concentrations in 2017 and 2018 (mg g–1 h–1)

4.2.Enhanced protein synthesis and reduced protein degradation both contributed to the increment of Bt protein under urea spraying

Bt cotton has a higher total N content in leaves and bolls than its parent cultivar (Donget al.2000;Chenet al.2004).High N also causes strong N metabolism physiology in Bt cotton (Yanget al.2005).In this study,we found that spraying urea on the cotton boll enhanced the seed soluble protein and amino acid contents,increased GPT and GOT activities,reduced protease and peptidase activities,and the greatest increments were all observed at the optimum urea concentration.Significant positive correlations were detected between Bt protein and amino acid contents,as well as between Bt protein content and GPT activity in cotton seeds.The results suggest that the significant increases in amino content and GPT activity were associated with higher Bt protein content;meanwhile,the significant decrease in peptidase may contribute to decreased degradation of the Bt protein.

These results indicated that external nitrogen application on the boll enhanced the seed protein synthesis and reduced protein degradation,which resulted in the increase of the seed proteins,including the Bt protein increment.This finding was consistent with previous studies which showed that enhanced boll nitrogen metabolic intensity increased boll Bt protein content in cotton (Jackson and Gerik 1990;Zhanget al.2007,2014).

5.Conclusion

Our results showed that external spraying 5–6% urea is the fastest way to increase insecticidal ability,control cotton bollworms and ultimately achieve higher seed cotton yield in Bt cotton.External nitrogen application on the boll enhanced the seed protein synthesis and reduced protein degradation,which resulted in an increase of the Bt protein increment.

Acknowledgements

This study was supported by the National Natural Science Foundation of China (31671613 and 31901462),the National Key Research and Development Program of China (2018YFD0100406 and 2017YFD0201306),the Natural Science Foundation of Jiangsu Province,China(BK20191439),the Natural Science Foundation of Jiangsu Higher Education Institutions,China (17KJA210003),the Priority Academic Program Development of Jiangsu Higher Education Institutions,China (PAPD),and the Brand Professional Construction Program of Jiangsu Higher Education Institutions,China.

Declaration of competing interest

The authors declare that they have no conflict of interest.