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Agronomy Journal 95:207-211 (2003)
© 2003 American Society of Agronomy

PRODUCTION PAPERS

Nitrogen Fertility Effects on Bt {delta}-Endotoxin and Nitrogen Concentrations of Maize during Early Growth

H. Arnold Bruns*,a and Craig A. Abelb

a USDA-ARS, Crop Genet. and Prod. Res. Unit, Box 345, Stoneville, MS 38776
b USDA-ARS, Southern Insect Manage. Res. Unit, Box 346, Stoneville, MS 38776

* Corresponding author (abruns{at}ars.usda.gov)

Received for publication March 4, 2002.

   ABSTRACT
TOP
 NOTES
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Nitrogen deficiencies interfere with protein synthesis and growth in general in maize (Zea mays L.). Transgenic maize hybrids with the Bacillus thuringiensis Berliner (Bt) gene produce proteins that are solubilized and become insecticidal in the highly alkaline midgut of certain lepidopteran insects. The effect N fertility has on Bt {delta}-endotoxin and how whole-plant N concentrations relate to Bt {delta}-endotoxin levels has yet to be documented in early-growth maize. Two Bt maize hybrids (‘Pioneer 33V08’1 with Bt event MON-810 and ‘Dekalb 626Bty’1 with Bt event DBT 418) were grown in pots in duplicate greenhouse experiments with N fertility rates of 0, 112, 224, and 336 kg ha-1 N as NH4NO3. Fertilizer was blended into a potting mixture of a 2:1:1 ratio of peat moss/sand/soil at planting. Plants were harvested at growth stage V5 and assayed for Bt {delta}-endotoxin using a commercial quantification plate kit. Whole-plant N concentrations were determined by semimicro-Kjeldahl. Whole-plant N concentrations were 25.8, 33.1, 35.1, and 37.7 mg g-1, and Bt {delta}-endotoxin concentrations were 350, 367, 486, and 534 µg kg-1 at N fertility levels of 0, 112, 224, and 336 kg ha-1, respectively. Increased available N likely increases Bt {delta}-endotoxin–synthesizing proteins and thus increases the Bt {delta}-endotoxin concentration. The response of the two Bt hybrids to increased N fertility was similar. Adequate levels of N fertility during early growth appear essential for Bt {delta}-endotoxin production by the plant and may affect the ability of maize plants to resist insect predation.

Abbreviations: Bt, Bacillus thuringiensis • PEPC, phosphoenolpyruvate carboxylase


   INTRODUCTION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
NITROGEN FERTILIZER is primarily responsible for the increases in maize grain yields observed during the past 50 yr (Olson and Sander, 1999). Nitrogen is essential in C flow and protein synthesis of higher plants (Yamazaki et al., 1986; Sugiharto et al., 1990). Being a constituent of proteins, purines, pyrimidines, and many coenzymes, a deficiency of N interferes with protein synthesis and thus growth in general (Epstein, 1972). Sugiharto et al. (1990) reported that in maturing photosynthetic leaf cells of maize, a reduced level of phosphoenolpyruvate carboxylase (PEPC) mRNA occurred due to a lack of N, which reduced the levels of PEPC protein.

The development and commercial release of transgenic maize hybrids with the Bt gene offers producers a means of controlling certain insect pests with reduced or no applications of chemical insecticides. Such hybrids are especially helpful in the control of European corn borer (Ostrinia nubilalis Hubner), southwestern corn borer (Diatraea grandiosella Dyar), corn earworm (Helicoverpa zea Boddie), and fall armyworm (Spodoptera frugiperda J.E. Smith) (Williams et al., 1998). The insecticidal properties of Bt are crystalline proteins ({delta}-endotoxin) that are solubilized and activated in the highly alkaline midgut environment of lepidopteran insects. These proteins, referred to as Cry proteins because of their crystalline nature, are encoded by the cry genes and classified according to their nucleotide sequence and insect specificity (Hofte and Whitely, 1989). More than 50 Cry proteins have been sequenced, but only four, from two cry genes, are currently commercially available in transgenic maize hybrids. Three of the proteins, referred to as events, are products of the Bt gene cryIAb, whereas one event, DBT 418, is a product of the Bt gene cryIAc (Bessin, 2000). Comparisons of Bt and non-Bt maize hybrids under temperature and drought stress have shown that Bt hybrids incurred less damage from second-generation European corn borers than did non-Bt hybrids (Traore et al., 2000).

Nitrogen fertilizer recommendations for maize in the Midsouth states (USA) generally are 26 kg of N per Mg of grain production expected under irrigated conditions (Larson and Oldham, 2001). These recommendations usually call for split applications of N fertilizer, with 30 to 50% of the total fertilizer being applied at planting. The remainder of the N will usually be applied at growth stage V6 as defined by Ritchie et al. (1997). Split applications are recommended for sandier soils to reduce the impact of possible early-season N losses caused by leaching during periods of heavy rainfall. Other planned farming operations, however, will occasionally force producers to apply all of the required N fertilizer at one time, usually before planting. Should leaching losses occur, the effectiveness of Bt hybrids at resisting insect damage later in the growing season might be impaired. Alinia et al. (2000) found that the stem borer (Chilo suppresalis Walker) had much lower larval survival rates on a Bt rice (Oryza sativa L.) cultivar with the cry1Ab gene than a non-Bt rice cultivar among all fertilizer treatments used in a greenhouse experiment.

Maize produced in the Midsouth states (USA) is subject to attack and suffers economic damage during very early growth stages by several of the lepidopteran insects previously mentioned (Metcalf et al., 1962, p. 471–475). Southwestern corn borer is known to heavily infest maize in the early growth stages and cause yield reductions of 25 to 50% (Williams and Davis, 1985, 1990). First-generation European corn borer has been shown to cause greater damage to maize in the Corn Belt states than succeeding generations (Lynch et al., 1980). The impact of N fertilizer application rates at planting on Bt {delta}-endotoxin quantity during early growth stages of maize has yet to be documented. This study is designed to ascertain any such possible differences and determine if further research may be needed on management strategies to maximize the benefit of the Bt gene.


   MATERIALS AND METHODS
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
This study was conducted at the USDA-ARS, MSA, Jamie Whitten Delta States Research Center, Stoneville, MS. Experiments were seeded on 15 Nov. 2000 and 9 Jan. 2001. Two maize hybrids, Pioneer 33V08 with Bt event MON-810 and Dekalb 626Bty with Bt event DBT 418, were planted in 20-cm plastic pots and grown in a greenhouse located at the research facility.

The plants were grown in a potting mixture consisting of a 2:1:1 peat moss/sand/soil ratio. Soil used in the study was a Beulah fine sandy loam (coarse-loamy, mixed thermic Typic Dystrochrepts). Sand, soil, and peat moss were thoroughly blended in 45.5-kg lots. Nitrogen fertilizer in the form of NH4NO3 was added to each lot during blending to the equivalent of 0, 112, 224, or 336 kg ha-1 N.

Pots were filled with potting mixture to within 3 cm of the top and arranged on tables in a greenhouse. Experimental design was a randomized complete block replicated four times. Individual experimental units consisted of 12 pots for each N fertility and hybrid treatment combination. The treatments had a factorial structure with hybrids and N fertility levels. The entire experiment was bordered with one row of pots planted with bulk non-Bt maize seed left over from previous field studies. Three kernels were planted in each pot, which were thinned to one plant per pot at growth stage V2 (Ritchie et al., 1997).

After planting, pots were carefully watered with tap water four times, at 2-h intervals, to thoroughly moisten the potting mixture but avoid leaching of the N fertilizer. Watering continued on an as-needed basis. Pots were monitored daily and watered sparingly when the surface of 50% of the pots appeared dry. Continued care was taken to avoid overwatering and possible leaching of applied N fertilizer.

Fourteen hours of supplemental light was provided using 24 General Electric1 F96T12.CW florescent bulbs placed in 12 two-bulb banks during the entire experiment. The lights were adjusted weekly to approximately 25 cm above the top of the plants. Greenhouse temperatures during the study were approximately 24 and 18°C (day and night, respectively).

At the V5 growth stage (Ritchie et al., 1997), the distal 5- to 7-cm tip of each leaf was removed with scissors and placed into a plastic bag and a cooler kept at approximately 4°C. Leaves were transported to the laboratory, and within an hour of harvest, one sample (about 20 mg) was taken from each leaf. The leaf samples were weighed to accurately determine the amount of starting material and combined into a 1.5-mL microcentrifuge tube. The samples were analyzed to quantify the amount of {delta}-endotoxin present (Adamczyk et al., 2001). A non-Bt hybrid (‘Pioneer 3394’) was used in all protocol steps as a control. To quantify the amount of Bt {delta}-endotoxin present for each cultivar, a commercial quantification plate kit was used (EnviroLogic,1 Portland, ME). This sandwich enzyme-linked immunosorbent assay (ELISA) uses a color development step where intensity of color production is proportional to cry1Ab concentration in the sample extract. Therefore, quantification of Bt {delta}-endotoxin is determined spectrophotometrically (Benchmark,1 Bio-Rad, Hercules, CA). The kit protocol was used for all steps, but the homogenization step was modified. To maximize uniformity during extraction, a high-speed homogenizer using stainless steel beads (6 mm) with extraction buffer (EnviroLogic, Portland, ME) to shear the tissue (Mini-Bead Beater,1 Biospec Prod., Bartlesville, OK) was used. The extraction process for each hybrid was replicated twice. The proper standard curve, dilution factors, positive and negative controls, and calculations were conducted as dictated in the kit protocol.

The remaining plant tissue of each pot was excised at soil level with a razor and oven-dried at 60°C for 96 h. The dried tissue from each plot was then weighed and ground to pass a 2-mm screen. Total N concentrations of individual experimental units were determined at the Mississippi Cooperative Extension Service Soil Testing and Plant Tissue Analysis Laboratory in Mississippi State, MS, via semimicro-Kjeldahl (AOAC, 1975). Analysis of variance was conducted on data combined across the two experiments where experiment x treatment interactions were treated as a random source of error. An additional analysis of variance was preformed using a linear trend to identify differences in Bt {delta}-endotoxin among N fertility levels. Data were analyzed using the PROC MIXED procedure (SAS Inst., 2000).


   RESULTS AND DISCUSSION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
No significant differences in either whole-plant N concentration or Bt {delta}-endotoxin concentration were observed among the hybrids or experiments of the study (Table 1), nor was the hybrid x fertility interaction statistically significant. This indicates that these two Bt events responded to N fertility in a similar way.


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  		Table 1. Combined analysis of variance of N concentration and Bacillus thuringiensis (Bt) {delta}-endotoxin concentration of two Bt maize hybrids (Pioneer 33V08 and Dekalb DK626Bt) at growth stage V5 grown with varying rates of N fertility (0, 112, 224, and 336 kg ha-1).

 
Whole-plant N concentrations increased with increasing levels of N fertilizer applied (Table 2). Correlations of whole-plant N concentration to N fertility level were positive and statistically significant (P < 0.001) (Fig. 1). Past research aimed at relating leaf N concentration to maize grain yield have reported increases in leaf N concentration with increased N fertility (Killorn and Zourarakis, 1992). However, plants were evaluated at a later growth stage in that study than in this research. Much of the early research on N concentration in maize has been directed toward relating it to final grain yield, grain quality, or both. These studies have basically demonstrated increases in grain yield and/or quality with increased levels of N in the plant tissue (Deckard et al., 1973). Early levels of whole-plant N concentration most likely will not directly influence grain yield although deficiencies during early growth adversely affect final leaf size, ear size, and other plant organ development, which will result in a reduced grain yield (Ritchie et al., 1997).


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  		Table 2. Nitrogen concentration and Bacillus thuringiensis (Bt) {delta}-endotoxin concentration of two Bt maize hybrids (Pioneer 33V08 and Dekalb DK626Bt) at growth stage V5 grown with varying rates of N fertility.

 


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  		Fig. 1. Whole-plant N concentration averaged over two Bacillus thuringiensis (Bt) maize hybrids (Pioneer 33V08 and Dekalb 626Bt) at growth stage V5 grown with varying rates of N fertility. *Significant at the 0.05 level.

 
The hybrid x fertility (linear) interaction for Bt {delta}-endotoxin was not significant (F = 0.31, P = 0.58). However, the effect of the fertility treatments (linear) across hybrids was significant (slope = 0.006, F = 6.72, P = 0.03) (Fig. 2). Increasing levels of available N are known to increase protein levels in most plant species, especially in vegetative cells (Tisdale and Nelson, 1975, p. 68). Much of this increase is in the form of enzymes used in further growth and development. Because of the increased availability of N to the plant, as N fertility levels are increased, greater quantities of the Bt {delta}-endotoxin–synthesizing enzymes and/or mRNA are likely produced; thus, greater quantities of the Bt {delta}-endotoxin protein will be synthesized as a result. Alinia et al. (2000) suggest that increased levels of N fertility may have the effect of increasing the levels of Bt {delta}-endotoxin in plants with the cry1Ab gene using the PEPC promoter. Both hybrids used in this study contain the cauliflower mosaic virus 35S promoter. It is unclear how N fertility may affect this promoter and its Bt {delta}-endotoxin production. Further study of this possible occurrence is warranted to better understand and utilize Bt {delta}-endotoxins.



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  		Fig. 2. Endotoxin concentrations averaged two Bacillus thuringiensis (Bt) maize hybrids (Pioneer 33V08 and Dekalb 626Bt) at growth stage V5 grown with varying rates of N fertility. *Significant at the 0.05 level.

 
The correlation of Bt {delta}-endotoxin concentration to whole-plant N concentration further supports the fact that an increase in the amount of available N increases the quantity of Bt {delta}-endotoxin produced (Fig. 3). The Bt {delta}-endotoxin concentration to whole-plant N concentration correlation was observed to be positive and statistically significant (P < 0.001). In this study, no attempt was made to separate protein N from nonprotein N contained in the tissue.



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  		Fig. 3. Relationship of whole-plant N concentration and endotoxin concentration of two Bt maize hybrids (Pioneer 33V08 and Dekalb 626Bt) at growth stage V5 grown under N fertility rates of 0, 112, 224, and 336 kg ha-1. **Significant at the 0.01 level.

 
How such an increase in Bt {delta}-endotoxin concentration in the plant would translate into insect feeding resistance has yet to be documented. If it is shown that the level of insect feeding resistance is affected by Bt {delta}-endotoxin concentration, then N fertility recommendations for Bt hybrids may need to be adjusted to provide maximum insect control. It is also unclear if the Bt {delta}-endotoxin levels in more mature plants would be affected in similar ways with differing N fertility levels, as were the juvenal plants in this study. Further research in the field would help determine if varying N fertility rates affect Bt {delta}-endotoxin levels and thus insect control. Research comparing Bt and non-Bt maize hybrids grown under temperature and moisture stress showed that Bt hybrids evidently remain effective in resisting feeding by European corn borer (Traore et al., 2000). Further research is also needed to determine if the Bt {delta}-endotoxin protein remains intact after synthesis or if it can be degraded and translocated and the N utilized in developing tissue, thus rendering the plant susceptible to insect attack.


   NOTES
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 NOTES
ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
1 Trade names are used in this publication solely for the purpose of providing specific information. Mention of a trade name, propriety product, or specific equipment does not constitute a guarantee or warranty by the USDA-ARS and does not imply approval of the named product to the exclusion of other similar products. Back


   REFERENCES
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 MATERIALS AND METHODS
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