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Published online 3 January 2006
Published in Agron J 98:100-106 (2006)
DOI: 10.2134/agronj2005.0143
© 2006 American Society of Agronomy
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Production Papers

Planting Date Effects on Bt and Non-Bt Corn in the Mid-South USA

H. Arnold Bruns* and H. K. Abbas

Crop Genetics and Production Research Unit, Box 345, Stoneville, MS 38776

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

Received for publication May 13, 2005.

   ABSTRACT
TOP
 NOTES
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Corn (Zea mays L.) planting dates are regional and vary across the contiguous USA. Improved technologies allow corn to be planted earlier. The objective of this research was to evaluate the effects of planting date on the agronomics of Bt and non-Bt hybrids grown in the Mid-South. Twelve hybrids, two Bt [Bacillus thuringiensis (Berliner)], and two non-Bt for three maturity groups [short-season (1180–1270 GDU 10's), mid-season (1445–1470 GDU 10's), and full-season (1540–1625 GDU 10's)] were evaluated for GDU 10's at silking and physiological maturity, yield, yield components, and mycotoxins in 2002, 2003, and 2004 at Stoneville, MS. Plots were planted in a split-plot of a randomized complete block replicated four times and furrow irrigated. Whole plots were plantings in early April, late April, or mid-May, while subplots were hybrids randomly assigned. Experimental units were four 102-cm rows, 9.1 m long. Lodging and dropped ears were inconsequential. Yields were greater for both April plantings (8.6 and 9.2 Mg ha–1 for early April and late April, respectively) than mid-May plantings (7.8 Mg ha–1). Short-season hybrids generally yielded less than mid-season or full-season hybrids. The Bt hybrids yielded more than non-Bt hybrids (9.1 Mg ha–1 vs. 7.9 Mg ha–1, respectively). Yields correlated with GDU 10's at silking [yield = 0.037x – 20.416 (r = 0.77)] but not physiological maturity. Aflatoxin was high in 2002 (224.0 mg Mg–1), and much less (28.8 and 7.4 mg Mg–1) in 2003 and 2004, respectively. The Bt hybrids had less fumonisin contamination than non-Bt hybrids (5.2 mg kg–1 vs. 8.5 mg kg–1) but less aflatoxin only in 2003 (12.4 mg Mg–1 vs. 45.3 mg Mg–1).

Abbreviations: GDU 10, growing degree unit based on 10°C


   INTRODUCTION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
OPTIMUM CORN PLANTING DATES are regional and vary across the contiguous USA because of differences in climate and length of growing seasons in areas where the crop is produced (Bruns, 2003). A map published by Sprague and Larson (1966) showed the mean date for corn planting ranged from 1 February in the Rio Grande Valley to 20 May for most of the Great Lakes region. Technological factors such as hybrids with better early season vigor and tolerance for germination in cool wet soils, better seed treatments to guard against damping off diseases and seedling insect pests, the advent of herbicides, plus improvements in planters that distribute seed more uniformly and function better under less than ideal planting conditions have contributed to corn planting today being done earlier throughout most of the USA than it was 30 yr ago (Shaw, 1988; Lauer, 2001). This trend is true in the Mid-South with the date for 50% of the corn planted in the early 1970s being early May, to it currently being early April (USDA-NASS, 2005; USDA-NOAA, 2005).

Research from the Corn Belt on planting dates has shown only slight or no yield reductions occur when seeding is done before the determined optimum date, but late planting can be detrimental (Nafziger, 1994; Johnson and Mulvaney, 1980; Lauer et al., 1999; Carter, 1984; Swanson and Wilhelm, 1996). In Illinois, Nafziger (1994) found corn yields generally increased as planting was delayed from 10 to 30 April, but then steadily declined from 9 to 29 May. Earlier, Johnson and Mulvaney (1980) found the optimum planting date in central Illinois was 6 May, and that planting 2 wk before or after that date reduced yields <5%. However, a rapid decline in grain yield occurred if planting was delayed past 20 May. Though the optimum planting dates differed, Lauer et al. (1999) later reported similar results from research at several locations in Wisconsin. In Wisconsin average grain yields for corn hybrids of similar maturities declined 31% when planting was delayed from 1 May to 1 June (Carter, 1984). Bauer and Carter (1986) later determined delaying planting from 1 to 30 May, for several hybrids of different maturities, resulted in increased levels of kernel breakage during handling. In simulated no-till planting conditions, Swanson and Wilhelm (1996) reported planting corn before or after the optimum date resulted in reduced leaf area index, leaf area duration, total dry matter production, and grain yield. Yields declined with both earlier and later planting dates but declined more rapidly when planting was delayed than when it was advanced.

Hybrid Bt corns have changed the management of the crop throughout the USA. When planting is delayed, Bt corn hybrids are often recommended over non-Bt hybrids to avoid yield losses that frequently occur due to feeding by several lepidopteran insects species (VanDyk, 2002; Flanders et al., 1999; Buntin et al., 2001; Wiatrak et al., 2004). VanDyk (2002) stated Bt hybrid corn should not be intentionally planted "late" but planting such hybrids last during the spring planting sequence provides the best opportunity for an economic benefit of growing them rather than non-Bt hybrids. Flanders et al. (1999) reported that in Alabama, Bt hybrids have a decided advantage in yield over non-Bt hybrids when grown in the southern part of the state. However, in central and northern sites such an advantage existed only when planting occurred late. Buntin et al. (2001) found Bt hybrids had reduced ear infestation of fall armyworm (Spodoptera frugiperda J.E. Smith) and corn earworm (Helicoverpa zea Boddie) larvae resulting in less kernel damage and thus greater grain yields than was observed in non-Bt hybrids. Wiatrak et al. (2004) reported insect feeding and disease contributed to grain yield declines in both Bt and non-Bt genotypes as planting was delayed. However, smaller yield reductions due to late planting were noted in Bt hybrids compared with non-Bt hybrids.

Mycotoxins can be a serious problem in corn, particularly in crops grown in the southern USA. The economic losses and expenditures in research and monitoring mycotoxins in all crops grown in the USA are estimated at between $0.5 and $1.5 billion annually (Robens and Cardwell, 2003). Drought and heat stress in 1977 and 1978 resulted in nearly 90% of the corn crop grown in South Carolina being contaminated with aflatoxin, a mycotoxin produced by the fungus Aspergillus flavus (Manwiller and Fortnum, 1979). Losses to aflatoxin in Texas, Arkansas, Mississippi, and Louisiana in 1998 were estimated to be $85 million (U.S.) (Williams et al., 2003; Abbas et al., 2002, 2005). Williams et al. (2004) later concluded that in areas with high southwestern cornborer (Diatraea grandiosella Dyar) infestations, Bt hybrids should effectively reduce aflatoxin contamination, but the reduction is due to control of the insects and not to any resistance to A. flavus.

Fumonisin, a mycotoxin produced by the fungus Fusarium moniliforme J. Sheld., is known to cause several diseases in livestock (Bruns, 2003). Corn with Bt genes has been shown to experience less Fusarium infection because of the association between the fungus and insect feeding (Munkvold et al., 1997). Hammond et al. (2004) observed lower levels of fumonisin in Bt hybrids than non-Bt hybrids and concluded the use of Bt hybrids can increase the percentage of corn grain that would be suitable for use in food and feed.

Planting date has been shown to facilitate mycotoxin contamination (Bruns, 2003). The general conclusion is planting should be done so that reproductive growth of the crop avoids as much drought and heat stress as possible. Current information on planting dates of newer hybrids with varying maturities and with Bt and non-Bt genetics is very limited for the Mid-South, USA. The objective of this research was to examine effects of varying planting dates on the agronomics of both Bt and non-Bt hybrids of different maturity ratings grown using furrow irrigation in the Mid-South. Growing degree units using a 10°C base (GDU 10's) for growth stage R1 (silking) and growth stage R6 (physiological maturity) as defined by Ritchie et al. (1997), yield, yield components, grain bulk density, lodging, and mycotoxin contamination are reported.


   MATERIALS AND METHODS
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
The research was conducted at Mississippi State University's Delta Branch Experiment Station at Stoneville, MS, from 2002 to 2004. Soil at the experimental site was a Beulah fine sandy loam (coarse-loamy, mixed, thermic Typic Dystrochrepts) prepared for planting by forming 50-cm ridges spaced 102 cm apart. The previous crop all 3 yr of the experiment was corn. The experimental design was a randomized complete block with a split-plot arrangement of treatments replicated four times. Whole plots were randomized within each replication each year and consisted of plantings in: early April (6 Apr. 2002, 1 Apr. 2003, and 1 Apr. 2004) late April (25 Apr. 2002, 22 Apr. 2003, and 22 Apr. 2004), and mid-May (16 May 2002, 13 May 2003, and 11 May 2004). Sub-plots were 12 hybrids (Table 1) assigned at random within each whole plot. The 12 hybrids represented three maturity groups: short-season (1180–1270 GDU 10's), mid-season (1450–1475 GSU 10's), and full-season (1540–1625 GDU 10's). Each maturity group was represented by two Bt hybrids and two non-Bt hybrids. With exception of the short-season hybrids, the remaining eight were grown in the Mid-South at the initiation of the experiment. Specific hybrid selections were based on seed availability and matching a Bt and non-Bt hybrid from the same company with similar maturities.


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  		Table 1. Sources and maturity ratings of corn hybrids evaluated in an irrigated planting date study at Stoneville, MS, from 2002 to 2004.

 
Individual experimental units were four rows 9.1 m long, spaced 102 cm apart. Seeding rate was 75 300 plants ha–1 for an expected final population of about 64 000 plants ha–1. Soil samples were analyzed before tillage each year for P and K fertility requirements at a yield goal of 12.5 Mg ha–1. Required P and K in a dry fertilizer form, as well as 112 kg ha–1 N as NH4NO3 were applied preplant to the experiment. An additional 112 kg ha–1 N as a urea–NH4NO3 solution was side-dressed at growth stage V6 (six leaves fully emerged) of the first planting date (Ritchie et al., 1997). The experiment was furrow irrigated using a schedule previously described and beginning at growth stage R1 (silking) to R2 (blister) of most of the hybrids in the early April planting (Bruns et al., 2003). Approximately 2.5 cm of water per irrigation was applied on 3 and 18 June 2002, 4 June and 2 July 2003, and 10 June 2004. Weed control was accomplished with a pre-emergence application of Bicep1 (Monsanto Co., St. Louis, MO) {atrazine [2-chloro-4-(ethylamino)-6-(isopropylamino)-s-triazine] (2.24 kg ha–1 a.i.) + metolachlor [2-chloro-N-(2-methoxy-1-methylethyl) acetamide] (1.68 kg ha–1 a.i.)} and by cultivating at growth stage V6.

Date of growth stage R1and growth stage R6 (physiological maturity) was determined for each experimental unit. These data were combined with weather data collected at the research station (Mississippi State Univ., 2005) to determine GDU 10's as previously described (Bruns and Abbas, 2005; Shaw, 1988). During reproductive growth the center 5.2 m of the two middle rows of each experimental unit were identified, and the end plants marked with flagging tape. Plant populations for each experimental unit were determined by counting plants within the marked areas. Just before harvests, these same areas were reexamined and counts made on any lodged plants and/or dropped ears. Individual experimental units were harvested beginning about 28 d after growth stage R6 (Bruns and Abbas, 2004). Ears of plants contained between marked sections of each experimental unit were hand harvested, then shelled using an Almaco1 (Allen Machine Co., Nevada, IA) gasoline-powered corn sheller, and the grain weighed. A sample of approximately 1.0 kg of grain was taken to determine moisture content, grain bulk density, kernel weight, and mycotoxin contamination in each experimental unit. Grain moisture content and bulk density were determined using a Seedboro1 Model GMA 128 Grain Moisture Analyzer (Seedboro Equipment Co., Chicago, IL). Grain yields were adjusted and reported at a moisture level of 155 g kg–1.[CONVERTED 1:1 TO PER-KG] Grain samples were then dried at 30°C for 18 h. Kernel weights for each experimental unit were determined by hand counting and weighing 100 sound kernels. Kernels ear–1 were estimated by dividing harvested grain weight per experimental unit, adjusted for moisture content, by population data, assuming one ear plant–1, and then dividing that product by the kernel weight.

Aflatoxin and fumonisin contamination levels were determined using Veratox1–Aflatoxin and Veratox1–Fumonisin Kits (Neogen Co., Lansing, MI), respectively. Specific procedures used for mycotoxin determinations have been previously published by Abbas et al. (2002).

Statistical analyses on grain yields, yield components, GDU 10's, and fumonisin contamination were performed on data combined over years using procedures outlined by McIntosh (1983), treating years as random effects and using PROC MIXED of the SAS procedure (SAS Institute, 2004). Pearson's correlations were calculated on some GDU 10 data. Data on GDU 10's were also analyzed for individual years and due to a lack of homogeneity, data on aflatoxin was analyzed and reported for individual years.


   RESULTS AND DISCUSSION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
CONCLUSIONS
 REFERENCES
 
Plant populations did not differ among years, hybrids, or planting dates (63c825 plants ha–1) and lodging and dropped ears were inconsequential in this experiment (≤0.1 plant per experimental unit). The mean maximum temperature for April 2002 was below normal while all other mean maximum temperatures during the experiment were normal (Table 2). Rainfall totals during June 2003 and June 2004 were above normal due to two large rain events in June 2003 and an unusually wet period during the last week of June 2004.


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  		Table 2. Normal monthly maximum temperature, total rainfall, and observed comparable weather data for the 2002, 2003, and 2004 corn growing seasons at Stoneville, MS (Mississippi State Univ., 2005).{dagger}

 
No statistically significant planting date x hybrid interactions were observed for the data analyzed (Table 3). Grain yields were greater (P ≤ 0.10) for early April and late April plantings than mid-May plantings (Tables 3 and 4). These observations are similar to previously reported data from the Corn Belt with regard to yield and planting date (Nafziger, 1994; Johnson and Mulvaney, 1980; Lauer et al., 1999; Carter, 1984; Swanson and Wilhelm, 1996). Grain moisture at harvest, mean kernels ear–1, kernel weights, and grain bulk density did not differ among the three planting dates (Tables 3 and 4).


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  		Table 3. Type three tests of fixed effects and covariance parameter estimates of a corn planting date experiment conducted at Stoneville, MS, in 2002, 2003, and 2004 using Bt and non-Bt hybrids of short-season, mid-season, and full-season maturities.

 

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  		Table 4. Grain yields, moisture at harvest, kernels per ear, kernel weight, and grain bulk density of furrow irrigated corn planted on different dates at Stoneville, MS.{dagger}

 
Except for 3394, grain bulk densities of full-season and mid-season hybrids were greater (P ≤ 0.01) than those of short-season hybrids (Tables 3 and 5). These data are similar to earlier research (Bruns and Abbas, 2005). Grain bulk densities for short-season hybrids 8830 and 9185Bt were below the 695 kg m–3 minimum requirement for U.S. no. 2 yellow corn, the most common grade traded on the world market, and would have likely been docked in price when sold (USDA, 1996).


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  		Table 5. Yield, kernels per ear, kernel weight, grain bulk density, grain moisture at harvest, growing degree units 10°C at R1 and R6, and fumonisin levels of corn hybrids evaluated in an irrigated planting date study at Stoneville, MS, from 2002 to 2004.{dagger}

 
Grain moisture at harvest differed among the hybrids in the experiment (Table 5). However, the grain moisture at harvest was greater (P ≤ 0.05) only for A6729Bt than all the earlier maturing hybrids in the experiment. Conversely both 8830 and A6257 had less (P ≤ 0.05) grain moisture at harvest than most of the later maturing hybrids. The low grain moisture levels observed for these two non-Bt hybrids was the likely reason greater (P ≤ 0.01) grain moisture levels were observed for the Bt hybrids than the non-Bt hybrids when averaged among all years and planting dates (Table 6). From a production standpoint, however, these differences would be minor and would not likely affect the price received for the grain on the market.


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  		Table 6. Mean yield, kernels per ear, kernel weight, grain moisture at harvest, and grain bulk density between Bt and non-Bt corn hybrids grown with furrow irrigation at Stoneville, MS.{dagger}

 
The Bt hybrids of this experiment averaged greater (P ≤ 0.01) grain yields than non-Bt hybrids (Table 6). This resulted from the Bt hybrids averaging more (P ≤ 0.01) kernels ear–1 that weighed more (P ≤ 0.01). However, unlike previous research, Bt hybrids in this experiment did not yield more grain than non-Bt hybrids when planted late (mid-May) (Table 3). No differences in mean grain bulk density were observed between Bt and non-Bt hybrids.

With exception of 9185Bt, all Bt hybrids yielded more grain than at least one non-Bt hybrid in their respective maturity group (Table 5). It cannot be stated these differences were due to less insect damage to the Bt hybrids because such data were not collected in this experiment. However, other studies have shown yield advantages for Bt hybrids over non-Bt genotypes that were related to less insect feeding on developing kernels (Flanders et al., 1999; Buntin et al., 2001; VanDyk, 2002; Wiatrak et al., 2004). Fall armyworm, corn earworm, and southwestern corn borer (Diatraea grandiosella Dyar) are known to reduce corn yields in the Mid South USA (C.A. Abel, personnel communication, 2005).

With exception of A6333Bt, both full-season and mid-season hybrids produced more grain than short-season hybrids in this experiment (Table 4). Averaged among all years and planting dates, short-season hybrids produced fewer (P ≤ 0.01) kernels ear–1 (458 vs. 523 and 539 for full-season and mid-season hybrids, respectively) that weighed less (P ≤ 0.01) (268 mg vs. 279 mg and 285 mg, respectively, for full-season and mid-season hybrids). Corn hybrids that utilize the greatest amount of the growing season generally yield more grain than those requiring fewer GDU 10's to reach growth stage R6 (Poehlman, 1959, p. 263; Larson, 2002a). However, Bruns and Abbas (2005) recently reported some modern short-season corn hybrids when grown in the Mid South USA using furrow irrigation, produce comparable yields to full-season and mid-season hybrids.

The GDU 10's required to achieve both growth stage R1 and growth stage R6, differed among hybrids of this experiment (Tables 3 and 5). Except for A6333Bt, short-season hybrids of this study required fewer GDU 10's (P ≤ 0.01) to achieve growth stage R1 than mid-season and full-season hybrids. For short-season hybrids, the GDU 10's required to reach growth stage R6 were greater than the stated requirement found in company sales literature, when grown in their adapted environments (Tables 1 and 5). Similar findings have been previously reported (Bruns and Abbas, 2005). Two mid-season hybrids (34B23 and 34B24Bt) and two full-season hybrids (A6670 and A6729Bt) required more GDU 10's to achieve growth stage R6 than any short-season hybrid except A6257 (Table 5). The short-season hybrid 9185Bt, which was one of the lower yielding hybrids in the experiment and also the only Bt hybrid not to yield more grain than at least one non-Bt hybrid in its maturity range, also had the least GDU 10 requirement to achieve growth stage R1 and one of the least to achieve R6 (Table 5).

The GDU 10's accumulated at growth stages R1 and R6, when analyzed by individual years, differed significantly (P ≤ 0.01) between planting dates all 3 yr but, no trend existed for any 2 yr (data not shown). When combined over years, no differences in GDU 10 requirements at any planting date neither growth stage R1 nor R6 were observed (Table 3).

Grain yields of hybrids in this experiment were found to be correlated (P ≤ 0.01) to GDU 10's required to achieve growth stage R1 [yield = 0.0371x – 20.416 (r = 0.77)]. However, no correlation with yield and growth stage R6 was evident. Grain yield was also found to be correlated (P ≤ 0.01) to kernels ear–1 (y = 0.0128x + 2.0737, r = 0.74) while no relationship was observed between yield and kernel weights. These data indicate the period of growth in which potential kernels ear–1 was being determined (growth stage V12 to growth stage R1) was more critical to grain yield in this experiment than the period of kernel filling (growth stage R2 to growth stage R6) (Ritchie et al., 1997). In the Mid-South USA a greater potential exists for heat and drought stress during growth stages R1 to R6 than during growth stages before R1 (Boykin et al., 1995). Drought and heat stress encountered during growth stages R2 to R6 most likely diminish the probability of detecting any potential relationship between GDU 10's required for physiological maturity (R6) and grain yield.

Mean aflatoxin contamination levels differed extensively among years (Table 7). Heat stress during kernel filling favors A. flavus infection and subsequent aflatoxin production (Bruns, 2003). Total days with maximum temperatures 32°C during June and July, when the bulk of kernel filling was occurring in all planting dates, was 41 in 2002 and 30 for both 2003 and 2004 (Mississippi State Univ., 2005).


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  		Table 7. Aflatoxin contamination of corn grain from Bt and non-Bt hybrids, representing three maturity groups, planted at three different planting dates, and grown with furrow irrigation at Stoneville, MS, in 2002, 2003, and 2004.{dagger}

 
Planting date did not affect grain contamination by aflatoxin any year of the experiment (Table 7). The Bt hybrids averaged less (P ≤ 0.01) aflatoxin contamination than the non-Bt hybrids in 2003 (12.4 mg Mg–1 vs. 45.3 mg Mg–1, respectively), but no such differences were noted in the other 2 yr. Full-season hybrids had more (P ≤ 0.01) aflatoxin contamination in 2002 than the mid-season or short-season hybrids (Table 7). No such differences were noted in the two succeeding years. Aflatoxin levels among individual hybrids were found not to differ.

Data on grain contamination by fumonisin did not vary among years and therefore were combined and analyzed. Non-Bt hybrids averaged among all years and planting dates had greater (P ≤ 0.01) amounts of fumonisin grain contamination than Bt hybrids (Table 6). As previously stated, insect damage was not quantified in this study. However, lower levels of fumonisin in Bt hybrids have previously been reported (Hammond et al., 2004).


   CONCLUSIONS
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
REFERENCES
 
These data support earlier recommendations for optimum corn planting dates in the Mississippi Delta (Larson, 2002b). Yields appear to rapidly decline as planting is delayed past 20 April. This observation is similar to previous reports from the Corn Belt regarding planting in relation to the identified optimum planting date for those regions (Nafziger, 1994; Johnson and Mulvaney, 1980; Lauer et al., 1999).

Mid-season hybrids appear to yield as well as full-season hybrids in the lower Mississippi River Valley. One short-season hybrid (A6333 Bt) in this experiment yielded as well as most of the mid-season and full-season hybrids. Grain bulk density of two of the short-season hybrids was below the minimum standard for no. 2 yellow corn and would have been docked in price when sold.

These data indicate that, using current plant populations, corn yields in the Mid-South and any possible genotypic differences in yield are influenced most by environmental conditions during growth stages when the kernels ear–1 are being determined. Potential genotypic differences in kernel filling are likely being masked by drought and/or heat stress in the region that is more probable during those growth stages (R2–R6) (Boykin et al., 1995). Additional heat stress during these growth stages in 2002 appears to have also greatly increased aflatoxin contamination. Such an increase has been demonstrated by others in previous research (Manwiller and Fortnum, 1979; Williams et al., 2003).

The Bt hybrids have a yield advantage over the non-Bt hybrids by producing more kernels ear–1 as well as less fumonisin contamination. However, only in 2003 did the Bt hybrids have less aflatoxin.


   ACKNOWLEDGMENTS
 
Appreciation is expressed to Mr. Rodrick R. Patterson, Mr. Roosevelt Johnson, Ms. Bobbie Johnson, and Ms. Jennifer Tonos of the USDA-ARS, MSA, CG&PR Unit at Stoneville, MS, for their technical assistance in conducting the research.


   NOTES
TOP
 NOTES
ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Trade names are used in this publication solely for the purpose of providing specific information.

1 Mention of a trade name, proprietary 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
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 




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