Progress 12/01/16 to 11/30/18
Outputs
Target Audience:
Nothing Reported
Changes/Problems:Acknowledgments The author would like to thank the Upper Coastal Research Station staff and Craig Diel with field preparation. Dan Mott, Lewis Braswell, Dannyel Nelson and Eric Crozier for aiding the study. The author thanks Alejandro Del-Pozo for their help in SAS programming and statistical consulting. References Adamczyk Jr., J. J., L. C. Adams, and D. D. Hardee. 2001. Field efficacy and seasonal expression profiles for terminal leaves of single and double Bacillus thuringiensis toxin cotton genotypes. J. Econ. Entomol. 94: 1589-1593. Aghaee, M. and Godfrey, L.D. 2014. Effect of Bacillus thuringiensis ssp. galleriae on rice water weevil in California rice. Journal of Economic Entomology 1(8): 45-52 Aghaee, M-A., L. Espino, K. Goding, E. Goldman, and L.D Godfrey. 2015a. Effects of Seeding Rates and Rice Water Weevil (Coleoptera: Curculionidae) Density on Damage in Two Medium Grain Varieties of Rice. Journal of Economic Entomology. 1-9. doi: 10.1093/jee/tov342 Aghaee, M., Olkowski, S., Shelomi, M., Klittich, D., Kwok, R., Maxwell, D., and M, Portilla. 2015b. Waiting on the Gene Revolution: Challenges for Adopting GM Crops in the Developing World. Trends in Food Science and Technology 46(1): 132-136 Bagwell, R. D. 1994. Monitoring the cotton plant for insecticide effects and late-season insecticide use termination. Ph.D. dissertation, University of Arkansas, Fayetteville. Carter, L. M., J. W. Jones, L. Berry, V. Burkett, J. F. Murley, J. Obeysekera, P. J. Schramm, and D. Wear, 2014: Ch. 17: Southeast and the Caribbean. Climate Change Impacts in the United States: The Third National Climate Assessment, J. M. Melillo, Terese (T.C.) Richmond, and G. W. Yohe, Eds., U.S. Global Change Research Program, 396-417. doi:10.7930/J0N- P22CB Chilcutt, C. F., L. T. Wilson, and R. J. Lascano. 2003. Field evaluation of a Helicoverpa zea (Lepidoptera: Noctuidae) damage simulation model: effects of irrigation, H. zea density, and time of damage on cotton yield. J. Econ. Entomol. 96: 1174-1183. Greenplate, J. T. 1999. Quantification of Bacillus thuringiensis insect control protein Cry1Ac over time in Bollgard cotton fruit and terminals. J. Econ. Entomol. 92: 1377-1383. Greenplate, J. T., J. W. Mullins, S. R. Penn, A. Dahm, B. J. Reich, J. A. Osborn, P. R. Rahn, L. Ruschke, and Z. W. Shappley. 2003. Partial characterization of cotton plants expressing two toxin proteins from Bacillus thuringiensis: relative toxin contribution, toxin interaction, and resistance management. J. Appl. Entomol. 127: 340-347. Gore, J., B. R. Leonard, G. E. Church, J. S. Russell, and T. S. Hall. 2000. Cotton boll abscission and yield losses associated with first-instar bollworm (Lepidoptera: Noctuidae) injury. J. Econ. Entomol. 93: 690-696. Gore, J., B. R. Leonard, G. E. Church, and D. R. Cook. 2002. Behavior of bollworm (Lepidoptera: Noctuidae) larvae on genetically engineered cotton. J. Econ. Entomol. 95: 763-769. Gore, J., J. J. Adamczyk, and C. A Blanco. 2005. Selective feeding of tobacco budworm and bollworm (Lepidoptera: Noctuidae) on meridic diet with different concentrations of Bacillus thuringiensis proteins. J. Econ. Entomol. 98: 88-94. Guinn, G. 1982. Causes of square and boll shedding in cotton. USDA Tech. Bulletin No. 1672. US Gov. Print Office, Washington DC. Gulzar, A., B. Pickett, A. H. Sayyed, and D. J. Wright. 2012. Effect of Temperature on the Fitness of a Vip3A Resistant Population of Heliothis virescens (Lepidoptera: Noctuidae). J. Econ. Entomol. 105: 964-970. Gutbrodt, B., K. Mody, and S. Dorn. 2011. Drought changes plant chemistry and causes contrasting responses in lepidopteran herbivores. Oikos. 120: 1732-1740. Head, G., R. E. Jackson, J. Adamczyk, J. R. Bradley, J. Van Duyn, J. Gore, D. D. Hardee, B. R. Leonard, R. Luttrell, J. Ruberson, J. W. Mullins, R. G. Orth, S. Sivasupramaniam, and R. Voth. 2010. Spatial and temporal variability in host use by Helicoverpa zea as measured by analyses of stable carbon isotope ratios and gossypol residues. J. App. Ecol. 47: 583-592. Jackson, R. E., J. R. Bradley, J. Van Duyn, B. R. Leonard, K. C. Allen, R. Luttrell, J. Ruberson, J. Adamczyk, J. Gore, D. D. Hardee, R. Voth, S. Sivasupramaniam, J. W. Mullins, and G. Head. 2008. Regional assessment of Helicoverpa zea populations on cotton and non-cotton crop hosts. Entomol. Exp. et Appl. 126: 86-106. Kranthi, K. R., S. Naidu, C. S. Dhawad, A. Tatwawadi, K. Mate, E. Patil, A. A. Bharose, G. T. Behere, R. M. Wadaskar, and S. Kranthi. 2005. Temporal and intra-plant variability of Cry1Ac expression in Bt-cotton and its influence on the survival of the cotton bollworm, Helicoverpa armigera (Hubner) (Noctuidae: Lepidoptera). Curr. Sci. 89: 291-298. Leigh, T. F., S. H. Roach, and T. F. Watson. 1996. Biology and ecology of important insect and mite pests of cotton, pp. 17-162. In E. G. King, J. R. Phillips, and R. J. Coleman [eds.], Cotton insects and mites: characterization and management. The Cotton Foundation Publisher, Memphis, TN. National Research Council. 2010. The impact of genetically engineered crops on farm sustainability in the United States. Washington, DC. National Academies Press. Olsen, K. M., J. C. Daly, R. J. Mahon, and E. J. Finnegan. 2005. Changes in Cry1Ac Bt Transgenic Cotton in Response to Two Environmental Factors: Temperature and Insect Damage. J. Econ. Entomol. 98: 1382-1390. Roe, R.M., A. Dhammi, D. Reisig and R.W. Kurtz. 2015. Caterpillar resistance to transgenic cotton: impact of increased feeding rates. In Proceedings, 2015 Beltwide Cotton Conferences, San Antanio, TX, Jan. 5-7, 2015. Cotton Council, Memphis, TN. Pp. 831-840. Roe, R.M., A. Dhammi, J. Zhu, D. Reisig and R.W. Kurtz. 2016. Multiple mechanisms for caterpillar resistance to Bt: Don't forget our history. In Proceedings, 2016 Beltwide Cotton Conferences, New Orleans, LA Jan. 5-7, 2016. Cotton Council, Memphis, TN. Sharma, H. C. 2005. Heliothis/Helicoverpa management: emerging trends and strategies for future research. Science Publishers, Inc., Plymouth, U.K. Siebert, M. W., T. G. Patterson, G. J. Gilles, S. P. Nolting, L. B. Braxton, B. R. Leonard, J. W. Van Duyn, and R. B. Lassiter. 2009. Quantification of Cry1Ac and Cry1F Bacillus thuringiensis insecticidal proteins in selected transgenic cotton plant tissue types. J. Econ. Entomol. 102: 1301-1308. Sivasupramaniam, S., M. J. Moar, L. G. Ruschke, J. A. Osborn, C. Jiang, J. L. Sebaugh, G. R. Brown, Z. W. Shappley, M. E. Oppenhuizen, J. W. Mullins, and J. T. Greenplate. 2008. Toxicity and characterization of cotton expressing Bacillus thuringiensis Cry1Ac and Cry2Ab2 proteins for control of Lepidopteran pests. J. Econ. Entomol. 101: 546-554. Stewart, S. D., and W. L. Sterling. 1989. Susceptibility of cotton fruiting forms to insects, boll rot, and physical stress. J. Econ. Entomol. 82: 593-598. Terry, I., J. R. Bradley, Jr., and J. W. Van Duyn. 1987. Survival and development of Heliothis zea (Lepidoptera: Noctuidae) larvae on selected soybean growth stages. Environ. Entomol. 16: 441-445. Williams, M. R. 2013. Cotton insect losses 2012, pp. 546-586. In Proceedings of the Beltwide Cotton Conferences, January 7-10, San Antonio, TX. National Cotton Council of America, Memphis, TN. Williams, M. 2016. Cotton insect losses - 2014. Mississippi State University- Crop Losses http://www.entomology.msstate.edu/resources/croplosses/2014loss.asp Accessed 2/3/2016 What opportunities for training and professional development has the project provided?Objectives The specific objectives that were completed before early termination of the project were to determine 1) howtemperaturecontributes to changes inH. zeafeeding, Bt expression, and yield and 2) howdroughtcontributes to changes inH. zeafeeding, Bt expression, and yield. Methods Irrigation Study Cotton was planted during May in randomized and replicated small plots (four rows about 0.9 m wide by 12.2 m long plots, four replicates per variety) at the Upper Coastal Research Station in Rocky Mount, NC in 2017. Cotton varieties included the following toxins: 1) Cry1Ac + Cry1F (WideStrike, PhytoGen, DowDuPont AgroSciences, Indianapolis, IN); 2) Cry1Ac + Cry2Ab (Bollgard II, Deltapine, Monsanto Company, St. Louis, MO); and 3) Cry1Ab + Cry2Ae (TwinLink, Stoneville, Bayer CropScience, Research Triangle Park, NC). North Carolina State University Extension recommendations were used to complete all weed, nutrient, and growth regulator management decisions. Irrigation lines (1.27 cm emitter tubing) with emitters about 30 cm apart were setup to irrigate plots in a split block design. Vinyl mainline hoses (15 cm diameter) were used to feed the irrigation lines. Half of the plots received irrigation starting in mid-June through mid-July, while the other half were left to be rain fed. Overall, irrigated plots received 5 cm of extra water than the non-irrigated plots. Orthene (acephate 1.05 kg AI/ha [Amvac Chemical Corporation Newport Beach, CA]) foliar spray was applied prior toH. zeaoviposition to eliminate natural enemies and encourageH. zealarval establishment across trials. Major flights were detected by sudden increases of adult catches in sentinel pheromone traps placed around the state. We inspected cotton terminals for hatched eggs and evidence of feeding. When feeding was observed in plots we tagged 40 injured squares and bolls in each plot. Feeding was apparent sooner in non-Bt, therefore non-Bt plots were tagged one week before the Bt plots. All tagged tissues were tracked and measured for injury in the following manner: bract injury (mm2), sepal injury (mm2), wall injury (mm2), and hole(s) into the ovary (mm2). Tissue injury to the bolls were tracked for four weeks until bollworms were no longer observed in large numbers. We categorized boll size in the following manner: small (< 23 mm diameter), medium (23 - 28 mm diameter), and large (> 28 mm) (Bacheler et al 2011). Tissues were tagged in the third week of July sinceH. zeabegan early that year. All tagged tissues were noted for date, plot, tissue identification, and growth stage and the progress of feeding was tracked for up to two weeks for squares and three weeks for bolls. How have the results been disseminated to communities of interest?Introduction Cotton expressing insect-specific toxin from Bacillus thuringiensis Berliner was introduced in the 1990s to manage the heliothine moth complex of Helicoverpa zea (cotton bollworm) and Heliothis viriscens (tobacco budworm). Since then, adoption has grown to over 99% in the southeastern U.S. (Williams 2013). As a result, the amount of cotton acres sprayed with any insecticide has dropped below 30% from before Bt cotton was introduced (NRC 2010). Today, H. virescens is effectively controlled with Bt cotton varieties, but metabolic and behavioral resistance in the related H. zea to some varieties of Bt toxins has made control of this insect difficult (Gore et al. 2005). Any additional foliar sprays for this pest complex are now focused on H. zea. In the U.S. in 2014, 1.49 million acres of Bt cotton were sprayed for H. zea with an average of 1 insecticide application per acre (Williams 2016), which is an increase from 2012 of 1.36 million acres treated with 0.95 applications per acre (Williams 2013). This sets up a worrisome trend as climate change will negatively impact cotton growers in the southeast. North Carolina is predicted to lower crop productivity because of droughts and higher temperatures according to the Third National Climate Change Assessment (Carter et al. 2014). Higher temperatures and drought can alter pest behavior and weaken expression of Bt toxin by changing plant architecture and physiology. In the southeastern US, the early H. zea generations occur on wild hosts and corn following diapause (Sharma 2005); later in the season they infest cotton and soybeans (Terry et al. 1987, Head et al. 2010). The adults usually oviposit in late June through July (Jackson et al. 2008). Larvae feed and move among terminals and the reproductive parts of the plant: pre-floral buds (squares), flowers and bolls (Leigh et al. 1996). H. zea destroy reproductive tissues or cause the abscission of squares or bolls (Guinn 1982). Cotton can compensate for injury through its indeterminate fruiting pattern, but that ability diminishes as the plant matures (Chilcutt et al. 2003). Although bolls become more resistant to penetration from H. zea (Bagwell 1994), 5th instar larvae can penetrate nearly-mature bolls, so bolls are never completely resistant to injury (Stewart and Sterling 1989, Adamczyk et al. 1998, Gore et al. 2000). The threat posed by climate change to the US cotton industry requires researchers to look at the effects of changes in temperatures and moisture and their interaction on H. zea feeding in cotton. It is known that environmental conditions, can drastically affect the efficacy of Bt cotton by reducing expression (Rochester 2006). For example efficacy of Cry1Ac against Helicoverpa armigera, the old world cotton bollworm, is reduced at higher temperatures (Olsen et al. 2005). Recent studies by Mike Roe (NCSU entomology) have shown that susceptibility to Bt decreases at higher temperatures (30-35°C) because of an increase in feeding rates for H. zea (Roe et al. 2015, Roe et al. 2016). This is contrary to what is normally expected, which is susceptibility to ingested toxins should increase with higher temperatures (Gulzar et al. 2012). Additionally, reduced irrigation and drought can increase susceptibility to pest pressure and reduce yields in cotton. Reduced irrigation and late season damage have strong negative impacts on yield (Chilcutt et al. 2013). Furthermore, Bt expression can vary depending on the type of Bt insertion type (Bt event), which is further affected by tissue type and plant age (Greenplate 1999, Greenplate et al. 2001, Kranthi et al. 2005, Sivasupramaniam et al. 2008, Siebert et al. 2009). In addition, it has been shown that H. zea larvae can detect the presence of Bt toxin within plants and will move to selectively feed on tissues that likely contain lower Bt concentrations (Gore et al. 2002, Gore et al. 2005). These combined factors will threaten US cotton production, as climate change creates stress for cotton plants and lowers Bt expression. Pests, like H. zea, will capitalize on this stress, selecting tissues with less Bt and develop resistance faster with reduced Bt susceptibility at higher temperatures. To keep US cotton competitive and to maintain insecticide susceptibility, we need to mitigate potential pest problems brought on by climate change. We have to fully understand the effect of interacting environmental factors on H. zea feeding behavior. These studies will help us maintain Bt crops, which benefit growers and the environment, through an overall reduction in inputs and provide the information needed for crop breeders and developers to construct hardier varieties. What do you plan to do during the next reporting period to accomplish the goals??Temperature Study This study was conducted at the USDA-ARS Plant Science Research Unit in Raleigh, NC using OPEC chambers. Cotton plants (Widestrike) that express Cry1Ac + Cry1F were planted and grown in Plymouth, NC. When the plants hit first flower they were transported to Raleigh, NC. Six plants were placed in each of 8 chambers. Plants were flood irrigated. Four chambers received the high daytime temperature treatment of 34°C while the other four received the low daytime temperature treatment of 30°C. All chambers were set to 28°C for nighttime temperatures. Helicoverpa zea neonates that had hatched the day prior were infested onto flowers and squares on all plants using a paintbrush. Approximately 150 neonates were infested in each chamber. Plants were huddled together to facilitate larval movement between plants to maximize infestation success. Bt Quantification Tissue Collection At beginning of oviposition, we collected five samples of uninjured leaf tissue (4th true leaf from the top), squares (square growth midpoint), white flowers and medium sized (23 - 28 mm diameter) bolls from each plot. Samples were stored at -80°C until ready to analyze. Tissues were collected again when 3rd stadium larvae are observed on Bt plants and at the end of when bollworm was observed on Bt plants. In 2014, tissues were collected on 8/14, 8/22, and 9/5. In 2015 tissues were collected on 8/21 and 9/15. In 2016, tissues were collected on 7/22, 8/24, 9/8, and 9/18. In 2017, tissues were only collected on 8/17. Bt toxins and formulations We tested Cry1Ac, Cry1Ab, Cry2Ab2, Cry 2Ae and Cry1F. Monsanto Company (St. Louis, MO) provided Cry1Ac and Cry2Ab2. Cry1Ac was from Monsanto sourced from the fermentation of Escherichia coli paste containing the expression plasmid pMON107800 and was in a purity concentration of 1.30 mg/ml. The activated protein was stored in a buffer of 50 mM CAPS at pH 10.25 with 1 mM EDTA, 2.5 mM DDT, and 1 mM benzamidine-HCl. Monsanto Cry2Ab2 was sourced from the fermentation of Escherichia coli containing the pMON70520 expression plasmid and was at a purity concentration of 0.31 mg/ml. The activated protein was stored in buffer composed of 50 mM CAPS at pH 11 with 2 mM DTT. Bayer CropScience (Morrisville, NC) provided Cry1Ab and Cry2Ae. Bayer Cry1Ab was expressed using an unnamed Bt vector and purified by extraction in 50 mM carb pH 10.5 with 5 mM DTT and DTT was later removed. It was in a concentration of 3.2 mg/ml. Bayer Cry2Ae protein was expressed using an unnamed Bt vector and purified by extraction in 50 mM carb pH 10.5 with 5 mM DTT and DTT was later removed. The protein was at 2.6 mg/ml concentration. DowDuPont (Indianapolis, IN) provided Cry1Ac and Cry1F. Dow Cry1F was in a lyophilized powder that was reconstituted in 10 mM CAPS buffer at pH 10.25, set on ice for 15 minutes to allow for solubilization. All proteins were stored -80°C until needed. All proteins with the exception of Cry 2Ab2 were reconstituted in tissue extraction buffer solution of 0.01M phosphate.0.15M saline/0.55% Tween-20 solution, pH 7.4. Measuring Bt Expression Tissues were freeze dried using a 2.5 L freeze drier (Labconco Kansas City, Missouri) run for 6 days to completely dry tissues. In early 2017, we switched devices to a SP Scientific Virtis freeze dryer (~ 40 L capacity) to expedite the process. Tissues were placed in 50 ml Nunc Centrifuge tubes (ThermoFischer Scientific Waltham, MA) with 2-3 9.5 mm stainless steel balls (MSC Industrial Direct Melville, NY). Tubes were loaded into a Geno/Grinder 2000 (SPEX CertiPrep Metuchen, NJ) and shaken at 1500 RPM. Bracts were ground for 1 min 30 seconds while boll walls had to be shaken for 20 minutes until at least 20 mg could pass through a 0.231 mm sieve. Approximately 10.5 mg of ground sample were loaded into 2 ml microcentrifuge tubes and filled with 0.5 ml of extraction buffer (0.01M phosphate.0.15M saline/0.55% Tween-20 solution, pH 7.4). Extraction tubes were loaded into a centrifuge (Sorvall Legend Micro21R Refrigerated Thermo Scientific) and spun for 10 minutes at 3000 RPM at 4?C. Approximately 0.4 ml of supernatant was extracted and stored at -80?C for further use. We used pure Cry protein samples to create our positive protein standards at the following concentrations 20, 10, 5, 2.5, 1.25, 0.625, 0.375, and 0.156 ng/ml. We used 96 well ELISA qualiplates (Envirologix Portland, ME) for Cry 2A, Cry 1Ac and Cry1Ab, and Cry 1F to measure Bt expression levels in tissues. Four total replicates of each tissue sample were duplicated twice on each plate for a total of 8 wells per tissue sample. Cry 2A and 1F plates were incubated for 1 hour, while Cry 1Ac and Cry 1Ab plates were incubated for 1.5 hours per kit instructions on an orbital plate shaker at 200 rpm (Torrey Pines Scientific Carlsbad, CA). The Bt protein toxin was analyzed in duplicate using a Spectramax Plus 384 (Molecular Devices Sunnyvale, CA). Each plate was analyzed at 450 nm with a 650-nm reference wavelength. Protein concentrations were calculated from the absorbance data by using logarithmic or power functions to best fit the data. Total Plant Protein Measurements The Bradford assay method was used to assess total protein content of tissue extracts. Standard curves were constructing using a two-fold dilution series of Bovine Serine Albumin (Sigma Aldrich) at a starting concentration of 1 mg/ml with the following concentrations: 1, 0.5, 0.25, 0.125, 0.0625, and 0.0375 mg/ml. 10 microliters of each extract was loaded onto an uncoated 96 well plate (Corning) followed with 200 ul preparation of 1:4 dilution of Bradford reagent to distilled water (DeerPark, Nestle Waters Issy les Moulineaux, Paris, France). Plates were incubated at room temperature 25°C for 15 minutes. Total plant protein was analyzed in duplicate using the plate reader at 595 nm with a 650-nm reference wavelength. Total protein was calculated using the BSA standard curve with the data fit to a logarithmic curve. Statistical Analyses We used PROC GLIMMIX to run one-way ANOVA to determine if there differences in cumulative feeding patterns for the season between all four varieties among squares and bolls using the individual square or boll as the experimental unit nested within variety. Data were log or square root transformed to fit assumptions of normality. If tests of normality failed, then data were fitted to lognormal or poisson distributions based on chi-square goodness of fit test. Bt expression data were analyzed with ANOVA using PROC GLIMMIX. Changes in Bt expression over time were analyzed by tissue type using PROC GLIMMIX.
Impacts
What was accomplished under these goals?
RESULTS Tissue Injury In the field, hole injury in Non-Bt was highest compared to the rest of the varieties (F= 68.14, df = 3, 274.6, P < 0.01). Bract injury in Non-Bt was highest compared to the TwinLink and Bollgard II (F= 4.64, df = 3, 277, P = 0.01) (Fig 1d). There were no differences detected in sepals (F= 2.26, df = 3, 277, P = 0.08) (Fig. 1). There were no significant differences between varieties among tissues in bolls (Fig. 2) In the greenhouse, there was reduced feeding injury on plants under the high temperature treatment compared to the low temperature treatment (F = 6.6, df = 1, 29, P < 0.01) (Fig. 3). Bt Protein Expression In the irrigation study, the interaction between tissue and protein was significant (F = 2.51, df = 34, 119, P < 0.01). Analyzing by each protein specific to variety, tissue differences were significant for all six proteins (Table 1). There was no effect of irrigation on protein expression as the trends were similar in irrigated (Fig. 4) and drought treatments (Fig. 5) In the temperature study, there was no interaction between protein and temperature even though overall Bt expression seemed lower at 34°C than at 30°C. Cry1Ac generally showing lower quantity of expression when compared to Cry1F (t = 5.399, df = 1, 65, P <0.01). Cry1F in the leaf was seven times higher than Cry1Ac in the same tissue (Fig. 6). Discussion The irrigation study only showed more feeding on sepals in WideStrike squares in irrigated plots compared to drought plots. There were no feeding differences between bolls across variety or treatment. Differences in Bt expression were not affected by treatment but influenced by the predetermined genetic expression between varieties with different proteins. The temperature study showed that at higher temperatures feeding by H. zea drops significantly, and Bt expression trends downward. The third component of this study that was needed was the temperature and irrigation interaction which could have been helpful in explaining the feeding trends of H. zea on bolls. The difference in square and boll feeding in the irrigation study can be explained by the maturity of the larvae feeding on those tissues. Generally, larger larvae can break through the tougher carpal wall of the cotton boll, while neonates are limited to feeding on softer cotton squares. By the time H. zea is a 3rd or 4th instar it also has an easier time overcoming the effects of Bt toxins. Even so, across all varieties, Bt expression is typically lowest in the boll wall. An appropriate analogy would be of a 15-year-old child clearing a 1-foot hurdle compared to a 4-year-old child. This may possibly be an issue The drawback of the temperature study was that more plants were needed to gather more data on tissue specific feeding by the larvae. The drawback with the irrigation study was that more tissue samples should have been taken to examine the effect of time on Bt expression over the period of irrigation and drought. The important conclusion from this study is that a higher temperature has a greater direct impact on H. zea larvae than irrigation. However, since moisture and temperature were not compared together in a factorial analysis this cannot be stated as a certainty. The moisture levels of the plant could have been measured to help understand if the irrigation treatment was having an effect. Visual observations in the field showed that irrigated treatments had greater rank growth and were taller than plants in the drought treatment, but this effect seemed to be limited to Twinlink and Bollgard2 to an extent (Fig. 7).
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