Keith L. Edmisten

Reproductive abnormalities in glyphosate-resistant cotton caused by lower CP4-EPSPS levels in the male reproductive tissue

Abstract Glyphosate treatments to glyphosate-resistant (GR) cotton have been associated with poor pollination and increased boll abortion. Anatomical studies were conducted to characterize the effect of glyphosate treatments on the development of male and female reproductive organs of cotton flowers at anthesis. In comparison with nontreated plants, glyphosate applied at both the four-leaf stage postemergence (POST) and at the eight-leaf stage POST directed inhibited the elongation of the staminal column and filament, which increased the distance from the anthers to the receptive stigma tip by 4.9 to 5.7 mm during the first week of flowering. The increased distance from the anthers to the stigma resulted in 42% less pollen deposited on stigmas of glyphosate-treated plants than in nontreated plants. Moreover, pollen from glyphosate-treated plants showed numerous morphological abnormalities. Transmission electron microscopy showed the presence of large vacuoles, numerous starch grains, and less organized pockets of the endoplasmic reticulum containing fewer ribosomes in pollen from glyphosate-treated plants than from nontreated plants. Pollen development in glyphosate-treated plants is likely inhibited or aborted at the vacuolate microspore and vacuolate microgamete stages of microgametogenesis, resulting in immature pollen at anthesis. Although stigmas from glyphosate-treated plants were 1.2 to 1.4 mm longer than those from nontreated plants, no other anatomical differences in stigmas were visibly evident. The presence of the GR 5-enolpyruvylshikimate-3-phosphate synthase (CP4-EPSPS) enzyme from Agrobacterium sp. strain CP4 was quantified in reproductive and vegetative tissues using enzyme-linked immunosorbent assay. The content of CP4-EPSPS in the stigma, anther, preanthesis floral bud (square), and flower petals was significantly less than that in the vegetative leaf tissue. Glyphosate effects on the male reproductive development resulting in poor pollen deposition on the stigma, as well as production of aborted pollen with reduced viability, provide a likely explanation for reports of increased boll abortion and pollination problems in glyphosate-treated GR cotton. Nomenclature: Glyphosate; cotton, Gossypium hirsutum L. ‘Delta Pine & Land 5415RR’, ‘Delta Pine & Land 50’, ‘Delta Pine & Land 90’, ‘SureGrow 125RR’.

Absorption and translocation of glyphosate in glyphosate-resistant cotton as influenced by application method and growth stage

Abstract The influence of herbicide placement and plant growth stage on the absorption and translocation patterns of 14C-glyphosate in glyphosate-resistant cotton was investigated. Plants at four growth stages were treated with 14C-glyphosate on a 5-cm2 section of the stem, which simulated a postemergence-directed spray (PDS) application, or on the newest mature leaf, which simulated a postemergence (POST) application. Plants were harvested 3 and 7 d after treatment and divided into the treated leaf or treated stem, mature leaves, immature leaves and buds, stems, roots, fruiting branches (including the foliage on the fruiting branch), squares, and bolls. The PDS versus POST application main effect on absorption was significant. Absorption of 14C-glyphosate applied to stem tissue was higher in PDS applications than in POST applications. Plants receiving PDS applications absorbed 35% of applied 14C-glyphosate, whereas those receiving POST applications absorbed 26%, averaged over growth stages at application. Absorption increased from the four-leaf growth stage to the eight-leaf stage in POST applications but reached a plateau at the eight-leaf stage. Plants with PDS applications showed an increase in absorption from the four- to eight- to twelve-leaf stages and reached a plateau at the 12-leaf stage. Translocation of 14C-glyphosate to roots was greater at all growth stages with PDS treatments than with POST treatments. Herbicide placement did not affect translocation of 14C-glyphosate to squares and bolls. Squares and bolls retained 0.2 to 3.7% of applied 14C-glyphosate, depending on growth stage. Separate studies were conducted to investigate the fate of foliar-applied 14C-glyphosate at the four- or eight-leaf growth stages when harvested at 8- or 10-leaf, 12-leaf, midbloom (8 to 10 nodes above white bloom), and cutout (five nodes above white bloom, physiological maturity) stages. Thirty to 37% of applied 14C-glyphosate remained in the plant at cutout in four- and eight-leaf treatment stages, respectively. The concentration of 14C-glyphosate in tissue (Bq g−1 dry weight basis) was greatest in mature leaves and immature leaves and buds in plants treated at the four-leaf stage. Plants treated at the eight-leaf stage and harvested at all growth stages except cutout showed a higher concentration of 14C-glyphosate in squares than in other plant tissue. Accumulation of 14C-glyphosate in squares reached a maximum of 43 Bq g−1 dry weight at harvest at the 12-leaf stage. This concentration corresponds to 5.7 times greater accumulation of 14C-glyphosate in squares than in roots, which may also be metabolic sinks. These data suggest that reproductive tissues such as bolls and squares can accumulate 14C-glyphosate at higher concentrations than other tissues, especially when the herbicide treatment is applied either POST or PDS during reproductive stages (eight-leaf stage and beyond). Nomenclature: Glyphosate; cotton, Gossypium hirsutum L. ‘Delta Pine 5415RR’.

Glyphosate-induced reductions in pollen viability and seed set in glyphosate-resistant cotton and attempted remediation by gibberellic acid (GA3)

Abstract Glyphosate treatments to glyphosate-resistant (GR) cotton can cause increased fruit loss compared with untreated plants, likely due to reductions in pollen viability and alterations in floral morphology that may reduce pollination efficiency. This study was conducted to determine whether both stamen and pistil are affected by glyphosate treatments by measuring seed set from reciprocal reproductive crosses made between glyphosate-treated GR, untreated GR, and conventional nontransgenic cotton. Pollen viability was 51 and 38% lower for the first and second week of flowering, respectively, in GR plants treated with a four-leaf postemergence (POST) treatment and an eight-leaf POST-directed treatment of glyphosate than in GR plants that were not treated. Seed set per boll was significantly reduced when the pollen donor parent was glyphosate treated vs. untreated for the first 2 wk of flowering. There were no significant differences between treatments applied to male parents as measured by seed set at Weeks 3 and 4 of flowering. Seed set was not influenced by glyphosate treatments applied to female parents at any time. Retention of bolls resulting from crosses was reduced by glyphosate treatment of male parents during the first and third week of flowering but was not affected by glyphosate treatment of female parents. The application of gibberellic acid (GA), which has been shown to reverse male sterility in tomato (Lycopersicon esculentum L.) and to enhance boll retention in cotton, was investigated for similar effects in glyphosate-treated GR cotton. The GA treatments to glyphosate-treated plants increased the anther–stigma distance 12-fold, stigma height, and pollen viability in the second week of flowering but decreased the number of seeds in second-position bolls on Fruiting branches 1 through 3, decreased the number of first-position bolls per plant, and increased the number of squares in comparison with glyphosate-treated GR plants not receiving GA. Although GA applications to glyphosate-treated GR cotton have some remedial effect on pollen viability, the GA-induced increase in the anther–stigma difference exacerbates the increase in anther–stigma distance caused by glyphosate, resulting in low pollination. Nomenclature: Glyphosate; cotton, Gossypium hirsutum L. ‘Delta Pine & Land 5415RR’, ‘Delta Pine & Land 5415’.

Weed-Free Yield Response of Seven Cotton (Gossypium hirsutum) Cultivars to CGA-362622 Postemergence1

Field studies were conducted in 1998 and 1999 to evaluate the response of seven cotton cultivars to CGA-362622 applied postemergence at 7.5 and 15 g ai/ha to three- to five-leaf cotton. The cultivars evaluated included Deltapine 51, Deltapine NuCotn 33B, Paymaster 1220 RR, Paymaster 1220 BG/RR, Stoneville bromoxynil-resistant 47, Stoneville 474, and Sure-Grow 125. At 1 to 2 wk after treatment (WAT), CGA-362622 at 7.5 and 15 g/ha injured all cotton cultivars 7 to 9% and 13 to 15%, respectively. Cotton injury symptoms included chlorosis and minor stunting. At 3 to 4 WAT, injury from CGA-362622 at 7.5 and 15 g/ha was 2 to 6% and 7 to 9%, respectively. Except for Paymaster 1220 RR, Deltapine NuCotn 33B, and Stoneville 474, all cotton cultivars were injured more by the higher rate than by the lower rate of CGA-362622. Injury was not visibly apparent 6 to 8 WAT. CGA-362622 at either rate had no effect on cotton lint yield. Nomenclature: CGA-362622 (proposed common name trifloxysulfuron), N-[(4,6-dimethoxy-2-pyrimidinyl)carbamoyl]-3-(2,2,2-trifluoroethoxy)-pyridin-2-sulfonamide sodium salt; cotton, Gossypium hirsutum L. ‘Deltapine 51’, ‘Deltapine NuCotn 33B’, ‘Paymaster 1220 RR’, ‘Paymaster 1220 BG/RR’, ‘Stoneville BXN 47’, ‘Stoneville 474’, ‘Sure-Grow 125’. Additional index words: Crop injury, crop yield. Abbreviations: ALS, acetolactate synthase; BXN, bromoxynil-resistant; LAYBY, late postemergence directed; POST, postemergence; WAT, weeks after treatment.

Glyphosate negatively affects pollen viability but not pollination and seed set in glyphosate-resistant corn

Abstract Experiments were conducted in the North Carolina State University Phytotron greenhouse and field locations in Clayton, Rocky Mount, and Lewiston-Woodville, NC, in 2002 to determine the effect of glyphosate on pollen viability and seed set in glyphosate-resistant (GR) corn. Varieties representing both currently commercial GR corn events, GA21 and NK603, were used in phytotron and field studies. All glyphosate treatments were applied at 1.12 kg ai ha−1 at various growth stages. Regardless of hybrid, pollen viability was reduced in phytotron and field studies with glyphosate treatments applied at the V6 stage or later. Scanning electron microscopy of pollen from affected treatments showed distinct morphological alterations correlating with reduced pollen viability as determined by Alexander stain. Transmission electron microscopy showed pollen anatomy alterations including large vacuoles and lower starch accumulation with these same glyphosate treatments. Although pollen viability and pollen production were reduced in glyphosate treatments after V6, no effect on kernel set or yield was found among any of the reciprocal crosses in the phytotron or field studies. There were also no yield differences among any of the hand self-pollinated (nontreated male × nontreated female, etc.) crosses. Using enzyme-linked immunosorbent assay to examine CP4-5-enolpyruvlshikimate-3-phosphate synthase expression in DKC 64-10RR (NK603) at anthesis, we found the highest expression in pollen with progressively less in brace roots, ear leaf, anthers, roots, ovaries, silks, stem, flag leaf, and husk. Nomenclature: Glyphosate; corn, Zea mays L.; ‘DK 662RR’; ‘DK 687RR’; ‘DKC 64-10RR/SIL’.

Measuring Canopy Coverage with Digital Imaging

Abstract Sampling plant canopies for their ability to intercept sunlight has traditionally been done with destructive or time‐consuming methods. Although nondestructive methods are available, they are either time consuming or subject to large variation. A commercially available software was utilized to analyze digital images of a cotton (Gossypium hirsutum L.) canopy in an effort to quantify canopy coverage. Digital images were obtained from a vertical perspective using a stationary camera stand. Images were analyzed using Adobe Photoshop 4.0 (Adobe Systems, Inc., Seattle, WA) software. Using functions within the software, plant material in the image was separated from the soil and converted to black. The soil surface was converted to white. The resulting black and white image was analyzed with Javascript software developed at North Carolina State University that counts the black and white pixels in each image. The resulting percentage of black pixels in the image was termed percent ground cover for the canopy. Percent ground cover was well correlated with leaf area index (LAI) over a low range of LAI with r2=0.74. This method provides a reasonable estimation of canopy coverage and proved to be a simple and efficient method of sampling a plant canopy. As image processing software becomes more refined, this and other techniques will become powerful tools for plant science research.

Winter annual small grain forage potential. I. Dry matter yield in relation to morphological characteristics of four small grain species at six growth stages

Abstract Small grains offer a potential animal feed at a time when moisture is usually available in the Southeast. The four major winter annual small grains, barley, oats, rye, and wheat, have not been compared as potential feed sources in the same study in the past. These four small grains were harvested at six stages of growth: vegetative, boot, heading, milk, soft dough, and hard dough. Harvest dates, dry matter (DM) yield, DM yield of regrowth following initial harvest, DM concentration, and the proportions of leaf, stem and inflorescence were measured and used to evaluate the four species for silage production and grazing potential. Rye and barley reached boot prior to the suggested planting date for corn in the Piedmont of North Carolina and dry matter yields ranged from 4.72 to 6.71 Mg ha‐1 and were harvested. Barley reached milk, soft dough, and hard dough earlier than the other species and was equal to or higher than the other species in proportion of DM in leaf DM yield and total DM yield in 198...

Winter annual small grain forage potential. II. Quantification of nutritive characteristics of four small grain species at six growth stages

Abstract Four small grain species, barley, oat, rye, and wheat were harvested at six growth stages to investigate their nutritive quality as a possible replacement for corn silage. Crude protein (CP), in vitro dry matter disappearance (IVDMD), neutral detergent fiber (NDF), acid detergent fiber (ADF) and lignin contents were measured on fresh and ensiled samples. The IVDMD, an estimate of forage ruminant digestibility, of all species generally decreased from the vegetative (765–854 g kg‐1) through the milk stage (505–662 g kg‐1) and then remained unchanged or increased slightly through hard dough with the exception of oats which often decreased during later stages of growth. The IVDMD of rye was usually lower than the other species from the milk to hard dough stage. The IVDMD of barley was generally higher than the other species at the soft and hard dough stages. The NDF, ADF, and lignin fractions usually increased from the vegetative to milk stages and remained unchanged or increased slightly through the...

Response of Ultra–Narrow and Conventional Spaced Cotton to Variable Nitrogen Rates

Abstract Insufficient nitrogen (N) uptake in cotton (Gossypium hirsutum L.) has been shown to delay maturity and decrease lint yields, while excessive amounts will cause a host of disadvantages, ranging from increased insect damage to defoliation difficulties. Proper nitrogen fertilization rates are therefore essential to maximizing lint production while minimizing input costs. This test was designed to determine if ultra–narrow row cotton (UNRC) has similar nitrogen requirements as conventional row cotton (CRC). Two sites, one in peanuts and the other fallow the previous growing season, and four nitrogen rates (45, 67, 90, and 112 kg N ha−1) were used to compare soil fertility and nitrogen effects on petiole nitrate concentrations, lint yields, and ginning percentages. Although petiole nitrate concentrations between early and peak bloom were lower in ultra–narrow row cotton with 45 and 67 kg N ha−1, there was no correlation between petiole nitrate concentrations and lint yield. Conventional spacing receiving 67 kg N ha−1 had the highest lint production at both locations, while application of 90 kg N ha−1 produced the highest yields in UNRC at both locations. Ultra–narrow row cotton produced numerically higher yields than conventional row at all nitrogen rates at the location without a previous legume crop, but no significant trend was observed. Ultra–narrow row cotton lint percentages were significantly lower than conventional spacing, independent of nitrogen application rate.

Yield and Physiological Response of Nontransgenic Cotton to Simulated Glyphosate Drift1

Field studies were conducted in 2001 in Lewiston, NC, and in 2002 at Clayton and Lewiston, NC, to investigate the response of nontransgenic cotton to simulated glyphosate drift in a weed-free environment. Nontransgenic cotton variety ‘Fibermax 989’ was planted in a conventional seedbed at all locations. Glyphosate treatments were applied early postemergence (EPOST) at the four-leaf growth stage of cotton at 0, 8.7, 17.5, 35, 70, 140, 280, 560, and 1,120 g ai/ha and represent 0, 0.78, 1.55, 3.13, 6.25, 12.5, 25, 50, and 100% of the commercial use rate, respectively. Rates as low as 140 g/ha caused lint yield reductions depending on year and location. When averaged over all locations, lint yield reductions of 4, 49, 72, and 87% compared with nontreated cotton were observed with glyphosate rates of 140, 280, 560, and 1,120 g/ha, respectively. Visual injury and shikimic acid accumulation were evident at glyphosate rates greater or equal to 70 g/ha. Collectively, visual injury and shikimic acid accumulation at 7 d after EPOST treatment might be used as a diagnostic indicator to predict potential yield reductions from simulated glyphosate drift. Nomenclature: Glyphosate; cotton, Gossypium hirsutum L. ‘Fibermax 989’. Additional index words: Shikimic acid. Abbreviations: DAT, days after early postemergence treatment; DD, degree-day; EPOST, early postemergence; EPSPS, 5-enolpyruvylshikimate-3-phosphate synthase [EC 2.5.1.19]; HPLC, high-performance liquid chromatography; PDS, postemergence-directed; POST, postemergence; PRE, preemergence.