Randy Wells

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’.

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.

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.

Effects of natural shade on soybean thylakoid membrane composition.

The effect of natural shade on chloroplast thylakoid membrane activity and composition was examined for soybean (Glycine Max. cv. Young) grown under field conditions. Plots with high (10 plants m−1 row) or low (1 plant m−1 row) plant density were established. Expanding leaves were tagged at 50, 58 and 65 days after planting (DAP). At 92 DAP, tagged leaves were used as reference points to characterize canopy light environments and isolate thylakoid membranes. Light environments ranged from a photosynthetic photon flux density (PPFD) of 87% of full sun to a PPFD of 10% of full sun. The decline in PPFD was accompanied by an increase in the far-red/red (735 nm/645 nm) ratio from 0.9 to approximately six. The major effects of shade on chloroplast thylakoid membranes were a reduction in chloroplast coupling factor and a shift in light-harvesting capacity from Photosystem I to Photosystem II. Photosynthetic electron transport capacity was not affected by differences in PPFD, but was 20 to 30% higher in the 1 plant m−1 row treatment. The plant density effect on electron transport was associated with differences in plastocyanin concentration, suggesting that plastocyanin is a limiting factor in soybean. Shade did not have a significant effect on the concentration of Photosystem II, Cyt b6f, or Photosystem I complexes.

Boll openers in cotton: effectiveness and environmental influences

Abstract The indeterminate growth of cotton ( Gossypium hirsutum L.) often results in a boll population at various stages of maturity when the crop is harvested. Boll opening compounds can be used to facilitate harvest by accelerating the opening of green bolls. Field trials were conducted at two locations in 1996 and 1997 to test the efficacy of four commercially available boll openers that stimulate ethylene production or desiccate the plant. Two of these boll openers are new to the marketplace: Finish™, ethephon+cyclanilide (2-chloroethyl phosphonic acid+1-(2,4-dichlorophenylaminocarbonyl)-cyclopropane carboxylic acid), and CottonQuick™, ethephon+AMADS (2-chloroethyl phosphonic acid+1-aminomethanamide dihydrogen tetraoxosulfate). Open bolls were hand harvested at 3–7 days intervals after application of the boll openers, and environmental conditions in conjunction with these harvests were recorded. The desiccant, paraquat (1,1′-dimethyl-4,4′-bipyridinium dichloride), was found to have erratic boll opening activity. Two products that contain ethephon plus the synergists cyclanilide or AMADS were found to have equal or higher rates of boll opening than ethephon alone. The rate of boll opening of all compounds was influenced more by the accumulation of heat units than by the amount of time following the application of boll openers. Although erratic, boll opening activity from paraquat application can be significant. Moreover, the reliability of ethephon based products, which are consistent boll openers, may be enhanced by formulations that contain a synergist. Heat unit accumulation may be used to predict and model the efficacy of boll opening with these harvest aides.

Glufosinate does not affect floral morphology and pollen viability in glufosinate-resistant cotton

Studies were conducted to determine whether glufosinate treatments to glufosinate-resistant cotton caused changes in floral morphology, pollen viability, and seed set. Four glufosinate treatments were included: (1) glufosinate applied postemergence over the top (POST) at the four-leaf stage, (2) glufosinate applied POST at the eight-leaf stage, (3) the first two treatments sequentially, and (4) a POST application at the four-leaf stage followed by (fb) a postemergence-directed stem application (PDS) at the eight-leaf stage. Glufosinate was consistently applied at 0.49 kg ai/ha. A nontreated control was included. Glufosinate treatments did not affect stigma height, length of the staminal column, or pollen viability. However, the distance from the top anther to the tip of the stigma was less in plants treated with an eight-leaf POST treatment than in nontreated plants, although this difference is not likely to influence pollen deposition because in both cases anthers reached above the stigma tip. Plants receiving four-leaf POST fb eight-leaf PDS treatment with glufosinate had eight seeds per boll less than nontreated plants; however, the more rigorous four-leaf POST fb eight-leaf POST treatment did not differ from the nontreated in seeds per boll. Nomenclature: Glufosinate; cotton, Gossypium hirsutum L. Additional index words: Glyphosate, herbicide-resistant crops, transgenic crops. Abbreviations: fb, followed by; PDS, postemergence-directed stem application; POST, postemergence over the top.