Additional fertilizer and nematicide combinations on upland cotton to manage Rotylenchulus reniformis and Meloidogyne incognita in Alabama

Ammonium sulfate, (NH₄)₂SO₄ and 28-0-0-5 are the standard nitrogen fertilizer blends utilized for cotton production in the southeast and are usually applied as a split application near planting and at pin head square (PHS). Max-In^® Sulfur (WinField United, Arden Hills, MN) is an additional sulfur application that maybe added to any spray application applied to cotton usually at PHS. We evaluated splitting fertilizer applications of (NH₄)₂SO₄, or 28-0-0-5 and Max-In^® Sulfur across the season at PHS or at PHS and first bloom (FB).

Aeris^® (Bayer CropScience, Research Triangle Park, NC) (Imidacloprid and Thiodicarb) was the seed treatment nematicide in 2019 and (Table 1). COPeO^™ Prime (BASF, Florham Park, NJ) (Fluopyram) was the seed treatment nematicide in 2020. Seed treatments were applied to the DP 1646 B2XF cotton seed before planting using a Gustafson laboratory tabletop seed treater (Pinckard, 1971). Seeds were air dried for 24 hr before planting. The additional nematicide Vydate^® C-LV (DuPont, Wilmington, DE) (Oxamyl) was applied as a foliar spray at pinhead square (PHS) and/or first bloom (FB) (Table 1).

Trials were conducted with 12 fertilizer and nematicide combinations with the idea of continually feeding and protecting the developing cotton plant as it grows and develops cotton bolls. All treatments received the initial at plant base fertilizer of (NH₄)₂SO₄ or 28-0-0-5. Treatment 1 was the control with no seed treatment nematicide on the cotton seeds at planting with an additional PHS application of the granular (NH₄)₂SO₄ fertilizer while treatment 2 is also the control again without a seed treatment nematicide on the cotton seeds with an additional PHS application of the liquid 28-0-0-5 fertilizer (Table 1). Treatments 3 and 4 were the same cotton seeds with a seed treatment nematicide (Aeris in 2019 and COPeO in 2020) with an additional PHS application of the granular (NH₄)₂SO₄ fertilizer for treatment 3 and an additional PHS application of the liquid 28-0-0-5 fertilizer for treatment 4. Treatment s 5 and 6 were the same as 3 and 4 with the addition of a sequential fertilizer application at FB. Treatments 7 and 8 were the same seed treatment nematicide and fertilizers as treatments 3 and 4 with the addition of the nematicide Vydate^® C-LV applied at PHS. Treatments 9 and 10 were the same as 7 and 8 with another addition of Max-In^® Sulfur added to the Vydate^® C-LV applied at PHS. Treatments 11 and 12 were the same as 9 and 10 with the addition of Max-In^® Sulfur and Vydate^® C-LV applied at PHS and sequentially at FB.

Microplot trials were conducted in 2019 and 2020 at the Plant Science Research Center (PSRC) in Auburn, AL. Four tests were conducted, one each year for R. reniformis and M. incognita. Microplots were 26.5 L plastic pots filled with Kalmia loamy sand (24% sand, 49% silt and 28% clay) from the Plant Breeding Unit (PBU) or Decatur silt loam (24% sand, 49% silt and 28% clay) from the Tennessee Valley Research and Extension Center (TVREC) and represent 0.3 m of row in the field. Each microplot was inoculated with 250 cm³ of soil containing approximately 50,000 eggs and vermiform life stages of either R. reniformis or M. incognita and placed in the base of the planting furrow. Rotylenchulus reniformis nematode was collected from Tennessee Valley Research and Extension Center near Belle Mina, AL and M. incognita from Plant Breeding Unit near Tallassee, AL. Cultures of these nematodes were maintained at the PSRC at Auburn University. The cotton cultivar “Phytogen 340 W3FE” (Corteva Agriscience, Wilmington, DE) was used to maintain R. reniformis and M. incognita populations were maintained on corn “Mycogen 2H723” (Dow AgroScience, Indianapolis, IN). Microplots received a pre-plant broadcast application of 13-13-13 applied at 0.13kg/m and hand tilled into the soil. Ten cotton seeds, “DP 1646 B2XF” (Bayer CropScience, Research Triangle, NC) pretreated with an insecticide/fungicide seed treatment by Bayer CropScience (metalaxyl, pyraclostrobin, myclobutanil, imidacloprid, fluxapyroxa), were planted 2.5 cm deep into a furrow in each microplot and thinned to five seedlings after germination. Irrigation was administered through a drip irrigation system at 30 ml/min and was adjusted throughout the season to run for 15 to 45 min twice a day.

All tests were arranged in a randomized complete block design (RCBD), with five replications. The first additional application of fertilizers and nematicides were applied at PHS. At PHS, (NH₄)₂SO₄ was applied by hand to the base of the plant. At PHS, 28-0-0-5 was pipetted into a narrow indention created with a hand spade 5 cm beside and 5 cm below the base of treated plants. Max-In^® Sulfur and Vydate^® C-LV were applied as foliar sprays via a handheld spray bottle. The second application of additional fertilizers was applied at FB. All fertilizers and nematicides were applied identically at PHS and FB. In 2020, microplots received the same management practices as in 2019 with the exception that the nematicide seed treatment Aeris^® was replaced with Copeo™ Prime.

Data were collected at PHS and FB. One cotton plant was excavated from each microplot for plant and nematode data at each of the sample data collection times. Plant parameters included plant height (PH), and root fresh weight (RFW) and seed cotton yield. Nematode parameters of R. reniformis and M. incognita population density included number of eggs per g of root. At plant maturity, cotton was hand harvested for all microplot trials.

To obtain nematode population levels, eggs were extracted from the cotton roots using a modified method of Hussey and Barker (1973). Eggs were collected for R. reniformis and M. incognita by placing roots in a 0.625% NaOCl solution and shaken for four minutes on a Barnstead Lab Line Max Q 5000 E Class shaker (Conquer Scientific: San Diego, CA). Roots were rinsed with water and scrubbed by hand; eggs were collected on a 25-μm sieve and poured into a 50mL centrifuge tube. The product was further processed by sucrose centrifugation following the modified methodology of Jenkins (1964). Contents were centrifuged at 220 g-forces for 1 min and then rinsed with water; eggs were collected on a 25-μm sieve. Eggs were enumerated via a Nikon TSX 100 inverted microscope at a ×40 magnification.

Field trials were conducted at TVREC near Belle Mina, AL, and at PBU near Tallassee, AL. Both research stations maintained plots throughout the growing season with standard herbicide, insecticide, and fertility practices. Fertility practices at both locations included a pre-plant application of 28-0-0-5 at 112 kg/ha in late April followed by a sidedress application of 28-0-0-5 at 34 kg/ha applied in mid-July. TVREC was artificially infested with R. reniformis in 2007, the initial population density at planting averaged 5000 vermiform life stages per 100 cm³ of soil in 2019 and 2020. The soil type in this field is a Decatur silt loam. PBU is naturally infested with M. incognita and the initial population density at planting was 77 J2 per 100 cm3 of soil in a Kalmia loamy sand soil type. The trials were arranged in a RCBD with 5 replications and the entire test was repeated within each year. Both sites were planted using a John Deere MaxEmerge planter (Moline, Illinois) equipped with Almaco cone planters (Nevada, Iowa). Trials were planted with DP 1646 B2XF at a rate of 100 seeds per 7.6 m. Plots at TVREC consisted of 2 rows that were 7.6 m long with 1.01 m row spacing and a 6 m wide alley. Plots at PBU consisted of 2 rows that were 7.6 m long with 0.9 m row spacing and a 6 m wide alley.

Additional fertilizer, nematicides, and seed treatment applications were identical to the microplot trials. Additional fertilizer and nematicide combinations were applied at PHS and FB. Ammonium sulfate was applied by hand to the base of the plant. The 28-0-0-5 was knifed into the soil 5cm beside and 5 cm below the plant with a liquid fertilizer applicator and fertilizer disc. WinField Max-In^® Sulfur and Vydate^® C-LV (Oxamyl) were applied as a foliar spray at 25 PSI with a Case IH 265 tractor equipped with a 4 boom sprayer at PHS and FB. Entire plots were machine harvested with a Case International Harvester 2555 cotton picker with Harvest Weigh Mobile by System Scales at TVREC and PBU.

Four representative cotton plants from each plot were randomly dug up from each plot to collect plant and nematode data at PHS and FB. The plant growth parameters included plant stand, plant height, root fresh weight and cotton yield. Nematode population density was collected as in the microplot trials after transport from the field to PSRC. Fifty mature bolls were hand harvested from the first rep of each test. Samples were ginned using a 10-saw table-top gin at the PSRC. The lint and seeds collected from the gin were weighed individually and these data were used to calculate the lint ratio for each treatment. All plots were machine harvested to determine seed cotton yields.

Data collected from microplot, and field trials were analyzed with SAS 9.4 (SAS Institute, Cary, NC) using the PROC GLIMMIX procedure. LS-means were compared using ANOVA, and Tukey–Kramer multiple pair wise comparison at a significance level of P ≤ 0.10. Dependent variables included plant stand, plant height, root fresh weight, R. reniformis and M. incognita eggs per gram of root (eggs/g of root), seed cotton yield (kg/ha) and mean ($/kg). Fixed effects comprised of nematicide and fertilizer treatments at PHS and/or FB. Random effects comprised of replication, test repetition and location.

There were no significant interactions between 2019 and 2020 thus the data from both years were combined into a single dataset. Different nematicide seed treatments were used in both years; therefore, we analyzed the effects of a general nematicide seed treatment and not a specific chemical.

Revenue was calculated using the most current price from the USDA upland cotton announcement (https://www.fsa.usda.gov/Internet/FSA_EPAS_Reports.pdf) of $1.32/kg in 2019 and $1.54/kg in 2020 and the lint ratio from each treatment. Fertilizer and nematicide input costs were acquired through a local sales agricultural (Stephen Till, personal communications) representative in 2019 and 2020. Input costs were subtracted from revenue to determine profit for individual treatment combinations. Upper, lower, and mean profit for each combination was determined using a confidence interval from an ANOVA test at P ≤ 0.10.

In the microplot setting with R. reniformis, the additional fertilizer and nematicide applications did not significantly increase plant height or root fresh weight when data collected at PHS and FB were combined (Table 2). Rotylenchulus reniformis eggs per gram of root were significantly reduced when data collected at PHS and FB were combined for the nematicide ST + 28-0-0-5 + Vydate^® C-LV + Max-In^® Sulfur at PHS application compared to the control application of (NH₄)₂SO₄ at PHS with no ST nematicide. A significant increase in seed cotton yield was measured with the maximum input combination of the nematicide ST + (NH₄)₂SO₄ + Vydate^® C-LV + Max-In^® Sulfur applied at PHS and FB when compared to the combination of the nematicide ST + (NH₄)₂SO₄ applied at PHS (P ≤ 0.10).

The M. incognita infested microplot trials found the additional fertilizer and nematicide combinations did not significantly increase plant height or root fresh weight sampled when data collected PHS and FB were combined (Table 3). Meloidogyne incognita eggs per gram of root were lowest in the application of 28-0-0-5 at PHS, and in the combinations of the nematicide ST + 28-0-0-5 + Vydate^® C-LV at PHS and a nematicide ST + (NH₄)₂SO₄ + Vydate^® C-LV + Max-In^® Sulfur at PHS and FB. Seed cotton yields were similar across all fertilizer and nematicide combinations in the microplot tests.

Plant stand was not affected by the additional fertilizer or Vydate^® C-LV combinations (Table 4). The additional fertilizer and nematicide combinations did not significantly increase root fresh weight at PHS + FB. Rotylenchulus reniformis eggs per gram of root were not significantly decreased when sampled at PHS + FB. The combination of a nematicide ST + (NH₄)₂SO₄ + Vydate^® C-LV + Max-In^® Sulfur at PHS and FB had the lowest R. reniformis eggs per gram of root when sampled at PHS + FB. The combination of a nematicide ST + (NH₄)₂SO₄ + Vydate^® C-LV at PHS supported a significantly larger lint yield than the treatments with no nematicide ST, which applied (NH₄)₂SO₄ at PHS, 28-0-0-5 at PHS and the combination of a nematicide ST + 28-0-0-5 at PHS and FB (P ≤ 0.10). Lint yield was increased by 546 kg/ha, 603 kg/ha, 580 kg/ha or 31%, 34% and 33% with the combination of a nematicide ST + (NH₄)₂SO₄ + Vydate^® C-LV at PHS when compared to the three lowest yielding combinations, (NH₄)₂SO₄ at PHS, 28-0-0-5 at PHS and a nematicide ST + 28-0-0-5 at PHS and FB.

Mean profits for fertilizer applications of (NH₄)₂SO₄ and 28-0-0-5 at PHS with no nematicides averaged $853.62 (Table 5). A nematicide ST with an additional fertilizer application at PHS increased mean profit by $46/ha from an additional fertilizer alone. A nematicide ST with an additional application of fertilizer at PHS and FB increased profit by $32/ha from an additional fertilizer alone. Combinations with Vydate^® C-LV applied at PHS had an increased profit of $137/ha from an additional fertilizer alone. Fertilizer and nematicide combinations that included Vydate^® C-LV at PHS and FB had an increased profit of $48/ha. All combinations were compared to the mean profit of treatments of an additional fertilizer with no nematicides to determine increased profit per hectare. The combination of a nematicide ST + (NH₄)₂SO₄ + Vydate^® C-LV at PHS had the greatest overall mean profit of $1175.87 which was significantly greater than the mean profit of (NH₄)₂SO₄ at PHS, 28-0-0-5 at PHS and a nematicide ST + 28-0-0-5 at PHS and FB.

Plant stand was not affected by the additional fertilizer or Vydate^® C-LV combinations (Table 6). The additional fertilizer and nematicide combinations did not significantly increase root fresh weight in samples taken at PHS + FB. Meloidogyne incognita eggs per gram of root were not significantly reduced in samples taken at PHS + FB. A nematicide ST + (NH₄)₂SO₄ at PHS and FB had the lowest M. incognita eggs per gram of root, represented by the combined sample. The combination of a nematicide ST + 28-0-0-5 + Vydate^® C-LV + Max-In^® at PHS and FB had a significantly higher lint yield when compared to application of (NH₄)₂SO₄ at PHS or 28-0-0-5 at PHS (P ≤ 0.10). Lint yield was increased by 433 kg/ha and 447 kg/ha or 35% and 38% with the combination of a nematicide ST + 28-0-0-5 + Vydate^® C-LV + Max-In^® at PHS and FB when compared to the lowest yielding treatments, (NH₄)₂SO₄ at PHS and 28-0-0-5 at PHS.

Mean profits for fertilizer applications of (NH₄)₂SO₄ and 28-0-0-5 at PHS with no nematicides averaged $548.53 (Table 7). A nematicide ST with an additional fertilizer application at PHS increased mean profit by $54/ha from an additional fertilizer application alone. A nematicide ST with an additional application of fertilizer at PHS and FB increased profit by $103/ha from an additional fertilizer application alone. Fertilizer combinations with Vydate^® C-LV applied at PHS had an increased profit of $93/ha from an additional fertilizer application alone. Fertilizer and nematicide combinations that included Vydate^® C-LV at PHS and FB had an increased profit of $131/ha. All combinations were compared to the mean profit of treatments of an additional fertilizer with no nematicides to determine increased profit per hectare. The combination of a nematicide ST + 28-0-0-5 + Vydate^® C-LV + Max-In^® Sulfur at PHS and FB had the greatest overall mean profit of $784.00 which was significantly greater than the mean profit of treatments of (NH₄)₂SO₄ or 28-0-0-5 at PHS.

A nematicide ST + 28-0-0-5 + Vydate^® C-LV + Max-In^® Sulfur at PHS was the most effective combination in reducing R. reniformis eggs per gram of root sampled at PHS + FB. The combination of a nematicide ST + (NH₄)₂SO₄ + Vydate^® C-LV + Max-In^® Sulfur at PHS and FB was the most effective treatment in increasing seed cotton yield. The combination of a nematicide ST + (NH₄)₂SO₄ + Vydate^® C-LV + Max-In^® Sulfur at PHS and FB increased seed cotton yields by 50% when compared to the combination of a nematicide ST + (NH₄)₂SO₄ at PHS. Research supports the use of Vydate^® C-LV on cotton during PHS and sequential treatments reducing R. reniformis population levels (Hammes et al., 1999). Applications of nematicides when analyzed individually increased lint yields by 8% with a nematicide ST, 19% with a single Vydate^® C-LV application and 29% with a double application of Vydate^® C-LV.

The combination of a nematicide ST + 28-0-0-5 + Vydate^® C-LV + Max-In^® Sulfur at PHS and FB was the most effective treatment in increasing root fresh weight sampled at PHS + FB. The application of 28-0-0-5 at PHS had the lowest M. incognita eggs per gram of root sampled at PHS + FB, followed by a nematicide ST + (NH₄)₂SO₄ + Vydate^® + Max-In^® Sulfur at PHS and FB. Studies found that plants which had increased nitrogen available had lower nematode population levels (Miller and Wihrheim, 1966; Rodriguez-Kabana, 1986). Seed cotton yield was greatest with the combination of a nematicide ST + 28-0-0-5 + Vydate^® C-LV + Max-In^® Sulfur at PHS and FB. The combination of a nematicide ST + 28-0-0-5 + Vydate^® C-LV + Max-In^® Sulfur at PHS and FB also supported the largest root fresh weight among treatments. Similarly, a study conducted by Bednarz et al. (2000) found that cotton yields were greatest using 28-0-0-5 in a loamy sand soil type. Analyzing nematicide applications individually indicated overall seed cotton yield was increased by 7% with a nematicide ST and 6% with two foliar applications of Vydate^® C-LV.

At TVREC, the combination of a nematicide ST + (NH₄)₂SO₄ + Vydate^® C-LV + Max-In^® Sulfur at PHS was the most effective at increasing root fresh weight from sample data combined at PHS + FB. A nematicide ST + (NH₄)₂SO₄ + Vydate^® C-LV + Max-In^® Sulfur at PHS and FB had the lowest R. reniformis eggs per gram of root, closely followed by the combination of a nematicide ST + (NH₄)₂SO₄ + Vydate^® C-LV at PHS. These findings are supported by Badra and Elgindi (1979), where foliar applications of Vydate^® C-LV significantly reduced R. reniformis population levels. Similarly, the use of a nematicide ST COPeO^™ Prime (Fluopyram) inhibited R. reniformis from increasing on cotton root systems (Faske and Hurd, 2015). The combination that supported the largest lint yield was a nematicide ST + (NH₄)₂SO₄ + Vydate^® C-LV at PHS. Nematicide applications were analyzed individually in response to lint yield. An application of a nematicide ST increased overall yield by 11%. A trial conducted by Groover et al. (2020) supports this conclusion and found that a nematicide ST (COPeO^™ Prime) increased lint yield by 14%. A single foliar application of Vydate^® C-LV increased overall lint yield by 13%, while two applications of Vydate^® C-LV increased overall lint yield by 2%. Increases in yield with single or multiple Vydate^® C-LV applications on cotton were also found in a study conducted by Hammes et al. (1999).

The combination with the largest mean profit in dollars/ha was a nematicide ST + (NH₄)₂SO₄ + Vydate^® C-LV at PHS. This combination also supported the largest lint yield. Based on the lower and upper profits, there is a 90% chance when using this fertilizer and nematicide combination in a R. reniformis infested field, the mean profit will fall between $991.15/ha and $1360.59/ha. The increased mean profit of this combination could be contingent on the moderate fertilizer and nematicide input costs. The overall input cost of this combination was $55.96/ha which was $46.17 cheaper than the most expensive (NH₄)₂SO₄ based combination. This evaluation is supported by Zimet et al. (2002) who reported financial returns in R. reniformis fields with lower chemical rates equating to reduced chemical input costs. Similarly, a study conducted by Koenning et al. (2007) saw an increase in yield with the use of nematicides in R. reniformis infested fields but when conducting an economic analysis determined the profit from the additional yield did not cover the increased chemical costs.

At PBU, the combination that supported the largest root fresh weight sampled at PHS + FB was a nematicide ST + (NH₄)₂SO₄ at PHS. Meloidogyne incognita eggs per gram of root combined were lowest in the treatment combination of a nematicide ST + (NH₄)₂SO₄ + Vydate^® C-LV + Max-In^® Sulfur at PHS. This combination decreased M. incognita eggs per gram of root by 15% when compared to the treatment of (NH₄)₂SO₄ at PHS, with no nematicide. A study conducted by Faske and Hurd (2015) also found that a nematicide ST reduced M. incognita population levels when compared to treatments with only a base fungicide. The combination of a nematicide ST + 28-0-0-5 + Vydate^® C-LV + Max-In^® Sulfur at PHS and FB was the most effective at increasing lint yield. This combination also supported the largest seed cotton yield in the M. incognita microplot trials in 2019 and 2020. The application of a nematicide ST (COPeO^™ Prime) provided the greatest yield protection against M. incognita in trials conducted by Faske et al. (2021). This finding contrasts with Anderson et al. (2012) who found applications of Vydate^® C-LV did not have an impact on cotton lint yield. Nematicide applications were analyzed individually to evaluate lint yield responses. The application of a nematicide ST increased overall lint by 20%, a single Vydate^® C-LV application increased overall lint by 11%, and sequential Vydate^® C-LV applications increased overall lint by 5%.

The largest mean profit was obtained with the combination of a nematicide ST + 28-0-0-5 + Vydate^® C-LV + Max-In^® Sulfur at PHS and FB. This combination also supported the largest lint yields, correlating increased lint yields with additional profit. Established on lower and upper profit, there is a 90% chance that if this combination is used in a M. incognita populated field, profit will fall between $692.42/ha and $875.58/ha. The input cost of this combination was $83.67/ha, making it one of the most expensive treatment combinations. A study conducted by Zimet et al. (2004) found that nematicide treatments in M. incognita fields with fewer input costs had reduced lint yields resulting in partial net returns.

There was a decrease in R. reniformis eggs per gram of root and an increase in seed cotton and lint yield with the combination of a nematicide and fertilizer at either PHS or PHS + FB. Greater profit per hectare was obtained in R. reniformis infested soil with combinations that included at least one nematicide in combination with an additional fertilizer at either PHS or PHS + FB. The three highest profiting treatments from field trials had an additional application of (NH₄)₂SO₄ at PHS or PHS + FB. The same trend was seen with M. incognita; eggs per gram of root were reduced and seed cotton and lint yield increased in combinations with a nematicide ST + an additional fertilizer combination at PHS or PHS + FB. Profit increased in combinations with a nematicide in combination with an additional application of fertilizer at PHS or PHS + FB. The three highest profiting treatments from field trials had an additional application of 28-0-0-5 at PHS or PHS + FB.

Our finding suggests that utilizing a nematicide with a fertilizer will increase yield and profit for growers with R. reniformis or M. incognita infested cotton fields. In R. reniformis field trials, 10 out of 10 fertilizer and nematicide combinations increased lint yields and 9 out of 10 combinations increased profits when compared to treatments with no nematicides. The three highest yielding combinations, a nematicide ST + (NH₄)₂SO₄ at PHS and FB, a nematicide ST + (NH₄)₂SO₄ + Vydate^® C-LV at PHS and a nematicide ST + (NH₄)₂SO₄ + Vydate^® C-LV + Max-In^® Sulfur at PHS also had the largest profits. Combinations with applications of (NH₄)₂SO₄ were the most economical in R. reniformis infested fields. These field trials suggested that the application of a nematicide ST increased lint yield by 8% and a single application of Vydate^® C-LV increased lint yield by 19%. The addition of a nematicide ST increased profit by $32/ha and an additional application of Vydate^® C-LV at PHS increased profit by $137/ha when compared to an additional fertilizer alone. In M. incognita field trials, 10 out of 10 fertilizer nematicide combinations increased lint yields and profit when compared to treatments with no nematicides. The three highest yielding combinations were a nematicide ST + Vydate^® C-LV + Max-In^® Sulfur at PHS and FB, a nematicide ST + 28-0-0-5 at PHS and FB, and a nematicide ST + 28-0-0-5 + Vydate^® C-LV at PHS also had the largest profits. Combinations with applications of 28-0-0-5 were the most economical in M. incognita infested fields. These trials suggested that the application of a nematicide ST increased lint yield by 20%, a single application of Vydate^® C-LV increased lint yield by 11% and sequential Vydate^® C-LV applications increased overall lint by 5%. The addition of a nematicide ST increased profit by $54/ha when compared to an additional fertilizer alone. The addition of a single application of Vydate^® C-LV increased profit by $93/ha and sequential applications of Vydate^® C-LV at PHS and FB increased profit by $131/ha. In conclusion, our hypothesis that combining nematicides with fertilizers at PHS and FB plant growth stages can provide a management system for R. reniformis or M. incognita infested cotton fields with potential for economic gains.