Exogenous application of bio‑stimulants and growth retardants improve nutrient absorption and fiber quality in upland cotton

Background Natural and synthetic plant growth regulators are essential for plant health, likewise these regulators also play a role in increasing organic production productivity and improving quality and yield stability. In the present study, we have evaluated the effects of foliar applied plant growth regulators, i


Introduction
Gossypium hirsutum L. is a cash crop cultivated for fiber and oil seed in many countries/territories, playing a vital role in the socio-economic activities.The textile industry relies on cotton plants to yield high-quality fiber (Dehghanisanij et al., 2022).Fiber quality is a range of assessable fiber properties that enhance the spinning performance during textile processing.Among these properties, fiber strength is the key fiber quality index and is measured as one of the imperative features defining yarn quality, particularly yarn strength (Sief et al., 2021).Fiber strength is primarily constituted during the thickening of the secondary fiber wall and is associated with the cellulose deposition (Mehran et al., 2023).The physiological effects of growth stimulants are diverse on various crops.They can directly affect the physiology and metabolism of the plant (Mosa et al., 2023) as well as growth and development from seed germination to maturity in various ways (Gupta et al., 2023).
Application of growth regulators enhances the plant metabolism efficiency, resulting in improved yield and product quality.These regulators also enhance the resistance to biotic and abiotic stresses, facilitate the absorption, transfer, and use of nutrients, improve the water consumption efficiency, and refine the physio-chemical properties of soil while promoting the growth of soil microorganisms (Irani et al., 2021).An exogenous application of different growth regulators boosted the phenolic, sugar, and total protein contents as well as chlorophyll biosynthesis (Arif et al., 2022a;Mashamaite et al., 2022).Furthermore, the foliar spray of various organic osmolytes improves the source-sink relationship which results in higher lint yield and oil seeds.Amongst various organic growth enhancers, leaves of moringa are rich sources of plant growth promoters (ascorbates and zeatin), minerals (K + and Ca 2+ ), and pigments (phenols and carotenoid), proved an ideal growth promoter (Shafiq et al., 2021).Foliar spray of moringa leaf extract (MLE) significantly increased the chemical composition, enzyme activities, growth, productivity, and fiber quality attributes (Ibrahim et al., 2021).
The growth pattern of the cotton crop is unique because it is a perennial with an indeterminate growth nature (Murtza et al., 2022).Excessive vegetative growth, poor bud development, shedding of squares and flowers, and imbalance between source and sink are reasons for the indeterminate growth behavior of cotton (Hussain et al., 2021).Numerous tactics have been tried to break the yield plateau, among which the use of plant growth retardants, especially mepiquat chloride (MC) has received greater attention among cotton researchers.Mepiquat chloride prevents cell expansion but not cell division.Its application channels the carbohydrates into reproductive organs and therefore diminishs vegetative growth (Abbas et al., 2022).This allows the plants to transfer the desired amount of photo-synthetase from the vegetative organs to the reproductive ones, enhancing the cotton yield and quality parameters (Hussain et al., 2021).
Considering the role and importance of plant growth stimulants in plant nutrition and health, especially in the production of healthy and organic products, an understanding of the characteristics and mechanisms of their effect on the plant, as well as the challenges to their application in field conditions, is necessary to optimize their use for cotton production.The worldwide use of growthpromoting substances has increased significantly in recent years, but there is still not enough documented information about these compounds, especially on the influence of moringa leaf extract and mepiquat chloride on cotton fiber quality parameters.This study was conducted to evaluate the role of MLE as a natural growth promoter and MC as a vegetative growth retardant for improving the seed cotton yield, fiber quality characteristics, and oil contents of both Bt and non-Bt cotton cultivars.The research was based on the hypothesis that combined application of MLE and MC might be more effective in obtaining optimum seed cotton yield and fiber quality attributes.

Results
Data regarding the relative growth rate was recorded at various stages of both cotton cultivars.Foliar spray of MLE and MC significantly improved the relative growth rate during 101-130 and 131-160 days after sowing (Fig. 1).A foliar spray of only MLE and combined application of MLE + MC on CIM 598 at 45 days after blooming produced a significantly higher relative growth rate than the control treatment with distilled water during 101-130 and 131-160 days after sowing.The net assimilation rate followed an increasing trend in the primary stages of cotton growth and then reduced subsequently (Fig. 2).Data regarding the net assimilation rate was recorded at various stages of both cotton cultivars.Foliar spray of MLE and MC significantly improved the net assimilation rate during 101-130 and 131-160 days after sowing.A foliar spray of MLE on CIM 598 at 45 days after blooming produced a significantly higher net assimilation rate than the control treatment with distilled water during 101-130 and 131-160 days after sowing.
The number of bolls per plant was recorded at different stages, and foliar spray with the growth regulators after blooming significantly affected the number of bolls per plant and ultimately the final seed cotton yield during both years (Table 1).The combined application of MLE and MC at 90 days after blooming produced a significantly greater number of bolls per plant which ultimately produced the highest seed cotton yield per hectare.
The growth regulators significantly affected the nutrient accumulation and fiber quality characteristics of the varieties during both growing seasons.Spraying on CIM 598 with MLE at 90 days after blooming significantly increased the nitrogen percentage in leaves in both growing seasons (Table 2).The combined spray of MLE and MC at 90 days after blooming increased the uptake of phosphorus and potassium  2).
The MLE + MC treatment at 90 days after blooming significantly increased the locule nitrogen percentage in the Bt cotton during both growing seasons (Table 3), while the conventional cotton (CIM 573) sprayed with distilled water showed the lowest locule N, P, and K uptake.Foliar spray with MLE at 90 days after blooming showed a significantly higher percentage of phosphorus in the locule of CIM 598 (Table 3).Treatment of CIM 573 Foliar spray on CIM 598 with MLE at 90 days after blooming produced the highest ginning percentage.The lowest ginning percentage was observed in CIM 573 control plots in 2016 (Table 4).However, the treatment with MLE + MC showed a non-significant impact on the ginning percentage in 2017.
Foliar spray on CIM 573 with MLE + MC at 90 and 45 days after blooming produced significantly higher micronaire values than other treatments (Table 4), while spraying on CIM 598 with distilled water and MC at the start of bloom produced the lowest micronaire values.Fiber strength is a key parameter that determines the yarn's spinnability.The combined application of MLE + MC and MLE on CIM 573 at 90 and 45 days after blooming produced significantly higher fiber strength while distilled water produced the lowest fiber strength in CIM 598 (Table 4).Foliar spray on CIM 573 with MLE at 45 and 90 days after blooming produced the highest fiber uniformity ratio in 2016 and 2017, respectively, whereas the lowest ratios were observed with MC and distilled water at 45 and 90 days after blooming in CIM 598 in 2016 and 2017, respectively (Table 4).Spraying on CIM 573 with MLE + MC at 90 days after blooming produced the longest fiber while the shortest was obtained from CIM 598 treated with distilled water at 0 days after blooming (Table 4).The MLE applied at 90 and 45 days after blooming produced the highest seed oil content for CIM 598 while distilled water applied to the conventional cultivar, CIM 573 at the beginning and 90 days after blooming produced the lowest seed oil content (Table 4).

Discussion
The use of bio-stimulants in agriculture altered the constituents of crop plants and enhanced their growth, yield, and quality parameters (Hu et al., 2023).In the current study, foliar application of MLE in combination with MC improved the relative growth rate and net assimilation rate and increased the number of bolls per plant which finally improved the seed cotton yield.This effect may be attributed to the foliar spray of MLE alters the endogenous cytokinin levels; the enhanced contents stimulate cell division resulting in significantly higher growth and yield (Khan et al., 2021;Shafiq et al., 2021).Moreover, the foliar spray of MLE prevents the abscission of squares and bolls and stimulates the mobilization and accumulation of photosynthates in newly formed bolls (Arif et al., 2019).Foliar spray with MC, being anti-gibberellic acid when absorbed by the plants, leads to a decrease in cell elongation, thereby inhibiting vegetative growth but improving the retention of early buds and bolls with higher productivity, particularly during the first pick period.This may be associated with higher availability of plant nutrients to reproductive parts through favorable photo-assimilate partitioning from the vegetative to the reproductive organs (Arif et al., 2022b).Moreover, foliar application of MC in combination with MLE maintains the balance between the vegetative organs and the reproductive ones and hence the overall productivity.They also play an important role in maintaining internal hormonal balance and effective source-sink relationship, which improves yield contributing parameters depending on the enhanced photosynthetic activity (Hussain et al., 2021).
Exogenously applied MLE significantly improved the nutrient uptake in cotton leaves and locules.Similarly, Yuniati et al. (2022) observed higher uptake and accumulation of various nutrient elements such as nitrogen, phosphorus, potassium, calcium, iron, and magnesium in different parts of numerous crop plants with the application of MLE, which was considered to enhance the absorption of mineral nutrients and translocation throughout the plant by improving the membrane permeability of roots for electrolytes, averting nutrient fixation, and enhancing their mobility in soil.Foliar spray of MLE remarkably improved the N, P, and K contents in a snap bean pod (Elzaawely et al., 2016) and brinjal fruit (Hoque et al., 2020).
The variation in fiber quality attributes between the two cotton cultivars is attributed to the use of different types of genetic material and efficient utilization of inputs and natural resources (Arif et al., 2019).Previous studies indicated similar reports and they concluded that conventional cultivars have produced better fiber quality attributes than Bt cultivars, possibly due to the superior genetic potential and optimum agroecological aspects (Yasmeen et al., 2018).Foliar spray of MLE and MC had a realized role in fiber development and hence the foliar spray of this combination improved the fiber properties of cotton and produced longer cellulose molecules and better cross-linkages between the fibers that enhance their quality traits (Hussain et al., 2021).
It might be due to the fact that the combined application of MLE + MC plays an important role in enzymatic activities, cell division, hormonal balance, photosynthesis, and translocation of photosynthates from leaves to bolls thus improving the fiber quality attributes (Arif et al., 2022b).

Conclusions
It is concluded from this study that the foliar application of MLE alone, and in combination with MC at 90 days after blooming can improve nutrient absorption and thereby improve plant growth, yield, and quality attributes of upland cotton.5.

Crop husbandry
The seedbed was prepared with a tractor-mounted ridge, and 6 m × 4.5 m beds were properly shaped.Seeds of CIM 573 and CIM 598 were dibbled manually, keeping a plant-to-plant distance of 30 cm and a row-to-row of 75 cm, resulting in an approximate plant population of 43 000 per hectare.The furrows were irrigated followed by dibbling to achieve the highest germination percentage.Following irrigations were applied with 6-10 days intervals depending on crop requirement, until the first week of September.Nitrogen fertilizer at 140 and 115 kg•ha −1 for CIM 598 and CIM 573 was applied in three identical splits at the time of seedbed preparation, the start of flowering, and the peak flowering phase.Phosphorus and potassium fertilizers were broadcast at 55 and 60 kg•ha −1 during seedbed preparation.

Data collection
Ten plants were randomly selected from each experimental unit and labeled to record the number of bolls per plant in each experimental plot.Manual harvesting of seed cotton was performed twice in the middle two rows.Cotton picking was first carried out at 60% boll opening, while the last picking was performed on 24th and 27th November of 2016 and 2017, respectively.
Relative growth rate (RGR) and net assimilation rate were computed using the standard procedures of Radford (1967).Leaf samples (the 6th and 7th from the top) were collected from the central part of ten randomly selected plants at 210 days after sowing.Locule samples (the 4th and 5th from the top) were collected from ten randomly selected plants at 225 days after sowing.Both the leaf and locule samples were washed with distilled water, air-dried, and then oven-dried at 72 °C till constant weight in the oven (SLN 32, POLEKO-APARA TUR A).Dry ashing was used to determine the nitrogen contents in cotton leaves and locules by the micro Kjeldahl method and phosphorus content by the Vanadomolybdophosphoric yellow colour method (Shimadzu UV-Vis spectrophotometer, model: UV-1280, wavelength range: 190 to 1 100 nm, Japan) on spectrophotometer after digestion in triacid (Jackson, 1967).Leaves and locules tissue were digested in a diacid mixture (HNO 3 and HClO 4 in 3:1, v/v), and the potassium content of aqueous extracts was determined by a flame photometer (FP910-4) (Miller, 1997).Ginning out turn was calculated using the following equation.
Seed cotton acquired from the net plot area of each treatment was uniformly mixed and a sample of 300 g was separated from each treatment, and then ginned to determine the fiber quality parameters.Lint samples were used to evaluate the fiber quality parameters using HVI-900, a computerized High-Volume Instrument that provides a comprehensive profile of raw fiber according to the International Trading Standards (Sundaram et al., 2002).
Oil extraction was carried out using n-hexane (1 000 mL) according to the method proposed by Atolani et al. (2016).A sample of cottonseed of 200 g was used for oil extraction using Soxhlet extractor at 55 °C for 7 h.The oil was obtained using a rotary evaporator at 40 °C, according to standard methods described by Zubair et al. (2018).

Statistical analysis
The data of each variable were subjected to analysis of variance (ANOVA) using the statistical package of MSTAT-C (Fareed et al., 1991).Treatment mean differences of each parameter were conducted using the least significant difference (LSD) (Steel et al., 1997).

Fig. 1
Fig. 1 Effect of foliar spraying with MLE (moringa leaf extract) and MC (mepiquat chloride) on the relative growth rate of two cotton cultivars during 2016 and 2017.DAB: days after blooming; DAS: days after sowing

Fig. 2
Fig. 2 Effect of foliar spraying with MLE (moringa leaf extract) and MC (mepiquat chloride) on net assimilation rate of two cotton cultivars during 2016 and 2017.DAB: days after blooming; DAS: days after sowing

Table 1
Effect of foliar spray with MLE and MC on number of bolls per plant and seed cotton yield of two cotton cultivars Values are means of three replications with ten plants per replication.Values with the same letter in a column do not differ significantly at P ≤ 0.05.MLE Moringa leaf extract, MC Mepiquat chloride, DAB days after blooming

Table 2
Effect of foliar spray with MLE and MC on nutrient uptake in leaves of two cotton cultivars Values are means of three replications with ten plants per replication.Values with the same letter in a column do not differ significantly at P ≤ 0.05.MLE Moringa leaf extract, MC Mepiquat chloride, DAB days after blooming

Table 3
Effect of foliar spray with MLE and MC on nutrient uptake in locules of two cotton cultivars Values are means of three replications with ten plants per replication.Values with the same letter in a column do not differ significantly at P ≤ 0.05.MLE Moringa leaf extract, MC Mepiquat chloride, DAB days after blooming

Table 4
Effect of foliar spray with MLE and MC on the ginning percentage, fiber quality parameters and oil contents of two cotton cultivars Values are means of three replications with ten plants per replication.Values with the same letter in a column do not differ significantly at P ≤ 0.05.MLE Moringa leaf extract, MC Mepiquat chloride, DAB days after

Table 5
Physi-chemical properties of the experimental soils pH Potential of hydrogen, EC Electrical conductivity, BD Bulk density, PWP Permanent wilting point, FC Field capacity, OC Organic carbon