Minimizing the Adverse Impact of Drought on Corn by Applying Foliar Potassium Humate Combined with Chitosan

Previous studies have demonstrated the impact of potassium humate (KH) and chitosan (CH) on ameliorating drought effects, but their combined applications in promoting these benefits are still unfound. It was found that separate foliar applications of KH or combined foliar applications of KH + CH had a significant impact on the most examined traits. However, compared to the control, adopting limited irrigation and applying combined applications thereof have significantly increased iron, zinc, manganese, oil, protein, yield, and water productivity. In addition, this combination decreased proline, and the maximum reduction was observed for the combined application with adopting full irrigation. In arid regions, the researcher recommends treating stressed plants with combined foliar applications of KH + CH, which could help plants overcome the negative effects of drought and attain the highest yield and water productivity.

Introduction

Agricultural production is impacted by a range of environmental factors, including drought. Water stress is considered one of the main obstacles to global agricultural production, as it inhibits leaf area, and vegetative and reproductive growth of crops. To define suitable water regimes for various crops in the agricultural sector, several technologies and methodologies are well developed. However, finding the optimum combination of water management with various agricultural inputs such as foliar applications needs more studies.

Insufficient water or increased transpiration may cause drought stress in plants, impacting the generation and accumulation of secondary metabolites. In this respect, Chitosan is a cationic polysaccharide with units of glucosamine Chitosan containing also N-acetylglucosamine. Through alkaline N-deacetylation of chitin, chitosan has been produced. Chitosan offers many advantages to agriculture, including high biodegradability, biocompatibility, polyelectrolyte characteristics, lowest toxicity, chemical versatility, gel- and film-forming ability, and high adsorption capacity. Chitosan also allows plants to tolerate a variety of abiotic stress, including stress water; its use can be limited by its low water solubility, and Chitosan derivatives improved the photosynthetic parameters of drought-sensitive corn, causing tolerance to this stress. Moreover, Chitosan has a unique bioactive property that can prevent serious injury to plants under stressful conditions by triggering the mechanisms of plant defense. Previous research has shown the use of CH as an antiperspirant. In this respect, divided antiperspirants into three main groups: (A) physical antiperspirants forming a thin layer on the stomata to prevent water evaporation; (B) reflective antiperspirants that improve the light reflectivity of leaf surfaces, reducing leaf temperature; (C) physiological antiperspirants containing certain compounds influencing the guard cells around the stomata and inducing plants to close it.

In addition, it is known that in a dry climate, the soil contents of organic matter decrease. Therefore, adding organic material to these soils not only raises organic levels but also provides available absorbable nutrients to plants. Furthermore, almost 35% of the agricultural lands suffer from salinity. Thus, applying KH to sandy soil represents a promising natural resource and a better alternative that could be used effectively to increase organic matter levels and improve soil nutrition status, crop growth, and productivity. It is a soluble organic material in water and it is used as an organic fertilizer. It can reduce abiotic stress by enhancing the activities of various antioxidant enzymes such as superoxide dismutase, catalase, and peroxidase. Moreover, KH increased root formation and plant growth parameters as they are applied to major vegetables and crops. Furthermore, Mohammed et al. referred to the possibility of replacing some of the potassium soil applications with foliar applications of KH without affecting tomato yield and growth, as well as the nutritional status of tomato plants grown in newly reclaimed sandy soil in Egypt. In the same context, Ataya et al. and Hegab et al. mentioned a similar result of the importance of applied foliar applications of KH on date palms and oat grown in sandy soil in Egypt. Also, in corn plants, humic compounds enhanced root growth, carbohydrates, nitrogen responses, and photosynthesis; it can lead to more improvements in soil physical properties, biological activities, and acceleration of nutrient transfer to plants.

Numerous mechanisms are directly affected by KH, including photosynthesis, cellular respiration, membrane permeability, protein synthesis, enzymatic activities, improved water, and nutrients absorption in tissues by increasing the membrane stability index and relative water content while electrical leakage decreases, which stimulates crop performance.

Corn (Zea mays L.) is a member of the Poaceae family. It is a major annual cereal crop in Egypt and other Mediterranean countries, which has the third largest crop after wheat and rice with a total area under cultivation. Moreover, according to Abaza et al. and Abaza, the average daily evaporation when corn planting in February in the study area of the experiment ranges between 10.45 and 0.89 mm per day, and the crop lasts for an average growth period, reaching 93 days, with a total gross water requirement up to 10,890 m^-3 ha^-1.

In the irrigation water rationalization programs, searching for an effective and easy way to ameliorate the negative impacts of drought on crop growth still attracts much more attention. As a result of the rapid drought impact on the aerial parts, foliar KH addition was chosen to unify the comparison methodology with CH. Both materials share the ability to supply plants with nutrients as well as both can reduce transpiration but in different ways. Hence, the prominent question that this research tries to answer is: When plants are exposed to drought, besides improving nutrition, is it preferable to choose a substance that reduces transpiration by formatting a thin protective layer or one that improves water relation? Therefore, this research was conducted to study the impact of drought on corn growth traits, yield, nutrient content, water productivity, and the influences of KH and CH in improving it.

Materials and Methods

2.1Site Description
A field experiment was conducted during the 2021 and 2022 growing seasons at the experimental farm located in the Abu Simbel City of Southern Egypt. The irrigation water source was a deep well dug in the studied area.

2.2Experimental Design and Treatments
An experiment was conducted in a split-plot design with three replicates, and each plot was 24 m2 (6 m long × 4 m wide). Two irrigation water levels, i.e., 100 and 70% of corn gross water requirements, were allocated in the main plots. The plants were irrigated by drip irrigation and each irrigation plot was equipped with a pressure gauge valve to maintain the operating pressure at 1 bar, and a flow emitter with a discharge of 4.0 L h^-1 which was used to measure the quantity of targeted irrigation water at each irrigation level. Also, the experimental units were bounded with a buffer zone (3 m width) to avoid interactions. While the subplots were devoted to the four foliar applications, namely 0 (spray with pure water-control), CH 500 mg l^-1, KH 3000 mg l^-1, and CH 500 mg l^-1 alongside KH 3000 mg l^-1.

2.2.1The Preparation of CH
Purified CH, its solubility reaches about 97% in 1.0% acetic acid under continuous stirring, and the pH was adjusted to 5.6 using 1N NaOH. Triton B as a wetting agent was added at a rate of 0.05% to all spraying solutions before the application.

Chitosan was sprayed as a foliar application four times every 15 days, initiated after 4 weeks of emergence. Also, foliar KH was purchased from Zain Fert Co. (75% humic acid+4% fulvic acid+2% iron (Fe)+10% K2O). It was applied twice at 45 and 60 days after the sowing date. Crops were planted as recommended by the region for fertilization and other field practices. On the 10 of February 2021 and 15 of February 2022, respectively, two grains of corn (Giza 352-triple hybrid) were sown directly in hills. It thinned to one plant per hill after 14 days of the sowing date. Calcium superphosphate (15.5% P₂O₅) was applied after 2 weeks of sowing at a rate of 475 Kg ha^-1, and potassium sulfate (48% K₂O) was applied in three equal portions at a rate of 235 Kg ha^-1 after 60, 75, and 90 days of cultivation. Meanwhile, ammonium nitrate (33.5% N) at a rate of 950 Kg ha^-1 was applied in equal doses, beginning with a 50 kg dose after 2 weeks of planting, along with repeating the same doses every 3-4 days up to the flowering stage. On the other hand, the harvest was on the 11 of June 2021 and 15 of June 2022, respectively.

2.3Irrigation Water Requirement
The gross irrigation water requirement was calculated by using the Toshka agrometeorological station. The obtained data were entered, using CROPWAT, version 8.0, which is a software program for planning and man- aging irrigation, to calculate reference evapotranspiration, using the Penman-Monteith equation. Consequently, crop evapotranspiration (ETc) was calculated according to Waller and Yitayew (2016) as follows:

ETc = (ETo × kc)

where:
ETc crop evapotranspiration (mm/day)
ETo reference evapotranspiration (mm/day)
kc single crop coefficient, dimensionless

The gross water requirement (GWR₁₀₀) was calculated according to AL-Omran et al. (2019):

The amount of GWR₇₀ treatment was proportionally obtained from the GWR₁₀₀. And the corn plants were irrigated with limited irrigation from the development stage onwards (after 35 days of sowing). The ETo, Etc, and GWR during the seasons of 2021 and 2022 are presented in Table 1.

		Growth stages				Total
		Seeding	Vegatative	Flowering	Maturation
ETO (mm)		149.1	466.0	335.0	98.0	1048.1
ETC (mm)		89.5	419.4	426.0	88.2	1023.1
Irrigation system efficiency %		0.85
GWR (mm)		105.3	493.4	501.2	103.8	1203.7
Leaching reaquirements		10.5	49.3	50.1	10.4	120.4
The total GWR (m3 ha-1)	GWR 100%	1158.0	5427.0	5513.0	1142.0	13,240.0
	GWR 70%	1158.0	3799	3859.1	799.0	9615

2.4Proline Content in Corn Leaves
At 65 days from the sowing date, proline was estimated, using the 4th corn leaf from the tip of the plant as described by (Sahin 2019), three fresh leaf samples were collected between 11:00 am and 2:00 pm and the leaf was immediately submerged into a cooling extraction solution that contained 3% aqueous sulfosalicylic acid solution and was stored in the refrigerator until the extraction process. Briefly, the extraction was done by taking (0.5 g) of the crushed frozen leaf, along with the same weight of dried corn leaf and extracting in10 ml of sulfuric acid (3%). Then the extraction solution was filtered, and for every 10 ml of the resulting essences, 2 ml of ninhydrin and 2 ml glacial acetic acid were added. The samples were then put into a Bonmary device for 1 h at 100℃. After that, to each sample, 4ml of toluene was added and then blended for 15-20s into a shaking machine. Finally, the photo absorbance capacity by toluene was estimated by a spectrophotometer at 520 nm. And to determine the proline content (as μg g-1 FW) in each sample, the following formula was used:

2.5 Leaf Relative Water Content (RWC)
It was measured at 65 days from the sowing date according to (Afzal 2017):

2.6 The Total Carbohydrates
After harvesting, the total carbohydrates were determined as described by El-Katony et al. (2019). The sample, 0.5 g of the powdered grain tissue was extracted overnight with 5 ml of 80% ethanol and then centrifuged for 10 min and the extract was replicated with fresh ethanol. The extracts were mixed and dried under the vacuum, and in 1 ml distilled water, the residue was redissolved, which was used for the determination of total soluble carbohydrates. The aliquots of the concentrated extract of soluble carbohydrates have been thoroughly blended with anthrone reagent (8.6 mM anthrone in 80% v/v H2SO4) and warmed for 10 min in a water bath at 80℃ and then cooled for 30 min on the ice. The absorbance was measured at 623nm, and the total soluble carbohydrate concentration was computed from a glucose calibration curve.

Results

3.1Effect of Irrigation Levels, KH and CH on Proline, RWC, and Total Carbohydrates
Based on the results, by comparing the control in (Fig. 1a), the proline was increased by adopting GWR70% of irrigation level compared to GWR100%. Furthermore, the application of KH and CH in the current study decreased the proline content compared to the control. By adopting the GWR100% irrigation level, the proline content decreased from 38.7 (μg g^-1 FW) for the control to 32.9 and 31.2 (μg g^-1 FW) for KH3000 and CH500 + KH3000, respectively. Adopting the GWR70% irrigation level, the proline content decreased from 49.7(μg g^-1 FW) for the control to 45.3 and 42.8 (μg g^-1 FW) for the same previous treatment, respectively. The maximum reduction of proline content was observed for the combined application of KH and CH with adopting GWR100% of irrigation level.

Compared to the control, RWC increased when applying separated or combined foliar applications of KH and CH and adopting GWR70% (Fig. 1b). It was found that the combined foliar applications of KH + CH significantly increased RWC compared to the control when adopting GWR100%. While there was a non-significant effect by applying separated foliar applications of CH and adopting GWR70% of irrigation level.

As can be seen in (Fig. 1c), adopting GWR70% of irrigation level decreased the carbohydrate content compared to GWR100%. In addition, separate or combined foliar applications of KH and CH surpassed the control for carbohydrates in the corn grains. Compared to the control, the maximum increase of carbohydrates was obtained for the separated foliar applications of KH and the combined application of KH + CH when adopting GWR100%. Likewise, adopting GWR70% and applying combined foliar applications of KH + CH significantly increased carbohydrate content com- pared to the control.

3.2Effect of Irrigation Levels, KH and CH on Macronutrients (N, P, and K) and Micronutrients (Fe, Zn, and Mn)
Concerning the interaction, there were insignificant varia- tions among the studied treatments and control for N when adopting GWR70%, while the opposite was right when adopting GWR100% (Fig. 2a). However, there were insignificant variations among the foliar applications when adopting GWR100% on N. On the other side, there was a significant difference from the control for P, K Fe, Zn, and Mn.

The results indicated that GWR70% led to lower P con- tent, although the foliar applications of KH and CH in each irrigation level improved P contents (Fig. 2b). The highest P content was obtained with GWR100% irrigation level and applying foliar applications of KH and CH. The following highest P content was seen with the same irrigation level by using a separated foliar application of KH.

The content of K was increased by adopting GWR100% compared to GWR70% (Fig. 2c). Although the separate appli- cations of KH and the combined foliar applications of KH + CH improved K content when adopting GWR100%. But when adopting GWR70%, K content improved by applying the combined foliar applications of KH + CH. The full irrigation (GWR100%) was pronounced with the combined foliar applications of KH and CH for attaining the highest K content in corn grains. However, GWR70% × combined application of KH + CH significantly equaled GWR100% × combined application of KH + CH.

On the other hand, the superiority of full irrigation (GWR100%) is still pronounced compared to GWR70% causing increases in Fe content, unlike for Zn and Mn. Also, compared to the control, Fe content improved when applying separated or combined foliar applications of KH and CH and adopting full irrigation GWR100% or limited irrigation level GWR70% (Fig. 1d).

The Zn content increased from 37.5 (μg kg^-1) for the control to 53.6 (μg kg^-1) in GWR70% treatment and applying CH500 + KH3000 (Fig. 2e). Also, adopting GWR100% of irrigation level and applying individual applications of KH or CH were significantly equaled. Likewise, adopting GWR70% and applying separate applications of KH significantly equaled GWR100% × combined application of KH + CH on Zn contents. The highest Zn contents were recorded by applying combined foliar applications of CH + KH and adopting GWR70%.

Compared to the control, the maximum increase of Mn content was obtained by applying the separated and the combined foliar applications of KH and CH when adopting GWR100% (Fig. 2f). Likewise, compared to the control, adopting GWR70% and applying the separate foliar applications of KH, the combined foliar applications of KH + CH attained a maximum increase of Mn content.

3.3Corn Agronomic Traits, Oil and Protein
By comparing the control in (Figs. 3, 4, and 5), it was found that the examined irrigation levels had significant difference in corn traits, except for the number of rows per ear, number of ears per plant, harvest index, and crop index.

Likewise, and compared to the control, the separate foliar applications of KH improved corn traits, oil, and protein when adopting GWR100%, except for plant height, harvest index, and crop index (Figs. 3, 4, and 5). While the combined applications of KH + CH improved corn traits, oil, and protein by adopting GWR100%, except for plant height and ear length.

Under GWR70%, all corn traits, oil, and protein spraying with KH applied as a sole foliar application recorded significantly higher values than the control treatment except for the number of rows per ear, ear length, and grain index. But spraying KH applied as combined with CH, achieved significantly higher values than the control treatment except (number of ears per plant, ear length, and grain index) under the same previous irrigation level.

By adopting GWR100%, the highest grain yield was observed by applying foliar KH applied with CH, although that significantly equaled the adoption of GWR70% × com- bined application of KH + CH, (Fig. 4b).

3.4Corn Water Productivity (WP)
Generally, WP was increased by adopting GWR70% of irrigation level compared to GWR100% (Fig. 6). By adopting GWR100% irrigation level, the WP increased from 0.25 (kg m^-3) for the control to 0.45 (kg m^-3) for CH500 + KH3000 mg l^-1. Applying the foliar applications of KH & CH, as separated applications in GWR100% irrigation level, was statistically similar. However, applying the separated foliar applications of CH significantly equaled the combined application of KH + CH in GWR100%.

Likewise, when adopting the GWR70% irrigation level, the WP increased from 0.27 (kg m^-3) for the control to 0.59 (kg m^-3) for CH500 + KH3000 mg l^-1. The highest WP was observed by applying the separate foliar applications of KH and the combined foliar applications of KH + CH.

Thus, in arid regions, the researcher recommends treating the stressed plants with separate foliar applications of KH or with the combined foliar applications of KH + CH, which could help plants overcome the negative effects of drought and attain the highest WP.

Discussion

Plants have a specific set of priorities for their life cycle which only break if exposed to outside influences, including drought. This leads plants to rearrange their priorities to mitigate or avoid periods of drought. The current study showed that irrigated corn plants with GWR70% increase proline levels, in contrast to GWR100%. From the results, I conclude that when adopting the limited irrigation schemes, the plants are exposed to some degree of drought that has contributed to a series of successive effects. Physiologically, this leads to a decline in soil moisture around the roots leading to a decrease in the absorption rate of water and nutrients from the soil sector, which is opposite for N, K, and Zn. These findings are in line with the results observed by Fahad et al., as they reported that drought has declined the uptake of P, leading to a reduction in the relative moisture content of the leaves; at par with the increase of the uptake of (N, K, and Zn).

The lack of absorbed water has also led to the decreased metabolic activity of leaf tissue and a decline in P uptake, which is reflected in the synthesis of carbohydrates and oil. While protein content has increased, as a consequence of the increase in N uptake. To alleviate the stressful conditions, the plants have increased proline levels, which perform multiple functions such as an osmotic compatible and adjusted osmotic potential, thus maintaining turgor pressure, and facilitating the water supply to the plant cell, leading to minimizing the adverse effects of drought.

While in terms of morphology, exposure of corn plants to drought contributed to inhibiting plant growth and other vegetative and yield traits. I hypothesized that due to the decrease in levels of leaf extension, cell division, photosynthetic capacity, and enlargement rates. Furthermore, Kulczycki et al. added, ‘although corn as another C4 plant is highly efficient in water use, it remains sensitive to water availability.’

Based on the above, the impact of drought on the plants was clear and represented in two prominent results: (A) reducing nutrient absorption, (B) reducing RWC as a result of water decrease and increasing evaporation. Once plants are exposed to different stressful conditions, they have defensive tools to counteract them. However, they fulfill their effective and desirable role within specific boundaries. Thus, the current study has hypothesized that providing plants with KH and Chitosan would allow them to expand the range of these boundaries and also improve their defensive mechanisms.

Accordingly, the results showed that KH and CH applications lead to improving yield and causing a decline in proline and this impact has remained unchanged under GWR100 and GWR70. In this regard, I have concluded that although separate applications were somewhat similar in their influences, they differ in the mechanisms. Applying KH to stressful leaves has caused the following:

Once K becomes within plant tissues, it works on raising its levels in guard cells, leading to their closure. Also, K increases the permeability of water in plant membranes, leading to an increase in RWC in leaves. Also, applying KH leads to increasing the absorption of Zn, Fe, and Mn than GWR70 control, which, in turn, promoted the enzymes of photosynthesis, and proteins led to improving the effectiveness of photosynthesis and increasing protein content.In addition, KH work on increasing the absorption of P which is an important nutrient in the process of carbohydrate biosynthesis and transport.

Moreover, humic absorption through the leaves worked on enhancing nutrient contents directly and as a result of the acidity of humic; this reduced the osmotic potential of the sap, which led to differences between the potentials within the plant and the alkaline soil solution in this study, which contributed to raising the nutrients transport, and thus improving yield and WP.

Due to that stressful plants quickly promote the production of proline. Therefore, as a result of the many benefits mentioned earlier of applying KH, plants work on reducing the level of proline, which is considered a guide for improving plant conditions. In this regard, this study is not consistent with other studies claiming that applying K to stress plants leads to an increase in proline levels. In this concern, previous studies demonstrated that the relationship between proline and K varies and depends on such factors as (the taken plants part, the application methodology, and the used concentrations of K). For instance, in a study by Bahrami and Hajiboland, the vegetative parts were split into three different fractions, including upper, middle-aged, and lower leaves. The results showed that the addition of K to stress plants caused decreases in proline content in the lower leaves than the others. Also, proline content in the lower leaves decreased by applying K through the leaf than the root. Moreover, according to Anokye et al., it was found that proline content decreased when applying a 3 g concentration of k on the plants put under ordinary growth conditions, while the same happened when applying a 3 g concentration of k on the plants put under drought stress conditions. However, this effect did not happen with other concentrations.

As a result of the poor Chitosan solubility, it seems that the absorbed amounts were small; thus, its effects did not exceed the individual effects of KH applications, especially under drought conditions.

In addition to the positive effect of Chitosan on RWC, it was regarded that the remaining Chitosan on the leaves was a sticky liquid attached to the vegetative parts that were sprayed, I suppose it forms a transparent layer reducing transpiration, which, in turn, led to raising RWC and worked on conserving water, especially under drought conditions. Also, the absorbed Chitosan amounts, as a result of the presence of the amino proton group, work on increasing the photosynthesis rate. Also, the results mention that Chitosan contributes to the increased absorption of (P, Fe, and Zn) nutrients than GWR70 control, where Zn is a major component in many enzymes and proteins and has effective roles in (water relations, stability of cell membrane, osmolyte accumulation, and stomatal regulation), which has led to the improvement of proteins and oil, and this was sup- ported by previous studies.

Furthermore, the absorbed Chitosan molecules have caused decreased proline levels, which was attributed to the improvements in the conditions surrounding the plant that was caused by drought. I assume that to be relying upon such factors as the density of drought stress and Chitosan concentration. For more clarification, Sheikha and AL- Malki tested the effect of Chitosan on proline underwater levels (100, 45, 25, and 15% of water holding capacity); their results showed that although proline was declining at different water levels, the decline was pronounced at 15% of water holding capacity, which agrees with the current results. Also, Attaran et al. studied the effect of three levels of Chitosan (30, 60, and 90 ppm) under irrigation levels (30, 50, and 100% of field capacity); they indicated that at 50% of field capacity and applying Chitosan at a rate of 30 ppm, it caused decreasing proline, contrariwise under 30 and 100% irrigation levels. This might point to the priorities of plants that require rearrangement according to the case.

Through the previous part, the benefits of applying foliar KH and CH as sole applications on corn were clear. But the current results pointed out that their positive effect has increased by applying combined foliar applications of KH and CH, particularly with plants exposed to drought. This was attributed to the aforementioned advantages as a result of applying the separate applications of KH and CH; in addition, those combined applications worked on prolonging the impacts of Chitosan: according to the aforementioned, as Chitosan has poor solubility. Thus, applying successive spraying of KH (which has an acidic pH) led to the dissolving some of the chito Chitosan molecules continuously, which have remained on the leaves.

Also, this combination supplies stressed plants with auxiliary applications for the longest possible time, approaching 90 days: whereas the successive foliar spraying of KH+CH beginning from the second month of cultivation and onwards (two and four times for both foliar substances, respectively) seems to be working on enhancing plants tolerance, especially in the sensitive periods of drought.

Moreover, this combined application helped to create an integration between Potassium Humate and Chitosan: it was noticed that spraying Chitosan on the non-stressed plants has shown a sort of superiority in increasing yield over the separate applications of Potassium Humate. However, with the combined application, it was noticed that it has improved the yield and attained the highest value. This notice was repeated when separate applications of Potassium Humate have been added, although it enhanced several traits than the control, particularly under limited irrigation, as the combined application promoted these enhancements.

Conclusion

This research showed the clear impacts of supplying stressed plants with auxiliary foliar applications of potassium humate and chitosan which resulted in enhancing nutrient absorp- tion and relative water content. Foliar potassium humate and chitosan causing a decline in proline content, and this impact has remained unchanged under full and limited irrigation levels. These findings conflict with the concept that the application of potassium humate and chitosan has the potential for increasing proline contents; nonetheless, further studies are required to observe this impact on other crops under different irrigation levels. As chitosan application has poor solubility, therefore, combining the application with potassium humate creates a sort of integration and prolongs the impacts of chitosan. It is recommend applying combined foliar applications of potassium humate and chitosan under 70% of gross irrigation water requirements as auxiliary foliar applications to water-stressed plants by which nutrient absorption and yield can be improved as well as those combined applications are beneficial to mitigate drought impact and rise water productivity of the corn crop.