Impact of Water Stress and Marine Algae Spraying on Vegetative and Biochemical Traits of Hibiscus tiliaceus

The Hibiscus tiliaceus plant is considered the latest type of tree that has spread in Iraq recently due to its ability to withstand harsh environmental conditions and the beauty of its large yellow flowers. Water scarcity in Iraq is considered one of the biggest pressures and problems facing plant growth, especially in the early stages of agriculture [1].

The method of treating plants with seaweed is one of the methods used recently in the agricultural field. It is a natural material that does not contain any chemical substances in its manufacture, which would increase pollution in the environment [2].

Algae are multicellular photosynthetic organisms that do not have true roots, stems, or leaves. They live in brackish ocean waters as well as in fresh and very cold waters. In return, these algae make their living conditions much more favorable by means of their mineral content. All algae vary in size, some have cell sizes so small that they are only visible, under a normal microscope while others can be so large, that can be seen by naked eyes [3].

The environmental circumstances found in ecosystems, among others, include saline media exposure, strong winds and water scarcity, which pose numerous challenges to the growth and resilience of vegetation that thrives in such regions [4]. Hibiscus with the commonly known as beach hibiscus has soft flowers and large leaves. One interesting thing is its ability to cope with stressful water conditions as it occurs in arid or semi-arid regions and that is the reason seaweed irrigation should be put under the microscope to reveal its influence on ecosystems in general and plant diversity and productivity in particular [5].

Coastal areas often face water shortages due to negative factors, like water salinization, bad water quality and high humidity. Water stress generally acts as a trigger for plants to develop adaptations like reduced leaf size and change in the root features or in growth patterms. Its impacts could range from simple changes in the ecosystem to more complex ones [6].

Seaweeds especially with their range variety have been found to emit an enormous amount of biomass; indeed, numerous studies have reported about the near future benefits of the seaweed for increasing crop yields and resistance together to stress [7]. The application of seaweed sprays or solutions to plants has been studied by researchers as a potential method to enhance plant performance and possibly mitigate negative environmental effects [8]. Having the purpose to look into the impacts of seaweed spraying on marine vegetation, the researchers will reveal the underlying metabolic changes in the main target plants in 1999, and their activities, including the changes occurring in the chemicals themselves, which may influence the ecological contacts of such plant with other species and thus their suitability for the use in various manufacturing processes [9].

The goals of the study were to disclose and categorize the effects of drought stress and a change in seaweed management of Hibiscus tiliaceus [10]. Through the study of this coastal plant's water stress and seaweed response, researchers will benefit from the detailed finding thereby providing information on the plant's adaptability mechanisms, possibilities of stress downstream, and what changes in the secondary metabolites [11]. The results of the study can add to the knowledge on how plant proliferate in marine environments and provide useful data to the sea coast safeguarding and sustainable use. Possibilities of the research findings being utilized in fields as diverse as agriculture, medicine, and even other disciplines are enormous [12].

The experiment was conducted in the lath house covered by saran belonging to preparatory school of agriculture in Abu Gharaq for the spring season 2022/2023. To study the effect of water stress and spraying with marine algae on the vegetative traits and active compounds on Hibiscus tiliaceus trees planted at a distance of (3 × 4 m), based on completely randomized block design (B.C.R.D) with three replicates and each tree representing one experimental unit.

Selection of 45 trees that were as homogeneous as possible in terms of the size of the shoots was done through this. The paper is a study of two factors, the first factor is the water stress and its effect on the growth and development of the Hibiscus tiliaceus plant and physiological processes, including the area of the plant leaf, the increase in the diameter of the branches, and the total chlorophyll in the leaves, which leads to changes in plant characteristics and active compounds [20]. The second factor is spraying coastal Hibiscus tiliaceus plants with seaweed. Seaweed, also known as seaweed, is used as a therapeutic method in this study. Spraying seaweed extracts or solutions on plants is being explored as a potential approach to enhance plant performance and mitigate the negative effects of water stress. Application of seaweed has effects Specific biological, physiological and biochemical effects on the plant, affecting its vegetative traits and increasing the production of active compounds.

The design of the study was a completely randomized design with three kinds of treatments and seven replicates per treatment. Before the start of the experiment, suitable plants were chosen after recording some initial vegetative and physiological traits. In order to have before and after measurements, all types of treatments were carried out in parallel. The groups that involve only the spraying of marine algae are not included in the design and do not run in parallel with the treatments, as this would not allow for the evaluation of the efficiency of marine algae under different water regimes [21]. This is fundamental for understanding the real feasibility of applying this innovative approach, especially when treatments start from seedlings, a very useful approach in the context of seawater agriculture to obtain a resistance trait from the seedling phase. Three groups were arranged: control (T0—1 daily irrigation per day), light water stress in terms of VOC decrease (T1—70% daily reposition), light-moderate water stress in terms of Fv/Fm decrease (T2—40% daily reposition), in compliance with the thresholds identified. Thus, these thresholds were suitable for inducing different levels of stress in the plants, negativity, and decline in the measured parameters, affecting their growth, as shown in the results.

The duration of the treatments was adequately decided, reasoning that the intervals between the repositioning of daily irrigations, except for T0, had to allow a progressive increase of water stress in the plants that, theoretically, would have reached very high values. The experiment was carried out in the experimental greenhouse of the Department of Agricultural Sciences, located in Portici. The treatments were performed while maintaining both agronomic and environmental conditions suitable for plant growth. The allocation of the treatments started in the evening and was carried out in such a way as to balance the possible bias due to the use of fertilizers and plants. After ten days from classifying the plants, the treatments were arranged and monitored every 7 days. The data collected served for statistical analysis to understand which treatment was most effective. The water flow index was analyzed using an analysis of variance.

3.1 Hibiscus tiliaceus

It is a relatively small tree with a height of about 10 meters. Its native habitat is Southeast Asia, the Pacific Islands, and, in general, the southern coastal areas below the equator. This genus includes many species exceeding 200 species. Its flowers are abundant in the spring, and appear intermittently throughout the year. The lifespan of the flower often does not exceed one or two days, after which it turns orange. The flowers consist of light yellow petals. The flower is marsupial-shaped, 15 cm wide, and the carpel is long, protruding from the center of the flower. The color is crimson, as shown in Figure 1. The flowers fall one or two days after they open.

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Figure 1. Hibiscus tiliaceus tree with large yellow flowers

3.2 Seaweed

Seaweed, also known as marine algae, is a diverse group of photosynthetic organisms that inhabit marine environments. They are classified as macroalgae and can be found in coastal areas, oceans, and even freshwater bodies. Seaweeds come in various shapes, sizes, and colors, ranging from microscopic unicellular forms to large, multicellular species that can reach several meters in length. Seaweeds play crucial roles in marine ecosystems and have significant ecological importance. In the spray application for this study, the seaweed extract was diluted to the required concentration using an appropriate diluent (distilled water). The concentrations of algae used in this study are (1.5, 4) ml^-1 as shown in Figure 2, the diluted extract is then applied to the Hibiscus tiliaceus plant using a hand sprayer or specialized spray device. Care is taken to ensure comprehensive and uniform coverage of plant surfaces with the extract solution. In control groups Control groups are created where seaweed plants are treated with diluted water without seaweed extract. These control groups allow comparison of effects between treated and untreated plants.

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Figure 2. Seaweed spraying system

During the trial period, the plants are monitored and cared for according to their specific requirements, including appropriate lighting, temperature and irrigation systems. The duration of the experiment may vary depending on the objectives of the study, but in this study it extended to 3 months.

A reliable and repeatable protocol is indispensable for comparison between the present data and those of similar investigations. New research systems should always be tested on one species in several laboratories before being widely accepted. The materials and methodology sections provide a clear and concise description of the test materials, how they were obtained, and the methods used. Two different types of marine algae were harvested [22]. Both of them were processed and received at the experimental station. Then diluted to a 2% stock preparation in water for tape. Plant treatment by spraying a solution rich in minerals and vitamins was dissolved in water for tape; after two simultaneous plant treatments, the pots were placed in the controlled environment cabinets. The growth of the plant was monitored, leveling the enviable condition until 93 days after the plants' sowing. The upper part of the plants was cut for chlorophyll analysis. Data have been analyzed using the F test. The critical level of significance was set at 5% or 1% using statistical software. The least significant difference test was used for calculation. The physiological data were subjected to one-way analysis of variance, and the means were tested by the least significant difference test. A correlation analysis was performed between the measured physiological parameters.

3.3 Characteristics studied

Plant leaf area cm², it was calculated using the (Digimizer software), as the papers were scanned using an Hp scanner, and it was done, and placed a white board behind the papers and drew on the board a colored line 10 cm long next to the papers and transferred the picture to the software calculated the paper area and took measurements in the month of May. Increase in branch diameter (mm), the measurement of the vegetative branches was taken using the (Vennier) measuring tool, from the base of the branches at the beginning and end of the experiment, by choosing four branches in different directions for each experimental unit, and the difference between the two readings represent the increase in branch diameter. Total chlorophyll in leaves, estimate the chlorophyll content of leaves in the field using Content Chlorophyll (CCM200) meter plus. The physical and chemical characteristics of the soil of the research field were arranged as in Table 1.

Table 1. Physical and chemical characteristics for soil of the study

Nine seedlings nurtured in a polybag were treated per treatment—namely water stress and seaweed. The seaweed of Gracilaria spp. was manufactured by a small-scale industry producing seaweed chips. We believe that the population of these algae is placed in a quite clean site. The stem/leaf biomass (50:50) was used, whereas the water extract was prepared at a range of 5000, 10000, and 20000 ppm, produced by soft and hard methods. Thus, there are two independent variables, i.e., the concentration and time of spray on the bio-stimulant [23]. The water stress treatment was merely used as a control plant. Therefore, distilled water of equal volume to the chemical applications was applied to the stressed trees and the control plant.

Measurement of morphological parameters: Hibiscus tiliaceus shoots after the first stage (30 days old) and the second stage (60 days old) were collected to measure shoot length (cm) and fresh plant weight. The collected samples were subjected to oven drying at a temperature of 70℃ until the weights were constant it was relied upon to measure the dry weight of the Hibiscus tiliaceus plant and the percentage of water content in the plant buds estimated according to the following equation:

$Water \,content$ $(\%)=\frac{M f-M d}{M f} \times 100$             (1)

where, Mf is fresh mass, Md is dry mass.

Concentration the percentage of chlorophyll in the tissues of Hibiscus tiliaceus was calculated with the following equations:

$\begin{gathered}\text { Chl }-\frac{a}{g} \text { plant tissue }=11.63(O D \text { at } 665) -2.39(O D \text { at } 649)\end{gathered}$              (2)

$\begin{gathered}\text { Chl }-\frac{b}{g} \text { plant tissue }=20.11(\text { OD at } 649) -5.18(\text { OD at } 665)\end{gathered}$           (3)

Total chlorophyll:

$\begin{gathered}\frac{a+b}{g} \text { plant tissue }=6.45(\text { OD at } 665) +17.72(\text { OD at } 649)\end{gathered}$               (4)