Effect of Seaweed and Zinc on Growth, Leaf Proline, Chlorophyll Content and Soil Attributes of Manilkara hexandra (Roxb.) cv. Thar Rituraj

Manilkara hexandra (Roxb.) belonging to the family Sapotaceae, is a socio-economically important underutilized fruit species of Western-Central India. It is locally known as ‘Khirni’, ‘Raina’ and ‘Rayan’ by tribal people of different states of India. It is believed to be originated in India (Stewart and Brandis, 1992). The tree is found as wild in the South, North and Central India mostly in the states of Rajasthan, Gujarat, Madhya Pradesh and Maharashtra (Malik et al., 2010). It is a commercially and medicinally important tropical tree species and is a significant source of livelihood and nutritional support for local tribal population (Mishra and Pareek, 2014). In India, this species is occasionally cultivated in backyards, homestead gardens, public parks as avenue tree and in farmer’s fields near villages due to its economic importance as fruit tree having nutritional and medicinal properties. Fruits of khirni have high economic value as mature fresh fruits which are sweet and a good source of minerals, sugars, protein, carbohydrate and vitamin A (Pareek et al., 1998). Its bark and fruits are also used for several medicinal purposes like the treatment of ulcers, dyspepsia, opacity of the cornea, bronchitis, urethrorrhea, leprosy, etc. (Anonymous, 1962; Warrier et al., 1995; Pareek et al., 1998; Hoareau, 1999; Raju and Reddy, 2005; Chanda and Parekh, 2010). The seeds contain approximately 25% oil, which is used for cooking purposes (Xian-zi, 1996). The bark also contains 10% tannin, which is used for the treatment of fever and may be utilized for tanning purposes (Anonymous, 1962).
       
The benefits of seaweed as a source of organic matter and fertilizer nutrients have led to their use as a soil conditioner for centuries. They are used as food for humans, fodder for cattle, substitutes for chemical fertilizers and as a source of various fine chemicals. Besides this, it is used for obtaining many industrial products such as agar and alginate (Khan et al., 2009). Seaweed extracts are marketed as liquid fertilizers and bio-stimulants because they contain multiple growth regulators such as cytokinins (Durand et al., 2003), auxins (Sahoo, 2000), gibberellins (Strik and Staden, 1997) and macronutrients such as nitrogen, phosphorus, potassium and micronutrients such as calcium, magnesium and sulfur optimally necessary for plant growth and development. Moreover, it helps in promoting and sustaining the growth of beneficial soil microorganisms (Khan et al., 2009), developing tolerance to environmental stress, increasing nutrient uptake from soil (Turan and Kose, 2004). Seaweed extracts are eco-friendly and help in mitigating environmental pollution and play a biologically active role in intrinsic plant systems. The foliar spray induces faster growth and increased yield in cereals, vegetables, fruit plants and horticultural crops (Mahima et al., 2018).
               
Additionally, thought to be essential for fruit growth and development is Zinc. It is one among the components needed to synthesise chlorophyll, which makes it helpful for photosynthetic activity (Chaudhuri et al., 2025). Zinc is a pivotal micronutrient for auxin enhancement, protein synthesis, seed production and fruit maturity. It plays an important role in a wide range of processes such as growth hormone productions, especially IAA and elongation of internode. The zinc sulphate is an important source of zinc for plants. Zinc acts as a co-factor of numbers of enzymes and is also involved in the production of growth regulator and chloroplast development (Hambidge et al., 2000). Foliar application of zinc is the most effective in controlling zinc deficiency and improvement of vegetative growth attributes, fruit morphological attributes and internal fruit quality attributes like total soluble solids and ascorbic acid contents. Physiological, morphological and biochemical abnormalities are usually initiated by Zn deficiency resulting in declined growth of plants (Nadeem et al., 2020). Zinc promotes enhancement of chlorophyll in leaves which is an important parameter of carboxylation efficiency. It is essential for water uptake (Disante et al., 2010). Zinc supports healthy root growth thereby leading to more conversion of assimilates for increased Vigor of plant system. Therefore, the effect of seaweed and zinc was studied on various growth parameters of khirni.

A field experiment was carried out at the Instructional Farm, Department of Fruit Science, College of Horticulture and Forestry, Jhalawar in khirni block during the years 2021-22 using plants having age of 2 years and planted at distance of 6m × 6m. The treatments under study were T₀- Control,  T₁- Seaweed extract 2 ml/l, T₂- Seaweed extract 4ml/l, T₃ -Seaweed extract 6 ml/l, T₄ -Zinc sulphate 200 mg/l, T₅- Zinc sulphate 400 mg/l, T₆- Zinc sulphate 600 mg/l, T₇- Seaweed 2 ml/l+ Zinc sulphate 200 mg/l, T₈-Seaweed 2 ml/l+Zinc sulphate 400 mg/l, T₉- Seaweed 2 ml/l+Zinc sulphate 600 mg/l, T₁₀- Seaweed 4 ml/l+Zinc sulphate 200 mg/l, T₁₁-Seaweed 4ml/l+Zinc sulphate 400 mg/l, T₁₂- Seaweed 4 ml/l + Zinc sulphate 600 mg/l, T₁₃- Seaweed 6 ml/l+Zinc sulphate 200 mg/l, T₁₄- Seaweed 6 ml/l+Zinc sulphate 400 mg/l, T₁₅- Seaweed 6 ml/l + Zinc sulphate 600 mg/l. The application of treatments were mode in the form of foliar spray. The spray was done during 1^st week of July 2021 after recording initial (base) growth parameters of plants.                

The plant growth parameters recorded during the period of experimentation were height of plants (cm), rootstock girth (mm), scion girth (mm), number of nodes per plant, number of shoots per branch, number of leaves per plant, leaf length, leaf width, leaf area, leaf perimeter and leaf area index which were measured initially in July and later at every two-month interval till March. Chlorophyll content and proline content were measured initially and at the end of the experiment.
       
Plant height was measured from the base of plant to the highest tip of the plant with the help of a measuring scale. The girth of rootstock and scion was measured separately by using digital vernier caliper, number of nodes per plant, number of shoots per branch and number of leaves per plant were counted manually at every two months interval from starting of experiment till the end of experiment.
       
The average increase in leaf length and leaf width was calculated on the basis of cumulative increase in initial value. The leaf area was calculated using formula as suggested by Montgomery (1911) for this purpose. The leaf area of five tagged plants was recorded using graph paper. For measuring leaf perimeter, selected tagged leaves under various treatments were measured by encircling the thread and thread length was ultimately recorded with the help of measuring scale. Leaf area index was computed by the formula as suggested by Watson (1947). Stress related parameter; the proline content was measured as per the method given by Bates et al., (1973), besides the chlorophyll content as per the method suggested by Sadasivam and Manickam (1997).

Effect on growth parameters
 
It was demonstrated from the Table 1 that the treatment T₁₅ (Seaweed 6 ml/L+Zinc Sulphate 600 mg/L) had maximal plant height (28.47%) rootstock girth (32.39%) and scion girth (20.08%). It was shown in Table 2 that topmost number of nodes/plant (33.62%), No. of shoots/branch (3.74%), No. of leaves/plant (27.91%) and Leaf perimeter (18.80%), Table 3 illustrated that the highest Leaf length (27.89%), Leaf width (13.65%), Leaf area (24.40%) and Leaf area index (0.40). Among the different treatments, T₁₅ (Seaweed 6 ml/L+Zinc sulphate 600 mg/L) was noted superior as compared to rest of the treatments and including control. This improvement can be attributed to the beneficial impact of seaweed extract, which contains essential macro elements (N, P, K and Mg) and microelements (Fe, Zn, Cu and Mn). These elements fulfill the plant’s mineral needs for processes such as photosynthesis, respiration, cell division and elongation. The improved growth of plants may be attributed to the increased availability of microelements such as Fe, Zn, Cu and Mn. These microelements are essential for protein synthesis, chlorophyll formation, as well as the production of the growth hormone IAA and cell division. These processes collectively contribute to the regulation of cell respiration, photosynthesis, protein synthesis and the uptake of nutrients and water, ultimately promoting enhanced plant growth. The growth attributes of khirni plants strongly align with those obtained by Rishitha (2023) in strawberry, Harhash et al., (2021) in grapevines cv. Flame Seedless, Al-Hchami, (2013) in peach trees, Al-Hadethi et al., (2015 and 2013) in apricot trees, Alam et al., (2013) in strawberry, along with beneficial effect in orange and mandarin as obtained by Rayorath et al., (2008), Sharaf et al., (2011), Abbas et al., (2012), Abdel Hamied-Sheven (2014) and Al-Hayani et al., (2016); and that of zinc with those as reported by Razzaq et al., (2013) and Dawood et al., (2001) in Kinnow mandarin.






       
Moreover, the maximum numbers of secondary branches (7.49 and 6.58) were observed in the treatment of 75 ppm Zn+0 ppm GA3 in the first and second seasons, respectively. The spraying of Zn may compensate for the lack of Zn and avoid deficiency of Zn in plants especially in poor micro-elements sandy soils. So, by increasing the level of Zn, the vegetative growth parameter will be improved (Atteya et al., 2018). In the case of zinc application, the plots which received 30 kg/ha of ZnSO₄ recorded the tallest plant height which were 10.56, 3.64, 4.71 and 4.72% taller than 20 kg/ha of ZnSO₄. 

However, it is found to be comparable with 40 kg/ha of ZnSO₄. Such kind of increment due to zinc application is a result of higher nitrogen uptake and enhanced enzymatic activity. (Bhattacharjee et al., 2022).
       
The results of study presented regarding beneficial effect of seaweed and zinc are congruous to the findings as reported by Ali and Mohamed (2016), Gomaa and Ibrahim (2020) in banana, Abdulrahman (2013) in Almond seedlings, AI-Rawi et al., (2016) in peach plants, Chhonkar and Singh (1981) in Guava, Banik and Sen (1997) in mango, Sarolia et al., (2007) in guava and Waskela et al., (2013) in guava.
 
Effect on proline and chlorophyll content
 
The data concerning chlorophyll content (0.98 mg/g) and proline content (5.10 µmol./g) are presented in Fig 1. Data reveals that there were significant variations among the various treatments and it was obvious that maximum increase in chlorophyll content and minimum increase in proline content was found in T₁₅ (Seaweed 6ml/l+Zinc sulphate 600mg/l).


       
The better increase in growth parameters, chlorophyll and reduction in proline content under T₁₅ (Seaweed 6 ml/l + Zinc sulphate 600 mg/g) may have association with the combined effect of seaweed and zinc. The effect may be explained in the light of the fact that auxins content in the seaweed extracts have an effective role in cell division and enlargement. This perhaps led to an increase in the shoot growth, leaves area and plant dry weight (Gollan and Wright, 2006). Seaweed extracts contain cytokinins which induce physiological activities like activating enzymes that are involved in photosynthesis and increase in total chlorophyll in the plant and via these ways it positively influenced shoots characteristics (Thomas, 1996). Besides, the favorable influence of applied zinc through foliar feeding might also play augmentory role in stimulating the stimulatory effect on most of the physiological and metabolic process governing chlorophyll synthesis, auxin enhancement, protein synthesis and acceleration of synergy in the plants.
 
Effect on soil parameters
 
The data showcased in Table 4 illustrates the influence of foliar application of seaweed extract and zinc sulphate in individual as well as well combination effect during experimental season 2021-22 in the Khirni cv. Thar Rituraj. The findings disclose that there were no discernible differences in soil parameters viz; soil EC, soil pH, organic carbon (%), available N (kg/ha.), P (kg/ha), K (kg/ha) and water holding capacity (%). However, statistically T₁₅ (Seaweed 6 ml/l+Zinc sulphate 600 mg/l) was the most effective treatment from the remaining treatments. It is due to that the seaweed and Zinc was applied as a foliar spray (directly on leaves), the nutrients have not reached the soil in significant amount. In other view, the applied substances have not leached into the soil or interacted with soil microorganisms and particles.


               
Zinc plays a pivotal role in the fundamental process involved in the cellular mechanism and respiration. The presence of sufficient available zinc in chloroplasts and whole plant tissue system may also be considered a possible reason for increase in growth attributes of the plants. Zinc is an essential component of enzymes responsible for nitrogen, thereby resulting in an increase in uptake of nitrogen by the plant. Along with, involvement of zinc in synthesis of tryptophan which is a precursor of indole acetic acid synthesis regulates tissue growth and development (Alloway, 2008) and thereby improvement of shoot parameters. From the result of investigation, it may be concluded that the application of T₁₅ (Seaweed 6 ml/l + Zinc sulphate 600 mg/l) emerged better in its effectiveness on growth and development of Khirni (Manilkara hexandra L.) cv. Thar Rituraj. All the plant growth and development parameters were found significantly superior over all other individuals as well as combined treatments.

From the result of investigation, it is concluded that T₁₅ (Seaweed 6ml/l + Zinc sulphate 600 mg/l) emerged better in its effectiveness on growth and development of Khirni (Manilkara hexandra L.) cv. Thar Rituraj. All the plant growth and development parameters were found significantly superior in T₁₅ (Seaweed 6ml/l + Zinc sulphate 600mg/l) over all other individual as well as combined treatments. In this treatment (T₁₅) the per cent increase in parameters like height of the plant (28.47%), rootstock girth (32.39%), scion girth (20.08%), No. of nodes/plant (33.62%), No. of shoots/branch (3.74%), No. of leaves/plant (27.91%) and leaf parameters (18.80%) like Leaf perimeter (18.80 cm), Leaf length (27.89cm), Leaf width (13.65cm) leaf area (24.40%). Leaf area index (0.40) and physiological parameters like proline content (5.10 µmol. /g) and chlorophyll content (0.98) were significant at the time of final observation during March.

I would like to thank my mentor Dr. Jitendra Singh for their guidance and support throughout this research. I am also grateful to my committee members for their valuable feedback and suggestions.

The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.