Chitosan Improves Morphological and Physiological Attributes of Grapevines Under Deficit Irrigation Conditions

Water deficit is considered the most common abiotic factor that limits plant growth and productivity of fruit trees in many areas of the world, especially in arid and semi-arid regions. Approximately one-third of the cultivated area of the world suffers from chronically insufficient supplies of water (Massacci et al. 2008). It has been reported that 70% of the total water consumption in the world occurs in the agricultural sector (FAO 2015), and the need for water is increasing in other sectors such as domestic consumption and industry. This will add more pressure to water availability for horticulture production. A periodical reduction in the yields of rain-fed crops due to drought and the continuing global climate change may increase the severity of the problem (IPCC 2013). Therefore, it is important to use wise irrigation such as deficit irrigation and to improve drought tolerance in horticultural crops. Water deficit can stimulate production of reactive oxygen species (ROS) in plants, which can cause damage to plants by oxidation of DNA, lipids, and proteins. Plants grown under drought stress are capable of introducing morphological modifications to cope with water scarcity, including reduction of leaf area, shoot elongation, and shoot-to-root ratio, as well as increasing root growth (Toscano et al. 2014; Khalil & El-Ansary 2015, 2020). Some plants can modify their structures that help them survive under drought conditions, such as the thickness of the upper and lower epidermis (Ennajeh et al. 2010) and thick cuticle (De Micco & Aronne 2012). They also established physiological strategies involved in leaf water potential maintenance, stomatal conductance control, and osmotic adjustment during drought (Karimi et al. 2012; Khalil 2015). It is widely known that the deliberate withholding of irrigation water by the deficit irrigation technique and the use of drought-adapted trees to cope with water shortage can be effective management strategies for controlling crop water use. Deficit irrigation is a useful production technique for fruit crops that can control their growth and improve fruit quality (Khalil & El-Ansary 2015). This is done by adding water at a rate or amount lower than evapotranspiration. Deficit irrigation at any growth stage could pose negative impacts on physiological, morphological, and biochemical processes in plants, which could include decreased stem elongation, leaf area, root size, and depth, and changes in stomatal formation and plant-water relations with reduced water use efficiency and crop production (Li et al. 2009).

Elicitation is an effective strategy to induce drought tolerance and physiological changes in plants (Baenas et al. 2014). Chitosan is one of the most common elicitors inducing cell defense reactions in plants (Shibuya & Minami 2001). Chitosan is a natural polysaccharide derived from a low-acetyl form of chitin, mainly composed of glucosamine and N-acetyl glucosamine and is commercially produced from crab shells, shrimp shells, lobster and squid, and filamentous fungi (Kumaresapillai et al. 2011). Previous studies have shown that chitosan can stimulate plant growth indices (Farouk et al. 2008), increase yields, induce plant resistance to bacterial, fungal, and viral infections, and reduce transpiration (Karimi et al. 2012). Moreover, chitosan-treated plants may be less susceptible to stress induced by adverse conditions, such as low or high temperature, salinity, and drought (Lizárraga-Paulín et al. 2011; Pongprayoon et al. 2013). Chitosan has decreased transpiration in pepper plants, and hence, reduction of water loss while maintaining biomass and yield. This result suggests that chitosan can effectively counteract transpiration, protecting water in drought conditions (Bittelli et al. 2001). Foliar application of chitosan may induce stomatal closure. However, the mechanisms of action of chitosan on plant growth remain unclear. Yang et al. (2009) reported that pretreatment of leaves of apple seedlings with chitosan solution (100 mg·dm⁻³) prior to drought stress significantly enhances leaf membrane stability and antioxidant enzyme activities. Górnik et al. (2008) found that chitosan significantly enhanced rooting of the cuttings, increased the number of new canes and their length as well as the number of nodes and chlorophyll content in the leaves developed on grapevines grown under drought stress conditions. El-Kenawy (2017) described morphological and physiological responses, such as the increase in shoot length, leaf surface area, and total chlorophyll content in grapevines subjected to chitosan. However, studies on the interaction effects of foliar application of chitosan and drought stress on grapevine growth are still lacking.

Grapevine (Vitis vinifera L.) is one of the most ancient and widely cultivated fruit trees grown in a temperate and semi-arid climate, where it may be afforded consecutive cycles of water deficit and re-watering either through rainfall or irrigation. Vines are considered drought-tolerant plants, characterized by diverse stomatal behaviors and hydraulics, depending on the cultivar and can be classified based on their water potential as tolerant to water limitations (Martorell et al. 2015). Grapevine is able to perform physiological drought avoidance mechanisms, such as effective stomatal management of transpiration and xylem embolus, and the potential for osmotic adjustment. Therefore, the aim of this study was to determine the morphological and physiological response of young grapevines to various doses of foliar applied chitosan under deficit irrigation conditions. The morphological indices chosen were leaf area, trunk cross-sectional area, plant dry weight, and root dry weight. Physiological stress-associated characteristics are leaf midday water potential (ψ), relative water content (RWC), crop evapotranspiration rate (ET_c), chlorophyll content, proline content, leaf total carbohydrates, and catalase activity.

The results of the present study show that irrigation levels alone and jointly with chitosan application had significant effects on most studied morphological indices (Fig. 1). There was a reduction in leaf area following decreased irrigation levels in control plants, which is in line with the previous investigations on grapes (Gómez-del-Campo et al. 2002; Buesa et al. 2017). The reduction in leaf area is considered as one of the adaptation mechanisms in plants to avoid drought stress conditions by minimizing the evapotranspiration rate and reducing water consumption (Toscano et al. 2014). The reduction in plant trunk cross-sectional area following decreasing irrigation levels is expected as a result of drought stress and lessen growth rate (Boland et al. 2000) and is also a morphological adaptation of plants to water stress (Bañon et al. 2006). A reduction in grapevine dry weight was found in this investigation following decreasing irrigation levels. This result has been shown in previous studies on the grapevines (Tehrani et al. 2016; Conesa et al. 2016). In contrast to our results, increased root dry weight under moderate deficit irrigation is considered as a defense mechanism to cope with drought conditions and induce water uptake in some plants (Toscano et al. 2014) and could be attributed to the accumulation of photoassimilates in the roots more than in the shoots. The redistribution of dry matter in favor of the root at the expense of the shoot is considered the plant's demand to keep enough surface leaf area under drought stress (Conesa et al. 2016). Drought stress reduced the plant vegetative growth, which appears to be the result of disrupted plant water relations in specific turgor potential (Hussain et al. 2009). The application of chitosan at 1, 5, and 10 g·dm⁻³ improved all the growth indices under well-watered and drought conditions and resulted in a significant increase in vegetative growth features compared to the untreated control plants. Malekpoor et al. (2016) found that foliar-applied chitosan increased plant growth (common bean and basil) under stressed or non-stressed conditions. Moreover, Ait Barka et al. (2004) reported that chitogel, a derivative of chitosan, improved the vegetative growth of grapevine plantlets. The increase in total dry weight due to chitosan application may be due to its effects in stimulating the photosynthetic process (Khan et al. 2002). Moreover, the stimulating effect of chitosan on plant growth under drought conditions might be explained by increasing nutrient and water uptake by adjusting cell osmotic pressure and reducing free radicals by stimulating antioxidant activity (Guan et al. 2009). Physiological determinations of grapevine leaves responding to drought have revealed that chlorophyll content decreased, which in turn inhibited the photosynthetic activity. Similar to the results of this study, the reduction in chlorophyll content (SPAD values) has been reported in grapevines in response to drought stress (Haider et al. 2017). This could be due to the destruction of the pigment-protein complexes, which defend the photosynthetic machinery (Lai et al. 2007), or to a reduction of specific enzymes such as HemA glutamyl-tRNA reductase 1 and magnesium chelatase H subunit, which are important for the construction of photosynthetic pigments (Murkute et al. 2006). The increase in chlorophyll content following chitosan application supports the previous suggestion that chitosan enhances the photosynthesis process by increasing photosynthetic pigments (Dzung et al. 2011). The proline level in grapevines increased substantially as drought stress intensified. Proline accumulation is considered as one of the initial responses of many plants to drought stress. It is well known that proline accumulation reduces the water potential of plant tissues, particularly in leaves, thereby enabling them to restrict water loss and/or continue to absorb water from soil under drought stress conditions. In contrast, foliar spraying of chitosan in both seasons showed a significant decrease in the proline content under normal or stressed conditions. The effect of chitosan on the reduction of the proline content was at least partly due to its role in reducing the water potential of plant tissues. The reduction in total carbohydrate content induced by water deficit treatments may be due to their inhibitory effect on photosynthetic pigment concentrations (Fig. 2) as well as the decrease in photosynthetic rate (Jie et al. 2010). Moreover, under drought conditions, the breakdown of polysaccharides caused an accumulation of osmolytes such as soluble sugars, which helped the plants to maintain the cell turgor (Nazarli et al. 2011), and this is considered as an adaptive mechanism to drought stress conditions. The effects of chitosan, especially at 10 g·dm⁻³, on increasing total carbohydrate content were confirmed by Farouk et al. (2008) on cucumber. The influence of chitosan on alleviating drought stress effects on photosynthetic pigments leads to stimulate photosynthetic activity and carbohydrate accumulation (Farouk et al. 2008).

In the current study, substantially lower CAT levels were detected in plants grown under drought stress than in those in well-watered conditions. These results are in agreement with the earlier reports indicating that drought stress decreased CAT activity in the leaves of pomegranate seedlings (Khalil 2015). Chitosan application to grapevines alleviated some of the negative impacts of drought and increased CAT activity (Fig. 2). This result supported the results of Guan et al. (2009), who indicated that the application of chitosan on maize plants significantly decreased lipid peroxidation, due to stimulation of some antioxidant enzymes.

The gradual reduction in ψ values following deficit water treatments found in this research is in agreement with those previously reported by Conesa et al. (2016) and Buesa et al. (2017) working on grapevines. A short period of mild water deficit may promote plants to reduce the leaf water potential substantially (Pérez-Pastor et al. 2014). Decreased leaf water potential acts as a hydraulic signal that triggers reduced leaf area expansion and partial closure of stomata (Shahnazari et al. 2007). Several reports on grapevines revealed that drought stress negatively affects leaf water potential due to stomatal closure and a decrease in stomatal conductance (Conesa et al. 2016; Tehrani et al. 2016). The reduction in stomatal conductance was strongly associated with a reduction in photosynthetic activity and leaf area, which are well known in plants grown under drought stress conditions (Medrano et al. 2002).

In this study, deficit irrigation treatments decreased RWC, while significantly higher RWC values were observed in chitosan-sprayed plants. The decrease in RWC values following water deficit treatments found in this study is consistent with those reported by Abdi et al. (2016). They agreed that with increasing moisture stress in the growing medium, a noticeable decrease in the RWC of the leaves was noticed. The results presented here showed that prolonged water deficit causes a decrease in RWC values, and these reductions may be lessened when spraying chitosan. Undoubtedly, this might be due to the role of chitosan in reducing plant transpiration. Bittelli et al. (2001) suggested that chitosan could be used as an effective anti-transpirant to reduce irrigation water use. The decrease in crop evapotranspiration (ET_c) following increased drought stress conditions of less irrigation water in fruit trees have been reported in several studies (Çamođlu 2013). The decrease in the ET_c values coupled with an increase in leaf water potential values in most treatments was recorded. The results generally indicated that the ET_c obviously decreased as a result of chitosan application. The magnitude of this reduction at 40% field capacity reached as much as 29.3% and 23.9% for the 5 and 10 g·dm⁻³ chitosan treatments, respectively. The influence of chitosan in reducing ET_c during drought stress conditions could be attributed to the induction of stomatal closure (Bittelli et al. 2001).

Deficit irrigation, as expected, markedly reduced leaf area, trunk cross-sectional area, plant dry weight, and root dry weight, which proved biomass production and distribution. Water deficit decreased leaf midday water potential (ψ), relative water content (RWC), and crop evapotranspiration rate (ET_c), causing changes in plant water status. Chitosan application under all irrigation levels increased leaf area, trunk cross-sectional area, plant dry weight, and root dry weight, relative chlorophyll content, leaf total carbohydrates, catalase activity, and relative water content, but reduced the proline content. Reduction of plant biomass under drought stress and the positive impacts of chitosan as anti-transpiring agent play the main role in drought stress avoidance mechanisms in potted-vines grown under moderate and severe deficit irrigation conditions.