Water stress changes on AMF colonization, stomatal conductance and photosynthesis of Dalbergia sissoo seedlings grown in entisol soil under nursery condition

Abstract Water stress significantly impacts the plants’ physiological activity. They are influenced by the stomata and photosynthesis of the plant. The main objectives of the experiments are to determine the stomata and photosynthetic activity changes in the Dalbergia sissoo seedlings under the entisol soil in nursery conditions and the water stress conditions on AMF colonization. The plant growth characteristics and physiological activities in D. sissoo were assessed under three conditions: WW (well watering), FW (fractionated watering), and SW (stopped/no watering), with the results revealing that the FW condition has a higher mycorrhizal dependency of 24.53% than the WW condition, which is 24.37%. AMF root colonization was also higher in D. Sissoo, at 56% and 47% under FW and WW conditions. These findings highlight the significance of AMF, especially when plants are experiencing water stress. When FW was used instead of WW, the photosynthetic rate of D. sissoo and AMF + plants increased by 17.85%. AMF inoculation changed the plant’s physiological activities, resulting in a significantly higher photosynthesis rate and stomatal conductance. However, higher transpiration, intercellular CO2 concentration, and a lower leaf temperature regardless of WW or FW conditions indicate that AM positively affects physiological activities. The findings support the use of AMF in entisol soil to improve plant growth and biomass by alleviating adverse edaphic conditions.


Introduction
The forest is worldwide shrinking due to the increasing aridity and climate change consequences of biodiversity loss and ecological disasters. The pressure on forests and deforestation gave birth to social forestry and agroforestry programs (Mogotsi et al. 2016). The changing scenario of forestry research priorities has emphasized improving biomass production to meet the needs of fuel wood, fodder, timber, and other forest resources (Carle et al. 2020). All of these programs necessitate high-quality seeds and seedlings for plantation and the development of effective planting technology for increasing biomass within a short rotation. The success of an afforestation program depends on the use of quality seedlings and healthy stocks. Plantation programmes typically fail due to insufficient planting stocks (Holl and Brancalion 2020). High-quality planting stock production is critical for large-scale plantation programs that require high-quality seeds and forest nurseries where seeds are grown under the supervision of experienced professionals because the nursery inputs ensure healthy plants, better survival, and subsequent growth commensurate with high productivity in the site.
Dalbergia sissoo is a large, fast-growing plant that grows in natural forests and agricultural fields in Chhattisgarh. It is a valuable species that provides wood for a variety of applications as well as fodder for livestock. It is an important agroforestry species that are used for multipurpose uses and trees' ability to fix nitrogen. It is mostly considered one of the highestpriority species in plantation programs. It prefers soils ranging from pure sand and gravel to the rich alluvium of river banks. D. sissoo can grow in slightly saline soils (Naqvi et al. 2019). Ensisols are widely spread in Bilaspur region. This soil is very compact and has a low water-holding capacity, which hinders plant root penetration as well as the availability of water, particularly in dry months. However, Arbuscular mycorrhizal fungi (AMF) have been reported to affect plant-water relationships (Auge 2004;Heidari and Karami 2014). AMF are closely related to the host plant that grows vigorously under stressful conditions. They mediate a series of complex communication events between the plant and the fungus, leading to enhanced photosynthetic rate and other gas exchange-related traits and increased water uptake (Birhane et al. 2012). 90% of plant species, including flowering plants, bryophytes, and ferns, can develop interdependent connections with AMF (Zhu et al. 2010;Ahanger et al. 2014). In the roots, AMF produces vesicles, arbuscules, hyphae, and spores and hyphae in the rhizosphere. The formation of a hyphal network by the AMF with plant roots improves root access to a large soil surface area, improving plant growth (Bowles et al. 2016). AMF improves plant nutrition by increasing the availability of various nutrients and their translocation (Rouphael et al. 2015). AMF improves soil quality by influencing the structure and texture of the soil, which enhances plant health (Zou et al. 2016;Thirkell et al. 2017).
The main aim of these experiments is to produce high-quality seedlings with functional characteristics and management strategies to enhance tree growth and productivity. However, the species is still poorly studied for its management in plantations and its physiological responses to AMF applications (Quatrini et al. 2003). Inoculation with mycorrhizal fungi in the nursery can increase seedling performance as many of the workers have reported consistently positive results of AMF inoculations in several forest species (Seema et al. 2000;Sharma et al. 2001;Chandra et al. 2012). Similarly, work done by Ranjan et al. (2020), seedlings inoculated with AMF in the forest nursery to produce superior planting stock of Swietenia mahagoni seedlings with improved drought resistance. AMF is nature's gift to plants and plays a vital role in plant growth and development, especially under adverse soil conditions. It may be exploited to produce quality nursery stock, particularly for different types of wastelands, mined soil, and dunes. Soil substrates, infected roots of AMF mycelium, and spores are commonly used as AMF inoculums (Klironomos and Hart 2002). They may be produced in form by using suitable trap host plants.

Material and method
This research was conducted in the nursery of the Forestry Department, Guru Ghasidas Vishwavidyalaya (GGV), Bilaspur, India. Implementation of the study began in October 2016 and ended in January 2017. The entisol soil was brought from the Bhataland of Chakarbhatha for poly pot experiments to GGV, Bilaspur (C.G.) This research was conducted in the Forestry Department, GGV, Bilaspur, India. Implementation of the study began in October 2016 to January, 2017. The climatological data during the study period i.e. October-January, 2016 was depicted 17 mm/month precipitation, max. temperature experienced 31.1 C in October while a minimum of 13.3 C in December -January. Evapotranspiration rate recorded was 3.05 mm/day in this period.
The experiment was conducted in the open (not controlled conditions) and plants were used from D. sissoo their seeds were collected from the Bilaspur provenance. To ensure the absence of preexisting mycorrhizal spores, seeds of these species were directly sown in polythene bags with 2.5 kg of sterilized entisol soil without any amendments at 15 psi/h for 45 min twice. Seeds were planted on October 4 th , and seedlings were completed one month later, with only one seedling per polythene bag.
The viable AMF spores were extracted by the sucrose centrifugation technique (Daniel and Skipper 1982). The 100 g of soil samples were suspended in one litter of water. The material was sieved by 1-45 mm sieve size and washed repeatedly to remove root beats and stones from the samples. The content from the sieves (45 mm) was transferred into a centrifuge tube and allowed to centrifuge for 4 min at 4000 rpm. The supernatant was transferred into a 45 mm sieve and dead and over-matured spores were separated. Subsequently, centrifuge tubes were filled with saturated sucrose solution, 80% concentrated, and made a suspension through stirring with a glass rod and centrifuged for 15 s at 4000 rpm. The supernatant was collected in a 45 mm sieve size and washed in tap water immediately and then finally transferred into a petri-dish for observation under a stereo zoom microscope. It was also used for mass multiplication.
In open conditions, marigold was chosen for mass multiplication and inoculum production of AMF. To eradicate soil-borne bacteria and fungi from the combination substrate, sand and soil were combined in a 1:2 ratio and autoclaved for 1 h at 15 psi twice. The sterile soil combination was then placed in a 15 kg capacity high-density plastic pot for mass multiplication.
AMF spores that had been surface sterilized were employed as inoculums by placing them near young, developing lateral roots to stimulate germ tube formation (Mosse 1962;Hepper 1983). The spore population and root infection were investigated after 60 days of plant growth. Wet sieving and decanting techniques (Gerdemann and Nicolson 1963) were used to recover 25 g of the substrate containing root pieces and AMF spores, and the Phillips and Hayman (1970) method was used to count the spore population. The same procedure was used to check and confirm the multiplication status of AMF at 120 and 180 days after inoculation.
Simultaneously, 20 g of AMF mixed inoculum was placed in the seedling's root zone by making holes around the plant and then refilling them with sterilized entisol soil.
The main goal of the experiment is to see how water stress affects plant growth parameters, physiology, and AMF development in D. sissoo plants grown in entisol soil under nursery conditions. There were two types of treatments in this study: AMF inoculation and an uninoculated control. Funneliformis mosseae mycorrhiza species were added to 20 g of inoculum, which consisted of infected roots, spores, and soil substrate, and contained approximately 1000 spores in each seedling of tree species. To ensure that control seedlings received the same amount of sterilized inoculum as control plants, they were also given the same amount of sterilized inoculums. Furthermore, water stress was investigated using three irrigation levels: well-watered [WW] and fractionated water [FW] and SW (stopped/no watering). Thus, four treatments, including an uninoculated control, covered the two experimental factors. Total of 160 seedlings was tested for AMF þ and AMF À treatments, including WW and FW conditions, with each treatment consisting of a block of eight seedlings with five replications.
After the plants had established themselves and AMF had been inoculated at the age of 40 days, half of the plants were given fractionated water (FW), while the other half was watered daily. The pots received the same treatment and were watered once a day before the drought treatment began. WW treatment (2 ml/plant, receiving 100% of the water estimated to have been lost through evapotranspiration) and the FW treatment (200 ml/plant at two-day intervals, receiving 60% of the water of the WW-treated plants) were randomly assigned to two irrigation lines. Watering was scheduled between 9 and 11 A.M.
When experiments A and B showed no significant signs of drought stress, a third experiment was conducted due to the WW experiment to increase stress on the drought plants. This experiment followed the same procedures as experiment A [WW], with inoculated AMF and regular watering of 2l/day/plant for 120 days, but there was no watering (no watering) until the plants died. Following the cessation of watering, daily measurements of photosynthesis, transpiration, other physiological parameters, and soil moisture were taken for five days in a row until the plant leaves had reached 100% senescence.
Five plants were randomly selected from each treatment, one from each replicate, after 120 days of plant age and 90 days of AMF inoculation, and the substrate was carefully washed from the root mass. Before harvesting the plants, physiological observations were taken, and the same plants were used for further growth and biomass measurements. The fifth leaf from the top to the bottom of each plant was chosen for photosynthesis variable measurements. From 9 to 11 a.m. on a sunny day, researchers measured gas exchange parameters such as net photosynthetic rate, stomatal conductance, intercellular CO 2 concentration, transpiration rate, and leaf temperature. Transpiration rates were measured by the METER SC-1 leaf porometer and leaf temperature was measured by the Leaf Thermocouple. Sensor K-type. Moisture meter model GB-2283656 was used to determine the moisture content of the soil. ADC Bioscientific Limited's LC Proþ photosynthetic analyzer, Serial No. 32480, recorded all physiological characteristics. Photosynthesis rate was determined by the rate at which a CO 2 concentration was assimilated by a known leaf area in a given formula: where C 0 ( C 1 ) outlet (inlet) CO 2 concentration (ppm/m mol), E¼ net photosynthesis rate (m mol/m 2 /s), W ¼ mass flow rate per leaf area (mol/m 2 /s).
Transpiration rate is the water vapour flux per one leaf area: where e 0 (e 1 ) outlet (inlet) water vapor, P¼ atmospheric pressure.

Statistical analysis
Statistical analyses were done using SPSS 16.0 version for the calculation and interpretation of the data. Pearson's correlation coefficients were employed to determine the relationships between fungal colonization parameters and environmental factors. Standard errors of means were calculated for all the parameter studied and Duncan Multiple Range (DMR) test was used for mean comparison at 5% P level.

Result and discussion
Experiment A: well water condition (WW)

Effect on plant growth and biomass
The well-watered seedlings inoculated with AMF thrive well with increased growth patterns and physiological functions (Tables 1 and 2 and Figure 1). The plant height of the AMF þ plant was recorded as 36.70 cm, and it was 31.18 cm in seedlings without AMF À which was 17.70% higher than AMF À plant (Table 1). Similarly, significant results were rendered by plants with AMF þ for diameter at F ¼ 26.51, p < .001. The collar diameter of D. sissoo with AMF þ was 3.28 mm compared to AMF À plants (2.04 mm/plant); thus, about 60% increment was observed in AMF þ seedlings against AMF À plants (Table 1). However, root length, shoot fresh weight, and root fresh weight value could not have significant results within treatment factors at p < .05. By analyzing Table 2, it is indicated that the root length of the un-inoculated plant was 5.17% higher than AMF þ plant, but other parameters such as fresh shoot weight and fresh root weight were enhanced by 20% and 11.83% in AMF þ plant, respectively compared to AMF À plants; however, the significant difference was at par with treatments at p < 0.05 (Table 1). Total fresh weight of AMF þ plant increased by 15% that that of AMF À plant (4.65 g/plant) ( Table 1) which was significant at F ¼ 7.0 and significance level p < .029. The total dry weight of the plant recorded 1.52 g/plant for AMF þ and 1.23 g/plant for AMF À showed a significant difference between treatment factors under WW condition at p < .05 (Table 2). A similar pattern was followed by shoot dry weight and root dry weight, showing their fresh weight attributes, which exhibited non-significant results with treatment factors, i.e. AMF þ and AMFÀ. The AMF colonization in roots of inoculated plants was found 47% and no colonization in AMF À plant under well-water conditions (Figure 1(F)). It was further proved with the higher mycorrhizal dependency in seedlings growing in well-watered conditions.

Effect on plant physiology
The plant physiology data of the experiment reveals that the AMF has a positive influence on plant physiology under WW conditions ( Figure 1). The photosynthesis value of the AMF þ plant was found to be 7.84 mmol/m 2 /sec, and it was 5.68 mmol/m 2 /s in plants without AMFÀ. So there was 38.02% higher photosynthesis with the inoculation of AMF þ in plants provided with WW ( Figure 1(A)). There was a significant difference in results between AMF þ and AMF À plants at F ¼ 23.33 and significance level p < .001. Stomatal conductance was also increased significantly by 58.33% as it was 0.20 mmol/m 2 /s for AMF À and 0.38 mmol/m 2 /s for AMF þ plants. Transpiration potential of plants with AMF inoculation was found to improve by 11.58% (non-significant at p < .05) (Figure 1(B)), but leaf temperature and intercellular CO 2 concentration between treatment factors did not show significant results under WW condition in D. sissoo. However, it was found to decrease the leaf temperature of plants with AMF þ compared to AMFÀ (Figures 1 (D,E)).

Effect on plant growth and biomass
The experiment data are tabulated in Tables 3 and 4, indicating higher plant growth and dry weight than non-inoculated plants under FW conditions. The plant height of AMF þ recorded 31.54 cm/plant and 26.41 cm/plant in AMFÀ, was significant at F ¼ 5.782 and p < .043. There was a 19.42% increment in the seedling height of AMF þ than AMF. Collar diameter also increased significantly by 70.52% in plants treated with AMFþ. However, as compared to other parameters that influenced positively with AMFþ, root length showed negative results than control plants (Table 4), resulting in non-significant difference due to treatment factors; however, the length of the root was increased in both AMF þ and AMF À plants in FW experiment compared to WW condition. Shoot length was also non-significant at p < .05, although a slight improvement was observed in AMF þ plants. The fresh root weight of AMF þ and AMF À was found 2.74 g/plant and 2.06 g/plant, respectively, with an increase of 33.0% root weight in the AMF À plant. Total fresh weight production of AMF þ enhanced by 19.95% significantly at F ¼ 8.42 and p < .02. In consequence of the non-significant value of fresh shoot weight, dry weight of the same parameter also produced non-significant results due to at par value between treatment factors. Root dry weight increased by 24.29%, while the total dry weight of plants with AMF þ and AMF À inoculation found 1.33 g/plant and 1.07 g/plant respectively showed a significant positive difference of AMF by 24.49% compared to AMF À plants (Table 4). AMF colonization in roots of inoculated plants found 56% and no colonization in AMF À plant under fractionated water condition (Figure 1(F)). Similar to WW condition, FW plants also showed 24.53% mycorrhizal dependency in furnishing higher growth and biomass compared to AMF À plants grown in entisol soil.
Effect on plant physiology AMF was found to improve physiological attributes of the D. sissoo under AMF þ followed by FW condition. Data of the results are summarized in the table (Figure 1 (A, B, C, D, & E)). The results reveal that the effect of AMF under the FW condition is excellent compared to the WW condition, as the photosynthesis rate was increased by 83.69% while it was 38.02% under the WW condition. The photosynthesis potential of the AMF þ plant was 9.24 mmol/m2/s, while the value of the same attribute for AMF À found 5.03 mmol/m 2 /s. This shows significance at F ¼ 30 and p < .001) (Figure 1(A)). Stomatal conductance increased significantly by 80% of the AMF þ seedling against AMF À seedling (Figure 1(B)). Under FW conditions, the transpiration rate of the AMF þ plant was 1.64 mmol/m 2 /s and in AMF À plant 1.38 mmol/m 2 /s (Figure 1(C)) depicts significantly higher transpiration (F ¼ 4.4, significance level p < .039) in the presence of AMF þ for maintaining higher physiological dynamics in the plant by providing adequate water to plant. Leaf temperature (Figure 1(D)) and intercellular CO 2 concentration (Figure 1(E)) rendered non-significant results under the FW experiment for D. sissoo.

Experiment C: no watering condition
Soil moisture Effect of no watering directly influenced soil moisture results in significantly decreasing soil moisture continuously with the increase in the day of no watering to both AMF þ and AMF À plants at F ¼ 35.04 and p < .05. AMF þ plants enable increased soil moisture in all the observations compared to non-inoculated AMF À plants experimented in entisol soil (Figure 2(A)). On the third day of no watering, soil moisture was found at 19.33, which decreased to 9.98% on the seventh day of no watering in AMF þ plants while it was 18.67% and 9.13% on the same days of no watering in plants without AMFÀ. The percent increase in soil moisture due to  AMF þ found 3.53% to 9.30% in 3 th day and seventh days of no watering to plants respectively was indicative of consistently increasing the effectiveness of AMF þ on this attribute which probably not derived by plants due to the absence of AM fungi under moisture stress condition. Overall the moisture loss was found less in AMF þ soil (93.68%) than non-AMF À plants (104.49%) in the last day observation as compared to their respective initial values recorded on 3rd days of no watering, thus saving soil moisture, mainly during adversities was found to be a great benefit of AMF þ plant. It was also observed that the decline in soil moisture was intense after the fifth day of no watering compared to 3th and 4th days, and due to this AMF þ role was found highly important. Similar to the other species of the experiment, soil moisture was found to decrease consistently with the increasing days of no watering also reflected and confirmed with regression correlation (Figure 2(A)) r 2 ¼ 0.980 and r 2 ¼ 0.984 for plants with AMF À and AMF þ respectively.

Photosynthesis
The results on this parameter of plants due to water stress is summarized and presented in (Figure 2(B)), revealing that AMF þ plants alleviated the rate of photosynthesis by 17.82% on the 3 rd day of no watering following increasing trends till the 6 th day where the increment was maximum of 91.34% and eventually started declining afterwards in the seventh day of no watering as compared to AMF À plants. The results were significantly higher in AMF þ inoculated plants than in AMF À plants at p < .05. It was recorded that the photosynthesis rate was 8.46 mmol/m 2 /s in AMF þ plants, which declined to its minimum level of 6.32 mmol/m 2 /s on the 7th day of no watering in plants with AMFþ. Similarly, plants without AMF À showed 7.18 mmol/m 2 /sec and 3.79 mmol/m 2 /sec in 3rd day and 7th day of no watering. Significantly positive relationship was observed in plants with AMF À and AMF þ with an increase in the days of no watering (r 2 ¼ 0.873 and r 2 ¼ 0.259 for AMF À and AMF þ respectively). The trend of linear lines and polygonal lines also depicts the positive role of AMF in the augmentation of plants' photosynthesis rate, especially under moisture-stress conditions (Figure 2(B)).

Stomatal conductance
These attributes also enhanced significantly due to AMF þ inoculation in plants under water stress conditions (Figure 2(C)). Stomatal conductance of plants AMF þ followed increasing trends from the third days (42.85%) to 5 days of no watering (80.95% after that, declining trends continued till the last observation, i.e. the 7th day of no watering as compared to AMF À AMF À plants. It was recorded that the stomatal conductance on 3rd day of no watering was 0.40 mmol/m 2 /s, reaching a maximum of 0.38 mmol/m 2 /s on the 5th day, and found 0.21 mmol/m 2 /s on the 7 th day of no watering plants. The results were significantly higher than AMF þ than AMF À AMF À treatment factor except in the 5th-day observation. Overall, AMF þ effectively increased the stomatal conductance of D. sissoo plants grown under water stress in entisol soil. Stomatal conductance showed declining trends with the increase in no-water days, resulting in a significantly positive correlation r 2 ¼ 0.939 and r 2 ¼ 0.609 for AMF À AMF À and AMF þ plants, respectively.

Transpiration rate
The effect of treatment factor, i.e. AMF found to follow inconsistent trends of transpiration, results in a non-significant difference of treatment factor at p < .05 level, however, this attribute on 6 th days of no watering rendered significant results at p < .01 (Figure 2(D)). The alleviated rate of transpiration in AMF þ plants was observed between 5.55 to 26.76% compared to AMF À AMF À plants, indicating the importance of AMF on regulating transpiration, especially under water stress conditions. On the 3rd day of observation after no watering, the rate of transpiration was recorded 2.39 mmol/m 2 /s, which decreased to 1.17 mmol/m 2 /s on the 7 th day of no watering in AMF þ plants while the same attribute was recorded 1.98 and 1.04 mmol/m 2 /s in 3rd and seven days of no watering respectively in AMF À plants. However, the transpiration rate of the plants followed declining trends continuously in both AMF þ and AMF À AMF À plants and showed significance at F ¼ 7.25, at p < .00. Plants with AMF þ rendered a higher rate of transpiration compared to AMF À AMF À plants also exhibited a significant positive correlation between transpiration and an increasing day of no watering (r 2 ¼ 0.981 and r 2 ¼0.970 for AMF À AMF À and AMF þ plants, respectively) Leaf temperature AMF showed moderating effects on leaf temperature results in lowering the temperature compared to AMF À AMF À plants without any inoculation (Figure 2(E)); however, the results due to treatment factor were statistically non-significantly. However, when increasing watering days were considered, it gave significant results at F ¼ 2.39 and a significance of p < .00. The plants inoculated with AMF þ experienced lower temperatures on the leaf ranging from the lowest of 0.35% to the highest of 0.94% compared to AMF À plants was indicative of the increasing effectiveness of AMF due to increasing water stress to plants. The initial leaf temperature was recorded 31.17 C and 31.28 C in plants with and without AMF on the third day of no watering, which increased from 32.74 to 33.01 C with the same treatment, respectively (AMF þ ad AMF À plant) after the seven th days of no watering. There was an increasing trend as leaf temperature was concerned about the increase in days of no watering to the plants. The correlation between leaf temperature and no watering days was found positive in both the plants grown with AMF þ and AMF À AMFÀ (r 2 ¼ 0.778 and r 2 ¼ 0.875 for inoculated and non-inoculated plants, respectively) Internal cellular CO 2 concentration This attribute was consistently found to decrease with the increasing days of no watering both in AMF þ and AMF À plants under water stress conditions of D. sissoo (Figure 2(F)); however, the difference in data is due to treatment effects was non-significant at p < .05. Similar to leaf temperature results, the internal cellular CO 2 concentration remarkably lowered due to inoculation of AMF þ as compared to AMF À plants. These attributes recorded 375.80 and 367.20 mmol/m 2 /s for AMF À and AMF þ plants respectively on the third day of no watering, which decreased to 332.0 and 315.05 mmol/m 2 /s respectively from their third-day values after the seventh day of no watering observation. This result reveals that the effectiveness of AMF þ increases with the increasing days of no watering, i.e. increasing stress to plant for water. The intercellular CO 2 content of AMF þ plants showed a net decrease of 2.34% on 3rd day of observation following increasing trends with the increasing days of no watering and reaching the highest level of 5.39% on the 7th day of no watering to the plant. The overall decline in internal CO 2 was 17.09% in AMF þ plants while it was 13.19% in AMF À plants, indicating the role of AMF in rapid modifying and lowering of internal CO 2 from leaves of plants compared to AMF À plants. Linear relation and a polygonal line drawn in both the AMF À and AMF þ showed a significant positive relation between inter Cellular CO 2 and days after no watering (r 2 ¼ 0.970 and r 2 ¼ 0.957 for AMF À and AMF þ plant, respectively).
Water plays a crucial role in the plant's function; they maintain cell turgidity for structure and growth, transports nutrients throughout the plant and serves as a raw material for various physiological processes, including photosynthesis and transpiration (McElrone et al. 2013). Water deficit influences plants' growth, development, and physiological activities (Barry et al. 2004;Pavel and de Villiers 2004;Ma et al. 2006;Luvaha et al. 2008). One of the major factors in plant mortality is water stress, which causes a decrease in leaf water potential and stomatal opening, resulting in down-regulation of photosynthesis-related genes and reduced CO 2 availability (Osakabe and Osakabe 2012;Ahanger et al. 2021;Khan et al. 2022). Non-nutritional mechanisms include hormone effects brought on by mycorrhizal colonization (via abscisic acid), improved soil-hyphal contact (particularly crucial during soil drying), more efficient water scavenging in micropores, direct water uptake by hyphae, and increased photosynthesis through sink stimulation (Smith et al. 2010;Wu et al. 2020;Gupta et al. 2021). Water stress harms plant growth. As a result, increasing plant survivability and growth during water stress is an important goal in the plant sciences (Osakabe et al. 2014). Plants with AM fungi help in improving growth and biomass development, especially under drought and moisture stress conditions (Smith et al. 2010;Zhu et al. 2012;. Entisol soil is known for its water stress and poor nutrients, causing a significant reduction in plants' survival, growth, development, and physiological activities and eventually failure of plantations. In the present investigation, the effects of water stress and its influence on the growth and physiological activities of D. sissoo were carried out to determine the effects of AMF mitigating the impact of water stress on the plant's average growth and physiological functioning under WW and FW conditions. Results showed that D. sissoo seedlings formed symbiotic relationships with AMF under WW and FW conditions, which resulted in significant improvement in the growth and biomass of the plants. It was also noticed that the AMF colonization was 9% higher in D. sissoo under the FW condition compared to the WW condition, which shows the strong symbiotic relationship with plants when experiencing water stress. However, the impact of adversities due to FW condition was exhibited in both the tree species on plant characteristics, viz. height, fresh weight, and dry weight, and the stress was mitigated via higher root colonization in plant roots grown in the presence of AMF þ more efficiently compared to AMF À plants under FW condition. It was confirmed by recording the higher mycorrhizal dependency of plants in FW conditions than in WW conditions in the species. Most of the parameters related to plant growth and biomass exhibited higher values in the presence of AMF þ under the WW condition in D. sissoo used in the experiment. The result of the significant positive role of AMF in plant growth by which more efficient uptake of water and nutrients was absorbed via the hyphal network (Smith and Read 2008;Zhu et al. 2012;Shi et al. 2016). The findings of the present study were also confirmed by Wu and Xia (2006), Fidelibus et al. (2001), Dell-Amico et al. (2002, Wu et al.(2008), Asrar andElhindi (2011), andShi et al. (2016). Moreover, enhanced biomass by mycorrhization could significantly increase the absorption surface and thus nutrient uptake capacity under FW conditions, as was also reported by Shi et al. (2016).
The influence of AM fungus on physiological activities was studied under WW and FW conditions. The results showed that the FW condition significantly decreased photosynthetic rate, stomatal conductance, transpiration rate, and intercellular CO 2 concentration but increased leaf temperature in plants without AMF in D. sissoo than the WW condition. In contrast to AMF À plants, mycorrhizal plants showed a significantly higher photosynthesis rate, stomatal conductance, and non-significant but higher transpiration and intercellular CO 2 concentration, while leaf temperature was lower regardless of water treatments. The positive effects of AMF in ameliorating physiological activities in any condition (Figure 1) indicate positive effect of AMF in any condition. Interestingly, D. sissoo photosynthetic rate increased by 17.85% in AMF þ plants treated with FW compared to WW plants, whereas D. sissoo's stomatal conductance and transpiration rate decreased by 5.55 and 11.58%, respectively, in AMF þ plants treated with FW as compared to WW plants treated with AMFþ. This influence of AMF þ plants was due to a higher degree of symbiosis in plant roots under the FW condition than the WW condition, which enables plants to absorb water optimally as per the plant's demand for normal physiological functioning even under water stress conditions such as the FW condition. Kong et al. (2020) also suggested that the rate of gas exchange plays an important factor in plant growth by increasing the rate of photosynthesis in mycorrhizal plants than in nonmycorrhizal plants, suggesting that AMF colonization increases the number of photosynthesis units and the rates of photosynthetic storage and export. Similar observations were also made by Zhu et al. (2012) in Zea mays under drought stress conditions and indicated that AM symbiosis could enhance the photosynthesis and increased transpiration fluxes indicated that AMF þ plants were able to keep the stomata open longer than AMFÀ. Our findings show that the efficiency of AMF increases with increasing levels of water stress, as demonstrated by WW and FW conditions, which is also supported by Porcel and Reiz-Lozano (2004), who reported that AM symbiosis could improve the water status of the host plant, resulting in higher leaf water potential under drought conditions compared to nonmycorrhizal plants.
In the present investigation, drought stress [FW] markedly increased leaf temperature in AMF À plants, while the same attribute decreased with AMF inoculation in D. Sissoo under WW conditions. The same might be due to high leaf water potential during FW conditions due to AM symbiosis, also ascribed by Porcel and Ruiz-Lozano (2004). Mycorrhizal plants' better water status may be due to external hyphal extraction of soil water, stomatal regulation through hormonal signals (Cheng et al. 2021), more significant osmotic adjustment (Kapoor et al. 2013), and higher hydraulic conductivity (Fan and Liu 2011) than non-AMF À plants. The current results also agree with previous reports (Ahanger et al. 2018;Ganugi et al. 2019;Mathur et al. 2019;Begum et al. 2019), emphasizing a higher rate of photosynthesis and stomatal conductance in the presence of AMF plants than non-AMF plants under water stress.
Further confirming the benefits of AMF on the regulation of physiological characteristics under acute water shortage, it was examined by stopping/no watering in D. sissoo plants grown in entisol soil under nursery conditions, and the results are presented graphically (Figure 2). High water stress was created by not watering the plants, and physiological activities were recorded from 3 days of no watering till the senescence of plants, i.e. the 8th day of no watering in both AMF þ and AMF À plants. Wilting symptoms occurred just after three days of no watering in AMF À plants, but in AMF þ plants, this stage was observed after the 5th day of no watering. These indicated the higher survival rate of mycorrhized plants with improved water use efficiency and drought resistance of AMF þ plants. Longer stomatal openings and higher photosynthesis rates of the plant may be due to hyphae that penetrate pores inaccessible to roots beyond the root zone (Izanloo et al. 2008) and the effectiveness of AMF for better exploitation of bound water in dry conditions (Smith and Read 2008), and AMF can sometimes provide soil water below the plant's permanent wilting point (Sun et al. 2018).
In the current experiment, regardless of the AMF treatment factor, no watering significantly reduced soil moisture percentage in the plants. However, soil moisture was enhanced with the inoculation of AMF þ to plants, significantly at p < .05 as compared to control plants. Soil moisture decrease significantly in a gradual manner with the increasing days of no watering and, overall, more than 85% of soil moisture was lost between 3 and 8 days of no watering, which showed a strong positive correlation also for the D. sissoo which consequently resulted in wilting of plants irrespective of treatment effect (Figure 2(A)). Concurrently, Li et al. (2019) and Hashem et al. (2019) have reported the importance of AMF on soil aggregate stability and high moisture content due to binding through an extensive hyphal network as well as the release of glomalin to the soil, which plays a paramount role in soil moisture conservation, especially under moisture stress conditions exploited efficiently by AMF þ plants as compared to non-AMF À plants.
Interestingly, no watering in AMF þ plants was found to enhance the rate of photosynthetic and stomatal conductance results in consistently increasing trends even on the 5th and 4th days of no watering, respectively, for these two attributes as compared to AMF À plants, which showed significantly decreasing trends in photosynthesis and stomatal conductance. Nonetheless, the strong positive correlation between photosynthesis and days without watering for AMF À plants (r 2 ¼ 0.873 and 0.875 for AMFÀ(control) and a comparatively weak relationship in plants with AMFþ (r 2 ¼ 0.259 and 0.278 for AMF þ plants) confirms the vital role of AMF during stress time in maintaining and regulating lifesaving activities, namely photosynthesis rate. Similarly, stomatal conductance also found similar trends in AMF þ and AMF À plants, which agrees with results reported by other researchers (Zhu et al. 2012;Shi et al. 2016). However, after five days of no watering, AMF þ plants also could not maintain the same pace of photosynthesis and started to decline gradually, while it was exponential in control plants. Iqbal et al. (2015); Hashem et al. (2019); Shi et al. (2016) also noticed similar results due to higher leaf chlorophyll, carotenoid, and photosynthesis under AMF association, making it possible to have higher C fixation and carbohydrate accumulation.
In contrast to the abovementioned attributes, the transpiration rate of AMF þ plants was, however, higher than that of AMF À plants but declined consistently with the increasing days of no watering. The whole experiment demonstrates the positive role of AMF in mitigating the adverse effects of water stress, thus alleviating the rate of transpiration in AMF þ plants. In the current study, leaf temperature was recorded lower in AMF þ plants than non-AM once they made a congenial environment for essential physiological activities during acute water stress conditions. However, after that, AMF was also unable to reduce the surface temperature of the leaf, resulting in a consistent increase in leaf temperature with increasing days of no watering in both AMF þ and AMF À plants. This may be the combined result of AMF symbiosis and the high water use efficiency of plants by maintaining enhanced water potential in plants and leaves, as also ascribed by Abbaspour et al. (2012). They have demonstrated how AMF þ plants tolerate drought stress by accumulating high organic solutes and sugars to regulate the osmotic potential of cells. This result reveals that AMF þ plants have better managerial skills than non-AMF plants during water stress times, resulting in a higher survival rate, drought resistance, and delayed senescence even after no watering. Intercellular CO 2 concentrations varied with AMF treatment under WW and FW conditions but declined with each day of no watering in D. sissoo regardless of the treatment factor. However, AMF-treated plants were found to have a lower rate of CO 2 concentration than values recorded from AMF À planted plants on the same day of no watering. These results were consistent with those from maize alfalfa, citrus, and Caucasian Hackberry regardless of water and salt stress conditions (Zhu et al. 2012;Campanelli et al. 2013;Wu et al. 2013;Sepahvand et al. 2021;Meddich et al. 2021) and even in rocky areas (Chen et al. 2014).

Conclusion
Water is scarce in entisol soil, and its scarcity impacts plant development, resulting in a high level of AMF interaction with the plant to develop a mechanism for efficient water use. The plant growth attributes and physiological activities in D. sissoo were evaluated under three conditions: WW (well watering), FW (fractionated watering), and SW (stopped/no watering), and the results show that the FW condition has a higher mycorrhizal dependency of 24.53% than the WW condition (24.37%). In D. Sissoo, AMF root colonization was higher at 56% and 47% under FW and WW conditions. These findings highlight the importance of AMF, mainly when plants are under water stress. Compared to WW, the photosynthetic rate of D. sissoo and AMF þ plants increased by 17.85% when FW was applied. The plant's physiological activities were altered by AMF inoculation, which showed significantly higher photosynthesis rate, stomatal conductance, and non-significant but higher transpiration and intercellular CO 2 concentration. At the same time, leaf temperature was lowered regardless of WW and FW conditions, indicating that AM positively affects physiological activities. The linear correlation between photosynthesis and days of no watering days showed very strong for AMF À plants (r 2 ¼ 0.875 for AMFÀ (control) and a comparatively fragile relationship in plants with AMFþ (r 2 ¼ 0.278 for AMF þ plants) confirms the vital role of AMF during stress time in maintaining and regulating lifesaving activities. The study confirms the benefits of AMF in entisol soil for ameliorating the adverse edaphic condition and improving plants' growth and biomass.