Water scarcity caused by global warming is becoming more common, posing a huge challenge to agricultural production, and resulting in large-scale crop losses and food shortages for mankind (Zhang et al., 2018; Rivero et al., 2022). Plants have evolved a series of mechanisms to cope with drought stress, such as prolonged root growth, regulating stomata movement, and maintaining the osmotic balance of cells (Mathur and Roy, 2021). In addition, plants also use a variety of enzymatic and non-enzymatic antioxidants to efficiently and rapidly remove reactive oxygen species (ROS) (Fang and Xiong, 2015; Khan et al., 2021).

Plant growth-promoting rhizobacteria, such as arbuscular mycorrhizal (AM) fungi, play an important role in alleviating drought stress in plants (Vurukonda et al., 2016; Porter et al., 2020; Abdelaal et al., 2021). AM, act as an extension of the plant root system, provide their host plants with access to soil water and nutrients through far-reaching extra radical hyphae, while fungi are provided with carbon from host plants (Gutjahr and Paszkowski, 2013; Frew et al., 2022). Most importantly, this symbiosis relationship creates a functionally active ecosystem that influence nutrient cycling, decomposition, soil aggregation, belowground biodiversity, and plant community ecology (Kaushal, 2019; Zhang et al., 2020). Under drought stress, AM reduced the damage of free radicals by promoting nutrient absorption capacity, improving root function, maintaining leaf photosynthetic efficiency (Lenoir et al., 2016). In addition, extrapular-root mycelium formed a huge mycelium network in the soil, increasing root xylem conductance and improving the adaptability of water transport (Abdelaal et al., 2021; Sun et al., 2022). Meanwhile, the polysaccharides produced by fungi also have the ability to increase mineral strength and retain water (Hooker et al., 2007).

Melatonin (N-acetyl-5-methoxy-tryptamine), as a master regulator, participant in plant development and protects plant against almost all abiotic stresses, such as drought, salt damage, heavy metals, UV radiation, high temperature, cold damage, etc. (Arnao and Hernández-Ruiz, 2019; Sun et al., 2021). Melatonin ameliorates the adverse effects of drought by regulating morphological, physiological and redox regulatory processes, and a variety of regulatory mechanisms have been explored in recent years (Tiwari et al., 2021). Most fundamentally, melatonin has the strong antioxidant activity to remove ROS and free radicals caused by stress, by improving antioxidant enzymes activities, such as ascorbic acid, ascorbate peroxidase, superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD), and the content of antioxidants such as flavonoids (Huang et al., 2019). Melatonin also affects the nutrients absorption from soil by regulating the activity and expression of nutrient transporters (Xia et al., 2021; Arnao et al., 2022; Du et al., 2022; Sun et al., 2022). Melatonin was also found can solve the replant problem by altering the composition of bacterial and fungal communities (Li et al., 2018), and promoted the primary root growth in Arabidopsis (Yang et al., 2021).

Kiwifruit (Actinidia chinensis Planch.) is an emerging fruit tree grown worldwide, but it is more vulnerable to drought than other fruit trees because of its shallow, fleshy root system and large, hairy leaves (Liang et al., 2019). It has been confirmed that melatonin pretreatment can increase the biomass of kiwifruit seedlings by improving leaf photosynthetic capacity and carbon fixation, and improve the drought tolerance of kiwifruit seedlings by promoting root elongation and growth and increasing antioxidant content (Liang et al., 2019; Xia et al., 2020). However, whether melatonin and AM have synergistic effects on drought resistance in plants is still unknown. In this study, AM fungi and melatonin were applied to kiwifruit seedlings alone or in combination to investigate the effect on plant growth, photosynthesis, antioxidant enzyme activity, nutrient absorption, in order to further find a better scheme to improve the drought tolerance of kiwifruit for field cultivation.

Improving crop drought resistance and reducing yield loss have always been the focus of global agricultural production (Zhang et al., 2021). In addition to breeding resistant varieties and mining resistance genes afford by scientists, improving plant water use efficiency and resistance by use of exogenous stimulants or bacterial agents seems to be more effective way to conquer abiotic stresses brought about by climate change and thus is favored by farmers (Farooq et al., 2019).

Increasing evidence studies have demonstrated that MT application and AM fungi inoculation have positive effects on plant growth under drought conditions (Liu et al., 2020; Bolin et al., 2022). AM fungi alleviate drought stress through several mechanisms (Abdel-Salam et al., 2017). One most important is that AM fungi induce changes in plant root structure, especially the increase of root length, root surface area, root volume and root tip number. Improvements in the root structure of mycorrhizal plants allows extraparal hyphae to extend beyond the barren areas of the plant rhizosphere, thus more efficiently absorbing water and low-mobility mineral nutrients under water scarcity conditions (Müller, 2021; Persi and Maherali, 2021). Melatonin, on the other hand, promotes the growth of lateral root (Bian et al., 2021). In this study, both melatonin and AM application alone reduced the decline of dry biomass ( Figure 2 ). However, inoculation with AM fungi had a greater effect on increasing root biomass than melatonin, melatonin increased shoot biomass significantly, and their combination had the strongest effect, which was consistent with previous study (Liu et al., 2020). These results indicated that AM fungi and melatonin may drive different mechanisms to alleviate drought stress (Yang et al., 2020).

Plants rely on chlorophyll for photosynthesis to accumulate biomass (Nozue et al., 2016). Under drought stress, their cells undergo plasmid lysis, chloroplast membrane rupture, ultrastructural damage, and chlorophyll synthesis decreases and degradation accelerates, leading to the reduction of its content and photosynthetic capacity, which further affects plant growth and yield (Tambussi et al., 2000; Munné-Bosch et al., 2001). In this study, melatonin treatment and AM fungi inoculation increased the content of chlorophyll a, and b in kiwifruit seedlings under drought stress ( Figure 4 ), which was inconsistent with previous studies (Liang et al., 2019; Xia et al., 2020).

Under drought stress, plants increase water use efficiency mainly by closing stomata to reduce transpiration, but preventing carbon dioxide from entering mesophyll cells leads to a decrease in photosynthetic rate Pn, which is known as a stomatal limiting strategy (Du et al., 2018). Our results also confirmed a synchronously decline of Pn, Gs, Tr, and Ci under drought stress. However, the application of melatonin and AM did not significantly promote Pn, Gs, Tr, and Ci under drought stress, which was different from previous results (Liang et al., 2019), possibly because the weakened effect due to prolonged treatment time. Interestingly, the combined application of MT and AM had a greater effect on increasing Pn, Gs, Tr, and Ci, indicating an obvious superposition effect, as in study of tobacco (Liu et al., 2020).

Recent studies have found that melatonin enhances plant uptake of nutrients. For example, the concentrations of nitrogen, potassium, copper, iron, and zinc in grape fruits were improved by MT supplemental (Xia et al., 2021). Mycorrhiza can obviously promote the absorption of N, P and Fe, make the seedlings more conducive to nutrient growth and alleviate the damage caused by stress to plants (Chandrasekaran, 2022). In this study MT and AM both increased significantly the concentration of P in seedling leaves under watering and drought condition, importantly, which contributed to drought tolerance enhancement (Begum et al., 2020; Bechtaoui et al., 2021). PHT1 (phosphate transporter 1) family genes play an important role in regulating plant growth and coping with stress (Cao et al., 2020). Our results showed that the expression of all six PHT1 genes significantly increased after MT and AM induction. It was speculated that MT and AMF treatment can alleviate drought damage by inducing high expression of PHT1s to improve P uptake.

In addition to promoting plant uptake of nutrients in plants, melatonin supplementation also altered the composition of bacterial and fungal communities in the soil, alleviating the effect of apple diseases (Li et al., 2018). Herein, we further confirmed that melatonin could not only promote the uptake of soil nutrients by itself, but also expand plant uptake of distal soil nutrients by strengthening the symbiosis between arbuscular mycorrhizal fungi and roots and increasing colonization. However, different from the previous study on tobacco (Liu et al., 2020), melatonin and mycorrhizal seem to have no additive effect on nutrient absorption, especially in the mineral elements Fe, Zn and Cu, whose contents in the mixed treatment group decreased instead. It is speculated that they may have some competitive or antagonistic mechanism in their nutrient absorption to avoid metal poisoning.

In our study, melatonin and mycorrhiza have additive effect on improving plant growth under drought, and increasing antioxidant enzyme activity, chlorophyll content, osmotic regulating substance, photosynthetic capacity, and reducing MDA and REL. This is largely due to their different mechanisms for performing functions. As a broad-spectrum antioxidant, melatonin itself has the ability to remove ROS and prevent its accumulation in cells (Yu et al., 2018; Arnao and Hernández-Ruiz, 2019). At the same time, it can improve the activity of antioxidant enzymes and the content of antioxidant substances under adversity conditions, thus greatly reducing the damage caused by drought stress (Xia et al., 2020). On the other hand, melatonin, as an auxin precursor, has a similar function of promoting plant growth (Hernandez-Ruiz et al., 2005; Szafrańska et al., 2013). While mycorrhiza reduced the stress damage mainly by promoting nutrient absorption capacity, improving root function (Lenoir et al., 2016). Because extrapular-root mycelium formed a huge mycelium network in the soil, increasing root xylem conductance and improving the adaptability of water transport (Abdalla and Ahmed, 2021; Sun et al., 2022). Most important, in this study, melatonin was found to promote the establishment of a symbiotic relationship between arbuscular mycorrhizal fungi and roots.

Under drought conditions, AM inoculation was more effective than MT in promoting root biomass, soluble sugar content, and absorption of N and Fe nutrients. MT has a better effect in improving shoot biomass and the activity of antioxidant enzymes to remove ROS accumulation. Moreover, combination of mycorrhizal and MT promotes the root systems to form a symbiotic relationship. In addition, the combination of AM and MT treatment had an additive effect on improving plant growth, antioxidant enzyme activity, chlorophyll content, osmotic regulating substance, photosynthetic capacity, and reducing MDA and REL. Our results indicated that MT and AM fungi have a synergistic effect on improving drought tolerance, which provides a new strategy for the widespread application of biological modulators to improve crop drought tolerance.

The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.

HX and DL conceived the idea and designed the research; CY, YL, ZH and YG performed the experiments with the help of YL, HD and XT; CY, JW and XL analyzed the data; CY and HX drafted the manuscript; YuL and DL revised the manuscript, other authors provided suggestions for the revision of the manuscript. All authors contributed to the article and approved the submitted version.

This work was supported by fund received from the Sichuan Science and Technology Program (2022YFH0049 and 2022-YF05-00655-SN). We also thank the fund support from the Engineering Technology Research Center of Biological Resources Conservation and Utilization of Inner Mongolia (21400-222526).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.