The Mitigating Effects of Biostimulant Amendments on the Response of Purslane Plants Grown under Drought Stress Conditions

Abstract

Portulaca oleracea L. is a wild edible plant with high potential for exploitation in commercial cropping systems due to its nutritional value and great adaptability to abiotic stress conditions. The present study aimed to investigate the response of purslane plants grown under drought stress conditions (100%, 80%, and 60% of field capacity (FC)) and the implementation of biostimulant amendments (control without amendment, plant growth-promoting rhizobacteria (PGPR), mycorrhiza, and effective microorganisms (EMs)) for two consecutive years. In the two-year experiment, the greatest height was recorded in plants grown under no-stress conditions and inoculated with PGPR. The highest branch number, and fresh and dry weight of aboveground and underground parts were observed under no-stress conditions at the mycorrhiza treatment. Moreover, mycorrhiza application in plants growing under 100% FC resulted in the highest N, P, total carbohydrates, and vitamin C and the lowest nitrate and proline contents in leaves. Purslane plants grown under 100% FC and inoculated with PGPR treatment resulted in the highest K and total chlorophyll leaf contents. Additionally, growing plants under mild drought stress (80% FC) combined with biostimulant application (e.g., inoculation with mycorrhiza, PGPR, and EM) may improve plant growth characteristics and mitigate negative stress effects. In general, the applied biostimulant amendments alleviated the adverse effects of drought on plant growth and leaf chemical composition indicating the importance of sustainable strategies to achieve high yield and sufficient quality within the climate change scenario.

1. Introduction

Purslane (Portulaca oleracea L.) is a wild edible plant widely distributed around the globe and it is among the three most frequently reported weeds [1]. However, it is also an easy to grow edible green owing to its heat and drought tolerance [2]. The leaves and shoots of the plant can be eaten fresh, cooked, or dried, and recently there has been an increasing interest in its commercial cultivation due to its high content of health-beneficial compounds such as omega-3 polyunsaturated fatty acids and antioxidants [3,4,5]. It also might have medicinal properties against cancer, heart disease, and several other chronic illnesses and ailments that affect modern people [6,7,8], while it can contribute to a balanced intake of biologically active compounds, minerals, and essential nutrients [9,10]. Purslane has been proven to be more salt and drought tolerant than conventional vegetable crops [11,12,13] due to effective protective physiological mechanisms that allow plants to cope with abiotic stress conditions [14].

Environmental stressors such as extreme temperatures, water stress, high salinity, flooding, and pollutant toxicity may negatively affect plant growth [15], while water stress has been identified as the most significant abiotic stressor with severe implications on crop production worldwide [16]. Drought or soil salinization due to anthropogenic activities are the main causes of water shortage, which consequently results in water stress. Every year, drought incidents affect various regions of the world, especially the arid and semi-arid regions, as well as other areas which exhibit irregular precipitation that cannot cover crop-irrigation requirements [17].

When plants are subjected to drought stress, they have lesser water potential and turgor; therefore, cell enlargement is reduced, resulting in growth inhibition and low crop yield [18]. Drought may hinder plant respiration, photosynthesis, and stomatal function with further implications on plant morphology, the expression of water stress-resistant genes, and the synthesis of hormones (e.g., abscisic acid—ABA), osmolytes and inorganic ions (e.g., sucrose, proline, and betaine) that allow plants to cope with drought stress [19,20,21]. Therefore, water deficiency affects various biochemical, morphological, and physiological processes, resulting in reduced growth and yield [20,22,23]. Considering that water shortage is a pivotal challenge for modern crop production, sustainable practices should be adopted to improve the productivity and quality of crops within the climate change scenario [24]. One recently adopted approach to attain crop resistance against environmental stressors, and ultimately to promote health and plant growth is microbial inoculation, fostering symbiotic relationships between beneficial microorganisms and plants [25,26].

Plant growth-promoting rhizobacteria (PGPR) are bacteria that colonize the roots of the plants [27], and they may trigger a range of direct and indirect mechanisms to counteract the impacts of water stress and allow plants to survive under stressful conditions [28]. In particular, PGPR facilitate plant growth and production through direct mechanisms such as nitrogen fixation, potassium and phosphorus solubilization, improved uptake of minerals by acquiring nutrients [27,29,30], the synthesis of hormones such as auxins, gibberellins, siderophores, the induction of osmolytes and antioxidant compounds production, and the regulation of stress-responsive genes [31]. Therefore, PGPR can function as biostimulants/biofertilizers that directly stimulate plant development through a wide range of various biochemical and physiological processes and facilitate plants to overcome and adapt to drought stress [32,33]. In addition, effective microorganisms (EMs) are an environmentally eco-friendly practice that refers to the application of a fermented mixed culture of coexisting and mutually compatible microorganisms in an acidic medium [34], such as Lactobacillus, Streptomyces, actinomycetes, and yeasts [33]. Effective microorganisms were firstly chosen as an alternative to crop pesticides; however, field studies established their effective uses in various areas including their use as biofertilizers. They have been used through soil and foliar applications to stimulate plant growth and production, while they can boost photosynthetic effectiveness by enhancing the availability of nutrients and increasing the root biomass [33]. Soil addition of EM also improves the physical, chemical, and biological properties of soil, since it increases soil organic matter content, cation exchange capacity, and the availability of mineral nutrients; furthermore, biofertilizers may reduce the excessive use of chemical fertilizers and other inputs [35].

Microbial formulations based on arbuscular mycorrhizal fungi (AMF) are also important biostimulants resulting in crop and yield improvement and in better, healthier, and higher-functionality foods in sustainable agricultural systems [36]. Arbuscular mycorrhizal fungi are beneficial for soil structure and they can increase the activity of soil-beneficial microorganisms, which can eventually facilitate resistance of plants to drought and disease [37]. For example, they may alter soil chemical and physical structure, enhance root surface area, maintain soil fertility, improve N fixation, increase water and nutrient uptake, improve plant enzymatic functions and photosynthesis, and enhance plant response to root pathogens among other functions [38,39,40]. Among the most significant impacts of AMF is the capacity to solubilize P and micronutrients in nutrient-deficient soils through its hyphae and improve their availability to growing plants [33,35], since fungal hyphae can penetrate small pores and uptake more nutrients than the root [41]. Moreover, several studies indicated that apart from plant growth AMF can induce plants’ secondary metabolism and enhance the content of health-promoting phytochemicals such as sulfides, polyphenols, phytosterols, stilbenes, vitamins, lignans, and terpenoids [42]. Therefore, the present study investigated the effect of biostimulant application (PGPR, EM, and mycorrhiza) on growth parameters and chemical composition of purslane plants cultivated under drought conditions aiming to assess the potential of using of these biostimulants as an eco-friendly and sustainable agronomic practice within the context of the climate change scenario.

2. Materials and Methods

2.1. Experimental Conditions and Layout

Pot experiments were carried out during the summer season of 2022 and 2023 at the experimental farm of Benha University, Qalubia Governorate, Egypt (30°16′17.2″ N 30°46′20.4″ E) aiming to study the response of purslane plants grown under water stress conditions combined with biostimulant amendments. The average temperature, precipitation, and relative humidity values throughout the experimental period ranged between 16–38 °C, 1.8–5.4 mm, and 55–69%, respectively (

Table 1. Meteorological data for the experimental area during the 2022 and 2023 growing seasons.

Months	2022
	Maximum Temperature (°C)	Minimum Temperature (°C)	Relative Humidity (%)
April	29	15	49.2
May	32	18	53.8
Jun	36	21	60.2
July	38	23	65.5
	2023
April	31	15	49.5
May	32	17	55.2
Jun	36	21	65.4
July	37	22	72.5

). The soil pH was 7.72, and the texture was clay loam. The physicochemical parameters of the soil are presented in

Table 2. Physical and chemical parameters of the soil used in the two growing seasons.

Physical Parameters		Chemical Parameters
		Cations		Anions
Coarse sand	7.1%	Ca2+	6.26 mEq/L	CO3−	0.00 mEq/L
Fine sand	17.3%	Mg2+	3.02 mEq/L	HCO3−	3.92 mEq/L
Silt	22.3%	Na+	5.36 mEq/L	Cl−	4.42 mEq/L
Clay	53.3%	K+	0.93 mEq/L	SO4−	7.38 mEq/L
Clay loam
Soil pH	7.72	Available N		26.4 mg/kg
E.C	1.48 dS/m	Available P		8.73 mg/kg
Organic matter	1.59%	Available K		119.9 mg/kg

. Mechanical analysis was conducted following Jackson [43], while the chemical analyses were carried out according to Black et al. [44]. Soil pH and EC were assessed in the soil paste, whereas organic matter content was evaluated using potassium chromate and then titrated by ferrous sulfate [44].

For pot experiments in both seasons, a local purslane (Portulaca oleracea L.) variety was used (Egyptian purslane, cv. Balady). Sowing of at least three seeds per pot was conducted in 25 cm plastic pots on 1 April for two successive growing seasons (2022 and 2023). Fifteen days after the sowing, the seedlings were thinned to leave one seedling per pot. For the next ten days, seedlings were irrigated with tap water (180 mg/L of CaCO₃) until the full establishment of plants without fertilization

A two-factor experiment was conducted in a complete randomized block design (CRBD) with three replicates (each replication consisted of twelve pots). Drought and biostimulants were the first and second factors, respectively. The drought stress levels were 100% of field capacity (FC), 80% of FC, and 60% of FC, while the biostimulant treatments included the control without soil amendments, PGPR, mycorrhiza, and EM. Therefore, the experiments included 12 treatments and 144 pots in total (3 drought stress levels × 4 biostimulant treatments; 12 pots per treatment).

2.1.1. Drought Stress Levels

The experimental treatments of drought stress were based on the Field Capacity (FC) and included the following:

• 100% of FC;
• 80% of FC;
• 60% of FC.

Soil FC was determined according to the modified funnel method [45]. In particular, soil samples were placed in a funnel (100 mL volume) with a filter paper at the bottom. The funnel was mounted on the top of a volumetric cylinder (500 mL volume). Then 100 mL of water was added to each funnel and left to drain for 72 h. The amount of water collected in the volumetric cylinder was recorded to calculate FC. In this respect, WFC of the used soil in this experiment was 29.32%, which means that every 100 g of the used soil needs 29.32 mL water to reach 100% WFC. The weight of soil used to fill each pot was 3.800 g. Therefore, in order to reach 100% WFC, the soil was irrigated with 1114 mL tap water every time at 4-day intervals, while 891 and 668 mL of tap water were used to reach 80% and 60% WFC, respectively. Drought stress initiated after seedling emergence (approximately 7 days after sowing). The experiment lasted 65 days, and each pot received 18.1 L, 14.5 L, and 10.9 L of tap water for the treatments of 100%, 80%, and 60% WFC, respectively.

2.1.2. Biostimulants

The biostimulant formulations (except for mycorrhiza) were added to the soil three times in all pots, firstly at sowing and at 15-day intervals. In each application, 200 mL of the formulations were added in each pot.

The treatments were as follows:

• Control: without soil amendment;
• Plant growth-promoting rhizobacteria (PGPR);
• Mycorrhiza;
• Effective microorganisms (EMs).

The plant growth-promoting rhizobacteria were kindly provided by the Microbiology Department, Faculty of Agriculture, Benha University, Benha, Egypt, and included potassium-dissolving bacteria (Potassine), such as Bacillus circulans, or potassium-mobilizing bacteria. The inoculum (Bacillus circulans) was prepared by growing the bacterial strains in a nutrient broth medium under continuous shaking (140 rpm) at 30 °C. Then, the inoculum was collected after centrifugation (4000 rpm) at 4 °C for 10 min. The collected cells were suspended using water peptone at 108 CFU/mL, and then applied directly in the soil near the seedling in each pot, at 15-day intervals.

The Mycorrhiza inoculum consisted of Glomus mosseae-NRC31 and Glomus fasciculata-NRC15, which were originally isolated from Egyptian soils from onion roots; the inoculum was grown in sterilized peat–vermiculite–perlite mixtures and then it was added at 0, 150, and 300 spores/g to the pots immediately after sowing of purslane seeds and this was repeated twice through irrigation, at 15-day intervals.

The effective microorganisms (EMs) used are a commercial brand name and include a mixture of microorganisms isolated from fertile soils. They were provided by the Egyptian Ministry of Agriculture and Land Reclamation. They included photosynthesis bacteria (Rhodopseudomonas palustris and Rhodobacter sphaeroides), milk bacteria (Lactobacillus casei and Streptococcus lactis), yeasts (Saccharomyces albus and Candida utilis), actinomycetes (Streptomyces albus and Streptomyces griseus), and molds (Aspergillus oryzae and Mucor hiemalis). EMs also include common aerobic and anaerobic microorganisms and nutrients.

2.2. Growth Traits

Vegetative and root growth parameters were determined at 65 days after sowing. In particular, plant height, branch number, and aboveground and underground fresh and dry weights were measured. In each pot, the whole plants were harvested, washed under tap water, and kept in a cool dry place for 3 days and then fresh weight was recorded. For dry weight measurement, the samples were left in an oven at 40 °C for three days to avoid sudden heat burning and then at 50 °C until constant weight.

2.3. Chemical Composition of Plant Foliage

The chemical composition of plant foliage was determined at plant tissues harvested at 60 days after sowing in both growing seasons.

2.3.1. Chlorophyll Content

Fresh mature leaves from the plants were used for measuring total chlorophyll content using the protocol described by the Association of Official Analytical Chemists [46]. The chlorophyll was extracted from the leaves using 20 mL of 80% acetone several times until the residue became colorless, and then the absorption was measured at 663 and 645 nm using a spectrophotometer (model UV752/UV754-single beam UV/Vis spectrophotometer, YK Scientific, Shanghai, China).

2.3.2. Total Nitrogen, Phosphorus, Potassium, and Carbohydrate Contents

Total N, P, and K leaf contents were determined after digestion with HClO₄ and H₂SO₄ (1/3 v/v) using the Micro-Kjeldahl spectrophotometer, and flame photometer apparatuses as described in Pregl [47], John [43], and Brown and Lilleland [48], respectively. Total carbohydrate content was determined in the dried samples according to Herbert et al. [49]. Briefly, 0.2 g of dry sample were extracted with HCl (0.1 M) in Eppendorf tubes (25 mL) for 4 h. Then, the extract was filtrated and placed into a flask. After that, 1 mL of the extract was added with 1 mL of concentrated sulphuric acid and 1 mL of phenol 5% and chlorophyll content was measured at 490 nm using a spectrophotometer (model UV752/UV754-single beam UV/Vis spectrophotometer, YK Scientific, Shanghai, China).

2.3.3. Ascorbic Acid Content

The ascorbic acid of leaves was determined by titration using the indicator 2,6 dichlorophenol indophenol as described by the Association of Official Analytical Chemists [46].

2.3.4. Proline Content

Leaf proline content was determined according to the Association of Official Analytical Chemists [46].

2.3.5. Nitrate Contents

The methods used for the determination of nitrate in plant leaves were adapted from methods used in the analysis of soils and fertilizers. The one most used at present involves reduction by Devarda’s alloy, and the determination by titration of the ammonia formed, according to the protocol described by the Association of Official Analytical Chemists [46].

2.4. Statistical Analysis

All data were subjected to Shapiro–Wilk and Levene tests to check the normal distribution and homogeneity of data followed by a two-way ANOVA using the M-stat v.4 program for Windows (Informer Technologies, Inc., Los Angeles, CA, USA). For mean comparisons, Duncan’s multiple range test was implemented at p < 0.05.

3. Results and Discussion

3.1. Vegetative Growth Parameters

Data presented in

Table 3. Effect of drought and biostimulant application on vegetative growth of purslane plants during 2022 and 2023 seasons.

Treatments		Height (cm)		Shoots/Plant		Fresh Weight/Plant (g)		Dry Weight/Plant (g)
Drought	Biofertilizer	2022	2023	2022	2023	2022	2023	2022	2023
100% FC		31.6 ± 0.39 a *	30.1 ± 0.44 a	13.1 ± 0.27 a	14.0 ± 0.21 a	270 ± 2.87 a	278 ± 1.85 a	40.8 ± 0.46 a	44.2 ± 0.40 a
80% FC		29.2 ± 0.39 b	27.9 ± 0.44 b	11.3 ± 0.27 b	12.1 ± 0.21 b	240 ± 2.87 b	248 ± 1.85 b	35.9 ± 0.46 b	39.2 ± 0.40 b
60% FC		23.7 ± 0.39 c	22.8 ± 0.44 c	9.3 ± 0.37 c	9.3 ± 0.21 c	186 ± 2.87 c	198 ± 1.85 c	27.2 ± 0.46 c	30.9 ± 0.40 c
	Control	25.8 ± 0.45 c	24.8 ± 0.5 c	9.9 ± 0.31 c	10.0 ± 0.24 d	213 ± 3.3 c	221 ± 2.1 d	31.6 ± 0.5 c	34.4 ± 0.46 d
	Mycorrhiza	29.3 ± 0.45 ab	27.6 ± 0.5 ab	12.2 ± 0.31 a	13.2 ± 0.24 a	250 ± 3.3 a	260 ± 2.1 a	37.4 ± 0.5 a	41.1 ± 0.46 a
	PGPR	29.4 ± 0.45 a	28.8 ± 0.5 a	11.5 ± 0.31 ab	12.4 ± 0.24 b	236 ± 3.3 b	247 ± 2.1 b	35.2 ± 0.5 b	39.2 ± 0.46 b
	EM	27.5 ± 0.45 b	26.9 ± 0.5 b	11.1 ± 0.31 b	11.5 ± 0.24 c	262 ± 3.3 b	238 ± 2.1 c	34.2 ± 0.5 b	37.6 ± 0.46 c
100% FC	Control	29.7 ± 0.8 bc	28.6 ± 0.96 b	11.7 ± 0.5 bcd	12.1 ± 0.44 d	257 ± 6.0 bc	262 ± 3.6 d	38.7 ± 0.9 bc	41.8 ± 0.74 d
	Mycorrhiza	32.9 ± 0.8 a	30.7 ± 0.96 ab	14.6 ± 0.5 a	15.4 ± 0.44 a	284 ± 6.0 a	293 ± 3.6 a	42.9 ± 0.9 a	46.3 ± 0.74 a
	PGPR	33.2 ± 0.8 a	32.3 ± 0.96 a	13.2 ± 0.5 ab	14.9 ± 0.44 ab	271 ± 6.0 ab	281 ± 3.6 b	40.8 ± 0.9 ab	44.6 ± 0.74 ab
	EM	31.6 ± 0.8 ab	29.8 ± 0.96 ab	12.7 ± 0.5 bc	13.6 ± 0.44 bc	267 ± 6.0 ab	276 ± 3.6 bc	40.6 ± 0.9 ab	43.9 ± 0.74 bc
80% FC	Control	26.4 ± 0.8 de	25.3 ± 0.96 cd	9.8 ± 0.5 ef	10.1 ± 0.44 e	219 ± 6.0 ef	224 ± 3.6 f	32.1 ± 0.9 e	35.4 ± 0.74 f
	Mycorrhiza	30.4 ± 0.8 abc	28.6 ± 0.96 b	12.2 ± 0.5 bc	13.8 ± 0.44 bc	261 ± 6.0 bc	267 ± 3.6 cd	39.5 ± 0.9 bc	42.2 ± 0.74 cd
	PGPR	31.7 ± 0.8 ab	29.8 ± 0.96 ab	11.8 ± 0.5 bcd	12.6 cd	246 ± 6.0 cd	259 ± 3.6 d	36.7 ± 0.9 cd	41.1 ± 0.74 d
	EM	28.4 ± 0.8 cd	27.8 ± 0.96 bc	11.2 ± 0.5 cde	11.8 ± 0.44 d	235 ± 6.0 de	243 ± 3.6 e	35.1 ± 0.9 d	38.2 ± 0.74 e
60% FC	Control	21.3 ± 0.8 f	20.4 ± 0.96 e	8.2 ± 0.5 f	7.9 ± 0.66 f	162 ± 6.0 i	176 ± 3.6 h	23.9 ± 0.9 g	26.0 ± 0.74 h
	Mycorrhiza	24.6 ± 0.8 e	23.4 ± 0.96 d	9.9 ± 0.5 def	10.3 ± 0.66 e	204 ± 6.0 fg	219 ± 3.6 f	29.7 ± 0.9 ef	34.7 ± 0.74 f
	PGPR	25.1 ± 0.8 e	24.3 ± 0.96 d	9.6 ± 0.5 ef	9.6 ± 0.66 e	192 ± 6.0 gh	202 ± 3.6 g	28.1 ± 0.9 f	32.0 ± 0.74 g
	EM	23.6 ± 0.8 ef	23.0 ± 0.96 de	9.3 ± 0.5 ef	9.2 ± 0.66 ef	184 ± 6.0 h	194 ± 3.6 g	27.0 ± 0.9 f	30.8 ± 0.74 g

* Means between treatments in the same column followed by the same letter were not significantly different according to Duncan’s multiple range test (DMRT) at p < 0.05.

showed that plant height, number of shoots per plant, and fresh and dry weight decreased with increasing water stress intensity (100% FC to 60% FC) in both growing seasons. In particular, plant height decreased by 6.3% (2022) and 7.3% (2023) at 80% FC and by 25% (2022) and 24.2% (2023) at 60% FC; number of shoots per plant decreased by 13.8% (2022) and 13.6% (2023) at 80% FC and by 29.8% (2022) and 33.5% (2023) at 60% FC; fresh weight decreased by 11.1% (2022) and 10.8% (2023) at 80% FC and by 25.9% (2022) and 28.8% (2023) at 60% FC; and dry weight decreased by 12.8% (2022) and 11.3% (2023) at 80% FC and by 33.3% (2022) and 30.1% (2023) at 60% FC. Moreover, in both growing seasons, the highest and lowest values of the tested vegetative parameters were observed in plants grown at 100% FC and 60% FC, respectively, while moderate water stress differed significantly from the rest of treatments. Similarly to our study, Hosseinzadeh et al. [50] mentioned that drought stress (at 50% of field capacity) decreased the dry matter content of purslane leaves by 21.1%, while Saheri et al. [51] reported a gradual and significant decrease in plant height, total fresh and dry weight, and leaf number of purslane plants grown at drought levels up to 30% FC. Moreover, Jin et al. [14] suggested that drought stress alone or combined with heat stress resulted in retarded growth of purslane plants as indicated by reduced leaf area, whereas leaf water content was not significantly affected due to the succulent texture of leaves. However, according to Jin et al. [52] purslane has high adaptability to drought stress conditions and plants can fully recover after rehydration following a prolonged period of water shortage.

All the tested biostimulants, e.g., PGPR, mycorrhiza, and EM, resulted in significantly higher plant height, number of branches per pot, and herb fresh and dry weight compared to the untreated control for both growing seasons, while no significant differences were recorded between the biostimulant treatments in most cases (Table 3). For instance, the highest height was recorded in plants inoculated with PGPR (29.4 and 28.8 cm, in 2022 and 2023, respectively; increase by 13.9% and 16.1% in 2022 and 2023, respectively), followed by plants treated with mycorrhiza (29.3 and 27.6 cm, in 2022 and 2023, respectively; increase by 13.6% and 11.3% in 2022 and 2023, respectively) and EM (27.5 and 26.9 cm, in 2022 and 2023, respectively; increase by 6.6% and 8.5% in 2022 and 2023, respectively), without significant differences between the treatments. Similarly, mycorrhiza application (EM) resulted in the highest overall number of shoots/plant (12.3 and 13.2, in 2022 and 2023, respectively; increase by 23.2% and 32.0% in 2022 and 2023, respectively), while the same trend was recorded for dry weight of plants (37.4 and 41.1 g, in 2022 and 2023, respectively; increase by 18.3% and 19.5% in 2022 and 2023, respectively). Finally, the highest fresh weight was observed for EM and mycorrhiza treatments in 2022 and 2023 (262 and 260 g, respectively; increase by 23.0% and 17.6% in 2022 and 2023, respectively). Previously, Hosseinzadeh et al. [50] suggested that inoculation with Rhizophagus irregularis (AMF) increased the total dry matter of purslane leaves, while similar results have been reported for lettuce plants where bacterial inoculum increased the biomass owing to the higher leaf area which resulted in a more effective photosynthetic apparatus and in increased CO₂ availability and assimilation [53]. Moreover, the inoculation with PGPR or AMF may improve the nutrients’ availability, e.g., N, P, and K and beneficially affects plant growth and productivity [54]. In other studies with stevia and pepper plants, EM inoculation increased plant height, number of brunches, and dry weight of plants [55,56]. On the other hand, Cunha et al. [25] reported that growth parameters did not significantly differ among different bacterial inoculation treatments, suggesting that the lack of significance indicates that the tested inoculants might have a non-detectable effect under the studied experimental conditions.

A significant interaction among the studied factors was recorded in terms of plant growth parameters. In general, plants grown under 100% field capacity and inoculated with PGPR had the highest overall height (33.2 and 32.3 cm, in 2022 and 2023, respectively, showing an increase by 11.8% and 16.0% over the untreated plants grown under 100% FC, in 2022 and 2023, respectively), while the highest number of shoots per plant, and fresh and dry weight of plants was recorded for the combination of 100% FC and mycorrhiza application. However, these treatments did not differ significantly from the other biostimulants at the same water stress level and mycorrhiza and PGPR application at 80% FC (for plant height and fresh weight per plant only). On the other hand, the highest water stress level (60% FC) resulted in significantly lower values for all the studied growth parameters, especially in the untreated plants where plant height was reduced by 28.3% (2022) and 28.7% (2023) compared to the untreated plants grown at 100% FC; number of shoots per plant was reduced by 29.9% (2022) and 34.7% (2023) compared to the untreated plants grown at 100% FC, fresh weight was reduced by 37.0% (2022) and 32.8% (2023) compared to the untreated plants grown at 100% FC; and dry weight was reduced by 38.2% (2022) and 37.8% (2023) compared to the untreated plants grown at 100% FC. According to the literature, the application of AMF improved the shoot dry weight of wheat under drought stress [57]. Various processes are responsible for AMF and PGPR mitigating effects under drought stress such as alteration in root architecture and hormonal signaling, enhanced water and nutrient uptake (particularly recalcitrant nutrients, like P, Zn, and Cu), increased photosynthetic activity, regulation of stomatal opening, production of antioxidants and osmolytes, and lastly the positive effects on the soil microbe near the rhizosphere [58,59,60,61,62]. In this respect, in arid and semi-arid regions, PGPR, mycorrhiza, and EM could be valorized as a novel agronomic practice to improve crop drought resistance and alleviate the negative effects of water stress on plant growth and yield parameters.

3.2. Root Growth Parameters

Root fresh and dry weight of purslane plants were significantly reduced when the intensity of water stress increased in both growing seasons with the lowest values being observed at 60% FC (

Table 4. Effect of drought and biostimulant application on root growth parameters of purslane plants during 2022 and 2023 seasons.

Treatments		Roots Fresh Weight/Plant (g)		Roots Dry Weight/Plant (g)
Drought	Biofertilizers	2022	2023	2022	2023
100% FC		51.0 ± 0.43 a *	51.9 ± 0.64 a	8.61 ± 0.11 a	9.25 ± 0.10 a
80% FC		44.7 ± 0.43 b	45.6 ± 0.64 b	7.48 ± 0.11 b	8.12 ± 0.10 b
60% FC		33.6 ± 0.43 c	35.7 ± 0.64 c	5.58 ± 0.11 c	6.34 ± 0.10 c
	Control	39.1 ± 0.50 d	40.0 ± 0.74 c	6.55 ± 0.13 c	7.12 ± 0.12 c
	Mycorrhiza	46.8 ± 0.50 a	48.0 ± 0.74 a	7.84 ± 0.13 a	8.59 ± 0.12 a
	PGPR	44.3 ± 0.50 b	45.3 ± 0.74 b	7.42 ± 0.13 b	8.03 ± 0.12 b
	EM	42.1 ± 0.50 c	43.8 ± 0.74 b	7.08 ± 0.13 b	7.86 ± 0.12 b
100% FC	Control	46.2 ± 0.87 c	48.6 ± 1.3 bcd	7.81 ± 0.24 cd	8.62 ± 0.21 cd
	Mycorrhiza	54.3 ± 0.87 a	56.9 ± 1.3 a	9.18 ± 0.24 a	10.11 ± 0.21 a
	PGPR	52.6 ± 0.87 ab	51.7 ± 1.3 b	8.82 ± 0.24 ab	9.17 ± 0.21 b
	EM	51.0 ± 0.87 b	50.2 ± 1.3 bc	8.64 ± 0.24 ab	9.08 ± 0.21 bc
80% FC	Control	41.6 ± 0.87 d	40.3 ± 1.3 e	6.92 ± 0.24 e	7.21 ± 0.21 e
	Mycorrhiza	48.3 ± 0.87 c	49.7 ± 1.3 bc	8.14 ± 0.24 bc	8.84 ± 0.21 bc
	PGPR	46.2 ± 0.87 c	46.9 ± 1.3 cde	7.72 ± 0.24 cd	8.31 ± 0.21 d
	EM	42.6 ± 0.87 d	45.3 ± 1.3 de	7.14 ± 0.24 de	8.11 ± 0.21 d
60% FC	Control	29.4 ± 0.87 g	31.2 ± 1.3 g	4.93 ± 0.24 h	5.54 ± 0.21 g
	Mycorrhiza	37.9 ± 0.87 e	38.4 ± 1.3 f	6.21 ± 0.24 f	6.82 ± 0.21 ef
	PGPR	34.2 ± 0.87 f	37.2 ± 1.3 f	5.72 ± 0.24 fg	6.62 ± 0.21 f
	EM	32.8 ± 0.87 f	36.0 ± 1.3 f	5.46 ± 0.24 gh	6.39 ± 0.21 f

* Means between treatments in the same column followed by the same letter were not significantly different according to Duncan’s multiple range test (DMRT) at p < 0.05.

). In particular, fresh weight was reduced by 12.3% and 12.1% at 80% FC in 2022 and 2023, respectively, and by 34.1% and 31.2% at 60% FC in 2022 and 2023, respectively; while dry weight decreased by 13.2% and 12.2% at 80% FC in 2022 and 2023, respectively, and by 35.2% and 31.4% at 60% FC in 2022 and 2023, respectively. All the tested biostimulants increased fresh and dry weight of roots over the control treatment (no biostimulants added), although no significant differences were recorded among the biostimulant treatment. However, it is worth noting that the application of mycorrhiza resulted consistently in the highest overall fresh and dry weight of roots compared to the rest of the treatments (fresh weight was increased by 19.7% and 20.0% in 2022 and 2023, compared to the untreated plants, while dry weight of roots increased by 19.7% and 20.6% in 2022 and 2023, compared to the untreated plants). It is well established that roots play a significant role in plant growth and are more prone to drought conditions [63]. Therefore, drought conditions may alter root morphology, increase root mortality, and reduce the overall root biomass [64]. Moreover, literature reports suggest that AMF inoculation increased the root length, providing better nutrient absorption and further affecting photosynthesis and cell metabolism [65]. Similarly to our study, PGPR application may also affect the architecture of root systems through phytohormone production or enzymatic activities [66].

Regarding the interaction effects between the tested factors, the highest fresh (54.3 and 56.9 g, in 2022 and 2023, respectively; increase by 17.5% and 17.1% in 2022 and 2023, respectively, and by 17.5% and 17.1% in 2022 and 2023, respectively) and dry (9.18 and 10.11 g, in 2022 and 2023, respectively; increase by 19.7% and 20.6% in 2022 and 2023, respectively) root weight were recorded in plants grown at 100% FC and inoculated with mycorrhiza. However, no significant differences were found in the rest of the biostimulant treatments at 100% FC (including the control treatment), as well as in the mycorrhiza and PGPR application at 80% FC. In contrast, the lowest fresh and dry weights of purslane were observed in the untreated plants (no biostimulants added) grown at 60% field capacity, showing a reduction by 36.3% (2022) and 35.8% (2023) for the fresh weight and by 36.9% (2022) and 35.7% (2023) for the dry weight. Mycorrhiza and PGPR can produce plant hormones, such as auxins and abscisic acid, which stimulate root growth and have a positive impact on plant–water relations and facilitate plant recovery and adaptation to water stress conditions [67,68]. Moreover, roots with larger surface may absorb more nutrients and water from the soils and translocate them to various organs, resulting in enhanced growth [69], while inoculation with Rhizobium sp. modified the soil structure around the root system, thus facilitating better nutrients and water uptake [70].

3.3. Chemical Composition

Data presented in

Table 5. Effect of drought and biostimulant application on macronutrients and total carbohydrate content (%) of purslane leaves during 2022 and 2023 seasons.

Treatments		N%		P%		K%		Total Carbohydrates%
Drought	Biofertilizer	2022	2023	2022	2023	2022	2023	2022	2023
100% FC		1.55 ± 0.05 a *	1.50 ± 0.40 a	0.242 ± 0.02 a	0.231 ± 0.02 a	1.40 ± 0.02 a	1.45 ± 0.02 a	17.8 ± 0.29 a	19.2 ± 0.34 a
80% FC		1.48 ± 0.05 ab	1.44 ± 0.40 ab	0.224 ± 0.02 b	0.211 ± 0.02 b	1.25 ± 0.02 b	1.27 ± 0.02 b	15.7 ± 0.29 b	16.9 ± 0.34 b
60% FC		1.31 ± 0.05 b	1.24 ± 0.40 a	0.199 ± 0.02 c	0.188 ± 0.02 c	1.03 ± 0.02 c	1.05 ± 0.02 c	11.8 ± 0.29 c	12.8 ± 0.34 c
	Control	1.27 ± 0.06 b	1.22 ± 0.4 b	0.199 ± 0.03 d	0.186 ± 0.03 c	1.02 ± 0.02 c	1.12 ± 0.02 c	13.3 ± 0.34 b	14.2 ± 0.40 c
	Mycorrhiza	1.56 ± 0.06 a	1.53 ± 0.4 a	0.244 ± 0.03 a	0.243 ± 0.03 a	1.31 ± 0.02 a	1.27 ± 0.02 b	16.8 ± 0.34 a	17.8 ± 0.40 a
	PGPR	1.51 ± 0.06 a	1.44 ± 0.4 a	0.216 ± 0.03 c	0.203 ± 0.03 b	1.33 ± 0.02 a	1.37 ± 0.02 a	15.9 ± 0.34 a	17.2 ± 0.40 a
	EM	1.44 ± 0.06 ab	1.38 ± 0.4 ab	0.226 ± 0.03 b	0.210 ± 0.03 b	1.22 ± 0.02 b	1.26 ± 0.02 b	14.3 ± 0.34 b	15.9 ± 0.40 b
100% FC	Control	1.37 ± 0.12 ab	1.32 ± 0.81 ab	0.214 ± 0.05 de	0.203 ± 0.05 de	1.16 ± 0.04 de	1.24 ± 0.05 de	15.8 ± 0.63 cd	16.4 ± 0.69 def
	Mycorrhiza	1.69 ± 0.12 a	1.64 ± 0.81 a	0.273 ± 0.05 a	0.261 ± 0.05 a	1.43 ± 0.04 b	1.48 ± 0.05 ab	19.8 ± 0.63 a	20.5 ± 0.69 ab
	PGPR	1.62 ± 0.12 a	1.56 ± 0.81 a	0.234 ± 0.05 bc	0.2250.05 c	1.59 ± 0.04 a	1.62 ± 0.05 a	18.7 ± 0.63 ab	21.2 ± 0.69 a
	EM	1.51 ± 0.12 ab	1.48 ± 0.81 ab	0.246 ± 0.05 b	0.236 ± 0.05 bc	1.41 ± 0.04 b	1.45 ± 0.05 b	16.9 ± 0.63 bc	18.7 ± 0.69 bc
80% FC	Control	1.32 ± 0.12ab	1.29 ± 0.81ab	0.203 ± 0.05ef	0.191 ± 0.05ef	1.08 ± 0.04e	1.18 ± 0.05def	13.8d ± 0.63ef	14.9 ± 0.69 efg
	Mycorrhiza	1.57 ± 0.12 a	1.58 ± 0.81 a	0.246 ± 0.05 b	0.249 ± 0.05 ab	1.36 ± 0.04 bc	1.26 ± 0.05 d	17.3 ± 0.63 bc	18.5 ± 0.69 bcd
	PGPR	1.54 ± 0.12 a	1.46 ± 0.81 a	0.219 ± 0.05 cde	0.201 ± 0.05 def	1.32 ± 0.04 bc	1.36 ± 0.05 bc	16.9 ± 0.63 bc	17.2 ± 0.69 cd
	EM	1.49 ± 0.12 ab	1.42 ± 0.81 ab	0.226 ± 0.05 cd	0.204 ± 0.05 de	1.24 ± 0.04 cd	1.29 ± 0.05 cd	14.7 ± 0.63 de	16.8 ± 0.69 cde
60% FC	Control	1.12 ± 0.12 b	1.04 ± 0.81 b	0.181 ± 0.05 g	0.164 ± 0.05 g	0.86 ± 0.04 f	0.94 ± 0.05 g	10.2 ± 0.63 h	11.4 ± 0.69 i
	Mycorrhiza	1.42 ± 0.12 ab	1.38 ± 0.81 ab	0.214 ± 0.05 de	0.218 ± 0.05 cd	1.14 ± 0.04 de	1.08 ± 0.05 efg	13.4 ± 0.63 efg	14.3 ± 0.69 fgh
	PGPR	1.38 ± 0.12 ab	1.31 ± 0.81 ab	0.195 ± 0.05 fg	0.182 ± 0.05 f	1.08 ± 0.04 e	1.14 ± 0.05 def	12.1 ± 0.63 fgh	13.1 ± 0.69 ghi
	EM	1.32 ± 0.12 ab	1.23 ± 0.81 b	0.206 ± 0.05 ef	0.189 ± 0.05 ef	1.02 ± 0.04 e	1.03 ± 0.05 fg	11.4 ± 0.63 gh	12.3 ± 0.69 hi

* Means between treatments in the same column followed by the same letter were not significantly different according to Duncan’s multiple range test (DMRT) at p < 0.05.

suggested that drought stress significantly decreased the macrominerals (N, P, K) and total carbohydrate content in purslane leaves, while the lowest values were observed for the most intense drought stress (e.g., 60% FC), where N, P, K and carbohydrates were reduced by 15.5%, 17.8%, 264% and 21.9% in 2022 and by 17.3%, 18.6%, 27.6% and 33.3% in 2023, respectively. Moreover, the control irrigation treatment (100% FC) resulted in significantly higher values compared to the water stress treatments for all the macronutrients and total carbohydrate content in both growing seasons. Regarding the biostimulant effect, mycorrhiza was the most beneficial treatment for all the studied parameters, resulting in the highest overall values (showing an increase of 22.8% (2022) and 25.4% (2023) in N content; an increase of 22.6% (2022) and 30.8% (2023) in P content; and an increase of 26.3% (2022) and 25.3% (2023) in carbohydrate content), except for K content where the highest content was observed for PGPR treatment (increased by 30.4% and 22.3% in 2022 and 2023, respectively), without significant differences from mycorrhiza and EM application. Similarly to our study, previous reports suggested that inoculation with PGPR and AMF increased the N, P, K content in various crops [29,54,71], while Hosseinzadeh et al. [50] reported that AMF inoculation improved the N and P in purslane leaves. The mitigating effects of mycorrhiza against drought stress might be owing to the fungal hyphae’s increased water absorption [54], as well as to the increased mineral-absorption capacity of roots which resulted in higher shoot and root dry weights and higher N and P content compared to the untreated plants, a finding which is also consistent with the findings of our study regarding the root growth parameters [72].

Regarding the interaction effect of the tested factors, the application of mycorrhiza and PGPR at 100% FC resulted in significantly higher N content (increased by 23.3% and 18.2% in 2022, respectively, and by 24.2% and 18.2% in 2022 and 2023, respectively), while P content was beneficially affected only by mycorrhiza (increased by 27.8% and 22.2% in 2022 and 2023, respectively) (Table 5). On the other hand, no significant differences among the biostimulant treatments were recorded for K and total carbohydrate content at 100% FC, while all of them were significantly higher compared to the untreated plants (increased by up to 37.1% (2022) and 23.4% (2023) in the case of K content, and by up to 25.3% (2022) and 29.4 (2023) for carbohydrate content). Moreover, the highest overall values were observed in mycorrhiza treatment at 100% FC for N, P and total carbohydrate content (only in 2022 growing season), while PGPR were more beneficial in K (both growing seasons) and total carbohydrate content of 2023. In contrast, the lowest overall values were recorded for the control treatment (no biostimulants added) at 60% FC (reduced by 18.2% (2022) and 21.2% (2023) for N content; 15.4% (2022) and 19.4% (2023) for P content; 25.9% (2022) and 24.2% (2023) for K content; and 35.4% (2022) and 30.5% (2023) for carbohydrate content, compared to the untreated plants at 100% FC), while biostimulant application mitigated the negative effects of severe drought stress, especially mycorrhiza which increased nutrient content under severe stress increased by up to 32.7%, 32.9%, and 32.6% for N, P, and K, respectively, compared with the control treatment (no biostimulants added). The beneficial impacts of mycorrhiza are associated with the activation of urease, glutamine synthetase, and arginase, which improve host plants’ ability to absorb and use nitrogen, while the mycelium of mycorrhiza has several ammonium and nitrate transporters with roles beneficial to nitrogen assimilation [73]. Mycorrhiza also provides the essential components N, K, P, and Mg to plant roots, as well as anabolized molecules needed for osmoregulatory and osmoprotective compound production [74]. Moreover, the activation of glutamine synthetase allows the conversion of nitrate or ammonium which is then taken up by mycelial N and converted into organic molecules [75]. According to Zhang et al. [76] and Ortiz et al. [77], root and leaf P content in Zenia insignis and Trifolium repens, respectively, dropped as drought stress increased, but the negative impact was lessened by mycorrhiza and PGPR treatments. Considering that the mycorrhiza hyphal network has a small diameter and can grow into tiny pores that fine roots cannot, this expands the active root surface of the plant and allows one to access a larger volume of soil [69]. Moreover, AMF has been demonstrated to increase water uptake by plants during drought, since the symbiosis with AMF enables plants to absorb more water and retain positive water relations for a larger variety of growing conditions [76].

In purslane plants grown under severe drought stress (60% FC), a significant decrease in total chlorophyll content and vitamin C was recorded (reduced by 22.7% (2022) and 34.5% (2023) in the case of total chlorophyll content and by 26.6% (2022) and 39.5% (2023) with regards to vitamin C content) (

Table 6. Effect of drought and biostimulant application on chemical composition of purslane leaves during 2022 and 2023 seasons.

Treatments		Total Chlorophyll Content (mg/100 g f.w)		Vitamin C Content (mg/100 g f.w)		Nitrate Content (mg/g f.w)		Proline Content (μg/g f.w)
Drought	Biofertilizer	2022	2023	2022	2023	2022	2023	2022	2023
100% FC		189 ± 2.08 a *	200 ± 2.07 a	77.9 ± 0.67 a	82.6 ± 0.59 a	2.43 ± 0.02 c	2.45 ± 0.03 c	483 ± 14.0 c	461 ± 18.2 c
80% FC		179 ± 2.08 b	180 ± 2.07 b	73.6 ± 0.67 b	70.7 ± 0.59 b	2.57 ± 0.02 b	2.77 ± 0.03 b	879 ± 14.0 b	804 ± 18.2 b
60% FC		146 ± 2.08 c	131 ± 2.07 c	57.2 ± 0.67 c	50.0 ± 0.59 c	3.14 ± 0.02 a	3.12 ± 0.03 a	1245 ± 14.0 a	1170 ± 18.2 a
	Control	136 ± 2.40 c	134 ± 2.39 c	65.2 ± 0.77 c	61.8 ± 0.69 d	2.97 ± 0.03 a	3.02 ± 0.04 a	959 ± 16.2 a	969 ± 21.1 a
	Mycorrhiza	178 ± 2.40 b	179 ± 2.39 b	72.9 ± 0.77 a	73.0 ± 0.69 a	2.56 ± 0.03 c	2.62 ± 0.04 c	810 ± 16.2 d	702 ± 21.1 d
	PGPR	186 ± 2.40 a	186 ± 2.39 a	70.9 ± 0.77 ab	69.4 ± 0.69 b	2.63 ± 0.03 bc	2.70 ± 0.04 bc	842 ± 16.2 c	761 ± 21.1 c
	EM	181 ± 2.40 ab	181 ± 2.39 b	69.3 ± 0.77 b	67.0 ± 0.69 c	2.70 ± 0.03 b	2.77 ± 0.04 b	865 ± 16.2 b	813 ± 21.1 b
100% FC	Control	154 ± 4.54 cd	161 ± 2.48 d	74.3 ± 1.23 cd	76.8 ± 0.93 d	2.64 ± 0.05 e	2.58 ± 0.06 fgh	514 ± 6.6 g	496 ± 6.4 i
	Mycorrhiza	196 ± 4.54 ab	207 ± 2.48 b	81.9 ± 1.23 a	87.9 ± 0.93 a	2.31 ± 0.05 g	2.36 ± 0.06 i	462 ± 6.6 h	438 ± 6.4 k
	PGPR	206 ± 4.54 a	218 ± 2.48 a	79.4 ± 1.23 ab	84.3 ± 0.93 b	2.36 ± 0.05 g	2.41 ± 0.06 hi	471 ± 6.6 h	447 ± 6.4 jk
	EM	199 ± 4.54 ab	212 ± 2.48 ab	76.2 ± 1.23 bc	81.4 ± 0.93 c	2.42 ± 0.05 fg	2.46 ± 0.06 ghi	483 ± 6.6 h	461 ± 6.4 j
80% FC	Control	141 ± 4.54 d	134 ± 2.48 e	71.0 ± 1.23 d	67.3 ± 0.93 g	2.82 ± 0.05 d	2.93 ± 0.06 bc	926 ± 6.6 d	1018 ± 6.4 d
	Mycorrhiza	187 ± 4.54 b	194 ± 2.48 c	75.6 ± 1.23 c	74.2d ± 0.93 e	2.42 ± 0.05 fg	2.61 ± 0.06 efg	818 ± 6.6 f	684 ± 6.4 h
	PGPR	195 ± 4.54 ab	198 ± 2.48 c	74.2 ± 1.23 cd	71.4 ± 0.93 ef	2.48 ± 0.05 efg	2.74 ± 0.06 def	869 ± 6.6 e	718 ± 6.4 g
	EM	191 ± 4.54 b	193 ± 2.48 c	73.6 ± 1.23 cd	70.0f ± 0.93 g	2.57 ± 0.05 ef	2.78 ± 0.06 cde	904 ± 6.6 d	794 ± 6.4 f
60% FC	Control	113 ± 4.54 e	108 ± 2.48 f	50.3 ± 1.23 f	41.2 ± 0.93 j	3.46 ± 0.05 a	3.54 ± 0.06 a	1436 ± 6.6 a	1394 ± 6.4 a
	Mycorrhiza	152 ± 4.54 cd	136 ± 2.48 e	61.2 ± 1.23 e	56.8 ± 0.93 h	2.94 ± 0.05 cd	2.89 ± 0.06 cd	1149 ± 6.6 c	984 ± 6.4 e
	PGPR	164 ± 4.54 c	142 ± 2.48 e	59.2 ± 1.23 e	52.4 ± 0.93 i	3.05 ± 0.05 bc	2.96 ± 0.06 bc	1186 ± 6.6 b	1117 ± 6.4 c
	EM	154 ± 4.54 cd	139 ± 2.48 e	58.1 ± 1.23 e	49.6 ± 0.93 i	3.12 ± 0.05 b	3.08 ± 0.06 b	1208 ± 6.6 b	1184 ± 6.4 b

* Means between treatments in the same column followed by the same letter were not significantly different according to Duncan’s multiple range test (DMRT) at p < 0.05.

). On the other hand, severe stress resulted in a significant increase in the content of nitrates (by 29.2% and 27.3% in 2022 and 2023, respectively) and proline (by 157.8% and 153.8% in 2022 and 2023, respectively) compared to 100% FC. The inoculation with PGPR increased total chlorophyll content by 36.7% and 38.8% in 2022 and 2023, respectively, while the application of mycorrhiza resulted in the accumulation of vitamin C (increased by 11.8% and 18.1% in 2022 and 2023, respectively), and a significant reduction in the content of nitrate (increased by 13.8% and 13.2% in 2022 and 2023, respectively) and proline (increased by 15.5% and 27.5% in 2022 and 2023, respectively) compared to the untreated plants (control treatment) (Table 6). Regarding the combined effect of the studied factors, the application of biostimulants mitigated the negative effects on chlorophyll and vitamin C content under moderate and severe water stress, while their application resulted in the highest values for plants treated with 100% FC (Table 6), especially PGPR, which increased total chlorophyll content by 33.7% and 35.4% (2022 and 2023, respectively) and mycorrhiza which resulted in an increase by up to 10.2% and 14.4% (2022 and 2023, respectively). On the other hand, biostimulants decreased nitrate and proline content over the control treatment for all the irrigation levels, while the lowest values were recorded when plants were grown at 100% FC and inoculated with mycorrhiza (reduced by 10.1 and 11.7 in 2022 and 2023, respectively).

Water stress is reduced by beneficial microbiomes connected to roots and plant tissues through several different processes [78,79]. For instance, under water stress conditions plants show a significant reduction in photosynthetic rate, stomatal conductance, and transpiration rate due to disruption of the photosynthetic apparatus [80], which could be associated with the decreased content of photosynthetic pigments [76]. Increased absorption of magnesium by mycorrhizal hyphae may also contribute to increased chlorophyll production [74]. Moreover, chlorophyll inhibition may be directly influenced by the reduced uptake of minerals and magnesium in particular [81]. PGPR inoculation may increase the chlorophyll content by mitigating drought stress effects due to the enhanced production of osmoregulatory secondary metabolites, as well as to the increased availability of nutrients by converting complex organic compounds into forms that plants may absorb more easily [68]. In addition, EM may enhance the accumulation of photosynthetic pigments through improved leaf characteristics and increased antioxidant capacity [82]. Inoculation with AMF may also maintain chlorophyll a, b, and carotenoids which are reduced in plants due to drought stress. This effect is mainly owing to the ability of AMF to improve plant water and nutrient uptake [80], which facilitates stomata opening and transpiration [83]. In addition, mycorrhiza secrete hormones like ABA that control stomatal function, thus increasing assimilation and biosynthesis of photosynthetic products that act as osmoregulators [84].

Drought leads to oxidative stress increasing hydrogen peroxide production, and lipid peroxidation [85,86]. Moreover, photosynthetic rate and stomatal conductance were reduced under drought conditions [86] and chlorophyll content was significantly decreased [82]. The reduced chlorophyll content under drought conditions indicates that stress affects purslane chlorophyll biosynthesis [52], while the significant increase of proline content also suggests the negative impacts of drought on purslane plants, since proline is considered a stress biomarker [52,82,86]. In particular, the biosynthesis of osmolytes such as proline is a defensive mechanism to cope with cellular hyperosmolarity and ion disequilibrium [14]. Moreover, plants inoculated with mycorrhiza showed increased ascorbic acid and reduced proline content in various crops, thus highlighting the protective role of mycorrhiza against abiotic stressors [29,54,71]. Therefore, PGPR, mycorrhiza, and EM tested in the present work may indirectly enhance the plant immune system against drought or directly improve nutrient and water absorption and phytohormone balance [26,87].

4. Conclusions

Purslane is classified as a noxious weed, while it is highly appreciated for being rich in omega-3 polyunsaturated fatty acids and antioxidants. Recent studies highlight the potential of cultivating purslane as an alternative vegetable crop, especially under harsh environmental conditions. Our results indicate that vegetative and root growth, as well as the chemical composition of the leaves are beneficially affected when plants are grown under non-drought conditions combined with the inoculation with mycorrhiza, PGPR, and EM. Additionally, biostimulant application in plants cultivated under moderate drought stress conditions (80% field capacity) mitigated the negative impacts of stress on vegetative and root biomass yield and improved the chemical composition of leaves compared to the plants that were not treated biostimulants. In conclusion, plant inoculation with the tested biostimulant formulations seemed to alleviate the negative effects of drought even under severe stress (60% FC), especially the inoculation with mycorrhiza and PGPR. Therefore, the tested soil amendments could be suggested as environmentally friendly agronomic practices to be adopted in purslane cultivation to facilitate the unrestricted plant growth and productivity. However, further research is needed with more genotypes and more biostimulant formulations added, while the extrapolation of these results in filed experiments would provide practical information for the cultivation of the species under drought stress.