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Article

Enhancing Almond Seed Germination and Growth Through Microbial Priming: A Biostimulation Strategy for Sustainable Agriculture

by
Zineb Bouabidi
1,
Najat Manaut
2 and
Mountasser Douma
1,2,*
1
Natural Resources Engineering and Environmental Impacts Team, Multidisciplinary Research and Innovation Laboratory, Polydisciplinary Faculty of Khouribga (FPK), Sultan Moulay Slimane University, Khouribga 25000, Morocco
2
Laboratory of Water Sciences, Microbial Biotechnologies, and Natural Resources Sustainability, Faculty of Science Semlalia, Cadi Ayyad University, Marrakech 40000, Morocco
*
Author to whom correspondence should be addressed.
Agronomy 2025, 15(10), 2434; https://doi.org/10.3390/agronomy15102434
Submission received: 13 March 2025 / Revised: 6 April 2025 / Accepted: 15 October 2025 / Published: 21 October 2025
(This article belongs to the Special Issue Selected Papers from the 4th International Electronic Conference on Agronomy (IECAG 2024))
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Abstract

Microbial priming is an emerging strategy in sustainable agriculture that involves the use of beneficial microorganisms to enhance agricultural productivity and sustainability. This innovative approach leverages the natural interactions between plants and microorganisms to promote plant growth and improve soil health. This study explores the application of microbial priming on almond seeds, focusing on the biostimulant effect of soil-based microbial extracts from a mediterranean shrub Pistacia lentiscus L. as an ecological strategy to improve the germination and seedling of almond (Prunus dulcis (Mill.)). The extraction process of soil differentiates three extracts: the first separates AMF spores (Myco) from all other bacterial and fungal consortia (MW), and the third combines the two previous extracts (MW + Myco). The experiment evaluated germination rates, seedling growth parameters, and conducted physico-chemical soil analyses. Arbuscular Mycorrhizal Fungi (AMF) colonization was also measured. Microbial priming significantly improved germination rates and enhanced seedling growth compared to untreated controls. The three microbial extracts showed significant effects on germination rate after 20 days, exceeding 90%. After 27 days, all treatments reach their maximum (100%). Seedling indicators allow MW + Myco extract to be considered as the most powerful extract on almond seedling growth. The combination of microbial and endomycorrhizal fungal extracts could be considered as a facilitator of seedling growth of almond. The AMF colonization was notably higher in treated plants. Overall, microbial priming effectively enhances almond seed germination and seedling growth, demonstrating its potential as a sustainable biostimulation strategy in agriculture. This practice boosts crop productivity and promotes soil health by enriching microbial communities and improving nutrient cycling. These results open up perspectives towards a natural-based strategy able to facilitate the germination and early seedling of almonds in both nurseries and in the field—and to enhance the productivity and health of almond cultivation in special Mediterranean area.

1. Introduction

Microbial priming is an emerging strategy in sustainable agriculture, which uses the natural symbiotic relationships between plants and microorganisms to improve crop productivity and enhance soil health [1]. This innovative approach contributes to sustainable agriculture by enhancing plant growth, improving nutrient availability, and increasing plant resilience [2]. By using microbial priming, plants are pre-exposed to beneficial microorganisms, which activate growth-promoting pathways and defense mechanisms, ultimately improving the overall crop performance [3].
Microbial priming refers to the process of exposing plants to beneficial microorganisms, which enhances its growth and resilience through the activation of defense mechanisms and the improvement of nutrient uptake [3]. Beneficial microbes interact with plant receptors, activating defense mechanisms by secreting proteins to enable plant infection [4]. Nonetheless, plants possess proteins that recognize these microbial efforts, triggering responses that provide enduring protection against pathogens [4]. Thus, microbial priming offers a promising approach to improve plant health in the agricultural sector [3,4].
The rhizosphere plays a crucial role in these microbial interactions, providing a habitat for a wide range of beneficial bacteria and fungi, including Arbuscular Mycorrhizal Fungi (AMF), which are essential for plant health and soil fertility [5]. These microorganisms help plants absorb water and nutrients more efficiently, improve soil structure, and provide protection against pathogens [6]. AMF, in particular, form a mutualistic relationship with plant roots, facilitating the uptake of essential nutrients like phosphorus and water, which are vital for healthy plant growth [7]. Therefore, microbial priming holds significant potential for improving plant health and stress tolerance in agriculture [3,4], particularly in the southern Mediterranean area where the search for resilient crops adapted to challenging climatic conditions is very desirable for sustainable agriculture [8].
In this sense, the search for ecological solutions to improve the adaptation and resilience of high value-added crops attracts the attention of agricultural managers. One of these cultures, almond (Prunus dulcis (Mill.) D.A. Webb) (Rosaceae), is considered as an economically important nut tree crop worldwide [9,10].
The almond is native to central Asia, and it is one of the oldest domesticated fruit trees [11]. More than 50% of world production comes from the USA, with more than 2.3 million tons. Mediterranean countries such as Spain, Turkey, Morocco, Syria, and Italy occupy advanced places in world production [12]. However, the Mediterranean region remains extremely vulnerable to climate change [13], particularly in southern countries where the dominant climates are arid and semi-arid [14]. This critical situation influences the production of almond, despite the intensive use of foreign almond cultivars, which are not necessarily acclimated and adapted to water scarcity in the southern Mediterranean area [15].
The return to autochthonous varieties could constitute a strategy to conserve native genetic resources [16] and enhance traditional farming systems in order to mitigate climate change [17]. The use of the bio-priming of almond seeds from the native soils of associative plants could constitute a strategy for both improving adaptation and productivity. Studies have shown that AMF can improve the drought tolerance of almond trees by increasing their water uptake efficiency [18] and enhancing the plant growth uptake of soil nutrients [19].
The current literature lacks information on the impact of microbial priming from Mediterranean native soils on almond germination and seedling growth. There is a gap in understanding how microbial priming from Mediterranean native soils specifically affects almond seedling growth. Understanding this influence is crucial for enhancing almond seedling growth, particularly in Moroccan agricultural practices.
Microbial priming, specifically the inoculation of seeds with beneficial rhizospheric microorganisms, can offer an effective way to enhance almond seed germination and seedling growth. Microbial priming, through the exposure of seeds to a microbial wash from native plants such as Pistacia lentiscus L., may facilitate beneficial plant–microbe interactions, which results in enhancing nutrient uptake, accelerating germination, and stimulating seedling development. Furthermore, microbial priming can improve soil health by increasing the population of beneficial microbes that support plant growth, soil fertility, and nutrient cycling [20].
Pistacia lentiscus L. was selected for this study due to its rich rhizosphere microbial community, which is known to enhance plant growth and soil health [21,22,23]. Studies have shown that its rhizosphere harbors diverse beneficial bacteria and fungi that can improve nutrient availability and support plant resilience [24,25]. Additionally, research has also associated Pistacia lentiscus L. with a diverse AMF population, supporting plant recovery and soil restoration [26]. Given its potential to promote beneficial plant–microbe interactions, P. lentiscus L. was chosen as a promising candidate for microbial priming in sustainable agriculture.
Despite the potential of microbial priming to enhance plant cultivation, its application to almonds using Moroccan native soils has been underexplored. This study aims to assess the effects of microbial priming on almond seed germination and seedling growth, with a particular focus on AMF colonization. The research uses a microbial wash extracted from the rhizosphere soil of Pistacia lentiscus L., a native Mediterranean plant, to evaluate its impact on seedling vigor, germination rates, and soil properties.

2. Materials and Methods

2.1. Soil Sampling and Seeds Collecting

Rhizospheric soil, from the indigenous plant species Pistacia lentiscus L., was sampled from Azilal area (31°57′51″ N, 6°34′27″ O), and used as substrate to prepare microbial extracts. Rhizospheric soils (25–35 cm depth) were collected using sterilized material according to Manaut et al. (2023) [27], with small modifications. Briefly, from a homogeneous and undisturbed zone, 5 shrubs, spaced 20 m apart from each other, were randomly selected for sampling. The mixed sample was used in experiment treatments for the production of replicates.
The used seeds were collected, in December 2022, from a farmer in the locality of AitAtab, Azilal (32°6′36″ N, 6°40′48″ W), where the decades-long production, harvesting and conservation of the wild local variety “Beldi” of almond tree was confirmed by the local population. The seeds were stored in a fresh and dry place. Then, they were sorted to choose those of good quality (weight, size, semi-open exocarp). The seed exocarps were removed. The endocarps were carefully cracked and removed so the kernels remain intact. Before being subjected to the priming process, the collected kernels were subsequently sterilized by sodium hypochlorite (6%) for 10 min, and then rinsed with sterile distilled water multiple times.

2.2. Treatments Preparation

The standard soil microbial wash method was applied to treat the seeds with the native soil microbiome, following the procedure outlined by Howard [28]. This involved mixing 40 g of soil with 160 mL of 0.85% NaCl solution using a magnetic stirrer (VELP Scientifica Srl., Usmate, Italy) at 180 rpm for 10 min. Soil particles were removed from the solution through vacuum filtration using Whatman filter papers (Merck KGaA, Darmstadt, Germany). To isolate the microbial fraction and eliminate water-soluble nutrients and chemicals, the filtrate was centrifuged at 3000 rpm for 30 min in 50 mL centrifuge tubes, and the resulting supernatant was discarded. The microorganisms collected at the tube’s bottom were then extracted in 200 mL of 0.85% NaCl solution and considered a consortium of microscopic bacteria and fungi (MW), free of AMF spores [29]. The second treatment comprised the extraction of AMF spores using the wet sieving extraction method [30]. The extracted spore surface was disinfected with a 2% bleach solution, rinsed multiple times with sterile DI water, and then placed in 200 mL of 0.85% NaCl solution (Myco). The third treatment combined both microbial extracts (Myco + MW).

2.3. Seeds Preparation for Germination Test

After conducting a water flotation test to separate the viable seeds from the empty or non-viable ones, the study selected 150 viable seeds. These seeds where collected from a local cultivar of the “Beldi” almond variety (Prunus dulcis (Mill.)). While the kernels remained intact and sterilized as previously mentioned (see Section 2.1), they were well rinsed and soaked in sterile DI water for 24 h. Thereafter, each group of 30 seeds was soaked for 24 h in the microbial wash (MW), the AMF spore solution (Myco), or a combination of both treatments (MW + Myco), with thirty seeds allocated to each treatment. A solution of 150 mg/L of gibberellic acid served as the positive control (T+), while seeds soaked in sterile DI water for an equivalent duration acted as the negative control (T−). The use of gibberellic acid as a positive control is based on its role as a plant bioregulator [31]. The treated seeds were then placed in sterile plastic containers filled halfway with sterilized perlite. These containers were kept in the dark at room temperature (24–26 °C) for 27 days, with the total number of germinated seeds assessed every day. Seeds were considered germinated upon the visible emergence of the radicle [32].
After incubation under different treatments, germinated seeds followed the evaluation of several germination parameters.
  • First day of germination (FDG)
FDG is the day on which the first germination event occurred. A lower FDG value indicates a faster initiation of germination [33].
  • Germination percentage (GP) (%)
The germination percentage was calculated by the following Equation (1) [33]:
GP = (Number of germinated seeds/Total number of seeds) × 100
  • Mean germination time (MGT)
The mean germination time (MGT) was calculated by Equation (2) according to Ellis and Roberts [34].
MGT = ∑(n × d)/N,
where n = number of seeds germinated on each day, d = number of days from the beginning of the test, and N = total number of seeds germinated at the termination of the experiment [34]. The lower MGT value indicates a faster germination of seed population.
  • Germination rate index (GRI)
GRI was calculated according to Kader (2005) [33].
GRI = G1/1 + … + Gx/x,
G1 = Germination percentage at the first day after sowing, Gx = Germination percentage at the x day after sowing. The GRI reflects the percentage of germination on each day of the germination period. Higher GRI values indicate higher and faster germination [35].

2.4. Growth Chamber Experiment

To explore the influence of native microbes on plant growth, seedlings randomly chosen from the previous germination experiment were transplanted into plastic cups filled with a sterilized mixture of topsoil and sand in a 1:1 ratio. Each seedling from every experimental group received an additional 1 mL of the same treatment applied to the seeds before germination. The experiment comprised 15 replicates for each treatment, totaling 75 plastic cups. These cups were positioned within a growth chamber under controlled conditions (maintained at 25 °C with a natural light/dark cycle) and watered every other day with sterile distilled water. Over the 7-week growth period, the seedling height and leaf number were measured at regular intervals to evaluate growth parameters, including seedling length (WSL) (cm), total leaf number (TNL), and vigor index (SVI).
SVI was calculated following the modified formula [36].
SVI = shoot length(cm) × germination percentage (%)
The seed group showing the higher seed vigor index is considered to be more vigorous.

2.5. Mycorrhization Assessment

2.5.1. Spore Extraction and Identification

Spore extraction was based on sieving soil samples with water and decantation [30]. Briefly, from carefully homogenized soil sample, 100 g is taken and sieved through a series of sieves (800 μm and 50 μm) under a water jet. Then, the spores are concentrated on a sucrose solution. Spores are finally counted under a binocular magnifying glass (Magnification: X40, Nikon Co., Tokyo, Japan). The different morphotypes are identified according the descriptions available at the International Culture Collection of Vesicular Arbuscular Mycorrhizal Fungi (INVAM) database [37].

2.5.2. AMF Colonization

To assess the arbuscular mycorrhizal fungi (AMF) colonization of roots, fragments of the lateral roots of almond were carefully washed with tap water and cleared with 10% of KOH at 90 °C for 30 min. Then, they were stained with Trypan blue at 90 °C for 20 min [38]. AMF infection frequency and intensity were evaluated in root fragments according to Trovelot (1986) [39] based on the following equations:
Frequency of mycorrhization (F):
F% = (N − n0)/N × 100,
where N denotes number of observed fragments; and n0 denotes number of fragments with no trace of mycorrhization.
Intensity of colonization (M):
M (%) = (95 × n5) + (70 × n4) + (30 × n3) + (5 × n2 + n1)/N,
where n5, n4, …, n1 are the numbers of fragments denoted 5, 4, …, 1, respectively; class 5: more than 91%, class 4: from 51 to 90%, class 3: from 11 to 50%, class 2: less than 10%, class 1: trace and class 0: no mycorrhization.

2.6. Soil Physicochemical Analysis

The physico-chemical properties of the soil, including pH, electrical conductivity (EC), total organic carbon (TOC), available phosphorus (AP), and total nitrogen (N), were analyzed. Soil pH and EC were measured in a 1:5 (v/v) soil-to-water suspension following the NF ISO 10390:2021 standard. Soil samples were dried and sieved through a 2 mm mesh in order to assess the other analysis. Total organic carbon and organic matter content were assessed using Anne’s method [40], which involves the oxidation of organic matter with potassium dichromate in the presence of sulfuric acid [41]. Available phosphorus was quantified using the Olsen method [42], and total nitrogen was determined via the Kjeldahl method [43].

2.7. Statistical Analysis

Data analysis was performed using the ANOVA test, with mean values compared using the Tukey post hoc test following a normality pre-test (p = 0.05). The results are presented as mean values ± standard deviation (SD). Statistical analyses were conducted using SPSS Statistics, IBM version 26.0. Additionally, biological parameters were converted into scores ranked from 1 to 5 based on their relative strength using Past software (version 4.11). Graphs were generated using GraphPad Prism 10 software.

3. Results

The effect of the application of Pistacia lentiscus L. soil extracts was evaluated by assessing several response parameters on soil fertility, germination, and seedlings growth of Prunus dulcis (Mill.).

3.1. Identification and Density of AMF SPORES

The taxonomic identification of Pistacia lentiscus L. Native Soil (PNS), source of extracts, showed the presence of 19 taxa belonging to 7 genera: Dentiscutata, Glomus, Rhizophagus, Septoglomus, Acaulospora, Cetraspora, and Scutellospora. After harvest, the soil from the Myco and MW + Myco treatments contained 10 taxa from 6 genera: Dentiscutata, Glomus, Rhizophagus, Septoglomus, Acaulospora, and Cetraspora (Figure 1).
A significant difference in spore density was observed among the soils. Compared to the initial source (PNS) with 935 spores/100 g, spore density significantly increased under microbial priming (p < 0.05) in both Myco (2099 spores/100 g) and Mw + Myco (1087 spores/100 g) treatments. The highest spore density was recorded in the Myco treatment within the harvested Prunus dulcis (Mill.) roots (Table 1).
To evaluate root colonization, we stained and analyzed the roots of Prunus dulcis (Mill.) plants after harvest. Microscopic examination revealed fungal infection structures among the priming treatments, Myco and Mw + Myco, including hyphae, vesicles, and spores (Figure 1), exhibited complete colonization (F% = 100%). The highest mycorrhizal intensity was recorded in the Myco treatment (86.17%), followed by Mw + Myco (52.67%), while the MW treatment showed the lowest intensity (3.37%) (Table 1). No spores were detected in the MW, T+, and T− samples. However, root staining in these samples revealed the presence of endophytic structures, which correspond to the M% and F% values presented in Table 1.

3.2. Germination Parameters

3.2.1. Germination Percentage (GP)

The results presented in Figure 2 indicate that seeds subjected to MW and Myco treatments exhibited germination patterns closely aligned with those of the positive control. All treatments demonstrated superior germination performance compared to the negative control.
As shown in Table 2, the priming application of almond seeds using microbial extracts showed a significant effect (p < 0.05) on the germination percentage (GP) during the test period (27 days). At day 10, the two highest rates were recorded for MW and Myco treatments, with 43.33 and 46.67%, respectively. At day 20, this effect appeared more remarkable, for Myco et MW treatments, when the GP reached the maximum (100%). After 27 days, all treatments reach their maximum (100%) and show significance difference in GP in comparison to the control (T−) that does not exceed 53.33%.

3.2.2. Germination Indicators

To assess the overall effect of microbial extract priming on almond seed germination, three indicators (FDG, MGT, GRI) were measured. A gradual numerical rating scale (GMR) from 1 to 5 was given to each germination indicator, depending on its hierarchically powerful effect. The results were presented in Table 3.
The results in Table 3 emphasize the significant effects of microbial priming treatments (MW, Myco, and MW + Myco) on almond seed germination compared to the controls. The GRI for MW (0.99), Myco (0.99) and their consortia (MW + Myco) (0.77) was considerably higher than T− (0.44).
Regarding the mean germination time (MGT), where a lower value signifies faster germination [44], with Myco (18.77 days) and MW (18.83 days) indicating faster germination than MW + Myco (19.63 days) and T− (19.34 days).
Based on the germination mean rank (GMR), and a part from T+, it appeared that the greatest effect on almond germination is MW, followed by Myco and MW + Myco treatments, which are manifested by important germination indicators, compared to the negative control. Thus, we can classify the extracts according to their gradually powerful effects on germination as follows: MW > Myco > MW + Myco > T− (Table 3). According to the germination results, it appears that there is a remarkable effect of MW treatment (MW), followed by Myco treatment, and the combination of the two consortia, MW + Myco treatment.

3.3. Seedling Growth Assessment

Seedling growth parameters, including whole seedling length (WSL) (cm), total number of leaves (TNL), and seedling vigor index (SVI), were measured over a 7-week period (Table 4). As in the previous assessment, a numerical rating scale from 1 to 5 was assigned to each seedling indicator based on its hierarchical effect and a seedling mean rank (SMR) is calculated.
As presented in Table 4, the mean values of WSL, TNL, SVI, and SMR were calculated. A significant effect of MW + Myco application was observed in the treated seedlings, with a notable increase in leaf number (TNL) (+36.76%) compared to untreated almond seedlings (T−). However, the SVI and WSL values for MW + Myco and Myco were very similar, with Myco showing the highest value for both indices. Based on these indicators, the treatments can be ranked according to their effects on seedling growth in the following hierarchical order: T+ > MW + Myco > Myco > MW > T− (Table 4). Apart from T+ treatment, the MW + Myco combination was identified as the most effective microbial priming for almond seedling growth, followed closely by Myco and MW, which showed quite similar results. In general, except controls, the effect of microbial extracts appears to gradually reverse between germination (MW > Myco > MW + Myco) and seedling growth (MW + Myco > Myco > MW). The most efficient treatment for germination is the least efficient for seedling growth.

3.4. Assessment of Physicochemical Properties of Soils

The results of the soil analysis after harvest are presented in Table 5. Compared to the initial state of the soil (S0) used for planting before treatment application, all parameters exhibited an overall increase after the microbial priming application. The soil pH remained slightly alkaline (7.97–8.29). Electrical conductivity (EC) values indicated non-saline soils (0.39–0.61 mS/cm). All treatments showed a slight increase in conductivity compared to the negative control (T−), with the highest value recorded in soil with MW + Myco treatment (0.7 mS/cm).
Regarding the total organic carbon (TOC), the highest value was observed in MW treatment soil (0.75%). The content of available phosphate (AP) was relatively low to moderate across all treatments, with the lowest value in Myco treatment soil (0.17 mg/g) and the highest in the positive control (T+) (0.21 mg/g). Thus, it can be seen that all treatments show very low nitrogen values (0.02–0.05%).
Overall, the five treatments demonstrated a slightly alkaline pH, moderate electrical conductivity, relatively high levels of TOC and AP, and a very low nitrogen level. However, no significant differences were observed between treatments for any of the measured parameters compared to the control.

4. Discussion

The obtained results demonstrated that the biopriming using microbial (MW) and mycorrhizal spore’s extracts (Myco), separately or in combination (MW + Myco), has beneficial effects on both germination and seedling growth indicators. However, no significant effect was detected on soil fertility (Table 5). This could be explained by the insufficient time in unpotting plants (7 weeks) to properly detect the evolution of the physicochemical parameters indicating soil fertility.
On the other hand, the taxonomic identification of arbuscular mycorrhizal fungi (AMF) spores extracted from Pistacia lentiscus L. native soil (PNS), which served as the source for treatment extracts, revealed a diverse community comprising 19 taxa distributed across 7 genera: Dentiscutata, Glomus, Rhizophagus, Septoglomus, Acaulospora, Cetraspora, and Scutellospora. This highlights the rich and diverse fungal biodiversity present in the native soil environment and sheds light on key roles’ interactions in plant growth promotion (PGP), such as phosphate solubilization, mineralization, as well as the production of compounds that include indole acetic acid (IAA), siderophores, and antibiotics [5,45].
In our experiment, a positive correlation was observed between spore density and mycorrhization intensity (Table 1), which can be explained by the significant germination of spores under the conditions of the rhizospheric soil, subsequently leading to a significant mycorrhizal infection. Both the highest density of AMF spores and mycorrhization intensity are recorded in Myco treatment. This result is in agreement with previous studies reporting that the density of spores has been defined as an early and useful indicator of the AMF colonization potential [27,46]. Moreover, spore density determines both the ability of AMFs to colonize the subsequent cultivated crops and capability to resist under ecological and physical disturbances [47]. These potentialities are well demonstrated in previous studies showing that the inoculation of seeds with AMFs is known to facilitate seed germination and enhance early seedling through mutualistic associations [48].
In fact, the overall colonization of roots under Myco and Mw + Myco treatments showed an important and significant (p < 0.05) intensity of colonization, 86.17 and 52,67%, respectively (Table 1). However, the relatively lowest one is recorded under MW treatment (3.37%). Both Myco and Mw + Myco treatments show maximum infectivity, with frequencies (F% = 100%). Studies have shown that the beneficial effects of AMFs on plants result from synergistic interactions within the mycorrhizosphere, which involve diverse microbial communities present in this environment [5,45]. These microbes, closely associated with AMF spores and extraradical mycelium, contribute to the stimulation of spore germination and promote mycorrhizal root colonization [49,50].
According to the classification based on germination indicators, MW as a microbial consortium, free of AMF spores, showed the most significant and powerful effect on germination on 27 days, compared to the control (Table 2 and Table 3). Furthermore, the effects of Myco and Mw + Myco extracts on germination are significant and important with a germination percentage (GP) exceeding 90% by day 20. This also implies the potential effect of AMF spores on the success of germination, which is very remarkable in Myco treatment. The results confirm the beneficial impact of microbial priming on almond seed germination. The MW and Myco treatments showed a consistently high germination performance across multiple parameters. In contrast, the T− treatment (negative control) showed significantly lower values across all metrics, emphasizing the role of microbial and biostimulant treatments in enhancing germination. The lower performance of MW + Myco compared to Myco and MW indicates that the combination of treatments may not have a synergic effect as much as the separate treatments.
The positive effects of MW and Myco applications can be explained by the positive involvement of both bacteria and fungi other than the AMFs of the indigenous rhizospheric microbiome of Pistacia lentiscus L. plant in improving the germination of the almond seeds. This result could be explained by the fact that the native soil of the associative plant offers a desirable rhizospheric environment cue for subsequent seed germination and therefore for natural regeneration [51,52]. Indeed, some seed microbes are inherited, whereas others are found in soil that can adhere to the seed coat to colonize seed surfaces [53]. These microorganisms are essential in protecting seeds from stressors and promoting seed germination [54]. These microbes attach to certain molecules on the seed coat via adhesion factors, creating biofilms made up of extracellular matrix and microbial cells [55]. The plant engages in a complex of interactions, involving chemical signals that controlled and regulated production of phytohormones, fatty acids, and phenolic acids that protect and promote seed germination [52,56].
In terms of seedling growth, the combination MW + Myco shows the most powerful effect (Table 4). The effect of the microbial extracts seems to gradually reverse between germination (MW > Myco > MW + Myco) and seedling growth (MW + Myco > Myco > MW), where the most efficient in germination is the least one in seedling growth. It seems that germination is faster and more successful under the effect of the microbial extract (MW), while the seedling is more vigorous and efficient under the effect of a combination of endomycorrhizal fungal spores and the other beneficial microbes (MW + Myco).
The combination of microbial and endomycorrhizal fungal extracts could be considered as a facilitator of early seedling growth for almond. Seedling growth is a later stage of germination, developed after the establishment of associated-microbe infection. However, other works also show a certain dependence on biotic factors, such as AMFs [57]. In our case, microbial extracts have been well demonstrated as a facilitator of the early seedling growth of almond. These potentialities constitute a new insight of Pistacia lentiscus L. rhizospheric soil as a source of microbial inoculation for almond culture.
Several research studies have explored the dynamics of seed microbiota during germination and early seedling stages [58]. Biopriming with PGPB strains demonstrated the ability to solubilize essential nutrients and produce plant growth hormones, thereby promoting seed germination and seedling growth [1,2,58]. Many studies found that the seed microbiota consists of major bacterial and fungal taxa [59], suggesting that seeds act as microbial reservoirs for other plant parts [60]. They offer significant potential for improving agricultural outcomes sustainably, including enhanced seed germination, seedling growth, and biotic stress management, while reducing reliance on chemical fungicides [1,61].
Barret et al. [62] investigated eight native alpine plant genotypes across three developmental stages: seed, germinating seed, and seedling. The plant genotype significantly shapes the composition and diversity of the seed microbial community [62,63].
According to Zulueta-Rodríguez et al. [64], biopriming treatments conducted on endangered Mexican native Abies species revealed promising results. They notably improved germination rates, reaching up to 91% for one species and up to 68% for another, while also promoting positive growth outcomes.
The study conducted by Yaqoob et al. [65] noted an enhanced growth and biochemical characteristics in maize plants treated with Ochrobactrum ciceri, particularly when combined with basal Zn supplementation. These findings suggest that biopriming maize seeds with Ochrobactrum ciceri and Zn holds promise for both disease control and promoting plant growth [65].
The current study also highlights an interesting shift in the treatment effectiveness between germination and seedling growth. MW demonstrates the best effect during germination but remains less effective for long-term growth compared to MW + Myco. This observation underscores the complexity of treatment interactions and the importance of considering both initial germination and subsequent seedling development when selecting treatments for crop improvement.
The successful germination and seedling establishment of almonds using a biopriming strategy offer multiple benefits. Firstly, it allows the conservation of local varieties, through natural regeneration under native floristic association of almond, not only those closest, considered as nurse plants, but also those distant, endowed as thus having a beneficial effect on the success of germination and establishment. Although the almond tree has often been found in locations with non-native flora throughout the world, data on its naturalization and accompanying wild flora has rarely been studied in the field [66,67].

5. Conclusions

The use of extracts from the associated flora as biopriming substrate has demonstrated a beneficial effect on both germination and seedling of almond. Microbial priming treatments, such as MW + Myco, provide substantial benefits for seedling growth, demonstrating their potential as effective bio-enhancements in agricultural practices. The combination of MW and Myco not only boosts seedling vigor, as indicated by higher values for growth parameters, but also achieves a balance in promoting both germination and subsequent seedling development. Thus, the obtained results will make it possible to consider biopriming using microbial consortiums, among them AMFs, as a green strategy to boost and facilitate the success of almond naturalization using direct sowing.
Its ability to improve key growth and germination parameters demonstrates its potential in boosting crop productivity. This makes it an excellent choice for eco-friendly and resilient agricultural systems.

Author Contributions

Conceptualization: Z.B., N.M., and M.D.; Methodology: Z.B., N.M., and M.D.; Formal analysis and investigation: Z.B., N.M., and M.D.; Writing—original draft preparation: Z.B.; Writing—review and editing: Z.B., N.M., and M.D.; Resources: N.M. and M.D.; Supervision: N.M. and M.D. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no funding.

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author.

Acknowledgments

This work was supported by the Multidisciplinary Research and Innovation Laboratory, Polydisciplinary Faculty of Khouribga (FPK), Sultan Moulay Slimane University, Khouribga, Morocco, and the Faculty of Sciences Semlalia, Cadi Ayyad University, Marrakesh.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. AMF analysis. (A) Identification of some AMF-spore taxa recorded in Pistacia lentiscus L. native soil and in Myco and MW + Myco treatments soil ((a) Rhizophagus sp., (b) Glomus sp., (c) Dentiscutata rubra., (d) Dentiscutata sp.). (B) Different structures of AMF colonization (inter and intraradical hyphae (H), vesicles (V), spores (S), mycelium (M), and endophyte (E)) of almond (Prunus dulcis (Mill.)) seedlings roots detected after root stain in different treatments ((e) Myco, (f) MW + Myco, (g) MW, (h) T−). Scale bar 100 µm (40× magnification).
Figure 1. AMF analysis. (A) Identification of some AMF-spore taxa recorded in Pistacia lentiscus L. native soil and in Myco and MW + Myco treatments soil ((a) Rhizophagus sp., (b) Glomus sp., (c) Dentiscutata rubra., (d) Dentiscutata sp.). (B) Different structures of AMF colonization (inter and intraradical hyphae (H), vesicles (V), spores (S), mycelium (M), and endophyte (E)) of almond (Prunus dulcis (Mill.)) seedlings roots detected after root stain in different treatments ((e) Myco, (f) MW + Myco, (g) MW, (h) T−). Scale bar 100 µm (40× magnification).
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Figure 2. Monitoring the number of germinated seeds over a 27-day period under different treatments.
Figure 2. Monitoring the number of germinated seeds over a 27-day period under different treatments.
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Table 1. Assessment of the AMF spore density, frequency, and intensity in PNS and soil samples from other treatments. The standard deviation of the mean was calculated from three replicates (n = 3) and defined by the number after ± symbol.
Table 1. Assessment of the AMF spore density, frequency, and intensity in PNS and soil samples from other treatments. The standard deviation of the mean was calculated from three replicates (n = 3) and defined by the number after ± symbol.
TreatmentsNumber of AMF Spores/100 g of SoilIntensity of Mycorrhization (M%)Frequency (F%)
PNS935 ± 194.17 ± 1.44100% ± 0.0
MW00 ± 0.0 a3.37% ± 0.23 a20 ± 0.0 a
Myco2099 ± 4.5 b86.17 ± 1.25 c100% ± 0.0 a
MW + Myco1087 ± 3 a52.67 ± 2.3 b100% ± 0.0 a
T+00 ± 0 a0.73% ± 0.23 a20.00 ± 0.0 a
T−00 ± 0 c0.23 ± 0.23 a10 ± 0.0 a
a,b,c Values with different letters indicate significant differences as determined by the T-Tukey test performed using one-way ANOVA (p = 0.05).
Table 2. The pursue of Germination Percentage (GP) over a 27-day period following the priming application of almond seeds.
Table 2. The pursue of Germination Percentage (GP) over a 27-day period following the priming application of almond seeds.
TreatmentDay 10Day 20Day 27
MW43.33 ± 0.33 c100 ± 0.0 c100 ± 0.0 b
Myco46.67 ± 0.68 d100 ± 0.0 c100 ± 0.0 b
MW + Myco26.66 ± 0.66 b90 ± 0.45 b100 ± 0.0 b
T+100 ± 0.0 e100 ± 0.0 c100 ± 0.0 b
T−20 ± 0.33 a50 ± 0.22 a53.33 ± 0.36 a
a,b,c,d,e Values with different letters indicate significant differences, as determined by T-Tukey test after conducting a one-way ANOVA (p = 0.05).
Table 3. The data includes various germination metrics, such as first day of germination (FDG), germination rate index (GRI), mean germination time (MGT), germination percentage (GP) and germination mean rank (GMR).
Table 3. The data includes various germination metrics, such as first day of germination (FDG), germination rate index (GRI), mean germination time (MGT), germination percentage (GP) and germination mean rank (GMR).
FDG (Day)GRIMGT (Day)GP (%)GMR
MW6 (2)0.99 (2)18.83 (3)100 (1)2
Myco7 (4)0.99 (2)18.77 (2)100 (1)2.25
MW + Myco7 (4)0.77 (4)19.63 (4)100 (1)3.25
T+4 (1)1.45 (1)17.00 (1)100 (1)1
T−6 (2)0.44 (5)19.34 (5)53.33 (5)4.25
(1), (2), (3), (4), and (5) reflect the hierarchical classification impact of each germination indicator after 27 days under almond seed priming treatment. The ranking method follows the software’s system, where identical values share the same rank, and the next rank adjusts accordingly.
Table 4. The performance of five different treatments (MW, Myco, MW + Myco, T+, and T−) on seedling growth and vigor, measured by whole seedling length (WSL) (cm), total number of leaves (TNL), seedling vigor index (SVI), and seedling mean rank (SMR). Values are shown with mean ± standard deviation.
Table 4. The performance of five different treatments (MW, Myco, MW + Myco, T+, and T−) on seedling growth and vigor, measured by whole seedling length (WSL) (cm), total number of leaves (TNL), seedling vigor index (SVI), and seedling mean rank (SMR). Values are shown with mean ± standard deviation.
WSL (cm)TNLSVISMR
MW21. 2 ± 6.67 a (4)32.66 ± 7.77 ab (3)21.2 (4)3.66
Myco23.47 ± 6.37 a (2)29.33 ± 7.23 bc (4)23.47 (2)2.66
MW + Myco23.46 ± 8.52 a (2)34 ± 9.5 bc (2)23.46 (2)2
T+37.9 ± 6.32 b (1)40.06 ± 7.66 c (1)37.9 (1)1
T−18 ± 10.53 a (5)21.5 ± 9.98 a (5)9.6 (5)5
a,b,c Values with different letters indicate significant differences, as determined by T-Tukey test after conducting a one-way ANOVA (p = 0.05). (1), (2), (3), (4), and (5) reflect the hierarchical classification impact of each seedling parameter after 7 weeks of growth experiment. Equal values receive the same rank, and the following rank is modified accordingly.
Table 5. Physicochemical parameters of the treatments before (S0) and after the application of microbial priming treatments. Treatments include MW, Myco, and MW + Myco, while T− and T+ represent the negative and positive controls, respectively. S0 refers to the initial state of the soil used for transplantation before the application of treatments.
Table 5. Physicochemical parameters of the treatments before (S0) and after the application of microbial priming treatments. Treatments include MW, Myco, and MW + Myco, while T− and T+ represent the negative and positive controls, respectively. S0 refers to the initial state of the soil used for transplantation before the application of treatments.
pHEC (mS/cm)TOC (%)N (%)AP (mg/g sol)
S08.05 ± 0.070.17 ± 0.040.06 ± 0.010.01 ± 0.000.1 ± 0.02
MW7.99 ± 0.16 a0.61 ± 0.2 a0.75 ± 0.1 a0.04 ± 0.03 a0.18 ± 0.03 a
Myco8.12 ± 0.15 a0.49 ± 0.19 a0. 58 ± 0.26 a0.05 ± 0.004 a0.17 ± 0.01 a
MW + Myco7.97 ± 0.07 a0.7 ± 0.32 a0.46 ± 0.26 a0.02 ± 0.009 a0.19 ± 0.03 a
T+8.22 ± 0.33 a0.5 ± 0.2 a0.4 ± 0.1 a0.03 ± 0.009 a0.21 ± 0.01 a
T−8. 29 ± 0.04 a0.39 ± 0.07 a0.64 ± 0.05 a0.04 ± 0.007 a0.2 ± 0.05 a
a Values sharing the same letters are not significantly different, as determined by Tukey’s post hoc test following one-way ANOVA at p = 0.05. Numbers following the ± symbol represent the standard deviation of the mean (n = 3).
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Bouabidi, Z.; Manaut, N.; Douma, M. Enhancing Almond Seed Germination and Growth Through Microbial Priming: A Biostimulation Strategy for Sustainable Agriculture. Agronomy 2025, 15, 2434. https://doi.org/10.3390/agronomy15102434

AMA Style

Bouabidi Z, Manaut N, Douma M. Enhancing Almond Seed Germination and Growth Through Microbial Priming: A Biostimulation Strategy for Sustainable Agriculture. Agronomy. 2025; 15(10):2434. https://doi.org/10.3390/agronomy15102434

Chicago/Turabian Style

Bouabidi, Zineb, Najat Manaut, and Mountasser Douma. 2025. "Enhancing Almond Seed Germination and Growth Through Microbial Priming: A Biostimulation Strategy for Sustainable Agriculture" Agronomy 15, no. 10: 2434. https://doi.org/10.3390/agronomy15102434

APA Style

Bouabidi, Z., Manaut, N., & Douma, M. (2025). Enhancing Almond Seed Germination and Growth Through Microbial Priming: A Biostimulation Strategy for Sustainable Agriculture. Agronomy, 15(10), 2434. https://doi.org/10.3390/agronomy15102434

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