Abstract

   Effective Microorganism (EM) is widely employed as a growth promoter in
   agricultural practices. The aging of oat seeds not only directly
   impairs agricultural production but also exerts adverse effects on
   biodiversity. The mechanism through which EM influence the germination
   of aging seeds remains unclear. In this experiment, the EM bacterial
   solution underwent pretreatment, which included the original-solution
   treatment (OrT), supernatant treatment (SuT), and sterile treatment
   (StT). Aging of oat seeds was induced using the pretreated EM bacterial
   solution. In this study, the EM bacterial solution facilitated the
   enhancement of the germination rate, germination index, and vitality
   index of aged seeds, with SuT demonstrating the most pronounced
   effects. Specifically, SuT resulted in a significant increase in APX
   and POD activities, while significantly reducing the malondialdehyde
   content. In addition, metabolic profiling highlighted the significance
   of 5-phosphoribosylamine in the purine metabolic pathway. Particularly
   in the SuT, the upregulation of 5-phosphoribosylamine facilitated the
   synthesis of (R)-Allantoin, consequently augmenting antioxidant enzyme
   activity.

   Keywords: Effective microorganisms, Age seeds, Seed germination,
   Metabolomics, Antioxidant enzymes, 5-Phosphoribosylamine

Introduction

   Effective Microorganisms (EM) is a composite microbial agent comprising
   over 80 beneficial bacteria from 10 genera, including lactic acid
   bacteria, photosynthetic bacteria, and actinomycetes. Widely recognized
   for its effectiveness, EM is extensively employed as a growth promoter
   in agriculture [[40]1, [41]2]. These microbial agents play a pivotal
   role in enhancing soil fertility, promoting plant growth, and
   consequently contributing to overall crop yield [[42]3, [43]4]. For
   instance, EM microbial agents facilitate the introduction of a plethora
   of beneficial bacteria into the soil. The actions of these bacteria can
   augment the solubility of nutrients within the soil, reverse soil
   oxidation to an antioxidant state, and catalyze the activation of
   inorganic elements present in the soil. Consequently, this process
   enhances the absorption rate of crops [[44]5]. Furthermore, EM agents
   also contribute to inhibiting harmful pathogens and pests while
   concurrently reducing the reliance on chemical pesticides [[45]6]. In
   practical application, whether applied to rice, vegetables, fruits, or
   ornamental plants, EM agents consistently demonstrate a significant
   increase in yields [[46]7–[47]9]. The utilization of EM inoculants has
   notably enhanced the resilience of plants against diseases and adverse
   environmental conditions Dourado discovered that the application of EM
   and their metabolites could significantly decrease the incidence of
   plant pathogenic fungi in corn seeds, reducing the rate from 67 to 21%.
   Additionally, the study isolated non-pathogenic microorganisms from
   treated corn seeds and assessed their interactions with Fusarium in
   paired cultures. Among the 42 strains isolated from EM-treated seeds,
   21 demonstrated the ability to inhibit Fusarium hyphal growth through
   antagonistic mechanisms, competition, and antimicrobial effects
   [[48]10]. This improvement has led to significant enhancements in
   agricultural production [[49]11, [50]12]. Given these benefits, EM
   inoculants have garnered widespread adoption in organic agriculture
   practices, thereby providing robust support for achieving more
   sustainable and environmentally friendly agricultural methods [[51]7,
   [52]13]. Certainly, improper utilization of various types of EM
   extracts may sometimes result in failure to enhance crop yield and soil
   quality. Additionally, it can lead to a decrease in soil organic carbon
   and water-stable aggregates, which is detrimental to agricultural
   production [[53]14, [54]15]. Therefore, it is imperative to conduct
   thorough and comprehensive research to elucidate the growth-promoting
   mechanism of EM bacterial solution. Consequently, three distinct
   treatment methods utilizing EM bacterial solution were employed in this
   experiment. In addition to the EM bacterial stock solution, the liquid
   was also subjected to centrifugation to isolate the supernatant, which
   is enriched with microbial metabolites, metabolic enzymes, and
   extracellular products. Furthermore, another group implemented a method
   to deactivate the EM bacterial fluid, thereby attenuating the influence
   of biological activity. These approaches aimed to investigate the
   effects of metabolites and microbial activity present in EM bacterial
   solution.

   Despite the widespread usage and research on EM inoculants in various
   agricultural aspects, our understanding of their impact on seed
   germination, particularly on aged seeds, remains limited. Seed aging
   typically results in a diminished germination rate. Even if aged seeds
   do germinate, the ensuing seedlings often exhibit growth limitations,
   including slower growth, inadequate root development, and decreased
   resistance to pests and diseases [[55]16, [56]17]. Aging seeds not only
   diminish production but also escalate planting costs [[57]18]. Aged
   seeds exhibit lower and uneven germination rates, necessitating the use
   of more seeds to achieve the desired planting density [[58]19]. This
   not only increases seed costs but also results in inconsistent crop
   growth, requiring additional labor and resources for management. To
   mitigate the low yields and address pest and disease issues associated
   with aged seeds, farmers often resort to increased use of pesticides
   and fertilizers [[59]20]. Consequently, this practice can lead to soil
   and water pollution [[60]21]. Therefore, Seed aging not only directly
   harms agricultural production but also has the potential to negatively
   impact the ecological environment and biodiversity [[61]22]. Seeds
   possessing specific rare or unique genetic traits may lose their
   germination ability as they age, leading to the potential loss of
   invaluable genetic resources [[62]23]. Enhancing the germination rate
   of aged seeds holds not only immediate economic and practical value for
   agricultural production but also carries profound implications for the
   environment, ecology, and scientific research. Hence, we posit a new
   hypothesis suggesting that EM may possess the capability to promote the
   germination of aged seeds. If this hypothesis is confirmed, it could
   present a novel strategy for agricultural production, particularly in
   enhancing the germination efficiency of aged seeds. This could lead to
   increased planting efficiency and income for farmers. The vitality and
   quality of seeds represent a pivotal aspect of global food production
   and agricultural sustainability [[63]24]. Seeds are susceptible to
   aging during storage, particularly in environments characterized by
   high temperatures and humidity [[64]23]. This aging process encompasses
   various morphological, physiological, and biochemical changes,
   including membrane damage, structural deterioration of organelles, loss
   of seed leachate, reduction in respiratory rate and ATP production, and
   enzyme inactivation [[65]25]. More significantly, the excessive
   generation of reactive oxygen species (ROS) serves as a primary
   contributor to seed aging, leading to severe oxidative damage to
   cellular components such as proteins, lipids, chromosomal
   abnormalities, and DNA lesions [[66]26]. Among these factors,
   mitochondrial dysfunction emerges as a key element in seed aging, with
   the excessive accumulation of ROS in mitochondria playing a decisive
   role [[67]27]. During seed aging, degradation of cell membrane
   phospholipids, disruption of protein and genetic material structure and
   function, and attenuation of antioxidant system enzyme activity are
   observed [[68]28]. In essence, seed aging is a multifaceted process
   encompassing a multitude of internal biochemical mechanisms and
   external environmental factors [[69]24, [70]29].

   Oat (Avena sativa L.) is recognized as an environmentally sustainable
   crop, cultivated extensively worldwide for its remarkable ability to
   thrive under diverse environmental stresses [[71]30]. However, oat
   grains tend to exhibit a high fat content, rendering them susceptible
   to rancidity or deterioration [[72]31]. The oat variety “Qinghai 444
   (QH444)” is extensively cultivated in China owing to its robust fibrous
   roots, vigorous growth, and resilience against lodging, drought,
   barrenness, and verticillium wilt. Therefore, in this study, we
   utilized seeds from the QH444 oat variety as materials for artificial
   aging treatment. Seeds age more rapidly when exposed to high
   temperatures (41 °C to 55 °C) and extremely high humidity (100%
   humidity) [[73]32–[74]34]. Subsequently, we employed an EM bacteria
   diluent as an initiator to revitalize the vigor of aging seeds. Our
   study aims to uncover the specific mechanisms responsible for the
   anti-aging properties of oat seeds and to evaluate the effectiveness of
   EM in this context. We investigate these mechanisms by analyzing the
   growth, physiological, and metabolic indicators of oat seeds. By
   examining growth dynamics, physiological changes, and metabolic
   activities, we seek to elucidate the mechanisms underlying the
   anti-aging characteristics of oat seeds. This research will provide
   valuable insights for advancing agricultural production and the seed
   industry.

Materials and methods

Test materials

   The experiment was conducted at the Pratacultural Laboratory of
   Northeast Agricultural University. The test seeds utilized in the study
   were Qinghai No. 444 oat seeds, sourced from the Pratacultural
   Laboratory of Northeast Agricultural University. Mature and fully
   developed oat seeds were carefully chosen for the study. The EM
   bacterial solution is prepared using three distinct methods, denoted as
   OrT (original-solution treatment), SuT (supernatant treatment), and StT
   (sterile treatment). The specific processing methods for each treatment
   are outlined in Table [75]1.

Table 1.

   Detailed information on the treatment method of EM microbial liquid
   Treatment EM bacterial solution processing method
   OrT Prepare the EM bacterial solution with a concentration of 0.5%
   (v/v) using distilled water
   SuT After centrifuging the EM bacterial solution, collect the
   supernatant and prepare it to a concentration of 0.5% (v/v) supernatant
   StT After subjecting the EM bacterial solution to high temperature
   (120℃, 2 h), prepare a solution with a concentration of 0.5% (v/v)
   using distilled water.”
   [76]Open in a new tab

Experimental design and process

Seed aging test

   First, the seeds underwent an artificial aging process. Thirty seeds
   were placed in a glass Petri dish and incubated in a chamber set to
   45 °C with 100% humidity. The artificial aging treatments were applied
   for durations of 24, 48, 72, 96, 120, and 144 h, with a control group
   of 0 h. Each treatment condition was conducted in triplicate. After the
   aging treatment, the seeds are transferred to the incubator (constant
   temperature 25℃, relative humidity 80%) in the absence of light.
   Germination indices were assessed and samples were collected upon seed
   germination (3 days). Meanwhile, the unsampled seeds were left to
   germinate unabated until the 7th day, with the daily growth data
   meticulously documented.

Seeds induced by EM bacteria liquid

   For this stage, a predetermined number of seeds aged for 144 h were
   selected, following the seed aging method described in Sect. [77]2.2.1.
   Subsequently, the seeds were subjected to treatment with three
   different EM bacteria diluents (OrT, SuT, StT) applied by spraying onto
   their surface, while distilled water (H[2]OT) served as the control.
   Each treatment condition was conducted in triplicate. Following this
   treatment, the seeds underwent a 4-hour period in an oven set at 35 °C.
   Afterward, they were removed and infiltrated with distilled water
   before being transferred to the incubator (constant temperature 25℃,
   relative humidity 80%) for a period of 7 days. Upon the germination of
   seeds (3 days post-treatment), germination indices were meticulously
   assessed, and samples were collected for further analysis. These
   samples were promptly frozen using liquid nitrogen and stored in a
   refrigerator at -80 °C for subsequent analysis. Meanwhile, unsampled
   seeds were allowed to germinate uninterrupted until the 7th day, during
   which daily growth data were meticulously documented. A portion of the
   frozen samples was allocated for the measurement of physiological
   indicators, while the remaining portion was forwarded to Hangzhou
   Lianchuan Biological Co., Ltd. for metabolomic analysis.

Determination of seed germination index

   The procedures for measuring seed germination rate, germination index,
   and vigor index adhere to the guidelines established by the ISTA
   (International Seed Testing Association) [[78]35]. Seed germination
   rate, germination index, and vitality index are calculated according to
   the following formula:
   graphic file with name M1.gif
   graphic file with name M2.gif
   graphic file with name M3.gif

Determination of physiological indicators of seeds

Hydrogen peroxide (H[2]O[2])

   To quantify the H[2]O[2] content in oat seeds post-priming with OrT,
   SuT, and StT, Solarbio’s kit was employed. Initially, 0.1 g of oat seed
   samples were accurately weighed and ground into a fine powder using
   liquid nitrogen. Subsequently, the provided reagents were added to the
   samples in accordance with the instructions provided by Solarbio.
   Following the addition of reagents, the sample-reagent mixtures were
   thoroughly blended and allowed to incubate for the prescribed duration,
   as outlined in the kit instructions. Subsequently, the absorbance
   values of the samples were measured at a wavelength of 415 nm using a
   K6600-A microplate reader. Prior to measurement, the microplate reader
   was calibrated to ensure precise readings. To calculate the H[2]O[2]
   content in the oat seed samples, the formula provided in the kit
   instructions was applied.

Superoxide anion (O[2]^-)

   The preparation of the standard curve, along with the measurement and
   calculation methods for O[2]^−, is based on the work of Elstner and
   Heupel [[79]36]. Accurately weigh a 0.1 g sample of oat seeds and grind
   it into a fine powder using liquid nitrogen. Next, prepare three test
   tubes for the sample analysis. Add 0.5 mL of the sample supernatant,
   0.375 mL of phosphate buffer, and 0.125 mL of hydroxylamine
   hydrochloride to each tube. Incubate the tubes in a 25 °C water bath
   for 20 min. Then, add the reagents as done for the standard curve
   preparation and measure the absorbance at 530 nm.

Malondialdehyde (MDA)

   The measurement and calculation of MDA in oat seeds treated with OrT,
   SuT, and StT follow the method outlined by Schmedes and Hølmer
   [[80]37]. Weigh 0.1 g of the sample and add 1 mL of pre-cooled 10%
   trichloroacetic acid. Centrifuge the mixture at 4000 rpm for 10 min.
   Transfer 0.2 mL of the supernatant to a new tube. To this, add 1.8 mL
   of distilled water and 0.6% thiobarbituric acid solution. Mix
   thoroughly and incubate in a boiling water bath for 15 min. After
   incubation, cool the mixture rapidly. Take 200 µL of the cooled
   solution from each treatment and measure the absorbance at 600, 532,
   and 450 nm.

Proline (PRO)

   The preparation of the standard curve, along with the measurement and
   calculation methods for proline, is based on the work of Ábrahám et al.
   [[81]38]. Weigh a 0.1 g sample and grind it with liquid nitrogen. Plot
   the standard curve with proline content on the horizontal axis and
   absorbance on the vertical axis. Add 2 mL of glacial acetic acid, 2 mL
   of ninhydrin, and 1.5 mL of distilled water to 0.5 mL of the sample
   solution. For the control tube, add 2 mL each of glacial acetic acid,
   ninhydrin, and distilled water. Mix thoroughly. Develop the color and
   extract in the same manner as described for the standard curve
   preparation. Measure the absorbance at a wavelength of 520 nm.

Antioxidant enzyme activity

   To measure Catalase (CAT), Superoxide dismutase (SOD), Peroxidase (POD)
   and Ascorbate peroxidase (APX), we utilized the kit provided by
   Solarbio, adhering strictly to the accompanying instructions. The
   absorbance of the specified band was measured using the K6600-A
   microplate reader, and calculations were performed according to the
   provided formula. For further details, please refer to the instructions
   available on the Solarbio website: [82]https://www.solarbio.com/.

Metabolomics detection of oat seeds under different treatments

   Based on the germination index and physiological index of seeds, SuT
   and H[2]OT were chosen for metabonomic analysis, while samples without
   any initiation and aging treatment were designated as the control (CK).
   Three biological replicates were established in each group, and
   subsequently, the samples were sent to Hangzhou Lianchuan Biological
   Co., Ltd for subsequent metabonomic analysis.

Data analysis

   The significance of differences between groups was analyzed using SPSS
   26.0. LSD was chosen for post-hoc testing, with a significance level
   set at P < 0.05. Line charts and bar charts were created using GraphPad
   Prism 9. Advanced volcano mapping was conducted using the OmicStudio
   tool ([83]https://www.omicstudio.cn/tool). Additionally, a high-level
   pie chart illustrating the number of common differential metabolites
   between samples was generated using the OmicStudio tool. Metabolic
   pathway enrichment analysis was performed using MBRole 2.0, and the
   results were downloaded from
   [84]http://csbg.cnb.csic.es/mbrole2/index.php. Subsequently, the
   OmicStudio tool was utilized to draw the KEGG enrichment pathway map.
   RDA analysis and Spearman correlation analysis between differential
   metabolites and physiological indicators were carried out using
   OmicStudio tools and visualized accordingly. The schematic diagram is
   drawn by figdraw ([85]https://www.figdraw.com/).

Results and analysis

Effect of aging on germination of oat seeds

   As illustrated in Fig. [86]1, subsequent to the aging treatment, there
   was a significant decline observed in the germination rate, germination
   index, and vigor index of oat seeds, correlating with the duration of
   aging. Following exposure to a 144-hour aging treatment, the oat seeds
   exhibited a reduction of 23.53% in the germination rate, a decrease of
   28.21% in the germination index, and a decline of 58.63% in the vigor
   index.

Fig. 1.

   [87]Fig. 1
   [88]Open in a new tab

   The impact of artificial aging duration on oat seed germination. (A)
   The variation in oat seed germination rate with different artificial
   aging time; (B) The variation in oat seed germination index with
   different artificial aging time; (C) The variation in oat seed vigor
   index with different artificial aging time. The ‘*’ symbol denotes
   significant differences between various processing times and 0 h (CK).
   (0.01≤*P<0.05, 0.001≤**P<0.01,0.0001≤***P<0.001, ****P<0.0001)

Effect of EM bacterial solution on germination of aged oat seeds

   Figure [89]2 depicts the influence of EM bacterial solution on the
   germination rate, germination index, and vigor index of aged seeds.
   Significantly, OrT demonstrated a notable increase in the seed
   germination rate by 15.38%, the germination index by 30.71%, and the
   vigor index by 296.38%. Similarly, SuT exhibited a remarkable increase
   in the seed germination rate by 53.85%, the germination index by
   90.36%, and the vigor index by 397.29%. StT displayed a noteworthy
   increase in the seed germination rate by 41.03%, the germination index
   by 44.31%, and the vigor index by 339.12%.

Fig. 2.

   [90]Fig. 2
   [91]Open in a new tab

   The impact of EM bacterial solution on aged oat seed germination. (A)
   The variation in oat seed germination rate with different types of EM
   bacterial solution; (B) The variation in oat seed germination index
   with different types of EM bacterial solution; (C) The variation in oat
   seed vigor index with different types of EM bacterial solution. The ‘*’
   symbol denotes significant differences between different types of EM
   bacterial solution treatments and the H[2]OT (CK), The same notation
   applies below for subsequent analyses. (0.01≤*P<0.05,
   0.001≤**P<0.01,0.0001≤***P<0.001, ****P<0.0001)

Effects of EM bacterial solution on physiology of aged oat seeds

   Figure [92]3 illustrates the impact of EM bacterial solution on oat
   seed physiology. Notably, OrT demonstrated a considerable decrease in
   the MDA content by 68.33% and a significant increase in APX activity by
   69.88%. SuT resulted in a significant reduction in MDA content by
   50.00%, a notable increase in POD activity by 68.25%, and a significant
   elevation in APX activity by 94.31%. Additionally, StT exhibited
   significant reductions in MDA content by 41.67% and H[2]O[2] content by
   42.28%, alongside a notable decrease in POD activity by 82.28%.

Fig. 3.

   [93]Fig. 3
   [94]Open in a new tab

   The impact of EM bacterial solution on aged oat seed physiology. (A)
   The variation in oat seed MDA content with different types of EM
   bacterial solution; (B) The variation in oat seed O[2]^- content with
   different types of EM bacterial solution; (C) The variation in oat seed
   H[2]O[2] content with different types of EM bacterial solution; (D) The
   variation in oat seed POD activity with different types of EM bacterial
   solution; (E) The variation in oat seed SOD activity with different
   types of EM bacterial solution; (F) The variation in oat seed CAT
   activity with different types of EM bacterial solution; (G) The
   variation in oat seed APX activity with different types of EM bacterial
   solution; (H) The variation in oat seed PRO content with different
   types of EM bacterial solution. The ‘*’ symbol denotes significant
   differences between different types of EM bacterial solution treatments
   and the H[2]OT (CK). (0.01≤*P<0.05, 0.001≤**P<0.01,0.0001≤***P<0.001,
   ****P<0.0001)

KEGG enrichment analysis of differential metabolites

   Figure [95]4 provides comprehensive details regarding the differential
   metabolites observed across various comparison groups. Specifically, a
   total of 122 differential metabolites were identified between the
   H[2]OT and CK comparison groups, consisting of 64 up-regulated and 58
   down-regulated metabolites (Fig. [96]4A). These metabolites are
   associated with 9 pathways, with Histidine metabolism being the most
   significantly enriched pathway (Fig. [97]4D). In the comparison group
   of SuT and CK, a total of 381 differential metabolites were identified,
   with 260 up-regulated and 121 down-regulated metabolites (Fig. [98]4B).
   These metabolites are associated with 51 pathways, with the
   Aminobenzoate degradation pathway being the most significantly enriched
   (Fig. [99]4D). Furthermore, it is noteworthy that 11 differential
   metabolites were consistently identified across all comparison groups
   (Fig. [100]4C).

Fig. 4.

   [101]Fig. 4
   [102]Open in a new tab

   Analysis of differential metabolites and KEGG function between
   comparison groups. (A) Volcano diagram of differential metabolites
   between H[2]OT and CK ; (B) Volcano plot of differential metabolites
   between SuT and CK ; (C) Wayne diagram of differential metabolites
   between different treatments ; (D) KEGG enrichment pathway of
   differential metabolites

Identification of key differential metabolites

   Physiological indicators of oat seeds were utilized as response
   variables, with significantly different metabolites from the three
   treatments selected as explanatory variables. Redundancy analysis
   unveiled the metabolites influencing physiological indicators of purple
   oat seeds. As depicted in Fig. [103]5A, the cumulative explained
   variation of the first and second axes reached 79.52%, effectively
   reflecting the impact of significantly different metabolites on
   physiological indicators. Notably, 5-phosphoribosylamine exhibited
   robust explanatory power on physiological indicators, with an r^2 value
   of 0.8585.

Fig. 5.

   [104]Fig. 5
   [105]Open in a new tab

   Correlation between differential metabolites and physiology of oat
   seeds. (A) Redundancy analysis of differential metabolites and oat seed
   physiology in a single sample of different treatments. The blue arrow
   represents the physiological index of oat seeds, the red arrow
   represents the differential metabolites, and the length of the arrow
   line represents the effect of differential metabolites on the
   physiology of oat seeds. The longer the arrow length, the greater the
   effect. (B) Spearman correlation between 11 common differential
   metabolites and physiological indexes of oat seeds, *0.01 ≤ P<0.05

   Furthermore, Spearman correlation analysis was conducted on
   significantly different metabolites and physiological indicators among
   the three treatments, as shown in Fig. [106]5B. The findings revealed
   that differential metabolites significantly influenced oat seed
   physiological indicators. Specifically, seven metabolites exhibited
   significant correlations with physiological indicators (rho > 0.8,
   P < 0.05). Notably, MDA demonstrated significant negative correlations
   with four differential metabolites, namely neg-3.266_145.05147,
   neg-3.267_135.06708, neg-3.267_147.03088, and 5-phosphoribosylamine. In
   this study, 5-phosphoribosylamine emerged as prominently associated
   with seed physiology in both statistical analyses. Subsequently, this
   experiment delves into investigating the specific role of
   5-phosphoribosylamine (5-PRA) within metabolic pathways, building upon
   prior research.

Effect of EM bacterial solution on metabolic pathways of aged seeds

   Figure [107]6 illustrates the principal metabolic pathways associated
   with 5-PRA. These pathways include Alanine, aspartate, and glutamate
   metabolism, as well as Purine metabolism. Interestingly, no significant
   alterations in metabolite levels were detected in Alanine, aspartate,
   and glutamate metabolism, with 5-PRA primarily deriving from the
   breakdown of L-Glutamine. In Purine metabolism, 5-PRA serves as a
   crucial intermediate, displaying significantly heightened levels under
   SuT conditions. Guanine and Adenine, both products of Purine
   metabolism, exhibit decreased levels in H[2]OT and SuT compared to CK.
   Furthermore, (R)-Allantoin, another product of Purine metabolism,
   demonstrates markedly increased levels in SuT compared to H[2]OT and
   CK. Additionally, there is a noteworthy rise in the content of the
   (R)-Allantoin synthetic precursor, 5-hydroxyisourate.

Fig. 6.

   [108]Fig. 6
   [109]Open in a new tab

   Changes in the main metabolic pathways in aged oat seeds after induced
   by SuT

Discussion

   Senescence is thought to delay germination and seedling emergence,
   hinder growth rates, and increase sensitivity to environmental stress.
   It can also lead to higher levels of genomic damage, which compromises
   germination potential, induces instability in the plant genome, and
   impedes overall growth [[110]17, [111]39]. In this study, following the
   exposure of seeds to 144 h of artificial aging, notable decreases were
   observed in the germination rate, germination index, and vigor index,
   with the most significant decline noted in the vigor index.
   Furthermore, the utilization of EM bacterial solution notably augmented
   the germination rate, germination index, and vigor index of aged oat
   seeds. Particularly noteworthy was the remarkable effectiveness of the
   SuT in alleviating the detrimental effects of seed aging. Shaffique’s
   research demonstrates that Bacillus enhances the germination rate,
   germination index, and vigor index of rice seeds [[112]40]. This
   finding underscores the capacity of certain microorganisms to promote
   seed germination, which aligns with the results obtained in this study.
   The application of three different EM bacterial extract treatments
   significantly enhances the vigor index of aged oat seeds. Enhancing
   seed vigor index is crucial for promoting the healthy growth of plant
   seedlings [[113]41]. Seeds with high vigor possess the ability to
   germinate and develop at an accelerated rate, enabling crops to
   establish robust root and plant systems during the early stages of
   growth [[114]42]. This capability aids in minimizing crop losses
   attributable to external environmental factors. OrT, SuT, and StT
   extracts all exhibit the ability to promote seed germination, albeit
   with varying effects. This indicates that the mere activity of
   microorganisms may not be the primary determinant in facilitating seed
   germination. Instead, microbial metabolites are likely to play a
   crucial role. Specifically, the presence of active microbial
   metabolites and an abundance of metabolic enzymes within the SuT
   extract may serve as key factors in promoting the germination of aged
   oat seeds. It is possible that in SuT, bacteria and larger cell debris
   are separated and deposited at the bottom of the tube due to
   centrifugal force, leading to the release of their metabolites into the
   supernatant as a result of cell rupture. This process could potentially
   increase the presence of metabolites beneficial for anti-aging effects
   compared to OrT. Conversely, subjecting StT to high temperatures may
   result in the inactivation of certain metabolites [[115]43].

   The enhancement of seed germination indicators may arise from
   improvements in seed physiology. In this study, OrT, SuT, and StT
   significantly reduced the MDA content of oat seeds. A study conducted
   by Li et al. demonstrated a similar effect, where immersion of maize
   seeds in suspension of strain HX-2 resulted in a decrease of 42.38% in
   MDA content, indicating a positive effect of bacterial treatment on
   reducing MDA levels in seeds [[116]44]. The decrease in malondialdehyde
   production can be attributed to the impact of microbial metabolites
   present in EM solutions on plant metabolic processes. This phenomenon
   likely involves the modulation of redox equilibrium within plants and
   the enhancement of antioxidant substance synthesis [[117]45]. Reactive
   oxygen species can induce oxidative stress in seeds; however, this
   stress can be mitigated by the action of antioxidant enzymes [[118]46,
   [119]47]. In this study, the H[2]O[2] content in StT increased
   significantly, but this was not accompanied by an increase in MDA. In
   contrast, the study by Yao et al. found a strong correlation between
   H[2]O[2] and MDA, which differs from the findings in this study
   [[120]48]. This discrepancy may be attributed to an increase in
   H[2]O[2]induced by cellular components or PAMPs produced during
   high-temperature inactivation [[121]49]. Additionally, the changes in
   MDA content may be attributed to the presence of active metabolites in
   the EM bacterial solution, which can modulate the levels of H2O2 and
   MDA by influencing the activity of antioxidant enzymes [[122]50,
   [123]51]. The successful germination of seeds hinges upon maintaining a
   delicate balance between the rate of ROS accumulation and the activity
   of the ROS neutralization system. These roles include weakening the
   endosperm, mobilizing seed reserves, providing defense against
   pathogens, and facilitating programmed cell death. Moreover, ROS can
   serve as messengers or transmitters of environmental signals during
   seed germination [[124]52]. In this study, it was observed that the
   levels of O[2]^− and H[2]O[2] in SuT samples decreased slightly,
   although not significantly, and the overall levels of active oxygen did
   not undergo drastic changes. This suggests that the SuT bacterial
   solution induces a more gradual alteration in active oxygen levels,
   which may potentially be more advantageous for seed germination.
   Conversely, the significant increase in H[2]O[2] content observed in
   StT indicates that microbial activity plays a crucial role in
   regulating and maintaining stable levels of reactive oxygen species.
   Additionally, all three EM bacterial solutions led to increased
   activities of POD and APX, further indicating their positive influence
   on antioxidant enzyme systems. In Shaffique’s study, SH-9 was found to
   enhance the growth of rice plants, coinciding with a significant
   increase in the activities of antioxidant molecules including CAT, SOD,
   and APX. This observation is consistent with the findings of the
   current study [[125]40]. The elevation in antioxidant enzyme activity
   may be attributed to microorganisms acting as a source of antioxidants
   [[126]53, [127]54]. Moreover, microbial metabolites could potentially
   trigger specific regulatory mechanisms related to antioxidant enzymes
   [[128]55]. The increase in APX activity signifies an enhanced ability
   of oat seeds to finely regulate the levels of reactive oxygen species
   [[129]56]. POD enzymes are involved in plant differentiation and are
   also known to respond to environmental stress [[130]57]. The observed
   highest POD activity in StT could be attributed to the fact that
   H[2]O[2] serves as the substrate for POD. Therefore, a significant
   increase in substrate content leads to a notable increase in enzyme
   activity [[131]58, [132]59]. Notably, the SuT significantly elevated
   POD and APX activities and exhibited the highest levels of SOD and CAT
   activities, although the differences were not statistically
   significant. SOD serves as the primary enzyme in the antioxidant
   system, responsible for catalyzing the dismutation of superoxide
   radicals into H[2]O[2] [[133]60]. CAT is primarily responsible for
   catalyzing the decomposition of H[2]O[2] into water and oxygen
   [[134]61]. The positive effect of SuT on the antioxidant enzyme
   activity of oat seeds helps maintain the stability of moderate levels
   of ROS, while simultaneously keeping the MDA content of oat seeds at a
   low level. This preservation of physiological quality in the seeds is
   more conducive to germination [[135]60].

   EM can enhance plant tolerance to various environmental stresses by
   inducing changes in metabolic pathways. Examples of these changes
   include the synthesis of osmolytes, antioxidants, and stress-related
   hormones, all of which aid plants in coping with adverse conditions
   [[136]62–[137]64]. In this study, it was observed that SuT altered the
   pathway with the most significant enrichment of differential
   metabolites from Histidine metabolism to the aminobenzoate degradation
   pathway. Histidine serves as a precursor for the biosynthesis of
   various secondary metabolites in plants, including alkaloids,
   flavonoids, and cyanogenic glycosides [[138]65]. These secondary
   metabolites play crucial roles in plant defense mechanisms, stress
   responses, and signaling pathways [[139]66]. The enrichment of
   aminobenzoate degradation pathways may be attributed to EM. The
   abundance of bacteria within EM provides significant biodegradation
   capabilities [[140]67]. Furthermore, plants and EM can utilize the
   metabolites resulting from aminobenzoate degradation as either carbon
   or energy sources [[141]68]. Moreover, by facilitating the breakdown of
   organic pollutants such as aminobenzoates, this process can mitigate
   soil and water contamination, thereby safeguarding the health of the
   ecological environment [[142]69].

   5-PRA serves as a pivotal intermediate in an alternative initial
   pathway of purine biosynthesis, rendering it a crucial metabolite
   within this biosynthetic process [[143]70]. In this study, the
   metabolites of 5-PRA that were consistently present across all
   comparison groups were found to exhibit a stronger correlation with
   seed physiology in statistical analyses. Consequently, these
   metabolites were identified as key differential metabolites. In the
   current investigation, a noteworthy elevation of 5-PRA was observed in
   the H[2]OT treatment group, albeit not as pronounced as in the SuT
   group. This underscores the collective impact of microbial metabolites,
   metabolic enzymes, extracellular products, and the water component
   present in the EM bacterial supernatant, which contributes to the
   upregulation of 5-PRA. 5-PRA is likely produced through the Alanine,
   aspartate, and glutamate metabolism pathways, arising from the
   breakdown of L-Glutamine [[144]71]. Notably, our experiment did not
   observe substantial alterations in other metabolites within the 5-PRA
   synthesis pathway, potentially attributed to feedback inhibition
   mechanisms [[145]72]. Following its upregulation, 5-PRA entered purine
   metabolism; however, it did not induce notable changes in the levels of
   guanine and adenine, which are the other two key products in purine
   metabolism. Moreover, there were no significant alterations observed in
   other intermediate products of the guanine and adenine synthesis
   pathways. This suggests that the synthesis of guanine and adenine
   remains unaffected by the upregulation of 5-PRA. Conversely, within the
   synthesis pathway of (R)-Allantoin, another metabolite of Purine
   metabolism, a significant upregulation of (R)-Allantoin and its
   associated intermediates was evident. Irani and Christopher D Todd
   highlighted that Arabidopsis thaliana seedlings exhibiting elevated
   levels of allantoin demonstrate enhanced tolerance to drought and
   sodium chloride stress. The heightened concentrations of allantoin are
   believed to mitigate oxidative damage, thereby enhancing the plant’s
   resilience to abiotic stress conditions in Arabidopsis thaliana
   [[146]73]. In another study, it was observed that exogenous allantoin
   upregulated the expression of several antioxidant-encoding genes, such
   as SOS1 and RCD1 [[147]74]. In this study, it was observed that SuT
   exhibited elevated antioxidant enzyme activity, a phenomenon that may
   be associated with the augmented levels of (R)-Allantoin. Hence, SuT
   likely induces a notable upregulation in the synthesis of (R)-Allantoin
   within Purine metabolism by facilitating the production of 5-PRA.
   Subsequently, (R)-Allantoin is presumed to augment the activities of
   key antioxidant enzymes such as POD, SOD, CAT, and APX by upregulating
   the expression of genes involved in oxidative stress response. This
   enhancement significantly boosts the antioxidant capacity of oat seeds,
   allowing for precise regulation of reactive oxygen species levels.
   Consequently, despite a considerable decrease in the MDA content of oat
   seeds, there is a steady decline in the concentration of reactive
   oxygen species. This consistent maintenance of cellular redox
   equilibrium ensures minimal fluctuations in cell redox states, thereby
   preserving the integrity of cellular processes and intracellular
   reactions [[148]75]. Consequently, this state renders oat seeds more
   conducive to successful germination.

   Future studies should focus on identifying the regulatory genes
   involved in 5-PRA synthesis and elucidating their regulatory
   mechanisms. Furthermore, there is an urgent need to clarify the
   specific components of microbial active metabolites in the EM solution
   that promote seed germination. This study also acknowledges certain
   limitations that may influence the interpretation of the results. While
   it demonstrated that effective microbial EM has beneficial effects on
   oat seeds, it did not confirm whether EM exhibits broad biological
   adaptability. Investigating this aspect is essential to uncover the
   underlying mechanisms of microbial growth and its potential
   applications in agricultural production.

Conclusion

   The detrimental effects of oat seed aging intensify with prolonged
   exposure to aging conditions. Significant reductions in germination
   rate, germination index, and vigor index of oat seeds were observed
   following aging. However, treatment with EM bacteria solution
   demonstrated a capacity to restore the germination ability of oat
   seeds, with the most notable enhancement observed in seed vigor. This
   improvement in germination ability is attributed to decreased levels of
   malondialdehyde and enhanced antioxidant capacity within the seeds.
   Moreover, analysis of three distinct EM treatments revealed that the
   active metabolites produced by EM bacteria played a pivotal role in
   promoting seed germination, rather than the microorganisms themselves.
   Metabolic profiling highlighted the significance of
   5-phosphoribosylamine in the purine metabolic pathway. Particularly in
   the SuT, the upregulation of 5-phosphoribosylamine facilitated the
   synthesis of (R)-Allantoin, consequently augmenting antioxidant enzyme
   activity. The findings of this study not only offer a theoretical
   foundation for enhancing the quality of aged seeds but also present a
   promising approach for investigating the molecular responses of various
   plants to EM bacteria solution. This research provides valuable
   insights into strategies for improving seed quality.

Acknowledgements