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Article

The Synergistic Effect of Plant Growth-Promoting Rhizobacteria and Spent Mushroom Substrate Improves Ginseng Quality and Rhizosphere Nutrients

by
Siyao Fan
1,
Qian Hu
1,
Qi Liu
1,
Wenman Xu
1,
Zixin Wang
1,
Yu Huang
1,
Yang Zhang
2,*,
Wenxiu Ji
1,* and
Weiwei Dong
1,*
1
College of Agricultural, Yanbian University, Yanji City 133002, China
2
Jilin Academy of Agricultural Sciences, Changchun City 130000, China
*
Authors to whom correspondence should be addressed.
Agriculture 2024, 14(11), 1880; https://doi.org/10.3390/agriculture14111880
Submission received: 13 September 2024 / Revised: 19 October 2024 / Accepted: 21 October 2024 / Published: 24 October 2024
(This article belongs to the Section Agricultural Soils)
Browse Figures
Versions Notes

Abstract

:
The ginseng industry’s reliance on chemicals for fertilizer and pesticides has adversely affected the environment and decreased the quality of ginseng; therefore, microbial inoculum is an effective way to restore the damaged soil in ginseng fields. To investigate the effects of plant growth-promoting rhizobacteria (PGPR) and spent mushroom substrate (SMS) on soil and plant quality in ginseng, high throughput sequencing was performed to examine the microbial community structures in ginseng rhizosphere soil. All treatments significantly increased soil nutrient, enzyme activity, and ginseng biomass compared to control (p < 0.05). The combination of PGPR and SMS notably enhanced soil enzyme activities: urease (7.29%), sucrase (29.76%), acid phosphatase (13.24%), and amylase (38.25%) (p < 0.05). All treatments had different effects on ginseng rhizosphere soil microbial diversity. Significantly, the combination treatments enhanced microbial diversity by increasing the abundance of beneficial bacteria such as Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium and Plectosphaerella, meanwhile suppressing harmful Klebsiella. The relative abundance of Fusarium was reduced to some extent compared with the application of SMS alone. The soil organic matter, available potassium, available phosphorus, and alkaline nitrogen, as key factors, influenced microbial community structures. Overall, the combination of PGPR and SMS positively impacted the rhizosphere environment and ginseng plant quality.

1. Introduction

Panax ginseng C. A. Meyer as a Chinese medicine is a perennial herb in the Araliaceae family, considered to be a valuable and traditional remedy [1]. The three northeastern provinces of China, North Korea, and Russia are the main regions that produce wild ginseng. However, due to unregulated excavation and ecological destruction, wild ginseng resources are on the verge of extinction, and cultivated ginseng has become the mainstream product in the market [2]. The rigorous soil conditions needed for ginseng cultivation result in soil degradation over time. This degradation is characterized by soil compaction, decreased fertility, reduced soil biological activity, a decline in the number of beneficial microorganisms in the soil, and lowered soil biodiversity. These factors negatively affect crop yield and quality [3,4,5,6]. Intensive use of fertilizers and pesticides to increase yields and combat plant diseases leads to the accumulation of their toxic residues and intermediates [7]. Therefore, the development of a green and healthy environmentally friendly soil remediation method is essential.
Microbial activity is associated with plant roots and soil fertility. Their application to soil improvement is characterized by low cost, high efficiency, and minimal pollution [8]. Microorganisms play an important role in soil improvement by enhancing the physical and chemical properties, reducing nutrient loss, increasing soil aggregation, boosting the organic matter content of soil, and providing certain essential nutrients for plants [9,10]. At present, PGPR is commonly used to improve the quality of ginseng rhizosphere soil and plants. It can enhance soil enzyme activity, increase the abundance of beneficial microorganisms, change the microbial community structure of soil, reduce soil compaction, and thus improve crop yield and quality [11,12]. Using single or combined microbial strains can achieve both soil fertilization and a reduction in the need for chemical fertilizers. Qi et al. showed that the addition of the microbial agent Frankia F1 to the soil planted with ginseng promoted the secretion of organic acids from secondary metabolites, dissolved and released soil nutrients, improved enzymatic activity, and increased the availability of effective soil nutrients [13]. Shi et al. found that Paenibacillus polymyxa NSY50 can prevent the growth of ginseng rust roots, increase nutrient content, and improve the microbial community structure of ginseng soil [14]. Mohamed et al. found that the addition of Bacillus subtilis and Pseudomonas fluorescens, either singly or in combination, could control plant pathogens while improving plant nutrient status and maintaining soil sustainability in rapeseed soil [15]. Current research on soil improvement using PGPR has mainly focused on single strains or combinations of bacterial agents applied directly as fertilizers [16]. Although the PGPR promotion effect is obvious in favorable environments, some inoculated microbes may not survive in the face of intense competition from native genera [17]. Therefore, PGPR are immobilized in specific carriers to increase the activity and effective concentration of microorganisms while promoting their survival and establishment in the soil [18].
Spent mushroom substrate (SMS) is an organic solid fertilizer derived from the residue of cultivating various edible fungi [19]. As a carrier, SMS has a loose texture and a good pore structure, which facilitates the attachment of microorganisms. Combined with PGPR, SMS can improve the survival and colonization of PGPR in soil due to its special structure. SMS provides a nutrient-rich environment that supports microbial activity, allowing PGPR strains to thrive and further improve the nutrient status and microbial diversity of ginseng rhizosphere soil [20,21].
In this experiment, two PGPR strains were screened from healthy ginseng plants to prepare a composite suspension of bacteria. The effects of PGPR, SMS, and their combination on rhizosphere soil nutrients, enzyme activity, microbial community structure, and the quality of ginseng plants were investigated. We hope to utilize the synergistic effect of PGPR and SMS to promote the growth of ginseng plants, boost the population of beneficial bacteria in the soil, and provide a new approach for the high-quality development of the ginseng industry.

2. Materials and Methods

2.1. Test Materials

Ginseng seedlings (2 years old) were purchased from Helong Ginseng Farm (Jilin, China). The soil for the potting experiment was taken from the experimental field at Yanbian University: 5.69 pH, 0.116 S·m −1 electronic conductivity (EC), 11.94% organic matter (OM), 360.27 mg·kg−1 available nitrogen (AN), 7.99 mg·kg−1 available phosphorus (AP), 149.33 mg·kg−1 available potassium (AK), 7.46 g·kg−1 total nitrogen (TN), 0.54 g·kg−1 total phosphorus (TP), and 37.96 g·kg−1 total potassium (TK). The soil was brought to the laboratory for centrifugal classification, and 41.21% sand particles, 36.24% silt particles, and 22.11% clay particles were obtained. SMS was obtained from black fungus produced in Tianqiaoling, Tumen, Yanbian Korean Autonomous Prefecture, Jilin Province, China. After drying in an oven at 50 °C, it was ground and sieved through a 40-mesh sieve before use. The physical and chemical properties included pH 7.0, 0.963 S·m−1 EC, 26.93% total organic carbon (TOC), 51.08% OM, 15.90 g·kg−1 TN, 778.33 mg·kg−1 AN, and 22.87% humic acid (HA).
In a previous experiment, two PGPR strains were isolated and identified from healthy ginseng roots as Ochrobactrum sp. and Lysinibacillus sp. Furthermore, the plant growth-promoting functions of these two strains were analyzed and they exhibited various functions (Table S1). The 16S rRNA gene segments of the two PGPR strains were added to the NCBI database with accession codes MH788960 and MH788964.

2.2. Preparation of PGPR Bacterial Suspension

Two strains were inoculated in LB liquid medium, rapidly centrifuged to remove the supernatant, and washed repeatedly with sterile water to remove the medium. Finally, the strains were dissolved in sterile water. The concentration was adjusted to 1.0 × 108 CFU·mL−1 to prepare a single bacterial suspension, and a volume ratio of 1:1 was used to prepare a composite bacterial suspension [22].

2.3. Pot Experimental Design

Four treatment groups were established as follows: no fertilizer control (CK), a combination of the two PGPRs (OL), SMS (BCK), SMS, and two PGPRs (BOL). The container used for ginseng cultivation was an earthenware pot with a height of 20 cm and an inside diameter of 25 cm. The BCK and BOL groups incorporated SMS at 2% of the total soil mass, while the CK and OL groups added only soil, for a total of 4.5 kg of substrate per pot [23]. Two ginseng seedlings were transplanted into each pot in May 2023 for a trial period of 90 days. The OL and BOL groups were watered with 50 mL of microbial solutions on ginseng roots every 14 days (on days 7, 21, 35, 49, 63, and 77), starting from the 7th day of planting. The CK and BCK groups received the same volume of distilled water at the same time, for a total of 6 applications. Each treatment was independently replicated four times. Throughout the trial, a constant soil water content of 70 ± 5% was maintained, with a 12 h photoperiod and a temperature of 24 ± 1 °C.
After the pot experiment, the 90th day marked the collection of all samples to assess the physicochemical factors of the ginseng rhizosphere soil, along with the microbial community changes in the soil samples following 90 days of cycling, soil enzyme action, agricultural features of the herb ginseng, the quantity of chlorophyll, and antioxidant levels of enzymes.

2.4. Sample Collection

A five-point sampling technique was employed to collect and mix soil samples uniformly. After bringing the soil samples back, they were split into two halves. One portion was directly used to determine soil enzyme activity, while the other portion was spread out in a well-ventilated and dry indoor space. After adequate air drying, the sample was passed through a 2 mm mesh sieve to evaluate the physicochemical characteristics of soil.
The root zone soil was collected using the shaking root method for the detection of rhizosphere microorganisms. A sterile shovel was used, the ginseng roots from about 15 to 30 cm around the plant were excavated, and the roots were completely and carefully separated from the soil. The roots were rinsed with distilled water, and then the root vitality was measured immediately, while another fraction was stored at −80 °C for subsequent experiments.

2.5. Determination of Soil Physical and Chemical Properties and Soil Enzymes

The soil pH was measured using a pen pH meter purchased from Xinwei Co., Ltd. (Shanghai, China). The potassium dichromate sulfuric acid method, the NaOH alkaline hydrolysis and diffusion method, the sodium bicarbonate colorimetric method, and the NH4OAc extraction flame photometer methods were used to determine the OM, AN, AP, and AK contents, respectively [24,25]. The sodium phenol-sodium hypochlorite colorimetric approach was employed to detect urease (URE) activity. Acid phosphatase (ACP) activity was measured using p-nitrophenol-linked substrates. The 3,5-dinitro-2-hydroxybenzoic acid colorimetric method was used to measure the enzyme activities of sucrase and cellulase, whereas the potassium permanganate titration method was used to evaluate the activity of catalase (CAT) [26,27].

2.6. Determination of Physiological and Biochemical Indexes of Ginseng Plants

Tetranitroblue tetrazolium chloride was used to measure the amount of malondialdehyde (MDA) and the activity of superoxide dismutase (SOD). 2, 3, 5-triphenyltetrazolium chloride (TTC) was used to assess the root vigor. The 2-methoxyphenol technique was utilized to determine the activity of peroxidase (POD), and phenylalanine ammonia-lyase (PAL) activity was measured using 2-hydroxy-1-ethanethiol. Agronomic traits of ginseng roots were measured using a straightedge and Vernier caliper from Yuyan Scientific Instrument (Shanghai, China) [28,29].

2.7. Total DNA Extraction and PCR Amplification from Ginseng Rhizosphere Soil

A total of 1 g of fresh ginseng rhizosphere soil was used to extract soil DNA through the Power Soil DNA Isolation Kit (Mobio, Carlsbad, CA, USA). Electrophoresis on a 1.8% agarose gel was performed to assess the quality and quantity of the extracted DNA. The extracted DNA was then stored at −20 °C and amplified by PCR using primers 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) for the 16S rDNA V3-V4 region. The ITS1–ITS2 region of the genomic DNA was amplified by PCR using fungal primers ITS1F (5′-CTTGGTCATTTAGAGGAAGTAA-3′) and ITS2R (5′-GCTGCGTTCTTCATCGATGC-3′). The PCR reaction mixture consisted of 5–50 ng of DNA template, 0.3 μL of forward primer (10 μM), 0.3 μL of reverse primer (10 μM), 5 μL of KOD FX Neo Buffer, 2 μL of dNTP (2 mM each), 0.2 μL of KOD FX Neo, and ddH2O up to 20 μL. The PCR conditions were as follows: initial denaturation (95 °C, 5 min), followed by one cycle of denaturation (95 °C, 30 s), annealing (55 °C, 30 s), extension (72 °C, 45 s), repeated for 20 cycles, and a final extension (72 °C, 10 min), performed 1 cycle. The amplification product was then quantified with the Qsep-400 (BiOptic, Inc., New Taipei City, Taiwan, China) and purified using the Omega DNA purification kit (Omega Inc., Norcross, GA, USA). The products of the PCR were analyzed using a 1% agarose gel examination [30].

2.8. High-Throughput Sequencing of Ginseng Rhizosphere Soil Samples

Through base recognition analysis, the raw picture data files from high-throughput genetic sequencing were transformed into raw sequencing reads. The output was saved in the Fastq file format, which contained the sequence information together with the matching sequencing quality information. The preprocessing of the acquired data involved using Trimomatic v0.33 software to filter the raw reads first, and then, using Cutadapt1.9.1 software to identify and remove primer sequences, produced clean reads free of primer sequences and finished the quality filtering of the data. The clean reads of each sample were then concatenated by overlap utilizing paired-end sequence merging, which was carried out using Usearch v10 software. Next, the aggregated information was cleaned according to the various regions’ length ranges. To obtain the final legitimate data for further research, chimeric sequences were discovered and removed using UCHIME v4.2 software. USEARCH (version 10.0) was used to cluster sequences that share greater than 97% similarity into operational taxonomic units (OTUs). QIIME v2.0 software was used to classify and annotate OTUs and ASVs at a 70% confidence threshold. With the accession numbers PRJNA1149570 for bacterial sequences and PRJNA1149667 for fungal sequences, the original reads were uploaded to the NCBI database.

2.9. Data Analysis and Processing

Using SPSS v26.0 software to compare the differences in microbial composition and microbial diversity index of two rhizosphere soils at different levels in one-way ANOVA. The diversity and abundance of microbial and fungal communities in ginseng rhizosphere soil under various treatment scenarios were evaluated. Using Uparsene v10.0 software, the soil OTU clustering was assessed, and the alpha and beta diversity indices were calculated. The alpha index analysis of each sample’s species diversity and complexity was determined using QIIME2 software. Principal coordinate analysis (PCoA) was performed in the R environment using the “vegan” package to analyze beta diversity, species complexity within the samples, and variations in the organization of the microbial community under various treatment conditions.
To assess the relationship between the physicochemical characteristics of ginseng rhizosphere soil, soil enzymes, and dominant bacterial genera in the microbial community, redundancy analysis (RDA) correlation analysis was conducted using the corrplot package. PERMANOVA analysis (ADONIS), multiple response permutation procedure (MRPP), and similarity analysis (ANOSIM) techniques were used to investigate how the various treatments affected the community structure. Additionally, to evaluate the direct or indirect correlations between prevalent bacterial genera and EC, pH, OM, AK, AN, AP, URE, ACP, SCL, CAT, and IA, structural equation modeling (SEM) was performed using AMOS v26.0 software (IBM SPSS AMOS 26).

3. Results

3.1. Effects of Physicochemical Characteristics of Ginseng Rhizosphere Soil Under Different Treatments

Alterations in the chemical and physical properties of ginseng rhizosphere soil under different treatments are denoted in Figure 1. Compared to the CK group, the physicochemical properties of the ginseng rhizosphere soil in each inoculation treatment group were improved. Among them, the components of OM, AN, AP, and AK in the BOL group’s rhizosphere soil were 15.56%, 97.62 mg·kg−1, 60.21 mg·kg−1, and 250.90 mg·kg−1, respectively. These contents increased significantly, and the improvement effect was the best among all treatments (p < 0.05) (Figure 1c–f). At the same time, SMS combined with PGPR could, to some extent, increase soil pH, reduce soil EC values, delay soil acidification, and maintain the healthy development of ginseng plants (Figure 1a,b).

3.2. Effects of Enzyme Activity of Ginseng Rhizosphere Soil Under Different Treatments

The activity of the enzymes in the ginseng rhizosphere generally improved under the influence of PGPR and SMS (Figure 2). In terms of URE, ACP, IA, and SCL activities, the improvement in the BOL group was the most significant among all treatment groups (p < 0.05). In comparison to the CK group, these activities increased by 7.29%, 13.24%, 38.25%, and 29.76%, respectively (Figure 2a–d). Although the activity of CAT in the OL group was 2.26% higher than that in the BOL group (Figure 2e), applying PGPR or SMS alone improved the activity of ginseng rhizosphere soil enzymes to a certain extent. However, the overall effect was not as pronounced as that observed in the BOL group.

3.3. Effects of Various Inoculants on the Agronomic Characters of Ginseng Plants

3.3.1. Chlorophyll Content in Ginseng

Measuring the physiological indicators and agronomic characteristics of ginseng can intuitively reflect its growth, development, and the efficacy of the soil nutrient supply [31]. As shown in Figure 3a, the chlorophyll content of ginseng plants in the OL, BCK, and BOL groups had a greater level than that in the CK group. The BOL group had the highest chlorophyll content among all groups, which was 73.87% higher than that of the CK group (p < 0.05).

3.3.2. Root Indexes of Ginseng Plants

It denotes the alterations in the root indexes of ginseng plants under different treatments in Figure 3b–f. Agronomic traits such as root length, root activity, root diameter, root weight, and fibrous root number were improved in the OL, BCK, and BOL groups compared to the CK group.

3.4. Antioxidant Enzyme Activity of Ginseng Plants in Response to Various Treatments

The reactive oxygen species (ROS) scavenging enzyme system plays a crucial role in plants [32]. Consequently, the function of antioxidant enzymes in ginseng plants was assessed by measuring the activities of SOD, POD, MDA, and PAL enzymes. Compared to that of the CK group, the OL, BCK, and BOL groups all significantly enhanced the activity of relevant antioxidant enzymes (p < 0.05), and the activities of SOD, POD, and PAL improved by 11.42%, 59.26%, and 26.51%, respectively, in the BOL group (Figure 4a,b,d). In contrast, the MDA content was reduced by 24.31% (Figure 4c).

3.5. Effects of the Abundance and Diversity of Ginseng Rhizosphere Soil Under Different Treatments

We conducted an Illumina Novaseq (Baimaike Biotechnology Co., Ltd, Beijing, China) sequencing analysis of bacterial 16S rRNA (V3–V4 region) and fungal 18S rDNA (ITS region) from the four treatment groups to understand the variety of microbes present in the soil of the ginseng rhizosphere. In total, 1,440,531 pairs of effective 16S rRNA and 1,437,870 pairs of effective 18S rDNA sequences were obtained from the 24 samples. After quality control, 1,434,584 and 1,432,524 clean sequences were obtained with averages of 79,699 and 79,585 sequences produced. The average lengths of the sequence reads were 423 bp and 254 bp.
Different treatments resulted in varying quantities of bacterial and fungal OTUs in the soil samples from the ginseng rhizosphere (Figure 5a,b). The bacterial community had 612 identical OTUs found in all groups, while the total number of OTUs specific to each group was 1, 2, 3, and 5, respectively. There were 361 identical OTUs in all groups in the fungal community, with characteristic OTU numbers of 14, 96, 11, and 74, respectively.
Alpha diversity was used to analyze species diversity and richness within a single sample, whereas ACE and Chao1 indices mostly demonstrated the sample’s richness in communities. Compared to the CK, OL, and BOL groups, the BCK group had significantly greater levels of both markers in the samples of the bacterial and fungal communities, indicating that the rhizosphere soil’s microbial, bacterial, and fungal communities’ richness of ginseng in the BCK group was higher (Table 1, Figure 5c,d). The Shannon and Simpson indices demonstrated the diversity of the community. Compared to the BCK group, these two indicators were significantly lower in the other three groups, indicating that the BCK group has the highest diversity of bacterial and fungal communities (Table 1, Figure 5e,f).

3.6. Variations in the Bacterial and Fungal Community Composition in Ginseng Rhizosphere Soil Under Different Treatments

The relative prevalence of each of the four treatments at the phylum and genus levels is displayed in a bar chart in Figure 6. All groups were similar at the bacterial community phylum level but with different abundances. Among these, Proteobacteria, Firmicutes, Actinobacteria, Bacteroidetes, and Chloroflexi were the five most abundant bacteria, accounting for over 97.04% of the total sequences. Compared with that of the CK, BCK, and BOL groups, the relative richness of Firmicutes decreased while the relative abundance of Proteobacteria increased in the OL group.
Additionally, compared to the CK group, each of the three groups displayed an increasing trend in their proportions of Actinobacteria (Figure 6a). The five genera with the highest abundances among all treatment groups were Klebsiella, Pseudomonas, Allorhizobium-Neorhizobium-Rhizobium, Ochrobactrum, and Achromobacter. Among these, the relative abundances of Klebsiella (p < 0.05) and Ochrobactrum in the BCK group were not as high as they were in the other three groups, while the abundances of Achromobacter and Allorhizobium-Neorhizobium-Rhizobium (p < 0.05) were higher (Figure 6b,e,f).
Figure 6c,d display the makeup of the fungal communities from each group in the soil of the ginseng rhizosphere. Each group’s community organization was comparable at the phylum level. Ascomycota, Basidiomycota, Mortiellomycota, and Chytidiomycota were the four most prevalent phyla. Phylogenetic relative richness, however, differed between the groupings. The relative abundances of Ascomycota and Mortiellomycota in the BCK group exhibited a growing trend in comparison to the other three groups, whereas the proportions of Basidiomycota and Chytridiomycota showed a declining trend (Figure 6c). The top five dominant fungal genera were Mortierella, Fusarium, Plectosphaerella, Leucoagallicus, and Erysiphe, respectively. Among all groups, the largest proportion of Mortierella was found in the BOL group. The relative abundance of Plectosphaerella increased (p < 0.05) in the BCK group compared to each of the other groups, while the relative abundance of Erysiphe declined (Figure 6d,g,h). The findings suggested that the community composition of bacterial and fungal microbes in the soil of the ginseng rhizosphere might have been changed by PGPR and SMS.

3.7. Correlation Investigation of Environmental Parameters of Microbial Populations in Ginseng Rhizosphere Soil

The correlations among soil microbes, soil physicochemical characteristics, and the function of soil enzymes in the ginseng rhizosphere across several treatment groups were investigated using redundancy analysis (RDA). On the first and second sorting axes, the variations in the explanations of soil physicochemical parameters by bacteria were 34.98% and 16.63%, respectively (Figure 7a), while the variations in soil enzyme activity were 22.09% and 14.63%, respectively (Figure 7b). The pH value was negatively correlated with the EC value and positively correlated with soil nutrients, such as OM, AN, AP, and AK. Achromobacter, Allorhizobium-Neorhizobium-Rhizobium, Bacillus, and Pseudomonas were positively correlated with soil nutrients. In addition, there was a positive correlation between soil enzyme activities and Paenibacillus, Klebsiella, and Acinetobacter, which showed positive correlations with all soil enzyme activities.
The differences in the explanations of soil physicochemical properties by fungi on the first and second sorting axes were 17.76% and 16.03%, respectively (Figure 7c), and the variations in soil enzyme activity were 17.28% and 15.43%, respectively (Figure 7d). There was a positive correlation among soil nutrients, all of which were positively correlated with Mortierella, Plectosphaerella, Fusarium, Pseudopyrochaeta, and Tetracladium. The activities of various enzymes in the soil were positively correlated with Paecilomyces, Fusarium, Plectosphaerella, Pseudopyrochaeta, and Aspergillus, whereas the activities of CAT and ACP soil enzymes were positively correlated with Leohumicola and Erysiphe.

3.8. Analysis of β-Diversity for the Bacteria and Fungi in Ginseng Rhizosphere Soil Under Different Treatment Conditions

We used principal coordinate analysis to analyze the differences in the microbial community structure of ginseng rhizosphere soil under different treatment conditions [33]. The PCoA results showed significant differences between different treatments in the bacterial and fungal communities of ginseng rhizosphere soil, with p-values of 0.001 for both (Figure S1a,b). In terms of bacterial community, principal components PC1 and PC2 accounted for 23.39% and 11.85% of the total, respectively. The CK and BCK groups were concentrated in the second quadrant, while the OL and BOL groups were concentrated in the third and fourth quadrants. The distance between groups was relatively large, indicating that there were some differences between groups (Figure S1a). In terms of the fungal communities, principal components PC1 and PC2 accounted for 37.70% and 16.14% of the total, respectively. The CK and BCK groups were concentrated in the second quadrant, while the OL and BOL groups were concentrated in the first and fourth quadrants. The distance between the groups was relatively large, indicating that there were some differences between the groups (Figure S1b).

3.9. Correlation Network Analysis of Bacterial and Fungal Microbial Communities in Ginseng Rhizosphere Soil

In order to analyze the correlation of microbial species and species interactions in ginseng rhizosphere soil, the Spearman correlation method was used to conduct network analysis between bacterial and fungal communities in the samples [34]. As shown in Figure 8, there was a good correlation between species at both the phylum and genus levels in the bacterial and fungal communities. In terms of bacterial communities, Klebsiella, Pseudomonas, and Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium had a higher abundance of bacterial genera, which was consistent with previous research on dominant bacterial genera at the phylum level. The thicker the line in the figure, the more significant the correlation, with red representing positive correlation and green representing negative correlation. In terms of fungal community richness, the top-ranked genera were Erysiphe, Plectosphaerella, Fusarium, and Mortierella, which was consistent with previous research. In the fungal communities, the positive and negative correlations between genera were balanced, with Erysiphe and Plectosphaerella showing the most significant correlation. This also supported the findings between them and indicated that there were variations in the bacterial and fungal microbiological communities in the ginseng rhizosphere soil under various treatment conditions.

4. Discussion

The quality of ginseng plants is strongly correlated with the surrounding environment. PGPR is an important resource in biological fertilizers that promote the utilization of microorganisms in the soil, increasing crop yields. The two PGPR strains Ochrobactrum sp. and Lysinibacillus sp. used in this experiment have unique characteristics and are widely used in agriculture and environmental protection [35]. Ochrobactrum sp. is renowned for its nitrogen fixation ability and plant hormone production, while Lysinibacillus sp. excels in phosphorus dissolution and the production of antibacterial compounds, helping to protect plants from pathogens [36,37]. According to the Supplementary Material shown in Table S1, this experiment also detected that two strains possess phosphorus solubilization and nitrogen-fixing functions, demonstrating good resistance against black spot disease and gray mold disease. Based on this, their combined application may improve nutrient supply and enzyme activity in ginseng soil, thereby enhancing soil quality and plant health. In this experiment, the application of PGPR increased the pH of the ginseng rhizosphere soil and delayed soil acidification compared with the CK group.
Nutrients, including cellulose, lignin, amino acids, minerals, microbes, and organic acids are plentiful in SMS. It has the effect of improving soil, promoting microbial activity, and enhancing plant quality. Additionally, its application in soil can trigger a series of microbiological changes, including increased diversity of bacteria and fungi, enrichment of specific microorganisms, production of antioxidant enzymes, and enhancement of metabolic pathways [38,39,40].
Moreover, PGPR and SMS can increase the accumulation rate of soil nitrogen, reduce ecosystem nitrogen cycling, and alleviate soil acidification in farmlands. The decrease in soil EC might be due to the fact that PGPR promoted more soil aggregate formation in the root system of ginseng plants. These soil aggregates were negatively correlated with salt concentration, indicating that PGPR was effective in mitigating the buildup of soluble salts in the soil [41]. Applying SMS increased the amount of organic carbon in the soil and intensified the growth of strains, promoting the release of nutrients and solubilization of insoluble nutrients, therefore raising the accessible potassium and phosphorus levels in the soil. Shi et al. found that inoculation with Pseudomonas chloriraphisH1 and Bacillus altitudinisY1 significantly increased the pH of soybean soil, delayed soil acidification, and promoted the growth of soybean plants [42]. Dumigan et al. showed that applying PGPR containing Providencia rettgeriP2, Advenella incentataP4, Acinetobacter calcoaceticusP19, and Serratia plymuthicaP35 increased the available nutrients in the soil, thereby promoting the growth of Avena sativa, Medicago sativa, and Cucumis sativa seedlings [43].
Studies conducted recently have also demonstrated that PGPR influences the physicochemical characteristics of plant soil, increases soil enzyme activity, and facilitates the conversion of solid nutrients into available nutrients. In this study, more extracellular enzymes were released after applying PGPR, and their microbial activity induced the secretion of certain compounds by rhizosphere microorganisms, thereby improving the rhizosphere soil environment. After the addition of SMS, the increase in soil enzyme activity may be due to changes in the soil pore structure, which provides a carrier for various enzyme reactions and energy for the growth and development of various microorganisms, thereby stimulating soil biological activity. Xu et al. found that applying PGPR enhanced the activity of related enzymes in soil samples and the tolerance of tobacco seedlings to salt stress [44].
The plant root system is the organ of plant absorption and synthesis, which primarily receives signals from environmental factors and has a very important influence on vital plant activities. In this study, ginseng plants treated with PGPR and SMS exhibited a significantly higher chlorophyll content compared to the CK group. At the same time, agronomic characteristics that were superior to those in the CK group included root weight, root length, root thickness, and the number of fibrous roots This finding is similar to the results of a previous study on PGPR in promoting soybean growth [45]. Both high and low chlorophyll content can affect the formation of photosynthetic products and promote the absorption of soil nutrients by plant roots, thus improving plant quality.
Superoxide dismutase and peroxidase form a complete antioxidant chain that can effectively eliminate excess oxidative free radicals in the plant body, protect the plant from oxidative damage, and improve its resistance [46]. This study found that the combined application of PGPR and SMS promoted antioxidant enzymes, which might be related to improved soil nutrition and the introduction of beneficial microorganisms. The changes in MDA content in plants reflect the degree of peroxidation; the higher the content, the greater the damage to the plant. After the combined use of PGPR and SMS, the MDA content was reduced compared to the CK group. PAL is a key enzyme in plant secondary metabolism and plays an important role as an antibacterial agent in plants. Nozari et al. found that PGPR-containing Streptomyces spp. could regulate the enzyme activity and osmotic pressure of the antioxidant system, thereby reducing the harmful effects of salinity on maize plants [47]. Afragan et al. showed that inoculation with PGPR and arbuscular mycorrhizal fungi (AMF) could significantly improve the redox state of rapeseed and enhance salt tolerance [48]. This study also showed that PGPR and SMS increased PAL activity to varying degrees, thereby improving the quality and disease-resistance capabilities of ginseng plants.
The diversity and composition of microorganisms significantly affect soil productivity, and plant growth and development, as well as plant quality, which may be related to fertilization methods, cropping systems, and rhizospheric exudates. In one study, the application of PGPR and SMS significantly increased the richness and diversity of microorganisms in the ginseng rhizosphere. This might be because the porous nature of SMS provides a good ecological niche for PGPR, facilitating its establishment in the soil, supporting the development and proliferation of beneficial microorganisms [49].
Regarding bacterial communities, the proportion of Actinobacteria was greater in all treatment groups, possibly because of the release of various soluble organic carbons by PGPR in the roots of ginseng plants, which stimulated the activity of Actinobacteria and promoted their rapid proliferation. Actinobacteria is a vital bacterial group in soil, essential for suppressing soil diseases, enhancing nutrient cycling, and producing various antibiotics. Our results indicated that the relative abundance of Actinobacteria was greater in each treatment group compared to the CK group, consistent with the results of Piromyou et al. Actinobacteria have important potential applications in alleviating obstacles to plant monoculture, regulating the balance of soil microbiota, and increasing the abundance of helpful bacteria [50]. In addition, Pseudomonas is an important bacterial genus in the plant body that acts as potassium and phosphorus-solubilizing bacteria that improve the efficiency of soil nutrient use. Sun et al. showed that PGPR isolated from tomato plants enhanced the relative abundance of Pseudomonas spp. in rootstocks [51]. In this study, PGPR and SMS also increased the relative abundance of the Pseudomonas spp. and the available phosphorus and potassium contents in the soil, further demonstrating the importance of the Pseudomonas spp. The relative abundance of Allorhizobium-Neorhizobium-Rhizobium also increased after the addition of PGPR with SMS, which aligns with the findings of a previous study. Balazs et al. reported that Sophora japonica could be symbiotic with Allorhizobium-Neorhizobium-Rhizobium, which helped improve the yield of the plant and enhance levels of its active ingredients. However, the mechanism of action is relatively complex [52]. Zhang et al. conducted a high-throughput 16S rRNA sequencing study of Allorhizobium-Neorhizobium-Rhizobium and endophytes in healthy and root rot-diseased pseudo-ginseng plants [6]. The relative abundance of Allorhizobium-Neorhizobium-Rhizobium was notably greater in healthy plants compared to those affected by root rot, promoting the healthy growth of pseudo-ginseng. Interestingly, in this study, it was found that the relative abundance of Ochrobactrum and Lysinibacillus in the CK group was higher than in other treatment groups. The reason for this result might be due to the complex relationships between microbial communities and the possibility of these two beneficial bacteria colonizing as endophytes within the host plant, which resulted in a decrease in the relative abundance of Ochrobactrum and Lysinibacillus in the rhizosphere soil [42]. In addition, Fusarium, a widespread genus of fungi found in plants, plays an important role in crop production and soil health due to its ecological functions. In this study, it was found that the addition of SMS introduced Fusarium, and its relative abundance decreased to some extent with the application of PGPR fungicide. This might be due to the synergistic effect of PGPR with SMS, which inhibited the activities of some specific microorganisms in SMS, thus attenuating the presence of Fusarium [53].
This study revealed that the relative abundance of Ascomycota and Basidiomycota in the horizontal fungal phylum community of ginseng rhizosphere soil showed opposite trends, likely attributed to the competitive interactions between these two fungal groups, aligning with the findings of Ding et al. [54]. At the genus level of fungal genera, Li et al. reported that Plectosphaerella is a fungal species that prevents and controls soil-borne plant and fungal diseases and has a high lethal activity against root-knot nematodes [22]. The addition of PGPR and SMS in this study made Plectosphaerella the dominant microbial community, reducing the likelihood of plant diseases occurring. Studies have shown that Erysiphe is a pathogen that affects plant growth and development and causes damage to leaf surfaces. In this study, despite a small quantity of Erysiphe in the CK group, its abundance decreased after applying PGPR and SMS [55]. Further research on the biological control of ginseng and its rhizosphere soil can be conducted by combining changes in the abundance of Plectosphaerella and Erysiphe. The RDA was used to analyze the association between the growth, physiological characteristics, and rhizosphere characteristics of ginseng in this study. The results indicated that soil enzyme activity, soil properties, and microbial genera are closely related to the growth of ginseng plants, and this experimental result has been proven in many previous studies. Gastélum et al. showed that after inoculating maize soil with spore-forming bacteria, they could interact with rhizosphere bacteria to promote crop growth [56]. Fountain et al. identified a crucial positive correlation between the Aspergillus abundance and enzyme activity in their research on cucumber biocontrol. This finding was consistent with the changes in soil properties of the ginseng rhizosphere after the addition of SMS and PGPR in this study, and it also showed a positive correlation with both Aspergillus abundance and soil physicochemical properties in the correlation analysis with environmental factors [57].

5. Conclusions

This study revealed that the use of PGPR alongside SMS significantly enhances the levels of nitrogen, phosphorus, potassium, and other nutrients in the soil, while also increasing enzyme activity and improving the overall soil environment. Furthermore, this synergistic effect seems to have a positive impact on the agronomic characteristics of ginseng roots, increasing the activity of antioxidant-related enzymes and promoting plant growth, ultimately enhancing ginseng quality. It is worth noting that the application of PGPR and SMS also significantly altered the microbial community structure in the ginseng rhizosphere soil. Various soil enzyme activities are associated with specific microorganisms, and the interactions between microorganisms are crucial. These findings suggest that the integration of PGPR and SMS may play an essential role in improving and remediating soil in ginseng fields. Further research should focus on the metabolic products, and their promoting mechanisms, of these two strains in the coming years.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agriculture14111880/s1, Table S1: The plant growth promoting function of two strains of bacteria. Figure S1: Analysis of bacterial and fungal community diversity in β—ginseng rhizosphere soil: (a) Bacteria, (b) Fungi.

Author Contributions

S.F. and Q.H. prepared the text, while W.D. thought out and planned the experiments. Thesis design and organizational implementation by Q.L., W.X., Z.W. and Y.H. The experiments and data analysis were carried out by S.F. and Q.H. The manuscript was revised by W.D. and S.F. The strain culture method was provided by W.J. Some of the supplies and tools required for the experiment were given by Y.Z. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by Jilin Provincial Science and Technology Department Project (YDZJ202201ZYTS635, 20230402028GH).

Institutional Review Board Statement

Not applicable.

Data Availability Statement

The original data presented in the study are openly available in NCBI at https://www.ncbi.nlm.nih.gov, accessed on 19 August 2024 and with login number PRJNA1149570 and PRJNA1149667.

Acknowledgments

The authors would like to thank Zhang Yang coming from Jilin Academy of Agricultural Sciences for providing the experimental base.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. The characteristics of physical and chemical in the rhizosphere soil of ginseng under different treatments: (a) pH, (b) EC, (c) OM, (d) AN, (e) AP, (f) AK. The values indicate the mean ± the standard deviation of four replicate tests (n = 4) for each group using Duncan multiple range test. a, b, c, and d denote significant variance at p < 0.05.
Figure 1. The characteristics of physical and chemical in the rhizosphere soil of ginseng under different treatments: (a) pH, (b) EC, (c) OM, (d) AN, (e) AP, (f) AK. The values indicate the mean ± the standard deviation of four replicate tests (n = 4) for each group using Duncan multiple range test. a, b, c, and d denote significant variance at p < 0.05.
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Figure 2. Changes in enzyme activity of ginseng rhizosphere soil: (a) URE activity, (b) ACP activity, (c) IA activity, (d) SCL activity, and (e) CAT activity. The values indicate the mean ± the standard deviation of four replicate tests (n = 4) for each group using Duncan multiple range test. a, b, c, and d denote significant variance at p < 0.05.
Figure 2. Changes in enzyme activity of ginseng rhizosphere soil: (a) URE activity, (b) ACP activity, (c) IA activity, (d) SCL activity, and (e) CAT activity. The values indicate the mean ± the standard deviation of four replicate tests (n = 4) for each group using Duncan multiple range test. a, b, c, and d denote significant variance at p < 0.05.
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Figure 3. The agronomic traits of ginseng plants in different treatments: (a) Chlorophyll content, (b) Root activity, (c) Root weight, (d) Root length, (e) Root diameter, and (f) Fibrous root number. The values indicate the mean ± the standard deviation of four replicate tests (n = 4) for each group using Duncan multiple range test. a, b, c, and d denote significant variance at p < 0.05.
Figure 3. The agronomic traits of ginseng plants in different treatments: (a) Chlorophyll content, (b) Root activity, (c) Root weight, (d) Root length, (e) Root diameter, and (f) Fibrous root number. The values indicate the mean ± the standard deviation of four replicate tests (n = 4) for each group using Duncan multiple range test. a, b, c, and d denote significant variance at p < 0.05.
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Figure 4. The antioxidant enzyme activity of ginseng plants under different treatments: (a) SOD, (b) POD, (c) MDA, and (d) PAL. The values indicate the mean ± the standard deviation of four replicate tests (n = 4) for each group using Duncan multiple range test. a, b, c, and d denote significant variance at p < 0.05.
Figure 4. The antioxidant enzyme activity of ginseng plants under different treatments: (a) SOD, (b) POD, (c) MDA, and (d) PAL. The values indicate the mean ± the standard deviation of four replicate tests (n = 4) for each group using Duncan multiple range test. a, b, c, and d denote significant variance at p < 0.05.
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Figure 5. The abundance curves, sparse curves, and Venn diagrams for bacteria and fungi in ginseng rhizosphere soil: (a) Bacterial Venn diagram, (b) Fungal Venn diagram, (c) Bacterial grade abundance curve, (d) Fungal grade abundance curve, (e) Bacterial sparse curve, (f) Fungal sparse curve.
Figure 5. The abundance curves, sparse curves, and Venn diagrams for bacteria and fungi in ginseng rhizosphere soil: (a) Bacterial Venn diagram, (b) Fungal Venn diagram, (c) Bacterial grade abundance curve, (d) Fungal grade abundance curve, (e) Bacterial sparse curve, (f) Fungal sparse curve.
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Figure 6. The phylum and genus levels of bacterial and fungal communities in the rhizosphere soil of ginseng: (a) Bacterial phylum, (b) Bacterial genus, (c) Fungal phylum, (d) Fungal genus, (e–h) Comparison of differential bacterial genera. *, ***, **** denote significant variance at p < 0.05, p < 0.05, and p < 0.001, respectively.
Figure 6. The phylum and genus levels of bacterial and fungal communities in the rhizosphere soil of ginseng: (a) Bacterial phylum, (b) Bacterial genus, (c) Fungal phylum, (d) Fungal genus, (e–h) Comparison of differential bacterial genera. *, ***, **** denote significant variance at p < 0.05, p < 0.05, and p < 0.001, respectively.
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Figure 7. RDA analysis of the correlation between bacterial and fungal genera in ginseng rhizosphere soil and environmental factors: (a,b) Bacteria, (c,d) Fungi.
Figure 7. RDA analysis of the correlation between bacterial and fungal genera in ginseng rhizosphere soil and environmental factors: (a,b) Bacteria, (c,d) Fungi.
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Figure 8. Network analysis of bacterial and fungal communities in the rhizosphere soil of ginseng: (a) Bacteria, (b) Fungi.
Figure 8. Network analysis of bacterial and fungal communities in the rhizosphere soil of ginseng: (a) Bacteria, (b) Fungi.
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Table 1. The alpha diversity index of bacterial and fungal communities in the rhizosphere soil of ginseng.
Table 1. The alpha diversity index of bacterial and fungal communities in the rhizosphere soil of ginseng.
CategoryTreatmentACE IndexChao1 IndexSimpson IndexShannon Index
bacterialCK683.5732 ± 1.32 b688.1639 ± 2.31 b0.8266 ± 0.02 d4.1577 ± 0.13 d
OL639.7686 ± 2.61 d638.5988 ± 1.65 d0.8483 ± 0.06 c4.4643 ± 0.16 c
BCK698.6491 ± 2.49 a708.9038 ± 2.54 a0.9336 ± 0.03 a5.5416 ± 0.11 a
BOL677.1414 ± 1.69 c682.9095 ± 2.76 c0.8783 ± 0.04 b5.1309 ± 0.12 b
fungalCK751.3808 ± 2.31 b699.6522 ± 1.32 c0.9436 ± 0.04 c6.4715 ± 0.14 b
OL754.0475 ± 2.14 b742.2826 ± 1.45 b0.9557 ± 0.03 b5.9319 ± 0.11 d
BCK783.2486 ± 3.15 a759.5000 ± 1.56 a0.9778 ± 0.08 a6.9699 ± 0.06 a
BOL664.1192 ± 2.45 c657.6742 ± 2.31 d0.9487 ± 0.02 c6.2180 ± 0.12 c
The values indicate the mean ± the standard deviation of four replicate tests (n = 4) for each group using Duncan multiple range test. a, b, c, and d denote significant variance at p < 0.05.
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Fan, S.; Hu, Q.; Liu, Q.; Xu, W.; Wang, Z.; Huang, Y.; Zhang, Y.; Ji, W.; Dong, W. The Synergistic Effect of Plant Growth-Promoting Rhizobacteria and Spent Mushroom Substrate Improves Ginseng Quality and Rhizosphere Nutrients. Agriculture 2024, 14, 1880. https://doi.org/10.3390/agriculture14111880

AMA Style

Fan S, Hu Q, Liu Q, Xu W, Wang Z, Huang Y, Zhang Y, Ji W, Dong W. The Synergistic Effect of Plant Growth-Promoting Rhizobacteria and Spent Mushroom Substrate Improves Ginseng Quality and Rhizosphere Nutrients. Agriculture. 2024; 14(11):1880. https://doi.org/10.3390/agriculture14111880

Chicago/Turabian Style

Fan, Siyao, Qian Hu, Qi Liu, Wenman Xu, Zixin Wang, Yu Huang, Yang Zhang, Wenxiu Ji, and Weiwei Dong. 2024. "The Synergistic Effect of Plant Growth-Promoting Rhizobacteria and Spent Mushroom Substrate Improves Ginseng Quality and Rhizosphere Nutrients" Agriculture 14, no. 11: 1880. https://doi.org/10.3390/agriculture14111880

APA Style

Fan, S., Hu, Q., Liu, Q., Xu, W., Wang, Z., Huang, Y., Zhang, Y., Ji, W., & Dong, W. (2024). The Synergistic Effect of Plant Growth-Promoting Rhizobacteria and Spent Mushroom Substrate Improves Ginseng Quality and Rhizosphere Nutrients. Agriculture, 14(11), 1880. https://doi.org/10.3390/agriculture14111880

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