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Article

Plant Growth-Promoting Rhizobacteria Microbial Fertilizer Changes Soils’ Microbial Structure and Promotes Healthy Growth of Cigar Tobacco Plants

by
Xianchao Shang
1,†,
Sha Fu
1,†,
Xiaomeng Guo
1,†,
Zheng Sun
2,
Fangyu Liu
1,
Qian Chen
1,
Tao Yu
1,
Yun Gao
1,
Li Zhang
1,
Long Yang
1 and
Xin Hou
1,*
1
College of Plant Protection, Shandong Agricultural University, Tai’an 271002, China
2
Tai’an Forestry Protection and Development Center, Tai’an 271002, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Agronomy 2023, 13(12), 2895; https://doi.org/10.3390/agronomy13122895
Submission received: 28 August 2023 / Revised: 8 November 2023 / Accepted: 14 November 2023 / Published: 24 November 2023
(This article belongs to the Special Issue Research Progress on Pathogenicity of Fungus in Crop)
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Abstract

:
Cigar tobacco, an economically important cash crop, holds a substantial role within the fiscal framework of the national economy. This crop, however, is characterized by a marked vulnerability to pathogenic bacteria, culminating in consequential financial loss throughout its cultivation phase. Plant growth-promoting rhizobacteria (PGPR), a salient class of advantageous bacterial flora, are recognized for their ability to enhance plant growth, inhibit deleterious pathogens, and synthesize compounds that either have a direct impact on plant morphogenesis or activate otherwise ineffectual soil components. Through these mechanisms, PGPR augments the soil’s nutritional profile, making it more receptive to plant uptake, thus stimulating vegetative growth. The Bacillus subtilis microbial fertilizer, the prime exemplar of PGPR, demonstrates not only a pathogen-suppressive effect but also an induction of the plant’s innate disease resistance mechanism. This bolsters the plant’s resilience to disease fosters a probiotic milieu within the soil, and catalyzes the formation of agglomerate structures, all of which contribute to enhanced soil fertility and moisture retention, increased soil friability, and the facilitation of root expansion. In this study, a controlled pot experiment was conducted to elucidate the mechanism through which inter-root probiotics rehabilitate the soil’s ecosystem and foster crop growth in cigar tobacco seedlings afflicted with root black rot bacteria. Four treatments were instituted, including CK: a blank control (no microbial application); A: probiotic only (Bacillus subtilis microbial fertilizer); B: both pathogenic and probiotic (the Bacillus subtilis microbial fertilizer together with root black rot pathogen); C: pathogenic only (the root black rot pathogen). Our empirical findings delineate that the presence of pathogenic bacteria deteriorates the soil environment, thereby constraining the transmutation of soil nutrients and their subsequent assimilation by plants. This severely impedes the vegetative development of cigar plants. By contrast, the application of a PGPR microbial fertilizer modified the soil microbial community structure, exhibiting an antagonistic interaction with the indigenous pathogenic bacterial species. Relative to the CK treatment, the application of the Bacillus subtilis microbial fertilizer was found to invigorate the catalytic conversion of soil enzymes, incrementing the peroxidase, acid phosphatase, urease, and sucrase activity by 12.98%, 19.55%, 13.57%, and 17.91%, respectively. Meanwhile, it was observed to ameliorate the soil’s physicochemical attributes, enhancing the available content of nitrogen, phosphorus, and potassium by 4.52%, 6.52%, and 15.14%, respectively, along with the augmentation of soil organic matter content by 17.33%. The fortification of soil physicochemical properties and the enrichment of soil fertility, as a result of the PGPR microbial fertilizer application, translated into a robust 57.23% enhancement of root vigor and a 60.47% extension of the root length of cigar tobacco seedlings. These soil amendments subsequently fueled an uptick in the growth parameters of cigar plants, including increases in plant height, stem girth, leaf count, maximal leaf dimensions, and both the fresh and dry weight of cigar tobacco.

1. Introduction

The cigar is derived from tobacco leaves that undergo a meticulous process of drying and fermentation [1] and constitute an essential economic commodity that is cultivated globally [2]. The growth of cigar tobacco is susceptible to a complex array of pathogens (Ralstonia solanacearum, Eurotium spp., and Talaromyces funiculosus), including but not limited to bacteria, fungi, viruses, and nematodes. Among these, tobacco root black rot, a pervasive soil-borne disease, manifests itself across various developmental stages of the plant, primarily targeting the root system. This pathogen can colonize non-host roots, infiltrating the plant’s roots via wounds, root hairs, or epidermal cells, and can subsequently permeate the entire cortex, instigating black rot. Such an infestation results in nutrient depletion, growth retardation, and delayed maturation, thereby directly impacting the plant’s overall vigor. Tobacco root black rot is widely distributed and found in many countries in all temperate tobacco-producing regions of the world, seriously affecting tobacco yield and quality [3,4].
Soils contain minerals, water, gases, and vital nutrients [5], serving as the primary source of sustenance for plants and thereby playing an instrumental role in shaping both yield and leaf quality in tobacco [6]. For example, the increase in potassium and available phosphorus content in soil is beneficial to improving tobacco leaf quality [5,6]. Although chemical fertilization has been employed as a rapid method to augment nutrient quantity and accessibility for plant assimilation [7], its prolonged use has been associated with several deleterious effects. These include the degradation of the soil structure, soil slumping, the diminution of soil organic matter, diminished soil nutrient utilization efficiency, and an imbalance of microbial diversity [8,9]. Consequently, the excessive use and application of chemical fertilizers can cause ecological damage to soil and environmental pollution, and its efficacy on crop yield decreases [10]. By contrast, microbial fertilizers are unique at solving this problem.
The microbial fertilizer, enriched with an abundance of microorganisms (Pseudomonas and Bacillus) that are beneficial to plant growth, can stimulate plant growth and enhance plant resistance to both biotic and abiotic stresses, facilitating the plant absorption of soil nutrients and resulting in a significant increase in physiological indicators such as plant height, root length, the root surface area and average root diameter [9,11,12]. Plant growth-promoting rhizobacteria (PGPR), a specific class of beneficial microorganisms, has garnered significant attention in contemporary agricultural paradigms as potential biofertilizers [13,14]. Recent scientific explorations into PGPR have emphasized their role in fostering plant growth, ameliorating soil quality, and mitigating crop disease [11,13,14]. Through various mechanisms such as phytohormone production, the enhancement of plant nutrient supply, the suppression of pathogenic entities, and modification of soil physicochemical properties, PGPR, when colonizing plant inter-roots, has demonstrated the capacity to significantly influence plant growth and health [9,15,16,17]. During the entire developmental phase of plants, there exists an alliance among the soil, plant, and microorganisms [18]; these interactions can activate plant defense mechanisms, stimulate plants to grow, increase crop yields, and increase disease resistance [9,16], and are considered an environmentally sound option to reduce chemical fertilizer inputs, improve soil quality and increase crop yields [19]. The widespread deployment of PGPR as inoculants may indeed revolutionize current agricultural practices, significantly curtailing the utilization of environmentally detrimental chemical fertilizers and pesticides [14].
Bacillus subtilis, as one of the members of the PGPR community, is characterized by its capacity to form spores and endure within soil under harsh environmental conditions [16]. As a biocontrol agent, B. subtilis exhibits antagonistic properties against specific phytopathogenic fungi, such as those affecting peanuts, enhancing crop dry weight and yielding healthier pods [20]. Its application to various crops, including wheat, cucumber, tomato, and pepper, has been shown to enhance bacterial colonization, microbial enzyme activity, and plant growth [21,22,23,24]. Furthermore, B. subtilis has been found to significantly ameliorate soil physicochemical properties, enhance microbial diversity, promote inter-root development, and mitigate soil-borne diseases in crops like ginger [25]. In the context of tobacco, a robust interaction exists between beneficial microorganisms in the root interstice and tobacco roots, fostering the colonization of probiotic bacteria [26]. The application of Bacillus sp. to tobacco plants has been demonstrated to stimulate a defense enzyme activity, substantially increase the pathogenesis-related protein content, and exert effective control over pathogens, including the tobacco mosaic disease and tobacco black shank [27,28,29].
Numerous studies have demonstrated that the application of PGPR can induce significant alterations in soil microbiota, thereby playing a vital role in enhancing soil attributes and averting soil-borne diseases [13,14,15,17,22]. This study aims to delve into the mechanisms underpinning the effects of Bacillus on soil properties in the context of cigar plants. The intention is to elucidate the specific impacts of exogenous bacterial application on soil microbial community architecture and soil attributes, thereby contributing to the ongoing discourse on sustainable agricultural practices. We hypothesized that Bacillus subtilis could alter the soil microbiota, increase soil microbial diversity, promote root development, and favor healthy tobacco growth.

2. Materials and Methods

2.1. Species, Plant Material and Growing Conditions

Cigar (Cigarro) seedlings of SNT60 were used in the potting experiments, and the soil was obtained from the Plant Protection Experiment Station of Shandong Agricultural University, Taishan District, Tai’an City, Shandong Province, Shandong Province, China. In total, 50 kg of soil from 0 to 20 cm was collected from five randomly selected plots without any disease, and the soil was removed from the surface of the soil and mixed thoroughly to be brought back to the laboratory for further microscopic experiments. When seedlings grew to 3–5 true leaves, seedlings of a similar size and health condition were selected and transplanted into pots (22 × 16 cm) with a total of 3 kg of substrate and soil to ensure consistent conditions in each pot, which were then watered after transplanting, and allowed to grow naturally outdoors and managed with regular watering.
The Bacillus subtilis microbial fertilizer and tobacco root black rot fungus (Thielaviopsis basicola (Berk. and Br.) Ferraris) was applied. The preserved root black rot pathogen was activated and incubated on a potato dextrose agar (PDA) medium at 28 °C for 7 days, and was then punched into 200 mL of a liquid fungal medium with a puncher (6 mm), shaken at 28 °C and 180 rpm for 48 h, followed by the preliminary filtration of the culture solution using gauze, and then the filtrate obtained was divided into centrifuge tubes and centrifuged at 10,000 rpm at 4 °C for 10 min. The supernatant was discarded to obtain the precipitation of the fungal spores, and the spore suspension was adjusted to the concentration of spores using sterile water and then inoculated with the pathogens.

2.2. Experimental Design

Three days after transplanting, the pots were inoculated with a Bacillus subtilis microbial fertilizer and a fungal spore solution of the tobacco black rot pathogen. Forty mL of the root black rot pathogen suspension was cultured as described above and added to the pots of treatments B and C, and 40 mL of sterilized water was added to the other groups to ensure the consistency of the test variables. At the same time, Bacillus subtilis microbial fertilizer was mixed with water (60%, v/v) at 200 mL and applied to the pots of treatments A and B, and 200 mL of sterile water was added to the other groups. The four treatments were designed as follows. (1) CK: the blank control was applied with clean water; (2) Treatment A applied the Bacillus subtilis microbial fertilizer only; (3) Treatment B: cigar tobacco seedlings were treated with both root black rot pathogenic suspension and Bacillus subtilis microbial fertilizer via root irrigation; (4) Treatment C: cigar tobacco seedlings were treated with root black rot pathogenic suspension only. The plants were placed under greenhouse conditions at 28 °C with a relative humidity of 70% and a 12 h light and dark cycle.

2.3. Determination of Plant Traits of Cigar Tobacco

Measurement of agronomic traits. After the experimental treatments, the distance from the base of the stem to the top growth point of the tobacco plant was measured using a tape measure for plant height; the circumference of the base of the stem was measured using a tape measure as the stem circumference; the number of leaves of each tobacco plant was recorded; the largest leaf of each cigar tobacco plant was selected to measure the leaf length and width using a tape measure; the weight of the fresh leaves was weighed using electronic balance as the fresh weight; and the leaves were dried in the drying oven at 80 °C for 48 h and weighed as the dry weight. The maximum root length of the root system for each treated tobacco plant was measured using a tape measure.

2.4. Collection and Determination of Inter-Root Soil Samples and Tobacco Plant Roots

Soil sample collection and measurement. One month after the cigar seedlings were treated, 1 cm of the topsoil was removed, the cigar plants were carefully pulled out, the soil at the periphery of the root system was shaken off, and inter-root soil was collected. The collected inter-root soil was sieved with 40 mesh and used to determine the soil’s physical and chemical properties and soil enzyme activity. The determination of soil physicochemical properties was conducted as follows: the soil’s alkaline nitrogen content was determined using the alkaline diffusion method; the soil‘s fast-acting phosphorus content was determined via the sodium bicarbonate leaching-molybdenum-antimony counterstain method; the soil’s fast-acting potassium content was determined using the ammonium acetate leaching-flame photometric method; the soil’s organic matter content was determined using the hydrated thermal potassium dichromate oxidation-colorimetric method; finally, the soil’s pH was determined via the potentiometric method. The determination of soil enzyme activities was performed as follows: soil urease, soil sucrase, soil peroxidase, and soil acid phosphatase activities were determined using the soil urease kit (Solarbio, BC0120), soil sucrase kit (Solarbio, BC0240), soil peroxidase kit (Solarbio, BC0100) and soil acid phosphatase kit (Solarbio, BC0100), respectively. phosphatase kit (Solarbio, BC0140). The determination methods are described in the relevant manuals (Solarbio Technology Co., Ltd., Beijing, China) [11].
Collection and determination of root samples. Cigar tobacco plants were carefully pulled out, the soil attached to the roots was gently shaken off, rinsed carefully with water, and then the triphenylmethyl hydrazone (TTF) in the roots was extracted using the Plant Root Viability Assay Kit (Shanghai Yuanye Biotechnology Co., Ltd., Shanghai, China), and the absorbance at 485 nm was measured with a spectrophotometer to calculate the reduction in root triphenyl tetrazolium chloride (TTC) as an indicator of root viability.

2.5. Analysis of 16S and ITS Full-Length Microbial Diversity in Inter-Root Soils

The collected soil samples were handed over to Beijing Bemec Biotechnology Co., Ltd. (Beijing, China) to extract the soil’s DNA; the extracted DNA was amplified using a PCR, and the amplified products were purified, quantified, and homogenized to form a sequencing library (SMRT Bell). The built library was first subjected to library quality control, and the library that passed the quality control was sequenced using PacBio Sequel, PacBio offline data were exported, and the CCS sequences were identified via a barcode using lima v1.7.0 software (Edgar, R. C., London, UK) to obtain Raw-CCS sequence data. The CCS sequences were identified via a barcode using lima v1.7.0 software to obtain Raw-CCS sequence data; the primer sequences were identified and removed via cutadapt 1.9.1 software (Marcel Martin, Dortmund, Germany) and filtered by length to obtain Clean-CCS sequences without primer sequences. Then, the chimeric sequences were identified and removed via UCHIME v4.2 software (Edgar, R. C., London, UK) to obtain effective-CCS sequences. The Effective-CCS sequence was obtained by removing the chimeric sequence with UCHIME v4.2 software. The Effective-CCS sequences were clustered at a 97.0% similarity level using Usearch v10 software (Edgar, R. C., London, UK), and OTUs were obtained; the sequences were taxonomically annotated using SILVA as the reference database, and a plain Bayesian classifier combined with a comparison method was used to obtain the taxonomic information of each feature before the taxonomic information of the species at each level (phylum, class, order, family, etc.) was obtained. The taxonomic information of each feature was obtained, and then at each level (phylum, class, order, family, genus, species), the community composition of each sample was counted; furthermore, α-diversity, β-diversity, significant species differences, and correlation analysis could be performed to explore the differences between soil samples.

2.6. Statistical Analysis

Data were collected in three replicates, and data were processed using IBM SPSS Statistics 26 (IBM, Armonk, NY, USA) and Excel 2019 (Microsoft, Redmond, DC, USA) for significance testing, along with a correlation analysis of experimental data on the agronomic traits of cigars, soil enzyme activity, and soil physicochemical traits. Plotting was performed using Origin 2021 (Northampton, MA, USA) and R software v3.6.1 (Robert Gentleman and Ross Ihaka, Auckland, New Zealand). The bacterial and fungal α-diversity index of soil samples was analyzed for group differences based on Student’s t-test at the operational taxonomic units (OTU) level; for the β-diversity of soil bacteria and fungi, the method of Non-Metric Multi-Dimensional Scaling (NMDS) was used based on the Bray–Curtis distance algorithm for between-group variance analysis; and Venn diagram based on OTU level. The relationship between soil properties and soil microorganisms was analyzed using redundancy analysis (RDA).

3. Results

The application of the root black rot pathogen altered the soil microbial environment and had a damaging effect on soil properties as well as the growth of cigar tobacco seedlings. Inoculation with inter-root promoting bacteria improved the soil environment and promoted plant growth, somewhat reducing the soil stress caused by pathogenic bacteria and providing a considerable improvement to plant traits.

3.1. Effect of PGPR Microbial Fertilizer and Root Black Rot Pathogen Application on Soil Nutrients and Agronomic Traits of Cigar Plants

Soil enzyme activities, including soil urease (urease), soil sucrase (sucrase), soil catalase (CAT), and soil phosphatase (ACP), are significantly affected by the application of microbial agents to the soil (Figure 1a), and the experimental results show that the application of root black rot fungus significantly reduced soil enzyme activities. Relative to the CK treatment, the soil catalase activity in treatment C decreased by 15.05%; for acid phosphatase activity compared to CK treatment, the soil peroxidase activity decreased by 15.05%; acid phosphatase activity decreased by 15.89%; soil urease activity decreased by 57.89%; and soil sucrose enzyme activity decreased by 36.16%. On the contrary, the application of Bacillus subtilis significantly increased the soil enzyme activities, with a 12.98% increase in soil catalase activity, a 19.55% increase in soil acid phosphatase activity, a 13.57% increase in soil urease activity, and a 17.91% increase in soil sucrose enzyme activity in relation to the CK treatment. By contrast, soil enzyme activities in the B treatment group were not significantly different from those in the CK group.
The addition of microbial agents to the soil improved the nutrient composition of the soil (Figure 1b), and the application of Bacillus subtilis significantly increased the levels of available nitrogen (AN), available phosphorus (AP), available potassium (AK) and soil organic matter (SOM) in the soil. Compared to the control, the AN content increased by 4.52%, the AP content increased by 6.52%, the AK content increased by 15.14%, and the SOM content increased by 17.33% in treatment A. By contrast, treatment C showed a significant decrease in the content of all components, with a 12.68% decrease in AN, a 11.26% decrease in AP, an 11.51% decrease in AK, and a 10.33% decrease in SOM. According to the analysis, it is known that the content of each soil component in the B treatment was not significantly different from that of the control group, and it can be seen from this figure that the content of soil components in treatment group B was similar to that of the CK treatment group.
To evaluate the effect of Bacillus subtilis application on the biomass of cigar tobacco plants under root black rot pathogen stress, the cigar tobacco plant height, stem circumference, leaf number, maximum leaf length, maximum leaf width, fresh weight, and dry weight were determined (Table 1). The experimental results show that plant height decreased by 40.24%, leaf number decreased by 21.59%, and maximum leaf length, maximum leaf width, dry weight, and fresh weight decreased to different degrees after the application of the root black rot pathogen compared with the CK treatment group. On the contrary, after the application of Bacillus subtilis, the cigar tobacco plant height increased by 81.21%, the number of leaves increased by 33.35%, and the maximum leaf length, maximum leaf width, dry weight, and fresh weight all increased significantly. In experimental treatment B, the simultaneous application of Rhizoctonia solani and Bacillus subtilis revealed that the cigar tobacco plant height, stem circumference, leaf number, maximum leaf length, maximum leaf width, fresh weight, and dry weight in this treatment were not significantly different from the untreated control. The results indicate that the tobacco plants with Bacillus subtilis alone had the best growth and had a significant promotion effect on the growth of cigar tobacco. The root black rot pathogen had a negative effect on the growth of tobacco seedlings, which was improved by the application of Bacillus subtilis, indicating that the Bacillus subtilis microbial fertilizer could promote plant growth and reduce the stressful effect of the pathogen on plant growth (Figure 1c).
The addition of exogenous bacteria had a significant effect on the root growth condition of cigar tobacco seedlings (Table 1). Compared with CK, the root vigor of cigar tobacco seedlings decreased by 27.58%, and the root length of these seedlings decreased by 40.12% under the treatment of black rot bacteria in the roots. On the contrary, the root vigor of cigar tobacco seedlings was enhanced by 57.23% in treatment group A with Bacillus subtilis application, while the root length increased by 60.47%. Experimental data show that there was no significant difference in the root vigor and root length of cigar tobacco seedlings in the B treatment group with the simultaneous application of Rhizoctonia solani and Bacillus subtilis compared to the CK treatment group. The experimental results show that the application of Bacillus subtilis microbial fertilizer could improve the persecution effect of root black rot bacteria on soil roots, enhance root vigor, and promote root growth of cigar tobacco (Figure 1c).

3.2. Effect of PGPR Microbial Fertilizer and Root Black Rot Pathogen Application on Inter-Root Soil Microorganisms

3.2.1. Microbial Diversity Characterization and Community Composition

A total of 157,210 bacterial CCS sequences and 139,070 fungal CCS sequences were obtained from all 12 soil samples, yielding an average of 13,101 bacterial CCS sequences and 11,589 fungal CCS sequences per sample. The average read length of bacterial sequences was 1447 bp (range 1445–1448 bp), and the average read length of fungal sequences was 587 bp (range 575–600 bp). Based on the clustering of these sequences at a 97.0% similarity level, a total of 3577 bacterial OTUs and 510 fungal OUTs were obtained. In this study, the distribution of bacterial and fungal OTUs in the inter-root soil of different treatments was analyzed using Venn diagrams, and the sequencing results show that the four treatments collectively contained 1912 bacterial OUTs and 133 fungal OTUs (Figure 2a,b).
Alpha diversity reflects the species abundance and species diversity of the samples. For the bacterial community (Figure 2c) compared with the CK treatment group, the Shannon index and Simpson index of treatment group B exhibited a certain degree of decrease, while the Chao1 index and Ace index exhibited a certain degree of increase. In terms of fungal communities (Figure 2d), the Shannon index and Simpson index were significantly lower in the C treatment group, and the Chao1 index and Ace index were significantly lower in the A treatment group compared to the CK treatment group. It is shown that the application of PGPR microbial fertilizers and root black rot pathogens affects the alpha diversity of inter-root soil microorganisms.
Non-Metric Multi-Dimensional Scaling (NMDS) is a sorting method that is applicable to ecological research, which is mainly a data analysis method that simplifies research objects in multi-dimensional space to a lower dimensional space for locating, analyzing, and categorizing while preserving the original relationship between objects. The model of NMDS is nonlinear, and the distribution of samples can be used to see the differences between or within groups, which can better reflect the nonlinear structure of ecological data. The NMDS analysis based on Bray–Curtis variability reflected the beta diversity of soil bacterial and fungal microorganisms among different treatments. The results show that the application of the PGPR microbial fertilizer and root black rot pathogens altered the community composition of inter-root soil bacteria and fungi, but different microbiota showed different responses. For bacterial microorganisms, the microbial communities in treatments A and C showed greater separation, and those in CK experienced some overlap with treatment B (Figure 2e). For fungal microorganisms, the fungal communities of all three treatment groups showed a greater separation from the control, with treatment A and B for flora showing some similarity (Figure 2f).

3.2.2. Relative Abundance of Inter-Root Soil Bacterial and Fungal Communities

The sequencing results show that different treatments alter the composition and relative abundance of bacterial and fungal communities in the soil. The bacteria identified in this study belonged to 31 phyla, 79 classes, 224 orders, 403 families, 672 genera, and 967 species. The fungi identified in this study belonged to 8 phyla, 21 classes, 49 orders, 101 families, 171 genera, and 213 species. At the phylum level, the dominant bacteria in the soil samples of each treatment were mainly distributed among Proteobacteria, Bacteroidota, Planctomycetota, Acidobacteriota, Verrucomicrobiota, Patescibacteria Gemmatimonadota, Myxococcota, Chloroflexi (Figure 3a); the dominant fungi were mainly distributed in Ascomycota, Basidiomycota, Mortierellomycota, Chytridiomycota, Rozellomycota, unclassified_Fungi, Glomeromycota and Zoopagomycota (Figure 3b). At the genus level, the dominant bacterial genera in the soil samples of each treatment were mainly Chryseolinea, Terrimonas, uncultured_soil_bacterium, unclassified_Saprospiraceae, unclassified_Gemmatimonadaceae, unclassified_Alphaproteobacteria, unclassified_Bacteria, uncultured_gamma_proteobacterium, etc. (Figure 3c), and the dominant fungal genera in each treatment soil sample were mainly Zopfiella, Mortierella, Mycothermus, unclassified_Sordariomycetes, Coprinellus, Iodophanus, unclassified_Fungi, Pseudallescheria, unclassified_Ascobolaceae, etc. (Figure 3d). The results of this study show that the relative abundance of bacterial communities in the different types of treatment soil differed less, while the relative abundance of fungal communities differed significantly. For the relative abundance of phylum-level fungal communities, the relative abundance of Ascomycota and Basidiomycota significantly decreased, and the relative abundance of Chytridiomycota and Rozellomycota significantly increased in the soil samples of treatment A. By contrast, in treatment C, the relative abundance of Ascomycota and Basidiomycota in the soil and the relative abundance of Ascomycota and Basidiomycota was significantly increased while the relative abundance of Chytridiomycota and Rozellomycota was significantly decreased in treatment C (Figure 3b), indicating that Chytridiomycota and Rozellomycota in the soil may have a better effect on the growth of cigar plants.

3.3. Correlation Analysis between Soil, Plants and Microorganisms

3.3.1. Correlation Analysis between Inter-Root Soil Microorganisms

Based on the analysis of the network diagram, the coexistence of species in environmental samples can be obtained, and the interaction of species in the same environment and important pattern information can be obtained to further explain the formation mechanism of phenotypic differences between samples. To explore the ecological interaction patterns of fungi and bacteria in inter-rhizosphere communities, a microbial community correlation network map of bacteria and fungi was established based on strong correlations at the genus level, which shows the top 50 genera with the highest correlations. From this figure, it is understood that the positive correlation between bacterial and fungal communities is higher than the negative correlation, and the network complexity of bacterial communities is higher than that of fungal communities. In the bacterial community network, most of the nodes belonged to Aspergillus and Bacteroidetaceae (Figure 4a), and in the fungal community network, most of the nodes belonged to Ascomycetaceae and Basidiomycetaceae (Figure 4b), which is consistent with the performance of the relative abundance map of soil bacterial and fungal communities. This network diagram suggests that microorganisms interact with each other and that the addition of exogenous bacterial agents changes the structure of the soil microbial community (Figure 4).

3.3.2. Analysis of the Correlation between Soil Properties and Microorganisms

To identify potential environmental drivers, the relative abundance of bacterial and fungal communities is correlated with environmental factors using RDA analysis. Among soil physicochemical properties, RDA explained 32.54% and 42.24% of the total variation in the soil bacterial and fungal community structure, respectively. RDA analysis showed that the abundance of AN, AP, AK, and SOM in the soil had a significant effect on the microbial community composition, with AK being the most important factor. Among the bacterial communities (Figure 5a), the abundance of Acidobacteriota was significantly and positively correlated with the content of AN, AP, AK, and SOM in the soil and was more strongly correlated with the sample distribution of treatment A. The abundance of Planctomycetota and Chloroflexi was significantly and negatively correlated with the content of AN, AP, AK, and SOM in the soil and was more strongly correlated with the sample distribution of treatment C. The correlation was strong. Among the fungal communities (Figure 5b), the abundance of Chytridiomycota and Rozellomycota were significantly and positively correlated with the content of AN, AP, AK, and SOM in the soil and was more correlated with the sample distribution of treatment A, while the abundance of Ascomycota was significantly and negatively correlated with the content of AN, AP, AK and SOM in the soil, and was more strongly correlated with the sample distribution of treatment C. The correlation was stronger with the sample distribution of treatment C (Figure 5).
The RDA analysis of soil enzyme activity and environmental factors explained 29.25% and 37.77% of the total variation in the soil bacterial and fungal community structure, respectively, and RDA analysis indicated that soil CAT, ACP, urease, and sucrase had significant effects on the microbial community’s composition. In the bacterial community (Figure 5c), the abundance of Planctomycetota was significantly and negatively correlated with the activities of soil CAT, ACP, urease, and sucrase and was strongly correlated with the sample distribution of treatment C. In the fungal community (Figure 5d), the abundance of Ascomycota was significantly and negatively correlated with the activities of soil CAT, ACP, urease, and sucrase, which is in agreement with the results of the soil’s physicochemical properties. It is speculated that bacteria such as Acidobacteriota and Verrucomicrobiota in the bacterial community and Chytridiomycota and Rozellomycota in the fungal community may have some promotion effect on plant growth; Planctomycetota and Chloroflexi in the bacterial community and Ascomycota in the fungal community may have some inhibitory effect on plant growth (Figure 5).
The correlation heat map shows the top 15 phyla of the bacterial community and all phyla of the fungal community. The correlation heat map shows that the dominant bacterial phylum Desulfobacterota and Acidobacteriota were significantly influenced by edaphic factors, with Desulfobacterota showing a significant negative correlation with soil environmental factors and Acidobacteriota showing a significant positive correlation with soil environmental factors (Figure 5e). The dominant fungal phylum Ascomycota, Rozellomycota, and Chytridiomycota were significantly affected by the edaphic factor; Ascomycota showed a significant negative correlation with soil environmental factor, while Rozellomycota and Chytridiomycota showed a significant positive correlation with the soil environmental factor (Figure 5f). The correlation heat map shows that multiple bacterial phyla were significantly correlated with soil urease, and the microbial community structure was weakly correlated with the pH.

4. Discussion

4.1. Exogenous Microbial Agents Improve Soil Nutrients and Promote Plant Growth

Soil contains nutrients that are essential for crop growth and can be manipulated and managed to improve soil fertility and reduce the spread of soil-borne diseases, which is important for improving crop yields [30]. The utilization of PGPR microbial fertilizers has been proven to amplify soil fertility, minimize the incidence of soil-borne pathogens, stimulate crop growth, and augment the caliber of agricultural products. Notably, these microbial inoculants facilitate the uptake of soil nutrients via plants and invigorate the vitality of plant roots through the augmentation of enzymatic activity and rapid-acting nutrient content within the inter-root soil [9,31]. This, in turn, promotes crop growth and potentially forestalls soil-borne diseases. Microbial fertilizers that fix nitrogen and dissolve phosphorus and potassium in the soil play a crucial role in increasing the availability of these essential elements [32,33,34].
Bacillus subtilis, a functional probiotic bacterium, constitutes a vital PGPR microbial fertilizer that is known for enhancing soil conditions and mitigating soil-borne diseases. Its multifaceted mechanisms lead to the promotion of crop growth and enhance nutrient utilization, making it a viable biofertilizer alternative to chemical counterparts [35,36,37]. Four treatments were set up in this study, and the experimental results show that the enzymatic activities of soil urease, soil sucrase, soil catalase, and soil phosphatase showed significant reductions in treatment group C where only the root black rot pathogen was applied, and the contents of AN, AP, AK, and SOM in the soil were also reduced when combined with the growth status of the cigar plants. The presence of the pathogenic bacteria reduced the content of nutrients in the soil and inhibited the absorption of soil nutrients via the crop, which significantly reduced agronomic traits such as plant height, leaf length, leaf width, and root vigor and seriously affected the healthy growth of the crop. In treatment group A, where only the B. subtilis microbial fertilizer was applied, the soil enzyme activity and the contents of AN, AP, AK, and SOM were significantly increased, indicating that the application of the B. subtilis microbial fertilizer promoted the conversion of soil nutrients and improved the nutrient composition of the soil [38] (Figure 1a,b). This, in turn, provided sufficient nutrients to the cigar plants, resulting in a significant increase in agronomic traits such as plant height, leaf length, leaf width, and root vigor (Table 1), which had a good promotional effect on the growth of cigar plants. The soil enzyme activities and contents of AN, AP, AK, and SOM in treatment B with the simultaneous application of the root black rot pathogen and Bacillus subtilis microbial fertilizer were not significantly different from those in the CK group, indicating that the Bacillus subtilis microbial fertilizer had a mitigating effect on the root black rot pathogen stress. This further confirms the importance of using PGPR microbial fertilizers in crop production: the application of PGPR microbial fertilizers improved soil physicochemical properties, reduced the risk of plant diseases, improved the vigor of plant roots, increased the biomass of above-ground plant parts, and promoted good plant growth [18,39,40,41,42].

4.2. Effect of Exogenous Microbial Agent Application on the Inter-Rhizosphere Soil Microbial Community of Cigar Plants

Root black rot constitutes a profound impediment in tobacco production, wherein the utilization of superfluous chemical fertilizers or pesticides may engender soil quality aberrations, encompassing soil nutrient disequilibrium, diminishing microbial heterogeneity, and soil-borne pathologies. These factors collectively contribute to the degradation of plant integrity and fecundity, exerting profound ramifications on sustainable soil productivity [43,44]. The utilization of biofertilizers replete with beneficial microorganisms has emerged as a propitious countermeasure to disease, fostering crop development through the modulation of the soil’s microbial architecture and taxonomy, thus attenuating complications engendered by imprudent fertilizer deployment [45,46]. Soil microbial communities and diversity are key to maintaining healthy soil ecosystems [47]. The introduction of Bacillus subtilis has been observed to metamorphose the relative prevalence of bacterial and fungal taxa within the soil, exhibiting a marked suppressive impact on pathogenic fungi in species such as poplar and wheat, thereby augmenting crop proliferation and affirming its efficacy as a biological control agent [36,48,49]. In the current investigation, the induction of the root black rot pathogen and Bacillus subtilis microbial fertilizers reshaped the soil microbial diversity and shifted the microbial community’s assemblage. Pertaining to bacterial communities, exogenous bacterial induction did not materially influence bacterial α-diversity (Figure 2c), whereas employing Non-Metric Multi-Dimensional Scaling (NMDS) to ascertain bacterial β diversity revealed a pronounced divergence between treatments within the NMDS analysis plot (Figure 2e), which is potentially attributable to the exogenous bacterial agent restructuring the inter-rhizosphere bacterial community and engendering a novel community construct. Among these emergent communities, treatments A and C demonstrate marked distinctions in their composition, contrasting treatment B and the CK group, which manifested nominal variances. Correlated with plant growth dynamics, this observation may be ascribed to the induction of Bacillus subtilis microbial fertilizers in treatment A, which harbored microorganisms conducive to plant development, while pathogenic fungi in treatment C harbored microorganisms inimical to plant growth, and the antagonistic interplay in treatment B neutralized this effect, resulting in a growth comparable to the untreated control. Considering fungal communities, both the Shannon and Simpson indices in the α-diversity of treatment group C displayed a significant diminution (Figure 2d), and within the NMDS plot illustrating β-diversity, the community composition in the C treatment group diverged markedly from that of the A treatment group (Figure 2f). It may be conjectured that the induction of pathogenic fungi altered the soil fungal community composition, suppressing microbial species’ heterogeneity in the inter-root soil and subsequently inhibiting crop growth [50,51].

4.3. Correlation Analysis between Soils, Plants, and Microorganisms

Soil harbors intricate networks of microorganism interactions that significantly influence plant productivity. Microbial correlation network analysis elucidates these connections, offering insights into cooperative and antagonistic relationships [52,53]. Microbial interactions in the soil can affect plant productivity, with positive interactions promoting cooperation among organisms, while negative interactions may lead to mutual antagonism. Although network analysis does not truly reflect microbial interactions, it highlights the associations between microbial species [54,55,56]. In this study, correlation network analysis was performed on inter-rhizosphere soil microorganisms, and the results show a strong correlation between soil microbial communities (Figure 4), which could lead to the hypothesis that the addition of exogenous bacterial agents can affect the community composition of soil microorganisms.
Alterations in the composition of inter-root soil microorganisms are anticipated to induce modifications in soil characteristics, subsequently exerting either a stimulating or inhibitory effect on plant growth. The results obtained from RDA analysis and species abundance distribution maps (Figure 3 and Figure 5a–d) indicate that the introduction of exogenous bacterial agents significantly alters the composition of soil microorganisms. Moreover, these changes exhibit a stronger association with soil parameters. In the bacterial community, the addition of the Bacillus subtilis microbial fertilizer caused the microorganisms of Acidobacteriota and Verrucomicrobiota to show a certain degree of elevation, and the addition of root black rot pathogens increased the content of Planctomycetota, decreased the content of Acidobacteriota and Verrucomicrobiota, and combined with the results of RDA analysis. The Acidobacteriota and Verrucomicrobiota showed a positive correlation with the contents of AN, AP, AK, SOM, urease, sucrase, catalase and acid phosphatase in the soil, while Planctomycetota showed a negative correlation with the contents of AN, AP, AK, SOM, urease, sucrase, catalase, and acid phosphatase in the soil and phosphatase content in the soil showed a negative correlation. Among the fungal communities, the addition of the Bacillus subtilis microbial fertilizer significantly increased the content of Rozellomycota and Chytridiomycota in the microorganisms, while the addition of root black rot pathogens significantly decreased the content of Rozellomycota and Chytridiomycota, and the treatment group with root black rot pathogens had a higher Ascomycota content; combined with the results of RDA analysis, the content of Rozellomycota and Chytridiomycota in the fungal community showed a significant positive correlation with the content of AN, AP, AK, SOM, urease, sucrase, catalase and acid phosphatase in the soil, while Ascomycota showed a significant positive correlation with the content of AN, AP, AK, SOM, urease, sucrase, catalase in the soil and acid phosphatase showed a significant negative correlation. The microbial community structure and the contents of AN, AP, AK, SOM, urease, sucrase, catalase and acid phosphatase in the soil were not significantly different from the control treatment for treatment B with the application of both root black rot pathogen and Bacillus subtilis microbial fertilizers. It can be inferred that Acidobacteriota and Verrucomicrobiota in the bacterial community and Rozellomycota and Chytridiomycota in the fungal community may play an important role in improving soil properties, thus effectively promoting the growth of cigar plants. The application of root black rot pathogens may lead to an increase in Planctomycetota, Ascomycota and other microorganisms in the soil microbial community, which, in turn, deteriorates the soil, causing soil nutrient loss, reducing the content of AN, AP, AK, SOM, urease, sucrase, catalase and acid phosphatase in the soil; this prevents the crop root system from absorbing sufficient nutrients from the soil, limits crop growth, and affects Agronomic traits such as plant height, stem circumference, leaf number, maximum leaf length, maximum leaf width, fresh weight and dry weight, which are used to measure the growth status of the plant (Figure 1c). Notably, a substantial reduction in these parameters has been seen, indicating a potentially detrimental impact on crop yield [57,58,59]. This finding provides additional evidence supporting the significance of employing Bacillus subtilis microbial fertilizers to promote crop growth. The introduction of exogenous bacterial agents has the potential to alter the composition of soil microbial communities. Additionally, certain microorganisms possess the ability to secrete enzymes or facilitate the conversion of soil nutrients. Consequently, this can enhance the activity of soil enzymes, improve the physical and chemical properties of the soil, facilitate the uptake of soil nutrients by crops, and promote crop growth. As a result, these interventions can effectively mitigate the detrimental impacts of root black rot bacteria on both soil and plants. Furthermore, they demonstrate promising outcomes in the restoration of soil that has experienced significant nutrient depletion [17,60,61,62,63].

5. Conclusions

The application of the Bacillus subtilis microbial fertilizer caused substantial shifts in the chemical properties, enzyme activities, microbial abundances, and bacterial community compositions of soil containing the root black rot pathogen. The utilization of the Bacillus subtilis microbial fertilizer effectively mitigated the adverse impact of root black rot pathogens on both the soil and plants. The root vigor, plant height, stem circumference, leaf number, maximum leaf length, maximum leaf width, fresh weight, and dry weight of cigars were significantly promoted. Additionally, the utilization of the Bacillus subtilis microbial fertilizer resulted in enhanced soil enzyme activity and elevated levels of AN, AP, AK, and SOM in the soil. This application demonstrates the ability to enhance soil conditions and subsequently stimulate plant growth, all while avoiding environmental contamination. Consequently, it presents a viable option for reducing reliance on chemical fertilizers and pesticides, thereby minimizing environmental pollution.

Author Contributions

X.H., L.Z. and L.Y. designed the experiments. S.F., X.G., F.L., Q.C. and T.Y. performed the experiments and analyzed the data. Z.S. and Y.G. validated the experiment. S.F., X.S., X.G. and X.H. wrote the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by the Shandong Province Modern Agricultural Technology System (SDAIT-25-02).

Data Availability Statement

The original contributions presented in the study are included in the article, and further inquiries can be directed to the corresponding authors.

Acknowledgments

We thank our colleagues for their valuable suggestions on the experimental protocols and for their great help during the experimental process.

Conflicts of Interest

The authors declare no conflict of interest.

Abbreviations

PGPR: plant growth promoting rhizobacteria; AN: available nitrogen; AP: available phosphorus; AK: available potassium; SOM: soil organic matter; CAT: peroxidase; ACP: acid phosphatase.

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Figure 1. The application of PGPR microbial fertilizers and root black rot pathogens changed the nutrient status of the soil (a,b) and affected the growth of cigar tobacco plants (c). Note: CK: blank control (no microbial application); A: application of probiotic bacteria only (Bacillus subtilis microbial fertilizer); B: application of both pathogenic and probiotic bacteria (Bacillus subtilis microbial fertilizer together with root black rot bacteria); C: application of pathogenic bacteria only (root black rot bacteria) *: multiply sign. Different letters on the bar graphs indicate significant differences in the mean of each treatment at p ≤ 0.05.
Figure 1. The application of PGPR microbial fertilizers and root black rot pathogens changed the nutrient status of the soil (a,b) and affected the growth of cigar tobacco plants (c). Note: CK: blank control (no microbial application); A: application of probiotic bacteria only (Bacillus subtilis microbial fertilizer); B: application of both pathogenic and probiotic bacteria (Bacillus subtilis microbial fertilizer together with root black rot bacteria); C: application of pathogenic bacteria only (root black rot bacteria) *: multiply sign. Different letters on the bar graphs indicate significant differences in the mean of each treatment at p ≤ 0.05.
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Figure 2. Effect of PGPR microbial fertilizer and root black rot pathogen application on the alpha diversity (c,d) and beta diversity (e,f) of the inter-root soil microbial community. Note: CK: blank control (no microbial application); (a) probiotic application only (Bacillus subtilis microbial fertilizer); (b) both pathogenic and probiotic applications (Bacillus subtilis microbial fertilizer together with root black rot); (c) pathogenic application only (root black rot). In NMDS analysis, each point in the graph represents a sample, different colors represent different groupings, and the oval circle indicates that it is a 95% confidence ellipse; the closer the samples on the coordinate graph, the higher the similarity and it is usually considered that when stress is less than 0.2, it indicates that NMDS analysis has some reliability (e,f).
Figure 2. Effect of PGPR microbial fertilizer and root black rot pathogen application on the alpha diversity (c,d) and beta diversity (e,f) of the inter-root soil microbial community. Note: CK: blank control (no microbial application); (a) probiotic application only (Bacillus subtilis microbial fertilizer); (b) both pathogenic and probiotic applications (Bacillus subtilis microbial fertilizer together with root black rot); (c) pathogenic application only (root black rot). In NMDS analysis, each point in the graph represents a sample, different colors represent different groupings, and the oval circle indicates that it is a 95% confidence ellipse; the closer the samples on the coordinate graph, the higher the similarity and it is usually considered that when stress is less than 0.2, it indicates that NMDS analysis has some reliability (e,f).
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Figure 3. Effect of the PGPR microbial fertilizer and root black rot application on the relative abundance of soil bacteria (a,c) and soil fungi (b,d) microorganisms. Note: CK: blank control (no microbial application); A: probiotic application only (Bacillus subtilis microbial fertilizer); B: both pathogenic and probiotic applications (Bacillus subtilis microbial fertilizer together with root black rot); C: pathogenic application only (root black rot).
Figure 3. Effect of the PGPR microbial fertilizer and root black rot application on the relative abundance of soil bacteria (a,c) and soil fungi (b,d) microorganisms. Note: CK: blank control (no microbial application); A: probiotic application only (Bacillus subtilis microbial fertilizer); B: both pathogenic and probiotic applications (Bacillus subtilis microbial fertilizer together with root black rot); C: pathogenic application only (root black rot).
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Figure 4. Correlation network analysis between inter-root soil microorganisms: between bacteria (a) and between fungi (b). Note: Spearman rank correlation analysis was performed and data with a correlation greater than 0.1 and a p-value less than 0.05 were screened to construct correlation network plots. The circles in the graph represent the species, the circle size represents the average abundance size of the species, the lines represent the correlation between the two species, the thickness of the lines represents the strength of the correlation, and the color of the lines are as follows: red represents positive correlation, green represents negative correlation.
Figure 4. Correlation network analysis between inter-root soil microorganisms: between bacteria (a) and between fungi (b). Note: Spearman rank correlation analysis was performed and data with a correlation greater than 0.1 and a p-value less than 0.05 were screened to construct correlation network plots. The circles in the graph represent the species, the circle size represents the average abundance size of the species, the lines represent the correlation between the two species, the thickness of the lines represents the strength of the correlation, and the color of the lines are as follows: red represents positive correlation, green represents negative correlation.
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Figure 5. Correlation analysis between soil microorganisms and soil traits based on the phylum level. RDA analysis of soil physico-chemical properties and bacterial communities (a); RDA analysis of soil physico-chemical properties and fungal communities (b); RDA analysis of soil enzyme activities and bacterial communities (c); RDA analysis of soil enzyme activities and fungal communities (d); heat map of correlation between soil nutrient status and bacterial communities (e); heat map of correlation between soil nutrient status and fungal communities (f). Note: The angle between species and environmental factors in the RDA plot represents the correlation between species and environmental factors (acute angle: positive correlation; obtuse angle: negative correlation; right angle: no correlation); the red color in the correlation heat map is the positive correlation, the blue color is negative correlation, and the darker color indicates a higher correlation, * is p < 0.05, ** is p < 0.001, *** is p < 0.0001.
Figure 5. Correlation analysis between soil microorganisms and soil traits based on the phylum level. RDA analysis of soil physico-chemical properties and bacterial communities (a); RDA analysis of soil physico-chemical properties and fungal communities (b); RDA analysis of soil enzyme activities and bacterial communities (c); RDA analysis of soil enzyme activities and fungal communities (d); heat map of correlation between soil nutrient status and bacterial communities (e); heat map of correlation between soil nutrient status and fungal communities (f). Note: The angle between species and environmental factors in the RDA plot represents the correlation between species and environmental factors (acute angle: positive correlation; obtuse angle: negative correlation; right angle: no correlation); the red color in the correlation heat map is the positive correlation, the blue color is negative correlation, and the darker color indicates a higher correlation, * is p < 0.05, ** is p < 0.001, *** is p < 0.0001.
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Table 1. PGPR microbial fertilizers and root black rot pathogens affect the agronomic traits of cigar plants.
Table 1. PGPR microbial fertilizers and root black rot pathogens affect the agronomic traits of cigar plants.
Plant TraitTreatments
CKABC
Cigar Tobacco plant
Plant height (cm)24.43 ± 0.23 b44.27 ± 0.43 a24.63 ± 0.37 b14.60 ± 0.46 c
Stem circumference (cm)2.67 ± 0.03 c4.20 ± 0.06 a3.23 ± 0.03 b2.43 ± 0.03 d
Number of leaves (pieces)17.00 ± 0.58 b22.67 ± 0.67 a16.00 ± 0.58 b13.33 ± 0.33 c
Leaf length (cm)28.50 ± 0.29 b34.77 ± 0.39 a28.53 ± 0.29 b24.70 ± 0.35 c
Leaf width (cm)9.83 ± 0.17 c14.03 ± 0.26 a11.60 ± 0.21 b9.43 ± 0.07 c
Fresh weight (g)6.18 ± 0.01 c10.39 ± 0.11 a6.71 ± 0.12 b5.44 ± 0.05 d
Dry weight (g)0.94 ± 0.01 c1.92 ± 0.01 a1.19 ± 0.02 b0.64 ± 0.02 d
Root system
Root length (cm)16.87 ± 0.26 b27.07 ± 0.22 a16.83 ± 0.18 b10.10 ± 0.23 c
Root vitality (μg/g ∗ h−1)130.75 ± 2.45 b202.58 ± 1.80 a133.47 ± 2.04 b94.69 ± 3.54 c
Note: CK: blank control (no microbial application); A: probiotic only (Bacillus subtilis microbial fertilizer); B: both pathogenic and probiotic (Bacillus subtilis microbial fertilizer together with root black rot pathogen); C: pathogenic only (root black rot pathogen). Data are expressed as the mean ± S.E (standard error) of three independent replicates. *: multiply sign. Different letters in the same row of the table indicate a significant difference in the mean of each treatment at p ≤ 0.05.
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MDPI and ACS Style

Shang, X.; Fu, S.; Guo, X.; Sun, Z.; Liu, F.; Chen, Q.; Yu, T.; Gao, Y.; Zhang, L.; Yang, L.; et al. Plant Growth-Promoting Rhizobacteria Microbial Fertilizer Changes Soils’ Microbial Structure and Promotes Healthy Growth of Cigar Tobacco Plants. Agronomy 2023, 13, 2895. https://doi.org/10.3390/agronomy13122895

AMA Style

Shang X, Fu S, Guo X, Sun Z, Liu F, Chen Q, Yu T, Gao Y, Zhang L, Yang L, et al. Plant Growth-Promoting Rhizobacteria Microbial Fertilizer Changes Soils’ Microbial Structure and Promotes Healthy Growth of Cigar Tobacco Plants. Agronomy. 2023; 13(12):2895. https://doi.org/10.3390/agronomy13122895

Chicago/Turabian Style

Shang, Xianchao, Sha Fu, Xiaomeng Guo, Zheng Sun, Fangyu Liu, Qian Chen, Tao Yu, Yun Gao, Li Zhang, Long Yang, and et al. 2023. "Plant Growth-Promoting Rhizobacteria Microbial Fertilizer Changes Soils’ Microbial Structure and Promotes Healthy Growth of Cigar Tobacco Plants" Agronomy 13, no. 12: 2895. https://doi.org/10.3390/agronomy13122895

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