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Article

Effects of Bacillus amyloliquefaciens QSB-6 on the Growth of Replanted Apple Trees and the Soil Microbial Environment

by
Yanan Duan
,
Yifan Zhou
,
Zhao Li
,
Xuesen Chen
,
Chengmiao Yin
* and
Zhiquan Mao
*
State Key Laboratory of Crop Biology, College of Horticultural Science and Engineering, Shandong Agricultural University, Taian 271018, China
*
Authors to whom correspondence should be addressed.
Horticulturae 2022, 8(1), 83; https://doi.org/10.3390/horticulturae8010083
Submission received: 23 November 2021 / Revised: 11 January 2022 / Accepted: 12 January 2022 / Published: 17 January 2022
(This article belongs to the Special Issue Sustainable Control Strategies of Plant Pathogens in Horticulture)
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Abstract

:
Apple replant disease (ARD), caused largely by soil-borne fungal pathogens, has seriously hindered the development of the apple industry. The use of antagonistic microorganisms has been confirmed as a low-cost and environmentally friendly means of controlling ARD. In the present study, we assessed the effects of Bacillus amyloliquefaciens QSB-6 on the growth of replanted apple saplings and the soil microbial environment under field conditions, thus providing a theoretical basis for the successful use of microbial biocontrol agents. Four treatments were implemented in three apple orchards: untreated replant soil (CK1), methyl bromide fumigation (CK2), blank carrier treatment (T1), and QSB-6 bacterial fertilizer treatment (T2). The plant height, ground diameter, and branch length of apple saplings treated with T2 in three replanted apple orchards were significantly higher than that of the CK1 treatment. Compared with the other treatments, T2 significantly increased the number of soil bacteria, the proportion of actinomycetes, and the activities of soil enzymes. By contrast, compared with the CK1 treatments, the phenolic acid content, the number of fungi, and the abundance of Fusarium oxysporum, Fusarium moniliforme, Fusarium proliferatum, and Fusarium solani in the soil were significantly reduced. PCoA and cluster analysis showed that soil inoculation with strain QSB-6 significantly decreased the Mcintosh and Brillouin index of soil fungi and increased the diversity of soil bacteria in T2 relative to CK1. The soil bacterial community structure in T2 was different from the other treatments, and the soil fungal communities of T2 and CK2 were similar. In summary, QSB-6 bacterial fertilizer shows promise as a potential bio-inoculum for the control of ARD.

1. Introduction

In the main apple planting regions of China, many old apple orchards are facing renewal but are limited by land resources, leading to the development of apple replant disease (ARD), which seriously affects the yield and quality of apples [1,2]. The disease is particularly harmful to young, replanted apple trees, causing slow growth, disease susceptibility, stopping root growth, below-ground necrosis, and even death in severe cases [3,4]. Previous studies have reported that replant disease does not arise from a single cause, but instead emerges from a combination of biotic and abiotic factors. The abiotic factors include allelopathy, autotoxicity, and soil physicochemical imbalances [5,6,7,8]. The biotic factors include the accumulation of soil-borne pathogens such as nematodes (Pratylenchus spp.), fungi (Rhizoctonia solani, Fusarium spp., and Cylindrocarpon spp.) and oomycetes (species of Pythium and Phytophthora). Although it is generally believed that replant disease results from a combination of biotic and abiotic factors, biotic factors play a leading role in disease development [2,3,8,9,10]. This has been widely demonstrated in other studies through soil pasteurization and biocide application [11,12,13].
Sheng et al. [14] demonstrated that the relative abundances of F. oxysporum and F. solani were significantly reduced after the replant soil was fumigated with methyl bromide. Quantitative analysis based on qPCR confirmed the wide distribution of F. oxysporum in replanted soil around the Bohai Gulf region, and F. verticillioides (formerly F. moniliforme), F. solani, F. oxysporum, and F. proliferatum isolated from apple replant soil around the Bohai Gulf region were highly pathogenic to Malus hupehensis seedlings. These findings demonstrate that Fusarium spp. are among the main soil-borne pathogenic fungi that cause ARD in China [15,16,17,18], and similar results have been reported from orchards in South Africa [10]. Fusarium spp. can survive saprophytically in soil or crop debris for more than six years. It infects the roots of susceptible hosts, colonizes the vascular system of plants, and produces toxins that kill plants by blocking xylem vessels and restricting water transport [19,20,21]. Traditional control methods, such as crop rotation and organic material application, are usually ineffective against this pathogen, and the cultivation of disease-resistant rootstocks and the application of chemical fumigants (e.g., methyl bromide) are the main measures used to control ARD. However, methyl bromide has been gradually eliminated because of its high environmental toxicity and time-consuming application [12,22,23,24]. The use of biological control agents (BCAs) is considered to be a safe, environmentally friendly, and sustainable means of protecting plants from soil-borne pathogenic fungi, and it has been widely used to control various fungal diseases of greenhouse and field crops. Therefore, the development of BCAs is one of the main directions for disease control research in the future [25,26,27].
Among the previously developed BCAs, Pseudomonas spp., arbuscular mycorrhizal fungi, Bacillus spp., and Trichoderma spp. have been reported to be effective antagonists against wilt caused by Fusarium spp. under field conditions [28,29,30,31,32,33]. Bacillus spp. offer several advantages over other BCAs for protection against root pathogens, including broad-spectrum activity of their antifungal secondary metabolites, plant growth promoting (PGP) properties, unique sporulation capacity, and ability to resist various stresses, which facilitates their long-term storage and commercialization [34,35]. Haddoudi et al. [36] revealed that the application of Bacillus amyloliquefaciens, Bacillus velezensis, Bacillus subtilis, and Bacillus mojavensis significantly reduced broad bean Fusarium wilt caused by Bacillus equisetum under greenhouse conditions and promoted plant growth. Zheng et al. [37] reported that B. velezensis D61-A had strong inhibitory activity against R. solani; its control efficacy reached 61.5% and 74.6% in the greenhouse and the field, respectively. Anusha et al. [38] obtained five strains of Bacillus spp. with strong antagonistic effects on F. oxysporum f. sp. ciceri from rhizosphere soils. Plants treated with these strains showed reduced disease incidence and delayed symptom development relative to non-inoculated controls under greenhouse and wilt-infested field conditions. Therefore, the introduction of disease-suppressing microorganisms is of great significance to the control of ARD [39,40].
In earlier work, we isolated a strain of B. amyloliquefaciens QSB-6 with broad-spectrum antifungal properties. Its fermentation broth was found to contain antibacterial substances (1,2-benzenedicarboxylic acid and benzeneacetic acid, 3-hydroxy-, methyl ester) with growth-promoting properties that significantly inhibited the mycelial growth and spore germination of F. oxysporum, F. moniliforme, F. proliferatum, and F. solani. These substances also protected the roots of M. hupehensis seedlings from F. oxysporum, F. moniliforme, F. proliferatum, and F. solani damage and promoted plant growth in a pot experiment [18]. Here, on the basis of these initial results, we evaluated the effects of B. amyloliquefaciens QSB-6 as a biological control agent of ARD under field conditions. Our specific aims were to evaluate the effects of QSB-6 on: (i) the growth of replanted apple saplings; (ii) the microbial diversity and community structure of the rhizosphere soil; and (iii) soil enzyme activities and the phenolic acid content. Lastly, we evaluated this new method in the renewal of aging apple orchards.

2. Materials and Methods

2.1. Bacterial Fertilizer Production

A strain of B. amyloliquefaciens QSB-6 was previously isolated from the rhizosphere soil of healthy apple trees in a replanted orchard and demonstrated to have good inhibitory effects on F. oxysporum, F. moniliforme, F. proliferatum, and F. solani [18].
QSB-6 bacterial fertilizer was produced by Chuangdi Microbial Resources Co., Ltd. (Dezhou, China). The production process was as follows: strain QSB-6 was first subjected to liquid fermentation for 12 h in an optimal flask fermentation medium that contained 20.0 g sucrose, 15.0 g yeast extract, 1.0 g MnSO4, 2.0 g NaH2PO4·2H2O, 4.0 g Na2HPO4·2H2O, and 1 L distilled water. The liquid inoculum was then thoroughly and evenly mixed with sterilized, decomposed carrier (3:1 cow dung:wheat straw by weight). The mixture was stored in a cool place, covered with a plastic sheet, and maintained at a temperature of 35–38 °C and a humidity of 45%. After 12–24 h, it was placed in a sealed container and used after 15 days of fermentation when the bacterial density was 5.0 × 109 CFU·g−1. The content of organic matter was 35.57%, available nitrogen was 0.36 mg·g−1, available phosphorus was 1.49 mg·g−1, and available potassium was 1.03 mg·g−1.

2.2. Test Materials

The apple seedlings used in the experiment were two-year-old grafted seedlings. The rootstock was T337, and the scion was Yanfu 3. The grafted seedlings had a stem thickness of approximately 10 mm and a stem height of approximately 1.4 m. They were purchased from Laizhou Nature Horticultural Technology Co., Ltd. (Laizhou, China). The row spacing of the plants was 1.5 m × 4 m, and the trees were pruned to a spindle shape.

2.3. Field Experiment

The field test was carried out in Wangtou Village, Laizhou City (119.81 longitude, 37.10 latitude), Sujiadian Town, Qixia City (120.83 longitude, 37.28 latitude), and Yiyuan, Zibo City (118.43 longitude, 36.09 latitude) in Shandong Province, China. The soil textures at the Laizhou (LZ), Qixia (QX), and Yiyuan (TY) sites were clay loam, sandy loam, and loam, respectively. Physicochemical properties of the soils are presented in Table 1. In March 2021, 30-year-old trees were removed from the orchards, and replanted orchards were established simultaneously.
The experiment consisted of four treatments: 30-year-old orchard soil (CK1), 30-year-old orchard soil fumigated with methyl bromide (CK2), bacterial fertilizer carrier treatment (T1), and QSB-6 bacterial fertilizer treatment (T2). The planting holes (80 cm3) were dug according to the row spacing, and the bacterial manure carrier or QSB-6 bacterial manure were mixed with the soil and backfilled. Each soil amendment was applied at a rate of 1 kg per tree, and there were 20 trees per treatment. All measurements (plant height, ground diameter, number of branches, and length of new branches) and soil sampling were performed on 20 October 2021. A Canon PowerShot G16 camera was used to photograph the saplings. The surface soil was removed, and multiple soil samples were collected within a 0.5-m radius around each sapling. Three replicate samples from each treatment were used for each measurement. First, impurities, such as roots, weeds, soil organisms, and stones, were removed from each soil sample. Next, each sample was divided into three portions: one portion was stored in a refrigerator at 4 °C for the determination of culturable microbes; one portion was air-dried for the measurement of soil enzyme activities, nutrient content, and phenolic acid content; and one portion was stored in a freezer at −80 °C for DNA extraction, real-time fluorescence quantitative PCR (qPCR), and terminal-restriction fragment length polymorphism (T-RFLP) analysis [14].

2.3.1. Soil Physical and Chemical Properties

The soil organic matter content was determined by potassium dichromate capacity-spectrophotometry as described in the Soil Physical and Chemical Analysis Experiment Guide [41]. Soil ammonium-nitroge and nitrate nitrogen contents were determined by colorimetric methods, soil available phosphorus content was determined by the 0.05 M NaHCO3 method, and soil available potassium content was determined by flame photometry. The soil bulk density was determined by the ring knife method, and the moisture content was determined by drying at 105 °C. Soil pH was measured in a water extract (1:2.5, w/v) using a Shanghai Lei PHS-3EJ Magnetic Benchtop pH Meter (Shanghai, China). The particle size distribution (the percentages of clay, silt, and sand) was determined by hydrometry [42].

2.3.2. Microbial Culture Methods

Soil microbial populations (bacteria, fungi, and actinomycetes) were assessed by the dilution method of plate counting described by Zhang et al. [43]. Bacteria, fungi, and actinomycetes were incubated in beef broth peptone substrate, potato dextrose agar (PDA; Difco, Detroit, MI, USA), and Gause No. 1 substrate, respectively. Five plates per dilution were used for each measurement of each soil sample, and the populations of bacteria, fungi, and actinomycetes were quantified as CFU per gram of dry soil.

2.3.3. Determination of Soil Enzyme Activities

Soil urease activity was measured by the indophenol blue colorimetric method as described in the instructions of the solid-urease (S-UE) activity kit. Soil neutral phosphatase activity was measured by the disodium phenyl phosphate method as described in the instructions of the soil neutral phosphatase (S-NP) activity kit. Soil sucrase activity was measured by the 3,5-dinitrosalicylic acid (DNS) method as described in the instructions of the soil sucrase (S-SC) activity kit. The measurement of soil catalase activity used H2O2, which has a characteristic absorption peak at 240 nm. The specific method is described in the instructions of the solid-catalase (S-CAT) activity kit. All kits were purchased from Suzhou Keming Biotechnology Co., Ltd. (Suzhou, China).

2.3.4. Quantitative Determination of Soil Phenolic Acids by HPLC

The soil phenolic acid content was measured using the method of Yin et al. [44]. A sample of dry soil (100 g) was passed through a 12-mesh size sieve, mixed with diatomaceous earth, and placed into a 100-mL extraction tank. The ASE 350 Fast Solvent Extractor (Sunnyvale, CA, USA) was used to perform the extraction. First, absolute ethanol was used as the extraction solvent, and static extraction was performed for 5 min at 120 °C and 10.3 MPa two times, followed by a purge volume of 60% and a purge time of 90 s. Next, the same sample was extracted again under the same conditions using methanol as the extraction solvent. After the extraction was completed, the two solvents were mixed and concentrated under reduced pressure at 34 °C to near dryness, and the sample was then reconstituted with 1 mL methanol and passed through a 0.22-μm organic phase filter membrane for HPLC analysis.
The HPLC procedure followed that described by Xiang et al. [45], with some modifications. An UltiMate 3000 HPLC system (Dionex) with an Acclaim 120 C18 column (3 μm, 150 mm × 3 mm) and a column temperature of 30 °C were used for quantification. The mobile phase A was acetonitrile, and the mobile phase B was water (adjusted to pH 2.6 with acetic acid). The flow rate was 0.5 mL·min−1, the automatic injection volume was 5 μL, and the detection wavelength was 280 nm. All reagents were chromatographic grade.

2.3.5. DNA Extraction from Four Species of Fusarium and Quantitative PCR

Sieved fresh soil (5.0 g) was used for DNA extraction with the DNeasy PowerMax Soil Kit (Qiagen, Hilden, Germany). Quantitative PCR amplifications for standard and environmental DNA samples were performed with a volume of 20 μL in each reaction using SYBR Premix Ex Taq (Takara, Dalian, China) and a CFX96 Touch Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) following the method of Duan et al. [18]. Each 20 μL PCR reaction contained 2 μL of genomic DNA, 10 μL of SYBR green PCR Master Mix (TaKaRa Biotech, Dalian, China), 0.4 μL of each primer pair, and 7.2 μL of ddH2O. Reactions were carried out using the following PCR cycling conditions: JR/JF and CHR/CHF primer pairs, 95 °C 30 s, 94 °C 5 s (40 cycles), 60 °C 30 s, 72 °C 1 min, followed by a 4 °C soak; CR/CF and FR/FF primer pairs, 95 °C 30 s, 94 °C 5 s (40 cycles), 65 °C 30 s, 72 °C 1 min, followed by a 4 °C soak. The primer pairs used in this experiment are as follows: JR (5′GGCCTGAGGGTTGTAATG-3′) × JF (5′CATACCACTTGTTGTCTCGGC-3′) for F. oxysporum; CHR (5′GACTCGCGAGTCAAATCGCGT-3′) × CHF (5′GGGGTTTAACGGCGTGGCC-3′) for F. moniliforme; CR (5′GATCGGCGAGCCCTTGCGGCAAG-3′) × CF (5′CGCCGCGTACCAGTTGCGAGGGT-3′) for F. proliferatum; FR (5′CGAGTTATACAACTCATCAACC-3′) × FF (5′GGCCTGAGGGTTGTAATG-3′) for F. solani. Four species of Fusarium DNAs were used as templates, and specific primers were used for PCR amplification. The purified PCR products were connected to pMD18-T vector (TaKaRa Biotech, China), and transformed into E. coli competent cells DH5α (TaKaRa Biotech, Dalian, China). After sequencing verification, the plasmid was extracted according to the method of the plasmid extraction kit (TaKaRa Biotech, Dalian, China), its concentration was measured and a gradient dilution with sterile water was carried out. The standard curves were generated by plotting the cycle threshold (Cq) values obtained for each specific DNA concentration versus the log of the initial concentration of species DNA. The standard curve is as follows: F. oxysporum: y = −2.291x + 36.396, R2 = 0.993; F. moniliforme: y = −3.495x + 12.421, R2 = 0.998; F. proliferatum: y = −3.675x + 9.128, R2 = 0.999; F. solani: y = −2.352x + 26.941, R2 = 0.994. The concentration of plasmid DNA was measured and converted to copy concentration using the following equation from Whelan et al. [46]: DNA (copy) = [6.02 × 1023 (copies mol−1) × DNA amount (g)]/[DNA length (bp) × 660 (g mol−1 bp−1)]. Sterile water was used as the negative control instead of the template. All real-time PCR reactions were performed in triplicate with three biological replicates, so that each treatment was analyzed nine times.

2.3.6. Terminal-restriction fragment length polymorphism (T-RFLP) Analysis

The DNA was amplified using the universal primers 27F-FAM/1492R and ITS1F-FAM/ITS4R that target the bacterial 16S rRNA gene and the fungal ITS region between 18S and 28S rRNA regions, respectively. The forward primers were labeled at the 5′ end with 6-carboxyfluorescein (FAM), which was synthesized by Sangon Biotech (Shanghai, China). The specific steps are described in Xu et al. [47]. The 50-μL PCR mixture contained 0.6 μL of 5 U/μL Ex Taq (TaKaRa), 5 μL of 10 × Ex Taq Buffer, 1 μL of 2.5 mM dNTP mixture, 2 μL of 0.5 mM forward and reverse primers, 12.6 μL of ddH2O, and 2.0 μL containing 100 ng of the extracted DNA template. The primer pairs used in this experiment are as follows: 27F-FAM (5′AGAGTTTGATCCTGGCTCAG-3′) × 1492R (5′GTTACCTTGTTACGACTT-3′) for bacteria; ITS1F-FAM (5′CTTGGTCATTTAGAGGAAGTAA-3′) × ITS4R (5′CAGGAGACTTGTACACGGTCCAG-3′) for fungi [48,49]. All PCR amplifications were performed on an Applied Biosystems 2720 Thermal Cycler (Applied Biosystems, Foster City, CA, USA). For bacteria, PCR conditions consisted of 94 °C for 3 min, followed by 30 cycles at 94 °C for 45 s, 52 °C for 45 s, and 72 °C for 1 min. A final extension was performed at 72 °C for 10 min. For fungi, the PCR conditions consisted of 95 °C for 5 min, followed by 30 cycles at 94 °C for 30 s, 50 °C for 30 s, and 72 °C for 1 min. A final extension was performed at 72 °C for 10 min. Prior to digestion, PCR products were cleaned with the EZNA PCR Purification Kit (OMEGA Bio-tek Inc., Doraville, GA, USA) following the manufacturer’s instructions and quantified using a DNAmaster Nucleic Acid and Protein Analyzer (Dynamica, Scientific Ltd., Newport Pagnell, UK). The purified PCR product (500 ng) was digested with MspI (TaKaRa, Tokyo, Japan) for 16S rRNA gene amplicons and HinfI (TaKaRa, Japan) for ITS amplicons in two separate reactions according to their protocols [50,51]. T-RFLP analysis was run on an ABI 3730 DNA Analyzer (Applied Biosystems, Melbourne, Australia) by Sangon Biotech Co., Ltd. (Shanghai, China) using LIZ-labeled GS500 (−250) as the internal size standard.

2.4. Statistical Analysis

All statistical analyses were performed with IBM SPSS 20.0 (IBM SPSS Statistics, IBM Corporation, Armonk, NY, USA). Different lowercase letters represent significant differences between treatments (one-way ANOVA, p < 0.05) according to Duncan’s multiple range test. The figures were plotted with Microsoft Excel 2013 (Microsoft Corporation, Redmond, WA, USA) and GraphPad Prism 7.0 (GraphPad software, Inc., San Diego, CA, USA). TBtools software was used for cluster analysis; similarities and differences among treatments are indicated by the color gradient, and the color intensity is directly proportional to substance content.
T-RFLP profiles were analyzed using Peak Scanner Software v1.0 (Thermo Fisher Scientific, Wilmington, NC, USA); T-RFs < 50 bp in length and T-RFs that contributed to < 0.5% of total peak area in each sample were excluded from subsequent analyses. The apparent T-RF sizes in capillary electrophoresis were compared against the MiCA database to determine the phylotype [52]. The R statistical platform (v.4.1.1) was used for principal component analysis (PCoA) and cluster analysis to study differences in community composition among samples. Differences among samples were calculated by Bray–Curtis dissimilarity, and analysis of similarity (ANOSIM) was performed to identify significant differences among the fungal communities [53,54]. The richness index (SR) and evenness index (E) were calculated using Bio-Dap software [18].

3. Results

3.1. Effect of Strain QSB-6 on the Biomass of Replanted Apple Saplings

The application of QSB-6 bacterial fertilizer (T2) increased the plant height, ground diameter, and branch length of young apple trees relative to CK1 (Table 2). In Yiyuan, Qixia, and Laizhou, T2 treatment increased plant height by 31.30%, 29.98%, and 24.78%, the ground diameter by 37.84%, 42.28%, and 46.94%, and the average branch length by 34.62%, 43.46%, and 30.34% relative to the replant soil control (CK1). In Yiyuan, T2 treatment increased the plant height and the ground diameter by 9.74% and 23.19% relative to the blank carrier (T1), and their growth was close to that of seedlings planted in methyl bromide-fumigated soil. In Qixia, T2 treatment increased the plant height, the ground diameter, and the average branch length by 9.05%, 10.85%, and 27.99% relative to T1.

3.2. Effect of Strain QSB-6 on Soil Microorganisms

In Laizhou, Qixia, and Yiyuan, the number of soil bacteria was significantly higher in the QSB-6 bacterial fertilizer treatment (T2) than in the other treatments. The number of soil bacteria was 2.21-, 2.82-, and 2.0-fold higher in T2 than in the replant soil control (CK1), and 1.91-, 1.69-, and 1.74-fold higher in T2 than in the blank carrier treatment (T1) (Table 3). The number of soil fungi was significantly higher in T1 than in T2. The T1 treatment also significantly increased the soil fungus in Laizhou (1.31-fold), Qixia (1.81-fold), and Yiyuan (1.53-fold) relative to the T2 treatment. In Laizhou, Qixia, and Yiyuan, the number of soil fungi was reduced by 40.38%, 68.45%, and 49.07% in CK2 relative to CK1 and by 34.62%, 53.40%, and 41.61% in T2 relative to CK1. The effects of T2 were similar to those of methyl bromide fumigation treatment in Laizhou and Yiyuan. The number of soil actinomycetes at all three sites could be ranked as: T2 > T1 > CK2. The T2 treatment also significantly increased the soil bacteria/fungi and actinomycetes/fungi ratios in Laizhou (2.55- and 2.05-fold), Qixia (3.06- and 2.73-fold), and Yiyuan (2.66- and 2.26-fold) relative to the T1 treatment.

3.3. Effect of Strain QSB-6 on Soil Enzyme Activities

The T2 treatment significantly increased the soil activities of urease, phosphatase, invertase, and catalase in Qixia, Yiyuan, and Laizhou (Figure 1). Urease activity was 1.86-, 1.74-, and 1.55-fold higher in T2 than in CK1. Phosphatase activity was 2.17-, 2.16-, and 2.16-fold higher in T2 than in CK1. Sucrase activity was 2.94-, 3.84-, and 3.79-fold higher in T2 than in CK1. Catalase activity was 1.90-, 1.85-, and 1.73-fold higher in T2 than in CK1. Compared with the T1 treatment, urease activity was 22.24%, 21.20%, and 20.38% higher in T2; phosphatase activity was 40.47%, 27.42%, and 23.02% higher in T2; sucrase activity was 49.21%, 35.86%, and 73.91% higher in T2; and catalase activity was 40.80%, 27.39%, and 26.97% higher in T2.

3.4. Effect of Strain QSB-6 on Soil Phenolic Acids

The contents of several phenolic acids were highest in the replant soil control (CK1) at all three field sites, whereas the phenolic acid contents were significantly reduced in soil treated with QSB-6 bacterial fertilizer (T2) (Figure 2). In Laizhou, the soil contents of cinnamic acid, phlorizin, benzoic acid, ferulic acid, vanillin, p-hydroxybenzoic acid, and caffeic acid were reduced by 53.58%, 67.64%, 61.45%, 62.62%, 71.31%, 60.50%, and 71.31% in T2 relative to CK1 (Table S1). In Qixia, the contents of these phenolic acids were reduced by 53.15–83.32% in T2 relative to CK1, and in Yiyuan, they were reduced by 52.65–72.73%. In Laizhou, Qixia, and Yiyuan, the soil total phenolic acid contents were higher in the T1 treatment than in the T2 treatment: cinnamic acid (1.59-, 1.98-, and 1.72-fold higher in T1), phenophyllin (1.66-, 1.11-, and 1.20-fold), benzoic acid (2.11-, 3.20-, and 2.03-fold), ferulic acid (2.21-, 1.99-, and 1.80-fold), vanillin (2.59-, 2.86-, and 1.54-fold), p-hydroxybenzoic acid (2.15-, 2.42-, and 2.62-fold), and caffeic acid (2.76-, 3.29-, and 1.50-fold).

3.5. Inhibitory Effect of Strain QSB-6 on Four Species of Fusarium in Rhizosphere Soil

The qPCR results showed that F. proliferatum, F. solani, F. verticillioides, and F. oxysporum abundance was significantly reduced in the CK2 and T2 treatments compared with the CK1 treatment at all three field sites (Figure 3). In Qixia, the abundances of F. proliferatum, F. solani, F. verticillioides, and F. oxysporum were 35.23%, 32.79%, 57.48%, and 36.60% lower in T2 than in CK1. In Yiyuan, the abundances of F. proliferatum, F. solani, F. verticillioides, and F. oxysporum were 32.05%, 33.16%, 32.62%, and 49.63% lower in T2 than in CK1. In Laizhou, the abundances of F. proliferatum, F. solani, F. verticillioides, and F. oxysporum were 28.26%, 43.56%, 55.86%, and 38.04% lower in T2 than in CK1. In Qixia, Yiyuan, and Laizhou, the abundances of F. proliferatum were 47.22%, 43.11%, and 31.33% lower in CK2 than in CK1. The abundances of F. solani were 41.94%, 50.27%, and 50.60% lower in CK2 than in CK1. The abundances of F. verticillioides were 64.94%, 37.88%, and 61.05% lower in CK2 than in CK1. The abundances of F. oxysporum were 45.49%, 59.51%, and 49.74% lower in CK2 than in CK1. The gene copy number of four species of Fusarium was significantly higher in T1 soil than in T2 soil. In Qixia, Yiyuan, and Laizhou, the relative abundance of F. proliferatum was 1.33-, 1.30-, and 1.26-fold higher in T1 soil than in T2 soil. The relative abundance of F. solani was 1.26-, 1.25-, and 1.41-fold higher; that of F. verticillioides was 1.48-, 1.22-, and 1.45-fold higher; and that of F. oxysporum was 1.25-, 1.41-, and 1.40-fold higher.

3.6. Effect of Strain QSB-6 on the Soil Microbial Community

Principal component analysis and cluster analysis showed that the soil microbial community structure in T2 and CK2 was significantly different from that in CK1 (Figure 4). The soil bacterial community structure in T2 was different from the other treatments, and the soil fungal and bacterial communities of T1 and CK1 were similar. Margalef, Mcintosh, Brillouin, and Shannon indices reflect the richness and diversity of soil microbial communities (Table 4). The Margalef index reflects the abundance of soil microbial communities; the Mcintosh index reflects the number of different types of carbon sources utilized; and the Brillouin and Shannon indices reflect the diversity of the soil microbial community [55]. Compared with CK1 treatment, the abundance of the soil fungal community was significantly increased after treatment with strain QSB-6. The Brillouin index of the soil fungal community and the utilized carbon sources were significantly reduced, whereas the bacterial community showed the opposite pattern.

4. Discussion

Replant disease is a complex, multifaceted disease that often occurs in crops and orchards and strongly constrains the sustainable development of global agriculture and fruit production [56,57]. In this study, strain QSB-6 acted as a biocontrol agent that significantly improved the growth and health of replanted young apple trees under field conditions. Multiple aspects of plant vegetative growth (height, ground diameter, and length of branches) were measured, and all were markedly increased by QSB-6 fertilizer application relative to the replanted soil control. The biomass of young apple trees was higher in Yiyuan than in Laizhou and Qixia, perhaps because of the better soil physicochemical properties and management practices [58,59]. These plant growth results indicate that treatment with strain QSB-6 was effective for the control of ARD under field conditions.

4.1. Effect of QSB-6 Fertilizer on the Soil Microbial Community

Bacteria, actinomyces, and fungi are three categories of the soil microbial community that constitute the majority of the soil microbial biomass; their community structure and abundances play important roles in plant growth and disease control [60,61]. Previous studies have found that long-term continuous cropping leads to a transformation of the soil microbial community structure from a “bacterial type” to a “fungal type”. The soil microbial community structure becomes unbalanced, leading to an increase in harmful pathogens and a decrease in beneficial microbiota, ultimately leading to poor soil health [2,62,63,64,65]. Soil microbial diversity is considered to be a key factor in disease prevention [66]. High microbial diversity and appropriate microbial composition play key roles in preventing pathogen invasion, maintaining soil health, and promoting plant growth [67,68]. Gadhave et al. [69] reported that the soil bacterial diversity and richness index increased significantly after application of Bacillus spp., and we reached a similar conclusion in this study. The application of QSB-6 bacterial fertilizer to three apple orchards significantly increased the diversity and number of bacteria. The results of the plate count test revealed that most of the cultured bacterial colonies showed morphologies consistent with that of strain QSB-6. A single colony with the same morphology was randomly selected and sequenced, and its similarity to strain QSB-6 was 100%. These results indicate that strain QSB-6 was able to reproduce normally after being inoculated into the soil. At the same time, the addition of vectors can also support the survival of the strain QSB-6 and improve its performance in preventing and controlling plant diseases [70,71]. The raw materials (cow dung and wheat straw) in the formula are cheap and easy to obtain, and the fermentation level is high, which can provide a good foundation for large-scale industrial production [72,73].
A decrease in the quantity of soil fungi may be related to fungistatic compounds (1,2-benzenedicarboxylic acid and benzeneacetic acid, 3-hydroxy-, methyl ester) produced by strain QSB-6. These compounds can significantly inhibit the reproduction of harmful F. oxysporum, F. moniliforme, F. proliferatum, and F. solani in rhizosphere soil, and they can also inhibit other harmful fungi to some extent [18]. Our results were consistent with those of Cao et al. [74]. Bacillus subtilis SQR 9 was able to survive well in the rhizosphere of cucumber, where it suppressed the growth of F. oxysporum and protected the host from the pathogen. Tao et al. [40] reported that the population densities of Bacillus spp. and Pseudomonas spp. were correlated with one another and negatively correlated with F. oxysporum density and wilt disease. Several well-known plant growth-promoting bacteria belong to the genus Pseudomonas spp. [75,76,77]. The increase in soil bacterial diversity reported here after QSB-6 inoculation may be due to the enrichment of some specific beneficial bacteria such as Pseudomonas spp., Gemmatimonas spp., and Sphingomonas spp., resulting in a significant decrease in bacterial richness [78], but a significant increase in the bacteria/fungi ratio, thus transforming the soil into a “bacterial type” and improving the soil microbial community structure, which may promote the healthy growth of plants [79,80]. Sheng et al. [14] demonstrated that soil fumigation treatment can effectively kill most fungi, especially F. oxysporum and F. solani. The relative abundances of F. solani and F. oxysporum are significantly reduced, but with prolonged time, some fungi are recruited to form new microbial communities, such as Ohtaekwangia spp., Opitutus spp., Mortierella spp., and Synchytrium spp., which improve the soil fungal community, thus effectively controlling plant diseases [81]. These results were consistent with the findings of this study. Methyl bromide fumigation treatment significantly reduced the relative abundance of F. oxysporum, F. moniliforme, F. proliferatum, and F. solani and promoted the growth of replanted apple trees. Cluster analysis showed that the soil fungal communities of T2 and CK2 were similar, indicating that the addition of strain QSB-6 may also promote the growth of replanted apple trees by improving the soil fungal community structure.

4.2. Effect of QSB-6 Fertilizer on Soil Phenolic Acids

There is evidence that allelochemicals, such as phenolic acids, in root exudates or decomposing residues contribute to apple replanting obstacles [23]. Yin et al. [17] showed that phlorizin can promote a rapid increase in the number of F. moniliforme and accelerate the speed of mycelial division, thus increasing apple replant challenges. The replanted soil contains a variety of phenolic acids such as p-hydroxybenzoic acid, phloroglucinol, syringic acid, benzoic acid, caffeic acid, and ferulic acid. Above a certain concentration, these compounds can impair the root antioxidant system of apple seedlings and inhibit their growth [44,66,82,83]. Here, we found that strain QSB-6 may have the ability to degrade phenolic acids. The addition of strain QSB-6 significantly reduced the contents of major phenolic acids in the soil, including phlorizin, cinnamic acid, benzoic acid, and p-hydroxybenzoic acid. At the same time, the abundance of F. oxysporum, F. moniliforme, F. proliferatum, and F. solani also declined significantly in the rhizosphere soil, consistent with the results of Yin et al. [17]. Bai et al. [65] demonstrated that phenolic acids significantly affected the biomass, diversity, and community structure of soil microbes, selectively increasing specific microbial species, such as soil-borne pathogenic microorganisms, and thus, increasing morbidity [84]. It is possible that the addition of strain QSB-6 increases the populations of some beneficial bacteria that can degrade phenolic acid, increase bacterial diversity, and improve the bacterial community structure of the rhizosphere soil, thus accelerating the decomposition and transformation of phenolic acids and promoting plant growth [85,86].

4.3. Effect of QSB-6 Fertilizer Treatment on Soil Enzyme Activity

Soil enzymes are an important component of the soil, catalyzing various reactions and processes of organic matter metabolism (e.g., soil organic matter formation and degradation; C, N, and P cycling; and plant nutrient transformation) and generating energy for microorganisms and plants. Therefore, the extent of soil enzyme activity can be used to characterize the extent of soil maturation and fertility [87,88]. Soil urease is involved in the soil N cycle. Phosphatases hydrolyze P-containing organic compounds into inorganic P that is required by plants. Invertase hydrolyzes sucrose to glucose and fructose for use as plant and microbial energy sources, whereas catalase is an oxidoreductase that protects organisms from H2O2 toxicity [89,90,91,92]. Sabaté et al. [93] showed that inoculation with Bacillus sp. P12 significantly increased soil enzyme activities and the number of beneficial microorganisms, improved soil quality, and reduced the incidence of Macrophomina phaseolina. Our results were similar, that is, inoculation with strain QSB-6 at three field sites significantly increased the rhizosphere activities of urease, phosphatase, invertase, and catalase, thus presumably promoting nitrogen, phosphorus, and potassium transformation in the soil, improving soil fertility, and strengthening plant resistance to abiotic and biotic stresses [94,95]. Yadav et al. [96] reported that the micro-organisms associated with rhizosphere soil also have profound impacts on plant health and soil fertility, as they strongly influence nutrient mineralization and soil organic matter decomposition. Thus, increases in soil enzyme activity may be related to increases in soil microbial richness and diversity, as well as changes in microbial community composition [97].

5. Conclusions

The results of our field experiments were similar to those of our previous pot study [18]. The addition of strain QSB-6 can significantly promote the height, ground diameter, and length of branches of apple plants under field conditions, reduce the abundance of F. oxysporum, F. moniliforme, F. proliferatum, and F. solani and the content of phenolic acids in rhizosphere soil, increase soil bacterial diversity and activity, improve the structure of the soil microbial community, and increase soil enzyme activities. Together, these effects help to mitigate ARD. In summary, QSB-6 bacterial fertilizer appears to offer a more sustainable approach to enhancing apple growth and soil health under replant conditions, thereby advancing the development of the apple industry.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/horticulturae8010083/s1, Table S1: Effects of different treatments on soil phenolic acids in three places.

Author Contributions

Conceptualization, Z.M. and C.Y.; methodology, Y.D.; software, Z.L.; validation, Y.D., Y.Z. and Z.L.; investigation, Y.D.; data curation, Y.D. and Y.Z.; writing—original draft preparation, Y.D.; writing—review and editing, C.Y. and X.C. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by China Agriculture Research System of MOF and MARA (CARS-27), the National Natural Science Foundation of China (32072510), Shandong Agricultural Major Applied Technology Innovation Project (SD2019ZZ008); Taishan Scholar Funded Project (NO.ts20190923); Qingchuang Science and Technology Support Project of Shandong Colleges and Universities (2019KJF020); and the Natural Science Foundation of Shandong Province (ZR2020MC131).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The study did not report any data.

Acknowledgments

We thank all the colleagues that helped with the development of different parts of this manuscript.

Conflicts of Interest

The authors declare no conflict of interest.

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Horticulturae 08 00083 g001 550
Figure 1. Effect of different treatments on soil enzyme activity in three places. LZ: Laizhou, QX: Qixia, TY: Yiyuan. CK1: replant control, CK2: methyl bromide fumigation, T1: blank carrier treatment, T2: QSB-6 bacterial fertilizer treatment. Columns with the same letter for each place are not significantly different based on the Duncan multiple range test at p < 5%.
Figure 1. Effect of different treatments on soil enzyme activity in three places. LZ: Laizhou, QX: Qixia, TY: Yiyuan. CK1: replant control, CK2: methyl bromide fumigation, T1: blank carrier treatment, T2: QSB-6 bacterial fertilizer treatment. Columns with the same letter for each place are not significantly different based on the Duncan multiple range test at p < 5%.
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Figure 2. Effects of different treatments on soil phenolic acids in three places. LCK1: replant control in Laizhou, LCK2: methyl bromide fumigation in Laizhou, LT1: blank carrier treatment in Laizhou, LT2: QSB-6 bacterial fertilizer treatment in Laizhou, QCK1: replant control in Qixia, QCK2: methyl bromide fumigation in Qixia, QT1: blank carrier treatment in Qixia, QT2: QSB-6 bacterial fertilizer treatment in Qixia, YCK1: replant control in Yiyuan, YCK2: methyl bromide fumigation in Yiyuan, YT1: blank carrier treatment in Yiyuan, YT2: QSB-6 bacterial fertilizer treatment in Yiyuan. The color intensity was proportional to the total phenolic acid content. Taxa relative abundances were log10-transformed, and the scale method (from zero to one) was used for the heatmap representation. Each treatment included three repetitions.
Figure 2. Effects of different treatments on soil phenolic acids in three places. LCK1: replant control in Laizhou, LCK2: methyl bromide fumigation in Laizhou, LT1: blank carrier treatment in Laizhou, LT2: QSB-6 bacterial fertilizer treatment in Laizhou, QCK1: replant control in Qixia, QCK2: methyl bromide fumigation in Qixia, QT1: blank carrier treatment in Qixia, QT2: QSB-6 bacterial fertilizer treatment in Qixia, YCK1: replant control in Yiyuan, YCK2: methyl bromide fumigation in Yiyuan, YT1: blank carrier treatment in Yiyuan, YT2: QSB-6 bacterial fertilizer treatment in Yiyuan. The color intensity was proportional to the total phenolic acid content. Taxa relative abundances were log10-transformed, and the scale method (from zero to one) was used for the heatmap representation. Each treatment included three repetitions.
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Figure 3. Effect of different treatments on the copy number of four species of Fusarium in three places. LZ: Laizhou, QX: Qixia, TY: Yiyuan. CK1: replant control, CK2: methyl bromide fumigation, T1: blank carrier treatment, T2: QSB-6 bacterial fertilizer treatment. Columns with the same letter for each place are not significantly different based on the Duncan multiple range test at p < 5%.
Figure 3. Effect of different treatments on the copy number of four species of Fusarium in three places. LZ: Laizhou, QX: Qixia, TY: Yiyuan. CK1: replant control, CK2: methyl bromide fumigation, T1: blank carrier treatment, T2: QSB-6 bacterial fertilizer treatment. Columns with the same letter for each place are not significantly different based on the Duncan multiple range test at p < 5%.
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Figure 4. Principal component analysis (A,C) and cluster analysis (B,D) of T-RFLP patterns of bacteria (A,B) and fungi (C,D) in the soil relative to treatment based on the Bray–Curtis method. CK1: replant control, CK2: methyl bromide fumigation, T1: blank carrier treatment, T2: QSB-6 bacterial fertilizer treatment. Each treatment includes three repetitions.
Figure 4. Principal component analysis (A,C) and cluster analysis (B,D) of T-RFLP patterns of bacteria (A,B) and fungi (C,D) in the soil relative to treatment based on the Bray–Curtis method. CK1: replant control, CK2: methyl bromide fumigation, T1: blank carrier treatment, T2: QSB-6 bacterial fertilizer treatment. Each treatment includes three repetitions.
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Table
Table 1. Basic physical and chemical properties in the rhizosphere soil in three orchards.
Table 1. Basic physical and chemical properties in the rhizosphere soil in three orchards.
PlaceAmmonium-Nitrogen (mg kg−1)Nitrate Nitrogen
(mg kg−1)		Organic Matter (%)Available Phosphorus (mg kg−1)Soil Bulk Density (g cm−3)Available Potassium (mg kg−1)Soil pHSoil Moisture Content (%)
LZ1.5 ± 0.0 b39.2 ± 1.1 b2.2 ± 0.1 a17.5 ± 0.0 c1.0 ± 0.1 a63.2 ± 8.8 c7.1 ± 0.0 a8.9 ± 0.2 a
QX1.6 ± 0.0 b42.8 ± 0.2 a2.2 ± 0.0 a19.2 ± 0.3 b1.2 ± 0.1 a108.4 ± 5.0 b6.7 ± 0.3 a10.3 ± 1.7 a
TY2.3 ± 0.2 a43.7 ± 0.2 a2.3 ± 0.0 a21.7 ± 0.5 a1.1 ± 0.1 a223.3 ± 11.7 a7.1 ± 0.0 a11.5 ± 1.1 a
LZ: Laizhou, QX: Qixia, TY: Yiyuan. Numbers followed by the same letter in each column are not significantly different based on the Duncan multiple range test at p < 5%.
Table
Table 2. Effects of different treatments on the biomass of apple replanting saplings in three areas.
Table 2. Effects of different treatments on the biomass of apple replanting saplings in three areas.
PlaceSoil TreatmentsPlant Height
(cm)		Ground Diameter
(mm)		Numbers of BranchesBranch Length
(cm)
LZCK1150.7 ± 3.0 c13.8±0.1 c7.7 ± 0.9 b44.5 ± 6.6 c
CK2206.0 ± 7.6 a24.3±1.1 a11.0 ± 0.6 a73.8 ± 2.4 a
T1179.3 ± 0.9 b18.4±0.9 b7.7 ± 0.7 b54.3 ± 17 bc
T2188.0 ± 0.6 b20.2±0.9 b10.0 ± 0.6 ab58.0 ± 3.1 b
QXCK1139.0 ± 5.9 c18.0 ± 0.1 d8.7 ± 1.5 b34.0 ± 1.7 b
CK2191.3 ± 6.3 a28.4 ± 0.6 a15.0 ± 1.0 a53.6 ± 2.7 a
T1165.7 ± 2.3 b23.1 ± 0.0 c11.7 ± 1.2 ab38.1 ± 1.6 b
T2180.7 ± 1.7 a25.6 ± 1.0 b12.0 ± 1.5 ab48.8 ± 3.0 a
TYCK1191.7 ± 12.1 c17.0 ± 1.5 b10.0 ± 1.5 b49.1 ± 4.7 b
CK2254.3 ± 4.3 a25.8 ± 0.1 a16.3 ± 0.7 a68.0 ± 5.6 a
T1229.3 ± 0.7 b19.1 ± 0.2 b12.0 ± 1.0 ab55.7 ± 3.8 ab
T2251.7 ± 4.9 a23.5 ± 0.6 a15.0 ± 2.1 a66.1 ± 1.1 a
LZ: Laizhou, QX: Qixia, TY: Yiyuan. CK1: replant control, CK2: methyl bromide fumigation, T1: blank carrier treatment, T2: QSB-6 bacterial fertilizer treatment. Numbers followed by the same letter in the columns for each place are not significantly different based on the Duncan multiple range test at p < 5%.
Table
Table 3. Effects of different treatments on the density of microorganisms in the rhizosphere of young apple trees in three places.
Table 3. Effects of different treatments on the density of microorganisms in the rhizosphere of young apple trees in three places.
PlaceSoil Treatments The Number of Soil Bacteria
(×105 CFU/g Soil)		The Number of Soil Fungi
(×103 CFU/g Soil)		The Number of Soil Actinomycete
(×104 CFU/g Soil)		The Ratio of Bacteria and FungiThe Ratio of Actinomycete and Fungi
LZCK140.3 ± 1.5 b52.0 ± 2.1 a60.7 ± 3.5 c78.0 ± 5.8 b11.7 ± 1.0 c
CK225.7 ± 1.2 c31.0 ± 0.6 c52.0 ± 1.0 c82.7 ± 2.4 b16.8 ± 0.4b c
T146.7 ± 1.5 b44.7 ± 1.8 b91.0 ± 1.2 b104.7 ± 3.7 b20.4 ± 0.9 b
T289.0 ± 3.8 a34.0 ± 2.5 c140.3 ± 4.2 a266.4 ± 31.7 a41.9 ± 4.2 a
QXCK138.0 ± 1.5 c68.7 ± 0.9 a92.3 ± 1.2 b55.4 ± 3.0 c13.5 ± 0.1 c
CK227.0 ± 3.0 d21.7 ± 0.9 d75.0 ± 2.3 c123.9 ± 9.6 b34.6 ± 0.4 b
T163.6 ± 1.8 b58.0 ± 2.1 b102.7 ± 4.1 b110.2 ± 6.0 b17.78 ± 1.2 c
T2107.3 ± 1.9 a32.0 ± 1.2 c154.7 ± 6.4 a336.7 ± 17.9 a48.54 ± 3.2 a
TYCK156.0 ± 0.6 c53.7 ± 2.0 a85.0 ± 2.5 c104.6 ± 3.7 c15.9 ± 0.3 b
CK224.3 ± 0.9 d27.3 ± 0.9 c65.3 ± 2.7 c89.0 ± 0.4 d24.0 ± 1.7 b
T166.0 ± 2.1 b48.0 ± 1.2 b114.3 ± 7.7 b137.5 ± 3.1 b23.9 ± 2.2 b
T2114.7 ± 3.7 a31.3 ± 0.9 c169.0 ± 9.3 a365.9 ± 2.5 a54.1 ± 4.0 a
LZ: Laizhou, QX: Qixia, TY: Yiyuan. CK1: replant control, CK2: methyl bromide fumigation, T1: blank carrier treatment, T2: QSB-6 bacterial fertilizer treatment. Numbers followed by the same letter in the columns for each place are not significantly different based on the Duncan multiple range test at p < 5%.
Table
Table 4. Effects of different treatments on microbial diversity in the rhizosphere of young apple trees in three places.
Table 4. Effects of different treatments on microbial diversity in the rhizosphere of young apple trees in three places.
MicroorganismTreatmentMargalef’s IndexShannon’s IndexMcintosh’s IndexBrillouin’s Index
FungiCK110.3 ± 0.1 b3.6 ± 0.0 ab16.6 ± 0.8 a3.0 ± 0.0 a
CK29.9 ± 0.7 b3.5 ± 0.1 ab8.7 ± 0.9 c2.7 ± 0.1 bc
T112.2 ± 0.2 a3.7 ± 0.0 a11.9 ± 0.4 b2.9 ± 0.1 ab
T211.7 ± 0.3 a3.4 ± 0.1 b8.5 ± 0.6 c2.5 ± 0.1 c
BacterialCK111.0 ± 0.2 b3.5 ± 0.0 b12.420.6 b2.8 ± 0.0 c
CK29.6 ± 0.1 d3.3 ± 0.0 c12.6 ± 0.5 b2.6 ± 0.0 d
T112.1 ± 0.1 a3.7 ± 0.0 a12.6 ± 0.4 b3.0 ± 0.0 b
T210.5 ± 0.0 c3.7 ± 0.0 a25.5 ± 1.2 a3.3 ± 0.0 a
CK1: replant control, CK2: methyl bromide fumigation, T1: blank carrier treatment, T2: QSB-6 bacterial fertilizer treatment. Numbers followed by the same letter in the columns for each microorganism are not significantly different based on the Duncan multiple range test at p < 5%.
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Duan, Y.; Zhou, Y.; Li, Z.; Chen, X.; Yin, C.; Mao, Z. Effects of Bacillus amyloliquefaciens QSB-6 on the Growth of Replanted Apple Trees and the Soil Microbial Environment. Horticulturae 2022, 8, 83. https://doi.org/10.3390/horticulturae8010083

AMA Style

Duan Y, Zhou Y, Li Z, Chen X, Yin C, Mao Z. Effects of Bacillus amyloliquefaciens QSB-6 on the Growth of Replanted Apple Trees and the Soil Microbial Environment. Horticulturae. 2022; 8(1):83. https://doi.org/10.3390/horticulturae8010083

Chicago/Turabian Style

Duan, Yanan, Yifan Zhou, Zhao Li, Xuesen Chen, Chengmiao Yin, and Zhiquan Mao. 2022. "Effects of Bacillus amyloliquefaciens QSB-6 on the Growth of Replanted Apple Trees and the Soil Microbial Environment" Horticulturae 8, no. 1: 83. https://doi.org/10.3390/horticulturae8010083

APA Style

Duan, Y., Zhou, Y., Li, Z., Chen, X., Yin, C., & Mao, Z. (2022). Effects of Bacillus amyloliquefaciens QSB-6 on the Growth of Replanted Apple Trees and the Soil Microbial Environment. Horticulturae, 8(1), 83. https://doi.org/10.3390/horticulturae8010083

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