Biocontrol Potential of The Phloridin-Degrading Bacillus Licheniformis XNRB-3 Against Apple Replant Disease

Background: Apple replant disease (ARD) is a common occurrence in many major apple-growing areas worldwide, seriously hindering the development of the apple industry. To avoid the shortcomings of chemical fungicides currently used to control ARD, it is necessary to nd sustainable and effective control methods. Here, an endophytic phloridin-degrading Bacillus licheniformis XNRB-3 was isolated from the root tissue of healthy apple trees, and its control effect on apple replant disease (ARD) and its how to alleviates the pathogen pressure via changes in soil microbiomes were studied. Results: The addition of strain XNRB-3 in Fusarium infested soils signicantly reduced the number of pathogens in the soil, thus resulting in a lower disease incidence, and the relative control effect reached more than 60%. The fermentation broth can also protect the roots of the plants from Fusarium infection. These antagonistic effects were further validated using an in vitro assay in which the pathogen control was related to growth and spore germination inhibition via directly secreted antimicrobial substances and and indirect interspecic competition for nutrients. The antifungal organic compounds in the fermentation metabolites were identied using GC-MS technology. Among them, alpha-bisabolol and 2,4-di-tert-butylphenol had signicant inhibitory effects on many planted pathogenic fungi. Butanedioic acid, monomethyl ester, and dibutyl phthalate can promote the root elongation and lateral root development of Arabidopsis plants. The potential of strain XNRB-3 to control ARD was later validated using microbial fertilizer inoculation in pot and eld experiment. The addition of strain XNRB-3 signicantly promoted the growth of plants, and the activity of enzymes related to disease resistance (SOD, POD, and CAT) was also signicantly enhanced. It also reduced the abundance of Fusarium and the content of phenolic acids in the rhizosphere soil, improved soil microbial community structure and nutritional conditions, and increased soil microbial diversity and activity, as well as soil enzyme activity. Conclusions: The incorporation of strain XNRB-3 in the soil alleviated the damage of soil-borne pathogens to plants by reducing the relative abundance of pathogenic fungi and the content of phenolic acids, and inducing disease resistance of plants. Taken together, B. licheniformis XNRB-3 could be developed into a promising biocontrol and plant-growth-promoting agent. This provides a new management strategy to control ARD. relative control effect seedlings at 33 and 50%, respectively. During test period, plants inoculated sterile distilled water remained healthy. The disease symptoms were the result of articial infection with Fusarium, which successfully re-isolated from inoculated plants the end of the experiment. According to RT-PCR, the size of the Fusarium populations of the two treatments signicantly 21 planting the infected soil. Compared with the CK × 10 5 the size of Fusarium population These results are consistent with the incidence of the aforementioned diseases. We also found that watering the strain XNRB-3 in soil pre-infected with Fusarium can signicantly reduce the abundance of Fusarium in rhizosphere soil. After 5 weeks, the relative control effect was as high as 51%. These ndings are consistent with the results of Cao et al. (2011) showing that the pathogen density in the rhizosphere of cucumber seedlings inoculated with Bacillus subtilis SQR9 was signicantly reduced. This result indicated that strain XNRB-3 can stably colonize the rhizosphere of apple seedlings and provide protection to plants. Similar results were obtained in the PAS staining test. The roots treated with the fermentation broth of strain XNRB-3 did not show symptoms of Fusarium infection, which demonstrated that strain XNRB-3 can colonize the roots of the plant and grow root epidermis, forming a biolm that prevents Fusarium infection and improves the resistance of plants to infection (Duan et al., 2021), Similar results were obtained by Benhamou et al. (1998): seed treatment of tomato with the endophytic B.

Genes related to the biosynthesis of lipopeptides, dipeptides and polyketides were detected by PCR using primers listed in Table S3 and following steps described by Hsieh et al. (2004), Cao et al. (2012), and Hussein et al. (2017). PCR reactions were carried out in a 25 µl reaction volume containing 1 µl genomic DNA, 2.5 µl 10× PCR buffer, 20 mM MgCl 2 , 0.2 mM of each dNTP, 0.5 µM of each primer and 1.25 U Taq DNA polymerase (Takara, Dalian). Ampli cation was performed with a Applied Biosystems 2720 Thermal Cycler (Applied Biosystems Inc., USA) programmed for one cycle of 94°C for 5 min, followed by 35 cycles of 94°C for 30 s, annealing for 30 s at 52°C for ituC, yndJ, bamC and sboA speci c primers, whereas those for srf, srfAB, ituD, fen, fenB and qk were set to 58°C and 72°C for 1 min, and extension at 72°C for 7 min. The ampli cations were analysed by electrophoresis of 5 µl in an 1.5% agarose gel followed by ultraviolet visualisation.

Optimization of fermentation conditions
Seed culture of B. licheniformis XNRB-3 was prepared by inoculating single colony into 100 ml of LB broth (tryptone 10 g, yeast extract 5 g, NaCl 10 g, pH 7.0) and incubated at 37°C for overnight with agitation (180 rpm).

Single factor test
Batch fermentation was carried out in 250-mL Erlenmeyer asks containing 100 ml of fermentation medium (glucose 22 g, trypone 5.4 g, KH 2 PO 4 4 . The density of the bacterial suspensions was adjusted to 1×10 8 CFU ml −1 , The spores of Fusarium obtained refers to the method of Duan et al. (2021). Each suspension (about 50 µL) were then placed on a microscope slide, and kept at 20 ℃ on moistened, sterilized lter paper placed in Petri dishes for 24 h. The germination percentages of conidia (200 conidia for each treatment) were measured using the Nikon BX-51 uorescence microscope. The conidium was considered germinated when the length of the germ tube length was at least equal to the diameter of the conidium. This experiment was repeated three times.

Effect of cell-free culture ltrate on Fusarium hyphae
Refer to the method of Abdelmoteleb et al. (2017) to obtain the cell-free culture ltrate (CFCF) of strain XNRB-3. Bacteria were grown on optimized liquid fermentation medium at 33 ℃, with constant shaking at 191 rpm and after 24 h of incubation to obtain fermentation broth (FB), FB were removed by centrifugation at 10,000 rpm for 10 min at 4 ℃. The supernatant (extracellular medium) was passed through a 0.22-µm Nylon66 microporous membrane to obtain CFCF. Effect of cell-free culture ltrate on Fusarium hyphae refers to the method of Duan et al. (2021). The samples were sent to Keshang Biotech Co., Ltd. for scanning electron microscope observation.

Plant growth promoting activities
Ability of strain XNRB-3 to promote plant growth was tested with reference to the method in Table S4. FB of B. licheniformis XNRB-3 were centrifuged at 13,400 g for 10 min. The supernatant (extracellular medium) was removed and frozen at 4 ℃ for analysis of amino acids. Amino acids were pre-column derivatized with PITC and were separated and quanti ed under the optimum condition (running buffer 30 mM phosphate and 3 mM β-CD at pH 7.0; voltage of 20 kV) by Biochrom 30 + amino acid analyzer (Biochrom, U.K) attached to a PA800 high performance capillary electrophoresis (HPCE) system equipped with an on-column ultra violet detector system (Genstech Biotechnology Co., Ltd, Shanghai, China) refer to the method of Ren et al. (2012). The capillaries were Bare Fused-Silica Capillary-50 µm ID, 375 µm OD, 67 cm (pkg of 3) (Shkmsw Biotechnology Co., Ltd, Shanghai, China). Individual amino acid concentrations were calculated by comparing the speci c amino acid peak area to a standard curve for that amino acid (Ren et al., 2012).
Phytohormones extraction and quanti cation by gas chromatography-mass spectrometry/selected ion monitoring (GC-MS/SIM) (Shimadzu, Japan) refer to the method of Shahzad et al. (2019). Brie y, B. licheniformis XNRB-3 was grown in optimized liquid fermentation medium for seven days. After seven days, the pure culture ltrate in which B. licheniformis XNRB-3 was grown was supplemented with [D5]-IAA, [2H2] GA, [(±)-3,5,5,7,7,7-d6]-ABA] as an internal standard, extracted, and subjected to GC-MS/SIM for determination and quanti cation. Further, the GC/MS used for quanti cation was equipped with a HP-5 capillary column HP-5 (30 m length, 0.25 mm ID, 0.25 µm lm, 325 ℃ maximum temperature) and used He (99.999 %,) was as the carrier gas with a head pressure of 30 kPa, an injector temperature of 200°C, and an ionizing voltage of 70 eV. Phytohormones concentrations were calculated from ratios of peak area of sample to a corresponding internal standard.

Tests of microbial growth and phenolic degradation
Measure the absorbance value of MSM solution with phloridin (10 mmol L −1 ) at each wavelength to determine that the maximum absorbance wavelength was 280 nm (Table S5), Prepare the MSM solution with a total of 11 concentrations of 0-10 mmol L −1 . Use MSM solution with 0 mmol L −1 phloridin as the reference solution for blank calibration. Determine the absorbance value at 280 nm. Take the absorbance value as the ordinate and the phlorizin concentration as the abscissa to make a standard curve ( Figure 2P).
Tests of microbial growth and phenolic acid degradation in MSM solution refer to the method of Wang et al. (2021), with some modi cations. Strain XNRB-3 was inoculated into MSM with 0~10 mmol L −1 phloridin and cultured at 33℃ in the dark to determine the maximum tolerance of phloridin ( Figure 2N). Strain XNRB-3 was inoculated with 6 ml of liquid fermentation medium in a 15 ml plastic tube with cover, followed by overnight incubation at 33°C. After centrifugation at 2500 g for 5 min, the supernatant was discarded and the pellet was diluted to an OD 600 value of 1.0 using ddH 2 O. Afterwards, 0.1/0.2 ml of the resuspended isolates was transferred to 10 ml MSM solution with phloridin (3 mmol L −1 ) and incubated for 60 h in the dark at 33°C with shaking (191 rev min −1 ). 1 ml of the bacterial suspension was taken from each treatment and was centrifuged at 13 000 g for 5 min, and the supernatant to detect OD 280 , Converted to the concentration of phloridin according to the standard curve. Degradation rate of phloridin = (phloridin concentration in uninoculated culture solution-phloridin concentration in inoculated culture solution)/phloridin concentration in uninoculated culture solution×100%.
To test the isolate's ability to utilize other phenolic acids, Strain XNRB-3 was inoculated into MSM solution with 0.5 g L −1 of CA, FA, BA or PHBA, and their growth was tested according to the previously described method, and 0.2 ml of the suspension was transferred to 10 ml of MSM solution with 0.5 g L −1 of CA, FA, BA or PHBA. After incubation for 12, 24, 36, 48 and 60 h at 33°C with shaking (190 rev min −1 ), 1 ml of the bacterial suspension was taken from each treatment and was centrifuged at 13 000 g for 5 min. The pellet was resuspended with 1 ml ddH 2 O to detect OD 600 , and the supernatant was mixed with an equal volume of methanol. Then, 0.1ml of the mixed solution was added to 1.9 ml of 50 % methanol solution, and was sterilized by ltering through 0.22 µm pore-size lter membranes prior to HPLC analysis. The control used was MSM solution of phenolic acids but without bacteria, all assays were performed in triplicate.
The concentrations of phenolic acids were detected based on peak areas using external standards using an UltiMate 3000 HPLC system (Dionex, USA). All separations were performed using Symmetry® C18 column (4.6 mm × 150 mm, 5.0 µm; Waters, Milford, MA, USA). Mobile phase solutions were 0.1% methanoic acid + 2 % methanol (A) and acetonitrile (B). The gradient elution composition used was as follows: 0 min, 96 % A plus 4 % B 10 min; 10 % A plus 90 % B 16 min; 96 % A plus 4% B 30 min; with a ow rate of 1.0 ml min −1 , an injection volume of 10 µl, a column temperature of 20°C and UV detection at 280 nm.

Soil treatment with microbes and analysis of phenolic acid degradation
The soil obtained from a 31-year-old apple orchard in Manzhuang Town, Taian, China (117.081039 longitude, 36.06682 latitude) was dried at 60°C for 6 h and and was passed through a 0.84-mm sieve. 30 µg g −1 of Phloridzin, 10 µg g −1 of CA, 90 µg g −1 of BA, 100 µg g −1 of FA, and 20 µg g −1 of PHBA was added to the dried soil. The soil was dried again at 60°C for 2 h. 200 mL with OD 600 =1.0 of bacterial suspension was added to 1kg of soil; sterile water was used as control. Each treatment consisted of three replicates. After incubation for 3, 6 and 9 days at 25°C/20°C (16 h/8h, light/dark), and ddH 2 O was added to keep the soil moist. 60 g of soil was obtained from each pot for HPLC analysis (Zhang ZY et al., 2010).
Analysis of phenolic degradation was conducted using the method of Yin et al., (2013), with few modi cations. A sample of dry soil (60 g) was passed through a 12-mesh sieve and mixed with diatomaceous earth, then placed into a 100-ml extraction tank. ASE 350 Fast Solvent Extractor (Dionex, 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 with 2 cycle times, a purge volume of 60 %, and a purge time of 90 s. Then 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, then reconstituted with 1 ml methanol and passed through a 0.22-µm organic phase lter membrane for HPLC analysis.
The HPLC analysis procedure followed that described by Xiang et al. (2021). An UltiMate 3000 HPLC system (Dionex, USA) was used for quanti cation with an Symmetry® C18 column (4.6 mm × 250 mm, 5.0 µm; Waters, Milford, MA, USA), and a column temperature of 30°C. Mobile phase A was acetonitrile, and mobile phase B was water (adjusted to pH 2.8 with acetic acid). The ow rate was 1.0 mL min − 1 , the automatic injection volume was 10 µL, and the detection wavelength was 280 nm. HPLC solvents are purchased from Burdick &Jackson Inc. (Muskegon, MI). The retention time was used for qualitative analysis, and the peak area external standard method was used for quanti cation 2.9 Root Colonization Assay To investigate root colonization refer to the method of Yu et al. (2011), a spectinomycin-and rifampin-resistant mutant of XNRB-3 was obtained by inoculation of XNRB-3 into LB (Luria-Bertani) medium containing gradually increasing concentrations of spectinomycin and rifampin (20,50,75,100,150,200,250, and 300mg mL −1 ), and a mutant resistant to 300mg mL −1 spectinomycin and rifampin was used in the study ( Figure 2O). In March 2017, Malus hupeheusis Rehd. seeds were strati ed at 4°C for about 30 days. After the seeds had become white, they were sown in nutrient bowls lled with seedling substrate (organic matter ≥45%, pH 5.8-6.5, EC 1.0-1.5 ms/cm). When the seedlings had grown six true leaves, disease-free plants of similar size were selected for use in subsequent experiments.
The ability of B. licheniformis XNRB-3 to colonize roots was determined according to the method of Sanei and Razav. (2011), with some modi cations. Malus hupeheusis Rehd. Seedlings were carefully uprooted from the substrate, their roots thoroughly washed in tap water without intentional wounding, and dipped in a bacterial suspension (1×10 8 CFU ml -1 ) for 10 min. For the control treatment, plants were treated similarly except that roots were dipped in 10 mM MgSO 4 .7H 2 O. Plants were then transplanted (one per pot) into pots (AC 140) lled with an autoclaved (121°C, 1 h, twice on consecutive days) soil mixture (vermiculite/soil/vermiculite, 1:3:1, vol/vol/vol). There were four replicated plants for each treatment in a randomized complete block design. Plants were incubated under greenhouse conditions. The air temperature during the experiment uctuated between 18°C and 33°C. Plants were watered as needed. To determine colonization of root tissue by bacteria, plants were uprooted delicately from pots and the root systems were thoroughly washed under running tap water, dried with sterile lter paper, and cut into 1-cm long pieces. For each plant, samples of 2 g of root pieces were surface-deinfested in 1% NaOCl for 3 min, washed three times in sterile distilled water, and ground in 10 ml of 10mM MgSO 4 .7H 2 O using an autoclaved pestle and mortar. Serial dilutions of the macerates were plated onto LB agar (LB supplemented with spectinomycin and rifampin at 300mg mL -1 ) and incubated at 37°C for 48 h. Then, bacterial colonies were counted and bacterial populations were expressed as colony-forming units (cfu)/g of fresh root tissue.

The protective effect of strain XNRB-3 on plant roots
According to the method of Duan et al. (2021), the root system of M. hupehensis Rehd. seedlings was treated with the FB of strain XNRB-3 and the spore suspension of Fusarium. The roots were placed in a sterile centrifuge tube that contained 2.5 % glutaraldehyde xative and sent to Keshang Biotech Co., Ltd. for para n sectioning and Periodic Acid Schiff (PAS) staining (Shao et al., 2019).

Biological control of the strain XNRB-3
The ability of strain XNRB-3 to control ARD was investigated in an Fusarium-infested sterilized soil following the method described by Wu et al. (2019), with some modi cations. Treatments included: a sterilized soil inoculated with sterile distilled water as a negative control, a sterilized soil inoculated with Fusarium as a positive control, and sterilized soil inoculated with Fusarium and the strain XNRB-3. Each treatment included 15 M. hupeheusis Rehd. Seedlings. The spore suspension of Fusarium (10 6 spores mL -1 ) was rst drenched into the sterilized soil, followed by a suspension of strain XNRB-3 (10 9 CFU ml -1 ). The nal concentration of Fusarium (10 5 spores g -1 ) and strain XNRB-3 (10 8 CFUg -1 ) in the growth substrates. Plant seedlings were then transplanted in the substrate trays (AC140: the outer diameter is 12.5 cm, the inner diameter is 11 cm, and the height is 9.5 cm), and then grown at 16 hr light/8 hr dark at 28°C. Plants were watered as required for plant growth and disease development.
Each pot contained one M. hupehensis seedling, and all pots were arranged randomly with three replicates per treatment and 15 pots per replicate. Disease severity was estimated over the course of 5 weeks starting 1 week after inoculation. Scoring of wilting symptoms on a 0-4 scale was performed using the criteria developed by Azabou et al. (2020). 0 = healthy plant or plant without symptoms; 1 = 1-33 % of plant tissue affected by chlorosis, leaf and shoot necrosis, or defoliation; 2 = 34-66 % affected tissue; 3 = 67-100 % affected tissue; and 4 = dead plant. The percentage of dead plants (PDP) was measured to estimate wilt severity and the ability of plants in different treatment groups to recover from the disease. The area under the disease progress curve (AUDPC) The cell-free culture ltrate was used to assess in uence of extracellular metabolites on Fusarium radial growth as described by Azabou et al. (2020), with some modi cations. Bacterial culture was grown in a shaker incubator at 100 rpm for 72 h. Culture was then centrifuged at 10,000 rpm for 5 min at 4°C. The supernatant was collected and ltrated through 0.22 µm membrane lters. Cell-free culture ltrate was added to a warm PDA medium (55°C) to nal concentrations (from 5-75%). PDA plates without culture ltrate were used as controls.Fungal mycelial plugs of 5 mm diameter were placed centrally in amended media and incubated at 25°C until negative control growth covers the whole surface of the plate. Growth inhibition of the pathogen was measured using the same formula previously described.

Identi cation of extracellular metabolites by GC-MS
The fermentation supernatant of strain XNRB-3 was extracted with ethyl acetate, and then concentrated under reduced pressure with Rotary Evaporator N-1300D-WB (Tokyo, Japan) to obtain a crude extract. A small amount of methanol was added to dissolve extracts, and passed through a Nylon66 0.22-µm lter membrane, stored in a refrigerator at 4 ℃. The compounds in the active extract were identi ed by gas chromatography-mass spectrometry (GC-MS) followed by an NIST17 database search. The GC-MS analysis was performed on a GCMS-QP2010 Plus instrument (Shimadzu, Japan). The peak area normalization method was used to calculate the relative content of each component. The chromatographic and mass spectrometry conditions according to the method of

Veri cation of synthetic compounds against plant fungal pathogen
Among the identi ed extracellular metabolites, 16 standard compounds were purchased from the reagent company (Table S6). The antifungal activity of the standard compounds was assessed using the I-plate system described by by Yuan et al. (2012). The I-plates added with methanol or distilled water were used as control. The colony diameter of plant fungal pathogen was recorded after 7 days incubating. The experiment was repeated three times.

Plant growth promotion activities of synthetic compounds
The plant growth promotion activities of the 16 compounds were measured by the modi ed method described by Wu et al. (2019). The synthetic compounds were diluted separately in alcohol, and 20 µl of the resulting suspension was applied to a sterile lter paper disk on the other side of the I-plate. A total of 10 µg, 100 µg, 500 µg, and 1,000 µg doses of each synthetic compounds were tested. Each treatment was repeated for three times. The fresh weight of the Arabidopsis thaliana Col-0 seedlings was measured after 10 days.

Carrier characteristics
15 carriers purchased from different Chinese commercial enterprises through taobao were used as formulation carriers for strain XNEB-3. The selected properties of these carrier candidates are listed in Table S7. The carriers were dried to a moisture content of 5 % in an oven (Shangdong, China) at 80°C for 24 h, nely ground in a hammer mill to pass through a 1-mm screen, and stored at room temperature for further studies.
The effect of the carrier as a substrate on the survival of strain XNEB-3 was evaluated using plastic bottles equipped with a 0.22-µm lter membrane in the cap to allow air exchange refer to the method of Wei et al. (2015), with some modi cations. 20 mL of fermentation broth of strain XNRB-3 was mixed with 100 g of each carrier (sterilization at 115°C for 30 min). Carriers treated with 20 mL of aseptic fermentation broth were used as controls. Each treatment included three replications (three bottles). In addition, these plastic bottles were covered with black plastic bags to avoid the in uence of light on the survival of strain XNRB-3. The bottles were stored at 25°C and periodically sampled at 0, 10, 20, 30, 60, 90, 120, and 180 days post-inoculation. The population of strain XNRB-3 in each carrier at each sampling time point was determined by the plate count method using LB medium.
Four key factors including Inoculation amount, pH, Temperature, and Rotating speed were selected to carry out the Box-Behnken test design with four factors and three levels. When the above fermentation conditions were optimized, except for the test single factor as a variable, the other conditions were unchanged, and the population number of strain XNRB-3 was determined for 180 days.   (Ma et al., 2018). Plants received normal watering and manure management, and samples from each treatment were obtained on July 15, August 15, and September 15, 2017. Three pots from each treatment were randomly selected, and the soil was removed from the surface layer and the pot.
Soil impurities were removed using a 2-mm sieve, and the soil samples were stored in separate sealed pockets.

Field experimental trials
In order to test the potential of XNRB-3 bacterial fertilizer to prevent and control ARD under eld conditions. The eld test was carried out in Wangtou Village, Laizhou City (Shandong China, Lon:119.814701, Lat:37.095159). Physicochemical properties of the tested soil are presented in Table S8. After the apple orchard was rebuilt, severe ARD occurred, the growth of fruit trees was weak, and the survival rate was less than 50%. In March 2020, 28-year-old trees were removed from the orchard, and the replanted orchard was simultaneously established. The apple seedlings used in the experiment were two-year-old grafted seedlings. The rootstock and spike combination was Yanfu 3/T337. The grafted seedlings had a stem thickness of about 10mm and a xed stem to 1.4 m.
They were purchased from Laizhou Nature Horticultural Technology Co., Ltd. The row spacing of the plants is 1.5 m×4 m, and the tree shape is pruned to a spindle shape. The production of XNRB-3 bacterial fertilizer is as above.
The experiment consisted of 4 treatments: 28-year-old orchard soil (CK1), 28-year-old orchard soil fumigated with methyl bromide (CK2), bacterial fertilizer carrier treatment (T1), and XNRB-3 bacterial fertilizer treatment (T2). Dig a planting hole of 80 cm 3 according to the row spacing, mix the bacterial manure carrier and XNRB-3 bacterial manure with soil and back ll. The amount of application for each young tree is controlled at 1kg, and 20 trees are treated for each. A new application will be carried out at the beginning of the second spring. All indexes were measured on 15 July and 20 October, 2020, 2021. The surface soil was removed, and multiple samples within a radius of 0.5 m around the apple saplings in different treatments were collected. Each treatment was repeated 3 times. Impurities such as the root system and weeds, soil animals and stones were removed, after which the samples were divided into 3 portions: one part was stored in a refrigerator at 4 ℃ for the determination of the soil microbial structure; one part was naturally dried for soil enzyme activity, soil nutrients, and soil phenolic acid detection; one part was stored in a refrigerator at -80 ℃ for DNA extraction and perform real-time uorescence quantitative analysis (Sheng et al., 2020). catalase, and sucrase activities were assayed by the colorimetric method, the disodium phenyl phosphate method, the permanganate titration method, and the 3,5-dinitrosalicylic acid (DNS) method, respectively (Guan, 1986). All enzyme activities are expressed based on soil dry weight.

DNA extraction and Real-time quantitative analysis
Sieved fresh soil (5.0 g) was used for DNA extraction with the PowerMax soil DNA isolation kit (MO BIO Laboratories Inc., Carlsbad, CA). Quantitative PCR ampli cations for standard and environmental DNA samples were performed with a total volume of 20 µl in each reaction using the SYBR ® Premix Ex Taq™ (TaKaRa, Japan) and a CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad, USA) following the method of Duan et al. (2021). The concentration of plasmid DNA was measured and converted to copy concentration using the following equation as described by Whelan et al. (2003): DNA (copy)= [6.02×1023 (copies mol -1 )×DNA amount (g)]/[DNA length (bp) ×660 (gmol -1 bp -1 )]. The primers and annealing temperatures are presented in Table S2. Sterile water was used as a negative control to replace the template. All real-time PCR reactions were done in technical triplicates such that each treatment was analyzed nine times.

Terminal-restriction fragment length polymorphism (T-RFLP) analysis
DNA was ampli ed using the universal primers 27F-FAM/1492R and ITS1F-FAM/ITS4R (Table S2)  (1) AWCD=[Σ(Ci-R)]/n. In formula (1), Ci is the absorbance value of each reaction well at 590 nm, R is the absorbance value of the control well, and n is the number of wells.
H′=-ΣPi.ln(Pi). The Pi represents the ratio of the absorbance value in the i th (1 to 31) well to the total absorbance values of all wells.
(3) Shannon evenness index (E): E=H′/Hmax=H′/lnS, S represents the total number of utilized carbon sources (31 carbon sources), the number of wells that vary in color.
(4) Simpson diversity index (D) re ects the most common species of the community, and was often used to assess the dominance degree of microbial community. D=1-Σ(Pi) 2 (5) McIntosh index was used to measure the homogeneity degree of the community.
U=√Σni 2 . ni was the relative absorbance of the i th hole (Ci-R). 3 Results

Isolation and identi cation of bacteria for biocontrol activity
The bacterium XNRB-3 was isolated from the root system of a healthy fruit tree in a replanted orchard in Jincheng City, Shanxi Province. This bacterium had the strongest inhibitory effect on plant pathogenic fungi, with an inhibition rate of more than 70%. F. oxysporum and Alternaria alternata had the strongest inhibition rates, reaching 84.52% and 84.59%, respectively ( Figure 1C).
Strain XNRB-3 was cultured on LB agar at 37 ℃ for 24 h; the surface of the colony was white with irregular edges, and the surface was at to convex, smooth, and opaque ( Figure S2 A). It formed bio lms when grown in liquid medium. Inspection under a uorescence microscope (100×/1.30 oil lens) revealed that individual bacteria were long and rod-shaped; they also formed ovoid spores ( Physiological and biochemical tests revealed that strain XNRB-3 could produce hydrogen peroxide and hydrogen sul de, hydrolyze starch, reduce nitrate, and use sucrose. The results of the contact enzyme, Voges-Proskauer reaction, and methyl red reaction tests were positive, whereas the results of the indole enzyme, arginine bihydrolysis reaction, citrate enzyme, and glucose fermentation reaction tests were negative. According to Bergey's Manual of Systematic Bacteriology (2nd edition) and the Common Bacterial Identi cation Manual, strain HSB-2 matched the physiological and biochemical properties of B. licheniformis (Table S9).
Tests of the carbon source utilization and chemical sensitivity using a Biolog GEN III MicroPlate™ revealed that strain XNRB-3 can utilize 32 carbon sources such as dextrin, D-maltose, and pentabiose and is sensitive to pH 6, 1% NaCl, 4% NaCl, and 8% NaCl conditions. The strain XNRB-3 belonged to B. licheniformis, which was identi ed based on carbon utilization (Table S10- 11). The values of PROB, SIM, and DIST were 0.689, 0.689, and 4.698, respectively, indicating that reliable matching results were obtained.
Approximately 555-938 bases were sequenced for rpoB and gyrA, 1,455 bases for 16S rDNA, and 1,136 bases for gyrB. Congruency analysis revealed no con ict between the 16S rDNA, gyrA, gyrB, and rpoB sequences; the sequences were therefore combined. Maximum likelihood (ML) analysis of identities based on the four gene sequence alignments revealed that strain XNRB-3 had the highest homology with Bacillus licheniformis ( Figure S3). In sum, strain XNRB-3 was identi ed as B. licheniformis.

Identi cation of antibiotic biosynthesis genes from XNRB-3
To determine the mechanisms underlying the effects of the antagonistic strain, polymerase chain reaction (PCR) was used to screen XNRB-3 for genes involved in the biosynthesis of antibiotics. Amplicons of the expected sizes detected included yndJ (involved in biosynthesis of Yndj protein), qk (involved in subtilisin synthesis), bamC (involved in bacillomycin synthesis), ituD (involved in iturin A synthesis), fen and fenD (involved in fengycin synthesis), and srf and srfAB (involved in surfactin synthesis) ( Figure S4).

Optimization of liquid fermentation conditions
The results of single-factor tests showed that the main carbon source affecting the growth of strain XNRB-3 was sucrose, the main nitrogen source was beef extract, and the inorganic salts were MgSO 4 , KH 2 PO 4 , and KCl (Figure S1 D,G,H). According to the orthogonal test results (Table S14), the organic salt that had the strongest effect on the growth of strain XNRB-3 was MgSO 4 , followed by KH 2 PO 4 and KCl. According to the K value, the optimal combination was F4I3E3 (MgSO 4 1.0 g L −1 , K 2 HPO 4 1.5 g L −1 , and KCl 1.0 g L −1 ). According to the results of the single-factor experiment ( Figure S1), the Plackett-Burman experiment was performed using the experimental factors and levels in Table S15, and Minitab 17 software was used for multiple regression analysis. The optimal equation with OD 600 as the response value was OD 600 = 0.
+0.00465G+0.001711H+0.000272I. According to the size of the P value, the key factor affecting OD 600 was A (temperature), followed by B (rotating speed), G  temperature, and rotating speed) and three levels (Table S18) where Y was the inhibition zone diameter (mm), A was sucrose, B was beef extract, C was temperature, and D was rotating speed. Analysis of variance and signi cance test of the regression model were conducted (Table S19, Table 1), The F value of the regression model was 94.09 (P<0.0001), which indicated that the regression model was robust. The coe cient of determination of the model was R 2 =0.9895 and R 2 Adj =0.9790, indicating a good t. Therefore, the regression model could be used to analyze and predict the abundance of the strain XNRB-3. In the regression model, the primary term C, D and the secondary term AC, CD signi cantly affected the diameter of the inhibition zone (P<0.0001); the signi cance of A, B, C, D was the same based on the results of the Plackett-Burman test.   Table 2). The F value of the regression model was 102.13 (P<0.0001), indicating that the regression model was robust. The coe cient of determination of the model was R 2 =0.9903 and R 2 Adj = 0.9806, indicating a good t. Therefore, the regression model could be used to analyze and predict the abundance of B. licheniformis XNRB-3. In the regression model, the effects of the rst term A, B, C and the second term BC on the diameter of the inhibition zone were signi cant (P<0.0001). The interactions between pH and inoculation amount, temperature and pH, and rotating speed and pH had the most signi cant effect on the population of strain XNRB-3. The optimal solid fermentation conditions for B. licheniformis XNRB-3 were predicted by the regression model: inoculation amount 20.25%, pH 8.25, temperature 39.23 ℃, and rotating speed 199.75 rpm. The predicted maximum theoretical value of the population of strain XNRB-3 was 8.91E+08. To verify the predicted value, three parallel experiments were performed using the optimized fermentation conditions, and the diameter of the inhibition zone was (22.03±0.24) mm; the error from the theoretically predicted value (8.91E+08) was only 0.86%, indicating that there was a good t between the predicted value and the measured value and that the optimized model was robust.

Determination of antifungal activity
Microscopic observations of hyphal and spore morphology revealed that the control Fusarium mycelium was uniform in thickness and slender with fewer branches; it was full of spores, its structure was complete, and its growth was strong ( Figure 1A A-D). The mycelia treated with the cell-free culture ltrate were irregularly reticulated, uneven in thickness, shrunken ( Figure  Fermentation broth, cell-free culture ltrate, and crude extract signi cantly inhibited the spore germination of Fusarium spores, and the spore germination rate decreased by more than 60% (Figure 1A B, E). The antifungal metabolites produced by XNRB-3 were extracted from 3-day-old cell-free culture ltrates. As the concentration of extracellular metabolites increased, the inhibitory effect on the growth of Fusarium became more pronounced. At higher concentrations (25%, 50%, and 75%), the inhibition rate reached more than 60% ( Figure 1D).

Identi cation of antifungal compounds
Our results showed that a 25% concentration of bacterial cell-free culture ltrate of B. licheniformis XNRB-3 showed high activity against fungal pathogens.

Phloridin degradation ability of strain XNRB-3
The phloridzin utilization e ciency by strain XNRB-3 in MSM solution was high ( Figure 2Q). After culture for 60 h, the degradation rate was 60.75% when the amount of inoculum added was 1%, and the degradation rate was 68.83% when the amount of inoculum added was 2%. The ability of strain XNRB-3 to degrade phloridin increased as the amount of inoculum added increased. Strain XNRB-3 could effectively utilize CA, BA, FA, and PHBA (Table S23), and the degradation rates ranged from 45.65-69.20%. Strain XNRB-3 could e ciently degrade phloridzin, CA, BA, FA, and PHBA in soil ( Table 4). The content of phloridzin in the soil decreased to 2.9292 µg g −1 at 9 days after inoculation. The content of BA and PHBA in the soil decreased to 18.4953 and 5.2882 µg g −1 , respectively, at 9 days after inoculation. FA and CA concentrations in the soil decreased to 13.3669 and 0.3785 µg g −1 at 9 days after treatment, respectively, which was only 13.31% and 3.63% of the control concentration. Table 4 Phenolic acids degradation of strain XNRB-3 in soil. The soil samples were added with Phloridzin, Cinnamic acid (CA), Ferulic acid (FA), Benzoic acid (BA), and P-hydroxybenzoic acid (PHBA) (µg g −1

The protective effect of strain XNRB-3 on plant roots
Strain XNRB-3 can form a thick bio lm in a static medium. After 14 days of planting, the population of strain XNRB-3 colonized on root tissue was approximately 7.30 × 10 6 CFU g -1 . The population sharply decreased to 6.47 × 10 4 CFU g -1 after 35 days (Figure S7 B). The total number of strain XNRB-3 cells measured on the LB medium with antibiotics was signi cantly higher than that on the roots of the control plants. Observation of plant root sections revealed that the root system was mainly composed of three parts from outside to inside: the epidermis, the cortex (the outer cortex, the cortical parenchyma, and the Kjeldahl belt), and the vascular column (the central sheath, phloem, and xylem). Root epidermal cells infected by Fusarium were deformed, broken, detached, and/or irregularly arranged. Fungal hyphae invaded the cortical cells through the intercellular layer and then entered the vascular column. New hyphae were visible close to the cell wall, and mature hyphae were scattered in the cells. There was also a large amount of cell contents (viscous substances and starch granules) in the cortical cells and vascular column (Figure 8, Figure S7A). The epidermal and cortical cells treated with both strain XNRB-3 and Fusarium appeared slightly shrunken and ruptured, and the conidia and hyphae of Fusarium were attached only to the root epidermal cells ( Figure 8A-D). The root systems from the control treatment were complete and neatly arranged (Figure 8, Mock).

Disease severity assessment
In the greenhouse test, the symptoms of Fusarium wilt appeared 7 days after the plant seedlings were transplanted into the infected soil. The incidence of Fusarium wilt increased rapidly over the next 28 days, and the disease severity index increased progressively over the experimental period and ultimately reached 4.00±0.00; a reduction in disease progress was noted in plants treated with strain XNRB-3 (Table 5). In the fth week, the disease index and relative control effect of plant seedlings inoculated with strain XNRB-3 were both stable at 33 and 50%, respectively. During the test period, plants inoculated with sterile distilled water remained healthy. The disease symptoms were the result of arti cial infection with Fusarium, which was successfully re-isolated from inoculated plants at the end of the experiment. According to RT-PCR, the size of the Fusarium populations of the two treatments signi cantly increased after 21 days of planting in the infected soil. Compared with the CK treatment (5.50 × 10 5 copies/g soil), the size of the Fusarium population treated with strain XNRB-3 (2.37 × 10 5 copies/g soil) was signi cantly reduced ( Figure S7 C). These results are consistent with the incidence of the aforementioned diseases. The same results were obtained in the outdoor pot experiment. The biomass of apple plants treated with strain XNRB-3 (T2) in September was signi cantly higher than the biomass of apple plants in CK1, and the plant height, ground diameter, fresh weight, and dry weight were increased by 88.07%, 61.28%, 181.35%, and 140.44%, respectively, which were second only to the methyl bromide fumigation treatment. (Figure S8). Treatment with strain XNRB-3 also signi cantly enhanced the root growth of apple plants ( Figure 7E, I, J, L, M). In September, the plants had grown considerably, and the root length, surface area, number of tips, and number of forks were signi cantly lower in CK1 than in CK2 and T2. The length, surface area, number of tips, and number of forks were 1.73, 2.62, 5.67, and 2.65 times higher in T2 than in CK1. The root respiration rate and the SOD, POD, and CAT activity increased from July to September in T2 and CK2 ( Figure 7E-H, K, N
The activity of urease, phosphatase, sucrase, and catalase increased steadily in T1 and T2 in July and October 2020, 2021 relative to CK1. The soil enzyme activity decreased in the rst year of the fumigation treatment; it then continued to increase and increased most signi cantly in T2 ( Figure 10G-J).In October 2020, 2021, the urease activity was 1.65-fold and 1.64-fold higher, the phosphatase activity was 1.56-fold and 1.93-fold higher, the sucrase activity was 2.03fold and 2.34-fold higher, and the catalase activity was 1.69-fold and 1.87-fold higher in T2 than in CK1, respectively. After two years of applying strain XNRB-3, the physical and chemical properties of plant rhizosphere soil were signi cantly improved. Compared with CK1, organic matter, total nitrogen, total phosphorus, total potassium, available potassium, available phosphorus, NH 4 + -N, nitrate nitrogen, and soil pH increased by 228.54%, 245.25%, 242.29%, 30.53%, 327.89%, 128.64%, 56.05%, 239.61%, and 7.43% respectively, in T1. Various nutrient indexes of the soil were also increased in T1, indicating that the addition of organic amendments could enhance the nutrient conditions of the soil.
In July and October 2020, 2021, the number of soil bacteria in the XNRB-3 bacterial fertilizer treatment (T2) increased signi cantly, and compared with CK1, the number of soil bacteria was increased by 107.21%, 45.13%, 100.79%, and 113.08% in T2, respectively ( Figure 10C). There were signi cant differences in the number of soil fungi between different treatments. Compared with CK1, the number of soil fungi in CK2, T1, and T2 was signi cantly reduced, the number of soil fungi in T1 was signi cantly higher than that in T2, and the effect of T2 was similar to the fumigation treatment. In October 2020, 2021, the number of soil fungi in CK2 and T2 was reduced by 58.55%, 50.26% and 62.74%, 52.83% compared with CK1, respectively ( Figure 10D). The number of actinomycetes and the ratio of bacteria/fungi in the soil were signi cantly higher in T2 than in CK1 ( Figure 10E-F).
The qPCR results showed that the abundance of Fusarium was signi cantly reduced in July and October 2020, 2021 in CK2 and T2 compared with CK1 and T1 ( Figure 10K-N) 3.13 Effect of strain XNRB-3 on the soil microbial community AWCD was used as an indicator of soil microbial activity. Variation in AWCD with incubation time is shown in Figure 11 (I, L). AWCD increased as the incubation time extended for all treatments in October 2020, 2021. Soil with strain XNRB-3 (T2) had a higher AWCD value than soil in other treatments, which indicated that the addition of strain XNRB-3 increased the activity of microorganisms. After 120 h, the AWCD values of T1 and CK1 were similar, and the AWCD value was lower in CK2, indicating that fumigation can inhibit the activity of microorganisms for a longer period.
Biolog-ECO plates have six categories of carbon sources: carbohydrates, carboxylic acids, amino acids, polymers, phenolic compounds, and amines (Guo et al., 2015). In October 2020 ( Figure 11J-K), the OD value of three types of carbon sources (polymers, miscellaneous, and amino acids) increased signi cantly in T1 and CK1 relative to CK1. In contrast, amines/amines and carbohydrates were signi cantly reduced in T2. The effect of strain XNRB-3 treatment (T2) on the utilization rate of the four substrate groups (polymers, miscellaneous, carboxylic acids, and amino acids) was stronger compared with the other treatments.
The utilization rate of these four substrate groups was 6.11%, 0.97%, 15.41%, and 10.24% higher in T2 than in T1, respectively. In October 2021( Figure 11M-N), the changes in the utilization rate of microbial substrates under different soil treatments varied starting in 2020. Compared with CK1, the OD values of the six types of carbon sources increased signi cantly in T2. With the exception of miscellaneous, the OD values of amines/amines, carboxylic acids, carbohydrates, polymers, and amino acids increased by 40.79%, 42.17%, 24.45%, 10.56%, and 7.91% in 2021, respectively, compared with 2020. These ndings indicated that the soil microorganisms treated by strain XNRB-3 mainly use polymers, carboxylic acids, and amino acids. The utilization rate of the six types of carbon sources in CK2 was signi cantly lower compared with the other treatments; this showed that fumigation greatly affected the soil microbial community.
Principal component analysis and cluster analysis showed that the soil microbial community structure in T2 and CK2 signi cantly differed from that in CK1 ( Figure 11A-H). The soil bacterial community structure in T2 differed signi cantly from that in other treatments ( Figure 11G-H), and the soil fungal and bacterial communities of T1 and CK1 were similar (Figure 11C-D, G-H). The Margalef, Mc intosh, Brillouin, Simpson and Shannon Index re ect the richness and diversity of soil microbial communities (Table S24- Strain XNRB-3 can also produce enzymes that dissolve fungal cell walls (cellulose, pectinase, β1,3-glucanase, chitosanase, and protease), antifungal compounds (2,4-di-tert-butylphenol and alpha-bisabolol), and low molecular weight metabolites (HCN), which limits the growth of pathogens and protects plants from phytopathogenic fungi. Among them, cellulases and pectinases are important for the intracellular root colonization of PGP bacteria, as these are hydrolytic enzymes with the ability to degrade cellulose/pectin material of the plant cell wall (Verma et al., 2001). This feature helps strain XNRB-3 better colonize the root system and continue to produce substances that are bene cial to plant growth to promote the development of the root system, which enhances the growth of the aboveground parts of plants. The above results indicate that the endophytic bacterium XNRB-3 has high potential to be used as a biofertilizer and biopesticide and could aid the development of a sustainable, safe, and effective agriculture system.
The production and transportation of BCAs are essential for successful biological control under eld conditions (Thangavelu et al., 2004). An appropriate carrier can support the survival of BCAs while inhibiting the growth of target pathogens, thereby improving the performance of BCAs for plant disease control (Ling et al., 2010;Wei et al., 2015). The selection of a suitable vector for strain XNRB-3 is thus necessary for the successful application of BCAs (Malusá et al., 2012). A carrier ideally possesses the following properties: high water-holding capacity, ease of processing, free of lump-forming materials, near-sterile or easy to sterilize by autoclaving or by other methods (e.g., gamma irradiation), high pH buffering capacity, low cost, available in adequate amounts, no toxicity, and environmental safety (Stephens and Rask, 2000;Ferreira and Castro, 2005). Smith (1992) found that dry inoculants can be produced using different types of soil materials (peat, coal, clays, and inorganic soil), organic materials (composts, soybean meal, wheat bran, and sawdust), or inert materials (e.g., vermiculite, perlite, kaolin, bentonite, and silicates). In this experiment, dry inoculants in Table S7 were used to optimize the fermentation conditions of strain XNRB-3 using response surface analysis (RSM), which signi cantly increased the survival rate and shelf life of strain XNRB-3 and complies with the Chinese bio-organic fertilizer production standard stipulating that the functional microorganism content should be greater than 2.0 × 10 7 CFU g −1 dry formulation after storage for 6 months at room temperature (Emmert and Handelsman, 1999). The raw materials (cow dung compost and wheat straw) in the formula are cheap and easy to obtain, and the fermentation level is high, which provides a good foundation for its large-scale industrial production. We veri ed the results under eld conditions and found that the addition of optimized XNRB-3 bacterial fertilizer can signi cantly promote the growth of replanted young apple trees and inhibit the growth of Fusarium in the soil. The abundance of Fusarium in the soil was signi cantly lower after treatment with the optimized XNRB-3 bacterial fertilizer compared with CK1. This ability to promote plant growth might also stem from the ability of XNRB-3 to enhance the soluble mineral nutrient content and produce indole-3-acetic acid (IAA), as well as its multiple PGP properties and antagonistic traits (Pii et al., 2015) . . The antibiotics (surfactin, fengycin, iturin, bacillomycin, and subtilosin) produced by B. subtilis SQR 9 signi cantly inhibit the growth of F. oxysporum, Verticillium dahliae, Phytophthora capsici, and Phytophthora nicotianae. The production of lipopeptides substances might also be one of the important reasons why strain XNRB-3 can form a bio lm on the surface of roots. Besson et al. (1990) found that asparagine appeared to be the optimal precursor among the α-amino acids in the peptidic part of iturin, indicating that the production of amino acids also affects the biosynthesis of peptide antibiotics. In this experiment, capillary electrophoresis was used to detect the content of free amino acids during fermentation by strain XNRB-3. The concentration of four free amino acids (aspartic acid, glutamic acid, proline, and tyrosine) in the extracellular matrix was the highest. This nding is consistent with the results of Ren et al. (2012). This indicates that the production of amino acids might be involved in the biosynthesis of peptide antibiotics. There is thus a need to evaluate the colonization ability of strain XNRB-3 in the root system. Given that strain XNRB-3 has a variety of lipopeptide biosynthetic genes and forms a thick bio lm in a static medium, we also evaluated the colonization ability of the root system by dilution-plate counting in a greenhouse and in the eld in non-sterile soil (Mendis et al., 2018). Strain XNRB-3 could colonize plant roots, and its fresh weight ranged from 10 5 to 10 7 cfu/g within 21 days. This is consistent with the results of Hallmann (2001) indicating that strain XNRB-3 can colonize the roots of apple seedlings, which is critical for its ability to become established in the soil environment after applying it in the eld. We also found that watering the strain XNRB-3 in soil pre-infected with Fusarium can signi cantly reduce the abundance of Fusarium in rhizosphere soil. After 5 weeks, the relative control effect was as high as 51%. These ndings are consistent with the results of Cao et al. (2011) showing that the pathogen density in the rhizosphere of cucumber seedlings inoculated with Bacillus subtilis SQR9 was signi cantly reduced. This result indicated that strain XNRB-3 can stably colonize the rhizosphere of apple seedlings and provide protection to plants. Similar  pumilus SE 34 prevents the entry of the vascular wilt fungus F. oxysporum f. sp. radicis-lycopersici into the vascular stele, and the mycelial growth is restricted to the epidermis and outer root cortex. Infected roots can also produce a large amount of sticky substances and result in the deposition of formed callose and starch granules to form a mechanical barrier that inhibits the invasion of pathogens (Lagopodi et al., 2002;Grunewaldt-Stöcker et al., 2020). A similar structure was also observed in this experiment.
Soil enzyme activity is often used to monitor changes in soil microbial activity and soil fertility because it is involved in all soil biochemical processes (e.g., soil organic matter formation and degradation; C, N, and P cycling; and plant nutrient transformation); it is also sensitive to changes in soil management (Kandeler et al., 2006;Song et al., 2012). Changes in soil attributes (e.g., SOC, TN, TP, AN, and AP concentrations) are also important indicators of changes in soil fertility and long-term ecosystem sustainability (Pan et al., 2013). Therefore, we evaluated soil microbial activity and fertility status by measuring the activity of soil-related enzymes and soil nutrient attributes after adding strain XNRB-3. The application of strain XNRB-3 signi cantly increased the activity of soil-related enzymes, and it increased after the second year of applying strain XNRB-3. This is consistent with the results of several previous studies. Phosphorus (P) plays a key role in crop productivity, and its availability depends on P mineralization from soil organic matter. This enzymatic process is performed by a group of phosphatases, such as AP, which provide inorganic P to the soil solution (Krämer, 2000). . Therefore, biological methods were used in this study to investigate the functional diversity and carbon source utilization of the rhizosphere soil microbial community after treatment with strain XNRB-3, as well as evaluate the safety of its use in the soil environment. The AWCD value of rhizosphere soil was signi cantly higher after the addition of strain XNRB-3 compared with other treatments; the addition of strain XNRB-3 also signi cantly enhanced the use of carbon sources such as polymers, carboxylic acids, and amino acids, which might be related to the increase in the number of soil bacteria and actinomycetes after the addition of strain XNRB-3. Biolog GEN III microplate identi cation revealed that strain XNRB-3 can use a wide range of carbon sources, which permits this strain to grow and reproduce in environments with different nutrient levels (Schutter and Dick, 2001).
Previous studies have shown that the occurrence of ARD is closely related to the structure and diversity of soil microbial communities. Increases in the number . Phlorizin is a unique phenolic acid substance of apples that mainly exists in the roots, stems, bark, tender leaves, and fruits of apples. The high concentration of phlorizin can signi cantly inhibit the growth of apple seedlings and reduce the rate of plant and these microbes could effectively degrade ferulic acid, p-hydroxybenzoic acid, and p-hydroxybenzaldehyde and promote seedling growth. Phloridin is degraded in the soil in two main ways. The rst is through the hydrolysis of phloretin into phloroglucinol and p-hydroxyphenylpropionic acid by secreting a phloretin hydrolase, followed by decomposition to phloretin and glucose by β-glucosidase, which is then used by bacteria (Chatterjee et al., 1969), Alternatively, it can be degraded to pyruvic acid by the protocatechuic acid pathway in Pseudomonas (Mohan and Phale, 2017), and pyruvic acid can be converted to acetyl CoA and enter the tricarboxylic acid cycle, which produces organic acids, such as citric acid, succinic acid, malic acid, and oxaloacetic acid (Priefert et al., 2001;Zhang Y et al., 2010), These substances play an important role in promoting the absorption and transportation of certain nutrients and improving the photosynthetic e ciency of plants and the accumulation of nitrogen, phosphorus, and potassium (Liu et al., 2005). Therefore, the method of phloridin degradation in the soil environment by the strain XNRB-3 is thought to be an effective approach for overcoming the obstacles of continuous apple cropping.

Conclusions
The phloridin-degrading bacterium B. licheniformis XNRB-3 was isolated from the roots of apple plants grown in a replanted orchard. Strain XNRB-3 features a variety of PGP characteristics and antagonistic traits, which confers it with high potential for practical use, including its ability to produce some antifungal substances and signi cantly inhibit the spore germination of Fusarium. XNRB-3 could effectively colonize the root surface of plant seedlings and even enter roots after it was inoculated on the roots of plant seedlings. The addition of strain XNRB-3 under potted and eld conditions can signi cantly promote the growth of apple plants; reduce the abundance of Fusarium and the content of phenolic acids in the rhizosphere soil; improve the structure of the soil microbial community; increase the available nitrogen, phosphate, and potassium in soil; and improve soil health( Figure 12). This study provided new insight as well as a strain resource that could be used to aid the prevention and control of ARD.

Declarations
Antifungal activity of strain XNRB-3 against plant fungal pathogen. (A): The mycelia and spore morphology of Fusarium oxysporum under the scanning electron microscope. A-D was the normal mycelium and spore, E-L was the mycelium treated with fermentation broth. (B): Effects of different treatments on spore germination of Fusarium. CK: Fusarium spore suspension was mixed with sterile water at 1:1, FB: Fusarium spore suspension was mixed with fermentation broth at 1:1, CFCF: Fusarium spore suspension was mixed with cell-free culture ltrate at 1: