Effects of Different Forms of Tagetes erecta Biofumigation on the Growth of Apple Seedlings and Replanted Soil Microbial Environment

Abstract

Apple replant disease (ARD) is a common soil disease that occurs in apple-growing areas around the world, causing root tip rot and necrosis, plant growth retardation and even plant death. Biofumigation is a promising strategy for controlling ARD due to its advantages of convenient application and being environmentally friendly. Tagetes erecta is an effective biological fumigant, but its effect on ARD is unclear. In the present study, we used Malus hupehensis Rehd. seedlings as the test material to detect the mitigating effects of different forms of T. erecta: air-dried sample (DS), fresh samples (FS) and fresh sample infusion solution (IS) on ARD. The effects of different forms of T. erecta on the growth of apple seedlings, leaf photosynthesis, root antioxidant enzyme, soil enzymatic activity and microbial environment were investigated. Compared with the CK treatments, DS, FS, and IS treatments all significantly increased the biomass of apple seedlings and promoted root growth under replanting conditions. Among them, DS showed the best results. The activity of antioxidant enzyme including superoxide dismutase, peroxidase and catalase were significantly increased in roots grown in soils treated with T. erecta. Moreover, T. erecta treatment also increased the activity of soil urease, phosphatase, sucrase and catalase enzyme, significantly altered the abundance of soil fungal communities and, in particular, reduced the abundance of Fusarium oxysporum, the main causal fungus of ARD. Therefore, our results suggest that biofumigation of different forms of T. erecta enhanced the resistance of ARD by regulating root reactive oxygen levels and improving the soil fungal communities.

1. Introduction

In China, many developed apple orchards are seriously aging and facing renewal. However, restricted by land resources, apple industrialization and regional development, many old orchards are renewed and renovated only in situ, leading to the widespread occurrence of apple replant disease (ARD), which seriously affects the healthy development of the apple industry. ARD is a root disease of apples (Malus domestica Borkh.) that occurs when apple is replanted on soil previously planted with apple or related species [1]. ARD often occurs within the first 2–3 years of replanting orchards, with young apple trees presenting slowed growth, increased pest and disease incidence, and even shortened life span and plant. Although the traditional chemical fumigation method can effectively control ARD, it has been phased out due it causing huge damage to the environment. Biofumigation can regulate soil enzyme activity, improve soil microbial community structure, stimulate plant defenses to alleviate plant replanting disease, and is considered a promising method to control ARD [2,3].

The application of green manure of Brassica spp. significantly reduced the amount of Gaeumannomyces graminisvartritici in wheat fields, and first referred to this measure as biofumigation [4]. Many studies have mentioned that Brassica spp. play an important role in suppressing soil-borne fungi and promoting healthy root growth [5,6,7]. Isothiocyanates produced during decay of Brassica spp. have excellent fungicidal effects, conferring biofumigation to Brassica spp. [7,8]. Allium plants, such as leeks and onions, release fungicidal substances propyl disulfide and methyl disulfide into the soil, reducing the occurrence of seedling rot in strawberries and asparagus [9]. Asteraceae contains a variety of fungicidal active ingredients, especially for fungi with large spore production, short reproductive cycles and easy to develop resistance [10]. Tagetes erecta L. is an annual herb of the genus Asteraceae, with antifungal, bacteriostatic, insecticidal and other biological activities [11,12]. Moreover, we noted the fungicidal effect of T. erecta on F. oxysporum [13], the main causative fungi of ARD, but the effect of its biofumigation on plant replanting disease has not been studied.

Therefore, we used M. hupehensis Rehd. as the test material to evaluate the control effect of T. erecta biofumigated soil in different forms (dry samples, fresh samples and fresh sample extracts) on ARD. The results showed that T. erecta biofumigation significantly promoted the growth of plants in the replanted soil, altered the abundance of soil fungal communities and increased soil enzyme activities. In short, T. erecta biofumigation significantly alleviated ARD symptoms in apple plants.

2. Materials and Methods

2.1. Experimental Materials

The soil used in the experiment was taken from a 25-year-old apple orchard in Manzhuang Town, Tai’an City, Shandong Province (119.81 longitude, 36.07 latitude). The rootstock was Malus micromalus Makino. Soil was taken from an area 80 cm away from the trunk, 10–40 cm deep after removing the topsoil, and randomly sampled from multiple points. Soil samples were mixed well for later use. The basic chemical properties of the soils are shown in

Table 1. The basic characteristics of the test soil.

Soil Type	pH	Nitrate Nitrogen (mg kg−1)	Ammonium Nitrogen (mg kg−1)	Available Potassium (mg kg−1)	Available Phosphorus (mg kg−1)	Organic (g kg−1)
brown loam	6.08	5.5	3.8	90.6	9.3	5.3

.

The test material was the apple rootstock variety M. hupehensis. Following cold stratification at 4 °C for 30 days, seeds were sown in the seedling matrix (peat: vermiculite: perlite = 1:1:1). When the seedlings reached 6 true leaves, those with consistent growth and free from diseases and pests were transplanted and treated. The whole flowering annual T. erecta of 20 cm in height were purchased for RMB 1 yuan from Chunmanyuan gardening center, Taishan District, Tai’an City for the experimental treatment. T. erecta was processed in three ways: (1) The whole plants were cleaned, dried naturally, cut and crushed, passed through a 30-mesh sieve and put into a sealing bag. (2) Fresh plants were cleaned and cut into small pieces of about 0.5 cm. (3) Fresh plants were cut into 0.5 cm pieces and soaked in water (1:20) for 24 h to obtain fresh sample soaking solution.

2.2. Experimental Design and Sampling

In April 2015, pot experiments were performed at the National Research Center for Apple Engineering and Technology, Shandong Agriculture University, Tai’an, Shandong, China (116°20′–117°59′, 35°38′–36°28′). There were four treatments: (1) replanting soil (untreated, CK); (2) T. erecta dry sample powder 6.0 g/kg (DS); (3) T. erecta fresh sample 12.0 g/kg (FS); (4) T. erecta fresh sample extract infusion solution (IS), pouring 2 L of extract in each pot (16.7 g/kg). The test concentration was set according to the results of the indoor bacteriostatic test. In the DS and FS treatments, the plant material was well mixed with the replanting soil and added to the clay pots (outer diameter, 27 cm; inner diameter, 23 cm; height, 18 cm), and the same amount of water was poured into each treatment (IS treatment with extracts). IS treatment involved filling the pot with replant soil and then irrigating it. A black plastic bag (thickness: 0.025 mm, width: 40 cm, height: 64 cm) was then covered over the pots for 15 days, then removed and aired out for 7 days. Two prepared seedlings of uniform size were planted in pots with 20 replicates conducted for each treatment. The treatments were arranged in a completely randomized design. Nutrients and water were managed following usual practices, and the N, P, and K contents in the soil were normalized among treatments.

In August 2015, approximately 500 g of inter-root soil was taken, and sieved through a 12-mesh sieve (1.70 mm). The soil samples were divided into three parts; one part was refrigerated at 4 °C for soil microbial count determination; the second was air-dried for soil enzyme determination; and the third was stored in the refrigerator at −20 °C for DNA extraction, terminal restriction fragment length polymorphism (T-RFLP) analysis and real-time fluorescence quantitative PCR (RT-qPCR) analysis.

Three plants were randomly selected for each treatment as biological replicates for biomass determination. The experiment was repeated 3 times, and the data presented were the average of 3 experiments. The white new growth roots of the plants were taken, rinsed with water and quickly placed in a liquid nitrogen for the determination of root respiration rate and protective enzyme activity.

2.3. Test Indexes

2.3.1. Measurements of the Plant Biomass and Root Morphology

The root morphology was analyzed using WinRHIZO software (version 2007, Guangzhou Hangxin Scientific Instrument Co., Ltd., Guangzhou, China), mainly including root length, root volume, root surface, root diameter and tips. Plants were dried at 105 °C and then at 60 °C to constant weight, and the shoot and root biomass was determined.

2.3.2. Determination of Plant Root Respiration Rate

The root respiration rate was measured according to the method of Mao [14]. Briefly, 0.1 g root sample is taken and cut into 2 mm pieces, and the root respiration rate measured in a 3 mL cuvette at 25 °C in a Clark-type oxygen electrode (standard Hansatech electrode disc) connected to a CB1D control box (Hansatech Instruments Ltd., Kings Lynne, UK).

2.3.3. Determination of Root Antioxidant Enzyme Activities and MDA Content

The superoxide dismutase (SOD), peroxidase (POD)and catalase (CAT) activities were determined by the method of Zhao [15]. For the extraction of protective enzymes, 1.0 g of fresh white root was taken, 8 mL of phosphate buffer solution was added, a small amount of quartz sand was added and ground into a homogenate, and the supernatant was the enzyme solution determined by centrifugation at 12,000 rpm/min at 4 °C for 20 min; all determinations were performed at 2–4 °C.

The malondialdehyde (MDA) content was determined by the thiobarbituric acid method and expressed as mmol/g FW [15].

2.3.4. Determination of Soil Enzyme Activities

The soil invertase, neutral phosphatase, and urease activities were determined with air-dried soils, following the method of Guan [16].

2.3.5. Determination of Soil Microbial Amounts

The numbers of bacteria, fungi and actinomycetes in soil samples were determined by the dilution method of plate counting. Fresh soil samples (10 g) were placed in a triangular flask and 90 mL of sterile water was added (this represented the 10⁻¹ dilution). The flask was sealed and shaken at room temperature for 10 min. Then, the soil was mixed well and left to stand for 20–30 s. Next, 1 mL of 10⁻¹ dilution was placed into a test tube and 9 mL of sterile water was added and mixed well (this represented the 10⁻² dilution). In this order, a series of soil dilutions including 10⁻³, 10⁻⁴ and 10⁻⁵ were prepared by successive dilutions. Beef extract-peptone agar medium was used for bacterial culture, Martin medium for fungal culture, and GAUZE’s medium for actinomycetes [17]. The numbers of bacteria, fungi and actinomycetes were quantified as CFU per gram of dry soil.

2.3.6. DNA Extraction and T-RFLP Analysis

The E.Z.N.A.™ Soil DNA Kit (Omega Bio-tek Inc., Norcross, GA, USA) was used to extract total soil microbial DNA. Total DNA was extracted using 0.5 g of sieved soil sample according to the instructions. Total extracted DNA was quantified (Nanodrop 2000 UV–Vis spectrophotometer, Nanodrop Scientific, Wilmington, DE, USA) and diluted to 20 ng μL⁻¹. The fungal internal transcribed spacer (ITS) was amplified using the ITS1F and ITS4 primers by polymerase chain reaction (PCR) as follows [18]: 94 °C, 3 min for initial denaturation; 34 cycles of 94 °C for 1 min, 51 °C for 1 min, 72 °C for 1 min; 72 °C for 10 min. The PCR system was as follows: 3 μL of each primer (ITS1F and ITS4, 10 μM), 2 μL DNA template, 25 μL 2 × Taq PCR MasterMix, and 17 μL RNAse-free water. PCR products were purified using the EasyPure PCR purification kit (TransGen Biotech Co., Ltd., Beijing, China). The purified samples were analyzed by the Sangon Biotech Co. Ltd. (Shanghai, China) using an ABI 3730LX DNA Analyzer. Three replicates of per treatment were analyzed.

2.3.7. qPCR Analysis

The qPCR analyses of F. oxysporum were conducted according to a previously described method by Wang [19]. The primer pairs used in this experiment were as follows: JR (5′GGCCTGAGGGTTGTAATG-3′) × JF (5′CATACCACTTGTTGTCTCGGC-3′). The qPCR was performed on a CF × 96™ Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). Each 25-μL PCR reaction mixture contained 12.5 μL SYBR green PCR Master Mix (Takara Biotech Co., Ltd., Dalian, China), 1 μL of JR (10 μM), 1 μL of JF (10 μM), 1.5 μL DNA, and 9 μL ddH₂O. Reactions were carried out in the following PCR cycling conditions: 95 °C pre-denaturation for 30 s; 39 cycles of 95 °C denaturation for 5 s, 60 °C annealing for 30 s, and 72 °C extension for 1 min; and a final 72 °C extension for 5 min.

2.4. Statistical Analysis

Statistical calculations were performed with Origin 8.5 (OriginLab Corporation) and IBM SPSS 20.0 (IBM SPSS Statistics, IBM Corporation, Armonk, NY, USA). Data are expressed as means and standard deviations of three biological replicates. Each experiment is repeated at least three times. Duncan’s test was used to detect significant differences in the data (p < 0.05). The percentage abundance of each terminal restriction fragment (TRF) was calculated as described by Thomas et al. (2000) [20]. The T-RFs with lengths between 50 and 500 bp were used for the T-RFLP profile analyses. Only T-RFs with peaks of RA ≥ 1% were considered.

Fungal diversity was evaluated by calculating the Margalef richness index (S), Brillouin evenness index (J′), Simpson dominance index (D) and Shannon’s diversity index (H′) using BIO-DAP software (NB, Canada). The T-RF profiles were also analyzed by principal component analysis (PCA) to describe differences in the structure and composition of the fungal communities among different treatments.

3. Results

3.1. Effects of Tagetes erecta Biofumigation on the Growth of Apple Seedlings

As shown in

Table 2. Effects of different forms of T. erecta biofumigation on the biomass of M. hupehensis seedlings.

Treatments	Plant Height (cm)	Ground Diameter (cm)	Fresh Weight (g)		Dry Weight (g)
			Above Ground	Root	Above Ground	Root
CK	21.67 ± 0.88 b	2.93 ± 0.14 c	5.60 ± 0.27 c	3.80 ± 0.28 c	2.09 ± 0.17 c	0.99 ± 0.11 c
DS	45.00 ± 1.73 a	5.49 ± 0.08 a	16.34 ± 0.62 a	11.19 ± 0.72 a	6.19 ± 0.23 a	2.15 ± 0.24 a
FS	44.33 ± 0.88 a	4.68 ± 0.21 b	13.17 ± 0.35 b	9.82 ± 0.74 b	4.42 ± 0.16 b	1.70 ± 0.25 b
IS	42.33 ± 0.88 a	5.04 ± 0.26 ab	14.29 ± 0.92 b	9.57 ± 0.33 b	4.59 ± 0.17 b	2.01 ± 0.14 ab

CK: replanted soil, DS: air-dried Tateges erecta sample, FS: fresh T. erecta sample, IS: infusion solution of fresh T. erecta. Data are the means of three replicates, and the different letters following the number indicate significant differences at p < 0.05.

, compared with the replanting soil (CK), different forms of T. erecta treatments all significantly increased the biomass of apple seedlings. Among them, the best effect was obtained with the dry sample treatment (DS). The plant height, ground diameter, above-ground and root fresh weight, and above-ground and root dry weight increased significantly by 107.7%, 87.3%, 192.0%, 194.1%, 196.5% and 117.6%, respectively.

3.2. Effect of Tagetes erecta Biofumigation on Gas Exchange Parameters of Seedling Leaves

Different forms of T. erecta treatments increased the net photosynthetic rate, stomatal conductance and transpiration rate of M. hupehensis seedlings (Figure 1). Compared with CK, the net photosynthetic rate in DS, IS and FS treatments increased significantly by 32.6%, 31.9% and 21.7%, respectively. Stomatal conductance in DS, IS and FS treatments increased by 40.7%, 27.6% and 12.2%, respectively, where FS treatment was not significantly different from CK. Transpiration rate in DS, IS and FS treatments increased significantly by 72.9%, 63.2% and 55.5%, respectively. There was no significant difference in the intercellular CO₂ concentration among the different treatments.

3.3. Effect of Tagetes erecta Biofumigation on Root Indexes of Seedlings

3.3.1. Root Indexes Analysis

All treatments of different forms of T. erecta promoted the root growth of M. hupehensis seedlings. The root length, root surface area, root volume and the number of root tips of M. hupehensis seedlings in DS treatment were 1.9 times, 1.8 times, 1.8 times and 2.0 times that of CK, respectively. The IS treatment was not as significant as DS, with 1.5 times, 1.6 times, 1.5 times and 1.7 times the root length, root surface area, root volume and the number of root tips of that of CK, respectively. The FS treatment had the least significant effect, affecting only root length and root surface area (

Table 3. Root growth of M. hupehensis seedling in soils biofumigated with different forms of Tateges erecta.

Treatments	Roots Length (cm)	Roots Surface Area (cm2)	Roots Volume (cm3)	Root Tips
CK	542.59 ± 1.87 c	315.57 ± 12.65 c	14.66 ± 1.24 c	2827.67 ± 28.31 c
DS	1022.95 ± 37.16 a	577.92 ± 11.98 a	26.58 ± 0.65 a	5553.67 ± 225.99 a
FS	788.22 ± 22.16 b	445.40 ± 14.53 b	17.63 ± 1.88 c	3607.67 ± 263.44 b
IS	828.95 ± 49.34 b	512.17 ± 37.49 ab	22.20 ± 1.30 b	4886.67 ± 323.18 a

CK: replanted soil, DS: air-dried Tateges erecta sample, FS: fresh T. erecta sample, IS: infusion solution of fresh T. erecta. Data are the means of three replicates, and the different letters following the number indicate significant differences at p < 0.05.

).

3.3.2. Root Respiration Rate

T. erecta treatments increased the root respiration rate of M. hupehensis seedlings. Compared to CK, the root respiration rate of M. hupehensis seedlings grown in DS-treated soil increased by 64.6%, and that of apple seedlings grown in IS-treated soil improved by 38.3%. However, the FS treatment showed no significant difference on the root respiration rate of the plants (Figure 2).

3.3.3. Root Protective Enzymes Activity

Different forms of T. erecta treatments all increased the activity of root protective enzyme in M. hupehensis seedlings (Figure 3). Compared with CK, the SOD activity of DS, IS and FS treatments increased by 62.7%, 44.6% and 14.3%, respectively. Among them, DS- and IS-treated plants were significantly different from CK. The POD activity of DS, IS and FS treatments increased by 39.55%, 31.23% and 18.14%, respectively. The CAT activities of DS, IS and FS treatments increased by 95.62%, 75.91% and 43.06%, respectively.

Compared with CK, DS, IS and FS treatments significantly reduced the content of MDA by 35.2%, 15.9% and 11.9%, respectively.

3.4. Effect of Tagetes erecta Biofumigation on Soil Enzyme Activity

The soil enzyme activities of different forms of Tagetes erecta treatments were significantly increased compared to CK. DS treatment increased the activities of urease, phosphatase, sucrase and catalase by 269.9%, 157.5%, 60.8% and 66.7%, respectively. IS increased by 123.9%, 121.7%, 37.0% and 53.8%, respectively. FS treatment was the least effective (Figure 4).

3.5. Effect of Tagetes erecta Biofumigation on Soil Microbial Environment

3.5.1. Effect on the Number of Soil Microorganisms

Tagetes erecta treatments increased the number of bacteria and actinomycetes, decreased the number of fungi in the replanted soil, and increased the ratio of bacterial/fungal (Figure 5). The number of bacteria when treated with DS was five times that of CK. The fungi number with the DS and FS treatments was 22.9% lower than that of CK. The number of actinomycetes in the replanted soil of DS, FS and IS treatments was 2.8, 1.9 and 1.8 times higher than that of CK, respectively; all of which were significantly different from CK. The bacterial/fungal ratios in the soils of the different treatments were elevated, with the DS, FS and IS treatments being 6.5, 2.0 and 1.1 times higher than CK, respectively.

3.5.2. Effects on Soil Fungal Community Diversity

Based on the number, species and abundance of OUT in T-RFLP profiles, the Shannon index, evenness index, Simpson’s index and richness index of soil fungi treated with different forms of T. erecta were calculated, respectively. Compared with CK, the Shannon and Simpson’s indices of soil fungal community decreased in different treatments. In addition, the Pielou and Margalef indices increased in the DS treatment, and the Pielou index decreased and the Margalef index increased in the FS and IS treatments (

Table 4. Fungal diversity in soils biofumigated with different forms of Tateges erecta.

Treatments	Shannon Index	Pielou Index	Simpson’s Index	Margalef Index
CK	2.11 ± 0.004 a	5.60 ± 0.004 b	0.65 ± 0.001 a	0.16 ± 0.001 c
DS	1.95 ± 0.005 b	5.63 ± 0.004 a	0.60 ± 0.001 b	0.23 ± 0.003 a
FS	1.85 ± 0.007 c	5.47 ± 0.010 d	0.57 ± 0.002 c	0.19 ± 0.002 b
IS	1.83 ± 0.012 c	5.52 ± 0.008 c	0.56 ± 0.004 c	0.19 ± 0.003 b

CK: replanted soil, DS: air-dried Tateges erecta sample, FS: fresh T. erecta sample, IS: infusion solution of fresh T. erecta. Data are the means of three replicates, and the different letters following the number indicate significant differences at p < 0.05.

), thus showing that the T. erecta treatment changed the community structure diversity of soil fungi.

3.5.3. Effects on Soil Fungal Community Richness

As shown in Figure 6, different forms of T. erecta treatment caused significant changes in the relative abundance of fungal communities. Among the treatments, the DS treatment showed the greatest variation in each fragment, with 146, 161, 162, 165, 329 and 342 bp T-RFs only in the DS treated soil. In contrast, 57, 81, 82, 90, 91, 106, 159 and 327 bp T-RFs, which were not present in the DS treatment, were present in the CK-treated soil. The changes in the relative abundance of fungal communities were similar between FS and IS treatments, increasing T-RFs at 54, 86, 94 and 159 bp, decreasing T-RFs at 50, 81, 82, 87 and 90 bp, and little change in T-RFs at 57 bp compared with CK, and other fragments did not appear in the soil of both treatments.

3.5.4. Effects on the Gene Copy Number of Fusarium Oxysporum

Real time qPCR was used to quantify the gene copy number of F. oxysporum in soil after treatment with different forms of T. erecta. The results showed that the gene copy numbers of F. oxysporum in different forms of T. erecta treatments were lower than CK, and the DS, FS and IS treatments were 51.6%, 10.5% and 20.2% lower than CK (Figure 7), suggesting that the T. erecta treatment reduced the number of F. oxysporum in the soil.

4. Discussion

Apple replant disease affects global apple yield and quality, and its control is of wide concern. Among the damage caused by ARD, the most prominent problems are low seedling survival, dwarf trees and poor fruit quality. Our experiments showed that different forms of T. erecta biofumigation promoted the growth of M. hupehensis seedlings, among which the air-dried powder biofumigation showed the best effect. Brassica, Allium and Asteraceae have been shown to have excellent fungicidal activity [7,8,9]. Among them, Brassica and Allium biofumigation or extracts had significant inhibitory effects on soil-borne diseases [6,7,9,21], and our study reported, for the first time, the biofumigation effect of Asteraceae. Biofumigation with T. erecta dry powder significantly increased the root respiration rate and enhanced the net photosynthetic rate and transpiration rate of M. hupehensis seedlings, which may promote the growth of seedlings and increase the plant height, fresh mass, and dry mass (Table 2).

Under normal conditions, the production and elimination of free radicals in plants are in dynamic balance. In the case of adversity stress, the production rate of free radicals becomes much higher than their elimination rate, and the plants will suffer from damage. Superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) are the main players in protecting plants from excess free radicals, which act synergistically to protect plant inner membrane structures from free radical damage. The addition of biochar could alleviate apple replant disease by activating antioxidant enzymes, decreasing lipid peroxidation and significantly reducing the phenolic acids content of replant soil [22]. In this study, T. erecta treatments increased the root respiration rate of M. hupehensis seedlings, and significantly increased the activities of SOD, POD and CAT. It may be that T. erecta biofumigation could significantly alter the abundance of soil fungal communities, create a more conducive environment for plant growth, and make the root growth more healthily and vigorously. Therefore, in the face of pathogen invasion, the apple seedlings could start their own defense systems more quickly, enhance the activity of antioxidant enzymes and resist the damage caused by pathogen infection. In the future research, we will pay more attention to the reports of biological fumigation and antioxidant enzyme activity, and formulate a reasonable plan to further explore. In addition, MDA, the end product of lipid oxidation that causes plant membrane damage, was significantly reduced in the roots of plants treated with T. erecta. Similar to previous results, low concentration of mustard infusion promoted the growth of cowpea seedlings, and alleviated the damage caused by replantation disease on cowpea seedlings by increasing the antioxidant enzyme activity and improving the root environment of cowpea seedlings [23]. The application of indigenous arbuscular mycorrhizal fungi can enhance the resistance of M. hupehensis seedlings to ARD by enhancing antioxidant enzyme activity in the root system [24].

Soil enzymes are a general term for some special protein compounds in the soil that have biocatalytic capacity. The activity of soil enzymes is one of the indicators for judging the intensity of soil biochemical processes and evaluating soil fertility. The reduction of catalase, urease, sucrase, and neutral phosphatase activities in apple replanting soils is one of the manifestations of soil environmental degradation due to continuous crop. The activities of sucrase, urease, phosphatase and catalase were significantly increased when treated with the addition of T. erecta powder or infusion in this study, indicating that T. erecta biofumigation significantly improved the soil physicochemical properties. We speculate that decaying plant tissues of T. erecta powder may improve soil nutrient conditions and activate soil enzyme activities, thereby promoting plant growth.

Long-term replanting will lead to changes in the soil physicochemical and biological properties, thus breaking the original inter-root microecological balance. The main manifestation is the disorder of soil microbial population structure, quantity and ratio, with a dramatic increase in the number of certain pathogenic microorganisms and a significant decrease in beneficial microorganisms [25]. T. erecta dry powder and infusion treatment in this study increased the number of bacteria and actinomycetes, decreased the number of fungi, and significantly increased the bacterial/fungal ratio (Figure 5), resulting in a shift of the replanting soil from a fungal to a bacterial type. In addition, the results of diversity and T-RFLP analysis showed that T. erecta treatment significantly changed the fungal community structure and optimized the microbial environment of the replanted soil (Figure 6). The Shannon index and Simpson’s index of the soil fungal community in different treatments decreased, the Pielou index and Margalef index of DS treatment increased, the Pielou index of FS and IS treatments decreased, and the Margalef index of FS and IS treatments increased. Mustard seed meal significantly affected the structure of soil fungal and bacterial communities, reduced fungal diversity by 60%, and increased the richness of bacterial groups related to fungal diseases (e.g., Bacillus, Pseudomonas, Streptomyces, etc.), effectively inhibiting the occurrence of fungal diseases [26]. In addition, qPCR analysis showed a significant reduction in the gene copy number of F. oxysporum, which was one of the main pathogenic fungi causing ARD (Figure 7). Numerous Actinomyces species, particularly those belonging to the genus Streptomyces, are known as antifungal biocontrol agents, inhibiting several fungal plant pathogens [27], suggesting its role in regulating the soil microbial community structure. The increase in the abundance of beneficial bacteria and the decrease in the abundance of harmful fungi in T. erecta treated soil helped to reduce the negative effects of replanting soil on plant growth and development.

5. Conclusions

In conclusion, our study showed that biofumigation of T. erecta improved the replanting soil environment, including enhancing soil enzyme activity, increasing the bacterial/fungal ratio, improving soil fungal community structure, and reducing the content of F. oxysporum. It also promoted the growth of M. hupehensis seedlings in the replanted soil and improved photosynthesis and plant root protection enzyme activity. In summary, air-dried samples of T. erecta appears to offer the best approach to enhancing apple growth and soil health under replant conditions, thereby advancing the development of the apple industry.