Effect of Bacillus methylotrophicus on Tomato Plug Seedling

Abstract

Tomato production is gradually shifting to modern production, which requires the factorization of tomato seedlings to shorten the seedling cycle and improve the seedling quality. Bacillus methylotrophicus, as a biofertilizer for plant growth-promoting rhizobacteria, can promote plant growth and enhance native plant defenses. However, reports on the role of this type of bacterial agent in horticultural crop seedlings are limited. We investigated the effects of different dosages of Bacillus methylotrophicus (0.00, 0.25, 0.50, 0.75, 1.00, 1.25, and 1.50 g/strain) on tomato plug seedlings and aimed to screen out the suitable dosage of Bacillus methylotrophicus for tomato seedlings in 50-hole cavity trays. In this experiment, with the increase in Bacillus methylotrophicus, the number of leaves, plant height, stem thickness, leaf area, dry matter accumulation in each organ, growth function (G value), and seedling strength index of tomato seedlings showed an increasing trend, followed by a decreasing one. The appropriate dosage (0.50–1.25 g/strain) of bacterial agent increased the activities of the substrates urease, sucrase, and catalase, thus forming a good microbial community to maintain the balance of organic and inorganic carbon and guaranteeing the normal development of the root system. Meanwhile, under the treatment of 1.00 and 1.25 g/strain of inoculum, the absorption range of tomato roots increased, more nitrogen, phosphorus, and potassium were absorbed from the substrate, and more nutrients were transported from the underground to the above-ground parts, which promoted shoot elongation and thickening of the shoots, increased the leaf number and dry matter accumulation, and improved the seedling quality. In this study, the mechanism of action of this microbial product on tomato seedlings was studied from the perspective of nutrient uptake and supply, and a sowing root application of 1.00 g/strain of Bacillus methylotrophicus in 50-hole cavity trays can improve the quality of tomato seedlings.

1. Introduction

Plant growth-promoting rhizobacteria (PGPR) are a group of microorganisms that exist within root systems or inter-root environments and can promote plant growth in a direct or indirect manner [1] (Zhang et al., 2014). In agroecosystems, inter-root microbes can directly contribute to energy conversion, material cycling, and plant growth [2,3,4] (Crowley, 2006; Egamberdieva et al., 2011; Mohite, 2013) and indirectly regulate plant systemic resistance and biocontrol-related pathways [5,6] (Pineda et al., 2010; Li et al., 2019), thereby regulating plant morphological development, improving the quality of the inter-root microenvironment, and promoting yield and quality improvement. The structure, diversity, and activity of microbial communities are important for inter-root nutrient supply, ecosystem stability, and improved crop yield [7] (Li et al., 2020). The addition of PGPR can facilitate improved crop growth [8,9,10] (Yu et al., 2011; Krishna et al., 2014; Ma et al., 2019). Ansari et al. [11] (2019) isolated a strain of Bacillus pumilus FBA10, which can effectively promote the growth of cucumber and improve the salt tolerance of wheat. Tomes et al. [12] (2019) isolated a strain of Bacillus velezensis XT1 with the ability for nitrogen fixation, phosphate solubilization, potassium solubilization, and IAA and ACC deaminase production, which significantly promoted the growth of horticultural crops, such as tomato, melon, and cucumber. Mohite et al. [4] (2013) isolated Bacillus megaterium, Bacillus subtilis, and Lactobacillus acidophilus strains from inter-rhizosphere soil of tomato, wheat, banana and cotton and found that they can secrete IAA efficiently, and their IAA secreting properties promote plant growth. Shahid et al. [13] (2018) observed that the beneficial bacterium Planomicrobium sp. MSSA-10 can produce IAA, reduce the level of reactive oxygen species in plants, and increase the activity of antioxidant enzyme systems, which resulted in a significant growth-promoting effect on peas.

Bacillus methylotrophicus is a PGPR isolated from rice rhizosphere soil and has been identified as a new species of the genus Bacillus [14] (Madhaiyan et al., 2010). At present, studies on the application of methylotrophic Bacillus in crop production at home and abroad are limited, and most of them focus on the study of its mechanism in crop pathogenic microorganisms [15] (Adeniji and Babalola, 2018). Bacillus methylotrophicus can produce two non-volatile mesostereoisomers, namely, 3S,4R-acetylbutanediol and 3R,4R-acetylbutanediol, which can effectively promote root elongation and improve the growth of corn and rice, when both are present in the inter-root soil at a 1:2 volume ratio [16] (Wang et al., 2020). Bacillus methylotrophicus can increase substrate enzyme activity, stimulate root development, and improve cucumber seedling quality [17] (Hu et al., 2020). Wang et al. [18] (2021) observed that Bacillus methylotrophicus effectively improved the inter-root environment of cucumber and melon grown in organic substrate bags, promoting element uptake and utilization and improving fruit yield and quality. Bacillus methylotrophicus, native to polluted estuaries, was able to improve photosynthesis in Spartina maritima and the tolerance of its roots to heavy metals, thus helping ecological recovery [19] (Mesa et al., 2015).

The tomato (Solanum lycopersicum L.) has a high nutritional value and is widely cultivated worldwide [20] (FAOSTAT 2022). Seedling is a key aspect of vegetable cultivation and a critical step toward modern vegetable production and improving vegetable yield and quality [21,22] (Guo et al., 2015; Cui et al., 2020). Seedling quality affects tomato yield and quality to a certain extent; thus, it has become especially important in shortening the nursery cycle and improving seedling quality [23] (Shi et al., 2021). Previous studies have found that exogenous application of exogenous hormones, as well as some natural substances, can improve the quality of tomato seedlings or enhance their resistance to abiotic stresses, but there are fewer studies on the application of bacterial agents in related areas [24] (El-Hady et al., 2021). PGPR can improve the physicochemical properties and biological traits of seedling substrates to achieve a better seedling effect, but the application of Bacillus methylotrophicus in seedlings of horticultural crops has rarely been reported. In this study, we investigated the effect of Bacillus methylotrophicus on the growth and robustness index of tomato seedlings by the addition of different doses of Bacillus methylotrophicus to tomato seedling substrates. To expand the application of Bacillus methylotrophicus in horticultural crop seedlings from a nutritional point of view, we analyzed the effects of the bacterium on the physical and chemical properties of substrates, their root growth distribution, and plant element accumulation to investigate the reason for improved seedling quality. A total of 1.00 g/strain of Bacillus methylotrophicus was screened to improve the quality of tomato seedlings in 50-hole cavity trays, and such an amount can be used for the industrial production of high-quality tomato seedlings.

2. Materials and Methods

2.1. Experimental Materials

The tomato variety “Jinpeng No. 1” and “VL-10” Bacillus methylotrophicus (powder, produced by Shandong Azure Biotechnology Co., Ltd., with an effective live count of 5 × 10¹⁰ CFU g⁻¹) were used as experimental materials. The physical and chemical indexes of the substrate (the soil in which tomato seedlings are grown) were as follows: pH 6.21, EC (electrical conductivity) value of 15 mS/m, and fast-acting N, P, and K contents of 57.72, 71.27 and 275.94 mg·kg⁻¹, respectively. The 50-hole black plastic cavity trays were used for seedling development.

2.2. Experimental Design

Tomato seeds soaked in warm broth, then germinated and exposed and were sown in substrates mixed with Bacillus methylotrophicus dosages of 0, 0.25, 0.50, 0.75, 1.00, 1.25, and 1.50 g/strain (recorded as CK, T1, T2, T3, T4, T5, and T6, respectively), with three replications of each treatment, that is, one for each cavity tray. During the nursery period, the average daily temperature was 18.7 ± 2.6 °C, the average day/night temperature was 23.3 ± 4.7 °C/14.4 ± 2.6 °C, the average daily air humidity ranged from 55.6% to 96.7%, and the average day/night air humidity was in the range of 43.2–94.9%/66.4–99.4%.

After sowing, the plants were irrigated with 10 mL of water every five days and 10 mL/strain of 1:1000 “Pro-Soil No. 1” compound fertilizer nutrient solution (powder, authorized by US Fulan Biotechnology Co., Ltd., produced by Jinzhengda Ecological Engineering Group Co., Ltd.; N + P₂O₅ + K₂O > 60%, 20-20-20, 4% nitrate N, 0.2–3.0% trace elements, 1 g compound fertilizer dissolved in 1000 mL distilled water to obtain a nutrient solution pH of 7.15, and EC value of 115 mS/m) was used instead of water on days 15, 20 and 25.

2.3. Measurement Items and Methods

2.3.1. Plant Morphology and Biomass

The number of leaves of all seedlings was counted every 5 days, starting from the 10th day after sowing, with a total count of 5 times (30 days after sowing). The number of seedling leaves was calculated using Equation (1).

$$\mathrm{NL}=\frac{\left(0.5\times {\mathrm{N}}_{\mathrm{c}}+{{\displaystyle \sum }}_1^{\mathrm{i}}\mathrm{i}\times {\mathrm{N}}_{\mathrm{i}}\right)}{{\mathrm{N}}_{\mathrm{c}}+{{\displaystyle \sum }}_1^{\mathrm{i}}{\mathrm{N}}_{\mathrm{i}}}$$

(1)

In Equation (1), NL is the average number of leaves, N_C is the number of plants with only cotyledons spreading, N_i is the number of plants with i true leaves, the number of leaves of seedlings with only cotyledons spreading is set to 0.5.

Three plants were collected from each cavity tray at thirty days, with a total of nine plants per treatment, to determine the above- and below-ground morphological indicators and biomass. Stem thickness at the base of the stem and plant height (stem base to growth point) were determined using digital vernier calipers, and the leaf area of the second true leaf at the lower end of the morphology was calculated using ImageJ 1.51j8 (National Institutes of Health, Bethesda, MD, USA). Seedling roots, stems, and leaves were washed and wiped dry of surface water, and the fresh weight of each part was determined. The root morphology was scanned using an EPSON Perfection V700N (Epson (China) Co., Ltd., Shanghai, China) scanner, and root morphological analysis was performed using WinRHIZO PRO 2012 (Regent, Canada) to obtain the root length (RL), root surface area (RA), root average diameter (RD), root volume (RV), root tips (RT), root forks (RF), and root data of different diameter classes. After the roots were scanned, the roots, stems, and leaves were divided into envelopes, killed at 105 °C for 30 min, and then dried at 65 °C to a constant weight. The dry weight of each part was measured, and the dried samples were crushed and sieved for elemental determination.

2.3.2. Chlorophyll Content

At 30 days, the second true leaf from the lower part of the seedling morphology was selected, and chlorophyll was extracted using a mixture method [25] (Chen and Chen, 1984). To exclude the interference caused by different dry-to-fresh weight ratios (water content) of the leaves, we calculated the chlorophyll a and b and total chlorophyll contents using unit fresh and dry weights [26,27] (Li, 2000; Jia et al., 2008).

2.3.3. Seedling Growth Function and Seedling Strength Index (SI)

The growth function and SI of tomato seedlings at 30 days were calculated from seedling height, whole plant dry weight, and chlorophyll a + b content per unit of fresh weight measured in Section 2.3.1 and Section 2.3.2, using Equations (2) and (3) [28,29] (Moore et al., 1981; Gong et al., 2019):

$$\mathrm{G}=\frac{\mathrm{M}}{\mathrm{D}}$$

(2)

$$\mathrm{S}=\frac{\mathrm{Total} \mathrm{Chl}}{\mathrm{PH}}\times \mathrm{M}$$

(3)

In Equations (2) and (3), G is the growth function, S is the SI, M is the plant dry weight, D is the number of seedling days, Total Chl is the chlorophyll a + b content per unit fresh weight, and PH is the plant height.

2.3.4. Root Development Strategy

The following root system metrics were calculated using the root morphological metrics and root dry weight (RDW) obtained in Section 2.3.1 [30,31] (Comas et al., 2013; Wang et al., 2017):

Specific root length (SRL):	SRL = RL/RDW	(4)
Specific root surface area (SRA):	SRA = RA/RDW	(5)
Root tip density (RTD):	RTD = RT/RL	(6)
Root branch density (RBI):	RBI = RF/RL	(7)
Root tissue density (RTID):	RTID = RDW/RV	(8)
Root fineness (RFN):	RFN = RL/RV	(9)

The root morphological indicators were divided into the following five classes based on the root diameter, in the order of Grade 1 (G1): RD ≤ 0.05 mm, G2: 0.50 mm < RD ≤ 1.00 mm, G3: 1.00 mm < RD ≤ 1.50 mm, G4: 1.50 mm < RD ≤ 2.00 mm, G5: 2.00 mm < RD, with G1–G4 being the absorbing roots and G5 being the transporter roots [32] (Arima et al., 2013); thus, root morphological indicators were analyzed at different diameter classes and functional divisions [33] (McCormack et al., 2015).

2.3.5. Physical and Chemical Properties of Substrates

At 30 days, the seedling substrates of each treatment were collected, naturally dried, ground through the 400-mesh sieve, and then stored. The EC and pH of the substrates, carbon content, and urease (URE), sucrase (SUC), catalase (CAT), and alkaline phosphatase (ALP) activities were measured. The substrate and ultrapure water (measured EC value of 0.973 mS/m and pH of 6.45) were mixed thoroughly at the ratio of 1:10, and the filtrate was collected. The EC and pH were determined using a Remagnet DDS-307A conductivity meter (Shanghai Yidian Scientific Instruments Co., Ltd., Shanghai, China) and a PB-10 PH meter (Sartorius Corporation, Göttingen, Germany), respectively. The total carbon (TC) and inorganic carbon (IC) contents of the substrate were measured using a total organic carbon (TOC) analyzer (Shimadzu Corporation, Kyoto, Japan), and the difference between the two indicators was the TOC content. The URE, SUC, CAT, and ALP activities of the substrates were determined by the phenol–sodium hypochlorite colorimetric method, 3,5-dinitrosalicylic acid colorimetric method, potassium permanganate titration method, and sodium benzene phosphate colorimetric method, respectively [34] (Li et al., 2019).

2.3.6. Determination of Elemental Content of Plants

Dried samples of the seedling roots, stems, and leaves obtained using the method in Section 2.3.1 were subjected to ablation using the H₂SO₄–H₂O₂ ablation method; the whole nitrogen and phosphorus contents of each part were determined using an Auto Analyzer 3 flow analyzer (SEAL Analytical, Ltd., Norderstedt, Germany), whereas the whole potassium content of each part was determined using a M410 BlueNotes flame photometer (Sherwood Scientific, Ltd., Cambridge, UK) [35] (Gallardo et al., 2021).

$$\mathrm{EAEO}=\mathrm{ECPUWEO}\times \mathrm{DWEO}$$

(10)

$$\mathrm{EDREO}=\frac{\mathrm{EAEO}}{\mathrm{EAWP}}$$

(11)

In Equations (10) and (11), EAEO is the elemental accumulation of each organ, ECPUWEO refers to the elemental content per unit weight of each organ, DWEO denotes the dry weight of each organ, EDREO represents the elemental distribution rate of each organ, and EAWP corresponds to the elemental accumulation of the whole plant.

2.4. Data Processing and Statistical Analysis

Data were organized using Excel 2016 (Microsoft, USA), plotted in Excel 2016, Word 2016 (Microsoft, USA), and Origin 2022 (OriginLab, USA), and analyzed using SPSS 26.0 (IBM, USA) software for analysis of variance and least significant difference analysis (p < 0.05), and data were expressed as “mean ± standard deviation” [36] (Meng et al., 2022). Correlation analyses of the substrates’ physical and chemical properties, root development strategy, nutrient accumulation distribution and plant morphology, growth function, and SI were performed using Pearson correlation. Systematic cluster analysis in Origin 2022 was used to classify the root morphological indicators and select the characteristic variables to investigate the relationship between substrate physical and chemical properties (SI) and nutrient uptake of tomato seedlings, using redundancy analysis (RDA) in Canoco 5 (http://www.canoco5.com/ accessed on 12 June 2021). The SI vectors were calculated using the eigenvectors of the matrix physical and chemical property indicators with significant effects (p < 0.05) in the RDA and weighted by the conditional effects (Ce). Equation (12) was applied as follows:

$$\overrightarrow{\mathrm{SI}}={{\displaystyle \sum }}_1^m\frac{C{e}_m}{{{\displaystyle \sum }}_1^{m}C{e}_m}\times \overrightarrow{S_m}$$

(12)

In Equation (12), $\overrightarrow{\mathrm{SI}}$ is the eigenvector of the substrate physical and chemical property representative indicators, $\overrightarrow{S_m}$ is the eigenvector of the “m”th substrate physical and chemical property indicators, with a significant effect on tomato seedling root system indicators and nutrient uptake, and $C{e}_m$ is the conditional degree of explanation of $\overrightarrow{S_m}$, where m ≥ 1.

3. Results Analysis

3.1. Effect of Bacterial Agentbacterial Agent Dosage on the Number of Leaves of Tomato Seedlings

The addition of Bacillus methylotrophicus to the substrate promoted the growth rate of tomato seedlings, and the number of leaves of tomato seedlings showed an increasing and then a decreasing trend with the amount of bacterial agent. At 30 days, the number of leaves under T4 treatment was optimal, with a significant increase of 60.65% compared with that in the CK. The effects of T5 and T4 were not significantly different, and the average number of leaves under T1, T2, T3, and T6 increased significantly by 27.69%, 41.88%, 49.21%, and 45.81%, respectively, compared with that in the CK (Figure 1).

The one-dimensional linear fit equation for the number of leaves of tomato seedlings and the number of days of growth had a high fit (R² = 0.9757–0.9956) (Table S1). At 30 days, the slope of the fitted equation for T4 was the highest and was 1.69 times higher than that of the CK, followed by that for T5, which was 1.66 times higher than that of the CK. The slopes of the fitted equations for T1, T2, T3, and T6 treatments were 1.27, 1.46, 1.52, and 1.51 times higher than that of the CK, respectively. These results indicated that the addition of bacterial agent to the substrate promoted the appearance of true leaves and facilitated the spread of true leaves in tomato seedlings, and the effect was influenced by the amount of bacterial agent administered.

3.2. Effect of Bacterial Agent Dosage on Above-Ground Morphological Indices of Tomato Seedlings

All Bacillus methylotrophicus treatments increased the plant height, stem diameter, and leaf area of tomato seedlings. T2, T3, and T4 treatments yielded the best plant height, which increased by 36.42%, 32.35%, and 36.07%, respectively, compared with that of the CK. Meanwhile, the plant height under T5, T6, and T1 treatments also increased by 14.22–27.34%, compared with that in the CK. The stem diameter and leaf area under T4 treatment were the largest, increasing by 21.33% and 51.31%, respectively, compared with those in the CK. Meanwhile, the stem diameter and leaf area under other treatments with added bacterial agents increased by 2.00–17.33% and 18.23–50.78%, respectively, compared with those in the CK (Figure 2).

3.3. Effect of Bacterial Agent Dosage on the Biomass of Tomato Seedlings

With the increase in Bacillus methylotrophicus dosage, the fresh weight of all organs and whole plant of tomato seedlings showed an increasing and then decreasing trend. Except under T1 and T6 treatments, the fresh weights of each organ and whole plant in all treatments were significantly greater than those in CK, with T4 showing the best effect (

Table 1. Effect of different inoculant dosages on the fresh weight of tomato seedlings.

Treatment	Fresh Weight (g/strain)				Fresh Weight Proportion (%)			Root–Shoot Ratio (Fresh)
	Root	Stem	Leaf	Total	Root	Stem	Leaf
CK	0.28 ± 0.01 cd	0.92 ± 0.25 e	0.73 ± 0.17 d	1.93 ± 0.41 e	15.16 ± 3.74 a	47.30 ± 3.06 d	37.54 ± 0.68 b	0.18 ± 0.05 a
T1	0.29 ± 0.02 cd	0.81 ± 0.04 e	1.14 ± 0.09 b	2.24 ± 0.14 e	12.95 ± 0.03 a	36.03 ± 0.67 e	51.03 ± 0.64 a	0.15 ± 0.00 ab
T2	0.31 ± 0.05 bcd	2.10 ± 0.04 ab	1.22 ± 0.04 ab	3.62 ± 0.13 ab	8.41 ± 1.04 b	57.91 ± 0.95 a	33.68 ± 0.09 d	0.09 ± 0.01 c
T3	0.32 ± 0.03 abc	1.81 ± 0.09 c	1.10 ± 0.06 bc	3.23 ± 0.17 c	9.79 ± 0.72 b	56.16 ± 0.94 abc	34.05 ± 0.22 d	0.11 ± 0.01 c
T4	0.36 ± 0.02 a	2.24 ± 0.09 a	1.35 ± 0.03 a	3.96 ± 0.07 a	9.04 ± 0.60 b	56.71 ± 1.57 ab	34.25 ± 1.04 d	0.10 ± 0.01 c
T5	0.35 ± 0.02 ab	1.90 ± 0.14 bc	1.23 ± 0.06 ab	3.48 ± 0.08 bc	10.07 ± 0.65 b	54.45 ± 3.01 bc	35.48 ± 2.36 cd	0.11 ± 0.01 bc
T6	0.26 ± 0.01 d	1.41 ± 0.11 d	0.98 ± 0.07 c	2.65 ± 0.18 d	9.85 ± 0.45 b	53.03 ± 0.39 c	37.12 ± 0.05 bc	0.11 ± 0.01 c

Note: Tomato seeds were sown in substrates mixed with seven Bacillus methylotrophicus dosages (0, 0.25, 0.50, 0.75, 1.00, 1.25, and 1.50 g/strain), which were recorded as CK, T1, T2, T3, T4, T5, and T6, respectively), with three replications of each treatment, that is, one for each cavity tray. Different lowercase letters after data mean significant difference at p < 0.05.

). The addition of bacterial agent also changed the ratio of fresh weight of different organs of tomato seedlings, and the root–shoot ratio (R:S) (fresh) decreased significantly, except under T1 treatment, where the percentage of fresh weight of leaves increased significantly compared with the CK. All other treatments with bacterial agent addition increased the percentage of the fresh weight of stems. Thus, the addition of bacterial agent increased the fresh biomass of below- and above-ground organs of tomato seedlings, but the promotion effect on above-ground organs was greater than on the below-ground ones, increasing the leaf fresh weight at lower dosages (T1) and stem fresh weight at medium to high dosages (T2–T6).

Meanwhile, with the increased amount of agents, the tomato seedlings showed the same trend of dry matter accumulation in all organs and the whole plant, with improved results in the T3, T4, and T5 treatments. The R:S (dry) was also significantly reduced compared with that in the CK. The addition of bacterial agent also promoted dry matter accumulation in the below- and above-ground organs of tomato seedlings, with a greater promotion effect on above-ground organs than below-ground ones (

Table 2. Effect of different inoculant dosages on the dry weight of tomato seedlings.

Treatment	Dry Weight (10−1 g/strain)				Dry Weight Proportion (%)			Root–Shoot Ratio (Dry)
	Root	Stem	Leaf	Total	Root	Stem	Leaf
CK	0.17 ± 0.01 cd	0.44 ± 0.17 c	0.74 ± 0.17 c	1.34 ± 0.29 c	12.94 ± 3.00 a	31.88 ± 4.60 d	55.18 ± 1.60 abc	0.15 ± 0.04 a
T1	0.17 ± 0.01 cd	0.53 ± 0.02 bc	0.89 ± 0.02 b	1.58 ± 0.04 bc	10.73 ± 0.32 ab	33.16 ± 0.03 cd	56.11 ± 0.29 ab	0.12 ± 0.00 b
T2	0.19 ± 0.02 bc	0.84 ± 0.00 a	1.17 ± 0.07 a	2.20 ± 0.03 a	8.44 ± 1.15 c	38.28 ± 0.88 a	53.28 ± 2.03 c	0.09 ± 0.01 b
T3	0.21 ± 0.01 ab	0.79 ± 0.03 a	1.19 ± 0.05 a	2.19 ± 0.04 a	9.48 ± 0.34 bc	36.01 ± 1.63 abc	54.50 ± 1.37 bc	0.10 ± 0.00 b
T4	0.20 ± 0.02 ab	0.87 ± 0.03 a	1.29 ± 0.04 a	2.37 ± 0.04 a	8.58 ± 0.43 bc	36.92 ± 1.30 ab	54.50 ± 1.30 bc	0.09 ± 0.01 b
T5	0.21 ± 0.02 a	0.76 ± 0.00 a	1.25 ± 0.09 a	2.22 ± 0.09 a	9.44 ± 0.54 bc	34.14 ± 1.75 bcd	56.41 ± 1.28 ab	0.10 ± 0.01 b
T6	0.16 ± 0.00 d	0.59 ± 0.05 b	1.00 ± 0.07 b	1.75 ± 0.09 b	9.15 ± 0.59 bc	33.63 ± 0.49 bcd	57.22 ± 0.10 a	0.10 ± 0.01 b

Note: Tomato seeds were sown in substrates mixed with seven Bacillus methylotrophicus dosages (0, 0.25, 0.50, 0.75, 1.00, 1.25, and 1.50 g/strain), which were recorded as CK, T1, T2, T3, T4, T5, and T6, respectively), with three replications of each treatment, that is, one for each cavity tray. Different lowercase letters after data mean significant difference at p < 0.05.

).

3.4. Effect of Bacterial Agent Dosage on Chlorophyll Content of Tomato Seedlings

A great difference was observed in the leaf dry-to-fresh weight ratio (Figure 3a), and only the chlorophyll b content per unit dry weight was significantly positively correlated with the chlorophyll b content per unit fresh weight (p ≤ 0.05). Meanwhile, the chlorophyll a and a + b contents per unit dry weight were only highly and significantly negatively correlated with the leaf dry and fresh weight ratio (p ≤ 0.01). This finding indicated that the difference in leaf water content had a great effect on the chlorophyll content and that the correlation between chlorophyll a and a + b contents strengthened at the dry weight, changing from a significant positive correlation at the fresh weight to a highly significant positive correlation at the dry weight. The correlation between chlorophyll b and a + b contents was significant in both cases and did not change significantly (Figure 3f).

The contents of chlorophyll a, b, and a + b demonstrated a consistent trend with the increase in Bacillus methylotrophicus dosage per unit fresh weight, i.e., they decreased from the CK to T2, increased from T3 to T4, and decreased again and leveled off from T5 to T6 treatments. Only the chlorophyll a, b, and a + b contents of T4 were always at a higher level (Figure 3b–d). The trend of chlorophyll content per unit dry weight was similar to that per unit fresh weight, and the contents of chlorophyll a, b, and a + b were always higher in T1 and T4 treatments than in the CK. Meanwhile, only the chlorophyll a/b values in T2 and T3 treatments were not significantly different from those in the CK, whereas the chlorophyll a/b values in all other treatments decreased substantially compared with those in the CK (Figure 3b–e).

In summary, significant differences were observed in the dry and fresh weights of leaves under different treatments, thus seriously affecting the magnitude of chlorophyll content. The addition of Bacillus methylotrophicus not only affected the chlorophyll content in tomatoes, but also changed the chlorophyll a/b values. Only T4 had a high chlorophyll content at fresh and dry weights.

3.5. Effect of Bacterial Agent Dosage on the G Value and Seedling Strength Index of Tomato Seedlings

With the increase in the amount of Bacillus methylotrophicus, the G value of tomato seedlings increased and then decreased, with the maximum value observed under T4 treatment and the minimum in the CK. The addition of bacterial agent increased the G value by 51.85% (17.78% to 75.56%) on average. The SI was smaller in T4 and T5 treatments and greater in T2. T4 and T5 treatments improved the SI by 15.77% and 15.35%, respectively, compared with that in the CK, and the magnitude of the SI was only influenced by the combination of each calculated component. Therefore, the combined G value and seedling index showed the best results under T4 treatment, when using 50-hole seedling trays (Figure 4).

3.6. Effect of Bacterial Agent Dosage on the Root Morphology of Tomato Seedlings

With increased dosage of Bacillus methylotrophicus, the trends in the root morphology of tomato seedlings can be divided into four classes as follows (Figure 5):

For class A, RL, RA, RD, RF, RBI, and RV all increased before decreasing, and all these indexes were higher than those in CK after the bacterial agent treatment. Based on the effects, these indexes were further subdivided into the following three categories: a: the bacterial agent treatment was more effective on RT, RF, and RBI at low concentrations, with the greatest effect being observed on RT at T1, increasing by 29.08% compared with that in the CK, and the T2 treatment showing the greatest effect on RF and RBI, which increased by 65.48% and 37.76% compared with those in the CK, respectively. b: The treatment was more effective on RA, RD, and RV at medium-to-high concentrations, with better results observed under T5 treatment and with values increasing by 55.22%, 27.41%, and 98.21%, respectively, compared with those in the CK. c: A significant effect was observed for RL under all treatments except T6, with better results detected in T1 and T3–T5 treatments and an average increase of 23.00% compared with that in the CK.

For class B, RTID, RFN, RT, and RTD all presented a decreasing trend, and these indexes were all lower than those in the CK after the addition of bacterial agent, with values decreasing by 1.61–38.31%, 14.00–42.40%, and 1.61–38.31%, respectively. Meanwhile, RT was promoted at low and medium bacterial agent concentrations, with the values under T1–T4 treatments increasing by 0.66–29.08%. RT was suppressed at high bacterial agent concentrations, with T5 and T6 treatments decreasing the values by 20.23% and 33.72%, respectively, compared with the CK.

For class C, increased SRA and RD were observed, with both showing the highest increase under T6 treatment compared with those in the CK (31.66% and 32.76%, respectively).

For class D, the effect on SRL was first inhibited and then promoted, with a maximum value observed in T1 treatment and an increase of 15.81% compared with the CK.

The evaluation of plant root development by simple root morphological indicators is difficult. Thus, the plasticity response of tomato seedling roots under the effect of different doses of Bacillus methylotrophicus must be determined in terms of the fine root structure at different diameter levels.

3.7. Plasticity of Fine Root Structure of Tomato Seedlings at Different Diameter Levels in Response to Bacterial Agent Dosage

From G1 to G4, the larger the diameter class, the smaller the RL, RA, and RT of tomato seedlings and the smaller their percentage in the total root system. The RL, RA, and RV of G1–G4 (RD < 2.00 mm) accounted for 99.41–99.73%, 92.60–96.68%, and 61.68–79.37% of the total root system (Figure 6), respectively, and the number of RT was mainly distributed in G1–G2 (RD < 1.00 mm) (99.99–100.00%), indicating that the roots of the tomato seedlings in the experiment were mainly absorbing roots (

Table 3. Ratio of G1–G2 to G5 root indicators of tomato seedlings.

Treatment	(G1-G2)/G5
	Root Length	Root Area	Root Volume
CK	373.17 ± 22.23 a	29.02 ± 1.21 a	3.27 ± 0.54 a
T1	373.70 ± 4.37 a	27.36 ± 2.41 a	2.95 ± 0.41 ab
T2	159.71 ± 2.48 c	11.31 ± 0.82 c	1.18 ± 0.07 d
T3	255.63 ± 18.52 b	19.58 ± 1.35 b	2.41 ± 0.09 bc
T4	251.43 ± 20.30 b	16.90 ± 0.70 b	1.85 ± 0.08 c
T5	236.03 ± 47.10 b	17.94 ± 0.96 b	2.01 ± 0.14 c
T6	284.85 ± 31.10 b	28.17 ± 3.73 a	3.48 ± 0.48 a

Note: Tomato seeds were sown in substrates mixed with seven Bacillus methylotrophicus dosages (0, 0.25, 0.50, 0.75, 1.00, 1.25, and 1.50 g/strain), which were recorded as CK, T1, T2, T3, T4, T5, and T6, respectively), with three replications of each treatment, that is, one for each cavity tray. Different lowercase letters after data mean significant difference at p < 0.05.

).

The trends of RL, RA, RV, and RT of G1 and G2 fine roots under different Bacillus methylotrophicus treatments were consistent with the total root system (Figure 5 and Figure 6). The addition of bacterial agent promoted the growth of all four indices, with RL, RA, and RV of G1 and G2 fine roots being the largest under T5 treatment and the smallest in the CK. Meanwhile, RT was the highest under T1 treatment. At RD < 1.00 mm, RL, RA, and RV were 1.26, 1.59, and 1.99 times higher under T5 treatment, 1.25, 1.46, and 1.75 times higher in T3 treatment, and 1.22, 1.41, and 1.68 times higher in T4 treatment, and all of these values were significantly higher than those observed in the CK. The RL, RA, and RV of G5 (RD ≥ 2.00 mm) transport roots were higher in all treatments with the addition of the bacterial agent, with T2–T5 promoting RL, RA, and RV of tomato seedlings most significantly with values 1.76–2.60, 2.07–3.13, and 2.18–3.77 times higher than those in CK, respectively, with T2 presenting the highest values. In summary, the root system of tomato seedlings was mainly composed of fine absorbing roots with RD < 1.00 mm, and the addition of bacterial agent can significantly increase the RL, RA, and RV, allowing seedlings to better absorb nutrients, whereas the application of bacterial agent also significantly promoted the development of transporting roots with RD ≥ 2.00 mm, which can well support the transport of nutrients from the lower-ground parts to the above-ground ones.

3.8. Effect of Bacterial Agent Dosage on the Accumulation and Distribution of Nitrogen, Phosphorus and Potassium in Tomato Seedlings

The accumulation of nitrogen, phosphorus, and potassium in all tomato organs and the whole plant showed an increasing, followed by a decreasing, trend as the dosage of bacterial agent increased (

Table 4. Mineral nutrient accumulation and distribution ratio in various organs of tomato seedlings under different treatments.

Nutrient	Treatment	Accumulation (mg/strain)			Total	Distribution Ratio (%)
		Root	Stem	Leaf		Root	Stem	Leaf
Total nitrogen	CK	0.48 ± 0.01 b	1.00 ± 0.12 c	3.36 ± 0.46 c	4.84 ± 0.57 c	9.98 ± 0.21 a	20.69 ± 2.45 bc	69.32 ± 9.40 a
	T1	0.40 ± 0.03 c	0.94 ± 0.02 c	3.34 ± 0.25 c	4.68 ± 0.25 c	8.56 ± 0.73 b	20.15 ± 0.36 bc	71.29 ± 5.36 a
	T2	0.44 ± 0.04 bc	1.39 ± 0.13 b	4.55 ± 0.41 b	6.38 ± 0.33 b	6.96 ± 0.65 cde	21.80 ± 2.07 b	71.24 ± 6.43 a
	T3	0.50 ± 0.02 b	1.39 ± 0.09 b	4.49 ± 0.22 b	6.37 ± 0.27 b	7.80 ± 0.25 bc	21.76 ± 1.36 b	70.44 ± 3.46 a
	T4	0.44 ± 0.01 bc	1.85 ± 0.29 a	4.49 ± 0.92 b	6.78 ± 0.67 b	6.55 ± 0.14 de	27.26 ± 4.26 a	66.19 ± 13.55 a
	T5	0.59 ± 0.07 a	1.68 ± 0.09 a	5.56 ± 0.38 a	7.83 ± 0.33 a	7.53 ± 0.93 bcd	21.43 ± 1.14 b	71.04 ± 4.84 a
	T6	0.39 ± 0.06 c	1.09 ± 0.16 c	4.83 ± 0.17 ab	6.31 ± 0.21 b	6.22 ± 0.88 e	17.26 ± 2.53 c	76.52 ± 2.64 a
Total phosphorus	CK	0.23 ± 0.01 a	0.51 ± 0.03 c	0.92 ± 0.12 d	1.66 ± 0.16 c	13.72 ± 0.40 a	30.66 ± 2.09 ab	55.62 ± 7.11 b
	T1	0.19 ± 0.02 b	0.56 ± 0.01 bc	1.04 ± 0.07 bcd	1.79 ± 0.07 bc	10.80 ± 0.89 b	31.24 ± 0.51 ab	57.96 ± 4.09 b
	T2	0.17 ± 0.02 bc	0.66 ± 0.05 a	1.22 ± 0.09 ab	2.05 ± 0.05 a	8.23 ± 0.74 cd	32.32 ± 2.58 a	59.45 ± 4.36 b
	T3	0.18 ± 0.00 b	0.60 ± 0.04 ab	1.18 ± 0.05 ab	1.95 ± 0.07 ab	9.31 ± 0.23 c	30.47 ± 1.83 ab	60.21 ± 2.37 b
	T4	0.16 ± 0.00 c	0.68 ± 0.10 a	1.12 ± 0.20 abc	1.96 ± 0.13 a	8.11 ± 0.12 d	34.57 ± 4.86 a	57.32 ± 10.22 b
	T5	0.18 ± 0.02 bc	0.53 ± 0.03 bc	1.26 ± 0.07 a	1.97 ± 0.08 a	9.08 ± 1.01 cd	27.03 ± 1.40 b	63.89 ± 3.69 ab
	T6	0.09 ± 0.01 d	0.30 ± 0.04 d	0.97 ± 0.03 cd	1.36 ± 0.04 d	6.71 ± 0.71 e	21.75 ± 2.63 c	71.54 ± 2.04 a
Total potassium	CK	0.71 ± 0.03 b	3.02 ± 0.36 c	2.87 ± 0.35 d	6.60 ± 0.16 d	10.71 ± 0.39 a	45.75 ± 5.41 ab	43.54 ± 5.27 bc
	T1	0.68 ± 0.04 b	3.60 ± 0.06 c	3.56 ± 0.21 c	7.84 ± 0.23 c	8.65 ± 0.55 b	45.92 ± 0.78 ab	45.43 ± 2.62 bc
	T2	0.70 ± 0.05 b	5.33 ± 0.42 ab	4.96 ± 0.41 a	10.99 ± 0.83 ab	6.36 ± 0.49 d	48.54 ± 3.78 a	45.11 ± 3.76 bc
	T3	0.78 ± 0.03 a	5.13 ± 0.29 b	4.94 ± 0.25 a	10.85 ± 0.43 ab	7.19 ± 0.24 c	47.30 ± 2.70 a	45.51 ± 2.32 bc
	T4	0.66 ± 0.00 b	6.14 ± 0.97 a	4.61 ± 0.72 ab	11.41 ± 0.43 a	5.81 ± 0.00 d	53.77 ± 8.46 a	40.41 ± 6.27 c
	T5	0.69 ± 0.05 b	4.88 ± 0.32 b	4.97 ± 0.24 a	10.54 ± 0.14 b	6.56 ± 0.47 cd	46.28 ± 3.06 ab	47.16 ± 2.26 b
	T6	0.50 ± 0.05 c	3.04 ± 0.35 c	4.25 ± 0.16 b	7.79 ± 0.24 c	6.45 ± 0.63 cd	39.04 ± 4.50 b	54.52 ± 1.99 a

Note: Tomato seeds were sown in substrates mixed with seven Bacillus methylotrophicus dosages (0, 0.25, 0.50, 0.75, 1.00, 1.25, and 1.50 g/strain), which were recorded as CK, T1, T2, T3, T4, T5, and T6, respectively), with three replications of each treatment, that is, one for each cavity tray. Different lowercase letters after data mean significant difference at p < 0.05.

). The tomato seedlings accumulated the highest total N and P in all leaves; The T5 and T6 treatments accumulated the most total K in the leaves, while the other treatments accumulated more total K in the stem. The whole plant N, P, and K accumulations in T2–T5 treatments were all higher than those in the CK, accumulating 31.60% to 61.75% more N, 17.86% to 23.43% more P and 59.63% to 72.96% more K. N accumulation was the highest in T5 treatment, followed by that under T4 treatment, with values 61.75% and 40.06% higher than those in CK, respectively. P accumulation in T2, T5, and T4 treatments increased by 23.43%, 19.03%, and 18.34%, respectively, compared with that in the CK. K accumulation was the highest in T4 treatment, increasing by 72.95% compared with that the CK. Meanwhile, the application of Bacillus methylotrophicus greatly changed the distribution rate of elements in the underground and aboveground parts, compared with the CK. A total of 1.43–3.76% N, 2.92–7.01% P, and 2.06–4.90% K were transferred from the underground to the aboveground parts. The proportions of N and P transported were the highest under T6 treatment, with values that were 3.76% and 7.01% higher than those in the CK, respectively, followed by those in T4 treatment. The proportion of K transported was the highest at T4, with a 4.90% higher value than that in the CK, followed by that in T2 treatment. The addition of bacterial agent promoted the accumulation of total N, P, and K in the tomato seedlings and induced increased nutrient transfer from the lower ground parts to the above-ground ones, which was beneficial to the above-ground growth of seedlings. N, P, and K all had a high percentage of translocation under T4 treatment, and the accumulation of the whole plant was also very high.

3.9. Effect of Bacterial Agent on the Physicochemical Properties of the Substrate

As shown in

Table 5. Effect of different inoculant dosages on physical and chemical properties of the substrate.

Treatment	EC (10−1 mS/m)	pH	Substrate Carbon Content (%)
			Total Carbon	Inorganic Carbon	Total Organic Carbon	Inorganic Carbon/Total Organic Carbon
Original substrate	150.00 ± 3.00 f	6.21 ± 0.03 d	29.65 ± 1.13 b	0.00 ± 0.00 d	29.65 ± 1.13 b	0.00 ± 0.00 e
CK	449.33 ± 6.11 d	5.18 ± 0.03 e	28.33 ± 0.95 bc	0.00 ± 0.00 d	28.33 ± 0.95 bc	0.00 ± 0.00 e
T1	346.00 ± 3.61 e	6.24 ± 0.15 d	26.37 ± 0.22 e	0.00 ± 0.00 d	26.37 ± 0.22 de	0.00 ± 0.00 e
T2	440.67 ± 6.11 d	7.44 ± 0.03 ab	31.50 ± 0.79 a	0.00 ± 0.00 d	31.50 ± 0.79 a	0.00 ± 0.00 e
T3	476.33 ± 13.05 c	7.13 ± 0.08 c	29.31 ± 0.56 b	0.05 ± 0.01 d	29.28 ± 0.53 b	0.15 ± 0.02 d
T4	490.33 ± 12.74 c	7.53 ± 0.06 a	27.83 ± 0.29 cd	0.14 ± 0.01 c	27.74 ± 0.22 cd	0.49 ± 0.02 c
T5	553.00 ± 16.09 b	7.54 ± 0.04 a	26.84 ± 0.42 de	0.57 ± 0.05 b	26.28 ± 0.37 e	2.15 ± 0.17 b
T6	868.00 ± 17.09 a	7.39 ± 0.03 b	23.77 ± 1.27 f	0.93 ± 0.07 a	23.14 ± 1.35 f	4.10 ± 0.01 a

Note: Tomato seeds were sown in substrates mixed with seven Bacillus methylotrophicus dosages (0, 0.25, 0.50, 0.75, 1.00, 1.25, and 1.50 g/strain), which were recorded as CK, T1, T2, T3, T4, T5, and T6, respectively), with three replications of each treatment, that is, one for each cavity tray. Different lowercase letters after data mean significant difference at p < 0.05.

, at 30 days, the EC values of all treated substrates were significantly higher than those before the start of the experiment, and the higher the amount of Bacillus methylotrophicus, the greater the EC values of the substrates, except under T1 and T2 treatments. The EC values of T3–T6 substrates were significantly higher than those in the CK by 6.01–93.18%. The pH of substrates in the CK were significantly lower than those at the start of the experiment, whereas the pHs of bacterial agent-treated substrates were all higher than that at the start of the experiment, except for those under T1 treatment, in which the pH was weakly acidic (6.24), and T2–T6, in which the pH was weakly alkaline (7.13–7.54). Meanwhile, with the increase in the bacterial agent dosage, the TC (organic carbon) content of the substrate showed an increasing and then decreasing trend, with the maximum value observed in T2 and the minimum under T6 treatment. The IC content of the substrate was detected only in the substrate with the highest bacterial agent content (T3–T6) and gradually increased with the dosage of bacterial agent, with the maximum value observed under T6 treatment and 0 in CK, T1, and T2 treatments, because the IC content in the substrate was notably lower than the organic carbon content. Thus, the trend of IC/TOC value was consistent with that of the IC content.

The addition of bacterial agent to the substrate significantly increased the activities of substrates URE, SUC, and CAT, but had no effect on the activity of substrate ALP. With the increase in bacterial agent dosage, the URE activity increased consistently and peaked under T6 treatment. The SUC and CAT activities increased first and then decreased. The SUC activity increased by 253.01–320.39% from T2 to T6 treatments compared with that with the CK, and the maximum value was recorded under T5 treatment. The CAT activity under bacterial agent treatment increased by 8.92–20.86% and reached the maximum under T3 treatment (

Table 6. Effect of different inoculant dosages on the enzyme activity of the substrate.

Treatment	URE (mg/g/d)	SUC (mg/g/d)	CAT (mg/g/d)	ALP (mg/g/d)
Original substrate	-	-	-	-
CK	0.18 ± 0.03 d	13.34 ± 1.53 b	7.84 ± 0.77 d	0.78 ± 0.03 a
T1	0.64 ± 0.06 c	11.78 ± 1.67 b	8.28 ± 0.33 cd	0.71 ± 0.02 a
T2	0.73 ± 0.06 bc	54.89 ± 2.41 a	9.00 ± 0.12 ab	0.73 ± 0.05 a
T3	0.72 ± 0.05 bc	47.29 ± 5.73 a	9.48 ± 0.29 a	0.75 ± 0.01 a
T4	0.73 ± 0.02 bc	51.74 ± 5.73 a	9.06 ± 0.19 ab	0.69 ± 0.09 a
T5	0.83 ± 0.04 b	56.07 ± 8.44 a	9.15 ± 0.37 ab	0.69 ± 0.06 a
T6	1.18 ± 0.13 a	52.06 ± 7.47 a	8.54 ± 0.19 bc	0.71 ± 0.11 a

Note: Tomato seeds were sown in substrates mixed with seven Bacillus methylotrophicus dosages (0, 0.25, 0.50, 0.75, 1.00, 1.25, and 1.50 g/strain), which were recorded as CK, T1, T2, T3, T4, T5, and T6, respectively), with three replications of each treatment, that is, one for each cavity tray. Different lowercase letters after data mean significant difference at p < 0.05. “-” indicates that the data were not measured.

). In summary, the substrates possessed high EC and pH, IC content, URE, SUC, and CAT activities at high dosages of Bacillus methylotrophicus (0.75–1.50 g/strain, i.e., T3–T6).

3.10. Effect of Substrate Physicochemical Properties on Root Morphological Characteristics and Element Uptake of Tomato Seedlings

RDA was used to determine the effect of the substrate’s physicochemical properties on root system indicators and nutrient uptake of tomato seedlings. To facilitate the description of the analytic process, we first subjected the root system indicators to cluster analysis. As shown in Figure 7, the root system characteristics of tomato seedlings were divided into four classes. Class A indicators (RL, RF, and RBI) are related to the extended absorption range of the root system. Class B indicators (RA, RV, RD, and SRA) are directly related to root absorption. Class C indicators (RT, RTD, RTID, and RFN) can be used to directly characterize the root system response to different environmental conditions. Class D indicators are related to the root development strategy and reflect the ratio of fine absorbing roots to transporting roots; they include (G1–G2)/G5 RL, (G1–G2)/G5 RA, and (G1–G2)/G5 RV. RF, RD, RTD, and (G1–G2)/G5 RA can be used as representative indicators of each type of root system indicators, respectively, and the indicators of classes A and B and classes C and D can be further clustered.

No significant correlation was observed between class A root indexes (RA-RBI) and leaf number, plant height, stem thickness, leaf area, plant dry weight, G value, and SI, whereas class B root indexes (RA-SRA) and whole plant element accumulation only had a significant positive correlation with the above indicators and a significant negative correlation with R:S. Classes C (RT-SRL) and D root indexes ((G1–G2)/G5) were only significantly negatively correlated with the above indicators, but were significantly positively correlated with the R:S (Figure 8).

As presented in

Table 7. Explanation and significance tests for substrate properties in RDA.

Substrate Properties	Simple Effects			Conditional Effects
	Explanation %	Pseudo-F	p	Explanation %	Pseudo-F	p
SUC	43.50	14.60	0.002	43.50	14.60	0.002
pH	41.00	13.20	0.002	2.00	1.40	0.162
CAT	30.20	8.20	0.002	1.20	0.90	0.502
URE	24.90	6.30	0.002	4.80	3.00	0.014
EC	23.80	5.90	0.002	24.50	13.80	0.002
IC/TOC	23.90	6.00	0.004	1.40	1.00	0.344
IC	24.20	6.10	0.002	1.00	0.70	0.644
TOC	17.50	4.00	0.014	3.80	2.60	0.022
TC	17.40	4.00	0.022	0.60	0.40	0.886
ALP	5.00	1.00	0.41	1.80	1.30	0.248

Note: “simple effects” is the degree of explanation for each substrate’s properties individually, whereas “conditional effects” is the degree of explanation increased by the addition of substrate properties in descending order of explanation.

, under separate effects, all nine substrate indicators, except ALP activity, had significant effects on seedling root system indicators and nutrient accumulation (p < 0.05), and the individual explanatory degrees were as follows: SUC activity > pH > CAT activity > URE activity > IC > IC/TOC > EC (p < 0.01) > TOC > TC (p < 0.05) in descending order. Given the correlation between the indicators, the duplicate part of the explanatory degree under the conditional effect and re-screened SUC (conditional explanatory degree of 43.50%, same as below), EC value (24.50%, p < 0.01), URE (4.80%), and TOC (3.80%, p < 0.05) must be eliminated as the components of the substrate representative index SI. The combined conditional explanatory degree of these four indicators explained 74.70%, accounting for 90.54% of the total 84.60% conditional explanation degree of all matrix indicators, and it can represent the matrix indicators in the experiment and be used to calculate the representative index of matrix physicochemical properties (SI) by the weight of the conditional explanation degree.

The eigenvectors of SUC, EC, URE, TOC, and SI were in the second and third quadrants, and positive correlations were detected between classes A (represented by RF) and B root indicators, which were represented by the RD, whole plant nitrogen (TNA), phosphorus (TPA), and potassium (TKA) accumulation in tomato seedlings and SI in the figure. Meanwhile, class C was represented by RTD and class D root indicators by (G1–G2)/G5 RA. SI mainly had negative correlations. In addition, the correlations between the experimental treatments and SI were in the following descending order: T5 > T4 > T3 > T2 > T6 > CK > T1 (Figure 9).

4. Discussion

Dry matter accumulation and morphological indicators of seedlings are the most intuitive indicators of their growth and development and robustness. The treatment of maize seeds with interplant root-promoting bacteria, such as Lysinibacillus sphaericus, Paenibacillus alvei, and Bacillus safensis, significantly increased the final maize dry matter accumulation [37,38] (Breedt et al., 2017; Da Salvo et al., 2018). Bacillus subtilis treatment of sugarcane seedlings increased their total dry matter, tiller number, and tiller diameter by 13%, 37%, and 48%, respectively [39] (Santos et al., 2018). Antunes et al. [40] (2017) treated sugarcane with seven similar agents isolated from Bacillus and obtained similar results, reporting that Herbaspirillum seropedicae, Paenibacillus sp., and Burkholderia sp. and other PGPR can increase N fixation, dry matter accumulation, and crop yield in sorghum and maize [41] (Aquino et al., 2021). In this experiment, the number of leaves, plant height, stem thickness, leaf area, dry matter accumulation in each organ, G value, and SI of tomato seedlings increased and then decreased with the increased concentration of Bacillus methylotrophicus. That is, the growth effect of Bacillus methylotrophicus on tomato seedlings also showed growth inhibition at high doses and growth promotion at low doses.

PGPR can promote plant growth and development directly or indirectly by fixing nitrogen, mineralizing elements, changing the soil microorganism group, improving soil physical and chemical properties, enhancing soil fertility, secreting phytohormones, and secreting related regulatory substances [42,43,44] (Souza et al., 2015; Compant et al., 2019; Da Silva et al., 2020). Organic carbon is an important component in providing plant nutrients and energy for microbial biotic activities, and organic carbon mineralization is related to the supply of nutrient elements, maintenance of soil or substrate quality and carbon emission, and mainly involves microorganisms; when the nutrient status of the substrate is good, microorganism activity is high and mineralization is significant [45,46,47] (Manzoni et al., 2012; Fang et al., 2018; Bahadori et al., 2021). The substrate organic carbon (TOC) consists mainly of the TOC fraction of the original substrate, plant-derived carbon, and microbial carbon [48] (Liu, 2020). In this experiment, no IC was detected in the substrates of the control or low mycorrhizal-dosage treatments (0–0.50 g/strain), probably because the mineralization of substrates in these treatments was not evident, and the soil nutrient supply cannot meet the plant and microbial demand. The IC content increased with the continuous increase in bacterial agent dosage when the mineralization was evident, and the microorganisms were active. The appropriate dosage (0.50–1.00 g/strain) can increase plant-derived carbon by promoting plant growth and forming a good microbial community to maintain the balance of TOC and IC, without drastically damaging soil physicochemical properties and by guaranteeing normal root development. Although the high dosage (1.25–1.50 g/strain) of agent produces more IC, the microbial activity may be extremely high, and the physicochemical properties of the substrate are imbalanced, which is manifested by the decrease in TOC, high EC value, and reduced buffering capacity, which affect the normal growth and development of seedlings. Soil microorganisms produce major soil enzymes and are associated with numerous soil quality indicators [49] (Ghorbani-Nasrabadi et al., 2013), and several researchers have suggested that soil enzymes, such as URE, SUC, CAT, and enzyme ALP, can be used as a class of soil fertility indicators [50,51,52] (Rietz and Haynes, 2003, Karlen et al., 2008; Liu et al., 2017). In the experiment, the URE activity increased continuously with the increase in the amount of bacterial agent, whereas the SUC and CAT activities showed an increasing and then decreasing trend. The ALP activity was not significantly affected (Table 5 and Table 6). The SUC, EC value, URE, and TOC of the substrate in this experiment were identified as the main indicators of its physical and chemical properties (Table 7).

The root system is the direct pathway and the front end of plant nutrient uptake from soil, and it is most sensitive to the soil’s physicochemical properties and nutrient status [53] (Urgenson et al., 2012). Plant roots exhibit plasticity, and changes in the soil’s physicochemical properties and nutrient status can lead to changes in root development strategies [54] (Lobet et al., 2018), which generally include thick and short roots, a high number of lateral roots, and high dry matter accumulation when nutrients are adequate, and vice versa, when nutrients are deficient, and when the root to crown ratio increases [55,56] (Forde and Lorenzo, 2001; Fujimura et al., 2012). In this experiment, the substrate SUC, EC values, URE, and TOC characteristic vectors were weighted by the degree of explanation of the condition effect to form the substrate physicochemical property characteristic vector SI, which was positively correlated with A and B root indicators and seedling nutrient accumulation and negatively correlated with C and D root indicators, indicating that when the substrate fertility was adequate, tomato seedling roots increased in the number of lateral roots and root diameter and had a large absorption range [57] (Henke et al., 2014), with a high overall absorption capacity, resulting in the accumulation of more elements in the seedlings and enhanced transport capacity above ground [58] (Poorter et al., 2012), whereas when the substrate nutrient supply was problematic, the absorption area was reduced and became more sensitive to nutrients, with enhanced in situ utilization of nutrients in the root system and reduced upward transport capacity [59] (Hummel et al., 2007). The R:S increased, and the rapid aboveground plant growth was difficult to sustain [60] (McCormack and Guo, 2014) (Table 2, Table 3 and Table 4, Figure 5). The substrates under T5 and T4 treatments had high SUC, URE, and CAT activities, higher EC values, contained a certain amount of IC, and were in an environment conducive to the functioning of the root system. The tomato root system had a large absorption range and a strong overall absorption capacity, absorbing high amounts of N, P and K from the substrate and transporting high levels of nutrients from the lower to the above-ground parts (Figure 9), thus promoting above-ground seedling stem elongation and thickening, increasing the number of leaves, increasing dry matter accumulation, and improving seedling quality (Figure 8). The increase in soil enzyme activity stimulated plant root development and influenced the morphological development of the below- and above-ground parts, thus promoting plant growth and development. Compared with those in T5 treatment (1.25 g/strain), the root development strategy and nutrient uptake and transport of seedlings under T4 treatment (1.00 g/strain) were more balanced and economical. Therefore, the addition of 1.00 g/strain of Bacillus methylotrophicus is appropriate for growing tomato seedlings in 50-hole cavity trays.

5. Conclusions

Bacillus methylotrophicus can stimulate tomato seedling root development by accelerating the mineralization of organic carbon substrates, maintaining the balance of organic substrate and IC, and increasing soil enzyme activity. The most effective dose of Bacillus methylotrophicus treatment for tomato seedlings in 50-hole cavity trays was 1.00 g/strain.