Screening of Rhizosphere Microbes of Salt-Tolerant Plants and Developed Composite Materials of Biochar Micro-Coated Soil Beneficial Microorganisms

Abstract

To develop composite materials of biochar micro-coated soil beneficial microorganisms, soil samples were collected from the saline-alkali land of the Penghu in Taiwan. After isolation, purification and identification, a total of one Bacillus amyloliquefaciens and two Bacillus megaterium were identified as the source of beneficial microorganisms. The agricultural waste is collected and initially crushed, and then made into biochar through a series of thermal cracking processes. The specific surface area of biochar is more than 100 m²/g, the fixed carbon is more than 80%, the proportion of medium and large pores is more than 30%, the pH after adjustment is 7.3 ± 0.2, and the pore size of beneficial microorganisms grows inward, which is one of the beneficial microorganisms. The biochar micro-coated soil-beneficial microorganism composite material developed in this experiment can indeed help crops to overcome the stress of salt damage to a certain extent, and Bacillus amyloliquefaciens can indeed promote plant growth and help crops effects of over-salting adversity.

1. Introduction

The loss of agriculture caused by soil salinization is one of the problems faced by many countries. About 2000 hectares of agricultural land worldwide is affected by soil salinization every day, including in the United States, China, Australia and many parts of the Middle East, which cannot maintain crop yields due to severe soil salinity [1]. When excess sodium or sodium chloride accumulates in the soil, the salt builds up in the roots of the plant, and the plant needs more energy to grow. Globally, over 62 million hectares of irrigated land has become uncultivated [2]. In recent years, the tolerance of plants to abiotic stress caused by plant growth-promoting rhizobacteria (PGPR) has been increasingly concerned [3,4]. Related studies demonstrate that inoculation with arbuscular mycorrhizal fungi (Arbuscular mycorrhizal, AM) improved plant growth under salt stress; synergistic effects of Pseudomonas mendocina strains on soil aggregates; P. alcaligenes (PsA15), Bacillus polymyxa (BcP26) and Mycobacterium phlei (MbP18) were sufficiently tolerant to high temperature and salt concentrations, which endows them with a relatively shallow competitive advantage and enables them to survive in arid and saline-alkali soils, etc. [5,6,7,8].

Inoculating plants with rhizosphere probiotic strains can help plants increase their tolerance to salt stress and promote plant growth [9,10,11,12]. Among them, five kinds of salt-tolerant bacteria that promote plant growth have been shown to have significant effects on wheat growth in experiments, and these salt-tolerant bacterial strains can alleviate the growth of wheat seedlings under salt stress (80, 160 and 320 mM), compared with the uninoculated control group, the root length increased by 71.7% [13]. In order to effectively improve soil degradation, the application of biochar is currently a countermeasure in many countries. Biochar refers to a carbon-rich material produced by pyrolysis of biomass under low oxygen conditions. Biochar has good adsorption function due to the functional groups existing on the surface, which can adsorb and remove pollutants in soil and water [14,15]. Many studies have pointed out that biochar-added soils have improved properties such as pH value, cation exchange capacity (CEC), and water holding capacity (WHC) [16,17]. In addition, a large number of studies have confirmed that when plants face drought and salt stress, the application of biochar not only helps plants absorb nutrients, but also promotes plant growth and increases biomass [18,19,20,21,22]. Biochar contains high concentrations of N, P, Ca and K, which can directly provide soil nutrients. When used as a soil conditioner, biochar can increase the porous density of the soil, provide space for microbial growth, and improve aeration, water retention, nutrient preservation and enhance the activity of microorganisms and the growth rate of plants.

Biochar is not an organic fertilizer, but it has been shown in many studies to improve soil matrix properties. Among them, the mechanism of action between biochar, soil organic matter and soil microbes is one of the research objects of many researchers. However, the large number and diversity of soil microbes means the role of biochar in this environment and its related mechanisms to microbes is not fully elucidated. In 2014, Hammer et al. demonstrated for the first time that Arbuscular mycorrhiza (AM) can utilize biochar as a physical growth substrate and nutrient source [23].

Using scanning electron microscopy, arbuscular mycorrhizal fungal hyphae were observed growing on two contrasting types of biochar particles, which could attach to the inner and outer surfaces, respectively. If nutrients are added to the surface of nutrient-deficient biochar, the mycelium can be stimulated to expand. In addition, bacteria may also adsorb to the surface of biochar, making it less susceptible to leaching in soil [24], and application of biochar may also increase plant disease resistance, which is presumably caused by the release of biochar itself. Plant resistance was caused by less toxic plant compounds such as ethylene and propylene glycol, or by the presence of plant resistance-inducing probiotic Trichoderma spp. in biochar-treated soil [25]. In this study, by screening soil-entraining probiotics and preparing biochar with bacterial-carrying effect, a composite material of biochar micro-coated soil beneficial microorganisms was developed, and a pot experiment was conducted to evaluate the effect of resisting salt damage, which is the correlation between biochar and probiotics in the follow-up mechanism laying the foundation. The purpose of this study was to determine whether biochar coating with Bacillus amyloliquefaciens could promote plant growth and help crops survive the stress of salinity.

2. Materials and Methods

2.1. Screening of Beneficial Microorganisms in the Rhizosphere of Salt-Tolerant Plants

Before the experiment, the beneficial microorganisms in the rhizosphere of salt-tolerant plants (Ice flower) were screened from Huxi Village of Huxi Township in Penghu County, and the area was divided into Site 1, Site 2, Site 3, and Site 4.

Table 1. Soil test results.

Location	Soil pH Value	Soil EC Value (dS/m)
Site 1	7.3	4.3
Site 2	7.6	1.2
Site 3	8.1	0.2
Site 4	8.4	0.4

shows that the soil pH at the collection site is 7.3, 7.6, 8.1 and 8.4, the soil is alkaline, and the EC value is Electrical conductivity (EC). 4.3, 1.2, 0.2 and 0.4 dS/m, respectively. In subsequent experiments, the soil collected in area Site 1 was used as the saline soil sample. The collected soil was subjected to microbial isolation, treatment A (95 °C, 5 min) and B treatment (85 °C, 15 min), and a total of 78 strains were isolated (

Table 2. Isolation and purification of strains.

Location	Treatment A	Treatment B	Total
Site 1	5	3	8
Site 2	13	25	38
Site 3	7	2	9
Site 4	10	13	23

Treatment A: 95 °C, 5 min; Treatment B: 85 °C, 15 min.

) [26].

2.2. Identification of Bacterial Species

Through the results of Gram staining, it was preliminarily determined that Bacillus amyloliquefaciens (Figure 1), Bacillus megaterium B (Figure 2) and Bacillus megaterium B (Figure 3) were Gram-positive. In Bacillus amyloliquefaciens, the colony morphology is milky white, the colony grows close to the surface of the medium (PDA), and the colony is forked around the colony. After the ITS (Internal transcribed spacer, ITS) sequence analysis, it was identified as Bacillus amyloliquefaciens after comparison with the database (Bacillus amyloliquefaciens). The colony morphology of Bacillus megaterium A and B are goose yellow, and the colony grows in the medium (PDA), showing a protruding shape. After the colony grows, the middle will appear concave, and the protruding part is Michelin-shaped. After ITS sequence analysis and the data after library alignment, it was identified as Bacillus megaterium [27].

2.3. Amylolytic Activity Assay

Beneficial microorganisms were cultured in liquid overnight, and 10 μL was dropped into solid medium (beef extract 3 g/L, starch 10 g/L, agar 12 g/L, pH 7.5 ± 0.2). After culturing at 30 °C for 48 h, the Gram’s iodine (2 g KI, 1 g iodine, 300 mL dH₂O) was stained for one minute and observed for permeabilization circles [27].

2.4. Proteolytic Activity Assay

Beneficial microorganisms were cultured in liquid overnight, and 10 μL was dropped into solid medium (skim milk powder 28 g/L, casein enzymichydrolysate 5 g/L, Yeast extract 2.5 g/L, dextrose 1 g/L, agar 20 g/L, pH 7). After culturing at 30 °C for 48 h, it was observed for a permeabilization circle [27].

2.5. Cellulytic Activity Assay

Beneficial microorganisms were cultured overnight in liquid state, and 10 μL was dropped into solid medium (CMC 1%, peptone 3%, KH₂PO₄ 0.1%, MgSO₄ 0.01%, agar 2%, pH 6.8), and incubated at 30 °C for 48 h. It was then stained with iodine (2 g KI, 1 g iodine, 300 mL dH₂O) for five minutes and observed for a permeabilization circle [27].

2.6. Lipolytic Activity Assay

The lipolytic activity was determined as follows: beneficial microorganisms were cultured in liquid overnight, and 10 μL was added dropwise to solid medium (peptone 5 g/L, beef extract 3 g/L, tributyrin 10 mL/L, agar 20 g/L), and after 48 h of incubation at 30 °C, then observed for a permeabilization circle [27].

2.7. Phosphate Solubilization

Beneficial microorganisms were cultured in liquid state overnight, and 10 μL was dropped into solid medium (Glucose 10 g/L, Ca₃(PO₄)₂ 5 g/L, MgCl₂·6H₂O 5 mL/L, MgSO₄·7H₂O 0.25 g/L, KCl 0.2 g/L, (NH₄)₂SO₄ 0.1 g/L, agar 20 g/L), after culturing at 30 °C for 7 days, it was observed for a permeabilization circle [27].

2.8. Pathogenic Microbial Inhibition Test

10 μL of fermented beneficial microorganisms were dropped into NA culture base for three days at 30 °C. The tomato wilt fungus (Fusarium oxysporum f. sp. Lycopersici), which had been cultivated for two weeks, was cut out with a 5 mm punch and placed in the pre-cultured beneficial microorganism medium for confrontation culture. Bacillus amyloliquefaciens has an antagonistic inhibitory effect.

2.9. Biochar Process

Biomass raw materials (agricultural residues after pruning and discarded fruit branches: such as Syzygium samarangense, Leucaena leucocephala, and Ziziphus jujuba) are roughly crushed and then carbonized in a carbonization furnace; the carbonization conditions are 10~12.23 °C/min, and the temperature is raised to 500 °C, and the carbonization is completed after two hours. After mixing with crushed oyster shells at 1:1 (w/w), heating to 900 °C for activation and modification, reprocessing, crushing and sieving, the 1–2 mm biochar was selected as the biochar used in subsequent experiments [28].

2.10. The Process of Embedded Beneficial Microorganisms with Biochar

Each biochar with a particle size of 1~2 mm and the prepared NA culture medium were deposited into the incubation container at a ratio of 1:20 (w/v) and mixed evenly, the pH value was adjusted to 7.1~7.2 with 1N HCl; Sterilize for 15 to 20 min, the amount of cultured beneficial bacteria diluted by about 50 to 100 times was added, then embedded and cultivated in an incubator at a temperature of 30 °C and 120 rpm for about seven days. One gram of biochar was sampled to detect the microorganisms contained in it. When the amount reaches more than 10⁸ CFU/g, the production of biochar coated with beneficial microorganisms is completed.

2.11. Scanning Electron Microscope (SEM)

SEM specimen preparation: The biochar porous cross-section test is to solidify the biochar-embedded resin; it is carried out by micro-section, and the whole is not cut off. In addition to sample fixation, it is also important to ensure complete dehydration and drying in the SEM specimen preparation process. SEM mainly uses a tiny focused electron beam (Electron Beam) to scan the surface of the sample. The interaction between the electron beam and the sample will excite various signals, and SEM mainly collects the signals of secondary electrons for imaging. The observed sample must be conductive, so the observation of metal samples can be observed without special treatment. Non-conductors such as minerals, polymers, etc., need to be vacuum evaporated and coated with a metal film or carbon film with good conductivity, making another observation. Bio-related samples must be dehydrated and dried or frozen with liquid nitrogen, and finally subjected to vacuum evaporation gold treatment (the model of the SEM machine used is LEO-1530).

2.12. Pot Experiment of Tomato Wilt Fungus

After the seeds were primed for 72 h, they were planted in plastic seedling trays containing peat soil, with one seed per hole. After colonization, they were cultivated in a LED plant light shelf at room temperature (27 ± 2 °C) for 2–3 weeks, until the seedlings grow 3–4 true leaves. The strain of tomato wilt was cultured in 1/2 sugar-reduced PDA for two weeks, and the spores of the pathogen were washed with sterile water, and the spores were observed to be conidia under a light microscope, and prepared into 106 conidia/mL. The spore suspension was then ready for use. Tomato seedlings with 3–4 true leaves were planted in three-inch pots and treated with the conditions of each group. The addition ratio of biochar and biochar + microbe is 2% (w/w) (microbe in biochar is 10⁸ CFU/g). Three days after the completion of each treatment, 10 mL of the above tomato wilt spore suspension was inoculated by watering. Plants were kept at room temperature (27 ± 2 °C) and placed under the LED grow light shelf with daily light and dark for 12 h each.

3. Results and Discussion

3.1. Beneficial Microorganisms

In order to verify that the salt-tolerant beneficial microorganisms isolated from Penghu have sufficient conditions for subsequent experiments, we conducted a series of physiological and biochemical assays on the beneficial microorganisms in the early stage of the experiment. The amylolytic activity, cellulolytic activity, proteolytic activity, lipolytic activity and phosphorus-dissolving activity of beneficial microorganisms were measured (Figure 4, Figure 5, Figure 6, Figure 7 and Figure 8). After testing Bacillus amyloliquefaciens, Bacillus megaterium A and Bacillus megaterium B the amylolytic activity, proteolytic activity, cellulolytic activity test, Lipolytic activity test and phosphorus-dissolving activity, the experimental results show that Bacillus amyloliquefaciens has amylolytic activity, proteolytic activity, cellulolytic activity test and phosphorus-dissolving activity; Bacillus megaterium A It has proteolytic activity, cellulolytic activity test and phosphorus-solubilizing activity; Bacillus megaterium B has amylolytic activity, proteolytic activity, cellulolytic activity test and phosphorus-solubilizing activity (

Table 3. Physiological and biochemical assay results of beneficial microorganism.

Activity	Bacillus amyloliquefaciens	Bacillus megaterium A	Bacillus megaterium B
Amylolytic	+	−	+
Cellulolytic	+	+	+
Proteolytic	+	+	+
Lipolytic	−	−	−
Phosphorus solubilizing	+	+	+

). Based on the experimental results, the follow-up research will use two microorganisms, Bacillus amyloliquefaciens and Bacillus megaterium B, as the target for the development of beneficial microorganisms.

3.2. Biochar Preparation

To produce biochar suitable for this research purpose, we used agricultural residues such as Syzygium samarangense, Leucaena leucocephala, and Ziziphus jujuba as crude materials. After initial crushing and carbonization at 500 °C, biochar and crushed oyster shells are mixed 1:1, and then re-modified and activated at 900 °C. After crushing and screening, 1–2 mm biochar was screened for subsequent follow-up. It can be known from the fertility analysis that the biochar fired in the experiment basically has basal fertilizer parts (

Table 4. Specification of biochar microbial carrier material for resistance to salt stress-1.

Elements	N %	P %	K %	Ca ppm	Mg ppm	Mn pm	Fe ppm	Cu ppm	Zn ppm	Na ppm
Syzygium samarangense	1.01	0.24	1.16	25,232	3471	25.4	145.2	14.7	12.9	1162
Leucaena leucocephala	1.07	0.05	0.74	13,633	1838	22.3	187.3	6.4	12	677
Ziziphus jujuba	0.87	0.29	2.05	36,618	4692	72.6	188.1	23.6	32.1	1213

and

Table 5. Specification of biochar microbial carrier material for resistance to salt stress-2.

Elements	B ppm	Cd ppm	Cr ppm	Ni ppm	Pb ppm	As ppm	Hg ppm	pH (1:10)	Conductivity (1:10) mS/cm	CEC (cmol(+)/kg soil)
Syzygium samarangense	46.8	<0.009	6.52	5.7	<0.027	<0.005	0.52	10.6	3.13	10.4
Leucaena leucocephala	53	<0.009	32.8	20.7	<0.027	<0.005	0.3	10.2	1.8	6.8
Ziziphus jujuba	31.8	<0.009	37.4	26.9	<0.027	<0.005	0.52	10.9	5.33	13.3

), but the contained amount still needs to be fertilized as an aid if the effect of increasing crop yield is to be achieved [28].

In addition, the suitable conductivity for the growth of general crops is about 0.7~2.0 mS/cm. The conductivity of the biochar fired in the experiment is high, and the pH value of the biochar is highly alkaline due to the high temperature firing. Therefore, the biochar must be upgraded before application and bacteria loading (Table 5). Cation exchange capacity (CEC) refers to the cations adsorbed by soil particles and the internal and external surfaces of organic matter, so the exchangeable cations in the soil (especially in the soil solution) can be freely exchanged. The soil exchangeable one-hundredth mole of cations is expressed in units (cmol (+)/kg soil). Because of the large surface area of clay and humus in the soil, they carry more charges, which dominate the cation exchange capacity of the soil. Different organic matter and clay minerals have different CEC values. The larger the value, the stronger the ability of soil to adsorb cations.

The factors that generally affect the soil CEC value are: (1) the content of organic matter, (2) the type of clay minerals, (3) the content of clay particles, and (4) the degree of weathering of soil minerals. Taking kaolinite, illite (fine mica), montanite (or bentonite), vermiculite and organic matter as examples, the CECs are 3–30, 10–40, 80–120, 100–150 and >500~1000 cmol (+)/kg soil; therefore, the cation exchange capacity of the biochar produced in this experiment is weak (10.4, 6.8, 13.3 cmol (+)/kg soil), and may not be effective as a soil conditioner. However, biochar produced from agricultural wastes focuses on whether the pore size is suitable for the ingrowth of beneficial microorganisms and serves as a breeding base for beneficial microorganisms in the soil. Therefore, the “heterogeneous material binding effect” between beneficial microorganisms and biochar is the focus of this experiment. At the same time, when helping crops to survive the stress of salinity, the biochar produced in this experiment is not primarily to improve the soil effect, so it can be indirectly confirmed that the main reason for improving the growth of crops is from beneficial microorganisms.

3.3. Composite Materials of Biochar Micro-Coated Soil Beneficial Microorganisms

The pH value of the three biochars was adjusted to 7.3 ± 0.2, and the specific surface areas (BET) were 315.09, 213.53 and 318.53 m²/g, respectively. The specific surface area of biochar produced in general is about 20–50 m²/g, and the specific surface area of the biochar fired in this experiment is all greater than 100 m²/g. The possible reason is that the firing temperature is high (900 °C), and during the firing process, oyster shells (calcium carbonate mixture) are mixed, and carbon dioxide is released through high temperature sintering, which collides with the surface of biochar in a microscopic environment, forming numerous small pores (<2 nm) and increasing the specific surface area. In addition, the fixed carbon contents of the three types of biochars were 92.16, 93.69 and 87.13 Wt%, respectively, with a higher proportion of carbon, which was presumably caused by the volatilization of most of the other elements during the carbonization process. The pore size of biochar was analyzed by Mercury intrusion porosimetry (MIP). The ratios were 2.58% and 97.42%, respectively, and the ratios of mesopore and macro-pore in Ziziphus jujuba were 2.19% and 97.81%, respectively. The three biochars have high pore size ratios, which are very suitable as a habitat for the growth of beneficial microorganisms and as a carrier for micro-coated beneficial microorganisms (

Table 6. Specification of biochar microbial carrier material for resistance to salt stress-3.

	pH	BET (m2/g)	Fixed Carbon (Wt%)	Pore Ratio (%)
Syzygium samarangense	Adjust to 7.3 ± 0.2	315.09	92.16	Mesopore (2~50 nm): 27.63
				Macropore (>50 nm): 72.37
Leucaena leucocephala	Adjust to 7.3 ± 0.2	213.53	93.69	Mesopore (2~50 nm): 2.58
				Macropore (>50 nm): 97.42
Ziziphus jujuba	Adjust to 7.3 ± 0.2	318.53	87.13	Mesopore (2~50 nm): 2.19
				Macropore (>50 nm): 97.81

).

Observed by Scanning Electron Microscope (SEM), the three biochars are all porous structures, and their pore sizes are about 13.31–23.44 × 16.88–23.06 μm, 15.94–25.31 × 20.91–26.63 μm, 8.53–16.68 × 11.44–17.16 μm, respectively. Taking Bacillus amyloliquefaciens as an example, the cell length is about 0.7–0.9 × 1.8–3.0 μm, and the cells are often linear. The size of other microorganisms of the genus Bacillus is similar. The pore size of biochar is suitable for the growth of beneficial microorganisms (Figure 9, Figure 10 and Figure 11).

According to the polycyclic aromatic hydrocarbons (PAHs) report, most of the polycyclic aromatic hydrocarbons in Syzygium samarangense biochar were not detected, except naphthalene (CAS No.: 91-20-3), which was detected at 0.289 ppm (Figure 7); the allowable amount was determined by comparing the two major international biochars—European Biochar Certificate (EBC) [29,30] and International Biochar Initiative (IBI) [31] Basic: <12 mg/kg, Premium grade: <4 mg/kg and 6–300 mg/kg; 3 mg/kg B(a)p-TEQ, respectively. In addition, the dioxin test result (PCDD/Fs) of Syzygium samarangense biochar was 0.208 pg/kg WHO2005-TEQ (Annex 2), while EBC and IBI set the tolerable levels to be <20 ng/kg I-TEQ and <17 ng/kg WHO-TEQ, shows that the dioxin content of this Syzygium samarangense biochar is far lower than the allowable amount set by the international mass organization. Therefore, based on the above biochar test results, the use of agricultural waste the biochar produced is very safe.

In previous studies, a multifunctional cyclic embedded technology was developed through the carbonization technology of recycled biomass materials and the microbial culture and fermentation technology, and a composite material of biochar micro-coated soil beneficial microorganisms was successfully prepared. Figure 12 shows the micro-coating results of Syzygium samarangense embedded with Bacillus amyloliquefaciens; it can be clearly seen that there are short rod-shaped Bacillus amyloliquefaciens in the pores. The presence of Bacillus amyloliquefaciens was also clearly observed in the SEM images of the Leucaena leucocephala and the Ziziphus jujuba biochar (Figure 13 and Figure 14). These results successfully confirmed that Bacillus amyloliquefaciens can indeed enter the pores of biochar for growth and reproduction. The micro-coated beneficial microorganisms with biochar using the multi-functional cycle embedded technology can have a bacterial count of more than 10⁸ CFU/g, and some of the culture medium up to 10¹⁰ CFU/mL.

To test whether beneficial microorganisms can inhibit pathogenic microorganisms and prevent crop diseases, the fermented beneficial microorganisms and tomato wilt fungus (Fusarium oxysporum f. sp. Lycopersici) were placed in the pre-cultured beneficial microorganism medium for confrontation in the experiment. The results showed that the results of the five-day confrontation culture were obtained (Figure 15). Bacillus amyloliquefaciens has inhibitory effect on tomato wilt pathogen. It can be seen from the figure that the inhibition circle is complete, and it should be used as a biological agent for future development. While Bacillus megaterium A and Bacillus megaterium B can make the edge of tomato wilt mycelium sparse, the inhibitory effect is not obvious, and other functions should be tested.

3.4. Pot Experiment of Tomato Wilt Fungus

With the demand for the reduction of fertilizer and pesticides, there is an urgent need to reduce the use of environmentally unfriendly pesticides and agrochemicals (Wei et al., 2019). Pathogen invasion can drive community-wide dynamics in tomato endophytic bacterial composition. Bacillus sp., which is enriched in the seedling phase, may play a key role for the suppression of F. oxysporum based on Lefse analysis [32,33]. We tested biochar blended with Bacillus amyloliquefaciens to see if the beneficial microbe-embedded biochar had a slowing effect on tomato wilt. The test seedlings were tomato seeds (Lycopersicon esculentum Mill.). The habit of this variety is a one or two-year-old herb. The optimum temperature for germination is 20~30 °C, and the optimum temperature for growth is 15~30 °C. It is a cow tomato with full red and large fruits, light green fruit shoulder and a high yield. The fruit weighs about 200 g, and the fruit is hard and not easy to crack. In the experiment, the disease severity of tomato was recorded on days 0, 7, 14, 21 and 28, and the wilting disease incidence index was divided into five grades [26]: grade 0 was healthy plants with no symptoms (0%); grade 1 is plant dwarfing and yellowing of cotyledons (less than 25% of leaves have symptoms); grade 2 is yellowing of lower leaves (26–50% of leaves have symptoms); grade 3 is death of lower leaves and some upper leaves are yellow with degradation or wilting (51–75% of leaves show signs of disease); grade 4 is the death of lower leaves and withering of upper leaves (76–100% of leaves show signs of disease); grade 5 is the death of the whole plant. The calculation formula is Disease severity (%) = (∑ni × i)/5N × 100. i: incidence grade; ni: the number of plants with disease i level; N: the total number of plants under investigation.

Observed for 28 days after inoculation and counting the morbidity of tomato wilt disease (Figure 16), it was found that the morbidity of the samples treated with the composite material (Bacillus amyloliquefaciens + biochar) was the lowest, and only lower leaf yellowing occurred. In the treatments of adding water and biochar, the lower leaves died, the upper leaves withered, and the plants were lodging, but no complete plant death was found after 28 days. In addition, the disease degree of the biochar group was slightly higher than that of the water-added control group in the early stage of disease. It is speculated that because the biochar is not coated with microorganisms, the nutrients absorbed by itself can be used and grown by pathogenic bacteria, resulting in a high disease degree in the initial stage of the biochar treatment. The verification test results and analysis of the crop control effect of biochar-based biological fertilizers or preparations showed that the composite material treatment can effectively reduce the disease degree of tomato by 69.43%, compared with water treatment (Figure 17).

In addition, the experiment also used the composite material made of Syzygium samarangense biochar and Bacillus amyloliquefaciens to test the anti-salt damage effect. In the experiment, CK (water) was used as the control group for the overall experiment, 100 mM NaCl was used as the source of salt damage, and leaf lettuce was used for comparison with CK, Syzygium samarangense biochar, beneficial microorganisms and composite materials (Figure 18) (

Table 7. The results of pot experiment testing salt-resistant stress materials.

	CK (Water)	100 mM NaCl
		CK	Biochar	Probiotics	Biochar & Probiotics
SPAD *	21.9	7.6	3.4	20.2	19
Fresh weight (g)	7.86	0.64	0.12	2.02	0.77
Dry weight (g)	0.83	0.05	0.01	0.2	0.1
Dry weight/Fresh weight (%)	10.56	7.81	8.33	9.9	12.99
EC (dS/m)	1.5	9.5	9.6	9.9	9.8
pH	7	7.3	7.1	7.2	7.1

* SPAD: (Soil and Plant Analyzer Development).

). Among them, 85 mM sodium chloride was added as the medium base when Bacillus amyloliquefaciens were cultured in NA (Nutrient Agar) medium. It can be seen that Bacillus amyloliquefaciens in this experiment can indeed grow under the stress of salt damage. The SPAD value (Soil and Plant Analyzer Development) is used to measure the relative greenness content or greenness degree of leaves at night. It shows that beneficial microorganisms can make the chlorophyll of plants relatively unaffected. The greenness of the plants under the untreated salt damage stress decreased by 65%, and the greenness of the plants with single addition of Syzygium samarangense biochar decreased by 84%. It is speculated that the reason for this is that the biochar has not been modified, and it is a strong alkali and high EC. The Syzygium samarangense biochar of value is caused by the direct growth of crops in multiple harsh environments during the seedling stage.

The fresh weight of the crops was weighed after about a month of growth. The fresh weights of CK (water), CK under salt damage, Syzygium samarangense biochar, beneficial microorganisms and composite materials were 7.86, 0.64, 0.12, 2.02 and 0.77 g, respectively. It can be seen that under the condition of salt damage, the plants shrank; the dry weights were 0.83, 0.05, 0.01, 0.2 and 0.1 g, and the conversion ratios were 10.56, 7.81, 8.33, 9.90 and 12.99%, respectively. Although the plants shrank under the stress of salt damage, the dry/fresh weight ratio of the composite material was found to be higher than that of the control group with only water added through the test results. It can be seen that the composite material is indeed effective for crops to survive the stress of salt damage. There is a preliminary effect, followed by beneficial microorganism treatment, and the performance is excellent in all values. In terms of soil, the EC values of CK (water), CK under salt stress, Syzygium samarangense biochar, beneficial microorganisms and composites were 1.5, 9.5, 9.6, 9.9 and 9.8 dS/m, respectively, and 100 mM NaCl was 9.4 dS/m, the soil pH value was roughly neutral, and this result showed that a large amount of salt accumulation was indeed the main cause of crop atrophy (Table 7).

The beneficial microorganisms isolated in this experiment performed well in various values. In the experiments of salt damage and tomato wilt disease, it was shown that the beneficial microorganisms and composite materials can effectively improve the degree of tomato wilt disease and the rate of leaf atrophy. The situation may be used as a good material for the future development of agricultural and industrial projects.

4. Conclusions

In this research, to develop composite materials of biochar micro-coated soil beneficial microorganisms, we isolated the salt-tolerant beneficial microorganism Bacillus amyloliquefaciens in salinized soil. In the production of biochar, the experimental data show that the biochar carbonized at 500 °C is the most suitable for mixing beneficial microorganisms; it can save 50% of the operating time in the process of biochar mixed medium and can enable beneficial microorganisms to effectively enter the organism. In the charcoal pores, the total amount of microorganisms >10⁸ CFU/g was achieved at the same time. The composite material produced after being inlaid with biochar can effectively improve plant growth, help crops resist adverse environments and slow down the occurrence of diseases, effectively reduce tomato wilt disease (69.43%) and improve the degree of chlorosis, which can be developed in the future. Biochar is embedded with probiotics to decompose cellulose, starch, protein and dissolved phosphorus to increase crop absorption, stabilize yield and reduce food crisis caused by climate change. With the demand for the reduction of fertilizer and pesticides, there is an urgent need to reduce the use of environmentally unfriendly pesticides and agrochemicals. We established novel composite materials of biochar micro-coated soil beneficial microorganisms.