Optimizing the Fermentation Conditions and Determining the Disease Control and Growth Promotion Function of Bacillus Amyloliquefaciens 35

Bacillus spp. are helping to develop towards sustainable agriculture and have become a research hotspot in the eld of plant pathology because they have great development potential such as control fungal plant diseases. Bacillus Amyloliquefaciens 3-5 was used as antagonistic bacteria in this experiment. The optimal medium for solid-state fermentation of B. amyloliquefaciens 3-5 contained bran (35%), rice hull powder (40%), corn gluten (20%), bean our (15%), corn starch (1.5%), beef extract (2.5%) and MgSO 4 (1.5%), and the optimal fermentation conditions included an inoculum of 6%, a solids content of 36 g/L, a feed-to-water ratio of 1:1, a fermentation temperature of 32 ℃ , an initial pH of 7.0 and fermentation time of 44 h. When the dosage of the B. amyloliquefaciens 3-5 agent was 10%, the thick stems, root length and plant height of cucumber were signicantly higher than those of the control (P(cid:0)0.05), and the growth rates were 77.45 %, 35.77 %, and 53.33 %, respectively in pot experiments. Compared with the control, and the preventive effect and therapeutic effect on cucumber Fusarium wilt were 72.09 % and 48.83 % by the application of B. amyloliquefaciens 3-5 agent(cid:0)which showed that cucumber Fusarium wilt was successfully controlled by a newly isolated strain of B. Amyloliquefaciens 3-5. These results suggested that the prevention and control effect of B. amyloliquefaciens 3-5 agent on cucumber Fusarium wilt could not be underestimated.


Introduction
Cucumber, a popular vegetable with young people, is an economically important crop. Vascular wilt of cucumber caused by Fusarium oxysporum f. sp. cucumerinum (FOC) is one of the most destructive soilborne diseases causing major economic losses worldwide (Qiu et al. 2012). Pathogens of the genus Fusarium have a particularly wide host range, including potato, tomato, pepper, bean, pea, chickpea, banana, strawberry, cotton, and melon, and are among the most damaging soilborne pathogens in crop production systems. They usually infect plants through the roots, and cause damping-off, root rot, and vascular wilt (Cha et al. 2016).
Chemical control with fungicides has been the most common control strategy for diseases caused by Fusarium spp. However, their extensive use has not only posed threat to pathogen resistance and the resurgence of the disease but endangered human health (Feng et al. 2021). Moreover, as a matter of common knowledge that the application of chemical fungicides leads to the selection for resistant strains, which limits their long-term e cacy (Janga et al. 2017). Furthermore, owing to the persistence or survival of F. oxysporum in the soil over long periods of time and the wide hosts range of F. oxysporum it is so di cult to control this disease using chemical control method (Park et al. 2020).
Therefore, safe and e cient methods for prevention and control of diseases caused by Fusarium wilt are badly needed. Biocontrol, as an eco-security, safe and sustainable method of controlling plant infections, represents a promising approach to protect crops from the pathogen (Compant et al. 2005; Haas and Keel. 2003; Hass and Défago. 2005; Lugtenberg and Kamilova. 2009). In particular biocontrol of Fusarium wilt by bacterial antagonists has been extensively studied. For example, B. subtilis SQR9 was found to control Fusarium wilt of cucumber by colonizing plant roots and producing lipopeptides (fengycin and bacillomycin) that are effective against F. oxysporum (Cao et al. 2011). B. velezensis RC 218 reduced disease severity of Fusarium head blight (FHB) caused by Fusarium graminearum and reduced accumulation of associated mycotoxins (Palazzini et al. 2016). "Am ssis" trees treated with Paenibacillus alvei K165 had signi cantly lower disease severity and lower relative AUDPC (Area Under the Disease Progress Curve) values than those of untreated, diseased plants (Markakis et al. 2016). Streptomyces griseorubens E44G was found to possess chitinolytic activity and was able to reduce the severity of Fusarium wilt of tomato and increase tomato growth and yield (Rashad et al. 2017). Finally, isolates of the fungus Trichoderma asperellum which had high levels of chitinase and β-1,3-glucanase activities, strongly inhibited the mycelial growth of F. oxysporum f. sp. lycopersici (El Komy et al. 2015). As everyone knows, microorganisms are an almost unlimited source of natural products, many of which have potential therapeutic uses. In the context of the current study, the bacterium B. amyloliquefaciens has been widely used as a probiotic in the eld of biological control that its antibacterial compounds play a vital role in the prevention and control of plant, livestock and poultry diseases (Wang et al, 2020), and it has the advantages of safe, e ciency and green environmental protection.
An increasing number of literature indicated a series of advantages of endophytes. Kang et al. (2007) detailed the growth-promoting characteristics of endophytes, while Kloepper et al. (2004) and Senthilkumar et al. (2007) demonstrated the disease-inhibiting traits of endophytes. The nature of endophytes in strengthening the defence mechanism of crops to various plant diseases was researched upon by Bargabus et al. (2002), Mishra et al. (2006) and Bakker et al. (2007). In the early stage of the laboratory, endophytic strain 3-5 with magni cent antagonistic effect against Streptomyces galilaeus, the pathogen of potato scab, was screened by plate confrontation method and identi ed as Bacillus amyloliquefaciens. B. amyloliquefaciens 3-5 has good antagonistic effect on many pathogens such as F. oxysporium f. sp. cucumerinum, F. solani, F. avenaceum, F. oxysporum f. sp. potato, F. oxysporum f. sp. melonis, F. oxysporum f. sp. chili, F. solani f. sp. chili, Alternaria solani, A. tenuissima and Colletotrichum coccodes, which showed B. amyloliquefaciens 3-5 has great application potential. It would be necessary to know "soild-state fermentation" from previous literature before the experiment started, which the level of production through solid-state fermentation depends not only on the performance of the strain itself, but on the provision of suitable fermentation conditions to fully utilize its production capacity (Kubiak et al, 2019). Research on fermentation process optimization is particularly important for its can make full use of the potential of strains,improve production e ciency and reduce production cost (Wang et al, 2018). Therefore, the solidstate fermentation conditions of B. amyloliquefaciens 3-5 were optimized, its antagonistic effect on FOC and other plant pathogenic fungi was determined, and its function of disease prevention and growth promotion was demonstrated in this experiment. The microbial agent was evaluated, and its growth promoting effect and disease control effect on cucumber Fusarium wilt were discussed. It not only provided the basis for the development of multifunctional B. amyloliquefaciens 3-5, but also laid the foundation for the development of preventive microbial agents.

Microorganisms growth conditions
In this study, a functional strain B. amyloliquefaciens 3-5 was transferred from − 20°C frozen stocks to NB agar plates at 28°C for pre-culture before being used as inoculum for solid substrate fermentations.

Preparation of seed cultures
To prepare seed cultures, B. amyloliquefaciens3-5 colonies were inoculated from fresh NB plates in 250-mL asks containing 50 mL of liquid NB medium. The asks were incubated at 28°C with shaking at 180 rpm for 24 h.

Single matrix screening
In this experiment, bran, corn stalk, rice bran, corn our, corn starch, soybean powder, oil residue and beet pulp residue were selected as the base medium, and the seed solution was inoculated into 20 g substrate with the ratio of material to water of 1 : 1 at 10 % ( v / m ). The seed solution was placed in an incubator at a constant temperature of 28°C, and after fermentation for 36 h, it was removed and dried in an oven at 65°C. 1 g of fermented substrates were mixed with 99 mL of sterilized water containing some beads in a 250-mL ask and stirred for 20 min at room temperature. The number of viable germs ( cfu / g ) was then counted using the standard dilution plate counting method (Turner and Backman 1991). All experiments were repeated three times, and the optimum substrate combination and hair were selected based on the number of viable bacteria fermentation conditions.

Base matrix optimization
In the selected single substrate, wheat bran and rice husk our were used as the main material, and corn our and soybean powder were used as auxiliary materials. According to the mass ratio of main and auxiliary materials, four levels were determined 90 % : 10 %, 80 % : 20 %, 70 % : 30 % and 60 % : 40 % ( Table   1 ). Count the number of viable bacteria after fermentation, and nally determine the best mass ratio of basic substrate.

Optimization of solid addition
In the optimal matrix, the seed solutions of 5 g, 7 g, 9 g, 11 g and 13 g were added as solids to a 250 mL triangular ask, and fermented in a constant temperature incubator at 28°C. After 36 h, the number of viable cells was determined. The other test methods were the same as in Sect. 2.2.3.

Material-water ratio optimization
In the optimum medium, the seed liquid was added with the ratio of material to water of 1 : 0.6, 1 : 0.8, 1 :

Optimization of fermentation temperature
Five temperature levels were set for the optimum substrate at 24°C, 28°C, 32°C, 36°C and 40°C, respectively.
After 36 h, the number of viable bacteria was determined, theother test methods were the same as in Sect.

2.2.3.
The above four factors were orthogonally designed as shown in Table 3. Table 3 Factors and levels of orthogonal experiment (L25(4 4 )) No. The substrate soil ( provided by Gansu Lvneng Agricultural Science and Technology Co., Ltd. ) and vermiculite were thoroughly mixed at a ratio of 1:1 (w/w) after autoclaved at 121°C for 2 h, and supplemented with fertilizers to which 3-5 bacteriocin was added according to the mass ratio of bacteriocin to culture medium ( the mixture of substrate soil and vermiculite ) of 0.25%, 0.5%, 1.0%, 5.0%, 10.0% and 20.0%. The soil (200 g) was distributed in plastic pots ( Ф = 12 cm ), and moisture was maintained at 90% of the maximum water-holding capacity of the soil by daily addition of sterilized water.
After mixing, it was placed in a 12 cm × 12 cm plastic ower pot ( 200 g per pot ), without B. amyloliquefaciens 3-5 agent as a control. Cucumber seeds with full grains were selected for germination ( soaked at 45°C for 2 hours ) and planted in each pot ( four plants per pot ) after germination. When the fourth true leaf of the cucumber seedlings had grown out, thefresh weight (g), dry weight (g), stem thickness (cm), root length (cm) and plant height (cm) of each plant were measured (after being killed in 105°C oven and dried at 70°C to constant weight). This experiment was repeated three times.  Mizumoto et al. 2006). The healthy cucumber seeds with equal growth vigor were planted in a plastic ower pot ( Ф = 12 cm ) containing a nutrient soil with the dosage of the microbial agent was 10.0 % ( nutrient soil was mixed with vermiculite at a ratio of1 : 1 ). When the cucumber seedlings had grown out of the fourth true leaf, the experiment was conducted.
( 1 ) Prevention experiment : The soil on the surface of the ower pot was plucked off with a sterile dissector, and the bacterial agent which constituted 10.0 % of the mass of the nutrient soil in the ower pot was applied to the surrounding plants. The bacterial agent was covered with the previously plucked soil.
After 3 days, the soil was inoculated and pricked 10 cm from the stem base of the plant, and the spore suspension of FOC with the concentration of 10 7 cfu / mL was dipped with defatted cotton and spread evenly on the microinjury site.
( 2 ) Treatment test : 10 cm from the stem base of the plant, a pinprick injury was inoculated and the spore suspension of FOC with a concentration of 10 7 cfu / mL was inoculated with defatted cotton. After 3 days, 10.0 % of nutrient soil was applied around the plant in the ower pot and the soil was covered with the previously released bacterial agent.
( 3 ) Control : The spore suspension of FOC containing 10 7 cfu / mL was used as control.
Each treatment set 10 seedlings, repeated three times. After inoculation, humidi cation with preservative lm for 48 hours, and the incidence of cucumber wilt was studied after 15 days, and the disease index and control effect of 3-5 on cucumber wilt were calculated.

Statistical analysis
Three independent experiments were performed to check the reproducibility of results. Data obtained were expressed as the mean ± standard deviation (SD) and analyzed statistically using the computer software package SPSS 24.0 program. Multiple comparisons were performed on the data using the one-way ANOVA program, with 0.05 set as the threshold signi cance level for Duncan's test (Li et al., 2010).

Single Matrix Screening
To explore the effect of the feeding ingredients on the fermentation, we have chosen three kinds of carriers, carbon sources and nitrogen sources respectively. As shown in the Fig. 1, in the single substrate base medium with bran as carrier, the number of viable cells of B. amyloliquefaciens 3-5 was the highest, which was 31.25 × 10 10 cfu / g, which was signi cantly higher than that of the carrier rice bran and corn straw. In the single substrate base medium with corn our as carbon source, the number of viable cells was the greatest ( 8.65 × 10 10 cfu / g ), which was signi cantly higher than that of corn starch and sugar beet residue. In the single substrate medium with soybean powder as nitrogen source, the number of viable bacteria was the greatest at 4 × 10 10 cfu / g, which was signi cantly higher than that of the nitrogen source oil residue. Therefore, wheat bran as carrier, corn our as carbon source and soybean our as nitrogen source were the best substrate combination for basic medium of B. amyloliquefaciens 3-5.

Base matrix optimization
Following optimization the base matrix, our results showed that at aratio of bran : rice husk powder : corn our : soybean powder of 3.5 : 4 : 2 : 1.5, the number of viable cells of strain 3-5, was the highest, was 9.97 × 10 12 cfu / g, which was signi cantly higher than that of the other composite substrate combinations, indicated that the optimum combination for solid-state fermentation of B. amyloliquefaciens 3-5 were 35 % bran, 40 % rice husk powder, 20 % corn our and 15 % soybean powder, respectively ( Table 4 ). Note: K1 represents the average value of the four tests at the rst level of each factor; K2 represents the second level of each factor and the mean value of the four tests; K3 represents the average value of the third level four tests of each factor; K4 represents the fourth level of each factor and the average of the four trials.

Carbon source optimization
For the different carbon sources that we tested, our results showed that B. amyloliquefaciens 3-5 had the highest number of viable cells ( 2.48 × 10 13 cfu / g ) in the solid matrix with corn starch as carbon source which was signi cantly higher than that of other carbon sources (Fig. 2).

Nitrogen source optimization
The in uence of different nitrogen sources on the visible counts was shown in the Fig. 3. The visible number of the strain in the solid matrix with yeast extract as nitrogen source was 0.87 × 10 13 cfu / g ; In the solid matrix with beef extract as nitrogen source, the number of viable cells of strain 3-5 was the largest, which was 1.21 × 10 13 cfu / g. In conclusion, the viable count of beef extract with nitrogen source was signi cantly higher than the visible counts of urea, (NH 4 ) 2 SO 4 , KNO 3 , NH 4 Cl and yeast extract with nitrogen source.

Inorganic salt optimization
The suitable inorganic salt was critical for the viable cells growth because of it is a useful ingredient in media for the cultivation microorganisms. As shown in the Fig. 4, the number of viable cells of strain 3-5 was the largest when in the solid medium with MgSO 4 as inorganic salt, was 2.13 × 10 13 cfu / g, which was signi cantly higher than that of NaCl, MnSO 4 , FeSO 4 and KH 2 PO 4 . Note: k1 represents the average value of the four tests at the rst level of each factor; K2 represents the second level of each factor and the mean value of the four tests; K3 represents the average value of the third level four tests of each factor; K4 represents the fourth level of each factor and the average of the four trials.
Results of optimization of parameters mentioned in the

Optimization of fermentation conditions
As we expected, when the inoculation amount was 6 %, the solid-solid addition amount was 36 g / L, the solid-water ratio was 1 : 1 and the temperature was 32°C, the viable count of B. amyloliquefaciens 3-5 was the largest, which was 4.545 × 10 13 cfu / g, which was signi cantly higher than that of other combinations. The results showed that the optimum fermentation conditions for strain 3-5 were as follows : a 6 % inoculation rate, a solid addition rate of 36 g / L, solid-liquid ratio of 1 : 1 and temperature of 32°C ( Table 6 ). Note: k1 represents the rst level of each factor and the mean value of the ve tests; K2 represents the second level of each factor and the mean value of the ve trials; K3 represents the third level of each factor and the average value of the ve trials; K4 represents the fourth level of each factor and the average value of the ve trials; K5 represents the fth level of each factor and the average of the ve trials. Note: k1 represents the rst level of each factor and the mean value of the ve tests; K2 represents the second level of each factor and the mean value of the ve trials; K3 represents the third level of each factor and the average value of the ve trials; K4 represents the fourth level of each factor and the average value of the ve trials; K5 represents the fth level of each factor and the average of the ve trials.

Optimization of initial pH of fermentation
The pH value was an essential parameter to ensure the normal reproduction and metabolism of microorganisms, having great in uence on fermentation. As shown in the Fig. 5, the number of viable cells of strain 3-5 increased with the initial pH of fermentation. When the pH value was 4.5, the viable count grew to reach a stable phase and the viable count grew at a faster speed when the pH value was 6; the number of viable cells, reached a maximum of 4.568 × 10 13 cfu / g, was signi cantly higher than that at other pH values when the initial pH of fermentation was 7. When the pH value was between 7.5 and 10, the number of viable count was obviously decreased. Therefor, the initial pH value of fermentation was 7, which was the optimal initial pH of the strain.

Optimization of fermentation time
The fermentation time also was one of the key of parameters. As shown in the Fig. 6, the number of viable cells of strain 3-5 changed as fermentation time increased. The number of viable bacteria was approach 0 when the fermentation time was less than 36 h. When the fermentation time was 40 h, the viable count was dramatic increase. Until the fermentation time was 44 h, the number of viable bacteria was 2.835 × 10 12 cfu / g, which was signi cantly higher than that of other fermentation times, and then signi cantly decreased. Obviously, 44 h was the strain's best fermentation time.  (Table 7). 3.4 Determination of the control effect of B. amyloliquefaciens 3-5 on cucumber fusarium wilt The results of pot control showed that the disease index of prevention and treatment was signi cantly lower in the pot control group than in thecontrol group. The treatment effect was 48.83 %, and the prevention effect was 72.09 %.The prevention effect overperformed the treatment effect (Table 8).

Discussion
Modern chemical fungicides were less harmful than those of older generations, but agrochemicals in These bioproducts with antifungal activities are called biofungicides, and they can be produced in several ways.
Solid-state fermentation (SSF) has enormous potential because of its lower production investment, shorter fermentation time and higher yield of secondary metabolites (Vandenberghe et al. 2021 (Ramachandran et al. 2004). Therefore, the development of low-cost and highly e cient technologies for the production of spore-forming probiotics became necessary. It was generally accepted that SSF has several biotechnological advantages; it was a simple and low-investment process as it requireed low energy consumption, cheap plant raw materials can be used as growth substrates, and after SSF, the product can be lyophilized directly without centrifugation.
It also provides higher productivity of fermentation and higher concentration of products (Holker et al. 2004 . The reasonable way to increase spore production is to create optimal cultivation conditions that promote maximum vegetative cell accumulation followed by effective sporulation. One of the valid approaches is to supplement the culture medium with a suitable nitrogen source at the optimal concentration. To achieve this goal, several abundantly available agro-industrial byproducts were tested. Among these growth substrates, bran followed by rice bran and corn straw, provided particularly high yields of spores. These results suggest that a distinctive feature of B. amyloliquefaciens 3-5 is its ability to use various low-cost lignocellulosic materials as growth substrates for high-yield spore production. Similar results were reported for submerged cultivation of B. subtilis where ammonium sulfate (4.54%) in combination with corn our (1.2%) resulted in maximum spore production (Khardziani et al. 2017). Furthermore, using a response surface method, Rao et al. (2007) optimized the composition of the medium for maximum spore production of B. amyloliquefaciens subsp. B128 and showed that a mixture of (NH 4 ) 2 SO 4 and peptone gave the highest yield of spores at concentrations of 1.8 and 8.0 g L −1 , respectively.
In this study, we attempted to reduce the cost of probiotics production by using low cost raw materials as medium components by optimizing of the medium for signi cant increase in spore yield.
Recently, several studies have investigated the antagonistic activity of different bacterial strains on phytopathogenic fungi in postharvest fruits, so their use can be considered as an alternative in controlling fungal diseases by chemical methods (Ma et al. 2019). B. amyloliquefaciens 3-5 as an endophytic bacterium with good biocontrol potential. In this study, its xed fermentation conditions were optimized.
The results showed that wheat bran was the carrier, corn our was the carbon source and soybean meal was the nitrogen source. 35% bran, 40% rice husk meal, 20% corn meal and 15% soybean meal were selected as the composite matrix for solid-state fermentation of strain 3-5. The best carbon source was 1.5% corn starch, 2.5% beef paste and 1.5% magnesium sulfate. The optimal fermentation conditions were 6% inoculation, 36 g/L solid addition, 1:1 ratio of material to water, 32℃ temperature, pH7 and 44 h fermentation time. The results of this study provided the basis for the production of the bactericide of B. amyloliquefaciens [3][4][5], and also provided the Micrabiae resources for the biological control of cucumber Fusarium wilt.
It is widely known that Fusarium wilt, as a destructive and economic disease, is very di cult to control by classical methods because there is no effective chemical treatments. In addition, it is not practical to control the soil-borne diseases with chemicals, biological control is a realistic approach for the management of Fusarium wilt. For all these reasons, there is an urgent need for plant disease control in agriculture.
In this research, B. amyloliquefaciens [3][4][5] showed that preventive effect on cucumber Fusarium wilt and therapeutic effect was 72.09 % and 48.83 % by applying B. amyloliquefaciens 3-5 agent by pot culture experiments under greenhouse conditions. B. amyloliquefaciens 3-5 agent developed in this laboratory signi cantly reduced the incidence and severity index of cucumber Fusarium wilt and can partially reduce the economic losses. Thus, B. amyloliquefaciens 3-5 agent can effectively control cucumber Fusarium wilt. Biocontrol agents developed by antagonistic strains do not pollute the environment, which is bene cial for maintaining the balance of the ecosystem and promoting agriculture. At present, this experiment only studied the development of B. amyloliquefaciens 3-5 indoor research for disease prevention, growth promotion. However, the application of B. amyloliquefaciens 3-5 agent in the eld needs further investigation.

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Availability of Data and Materials
Data and materials available on request from the authors. The data and materials that support the ndings of this study are available from the corresponding author upon reasonable request.
Competing Interests Figure 1 Single substrate screening for solid fermentation of strain 3-5 Note: Different lowercase letters in the g mean signi cantly different at p 0.05 level with Duncan's new multiple range least, the same below.

Figure 2
The effect of carbon source on the number of viable bacteria Effects of inorganic salts on the number of viable bacteria Figure 5 Effect of initial pH on the number of viable bacteria Effect of fermentation time on the number of viable bacteria