Initial Medium Optimization of Nigrospora oryzae JL-4 and Its Biocontrol Potential on Solanum rostratum

Abstract

To assess the biocontrol potential of Nigrospora oryzae against Solanum rostratum, the effects of different medium components and fermentation conditions on the biomass of N. oryzae were investigated to determine the optimal medium composition and fermentation conditions. Subsequently, the pathogenicity of S. rostratum increased after artificial inoculation of S. rostratum with N. oryzae. Additionally, the safety of N. oryzae fermentation on seven crops were evaluated. The results revealed that the optimal shake flask culture ratio for N. oryzae was lactose:glycerol:yeast extract:(NH₄)₂SO₄ = 2:2:1:2. The optimal fermentation conditions were as follows: 15 mL of inoculum, 75 mL of fermentation liquid in a 150 mL shake flask, an initial pH of 5.0, a culture temperature of 20 °C, and 4 days of culture. The disease index of S. rostratum after spraying with N. oryzae fermentation agent was 60.00, indicating strong pathogenicity to S. rostratum. Safety evaluation revealed that N. oryzae exhibited no symptoms on Zea mays, S. melongena, Festuca arundinacea, Bromus inermis, or Medicago sativa but showed moderate susceptibility to Triticum aestivum and S. lycopersicum. This study represents the first exploration of the biocontrol potential of N. oryzae against S. rostratum. These findings suggest the potential of N. oryzae for development as a microbial herbicide targeting S. rostratum.

1. Introduction

Solanum rostratum is an annual malignant invasive weed of the Solanum genus within the Solanaceae family. It can invade grasslands, riparian grasslands, and roadsides, causing severe damage to agriculture, animal husbandry, and biodiversity in China [1,2]. S. rostratum is the primary host for potato leaf-roll virus and Leptinotarsa decemlineata, both of which inflict damage on agriculture [3,4]. Moreover, it was treated as a noxious weed because it grows aggressively following habitat disturbance [5,6], and livestock were discouraged from grazing on vegetation where it grows as thorns cover all the plant except the flowers and can cause poisoning if ingested [6]. The roots of S. rostratum produce chemical allelopathy, which inhibits the growth of surrounding plants and poses substantial threats to biodiversity and the environment in China [7]. Because S. rostratum causes severe damage to agriculture, animal husbandry, and biodiversity in China, preventing the spread of S. rostratum is necessary. Although current control methods involve physical, chemical, and replacement strategies for S. rostratum [8], there is a lack of research on the biological control of S. rostratum. Consequently, exploring and implementing biological control strategies for the integrated management of S. rostratum are necessary.

Pathogenic microorganisms have been primarily employed in the biological control of weeds. It was isolated from diseased plant leaves, roots, or fruits. Notably, Alternaria alternata [9], potato leafroll virus [10], tomato brown rugose fruit virus [11], and yellow tailflower mild mottle virus [12] have been identified as infecting S. rostratum and inducing diseases. In a prior study, five species of pathogenic fungus were isolated from the spot area of diseased leaves by S. rostratum. Through pathogenicity testing, we found that N. oryzae was strongly pathogenic to S. rostratum. N. oryzae was initially isolated from Oryza sativa leaves [13]. Subsequent research revealed the infection of economic crops such as Triticum aestivum, Gossypium hirsutum, Zingiber officinale, and Actinidia chinensis [14,15]; ornamental plants such as Ficus religiosa and Nelumbo nucifera [16,17]; and Gramineae weeds such as Poa annua [18] by N. oryzae. In contrast, Solanaceae plants were not susceptible to this pathogen.

Biologically controlling weeds using pathogenic fungi is crucial for enhancing the production of spores, mycelia, or other active substances [19]. The fermentation of pathogenic fungi can be used for the biological control of these weeds. For example, Li et al. [20] applied fermentation broth to 10 species of the pathogenic fungus Amaranthus retroflexus in Sorghum bicolor fields, and they discovered that the fermentation broth of the pathogenic fungus had better effects on controlling the weed. When the fermentation broth of Phoma herbarum was used to control Commelina communis, liquid fermentation was better than solid fermentation [21]. The optimization of medium components has been shown to promote biomass accumulation [22].

The carbon source, nitrogen source, and other nutritional components improved the growth of the fungus [23]. In addition, suitable fermentation conditions, such as inoculation amounts, fermentation liquid volumes, temperature, pH, and cultivation days, improved fungal growth and biocontrol [24]. The selection of carbon: nitrogen ratio was important during the biocontrol fungus. Orthogonal experiments were applied in the screening of carbon: nitrogen ratio for weed biocontrol fungus [25] and pathogen biocontrol fungus [26].

In a previous study, we discovered that N. oryzae exhibited strong pathogenicity to S. rostratum, indicating its significant potential for biological control. The fermentation conditions of N. oryzae were consequently optimized to produce fermentation broth. Pathogenicity was assessed through artificial inoculation of the fermentation from N. oryzae onto S. rostratum, and the safety of N. oryzae fermentation for seven crops was also evaluated. This study represents the first investigation into the pathogenicity of S. rostratum and a safety evaluation of crops subjected to fermentation broth supplemented with N. oryzae. This study aimed to elucidate the biocontrol potential of N. oryzae and provide a reference for the biological control of S. rostratum.

2. Materials and Methods

2.1. Test Materials

2.1.1. Tested Strains

N. oryzae JL-4 was isolated from the leaves of S. rostratum in Jilin, China.

2.1.2. Tested Plants

Seeds of S. rostratum, Zea mays, T. aestivum, S. lycopersicum, S. melongena, Festuca arundinacea, Bromus inermis, and Medicago sativa were used.

2.1.3. Instruments and Reagents

The following instruments and reagents were used: climatic cabinet (Ningbo Jiangnan-1500A-L, Ningbo Jiangnan Istrument Factory, Ningbo, China), electron microscope (Stellar 1 Pro, Motic China Group Co., Xiamen, China), orbital shaker (FA2004, Shanghai Puchun, Shanghai, China), superclean bench (SW-CJ-1D, Lichen Technology, Shanghai, China), high-pressure sterilization pot (XFH-30MA, Xinfeng, Shaoxing, China), oscillation incubator (THZ-98C, Shanghai Yiheng, Shanghai, China), glycerol, sucrose, maltose, soluble starch, lactose, glycerol, peptone, beef extract, yeast extract, NH₄Cl, (NH₄)₂SO₄, urea, MgSO₄·H₂O, K₂HPO₄, NaOH, HCl, and HClO.

2.2. Initial Medium Optimization for N. oryzae

2.2.1. Preparation of Fermentation Filtrate by N. oryzae

Ten 7-day-old fungal discs (d = 5 mm) of N. oryzae were collected from potato dextrose agar media and inoculated into PDB. The medium volume was 200 mL for each 500 mL shake flask, and the cultivation conditions involved shaking at 120 rpm for 72 h at 30 °C. The mycelia were separated using four layers of gauze, and the seed liquid was prepared.

2.2.2. Screening Significant Carbon Source (C) Components for N. oryzae

The nutrient media used for submerged cultivation contained (g/L of tap water): 1.2 g of carbon sources, 1.2 g of peptone, 0.06 g of KH₂PO₄, and 0.03 g of MgSO₄·H₂O. In the liquid fermentation medium, 1.2 g each of glucose, sucrose, maltose, soluble starch, lactose, and glycerol were selected as carbon sources. The fermentation medium was sterilized at 121 °C for 20 min. Submerged cultivation was conducted on a rotary shaker at 120 rpm and 30 °C for 6 days in 150 mL conical flasks containing 60 mL of nutrient medium inoculated with 3 mL of seed liquid. After cultivation, the mycelia were separated using four layers of gauze during N. oryzae fermentation. The mycelia were subsequently poured onto constant-weight filter paper and dried to a constant weight at 80 °C. The biomass was calculated using the formula below, and two optimum carbon sources were selected. The abovementioned treatments were repeated three times.

Biomass (g/L) = (Total mass of the filter paper and mycelium − Mass of the filter paper)/Total volume of the medium

2.2.3. Screening Significant Nitrogen Source (N) Components for N. oryzae

The nutrient media used for submerged cultivation contained (g/L of tap water): 1.2 g of sucrose, 1.2 g of nitrogen sources, 0.06 g of KH₂PO₄, and 0.03 g of MgSO₄·H₂O. In the liquid fermentation medium, 1.2 g each of peptone, beef extract, yeast extract, NH₄Cl, (NH₄)₂SO₄, and urea were selected as nitrogen sources. The fermentation medium was sterilized at 121 °C for 20 min. Submerged cultivation was conducted on a rotary shaker at 120 rpm and 30 °C for 6 days in 150 mL conical flasks containing 60 mL of nutrient medium inoculated with 3 mL of seed liquid. After cultivation, the mycelia were separated using four layers of gauze during N. oryzae fermentation. The mycelia were subsequently poured onto constant-weight filter paper and dried to a constant weight at 80 °C. The biomass was calculated using the formula below, and two optimum nitrogen sources were selected. The abovementioned treatments were repeated three times.

2.2.4. Screening Significant Carbon: Nitrogen Ratio Components for N. oryzae

The two optimal carbon and nitrogen sources with the highest biomass were selected, and the other components were not added to the nutrient medium. Using an L₉(3⁴) orthogonal array, nine distinct combinations of experimental conditions were evaluated, considering each level and factor. Submerged cultivation was conducted using a rotary shaker at 120 rpm and 30 °C for 6 days in 150 mL conical flasks containing 60 mL of nutrient media inoculated with 3 mL of seed liquid. The abovementioned treatments were repeated three times. Further optimization of N. oryzae fermentation was performed using an L₉(3⁴) orthogonal table (

Table 1. L₉(3⁴) Orthogonal experimental factors and levels.

Levels	Factors
	A Yeast Extract/g	B (NH4)2SO4/g	C Lactose/g	D Glycerol/mL
1	0.6	0.6	0.6	0.6
2	0.6	1.2	1.2	1.2
3	0.6	1.8	1.8	1.8
4	1.2	0.6	1.2	1.8
5	1.2	1.2	1.8	0.6
6	1.2	1.8	0.6	1.2
7	1.8	0.6	1.8	1.2
8	1.8	1.2	0.6	1.8
9	1.8	1.8	1.2	0.6

).

2.2.5. Screening Significant Fermentation Conditions for N. oryzae

Various factors, including the concentration of inoculation, fermentation volume, temperature, pH, and cultivation days, were evaluated to assess the effect on biomass and determine the optimal fermentation conditions. The inoculation volumes were 3 mL, 6 mL, 9 mL, 12 mL, and 15 mL (v/v). The fermentation liquid volumes were 15 mL, 30 mL, 45 mL, 60 mL, and 75 mL (v/v). Before sterilization, the initial pH values of the media were 5.0, 6.0, 7.0, 8.0, and 9.0. The culture temperatures were 15 °C, 20 °C, 25 °C, 30 °C, and 35 °C. The culture durations were 2 days, 4 days, 6 days, 8 days, and 10 days. All the conditions were tested in triplicate.

2.3. Pathogenicity Test of Fermentation Broth on S. rostratum

Seeds of S. rostratum were disinfected with a 1% NaClO solution for 10 min, rinsed with sterile water three times, and soaked in 0.83 g/L gibberellin at 30 °C for 24 h in the dark to break dormancy. Twenty-one seeds of S. rostratum were planted in a 15-cm diameter plastic pot filled with soil. When four true leaves of S. rostratum grew, 2 mL of fermentation broth was sprayed onto the leaves. An equal amount of sterile water was inoculated as the negative control, and each treatment was repeated three times. Following inoculation, the plants were incubated at 28 °C ± 1 °C with a relative humidity of 90% and a photoperiod of 14:10 (L:D) h. The diseased areas of the leaves were evaluated and recorded at 3 days, 7 days, 11 days, and 14 days. The incidence area rate was calculated based on the proportion of diseased areas among the total leaf area. Then, the disease index was calculated using the incidence area rate. The pathogenicity of N. oryzae to S. rostratum was determined by a disease index.

Disease index = (Number of vaccination points for disease grade × the corresponding grade)/(Total number of vaccination points × The highest grade) × 100%

Pathogenicity was categorized as follows: disease index of 0, not pathogenic; disease index of 0–25, weakly pathogenic; disease index of 25–50, medium pathogenic; and disease index of 50–100, strongly pathogenic.

2.4. Evaluation of the Fermentation Broth for Seven Crops

The seeds of Z. mays, T. aestivum, S. lycopersicum, S. melongena, F. arundinacea, B. inermis, and M. sativa were disinfected with 1% NaClO for 10 min, rinsed with sterile water three times, and soaked in sterile water at 30 °C for 24 h in the dark to prevent dormancy. When four true leaves were grown on the plants, 2 mL of fermentation broth was sprayed onto the leaves, and an equal amount of sterile water was sprayed onto the leaves to establish a negative control. Each treatment was repeated three times. After inoculation, the plants were incubated at 28 ± 1 °C with a relative humidity of 90% and a photoperiod of 14:10 (L:D) h. The diseased areas on the leaves were investigated and recorded after 3, 7, 11, and 14 days. The incidence area rate was calculated based on the proportion of diseased areas among the total leaf area. Then, the disease index was calculated using the incidence area rate. The safe evaluation of N. oryzae for crops was determined by the disease index.

At disease indices of 0~5, 5~10, 10~50, and 50~100, the safety grades were no symptom (NS), slightly susceptible (LS), moderately susceptible (MS), and severely susceptible (SS), respectively.

2.5. Statistical Analysis

The data were analyzed for significance by one-way analysis of variance, followed by Duncan’s multiple range test (p ≤ 0.05) using SPSS 26.0.

3. Results

3.1. Optimization Results of the Fermentation Medium Components of N. oryzae

3.1.1. The Biomass of N. oryzae Was Affected by Different Carbon and Nitrogen Sources

The mycelia of N. oryzae were grown in media supplemented with six carbon sources. The biomass of N. oryzae was the greatest in media supplemented with glycerol. This biomass was significantly greater than that in media in which the carbon sources were maltose, glycerol, soluble starch, and sucrose (F = 11.03, df = 5, 12, p < 0.05), and the maximum biomass was 0.78 g/L. This indicated that using glycerol as a carbon source promoted the mycelial growth of N. oryzae. The second-highest biomass was observed in the medium with lactose as a carbon source, with a biomass of 0.58 g/L. In the fermentation media with maltose and glucose as carbon sources, the biomass was similar and significantly lower than that with glycerol and lactose, and the biomass was 0.35 g/L and 0.36 g/L, respectively. However, the biomass of N. oryzae was lowest in media supplemented with sucrose, at 0.01 g/L. This indicated that the use of sucrose as a carbon source inhibited the mycelial growth of N. oryzae. Therefore, glycerol and lactose were selected as the carbon sources for the media (Figure 1a).

The mycelia of N. oryzae grew in nitrogen sources such as NH₄Cl, (NH₄)₂SO₄, yeast extract, beef extract, and peptone. The biomass of N. oryzae was the greatest in media supplemented with (NH₄)₂SO₄. This biomass was significantly greater than that in media in which the nitrogen sources were NH₄Cl, yeast extract, beef extract, and peptone (F = 6.78, df = 5, 12, p < 0.05), and the maximum biomass was 4.58 g/L. The second-highest biomass was observed in the medium with yeast extract as the nitrogen source, and the biomass was 3.17 g/L. Biomass (2.21 g/L) was observed in the media supplemented with NH₄Cl as the nitrogen source. The biomass growth of N. oryzae was relatively lower than that with beef extract and peptone as nitrogen sources. However, when urea was used as the nitrogen source, the fungus did not grow, and the biomass was 0 g/L. Therefore, (NH₄)₂SO₄ and yeast extract were selected as the nitrogen sources for the media (Figure 1b).

3.1.2. The Biomass of N. oryzae Was Affected by Different Carbon: Nitrogen Ratios

The results of the orthogonal test are shown in Figure 2. Mycelia of N. oryzae grew in the nine treatment groups; however, no significant differences were detected among the groups (p > 0.05). The biomass of N. oryzae in group 2 was greater than that in the other treatment groups, and the carbon:nitrogen ratio for N. oryzae was 2:2:1:2 (glycerol:lactose: yeast extract:(NH₄)₂SO₄). The maximum biomass was 1.08 g/L. The biomass of N. oryzae was greater than that in the other seven treatment groups in group 6, the carbon: nitrogen ratio for N. oryzae was 2:3:1:2 (glycerol:lactose:yeast extract:(NH₄)₂SO₄), and the biomass was 1.05 g/L. The biomass of N. oryzae was lower than that in the other eight treatment groups in group 1; the carbon: nitrogen ratio for N. oryzae was 1:1:1:1 (glycerol:lactose:yeast extract:(NH₄)₂SO₄), and the biomass was 0.13 g/L. The optimal carbon: nitrogen ratio for N. oryzae was 2:2:1:2 (glycerol:lactose:yeast extract:(NH₄)₂SO₄) (Figure 2).

3.1.3. Optimization of Fermentation Conditions in N. oryzae

When the inoculation volume was 3~15 mL, the biomass increased as the amount of fermentation broth inoculation increased. When the inoculation amount was 15 mL, the biomass of N. oryzae was significantly greater than those observed at other inoculation amounts (F = 138.87, df = 4, 10, p < 0.05), and the maximum biomass was 1.89 g/L. The second-highest biomass was observed in the medium with 3 mL as the inoculation amount, and the biomass was 0.69 g/L. There were no significant differences in biomass between the 9 mL and 12 mL inoculation amounts, and the biomasses were 0.45 g/L and 0.54 g/L, respectively. However, the biomass of N. oryzae was lowest in the medium in which the inoculation amount was 6 mL, and the biomass was 0.27 g/L. The optimal inoculation amount was 25% (Figure 3a).

There were no significant differences in biomass among the five fermentation liquid volumes. The biomass of N. oryzae was affected by different fermentation liquid volumes. When this volume was 75 mL, the biomass was greater than that observed at other volumes; however, this difference was not significant (p > 0.05). The maximum biomass was 0.40 g/L. The second-highest biomass was observed in the medium with 30 mL of fermentation liquid, and the biomass was 0.32 g/L. There were no significant differences in biomass among the inoculation amounts of 15 mL, 45 mL, and 60 mL, and the biomasses were 0.25 g/L, 0.28 g/L, and 0.27 g/L, respectively. The optimal fermentation liquid volume was 75 mL (Figure 3b).

When the initial culture temperature was 15~20 °C, the biomass of N. oryzae increased. When the culture temperature was greater than 20 °C, the biomass of N. oryzae decreased. When the culture temperature was 20 °C, the biomass was greater than that observed at other culture temperatures (F = 118.23, df = 4, 10, p < 0.05). The maximum biomass was 5.80 g/L. At 15 °C, 25 °C, 30 °C, and 35 °C, the biomass was less than 2.00 g/L, and there was no significant difference in biomass growth. The biomass of N. oryzae was lowest in the medium in which the culture temperature was 15 °C, and the biomass was 0.07 g/L. The optimal culture temperature was 20 °C (Figure 3c).

The biomass of N. oryzae was affected by different pH values. At a pH of 5.0, the biomass of N. oryzae increased significantly compared with that at other pH values (F = 261.04, df = 4, 10, p < 0.05). When the pH was greater than 5.0, the biomass of N. oryzae decreased rapidly. The maximum biomass was 14.92 g/L. At pH values of 6, 7, 8, and 9, the biomass was less than 5.00 g/L. The second-highest biomass was observed in the medium with a pH of 7, and the biomass was 3.97 g/L. The biomass at pH 7 was significantly greater than that at pH 6, 8, and 9. The optimal initial pH was 5.0 (Figure 3d).

When N. oryzae was cultured for 2~4 days, its biomass increased. When the plants were cultured for more than 4 days, their biomass decreased. After 4 days of culture, the N. oryzae biomass increased significantly compared with that observed during other culture periods (F = 37.12, df = 4, 10, p < 0.05). The maximum biomass was 1.27 g/L. The second-highest biomass was observed in the medium after 8 days of culture, and the biomass was 0.78 g/L. The third-highest biomass was observed in the medium after 6 days of culture, and the biomass was 0.69 g/L. At 4 days, the biomass was significantly greater than that observed at 6 days and 8 days. The optimal culture period was 4 days (Figure 3e).

3.2. Pathogenicity Test Results of Fermentation Broth on S. rostratum

The results of the spray fermentation broth for S. rostratum leaves for 3~14 days are shown in Figure 4. No obvious disease spots were observed on the leaves after 3 days of inoculation. The leaf edges were circular disease spots. By 7 days, the main veins of the leaves showed noticeable disease spots and were chlorotic. After 11 days, the leaves died, and the color of the veins changed from green to red. After 14 days, the dead leaves had fallen out (Figure 4).

With increasing duration of spraying, the area affected by S. rostratum increased gradually. S. rostratum invaded after 3 days of inoculation. The incidence area of S. rostratum was 0.08% after 3 days, and the disease index was 20.00. By 7 days, the incidence of S. rostratum was 4.54%, and the disease index was 30.00. After 11 days, the incidence of S. rostratum was 22.60%, and the disease index was 45.00. After 7~11 days, the disease spot area on S. rostratum expanded greatly. After 14 days, the disease index was highest, reaching 60. N. oryzae exhibited strong pathogenicity against S. rostratum (

Table 2. Pathogenicity of S. rostratum after spraying N. oryzae fermentation solution.

Spray Days	Incidence Area Rate %	Disease Index	Pathogenicity
3 d	0.08 ±0.04 b	20.00 ± 0.00 c	Weak
7 d	4.54 ± 1.58 a	30.00 ± 5.77 bc	Moderate
11 d	22.60 ± 6.40 a	45.00 ± 5.00 ab	Moderate
14 d	30.81 ± 6.93 a	60.00 ± 8.16 a	Strong

Note: Data with the different lowercases letters in same column indicated significantly different p < 0.05.

).

3.3. The Safe Evaluation Result of the Fermentation Broth on Seven Crops

Z. mays, T. aestivum, S. lycopersicum, and F. arundinacea were invaded by N. oryzae after 14 days of spraying. Z. mays, S. melongena, F. arundinacea, B. inermis, and M. sativa disease indices did not exceed 5 in the case of N. oryzae, and the safety evaluation was NS. The T. aestivum and S. lycopersicum disease indices were 11.11 and 17.78, respectively, in the case of N. oryzae, and the disease indices were between 11 and 50. The safety evaluation was MS (

Table 3. Sensibility of the fermentation broth of N. oryzae to different crops.

Crops	Incidence Area Rate %	Disease Index	Disease Severity
Z. mays	1.67 ± 0.07 b	4.44 ± 0.07 b	NS
T. aestivum	2.11 ± 0.40 b	11.11 ± 0.13 a	MS
S. lycopersicum	6.00 ± 2.52 a	17.78 ± 0.07 a	MS
S. melongena	0.00 ± 0.00 b	0.00 ± 0.00 b	NS
F. arundinacea	0.22 ± 0.22 b	2.22 ± 0.07 b	NS
B. inermis	0.00 ± 0.00 b	0.00 ± 0.00 b	NS
M. sativa	0.00 ± 0.00 b	0.00 ± 0.00 b	NS

NS: no symptoms. MS: moderately susceptible. Note: Data with the different lowercases letters in same column indicated significantly different p < 0.05.

).

4. Discussion

The present optimal medium composition and fermentation conditions improved the control efficiency [27]. The biomass of N. oryzae increased when glycerol and lactose were used as carbon sources. Previous studies have shown that N. oryzae biomass is greatest when soluble starch, mannitol, and sucrose are used as carbon sources [28,29]. The present results were inconsistent with previous results, and this discrepancy may be attributed to differences in host plants and collection locations. The N. oryzae biomass increased when (NH₄)₂SO₄ and yeast extract were used as nitrogen sources. Previous studies have shown that N. oryzae biomass is greatest when NH₄NO₃ and yeast are used as nitrogen sources [30]. The NH₄NO₃ was not selected as the test nitrogen for safety considerations in the experiment. To determine the optimal carbon:nitrogen ratio, two types of carbon sources and two types of nitrogen sources were used. This approach was chosen because using mixed carbon and nitrogen sources enhanced the content of microbial metabolites [31]. Previous studies have shown that the growth of N. oryzae is greatest at a pH of 5~6 and a temperature of 20~30 °C [21,32]. This result was consistent with previous study results.

The effectiveness and safety of microorganisms are crucial for controlling weeds [33,34]. N. oryzae exhibited strong pathogenicity against S. rostratum. Furthermore, this pathogenicity was affected by environmental factors in the control and application fields, especially humidity and temperature, as weeds were sprayed on cloudy days, after rain, or under other high-humidity conditions [35]. The fermentation broth was added to appropriate dispersants and UV protectors to improve control efficiency [36]. The safety evaluation indicated that T. aestivum and S. lycopersicum were moderately susceptible to N. oryzae. N. oryzae fermentation broth was sprayed on S. rostratum without T. aestivum or S. lycopersicum. Therefore, we can apply the fermentation broth of N. oryzae to riverbanks, grasslands, wastelands, and other habitats where S. rostratum grows. Or the N. oryzae fermentation broth was sprayed in S. rostratum area of T. aestivum and S. lycopersicum. For example, when the fermentation broth of N. oryzae is used in forests and farmlands where S. rostratum grows, cardboard, fences or shields are needed, and the drift of the fermentation broth is minimized. In a previous study, the biocontrol potential of N. oryzae against S. rostratum was demonstrated [37]. To confirm the potential for biocontrol of the N. oryzae fermentation broth on S. rostratum, field trials will be conducted in the future to determine its control effect of the N. oryzae fermentation broth on S. rostratum. The N. oryzae fermentation broth will be utilized for development of a microbial agent.

5. Conclusions

The fermentation media used for N. oryzae were lactose:glycerol:yeast extract:(NH₄)₂SO₄ = 2:2:1:2. The recommended inoculum amount and fermentation liquid volume were 25% and 25%, respectively, with an initial pH value of 5.0, and the samples were incubated at 20 °C for 4 days. The biomass significantly increased, and the fermentation broth of N. oryzae was strongly affected by S. rostratum. Safety evaluation revealed that N. oryzae exhibited no symptoms on Z. mays, S. melongena, F. arundinacea, B. inermis, or M. sativa but showed moderate susceptibility to T. aestivum and S. lycopersicum. In the future, we will perform more studies on biocontrol by N. oryzae in the field, pesticide formulation, and mechanism of action, and we will provide a basis for the biological control of S. rostratum.