Preparation and Herbicidal Activity of a Microbial Agent Derived from Alternaria gaisen Strain GD-011

Abstract

Microbial herbicides, recognized for their target specificity, environmental compatibility, and simple production processes, hold promising potential for sustainable agriculture. This study isolated a strain of Alternaria gaisen (designated GD-011) from infected Medicago sativa L. in Qinghai Province, China, and evaluated its herbicidal potential through systematic development and efficacy assessment. Using single-factor and orthogonal experimental designs, the optimal sporulation substrate was identified as wheat bran, and the fermentation medium was optimized to consist of 14.5 g wheat bran, 19.4 g wheat middlings, 1.5 g rapeseed cake, and 14.6 g corn flour. Based on colony diameter and OD₆₀₀ measurements, diatomite was selected as the most suitable carrier, while bentonite, humic acid, and polyvinyl alcohol were chosen as the stabilizer, protectant, and dispersant, respectively. Pot trials under controlled conditions demonstrated strong herbicidal activity of GD-011 against three common weed species: Chenopodium album L., Elsholtzia densa Benth., and Galium aparine L. The highest efficacy was observed against C. album, with disease incidence and fresh weight inhibition reaching 80.83% and 79.87%, respectively. Inhibition rates for both E. densa and G. asparine exceeded 60%. A wettable powder formulation developed from GD-011 showed particularly effective control of C. album and E. densa, providing a practical foundation for the application of GD-011 as a novel bioherbicide.

1. Introduction

The Qinghai–Tibet Plateau, being the headwater region of China’s three major rivers (the Yangtze, Yellow, and Lancang Rivers), holds strategic significance for national ecological security. The alpine conditions of this region—characterized by high altitude, low temperature, intense solar radiation, and low precipitation [1]—result in fragile ecosystems with 40–60% lower environmental carrying capacity per unit area compared to plains [2]. Under these conditions, weeds exhibit polymorphic distribution patterns, predominantly including annual weeds (e.g., Chenopodium album L., Galium aparine L.) and invasive alien species (e.g., Amaranthus retroflexus L.), forming complex community structures.

According to the Weed Science Society of America (WSSA), there are 1118 taxonomically confirmed weed species worldwide, distributed across 2847 genera in 177 plant families [3]. Weeds significantly constrain sustainable agricultural development by competing for essential resources such as light, water, and nutrients, reducing crop quality, and impeding mechanized operations, thereby exerting multi-dimensional impacts on agricultural ecosystems. Particularly challenging are perennial C4 weeds, which establish persistent soil seed banks through vegetative propagation and exhibit strong resistance to conventional control measures. Their ecological plasticity and environmental adaptability pose major challenges for effective management [4]. Current weed control strategies predominantly rely on manual weeding and chemical herbicides; however, the former is inefficient and the latter involves high economic costs, both presenting considerable limitations [5].

Given the limitations of chemical control, the development of environmentally friendly bioherbicides utilizing microbial resources has become a major research focus. Among various biocontrol strategies, fungi have garnered significant attention due to their diverse secondary metabolites and highly efficient infection mechanisms [6]. Currently, bioherbicides are primarily categorized into two groups: live microbial formulations (including spore suspensions and mycelial cultures) and fungal metabolite-based formulations (such as antibiotics and enzymes). Based on their physical forms, they can be further classified into solid formulations (e.g., dustable powders [DP] and wettable powders [WP]) and liquid formulations (e.g., emulsifiable concentrates [EC] and suspension concentrates [SC]). Among these, wettable powders (WP) have become the dominant formulation in China’s bioherbicide market due to their excellent stability and user-friendly application [7].In their study, Luo Z et al. [8] developed an innovative wettable powder using Sophora alopecuroides L., Hippophae rhamnoides L., and Juniperus sabina L. as raw materials. Through systematic single-factor and orthogonal experiments, the researchers optimized the preparation parameters: bentonite as the carrier, calcium lignosulfonate as the dispersant (at a ratio of 1:9 with wetting agent), and 12% additive content. The resulting formulation met all quality standards, establishing a novel approach for pesticide innovation while demonstrating the potential for resource utilization of botanical ingredients. Wen et al. [9] reported that the F0b wettable powder, applied at a dosage of 300 g/667 m², achieved a control efficacy of 73.14% against Fragaria × ananassa gray mold (Botrytis cinerea) 14 days after the second application, significantly outperforming the Trichoderma-based WP control group (63.71%). The efficacy further increased with higher application rates. The exploration of distinctive bioherbicides and the development of their commercial application systems hold considerable promise for future herbicide development [10]

In this study, the Alternaria gaisen GD-011, isolated from diseased Medicago sativa in Qinghai, was employed. Previous research by Zhang et al. [11] demonstrated that this herbicidal strain exhibited optimal growth at 25 °C, pH 7–8, and 16 h/day light exposure when cultured in a 200 r/min shaker for 4 days with 6% inoculation volume and 200 mL liquid medium. The combination of NaCl and glucose (C₆H₁₂O₆) constituted the most favorable formulation for the optimal growth medium, while a combination of NaCl and MgSO₄ maximized sporulation. The mycelia exhibited a lethal temperature of 51 °C for 10 min. Further studies by He et al. [12] revealed that GD-011 fermentation products demonstrated broad-spectrum herbicidal activity, achieving up to 94.83% weed control efficacy within 7 days against nine weed species, including Chenopodium album L and Elsholtzia densa Benth. Notably, the strain exhibited no pathogenicity toward wheat (Triticum aestivum L.) and faba bean (Vicia faba L), while only mild pathogenicity was observed in barley (Hordeum vulgare L).

This study aims to develop broad-spectrum microbial herbicides to address the research gap in this field in Qinghai Province. By optimizing solid-state fermentation substrates, screening carriers and adjuvants, preparing wettable powder formulations, and evaluating their efficacy in pot experiments, a stable and high-performance formulation was successfully developed. These findings provide theoretical support for microbial herbicide research and establish a foundation for the field application of green weed control technologies.

2. Materials and Methods

2.1. Field Sampling, Microbial Isolation and Spore Inoculum Preparation

This study was conducted in the experimental farmland of Qinghai University (36°43′51.6″ N, 101°44′06.0″ E, altitude 2275 m) in Chengbei District, Xining City, Qinghai Province. Field surveys of weed infestation were performed across croplands of wheat (Triticum aestivum L.), highland barley (Hordeum vulgare L.), faba bean (Vicia faba L.), rapeseed (Brassica napus L.), pea (Pisum sativum L.), and potato (Solanum tuberosum L.). The GD-011 strain preserved by the Qinghai Provincial Key Laboratory of Agricultural Pest Integrated Management was activated and inoculated into PDB liquid medium using agar block method (6 mm mycelial plugs) under a laminar flow hood (SW-CJ-1D, Suzhou Purification Equipment Co., Ltd., Suzhou, China). Cultivation was carried out in a shaking incubator (THZ-82A, Shanghai Yiheng Scientific Instruments Co., Ltd., Shanghai, China) at 180 rpm for 5 days. The resulting culture was then diluted to prepare spore suspensions at a concentration of 1 × 10⁶ spores/mL for subsequent experiments. Preliminary tests demonstrated that 1 × 10⁶ spores/mL represented the optimal infection concentration. Lower concentrations resulted in failed infection, while higher concentrations led to inconsistent infection outcomes. Therefore, all subsequent experiments used hemocytometer-adjusted inoculum precisely standardized to 1 × 10⁶ spores/mL.

2.2. Optimization of Solid-State Fermentation Substrates Through Single-Factor Experiments and Orthogonal Array Design

Strain GD-011 was cultured on PDA plates at 28 °C for 7 days. Under aseptic conditions, 10 mL of sterile water was added to each plate, and the surface mycelia and spores were gently scraped using a sterile spreader. The resulting suspension was filtered through four layers of sterile gauze to obtain a spore suspension. The initial spore concentration was determined using a hemocytometer under a microscope, and the suspension was subsequently diluted with sterile water to a final concentration of 1 × 10⁶ spores/mL for subsequent use. Selected agricultural by-products—including corn meal, rapeseed cake, wheat straw, sheep manure, and wheat bran—were used as solid fermentation substrates. Each 25 g of substrate was inoculated with 5 mL of the prepared spore suspension (1 × 10⁶ spores/mL), thoroughly mixed, and incubated statically at 25 °C in a biochemical incubator for 8 days. After fermentation, the products were naturally dried and ground into powder [13]. The dried powder was then resuspended in sterile distilled water at a ratio of 1:10 (w/v), vortexed for 3 min to achieve a homogeneous dispersion, and the optical density at 600 nm (OD₆₀₀) was measured using a spectrophotometer with sterile distilled water as the blank control. All treatments were performed in triplicate.

Based on preliminary screening of five single-substrate fermentations, wheat bran, straw bran, rapeseed cake, and corn meal were selected as the four fundamental components. Each factor was set at three levels according to different ratios, and nine formulations were generated using an L9(3⁴) orthogonal design (

Table 1. Solid-state fermentation test design of the L₉(4³) formulation combinations.

	4-Factor Quality(g)
Recipe Number	Wheat Straw Chaff	Wheat Bran	Rapeseed Cake	Corn Flour
1	24.5	16.5	0.8	8.2
2	18.3	18.3	1.2	12.2
3	14.5	19.4	1.5	14.6
4	21.7	10.9	1.1	16.3
5	24.1	18.1	1.8	6
6	19.8	19.8	0.5	9.9
7	26.8	10.8	1.6	10.8
8	22.3	13.4	0.9	13.4
9	24.7	19.8	0.5	5

). The solid fermentation substrates were prepared accordingly, supplemented with 2% (w/w) of the optimal carbon and nitrogen sources, and adjusted with sterile water. The mixtures were loaded into 250 mL conical flasks, sterilized at 121 °C for 30 min, and cooled. Each flask was then inoculated with 10 mL of spore suspension (1.00 × 10⁶ spores/mL), sealed, and subjected to fermentation under the aforementioned conditions. Finally, conidia were eluted and collected, and both sporulation yield and OD₆₀₀ nm were measured.

2.3. Screening of Carriers and Additives

To find the optimal medium for strain GD-011, the following components were screened: carrier (kaolin, limestone, diatomaceous earth, lakai powder, and clay), dispersant (tween, carboxymethylcellulose, sodium lignosulphonate, sodium carboxymethylcellulose, SDS, and polyvinyl alcohol), stabilizer (calcium carbonate, and bentonite, silica), protectant (humic acid, sodium alginate, dextrin, and soluble starch), and wetting agent (SDS, and starch). These substances were added in an amount of 2%, and then the culture media containing different carriers and auxiliaries were made into cakes with a diameter of 8 mm. The cakes were inoculated to observe the growth of the GD-011 strain on the different carriers and additives. According to their physicochemical properties, the biofungal agents, suitable carriers, stabilizers, wetting agents, dispersants and other auxiliaries were screened [14].

2.4. Preliminary Preparation of Wettable Powders

The prepared spore culture was added to a solid medium containing maltose (optimal carbon source) and potassium nitrate (optimal nitrogen source) [11], followed by incubation at 25 °C for 10 days. The resulting solid fermentation product was dried and ground into a fine powder. The fungal powder from each strain was then blended with a selected carrier and auxiliary agents at the following ratios: 50% carrier, 6% dispersant, 1% stabilizer, and 3% wetting agent. This mixture was processed using a standardized protocol to produce a wettable powder formulation [14]. Subsequently, the carrier and fungal powder were uniformly mixed, combined with 1% sodium alginate solution at a 1:3 (w/v) ratio, and then dropwise added into a calcium chloride (CaCl₂) solution. After forming into granules, the beads were collected using sterile gauze to remove excess moisture and allowed to air-dry at room temperature [15].

2.5. Valuation of Herbicidal Activity of GD-011 Microbial Inoculant Against Weeds in Pot Experiments

The weed seeds used in this study were collected from field-grown mature plants in 2022 and subsequently sown in 15 cm diameter pots containing a sterilized growth substrate (leaf mold:garden soil:perlite:plant ash = 2:2:1:1, v/v). When the plants reached the 6–8 leaf stage under greenhouse conditions, they were inoculated by uniformly spraying 30 mL of spore suspension (10⁶ spores/mL) per pot, while control plants received sterile water. The greenhouse environment was maintained at 25 °C with >80% relative humidity and a 12 h photoperiod. Disease assessment was conducted 7 days post-inoculation by calculating both disease incidence and disease index. Disease severity was evaluated daily according to the established rating scale [16]: Grade 0 (no lesions), Grade 1 (visible leaf lesions), Grade 2 (lesions covering 1/3–2/3 of leaf area), Grade 3 (necrosis affecting >2/3 of leaf area), and Grade 4 (complete leaf wilting and yellowing). Lesion progression was monitored and recorded daily throughout the experimental period.

$$ncidence rate={\displaystyle \frac{number of leaves affected}{total number of leaves surveyed}}\times 100\%$$

$$Fresh weight control efficacy={\displaystyle \frac{Control group fresh weight-Treatment group fresh weight}{Control group fresh weight}}\times 100\%$$

2.6. Statistical Analyses

The data were processed and analyzed using Excel 2016 and IBM SPSS Statistics 25.0 software. Experimental data are presented as mean ± standard error (SE). Statistical significance of differences among mean values was determined by Duncan’s new multiple range test at a significance level of p ≤ 0.05. The raw data were subjected to normality testing (Shapiro–Wilk test) and homogeneity of variance testing (Levene’s test), with results indicating that all significance levels exceeded 0.05. Consequently, the data satisfied the normality and homogeneity of variance assumptions for parametric testing (ANOVA), thereby eliminating the need for data transformation.

3. Results

3.1. Results of the One-Factor Screening and Orthogonal Combination Optimization of the Solid State Fermentation Substrates

From the Lambert-Beer law, a positive relationship exists between the amount of spores in a spore suspension and the OD value, and the concentration of the spore suspension reflects the amount of spore production. The data in Figure 1 and

Table 2. Effects of different solid fermentation substrates on the spore content and light absorption (OD) value of the spore solution of A. gaisen GD-011.

		Strain GD-011
Substrate		OD Value	Spore Production × 1010 (Spores/mL)
Straw bran		0.47 ± 0.04c	0.69 ± 0.07c
Wheat bran		0.79 ± 0.02a	1.14 ± 0.10a
Maize flour		0.70 ± 0.02b	0.87 ± 0.03b
Rapeseed cake		0.21 ± 0.02d	0.43 ± 0.05d
Sheep manure		0.40 ± 0.02c	0.33 ± 0.04d

Note: Data are the “mean ± standard error”. Different letters in the same column indicate significant differences (p < 0.05) by Duncan’s new complex polarity test.

and

Table 3. Screening results of orthogonal combinations of components to optimize the solid substrate fermentation formulation.

		Strain GD-011
Combination		OD Value	Spore Production × 1010 (Spores/mL)
1		0.43 ± 0.12d	1.12 ± 0.17d
2		0.85 ± 0.28b	0.92 ± 0.12d
3		1.10 ± 0.32a	4.8 ± 0.39a
4		0.85 ± 0.03b	1.92 ± 0.24c
5		0.61 ± 0.03c	3.01 ± 0.19b
6		0.51 ± 0.14d	3.37 ± 0.39b
7		0.60 ± 0.20c	1.28 ± 0.20cd
8		0.84 ± 0.23b	3.12 ± 0.26b
9		0.74 ± 0.01bc	3.49 ± 0.25b

Note: Data are the “mean ± standard error”. Different letters in the same column indicate significant differences (p < 0.05) by Duncan’s new complex polarity test.

show that there were differences in the spore production of the GD-011 strain in the different fermentation substrates. Under the premise of equal amounts of fermentation substrate and seed liquid access, the spore production and OD value of the GD-011 strain on wheat bran were significantly larger than the other treatments in the one-factor screening of solid fermentation substrates. The orthogonal test showed that strain GD-011 grew better on combination 3 (straw bran 14.5 g + wheat bran 19.4 g + rapeseed cake 1.5 g + corn flour 14.6 g), with large colonies and vigorous mycelial growth.

3.2. Screening of GD-011 Strain Carriers and Auxiliaries

All the carriers were added into the optimal solid medium corresponding to each strain at a ratio of 2%, and the 8 mm diameter cakes were placed in the center of the plate for cultivation. The colony diameters and spore production of the GD-011 strain on the different carriers and auxiliaries were measured at the 3rd and 6th days, respectively. As can be seen in Figure 2 and

Table 4. Comparison of colony diameters and OD₆₀₀ values of A. gaisen GD-011. under different carrier and auxiliary conditions.

Carrier or Auxiliary	Colony Diameter		OD600 Value
	3d	6d
Potato Dextrose Agar medium	3.83 ± 0.17 b	7.13 ± 0.09 ab	0.22 ± 0.01 c
Diatomaceous earth	4.10 ± 0.16 ab	7.53 ± 0.21 a	0.41 ± 0.13 a
Nekal	0 ± 0 d	0 ± 0 e	0 ± 0 d
Limestone	4.03 ± 0.13 a	6.97 ± 0.13 ab	0.25 ± 0.09 c
Ball clay	3.73 ± 0.21 b	7.14 ± 0.17 ab	0.20 ± 0.06 c
Sodium dodecyl sulfate	1.27 ± 0.09 c	1.73 ± 0.13 d	0.28 ± 0.03 c
Humic acid	4.27 ± 0.13 a	7.13 ± 0.13 ab	0.33 ± 0.0 b
Dextrin	3.43 ± 0.21 bc	6.77 ± 0.21 ab	0.30 ± 0.03 b
Algin	3.57 ± 0.05 bc	6.27 ± 0.38 b	0.32 ± 0.04 b
Amylogen	4.17 ± 0.13 ab	6.07 ± 0.25 b	0.28 ± 0.01 c
Bentonite	3.80 ± 0.22 b	7.67 ± 0.60 a	0.37 ± 0.18 b
Light calcium carbonate	3.83 ± 0.13 b	7.47 ± 0.42 a	0.30 ± 0.02 b
Carbon-white	3.97 ± 0.05 b	7.03 ± 0.17 ab	0.35 ± 0.05 b
Heavy calcium carbonate	3.96 ± 0.05 ab	6.83 ± 0.13 ab	0.26 ± 0.03 c
Carboxymethylcellulose	3.43 ± 0.17 bc	6.47 ± 0.17 b	0.26 ± 0.07 c
Carboxymethylcellulose sodium	3.70 ± 0.08 b	7.13 ± 0.34 ab	0.28 ± 0.04 c
Kaolinite	4.47 ± 0.05 a	7.47 ± 0.09 a	0.29 ± 0.05 c
Poval	3.60 ± 0.23 b	5.80 ± 0.16 c	0.45 ± 0.14 a
Sodium lignin sulfonate	3.63 ± 0.13 b	7.30 ± 0.14 a	0.32 ± 0.07 b

Note: Data are the “mean ± standard error”. Different letters in the same column indicate significant differences (p < 0.05) by Duncan’s new complex polarity test.

, the overall growth status of the GD-011 strain is better in the media containing different carriers and auxiliaries, but most of the differences are not very large. The effects of the powder and SDS carrier on growth were slower and assessed after 6 d of basic growth, when the plate was about two-thirds full. After adding the carrier bentonite, the average value of OD₆₀₀ was 3.80, the diameter of the colony at 6 d was 7.67 cm, and the other carrier treatments differed significantly (p < 0.05). By analogy, diatomaceous earth, bentonite, humic acid and polyvinyl alcohol were selected as the best carriers and auxiliaries for the GD-011 strain.

3.3. Preliminary Preparation of Wettable Powders

The optimized solid-state fermentation substrate formula components above were used to determine the optimal substrate ratios based on batch solid fermentation, and the GD-011 strains of white mycelia in this ratio generated vigorous growth for 10 d. After growth, the fungi were naturally dried, crushed into a Microbial Powder, and screening indicated the appropriate carriers and auxiliaries in accordance with the proportions of 50% carrier, 6% dispersing agent, 1% stabilizer and 3% wettable agent. These were mixed to make the wettable powders and stored in the refrigerator at 4 °C prior to use.

3.4. Determination of the Pathogenicity of Wettable Powders on Potted Weeds

As shown in

Table 5. Effectiveness of A. gaisen GD-011. wettable powder for weed control in different potted plants.

Weed Name	Incidence of Disease (%)	Control Effect of Fresh Weight (%)
C. album	80.83 ± 5.14 a	79.87 ± 0.87 a
E. densa	65.66 ± 4.16 b	61.39 ± 3.69 c
G. aparine	78.38 ± 4.45 ab	74.64 ± 4.39 b

Note: Data are the “mean ± standard error”. Different letters in the same column indicate significant differences (p < 0.05) by Duncan’s new complex polarity test.

and Figure 3, after spraying GD-011 wettable powder, the control effects on Chenopodium album L and Galium aparine L. bane were outstanding. The incidence and fresh weight preventive effect on Chenopodium album L. reached 80.83 and 79.87%, respectively, and Chenopodium album L. plants showed yellowing, drying and wilting until the whole plant died. The incidence and fresh weight preventive effect on Galium aparine L. were 78.38 and 74.64%, respectively, and the leaves of the plants produced black spots, which gradually faded to black-brown and the plants withered in serious cases. The incidence and fresh weight preventive effect on Elsholtzia densa Benth. were 65.66 and 61.39%, respectively, and the plants showed large leaf spots, which gradually faded to dark brown, and 61.39% Chenopodium album L plants showed large leaf spots and dwarfing.

4. Discussion

With increasingly stringent restrictions on the use of chemical pesticides, microbial pesticides have emerged as a major category of biopesticides due to their significant advantages, including high efficacy, broad-spectrum activity, safety, and good environmental compatibility, demonstrating broad application prospects [17]. However, compared to chemical pesticides, although various microbial herbicides have been developed, only a few products have achieved commercial availability due to limitations in formulation technology, application methods, and commercialization challenges [18].

In the development of fungal herbicides, given that their efficacy is easily affected by external environmental conditions, optimizing formulation design and selecting appropriate carriers and adjuvants to maintain the stability and infectivity of microbial agents have become key factors for the successful development of biopesticides [19,20]. Currently, large-scale fermentation equipment serves as the primary method for the industrial production of biocontrol fungi. Standard production typically begins with small-scale laboratory trials to determine the optimal culture medium composition and growth conditions that promote sporulation. This preparatory phase aims to achieve spore yields that meet industrial production standards, thereby scaling up the system and laying the technical foundation for mass fermentation and commercial production [21,22,23,24,25]. Research on microbial fermentation media and culture conditions represents a key methodological approach for enhancing spore yield [22,24]. Microbial fermentation is influenced by multiple factors, including the type of carbon source, fermentation temperature, substrate moisture content, incubation duration, agitation speed, inoculum size, and initial pH, all of which directly affect microbial growth and metabolite synthesis [25]. A crucial pathway for increasing spore production lies in the optimization of medium composition and fermentation parameters. Lin et al. [26] demonstrated that optimizing the medium formulation significantly enhanced the sporulation capacity of Botrytis elliptica HZ-011, highlighting its potential for weed control applications. Zhu Haixia et al. [27] achieved increased sporulation in the herbicidal Trichoderma polysporum HZ-31 through fermentation condition optimization. Jiang et al. [28] reported that optimized fermentation conditions improved the efficacy of the sclerotial fungus Aspergillus sclerotiorum As-68 against Xanthomonas oryzae pv. oryzae. Yang Ying et al. [29] employed single-factor experiments and response surface methodology to optimize the liquid fermentation medium of Aureobasidium pullulans PA-2, resulting in a significant increase in spore yield and providing 75.0% fresh weight control efficacy against Chenopodium album. Yin Yanan et al. [30] optimized Bacillus cereus NJSZ-13, achieving a 4.1-fold increase in viable cell count compared to pre-optimization levels. Attaining industrial-standard spore production is a fundamental prerequisite for the formulation, processing and commercial application of microbial products.

This study utilized spore yield and OD600 as key evaluation indicators to determine the optimal solid-state fermentation (SSF) substrate for the strain GD-011. An orthogonal experimental design was applied to screen the best substrate composition for maximizing fungal proliferation. Subsequently, suitable carriers and adjuvants were systematically screened to preliminarily develop a GD-011 wettable powder (WP) formulation. The research systematically optimized the fermentation process of Alternaria alternata GD-011 and successfully prepared a wettable powder with high herbicidal activity, with results being highly consistent with existing literature. This study identified wheat bran as the optimal substrate for mycelial growth, which aligns with the findings of Wan et al. [31] that strain H116 exhibited the fastest and most robust mycelial proliferation on PDA supplemented with wheat bran. The optimized substrate formulation (14.5 g straw powder + 19.4 g wheat bran + 1.5 g rapeseed cake + 14.6 g corn flour) significantly enhanced mycelial density and sporulation, supporting the hypothesis proposed by Duré et al. [32] that composite substrates provide balanced nutrient availability. The results further confirm the crucial role of carbon-to-nitrogen (C/N) ratio in fungal growth: wheat bran and corn flour served as efficient carbon sources, while rapeseed cake supplemented nitrogen, consistent with the study by Chen et al. [33]. The developed wettable powder (50% carrier + 6% dispersant + 1% stabilizer + 3% wetting agent) integrated the functional advantages of each component, adhering to established principles of solid-state fermentation formulations. The GD-011 strain demonstrated remarkable herbicidal efficacy, achieving a disease incidence rate of 80.83% and a fresh weight inhibition rate of 79.87% against Chenopodium album. Notably, it also exhibited >60% inhibition efficacy against Galium aparine and Elsholtzia densa, consistent with the findings of Cheng et al. [34] that Alternaria alternata DT-DYLC provided 85.99% fresh weight control against Elsholtzia densa.

5. Conclusions

This study successfully optimized the solid-state fermentation process for the Alternaria sp. GD-011 strain, identifying wheat bran as the optimal substrate and developing a composite formulation (14.5 g straw chaff + 19.4 g wheat bran + 1.5 g rapeseed cake + 14.6 g corn flour) that significantly enhanced mycelial growth (OD₆₀₀) and sporulation yield (p < 0.05). Utilizing diatomaceous earth as the carrier and bentonite as a stabilizer among other additives, a wettable powder formulation was developed (50% carrier + 6% dispersant + 1% stabilizer + 3% wetting agent). The formulation demonstrated promising herbicidal activity, achieving fresh weight control efficiencies of 79.87% against Chenopodium album L. and 74.64% against Galium aparine, with an incidence rate of 80.83% for C. album. These findings provide crucial technical support for the industrial-scale production of the GD-011 strain. Future research should prioritize addressing challenges such as room-temperature storage stability and formulation optimization through compound mixtures to enhance field application performance.