Typical anthracnose-infected sorghum leaves were collected from Da’an District, Zigong City, Sichuan Province, China, in 2024. The leaf samples were surface-sterilized by sequential immersion in 75% ethanol for 30 s and 1% sodium hypochlorite for 1 min, followed by three rinses with sterile distilled water. Small pieces (approximately 5 mm²) of sterilized tissue from the lesion margins were plated on potato dextrose agar (PDA) medium and incubated at 28°C for 3 days in the dark. Emerging fungal colonies were purified by subculturing on fresh PDA. Conidial morphology was examined under a light microscope (Olympus BX53, Japan) at 400× magnification. For molecular identification, genomic DNA was extracted from 7-day-old mycelial cultures using a commercial fungal DNA extraction kit (Solarbio, Beijing, China) following the manufacturer’s instructions. The internal transcribed spacer (ITS) region of the ribosomal DNA was amplified using the universal primers ITS1 (5’-TCCGTAGGTGAACCTGCGG-3’) and ITS4 (5’-TCCTCCGCTTATTGATATGC-3’) (White et al., 1990). PCR reactions were performed in a 25 μL volume containing 12.5 μL 2× Taq Master Mix (Vazyme, Nanjing, China), 1 μL each primer (10 μmol/L), 1 μL template DNA (50 ng/μL), and 9.5 μL ddH₂O. The thermal cycling protocol comprised an initial denaturation at 94°C for 5 min; 35 cycles of denaturation 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 1 min; followed by a final extension at 72°C for 10 min. The resulting PCR products were sequenced bidirectionally by Sangon Biotech (Shanghai, China). Sequences were analyzed using BLASTn against the NCBI GenBank database for species identification. The isolated strain, designated ZG-DA-20, was preserved on PDA slants at 4°C for subsequent studies.

Pathogenicity of the isolated strain ZG-DA-20 was verified in accordance with Koch’s postulates. Healthy sorghum seedlings (cv. ‘GJH’, susceptible to anthracnose) at the 5-leaf stage were inoculated with a conidial suspension (1 × 10⁶ conidia/mL) prepared in sterile distilled water with 0.1% Tween-20. The suspension was sprayed onto leaves until runoff using a handheld atomizer. Control plants were treated similarly with sterile distilled water containing 0.1% Tween-20. All inoculated plants were maintained in a growth chamber at 28°C with 90% relative humidity and a 12-h photoperiod for 7 days. The pathogen was subsequently re-isolated from the developing lesions on the inoculated leaves and confirmed to be identical to the original strain ZG-DA-20 through both morphological and molecular characterization.

Eleven agents were evaluated for their efficacy in controlling sorghum anthracnose caused by C. sublineola strain ZG-DA-20 (Supplementary Table S1). Three chemical fungicides served as positive controls: Pyraclostrobin (250 g/L EC, BASF Crop Protection, Jiangsu, China), Carbendazim (50% WP, Anhui Guangxin Agrochemical Co., Ltd., China), and Tebuconazole (430 g/L SC, Jiangsu Qizhou Green Chemical Co., Ltd., China). Six biofungicides were tested: S-abscisic acid (5% SL, Sichuan Longmang Fusheng Technology Co., Ltd., China), Alexin (0.3% Matrine AS, Xiangyu Agricultural Technology Co., Ltd., China), Atailing (0.5% Physcion AS, Hebei Zhongbao Lunong Crop Technology Co., Ltd., China), Iron chlorin (also known as dihydroporphyrin iron, 0.5% SL, Anqing Baite Biological Engineering Co., Ltd., China), HhrpEcc (0.5% Osthole AS, Sichuan Haiboshi Biological Technology Co., Ltd., China), and Pterostilbene (99.5% SC, Beijing Solebo Technology Co., Ltd., China). Additionally, two microbial antagonists were incorporated: Bacillus subtilis (10¹⁰ CFU/g WP, Hebei Guanlong Agrochemical Co., Ltd., China) and Trichoderma ha0rzianum (2 × 10⁹; CFU/g WP, Hubei Qiming Biological Engineering Co., Ltd., China). All agents were initially prepared as stock solutions according to the field concentrations recommended by the manufacturers, followed by serial dilutions. Based on these tests and preliminary in vitro experiments, we determined the final concentration ranges (Supplementary Table S2) used for EC₅₀; estimation (Abo-Zaid et al., 2025). Sterile distilled water served as the negative control throughout the study.

Stock solutions of each agent were serially diluted to obtain a graded concentration series, as determined by preliminary range-finding tests. One milliliter of each diluted solution was mixed with 99 mL of molten potato sucrose agar (PSA) medium (cooled to 45-50°C) to prepare agent-amended plates. Control plates received 1 mL of sterile distilled water. Mycelial plugs (5 mm diameter) were excised from the actively growing margins of 7-day-old ZG-DA-20 colonies on PDA and placed centrally on the amended PSA plates. Plates were incubated at 28°C for 6 to 8 days in the dark. Colony diameters were measured using the cross-measurement method (two perpendicular diameters, subtracting the plug diameter). Each treatment consisted of four times. Mycelial growth inhibition rate was according to the following formula (Lü et al., 2023):

where Dc is the average colony diameter on control plates, and Dt is the average colony diameter on treatment plates.

Stock solutions of each agent were diluted to the desired concentrations and incorporated into water agar (WA) medium at a 1% (v/v) ratio. Control plates were prepared by adding an equivalent volume of sterile distilled water. Conidial suspensions of ZG-DA-20 were prepared by flooding 7-day-old PDA cultures with sterile distilled water containing 0.1% Tween-20, scraping the surface, and filtering through double-layer cheesecloth. The concentration was adjusted to 1 × 10⁶ conidia/mL using a hemocytometer. Fifty microliters of the suspension were spread evenly onto each WA plate. Plates were incubated at 28°C for 9 h in darkness. Germination was assessed microscopically by examining 100 randomly selected conidia per plate; a conidium was considered germinated if the germ tube length exceeded half the conidial length. Each treatment was replicated four times. Conidial germination rate was calculated as follows:

Inhibition rate (%) was then determined as (Hu et al., 2021):

where Gc is the germination rate in the control, and Gt is the germination rate in the treatment.

Virulence regression equations were constructed using the agent concentration as the x-axis and the probit-transformed inhibition rate as the y-axis. Regression parameters, correlation coefficients (r), and median effective concentrations (EC₅₀;) with 95% confidence intervals were estimated using probit analysis in SPSS 26.0 software. EC₅₀; values were used to compare inhibitory potencies and select agents with high efficacy at low doses.

Sorghum seeds (cv. ‘GJH1’) were sown in plastic pots (15 cm diameter) filled with sterilized potting mix (peat:vermiculite:perlite = 2:1:1, v/v/v) and grown in a greenhouse at 28 °C with a 12-h photoperiod. At the six-leaf stage, uniform seedlings were selected and transferred to an inoculation chamber maintained at approximately 75% relative humidity. To sustain high humidity, the chamber was covered with white plastic film. Plants were inoculated by spraying their leaves with a conidial suspension of ZG-DA-20 (1 × 10⁶ conidia/mL in 0.1% Tween-20) until runoff occurred. After 36 h, agents were applied at their respective effective concentrations (from in vitro assays) using a handheld sprayer. The experimental design comprised the following treatments: sterile water (blank control), chemical fungicides (positive controls), and pathogen inoculation without agent (negative control). Each treatment had 10 pots with 3 plants per pot, arranged in a completely randomized design. After 7 days, plant growth parameters (fresh weight, root length, and plant height) were measured using a digital balance and ruler. Specifically, root length was measured as the distance from the stem base to the tip of the longest root using a ruler, with each plant measured individually. Mean values for each treatment were used for analysis. Disease severity was rated on a 1–5 scale (Zhang et al., 2025): 1 = no symptoms (healthy); 2 = hypersensitive lesions on lower leaves; 3 = acervuli on bottom leaves; 4 = acervuli on middle to bottom leaves; 5 = acervuli on whole plant including flag leaf. Disease index (DI) was calculated as:

Control efficacy (%) was determined as (Egel et al., 2019):

Field trials were conducted during May 2025 in experimental sorghum fields in Chouzhou (103.66°E, 30.57°N) and Luhzhou (105.35°E, 28.96°N), Sichuan Province, China. Sorghum plants (cv. ‘GJH1’) were cultivated at a spacing of 40 cm × 20 cm in plots of 20m², arranged in a randomized complete block design with four replicates per treatment. Five selected agents (Atailing, Iron chlorin, T. harzianum, pterostilbene, and B. subtilis) were tested, along with a water control, resulting in 50 plots (2 plants per plot). A conidial suspension of ZG-DA-20 (1 × 10⁶ CFU/mL) was applied to leaves using a backpack sprayer, followed 30 min later by agent application at manufacturer-recommended field concentrations. Standard agronomic practices were followed, without additional fungicide applications. For field evaluations, however, the rating standards were manually adjusted by taking into account local meteorological data (rainfall and average daily temperature) recorded after inoculation, the rate of disease progression, and the proportional distribution of severity levels observed under controlled greenhouse conditions. Fourteen days post-inoculation, disease incidence (percentage of infected plants), severity (1–5 scale as in greenhouse), and disease index were recorded for all plants per plot. Control efficacy was calculated as in the greenhouse trials. Grading criteria for anthracnose severity were adjusted based on natural field conditions, incorporating environmental factors such as rainfall and temperature.

All data were analyzed with SPSS 26.0 software (IBM Corp., Armonk, NY, USA). Means of plant growth parameters, inhibition rates, EC₅₀; values, disease indices, and control efficacies were compared using one-way analysis of variance (ANOVA) followed by the least significant difference (LSD) test at P< 0.05.

A highly virulent fungal strain of C. sublineola, designated ZG-DA-20, was isolated from symptomatic sorghum leaves collected in Da’an District, Zigong City, in southwestern China. The disease symptoms and morphological characteristics of the pathogen confirmed its identity as C. sublineola (Figure 1A). When cultivated on PDA at 28°C for 7 days, the colonies initially exhibited a white coloration that gradually turned gray, forming abundant acervuli with setae (Figure 1B). The conidia were crescent-shaped, hyaline, and aseptate, measuring 15-25 μm in length and 3-5 μm in width, aligning with previously described characteristics for C. sublineola (Figure 1C). Molecular identification based on the ITS region showed that the sequence of ZG-DA-20 (539/539 bp) was 100% identical to the reference strain C. sublineola GD202206 (GenBank: ON680862.1). Pathogenicity was then confirmed via Koch’s postulates. Sorghum seedlings inoculated with a conidial suspension developed characteristic anthracnose symptoms, including red-brown lesions bearing acervuli at 7 dpi. In contrast, mock-inoculated control plants remained healthy and symptom-free (Figure 1D). The same fungus was successfully re-isolated from the diseased tissues, thereby fulfilling Koch’s postulates and confirming C. sublineola as the causal agent of the observed symptoms.

The three chemical fungicides and five biofungicides (Iron chlorin, HhrpEcc, Alexin, S-abscisic acid, and Pterostilbene) exhibited varying degrees of inhibitory activity against the mycelial growth and spore germination of C. sublineola in vitro (Figures 1, 2; Tables 1, 2). For mycelial growth inhibition, all agents significantly reduced growth compared with the control, with the three chemical agents showing particularly strong inhibition (Figures 1, 2). Among the five biofungicides, iron chlorin was the most effective, with the lowest median effective concentration (EC₅₀ = 0.0790 µg/mL), followed by Pterostilbene (EC₅₀ = 113.3241 µg/mL). Notably, HhrpEcc displayed a steep regression slope (k = 3.2209), indicating a rapid decline in pathogen viability with increasing concentrations. The correlation coefficients (r) ranged from 0.9113 to 0.9969, implying that all agents exhibited significant dose-response relationships (Table 1).

Spore germination assays yielded results consistent with those observed for mycelial growth inhibition. Among the five biofungicides tested, iron chlorin again exhibited the strongest inhibitory effect, showing with the lowest EC₅₀; (0.0475 µg/mL), followed by pterostilbene (65.3901 µg/mL). HhrpEcc and S-abscisic acid also displayed moderate effects, with EC₅₀; values of 110.7003 and 68.0103 µg/mL, respectively (Table 2). Notably, the EC₅₀; values for spore germination were consistently lower than those for mycelial growth across all tested agents.

In greenhouse trials, all agents reduced anthracnose severity compared with the inoculated control, which exhibited a disease index of 88.89 and extensive leaf lesions covering over 70% of the leaf area (Figure 3; Table 3). Among the six test biocontrol agents, pterostilbene and iron chlorin provided the most effective protection, achieving disease indices of 51.85 and 42.96, respectively, and preventing the formation of visible red spots on leaves (Figure 3). These results corresponded to control efficacies of 41.3% for pterostilbene and 51.7% for Iron chlorin. Moderate efficacy was observed for HhrpEcc (33.8% efficacy; disease index 58.89), S-abscisic acid (36.7%; 56.30), and Atailing (25.8%; 65.93). Alexin showed the lowest control efficacy (16.7%; 87.40) (Table 3). Regarding plant growth parameters, none of the treatments had a significant impact on plant height or root length. As expected, pathogen inoculation significantly reduced fresh weight, indicating that anthracnose significantly inhibits plant growth. After treatment with biocontrol agents, only pterostilbene restored plant fresh weight to a level statistically comparable to that of the healthy mock-inoculated control, while other treatments still significantly reduced plant fresh weight (Table 3).

A separate greenhouse pot experiment was also conducted to evaluate the biocontrol efficacy of T. harzianum and B. subtilis against sorghum anthracnose. At 7 dpi after foliar application of spore suspensions at the manufacturer-recommended concentrations, B. subtilis and T. harzianum exhibited high relative control efficacies of 73.0% and 65.5%, respectively (Table 4). To further investigate their modes of action, dual-culture assays were performed, showing that T. harzianum and B. subtilis significantly inhibited the mycelial growth of strain ZG-DA-20, leading to restricted colony expansion (Figure 4). These results demonstrate that T. harzianum and B. subtilis achieve effective control through direct antagonism and possibly by inducing host resistance. Their demonstrated performance underscores the potential of these microbial agents as sustainable alternatives to chemical fungicides, which could help reduce pesticide residues and mitigate the risk of resistance development.

Field trials conducted in Luzhou and Chongzhou confirmed the consistency of greenhouse results, with pterostilbene and B. subtilis providing consistent control across locations. Disease severity on sorghum leaves was assessed using a 0–4 grading scale, where 0 represented no symptoms and 4 indicated severe lesions covering most of the leaf surface (Figure 5). This grading system highlights progressive deterioration with increasing infection severity. Across the two trial sites, pterostilbene-treated plants exhibited fewer instances of grades 2-4, indicating generally milder disease severity, followed by those treated with B. subtilis, which showed a similar but slightly less pronounced reduction in higher-grade symptoms (Figure 5).

In Luzhou, the inoculated control treatment showed a disease incidence of 81.24% and a disease index of 34.1 (Table 5). Application of pterostilbene reduced the disease incidence to 53.69% and the disease index to 18.98, achieving 44.33% efficacy. B. subtilis achieved 27.43% efficacy, with incidence disease at 71.93% and disease index at 23.89. Iron chlorin and Atailing achieved 26.78% and 19.9% efficacy, respectively. In Chongzhou, where disease pressure was higher, with the inoculated control showing a disease incidence of 97.44% and a disease index of 50.1. However, pterostilbene treatment excelled again at 45.01% efficacy and a disease index of 27.5, followed by B. subtilis at 34.20% efficacy and disease index of 32.7, then Atailing at 34.48% efficacy and disease index of 32.8. T. harzianum demonstrated limited efficacy at 13.28% (Table 5). Statistical analysis confirmed significant differences among treatments, with pterostilbene and B. subtilis outperforming the others in both locations, as evidenced by the skewed distributions toward lower severity grades in the stacked bar charts.