Application of Difenoconazole and Trichoderma Broth Combination for Synergistic Control of Corn Leaf Blight and Stalk Rot in Straw-Returned Fields in Liaoning Province, China

Abstract

Maize production in Fuxin City, Liaoning Province, China, is threatened by northern corn leaf blight (NCLB) and Fusarium stalk rot, with straw return under conservation tillage exacerbating the NCLB severity by 20% in local fields. This study evaluated the efficacy of combining difenoconazole, a commonly used fungicide, with a Trichoderma bioagent for disease control in straw-incorporated soils. Field trials in Fuxin showed that applying 300 g/ha difenoconazole with 1.5 L/ha Trichoderma fermentate achieved superior results: a 72.4% reduction in the NCLB disease index and a stalk rot incidence of only 0.61%. These outcomes significantly outperformed single-component treatments like difenoconazole alone (56.2% NCLB suppression) or other fungicides (e.g., carbendazim, triadimefon). The combined treatment also outperformed the single treatments with biocontrol agent (67.1% NCLB inhibition). The results highlight the synergistic potential of integrating chemical and biological agents to manage residue-borne diseases, offering a practical strategy for sustainable disease control in conservation agriculture systems with straw return in Liaoning, China.

1. Introduction

Maize (Zea mays L.) is a globally vital cereal crop for food security and agricultural economies [1]. In China, it represents over 30% of total grain production, with the northeast region being the primary cultivation area [2].

Two fungal diseases significantly threaten maize productivity. Northern corn leaf blight (NCLB), caused by Exserohilum turcicum, reduces yields by 30–50% through foliar damage [3]. The pathogen persists in crop residues, increasing the disease risk in conservation tillage systems [4]. Fusarium stalk rot, primarily caused by Fusarium graminearum and F. verticillioides, leads to lodging and yield losses while producing hazardous mycotoxins [5]. Both pathogens demonstrate strong survival in soil and plant debris [6].

Conservation tillage has emerged as a sustainable alternative to conventional practices, offering significant potential for soil quality improvement, carbon sequestration, and environmental impact mitigation. However, its complex relationship with disease risk presents a dual challenge that requires careful consideration. The reduction in soil disturbance and retention of crop residues characteristic of conservation tillage can alter the soil microbial community structure and enzymatic activity, potentially modifying the disease dynamics. For instance, long-term conservation tillage (e.g., no-till with straw mulching) may lead to shifts in the abundance of specific pathogenic or beneficial microorganisms. While some studies report an increased abundance of beneficial rhizosphere nitrogen-fixing bacteria under conservation tillage, altered soil conditions (e.g., water saturation) could simultaneously elevate the risks of N₂O and NH₃ emissions, indirectly compromising soil health [7,8,9].

The modification of soil physicochemical properties via conservation tillage may further exacerbate disease risks. Long-term no-till practices can result in surface organic matter accumulation but uneven nutrient distribution in deeper soil layers, creating microenvironments conducive to pathogen proliferation. Additionally, changes in soil pH and C/N ratio under conservation tillage may compromise plant disease resistance, particularly when nutrient availability (e.g., ammonium and nitrate nitrogen) becomes misaligned with microbial metabolic activity [10,11]. The heterogeneous effects on soil enzyme activities, with differential responses among enzyme classes (e.g., hydrolases vs. oxidases), may disrupt organic carbon degradation processes and subsequently affect soil suppressiveness [7,12,13,14].

Practical implementation challenges are compounded by limited farmer awareness of tillage–disease interactions. Many growers hesitate to adopt conservation practices due to concerns about yield penalties or increased disease management complexity, especially in the absence of precision agriculture support [15,16,17]. Current research remains inconclusive regarding conservation tillage–disease relationships, with studies reporting both suppressive and promotive effects of straw retention depending on climatic conditions and crop types [10,18,19], underscoring the need for long-term monitoring and region-specific management strategies.

Our investigation focused on maize straw return effects in Liaoning Province, where this practice has significantly exacerbated the northern corn leaf blight severity (20% disease index increase in Fumeng County) while showing neutral effects on Fusarium stalk rot. Building on local farmers’ predominant use of difenoconazole, we demonstrated that its integration with Trichoderma fermentate achieved superior disease control (72.4% suppression of leaf blight, 0.61% stalk rot incidence), outperforming conventional fungicides. These findings provide actionable insights for balancing conservation agriculture benefits with sustainable disease management.

This study aimed to (1) quantify the disease-enhancing effects of straw return on NCLB and Fusarium stalk rot in Fuxin’s conservation tillage systems, and (2) develop a novel control strategy by synergizing difenoconazole with Trichoderma-derived antagonistic metabolites to target residue-borne pathogens. We hypothesized that:

Straw retention would selectively increase the NCLB severity by preserving foliar pathogen inoculum, while minimally affecting stalk rot due to vascular tissue colonization barriers. The combined application would outperform solo treatments by coupling difenoconazoles’ immediate fungicidal action with Trichoderma metabolites’ long-term antagonism, disrupting pathogen survival through both chemical and microbial interference.

2. Materials and Methods

2.1. Description of the Study Sites

Field experiments were conducted at multiple sites across Liaoning Province, China, including northern (Kangping County, Faku County), western (Beipiao County, Fumeng County), central (Qingyuan County), and southern (Fengcheng County) regions (see

Table 1. Test locations and information on longitude and latitude.

City	County (Latitude and Longitude)	Agrotype
Shenyang	Kangping (42.75° N, 123.35° E)	Loam soil
	Faku (42.50° N, 123.40° E)	Loam soil
Fushun	Qingyuan (42.10° N, 124.92° E)	Loam soil
Chaoyang	Beipiao (41.80° N, 120.75° E)	Loam soil
Fuxin	Fumeng (42.05° N, 121.75° E)	Sandy loam soil
Dandong	Fengcheng (40.45° N, 124.07° E)	Loam soil

for the GPS coordinates). The soils were predominantly brown soil, except for Fumeng County, which had sandy soil. During the 2023–2024 growing seasons (spring and summer), the mean temperatures were 17.7 °C and 17.8 °C, with precipitation averaging 483 mm and 740 mm, respectively (Liaoning Meteorological Bureau, available at http://ln.cma.gov.cn/, accessed on 9 July 2025). No extreme weather events occurred during the experimental period. All experimental plots were located on flat cultivated land.

The fungicide application study was conducted at the Fuxin Experimental Station of Liaoning Academy of Agricultural Sciences in Fumeng County. The experimental fields had been under continuous maize cultivation for more than five years, with the surrounding farmland also dominated by maize production.

2.2. Maize Cultivation and Experimental Design

2.2.1. Experimental Design for Investigating the Effects of Straw Returning on Northern Corn Leaf Blight and Stalk Rot Incidence

The experiment was conducted using a randomized complete block design (RCBD) with three replications to account for potential field variability. The test crop was maize (Zea mays L.), cultivar “Tieyan 58” (Liaoning Tieyan Seed Technology Co., Ltd., Changtu County, China). The experimental sites and their respective soil types are presented in Table 1, with each plot covering an area of ≥30 hectares (hm²). This study was carried out on farmer-leased croplands.

Maize was planted at a row spacing of 60 cm and an intra-row spacing of 30 cm. For weed control, a pre-emergence herbicide containing nicosulfuron (2%), mesotrione (5%), and atrazine (20%) (suspension concentrate, Shandong Jinnonghua Pharmaceutical Co., Ltd.) was uniformly applied at a rate of 2250 mL per hectare. Additionally, N-P-K compound fertilizer (Shandong Klynor Company, Zibo, China) was applied at 450 kg per hectare. All plots followed a no-till system with full straw retention.

2.2.2. Field Plot Layout Design for Studying Fungicide Application on Crop Residues

The experiment followed a randomized complete block design (RCBD) with three replications to minimize spatial variability effects. All treatments, including the blank control (CK), were randomly assigned within each block. Seeds were not treated with coating agents before sowing.

Each experimental plot measured 4.0 m in length and 2.2 m in width, with three rows planted per plot. The row spacing was maintained at 60 cm, while the plant spacing within rows was 30 cm. Field trials were conducted at the Fuxin Experimental Station of Liaoning Academy of Agricultural Sciences. All field management practices, including fertilization, pesticide application, and tillage methods, were consistent with those described in Section 2.2.1.

2.3. Fungicides and Dosages

The following were all commercially purchased: 10% difenoconazole wettable powder (WP), 15% triadimefon WP, 0.3% tetramycin aqueous solution, 250 g/L azoxystrobin suspension concentrate, 50% carbendazim WP, etc. The biological agent used was Trichoderma fermentation broth, provided by Shanghai Jiao Tong University. The Trichoderma fermentate was prepared by culturing the fungal strain in a 300 L liquid fermentation bioreactor for 5 days. The fermentation broth was centrifuged to obtain the supernatant, which was then heat-inactivated at 50–55 °C to eliminate viable Trichoderma propagules, resulting in a metabolite solution containing no live fungal cells. (Note: the specific Trichoderma species and fermentation parameters are currently undisclosed as they are under a patent application.)

The dosage of each fungicide can be found in

Table 2. The dosage of the fungicide used in the research.

Treatment Number	Treatment Combinations	Formulation	Dosage per Hectare
1	10% Difenoconazole	WP (Wettable powder)	300 g
2	10% Difenoconazole + Biocontrol agent	WP (Wettable powder) + AS (Aqueous solution)	300 g + 1.5 L
3	15% Triadimefon	WP (Wettable powder)	1200 g
4	0.3% Tetramycin	AS (Aqueous solution)	1050 g
5	250 g/L Azoxystrobin	SC (Suspension concentrate)	300 mL
6	50% Carbendazim	WP (Wettable powder)	1500 g
7	Biocontrol agent	AS (Aqueous solution)	1.5 L
8	CK	-	-

. Before spring sowing, the tested fungicides or Trichoderma fermentation broth were sprayed onto the surface of retained crop residues (straw mulch) in the field.

2.4. Investigation Methods

2.4.1. Investigation on Farmers’ Preference for Fungicide Types in Corn Planting in Fuxin City

The survey covered Zhangwu County, Fumeng County, and Xihe District under Fuxin City—the primary corn-producing regions of Fuxin. A total of 76 questionnaires were distributed to local farmers to investigate the types of fungicides applied in cornfields over the past two years. After excluding questionnaires reporting the use of mixed-formulation fungicides, 45 valid responses were retained. Data analysis and visualization were performed using Microsoft Excel 2010.

2.4.2. Investigation on Disease Incidence, Disease Index, and Control Efficacy

Investigations were conducted in mid-to-late September during the maize wax-ripening stage.

Disease investigation protocol in 2023: In each region where disease outbreaks occurred, one representative plot (5–8 hm²) was selected per county. Five sampling points were established per plot using a diagonal sampling pattern (four corners and center). To minimize edge effects, sampling avoided the outermost five corn rows along plot boundaries. At each sampling point, ≥20 plants were examined, ensuring a minimum total of 100 plants surveyed per plot.

Disease investigation protocol in 2024: Each treatment contained three biological replicates, with >25 plants examined per replicate (minimum 75 plants per treatment). The disease evaluation protocols included the following: (1) Fusarium stalk rot—quantification of infected plants among total sampled plants at each sampling point; (2) northern leaf blight—severity scoring based on the established classification system (

Table 3. Grading criteria for northern corn leaf blight severity.

Disease Severity Level	Symptom Description
1	Few lesions on leaves, lesion area ≤5% of the total leaf area
3	Sparse lesions, lesion area accounting for 6–10% of the leaf area
5	Moderate lesions, lesion area accounting for 11–30% of the leaf area
7	Abundant lesions, lesions merging, lesion area accounting for 31–70% of the leaf area
9	Leaves nearly covered by lesions, leading to withering

).

The following formulas were used to calculate the disease incidence and inhibition rate [20]:

Disease incidence (%) = (Number of infected plants/Total number of investigated
plants) × 100;

Disease index = [∑(Number of diseased leaves at each severity level ×
Corresponding severity value)/(Total number of investigated leaves ×
Maximum severity value)] × 100;

The inhibition rate of NCLB = [(Disease index of control group − Disease index
of treatment group)/Disease index of control group] × 100.

The inhibition rate of Fusarium stalk rot = [(Disease incidence of control group − Disease incidence of treatment group)/Disease incidence of control group] × 100. When treatment groups demonstrated non-significant inhibitory effects, the inhibition rate was recorded as 0.

2.5. Data Analysis

The disease incidence was assessed following the treatment of straw with chemical or biological agents. The significance of differences in disease index between the treated groups and the control was determined using Student’s t-test in Excel 2010, with statistical significance set at p < 0.05. Differences in inhibition rates were analyzed for significance using Duncan’s multiple range test in SPSS 26.

3. Results

3.1. Straw Return Aggravates Northern Corn Leaf Blight in Fuxin Region with Minimal Effects on Fusarium Stalk Rot

To investigate the impact of conservation tillage with maize straw return on the disease incidence in subsequent crops, we selected several fields where this practice had been implemented since 2020 as the study sites (see Table 1 for the specific locations). Fields without straw return served as the control group. Maize was planted in spring 2023, and disease surveys for northern corn leaf blight and Fusarium stalk rot were conducted in autumn at the locations listed in

Table 4. Disease investigation (northern leaf blight and stalk rot) in selected areas of Liaoning, China.

City	County (Latitude and Longitude)	Northern Corn Leaf Blight (Disease Index)		Fusarium Stalk Rot (Disease Incidence, %)
		Non-Straw Return	Straw Return	Non-Straw Return	Straw Return
Shenyang	Kangping (42.75° N, 123.35° E)	11.11	12.25	0	0
	Faku (42.50° N, 123.40° E)	12.04	13.08	13.89	11.52
Fushun	Qingyuan (42.10° N, 124.92° E)	23.82	26.54	19.00	18.75
Chaoyang	Beipiao (41.80° N, 120.75° E)	48.58	51.52	6.00	7.52
Fuxin	Fumeng (42.05° N, 121.75° E)	35.64	42.78	12.00	12.80
Dandong	Fengcheng (40.45° N, 124.07° E)	32.86	28.95	7.00	8.00

. Northern corn leaf blight was prevalent across all the surveyed regions. The disease index was notably high in Beipiao City, Chaoyang (48.58 for non-straw return vs. 51.52 for straw return) and Fumeng County, Fuxin (35.64 for non-straw return vs. 42.78 for straw return), indicating severe disease occurrence in these areas. In other regions, the disease index showed minor increases or remained relatively unchanged after straw return. Notably, Fumeng County, Fuxin, exhibited the most significant increase in the disease index for northern corn leaf blight, with a nearly 20% rise under straw return conditions.

For Fusarium stalk rot, infected plants were observed at all the survey sites except Kangping County, Shenyang, where no incidence was recorded. However, in these regions, stalk rot remains prevalent, with a slight increase in disease incidence observed in Fumeng County and Fengcheng areas, indicating its persistent impact.

In conclusion, straw return was associated with an aggravation of northern corn leaf blight in Fuxin, highlighting a potential drawback of this conservation practice in certain regions.

3.2. Survey of Commonly Used Fungicides in the Corn Fields of Fuxin Region

Currently, chemical fungicides remain the predominant approach for disease control. To investigate fungicide usage patterns, we conducted a survey among farmers in Fuxin City where the northern corn leaf blight severity was exacerbated under straw incorporation practices. The survey revealed five main fungicides employed in this region: difenoconazole, triadimefon, tetramycin, azoxystrobin, and carbendazim. Notably, difenoconazole was the most widely adopted, being utilized by over 40% of the respondents (Figure 1).

Based on these findings, we selected these fungicides to evaluate their efficacy in controlling the primary pathogen infections associated with straw incorporation. Particular emphasis was placed on investigating difenoconazole’s performance, as optimizing the application methods for this conventional fungicide could significantly improve local disease management practices.

3.3. Synergistic Effect of Difenoconazole and Trichoderma Fermentate in Controlling Northern Corn Leaf Blight and Stem Rot

The results demonstrated that the combined application of fungicides with biocontrol agents exhibited superior efficacy in disease control. In spring 2024, prior to sowing, we conducted a field experiment in Fumeng County, Fuxin City, where straw mulch was treated with the aforementioned fungicides according to the commercial product specifications, followed by normal sowing. Additionally, we incorporated a Trichoderma fermentate biocontrol agent developed by Shanghai Jiao Tong University (application followed the developer’s guidance; see Table 2) to explore the potential synergistic effects when combined with the widely used difenoconazole. Untreated plots served as controls. For leaf blight control, Treatment 2 (difenoconazole + biocontrol agent) showed optimal performance with a disease index of 14.96 and inhibition rate of 72.40 ± 0.52%, significantly outperforming other treatments. Treatment 1 (difenoconazole alone) achieved 56.21 ± 2.73% inhibition (disease index 23.74), while Treatment 5 (azoxystrobin) and Treatment 7 (biocontrol agent) showed moderate efficacy with 69.91 ± 2.24% and 67.11 ± 1.44% inhibition, respectively. Treatment 6 (carbendazim) demonstrated the weakest effect (46.11 ± 5.27%). The control (CK) showed severe infection (disease index 54.22) (

Table 5. Inhibitory effects of different fungicides on northern corn leaf blight after straw spray application.

Treatment Number	Treatment Combinations	Disease Index	Disease Inhibition Rate (%)
1	10% Difenoconazole	23.74 ± 1.47 ***	56.21 ± 2.73 c
2	10% Difenoconazole + Biocontrol agent	14.96 ± 0.28 ***	72.40 ±0.52 a
3	15% Triadimefon	24.70 ± 1.91 ***	54.44 ± 3.53 c
4	0.3% Tetramycin	23.94 ± 2.51 **	55.84 ± 4.62 c
5	250 g/L Azoxystrobin	16.31 ± 1.21 **	69.91 ± 2.24 b
6	50% Carbendazim	29.22 ± 2.86 **	46.11 ± 5.27 d
7	Biocontrol agent	17.83 ± 0.78 ***	67.11 ± 1.44 b
8	CK	54.22 ± 0.71	-

Asterisks indicate statistically significant differences between treatment and control groups as determined by Student’s t-test (***, p < 0.001; **, p < 0.01). Duncan’s multiple range test was used to compare significant differences among various treatment groups (excluding the control), with different lowercase letters (a, b, c, d) denoting significant differences at p < 0.05.

).

For stem rot management, Treatment 2 again exhibited outstanding performance with the lowest incidence rate (0.61 ± 0.70%) and highest disease inhibition rate (%): 71.03 ± 71.51%, significantly superior to that of other treatments. Notably, Treatment 1 (2.15 ± 1.18%) showed comparable efficacy to CK (2.11 ± 2.47%), indicating the limited effect of solo difenoconazole application. Treatments 5 (2.34 ± 2.47%) and 4 (tetramycin, 2.70 ± 1.76%) showed intermediate performance, while Treatments 3 (triadimefon, 4.61 ± 6.01%), 6 (3.28 ± 2.51%), and 7 (3.97 ± 0.64%) demonstrated relatively poor control (

Table 6. Inhibitory effects of different fungicides on Fusarium stalk rot after straw spray application.

Treatment Number	Treatment Combinations	Disease Incidence (%)	Disease Inhibition Rate (%)
1	10% Difenoconazole	2.15 ± 1.18	2.09 ± 2.13
2	10% Difenoconazole + Biocontrol agent	0.61 ± 0.70 *	71.03 ± 71.51
3	15% Triadimefon	4.61 ± 6.01	0
4	0.3% Tetramycin	2.70 ± 1.76	0
5	250 g/L Azoxystrobin	2.34 ± 2.47	0
6	50% Carbendazim	3.28 ± 2.51	0
7	Biocontrol agent	3.98 ± 0.64	0
8	CK	2.11 ± 2.47	-

Asterisks indicate statistically significant differences between treatment and control groups as determined by Student’s t-test (*, p < 0.05).

). These findings highlight that the combination of difenoconazole with Trichoderma fermentate achieved synergistic effects for both leaf blight and stem rot control, whereas carbendazim and triadimefon showed limited efficacy under experimental conditions.

4. Discussion

Crop diseases remain a critical constraint to yield and quality in modern agricultural production. Our disease surveys in Liaoning Province revealed significant regional variations in the disease prevalence. Beipiao City (Chaoyang) exhibited notably high disease indices (48.58 for non-straw return vs. 51.52 for straw return), followed by Fumeng County, Fuxin (35.64 vs. 42.78), indicating severe disease pressure in these areas. Particularly striking was the 20% increase in the northern corn leaf blight disease index under straw return conditions in Fumeng County. Additionally, at the trial site of Fumeng County, modest increases in Fusarium stalk rot incidence were observed with straw retention, providing field evidence for subsequent analyses.

The deployment of disease-resistant cultivars represents the most cost-effective control strategy by eliminating additional inputs. However, widespread adoption faces constraints from limited genetic resources. While advances in genetic engineering, such as the discovery of ZmLecRK1 enhancing the resistance to soil-borne diseases [21], offer potential solutions, identifying broad-spectrum resistance genes remains time- and resource-intensive. Moreover, rapid pathogen evolution threatens the durability of resistance [22].

Conservation tillage systems present unique challenges through straw retention. Although beneficial for soil health, surface residues modify pathogen survival environments, potentially accelerating adaptive evolution and resistance breakdown [23,24]. Our findings demonstrate that straw return significantly exacerbates both northern corn leaf blight (Exserohilum turcicum) and stalk rot (primarily Fusarium spp.), consistent with previous reports on residue-mediated pathogen survival and corroborating our Liaoning observations. This phenomenon is well-documented in other pathosystems; for instance, Goss’s bacterial wilt (Clavibacter michiganensis subsp. nebraskensis) prevalence strongly correlates with residue retention in reduced tillage systems [25]. We hypothesize that straw return increases the initial inoculum loads and improves the overwintering conditions, while continuous maize monoculture may further intensify pathogen accumulation, creating a detrimental pathogen–residue–host feedback loop [25].

Chemical control remains an important component of integrated management. Camera et al. demonstrated 60% control efficacy against NCLB using prothioconazole + trifloxystrobin at pre-tasseling [26]. Our trials with locally used fungicides (e.g., difenoconazole, triadimefon) in Fuxin confirmed their effectiveness for residue-borne disease suppression. Building on previous work with live Trichoderma harzianum SH2303 and fungicide combinations showing 60% field efficacy against Southern Leaf Blight [27], we innovatively employed inactivated Trichoderma fermentate with difenoconazole for residue treatment.

We developed a novel chemo-biological approach for pre-sowing straw treatment. Chemical pretreatments (e.g., urea–NaOH) disrupt lignin matrices to accelerate decomposition [28], while biocontrol agents (e.g., Trichoderma asperellum) enhance cellulase activity to competitively exclude pathogens [29,30]. Their synergy may establish physicochemical barriers while stimulating functional microbiota—a mechanism requiring further elucidation in residue-mediated disease control [29,31].

Pathogen niche differentiation explained the treatment efficacy variations. Vascular colonization by stalk rot pathogens (e.g., Fusarium spp.) [32] reduces the exposure to surface-applied agents, whereas foliar pathogens’ external localization [30,33] enhances fungicide accessibility, accounting for superior leaf disease control.

Our study demonstrates the synergistic efficacy of difenoconazole and Trichoderma combinations against residue-borne diseases in conservation systems. Limitations include single-region/cultivar focus, unresolved molecular mechanisms, and unassessed long-term impacts. Future priorities should encompass: multi-region validation, omics-driven mechanism elucidation, long-term pathogen/soil health monitoring, optimized formulations with risk assessment, and integration with smart monitoring/resistant cultivars to develop comprehensive management frameworks—critical steps toward sustainable conservation agriculture.

5. Conclusions

This study demonstrates that combining difenoconazole (300 g/ha) with Trichoderma fermentate (1.5 L/ha) effectively controls northern corn leaf blight (72.4% reduction) and Fusarium stalk rot (0.61% incidence) in straw-return systems of Liaoning Province, with Fuxin as a representative test area. The integrated approach outperformed single treatments and mitigated the 20% disease exacerbation typically associated with straw retention. However, the current single-region/single-cultivar design and limited mechanistic insights necessitate further validation across the diverse agroecological zones of Liaoning and other regions. Future studies should employ omics technologies to elucidate the molecular interactions, evaluate the long-term field efficacy, and develop regionally optimized formulations for broader adoption in conservation agriculture.