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Article

Evaluation of the Potential of Pyrimidine Nucleoside Antibiotics Against Alternaria spp. Resistant to QoIs Fungicides: Insights for the Management of Ginseng Alternaria Leaf and Stem Blight Disease

by
Shuai Shao
1,
Mingyuan Hu
1,
Xiaolin Chen
2,
Ming’en Jiang
1,
Changqing Chen
1,
Baohui Lu
1 and
Jie Gao
1,3,*
1
College of Plant Protection, Jilin Agricultural University, Changchun 130118, China
2
Jilin Provincial Ginseng and Deer Antler Department (Jilin Provincial Traditional Chinese Medicine Material Development Center), Changchun 130118, China
3
State-Local Joint Engineering Research Center of Ginseng Breeding and Application, Changchun 130118, China
*
Author to whom correspondence should be addressed.
Agriculture 2025, 15(8), 875; https://doi.org/10.3390/agriculture15080875
Submission received: 3 March 2025 / Revised: 8 April 2025 / Accepted: 14 April 2025 / Published: 16 April 2025
(This article belongs to the Section Crop Protection, Diseases, Pests and Weeds)
Browse Figures
Review Reports Versions Notes

Abstract

:
To manage the developing resistance of Alternaria spp. [the causal fungi of ginseng Alternaria leaf and stem blight (GALSB)] to QoIs fungicides, the toxicity and biochemical activity of pyrimidine nucleoside antibiotics (PNA) against Alternaria spp., cross-resistance between PNA and eight other fungicides currently used to control GALSB disease, and the efficacy of PNA for controlling GALSB in vitro and in vivo were investigated. The distributions of EC50 values of PNA against the mycelial growth (115 isolates) and conidia germination (89 isolates) of A. alternata were unimodal, with mean EC50 values of 10.192 ± 4.961 μg/mL and 0.828 ± 0.101 μg/mL, respectively. There were no significant correlations between the sensitivity of A. alternata to PNA and eight other fungicides (p < 0.05). PNA caused morphological changes in A. alternata mycelia and germ tubes, increased cell membrane permeability, and reduced intracellular DNA and protein levels. On detached ginseng leaves, 300 μg/mL PNA achieved mean protective and curative effects of 87.93% and 94.77% against A. alternata 7 days post-inoculation, outperforming that of 300 μg/mL kresoxim-methyl. Field trial results showed that PNA (180 g a.i./hm2) achieved mean efficacies of 85.63%, 84.07%, and 72.55% at three sites 7, 15, and 30 days after the last spray, which were 5.28–37.74% higher than those of control fungicides pyraclostrobin, azoxystrobin, and kresoxim-methyl at corresponding time points. Overall, our findings indicate that PNA are effective agents for the management of Alternaria spp. resistance to QoIs fungicides.

Graphical Abstract

1. Introduction

Ginseng Alternaria leaf and stem blight (GALSB) caused by Alternaria spp. ranks among the most prevalent and severe diseases in ginseng cultivation. It affects various parts of the ginseng plant, leading to excessive drying and withering of leaves, plant lodging, and even death. When infecting fruits, it may cause seed contamination by the pathogen. GALSB typically manifests as circular or irregular necrotic lesions encircled by chlorotic zones on host leaves. It spreads rapidly via copious conidia production, leading to secondary infections [1,2]. Typically, it has a normal annual incidence rate of 20% to 30%, which can reach up to 70% in severe cases, which lead to serious economic losses to ginseng production [1,3]. In addition to A. panax and A. tenuissima, A. alternata is one of the main pathogens causing GALSB disease, which has been frequently isolated from ginseng leaves in most regions [2]. A. alternata is an important pathogenic fungus with a broad host range. Besides infecting ginseng/Korea ginseng and American ginseng [2,4], it can also infect a variety of crops such as tobacco, quinoa, and other plants [5,6].
Chemical fungicide application is the main approach used for the control of GALSB disease. In China, various fungicides, including sterol demethylation inhibitors (DMIs), quinone outside inhibitors (QoIs), and certain protectants, have been officially approved for the management of GALSB disease (http://www.chinapesticide.org.cn, accessed on 6 April 2025). The main fungicides used for the control of diseases caused by Alternaria spp. include kresoxim-methyl [1] and azoxystrobin [7,8], pyraclostrobin [9,10], tebuconazole [11,12], mancozeb [13,14], and copper-based fungicides. Positive cross-resistance was observed between pyraclostrobin and tebuconazole in Alternaria spp. [15]. Research reports indicate that several Alternaria spp., including A. alternata, A. tenuissima, and A. arborescens, which cause Alternaria rot in citrus, Alternaria late blight in pistachios, and early blight in potatoes, have developed varying degrees of resistance to QoIs fungicides including azoxystrobin, kresoxim-methyl, pyraclostrobin, and so on [16,17,18]. Additionally, the efficacy of these fungicides, such as azoxystrobin, pyraclostrobin, and tebuconazole, has decreased over time due to the development resistance in Alternaria spp., and this has led to increases in the dosage and application frequency of fungicides [19,20]. Consequently, the fungicide residues associated with the excess application of fungicides can result in environmental pollution and residues in plants [21]. There is thus a need to characterize the efficacy of alternative fungicides, especially highly effective and low-toxicity fungicides and cross-resistance between fungicides for mitigating the development of resistance, overcoming the challenges associated with the current levels of resistance of Alternaria spp., and controlling GALSB disease.
Pyrimidine nucleoside antibiotics (PNA), derived from Streptomyces hygrospinosus var. beijingensis [22], are natural compounds with a nucleoside-modified structure that are widely used in agriculture and medicine [23]. In China, the PNA in use mainly contain anisomycin, tetramycin A, tetramycin B, toyocamycin, and nystatin [24,25]. All these components are approved for crops production. A total of 13 domestic and international manufacturers produce 17 PNA products, which were registered for 11 crops. These products combat seven major categories of diseases, such as black spot of Chinese cabbage and early blight of tomato caused by Alternaria spp. (http://www.chinapesticide.org.cn, accessed on 6 April 2025). Nevertheless, the inhibitory activity of PNA against Alternaria spp., causing GALSB disease and its underlying biochemical mechanisms, has not been reported. Whether there is cross-resistance between PNA and fungicides currently used to control GALSB disease in Alternaria spp. remains unclear. Therefore, additional research is needed to determine whether PNA could be used to effectively control Alternaria spp. resistance. In order to explore whether PNA can be used to manage the resistance of Alternaria spp. to QoIs fungicides, we conducted the following investigations. We investigated the inhibitory and biochemical activity of PNA against three species of GALSB fungi in vitro. The protective and curative effects of PNA against GALSB on detached ginseng leaves were evaluated by performing inoculation tests. Cross-resistance relationships between PNA and three methoxypropyl acrylate fungicides (pyraclostrobin, azoxystrobin, and kresoxim-methyl), three sterol demethylation inhibitors (difenoconazole, propiconazole, and tebuconazole), one dimethylformamide-derived fungicide (iprodione), and one thiocarbamate fungicide (mancozeb) were evaluated by the mycelial growth rate method. Field experiments were conducted to evaluate the efficacy of PNA for controlling GALSB disease in three ginseng-growing areas. The aim of this study was to evaluate the inhibitory effect of PNA against Alternaria spp. and its application potential in the field, so that it provided a rationale for the field control of GALSB disease and resistance management of GALSB fungi.

2. Materials and Methods

2.1. Fungicides

Technical-grade (TA) pyrimidine nucleoside antibiotics (PNA 40% active ingredient, a.i.) were obtained from Shaanxi Macro Bio-Tech Co., Ltd. (Xi’an, China); azoxystrobin (98% a.i., TA), kresoxim-methyl (97% a.i., TA), and pyraclostrobin (96% a.i., TA) were obtained from Hebei Ruiyao Biotechnology Co., Ltd. (Shijiazhuang, China); mancozeb (97% a.i., TA) was obtained from Jiangsu Limin Chemical Co., Ltd. (Xinyi, China); difenoconazole (96% a.i., TA) was obtained from Hubei Jiufeng Long Chemical Co., Ltd. (Wuhan, China); propiconazole (95% a.i., TA) was obtained from Zhejiang Heben Technology Co., Ltd. (Hangzhou, China); tebuconazole (98% a.i., TA) was obtained from Qingdao Hailier Chemical Co., Ltd. (Qingdao, China); and iprodione (97% a.i., TA) was obtained from Shandong Union Pesticide Industry Co., Ltd. (Jinan, China) Prior to use, the aforementioned fungicides, except PNA and mancozeb, were dissolved in acetone, and a stock solution of 10 mg/mL was prepared by diluting with a 0.1% (v/v) Tween-80 solution; the fungicides were then stored at 4 °C for subsequent assays. In addition, the fungicides used in the field trials included PNA 4% aqueous solution (AS) obtained from Shaanxi Hongda Science and Technology Co., Ltd. (Xi’an, China); pyraclostrobin 25% suspension concentrate (SC) provided by Syngenta Nantong Crop Protection Co., Ltd. (Nantong, China); azoxystrobin 25% suspension concentrate (SC) provided by Limin Chemical Co., Ltd. (Xinyi, China); and kresoxim-methyl 30% wettable powder (WP) provided by Shandong Jingbo Agrochemical Science and Technology Co., Ltd. (Jingbo, China).

2.2. Isolates

A total of 170 isolates of Alternaria spp. were isolated from ginseng stem, leaf, and seed samples collected from Baishan City, Tonghua City, Yanbian Prefecture, Jilin City and Changchun City, Jilin Province, from 2018 to 2022. These consisted of 115 isolates of A. alternata, 29 isolates of A. tenuissima, and 26 isolates of A. panax. All isolates were identified through morphological approaches, and some isolates were further distinguished via molecular biological methods. PNA were not previously applied in any of the five regions from which samples were collected. All isolates were stored at −20 °C using filter paper blocks.

2.3. Sensitivity of Alternaria spp. Isolates to PNA

The sensitivity of the mycelia of A. alternata (n = 115), A. tenuissima (n = 29), and A. panax (n = 26) was tested using the mycelial growth rate method [26]. The isolates were cultivated on potato dextrose agar (PDA) medium (consisting of 200 g of potato, 16 g of agar, 20 g of glucose, and 1 L of deionized water per L of the medium) at 25 °C for 7 days. Mycelial plugs measuring 8 mm in diameter were inoculated onto PDA medium containing PNA at final concentrations of 0, 0.1, 0.5, 1, 5, 10, 50, and 100 µg/mL PDA medium containing the same concentration of Tween-80 was used as a control. Colony diameter was measured using the criss-cross method after 7 days of incubation at 25 °C in the dark. The sensitivity of A. alternata (n = 89) and A. tenuissima (n = 29) conidia was tested using the conidia germination method [27]. The isolates cultivated on PDA at 25 °C for 5 days were rinsed with a 0.1% (v/v) Tween-80 solution to facilitate conidia release, which resulted in the preparation of a conidia suspension with a final concentration of 1.0 × 106 conidia·mL−1. The PNA storage concentrations were 0, 0.2, 1.0, 2.0, 10, and 20 μg/mL. Three replicates of 25 μL of conidia suspension and PNA storage solution were added to concave glass slides for each treatment. The treated conidia suspension was incubated under moisturizing conditions at 25 °C for 3 h, and conidia germination was determined. Germination was considered to occur when the length of the germ tube exceeded the short radius of the conidia. Conidia germination was determined in three randomly selected areas, ensuring that the total number of conidia was not less than 300, and the number of germinated conidia was recorded. The relative inhibition rate was calculated based on measured colony diameters and conidia germination rates using Formulas (1) and (2). Calculations of EC50 (the concentrations at which 50% of mycelial growth or conidia germination was inhibited) were made using SPSS software (version 27.0 for Windows).
The sensitivity level was evaluated by EC50. In addition, the baseline sensitivity of A. alternata to PNA was established for 115 isolates of A. alternata using the mycelium growth method and 89 isolates of A. alternata by the conidia germination method. Among the A. alternata isolates, only 89 isolates capable of producing conidia were used in subsequent experiments.
Relative inhibition rate (%) = 1 − (colony diameter of treatment group − disk diameter)/(colony diameter of control group − disk diameter)) × 100
Relative inhibition rate (%) = 1 − (conidia germination rate of treatment group/conidia germination rate of control group) × 100

2.4. Effects of PNA on the Morphology of Mycelia and Conidia Germ Tubes of A. alternata

Fungal mycelia of isolates DH3-1, XJ2-5, and JY39 were introduced into PDB medium supplemented with varying concentrations (0, 10, and 100 μg/mL) of PNA. The cultures were incubated at 25 °C and 160 rpm for 48 h on a shaker, followed by an additional static incubation period of 24 h. After incubation, the fungal mycelia were sampled and examined microscopically for any morphological changes, with each treatment consisting of three replicates.
A modified staining method [28] was used in which isolates DH3-1, XJ2-5, and JY39 were cultivated on PDA medium at 25 °C for 5 days. Subsequently, the colonies were washed in 0.1% Tween-80 solution to prepare a conidia suspension with a final concentration of 1 × 106 conidia·mL−1; they were then combined with a PNA solution at 20 μg/mL. The conidia suspension and reagent were applied to concave slides in triplicate and incubated at 25 °C for 3 h for observations of conidia germ tube morphology. Following dehydration, fixation, permeabilization, DAPI staining, and washing, the slides were examined under a fluorescence microscope (Zeiss Axio Imager 2 Pol, Carl Zeiss AG, Oberkochen, Germany) for nuclear structure analysis.

2.5. Test for Biochemical Activity of PNA on A. alternata

2.5.1. Determination of Cell Membrane Permeability

Five mycelial plugs from each isolate of DH3-1, XJ2-5, and JY39 were inoculated separately into shaking flasks containing 100 mL of potato dextrose broth (PDB). In the PDB medium, 0, 0.1, 1, and 10 μg/mL PNA were added individually; subsequently, the mycelia were incubated in the dark at 25 °C with shaking at 160 rpm for 48 h. Next, the mycelium was washed three times with deionized water, vacuum-filtered for 5 min, and finally resuspended in sterile double-distilled water (ddH2O) to prepare a suspension of 40 mL/g mycelium. Conductivity was measured after 0, 30, 60, 90, 120, 150, 180, 210, 240, and 270 min using a conductivity meter (DDS-307A, Shanghai Precision Scientific Instrument Co., Ltd., Shanghai, China). The entire experiment was repeated three times, with three replicates for each treatment [29].

2.5.2. Determination of the Ergosterol Content

The mycelia were collected according to the method in Section 2.5.1 after 3 days, and the net wet weight was recorded. For each treatment, 0.1 g of mycelium was transferred to a centrifuge tube containing ethanol-potassium hydroxide solution (5 mL of 25%). The solution was thoroughly mixed using a vortex mixer and incubated in a water bath at 85 °C for 4 h. Subsequently, 2 mL of distilled water and 5 mL of n-butane were added and mixed thoroughly; the mixture was left at room temperature for phase separation. The n-hexane layer was analyzed using a UV-Vis spectrophotometer (UV-1780, Shimadzu Corporation, Kyoto, Japan) within the wavelength range of 230 to 300 nm. In the n-hexane layer, characteristic peaks corresponding to ergosterol (282 nm) and the late-stage sterol intermediate 24(28) dehydroergosterol (DHE) (230 nm and 282 nm) were observed. The ergosterol content was calculated using Formulas (3)–(5).
Ergosterol + 24(28) DHE = (A282/290)/pellet weight
Concentration of 24(28) DHE = (A230/518)/pellet weight
Concentration of ergosterol = (A282/290)/pellet weight − (A230/518)/pellet weight
where 290 and 518 are the E-values (in percentages per cm) determined for crystalline ergosterol and 24(28) DHE, respectively, and the pellet weight is the net wet weight (g) [30].

2.5.3. Determination of the DNA and Protein Content

DNA was extracted from mycelium using the CTAB method, and the biomass of DNA was determined. Proteins were extracted using the TCA/acetone method combined with the SDS/phenol extraction technique.
The CTAB method was used for DNA extraction [31]. Subsequently, sequential extractions were performed with phenol:chloroform:isopentanol (25:24:1) followed by chloroform:isopentanol (24:1) to eliminate impurities. Next, the DNA was precipitated by the addition of isopropanol and sodium acetate. The precipitated DNA was washed with both 70% and 100% ethanol, centrifuged to remove the supernatant, and allowed to dry. Finally, the DNA was dissolved in double-distilled water (50 μL), and its concentration was measured.
The mycelium (0.1 g) was weighed and ground in liquid nitrogen using the TCA/acetone method along with SDS/phenol extraction [32]. A total of 1 mL of precooled 10% TCA-acetone solution (with 0.2% DTT) was added and vortex-mixed. The mixture was left to precipitate overnight at −20 °C, then centrifuged at 4 °C, 12,000 rpm for 15 min. The precipitate underwent one wash with 100 mmol/L ammonium acetate, 80% methanol, and 80% acetone solutions. Next, 1 mL of SDS/phenol extraction solution (1:1 saturated phenol and SDS buffer) was added, vortex-mixed, and incubated on ice for 5 min. After centrifugation at 4 °C, 12,000 rpm for 15 min, the upper phenolic phase was transferred to five volumes of precooled 100 mmol/L ammonium acetate-methanol solution. The solution was precipitated at −20 °C for 4 h and centrifuged again at 4 °C for 15 min. The resulting precipitate was washed with precooled methanol and 80% acetone, freeze-dried (Alpha 1-2 LD plus, Shanghai Bajiu Industrial Co., Ltd., Shanghai, China) to obtain a powder. The powder was dissolved in 1 mL of ddH2O to determine protein concentration and content. Standard solutions of bovine serum albumin at 0, 100, 200, 300, 400, and 500 μg/mL were prepared in triplicate. OD values at 280 nm were measured to create a standard curve for calculating protein content in different treatments. Substance content calculation Formula (6):
DNA or protein content (mg/g) = (DNA or protein concentration (mg/mL) × volume of ddH2O (mL))/mass of mycelium (g)

2.6. Protective and Curative Efficacy

The detached leaves with similar growth and size from ginseng of the same age (cv. Damaya, susceptible to Alternaria spp.) were used to evaluate the protective and curative efficacy of PNA against A. alternata in vitro. Leaves were immersed in 75% ethanol for 10 s; they were then rinsed three times with sterile distilled water and dried naturally. The sterile culture dish (long × high × width = 50 cm × 6 cm × 35 cm) with sterilized filter paper was impregnated with sterile distilled water. A handheld sprayer was utilized to administer 150 μL of PNA solution of various concentrations (0, 10, 100, 200, 300, and 500 μg/mL) onto each ginseng leaf. Three incisions on the adaxial surface of the leaf were made using a needle; this was followed by inoculation with A. alternata mycelial plugs (8 mm in diameter) cultivated on PDA at 25 °C for 5 days. Evaluations of the protective and curative treatments of PNA on fungal isolates DH3-1, XJ2-5, and JY39 with varying sensitivity levels according to the conidial germination method were conducted. In the protective treatments, leaves were inoculated with A. alternata mycelial plugs 24 h after PNA were sprayed. In the curative treatments, leaves were inoculated 24 h before PNA were sprayed. The test was performed three times, and three independent replications of the entire test were performed [33]. The treated leaves were incubated at 25 °C for 7 days. Protective efficacy and curative efficacy were evaluated and calculated using the following Formula (7).
Protective/curative efficacy (%) = (diameter of control lesions (mm) − diameter of treated lesions (mm))/(diameter of control lesions (mm)) × 100

2.7. Test for Field Efficacy of PNA Against GALSB Disease

Field tests were conducted at three ginseng cultivation centers in Jingyue District, Changchun City (125.4° N, 43.8° E), Songjianghe Town, Fusong County (127.5° N, 42.2° E), and Yanminghu Town, Dunhua City (129.4° N, 42.8° E), Jilin Province. The experimental areas we selected are the regions where GALSB disease prevalence often happens, and the GALSB disease occurred severely in the selected experimental plots in the last year. Three field trials were performed in a completely randomized block design, with an area of 2–3 m2 per plot. A total of seven treatments were performed, including PNA 4% AS at dosages of 108, 144, and 180 g a.i./hm2, pyraclostrobin 25% SC at a dosage of 112 g a.i./hm2, azoxystrobin 25% SC at a dosage of 94 g a.i./hm2, kresoxim-methyl 30% WP at a dosage of 220 g a.i./hm2, and a clear water control (CK); each treatment was replicated three times in each test site. In the early stages of GALSB disease, a foliar spray application method was utilized. The nozzle was positioned 20 cm from the leaf surface to ensure that the PNA were evenly applied. Consistent volumes of fungicide were sprayed in all plots. Fungicides were applied three times with 7 to 10 days between each application during the flowering stage (GALSB disease was in the initial stage) to the fruit-ripening stage (the disease was in the decline period of the occurrence) of the ginseng. Surveys were performed 7 days, 15 days, and 30 days after the final fungicide application. In each plot, samples were taken at five random points, and all the leaves of approximately five ginseng plants were investigated at each point. The grading method (based on leaves) was as follows [34]: level 0: no lesions; level 1: the lesion area accounts for 1% to 5% of the entire leaf area; level 3: the lesion area accounts for 6% to 10% of the entire leaf area; level 5: the lesion area accounts for 11% to 20% of the entire leaf area; level 7: the lesion area accounts for 21% to 50% of the entire leaf area; and level 9: the lesion area accounts for more than 50% of the entire leaf area. The disease index and control efficacy were calculated using Formulas (8) and (9), respectively.
Disease index = Σ (Number of infected leaves at each disease level × corresponding severity value)/(Total number of surveyed leaves × maximum severity value) × 100
Control efficacy (%) = (disease index of the blank control area post-treatment − disease index of the treatment area post-treatment)/disease index of the blank control area post-treatment) × 100

2.8. Cross-Resistance Tests Between PNA and Other Fungicides

A total of 81 isolates of A. alternata were selected to evaluate their sensitivity to PNA and various fungicides (including pyraclostrobin, azoxystrobin, kresoxim-methyl, mancozeb, difenoconazole, propiconazole, tebuconazole, and iprodione) using the mycelial growth rate method. The final concentrations of seven tested fungicides (pyraclostrobin, azoxystrobin, kresoxim-methyl, difenoconazole, propiconazole, tebuconazole, and iprodione) were 0, 0.001, 0.01, 0.1, 1, 10, 100, and 1000 μg/mL. The final concentrations of mancozeb were 0, 10, 50, 100, 500, 1000, and 2500 μg/mL. Correlations in the sensitivity levels to various fungicides were analyzed. The experiment was carried out three times, and three replicate plates were used for each concentration. Cross-resistance between PNA and other fungicides was analyzed on the basis of Spearman’s rank correlation coefficient. Cross-resistance between the two fungicides was categorized as strong (r > 0.8), moderate (0.5 ≤ r < 0.8), low (0.3 ≤ r < 0.5), or absent (r < 0.3) and was considered significant when p < 0.05 [35].

2.9. Statistical Analysis

All data were analyzed using SPSS software (version 27.0 for Windows). Values are presented as mean ± standard deviation (SD). Least significant difference (LSD) tests were used to determine differences among means, with the significance threshold set at p < 0.05.

3. Results

3.1. Baseline Sensitivity of Alternaria spp. to PNA

PNA had pronounced inhibitory effects against all three species of Alternaria spp., as listed in Table 1. The inhibitory activity against conidial germination was found to exceed that of mycelial growth. Concentrations of PNA between 5.0 and 10.0 μg/mL completely inhibited conidia germination, whereas a concentration of 10.0 μg/mL resulted in only a 50% inhibition rate of mycelial growth. The mean EC50 value of the mycelium of A. panax isolates to PNA was 31.448 ± 12.846 μg/mL, which was significantly higher than the EC50 values of A. tenuissima and A. alternata. The mean EC50 values of the mycelia and conidia of A. tenuissima isolates to PNA were 11.494 ± 3.379 μg/mL and 0.666 ± 0.199 μg/mL, respectively. The mean EC50 values of the mycelia and conidia of A. alternata isolates to PNA were 10.192 ± 4.961 μg/mL and 0.828 ± 0.101 μg/mL, respectively (Table 1). The frequency distribution of EC50 values of A. alternata isolates to PNA approximated a unimodal curve (Figure 1). This pattern suggested that PNA-resistant isolates were absent from field-collected samples. Consequently, the mean EC50 values were used to establish the baseline sensitivity of the mycelia or conidia of A. alternata associated with GALSB to PNA in Jilin Province.

3.2. Effects of PNA on the Morphology of Mycelia and Conidia Germination

PNA more strongly inhibited conidia germination and germ tube elongation than mycelial growth. Additionally, at a concentration of 50 μg/mL, PNA completely inhibited both conidia germination and germ tube elongation. Microscopic examinations were conducted to observe alterations in A. alternata mycelia treated with PNA at final concentrations of 0, 10, and 100 μg/mL, as well as changes in the germ tubes of A. alternata conidia treated with 0 and 10 μg/mL. These observations revealed that both the mycelial tips and germ tubes exhibited swelling post-treatment. Increased concentrations led to a reduction in mycelial branching, which was accompanied by an increase in swollen tips (Figure 2A). Moreover, the number of nuclei within the germ tubes also increased (Figure 2B).

3.3. Biochemical Activity of PNA on A. alternata

3.3.1. Cell Membrane Permeability of A. alternata Isolates

Following the treatment of A. alternata isolates DH3-1, XJ2-5, and JY39 with PNA at final concentrations of 0, 0.1, 1, and 10 μg/mL, an increase in conductivity was observed for all three isolates based on the treatment concentration and duration. The conductivity levels in the treatment groups consistently exceeded those of the control group over time (Figure 3). This finding indicates that there was a positive correlation between the permeability of cell membranes in A. alternata mycelia and the concentrations of PNA.

3.3.2. Ergosterol Content of A. alternata Isolates

The experimental results demonstrated that PNA significantly influenced the ergosterol content in the plasma membrane of A. alternata. A reduction in the ergosterol content was observed following treatment with PNA, which was inversely correlated with the treatment concentration (Figure 4). The ergosterol content of the plasma membrane of untreated mycelial tissue was 247.34 ± 35.12 μg/g; after treatment with 10 μg/mL PNA, it decreased to 84.86 ± 7.36 μg/g.

3.3.3. DNA and Protein Content of A. alternata Isolates

Quantification of the protein and DNA content in A. alternata mycelium treated with various concentrations of PNA revealed that both protein and DNA levels decreased as the treatment concentration increased. The untreated mycelium exhibited a protein content of 134.64 ± 3.78 mg/g and a DNA content of 45.15 ± 5.22 mg/g. Following treatment with 10 μg/mL PNA, the protein and DNA content in the mycelium decreased to 84.86 ± 7.36 mg/g and 26.25 ± 3.96 mg/g, respectively, indicating that PNA disrupt the biosynthesis of both proteins and DNA within the mycelium; this resulted in reductions of protein and DNA synthesis of approximately 37% and 42%, respectively (Figure 5).

3.4. In Vitro Efficacy of PNA for Controlling GALSB Disease

The protective and curative efficacy of PNA against GALSB in vitro improved with the treatment concentration (Figure 6).
DH3-1 showed the highest sensitivity to PNA according to the conidia germination method, followed by XJ2-5 and JY39. The protective efficacy of PNA decreased as the sensitivity of the isolates decreased. At a concentration of 300 μg/mL, no significant difference was observed between the protective efficacy of PNA and that of kresoxim-methyl on ginseng leaves inoculated with isolates DH3-1, XJ2-5, and JY39, and an average protective efficacy exceeding 82% at 7 days after inoculation was observed in both treatments. The protective efficacy of PNA against GALSB disease significantly increased as the sensitivity of A. alternata to PNA increased (p < 0.05). However, the curative efficacy of PNA (an average curative efficacy of 94.77%) was significantly higher than that of kresoxim-methyl (69.31%) on pathogen-inoculated ginseng leaves (Figure 7, Table 2). The curative efficacy of PNA against GALSB disease increased as the sensitivity of A. alternata to PNA decreased, although this result was not significant (p > 0.05). This indicates that the protective and curative effects of PNA are stronger than those of kresoxim-methyl.

3.5. Field Efficacy of PNA Against GALSB Disease

Field trials across Changchun, Baishan, and Yanbian in Jilin Province demonstrated that the control efficacy of PNA 4% AS against GALSB disease increased with higher dosages. Specifically, at 180 g a.i./hm2, PNA consistently outperformed other fungicides over 7 days, 15 days, and 30 days post-last spray (PLS) (Figure 8, Table 3 and Tables S1–S3).
7 days PLS: High-dose PNA achieved a mean control efficacy of 85.62%. This was substantially higher than pyraclostrobin (78.65%), azoxystrobin (70.81%), and kresoxim-methyl (74.74%).
15 days PLS: PNA maintained a high mean control efficacy of 84.07%, again surpassing the comparison fungicides (pyraclostrobin: 72.17%; azoxystrobin: 64.58%; kresoxim-methyl: 73.82%).
30 days PLS: PNA still showed a mean control efficacy of 72.55%, while the efficacies of other fungicides dropped below 54% (pyraclostrobin: 52.67%; azoxystrobin: 53.18%; kresoxim-methyl: 53.56%).
There were significant differences between the control efficacy of PNA and that of control fungicides at 30 days PLS (p = 0.05), which indicates that PNA is more effective for preventing GALSB disease and has effects that last longer than 30 days. Overall, high-dose PNA provided both superior efficacy and a longer duration compared to pyraclostrobin, azoxystrobin, and kresoxim-methyl.

3.6. Cross-Resistance

A total of 81 A. alternata isolates were selected to determine the EC50 values of various fungicides. A Pearson correlation analysis of log-EC50 was conducted using SPSS 27 software, which revealed that the sensitivity of A. alternata to PNA was not significantly correlated with its sensitivity to eight other fungicides: pyraclostrobin, azoxystrobin, kresoxim-methyl, mancozeb, difenoconazole, propiconazole, tebuconazole, and iprodione (p > 0.05, |r| < 0.3) (Figure 9).

4. Discussion

Sensitivity analyses of the mycelium and conidia of 170 isolates of GALSB fungi Alternaria spp. collected from various regions in Jilin Province, China, to PNA were conducted. PNA more strongly inhibited conidia germination than mycelial growth within the same species. A. panax was significantly less sensitive to PNA compared with A. alternata and A. tenuissima. The results revealed that there was no significant difference in the sensitivity of A. alternata and A. tenuissima isolates to fungicides. A. alternata and A. tenuissima shared many similarities in terms of morphology and sensitivity to fungicides, and the synonymization of A. tenuissima under A. alternata was once put forward by Woudenberg et al. [36]. Furthermore, the sensitivity frequencies for both mycelial growth and conidia germination exhibited an unimodal distribution, indicating that there were no resistant subpopulations in the wild A. alternata population [37]. Consequently, the results of these sensitivity tests could provide a reliable baseline for monitoring changes in the sensitivity of A. alternata to PNA. In addition, A. panax and A. tenuissima were infrequently isolated as pathogens of GALSB disease, as they could only be isolated from some areas of the sample. Although sensitivity to PNA was determined for A. panax and A. tenuissima, the PNA sensitivity levels of A. panax and A. tenuissima to PNA were not fully representative of the PNA sensitivities of the populations of these two fungal species.
The physiological and biochemical effects of PNA on A. alternata were determined. A. alternata primarily utilizes conidia and mycelia to infect host plants, and the effects of PNA on the morphology of A. alternata mycelium and germ tubes were significant, indicating that they potentially impair fungal infectivity. The increase in mycelial conductivity reflects the exudation of electrolytes from within the cells, and the reduction in the ergosterol content, a sterol component of the fungal plasma membrane, further indicates that PNA influences the structure and function of the cell membrane. Furthermore, the intracellular DNA and protein content decreased as concentrations of PNA increased; this might stem from their effects on DNA synthesis as well as RNA and protein metabolism, which may disrupt ergosterol synthesis along with the expression of related genes and encoded proteins in A. alternata [38,39]. The decrease in mycelial DNA and protein levels further confirmed the mechanism of action of fungicides [40]. However, changes in mycelial and germ tube morphology suggested that some aspects of the mechanism of these fungicides have yet to be explored.
Assessing the cross-resistance of A. alternata isolates to fungicides is essential for developing strategies for the application of fungicides and delaying the development of resistance [41,42,43]. PNA effectively inhibited conidia germination and mycelial growth. Furthermore, the sensitivity of A. alternata to different fungicides varied considerably, and no significant correlation was observed between its sensitivity to PNA and the two main classes of fungicides. These findings suggest that cross-resistance between fungicides is not typically observed in isolates and that these fungicides show distinct mechanisms of action [44]. Correlation analysis of EC50 values for A. alternata in response to various fungicides demonstrated a positive cross-resistance relationship between fludioxonil and iprodione [14]. A comparative analysis of the sensitivity of A. alternata to difenoconazole, tebuconazole, and propiconazole revealed significant positive cross-resistance among these three fungicides [11]. Additionally, positive cross-resistance was observed between mefentrifluconazole and either difenoconazole or fenbuconazole in A. alternata, whereas no cross-resistance was detected with three non-DMI fungicides [37]. The above findings demonstrated cross-resistance between fungicides with the same mechanism of action. Additionally, a previous study of A. alternata exhibiting single or dual resistance to respiratory inhibitors of types II and III revealed positive cross-resistance between cyproconazole and mancozeb, as well as between pyraclostrobin and tebuconazole [15]. Although some pathogenic fungi were found to be resistant to multiple fungicides, the findings of our study demonstrate that no cross-resistance of PNA with pyraclostrobin, tebuconazole, or mancozeb was observed. PNA are biopesticides with a unique chemical structure and mode of action, and there are no reports of cross-resistance between PNA and other fungicides in pathogenic fungi [23]. Furthermore, previous studies have indicated that isolates from different host sources, even the same species, could exhibit different patterns of resistance due to differences in growth characteristics and host plant fungicide application [45]. Therefore, the fungicide resistance mechanism of the pathogenic fungi against PNA requires further study.
Fungicides exhibiting both protective and curative efficacy are effective in preventing disease onset and controlling disease progression (Ref). Kresoxim-methyl, a widely used fungicide for ginseng, demonstrated indoor protective and curative efficacy at concentrations comparable to those of PNA. Compared with 300 μg/mL kresoxim-methyl, PNA displayed superior protective and curative effects against A. alternata infection on ginseng leaves, which highlighted their potential efficacy in the field. The protective effect of PNA against the three isolates varying in sensitivity increased with the sensitivity level. Kresoxim-methyl is a methoxyacrylate fungicide that is generally thought to be highly effective [46]. PNA had an excellent curative effect; the curative effect of PNA was stronger than their protective effect. Furthermore, the climatic conditions and GALSB incidence of the three regions in the field trials differed, but PNA showed excellent control efficacy and sustainable performance in all regions.
QoIs fungicides are currently commonly used to control GALSB. However, due to the resistance development of Alternaria spp., their control efficacy has been continuously declining. The results of three consecutive investigations at three time points in this study all showed that the high-dose PNA was more effective in controlling GALSB than those of the three QoIs fungicides, and it still had a relatively high control efficacy 30 days post-last spray. This indicates that PNA can not only effectively control GALSB disease but also has a relatively long effect period, suggesting that it has the potential to address the development of resistance in Alternaria spp.
Natural products have diverse structures, such as cinnamic acid derivatives, and high antifungal activity, and biofungicides, such as Double Nickel and Stargus, are effective because crops can easily develop tolerance to them and they do not generate large amounts of residues, which improves the quality of agricultural products [47,48]. The extensive use of chemical fungicides increases the risk of pathogen resistance in addition to environmental pollution. Previous research on turfgrass fungal disease has shown that the pathogen can become resistant to thiophanate-methyl and propiconazole, but the use of QST713 in combination with or as a replacement of the original agents could greatly improve the control efficacy [49]. PNA are biological fungicides that substantially differ from traditional chemical fungicides and show great potential. PNA had high protective and curative efficacy compared with control fungicides (kresoxim-methyl); PNA were also shown to be effective in the field, and their effects were long-lasting. PNA extracted from microorganisms have been shown to have growth-promoting effects on crops, which was consistent with the results of our study [50]. Furthermore, bio-pesticides usually have low environmental toxicity and generate fewer environmental residues, which promotes ecological balance and biodiversity [51,52]. The water solubility of PNA facilitates their dissolution for spray application. No pathogenic fungi have been documented to show resistance to PNA. PNA will likely play a more prominent role in agricultural production as more research is conducted and will positively contribute to agricultural practices.

5. Conclusions

In this study, we systematically investigated the potential of pyrimidine nucleoside antibiotics (PNA) against A. alternata. First, we successfully established the baseline sensitivity of A. alternata to PNA. Significantly, its sensitivity to PNA followed a unimodal distribution. This finding offered a crucial method for monitoring the development resistance of Alternaria alternata to PNA in field populations, enabling early intervention in resistance management.
Experiments in vitro demonstrated that PNA exhibited remarkable antifungal and biochemical activity against A. alternata. PNA effectively inhibited the growth and metabolic activities of A. alternata, highlighting its potential as a potent antifungal agent. Importantly, no cross-resistance between PNA and fungicides used on ginseng were found. This unique characteristic allowed for more flexible and effective fungicide rotation strategies in agricultural settings.
Finally, PNA demonstrated high protective and curative efficacy. Field trials further validated its effectiveness, showing longer duration in controlling GALSB diseases. These findings were of great significance for the green management of GALSB disease. They also provided valuable insights into managing the fungicide resistance of Alternaria spp. responsible for GALSB disease, offering new approaches to sustainable disease control.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agriculture15080875/s1, Table S1: Comparison of the control efficacy of PNA and three other fungicides against GALSB disease in the field (Jingyue District, Changchun City, 2024). Table S2: Comparison of the control efficacy of PNA and three other fungicides against GALSB disease in the field (Songjianghe Town, Fusong County, Baishan City, 2024). Table S3: Comparison of the control efficacy of PNA and three other fungicides against GALSB disease in the field (Yanminghu Town, Dunhua City, 2024). Figure S1 Precipitation and temperature during the phenological period in the ginseng production process in different regions of Jilin Province. Jingyue District, Changchun City (125.4° N, 43.8° E) (A), Songjianghe Town, Fusong County (127.5° N, 42.2° E) (B), Yanminghu Town, Dunhua City (129.4° N, 42.8° E) (C). The fungicides treatment was carried out from late June to late July. The field investigation was conducted from early August to early September.

Author Contributions

Conceptualization, J.G.; Methodology, S.S., B.L. and J.G.; Software, S.S.; Validation, M.H.; Formal analysis, S.S., M.H. and B.L.; Investigation, S.S., X.C., M.J., C.C. and B.L.; Resources, J.G.; Data curation, S.S., M.H., M.J., C.C. and B.L.; Writing-original draft preparation, S.S.; Writing-review and editing, J.G.; Visualization, S.S.; Supervision, J.G.; Project administration, J.G.; Funding acquisition, J.G. All authors have read and agreed to the published version of the manuscript.

Funding

This study was supported by the Program for Science and Technology of Jilin Province (20220202110NC).

Data Availability Statement

The original contributions presented in this study are included in the article/Supplementary Materials. Further inquiries can be directed to the corresponding authors.

Acknowledgments

We are grateful to Jilin Ginseng King Plant Protection Co. Ltd., who generously allowed us to complete our experiments in their ginseng base.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
PNAPyrimidine nucleoside antibiotics
GALSB Ginseng Alternaria leaf and stem blight
TATechnical grade
ASAqueous solution
SCSuspension concentrates
WPWettable powder
DHEDehydroergosterol
ODOptical density
CEControl efficacy

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Agriculture 15 00875 g001
Figure 1. The distribution of EC50 values of 115 isolates of Alternaria alternata in response to PNA based on mycelial growth (A) and conidia germination (B). The isolates were collected from various locations in Jilin Province from 2018 to 2022. EC50 represents the concentrations at which mycelial growth or conidia germination was inhibited by 50%.
Figure 1. The distribution of EC50 values of 115 isolates of Alternaria alternata in response to PNA based on mycelial growth (A) and conidia germination (B). The isolates were collected from various locations in Jilin Province from 2018 to 2022. EC50 represents the concentrations at which mycelial growth or conidia germination was inhibited by 50%.
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Figure 2. Changes in mycelium tips (A) of the isolates Alternaria alternata DH3-1, XJ2-5, and JY39 caused by 0, 10, and 100 μg/mL concentrations of PNA, respectively, and conidia germination, germ tube morphology, and the number of nuclei in germ tubes (B) of isolates DH3-1, XJ2-5, and JY39 at a concentration of 10 μg/mL. Bright field: a common microscopy mode. DAPI is the abbreviation of 4’,6-diamidino-2-phenylindole. It binds specifically to double-stranded DNA and emits blue fluorescence under excitation light; it is used to reveal the location and morphology of cell nuclei. The observations were documented through microscopy. Bar = 50 μm.
Figure 2. Changes in mycelium tips (A) of the isolates Alternaria alternata DH3-1, XJ2-5, and JY39 caused by 0, 10, and 100 μg/mL concentrations of PNA, respectively, and conidia germination, germ tube morphology, and the number of nuclei in germ tubes (B) of isolates DH3-1, XJ2-5, and JY39 at a concentration of 10 μg/mL. Bright field: a common microscopy mode. DAPI is the abbreviation of 4’,6-diamidino-2-phenylindole. It binds specifically to double-stranded DNA and emits blue fluorescence under excitation light; it is used to reveal the location and morphology of cell nuclei. The observations were documented through microscopy. Bar = 50 μm.
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Figure 3. Efficacy of different concentrations (0, 0.1, 1, and 10 μg/mL) of PNA on the mycelial conductivity of DH3-1 (A), XJ2-5 (B), and JY39 (C) isolates. Results were expressed as mean ± SD (standard deviation, n = 3).
Figure 3. Efficacy of different concentrations (0, 0.1, 1, and 10 μg/mL) of PNA on the mycelial conductivity of DH3-1 (A), XJ2-5 (B), and JY39 (C) isolates. Results were expressed as mean ± SD (standard deviation, n = 3).
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Figure 4. Effect of PNA at different concentrations (0, 0.1, 1.0, and 10.0 μg/mL) on the ergosterol levels of DH3-1, XJ2-5, and JY39 isolates after 3 days of cultivation. The results were expressed as mean ± SD (n = 6). According to Fisher’s least significant difference test at a significance level of p = 0.05, values with the same letters did not significantly differ.
Figure 4. Effect of PNA at different concentrations (0, 0.1, 1.0, and 10.0 μg/mL) on the ergosterol levels of DH3-1, XJ2-5, and JY39 isolates after 3 days of cultivation. The results were expressed as mean ± SD (n = 6). According to Fisher’s least significant difference test at a significance level of p = 0.05, values with the same letters did not significantly differ.
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Figure 5. Effects of PNA at different concentrations (0, 0.1, 1.0, and 10.0 μg/mL) on the DNA content (A) and protein content (B) of DH3-1, XJ2-5, and JY39 isolates after 3 d of culture. The findings were presented as mean ± SD (standard deviation, n = 6). Statistical analysis using Fisher’s least significant difference test at a significance level of p = 0.05 revealed that values with the same letters did not significantly differ.
Figure 5. Effects of PNA at different concentrations (0, 0.1, 1.0, and 10.0 μg/mL) on the DNA content (A) and protein content (B) of DH3-1, XJ2-5, and JY39 isolates after 3 d of culture. The findings were presented as mean ± SD (standard deviation, n = 6). Statistical analysis using Fisher’s least significant difference test at a significance level of p = 0.05 revealed that values with the same letters did not significantly differ.
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Agriculture 15 00875 g006
Figure 6. Protective and curative efficacy of fungicides on ginseng leaves. The efficacy of PNA and kresoxim-methyl (Kre) against Alternaria alternata isolates DH3-1, XJ2-5, and JY39.
Figure 6. Protective and curative efficacy of fungicides on ginseng leaves. The efficacy of PNA and kresoxim-methyl (Kre) against Alternaria alternata isolates DH3-1, XJ2-5, and JY39.
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Agriculture 15 00875 g007
Figure 7. The protective efficacy (A) and curative efficacy (B) of different concentrations of PNA and Kre on ginseng leaves. The sensitivity level of the isolates to PNA by the conidia germination method was highest for DH3-1, followed by XJ2-5 and JY39. The data were expressed as mean ± SD (standard deviation, n = 6). The mean values followed by the same letter in the columns were not significantly different according to Fisher’s LSD test at p = 0.05.
Figure 7. The protective efficacy (A) and curative efficacy (B) of different concentrations of PNA and Kre on ginseng leaves. The sensitivity level of the isolates to PNA by the conidia germination method was highest for DH3-1, followed by XJ2-5 and JY39. The data were expressed as mean ± SD (standard deviation, n = 6). The mean values followed by the same letter in the columns were not significantly different according to Fisher’s LSD test at p = 0.05.
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Agriculture 15 00875 g008
Figure 8. The efficacy of pyrimidine nucleoside antibiotics 4% AS (PNA), pyraclostrobin 25% SC (Pyr), azoxystrobin 25% SC (Azo), and kresoxim-methyl 30% WP (Kre) against GALSB disease in the three regions at 7 d, 15 d, and 30 d post-last spray. The average control effects in Jingyue District, Changchun City (A), Songjianghe Town, Fusong County, Baishan City (B), and Yanminghu Town, Dunhua City (C) and average control efficacy for the three sites (D). PNA-104, PNA-144, and PNA-180 indicate the application of PNA at doses of 104, 144, and 180 g a.i./hm2, respectively. Pyr-112 indicates the application of pyraclostrobin at a dose of 112 g a.i./hm2. Azo-94 indicates the application of azoxystrobin at a dose of 94 g a.i./hm2. Kre-220 indicates the application of kresoxim-methyl at a dose of 220 g a.i./hm2. The data were expressed as mean ± SD (standard deviation) for three regions (n = 9). The mean values followed by the same letter in the columns are not significantly different according to Fisher’s LSD test at p = 0.05.
Figure 8. The efficacy of pyrimidine nucleoside antibiotics 4% AS (PNA), pyraclostrobin 25% SC (Pyr), azoxystrobin 25% SC (Azo), and kresoxim-methyl 30% WP (Kre) against GALSB disease in the three regions at 7 d, 15 d, and 30 d post-last spray. The average control effects in Jingyue District, Changchun City (A), Songjianghe Town, Fusong County, Baishan City (B), and Yanminghu Town, Dunhua City (C) and average control efficacy for the three sites (D). PNA-104, PNA-144, and PNA-180 indicate the application of PNA at doses of 104, 144, and 180 g a.i./hm2, respectively. Pyr-112 indicates the application of pyraclostrobin at a dose of 112 g a.i./hm2. Azo-94 indicates the application of azoxystrobin at a dose of 94 g a.i./hm2. Kre-220 indicates the application of kresoxim-methyl at a dose of 220 g a.i./hm2. The data were expressed as mean ± SD (standard deviation) for three regions (n = 9). The mean values followed by the same letter in the columns are not significantly different according to Fisher’s LSD test at p = 0.05.
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Agriculture 15 00875 g009
Figure 9. The correlation (r) between the sensitivity of 81 isolates of Alternaria alternata to PNA and eight other fungicides, including pyraclostrobin (A), azoxystrobin (B), kresoxim-methyl (C), mancozeb (D), difenoconazole (E), propiconazol (F), tebuconazole (G), and iprodione (H), was investigated. The sensitivity assessment of these isolates was conducted using the mycelial growth rate method on PDA media.
Figure 9. The correlation (r) between the sensitivity of 81 isolates of Alternaria alternata to PNA and eight other fungicides, including pyraclostrobin (A), azoxystrobin (B), kresoxim-methyl (C), mancozeb (D), difenoconazole (E), propiconazol (F), tebuconazole (G), and iprodione (H), was investigated. The sensitivity assessment of these isolates was conducted using the mycelial growth rate method on PDA media.
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Table 1. The sensitivity of the mycelia of three Alternaria spp. and conidia of two Alternaria spp. to PNA.
Table 1. The sensitivity of the mycelia of three Alternaria spp. and conidia of two Alternaria spp. to PNA.
SpeciesTest MethodsIsolates NumberEC50 (μg/mL) z
RangeMean ± SD y
A. alternataMycelial growth rate1151.284–27.28410.192 ± 4.961 b
A. tenuissima296.355–20.22711.494 ± 3.379 b
A. panax2611.398–57.57331.448 ± 12.846 a
A. alternataConidia germination890.644–1.0770.828 ± 0.101 c
A. tenuissima290.419–1.2510.666 ± 0.199 c
z EC50, the concentrations at which the mycelial growth or conidia germination is inhibited by 50%. Mean values followed by the same letter in the columns are not significantly different according to Fisher’s LSD test at p = 0.05. y SD = standard deviation.
Table 2. Protective and curative efficacy of PNA and kresoxim-methyl against Alternaria alternata isolates DH3-1, XJ2-5, and JY39 on ginseng leaves.
Table 2. Protective and curative efficacy of PNA and kresoxim-methyl against Alternaria alternata isolates DH3-1, XJ2-5, and JY39 on ginseng leaves.
FungicidesTreatment (μg/mL)Protective Efficacy (%)Curative Efficacy (%)
DH3-1XJ2-5JY39Average
Efficacy		DH3-1XJ2-5JY39Average
Efficacy
Pyrimidine nucleoside antibiotics10068.63 ± 2.43 c75.13 ± 2.61 b43.45 ± 6.31 c62.40 ± 16.73 b69.38 ± 4.65 b82.26 ± 2.50 ab98.61 ± 2.41 a83.42 ± 14.65 ab
20081.18 ± 1.46 ab78.99 ± 4.11 b62.67 ± 4.69 b74.28 ± 10.11 ab82.07 ± 2.11 a91.49 ± 0.18 a97.86 ± 2.17 a90.47 ± 7.94 a
30091.06 ± 1.85 a91.73 ± 2.09 a80.99 ± 1.72 a87.93 ± 6.02 a90.04 ± 1.17 a94.32 ± 1.24 a100.00 ± 0.00 a94.79 ± 5.00 a
Kresoxim-methyl30076.44 ± 2.98 bc92.66 ± 4.28 a83.05 ± 2.80 a84.05 ± 8.16 ab60.30 ± 1.37 c78.74 ± 1.25 b68.88 ± 2.01 b69.31 ± 9.23 b
Note: The data were expressed as mean ± SD (standard deviation) (n = 6). The mean values followed by the same letter in the columns were not significantly different according to Fisher’s LSD test at p = 0.05. By the conidia germination method, DH3-1 isolates showed the highest sensitivity to PNA, followed by XJ2-5 and JY39 isolates.
Table 3. The mean control efficacy of PNA 4% AS, pyraclostrobin 25% SC, azoxystrobin 25% SC, and kresoxim-methyl 30% WP against GALSB disease in the three regions at 7 days, 15 days, and 30 days post-last spray.
Table 3. The mean control efficacy of PNA 4% AS, pyraclostrobin 25% SC, azoxystrobin 25% SC, and kresoxim-methyl 30% WP against GALSB disease in the three regions at 7 days, 15 days, and 30 days post-last spray.
FungicideDosage
(g a.i./hm2)		Control Efficacy (%)
7 Days Post Last Spray		Control Efficacy (%)
15 Days Post Last Spray		Control Efficacy (%)
30 Days Post Last Spray
Range zMean ± SD yRange zMean ± SD yRange zMean ± SD y
Pyrimidine nucleoside antibiotics 4% AS10879.01–90.3683.77 ± 6.08 a74.51–79.2577.72 ± 2.78 ab51.92–79.0164.20 ± 12.93 ab
14483.77–86.6184.78 ± 1.59 a78.82–83.0181.15 ± 2.13 ab56.73–68.5164.26 ± 6.54 ab
18081.68–89.2985.62 ± 4.13 a83.43–85.1184.07 ± 0.91 a67.15–76.1072.55 ± 4.75 a
Pyraclostrobin 25% SC11274.55–81.1578.65 ± 3.58 ab62.79–83.7972.17 ± 10.68 bc52.48–52.8152.67 ± 0.17 c
Azoxystrobin 25% SC9464.51–77.7870.81 ± 6.66 b56.62–73.2064.58 ± 8.50 c45.61–62.0053.18 ± 8.27 c
Kresoxim-methyl 30% WP22069.63–82.7274.74 ± 7.00 ab66.79–83.6673.82 ± 8.78 abc47.12–60.0653.56 ± 6.47 bc
Note: The data were expressed as mean ± SD (standard deviation) for three sites (n = 9); The mean values followed by the same letter in the columns are not significantly different according to Fisher’s LSD test at p = 0.05. z Range indicates the control efficacy (CE) of the fungicides from low to high CE at three sites. y Mean ± SD means the mean CE of different fungicides at different dosages in three sites.
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MDPI and ACS Style

Shao, S.; Hu, M.; Chen, X.; Jiang, M.; Chen, C.; Lu, B.; Gao, J. Evaluation of the Potential of Pyrimidine Nucleoside Antibiotics Against Alternaria spp. Resistant to QoIs Fungicides: Insights for the Management of Ginseng Alternaria Leaf and Stem Blight Disease. Agriculture 2025, 15, 875. https://doi.org/10.3390/agriculture15080875

AMA Style

Shao S, Hu M, Chen X, Jiang M, Chen C, Lu B, Gao J. Evaluation of the Potential of Pyrimidine Nucleoside Antibiotics Against Alternaria spp. Resistant to QoIs Fungicides: Insights for the Management of Ginseng Alternaria Leaf and Stem Blight Disease. Agriculture. 2025; 15(8):875. https://doi.org/10.3390/agriculture15080875

Chicago/Turabian Style

Shao, Shuai, Mingyuan Hu, Xiaolin Chen, Ming’en Jiang, Changqing Chen, Baohui Lu, and Jie Gao. 2025. "Evaluation of the Potential of Pyrimidine Nucleoside Antibiotics Against Alternaria spp. Resistant to QoIs Fungicides: Insights for the Management of Ginseng Alternaria Leaf and Stem Blight Disease" Agriculture 15, no. 8: 875. https://doi.org/10.3390/agriculture15080875

APA Style

Shao, S., Hu, M., Chen, X., Jiang, M., Chen, C., Lu, B., & Gao, J. (2025). Evaluation of the Potential of Pyrimidine Nucleoside Antibiotics Against Alternaria spp. Resistant to QoIs Fungicides: Insights for the Management of Ginseng Alternaria Leaf and Stem Blight Disease. Agriculture, 15(8), 875. https://doi.org/10.3390/agriculture15080875

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