In 2023, a total of 20 leaves and stems of blight on P. auriculata with typical symptoms were collected in Wanzhou District (108°26.4′N,30°45′E), Chongqing City, China. Wash with running water, dry naturally. The disease samples of 5 mm × 5 mm at the junction of disease and healthy were cut with a sterile scalpel, surface sterilized in 75% ethanol for 1 min and then in 3% NaClO for 4 min, rinsed three times in sterilized distilled water. Air-dried on sterilized filter paper, the samples were then placed on antibiotic (streptomycin sulphate, 50 μg/mL) amended potato dextrose agar (PDA) plates and incubated for 5 days at 25°C (Lyu et al., 2024). According to the characteristics of the colony and morphological characteristics, several representative strains were purified and cultured at 25°C on PDA. Inoculate the isolates onto a PDA tube ramp and store at 4°C (Wang et al., 2022).

In vitro inoculation was carried out using the mycelium plugs inoculation method and the conidia suspension inoculation method to inoculate detached leaves and stems, respectively (Tang et al., 2022; Ao et al., 2024). In vivo inoculation was carried out using the conidia suspension inoculation method. Inoculate healthy P. auriculata potted plants (Tang et al., 2022).

Prior to in vitro inoculation, rinse the healthy leaves and stems with 75% alcohol for 1 min, wash them with 0.4% sodium hypochlorite solution for 30 s, and then rinse them three times with sterile water (each time for 1 min). Dry naturally. On a healthy leaf, prick the leaf symmetrically with a sterilized needle (the main leaf vein is symmetrical on both sides). The mycelium plugs were dispensed from the representative strain L9 with a sterile punch (5 mm in diameter) and placed in P. auriculata healthy leaves flanking the main veins. Another healthy leaf was taken and inoculated with sterile potato dextrose agar medium as a control. After culturing the representative strain L9 on PDA for 7 days, a 1 × 10⁶ mL conidia suspension was prepared. Spray conidia suspension (1 × 10⁶ conidia/mL) onto P. auriculata healthy stems while using the same procedure with sterile distilled water as a control. All inoculated leaves and stems were placed in 25°C Petri dishes covered with moist sterile filter paper (80% relative humidity).

The potted plants used for in vivo inoculation were healthy P. auriculata that were 3 months old. Before in vivo inoculation, disinfect the surface of the P. auriculata plants with 75% alcohol, rinse them with sterile water, and allow them to dry naturally. Inoculate P. auriculata plants with a conidia suspension by applying an average of 5 mL of conidia suspension (1 × 10⁶ conidia/mL) to each plant until the plant surfaces are completely moist. Healthy plants sprayed with sterile distilled water served as negative controls.

The inoculated materials were placed in a temperature incubator and cultivated at 25°C, with a photoperiod of 12 h and a relative humidity (RH) of 80%. Both the in vitro inoculation experiment and the in vivo inoculation experiment comprised five repetitions. To observe whether the disease symptoms of plants in the temperature incubator were consistent with the field symptoms regularly. After typical symptoms appeared in stems and leaves, the pathogenic fungus was reisolated from the infected leaves and stems. If the isolated strain was consistent with the inoculated strain, it was identified as the pathogen.

Two representative isolates (L9 and L11) obtained were cultured on potato dextrose agar (PDA) at 25°C in the dark for 5 days to observe the morphological characteristics of colonies by optical microscope. Preliminary identification of species was based on morphological characteristics of the pathogenic strains on PDA, including colony color, texture, pigment production, and the morphology of conidiophores and chlamydospores. For each representative fungal isolate, 50 conidia and chlamydospores were randomly selected for measurement.

Representative fungal strains were cultured, and mycelium was collected. Plant genomic DNA extraction kit (Cwbio, Jiangsu, China) was used to extract DNA representing strains L9 and L11. Primers ITS1/ITS4 (White et al., 1990), 5F2/7CR (Reeb et al., 2004), EF1/EF2 (O'Donnell et al., 1998), CL1/CL2A (O’Donnell et al., 2000) were used to amplify four loci of L9 and L11, including the internal transcribed spacers (ITS), RNA polymerase II second largest subunit (RPB2), translation elongation factor 1 alpha (EF–1α), and calmodulin (CAMD).

The PCR amplification reaction system was 20 μL, including Taq Master Mix (K1071, Sangon Biotech (Shanghai) Co. Ltd) 10 μL, 10 μmolL⁻¹ upstream and downstream primers 0.3 μL each, DNA template 2 μL, and ddH₂O added to 20 μL. The amplification conditions used for the ITS1/ITS4, 5F2/7CR, EF1/EF2, CL1/CL2A, and primer pairs are illustrated in Table 1. After amplification, part of the reactants was detected by 1% agarose gel electrophoresis. The remaining reactants were sent to Sangon Biotech (Shanghai) Co., Ltd. (Chengdu) for sequencing. The obtained sequences were compared and analyzed using GenBank¹ to download high-consistency sequences (Table 2). These sequences were combined in PhyloSuite to form the ITS-RPB2-EF–1α-CAMD sequence, and a phylogenetic tree was constructed using the Maximum Likelihood method in MEGA11.0 software, with the repetition value set to 1,000 times. The obtained DNA sequences were uploaded to the National Center for Biotechnology Information to acquire accession numbers.

Healthy detached leaves were inoculated by mycelium plug inoculation to determine whether the representative strain L9 was also pathogenic to other horticultural plants (Ao et al., 2024). The selected two horticultural plants are Prunus serrulata Lindl. (which belongs to the family Rosaceae and the genus Prunus L.) and Heptapleurum arboricola Hayata (which belongs to the family Araliaceae and the genus Schefflera).

The mycelium plugs inoculation method was carried out as described in section 2.2. The experiment comprised five repetitions. The inoculated leaves were placed in a temperature-controlled incubator and cultivated at 25°C, with a 12-h photoperiod and 80% relative humidity (RH). When re-isolating the pathogen, it matched the pathogen from leaves with lesion symptoms. If the re-isolate was consistent with isolate L9, it confirmed that L9 was pathogenic to P. serrulata and H. arboricola.

The inhibitory effect of seven types of biological pesticides on pathogenic fungi was measured using the mycelium growth rate method. Each biological pesticide was made into pharmaceutical solutions with different concentration gradients using sterile water (Table 3). The different pharmaceutical solutions concentrations were added to the sterilized PDA medium according to a certain volume ratio. The mixture was thoroughly mixed, and medicine plates were prepared with different test concentrations. Each treatment was repeated five times for each concentration. A sterile punch (5 mm in diameter) was used to extract the mycelium plugs of a representative strain L9 at the edge of the colony. The mycelium plugs were inoculated in the center of the medicine-prepared plates. PDA medium with an equal amount of sterile water served as a control. The plates were incubated at 25°C in darkness within a constant temperature incubator for 5 days. The colony diameter was measured using the cross-shaped intersecting method.

The percent inhibition of mycelial growth (PIMG) was calculated using the following formula (where F is the diameter of the fungal plug, C is the radial growth diameter of the fungus in the control, and T is the radial growth diameter of the fungus in the treatment group) (Ao et al., 2024).

Microsoft Excel 2019 was used to calculate a toxicity regression equation (where x is the logarithm of the fungicide concentration (μg/mL) and y is the inhibition rate) and correlation coefficient for each fungicide. SPSS 26.0 was used to calculate the half maximal effective concentration (EC₅₀) value of each fungicide. We used ANOVA with post-hoc Duncan’s test in SPSS 26.0 to compare inhibition rates across treatments at different concentrations among the same fungicides. And mark the statistical differences with lowercase letters.

Based on the indoor toxicity test results, the fungicide 0.4% osthole SL, which showed an obvious antibacterial effect, was selected for the pot control effect test. The treatment was carried out with three concentration gradients of 0.4% osthole SL EC₅₀, EC₇₀, and EC₉₀ (1.089 μg/mL, 2.071 μg/mL, and 5.244 μg/mL, respectively). The treatment without any pharmaceutical application, but inoculated with the isolate L9, served as the negative control. Each treatment was set up with five replicates.

After culturing the representative strain L9 on PDA for 7 days, 1 × 10⁶ mL of conidia suspension was prepared in advance. Sterilize the soil in the potted plants in advance. Select healthy P. auriculata plant seedlings of the same size and age of 3 months and transfer them into seedling pots. After they stand upright and grow normally, disinfect the surface of P. auriculata plants with 75% ethanol and rinse with sterile water. Inoculate an average of 5 mL of the conidia suspension per plant until the surface of the plants is completely moist. The potted plants were inoculated and sprayed with 0.4% osthole SL early in the disease for prevention and control. Fungicides were applied once every 7 days for a total of 4 times. Spray each plant with a micro sprayer to ensure even application of the medicine. The potted plants were placed in a greenhouse and cultivated at 25°C, with a photoperiod of 12 h and a relative humidity (RH) of 80%. The disease severity was investigated 1 day before the application of the pesticide and on the 7th day after the application ended, and the disease severity (DS) and control efficacy (CE) were calculated, respectively (Wang et al., 2024).

The DS was scored for each plant according to the percentage of leaves and stems with yellowing or necrosis where 0 = No evidence of lesions, 1 = 33% of leaf and stem area infected, 2 = 34–66% of leaf and stem area infected, 3 = 67–100% of leaf and stem area infected and 4 = dead plant (Díaz-Gutiérrez et al., 2021). The disease severity index (DSI) was calculated for each treatment using the formula of Promwee et al. (2017).

The control efficacy (CE) was calculated for each treatment using the formula of Wang et al. (2024) (where D is the disease severity index in the blank control area, and d is the disease severity index of the treatment area).

Data were collated using Microsoft Excel 2019 software, and the Duncan’s test in SPSS 26.0 statistical software was used for the significance analysis of differences.

According to the investigation, the incidence of wilt disease of P. auriculata was 50–60% in the field. The disease started in April; the worst onset was in September to October. The initial symptoms of the disease are the yellowing and wilting of the top stems and young leaves. The leaves lost water, curled, and the junction between disease and health appeared dark brown (Figure 1a). The symptoms gradually spread downwards. The leaves became dry, curly, drooping, and dying. The later symptoms were manifested as the withering and death of the entire plant (Figure 1b).

Fungal colonies have white cotton flocculent aerial mycelium after 7 days of incubation under dark conditions on PDA. The back of the colony is pale pink. The edges of the colony are uneven (Figure 1c). The macroconidia are falcate, prominently cell papillate or hooked, and have 4–6 septa, measuring 40–68 × 4–7 μm (Figures 1p,q). Microconidia are oval and have either one or no septa. Microconidia 8–10 × 3–5 μm (Figure 1r). A lot of chlamydia spores are observed on PDA. Chlamydospores were produced in chains or pairs, globose, and thick-walled (Figures 1s,t). The conidiophores in the sporodochia vary in length and are branched verticillately. The hyphae are thin-walled and hyaline (Figure 1u). These characteristics suggest the pathogenic fungus was Fusarium spp.

Four to five days after inoculation with the representative strain L9, the detached stems showed chlorosis and yellowing (Figure 1f). Brown spots were observed on the detached leaves (Figure 1i). Wilting and yellowing occurred in leaves and at the tips of the stems of P. auriculata potted plants (Figure 1m). After 8–10 days, the outer layer of the stems turned light brown (Figure 1g). The browning area of the detached leaves expanded, and some gradually died (Figure 1j). The leaves of potted plants have become dry and curly (Figure 1n). After 12–15 days, the outer layer of the detached stems turned dark brown (Figure 1h). The inoculated site of the detached leaves completely turned brown and withered (Figure 1k). The symptoms of withering in potted plants gradually spread downward, and the drooping leaves completely withered and died. White mycelial layers appeared on the surface of diseased plant tissues (Figure 1o). The boundary between diseased and healthy plant tissues appeared dark brown. The symptoms of the indoor pathogenicity test were consistent with those of the field diseases. In contrast, all the control groups remained healthy on the 15th day (Figures 1d,e,l).

The inoculated and diseased P. auriculata leaves and stems were subjected to pathogen re-isolation and identification. The re-isolated pathogens were consistent with the inoculated pathogens. Thus, we identified L9 as the pathogen causing blight in P. auriculata.

The gene regions of ITS, RPB2, EF–1α and CAMD were PCR amplified and sequenced. The obtained sequences of ITS, RPB2, EF–1α and CAMD were deposited in GenBank. The combined dataset of ITS, RPB2, EF–1α and CAMD genes was used for phylogenetic analysis by MEGA11.0. Alternaria alternata strain REIS 68 was used as an outgroup. The ML tree demonstrated that L9 and L11 isolates were placed in the F. ipomoeae (GZAX 307) group, supported by a 100% bootstrap value (Figure 2). Representative isolates L9 and L11 were confirmed as F. ipomoeae based on morphological characteristics and molecular identification.

After in vitro inoculation of the isolated plant leaves of P. serrulata and H. arboricola, the inoculated parts of the plant leaves showed symptoms of disease to varying degrees. Strain L9 had a relatively strong pathogenicity to P. serrulata. Four days after inoculation, 3–4 mm brown small lesions appeared at the pinhole. After 12 days of inoculation, the lesion size reached 17–25 mm (Figures 3a–d). Conversely, the strain L9 had weaker pathogenicity against H. arboricola. Twelve days after inoculation, the size of the lesion was 9–11 mm (Figures 3e–h). Twelve days later, none of the control group showed any symptoms of the disease. Re-isolate the pathogen from the diseased leaves, and the resulting pathogen was consistent with that of L9. The isolate L9 was pathogenic to both P. serrulata and H. arboricola. Fusarium ipomoeae isolated from P. auriculata blight can infect other horticultural plants.

The biological pesticides treated with different fungicide types and concentrations could inhibit the mycelial growth of F. ipomoeae to different degrees (Figure 4). For each biopesticide used in the fungicide assays, the original fungicide concentration, formulation type, toxicity regression equation, correlation coefficient, EC₅₀ value, and 95% confidence intervals are shown in Table 4.

Among the seven biological pesticides, the inhibitory effect of 0.4% osthole SL on F. ipomoeae is the best, with an EC₅₀ value of 1.089 μg/mL. 1% phenazino-1-carboxylic acid SC and 80% ethylicin EC show good inhibitory effect on mycelia growth, with EC₅₀ values of 9.538 μg/mL and 11.802 μg/mL, respectively. Followed by 5% avermectin EC and 3% zhongshengmycin AS, with EC₅₀ values of 68.449 μg/mL and 92.720 μg/mL, respectively. The inhibitory effect of 4% berberine AS and 8% ningnanmycin AS is poor, with EC₅₀ values of 104.104 μg/mL and 124.261 μg/mL, respectively.

The control effects of different concentrations of 0.4% osthole SL on the blight of P. auriculata potted plants caused by F. ipomoeae were evaluated (Table 5; Figure 5). The results showed that 0.4% osthole SL has a good control efficacy on P. auriculata blight caused by F. ipomoeae. However, three different concentrations of osthole treated had significantly different control efficacy on P. auriculata blight. Before the fourth application, 0.4% osthole SL had the highest control efficacy at a concentration of 5.244 μg/mL (EC₉₀), reaching 91.10%, significantly higher than 2.071 μg/mL (EC₇₀) and 1.089 μg/mL (EC₅₀). After the 4th application for 7 days, the control efficacy remained good at a concentration of 5.244 μg/mL of 0.4% osthole SL, reaching 88.00%. In contrast, the control effects of 0.4% osthole SL concentrations of 2.071 μg/mL (EC₇₀) and 1.089 μg/mL (EC₅₀) were weaker, with 66.01 and 43.62%, respectively. 0.4% osthole SL can be used as a biological pesticide for the field control of P. auriculata blight caused by F. ipomoeae, and the recommended application concentration is 5.244 μg/mL.