In January 2022, a serious leaf blight disease was observed on the leaves of A. japonica in the outdoor gardens of Guizhou University, Huaxi District, Guiyang City, Guizhou Province, China (26°44′58”N, 106°65′91″E). On the leaf surface of A. japonica, a burn-like black blight with wilting signs was frequently observed, leading to rapid and extensive foliage death. The disease affected an area of approximately 0.52 hectares, with disease incidence ranging from 60 to 70%, resulting in a mortality rate of 20 to 30% in plants. For fungal isolation, every two symptomatic leaves were collected from ten different plants using disposable gloves. The diseased leaves were first soaked in 75% v/v ethanol for 1 min, then in a 4% w/v sodium hypochlorite (NaClO) solution for 3 min, and finally washed thrice with sterile distilled water (dH₂O). Subsequently, the leaves were placed on potato dextrose agar (PDA) medium and incubated at 25 ± 2°C for 7 days. The fungal colonies were purified by transferring single spores, pure cultures were obtained. Representative isolates were selected for further identification.

The morphological characteristics of five representative isolates were examined under the microscope. Fungal conidia were observed and photographed using a Zeiss microscopic system (Carl Zeiss-Axioscope 5, Germany). The length and width of 60 randomly selected conidia were measured and recorded. For molecular identification and phylogeny analysis, the genomic DNA of the representative isolates was extracted from mycelia of seven-day-old cultures according to the manufacturer’s instructions (Biomiga Fungal DNA Extraction Kit, CA, United States). Subsequently, polymerase chain reaction (PCR) amplification of internal transcribed spacer (rDNA-ITS), a partial sequence of the chitin synthase (CHS) gene and actin (ACT) gene were performed using the following primers: ITS1 (5’-TCCGTAGGTGAACCTGCGG-3′) and ITS2 (5′-GCTGCGTTCTTCATCGATGC-3′; White et al., 1990), CHS-79F (5′-TGGGGCAAGGATGCTTGGAAGAAG-3′) and CHS-345R (5′-TGGAAGAACCATCTGTGAGAGTTG-3′; Carbone and Kohn, 1999), and ACT-512F (5′-ATGTGCAAGGCCGGTTTCGC-3′) and ACT-783R (5′-TACGAGTCCTTCTGGCCCAT-3′; Carbone and Kohn, 1999), respectively. The PCR amplifications were carried out in a Bio-Rad S1000 Thermal Cycler in a 25 μl reaction mixture containing 1 μl of DNA sample, 1 μl of each primer, 12.5 μl 2 × SanTaq PCR Mix (Sangon Biotech, Shanghai, China), and 9.5 μl double distilled water (ddH₂O). The PCR conditions were as follows: initial denaturation at 94°C for 3 min, followed by 35 cycles of denaturation at 95°C for 30 s, annealing for 30 s at the corresponding temperatures (53°C for ITS, 58°C for CHS gene, and 61°C for ACT gene), extension at 72°C for 45 s, then a final extension for 10 min.

The DNA sequences obtained in this study were queried against other DNA sequences in the GenBank database in the National Center for Biotechnology Information (NCBI).¹ Sequences of the sequenced DNA regions of strains have been deposited with GenBank. The phylogenetic tree was constructed by the neighbor-joining algorithms based on concatenated ITS region, CHS gene, and ACT gene sequences using MEGA 6.05 software (Tamura et al., 2013).

To confirm Koch’s postulates, the pathogenicity of the representative strain C. aenigma BY827 was tested on healthy leaves of A. japonica. Firstly, inoculation was performed in a laminar flow workstation (Haier, Qingdao, China) using sterilized needles to perform pathogen inoculation on healthy A. japonica detached leaves (n = 9; Bajwa et al., 2010). Then, strain C. aenigma BY827 plugs (d = 5 mm) were inoculated onto the leaves, obtained from active growing C. aenigma BY827 colonies that were 7 days old. As a control, nine leaves were mock-inoculated with sterile non-inoculated PDA medium plugs. After inoculation, all leaves were incubated at 25 ± 2°C, with a 16 h: 8 h photoperiod, and 70 ± 2% relative humidity (RH). In parallel, the pathogenicity of the representative strain C. aenigma BY827 was verified on each of six leaves of three healthy 5-year-old A. japonica plants grown in the campus green space of Guizhou University, Guiyang, Guizhou, China. Each leaf was spray inoculated with 500 μL conidial suspension (1 × 10⁵ conidia/mL) of C. aenigma BY827. Another three plants sprayed with equal amounts of sterile distilled water served as controls. The pure cultures of the pathogen were reisolated from diseased A. japonica leaves and confirmed through the molecular analysis mentioned above. The experiment was repeated three times.

To study the mycelial growth and conidia production of C. aenigma BY827 at different temperatures, a 5 mm diameter mycelial plug was taken from the edge of a seven-day-old colony and placed in the center of a petri dish with PDA medium and then incubated at different temperatures. The petri dishes were then incubated at temperatures, ranging from 15°C to 35°C at 5°C intervals. Mycelial growth was recorded on the 5th day after inoculation. Three repetitions were conducted for each treatment, and the fungal colony’s diameters were measured by the criss-cross-method (Hu et al., 2022). Ten agar-mycelium plugs (5 mm diameter) were taken from the edge of the active growing colonies simultaneously and transferred into a centrifuge tube containing sterile water. The solution was vigorously vortexed and then filtered through a double-layer gauze. Afterwards, the solution was diluted, and a hemocytometer was used to count the number of conidia per square centimeter of the colony, which was calculated using the formula below. For the germination rate analysis, conidia from seven-day-old colonies were washed with sterile distilled water, and the conidia concentration was adjusted to 1 × 10⁵ conidia/mL using a hemocytometer. Subsequently, 0.2 mL of conidial suspension was added to 0.2 mL potato dextrose broth (PDB). After 12 h, the germination rate was calculated by microscopy.

X: dilution fold, N: the number of conidia in five squares of the hemacytometer, n: the number of perforated agar-mycelium plugs, r: the inner diameter of the perforator (Deng et al., 2012; Cao et al., 2022).

To assay the effects of photoperiod on C. aenigma BY827, inoculated growth medium (PDA for mycelial growth and conidia production; PDB for conidial germination rate) was placed in incubators at 25 ± 2°C with the following settings: (1) 6 h light and 18 h dark; (2) 10 h light and 14 h dark; (3) 14 h light and 10 h dark; (4) 18 h light and 6 h dark. Each treatment was performed in triplicates.

The experiment aimed to determine the effect of humidity on the conidial germination rate of C. aenigma BY827. A conidial suspension (1 × 10⁵ conidia/mL) was smeared onto glass slides, dried with sterile air, and placed in a closed petri dish. The relative humidity was set to 60, 70, 80, and 90%, respectively. The plates were incubated for 24 h at 25 ± 2°C in the dark, and the conidial germination rate was determined by microscopic observation after 12 h. Each treatment was repeated three times.

Antifungal activities of eight chemical fungicides and two biopesticides against a representative strain of C. aenigma BY827 were screened to explore sustainable control resources for managing the pathogen. The mycelial growth rate method was used (Xin et al., 2020). The employed fungicides were formulations of tebuconazole (43% suspension concentrate; SC), mancozeb (43% SC), difenoconazole (10% water-dispersible granule; WG), pentachloronitrobenzene (40% dust powder; DP), myclobutanil (12.5% emulsifiable concentrates; EC), dimethachlone (40% wettable powder; WP), hymexazol (70% WP), thiram (50% WP), carvacrol (5% aqueous solutions; AS), and kasugamycin (2% AS). They were purchased from a local distributor and diluted to various concentrations for further experiments (Table 1). Different fungicides were dissolved in organic solvents or water (myclobutanil was dissolved in acetone, and all other fungicides were dissolved in water). For those fungicides dissolved in acetone, a comparable final concentration of the indicated solvents was added into the PDA medium, which served as control plates (CK). Different concentrations of fungicides were mixed with PDA medium, and 10 mL was dispensed into sterile 9 cm diameter petri dishes. Pathogenic mycelial plugs of representative strain C. aenigma BY827 (5 mm in diameter) were inoculated into the center of PDA plates containing varying concentrations of gradient agents, and each treatment was repeated three times. The plates were then cultured at 25 ± 2°C under dark conditions. After 7 days, the colony diameter of each treatment was measured. The following equation was used to determine the inhibition efficiency of the fungicides, and EC₅₀ (concentration for 50% of maximal effect) values were calculated.

R: relative inhibition rates, C: diameter of the fungus in control, T: diameter of the fungus in the treatment, F: diameter of fungus plugs (Luo et al., 2021).

Four pesticides, namely difenoconazole, tebuconazole, pentachloronitrobenzene, and mancozeb, were selected to determine their effectiveness in controlling the leaf blight disease caused by C. aenigma in A. japonica. The experiments were carried out at the campus green space of Guizhou University, Guiyang, Guizhou, China, which was naturally infested with A. japonica leaf blight. The pesticides were formulated according to the recommended concentration for field use. To conduct the experiment tebuconazole (at a concentration of 2.87 μg/mL in sterile water containing 43% active pesticide ingredients), mancozeb (at a concentration of 10.75 μg/mL in sterile water containing 43% active pesticide ingredients), difenoconazole (at a concentration of 1.67 μg/mL in sterile water with 10% active pesticide ingredients), and pentachloronitrobenzene (at a concentration of 266.67 μg/mL in sterile water with 40% active pesticide ingredients) were used as the pesticide treatments, while sterile water was used as the control treatment. Each treatment was repeated three times. A pump sprayer was used to evenly spray the 5-year-old plants of A. japonica. The volume of sprayed liquid was 5 mL per plant. Leaf blight was investigated before and 14 days after spraying. A total of 100 plants were randomly selected in each treatment (6 m²). According to Fang (1998), the disease grading was based on the percentage of disease spots on leaves in the whole leaf area: Grade I, healthy, representative value was 0; Grade II, diseased area ≤ 25%, representative value was 1; Grade III, diseased area > 25 and ≤ 50%, representative value was 2; Grade IV, diseased area > 50 and ≤ 75%, representative value was 3; and Grade V, diseased area > 75%, representative value was 4. Meanwhile, the disease incidence, disease index, and control efficiency were calculated using the following formula (Henderson and Tilton, 1955; Sun and Song, 2002):

pt₀: disease incidence in pesticide treatment plot before spraying, pt₁: disease incidence in pesticide treatment plot after spraying.

CK₀: disease index in control plot before spraying, CK₁: disease index in control plot after spraying, PT₀: disease index in pesticide treatment plot before spraying, PT₁: disease index in pesticide treatment plot after spraying.

All percentage data were subjected to arcsine transformation and expressed as the mean ± SE (standard error of the mean). The statistical significance of the results was calculated using a one-way analysis of variance followed by Duncan’s new complex polar difference method at a significant level of p < 0.05 using IBM SPSS Statistics software version 25.0. The EC₅₀ of different fungicides was calculated with a toxicity regression equation using the Data Processing System (DPS) v7.05 software (Pasche et al., 2004). Figures were created using GraphPad Prism (v8.0.2) software.

A total of 31 Colletotrichum-like isolates were obtained from 20 diseased A. japonica leaves (Figure 1A). The mycelial growth rate ranged from 4.7 to 5.0 mm/day. Initially, the colonies appeared white, but as time progressed, the reverse side of the colonies gradually turned pale gray from the center. The colonies were velutinous to woolly and had a regular circular shape (Figures 1B,C). Five representative strains were selected from the obtained isolates for microscopic observation. Conidia were cylindrical, with one end slightly acute or broadly rounded, and ranged in size from 13.67 to 21.09 μm × 4.24 to 7.30 μm (n = 60; Figures 1D,E). Based on these morphological characteristics, the isolates matched the description of the genus Colletotrichum spp. (Weir et al., 2012).

Nucleotide sequences were submitted for BLAST analysis using the NCBI-BLAST program. BLAST searches of the sequenced fragments resulted in the best match to C. aenigma sequences (Table 2). The sequences of the DNA regions of strains BY825, BY826, BY827, BY828, and BY829 were deposited in GenBank and are included in the Supplementary Material (Table 2). In addition, a phylogenetic analysis of the 5 strains was conducted, and the phylogenetic tree was constructed based on the ITS region, CHS, and ACT genes sequences (Figure 2). The results confirmed that the strains BY825, BY826, BY827, BY828, and BY829 belong to the C. aenigma group when compared with sequences of the type strains.

Seven days after inoculation, typical disease symptoms (early stage of blight: black spots) were visible on all inoculated plants (Figure 3). In contrast, leaves in the control group did not show any disease symptoms. The pathogenicity test was repeated and confirmed three times. Pure cultures were re-isolated from diseased leaves and confirmed to be C. aenigma BY827 based on the morphological and molecular methods mentioned above (ITS region, CHS, and ACT sequences).

The experimental results indicated that the pathogen was capable of growing within a temperature range of 15–35°C. The highest mycelial growth rate of C. aenigma BY827 was observed at 25°C and 30°C (Figure 4A). However, neither mycelium nor conidia could grow at 10°C and 40°C. The number of conidia produced by the pathogen was significantly higher at 15°C and 35°C, as shown in Figure 4B. Furthermore, the results showed that the conidial germination rate at 15°C was significantly higher at 30°C (Figure 4C).

The result indicated that 10-h/14-h alternating light increased the mycelial growth of C. aenigma BY827 (Figure 5A). There was no significant difference in conidia production under different photoperiods (Figure 5B). However, the alternation of 10 h light and 14 h dark, as well as 14 h light and 10 h dark, increased the conidial germination of C. aenigma BY827 (Figure 5C).

The results showed that at 90% humidity, the conidial germination rate of C. aenigma BY827 was significantly higher than that of other humidifies (Figure 6).

The sensitivities of the representative isolate C. aenigma BY827 to ten selected fungicides are presented in Table 3. Except for mancozeb, increasing concentrations of the fungicides inhibited the mycelial growth of C. aenigma BY827, as illustrated in Figure 7. Among the ten tested fungicides, the EC₅₀ values of nine fungicides were less than 1 μg/mL. Difenoconazole showed the highest inhibition rate, with an EC₅₀ value of 0.0148 μg/mL, followed by tebuconazole, pentachloronitrobenzene, myclobutanil, carvacrol, and kasugamycin, which also showed an effective inhibitory effect against C. aenigma BY827, with EC₅₀ values of 0.0388 μg/mL, 0.1890 μg/mL, 0.1906 μg/mL, 0.2081 μg/mL, and 0.2996 μg/mL, respectively. In contrast, mancozeb did not show effective inhibitory effects against C. aenigma BY827, with EC₅₀ values of 202.1461 μg/mL.

The results of the field experiment were presented in Table 4. Difenoconazole exhibited the highest control efficiency with a decreasing rate of disease incidence of 44.60% and decreasing rate of disease index of 47.75%, respectively, which were significantly higher than other treatments. Tebuconazole and pentachloronitrobenzene showed no differences in disease incidence and disease index. Moreover, mancozeb exhibited the lowest control efficiency with decreasing rate of disease incidence of 3.53% and decreasing rate of disease index of 3.77%, respectively, which were significantly lower than other treatments.