First report of soybean leaf spot caused by in China.

Soybean (Glycine max L. Merr.) is a highly economically valuable crop grown extensively worldwide. However, it is prone to reduced yield and quality due to pests and diseases. A novel leaf spot disease was discovered during a soybean disease survey conducted in 2022 within a field situated at N 46°50'5.05" E 126°30'9.77" (Liu et al., 2023). The disease has a high incidence in the field, with an incidence rate of 23%. Affected areas turn yellow and are distributed in spots on the leaves. Severe cases develop into patches of dark green tissue lesions, and the leaves may become yellow, wilt, or even fall off. It was observed that the incidence rate in the shade under the trees was higher than that in direct sunlight, which affects soybean leaves and severely impacts their photosynthetic ability. To investigate this disease, a total of 30 diseased soybean plants were sampled from a continuous field. The infected leaf tissues were disinfected and rinsed, after which the pathogen was isolated using the single spore isolation method. After obtaining the purified pathogen, it was cultured on a PDA plate at 28°C for 7 days for future use. Molecular identification was conducted using complete rDNA-ITS sequences with primer pairs ITS1/ITS4, ACT1/ACT2, and MS1/MS2 (Raja et al., 2017). The amplification system comprised 25 µL: 12.5 µL PCR Mix, 1.5 µL DNA template, 1 µL of each upstream and downstream primer, and 9 µL ddH2O. The PCR amplification reaction conditions were as follows: pre-denaturation at 95°C for 2 minutes, denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, extension at 72°C for 45 seconds, final extension at 72°C for 6 minutes, and storage at 4°C, with a total of 30 cycles. The PCR products were sent to Shenggong Bioengineering (Shanghai) Co., Ltd. for sequencing. The sequences were deposited in GenBank with accession numbers OR237554 (ITS), PQ336777 (ACT), and OR137984 (MS), respectively. In this study, three diffeent genes were used to identify the species of this fungus, confirming the reliability of the strain identification results. The maximum likelihood tree revealed that the isolate clustered with representative isolates of Cladosporium cladosporioides with 96% bootstrap support (Supplement Figure 1). Thus, the isolate was identified as Cladosporium cladosporioides. based on these results. This fungus grows relatively slowly and changes from light green to dark green (Figure 1). The fungus was cultured on PDA medium for microscopic observation. Conidia were abundant, transparent, light green, oval or fusiform, measuring 2 to 3 µm × 6 to 7 µm (n=50); macroconidia were less numerous, mostly two-septate, cylindrical, measuring 2 to 3 µm × 48 µm to 50 µm (n=50). The results showed various spore types and mycelial conditions. Pathogenicity experiments were first conducted using leaf. Leaves were disinfected, inoculated with fungal cakes, and incubated at 28°C for five days. The results showed a 100% leaf incidence, with symptoms consistent with leaf spot disease observed in the field. Another experiment was conducted using potted plants with three different inoculation concentrations. After the potted plants grew for two weeks, fungal spore suspensions with concentrations of 1×106, 1×107, and 1×108 were sprayed on the leaves of soybean seedlings every two days, at a volume of 1 mL per pot each time. Symptoms began to appear after three sprays. There were no symptoms observed in the leaves of the blank control group. Six pots of potted plants were planted at each concentration, with six soybean plants per pot, resulting in a total of 36 soybean plants sprayed seven times. The three concentrations of soybean potted plants began to develop diseases, which were similar to those observed in the field. According to statistics, the incidence rate was 60%. Therefore, the pathogenicity of this pathogen is high and seriously affects the growth and yield of soybean plants, necessitating further research on this pathogen. The experimental results were consistent with Koch's postulates. This study is the first to report Cladosporium cladosporioides causing leaf spot disease in soybean in China.