Molecular Microbiology Research 2024, Vol.14, No.6, 259-270 http://microbescipublisher.com/index.php/mmr 266 After the diseased leaves were cleaned with sterile water, the leaves at the junction of necrotic spots and healthy tissues were cut into 5 mm×5 mm pieces and disinfected in 75% alcohol for 30 s and in a 2% commercial sodium hypochlorite solution (4% active chlorine) for 2 min. After each disinfection, the samples were washed several times with distilled water. Then, surface moisture was sucked up, and the samples were placed on potato dextrose agar (PDA) plates, with 3 pieces per plate. Mycelia around the disinfected tissue were selected for purification, and the pure cultured isolates were obtained through continuous transfer and stored at 4 ℃. 4.2 Pathogenicity tests After the healthy G. jasminoides leaves were wiped clean with 75% alcohol, a wound was made on each side near the middle main vein using a 1 mL sterilized syringe. Then, agar plugs (5 mm in diameter) of the isolates were inoculated into these wounds. The leaves were then moisturized and placed in an incubator at 25 ℃. Five leaves were treated per isolate. The agar plugs (5 mm in diameter) of the PDA plate were used as the negative control. After 72 h of moisturizing culture, the agar plugs were removed to continue observing and recording the disease symptoms that were captured with a digital camera (Nikon, D750, Japan). Subsequently, the tissues with obvious symptoms were re-isolated from the inoculated leaves to determine whether the inoculated isolates were the causal agents of the G. jasminoides leaf spots. 4.3 Identification based on cultural and morphological characteristics The pathogenic strains were inoculated on a PDA plate and cultured at 25 ℃ in the dark. For each isolate, three replicates were used. After 7 days, all cultures were assessed for colony color, margin, and texture. The colony morphology was photographed. To observe the spore morphological characterization, isolates from PDA plates were transferred to potato–carrot agar (PCA) and incubated for 14 days. For each isolate, conidia were harvested from the same plates and suspended in distilled water. The conidial suspension was observed under a Nikon Eclipse microscope (Japan), and more than 10 pictures were taken for each isolate. These pictures were analyzed using ImageJ 1.47 software (National Institutes of Health, Bethesda, MD) to measure the length and width of each conidia per isolate. 4.4 Identification based on molecular characteristics The DNA of pathogenic fungi was extracted from the mycelia that had been growing for 7 days, using the plant Genomic DNA kit (Tiangen, China), according to the instructions. Polymerase chain reaction (PCR) amplification of the internal transcribed spacer (ITS, ITS1/ITS4), the Alternaria major allergen sequence (Alt a 1), the second largest subunit of RNA polymerase II gene (rpb2), and the glyceraldehyde-3-phosphate dehydrogenase (gapdh) gene was conducted using specific primers: ITS1/ITS4 (White, 1990), Alt1-F/Alt1-R (Chruszcz et al., 2012), RPB2-5f2/RPB2-7cr (Liu et al., 1999), and gpd1/gpd2 (Berbee et al., 1999). The extracted pathogenic DNA served as a template for this process. The PCR was carried out using 2×Phanta® Flash Master Mix Dye Plus (Vazyme, Nanjing, China). The volume of each reaction system was 30 μL, including 15 μL of Taq enzyme, 1 μL of each primer, 1 μL of DNA template, and 12 μL of sterile water; the PCR system without a DNA template served as a negative control. The PCR amplification conditions were set as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 98 ℃ for 10 s; annealing at 54 ℃ (ITS) for 30 s, 59 ℃ (rpb2) and 57℃(Alt a 1) for 45 s, and 61℃(gapdh) for 40 s. This process depended on the fragment size, with an extension time of 72 ℃ for 10 s, followed by 30 cycles, and concluded with a final extension of 72 ℃ for 5 min. Finally, 5 μL of PCR products were taken and detected by 1% (v/v) agarose gel electrophoresis, and the products with correct bands obtained were sequenced by Sangon Biotech Co., Ltd. All sequences in this study were uploaded to GenBank of the National Center for Biotechnology Information (NCBI), and the accession numbers are listed in Table 1. The multi-locus sequences were aligned with the previously deposited sequences in the GenBank database using BLAST, and a phylogenetic tree was constructed using the maximum likelihood method in MEGA 7.0 (https://www.megasoftware.net) software to clarify the pathogenic fungi.
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