Molecular Microbiology Research 2024, Vol.14, No.6, 259-270 http://microbescipublisher.com/index.php/mmr 267 Table 1 GenBank accession numbers of isolates obtained fromG. jasminoides with leaf spot disease Isolate GenBank accession number ITS Alt a1 gapdh rpb2 ES1-1 PQ119762 PQ148990 PQ148996 PQ149002 ES2-1 PQ119763 PQ148991 PQ148997 PQ149003 ES2-2 PQ119764 PQ148992 PQ148998 PQ149004 ES2-3 PQ119765 PQ148993 PQ148999 PQ149005 ES4-2 PQ119766 PQ148994 PQ149000 PQ149006 ES4-3 PQ119767 PQ148995 PQ149001 PQ149007 4.5 Melatonin control of G. jasminoides leaf spot disease To investigate the inhibitory effect of melatonin on the mycelial growth of A. alternata, we added various concentrations of melatonin to the PDA. In brief, 2.32 g of melatonin (SIGMA; Chemical Abstracts Service (CAS) Number: 73-31-4; total chromatographic limit (TCL)≧98%) was dissolved in dimethyl sulfoxide (DMSO), and then water was added to reach a volume of 10 mL (DMSO: H2O=5:3/v:v) to create a mother liquor for storage. The PDA medium (potato, 200 g/L; glucose, 20 g/L; agar, 15 g/L) was configured, and different concentrations of the melatonin solution (based on the mother liquor, diluted to concentrations of 0.05 mM, 0.1 mM, 0.5 mM, 1 mM, 5 mM, and 10 mM) were added before sterilization, and DMSO and sterile water were added to the PDA medium as a control. We used ES4-2 (one of the six Alternaria isolates) for the melatonin control experiment; the isolate was cultured on PDA medium for approximately 7 d. Then, we took agar plugs measuring approximately 5 mm to inoculate each treatment, using 4~5 dishes for each treatment. The colony diameters were measured at 7 d and 14 d, the inhibition rate was calculated, and the experiment was repeated three times. The effects of melatonin on leaf spot disease were tested by applying melatonin both before and after inoculation with ES4-2. We collected G. jasminoides leaves and rinsed the leaf surface using deionized water; then, we wiped the leaves clean with skimmed cotton dipped in alcohol. These leaves were placed in a square Petri dish with two pieces of filter paper. Before treatment, we wrapped the injured part of the petiole with skimmed cotton and added 5 mL of sterile water to the Petri dish. Melatonin was configured in the same way as before and diluted to concentrations of 0.1 μM, 0.5 μM, 1 μM, 5 μM, 10 μM, 100 μM, and 1 mM, with DMSO and sterile water as controls. To inoculate the G. jasminoides leaves, two small holes were made using a syringe in the middle of both sides of the large leaf veins; ES4-2 was then inoculated into the small holes. The agar plug was removed three days after inoculation, and the leaves were sprayed with different concentrations of melatonin (0.1 μM, 0.5 μM, 1 μM, 5 μM, 10 μM, 100 μM, and 1 mM), with DMSO and sterile water sprayed as controls. Spraying was repeated on the 7th d after the initial application, and the incidence of disease was assessed 14 d after melatonin spraying. The experiment was repeated three times. For melatonin treatment before inoculation, two small holes were created on both sides of the large leaf veins in the middle of leaves and then sprayed with different concentrations of melatonin (0.1 μM, 0.5 μM, 1 μM, 5 μM, 10 μM, 100 μM, and 1 mM), with DMSO and sterile water sprayed as controls. All these leaves were incubated under dark conditions for 12 h, ES4-2 was inoculated into the positions of the small holes, and the agar plugs were carefully removed after 3 d. Then, the leaves were incubated in an incubator and were sprayed again on the 7th d. The disease incidence was investigated 14 d after melatonin spraying. This experiment was repeated three times. 4.6 Statistical analysis All data in this study are presented as averages±standard deviations (SDs). One-way analysis of variance (ANOVA) and Duncan's multiple tests were used for the statistical analysis by SPSS 18.0 (IBM, Armonk, USA), and significant differences at P<0.05 or P<0.01were marked using distinct letters.
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