Molecular Microbiology Research 2024, Vol.14, No.6, 259-270 http://microbescipublisher.com/index.php/mmr 260 Woudenberg et al., 2015). Molecular sequence analysis of nuclear genes such as intron spacer regions (ITSs) and the mitochondrial ribosomal large subunit (mtLSU), Alternaria major allergen (Alt a 1), glyceraldehyde-3-phosphate dehydrogenase (gapdh), and the RNA polymerase II second largest subunit (rpb2) is useful for evaluating phylogenetic relationships among species (Zhu et al., 2015; Ma et al., 2021). Chemical fungicides are an effective strategy for managing diseases caused by Alternaria spp. in many plants. Hou et al. (2024) reported that flutolanil, phenamacril, pyraclostrobin, and boscalid can effectively prevent and control black spot disease in tree peony, which is caused by Alternaria suffruticosae. Pyraclostrobin can especially suppress the conidial germination, mycelial growth, germ tube elongation, and sporulation quantity of A. suffruticosae. Moreover, prochloraz was shown to have a stronger effect on leaf spot disease in blue honeysuckle, which was caused by A. tenuissima (Liu et al., 2024a). However, over-reliance on fungicides results in the pathogens becoming resistant to these fungicides, posing dangerous health and environmental safety concerns. Therefore, Alternaria spp. could be controlled in an environmentally friendly way by using beneficial fungi or bacteria and harmless chemicals. Melatonin (nacetyl-5-methoxytryptamine) was first identified in 1985 and given this name because of its role in the darkening effects of melanocyte-stimulating hormones (Lerner et al., 1958). It is a naturally occurring small-molecule indole neurohormone, which contains no obvious toxicity to vertebrates, including humans, and is safe and harmless to plants and the environment, so it has good potential for its application in plants. Melatonin also plays an important role in plant defense against biotic stresses. Exogenous melatonin treatment of apple fruits that were inoculated with Botrytis cinerea effectively reduced the incidence of gray mold and the area of spots, resulting in significant resistance to gray mold (Zhu et al., 2021). Root application of 0.1 mmol/L melatonin significantly enhanced the resistance of apples to brown spot disease (caused by Diplocarpon mali) (Yin et al., 2013). Melatonin inhibited the expansion of Litchi chinensis fruit spots infected with Peronophythora litchii and induced the resistance of litchi to the pathogenic fungus (Zhang et al., 2021). The treatment of fresh pistachios (Pistacia vera) inoculated with Aspergillus flavus and treated with exogenous melatonin inhibited Aspergillus flavus spore germination and shoot tube elongation; this effectively slowed down the fruit rot caused by the fungus and reduced the accumulation of aflatoxins (Aghdam et al., 2020). In addition, exogenous melatonin treatment reduced the incidence of leaf blight in rice (Chen et al., 2020). Sun et al. (2020) sprayed strawberry leaves and stolons inoculated with Alternaria alternata with 0.5 mmol/L of melatonin to effectively inhibit the growth of the mycelium of the pathogen. However, although these studies have shown that melatonin can alleviate plant diseases by increasing the level of plant autoimmunity and inhibiting the pathogenicity of plant pathogenic microorganisms, the specific mechanism of melatonin in the plant immune response is still unclear, and the pathogenic mechanisms of fungi, bacteria, viruses, and other pathogenic bacteria are different. Considering the significant influence of leaf spot disease in the G. jasminoides industry and the limited understanding of its causal agent, it has become imperative to identify and control the phytopathogenic agent. The key objective of this research was to isolate and identify Alternaria spp. which causes leaf spot disease in G. jasminoides. Furthermore, alternative approaches using melatonin as an environmentally friendly chemical to control leaf spot disease were also investigated. 2Results 2.1 Sample collection In June 2023, high temperature, high humidity, and poor ventilation accelerated the incidence rate of leaf spot disease in G. jasminoides Ellis in Enshi, Hubei Province, China. According to the field observations, 40%~60% exhibited leaf spot symptoms, as the infected leaves of G. jasminoides Ellis had dark brown or black lesions with a surrounding concentric ring. The diameter of the lesions ranged from 3 mm to 2 cm, and larger lesions had a chlorotic halo (Figure 1).
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