Carvacrol expresses a wide range of biological activities, but the studies of its mechanisms focused on bacteria, mainly involving the destruction of the plasma membrane. In this study, carvacrol exhibited strong activities against several phytopathogenic fungi and demonstrated a novel antifungal mechanism against Lasiodiplodia theobromae. RNA sequencing indicated that many genes of L. theobromae hyphae were predominately induced by carvacrol, particularly those involved in replication and transcription. Hyperchromic, hypsochromic, and bathochromic effects in the UV-visible absorption spectrum were observed following titration of calf thymus DNA (ctDNA) and carvacrol, which indicated the formation of a DNA–carvacrol complex. Circular dichroism (CD) spectroscopy indicated that the response of DNA to carvacrol was similar to that of 4′,6-diamidino-2-phenylindole (DAPI) but different from that of ethidium bromide (EB), implying the ionic bonds between carvacrol and ctDNA. Fluorescence spectrum (FS) analysis indicated that carvacrol quenched the fluorescence of double-stranded DNA (dsDNA) more than single-stranded DNA, indicating that carvacrol mainly bound to dsDNA. A displacement assay showed that carvacrol reduced the fluorescence intensity of the DNA–DAPI complex through competition with DAPI, but this did not occur for DNA–EB. The FS assay revealed that carvacrol bound to the AAA sequence on the minor groove of ds-oligonucleotides. The hydroxyl of carvacrol was verified to bind to ctDNA through a comparative test in which structural analogs of carvacrol, including thymol and 4-ethyl-1,2-dimethyl, were analyzed. The current study indicated carvacrol can destruct plasma membranes and bind to the minor groove of DNA, inhibiting fungal proliferation by disturbing the stability of dsDNA.

Because effective control measures are lacking, tea leaf spot caused by Didymella segeticola results in huge tea ( Camellia sinensis) production losses on tea plantations in Guizhou Province, southwestern China. Screening for natural antimicrobial agents with higher control effects against this pathogen and studying their modes of action may contribute to disease management. Here, Penicillium griseofulvum-derived antimicrobial griseofulvin (GSF) can inhibit the hyphal growth of D. segeticola strain GZSQ-4, with a half-maximal effective concentration of 0.37 μg/ml in vitro and a higher curative efficacy at a lower dose of 25 μg/ml for detached tea twigs. GSF induces deformed and slightly curly hyphae with enlarged ends, with protoplasts agglutinated in the hyphae, and higher numbers of hyphal protuberances. GSF alters hyphal morphology and the subcellular structure's order. The integrated transcriptome and proteome data revealed that the transport of materials in cells, cellular movement, and mitosis were modulated by GSF. Molecular docking indicated that beta-tubulin was the most potent target of GSF, with a binding free energy of −13.59 kcal/mol, and microscale thermophoresis indicated that the dissociation constant (Kd) value of GSF binding to beta-tubulin 1, compared with beta-tubulin 2, was significantly lower. Thus, GSF potentially targets beta-tubulin 1 to disturb the chromosomal separation and fungal mitosis, thereby inhibiting hyphal growth.

In order to discover new antifungal agents, twenty novel benodanil-heterocyclic carboxamide hybrids were designed, synthesized, and characterized by ¹H NMR and HRMS. In vitro, their antifungal activities against four phytopathogenic fungi were evaluated, as well as some of the target compounds at 50 mg/L demonstrated significant antifungal activities against Rhizoctonia solani. Especially, compounds 17 (EC₅₀ = 6.32 mg/L) and 18 (EC₅₀ = 6.06 mg/L) exhibited good antifungal activities against R. solani and were superior to the lead fungicide benodanil (a succinate dehydrogenase inhibitor, SDHI) (EC₅₀ = 6.38 mg/L). Furthermore, scanning electron microscopy images showed that the mycelia on treated media with the addition of compound 17 grew abnormally as compared with the negative control with tenuous, wizened, and overlapping colonies, and compounds 17 (IC₅₀ = 52.58 mg/L) and 18 (IC₅₀ = 56.86 mg/L) showed better inhibition abilities against succinate dehydrogenase (SDH) than benodanil (IC₅₀ = 62.02 mg/L). Molecular docking revealed that compound 17 fit in the gap composed of subunit B, C, and D of SDH. Furthermore, it was shown that the main interaction, one hydrogen bond interaction, was observed between compound 17 and the residue C/Trp-73. These studies suggested that compound 17 could act as a potential fungicide to be used for further optimization.

At present, chayote (Sechium edule (Jacq.) Swartz) have been widely planted in Guizhou Province, southwestern China, and the cultivation area in Huishui county ranks first among all the counties or...

Duyun Maojian Tea mainly cultivated in Duyun City is one of the ten famous teas in China. A brown leaf spot was discovered in March 2023 in Qiannan Normal University for Nationalities' Maojian Tea experimental field (26.29°N, 107.52°E). The incidence of the disease is about 40% surveyed on about 300 m2 of tea plants. Infected leaves showed light brown, round or irregular lesions. With disease progression, they enlarged and merged, creating dark brown necrotic zones along leaf edges. To identify the responsible pathogen, three symptomatic leaves from the tea nursery base were collected. The samples were cut 3 mm x 3 mm tissues with 75% ethanol for 30 s and 5% sodium hypochlorite for 60 s, rinsing three times with sterile water. Tissues were taken to potato dextrose agar (PDA) medium and incubated dark at 28 °C for 3 days. Three isolates (TF1, TF3 and TF5) with similar morphology were obtained from three samples, respectively. The colony showed white, with short, villous hyphae expanding radially. Notably, mycelium near the disc was erect and sparse 2 cm away. After 30 days, conidia were observed. Alpha conidia (5.9 to 8.6 μm × 2.0 to 2.8 μm (n = 50)) were colorless and transparent, no septum, ellipsoid to spindle-shaped. Beta conidia (11.1 to 25.3 μm × 1.0 to 1.8 μm (n = 50)) were transparent, no septum, filiform, hooklike, tapering towards the ends. The isolates were identified as Diaporthe pescicola based on the morphology (Zhang et al. 2022). Genomic DNA was extracted, the internal transcribed spacer region (ITS), β-tubulin (tub2), and translation elongation factor 1-alpha (TEF) genes were amplified with primer pairs ITS1/ITS4 (White et al. 1990), Bt2a/Bt2b (Glass and Donaldson 1995), and EF1-728F/EF1-986R (Carbone and Kohn 1999), respectively, and sequenced. The ITS (PQ163865, PQ685064 and PQ685065), tub2 (PQ197032, PV055438 and PV055439), and TEF (PQ197029, PV055440 and PV055441) sequences of isolates (TF1, TF3 and TF5) were deposited in GenBank, respectively. BLAST searches showed >97% nucleotide identity to sequences of D. pescicola MFLUCC 16-0105 (ITS, 98.40% to KU557555.1; tub2, 97.40% to KU557579.1; and TEF, 97.63% to KU557623.1). The phylogenetic tree of ITS, tub2, and TEF gene sequences was constructed using MEGA 6 software with the neighbour-joining method (NJ), and the bootstrap method was employed for 1,000 repeated tests. Isolates TF1, TF3, TF5, and D. pescicola DT 1-1 were grouped together in a node with a bootstrap value of 99. Isolates were identified as D. pescicola. Since large quantities of conidia are difficult to produce in cultures, pathogenicity on the wounded leaves of 5-year-old tea plants of Duyun Maojian Tea local population was conducted by inoculating 6 mm mycelium discs of TF1. The control group and the treatment group were inoculated with 3 tea plants, repeated 2 times. All tea plants were incubated at 25 ± 1 °C, 70% humidity, and natural daylight. The onset of symptoms appeared 7 days later. Lesions were brown, similar to field symptoms. In contrast, the control were healthy. A strain exhibiting the same morphology as TF1 was re-isolated from the diseased portion. ITS, tub2, and TEF gene sequences were consistent with TF1, completing Koch's postulates. D. pescicola as a pathogen has caused leaf spot on Kerria japonica (Zhang et al. 2022), dieback on Prunus persica (Dissanayake et al. 2017) and Vitis vinifera (Manawasinghe et al. 2019), and shoot canker on Pyrus plants (Guo et al. 2020). To our knowledge, this is the first report of tea leaf spot caused by D. pescicola in China and worldwide. It is imperative to develop and implement control measures to manage the disease.

Rice sheath blight caused by Rhizoctonia solani is one of the most destructive diseases of rice. Bixafen has been proposed as a promising control agent with moderate resistance risk; however, its cellular mode of action remains unclear. Therefore, this study investigated the antifungal mechanism of bixafen from the perspective of programmed cell death (PCD). Bioassays showed that bixafen strongly inhibited R. solani, with a median effective concentration (EC₅₀) of 1.16 μg/mL. Morphologically, bixafen induced hyphae collapse, vacuolization, chromatin aggregation, and mitochondrial disruption. Transcriptome analysis further revealed that bixafen significantly altered the expression of genes involved in the tricarboxylic acid cycle and PCD pathways. In addition, bixafen, at the concentration of EC₅₀, triggered ROS accumulation accompanied by increased malondialdehyde (MDA) levels. These oxidative effects led to mitochondrial damage, characterized by loss of membrane potential, reduced Tomm20 expression, and decreased Aco-2 activity. Subsequently, bixafen activated apoptosis, as evidenced by induction of the mitochondria-associated inducer of death (AMID), down-regulation of Bcl-2, and DNA fragmentation. Moreover, bixafen also induced autophagy by reducing p62 and increasing Beclin-1 expression, which suggests the clearance of damaged mitochondria. Collectively, these results demonstrated that bixafen induced mitochondrial-dependent apoptosis and autophagy in R. solani, which provided novel insights into its cellular antifungal mechanism and supported its potential as a PCD-targeted fungicide.