Evolution from Natural β-Carboline Alkaloids to Obtain 1,2,4,9-tetrahydro-3-thia-9-aza-fluorene Derivatives as Potent Fungicidal Agents against Rhizoctonia solani

Abstract

Rice sheath blight, caused by Rhizoctonia solani, is a globally important rice disease and the increasing resistance of this pathogen highlights the need for new active compounds against rice sheath blight. In this study, natural β-carboline alkaloids were optimized to obtain a series of 1,2,4,9-tetrahydro-3-thia-9-aza-fluorene derivatives and evaluated for their fungicidal activity and mode of action against R. solani. Of these compounds, 18 exhibited significant in vitro fungicidal activity against R. solani, with an EC₅₀ value of 2.35 μg/mL, and was more active than validamycin A. In vivo bioassay also demonstrated that 18 displayed superior protective and curative activities as compared to validamycin A. Mechanistically, 18 not only induced the loss of mitochondrial membrane potential and accumulation of reactive oxygen species, but also interfered with DNA synthesis. Therefore, compound 18 displayed pronounced in vitro and in vivo fungicidal activity against R. solani and could be used as a potential candidate for the control of rice sheath blight.

Keywords: 1,2,4,9-tetrahydro-3-thia-9-azafluorene; Rhizoctonia solani; fungicidal activity; β-carboline.

Figure 1

Representative structures of the β-carboline alkaloids.

Figure 2

Synthesis of target compounds 9–39.

Figure 3

The in vivo protective effect of selected compounds against R. solani using detached leaf assay.

Figure 4

The in vivo protective and curative activities of compounds 17–19 against R. solani.

Figure 5

SEM of R. solani hyphae treated with 0 (a,b) or 50 μg/mL 18 (c,d).

Figure 6

TEM of R. solani hyphae treated with 0 (a–d) or 50 μg/mL 18 (e–h). (a) Transverse of control hyphae and many organelles were observed such as mitochondria (M) and vacuole (V); (b) cell wall (CW) and plasma membrane (PM) of untreated hyphae; (c) mitochondria of untreated hyphae; (d) longitudinal of untreated hyphae, and spectra (S) was uniform; (e,f) transverse of 18-treated hyphae; (g) longitudinal of 18-treated hyphae (loss of matrix in vacuoles and obvious vacuolization); (h) mitochondria of 18-treated hyphae was swollen.

Figure 7

The mode of action of compound 18 against R. solani. (a,b) Fluorescent micrographs of the hyphae stained with DCFH-DA to assess endogenous reactive oxygen species (ROS) production; (c,d) fluorescent micrographs of the hyphae stained with Rhodamine 123 to evaluate mitochondrial membrane potential (MMP); (e,f) nuclear morphology of the hyphae stained by Hoechst 33258; (g) the number of nuclei per cell of somatic hyphae treated with 0 or 50 μg/mL 18; (** p < 0.01); (h) the conductivity of the hyphae suspensions during different time exposure to 18 was measured to assess cell membrane permeability.