Isolation, Identification and Molecular Mechanism Analysis of the Nematicidal Compound Spectinabilin from Newly Isolated Streptomyces sp. DT10

Abstract

Plant parasitic nematodes (PPNs) are highly destructive and difficult to control, while conventional chemical nematicides are highly toxic and cause serious environmental pollution. Additionally, resistance to existing pesticides is becoming increasingly common. Biological control is the most promising method for the controlling of PPNs. Therefore, the screening of nematicidal microbial resources and the identification of natural products are of great significance and urgency for the environmentally friendly control of PPNs. In this study, the DT10 strain was isolated from wild moss samples and identified as Streptomyces sp. by morphological and molecular analysis. Using Caenorhabditis elegans as a model, the extract of DT10 was screened for nematicidal activity, which elicited 100% lethality. The active compound was isolated from the extracts of strain DT10 using silica gel column chromatography and semipreparative high-performance liquid chromatography (HPLC). The compound was identified as spectinabilin (chemical formula C₂₈H₃₁O₆N) using liquid chromatography mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR). Spectinabilin exhibited a good nematicidal activity on C. elegans L1 worms, with a half-maximal inhibitory concentration (IC₅₀) of 2.948 μg/mL at 24 h. The locomotive ability of C. elegans L4 worms was significantly reduced when treated with 40 μg/mL spectinabilin. Further analysis of spectinabilin against known nematicidal drug target genes in C. elegans showed that it acts via target(s) different from those of some currently used nematicidal drugs such as avermectin and phosphine thiazole. This is the first report on the nematicidal activity of spectinabilin on C. elegans and the southern root-knot nematode Meloidogyne incognita. These findings may pave the way for further research and application of spectinabilin as a potential biological nematicide.

Keywords: biological control; drug target; natural products; nematicide; plant parasitic nematode.

Figure 1

Morphological characters of DT10 strain. (a) Representative images of the DT10 colony on ISP2 medium. Image was photographed using a Nikon Digital Single Lens Reflex camera. (b) The morphology of DT10 mycelia. Image was captured with a Leica DM6B fluorescence microscope using 40× objective (scale bar = 25 µm).

Figure 2

Unrooted phylogenetic tree of strain DT10 based on 16S rRNA sequences. Bootstrap values are expressed as percentages. Numbers at branching points refer to bootstrap value based on neighbor-joining analysis of 1000 resample data sets. The scale bar presents sequence divergence. The strain marked with “T” represents type strain.

Figure 3

Isolation and nematicidal activity of main active compound 1 of strain DT10. (a) Flow chart showing extraction and separation procedure of compound 1 from strain DT10. (b) Chemical structure of compound 1 (Spectinabilin).

Figure 4

Dose–response curve of spectinabilin’s nematicidal activity on C.elegans L1 worms. The curve shows that the nematicidal activity was positively correlated with the concentration of spectinabilin. The assay was employed to calculate the half-maximal inhibition concentration (IC₅₀) after (a) 24 h of incubation with spectinabilin at 2.5, 3, 4, 5 and 10 μg/mL and (b) 2 h of incubation with spectinabilin at 3.125, 6.25, 12.5, 25, 50, 100, 200 and 400 μg/mL. The data were analyzed using Prism 8.0.2. Data points represent experiments conducted in triplicate; mean ± standard error of the mean (SEM).

Figure 5

Effect of spectinabilin on locomotive ability of C. elegans L4 worms. Relative locomotive activity of the worms incubated with 40 μg/mL spectinabilin was captured using WMicrotracker ARENA unit and analyzed using Prism 8.0.2. Data points represent four independent experiments; mean ± standard error of the mean (SEM).

Figure 6

Analysis of spectinabilin on known drug target genes. The L1 worms of C. elegans mutants of known nematicidal drug target genes were treated with 4 μg/mL spectinabilin for 24 h. The lethality of worms was counted, and the images were captured using Leica DMi8 inverted microscope using 10× objective (scale bar = 100 μm).

Figure 7

Morphological observation of M. incognita incubated with spectinabilin. (a) M. incognita in the control; (b) M. incognita treated with 12.5 μg/mL spectinabilin for 72 h. Images were captured using Leica DMi8 inverted microscope using 10× objective (scale bar = 100 μm).