Identification of Nematicidal Metabolites from Purpureocillium lavendulum

Abstract

Purpureocillium lavendulum is a fungus with promising biocontrol applications. Here, transcriptome data acquired during the infection of Caenorhabditis elegans by Purpureocillium lavendulum showed that the transcription of metabolite synthesis genes was significantly up-regulated after 24 and 48 h of the fungus-nematode interaction. Then, the up-regulated transcription level of lipoxygenase was confirmed by RT-qPCR. The ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) analysis of differential metabolites revealed that this interaction resulted in the emergence of new metabolites or enhanced the production of metabolites. The results of the UPLC-MS analysis and the nematicidal assay were used to establish optimal culturing conditions under which 12 metabolites, including 3 hydroxylated C₁₈ fatty acids and 9 steroids, were isolated and identified. Among them, hydroxylated fatty acids showed pronounced nematicidal activity against Meloidogyne incognita, and two degradative sterols showed chemotaxis activity to M. incognita. This study lays a foundation for the function of lipoxygenase and its products during the infection of Purpureocillium lavendulum.

Keywords: Meloidogyne incognita; Purpureocillium lavendulum; hydroxylated fatty acids; lipoxygenase; nematicidal activity; transcriptome data.

Figure 1

KEGG enrichment of differential genes for (A) 24 h, (B) 48 h, (C) 72 h, and (D) 96 h co-culturing.

Figure 2

Relative transcription levels of three genes quantified using RT-qPCR at different interaction times. β-Tubulin was used as the standard for the statistical analysis of the transcription level of three genes in different co-culture times relative to those in the wild-type strain under a given condition. Error bars indicate the standard deviations.

Figure 3

Chromatograms of interaction and control samples. (A) Base peak chromatograms of samples at 24 h: 24PC = experimental group, 24P and 24C = control groups. Red box shows chromatogram expansions. (B) Base peak chromatograms of samples at 48 h: 48PC = experimental group, 48P and 48C = control groups. Red box shows chromatogram expansions.

Figure 4

Results of culture medium screening based on UPLC profiling and nematicidal activity assessment. (A) Profiles of crude extracts produced through the fermentation of P. lavendulum on five solid media. (B) Nematicidal activities of crude extracts obtained from different media.

Figure 5

The MS/MS spectra of compounds 1–3. (A). The HR-ESI-MS and MS/MS spectra and fragmentation pathway for compound 1. The HR-MS/MS spectrum of 1 showed the parent ion with m/z 299.2579 and two product ions with m/z 253.2529 and 141.1269. And Proposed fragmentation pathways for compound 1 producing m/z 253.2529 and 141.1269. (B). The HR-ESI-MS and MS/MS spectra and fragmentation pathway for compound 2. The HR-MS/MS spectrum of 2 showed the parent ion with m/z 315.2535 and several key product ions with m/z 201.1108, 141.1273 and 127.1115. And Proposed fragmentation pathways for compound 2 producing m/z 201.1108, 141.1273 and 127.1115. (C). The HR-ESI-MS and MS/MS spectra and fragmentation pathway for compound 3. The HR-MS/MS spectrum of 3 showed the parent ion with m/z 297.2428 and the key product ion with m/z 185.1169. And Proposed fragmentation pathways for compound 3 producing m/z 185.1169.

Figure 6

The structures were identified from P. lavendulum.

Figure 7

Nematicidal activities of metabolites isolated from P. lavendulum. Mortality (%) of M. incognita J2s treated with OA, SA, and 1–3.

Figure 8

Results of the nematode chemotaxis assay. (A) Avoidance activity of 8 at different times and concentrations. (B) Attractive activity of 9 at different times and concentrations.