Identification of Nematicidal Metabolites from Purpureocillium lavendulum

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.


Introduction
As agricultural pests of global significance, plant-parasitic nematodes, particularly Meloidogyne spp. and Heterodera spp., cause large crop losses annually [1]. Nematophagous fungi are a diverse group of fungal species that can capture or infect nematodes. These fungi represent an important source of potential biocontrol agents against plant-parasitic nematodes [2,3]. Purpureocillium lavendulum [4] is a fungal species discovered recently and is a close relative of the nematophagous fungus P. lilacinum (formerly known as Paecilomyces lilacinus), which has been the most widely used species for controlling nematodes and harmful insects [5][6][7]. One obvious difference between the two species is that P. lavendulum cannot grow at above 35 • C and is therefore not considered an infectious threat to humans, while P. lilacinum can grow well at above 35 • C [8]. Lacking growth above 35 • C makes P. lavendulum safer as a biological control agent.
Research on the mechanism of such biocontrol has mainly focused on the activity of extracellular enzymes [9,10] and metabolites profiling. Tadashi isolated leucinostatins (nonribosomal peptides) from the fermentation broth of Purpureocillium spp. and demonstrated that the members of this novel antibiotics class inhibit the proliferation of certain grampositive bacteria, fungi, and even tumor cells [11]. Another work showed that leucinostatins can be produced by different strains of P. lilacinum and are highly toxic to nematodes [12]. In addition, other compounds including fatty acids [12], phenolic acids, sesquiterpenoids [13], with ddH 2 O. Three replicates were conducted for each time point. The transcriptome sequencing was assisted by the BioMarker Company (Beijing, China).

UPLC-MS Analysis
UPLC-MS analysis was performed on a Dionex UltiMate 3000 LC system coupled with a Q-Exactive Orbitrap mass spectrometer (San Jose, CA, USA) (Thermo, Bremen, Germany) with an electrospray ionization (ESI) source. Separation was performed on a Hypersil Gold column (100 mm × 2.1 mm, Thermo Fisher Scientific, Waltham, MA, USA) with a particle size of 1.9 µm at an LC flow rate of 300 µL min −1 and a column temperature of 40 • C. Mobile phase A was 0.1% formic acid in water, and mobile phase B was 0.1% formic acid in methanol. The 30 min gradient for the positive ESI mode was set as follows: 0-2 min, 5% solvent B; 2-22 min, 5-95% solvent B; 22-25 min, 95% solvent B; and 25-30 min, 5% solvent B. The injection volume was 10 µL, and each sample was injected in triplicate. For normal detection, the ESI source parameters were set to capillary temperature = 350 • C, sheath gas flow rate = 35 arbitrary units (a.u.), auxiliary gas flow rate = 8 a.u., spray voltage = 3.5 kV, and a full MS resolution of 70,000, whereas fatty acid detection was performed at sheath gas = 25 a.u., auxiliary gas =10 a.u., auxiliary gas heater temperature = 220 • C, spray voltage = −2.8 kV, and capillary temperature = 350 • C. For the full scan, the automatic gain control (AGC) target and maximum injection time (IT) were 3 × 10 6 and 100 ms, respectively, with a resolution of 70,000. For parallel reaction monitoring (PRM), the resolution was set at 17,500, and the AGC target and maximum IT were 2 × 10 5 and 100 ms, respectively. The normalized collision energy (CE) was set as 30%. The LC-MS instrument was controlled using Thermo Scientific Xcalibur 4.1 software (San Jose, CA, USA).

Metabolite Isolation and Purification
Based on the results of the culturing condition optimization, we selected oat agar as the medium for the culturing of P. lavendulum at 28 • C for 21 days, with the total fermentation volume equaling 30 L. After soaking, the soaking solution was filtered out from four layers of gauze and then decompressed and dried at 45 • C to obtain the extract. The extract was dissolved in distilled water (6 L), and the solution was successively extracted with the same volumes of petroleum ether, ethyl acetate, and n-butanol. Each of the three extracts was concentrated, and the mass of the ethyl acetate fraction was determined as 252.4 g.

Assay of Nematicidal Activity against M. incognita
The twelve metabolites isolated from the P. lavendulum YMF1.00683 were used to test for nematicidal activity [19], with stearic acid and linoleic acid purchased from Macklin used as references. The metabolites were dispersed in MeOH, while stearic acid (SA) and oleic acid (OA) were dispersed in acetone. Two hundred J2s (10 µL) of M. incognita were added to each sample, and the concentration of the working solutions of the tested compounds was set to 400 ppm. The total and dead nematode numbers were determined every 24 h. Three replicates were conducted for each test.

Assay of Chemotaxis Activity against M. incognita
The nematode chemotaxis assay was conducted for the 12 isolated metabolites using the plate method [20]. The pure compounds were dispersed in MeOH to afford stock solutions with concentrations of 400 µg mL −1 . These stock solutions were used to prepare working solutions (40, 20, and 10 ppm). After air drying, the sample was treated with 5 µL of the working solution, and MeOH (5 µL) was used as the control. Typically, 10 µL (~200 nematodes) of the solution was added to the middle of the plate. The chemotaxis index (CI) was calculated as reported elsewhere [20].

Transcriptomics Analysis of C. elegans Infection by P. lavendulum
To determine the changes induced by the interaction of P. lavendulum with C. elegans, we performed RNA sequencing experiments on isolated P. lavendulum and P. lavendulum interacting with C. elegans. As a result, 1160, 1533, 1012, and 1580 differentially expressed genes (DEGs) were identified in the latter (interaction) sample at 24, 48, 72, and 96 h ( Figure 1). Among them, 434, 857, 482, and 706 genes were upregulated, respectively. The DEGs were divided into different categories. The GO classification of the DEGs showed that most of them were responsible for the basic processes related to biological regulation and metabolism, revealing significant differences in the transcription of genes involved in energy metabolism and transport ( Figure 1). Notably, 45 of the DEGs were different at four stages from the beginning to the end of co-culturing, among which the genes encoding chitin synthase and phosphatidyltransferase showed differences in expression at each stage. In the early stage of infection, the expression of the chitin synthase and phosphatidyltransferase genes increased, as did the secretion of chitinase and phospholipase, which enhanced the degradation of the nematode epidermis. At the late stage of infection, epidermal degradation was complete, and the expression level decreased.
interacting with C. elegans. As a result, 1160, 1533, 1012, and 1580 differentially expressed genes (DEGs) were identified in the latter (interaction) sample at 24, 48, 72, and 96 h ( Figure 1). Among them, 434, 857, 482, and 706 genes were upregulated, respectively. The DEGs were divided into different categories. The GO classification of the DEGs showed that most of them were responsible for the basic processes related to biological regulation and metabolism, revealing significant differences in the transcription of genes involved in energy metabolism and transport ( Figure 1). Notably, 45 of the DEGs were different at four stages from the beginning to the end of co-culturing, among which the genes encoding chitin synthase and phosphatidyltransferase showed differences in expression at each stage. In the early stage of infection, the expression of the chitin synthase and phosphatidyltransferase genes increased, as did the secretion of chitinase and phospholipase, which enhanced the degradation of the nematode epidermis. At the late stage of infection, epidermal degradation was complete, and the expression level decreased. Whereas carbohydrate decomposition-related genes were significantly downregulated during predation, ON184318, encoding a linoleate diol synthase, was significantly upregulated at 24 h. The linoleate diol synthase is involved in lipid metabolism, catalyzing the oxygenation of linoleate, and plays important roles in plant development and defense responses under various environmental stresses. Much evidence suggests that oxylipin is an important factor that regulates biological development, participates in cellular signaling pathways [21,22], and plays an important role in the defense response against pathogens [23][24][25]. The transcriptomic data analysis of C. elegans infection by P. lavendulum showed that the transcription of the gene encoding chitinase (ON184319) was upregulated, which agreed with the known upregulation of chitinases at the transcriptome level during the fungal infection of nematodes [26,27]. Another gene (ON184320) encoding 3-hydroxy-3-methylglutaryl-coenzyme A reductase, which is a rate-limiting enzyme in the mevalonate pathway, plays a key role in the biosynthesis of terpenoids Whereas carbohydrate decomposition-related genes were significantly downregulated during predation, ON184318, encoding a linoleate diol synthase, was significantly upregulated at 24 h. The linoleate diol synthase is involved in lipid metabolism, catalyzing the oxygenation of linoleate, and plays important roles in plant development and defense responses under various environmental stresses. Much evidence suggests that oxylipin is an important factor that regulates biological development, participates in cellular signaling pathways [21,22], and plays an important role in the defense response against pathogens [23][24][25]. The transcriptomic data analysis of C. elegans infection by P. lavendulum showed that the transcription of the gene encoding chitinase (ON184319) was upregulated, which agreed with the known upregulation of chitinases at the transcriptome level during the fungal infection of nematodes [26,27]. Another gene (ON184320) encoding 3-hydroxy-3-methylglutaryl-coenzyme A reductase, which is a rate-limiting enzyme in the mevalonate pathway, plays a key role in the biosynthesis of terpenoids and steroids in fungi. Therefore, the above three genes were selected to verify changes in their transcription level by RT-qPCR. The results showed that the mRNA levels of the linoleate diol synthase in the interaction sample increased to 149.3, 158.0, 131.1, and 141.7% at 24, 48, 72, and 96 h, respectively ( Figure 2). The mRNA levels of chitinase in the interaction sample also slightly increased at 24, 48, and 72 h but decreased at 96 h ( Figure 2). and steroids in fungi. Therefore, the above three genes were selected to verify change their transcription level by RT-qPCR. The results showed that the mRNA levels of linoleate diol synthase in the interaction sample increased to 149.3, 158.0, 131.1, 141.7% at 24, 48, 72, and 96 h, respectively (Figure 2). The mRNA levels of chitinase in interaction sample also slightly increased at 24, 48, and 72 h but decreased at 96 h ( Fig  2).

UPLC-MS Analysis of Interaction Samples between P. lavendulum and C. elegans
In order to investigate whether the metabolites of P. lavendulum in the process of infec nematodes are the same as those under the same culture conditions, samples were lected at 24, 48, 72, and 96 h after infection (PC group) and analyzed by UPLC-MS, w the group P and group C samples used as controls. To better visualize the difference the changes of the main components, we used base peak chromatograms. As a re several peaks appeared (17.9 and 19.8 min) or gained intensity (17.9, 18.3, 19.1, 19.8, 20.7

UPLC-MS Analysis of Interaction Samples between P. lavendulum and C. elegans
In order to investigate whether the metabolites of P. lavendulum in the process of infecting nematodes are the same as those under the same culture conditions, samples were collected at 24, 48, 72, and 96 h after infection (PC group) and analyzed by UPLC-MS, with the group P and group C samples used as controls. To better visualize the differences in the changes of the main components, we used base peak chromatograms. As a result, several peaks appeared (17. As the transcriptomic and RT-qPCR analyses showed that the transcription of the lipoxygenase gene was significantly upregulated at 24 h, we analyzed the possible fatty acid metabolites. Compared with the control (C and P) groups, the PC group showed different quasi-molecular ion peaks (m/z 299.2576 and 339.2494), which were ascribed to the products of fatty acid oxidation. After repeated experiments, some specific small molecules were stably identified. Although the relative abundances of some metabolites significantly increased during the infection process, these metabolites could not be obtained in large amounts at this stage. The results of the UPLC-MS analysis showed that, although some metabolites in the PC group were obviously produced, the metabolites also accumulated and changed with time in the P group. Therefore, in the next step, we screened culturing conditions to identify those favoring the production of metabolites corresponding to oxidized fatty acids.  As the transcriptomic and RT-qPCR analyses showed that the transcription of the lipoxygenase gene was significantly upregulated at 24 h, we analyzed the possible fatty acid metabolites. Compared with the control (C and P) groups, the PC group showed different quasi-molecular ion peaks (m/z 299.2576 and 339.2494), which were ascribed to the products of fatty acid oxidation. After repeated experiments, some specific small molecules were stably identified. Although the relative abundances of some metabolites significantly increased during the infection process, these metabolites could not be obtained in large amounts at this stage. The results of the UPLC-MS analysis showed that, although some metabolites in the PC group were obviously produced, the metabolites also accumulated and changed with time in the P group. Therefore, in the next step, we screened culturing conditions to identify those favoring the production of metabolites corresponding to oxidized fatty acids.

Results of Culturing Conditions Screening
As the interaction experiment was carried out using a solid medium, we selected five solid media to ferment P. lavendulum for screening culturing conditions, producing compounds similar to those in the PC group. After the 21-day culturing of P. lavendulum on solid media, the extracts were analyzed by LC-MS and assayed to determine nematicidal activity. The results of the LC-MS analysis showed that the chromatogram profiles of the samples cultured on oat and PDA media resembled those of the PC group, featuring peaks corresponding to fewer polar compounds with retention times of 16-25 min. In the case of the oat solid medium, quasi-molecular ion peaks with m/z 299.2576, 321.2399, and 339.2494 were detected, gaining intensity in the PC group. Moreover, the metabolites produced on the former medium were more uniformly distributed on the chromatogram ( Figure 4A). Finally, the nematicidal activity of the five extracts was highest in the case of the oat medium ( Figure 4B). Therefore, we chose the oat solid me-

Results of Culturing Conditions Screening
As the interaction experiment was carried out using a solid medium, we selected five solid media to ferment P. lavendulum for screening culturing conditions, producing compounds similar to those in the PC group. After the 21-day culturing of P. lavendulum on solid media, the extracts were analyzed by LC-MS and assayed to determine nematicidal activity. The results of the LC-MS analysis showed that the chromatogram profiles of the samples cultured on oat and PDA media resembled those of the PC group, featuring peaks corresponding to fewer polar compounds with retention times of 16-25 min. In the case of the oat solid medium, quasi-molecular ion peaks with m/z 299.2576, 321.2399, and 339.2494 were detected, gaining intensity in the PC group. Moreover, the metabolites produced on the former medium were more uniformly distributed on the chromatogram ( Figure 4A). Finally, the nematicidal activity of the five extracts was highest in the case of the oat medium ( Figure 4B). Therefore, we chose the oat solid medium to expand the mycelium of P. lavendulum and isolate and identify target compounds. The 13 C-NMR and DEPT spectra (Table 1) revealed one quaternary carbon (δ C 178.8), one oxygen-substituted methine, fifteen methylene groups, and one methyl group (δ C 14.1). According to MS and NMR data, compound 1 was presumed to be an 18-carbon straightchain fatty acid bearing one hydroxyl group. Despite the availability of 2D-NMR data, we could not identify the position of the hydroxyl group because of the overlap of numerous NMR signals and therefore resorted to HR-ESI-MS and MS/MS data to obtain structural clues. The MS/MS spectrum of 1 ( Figure 5A) featured the parent ion and two product ions with m/z 253.2529 and 141.1269. The ion with m/z 253.2529 was assumed to have the composition of C 17 H 33 O − ([M − H] − calc. for 253.2526) and was produced through the sequential loss of CO 2 and H 2 from the parent ion (m/z 299.2579), which afforded a stable alkoxide anion. This fragmentation is believed to be typical of hydroxylated fatty acids [28]. The ion with m/z 141.1269 was assumed to have the composition of C 9 H 17 O − (calc. for 141.1274) and could be produced via (i) homolytic C9-C10 cleavage with subsequent C11-H bond homolysis or (ii) the loss of CO 2 from the parent ion (m/z 299.2581) followed by C10-C11 bond homolysis and the loss of a C9 hydrogen radical to form m/z 141.1274 [28,29]. Thus, the hydroxyl group was located at C10, i.e., 1 was identified as 10-hydroxyoctadecanoic acid ( Figure 6).   The 13 C-NMR and DEPT spectra (Table 1) revealed one quaternary carbon (δC 178.8), one oxygen-substituted methine, fifteen methylene groups, and one methyl group (δC stable alkoxide anion. This fragmentation is believed to be typical of hydroxylated fatty acids [28]. The ion with m/z 141.1269 was assumed to have the composition of C9H17O − (calc. for 141.1274) and could be produced via (i) homolytic C9-C10 cleavage with subsequent C11-H bond homolysis or (ii) the loss of CO2 from the parent ion (m/z 299.2581) followed by C10-C11 bond homolysis and the loss of a C9 hydrogen radical to form m/z 141.1274 [28,29]. Thus, the hydroxyl group was located at C10, i.e., 1 was identified as 10-hydroxyoctadecanoic acid ( Figure 6).  hydroxyl groups involves the cleavage of bonds between the hydroxyl-substituted C and its neighboring (α) atoms [28,30,31]. The key product ion with m/z 201.1108, identified as C 10 H 17 O 4 − (calc. for 201.1121), could be produced through C10-C11 bond homolysis followed by C10-H homolytic cleavage, while the ion with m/z 171.1008 (C 9 H 15 O 3 − , calc. for 171.1016) was possibly produced through C9-C10 bond cleavage. Moreover, the ion with m/z 127.1114 (C 8 H 15 O − , calc. for 127.1117) was produced by the loss of CO 2 from the parent ion (m/z 315.2535) followed by C9-C10 bond homolysis and the subsequent loss of a C9 hydrogen radical. According to the above MS/MS data, 2 was identified as 9,10-dihydroxyoctadecanoic acid ( Figure 6).  Figure 5B) featured the peak of the parent ion as well as those of five product ions with m/z 297.2434, 201.1108, 171.1017, 141.1273, and 127.1115. Among them, the ion of m/z 297.2334 was produced through the loss of one hydroxyl group not involving C-C bond cleavage, whereas the other four ions could be formed through C-C bond cleavage and provide clues for determining the hydroxyl group positions. According to the literature, the fragmentation of fatty acids with multiple hydroxyl groups involves the cleavage of bonds between the hydroxyl-substituted C and its neighboring (α) atoms [28,30,31]. The key product ion with m/z 201.1108, identified as C10H17O4 − (calc. for 201.1121), could be produced through C10-C11 bond homolysis followed by C10-H homolytic cleavage, while the ion with m/z 171.1008 (C9H15O3 − , calc. for 171.1016) was possibly produced through C9-C10 bond cleavage. Moreover, the ion with m/z 127.1114 (C8H15O − , calc. for 127.1117) was produced by the loss of CO2 from the parent ion (m/z 315.2535) followed by C9-C10 bond homolysis and the subsequent loss of a C9 hydrogen radical. According to the above MS/MS data, 2 was identified as 9,10-dihydroxyoctadecanoic acid ( Figure 6).

Identification of Compound Structures
Compound 3 was obtained as a colorless, amorphous solid. Negative HR-ESI-MS data suggested a molecular formula of C18H33O3 based on the [M − H] − ion with m/z 297.2427 (calc. 297.2424). According to MS and NMR data, 3 was identified as octadecanoic acid containing one hydroxyl group and one double bond. Further structural insights were provided by HSQC and HMBC experiments. In particular, the HMBC experiment showed that the methylene protons at δH 2.19 were correlated with the carbons  Compound 4 was very similar to 6, except for an additional group appended to the 3-OH of the latter compound ( Figure 6), which resulted in a downfield shift of the corresponding signals (from δ H 4.62 and δ C 66.8 in 6 to δ H 5.25 and δ C 70.5 in 4) [35]. HMBC data showed that the proton at δ H 8.02  was correlated with the carbon at δ C 70.5 (C-3). Based on the similarity of the NMR data and the biogenetic perspective [35], 4 was proposed to have the absolute configuration shown in Figure 6 ((22E,24R)-3β,5α,9α-trihydroxyergosta-7,22-dien-6-one-3-yl formate).
Compound 5 was obtained as a colorless, amorphous solid. Positive HR-ESI-MS data indicated a molecular formula of C 29 H 44 O 4 Na based on the [M + Na] + ion with m/z 479.3128 (calc. 479.3132). The acquired NMR data suggested that 5 was a steroid featuring a formate group attached to the 3-hydroxyl group ( Table 2). HMBC data showed that the proton at δ H 8.01  was correlated with the carbon at δ C 71.0 (C-3). Based on the similarity of the NMR data and the biogenetic perspective [36], 5 was proposed to have the absolute configuration shown in Figure 6 (5α,6α-epoxy-(22E,24R)-ergosta-8,22-diene-3β,7α-diol-3-yl formate).

Effect of Metabolites on the Mortality of M. incognita J2s
Compounds 1-3 isolated from P. lavendulum YMF1.00683 had different nematicidal activities against the J2s of M. incognita (Figure 7). At a test concentration of 400 ppm, 2 and 3 showed weak nematicidal activity at 24 h, while compound 1, SA, and OA had no obvious nematicidal activity. At 48, 72, and 96 h, the nematicidal activities of 1 and 3 exceeded those of SA and OA. At 96 h, mortalities of 70.4, 48.6, 69.1, 30.0, and 15.0% were observed for 1, 2, 3, OA, and SA, respectively. Fatty acids, which are ubiquitous in nature and play a key role in life processes, show antifungal activities, inhibit pathogenic bacteria, and activate plant disease resistance. In addition, fatty acids (e.g., caproic, caprylic, capric, lauric, myristic, and palmitic acids) are known to be toxic to the J2s of M. incognita [19]. Moreover, a mixture of fatty acids containing linoleic acid, OA, and palmitic acid isolated from Pleurotus pulmonarius was reported to exhibit nematicidal activity [41].

Chemotactic Activities of Metabolites
The M. incognita J2s exhibited negative chemotaxis toward 8, whereas the opposite effect was observed for 9, and the other tested metabolites featured no pronounced activity. At a concentration of 40 ppm, 8 exhibited a CI of −0.65 at 4 h, losing activity at 8 h; meanwhile, the effect of 8 became less pronounced as its concentration decreased from 40

Chemotactic Activities of Metabolites
The M. incognita J2s exhibited negative chemotaxis toward 8, whereas the opposite effect was observed for 9, and the other tested metabolites featured no pronounced activity. At a concentration of 40 ppm, 8 exhibited a CI of −0.65 at 4 h, losing activity at 8 h; meanwhile, the effect of 8 became less pronounced as its concentration decreased from 40 to 10 ppm ( Figure 8A). At 40 ppm and 2 h, 9 showed a CI of 0.21, and the activity of the compound decreased with decreasing time and concentration ( Figure 8B).

Chemotactic Activities of Metabolites
The M. incognita J2s exhibited negative chemotaxis toward 8, whereas the opposite effect was observed for 9, and the other tested metabolites featured no pronounced activity. At a concentration of 40 ppm, 8 exhibited a CI of −0.65 at 4 h, losing activity at 8 h; meanwhile, the effect of 8 became less pronounced as its concentration decreased from 40 to 10 ppm ( Figure 8A). At 40 ppm and 2 h, 9 showed a CI of 0.21, and the activity of the compound decreased with decreasing time and concentration ( Figure 8B).

Discussion
The microbial control of nematodes is a sustainable and environmentally friendly strategy. However, the effects and persistence of some biocontrol microorganisms with potential application value vary greatly with region, soil characteristics, and agricultural practice. However, a more important reason may be that the molecular mechanism of the microbial infection of nematodes is not fully understood [42]. Currently, it is accepted that nematophagous fungi need to break through the cuticle or eggshell of plant-parasitic nematodes during infestation to digest the internal tissues of their hosts. The invasion mechanism may be completed by mechanical pressure and enzymatic hydrolysis. Proteases such as chitinase, protease, and lipase, which are often the determinants of host-infected pathogenic strains, may play key roles in fungal infection. In the transcriptome of the infection of C. elegans by P. lavendulum, the transcription of genes corresponding to chitinase, protease, and esterase was significantly upregulated.
In addition, some microorganisms with biocontrol potential secrete small molecular compounds to attract nematodes and further kill them. For example, Bacillus nematocida B16, which has good application prospects, produces 2-heptanone to attract nematodes, subsequently infesting and killing them through protease secretion [43,44], while Arthrobotrys oligospora produces volatile methyl 3-methyl-2-butenoate to attract C. elegans and realizes a nematode trapping strategy [45]. Two Zn(2)-C6 transcription factors responsible for the regulation of fungal metabolism were significantly upregulated during the interaction between P. lavendulum and C. elegans. The metabolite difference analysis of the interaction between fungi and nematodes showed that some small molecular metabolites were involved. Based on the transcriptome data and metabolite differences corresponding to the interaction between P. lavendulum and nematodes, 12 compounds were obtained using an optimized oat solid medium. The experiments evaluating the effect of the secondary metabolites of P. lavendulum on the chemotaxis of M. incognita revealed that 9 had a moderate attractive activity at 40, 20, and 10 ppm, with the respective CI values equaling 0.21, 0.15, and 0.16. Compound 8 showed avoidance activity at 40, 20, and 10 ppm, with the respective CI values equaling −0.64, −0.23, and −0.18. Although 8 and 9 are both steroid degradation products [37,46] with structural differences limited to substituents at the 4-position, these differences are sufficient to afford opposite activities.
In the nematicidal activity assay, 1 and 3 showed toxicity against M. incognita (70.4 and 69.1% mortalities, respectively) at 400 ppm. These compounds are the precursors of oxylipins, which are a large class of oxidized fatty acids and their derived metabolites widely found in animals, plants, bacteria, and fungi. In plants, oxylipins act as signaling molecules to regulate developmental processes such as pollen formation and mediate responses to biotic and abiotic stresses such as herbivore or pathogen attack and desiccation [47,48]. Oxylipins are enzymatically formed by the initial peroxidation of polyunsaturated fatty acids catalyzed by lipoxygenase. However, some studies report the direct production of jasmonic acid and its derivatives by fungi such as the phytopathogenic fungus Botrydiplodia theobromae [49], while this acid and its derivatives were also found in Fusarium oxysporum [50]. Although numerous hydroxylated fatty acids and their derivatives have been isolated from microorganisms, their functions remain unclear [51]. During the interaction between P. lavendulum and C. elegans, lipoxygenase was upregulated to varying degrees at 48 and 72 h, which indicated that, when infecting nematodes, the fungus could also produce hydroxylated fatty acids through the oxygen-lipid pathway. Thus, mycotoxins or other secondary metabolites harmful to nematodes may be a selective strategy of fungi against prey.