Pathogenicity and Metabolites of Purpureocillium lavendulum YMF1.00683 against Meloidogyne incognita

Purpureocillium lavendulum is a biological control agent with several registered products that can parasitize the eggs and larvae of various pathogenic nematodes. In this study, the pathogenicity and secondary metabolites of the fungus P. lavendulum YMF1.00683 were investigated. The strain YMF1.00683 had infection efficiency against the plant root-knot nematode Meloidogyne incognita. The strain’s process of infecting nematodes was observed under a microscope. Moreover, seven metabolites, including a new sterol (1), were isolated and identified from cultures of YMF1.0068 in Sabouraud’s dextrose agar. A bioassay showed that 5-methoxymethyl-1H-pyrrole-2-carboxaldehyde (7) is toxic to M. incognita and affects the egg hatching. It caused 98.23% mortality in M. incognita and could inhibit 80.78% of the hatching eggs at 400 μg/mL over a period of 96 h. Furthermore, 5-methoxymethyl-1H-pyrrole-2-carboxaldehyde (7) showed a strong avoidance effect at 40 ppm, and its chemotactic index value was −0.37. The results indicate that P. lavendulum could produce active metabolites against M. incognita.


Introduction
The damage caused by plant-parasitic nematodes to agriculture reaches USD157 billion every year, and the most serious damage is caused by the plant root-knot nematode belonging to the genus Meloidogyne [1,2]. Chemicals are still the most widely used pesticides in agriculture and forestry. However, long-term use of chemical nematicides may lead to the development of resistance among pathogens and may have detrimental effects on the environment and human health [3]; for example, methyl bromide is a broad-spectrum fumigant that is toxic to nematodes and has thus been banned. Currently, there is an urgent need for environmentally friendly biopesticide formulations, but their successful application is often affected by factors such as environmental conditions, storage time, and the degradation of the biocontrol strain. Therefore, one key measure to improve the effect and stability of biological pesticides is to analyze the control mechanism.
With the rapid development of whole-genome sequencing technology and informatics biology, the sequencing of numerous fungal genomes shows that most fungal metabolic biosynthetic potential has not been fully exploited. By analyzing the genomic data of P. lavendulum YMF1.00683 (which have not been released yet), it was found that this strain strain has the potential to produce abundant secondary metabolites. In general, the metabolites of microorganisms often play an important role in biological control of nematode. For example, 5-hydroxymethylfuran-2-carboxylic acid was isolated from Drechmeria coniospora and possessed nematocidal activity against M. incognita [10], and leucinostatins obtained from Paecilomyces lilacinus showed nematocidal activity [11], while other compounds such as phenolic acids and sesquiterpenoids were obtained from Paecilomyces lilacinus and exhibited nematocidal activity [11,12]. Therefore, it is necessary to carry out research on active metabolites from biocontrol potential fungus P. lavendulum YMF1.00683. In the present work, we observed the nematodes infected by P. lavendulum YMF1.00683 under a microscope and found that lethality gradually increased over time ( Figure 1) during co-cultivation. We speculate that P. lavendulum YMF1.00683 produced small molecule compounds to kill the nematodes. Therefore, in this study, the metabolites were purified from culture extracts of P. lavendulum YMF1.00683, and we tested the compounds isolated for their activity against M. incognita. The results indicated that the metabolites from P. lavendulum YMF1.00683 showed multiple activities against nematodes.

Pathogenicity of P. lavendulum YMF1.00683 against M. incognita
Many reports on fungi of the genus Purpureocillium (formerly Paecilomyces) have shown that they can control and antagonize root-knot nematodes of M. incognita [6,7]. Here we provide the microscopic observation of P. lavendulum YMF1.00683 infestations and the decomposition process of M. incognita in the laboratory. As shown in Figure 1, it can be seen that after three days of spore germination, the hyphae had overgrown the plate. At this time, approximately 50 M. incognita worms were added to each plate. After 24 h of M. incognita addition, the nematodes still had good viability. After 48 h, the vigor had been significantly reduced, and 20% of the M. incognita had died. At 72 h, 34% of M. incognita had died, and the body walls of the dead nematodes had ruptured. At 96 h, 41% of the nematodes had died, and the bodies of the dead nematodes had been degraded.

Pathogenicity of P. lavendulum YMF1.00683 against M. incognita
Many reports on fungi of the genus Purpureocillium (formerly Paecilomyces) have shown that they can control and antagonize root-knot nematodes of M. incognita [6,7]. Here we provide the microscopic observation of P. lavendulum YMF1.00683 infestations and the decomposition process of M. incognita in the laboratory. As shown in Figure 1, it can be seen that after three days of spore germination, the hyphae had overgrown the plate. At this time, approximately 50 M. incognita worms were added to each plate. After 24 h of M. incognita addition, the nematodes still had good viability. After 48 h, the vigor had been significantly reduced, and 20% of the M. incognita had died. At 72 h, 34% of M. incognita had died, and the body walls of the dead nematodes had ruptured. At 96 h, 41% of the nematodes had died, and the bodies of the dead nematodes had been degraded.

Isolation and Structural Identification of Compounds
In order to investigate the metabolites and their function of P. lavendulum YMF1.00683, a systematic isolation of the extract of the strain with different chromatographic techniques is achieved. The extract (200.5 g) from the SD solid fermentation of YMF1.00683 was Pathogens 2022, 11, 795 3 of 11 chromatographed on various columns (such as RP-18, Sephadex LH-20, and silica gel), and seven compounds (1-7) were obtained ( Figure 2). Their structures were identified by NMR and MS data.

Isolation and Structural Identification of Compounds
In order to investigate the metabolites and their function of P. lavendulum YMF1.00683, a systematic isolation of the extract of the strain with different chromatographic techniques is achieved. The extract (200.5 g) from the SD solid fermentation of YMF1.00683 was chromatographed on various columns (such as RP-18, Sephadex LH-20, and silica gel), and seven compounds (1-7) were obtained ( Figure 2). Their structures were identified by NMR and MS data. Compound 1 was obtained as a colorless solid, and its molecular formula was found to be C29H44O4 using high-resolution mass spectrometry 457.3311 ([M + H] + , Calcd. 457.3312); it contained eight unsaturation degrees. According to the nuclear magnetic resonance (NMR) spectrum data of compound 1 (Table 1), it contained 29 carbon signals, including 5 quaternary carbons, 11 methines, 7 methylenes, and 6 methyls.  Compound 1 was obtained as a colorless solid, and its molecular formula was found to be C 29 H 44 O 4 using high-resolution mass spectrometry 457.3311 ([M + H] + , Calcd. 457.3312); it contained eight unsaturation degrees. According to the nuclear magnetic resonance (NMR) spectrum data of compound 1 (Table 1), it contained 29 carbon signals, including 5 quaternary carbons, 11 methines, 7 methylenes, and 6 methyls.

Evaluation of the Nematocidal Activity of Compounds against M. incognita
As shown in Table 3, at 400 ppm, the nematocidal activities of 5-(methoxymethyl)-1H-pyrrole-2-carboxaldehyde (7) were gradually enhanced with the increase in time, and compound 7 showed strong activity at 96 h (corrected mortality rate > 90%). We found, using multiple comparisons in SPSS (Table 3), that other compounds were not significantly different at the 0.05 level during all observation periods, and they showed moderate activity (30-40%) at 96 h. Meanwhile, the adjusted mortality of avermectin was 81% at a concentration of 100 µg/mL over a period of 48 h.  (7) showed obvious activity in inhibiting egg germination and nematocidal activity against M. incognita J2. Therefore, it was selected for the analysis of its chemotactic effect on M. incognita. The results showed that when the compound's concentration was 40 ppm (Figure 3A), it exhibited a strong avoidance effect towards root-knot nematodes of M. incognita (CI < 0). When the compound's concentration decreased to 5 ppm ( Figure 3B), it showed a weaker attractive effect (0 < CI < 0.2).  (7) showed obvious activity in inhibiting egg germination and nematocidal activity against M. incognita J2. Therefore, it was selected for the analysis of its chemotactic effect on M. incognita. The results showed that when the compound's concentration was 40 ppm (Figure 3A), it exhibited a strong avoidance effect towards root-knot nematodes of M. incognita (CI < 0). When the compound's concentration decreased to 5 ppm ( Figure 3B), it showed a weaker attractive effect (0 < CI < 0.2).

Discussion
Root-knot disease caused by M. incognita is a serious concern because it affects several economically important crops globally. Researchers found that Paecilomyces lilacinus (currently valid name: Purpureocillium lilacinum) showed a parasitic effect on root-knot nematodes [6][7][8]. Several experts have conducted numerous studies on the fungus and have confirmed that Paecilomyces lilacinus has control effects on various plant-parasitic nematodes [3,21]. Paecilomyces lilacinus can parasitize the eggs and larvae of important pathogenic nematodes, including root-knot nematodes and cyst nematodes. Paecilomyces lilacinus was subsequently found to disrupt the lipid and chitin layers of nematode eggshells [22]. Additionally, Paecilomyces lilacinus can also produce a variety of biologically active secondary metabolites, including polyketides and non-ribosomally synthesized peptides, such as leucinostatins, which are nematocidal, antiviral, and phytotoxic and possess a series of biological activities [11,23,24]. By comparing the 18S rRNA gene, internal transcribed spacer, and partial translation elongation factor 1-a of the isolates of P. lilacinus from the environment and organisms, the genus Purpureocillium was proposed [4], which includes P. lilacinum, P. lavendulum, and P. takamizusanense [4,25].
While studying the infection of M. incognita by P. lavendulum, a certain percentage of nematodes died gradually over time (Figure 1) after the co-cultivation of Pur. lavendulum with M. incognita. Through the isolation, purification, and activity assaying of the compounds from the solid fermentation medium, two compounds showed activity against M. incognita. Among them, 7,8-dimethylalloxazine (6) exhibited weak egg hatching inhibitory activity; this is the main photodegradation product of riboflavin under neutral or acidic conditions and is known as an effective photosensitizer [26][27][28]. Some microorganisms and plants also produce 7,8-dimethylalloxazine [29,30] and show various types of activity: 7,8-dimethylalloxazine was isolated from culture filtrates of Chlamydomonas as a quorumsensing signal-mimicking compound capable of activating Pseudomonas aeruginosa LasR receptor [31], and it was also purified and identified from a culture of the bacterium Sinorhizobium meliloti and found to be responsible for stimulating root respiration and plant

Discussion
Root-knot disease caused by M. incognita is a serious concern because it affects several economically important crops globally. Researchers found that Paecilomyces lilacinus (currently valid name: Purpureocillium lilacinum) showed a parasitic effect on root-knot nematodes [6][7][8]. Several experts have conducted numerous studies on the fungus and have confirmed that Paecilomyces lilacinus has control effects on various plant-parasitic nematodes [3,21]. Paecilomyces lilacinus can parasitize the eggs and larvae of important pathogenic nematodes, including root-knot nematodes and cyst nematodes. Paecilomyces lilacinus was subsequently found to disrupt the lipid and chitin layers of nematode eggshells [22]. Additionally, Paecilomyces lilacinus can also produce a variety of biologically active secondary metabolites, including polyketides and non-ribosomally synthesized peptides, such as leucinostatins, which are nematocidal, antiviral, and phytotoxic and possess a series of biological activities [11,23,24]. By comparing the 18S rRNA gene, internal transcribed spacer, and partial translation elongation factor 1-a of the isolates of P. lilacinus from the environment and organisms, the genus Purpureocillium was proposed [4], which includes P. lilacinum, P. lavendulum, and P. takamizusanense [4,25].
While studying the infection of M. incognita by P. lavendulum, a certain percentage of nematodes died gradually over time (Figure 1) after the co-cultivation of Pur. lavendulum with M. incognita. Through the isolation, purification, and activity assaying of the compounds from the solid fermentation medium, two compounds showed activity against M. incognita. Among them, 7,8-dimethylalloxazine (6) exhibited weak egg hatching inhibitory activity; this is the main photodegradation product of riboflavin under neutral or acidic conditions and is known as an effective photosensitizer [26][27][28]. Some microorganisms and plants also produce 7,8-dimethylalloxazine [29,30] and show various types of activity: 7,8-dimethylalloxazine was isolated from culture filtrates of Chlamydomonas as a quorum-sensing signal-mimicking compound capable of activating Pseudomonas aeruginosa LasR receptor [31], and it was also purified and identified from a culture of the bacterium Sinorhizobium meliloti and found to be responsible for stimulating root respiration and plant growth. Moreover, 5-methoxymethyl-1H-pyrrole-2-carboxaldehyde (7), which was isolated from plants and other fungi [32,33], showed multiple activities in the present work: at a low concentration of 5 ppm, the metabolite showed a certain attraction to M. incognita, while at 40 ppm, it had a strong avoidance effect. Lastly, this compound showed nematocidal activity and inhibited egg hatching activity in M. incognita at a concentration of 400 ppm.

Materials
Optical rotations were measured with a Jasco DIP-370 digital polarimeter (JASCO, Tokyo, Japan). Ultraviolet (UV) spectra were recorded on a Shimadzu UV-2401PC spectrophotometer. The nuclear magnetic resonance (NMR) spectra were recorded on Avance III-600 spectrometers, with tetramethylsilane (TMS) as an internal standard. The electrospray ionization mass spectra (ESI-MS) and high-resolution electrospray ionization mass spectra (HR-ESI-MS) were recorded on a high-resolution Thermo Q Exactive Focus mass spectrometer (Thermo Fisher Scientific, Bremen, Germany).
P. lavendulum YMF1.00683 was isolated from a soil sample in Yunnan. After 8 days of growth on PDA at 28 • C, the hyphae of P. lavendulum YMF1.00683 have covered 6 cm plates, and it is no growth at 35 • C. Colonies consist of dense basal mats composed of many conidiophores and sparse aerial hyphae, and they fold radially toward the periphery. Conidiophores grow from the mycelium 3-4 days at 28 • C, are long stem chains, unicellular, subglobose, apically or lemon-colored at the base, and the clumps are pinkish-purple [34]. P. lavendulum YMF1.00683 was stored in glycerol at −80 • C at the State Key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan, China. The YMF1.00683 strain was inoculated in PDA medium and incubated at 28 • C for 6 days, and then was transferred into a fresh medium for 5 days as a seed strain. Meloidogyne incognita was obtained from the roots of tomatoes grown in E'shan County in Yunnan Province.

4.2.
Infection of M. incognita by P. lavendulum P. lavendulum grown on PDA plates for seven days, was harvested by cutting the sample into pieces, shaking it at 100 RPM in 0.1% Tween−80 for 30 min, and filtering it through sterilized four-layer filter paper to obtain a spore suspension. The spores were counted, and their concentration was adjusted to 10 6 mL −1 . A 200 µL aliquot of the spore suspension was dispersed on a cellophane-covered water agar plate, which was then cultured at 28 • C for three days to germinate spores. M. incognita was washed with sterile ddH 2 O, and then approximately 50 M. incognita worms were added to the germinated spore plates. The infection process and number of dead nematodes were observed and recorded at 24, 48, 72, and 96 h.

Extraction and Separation of Metabolites
The mycelium of Pur. lavendulum was cultivated in SD solid medium (10 g tryptone, 40 g glucose, 15 g agar, 1 L water) for 21 days at 28 • C. The culture (30 L) was cut into small pieces and extracted by EtOAc/MeOH/AcOH (80:15:5, v/v/v) 3 times, and then concentrated under reduced pressure to obtain a crude extract. The crude extract was dissolved with 2.5 L of pure water, extracted with EtOAc to obtain the EtOAc crude extract (100.2 g), and then extracted with n-butanol to obtain the n-butanol extract (100.3 g). After LC-MS analysis, both extracts were combined.
The whole crude extract (200.5 g) was placed on a reversed-phase silica gel column    Assays were performed in 24-well cell culture plates. M. incognita egg acquisition [10]: oocysts were picked from the root knots of susceptible tomato and washed of impurities with ddH 2 O, shaken with 0.5% NaClO for 3 min, and then filtered with a 500-mesh sieve to remove NaClO, rinsed with sterile water 3 times, shaken with 2% NaClO solution for 3 min to obtain eggs, and subjected to 10 µL microscopic examinations after completion to prevent excessive cracking. For the second stage juveniles of M. incognita (J2), the obtained eggs were hatched in a 25 • C incubator, and M. incognita (J2) were collected for later use.

Spectroscopic Data
The experimental operation of the effect on the egg hatching of M. incognita [35]: we used sterile water to prepare the egg solution; we prepared a compound solution with methanol sterile water, and the final concentration of methanol was 1%; egg-containing solution and compound solution were added to each well for a final total volume of 500 µL, containing approximately 150 eggs, and the tested compound working concentration was 400 ppm. Sterile water containing 1% methanol was used as a control, and each treatment was repeated thrice.

Assay of Nematocidal Activity against M. incognita
The tested compounds were dispersed in MeOH. Two hundred M. incognita J2s (100 µL) were added to each sample, and the final concentration of the tested compounds was set at 400 ppm. The total and dead nematode numbers were determined every 24 h [36]. The worms were considered dead if they were flat or cracked, and then the nematode mortality was calculated. According to the nematocidal activity strength classification standard, the five-level grading criteria are as follows: inactive, adjusted mortality rate ≤10%; weak activity, adjusted mortality rate of 10% to 30%; moderate activity, adjusted mortality rate of 30% to 70%; strong activity, adjusted mortality > 70%. Avermectin was used as a positive control; the test solution without compound was used as a negative control. Three replicates were conducted for each test.

Assay of Chemotaxis Activity against M. incognita
The nematode chemotaxis assay was conducted for compound 7 using the fourpoint plate method [37]. The 90 mm plate contained 1% agarose for the chemotaxis assay. Compound 7's working concentration was set to 40, 20, 10, 5, 1, 0.5, and 0.01 ppm using 1% methanol sterile water, and sterile water containing 1% methanol was used as a control.