Secondary Metabolites from the Endoparasitic Nematophagous Fungus Harposporium anguillulae YMF 1.01751

Harposporium anguillulae, an endoparasitic nematophagous fungus (ENF), is a model fungus from which the genus Harposporium was established. It can infect nematodes via ingested conidia. In this paper, the morphology and nematode–fungus interaction between Panagrellus redivivus and H. anguillulae were observed by scanning electron microscopy (SEM). The secondary metabolites of H. anguillulae were also studied. Seven metabolites were purified and identified from an ethyl acetate extract of broth and a methanol extract of mycelium. These include a new polyketone 5-hydroxy-3-(hydroxymethyl)-6-methyl-2H-pyran-2-one (1) and six known metabolites (17R)-17-methylincisterol (2), eburicol (3), ergosterol peroxide (4), terpendole C (5), (3β,5α,9β,22E)-3,5-dihydroxy-ergosta-7,22-dien-6-one (6), and 5α,6β-epoxy-(22E,24R)-ergosta-8,22-diene- 3β,7α-diol (7). These metabolites were assayed for their activity against plant root-knot nematode, Meloidogyne incognita, and the results showed that terpendole C (5) had weak nematicidal activity but also that other compounds did not have evident activity at a concentration of 400 μg mL−1. Compound 1 exhibited an attractive effect towards P. redivivus.


Introduction
The diseases caused by plant parasitic nematodes lead to huge economic losses to crops every year [1]. Traditional chemical pesticides to control nematode diseases have irreversible damaged the ecological environment, so biological control has gradually become a hot topic of research. Nematophagous fungi play an important role as the main source of nematode biological control management [2]. Nematophagous fungi include trapping fungi, endoparasitic nematophagous fungi (ENF), opportunistic fungi, and toxic fungi [3]. Among them, ENF are a type of nematophagous fungi that can parasitize nematodes through special spores.
Fungi of the genus Harposporium are a class of endoparasitic nematophagous fungi that can produce conidia to parasitize nematodes, and their unique spore morphology provides more possibilities for them to exhibit nematicidal effects [4]. Harposporium was established by Lohde (1874) [5], and the genus is an important group of endoparasitic nematophagous fungi. Harposporium species are widely distributed and can be isolated from soil and substrate nematodes [6,7]. Up to now, 31 species of Harposporium have been reported all over the world [8]. In addition to producing infection conidia, some species can also produce arthroconidia, chlamydospores, and accessory conidia [9]. In studies of Harposporium metabolites, only one paper has reported a new furan, harposporin A, along with a known aureonitol that was found in Harposporium sp. YMF 1.01735 [10].
H. anguillulae was the first fungus described as a parasite of nematodes. It is also the model fungus from which the genus Harposporium was established. It can infect nematodes by producing abundant ingested conidia. The process from ingesting conidia to 2 of 9 infecting nematodes is divided into three processes: ingestion, germination, and penetration. H. anguillulae can form spores and chlamydospores in water-agar petri dishes. In an investigation of phospholipids on growth and sporulation of H. anguillulae, the result showed that the mycelial growth and sporulation of the species was not affected by the addition of phospholipids [11]. H. anguillulae can significantly reduce the free living stages of trichostrongylid nematodes, indicating that the species could be a candidate for the development of a biocontrol agent for trichostrongylid nematodes [12].
In the present investigation, research on H. anguillulae mainly focuses on strain isolation, identification, and pathogenicity to nematodes, in addition to studying secondary metabolites, which have not been reported for H. anguillulae. In this study, the strain H. anguillulae YMF 1.01751 was selected as the research object for its morphology and secondary metabolites, in which seven compounds, including one new polyketone, were identified.

Normal Materials
H. anguillulae YMF 1.01751 was isolated from soil in Yunnan Province, China, and is now deposited in the microbial library of the germplasm bank of wild species from Southwest China, Yunnan University, Kunming, China. It was stored in glycerol at -80 • C.
The plant root-knot nematode Meloidogyne incognita was obtained from the roots of tomatoes grown in E'shan County in Yunnan Province. The method of obtaining M. incognita is referred to in the literature [13].
Panagrellus redivivus: An appropriate amount of oat (25 g) and water (60 mL) was added to a 250 mL conical flask for sterilization. After cooling, an appropriate amount of P. redivivus seeds were picked and inoculated into oat medium and cultured at 25 • C for one week. The culture and preparation of P. redivivus is referred to in the literature [14].
2.3. The Morphology and Pathogenicity against Nematodes of H. anguillulae YMF 1.01751 Observed by Scanning Electron Microscopy H. anguillulae YMF 1.01751 was cultured on improved potato dextrose agar (PDA) (200 g potato, 20 g glucose, 3 g KH 2 PO 4 , 1.5 g MgSO 4 , 5 g yeast extract, 2 g peptone, and 15 g agar) plates for 14 days. P. redivivus was cultured in oats for about 7 days, and the oats containing nematodes were placed into three layers of sterilized lens paper, which were then placed into sterile water to allow the nematodes separate from the oats. The nematodes were prepared as a suspension in water corresponding to about 100 nematodes/10 µL.
After growth of H. anguillulae YMF 1.01751 on improved PDA plates for 14 days, 20 µL P. redivivus suspension was added to the cultured strains for interaction. Samples were taken after 12, 24, 36, 48, 60, and 72 h of nematode-fungus coculture on plates. These samples were cut into small pieces and fixed for 30 min in 4% glutaraldehyde, then dehydrated with 30%, 50%, 70%, 80%, and 90% ethanol for 15 min each, respectively, and then dehydrated twice with 100% ethanol for 15 min each time, then, after that, in ethanol/isoamyl acetate (1:1, v/v) and in 100% isoamyl acetate liquor for 10 min, respectively. The samples were freeze-dried, gilded, and observed by scanning electron microscopy (SEM) [15].

Nematicidal and Chemotaxis Activity of Metabolites
The tested compounds (1, 2, 3, and 5) were dissolved in methanol and diluted with sterile water. Approximately 200 M. incognita nematodes J2s were added to each plate, and the final concentration of compounds was 400 µg mL −1 . M. incognita was considered dead when no movement occurred after touching it with a needle [16]. The mortality was calculated at different times, and the experiment was repeated 3 times.
The nematode chemotaxis assay for compounds 1, 2, 3, and 5 was conducted using the four-point plate method [17]. The compounds' concentrations were set to 4, 8, 16, and 32 µg. Approximately 200 worms were added to the center of the petri dish. Compounds and control (methanol) were added. After 2 h at 25 • C, the worms were counted under the microscope, and the chemotaxis index (CI) was calculated.

Scanning Electron Microscopy Observation of Interactions between H. anguillulae YMF 1.01751 and P. redivivus
After growth on the improved PDA medium for two weeks at 28 • C, the hyphae of H. anguillulae YMF 1.01751 had overspread 6 cm on the plate. The mycelia of H. anguillulae YMF 1.01751 were septate and colorless. SEM results show that the phialides (sporogenous cell) of H. anguillulae YMF 1.01751 were globose or flask-shaped, and there was a conidial peduncle on the tip of phialides. Conidia were colorless, curved, bow-shaped, and had hooked ends ( Figure 1A-D).

Scanning Electron Microscopy Observation of Interactions between H. anguillulae YMF 1.01751 and P. redivivus
After growth on the improved PDA medium for two weeks at 28 °C, the hyphae of H. anguillulae YMF 1.01751 had overspread 6 cm on the plate. The mycelia of H. anguillulae YMF 1.01751 were septate and colorless. SEM results show that the phialides (sporogenous cell) of H. anguillulae YMF 1.01751 were globose or flask-shaped, and there was a conidial peduncle on the tip of phialides. Conidia were colorless, curved, bow-shaped, and had hooked ends ( Figure 1A-D).
H. anguillulae produces ingested conidia to infect nematodes. While studying the infection of P. redivivus by H. anguillulae YMF 1.01751, approximately 200 worms were added to each plate. In the process of infesting nematodes, the conidia were swallowed by nematodes, after which the spores underwent germination to produce hyphae, which penetrated the body wall of the nematodes and stuck there for further growth to produce new conidia again. If nematodes were present, a new round of the infestation process could be carried out. Endoparasitic nematophagous fungi can infect nematodes by producing adhesive conidia or ingested conidia. Adhesive conidia were specifically identified and adhered to the body wall, head, and genital pore of the nematode, then grew and reproduced using nematodes. Previous studies have shown that treating the nematode epidermis with pronease E can cause the conidia to fail to adsorb, which leads to the inference that H. anguillulae produces ingested conidia to infect nematodes. While studying the infection of P. redivivus by H. anguillulae YMF 1.01751, approximately 200 worms were added to each plate. In the process of infesting nematodes, the conidia were swallowed by nematodes, after which the spores underwent germination to produce hyphae, which penetrated the body wall of the nematodes and stuck there for further growth to produce new conidia again. If nematodes were present, a new round of the infestation process could be carried out.
Endoparasitic nematophagous fungi can infect nematodes by producing adhesive conidia or ingested conidia. Adhesive conidia were specifically identified and adhered to the body wall, head, and genital pore of the nematode, then grew and reproduced using nematodes. Previous studies have shown that treating the nematode epidermis with pronease E can cause the conidia to fail to adsorb, which leads to the inference that nematode surface proteins play a key role in the inter action process of fungi and nematodes. Similarly, trypsin treatment for conidia eventually resulted in a reduction in the ability of the conidia to adsorb onto nematodes. It has been speculated that the interaction process of nematode and adhesive conidia is a protein-with-protein process of interaction [18]. The process by which ingested conidia infect nematodes is the same as the interaction between H. anguillulae YMF 1.01751 and nematodes, which is divided into swallowing conidia, parasitism, germination, and penetration. This interaction begins when the nematode ingests conidia [9]. In addition to being swallowed, some spores will attach to the surface of the nematode (Figure 2A). The conidia began to germinate and the hyphae grew through nematode body about 48 h after being swallowed ( Figure 2B), and some nematode had already begun to die. Subsequently, a lot of hyphae would continue to grow through the body wall ( Figure 2C,D) until the nematode mass was finally surrounded ( Figure 2E,F), and new conidia were then produced. A new round of infestation was performed in the case of nematodes.
todes. Similarly, trypsin treatment for conidia eventually resulted in a reduction in the ability of the conidia to adsorb onto nematodes. It has been speculated that the interaction process of nematode and adhesive conidia is a protein-with-protein process of interaction [18]. The process by which ingested conidia infect nematodes is the same as the interaction between H. anguillulae YMF 1.01751 and nematodes, which is divided into swallowing conidia, parasitism, germination, and penetration. This interaction begins when the nematode ingests conidia [9]. In addition to being swallowed, some spores will attach to the surface of the nematode (Figure 2A). The conidia began to germinate and the hyphae grew through nematode body about 48 h after being swallowed ( Figure 2B), and some nematode had already begun to die. Subsequently, a lot of hyphae would continue to grow through the body wall ( Figure 2C-D) until the nematode mass was finally surrounded ( Figure 2E-F), and new conidia were then produced. A new round of infestation was performed in the case of nematodes.

Structure Identification
Seven compounds (1-7) were isolated from an ethyl acetate extract of broth and a methanol extract of the mycelium of H. anguillulae YMF 1.01751. Their structures were identified by nulear magnetic resonance (NMR) and mass spectrometry (MS) data.
Compound 1 was obtained as colorless solid. The molecular formula of compound 1 is C7H8O4 (157.0494 [M + H] + , Calcd. 157.0495), which was calculated with positive ion mode high-resolution mass spectrometry. It contains four unsaturation degrees. According to 13 C-nulear magnetic resonance and distortionless enhancement by polarization transfer (DEPT) ( Table 1)

Structure Identification
Seven compounds (1-7) were isolated from an ethyl acetate extract of broth and a methanol extract of the mycelium of H. anguillulae YMF 1.01751. Their structures were identified by nulear magnetic resonance (NMR) and mass spectrometry (MS) data.

The Nematicidal and Chemotaxis Activity of Compounds
Compounds 1, 2, 3, and 5 were tested for their nematicial activity against root-knot nematode M. incognita. The results showed that terpendole C (5) caused 37.4% mortality of M. incognita at 400 μg mL −1 during 48 h. Other compounds have no evident nematicidal activity at the same concentration. Terpendole C (5) is an indole diterpene. This type of compound has been reported to have nematicidal activity. A similar compound, gymnoascole acetate, was obtained from fungus Gymnoascus reessii za-30 and caused 100% paralysis of M. incognita at 36 μg mL −1 for 24 h [25].
In the chemotaxis assay, the new polyketone 1 exhibited an attractive effect towards P. redivivus. With the concentration increased (4, 8, 16 μg), the attraction activity of compound 1 was enhanced. At 16 μg, it showed a chemotaxis index of 0.21 after 2 h. The attractive effect of compound 1 decreased with increasing concentration (32 μg) after 2 h (Figure 4). The other tested metabolites (1, 2, and 3,) displayed no pronounced activity.

The Nematicidal and Chemotaxis Activity of Compounds
Compounds 1, 2, 3, and 5 were tested for their nematicial activity against root-knot nematode M. incognita. The results showed that terpendole C (5) caused 37.4% mortality of M. incognita at 400 µg mL −1 during 48 h. Other compounds have no evident nematicidal activity at the same concentration. Terpendole C (5) is an indole diterpene. This type of compound has been reported to have nematicidal activity. A similar compound, gymnoascole acetate, was obtained from fungus Gymnoascus reessii za-30 and caused 100% paralysis of M. incognita at 36 µg mL −1 for 24 h [25].
In the chemotaxis assay, the new polyketone 1 exhibited an attractive effect towards P. redivivus. With the concentration increased (4, 8, 16 µg), the attraction activity of compound 1 was enhanced. At 16 µg, it showed a chemotaxis index of 0.21 after 2 h. The attractive effect of compound 1 decreased with increasing concentration (32 µg) after 2 h (Figure 4). The other tested metabolites (1, 2, and 3,) displayed no pronounced activity.

Discussion
In this study, the morphology and pathogenicity of H. anguillulae YMF 1.01751 was observed by scanning electron microscopy. It produces abundant crescent-shaped conidia. This kind of long and curved spore is characteristic of Harposporium. Most Herposporiums infect nematodes by ingested spores that germinate inside the nematode and produce septate and branching hyphae that absorb the nutritious of the nematode. At last, the hyphae break out through the nematode cuticle [26].
Other structures of the known metabolites are triterpene, indole diterpene, and steroid. (17R)-17-Methylincisterol (2) was identified from Dictyonella incisa for the first time as a new natural product. It is believed that the biosynthesis pathway of this compound might begin with the unique oxidation process of cholestatrien-3β-ols in vivo [15]. In addition, the compound can also be obtained from the fungus Aspergillus terreus TZS-201607 and has certain cytotoxic activity against A549, MCF-7, and THP-1 with IC50 values of 48.9, 16.8, and 23.6 μM, respectively [28]. Compounds 4, 6, and 7 are sterols. Steroids are a kind of important natural organic compounds that are widespread in biological tissues. With cyclopentane polyhydrophenanthrene as the mother nucleus, steroids can be divided into many types according to their substituents, double-bond positions or stereoconfigurations. Steroids have a variety of biological activities because of their structural specificity, such as antitumor, anti-inflammatory, antibacterial, antiviral, and enzyme-inhibitory activities [29]. Compound 6 showed no activity in an experiment evaluating neutrophil elastase (HNE) activity [30]. Compound 7 exists in a variety of fungi and has moderate cytotoxic activity on tumor cells such as A549 (non-small cell lung adenocarcinoma), SK-OV-3 (ovarian cancer), SK-MEL-2 (skin melanoma), XF498 (central nervous system), and HCT15 (colon) cultured in vitro. In addition, compound 7 can inhibit the growth of the hypocotyl of lettuce and promote the growth of lettuce root at the same concentration [31].
Terpendole C (5) is an indole diterpene, and many indole diterpenoids have been reported to be metabolites of fungi. Terpendole C (5) showed acyl-CoA: cholesterol acyltransferase (ACAT)-inhibitory activity, with an IC50 value of 2.1 μM, in an vitro assay [32]. Terpendole C (5) can specifically inhibit the synthesis of cholesterol esters in peri-

Discussion
In this study, the morphology and pathogenicity of H. anguillulae YMF 1.01751 was observed by scanning electron microscopy. It produces abundant crescent-shaped conidia. This kind of long and curved spore is characteristic of Harposporium. Most Herposporiums infect nematodes by ingested spores that germinate inside the nematode and produce septate and branching hyphae that absorb the nutritious of the nematode. At last, the hyphae break out through the nematode cuticle [26].
Other structures of the known metabolites are triterpene, indole diterpene, and steroid. (17R)-17-Methylincisterol (2) was identified from Dictyonella incisa for the first time as a new natural product. It is believed that the biosynthesis pathway of this compound might begin with the unique oxidation process of cholestatrien-3β-ols in vivo [15]. In addition, the compound can also be obtained from the fungus Aspergillus terreus TZS-201607 and has certain cytotoxic activity against A549, MCF-7, and THP-1 with IC 50 values of 48.9, 16.8, and 23.6 µM, respectively [28]. Compounds 4, 6, and 7 are sterols. Steroids are a kind of important natural organic compounds that are widespread in biological tissues. With cyclopentane polyhydrophenanthrene as the mother nucleus, steroids can be divided into many types according to their substituents, double-bond positions or stereoconfigurations. Steroids have a variety of biological activities because of their structural specificity, such as antitumor, anti-inflammatory, antibacterial, antiviral, and enzyme-inhibitory activities [29]. Compound 6 showed no activity in an experiment evaluating neutrophil elastase (HNE) activity [30]. Compound 7 exists in a variety of fungi and has moderate cytotoxic activity on tumor cells such as A549 (non-small cell lung adenocarcinoma), SK-OV-3 (ovarian cancer), SK-MEL-2 (skin melanoma), XF498 (central nervous system), and HCT15 (colon) cultured in vitro. In addition, compound 7 can inhibit the growth of the hypocotyl of lettuce and promote the growth of lettuce root at the same concentration [31].
Terpendole C (5) is an indole diterpene, and many indole diterpenoids have been reported to be metabolites of fungi. Terpendole C (5) showed acyl-CoA: cholesterol acyltransferase (ACAT)-inhibitory activity, with an IC 50 value of 2.1 µM, in an vitro assay [32]. Terpendole C (5) can specifically inhibit the synthesis of cholesterol esters in peritoneal macrophages by inhibiting cholesterol acyltransferase activity [33]. In addition to enzymeinhibitory activity, the compound also has cytotoxic activity and antibacterial activity. It has a cytotoxic effect on NCI-H187 and Vero cells, with IC 50 values of 35.69 µM and 23.56 µM, respectively, and it has anti-Mycobacterium tuberculosis H37Ra activity, with an MIC value of 25.00 µg mL −1 [34]. According to reports, some indole diterpenes have nematicidal activity. For example, gymnoascole acetate, from G. reessii za-30, has activity against M. incognita, with an EC 50 value of 47.5 µg mL −1 for 24 h [25]. This type of metabolite may be a potential biocontrol resource for nematodes. In this study, compound 5 showed weak activity against nematodes. In the isolation process of metabolites from H. anguillulae YMF 1.01751, some other indole diterpenes had not been identified due to their presence in minor amounts. These compounds that have not been isolated may be potential active substances. Therefore, the metabolites of H. anguillulae deserve further study.
H. anguillulae is a typical endoparasitic fungus of nematodes. It infects nematodes by producing ingested conidia. This is the first report on the metabolites of the species. The results state that terpendole C (5) has weak nematicidal activity against root-knot nematode M. incognita, and polyketide 1 exhibites an attractive effect towards the nematode. This result indicated that some secondary metabolites were involved in the pathogenicity process of H. anguillulae infecting nematodes.