Secondary Metabolites from the Nematode-Trapping Fungus Dactylellina haptotyla YMF1.03409

As a representative nematode-trapping fungus, Dactylellina haptotyla can capture and kill nematodes by producing traps, known as adhesive knobs. In this paper, the strain of D. haptotyla YMF1.03409 was studied by means of medium screening, fermentation, and purification and identification of crude extracts. Eighteen compounds were obtained from D. haptotyla YMF1.03409, including two new metabolites, nosporins C (1) and D (2). The known metabolites were identified to be 3-chloro-4-methoxybenzaldehyde (3), 3-chloro-4-methoxybenzoic acid (4), 2-chloro-1-methoxy-4-(methoxymethyl)benzene (5), 3-hydroxy-3-methyloxindole (6), nicotinic acid (7), succinic acid (8), 3,4-dihydroxybutanoic acid (9), 5′-O-methyladenosine (10), uridine (11), 2′-deoxyuridine (12), thymidine (13), 3-(phenylmethyl)-2,5-morpholinedione (14), methyl-β-D-glucopyranoside (15), 1,2-benzenedicarboxylic acid bis(2-methyl heptyl) ester (16), β-sitosterol (17), and 3β,6α-diol-stigmastane (18). The bioactive assay showed that these compounds had no obvious nematicidal activity against the nematodes Meloidogyne incognita and Panagrellus redivivus.


Introduction
Plant pathogenic nematodes can wreak havoc on crop productivity [1].Root-knot nematodes are the most prominent pathogenic nematodes, which have numerous hosts and a wide distribution.Their quick reproduction, environmental adaptability, and ease of dissemination make them challenging to control.The current methods for controlling plant pathogenic nematodes mainly include chemical control, agricultural control, and biological control.Chemical control has the advantages of short cycle and quick effect, and plays an important role in ensuring a high and stable yield of crops [2].Despite their efficiency, chemical pesticides can easily contaminate the environment.Pesticide residues that are left on agricultural products have the potential to harm soil structure, create soil erosion, and pose a concern to food safety [3].Agricultural control conquers nematode infestation mainly via crop rotation, flooding, and culturing anti-nematode plants.These methods may be time-consuming and incomplete in their control of pathogenic nematodes [4].
Due to the aforementioned disadvantages, biological control has been widely emphasized in recent years.Research on the development of biological control agents using nematophagous microbial resources, such as nematode-trapping fungi, has emerged as a popular topic in the control of nematodes [3].Nematode-trapping fungi constitute a specialized group of fungi that can catch and kill nematodes by producing traps [5].Dactylellina haptotyla is a canonical model of nematode-trapping fungi, and current research on the species has mainly focused on the physiological mechanisms underlying how it controls nematodes, but research on its secondary metabolites is rarely reported [6][7][8].In the present study, eighteen compounds were obtained from the strain D. haptotyla YMF1.03409, and their structural types were found to mainly include polyketides, aromatics, and nucleosides.

Screening of Culture Conditions
D. haptotyla YMF1.03409 was inoculated in the nine media listed in Table 1.The culture volume was 300 mL, and the solid-state culture conditions (#5, #6, #7, #8, and #9) were 28 • C for 31 days.The liquid-state culture conditions (#1, #2, #3, and #4) were 28 • C at 180 rpm for 14 days.After the completion of the culture, the fermentation products were processed as follows: the liquid culture products were filtered through eight layers of gauze to remove mycelia; equivalent amounts of ethyl acetate were used to extract the fermentation broth; and the extracts were combined and evaporated under reduced pressure on a rotary evaporator to obtain the crude extracts.The solid culture products were pounded into small pieces and extracted using ethyl acetate/methanol/glacial acetic acid (80:15:5, v/v/v) for three, two, and one days, respectively, and finally, the soaking solutions were combined and evaporated under reduced pressure to obtain the crude extracts.These crude extracts were then weighted.

Fermentation and Isolation of Compounds
Medium #9 was selected to culture D. haptotyla YMF1.03409 at 28 • C for 31 days in a total volume of 40 L. The solid fermentation products were cut into small pieces and extracted exhaustively using the mixture solution (ethyl acetate/methanol/acetic acid = 80:15:5, v/v/v) six times.The filtrate was evaporated using a rotary evaporator under reduced pressure to obtain the crude extracts (35.64 g).

Nematicidal Activity of Compounds
The nematicidal activity test consisted of the following steps: (1) the compounds were dissolved in a methanol-water solution that contained 3% methanol, and the concentration of the compounds was measured at 400 ppm.As a control, the methanol-water solution that contained 3% methanol was used.(2) About 150 juveniles (J2) of M. incognita or P. redivivus were transferred to 3.5 cm Petri dishes containing either the compound solution or the control solution.(3) The assay was performed in triplicate, and each replicate was performed with three Petri dishes.The numbers of dead and live nematodes were counted after 12, 24, and 48 h using a light microscope (Olympus, Tokyo, Japan) [9].

Culture and Fermentation of D. haptotyla YMF1.03409
D. haptotyla is one of the representative nematode-trapping fungi (Figure 1A), which belongs to the genus Dactylellina of the family Orbiliaceae (Ascomycota).Its conidiospores are fusiform or teardrop-shaped (Figure 1B,C).Its trap is an adhesive knob, which is ovoid and grows on vegetative mycelia (Figure 1D,E).

Nematicidal Activity of Compounds
The nematicidal activity test consisted of the following steps: (1) the compounds were dissolved in a methanol-water solution that contained 3% methanol, and the concentration of the compounds was measured at 400 ppm.As a control, the methanolwater solution that contained 3% methanol was used.(2) About 150 juveniles (J2) of M. incognita or P. redivivus were transferred to 3.5 cm Petri dishes containing either the compound solution or the control solution.(3) The assay was performed in triplicate, and each replicate was performed with three Petri dishes.The numbers of dead and live nematodes were counted after 12, 24, and 48 h using a light microscope (Olympus, Tokyo, Japan) [9].

Culture and Fermentation of D. haptotyla YMF1.03409
D. haptotyla is one of the representative nematode-trapping fungi (Figure 1A), which belongs to the genus Dactylellina of the family Orbiliaceae (Ascomycota).Its conidiospores are fusiform or teardrop-shaped (Figure 1B,C).Its trap is an adhesive knob, which is ovoid and grows on vegetative mycelia (Figure 1D,E).The optimal fermentation conditions were determined based on the mass of crude extracts.Firstly, by comparing the mass of crude extracts under different conditions, it was found that when rice was included in the media's composition (#5, #6, #7, #8, and #9), the mass of crude extracts under the corresponding culture conditions was significantly higher than when rice was not included (#1, #2, #3, and #4).Detailed information on the mass of crude extracts under the corresponding culture conditions is shown in Figure 2.
The optimal fermentation conditions were determined based on the mass of crude extracts.Firstly, by comparing the mass of crude extracts under different conditions, it was found that when rice was included in the media's composition (#5, #6, #7, #8, and #9), the mass of crude extracts under the corresponding culture conditions was significantly higher than when rice was not included (#1, #2, #3, and #4).Detailed information on the mass of crude extracts under the corresponding culture conditions is shown in Figure 2.Among the media #5, #6, #7, #8, and #9, medium #9 showed the highest mass of crude extracts; thus, medium #9 was finally determined as the amplified fermentation medium, and the culture condition was 28 °C for 31 days of static incubation.

Structural Identification of Compounds
Medium #9 was selected to culture D. haptotyla YMF1.03409.Eighteen compounds were isolated from the crude extracts.Their structures were identified based on the obtained NMR and MS data.An analysis of the HR-ESI-MS data revealed a molecular formula of C10H12O6 based on the [M + Na] + ion signal at m/z 251.0524 (calcd.for C10H12O6Na, 251.0526).The spectroscopic data (Table 2) of compound 1 are basically the same as those of nosporin A, except that the methyl group at 9-OH is changed to a formate group in compound 1 [10].
Compound 18: Colorless oil, the molecular formula of compound

Nematicidal Activity of Compounds
Compounds 1, 2, 4, 5, 6, 8, and 9 were tested for their nematocidal activity against M. incognita and P. redivivus.The results showed that all seven compounds caused less than 15% nematode mortality at 48 h (Figure S1) when the concentration of tested compounds was 400 ppm, and also did not show significant differences compared to the control.

Discussion
Nosporin C (1) and nosporin D (2) are newly discovered metabolites in this study, and their structure types are polyketides.In previous research, nosporins A and B, which are the structural analogues of nosporins C (1) and D (2), were isolated from the filamentous fungus VKM-3750, and possessed cytotoxic effects on the sea urchin Strongylocentrotus intermedius and antibacterial effects on Gram-positive bacilli [10].
3-Chloro-4-methoxybenzaldehyde (3) was obtained from the white-rot basidiomycete Pleurotus ostreatus.4-Methoxybenzaldehyde is the structural analog of 3, which has one less chlorine substituent group than 3 and has efficacy against Bacillus subtilis, Pseudomonas aeruginosa, Aspergillus niger, and Fusarium oxysporum [26].This compound was also discovered in Anthracophyllum discolor, showing antibacterial activity [27].3-Chloro-4-methoxybenzoic acid (4) was isolated from Bjerkandera adusta, which can promote the activity of two key protein degradation systems in human foreskin fibroblasts, the autophagy-lysosomal pathway (ALP), and the ubiquitin-proteasome pathway (UPP).It is important in the development of new regulators of the proteostasis network and has the potential to be an anti-aging agent [28].In addition, 4 has been reported to possess anti-Escherichia coli and anti-Candida albicans activities [29].2-Chloro-1-methoxy-4-(methoxymethyl)benzene ( 5) is an aromatic compound [12] whose structural analog (methoxymethyl)benzene is a major constituent of the floral scents of Nymphaea lasiophylla and Nymphaea lingulata [30].
The organic synthesis process of 3-hydroxy-3-methyloxindole (6) [31] has been reported.This imine may be oxidized to 6 by a cytosolic enzyme, aldehyde oxidase [31].The substitution and cyclization processes with indole ring-related compounds have subsequently been reported [13].The vitamin B group's well-known pharmaceutical compound, nicotinic acid (7), has garnered a lot of attention in recent years due to its crucial function in the treatment of human disorders like pellagra.This compound has anti-tuberculosis activity [32] and fibrinolytic activity [33].In the chemical industry, succinic acid (8) is a highly valued biological raw ingredient.It serves as a precursor for a variety of other compounds [34], such as 1,4-butanediol, tetrahydrofuran, biodegradable polymers, and fumaric acid.In a previous study, the concentration of this compound was positively correlated with the area of colonic mucosal erosion formation in rats [35].
Uridine (11) is the precursor substance for uracil, which is widely produced in nature via the decarboxylation of uronic acid that is catalyzed by the enzyme uridine decarboxylase [36].It has been shown that uracil can be used as a nutrient source in the tumor microenvironment, and studies targeting the uracil synthesis pathway suggest that uracil may become a new target for cancer and immunotherapy in the future [36]. 2 -Deoxyuridine ( 12) is a nucleoside analog that has a very similar chemical composition to uracil but lacks the 2 hydroxyl group, which is used in antiviral medicines that are derivatives of deoxyuridine, and the application of 12 is as a precursor in the production of edoxuridine [19].Thymidine (13) is also isolated from Hydrilla verticillata [20], and the structural analog of 13, azidothymidine (AZT), is commonly used to treat HIV infection [37].1,2-Benzenedicarboxylic acid bis(2-methyl heptyl) ester ( 16) is obtained from Phellinus linteus, and previous in silico and in vitro results have validated that 16 could be exploited as a promising pancreatic lipase inhibitor [38].β-Sitosterol (17) has been reported to be present in different parts of plants, such as fruits, leaves [39], and rhizomes [40], and possesses anti-inflammatory and immunomodulatory activities [41].

Conclusions
Nematode-trapping fungi can capture nematodes by producing traps.Some recent studies have shown that metabolites play a role in the process of these fungi capturing nematodes, such as 3-methoxy-3-methyl-1-butanol [42] and 6-methylsalicylic acid [43], with nematode attraction activity identified from Orbilia oligospora and Arthrobotrys flagrans, and C-280 [44] with nematicidal activity isolated from O. oligospora.These results indicate that nematode-trapping fungi have the potential to produce a variety of active secondary metabolites.
In our previous studies, the genome of D. haptotyla YMF1.03409 was found to contain relatively rich information on biosynthetic gene clusters, and a compound with broadspectrum nematicidal activity, 2-furoic acid, was identified.In addition to being isolated from its fermentation products, 2-furoic acid could increase production during the process of D. haptotyla YMF1.03409 infection with nematodes.These results suggest that D. haptotyla YMF1.03409 possesses the ability to produce abundant metabolites [45].Therefore, in this study, we continued to carry out further investigation on the metabolites of D. haptotyla YMF1.03409.By extracting and isolating the fermentation products, a total of eighteen compounds were purified and identified as polyketides, steroids, aromatic compounds, organic acids, and nucleosides, including two new polyketides, nosporins C (1) and D (2).Some of these compounds have also been reported to possess diverse activities in previous research.In the future, metabolic regulation can be employed to boost the active secondary metabolites of D. haptotyla YMF1.03409 and apply them to biological control.Alternatively, the secondary metabolites information in D. haptotyla YMF1.03409 could be further mined via the heterologous expression technique.Our study deepens the understanding of the secondary metabolites of D. haptotyla YMF1.03409 and also lays the foundation for the application of this species in the future.

Figure 2 .
Figure 2. Mass of crude extracts under nine culture conditions.The horizontal coordinates represent the numbers of the nine culture conditions.**** represents p-value < 0.0001, which was calculated using the Student's t-test.

Figure 2 .
Figure 2. Mass of crude extracts under nine culture conditions.The horizontal coordinates represent the numbers of the nine culture conditions.**** represents p-value < 0.0001, which was calculated using the Student's t-test.

Table 1 .
The formulation of screening media.