Lipids and Terpenoids from the Deep-Sea Fungus Trichoderma lixii R22 and Their Antagonism against Two Wheat Pathogens

Five new lipids, tricholixins A–E (1–5), and two known terpenoids, brasilane A (6) and harzianone A (7), were discovered from a deep-sea strain (R22) of the fungus Trichoderma lixii isolated from the cold seep sediments of the South China Sea. Their structures and relative configurations were identified by meticulous analysis of MS and IR as well as NMR data. The absolute configuration of 5 was ascertained by dimolybdenum-induced ECD data in particular. Compounds 1 and 2 represent the only two new butenolides from marine-derived Trichoderma, and they further add to the structural diversity of these molecules. Although 6 has been reported from a basidiomycete previously, it is the first brasilane aminoglycoside of Trichoderma origin. During the assay against wheat-pathogenic fungi, both 1 and 2 inhibited Fusarium graminearum with an MIC value of 25.0 μg/mL, and 6 suppressed Gaeumannomyces graminis with an MIC value of 12.5 μg/mL. Moreover, the three isolates also showed low toxicity to the brine shrimp Artemia salina.


Introduction
As one of the main grains, wheat (Triticum aestivum L.) is cultivated worldwide and taken as a staple food by over 35% of the global population [1].Although the current wheat production (>700 million tons per year) is increasing all over the world, many pathogenic fungi can threaten wheat growth and lead to 15-20% yield losses every year [2].Among the fungal pathogens, Fusarium graminearum is the causal agent of Fusarium head blight that occurs at floret and glume parts of wheat [3].This wheat disease has ever outbroken in Asia, Europe, North America, and South America and resulted in devastating economic losses [2,3].As a wheat-rhizospheric pathogen, the soil-borne fungus Gaeumannomyces graminis can give rise to the notorious take-all disease.The infection may result in stunting and premature ripening, with the observed symptoms inclusive of dark roots, yellow leaves, white heads, and shrivelled grains [4].Fusarium head blight and take-all disease can decrease both the yield and quality of wheat and their pathogens also have the ability to infect other grains, such as barley and oats.Besides the alteration of tillage practices, synthesized fungicides have been widely used to control these diseases.However, their efficacy has been decreased by the emergence of drug resistance due to long-time utilization [5].Natural antibiotics that are more ecologically and environmentally preferable than synthesized ones have been continuously discovered [6].
Molecules 2023, 28, x FOR PEER REVIEW 2 of 11 from Trichoderma species of terrestrial and marine origin [7][8][9][10].Recently, deep-sea Trichoderma fungi have attracted the attention of some researchers, and three unidentified strains have been chemically surveyed [11][12][13][14].Although a total of 18 new metabolites have been obtained from these deep-sea Trichoderma strains, no antagonism against plant-pathogenic fungi has been detected.During our research on functional metabolites from deep-sea fungi [15][16][17], another isolate designated Trichoderma lixii R22 was obtained from a cold-seep sediment sample (−1217 m) collected in May 2020.Chemical investigation of this strain led to the discovery of five new lipids (1-5) and two known terpenoids (6 and 7) (Figure 1).

Results and Discussion
The organic extracts of deep-sea fungus Trichoderma lixii R22 isolated from the cold seep sediments of the South China Sea were subjected to a series of column chromatography, Sephadex LH-20, preparative TLC, and semipreparative HPLC to yield five new lipids, tricholixins A-E (1-5), and two known terpenoids, brasilane A (6) and harzianone A (7) (Figure 1).

Structural Elucidation
Tricholixin A (1) has a molecular formula of C13H18O4, which was deduced from the deprotonated molecular ion peak at m/z 237.1123 in negative HRESI(−)MS.The IR absorption bands at 3440 and 1738 cm −1 demonstrated the presence of hydroxy and carbonyl groups.The 1 H NMR spectrum, recorded in DMSO-d6 (Table 1), showed a methyl (C-1) doublet at δH 1.02 and a hydroxy doublet at δH 4.63, both of which exhibited COSY correlations with a multiplet at δH 3.77 for a methine group (C-2).This information defined a hydroxyethyl group, which was elongated to C-3 to form a 2-hydroxypropyl unit based on the COSY correlation between H-2 and H-3b and the HMBC correlations from H-3b to C-1 and C-2 (Figure 2).On the other hand, a trans double bond at C-6 was established by the chemical shifts of H-6 (δH 6.54) and H-7 (δH 6.14) and their mutually coupling constant (J = 16.1 Hz), and an acetonyl group was defined by the deshielded chemical shift of C-10 (δC 207.4) and its HMBC correlations with H-9 and H-11.These two parts were linked together with a methylene group (C-8) to form a 5-oxohexenyl group based on the HMBC correlations from H-6 to C-8 and from H-7 to C-9.The remaining 1 H and 13 C NMR signals, especially in CDCl3 (Tables 1 and 2), corresponded to an

Results and Discussion
The organic extracts of deep-sea fungus Trichoderma lixii R22 isolated from the cold seep sediments of the South China Sea were subjected to a series of column chromatography, Sephadex LH-20, preparative TLC, and semipreparative HPLC to yield five new lipids, tricholixins A-E (1-5), and two known terpenoids, brasilane A (6) and harzianone A (7) (Figure 1).

Structural Elucidation
Tricholixin A (1) has a molecular formula of C 13 H 18 O 4 , which was deduced from the deprotonated molecular ion peak at m/z 237.1123 in negative HRESI(−)MS.The IR absorption bands at 3440 and 1738 cm −1 demonstrated the presence of hydroxy and carbonyl groups.The 1 H NMR spectrum, recorded in DMSO-d 6 (Table 1), showed a methyl (C-1) doublet at δ H 1.02 and a hydroxy doublet at δ H 4.63, both of which exhibited COSY correlations with a multiplet at δ H 3.77 for a methine group (C-2).This information defined a hydroxyethyl group, which was elongated to C-3 to form a 2-hydroxypropyl unit based on the COSY correlation between H-2 and H-3b and the HMBC correlations from H-3b to C-1 and C-2 (Figure 2).On the other hand, a trans double bond at C-6 was established by the chemical shifts of H-6 (δ H 6.54) and H-7 (δ H 6.14) and their mutually coupling constant (J = 16.1 Hz), and an acetonyl group was defined by the deshielded chemical shift of C-10 (δ C 207.4) and its HMBC correlations with H-9 and H-11.These two parts were linked together with a methylene group (C-8) to form a 5-oxohexenyl group based on the HMBC correlations from H-6 to C-8 and from H-7 to C-9.The remaining 1 H and 13 C NMR signals, especially in CDCl 3 (Tables 1 and 2), corresponded to an α,β-unsaturated γ-lactone ring by comparison with harzianolide [18,19], and its connectivity with the 2-hydroxypropyl and 5-oxohexenyl groups was confirmed by the HMBC correlations from H-3b to C-4 and from H-6 to C-5.The above evidence established the planar structure of 1, verified by other COSY and HMBC correlations as shown in Figure 2. C-2 is the only chiral center in this molecule, and its absolute configuration was deduced to be 2S by comparison of the specific rotation value with that of harzianolide ([α] D 6.6) [18].Thus, compound 1 was named (S,E)-3-(2-hydroxypropyl)-4-(5-oxohex-1-en-1-yl)furan-2(5H)-one.The molecular formula of tricholixin B (2) was determined to be C11H16O4, two fewer carbon atoms than for 1, on the basis of HRESI(+)MS data.The hydroxy and carbonyl groups were indicated by the IR absorption bands at 3430 and 1740 cm −1 , similar to those of 1.The 1 H and 13 C NMR data (Tables 1 and 2) resembled those for 1, but some signals  The molecular formula of tricholixin B (2) was determined to be C 11 H 16 O 4 , two fewer carbon atoms than for 1, on the basis of HRESI(+)MS data.The hydroxy and carbonyl groups were indicated by the IR absorption bands at 3430 and 1740 cm −1 , similar to those of 1.The 1 H and 13 C NMR data (Tables 1 and 2) resembled those for 1, but some signals lacked or altered.Analysis of those data revealed that a hydroxymethyl group (C-9), instead of an acetonyl group in 1, should be present in 2. This functionality was linked to a methylene group (C-6) via a trans double bond to form a 4-hydroxy-2-butenyl group, as seen from the large coupling constant (J = 15.3) between H-7 and H-8 and the COSY correlations of H 2 -9/H-8/H-7/H 2 -6 (Figure 2).The remaining NMR signals were identical to those of 1, suggesting the same connectivity of corresponding parts in 1 and 2. The whole structure was further validated by other COSY and HMBC correlations as shown in Figure 2. Finally, a 2S configuration was assigned to C-2 by the identical specific rotation value with that of HRESI(+)MS analysis gave tricholixin C (3) a molecular formula of C 13 H 22 O 3 , with three degrees of unsaturation.The high-frequency chemical shifts at δ H 4.9-6.4 and δ C 115-139 in 1 H and 13 C NMR spectra (Tables 3 and 4), respectively, along with their coupling constants and COSY correlations (Figure 2), indicated the presence of a butadienyl group (C-10 to C-13), accounting for two degrees of unsaturation.To satisfy the remaining one degree of unsaturation, this compound should contain a ring unit.Four oxygenated methine groups (C-2, C-3, C-6, and C-7) were proposed by the four 1 H NMR signals at δ H 3.5-3.9and the four 13 C NMR signals at δ C 70-85.However, only three oxygen atoms existed in the molecular formula.To match the above data, one cycloether ring and two hydroxy groups were suggested in the molecule, and the hydroxy groups were supported by the IR absorption at 3425 cm −1 .A comparison of the NMR signals for oxygenated methine groups with literature data revealed the presence of a 2,5-dihydroxymethyltetrahydrofuran unit [20], which was supported by the COSY correlations between H-2 and H-3 and between H-6 and H-7 and the HMBC correlations from H-2 to C-3 and C-4 and from H-7 to C-5 and C-6.One terminal was methylated according to the COSY correlation between H 3 -1 and H-2 and the HMBC correlations from H 3 -1 to C-2 and C-3, and the other was linked to the butadienyl group via an ethylene group based on the COSY correlations of H-7/H 2 -8/H 2 -9/H-10.The above information secured the planar structure of 3, of which the double bond at C-10 was trans in view of the large coupling constant (J = 15.2) between H-10 and H-11.Moreover, H-3 and H-6 were located on the opposite face of the tetrahydrofuran ring, as supported by the NOESY correlation between H-3 and H-7.The relationships of H-2/H-3 and H-6/H-7 were deduced to be erythro and threo, respectively, by comparison of NMR data with those for annonacin A [21].The systematic name of compound 3 was (E)-1-(5-(1-hydroxyethyl)tetrahydrofuran-2-yl)hepta-4,6-dien-1-ol.
Tricholixin D (4) was assigned a molecular formula of C 14 H 24 O 4 by interpretation of HRESI(+)MS data.Two propenyl groups were deduced in the molecule by comparing NMR data (Tables 3 and 4) with literature ones [22], and they were also confirmed by the HMBC correlations from H 3 -1 to C-2 and C-3 and from H 3 -13 to C-11 and C-12.One propenyl group was extended to the nonprotonated C-8 via an ethylene group according to the COSY correlations from H-9b to H 2 -10 and the HMBC correlations from H-9b to C-8 and from H 2 -10 to C-11 and C-12.The other was bonded to a hydroxylated methine group based on the HMBC correlations from H-4 to C-2 and C-3 and then elongated to C-8 based on the COSY correlations from OH-4 through to HO-7 and the HMBC correlations from OH-7 to C-6, C-7, and C-8.The remaining NMR signals corresponded to a methoxy group [23], which was attached to C-8 on the basis of their HMBC correlation.To satisfy the molecular formula, an ether linkage was situated between C-5 and C-8.Taken together, the planar structure of 4 was completed.As indicated by the signal of H-2, the large coupling constant (J = 15.4) between H-2 and H-3 suggested the double bond at C-2 to be trans.Although the coupling constants of the other two olefinic methines (C-11 and C-12) failed to be diagnosed, their chemical shifts demonstrated the double bond at C-11 to be trans [22].Moreover, H-5, OH-7, and CH 3 O-8 were oriented on the same face of the tetrahydrofuran ring by the NOESY correlations between H-5 and CH 3 O-8 and between H-7 and H-9b.The relationship between H-4 and H-5 was speculated to be threo according to the similar coupling constants of H-5 with that of (6S,7R,9R,10R)-6,9-epoxynonadec-18-ene-7,10-diol [24].Despite the wide distribution of the tetrahydrofuran motif in marine natural products, ketal-bearing ones have rarely been discovered so far [25].Therefore, compound 4 was named 5-((E)-1hydroxybut-2-en-1-yl)-2-methoxy-2-((E)-pent-3-en-1-yl)tetrahydrofuran-3-ol.Tricholixin E (5) possesses a molecular formula of C 13 H 20 O 3 , established by HRESI(−)MS data.Its IR spectrum exhibited absorption bands for hydroxy and carbonyl groups at 3439 and 1735 cm −1 .The doublet at δ H 1.64 (H 3 -13) in the 1 H NMR spectrum (Table 3) and the low-frequency signal at δ C 18.1 in the 13 C NMR spectrum (Table 4) were attributed to a methyl group by analysis of HSQC and DEPT data.Its linkage with a double bond was deduced by the HMBC correlations from H 3 -13 to C-11 and C-12 and further verified by comparison of NMR data with those for 4. The high singlet at δ H 2.28 (H 3 -1) in the 1 H NMR spectrum was also ascribed to a methyl group, which was then connected to a carbonyl group to form an acetyl unit based on the HMBC correlation from H 3 -1 to C-2.In addition, a large spin system from C-3 to C-10 was established by the COSY correlations as shown in Figure 2 and was further validated by the identical NMR data with those for the same unit reported in the literature [26].These three moieties were linked together according to the HMBC correlation from H 2 -10 to C-11 and C-12, from H-9b to C-11, and from H 3 -1 to C-3.To ascertain the absolute configurations at C-7 and C-8, a dimolybdenum-induced ECD spectrum (Figure 3) was determined by the addition of Mo 2 (OAc) 4 .The curve with a negative Cotton effect at 310 nm matched well with that of neocyclocitrinol A, which features a threo vicinal diol unit with R,R configurations according to the empirical helicity rule [27].Thus, compound 5 was named (3E,5E,7R,8R,11E)-7,8-dihydroxytrideca-3,5,11-trien-2-one.The two known compounds, brasilane A (6) and harzianone A (7), were ide by comparison of their spectroscopic data with those reported in the literature [2 Compound 6 features a brasilane skeleton that has been rarely discovered from T derma [31,32].It is the first brasilane aminoglycoside from Trichoderma.Compo possesses a harziane scaffold, and its discovery further verifies the universality class of diterpenoids in Trichoderma [33].

Antifungal Activity of Isolated Compounds
Compounds 1-7 were evaluated for their inhibition of two wheat-pathogenic Fusarium graminearum ACCC39334 and Gaeumannomyces graminis ACCC38864.T sults showed only compounds 1, 2, and 6 were active against one of the two path  The two known compounds, brasilane A (6) and harzianone A (7), were identified by comparison of their spectroscopic data with those reported in the literature [28][29][30].Compound 6 features a brasilane skeleton that has been rarely discovered from Trichoderma [31,32].It is the first brasilane aminoglycoside from Trichoderma.Compound 7 possesses a harziane scaffold, and its discovery further verifies the universality of this class of diterpenoids in Trichoderma [33].

Antifungal Activity of Isolated Compounds
Compounds 1-7 were evaluated for their inhibition of two wheat-pathogenic fungi, Fusarium graminearum ACCC39334 and Gaeumannomyces graminis ACCC38864.The results showed only compounds 1, 2, and 6 were active against one of the two pathogens (Table 5).Compounds 1 and 2 inhibited F. graminearum ACCC39334 with an MIC value of 25.0 µg/mL, and 6 suppressed G. graminis ACCC38864 with an MIC value of 12.5 µg/mL.Although harzianolide and its 2-oxo derivative effectively inhibited G. graminis at 200 and 100 µg/plug [34], no activities were detected at a concentration of 50 µg/mL for 1 and 2. This might result from the low concentration of the two new compounds or the structural discrepancy of these butenolides.The inhibition effect of 1 and 2 on F. graminearum further demonstrated the potency of butenolides to antagonize plant-pathogenic fungi.Additionally, terpenoid aminoglycosides are a family of metabolites that have often been discovered from marine-derived Trichoderma in recent years [10,35,36].The high inhibition effect of 6 on G. graminis may afford a new target for the application of this class of molecules.On the other hand, the brine shrimp lethality of 1-7 was also evaluated, and only <24% inhibition rates were detected for these compounds at 100 µg/mL.The brine shrimp Artemia salina was widely used to predict human toxicity to environmental chemicals and natural products [37].The low toxicity of compounds 1, 2, and 6 may suggest their prospect in the development of antifungal agents in agriculture.

Fungal Material and Fermentation
Trichoderma lixii R22 was obtained from the sediments collected from a deep-sea (−1217 m) cold seep off southwestern Taiwan Island in May 2020.Its identification was fulfilled by analysis of the internal transcribed spacer rDNA sequence data (GenBank no.OQ745814, Bethesda, MD, USA).The static cultivation was completed in 150 × 1 L Erlenmeyer flasks at 25 • C for 40 days.Each flask mainly harbored 25.0 g of rice, 1.0 g of glucose, 0.25 g of peptone, 0.25 g of yeast extract, 0.25 g of monosodium glutamate, 0.05 g of NaBr, 25.0 mL of pure water, and 25.0 mL of natural seawater (Yantai coast).

Extraction and Isolation
Some 50 mL EtOAc was poured into each flask to end the cultivation.Mycelia were collected by filtration, which were further dried and then extracted with CH 2 Cl 2 /MeOH (1:1, v/v) three times.After evaporating organic solvents under negative pressure, the mycelial extract (308 g) was obtained.The filtrate was extracted with EtOAc and then concentrated to afford the broth extract (38 g).The two parts (346 g) were combined and then subjected to silica gel CC with step-gradient solvent systems comprising petroleum ether (PE)/EtOAc and CH 2 Cl 2 /MeOH to give 9 fractions (Frs.1-9).Fr. 5 3 and 4; HRESI(+)MS m/z 279.1561 [M + Na] + (calcd for C 14 H 24 O 4 Na, 279.1572).

ECD Determination for Mo 2 -Complex of 5
Solutions of 5 and dimolybdenum tetraacetate, Mo 2 (OAc) 4 , were prepared by dissolving appropriate amounts of them in 1.0 mL of DMSO solvent of analytical grade, respectively, and both the concentrations were 1.5 mg/mL.Subsequently, 0.15 mL of the two solutions were mixed and then poured into a quartz cuvette with a 1.0 mm optical path length.In the mixture, the molar ratio of 5 to Mo 2 (OAc) 4 was 1.9:1.Within the first 40 min, ECD signals were continually determined until the emergence of an invariant spectrum.

Assay for Antifungal Activity
Antifungal assay toward the wheat-pathogenic Fusarium graminearum ACCC39334 and Gaeumannomyces graminis ACCC38864, which were purchased from the Agricultural Culture Collection of China, was carried out through the microdilution method in a 96-well plate as described previously [10].During the antifungal test, carbendazim was taken as the positive control.Following a previous procedure [38], the brine shrimp lethality was assayed against Artemia salina, with CuSO 4 being a positive control.

Conclusions
Chemical investigation of the deep-sea fungus Trichoderma lixii R22 resulted in the isolation and identification of five new lipids, tricholixins A-E (1-5), and two known terpenoids, brasilane A (6) and harzianone A (7), and compounds 1 and 2 represent the only two new butenolides from marine-derived Trichoderma.In addition, compound 6 is the first brasilane aminoglycoside of Trichoderma origin.Compounds 1-7 were evaluated for inhibition against two plant-pathogenic fungi and one marine plankton species.Among them, compounds 1 and 2 inhibited F. graminearum with an MIC value of 25.0 µg/mL, and the structure-activity relationship suggested that butenolides could enhance the antifungal activity of these lipids.On the other hand, the high inhibition effect of 6 on G. graminis may afford a new target for the application of this class of molecules.The low toxicity of compounds 1, 2, and 6 may suggest their prospect in the development of antifungal agents
α,β-unsaturated γ-lactone ring by comparison with harzianolide[18,19], and its connectivity with the 2-hydroxypropyl and 5-oxohexenyl groups was confirmed by the HMBC correlations from H-3b to C-4 and from H-6 to C-5.The above evidence established the planar structure of 1, verified by other COSY and HMBC correlations as shown in Figure2.C-2 is the only chiral center in this molecule, and its absolute configuration was deduced to be 2S by comparison of the specific rotation value with that of harzianolide ([α] D 6E)-3-(2-hydroxypropyl)-4-(5-oxohex-1-en-1-yl)furan-2(5H)-one.

Figure 2 .
Figure 2. Key COSY and HMBC correlations of 1-5 (bold lines for COSY and arrows for HMBC).

Figure 2 .
Figure 2. Key COSY and HMBC correlations of 1-5 (bold lines for COSY and arrows for HMBC).