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Article

Metabolic Profiling of Water-Soluble Compounds from the Extracts of Dark Septate Endophytic Fungi (DSE) Isolated from Scots Pine (Pinus sylvestris L.) Seedlings Using UPLC–Orbitrap–MS

by
Jenni Tienaho
1,2,*,
Maarit Karonen
3,
Riina Muilu–Mäkelä
2,
Kristiina Wähälä
4,
Eduardo Leon Denegri
4,
Robert Franzén
5,
Matti Karp
1,
Ville Santala
1 and
Tytti Sarjala
2
1
Faculty of Natural Sciences and Engineering, Tampere University, FI-33101 Tampere, Finland
2
Natural Resources Institute Finland (Luke), FI-00791 Helsinki, Finland
3
Natural Chemistry Research Group, Department of Chemistry, University of Turku, FI-20014 Turku, Finland
4
Department of Chemistry, University of Helsinki, FI-00014 Helsinki, Finland
5
School of Chemical Engineering, Department of Chemistry and Materials Science, Aalto University, FI-00076 Espoo, Finland
*
Author to whom correspondence should be addressed.
Molecules 2019, 24(12), 2330; https://doi.org/10.3390/molecules24122330
Submission received: 24 May 2019 / Revised: 14 June 2019 / Accepted: 22 June 2019 / Published: 25 June 2019
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Abstract

:
Endophytes are microorganisms living inside plant hosts and are known to be beneficial for the host plant vitality. In this study, we isolated three endophytic fungus species from the roots of Scots pine seedlings growing on Finnish drained peatland setting. The isolated fungi belonged to dark septate endophytes (DSE). The metabolic profiles of the hot water extracts of the fungi were investigated using Ultrahigh Performance Liquid Chromatography with Diode Array Detection and Electronspray Ionization source Mass Spectrometry with Orbitrap analyzer (UPLC–DAD–ESI–MS–Orbitrap). Out of 318 metabolites, we were able to identify 220, of which a majority was amino acids and peptides. Additionally, opine amino acids, amino acid quinones, Amadori compounds, cholines, nucleobases, nucleosides, nucleotides, siderophores, sugars, sugar alcohols and disaccharides were found, as well as other previously reported metabolites from plants or endophytes. Some differences of the metabolic profiles, regarding the amount and identity of the found metabolites, were observed even though the fungi were isolated from the same host. Many of the discovered metabolites have been described possessing biological activities and properties, which may make a favorable contribution to the host plant nutrient availability or abiotic and biotic stress tolerance.

Graphical Abstract

1. Introduction

Endophytes are bacterial or fungal microorganisms that colonize a wide variety of plant tissues during at least some period of their lifecycle. Endophytic infection is considered inconspicuous, the infected host tissues are at least transiently symptomless, and the microbial colonization is internal [1,2]. Endophytes have been isolated from all of the studied plant species [3]. However, it has been estimated that only 1–2% of the known 300,000 plant species have been studied for their endophytes [4]. The relationship between the host and the endophyte can have many forms ranging from saprobic to parasitic and from exploitative to mutualistic. Endophytic fungi and bacteria have been shown to improve the health of the host plant by improving the biotic and abiotic stress tolerance due to phytohormone production, and host’s nutrient uptake [5,6,7,8]. The endophytes have also been shown to produce toxic chemicals preventing attacks by insects and herbivores [9,10].
Dark septate endophytic fungi (DSE) are often dominant in the roots of tree species [11] and characterized with melanized and septate hyphae. These Ascomycetous conidial or sterile endophytic fungi colonize the roots of many higher plant species widely in the northern hemisphere and are extensively distributed in coniferous boreal forests [12]. The most frequent DSE in natural forest ecosystems in the northern hemisphere belong to the Phialocephala fortinii s.l.–Acephala applanata species complex (PAC) and up to 80% of fine roots in forest stands can be colonized by them [11]. Studies have shown that DSE and PAC species induce resistance to abiotic stress, accelerate root turnover and mineralization, and suppress root pathogens [11,13,14,15].
The metabolic profiling of endophytes has revealed novel compounds possessing interesting bioactive properties to be utilized in future. For example, during the years 1995 to 2011, at least 313 novel compounds were isolated and identified from bacterial and fungal endophytic microorganisms [16]. These were found possessing interesting properties, for example as agrochemicals, antiparasitics and in the field of pharmacology. Additionally, in the previous studies of Scots pine bacterial endophytes, they have been shown to produce efficient antioxidant and antimicrobial compounds [17]. Water extraction has been previously found effective in yielding bioactive principles from microorganisms. For example, water-soluble nucleosides, exopolysaccharides, peptides, proteins and sterols showed also bioactive properties in the caterpillar parasitic fungus Cordyceps sinensis [18].
In this study, we explored three common endophytic fungal isolates from conifer roots and their aqueous extracts using LC–MS methodologies. The endophytic fungal species used in this study were isolated from the roots of eight-year-old Scots pine seedlings growing in a Finnish drained peatland setting. The growth conditions for trees and especially young seedlings in drained peatlands are harsh due to the extreme variability in temperature, solar radiation, variability in soil ground water level (drought/flood) and poor nutrition. The northern peatlands are rather unexplored environments as regards their endophytes. Scots pine is one of the most economically important and common tree species in Finland and the boreal zone in general. In spite of that, a limited number of studies has been published about its endophytic symbionts. In fact, to our knowledge, this is the first time that the metabolic profile of water extracted Scots pine root associated endophytic fungi belonging to DSE is investigated. However, under this kind of continuously strenuous growth conditions, the associated endophytes may play a role in enhancing the survival of the host trees by producing effective metabolites with interesting bioactivities. Moreover, we decided to use UPLC–Orbitrap–MS as UPLC enables fast and sensitive analyses with ultra-high performance for complex samples, and Orbitrap has a high resolving power and is thereby suitable for the accurate mass measurements and characterization of compounds.

2. Results and Discussion

2.1. Identification of the Endophytic Fungal Isolates

The taxonomic identification of the isolated fungal strains (A, R, and S16) was made by comparing the ITS (Internal Transcribed Spacer) region with the best GenBank Blast matches (Table 1). According to the DNA sequencing of ITS1, 5.8S and ITS2 rDNA regions, A and R strains belong to PAC. ITS region of the S16 strain matched with the two species Humicolopsis cephalosporioides and Coniochaeta mutabilis. The alignments of the S16 strain together with its best GenBank matches are presented in Supplementary Figure S1. PAC species cannot be reliably separated using only the ITS regions which emphasizes the need for complementing sequencing methods in order to validate the results [11]. However, the strain A had 100% identity together with Acephala applanata strain (AY078147.1), whereas strain R was more similar with Phialocephala fortinii strains (see alignments in Supplementary Figure S2). Humicolopsis cephalosporioides and Coniochaeta mutabilis can be considered as DSE-like fungi [19]. Previously, Coniochaeta were considered as Lecytophora sp. [20]. The phylogenetic tree (Figure 1) is rooted with Coniochaeta mutabilis. Fungal species used in this study are to be joined into the microbe and yeast library collection of Natural Resources Institute Finland.

2.2. Identification of the Metabolites

We were able to identify 220 metabolites from three fungal extracts (Table 2) and 98 compounds were left unidentified (Supplementary Table S1). Identification was mainly based on the exact masses and the molecular formula observed. It was performed with Thermo Compound Discoverer software and SciFinder Scholar database with the substance role Occurrence and the highest number of references to scale down possible compound hits. Compound Discoverer hits were also run through SciFinder database to verify that the identified products have been found in natural sources. The references shown in Table 2 were used to complement the identification and additionally 39 authentic standards were examined. Metabolites, whose presence and identity were verified with authentic standards, are marked with the reliability of three stars. The majority of the identified metabolites belonged to the group of amino acids, dipeptides and peptides. In addition to the main metabolites, minor ones were also present but the intensity limit of the peaks in the ion chromatogram was set at 1 × 107, and the peaks with intensities lower than that were not included.

2.2.1. Amino Acids, Dipeptides and Peptides

Amino acids, dipeptides and tripeptides are the most abundant group of identified metabolites in Table 2, as can be expected with water extraction. With the used m/z range 150–2000, we were able to detect five amino acids: arginine, valine, tyrosine, phenylalanine and tryptophan (Figure 2). All except valine were verified with authentic standards and their retention time order is similar to that found in the literature [23,24,55]. Valine was detected by the [2M + H]+ ion as its molecular weight is too low for the scan range used. Arginine was found to be the most dominant compound and it was detected with multiple retention times and the intensities up to 1 × 109 in the mass spectrum. It was also detected as a degradation product of many peptides or other structures with arginine backbone, which explains the multiple retention times. Amino acids formed potassium adducts and [2M + H]+ and [2M − H]− ions were also observed for them. In addition, arginine was detected by [3M + H]+ and [3M − H]⁻ ions and tryptophan with ammonium fragment ions.
Dipeptides were the most abundant class of compounds in our samples (Table 2) with 122 possible identifications, which represents 55% of the identified metabolites. Out of the dipeptides, 16 were verified with autentic standards (Figure 3). In addition, three tentative identifications of cyclic dipeptides were made using databases and listed references in Table 2: Cyclo(Glu-Tyr); Cyclo(3-OH-Pro-Tyr) and Cyclo(Glu-Leu) or Cyclo(Glu-Ile). Plant associated microorganisms are known to produce a variety of N-containing compounds, such as cyclic peptides and peptides [6,83,84,101].
Furthermore, 29 tripeptides and other oligopeptides were tentatively identified in our samples (Table 2) together with 14 amino acid derivatives. Small peptides and amino acids have also been identified from the tubers of Pinellia ternate roots [26] and some of the findings confirm the retention order of the compounds identified by databases and literature in our study. For some compounds, additional ions were detected. Ammonium adducts were typical for aceglutamide and acetylcitrulline type compounds and the sodium adduct was detected for acetylleucine and acetylisoleucine. Glutathione yielded an additional doubly charged ion. It is a peptide produced in response to several stress situations in endophytic microbes [17,65]. Its retention order in comparison to acetylcarnitine, tyrosine, adenosine, phenylalanine and tryptophan is the same as reported by Ibanez et al. [55]. Additional ions of tryptophan and its ammonium fragment were detected for acetyltryptophan and for Trp-Ala dipeptide, which strengthens the tentative identifications. The retention order of arginine, dimethylarginine, tyrosine and tryptophan was similar to that found in Liang et al. [25]. Dimethylarginine was also detected with ESI–MS by Gamal–Eldin et al. [33]. In addition, methionine and its derivatives, such as acetylmethionine have an important role in the biochemistry of plan tissues [86].
The proteins and enzymes produced by the endophytes have been reported to increase thermostability, pH-stability, UV tolerance and products with activity against pathogenic microorganisms [6]. Peptides produced by the endophytes have also been searched for new antibiotic compounds and other bioactive properties [102,103]. Antimicrobial peptides and proteins have been found to be biosynthesized immediately in response to pathogenic microorganism assault [104,105,106]. In this study, we detected large amounts of arginine, which is commonly used as nitrogen storage because it has the highest nitrogen to carbon ratio out of all 21 proteinogenic amino acids [107]. Nitrogen is often a limiting resource for the plant growth since it is needed for nucleic acid and protein synthesis. Arginine is used in the production of nitric oxide and polyamines in plants as well and both play a crucial role in the responses to abiotic and biotic stress [107]. One of the cyclic dipeptides found, Cyclo(3-OH-Pro-Tyr) was reported with acaricidal activity against Tetranychus urticae and cyclic dipeptides have been described being active towards plant pathogens [84,108].

2.2.2. Opine Amino Acids

Four possible identifications in Table 2 were opines or N2-(1-carboxyethyl)-amino acids. Heliopine is a conjugate of glutamine and pyruvate, whereas rideopine is a product of the reductive amination of polyamine putrescine with α-ketoglutaric acid [34]. Lysopine is a condensation product of lysine and pyruvate [43]. Heliopine, rideopine and lysopine have all been detected from crown gall tumours produced by rhizosphere bacteria Agrobacterium tumefaciens [34,43]. Valinopine has been detected from a poisonous mushroom Clitocybe acromelalga and is suggested being a fungal toxin [92]. In addition, we were able to observe a compound tentatively identified as saccharopine, which is a precursor of lysine in the fungal α-aminoadipate pathway [109]. However, the intensities were under 1 × 107 and, thus, it was not included in Table 2.

2.2.3. Amino Acid Quinones and Amadori Compounds

Abenquine C or its enantiomer and Abenquine B1 and B2 are tentatively identified amino acid quinone derivatives in Table 2. Abenquine C or N-[4-(acetylamino)-3,6-dioxo-1,4-cyclo-hexa- dien-1-yl]-l-valine and N-[4-(acetylamino)-3,6-dioxo-1,4-cyclohexadien-1-yl]-leucine (Abenquine B1) and -isoleucine (Abenquine B2) have been isolated from the rhizosphere bacteria Streptomyces sp. strain DB634 [69,70]. Because more than one possible amino acid quinone masses were detected and they have been isolated from the rhizosphere, there is a possibility that root-colonizing fungi of the rhizosphere could produce these metabolites. However, the confidence level is putative identification.
Furthermore, Amadori compounds were detected. They are Maillard reaction products where amino acid is attached to a pentose or hexose sugar. Namely, hexosearginine, hexosevaline, pentoseproline, hexoseaminobutyric acid and deoxyhexosethreonine were among the tentatively identified compounds in Table 2. Out of these, the presence of hexosearginine (Figure 2) was verified with a synthetic reference compound (purity 99%) [110]. Additionally, hexosearginine’s fragmentation into arginine was detected in UPLC-MS. Amadori compounds have previously been identified from fungal cultures [28] and characteristic ions similar to our findings have also been detected by Davidek et al. [93] and Wang et al. [29]. Hexoseaminobutyric acid was detected with a shorter retention time than the hexose sugar structure as also shown by Lamberts et al. [42]. Additionally, deoxyhexose amino acids have been previously detected from eukaryotic cells [79].

2.2.4. Cholines

Discovered cholines presented in Table 2 are choline-O-sulphate and glycerophosphorylcholine (Figure 2). Choline-O-sulphate has been found in relatively large amounts in fungal mycelia and has been suggested to act as a storage of sulphur, which is an essential metabolite for growth in filamentous fungi [41,44]. Glycerophosphorylcholine is a part of phosphatidylcholine, which is a type of phospholipid in lecithin. Lecithin is a major component of the phospholipid membrane also found in plant tissues [41]. The occurrence of both of these compounds was confirmed with authentic standards and they were observed as potassium adducts in the positive ionization. Glycerophosphorylcholine retention time with respect to arginine, tyrosine, adenosine and tryptophan was same as found by Liang et al. [25].

2.2.5. Nucleobases, Nucleosides, Nucleotides, and their Derivatives

Out of nucleobases, we were able to detect guanine and isoguanine or oxyadenine, which are the only ones with molecular mass over 150 Da (Table 2). The identification of guanine was confirmed by an authentic standard (Figure 2). Isoguanine is a purine analog, which is formed as a result of direct oxidation of adenine [71,72].
Nucleosides contain a nucleobase with a pentose sugar unit: ribose or deoxyribose. Cytidine, pseudouridine, uridine, adenosine, guanosine isomer, deoxyguanosine, methylthymidine, and deoxythymidine (Table 2) were tentatively identified, and the presence of cytidine, uridine and adenosine was verified with authentic standards (Figure 2). Nucleosides were discovered to form formate adducts and [2M − H]− cluster ions in negative ESI, and guanosine isomer also yielded a fragment ion responding to the detachment of pentose sugar unit. Methylthymidine has been used as an indicator of microbial presence in wastewaters [78].
Nucleotides are nucleosides joined with at least one phosphate group. We were able to tentatively identify adenosine monophosphate (AMP) or deoxyguanosine monophosphate (dGMP), cyclic uridine monophosphate (cUMP), deoxyribose adenosine monophosphate (dAMP), cyclic adenosine diphosphate ribose (cyclic ADP-ribose), cyclic guanosine monophosphate (cGMP) and two exact masses and molecular formulae corresponding to dinucleotides (Table 2). The dinucleotides exhibited a UV maximum at 258–261 nm, which in addition to the shape of the UV spectrum correlates with the literature [111]. The absorption maximum in our study was, however, broader and continued until 300 nm, which is likely caused by other compounds eluting simultaneously. Cyclic nucleotides are used as signaling metabolites in almost all organisms and they regulate a vast number of cellular processes [59,60,61,62]. The presence of the main fragment ion at m/z 152.1 was also detected with cGMP as reported in the literature [59]. ADP-ribosyl groups are formed on target proteins as a response to DNA damage and poly(ADP-ribose) polymerase enzyme homologs, which catalyze the reaction, have also been found in fungi [66]. In addition, nicotinamide riboside and nicotinamide adenine dinucleotide (NAD) were tentatively identified. NAD produced a fragment ion at m/z 540.1 in the negative ESI mode corresponding to the cleavage of nicotinamide. The retention order of the above mentioned metabolites was similar to that found in the literature [25,26,36,37,38,39,40,48,51,52,53,55,63,64,85]. Shiao et al. [35] detected nucleosides and nucleobases from the pathogenic fungus Cordyceps sinensis and their retention order is same as ours.
Additionally, sugar-nucleotides, such as uridine diphosphate (UDP)-glucose and UDP-galactose as well as UDP-galactosamine and UDP-glucosamine, were discovered (Table 2). UDP-glucosamines and UDP-galactosamines are important precursors of the bacterial and fungal cell wall [49]. Sugar nucleotides are donors of sugar groups in the biosynthesis of glycosides, polysaccharides and glycoconjugates, and they are abundant in microorganisms and plants [46]. They also possess many important roles in fungi [47,54].

2.2.6. Siderophores

One exact mass corresponding to cis- and/or trans-fusarinine siderophore was found with two retention times (Table 2). The cis- and trans-fusarinine backbones are very common in many fungal siderophores [75,76]. Siderophores are low molecular weight compounds that are used for iron uptake and storage and they have, for example, been found to have importance in the maintenance of plant–fungi symbioses [74,77]. Fungi and other microorganisms have been found to produce siderophores under aerobic growth conditions, where low iron availability is detected [75]. Iron is essentially required for the growth and proliferation in both bacteria and fungi and siderophores provide cells with nutritional iron [102]. In DSE fungi, it was found that these species have the ability to acidify the environment and produce siderophores to increase the micronutrient uptake to both members of the symbiont, indicating the association to be mutualistic rather than pathogenic [73]. Fusarinine monomers where also discovered with characteristic formic acid and water fragment ions.

2.2.7. Other Common Metabolites

Pentonic and hexonic acids in Table 2 were identified by their exact masses and molecular formulae according to Sun et al. [26], where they had further identified the species being ribonic and gulonic acids using MS/MS data. Glycerophosphoinositol is closely related to glycerophosphorylcholine. It is found in both plants and fungi and is a major deacylation product of lipid metabolism [30,31,55]. In addition, as with cholines, the potassium adduct was observed for it.
Acetyl coenzyme A is a central carbon and energy cycle metabolite, which is bulky and amphibilic and, thus, cannot readily transverse biological membranes. Acetylcarnitine is used in fungi to transport the acetyl unit [50]. The retention order of acetylcarnitine in relation to tyrosine, glutathione dimer, adenosine, phenylalanine and tryptophan is also similar to that found by Ibanez et al. [55].
Isocitrate and citric acid are isomers with the same molecular formula as well as methylisocitric acid and methylcitric acid. Citric acid and isocitrate are both important intermediates in the Krebs cycle, which is the metabolic route to produce energy to the eukaryotic cells, such as in plants and fungi. The presence of citric acid was confirmed with an authentic standard (Figure 2). In addition, we observed potassium adducts with both citric acid and isocitrate, which strengthened the tentative identification of isocitrate. Methylcitrate cycle catabolizes propionate in yeast and filamentous fungi [57]. Propionate is produced during the catabolism of amino acids and fatty acid oxidation in higher eukaryotes and is toxic, thus, inhibiting the cell growth [112]. Methylcitrate cycle metabolizes it into pyruvate, which can be used as a source of carbon [58]. Methylcitric acid and methylisocitric acid are important intermediates in this cycle.

2.2.8. Sugars, Sugar Alcohols, Disaccharides

The presence of mannitol and fucose (Table 2) was confirmed using authentic standards (Figure 2). Mannitol is widely distributed in filamentous fungi and stored in the fungal hyphae as a carbon source [32]. Fucose appears to represent a prominent feature in protein-linked glycans in the fungal kingdom [45]. Additionally, disaccharides, such as the one isolated from pathogenic fungal species Claviceps africans, with fructofuranose and arabinose backbone [56] were found. Deoxyhexoses, then again, are produced in fungi by pyranose oxidases, which have been reported among lignin-degrading fungi [68] for example. Deoxyhexose yielded fragment ions corresponding to the cleavage of water, whereas dehydrohexose structure was detected by its sodium and ammonium adducts and by [2M + H]+ and [2M − H]− ions. Dehydrohexose has also been previously reported from evergreen trees [67].

2.2.9. Endophyte or Plant Metabolites

Phomone A and B are enantiometric α-pyrone dimers isolated from the endophytic fungus Phoma sp. YN02-p-3 [81,82]. Blumeoside C, which is an iridoid glucoside isolated from Fagraea blumei [80] has the same molecular formula. Cuendet et al. [80] discovered that Blumeoside A elutes later than Blumeoside C, which is in accordance with our findings (Table 2).
Asperulosidic acid and its stereoisomer were isolated from the plant Hedyotis diffusa using water extraction [89]. According to Friscic et al. [90], asperulosidic acid elutes later than mannitol using reversed-phase liquid chromatography as in our study (Table 2). Asperulosidic acid has also been isolated from Vernonia cinerea with ethanol [91] and its structural isomers from Morinda coreia and Saprosma scortechinii with methanol [87,88].
Furthermore, we were able to find exact masses corresponding to orsellinic acid esters, which have been isolated from the endophytic Chaetomium sp. fungus [16,95,96,97]. However, orsellinic acid ester Globosumone B was not included in the Table 2 because of the chosen intensity limit 1 × 107.
Two possible triterpene saponin structures were obtained with the molecular formula C35H50O12, one could be Dianthosaponin F, which has been isolated from Dianthus japonicus with methanol [98], and the other Celosin F, which has been isolated from Celosia argentea with 50% ethanol [99].
Linamarin is a cyanogenic glucoside isolated from the cassava (Manihot esculenta) roots [94]. Ramulosin derivatives have been previously isolated from endophytic fungi Nigrospora sp. Present in the branches of Garcinia nigrolineata tree [16,100].

2.3. Metabolites in Fungal Extracts

We conducted a qualitative study on the screening and identification of the water-soluble metabolites from the endophytic fungi extracts. There was a high number of primary metabolites in the aqueous extracts as expected. The number of identified metabolites was almost the same with all of the fungal species, and a majority of the metabolites, 141 compounds, were detected from all of the fungal extracts (Figure 4). From the extract A (A. applanata), we identified 177 metabolites and out of these 12 metabolites were exclusively found in fungus A (Figure 4). These twelve were all dipeptides or peptides except the nucleoside derivative 5-methoxycarbonylmethyluridine (Table 2). From the extract of fungus R (P. fortinii) 184 metabolites were identified and 15 of the metabolites were found only in this extract. These included fucose, guanine, acetylcitrulline, disaccharides, dinucleotides, acetylleucine or acetylisoleucine, the endophytic fungi metabolite orsellinic acid ester and the plant metabolites blumeoside A and asperulosidic acid as well as dipeptides and peptides. From the extract of fungus S16 (H. cephalosporioides or C. mutabilis) we identified 177 metabolites and 16 of these were found in the S16 extract only. These included hexosevaline, pseudouridine, acetylglutamic acid, cUMP, NAD and saponin as well as Ala-Glu or Glu-Ala or heliopine and other dipeptides and peptides.
In this study, we identified a large number of water-soluble metabolites that may make a contribution to nutrient intake or stress-resistance of the host plant. Many of the identified compounds have been previously reported possessing interesting bioactivities. Thus, the bioactive properties of the fungal isolates and their sub-fractions are to be investigated in the future for their antimicrobial and antioxidant properties to evaluate their potential for the host plant vitality and other applications. To our knowledge, this is the first time that metabolic profiling is conducted on these Scots pine associated endophytic fungi species using water extracts. Thus, this work offers valuable reference about the metabolites of similar endophytes, which are to be discovered in the future.

3. Materials and Methods

3.1. Reagents

Ala-Phe, Ala-Tyr, Asp-Phe (methyl ester), Leu-Leu (acetate), Leu-Pro (hydrochloride), Phe-Ala, Pro-Gly, Pro-Leu, Tyr-Ala, Val-Tyr, guanine, cytidine, uridine, guanosine (hydrate), β-(–)-adenosine, d-mannitol, l-tyrosine, l-arginine (hydrochloride), l-phenylalanine, l-(–)-fucose and trans-3-indoleacrylic acid were obtained from Sigma-Aldrich (Saint Louis, MO, USA) each with purity ≥98%. l-α-Glycerophosphorylcholine (purity 99%) and Choline-O-sulphate (D13, purity 98%) were purchased from Carbosynth Limited, Compton, UK. DL-Ala-DL-Leu (purity ≥95%), DL-Ala-DL-Met, DL-Ala-DL-Val, l-Ala-l-Gln (HPLC grade), l-Ala-l-Trp, l-Ala-l-Pro (purity >96%), Gly-l-Ile (purity >99%), Gly-DL-Leu, Gly-l-Phe (HPLC grade), Gly-l-Pro (HPLC grade), DL-Leu-Gly, l-Leu-l-Tyr, DL-Leu-DL-Val, l-Leu-l-Ala (hydrate), and Salicin (HPLC grade) were obtained from TCI Europe, Zwijndrecht, Belgium, with purity >98% unless specified. l-(–)-tryptophan (purity >99%) was purchased from Acros Organics/Thermo Fisher Scientific, Waltham, MA, USA. Hydrogen peroxide and citric acid (monohydrate, purity >99%) were obtained from Merck KGaA, Darmstadt, Germany, and ethanol from Altia, Helsinki, Finland.

3.2. Endophytic Fungi Isolation and Identification

The fungal endophytes were originally isolated from the roots of eight-year-old Scots pine (Pinus sylvestris L.) trees grown on a drained peatland forest site in western Finland. Scots pine roots were washed and the root tips were examined under a dissecting microscope. The root tips showing signs of potential fungal association or mycorrhizal features were selected for surface sterilization with a short bath in 70% ethanol and 30% H2O2 and followed by laying on sterile Petri dishes on agar. The pure cultures of the fungus mycelium were cultivated on a solid Hagem agar [113] on Petri dishes.
The species of the fungus isolates were identified with molecular methods. DNA from the fungus mycelium was extracted using E.Z.N.A. Fungal DNA Mini Kit (Omega bio-tek, Norcross, GA, USA) according to the manufacturer’s instructions. The nucleotide sequence of the Internal Transcribed Spacer (ITS) region of fungal ribosomal DNA (rDNA) was analysed in Macrogen Inc. (Amsterdam) from polymerase chain reaction (PCR) product amplified with ITS1 and ITS4 primer pair [114]. The reaction mixture of 50 µL included, 10x enzyme buffer (Biotools B&M Labs, S.A. Madrid, Spain), 0.5 µM each primers, 0.2 µM dNTP mix, DNA Polymerase (5 U/µL) (Biotools B&M Labs, S.A. Madrid, Spain) and 1 µL DNA template. The PCR were performed with the following conditions: initial incubation at 94 °C for 5 min followed by 25 cycles of 1 min at 94 °C, 1 min at 58 °C and 1.5 min at 72 °C. Sequences with a similarity of >99% to ITS1, 5.8S and ITS2 rDNA regions were considered as identical species [115]. ITS1, 5.8S and ITS2 regions were extracted from the fungal ITS sequences and the cleaned sequences were used for BLAST searches against GenBank/NCBI to provide taxonomic identification. The best matches from GenBank were aligned and a phylogenetic tree was generated in Geneious 6.0.6 using the Neighbor-Joining analysis (Figure 1). The sequences were deposited in GenBank with accession numbers KM068384, KJ649992 and KJ649998.

3.3. Fungal Extract Preparation

The fungal mass was collected from the surface of a cellophane membrane on agar [113] with a scalpel, stored at −80 °C, and ground in a mortar before adding to sealed, sterile and previously weighed polypropylene test tubes (BD Falcon™, VWR International Oy, Helsinki, Finland). The extraction was executed with boiling deionized and filtered (0.2 µm, Nylon 66 Filter Membrane from Supelco by Sigma–Aldrich Co, Saint Louis, MO, USA) water. The fungal mass was mixed with equal amount of boiling deionized and filtered (0.45 µm Nylon membrane, Supelco Analytical/Sigma Aldrich, Saint Louis, MO, USA) water (1 mL = 1 g) by vortexing. Extraction tubes were then shaken in +95–100 °C water bath (SW22, JULABO Labortechnik GmbH, Seelbach, Germany) for 15 minutes after which the tubes were cooled in an ice bath before vortexing again. Extraction tubes were then centrifuged at +4 °C and 8200 g for 10 minutes (Eppendorf Centrifuge 5804R, Hamburg, Germany). The supernatants were collected into new polypropylene tubes and centrifuged again with the same settings. Finally, the supernatants were filtered through nylon syringe filters (0.2 µm, Cronus Filter from SMI–LabHut Ltd., Gloucester, UK) to new polypropylene tubes. Aliquots of water without fungal material were extracted simultaneously as control samples. The pH of the extracts was measured with indicator paper (scale 1–14) and it ranged from 5.5 to 7. Extracts were dried with freeze-drying equipment (VirTis BenchTop 6K with Trivac E2, D 2,5E Vacuum pump, SP Industries, Warminster, PA, USA) before storing at −80 °C. The dried extracts were dissolved in sterile purified water before analysis.

3.4. UHPLC-DAD-ESI-Orbitrap-MS

After vacuum drying, 20 µL of ethanol and 980 µL of water were added to the samples. The samples were then mixed with a vortex and filtered using 0.2 µm PTFE syringe filter prior to analyses. The samples were analyzed using an ultra-high performance liquid chromatograph coupled to a photodiode array detector (UHPLC–DAD, Acquity UPLC, Waters Corporation, Milford, MA, USA) and a hybrid quadrupole-Orbitrap mass spectrometer (Q Exactive™, Thermo Fisher Scientific GmbH, Bremen, Germany). The column was Acquity UPLC® BEH Phenyl (100 × 2.1 mm i.d.; 1.7 µm; Waters Corporation, Wexford, Ireland). The mobile phase consisted of (A) acetonitrile and (B) water and formic acid (99.9:0.1, The elution profile was as follows: 0–0.5 min, 0.1% A; 0.5–5.0 min, 0.1–30% A (linear gradient); 5.0–5.1 min, 30–90% A (linear gradient); 5.1–7.1 min, 90% A; 7.1–7.2 min, 90–0.1% A (linear gradient); 7.2–8.5 min, 0.1% A. The injection volume was 5 µL and flow rate 0.5 mL/min. The UV data was collected at 190–500 nm. The heated ESI source (H–ESI II, Thermo Fisher Scientific GmbH, Bremen, Germany) was operated both in negative and positive ion modes. The parameters for negative ionization were as follows: spray voltage was set at –3.0 kV, sheath gas (N2) flow rate at 60 (arbitrary units), aux gas (N2) flow rate at 20 (arbitrary units), sweep gas flow rate at 0 (arbitrary units), capillary temperature at +380 °C and S-lens RF level at 60. The parameters for positive ionization were similar, except that spray voltage was set at 3.8 kV. Orbitrap was set at a resolution of 70,000 and an automatic gain of 3 × 106 was used. Masses were scanned at m/z 150–2000. Pierce ESI Negative Ion Calibration Solution (Thermo Fischer Scientific Inc., Waltham, MA, USA) was used to for the calibration. The data was processed with Thermo Xcalibur Qual Browser software (Version 3.0.63, Thermo Fisher Scientific Inc., Waltham, MA, USA).

3.5. Identification

Orbitrap data was processed using Compound Discoverer 2.1 SP1 (Thermo-Fisher Scientific, Waltham, MA, USA). The processing flow ‘Untargeted Metabolomics Workflow’ was utilized. The following general settings were used for the workflow: mass tolerance = 5 ppm, intensity threshold = 30%, S/N threshold = 3, minimum peak intensity = 1 × 106, maximum element counts = 100 × C, 200 × H, 10 × N, 100 × O, 10 × S and 10 × P. The following settings were used for the peak detection: filter peaks = true, maximum peak width = 0.5 min, remove singlets = true, minimum # scans per peak = 5 and minimum # isotopes = 1. ChemSpider and KEGG databases were used for the identification. In addition, we used SciFinder Scholar database (American Chemical Society, CAS, Columbus, OH, USA) with substance role Occurrence and highest number of references to scale down possible compound hits.

Supplementary Materials

The following are available online. Supplementary Figure S1: Alignment of ITS region of S16 strain (KJ649998) with its best GenBank matches Coniochaeta mutabilis (DQ93680) and Humicolopsis cephalosporioides (KC128659). Supplementary Figure S2: Alignment of ITS region of A (KM068384) and R (KJ649992) strains with their best GenBank matches Acephala applanata (AY078147) and Phialocephala fortinii (AB671499.2 and AY033087), respectively. Supplementary Table S1: The unidentified metabolites.

Author Contributions

Conceptualization, M.K. (Maarit Karonen), K.W., R.F., M.K. (Matti Karp), V.S. and T.S.; Data curation, J.T. and M.K. (Maarit Karonen); Formal analysis, J.T., M.K. (Maarit Karonen), R.M.-M. and E.L.D.; Funding acquisition, R.F., M.K. (Matti Karp) and T.S.; Investigation, J.T., M.K. (Maarit Karonen), R.M.-M. and E.L.D.; Methodology, M.K. (Maarit Karonen), K.W. and T.S.; Project administration, R.F., M.K. (Matti Karp), V.S. and T.S.; Resources, M.K. (Maarit Karonen), K.W., M.K. (Matti Karp), V.S. and T.S.; Supervision, M.K. (Maarit Karonen), K.W., R.F., M.K. (Matti Karp), V.S. and T.S.; Validation, J.T., M.K. (Maarit Karonen), K.W. and T.S.; Visualization, J.T., M.K. (Maarit Karonen) and R.M.-M.; Writing—original draft, J.T. and M.K. (Maarit Karonen); Writing—review & editing, J.T., M.K. (Maarit Karonen), R.M.-M., K.W., E.L.D., R.F., M.K. (Matti Karp), V.S. and T.S.

Funding

This research was partly funded by COST Action FA1103: Endophytes in Biotechnology and Agriculture in the form of a short-term scientific mission fund of J. Tienaho. This work has also been funded by the European Regional Development Fund (project code A71142) as well as the town of Parkano and SASKY municipal education and training consortium. Natural Resources Institute Finland and Tampere University are also warmly acknowledged for their financial support. In addition, J. Tienaho is grateful for the personal fund by Kone Foundation.

Acknowledgments

A. Käenmäki, H. Leppälammi and E. Pihlajaviita are kindly acknowledged for their technical assistance. J. Tienaho also thanks Jesús Martín and Olga Genilloud from Fundación MEDINA, Granada, Spain as well as other team members, who welcomed and guided her warmly to their daily work during her short-term scientific mission. Their help with the initial metabolite identification is also respectfully valued.

Conflicts of Interest

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision of publishing the results.

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Sample Availability: Samples of the compounds are not available from the authors.
Molecules 24 02330 g001 550
Figure 1. The Neighbor-Joining topology of ITS1, 5.8S and ITS2 rDNA sequences of root endophyte strains A, R and S16 from Scots pine and those obtained from GenBank. The phylogenetic tree was rooted with the Coniochaeta mutabilis.
Figure 1. The Neighbor-Joining topology of ITS1, 5.8S and ITS2 rDNA sequences of root endophyte strains A, R and S16 from Scots pine and those obtained from GenBank. The phylogenetic tree was rooted with the Coniochaeta mutabilis.
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Molecules 24 02330 g002 550
Figure 2. Structures of some of the identified compounds with #ID from Table 2.
Figure 2. Structures of some of the identified compounds with #ID from Table 2.
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Molecules 24 02330 g003 550
Figure 3. Dipeptides, whose presence was verified with authentic standards and #ID from Table 2.
Figure 3. Dipeptides, whose presence was verified with authentic standards and #ID from Table 2.
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Molecules 24 02330 g004 550
Figure 4. A Venn diagram of the 220 identified metabolites and how they are distributed among the fungal species A (A. applanata), R (P. fortinii) and S16 (H. cephalosporioides or C. mutabilis).
Figure 4. A Venn diagram of the 220 identified metabolites and how they are distributed among the fungal species A (A. applanata), R (P. fortinii) and S16 (H. cephalosporioides or C. mutabilis).
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Table
Table 1. The endophytic fungus isolates and NCBI information about the best match and our identification.
Table 1. The endophytic fungus isolates and NCBI information about the best match and our identification.
Strain (GenBank Accession NO.)GenBank Accession NO. for the Best MatchMax Identity (%)/Query Coverage (%)Our Description for the StrainOrderClassPhylum
A (KM068384)AY078147.1100/98Acephala applanataHelotialesLeotiomycetesAscomycota
R (KJ649992)AB671499.2100/100Phialocephala fortiniiHelotialesLeotiomycetesAscomycota
S16 (KJ649998)KC12865999/98Humicolopsis cephalosporioides Ascomycota
DQ9368099/98Coniochaeta mutabilisConiochaetalesSordariomycetesAscomycota
Table
Table 2. The identified metabolites from the aqueous endophytic fungi extracts. Metabolomics Standards Initiative (MSI) was used to define the metabolite identification confidence [21,22]. Level 1 is for confidently identified compounds, where an authentic chemical standard was analyzed under same analysis conditions. Level 2 is for putatively annotated compounds, where physicochemical properties and spectral similarities with public spectral libraries as well as listed references were used. Level 3 is for putatively annotated compound classes, where characteristic physicochemical properties or spectral similarities of compound classes are used to confirm identity. Unidentified compounds are shown in the Supplementary Table S1 and their identification is level 4: unidentified and unclassified but can be differentiated based upon spectral data. Listed references were used to confirm the identification for example by similar findings from natural sources and further justifications are described after the table. If the observed intensity of the metabolite in the total ion chromatogram is over 1 × 107, metabolite is marked with x under the corresponding fungal extract. nd = not detected. RDB = ring-double bond equivalent.
Table 2. The identified metabolites from the aqueous endophytic fungi extracts. Metabolomics Standards Initiative (MSI) was used to define the metabolite identification confidence [21,22]. Level 1 is for confidently identified compounds, where an authentic chemical standard was analyzed under same analysis conditions. Level 2 is for putatively annotated compounds, where physicochemical properties and spectral similarities with public spectral libraries as well as listed references were used. Level 3 is for putatively annotated compound classes, where characteristic physicochemical properties or spectral similarities of compound classes are used to confirm identity. Unidentified compounds are shown in the Supplementary Table S1 and their identification is level 4: unidentified and unclassified but can be differentiated based upon spectral data. Listed references were used to confirm the identification for example by similar findings from natural sources and further justifications are described after the table. If the observed intensity of the metabolite in the total ion chromatogram is over 1 × 107, metabolite is marked with x under the corresponding fungal extract. nd = not detected. RDB = ring-double bond equivalent.
Exact MassError Fungal Extract
#IDRT[M + H]+[M − H]−MeasuredCalculatedΔm (ppm)Molecular FormulaRDBCompoundClassMSIReferencesARS16
10.46175173174.11199174.111681.8C6H14O2N42ArginineAmino acid1[23,24,25,26]xxx
20.49337335336.16316336.16450−4.0C12H24O7N43HexosearginineAmadori1[27,28,29]xxx
30.51335333334.06709334.066501.8C9H19O11P2Glycerophospho-inositolCommon metabolite2[30,31]x-x
40.52372370371.22764371.22810−1.2C15H29O4N75Ace-Ala-Arg-Ala-NMePeptide2 xxx
50.53183181182.07875182.07904−1.6C6H14O61MannitolHexitol1[24,26,32]xxx
60.53246244245.14851245.14879−1.1C9H19O3N53Ala-Arg or Arg-AlaDipeptide2 -xx
70.54nd165166.04668166.04774−6.4C5H10O61Pentonic acidPentonic acid3[26]xxx
80.54nd195196.05748196.05831−4.2C6H12O71Hexonic acidHexonic acid3[26]xxx
90.54218216217.10586217.10626−1.8C8H15O4N33Ala-GlnDipeptide1 --x
100.55203201202.14253202.14298−2.2C8H18O2N42DimethylarginineAmino acid derivative2[25,33]xxx
110.55219217218.08984218.09027−2.0C8H14O5N23Ala-Glu or Glu-Ala or HeliopineDipeptide or Opine amino acid2[34]--x
120.55244242243.08558243.085520.2C9H13O5N35CytidineNucleoside1[25,26,35,36,37,38,39,40]xxx
130.55248246247.11641247.11682−1.7C9H17O5N33Gln-Thr or Thr-GlnDipeptide2 --x
140.55258nd257.10240257.10282−1.6C8H20O6NP1Glycerophosphoryl-cholineCholine1[25,41]xxx
150.56191189190.09479190.09536−3.0C7H14O4N22Ala-Thr or Thr-AlaDipeptide2 xxx
160.56235°nd234.15747234.15796−2.1C10H22O4N21ValineAmino acid2[23,24,26]-xx
170.56253251252.12182252.12224−1.7C11H16O3N46His-Pro or Pro-HisDipeptide2 xxx
180.56266264265.11662265.116151.8C10H19O7N2Hexoseaminobutyric acidAmadori3[42]xx-
190.57205203204.11052204.11101−2.4C8H16O4N22Ser-Val or Val-SerDipeptide2 -xx
200.58187185186.10007186.10044−2.0C8H14O3N23Pro-AlaDipeptide2 --x
210.58198196197.07971197.08004−1.7C8H11O3N35AcetylhistidineAmino acid derivative2 -xx
220.58219217218.12640218.12666−1.2C9H18O4N22Dipeptidea or Lysopine or RideopineDipeptide or Opine amino acid3/2[34,43]xxx
230.58246244245.13727245.13756−1.2C10H19O4N33DipeptidebDipeptide3 xxx
240.58260258259.18913259.18959−1.8C12H25O3N3 2DipeptidecDipeptide3 xxx
250.59184nd183.05615183.05653−2.1C5H13O4NS2Choline-O-sulphateCholine1[41,44]xxx
260.60233231232.10553232.10592−1.7C9H16O5N23Asp-Val or Val-AspDipeptide2 xxx
270.60335333334.06584334.06650−2.0C9H19O11P2Glycerophospho-inositolCommon metabolite2[30,31]xxx
280.61165163164.06831164.06848−1.0C6H12O51FucoseHexose1[45]-x-
290.64191189190.09482190.09536−2.8C7H14O4N22Ala-Thr or Thr-AlaDipeptide2 xx-
300.64219217218.12619218.12666−2.2C9H18O4N22Dipeptidea or Lysopine or RideopineDipeptide or Opine amino acid3/2[34,43]-xx
310.64219217218.08980218.09027−2.2C8H14O5N23Ala-Glu or Glu-Ala or HeliopineDipeptide or Opine amino acid2[34]--x
320.64nd565566.05592566.055021.6 C15H24O17N2P28UDP-galactoseNucleotide sugar2[46,47]xxx
330.65217215216.12187216.12224−1.7C8H16O3N43AcetylarginineAmino acid derivative2[26]xxx
340.66255nd254.08980254.09027−1.8C11H14O5N26Nicotinamide ribosidePyridine nucleoside2[48]xx-
350.67152150151.04916151.04941−1.7C5H5ON56GuanineNucleobase1[35,38]-x-
360.67247245246.12114246.12157−1.7C10H18O5N23DipeptidedDipeptide3 xxx
370.68219217218.08995218.09027−1.5C8H14O5N23Ala-Glu or Glu-Ala or HeliopineDipeptide or Opine amino acid2[34]--x
380.68608606607.08235607.081571.3C17H28O17N3P28UDP-galactosamineNucleotide sugar2[45,49]xxx
390.69247245246.12112246.12157−1.8C10H18O5N23DipeptidedDipeptide3 xxx
400.69288286287.19514287.19574−2.1C12H25O3N53DipeptideeDipeptide3 xxx
410.71189187188.07948188.07971−1.2C7H12O4N23AceglutamideAmino acid derivative2 xxx
420.72193191192.02628192.02701−3.8C6H8O74IsocitrateCommon metabolite2[50]xxx
430.72280278279.13127279.1318−1.9C11H21O7N2HexosevalineAmadori2[28,29]--x
440.73246244245.13705245.13756−2.1C10H19O4N33DipeptidebDipeptide3 xxx
450.74348346347.06239347.06309−2.0C10H14O7N5P8AMP or dGMPNucleotide2[51,52,53]-xx
460.74nd565566.05580566.055021.4C15H24O17N2P28UDP-glucoseNucleotide sugar2[46,47,53,54]xxx
470.77204nd203.11554203.11576−1.1C9H17O4N2AcetylcarnitineCommon metabolite2[50,55]xxx
480.77219217218.12622218.12666−2.0C9H18O4N22Dipeptidea or Lysopine or RideopineDipeptide or Opine amino acid3/2[34,43]xxx
490.77333331332.13261332.13320−1.8C12H2O7N45Tripeptide/peptidefTripeptide or peptide3 xx-
500.78205203204.11084204.11101−0.8C8H16O4N22Ser-Val or Val-SerDipeptide2 xxx
510.78608606607.08248607.081571.5C17H27O17N3P28UDP-glucoseamineNucleotide sugar2[46,49]xxx
520.80187185186.10017186.10044−1.5C8H14O3N23Ala-ProDipeptide1[26]xxx
530.80193191192.02628192.02701−3.8C6H8O74Citric acidCommon metabolite1[24,26]xxx
540.80260258259.18922259.18959−1.4C12H26O3N3 2DipeptidecDipeptide3 xxx
550.80315313314.12205314.12132.4C11H22O101DisaccharideDisaccharide3[56]-x-
560.81189187188.11563188.11609−2.4C8H16O3N22DipeptidegDipeptide3 xxx
570.81288286287.19523287.19574−1.8C12H25O3N53DipeptideeDipeptide3 -xx
580.81333331332.13261332.13320−1.8C12H20O7N45Tripeptide/peptidefTripeptide or peptide3 xx-
590.82219217218.12630218.12666−1.7C9H18O4N22Dipeptidea or Lysopine or RideopineDipeptide or Opine amino acid3/2[34,43]x-x
600.84260258259.15272259.15321−1.9C11H21O4N33Di-/TripeptidehDi-/Tripeptide3 xxx
610.85219217218.12633218.12666−1.5C9H18O4N22Dipeptidea or Lysopine or RideopineDipeptide or Opine amino acid3[34,43]xxx
620.85233231232.14192232.14231−1.7C10H20O4N22Dipeptidei Dipeptide3 xxx
630.85260258259.18870259.18959−3.4C12H26O3N3 2DipeptidecDipeptide3 xxx
640.86221219220.08778220.08817−1.8C8H16O3N2S2Cys-Val or Val-Cys or Met-AlaDipeptide2 xxx
650.86245243244.06948244.06954−0.2C9H12O6N25PseudouridineNucleoside2[25,35]--x
660.86247245246.12114246.12157−1.7C10H18O5N23DipeptidedDipeptide3 xxx
670.87182180181.07374181.07389−0.8C9H11O3N5TyrosineAmino acid1[23,24,25,26,55]xxx
680.87nd205206.04209206.04266−2.8C7H10O73Methylisocitric acidCommon metabolite2[57,58]xxx
690.89260258259.15288259.15321−1.3C11H21O4N33Di-/TripeptidehDi-/Tripeptide3 xx-
700.91190188189.06334189.06372−2.0C7H11O5N3Acetylglutamic acidAmino acid derivative2 -xx
710.91233231232.14152232.14231−3.4C10H20O4N22Dipeptidei Dipeptide3 xxx
720.91260258259.18907259.18959−2.0C12H26O3N3 2DipeptidecDipeptide3 xxx
730.93233231232.10574232.10592−0.8C9H16O5N23Asp-Val or Val-AspDipeptide2 xxx
740.95nd205206.04215206.04266−2.5C7H10O74Methylcitric acidCommon metabolite2[57,58]xxx
750.95218216217.10599217.10626−1.2C8H15O4N33AcetylcitrullineAmino acid derivative2 -x-
760.97190188189.06359189.06372−0.7C7H11O5N3Acetylglutamic acidAmino acid derivative2 --x
770.97307305306.02467306.02530−2.1C9H11O8N2P72’,3’-cUMP or 3’,5’-cUMPNucleotide2[59,60,61,62]--x
780.99245243244.06915244.06954−1.6C9H12O6N25UridineNucleoside1[35,36,38,39,63]xxx
790.99288286287.19481287.19574−3.2C12H25O3N53DipeptideeDipeptide3 xxx
800.99332330331.06749331.06817−2.0C10H14O6N5P8dAMPNucleotide2[52,53,64]-xx
811.01253251252.11056252.11101−1.8C12H16O4N26Ala-TyrDipeptide1 xxx
821.03261259260.13679260.13722−1.6C11H20O5N23DipeptidejDipeptide3 xxx
831.04203201202.13162202.13174−0.6C9H18O3N22Leu-AlaDipeptide1 xxx
841.04215213214.13141214.13174−1.5C10H18O3N23Pro-Val or Val-ProDipeptide2 xxx
851.08221219220.08783220.08817−1.5C8H16O3N2S2Cys-Val or Val-Cys or Met-AlaDipeptide2 xxx
861.10261259260.13679260.13722−1.6C11H20O5N23DipeptidejDipeptide3 xxx
871.10664662663.10777663.10912−2.0C21H27O14N7P2 15Nicotinamide adenine dinucleotideDinucleotide2[48]--x
881.11260258259.15306259.15321−0.6C11H21O4N33Di-/TripeptidehDi-/Tripeptide3 x--
891.17202nd201.11101201.11134−1.6C8H15O3N33Acetylmethyl-glutaminamideAmino acid derivative2 x--
901.17403401402.22194402.22268−1.8C16H30O6N65Tripeptide/peptidek Tripeptide or peptide3 x--
911.18613611612.15080612.15197−1.9C20H32O12N6S28Glutathione dimerTripeptide2[17,55,65]x-x
921.23315313314.12193314.121302.0C11H22O101DisaccharideDisaccharide3[56]xx-
931.24189187188.11571188.11609−2.0C8H16O3N22DipeptidegDipeptide3 xxx
941.24269267268.10536268.10592−2.1C12H16O5N26Ser-Tyr or Tyr-SerDipeptides2 xxx
951.30268266267.09608267.09676−2.5C10H13O4N57AdenosineNucleoside1[25,26,35,36,37,38,40,55,63]xxx
961.33315313314.12166314.121301.2C11H22O101DisaccharideDisaccharide3[56]-x-
971.33542540541.05998541.06111−2.1C15H21O13N5P211Cyclic ADP-riboseNucleotide2[66]xx-
981.40179177178.04742178.04774−1.8C6H10O62DehydrohexoseDehydro-hexose3[67]xxx
991.42221219220.08820220.088170.1C8H16O3N2S2Ala-MetDipeptide1 --x
1001.47346344345.04695345.04744−1.4C10H12O7N5P92’,3’-cGMP or 3’,5’-cGMPNucleotide2[25,38,51,59,60,61,62]xxx
1011.48219217218.12633218.12666−1.5C9H18O4N22Dipeptidea or Lysopine or RideopineDipeptide or Opine amino acid2/3[34,43]xxx
1021.50267265266.12611266.12666−2.1C13H18O4N2 6DipeptidelDipeptide3 xxx
1031.50284282283.09056283.09167−3.9C10H13O5N57Guanosine isomerNucleoside2[26,35,37,39]xxx
1041.54346344345.04701345.04744−1.2C10H12O7N5P92’,3’-cGMP or 3’,5’-cGMPNucleotide2[25,38,51,59,60,61,62]x-x
1051.57165163164.06821164.06848−1.6C6H12O51DeoxyhexoseDeoxyhexose3[68]xx-
1061.61246244245.13713245.13756−1.7C10H19O4N33DipeptidebDipeptide3 xxx
1071.64219217218.12628218.12666−1.7C9H18O4N22Dipeptidea or Lysopine or RideopineDipeptide or Opine amino acid3/2[34,43]xxx
1081.64267265266.12620266.12666−1.7C13H18O4N2 6DipeptidelDipeptide3 xxx
1091.67260258259.15294259.15321−1.0C11H21O4N33Di-/TripeptidehDi-/Tripeptide3 xxx
1101.68294292293.13692293.13756−2.2C14H19O4N37Di/tripeptidemDi-/Tripeptide3 xxx
1111.69233231232.14200232.14231−1.3C10H20O4N22Dipeptidei Dipeptide3 xxx
1121.71281279280.10530280.10592−2.2C13H16O5N27Abenquine C or enantiomerAA quinone2[69,70]xxx
1131.72189187188.11572188.11609−2.0C8H16O3N22DipeptidegDipeptide3 xxx
1141.74152150151.04925151.04941−1.0C5H5ON56Isoguanine or OxyadenineNucleobase2[71,72]-xx
1151.74246244245.13727245.13756−1.2C10H19O4N33DipeptidebDipeptide3 xxx
1161.74279277278.12642278.12666−0.9C14H18O4N27Pro-Tyr or Tyr-ProDipeptide2 xxx
1171.74288286287.19527287.19574−1.6C12H25O3N53DipeptideeDipeptide3 --x
1181.75261259260.13698260.13722−0.9C11H20O5N23Fusarinine monomerSiderophore2[73,74,75,76,77]xxx
1191.75268266267.09639267.09676−1.4C10H13O4N57DeoxyguanosineNucleoside2[35]xxx
1201.75318316317.15840317.15869−0.9C13H23O6N34Tripeptide/peptidenTripeptide/ peptide3 xxx
1211.76166164165.07893165.07898−0.3C9H11O2N5PhenylalanineAmino acid1[23,24,26,55]xxx
1221.76189187188.11574188.11609−1.8C8H16O3N22DipeptidegDipeptide3 --x
1231.76260258259.15306259.15321−0.6C11H21O4N33Di-/TripeptidehDi-/Tripeptide3 xxx
1241.78257255256.10554256.10592−1.5C11H16O5N25MethylthymidineNucleoside derivative2[78]xxx
1251.78266264265.11577265.11615−1.4C10H19O7N2Deoxyhexose-threonine Amadori3[79]xx-
1261.80203201202.13164202.13174−0.5C9H18O3N22Ala-LeuDipeptide1 xxx
1271.80260258259.15303259.15321−0.7C11H21O4N33Di-/TripeptidehDi-/Tripeptide3 xxx
1281.82189187188.11586188.11609−1.2C8H16O3N22DipeptidegDipeptide3 xxx
1291.82215213214.13157214.13174−0.8C10H18O3N23Pro-Val or Val-ProDipeptide2 xxx
1301.82233231232.14207232.14231−1.0C10H20O4N22Dipeptidei Dipeptide3 xxx
1311.85307305306.02538306.025300.3C9H11O8N2P72’,3’-cUMP or 3’,5’-cUMPNucleotide2[59,60,61,62]xxx
1321.86260258259.15312259.15321−0.3C11H21O4N33Di-/TripeptidehDi-/Tripeptide3 x-x
1331.86348346347.16926347.169250.0C14H25O7N34TripeptideoTripeptide3 x--
1341.87249247248.11934248.11947−0.5C10H20O3N2S2Met-Val or Val-MetDipeptide2 xxx
1351.89261259260.13682260.13722−1.5C11H20O5N23Fusarinine monomerSiderophore2[73,74,75,76,77]xxx
1361.89281279280.14177280.14231−1.9C14H20O4N26Val-TyrDipeptide1 xxx
1371.89295293294.12105294.12157−1.8C14H18O5N27Peptide type compoundpDipeptide or peptide derivative3[69,70]xxx
1381.90318316317.15846317.15869−0.7C13H23O6N34Tripeptide/peptidenTripeptide/ peptide3 x--
1391.90317315316.09129316.090672.0C12H16O8N275-methoxycarbonyl-methyluridineNucleoside derivative2[39,40]x--
1401.91229227228.14731228.14739−0.3C11H20O3N23Pro-LeuDipeptide1 xxx
1411.91247245246.12143246.12157−0.6C10H18O5N23DipeptidedDipeptide3 xxx
1421.95249247248.11934248.11947−0.5C10H20O3N2S2Met-Val or Val-MetDipeptide2 xxx
1431.96231229230.16318230.163040.6C11H22O3N22DipeptideqDipeptide3 xxx
1441.96237235236.11595236.11609−0.6C12H16O3N26Ala-PheDipeptide1 xxx
1451.96348346347.16933347.169250.2C14H25O7N34TripeptideoTripeptide3 xx-
1461.99223221222.10043222.100440.0C11H14O3N26Gly-PheDipeptide1 xxx
1471.99246244245.13786245.137561.2C10H19O4N33DipeptidebDipeptide3 x--
1482.02541539540.14854540.147911.2C24H28O1411Phomone A or B or Blumeoside CEndophyte or plant metabolite 2[80,81,82]-xx
1492.06355353354.15419354.153940.7C15H22O6N47PeptiderPeptide3 --x
1502.07581579580.15391580.15436−0.8C20H25O9N10P15DinucleotideDinucleotide3 -x-
1512.08229227228.14738228.147390.0C11H20O3N23Ile-Pro or Pro-IleDipeptide2 xxx
1522.09231229230.16290230.16304−0.6C11H22O3N22DipeptideqDipeptide3 xxx
1532.10247245246.12100246.12157−2.3C10H18O5N23DipeptidedDipeptide3 xxx
1542.10306304305.13731305.13756−0.8C15H19O4N38Thr-Trp or Trp-ThrDipeptide2 xxx
1552.10581579580.15428580.15436−0.1C20H25O9N10P15DinucleotideDinucleotide3 -x-
1562.14318316317.15843317.15869−0.8C13H23O6N34Tripeptide/peptidenTripeptide/ peptide3 xxx
1572.15253251252.11087252.11101−0.5C12H16O4N26Phe-Ser or Ser-PheDipeptide2[55]xxx
1582.15279277278.12630278.12666−1.3C14H18O4N27Pro-Tyr or Tyr-ProDipeptide2 xxx
1592.19455453454.15532454.15560−0.6C16H30O7N4S2 4PeptidesPeptide3 xx-
1602.20280278279.12166279.12191−0.9C13H17O4N37Di/tripeptidetDi-/Tripeptide3 xxx
1612.20318316317.15861317.15869−0.2C13H23O6N34Tripeptide/peptidenTripeptide/ peptide3 xxx
1622.22229227228.14723228.14739−0.7C11H20O3N23Leu-ProDipeptide1 xxx
1632.22345343344.13676344.13722−1.3C18H20O5N210Tyr-TyrDipeptide2 xxx
1642.22348346347.16899347.16925−0.7C14H25O7N34TripeptideoTripeptide3 x--
1652.24223221222.10048222.100440.2C11H14O3N26Phe-GlyDipeptide2 xxx
1662.26277275276.11095276.11101−0.2C14H16O4N28Peptide type compounduDipeptide/ cyclodipeptide3[83,84]xxx
1672.27294292293.13713293.13756−1.5C14H19O4N37Di/tripeptidemDi-/Tripeptide3 xxx
1682.27295293294.15724294.15796−2.4C15H22O4N26Leu-TyrDipeptide1 xxx
1692.28247245246.12108246.12157−2.0C10H18O5N23DipeptidedDipeptide3 xxx
1702.29231229230.16275230.16304−1.2C11H22O3N22DipeptideqDipeptide3 xxx
1712.29237235236.11557236.11609−2.2C12H16O3N26Gly-Phe or Phe-Gly methyl esterDipeptide derivative2 xxx
1722.30267265266.12630266.12666−1.3C13H18O4N2 6DipeptidelDipeptide3 xxx
1732.32229227228.14734228.14739−0.2C11H20O3N23Ile-Pro or Pro-IleDipeptide2 xxx
1742.32557555556.15074556.1428314.2 C24H28O15 11Blumeoside APlant metabolite2[80]-x-
1752.34205203204.08986204.08988−0.1C11H12O2N27TryptophanAmino acid1[23,24,25,26,55]xxx
1762.34227225226.09527226.09536−0.4C10H14O4N25DeoxythymidineNucleoside2[85]x-x
1772.34263261262.13484262.13512−1.1C11H22O3N2S2DipeptidevDipeptide3 xxx
1782.35261259260.13704260.13722−0.7C11H20O5N23DipeptidejDipeptide3 xxx
1792.37295293294.15764294.15796−1.1C15H22O4N26DipeptidewDipeptide3 xxx
1802.38281279280.10551280.10592−1.5C13H16O5N27Abenquine C or enantiomerAA quinone2[69,70]xxx
1812.39277275276.11095276.11101−0.2C14H16O4N28Peptide type compounduDipeptide/ cyclodipeptide3[83,84]xxx
1822.39295293294.12163294.121570.2C14H18O5N27Peptide type compoundpDipeptide or peptide derivative3[69,70]xxx
1832.40160158159.08949159.08954−0.3C7H13O3N2AcetylvalineAmino acid derivative2[26]xx-
1842.41276274275.12709275.126990.4C14H17O3N38Ala-TrpDipeptide1 xxx
1852.45261259260.13728260.137220.2C11H20O5N23DipeptidejDipeptide3 xxx
1862.45263261262.13499262.13512−0.5C11H22O3N2S2DipeptidevDipeptide3 xxx
1872.46295293294.15803294.157960.2C15H22O4N26DipeptidewDipeptide3 xxx
1882.50281279280.10603280.105920.4C13H16O5N27α-Asp-Phe Dipeptide1 xxx
1892.54245243244.17876244.178690.3C12H24O3N22Leu-LeuDipeptide1 xxx
1902.54281279280.10582280.10592−0.3C13H16O5N27β-Asp-Phe Dipeptide1 xx-
1912.55263261262.13402262.13512−4.2C11H22O3N2S2DipeptidevDipeptide3 xxx
1922.55265263264.14735264.14739−0.1C14H20O3N26Val-Phe or Phe-ValDipeptide2 xxx
1932.58253251252.12127252.12224−3.8C11H16O3N46His-Pro or Pro-HisDipeptide2 xxx
1942.59382380381.15388381.153600.7C17H23O7N38TripeptidexTripeptide3 xx-
1952.60293291292.10606292.105920.5C14H16O5N28Pyr-Tyr or Cyclo(Glu-​Tyr)​Dipeptide/ cyclo-dipeptide2[83]x--
1962.61192190191.06101191.06162−3.2C7H13O3NS2AcetylmethionineAmino acid derivative2[26,86]-xx
1972.66281279280.10557280.10592−1.2C13H16O5N27Phe-AspDipeptide2 xxx
1982.66295293294.15748294.15796−1.6C15H22O4N26DipeptidewDipeptide3 x-x
1992.69263261262.13505262.13512−0.3C11H22O3N2S2DipeptidevDipeptide3 xxx
2002.75276274275.12715275.126990.6C14H17O3N38Trp-AlaDipeptide2 xxx
2012.75433431432.12736432.126781.3C18H24O127Asperulosidic acid or isomerPlant metabolite2[87,88,89,90,91]-x-
2022.75518516517.23808517.23840−0.6C21H35O10N57Peptidey Peptide3 xx-
2032.77306304305.13740305.13756−0.5C15H19O4N38Thr-Trp or Trp-ThrDipeptide2 xxx
2042.78295293294.12150294.12157−0.2C14H18O5N27Peptide type compoundpDipeptide or peptide derivative3[69,70]xxx
2052.81265263264.14732264.14739−0.3C14H20O3N26Val-Phe or Phe-ValDipeptide2 xxx
2062.91248246247.10475247.10559−3.4C10H17O6N3Pentoseproline or Valinopine or LinamarinAmadori or Opine amino acid or Plant metabolite2[92,93,94]xxx
2072.99287285286.10512286.10526−0.5C13H18O75Orsellinic acid esterEndophytic fungi metabolite3[16,95,96,97]-x-
2083.01248246247.10585247.105591.1C10H17O6N3Pentoseproline or Valinopine or LinamarinAmadori or Opine amino acid or Plant metabolite2[92,93,94]xxx
2093.02528526527.29613527.295521.2C24H41O8N57PeptidezPeptide3 -x-
2103.11243241242.12691242.126661.0C11H18O4N24Dipeptide#Dipeptide3 x--
2113.22174172173.10513173.10519−0.3C8H15O3N2Acetylleucine or acetylisoleucineAmino acid derivative2[26]-x-
2123.29243241242.12608242.12666−2.4C11H18O4N24Dipeptide#Dipeptide3 x--
2133.37174172173.10538173.105191.1C8H15O3N2Acetylleucine or acetylisoleucineAmino acid derivative2[26]-x-
2143.37663661662.33214662.330232.9C35H50O1211SaponinSaponin3[98,99]--x
2153.50282280281.08955281.08994−1.4C13H15O6N7Phenylacetyl-glutamineAmino acid derivative2 xxx
2163.76277275276.11052276.11101−1.8C14H16O4N28Peptide type compounduDipeptide/ cyclodipeptide3[83,84]x--
2174.07517515516.27897516.276854.1C19H36O7N107Peptide¤Peptide3 -x-
2184.23247245246.10060246.100440.7C13H14O3N28AcetyltryptophanAmino acid derivative2 x-x
2194.30397395396.21529396.212128.0C17H28O5N66Tripeptide/peptide§Tripeptide/ peptide3 -x-
2204.95201199200.10441200.10486−2.2C10H16O42Ramulosin derivativeendophytic fungi metabolite3[16,100]xxx
a = Dipeptide containing Leu or Ile and Ser or Thr and Val; b = Dipeptide containing Leu or Ile and Asn or Gln and Val; c = Dipeptide containing Leu or Ile and Lys; d = Dipeptide containing Leu or Ile and Asp or Glu and Val; e = Dipeptide containing Leu or Ile and Arg; f = Tripeptide containing Ala, Asp and Gln or Gln, Glu and Gly or Ala, Asn and Glu or a tetrapeptide containing Ala, Ala, Asp and Gly or Ala, Glu Gly and Gly or the methyl ester of acetylated tripeptide containing Asn, Gly and Ser; g = Dipeptide containing Leu or Ile and Gly or Ala and Val; h = Dipeptide containing Leu or Ile and Gln; or a tripeptide containing Ala, Gly and Leu or Ile; or Ala, Ala and Val; or the methyl ester of an acetylated dipeptide containing Gly or Ala and Lys; or the ethyl ester of Ala-Ala-Ala or a tripeptide containing Gly, Gly and Val; or an acetylated dipeptide containing Thr and Val; or the methyl ester of a tripeptide containing Gly, Gly and Leu or Ile; or the methyl ester of a dipeptide containing or Asn and Leu or Ile; or Val and Gln; or the methyl ester of a tripeptide containing Ala, Gly and Val; i = Dipeptide containing Leu or Ile and Thr; j = Dipeptide containing Leu or Ile and Glu; k = Tripeptide containing Arg, Glu and Val; or Arg, Asp and Leu or Ile; or Gln, Gln and Lys or a tetrapeptide containing Ala, Ala, Asn and Lys; or Ala, Gln, Gly and Lys or a pentapeptide containing Ala, Ala, Gly, Gly and Lys; l = Dipeptide containing Phe and Thr or the methyl ester of a dipeptide containing Phe and Ser or Ala and Tyr; or the ethyl ester of a dipeptide containing Gly and Tyr; or the phenyl methyl ester of a dipeptide containing Ala and Ser; m = Dipeptide containing Gln and Phe or a tripeptide containing Ala, Gly and Phe or the methyl ester of a dipeptide containing Asn and Phe or the methyl ester of a tripeptide containing Gly, Gly and Phe or the phenyl methyl ester of a dipeptide containing Gln and Gly or the phenyl methyl ester of a tripeptide containing Ala, Gly and Gly; n = Tripeptide containing Ala, Glu and Val; or Glu, Gly and Leu or Ile; or Pro, Thr and Thr; or Ala, Asp and Leu or Ile; or an acetylated tripeptide containing Gly, Ser and Leu or Ile; or Gly, Thr and Val; or the methyl ester of a tripeptide containing Ala, Asp and Val or Asp, Gly and Leu or Ile; o = Tripeptide containing Asp, Thr and Leu or Ile; Or Glu, Ser and Leu or Ile; or Glu, Thr and Val; or the methyl ester of a tripeptide containing Asp, Thr and Val; or an acetylated tripeptide containing Ser, Thr and Val; p = Dipeptide containing Glu and Phe or Abenquine B1 or Abenquine B2; q = Dipeptide containing Leu or Ile and Val; r = Tripeptide-amide containing Pyr, Glu and Pro; s = Tetrapeptide containing Cys, Met, Thr and Thr or Met, Met, Ser and Ser; t = Dipeptide containing Asn and Phe or a tripeptide containing Gly, Gly and Phe; or a dipeptide-amide containing Asp and Phe; or an acetylated dipeptide-amide containing Gly and Tyr or the phenyl methyl ester of Gly-Gly-Gly; u = Pyr-Phe or Cyclo(3-OH-Pro-Tyr) or Acetylmethoxy-Trp; v = Dipeptide containing Leu or Ile and Met; w = Dipeptide containing Leu or Ile and Tyr; x = Tripeptide containing Ala, Glu and Tyr; or Asp, Phe and Thr; or Glu, Phe and Ser or the methyl ester of a tripeptide containing Asp, Phe and Ser; y = Tetrapeptide containing Gln, Glu, Glu and Leu or Ile; or a pentapeptide containing Ala, Asp, Asp, Val and Val; or Asp, Pro, Ser, Ser or Leu or Ile; or Ala, Ala, Glu, Glu And Val; or Ala, Ala, Asp, Glu and Leu or Ile; or Ala, Glu, Glu, Gly and Leu or Ile; or Asp, Asp, Gly, Val and Leu or Ile; or Asp, Pro, Thr, Ser, Val; or Glu, Pro, Ser, Ser and Val; or Ala, Glu, Pro, Thr and Thr; z = Pentapeptide containing Pro, Pro, Thr, Thr and Leu or Ile; or Glu, Gly, Pro, Leu or Ile and Leu or Ile; or Ala, Asp, Pro, Leu or Ile and Leu or Ile; or Ala, Glu, Pro, Val and Leu or Ile; # = Cyclo(Glu-Leu); or Pyr and Leu or Ile; or Pyr-Val methyl ester; ¤ = Tetrapeptide containing Ala, Arg, Arg and Asp; or Arg, Arg, Glu and Gly; or an acetylated tetrapeptide containing Arg, Arg, Gly and Ser; § = Tripeptide containing Gln, His and Leu or Ile; or a tetrapeptide containing Ala, Gly, His and Leu or Ile; or Ala, Ala, His and Val; or an acetylalted tripeptide containing Ala, His and Lys; or the methyl ester of a tripeptide containing Pyr, Arg and Pro; or a pentapeptide-amide containing Ala, Gly, Gly, Pro and Pro; ° = Is detected by the [2M + H]+ ion due to the used m/z range.

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MDPI and ACS Style

Tienaho, J.; Karonen, M.; Muilu–Mäkelä, R.; Wähälä, K.; Leon Denegri, E.; Franzén, R.; Karp, M.; Santala, V.; Sarjala, T. Metabolic Profiling of Water-Soluble Compounds from the Extracts of Dark Septate Endophytic Fungi (DSE) Isolated from Scots Pine (Pinus sylvestris L.) Seedlings Using UPLC–Orbitrap–MS. Molecules 2019, 24, 2330. https://doi.org/10.3390/molecules24122330

AMA Style

Tienaho J, Karonen M, Muilu–Mäkelä R, Wähälä K, Leon Denegri E, Franzén R, Karp M, Santala V, Sarjala T. Metabolic Profiling of Water-Soluble Compounds from the Extracts of Dark Septate Endophytic Fungi (DSE) Isolated from Scots Pine (Pinus sylvestris L.) Seedlings Using UPLC–Orbitrap–MS. Molecules. 2019; 24(12):2330. https://doi.org/10.3390/molecules24122330

Chicago/Turabian Style

Tienaho, Jenni, Maarit Karonen, Riina Muilu–Mäkelä, Kristiina Wähälä, Eduardo Leon Denegri, Robert Franzén, Matti Karp, Ville Santala, and Tytti Sarjala. 2019. "Metabolic Profiling of Water-Soluble Compounds from the Extracts of Dark Septate Endophytic Fungi (DSE) Isolated from Scots Pine (Pinus sylvestris L.) Seedlings Using UPLC–Orbitrap–MS" Molecules 24, no. 12: 2330. https://doi.org/10.3390/molecules24122330

APA Style

Tienaho, J., Karonen, M., Muilu–Mäkelä, R., Wähälä, K., Leon Denegri, E., Franzén, R., Karp, M., Santala, V., & Sarjala, T. (2019). Metabolic Profiling of Water-Soluble Compounds from the Extracts of Dark Septate Endophytic Fungi (DSE) Isolated from Scots Pine (Pinus sylvestris L.) Seedlings Using UPLC–Orbitrap–MS. Molecules, 24(12), 2330. https://doi.org/10.3390/molecules24122330

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