In Vitro Assessment of Organic and Residual Fractions of Nematicidal Culture Filtrates from Thirteen Tropical Trichoderma Strains and Metabolic Profiles of Most-Active

Abstract

The nematicidal properties of Trichoderma species have potential for developing safer biocontrol agents. In the present study, 13 native Trichoderma strains from T. citrinoviride, T. ghanense (2 strains), T. harzianum (4), T. koningiopsis, T. simmonsii, and T. virens (4) with nematicidal activity were selected and cultured in potato dextrose broth to obtain a culture filtrate (CF) for each. Each CF was partitioned with ethyl acetate to obtain organic (EA) and residual filtrate (RF) fractions, which were then tested on second-stage juveniles (J2s) of the nematodes Meloidogyne javanica and M. incognita in a microdilution assay. The most lethal strains were T. harzianum Th43-14, T. koningiopsis Th41-11, T. ghanense Th02-04, and T. virens Th32-09, which caused 51–100% mortality (%M) of J2s of both nematodes, mainly due to their RF fractions. Liquid chromatography–diode array detector-electrospray-high resolution mass spectrometry analysis of the most-active fractions revealed sesquiterpene and polyketide-like metabolites produced by the four active strains. These native Trichoderma strains have a high potential to develop safer natural products for the biocontrol of Meloidogyne species.

1. Introduction

Fungi belonging to the Trichoderma genus are cosmopolitan species, with 488 species identified [1]. Several of these species have been widely studied as biocontrol agents against phytopathogenic fungi [2] and nematodes [3,4], insects [5], and weeds [6], and as plant growth promoters [7,8]. The nematicidal potential of Trichoderma species is increasingly being harnessed to develop new and safer biocontrol agents against parasitic nematodes such as Globodera pallida, Heterodera avenae, Meloidogyne incognita, M. javanica, M. hapla, and Pratylenchus brachyurus [8,9]. In particular, Meloidogyne root-knot nematodes are considered the most harmful because they can affect a wide range of crops, causing production losses between 25% to 50% and millions of dollars. They thus continue to be controlled mainly with synthetic agrochemicals despite recognized problems for the environment and organisms [10,11,12] because of with lack of safer products and eco-friendly and holistic strategies. As harmful synthetic chemicals are withdrawn from the market, the search for alternatives such as crop rotation, resistant plant varieties, and biological control agents or their derivatives to control nematodes has intensified [4,11,13,14] Trichoderma species that have lethal effects against Meloidogyne species include T. harzianum, T. koningii, T. koningiopsis, T. longibrachiatum, T. citrinoviride and T. viride [15,16,17] against M. incognita; T. hamatum, T. harzianum, T. koningii, T. koningiopsis, and T. viridae against M. javanica [15,18,19]; T. asperellum, T. harzianum, T. viride and T. viride on M. hapla [9]; and T. harzianum against M. enterolobii [20].

The main mechanisms of action known for Trichoderma species are antibiosis, competition for space and nutrients, mycoparasitism, and induction of defense mechanisms. The antibiosis involves the production and secretion of secondary and primary metabolites that inhibit the growth and development of root-knot nematodes [19,21,22]. Regarding the production and secretion of metabolites, approximately 390 non-volatile metabolites have been identified from Trichoderma spp. [2,23], but only a few, such as gliotoxin, gliovirin, heptelidic acid, and viridin identified from T. virens [3,24], have been reported to have nematicidal activity. Therefore, a systematic bio-guided screening of Trichoderma species is a promising option to find novel nematicidal products.

During our ongoing bioprospecting studies in search of natural agrochemical products in the Yucatán península (Mexico), our group isolated 56 native Trichoderma species from soils. The foregoing is in response to the need to develop nematicides based on native species adapted to the areas where they are intended to be applied, with the knowledge of the active metabolites produced by fungal strains. These strains were tested for control of M. incognita, and 29 of these strains affected the viability of eggs and second-stage juveniles (J2s) in vitro and acted as plant growth promoters [25,26,27]. Moreover, these active Trichoderma strains decreased the severity of nematode damage on tomato and improved yields in the greenhouse [18,25].

In the present study, 13 Trichoderma strains from Yucatán were selected due to their activity against phytopathogenic fungi, nematodes, and as plant growth promoters. All selected Trichoderma strains were cultured in the liquid media potato dextrose broth. From each fungal strain, the culture filtrate, ethyl acetate fraction, and residual filtrate fraction were obtained and tested against the J2s of M. incognita and M. javanica. The chemical profiles of the most-active filtrates or fractions were analyzed by liquid chromatography– diode array detector-electrospray-high resolution mass spectrometry (LC-DAD-ESI-HRMS).

2. Materials and Methods

2.1. Trichoderma Strains

The 13 Trichoderma spp. strains, obtained from cultivated and non-cultivated soil in Yucatán state in Mexico [25,26] and supplied by the Phytopathology Laboratory of Tecnológico Nacional de México, Campus Conkal (Table 1), belonged to six Trichoderma species: T. citrinoviride (Th33-58), T. ghanense (Th02-04, Th26-52), T. harzianum (Th02-01, Th20-07, Th43-14 and Th33-59), T. koningiopsis Th41-11, T. simmonsii Th09-06; and T. virens (Th05-02, Th27-08, Th32-09, and Th43-13). The 5.8S-ITS regions of each strain were sequenced previously and are available in the GenBank database [27,28]. The fungal strains were reactivated in Petri dishes with potato dextrose agar (PDA, Dibico, Edo. Mex., MX) and incubated at 25 ± 2 °C, 12/12 h of light/dark for 8 days (Table 1, Figure 1).

Table 1.

Trichoderma strains selected and their biological activity.

Trichoderma Species	Key	GenBank Number	Activity	Place of Collection
T. citrinoviride Bissett 1984	Th33-58	MF078653	A/B	Ticul
T. ghanense Yoshim. Doi, Y. Abe & Sugiy 1987	Th02-04	MF078652	A/B	Tizimín
	Th26-52	MF078651	A/B	Tahdziú
T. harzianum Rifai 1969	Th02-01	MF952887	A/B	Tizimín
	Th20-07	MF078650	A	Tzucacab
	Th43-14	MF078649	A	San Felipe
	Th33-59	MF078648	A/B	Ticul
T. koningiopsis Samuels, Carm. Suárez & H.C. Evans 2006	Th41-11	MF952888	A/B	Sanahcat
T. simmonsii P. Chaverri, F.B. Rocha, Samuels, Degenkolb & Jaklitsch 2015	Th09-06	MF078647	A/B	Dzidzantun
T. virens (J.H. Mill., Giddens & A.A. Foster) Arx 1987	Th05-02	MF952889	A/B	Dzilam González
	Th27-08	MF078646	A	Chacsinkin
	Th32-09	MF078645	A	Oxkutzcab
	Th43-13	MF078644	A	San Felipe

A: Meloidogyne incognita antagonist; B: plant growth promoter.

Figure 1.

Cultures of the 13 study strains of Trichoderma on potato dextrose agar incubated at 25 ± 2 °C, 12 h light/12 h dark for 8 days.

Liquid Culture of Trichoderma Strains

The strains were grown in potato dextrose broth (PDB), which was made by adding 200 g of potato fragmented in distilled water at the boiling point (1000 mL) for 15 min, then filtered and 20 g of dextrose (Difco, Baltimore, MD, USA) added. A volume of 200 mL of the medium was deposited in Roux bottles and sterilized in an autoclave at 121 °C, 15 lb pressure, for 15 min. For each Trichoderma strain, cultured in PDA (8 days), a mycelial disk (7 mm diameter) was added to the medium in each of three Roux bottles. PDB without a Trichoderma strain was used as a control (blank). Three replicates of these cultures were incubated at 25 ± 2 °C, 12/12 h light/dark for 31 days. The mycelium was then removed from the culture broth by filtration through a double layer of cheesecloth. Each culture filtrate (CF), designated as 100% concentration, was then diluted with distilled water to 50% and 25% concentrations. The pH of 5 mL of the 100% CF was measured, then stored at 4 ± 2 °C until organic extraction (1–3 days).

2.2. Preparation of Fractions from Culture Filtrates

Each CF was liquid–liquid extracted with ethyl acetate three times (2:1, 1:1, 1:1 v/v), obtaining an ethyl acetate (EA) and residual filtrate (RF) fractions. The EA fraction was dried over sodium sulfate (Merck, New Jersey, USA), and the solvent was vacuum-evaporated at 40 °C in a rotary evaporator (IKA model RV-10, Staufen, Germany). The residual solvent in the RFs was also removed by evaporation, and the residue was designated as 100% concentration. The PDB control was processed the same way. All EAs were stored at 4 °C, and the RF fractions were frozen.

2.3. Nematicidal Bioassay

2.3.1. Nematode Inoculum

The population of M. incognita was obtained from the Tecnológico Nacional de México/ campus Conkal, Yucatán, México (30 ± 2 °C, 90% relative humidity) and M. javanica from the Instituto de Ciencias Agrarias, CSIC in Madrid, Spain (25 ± 1 °C, 70% relative humidity). Both nematodes were maintained on tomato plants (variety Marmanded) growing in pots in a greenhouse. Egg masses were collected from infested tomato roots and incubated for 72 h in sterile distilled water at 25 ± 2 °C for M. javanica and 30 ± 2 °C for M. incognita. The hatched J2s of Meloidogyne were adjusted to a final concentration of 100 J2 nematodes/100 μL distilled water to test CFs and RFs fractions (aqueous samples) and to 100 J2 nematodes/95 μL of distilled water solution to test EA samples [29,30].

2.3.2. Sample Preparation and Nematicidal Bioassay

Aqueous samples (100 µL) of a serial dilution (100, 50, or 25%) of either the CF or RF samples of a Trichoderma strain or a blank were deposited in wells of 96-well plates with U-bottom (BD Falcon, San Jose, CA, USA). The J2s suspended in distilled water (100 μL) that had been filtered through a 25 µm mesh screen were then transferred into each well. The negative controls consisted of CF or RF blanks, distilled water (DW), and 100 J2s. EA samples (5 µL) dissolved in DMSO:0.6%-Tween 20 (DT) were transferred to each well containing nematode suspension (95 µL), with a final concentration of 1 µg/µL. In this case, the negative control consisted of blank extracts, a mixture of water-DT 95:5 (WDT), and 100 J2s. Four replicates for each treatment were performed. The experimental plates were sealed with parafilm to prevent evaporation and maintained at the same conditions described above for egg masses in the dark [29,31].

After 72 h, immobile and rigid J2s that lacked intestinal contents were counted as dead, using a binocular microscope, and expressed as the percentage of juvenile mortality (%M). The nematicidal activity data were corrected using Schneider–Orelli’s formula [32]. A completely randomized design was used, and means were compared using the Scott-Knott test (p ≤ 0.05) in the statistical package Infostat Ver. 2018 [33].

2.4. Liquid Chromatography-Diode Array Detector-Electrospray-High Resolution Mass Spectrometry

The active EA and RF fractions were freeze-dried (Labconco FreeZone 2.5, model 7670520, Houston, TX, USA) and dissolved to 1% w/v with methanol, then 3 µL was injected onto a C8 column (Zorbax SB, 2.1 × 30 mm) in an Agilent 1200 liquid chromatograph (LC, Santa Clara, CA, USA) coupled to an Agilent diode array detector and a Bruker Maxis HR-QTOF mass detector (HRMS Bruker GmbH, Bremen, Germany). The samples were separated at 40 °C with a flow of 300 μL/min. The mobile phase was a mixture of water–acetonitrile 90:10 v/v 0.01% trifluoroacetic acid and 1.3 mM ammonium formate (solvent A) and 10:90 v/v 0.01% trifluoroacetic acid and 1.3 mM ammonium formate (solvent B). The gradient was from 10% to 100% of solvent B in 6 min, maintained in 100% B for 2 min, then returned to 10% B for 2 min [34]. Mass spectra (50 to 2000 m/z) in the positive mode were acquired, and components detected were compared against the MEDINA Microbial Dereplication Databases (with approximately 900 known bioactive molecules), the Chapman & Hall Dictionary of Natural Products (v25.1, CRC Press, Boca Raton, FL, USA), and a database available in the literature.

3. Results

3.1. Nematicidal Activity

The lethality of the CFs of the 13 species of Trichoderma against M. incognita differed (p ≤ 0.05) from that of the negative controls at the 100% concentration (Figure 2, Table 2). All the CFs were mortal to M. incognita at 100% concentration, except for that of T. citrinoviride (M = 62%) after 72 h. The most mortal CFs at 50% concentration against M. incognita after 72 h were from T. ghanense Th02-04 (M = 100%), T. virens Th27-08 (M = 71%), and T. harzianum Th43-14 (M = 66%). The activity of the CFs decreased to M < 50%) at 25% dilution, so they were considered non-nematicidal at this dilution. Against J2s of M. javanica, however, only CFs from T. ghanense Th02-04, T. harzianum Th20-07, and T. virens Th27-08 (M values of 85–99%) were effective at 100% concentration after 72 h (Table 2). The nematicidal activity of the CFs at 50% and 25% dilution against M. javanica did not differ from the DW. Therefore, the 100% concentration CF from T. ghanense Th02-04 was the most effective against both nematodes (M = 99–100%).

Figure 2.

Second-stage juveniles (J2s) of Meloidogyne incognita in (A) control in distilled water, (B) dead and living in culture filtrate of Trichoderma virens Th27-08.

Table 2.

Mortality of second-stage juveniles of Meloidogyne spp. after 72 h exposure to culture filtrate (CF), ethyl acetate (EA), or residual filtrate (RF) fractions from tropical Trichoderma strains.

Trichoderma Species	Strains	Mean Percentage Mortality
		Meloidogyne incognita						Meloidogyne javanica
		CF		EA	RF			CF	EA	RF
		100%	50%	1 µg/µL	100%	50%	25%	100%	1 µg/µL	100%	50%
T. citrinoviride	Th33-58	62 b	21 c	30 c	100 a	66 c	13 c	15 d	8 b	100 a	1 h
T. ghanense	Th02-04	100 a	100 a	20 d	100 a	63 c	24 c	99 a	0 c	100 a	16 e
	Th26-52	100 a	23 c	22 d	100 a	100 a	20 c	3 f	11 b	100 a	63 b
T. harzianum	Th02-01	100 a	42 c	0 e	100 a	23 d	15 c	4 f	0 c	98 a	34 d
	Th20-07	100 a	6 d	0 e	98 a	66 c	13 c	97 a	0 c	7 e	0 h
	Th43-14	100 a	66 b	51 b	100 a	100 a	57 b	45 c	66 a	93 b	4 g
	Th33-59	100 a	21 c	30 c	100 a	72 b	22 c	9 e	9 b	10 a	0 h
T. koningiopsis	Th41-11	100 a	38 c	54 b	100 a	100 a	82 a	7 e	1 c	93 b	51 c
T. simmonsii	Th09-06	100 a	6 d	37 c	100 a	96 a	53 b	6 e	0 c	14 d	0 h
T. virens	Th05-02	100 a	22 c	18 d	100 a	19 d	18 c	8 e	0 c	100 a	10 f
	Th27-08	100 a	71 b	0 e	100 a	100 a	64 b	85 b	9 b	100 a	94 a
	Th32-09	100 a	20 c	100 a	100 a	100 a	83 a	7 e	3 c	86 c	5 g
	Th43-13	100 a	23 c	31 c	100 a	86 b	49 c	7 e	8 b	95 b	0 h
Control	PDB	0 c	0 d	0 e	0 b	0 d	0 c	0 f	0 c	0 f	0 h
	DW/WDT *	0 c	0 d	0 e*	0 b	0 d	0 c	0 f	0 c*	0 f	0 h
	Blank	0 c	0 d	0 e	0 b	0 d	0 c	0 f	0 c	0 f	0 h
	SD	0.2	125	5.7	0.5	15.2	12.9	2.4	1.4	2.6	2.4

PDB: potato dextrose broth, DW: distilled water, WDT *: water-DMSO–0.6% Tween 20 (95:5); Blank: unfermented medium. SD: standard deviation, calculated from the analysis of variance for treatments (CFs, EA, RFs from each strain). Values with different letters within a column differed significantly in Scott–Knott test (p ≤ 0.05).

After the ethyl acetate extraction of the CFs, the activity of all the RF fractions differed greatly (p ≤ 0.05) from that of the negative control. All the residual filtrates generated from the ethyl acetate extraction of the CFs of the 13 strains were mortal against M. incognita, and all but those from T. harzianum Th20-07 and T. simmonsii Th09-06 were mortal against M. javanica (Table 2). The highest mortality against J2s of M. incognita at 25% dilution of the RFs after 72 h was caused by T. koningiopsis Th41-11 (M = 82%) and T. virens Th32-09 (M = 83%). Against J2s of M. javanica, the highest mortality was achieved with 50% dilutions of the RFs from T. ghanense Th26-52 (M = 63%), T. koningiopsis Th41-11 (M = 51%) and T. virens Th27-08 (M = 94%). After a 72 h exposure to EA fractions, the most active against M. incognita were from T. harzianum Th43-14 (M = 51%), T. koningiopsis Th41-11 (M = 54%), and T. virens Th32-09 (M = 100%) at 1 µg/µL; the EA fraction from T. harzianum Th43-14 strain was the only EA fraction active (M = 66%) against M. javanica at 1 µg/µL (Table 2).

In general, against both nematodes, the CFs and their RF and EA fractions from T. harzianum Th43-14 and T. virens Th27-08 were the most active. In addition, the RF fraction from CF of T. koningiopsis induced J2s mortality of both nematodes at the lowest dilution tested.

3.2. Identification of Components in Active Fractions from Trichoderma Strains by LC-DAD-ESI-HRMS

The results obtained from analyses of the liquid chromatograms and ultraviolet and high-resolution mass spectrometry data of the RF fraction from T. harzianum Th43-14, T. ghanense Th26-52, and T. virens Th27-08 and the EA fraction of T. koningiopsis are shown in Table 3, Table 4, Table 5 and Table 6 No significant differences were observed between the chromatograms from the active strains T. ghanense Th02-04 and the T. virens Th32-09 and negative control unfermented culture medium.

Table 3.

Dereplicated metabolites from residual filtrate fraction of Trichoderma harzianum Th43-14.

No.	Rt (min)	UV (nm)	[M + H]+ m/z		Molecular Formula	Putative Metabolite
			Experimental	Theoretical
1	0.88	200, 215	317.1599	317.1595	C15H24O7	unknown
2	1.35	205, 210	299.1492	299.1489	C15H22O6	3,7,8,15-Scirpenetetrol
3	2.05	200, 220	249.1488	249.1485	C15H20O3	Illudin M
4	2.69	200, 220	255.1591	255.1591	C14H22O4	unknown
5	2.86	200, 220	283.1541	283.1540	C15H22O5	3,4,15-Scirpenetriol
6	3.09	200, 218, 270	309.1692	309.1697	C17H24O5	Naematolin
7	3.66	200, 220	553.3000	553.3007	C29H44O10	unknown
8	3.84	200, 220	251.1644	251.1642	C15H22O3	Trichodermol

No.: Component number; Rt: Retention time; UV: Ultraviolet.

Table 4.

Dereplication metabolites from ethyl acetate fraction of Trichoderma ghanense Th26-52.

No.	Rt (min)	UV (nm)	[M + H]+ m/z		Molecular Formula	Putative Metabolite
			Experimental	Theoretical
1	0.63	200, 218	261.0966	261.0969	C11H16O7	unknown
2	0.86	218, 230, 300	168.0656	168.0655	C8H9NO3	unknown
3	1.22	205, 290	155.0700	155.0703	C8H10O3	Unknown
4	2.16	200, 220, 310	169.0493	169.0495	C8H8O4	atrichodermone D
5	2.40	200, 210, 240	225.0756	225.0758	C11H12O5	Unknown
6	2.64	200, 230, 270	235.1328	235.1329	C14H18O3	Unknown
7	2.89	200, 220, 280	195.1379	195.1380	C12H18O2	6-heptyl-2H-pyron-2-one

No.: Component number; Rt: Retention time; UV: Ultraviolet.

Table 5.

Dereplicated metabolites from ethyl acetate fraction of Trichoderma koningiopsis Th41-11.

No.	Rt (min)	UV (nm)	[M + H]+ m/z		Molecular Formula	Putative Metabolite
			Experimental	Theoretical
1	2.81	200, 255	281.1750	281.1747	C16H24O4	Koninginin L
2	3.04	200, 255	281.1749	281.1747	C16H24O4	Koninginin T
3	3.68	200, 260	283.1906	283.1904	C16H26O4	Koninginin B

No.: Component number; Rt: Retention time; UV: Ultraviolet.

Table 6.

Dereplicated metabolites from residual fractions of Trichoderma virens Th27-08.

No.	Rt (min)	UV (nm)	[M + H]+ m/z		Molecular Formula	Putative Metabolite
			Experimental	Theoretical
1	0.78	205, 240, 290	207.0653	207.0657	C11H10O4	unknown
2	1.00	210, 225, 295,	225.0757	225.0762	C11H12O5	sepedonin
3	1.57	205, 240, 290	207.0652	207.0657	C11H10O4	unknown
4	3.35	215, 255, 300	277.1435	277.1438	C16H20O4	unknown

No.: Component number; Rt: Retention time; UV: Ultraviolet.

The compounds were tentatively identified by taking into account the species of the producing strain, the UV spectrum, and the molecular formula assigned through analysis of the protonated and ammonium adducts of each molecule, helped in some cases by the presence of dimers and dehydration products, and through searches of in the internal MEDINA database of HRMS spectra, dictionary of natural products and other natural product databases.

3.2.1. Metabolites from Residual Filtrate Fraction of Trichoderma harzianum Th43-14

The chromatogram of the RF fraction from T. harzianum Th43-14 showed eight main components (Figure 3, Table 3). Five of these were tentatively identified as sesquiterpenes according to their UV and HRMS data. Component 5 (Rt = 2.86 min) presented in its HRMS an ammonium adduct (m/z 300.1807), a protonated molecular ion at m/z 283.1541, indicative of a molecular formula C₁₅H₂₂O₅ (calc. for C₁₅H₂₃O₅⁺_, 283.1540), which was tentatively assigned as 3,4,15-scirpenetriol. The HRMS of component 2 (Rt = 1.35 min) exhibited an ammonium adduct (m/z 316.1758), and a protonated molecular ion at m/z 299.1492 consistent with the molecular formula C₁₅H₂₂O₆ (calc. C₁₅H₂₃O₆⁺, 299.1489) and was putatively identified as 3,7,8,15-scirpenetetrol. All compounds tentatively identified (2, 5, and 8) have the same structural skeleton.

Figure 3.

UV_{210 nm} chromatograms of residual filtrate fraction from Trichoderma harzianum Th43-14 and unfermented culture medium. 1: Not identified (C₁₅H₂₄O₇), 2: 3,7,8,15-scirpenetetrol, 3: illudin M, 4: not identified (C₁₄H₂₂O₄), 5: 3,4,15-scirpenetriol, 6: naematolin, 7: not identified (C₂₉H₄₄O₁₀), 8: trichodermol.

The HRMS spectrum of the minor component 3 (Rt = 2.05 min) displayed several protonated fragments (m/z, 231.1383, 221.1544, 213.1272) and a protonated molecular ion at m/z 249.1488 in agreement with the molecular formula C₁₅H₂₂O₆ (calc. for C₁₅H₂₃O₆⁺, 249.1489) and was putatively identified as illudin M. The HRMS spectrum of another minor component (6, Rt = 3.09 min) revealed a protonated molecular ion at m/z 249.1488 and was given a molecular formula C₁₅H₂₀O₃ (calc. for C₁₅H₂₁O₃⁺, 249.1485) and tentatively identified as naematolin.

The largest peaks were from two unknown molecules (1 and 4), which according to their protonated molecular adducts (m/z 317.1599 and 255.1591, respectively), ammonium (m/z 334.1866 and 272.186, respectively), and [2M+ H]⁺ (m/z 650.3387 and 509.3114, respectively) ions, have molecular formulae of C₁₅H₂₄O₇ (Rt = 0.88 min) and C₁₄H₂₂O₄ (Rt = 2.69 min). In addition, another non-identified minor component (7, Rt = 2.16 min) was assigned a molecular formula of C₂₉H₄₄O₁₀ (Rt = 3.66 min) based on the analysis of its protonated (m/z 553.3000) and ammonium adducts (m/z 570.3270).

3.2.2. Metabolites from Residual Filtrate Fraction of Trichoderma ghanense Th26-52

Seven components were detected in the chromatogram of the residual fraction of T. ghanense Th26-52 (Figure 4, Table 4). The most abundant component, 1 (Rt = 0.63 min) displayed in its HRMS spectrum an ammonium adduct (m/z 278.1233) and a protonated molecular ion at m/z 261.0966, indicative of a molecular formula of C₁₁H₁₆O₇ (calc. C₁₁H₁₆O₇⁺, 261. 0973); this polar metabolite could not be identified. The HRMS of component 4 (Rt = 2.16 min) with a protonated molecular ion at m/z 169.0493 has a molecular formula C₈H₈O₄ (calc. C₈H₉O₄⁺, 169.0500) and was putatively identified as atrichodermone D. The analysis of the HRMS data of the minor component 7 (Rt = 2.89 min) displayed an ammonium adduct (m/z 212.1640), and a molecular protonated ion at m/z 195.1379 supporting the molecular formula C₁₂H₁₈O₂ (calc. C₁₂H₁₉O₂⁺, 195.1384) and tentatively assignment as the unsaturated lactone 6-heptyl-2H-pyron-2one. The other four unknown components included small metabolites with the molecular formulae of C₈H₉NO₃ (Rt = 0.86 min), C₈H₁₀O₃ (Rt = 1.22 min), C₁₁H₁₂O₅ Rt = 2.40 min), and C₁₄H₁₈O₃ (Rt = 2.64 min) according to their UV and HRMS data (Table 4).

Figure 4.

UV_{210 nm} chromatograms of residual filtrate fraction from Trichoderma ghanense Th26-52 and the unfermented culture medium. 1: Not identified (C₁₁H₁₆O₇), 2: not identified C₈H₉NO₃, 3: not identified C₈H₁₀O₃, 4: atrichodermone D, 5: Not identified (C₁₁H₁₂O₅), 6: not identified (C₁₄H₁₈O₃). 7: 6-heptyl-2H-pyron-2-one.

3.2.3. Metabolites from Ethyl Acetate Fraction of Trichoderma koningiopsis Th41-11

The chromatogram of the ethyl acetate extract from T. koningiopsis Th41-11 (Figure 5) showed three components, which tentatively were assigned as koninginin isomers. The HRMS of component 1 (Rt = 2.81 min) and 2 (Rt = 3.04 min) exhibited similar dehydration adducts (m/z 263.1642 245.1535) and protonated adducts (m/z 281.1750 and 281.1749, respectively), indicative of a molecular formula of C₁₆H₂₄O₄ (calc. for C₁₆H₂₅O₄+, 281.1752) for both molecules. Component 3 (Rt = 3.68 min) in its HRMS data showed dehydrated species (m/z 265.1796, 247.1668, 237.1847), a dimer adduct ([2M+H]⁺, m/z 565.3759), and a protonated molecular ion at m/z 283.1906 in accordance with a molecular formula of C₁₆H₂₆O₄ (calc. for C₁₆H₂₇O₄⁺, 283.1908). An extensive search of the literature based on spectral data and previous reports led to their tentatively identification as koninginin L (1), T (2), and B (3) (Table 5).

Figure 5.

UV_{210 nm} chromatogram of residual filtrate fraction from Trichoderma koningiopsis Th41-11 and the unfermented culture medium. 1: Koninginin T, 2: koninginin L, 3: koninginin B.

3.2.4. Metabolites from Residual Filtrate Fraction of Trichoderma virens Th27-08

The chromatogram of RF fraction of T. virens Th27-08 displayed four components not present in the blank sample (Figure 6, Table 6). Component 3, eluting at Rt of 1.00 min exhibited an ammonium adduct (m/z 242.1020), dehydration fragments (m/z 207.0652, 191.1424), and a protonated ion at m/z 225.0757 indicative of a molecular formula of C₁₁H₁₂O₅ (calc. for C₁₁H₁₃O₅⁺, 225.0762). Based on HRMS and UV data, compound 1 was tentatively identified as sepedonin. The major (1) and two minor components (2 and 4) were not identified, and molecular formulae were assigned as C₁₁H₁₀O₄ (Rt = 0.78 and 1.57 min) and C₁₆H₂₀O₄ (Rt = 3.35) based on their protonated HRMS ion [M+H]⁺ and additionally supported by its ammonium and dimer adducts.

Figure 6.

UV_{210 nm} chromatogram of residual filtrate fraction from Trichoderma virens Th27-08 and the unfermented culture medium. 1: Not identified (C₁₁H₁₀O₄), 2: sepedonin, 3: not identified (C₁₁H₁₀O₄), 4: not identified (C₁₆H₂₀O₄).

4. Discussion

The results of this study complement our previous discoveries on native Trichoderma strains with nematicidal properties against two Meloidogyne species as part of our continuing work to find and develop safer biocontrol agents. Herein, we demonstrated that 92% of the screened tropical Trichoderma strains (all 13 except T. simmonsii) are mortal to J2s of Meloidogyne javanica and confirmed that the CFs and the EA or RF fractions of all strains were highly mortal to M. incognita. T. koningiopsis UFSMQ40 [12], T. harzianum Th6, JX1614550 [18,19,35], and Trichoderma sp. EF1671 [5] have been shown to have nematicidal effects against J2s of M. javanica, but our report is the first to demonstrate the nematicidal activity of T. citrinoviride, T. ghanense, and T. virens against M. javanica.

The mortality data revealed that M. javanica was less sensitive than M. incognita to the CFs and EA fraction from Trichoderma strains. This differential sensitivity behavior of both Meloidogyne species against extracts, compounds, or fungal strains has been previously reported. For example, fungus Arthobortys sp. MVD18 caused less mortality against M. javanica (92.9%) than against M. incognita (99.0%) after a 48 h exposure. However, two Trichoderma sp. strains (KAV2 and KAV3) were more active against M. javanica than M. incognita [36]. Acetic acid and hexanoic acid yielded an EC₅₀ of 162.4 and 339.3 µg/mL, respectively, against M. javanica but EC₅₀ of 38.3 and 41.1 µg/mL against M. incognita, respectively, after 1 day [37,38]. Sensitivity differences between different species and even different populations of the same species have been attributed to the habitat and environmental conditions to which the organisms are exposed. Other saprophytic fungi have been reported to have low nematicidal effects against J2s of M. javanica; for example, a CF of Arthrobotrys oligospora, A. conoides, and Hypocrea lixii (sexual state of T. harzianum) at 100% concentration [16,39] caused from 16.14 to 64.5% mortality. An EA extract from Trichoderma sp. EFI 671, however, was not active [5].

The present study is also the first nematicidal bio-guided fractionation of the CFs from Trichoderma species and screening the organic and residual aqueous fractions against J2s. After the fractionation, 69% of the RF fractions from the non-nematicidal CFs had a higher mortal effect on M. javanica. This effect could be attributed to antagonistic action between metabolites and the concentration of the metabolites in the RFs of the strains. By contrast, the nematicidal effect of the CF from T. harzianum strain Th20-07 was not confirmed, suggesting that enzymes were mainly involved in the nematicidal activity of strain Th20-07 and were subsequently denatured by the solvent during the fractionation. The antibiosis produced by chitinase and protease action from Trichoderma species has been widely described. For example, an enzymatic filtrate obtained from T. koningiopsis UFSMQ40 is mortal to J2s of M. incognita (90.4% mortality) and M. javanica (63.2%) after 24 h of exposure [15]. Chitinase (51.42 U/mL) and protease (4.27 U/mL) from T. harzianum ITCC 6888 caused high mortality (93%) against M. incognita [40].

On the other hand, in our study, the highest lethal activity against both Meloidogyne species was found for T. koningiopsis Th41-11, T. harzianum Th43-14, T. virens Th27-08, and the two T. ghanense strains tested. The most investigated of these species has been T. harzianum for its properties as a biological control agent and its metabolites [2,4,14]. Other in vitro studies of CFs from T. harzianum strains grown in PDB have shown a significant mortal effect on the J2s of M. incognita. For example, CFs from T. harzianum strains ThU, JX1614550 and Th.6 caused a mortality of 33, 64.5 and 75%, respectively, at 100% concentrations after 72 h [19,41,42].

The residual fraction of T. harzianum Th43-14 revealed a mixture of illudin, naematolin, 4β-scirpenol (syn. trichodermol and roridin C), 3,4,15-scirpenetriol, and 3,7,8,15-scirpenetetrol were tentatively identified based on UV and MS data. Scirpenes have a trichothecene skeleton with a characteristic 9-double-bond and C12-C13 epoxide in their structure and are potent inhibitors of protein synthesis [43,44]. Semisynthetic derivatives of trichodermol have potent cytotoxic and antifungal activity against the fungus Ceratocystiopsis crassivaginata [45,46]. This metabolite has been isolated from several Trichoderma species, and 3,4,15-scirpenetriol has been isolated from Fusarium equiseti, F. roseum, and F. sporotrichiella, whereas 3,7,8,15-scirpenetetrol has only been isolated from F. graminearum [47]. Illudin M, isolated from several Omphalatus species and from Granulobasidium vellereum, has no activity against M. incognita or Caenorhabditis elegans at 100 µg/mL, but it has high antitumor and antimicrobial activities [48,49,50]. Naematolin was isolated from Hypholoma fasciculare (syn. Naematoloma fasciculare), a poisonous basidiomycete [51]. This caryophyllenediol derivative has antitumor, antiviral, and weak antibacterial properties [52,53] and reduced 97% of bloodstream forms of Trypanosoma cruzi at 250 µL/mL [54].

The two studied strains of T. ghanense (Th02-04 and Th26-52) were active on both Meloidogyne species. The data obtained from the UV and ESIMS spectra of its RF fraction allowed us to tentatively identify atrichodermone D and 6-heptyl-2H-pyron-2-one in the RF fraction of T. ghanense Th26-52. To date, metabolites detected from T. ghanense include the phenolics catechin, ferulic acid, and cinnamic acid from the CF of two strains from different areas in India [55]. Atrichodermone D is a cyclopentenone previously reported only from T. atroviride and has no known biological activity [56]. 6-Heptyl-2H-pyran-2-one is an alkyl pyrone isolated from T. koningii strain IMI-308475 and from T. asperellum and T. viride, but no nematicidal activity was reported [57,58]. Related lactones from Trichoderma species include 6-(1-heptenyl)-2-pyran-2-one, 6-(3-hydroxypent-1-en-1-yl)-2H-pyran-2-one and 6-pentyl-2H-pyran-2-one. 6-(3-Hydroxypent-1-en-1-yl)-2H-pyran-2-one was isolated from T. koningiopsis QA3 and has strong antibacterial activity against Micrococcus luteus [59]. 6-Pentyl-2H-pyran-2-one is a recognized plant growth promoter and produced by several Trichoderma species [60].

From the results of the present study, T. koningiopsis Th41-11 is another promising strain that produces nematicidal metabolites. The LC chromatogram of its EA fraction revealed small amounts of koninginins B, L, and T, tentatively identified based on UV, ESIHRMS data analyses, and comparisons with the literature. Koninginin B is a bicyclic polyketide that has also been reported from T. koningii [61], T. neokoningin [62], and T. applanatum [63]. Koninginin L and T are tricyclic polyketides with an oxygen bridge (C7 and C9) at C2 and an alcohol group at C4. Koninginins B, L, and T from T. koningiopsis QA3 and YIM PH30002 were recently reported to be weakly antibacterial [64,65,66].

Among the four T. virens strains studies, only Th27-08 had activity against both nematodes, with the RF fraction achieving the highest mortality. In the LC chromatogram of the RF fraction, we detected sepedonin, a tropolone first isolated from Sepedonium chrysospermum Fries (teleomorph Hyphomyces chrysospermus Tul.) and later from S. ampullosporum, S. chalcipori, S. microspermum, and S. chrysospermum and having antimicrobial activity against several bacterial and fungal pathogens [67]. The artifact anhydrosepedonin (C₁₁H₁₀O₄) is produced during the isolation process due to the instability of sepedonin [68,69]. We additionally detected three unknown metabolites in the RF fraction of T. virens Th27-08. Other metabolites previously reported from T. virens include cathequin, caffeic acid, ferulic acid, and 33 other non-volatile metabolites [2,55,70,71]. Our report of sepedonin is thus a new contribution to the chemical composition of this species.

Except for illudin M, the metabolites reported from our native Trichoderma species were not previously tested on nematodes [48]. The nematicidal efficacy of the metabolites tentatively identified from Trichoderma species herein studied are likely due to the alcohol or carboxylic acid groups in their structure. Ntalli et al. [38] reported that acetic acid and hexanoic acid, furfurol (syn. furfuryl alcohol), and furfural paralyzed J2s an EC_{50/1 h} of 1–100 µg/mL or less after 24 h, and the alcohols and aldehyde were more effective than the organic acids. They also demonstrated that acetic acid damages the cuticle and, the nuclei of pseudocoel cells and vacuolizes the cytoplasm of M. incognita, while (E)-2-decenal, and undecanone induced malformation of somatic muscles of the nematodes [72].

In general, few metabolites with nematicidal properties have been isolated from Trichoderma species; some of these are acetic acid, gliotoxin, trichorzianine, viridin [3], trichodermin [73], and cyclonerodiol [65,74]. Therefore, more studies should focus on bioassay-guided isolation and characterization of metabolites responsible for nematicidal activity in the fractions from Trichoderma strains. In addition, studies to optimize the production of the extracts with the most promising active compounds, evaluate their efficacy in greenhouses, and assess their toxicity on plants and beneficial organisms must be carried out before the compounds can be evaluated in the field and further developed as safe bionematicide products.

5. Conclusions

The present contribution enriches our knowledge of the nematicidal potential of 13 tropical Trichoderma species isolated from the soils of Yucatán state. The most effective species against M. incognita and M. javanica were T. ghanense strains Th02-04 and Th26-52, T. harzianum Th43-14, T. koningiopsis Th41-11, and T. virens Th27-08. The LC-DAD-ESIMS chemical profiles of the residual filtrate fractions and ethyl acetate fractions of culture filtrate from Trichoderma spp. revealed the presence of novel metabolites for the genus and others with molecular formulas not found in natural products databases. These results highlight the ability of Trichoderma strains to produce bioactive secondary metabolites that could be developed to manage M. incognita and M. javanica. However, more studies are needed to determine activity and potential phytotoxicity in plant applications and doses for effective, efficient biocontrol effect.

Author Contributions

Design and supervision of the study, J.C.-A. and M.G.-A.; fungal cultures, organic extraction and nematicidal bioassays, F.A.M.-K.; academic support and supervision of the nematicidal bioassays, M.F.A. and J.C.-A.; LC-DAD-ESI-HRMS data acquisition, analyses, interpretation, J.M., F.R. and M.G.-A.; financial support of the project, J.M.T.-S. and M.G.-A.; project coordination, J.C.-A.; writing and review of the manuscript, all authors. All authors have read and agreed to the published version of the manuscript.

Funding

Consejo Nacional de Ciencia y Tecnología (CONACYT)- International Development Research Centre (IDRC) financed project No. CEAR2019-01; Ministerio de Ciencia e innovación/ Agencia Estatal de Investigación (MCIN/AEI) financed project 10.13039/501100011033, and Tecnológico Nacional de México (TECNM) financed project 10759.21P.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.