Fungal Material. Fungal plant pathogens from the southern United States were acquired from the Department of Plant Pathology and Crop Physiology at Louisiana State University (LSU) and recovered on potato dextrose agar (PDA: Difco, Sparks, MD) for screening (Table 1). Pure cultures of all fungi in this study are preserved on cornmeal agar (CMA: Difco) slants at 4°C and 15% glycerol at -80°C in the Doyle Mycology Lab at LSU.
Table 1
Isolates for testing the antimicrobial potential of SMs produced by Xylaria necrophora.
Fungi | Crop source | Source code | DMCC |
Cercospora janseana | Rice | NA | 456 |
Rhizoctonia solani | Soybean, Rice | RSTP2015 LA Rhizoctonia | 3393 |
Cercospora sojina | Soybean | TN 209 | 3773 |
Magnoporthe oryzae | Rice | IH-174L2 Magnoporthe STS | 3394 |
Sclerotium rolfsii | Soybean | SRFG2018 LA STS | 3395 |
Ceratocystis fimbriata | Sweet potato | 18-BIP-1 | 3703 |
Monilochaetes infuscans | Sweet potato | NA | 2614 |
Rhizopus stolonifer | Sweet potato | 92-RS-02 | 3390 |
Cercospora zeae-maydis | Corn | CBS117757 | 2701 |
Exserohilum tursicum | Corn | ETST2019LA2 STS | 3397 |
Macrophomina phaseolina | Corn | WSM-10 | 3398 |
Cercospora cf. flagellaris | Soybean | NA | 2926 |
Curvularia lunata | Grain Sorghum | NA | 2087 |
Glomerella cingulata | Pecan | NA | 3140 |
Aspergillus flavus | Corn | AF-13ToxAF | 3704 |
NA: Fungi originated in Doyle Mycology Lab. DMCC = Doyle Mycology Lab Culture Collection accession number. The crop source is the host species from which the organism was obtained. |
Table 2
Treatments to assess the phytotoxicity stability of SMs produced by X. necrophora.
Treatment condition | Treatment § | Code |
X. necrophora | Cell-free culture filtrates (CFs) | CFs |
| CFs + MOPS | CFsM |
| CF + MOPS + Pronase | CFsMP |
Control | Filtered potato dextrose broth (control broth) | CBroth |
| Control broth + MOPS | CBrothM |
| Control broth + MOPS + Pronase | CBrothMP |
| MOPS + Pronase | MP |
§ Combination of the components denominated as the treatment. |
Fermentation. Xylaria necrophora (DMCC 2127; Table 1) was used for experiments in this study. The fungus was isolated on June 15, 2017, from soybean roots in West Carroll Parish, Louisiana, USA, (see Garcia-Aroca et al., 2021) and grown in potato dextrose broth (PDB: Difco) to produce cell-free culture filtrates (CFs) in sterile flasks (500 mL, regular and sidearm). Mock-inoculated PDB served as a control (control broth). CFs and control broth flasks were incubated in the dark in a rotatory incubator at 150 rpm at 25 ± 2°C for 14 days.
Plant material. Soybean seeds of the cultivar AG4632 were planted in 72-cell plastic trays containing Sungro Horticulture Metro Mix 360 RSi potting mix. Trays were placed on growth shelves equipped with lights (12:12 h dark: light cycle) in the lab and were irrigated daily using a watering can. Plants were used in experiments once they developed the first trifoliate.
Phytotoxicity of CFs. To validate previously reported phytotoxic effects of CFs from X. necrophora on soybean leaves (Garcia-Aroca et al., 2022) and confirm the presence of SMs in CFs, we conducted a preliminary assay comparing chlorophyll content in leaves of stem cuttings treated with CFs from the pathogen against control broth. Soybean plants were excised ~ 1 cm above the substrate using a scalpel and stem cuttings were rinsed with deionized water (DIH2O) and placed in test tubes (50 mL Falcon tubes) containing 50 mL of 1 mM potassium phosphate buffer (KH2PO4 + K2HPO4) pH 7 for five days to allow the development of adventitious roots. Separately, CFs and control broth were mixed with 1 mM KH2PO4 + K2HPO4 buffer pH 8 to make 25-fold dilutions (Garcia-Aroca et al., 2022). One set of stems was transferred to test tubes containing 25-fold CFs and another set was transferred to test tubes containing 25-fold control broth. There were 108 stem-cutting repetitions per treatment and two technical repetitions of this bioassay. Tubes containing stem cuttings were arranged in a completely randomized design.
To assess the phytotoxic effect of CFs on soybean roots, we evaluated the root growth from stem cuttings used in the preliminary phytotoxicity assay. We measured root biomass and root growth from stem cuttings 10 days after exposure to 25-fold CFs and 25-fold control broth. Roots were blot-dried and photographed. To measure root length, we randomly selected three lateral roots within 1 cm of the original stem cut and used ImageJ v.1.50i (Schneider et al., 2012) to collect root measurements. Photographed stems were excised 4 cm away from the base of the original stem cut for weighing. To document cell death, excised roots were stained using Trypan Blue 0.4% and were preserved in 70% glycerol at 25 ± 2°C.
CFs were subjected to protein digestion to evaluate phytotoxic stability and to determine if the phytotoxic compounds were proteinaceous. For this, CFs were mixed in a digestion solution [Pronase diluted in 50 mM 3-(N-morpholino) propane sulfonic acid (MOPS) buffer pH 7] at a concentration of 1 mg Pronase per mL of CF. Additionally, we assessed the effect of the components in the digestion solution by designing a set of negative controls that consisted of 1) control broth mixed with the digestion solution at 1 mg/mL, 2) 1 mg/mL Pronase in 50 mM MOPS pH 7, and 3) MOPS buffer pH 7. All the dilutions were incubated at 25 ± 2°C for four hours and exposed to 100°C for one hour if they included Pronase.
To assess the phytotoxic stability of CFs, we phenotyped leaflets from the first trifoliate of soybean plants. To conduct this assessment, we moved the trays with soybean plants from the growth shelves to a bench to facilitate treatment application. Approximately 200 µL of each treatment were infiltrated into the underside of soybean leaflets using 1 mL sterile needle-less syringes (Fisherbrand™ Sterile Syringes, PA). There were six leaflet repetitions per treatment and three technical repetitions of this bioassay. After infiltration, plants were returned to the growth shelves.
Antimicrobial activity of CFs. To evaluate the antimicrobial potential of CFs, we compared the growth of fungi in Table 1 on CFs to growth in the control broth. For this, we amended CFs and control broth to PDA+ (Difco; strengthen with 5 g of agar; 20 g of agar per liter total) to make v/v dilutions of 50% (50d) and 10% (10d). Fungal plugs (5 ± 1 mm diameter) were inoculated onto 90 mm diameter Petri plates containing 25 mL of the amended media. There were three plate repetitions per treatment dilution and fungus, and three technical repetitions of this bioassay. Inoculated plates were incubated in a completely randomized design at 25 ℃ in darkness for five days. Fungal growth was measured every 24 hours by delimiting the colony edge at the bottom of the plate with a fine-point Sharpie marker under a Zeiss stereoscope. Pictures of the recorded growth were taken using a Nikon D5300 camera and data was collected using ImageJ v.1.50i (Schneider et al., 2012). The antimicrobial effect of CFs was calculated using Eq. (1):
I = [(r1-r2)/r1] × 100 (Hajieghrari et al., 2008)
where I represents the inhibition percentage, “r1” is the mean radial growth in the control broth, and “r2” represents the mean radial growth of the fungus in CFs.
Antimicrobial activity of Xylaria necrophora in direct interaction. To evaluate the production of SMs by X. necrophora during direct interaction, we challenged X. necrophora with the 15 fungi in Table 1. We designed each interaction on 90 mm diameter plates containing 25 mL of PDA. For the interactions, one mycelium plug (5 ± 1 mm in diameter) of X. necrophora was cultured against one mycelium plug of each one of the 15 fungi. The plugs were placed 2.5 cm away from the edge of the plates and approximately 3.5 cm apart from each other. Plugs were placed onto the plates at the same time unless fungi were of slow or fast growth compared to X. necrophora.
To determine if a fungus was a fast or slow grower, we grew them on PDA at 25 ℃ in the dark to record radial growth measurements for five days (see Supplementary Fig. 1 in the Supplementary Information). If fungi were found to grow faster than X. necrophora, they were placed in group 1 (fast growth), if fungi grew at the same rate as X. necrophora, they were placed in group 2. If fungi grew slower than X. necrophora, they were placed in group 3 (slow growth). For group 1, X. necrophora was grown until it reached 1 cm diameter before adding the other fungal pathogen. For group 2, both X. necrophora and pathogen plugs were inoculated at the same time. Fungi of slow growth (group 3) were grown in advance for five days before inoculating the plate with X. necrophora. The dual interaction assay began once both fungi were on the plate. Controls consisted of each of the fungi from Table 1 growing alone on the center of Petri dishes containing 25 mL of PDA and were replicated three times. Each confrontation was replicated six times. Confrontation plates and controls were incubated at 25 ± 2°C in the dark. The experiment was stopped 10 days after the dual confrontation started with X. necrophora.
The antimicrobial effect of X. necrophora was determined by calculating the radial growth of the fungal mycelium of both fungi growing together on each Petri dish. Radial measurements on the side of interaction were taken every 24 hours for 10 days. Four radial measurements were taken from fungi growing alone every 24 hours for 10 days. Plates were photographed and analyzed with ImageJ v.1.50i (Schneider et al., 2012) to measure radial growth. The antimicrobial effect of X. necrophora was calculated using Eq. (1).
Fractionation. After confirming the phytotoxic and antimicrobial bioactivity of CFs, CFs, and the control broth were subjected to fractionation through extraction with hexane [Hex (C6H14)], diethyl ether [Et2O (C4H10O)], dichloromethane [DCM (CH2Cl2)], and ethyl acetate [EtOAc (C4H8O2)] four times each. The organic layers were dried with ~ 10 g of sodium sulfate (Na2SO4) and concentrated at 25 ± 2°C using a Heidolph rotary evaporator (Heizbad Hei-VAP, SN: 071106137). The viscous oil obtained was transferred into 20 mL scintillation vials. Each vial was vacuum-dried overnight and subsequently dried over a gentle flow of nitrogen for two days to remove residual solvent. The same drying process was conducted for the aqueous layers of the CFs and control broth. Aqueous layers were the remnants of control broth after extraction and X. necrophora CFs after extraction. Additional control broth and CFs not subjected to solvent extraction were used as controls. Organic extracts were kept at -80°C until use.
Phytotoxicity of extracted fractions. To determine the presence of SMs in extracted fractions, we compared the phytotoxic potential of fractions from CFs to extracts from control broth. Extracted fractions were diluted to 100 µg/mL in 5% dimethyl sulfoxide [DMSO (C2H6OS)]. We used control broth, CFs, and the aqueous phase of both after extraction and 5% DMSO as controls.
The phytotoxic potential of each fraction was tested on soybean leaf disks. We obtained 8 mm leaf disks from surface sterilized leaflets [60 s in 0.5% (v/v) sodium hypochlorite, 60 s in 70% (v/v) ethanol, and rinsed for 60 s with DIH2O] using a hole puncher. Leaf disks were placed in 48-well plates (VWR North American, Cat. No. 10062-898) and 200 µL of the treatments were pipetted to the wells. There were six disk repetitions per treatment and three technical repetitions of this bioassay. Plates were incubated at 25°C for 48 hours in a 12:12 dark: light cycle and arranged in a completely randomized design. To avoid contamination, the leaf disks were transferred to 48-well plates containing 500 µL of DIH2O in each well and incubated at 25°C in a 12:12 dark: light cycle for five days.
The phytotoxic effect of the treatments on stem cuttings, leaflets, and leaf disks was measured based on changes in chlorophyll content. Leaf disks and leaflets were photographed to measure chlorophyll content digitally (Liang et al., 2017) which was recently validated for soybean in this TRD system (Garcia-Aroca et al., 2022). For the leaflets, ImageJ v1.50i (Schneider et al., 2012) was used to extract the area of interest, and the central lesion was removed from the center of the primary extracted area. To measure chlorophyll content, the Chloropyll_Imager plugin was adapted for this experiment.
Antimicrobial activity of extracted fractions. Extracted fractions were screened to identify the most bioactive fraction containing SMs once the antimicrobial activity of CFs was confirmed. Extracts in Table 3 were dissolved using DMSO (Ramesh et al., 2012; Sharma et al., 2016) and 1 mL of each solution was diluted in 9 mL of DIH2O to make 10% DMSO dilutions and re-diluted to make 1 mg of extract per mL of 10% DMSO. The latter was the extract stock solution. For this assay, we filled 90 mm diameter plates with 25 mL of PDA. Once solidified, a 2.6 cm diameter well (2 mL) was perforated in the center of the medium using a sterile test tube cut in half. Wells were filled up with an extract/PDA mixture (200 µL of extract and 1800 µL of PDA at 100 µg/mL 1% DMSO). The negative control was 1% DMSO (Table 3).
Table 3
Treatment names and codes to assess the phytotoxic potential of SMs from Xylaria necrophora in extracted fractions from cell-free culture filtrates.
Treatment condition | Treatment name | Extract code |
X. necrophora | Cell-free culture filtrates (CFs) | CFs |
| Hexane extract from CFs | Hex |
| Dichloromethane extract from CFs | DCM |
| Diethyl ether extract from CFs | Et2O |
| Ethyl acetate extract from CFs | EtOAc |
| CFs after extraction | CFs After |
Control | § Filtered potato dextrose broth (control broth) | CBroth |
| Hexane extract from control broth | Ctrl Hex |
| Dichloromethane extract from control broth | Ctrl DCM |
| Diethyl ether extract from control broth | Ctrl Et2O |
| Ethyl acetate extract from control broth | Ctrl EtOAc |
| Control broth after extraction | Ctrl After |
| *Dimethyl sulfoxide | DMSO |
§ Used as the “r1” in Eq. (1) to calculate percentage of growth inhibition. |
*DMSO5% used for phytotoxicity assay. |
*DMSO1% used for microbial inhibition assay. |
When the media solidified, the mycelium plugs (5 ± 1 mm diameter) of the selected fungus (S. rolfsii) were placed in the center of the well filled with PDA amended with treatments. Each treatment was replicated three times and incubated at 25 ℃ in the dark for three days. Radial growth was measured as described previously.
Mass spectrometric analysis of the extracted fractions. We conducted liquid chromatography-mass spectrometry (LC-MS) tandem mass spectrometry (LC-MS/MS) to separate and identify SMs produced by X. necrophora and assessed the chromatogram of DCM and Et2O, the most bioactive fractions from previous experiments. For both LC-MS/MS and LC/MS analysis, extracted fractions were dried under a gentle stream of nitrogen and resuspended in 30% acetonitrile. As a control measure, the DCM and Et2O fractions from the control broth were also evaluated for comparison in the LC-MS experiments. LC-MS analyses were conducted on an Agilent 1,260 Infinity II quaternary liquid chromatograph coupled to an Agilent 6230 Electrospray Time-of-Flight mass spectrometer (Agilent, Santa Clara, CA). Samples were run in positive ionization mode with a capillary voltage of 4000V. Nitrogen was used as drying gas delivered at 10 L/min at a temperature of 325 ℃ and the fragmentor voltage was set to 150 V. The mass range used was 100-3,000 m/z. An Agilent Poroshell 120 EC-C18 column (3 mm ID, 100 mm length, 2.7 µm pores, end-capped) was used for chromatographic separation with a gradient program using a binary mixture of mobile phases at a fixed flow rate of 400 µL/min. The mobile phase composition was as follows: A = 0.1% formic acid in H2O and B = acetonitrile. The gradient program was as follows: 0–5 min = 5% B, 5–30 min 90% B, 30–35 min 90% B, 35–45 min 5% B.
LC-MS samples were analyzed with MassHunter Workstation module Qualitative Analysis Navigator v.B.08.00, Build 8.0.8208.0. The DCM and Et2O LC-MS runs were subtracted with their corresponding broth controls to exclude matrix peaks and other interferences. Initially, manual observation and integration of the peaks were performed to provide masses of the most intense peaks. Further analysis was performed with an automated approach. Chromatographic peak picking was performed with an absolute intensity filter of 30,000 counts and excluding spectra exceeding 10% of saturation. Mass spectrometric peak picking was performed with an absolute intensity filter of 1,000 counts and the maximum number of peaks set to 5,000.
Preliminary LC-MS analyses revealed relatively high concentrations of masses matching cytochalasin C and D (see Supplementary Fig. 4 in the Supporting Information). To corroborate the presence of cytochalasin C and D, we proceeded to conduct a targeted analysis. For this, we performed LC-MS/MS on a Bruker amaZon Speed ion trap (Bruker, Billerica, MA) coupled to a Thermo Scientific Ultimate 3000 HPLC system (Thermo Scientific, Waltham, MA). Column, mobile phase composition, flow rate, and gradient programs were the same as those used for the LC-MS analyses. The ion trap was operated in positive ion mode with a spray voltage of 4,500 V and an endplate voltage of 500 V. Nitrogen was used as auxiliary spray gas at 35 psi and as drying gas at 10 L/min and 250 ℃. The system was operated in “ultra-scan” mode, which results in an acquisition speed of 32,500 m/z per sec. A 2 Da mass window centered around 508.2 m/z was selected to isolate the compounds of interest (cytochalasin C and D). Fragmentation was induced with 1 V energy and using helium as gas. Selected fragments were used to build the output chromatograms comparing them with standards of cytochalasin C and D. LC-MS/MS samples were analyzed with Bruker Compass Data Analysis (v.5.3, Build 342.363.649). Chromatograms were built using windows of 0.1 m/z centered around four fragments: 412.1, 430.15, 448.1, and 490.2 to capture the fragmentation pattern of the cytochalasins C and D. A 2 µL aliquot was injected into each system. For LC-MS/MS, aliquots of cytochalasin C and D standards were used as references (see Supplementary Fig. 5 to Fig. 7 in the Supporting Information).
Finally, LC-MS data were used to conduct a database search against known SMs produced by species in the family Xylariaceae conducted using a mass tolerance of 20 ppm. The database was prepared by collecting selected SMs from (Song et al., 2014) and (Ibrahim et al., 2020). Masses and molecular formulas were obtained from ChemSpider (http://www.chemspider.com/Chemical-Structure.1906.html) and PubChem (https://doi.org/10.1093/nar/gkaa971). We retrieved the monoisotopic mass of the chemical compounds, and subsequently, the ions [M + H] +, [M + Na] + [M + K] +, and [M-H2O + H] + were added to create the database (available online at: https://github.com/jsolorzano734/Xylaria_necrophoraSMs). For identification, Only SMs that showed masses in addition to [M + H] +, [M + Na] + ions were considered matches with 100% confidence (see Supplementary Fig. 8 to Fig. 67 in the Supporting Information).
Statistical analysis. Statistical analyses were conducted using The R project software v.4.2.2 (R. C. Team, 2018; Wickham et al., 2019) and RStudio v.1.4.1717 (Rs. Team, 2018). For assays in this study, all the data was arranged in a completely randomized design. The `tidyverse` package v.1.3.1.9000 was used to organize the data. For analysis, functions within the R-based documentation package v.3.6.2 were used. First, the data were fitted into a `lm` (linear model) and the normality of the residuals was evaluated using the Kolmogorov-Smirnov test with the function `ks.test`. Analysis of variance (ANOVA) was conducted with the function `aov` using the data from the linear model. Finally, if differences were noted from ANOVA, a pairwise (Tukey Honest Significant Differences) posthoc analysis was conducted using the function `TukeyHSD`. Additionally, the function `t.test` was used to conduct a two-tailed Student’s t-test for the following assays: root weight, root length, and digital chlorophyll quantification from stem cuttings. Prior to the t-tests, the variance comparison of the groups was calculated using the `var.test` function. Code and data are available online at https://github.com/jsolorzano734/Xylaria_necrophoraSMs.