Antimicrobial and phytotoxic secondary metabolites produced by Xylaria necrophora, an emerging pathogen of soybean, play key roles in infection biology

Abstract

Xylaria species are recognized globally given their common occurrence as wood-degrading saprophytes in forest ecosystems. They are known for their ability to produce secondary metabolites with diverse bioactivity. A few are pathogens, but Xylaria necrophora is the only species known to be a pathogen of an annual crop, causing taproot decline (TRD) on soybean [ Glycine max (L) Merr.]. Recent work determined that culture filtrates produced by X. necrophora are phytotoxic and likely responsible for the foliar symptoms of the disease. We demonstrate that the foliar symptoms may be the result of root inhibition as culture filtrates also stop root development. Xylaria necrophora also produces antimicrobial secondary metabolites (SMs) that likely mediate interactions with other soil microbes to set the stage for plant infection. Bioassay-guided fractionation and extracted fractions from cell-free culture filtrates (CFs) led to the identification of SMs using LC-MS and LC-MS/MS analyses: 1 . 18-Deoxy-19,20-epoxycytochalasin Q, 2 . 19,20-epoxycytochalasin Q, 3 . 5-(1-Hydroxybutyl)-6-(hydroxymethyl)-2H-pyran-2-one, 4 . 6-[(1R)-1-Hydroxypentyl]-4-methoxy-2H-pyran-2-one, 5 - 6 . Cytochalasin C and D, 7 . Xylopimarane, 8 . Hirsutatin A, 9 . Xylaric acid C and 10 . Zygosporin E. SMs 1 - 7 presented antimicrobial activity against fungi and 1 , 2 , 5 , 6 , 8 , 9 , and 10 were phytotoxic to soybean. SMs 1 , 2 , 5 , and 6 , were both phytotoxic and antimicrobial. This is the first report identifying SMs produced by X. necrophora . SMs capable of both causing phytotoxicity and inhibiting a diversity of fungal pathogens suggests an important role for these SMs in the etiology of TRD.

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Antimicrobial and phytotoxic secondary metabolites
produced by Xylaria necrophora, an emerging
pathogen of soybean, play key roles in infection
biology
José E. Solórzano
University of Minnesota
Moshood O. Ganiu
Louisiana State University
Fabrizio Donnarumma
Louisiana State University
Teddy Garcia-Aroca
University of Nebraska-Lincoln
Rendy Kartika
Louisiana State University
Jonathan K. Richards
Louisiana State University
Joshua P. Van Houten
Louisiana State University
Michelle R. Gremillion
Louisiana State University
Paul P. Price
Louisiana State University
Vinson Doyle (  VDoyle@agcenter.lsu.edu )
LSU https://orcid.org/0000-0002-2350-782X
Research Article
Keywords: competition, cytotoxicity, fungal, mycotoxins, natural products, plants
Posted Date: June 6th, 2023
DOI: https://doi.org/10.21203/rs.3.rs-3002498/v1
License:   This work is licensed under a Creative Commons Attribution 4.0 International License. 
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Abstract
Xylaria
species are recognized globally given their common occurrence as wood-degrading saprophytes
in forest ecosystems. They are known for their ability to produce secondary metabolites with diverse
bioactivity. A few are pathogens, but
Xylaria necrophora
is the only species known to be a pathogen of an
annual crop, causing taproot decline (TRD) on soybean [
Glycine max
(L) Merr.]. Recent work determined
that culture ltrates produced
by
X.
necrophora are phytotoxic and likely responsible for the foliar
symptoms of the disease. We demonstrate that the foliar symptoms may be the result of root inhibition
as culture ltrates also stop root development.
Xylaria necrophora
also produces antimicrobial secondary
metabolites (SMs) that likely mediate interactions with other soil microbes to set the stage for plant
infection. Bioassay-guided fractionation and extracted fractions from cell-free culture ltrates (CFs) led to
the identication of SMs using LC-MS and LC-MS/MS analyses: 1. 18-Deoxy-19,20-epoxycytochalasin Q,
2. 19,20-epoxycytochalasin Q, 3. 5-(1-Hydroxybutyl)-6-(hydroxymethyl)-2H-pyran-2-one, 4. 6-[(1R)-1-
Hydroxypentyl]-4-methoxy-2H-pyran-2-one, 5-6. Cytochalasin C and D, 7. Xylopimarane, 8. Hirsutatin A, 9.
Xylaric acid C and 10. Zygosporin E. SMs 1-7 presented antimicrobial activity against fungi and 1, 2, 5, 6,
8, 9, and 10 were phytotoxic to soybean. SMs 1, 2, 5, and 6, were both phytotoxic and antimicrobial. This
is the rst report identifying SMs produced by
X. necrophora
. SMs capable of both causing phytotoxicity
and inhibiting a diversity of fungal pathogens suggests an important role for these SMs in the etiology of
TRD.
Introduction
Xylaria necrophora
(Garcia-Aroca et al., 2021) causes taproot decline (TRD) of soybean [
Glycine max
(L.)
Merr.], an important global commodity.
Xylaria
species can be saprophytes, endophytes (Rogers, 1979),
and/or pathogens, however,
X. necrophora
is the only known pathogen of an annual crop among its
congeners.
X. necrophora
is also a saprophyte of soybean, cotton, corn, and woody debris. Symptoms of
TRD develop on leaves at any growth stage as interveinal chlorosis and necrosis, damage to the vascular
tissue of the taproot, and root necrosis (Allen, 2017). Colonization of living soybean appears restricted to
the taproot and crown, despite observing foliar symptoms, but the mechanisms of infection and
symptom development have yet to be dened.
Foliar symptoms of TRD resemble those of Sudden Death Syndrome (SDS), caused by
Fusarium
virguliforme
, and Brown Stem Rot caused by
Cadophora gregata
(=
Phialophora gregata
), as well as
nutritional deciencies of soybean such as zinc and manganese (Allen, 2017). Similar to
X. necrophora
,
F.
virguliforme
is restricted to the lower parts of plants and causes root rot, chlorosis, necrosis, and damage
to leaet margins. Signs of SDS are observed on and in the roots, while symptoms in the leaves are
associated with phytotoxic secondary metabolites (SMs) translocated from infected roots to the leaves
(Jin et al., 1996; Hartman et al., 2004; Brar et al., 2011; Wang et al., 2015; Chang et al., 2016; Sahu et al.,
2017). A recent study suggests that
X. necrophora
produces phytotoxic SMs to cause TRD (Garcia-Aroca
et al., 2022), but with unknown roles in pathogenicity or virulence (Purvis, 2019).

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High isolation rates (~ 80% compared to other microbes) of
X. necrophora
from the roots of symptomatic
plants indicate that phytotoxicity may not be the only bioactivity of its produced SMs. This high recovery
rate of
X. necrophora
from soybean roots raises the prospect that it is producing antimicrobial SMs for
competition prior to and during root colonization. Hence, identifying and characterizing both phytotoxic
and antimicrobial SMs will improve our understanding of the etiology of TRD and may provide new
avenues for management.
Fungi in the family Xylariaceae produce bioactive SMs (Song et al., 2014; Helaly et al., 2018; Becker &
Stadler, 2021), i.e., phytotoxic, antifungal, antibacterial, antiviral, and cytotoxic SMs (Song et al., 2014;
Helaly et al., 2018). Cytochalasins, for example, are a large group of SMs with multiple bioactivities (Song
et al., 2014; Helaly et al., 2018; Elias et al., 2018; Lambert et al., 2023). Cytochalasins produced by
Xylaria
abelliformis
(=
Xylaria cubensis
) (Helaly et al., 2018; Biasettoa et al., 2019),
Xylaria mali
(Elias et al.,
2018), and
Dematophora necatrix
(=
Rosellinia necatrix
) (Arjona-Girona et al., 2017) are known to have
both phytotoxic and antifungal effects. Other phytotoxins produced by species in the Xylariaceae include
hymatoxin A (Bodo et al., 1987), xylobovide (Song et al., 2014; Abate et al., 1997), and seiricuprolide
(Helaly et al., 2018; Ballio et al., 1988) to name a few. There is also a suite of antimicrobial compounds
other than cytochalasins produced by the Xylariaceae, including xylarenones A and B, and xylarenic acid
(Hu et al., 2010), phomenone (Silva et al., 2010), xylopimarane (Isaka et al., 2011), sordaricin
(Pongcharoen et al., 2008), xylarin (Schneider et al., 1995), and piliformic acid (2-hexyl-3-methyl-
butanodioic acid) (Cafêu et al., 2005), among others (Song et al., 2014). Phytotoxic and antimicrobial
SMs produced by fungal pathogens within the Xylariaceae reect the potential of
X. necrophora
to
produce SMs involved in the etiology of TRD.
Due to the known ability of fungal pathogens in the family Xylariaceae to produce bioactive SMs,
apparent restriction of
X. necrophora
to the roots while producing symptoms in leaves of soybean plants,
and high rates of isolation, we hypothesized that
X. necrophora
produces phytotoxic and antimicrobial
SMs. With this study, we aimed to identify the specic bioactive compounds produced by
X. necrophora
as SMs while providing insight into their role in the biology of
X. necrophora
as a pathogen of soybean.
Specically, we sought to determine: 1) the identity of SMs producing foliar symptoms in soybean, 2) the
identity of SMs responsible for the inhibition of other microorganisms, and 3) the impact of SMs on
soybean root development.
Materials and methods
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.

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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 mbriata
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.
agellaris
Soybean NA 2926
Curvularia lunata
Grain Sorghum NA 2087
Glomerella cingulata
Pecan NA 3140
Aspergillus avus
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.

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Table 2
Treatments to assess the phytotoxicity stability of SMs produced by
X.
necrophora.
Treatment
condition
Treatment § Code
X. necrophora
Cell-free culture ltrates (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
ltrates (CFs) in sterile asks (500 mL, regular and sidearm). Mock-inoculated PDB served as a control
(control broth). CFs and control broth asks 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 rst trifoliate.
Phytotoxicity of CFs. To validate previously reported phytotoxic effects of CFs from
X. necrophora
on
soybean leaves (Garcia-Aroca et al., 2022) and conrm 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 ve 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.

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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 leaets from the rst 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 inltrated into
the underside of soybean leaets using 1 mL sterile needle-less syringes (Fisherbrand™ Sterile Syringes,
PA). There were six leaet repetitions per treatment and three technical repetitions of this bioassay. After
inltration, 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 ve days. Fungal growth was measured every 24 hours by
delimiting the colony edge at the bottom of the plate with a ne-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.

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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 ve 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 ve 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 conrming 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
ow 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

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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 leaets [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 ve days.
The phytotoxic effect of the treatments on stem cuttings, leaets, and leaf disks was measured based on
changes in chlorophyll content. Leaf disks and leaets 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 leaets, 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 conrmed. 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 lled 90 mm diameter plates
with 25 mL of PDA. Once solidied, 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 lled 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).

Page 10/29
Table 3
Treatment names and codes to assess the phytotoxic potential of SMs from
Xylaria necrophora
in extracted fractions from cell-free culture ltrates.
Treatment
condition
Treatment name Extract code
X. necrophora
Cell-free culture ltrates (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 solidied, the mycelium plugs (5 ± 1 mm diameter) of the selected fungus (
S. rolfsii
)
were placed in the center of the well lled 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 Innity II quaternary liquid chromatograph coupled to an

Page 11/29
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 xed ow 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 lter of 30,000 counts and excluding spectra exceeding 10% of saturation. Mass
spectrometric peak picking was performed with an absolute intensity lter 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 Scientic Ultimate 3000
HPLC system (Thermo Scientic, Waltham, MA). Column, mobile phase composition, ow 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

Page 12/29
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 identication, Only SMs that showed
masses in addition to [M + H] +, [M + Na] + ions were considered matches with 100% condence (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 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 tted into a (linear model) and the normality of the residuals was evaluated using
the Kolmogorov-Smirnov test with the function . Analysis of variance (ANOVA) was conducted
with the function using the data from the linear model. Finally, if differences were noted from ANOVA,
a pairwise (Tukey Honest Signicant Differences) posthoc analysis was conducted using the function
. Additionally, the function was used to conduct a two-tailed Student’s t-test for the
following assays: root weight, root length, and digital chlorophyll quantication from stem cuttings. Prior
to the t-tests, the variance comparison of the groups was calculated using the function. Code
and data are available online at https://github.com/jsolorzano734/Xylaria_necrophoraSMs.
Results
Bioassay-guided fractionation of culture ltrates from
X. necrophora
demonstrated that diethyl ether
(Et2O) was the most phytotoxic fraction and dichloromethane (DCM) was the most antimicrobial fraction.
From these bioactive fractions, we identied 10 phytotoxic and antimicrobial SMs using LC-MS and LC-
MS/MS analyses: 1. 18-Deoxy-19,20-epoxycytochalasin Q, 2. 19,20-epoxycytochalasin Q, 3. 5-(1-
Hydroxybutyl)-6-(hydroxymethyl)-2H-pyran-2-one, 4. 6-[(1R)-1-Hydroxypentyl]-4-methoxy-2H-pyran-2-one,
5–6. Cytochalasin C and D, 7. Xylopimarane, 8. Hirsutatin A, 9. Xylaric acid C and 10. Zygosporin E
(Fig.1). Further investigation of the phytotoxic fraction indicates that it may contain putative
cytochalasin homologs (see Supplementary Fig.7 in the Supporting Information).
Phytotoxicity of cell-free culture ltrates. CFs at 25-fold dilution signicantly reduced the chlorophyll
content of soybean leaves (102.21 ± 4.05 ng/mm2) compared to control broth (165.18 ± 5.53 ng/mm2) 10
days after treatments were applied (
P
< 0.05; Fig.2b). Around three days after application (daa), leaets
started to develop the rst chlorotic symptoms. The symptoms started to appear as patchy and mild
interveinal chlorosis, which in most cases led to total chlorosis of the leaves. Necrosis was not observed
10 daa. In contrast, patterns of reduction in chlorophyll content were not observed in stem cuttings
exposed to control broth (Fig.2a to 2d).
Cytotoxic effect of cell-free culture ltrates on roots. CFs at 25-fold dilution were applied to soybean stem
cuttings and reduced root growth and root elongation. Root growth was halted immediately (< 72 hours)
after exposure to CFs and chlorotic symptoms appeared on leaets. In contrast, stem cuttings exposed to
tverse
lm ks
.
test
aov
TukeyHSD t
.
test
var
.
test

Page 13/29
control broth developed roots that continued growing throughout the assay (Fig.2a to 2d). CFs
signicantly reduced fresh root biomass (0.62 ± 0.02 g;
P
< 0.05) in comparison to control broth (0.93 ±
0.03 g) and root length (6.75 ± 0.35 mm) compared to control broth (10.97 ± 0.47 mm, Fig.2c and 2d).
Trypan blue staining of roots shows the root cells exposed to the control broth are living while exposure
to CFs led to severe cell death (Fig.2a).
CFs were exposed to a digestion solution of Pronase to test for proteinaceous phytotoxins. MOPS and
Pronase were evaluated in combination and independently since they were used to make the digestion
solution (Fig.2e). Compared to the control broth (113.1 ± 5.1 ng/mm2), the addition of MOPS to any
treatment seemed to lead to a reduction in chlorophyll content (MP = 109.6 ± 7.9 ng/mm2; CBrothMP =
94.9 ± 5.2 ng/mm2; CBrothM – 88.4 ± 6.9 ng/mm2). The exception to this trend was when CFs were
amended with MOPS (CFsM = 101.1 ± 4.9 ng/mm2). The largest reductions in chlorophyll content,
however, were among both soybean leaves treated with CFs (84.1 ± 3.1 ng/mm2,
P
< 0.05; Fig.2e) and
CFs exposed to digestion with Pronase (CFsMP = 78.9 ± 3.6 ng/mm2,
P
< 0.05), suggesting that non-
proteinaceous compounds are responsible for symptoms of TRD.
Phytotoxic potential of fractionated cell-free culture ltrates (CFs). After conrming CFs’ phytotoxicity,
fractions were extracted from CFs to identify phytotoxic SMs. Extracted fractions were compared to
control broth extracts and the aqueous phases of CFs (“CFs After”) and the remaining layer of control
broth after extraction (“Ctrl After”; Table3). All studied extracted fractions obtained from CFs reduced
chlorophyll of soybean leaf disks (Fig.1f). However, the diethyl ether extracted fraction (Et2O = 37.74 ±
1.48 ng/mm2) exerted the greatest impact, signicantly reducing the chlorophyll content beyond that of
the CFs alone (CFs = 97.4 ± 4.67 ng/mm2) and all other extracts. The reduction in chlorophyll content by
CFs was not signicantly different from the CFs fractions extracted with dichloromethane (DCM = 71.32
± 3.95 ng/mm2), ethyl acetate (EtOAc = 75.76 ± 4.59 ng/mm2), and hexane (Hex = 102.12 ± 4.48 ng/mm2).
The aqueous phase remaining after extraction of CFs (CFs After = 116.63 ± 4.24 ng/mm2) elicited the
lowest reduction of chlorophyll content in comparison to CFs and extracts from CFs and was not
statistically different from the control broth (
P
> 0.05). Control broth after extraction exhibited the least
impact, and the hexane extract from control broth was the least effective at reducing chlorophyll content
(Ctrl Hex = 144.79 ± 5.85 ng/mm2) (Fig.1f).
Antimicrobial activity of Xylaria necrophora. To identify SMs with antimicrobial potential, we used two
approaches. First, CFs were amended to PDA + at 10d and 50d to test for antimicrobial activity without
morphological structures of the pathogen (chemical warfare alone). Second, a dual interaction assay was
conducted to challenge
X. necrophora
with 15 fungal plant pathogens (Table1) for
in situ
production of
SMs (chemical and physical warfare).
Antimicrobial activity induced by cell-free culture ltrates (CFs). Despite dilution, CFs amended to PDA +
successfully inhibited the growth of 15 fungi (Fig.3a). Inhibition percentages ranged from 0.3–93% at
10d, and 19–92% at 50d. The greatest inhibitions were observed against
Cercospora zeae-maydis
(
I
at

Page 14/29
10d = 93 ± 2% and at 50d = 92 ± 5%),
Ceratocystis mbriata
(
I
at 10d = 79 ± 10% and at 50d = 92 ± 6%) and
Exserohilum tursicum
(
I
at 10d = 67 ± 3% and at 50d = 85 ± 1%). In contrast, the lowest inhibitions were
observed against
Monilochaetes infuscans
(
I
at 10d = 8 ± 5% and at 50d = 29 ± 11%),
Rhizoctonia solani
(
I
at 10d = 1 ± 0.6% and at 50d = 19 ± 3%) and
S. rolfsii
(
I
at 10d = 0.3 ± 0.2% and at 50d = 20 ± 10%) (see
Supplementary Fig.2 in the Supporting Information).
Fungal growth inhibition by direct interaction with X. necrophora. For the dual interaction assay, 15 fungal
plant pathogens (Table1) were divided into three groups based on growth rates in relation to
X.
necrophora
. Regardless of growth type,
X. necrophora
directly inhibited the growth of the 15 fungi
(Fig.3b). The percentages of growth inhibition ranged from 34–64% at 10 days of interaction. The three
highest inhibited fungi were
Exserohilum tursicum
(64 ± 1%),
Ceratocystis mbriata
(61 ± 8%), and
Magnaporthe oryzae
(60 ± 2%). The least inhibited fungi were
Rhizopus stolonifer
(34 ± 1%),
Glomerella
cingulata
(37 ± 1%), and
Aspergillus avus
(38 ± 1%) (see Supplementary Fig.3 in the Supporting
Information).
Interestingly, both antimicrobial assay approaches resulted in a high percentage of growth inhibition for
each of the 15 fungi. However, growth inhibition percentages were lower in the dual interaction assay (10
days of duration) compared to the assay using CFs amended to PDA+ (ve days of duration). This is
likely related to the number or type of SMs produced by
X. necrophora
while directly competing with the
tested organisms compared to the number and type of SMs produced while cultured in liquid media. This
is supported by the fact that growth inhibition percentages that resulted from the CFs assay were
dependent on the dilution concentration of the CFs (Fig.3a).
Antimicrobial potential of cell-free culture ltrates (CFs) fractions. CFs were extracted with four solvents
to generate fractions (Table3). The DCM fraction was the most bioactive with signicantly greater
inhibition than all others (Fig.3c), exerting 35 ± 2% growth inhibition three days after culturing. Compared
to DCM, the next strongest inhibitors were the aqueous phase of the CFs after extraction (CFs After = 11 ±
0.1%), and CFs alone (CFs = 10 ± 3%) was the least effective CFs fraction (Fig.3c). All CFs fractions were
effective at growth inhibition, although not signicantly different from the CFs alone (Et2O = 5 ± 1%;
EtOAc = 6 ± 2%; Hex = 8 ± 1%). The lowest inhibition percentages were induced by the aqueous phase of
the control broth (CBroth After = 3 ± 2%) and the diethyl ether extract from the control broth (Ctrl Et2O = 4
± 2%), similar to the phytotoxicity assays. Interestingly, the DCM fraction reduced the aerial mycelium of
S. rolfsii
, which is similar to when
S. rolfsii
was exposed to PDA + amended with CFs at 50d.
Discussion
The fungus
X. necrophora
, a saprophyte and recently emerged plant pathogen of soybean, produces
antimicrobial and phytotoxic SMs to mediate ecological interactions and cause TRD. A preliminary study
attempting to uncover underlying mechanisms associated with the etiology of TRD conrmed the ability
of
X. necrophora
to produce toxins, likely SMs, that induce phytotoxicity at low concentrations (Garcia-
Aroca et al., 2022). However, we also suspected these or a different set of SMs facilitated microbial

Page 15/29
antagonism as a mechanism prior to and during colonization because of the high isolation rates from
eld-infected roots. Therefore, this study aimed to determine the role and identity of SMs producing foliar
symptoms in soybean, the identity of SMs responsible for the inhibition of other microorganisms, and the
impact of SMs on soybean root development. Below we additionally discuss the potential role that these
SMs may have in the disease cycle of taproot decline.
Phytotoxicity.
X. necrophora
produces phytotoxic SMs (Figs.1 and 2) that are non-proteinaceous. While
SDS symptoms in soybean are similar to those of TRD and are the result of fungal proteinaceous
phytotoxins translocated from roots to leaves (Chang et al., 2016), Pronase digestion of CFs from
X.
necrophora
did not modify the toxicity of CFs. While some reduction in chlorophyll content was observed,
this is due to the addition of MOPS. The effects of MOPS in metabolite dilutions are unknown, but MOPS
is thought to bind to metals such as Fe, Mn, and Cu elements needed for chlorophyll production, which
may account for the observed behavior of MOPS in this study (Ferreira et al., 2015; Schmidt et al., 2020).
In a different study, when Pronase diluted in MOPS was applied to CFs of
Cercospora beticola
and
inltrated into sugar beet leaves, CFs did not produce any symptoms. They determined that the effector
protein
CbNip1
was responsible for enhancing the development of necrotic symptoms on leaves (Ebert et
al., 2021). The effect of MOPS was not evaluated in that study. Similarly, when the CFs of
Phaeosphaeria
nodorum
(anamorph =
Stagonospora nodorum
) were exposed to Pronase with MOPS and inltrated into
susceptible wheat lines, necrotic symptoms were not observed on leaves, and the virulence factors were
determined to be proteinaceous effectors that were destroyed by the proteinase treatment (Friesen et al.,
2012). The persistence of symptoms induced by CFs after treatment with Pronase eliminates
proteinaceous compounds as being responsible for phytotoxicity.
The phytotoxicity observed in leaves of plants infected with
X. necrophora
may result from the
translocation of phytotoxic metabolites, however the CFs also signicantly inhibit root development of
soybean and cause cell death (Fig.2a). Similar to
X. necrophora
,
X. feejeensis
strain SM3e-1b, an
endophyte of
Sapium macrocarpum
and causal agent of dry rot of raa palm (
Raphia hookeri
) fruits,
produces SMs that inhibit root growth of
Trifolium pratense
,
Panicum miliaceum, Amaranthus
hypochondriacus
, and
Medicago sativa
(García-Méndez et al., 2016).
Dematophora necatrix
, a fungus of
the family Xylariaceae, colonizes the roots of avocado trees to cause root rot and produces low
concentrations of cytochalasin E damaging the foliage (Arjona-Girona et al., 2017). In our study, soybean
root growth was halted before foliar symptoms were observed, indicating that SMs of
X. necrophora
damage soybean roots to enable colonization. It may be that these SMs are also translocated to the
leaves where chlorotic symptoms develop, but the translocation of SMs from roots to leaves has not been
conrmed.
Phytotoxicity caused by SMs of
X. necrophora
suggests new bioactivity for previously known SMs. Seven
SMs of
X. necrophora
were identied to be phytotoxic (Fig.1, Table4). Five of these SMs are
cytochalasins (1, 2, 5, 6, and 10), SM-8 is a non-ribosomal peptide and SM-9 is a sesquiterpene acid
(Table4). All seven SMs have been isolated from other species in the family Xylariaceae, but only three of
the ve cytochalasins are known cytotoxic or antimicrobial while the remaining SMs have not been tested

Page 16/29
for phytotoxicity. We additionally identied the presence of putative cytochalasin homologs, but their
structure is unknown (see Supplementary Fig.7 in the Supporting Information). 18-deoxy-19,20-
epoxycytochalasin Q (SM-1), also known as xylobovatin, has been previously isolated from the wood-
inhabiting fungus
X. abovata
(strain ADA 228), but bioactivity was not evaluated (Abate et al., 1997).
19,20-epoxycytochalasin (SM-2) is produced by the wood-inhabiting fungus
Xylaria
sp. (strain BCC 1067)
and is known to inhibit the growth of
Saccharomyces cerevisiae
strains (Somboon & Soontorngun, 2021).
Similar cytochalasins and closely related to cytochalasin C (SM-5) recently isolated from
Xylaria
karyophthora
isolate NRRL 66613, a seed pathogen of greenheart (
Chlorocardium
sp.) (Lambert et al.,
2023), reduced biolm formation of
Staphylococcus aureus
and
Candida albicans
. Phytotoxicity was not
evaluated by Lambert et al. (2023). SM-5 has been isolated from
Hypoxylon terricola
J.H. Mill, a
saprophyte of senesced conifer needles (Edwards et al., 1989). In a recent study, SM-5 was cytotoxic to
mammalian THP-1 cells (Boguś et al., 2021) but no phytotoxicity has been reported. Cytochalasin D (SM-
6) is produced by
X. arbuscula
(Amaral et al., 2014)d
cubensis
and is known to be cytotoxic to cancer cell
lines NCI-H187 and KB (Sawadsitang et al., 2015). In other studies, SM-6 inhibited the growth of
Cladosporium cladosporioides
,
Cladosporium sphaerospermum
, and
Colletotrichum gloeosporioides
(teleomorph =
Glomerella cingulata
) (Cafêu et al., 2005; Elias et al., 2018).
G. cingulata
was inhibited in
the current study. Hirsutatin A (SM-8), has been isolated from the entomopathogenic fungus
Hirsutella
nivea
(BCC 2594) (Isaka et al., 2005), X.
ellisii
(Ibrahim et al., 2020), and from
X. cubensis
when co-
cultivated with
Aspergillus scheri
(Knowles et al., 2019). SM-8, however, did not exhibit bioactivity
against the malarial parasite
Plasmodium falciparum
and its role in phytotoxicity is unknown (Isaka et
al., 2005). SM-9 (Xylaric acid C) was previously isolated from a
Xylaria
species associated with termites
but it did not exhibit any bioactivity when tested by Yan et al., (2011) and its role in phytotoxicity is also
unknown. Zygosporin E (SM-10) was previously identied from
X. ellisii
, an endophyte of wild lowbush
blueberry (
Vaccinium angustifolium
Komatsu); however, no bioactivities were reported (Ibrahim et al.,
2020).

Page 17/29
Table 4
Classication and identication parameters of bioactive secondary metabolites produced by
Xylaria
necrophora
.
Structure Class Solvent for
extraction Retention time
(
rt) m/z
formula
m/z
value
1, 3 and
4
1, 3 and
4
Cytochalasins Dichloromethane 11.794, 11.81,
13.72, 13.737,
13.99, and
14.666
(M +
H)+
508.2632,
508.2632,
508.2631,
508.2628 and
508.2649
12.943, 12.96,
13.72, 13.737,
and 14.007
(M +
H)+[-
H2O]
490.2525,
490.2523,
490.2528,
490.2527 and
490.2539
13.99, 14.007,
and 14.666 (M +
Na)+
530.245,
530.2451 and
530.2468
Diethyl ether 13.516, 13.533,
13.786, and
13.803
(M +
H)+
508.2658,
508.2658,
508.2657 and
508.2656
12.84, 12.857,
13.516, 13.533,
13.786, and
13.803
(2M +
H)+
1015.523,
1015.5228,
1015.5257,
1015.5251,
1015.5288 and
1015.5285
12.84, 12.857,
13.043, 13.178,
13.516, 13.533,
13.786, and
13.803
(M +
H)+[-
H2O]
490.255,
490.2548,
490.255,
490.2558,
490.2557,
490.2557,
490.2555, and
491.2588
12.84, 13.178,
13.516, and
13.533
(M +
Na)+
530.2477,
530.2493,
530.2481 and
530.2482
12.84, 13.516,
and 13.533 (2M +
Na)+
1037.5059,
1037.5087 and
1037.5083
2 Cytochalasins Dichloromethane 12.537 and
12.554 (M +
H)+
524.2582 and
524.258
*SMs identied using both the ion trap method and a custom database
(https://github.com/jsolorzano734/Xylaria_necrophoraSMs). See Supporting Information.

Page 18/29
Structure Class Solvent for
extraction Retention time
(
rt) m/z
formula
m/z
value
11.472 and
12.554 (M +
H)+[-
H2O]
506.2463 and
506.2472
12.537 (M +
Na)+
546.2391
Diethyl ether 13.043, 13.06,
and 13.33 (M +
H)+
524.2608,
524.2608 and
524.2609
12.468 (2M +
H)+
1047.5153
12.468 (M +
H)+[-
H2O]
506.2493
13.043, 13.06,
and 13.33 (M +
Na)+
546.2422,
546.2419 and
546.2429
12.468, 12.485,
13.043, 13.06,
and 13.33
(2M +
Na)+
1069.4949,
1069.4942,
1069.4958,
1069.4962 and
1069.4965
5 Diterpenoids
and diterpene
glycosides
Dichloromethane 12.791 (M +
H)+
465.2426
12.774 and
12.791 (M +
Na)+
487.2244
6 Nonribosomal
peptides Diethyl ether 14.496, 14.513,
16.7610, and
16.7770
(M +
H)+
677.3716,
677.3718,
677.3674 and
677.3668
13.178, 13.195,
13.313, and
13.33
(M +
H)+[-
H2O]
659.3555,
659.3592,
659.3541 and
659.3538
14.513 (M +
Na)+
699.3533
7 Sesquiterpene
acids Diethyl ether 11.133 and
11.674 (M +
H)+
345.1857 and
345.1854
*SMs identied using both the ion trap method and a custom database
(https://github.com/jsolorzano734/Xylaria_necrophoraSMs). See Supporting Information.

Page 19/29
Structure Class Solvent for
extraction Retention time
(
rt) m/z
formula
m/z
value
10.88, 10.897,
12.265, and
12.282
(M +
Na)+
367.1733,
367.1733,
367.1722 and
367.1727
10.88 (M +
Na)+[-
H2O]
349.1631
8 Cytochalasins Diethyl ether 14.9520 (M +
H)+
492.2732
14.344, 14.699,
and 14.7160 (2M +
H)+
983.5352,
983.5356 and
983.5368
14.699, 14.7160,
and 15.2400 (M +
Na)+
514.254,
514.2537 and
514.2585
14.344, 14.496,
14.699, and
14.7160
(2M +
Na)+
1005.5179,
1005.5179,
1005.5191 and
1005.5195
9 Pyrone
derivatives Dichloromethane 7.096 (M +
H)+
199.0955
5.203 (M +
H)+[-
H2O]
181.0838
7.096 and 7.112 (M +
Na)+
221.0788 and
221.0789
10 Pyrone
derivatives Dichloromethane 5.71 and 10.036 (M +
H)+
213.1096 and
213.1115
5.727, 7.096, and
10.036 (M +
H)+[-
H2O]
195.0989,
195.1036 and
195.1003
5.727 (M +
Na)+
235.0914
*SMs identied using both the ion trap method and a custom database
(https://github.com/jsolorzano734/Xylaria_necrophoraSMs). See Supporting Information.
Antimicrobial activity.
X. necrophora
mediates interactions with other organisms to enhance colonization
by producing specialized SMs for microbial antagonism. As indicated above,
Xylaria
species produce

Page 20/29
SMs known to inhibit not only fungi but multiple economically important bacteria. In the current study, LC-
MS and LC-MS/MS analyses of the DCM fraction, the extracted fraction from CFs with the most
antifungal activity, led to the identication of seven antimicrobial SMs (Fig.1, Table4): Four
cytochalasins (SM 1, 2, 5 and 6) along with two pyrone derivatives (SM 3 and 4), and a diterpenoid and
diterpene glycoside (SM-7). Interestingly, SMs 1, 2, 5, and 6 were identied from both the phytotoxic and
antimicrobial fractions and are discussed above. In previous studies, 5-(1-Hydroxybutyl)-6-
(hydroxymethyl)-2H-pyran-2-one (SM-3), also known as taiwapyrone, was isolated from
Xylaria
sp. isolate
NCY2, an endophyte of Jack torreya (
Torreya jackii
Chun) (Hu et al., 2010). Antimicrobial activity of SM-3
was only conrmed against the bacteria
Escherichia coli
ATCC 25922,
Bacillus subtilis
ATCC 9372, and
Staphylococcus aureus
ATCC 25923, but no antimicrobial activity was observed against
Candida
albicans
and
Saccharomyces cerevisiae
(Hu et al., 2010). 6-[(1R)-1-Hydroxypentyl]-4-methoxy-2H-pyran-2-
one (SM-4) was identied from the marine-associated fungus
Xylaria
sp. isolate PSU-F100 isolated from
a gorgonian sea fan (
Annella
sp.). SM-4 was also observed to have mild antimicrobial activity against
Staphylococcus aureus
ATCC 25923 (Rukachaisirikul et al., 2009). Xylopimarane (SM-7) has been
isolated from
Xylaria
sp. isolate BCC 21097 and was cytotoxic to cancer cell lines NCI-H187, KB, and
MCF-7 (Isaka et al., 2011), but its activity against microbes has not been demonstrated. Given the
antimicrobial activity observed from the dual interaction assay, PDA + amended with CFs, PDA amended
with extracts, and the known bioactivities of the SMs identied (1–7), it is likely that
X. necrophora
produces SMs to inhibit the growth of other organisms while causing TRD.
Role of secondary metabolites in the disease cycle of TRD. Phytotoxic and antimicrobial SMs produced
by
X. necrophora
contribute to the etiology of TRD. Our results suggest that
X. necrophora
produces
antimicrobial SMs in the rhizosphere during competition with other soil microbes (Fig.3). The presence of
these SMs in the rhizosphere leads to root cell death (Fig.2a to 2d) and the subsequent colonization of
the roots as a necrotroph. It is likely that as roots are colonized,
X. necrophora
continues producing SMs
that are translocated to the foliage causing chlorosis and necrosis (Fig.2 and Fig.3), but translocation
needs to be conrmed.
X. necrophora
shifts from being a necrotrophic pathogen to a saprophytic lifestyle
on decomposing plant debris (soybean, corn, cotton, woody tissues) for survival and reproduction. New
infections of soybean by
X. necrophora
are likely initiated from this infested debris that is proximate to
soybean seeds or seedlings. This proposed model is supported by the identication of SMs that are
phytotoxic, antimicrobial, or both. Overall, SMs produced by
X. necrophora
aid in mediating ecological
interactions in the rhizosphere and inside soybean plants leading to TRD. The utilization of SMs for
infection is likely an exaptation of SMs that evolved to compete with other microbes involved in
decomposition rather than a specialized mechanism for plant infection.
Declarations
Funding
This work was supported by theLouisiana Soybean and Grain Research and Promotion Board with a
grant to Vinson Doyle.

Page 21/29
Competing interests
The authors have no relevant nancial or non-nancial interests to disclose.
Availability of data and material
The code for analyses and data supporting the results of this study are available online at:
https://github.com/jsolorzano734/Xylaria_necrophoraSMs.
Author Contributions
José E. Solórzano and Vinson P. Doyle conceptualized this study. All authors contributed to the study
design and interpretation of results. Material acquisition and preparation, data collection, and analyses
were performed by José E. Solórzano,Moshood O. Ganiu, Fabrizio Donnarumma,Joshua P. Van Houten,
Michelle R. Gremillion, Vinson P. Doyle, Paul Price, Rendy Kartika, and Teddy Garcia-Aroca. The
manuscript was written by José E. Solórzano and Vinson P. Doyle. All authors commented on and edited
the manuscript. All authors read and approved the nal manuscript.
Acknowledgments
The authors thank the Louisiana Soybean and Grain Research and Promotion Board for funding this
study, and Connie David and Elaisa Tubana for technical assistance.
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Figures

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Figure 1
Secondary metabolites produced by
Xylaria necrophora

Page 27/29
Figure 2
Representation of the phytotoxic potential of secondary metabolites produced by
Xylaria necrophora,
(a)
phytotoxicity of cell-free culture ltrates (CFs) at 25-fold dilution on soybean; squares on roots represent
section transversally cut for staining root cell death, (b)reduction of chlorophyll content, (c) root biomass,
and (d) root length 10 days after treatment exposure; (e) CFs exposed to Pronase digestion conserve
phytotoxic potential on soybean leaves, and (f)phytotoxicity of extracted CFs fractions (100 µg/mL) on

Page 28/29
soybean leaf disks ve days after application. Boxplots represent the lower percentile and upper limit
percentile and the median at the center. The treatment condition refers to the preparation source.
Whiskers represent values 1.5 above and below the interquartile range and outliers are represented by
dots. Asterisks in (b)to (d) represent statistical differences at
P
≤ 0.05 by a two-tailed
t
-test. Means
followed by the same letter in (e) and (f) indicate no signicant statistical difference (Tukey’s Honest
Signicant Difference
P
≤ 0.05)

Page 29/29
Figure 3
Representation of the antimicrobial potential of secondary metabolites produced by
Xylaria necrophora
,
(a)proportion of percentage growth inhibition after 5 days on media amended with cell-free culture
ltrates (CFs) at 10% and 50 % (v/v); (b) fungal growth inhibition after 10 days of direct interaction with
X. necrophora
, and (c)fungal growth inhibition after 3 days on media containing extracted CFs fractions
at 100 µg/mL. The growth type grouping in (b) represents the growth rate of evaluated fungi in relation to
X. necrophora
. The treatment condition in (c) refers to the preparation source. In (b)and (c), bars represent
the mean ± stand error of the mean. Means followed by the same letter in (b) and (c) indicate no
signicant statistical difference (Tukey’s Honest Signicant Difference
P
≤ 0.05)
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