Mycologia

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Identification and distribution of a fungal

endosymbiotic Alternaria species (Alternaria
section Undifilum sp.) in Astragalus garbancillo
tissues

María E. Pistán, Daniel Cook, Susana A. Gutiérrez, Leonhard Schnittger, Dale

R. Gardner, Luciana A. Cholich & Ana M. Gonzalez

To cite this article: María E. Pistán, Daniel Cook, Susana A. Gutiérrez, Leonhard Schnittger,
Dale R. Gardner, Luciana A. Cholich & Ana M. Gonzalez (31 Jan 2024): Identification and
distribution of a fungal endosymbiotic Alternaria species (Alternaria section Undifilum sp.) in
Astragalus garbancillo tissues, Mycologia, DOI: 10.1080/00275514.2023.2299191

To link to this article: https://doi.org/10.1080/00275514.2023.2299191

Published online: 31 Jan 2024.

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 MYCOLOGIA
https://doi.org/10.1080/00275514.2023.2299191

Identification and distribution of a fungal endosymbiotic Alternaria species

(Alternaria section Undifilum sp.) in Astragalus garbancillo tissues
María E. Pistán a,b, Daniel Cook c, Susana A. Gutiérrez a
, Leonhard Schnittger b,d
, Dale R. Gardner c
,
Luciana A. Cholich b,e, and Ana M. Gonzalez b,f
a
Facultad de Ciencias Agrarias, Universidad Nacional del Nordeste, Corrientes 3400, Argentina; bConsejo Nacional de Investigaciones
Científicas y Técnicas (CONICET), Corrientes 3400, Argentina; cPoisonous Plant Research Laboratory, Agricultural Research Service, United
States Department of Agriculture, Logan, Utah 84341; dInstituto de Patobiología Veterinaria, Instituto Nacional de Tecnología Agropecuaria
(INTA), Hurlingham 1686, Argentina; eFacultad de Ciencias Veterinarias, Universidad Nacional del Nordeste, Corrientes 3400, Argentina;
f
Instituto de Botánica del Nordeste, Universidad Nacional del Nordeste, Consejo Nacional de Investigaciones Científicas y Técnicas (IBONE-
CONICET-UNNE), Corrientes 3400, Argentina

ABSTRACT ARTICLE HISTORY

Plants belonging to the genera Astragalus, Oxytropis, Ipomoea, Sida, and Swainsona often contain Received 13 July 2023
the toxin swainsonine (SW) produced by an associated fungal symbiont. Consumption of SW- Accepted 20 December 2023
containing plants causes a serious neurological disorder in livestock, which can be fatal. In this KEYWORDS
study, a fungal endophyte, Alternaria section Undifilum, was identified in Astragalus garbancillo Astragalus garbancillo;
seeds, using polymerase chain reaction (PCR) followed by direct sequencing. In seeds, the SW endosymbiont; microscopy;
concentrations were about 4 times higher than in other parts of the plant. Furthermore, micro­ swainsonine; Undifilum;
scopic examination demonstrated that the fungus mycelium grows inside the petioles and stems, vertical transmission
on the outer surface and inside the mesocarp of the fruit, in the mesotesta and endotesta layers of
the seed coat, and inside the endosperm of the seeds. Our results support the notion that the SW-
producing fungus is vertically transmitted in the host plant A. garbancillo.

INTRODUCTION 2019a; Micheloud et al. 2017). On the other hand, non­

Plants belonging to the genera Astragalus, Oxytropis,
Ipomoea, Sida, and Swainsona may contain poisonous
species that induce a lysosomal storage disease in live­
stock after prolonged grazing (Cook et al. 2009a; de
Balogh et al. 1999; Furlan et al. 2009; Huxtable and
Dorling 1982; Lu et al. 2013; Pfister et al. 2007;
Stegelmeier et al. 1999; Takeda et al. 2014). The toxic
principle of these plants is swainsonine (SW), a fungus-
produced indolizidine alkaloid that inhibits lysosomal
α-mannosidase and Golgi mannosidase II, leading to
impaired degradation of oligosaccharides and incom­
plete processing of glycoproteins (Elbein 1989; Malm
and Nilssen 2008).
The genus Astragalus is widely distributed in tempe­
rate regions of Europe, Asia, and in the Americas
(Molyneux and Gomez-Sosa 1991; Rios and Waterman
1997). In South America, 16 Astragalus species have been
reported to contain SW, suggesting that grazing of these
plant species represents a potential intoxication risk to
livestock (Cook et al. 2017). In Argentina, some of these
species, namely, Astragalus garbancillo, A. punae,
A. pehuenches, and A. illini, have been reported to cause
toxicity in livestock (Marin et al. 2022; Martinez et al.
toxic species of this genus are considered excellent forage
for cattle (Allred 1991; Cook et al. 2009a).
Several SW-containing Astragalus and Oxytropis spe­
cies have been found to be associated with a fungal
symbiont belonging to Alternaria section Undifilum
spp., which is responsible for the production of this
toxin (Baucom et al. 2012; Braun et al. 2003; Martinez
et al. 2019b; Yu et al. 2010). However, the plant
A. garbancillo was not included in these studies and is
therefore subject of the present investigation. Although
isolation and culture methods are commonly used to
identify and characterize endophytic fungi, this
approach provides no information on the location and
distribution of endophytes within the plant host tissue
(Bernstein and Carroll 1977). In this sense, the micro­
scopic examination of the presence of the fungus in its
host is indispensable, as it allows the localization of the
endophytic mycelia inside the plant tissues, providing
important insights (Verma et al. 2012). Unfortunately,
studies using microscopy techniques to demonstrate
endophytic localization are scarce. Fungal endophytes
have been observed in the leaves, petioles, and stems of
Oxytropis sericea (Noor et al. 2018; Reyna et al. 2012),

CONTACT Luciana A. Cholich lucianaandreacholich@gmail.com

© 2024 The Mycological Society of America

Published online 31 Jan 2024

2 PISTÁN ET AL.: ALTERNARIA IN ASTRAGALUS GARBANCILLO TISSUES
A. mollisimus and A. lentiginosus (Noor et al. 2018), and
in the seeds of Ipomoea carnea (Neyaz et al. 2022; Pistán
et al. 2022). Importantly, vertical transmission of the
fungal endophyte of locoweeds was demonstrated
(Ralphs et al. 2011) and the presence of mycelia in the
seed coat was identified by light microscopy (Oldrup
et al. 2010).
To date, there have been no microscopic studies
among the SW-containing legumes that have investi­
gated all the different organs of the same plant simulta­
neously. The objective of this study was to identify the
fungus from the seeds of A. garbancillo by polymerase
chain reaction (PCR) and to demonstrate the presence
of the endosymbiont in different aerial organs of the
plant using light and scanning electron microscopy.
MATERIALS AND METHODS
Plant material.—Samples of Astragalus garbancillo
(n = 30) were collected from El Infiernillo (San
Miguel de Tucumán Province, Argentina).
Voucher herbarium specimens were deposited in
the herbarium CTES of the Institute of Botany,
Faculty of Agricultural Sciences of Northeast
(UNNE-CONICET) in Corrientes, Argentina,
under number Pistán ME 02.
Identification and determination of SW
concentration in different tissues of A. garbancillo.—
Seeds (n = 15), fruit walls (10 g), and portions of stems
with leaves (20 g) were dried at 37 C to a constant weight
and finely ground using a FW100 mill (BioSmartest,
China). SW concentrations were measured using high-
performance liquid chromatography and mass spectro­
metry (HPLC-MS) according to previously described
methods (Gardner et al. 2001).
Identification of the fungal endosymbiont in seeds
of A. garbancillo.—DNA was extracted from seeds
using the DNeasy Plant Mini kit (Qiagen, Valencia,
California) as described by the manufacturer. The
PCR primers ITS 5 and OR1a were used to amplify
a region of the internal transcribed spacer (ITS)
(Cook et al. 2009b). Thermal cycling conditions
for amplification of the ITS region from fungal
isolates as well as plant samples were set as fol­
lows: an initial denaturation step at 94 C for
3 min, followed by 40 cycles at 94 C for 33s, 57
C for 50s, and 72 C for 30s, with a final extension
step at 72 C for 5 min. To each reaction 50 ng of
total DNA isolated from the seeds were added as
template. GoTaq DNA polymerase was used as
recommended by the manufacturer (Promega,
Madison, Wisconsin). Amplified PCR products
were directly sequenced with the forward and
reverse amplification primers, respectively. DNA
sequencing was performed at Eton Biosciences
(San Diego, California).
Light microscopy (LM).—To determine the presence of
mycelium on the surfaces of fruits, seed coats, and
embryos, the corresponding materials were soaked in
sterile water for 30 min, then dissected to expose the
different parts of the fruit and seed coat, and stained by
immersion in trypan blue for 3 min at 100 C (modified
from Oldrup et al. 2010).
For the localization of endophytic hyphae, histologi­
cal sections obtained from seeds, stem, and leaves were
fixed in FAA (formalin, 70% alcohol, and acetic acid,
90:5:5). The material was dehydrated through
a histological dehydrating series and subsequently
embedded in paraffin (Gonzalez and Cristóbal 1997;
Johansen 1940). Serial transverse (TS) and longitudinal
(LS) sections 10–12 µm thick were cut with a rotary
microtome and stained with astra blue and safranin
(Luque et al. 1996). Safranin stains lignin and nuclei
red, and astra blue stains cellulose blue. Finally, the
sections were mounted in synthetic Canada balsam.
Observations and photographs were made using
a Leica DM LB2 light microscope, equipped with
a Leica ICC50 HD digital camera (Leica Microsystems,
Germany).
Scanning electron microscopy (SEM).—For conven­
tional observation, portions of FAA-fixed fruits,
leaves, stems, and seeds were dehydrated in an
ascending series of acetone, then dried to the cri­
tical point with CO 2, and finally metallized with
gold-palladium (Bozzola 2014).
Additionally, paraffin-embedded and cut materials
were observed by SEM using the Gaudet and Kokko
(1984) technique with modifications as described
below. To this aim, the material was dehydrated and
embedded in paraffin and then cut into 50-µm-thick
serial sections as previously described (see Light
Microscopy (LM)). Sections were affixed to a glass
slide with Haupt’s adhesive (Bissing 1974) and dried
for 48 h. The slides used for SEM observation were
previously cut into 4-cm-long sections, so that they
would fit into the critical point drying chamber. The
slide-mounted sections were deparaffinized with xylol,
dehydrated in an ascending series of acetone, and dried

to the critical point with CO2. The pieces of glass slides
with several sections were attached with double-
adhesive tapes to aluminum stubs and sputter-coated
with gold-palladium and examined with a scanning
electron microscope.
The materials were observed and documented by
photomicrographs using a scanning electron micro­
scope (JEOL 5800 LV) at the Electronic Microscopy
Service of Corrientes (UNNE), Argentina.
RESULTS
Identification and determination of SW
concentration in different tissues.—Swainsonine was
detected in all the tissues at concentrations of 0.08% of
dry weight (DW) in the seeds (100 mg DW), 0.02% of
DW in fruit walls, and 0.02% of DW in leaves and stems
of A. garbancillo.
Identification of the fungal endosymbiont in seeds
of A. garbancillo.—Two unique DNA sequences were
amplified, sequenced, and subsequently deposited in
GenBank under accession numbers OR072798 and
OR072803. The percent identity between the two
sequences was 99.61%. Using the ITS sequence
OR072798 (510 nucleotides [nt]) as a query, a BLASTn
search showed as a best hit the sequence OR072803
(99.61% identity, E value of 0.0), followed by several
additional sequences (99.41% identity, E value of 0.0)
representing isolates of Alternaria section Undifilum
spp. Among these were sequences representing isolates
of Alternaria section Undifilum spp. from the South
American species A. pehuenches (MN317300) and
A. illini (MN322830) (Martinez et al. 2019b). Using the
ITS sequence OR072803 yielded similar results. Notably,
Figure 1. a. General appearance of the plant. b. Surface view of leaf. c. Fruit and seeds. Bars: a = 20 cm; b–c = 1 mm.
MYCOLOGIA 3
both amplified ITS sequences were highly similar (>99%
identity, E value of 0.0) to the ITS sequences character­
ized from isolates of A. bornmuelleri, A. cinerea, A. fulva,
and A. oxytropis, previously described as Undifilum spe­
cies. These results suggest that A. garbancillo is infected
by a fungal symbiont belonging to Alternaria section
Undifilum, potentially representing an undescribed
species.
Anatomy of A. garbancillo.—Examination of the
sampled plants from the field revealed the absence
of visible hyphae on the different surfaces (leaves,
stems, fruits, and seeds) (FIG. 1). However, the
leaflets and fruits are covered with nonglandular
trichomes (FIG. 1b–c).
Vegetative organs
Longitudinal sections of petioles and stems revealed
endophytic hyphae growing intracellularly inside par­
enchyma cells of the pith and cortex. The hyphae have
hyaline walls and cytoplasm that is stained with safranin.
The fungi did not maintain a uniform direction or precise
orientation; some hyphae were aligned parallel to the
longitudinal axes of the plant stem (FIG. 2a–e), whereas
others such as branched hyphae crossed transversely.
Necrosis or degradation of plant cells was not observed.
No hyphae were found inside leaflets (FIG. 2f).
Fruit and seed
The fruit wall is divided into exocarp, mesocarp, and
endocarp (FIG. 3a). The exocarp or outer skin was com­
posed of a layer of thin-walled cells covered by thickened
cuticle, the mesocarp consists of several layers of parench­
yma cells, and the endocarp is made up of 4–6 layers of
sclerenchyma cells, which are arranged in several planes. In
4 PISTÁN ET AL.: ALTERNARIA IN ASTRAGALUS GARBANCILLO TISSUES
Figure 2. a–f. Light microscope photographs stained with astra blue–safranin (a–c) and scanning electron microscope photographs
(d–f) from stem and leaf of A. garbancillo showing mycelia of fungal endosymbiont (arrows). a–e. Longitudinal sections of petiole (a–b)
and stem (c–e). f. Transversal section of leaflet without hyphae. Bars: a–e = 25 µm; f = 100 µm.
fully ripe fruits, the outer layers dry out and the mesocarp
is compressed. Hyphae were observed on the external
surface of the fruit (FIG. 3a, b). In addition, the presence
of septate hyphae with a sinuous course in the mesocarp is
very abundant (FIG. 3b–e).
Seeds have a three-layered seed coat. The outer coat is
referred to as exotesta and is composed of a layer of
thick-walled, palisade-like macrosclereids, common to
all legumes (FIG. 3f). Adjacent to the exotestal cells is
the mesotesta, which is followed by the endotesta; both
these inner cellular layers are formed by thin-walled
parenchyma cells. In these latter two layers, hyphae
were frequently present (FIG. 3f–h). Hyphal growth
was also visible on the inner side of the fruit and seed.
Within the seed, hyphae were abundant in the endo­
sperm (FIG. 3i) but not in the embryo tissues.
DISCUSSION
The presence of the SW-producing fungus is
a prerequisite for the presence of SW in plants (Braun
et al. 2003; Pistán et al. 2022; Ralphs et al. 2011).
However, it has been observed that the SW concentra­
tion varies widely between plant species and within the
same plant (leaves, scape(s), and flowers/pods) (Cook
et al. 2009b). The SW concentration has been estimated
in leaves and stems (0.03% DW) of A. garbancillo, but
not in seeds (Marin et al. 2020; Micheloud et al. 2017).
In contrast, in this study, we determined the SW
concentration in seeds (0.08% DW) and fruit walls
(0.02% DW), besides other plant organs, and observed
that the SW content in seeds was about 4 times higher
than in any other studied tissue. The different SW con­
centrations among species and within different organs
of a species is consistent with observations in popula­
tions of A. pehuenches, A. illinii, and A. chamissonis,
where it has been reported to range from under 0.001%
to over 0.08% DW (Martinez et al. 2018, 2019b).
The association of the SW-producing endosymbiotic
fungus Alternaria section Undifilum spp. with several
Astragalus species has been demonstrated by in vitro
culture (Martinez et al. 2019b; Noor et al. 2020).
However, it has been reported that from the species
A. mollisimus var. thompsonii and A. amphioxys, detec­
tion of the fungus Undifilum spp. was faster and less
laborious by PCR than by culturing (Ralphs et al. 2008).
Additionally, Martinez et al. (2019b) reported that when
SW concentrations were lower than 0.01% DW, the
endophyte could not be in vitro cultured but was con­
sistently detected in Astragalus samples by PCR.
Interestingly, these observations may suggest a higher
sensitivity of endophyte detection by PCR detection
than by in vitro culturing. In the present study, PCR
amplification followed by sequencing allowed us to
determine that the fungal symbiont belongs to
Alternaria section Undifilum.
The use of different microscopic techniques has
demonstrated the presence of the SW-containing fungal
 MYCOLOGIA 5

Figure 3. Fruit and seed of A. garbancillo showing mycelia (arrows). a, d–h. Light microscope photographs; stained with astra blue–
safranin (a, d, f, g) and with trypan blue (e, h). b–c. Scanning electron microscope photographs. a–e. Fruit. a. Cross-section of pericarp,
hyphae over exocarp (ec) and in mesocarp (mc); endocarp (nc) formed by sclerified cells. b. Mycelia over exocarp. c–d. Detail of
mesocarp with hyphae. e. Inner face of fruit with mycelia. f–i. Seed. f. Transversal section of seed coat composed by exotesta (et)
formed by macrosclereids, mesotesta (mt) and endotesta (nt), and hyphae. g. Detail of f showed one hypha in mesotesta. h. Inner face
of seed coat with mycelia. i. Detail of endosperm with hyphae. Bars: a, c, e, f = 50 µm; b, d, g–i = 10 µm.

endosymbiont of the order Chaetothyriales in Ipomoea
carnea (Cook et al. 2013; Neyaz et al. 2022; Pistán et al.
2022). However, only a few species of locoweeds from
the genus Astragalus and Oxytropis have been studied
microscopically for the SW-producing endophytes.
Reyna et al. (2012) and Noor et al. (2018) mention the
location of hyphae of the fungus Alternaria section
Undifilum spp. in leaves, stems, and petioles of
Oxytropis sericea, A. lentiginosus, and A. mollissimus
using different imaging methods. Applying SEM, trans­
mission electron microscopy (TEM), and laser scanning
confocal microscopy, these authors identified the exact
location of the fungal endosymbionts at the level of the
vascular bundle in the leaves and the pith tissue of
petioles and stems. In the present study, the location
of endophytes in the pith and cortex of petioles and
stems in A. garbancillo was found to be similar to that
reported by Reyna et al. (2012) and Noor et al. (2018).
No alteration of morphology or changes at the cellular
level were observed in these regions and organs.
Oldrup et al. (2010) showed by LM that the endo­
phyte of A. lentiginosus is present inside the seed,
attached to or within the seed coat, but not to the seed
embryo. Our analysis of A. garbancillo seeds is consis­
tent with the results reported by Oldrup et al. (2010),
since we observed the fungus to be located in the meso­
testa and endotesta layers of the seed coat, but not inside
the embryo. Importantly, our study also showed that the
fungus grows inside the endosperm. Interestingly, this
can be seen also in FIG. 2 of Oldrup et al. (2010), but it
seems to have escaped the notice of the authors because
it is not further mentioned. The endosperm, an embryo-
nourishing tissue, either is consumed during the devel­
opment of the embryo or remains in the mature seed to
be used in the germination process, during which the
embryo develops into a seedling (Baroux and
6 PISTÁN ET AL.: ALTERNARIA IN ASTRAGALUS GARBANCILLO TISSUES
Grossniklaus 2019). In Astragalus, traces of endosperm
remain in mature seeds and contain abundant myce­
lium. In addition to storage of nutrients, the endosperm
has been shown to regulate seed germination and pro­
mote seedling establishment (Baroux and Grossniklaus
2019; Doll and Ingram 2022). We hypothesize that dur­
ing these developmental stages, the hyphae come into
contact with the embryo, and subsequently with the
seedling, thus transferring the fungus to the next gen­
eration. We also detected the presence of the endophytic
fungus in the external surface of the fruit, and inside the
mesocarp, which has not been demonstrated in other
SW-containing legumes.
The finding of the endophytic fungi in seeds and seed-
related tissues strongly suggests vertical host transmis­
sion of the endophyte (Hardoim et al. 2015; Yan et al.
2019). Thus, in agreement with other authors (Cook
et al. 2011; Guan et al. 2021; Noor et al. 2018;
Nzabanita et al. 2018; Oldrup et al. 2010; Ralphs et al.
2011; Reyna et al. 2012), our observation of endophyte in
seeds and fruits in A. garbancillo corroborates that the
endophyte is also vertically transmitted in this plant host.
In conclusion, our results affirm by PCR that the
fungus associated with A. garbacillo seeds belongs to
the genus Alternaria section Undifilum. Furthermore,
through analysis of different plant tissues using various
microscopy techniques, we describe the tissue location
and distribution of the fungus in this plant host,
strongly suggesting vertical transmission.
ACKNOWLEDGMENTS
We thank the two anonymous reviewers for their timely
suggestions that helped to improve the manuscript.
DISCLOSURE STATEMENT
No potential conflict of interest was reported by the author(s).
FUNDING
This study was supported by grants from the Secretaría
General de Ciencia y Técnica, Universidad Nacional del
Nordeste [UNNE; grant no. PI17B007] and by the Agencia
Nacional de Promoción Científica y Tecnológica (PICT 2020/
01484).
ORCID
María E. Pistán http://orcid.org/0000-0001-8210-7060
Daniel Cook http://orcid.org/0000-0001-8568-113X
Susana A. Gutiérrez http://orcid.org/0000-0001-8050-9322
Leonhard Schnittger http://orcid.org/0000-0003-3484-
5370
Dale R. Gardner http://orcid.org/0000-0003-3218-6893
Luciana A. Cholich http://orcid.org/0000-0002-7936-8806
Ana M. Gonzalez http://orcid.org/0000-0002-9311-0967
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