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es el 03-08-2016

Rev Argent Microbiol. 2016;48(2):154---160

REVISTA ARGENTINA DE

MICROBIOLOGA
www.elsevier.es/ram

ORIGINAL ARTICLE

Endophytic fungi from selected varieties of soybean

(Glycine max L. Merr.) and corn (Zea mays L.) grown
in an agricultural area of Argentina
Mara L. Russo a, , Sebastin A. Pelizza a,b , Marta N. Cabello a,d ,
Sebastin A. Stenglein c , Mara F. Vianna a , Ana C. Scorsetti a
a

Instituto de Botnica Carlos Spegazzini (Facultad de Ciencias Naturales y Museo-Universidad Nacional de La Plata), La Plata,
Argentina
b
Centro de Estudios Parasitolgicos y de Vectores (CEPAVE), CCT La Plata CONICET-UNLP, La Plata, Argentina
c
Laboratorio de Biologa Funcional y Biotecnologa (BIOLAB)-CICBA-INBIOTEC, Facultad de Agronoma de Azul, Ctedra de
Microbiologa, UNCPBA, Repblica de Italia 780, 7300 Azul, Argentina
d
Comisin de Investigaciones Cientcas (CIC) de la Provincia de Buenos Aires, Argentina
Received 12 September 2015; accepted 11 November 2015
Available online 1 April 2016

KEYWORDS
Endophytic fungi;
Soybean;
Corn;
Diversity

Abstract Endophytic fungi are ubiquitous and live within host plants without causing any
noticeable symptoms of disease. Little is known about the diversity and function of fungal
endophytes in plants, particularly in economically important species. The aim of this study
was to determine the identity and diversity of endophytic fungi in leaves, stems and roots of
soybean and corn plants and to determine their infection frequencies. Plants were collected in
six areas of the provinces of Buenos Aires and Entre Ros (Argentina) two areas were selected
for sampling corn and four for soybean. Leaf, stem and root samples were surface-sterilized,
cut into 1 cm2 pieces using a sterile scalpel and aseptically transferred to plates containing
potato dextrose agar plus antibiotics. The species were identied using both morphological and
molecular data. Fungal endophyte colonization in soybean plants was inuenced by tissue type
and varieties whereas in corn plants only by tissue type. A greater number of endophytes were
isolated from stem tissues than from leaves and root tissues in both species of plants. The most
frequently isolated species in all soybean cultivars was Fusarium graminearum and the least
isolated one was Scopulariopsis brevicaulis. Furthermore, the most frequently isolated species
in corn plants was Aspergillus terreus whereas the least isolated one was Aspergillus avus.
These results could be relevant in the search for endophytic fungi isolates that could be of
interest in the control of agricultural pests.
2016 Asociaci
on Argentina de Microbiologa. Published by Elsevier Espa
na, S.L.U. This is an
open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/bync-nd/4.0/).

Corresponding author.
E-mail address: leticiarusso@conicet.gov.ar (M.L. Russo).

http://dx.doi.org/10.1016/j.ram.2015.11.006
0325-7541/ 2016 Asociaci
on Argentina de Microbiologa. Published by Elsevier Espa
na, S.L.U. This is an open access article under the CC
BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

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Endophytic fungi from selected varieties of soybean and corn grown in Argentina

PALABRAS CLAVE
Hongos endtos;
Soja;
Maz;
Diversidad

155

Hongos endtos aislados de cultivares de soja (Glycine max L. Merr) y maz (Zea
mays L.) presentes en reas agrcolas argentinas
Resumen Los hongos endtos son ubicuos y se encuentran en el interior de los tejidos de las
plantas de manera asintomtica. Se sabe muy poco acerca de la diversidad y la funcin de estos
hongos, particularmente en especies de importancia econmica. El objetivo de este trabajo fue
determinar la diversidad y la frecuencia de colonizacin de hongos endtos en races, tallos
y hojas de 2 variedades de maz y de 4 variedades de soja; las muestras se tomaron de 6 reas
diferentes ubicadas en las provincias de Buenos Aires y Entre Ros (Argentina). Con un bistur
estril se obtuvieron porciones de 1 cm2 de raz, tallo y hoja, que fueron colocados en placas
con agar papa dextrosa ms antibitico.
Las especies de hongos fueron identicadas a partir de caractersticas morfolgicas y moleculares. La colonizacin de hongos endtos en soja estuvo inuenciada por la variedad y por
el tipo de tejido, en tanto que en el maz solo hubo inuencia del tipo de tejido. El mayor
nmero de endtos se encontr en los tallos de ambas especies. El aislamiento ms frecuente
en todas las variedades de soja fue Fusarium graminearum y el menos frecuente Scopulariopsis
brevicaulis. En ambas variedades de maz la especie con mayor frecuencia de aislamiento fue
Aspergillus terreus y la de menor fue Aspergillus avus. Estos resultados son relevantes para
la bsqueda de especies de hongos endtos que podran ser de inters en el control de plagas
agrcolas.
2016 Asociaci
on Argentina de Microbiologa. Publicado por Elsevier Espa
na, S.L.U. Este es un
artculo Open Access bajo la CC BY-NC-ND licencia (http://creativecommons.org/licencias/bync-nd/4.0/).

Introduction
By the year 2020, the supply of food especially of cereals,
will have to increase about 70% in developing countries to
secure food for the projected population of 6500 million
people. It is expected that most of this increase in food supply will come from developing countries33 . Soybean and corn
are the extensive major crops in Argentina, providing a high
percentage of the basic food needs of the population. The
most important crops in Argentina are soybean and corn with
18 and 3.4 million sown hectares, respectively19 .
Symptomless internal colonization of healthy plant
tissues by fungi is a widespread and well-documented phenomenon. Increasing interest in the ecological roles of these
fungi has stimulated research in recent years since they
might have plant growth --- promoting activity13 . Endophyte
is an all-encompassing topographical term that includes
all those organisms that during a variable period of their
life symptomlessly colonize the living internal tissues of
their hosts24 . It is hypothesized that fungal endophytes, in
contrast to known pathogens, generally have far greater
phenotypic plasticity and thus more options to interact
with their host than pathogens27 . Since the 1970s several reports have shown that these fungal endophytes play
important roles in protecting their host against predators
and pathogens25 . Endophytic fungi that infect plants are
ubiquitous in all environments studied7,24,28 . Although the
diversity and function of fungal endophytes that infect
grasses are well documented, little is known about the diversity and function of fungal endophytes in plants, particularly
in economically important species13,26 . Some fungal endophytes can reportedly reduce plant diseases and enhance
plant growth and may be the basis for emerging methods to
improve plant growth and production12,17,18,20 . For example

treatment of soybean [Glycine max L. (Merr)] with culture

ltrate from the endophyte Cladosporium sphaerospermun
increased plant height3,11,20,21 . Although soybean and corn
are major world crops, there is very limited knowledge of
their fungal endophyte community.
The goal of this study was to isolate fungal endophytes
from leaves, stems and roots of four soybean and two corn
varieties grown in agricultural sites of the provinces of
Buenos Aires and Entre Rios, Argentina, and to determine
their colonization frequencies.

Materials and methods

Sample collection
The plants were collected during January and February 2013
in six locations in the soybean and corn cropping area of
the provinces of Buenos Aires and Entre Rios (Argentina)5
(Table 1). The regions climate is temperate with an average temperature of 17 C and an average annual rainfall of
1000 mm. Two areas were selected for sampling corn and
four for soybean sampling, since soybean varieties are more
predominant in Argentina. Plants of both species were grown
in monoculture elds with a history of annual corn and
soybean rotation. Ten plants without symptoms of disease
were randomly selected from each plot in soybean cultivars
DM 3810, DM 4210, DM 4670 (Don Mario Co., Buenos Aires
province, Argentina), NA 5009 (Nidera Semillas Co., Buenos
Aires province, Argentina) and ten plants were selected
from corn cultivars NK 900 (Sygenta Semillas Co., Argentina)
and DK 747 (Dekald , Argentina). All samples were collected at 60---70 days after germination, cut at the soil line,

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156

M.L. Russo et al.

Table 1

Locations where different soybean and corn plants were sampled

Variety

Locality

GPS coordinate

Soybean
DM3810 (Don Mario, Argentina)

Alberti, Buenos Aires

35
60
35
60
36
62
32
60

DM4210 (Don Mario, Argentina)

Bragado, Buenos Aires

DM4670 (Don Mario, Argentina)

Salliquel, Buenos Aires

NA5009 (Nidera, Argentina)

Victoria, Entre Ros

Corn
DK747 (Monsanto)
NK900 (Syngenta)

Olascoaga, Buenos Aires

Las Cuevas, Entre Ros

immediately placed on ice and stored up for 72 h at 4 C until

processed according to Impulliti and Malvick13 .

Isolation and identication of endophytic fungi

Endophytic fungi were isolated according to the protocols
described by Pimentel et al.25 . All leaf, stem and root
samples were washed twice in distilled water, then surfacesterilized by immersion for 1 min in 70% (v/v) ethanol, 4 min
in sodium hypochlorite (3%, v/v available chlorine) and then
washed three times in sterilized distilled water for each
time. After surface sterilization, samples were cut into 1 cm2
pieces with a sterile scalpel and aseptically transferred to
plates containing potato dextrose agar (PDA, Britania S.A.,
Buenos Aires, Argentina) to which a 0.1% stock consisting
of 0.02 g of each of two antibiotics (chloramphenicol and
streptomycin) dissolved in 10 ml sterile distilled water was
added, followed by lter sterilization through a 0.2-m lter (Syringe lter sterile, E-Chrom Tech, Taiwan); 1 ml of
this was added to each litre of medium, to suppress bacterial growth31 . Aliquots from the third wash were plated onto
PDA to check that surface sterilization had been effective.
A total of 1080 fragments were plated (18 from each of the
60 plants investigated). To facilitate isolation of endophytic
fungi, the plates were incubated in the dark at 25 C. The
plates were checked everyday for up to ten days after incubation and any fungi present was isolated, puried and then
maintained at 4 C on PDA slopes for further identication.
Percentage colonization was dened for each variety as the
total number of fragments colonized by fungi in relation to
the total number of fragments 10025 .
The species were identied using both morphological and
molecular data.
Morphological identication of the isolates was done by
growing them on PDA plates or in microculture14 and examining the colonies for asexual or sexual reproductive structures
using optical microscopy and taxonomic keys. Species were
identied according to Leslie and Summerell15 and Domsch
et al.8 .

1 53 S
16 49 O
6 59 S
28 45 O
45 5 S
57 32 O
37 0 S
10 0 O

35 14 14 S
60 36 36 O
32 21 00 S
60 28 60 O

Genomic DNA of monosporic cultures was obtained

according to Stenglein and Balatti29 . To conrm morphological identications, a PCR was carried out in an XP
thermal cycler (Bioer Technology Co, Hangzhou, China) to
amplify the ITS rDNA region using primer pairs ITS5 (5 GGAAGTAAAAGTCGTAACAAG G-3 ) and ITS4 (5 -TCCTCC GCT
TATTGATATGC-3 )32 . For Fusarium species conrmation the
translation elongation factor (EF-1) region was amplied
using primers EF1 (5 -ATGGGTAAGGA(A/G)GACAAGAC-3 )
and EF2 (5 -GGA(A/G)GTACCAGT(G/C)ATCATGTT-3 )22 . The
PCR reactions, the fragment purications and sequencing
were performed according to Canel et al.6 and Stenglein
et al.30 .
The similarities of the fragment with previously published
sequence data were examined with BLASTn1 on the NCBI web
page.
Diversity was assessed using the Shannon Index16 (for the
cultivars and tissue types).
The differences between fungi isolates and frequency of
colonization for the different varieties were tested using
two-way analysis of variance (ANOVA) and their means were
compared by the LSD test (p < 0.05) using the Infostat software.

Results
From the soybean plants sampled, 11 fungal species were
isolated and identied using both morphological and molecular data. In all soybean cultivars, Fusarium graminearum
was the most frequently isolated species sampled while
Scopulariopsis brevicaulis was the least frequently isolated
one (Table 2). Furthermore, in the corn plants sampled,
7 fungal species were isolated (six species belonging to
Ascomycota and one to Zygomycota (Table 3)), being
Aspergillus terreus the most frequently isolated species
and Aspergillus avus the least frequently isolated one
(Table 3). All endophytic fungal species were deposited in
the strain culture collection of the Spegazzini Institute, La
Plata, Argentina (LPSC) (Tables 2 and 3).

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Endophytic fungi from selected varieties of soybean and corn grown in Argentina

157

Table 2 Colonization percentage of different fungal species isolated from roots, stems and leaves from four different soybean
cultivars sampled
Fungal species

Colonization percentage (%)

DM3810

LPSC1187 Alternaria
alternata (Fr.) Keissl
LPSC1186 Arthrinium
phaeospermun (Corda)
Ellis
LPSC1181 Aspergillus
niger Tiegh
LPSC1182 Clonostachys
rosea (Link) Schroers
LPSC1178 Curvularia
lunata (Wakker)
Boedijn
LPSC1188 Fusarium
graminearum Schwabe
LPSC1184 Fusarium
equiseti (Corda) Sacc
LPSC1191 Fusarium
oxysporum Schlecht
LPSC1185 Macrophomina
phaseolina (Tassi) Goid
LPSC1189 Scopulariopsis
brevicaulis(Sacc)
Bainier
LPSC1179 Trichoderma
saturnisporum
Hammill

DM4210

DM4670

NA5009

Root

Stem

Leave

Root

Stem

Leave

Root

Stem

Leave

Root

Stem

Leave

---

---

---

---

25

26.6

---

---

---

---

---

---

---

---

---

---

20

33.3

---

---

---

---

---

---

---

10

38.3

---

13.3

41.6

---

---

---

---

---

---

16

---

---

---

---

---

---

---

---

---

15

30

46.6

---

38.3

---

---

---

---

---

38.3

3.3

10

30

20

20

10

11.6

25

31.6

---

---

---

---

---

6.6

---

20

45

---

---

---

58.3

---

---

---

---

---

---

10

21.6

---

---

---

---

33.3

8.3

---

---

---

13.3

28.3

---

---

---

---

10

13.3

28.3

---

---

---

---

---

---

---

---

---

---

41.6

---

---

---

---

---

---

---

---

---

---

---

---

---

10

16.6

38.3

With regard to fungal diversity in soybean plants, cultivar DM3810 showed the highest diversity while in cultivar
NA5009 we observed the lowest diversity, with a Shannons
index of 3.09 and 1.93, respectively. Moreover, corn cultivar DK747 showed the highest fungal diversity whereas
the lowest diversity was observed in cultivar NK900, with
a Shannons index of 1.84 and 1.67, respectively. Furthermore, based on the Shannons index, the greatest fungal

diversity in all the soybean cultivars sampled was observed

in the stems and then in the leaves whereas the lowest diversity occurred in the roots, except for cultivar DM4670, where
we observed increased diversity in the roots rather than
in the leaves with a Shannons diversity index of 1.35 and
1.18, respectively. With respect to the fungal diversity in the
roots, stems and leaves of corn plants, most fungal diversity
was observed in the stems of both DK747 and NK900 cultivars

Table 3 Colonization percentage of different fungal species isolated from roots, stems and leaves from two different corn
cultivars sample
Fungal species

Colonization percentage (%)

DK747

LPSC1183
LPSC1180
LPSC1193
LPSC1223
LPSC1224
LPSC1190
LPSC1192

Aspergillus avus Link

Aspergillus terreus Thom
Bipolaris tetramera (McKinney) Shoemaker
Clonostachys rosea (Link) Schroers
Fusarium graminearum Schwabe
Fusarium proliferatum (Matsush) Nirenberg
Mucor circinelloides Tiegh

Root

Stem

8.3
8.3
------26.6
---

35
30
31.6
----43.3
25

NK900
Leaf
--28.8
------28.3
---

Root
--10
21.6
------10

Stem

Leaf

--43.3
23.3
18.3
28.3
25
33.3

------31.6
18.3
26.6
---

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158

M.L. Russo et al.

DM3810

DM4670

DM4210

NA5009

25
bc

Colonization (%)

c
bc

20

bc

bc

bc

bc

bc

bc

bc
bc

15

bc

bc

bc

bc

bc bc
b

10
b

a a

Alternaria
alternata

a a

a a

Arthrinium
Aspergillus niger Clonostachys
phaeospermun
rosea

a a a

Curvularia
lunata

Fusarium
graminearum

a a

Fusarium
equiseti

Fusarium
oxysporum

a a

a a

Macrophomina Scopulariopsis
phaseolina
brevicaulis

a a a
Trichoderma
saturnisporum

Endophytic fungi isolated

Figure 1 Colonization percentage of endophytes isolated from soybean plants of four cultivars DM3810, DM4670, DM4210, NA5009.
Different letters indicate statistically signicant differences between the groups (LSD test, p < 0.05).

NK900

DK747

35

Colonnization (%)

30
25

bc

20
15

bc

bc

bc

bc

ab
10

ab

5
0

Aspergillus
flavus

a
Aspergillus
terreus

Clonostachys
rosea

Bipolaris
tetramera

Fusarium
graminearum

Fusarium
proliferatum

Mucor
circinelloides

Endophytic fungi isolated

Figure 2 Colonization percentage of endophytes isolated from corn plants of two cultivars DK747, NK900. Different letters indicate
statistically signicant differences between the groups (LSD test, p < 0.05).

with a Shannons diversity index of 2.27 and 2.49, respectively, then in leaves and the lowest diversity occurred in
roots.
Fungal isolates identication was conrmed at molecular level and submitted to GenBank (Accession numbers:
KF753941---KF753956). Fungal endophyte colonization in
soybean plants was inuenced by the cultivars, showing
signicant differences between varieties (F = 4.17, df = 3,
p = 0.0063), fungi isolates (F = 6.93, df = 10, p < 0.0001) and
in the interaction among them (F = 7.12, df = 30, p < 0.0001)
(Fig. 1). Corn plants showed no signicant differences
between cultivars (F = 1.34, df = 1, p = 0.2500), however,
they did instead among fungi isolates (F = 4.07, df = 6,
p = 0.0009) and in the interaction among cultivars and fungi
isolates (F = 4.12, df = 6, p = 0.0008) (Fig. 2).

Discussion
Studies of fungal endophytes in many environments are
an active area for research; however, the endophytes in
soybean and corn have never been systematically characterized. This work expands our understanding of endophytic
fungi in soybean and corn plants. We have focused on roots,
stems and leaves because many soybean and corn pathogens

commonly colonize these organs. The fungal endophytes

identied in this study are not known to be soybean and corn
pathogens, and the functional associations between these
fungi and soybean and corn plants are unknown. Furthermore, it is important to mention that none of the plants used
in this study had symptoms of disease. The most prevalent
endophytic fungal species isolated in the organs (root, stem
and leaf) in all soybean cultivars was F. graminearum and
in the two corn cultivars was A. terreus. Pimentel et al.25 ;
Impullitti and Malvick13 found that Cladosporium was the
endophytic fungal genus most frequently identied from
leaves and stems of soybeans grown in Brazil and Minnesota,
USA whereas Pan et al.23 found this genus in leaves and stems
from corn in Minnesota, USA. In this study only one species of
Zygomycota was isolated in maize plants; species from this
Phylum were isolated as endophytes in Dactylis glomerata
L. and other plants18 .
The endophytic fungal species detected in plants may
be inuenced by many factors, including the type of tissue
sampled, the time when plants were assayed, perhaps the
climate and location in which they were grown13 , whether
the plant is grown in a monoculture or polyculture, the plant
age or cropping history of the eld2,4,9,27 . Soybean and maize
used in this study were grown in a monoculture and in elds
that had a history of corn, wheat and soybean rotations.

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Endophytic fungi from selected varieties of soybean and corn grown in Argentina
Fisher et al.10 observed that parts of corn-stems nearer
to the soil showed a lower incidence of fungal infection
and explained that this probably was due to these parts of
the stem having an increased frequency of bacteria that
inhibited fungal colonization. This could explain why we
obtained the greatest number and diversity of isolates from
stems in different soybean and corn cultivars, than in leaves
and roots.
A greater number of fungi such as endophytes in stems
were also observed in soybean in Brazil whereas endophytic
bacteria in maize were found in the USA10,25 . These studies also suggested that endophytes may exclusively colonize
certain tissues, for example, Colletotrichum was only isolated from soybean leaves and not from the stems cultivated
in Brazil25 . In our study, Fusarium equiseti was only isolated
from leaves of soybean plants and S. brevicaulis only from
stems.
Endophytes may be important organisms to improve a
sustainable production of crops, although their identities
and functions in a range of plants are just beginning to
be revealed. This is the rst time that we study the natural endophytes placed in roots, stems and leaves of the
main soybean and maize cultivars in Argentina. Species
could be determined by classical taxonomy and the use
of molecular techniques for each of the isolates obtained.
In addition, we determined the colonization percentage,
the fungal diversity in the different organs of every plant
studied, the differences between fungi isolates and the
frequency of colonization for different varieties using the
ANOVA analysis. Future research should be conducted to
determine which of these fungal natural endophytes could
be used for both biological control and plant growth
promotion.

Ethical disclosures
Protection of human and animal subjects. The authors
declare that no experiments were performed on humans or
animals for this investigation.
Condentiality of data. The authors declare that no patient
data appears in this article.
Right to privacy and informed consent. The authors
declare that no patient data appears in this article.

Conict of interest
The authors declare that they have no conicts of interest.

Acknowledgements
This study was partially supported by Consejo Nacional
de Investigaciones Cientcas y Tecnolgicas (CONICET
PIP0009), Agencia de Promocin Cientca y Tecnolgica
(PICT 2013-0543), Comisin de Investigaciones Cientcas
de la Provincia de Buenos Aires (CICPBA), Universidad
Nacional de La Plata (UNLP, 11/N 773) and Rizobacter
Argentina S.A.

159

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