ISSN 0373-580
X
M. I. Mercado et al. - Mycorrhizal associations in Smallanthus
species
Bol. Soc. Argent. Bot. 48 (2): 193-200. 2013
ArbusculAr mycorrhizAl AssociAtions And dArk septAte
endophytes in yAcon (smAllAnthus sonchifolius) And A wild
relAtive (smAllAnthus mAcroscyphus)
MARIA I. MERCADO1,3*, MARIA V. COLL ARÁOZ1, CELIA I. BRANDÁN DE WEHT2,
GRACIELA I. PONESSA3 and ALFREDO GRAU1
Summary: Mycorrhizal associations in Smallanthus sonchifolius (Poepp. & Endl.) H. Robinson
(Asteraceae), Yacon, an ancient Andean crop and Smallanthus macroscyphus (Baker ex Martius)
A. Grau, wild yacon, a close wild relative are described for the irst time. Yacon ibrous roots growing
under ield conditions have high levels of colonization by arbuscular mycorrhizal fungi (86 %). Other
fungi colonizing roots included dark septate endophytes (45 %) and unidentiied fungi that are probably
saprophytic (25 %) were observed. Only 9% of the samples analyzed were not colonized by any type of
fungi. Glomus, Acaulospora, Scutellospora, Gigaspora and Pacispora were the main genera of arbuscular
mycorrhiza identiied. A similar high degree of mycorrhizal colonization was observed in Smallanthus
macroscyphus, in natural populations associated with Juglans australis native forest. The high level of
mycorrhizal colonization, the low number of ine absorbing roots and the large roots diameters observed,
suggest that both Smallanthus species are likely dependent on mycorrhiza.
Key words: Smallanthus sonchifolius, Smallanthus macroscyphus, mycorrhizas, arbuscular mycorrhizas,
dark septate endophytes.
Resumen: Micorrizas arbusculares y endóitos septados oscuros en yacón (Smallanthus sonchifolius)
y un pariente silvestre (Smallanthus macroscyphus). Se describen por primera vez las asociaciones
simbióticas micorrícicas en Smallanthus sonchifolius (Poepp. & Endl.) H. Robinson (Asteraceae),
yacón, un cultivo andino precolombino, y en el yacón del campo, Smallanthus macroscyphus (Baker ex
Martius) A. Grau, especie silvestre estrechamente emparentada. Las raíces ibrosas de yacón creciendo
en un cultivo presentaron un alto nivel de colonización por hongos micorrícicos arbusculares (86 %).
Además se observaron endóitos septados oscuros colonizando las raíces (45 %) y otros hongos no
identiicados, probablemente saprofíticos (25 %). Solo el 9% de las muestras analizadas no presentaba
colonización. Los hongos micorrícicos arbusculares identiicados correspondíeron mayormente a los
géneros Glomus, Acaulospora, Scutellospora, Gigaspora y Pacispora. Ejemplares de poblaciones
naturales de Smallanthus macroscyphus creciendo asociados a Juglans australis presentaron un nivel
de colonización semejante. El alto nivel de colonización y las pocas raíces ibrosas observadas sugiere
que ambas especies se comportan como micótrofas dependientes.
Palabras clave: Smallanthus sonchifolius, Smallanthus macroscyphus, micorrizas, micorrizas
arbusculares, endóitos septados oscuros.
1
Instituto de Ecología Regional (IER), Facultad de Ciencias Naturales, Universidad Nacional de Tucumán, CC34, (4107)
Yerba Buena, Tucumán, Argentina.
2
Cátedra de Microbiología Agrícola, Facultad de Agronomía y Zootecnia, Universidad Nacional de Tucumán, Florentino
Ameghino s/n, Barrio Mercantil, El Manantial (4105), Tucumán, Argentina.
3
Instituto de Morfología Vegetal, Fundación Miguel Lillo, Miguel Lillo 251. S.M. de Tucumán (4000), Tucumán, Argentina.
* Corresponding author: mainesmer@yahoo.com.ar
193
Bol. Soc. Argent. Bot. 48 (2) 2013
introduction
Plant roots interact with a wide variety of
rhizospheric microorganisms, including bacteria
and fungi, which greatly affect plant growth and
productivity (Barea & Jeffries, 1995; Akiyama &
Hayashi, 2002). Arbuscular mycorrhizae (AM)
are the most widespread form of symbiotic
associations known to occur between the roots
of more than 80 % of higher plants and a
group of fungi that belongs mainly to order
Glomerales, phylum Glomeromycota (Schüßler et
al., 2001; Koide & Mosse, 2004). In addition to
the widely studied AM fungi, increased attention
has recently been given to a ubiquitous group of
miscellaneous fungi designated as dark septate
endophytes (DSE) characterized by melanized
septate hyphae and microsclerotia (Peterson et al.,
2004). These fungi are frequent root colonizers of
a broad range of plants in temperate and tropical
habitats (Jumpponen & Trappe, 1998) including
mycorhizal macrophytes (Marins et al., 2009) and
may function as mutualistic fungi (Jumpponen,
2001; Barrow & Osuna, 2002).
Yacon, Smallanthus sonchifolius (Poepp. &
Endl.) H. Robinson (Asteraceae), is an ancient
Andean crop (cultivated from Colombia to NW
Argentina) used for centuries by the native
inhabitants as food (Fig.1 A-B). Yacon tuberous
roots are rich in oligofructans (Goto et al.,
1995; Lachman et al., 2003) and antioxidants
(Simonovska et al., 2003; Yan et al., 1999) and are
potentially useful as prebiotics, natural sweetener,
functional food, and dietary supplements
(Seminario et al., 2003; Genta et al., 2005; Genta
et al., 2009). In the last two decades the use of
yacon leaves to treat diabetes has increased.
Infusions made from dry yacon leaves produce a
remarkable hypoglycemic effect on normal and
diabetic rats (Aybar et al., 2001). Yacon popularity
has increased and it is cultivated and sold in
markets of Peru, Brasil, NW Argentina, Japan,
China, Corea, New Zealand, and some European
countries.
Smallanthus macroscyphus (Baker ex Martius)
A. Grau, is a wild species found in deciduous
forests and riverbanks in Southern Bolivia and
NW Argentina and is taxonomically related to
yacon (Grau & Rea, 1997) (Fig.1 C-D).
Yacon and S. macroscyphus have a large aerial
194
biomass of leaves and stems (Kortzart Gonzales,
2009) and subterranean organs consisting of
rhizomes with large adventitious tuberous roots and
few ibrous absorbent roots (Machado et al., 2004;
Coll Aráoz et al., 2008). Plant species with root
systems of this nature are frequently dependent on
mycorrhizal fungi for adequate mineral and water
uptake (Varma, 1999; Jakobsen et al., 2003). Our
main objective was to determine the presence of
mycorrhizal fungal symbiotic interactions as well
as spore populations in rhizosphere soils in yacon
under cultivated conditions and S. macroscyphus
in its natural habitat. A second objective was to
screen roots for other fungal endophytes.
mAteriAls And methods
Sample collection
Soil samples, roots and rhizomes were collected
from ten individuals of yacon randomly selected
cultivated at Horco Molle University Center (600
masl, 27ºS, 65ºW, Tucumán, Argentina) in April
2007 at the lowering stage. Ten individuals of
S. macroscyphus in lowering stage growing in a
Juglans australis dominated forest were collected
in February of 2008 in Rearte, Trancas, Tucumán,
Argentina. Voucher specimens of both species
were deposited in the herbarium of “Fundación
Miguel Lillo”, San Miguel de Tucumán,
Tucumán, Argentina LIL Mercado and Ponessa s/
nº LIL 607175, 607375. Roots and rhizomes from
both species were treated individually and were
carefully cleaned and preserved in FAA (formalin:
glacial acetic acid: 70% ethanol, 5:5:90) until
processing. Three samples of rhizosphere soil
(1 Kg each) were collected from both localities
(Horco Molle and Rearte). From each sample 100
g were dried at 65ºC for three days until constant
weight and passed through a 2 mm sieve and used
for physical chemical analyses. The rest of the
soil samples were used for isolation of fungal
spores.
Soil properties of the study sites
The soils at the study sites were classiied as
Ustiluvens (Mon & Suayter, 1972; Toselli et al.,
1975), slightly acid and very rich in available
nutrients and organic matter, particularly Rearte
(Table 1).
M. I. Mercado et al. - Mycorrhizal associations in Smallanthus species
Fig.1. General aspect, panels A and C and detail of tuberous (Tr) and ibrous (Fr) roots, panels B and D..of
S. sonchifolius.. S. macroscyphus respectively.
195
Bol. Soc. Argent. Bot. 48 (2) 2013
Table 1: Physical chemical analysis of soils in two regions of the Yungas. Tucuman, Argentina. 2008.
Soil sample
Horco Molle
(S. sonchifolius)
Rearte
(S. macroscyphus)
Granulometry
S%
s%
C%
Texture
pH1
Organic
mater2 %
N3
%
P4
ppm
K5
me.100g-1
58.1
33.1
8.8
Sandy loam
5.6
5.33
0.31
62.5
0.86
71.9
24.2
3.9
Sandy loam
6.6
12.23
0.63
46.8
1.57
References: S, sand; s, silt; C, clay; (1) potentiometry ratio soil: water 1:2.5; (2) Walkey-Black method; (3)
total nitrogen Kjeldahl method; (4) available phosphor Bray-Kurtz method; (5) Morgan method.
Assessment of roots and rhizomes for colonization
Due to the limited availability of fibrous
roots in each plant, particularly S. macroscyphus
(Coll Aráoz et al., 2008), a composite sample
was prepared from the ten individuals of each
specie to estimate AM and DSE colonization.
Fibrous roots fixed in FAA were cleared in
10% KOH solution at 80 °C, acidified with
HCl 5 % and stained with Gueguén (lactic acid:
100 g, Trypan blue: 0,1 g, Sudan IV Or: 0,1
g, and Iodine tincture: 10-30 droplets) (Verna
& Herrero, 1952). Roots that remained dark
after clearing were bleached with an alkaline
solution (169 ml distilled water: 3 mL NH3: 20
mL H2O2) before staining. Four replicates each
consisting of twenty ive stained ibrous root 1
cm long were examined for each species. Root
segments were mounted on microscope slides in
lactoglycerol and examined for fungal structures.
A root segment was considered as AM positive
if it showed extraradical mycelium, intraradical
hyphae, arbuscules or vesicles and DSE positive
if pigmented dematiaceous septate hyphae or
microsclerotia were observed. Other unidentiied
fungal structures where classiied as “other”.
Percent of root colonization by AM fungi or
DSE was estimated according to the magniied
intersect method (McGonigle et al., 1990).
The rhizomes of the ten individuals of both
species were cleared and bleached in an identical
manner as the roots and analyzed separately.
After clearing they were free-hand sectioned,
stained with Gueguen, mounted in lactoglycerol
and examined with a compound light Karl Zeiss
Axiostar plus microscope with a Canon compact
digital camera, model PowerShot A 620 IS M52
(O) 12.1 Megapixel, for assessment of fungal
colonization.
196
Isolation and identiication of spore population
Three dry soil samples from each species
weighing 100 g were analyzed and considered
as three replications. The spore population
was assessed by the wet sieving and decanting
method (Gerdeman & Nicolson, 1963) followed
by centrifugation on a 80 % sucrose gradient
(Sieverding, 1991). All the spores were isolated
from the supernatant and extracted with a brush
into a watch glass with a small quantity of water.
Intact fungal spores were mounted in polyvinyl
alcohol-lactoglycerol with Melzer’s reagent and
were identiied using INVAM keys (http://www.
invam.caf.wvu.edu) and Schenck & Perez (1990)
by Dra. Marta Cabello, Instituto Spegazzini,
Facultad de Ciencias Naturales y Museo, La Plata,
Argentina. Because of the low abundance of certain
morphotypes, species identiication was performed
only with spores in good condition (no sign of
degradation or parasitism) and some could only be
identiied to genus.
results
A high level of mycorrhizal colonization was
observed with simultaneous occurrence of AM
fungi and DSE in most cases. AM associations were
observed in 75 % of the samples of S. macroscyphus
and 82 % of the samples of S. sonchifolius. DSE
colonized more than 40 % of the samples in both
species. The extent of unidentiied fungal structures
colonization, probably saprophytic fungi, ranged
between 5 % for S. macroscyphus and 25 % for
S. sonchifolius. Only 9 % to 11% of the samples
analyzed were not colonized by any type of fungal
structures (Fig. 2).
In both species AM associations were typiied
M. I. Mercado et al. - Mycorrhizal associations in Smallanthus species
as Arum-type (Fig. 3 A, B) characterized by root
entry with formation of an appressorium (Fig. 3 G),
intercellular hyphal growth in the root cortex, with
short lateral branches into cortical cells forming
arbuscules (Fig.3 A, B,C), and vesicles illed with
several types of lipid droplets (Fig. 3 D, E). On the
other hand DSE were frequently characterized by
brown, narrow, septate, runner hyphae commonly
occurring on the root surface and typically running
parallel to the long axis of roots (Fig. 3A, G).
Individual hyphae sometimes grew along the
furrows between adjacent epidermal cells and
colonized roots intercellularly. Penetration through
root hairs was not observed. Once in the epidermis,
hyphae grew from cell to cell within the epidermis
parallel to the main axis of the host root, causing
no distortion of host root. At regions the hyphae
penetrated the cortical cells illing each cell with
microsclerotia (Fig. 3 F). The root stele was not
colonized in any of the roots that had DSE fungal
associations and there was no evidence of damage
to host root tissues arising from fungal colonization.
The rhizome samples analyzed were not
colonized by any fungal structure.
In the rhizospheric soil samples of both species,
five AM fungal genera were identified on the
basis of spore morphology and Melzer’s reaction:
Glomus, Acaulospora, Scutellospora, Gigaspora
and Pacispora. In S. sonchifolius rhizospheric
soil these included, four morphotypes belonging
to Acaulospora (A. scrobiculata, A. elegans, A.
denticulata and Acaulospora sp.), two Glomus
(G. clarum and Glomus sp.) two Scutellospora
(S. pellucida and Scutellospora sp.), two
Gigaspora and one Pacispora morphotypes. In S.
macroscyphus rhizospheric soil three Acaulospora
(A. scrobiculata, A. foveata and A. bireticulata), ive
Glomus (G. clarum, G. etunicatum, G. mosseae,
Glomus sp.1 and Glomus sp.2), two Scutellospora,
one Gigaspora and one Pacispora morphotype
were observed.
discussion
Smallanthus sonchifolius and S. macroscyphus
show high productivity and development under
cultivation and in its natural habitat respectively
(Grau & Rea, 1997; Seminario et al., 2003;
Kortzart Gonzales, 2009). Natural mycorrhizal
Fig. 2. Ratio of different types of root-associatedfungi colonizing Smallanthus sonchifolius and S.
macrocyphus ibrous roots. References: Percent
of root colonization by AM fungi, DSE or other
unidentiied fungi (Others) was estimated according
to the intersect method of McGonigle et al., 1990.
*Detection of AM, DSE and other unidentiied fungi
is expressed as percentages for each fungus in
relation to the total number of samples observed
(n=100).
symbiotic associations may enhance plant growth
and production through improved access to mineral
and water resources, among others beneficial
effects (Finlay, 2004).
Simultaneous occurrence of AM fungi and
DSE was observed in both species. Simultaneous
colonization by AM and DSE was previously
reported for other medicinal and aromatic plants
(Jupponen & Trappe, 1998; Muthukumar et al.,
2006). DSE are common plant colonizers in
temperate and tropical habitats (Jumpponem &
Trappe, 1998), while Arum-type AM seems to be
abundant among herbaceous plants such as yacon
and Paris-type AM seems to be more frequently
found among trees (Muthukumar et al., 2006).
According to Brundrett (2004, 2009), the high
level of mycorrhizal colonization associated with a
poorly developed absorbing root system with large
roots diameters and a large biomass conformed by
tuberous roots, leaves and aerial stems, suggests a
mycotrophic relationship dependent on mycorrhiza.
All this characteristic of both Smallanthus species
(Machado et al., 2004; Coll Araoz et al., 2008;
Kortzart Gonzales, 2009).
Rhizomes of obligate mycorrhizal plant species
such as orchids and Psilotum can be colonized by
AM (Brundett, 2002). These associations were not
observed in any of the Smallanthus species under
study.
197
Bol. Soc. Argent. Bot. 48 (2) 2013
Fig. 3. Light micrographs. A and B: Arbuscular mycorrhizal Arum-type hyphae (h), vesicles (v) and dark
septate endophytic intraradical septate hyphae (DSEh) stained with Gueguén. Scale bar = 100 µm. C:
AM arbuscule (arb). D and E: AM vesicle (v) with fatty acids stained in red. F: DSE Microsclerotia (m). G:
Appressorium (ap) and DSE hyphae (DSEh) on the root surface of Smallanthus sonchifolius. Scale bar =
50 µm.
198
M. I. Mercado et al. - Mycorrhizal associations in Smallanthus species
Further studies are ongoing to characterize the AM
and DSE involved in this mycorrhizal association in
order to investigate the effect of nutrition mediated
by these fungi in the production of secondary
metabolites and determine their potential promoting
growth in both Smallanthus species.
Acknowledgements
Dra. Marta Cabello, Instituto Spegazzini, Fac.
Ciencias Naturales y Museo, La Plata, Argentina
contributed to the identiication of fungi spores. Dr.
R. Larry Peterson made valuable improvements to
the irst draft. Funding: This research was supported
by ANPCyT (Grant PICTO 2004-503), CIUNT
A26/403 and CONICET from Argentina.
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Recibido el 12 de abril de 2012, aceptado el 1 de marzo
de 2013.