CRECIMIENTO EN SIETE MEDIOS NUTRITIVOS

Y SNTESIS in vitro DE UNA CEPA DE Laccaria bicolor

GROWTH ON SEVEN NUTRITIVE MEDIA AND in vitro SYNTHESIS

OF ONE STRAIN OF Laccaria bicolor

Guadalupe Santiago-Martnez1, Arturo Estrada-Torres1, Luca Varela2 y Tefilo Herrera3

1
Centro de Investigacin en Ciencias Biolgicas, UAT, km 10.5 Autopista Texmelucan-Tlaxcala,
Ixtacuixtla. 90122. Tlaxcala. (gsantia@cci.uatx.mx). 2Escuela Nacional de Ciencias Biolgicas, IPN,
Carpio y Plan de Ayala, Mxico, D. F. 3Laboratorio de Micologa. Departamento de Botnica. Institu-
to de Biologa, UNAM. Apartado Postal 70-233. 04510. Coyoacn, Mxico, D. F.

RESUMEN ABSTRACT

Con el propsito de establecer las condiciones adecuadas de culti-
vos de Laccaria bicolor y su capacidad de asociarse con Pinus
montezumae, en este trabajo se evalua el crecimiento de una cepa
de este hongo en siete medios de cultivo, y se describen las estruc-
turas formadas en plntulas inoculadas in vitro. La mayor veloci-
dad de crecimiento y dimetro colonial se obtuvo en extracto de
malta-agar (EMA) y biotina-aneurina-cido flico-agar (BAF), y
la mayor produccin de biomasa en los medios de BAF y
Sabouraud agar (SAB). La sntesis in vitro de la micorriza de este
hongo con Pinus montezumae fue inducida en botellas de 1 L con
vermiculita, turba y medio de cultivo lquido BAF. Despus de 15
meses se extrajo el sistema radical para su caracterizacin, obser-
vando una micorriza de simple a dicotmica, con una coloracin
caf clara, un manto delgado y plectenquimatoso y una red de
Hartig que penetr de dos a tres capas de clulas corticales.
To determine the appropiate conditions for the culture of Laccaria
bicolor and its potential to infect Pinus montezumae seedlings, the
growth of one strain of this fungus was tested in seven culture
media, and structures formed on in vitro inoculated seedlings are
described. The highest growth rate and colony diameter were
obtained in malta-agar extract (MAE), and biotin-aneunin-folic
acid-agar (BAF) media, whereas the highest biomass production
was observed in BAF and Sabouraud-agar (SAB) media. In vitro
synthesis of the mycorrhizae of this fungus with Pinus montezumae
was induced in 1 L flasks containing a mix of vermiculite, peat-
moss and BAF broth medium. The root system was extracted for
characterization after 15 months. The mycorrhizae had a simple
to dichotomous morphology, with a pale brown color, a thin and
plectenquimatic sheath, and a Hartig net surrounding two or three
layers of the cortical cells.

Palabras clave: Pinus montezumae, ectomicorrizas, hongos Key words: Pinus montezumae, ectomycorrhizas, symbiotic fungi,
simbiontes, pruebas de crecimiento. growth test.

INTRODUCCIN INTRODUCTION

L L
accaria bicolor, un hongo que se distribuye en
Europa y Norteamrica (Mueller, 1992), se aso-
cia frecuentemente con pinceas, encontrndose
en lugares perturbados y en rodales jvenes (Danielson,
1984; Kropp y Mueller, 1999). Su carcter pionero y la
facilidad para manipularlo en cultivo puro, as como su
capacidad para formar micorrizas, facilitan el estudio de
su biologa (Kropp y Fortin, 1988; Godbout y Fortin,
1992; Baar et al., 1994; Kropp y Mueller, 1999). Se han
obtenido buenos resultados en ensayos de inoculacin
de plantas con este hongo, ya que incrementa el creci-
miento y la supervivencia de las plantas durante el tras-
plante. Adems compite favorablemente con Thelephora
terrestris en el vivero y con los hongos indgenas des-
pus del trasplante (Kropp y Mueller, 1999).
Recibido: Septiembre, 2002. Aprobado: Octubre 2003.
Publicado como ARTCULO en Agrociencia 37: 575-584. 2003.
accaria bicolor, found in Europe and North
America (Mueller, 1992), is frequently associated
with pine trees, and is generally found in disturbed
areas and stands of young trees (Danielson, 1984; Kropp
and Mueller, 1999). The mushrooms character as a
pioneer species, the fact that it is easy to work with in a
pure culture medium, as well as its capacity to form
mycorrhizae, facilitate the study of its biology (Kropp and
Fortin, 1988; Godbout and Fortin, 1992; Baar et al., 1994;
Kropp and Mueller, 1999). Good results have been
obtained in tests where plants were inoculated with this
mushroom: the growth rate of the inoculated plants
increased, as did the survival rate of transplanted plants.
Besides, this mushroom competes efficiently with
Thelephora terrestris in nurseries and indigenous
mushrooms after transplanting (Kropp and Mueller, 1999).
The various species of Laccaria, especially L. bicolor,
offer a great potential for use in nursery plant inoculation

575
576 AGROCIENCIA VOLUMEN 37, NMERO 6, NOVIEMBRE-DICIEMBRE 2003
Las especies del gnero Laccaria, en especial L.
bicolor, tienen un alto potencial para ser usadas en pro-
gramas de inoculacin de plantas en vivero debido a las
siguientes caractersticas: Se asocian con una amplia
gama de hospederos, entre los que estn especies de los
gneros Eucalyptus, Larix, Picea, Pinus y Pseudotsuga
(Baar et al., 1994; Danielson, 1984; Wong et al., 1989);
son antagonistas de patgenos de la raz que habitan en
el suelo, por lo que son potencialmente tiles como agen-
tes de control de ciertas enfermedades (Hung y Molina,
1986); pueden sobrevivir en lugares talados (Baar et al.,
1994); y son fciles de aislar.
No obstante, poco se sabe de las caractersticas de
crecimiento de las cepas mexicanas de L. bicolor por lo
que es necesario evaluar su crecimiento en diferentes
medios de cultivo, determinar su afinidad con las espe-
cies de pino usadas comnmente en los programas de
reforestacin y caracterizar la morfologa de su
micorriza para reconocerla fcilmente en campo. Por
ello, se llev a cabo el presente trabajo con el objetivo
de evaluar el crecimiento de un aislamiento de Laccaria
bicolor en siete medios de cultivo, as como obtener y
caracterizar la micorriza que forma con Pinus
montezumae.
MATERIALES Y MTODOS
Aislamiento del material biolgico
La cepa se aisl utilizando medio de cultivo papa-dextrosa-
agar (PDA) a partir de cuerpos fructferos procedentes de un bos-
que de Pinus-Abies, del volcn La Malintzi. El sitio de estudio est
en la regin SE del Estado de Tlaxcala y al N de la ciudad de Pue-
bla, entre 19o 12 y 19o 17 N, y entre 98o 08 y 97o 57 O, a una
altura de 2700 a 4461 m, con una temperatura anual de 3 a 12 oC y
una precipitacin anual de 800 a 1 000 mm. Parte de los especimenes
recolectados se caracterizaron de acuerdo con Cifuentes et al.
(1986), se herborizaron con el nmero de respaldo de Herbario
Cuaxilo-Limn 55 y se depositaron en el Herbario TLXM del Cen-
tro de Investigacin en Ciencias Biolgicas de la Universidad Au-
tnoma de Tlaxcala. La cepa est depositada en la coleccin de
hongos ectomicorrizgenos del mismo Centro con la clave TLAX
30.
Pruebas de crecimiento y caracterizacin de las colonias
Para las pruebas de crecimiento en diferentes medios nutriti-
vos, el micelio crecido durante tres semanas en cajas de Petri con
PDA (Bioxon, Becton Dickinson de Mxico) se cuadricul con un
bistur estril, cortando fragmentos de 5 mm por lado. Estos frag-
mentos se transfirieron a cajas de Petri de 90 mm de dimetro con
los siguientes medios de cultivo (tratamientos): (1) papa-dextrosa-
agar (PDA); (2) extracto de malta-agar (EMA); (3) Sabouraud (SAB)
(Bioxon, Becton Dickinson de Mxico); (4) Ingestad (ING) (Mason,
programs because of the following characteristics: they
are associated with a wide range of hosts, including
species of genus Eucalyptus, Larix, Picea, Pinus and
Pseudotsuga (Baar et al., 1994; Danielson, 1984; Wong
et al., 1989); they are antagonists of soil-borne pathogens
of roots, making them potentially useful as control agents
for certain diseases (Hung and Molina, 1986); they can
act as saprotrophs in areas that have been clear-cut (Baar
et al., 1994); and they are easy to isolate.
Nevertheless, there is little information about the
growth characteristics of the Mexican strains of L. bicolor.
That is why it is necessary to evaluate the strains growth
in different culture media, to determine its association
with the species of pines commonly used in reforestation
programs and to describe the morphology of its
mycorrhizae to more easily identify it in the field.
Therefore, this study was carried out with the objective
of evaluating the growth of one isolated strain of L.
bicolor in seven culture media, as well as to obtain and
describe the mycorrhizae that form on Pinus montezumae.
MATERIALS AND METHODS
Isolation of the biological material
The strain was isolated using the potato-dextrose-agar (PDA)
medium from fruitbodies collected in a Pinus-Abies forest on the volcano
La Malintzi. The study was undertaken in the south-eastern part of the
State of Tlaxcala, north of the city of Puebla, between 19o 12 and 19o
17 N, and 98o 08 and 97o 57 W, at an altitude of 2700 to 4461 m, with
annual temperatures of 3 to 12 oC and annual precipitation of 800 to
1000 mm. Some of the specimens collected were described according
to Cifuentes et al. (1986), dried for herbarium purposes, labeled Cuaxilo-
Limn 55 Herbarium and deposited in the TLXM Herbarium in the
Center of Research in Biological Sciences at the Universidad Autnoma
of Tlaxcala. The strain was deposited in the collection of ectomycorrhizal
mushrooms in that same Center with the key TLAX 30.
Growth trials and characterization of the colonies
For the growth trials of the mushroom in different nutritive media,
its mycelium was grown for three weeks in Petri dishes with PDA
(Bioxon, Becton Dickinson of Mxico), put on a grid with a sterile
scalpel and then cut into pieces of 55 mm. These fragments were
transferred into Petri dishes 90 mm in diameter containing the
following culture media (treatments): (1) potato-dextrose-agar (PDA);
(2) malt extract agar (EMA); (3) Sabourauds agar (SAB) (Bioxon,
Becton Dickinson of Mxico); (4) Ingestads agar (ING) (Mason,
1980); (5) modified Melin-Norkrans agar (MNM); (6) Hagems agar
(HG) (Molina and Palmer, 1982); (7) biotin-aneurin-folic acid agar
(BAF) (Moser, 1960). Once the fragments had been transferred to
their Petri dishes with their respective media, they were kept in an
incubator at a temperature of 25 oC for 40 d. Every third day, a reading
was taken of the diameter of the colony growing in the dish. The
 SANTIAGO-MARTNEZ et al.: CRECIMIENTO Y SNTESIS in vitro DE Laccaria bicolor
1980); (5) Melin y Norkrans modificado (MNM); (6) Hagem (HG)
(Molina y Palmer, 1982); (7) biotina-aneurina-cido flico-agar
(BAF) (Moser, 1960). Una vez transferidos en las cajas de Petri con
el medio, se conservaron en una incubadora a 25 oC durante 40 d.
Cada tercer da se tomaron lecturas del dimetro colonial y al final
se valor la biomasa producida.
Para caracterizar la morfologa colonial del hongo en los medios
de cultivo, se tomaron los siguientes datos: Color, comparando la ta-
bla de colores de Methuen, de la cual se utiliz su notacin alfa-nu-
mrica (Kornerup y Wanscher, 1978); textura de la colonia; color que
present la parte inferior de la colonia (Figura 1). Estos datos se re-
gistraron a los 10 y 40 d de incubacin.
Al final del ensayo se evalu la velocidad de crecimiento, el
dimetro colonial y la biomasa. Para obtener la velocidad de creci-
miento se tomaron los datos de dimetro colonial cada tercer da
durante 40 d. Estos datos se ajustaron mediante una ecuacin de
regresin (Glover y Mitchell, 2002) para calcular la pendiente de la
curva de crecimiento y el promedio de crecimiento del hongo en
milmetros por da. Al final de este periodo se midi el dimetro y
la biomasa a travs del peso seco de la colonia, modificando la
tcnica propuesta por Chapman et al. (1990) y Oort (1981). Para
ello se elimin el agar por medio de calentamiento en bao mara,
enjuagando la colonia con agua caliente y secando posteriormente
a 60 oC hasta obtener peso constante.
Se utilizaron seis repeticiones por tratamiento. Se aplic un an-
lisis de varianza para un diseo completamente aleatorizado y una
prueba de Tukey (Montgomery, 1984) con p0.05.
EMA
SAB
PDA
ING
HG MNM
Figura 1. Prueba de crecimiento de la cepa de Laccaria bicolor en
diferentes medios nutritivos (PDA: Papa dextrosa agar;
EMA: Extracto de malta agar; SAB: Medio de
Sabouraud agar; ING: Medio de Ingestad agar; MNM:
Medio de Melin y Norkrans modificado; HG: Medio de
Hagem; BAF: Biotinaaneurina-cido flico agar).
Figure 1. Growth tests the Mexican of Laccaria bicolor strain in
different nutritive media (PDA: potato dextrose agar;
EMA malt extract agar; SAB: Sabourauds agar; ING:
Ingestads agar media; MNM: modified Melin-
Norkrans agar; HG: Hagems agar; BAF: biotin-
aneurin-folic acid agar).
577
biomass production of the colony was measured at the end of the
trial.
To describe the morphology of each colony in the nutritive
media, the following information was recorded: the colonys color,
using Methuens table of colors, based on his alphanumeric notation
(Kornerup and Wanscher, 1978); the colonys texture; the color of
the colonys underside as seen through the bottom of the Petri dish
(Figure 1). This information was documented on the 10th and 40th
days of incubation.
At the end of the trial, the colonys rate of growth, its diameter
and its biomass were measured. To determine the rate of growth, the
diameter of the colony was measured every third day for 40 d. The
results were adjusted by means of a regression equation (Glover and
Mitchell, 2002) to calculate the slope of the growth curve and the
daily average growth of the mushroom in millimeters. At the end of
this period, the diameter and the biomass were measured using the
dry weight of the colony, using the technique proposed by Chapman
et al. (1990) and Oort (1981). For this purpose the agar was eliminated
by heating the colony in a warm water bath, rinsing it with hot water
and drying it afterwards at 60 oC until it reached a constant weight.
Each trial was repeated six times. A variance analysis for a
completely random design was applied, and Tukey test (Montgomery,
1984) with p0.05.
In vitro synthesis
To obtain the micorrhizae, the methods proposed by Trappe
(1967) and Mason (1980) were modified, using 15 glass 1 L bottles.
300 mL of vermiculite, 30 mL of peat moss and 200 mL of BAF
(biotin-aneurin-folic acid agar) liquid nutritive medium, containing
10 g of dextrose (Figure 2), were placed into each bottle. These
growth media were sterilized for 1 h at 120 oC. Once cooled, the
substrate was inoculated with the strain grown in the BAF liquid
medium and sowed with a Pinus montezumae seedling. The test units
were maintained at a room temperature (Figure 2). Every three
months, three bottles were selected at random to review the radical
system (Figure 3). The developing mycorrhizae were fixed in FAA
(formalin-acetic acid-alcohol). To describe the mycorrhizae, the
criteria developed by Agerer (1987) were followed, taking into
account their form, the size of the system, of the axis and the non-
branched tips, and the surface of their mantle. The colors were defined
based on the Methuens table of colors, using his alphanumeric
notation (Kornerup and Wanscher, 1978). To observe their anatomy,
the mycorrhizae were sliced manually. Afterwards, the mantle and
the Hartig net were observed with a Nikon Optiphot microscope,
with Normarskis interference contrast system.
RESULTS AND DISCUSSION
Characterization of the strain in the different
culture media
Figure 1 illustrates the growth and characteristics of
the colonies in each culture medium, as described below.
578 AGROCIENCIA VOLUMEN 37, NMERO 6, NOVIEMBRE-DICIEMBRE 2003
Sntesis in vitro
Para obtener la micorriza se modificaron los mtodos propues-
tos por Trappe (1967) y Mason (1980), utilizando 15 frascos de vi-
drio de 1 L, en los cuales se adicion 300 mL de vermiculita, 30 mL
de turba y 200 mL de medio nutritivo lquido BAF (biotina-aneurina-
cido flico) conteniendo 10 g de dextrosa (Figura 2); estos medios
de crecimiento se esterilizaron durante 1 h a 120 oC. Una vez fro, el
sustrato se inocul con el hongo crecido en medio BAF lquido y se
sembr una plntula de Pinus montezumae; los dispositivos se man-
tuvieron a temperatura ambiente (Figura 2). Cada tres meses se to-
maron tres frascos al azar, para revisar el sistema radical (Figura 3).
Las micorrizas se caracterizaron y se fijaron en FAA (formol-cido
actico-alcohol). Para caracterizar la micorriza se siguieron los cri-
terios de Agerer (1987), tomando en cuenta la forma, tamao del
sistema, de los ejes y de las puntas no ramificadas y superficie del
manto. Los colores se definieron con base en la tabla de colores de
Methuen, de la cual se utiliz su notacin alfa-numrica (Kornerup
y Wanscher, 1978). Para observar la anatoma de la micorriza se
hicieron cortes a mano de las micorrizas. Posteriormente se observ
el manto y la red de Hartig con un microscopio Nikon Optiphot con
sistema de iluminacin de contraste diferencial de Nomarski.
RESULTADOS Y DISCUSIN
Caracterizacin de la cepa en los
diferentes medios de cultivo
La Figura 1 ilustra el tipo de crecimiento y caracte-
rsticas en cada medio de cultivo, los cuales se describen
a continuacin.
En el medio PDA la colonia present a los 10 d creci-
miento postrado en el agar, una coloracin de blanqueci-
na a lila (2A2) y una textura butircea. El reverso fue de
coloracin blanquecina. A los 40 d, el micelio continu
Figura 2. Dispositivo de sntesis in vitro en frasco de vidrio de 1 L
con turba, vermiculita y medio nutritivo lquido de BAF.
Figure 2. In vitro synthesis glass bottles, with peat moss,
vermiculite and liquid nutritive BAF medium.
At day 10, the colony in the PDA medium showed a
prostrate growth in the agar, off-white to lilac (2A2) color
and a butyraceous texture. The underside of the colony
was off-white in color. At day 40, the mycelium had
continued its prostrate growth, but its color had changed
to yellowish-white (3A2) and had a loose velvety texture.
The colony showed a somewhat irregular growth pattern,
with a few lilac spots (16B4), while its underside was
yellowish-white (3A2).
The EMA culture medium produced a prostrate growth
in the agar by the 10th day, the mycelium was lilac in
color (16B3), while the center and margins were off-white.
This colony had a butyraceous texture; as well, some
mycelial strands had formed over the original inoculum.
The underside of the colony was a grayish-purple (14B2)
in the center and off-white at the margins. At day 40, the
colony was showing both regular and prostrate growth in
the agar, had a violet-gray center (17C3) with yellowish-
white margins (1A2) and a loose velvety texture. The
underside of the colony was orange-gray in the center
(5B5), surrounded by opaque lilac (15C3) and yellowish-
white (3A2) margins.
The colony in the SAB medium, by day 10, showed
prostrate growth in the agar, was a dark lilac in color
(17C7), with off-white margins and a velvety texture.
Some not very well-defined concentric rings had formed
on the underside of the colony. By day 40, there were
concentric rings, not well defined, of different colors:
purple-gray (14B2) in the center, followed by a violet-
gray (18D4), pale violet (18A3), another shade of violet-
gray (18B3) and with violet-gray (17E5) and grayish-
violet (17B2) margins. The colony had a dense velvety
texture, with slightly curly mycelia, and an elevated
center, while the margins displayed prostrate mycelia on
the agar. The underside of the colony had some not very
Figura 3. Plntula de Pinus montezumae al momento de ser ex-
trada del dispositivo de sntesis in vitro.
Figure 3. Pinus montezumae seedling after dismissal from the in
in vitro synthesis glass bottle.
 SANTIAGO-MARTNEZ et al.: CRECIMIENTO Y SNTESIS in vitro DE Laccaria bicolor
con crecimiento postrado, pero la coloracin cambi a
blanco amarillento (3A2), con una textura aterciopelada
laxa, un crecimiento ligeramente irregular, ocasionalmen-
te con manchas de color lila (16B4) y reverso de la colo-
nia blanco amarillento (3A2).
El medio de cultivo EMA tuvo un crecimiento pos-
trado en el agar a los 10 d, con una coloracin lila (16B3)
en el centro y el margen blanquecino. La colonia presen-
t una textura butircea; adems se formaron cordones
areos sobre el inculo; el reverso fue de color gris pr-
pura (14B2) en el centro y blanquecino en el margen. A
los 40 d, la colonia present crecimiento regular y pos-
trado en el agar con una coloracin violeta griscea
(17C3) en el centro y blanco amarillento (1A2) en el
margen, textura aterciopelada laxa. El reverso de la co-
lonia fue de color naranja grisceo (5B5) en el centro,
luego lila opaco (15C3) y hacia el margen, blanco ama-
rillento (3A2).
En el medio SAB la colonia present crecimiento pos-
trado en el agar a los 10 d, con una coloracin lila oscura
(17C7) con el margen blanquecino y una textura
aterciopelada; en el reverso de la colonia se formaron
anillos concntricos no bien definidos. A los 40 d se ob-
servaron anillos concntricos no bien definidos que to-
maron diferentes coloraciones: Gris prpura (14B2) en
el centro, enseguida violeta grisceo (18D4), violeta p-
lido (18A3), violeta grisceo (18B3) y con el margen
violeta grisceo (17E5) y gris violceo (17B2). La colo-
nia tena textura aterciopelada densa con micelio poco
rizado y elevado en el centro, mientras que el margen
tena micelio postrado sobre el agar. En el reverso se ob-
servaron anillos concntricos no bien definidos de dife-
rentes coloraciones: Gris violeta (17D2) en el centro, vio-
leta opaco (17E4), lila rojizo (14C3), violeta grisceo
(17D4) y gris (17D1).
En el medio ING se observ una coloracin de blan-
quecina a violeta plida (18A3) a los 10 d, con micelio
areo blanquecino, textura de algodonosa laxa a afelpada,
con micelio ligeramente elevado. El reverso de la colo-
nia tom una coloracin gris violeta (17E6) en el centro,
con el margen blanquecino. A los 40 d, la colonia pre-
sent coloraciones desde violeta opaca (17D3) a gris vio-
leta (17B2), textura aterciopelada densa, con micelio
areo corto en el centro y el margen algodonoso poco
denso con micelio areo ms largo. El reverso de las co-
lonias present coloraciones magenta griscea (14E3) en
el centro, naranja griscea (6B4) y blanco amarillenta
(2A2) en el margen, con algunas manchas de color vio-
leta grisceo (17D7).
En el medio MNM se observ el micelio con colora-
cin violeta griscea (17B3) a los 10 d, textura
algodonosa laxa a afelpada con micelios areos blanque-
cinos; el reverso de la colonia con el centro de color gris
violeta (17B2) y el resto blanquecino, con tonos rosados
579
well defined concentric rings of different colors: grayish-
violet (17D2) in the center, opaque violet (17E4), reddish
lilac (14C3), violet-gray (17D4) and gray (17D1).
At day 10, the colony in ING medium was off-white
to pale lilac (18A3), displaying aerial, off-white mycelia
with a loose cottony to velvety texture. The mycelia were
somewhat elevated. The underside of the colony was
grayish-violet (17E6) in the center, with off-white
margins. By day 40, the colony showed colors ranging
from opaque violet (17D3) to grayish-violet (17B2), had
a dense velvety texture, with short, aerial mycelia in the
middle and margins of a slightly dense cottony texture,
with longer aerial mycelia. The underside of the colony
was magenta-gray (14E3) in the center, with orange-gray
(6B4) and whitish-yellow (2A2) margins that had some
violet-gray (17D7) spots on them.
In the MNM medium, the colony was violet-gray
(17B3) by day 10, with a loose cottony to velvety texture,
with aerial, off-white mycelia. The underside of the
colony had a center of grayish-violet (17B2) to off-white,
with pink tones in the margins. By day 40 of incubation,
the colony had a pinkish-white color (7A2) in the center,
and grayish-pink (8B2) margins and a loose velvety
texture, with the mycelia prostrate on the agar. The
underside of the colony had a reddish-brown (9E4) center,
with orange-brown (6C4) adjoining parts and whitish-
yellow (2A2) margins.
The colony formed in the HG medium was off-white
to lilac-gray (18A2) by the 10th day of incubation, with
off-white mycelia in the center, a loose cottony texture
and hairy, aerial mycelia. The underside of the colony
had a lilac color (17B2). By day 40, the colony showed a
whitish-purple color (14A2), with whitish-pink (10A2)
margins, a loose to dense velvety texture, with hairy, aerial
mycelia on the margins. The underside of the colony was
dark brown (8F7), with an off-white margin and some
spots of opaque lilac (16C3).
The BAF culture medium produced a lilac-colored
colony by day 10, with off-white margins also showing
tones of pale violet (18A3). The new mycelia it had a
velvety texture, with very short aerial mycelia. The
underside of the colony was gray, with white margins.
By day 40 of incubation, the colony had turned lilac-
gray (16B2), with white spots in the center and forming
poorly defined concentric rings of pale violet (18A4).
The margins were gray (18B1), and the colony had a
dense velvety texture. On top of the inoculated area, loose,
prostrate mycelia were visible on the margins. The
underside of the colony was a grayish-purple in color
(14B2), with a violet-gray in the middle (18B3) and gray
(18B1) margins.
In most of the media the strain studied displayed a
lilac to violet color, except for the MNM medium, which
produced pink tones. Besides, in the SAB medium, the
580 AGROCIENCIA VOLUMEN 37, NMERO 6, NOVIEMBRE-DICIEMBRE 2003
en el margen. A los 40 d de incubacin la colonia tom
una coloracin blanco rosada (7A2) en el centro y gris
rosada (8B2) hacia el margen, textura aterciopelada laxa
con micelio postrado en el agar. El reverso tena color
caf rojizo (9E4) en el centro, naranja caf (6C4) en la
parte media y blanco amarillento (2A2) en el margen.
En el medio HG la colonia tena coloracin de blan-
quecina a lila griscea (18A2) a los 10 d de incubacin,
con micelio blanquecino en el centro, textura algodonosa
laxa con micelio areo hirsuto y corto; el reverso de la
colonia tena color lila (17B2). A los 40 d la colonia pre-
sentaba una coloracin blanco prpura (14A2) con el
margen blanco rosado (10A2), textura aterciopelada laxa
a densa, con micelios areos hirsutos en el margen. El
reverso de la colonia tuvo color caf oscuro (8F7) con el
margen blanquecino, en ocasiones con manchas de co-
lor lila opaco (16C3).
En el medio de cultivo BAF la colonia present una
coloracin lila a los 10 d de incubacin, con el margen
blanquecino con tonos violeta plido (18A3). El micelio
nuevo present una textura aterciopelada con micelio a-
reo muy corto. El reverso de la colonia fue grisceo con
margen blanquecino. A los 40 d de incubacin la colonia
present una coloracin lila griscea (16B2) con man-
chas blancas en el centro y formando anillos concntricos
no bien definidos de color violeta plido (18A4). El mar-
gen era gris (18B1), textura aterciopelada densa; sobre
el inculo se observ micelio laxo y postrado en el mar-
gen. El reverso de la colonia fue de color gris prpura
(14B2), violeta grisceo en la parte media (18B3) y el
margen gris (18B1).
En la mayora de los medios la cepa evaluada presen-
t una coloracin de lila a violeta, con excepcin del
MNM, el cual mostr tonos rosados. Adems, en el me-
dio de cultivo SAB se observ que la colonia form ani-
llos concntricos de diferentes tonalidades violetas. Tam-
bin se detect que esta cepa no produce exudados que
pigmenten el medio de cultivo como en el caso de algu-
nas cepas de Pisolithus tinctorius (Santiago-Martnez et
al., 1995). El color violeta de la colonia estudiada con-
cuerda con lo descrito previamente por Hutchison (1991)
en los medios MN, HG y BAF.
Pruebas de crecimiento en los
medios de cultivo
La mayor velocidad de crecimiento de la colonia se
obtuvo en el medio de EMA, la cual no fue diferente
(p>0.05) del valor obtenido en el medio BAF (Cuadro
1). La velocidad de crecimiento en el medio BAF no pre-
sent diferencias significativas (p>0.05) con los valores
de los medios SAB y PDA, los cuales fueron diferentes
(p0.05) respecto al valor ms alto (EMA) y a los ms
bajos (MNM, ING y HG).
colony formed concentric rings of different violet tones.
It was also observed that this strain does not produce
exudates that stain the culture media, as is the case with
some strains of Pisolithus tinctorius (Santiago-Martnez
et al., 1995). The violet color of the colonies studied
agrees with that previously described by Hutchison
(1991) in the MN, HG and BAF media.
Growth trials in different culture media
The greatest rate of colony growth was produced by
the EMA medium, which was not different (p>0.05) from
the value obtained in the BAF medium (Table 1). The
growth rate produced by the BAF medium did not show
significant differences (p>0.05) from the values of the
SAB and PDA media, which differed (p0.05) from the
highest (EMA) and the lowest (MNM, ING and HG)
values.
The EMA and BAF media produced the highest
values for the final diameter of the colonies, and the
lowest were produced by the MNM, ING and HG media
(Table 1). The highest biomass values were reached in
the BAF and SAB media, and the lowest in the MNM
and HG media (Table 1).
In terms of growth, the ING, HG and MNM media
produced the lowest values for diameters and biomass
production of the colonies. The PDA and EMA media,
however, produced relatively large colonies even though
their biomass production was very low compared to that
of the SAB and BAF media. The correlation between the
colonies diameters and biomass production was 0.55.
Description of the mycorrhizae
The roots of the plants were sampled at months 3, 6,
9, 12 and 15 and, up to the final date, the presence of
mycorrhizae was detected in two of the three glass bottles.
The mycorrhizal system ranged from simple (Figure 4)
to dichotomous (Figure 5) and were occasionally
irregular, measuring from 2.5 to 3.6 mm; the non-
branched tips ranged from 0.4 to 0.5 mm; and the
diameter of the axis ranged from 0.4 to 0.5 mm. The
surface of the mantle was granular, with abundant
protruding hyphae, and displayed an irregularly
distributed metallic shine. The color was a light brown
(7D4) at the non-branched tips, orange-brown (7C3) at
the apices and brown (7E6) at the axis. The old
mycorrhizae were a dark brown (7F6). The mycorrhizae
had abundant rhizomorphs and protruded hyaline hyphae.
The mantle had a width of 23.5 to 31.4 mm, was
plectenchymatous, with the hyphae tending to form a net,
and also showing many protruding clamped hyphae
(Figure 6). The Hartig net (Figure 7) penetrated two to
three layers of cortical cells.
 SANTIAGO-MARTNEZ et al.: CRECIMIENTO Y SNTESIS in vitro DE Laccaria bicolor 581

Cuadro 1. Valores promedio ( x ) y error estndar de las variables de crecimiento de la cepa de Laccaria bicolor en siete medios de cultivo.
Table 1. Average values ( x ) and standard error of the growth variables at the Laccaria bicolor strain in seven nutritive media.

Medio de cultivo
Variable

PDA EMA SAB ING MNM HG BAF

Velocidad media (mm d-1) x 1.47b 2.31a 1.59b 0.64c 0.65c 0.22c 2.03ab
s 0.18 0.05 0.06 0.16 0.10 0.07 0.07
Dimetro final (mm)a los 40 d x 69.6bc 86.6a 62.0c 25.8de 33.0d 12.5e 79.2ab
s 0.8 0.49 0.29 0.59 0.54 0.42 0.35
Biomasa (mg) x 52.1bc 105.2b 188.0a 61.5bc 22.1c 12.0c 208.3a
s 1.09 0.93 0.68 1.05 0.49 0.28 1.05

(PDA: Papa dextrosa agar; EMA: Extracto de malta agar; SAB: Medio de Sabouraud agar; ING: Medio de Ingestad agar; MNM: Medio de Melin
y Norkrans modificado; HG: Medio de Hagem; BAF: Biotina-aneurina-cido flico agar) v (PDA: potato dextrose agar; EMA malt extract agar;
SAB: Sabourauds agar; ING: Ingestads agar media; MNM: modified Melin-Norkrans agar; HG: Hagems agar; BAF: biotin-aneurin-folic
acid agar).
Promedios con diferente letra en una lnea, son diferentes (Tukey, p0.05) v Average with different letter on a line, are different (Tukey, p0.05).

Los valores ms altos de dimetro final de la colonial
se encontraron en los medios EMA y BAF y los ms
bajos en los medios MNM, ING y HG (Cuadro 1). En
cuanto a la biomasa, los valores ms altos se presentaron
en los medios BAF y SAB, y los ms bajos en los medios
MNM y HG (Cuadro 1).
Respecto al crecimiento, los medios ING, HG y
MNM tuvieron los valores ms bajos de dimetro colo-
nial y produccin de biomasa. Sin embargo, en los me-
dios PDA y EMA hubo colonias relativamente gran-
des, aunque la produccin de biomasa fue muy baja
comparada con la de los medios SAB y BAF. La corre-
lacin entre el dimetro de la colonia y la produccin
de biomasa fue 0.55.
Caracterizacin de la micorriza
The mycorrhizae produced in these tests required the
symbionts to remain in their glass bottles for 15 months,
more time than the three to four months described by
Trappe (1967). Even though the sugar content of the
culture media was reduced for this study, the presence of
this carbohydrate might have influenced the formation
of the mycorrhizae. Duddridge (1986a and 1986b)
mentioned that high concentrations of glucose in culture
media used to obtain mycorrhizae could cause a defensive
response in the host. This, in turn, would increase the
production of phenols and lead to the lignification of the
roots. Moreover, an excess of glucose could affect the
interphase between the mushroom and its host, cause
abnormalities in the mantle and the Hartig net, as well as
produce intracellular hyphae.

Las races de las plantas se muestrearon a los 3, 6, 9,

12 y 15 meses y hasta la ltima fecha se detect la presen-
cia de micorrizas en dos de los tres frascos. La micorriza
present una morfologa de simple (Figura 4) a dicotmica
HE
(Figura 5), ocasionalmente irregular. El largo del sistema
ramificado fue 2.5 a 3.6 mm; las puntas no ramificadas
midieron de 0.4 a 0.5 mm, y el dimetro de los ejes de 0.4
a 0.5 mm. La superficie del manto fue granulosa con abun-
dantes hifas emanantes, con brillo metlico distribuido
irregularmente. La coloracin fue caf clara (7D4) en las
puntas no ramificadas, naranja caf (7C3) en los pices
de las puntas, y caf (7E6) en los ejes; las micorrizas vie-
jas con color caf oscuro (7F6). La micorriza tuvo abun-
dantes rizomorfos e hifas emanantes hialinas.
El manto tuvo una anchura de 23.5 a 31.4 m,
plectenquimatoso, con las hifas dispuestas en forma de Figura 4. Micorriza simple de P. montezumae micorrizado con L.
bicolor TLAX 30 (12.5 x). (HE): hifas emanantes.
red y con abundantes hifas emergentes fibuladas (Figura Figure 4. Simple mycorrhizal system of Pinus montezumae and
6). La red de Hartig (Figura 7) mostr una penetracin Laccaria bicolor TLAX 30 (12.5 x). (HE): protruding
de dos a tres capas de clulas corticales. hyphae.
582 AGROCIENCIA VOLUMEN 37, NMERO 6, NOVIEMBRE-DICIEMBRE 2003

HE

Figura 5. Micorriza dicotmica de P. montezumae (6.8 x). Figura 6. Hifas emanantes (HE) de la micorriza obtenida in vitro
Figure 5. Dichotomous mycorrhizal systems of P. montezumae and (250 x).
Laccaria bicolor (6.8 x). Figure 6. Protruding hyphae (HE) of the in vitro obtained
mycorrhizae (250 x).

La micorriza obtenida en este ensayo requiri que

los simbiontes permanecieran 15 meses en el dispositi- The dichotomous morphology and the penetration of
vo, tiempo mayor que los tres a cuatro meses descritos the Hartig net by two to three cortical cells (Figure 7)
por Trappe (1967). A pesar de que se redujo el contenido has already been described for Pinus pinea mycorrhizae
de azcar en el medio empleado en el presente trabajo, with Laccaria laccata (Branzati et al., 1985). Despite
el contenido de este carbohidrato pudo influir para que the fact that the colonies produced by this mushroom
las micorrizas no se formaran antes. Duddridge (1986a y show violet colors in most of the culture media used in
1986b) mencion que altas concentraciones de glucosa this study, and that this coloration has been observed in
en los dispositivos para obtener micorrizas pueden pro- field mycorrhizae of other species of Laccaria (Agerer,
vocar una respuesta de defensa en el hospedero, el cual 1987), the mycorrhizae obtained in vitro did not show
incrementa la produccin de fenoles y la lignificacin de this coloration. Nor did Ingleby et al. (1990) observe a
las races. Adems, el exceso de glucosa puede tener efec- violet color in the mycorrhizae of L. proxima and L.
tos sobre la ultraestructura de la interfaz del hongo y el tortilis obtained in an in vitro synthesis.
hospedero, as como anormalidades en el manto y en la The morphology of the mycorrhizal structures might
red de Hartig y la presencia de hifas intracelulares. vary among isolates of the same fungal species. Thus, in
La morfologa dicotmica y la penetracin de la red
de Hartig de 2 a 3 clulas corticales (Figura 7) ya se ha-
ban descrito para micorrizas de Pinus pinea con Laccaria
laccata (Branzati et al., 1985). A pesar de que las colo-
nias de esta cepa mostraron coloraciones violeta en la
mayora de los medios de cultivo utilizados en el ensa-
yo, y esta coloracin se ha observado en micorrizas de
campo de otras especies de Laccaria (Agerer, 1987), la
micorriza obtenida in vitro no present dichas
H
coloraciones. Ingleby et al. (1990) tampoco observaron
una coloracin violeta en micorrizas de L. proxima y L.
tortilis obtenidas en sntesis in vitro.
La morfologa de las estructuras micorrzicas puede
variar entre aislamientos de la misma especie fngica.
As, en el caso de Laccaria, se han observado diferen-
cias morfolgicas en la colonizacin de Pinus banksiana
con diferentes cepas de Laccaria bicolor (Wong et al.,
Figura 7. Red de Hartig (H) de la micorriza obtenida in vitro
1989). Sin embargo, es importante describir las (500 x).
micorrizas obtenidas para diferenciar la cepa evaluada Figure 7. Hartig net (H) of the in vitro obtained mycorrhizae
en condiciones de vivero y campo. (500 x).
 SANTIAGO-MARTNEZ et al.: CRECIMIENTO Y SNTESIS in vitro DE Laccaria bicolor
El sistema de micorrizacin in vitro utilizado en este
ensayo presenta pocos problemas de contaminacin. Sin
embargo, la evaluacin es destructiva, por lo cual se re-
quiere un gran nmero de unidades experimentales.
CONCLUSIONES
Los resultados muestran que es recomendable utili-
zar los medios de BAF y SAB para obtener una buena
cantidad de biomasa de la colonia, o el medio extracto
de malta-agar (EMA), si se requiere que la colonia tenga
un rpido crecimiento radial. La comprobacin de la ca-
pacidad de esta cepa para asociarse con Pinus
montezumae, una confera muy utilizada en los viveros
mexicanos para reforestacin, le confiere un gran poten-
cial para la produccin de plantas forestales.
AGRADECIMIENTOS
Este trabajo forma parte del proyecto Seleccin de hongos
ectomicorrizgenos para la produccin de inoculantes en el Estado
de Tlaxcala, financiado por CONACyT, convenio nmero
4690-N9406.
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583
the case of Laccaria, different morphologies have been
observed in the colonized roots of Pinus banksiana by
different Laccaria bicolor strains. Nevertheless, it is
important to describe the mycorrhizae obtained in order
to distinguish the strain studied in nursery and field
conditions.
The glass bottles used in this study for the in vitro
mycorrhizae synthesis had few problems of contamination.
Nevertheless, because the evaluation is destructive, a large
number of experimental units is required.
CONCLUSIONS
The results of this study indicate that the BAF and
SAB are the best culture media to produce a good quantity
of mycelial biomass, but EMA is the best medium for
having a rapid radial growth rate. The evidence of the
strains ability to associate with Pinus montezumae, a
conifer tree commonly used by Mexican nurseries to
reforest the countryside, demonstrates that it has a great
potential for promoting the production of forest plants.
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