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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- To determine the appropiate conditions for the culture of Laccaria
vos de Laccaria bicolor y su capacidad de asociarse con Pinus bicolor and its potential to infect Pinus montezumae seedlings, the
montezumae, en este trabajo se evalua el crecimiento de una cepa growth of one strain of this fungus was tested in seven culture
de este hongo en siete medios de cultivo, y se describen las estruc- media, and structures formed on in vitro inoculated seedlings are
turas formadas en plntulas inoculadas in vitro. La mayor veloci- described. The highest growth rate and colony diameter were
dad de crecimiento y dimetro colonial se obtuvo en extracto de obtained in malta-agar extract (MAE), and biotin-aneunin-folic
malta-agar (EMA) y biotina-aneurina-cido flico-agar (BAF), y acid-agar (BAF) media, whereas the highest biomass production
la mayor produccin de biomasa en los medios de BAF y was observed in BAF and Sabouraud-agar (SAB) media. In vitro
Sabouraud agar (SAB). La sntesis in vitro de la micorriza de este synthesis of the mycorrhizae of this fungus with Pinus montezumae
hongo con Pinus montezumae fue inducida en botellas de 1 L con was induced in 1 L flasks containing a mix of vermiculite, peat-
vermiculita, turba y medio de cultivo lquido BAF. Despus de 15 moss and BAF broth medium. The root system was extracted for
meses se extrajo el sistema radical para su caracterizacin, obser- characterization after 15 months. The mycorrhizae had a simple
vando una micorriza de simple a dicotmica, con una coloracin to dichotomous morphology, with a pale brown color, a thin and
caf clara, un manto delgado y plectenquimatoso y una red de plectenquimatic sheath, and a Hartig net surrounding two or three
Hartig que penetr de dos a tres capas de clulas corticales. 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 accaria bicolor, found in Europe and North
Europa y Norteamrica (Mueller, 1992), se aso- America (Mueller, 1992), is frequently associated
cia frecuentemente con pinceas, encontrndose with pine trees, and is generally found in disturbed
en lugares perturbados y en rodales jvenes (Danielson, areas and stands of young trees (Danielson, 1984; Kropp
1984; Kropp y Mueller, 1999). Su carcter pionero y la and Mueller, 1999). The mushrooms character as a
facilidad para manipularlo en cultivo puro, as como su pioneer species, the fact that it is easy to work with in a
capacidad para formar micorrizas, facilitan el estudio de pure culture medium, as well as its capacity to form
su biologa (Kropp y Fortin, 1988; Godbout y Fortin, mycorrhizae, facilitate the study of its biology (Kropp and
1992; Baar et al., 1994; Kropp y Mueller, 1999). Se han Fortin, 1988; Godbout and Fortin, 1992; Baar et al., 1994;
obtenido buenos resultados en ensayos de inoculacin Kropp and Mueller, 1999). Good results have been
de plantas con este hongo, ya que incrementa el creci- obtained in tests where plants were inoculated with this
miento y la supervivencia de las plantas durante el tras- mushroom: the growth rate of the inoculated plants
plante. Adems compite favorablemente con Thelephora increased, as did the survival rate of transplanted plants.
terrestris en el vivero y con los hongos indgenas des- Besides, this mushroom competes efficiently with
pus del trasplante (Kropp y Mueller, 1999). Thelephora terrestris in nurseries and indigenous
mushrooms after transplanting (Kropp and Mueller, 1999).
Recibido: Septiembre, 2002. Aprobado: Octubre 2003. The various species of Laccaria, especially L. bicolor,
Publicado como ARTCULO en Agrociencia 37: 575-584. 2003. 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. programs because of the following characteristics: they
bicolor, tienen un alto potencial para ser usadas en pro- are associated with a wide range of hosts, including
gramas de inoculacin de plantas en vivero debido a las species of genus Eucalyptus, Larix, Picea, Pinus and
siguientes caractersticas: Se asocian con una amplia Pseudotsuga (Baar et al., 1994; Danielson, 1984; Wong
gama de hospederos, entre los que estn especies de los et al., 1989); they are antagonists of soil-borne pathogens
gneros Eucalyptus, Larix, Picea, Pinus y Pseudotsuga of roots, making them potentially useful as control agents
(Baar et al., 1994; Danielson, 1984; Wong et al., 1989); for certain diseases (Hung and Molina, 1986); they can
son antagonistas de patgenos de la raz que habitan en act as saprotrophs in areas that have been clear-cut (Baar
el suelo, por lo que son potencialmente tiles como agen- et al., 1994); and they are easy to isolate.
tes de control de ciertas enfermedades (Hung y Molina, Nevertheless, there is little information about the
1986); pueden sobrevivir en lugares talados (Baar et al., growth characteristics of the Mexican strains of L. bicolor.
1994); y son fciles de aislar. That is why it is necessary to evaluate the strains growth
No obstante, poco se sabe de las caractersticas de in different culture media, to determine its association
crecimiento de las cepas mexicanas de L. bicolor por lo with the species of pines commonly used in reforestation
que es necesario evaluar su crecimiento en diferentes programs and to describe the morphology of its
medios de cultivo, determinar su afinidad con las espe- mycorrhizae to more easily identify it in the field.
cies de pino usadas comnmente en los programas de Therefore, this study was carried out with the objective
reforestacin y caracterizar la morfologa de su of evaluating the growth of one isolated strain of L.
micorriza para reconocerla fcilmente en campo. Por bicolor in seven culture media, as well as to obtain and
ello, se llev a cabo el presente trabajo con el objetivo describe the mycorrhizae that form on Pinus montezumae.
de evaluar el crecimiento de un aislamiento de Laccaria
bicolor en siete medios de cultivo, as como obtener y MATERIALS AND METHODS
caracterizar la micorriza que forma con Pinus
montezumae. Isolation of the biological material

MATERIALES Y MTODOS The strain was isolated using the potato-dextrose-agar (PDA)
medium from fruitbodies collected in a Pinus-Abies forest on the volcano
Aislamiento del material biolgico 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
La cepa se aisl utilizando medio de cultivo papa-dextrosa- 17 N, and 98o 08 and 97o 57 W, at an altitude of 2700 to 4461 m, with
agar (PDA) a partir de cuerpos fructferos procedentes de un bos- annual temperatures of 3 to 12 oC and annual precipitation of 800 to
que de Pinus-Abies, del volcn La Malintzi. El sitio de estudio est 1000 mm. Some of the specimens collected were described according
en la regin SE del Estado de Tlaxcala y al N de la ciudad de Pue- to Cifuentes et al. (1986), dried for herbarium purposes, labeled Cuaxilo-
bla, entre 19o 12 y 19o 17 N, y entre 98o 08 y 97o 57 O, a una Limn 55 Herbarium and deposited in the TLXM Herbarium in the
altura de 2700 a 4461 m, con una temperatura anual de 3 a 12 oC y Center of Research in Biological Sciences at the Universidad Autnoma
una precipitacin anual de 800 a 1 000 mm. Parte de los especimenes of Tlaxcala. The strain was deposited in the collection of ectomycorrhizal
recolectados se caracterizaron de acuerdo con Cifuentes et al. mushrooms in that same Center with the key TLAX 30.
(1986), se herborizaron con el nmero de respaldo de Herbario
Cuaxilo-Limn 55 y se depositaron en el Herbario TLXM del Cen- Growth trials and characterization of the colonies
tro de Investigacin en Ciencias Biolgicas de la Universidad Au-
tnoma de Tlaxcala. La cepa est depositada en la coleccin de For the growth trials of the mushroom in different nutritive media,
hongos ectomicorrizgenos del mismo Centro con la clave TLAX its mycelium was grown for three weeks in Petri dishes with PDA
30. (Bioxon, Becton Dickinson of Mxico), put on a grid with a sterile
scalpel and then cut into pieces of 55 mm. These fragments were
Pruebas de crecimiento y caracterizacin de las colonias transferred into Petri dishes 90 mm in diameter containing the
following culture media (treatments): (1) potato-dextrose-agar (PDA);
Para las pruebas de crecimiento en diferentes medios nutriti- (2) malt extract agar (EMA); (3) Sabourauds agar (SAB) (Bioxon,
vos, el micelio crecido durante tres semanas en cajas de Petri con Becton Dickinson of Mxico); (4) Ingestads agar (ING) (Mason,
PDA (Bioxon, Becton Dickinson de Mxico) se cuadricul con un 1980); (5) modified Melin-Norkrans agar (MNM); (6) Hagems agar
bistur estril, cortando fragmentos de 5 mm por lado. Estos frag- (HG) (Molina and Palmer, 1982); (7) biotin-aneurin-folic acid agar
mentos se transfirieron a cajas de Petri de 90 mm de dimetro con (BAF) (Moser, 1960). Once the fragments had been transferred to
los siguientes medios de cultivo (tratamientos): (1) papa-dextrosa- their Petri dishes with their respective media, they were kept in an
agar (PDA); (2) extracto de malta-agar (EMA); (3) Sabouraud (SAB) incubator at a temperature of 25 oC for 40 d. Every third day, a reading
(Bioxon, Becton Dickinson de Mxico); (4) Ingestad (ING) (Mason, 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 577

1980); (5) Melin y Norkrans modificado (MNM); (6) Hagem (HG) biomass production of the colony was measured at the end of the
(Molina y Palmer, 1982); (7) biotina-aneurina-cido flico-agar trial.
(BAF) (Moser, 1960). Una vez transferidos en las cajas de Petri con To describe the morphology of each colony in the nutritive
el medio, se conservaron en una incubadora a 25 oC durante 40 d. media, the following information was recorded: the colonys color,
Cada tercer da se tomaron lecturas del dimetro colonial y al final using Methuens table of colors, based on his alphanumeric notation
se valor la biomasa producida. (Kornerup and Wanscher, 1978); the colonys texture; the color of
Para caracterizar la morfologa colonial del hongo en los medios the colonys underside as seen through the bottom of the Petri dish
de cultivo, se tomaron los siguientes datos: Color, comparando la ta- (Figure 1). This information was documented on the 10th and 40th
bla de colores de Methuen, de la cual se utiliz su notacin alfa-nu- days of incubation.
mrica (Kornerup y Wanscher, 1978); textura de la colonia; color que At the end of the trial, the colonys rate of growth, its diameter
present la parte inferior de la colonia (Figura 1). Estos datos se re- and its biomass were measured. To determine the rate of growth, the
gistraron a los 10 y 40 d de incubacin. diameter of the colony was measured every third day for 40 d. The
Al final del ensayo se evalu la velocidad de crecimiento, el results were adjusted by means of a regression equation (Glover and
dimetro colonial y la biomasa. Para obtener la velocidad de creci- Mitchell, 2002) to calculate the slope of the growth curve and the
miento se tomaron los datos de dimetro colonial cada tercer da daily average growth of the mushroom in millimeters. At the end of
durante 40 d. Estos datos se ajustaron mediante una ecuacin de this period, the diameter and the biomass were measured using the
regresin (Glover y Mitchell, 2002) para calcular la pendiente de la dry weight of the colony, using the technique proposed by Chapman
curva de crecimiento y el promedio de crecimiento del hongo en et al. (1990) and Oort (1981). For this purpose the agar was eliminated
milmetros por da. Al final de este periodo se midi el dimetro y by heating the colony in a warm water bath, rinsing it with hot water
la biomasa a travs del peso seco de la colonia, modificando la and drying it afterwards at 60 oC until it reached a constant weight.
tcnica propuesta por Chapman et al. (1990) y Oort (1981). Para Each trial was repeated six times. A variance analysis for a
ello se elimin el agar por medio de calentamiento en bao mara, completely random design was applied, and Tukey test (Montgomery,
enjuagando la colonia con agua caliente y secando posteriormente 1984) with p0.05.
a 60 oC hasta obtener peso constante.
Se utilizaron seis repeticiones por tratamiento. Se aplic un an- In vitro synthesis
lisis de varianza para un diseo completamente aleatorizado y una
prueba de Tukey (Montgomery, 1984) con p0.05. 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
EMA 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
SAB 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
PDA
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
ING
based on the Methuens table of colors, using his alphanumeric
HG MNM notation (Kornerup and Wanscher, 1978). To observe their anatomy,
the mycorrhizae were sliced manually. Afterwards, the mantle and
Figura 1. Prueba de crecimiento de la cepa de Laccaria bicolor en the Hartig net were observed with a Nikon Optiphot microscope,
diferentes medios nutritivos (PDA: Papa dextrosa agar; with Normarskis interference contrast system.
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
RESULTS AND DISCUSSION
Hagem; BAF: Biotinaaneurina-cido flico agar).
Figure 1. Growth tests the Mexican of Laccaria bicolor strain in Characterization of the strain in the different
different nutritive media (PDA: potato dextrose agar; culture media
EMA malt extract agar; SAB: Sabourauds agar; ING:
Ingestads agar media; MNM: modified Melin-
Norkrans agar; HG: Hagems agar; BAF: biotin- Figure 1 illustrates the growth and characteristics of
aneurin-folic acid agar). the colonies in each culture medium, as described below.
578 AGROCIENCIA VOLUMEN 37, NMERO 6, NOVIEMBRE-DICIEMBRE 2003

Sntesis in vitro At day 10, the colony in the PDA medium showed a
prostrate growth in the agar, off-white to lilac (2A2) color
Para obtener la micorriza se modificaron los mtodos propues- and a butyraceous texture. The underside of the colony
tos por Trappe (1967) y Mason (1980), utilizando 15 frascos de vi- was off-white in color. At day 40, the mycelium had
drio de 1 L, en los cuales se adicion 300 mL de vermiculita, 30 mL continued its prostrate growth, but its color had changed
de turba y 200 mL de medio nutritivo lquido BAF (biotina-aneurina- to yellowish-white (3A2) and had a loose velvety texture.
cido flico) conteniendo 10 g de dextrosa (Figura 2); estos medios The colony showed a somewhat irregular growth pattern,
de crecimiento se esterilizaron durante 1 h a 120 oC. Una vez fro, el with a few lilac spots (16B4), while its underside was
sustrato se inocul con el hongo crecido en medio BAF lquido y se yellowish-white (3A2).
sembr una plntula de Pinus montezumae; los dispositivos se man- The EMA culture medium produced a prostrate growth
tuvieron a temperatura ambiente (Figura 2). Cada tres meses se to- in the agar by the 10th day, the mycelium was lilac in
maron tres frascos al azar, para revisar el sistema radical (Figura 3). color (16B3), while the center and margins were off-white.
Las micorrizas se caracterizaron y se fijaron en FAA (formol-cido This colony had a butyraceous texture; as well, some
actico-alcohol). Para caracterizar la micorriza se siguieron los cri- mycelial strands had formed over the original inoculum.
terios de Agerer (1987), tomando en cuenta la forma, tamao del The underside of the colony was a grayish-purple (14B2)
sistema, de los ejes y de las puntas no ramificadas y superficie del in the center and off-white at the margins. At day 40, the
manto. Los colores se definieron con base en la tabla de colores de colony was showing both regular and prostrate growth in
Methuen, de la cual se utiliz su notacin alfa-numrica (Kornerup the agar, had a violet-gray center (17C3) with yellowish-
y Wanscher, 1978). Para observar la anatoma de la micorriza se white margins (1A2) and a loose velvety texture. The
hicieron cortes a mano de las micorrizas. Posteriormente se observ underside of the colony was orange-gray in the center
el manto y la red de Hartig con un microscopio Nikon Optiphot con (5B5), surrounded by opaque lilac (15C3) and yellowish-
sistema de iluminacin de contraste diferencial de Nomarski. white (3A2) margins.
The colony in the SAB medium, by day 10, showed
RESULTADOS Y DISCUSIN prostrate growth in the agar, was a dark lilac in color
(17C7), with off-white margins and a velvety texture.
Caracterizacin de la cepa en los Some not very well-defined concentric rings had formed
diferentes medios de cultivo on the underside of the colony. By day 40, there were
concentric rings, not well defined, of different colors:
La Figura 1 ilustra el tipo de crecimiento y caracte- purple-gray (14B2) in the center, followed by a violet-
rsticas en cada medio de cultivo, los cuales se describen gray (18D4), pale violet (18A3), another shade of violet-
a continuacin. gray (18B3) and with violet-gray (17E5) and grayish-
En el medio PDA la colonia present a los 10 d creci- violet (17B2) margins. The colony had a dense velvety
miento postrado en el agar, una coloracin de blanqueci- texture, with slightly curly mycelia, and an elevated
na a lila (2A2) y una textura butircea. El reverso fue de center, while the margins displayed prostrate mycelia on
coloracin blanquecina. A los 40 d, el micelio continu the agar. The underside of the colony had some not very

Figura 2. Dispositivo de sntesis in vitro en frasco de vidrio de 1 L Figura 3. Plntula de Pinus montezumae al momento de ser ex-
con turba, vermiculita y medio nutritivo lquido de BAF. trada del dispositivo de sntesis in vitro.
Figure 2. In vitro synthesis glass bottles, with peat moss, Figure 3. Pinus montezumae seedling after dismissal from the in
vermiculite and liquid nutritive BAF medium. in vitro synthesis glass bottle.
SANTIAGO-MARTNEZ et al.: CRECIMIENTO Y SNTESIS in vitro DE Laccaria bicolor 579

con crecimiento postrado, pero la coloracin cambi a well defined concentric rings of different colors: grayish-
blanco amarillento (3A2), con una textura aterciopelada violet (17D2) in the center, opaque violet (17E4), reddish
laxa, un crecimiento ligeramente irregular, ocasionalmen- lilac (14C3), violet-gray (17D4) and gray (17D1).
te con manchas de color lila (16B4) y reverso de la colo- At day 10, the colony in ING medium was off-white
nia blanco amarillento (3A2). to pale lilac (18A3), displaying aerial, off-white mycelia
El medio de cultivo EMA tuvo un crecimiento pos- with a loose cottony to velvety texture. The mycelia were
trado en el agar a los 10 d, con una coloracin lila (16B3) somewhat elevated. The underside of the colony was
en el centro y el margen blanquecino. La colonia presen- grayish-violet (17E6) in the center, with off-white
t una textura butircea; adems se formaron cordones margins. By day 40, the colony showed colors ranging
areos sobre el inculo; el reverso fue de color gris pr- from opaque violet (17D3) to grayish-violet (17B2), had
pura (14B2) en el centro y blanquecino en el margen. A a dense velvety texture, with short, aerial mycelia in the
los 40 d, la colonia present crecimiento regular y pos- middle and margins of a slightly dense cottony texture,
trado en el agar con una coloracin violeta griscea with longer aerial mycelia. The underside of the colony
(17C3) en el centro y blanco amarillento (1A2) en el was magenta-gray (14E3) in the center, with orange-gray
margen, textura aterciopelada laxa. El reverso de la co- (6B4) and whitish-yellow (2A2) margins that had some
lonia fue de color naranja grisceo (5B5) en el centro, violet-gray (17D7) spots on them.
luego lila opaco (15C3) y hacia el margen, blanco ama- In the MNM medium, the colony was violet-gray
rillento (3A2). (17B3) by day 10, with a loose cottony to velvety texture,
En el medio SAB la colonia present crecimiento pos- with aerial, off-white mycelia. The underside of the
trado en el agar a los 10 d, con una coloracin lila oscura colony had a center of grayish-violet (17B2) to off-white,
(17C7) con el margen blanquecino y una textura with pink tones in the margins. By day 40 of incubation,
aterciopelada; en el reverso de la colonia se formaron the colony had a pinkish-white color (7A2) in the center,
anillos concntricos no bien definidos. A los 40 d se ob- and grayish-pink (8B2) margins and a loose velvety
servaron anillos concntricos no bien definidos que to- texture, with the mycelia prostrate on the agar. The
maron diferentes coloraciones: Gris prpura (14B2) en underside of the colony had a reddish-brown (9E4) center,
el centro, enseguida violeta grisceo (18D4), violeta p- with orange-brown (6C4) adjoining parts and whitish-
lido (18A3), violeta grisceo (18B3) y con el margen yellow (2A2) margins.
violeta grisceo (17E5) y gris violceo (17B2). La colo- The colony formed in the HG medium was off-white
nia tena textura aterciopelada densa con micelio poco to lilac-gray (18A2) by the 10th day of incubation, with
rizado y elevado en el centro, mientras que el margen off-white mycelia in the center, a loose cottony texture
tena micelio postrado sobre el agar. En el reverso se ob- and hairy, aerial mycelia. The underside of the colony
servaron anillos concntricos no bien definidos de dife- had a lilac color (17B2). By day 40, the colony showed a
rentes coloraciones: Gris violeta (17D2) en el centro, vio- whitish-purple color (14A2), with whitish-pink (10A2)
leta opaco (17E4), lila rojizo (14C3), violeta grisceo margins, a loose to dense velvety texture, with hairy, aerial
(17D4) y gris (17D1). mycelia on the margins. The underside of the colony was
En el medio ING se observ una coloracin de blan- dark brown (8F7), with an off-white margin and some
quecina a violeta plida (18A3) a los 10 d, con micelio spots of opaque lilac (16C3).
areo blanquecino, textura de algodonosa laxa a afelpada, The BAF culture medium produced a lilac-colored
con micelio ligeramente elevado. El reverso de la colo- colony by day 10, with off-white margins also showing
nia tom una coloracin gris violeta (17E6) en el centro, tones of pale violet (18A3). The new mycelia it had a
con el margen blanquecino. A los 40 d, la colonia pre- velvety texture, with very short aerial mycelia. The
sent coloraciones desde violeta opaca (17D3) a gris vio- underside of the colony was gray, with white margins.
leta (17B2), textura aterciopelada densa, con micelio By day 40 of incubation, the colony had turned lilac-
areo corto en el centro y el margen algodonoso poco gray (16B2), with white spots in the center and forming
denso con micelio areo ms largo. El reverso de las co- poorly defined concentric rings of pale violet (18A4).
lonias present coloraciones magenta griscea (14E3) en The margins were gray (18B1), and the colony had a
el centro, naranja griscea (6B4) y blanco amarillenta dense velvety texture. On top of the inoculated area, loose,
(2A2) en el margen, con algunas manchas de color vio- prostrate mycelia were visible on the margins. The
leta grisceo (17D7). underside of the colony was a grayish-purple in color
En el medio MNM se observ el micelio con colora- (14B2), with a violet-gray in the middle (18B3) and gray
cin violeta griscea (17B3) a los 10 d, textura (18B1) margins.
algodonosa laxa a afelpada con micelios areos blanque- In most of the media the strain studied displayed a
cinos; el reverso de la colonia con el centro de color gris lilac to violet color, except for the MNM medium, which
violeta (17B2) y el resto blanquecino, con tonos rosados 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 colony formed concentric rings of different violet tones.
una coloracin blanco rosada (7A2) en el centro y gris It was also observed that this strain does not produce
rosada (8B2) hacia el margen, textura aterciopelada laxa exudates that stain the culture media, as is the case with
con micelio postrado en el agar. El reverso tena color some strains of Pisolithus tinctorius (Santiago-Martnez
caf rojizo (9E4) en el centro, naranja caf (6C4) en la et al., 1995). The violet color of the colonies studied
parte media y blanco amarillento (2A2) en el margen. agrees with that previously described by Hutchison
En el medio HG la colonia tena coloracin de blan- (1991) in the MN, HG and BAF media.
quecina a lila griscea (18A2) a los 10 d de incubacin,
con micelio blanquecino en el centro, textura algodonosa Growth trials in different culture media
laxa con micelio areo hirsuto y corto; el reverso de la
colonia tena color lila (17B2). A los 40 d la colonia pre- The greatest rate of colony growth was produced by
sentaba una coloracin blanco prpura (14A2) con el the EMA medium, which was not different (p>0.05) from
margen blanco rosado (10A2), textura aterciopelada laxa the value obtained in the BAF medium (Table 1). The
a densa, con micelios areos hirsutos en el margen. El growth rate produced by the BAF medium did not show
reverso de la colonia tuvo color caf oscuro (8F7) con el significant differences (p>0.05) from the values of the
margen blanquecino, en ocasiones con manchas de co- SAB and PDA media, which differed (p0.05) from the
lor lila opaco (16C3). highest (EMA) and the lowest (MNM, ING and HG)
En el medio de cultivo BAF la colonia present una values.
coloracin lila a los 10 d de incubacin, con el margen The EMA and BAF media produced the highest
blanquecino con tonos violeta plido (18A3). El micelio values for the final diameter of the colonies, and the
nuevo present una textura aterciopelada con micelio a- lowest were produced by the MNM, ING and HG media
reo muy corto. El reverso de la colonia fue grisceo con (Table 1). The highest biomass values were reached in
margen blanquecino. A los 40 d de incubacin la colonia the BAF and SAB media, and the lowest in the MNM
present una coloracin lila griscea (16B2) con man- and HG media (Table 1).
chas blancas en el centro y formando anillos concntricos In terms of growth, the ING, HG and MNM media
no bien definidos de color violeta plido (18A4). El mar- produced the lowest values for diameters and biomass
gen era gris (18B1), textura aterciopelada densa; sobre production of the colonies. The PDA and EMA media,
el inculo se observ micelio laxo y postrado en el mar- however, produced relatively large colonies even though
gen. El reverso de la colonia fue de color gris prpura their biomass production was very low compared to that
(14B2), violeta grisceo en la parte media (18B3) y el of the SAB and BAF media. The correlation between the
margen gris (18B1). colonies diameters and biomass production was 0.55.
En la mayora de los medios la cepa evaluada presen-
t una coloracin de lila a violeta, con excepcin del Description of the mycorrhizae
MNM, el cual mostr tonos rosados. Adems, en el me-
dio de cultivo SAB se observ que la colonia form ani- The roots of the plants were sampled at months 3, 6,
llos concntricos de diferentes tonalidades violetas. Tam- 9, 12 and 15 and, up to the final date, the presence of
bin se detect que esta cepa no produce exudados que mycorrhizae was detected in two of the three glass bottles.
pigmenten el medio de cultivo como en el caso de algu- The mycorrhizal system ranged from simple (Figure 4)
nas cepas de Pisolithus tinctorius (Santiago-Martnez et to dichotomous (Figure 5) and were occasionally
al., 1995). El color violeta de la colonia estudiada con- irregular, measuring from 2.5 to 3.6 mm; the non-
cuerda con lo descrito previamente por Hutchison (1991) branched tips ranged from 0.4 to 0.5 mm; and the
en los medios MN, HG y BAF. diameter of the axis ranged from 0.4 to 0.5 mm. The
surface of the mantle was granular, with abundant
Pruebas de crecimiento en los protruding hyphae, and displayed an irregularly
medios de cultivo distributed metallic shine. The color was a light brown
(7D4) at the non-branched tips, orange-brown (7C3) at
La mayor velocidad de crecimiento de la colonia se the apices and brown (7E6) at the axis. The old
obtuvo en el medio de EMA, la cual no fue diferente mycorrhizae were a dark brown (7F6). The mycorrhizae
(p>0.05) del valor obtenido en el medio BAF (Cuadro had abundant rhizomorphs and protruded hyaline hyphae.
1). La velocidad de crecimiento en el medio BAF no pre- The mantle had a width of 23.5 to 31.4 mm, was
sent diferencias significativas (p>0.05) con los valores plectenchymatous, with the hyphae tending to form a net,
de los medios SAB y PDA, los cuales fueron diferentes and also showing many protruding clamped hyphae
(p0.05) respecto al valor ms alto (EMA) y a los ms (Figure 6). The Hartig net (Figure 7) penetrated two to
bajos (MNM, ING y HG). 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 The mycorrhizae produced in these tests required the
se encontraron en los medios EMA y BAF y los ms symbionts to remain in their glass bottles for 15 months,
bajos en los medios MNM, ING y HG (Cuadro 1). En more time than the three to four months described by
cuanto a la biomasa, los valores ms altos se presentaron Trappe (1967). Even though the sugar content of the
en los medios BAF y SAB, y los ms bajos en los medios culture media was reduced for this study, the presence of
MNM y HG (Cuadro 1). this carbohydrate might have influenced the formation
Respecto al crecimiento, los medios ING, HG y of the mycorrhizae. Duddridge (1986a and 1986b)
MNM tuvieron los valores ms bajos de dimetro colo- mentioned that high concentrations of glucose in culture
nial y produccin de biomasa. Sin embargo, en los me- media used to obtain mycorrhizae could cause a defensive
dios PDA y EMA hubo colonias relativamente gran- response in the host. This, in turn, would increase the
des, aunque la produccin de biomasa fue muy baja production of phenols and lead to the lignification of the
comparada con la de los medios SAB y BAF. La corre- roots. Moreover, an excess of glucose could affect the
lacin entre el dimetro de la colonia y la produccin interphase between the mushroom and its host, cause
de biomasa fue 0.55. abnormalities in the mantle and the Hartig net, as well as
produce intracellular hyphae.
Caracterizacin de la micorriza

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 583

El sistema de micorrizacin in vitro utilizado en este the case of Laccaria, different morphologies have been
ensayo presenta pocos problemas de contaminacin. Sin observed in the colonized roots of Pinus banksiana by
embargo, la evaluacin es destructiva, por lo cual se re- different Laccaria bicolor strains. Nevertheless, it is
quiere un gran nmero de unidades experimentales. important to describe the mycorrhizae obtained in order
to distinguish the strain studied in nursery and field
CONCLUSIONES conditions.
The glass bottles used in this study for the in vitro
Los resultados muestran que es recomendable utili- mycorrhizae synthesis had few problems of contamination.
zar los medios de BAF y SAB para obtener una buena Nevertheless, because the evaluation is destructive, a large
cantidad de biomasa de la colonia, o el medio extracto number of experimental units is required.
de malta-agar (EMA), si se requiere que la colonia tenga
un rpido crecimiento radial. La comprobacin de la ca- CONCLUSIONS
pacidad de esta cepa para asociarse con Pinus
montezumae, una confera muy utilizada en los viveros The results of this study indicate that the BAF and
mexicanos para reforestacin, le confiere un gran poten- SAB are the best culture media to produce a good quantity
cial para la produccin de plantas forestales. of mycelial biomass, but EMA is the best medium for
having a rapid radial growth rate. The evidence of the
AGRADECIMIENTOS 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
Este trabajo forma parte del proyecto Seleccin de hongos
potential for promoting the production of forest plants.
ectomicorrizgenos para la produccin de inoculantes en el Estado
de Tlaxcala, financiado por CONACyT, convenio nmero
End of the English version
4690-N9406.

pppvPPP
LITERATURA CITADA
inoculum on container-grown Douglas fir and Ponderosa pine
Agerer, R. 1987. Colour Atlas of Ectomycorrhizae. Einhorn-Verlag- seedlings. Can. J. For. Res. 16: 802-806.
Schwbisch Gmiind. 212 p. Hutchison, L. J. 1991. Description and identification of cultures of
Baar, J., W. A. Ozinga, and Th. W. Kuyper. 1994. Spatial distribution ectomycorrhizal fungi found in North America. Mycotaxon 42:
of Laccaria bicolor genets reflected by sporocarps after removal 387-504.
of litter and humus layer in a Pinus sylvestris forest. Mycol. Res. Ingleby K., P. A. Mason, F. T. Last, and L. V. Fleming. 1990.
98: 726-728. Identification of Ectomycorrhizas. Institute of Terrestrial Ecology.
Branzati, B., A. Zambonelli, and G. Govi. 1985. Micorrizazione di London, 112 p.
Pinus pinea con Laccaria laccata ed Hebeloma crustuliniforme. Kornerup, A., and J. H. Wanscher. 1978. Methuen Handbook of Colour.
Mic. Ital. 3: 3-10. Eyre Methuen. London, 252 p.
Cifuentes J., M. Villegas, y L. Prez-Ramrez. 1986. Hongos. In: Kropp, B. R., and J. A. Fortin. 1988. The incompatibility system and
Manual para Herbario. Chiang, A. (comp.) Consejo Nacional de relative ectomycorrhizal performance of monokarions and
la Flora de Mxico. Inst. Biol. UNAM, Mxico, D. F. pp: 55-64. reconstituted dicaryons of Laccaria bicolor. Can. J. Bot. 66: 289-
Chapman, W. K., S. M. Berch, and T. M. Ballard. 1990. In vitro growth 294.
of ectomycorrhizal fungi on dilute agar. Mycologia 82: 526-527. Kropp, B. R., and G. M. Mueller. 1999. Laccaria. In: Ectomycorrhizal
Danielson, R. M. 1984. Ectomycorrhizal associations in Jack Pine Fungi. Key Genera in Profile. Cairney J. W. G. and S. M. Chambers
stand in Northeastern Alberta. Can. J. Bot. 62: 932-939. (eds.). Springer, New York. pp: 65-88.
Duddridge, J. A. 1896a. The development and ultrastructure of Mason, P. A. 1980. Aseptic synthesis of sheathing (ecto) mycorrrhizas.
ectomycorrhizas. III. Compatible and incompatible interactions In: Tissue Culture Methods for Plant Pathologist. Ingram D. S.
between Suillus grevillei (Klotzsch) Sing. and 11 species of and J. P. Helgeson (eds.). Blackwell Scientific Publications,
ectomycorrhizal host in vitro in the absence of exogenous London, pp: 173-178.
carbohidrate. New Phytol. 103: 457-464. Molina, R., and J. G. Palmer. 1982. Isolation, maintenance, and pure
Duddridge, J. A. 1896b. The development and ultrastructure of culture manipulaton of ectomycorrhizal fungi. In: Methods and
ectomycorrhizas. IV. Compatible and incompatible interactions Principles of Mycorrhizal Research. Schenk, N. C. (ed.) American
between Suillus grevillei (Klotzsch) Sing. and a number of Phytopathological Press, St. Paul. pp: 115-129.
ectomycorrhizal hosts in vitro in the presence of exogenous Montgomery, D. C. 1984. Design and Analysis of Experiments. John
carbohydrate. New Phytol. 103: 465-471. Wiley & Sons, New York. 649 p.
Glover, T., and K. Mitchell. 2002. An Introduction to Biostatistics. Moser, M., 1960. Die Gattung Phlegmacium. Die Pilze Mitteleuporas
Mc Graw Hill, New York. 416 p. 4. J. Bad Heilbrunn. 440 p.
Godbout, C., and J. A. Fortin. 1992. Effects of nitrogen fertilization Mueller, G. M. 1992. Systematics of Laccaria (Agaricales) in the con-
and photoperiod of basidiome formation of Laccaria bicolor tinental United States and Canada, with discussions on extralimital
associated with container-grown Jack Pine seedlings. Can. J. Bot. taxa and descriptions of extant types. Fieldiana Bot NS 30: 1-58.
70: 181-185. Oort, A. J. P. 1981. Nutritional requirements of Lactarius species, and
Hung, L. L., and R. Molina. 1986. Use of ectomycorrhizal fungus cultural characters in relation to taxonomy. North-Holland, New
Laccaria laccata in forestry. III. Effects of commercially produced York. 95 p.
584 AGROCIENCIA VOLUMEN 37, NMERO 6, NOVIEMBRE-DICIEMBRE 2003

Santiago-Martnez G., L. Varela, A. Estrada-Torres, y V. Cuaxilo. 1995. Wong, K. K. Y., Y. Pich, D. Montpetit, and B. R. Kropp. 1989.
Efecto de seis medios de cultivo sobre el crecimiento de tres ce- Differences in the colonization of Pinus banksiana roots by sib-
pas de Pisolithus tinctorius. Rev. Mex. Mic. 11: 57-68. monokaryotic and dikaryotic strains of ectomycorrhizal Laccaria
Trappe, J. M. 1967. Pure culture synthesis of Douglas-fir mycorrhizae bicolor. Can. J. Bot. 67: 1717-1726.
with species of Hebeloma, Suillus, Rhizopogon and Astraeus. For.
Sci. 13: 121-130.