diversidad y funciÓn de hongos micorrÍzicos …
TRANSCRIPT
DIVERSIDAD Y FUNCIÓN DE HONGOS MICORRÍZICOS ARBUSCULARES ASOCIADOS A CUATRO MAÍCES CRIOLLOS Y UN MEJORADO EN MILPAS POPOLUCAS
TESIS QUE PRESENTA LA M. EN C. WENDY SANGABRIEL CONDE
PARA OBTENER EL GRADO DE DOCTOR EN CIENCIAS
Xalapa, Veracruz, México 2014
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Agradecimientos
Al Consejo Nacional de Ciencia y Tecnología (CONACYT), por la beca otorgada durante el
periodo Septiembre de 2008 - Julio de 2013, para realizar mis estudios de doctorado.
Al instituto de Ecología A.C. por admitirme dentro de su programa de Doctorado en Ciencias y
otorgar el apoyo económico para realizar estancias de investigación.
A los proyectos BioPop (FOMIX 94427, CONACYT-Veracruz) y ECOS-ANUIES M08A01 por
el soporte económico y logístico para realizar la investigación.
Al personal del Laboratorio de Organismos Benéficos de la Facultad de Ciencias Agrícolas,
Universidad Veracruzana, por las facilidades otorgadas para realizar el experimento.
Al personal del Laboratorio de Ecología Molecular de la Rizósfera del Instituto Politécnico
Nacional-CIIDIR-Sinaloa, por el apoyo y asesoría durante el proceso de análisis molecular.
Al Dra. Simoneta Negrete Yankelevich por la dirección y asesoría constante durante mi proceso
de formación doctoral, gracias por el apoyo y la paciencia, y a los miembros de mi comité tutorial
Dra. Luciana Porter Bolland y Dr. Eduardo E. Maldonado Mendoza, por su continua
participación, enseñanzas y consejos para llevar a buen término mi doctorado.
A la Dra. Dora Trejo Aguilar por apoyarme durante mi formación doctoral y a lo largo de mi
carrera, muchas gracias.
A la M. en C. Lourdes Cruz por su apoyo en el análisis de las muestras de suelo, y por estar
siempre pendiente de mi.
A mis compañeras Leonor, Beatriz e Isis, gracias por su amistad y por su apoyo incondicional.
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Dedicatoria
Con amor a mis padres Julián y Eloisa por creer en mi y apoyarme incondicionalmente.
Al solecito que ilumina mi vida, mi hijo, “Toto” te quiero mucho, pero mucho, mucho!.
A mi esposo Antonio, con amor, por estar a mi lado en todo momento y apoyarme siempre.
A mis hermanos Julian, Edith y Leticia.
A mis suegros Gaudencio y Miriam, por todas sus porras y cariño, gracias!
A toda mi extensa familia, con todo mi agradecimiento.
A Dios.
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ÍNDICE
LISTA DE TABLAS……………………………………………………………………… 8
LISTA DE FIGURAS…………………………………………………………………….. 9
Resumen …………………………………………………………………………………. 10
I. Introducción general……………………………………………………………………. 11
Introducción ……………………………………………………………............... 12
Milpa y agrodiversidad ………………………………………………………....... 12
Simbiosis micorrízica: función e identidad …………………………………......... 15
Dependencia micorrízica ……………………………………………………......... 16
Diversidad de hongos micorrízicos arbusculares (HMA)……………………........ 16
Antecedentes…………………………………………………………………........ 17
Objetivo, planteamiento de hipótesis y estructura del documento ……………….. 21
Literatura citada………………………………………………………………........ 23
II. Native maize landraces from Los Tuxtlas, Mexico show varying dependency on mycorrhiza for P uptake…………………………………………………………………... 29
Abstract…………………………………………………………………………… 30
Introduction……………………………………………………………………..… 30
Materials and methods…………………………………………………………….. 32
Study sites and soil collection………………………………………………... 32
Establishment of the greenhouse experiment………………………………… 32
Acquisition of P and percentage of mycorrhizal colonization……………….. 32
Increases in root volume and mycorrhizal dependency……………………… 32
Data analysis…………………………………………………………………. 33
Results…………………………………………………………………………….. 33
Acquisition of P and percentage of mycorrhizal colonization……………….. 33
Increases in root volume, dry biomass and mycorrhizal dependency………... 33
Discussion………………………………………………………………………… 33
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ÍNDICE
Acknowledgements…………………………………………………………….…. 37
References………………………………………………………………………… 37
III. Glomeromycota associated with Mexican native maize landraces in Los Tuxtlas, Mexico……………………………………………………………………………………
40
Abstract…………………………………………………………………………… 41
Introduction……………………………………………………………………...... 42
Materials and methods…………………………………………………………...... 45
Extraction of AMF spores from soil samples………………………………… 45
Greenhouse experiments……………………………………………………... 45
Root samples…………………………………………………………………. 46
Molecular diversity analysis………………………………………………….. 46
PCR cloning………………………………………………………………….. 48
Sequencing analysis and comparison of sequences………………………….. 48
Results…………………………………………………………………………….. 49
Discussion………………………………………………………………………... 51
Acknowledgements…………………………………………………………….…. 54
References………………………………………………………………………… 61
IV. Discusión General y Conclusiones…………………………………………………... 69
Simbiosis micorrízica: ¿alternativa para la agricultura de subsistencia en las milpas?......................................................................................................................
70
La milpa ¿Reservorio de comunidades de HMA funcionalmente diversas?.......... 71
Perspectivas para futuras investigaciones………………………………………... 73
Conclusiones…..………………………………………………………………….. 79
Literatura citada …………………………………………………………………... 80
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LISTA DE TABLAS
I. Introducción general
Cuadro 1. Características del manejo actual del sistema de milpa de Ocotal Chico y
Mazumiapan, Veracruz, México………………………………………..…..……..
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II. Native maize landraces from Los Tuxtlas, Mexico show varying dependency on
mycorrhiza for P uptake
Table 1. ANOVA analysis of plants hoot P concentration, root P concentration, total
P content, mycorrhiza colonization in the root, increase in root volume, total dry
biomass and mycorrhizal dependency explained by the input level of P, type of
maize, inoculation with AMF and mycorrhizal colonization .................................. 34
Table 2. Increases in root volume in mycorrhizal plants (compared to non-
mycorrhizal plants), total dry biomass and total P content in mycorrhizal plants
and mycorrhizal dependency (in terms of total dry biomass) of the different
maize types............................................................................................................... 35
III. Glomeromycota associated with Mexican native maize landraces in Los Tuxtlas,
Mexico
Table 1. Abundance of AMF sequences recovered from maize roots. Classified
according to taxonomic affiliation, number assigned to OTU, maize type and
phosphate treatment. B = Black, Y = Yellow, R = Red, W = White, T = Texcoco
hybrid maize types. Numbers in subscript denote maize fertilization treatments at
5 and 65 mg kg-1 Pi…………………………………………………………........... 58
Table 2. Number of OTUs associated with the roots of different maize types and
under two phosphorus levels………………………………………………..…….. 60
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LISTA DE FIGURAS
II. Native maize landraces from Los Tuxtlas, Mexico show varying dependency on
mycorrhiza for P uptake
Figure 1. Relationship between root colonization percentage and P concentration in: a
shoot and b root in different maize landraces or Texcoco hybrid associated to
AMF. Empty symbols represent low P input of 5 mg kg−1 and filled symbols
represent medium P input of 65 mg kg−1. Lines represent the linear models of
the shoot and root P concentrations (for statistical details, see Table 1)………... 35
III. Glomeromycota associated with Mexican native maize landraces in Los Tuxtlas,
Mexico
Figure 1. Change in mycorrhizal OTU richness with the cumulative number of
sequences (sampling effort in all maize types), obtained through rarefaction
analysis where percentage indicates the homology level at which sequences
were compared………………………………………………………………..…. 55
Figure 2. Phylogenetic tree displaying the relationship of AMF sequences recovered
from maize roots in the present study (bold type) and 21 reference sequences.
Bootstrap values are shown on the lines. Reference sequences are labeled with
the name provided by the database entry and the corresponding accession
number. Phylogenetic group and subgroup assignments are shown on the right
side of the tree (the total number of sequences belonging to each specific OTU
is given in Table 1)…………………………………………………………......... 56
Figure 3. Percentage of AMF sequences pertaining to species in the different
experimental treatments. Total number of sequences is shown above each bar.
B = Black, Y = Yellow, R = Red, W = White, T = Texcoco hybrid maize types.
Numbers in subscript denote maize fertilization treatments at 5 and 65 mg kg-1
Pi............................................................................................................................ 57
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RESUMEN
En la naturaleza, la mayoría de las plantas captan los nutrientes por medio de interacciones
simbióticas establecidas con microorganismos del suelo que viven en la rizósfera. La simbiosis
micorrízica es la asociación más frecuente en los ecosistemas naturales y agrícolas. Para los
sistemas agrícolas, los hongos micorrízicos arbusculares (HMA) (Glomeromycota) son los más
importantes porque colonizan la mayoría de los cultivos y favorecen la captación de nutrientes
poco disponibles como el fósforo. Mediante un experimento en invernadero en esta tesis se
evaluó la respuesta de variedades de maíz (cuatro criollos popolucas y un híbrido) a la presencia
de HMA nativos en condiciones de fósforo bajo y medio (5 y 65 mg·kg-1) y se determinó la
diversidad genética de HMA asociada a las raíces de cada variedad de maíz. Los resultados
mostraron que los HMA nativos de policultivos tradicionales como la milpa son capaces de
establecer eficientemente la simbiosis micorrízica y promover un incremento en la incorporación
de fósforo a la planta. Sin embargo se observó que existe una respuesta distinta a la micorrización
entre maíces criollos y el híbrido, y que este último tiene una limitada capacidad para adquirir
fósforo aún en simbiosis con HMA. Además fue posible determinar que la identidad y la
abundancia de Unidades taxonómicas operacionales (OTUs) de HMA asociados con cada
variedad de maíz y nivel de fósforo no fueron los mismos y que la variedad más eficiente en
términos de adquisición de fósforo también fue colonizada por el mayor número de OTUs,
independientemente del tratamiento. Los resultados obtenidos mostraron una respuesta
diferencial a la micorrización entre las cuatro variedades criollas de maíz y con el híbrido,
indicando que el genotipo de maíz puede tener una fuerte influencia sobre la funcionalidad y la
estructura de la comunidad de HMA que coloniza sus raíces y que la reducción de la diversidad
de maíces criollos en las milpas popolucas podría disminuir la diversidad de las comunidades de
HMA nativos y los beneficios que dichos simbiontes proporcionan a los cultivos.
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CAPÍTULO I
INTRODUCCIÓN GENERAL
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Introducción
La presencia de microorganismos benéficos en el suelo juega un papel importante para la
sostenibilidad de los ecosistemas naturales y agrícolas (Altieri y Nicholls 2005). La gran mayoría
de las plantas captan los nutrientes por medio de interacciones establecidas con microorganismos
del suelo que viven en la rizósfera, especialmente con aquellos denominados simbiontes. Los
hongos micorrízicos arbusculares (HMA) son microorganismos simbiontes casi tan ubicuos en el
suelo como las raíces de las plantas con las que se asocian (Rosendahl 2008). Los HMA son
objeto de una creciente atención por considerarse componentes esenciales para potenciar la
sostenibilidad de los sistemas agrícolas (Jeffries et al. 2003; Van der Heijden et al. 2006).
Aunque están comprobados los efectos benéficos que dichos micosimbiontes les proporcionan,
las plantas pueden presentar diferencias en su respuesta a la micorrización en función de la
identidad de ambos simbiontes (Klironomos 2003; Zhu et al. 2003).
La intensificación agrícola que se ha producido a lo largo de las últimas décadas ha ocasionado
graves consecuencias para el medio ambiente, entre las que destaca, la pérdida de biodiversidad
(arriba y bajo el suelo) (Thrupp 2004). Los sistemas agrícolas que resultan de esta intensificación
se caracterizan por una disminución en el número de variedades locales cultivadas, uso de
variedades mejoradas y una elevada dependencia de insumos externos como fertilizantes y
herbicidas (Frow et al. 2009; von Braun 2009). Poco se ha explorado sobre qué consecuencias
tiene la reducción en la riqueza arriba del suelo y el simultáneo uso de fertilizantes fosfatados
para la integridad de la simbiosis de los cultivos con los hongos micorrízicos arbusculares
(Glomeromycota) (Verbruggen y Kiers 2010). Esta tesis se centra en evaluar la respuesta de
cuatro variedades de maíces criollos popolucas y un híbrido en cuanto a la micorrización con
HMA nativos y en condiciones distintas de disponibilidad de fósforo.
Milpa y agrodiversidad
Mesoamérica es uno de los principales centros de domesticación de plantas que condujo al
desarrollo de la agricultura. Al menos una séptima parte de las principales especies de plantas
utilizadas como alimento tuvieron origen en esta región (Harlan 1971). De acuerdo con Kirchhoff
(1943), Mesoamérica se extiende desde el sureste de México hasta la región del Valle central en
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Costa Rica, caracterizada por una multiculturalidad en donde sus habitantes coexistieron y
compartieron creencias religiosas, arte y tecnología durante miles de años (MacNeish 1964). En
esta región, especies como el maíz (Zea mays L.), frijol (Phaseolus spp.) y calabaza (Cucurbita
spp.) fueron domesticados e integrados en un sistema de policultivo conocido como milpa. La
milpa se convirtió en un sistema clave para la alimentación y el desarrollo de sociedades
altamente complejas como los pueblos mayas, olmecas, aztecas y zapotecas, entre otros (Gepts
2004).
En México, la milpa mesoamericana como sistema productivo sigue vigente, y está conformada
por una compleja combinación de asociaciones y rotación de cultivos, manejados bajo el sistema
denominado roza-tumba y quema (Emerson 1953; Parsons et al. 2009). Anualmente se cultivan
más de seis millones de hectáreas de milpa en México, en la mayoría de los casos para auto-
subsistencia, pues las familias dependen de la producción de la milpa para su alimentación (Nadal
2000). Uno de los principales productos que se obtienen de la milpa es el maíz, con el que se
hace la tortilla, la cual prevalece como alimento básico que acompaña diariamente las comidas de
los mexicanos (Perales et al. 2005). Particularmente las tortillas hechas con maíz criollo aportan
beneficios extras para la salud, pues comparadas con tortillas de variedades mejoradas presentan
mayor contenido de fenoles totales y antocianinas, antioxidantes relacionados con efectos anti-
carcinogénicos (Del Pozo-Insfran et al. 2006; Aguayo-Rojas et al. 2012).
Además de su importancia nacional, el sistema de milpa mexicano tiene un valor de importancia
mundial, ya que dicho sistema constituye un reservorio único de germoplasma para la humanidad
(Bellon y Berthaud 2004; Van Dusen y Taylor 2005). Es agrodiverso intra e interespecíficamente,
a la fecha se han documentado 59 variedades criollas de maíz (Vielle-Calzada y Padilla 2009) y
se han registrado 32 cultivos distribuidos en la milpa (Foster y Hallowell 1942; Terán y
Rasmussen 1994; Blanco 1999, 2006). La agrodiversidad es considerada esencial para la
estabilidad de cualquier agroecosistema (Altieri y Nicholls 2005; Gliessman et al. 2007). Algunos
estudios reportan que la agrodiversidad de milpa es importante alimenticia y ecológicamente,
pues las diversas especies cultivadas proporcionan una cubierta protectora que mantiene la
humedad, previene la erosión e incrementa la fertilidad y contenido de nutrientes en el suelo
(Makinde et al. 2006). Además, se sabe que las variedades criollas de maíz ofrecen una mayor
capacidad de regulación y control de plagas y enfermedades que los híbridos comerciales
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mediante complejos mecanismos de defensa basados en la producción de toxinas y compuestos
volátiles conocidos como HIPVs (herbivore-induced plant volatiles, en inglés;Tamiru et al.
2011). Por ello, el sistema de la milpa es uno de los más importantes patrimonios culturales,
agroecológicos y tecnológicos heredado de los pueblos mesoamericanos.
La milpa es particularmente importante en las regiones de México donde hay población indígena.
Este es el caso de la población Nahua y Popoluca que habita la Sierra de Santa Marta en
Veracruz. Esta zona del trópico mexicano, es una de las áreas más ricas en biodiversidad, y está
catalogada como área natural protegida (Guevara et al. 2004). Habitada en su mayoría por
población indígena, esta región es representativa de cómo el conocimiento tradicional empírico se
ha preservado a lo largo del tiempo en el complejo sistema de la milpa (Blanco 2006). En las
milpas popolucas aún es posible encontrar una gran diversidad intraespecífica (hasta 15
variedades criollas de maíz, 10 de frijol y tres de calabaza), resultado de muchos años de
selección, adaptación y domesticación a las condiciones de suelo, clima y ecología de la región
(Blanco 2006; Negrete-Yankelevich et al. 2013a). Sin embargo, en los últimos 15 años este
sistema ha sufrido cambios en la forma de manejo, trayendo como consecuencia una disminución
que podría ser de más del 60% de las especies y variedades cultivadas, entre ellas las variedades
de maíz criollo (Negrete-Yankelevich et al. 2013b). Investigaciones recientes indican que aunque
todavía existen en la región elementos de la milpa tradicional, ha habido una tendencia a la
reducción en las escalas espaciales, temporales y de diversidad con que se toman las decisiones
de manejo, y con ello se han reducido los tamaños de las unidades productivas, los periodos de
descanso de la milpa (uno a dos años en vez de cinco o siete) y la diversidad de cultivos, con una
clara tendencia al monocultivo (Negrete-Yankelevich et al. 2013b).
Numerosas investigaciones han sido desarrolladas en torno a las milpas popolucas, la mayoría de
ellas enfocados a diagnosticar los patrones culturales y las causas socio-políticas de la
trasformación del paisaje y uso de agrodiversidad. En dichas investigaciones se reporta la
importancia de la milpa para los agricultores (Blanco 1999; 2006), la dinámica de su economía
campesina de autosubsistencia (Paré et al. 1997), el apego a sus ritos y tecnología (Blanco 1999;
2006), las transformaciones que ha sufrido en relación a la reducción de tiempos de descanso y
variedades de maíces criollos (Durand y Lazos 2004; Blanco 2006; Zurita-Benavides et al. 2012)
y el efecto que tiene el uso del suelo sobre las comunidades microbianas (Varela et al. 2009). No
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obstante, poco se conoce sobre las consecuencias que han tenido la reducción de la
agrodiversidad (intra e interespecífica) y la incorporación de fertilizantes fosfatados sobre las
asociaciones simbióticas planta-microorganismo (Negrete-Yankelevich et al. 2013; Sangabriel-
Conde et al. 2014). Tomando en cuenta que la asociación micorrízica permite a los cultivos
desarrollarse exitosamente en suelos de baja fertilidad, como los de las milpas popolucas, es
necesario analizar cómo los cambios en el manejo han afectado el desarrollo de la simbiosis
micorrízica.
Simbiosis micorrízica: función e identidad
Dentro de las interacciones simbióticas benéficas planta-microorganismo, destacan por su gran
relevancia ecológica y su amplia distribución los hongos micorrízicos, que colonizan las raíces de
las plantas y establecen una simbiosis mutualista denominada micorriza (del griego mikos,
hongo, y rhiza, raiz). Aproximadamente el 90% de las especies vegetales forman simbiosis
micorrízica, las cuales se han diferenciado los siguientes siete tipos: ectomicorrizas,
endomicorrizas o micorrizas arbusculares, ectendomicorrizas, arbutoides, monotropoides,
ericoides y orquidioides (Brundrett 2004). Estos difieren en aspectos estructurales, funcionales y
taxonómicos. Con base en las estructuras formadas, tipo de colonización y cantidad de especies
fúngicas y vegetales implicadas, se puede decir que las micorrizas arbusculares son las más
extendidas y las que presentan mayor importancia desde el punto de vista de beneficios para la
planta (Smith y Read 2008). Los hongos que conforman este tipo de asociación pertenecen al
phylum Glomeromycota y se les conoce como hongos micorrízico-arbusculares (HMA)
(SchüBler et al. 2001).
Con el respaldo de evidencias fósiles y moleculares que datan su origen en hace más de 450
millones de años (Remy et al. 1994), la asociación planta-HMA constituye probablemente la
simbiosis más antigua y más ampliamente distribuida en la naturaleza (Smith y Smith 2011). Se
asume que esta asociación no presenta especificidad, sin embargo existe evidencia de diferencias
en la compatibilidad funcional dependiendo de la identidad de ambos simbiontes (van der
Heijden et al. 1998; Klironomos et al. 2000; Vandenkoornhuyse et al. 2002). Se sabe que géneros
de HMA asociados a una misma especie de hospedero presentan diferencias en relación al nivel y
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capacidad de esporulación (Bever 2002), habilidad para colonizar las raíces (Klironomos y Hart
2002) y para adquirir fósforo (Smith et al. 2000; Hao et al. 2008), y que a su vez las variedades
de una misma planta pueden presentar diferentes grados de respuesta en el porcentaje
colonización, adquisición de fósforo y dependencia micorrízica (Zhu et al. 2001; Zhu et al. 2003).
Dependencia micorrízica
La dependencia micorrízica se ha definido como el grado en el cual la planta es dependiente de la
condición micorrízica para alcanzar su máximo crecimiento para un nivel dado de fertilidad en el
suelo (Menge et al. 1978; Plenchette et al. 1983). La dependencia micorrízica puede estar
determinada por el genotipo de la planta y la variedad o híbrido a que pertenece (Zhu et al. 2001;
Hess et al. 2005).
En suelos con limitado contenido de fósforo las plantas pueden depender completamente de la
asociación micorrízica para adquirir dicho nutriente (Smith et al. 2003), pero cuando el contenido
de P disponible se incrementa, las diferencias en desarrollo entre plantas micorrizadas y aquellas
que no lo están son menores (Smith y Read 2008).
Se cree que los beneficios de la simbiosis micorrízica son mayores cuando tanto la planta, como
el hongo han evolucionado y se seleccionaron en conjunto y en las mismas condiciones locales
(Johnson et al. 2010). En el caso del maíz, la mayoría de los híbridos se han desarrollado para
obtener máximos rendimientos en sistemas con elevado uso de agroquímicos y sin la presencia de
microorganismos locales del suelo, mientras que los maíces criollos son producto de un largo
proceso de domesticación local (Sanchez et al. 2000), motivo por el cual se esperaría que los
maíces criollos hayan desarrollado mayor dependencia micorrízica en comparación con
variedades introducidas (Johnson et al. 2010; Martinez y Johnson 2010).
Diversidad de hongos micorrízicos arbusculares (HMA)
Más de 200 especies de hongos micorrízicos arbusculares han sido descritas por la morfología de
sus esporas (INVAM 2014), sin embargo, es muy probable que existan mucho mas especies dado
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que es bien sabido que una alta proporción de los HMA solo pueden reproducirse
vegetativamente, sin producción de esporas. Además, existe una gran variación en la morfología
de esporas incluso dentro de la misma especie de HMA, y algunos linajes recientemente
caracterizados no se distinguen con procedimientos estándar como el aislamiento en cultivos o la
identificación mediante caracteres morfológicos (Lee et al. 2008). En las últimas décadas, esta
limitante se ha reducido en gran medida mediante el uso de las técnicas moleculares como es el
caso de la reacción en cadena de la polimerasa (PCR) (Redecker et al. 2000; Lee et al. 2008).
Desde principios de la década pasada, los HMA se han investigado utilizando el DNA a través de
PCR, inicialmente para determinar su filogenia y taxonomía, pero recientemente son de gran
utilidad para identificar y monitorear la presencia de dichas especies asociadas a las raíces de las
plantas y en el suelo (Vandenkoornhuyse et al. 2003; Wright et al. 2005).
Antecedentes
Manejo actual de las milpas popolucas. Como primer paso para el desarrollo de esta tesis, se hizo
un diagnóstico del manejo actual que los productores popolucas hacen en sus milpas. En este
análisis se realizó una entrevista semi-estructurada a 76 productores que habitan los ejidos de
Ocotal Chico y Mazumiapan. Las entrevistas se aplicaron directamente en la comunidad, en las
casas o bien en las parcelas de trabajo. Además, como se reporta en Negrete-Yankelevich et al.
(2013a) se realizó un muestreo de suelo para determinar el contenido de nutrientes y obtener el
inóculo de HMA nativos.
Los resultados de las entrevistas arrojaron que el sistema productivo de la milpa sigue presente en
la mayor parte de la superficie destinada a la agricultura de los ejidos de Ocotal Chico y
Mazumiapan de la Sierra de Santa Marta, es una práctica generalizada y juega un papel muy
importante en la economía familiar al aportar los ingresos de especies comestibles para
autoconsumo (Cuadro 1). Un aspecto importante y que da pie al desarrollo de esta tesis es la
percepción generalizada que tienen los productores popolucas (98 % de los entrevistados) de que
la fertilidad del suelo y los rendimientos de las milpas, principalmente del maíz, decrecen
conforme avanzan los años seguidos de cultivo.
Se sabe que la simbiosis micorrízica permite a los cultivos desarrollarse exitosamente en suelos
de baja fertilidad, como los de las milpas popolucas y que en el caso del maíz, puede aportar
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hasta el 60% del total del P requerido (en función del manejo agrícola), favoreciendo el
crecimiento y el incremento en la producción de biomasa (Nurlaeny et al. 1996). Partiendo de la
importancia del maíz como elemento principal en torno al cual se toman las decisiones del
establecimiento y manejo de la milpa y considerando los beneficios que los HMA pueden aportar
en la productividad de los sistemas agrícolas, se hace necesario determinar el efecto que puede
tener la reducción de la agrodiversidad intraespecífica del maíz criollo y el uso de variedades
mejoradas sobre la funcionalidad y diversidad de los HMA, y el concomitante efecto en la
productividad de dicho cultivo.
19
Cuadro 1. Características del manejo actual del sistema de milpa de Ocotal Chico y Mazumiapan, Veracruz, México.
Factor o actividad de manejo Productores que realizan la actividad (%) Observaciones
Persona que toma las decisiones del manejo de la milpa
86% hombres
14% mujeres
Las mujeres que toman decisiones son viudas, madres solteras o aquellas que son miembros de familias donde el padre está trabajando fuera de la comunidad en otra actividad que no es la agricultura.
Mano de obra empleada en la preparación del terreno
100% familiar En algunos casos también se utiliza mano de obra externa mediante pago de jornales, mismos que se pagan en efectivo o con trabajo recíproco.
Sistema utilizado en la preparación del terreno
100% Roza-tumba y media quema
La roza consiste en limpiar el terreno de arbustos y plantas no leñosas, y la tumba consiste en derribar los árboles que los productores destinan, por lo general, a la construcción de casas, corrales, cercos y leña. La quema se ha modificado y actualmente realizan lo que ellos llaman media quema, que ha sido recomendada por las autoridades con el fin de evitar la propagación de incendios, y que consiste en reducir el tiempo de quema y no dejar quemar en su totalidad los residuos vegetales.
Aplicación de herbicidas 86.8% si aplica
13.2 % no aplica
Aunque se realizan deshierbes con instrumentos manuales como machete y azadón, la aplicación de herbicidas se realiza generalmente 20 días antes de la siembra. Los productos aplicados son Glifosato, Paraquat y/o Picloram (Methyl parathion / Cypermethrina), con dosis que van de 1 a 2.5 l/ha
Diversidad de cultivos en la milpa
63% siembra menos de 3 cultivos
29% siembra entre 4 y 6 cultivos
8% siembra entre 7 y 8 cultivos
La reducción de la agrodiversidad en la milpa es notoria, pues tan sólo se registraron siete variedades de maíz criollo, siete de frijol y tres de calabaza, además de algunos cultivos como mango, plátano, caña de azúcar, chayote, rábano, cebollín y yuca. En contraste con lo encontrado por Blanco (1999; 2006) en la misma región, donde reportó 15 variedades de maíz, siete de frijol, cuatro de calabaza, y 15 cultivos sembrados simultáneamente
20
Variedades de maíz 65% siembran maíz criollo
35% siembran variedades mejoradas
El número máximo de variedades criollas de maíz simultáneamente sembradas en las milpas fue de hasta cuatro (17% de los productores), 29% de los productores siembra tres variedades, 32% siembra dos y 21% siembra únicamente una variedad
Variedades de frijol 86 % si siembran
13 % no siembra
Entre los productores que siembran frijol, el 8 % siembra cinco variedades en una milpa, el 4 % siembra cuatro variedades, un 2% siembra tres variedades, el 35% cultiva dos variedades y el 24% cultiva una variedad
Variedades de calabaza 71% si siembran
29% no siembra
De los productores que siembran, el 61% siembra una variedad, el 32% siembra dos variedades y el 7% una variedad
21
Objetivo, planteamiento de hipótesis y estructura del documento
En esta tesis se evaluó en invernadero la respuesta de distintas variedades de maíz (cuatro criollos
popolucas y un híbrido) a la presencia de HMA nativos en niveles bajos de fósforo (5 mg.kg-1)
como los encontrados naturalmente en la región y medios (65 mg.kg-1) como los obtenidos con
una fertilización moderada como la que es practicada recientemente por los agricultores locales,
además se determinó la diversidad de HMA asociados a las raíces de cada variedad de maíz.
Las hipótesis planteadas se abordan en dos trabajos de investigación incluidos en los capítulos II
y III; en el capitulo dos se analizan las siguientes hipótesis: (1) el porcentaje de colonización
micorrízica, el contenido de fósforo en raíz y parte aérea, la biomasa seca total y la dependencia
micorrízica serían similares entre maíces criollos popolucas y mayores que en la variedad híbrida;
(2) en condiciones de bajas concentraciones de fósforo, la simbiosis micorrízica se desarrollaría
mejor en los maíces criollos que en la variedad híbrida y (3) en condiciones de concentraciones
de fósforo medio, la colonización y dependencia micorrízicas disminuirían y el contenido de
nutrientes en biomasa aérea y de raíz se incrementaría en todas las variedades de maíz respecto a
lo encontrado en condiciones de bajo fósforo, sin embargo, esta diferencia sería mayor en los
maíces criollos que en la variedad híbrida.
En el tercer capítulo mediante técnicas moleculares se analiza la composición taxonómica de las
comunidades de HMA asociadas a los distintos tipos de maíz y se plantean las siguientes
hipótesis: (1) los diferentes tipos de maíz serán colonizados en sus raíces por comunidades de
HMA distintas, (2) las raíces de las variedades criollas serán colonizadas por la mayor riqueza de
HMA y el híbrido de Texcoco por la más baja, y (3) la fertilización moderada de P reducirá la
riqueza de HMA en todos los tipos de maíz, en respuesta a que la planta tiene una mayor
posibilidad para obtener P por la vía no micorrízica.
Finalmente, en el cuarto capítulo se discute cómo la integración de los hallazgos del segundo y
tercer capítulos sustentan la importancia del papel que juega la diversidad de cultivos para
sostener la integridad de la simbiosis micorrízica y el funcionamiento de estos agroecosistemas
ancestrales. En este capítulo, también se analizan las perspectivas, desde el punto de vista de la
simbiosis micorrízica, para el manejo exitoso de las milpas ante las presiones de productividad y
de transición de sistemas de autoconsumo a sistemas comerciales.
22
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29
CAPÍTULO II
NATIVE MAIZE LANDRACES FROM LOS TUXTLAS, MEXICO SHOW
VARYING DEPENDENCY ON MYCORRHIZA FOR P UPTAKE
Publicado en:
Biology and Fertility of Soils (2014) 50:405–414
SHORT COMMUNICATION
Native maize landraces from Los Tuxtlas, Mexico showvarying mycorrhizal dependency for P uptake
Wendy Sangabriel-Conde & Simoneta Negrete-Yankelevich &
Ignacio Eduardo Maldonado-Mendoza & Dora Trejo-Aguilar
Received: 7 May 2013 /Revised: 25 July 2013 /Accepted: 5 August 2013 /Published online: 22 August 2013# Springer-Verlag Berlin Heidelberg 2013
Abstract Different degrees of dependency on the activity ofarbuscular mycorrhizal fungi (AMF) exist between nativemaize landraces and hybrids. In Los Tuxtlas, Mexico, thePopoluca people maintain a traditional polycultural land man-agement with more than 15 native landraces of maize; how-ever, it is not known whether the recent substitution of localmaize for improved hybrids and fertilization has affected theintegrity of the mycorrhizal symbiosis in these naturallyphosphorus-poor systems. A greenhouse experiment wasconducted to evaluate the response of four Popoluca maizelandraces and the hybrid Texcoco to the presence of nativeAMF in conditions of low and medium P input (5 and65 mg kg−1, respectively). After 120 days in both P treat-ments, the native landraces Black and Yellow presented highercolonization and had acquired more P in their shoot biomassthan the hybrid. The moderate fertilization did not appear tohave affected the integrity of the mycorrhizal symbiosis, sinceall of the maize types presented a positive mycorrhizal depen-dency (2–14 %). Under low P conditions, the Texcoco hybridmaize presented one of the highest mycorrhizal dependencies;however, unlike the local landraces, this was not reflected in ahigher tissue P concentration. The results obtained indicatethat the nativemaize Blackwas the best at capturing symbioticand direct P, which makes this landrace an important geneticand cultural heritage for the Popoluca and for the world.
Keywords Arbuscular mycorrhizae . Phosphorusavailability .Traditionalpolyculturemanagement .Zeamays .
milpas
Introduction
Traditional polycultures around the world are being replacedby less diverse systems (with one or two crop plants ofagronomically or genetically improved varieties) that requireexternal agrochemical inputs (Thrupp 2004). Little is knownregarding the consequences of this reduction in above-groundrichness and concomitant use of phosphate fertilizers for theintegrity of the symbiosis between crops and arbuscular my-corrhizal fungi (AMF) (Glomeromycota) (SchüBler et al.2001; Martinez and Johnson 2010).
AMF have existed for more than 450 million years (Remyet al. 1994) and have been studied closely over the last decadelargely because of the benefits they provide in terms of plantnutrition (Smith and Smith 2011, 2012). The presence of AMFin agricultural systems where mycorrhizal dependency existsaugments crop productivity (Nwaga et al. 2004; Poomipan et al.2011), tolerance to drought (Boomsma and Vyn 2008) and todisease (Liu et al. 2007) and the capture and transfer of low-mobility nutrients, particularly P, to the plant (Yao et al. 2001).
A wide genetic and functional diversity of AMF speciesexists (Bever et al. 2001; Helgason and Fitter 2009), andarbuscular mycorrhizal fungal genera associated with thesame host species can present differences relating to leveland capacity of sporulation (Bever 2002), root colonizingability (Klironomos and Hart 2002) and P acquisition (Haoet al. 2008; Smith et al. 2000). Moreover, different varieties ofthe same plant can present differences in their response toAMF colonization. In wheat, colonization by Glomusintraradices varies between 16 % and 37 % among cultivars(Zhu et al. 2001) and in barley, two varieties inoculated withG. intraradices differed by more than 100 % in their acquisi-tion of Pi (Zhu et al. 2003).
W. Sangabriel-Conde : S. Negrete-Yankelevich (*)Red de Ecología Funcional, Instituto de Ecología A.C. (INECOL),Carretera Antigua a Coatepec 351, El Haya,91070 Xalapa, VER, Mexicoe-mail: [email protected]
I. E. Maldonado-MendozaDepartamento de Biotecnología Agrícola. CIIDIR-Unidad Sinaloa,Instituto Politécnico Nacional, Guasave, Veracruz, Méxicoe-mail: [email protected]
D. Trejo-AguilarLaboratorio de organismos benéficos. Facultad de CienciasAgrícolas, Universidad Veracruzana, Xalapa, Veracruz, México
Biol Fertil Soils (2014) 50:405–414DOI 10.1007/s00374-013-0847-x
Mexico is considered the center of origin for maize(Matsuoka et al. 2002) with a great diversity of native land-races (Sanchez et al. 2000). At present, the diversity of nativecultivated maize landraces is in decline due to the creation andintroduction of hybrids or improved varieties that displace thelocal landraces (Dyer and Taylor 2008). In the BiosphereReserve of Los Tuxtlas, the Popoluca farmers maintain tradi-tional polycultural land management (known as the milpasystem) that includes more than 15 local landraces of maize,associated mainly with crops such as common bean(Phaseolus vulgaris ) and pumpkin (Cucurbita pepo )(Negrete-Yankelevich et al. 2013). Little is known regardingthe consequences of this reduced agrodiversity (intra andinterspecific) and the incorporation of phosphate fertilizerson the plant–microorganism symbiotic associations in thesenaturally P-poor systems. In a previous study, we found thatmycorrhizal colonization levels and soil P concentration bothdecline with the loss of species cultivated in the milpa(Negrete-Yankelevich et al. 2013). In this study, we assessthe differences between four local maize landraces and theTexcoco hybrid (the most commonly planted hybrid in theregion) in terms of colonization, mycorrhizal dependency andthe incorporation of P in the root and shoot biomass. This wasconducted in conditions of low P input (5 mg kg−1), such asthose found naturally in the region, as well as medium P input(65 mg kg−1), such as that provided by the moderate fertiliza-tion recently practiced by the local farmers.
Phosphorus is one of the most important nutrients for crops(Raghothama 1999); but the majority requires P in concentra-tions (in maize >0.25 mg l−1) higher than those that exist in thesoil (0.02 to 0.1 mg l−1) for optimal development (Chesworth2008; Hue and Fox 2010). Plants can acquire P by (1) directabsorption , which occurs via transporters in the root epider-mis and (2) mycorrhizal absorption , which translocates Pfrom the extraradical hyphae to the root interior (Smith et al.2004). In the first case, P transport is regulated mainly by plantgenes of the Pht1 family (Chen et al. 2007; Gordon-Weekset al. 2003; Harrison and Van Buuren 1995); the transcriptionlevels of these genes frequently decrease with increased soil P.In certain plants, expression of these genes is inhibited bymycorrhizal symbiosis (reviewed in Javot et al. 2007). In thecase of mycorrhizal absorption, expression of the transportergenes of the fungus decreases when phosphate concentrationincreases around the extraradical hyphae and in the interior ofthe mycorrhiza (Maldonado-Mendoza et al. 2001).
In maize, mycorrhizal symbiosis provides up to 60% of thetotal requirement of P (Nurlaeny et al. 1996). However, land-races and hybrids differ in terms of colonization percentage(An et al. 2010) and P absorption capacity (Ortas and Akpinar2011; Tchameni et al. 2009). Mycorrhizal dependency hasbeen defined as the degree to which a plant is dependent uponthe mycorrhizal condition to reach its maximum growth for agiven level of soil fertility (Menge et al. 1978; Plenchette et al.
1983). In P-limited soils, plants may depend entirely upon themycorrhizal association to acquire this nutrient (Smith et al.2003) but when available P concentration increases in the soil,developmental differences between mycorrhizal and non-mycorrhizal plants tend to disappear (Smith and Read 2008).Mycorrhizal dependency can be determined by plant genotypeand the landrace or hybrid to which it belongs (Hess et al.2005; Zhu et al. 2001). In the case ofmaize, most hybrids havebeen developed to produce maximum yields in systems fea-turing an elevated use of agrochemicals (Evans 1980), whilelocal landraces are generally selected under conditions of lowor zero chemical inputs (Hess et al. 2005). It is thereforeexpected that local landraces have developed greater mycor-rhizal dependency, although this remains to be explored(Martinez and Johnson 2010). To date, most studies haveaddressed the response to AMF colonization in improvedvarieties and hybrids of maize, using individual strains ofAMF (Hao et al. 2008; Nurlaeny et al. 1996; Ortas andAkpinar 2011; Pitakdantham et al. 2007; Wright et al. 2005)and few studies have reported the effects of AMF colonizationon native landraces (Gavito and Varela 1995; Hess et al. 2005;Martinez and Johnson 2010) or of mixed inocula originatingfrom the same agroecosystems where these landraces weredeveloped (Pitakdantham et al. 2007). Moreover, little isknown regarding the difference in response of local maizelandraces and hybrids to mixed inoculation with native AMF(Martinez and Johnson 2010).
It is thought that the benefits of mycorrhizal symbiosis aregreater when both members (AMF and plants) evolved andwere selected under the local conditions (Johnson et al. 2010).Due to the fact that native maize landraces are the product of along process of local domestication (Sanchez et al. 2000) andthat the AMF have subsisted and evolved in concert with theplants in this process (Brundrett 2002), it is possible thatnative maize landraces could benefit more from the mycorrhi-zal association than introduced varieties that have been devel-oped under controlled conditions and without the presence oflocal soil microorganisms (Wissuwa et al. 2009). We thereforepose the following hypotheses: When growing maize in soilsfeaturing the native inocula of milpas , (1) the percentage ofmycorrhizal colonization (PMC), P concentration (root andshoot biomass) and total content per plant, total dry biomassand mycorrhizal dependency will be similar between nativePopoluca maize landraces and greater than in the Texcocohybrid; (2) in low P conditions, mycorrhizal colonization willdevelop to a greater extent in the native maize landraces thanin the hybrid, and (3) under medium P input conditions,colonization and mycorrhizal dependency will decline andthe nutrient concentration (root and shoot biomass) and totalcontent will increase in all the maize landraces and hybrid,compared to that found in low P conditions, with the differ-ence being greater in the native maize landraces than in thehybrid.
406 Biol Fertil Soils (2014) 50:405–414
Materials and methods
Study sites and soil collection
Soil was collected from nine Popolucamilpas in the ejidos ofOcotal Chico andMazumiapan of the Sierra de Santa Marta inLos Tuxtlas in Veracruz, Mexico. The selected milpas featuresimilar fallow periods of 1–2 years and have had at least 5consecutive years under cultivation and receiving an applica-tion of the herbicides glyphosate 45 % and Paraquat 25 % (1–2.5 l/ha) and N–P–K fertilization (18–12–06) or with urea(100–200 kg/ha) (see Negrete-Yankelevich, et al. 2013 formore detail). The milpa commonly features five maize types:the local maize landraces Yellow Pu´uch mok , White Poopmok , Black Luk mok , and Red Tsabats mok and the intro-duced hybrid Texcoco (S-8929, CIMMYT), the product of across between Tuxpeño, Cuban flints and ETO (Rice et al.1997). Although other hybrids are found in the region,Texcoco has been the most generally adopted by thePopoluca farmers. In each milpa , 49 soil samples were taken(distributed uniformly throughout the plot) with a soil corer ofdiameter 5 cm and depth 15 cm. All samples collected in thenine milpas were mixed to create a compound sample fromwhich to obtain the experimental inoculum. The soil was aLuvisol, with a clay–sand texture, strong to moderate acidity(pH 4.3 to 5.6) and a P concentration of 1 to 5 mg kg−1,estimated following the method of Bray and Kurtz (1945).
Establishment of the greenhouse experiment
AMF spores were extracted from the compound soil sampleusing the wet sieving and decanting technique (Sieverding1991). Material collected in a 53-μm mesh was dried onplastic trays in the greenhouse for 3 days at a temperature of28–30 °C and then used as an inoculum in the experiment(spore content was 365 per 50 g of inoculum).
A factorial design was used; featuring two P levels (5 and65 mg kg−1) with five replicates each, two mycorrhizal treat-ments (inoculated vs. non-inoculated) and five maize types(four Popoluca landraces: Yellow, White, Black and Red, andthe hybrid Texcoco). Seeds from each type of maize, belong-ing to the same plots from which the inoculum was extracted,were disinfected with a 10 % NaOCl solution for 15 min,rinsed with distilled water and germinated for 3 days in Petridishes with moist filter paper at 28 °C in a natural convectionoven.
Germinated seeds of each maize type with a radicle length1–2 cm were sown in 20 litre plastic containers, with washedand sterilized silica sand as a substrate. Two maize seeds weresown in each container and 150 g of inoculum (approx. 1,100spores) was applied. The containers were randomly organizedon the greenhouse tables. One week afterwards, thinning wascarried out leaving one plant per container. One hundred and
fifty ml of the corresponding dose of P (orthophosphate assoluble NaH2PO4·H2O) were applied weekly to each contain-er. Since the pH in the fertilized containers was close to 6.0,orthophosphate availability was favored. In addition, basenutrients were added each week by applying 200 ml of LongAshton solution, with pH adjusted to 6.0 and without P(Hewitt 1966). Irrigation was carried out using potable water.To sustain environmental conditions as similar as possible tothose in the sampling sites, the greenhouse experiment wasestablished in May and maintained with natural daylight, at25–35 °C and with 60–80 % relative humidity.
Acquisition of P and percentage of mycorrhizal colonization
After 120 days the plants were harvested and root volumemeasured (by water displacement), as well as total dry bio-mass, colonization percentage and P concentration in theshoot and root biomass. At this time all maize types were invegetative state and at a very similar stage of development.Plant shoot and root material was washed with distilled waterand rinsed with deionized water before drying for 72 h at70 °C. Plant P concentration was determined using the wetdigestion method (Thomas et al. 1967) and colorimetry withvanadate–molybdate (Murphy and Riley 1962). Plant P con-tent was calculated by multiplying the total P concentration ofthe plant and total plant biomass. All measurements werecarried out in duplicate.
Fresh root samples (from the thinnest root of each system)were washed and cut into fragments of length 2 cm. Onehundred root fragments for each sample were analyzed forthe presence of AMF structures. Prior to dyeing, roots werecleared with a 10 % KOH solution for 10 min at 120 °C,followed by acidification with 1 % HCl for 1 min at ambienttemperature. Roots were dyed with a 0.05 % trypan bluesolution for 5 min at 120 °C. Roots were then placed inlactoglycerol and examined under an optical microscope(100× magnification, Nikon PFX Optiphot-2) to verify thepresence of AMF structures, then under a stereoscopic micro-scope (60× magnification, Nikon SMZ645) to determine thePMC using the intersect method (Giovannetti and Mosse1980) and applying the formula: PMC=(total mycorrhizalroot intercepts/total root intercepts)×100. The presence ofany fungal structure (arbuscules, vesicles or hyphae) in theinterior of the root was considered to constitute colonization.
Increases in root volume and mycorrhizal dependency
Percentage increase in root volume (IRV) and mycorrhizaldependency were calculated; for root volume, the followingformula was used: IRV=[(RVM−RVN)/RVN]×100, whereRVM is the root volume in the mycorrhizal treatment andRVN is the root volume in the non-mycorrhizal treatment. Formycorrhizal dependency in total dry biomass (MD), the
Biol Fertil Soils (2014) 50:405–414 407
following formula was used: MD=[(BM−BNM)/BM]×100,where BM is the total dry biomass in the mycorrhizal treat-ment and BNM is the total dry biomass in the non-mycorrhizal treatment (Plenchette et al. 1983).
Data analysis
A linear model was used to evaluate the effect of P input level,maize type and mycorrhizal inoculation (and their second-order interactions) on the P concentration of the shoots androots, total P content per plant and the total dry biomass. Astepwise modeling procedure was conducted, featuring bothinclusion and elimination of variables in order to ensure theselection of the most explicative variables and avoid redun-dancy in the final model. A partial analysis of variance(ANOVA) was conducted to determine the significance ofthe contribution of each explicative variable, while thestrength of the evidence for the best model was estimated withthe R2 and the overall significance of the model with anomnibus ANOVA test. For the post-hoc comparisons, orthog-onal contrasts were conducted that enabled the reduction oftype I error (Crawley 2005). A similar modeling process wasused to evaluate the effect of P input level and maize type onmycorrhizal dependency, in terms of biomass and increases inroot volume. To fulfill the assumptions of homogeneity ofvariance and normality of residuals, P concentration valueswere transformed following the equation: y =(x (0.7)−1)/0.7;PMC and mycorrhizal dependency values were transformedby the arcsine of the square root. All analyses were conductedwith the statistical package R 2.1.1 (http://R-project.org/).
Results
Acquisition of P and percentage of mycorrhizal colonization
The native AMF colonized the roots of all the maize types butno colonization was found in the non-inoculated treatments.Non-inoculated plants acquired, on average, 30 % less P intheir shoots and 40% less P in the roots, than was found in theinoculated treatments (data not shown). The P concentrationsin the maize shoots and roots were significantly explained bythe two-way interactions of P input level with type of maize,inoculation and mycorrhizal colonization (Table 1). All maizetypes associated with AMF presented higher total P contentvalues than the non-inoculated plants (Table 1). Total P con-tents in all maize types were higher when P input was medium(Table 2). The Black landrace accumulated more total Pcontent in mycorrhizal plants (ca. 3.7–4.3 mg) than the othermaize types at both P input levels (Table 2).
In both the medium and low P input levels, the maizelandraces with the highest colonization percentages werethose that acquired more P in their shoots (Fig. 1a). At both
input levels of P, the native landraces Black and Yellow werecolonized to a higher extent (ca. 60–80 %) and acquired moreP in their shoots (ca. 4.5–6 mg kg−1) than the hybrid Texcoco(ca. 45 % colonization and ca. 4–4.5 mg kg−1 of P) (Fig. 1a).The Black landrace presented the highest percentages of col-onization (greater than 75 % at both P input levels). Only theWhite landrace presented a greater colonization percentagewhen P input was low (Fig. 1a). For the other landraces andhybrid, there were no differences in colonization percentagebetween P input levels. All of the maize types, except forYellow, presented higher P concentrations in the roots in themedium dose of P (Fig. 1b).
Increases in root volume, dry biomass and mycorrhizaldependency
The variance in root volume was significantly explained bythe interaction between P input level and maize type (Table 1).Only the Yellow and Black landraces showed a greater per-centage IRV when the P input level was low (Table 2). TheBlack landrace presented the highest IRVat both P input levels(Table 2).
All maize types associated with AMF presented highertotal dry biomass values than the non-inoculated plants(Table 1). No differences were observed between the non-inoculated treatments. Only the Black landrace accumulatedmore total dry biomass (ca. 440–460 g) in mycorrhizal plantsthan the other maize types at both P input levels (Table 2).
All maize types presented a positive AMF dependency,with means of between 2 % and 14 % (Table 2). Underconditions of low P, all of the landraces increased their depen-dency, except Red (Table 2). The hybrid Texcoco presentedthe greatest difference in dependency between the two P inputlevels. Its dependency was among the highest in the low Pdose and among the lowest in the medium P dose (Table 2).
Discussion
In this study, we expected that the parameters evaluated in themaize plants (PMC, P in the roots and shoots, total P content,total dry biomass and mycorrhizal dependency) would besimilar between the native maize landraces and greater in thehybrid. In contrast, we found that the native maize landracesvaried widely and, compared to the hybrid Texcoco, no nativelandrace presented greater mycorrhizal dependency (at least inthe low P input treatment). Only one of the native landraces(Black) presented a higher total dry biomass and total Pcontent, two (Yellow and Black) presented higher coloniza-tion percentages, and three incorporated more P in their rootand shoot biomass. These results are consistent with thedifferences in compatibility reported between AMF and hostplant, attributed to the genetic variation and ecological
408 Biol Fertil Soils (2014) 50:405–414
Tab
le1
ANOVAanalysisof
plantshootPconcentration,rootPconcentration,totalP
content,mycorrhizalcolonizatio
nintheroot,increaseinrootvolume,totaldry
biom
assandmycorrhizaldependency
explainedby
theinputlevelof
P,type
ofmaize,inoculatio
nwith
AMFandmycorrhizalcolonizatio
n
PHOS
TYPE
INO
COL
PHOSxC
OL
PHOS×TYPE
PHOS×IN
OTYPE
×IN
OOmnibustest
SS
FSS
FSS
FSS
FSS
FSS
FSS
FSS
FR2
F
Pconcentrationin
shootb
iomass
(mgkg
−1)
34.65
306.20***
39.60
87.48***
135.24
1,194.89***
8.38
74.06***
1.86
16.49***
3.57
7.89***
7.07
62.54***
3.49
7.72***
0.96
121.60***
Pconcentrationin
root
biom
ass
(mgkg
−1)
22.03
936.49***
2.07
22.05***
75.31
3,200.60***
0.00
0.01
(ns)
1.09
46.53***
0.73
7.80***
7.01
297.84***
1.66
17.68***
0.98
274.80***
TotalP
content(mg
plant−1)
10.70
354.66***
20.47
169.63***
27.05
896.50***
1.51
50.19***
0.20
0.94
(ns)
0.63
5.24***
1.63
54.29***
0.16
1.37
(ns)
0.96
121.3***
Mycorrhizal
colonizatio
nin
the
root
(%)
0.01
6.42*
0.69
141.89***
––
––
––
0.01
3.72*
––
––
0.93
65.43***
Increase
inroot
volume(%
)879.80
5.19*
24,547.10
36.26***
––
––
––
3,543.30
5.23**
––
––
0.81
19.02***
Totaldry
biom
ass(g)
20,598
45.36**
192,434
105.95**
16,121
35.50***
541
1.19
(ns)
2.00
0.01
(ns)
5,757
3.16*
789
1.73
(ns)
811
0.44
(ns)
0.86
30.71***
AMFdependency
(%)
203.07
34.12***
246.29
10.34***
––
––
––
139.08
5.84***
––
––
0.71
10.99***
PHOSPinputlevel,TYPEtype
ofmaize,INO
inoculationwith
AMF,
COLmycorrhizalcolonizatio
n,SS
sum
ofsquares,FANOVA
F-value,R
2coefficientof
determ
inationof
themodel,n
snon-
significant,–notincludedin
themodel
*P<0.05;*
*P<0.01;*
**P<0.001
Biol Fertil Soils (2014) 50:405–414 409
specificity of both symbionts (An et al. 2010; Liu et al. 2000).The variation in response to AMF colonization found in thisstudy could be due to the fact that the different maize types donot associate with the same consortium of AMF species(Maldonado-Mendoza et al., personal communication) or per-haps that they respond differentially when associated to AMF(Oliveira et al. 2009).
It is possible that the observed differences in root volume, Pconcentration and total P content are related to the inherentgenotypic characteristics of each maize type (Gaume et al.2001; Zhu et al. 2005); when are in a mycorrhizal symbiosis,some maize varieties produce more roots (Zhu et al. 2005),while others increase nutrient absorption levels (Ortas andAkpinar 2011). In our study, mycorrhizal inoculation signifi-cantly increased P concentrations and total P content, howev-er, it is known that AMF species differ widely in their Pacquisition strategies (Jansa et al. 2005; Munkvold et al.2004; Thonar et al. 2011) and that the maize genotypes canassociate with different mycobiont species (Oliveira et al.2009). For this reason, it is feasible that the landraces thataccumulated most P in shoot tissues (Black and Yellow) areassociated with more efficient AMF, in terms of the assimila-tion and translocation of the nutrient to the plant (Thonar et al.2011), and those that accumulated less P (White, Red and
Texcoco) are associated with more carbon-demandingmycobionts (Jakobsen et al. 2002) that provide the plant withlittle phosphate (Lendenmann et al. 2011). In this study, themaize landrace that presented the highest colonization per-centages also accumulated more P in their shoot tissues andtotal P content, suggesting that the degree of symbiosisestablished was proportional to the benefit for the plant. It ispossible that the phosphate transporter genes in these landracemay be regulated by the mycorrhizal colonization (Nagy et al.2006); however, given that no molecular studies were carriedout to determine the phosphate transporter genes expressed ineachmaize type, further research would be required in order tofully understand the mechanisms involved in the response toAMF colonization and the availability of P in native maizelandraces.
Maize is considered to be a plant with high mycorrhizaldependency, especially in low P conditions (Ortas 2012).Several studies show differences in the AMF dependencybetween maize genotypes (Hao et al. 2008; Kaeppler et al.2000; Ortas and Akpinar 2011; Tawaraya 2003; Wright et al.2005), but we also found differences between native maizelandraces and also between these and the hybrid Texcoco. Thelocal landraces (Yellow and Black) presented the greatestdevelopment in low P conditions (in biomass and/or root
Fig. 1 Relationship between rootcolonization percentage and Pconcentration in: a shoot and broot in different maize landracesor Texcoco hybrid associated toAMF. Empty symbols representlow P input of 5 mg kg−1 andfilled symbols represent mediumP input of 65 mg kg−1. Linesrepresent the linear models of theshoot and root P concentrations(for statistical details, see Table 1)
Table 2 Increases in root volume in mycorrhizal plants (compared to non-mycorrhizal plants), total dry biomass and total P content in mycorrhizalplants and mycorrhizal dependency (in terms of total dry biomass) of the different maize types
Maize type Increases in root volume (%) Total dry biomass ofmycorrhizal plants (g)
Total P content of mycorrhizalplants (mg plant−1)
Mycorrhizaldependency (%)
Low P Medium P Low P Medium P Low P Medium P Low P Medium P
Yellow landrace 29.8 (3.2) bA 14.8 (3.4) eB 339.2 (6.9) bA 354.2 (12.1) bA 2.6 (0.2) bB 2.9 (0.3) bA 12.5 (1.7) aA 7.8 (0.5) aB
White landrace 28.2 (6.2) cA 25.9 (4.3) cA 362.2 (6.5) bA 373.9 (8.2) bA 2.5 (0.1) bB 2.8 (0.2) bA 13.3 (0.6) aA 8.7 (1.9) aB
Black landrace 95.5 (11.7) aA 57.8 (6.5) aB 445.6 (12.4) aA 462.9 (8.5) aA 3.7 (0.3) aB 4.3 (0.2) aA 7.2 (1.5) bA 3.0 (0.8) cB
Red landrace 14.3 (4.5) eA 16.5 (2.8) dA 298.8 (24.8) bB 354.7 (13.2) bA 2.2 (0.4) dB 2.7 (0.2) bA 5.5 (1.4) cA 7.4 (0.8) aA
Hybrid Texcoco 18.1 (1.6) dA 28.8 (6.8) bA 337.5 (10.8) bA 354.7 (8.1) bA 2.4 (0.2) cB 2.7 (0.1) bA 12.8 (0.9) dA 4.3 (0.7) bB
Means presented with standard errors in parenthesis (n =5). Lowercase letters indicate significant differences between maize types and capital lettersbetween P levels
410 Biol Fertil Soils (2014) 50:405–414
volume), possibly as a result of the long selection process towhich they have been subjected over time. These landracesare the most common in the region and, although their selec-tion has been directed largely by cultural factors, it is probablethat they were selected in order to maximize their develop-ment and productivity under local conditions with nativeAMF species and low nutrient availability (Smith et al.2004; Tchameni et al. 2009; Yao et al. 2001).
The landrace Black in particular presented increases in rootvolume and mycorrhizal colonization percentages that werefar superior to the other maize types, suggesting the develop-ment of a strong adaptation to the local mycosymbionts andthe benefits of AMF colonization (Smith and Smith 2012).The colonization percentages presented by this landrace (75–80 %) are among the highest reported to date for native maize(Hess et al. 2005; Martinez and Johnson 2010). It is notablethat, although this landrace had elevated mycorrhizal coloni-zation, its AMF dependency was among the lowest because itis capable of acquiring P even in the absence of symbionts. Itis therefore possible that the landrace Black utilizes variousadaptation strategies (exudation of organic acids, phospha-tases, nucleases and modification of its root structure)(Bayuelo-Jiménez et al. 2011; Javot et al. 2007; Machadoand Furlani 2004; Zhu et al. 2005) that make it highly efficientin both the symbiotic and direct capture of P, depending on theprevailing conditions (Bucher 2007; Smith and Smith 2012;Smith et al. 2004). This characteristic makes this landracegenetically and culturally important to the Popoluca peopleand to the world.
Our second hypothesis suggested that, in low P conditions,mycorrhizal symbiosis would develop better in native maizelandraces than in the hybrid. However, instead we found thatthe hybrid presented greater AMF dependency than some ofthe native landraces and the attained colonization percentagesindicate that the improvement process did not affect the ca-pacity of the hybrid to respond to the AMF colonization (Anet al. 2010; Sawers et al. 2010). It was observed that AMFcolonization is very important for the hybrid when there islittle P, since under these conditions its dependency wasamong the highest. However, this was not reflected in itsdevelopment and its tissue P concentrations and total P con-tent were among the lowest. Possibly the AMF species thatcolonized the hybrid were inefficient in terms of providing Pto the plant (Lendenmann et al. 2011), but they stimulated agreater AMF dependency. Certain maize hybrids behave in-efficiently in terms of absorption of nutrients when associatedwith AMF (Ortas and Akpinar 2011). For example, G .etunicatum can extensively colonize maize roots but causesa negative response to mycorrhizal association, while G .mosseae , colonizes at a lower percentage but elicits a positiveresponse to AMF colonization (Hao et al. 2008; Ortas 2012).
Some studies report that increased P reduces the develop-ment of mycorrhizal symbiosis; nevertheless, the levels at
which this reduction occurs are variable and are a functionof both the identity of the AMF species and the genotype ofthe maize. For example, the percentage of root colonizationvaried among 12 maize inbred lines grown at low(17 mg kg−1) or high (87 mg kg−1) P, but was consistentlylower when the dose was higher (Kaeppler et al. 2000). In ourcase, P concentration in root and total P content in all maizetypes increased when the dose of P was 65 mg kg−1,suggesting that when P concentration was 5 mg kg−1 maizeplants were P limited. Contrary to that suggested in our thirdhypothesis, only one native landrace (White) presented areduced colonization percentage with increased P concentra-tion, indicating that the medium P levels obtained with amoderate fertilization, such as that practiced recently by thePopoluca farmers, do not affect the integrity of the mycorrhi-zal symbiosis. Fries et al. (1998) report that concentrationsabove 200 mg kg−1 of P are required for inhibition of mycor-rhizal symbiosis in maize. Notwithstanding, it is possible thatthe White maize landrace is associated with AMF species thatare sensitive to P availability, as has been reported forGigaspora rosea (Nogueira and Nogueira-Cardoso 2007). Itis known that AMF species present different sensitivities tothe concentration of available P.
In contrast to colonization, increased P soil concentrationreduced the AMF dependency in three native maize landrace(with the maize landrace Red being the exception) and thehybrid Texcoco. The hybrid showed a very notable decrease,which suggests that in conditions of greater P availability,mycorrhizal symbiosis does not foment increased biomass,but rather that the plant is capable of obtaining the nutrientsthrough its roots (Ortas 2012; Smith et al. 2004). The fact thatthe landraces accumulated more P in the medium input level isprobably due to greater activation of the phosphate transportergenes (Nagy et al. 2006) and, having accumulated more P inthe shoot biomass and not in the roots, is possibly due toeffective translocation to the aerial part of the plant (Daramet al. 1999).
It is important to highlight that our results represent a pointin time and that we do not know the variation in the responseof these maize types to inocula from different cultivationcycles or under different field conditions. Also, we onlyevaluated one maize hybrid and therefore it is not possible toextrapolate the differences with local landraces to other hy-brids. However, this study does show for the first time differ-ences in response to AMF colonization among local maizelandraces and with the most widespread hybrid in milpapolycultures, a traditional system at serious risk of disappear-ance. It is possible that these differences are caused by differ-ences in the AMF consortia that colonize each maize type, oreven, by the presence of other soil microorganisms associatedto AMF spores, on their surface (Hetrick et al. 1988) or livingas intracellular endosymbionts (Alonso et al. 2008; Minerdiet al. 2002). Currently, our research group is conducting a
Biol Fertil Soils (2014) 50:405–414 411
ribosomal DNA survey of the AMF communities in order todetermine which AMF species interact with each native maizelandrace and the hybrid. One of the most important results ofthis study is that, despite showing a high mycorrhizal depen-dency in low P conditions, the capacity of the improvedhybrid Texcoco to acquire this nutrient under these conditionsis very low. In contrast, the Black landrace appears to haveadaptive mechanisms to efficiently obtain P either directly orthrough mycorrhizal symbiosis. For this reason, we concludethat the substitution of local maize landraces for the Texcocoimproved hybrid constitutes a risk of losing a genetic heritageof great importance both above and below the soil, and onethat has been developed over thousands of years of selection.
Acknowledgments We thank Claude Plassard for invaluable sugges-tions during the development of this research; to Isis de la Rosa and OmarLázaro for their invaluable help in the field; to the farmers and authoritiesof Ocotal Chico and Mazumiapan for their active participation in thisstudy; to Liliana Lara and the staff of Laboratorio de OrganismosBenéficos of the Faculty of Agronomy of the Universidad Veracruzanafor provision of experimental facilities. This study was financed by theBioPop project (FOMIX 94427, CONACYT-Veracruz) and by the pro-ject ECOS-ANUIES M08A01. We dedicate this study to the memory ofJosé Luis Blanco Rosas, close friend and enthusiastic collaborator in thisproject.
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40
CAPÍTULO III
GLOMEROMYCOTA ASSOCIATED WITH MEXICAN NATIVE MAIZE
LANDRACES IN LOS TUXTLAS, MEXICO
Artículo enviado a
Applied Soil Ecology
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Glomeromycota associated with Mexican native maize landraces in Los Tuxtlas, Mexico
Wendy Sangabriel-Conde1*, Ignacio E. Maldonado-Mendoza2*, Maria Elena Mancera-López2, Jesús Damián Cordero-Ramírez2, Dora Trejo-Aguilar3, Simoneta Negrete-Yankelevich1** 1Red de Ecología Funcional. Instituto de Ecología A. C. Carretera Antigua a Coatepec No. 351, El Haya, Xalapa 91070 Veracruz, Mexico 2Instituto Politécnico Nacional (IPN). CIIDIR-Sinaloa. Departamento de Biotecnología Agrícola. Boulevard Juan de Dios Bátiz Paredes No. 250. CP 81101. AP280. Guasave, Sinaloa, México. 3Laboratorio de Organismos Benéficos. Facultad de Ciencias Agrícolas, Universidad Veracruzana, Xalapa, Veracruz, México. *Both are considered as first authors. **Correspondence author: S. Negrete-Yankelevich e-mail: [email protected]
Abstract
Many native maize landraces are still cultivated by different ethnic groups in Mexico as part of
traditional polyculture systems (milpas). However, these systems and landraces are being steadily
replaced by fertilized monocultures of genetically improved varieties or maize hybrids. Little is
known about how such changes may affect the belowground community of maize symbionts. A
greenhouse experiment was conducted to assess the composition of the indigenous arbuscular
mycorrhizal fungi (AMF) communities colonizing four Popoluca maize landraces and the hybrid
Texcoco, and to determine how these communities are influenced by the level of phosphorus
fertilization. Fragments of AMF ribosomal DNA (rDNA) were amplified using nested PCR,
producing 327 Glomeromycota sequences. Phylogenetic analysis assigned the Glomeromycota
sequences into 84 operational taxonomic units (OTUs). This number of OTUs was more than
double that reported for maize exposed to native inoculum. Exclusive and promiscuous
associations were rare. Most AMF species colonized 2 to 4 maize types. This suggests that the
high diversity of AMF in milpas is related to the presence of several native maize landraces and
that replacement of these landraces with a single hybrid may diminish AMF community richness.
All landraces were found to associated with more OTUs under moderate P fertilization,
suggesting that low inorganic phosphate (Pi) in milpas is limiting symbiotic interactions.
However, only 0-20 % of OTUs were conserved between different P conditions for each
landrace. The Black landrace, which presented the highest AMF colonization and Pi uptake, is
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associated with the highest number of AMF OTUs, supporting previous suggestions regarding
this functional relationship with AMF richness.
Key words: arbuscular mycorrhizal fungi, traditional polyculture management, Zea mays, milpas
Introduction
Maize (Zea mays L.) is grown worldwide as a staple food and to manufacture secondary products
such as sugar, oil, and protein (FAO, 2006). Mexico is considered the center of origin for maize
(Matsuoka et al., 2002) and many native landraces are still cultivated in that country by different
ethnic groups as part of traditional polycultures (milpas). These systems constitute important
genetic resources, not only in terms of different varieties of maize, but also of belowground
symbionts, which have co-evolved with plants in a variety of nutrient deficient and harsh
environmental conditions. However, these systems and landraces are being replaced by fertilized
monocultures of genetically improved varieties or maize hybrids (Turrent-Fernández et al., 2012)
and little is known about how such management changes may affect the belowground community
of mycorrhizal symbionts.
Arbuscular mycorrhizal fungi (AMF) are ubiquitous soil organisms of the phylum
Glomeromycota that establish mutualistic symbiotic associations with most land plants (Smith
and Read, 2008). AMF are most influential for plants in nutrient deficient soils, in particular
where P is limiting (Antunes et al., 2011). They facilitate mineral nutrient uptake from the soil in
exchange for plant assimilated C (Smith and Read, 2008). In addition to improving plant
nutrition, AMF protect their hosts from pathogens (Pozo and Azcón-Aguilar, 2007) and
contribute to the maintenance of variability, stability, diversity and productivity of the plant cover
and soil structure (van der Heijden et al., 2006), as well as affecting plant growth traits
(Klironomos, 2003).
Despite the considerable role of AMF in terrestrial ecosystems, the relationship between their
taxonomic and functional diversity is little understood (Krüger et al., 2009). While AMF are not
strictly host-specific, it is known that they can exhibit a degree of host-preference (Helgason and
Fitter, 2009; Johnson et al., 2003; Scheublin et al., 2004). Plants, on the other hand, may favor
43
specific subgroups of these mycosymbionts (McGonigle and Fitter, 1990; Oliveira et al., 2009).
In addition, it is known that the relative abundance of several Glomus species decreases
significantly in response to chemical fertilization, while other species, including Entrophospora
schenckii, Glomus mosseae, and Scutellospora fulgida, remain unaffected (Bhadalung et al.,
2005) and maintain their provision of ecosystem services and life history strategies (van der
Heijden et al., 1998b).
Around 240 AMF species have been described to date (Oehl et al., 2011), mainly on the basis of
spore morphology. However, it is impossible to correlate spore information with specific
colonization events in plant roots since spores are typically produced on external hyphae that can
easily become detached from the root, and the level of spore production does not reflect the
abundance of species in the roots (Clapp et al., 1995). Molecular techniques are currently the best
option for characterization of AMF diversity associated with the roots. The use of DNA
sequencing methods has improved our understanding of AMF communities, particularly because
they allow the direct detection of AMF from environmental samples (Öpik et al., 2009; Öpik et
al., 2013).
AMF fungi associated with maize grown under different management conditions have been
studied through morphological identification of spores (Daniell et al., 2001; Jansa et al., 2003)
and molecular sequencing (Oliveira et al., 2009; Sasvári et al., 2011). Most of the recent studies
based on molecular techniques have found that AMF diversity in maize roots is reduced by
agricultural practices such as tillage, fertilization and the introduction of phosphorous-inefficient
genotypes (Franke-Snyder et al., 2001; Jansa et al., 2002; Oehl et al., 2003; Oliveira et al., 2009;
Sasvári et al., 2011; Toljander et al., 2008), while positive correlations have been found with crop
rotation and maize productivity (Maherali and Klironomos, 2007; van der Heijden et al., 1998a;
Wagg et al., 2011). Maize varieties differ in terms of the diversity and composition of AMF
communities colonizing their roots (Oliveira et al., 2009).
Low P availability limits plant growth in many acid soils of the tropical mountains, such as those
where the Popoluca people manage their milpas in the Biosphere Reserve of Los Tuxtlas, Mexico
(Sangabriel-Conde et al., 2014). Despite this limitation, more than 18 local maize landraces have
been developed in this region and are still cultivated in very low input subsistence agricultural
systems (Negrete-Yankelevich et al., 2013b) Our research has previously demonstrated that the
44
loss of cultivated plant species in Popoluca milpas may have a negative impact on the ability of
the mycorrhizal community to colonize maize roots, as well as reducing the availability of P
(Negrete-Yankelevich et al., 2013a).
In maize, mycorrhizal symbiosis provides up to 60% of the total P requirement (Nurlaeny et al.,
1996). However, AMF species that colonize the roots of maize or soybean exhibit strong host
specificity (Gosling et al., 2013). Landraces and hybrids differ in terms of colonization
percentage (An et al., 2010) and P absorption capacity (Ortas and Akpinar, 2011; Tchameni et al.,
2009), and different AMF species exhibit functional diversity in terms of colonization, maize
growth, Pi uptake as well as Pi transporter gene expression (Hao et al., 2008; Klironomos and
Hart, 2002; Smith et al., 2000; Tian et al., 2013); nevertheless, increasing the diversity of AMF in
maize roots through co-inoculation leads to higher Pi uptake in shoots (Tian et al., 2013). It has
been suggested that if a plant is colonized by several species of AMF that are complementary in
their functions (e.g. the uptake of nutrients from different soil pools), they may together prove to
be more beneficial to the plant than colonization by any of these species individually (Alkan et
al., 2004; Gustafson and Casper, 2006; Koide, 2000). These findings suggest that different maize
landraces or hybrids exposed to a diverse native AMF inoculum may associate with specific
assemblages of AMF, which could lead to differences in plant Pi uptake.
In a previous study we evaluated the response of four Popoluca native maize landraces, and the
recently introduced hybrid Texcoco, to the presence of native AMF in conditions of low and
moderate P input (5 and 65 mg kg-1) (Sangabriel-Conde et al., 2014). The native landrace Black
presented colonization percentages (75–80 %) that were among the highest reported to date for
native maize (Hess et al., 2005; Martinez and Johnson, 2010), as well as the highest shoot P
uptake compared with the hybrid Texcoco and the Yellow, White and Red landraces. The hybrid
Texcoco, despite showing a high mycorrhizal dependency, exhibited a very low capability to
acquire P in low conditions of this nutrient. In contrast, the Black landrace appears to have
adaptive mechanisms by which it efficiently obtains P, either directly or through mycorrhizal
symbiosis (Sangabriel-Conde et al., 2014). We postulated that these differences in response to
AMF colonization among local maize landraces and the hybrid from the Popoluca milpas are
related to differences in the AMF consortia that colonize each maize type. Since AMF species
exhibit functional diversity, maize landraces that are capable of higher Pi uptake may be
45
interacting with a richer fungal community (Tian et al., 2013). In this study, we hypothesize that
(1) maize types will differ in terms of the AMF community colonizing their roots, (2) the roots of
the Black landrace will be colonized by the highest richness of AMF and the Texcoco hybrid by
the lowest, and (3) moderate P fertilization will reduce AMF richness in all the maize types,
given the higher possibility for the plant to obtain Pi through the non-mycorrhizal route. To the
best of our knowledge, no previous study has tested the effect of indigenous AMF communities
on several native maize landraces and under different conditions of P availability.
Materials and methods
Extraction of AMF spores from soil samples
Soil samples were taken from nine Popoluca milpas in the communities of Ocotal Chico and
Mazumiapan of the Sierra de Santa Marta in Los Tuxtlas, Veracruz, Mexico. AMF spores were
extracted from a compound soil sample using the wet sieving and decanting technique
(Sieverding, 1991). Briefly, material collected on a 53 µm sieve was dried on plastic trays in the
greenhouse for three days at 28-30ºC and subsequently used as an inoculum in a greenhouse
experiment (full sampling and extraction details described in Sangabriel-Conde et al. (2014).
Greenhouse experiments
A complete factorial experimental design was implemented with maize type, inoculation and P
fertilization as factors. The most common maize types in Popoluca milpas were used: the local
maize landraces Yellow (Pu´uch mok), White (Poop mok), Black (Luk mok), and Red (Tsabats
mok) and the introduced hybrid Texcoco (S-8929, CIMMYT). Two P levels were chosen based
on the current levels of P availability in the milpas with (moderate P: 65 mg kg-1) or without (low
P: 5 mg kg-1) fertilization regimes. Two mycorrhizal treatment levels were applied (inoculated vs.
non-inoculated). The experiment included five replicate plants per combination of treatment
levels. Three plants were randomly selected from each of the inoculated combinations for
molecular analysis of AMF diversity.
46
To set up the experiment, seeds from each type of maize, belonging to the same plots from which
the inoculum was extracted, were disinfected with a 10% NaOCl solution for 15 min, rinsed with
distilled water and germinated for three days in Petri dishes with moist filter paper at 28ºC in a
natural convection oven. Germinated seeds of each maize type with a radicle of length 1-2 cm
were sown in 20 liter plastic containers, with washed and sterilized silica sand as a substrate. Two
maize seeds were sown in each container and 150 g of inoculum (approx. 1,100 spores) was
applied to the pots assigned for inoculation. The containers were randomly organized on the
greenhouse tables. One week afterwards, thinning was carried out, leaving one plant per
container. Each week, 150 ml of the corresponding dose of soluble NaH2PO4·H2O was applied to
each container. In addition, base nutrients were added weekly by applying 200 ml of Long
Ashton solution, without P (Hewitt, 1966). Irrigation was carried out using potable water.
Root samples
Three to five root pieces (0.5 to 1 cm in length) were taken from each of the 30 selected plants
(three plants × five maize types × two P levels) and prepared for use in the molecular analysis.
Roots from the three replicate plants were pooled and examined under the microscope. Segments
colonized by AMF were cut into pieces and collected in a single 1.5 mL tube where they were
crushed in DNAzol buffer with a pestle. Genomic DNA was extracted in DNAzol® following the
instructions of the manufacturer.
Molecular diversity analysis
Genomic DNA was used to conduct molecular identification using the polymerase chain reaction
(PCR) technique. Concentration of DNA in the samples was estimated by the Quant-it DNA
Quantitation kitTM (Cat. No. Q32854; Quant-it, Oregon, USA), following the manufacturer
instructions.
Oligonucleotides used for the nested PCR were previously described by Krüger et al., (2009). For
the first PCR reaction, the mixture contained 1 μL of eluted genomic DNA (100 ng), 1x reaction
buffer, 1.5 mM MgCl2, 0.2 μM of each primer mix SSU Af1-2 (SSU Af1: 5´ TGG GTA ATC
47
TTT TGA AAC TTY A 3´; SSUAf2: 5´ TGG GTA ATC TTR TGA AAC TTC A 3´) and
LSUmAr1-4 (LSUmAr1: 5´ GCT CAC ACT CAA ATC TAT CAA A3´; LSUmAr2: 5´ GCT
CTA ACT CAA TTC TAT CGA T 3´; LSUmAr3: 5´ T GCT CTT ACT CAA ATC TAT CAA A
3´; LSUmAr4: 5´ GCT CTT ACT CAA ACC TAT CGA 3´), 0.2 mM each of deoxynucleotide
triphosphates (dNTPs), and 1.0 U Platinum® Taq DNA polymerase (Invitrogen/Life
Technologies, Cat. No. 10966-030, Eugene, OR, USA), in a total reaction volume of 50 μL. The
DNA templates were first subjected to an initial denaturation step at 94°C for 2 min, followed by
35 cycles of a 30 s denaturation step at 94°C, 30 s annealing step at 55°C, and a 2 min extension
step at 72°C. Finally, there was a 10 min extension at 72°C. The PCR was performed using a
MultiGene Thermal Cycler (Model TCP600-G, Edison, NJ, USA). The first PCR product was
1800 bp and was diluted 1:10 with ultrapure water. The dilutions were used as template DNA in a
second PCR reaction, performed using the primer mixtures SSUmCf1-3 (SSUmCf1: 5´ T CGC
TCT TCA ACG AGG AAT C 3´; SSUmCf2: 5´ TAT TGT TCT TCA ACG AGG AAT C 3´;
SSUmCf3 5´ TAT TGC TCT TNA ACG AGG AAT C 3´) and LSUmBr1-5 (LSUmBr1: 5´ DAA
CAC TCG CAT ATA TGT TAG A 3´; LSUmBr2: 5´ AA CAC TCG CAC ACA TGT TAG A
3´; LSUmBr3: 5´ AA CAC TCG CAT ACA TGT TAG A 3´; LSUmBr4: 5´ AAA CAC TCG
CAC ATA TGT TAG A 3´; LSUmBr5: 5´ AA CAC TCG CAT ATA TGC TAG A 3´) to obtain
a 1500 bp PCR product. The same PCR conditions were used, except that the annealing
temperature was reduced to 50ºC. Products were visualized by electrophoresis on a 1% agarose
gel in 0.5x Tris-acetate-EDTA (TAE) buffer and stained with ethidium bromide, using a
Chemidoc photodocumentation system (Biorad, Philadelphia, PA, USA) to verify the product
size.
Amplification bands of 1500 bp for SSUmCf/LSUmBr were cut from the gel and DNA was
extracted with the QIAquick Gel Extraction kit, following the instructions of the manufacturer
(Cat. 28704, Qiagen, Oregon, USA).
48
PCR cloning
The purified 1500 bp PCR products were cloned in pGEM-T Easy Vector System II kit
(Promega, Cat No. A3600, Madison, WI, USA) as described by the manufacturer. Ligation
reactions (10 µl) were incubated at room temperature overnight. Transformation of circularized
plasmids into E. coli JM-109 competent cells was performed following the procedure described
by the supplier. Colonies were selected to take 96 clones per sample. Plasmid minipreps were
made following the manufacturer’s instructions (QIAprep® Spin Miniprep Kit, Cat. No. 27106).
The plasmid DNA was stored at -20°C for subsequent analysis. To verify clone insertion into the
vector, EcoRI restriction digests were performed (Cat. No. 15202013, NEB, Carlsbad, CA, USA)
for each sample. DNA from clones containing the correct length of inserts was quantified using
the Quant-it DNA Quantitation kitTM (Cat. No. Q32854, Qiagen, Oregon, USA) as described by
the manufacturer.
Sequencing analysis and comparison of sequences
The clones were sequenced with the help of an ABI Prism 3100 Automated Sequencer at the
National Laboratory of Genomics (Langebio, CINVESTAV-Irapuato, Mexico). Sequencing was
unidirectional and conducted using the T7 primer. To identify the DNA sequences obtained, the
software DNASTAR was first used to eliminate vector sequences. Sequences were screened for
chimeras using the CHIMERA_CHECK program, available from the Ribosomal Database
Project II (RDP II) website. All chimeric sequences were discarded. The sequences were then
compared with the existing sequences in Genbank from the National Center for Biotechnology
Information (NCBI) (http://www.ncbi.nlm.nih.gov/) using the BLAST-N program and the Mega
Blast algorithm. Sequences that did not belong to Glomeromycota were discarded. The partial
rDNA gene sequences of Glomeromycota produced by this study have been deposited in the
NCBI Genbank database under accession numbers KM041704 to KM042030.
The diversity of Glomeromycota associated with maize root samples was examined by
performing a rarefaction analysis. For this process, sequences were aligned using the Clustal-W
program (Thompson et al., 1994). The distance matrix was calculated with the Dnadist Phylip
Program (v. 3.69), using the Jukes-Cantor substitution model. The sequences were grouped into
operational taxonomic units (OTUs) with the Cluster program using the Average Neighbor
49
algorithm from the Mothur program v. 1.20.1 (http://www.mothur.org) (Schloss et al., 2009). The
OTUs were defined as a group of sequences sharing at least 97% pairwise similarity.
Representative sequences of these OTUs and 21 reference sequences from public databases were
used to construct a phylogenetic tree. Phylogenetic analyses were performed by the neighbor-
joining algorithm (Saitou and Nei, 1987), with the Kimura 2-parameter substitution model
(Kimura, 1980) and bootstrapping of 1,000 replicates in the Molecular Evolutionary Genetics
Analysis MEGA 5 software (Tamura et al., 2011). Rhodotorula hordea (Basidiomycota) was
used as the outgroup (Krüger et al., 2012).
A generalized linear modelling procedure was used to determine whether P level and maize type
could explain the number of OTUs found. We included the number of sequences in the model as
a first explanatory variable, since the number of sequences extracted per combination of
treatments was variable. In this model, a Poisson error distribution and a log link function were
used. To determine the significance of the contribution of each explanatory variable to the model,
we used a deviance analysis based on the Chi-square (X2) statistic.
Results
The level of Glomeromycota diversity represented in this study was estimated to be 84 OTUs, 65
singletons (containing only one sequence per OTU) and 19 non-singleton OTUs (≥2 sequences
per OTU). Rarefaction analysis revealed that the species sampling effort curves calculated with
95 and 97% sequence identity had a positive slope, although almost reaching a plateau, showing
a tendency to approach saturation (Figure 1). While a larger sequencing effort may be needed to
cover the entire diversity, not many more OTUs would be expected.
All rhizospheres showed association to AMF regardless of maize type or P treatment, AMF
colonization ranged from 45 to 80% (see Sangabriel et al., 2014). Plants that were not inoculated
showed no colonization. Of the 327 Glomeromycota sequences analyzed, 95.1% belonged to the
order Glomerales, followed in abundance by Diversisporales (4.59%) and Archeosporales
(0.31%). All the identified Glomeromycota sequences shared a high similarity (92-100%) with
sequences previously deposited at Genbank. The order Glomerales included many sequences that
were not allocated to any species (33.94% uncultured Glomus; Glomus sp. 0.61%), as well as
50
sequences that were identified as two Glomus species (G. versiforme [7.95%] and G. aggregatum
[0.31%]), two Rhizophagus species (R. intraradices [26.61%] and R. irregularis [24.46%]) and
Clareidoglomus claroideum (1.22%). Diversisporales, the second most abundant order, consisted
exclusively of Acaulospora mellea (4.59%). The order Archaeosporales consisted of the species
Archaeospora schenkii (0.31%).
A neighbor-joining tree was constructed using 22 sequences of representative OTUs of
Glomeromycota sequences obtained from the maize roots in this study and 21 reference
sequences downloaded from GenBank. The sequence groups covered four families of
Glomeromycota: Glomeraceae, Acaullosporaceae, Archaeosporaceae and Claroideoglomeraceae
(Figure 2). Rhizophagus intraradices, R. irregularis and uncultured Glomus were the groups that
were found in all the maize types (Table 1). OTU 1 (uncultured Glomus), represented by 43
sequences, was the most abundant and was present in all five types of maize and in eight out of
ten rhizosphere samples.
We found very few cases of fungal species exclusively associated to a maize type.
G. versiforme (26 sequences) and Glomus sp. (2 sequences) colonized Red maize only. A single
sequence each of A. schenkii and of Glomus aggregatum was found in Black and White maize,
respectively. However, different maize types were colonized by different AMF communities
(Figure 3). Four to 28 OTUs were associated with each combination of treatments and 10 to 38
OTUs were found per maize type (Tables 1 and 2). Some OTUs were present in four maize types
(OTUs 3 and 5, both identified as R. intraradices), three maize types (OTUs 2 [Rhizophagus
intraradices] and 7 [Uncultured Glomus]), or two maize types (OTUs 9 [Rhizophagus
irregularis], 10, [Acaulospora mellea], 11 [Uncultured Glomus] and 13 [Claroideoglomus
claroideum]). All other OTUs (75 out of 84) were present in a single rhizosphere sample at a
frequency of 1 to 23 sequences (Table 1). Singletons belonged to taxonomic groups already
represented in the most abundant OTUs (R. intraradices, R. irregularis, Glomus versiforme and
uncultured Glomus), except for the individual sequences identified as Archaeospora schenkii
(OTU 42) and Glomus aggregatum (OTU 59; Table 1).
51
All five maize types presented a higher number of sequences (Table 1) and OTUs (Table 2) in
moderate P compared to the low P treatment. Black maize was colonized by the highest number
of OTUs. However, there was a strong positive correlation between the numbers of sequences
and OTUs extracted per sample (Pearson’s correlation of 0.5). For this reason, P treatment and
maize type had no explanatory power in the generalized linear model (X2(1,7)=5.23, P> 0.1;
X2(4,3)=2.35, P> 0.1, respectively) once the effect of the number of sequences was considered as a
covariate (X2(1,8)=5.23, P< 0.001). Nevertheless, we did find a different community composition
in each P treatment (Figure 3). Of the 46 OTUs that were identified to species, 34 were
singletons. Of these, 11 occurred in the low and 23 in the moderate P treatment. Glomus
aggregatum and G. versiforme only occurred under moderate P, while Archaeospora trappei was
only detected under low P. Uncultured Glomus and Acaulospora mellea were dominant under
low P, while their proportion was reduced under moderate P. In contrast, the proportion of R.
irregularis and G. versiforme increased under moderate P (Figure 3). Other taxa seemed
unaffected by P fertilization. The proportion of Rhizophagus intraradices was generally high in
both P treatments.
Discussion
Previous studies evaluating the diversity of indigenous AMF in maize rhizosphere have been
conducted using a single variety of maize (Sasvári et al., 2011) or hybrid genotypes (Oliveira et
al., 2009; Picard and Bosco, 2008). To our knowledge, this study shows for the first time the
differential composition of AMF communities colonizing local maize landraces and a hybrid
when exposed to a mixed inoculation of native AMF.
We found twice the number of AMF OTUs (84) than has been reported for maize in similar
studies [33 by Moebius-Clune et al. (2013) and 22 by Sasvári et al. (2011)]. Three of the four
orders that comprise the current AMF taxonomic classification were found in our maize
rhizospheres (Table 1). This is consistent with the reported rarity of Paraglomeraceae in
agricultural soils (Alguacil et al., 2008). In this study, as in many others performed in tropical
forests (Husband et al., 2002; Onguene and Kuyper, 2001; Wubet et al., 2003), agricultural sites
(Daniell et al., 2001; Hijri et al., 2006) and the maize rhizosphere (Daniell et al., 2001; Jansa et
52
al., 2003; Sasvári et al., 2011), the order Glomerales was the most abundant and was present in
all five maize types. The dominance of Glomerales is thought to be due to their known
adaptations to survive under adverse soil conditions, such as the acid and nutrient deficient soils
found in the Popoluca milpas (Daniell et al., 2001; Oehl et al., 2003).
Although AMF have traditionally been thought to be non-specific, there is evidence suggesting
that some symbionts may exhibit preference (Hendrix et al., 1995). In our study, we consistently
found little evidence of either exclusive association or complete promiscuity. Only two identified
species (R. intraradices and R. irregularis) were found in all maize types. Rhizophagus
intraradices is the sequence type most commonly identified in field studies (Börstler et al., 2010;
Oehl et al., 2010). Two OTUs (identified as Glomus sp. and G. versiforme) were present only in
the Red landrace. Glomus aggregatum and Archaeospora schenkii each had a single sequence in
the roots of the Black and White maize, respectively. The occurrence of Archaeospora sp. in the
maize rhizosphere soil has been rarely reported (Oliveira et al., 2009; Sasvári et al., 2011) and
Archaeospora schenkii has never been previously reported in roots of maize inoculated with
native AMF inoculum. Of all the OTUs found for Acaullospora mellea, only one was found
associated with the White maize landrace but all were found to be associated with the hybrid
Texcoco. The Acaulosporaceae group is reported to be particularly well adapted to interaction
with the roots of hybrid maize (Hijri et al., 2006; Picard and Bosco, 2008).
In this study, diverse AMF groups simultaneously colonized all of the landraces and the hybrid
maize, as is often the case for most plants (Gollotte et al., 2004; Helgason et al., 2002; van
Tuinen et al., 1998). However, as expected, the identity and abundance of OTUs associated with
each maize type and level of P were not the same. As hypothesized, the most efficient maize type
in terms of P acquisition (Black) (Sangabriel-Conde et al., 2014) was also colonized by the
highest number of OTUs, regardless of P treatment. The hybrid Texcoco was consistently
colonized by the second lowest number of OTUs. However, the results for the Yellow landrace
were not consistent with this trend; while this landrace presented the second highest Pi uptake
and colonization (Sangabriel-Conde et al., 2014), its roots presented the lowest number of OTUs.
This may be due to differences in the strategies for establishment of mycorrhizal symbiosis that
have evolved in each maize genotype. It is known that the presence of AMF species may depend
upon the root exudations or signaling of each maize genotype under P stress (Oliveira et al.,
53
2009). Plants grown in P-deficient soils can exudate functional substances, such as phosphatases,
nucleases, organic acids and phenolic compounds that can stimulate the development of
mycorrhizal associations with particular AMF species (Bayuelo-Jiménez et al., 2011; Javot et al.,
2007; Machado and Furlani, 2004; Zhu et al., 2005) and these root exudates are crucial
determinants of rhizosphere microorganism diversity (Barea et al., 2005; Marschner, 1998). For
the Black maize landrace, this strategy may involve increasing the diversity of AMF in its roots
to achieve higher Pi uptake (Tian et al., 2013), while for the Yellow landrace it may consist of the
selection of more efficient fungal genotypes (Thonar et al., 2011).
Contrary to expectation, more OTUs in general and more identified singletons were found in the
moderate than in the low P treatment, suggesting that low Pi in milpas is limiting symbiotic
interactions. However, for all native landraces (except Yellow), the uncultured Glomus was more
abundant under low P treatment than under moderate fertilization. For the Texcoco hybrid, the
pattern was inverse and similar to the Yellow landrace. It is worth noting that only between 0 and
20 % of OTUs were conserved between P treatments for each landrace. This result is consistent
with the observation that indigenous assemblages of AMF found in plant roots are specific to the
local soil environment, at community composition scale and even in terms of the type of
sequences within the OTUs (Jie et al., 2013). Our results therefore suggest that a high OTU
richness of AMF in milpas is related to the presence of several native maize landraces.
Substitution for a single hybrid may reduce, while moderate P fertilization may increase AMF
community richness at the root level. However, the very high turnover of OTUs found in our
experiment, together with the lack of replication at the level of treatment interactions, limit our
ability to infer the combined effect of P fertilization and maize type on AMF diversity.
Furthermore, there was a high correlation in our study between the number of sequences
extracted and the number of OTUs, and it is therefore not possible to entirely disregard the
contribution of this artefact to our OTU richness results. Nevertheless, our results with the Black
landrace are consistent with the work by Tian et al. (2013), who found that an experimental
increase in diversity of AM fungi in maize roots led to higher expression of the mycorrhizal
inducible gene ZEAma:Pht1;6 and increased Pi uptake in shoots. Moreover, the higher number of
OTUs under moderate P conditions is consistent with our previous observation that low P was
limiting. AMF symbiotic interactions under moderate P fertilization also increased colonization
in all maize types except the White landrace (Sangabriel-Conde et al., 2014).
54
The present study confirms and extends previous findings that Glomerales fungi predominate in
the colonization of maize roots. Our results suggest that maize genotypes may have a strong
influence on the structure of the AMF community that colonizes the roots, since the identity and
abundance of OTUs associated with each maize type and P level differed greatly. Reduction of
the maize landrace diversity in the milpas may reduce native AMF community richness at root
level. In addition, a very low availability of P in soils may limit the genetic richness of the AMF
colonizing each root system. Findings for the Black maize landrace support the previous
observation that high P uptake ability may be correlated with AMF community richness in the
roots, which further demonstrates the genetic and cultural importance of this landrace for the
Popoluca and for the world.
Acknowledgements
We are grateful to Isis de la Rosa and Omar Lázaro for their invaluable help in the field; to the
staff of Laboratorio de Organismos Benéficos of the Faculty of Agronomy of the Universidad
Veracruzana for provision of experimental facilities; to the staff of Laboratorio de Ecología
Molecular de la Rizósfera of Instituto Politécnico Nacional-CIIDIR-Sinaloa for support in
molecular analysis. This study was financed by the BioPop project (FOMIX 94427, CONACYT-
Veracruz) and by the project ECOS-ANUIES M08A
55
Figure 1. Change in mycorrhizal OTU richness with the cumulative number of sequences (sampling effort in all maize types), obtained through rarefaction analysis where percentage indicates the homology level at which sequences were compared.
56
Figure 2. Phylogenetic tree displaying the relationship of AMF sequences recovered from maize roots in the present study (bold type) and 21 reference sequences. Bootstrap values are shown on the lines. Reference sequences are labeled with the name provided by the database entry and the corresponding accession number. Phylogenetic group and subgroup assignments are shown on the right side of the tree (the total number of sequences belonging to each specific OTU is given in Table 1).
57
Figure 3. Percentage of AMF sequences pertaining to species in the different experimental treatments. Total number of sequences is shown above each bar. B = Black, Y = Yellow, R = Red, W = White, T = Texcoco hybrid maize types. Numbers in subscript denote maize fertilization treatments at 5 and 65 mg kg-1 Pi
46 77 13 25 19 48 19 53 13 14
58
Table 1. Abundance of AMF sequences recovered from maize roots. Classified according to taxonomic affiliation, number assigned to OTU, maize type and phosphate treatment. B = Black, Y = Yellow, R = Red, W = White, T = Texcoco hybrid maize types. Numbers in subscript denote maize fertilization treatments at 5 and 65 mg kg-1 Pi.
Order Molecular ID and
OTU assigned number
B5 B65 Y5 Y65 R5 R65 W5 W6
5 T5 T65 Total No.
of sequence
s
Glomerales Uncultured Glomus (1, 7, 11, 12, 14, 22, 23, 25, 27, 29, 32-39, 43, 46, 48, 50, 51, 53, 54, 56-58, 60, 63, 64, 66, 68)
32 12 10 21 5 3 15 7 1 5 111
Glomerales Rhizophagus intraradices (2) - - - - - 7 - 32 3 - 42
Glomerales Rhizophagus intraradices (3) 10 - - 2 9 - - - - 4 25
Glomerales Glomus versiforme (4) - - - - - 23 - - - - 23
Glomerales Rhizophagus intraradices (5) 1 5 - - - 9 2 - - 1 18
Glomerales Rhizophagus irregularis (6) - 19 - - - - - - - - 19
Glomerales Rhizophagus irregularis (8) - 15 - - - - - - - - 15
Glomerales Rhizophagus irregularis (9) - 14 1 - - - - - - 15
Diversisporales Acaulospora mellea (10) - - - - - - - 6 7 - 13
Glomerales Claroideoglomus claroideum (13) 2 - - - - - - - - 1 3
Glomerales Rhizophagus intraradices (15) - - 3 - - - - - - - 3
Glomerales Rhizophagus irregularis (16) - 2 - - - - - - - - 2
Glomerales Glomus sp. (17) - - - - 1 1 - - - - 2
Glomerales Rhizophagus intraradices (18) - - - - - - - - - 2 2
Glomerales Rhizophagus irregularis (19) - 2 - - - - - - - - 2
Glomerales Rhizophagus intraradices (20) - - - - - - - 1 - - 1
Glomerales Rhizophagus irregularis (21) - 1 - - - - - - - - 1
Glomerales Rhizophagus intraradices (24) - - - - - - - 1 - - 1
Glomerales Rhizophagus irregularis (26) - 1 - - - - - - - - 1
Glomerales Rhizophagus intraradices (28) - 1 - - - - - - - - 1
Glomerales Glomus clarum (29) 1 - - - - - - - - - 1
Glomerales Rhizophagus irregularis (30) - 1 - - - - - - - - 1
Glomerales Rhizophagus intraradices (31) - - - - 1 - - - - - 1
Glomerales Rhizophagus intraradices (40) - - - - 1 - - - - - 1
59
Glomerales Rhizophagus irregularis (41) - 1 - - - - - - - - 1
Archaeosporales Archaeospora schenkii (42) 1 - - - - - - - - - 1
Diversisporales Acaulospora mellea (44) - - - - - - - - 1 - 1
Diversisporales Acaulospora mellea (45) - - - - - - - - 1 - 1
Glomerales Rhizophagus intraradices (49) - - - - - - 1 - - - 1
Glomerales Rhizophagus intraradices (52) - - - - - - - 1 - - 1
Glomerales Rhizophagus intraradices (55) - - - - - - - 1 - - 1
Glomerales Glomus clarum (56) 1 - - - - - - - - - 1
Glomerales Glomus aggregatum (59) - - - - - - - 1 - - 1
Glomerales Rhizophagus intraradices (61) - 1 - - - - - - - - 1
Glomerales Rhizophagus irregularis (62) - 1 - - - - - - - - 1
Glomerales Claroideoglomus claroideum (65) - - - 1 - - - - - - 1
Glomerales Rhizophagus irregularis (67) - - - - - 1 - - - - 1
Glomerales Rhizophagus irregularis (69) - 1 - - - - - - - - 1
Glomerales Glomus versiforme (72) - - - - - 1 - - - - 1
Glomerales Rhizophagus intraradices (73) - - - - 1 - - - - - 1
Glomerales Rhizophagus intraradices (75) - - - - - - - 1 - - 1
Glomerales Rhizophagus irregularis (76) - - - - - - 1 - - - 1
Glomerales Rhizophagus irregularis (77) - - - - - 1 - - - - 1
Glomerales Glomus versiforme (78) - - - - - 1 - - - - 1
Glomerales Rhizophagus intraradices (79) - - - - - - - - - 1 1
Glomerales Rhizophagus intraradices (80) - - - - - - - 1 - - 1
Glomerales Rhizophagus intraradices (81) - - - - 1 - - - - - 1
Glomerales Rhizophagus intraradices (82) - - - - - - - 1 - - 1
Glomerales Glomus versiforme (83) - - - - - 1 - - - - 1
Total 46 77 13 25 19 48 19 53 13 14 327
60
Table 2. Number of OTUs associated with the roots of different maize types and under two phosphorus levels.
Maize type Number of OTUs associated with maize roots
Low P
(5 mg kg-1)
Moderate P
(65 mg kg-1)
Pooled Present in both conditions (%)
Yellow 4 8 10 20.0
White 11 17 27 3.7
Black 11 28 38 2.6
Red 9 10 17 11.7
Hybrid Texcoco 5 7 12 0
61
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CAPÍTULO IV
DISCUSIÓN GENERAL Y CONCLUSIONES
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Discusión General
Simbiosis micorrízica: ¿alternativa para la agricultura de subsistencia en las milpas?
Se estima que la demanda de alimentos en el nivel mundial se duplicará para el año 2050 (Tilman
et al. 2011), y algunos autores pronostican que el área para la agricultura intensiva se
incrementará aproximadamente a 1 billón de hectáreas en ese intervalo de tiempo y que los
impactos serán mayores en las zonas tropicales donde se desarrolla la agricultura de subsistencia,
la cual se caracteriza por preservar una amplia diversidad de variedades nativas (Balmford et al.
2005; Zimmerer 2010; Tilman et al. 2011). Esta situación traerá como consecuencia la inminente
pérdida de una importante cantidad de biodiversidad y de los servicios asociados a ella (Norris
2008). Ante tal escenario, el desarrollo de tecnologías que concilien la producción de alimentos
con la conservación de la biodiversidad arriba y bajo el suelo es un tema prioritario.
Se sabe que el fósforo es uno de los macronutrientes más influyentes en el crecimiento vegetal y
la productividad de los cultivos, incluido el maíz (Calderón-Vázquez et al. 2009). Dado que el
mayor beneficio que las plantas obtienen de la micorriza es un incremento en la absorción de P
cuando este elemento es limitante (Smith et al. 2011) y que la mayor parte de los suelos
tropicales donde se desarrolla la agricultura de subsistencia tienen poca disponibilidad de P, es
factible pensar que la integración de los hongos micorrízicos arbusculares (HMA) como parte del
manejo de estos sistemas puede ser una opción viable para incrementar el rendimiento de los
diversos cultivos ahí sembrados.
Usualmente la integración de los HMA en los sistemas agrícolas se hace por dos métodos; el
primero es mediante la introducción de HMA seleccionados previamente como promiscuos y con
una eficiencia alta tanto en condiciones de laboratorio como de invernadero (Koide y Mosse
2004), y el segundo es con hongos nativos con el fin de potenciar los beneficios co-evolutivos
entre los micosimbiontes y las plantas (Johnson et al. 2010). El primero es ampliamente utilizado,
a pesar de que la introducción de especies exóticas puede alterar la eficiencia y reducir la
diversidad de los HMA nativos, principalmente en condiciones de policultivo (Yao et al. 2008).
En consecuencia, en aras de conservar la biodiversidad, lo más apropiado es aplicar el segundo
método para lo cual se requiere seleccionar cepas nativas de HMA altamente eficientes.
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Los estudios de diversidad y funcionalidad de HMA asociados a raíces de variedades criollas de
maíz son escasos. En esta tesis se analizó a nivel de invernadero en cuatro variedades criollas y
un híbrido de maíz, la diversidad y funcionalidad de HMA nativos bajo distintas condiciones de
disponibilidad de fósforo.
Los resultados obtenidos en esta tesis ponen de manifiesto tres aspectos importantes (al menos en
el contexto de invernadero): (1) los HMA nativos de policultivos tradicionales como la milpa son
capaces de establecer eficientemente la simbiosis micorrízica y promover un incremento en la
incorporación de fósforo en variedades locales de maíz, (2) las variedades locales como el criollo
Negro, son capaces de albergar en sus raíces una elevada diversidad de HMA y (3) el híbrido
introducido Texcoco presenta una limitada capacidad para adquirir fósforo aún en simbiosis con
HMA (indicando su naturaleza de dependencia a la fertilización química). Estos resultados
apuntan a que la sustitución de variedades criollas de maíz por el híbrido Texcoco puede no ser
una opción viable para incrementar la productividad de los sistemas de milpa, y que dada: a) la
poca disponibilidad de P en los suelos, b) el constante incremento en los precios de los
fertilizantes fosfatados (Cordell et al. 2009) y c) la dificultad de reproducir masivamente
consorcios altamente eficientes de HMA en invernadero (Trejo-Aguilar et al. 2013), el manejo de
las poblaciones nativas de HMA in situ mediante arreglos espaciales de los cultivos, por ejemplo
crecer en vecindad cultivos poco eficientes con variedades micorrizadas altamente eficientes en
captar fósforo como la variedad criolla Negro, puede ser una alternativa para apoyar la
agricultura de subsistencia donde los productores no cuentan con recursos para acceder a
fertilizantes inorgánicos.
La milpa ¿Reservorio de comunidades de HMA funcionalmente diversas?
Un aspecto único de la simbiosis HMA-planta es que ambos simbiontes son capaces de
interactuar de forma simultánea con varios socios, aunque también pueden darse asociaciones
específicas (Smith y Smith 2011). Se sabe que en los sistemas de policultivos se incrementa la
producción de exudados radicales y la diversidad de especies de HMA, en comparación con los
monocultivos (Zarea et al. 2009; Bainard et al. 2011). El incremento en la diversidad de especies
de HMA maximiza la eficiencia de absorción de nutrientes en la planta, no solo a nivel
individual, sino entre plantas vecinas, gracias a la red de hifas que actúa como un sistema de
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canales donde se transfieren nutrientes entre plantas eficientes en captar nutrientes (por ej.
leguminosas) y otras que no lo son (Martins 1993; Cruz et al. 2000). En esta tesis se encontró que
las distintas variedades de maíz fueron colonizadas por diversos OTUs de HMA, formando
comunidades distintas y en algunos casos únicas en las diferentes variedades de maíz estudiadas.
Aunque sabemos que las milpas con una diversidad alta de especies cultivadas tienen una
disponibilidad mayor de P en sus suelos y mayor colonización micorrízica en el maíz (Negrete-
Yankelevich et al. 2013), y que distintos maíces tienen distintas capacidades de absorción de P y
de asociación micorrízica, aún no sabemos si la diversidad de asociaciones HMA-variedades de
maíz, conlleva a una diversidad funcional equivalente o a una maximización funcional (de
absorción de nutrientes) en el nivel de comunidad de plantas. Es posible que las asociaciones
específicas encontradas estén determinadas por los exudados radicales de cada variedad de maíz
(Giovannetti et al. 1994; Jones et al. 2004)). Dado que las variedades de maíz difieren en la
producción de flavonoides (Casati y Walbot 2003) y que los flavonoides generan especificidad de
asociación entre géneros de HMA e incluso entre especies de un mismo género (Vierheilig et al.
1998), posiblemente las variedades que desarrollaron simbiosis específica produzcan flavonoides
que favorezcan la asociación con ciertos micosimbiontes. Por ejemplo, se sabe que el flavonoide
rutin estimula el desarrollo de hifas y la colonización radical de Gigaspora margarita, pero no
tiene efecto alguno en Gigaspora rosea (Scervino et al. 2007). Flavonoides como chrysin,
luteolin y morin incrementan la ramificación y elongación hifal en Glomus mosseae y G.
intraradices, mientras que otros como isorhamnetin y rutin no generan ningún efecto (Scervino et
al. 2005). Esto permite pensar que dada la elevada agrodiversidad (intra e interespecífica)
manejada en la milpa, es posible que la producción de exudados radicales también sea elevada
(Zarea et al. 2009) y favorezca una mayor diversidad de HMA, convirtiendo a la milpa en un
reservorio natural de comunidades de HMA funcionalmente más diversas que las que podríamos
encontrar en agroecosistemas convencionales como los monocultivos.
Del total de secuencias de HMA identificadas en esta tesis, el 95.1% corresponde al orden
Glomerales, resultado que coincide con otras investigaciones donde se indica la ubicuidad de
dichos micosimbiontes como principales colonizadores del cultivo de maíz (Daniell et al. 2001;
Oehl et al. 2003; Borriello et al. 2012). No es sorprendente que Rhizophagus intraradices y R.
irregularis se encontraran asociadas a todas las variedades de maíz ya que se sabe que los hongos
del género Rhizophagus son considerados “generalistas” y colonizan a numerosos cultivos,
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incluido el maíz (Oehl et al. 2003; Öpik et al. 2006; Borriello et al. 2012), además de ser capaces
de adaptarse a suelos con muy bajo contenido de nutrientes particularmente de fósforo (Kahiluoto
et al. 2001), como los suelos de las milpas popolucas. De igual forma, el OTU asignado como
uncultured Glomus colonizó todas las variedades de maíz, pero la ausencia de secuencias
plenamente identificadas en bases de datos públicas, impidió asociar su identidad con la de
alguna especie conocida. Dado que este micosimbionte aportó el mayor porcentaje de secuencias
con relación a las demás especies (33.94%) y que desconocemos cuál puede ser su contribución
en el desarrollo de las variedades de maíz (absorción y asimilación de fósforo, producción de
biomasa, incrementos de volumen radical), futuras investigaciones podrían encaminarse a
determinar su identidad y eficiencia para promover la nutrición y el desarrollo de las plantas.
Para profundizar en el entendimiento de las milpas como reservorios de HMA, es imprescindible
que el conocimiento taxonómico a nivel molecular de estos hongos también avance.
Perspectivas para futuras investigaciones
Es claro que la simbiosis micorrízica juega un papel importante en el funcionamiento y
productividad de los sistemas agrícolas (Verbruggen y Kiers 2010; Smith et al. 2011), sin
embargo muy poco se conoce sobre las comunidades que se desarrollan en sistemas de
policultivos tradicionales como la milpa. De acuerdo con las hipótesis planteadas, en el capítulo
II de esta tesis se esperaba que: Al crecer maíces en suelos con inóculos nativos de milpas (1) el
porcentaje de colonización micorrízica, el contenido de fósforo en raíz y parte aérea, la biomasa
seca total y la dependencia micorrízica serían similares entre maíces criollos popolucas y
mayores que en la variedad híbrida. En contraste, se encontró que existen diferencias entre
variedades criollas y con el hibrido Texcoco en relación con los parámetros evaluados, y que
comparadas con el híbrido, ninguna variedad criolla registró mayor dependencia micorrízica (al
menos en dosis bajas de fósforo), solo una de ellas (Negro) registró mayor producción de
biomasa seca total, dos (Amarillo y Negro) presentaron mayores porcentajes de colonización, y
tres de ellas incorporaron más fósforo en biomasa aérea y en raíz, se piensa que las diferencias
observadas pueden ser resultado de la variación genética y especificidad ecológica de ambos
simbiontes; (2) en condiciones de bajas concentraciones de fósforo, la simbiosis micorrízica se
desarrollaría mejor en los maíces criollos que en la variedad híbrida. Contrario a lo esperado, el
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híbrido Texcoco registró mayor DM que algunos criollos, y se observó que para dicho híbrido la
micorrización es muy importante cuando hay poco P, pues bajo estas condiciones su DM fue de
las más altas, sin embargo, esto no se vio reflejado en su desarrollo y el contenido de P en sus
tejidos fue de los más bajos; y (3) en condiciones de concentraciones de fósforo medio, la
colonización y dependencia micorrízicas disminuirían y el contenido de nutrientes en biomasa
aérea y de raíz se incrementaría en todas las variedades de maíz respecto a lo encontrado en
condiciones de bajo fósforo, siendo mayor la diferencia en los maíces criollos que en la variedad
híbrida. Coincidiendo con la hipótesis, el incremento en la concentración de P sí disminuyó la
DM de tres variedades criollas (exceptuando la variedad Rojo) y del híbrido Texcoco,
particularmente este último mostró un decremento notorio, indicando que en condiciones de
mayor disponibilidad de fósforo, la simbiosis micorrízica ya no fomenta el incremento en
biomasa. Además, el contenido de nutrientes en biomasa aérea sí se incrementó en todas las
variedades de maíz (excepto en la Amarilla) al incrementar la dosis de fósforo. Sin embargo, los
resultados mostraron que solo una variedad criolla (Blanco) registró una disminución del
porcentaje de colonización cuando se incrementó la dosis de P, indicando que los niveles de
fósforo medio obtenidos con una fertilización moderada como la practicada recientemente por los
agricultores popolucas no afectan la integridad de la simbiosis micorrízica.
En el tercer capítulo de esta tesis se postularon tres hipótesis: (1) los diferentes tipos de maíz
serían colonizados en sus raíces por comunidades de HMA distintas. Los resultados concordaron
con lo planteado en la hipótesis, y mostraron que la identidad y la abundancia de OTUs asociados
con cada variedad de maíz y nivel de P no fueron los mismos y que la variedad más eficiente en
términos de adquisición de P (Criollo Negro) también fue colonizada por el mayor número de
OTUs, independientemente del tratamiento de fósforo; (2) las raíces de las variedades criollas
serían colonizadas por la mayor riqueza de HMA y el híbrido de Texcoco por la más baja. En
contraste, se encontró que la variedad criolla (Amarillo) presentó el número más bajo de OTUs
en sus raíces, mientras que el híbrido Texcoco fue colonizado por el segundo número más bajo de
OTUs; y (3) la fertilización moderada de P reduciría la riqueza de HMA en todos los tipos de
maíz, en respuesta a que la planta tiene una mayor posibilidad para obtener P por la vía no
micorrízica. Contrario a lo planteado, se identificaron más OTUs en la fertilización moderada
que en la dosis baja de P, lo que sugiere que el bajo contenido de P en las milpas podría estar
limitando las interacciones simbióticas.
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Los resultados obtenidos apuntan a que existen estrategias distintas de colonización entre las
especies de HMA y diferencias en la translocación de nutrientes entre las variedades de maíz
(Kaeppler et al. 2000; An et al. 2010). Las futuras investigaciones deberán avanzar
principalmente en 4 temas:
(1) Evaluar si en las milpas existen otros cultivos en los que la simbiosis micorrízica
proporcione grandes beneficios en términos de una mayor asimilación de nutrientes y de
incremento en el rendimiento. Se sabe que la respuesta diferencial a la micorrización no
es exclusiva de variedades de maíz, y que otras especies comúnmente sembradas en
policultivos como la milpa, difieren en su respuesta a inoculación micorrízica. Por
ejemplo, genotipos de frijol (Phaseolus vulgaris) difieren en la producción de biomasa y
reflectancia de la clorofila al inocularse con HMA (Hacisalihoglu et al. 2005). Variedades
de calabaza (Cucurbita pepo) acumulan contenidos distintos de nutrientes (P, K, Ca, Fe y
Zn) en función de la identidad de los micosimbiontes con los que se asocian (Sensoy et al.
2011). Otros cultivos como chile (Capsicum annuum), poro (Allium porrum), cilantro
(Coriandrum sativum) y camote (Ipomoea batata) también responden de manera
diferencial a la micorrización (Schroeder y Janos 2005; Farmer et al. 2007; Jansa et al.
2008). Debido a que en esta tesis se analizó la diversidad y función de HMA nativos en la
diversidad intraespecífica de maíz a nivel de invernadero, no es posible extrapolar los
resultados a todo el sistema de milpa y en condiciones de campo.
(2) Establecer experimentos en campo para determinar si la respuesta diferencial a la
micorrización encontrada en las variedades criollas de maíz y el híbrido Texcoco bajo
condiciones de invernadero, se mantiene en un ambiente no controlado y en escalas
temporales de varios ciclos de cultivo. La evaluación en campo de la simbiosis
micorrízica en sistemas agrícolas es crucial para encontrar estrategias que permitan
incrementar la productividad de dichos sistemas (Smith et al. 2011). En el caso del maíz,
la mayoría de las investigaciones se han desarrollado en condiciones de microcosmos o
invernadero (Nurlaeny et al. 1996; Ibrikci et al. 2004; Pitakdantham et al. 2007; Hao et al.
2008; Ortas y Akpinar 2011; Wright et al. 2005). Los resultados obtenidos bajo
condiciones controladas son difíciles de extrapolar a condiciones de campo (Johnson et al.
1997); en el caso del maíz existen avances en el conocimiento de su interacción con HMA
76
en campo, sin embargo estos estudios se han centrado en medir parámetros como
porcentaje de colonización micorrízica o absorción de fósforo en periodos cortos de
tiempo (Gavito y Miller 1998; Hess et al. 2005; Ortas 2012). Futuras investigaciones
deberán enfocarse en diseñar experimentos en campo, haciendo uso de herramientas como
cámaras de observación de raíces in situ (Hertel y Leuschner 2006), divisiones de malla
(Schweiger y Jakobsen 1999) o marcadores isotópicos (Lerat et al. 2002), con el objetivo
de evaluar la funcionalidad de la simbiosis micorrízica no solo en maíz sino a nivel de
interacciones multiespecíficas durante varios ciclos de cultivo.
(3) Caracterizar los tipos de exudados radicales en variedades criollas e híbridos de maíz
y evaluar el efecto de dichos exudados en la composición de la comunidad microbiana
rizosférica y su función para proveer de nutrientes a las plantas de maíz. La rizósfera es la
zona biológicamente activa del suelo donde las raíces y los microorganismos interactúan
(Singh et al. 2004). Debido a la liberación de exudados radicales, la rizósfera se
caracteriza por una compleja densidad microbiana, donde algunos microorganismos
favorecen la captación y asimilación de nutrientes en las plantas (Paterson et al. 2007). Se
sabe que las variedades de maíz difieren en la producción de exudados radicales (Casati y
Walbot 2003) y que algunos exudados favorecen la selección de microorganismos
benéficos como cepas bacterianas del género Pseudomonas (Picard y Bosco 2005, 2006;
Roesch et al. 2006) y de especies de HMA como Gigaspora margarita Glomus mosseae y
Glomus intraradices (Scervino et al. 2005; 2007) que incrementan en la planta la
asimilación de nutrientes. A su vez, especies de HMA como Glomus mosseae interactuan
sinérgicamente con otros microorganismos como micromicetos saprobios que solubilizan
fosfatos (Khan et al. 2010; Posada et al. 2013) y que estimulan la nutrición de las plantas
(Fracchia et al. 2004; Aranda et al. 2007). Debido a que los exudados radicales son muy
diversos (Uren 2007) y a que están determinados por factores como la especie y el
genotipo de la planta (Appuhn y Joergensen 2006; Rengel y Marschner 2005), futuras
investigaciones se requieren para precisar el efecto que pueden tener los diversos tipos de
exudados radicales de variedades criollas e híbridos de maíz en la composición de los
microorganimos de la rizósfera y su función en la nutrición de las plantas.
77
(4) Establecer los mecanismos que permiten al maíz negro ser más eficiente en la
adquisición de P, comparado con las otras variedades. Los hallazgos de esta tesis sobre el
maíz Negro abren paso a nuevas investigaciones que contribuirán a un mejor
entendimiento y aprovechamiento de la evolución conjunta de los HMA con el maíz en
suelos de extrema deficiencia de P. La variedad criolla Negro fue la más eficiente en
términos de adquisición de P y también fue colonizada por el mayor número de OTUs.
Estos resultados pueden estar relacionados con los siguientes factores: (1) es posible que
el criollo Negro cuente con diversos rasgos adaptativos (como exudación de ácidos
orgánicos, fosfatasas y modificación de su estructura radical) que le permitan una mayor
eficiencia en la captación directa de P (Zhu et al. 2005; Javot et al. 2007; Bayuelo-
Jiménez et al. 2011). Se sabe que este tipo de rasgos adaptativos son comunes en
germoplasmas criollos (Bayuelo-Jiménez et al. 2011); y (2) también es posible que la
eficiencia de dicha variedad esté en función de la identidad de los HMA asociados a sus
raíces. Por ejemplo, en condiciones de bajo fósforo las especies Glomus clarum y
Archaeospora schenkii se asociaron de manera exclusiva con el criollo Negro. Se sabe
que Glomus clarum es una especie que, particularmente en condiciones de bajas
concentraciones de fósforo, activa en la planta la secreción de fosfatasa, incrementa la
absorción de fósforo y maximiza el crecimiento vegetal (Balota et al. 2011), además de
ser altamente eficiente para promover el rendimiento en maíz cuando el fósforo es escaso
(Stephen et al. 2013).
Por otro lado, fue en condiciones de fósforo medio que se asoció con Rhizophagus
irregularis, se sabe que este micosimbionte optimiza su capacidad de captación de fósforo
en función de la disponibilidad externa de dicho nutriente, y transfiere más fósforo a la
planta cuando la disponibilidad se incrementa (Fiorilli et al. 2013), por lo que es factible
que las especies de HMA arriba mencionadas estén acopladas de manera muy eficiente
con esta variedad en comparación con las demás, sin embargo, dado que se realizó una
inoculación mixta y no se evaluaron especies de HMA individualmente, futuras
investigaciones podrían encaminarse a determinar si las especies que se asociaron de
forma exclusiva con la variedad criolla Negro contribuyen individualmente a una mayor
eficiencia de dicha variedad o si sus beneficios sólo se potencializan estando en presencia
de otras especies de HMA.
78
Si bien los objetivos de esta tesis se centraron en evaluar algunos aspectos de la ecología
(diversidad y función) de los HMA asociados a variedades de maíz en invernadero, en la
investigación desarrollada subyace un aspecto orientado a la aplicación en campo, dadas las
diferencias en la respuesta a la micorrización entre variedades criollas de maíz y con el híbrido
Texcoco y la disparidad en las comunidades de HMA asociadas a sus raíces. Los resultados aquí
encontrados hacen referencia a la posibilidad de encontrar in situ combinaciones óptimas de
micorrizas (mediante el manejo de poblaciones nativas de HMA y arreglos espaciales de cultivos)
que contribuyan a mejorar la eficiencia de los diferentes cultivos que conforman la milpa y que
alberguen la mayor diversidad funcional de HMA con miras a incrementar la producción de estos
sistemas milenarios y conservar al mismo tiempo su biodiversidad.
79
Conclusiones
1. Todas las variedades formaron asociación con los HMA nativos, sin embargo se observó
una respuesta diferencial a la micorrización entre variedades criollas popolucas y con el
híbrido Texcoco.
2. La variedad criolla Negro fue la que mostró incrementos en volumen radical, porcentaje
de colonización micorrízica y captación de fósforo superiores a las demás variedades, y
fue altamente eficiente en la captación de P por vía simbiótica y por vía directa lo que la
hace un acervo genético y cultural importante para los popolucas y el mundo.
3. El híbrido Texcoco registró una dependencia micorrízica alta, sin embargo esto no se vio
reflejado en su desarrollo y contenido de fósforo, pues fue de los más bajos, esto se
atribuye a su naturaleza de dependencia a la fertilización química.
4. Los niveles de fósforo medio (65 mg.kg-1) obtenidos con una fertilización moderada como
la practicada recientemente por los agricultores popolucas no afectan la integridad de la
simbiosis micorrízica, dado que solo una variedad criolla (Blanco) registró una
disminución del porcentaje de colonización cuando se incrementó la dosis de fósforo.
5. Las variedades de maíz se asociaron con ensambles micorrízicos distintos y se
identificaron 84 OTUs de HMA, entre los cuales el orden predominante fue Glomerales,
seguido por Diversisporales y Archeosporales, estos resultados concuerdan con los de
otras investigaciones donde se reporta la dominancia y ubicuidad de dicho orden.
6. Los resultados obtenidos en esta tesis apuntan a que el genotipo de maíz puede tener una
fuerte influencia sobre la estructura de la comunidad de HMA que coloniza sus raíces, ya
que la identidad y abundancia de OTUs asociados con cada tipo de maíz y nivel de
fósforo fueron diferentes, por lo que se concluye que la reducción de la diversidad de
maíces criollos en las milpas popolucas podría disminuir la diversidad de las comunidades
de HMA nativos y mermar los beneficios que dichos simbiontes proporcionan a los
cultivos.
80
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