Arbuscular mycorrhizal colonization in Asteraceae from white sand savannas, in Pinar del Río, Cuba

Colonización micorrízica arbuscular en Asteraceae de las sabanas de arenas blancas, de Pinar del Río, Cuba

Abstracts

Asteraceae family shows a great diversity in Cuba and presents threatened taxons in high number. Pinar del Río has 19 threatened species belonging to this family and three of them are characteristic of a sandy (quartzitic) soil at the Reserva Florística Manejada San Ubaldo-Sabanalamar (Floristic Reserve San Ubaldo-Sabanalamar). The arbuscular mycorrhizal status and colonization rate in Aster grisebachii, Erigeron bellidiastroides, Erigeron cuneifolius, Pectis juniperinaand Sachsia polycephala (Asteraceae) from this Reserve was determined in October 2009 and April 2010, during hydrologic rainy and dry period, respectively. All species were mycotrophic in both periods with a high percentage of colonization rates, between 66 and 100% in rainy period and between 84 and 100% in dry period. Vesicles, arbuscules, spores, hyphal and arbuscular coils of arbuscular mycorrhizae were observed. Paris -type andArum -type morphologies (both) were observed indistinctly in all the species, and Intermediate type was observed only in Aster grisebachii andErigeron bellidiastroides, which could be a hint that these species are colonized by more than one fungal species, and therefore enhancing the relevance of fungal identity as a considerable character in the morphology association. Also, septate hyphae and microsclerotium of dark septate endophytes were identifyed inside the roots of all the species.

internal morphology; Paris-type; Arum-type; Intermediate type; quartzitic sands; dark septate endophytes

La familia Asteraceae muestra una gran diversidad en Cuba y presenta un elevado número de taxones amenazados. Pinar del Río cuenta con 19 especies amenazadas pertenecientes a esta familia, de las cuales tres son características de los suelos arenosos cuarcíticos de la Reserva Florística Manejada San Ubaldo-Sabanalamar. Se determinó el estatus micorrízico arbuscular y la tasa de colonización deAster grisebachii, Erigeron bellidiastroides, Erigeron cuneifolius, Pectis juniperina y Sachsia polycephala(Asteraceae) presentes en esta Reserva, en octubre del 2009 y abril del 2010, durante los períodos hidrológicos húmedo y seco, respectivamente. Todas las especies estaban micorrizadas en ambos períodos con elevados porcentaje de colonización micorrízica, entre 66 y 100% en el período lluvioso y entre 84 y 100% en el período seco. Se identificaron vesículas, arbúsculos, esporas, enrollados hifales y arbusculares característicos de las micorrizas arbusculares. Los tipos morfológicos Paris y Arum (ambos) fueron observados indistintamente en todas las especies, y el tipo Intermedio solo se observó enAster grisebachii y Erigeron bellidiastroides, lo cual sugiere la colonización de estas especies por más de una especie fúngica y con ello la importancia de la identidad fúngica en la determinación de la morfología de la asociación. Se identificaron además, hifas septadas y microesclerosios de endófitos oscuros septados en el interior de las raíces de todas las especies estudiadas.

morfología interna; tipo Paris; tipo Arum; tipo Intemedio; arenas cuarcíticas; endófitos oscuros septados

Introduction

Around 80% of species and 92% of families of terrestrial plants are known to be mycorrhizal (Wang & Qiu 2006) and the arbuscular type being the most common and ancestral (Trappe 1981, Brundrett 2002, Wang & Qiu 2006).

According to their internal morphology, arbuscular mycorrhizae are divided in two main types: Arum-Type and Paris-Type, which were described in these plant genera by Gallaud (1904, 1905) (cited by Dickson 2004 and Dickson et al. 2007). Smith & Smith (1997) recognize “Intermediate” morphology when both structural types are recorded in the same plant species (mixture of Arum and Paris types) and “Both” when they are observed in different species or genera within the same family. However Dickson (2004) use intermediate morphology to describe structures which are between Arum and Parismorphologies and claimed the existence of continuum of mycorrhizal structures fromArum to Paris, depending upon both the host plant and the fungus.Dickson et al. (2007) use “Intermediate” for families with characteristics of both Arum andParis structures within the same genera, whereas “Both” relates to the situation when Arum and Paris type morphologies, as strictly defined, have been observed within that family.

Cuban research on the mycorrhizal status of terrestrial plants are restricted to works by Ferrer & Herrera (1985, 1988), also in the province of Pinar del Río (nowadays Artemisa and Pinar del Río). So far there are no reports about the structural diversity of arbuscular mycorrhizae in Cuban plants.

The family Asteraceae shows a great diversity in Cuba and in turn presents elevated number of threatened taxons. Pinar del Río has 19 threatened species inside this family, three of them characteristic in the Reserva Florística Manejada (RFM) San Ubaldo-Sabanalamar: Aster grisebachii Britton, Erigeron bellidiastroides Griseb. and Pectis juniperina Rydb. These species, in addition to Erigeron cuneifolius DC and Sachsia polycephalaGriseb, also presents in this ecosystem, were evaluated in this study.

Our objective was determine arbuscular mycorrhizal status and describe the internal morphology of the AM symbiosis in five species of Asteraceae (Compositae) from the white sand (quartzitic) savannas in Southwestern Pinar del Río and their relation to previous reports in this family.

Material and methods

1.

Characterization of research site

RFM San Ubaldo-Sabanalamar (W 84°3′15″, N 22°9′2″ y W 84°57′23″, N 22°3′54″), in Southwestern Pinar del Río, Cuba, is included in the District Sabaloense according to Borhidi (1996) and the main characteristic of which is a sandy (quartzitic) soil with a low nutrient contents and very acid. This, together with alternating rainy and drought seasons, are the contributing factors for the establishment of a highly specialized flora and very high endemism (Urquiola et al. 2010). Field work took place in October 2009 and April 2010 at a disturbed zone inside the Reserve during hydrologic rainy and dry period, respectively.

2.

Species tested

Five herbaceous species of the family Asteraceae were selected, three of them endemics of Western Cuba: Aster grisebachii, Erigeron bellidiastroides(both present in Pinar del Río and Isla de la Juventud) and Pectis juniperina (only from Pinar del Río) and two non-endemics:Erigeron cuneifolius (also in West Indies) and Sachsia polycephala (also recorded from Bahamas and Hispaniola). Aster grisebachii and Erigeron bellidiastroides are considered endangered (EN) and Pectis juniperina in critical threat (CR) according to IUCN criteria. Erigeron bellidiastroides is considered near threatened (NT) and Erigeron cuneifolius and Sachsia polycephalaleast concern (LC) according to the Group of Experts on Cuban Plants (GEPC) of the Commission for the Survival of Species (CSE).

3.

Mycorrhizal variables assessed

Samples from root systems of five individuals x species were taken. Roots less than 2 mm thick were washed, dried and cut into pieces ca. 1 cm long and mixed so as to have a single sample/species. Fractions ca. 0.5 g were cleared and stained following Phillips & Hayman (1970). We took only five individuals x species because their populations are only a few and of restricted distribution.

The arbuscular mycorrhizal status and colonization rate under the stereoscopic microscope were determined. Mycorrhizal colonization was estimated in accordance with the intersection method (Giovannetti & Mosse 1980). Characteristic structures of MA inside the roots were examined and identified under compound microscope with magnifying from 200 up to 1000x (both microscopes belong to the trade mark CARL ZEISS model AXIOSKOP 2 Plus). Based upon the structures, the internal morphology was determined.

Results

All the species were mycorrhizal in both periods and colonization rate were between 65 and 100% in October and between 85 and 100% in April (Table 1.). The colonization average of all species in both seasons was higher than 60%; however, the values of mycorrhizal colonization in all species during the dry season were equal or bigger than the values observed during the rainy season. The characteristic structures of arbuscular mycorrhizae were found in all species: arbuscules, hyphal coils and intraradical vesicles, three of them showed intraradical spores and arbuscular coils (Figure 1). In addition, dark septate endophytes (DSE) not corresponding to the type of arbuscular mycorrhizal structure were found in all root samples.

Figure 1.Figura 1.
Arbuscular mycorrhizal colonization in studied species: a-b A.grisebachii, c-d E.bellidiastroides, e-f E. cuneifolius. AM structures: (A) arbuscules, (HC) hyphal coils, (AC) arbuscular coils and (V) vesicles; and microsclerotium (MS) characteristic of dark septate endophytes. Colonización micorrízica arbuscular en las especies estudiadas: a-b A.grisebachii, c-d E.bellidiastroides, e-fE. cuneifolius. Estructuras micorríco arbusculares: (A) arbúsculos, (HC) enrollados hifales, (AC enrollados arbusculares), vesículas; y (MS) microesclerosios de Endófitos Oscuros Septados.

Table 1.Tabla 1.
Percentage of Arbuscular Mycorrhizal Colonization (%CM) and intraradical fungal structures of arbuscular mycorrhizae in tested species: (A) arbuscules, (HC) hyphal coils, (AC) arbuscular coils, (S) spores, (V) vesicles and (LH+HC) longitudinal hypha+ hyphal coils. Intraradical fungal structures of dark endophyte: (SH) septate hyphae and (MS) microsclerotium. Morphological type (MT) of arbuscular mycorrhizal colonization: both (Arum and Paris Type) and Intermediate Type. Porcentajes de colonización micorrízico arbuscular (%CM) y estructuras fúngicas intrarradicales de las micorrizas arbusculares en las especies evaluadas: (A) arbúsculos, (HC) enrollados hifales, (AC) enrollados arbusculares, (S) esporas, (V) vesículas, (LH+HC) hifas longitudinales+enrollados hifales. Estructuras fúngicas intrarradicales de endófitos oscuros septados: (SH) hifa septada, (MS) microesclerosio. Tipo morfológico de la colonización micorrízica arbuscular: ambos (Tipos Arum y Paris) y Tipo Intermedio.

SPECIES	%CM (oct)	%CM (abr)	A	HC	AC	S	V	HL+HC	HS	MS	MT
Aster grisebachii	66	84	x	x			x	x	x	x	Both+ Intermediate
Erigeron bellidiastroides	90	95	x	x	x	x	x	x	x	x	Both + Intermediate
Erigeron cuneifolius	89	98	x	x	x	x	x		x	x	Both
Pectis juniperina	100	100	x	x			x		x		Both
Sachsia polycephala	75	100	x	x	x	x	x		x	x	Both

The identification of distinctive characteristics involved in the internal morphology of MA, hyphal coils and arbúsculos, showed that they belong to theParis-Type and Arum-Type respectively, and both were present simultaneously in every species. Aster grisebachii and E. bellidiastroides showed longitudinal hyphae connected with hypal coils, structures similar to Intermediate Type according to Dickson (2004).

Discussion

The fact that all species turned out to be mycotrophic is in close correspondence with previous reports in Asteraceae, a plant family in which around to 233 species have been assessed. Form these 84% are mycotrophic and 100% developed AM (Wang & Qiu 2006).

Mycorrhizal condition from three of our genera was previously reported:Aster always AM, Erigeron either AM or NM (non-mycorrhizal) and Pectis AM, however, their number was small (Wang & Qiu 2006). A. grisebachii and E. bellidiastroides were classified as AM by Ferrer & Herrera (1985) near Cortés, Pinar del Río (not quantified), a which was corroborated by our assessment.

The high colonization rate in all species in both period is in agreement with the suggestion of Read et al. (1976): in environments where competitive situations are dominant, species are strongly mycorrhizal and values of mycorrhizal colonization over and higher than 50% should be usual, at both (i) level of species and (ii) individual plants in any habitat and as a general rule.

The simultaneous occurrence of both morphologies (Arum-Type andParis-Type) in the same species has been reconsidered as intermediate by Dickson (2004), who has claimed that a continuum of structures exists between these two types. However, consistent reports about this subject generally refer to studies done with a single species of AMF (inoculated) where intermediate morphology is well defined. Thus we sugest to keep both types apart since in our study all species were growing under natural conditions with mixed source of AMF. Therefore we cannot be sure that they belong to a single fungus. Only species with longitudinal hypha connected to hyphal coils, structures similar to the Intermediate Type were considered as such, but both types (Arum and Paris) were observed simultaneously in all species. Experiments of Kubota et al. (2005) proved that both morphologies can occur in a single root system when plants are grown in soils with mixed fungal inoculum.

Yamato (2004) found in six Asteraceae species in vacant lands, a typical Arum-type morphology and includedErigeron genus, the same as Shah et al. (2009) that found that in 12 invasive Asteraceae in the Himalayas showedArum-Type only. In contrast with our results, the most studies performed so far this family has been reported as having the Arum-Type (49 out of the 51 genera which they had revised), and only one genus had both morphologic types (Dickson et al. 2007).

Several factors influence the AM structure: plant genome, fungus genome, environmental conditions and the combination of all these (Smith 1995, Bedini et al. 2000, Cavagnaro et al. 2001, Ahulu et al. 2005). In our assessment every species from a single family under similar edaphic-climatic conditions and with a mixed source of AMF exhibited both morphologies simultaneously and two Intermediate Type. The experiments of Cavagnaro et al. (2001) in tomato with six species of AMF showed that both morphologies can be observed in a single host depending on the guest fungal species. Husband et al. (2002) and Geue & Hock (2004) showed that a plant species can be colonized by more than one AMF in a given area or ecosystem. Even though the determinant factor in the formation of AM structures is not at all clear, our research shows that the fungus can play a major role in AM establishment and also that a plant genus or species can have both morphologies simultaneously in a given ecosystem. Dickson (2004) shows that the division of mycorrhizal structure in Arum and Paris based on plant families can be confusing and Dickson et al. (2007)recognize that both types and their combinations occur frequently in the mayor taxa, although one type is predominant in many individual family. However, we considered that the categorical classification of the botanical families according to the MA morphologic types is yet unreliable most of all when the number evaluated genera and/or species are not representative of the family and/or the number of habitat or of individuals is equally limited.

In this study DSE were observed co-occurring with AM fungi in all species, but they were not quantified. Recent studies report the co-occurrence of DSE and AMF and suggest that it is common in plant roots in nature (Rains et al. 2003,Muthukumar et al. 2006, Santos-Gonzalez et al. 2007, Mandyam & Jumpponen 2008, Khidir et al. 2010, Yang et al. 2010) and some of them suggest that colonization of roots can be interfered by non-mycorrhizal endophytes, especially during the hot season which corresponds to the plant growing season when water availability is low and that a competition between both endophytes during colonization could limit the distribution of AM in dry region (Mandyam & Jumpponen 2008, Khidir et al. 2010, Yang et al. 2010).

This relation has not been clarified yet; therefore, further studies are needed for resolving the existing relations between both fungal endophytes.

This is a first report of morphological AM types and DSE in Cuban plants and further studies are also necessary to know a diversity, co-occurrence and distribution of both fungal communities in plants and Cuban ecosystems.

The authors thank Lázaro Hernández (conservation's worker) of RFM San Ubaldo-Sabanalamar. We acknowledge the financial support of frame of the net AMFOODS “Functional links between aboveground changes and belowground activity with land use in the Americas” between 2006-2012.

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Publication Dates

• Publication in this collection
  Sept 2013

History

• Received
  19 Oct 2012
• Reviewed
  2 July 2013
• Accepted
  4 Sept 2013