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Anales de Biología 33: 127-134, 2011 ARTICLE
Morphology, anatomy and mycorrhizae in subterranean parts
of Zeuxine gracilis (Orchidaceae)
Thangavelu Muthukumar1, Eswaranpillai Uma1, Arumugam Karthikeyan2, Kullaiyan Sathiyadash1, Sarah Jaison1,
Perumalsamy Priyadharsini1, Ishworani Chongtham3 & Vellaisamy Muniappan1
1 Root and Soil Biology Laboratory, Department of Botany, Bharathiar University, Coimbatore 641 046, Tamil Nadu, India.
2 Institute of Forest Genetics and Tree Breeding, R. S. Puram, Coimbatore 641 002, Tamil Nadu, India
3 Department of Life Sciences, Manipur University, Canchipur, Imphal 795 003, India
Resumen
Correspondence
T. Muthukumar
E-mail: tmkum@yahoo.com
Received: 27 July 2011
Accepted: 23 November 2011
Published on-line: 14 December 2011
Morfología, anatomía y micorrizas en las partes subterráneas de
Zeuxine gracilis (Orchidaceae)
Zeuxine gracilis (Berda) Bl. es una orquídea terrestre endémica cuya
morfología, anatomía y micorrización es desconocida. A partir de plan-
tas colectadas en la región de los Ghats occidentales se investigó: (a)
anatomía de la raíz y el rizoma; (b) características de los pelos radicu-
lares y patrones de colonización de micorrizas. Los caracteres más re-
levantes en raices fueron: ausencia de velamen y espirantosomas;
exodermis simple y nueve protoxilemas arqueados. Rizoma con epi-
dermis uniseriada, abundantes espirantosomas en células corticales
internas, endodermis con bandas de Caspary y paquetes vasculares
biseriados. Se descubrió la presencia de hongos micorrícicos tanto en
las raíces como en los rizomas. Su entrada es principalmente a través
de pelos radiculares y epidermis del rizoma. Los hongos forman pelo-
tones y células monilioides en el cótex radicular. Ocasionalmente apa-
recieron micorrízas arbusculares (AM), caracterizadas por hifas sifona-
les, vesículas y esporas. La falta de arbúsculos en Z.gracilis indica que
AM no son funcionales.
Palabras clave: Hongos micorrícicos arbusculares, Células
monilioides, Pelotones, Rizoma, Raíz.
Abstract
Zeuxine gracilis (Berda) Bl., is an endemic, terrestrial green orchid
whose morphology, anatomy and mycorrhizal status is unknown. So
we investigated: (a) root and rhizome anatomy; (b) root hair character-
istics and mycorrhizal colonization patterns in Z. gracilis plants collec-
ted from Western Ghats region of southern India. The prominent ana-
tomical characters in the roots were: absence of velamen, spirantho-
somes, and the presence of single layered exodermis and nine arched
protoxylem. The rhizome had an uniseriate epidermis, abundant spir-
anthosomes in the inner cortical cells, a distinct endodermis with cas-
parian strips and biseriate vascular bundles. The presence of fungi
both in the roots and rhizomes was revealed. The entry of fungi was
chiefly through root hairs and through epidermis in the rhizome. Fungi
formed pelotons and monilioid cells in the root cortex. Additionally, ar-
buscular mycorrhizal (AM) fungi characterized by the presence of
aseptate hyphae, vesicles and spores were present occasionally in
roots. The lack of arbuscules in Z. gracilis indicated the AM to be non
functional.
Key words: Arbuscular mycorrhizal fungi, Monilioid cells, Pelotons,
Rhizome, Root.
128 T. Muthukumar et al. Anales de Biología 33, 2011
Introduction
Orchidaceae with around 25,000 to 30,000 species
is one of the largest families among flowering
plants. One of the peculiar features of this family
includes the production of dust-like non endo-
spermous seeds. Mode of seed germination and
plant development in Orchidaceae is unique
among flowering plants, which require association
with the mycorrhizal fungi. Orchids have varied
life-forms like epiphytes, lithophytes, or terrestri-
al. Among these varied life-forms terrestrial orch-
ids constitute less than one fourth number of the
total orchid species.
Zeuxine belonging to the subfamily Orch-
idoideae and tribe Cranichideae is one of the
largest genus among Orchidaceae. Taxa in Zeux-
ine are widely distributed in Asia with a few in
Africa and Australia. In India Zeuxine is represen-
ted by 11 species of which Zeuxine gracilis
(Berda) Bl., and Zeuxine longilabris (Lindl.)
Benth. ex Hook.f., are found in South India
(Hooker 1895). Zeuxine gracilis is a perennial
succulent herb found in shaded woodlands, with
brown creeping rhizome, ascending to erect aerial
shoot with petiolate velvety green leaves (Abra-
ham & Vatsala 1981). Zeuxine gracilis is endemic
to South India.
Published work on the anatomy of Zeuxine is
limited. Stern et al. (1993b) studied the anatomy
of Zeuxine oblonga R. Rogers & C. White and
Zeuxine strateumatica (L.) Schltr., while examin-
ing the vegetative anatomy and systematics of the
subfamily Spiranthoideae in which the tribe
Cranichideae was included at that time. These au-
thors found the vascular bundles in Z. oblonga
and Z. strateumatica arranged in one and two
series respectively and the absence of stored
starch in the former. In this study, Stern et al.
(1993b) also reported ‘spiranthosomes’, a special-
ized type of amyloplast in many species of
Cranichideae including Z. strateumatica.
Under natural conditions orchids are associ-
ated with mycorrhizal fungi belonging to basidio-
mycota and ascomycota (Elena et al. 2010). The
fungal hyphae invade the cortical cells and form
tightly interwoven coils called pelotons character-
istic of orchid mycorrhizae (Smith & Read 2008).
Reports in literature also indicate the association
of arbuscular mycorrhizal (AM) fungi (Glomero-
mycota) with terrestrial orchids. Hall (1976) re-
ported AM-like colonization in the terrestrial
orchid Corybas macranthus (Hook.f.) Reichb.f.,
from New Zealand. Raman & Nagarajan (1999)
surveyed the occurrence of mycorrhizae in six
epiphytic and five terrestrial orchids of Kodaik-
anal tropical forest of Western Ghats, South India.
They found exclusive occurrence of AM associ-
ations in all five terrestrial orchids examined. In-
formation on mycorrhizal association in Zeuxine
is limited. Burgeff (1932) reported mycorrhizal
association in Zeuxine clandestine Bl., Zeuxine
sp., and Zeuxine purpurascens Bl., and indicated a
trend from the autotrophic condition to saprophyt-
ism among these species which were strongly re-
lated to mycorrhizal dependence. Porter (1942) re-
ported mycorrhizal association in Z. strateumat-
ica growing in Florida, USA. Apart from these
there appear to be no information on mycorrhizal
status of other species of Zeuxine.
The objective of the present study was to in-
vestigate the morphology and anatomy of roots
and rhizomes of Z. gracilis and also to record the
mycorrhizal incidence and morphology.
Material and methods
Fresh plant materials of Z. gracilis for our
study were obtained from Top Slip, Indira Gandhi
Wildlife Sanctuary and National Park, Tamil
Nadu, India. This site lies in the Western Ghats
south of the Palaghat Gap, known as Anaimalais
with an area of 958 sq. km. The area lies between
10.13’-10.33’N and 76.49’-77.21’E, 800 m a.s.l.
Rainfall varies between 800-4500 mm.
Fresh plant materials of five individuals col-
lected during May 2011, were preserved in FAA
(9 parts 70% ethanol, 0.5 parts formalin and 0.5
parts glacial acetic acid) for 24h and stored in
70% ethanol before sectioning. Transverse sec-
tions of roots and rhizomes were prepared by free
hand sections and stained with safranin or trypan
blue. Observations were made using an Olympus
BX-51 microscope and images were recorded us-
ing Proges 2 camera.
For mycorrhizal assessment the roots and
rhizomes were cut into 1-cm long bits, cleared in
2.5% aqueous potassium hydroxide at 90 ºC for
45 min., washed in running water, acidified with 5
N HCl and stained with 0.05% trypan blue in
lactoglycerol. The extent of mycorrhizal coloniza-
Anales de Biología 33, 2011 Zeuxine gracilis: anatomy of mycorrhizal subterranean parts 129
tion was estimated according to magnified inter-
section method (McGonigle et al. 1990). Pelotons
were considered intact if the constituting hyphae
were distinguishable and considered degenerating
if the fungal hyphae could not be clearly distin-
guished or found as an amorphous mass. Twenty
1-cm long root bits were floated in water on a
glass slide to measure root thickness and to count
root hairs. Length and width of 50 root hairs, and
fungal variables (pelotons, monilioid cells, and in-
tracellular hyphae) were measured using an ocular
micrometer. We measured only 10 to 12 AM
fungal vesicles and spores due to their infrequent
occurrence. Measurements were presented as
(minimum value-) mean ±S.E. (-maximum value).
Results
Root
Roots arise from the nodal region, off-white,
(1.98-) 2.12 ± 0.001 (-2.29) mm in diameter (Fig.
1A). At the point of attachment to the rhizome, the
roots were enlarged (2.81-) 3.02 ± 0.16
(- 3.33) mm. Epidermis uniseriate bearing unicel-
lular hairs numbering (7-) 20.06 ± 2.77 (-36) mm-1
of root (Fig. 1B). Root hair's single, unbranched
cells (86-) 191.80 ± 19.73 (-270) µm long and 3-4
µm wide. Root hairs were swollen at the base
(Fig. 1C). Exodermis uniseriate with spirally
thickened radial walls (Fig. 1D). Cortex: 16 cells
wide, parenchymatic, and distinguishable into two
layers. Three to four cells wide cortical layer sub-
tending the exodermis constitute the outer layer of
the cortex. This layer is composed of smaller cells
and lacks intercellular spaces, whereas the inner
layer is characterized by triangular intercellular
spaces. Endodermis uniseriate with thin walls;
casparian strips present (Fig. 1G). Pericycle:
uniseriate with evenly thin walled cells. Vascular
cylinder solitary, nine arched. Conductive strands
few celled; vascular elements embedded in paren-
chymatous cells. Pith cells; polygonal, paren-
chymatous, lacking intercellular spaces.
Rhizome
Subterranean, smooth, brown (4.17-) 4.42 (- 4.80)
mm in diameter. Epidermis uniseriate, cells oval
with outer walls thickened. Hypodermis one to
two cells wide. Cortex: 18-20 cells wide, paren-
chymatous, cells rounded with small triangular in-
tercellular spaces. Cells of the inner cortical layer
contain abundant spiranthosomes (Fig. 1E). Endo-
dermis uniseriate, cells tangentially flattened with
casparian strips (Fig. 1F). Vascular cylinder 13
arched with xylem elements arranged in two con-
centric rings. Vascular tissue embedded in the par-
enchyma. Pith cells: parenchymatous, thin walled,
polygonal, with small intercellular spaces.
Mycorrhizal association
Mycorrhizal colonization occurred uniformly
throughout the cortex in roots and rhizomes (Fig.
2A). Fungal hyphae entered the roots by penetrat-
ing root hairs or the rhizodermal cells in roots and
rhizomes (Fig. 2B). Although single fungal hypha
entered each root hair, occasionally more than one
could be seen in a root hair (Fig. 2B). Fungal
entry into roots or rhizomes was not characterized
by the presence of vesicular or appresorial struc-
ture. No fungal structures were found in the endo-
dermis or in the stellar portion of roots and
rhizomes. Intact pelotons were abundant in the
young regions of the roots and rhizomes (Fig.
2C). Colonization in older parts of the roots and
rhizomes were disintegrating, the pelotons col-
lapsing into clumps and no recolonization of the
host cell was found (Fig. 2F). Sizes of the intact
pelotons were (109-) 158.04 ± 10.33 (-264)
× (23-) 56.42 ± (-91) µm. The fungal hyphae
within the root cortex were, hyaline, smooth, (2-)
3.0 ± 0.58 (-4) µm wide and those of the rhizomes
were (6-) 4.67 ± 0.67 (-8) µm wide. Major part of
the pelotons in the roots and rhizomes were trans-
formed into monilioid cells (Fig. 2D, E). The di-
mensions of the fungal monilioid cells were (12-)
14 ± 1.15 (-16) × (8-) 9.33 ± 1.33 (-12) µm in
roots, and (14-) 15.33 ± 0.67 (-16) × (10-) 12.67 ±
1.76 (-16) µm in rhizomes. The nucleus of colon-
ized cells was enlarged and distorted (data not
shown). Roots of Z. gracilis showed a uniform
and high degree of colonization of (58-) 67.33
± 7.42 (-82) % compared to (24-) 28.67 ± 2.90
(- 34) % in the rhizome. In root non-septate inter-
cellular hyphae and intracellular Glomus-like ves-
icles were evident (Fig. 2G). The vesicles were
oval (80-) 92.53 ± 6.52 (-105) × (30-) 38.26 ±5.67
(-45) µm. AM fungal spores measuring (45-)
62.01 ± 4.08 (-89) × (50-) 61.92 ± 4.17 (-94) µm
were also observed in roots containing abundant
130 T. Muthukumar et al. Anales de Biología 33, 2011
Figura 1. Hábito, raíz y rizoma de Zeuxine gracilis. A: Hábito mostrando el rizoma (r), raíz (ro) y alargamiento radicular (re). B: Sección
transversal de la raíz mostrando los pelos radiculares (rh), la rizodermis (ep), la exodermis (ex) y el córtex (cr). C: Un pelo radicular con base
hinchada (rhb). D: Celulas de la exodermis (ex) en la raíz con espesamientos espirales (flechas). E: Espirantosomas (s) en las células
corticales del rizoma; las flechas indican granos de almidón procedentes de espirosomas rotos. F: Corte transversal del rizoma mostrando el
córtex interno con las ceĺulas llenas (cr) de los espirantosomas (s), la endodermis (en) con haces vasculares agrupados en dos series (flechas
simples y dobles). G: Corte trnasversal de la raíz, cerca del cilindro central, mostrando el córtex (cr), la endodermis con bandas de Caspary
(en) y el cilindro vascular (vb). Líneas de escala: A=55cm, B-E = 50µm, F, G = 100 µm.
Figure 1. Habit, root and rhizome of Zeuxine gracilis. A: Habit showing rhizome (r), root (ro) and root enlargement (re). B: Cross-section of
the root showing root hairs (rh), rhizodermis (ep), exodermis (ex) and cortex (cr). C: A root hair with a swollen base (rhb). D: Exodermis
cells (ex) in the root with spiral thickenings (arrow heads). E: Spiranthosomes (s) in rhizome cortical cell, arrow heads indicate starch grains
from ruptured spiranthosomes. F: Cross-section of the rhizome showing inner cortex with spiranthosomes (s) filled cells (cr), endodermis
(en) with vascular bundles arranged in two series (single and double arrowheads). G: Cross-section of the root near the central cylinder
showing cortex (cr), endodermis with casparian strips (en) and vascular cylinder (vb). Scale Bars: A=5cm, B-E = 50µm, F, G = 100 µm
Anales de Biología 33, 2011 Zeuxine gracilis: anatomy of mycorrhizal subterranean parts 131
Figura 2. Colonización micorrícica en Zeuxine gracilis. A: Sección trnasversal del rizoma mostrando la zona de colonización. B: Un pelo
radicular (rh) con hifa (h). C: Pelotones (p) intactos en las células corticales de la raíz. D: Células monilioides (ms) e hifas pelotónicas
normales (flechas) en rizoma. F: Petotones degenerados (dp) en las células corticales de la raíz. G: Vesículas (v) de micorrízas arbusculares
en las células corticales de la raíz. H: Esporas (sp) de micorrízas arbusculares y pelotones degenerados (dp) en raíz. Líneas de escala: A, C,
D, F = 100 µm, B, E, G, H = 50 µm
Figure 2. Mycorrhizal colonization in Zeuxine gracilis. A: Cross-section of the rhizome showing colonization zone (iz). B: A root hair (rh)
with fungal hyphae (h). C: Intact pelotons (p) in root cortical cells. D: Monilioid cells (ms) in the cortical cells of rhizome. E: Monilioid
cells (ms) and normal pelotonic hyphae (arrow heads) in rhizome. F: Degenerating pelotons (dp) in root cortical cells. G: Vesicles (v) of
arbuscular mycorrhizal fungi in root cortical cell. H: Spores (sp) of arbuscular mycorrhizal fungi and degenerating peloton (dp) in root. Scale
Bars: A, C, D, F = 100 µm, B, E, G, H = 50 µm
132 T. Muthukumar et al. Anales de Biología 33, 2011
degenerating pelotons (Fig. 2H). The extent of
such AM fungal colonization was <5% of the root
length. Neither vesicles nor spores of AM fungi
were observed in rhizomes.
Discussion
Substantial gap in our knowledge on orchid mor-
phology, anatomy and mycorrhizal association
persist in spite of several decades of intense re-
search. For example, very limited taxa from each
genus have been examined for their anatomy or
mycorrhizal status. Root hair characteristics are
unknown in many orchids. Root hairs of Z. gra-
cilis with wider base resemble the root hairs of
Stenorrhynchos speciosus (Jacq.) L. C. Rich. ex
Spreng., of the same tribe (Stern et al. 1993b).
While the roots of Z. gracilis resemble Phaius
tankervilliae (Banks ex Ľ Herit.) Blume., in their
size, the root hairs of P. tankervilliae are much
longer and thicker compared to Z. gracilis
(Muthukumar & Sathiyadash 2009). Root hair
length and density are controlled by both environ-
mental and genetical factors of the plant (Datta et
al. 2011). Among the different environmental
factors, nutrient availability appears to be a major
factor in determining root hair abundance (Datta
et al. 2011). It is well acknowledged that plant
with long and dense root hairs absorb more nutri-
ents from the soil than those with relatively short
root hairs.
Root anatomy of Z. gracilis resembles those of
Z. strateumatica in the lack of velamen and the
presence of nine arched protoxylem arms (Porter
1942, Stern et al. 1993b). There appears to be sub-
stantial variation in these characters in the genus
Zeuxine. For example, one layered velamen and
14 armed protoxylem have been reported in Z. ob-
longa (Stern et al. 1993b). Our observation of an
uniseriate exodermis with scalariform thickenings
on the radial walls of Z. gracilis agrees with the
observations of Figueroa et al. (2008) in several
members of Cranichideae. The cortical layers in
the root are within the range of 8-25 reported for
Cranichideae. However, the heterogeneous cortex
resembles those of Goodyera pubescens (Willd.)
R. Br. (Stern et al. 1993b). Stern et al. (1993b) in-
dicated the embedment of one or two strands of
vascular tissue in the pith region of Z. strateumat-
ica, whereas such strands were not evident in Z.
gracilis. The supraendodermal spaces that has
been reported in roots of several members of
Cranichideae (Figueroa et al. 2008) were absent in
roots of Z. gracilis.
The arrangement of vascular bundles in two
series in the rhizome of Z. gracilis is in line with a
previous study by Stern et al. (1993b), who also
reported a similar arrangement in the aerial stems
of Z. oblonga. The rhizome of Z. gracilis differs
from the aerial stems of Cranichideae members in
the presence of a distinct endodermis with cas-
parian strips. This is in contrast to the aerial stems
of Zeuxine spp., where an identifiable endodermal
layer or endodermis is absent (Stern et al. 1993b).
The rhizome of Erythrodes hirtella (Sw.) Fawc. &
Rendle also has a true endodermis with casparian
strips though, the aerial stem has only endo-
dermoid layer (Stern et al. 1993b). Although raph-
ides have been reported in cortical cells of Z. ob-
longa, these were absent in the cortex of Z. gra-
cilis.
Stern et al. (1993a) described spiranthosomes,
a unique form of amyloplasts from cortical cells
of Spiranthoideae. Spiranthosomes were described
in the root cortical cells of several tribes of the
sub family Spiranthoideae, including Cranichi-
deae. In Z. gracilis, we did not observe any spir-
anthosomes in the cortical cells of the root. There
appears to be some inconsistency in the occur-
rence of spiranthosomes in the roots of Zeuxine
spp. For example, Stern et al. (1993a,b) noted the
presence of spiranthosomes in roots of Z. strateu-
matica, but not in Z. oblonga. However, spir-
anthosomes occurs in abundance in the inner cor-
tical cells of the rhizomes in Z. gracilis. This in
accordance with Schmucker (1927) who also re-
ported the occurrence of spherical bodies resem-
bling spiranthosomes in the rhizomes of Hae-
maria discolor A. Rich. Spiranthosomes have also
been reported in the aerial stems of Vrydagzynea
pachyceras Schltr., Prescottia stachyodes (Sw.)
Lindl., and Pelexia laxa (Poepp. & Endl.) Lindl.
(Stern et al. 1993b).
The entry of fungi into roots and rhizomes is
similar to those observed in earlier studies (Sen-
thilkumar et al. 2001, Látr et al. 2008, Muthuku-
mar & Sathiyadash 2009). The presence of my-
corrhizal fungi in the rhizome of Z. gracilis agrees
with the studies (Warcup 1985, Yagame et al.
2008) where the occasional presence of mycor-
rhizal fungi had been found in this plant part.
Anales de Biología 33, 2011 Zeuxine gracilis: anatomy of mycorrhizal subterranean parts 133
Porter (1942) also found abundant colonization in
the rhizomes of in vitro raised Z. strateumatica
plantlets. In contrast, Látr et al. (2008) reported
the lack of mycorrhizal fungi in unbranched
rhizomes of Cephalanthera longifolia (L.) Fritsch.
The fungi formed monilioid cells in the cortical
cells of rhizomes and roots in addition to pelotons.
An important event in the orchid mycorrhizal as-
sociation is the lysis of the fungal pelotons. Lysis
is initiated sequentially in the oldest colonized
cells followed by cells that have been sub-
sequently colonized (Senthilkumar et al. 2001).
Peloton lysis in Z. gracilis occurs in distinct zones
and not randomly as reported in Spathoglottis
plicata Blume (Senthilkumar et al. 2001). Like
many autotrophic orchids, the mycorrhiza in Z.
gracilis is of tolypophagy in which the fungal
hyphae are digested, and the organic products
from the fungal mycelium are transferred to the
plant (Burgeff 1932). Recolonization of cells after
peloton digestion has been observed in earlier
studies. Senthilkumar et al. (2001) reported the
presence of pelotons of up to three generations in
the root cortical cells of S. plicata. Kristiansen et
al. (2001) also reported recolonization of Neuwie-
dia veratrifolia protocorm cells containing disin-
tegrating pelotons. However, such recolonization
of cells containing disintegrating pelotons was not
observed in the present study. Average coloniza-
tion in Z. gracilis is higher compared to those re-
ported for other ground orchids like P. tankervilli-
ae (51%), S. plicata (57%), C. longifolia (<5%)
and Dactylorhiza majalis (Rchb.f.) P.F. Hunt &
Summerh. (12%) (Senthilkumar et al. 2001, Látr
et al. 2008, Muthukumar & Sathiyadash 2009).
Katiyar et al. (1985) recorded a colonization dens-
ity of 69-97% in the 12 species of terrestrial orch-
ids they examined from north eastern India. As
methods used for estimating colonization in these
studies are different comparisons are difficult.
Mycorrhizal fungi that colonize the roots of
orchids belong to basidiomycota or ascomycota
(Elena et al. 2010). However, the presence of AM
fungal structures in roots of Z. gracilis is in ac-
cordance with studies (Hall 1976, Raman &
Nagarajan 1999) where their presence in orchid
roots was noted. The lack of arbuscules in AM
roots of Z. gracilis is in accordance with Hall
(1976) who also found only Glomus-type vesicles
and hyphae, but not arbuscules in the terrestrial
orchid C. macranthus. However, these observa-
tions contradict those of Raman & Nagarajan
(1999) who reported the occurrence of arbuscules
in Anoectochilus elatus Lindl., and Habenaria el-
liptica Wight growing in the Kodaikanal forests of
the Western Ghats in southern India. Though the
absence of arbuscules suggests that AM in Z. gra-
cilis is non-functional, further observations in-
volving more number of plants and over different
seasons are essential to ascertain this. DeMars &
Boerner (1995) proposed that the presence of AM
in non-host root is the primary consequence of
progressive root senescence and colonization
spread from adjacent AM plants. In addition, sen-
escing roots and organic matter are known to act
as a niche for AM fungal sporulation (Nasim
2010).
In conclusion, our observations on the root and
rhizome anatomy of Z. gracilis agree in general
with observations on anatomy in members of
Cranichideae by Stern et al. (1993b) and Figueroa
et al. (2008). Nevertheless, the role of root hairs in
plant nutrient uptake in terrestrial orchids needs to
be explored. Similarly, the role of mycorrhizal
fungi on plant growth and nutrient uptake in Z.
gracilis needs to be ascertained experimentally.
References
Abraham A & Vatsala P. 1981. Introduction to orchids.
Trivandrum, India: Tropical Botanic Garden and Re-
search Institute.
Burgeff H. 1932. Saprophytismus und Symbiose. Jena.
Datta S, Kim CM, Pernas M, Pires ND, Proust H, Tam T,
Vijayakumar P & Dolan L. 2011. Root hairs: develop-
ment, growth and evolution at the plant-soil interfa-
ce. Plant and Soil 346: 1-14.
DeMars B & Boerner REJ. 1995. Arbuscular mycorrhizal
development in three crucifers. Mycorrhiza 5: 405-
408.
Elena V, Gastaldo A, Tondello A, Baldan B, Villani M &
Squartini A. 2010. Identification of two fungal endo-
phytes associated with the endangered orchid Or-
chis militaris L. Journal of Microbiology and Biotech-
nology 20: 630-636.
Figueroa C, Salazar GA, Zavaleta HA & Engleman EM.
2008. Root character evolution and systematic in
Cranichidineae, Prescottiinae and Spiranthinae (Or-
chidaceae, Cranichideae). Annals of Botany 101:
509-520.
Hall IR. 1976. Vesicular arbuscular mycorrhizas in the
orchid Corybas macranthus. Transactions of the Bri-
tish Mycological Society 66: 160.
Hooker JD. 1895. A century of Indian orchids. Calcutta,
India: Annals of the Royal Botanical Garden Calcu-
tta.
134 T. Muthukumar et al. Anales de Biología 33, 2011
Katiyar RS, Sharma GD & Mishra RR. 1985. Studies on
mycorrhizal associations in terrestrial orchids. In Bio-
logy, conservation and culture of orchids. (Vij SP,
ed.).New Delhi: East - West Press Ltd, pp. 63-70.
Kristiansen KA, Rasmussen FN & Rasmussen HN.
2001. Seedlings of Neuwiedia (Orchidaceae subfa-
mily Apostasioideae) have typical orchidaceous my-
cotrophic protocorms. American Journal of Botany
88: 956-959.
Làtr A, Čuřìkovà M, Balàž M & Uurčàk J.2008. Mycorrhi-
zas of Cephalanthera longifolia and Dactylorhiza
majalis, two terrestrial orchids. Annales Botanici
Fennici 45: 281-289.
McGonigle TP, Miller MH, Evans DG, Fairchild GL &
Swan JA. 1990. A method which gives an objective
measure of colonization of roots by vesicular–arbus-
cular mycorrhizal fungi. New Phytologist 115: 495–
501.
Muthukumar T & Sathiyadash K. 2009. Mycorrhizal mor-
phology of Nun’s orchid [Phaius tankervilliae (Banks
ex L’ Herit.) Blume]. Mycorrhiza News 21: 9-11.
Nasim G. 2010. Decaying leaf sheaths of wheat-an unu-
sual niche for Glomus monosporum. Pakistan Jour-
nal of Botany 42: 1313-1316.
Porter JN. 1942. The mycorrhiza of Zeuxine strateumati-
ca. Mycologia 34: 380-390.
Raman N & Nagarajan N. 1999. Mycorrhizal association
of orchids in a tropical forest of southern India. Jour-
nal of Tropical Forest Science 11: 548-553.
Schmucker T. 1927. Beiträge zur Kenntnis einer me-
rkwürdigen Orchidee, Haemaria discolor Lindl. Flora
121: 157-171.
Senthilkumar S, Saravanakumar P, Krishnamurthy KV &
John Britto. 2001. Morphological and structural fea-
tures of mycorrhizal roots of Spathoglottis plicata
and Dendrobium species. Phyta 5: 1-6.
Smith SA & Read DJ. 2008. Mycorrhizal symbiosis.
Cambridge: Academic Press.
Stern WL, Aldrich HC, McDowell LM, Morris MW & Pri-
dgeon AM. 1993a. Amyloplasts from cortical root ce-
lls of Spiranthoideae (Orchidaceae). Protoplasma
172: 49-55.
Stern WL, Morris MW, Pridgeon AM & Dressler RL.
1993b. Comparitive vegetative anatomy and syste-
matics of Spiranthoideae (Orchidaceae). Botanical
Journal of the Linnean Society 113: 161-197.
Warcup JH. 1985. Rhizanthella gardneri (Orchidaceae),
its Rhizoctonia endophyte and close association with
Melaleuca uncinata (Myrtaceae) in Western Austra-
lia. New Phytologist 99: 273-280.
Yagame T, Yamato M, Suzuki A & Iwase K. 2008. Cera-
tobasidiaceae mycorrhizal fungi isolated from non-
photosynthetic orchid Chamaegastrodia sikokiana.
Mycorrhiza 18: 97-101.
