LANKESTERIANA


and Scrugli (1987) suggested to classify orchid
endophytes in four morphometrical classes: A, B,
C, and D. This classification has no taxonomic
value, but because fungi are studied when they are
into cortical cells, it gives information on fungal
ecology. Three ascomycetes strains with
Rhizoctonia-like anamorphs were isolated from
Pterostylis sp. but no seeds germinated when they
were inoculated with these fungi (Warcup 1975).
Some tropical orchids have ascomycetes as
symbiont (Dreifuss & Petrini 1981 and 1984);
formerly these fungi were described as
Ascorhizoctonia Chin S. Yang & Korf, but since
they are saprobe fungi and do not produce sclerotia
they can not be ascribed to the genus Rhizoctonia
DC. (Andersen 1986). Septum ultrastructure of
Leptodontidium orchidicola Sigler & Currah
(Mitosporic Fungi) isolated from tropical orchids
reveals that this anamorph is related to ascomycetes
in having Woronin bodies near its septum pore.
 One of the first questions about the relationship
between orchids and endophytes deals with the
specificity. Bernard (1909) hypothesised a high
level of specificity but such close relationship was
soon rejected. Burgeff (1936) proposed that
specificity existed between some fungi and
ecological host groups, and Curtis (1937) suggested
a closer relationship between fungus and habitat.
Hadley and Harvais (1967) questioned Curtis'
ecological specificity because not all fungi isolated
from ripe plants were able to support host seed
germination. Further works built up evidence in
favour of the absence of specificity (Downie 1959,
Hadley 1970). Riess and Scrugli (1987) observed
that some orchid species (Ophrys bombyliflora
Link. and Ophrys tenthredinifera Willd.) had dif-
ferent endophytes when collected in different sites.
They also observed, in the same work, that in
Limodorum abortivum (L.) Sw. there were simul-
taneously two endophytes with different morpho-
logical characters. Similar results were obtained
from Curtis (1937), Downie (1943), Talbot and
Warcup (1967), and Harley (1969). Masuhara and
Katsuya (1989, 1994), by studying Spiranthes
sinensis (Persoon) Ames var. amoena (M.
Bieberstein) Hara, suggested two kinds of specifici-
ty: 1) "ecological specificity", i.e. when pelotons


are into root cortical cells or into the protocorms
in nature (in situ); and 2) "potential specificity", i.e.
associations between orchids and fungi in other
conditions, both in vitro or ex vitro. Masuhara and
others (1993) observed ecological specificity only
in some fungi with potential specificity. For
example, Microtis parviflora R. Br. (Orchidaceae)
has a narrow ecological specificity in the field,
while showing a broad potential specificity in
vitro. The factors that contribute to ecological
specificity could be fungal growth and survival in
the soil, which are influenced by environmental
factors, or fungal density in the field (Masuhara
and others 1995). Milligan and Williams (1988; in
Masuhara & Katsuya 1995) suggested that differ-
ences between ecological and potential specificity
could be due to a succession of fungi in orchid tis-
sues, but further investigations are necessary to
confirm this hypothesis.
 Our study was carried out in two steps: 1) isola-
tion and description of Serapias parviflora
endomycorrhizal fungi; 2) description of associa-
tions, in vitro, between S. parviflora and fungi, and
between Spiranthes spiralis and fungi. S. parviflora
was used as control and S. spiralis was used to ver-
ify the existence of specificity between host and
endophyte.

 Materials and methods. Serapias parviflora
roots samples were collected at Allerona Scalo
(Umbria, Italy) on 05/16/1999. The whole plant,
together with a clump of soil, was collected, in
order to prevent root damage. Samples were pre-
served in sterilised envelopes at 50 C until fungal
isolation (two days after collection). To remove
fungi and bacteria from external surfaces, roots
were sterilised by immersion on H202 (30%) for 4
minutes; then they were rinsed 4 times in sterile
distilled water. Roots were cut, 1 cm segments
were sowed on Petri dishes with PDA and strepto-
mycin (a broad-spectrum antibiotic) and then incu-
bated at 20 C. Fungi were observed under phase
contrast microscope, scanning and transmission
electron microscope and confocal laser microscope.
For the latter there is no need of a particular prepa-
ration but fungi stained better when coloured with
acid fuchsin. Fungi observed under TEM were