Octubre 2001 PACETTI and RIESS Endophytes of Serapias paiiillora and Spiranthes spiralis


 Figure 1. B-Sepa-1 monilioid cells (SEM, x 2000).

treated as follows: growth on liquid medium, fixa-
tion for 2 hours in para-formaldehyde/glutaralde-
hyde (2%/2.5% v/v) in phosphate buffer 0,025 M
pH 7 at 25 C, rinsed 3 times in phosphate buffer
0,025 M, postfixation in OsO4 1% in 0,05 M phos-
phate buffer v/v for 12 hours at 40 C, dehydration
in a graded ethyl alcohol series for 15 minutes each
(10%, 30%, 50%, 75%) and for 1 hour in pure ethyl
alcohol, embedding on Spurr's epoxy resin and
polymerisation for 8 hours at 70 C. Thin sections
(70 nm), stained with uranil acetate, 10% v/v in
50% ethanol (v/v) for 7 minutes at 70 C and fresh-
ly prepared lead citrate (1% v/v on 50% ethanol
v/v) for 12 minutes at 25 C were observed.
 Fungi were inoculated in S. parviflora plants to
confirm symbiosis and in Spiranthes spiralis
plants to establish specificity. All plants were
asymbiotically grown from seeds (modified Frosch
medium) and transplanted on medium for symbiot-
ic growth (modified Basic Oats medium, Riess and
Pacetti, 2001) before inoculation.

 Results. Many fungi were isolated from roots of
Serapias parviflora but only two strains had char-
acters of symbiotic fungi: mycelium with septa,
monilioid cells, sclerotia, without asexual spores
(grown on PDA until substrate exhaustion). Two
fungi were able to form, in vitro, typical pelotons in
S. parviflora and Spiranthes spiralis root cortex
cells. They were called B-Sepa-1 and A-Sepa-1.
 B-Sepa-1 (PDA) produces floccose to veluti-
nous colonies, without water-soluble pigments and
substrate pigmentation. Mycelium superficial and


 Figure 2. B-Sepa-1 sclerotium (SEM, x 1000).

from white to light grey. On PDA, monilioid cells
(fig. 1) and sclerotia (fig. 2) are differentiated.
Vegetative hyphae hyaline, septated, with constric-
tion on branched point and with smooth wall (fig.
3). Thin wall and hyaline monilioid cells, from
ellipsoidal to spherical, 4,5 x 6,5 mm, organised in
septated chains and branched or linear chains (fig.
4). Sclerotia torulose, 120 x 70 mm. Dolipores
always present under TEM (fig. 5).
A-Sepa-1 (PDA) consisting of floccose and from
light yellow to yellow colonies with white to light
grey micelium on peripheral area. Uncoloured exu-
date, from dark grey to black water-soluble pig-
ments and monilioid cells are produced. Vegetative
hyphae hyaline, septated, with smooth walls.
Hyaline monilioid cells are differentiated. Spherical
to irregular, 4,5-15 x 6,5-15 mm, organised in lin-
ear and never branched chains. Thick and irregular
electron dense layer around hyphae (fig. 6),
Woronin bodies near septum pore (fig. 7).
 Symbiosis between S. parviflora and B-Sepa-1
was observed under microscope. In these associa-
tions there is a massif fungal penetration of roots,
preferentially through hairs (fig. 8) but in some
case there is epidermal penetrations too.
Subepidermal invasion is confined to the first two
cortex layers. Pelotons and digested pelotons can
be observed from the third to the eighth cellular
layer. In some sections we can find both pelotons
(fig. 9) and digested pelotons (fig. 10) in the same
cell. We can also assume an infective cyclical pat-
tern by observing mycelium intercellular connec-
tions (fig. 11).


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