This paper reports the discovery of circadian rhythm in Omphalina Quelet, in pure culture under laboratory conditions. The species was collected as a mycobiont from an unidentified basidiolichen

Jyoti Kalangutkar

Nandkumar Kamat

English

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Some Studies on Circadian Rhythm in the Culture of Omphalina Quelet sp. (Basidiomycota, Agaricales) A Mycobiont of an Unidentified Basidiolichen

Nature Precedings

  
SOME STUDIES ON CIRCADIAN RHYTHM IN THE CULTURE OF 
OMPHALINA QUELET SP. (BASIDIOMYCOTA, AGARICALES) A 
MYCOBIONT OF AN UNIDENTIFIED BASIDIOLICHEN
JYOTI R. KALANGUTKAR AND NANDKUMAR M. KAMAT, 
DEPARTMENT OF BOTANY, GOA UNIVERSITY
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INTRODUCTION
• Circadian rhythms have been well studied in fungi. So 
far it has been reported only in two basidiomycetes, 
Pellicularia filamentosa and Spaerobolus stellatus.
• It has been reported that fungi exhibiting circadian 
rhythms do so to avoid injury from dessication and 
harmful UV radiations.
• This paper reports the discovery of circadian rhythm in 
Omphalina Quelet, in pure culture under laboratory 
conditions.
• Genus Omphalina includes approximately 40 species, 
eight of which are lichenized.
• Although lichen mycobiont are selective in choice of 
Nostoc symbionts, several fungi may often share 
identical cyanobiont strain.
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Objectives
• Collection and identification of the mushroom 
specimens associated with wet attached algal 
mats
• Confirmation of the phyco and mycobionts
• Tissue culture of Omphalina sp.
• Study of growth and colony morphology
• Assessment of the  effect of light on the culture 
and the spectral characteristics of the pigment
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Materials and Methods
Fresh fruit bodies of Omphalina sp. collected (Goa University 
campus) 
Cleaned with wet cotton 
Specimens microscopically examined to identify the 
phycobiont associated with mycobiont
Pileal tissue from mycobiont removed aseptically
Washed with sterile distilled water
Inoculated on MEA media plates
Incubated at room temperature (28-300 C )
Fresh growth from the tissues subcultured on fresh plates for 
purification and the cultures were maintained on MEA without 
antibiotics and subcultured every month. After screening the 
isolates, fast growing strains were used for further studies.
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Studies on Colony morphology
Sections of  7 day old colonies from three different 
regions, i.e. central, middle and peripheral growth 
zones were asceptically dissected. 
Slides were made in plain Lactophenol, Lactophenol 
with cotton blue and Ammoniacal congo red (glycerol 
as mounting medium) and observed microscopically.
Studies on the pigment:- The pink pigment from the 
colonies was extracted by macerating the agar blocks 
in Ethyl acetate and Methanol and the UV-Visible 
spectra was recorded using Shimadzu double UV-
Visible Spectrophotometer in 190-750 nm range in 
Quartz cuvettes
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Detection of circadian rhythm
The colonies were initially studied on MEA to see 
the response to changing light conditions. Plates 
were then kept under light and dark conditions to 
verify the growth form. Colonies which 
consistently showed concentric growth zones with 
thick and thin bands were scored as positive for 
circadian rhythm. This experiment was repeated 
3-4 times using inoculum of different age and 
from different zones of the colony to verify 
“circadian response” as a dominant character of 
the Omphalina culture.
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Habit & Habitat of the presumed basidiolichen showing 
fructifications of Omphalina sp.
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Results –1. The basidiolichen
Habit: single or groups of two-three
Habitat: Mycenoid. Found growing on greenish, black, 
soft, wet moss, under unidentified grasses and appear only 
between last week of June and first week of July. 
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 Taxonomic diagnosis of Omphalina Quelet tax. sp. 
PILEUS  1-2 cm, convex at first, with disc depressed and margin reflexed, expanding 
to broadly funnel-shaped, but often remaining with a spreading margin; hygrophanous, 
milky white, smooth, prominent striations remaining  moist, context opaque, thin, pliant 
or fragile, whitish, fibrous
GILLS  long decurrent, close to subdistant, rarely distant, narrow at mid-portion to 
moderately broad, forked at times, usually not intervened; white, decurrent, widely 
spaced; fade to  creamy in age 
STIPE  1-2 cm x 0.1-0.4cm, equal or nearly so, central or eccentric, slightly enlarged 
and bulbous at base, stuffed soon hollow, rather brittle, compressed at times; waxy 
white, faintly pruinose at first but soon naked and polished, base with slight white 
mycelium, smoothly transiting and embeded  into a greenish slimy algal mat/moss
SPORE PRINT:- (ON GLASS)Thin, white
BASIDIOSPORES:- (6)7-9(11) x 4-5(6) μms, elliptic to broadly elliptic or oval, 
smooth, inamyloid,  
BASIDIA :-mostly 4-spored but sometimes 1-, 2-, or 3-spored, 21-31(35) x 5-8 μms; 
clamp connections present, spores 7-10 x 4.5-6 μms, almond-shaped
Specimens examined:-GUMH-omph1/04,omph2/04,omph3/04, Coll. By N.Kamat, near 
ponds, Goa University campus, Taleigao, Goa, July 2004
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A mature specimen of 
Omphalina Quelet tax. Sp. 
with attached algal mat 
and soil
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Results 
Micro-morphological features of Omphalina vegetative 
mycelial cultures
All the three zones central, middle and peripheral growth zones 
showed two types of hyaline hyphae, narrow and wide, with narrow 
being the dominant form.
1. Central zone: slow dense growth zone
Some hyphae with large and a few with short cells, associated with 
different types of chlamydospores such as intercalary and terminal. 
Septa with clamp connections. Some special modifications of hyphae 
are seen such as ringlets, spiral and knots.
2.The middle zone showed two growth forms, ”slow 
dense” and “fast effuse”N
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Peripheral 
growth 
zone
Middle 
zone
Central 
zone
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Results 
Middle slow dense zone: 
Hyphae hyaline. Chlamydospores present, but number of 
chlamydospores are less as compared to central slow dense zone. 
Special modifications of hyphae are seen such as knots, ring, H-
shape junctions etc. 
Middle fast effuse zone:  Chlamydospores are completely 
absent in this zone. Special modifications of hyphae such as 
rings, cords and various types of hyphal junctions are seen.
Peripheral slow dense zone: 
Chlamydospores absent. Knots and cords are seen.
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Certain sectors 
of  Omphalina 
colonies 
produce a dark 
pink pigment 
after seven 
days on MEA, 
whereas the 
non productive 
or pigment less  
sectors do not 
show this 
property and 
remain so 
despite 
subculturing on 
fresh medium
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Omphalina cultures respond to 
natural day-night cycle
• Omphalina colonies consistently incubated 
under ambient light showed typical 
concentric zones which corresponded to a 
24 hours day-night rhythm
• The colonies seem photosensitive and 
produce pigment with higher intensity when 
exposed to light as compared to cultures 
grown in dark or in less light
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The effect of light and darkness on Omphalina colonies. A distinct 
concentric growth pattern is seen in the plate incubated under ambient 
light 
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Concentric growth zones in response to circadian rhythm
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A clear, distinct and remarkably 
stable and reproducible circadian 
growth pattern was identified in 
Omphalina colonies
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RESULTS
UV-Visible spectral absorption profile of the pigment in 
Ethyl Acetate and Methanol
TABLE 1. WAVELENGTH AND ABSORPTION (ETHYL ACETATE)
Sr. No Wavelength (nm) Absorbance
1 275.50 2.494
2 253.00 2.288
3 261.50 2.018
4 199.50 0.498
TABLE 2. WAVELENGTH AND ABSORPTION (METHANOL)
Sr.No Wavelength Absorbance
1 274.00 0.216
2 220.50 1.302
3 205.50 1.677
4 531.50 0.003
5 258.50 0.187
6 231.50 1.171Na
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In Ethyl 
acetate
In 
Methanol
UV-Visible spectral profile of the pink pigment from Omphalina sp.
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Discussion 
•This is the first report of an Omphalaceous  
cyano-lichen from India and Goa
•Although specific name has not been assigned 
to the taxa the mycobiont shows close affinity to 
Omphalina ericetorum  (Fr.) M.Lange  
Macromycetes Pt. II, Greenland Agaricales, 
Meddel. om Groenland 147: 25. 1955;  
Phytoconis ericetorum  (Persoon:Fries) 
Redhead & Kuyper,  Gerronema ericetorum  
(Pers.:Fr.) Singer
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The basidiolichen is a novel finding
• It has not been possible to establish the bi/tripartite nature of 
the basidiolichen. The basal mycelium of Omphalina fruit 
bodies was found to be interwoven and ramified in the 
matrix of an unidentified algal species Nostoc. We do not 
rule out the possibility of one more phycobiont. We have 
also noticed the  ecological, evolutionary and 
mycogeographical significance of the basidiolichen and 
would plan more exhaustive studies in future. 
• Very few basidiomycetous mycobionts have been cultured. 
This is first report in the world on a photosensitive 
Omphalina culture in which we have also observed 
mitospores as terminal and intercalary chlamydospores 
Whether such asexual propagules are formed in nature is an 
unanswerable question at present.
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First report in the world on Circadian rhythm in 
a mycobiont basidiomycetes culture
• This is also the first report on Circadian rhythm in 
vegetative stage of cultures of  mycobiont 
basidiomycetes species
• We postulate that Omphalina culture may have a 
dominant ‘Clock gene’ which regulates the circadian 
rhythm  
• The photosensitive cultures also produced a 
characteristic pink pigment which showed higher 
intensity after  exposure to light. This process is 
biochemically and ecologically important.
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Pigment synthesis is related to 
photosensitivity
•The UV absorption of pigment in ethyl acetate at 200, 253, 
262 and 276 nms clearly showed that its synthesis may be a 
protective defence mechanism against natural UV radiation to 
which the mycobiont mycelium might be getting exposed in 
its’ natural habitat
•The circadian clock in Omphalina sp. might exhibit an 
ecologically tuned response considering its symbiotic 
associationship with the phycobiont.
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Thank You
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Some Studies on Circadian Rhythm in the Culture of Omphalina Quelet sp. (Basidiomycota, Agaricales) A Mycobiont of an Unidentified Basidiolichen

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