Indian Journal of Chemistry

Vo1.39A, July 2000, pp. 697-702

Growth patterns and light induced kinetics of the fungi

Cephalosporium sacchari on agar plates
Ishwar Das* & Alpana 8ajpai
Chemistry Department, D.D.U. Gorakhpur University
Gorakhpur 273 009, India

Received 15 December 1999; revised 10 May 2000

Growth patterns and growth kinetics of the fungi Cephalosporium sacchari on agar surface have been investigated. The
mycelium of fungi grows similar to non-living systems. The growth patterns have been developed on different culture media. The
kinetic data obey the simple equation d = mt +c where m and c are slope and intercept respectively and t is the time. The growth
was found to be light induced. Dependence of pH of media on morphology and growth of the microorganism has been studied.
Two similar and dissimilar mycelia discs have been inoculated together at a fixed distance and the influence on patterns have
been studied. The contact zone has been characterised by a lack of spores abbreviated as inhibition pattern. The suppressed
sporulation at the contact zone is due to competition over nutrients.

Chemical reactions far from equilibrium may produce

interesting temporal and spatial structures such as chemi
cal waves and periodic precipitation processes 1 . 1 1 , oscil
latory chemical reactions and multiple stationary states 1 2 .
Periodicity has been observed in a great variety such as
rhythmic bands in agates, concentric layers in gallstones,
periodic depos i t i on i n polymeric systems, and
rhythmicity in bacterial growth 13 The process of rhyth
mic growth and reproduction is also important ecologi
cally. Macroscopic zonation patterns in culture of
filamentous fungi are known and reported in the litera
ture 1 4 . Fungi are of considerable interest due to their
uses in medicine including the synthesis of vitamins,
antibiotics, production of plastic materials, destruction
of organic wastes and utilization of fungi as food 15 and
processing of food products. Filamentous fungi due to
their ability to secrete large amount of protein are used
extensively for the production of industrial enzymesl6.
In particular concentric rings and spirals have been re
ported for certain fungi e.g. Necteria cinnabarina and
Penicillium diversumI 7IK similar to those observed in
non-living systems. In view of this wide variety of ap
plications, interest in the non-equilibrium growth pat
terns and similarities with bacterial growth, we report in
the present paper the morphologies and growth of the
fungi Cephalosporium sacchari which has relevance
from the view point of certain disease of sugarcane at
different experimental conditions including pH of the
medium, light and interaction of other mycelium present
in its neighbourhood.

Materials and Methods

Preparation ofculture media : Five different media were

prepared with the following compositions (i) potato-dex

trose-agar (PDA) medium was prepared by boiling po
tato (250 gil) in water containing dextrose (20 gil) and
agar agar ( 1 5 gil) ; (ii) tomato-dextrose-agar (TDA) me
dium contained tomato (250 gil) in place of potato, other
ingredients were the same; (iii) maize medium contained
maize (60 gil) and agar agar (20 gil) in water; (iv) czapek
medium was prepared by dissolving the following in
water NaN0 (3.0 gil), HP04 ( 1 .0 gil), MgS04 .7Hp
3
(0.5 gil), KCI (0.5 gil), FeS04 .7Hp (0.01 gil), sucrose
(30 gil) and agar agar ( 1 5 gil), pH was adjusted to 7.3
and (v) oat meal agar contained oat meal (60 gil) and
agar agar (20 gil).
All ingredients of above media were mixed thoroughly
and sterilized in an autoclave for 30 minutes. After steri
lization, media were allowed to cool, antibiotic penicil
lin (ampicillin) was added to each medium and poured
into petri dishes.

Development of growth patterns in different media

Discs of 4.0 mm diameter were cut from the myc

elium. Each disc was transferred to a glass petridish (90
mm diameter) containing 20 m!. of medium and put in
an incubator maintained at 30 1 .0 "c. Experiments were
performed in triplicate. Growth patterns observed were
shown in Fig. 1 .

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INDIAN J CHEM, SEC. A, JULY 2000

Fig I -

Growth patterns of Cephalosporium sacchari developed on different media at 371 "C.

(a) Czapek medium; (b) PDA medium; and (c)TDA medium

Table I Values of slope (m), intercept (c) and correlation coefficient (R) used in the equation d = m t + c obtained at different experimen
tal conditions at 37OC.
-

Medium

pH

Slope
(m)

Intercept
(c)

Correlation
coefficient (R)

em/day

cm

Potato - dextrose - agar agar

0.390

-0. 108

0.996

Tomato- dextrose-agar agar

0.357

-0. 1 1 8

0.998

Czapek-agar agar

0.334

-0.009

0.998

Maize-agar agar

0.332

-0. 1 53

0.998

0.30 1
0.558
0.609
0.576

-0. 101
-0.384
-0.400
-0.484

0.993
0.990
0.996
0.995

Oat meal-agar agar

6.80
3.96*
6.80*
1 1 .0*

*Observations taken at 3 1 .0 C

Growth kinetics

After inoculation of fungi Cephalosporium sacchari

into different media, growth kinetics were studied with
the help of a travelling microscope accurate to 0.001
cm. Results are recorded in Fig. 2

Influence ofpH on the morphology and growth kinetics

Influence of pH of the medium on growth patterns
and kinetics has been studied. OMA medium was se
lected for this purpose: Experiments were carried out on

OMA medium of various pH viz. 3 .96, 6.8, 8.0 and 1 1 .0.

pH of culture medium was measured with a digital pH
meter. Radii of the circular growth envelop was meas
ured at different time intervals with the help of a travel
ling microscope and growth patterns are recorded in Figs
3 and 4.

Influence of Light

Light is absolutely essential for the formation of vari

ous types of reproductive organs. To study the influence

DAS et al.: GROWTH KINETICS OF Cephalosporium sacchari ON AGAR PLATE

[, .0

699

3.5
3.0
E

2 .5

'i0

2.0

..

(!)

1.5
1 .0
0.5
0.0

Fig 2 -

Ii m e ( days )

Growth kinetics data for Cephalosporium sacchari on different media.[Il)

TDA, [0) PDA, [()OMA, [0 )Maiz agar IrD Czapek media.

of light on the growth behaviour and pattern of the fungi,

TDA medium was taken into petri dishes inoculated and
kept in complete darkness, while other petri dish was
kept in the presence of visible light at the same tempera
ture. Growth was measured employing the usual tech
nique as described earlier. Results are recorded in Fig.5.

Inhibition patterns

To study the dependence of hyphal growth and dif

ferentiation on adjacent hyphae, interacting hyphae are
experimentally generated by choosing two inoculating
centers at a fixed distance. Similar and dissimilar mi
croorganism discs were inoculated on a plate at a 3.0
cm. apart. Growth patterns were observed after 7 days
and photographed. Results are recorded in Fig.6.
Results and Discussion

We have examined the role of experimental condi

tions with respect to macroscopic pattern formation of
the fungi Cephalosporium sacchari on agar plates con
taining (i) potato and dextrose (ii) tomato and dextrose
(iii) NaN0 , K 2HP04 , MgS04 .7Hp FeS04 .7Hp, KCI
3
and sucrose (Czapek's medium)(iv) maize and (v) oat

meal. The media contained essentially carbon source and

small amount of inorganic nutrients. Carbon compounds
serve two essential functions in the metabolism offungi:
(i) It supplies the carbon needed for the synthesis of pro
teins, nucleic acid, cell wall membrane, reserve food etc;
and (ii) the sole source of appreciable amount of energy
is the oxidation of carbon compounds. In addition to car
bon source, certain i norganic micronutrients are also re
quired depending on the nutrients supplied. Fungi can
produce a great variety of complex organic compounds.
Typical growth patterns developed on PDA, TDA, and
Czapek agar media are shown in Fig. 1 . During the myc
elium development, nutrients are taken up and fungus
changes the ionic and molecular profile in the surround
ing medium. Periodicity is observed in almost all cases.
The growth occur linearly as evident by the growth ki
netic results shown in Fig. 2. The data obey simple em
pirical equation d = m t +c where d is the distance of
the hyphal tips from the centre (radius of the circular
envelop) which varies with till1;e t and c is the intercept
of the straight line on the y axis. Values of slope (m)
intercept (c) and correlation coefficient (R) for different
media are recorded in Table 1 . Figure 2 shows the re-

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INDIAN J CHEM, SEC. A, JULY 2000

Fig 3 -

Growth patterns of Cephalosporium sacchari developed on OMA plates contain

ing media of different pH at 301"C. pH [a] 3.96 , [b) 6.8 and [c ) 1 1 .0
4.0

3,0

,Ii '

i
.s:

2.0

, 1 .0

Tl m a

( d oys )

Growh kinetics data for Cephalosporium sacchari on

OMA plates containing media of different pH : [0 ] 3.96,
[A ] 6.8, I)] I 1 .0
suits obtained on different culture media. Results re
corded in Table 1 shows that the growth is maximum in
PDA medium
Growth patterns at different pH of the OMA medium
are shown in Fig 3. Plates i-iii show the morphology at a
lower pH value (3 .96), normal value (6.8) and t rla
tively higher value ( 1 1 .0) respectively. Dense radial like
colony is observed at low and normal pH (plates a,b)
while sharp transition to rhythmic pattern is noticed at
pH 1 1 (plate c). It grows best in the mediu having pH
.
.
in the normal range and declines 10 the aCidiC (3.96) or
in alkaline medium (PH 1 1 .0). Growth kinetic data at
different pH are recorded in Fig. 4. The data obey the
simple relation d = m t +c as evident by correlation coFig'. 4 -

Fig 5 -

Time

(td

Growth kinetic data for Cephalosporium sacchari

in presence of light [0] and in the complete dark
ness [ 0 ]at 30.IoC

efficient values. The values of m and c depend on pH of

medium. Results are recorded in Table 1 .
Growth kinetics of the fungi Cephalosporium sacchari
on IDA medium in the presence and absence of light
have been studied and results are shown in Fig.5. Re
sults show that the growth is linear and light induced.
Further the zonation is observed in the presence of light
at the given experimental condition. It suggests that the
fungus sporulats relatively faster in the light.
When petridishes were inoculated with two similar
or dissimilar mycelia at a certain distance on a petridish,
they grow and eventually meet each other as shown in
Fig.6. Plates show the growth patterns when the two in-

DAS et al.: GROWTH KINETICS OF Cephalosporium sacchari ON AGAR PLATE

a
Fig 6 -

70 1

Inhibition patterns of Cephalosporium sacchari on TDA medium when inoculated with (a) Cephalosporium
sacchari, (b) Fusarium oxysporum and (c) Curvularia pallescens at a fixed distance of 3.0 cm.

oculating discs were of similar pathogen, Cephalospo

rium sacchari (Plate i) or the two discs of dissimilar
pathogens Cephalosporium sacchari-Fusarium
oxysporum and Cephalosporium sacchari - Curvularia
pallescens respectively (plates ii and iii, Fig. 6). The con
tact zone is characterised by a lack of spore due to com
petition over nutrients. Plate (iii) does not show such a
lack of sporulation at a contact zone, probably there is
no such competition for nutrients.
Influence of additional hypha on the development of
a mycelium was studied. Inoculating a petridish with two
or more discs is giving rise to two or more mycelia grow
ing towards and eventually meeting each other. The con
tact zone is characterised by a lack of spore (inhibition
pattern). The suppressed sporulation is due to competi
tion over nutrients.

Physical explanation of ring-type structure

Rings of this general type have been observed in great

variety which include, periodic precipitation of sparingly
soluble salts l -? , growth of the bacteria Pr vulgaris13 and
several others. Explanation for the periodic precipita
tion phenomena was given by several workers based on
a) the supersaturation theory l 9, b) competitive particle
growth modeFo and c) sol coagulation modeFI . Accord
ing to the sper saturation theory which is most com
mon amongst the three, the two reactants A and B dif
fuse from opposite directions and coexist in the gel until
the solubility product reaches a critical value above which
the nucleation occurs according to the reaction,
A +B

AB (solid)

Once the nucleation has started, a depletion of con

centration of A and B occurs in the surrounding. Hence
at this stage, the formation of precipitate stops nuclea
tion in the neighbourhood, precipitation is also stopped
as diffusion continues further. This is followed by the
formation of clear spaces. Diffusion of ions persists and
after a certain stage, the critical supersaturation exceeds
and another band is formed in a similar manner. The
process is repeated until the supply of one of the electro
lytes is exhausted.
In a more recent version of the theory22 the picture is
modified. The two species A and B are assumed to react
to produce a new species C which also diffuses in to the
gel. Subsequently nuclei are formed followed by crys
tallisation according to the following scheme,
A+B C
C nuclei (n)
nuclei crystal (D)
when the local concentration of C reaches some thresh
old value, nucleation occurs and subsequently D clus
ters are formed. Modification of the above model was
made by Polezhaev and Mullerl l .
Colonies of Pr vulgaris were also observed to pos
sess concentric rings. However, a different mechanism
was suggested. The bacteria which is normally sparsely
flagellated alters itself to acquire large number of flagella.
In addition there is cessation of cell division due to inhi
bition of septum formation leading to formation of multi
nucleate, densely flagellate, nonseptate cells. The growth
could also be thought of primarily as a consequence of
nutrient diffusion and local exhaustion.

INDIAN J CHEM, SEC. A, JULY 2000

702

B acteria first spread in search of nutrient contained

in the medium and would consume that nutrients not only
within the circle of residence but by creating a diffusion
gradient, within a ring of certain thickness. Within that
ring bacteria could not subsequently flourish. The proc
ess would then be repeated.
In the case of fungi the growth occurs only at hyphal
tips. A hyphal element arises from the out growth of a
single spore. Upon germination the growth of the

Acknowledgement
Thanks are due to the U.G.C., New Delhi for sup
porting the investigation. Authors also wish to thank Prof.
N. B . Singh, Head, Chemistry Department for providing
necessary facilities and Dr. G. P. Rao, Sugar Cane Insti
tute, Kuraghat, Gorakhpur for providing the pathogens
and helpful discussions.
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