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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.

I

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 7•IK• 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 .

To study the influence . Influence of Light Light is absolutely essential for the formation of vari­ ous types of reproductive organs.80* 1 1 . (a) Czapek medium. and (c)TDA medium c Table I Values of slope (m).0* *Observations taken at 3 1 .993 0.0 and 1 1 . (b) PDA medium. intercept (c) and correlation coefficient (R) used in the equation d = m t + c obtained at different experimen­ tal conditions at 37OC. JULY 2000 a b Fig I - Growth patterns of Cephalosporium sacchari developed on different media at 37±1 "C.609 0.332 -0.558 0.96* 6.30 1 0. pH of culture medium was measured with a digital pH meter. 101 -0.001 cm. 2 Influence ofpH on the morphology and growth kinetics Influence of pH of the medium on growth patterns and kinetics has been studied. 1 53 0. 6. - Medium pH Slope (m) Intercept (c) Correlation coefficient (R) em/day cm Potato . 108 0.390 -0.8. 3 .dextrose .998 Maize-agar agar 0.96.998 0.agar agar 0.698 INDIAN J CHEM. Results are recorded in Fig.0 °C Growth kinetics After inoculation of fungi Cephalosporium sacchari into different media.334 -0.384 -0.0.dextrose-agar agar 0.009 0.996 0.80 3. OMA medium was se­ lected for this purpose: Experiments were carried out on OMA medium of various pH viz.576 -0. 1 1 8 0.996 Tomato.998 Czapek-agar agar 0.400 -0.990 0. SEC. 8.484 0.357 -0. growth kinetics were studied with the help of a travelling microscope accurate to ± 0. 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 4.995 Oat meal-agar agar 6. A.

0 699 o 3. Growth was measured employing the usual tech­ nique as described earlier.5 3. (!) 1. Results are recorded in Fig. . Growth patterns were observed after 7 days and photographed.7Hp FeS04 .6. and Czapek agar media are shown in Fig. 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 .5 'i0 2. of light on the growth behaviour and pattern of the fungi. Results are recorded in Fig. 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. KCI 3 and sucrose (Czapek's medium)(iv) maize and (v) oat meal. MgS04 . certain i norganic micronutrients are also re­ quired depending on the nutrients supplied. Inhibition patterns To study the dependence of hyphal growth and dif­ ferentiation on adjacent hyphae. TDA. [0) PDA. Figure 2 shows the re- .: GROWTH KINETICS OF Cephalosporium sacchari ON AGAR PLATE [. [0 )Maiz agar IrD Czapek media. apart. Carbon compounds serve two essential functions in the metabolism offungi: (i) It supplies the carbon needed for the synthesis of pro­ teins. Similar and dissimilar mi­ croorganism discs were inoculated on a plate at a 3.0 cm. TDA medium was taken into petri dishes inoculated and kept in complete darkness. Periodicity is observed in almost all cases. Fungi can produce a great variety of complex organic compounds. nucleic acid.0 � . Values of slope (m) intercept (c) and correlation coefficient (R) for different media are recorded in Table 1 . reserve food etc.e t and c is the intercept of the straight line on the y axis. The growth occur linearly as evident by the growth ki­ netic results shown in Fig.0 0. interacting hyphae are experimentally generated by choosing two inoculating centers at a fixed distance. cell wall membrane. The media contained essentially carbon source and small amount of inorganic nutrients. and (ii) the sole source of appreciable amount of energy is the oxidation of carbon compounds. 2.[Il) TDA. nutrients are taken up and fungus changes the ionic and molecular profile in the surround­ ing medium.0 0 Fig 2 - L 2 3 5 6 Ii m e ( days ) 7 8 9 Growth kinetics data for Cephalosporium sacchari on different media.5 1 . In addition to car­ bon source.5. K 2HP04 . [(»)OMA.7Hp.5 0. 1 .0 E u 2 . while other petri dish was kept in the presence of visible light at the same tempera­ ture. During the myc­ elium development.DAS et al. Typical growth patterns developed on PDA..

Plates i-iii show the morphology at a lower pH value (3 . pH [a] 3.96).IoC efficient values. Growth kinetic data at different pH are recorded in Fig.Ii ' i .0 3. Dense radial like colony is observed at low and normal pH (plates a.0 suits obtained on different culture media. Results are recorded in Table 1 . 1 . . normal value (6. [A ] 6. 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. JULY 2000 b a Fig 3 - c Growth patterns of Cephalosporium sacchari developed on OMA plates contain­ ing media of different pH at 30±1"C.0). It grows best in the mediu� having pH . It suggests that the fungus sporulats relatively faster in the light. [b) 6.700 INDIAN J CHEM. they grow and eventually meet each other as shown in Fig. The data obey the simple relation d = m t +c as evident by correlation coFig'. When petridishes were inoculated with two similar or dissimilar mycelia at a certain distance on a petridish.5.96) or in alkaline medium (PH 1 1 . in the normal range and declines 10 the aCidiC (3. Further the zonation is observed in the presence of light at the given experimental condition.8 and [c ) 1 1 .8. I)] I 1 .96 .b) while sharp transition to rhythmic pattern is noticed at pH 1 1 (plate c).96. 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. A.0) respectively.s: 2.0 4.0 u .6.8) and �t r�la­ tively higher value ( 1 1 . Re­ sults show that the growth is linear and light induced. SEC. Plates show the growth patterns when the two in- .0 Tl m a ( d oys ) Grow�h kinetics data for Cephalosporium sacchari on OMA plates containing media of different pH : [0 ] 3. 4.0 � � . The values of m and c depend on pH of medium. 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±.

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. Accord­ ing to the s�per saturation theory which is most com­ mon amongst the three. probably there is no such competition for nutrients. nucleation occurs and subsequently D clus­ ters are formed. Diffusion of ions persists and after a certain stage.: GROWTH KINETICS OF Cephalosporium sacchari ON AGAR PLATE b a Fig 6 - 70 1 c Inhibition patterns of Cephalosporium sacchari on TDA medium when inoculated with (a) Cephalosporium sacchari. The growth could also be thought of primarily as a consequence of nutrient diffusion and local exhaustion. In a more recent version of the theory22 the picture is modified. Fig. However. Subsequently nuclei are formed followed by crys­ tallisation according to the following scheme. densely flagellate. The process is repeated until the supply of one of the electro­ lytes is exhausted. The suppressed sporulation is due to competi­ tion over nutrients. A+B � C C � nuclei (n) nuclei � crystal (D) when the local concentration of C reaches some thresh­ old value. 6). Explanation for the periodic precipita­ tion phenomena was given by several workers based on a) the supersaturation theory l 9.0 cm. precipitation is also stopped as diffusion continues further. the formation of precipitate stops nuclea­ tion in the neighbourhood. Colonies of Pr vulgaris were also observed to pos­ sess concentric rings. a depletion of con­ centration of A and B occurs in the surrounding.Curvularia pallescens respectively (plates ii and iii. a different mechanism was suggested. The con­ tact zone is characterised by a lack of spore due to com­ petition over nutrients. In addition there is cessation of cell division due to inhi­ bition of septum formation leading to formation of multi­ nucleate. The two species A and B are assumed to react to produce a new species C which also diffuses in to the gel. oculating discs were of similar pathogen. 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. Cephalospo­ rium sacchari (Plate i) or the two discs of dissimilar pathogens Cephalosporium sacchari-Fusarium oxysporum and Cephalosporium sacchari . Hence at this stage. periodic precipitation of sparingly soluble salts l -? . Physical explanation of ring-type structure Rings of this general type have been observed in great variety which include. growth of the bacteria Pr vulgaris13 and several others.DAS et al. The con­ tact zone is characterised by a lack of spore (inhibition pattern). . This is followed by the formation of clear spaces. (b) Fusarium oxysporum and (c) Curvularia pallescens at a fixed distance of 3. Plate (iii) does not show such a lack of sporulation at a contact zone. Modification of the above model was made by Polezhaev and Mullerl l . A +B � AB (solid) Once the nucleation has started. nonseptate cells. b) competitive particle growth modeFo and c) sol coagulation modeFI . The bacteria which is normally sparsely flagellated alters itself to acquire large number of flagella. the critical supersaturation exceeds and another band is formed in a similar manner.

N. 21. 17. 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. Bourret J A. Upon germination the growth of the Acknowledgement Thanks are due to the U. Mechanism of development. edited by Y W Cochrane. Physiology offungi. Gorakhpur for providing the pathogens and helpful discussions.p 463. A cellular automation is a theoretical concept that can be applied to certain spatially distributed systems in which macroscopic behaviour results. NewYork) 1 993.C. 8. 94 ( 1 989). SEC. Macroscopic morphology is deter­ mined by the growth kinetics of the individual hyphal element within the pellets. Das I. 39( 1 925) 2. 15. (Cam­ bridge Univ. J phys Chem. 76( 1994) 66 1 . Lall R S & Pushkarna A. Muller S C. New York). 1 8. Indian J exp Bioi. Nielsen J. Deutsch A. Sugar Cane Insti­ tute. Sharma A & Sing U K. Dhar N R & Chatterjee A C. II. 4. branching and differentiation of a fungal myc­ elium. 1 9. 60 ( 1 974) 3458. 93 ( 1 989). branches are formed resulting in a hyphal element consisting of a branched network. Henisch H K. 1 4. Singh. G. (Henry Hold. Chand S & Puskarna A. In the case of fungi the growth occurs only at hyphal tips. Das S S. Cyrstals in gels and Liesengang rings. Das I . 1 6. Rensing L. Spore Germ tube Densely branched hyphal element Agglomarate of hyphal element spore rapidly results in the formation of a tube which extends its length by growth at the tip. J phys Chem. 1 3. Pushkarna A & Agrawal N R. J chem Phys. Kuraghat. Das I. 5. Science. J chem Phys.INDIAN J CHEM. 25 (1 956) 279. ( 1 988). 7269. Polezhaev A A & Muller S C. 71 ( I ) ( 1 994) 58. 84 ( 1 987) 23 1 . The relation offungi to human affairs. 9 1 ( 1 987) 747. 20. J chem Educ. Press. B . 1 66 ( 1969) 763. Rao. Authors also wish to thank Prof. ( 1 993) 1 7. We have observed similar zonation patterns during the fungal growth. Carpenter B H. J cryst Growth. J colloid interface Sci. within a ring of certain thickness. 1 959. Das I . Lall R S & Pushkarna A. Within that ring bacteria could not subsequently flourish. P. Pushkarna A & Bhattacharjee A. Das I. 29 ( 1 99 1 ) 1 1 09. 9. References I .. 1 30 ( 1 989) 1 76. 1 2. CHAOS. Das I. J colloid interface Sci. J phys Chern. Pushkarna A & Chand S. Flicker. Dress A. Das I. Lincoln R G. New York). TIBTECH. Statist Phys. J phys Chem. 1 50 ( 1 992) 1 78. Various hypotheses were te sted with the help of a cellular automation model which mimicks growth. 14 ( 1 996) 438. New Delhi for sup­ porting the investigation. exceeds a certain length. The proc­ ess would then be repeated. edited by L Rensing (Marcel Dekker. The germ tube develops into a hypha. J phys Chem 95 ( 1 99 1 ) 2. London) 1958.&Ross J. Oscillations andmorphologies. Prager S. (John Wiley. 3866. Differentiation may be incorporated by means of various activator-in­ hibitor mechanisms. M. Gray W D. 6. 22. 4 (1 994) 63 1 . Pushkarna A & Chand S. A. Luthi P & Droz M J. Pushkarna A & Bhattacharjee A. 94 ( 1 990) 896 . Chopard B. 1 0. Das I. Das I. 44. 1 • . A hyphal element arises from the out growth of a single spore.G. Head. Talanquer Y. • 3. 7. Kolloid Z. Chemistry Department for providing necessary facilities and Dr.