U T I L I Z A T I O N OF S O M E M O N O S A C C H A R I D E S S P E C I E S OF C O L L E T O T R I C H U M
BY T W O
by A. K. GHos~
Botany Department, Allahabad University, Allahabad, India
(5.x.1965) INTRODUCTION
Monosaccharides comprise a group of carbohydrates most readily utilized by fungi. Some of these sugars are most commonly encountered by fungi in nature, either as such or as component units of di-, oligo- and polysaccharides. Usually, the complex carbohydrates are broken down into their corresponding monosaccharide components before they can enter into various metabolic processes. The utilization of monosaccharides by fungi has been studied by m a n y investigators. Some of these workers including STEINBERG (1942), CANTINO (1949) and LILLY & BARNETT (1956) have shown that the capability of a fungus to utilize a particular monosaccharide is dependent chiefly upon the structural configuration of the sugar in question. In recent years chromatographic detection of sugars in the culture medium coupled with dry weight studies have rendered it possible to visualize clearly the efficiency with which fungi utilize different monosaccharides. In the present study the utilization of monosaccharides by some plant pathogenic isolates of Colletotrichum gloeosporioides and Colletotrichum dematium has been studied. MATERIALS AND METHODS
Pure single-spore cultures of Colletotrichum gloeosporioides PENZ. isolated from the diseased leaves of Carissa carandas L. (Isolate 1), Eucalyptus robusta SsL (Isolate 2) and Bougainvillaea glabra CHOlZY (Isolate 3); and Colletotrichum dematium (PERS. ex FR.) GROVE from the leaves of Porana paniculata BuR~. (Isolate 1) and Bombax malabaricum DE CAND. (Isolate 2) were employed. The basal Present address: Central Research Laboratory, Antibiotics Project, Rishikesla (U.P.), India.
TABLE
Dry weight of mycelium and utilization of different monosaecharides by the isolatesof Colletotrtehum glpeosporiolde ~ a n d C. d e ~ a t i u m Days
Sugar
Col]etotrJchum gloeosporioldes
of Isolate i inDry Frecuba- wt. senee tion in of m~ sugar Days
GLUCOSE
Isolate 2 Dr), Prewt. senee in of mg sugar Days
4 3
17.0
5 12.8
Colletotriohum de~.atium
Isolate 3 Dry Prewt. sence in of mg sugar Days
Isolate I Dry Prowt. senoe in of mg sugar Days
4 20,8
Isolate 2 Dry Prewt. sence in of mg sugar Days
8 15.9
5 19,5
6
56.3
65.6
84.3
29,7
32.9
9
95.0
107.6
100.9
73.3
80.8
12
102.8
113.7
100.9
85.2
95,6
t5
92.6
105.4
98.1
86.8
9t.6
FRUCTOSE
6 3
20.8
5 18.2
6
7
20.I
II.7
5 14.9
6
62.7
56.3
62.9
44.2
50.4
9
96.6
71.0
74.1
74.0
75.0
78.8
12
112.8
78.3
85,2
80.2
15
103.5
76.8
80.9
80.7
GALACTOSE
9
~l
12
78.6 9
10.7
9.0
8.1
6
32.6
27.9
23.7
20.2
18.8
9
67.1
59.9
50.0
36.4
32.6
i2
98.7
81.8
72.2
47.1
38.2
15
93.4
85.7
78,3
48.4
MA~FNOSE
6 3
8.1
5 7.2
6.8
9
3
37.1 6
7
7.8
5.9
10.0
6 10.5
6
16.3
17.7
17.6
25.6
27.9
9
38.7
40.1
38.9
45.8
48.8
12
52.3
54.3
59.5
60.9
67.1
15
50.8
50.9
59.3
58.5
S0RBOSE
12
16 3
O.O
0.O
6
4.6
5.2
9
22.2
25.1
14
O.O
64.3 18
0.O
17
0.0
2.8
0.0
0.0
20.7
11.1
13.5
12
48.8
46.3
39.7
35.1
37.2
15
60.1
48.0
40.9
40.6
41.4
18
60.5
44.8
43.5
43.5
43.9
21
55.7
39.3
42.8
42.8
XYLOSE
8 3
6.0
8
8
8.2
6.5
43.9 9
3.7
7 IO.2
6
25.9
33.8
24.i
20.3
34.0
9
56.8
48.0
59.7
41.4
46.9
12
78.2
66.1
58.9
43.4
48.1
15
80.9
69,8
60.7
46.2
48.8
ARABINOSE
6
8 3
18.6
6
16.0
20.5
7
6
17.6
19.7
6
41.6
35.6
48.9
39.8
45.9
9
60.4
49.6
50.9
48.7
53.5
12
70.8
68.5
62.7
49.1
56.3
15
63.3
60.0
58.1
41.7
52.1
3
8,1
9
RFLk~OSE
8 6.7
7
8 6,2
10.2
13 4.5
6
20.7
24,4
21.0
32.5
13.0
9
48.7
39.2
42.2
48.4
20.~
12
59.5
47.1
44.6
49.0
32.8
15
74.7
50,7
42.2
50.6
41.6
UTILIZATION OF MONOSACCHARIDES BY COLL~ETOTRICHUM
77
medium consisted of KNO a, 3.5 g; KH~POt, 1.75 g; MgSO4.7H20, 0.75 g; and distilled water 1000 cc. To this each monosaccharide was added singly in such a quantity so as to furnish 4 grams of carbon per litre. The monosaccharides used were D-glucose, Dfructose, D-galactose, D-mannose, L-sorbose, D-xylose, L-arabinose and L-rhamnose. All the chemicals employed were of guaranteed purity. In each case the pH of the medium was adjusted to 6. 25 cc of the medium was aportioned in each of the 150 cc Pyrex Erlenmeyer Ilasks. The media containing xylose or k e t o s e s - the sugars liable to change their configuration on autoclaving, were sterilized b y subiecting them to steaming for 30 minutes per day for three consecutive days. The media containing the rest of the sugars were autoclaved at 15 lbs pressure for 15 minutes. The flasks containing sterilized media were inoculated with mycelial bits of approximately equal size of 10 day old culture of the respective organisms and were incubated at 25 ° C. The experiments were conducted in triplicate sets. Each day 0.005 cc of the medium from a flask belonging to each set was analysed b y the circular paper chromatographic technique adopted b y RANJAN et al. (1955). The chromatograms were run in n-butanol-acetic acid-water (4:1:5, V/V; upper phase). They were subsequently sprayed with aniline - diphenylamine phosphate reagent (5 vols. 4% aniline, 5 vols. 4% diphenylamine and 1 vol. orthophosphoric acid; BUCHAN & SAVAGE, 1952). The bands were developed b y heating the chromatograms in electric oven at 110 ° C for 90 seconds. Simultaneously, at the end of each incubation period the mycelial mats of different fungi were harvested on previously dried and weighed Whatman filter paper No. 42. Incubation periods of 3, 6, 9, 12 and 15 days were used. In case of sorbose, on which the initial rate of growth was slow, two more incubations (18 and 21 days) were taken. The filter papers with the mycelial mats were dried to constant weight in an oven at 60 to 62 ° C and were subsequently weighed after cooling them in a desiccator. The average dry weight of mycelium was taken as the criterion for growth. RESULTS
Tile results obtained have been summarized in the Table. It would be evident from the Table that most of the monosaccharides were readily utilized by the organisms under study. The rate of utilization of sorbose b y these fungi was markedly different from that of other sugars. The Isolate 1 of Colletotrichum gloeosporioides and the two isolates of C. dematium failed to consume sorbose within 15 days of incubation. On prolonging the incubation period upto 21 days, however, sorbose was completely exhausted from the medium in all cases. Glucose, fructose and mannose were rapidly utilized b y all these organisms, whereas galactose persisted in the medium for a comparatively longer period. The pentoses
78
A. K. GHOSH
were also utilized at a fairly rapid rate. C. dematium Isolate 2 distinctly differed from the rest of the isolates in its comparatively slow rate of assimilation of rhamnose. It would also be observed from the Table that, in general, the dry weight of mycelial mat recorded an increase upto 9 to 12 days, after which it became more or less constant or there was slight decrease. This decline was marked in case of some organisms, and with some sugars - - especially those which were consumed within a short period. DISCUSSION
AND
CONCLUSIONS
It is evident from the above results that the rate of utilization of monosaccharides varied with different species of a fungus, as well as with different isolates of the same species. Glucose, fructose and mannose were consumed within a few days by all the organisms included in the present study. CHANDRA (1961) and CHATURVEDI (1961 ) working with Colletotrichum gloeosporioides have also reported rapid utilization of glucose and fructose. The Punica-leaf isolate of C. gZoeo@orioides studied b y CItA~'nRA (1961) exhibited a slow rate of utilization of mannose, and in this respect differed from the present isolates. It has been shown by numerous workers that monosaccharides undergo phosphorylation prior to their entering into other metabolic processes. The presence of phosphorylated derivatives of glucose and fructose in fungal mycelium has also been shown. CAPUTTO et al. (1949) have demonstrated galactose-l-phosphate in Saccharomyces/ragilis and KITA & PETERSON (I953) have shown the presence of mannose-l-phosphate in the mycelium of Penicillium chrysogenum. According to COCHRANE (1958), 'the ability of an organism to use fructose, mannose or gatactose depends upon its ability to convert the sugar in question into a phosphorylated derivative of glucose.' It is plausible that the differences shown b y the organisms included in the present investigation in their rates of utilization of different sugars might have been due to differences in kinase activity. Poor utilization of sorbose b y fungi has been reported by many workers. TATUlVf et al. (1949) recorded only restricted colonial growth of Neuro@ora Crassa on this sugar. LILLY & BARNETT (1953) have attributed this phenomenon to the killing of growing hyphal tips in sorbose medium, followed by branching of mycelium below the killed portion. In the present investigation microscopic examination failed to reveal such killing of hyphal tips. The initial lag period observed during the growth of the present fungi on sorbose, obviously denoted the time taken by these organisms to acclimatize themselves to the sorbose atmosphere or to produce adequate amount of an adaptive enzyme. A perusal of the Table would reveal that in those cases where
UTILIZATION
OF MONOSACCttARIDES
BY
COLL]~TOTRICHUNI
79
the rate of assimilation of a sugar was slow, there was an increase in dry weight upto the end of the final incubation period (15 days). On the other hand, whenever the sugar was consumed within a short period, the dry weight at later stage of incubation either remained more or less stationary or recorded a fall. Apparently, this decline in dry weight was the manifestation of the mobilization of reserve materials which subsequently entered the respiratory- pool, when the external supply of sugar was exhausted. The present investigation has shown that in some cases the dry weight of mycelium recorded an increase for few days, even after the sugar was exhausted from the medium. This phenomenon is not inexplicable, since in addition to carbohydrates, other essential elements like nitrogen, phosphorus and sulphur also contribute to the structure of fungal protoplasm. Slower rate of assimilation of any of these elements or presence of any 'bottle-neck' in the enzymic systems affecting protein synthesis would, therefore, result in the increase in protoplasmic matter, even after the carbohydrate uptake was complete. The rate of consumption of the monosaccharides from the medium, could not always be correlated with the amount of growth. For instance, in the present study most of the isolates of Colletotrichum attained poor mycelial growth on mannose, although this sugar was consumed from the medium within a short period. This probably indicated comparatively lesser efficiency of this sugar or its derivatives to participate in different metabolic processes.
Summary Utilization of 8 monosaccharides, viz., glucose, fructose, galactose, mannose, sorbose, arabinose, xylose and rhamnose, b y some plant pathogenic isolates of Colletotrichum gloeosporioides and C. dematium has been studied with the help of paper chromatography. Among hexoses, the rate of utilization of glucose, fructose and mannose was fast, whereas, that of galactose was comparatively slow. The rate of assimilation of sorbose was very slow at early stages of incubation, although at later stages this rate showed marked enhancement. The pentoses were utilized readily. The dry weight of mycelial mats showed an increase up to the end of final incubation period (15 days), on sugars which were slowly assimilated. In cases where the sugars were consumed up rapidly, the dry weight at later stages of incubation either became nearly stationary or recorded slight fall.
Acknowledgment The author is grateful to Prof. R. N. TANDON for his encouragement and guidance.
80
A. K. GHOSH
Literature BUCHAN, J. L. & R. I. SAVAaE. 1952. Paper chromatography of some starch coilversion products, Analyst 77: 401. CANTINO, E. C. 1949. The physiology of the aquatic Phycomycete Blastoeladia pringsheimi with emphasis on its nutritiml and metabolism. Amer. J. Bot, 36: 95--112. CAPUTTO, l~., L. F. LELOIR, i~. E. TRUCCO, E. C. CARDINI & A. C. PALANDINI. 1949. The enzymatic transformation of galactose into glucose derivatives. J. biol. Chem. 179: 497--498. CHANDRA~ S. 1961. D. Phil. Thesis, Allahabad University. CHATURVEDI, C. 1961. D, Phil. Thesis, Allahabad University. COCH~ANE, ¥. \¥. 1958. Physiology of fungi. John Wiley & Sons, Inc., New York, KIT.~, D. A. & W. H. PETERSON. 1953. Forms of phosphorus in the penicillinproducing mold Penieillium ~hrysogenum Q-176. J. biol. Chem. 203: 861-868. LILLY, V, G. & H, L. BARNETT. 1953, The utilization of sugars by fungi. Bull, \V. Virginia agric. Exp. Sta. 362 T. LILLY, V. G. ~¢ H. L. BARNETT, 1956. The utilization of D- and L-arabinose by fungi. Amer, J. Bot. 43: 709--714, RANJAN, S., GOVlNDJEB & M. M. LALORAYA. 1955. Chromatographic studies on amino acid metabolism of healthy and diseased leaves of Croton sparsi]torus. Proc. nat. Inst. Sci. (India) 21: 42--47. STEINBERG, R. A. 1942. The process of amino acid formation from sugars in Aspergillus niger. J. agric. Res. 64: 615--633. TATUM, 1~. L., R, W, BARRATT • V. M. CUTTER. 1949. Chemical induction of colonial paramorphs. Science 109: 509--511.
