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II.
PHYSIOLOGICAL STUDIES ON THE GENUS SCLEROTIUM
Utilization of Inorganic Nitrogen Sources by Sclerotium rolfsii (Sace.) and Sclerotium oryzae (Catt.) under Protracted Incubation
BY R. NARASIMHAN* (University Botany Laboratory, Madras-5)
Received February 18, 1969
(Communicated by Prof. T. S. Sadasivan, F.A.SC.)
ABSTRACT
The utilization of nitrate and ammoniacal sources of nitrogen by S. rolfsii and S. oryzae have been compared with reference to growth rate over a period of time and drift in pH of the culture solutions. The results indicate that utilization of ammoniacal nitrogen by the two Sclerotium spp. is governed more by the availability of ammonia accep- tors rather than by critical pH, limiting growth.
INTRODUCTION
EARLIER work of Sivapalan (1958) in this laboratory indicated that nitrate and ammoniacal sources of nitrogen were equally favourable for the growth of Sclerotium rolfsii (Sacc.). Sethunathan (1964) reported that ammoniacal sources were poorly utilized by Sclerotium oryzae (Catt.). Both these workers, however, compared the utilization of nitrogen at a fixed incubation period. It is now well recognised that growth measurements, to be objective, should be carried over a period of time (Lilly and Barnett, 1951; Cochrane, 1958). In light of this, the utilization of inorganic nitrogen by the two species of Sclerothtm has been re-investigated.
MATERIAL AND METHODS
Czapek's medium supplemented with trace elements, thiamine and biotin was used, the composition being, MgSO~.7H20 0.5g. , KH2PO4 1.0g., KC1 0.5g. , FeSO4.7H20 0-01 g., NaNOz 2.0g. , sucrose 30.0g. ,
* Present address: National Chemical Laboratory, Poona-8. Centre of Advanced Study in Botany.
42
Memoir No. 70 from the
fused and (Paterson, visually.
Physiological Studies on the Genus Sclerotium--/ / 43
thiamine 100 t~g., biotin 5 t~g. and 1 ml. of trace element mixture consist- ing of 0 .2mg . Zn (as ZnSO4.7HzO), 0.05 rng. Cu (as CuSO4.5HzO), 0.025rag. Mn (as MnSO4.4H20) and 0.02 nag. [Mo as (NH~)6MoTOz4. 4H20] and glass-distilled water to make 1,000 ml. The vitamins were inclu= ded in the medium since S. oryzae has been shown to be heterotrophic to thiamine by Suryanarayanan (1958) and S. rolfsii for thiamine by Robbins and Kavanagh (1938, 1942) and Sivapalan (1958) and also for biotin by Steinberg (1950). An aliquot of 25 ml. of the medium was pipetted out into each flask. Sodium nitrate of this medium was substituted by other nitrogen sources on an equal nitrogen basis.
Chemicals of utmost purity, B . D . H . AnalaR, or Merck pro analysi were used in all experiments.
Evaluation and adjustments of pH were done by using the original Dr. Lange's acidometer. Adjustments of the initial pH of the media were done with N. HC1 and N. Na OH.
The media were sterilized by autoclaving at 15 lb. pressure for 15 minutes.
The inoculum was prepared by growing the fungus initially i~1 10 ml. of the basal medium in a flask of 100 ml. capacity for 6 days. Subsequently, the mat was washed five times with sterile glass-distilled water and about 1 cm. diameter of the mat transferred to a 250 ml. pyrex flask with 100 pyrex glass beads immersed in 25 ml. of water. The entire process was done aseptically. The flask was hand-shaken for thirty minutes to macerate the mat. The experimental flasks were inoculated by means of a drop through a one ml. pipette. The cultures were incubated in glass shelves at 30 ° to 32 ° C.
The mycelial mats were filtered under vacaum on tared fitter-paper discs~ washed thoroughly with distilled water. The filter-paper with the mats were dried at 60 ° C. for 48 hours, subsequently cooled in a desiccator over CaC12,
quickly weighed. The data were subjected to statistical analysis 1939). The intensity of sclerotial production was determined
RESULTS
1. Effect of nitrate nitrogen sources on the rate o f growth am; sclerotial production o f S. rolfsii and S. oryzae.--The following nitrogen sources have been used in the present experiment on equal nitrogen basis: N a N Q .
B 5
44 R. NARASIMHAN
KNO3, Ca(NOa)~.4H~ O and NH4NOa. The initial pH of the media were adjusted to pH 6.0. Mycelial dry weights, sclerotial production and the final pH of the media were assessed everyday from the third day to the seventh day after inoculation and at 3-day intervals thereafter upto 22 days. The data are presented in Figs. 1 to 3 and Tables I and III.
500
e NeNO 3
• KNO 3
• , Ca{NO;s] 4H20
40( • NH4 NO$
50C
Z
~ 20C
I00 t
I o I ' 115 ,~ 119 z~ 3 7 10 INCUBATION IN DAYS
FIG. 1. Effect of nitrate nitrogen sources on the rate of growth of S. rolfsiL
It is clear from Figs. 1 and 3 that S. rolfsii accumulated more dry matter than S. oryzae. Interesting differences were evident even from the very beginning on the utilization of four nitrate sources by S. rolfsii (Fig. 1). While no appreciable growth was observed with NaNOa and KNOB upto 4 days, considerable mycelial yields were obtained with NH4NOa and Ca (NO 3)2.4H,O even on the third day. With the latter two sources growth rate was almost linear between 3 and 7 days. The rate of growth was highest between 4 and 10 days in the case of KNO3 and 5 and 16 days with NaNO3. During the period of active growth the highest growth rate was observed with NH,NO3 followed by Ca (NO3)2.4H20 and KNO3 and NaNO3.
Inspection of Fig. 2 will reveal that regardless of the nitrogen source the pH of the culture solutions dropped to about 2.0 by 7 days at which it was maintained thereafter. However, the rate of fall differed with the nitrogen sources during the early phase of growth. With NH4NOa the pH fell to 1.9 even by the fourth day. With Ca (NOa)~.4H20 this decline in
Physiological Studies on the Genus Selerotium--H 45
g,1
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46 R. NARASI MHAN
pH was less steep and the lowest pH of 1.9 was recorded only on the seventh day. With NaNO3 and KNO3 fail in pH was more gradual and the lowest pH 2.1 was recorded on the seventh day.
4
3
2
o 7
• NoNO 3
INITIAL DH g 0
. , = , , .A~ ,o . , . OAY=
FIG. 2. Changes in the final pH due to the growth of S. rolfsii in nitrate nitrogen sources.
Regardless of the nitrogen source, S. oryzae showed no appreciable growth upto a period of 3 days and rapid growth occurred between 3 and 7 days (Fig. 3). During this period no appreciable difference was noticeable in regard to the different nitrate sources. However, differences in mycelial yields were evident after 7 days. At tliis time culture on NaNO3 showed
~OC
~5o
• NH4NO 3
INiTiAL pH 6 " 0
INCUgATfON IN DAYS
FIG. 3. Effect of nitrate nitrogen sources on the rate of growth and changes in the final pH of S. oryzae,
Physiological Studies on the Genus Sclerotium--H 47
¢.
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48 R. NARASIMHAN
the highest mycelial dry weight followed by KNO3, NH4NO3 and Ca (NO3)2. 4H20.
It will be seen from Fig. 3 that with NaNO3 and KNO3 the pH of the culture solutions increased gradually from 6.0 to 8.7 and 8.4 respectively with increasing incubation. With Ca (NO3)2.4H~O the pH tended to fall upto 3 days but increased to about 6.0 on the fourth day which was maintained almost at that level throughout the incubation period. With NH4NO3 a sharp fall in pH to 3.5 was noticeable upto 4 days which was maintained thereafter.
40C
~30( E
~0¢
500
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& (NH4)2HPO 4 • |NH4] 2 $04
f /
, , , , , , , , 3 7 10 13 16 Ig 22
INCUgATION IN OAY~
l~o. 4. Effect of ammoniacal nitrogen sources on the rate of growth of $. rolfsii.
2. Effect of ammoniacal nitrogen sources o n the rate of growth and sclerotial production o f S. rolfsii and S. oryzae.--In this experiment NH~ 1'403, (NH4)~HFO~ and (NH4)2SO4 were used along with NaNO3 for com- parison. The results are presented in Figs. 4 to 6 and Tables II and IV.
It is evident from Figs. 4 and 6 that S. rolfsii produces more dry matter than S. oryzae with ammoniacal nitrogen sources also. Unlike with nitrate, no initial lag in growth was observed in the case of S. rolfsii (Fig. 4). The fungus utilized all the three anamoniacal sources equally well and, in fact, in the near linear growth period of 3 to 7 days the growth rate was almost similar. After 7 days, however, certain differences in the growth rate was noticed. Examination of Fig. 5 will show that regardless of the nitrogen source the pH of the media dropped to about 2.0 during the first 7 days
Physiological St,dies on the Genus Sclerotium--H 49
around which it remained during the latter stages of growth. It might also be noticed that the rate of fall in pH was almost similar with all the nitrogen sources during the early stages of growth.
4
= ~ o ~oNO 3 • NH4NO 3 6 (NN4)2HPO 4
i 3 • (NH6)2504
t INI'Tt~L. ON G C
INCUBATION IN DAYS
Fro. 5. Changes in the final pH due to the growth of S. rolfsii in ammoniacal nitrogen S o u r c e s .
In contrast to S. rolfsii, considerable differences were noticed between the utilization of the three ammoniacal sources of nitrogen by S. oryzae (Fig. 5). (NH~)2HPOa was utilized best followed by NHaNO3 and (NH4)2 SO4. The rate of growth between 3 and 7 days was also highest with (NH4)~HPO 4 as compared to the other two sources. The fall in pH was
100 ~ ;
z
r- 5 0
INITIAL pH 6 0
INCUBATION IN DAYS
Fro. 6. Effect of ammoniacal nitrogen sources on the rate of growth and changes in the ~a l pH of S. oryzae.
50 R. NARASIMHAN
O
O t...¢
° ~
t~ O
I I I ~ ~ ~
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Physiological Studies on the Genus Sclerotium--H 51
0
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= Z
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52 R. NARASIMHAN
also evident with all the three ammoniacal sources (Fig. 6). Although the rate of fall was nearly equal with the three sources upto 5 days, this was less pronounced thereafter with NH4NO3. The lowest pH 2.7 was re- corded both in the case of (NH4)~SO4 and (NH4)2HPOv In the medium containing NHaNO3 the pH dropped to a minimum value of 3.4.
The effect of nitrate and ammoniacal sources of nitrogen on the pro- duction of sclerotia by the two fungi is summarized in Tables I to IV. The visual ratings indicate that in general, all the nitrogen sources, both nitrate and ammoniacal, promoted fair to good sclerotial production by S. rolfsii (Tables I and II). However, Ca (NO3)2.4H~O did not appear to be favourable for sclerotial production (Table I),
For S. oryzae, all the nitrate sources appeared to be good for sclero- tim production (Table III). While (NH4)2HPO4 favoured good sclerotial production, (NH,),SO4 seemed to be unfavourabIe (Table IV).
DISCUSSION
An overall consideration of the data shows that both nitrate and ammo- niacal sources are equally favourable for the growth of S. ro~'ff (Figs. 1 and 4). Regardless of the source of nitrogen, the pH of the culture solu- tion dropped to about 2-0 during the active phase of the fungus growth (upto 7 days) (Figs. 2 and 5). While the rate of growth was nearly identical with ammoniacal sources (Fig. 4) during the active growth phase, certain differences were evident in regard to the nature of the nitrate sources (Fig. 1). The lag in the utilization of NaNO3 and KNO3 upto 4 days was not notice- able in the case of Ca (NO3)~.4H~O. This does not appear to be correlated with the fall in pH for the difference in the rate of pH drop is not very different with the three nitrate sources. A beneficial effect of calcium for nitrate reduction is therefore indicated. Johnson (1953) reported that S. rolfsii tolerated 3,000 ppm. of calcium. In view of this report it should be interesting to investigate the calcium requirement of this fungus with parti- cular reference to nitrate reduction.
In contrast to S. rolfsii, S. oryzae showed considerable differences in the utilization of nitrate and ammoniacal nitrogen, as well as in the drift in pH of the medium. Typically the same growth was observed with all the four sources of nitrate nitrogen including NH4NO3 (Fig. 3) during the active period (3 to 7 days). Nevertheless, the reaction of the medium turned alkaline with growth in the case of NaNO~ and KNO3 and acidic (pH 3"4) in the case of NH4NOa (Fig. 3). With Ca (NOa)z 4H20 the pH
Physiological Studies on the Genus Sclerotiummll 53
tended to fall initiall~¢ but was stabilized at about 6.0 by the fourth day. Thus, no direct correlation could be observed between nitrate utilization and drift in pH~ nor was the beneficial effect of calcium on nitrate utilization by S. rolfsii was evident in S. oryzae.
The utilization of ammoniacal nitrogen by S. oryzae varied with the nature of the source (Fig. 6), whereas (NH4)2HPO4 promoted the best growth and growth rate of the fungus. NH4NO3 and (NH4)2SO4 were definitely inferior. This confirms the earlier work of Sethunathan (1964) in this laboratory. Regardless of the source, however, a fall in pH was observed with the utilization of the ammoniacal nitrogen (Fig. 6). This fall in pH was considerably greater in the cases of (NH4)~SO~ and (NH4)~ HPOa than with NH4NO3. In spite of this, the fungus grew to a consi- derable extent with (NH~)2HPO4, and in fact, the growth rate was compa- rable to that on nitrate in the early stage (Fig. 6). This raises the question whether failure to utilize ammoniacal nitrogen could be attributed to a decrease in pH as generalised by Morton and Macmillan (1954). It is apparent that the beneficial effect of phosphorus is not due to a buffering effect sirlce the fall in pH was not affected (Fig. 3). It would rather indicate the high phosphorus requirement for sugar dissimilation with ammoniacal sources of" nitrogen providing carbon skeletons as acceptors of ammonia. This view is in line with Brian et aL (1947), who indicated a specific require- ment of organic acids in the assimilation of ammonia by Myrothecium verru- carla and Scopulariopsis brevicaulis. The response of S. rolfsii to ammonia- cal sources lends further support to this view. In spite of the considerable fall in pH to ~ about 2.0, this fungus utilized ammoniacal sources equally well as nitrate sources (Figs. 1 and 4). Higgins (1927) reported that S. rolfsii produced oxalic acid in culture and the considerable development of acidity with all nitrogen sources is very likely to be due to high con- centration of this acid. The production of this organic acid in sizable quan- tity appears to favour the utilization of ammoniacal nitrogen. In this context the report of Sethunathan (1964) that S. oryzae produced higher. mycelial yield with ammonium oxalate than with NaNO3 is a case in point. There is thus every reason to believe that ammonium utilization by the two Sclerotium spp. appears to be limited more by the availability of carbon acceptors rather than by a limiting pH, restricting growth. Further evi- dence to this view will be presented elsewhere.
ACKNOWLEDGEMENT
] have great pleasure in recording my indebtedness to Prof. T. S. Sada- sivan, Director, University Botany Laboratory, Madras, for suggesting the
54 R. NARASIMHAN
problem and guidance. I am also grateful to Dr. S. Suryanarayanan, Reader, University Botany Laboratory, Madras, for help in editing this paper.
REFERENCES
Brian, P.W., Curtis, P.J. and Hemming, H. G.
Cochrane, V.W. .. Higgins, B.B. . .
Johnson, S.P. . .
Lilly, V.G. and Barnett, H.L. . .
Morton, A. G. and Macmillan, A.
Patterson, D .D.
Robbins, W. J. and Kavanagh, F . . .
Sethtmathan, N.
Sivapalan, V.
Steinberg, R. A.
Suryanarayatmn, S.
"Glutinosin: a fungistatic metabolic product of the mold Metarrhiziumglutinosum S. Pope," Proc. R. Soc., 1947, 135B, 106-32.
Physiology of Fungi, John Wiley and Sons, Inc., N.Y., 1958. "Physiology and parasitism of Sclerotium rolfsii Sacc.,"
Phytopathology, 1927, 17, 417-88. "Some factors in the control of the southern blight orga-
nism, Sclerotium rolfsii," Ibid., 1953, 43, 363-68. Physiology of Fungi, McGraw-Hill Book Co., Inc., New
York, 1951. "Tl':e assimilation of nitrogen from ammonium salts and
nitrate by fungi," d. exp. Bot., 1954, 5, 232-52. Statistical Technique in Agricultural Research, McGraw-
Hill Book Co., Inc., New York, 1939. "Vitamin Bx or its intermediates and growth of certain
fungi," Am. d. Bot., 1938, 25, 229-36. "Vitamin deficiencies of the filamentous fungi," Bot.
Rev., 1942, 8, 411-71. "' Sclerotium oryzae. II. Nitrogen Utilization," Phyto-
path. Z., 1964, 50, 23-42. "Studies on Sclerotium rolfsii," Thesis approved for
the M.Sc. degree of the University of Madras, 1958.
"Growth on synthetic nutrient solution of some fungi pathogenic to tobacco," Am. J. Bot., 1950, 37, 711-14.
"Growth factor requirements of Piricularia spp. and Sclerotium oryzae," Proc. Ind. Acad. Sci., 1958, 48 B, 154--88.
1304-69. Printed at The Bangalore Press, Bangalore-18, by M. S. Narayana Murthy, Secretary, Published by B. S. Venketachar, Editor, "Proceedings of the Indian
Academy of Sciences", Bangalore
