1. Effect of different media.--the following complex organic media ~vere examined for the mycelial growth and sclerotial production by S.

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1 FACTORS AFFECTING THE GROWTH AND SCLEROTIAL PRODUCTION IN SCLEROTIUM ROLFSII SACC., CAUSING STORAGE ROT OF POTATO BY A. P. MISRA AND S. Q. HAQUE Received January 22, 1962 (Communicated by Dr. P. Maheshwali, ~.A.sc.) AJREKAR (1924) and Patwardhan (1926) reported Sclerotium rolfsii Sacc. as the cause of rottage of potato tubers in storage in Western India. In Bihar the authors first observed it at Sabour in Grover and Chona (1960) made a comparative study of nine isolates of S. rolfsii and one of Ozonium texanum Neal and Webster var. parasiticum Thirumalachar. The isolates of S. rolfsii studied by them in detail were obtained from pigeon pea, piper betel and sugarcane. Earlier Higgins (1927), Curzi (1931, 1931 a), Epps etal. (1951) and Endo (1940) studied the physiology and pathogenicity of S. rolfsii and made valuable contributions. With a view to examine the differences, if any, between different isolates of S. rolfsii, the present studies were carried out. MATERIAL AND METHODS The fungus was isolated from diseased potato tuber, collected at Sabour in May The various solid and liquid media were prepared according to standard formulae and their ph was adjusted to 6.0, which was found to be optimum in earlier experiments. Inoculations were carried out after the method of Paul (1929) by using equal pieces of mycelium. The size of the inoculum was standardized by cutting the mycelial mats, grown on PDA for 4 days, by a cork borer having an internal diameter of 5 mm. The cultures were incubated at 30 C. For estimation of the dry weight of mycelium, the flask cultures were incubated for 20 days. The number and dry weight of the sclerotia were determined by growing the fungus in petridishes with equal quantities of agar media for a period of 30 days. A. Nutritional Factors EXPERIMENTAL RESULTS 1. Effect of different media.--the following complex organic media ~vere examined for the mycelial growth and sclerotial production by S. rolfsii: 157

2 158 A.P. MISRA AND S. Q. HAQUE (1) Oat meal agar. (2) Potato dextrose agar. (3) Maize meal agar. (4) Host extract agar. Maximum growth was observed after 4 days in PDA, followed closely by oat meal agar, maize meal agar and host extract agar. The maximum number of sclerotia were produced in potato dextrose agar, which was followed in order by oat meal agar, maize meal agar and host extract agar (Table I). TABLE I Effect of different solid media on growth and sclerotial production in Sclerotium rolfsii* Media Dry Radial growth in mm. after Number weight of of 1,000 2 days 3 days 4 days sclerotia sclerotia in mg. Oat mealagar.. 45"6 90"3 90" Potato dextrose agar.. 39" 3 70' Maize mealagar.. 38" Host extract agar " " * Average of 3 replicates. 2. Growth rate on different synthetic media. The fungus was grown on several liquid media, viz., Richard's, Czapek's, Brown's, glucose-peptone and Knopp's solutions. The mycelial yield after 20 days' incubation at 30 C. was maximum in Richard's medium (762.0 mg.), followed in order by glucose-peptone (215-0rag.) and Czapek's (202.0mg.), Brown's (63.0 mg.) and Knopp's solution (44.0 mg.); mycelial growth on glucose-peptone and Czapek's medium did not differ significantly when put to statistical test. 3. Effect of different constituents of Richard's medium.--the effect of different constituents of the Richard's medium on the growth of S. rolfsii was investigated by omiting one of the constituents from each of the

3 Growth and Sclerotial Production in Sclerotium rolfsii Sacc. 159 treatments, except the control. In the absence of any one of the five constituents, viz., sucrose, potassium nitrate, potassium di-hydrogen phosphate, magnesium sulphate or ferric chloride, the growth was poor as compared to complete Richard's medium. The reduction in growth was maximum in the absence of sucrose and was followed in order by potassium nitrate, potassium di-hydrogen phosphate, ferric chloride and magnesium sulphate. 4. Effect of different carbohydrates.--in order to determine the effect of different carbon compounds, the following carbohydrates were incorporated in the basal Richard's medium (devoid of sucrose): Hexoses: Glucose, Levulose. Disaccharides: Sucrose, Lactose, Maltose. Pentoses: Xylose. Sugar alcohol: Mannitol. The carbon level was maintained at the original level of the standard Richard's medium. The results are summarized in Table II. The maximum growth occurred with glucose as the carbon source and the maximum number of sclerotia also formed in this medium. This was followed by rest of the treatments in the order given in Table II. Statistically, however, there TABLE II Effect of different carbon sources on growth and sclerotial production of S. rolfsii Weight of Dry Number Dry carbo- weight of weight of Carbohydrate hydrate in g. per of mycelium sclerotia 1,000 sclerotia 100 ml. in mg. in mg. Glucose " 990 Sucrose " Levulose Lactose Xylose.. 5" Mannitol Control* "686 * No carbohydrate added, but slight amount introduced with the inoculum which was common for all troatmonts.

4 160 A. P. MISRA AND S. Q. HAQUE was no significant difference in yield between glucose and sucrose. Some mycelial growth and sclerotial production in control treatment is attributable to the small amount of PDA introduced with the inoculum which is common for all treatments. 5. Effect of different concentrations of glucose.--six different concentrations of glucose with 0-2.5~o carbon level were tried. The requisite quantifies of glucose were incorporated in Richard's medium (devoid of sucrose). The results are summarized in Table III. TABLE III Effect of different concentrations of glucose on growth and sclerotial production of S. rolfsii Weight Dry Dry weight Carbon of glucose weight of Number of of 1,000 in g. per mycelium sclerotia sclerotia 100 ml. in mg. in mg L.S.D. for mycelial growth: L.S.D. fox numbei of sclerotia : (P = 0.05) 25-7 (P = 0"05) 19.6 (P = 0"01) 36-0 (P = 0.01) 27"9 The growth increased with increase in the concentration of glucose from 0-2.5~ carbon level. The number of sclerofia also increased with increase in the concentration of glucose. Statistically the treatment differences were highly significant. 6. Effect of different nitrogen sources.---the following nitrogenous compounds were incorporated separately in Richard's medium (devoid of potassium nitrate), the nitrogen level being maintained at the original concentration in the standard Richard's medium.

5 Growth and Sclerotial Production in Sclerotium rolfsii Sacc. 161 A. Ammonium sulphate. B. Ammonium nitrate. C. Potassium nitrate. D. Sodium nitrate. E. Calcium nitrate. F. Urea. G. Asparagine. H. Peptone (16~o N as given by the manufacturing firm). The mycelial yield and sclerotial formation were recorded after 20 and 30 days' incubation at 20 C. respectively. The results are summarized in Table IV. TABLE IV Effect of different nitrogen sources on growth and sclerotial production of S. rolfsii Weight Dry Dry weight Nitrogen of salt weight of Number of of 1,000 source in mg. mycelium sclerotia sclerotia per 100 ml. in mg. in mg. Control Peptone Urea "068 Ammonium sulphate " Asparagine Ammonium nitrate Sodium nitrate " Calcium nitrate Potassium nitrate L.S.D. for mycelial growth : (P = o.o5) 2o.5 (p = o.oi) 28.2 L.S.D. fox number of sclerotia : (P = 0-05) 12"7 (P = 0"01) 17"4

6 162 A. P. MISRA AND S. Q. HAQUE Peptone proved to be the best source of nitrogen, both for myeelial growth and sclerotial production, which was followed in order by Asparagine, Calcium nitrate, Ammonium nitrate, Potassium nitrate, Sodium nitrate, Ammonium sulphate and Urea. Statistically the result was significant. 7. Effect of different concentrations of peptone. The effect of different levels of nitrogen was next investigated. Six different concentrations of peptone, ranging between 0 and 0.257o nitrogen level, were incorporated in the basal medium devoid of potassium nitrate. The results are summarised in Table V. TABLE V Effect of different concentrations of peptone on growth and sclerotial production in S. rolfsii Weight Dry Dry weight Nitrogen of peptone weight of Number of of 1,000 percentage in g. per m.ycelium sclerotia sclerotia 100 ml. in mg. in mg L,S.D. for mycelial growth: L.S.D. for number of sclerotia : (P = 0-05) 29" 6 (P = 0" 05) 18-1 (P = 0.01)41-3 (P = 0.01)25.7 Both the mycelial growth and sclerotial production increased with an increase in the concentration of peptone and were maximum at 0"25~o nitrogen level. Statistically the results were highly significant. B. Environmental Factors 1. Effect of temperature.--the effect of temperature on mycelial growth and sclerotial production was also studied. Maximum growth and sclerotial production occurred at 30 C. This was followed in order by 32.5 C.,

7 Growth and Sclerotial Production in Sclerotium rolfsii Sacc C., 35 C. and 25 C. (Table VI). Statistically the treatment differences were highly significant. TABLE VI Effect of temperature on growth and sclerotial production in Sclerotium rolfsii Temperature Radial Number Dry.growth of weight of m ram. sclerotia 1,000 after after sclerotia 6 days 30 days in mg. 25 C " C C "5 C " C L.S.D. for mycclial growth: L.S.D. for number of sclerctia: (P=0.05) 0.99 (P= 0.05) 16.8 (P=0.01) 1.65 (P ) Effect of relative humidity.--to investigate the effect of relative humidity, the organism was grown on potato dextrose agar and exposed to different humidity levels which were controlled after the method of Buxton and Mellanby (1934). The results are summarized in Table VII. The mycelial growth increased with increase in relative humidity from o. Statistically the treatment differences were highly significant. Visually there were no marked differences in the thickness of the mycelium and the type of growth. 3. Effect of ph.--the effect of ph on mycelial growth and sclerotial production was later investigated. The fungus was grown on Richard's agar medium, adjusted to different ph levels. The results are summarized in Table VIII. Maximum growth occurred at ph 6. Maximum number of sclerotia were also produced at this level. Below and above ph 6 the number of sclerotia decreased. Statistically the treatment differences were highly significant. B3

8 164 A. P. MISRA AND S. Q. HAQUE TABLE VII Effect of relative humidity on growth of Sclerotium rolfsii Relative Radial growth humidity in mm. % after 5 days L.S,D. (P=0"05) 0.70; L.S.D, (P=0.01) Effect TABLE VIII of ph on mycelial growth and sclerotial production in S. rolfsii Dry weight *ph Radial growth in Number of of 1,000 mm. after 4 days sclerotia sclerotia in mg " t? * Square-root transformed data of growth (after 4 days) after adding 0"5 to each, nec~,,itated because of zero observation in one treatment, for statistical analysis. t Sclerotia not produced. L.S.D. for square-root transformed data of mycelial growth (P = 0.05) L.S.D. for number of sclerotia (P = 0.05) 21.3.

9 Growth and Sclerotial Production in Sclerotium rolfsii Sacc. 165 C. Viability of Sclerotia 1. Effect of storage.--mature sclerotia of S. rolfsii were stored in the laboratory in specimen tubes at room temperature and their viability tested at different intervals, for one year. At the end of one year 82% sclerotia remained viable. 2. Effect of soil depth.--mature sclerotia (50 in each treatment) were placed at the bottom of glass beakers and sterilized, oven-dried soil was filled up to the height of 3, 4, or 6 inches. The beakers were then kept inside incubator at 30 C. for four months. The viability of sclerotia was tested at the end of this period. 82.6% sclerotia buried at 3 inches depth germinated as against 55.2% at 4 inches and 11.2% at 6 inches depth. Thus the germination of sclerotia decreased with increase in soil depth. Statistically the results were highly significant. 3. Effect of temperature.--sclerotia, collected from 30 day-old PDA culture, and with an initial viability of 100%, were immersed in hot water at 50 C., 55 C. and 60 C. in cloth bags for 3, 5 and 10 minutes and the loss of viability was tested by planting each lot on potato dextrose agar plates incubated at 30 C. for 10 days. The number of sclerotia that failed to germinate was recorded. Data are summarized in Table IX. TABLE IX Effect of temperature on the loss of viability of sclerotia* Temperature Percentage of non-viable sclerotia after 3 minutes 5 minutes 10 minutes 50 C " 3 55 C C "0 * Average of 3 replicates. The sclerotia lost viability completely after 10 minutes treatment at 55 C. and after 5 minutes at 60 C. 4. Effect of chemicals. The effect of different concentrations of mercuric chloride and formalin on the viability of sclerotia was investigated.

10 166 A.P. MISRA AND S. Q. HAQUE Sclerotia in lots of 25 each were treated with different concentrations of the chemical for 30, 45 and 60 minutes and the loss of viability was tested as in earlier experiments. Data are summarized in Table X. TABLE X Effect of different concentrations of mercuric chloride and formalin on the loss of viability of sclerotia* Chemical Concentration Percentage of non-viable sclerotia after 30 minutes 45 minutes 60 minutes Mercuric chloride 0" " Formalin * Average of three replicates. 100~o sclerotia lost viability in 45 minutes in 0-3~o mercuric chloride solution and in 60 minutes in 0.1 and 0.2Yo solutions respectively. Under formalin treatment 100yo sclerotia lost viability in 30 minutes in 3~ formalin solution and in 45 and 60 minutes in 2~o and 1~ solutions respectively. DISCUSSION The mycelial growth and sclerotial formation in S. rolfsii were greatly affected by the nutrients in the medium and the environmental factors, viz., the temperature, relative humidity and the ph of medium. Maximum growth and sclerotial production were observed on PDA amongst the solid media. Of the liquid media, Richard's medium gave the maximum mycelial yield. The different constituents of the Richard's medium had a marked influence on the growth of fungus. The reduction in growth was maximum in the absence of sucrose and this was closely followed by potassium nitrate. The other ingredients in order of importance were potassium dihydrogen

11 Growth and Sclerotial Production in Sclerotium rolfsii Sacc. 167 phosphate, ferric chloride and magnesium sulphate. Grover and Chona (1960) working with three different isolates of S. rolfsii observed that the CHOs were of little use in the growth of these fungi. The difference may be due to a difference in isolates. Of the various carbohydrates tried, glucose supported the highest mycelial output and the yield increased progressively with the increase in the concentration of glucose from 0-2-5y o carbon level. Glucose also proved to be the best source of carbon for sclerotial production. Of the different nitrogenous compounds peptone recorded the highest mycelial yield and the maximum number of sclerotia. In studies on the effect of temperature, it was observed that the fungus grew over a temperature range of C. with the maximum growth at 30 C. This was also the optimum for sclerotial production. According to Grover and Chona (1960), however, the temperature requirements for the optimum growth and sclerotial formations were different in isolates studied by them. With regard to the effect of humidity on the growth of S. rolfsii it was observed that the fungus grew over a humidity range of ~ with maximum growth at 100~o relative humidity. In studies on the effect of ph, maximum growth was recorded at ph 6 and maximum number of sclerotia were also formed at this level. It is, thus, observed that the conditions which favoured mycelial growth also favoured sclerotial formation. SUMMARY The effect of certain environmental and nutritional factors on growth and sclerotial production of Sclerotium rdfsii, isolated from rotten potato tubers, was investigated. The results were as follows: Amongst the solid media, the fungus grew best on potato dextrose agar closely followed by oat meal and maize meal agar. Amongst liquid media Richard's medium supported the maximum mycelial growth. Of six carbohydrates tried maximum growth occurred with ghicose as the source of carbon. The yield increased with increase in the concentration of glucose from 0-2.5~ carbon level. Amongst nitrogenous compounds, peptone proved to be the best source of nitrogen for fungal growth, the yield increased with increase in the concentration of nitrogen from Yo. Maximum growth of the fungus occurred at 30 C., which was followed in order by 32"5 C., 28 C., 35 C. and 25 C.

12 168 A.P. MISRA AND S. Q. HAQUE Growth also increased with increase in relative humidity from %. The fungus grew over a ph range of 3-8, with maximum growth at ph 6. Thus the conditions which favoured maximum mycelial growth also favoured maximum sclerotial production. The longevity of sclerotia was different at different soil depths and their viability was not much affected after one year's storage at room temperature, but exposure to hot water treatment for 5 minutes at 60 C. or 10 minutes at 55 C. killed all sclerotia. Treatment with 0.1-0"37o mercuric chloride solution or 1-3% formalin also killed all sclerotia within one or less than one hour. ACKNOWLEDGEMENT Thanks are due to Sri. R. S. Roy, Principal, Bihar Agricultural College, Sabour, for providing the necessary facilities for this work and for his invaluable encouragement. 1. Ajrckar, S. L. 2. Buxton, P. A. and Mellanby, K. 3. Curzi, M Grover, R. K. and Chona, B. L. 8. Higgins, B. B. 9. Patwardhan, G.B Paul, W. R. C. REFERENCES.. " The problem of potato storage in Western India," Agtie. J. India, 1924, 19, "The measurement and control of humidity," Bull. Ent. Rea., 1934, 25, "Studi su Lo "Sclerotium rolfmi' Boll.," R. Staz. Pat. Veg. Ann., 1931, 11, "Studi su Lo 'Sclerotium rolfssii' Boll.," Ibid., 1931 a, N.S. 11, Endo, S... "Physiological studies on the causal fungi of sclerotium disease of rice plants, with special reference to some factors controlling the occurrence of the disease," Bull. Mivazaki Coll. Agri. and For., 1940, 11, Epps, W. M.,Patterson, "Physiology and paiasitism of Scletoti~m rolfsii Sacc.," J. C. and Freeman, I. E. Phytopathology, 1951, 41, "Comparative studies on Sclerotinm rolfiii and Ozonium texanum Neal and Webster var. patasiticum Thirumalachar," lnd. Phytopath., 1960, 13(2), "Physiolosy and parasitism of Sclerotium rolfiii Sate.," Phytopathology, 1927, 17, "Appendix M. Annual report of the Plant Pathologist to the Government of Bombay, Poona, for the year ," Ann. Rept. Dept. dgric. Bombay PteMdency, , 1926, pp "A comparative morphological and physiological stud:y ef a number of strains of Bottytis cinera Pets, with special reference to their virulence," Trans. Brit. Myco. Sot., 1929, 14~ ,