EFFECT OF DIFFERENT CARBON AND NITROGEN SOURCES ON SCLEROTIUM ROLFSII SACC. MYCELIAL GROWTH AND SCLEROTIAL DEVELOPMENT

Article history Received: November 20, 2019 Revised: March 28, 2020 Accepted: April 20, 2020 In vitro studies were conducted on Potato Dextrose Agar using different carbon (C) and nitrogen (N) sources to evaluate their effects on the mycelial growth, and the sclerotial development of three Tunisian Sclerotium rolfsii Sacc. isolates. Radial growth was optimum on basal medium supplemented with ammonium chloride (0.48 gram of nitrogen per liter (g of N.L-1)) as N source but was restricted on LArginine and completely inhibited on ammonium acetate amended media (0.48 g N.L-1). Sclerotial initiation occurred from the 3rd to the 12th day of incubation for all tested isolates. Potassium nitrate was the most suitable N source for sclerotial formation whereas sclerotial development was completely inhibited on ammonium acetate amended medium. Optimal sclerotial germination was recorded using LArginine (78-80%) followed by L-Asparagine (46-94%) and ammonium chloride (46-88%) as N sources. Nevertheless, the lowest sclerotial germination rate was noted on sodium nitrate and ammonium acetate amended media. As for C sources (16 gram of carbon per liter (g of C.L-1)), optimal radial growth occurred using Dmannitol for Sr1 and Sr2 isolates and maltose for Sr3, but no mycelial growth was recorded using sodium citrate for all isolates. All C sources tested, except sodium citrate, were suitable for sclerotial formation, production, and germination. Mature sclerotia became brownish after 6 to 12 days of incubation and sclerotial production was highest using D-mannitol, maltose, and D-glucose, depending on isolates used, as C sources. Optimal germination of sclerotia was noted using D-glucose, D-mannitol and maltose for Sr1 isolate, maltose for Sr2 and D-glucose and maltose for Sr3. It was concluded that N and C sources are both important factors for the growth of S. rolfsii and its survival.


INTRODUCTION
Sclerotium rolfsii Sacc. is a serious ubiquitous soilborne phytopathogenic fungus, causing Southern blight of a wide range of agricultural and horticultural crops (Aycock, 1966and Anahosur, 2001and Galdames and Diaz, 2010and Kwon et al., 2013and Shen et al., 2014and Mahadevakumar et al., 2015. The fungus is notorious for its ability to induce dark stem rot, during any plant growth stages, followed by drooping and wilting of leaves and gradually wilting of the whole plant. Such wilted plants show white cottony fungal thread girdling the basal part of the stem and moving below the stem to roots (Kator et al., 2015and Sun et al., 2020. This pathogen survives as sclerotia on decayed plant material in the soil which germinate later and attack surrounding host plants (Sachslehner et al., 1997 andLudwig andHaltrich, 2002). During its pathogenesis, S. rolfsii secretes pectinolytic enzymes and oxalic acid (Ferrar and Walker, 1993and Ansari and Agnihotri, 2000. These compounds affect cell walls, hydrolyze pectin and alter host defensive responses (Bateman and Beer, 1965and Kritzman et al., 1977and Ferrar and Walker, 1993. This fungus is widely distributed and causes heavy economic losses on many crops (Aycock, 1966and Gurha and Dubey, 1983and Fery and Dukes, 2002and Billah, 2017and Sun et al., 2020. Yield losses are estimated as 53.4% on cowpea (Fery and Dukes, 2002), 5-20% on peppermint (Singh and Singh, 2004), 10-45% on tomato and artichoke (Banyal et al., 2008), 25-90% on potato (Anahosur, 2001), and 60% as plant losses (McCarter and Kays, 1984). To determine the optimal environmental and nutritional conditions for S. rolfsii growth and survival, Ayed et al. (2018a) and Ayed et al. (2018b) demonstrated under laboratory conditions that temperatures, pH, aeration and culture media have significant effects on these parameters. However, there is little information on pathogen nutritional requirements in semi-synthetic media. Therefore, this study was undertaken to examine the effect of different nitrogen and carbon sources on the mycelial growth, sclerotial production and germination of three Tunisian S. rolfsii isolates.

MATERIALS AND METHODS
Pathogen culture and sclerotial production Three S. rolfsii isolates, originally isolated from artichoke stem rot (Sr1) and rotted potato tubers (Sr2 and Sr3), were used in the current investigation. They were identified in a previous study (Ayed et al., 2018a). They were maintained in the laboratory of Phytopathology at the Regional Research Centre on Horticulture and Organic Agriculture of Chott-Mariem, Tunisia. Pathogen cultures used in the current study were previously grown for one week on Potato Dextrose Agar (PDA) at 30 °C and in the dark. Mature sclerotia were harvested from 21-day-old cultures, placed into fine nylon bags and washed frequently in sterile distilled water to remove excess agar. They were then filtered through Whatman n°1 sterile filter paper, placed in sterile plastic Petri plates and dried in a laminar flow cabinet for 4 hours. After drying, sclerotia were sized by dry sieving (425-1000 µm) and collected before being used in the trials (Ritchie et al., 2009).

Nitrogen and Carbon sources
The effects of nitrogen and carbon sources were carried out on basal medium consisting of 1.75 g KH2PO4, 0.75 g MgSO4.7H2O, 15 g agar and 1 L of distilled water (Townsend, 1957). For nitrogen sources, inorganic (Ammonium chloride, Potassium nitrate, Sodium nitrate, Ammonium acetate) and organic (L-Arginine, L-Asparagine) sources were autoclaved separately and added to batches of sterile medium, with D-glucose (40 g.L -1 : 16 g of C.L -1 ) as carbon source, to give a nitrogen level of 0.48 g.L -1 . Carbon sources (D-glucose, Glycerol, D-Mannitol, Maltose, and Sodium citrate) were tested on the basal medium supplemented with potassium nitrate (KNO3: 3.5 g.L -1 (0.48 g of N.L -1 )) as a nitrogen source. All carbon sources were autoclaved separately and added to the batches of sterile medium, except for disaccharides which were added to the medium after filter sterilization. The total level of the carbon added in each case was 16 g.L -1 . The tested media was adjusted to pH 6 and poured into Petri plates before pathogen inoculation.

Mycelial growth and sclerotial production
To examine the effect of different N and C sources on S. rolfsii mycelial growth, mycelial plugs (6 mm in diameter), cut from the margin of 7-day-old cultures, were placed in the centre of Petri plates containing agar media adjusted with the tested N and C sources and supplemented with streptomycin sulfate (300 mg.L -1 ). Inoculated plates were incubated at 30 °C in darkness (Ayed et al., 2018a(Ayed et al., , 2018b. The diameters of the developing colonies were measured after 24, 48 and 72 h of inoculation and the radial growth rate (mm/day) was calculated. The same cultures were further incubated for additional 21 days. During this period, sclerotial production was monitored and determined at 3-day intervals (Maurya et al., 2010). For the monitoring of sclerotial production, brown sclerotia were removed with a sharp scalpel, placed in fine mesh nylon bags and washed with sterile distilled water to remove adhering agar. They were counted and the average number of mature sclerotia produced per plate was determined. After counting, sclerotia were placed on pre-dried and weighed Whatman n°1 filter papers and incubation at 70 °C for 48 h. The dry weight of 100 sclerotia formed per plate was determined. For all parameters noted, ten replicate plates were used per individual treatment (per isolate and per treatment (tested C or N sources).

Sclerotial germination
Sclerotia of a similar size (21-day-old) were used in this study. Ten sclerotia were placed onto Petri plates containing culture media supplemented with the different N and C sources tested and incubated at 30 °C in darkness (Ayed et al., 2018a andAyed et al., 2018b). Germination was determined after 24, 48 and 72 h of incubation by examining each sclerotia for any outgrowing hyphae observed under a binocular microscope. A sclerotium was considered as germinated when outgrowing hyphae were equal to or greater than its diameter. Ten replicate plates, containing 10 sclerotia, were used per individual treatment and the percentage of germinated sclerotia per plate was calculated (Ayed et al., 2018a andAyed et al., 2018b).

Statistical analysis
Data analyses were performed following a completely randomized factorial design where fungal treatment (S. rolfsii isolates) and the tested factors (N or C sources) were the two fixed factors. Mean values were evaluated and separated using Fisher's protected LSD and/or Duncan's Multiple Range tests (at P ≤ 0.05). Statistical analyses were carried out using SPSS software version 20. All the experiments were repeated twice and for each test, the mean data was presented in the current study.

Effect of nitrogen sources on S. rolfsii mycelial growth and survival Effect on radial mycelial growth
The mean diameter of S. rolfsii colonies, noted after 3 days of incubation at 30 °C, varied significantly (at P ≤ 0.05) depending on N sources only but no significant differences were noted between the three tested isolates. Furthermore, as no significant interaction was noted between both factors, results were presented and commented considering the mean radial growth per N source only as given in Figure 1. All S. rolfsii isolates showed optimum mycelial growth on the basal medium amended with ammonium chloride (20.08 -20.45 mm/day) but complete growth inhibition occurred on ammonium acetate treated medium. Sodium nitrate and potassium nitrate supported also good radial growth of all isolates with 18.7-18.94 mm/day and 17.8-18.52 mm/day, respectively. The poorest mycelial growth was recorded on basal medium amended with L-arginine (8.67-10.34 mm/day) ( Figure 1).

Effect on sclerotial formation and production
As indicated in Table 1, sclerotial formation was affected by N sources and S. rolfsii isolates. Sclerotial initiation of all isolates started after 3 to 12 days of incubation depending on tested treatments. It started on the 3 rd day after incubation on basal medium amended with potassium nitrate and L-asparagine, and on the 6-12 th days using the remaining nitrogen sources. The network developed to so-called initial and grew to white immature sclerotium on the 6-15 th day of incubation and brown mature sclerotia were observed only after 12-21 days. Potassium nitrate and sodium nitrate were the most suitable N sources for S. rolfsii sclerotial formation as sclerotia became brownish at the 9-15 th and 12-18 th day of incubation, respectively. However, a complete inhibition of any sclerotial development, till 21days of culture, was recorded on basal growth medium amended with ammonium acetate . The average number of mature sclerotia produced per plate, after 21 days of incubation, varied significantly (at P ≤ 0.05) depending on N sources tested and isolates used. A significant interaction was also observed between these two factors. As indicated in Table 2, the highest sclerotial yields were noted on Sr2 and Sr3 cultures grown on potassium nitrate amended medium (102.6 and 33.2 sclerotia/plate, respectively), while Sr1 formed significantly more sclerotia on ammonium chloride (6.6 sclerotia/plate), potassium nitrate (21.4 sclerotia/plate) and sodium nitrate (22.6 sclerotia/plate) amended media followed by those modified using the other N sources (Table 2). For all N sources tested (pooled data of all N sources), Sr2 isolate produced significantly (at P ≤ 0.05) more sclerotia than Sr1 and Sr3.

Effect on sclerotial germination
After 24 h of incubation at 30°C, the germination of sclerotia of all of S. rolfsii isolates occurred on all N source-amended media and varied significantly (at P ≤ 0.05) depending on tested N sources and isolates as indicated by the significant interaction noted between these two fixed factors. As shown in Table 3, optimal germination was recorded on ammonium chloride, L-Arginine and L-Asparagine amended basal media, for Sr1 and Sr2 isolates, while that of Sr3 was noted only using L-Arginine. However, the lowest sclerotial germination rate was observed on sodium nitrate added to the basal medium for all tested isolates (Table 3). For the data of all N sources combined, the highest number of germinated sclerotia was recorded for Sr1 isolate (76.8%) followed by Sr2 (68.4%) and Sr3 (48.4%). Table 2. Effect of various nitrogen sources (0.48 g of N.L -1 ) on the number of sclerotia produced by three Sclerotium rolfsii isolates after 21 days of incubation at 30°C on basal growth medium amended with D-glucose (16 g of C.L -1 ).

N Sources
Number  Mean sclerotial germination per N source for the three isolates combined. 2 Mean sclerotial germination per isolate for all N sources combined. * Mycelial growth and sclerotial formation were completely inhibited on ammonium acetate amended basal medium. Therefore, sclerotial germination was not recorded in this study. *LSD (N sources × S. rolfsii isolates) = 8.43% at P ≤ 0.05. *For each isolate and each mean of sclerotial germination (per N source or per isolate), values followed by the same letter are not significantly different according to Duncan Multiple Range test (at P ≤ 0.05).

Effect of carbon source on S. rolfsii growth and survival Effect on radial mycelial growth
The different S. rolfsii isolates responded differently to C sources as illustrated by the significant (at P ≤ 0.05) interaction recorded between isolates and C sources tested for their effects on pathogen mycelial growth. Optimal radial growth occurred on D-mannitol amended basal medium for Sr1 and Sr2 isolates with an average rate of 22.5 and 22.8 mm/day, respectively, whereas Sr3 isolate grew faster (20.6 mm/day) on maltose-amended medium. D-glucose supported also an important radial growth of all isolates (19-20.7 mm/day), but the mycelial growth was restricted on glycerol-amended medium with 15.7-18.1 mm/day). Nevertheless, no mycelial growth was observed after 3 days of incubation at 30°C on culture medium modified using sodium citrate as C source (Table 4).
For all C sources combined, the highest mycelial growth was recorded on Sr1 and Sr2 cultures.

Effect on sclerotial formation and production
As for the mycelial growth, all C sources tested, except sodium citrate, were suitable for the sclerotial formation in S. rolfsii as sclerotial initiation started on the 3 rd day after incubation and mature sclerotia became brownish at the 6 th to 12 th day of incubation. However, sclerotial development was completely inhibited on sodium citrate even after 21 days of incubation (Table 5). Table 4. Effect of various carbon sources (16 g of C.L -1 ) on the mycelial growth of three Sclerotium rolfsii isolates recorded after 3 days of incubation at 30 °C on basal medium with amended with potassium nitrate (0.48 g of N.L -1 ).

C Sources
Radial growth (  Sodium citrate The average number of brown sclerotia per plate, produced after 21 days of incubation at 30 °C on basal medium, varied significantly (at P ≤ 0.05) depending on tested C sources and S. rolfsii isolates and their interactions. As given in Table 6, Sr1 isolate showed optimal sclerotial production on D-mannitol or maltose amended basal media, estimated at 11.2 and 8.5 sclerotia/plate, respectively, but was significantly reduced using the other C sources. For Sr2 and Sr3, sclerotial yield was high using D-glucose with an average of 35 and 70 mature sclerotia/plate, respectively. Nevertheless, when grown on sodium citrate, sclerotial production was very restricted and inhibited. For all the C sources pooled, the number of sclerotia produced by Sr3 (31.3 sclerotia/plate), 21 days after incubation on amended basal medium, was significantly higher than that of the Sr2 (16.5 sclerotia/plate) and Sr1 (5.3 sclerotia/plate) isolates (Table 6).

Effect on sclerotial germination
The germination of S. rolfsii sclerotia, after 24 h of incubation on C source amended PDA media at 30 °C, varied significantly (at P ≤ 0.05) depending on tested C sources and pathogen isolates and on their interactions (Table 7). 5.3 c 16.5 b 31 . 3 a -1 Mean number of sclerotia per C source for the three isolates combined. 2 Mean number of sclerotia per isolate for all C sources combined. *LSD (C sources × S. rolfsii isolates) = 7.59 sclerotia at P ≤ 0.05. *For each isolate and each mean number of sclerotia (per C source or per isolate), values followed by the same letter are not significantly different according to Duncan Multiple Range test (at P ≤ 0.05). Table 7. Effect of various C sources (16 g of C.L -1 ) on the sclerotial germination of three Sclerotium rolfsii isolates on basal medium, with potassium nitrate (0.48 g of N.L -1 ), noted after 24 h of incubation at 30°C. 42.5 c -1 Mean sclerotial germination per C source for the three isolates combined. 2 Mean sclerotial germination per isolate for all C sources combined. *Mycelial growth and sclerotial formation were completely inhibited on sodium citrate amended basal medium. Therefore, sclerotial germination was not recorded in this study. *LSD (C sources × S. rolfsii isolates) = 7.81% at P ≤ 0.05 *For each isolate and each mean of sclerotial germination (per C source or per isolate), values followed by the same letter are not significantly different according to Duncan Multiple Range test (at P ≤ 0.05).

DISCUSSION
Sclerotium rolfsii, the causal agent of the Southern blight disease, is a soilborne fungus attacking a wide host plants (Galdames and Diaz, 2010and Kwon et al., 2013and Shen et al., 2014and Mahadevakumar et al., 2015. It occurs worldwide and has a great survival ability under varied environmental and host conditions (Punja, 1985). Previous studies have concentrated on its behavior and on the effect of abiotic factors on its growth and survival (Ayed, 2019and Ayed et al., 2018aand Ayed et al., 2018b. In the present study, our investigation focused on the effect of nitrogen and carbon sources on the mycelial growth and the sclerotial production and germination of three Tunisian S. rolfsii isolates. All tested isolates showed optimum mycelial growth on ammonium chloride-amended medium. However, their growth was significantly restricted using L-Arginine as N source and was completely inhibited with ammonium acetate. Potassium nitrate, Sodium nitrate, and L-Asparagine were found to be suitable for pathogen growth. These results are in agreement with those of Divya and Narayanba (2017) for the suitability of ammonium chloride, and those of Khattabi et al. (2004) for potassium nitrate. Hussain et al. (2003) found that potassium nitrate had the ability to enhance S. rolfsii mycelial growth. Concerning sodium nitrate, Mostafa and Mohamed (2018) demonstrated that this N source allowed faster growth of Rhizoctonia solani. However, L-Arginine was unfavorable unsuitable nitrogen source for the mycelial growth of some fungal pathogens (Shim et al., 2005 andJayasinghe et al., 2008). Nevertheless, this finding did not confirm a previous study (Muthukumar and Venkatesh, 2013) reporting the lowered mycelial growth of S. rolfsii using ammonium chloride as N source. Furthermore, Fariña et al. (1999) and Liu and Guo (2009) indicated that when nitrate was used as nitrogen source, fungal growth led to biomass concentrations higher than those obtained with ammonium. Sclerotial initiation for all S. rolfsii isolates started after 3 to 12 days of incubation. Potassium nitrate and sodium nitrate were found to be the most suitable N sources for S. rolfsii sclerotial formation as sclerotia became brownish at the 9-15 th and 12-18 th day of incubation, respectively. However, a complete inhibition of any sclerotial development was recorded using ammonium acetate. The optimal sclerotial production was recorded on potassium nitrate-amended medium. These results confirmed Pany and Apparao (1963) findings reporting that S. rolfsii sclerotia were more abundant when potassium nitrate was used as N source. However, Elgorban et al. (2014) reported no differences in the number of sclerotia of Sclerotinia sclerotiorum grown on culture media supplemented with ammonium chloride and L-Arginine. Optimal germination occurred media amended with L-Arginine, L-Asparagine, and ammonium chloride. Nevertheless, the lowest sclerotial germination rate was recorded using sodium nitrate and ammonium acetate.
In the present study, we also evaluated the suitability of five carbon sources added to a basal medium for the growth of three S. rolfsii isolates. Optimal radial growth was noted using D-mannitol for Sr1 and Sr2 isolates and maltose for Sr3. D-glucose supported also an important radial growth of all isolates whereas no mycelial growth was noted using sodium citrate. These findings are in accordance with those of Al-Noimi and Kassim (2006) and Survase et al. (2006) who reported a significant mycelial growth of S. rolfsii using maltose and glucose. Furthermore, Bhagat (2013) mentioned that maltose and glucose supported a good growth of S. rolfsii. Divya and Narayanba (2017) recorded a maximum growth of the fungus when glucose was used as the main carbon source. Chandra and Purkayastha (1977) reported that most of the tropical edible macrofungi were in favor of utilizing glucose than other carbon sources. The preference of glucose over other carbon compounds may be due to its fast metabolization by fungi (Garraway and Evans, 1984). The utilization of various carbon compounds may depend on the activity of the fungus to utilize simpler forms or on its ability to convert the complex carbon compounds into simpler forms, which may be easily utilized (Muthukumar and Venkatesh, 2013). Moreover, the C source not only acts as a major constituent for building of cellular material, but is also used in synthesis of polysaccharide, and as energy source (Dunn, 1985 andDube, 1983). All C sources tested, except sodium citrate, were suitable for sclerotial formation, production, and germination. Mature sclerotia became brownish after 6 to 12 days of incubation with an optimal sclerotial production occurring on D-mannitol and maltose modified media for Sr1 isolate and on D-glucose for Sr2 and Sr3. These results are in agreement with other studies reporting that glucose and maltose were the best C sources supporting the formation of sclerotia (Zoberi, 1980). However, Liu and Guo (2009) showed that glucose and maltose, allowing optimal mycelial growth, could not induce sclerotial formation of Polyporus umbellatus. The optimum sclerotial germination, noted after 24 h of incubation at 30°C on basal medium, was noted on growth media supplemented with D-glucose, D-mannitol and maltose for Sr1, with maltose for Sr2, and with Dglucose and maltose for Sr3. Punja et al. (1984) demonstrated that the addition of metabolizable carbon sources to substrates low in nutrients inhibits eruptive germination, possibly through catabolite repression. Moreover, Hyakumachi et al. (2014) reported that sclerotia become dependent on exogenous nutrients for germination when endogenous carbon loss reached 20% of available 14 C label. They become nearly completely dependent on exogenous supply of nutrients for germination, when carbon loss accounted for 40% of labeled carbon and death of sclerotia occurred when this reached about 50%.

CONCLUSION
In conclusion, considerable variations in mycelial growth, sclerotial development, and germination were observed using different N and C nutritional sources for S. rolfsii culture. Carbon sources such as ammonium chloride, potassium nitrate and L-Arginine were found to be suitable for mycelial growth, sclerotial production, and germination, respectively. For nitrogen sources, D-mannitol, maltose and D-glucose were found to be the most suitable for radial growth and sclerotial germination. However, further investigations into the effect on pathogen development under field conditions would provide a greater understanding of the biology of S. rolfsii isolates. Such studies would improve our understanding of the pathogen's population dynamics in soil and help to implement effective disease control methods.

ACKNOWLEDGMENTS
This work was funded by the Ministry of Higher Education and Scientific Research in Tunisia through the budget assigned to UR13AGR09-Integrated Horticultural Production in the Tunisian Centre-East, The Regional Research Centre on Horticulture and Organic Agriculture of Chott-Mariam, University of Sousse, Tunisia.