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Mycologia, 94(6), 2002, pp. 1051-1058.
© 2002 by The Mycological Society of America

Variability in Indian isolates of Sclerotium rolfsii


B. K. Sarma
U. P. Singh 1

     Department of Mycology and Plant Pathology, Institute of Agricultural Sciences, Banaras Hindu University, Varanasi-221005, India

K. P. Singh

     College of Forestry & Hill Agriculture, G. B. Pant University of Agriculture and Technology, Hill Campus, Ranichauri-249199, India

   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 

Variability among 26 isolates of Sclerotium rolfsii collected from various hosts/soil samples and localities in India is reported. The isolates varied in colony morphology, mycelial growth rate, sclerotium formation, teleomorph production and sclerotial size and color. Out of 26 isolates, only 4 produced the teleomorph stage on Cyperus rotundus rhizome meal agar medium. Mycelial incompatibility among the isolates was also seen, and out of 325 combinations, only 29 combinations (8.9%) showed compatible reactions. Based on mycelial compatibility, 13 vegetative incompatibility groups (VCG) were identified among the isolates. HPLC analysis of the ethyl acetate fraction of culture filtrates of the isolates revealed 10–22 peaks. Six peaks were identified as gallic, oxalic, ferulic, indole-3-acetic acid (IAA), chlorogenic, and cinnamic acids. Oxalic, IAA, and cinnamic acids were present in the culture filtrates of all the isolates in varying amounts. The other three phenolic acids were not detected in some of the isolates. A comparative HPLC analysis of sclerotial exudate, sclerotia, mycelia, and culture filtrates of two S. rolfsii isolates (leaf spot- and collar rot-causing) producing different symptoms on their respective hosts revealed variation in the content of phenolic acids, IAA, and oxalic acid.

Key words: Indian isolates, Sclerotium rolfsii, variability


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Sclerotium rolfsii Sacc. (teleomorph Athelia rolfsii (Curzi) Tu & Kimbrough) is a devastating soil-borne plant pathogenic fungus with a wide host range (Aycock 1966Citation, Punja 1988Citation). The fungus was placed in the form genus Sclerotium by Saccardo (Saccardo 1913Citation), as it forms differentiated sclerotia and sterile mycelia. Although there are several other sclerotium-producing fungi, the fungi characterized by small tan to dark-brown or black spherical sclerotia with internally differentiated rind, cortex, and medulla were placed in the form genus Sclerotium (Punja and Rahe 1992Citation). However, the teleomorphic state was discovered later (Punja 1988Citation), confirming that the fungus was a basidiomycete. Sclerotium rolfsii usually causes collar rot, but spotted leaf rot with a single tiny sclerotium in the center has also been reported (Singh and Pavgi 1965Citation).

Geographical variability among S. rolfsii populations was demonstrated by earlier workers (Harlton et al 1995Citation, Nalim et al 1995Citation, Okabe et al 1998Citation). Studies of variability within the population in a geographical region are important because these also document the changes occurring in the population. The importance could be realized in the light of the discovery of PCNB-tolerant strains of S. rolfsii isolated from a Texas peanut field in 1985 (Nelin 1992Citation). In previous studies, S. rolfsii isolates of the Indian sub-continent were not included, and, therefore, not much is known about the diversity of the Indian population of the fungus. The purpose of the present study was to understand the variability in cultural morphology, sclerotium formation, basidiocarp induction, mycelial compatibility, and mainly phenolic constituents of sclerotial exudate, culture filtrate, sclerotia, and mycelia of some isolates of S. rolfsii collected from different hosts and geographic locations of India.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
Fungal isolates and culture maintenance – Twenty-six isolates of S. rolfsii (22 causing collar rot and 4 causing leaf spot) used in this study were collected from various hosts/soil samples from diverse geographic origins (Table I). The isolates were further purified by growing single sclerotia from each colony on potato dextrose agar (PDA) (peeled potato 200 g, dextrose 20 g, agar 15 g, distilled water 1 L) slants.


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  		TABLE I. Characters of the isolates of Sclerotium rolfsii

 
Cultural variation – Radial growth (colony diameter, cm), colony morphology, and sclerotial production (total number per plate and size) were evaluated on PDA, whereas basidiocarp induction was observed on Cyperus rotundus rhizome meal agar (CRMA) medium (Prithiviraj et al 2000Citation). At least three plates each of PDA and CRMA were inoculated with a 5-mm-diameter mycelial disc taken from the margin of an actively growing colony (3- to 4-d-old) of each isolate. The inoculated plates were incubated at 25 ± 2 C under diffuse light, whereas CRMA plates were incubated at the same temperature under darkness. The colony diameter was measured every day to a maximum of 5 d by taking two measurements at right angles. The number of sclerotia per colony was counted after 20–25 d of incubation. Diameter of 25 sclerotia was measured. The data from the replicated plates were averaged. Morphological variation of the colonies on PDA and basidial stage formation on CRMA medium were observed after 4 and 7–10 d of incubation, respectively.

Mycelial compatibility – Mycelial discs (5 mm diameter) taken from the edge of an actively growing colony (3- to 4-d-old) of each isolate were placed approximately 25 to 35 mm apart on opposite sides of 100 x 15 mm petri dishes and incubated at 25 ± 2 C. Three isolates were usually paired on one dish and the test was repeated at least twice. The pairings were examined macroscopically after 5–15 d for the presence of an antagonistic (barrage or aversion) zone in the region of mycelial contact as described by Punja and Grogan (1983)Citation.

Ethyl acetate fractionation of culture filtrate – Fifty mL of potato dextrose broth was poured in each of several conical flasks (150 mL). Mycelial bits from young, growing cultures of S. rolfsii were inoculated separately and allowed to grow for 7 d at room temperature (27 ± 2 C). The culture filtrate was then filtered through sterilized Whatman No. 1 filter paper. An equal volume of ethyl acetate was mixed with culture filtrate separately, and after vigorous shaking in a separatory funnel, the ethyl acetate fractions of the culture filtrate were collected separately. The residue was extracted a second time and the ethyl acetate fractions were pooled with the previous extract. The fractions were then evaporated under vacuum. Dried samples were resuspended in 1.0 mL of HPLC grade methanol by vortexing and stored at 4 C for further analysis.

Ethyl acetate fractionation of the constituents of sclerotial exudate, sclerotia, mycelia, and culture filtrate of S. rolfsii – Two representative isolates each from collar rot causing (isolate 1) and leaf spot causing (isolate 14) were used for the study. Ethyl acetate fractions of culture filtrates of the isolates were obtained as described earlier. The mycelial mats of the two isolates from potato dextrose broth were harvested and washed three times in distilled water and placed on a pad of sterile filter paper to remove excess water. Two g of the mat from each isolate was thoroughly macerated separately in the presence of ethyl acetate in a pestle-mortar. The finely crushed material was collected in screw-capped bottles with an additional 5 mL of ethyl acetate and kept overnight.

For collection of sclerotial exudate and sclerotia, the two isolates were allowed to grow on PDA, and the plates were regularly observed for sclerotium formation as well as exudation from the developing sclerotia. The exudate was formed after 7–10 d. When exudate was visible on sclerotial primordia, it was removed with a sterilized capillary tube, collected in sterile screw-capped specimen tubes and stored at 4 C. The ethyl acetate fractions of the exudate from the two isolates were collected in a similar way as that of culture filtrate described earlier. The mature sclerotia produced on such plates were collected separately, and one g from each isolate was taken separately and crushed in a pestle-mortar in the same way as mycelia. The ethyl acetate fractions of exudate, culture filtrate, sclerotia, and mycelia were re-extracted twice, the pooled fractions of the same samples were then filtered through Whatman No. 1 filter paper, and the filtrate was evaporated under vacuum (Buchi Rotavapor Re Type). Dried samples were suspended in 1.0 mL of HPLC grade methanol by vortexing and stored at 4 C for further analysis.

HPLC analysis – High performance liquid chromatography (HPLC) of the samples was performed with an HPLC system (Shimadzu Corporation, Kyoto, Japan) equipped with two Shimadzu LC-10 ATVP reciprocating pumps, a variable UV-VIS detector (Shimadzu SPD-10 AVP) and a Winchrom integrator (Winchrom). Reverse phase chromatographic analysis was carried out in isocratic conditions using C-18 reverse phase HPLC column (250 x 4.6 mm id, particle size 5 µm Luna 5µ C-18 (2), Phenomenex, USA) at 25 C. Running conditions included injection volume 5 µL, mobile phase methanol: 0.4% acetic acid (80:20 v/v), flow rate 1 mL/min, and detection at 290 nm. Samples were filtered through a membrane filter (pore size 0.45 µm, Merck) prior to injection. Gallic, chlorogenic, ferulic, cinnamic, oxalic, and indole-3-acetic acids (IAA) were used as internal and external standards. Phenolic compounds, oxalic acid, and IAA in the samples were identified by comparing retention time (Rt) of standards and by co-injection. Concentrations were calculated by comparing peak areas of reference compounds with those in the samples run under the same conditions.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
Variability in growth characters and basidial stage production – The isolates of S. rolfsii varied in all of the characters evaluated, e.g., colony morphology, mycelial growth rate, sclerotial production, basidiocarp induction, sclerotial size, and color. Out of 26 isolates, colonies of 18 isolates were fluffy, whereas 8 were compact. The growth rate of the isolates varied substantially. While 9 isolates were the fastest growing (31 mm/d), the isolate from soybean was the slowest (23 mm/d). Others varied from 25 to 30 mm/d. Production of sclerotia among isolates varied significantly. Most of the isolates produced a very large number of sclerotia (>300 to 500 sclerotia/plate), while others produced fewer (<80 to 200 sclerotia/plate). Similarly, the size and color of sclerotia varied in different isolates. The average size of sclerotia of most of the isolates varied within 1–1.2 mm in diameter, whereas the largest (up to 2.2 mm in diameter) were produced by the Pogostemon cablin isolate. The color of sclerotia was mostly dark to reddish brown at maturity with an exception in the soil isolate from Varanasi (BHU campus) being very light brown even after maturity. Only four isolates (1, 2, 3, and 4) out of 26 produced the teleomorph on CRMA medium (Table I).

Mycelial compatibility – There were 325 pairings of the 26 isolates and out of all, only 29 combinations showed a compatible reaction (8.9% of all the combinations) where mycelia of the two isolates intermingled at the zone of interaction (Table II). The remainder of the combinations showed antagonistic reactions with each other, forming a thin band of living or dead mycelia (Fig. 1). Based on mycelial compatibility, 13 vegetative incompatibility groups (VCG) were found among the isolates. In all the antagonistic reactions, sclerotia were not formed at the interaction zone. Sclerotia were formed only in the border of the lytic zone of the two isolates in only 23.6% combinations. However, a few sclerotia produced later on such lytic zone in some combinations failed to develop to the full size as those produced on the border of such barrages. On prolonged incubation, the antagonistic site, in some combinations, was broadened at the interaction zone either parallel to both sides traversing to almost 2/3 of the mycelial growth, or in some cases lysis occurred completely in one isolate only (Fig. 2). Interestingly, sclerotia were not formed in such combinations. However, in some combinations, the interacting zone did not widen even after prolonged incubation.


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  		TABLE II. Mycelial compatibility of some isolates of Sclerotium rolfsii

 


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  		 Fig(s). 1, 2. Mycelial compatibility reactions between isolates of Sclerotium rolfsii. 1. Normal intermingling (NI) and barrage (Br) formation between compatible (a and b) and incompatible (c with both a and b) isolates. 2. Unlysed (a) and gradual lysis of mycelia in another isolate (b) after 20 d of inoculation

 
HPLC analysis of culture filtrate of S. rolfsii – HPLC analysis revealed 10–22 peaks in the culture filtrates of S. rolfsii isolates. Out of these, 6 were identified on the basis of their retention time (Rt) as well as by co-injection. All these 6 peaks consistently appeared in culture filtrates of most of the isolates. The peaks identified were of gallic (Rt 2.86 min), oxalic (Rt 3.03 min), ferulic (Rt 3.30 min), indole-3-acetic (Rt 3.60 min), chlorogenic (Rt 4.16 min) and cinnamic acids (Rt 4.46 min). Gallic, ferulic, chlorogenic, and cinnamic acids are phenolic acids synthesized following the shikimic acid pathway (Salisbury and Ross 1986Citation). Out of 26 isolates, gallic acid was detected in 14 isolates, ferulic acid in 16 isolates, and chlorogenic acid in 14 isolates. Oxalic acid, IAA, and cinnamic acid were detected in all the isolates. The major component in culture filtrates of all the isolates was oxalic acid, and its amount varied from 10.20 to 0.29 µg/mL of the total ethyl acetate fractions followed by IAA (0.52 to 0.09 µg/mL) and cinnamic acid (0.001 to 0.03 µg/mL), respectively (Table III).


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  		TABLE III. Phenolic, oxalic and Indoli-3-acetic acid (µg/ml) contents in culture filtrates of different isolates of Sclerotium roflsii

 
HPLC analysis of sclerotial exudate, sclerotia, mycelia, and culture filtrates of S. rolfsii – HPLC analysis revealed that the phenolic acids of mycelia, sclerotia and exudates, and culture filtrates have wide variation in their content among the isolates. In isolate 1, gallic acid was present in sclerotia, mycelia, exudates, and culture filtrate, whereas oxalic, ferulic, and cinnamic acids were present in all the samples except in mycelia, culture filtrate, and sclerotia, respectively. Chlorogenic acid was detected in the exudate, whereas IAA was detected in the culture filtrate only (Fig. 3A–D).



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  		 FIG. 3. HPLC analysis of ethyl acetate fractions of sclerotial exudate (A), mycelia (B), sclerotia (C) and culture filtrate (D) of a collar rot-causing and sclerotial exudate (E), mycelia (F), sclerotia (G) and culture filtrate (H) of a leaf spot-causing Sclerotium rolfsii (peak nos. 1 = gallic acid, 2 = oxalic acid, 3 = ferulic acid, 4 = indole-3-acetic acid, 5 = chlorogenic acid, 6 = cinnamic acid)

 
On the contrary, in isolate 14, gallic acid was detected only in sclerotia and mycelia. Ferulic acid was detected in all the samples except culture filtrate, whereas oxalic, chlorogenic, and IAA were detected only in exudate and culture filtrate. Cinnamic acid was detected in all samples (Fig. 3E–H).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
The results of the present study reveal wide variation among Indian isolates of S. rolfsii. Since the sexual stage of S. rolfsii is rare in nature and its role in the life cycle of the fungus is unknown, genetic exchange in mycelia of S. rolfsii isolates is largely thought to be limited to mycelial compatibility (Nalim et al 1995Citation). However, consistent production of the teleomorph stage in four isolates of S. rolfsii on CRMA medium may strengthen the claim that genetic exchange may occur through normal genetic recombination, i.e., meiosis discernible in the progeny. The absence of the teleomorph stage in most of the isolates may be because they have lost the ability to produce basidiospores during the course of evolution or they require specific conditions. Alternatively, the genetic factor responsible for sexual reproduction may be triggered in some isolates by components in CRMA medium. However, according to Nalim et al (1995)Citation, nuclear exchange through anastomosis in hyphae may be responsible for normal genetic recombination in this fungus.

The high rate of antagonistic reactions in the mycelial compatibility test further shows the extent of the diversity among these isolates of S. rolfsii. Interestingly, all of the four leaf spot-causing isolates (14, 15, 16, and 17) exhibited mycelial compatibility with each other but antagonistic reactions with collar rot- or foot rot-causing isolates. This is an important observation that distinguishes the leaf spot-causing isolates from others. The death of mycelia at the interaction zone is attributed to the heterokaryotic condition of the nuclei (Punja 1985Citation), but the involvement of toxin(s) cannot be ruled out (Punja 1985Citation). A detailed study in this regard may reveal more information about the cause of mycelial death in the incompatible reactions.

Ethyl acetate fractions of culture filtrates of different isolates of S. rolfsii show the qualitative and quantitative variation in their organic acid composition. No definite pattern of the occurrence of phenolic acids in culture filtrates was observed. Although oxalic acid was present in varying amounts in culture filtrates of all isolates, no relationship could be drawn between the leaf spot- and collar rot-causing S. rolfsii on the basis of the phenolic acids in culture filtrates. The presence of high amounts of IAA in the culture filtrates of all isolates is significant. There are several reports on the IAA production by fungi and bacteria that cause plant diseases (Gruen 1959Citation, Sequeira 1973Citation, Chauhan et al 2000Citation). In many cases, IAA production is related to gall formation in the host plants (Chauhan et al 2000Citation). But there is no such gall formation at the site of infection caused by S. rolfsii. More detailed studies are needed to define the exact role of IAA in infection.

The HPLC profiles of the exudate and culture filtrate of any individual isolate indicates that exudate and culture filtrate are different in their composition. Similarly, the two forms of mycelia, active and inactive (sclerotium), are also different in their phenolic composition. Moreover, the different HPLC profiles of exudate, sclerotia, mycelia, and culture filtrates of the leaf spot- and collar rot-causing S. rolfsii further demonstrate the variation in constituents in the two groups of isolates of S. rolfsii differing in pathogenic specificity. Phenolic compounds in sclerotia of S. rolfsii were reported by Punja and Damiani (1996)Citation, but identification of some of the phenolics is reported for the first time in the present investigation. Phenolic acids are believed to contribute to resistance in host plants against certain pathogens. The presence of phenolic acids in S. rolfsii may be attributed to its self-defense against other microbes during its survival in soil. The presence of relatively higher amounts of individual phenolic acids in sclerotia than in mycelium further suggests that sclerotia need higher amounts of phenolic acids for their survival than mycelia.


   ACKNOWLEDGMENTS
 
The authors are thankful to Prof. Y. Rathaiah, Department of Plant Pathology, Assam Agricultural University, Jorhat 785013, India for providing three isolates of Sclerotium rolfsii. B. K. Sarma is grateful to the Council of Scientific and Industrial Research, New Delhi, India for the award of Senior Research Fellowship.


   FOOTNOTES
 
1 Corresponding author, ups{at}banaras.ernet.in Back

Accepted for publication March 18, 2002.


   LITERATURE CITED
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Aycock R., 1966 Stem rot and other diseases caused by Sclerotium rolfsii. NC Agric Exp Stn Tech Bull 174: 202 p

Chauhan VB, Singh PN, Singh UP., 2000 Role of indole-3-acetic acid in gall formation in Phytophthora-infected stem of pigeonpea. J Pl Dis Prot 107:637-642

Gruen HE., 1959 Auxins and fungi. Ann Rev Pl Physiol 10:405-440

Harlton CE, Lévesque CA, Punja ZK., 1995 Genetic diversity in Sclerotium (Athelia) rolfsii and related species. Phytopathology 85:1269-1281

Nalim FA, Starr JL, Woodard KE, Segner S, Keller NP., 1995 Mycelial compatibility groups in Texas peanut field populations of Sclerotium rolfsii. Phytopathology 85:1507-1512

Nelin M., 1992 White mold in peanuts. Peanut Grower June:18–20

Okabe I, Morikawa C, Matsumoto N, Yokoyama K., 1998 Variation in Sclerotium rolfsii isolates in Japan. Mycoscience 39:399-407

Prithiviraj B, Sarma BK, Srivastava JS, Singh UP., 2000 Effect of Ca2+, Mg2+, light, temperature and pH on athelial stage formation in Sclerotium rolfsii Sacc. J Pl Dis Prot 107:274-278

Punja ZK., 1985 Biology, ecology and control of Sclerotium rolfsii. Ann Rev Phytopathol 23:97-127

———. 1988 Sclerotium (Athelia) rolfsii, a pathogen of many plant species. In: Sidhu GS, ed. Genetics of plant pathogenic fungi. Vol. 6. London: Academic Press. p 523–534

———, Damiani A., 1996 Comparative growth, morphology and physiology of three Sclerotium species. Mycologia 88:694-706

———, Grogan RG., 1983 Basidiocarp induction, nuclear condition, variability and heterokaryon incompatibility in Athelia (Sclerotium) rolfsii. Phytopathology 73:1273-1278

———, Rahe JE., 1992 Sclerotium. Pp. 166–170. In: Singleton, LL, Mihail JD, Rush CM, eds. Methods for research on soilborne phytopathogenic fungi. St. Paul: APS Press

Saccardo PA., 1913 Sclerotium rolfsii. Sylloge Fungorum XXII. Pavia, Italy. 1500 p

Salisbury FB, Ross CW., 1986 Lipids and other natural products. In: Plant physiology. Shahdara, Delhi: CBS Publishers and Distributors. p 268–289

Sequeira L., 1973 Hormone metabolism in diseased plants. Ann Rev Pl Physiol 24:353-380

Singh UP, Pavgi MS., 1965 Spotted leaf rot of plants—a new sclerotial disease. Pl Dis Reptr 49:58-59





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