Variation in Biochemical Composition among Indian Isolates of Sclerotinia sclerotiorum.

Biochemical variability among 20 Indian isolates of Sclerotinia sclerotiorum collected from different hosts/soil samples from different localities in India is reported. High Performance Liquid Chromatographic (HPLC) analysis of ethyl acetate fraction of culture filtrate, mycelia, sclerotia and sclerotial exudate showed 15~23 peaks but only 11 could be identified. They were tannic, gallic, oxalic, caffeic, vanillic, ferulic, O-coumeric, chlorogenic, cinnamic, salicylic and gentisic acids. The amount of phenolic compounds varied among the culture filtrates, mycelia, sclerotia and sclerotial exudates of S. sclerotiorum.

Sclerotinia sclerotiorum (Lib.) de Bary is a necrotrophic pathogen with cosmopolitan in distribution and wide host range (Purdy, 1979). Some workers (Corsini and Tourneau, 1973; Jones and Cooke, 1977) reported biochemical variability in S. sclerotiorum particularly in the amount and kind of organic acids (oxalic, fumaric, mallic and succinic acids) which vary with the isolates and the medium. According to some workers (Chet and Henis, 1975; Willetts and Bullock, 1992; Willetts and Wong, 1980) the importance of sclerotia for survival and propagation of S. sclerotiorum and other sclerotium-forming fungi has stimulated numerous investigations into the structural makeup and developmental regulation of sclerotia.

Keates et al. (1998) reported that extracts of sclerotia of S. sclerotiorum contained 2 active constituents, D-glyceropent-2-enono-1,4-lactone (D-erythroascorbic acid), and 5-O-(alpha-D-galactopyranosyl)-D-glycero-pent-2-enono-1,4-lactone. Starratt et al. (2002) demonstrated that young cultures of S. sclerotiorum isolate SS7 produced 1,8-dihydroxynaphthalene monoglucoside, a new natural product. It was previously shown to produce and excrete into agar medium in copious amounts of melanin precursor 1,8-dihydroxynaphthalene. When cultured in the presence of tricyclazole, such young cultures also accumulated two new monoglucosides of 1,3,8-trihydroxynaphthalene, as well as 1,8-dihydroxynaphthalene monoglucoside. Sharma et al. (2001) reported the activities of pectinases and pectin methylesterase enzymes in culture medium of S. sclerotiorum which were inhibited by phenolic compounds (m-coumaric, homovanillic and protocatechuic acid) present in the culture medium. Singh et al. (2004) analyzed ethyl acetate fraction of exudate of S. sclerotiorum with the help of High Performance Liquid Chromatography (HPLC) and showed that it consisted of tannic, gallic, ferulic and cinnamic acids along with many other unidentified compounds and the exudate showed antifungal activity against some parasitic as well as saprophytic fungi.

As much work has not been done about biochemical variability among the Indian isolates, the present experiments were conducted on the above aspects and the results are presented here.

Materials and Methods

Maintenance of Sclerotinia sclerotiorum isolates

Twenty isolates of S. sclerotiorum used in this study were collected from various hosts/soil samples from diverse geographic origins (Table 1). The isolates were further purified by growing single sclerotia from each colony grown on potato dextrose agar (PDA) (peeled potato 200 g, dextrose 20 g, agar 15 g, distilled water 1 l) slants.

Table 1

Phenolic acid content (µg/ml) in culture filtrates of 20 isolates of Sclerotinia sclerotiorum

Ethyl acetate fractionation of sclerotia, mycelia and culture filtrate of S. sclerotiorum

Twenty-five ml potato dextrose broth was poured in each of 100 ml capacity flasks. The mycelial disc from actively growing cultures of S. sclerotiorum was inoculated and allowed to grow for a week at 25 ± 2℃. The mycelial mat of the isolates from potato dextrose broth was harvested and washed three times with 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 ethyl acetate in a pestle-mortar. The finely crushed material was collected in screw-capped bottles containing 5 ml ethyl acetate and kept overnight. The culture filtrate was filtered through 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 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 ml HPLC grade methanol by vortexing and stored at 4℃ for further analysis.

Exudate depletion experiment

Mycelium of an actively growing (5-day-old) culture of S. sclerotiorum grown PDA medium was cut with a 5 mm diameter sterile cork borer and transferred to the center of Petri dishes containing PDA and incubated at 25 ± 2℃. The plates were observed regularly for the formation of exudate destined for sclerotium formation. The exudate in the form of fine droplets was discerned after 6~10 days. They were removed by sucking with a sterilized capillary tube, collected in sterile culture tubes and stored at 4℃. Sclerotial development following exudate depletion was consistently observed and the fresh weight and size of 100 mature exudate-depleted sclerotia were measured and compared with the completely matured non-depleted sclerotia of the same age. The whole experiment was repeated thrice.

Collection of sclerotia was done from different isolates grown on PDA and the matured sclerotia was collected after a week. One g of sclerotia from each isolate was taken separately and crushed in a pestle-mortar in the same way as the ethyl acetate fractions of mycelia described earlier.

HPLC analysis

High performance liquid chromatography (HPLC) of the samples was performed with HPLC system (Shimadzu Corporation, Kyoto, Japan) equipped with two Shimadzu LC-10 ATVP reciprocating pumps, a variable UV-VIS detector (Shimadzu SPD-10 ATVP) and a Winchrom integrator (Winchrom). Reverse phase chromatographic analysis was carried out in isocratic conditions using RP C-18 HPLC column (250 × 4.6 mm id, particle size 5 µm, Luna 5 µ C-18 (2), Phenomenex, USA) at room temperature. Running conditions included injection volume: 5 µl, mobile phase: methanol-0.4% acetic acid (80 : 20, v/v), flow rate: 1 ml/min, detection at 290 nm and attenuation response 0.03 AUFS. Samples were filtered through membrane filters (Pore size 0.20 µm, Millipore) prior to injection in sample loop. Tannic, gallic, oxalic, caffeic, vanillic, ferulic, chlorogenic and cinnamic acids were used as internal and external standards. Phenolic compounds present in the samples were identified by comparing retention time (Rt) of the standards and by the co-chromatography. Contents of phenolic acids were calculated by comparing peak areas of reference compounds with those in the samples run under similar elution conditions.

Results

HPLC analysis of culture filtrate

HPLC analysis of the culture filtrate of different isolates of S. sclerotiorum revealed 4~14 peaks consistently. Out of these, some were identified on the basis of their retention time (Rt.) as well as by co-injection with the standard compounds. Among these peaks, six consistently appeared in culture filtrates of most of the isolates. The peaks identified were of tannic (Rt.2.76 min), gallic (Rt.2.86 min), oxalic (Rt.3.03 min), caffeic (Rt.3.12 min), vanillic (Rt.3.26 min), ferulic (Rt.3.30 min), o-coumeric (Rt.3.55), chlorogenic (Rt.4.16 min), cinnamic acids (Rt.4.46 min) and gentisic acids (Rt.6.62). Out of 20 isolates, gallic acid was detected in 4 isolates, oxalic in all, vanillic in 13, ferulic in 17, o-coumeric in 16, cinnamic in 8, caffeic in 3, tannic in 7 and chlorogenic acid in 12 isolates. The major component of culture filtrates was oxalic acid and its amount varied from 21.42 to 998.76 µg/ml in ethyl acetate fractions. Ferulic acid ranged from 0.39 µg/ml (Phaseolus vulgaris) to 556.47 µg/ml (Brassica oleracea var.. botrytis) while, it was absent in 3 isolates. Caffeic acid ranged from 3.11 µg/ml (Mentha arvevs) to 31.97v (Cicer arietinum) being absent in 16 isolates. Cinnamic acid was 0.101 µg/ml in Mentha arvevs collected from Ranichauri and 9.70 µg/ml in Pisum sativum isolate from Lucknow and not detected in 8 isolates. Gallic acid was 1.30 µg/ml in Brassica campestris var. toria isolate and 3.63 µg/ml in Pisum sativum isolate. Vanillic acid was 0.29 to 461.96 µg/ml in some isolates. Eleven isolates showed chlorogenic acid while six isolates had tannic acid. Gentisic acid was found in 10 isolates (1.86 to 184.73v) whereas salicylic acid was present in very low amount (0.42 to 2.90 µg/ml) in three isolates (Table 1).

HPLC analysis of mycelium

Out of 20 isolates, gallic acid was detected in all the isolates except in two, oxalic in 8, vanillic in 12, ferulic in 17. o-coumeric in 8, cinnamic in 7, caffeic in 4 and tannic acid in all the isolates and chlorogenic in 14 isolates. The major component of mycelium was oxalic acid (425.51 µg/ml) in isolate of Coriendrum sativum. Ferulic acid was 23.48 µg/ml in isolate of Amaranthus tristis and 330.19 µg/ml in Papaver somniferum isolate while absent in 2 isolates isolated from Phaseolus vulgaris. Caffeic acid amounting to 1.92 µg/ml was observed in isolate from Gaillardia pulchella and 4.61 µg/ml in Pisum sativum isolate. However, caffeic acid was absent in rest of the isolates. Cinnamic acid was 0.039 µg/ml in the soil isolate and 3.50 µg/ml in Gaillardia pulchella isolate but absent in 13 isolates. Gallic acid was 1.93 µg/ml in isolate from Solanum melongena and 40.06 µg/ml in Cicer arietinum isolate. Vanillic acid was 0.02 µg/ml in isolate from Solanum melongena and 23.21 µg/ml in Phaseolus vulgaris. Tannic acid was present in all the isolates of S. sclerotiorum. Gentisic acid was detected in 9 isolates. It ranged from 0.12 µg/ml to 120.83 µg/ml whereas salicylic acid was discerned in very low amount (0.17 to 5.57 µg/ml) in 7 isolates (Table 2).

Table 2

Phenolic acid contents (µg/g) in mycelium of 20 isolates of Sclerotinia sclerotiorum

HPLC analysis of sclerotia

HPLC analysis of sclerotia of different isolates of S. sclerotiorum revealed 4~15 peaks consistently. Out of these, 11 were identified as mentioned above. Gallic and ferulic acids were present in all the isolates, oxalic in 7 and vanillic acid in 6 isolates, o-coumeric in 11 isolates, caffeic in 9 and chlorogenic acid in 11 isolates, tannic in all the isolates except in one isolate infecting Pisum sativum, salicylic and gentisic acids in 6 and 4 isolates, respectively. Major component of sclerotia was ferulic acid. Most of the phenolic compounds varied in quantity in different isolates (Table 3).

Table 3

Phenolic acid contents (µg/g) in sclerotia of 17 isolates of Sclerotinia sclerotiorum

HPLC analysis of exudates

HPLC analysis of sclerotial exudates of different isolates of S. sclerotiorum revealed 4~10 peaks consistently. Out of these, 7 were identified as mentioned above. Gallic acid was present in exudates of 11 isolates, oxalic acid in all except in two, vanillic in all except 4 isolates, ferulic acid in all the isolates, o-coumeric in 8, chlorogenic and cinnamic acids in 4 and 2 isolates respectively. Most of the phenolic compounds varied in quantity in different isolates (Table 4).

Table 4

Phenolic acid content (µg/ml) in sclerotial exudates of 17 isolates of Sclerotinia sclerotiorum

Discussion

Several workers (Cruikshank, 1983; Kohn, 1979; Petersen et al., 1982; Russo and Van Etten, 1985; Scott, 1981; Tariq et al., 1985; Willetts and Wong, 1980) have taken several characters like morphological, cytological, biochemical, cultural and epidemiological into consideration to characterize different species of Sclerotinia. The results on biochemical characteristics of the present experiment also revealed wide variation among Indian isolates of S. sclerotiorum. HPLC analysis of the exudate, mycelia, sclerotia and culture filtrate of all the individual isolate showed difference in phenolic and oxalic acid composition of exudate, mycelia, sclerotia and culture filtrate. Varied amounts of phenolic acids in different components of the S. sclerotiorum isolates further shows that this criterion can also be taken into consideration for studying variability. It is interesting to note that salicylic acid which is also reported to induce resistance in host plants against pathogen attack (Gaffney et al., 1993) was not detected in sclerotial exudates. In contrast, oxalic acid which is described to be responsible for pathogenicity of S. sclerotiorum (Ferror and Walker, 1993; Godoy et al., 1990; Maxwell and Lumsden, 1970; Margo et al., 1984; Marcino et al., 1983; Noyes and Hanecock, 1981) was commonly found in the culture filtrates as well as in sclerotial exudates in most of the isolates. The presence of oxalic acid in culture filtrates and sclerotial exudates of S. sclerotiorum has also been reported by some other workers (Ferror and Walker, 1993; Keets et al., 1998; Maxwell and Lumsden, 1970; Tahmasebi et al., 1998; Zhau, 1999).

Similarly, ferulic acid which is reported to be highly antifungal against another sclerotia forming pathogen Sclerotium rolfsii (Sarma and Singh, 2003) were also found in higher amounts in sclerotia compared to mycelia, culture filtrates and exudates of the isolates. However, its concentration was less than the inhibitory concentration (>500 µg/ml) (Singh et al., 2002) except in the isolate from Amaranthus tristis, which may be considered more resistant towards ferulic acid than the rest of the isolates. Apart from the other criteria, the variation in phenolic acid profile in different parts of the pathogen may also be taken as a criterion for assessing variability in S. sclerotiorum isolates. It will be interesting to see if there is any correlation between the phenolic acid content in the pathogen and its pathogenicity as well as its ability to survive in soil.