Influence of Medium Composition on Mycelial Growth and Oxalic Acid Production in Sclerotium Rolfsii

Abstract

Several fungi produce oxalic acid (OA) during growth in liquid culture (2, 8, 9). In the plant pathogenic fungus Sclerotium rolfsii Sacc. [teleomorph: Athelia rolfsii (Curzi) Tu and Kimbrough] (13), OA is a product ofthe glyoxylate bypass ofthe tricarboxylic acid cycle (7). Production of OA in plant tissues was reported to facilitate infection by the fungus and enhance disease development (1, 3, 5). In a study by Higgins (3), some factors influencing OA production by S. rolfsii in culture media were described. Maxwell and Bateman (6) investigated the effect of selected carbon sources and pH on OA production. Although S. rolfsii can utilize numerous carbon sources for growth (4), abundant mycelial growth does not insure high OA production (1, 6). The standard liquid basal salts medium (LSM) for growth of S. rolfsii contains the following: K2HP04, 0.7 g; KC1, 0.15 g; MgS04-7H20, 0.4 g; ZnS04 7H20, 12 mg; MnS04H20, 14 mg; FeCl3H20, 14 mg; thiamine HC1, 1 mg; distilled water, 1 liter (6). Dextrose (19.8 g/1) and NH4N03 (1 g/1) are added to provide the carbon and nitrogen sources, respectively. While this complete medium (C-LSM) supports good mycelial growth, OA production is negligible (6). A defined medium that would support abundant OA production without affecting mycelial growth would greatly facilitate studies on factors regulating OA production in S. rolfsii. The objective of this study was to define the constituents of such a medium. The following additions to LSM were tested: a) carbon sources (with NH4N03 as the nitrogen source)?19.8 g/1 dextrose, potato starch, citrus pectin, carboxymethylcellulose (CMC), peptone, or Na salts of succinate, citrate, and acetate; b) mixtures of carbon sources?9.9 g/1 dextrose mixed with 9.9 g/1 pectin, CMC, yeast extract, peptone, or with 20 g/1 Na succinate (0.074 m), 16.2 g/1 Na citrate (0.055 m), or citric acid-tris buffer (0.1 m, pH 6); c) inorganic nitrogen sources (with dextrose as the carbon source)?NH4N03, NH4H2P04, Ca(N03)2, (NH4)2S04, NH4C1, NaN03, KN03, and urea, all tested at a nitrogen level equivalent to that in 1 g/1 NH4N03 (12.5 mM of N); d) organic nitrogen sources?DL-asparagine, L-arginine, DL-threonine, DL-tryptophan, DL-phenylalanine, DL-leucine, L-alanine, and L-histidine, all tested at a N level of 12.5 mM; or 0.1% yeast extract; e) buffersC-LSM was buffered at pH values ranging from 2.1 to 6.9 with 0.1 m citric acidtris or at pH 5 with 12.5 mM KH2P04. Isolate 1132-8 of S. rolfsii from bentgrass was used throughout this study. Six additional isolates (1094, 1126, 2672, 2394, 2823, 3078), described in detail elsewhere (13), were grown in C-LSM to compare differences among isolates in dry weight (DW) and OA production. Each carbon and nitrogen source (in distilled water) was sterilized separately by autoclaving at 121 C for 15 min or by passage through a 0.45 p,m Millipore filter before mixing aseptically with cooled, autoclaved, double-strength LSM. The pH was adjusted to 5.7-5.8 with sterile 1 n NaOH or HC1. The media were dispensed in 30 ml aliquots into 125 ml Erlenmeyer flasks. Each flask was in-