A Shake Culture Method for Pythiaceae Applicable to Rapid, Small-Scale Assay of Vegetative Physiology

Abstract

A method is described for growing uniform mycelial pretreated for 10 min with a variety of inhibitors known to suspensions of Pvthium ultimum and Phytophthora impair specific cytoplasmic and mitochondrial functions. tnegasperma var. sojae in a defined medium in shake culture. The cultures were maintained easily, and after about 12 hr Dry weight doubling times of these fungi were 5 to 6 hr and from the seeding of fresh cultures they were ready for use. The about 12 hr, respectively. Incorporation of radioactive rapid growth and the ease and reproducibility of sampling leucine, adenine, glycerol, and acetate into proteins, nucleic make possible short-term manipulations of small amounts of acids, and lipids by 3-ml cultures could be measured easily cell material as well as the rapid production of large amounts with 10-min pulse periods. These synthetic activities were of uniform, actively growing mycelium. blocked in a predictable fashion if the cell samples were The utility of liquid culture methods for physiological In our work with an isolate of Pvthium ultimum Trow, studies of filamentous fungi often is restricted by one or one of the species used by Tolmsoff (9), we found the more factors (5, 8). Preparation of test cultures from a establishment of such cultures to be possible but timemycelial mat or tight mycelial clumps leads to sampling consuming and their maintenance was especially difficult difficulties owing to nonuniformity of culture material. In at high cell densities. Further, we were unable to adapt a addition, many fungi grow slowly in liquid culture. Phytophthora megasperma Drechs. var. sojae Hildb. Although slow growth and cell heterogeneity may be isolate to such hyphal suspension growth owing to overcome to some extent by seeding media with hyphal difficulty in satisfactorily fragmenting the mycelium fragments and mechanically shaking the cultures, many without extensive destruction. Even though Tolmsoff (9) fungi still exhibit extensive mycelial aggregation when did not study Phytophthora spp., he noted that the grown in shake culture. blending conditions and transfer interval were critical and In particular, these problems are common with that they varied with the organism. In this report we Pythium spp. and Phytophthora spp. Many of the latter describe a shake culture method which is applicable to a species typically grow very slowly, and in shake culture fast-growing P. ultimum isolate and a slow-growing P. hyphal seed pieces of both groups of fungi give rise to megasperma var. sojae isolate, that provides for rapid tight pellets of mycelium. Information regarding some of growth in a defined medium, ease of culture production the unusual features of these fungi, such as their and maintenance, near-uniformity of culture material, sensitivity to antibacterial drugs (3), is limited, in part sampling ease with good reproducibility, and rapid assay because their growth habit precludes certain types of of metabolic activities. A brief report of part of this work experimentation. The results of sev...

Mechanism of Antibacterial Antibiotic Sensitivity in Pythium ultimum

“…Pythium idtimum Trow was grown in shake culture in a defined medium as previously described (Rawn & Van Etten, 1978). It grew as a uniform suspension of individual hyphae and small pieces of mycelium, with a doubling time of about 5 h. Reproducible sampling of small volumes of actively growing cell material was therefore possible.…”

“…The procedure for measuring incorporation of [3H]leucine or [Wlacetate into trichloroacetic acid (TCA)-insoluble material, as well as the precursor stocks used, have been described previously (Rawn & Van Etten, 1978). Briefly, 3 ml culture samples were incubated with various antibiotics or respiratory inhibitors and then pulse-labelled with the radioactive precursors.…”

“…The TCA-insoluble radioactivity on the discs was measured as previously described (Rawn & Van Etten, 1978). This TCA-insoluble radioactivity was subtracted from the total ethanol-soluble radioactivity to give the reported values of free intracellular leucine.…”

“…In 10 to 15 rnin pulse periods, P. ultimum incorporated [3H]leucine selectively into protein, which could be measured conveniently as the total TCA-insoluble radioactivity (Rawn & Van Etten, 1978). TET and CAM reduced that incorporation by 40 to SO0{, within 20 min; CHI inhibition was essentially complete in this period (Table 2).…”

Cyanide-Insensitive Respiration in Relation to Growth of a Low-Temperature Basidiomycete

“…To eliminate the excessive lag phase and lack of uniformity in size of mycelial fragments in the homogenate, 100-ml continuous shake cultures of the type described by Rawn and VanEtten (14) were established. Cultures started as above were homogenized after 48 to 72 h and 10-or 20-ml portions of the homogenate were used to start new cultures.…”