Isolation & characterization of malformin

Abstract

Papers (5. 6) from this laboratory have reported the in(luction, by culture filtrates of the fungus, Asper-gillums niger, of curvatures and malformations of bean plants an(l curvatures of corn roots. The name, mal-fornmin, was propose(d for the active compound (7). We now present a complete isolation procedure for malformin and describe, in part. the character of the molecule. Isolation of nmalformin has made it possible to conduct quantitative experinments on its activity (see following report). MATERIALS & METHODS Aspergillts niger van Tiegh. strain 56-39 was use(l in these andl the original studies. The culture mle(lium, corn steep-glucose, and shake-flask methods were described earlier (5). Culturing wvas done at 25 to 260 C. Cultures of 56-39 were obtained fronm soil stocks by inoculating agar slants of potato-dextrose medium. After 7 to 10 days, one ml spore suspensions were use(I to inoculate 100 ml aliquots of corn steep-glucose me(lium in 500 ml erlenmiieyer flasks. After 24 hours on a reciprocating shaker, the cultures were transferred to addlitional 100 ml ali(luots of the saame medium in 1 liter erlenmeyer flasks andl again incubated for 24 hours onl the shaker. Each of these cultures was transferred to New Brunswvick M\To(lel FS-314 fer-nmentationi jars containing 10 liters of corn steep-dextrose me(liunm. The jars were incubated for 4 to 5 days on a Newv-Brunlswick fermiientor (lrive assembly (air flow = 6 liters/mmin,jar; agitation speed = 420 rpnm). Antifoam (Dow Polyglycol P-2000) was added as needed. At the end of the fermentation perio(l the miiyceliunm was remiioved by filtration and (li scar(le(l. To folloxv the isolation of malformin from the filtrates, one of two nmethods of assay was employed, the bean test (5) or the corn root test (6). The fractionis were usually driedl an(d then (lissolved in water (containing 4 drops of Txveen 80/100 ml for the bean test). In some cases a few drops of acetone wvere ad(led to the drie(l residue to assist in dissolving thle material. Additional mletlhods are given later.