HISTOLOGY OF MALFORMATIONS PRODUCED ON BEAN PLANTS BY CULTURE FILTRATE OF ASPERGILLUS NIGER

POSTLETHWAIT, S. N., AND RoY W. CURTJS. (Purdue U., Lafayette, Imid.) Histology of malformations produced on bean plants by culture filtrate of Aspergillus niger. Amer. Jour. Bot. 46(1): 31-35. Illus. 1959.-The stem and leaf petioles of bean seedlings treated with culture filtrates of A. niger exhibit severe curvatures, increased diameter, reduced length, and irregular protrusions. These malformations result from an apparent partial loss of polarity of the cells in the actively growing region of young internodes and petioles. Enlargement and multiplication of cells in the affected regions occur mostly at an angle to the longitudinal axis of the plant. The effect is localized and the portion of the plant produced from the apical meristem subsequent to treatment matures in a normal way. ONE OF THE writers has reported that culture filtrates of the fungus, Aspergillus niger 56-39, induce severe curvatures and grotesque malformations on bean plants (Curtis, 1958). The plants were treated with a drop of the culture filtrate on each side of the unexpanded bud between the simple leaves above the cotyledons. After 5-10 days, malformations commonly appeared on the first and second internodes above the simple leaves. The stems and petioles were short and curved, greatly enlarged in diameter, relatively flat in transverse section, and somewhat irregularly ridged longitudinally. It was found subsequently that ether extracts of the culture filtrate of A. niger caused pronounced curvatures on corn roots (Curtis, in press) . Evidence obtained through the use of paper chromatography indicated that the compound which caused malformations on the stems of bean plants was the same as the one which induced curvatures on corn roots. Although early, indirect evidence suggested that A. niger produces indoleacetic acid (BoysenJensen, 1932; Kogl and Kostermans, 1934), the work of Curtis (1958) indicated that the compound active on corn and bean plants was not IAA. The present paper is an attempt to describe in more detail morphological and histological changes in plants treated with the culture filtrate of A. niger. Special emphasis has been placed on the histology of the irregular stem enlargements. MATERIALS AND METHODS.-The methods used to obtain culture filtrates of A. niger have been described earlier (Curtis, 1957). The crude culture filtrates (pH 5) were extracted 3 times with ethyl ether and then evaporated to dryness; the residue was taken up in water containing 2 drops tween 80/100 ml. This preparation, referred to as an ether extract, was used without further purification for treating seedlings of bean plants (Phaseolus vulgaris var. 'Black Valentine') grown in vermiculite. Usually, the concentration of the active compound used in treating plants was twice that in the crude culture filtrate. The treatment was made 'Received for Publication June 19, 1958. 2Journal Paper No. 1297 of the Purdue Agricultural Experiment Station, Lafayette, Indiana. by placing a drop of the ether extract on both sides of the young apical bud when the laminae of the simple leaves above the cotyledons were approximately 4-5 cm. long. This was done on each of 2 alternate days. Samples for study were collected at various intervals up to 14 days after treatment from treated and untreated plants. Dissection and observation of the apex were made with the aid of a stereomicroscope. Transverse and longitudinal sections were studied from both free-hand sections and paraffin sections. Permanent sections were made from material killed and fixed in FAA, dehydrated in the ethyl alcohol series, embedded in paraffin, sectioned at 20 microns and stained with safranin and fast green. RESULTS.-Untreated bean stem.-The development of a young internode of the bean stem from a short internode between leaf primordia at the apex to a greatly elongated internode at maturity is very complex due to the initiation of the vascular system before primary enlargement has been completed (Esau, 1953). Three internodes are differentiated in the apex before any appreciable elongation occurs. When the simple leaves above the cotyledons are about 4-5 cm. in length (treatment was applied at this stage of ontogeny), the apex consists of three internodes and three trifoliolateleaf primordia. For convenience of reference in this paper, the first internode above the cotyledons will be referred to as internode 1 and internodes constituting the young apex will be numbered successively in an acropetal sequence as internode 2, internode 3, and internode 4. Internode 2, which is 3-5 mm. long and the most basal of the three internodes in the apex, elongates rapidly, reaching its maximum length in 7-10 days under average greenhouse conditions. Internode 3, which is about 1 mm. long, and internode 4, which is less than 1 mm. long, elongate progressively later. Internode elongation occurs as a result of both cell division and cell enlargement. This activity takes place throughout the length of the younger internodes and throughout most of the length of internode 2 except for the more rnature basal portion. During