791 - 795 2 , 4 - Dihydroxy - 7 - methoxy - 2 H - 1 , 4 - benzoxazin - 3 ( 4 H ) - one , an Inhibitor from Zea mays with Differential Activity against Soft

12,4-Dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-onel DIMBOA was extracted with ethyl acetate from acidified water homogenates of corn (Zea mays L.) seedlings. Pure DIMBOA or ethyl acetate extracts of corn tissue were added to bacterial growth medium at five concentrations (measured as hydroxamates). DIMBOA and corn extracts were more inhibitory to soft rot bacteria (Erwinia spp.) that are nonpathogenic to corn than to soft rot bacteria that are corn pathogens. The inhibitory activity of DIMBOA was similar to that of the ethyl acetate extracts. Both corn extracts and DIMBOA prolonged the lag phase of bacterial growth without significantly changing log phase growth rates. At various concentrations of the inhibitor, 50 to 100% of the activity of corn extracts inhibitory to different bacterial isolates was attributable to DIMBOA. Extracts of DIMBOA-deficient plants (genotype bxbx) were not inhibitory to Erwinia spp. It was concluded that DIMBOA is the major active component in those corn extracts which are inhibitory to soft rot Erwinia species. Bacterial stalk rot of corn (Zea mays L.) is caused by certain strains of Erwinia chrysanthemi (13). Many other isolates of E. chrysanthemi and other soft rotting Erwinia spp. (e.g. Erwinia carotovora) have many physiological and pathological characteristics in common with ECZ5 (e.g. rotting of potato tubers and carrot roots), but are typically unable to rot corn (9-11). The reasons that corn is resistant to most soft rotting Erwinia species have not been determined. Hartman et al. (11) reported that a lyophilized water extract from corn was only slightly inhibitory to most isolates of ECZ; however, it was strongly inhibitory to most other soft rot bacteria. The observed effect was an extension of the duration of the lag phase ofgrowth. No significant effect on growth rate was detected after the lag phase. It was suggested that the differential inhibitor(s) to soft rotting Erwinia species present in the extract could be involved in the resistance of corn to these bacteria. 'Research cooperative with the College of Agricultural and Life Sciences, University of Wisconsin, Madison, and the Agricultural Research Service, United States Department of Agriculture. 'Present address: Departamento de Biologia, Facultad de Ciencias, Universidad de Chile, Casilla 653, Santiago, Chile. 3Present address: Division of Plant Industry, CSIRO, P.O. Box 1600, Canberra City, ACT 2061, Australia. 4Also, Plant Disease Resistance Research Unit, ARS, U.S. Department of Agriculture. 'Abbreviations: DIMBOA: 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one; ECZ: strains of Erwinia chrysanthemn causing bacterial stalk rot in Zea mays L.; MBOA: 6-methoxy-2-benzoxazolinone; CFU: colony-forming units. Several closely related cyclic hydroxamic acids have been isolated as glucosides from corn (12, 20). The principal hydroxamic acid in aqueous extracts of corn is 2,4-dihydroxy-7-methoxy-2Hl,4-benzoxazin-3(4H)-one (DIMBOA), which decomposes to 6methoxy-2-benzoxazolinone (MBOA) in aqueous solutions (19, 23). DIMBOA and MBOA (Fig. 1) have been shown to be inhibitory to several plant pathogenic fungi and bacteria (3-6, 19) and to the insect pests of corn, Ostrinia nubilalis (Hubner) and Rhopalosiphum maidis (Fitch) (2, 15-17). Klun and co-workers (16) established a linear relationship between the logarithm of the DIMBOA content of the plant and field resistance to 0. nubilalis. However, in exotic maize varieties, Sullivan et al. (18) could find no such relationship between DIMBOA content and resistance to 0. nubilalis. Based on the stability and extractability properties of the inhibitory fraction present in corn extracts, Hartman et al. (11) suggested that the inhibitory activity could be due to DIMBOA. The purpose of this study was to determine ifDIMBOA was the major component in corn extracts with the inhibitory activity toward soft rotting Erwinia. A preliminary report of this work has appeared (4). MATERIALS AND METHODS Bioassays. Bacterial isolates (Table I) were stored in capped tubes in glass-distilled H20 at room temperature (20-26 C). To prepare inoculum for bioassays, the bacteria were streaked on a medium containing 10 g of peptone, I g of casein hydrolysate, 0.5 g of sucrose, and 17 g of agar/l (PCS medium). After incubation at 28 C for 60 hr, the bacteria were suspended in sterile, glassdistilled H20 prior to addition to the bioassay flasks. The liquid bacterial growth medium contained the salts described by Husain and Kelman (14), supplemented with 1 g/l casamino acids (Difco),6 10 g/l sucrose, and NaH2PO4-Na2HPO. buffer (pH 6.75; 0.1 M final concentration). The sample to be assayed was dissolved in ethanol, portions of this solution were added to sterile 25-ml Erlenmeyer flasks, and the ethanol was removed under a stream of N2. For turbidimetric assays, 4 ml of culture medium and 0.1 ml of bacterial suspension (4 x 109 CFU/ml) were added to each flask. Two flasks/treatment were prepared in each experiment. The flasks were placed in a reciprocating shaker (150 cycles/min) at 28 C. Turbidity readings were made at 2-hr intervals with a Klett-Summerson colorimeter. For viable cell count experiments, samples were taken every 4 hr and serial dilutions were made in saline solution (8 g NaCI/1). Bacteria were spread on PCS medium. Colonies were counted after a 48-hr incubation at 28 C. 6Mention of companies or commercial products does not imply recommendation or endorsement by the U.S. Department ofAgriculture over others not mentioned. 791 www.plantphysiol.org on July 27, 2017 Published by Downloaded from Copyright © 1978 American Society of Plant Biologists. All rights reserved. Plant Physiol. Vol. 61, 1978