Hemp sesbania [Sesbania exaltata (Raf.) Rybd. ex A.W. Hill] and sicklepod [Sennaobtusifolia (L.) Irwin and Barnaby] seeds were harvested from weed plots grown at the Crop Production Systems Research Unit, Stoneville, MS. Seeds were mechanically scarified, planted in paper towel cylinders, and grown hydroponically in the dark as described previously [34]. After 96 h growth, uniform seedlings were selected for test, and again placed in paper towel cylinders. Then seedlings were used in hydroponic bioassays [34], after treatment with chemicals, pathogen spores, or the combination of chemical and spores.

Cultures of Colletotrichum truncatum (Schwein.) Andrus & W.D. Moore and Alternaria cassiae Jurair and Khan are pathogens of hemp sesbania and sicklepod, respectively. Cultures of these fungi from isolates discovered and maintained in our laboratory at Stoneville were propagated on potato dextrose agar as described elsewhere [32] [30] in Petri dishes and the spores produced were collected by brushing from the surface of the colonies. Spores were suspended in deionized water and diluted (using a hemocytometer) to achieve concentrations of 1.0 × 10⁵ and 1.5 × 10⁵ spores ml⁻¹ for A. cassiae and C. truncatum, respectively. These spores were used directly for the preparation of spray inoculum for application to the seedlings.

Aminooxyacetate (AOA) and Boc-AOA, each at 1.0 mM, were applied (hand-held sprayer or root-fed) to 96 h-old, dark-grown hemp sesbania and sicklepod seedlings in paper towel cylinders as described above. Control seedlings received only a water spray. Shoot elongation in continuous darkness was measure after an additional 72 h after treatment. AOA and Boc-AOA were also applied to seedlings, alone or combined with the C. truncatum or A. cassiae for testing possible interactions. Each replication consisted of a minimum of four seedlings and each treatment was triplicated.

All reagent grade quality chemicals were used and were obtained from Sigma Chemical Co., St. Louis, MO.

After removal from the dark growth chamber 72 h following treatment, plant seedling shoot tissue was homogenized in an electric blender with 200 mM potassium phosphate buffer (pH 7.8), containing 1.0 mM dithiothreitol, and 1.0 mM ethylenediamine-tetra-acetic acid (EDTA). Assay mixtures contained 200 mM potassium phosphate buffer (pH 7.8), O-acetyl-L-serine(5 mM), sodium sulfate (1.0 mM), pyridoxyl-phosphate (0.05 mM), dithiothreitol (1.0 mM) and enzyme extract in a total of 1.0 ml. Reactions were run at 25˚C and were terminated with 2.0 ml HCl:glacial acetic acid (1:1, v:v) as outlined previously [27]. Extractable activity of cysteine synthase was determined spectrophotometrically (A₅₆₀), based on formation of the ninhydrin-cysteine reaction complex [35]. Enzyme activity values were based on a standard curve of absorbance of the ninhydrin-cysteine complex formed from the reaction of various concentrations of cysteine. Protein in enzyme extracts was determined using the Bradford reagent [36]. Each extraction utilized 4 to 6 seedlings per treatment and extractions of each treatment were performed in triplicate.

Excised cotyledons of 96 h –old, dark-grown seedlings were placed in solutions of AOA, Boc-AOA, or water (control) in plastic well-plates (16 mm wide, 18 mm deep) for 2 h in the dark. The plates were then transferred to low light (90 - 100 μE∙m⁻²∙s⁻¹, supplied by incandescent and florescent lightbulbs) for 72 h, and chlorophyll that accumulated in the greening excised cotyledons was determined using dimethyl sulfoxide (DMSO) extraction and spectrophotometry [37]. Each well contained a minimum of 4 cotyledon pairs, and each treatment was performed in triplicate.

The experiment was set up as a randomized complete block, all treatments were performed in triplicate and the experiment was repeated. Data were analyzed and compared using analysis of variance at the P = 0.05 level. Significant differences were detected using the F-test and means were separated (Fisher’s protected LSD at 0.05).

Root-fed AOA inhibited shoot elongation in hemp sesbania slightly more than Boc-AOA up to 72 h after treatment. Both AOA and Boc-AOA were equally inhibitory to sicklepod shoot growth (Figure 2). There was little or no effect of foliar application of AOA or Boc-AOA on hemp sesbania shoot elongation (Figure 3), but both compounds reduced shoot elongation by 20% to 25% in sicklepod (Figure 4). The reduction of growth in hemp sesbania was not altered when C. truncatum and AOA were applied simultaneously (Figure 3). However, Boc-AOA combined with C. truncatum did produce a reduction in elongation of hemp sesbania that was significantly greater than that of the pathogen alone or the pathogen combined with AOA (Figure 3). The reason(s) for this differential

effect of the two compounds are presently unexplainable. In sicklepod seedlings, treatments of the chemicals alone, pathogen (A. cassiae) alone, and the combination of pathogen plus AOA all caused similar growth reductions, i.e., 20% to 25% below control levels (Figure 4). Boc-AOA plus A. cassiae caused a growth reduction of ca. 30% compared to control seedlings and was also slightly greater than the AOA plus pathogen treatment. In hemp sesbania and sicklepod, total chlorophyll content of greening cotyledons was inhibited by the compounds, but levels of this pigment were reduced to a greater extent in sicklepod than in hemp sesbania (Figure 5). Furthermore, the chlorophyll inhibition was greater for AOA than Boc-AOA in hemp sesbania, but greater for Boc-AOA than AOA in sicklepod.

AOA and Boc-AOA reduced extractable CS activity by 30% in seedlings of both weed species, 72 h after treatment (Figure 6).

C.truncatum infection in hemp sesbania also lowered CS activity (15%), but A. cassiae infection in sicklepod increased activity 20% above untreated control levels. In vitro enzyme assays resulted in equal inhibition by 15% when either chemical and the substrates were added simultaneously to the enzyme (Figure 7). When either inhibitor was pre-incubated (10 min. at 25˚C) with enzyme without substrate, and then followed by substrate addition and assay, inhibition by Boc-AOA was not increased to any measurable extent, whereas the inhibition by AOA increased by an additional 20%.

Overall results indicate that hemp sesbania was generally more resistant than sicklepod to the phytotoxicity of AOA and Boc-AOA under these continuous dark-growth conditions and the greening of excised cotyledon test. The fact that these compounds had little effect after foliar application may be due to the waxy characteristics of these seedlings that could have slowed absorption of the compounds into the cytoplasm. Since cysteine synthase was inhibitedin vivo and in vitro by AOA and Boc-AOA, and because there was no major difference in phytotoxicity of the two compounds in these young plants, apparently the N-t-butoxycarbonyl group does not alter the activity of the AOA molecule. An exception however, was the greater growth reduction caused by Boc-AOA plus A. cassiae compared to the AOA plus A. cassiae treatment. Furthermore, these chemicals had no apparent interactions with C.truncatum and A. cassiae efficacy on their respective weed hosts. The increase in extractable cysteine synthase activity in sicklepod following infection byA. cassiae, may be related to defense mechanisms of this weed against this pathogen.

There may be some inherent differences in the overall action of AOA and Boc-AOA, but it is not possible to tell from these results and the scope of this study. It is also not known if Boc-AOA is converted to AOA by enzyme hydrolysis of the substituted amide bond by these plants and their respective pathogens. A herbicidal analog of AOA, benzadox, is thought to be metabolized in planta to form AOA [38]. Also interesting is the naturally occurring compound irpexil [methyl-2-(acetamidooxy)acetate], a herbicidal compound produced by the basidiomycete Irpexpachyodon [39]. This compound has structural features analogous

to AOA and the herbicide benzadox (Figure 1). Benzadox is an inhibitor of alanine aminotransferase [38], but the mechanism of action of irpexil, is unknown. Benzadox has been shown to be metabolized in plants to produce AOA [4]. Similar compounds such as aryl-substituted α-aminooxycarboxylic acids are also phytotoxic and act as auxin transport inhibitors [40]. AOA has been tested as a synergist with C. coccodes a bioherbicide of velvetleaf (Abutilon theophrasti) [41]. However, AOA was found to be inhibitory to the growth of this fungus on PDA in Petri dishes, when applied together with the fungus, or if applied via vacuum infiltration on velvetleaf leaves and furthermore, no synergy was observed in those studies.

Although the interactions that we found during these experiments resulted in no major synergistic effects, novel information was generated on the important enzyme CS and on AOA interactions with two bioherbicides that have received major attention as weed control agents.