Glufosinate (Basta) (99.8% pure, free acid) was a product of Plant Media (Division of BioWorld; Dublin, OH, USA). Hemp sesbania seeds were collected from plants grown in a field nursery at USDA-ARS, CPSRU, Stoneville, MS. Potato dextrose agar (PDA) was a Difco product (Difco Laboratories, Detroit, MI, USA). All other chemicals were of reagent grade or higher purity.

A single-spore strain of C. truncatum (NRRL No. 18434), originally isolated from infected hemp sesbania collected near Stoneville, MS, USA [20] , was used throughout these experiments. The fungus, which had been preserved in capped test tubes containing sterilized soil [37] , was grown for 5 to 7 days on PDA in 10 cm plastic Petri dishes. The dishes were incubated (Precision Scientific Inc., Chicago, IL, USA) on open-mesh wire shelves [25˚C, cool, white fluorescent lighting (12 h photo-period) providing 165 μmol m⁻² s⁻¹]. C. truncatum spores (conidia) were harvested by rinsing the conidia from the culture plates with water and straining through two layers of cheesecloth. Conidial densities were determined with hemacytometers and dilutions were made in sterile, distilled water. Conidia used in these tests were no older than 4 h after collection from PDA plates.

Hemp sesbania seeds were mechanically scarified, then surface sterilized (5% sodium hypochlorite; 10 min) and rinsed thoroughly with distilled water. Seeds were planted in paper toweling as described previously [38] and placed in a high-humidity (100% relative humidity) chamber under continuous darkness (78˚C). After seed germination and seedling growth for 3 days under these conditions, seedlings with uniform hypocotyl lengths (10 - 13 mm) were selected and transferred to fresh paper toweling and placed in a high-humidity chamber under continuous light (80˚C, 170 µEm⁻² s⁻¹) for 24 h prior to treatment, to acclimate seedlings to the environmental treatment conditions used in these tests.

After 24 h growth under continuous light (seedlings in cotyledonary growth stage), the plants were divided into four treatment groups for spray applications: control = water; C. truncatum (8.0 × 10⁴ conidia ml⁻¹); glufosinate (1.0 mM); and C. truncatum (8.0 × 10⁴ conidia ml⁻¹) plus glufosinate (1.0 mM). The glufosinate-treated groups were sprayed at the same time, and the herbicide spray allowed to partially dry (~30 min) prior to application of conidia to the plants in the glufosinate plus conidial treatments. Plants were spray-inoculated with conidial suspensions of aqueous preparations of C. truncatum and/or glufosinate using hand-held aerosol sprayers (Spra-tool Power Pack, Aervoe Industries, Inc., Gardnersville, NV). Spray delivery rates were ~200 L・ha⁻² [39] . After all spray applications were made, the plants were placed in a high-humidity chamber for 16 h at 78˚C under continuous darkness and then plants were transferred to continuous light in an environmental chamber (28 C, 600 μE) for the duration of the tests.

At various intervals over a 0 to 88 h time-course, hypocotyls were harvested, weighed and their lengths measured, and then the tissue was frozen (−80˚C) for further analysis. This plant organ was used for analysis in subsequent tests: (NH₃ content, extractable GS activity, protein levels, PAGE electrophoresis, in-gel GS localization and in-gel protein analysis), as described below.

Frozen (−80˚C) hypocotyls from the treated plants were ground/homogenized in cold phosphate buffer (0.05 M, pH 7.0) using chilled mortar/pestles. Each homogenate was collected and centrifuged (15 min, 20,000 x g, 4˚C). The supernatant was used to quantify the ammonia and ammonium ion concentration in hemp sesbania hypocotyl tissue using a salicylate-nitroprusside assay [40] .

Weighed portions of frozen (−80˚C) hypocotyls from the treated plants were ground/homogenized with extraction buffer (100 mM Tris base, 1 mM EDTA, 1 mM MgCl₂ and 10 mM β-mercapto-ethanol, pH 7.0) in chilled mortar/pestles and the homogenates were collected and centrifuged (20 min, 21,000 x g, 4˚C). The supernatants were used directly to determine total GS activity, as measured via the γ-transferase reaction [41] . The assay mixture contained 100 mM Tris base, 1.33 mM EDTA, 20 mM MgSO₄, 20 mM sodium arsenate, 0.5 mM ADP, 25 mM hydroxylamine and 50 mM L-glutamine, adjusted to pH 7.0. The mixture was equilibrated in a 96-well plate at 30˚C for 5 min, and the reaction was initiated by adding the enzyme extract. After appropriate times, the reaction was stopped with 100 µl of stop reagent [5.5% FeCl₃・6H₂O (w/v), 2% trichloroacetic acid (w/v), and 2.1% conc. HCl], followed by centrifugation (5 min; 10,000 × g) to remove any precipitate. The product (γ-glutamylhydroxamate) was measured spectrophotometrically (A₅₃₅ ηm) using a plate reader (BioTek Synergy HT, Winooski, VT), and a standard curve of γ-glutamylhydroxamate concentration was generated. A reaction mixture without GS enzyme served as the blank. Protein in extracts was quantified spectrophotometrically (A₆₆₀ ηm) (Pierce^TM 660 ηm Protein Assay; Pierce Biotechnology, Rockford, IL, USA).

Aliquots of the protein samples were loaded on gels (Criterion TGX, 4% - 20%, Bio-Rad), and were run 180 min at constant voltage (200 V, 4˚C - 5˚C). Molecular weight marker protein standards (NativeMark^TM; Thermo Fisher Scientific, Waltham, MA, USA) were electrophoresed in separate lanes on each gel. Gels were stained using Biosafe Coomassie stain (Bio-Rad Corp., Van Nuys, CA, USA), de-stained and then photographed. Two sets of the extracted samples (20 μg) from the four treatments were loaded on a 12.5% Tris-HCl gel (Bio-Rad, Van Nuys, CA). Standard protein markers were also included in separate lanes and the gel electrophoresed for 45 min at constant voltage (200 V; 4˚C - 5˚C). After electrophoresis the gels were removed and cut. Half of the gel was stained with Biosafe Coomassie stain (Bio-Rad) as described and half was used for in-gel localization of GS enzyme activity (see below).

To examine possible toxic effects of glufosinate on C. truncatum, the herbicide was incorporated into PDA to achieve various concentrations (0 to 2.0 mM) in this growth medium. Aliquots (3 µl) of C. truncatum conidia (5 × 10⁴ conidia ml⁻¹) were pipetted onto the center agar surface of each concentration and plates were placed in an incubator (Precision Scientific Inc.) at 28˚C under a 12 h alternating light-dark cycle. The test was set up in triplicate and radial growth (colony diameter) of each colony was measured at 24 h intervals over a 5-day growth period.

A randomized complete block experimental design with four replications was utilized in these experiments. All tests were performed in triplicate and experiments were repeated in time. Means from duplicate tests were pooled and values are presented with standard error of means (SE).

The analysis of growth of hemp sesbania seedlings over the time course under continuous light, showed that spray application of C. truncatum (8.0 × 10⁴ conidia ml⁻¹) caused no major effects on hypocotyl elongation, but glufosinate treatment (1.0 mM) caused dramatic reductions in elongation of this plant organ as early as 16 h after treatment (Figure 1(a)). This indicated that the herbicide is rapidly absorbed into the seedling tissues and it essentially caused a cessation of growth. Elongation caused by C. truncatum combined with glufosinate was generally equal to the effect caused by glufosinate alone. Fresh weight of this plant organ increased slightly with time in the untreated control plants over the time course, as did this parameter for the C. truncatum treatment, where it was unchanged until about 40 h after treatment when there was a strong decline (Figure 1(b)). This sharp decrease correlated with the beginning of hypocotyl collapse caused by the pathogen. Figure 2(a) and Figure 2(b) visually demonstrate these effects at 48 h and 80 h after treatment.

C. truncatum treatment of hemp sesbania did not cause significant effects on GS activity until after 40 h, a time when hypocotyls lose their structural integrity (unable to support themselves) during the initial stages of collapse (Figure 3). This effect has been previously described in another study [38] . At 64 h after treatment, the pathogen lowered GS activity by about 10%, but at 88 h, GS was inhibited by ~65%. Glufosinate and the glufosinate plus C. truncatum treatments both caused substantial inhibition of GS throughout the 16 to 88 h period. However, GS activity in the herbicide/pathogen combination treatment was slightly higher than the glufosinate alone treatment at most time points.

Electrophoretic separation of the hemp sesbania proteins after the various treatments offered the chance to visually localize GS activity on the gels and to qualitatively compare the activity and mobility of GS with that of other proteins (Figures 4(a)-4(c)). Figure 4(a) depicts a color image of GS activity as affected by the various treatments. Activity was present in the 0-time control, the 30-h control and in the C. truncatum-treated hypocotyls, 30 h after treatment. However, no activity was present in the glufosinate- or in the glufosinate plus C. truncatum-treated tissue, which indicated the enzyme was inhibited. As expected, no other proteins (standard markers and hemp sesbania proteins) exhibited activity (Figure 4(a) and Figure 4(b)). These red-brown color bands that fade rapidly, were subsequently imaged, to corroborate results (Figure 4(b)). A matching gel, stained with protein stain (Coomassie) showed the location of the GS protein (red arrow), as well as the profiles of other proteins from the treatments (Figure 4(c)).

Generally, ammonia levels in hemp sesbania tissues under these various treatments (Figure 5) were inversely correlated with the GS activity (Figure 3). The untreated control tissues contained much lower ammonia levels at 24, 64, and 88 h after treatment than the C. truncatum, glufosinate or the combination of the herbicide and bioherbicide treatments. C. truncatum alone caused elevated ammonia levels only at 64 and 88 h after treatment, and this is during tissue collapse.

The toxicity of various concentrations of glufosinate (0 to 2.0 mM), incorporated into PDA was examined on C. truncatum over a 144-h time-course. After inoculation on PDA dishes, radial growth of C. truncatum colonies was found to be negatively affected by the herbicide at all herbicide concentrations tested (Figure 6). When measured at the end of the time-course, the 0.25 mM, 1.0 mM and 2.0 mM glufosinate concentrations caused a 10%, 25% and 45% reduction of colony diameter, respectively as compared to fungal growth on PDA without glufosinate. Furthermore, glufosinate inhibited sporulation of the bioherbicide in this growth bioassayon PDA as noted by lack of dark pigmentation (probably melanin) on culture surfaces (Figure 6).

Glufosinate is produced naturally by several species of the Streptomycetaceae family (gram-positive bacteria) and has antibacterial, antifungal and potent herbicidal properties [11] . Antibacterial activity is generally weak since many bacteria can metabolize (detoxify) this compound [42] , although the herbicide was

antibacterial against P. syringae pv. glycinea [soybean (Glycine max) blight disease] [43] . Many reports are related to the activity of glufosinate on fungi. Similar to the present results, glufosinate also inhibited spore production of Penicillium expansum, a fungal pathogen (blue mold) of fruits and vegetables [44] . Furthermore, low doses of glufosinate in bean (Phaseolus vulgaris) roots resulted in enhanced sporangial germination of the fungal pathogen, Pythium ultimum [45] . At a low concentration (100 μg・mL⁻¹), the herbicide inhibited appressorium formation of Magnaporthe grisea [rice (Oryza sativa) blast disease] and Cochliobolus miyabeanus (brown spot disease in rice), but conidial germination was unaffected. Similar results were reported for M. grisea in vitro [46] [47] . It was also antifungal against pathogens of grapes (Vitis spp.) [44] [48] . Glufosinate significantly inhibited mycelial growth of Rhizoctonia solani (sheath blight disease) and S. homoeocarpa (dollar spot disease) infecting bentgrass (Agrostis palustris) [49] and also inhibited some beneficial plant soil fungi and bacteria [50] . Several disease-causing plant fungi were among the most resistant to glufosinate [42] . Compatibility tests of C. truncatum (different strain than used in present studies) conidia with herbicides in vitro, resulted in a wide range of effects on conidial germination with the different herbicides [51] . In that report, glufosinate caused only a slight inhibition of germination after incubation of conidia for 24 h in a 1X concentration of commercially formulated herbicide product.

Our studies on C. truncatum presented here centered around ammonia, i.e., increased ammonia levels produced and/or caused by the fungus, and ammonia produced in hemp sesbania plants due to the inhibition of GS by glufosinate. We also anticipated a possible synergistic interaction of this pathogen and glufosinate. It should be considered that, mechanisms other than GS inhibition can also lead to ammonia production in plants. For example, phenylalanine ammonia lyase (PAL), amidases, amidohydrolases, and nitralases [52] [53] . These enzymes can produce ammonia as a product via catalysis of their respective substrates. Furthermore, these enzymes have been implicated in plant-microbe interactions related to defense. GS has been implicated as having a defense role in plants-pathogen interactions and may play a role in the NO signaling response in root nodules by contributing to the antioxidant defenses [54] . Cytosolic GS genes were induced following pathogen infection in tobacco (Nicotiana tabacum) [55] and similar induction occurred in Arabidopsis following infectivity by E. amylovora [56] . Glutamine levels have been also correlated with the activation of defense responses in plants [57] .

PAL catalyzes the non-oxidative conversion of phenylalanine to cinnamic acid (precursor of the phenylpropanoids) and ammonia. Also, amidases (acylamide amidohydrolases, EC 3.5.1.4) catalyze the hydrolysis of carboxylic amide bonds to liberate carboxylic acids and ammonia. Presently it is unknown if these defense enzymes are involved in the CT and glufosinate interactions as studied here, but it would be a large and interesting undertaking to study.

Weed defense mechanisms against pathogens are important to bioherbicidal efficacy and to interactions of bioherbicides and herbicides. PAL activity can increase dramatically in response to various stimuli (light, mechanical injury, action of some chemicals, elicitors, and challenge by pathogens) [58] . This increased enzyme activity leads to increased production of cinnamic acid, phenolic products and phytoalexins, some of which are toxic to bacteria and fungi. Another product of PAL catalysis is ammonia. This increased enzyme activity leads to increased production of cinnamic acid, phenolic products and phytoalexins, some of which are toxic to bacteria and fungi. Another product of PAL catalysis is ammonia. Furthermore, PAL was suggested as a target site for herbicides [59] and PAL activity was shown to be elevated or inhibited by various herbicides [60] . The fungal pathogen Alternaria cassiae, has bioherbicidal activity against sicklepod (Senna obtusifolia) [61] [62] [63] , and alters phenylpropanoid metabolism in this weed [64] . PAL activity levels were increased prior to visual symptoms of pathogenesis when A. cassiae spores were applied to seedlings. PAL activity was elevated 3-fold above uninoculated plants and remained high for several days. Similar results were reported for Alternaria crassa, a fungal bioherbicide that controls jimsonweed (Datura stramonium) [65] . Many examples have been reported and summarized with respect to the correlation of PAL activity with pathogen challenge and plant defense e.g. [66] . Nitrilases are involved in a wide variety of biological processes, some related to plant defense [67] and certain amidohydrolases are reported to contribute to the biosynthesis of auxin, the ubiquitous plant growth hormone [68] .

Ammonia and creating an alkaline environment are important in the pathogenisis of some plant pathogens. For example, during colonization of a fungus (C. gloeosporioides) related to CT, ammonia accumulates in the host plant making it more alkaline, thus promoting the activities of extracellular enzymes such as pectate lyase [69] . Similarly, ammonia secretion, modulation of pH to alkaline conditions and ammonia accumulation have been shown to be critical factors related to the pathogenicity of C. coccodes on tomato (Lycopersicon esculentum) [70] . Plant pathogens and compounds that alter ammonia in a given host, may interact with pathogenesis.

Although some typical effects of CT (collapsed stems) [38] were observable in the combined treatment of glufosinate and CT, the degree of toxicity of glufosinate on this bioherbicide may have prevented a synergistic interaction response when these two weed control agents were applied at these concentrations together on hemp sesbania. Possibly, by modifying the relative concentrations of glufosinate and CT, i.e., so that the herbicide concentration is lowered (less toxicity to CT), while the CT concentration is raised, might result in synergy. Also, glufosinate could be applied and allowed to be absorbed by the weed for a more extended time, which would effectively reduce the herbicide concentration on the weed surface, and perhaps lower toxicity to CT, thus allowing a higher conidial germination rate, formation of appresoria and infection of this weed host. Synergy is also a concentration dependent phenomenon, and thus altering the concentration ratio of the components may promote or suppress this outcome. Several Colletotrichum species, (C. truncatum, C. gloeosporioides, and C. coccodes) have been reported to have synergistic interactions with various herbicides [51] [71] [72] . Synergy was also reported in combinations of glufosinate and two bacterial bioherbicides on some weeds [29] .