Biological Control 51 (2009) 181–190

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Biological Control

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Germination-Arrest Factor (GAF): 3. Determination that the herbicidal activity of GAF is associated with a ninhydrin-reactive compound and counteracted by selected amino acids q

Donald Armstrong a, Mark Azevedo b, Dallice Mills a, Bonnie Bailey a, Brian Russell a, Aleta Groenig a, Anne Halgren b, Gary Banowetz b,*, Kerry McPhail c a Department of Botany and Plant Pathology, Oregon State University, Corvallis, OR 97331, United States b USDA-ARS, NFSPRC National Forage Seed Production Research Center, Corvallis, OR 97331, United States c College of Pharmacy, Oregon State University, Corvallis, OR 97331, United States article info abstract

Article history: A novel, naturally-occurring herbicide (Germination-Arrest Factor, GAF), produced by Pseudomonas fluo- Received 17 March 2009 rescens WH6 and several related isolates of rhizosphere bacteria, irreversibly arrests germination of the Accepted 10 June 2009 seeds of a wide range of graminaceous species, including a number of important grassy weed species. GAF Available online 14 June 2009 activity has been shown previously to be associated with a hydrophilic, low molecular weight compound that contains an acid group. In the present study, thin-layer chromatography (TLC) of extracts of WH6 Keywords: culture filtrate demonstrated that GAF activity migrates on TLC plates with a particular ninhydrin-reac- Amino acids tive compound. This compound was found to be present in GAF-producing P. fluorescens isolates and Annual bluegrass absent in P. fluorescens strains that lack the ability to produce GAF. Treatments, including mutagenesis, Biocontrol Grassy weeds which resulted in the loss of GAF activity in culture filtrates from P. fluorescens WH6 were shown to result Herbicide in the disappearance of this ninhydrin-reactive compound from extracts of WH6 culture filtrates or in Poa annua alteration of its appearance on TLC chromatograms. The ninhydrin-reactivity of GAF indicates that it Pseudomonas fluorescens probably contains an amino group, as well as the acid group previously demonstrated, and suggests that Rhizobacteria GAF may be a small peptide or amino acid analog. Biological investigations motivated by this conclusion demonstrated that the effects of GAF in inhibiting the germination of seeds of annual bluegrass (Poa annua L.) could be counteracted by treatment with alanine or glutamine and, to lesser extent, by several other amino acids, suggesting that this compound may act by interfering with some aspect of amino acid metabolism or function. Published by Elsevier Inc.

1. Introduction cases, the compounds responsible for the effects of these organ- isms on higher plant species have yet to be identified. Rhizosphere bacteria that exert inhibitory effects on the growth The development of biological or chemical agents for the and development of various higher plant species have been iso- control of grassy weeds in the grass seed production systems that lated from the soil environment by a number of investigators (Nehl constitute an important element of agricultural activity in the et al., 1996). The potential of such bacteria (Deleterious Rhizo- Pacific Northwest has been a focus of our investigations. To this sphere Bacteria, DRB) to serve as agents for the biocontrol of weed end, we have been interested in identifying DRB that specifically species has received considerable attention, but met with limited target germination of the seeds of grassy weeds and in character- practical success to date. Many DRB have been identified as Pseudo- izing the biologically active compounds produced by such DRB. monas species, and pseudomonads are known to produce a wide We have previously described the isolation and characterization range of secondary products, including a number of compounds of five strains of rhizosphere bacteria that produce and secrete a that have antimicrobial and/or antifungal activity (Lesinger and novel, naturally-occurring herbicide that irreversibly arrests the Margraff, 1979; Dowling and O’Gara, 1994). However, in most germination of seeds of a large number of graminaceous species, including those of a number of important grassy weeds (Banowetz et al., 2008). These bacterial isolates were identified as strains of Pseudomonas fluorescens. The herbicide produced by these strains q The first paper in this series was Banowetz et al. (2008). arrests seed germination in a developmentally-specific manner, * Corresponding author. Address: USDA/ARS, 3450 S.W. Campus Way, Corvallis, OR 97331, United States. Fax: +1 541 738 4127. halting the germination process immediately after the emergence E-mail address: [email protected] (G. Banowetz). of the plumule and coleorhiza. Because germination is initiated

1049-9644/$ - see front matter Published by Elsevier Inc. doi:10.1016/j.biocontrol.2009.06.004 182 D. Armstrong et al. / Biological Control 51 (2009) 181–190 in the presence of the herbicide and then subsequently arrested, 2.3. Growth of bacterial cultures and preparation of bacterial culture we have described this compound as a Germination-Arrest Factor filtrates (GAF) rather than a germination inhibitor. The effects of GAF were found to be limited primarily to germination, with little effect ob- P. fluorescens WH6 and the other isolates and strains of P. fluo- served on the growth of established seedlings or mature plants. rescens used in investigations of GAF activity were taken from cryo- Moreover, the seeds of dicot species were shown to be less sensi- vial storage in 50% glycerol at À60 °C and inoculated into Wheaton tive to GAF than seeds of graminaceous species. A quantitative bio- bottles half-filled with sterile Pseudomonas Minimal Salts Medium assay for GAF activity was developed (Banowetz et al., 2008) using (PMS). The PMS Medium was modified from that described by Bol- seeds of annual bluegrass (ABG, Poa annua L.), a grassy weed of ton et al. (1989) by supplementation with iron as described by economic significance in grass seed production. Banowetz et al. (2008). The tops of the bottles were loosely capped An initial characterization of the physical and chemical proper- and secured with tape. The inoculated bottles were placed on a ro- ties of the GAF compound produced by one of our isolates, P. fluores- tary shaker (200 rpm) in a 27 °C chamber. Cells were harvested cens WH6, was reported in a previous publication (Banowetz et al., after 7 days in culture. 2009). GAF activity was found to be associated with a small (molec- To prepare culture filtrates, the Pseudomonas culture fluid ular weight less than 1000), hydrophilic compound that contained recovered from 7-day cultures was centrifuged (3000 g, 15 min), an acid group. GAF activity could be recovered from dried culture fil- and the supernatant was passed through a bacteriological filter trates by extraction with appropriate concentrations of aqueous eth- (Millipore GP Express Steritop, 0.22 lm pore size). The resulting anol, but it was essentially insoluble in other organic solvents with sterile culture filtrate was stored at 4 °C. the exception of methanol, in which it was moderately soluble. For molecular genetic studies, P. fluorescens WH6 was main- GAF activity was found to be destroyed by heating at temperatures tained on solid Fluorescent Pseudomonas Medium (FPM) (Sands above 65 °C, and there was a significant loss of activity in sterile and Rovira, 1970; Simon et al., 1973) at room temperature and WH6 culture filtrates after storage for several months at 4 °C. grown in Luria broth (LB) medium at 28 °C for transformation with The chemical and chromatographic properties of GAF have been pUTmini-Tn5gfp. E. coli were grown in LB medium at 37 °C(Sam- further examined in the current study. We present evidence here brook and Russell, 2001). All solid media contain 1.5 percent Difco that GAF activity is associated with a particular ninhydrin-reactive Bacto-Agar (Difco Laboratories, Detroit, MI). Tetracycline compound that can be recovered from WH6 culture filtrate solids (15 lgmLÀ1) and ampicillin (50 lgmLÀ1) were added when by extraction with aqueous ethanol solutions. This result indicates appropriate for selection. that GAF is likely to contain an amino group as well as the acid group detected earlier. In addition, the inhibitory effects of GAF 2.4. Extraction of culture filtrate solids with 90% (v/v) ethanol on seed germination were found to be reversed in the presence of particular amino acids. These results, taken together and with Measured volumes of bacterial culture filtrate were taken to our earlier data, suggest that the compound responsible for GAF dryness in vacuo at a temperature 645 °C. The dry solids remaining activity may be a small peptide or an amino acid analog. after evaporation of the filtrate were extracted three times (5 min per extraction) with 90% (v/v) ethanol. Each of these three extrac- tions was performed by swirling the solids with a volume of sol- 2. Materials and methods vent equal to one-third of the original volume of culture filtrate. The three extracts prepared in this manner were combined, taken 2.1. Biological materials to dryness in vacuo as described above, dissolved in a volume of 76% (v/v) ethanol equal to one-twentieth of the original volume The isolation and characterization of the GAF-producing isolates of culture filtrate (20Â concentration), and either applied immedi- of P. fluorescens used here (Isolates WH6, AD31, AH4, E34, and ately to thin-layer chromatography (TLC) plates (as described WH19) have been described previously (Banowetz et al., 2008). below) or stored at 4 °C or later use. The strains of P. fluorescens used as controls were obtained as fol- lows: P. fluorescens Pf-5 (Howell and Stipanovic, 1979; Loper and 2.5. Bioassay of GAF activity Gross, 2007) and P. fluorescens A506 (Wilson and Lindow, 1993) were obtained from Dr. Joyce Loper (USDA-ARS Horticultural Crops Bioassays for GAF activity were performed with ABG seeds Research Laboratory, Corvallis, OR). P. fluorescens PfO-1 (Compeau using the standard GAF bioassay protocol described by Banowetz et al., 1988) was kindly provided by Dr. Mark Silby (Department of et al. (2008). Culture filtrates and other solutions to be tested for Molecular Biology and Microbiology, Tufts University School of GAF activity were distributed to the wells of sterile 48-well plates Medicine, Boston, MA). P. fluorescens D7 (NRRL B-18293) (Corning Costar 3548). Each well received 200 lL of sterile test (Gurusiddaiah et al., 1994; Kennedy et al., 1991, 2001) was obtained solution and three ABG seeds from seed lots that had been from Dr. Ann Kennedy (USDA-ARS, Pullman, WA). The transposon surface-sterilized according to the procedure described in Bano- vector pUTmini-Tn5gfp (Tn5gfp), in its host Escherichia coli S17-1 k wetz et al. (2008). Controls consisted of seeds incubated in sterile pir (Matthysse et al., 1996), was obtained from the American Type water or sterile, un-inoculated PMS medium. Both control treat- Culture Collection (ATCC 87454) and as a gift from A. Matthysse. ments were without effect on the germination of ABG seeds. Three Annual bluegrass seeds (ABG, Poa annua L.) were obtained from replicate wells (9 seeds) were prepared for each concentration of 1996 mid-Willamette Valley grass seed screenings and were pro- each treatment tested. The plates were sealed with Parafilm and vided by International Seeds, Halsey, OR, and C and R Farm, Tan- incubated in a growth chamber at 20 °C with a photoperiod of gent, OR. The seeds were cleaned to remove straw and seeds of 8 h light (50 lmol mÀ2 sÀ1) and 16 h dark. Germination scores other species prior to use. were determined after 7 d incubations. The germination scoring system used to evaluate GAF bioassays 2.2. Solvents has been described in detail by Banowetz et al. (2008). In an abbre- viated outline, germination scores in this system range from 1 to 4, All aqueous ethanol solutions were prepared from 95% (v/v) with a score of 4 representing normal development (no GAF activ- ethanol that had been redistilled before use. All other solvents ity) at the end of the 7-day assay period. A score of 1 is assigned to were purchased as spectrophotometric grade reagents. seeds where the plumule and coleorhiza have emerged from the D. Armstrong et al. / Biological Control 51 (2009) 181–190 183 seed but both are shorter than the length of the seed. A score of 2 For bioassay of the distribution of GAF activity on cellulose TLC indicates the primary root is visible and the plumule is slightly chromatograms, the cellulose was scraped from developed TLC longer than the seed, but typically chlorotic. A score of 3 indicates plates as 1 cm bands located between the origin and solvent front. that the first true leaf has emerged from the coleoptile and is green The cellulose from each zone (1 Â 5 cm) was suspended and vor- in color, but the emerged portion of the leaf is obviously shorter texed in 1.0 mL of water in a 2.0 mL Minifuge tube. The cellulose than the length of the coleoptile. A score of 4 is assigned to seeds was then pelleted by centrifugation (15,000 rpm, 10 min, Mini- exhibiting normal development, equivalent to that of controls, fuge). The supernatant from each tube was filter-sterilized which at this stage of germination means that the first true leaf (0.2 lm filter), diluted to an appropriate concentration with sterile is fully emerged, green in color, and the emerged portion of the leaf water, and tested for GAF activity in the standard GAF bioassay is obviously longer than the coleoptile. A score of zero (no visible system as described above. evidence of seed germination) is possible in this system, but GAF concentrations equivalent to that of full-strength WH6 culture 2.8. Transposon mutagenesis of P. fluorescens WH6 filtrate typically result in permanent arrest of development at a germination score of 1. The plasmid pUTmini-Tn5gfp was extracted from E. coli S1701 Quantitative estimates of the relative amounts of GAF present cells using standard procedures (Sambrook and Russell, 2001). in various extracts are expressed in terms of GAF milliequivalents, Overnight cultures of P. fluorescens WH6 cells grown in 20 mL of where one GAF milliequivalent is defined as the minimum quantity LB at 28 °C with shaking (200 rpm) were centrifuged at 3330g for of GAF required to give a germination score of 1 when dissolved in ten minutes and washed. The cell pellets were cooled on ice, 1 mL of bioassay test solution. GAF milliequivalents were deter- suspended in 1 mL of sterile double deionized water, and 600 lL mined from a standard graph prepared by plotting germination ice-cold LB was then added to each. Cell suspensions were score against various dilutions of a WH6 culture filtrate. (For an transferred to electroporation cuvettes and transformed by pUT- illustration of such a graph, see Banowetz et al., 2009.) mini-Tn5gfp using a BTX Electro Cell Manipulator 600 (Harvard Apparatus, Inc., Holliston, MA) according to the manufacturer’s 2.6. Amino acid and GAF competition experiments protocol for P. putida transformation. Both a non-transformed con- trol and the electroporated cell suspension were transferred to ice- Amino acid stock solutions were prepared at concentrations cold, sterile 50 mL centrifuge tubes, which were then incubated on two-fold higher than those to be tested and adjusted to pH 6.5 with a shaker (200 rpm) at 28 °C for 17 h. A library of TetR colonies was HCl or NaOH. The resulting solutions were filter-sterilized (0.2 lM selected on solid LB medium containing tetracycline. These colo- filter). Similarly, sterile culture filtrate from P. fluorescens WH6 was nies were inoculated into PMS medium for the production of diluted to a concentration two-fold higher than that to be tested. culture filtrates, which were subsequently screened for GAF activ- Equal volumes (100 lL each) of the appropriate amino acid and ity in the standard GAF bioassay system described above. culture filtrate stock solutions were then mixed and distributed to assay plate wells for testing in the standard GAF bioassay system as described above. 3. Results

2.7. Thin-layer chromatography (TLC) materials and procedures 3.1. TLC analysis of the GAF activity in extracts of P. fluorescens WH6 culture filtrate TLC separations were performed on AvicelÒ Microcrystalline Cellulose Uniplates (5 Â 20 cm, 250 lm layer, glass-backed) and The hydrophilic character of the compound responsible for GAF on Hard-Layer Silica Gel GHL Uniplates (5 Â 20 cm, 250 lm layer, activity limits options for its purification. To achieve an initial glass-backed, with an inorganic binder), both purchased from separation of GAF from some of the salts in the culture filtrate, the Analtech, Inc. The TLC chromatograms were developed in cylindri- solids recovered after evaporation of culture filtrate from the GAF- cal chromatography tanks (6 Â 23 cm) containing 25 mL of solvent. producing isolate P. fluorescens WH6 were extracted with 90% etha- For chromatography on the Avicel cellulose TLC plates, the chro- nol as described in Section 2. As shown previously (Banowetz et al., matographic solvent consisted of ethyl acetate:isopropanol:water 2009), this procedure leaves behind 30–40% of the GAF activity, but (7.5:15:10). For chromatography on the silica gel GHL plates, the the resulting extract is more amenable to fractionation procedures solvent system consisted of ethyl acetate:isopropanol:metha- than the culture filtrate itself and proved to be a useful starting point nol:water (5:5:18:2). for subsequent fractionation and analysis of GAF activity. The chromatographic samples consisted of 200 lL aliquots of The GAF activity recovered from the WH6 culture filtrate solids the 76% ethanol solutions (20Â solutions) prepared from 90% eth- by extraction with 90% ethanol was examined by TLC fractionation. anol extracts of dried culture filtrates as described above. The As shown in Fig. 1, when these extracts were chromatographed on 200 lL aliquots of the extracts to be analyzed were each streaked microcrystalline cellulose TLC plates using an appropriate solvent across a TLC plate by repeated applications to an origin line located system, GAF activity could be recovered from the chromatograms 3 cm from one end of the plate. The sample was applied with a as a single broad band of activity in a zone located in the lower half 50 lL Lang–Levy micropipette equipped with a screw-type micro- of the plate (Fig. 1A). This zone contained no obvious UV-absorbing pipette-control device and using a filtered air-stream to dry the or fluorescent compounds when examined under UV-light. Tests of origin for 20–30 s between applications. Following the final appli- various color reagents revealed that this band of GAF activity was cation, the chromatograms were allowed to air dry for 4 or 8 min coincident with a zone on the chromatograms that gave a strongly (silica gel or cellulose plates, respectively) and then developed over positive color reaction when the chromatograms were sprayed a distance of 12 cm from the origin. with ninhydrin (Fig. 1B). For ninhydrin-staining of developed chromatograms, the dry chromatograms were sprayed with a ninhydrin solution consisting 3.2. TLC comparisons of GAF-producing and non-producing strains of 0.25% (w/v) ninhydrin (Sigma) dissolved in 95% (v/v) ethanol of P. fluorescens containing 3.0 mL of glacial acetic acid per 100 mL of solution. Color development was achieved by heating the sprayed chro- To determine whether the ninhydrin-positive band that co- matograms in an oven at 80–90 °C for 15 min. chromatographed with GAF activity in TLC fractionations of 184 D. Armstrong et al. / Biological Control 51 (2009) 181–190

ity (Fig. 2D). The salmon/rose-colored band observed in GAF-pro- ducing strains of P. fluorescens was immediately preceded by a narrow bright-blue band that was absent from the Pseudomonas strains that did not produce GAF. GAF activity could not be recov- ered from the silica gel plates, because aqueous extracts of the silica gel inhibited the germination of ABG seeds and interfered with the standard GAF bioassay. However, when GAF activity was extracted from the appropriated zone of cellulose TLC plates and rechromatographed on silica, ninhydrin-staining revealed the presence of the expected salmon/rose and narrow blue bands that had been shown to correlated with the presence of GAF activity.

3.3. Alterations in the ninhydrin-staining patterns of TLC chromatograms in association with changes in GAF activity

Additional confirmation of the identification of GAF with the ninhydrin-reactive compound in question was obtained by observ- ing the changes in TLC profiles associated with the loss of GAF activity observed during prolonged storage of WH6 culture filtrates or brought about by exposure of these culture filtrates to brief autoclaving or to mildly acidic conditions (pH2) for extended peri- ods of time at room temperature. The recovery of GAF activity from WH6 culture filtrates subjected to these treatments is summarized in Table 1. As shown in Fig. 3A, although a ninhydrin-reactive band was still visible at the same Rf in cellulose TLC chromatograms of extracts prepared from aged WH6 culture filtrates, the color of this band shifted from purple-blue to gray-green as GAF activity decreased in the culture filtrates. Moreover, on silica gel plates (Fig. 3B), the salmon/rose-colored band normally seen in extracts of GAF-producing pseudomonads largely disappeared with time and loss of activity in storage, while the narrow blue band seen in these extracts increased in intensity in the older filtrates. Thus, Fig. 1. Comparison of the distribution of GAF activity and ninhydrin-staining in TLC the loss of GAF activity seen in aged WH6 culture appears to be chromatograms of extracts of Pseudomonas fluorescens WH6 culture filtrate. associated with chemical changes in the original ninhydrin-reac- Extracts (90% ethanol) of the solids recovered from WH6 culture filtrate were tive compound that results in color shifts in the product obtained prepared and chromatographed on cellulose TLC plates as described in Section 2. with ninhydrin treatment. A similar change in ninhydrin-staining The GAF activity recovered by aqueous extraction of the various zones from one TLC plate is shown in 1A. (GAF activity is expressed in GAF milliequivalents as defined in was observed in TLC chromatograms prepared from sterile WH6 Section 2 and measured in the standard Poa bioassay for GAF activity.) The culture filtrates that had been adjusted to pH2 with 1 N HCl and corresponding ninhydrin-staining pattern for a replicate TLC fractionation of WH6 maintained at this pH for 7 days at room temperature (Fig. 3C) culture filtrate is shown in 1B. prior to neutralizing the pH and preparing extracts for bioassay. As shown in Table 1, transient exposure to this pH (for the few extracts of WH6 culture filtrate was indeed GAF, the ninhydrin- minutes required to acidify and then immediately neutralize the banding patterns obtained with WH6 were compared with those filtrate) had little effect on GAF activity, but extended exposure obtained with culture filtrates from other P. fluorescens isolates to these mildly acidic conditions resulted in a sharp drop in GAF and strains. In addition to P. fluorescens WH6, we have previously activity. Moreover, the loss of GAF activity brought about by this identified and characterized four other P. fluorescens isolates treatment was associated with color changes in the ninhydrin- (AD31, AH4, E34, and WH19) that produce GAF activity (Banowetz banding patterns similar to those observed to occur in aged WH6 et al., 2008). The results of cellulose TLC analysis of WH6 and these culture filtrates. Autoclaving had been shown previously (Bano- four additional GAF-producing P. fluorescens isolates are shown in wetz et al., 2009) to bring about a sharp drop in GAF activity, Fig. 2A. All of these isolates exhibited a ninhydrin-positive band and this result was confirmed in the present study (Table 1). More- at an Rf identical to that associated with GAF activity in fractiona- over, TLC analysis of WH6 culture filtrates that had been subjected tions of WH6 culture filtrate. In contrast to these results, cellulose to autoclaving revealed that all the ninhydrin-reactive compounds TLC analysis of culture filtrates from four strains of P. fluorescens present in the filtrate were destroyed by this treatment (Fig. 3D). (A506, D7, Pf-5, and PfO-1) previously shown to lack GAF activity Following this treatment, no ninhydrin-staining bands were evi- (Banowetz et al., 2008) failed to detect a ninhydrin-reactive com- dent on either the cellulose or silica gel TLC chromatograms. The pound at this Rf (Fig. 2B). loss of ninhydrin-reactivity with autoclaving is probably due to When the same culture filtrates were subjected to TLC analysis cleavage of the amino group and its loss as ammonia, although it on hard-layer silica gel plates using a different solvent system, the is possible that at high temperature the amino group may react results (as shown in Fig. 2C and D) paralleled those obtained with with some other chemical group in the culture filtrate with the the cellulose plates. On the silica TLC plates, the extract from WH6 result that the amino group is no longer available for reaction with culture filtrate gave rise to a large salmon or rose-colored band ninhydrin. when the developed chromatogram was sprayed with ninhydrin. This band was also present in chromatograms of the culture 3.4. Mutational confirmation of the identity of GAF filtrates from all four of the other GAF-producing P. fluorescens isolates (Fig. 2C), but it was not present in the chromatograms The association of GAF activity with the particular ninhydrin- developed from the four P. fluorescens strains that lacked GAF activ- reactive compound identified above was confirmed by mutagene- D. Armstrong et al. / Biological Control 51 (2009) 181–190 185

Fig. 2. TLC analysis of culture filtrates from Pseudomonas fluorescens isolates and strains. Extracts (90% ethanol) of the solids recovered from Pseudomonas culture filtrates were prepared and chromatographed on cellulose TLC plates as described in Section 2. The developed chromatograms were sprayed with ninhydrin reagent as described in the same source. The position of the Germination-Arrest Factor (GAF) band is indicated for WH6. (A) TLC analysis (on cellulose plates) of extracts from WH6 and other GAF- producing bacterial isolates. (B) TLC analysis (on cellulose plates) of extracts from WH6 and non-GAF-producing bacterial strains. (C) TLC analysis (on GHL silica plates) of extracts from WH6 and other GAF-producing bacterial isolates. (D) TLC analysis (on GHL silica plates) of extracts from WH6 and non-GAF-producing bacterial strains.

sis of P. fluorescens WH6. Transposon mutagenesis of WH6 with the genic loss of GAF activity also resulted in loss of the particular nin- Tn5 transposon coding for tetracycline resistance resulted in the hydrin-reactive compound present in culture filtrates of this GAF- generation of 1,214 tetracycline-resistant transformants. Screening producing line of P. fluorescens. of culture filtrates from these transformants in the standard GAF bioassay system resulted in the identification of three transfor- 3.5. Influence of amino acids on GAF activity in the Poa bioassay mants (WH6-1::Tn5, WH6-2::Tn5, and WH6-3::Tn5) that ap- peared to have lost the ability to produce GAF. The apparent loss The ninhydrin-reactivity of GAF indicates that it probably con- of GAF activity in the WH6-1::Tn5 mutant proved to be difficult tains an amino group. This fact, together with our previous demon- to reproduce, and additional work indicated that this mutation stration that GAF is a low molecular weight compound that had affected growth characteristics rather than GAF production. contains an acid group (Banowetz et al., 2009) suggested that Culture filtrates from the other two mutants (WH6-2::Tn5 and GAF might be some type of small peptide or an amino acid analog. WH6-3::Tn5), however, were confirmed to lack GAF activity. As The latter hypothesis led us to suspect that it might be possible to shown in Table 2, the GAF activity in culture filtrates from these counteract the effects of GAF by treatment with particular amino two mutants was essentially eliminated. TLC analyses of the cul- acids. To explore this possibility, the effects of the twenty protein ture filtrates from WH6-2::Tn5 and WH6-3::Tn5 are shown in amino acids on the response of ABG seeds to WH6 culture filtrate Fig. 4. The prominent ninhydrin-positive band visible on the TLC were examined in the standard GAF bioassay system. For purposes plate generated from wild type WH6 culture filtrate was absent of testing this interaction, the WH6 culture filtrate concentration from both the WH6-2 and WH6-3 culture filtrates. Thus, muta- used in the bioassay was reduced to the minimal level that would cause complete, or nearly complete, germination arrest (about 0.1Â in this case), and the response of ABG seeds to this filtrate Table 1 concentration was examined in the presence of varying concentra- GAF activity in P. fluorescens WH6 culture filtrates after aging or other treatments. tions of the test amino acid. The results are summarized in Table 3. Sterile WH6 culture filtrates were stored at 4 °C for varying periods of time or Several amino acids were found to significantly alter the re- subjected to the indicated treatments and then tested and scored for GAF activity in sponse of ABG seeds to GAF under the described test conditions. the standard GAF bioassay as described in Section 2. GAF activity is expressed as GAF The effects observed with alanine and glutamine were particularly milliequivalents, where 1 GAF milliequivalent is defined as the minimum amount of GAF required to give a germination score of 1.0 when dissolved in 1.0 mL and tested dramatic. At appropriate concentrations, both of these amino acids in the standard GAF bioassay. were able to completely counteract the effects of the 0.1Â culture filtrate on ABG germination (yielding bioassay scores of 4), with Treatment Duration of treatment GAF activity (Milliequivalents/mL) alanine being the most active of the two. As shown in Fig. 5, alanine None (Control) 0 (Initial activity) 9.6 was still able to counteract the effects of GAF when the concentra- Sterile storage (4 °C) 4 Months 6.5 10 Months 0.8 tion of the latter was increased two- to four-fold. Phenylalanine, 16 Months 0.1 tyrosine, leucine, serine, methionine, isoleucine, and glutamic acid 32 Months 0.1 also significantly counteracted the effects of GAF. With the excep- Autoclaving 30 Minutes 0.1 tion of tyrosine, all yielded germination scores of 3 or greater in the pH 2 Transient 9.6 7-Days 0.6 presence of 0.1Â culture filtrate. Tyrosine appeared to be relatively effective at counteracting the effects of GAF at low concentrations, 186 D. Armstrong et al. / Biological Control 51 (2009) 181–190

Fig. 3. TLC analysis of Pseudomonas fluorescens WH6 culture filtrates exhibiting degradation of GAF activity. Extracts (90% ethanol) of the solids recovered from P. fluorescens WH6 culture filtrates that had been subjected to various treatments resulting in a reduction in GAF activity were prepared as described in Section 2. (A) TLC analysis (on cellulose plates) of extracts prepared from WH6 culture filtrates stored under sterile conditions at 4 °C for various time intervals as indicated. (B) TLC analysis (on GHL silica plates) of extracts prepared from WH6 culture filtrates stored under sterile conditions at 4 °C for various time intervals as indicated. (C) TLC analysis of extracts of WH6 culture filtrates exposed to either transient (0 d) or prolonged (7 d) exposure to pH2. (D) TLC analysis (on cellulose and GHL silica plates) of extracts prepared from untreated and autoclaved WH6 culture filtrates. The chromatograms were developed in the appropriate solvent systems and sprayed with ninhydrin reagent as described in Section 2.

Table 2 GAF activity of culture filtrates from mutant P. fluorescens WH6 genotypes. Culture filtrates from wild type and mutant WH6 cell lines were tested for GAF activity in the standard GAF bioassay system as described in Section 2. (A germination score of 1.0 indicates high GAF activity, with germination completely arrested immediately after emergence of the coleorhiza and plumule. A germination score of 4.0 indicates no GAF activity, with germination and seedling development equivalent to that of controls.)

WH6 genotype Mutation designation Germination score (±Standard error of the mean) at the indicated culture filtrate concentration (1Â = No dilution) 0 0.1Â 0.3Â 1.0Â WH6 (Wild-Type) — 4.0 ± 0.0 1.1 ± 0.0 1.0 ± 0.0 1.0 ± 0.0 WH6-2::Tn5 gaf2 4.0 ± 0.0 4.0 ± 0.0 4.0 ± 0.0 3.8 ± 0.1 WH6-3::Tn5 gaf3 4.0 ± 0.0 4.0 ± 0.0 4.0 ± 0.0 3.7 ± 0.1

the exception of glutamic acid, were themselves somewhat inhib- itory to germination when tested in the absence of GAF. Interest- ingly, in these cases, the inhibitory effects of the amino acid itself appeared to be somewhat ameliorated in the presence of GAF, as shown in Fig. 6 for leucine. A number of other amino acids (threonine, histidine, valine, trypto- phan, asparagine, aspartic acid, and proline) exhibited somewhat weaker interactions with GAF in the Poa bioassay as shown in Table 3. Among the amino acids in this group, only tryptophan was able to give a germination score above 2.5 in the presence of 0.1Â culture filtrate. Four amino acids (glycine, cysteine, lysine, and arginine) were inhibitory to germination in their own right and exhibited slight or no effect on the activity of GAF in the Poa bioassay. Arginine Fig. 4. TLC analysis of culture filtrates from mutants of P. fluorescens WH6. Extracts was particularly inhibitory to the germination of ABG seeds, as (90% ethanol) of the solids recovered from culture filtrates of P. fluorescens WH6 and was lysine, the other basic amino acid. the mutants WH6-2::Tn5, and WH6-3::Tn5 were prepared and chromatographed on cellulose and on GHL silica gel TLC plates as described in Section 2. The developed chromatograms were sprayed with ninhydrin reagent as described in the 4. Discussion same source. The ninhydrin-stained cellulose plates are shown on the left, and the silica gel plates are shown on the right. The GAF activity present in P. fluorescens WH6 culture filtrates has been shown in the present study to chromatograph with a par- but its poor solubility limited the concentrations at which it could ticular ninhydrin-reactive compound that is visible on cellulose be tested. At higher concentrations, all of these amino acids, with TLC plates as a purple–blue band when chromatograms of culture D. Armstrong et al. / Biological Control 51 (2009) 181–190 187

Table 3 Interactions of GAF and amino acids in the standard GAF bioassay. A germination score of 1.0 indicates high GAF activity (germination completely arrested immediately after emergence of the coleorhiza and plumule). A germination score of 4.0 indicates no GAF activity (germination and seedling development equivalent to that of controls). See Section 2 for details of the bioassay procedure and the scoring system.

Amino acid WH6 culture filtrate concentration (1Â = No dilution) Germination score ( ± Standard error of the mean) at indicated amino acid concentration (mM) 0 0.3 1 3 10 30 Alanine 0 3.8 ± 0.1 4.0 ± 0.0 3.9 ± 0.1 4.0 ± 0.0 3.9 ± 0.1 3.2 ± 0.1 0.1Â 1.3 ± 0.1 1.9 ± 0.1 3.3 ± 0.3 4.0 ± 0.0 4.0 ± 0.0 4.0 ± 0.0 Arginine 0 4.0 ± 0.0 3.6 ± 0.1 2.6 ± 0.1 2.1 ± 0.1 1.5 ± 0.1 1.0 ± 0.0 0.1Â 0.8 ± 0.1 1.0 ± 0.0 0.9 ± 0.1 0.9 ± 0.1 1.0 ± 0.0 1.0 ± 0.0 Asparagine 0 4.0 ± 0.0 4.0 ± 0.0 3.6 ± 0.3 4.0 ± 0.0 3.9 ± 0.1 4.0 ± 0.0 0.1Â 1.0 ± 0.0 1.1 ± 0.1 1.7 ± 0.1 1.6 ± 0.1 2.1 ± 0.4 1.8 ± 0.3 Aspartic acid 0 3.9 ± 0.1 3.8 ± 0.1 4.0 ± 0.0 4.0 ± 0.0 4.0 ± 0.0 4.0 ± 0.0 0.1Â 1.3 ± 0.1 1.0 ± 0.0 1.1 ± 0.1 1.4 ± 0.1 1.6 ± 0.1 2.1 ± 0.3 Cysteine 0 4.0 ± 0.0 4.0 ± 0.0 4.0 ± 0.0 3.3 ± 0.1 1.4 ± 0.1 1.2 ± 0.1 0.1Â 1.0 ± 0.0 1.4 ± 0.2 1.4 ± 0.1 1.5 ± 0.2 1.3 ± 0.1 1.2 ± 0.1 Glutamic acid 0 4.0 ± 0.0 4.0 ± 0.0 4.0 ± 0.0 4.0 ± 0.0 3.9 ± 0.1 3.6 ± 0.2 0.1Â 1.6 ± 0.1 1.5 ± 0.0 1.5 ± 0.0 1.6 ± 0.1 1.5 ± 0.0 3.0 ± 0.3 Glutamine 0 4.0 ± 0.0 4.0 ± 0.0 4.0 ± 0.0 3.7 ± 0.3 3.7 ± 0.3 4.0 ± 0.0 0.1Â 1.3 ± 0.1 1.4 ± 0.1 1.8 ± 0.2 3.5 ± 0.3 4.0 ± 0.0 4.0 ± 0.0 Glycine 0 4.0 ± 0.0 4.0 ± 0.0 4.0 ± 0.0 3.7 ± 0.2 3.1 ± 0.1 2.2 ± 0.1 0.1Â 1.0 ± 0.0 1.3 ± 0.1 1.5 ± 0.1 1.5 ± 0.1 1.5 ± 0.1 1.6 ± 0.1 Histidine 0 4.0 ± 0.0 3.8 ± 0.2 3.5 ± 0.1 2.9 ± 0.1 1.8 ± 0.1 1.0 ± 0.0 0.1Â 1.0 ± 0.0 1.6 ± 0.2 1.7 ± 0.1 1.8 ± 0.2 2.5 ± 0.1 1.1 ± 0.1 Isoleucine 0 4.0 ± 0.0 4.0 ± 0.0 4.0 ± 0.0 4.0 ± 0.0 3.0 ± 0.1 1.5 ± 0.0 0.1Â 1.0 ± 0.0 1.2 ± 0.1 1.8 ± 0.1 2.1 ± 0.2 3.5 ± 0.0 2.3 ± 0.3 Leucine 0 4.0 ± 0.0 3.9 ± 0.2 4.0 ± 0.0 3.6 ± 0.2 3.0 ± 0.0 1.7 ± 0.1 0.1Â 1.0 ± 0.0 1.5 ± 0.0 1.5 ± 0.3 3.6 ± 0.1 3.4 ± 0.1 2.8 ± 0.1 Lysine 0 4.0 ± 0.0 3.9 ± 0.1 2.8 ± 0.1 2.1 ± 0.1 1.5 ± 0.1 1.0 ± 0.0 0.1Â 1.0 ± 0.0 1.6 ± 0.1 1.6 ± 0.1 1.6 ± 0.1 1.3 ± 0.1 1.1 ± 0.1 Methionine 0 4.0 ± 0.0 4.0 ± 0.0 4.0 ± 0.0 3.5 ± 0.1 2.3 ± 0.1 2.1 ± 0.1 0.1Â 1.0 ± 0.0 1.5 ± 0.1 2.2 ± 0.2 3.0 ± 0.2 3.0 ± 0.1 2.1 ± 0.2 ± Phenylalanine 0 4.0 ± 0.0 4.0 ± 0.0 4.0 ± 0.0 4.0 ± 0.0 2.6 ± 0.2 1.5 ± 0.0 0.1Â 1.0 ± 0.0 1.7 ± 0.1 3.0 ± 0.2 3.3 ± 0.2 3.3 ± 0.1 1.4 ± 0.1 Proline 0 4.0 ± 0.0 3.9 ± 0.1 4.0 ± 0.0 3.9 ± 0.1 3.7 ± 0.3 3.9 ± 0.1 0.1Â 1.2 ± 0.1 1.7 ± 0.1 1.5 ± 0.1 1.4 ± 0.1 1.7 ± 0.1 2.1 ± 0.3 Serine 0 4.0 ± 0.0 4.0 ± 0.0 3.7 ± 0.3 3.6 ± 0.2 3.1 ± 0.1 2.6 ± 0.2 0.1Â 1.3 ± 0.1 1.7 ± 0.1 2.1 ± 0.2 3.4 ± 0.3 3.4 ± 0.2 2.9 ± 0.1 Threonine 0 4.0 ± 0.0 4.0 ± 0.0 4.0 ± 0.0 4.0 ± 0.0 3.4 ± 0.2 2.9 ± 0.1 0.1Â 1.2 ± 0.1 1.7 ± 0.1 2.0 ± 0.2 1.9 ± 0.1 2.2 ± 0.2 2.2 ± 0.1 Tryptophan 0 4.0 ± 0.0 4.0 ± 0.0 4.0 ± 0.0 3.1 ± 0.2 2.6 ± 0.2 1.0 ± 0.0 0.1Â 1.0 ± 0.0 1.7 ± 0.1 1.0 ± 0.0 2.1 ± 0.2 2.8 ± 0.2 1.0 ± 0.0 Tyrosine 0 4.0 ± 0.0 3.9 ± 0.1 4.0 ± 0.0 — — — 0.1Â 1.1 ± 0.1 2.2 ± 0.2 2.9 ± 0.2 — — — Valine 0 4.0 ± 0.0 4.0 ± 0.0 4.0 ± 0.0 3.8 ± 0.1 3.3 ± 0.1 3.0 ± 0.0 0.1Â 1.1 ± 0.1 1.6 ± 0.1 1.7 ± 0.2 1.7 ± 0.2 2.2 ± 0.4 1.9 ± 0.2

filtrate extracts are sprayed with ninhydrin. This ninhydrin-reac- ing ninhydrin-reactive bands disappeared following autoclaving of tive band was present in TLC chromatograms prepared from four WH6 culture filtrates. A pronounced decrease in GAF activity that other GAF-producing P. fluorescens isolates, and it was absent in occurred with prolonged storage of WH6 culture filtrates was asso- TLC chromatograms prepared from culture filtrates of four strains ciated with a shift in color of the corresponding ninhydrin band of P. fluorescens that lack the ability to produce GAF. GAF activity visible on cellulose plates (from purple-blue to green-gray). On could not be recovered from silica gel TLC plates following frac- silica gel plates, aging of WH6 culture filtrates was associated with tionation of WH6 culture filtrates, because extracts of the silica a decrease in the salmon–rose band and a corresponding increase gel itself were toxic in the GAF bioassay. However, on silica gel in the bright-blue ninhydrin band running at a slightly higher Rf. chromatograms, a broad salmon/rose-colored band that was Similar color changes were associated with a loss of biological closely associated with a narrow blue ban running at a slightly activity associated with exposure of GAF to acid pH (pH 2) for higher Rf could be seen with ninhydrin-staining of TLC chromato- several days at room temperature. Thus, the narrow blue band vis- grams prepared from culture filtrates of WH6 and the four other ible on ninhydrin-stained silica gel TLC chromatograms and the GAF-producing P. fluorescens isolates. These bands were absent in green–gray band visible on ninhydrin-stained cellulose TLC chro- chromatograms prepared from the four P. fluorescens strains that matograms of aged culture filtrates appear to represent a degrada- lack the ability to produce GAF. Thus, in addition to the demonstra- tion product derived from GAF. This product must still retain the tion that GAF activity migrates with the ninhydrin-reactive band ninhydrin-reactive group of GAF, but based on its chromatographic visible on cellulose TLC plates, the presence or absence of GAF behavior on silica gel, it must be slightly more hydrophobic (less activity correlated precisely with the presence or absence of the hydrophilic) than GAF itself. This conclusion is based on the fact specific ninhydrin-reactive bands described above in comparisons that the product moves further than GAF in the relatively hydro- of GAF-producing and non-producing strains of P. fluorescens. phobic solvent mobile phase, while GAF exhibits a greater prefer- Further evidence of the identity of GAF and the ninhydrin-reac- ence for the hydrophilic silica gel matrix and moves more slowly tive compound visible on TLC chromatograms prepared from WH6 in the mobile phase. and other GAF-producing strains of P. fluorescens was obtained in Mutational analysis of GAF production by P. fluorescens WH6 studies of GAF degradation. Both GAF activity and the correspond- provided final confirmation of the identification of GAF as the 188 D. Armstrong et al. / Biological Control 51 (2009) 181–190

Fig. 5. Interaction of alanine with varying concentrations of WH6 culture filtrate in the standard GAF bioassay. A germination score of 1.0 indicates high GAF activity (germination completely arrested immediately after emergence of the coleorhiza and plumule). A germination score of 4.0 indicates no GAF activity (germination and seedling development equivalent to that of controls). See Section 2 for details of the bioassay procedure and the scoring system. ninhydrin-reactive compound visible on TLC plates. Transposon of the corresponding ninhydrin-reactive band from the culture fil- mutagenesis of WH6 produced two mutations of WH6 that re- trates of the mutant lines of WH6. Molecular genetic characteriza- sulted in the loss of GAF activity in the WH6 culture filtrates, and tions of the mutations in WH6-2 and WH6-3 are underway and in both cases, the loss of GAF activity was associated with a loss will be reported in a subsequent publication. Preliminary results

Fig. 6. Interaction of leucine with varying concentrations of WH6 culture filtrate in the standard GAF bioassay. A germination score of 1.0 indicates high GAF activity (germination completely arrested immediately after emergence of the coleorhiza and plumule). A germination score of 4.0 indicates no GAF activity (germination and seedling development equivalent to that of controls). See Section 2 for details of the bioassay procedure and the scoring system. D. Armstrong et al. / Biological Control 51 (2009) 181–190 189 indicate that these mutations have occurred at two different sites syringae 22d/93 to selectively inhibit the plant pathogen P. syringae in the WH6 genome (Mills, unpublished), and they should provide pv. glycinea. However, as shown here, arginine did not reverse the a number of opportunities for examining the regulation and bio- herbicidal activity of GAF, and we have not observed any effect of synthesis of GAF. culture filtrates from P. fluorescens WH6 on three strains of P. syrin- The ninhydrin-reactivity of the compound responsible for GAF gae pv. glycinea we have tested (Halgren, unpublished). Therefore, activity is most easily interpreted by assuming that GAF contains it appears unlikely that GAF is identical to this particular amino an amino group. This interpretation, together with our previous acid analog. demonstration (Banowetz et al., 2009) that GAF activity is associ- The novel biological properties we have demonstrated for GAF ated with a low molecular weight compound that contains an acid suggest that this compound may represent a new and potentially group, suggests that GAF may be a small peptide or an amino acid valuable type of herbicide. The very polar character of GAF renders analog. Based on the latter hypothesis, we have investigated the its purification a significant challenge, but the availability of the interactions of GAF with the various protein amino acids to deter- quantitative bioassay for GAF activity developed in our earlier mine if the effects of GAF might be ameliorated by one or more of work, combined with our current demonstration that this activity these compounds. The action of GAF in arresting the germination is associated with a particular ninhydrin-reactive compound, pro- of ABG seeds was found to be counteracted by alanine and gluta- vides encouragement that we will ultimately be able to purify mine and, somewhat less effectively, by several other amino acids. and chemically characterize the active compound. If GAF interferes with normal amino acid functions, the central role of glutamine in nitrogen assimilation in plant systems provides a Acknowledgments ready explanation for why this amino acid would be effective in counteracting the effects of GAF. In the case of alanine, which Support from the USDA CSREES Grass Seed Cropping Systems was the most effective of all the amino acids tested in counteract- for Sustainable Agriculture Special Grant Program is gratefully ing the effects of GAF, this amino acid is known to accumulate to acknowledged. The authors also gratefully acknowledge the excel- relatively high levels in plant tissues exposed to hypoxic conditions lent technical assistance of Donald D. Chen in the extraction and (Good and Crosby, 1989; De Sousa and Sodek, 2003), such as might chromatography of GAF activity. The use of trade, firm, or corpora- be expected to prevail in the fluid filled wells used in our bioassay tion names in this publication is for the information and conve- system or during germination in water-soaked grass seed produc- nience of the reader. Such use does not constitute an official tion fields. Presumably, this amino acid plays a particularly impor- endorsement or approval by the United States Department of Agri- tant role in nitrogen-shuttling under hypoxic conditions via its culture or the Agricultural Research Service of any product or production by transamination of pyruvic acid produced in glycoly- service to the exclusion of others that may be suitable. sis. The ability of these amino acids to counteract GAF inhibition of seed germination is certainly consistent with the hypothesis that References GAF may be affecting some aspect of nitrogen metabolism. Pseudomonads are known to produce a broad spectrum of sec- Banowetz, G.M., Azevedo, M.D., Armstrong, D.J., Halgren, A.B., Mills, D.I., 2008. ondary metabolites, a number of which have been shown to have Germination-Arrest Factor (GAF): biological properties of a novel, naturally- occurring herbicide produced by selected isolates of rhizosphere bacteria. inhibitory effects on higher plant growth or to have antimicrobial Biological Control 46, 380–390. properties (Lesinger and Margraff, 1979; Dowling and O’Gara, Banowetz, G.M., Azevedo, M.D., Armstrong, D.J., Mills, D.I., 2009. Germination- 1994). However, to our knowledge, none of the Pseudomonas iso- Arrest Factor (GAF): part 2. Physical and chemical properties of a novel, naturally-occurring herbicide produced by selected isolates of rhizosphere lates described to date exert the precise spectrum of biological bacteria. Biological Control 50, 103–110. effects exhibited by the GAF-producing P. fluorescens isolates that Bolton, H., Elliott, L.F., Gurusiddaiah, S., Fredrickson, J.K., 1989. Characterization of a we have characterized. The failure of GAF activity to partition into toxin produced by a rhizobacterial Pseudomonas species that inhibits wheat growth. Plant and Soil 114, 279–287. ethyl acetate or other organic solvents immiscible with water Braun, S.D., Vollksch, B., Nuske, J., Spiteller, D., 2008. 3-Methylarginine from (Banowetz et al., 2009) serves to distinguish it from a number of Pseudomonas syringae pv. syringae 22d/93 suppresses the bacterial blight caused secondary metabolites, such as the phenazines or the cyclic lipo- by its close relative Pseudomonas syringae pv. glycinea. ChemBioChem 9, 1913– peptides, isolated from other pseudomonads (Mavrodi et al., 1920. Compeau, G., Al-Achi, B.J., Platsouka, E., Levy, S.B., 1988. 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