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Keywords:

  • aminoethoxyvinylglycine;
  • aminooxyacetic acid;
  • antibiosis;
  • Erwinia amylovora;
  • fire blight;
  • formylaminooxyvinylgycine;
  • germination-arrest factor;
  • Pseudomonas fluorescens WH6

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Aims:  The germination-arrest factor (GAF) produced by Pseudomonas fluorescens WH6, and identified as 4-formylaminooxyvinylglycine, specifically inhibits the germination of a wide range of grassy weeds. This study was undertaken to determine whether GAF has antimicrobial activity in addition to its inhibitory effects on grass seed germination.

Methods and Results:  Culture filtrate from Ps. fluorescens WH6 had little or no effect on 17 species of bacteria grown in Petri dish lawns, but the in vitro growth of Erwinia amylovora, the causal agent of the disease of orchard crops known as fire blight, was strongly inhibited by the filtrate. The anti-Erwinia activity of WH6 culture filtrate was shown to be due to its GAF content, and a commercially available oxyvinylglycine, 4-aminoethoxyvinylglycine (AVG), exhibited anti-Erwinia activity similar to that of GAF. The effects of GAF on Erwinia were reversed by particular amino acids.

Conclusions:  The biological properties of GAF include a rather specific antimicrobial activity against Erw. amylovora. This may be a general property of oxyvinylglycines as AVG exhibited similar activity. The ability of particular amino acids to reverse GAF inhibition is consistent with a potential effect of this compound on the activity of aminotransferases.

Significance and Impact of the Study:  The results presented here demonstrate a novel antimicrobial activity of oxyvinylglycines and suggest that GAF and/or GAF-producing bacteria may have potential for the control of fire blight.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The genus Pseudomonas comprises a group of ubiquitous bacteria that frequently play a role in plant health (Babalola 2010). For example, some of the bacteria in this group act as pathogens (Buddenhagen and Kelman 1964; Hirano and Upper 2000), while others are active in disease suppression (Howell and Stipanovic 1980; Lemanceau and Alabouvette 1991; Weller et al. 2002).

A variety of small molecules are produced by pseudomonads, and a number of these compounds have been isolated and demonstrated to have antimicrobial or herbicidal activity (Haas and Défago 2005; Gross and Loper 2009). Examples of antimicrobial compounds produced by pseudomonads include phenazine derivatives, hydrogen cyanide and cyclic lipopeptides (Handelsman and Stabb 1996; Raaijmakers et al. 2002; Gross et al. 2007). Pseudomonad secondary metabolites reported to have herbicidal activity include the hydrophilic compounds produced by Pseudomonas fluorescens D7 (Gurusiddaiah et al. 1994) and Ps. fluorescens RC-1 (Fredrickson and Elliott 1985; Bolton et al. 1989), which have been shown to inhibit downy brome (Bromus tectorum) and winter wheat (Triticum sativum), respectively. More recently, we isolated and characterized strains of rhizosphere bacteria that produce a compound that arrests germination of the seeds of grassy weeds (Banowetz et al. 2008, 2009; Armstrong et al. 2009). The bacteria that produce this germination-arrest factor (GAF) were identified as particular strains of Ps. fluorescens. GAF irreversibly arrests germination of the seeds of a large number of graminaceous species at a stage immediately after the plumule and coleorhiza have emerged from the seed, while having little effect on established grass seedlings and mature plants. For the most part, the seeds of dicot plant species proved to be less sensitive to GAF than those of graminaceous species. We have identified the compound responsible for GAF activity as 4-formylaminooxyvinylglycine (S-2-amino-4-formylaminooxy-trans-3-butenoic acid) (McPhail et al. 2010). This compound was purified from culture filtrates of Ps. fluorescens WH6, and it was also shown to be responsible for the GAF activity associated with a number of other isolates we have examined (McPhail et al. 2010).

GAF is a new addition to the small class of naturally occurring compounds known as oxyvinylglycines. Only a few such compounds have been described previously. The oxyvinylglycine known as rhizobitoxine [4-(2-amino-3-hyroxypropoxy)vinylglycine] was isolated from the bacterium originally called Rhizobium japonicum, now Bradyrhizobium elkanii (Owens et al. 1972). Methoxyvinylglycine was isolated from Pseudomonas aeruginosa ATCC-7700 (Scannell et al. 1972), and aminoethoxyvinylglycine (AVG) was isolated from Streptomyces sp. X11,085 (Pruess et al. 1974). Oxyvinylglycines are known to block reactions catalysed by enzymes that are dependent upon pyridoxal phosphate as a cofactor (Berkowitz et al. 2006). These enzymes include aminotransferases that perform vital functions in nitrogen metabolism (Eliot and Kirsch 2004). In plants, the enzyme aminocyclopropanecarboxylic acid synthase (ACC synthase), which catalyses a crucial step in the biosynthesis of the plant hormone ethylene, also requires pyridoxal phosphate and is sensitive to the inhibition by oxyvinylglycines (Amrhein and Wenker 1979; Yu et al. 1979).

We were interested in determining whether GAF, in addition to its inhibitory effects on grass seed germination, might also exhibit some type of antimicrobial activity. This study was motivated in part by the potential practical utility of any such activity for the biocontrol of bacterial plant pathogens, but the identification of a bacterial strain sensitive to GAF was also anticipated to facilitate our molecular genetic investigations of the regulation of GAF production. The results of our tests of the antimicrobial properties of GAF and GAF-producing bacteria are reported here. These results are compared with the results of similar tests with AVG (a commercially available oxyvinylglycine) and with 2-aminooxyacetic acid (AOA), a compound that is also reported to inhibit pyridoxal phosphate-dependent enzyme reactions.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Bacterial cultures

The origins of the five GAF-producing strains of Ps. fluorescens (AD31, AH4, E34, WH6 and WH19) used here have been described previously (Elliott et al. 1998; Banowetz et al. 2008). WH6 has been used as our type strain in the studies of GAF production. Non-GAF-producing mutants of WH6 (WH6-2::Tn5 and WH6-3::Tn5) were obtained in a previous study (Armstrong et al. 2009). The genomic sites of these mutations have been identified (Kimbrel et al. 2010). Sources of all other bacterial species and strains used in this study are indicated in Table 1.

Table 1.   Bacterial species tested for response to Pseudomonas fluorescens WH6 culture filtrate
Test speciesStrains/pathovarsSource of bacteria (footnote reference)
  1. 1Dr Joyce Loper (USDA-ARS, Horticultural Crops Research Laboratory, Corvallis, OR, USA).

  2. 2Dr Virginia Stockwell (Department of Botany & Plant Pathology, Oregon State University, Corvallis, OR, USA).

  3. 3Marilyn Miller (Plant Clinic, Department of Botany & Plant Pathology, Oregon State University, Corvallis, OR, USA).

  4. 4Dr Walt Ream (Department of Microbiology, Oregon State University, Corvallis, OR, USA).

  5. 5Dr Jeff Chang (Department of Botany & Plant Pathology, Oregon State University, Corvallis, OR, USA).

  6. 6Dr Mark Silby (Department of Molecular Biology & Microbiology, Tufts University School of Medicine, Boston, MA, USA).

  7. 7Dr Ann Kennedy (USDA-ARS Land Management/Water Conservation Unit, Pullman, WA, USA).

Bacteria sensitive to WH6 culture filtrate
 Bacillus megateriumKm1
 Erwinia amylovora153, LA530, LA512, LA475, JL11961, 2, 2, 2, 2
 Pantoea agglomeransC9-11
Bacteria resistant to WH6 culture filtrate
 Agrobacterium radiobacterK1026bv23
 Agrobacterium tumefaciensA348, C58bv1, B49c/83bv24, 3, 3
 Agrobacterium vitisIL20bv33
 Bradyrhizobium japonicumUSDA1105
 Curtobacterium flaccumfaciensNE213
 Escherichia coliDH5αCommercial source
 P. agglomerans (syn. Erwinia herbicola)EH2521
 Pectobacterium carotovorum (syn. Erwinia carotovora)cc1011
 Pseudomonas agariciATCC259413
 Ps. fluorescensPfO-1, Pf-5, A506, D76, 1, 1, 7
 Pseudomonas marginalisPM-73
 Pseudomonas putidaNIR1
 Pseudomonas syringaeglycinea race 0, 4, 5;5
phaseolicola 1448A,5
syringae PSS61, syringae B728a1, 5
tomato DC30005
 Rhodococcus fasciansA3b, A44a rif, 02-815, A25 rif3, 3, 3, 3
 Stenotrophomonas maltophiliaRM1453
 Xanthomonas campestrisATCC33919, KXCC11, 1

All strains used in this study were maintained at −80°C in Luria–Bertani liquid medium (LB medium) (Sambrook and Russell 2001) with a final concentration of 15% (v/v) glycerol. For short-term use, strains were transferred onto LB medium supplemented with 1·5% agar, incubated at 28°C for 16 h, and maintained at 4°C.

Chemicals

4-Aminoethoxyvinylglycine (AVG) (Sigma-Aldrich 359629; Sigma-Aldrich, St Louis, MO) in the hydrochloride form and AOA (Sigma C13408) were purchased from the indicated commercial source. Synthesis of S-2-amino-4-aminoethoxybutyric acid (dihydroAVG) was accomplished by catalytic hydrogenation of commercial AVG-hydrochloride (12 mg, 60·7 μmol) using excess hydrogen gas over palladium on carbon (3 mg) and purified on a cation exchange resin (AG50-WX4) following the method of Scannell et al. (1976).

Culture filtrate production

Pseudomonas fluorescens cells were inoculated into the modified Pseudomonas minimal salts medium (PMS medium) described by Banowetz et al. (2008) and cultured and harvested as described in the same reference. To prepare culture filtrates, the Pseudomonas culture fluid recovered from 7-day cultures was centrifuged (3000 g, 15 min), and the supernatant was passed through a bacteriological filter (Millipore GP Express Steritop, 0·22 μm pore size; Millipore, Billerica, MA). The resulting sterile culture filtrate was stored at 4°C.

Testing for antimicrobial activity

To test bacteria for their response to GAF, each strain was grown overnight in 4 ml LB medium at 28°C (except Escherichia coli, which was grown at 37°C) with shaking (200 rev min−1). The following morning, the stationary phase bacterial suspensions were adjusted with sterile water to an optical density of 0·2 at 600 nm (or 0·8 in the case of E. coli) as measured using a Spectronic 20 spectrophotometer (Milton Roy Co., Rochester, NY, USA). Three hundred microlitres of suspension was spread onto the surface of a ‘925 Minimal Medium’ plate (100 × 15 mm, containing 25 ml of medium). The medium was made as described by Langley and Kado (1972), with the following exceptions: After the pH was adjusted to 6·8, the solution was supplemented with agar (15 g l−1) and autoclaved. After cooling, each litre of medium was amended with 20 ml of filter-sterilized 10% (w/v) glucose solution, 1 ml of filter-sterilized 100 mmol l−1 ferric chloride, and 10 ml of a filter-sterilized vitamin stock containing 3·37 g l−1 thiamine HCl and 1·23 g l−1 nicotinic acid. The appropriate bacterial suspension was spread onto the agar surface, and the plate was dried briefly in a sterile hood. Central wells were punched with a no. 9 cork borer, and 300 μl of test filtrate was dispensed into the wells. The plates were incubated for 48 h at 28°C and then examined and scored. Zones of inhibition in the area surrounding the well containing WH6 filtrate were quantified using AbleImage Analyzer® software (MU Labs, Ljublijana, Slovenia). A similar procedure was followed in comparing the anti-Erwinia effects of GAF-producing and non-producing strains of Pseudomonas and for examining the effects of culture filtrates from mutant strains of Ps. fluorescens WH6. All quantitative data reported for these and the other tests described below were replicated in two experiments using three replicate plates per experiment.

To test the antimicrobial activities of AVG, AOA and dihydroAVG, solutions of these test compounds were adjusted to pH 6·5, filter-sterilized and 300 μl of the indicated test concentrations were added to the central well of plates spread with lawns of the appropriate bacteria as described earlier.

For direct antibiosis tests with colonies of Ps. fluorescens WH6 and WH6 mutants, live bacteria were taken from a fresh plate of WH6 or WH6 mutant strains and spotted onto a plate freshly spread with an Erwinia lawn as described earlier. Plates were incubated at 28°C and examined for antibiosis 48 h after sample application.

Thin-layer chromatographic (TLC) analysis of anti-Erwinia activity in extracts of Pseudomonas fluorescens WH6 culture filtrate

WH6 culture filtrate was taken to dryness in vacuo at a temperature ≤45°C. The dry solids recovered in this manner were extracted with 90% (v/v) ethanol as described previously (Armstrong et al. 2009). The resulting extract was taken to dryness as described earlier, and the recovered solids were dissolved in a volume of 76% (v/v) ethanol equal to one-twentieth of the original volume of culture filtrate (20× concentration).

Aliquots (200 μl) of the 20× extract solution were applied to Avicel Microcrystalline Cellulose TLC Plates (5 × 20 cm, 250-μm layer, glass-backed with an inorganic binder), purchased from Analtech Inc. (Newark, DE, USA). The chromatograms were developed over a distance of 12 cm with a chromatographic solvent consisting of ethyl acetate : isopropanol : water (7·5 : 15 : 10). The developed chromatograms were allowed to air dry for 30 min prior to further processing.

For analysis of the distribution of anti-Erwinia activity on TLC plates, a developed chromatogram was divided into 1-cm bands located between the origin and solvent front. The cellulose from each zone (1 × 5 cm) was scraped into 2·0-ml minifuge tubes, suspended and vortexed in 1·33 ml of water. The cellulose was pelleted by centrifugation (minifuge, 10 000 rev min−1, 10 min). The supernatant from each tube was filter-sterilized (0·2-μm filter), diluted to a volume equivalent to that of the original culture filtrate and tested in the Erwinia bioassay by the addition of 300 μl of the solution to the central well of an Erwinia amylovora lawn prepared as described earlier.

The location of GAF on the developed plates was determined by ninhydrin staining of a control plate developed under the same conditions. The dry chromatogram was sprayed with a ninhydrin solution consisting of 0·25% (w/v) ninhydrin dissolved in 95% (v/v) ethanol containing 3·0 ml of glacial acetic acid per 100 ml of solution. Colour development was achieved by heating the sprayed chromatogram in an oven at 80–90°C for 15 min.

Amino acid effects on the anti-Erwinia activity of WH6 culture filtrate

The 20 proteinogenic amino acids were tested for their capacity to antagonize the antimicrobial activity of GAF for Erw. amylovora. Each amino acid was dissolved in distilled water at a concentration of 30 mmol l−1. The pH was adjusted to between 6·4 and 6·8, and the solution was sterilized through a 0·2-μm filter. Tyrosine was tested at 3 mmol l−1 owing to low solubility. Erwinia amylovora was spread on the surface of a 925 Minimal Medium plate (as described earlier). One central well and two adjacent wells spaced equidistant from the central well were punched in the inoculated Erwinia lawn with a sterile no. 9 cork borer. Three hundred microlitres of GAF-containing WH6 filtrate was dispensed into the central well, and 300 μl of amino acid solution was dispensed into both the left and right adjacent wells. A plate with sterile water in place of the amino acid was used as a control. Plates were incubated at 28°C and examined for the effects on the zone of inhibition 48 h after sample application.

A subset of amino acids (alanine, glutamine, serine and leucine) was tested more extensively over a range of concentrations (3–30 mmol l−1) in combination with WH6 filtrate. In these assays, varying concentrations of the test amino acid were added directly to WH6 culture filtrate, and the resulting solutions were filter-sterilized and transferred to the central well cut in Erwinia lawns prepared as described earlier.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Tests of Pseudomonas fluorescens WH6 culture filtrate for antimicrobial activity

GAF itself is not available in either purified or synthetic form in amounts adequate for extensive biological testing. Therefore, the antimicrobial activity of Ps. fluorescens WH6 culture filtrate was tested against 18 different species of bacteria, including multiple strains, pathovars and races of some of these species. Fifteen of the 18 species tested exhibited little if any response to the culture filtrate in tests where the filtrate was added to a central well cut in a recently spread bacterial lawn (Table 1). Representative assays for the three bacterial species showing a response to the WH6 culture filtrate are illustrated in Fig. 1. Of these, only Erw. amylovora exhibited a large, clear zone of inhibition in the bacterial lawn surrounding the central well containing the culture filtrate. Much smaller zones of inhibition were evident in the lawns of Bacillus megaterium and one strain (C9-1) of Pantoea agglomerans (formerly Erwinia herbicola), although another strain of P. agglomerans (EH252) did not give a response. The results with Erw. amylovora shown in Fig. 1 were obtained with Strain 153, but the other Erw. amylovora strains listed in Table 1, including two strains from Oregon (LA512 and LA474) and two from Washington (LA530 and JL1196), gave similar results. It may be worth noting that small zones of somewhat reduced lawn density (as opposed to clear zones of inhibition) were occasionally observed in the lawns of Pectobacterium carotovorum (formerly Erwinia carotovora) and E. coli DH5α.

image

Figure 1.  Effects of 7-day culture filtrate from Pseudomonas fluorescens WH6 on selected bacterial species. The response of lawns of Erwinia amylovora 153, Bacillus megaterium Km and Pantoea agglomerans C9-1 to WH6 culture filtrate (contained in the central well). Plates are shown after 48-h incubation at 28°C.

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Demonstration that the anti-Erwinia effects of WH6 culture filtrate are owing to GAF

To investigate whether the anti-Erwinia activity of WH6 culture filtrate was attributed to GAF or some other component of the culture filtrate, a 90% ethanol extract of the solids from dried WH6 culture filtrate was fractionated by TLC, and the distribution of anti-Erwinia activity in various zones of the chromatogram was determined by extraction and bioassay as described in Materials and Methods. As shown in Fig. 2a, anti-Erwinia activity was recovered as a single broad band of activity that was coincident with the position of the ninhydrin-staining band (Fig. 2b) previously shown to correspond to GAF (Armstrong et al. 2009).

image

Figure 2.  Comparison of the distribution of antimicrobial activity against Erwinia amylovora and ninhydrin staining on TLC chromatograms after fractionation of extracts of Pseudomonas fluorescens WH6 culture filtrate. Extracts (90% ethanol) of the solids recovered from evaporation of WH6 culture filtrate were prepared and chromatographed on cellulose TLC plates as described in Materials and Methods. (a) Distribution of antimicrobial activity recovered by aqueous extraction of the various zones from one TLC plate. (b) Distribution of ninhydrin staining in a replicate TLC chromatogram, showing the position of germination-arrest factor on the chromatogram. TLC, thin-layer chromatography.

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Further evidence that the anti-Erwinia activity of WH6 culture filtrate was associated with GAF was obtained by comparing the biological activities of culture filtrates from four additional GAF-producing Ps. fluorescens isolates with filtrates from four Ps. fluorescens strains previously shown to lack the ability to produce GAF (Armstrong et al. 2009). As shown in Table 2, culture filtrates from all the Ps. fluorescens strains that produce GAF, including WH6, produced zones of inhibition in Erw. amylovora lawns when tested in the Erwinia bioassay. Conversely, filtrates from Ps. fluorescens strains Pf-5, PfO-1, D7 and A506 that had been shown previously to lack GAF activity (Armstrong et al. 2009) failed to generate zones of inhibition against Erw. amylovora in this assay. It should be noted that in other assays, culture filtrates from the non-GAF-producing strains Ps. fluorescens D7 and A506 did occasionally produce very small zones of inhibition against Erw. amylovora, but these effects must be due to compounds other than GAF.

Table 2.   Effects on Erwinia amylovora of culture filtrate from selected strains of Pseudomonas fluorescens*
Ps. fluorescensArea of zone of inhibition† in Erw. amylovora 153 lawn
(cm2 ± standard error of the mean)
Isolates and strainsExperiment 1Experiment 2
  1. GAF, germination-arrest factor.

  2. *To test the anti-Erwinia activity of culture filtrates from the indicated strains of Ps. fluorescens, plates were spread with a lawn of Erw. amylovora, and the culture filtrate to be tested was placed in a central well cut in the agar immediately after spreading the bacterial lawn.

  3. †Areas of zones of inhibition were measured after 48-h incubation at 28°C. All data for each experiment are the average of three replicate plates.

GAF-producing isolates
 AD3113·8 ± 0·1314·1 ± 0·10
 AH415·6 ± 0·2716·5 ± 0·04
 E3415·9 ± 0·4316·2 ± 0·23
 WH1915·8 ± 0·5115·7 ± 0·36
 WH614·7 ± 0·5115·5 ± 0·32
Non-GAF-producing strains
 A506, D7, PfO-1, Pf-50·00·0
Mutants of WH6
 WH6-2::Tn50·00·0
 WH6-3::Tn50·00·0

Final confirmation that the anti-Erwinia activity of WH6 culture filtrate is owing to GAF itself, rather than some other component of the filtrate, was obtained in tests of culture filtrates from mutant lines of WH6. We have previously described the production and selection of two mutants of WH6 that have lost the ability to produce GAF (Armstrong et al. 2009; Kimbrel et al. 2010). Culture filtrates from these mutants (WH6-2::Tn5 and WH6-3::Tn5) no longer arrest the germination of Poa annua or exhibit the ninhydrin-reactive band corresponding to GAF in TLC analysis. Neither of the culture filtrates from these mutants produced a zone of inhibition in Erw. amylovora lawns (Table 2). Thus, mutagenic loss of GAF activity resulted in the concurrent loss of anti-Erwinia activity in the corresponding culture filtrates. Similarly, when colonies of WH6 and the two mutants, WH6-2::Tn5 and WH6-3::Tn5, were spotted on Petri plates inoculated with lawns of Erw. amylovora, a zone of inhibition was clearly evident around the colonies of WH6, and no zones were evident in the areas surrounding the two mutant colonies.

Antimicrobial activity of compounds structurally or functionally related to GAF

We have as yet been unable to synthesize or purify sufficient quantities of GAF to permit rigorous testing of the absolute activity of this compound. However, another oxyvinylglycine, AVG (S-2-amino-4-aminoethoxybut-2-enoic acid), is available commercially, as is the compound AOA, which is reported to inhibit pyridoxal phosphate-dependent enzyme reactions in a manner similar to the mode of action of oxyvinylglycines (Amrhein and Wenker 1979; Yu et al. 1979). The effects of these compounds on the growth of Erw. amylovora are compared in Table 3. Both compounds inhibited the growth of Erw. amylovora, but on a molar basis, AVG was considerably more active than AOA. As shown in Table 3, reduction of the vinyl double bond in AVG to produce S-2-amino-4-aminoethoxybutyric acid (dihydroAVG) resulted in elimination of the anti-Erwinia activity associated with AVG. In the case of AOA, removal of the amino group to give 2-methoxyacetic acid resulted in the complete loss of anti-Erwinia activity, as did replacement of the oxygen bridge in AOA with a carbon atom to form β-alanine. Both 2-methoxyacetic acid and β-alanine also differed from AOA in their failure to exhibit any GAF-like herbicidal activity in tests of the effects of these compounds on grass seed germination (data not shown).

Table 3.   Effects of structural and functional analogues of germination-arrest factor on Erwinia amylovora*
Test compoundArea of zone of inhibition† in Erw. amylovora 153 lawn at indicated concentration of test compound
(cm2 ± standard error of the mean)
0 mmol l−10·3 mmol l−11·0 mmol l−13·0 mmol l−1
  1. *Test plates were spread with a lawn of Erw. amylovora, and solutions of the test compounds at the indicated concentrations were placed in central well cut in the agar immediately after spreading the bacterial lawn.

  2. †Areas of zones of inhibition were measured after 48-h incubation at 28°C. All data for each experiment are the average of three replicate plates.

4-Aminoethoxyvinylglycine (AVG)
 Experiment 10·020·9 ± 0·1029·6 ± 0·1337·3 ± 0·60
 Experiment 2 24·9 ± 0·9633·8 ± 0·4940·5 ± 0·40
2-Amino-4-aminoethoxybutyric acid (dihydroAVG)
 Experiment 10·0
 Experiment 20·0
2-Aminooxyacetic acid (AOA)
 Experiment 10·00·08·2 ± 0·2315·7 ± 0·55
 Experiment 20·00·08·3 ± 0·1015·7 ± 0·21
2-Methoxyacetic acid
 Experiment 10·0
 Experiment 20·0
β-Alanine (3-aminopropanoic acid)
 Experiment 10·0
 Experiment 20·0

The antimicrobial activities of AVG and AOA relative to other bacterial species resembled that of GAF in the sense that most of the species tested were insensitive to either compound (Table 4). However, AVG was particularly effective in inhibiting the growth of B. megaterium, and AOA (in contrast to GAF and to AVG) gave large, clear zones of inhibition with E. coli DH5α but had little if any effect on B. megaterium.

Table 4.   Effects of aminoethoxyvinylglycine (AVG) and aminooxyacetic acid (AOA) on selected bacterial species*
Bacterial speciesArea of zone of inhibition† induced in a lawn of the indicated bacteria
(cm2 ± standard error of the mean)
AVG (3 mmol l−1)AOA (3 mmol l−1)
  1. *Test plates were spread with a lawn of the indicated bacterial species, and solutions of the test compounds were placed in central well cut in the agar immediately after spreading the bacterial lawn.

  2. †Areas of zones of inhibition were measured after 48-h incubation at 28°C. All data for each experiment are the average of three replicate plates.

Bacillus megaterium Km
 Experiment 130·8 ± 0·300·0
 Experiment 227·9 ± 0·400·0
Escherichia coli DH5α
 Experiment 10·013·7 ± 0·32
 Experiment 20·013·6 ± 0·19
Bacteria exhibiting no response to either test compound
 Agrobacterium tumefaciens C58Pseudomonas fluorescens WH6
 Pantoea agglomerans EH252Pseudomonas syringae pvr Syringae PSS61
 Pectobacterium carotovora cc 101Xanthomonas hortorum ATCC 33919
 Ps. fluorescens WH6 

Amino acid reversal of the inhibition of Erwinia amylovora by WH6 culture filtrate

Our earlier work (Armstrong et al. 2009) demonstrated that the ability of GAF to arrest the germination of annual bluegrass seeds could be reversed by specific amino acids. To determine whether amino acids also altered the response of Erw. amylovora to GAF, the 20 proteinogenic amino acids were screened for their ability to reverse the inhibitory effects of WH6 culture filtrate on Erw. amylovora. For this purpose, solutions of the amino acid to be tested in the Erwinia bioassay were distributed to wells placed on either side of the central well containing WH6 culture filtrate. Any resulting effect on the shape of the zone of inhibition around the central well in the Erwinia lawn was taken as evidence of interaction between the amino acid and the culture filtrate. As shown in Fig. 3, seven of the amino acids tested in this manner (alanine, asparagine, cysteine, glutamine, glycine, histidine and serine) produced strong lateral compression of the central zone of inhibition, indicating that these amino acids were reversing the inhibitory effects of GAF on the growth of Erwinia. In fact, in the case of serine, the zone of inhibition was completely overgrown by Erwinia. Leucine, which is included in Fig. 3 at the lower right in the panel, is illustrative of the 13 other amino acids that had little or no effect in compressing the zone of inhibition, indicating that they had little effect on the anti-Erwinia activity of the WH6 culture filtrate.

image

Figure 3.  Amino acid effects on the response of Erwinia amylovora to Pseudomonas fluorescens WH6 culture filtrate. Test plates were spread with lawns of Erw. amylovora. Culture filtrate from Ps. fluorescens WH6 was placed in the central well cut in the plate agar, and 30 mmol l−1 solutions of the indicated amino acid to be tested were prepared and distributed to the side wells. Results for eight amino acids, seven of which exhibited evidence of counteracting the effects of germination-arrest factor, are shown after 48-h incubation at 28°C.

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The ability of selected amino acids to reverse the inhibitory activity of WH6 culture filtrate on the growth of Erwinia lawns was examined in more detail by direct addition of representative amino acids to the wells containing the culture filtrate. The effects of varying concentrations of alanine, serine and glutamine in reducing the size of the zone of inhibition induced by the culture filtrate are illustrated in Fig. 4. Leucine, which showed little or no interaction with the culture filtrate in the tests illustrated in Fig. 3, is included for comparison. As expected, increasing concentrations of the first three amino acids resulted in the reduction in size and elimination of the zone of inhibition, while varying concentrations of leucine had little effect on the size of the zone.

image

Figure 4.  Quantitative tests of the interactions of selected amino acids with Pseudomonas fluorescens WH6 culture filtrate. Test plates were spread with lawns of Erwinia amylovora. The indicated concentrations of amino acids were dissolved in WH6 culture filtrate, and the resulting solutions distributed to central well cut in the agar plates. Areas of zones of inhibition were measured after 48-h incubation at 28°C. All data are the average of two experiments using three replicate plates per experiment. (inline image) alanine; (inline image) glutamine; (inline image) leucine and (inline image) serine. Standard errors of the mean were small and are obscured by the data points.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Culture filtrate from Ps. fluorescens WH6, a bacterial isolate that produces and secretes the naturally occurring herbicide GAF, previously identified as 4-formylaminooxyvinylglycine (McPhail et al. 2010), has been shown in the present study to rather specifically inhibit the bacterial plant pathogen Erw. amylovora. Although pure GAF was not available for testing, the evidence presented here indicates that the anti-Erwinia activity of WH6 culture filtrate is owing to its GAF content. During TLC fractionation of extracts of WH6 culture filtrate, the anti-Erwinia activity in the filtrate migrated with the ninhydrin-reactive compound previously shown to correspond to GAF. Moreover, filtrate from four other Ps. fluorescens strains that had been previously shown to produce GAF also exhibited antimicrobial activity against Erw. amylovora, while antimicrobial activity was completely absent or very nearly so in culture filtrates prepared from four Ps. fluorescens strains that lack the ability to produce GAF (strains Pf-5, PfO-1, D7 and A506). Tests of two mutants of Ps. fluorescens WH6 provided additional confirmation that GAF is the component of WH6 culture filtrate responsible for the anti-Erwinia activity of the filtrate. These two mutants are deficient in GAF production, as measured by both the GAF bioassay for herbicidal activity and by TLC analysis of their culture filtrates (Armstrong et al. 2009), and both failed to produce a zone of inhibition in lawns of Erw. amylovora. Thus, the antimicrobial activity exhibited by WH6 culture filtrate may be directly attributed to GAF. This finding identifies a new biological property of GAF, in addition to its herbicidal activity in irreversibly arresting the germination of the seeds of a wide variety of graminaceous species.

The antimicrobial activity of WH6 and other GAF-producing pseudomonads appears to be very selective. WH6 culture filtrate produced small zones of inhibition in lawns of B. megaterium and in lawns of one of two strains of P. agglomerans, but among the bacteria tested, the response of Erw. amylovora was unique in its consistency and scale. No Pseudomonas species or any of the other bacterial species tested exhibited any response to being challenged with the WH6 culture filtrate, other than an occasional and variable reduction in lawn density observed with E. coli and Pectobacterium carotovorum. Such selectivity may potentially prove useful in a biological setting, where precise targeting of the pathogen and preservation of beneficial microbes are critical.

The ability of GAF to inhibit Erw. amylovora may be a general property of oxyvinylglycines. Commercial preparations of the oxyvinylglycine AVG exhibited relatively specific anti-Erwinia activity similar to that of GAF in the work reported here. The simple compound AOA, which shares with oxyvinylglycines an ability to inhibit pyridoxal phosphate-dependent enzyme reactions, and exhibited weak herbicidal activity analogous to that of GAF in our tests (McPhail et al. 2010), also inhibited the growth of Erw. amylovora in our test systems. As in tests of its herbicidal activity, this compound was considerably less active on a molar basis than AVG. In addition, there were interesting, if subtle, differences in the biological specificity of these compounds in relation to GAF and each other. For example, at the concentrations tested, AVG strongly inhibited B. megaterium, but AOA had little if any effect on this organism while strongly inhibiting E. coli DH5α, which was unaffected by AVG. The chemistry of oxyvinylglycines has proven difficult, and the structure/activity relationships of these compounds have yet to be explored in any detail, but we report here that reduction of the vinyl double bond in AVG appeared to eliminate anti-Erwinia activity, and we have obtained similar results in tests of this compound for the corresponding herbicidal activity.

Oxyvinylglycines are known to inhibit pyridoxal phosphate-dependent enzyme reactions (Eliot and Kirsch 2004), including those catalysed by aminotransferases. Thus, the latter are potential targets for inhibition by GAF. In agreement with this hypothesis, our previous work has shown that particular amino acids are capable of reversing the inhibitory effects of GAF on the germination of seeds of annual bluegrass in our standard GAF bioassay (Armstrong et al. 2009). The action of GAF in arresting the germination of annual bluegrass seed was reversed by alanine and glutamine in particular and less effectively by several other amino acids. In the present study, we have shown that 7 of 20 amino acids tested caused a strong reversal of the anti-Erwinia effects of WH6 culture filtrate. Although the amino acids effective in reversing the effects of GAF on the growth of Erw. amylovora did not precisely mirror those effective in counteracting the herbicidal activity of GAF, both glutamine and alanine were effective in both assay systems. The ability of selected amino acids to reverse the inhibitory effects of GAF in both seed germination and Erw. amylovora growth is supportive of the idea that GAF may be exerting its effects by interfering with the aminotransferases that are important in nitrogen metabolism. Moreover, the effects of particular amino acids on the expression of GAF activity need to be taken into account in any attempt to test for GAF or to grow and utilize WH6 for biocontrol purposes.

Pseudomonas fluorescens WH6 and other GAF-producing pseudomonads with the ability to antagonize the growth of Erw. amylovora may have potential as biocontrol agents for the suppression of fire blight in orchard crops. Several other bacteria have already been tested or employed for this purpose. Among these, the antimicrobial properties of P. agglomerans (syn. Erw. herbicola) are probably the best characterized, as this organism has been shown to produce antibiotics effective against Erw. amylovora in vitro (Vanneste et al. 1992; Wodzinski et al. 1994). Several of the characterized strains of P. agglomerans produce a single antibiotic, while others produce multiple antibiotics (Ishimaru et al. 1988; Wright et al. 2001). Pantoea agglomerans strain E325 is marketed as a biocontrol agent for fire blight under the name Bloomtime Biological®, while Ps. fluorescens A506, first isolated from a California pear (Lindow 1984), is also marketed as a biocontrol agent for fire blight, using the name BlightBan®. A506 appears to act by competing for limited growth resources on the blossom entry sites of the fire blight pathogen (Johnson and Stockwell 1998). As mentioned earlier, this strain has been tested previously by us and shown not to produce GAF (Armstrong et al. 2009). The Gram-positive bacteria B. megaterium (Jock et al. 2002), B. subtilis (El-Goorani and Hassanein 1991) and B. cereus (Emmert and Handelsman 1999) also have been shown to antagonize Erw. amylovora, the latter via production of two antibiotics (Emmert and Handelsman 1999). Because the anti-Erwinia activity of our GAF-producing Ps. fluorescens isolates appears to be due to a different mechanism than any previously described activity, it is possible that our isolates may offer potential for synergistic interactions with other biocontrol agents in an integrated approach to the biocontrol of fire blight.

Most of the antibiotics currently marketed for the control of plant pathogens have originated from natural products isolated from micro-organisms, but owing to the emergence of antibiotic-resistant pathogens, there is a continuing need for the development of new antibiotic classes. For example, streptomycin was formerly an effective control for the management of fire blight, but the emergence of pathogenic strains resistant to the antibiotic has compromised its effectiveness (Moller et al. 1981; Loper et al. 1991). The only other antibiotic registered in the United States for control of fire blight, oxytetracycline, is registered for use only on pear and is considerably less effective than streptomycin at suppressing Erw. amylovora (McManus and Jones 1994). In this context, given the apparent effectiveness of the oxyvinylglycine AVG in inhibiting the in vitro growth of Erw. amylovora, it may be worth noting that a crude preparation of AVG is marketed commercially as a plant growth regulator under the name ReTain®. ReTain®, which contains 15% AVG, is produced by fermentation processes and used to delay fruit ripening in orchard crops. The growth regulatory properties of this preparation are based on the ability of AVG to inhibit the biosynthesis of ethylene through its effect on the enzyme ACC synthase. It appears likely that the growth regulatory properties of ReTain®, as well as its expense, would complicate any attempt to utilize this preparation as a disease control agent, but we have tested this growth regulator preparation and found it to be effective in the in vitro inhibition of the growth of Erw. amylovora (data not shown).

The selective antimicrobial activity of GAF demonstrated in this study provides evidence of both a new property of this compound and a potential innovative means of controlling Erw. amylovora, either through biocontrol or by the development of effective GAF analogues that can be produced by chemical synthesis. Future field experiments with GAF-producing bacteria may address whether the observed in vitro inhibition of Erw. amylovora can be translated to the suppression of fire blight disease on blossoms. More immediately, the response of Erw. amylovora to GAF and GAF-producing organisms is expected to find utility in our research as a fast and easy screen for mutant GAF phenotypes.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Support from the USDA-CSREES Grass Seed Cropping Systems for Sustainable Agriculture Special Grant Program and from the OSU Agricultural Research Foundation is gratefully acknowledged. The use of trade, firm or corporation names in this publication is for the information and convenience of the reader. Such use does not constitute an official endorsement or approval by the United States Department of Agriculture or the Agricultural Research Service of any product or service to the exclusion of others that may be suitable.

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  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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