Germination arrest factor (GAF): Part 2. Physical and chemical properties of a novel, naturally occurring herbicide produced by Pseudomonas fluorescens strain WH6

Biological Control 50 (2009) 103–110

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Germination arrest factor (GAF): Part 2. Physical and chemical properties of a novel, naturally occurring herbicide produced by Pseudomonas fluorescens strain WH6 q Gary M. Banowetz a,*, Mark D. Azevedo a, Donald J. Armstrong b, Dallice I. Mills b a b

USDA/ARS, 3450 S.W. Campus Way, Forage Seed, Corvallis, OR 97331, USA Department of Botany and Plant Pathology, Oregon State University, Corvallis, OR 97331, USA

a r t i c l e

i n f o

Article history: Received 19 August 2008 Accepted 24 March 2009 Available online 31 March 2009 Keywords: Annual bluegrass Biocontrol Herbicide Rhizobacteria Poa annua Pseudomonas fluorescens

a b s t r a c t Pseudomonas fluorescens isolate WH6 and several related isolates have been shown previously to produce and secrete a novel, naturally occurring herbicide that arrests germination of the seeds of a large number of grassy weed species. The physical and chemical characteristics of this Germination Arrest Factor (GAF) have been investigated in the present study. GAF was insoluble in all organic solvents tested with the exception of methanol, in which it was moderately soluble. However, appropriate concentrations of aqueous ethanol solutions could be used to extract GAF from dried WH6 culture filtrates. GAF activity was destroyed by heating at temperatures in excess of 65 °C, but no obvious loss of activity was observed after exposure for several hours at room temperature to either acid or alkaline conditions within the pH range 2–12. GAF activity in the culture filtrate gradually declined during prolonged storage at 4 °C. Ultrafiltration and gel filtration studies indicated that GAF activity was associated with a compound or compounds having a molecular weight less than 1000. As expected from its solubility properties, GAF activity did not bind to reverse-phase materials (e.g., silica-C18 cartridges). The very hydrophilic character of the GAF molecule suggests that it does not contain an aromatic ring structure. GAF was retained on an anion exchange column, indicating that the active molecule must contain an acid group. Published by Elsevier Inc.

1. Introduction The presence of grassy weeds like annual bluegrass (ABG, Poa annua L.) in grass seed crops decreases seed purity and impacts the suitability of the crop for many export markets (Barnes et al., 1995). Annual bluegrass also is a serious weed in professional and urban turfs where it is difficult to control because turf grasses are susceptible to the herbicides that are used to control the weed (Mueller-Warrant et al., 1995). The number of herbicides registered for use in specialty crops like grass seed production has diminished in recent years. The cost of developing new products is great (Ivany et al., 2002), but new and more effective agents for the control of grassy weeds are needed in grass seed production systems and in a variety of turf management settings. Microbes that produce naturally occurring herbicides represent an alternative to synthetic formulations and have potential to control weeds in a wide variety of crops (Elliott and Lynch, 1985; Schippers et al., 1987; Kremer et al., 1990; Kremer and Kennedy, 1996; Kremer, 2000; Kennedy et al., 2001; Li et al., 2003; FloresVargas and O’Hara, 2006; Kennedy and Stubbs, 2007). Deleterious rhizobacteria (DRB), usually Pseudomonas sp., which retard or inhiq

The first paper in this series was Banowetz et al. (2008). * Corresponding author. Fax +1 541 738 4127. E-mail address: [email protected] (G.M. Banowetz).

1049-9644/$ - see front matter Published by Elsevier Inc. doi:10.1016/j.biocontrol.2009.03.011

bit the growth or development of weeds have been identified, although their effectiveness in controlling ABG is not well demonstrated (Suslow and Schroth, 1982; Cherrington and Elliott, 1987; Boyetchko, 1997). Nevertheless, a particular strain (JT-P482) of Xanthomonas campestris pv poae is registered in Japan for the control of ABG in golf turfs (Imaizumi et al., 1997; Tateno, 2000). This bioherbicide is applied to cut grass in the form of viable bacteria, which apparently produce a polysaccharide material that prevents water transport and causes extensive wilting in ABG with minimal effect on other turf grasses (Fujimori, 1999). The efficacy of this approach depends upon temperature (Imaizumi et al., 1999a) and other environmental factors (Imaizumi et al., 1999b) that influence the successful survival and growth of the bacteria. An alternative to the use of live microbes as bioherbicides, and one that has interested us because it should be less dependent upon environmental conditions, is to identify microbially-derived compounds that can be used in place of the bacteria themselves and applied with delivery systems similar to those used with synthetic herbicides. We recently identified and described several isolates of Pseudomonas fluorescens that produce a compound that irreversibly arrests the germination of the seeds of a wide range of graminaceous plants, including a large number of grassy weeds such as ABG (Banowetz et al., 2008). Because of the developmentally-specific manner in which this compound acts to block germination, we have termed it a Germination Arrest Factor (GAF). GAF arrests the

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germination process in grasses immediately after the coleorhiza and plumule have emerged from the seed coat and before the length of these emergent structures have exceeded that of the seed. At the concentration of GAF present in these bacterial culture filtrates, no further growth or development occurs. Moreover, as demonstrated in our original publication, after about 24 h of exposure to GAF, the effect is irreversible. No further development takes place even if the grass seeds are removed from contact with GAF. On the other hand, once the process of germination has proceeded to a stage where the first true leaf has emerged from the coleoptile, treatment with GAF somewhat slows, but does not stop, further seedling development. In the case of mature plants, we have been unable to detect any significant effect of GAF treatment. The development of a quantitative bioassay for GAF, using seeds of ABG, and the results of a comprehensive survey of the plant species affected by this compound have been reported in our earlier publication (Banowetz et al., 2008). With rare exceptions, all graminaceous species we have tested are sensitive to GAF, including, in addition to ABG, most other Poa species and such significant grassy weeds as downy brome (Bromus tectorum) and jointed goatgrass (Aegilops cylindrica). The fact that GAF arrests germination in a developmentally-specific manner suggests that it, or the bacteria that produce this compound, may have utility for use as herbicides where stands of grasses are already established and germination of new seed is undesirable, such as in turfs and grass seed production fields. As a natural product, GAF might be expected to poise minimal risks to the environment, and both GAF and GAF-producing bacteria appear to be likely candidates for development and classification as biorational (minimum risk) herbicides. The US Environmental Protection Agency already lists several strains of Pseudomonas species as minimum risk biopesticides (http:// www.epa.gov/oppbppd1/biopesticides/regtools/25b_list.htm), but none of these strains exhibits the biological properties of our GAF producing isolates. The purification and identification of the compound responsible for the GAF activity present in culture filtrates from our Pseudomonas isolates is a primary focus of our current investigations. To this end, we have examined a number of the physical and chemical properties of GAF that might be expected to influence strategies for its purification. The solubility, molecular size, and ionic properties of the GAF compound produced by P. fluorescens WH6, one of the GAF-producing strains described by us earlier, have been examined in the present study. Conditions affecting the stability of the GAF molecule have also been determined. The results reported here establish that GAF is a very hydrophilic, low molecular weight compound that possesses an acid group.

2. Materials and methods 2.1. Biological materials Seeds of annual bluegrass (ABG, P. annua L.) were obtained from 1996 mid-Willamette Valley grass seed screenings and were provided by International Seeds, Halsey, OR, and C and R Farm, Tangent, OR. The seeds were cleaned to remove straw and seeds of other species. The isolation and characterization of P. fluorescens Strain WH6 were previously described (Banowetz et al., 2008). 2.2. Growth of bacteria and preparation of bacterial culture filtrates Pseudomonas fluorescens Strain WH6, which had been stored in cryovials in 50% glycerol at 60 °C, was inoculated into Wheaton bottles half-filled with sterile Pseudomonas Minimal Salts Medium (PMS) that was modified from that described by Bolton et al. (1989) by supplementation with iron as described (Banowetz

et al., 2008). The tops of the bottles were loosely capped and secured with tape. The inoculated bottles were placed on a rotary shaker (200 rpm) in a 27 °C chamber. Cells were harvested by centrifugation (3000 g, 15 min) after 7 days in culture, and the supernatant was passed through a bacteriological filter (Millipore GP Express Steritop, 0.22 lM pore size). The resulting sterile culture filtrate was stored at 4 °C. 2.3. Solvents All aqueous ethanol solutions were prepared from 95% (v/v) ethanol that had been redistilled before use. Absolute ethanol was purchased as USP grade. All other solvents listed in Table 2 were purchased as spectrophotometric grade reagents. 2.4. Chromatographic and ultrafiltration materials QAE-Sephadex, SP-Sephadex, and Sephadex G-10 were purchased from Sigma. The QAE-Sephadex column was packed and washed overnight in 0.025 M Tris–HCl buffer (pH 7.9, HCl) containing 0.5 M KCl. The column was then washed extensively with the same buffer without KCl prior to loading the sample. The SP-Sephadex column was packed and washed overnight with 0.025 M KH2PO4 buffer (pH 3.8, KOH) prior to loading the sample. The Sephadex G-10 column was packed and washed in deionized water prior to loading the sample. For experiments involving chromatography on silica-C18, SepPakÒ Plus C18 cartridges were purchased from Waters Corporation. Prior to loading a sample, the silica-C18 cartridges were prepared for use by sequential washes with 5 mL 76% ethanol, 5 mL 95% ethanol, 5 mL 76% ethanol, and finally with three 5 mL aliquots of deionized water to remove all traces of ethanol. Ultrafiltration studies were performed with 15 mL MacrosepÒ Centrifugal Concentrators (Pall Filtron Corporation) having molecular weight cut-off values of 3000 and 1000. The concentrators were prepared for use according to the instructions supplied with the units. 2.5. Solvent extraction of culture filtrate solids Measured volumes of bacterial culture filtrate were taken to dryness in vacuo at a temperature 6 45 °C. (Typically, 150 mL aliquots of culture filtrate were taken to dryness in 2-liter evaporation flasks.) The dry solids remaining after evaporation of the filtrate were extracted three times (5 min per extraction) with the test solvent. Each of these three extractions was performed by swirling the solids with a volume of solvent equal to one-third of the original volume of culture filtrate. The three extracts prepared in this manner were combined and either stored at 4 °C for later use or immediately taken to dryness in vacuo at 6 45 °C and redissolved in a solvent appropriate to the planned experimentation. 2.6. Bioassay of GAF activity Bioassays for GAF activity were performed with ABG seeds using the standard GAF bioassay protocol previously described (Banowetz et al., 2008). Culture filtrates and other solutions to be tested for GAF activity were distributed to the wells of sterile 48well plates (Corning Costar 3548). Each well received 200 lL of sterile test solution and three ABG seeds from seed lots that had been surface-sterilized as described (Banowetz et al., 2008). Three replicate wells (nine seeds) were prepared for each concentration of each treatment. The plates were sealed with Parafilm and incubated in a growth chamber at 20 °C with a photoperiod of 8 h light (50 lmol m 2 s 1) and 16 h dark. Germination scores

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were determined after 7 days incubations. Controls consisted of seeds incubated in wells containing sterile water or fresh sterile (uninoculated) culture medium, which had been prepared with glucose reduced to one-fifth of the normal concentration (to coincide with the glucose levels determined to remain in culture filtrates at the time of harvest). No difference was observed in the two types of controls. The germination scoring system used to evaluate GAF bioassays has been described in detail by Banowetz et al. (2008). In abbreviated outline, germination scores in this system range from 1.0 to 4.0, with a score of 4.0 representing normal development (no GAF activity) at the end of the 7 days assay period. A score of 1.0 is assigned to seeds where the plumule and coleorhiza have emerged from the seed but both are shorter than the length of the seed. A score of 2.0 indicates the primary root is visible and the plumule is slightly longer than the seed, but typically chlorotic. A score of 3.0 indicates that the first true leaf has emerged from the coleoptile and is green in color, but the emerged portion of the leaf is obviously shorter than the length of the coleoptile. A score of 4.0 is assigned to seeds exhibiting normal development, equivalent to that of controls, which at this stage of germination means that the first true leaf is fully emerged, green in color, and the emerged portion of the leaf is obviously longer than the coleoptile. The primary root is visible and elongated. A score of zero (no visible evidence of seed germination) is possible in this system, but GAF concentrations equivalent to that of full-strength WH6 culture filtrate typically result in permanent arrest of development at a Germination Score of 1.0. For quantitative comparisons, the relative amounts of GAF compound(s) present in various solutions may be expressed in terms of GAF-equivalents, where one GAF-equivalent is defined as the minimum quantity of GAF required to give a score of 1.0 when dissolved in 1 L of solution and tested in the standard GAF bioassay system. GAF-equivalents, or GAF-milliequivalents (the minimum quantity of GAF required to give a bioassay score of 1.0 when dissolved in 1 mL) were calculated from a standard curve prepared by first adjusting a WH6 culture filtrate to the minimum concentration required to give a score of 1.0 and then determining the variation of germination score with appropriate dilutions of this solution.

Table 1 Distribution of GAF activity in aqueous and organic phases after partitioning WH6 culture filtrate against organic solvents immiscible with water. Aliquots of P. fluorescens WH6 culture filtrate were mixed with two volumes of the indicated solvent, and the mixture was shaken in a separatory funnel. The aqueous and organic phases from each partition were dried separately in vacuo at 45 °C, and solids recovered from each phase were dissolved in a volume of deionized water equal to that of the original culture filtrate. The resulting solutions, designated as 1.0 concentrations, were tested for GAF activity in the standard GAF bioassay (see Section 2), where a germination score of 1.0 indicates germination was completely arrested, and a score of 4.0 indicates normal germination (no GAF activity). Solvent

Partition phase

0.1

0.3

1.0

Chloroform

Organic phase Aqueous Phase

4.0 ± 0.0 1.3 ± 0.1

4.0 ± 0.0 1.0 ± 0.0

4.0 ± 0.0 1.0 ± 0.0

Ethyl acetate

Organic phase Aqueous phase

4.0 ± 0.0 1.3 ± 0.1

4.0 ± 0.0 1.0 ± 0.0

4.0 ± 0.0 1.0 ± 0.0

None

WH6 culture filtrate

1.7 ± 0.1

1.1 ± 0.1

1.0 ± 0.0

Table 2 Solubility of GAF extracted from WH6 culture filtrates in selected organic solvents. Aliquots of P. fluorescens WH6 culture filtrate were dried in vacuo at 45 °C. The dry solids from each sample were extracted three times with the indicated solvent as described in Section 2, and the combined extracts from each solvent were dried in vacuo at 45 °C. Residual solids were dried in a similar manner. The dried extract and the residue from each extraction were dissolved in separate volumes of deionized water, each equal to the original volume of culture filtrate. The resulting solutions, designated as 1.0 concentrations, were tested for GAF activity in the standard GAF bioassay (see Section 2), where a score of 1.0 indicates germination was completely arrested, and a score of 4.0 indicates normal germination and seedling development (no GAF activity). Solvent

Fraction

0.1

0.3

1.0

Acetonitrile

Extract Residue

4.0 ± 0.0 2.0 ± 0.1

4.0 ± 0.0 1.0 ± 0.0

4.0 ± 0.0 1.0 ± 0.0

Acetone

Extract Residue

4.0 ± 0.0 1.8 ± 0.1

4.0 ± 0.0 1.2 ± 0.0

4.0 ± 0.0 1.0 ± 0.0

Chloroform

Extract Residue

4.0 ± 0.0 1.7 ± 0.2

4.0 ± 0.0 1.2 ± 0.1

4.0 ± 0.0 1.0 ± 0.0

Ethanol (absolute)

Extract Residue

4.0 ± 0.0 1.8 ± 0.1

4.0 ± 0.0 1.1 ± 0.0

4.0 ± 0.0 1.0 ± 0.0

Ethyl Acetate

Extract Residue

4.0 ± 0.0 1.7 ± 0.1

4.0 ± 0.0 1.0 ± 0.0

4.0 ± 0.0 1.0 ± 0.0

Isopropanol

Extract Residue

4.0 ± 0.0 2.2 ± 0.1

4.0 ± 0.0 1.0 ± 0.0

4.0 ± 0.0 1.0 ± 0.0

Methanol

Extract Residue

4.0 ± 0.0 1.8 ± 0.1

1.7 ± 0.0 1.2 ± 0.1

1.0 ± 0.0 1.0 ± 0.0

3. Results 3.1. Solubility of GAF in organic solvents The solubility of GAF in organic solvents was tested by examining the partitioning of GAF activity between aqueous and organic phases when WH6 culture filtrates were extracted with immiscible organic solvents, and by measuring the recovery of GAF activity following direct extraction of dried culture filtrates with a variety of solvents. The results of the partitioning experiments are shown in Table 1. All of the biological activity associated with GAF was retained in the aqueous phase when WH6 culture filtrate was partitioned against either ethyl acetate or chloroform. The results of direct extractions of dried WH6 culture filtrates with a range of organic solvents, both miscible and immiscible in water, are summarized in Table 2. Among the solvents tested, only methanol was effective in extracting GAF from the dried culture filtrates, and in even this case, the extraction of activity was incomplete under the conditions tested. The solubility of GAF was further investigated by direct extraction of dried WH6 culture filtrates with aqueous ethanol solutions. Although extraction with absolute ethanol failed to remove any GAF activity from the dried filtrates (Table 2), aqueous ethanol solutions were found to extract varying amounts of GAF activity

Mean germination score (±Standard error of the mean) relative sample concentration ()

Mean germination score (±standard error of the mean) relative sample concentration ()

(Table 3). Over 50% of the activity was recovered in extracts prepared with 90% (v/v) ethanol, and essentially complete recovery of GAF activity was observed in extracts prepared using 75% (v/v) ethanol. Varying amounts of solids, presumably consisting primarily of inorganic salts from the culture medium, but including some precipitated macromolecules derived from the bacteria, were left behind in the residue following these extractions. 3.2. Stability of GAF activity The heat stability of GAF was tested by measuring the recovery of GAF activity from WH6 culture filtrates exposed to a range of temperatures for periods of 1 h (Table 4). Under these conditions, no loss of GAF activity was detected at temperatures up to 65 °C, but some reduction in activity was evident after 1 h incubation

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Table 3 Solubility of GAF activity in aqueous ethanol solutions. Aliquots of Pseudomonas fluorescens WH6 culture filtrate were dried in vacuo at 45 °C, and the dry solids from each sample were extracted three times with the indicated concentration of aqueous ethanol as described in Section 2. The combined extracts from each ethanol concentration were dried in vacuo at 45 °C, as were the residual solids remaining after the extraction procedure. The extract and residue from each extraction were dissolved in separate aliquots of deionized water, each equal to the original volume of culture filtrate. The resulting solutions, designated as 1.0 concentrations, were tested for GAF activity in the standard GAF bioassay (see Section 2), where a score of 1.0 indicates germination completely arrested, and a score of 4.0 indicates normal germination and seedling development (no GAF activity).

Table 5 pH Stability of GAF activity. Aliquots of P. fluorescens WH6 culture filtrate were adjusted to the indicated pH values with 1 N HCl or 1 N NaOH and incubated at the indicated pH for 3 h before readjusting to the starting pH. Each aliquot was diluted as indicated below (1.0 = undiluted culture filtrate) and tested for GAF activity in the standard GAF bioassay (see Section 2), where a score of 1.0 indicates germination was completely arrested, and a score of 4.0 indicates normal germination and seedling development (no GAF activity). pH

Mean germination score (±standard error of the mean) culture filtrate concentration () 0.03

0.10

0.3

1.0

Solvent

Fraction

0.03

0.1

0.3

None

WH6 Culture Filtrate

2.0 ± 0.0

1.44 ± 0.0

1.0 ± 0.0

1.0 ± 0.0

95% Ethanol

Extract Residue

4.0 ± 0.0 2.6 ± 0.1

4.0 ± 0.0 1.7 ± 0.1

2.2 ± 0.2 1.0 ± 0.0

1.2 ± 0.1 1.0 ± 0.0

WH6 Culture filtrate (pH 6.8) 2 4 6 8 10 12

2.3 ± 0.2 2.7 ± 0.1 3.0 ± 0.2 2.5 ± 0.2 2.5 ± 0.3 2.3 ± 0.2 2.5 ± 0.2

1.7 ± 0.1 1.9 ± 0.1 1.8 ± 0.2 1.8 ± 0.1 1.8 ± 0.1 1.8 ± 0.1 1.9 ± 0.2

1.0 ± 0.0 1.1 ± 0.0 1.1 ± 0.0 1.0 ± 0.0 1.0 ± 0.0 1.0 ± 0.0 1.0 ± 0.0

1.0 ± 0.0 1.0 ± 0.0 1.0 ± 0.0 1.0 ± 0.0 1.0 ± 0.0 1.0 ± 0.0 1.0 ± 0.0

90% Ethanol

Extract Residue

2.7 ± 0.1 3.8 ± 0.2

2.0 ± 0.0 2.5 ± 0.1

1.0 ± 0.0 1.0 ± 0.0

1.0 ± 0.0 1.0 ± 0.0

85% Ethanol

Extract Residue

2.7 ± 0.1 4.0 ± 0.0

1.6 ± 0.2 3.6 ± 0.2

1.0 ± 0.0 2.0 ± 0.0

1.0 ± 0.0 1.0 ± 0.0

75% Ethanol

Extract Residue

2.5 ± 0.1 4.0 ± 0.0

1.5 ± 0.1 4.0 ± 0.0

1.0 ± 0.0 4.0 ± 0.0

1.0 ± 0.0 4.0 ± 0.0

Mean germination score (±standard error of the mean) relative sample concentration () 1.0

Table 4 Heat stability of GAF activity. Aliquots of P. fluorescens WH6 culture filtrate were incubated at the indicated temperatures for 1 h unless denoted otherwise. Each aliquot was diluted (1.0 = undiluted culture filtrate) and tested for GAF activity in the standard GAF bioassay (see Section 2), where a score of 1.0 indicates germination was completely arrested after emergence, and a score of 4.0 indicates normal germination and seedling development (no GAF activity). Heat treatment (°C)

22 35 45 55 65 75 85 95 100 (30 min) Autoclave (30 min)

Mean germination score (±standard error of the mean) culture filtrate concentration () 0.03

0.10

0.3

1.0

3.3 ± 0.2 3.6 ± 0.2 3.2 ± 0.3 3.8 ± 0.1 4.0 ± 0.0 4.0 ± 0.0 4.0 ± 0.0 4.0 ± 0.0 4.0 ± 0.0 4.0 ± 0.0

1.8 ± 0.1 1.7 ± 0.1 1.6 ± 0.1 1.8 ± 0.1 1.9 ± 0.2 3.9 ± 0.1 4.0 ± 0.0 4.0 ± 0.0 4.0 ± 0.0 4.0 ± 0.0

1.1 ± 0.0 1.0 ± 0.0 1.1 ± 0.0 1.0 ± 0.0 1.0 ± 0.0 1.8 ± 0.2 3.5 ± 0.0 3.0 ± 0.0 3.0 ± 0.0 3.0 ± 0.0

1.0 ± 0.0 1.0 ± 0.0 1.0 ± 0.0 1.0 ± 0.0 1.0 ± 0.0 1.2 ± 0.1 3.0 ± 0.0 2.6 ± 0.2 2.9 ± 0.1 2.9 ± 0.1

at 75 °C. Activity declined rapidly with exposure to higher temperatures. Only slight activity was observed after incubations at temperatures of 85 °C or higher, or exposure to autoclaving for 30 min. In separate tests of the effects of the temperatures used in drying culture filtrates on GAF activity, GAF proved to be somewhat more temperature sensitive with some loss of activity detectable at about 55 °C, under conditions used for drying (data not shown). For this reason, the temperatures routinely used in evaporation and concentration of culture filtrates have not been permitted to exceed 45 °C. To test the pH stability of GAF, aliquots of WH6 culture filtrate were adjusted to specific pH values by the addition of HCl or NaOH and incubated at those values for 3 h at room temperature. After the pH was adjusted back to the original pH of the culture filtrate (pH 6.8), each aliquot was tested for GAF activity in the standard GAF bioassay. As shown in Table 5, under these conditions, little if any loss of biological activity was observed over the pH range tested (pH 2–12). However, when the pH 2 treatment was extended overnight or when a sample of culture filtrate adjusted to pH 2 was taken to dryness in vacuo (45 °C) without adjusting

the pH 6.5 prior to evaporation, over 80% of the original GAF activity was lost (data not shown). Thus, although GAF appears to be reasonably stable to pH changes at room temperature, extended exposure to low pH conditions or to low pH at somewhat elevated temperatures must be avoided if GAF activity is to be preserved. The stability of GAF activity in sterile WH6 culture filtrates stored at 4 °C for prolonged periods of time was examined, and the results are shown in Table 6. After 11 months, approximately 25% of the original activity remained in the culture filtrate. The activity continued to decline with time so that less than 10% remained at the end of 17 months. 3.3. Estimation of the molecular weight of GAF The molecular weight of GAF was evaluated by centrifugation of WH6 culture filtrate through ultrafiltration membranes with decreasing molecular weight cut-off values. All of the GAF activity in the culture filtrate appeared to pass through an ultrafiltration membrane with a molecular weight cut-off (MWCO) of 3000 (Table 7). No activity was recovered in the retentate after that fraction was washed by dilution and recentrifugation, indicating that GAF clearly has a molecular weight less than 3000. Ultrafiltration of WH6 culture fluid with a membrane having a MWCO of 1000 gave more ambiguous results. About half of the GAF activity was recovered in the original ultrafiltrate, but a significant amount of activity remained in the washed retentate. On the basis of this result, at least half of the GAF activity present in WH6 culture filtrate could be assigned a molecular weight less than 1000, but the presence of significant activity in the 1000 MWCO retentate left open the possibility that some fraction of the activity might be associated with a higher molecular weight component of the filtrate. Interpretation of this result was complicated by the fact that filtration with the

Table 6 Stability of GAF activity in storage. Sterile WH6 culture filtrates were stored at 4 °C for the indicated period after which GAF activity was measured in the standard GAF bioassay (as described in Section 2). Bioassay scores were converted to GAF milliequivilents using the standard curve shown in Fig. 1, 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 of solution and tested in the standard GAF bioassay. Storage duration

GAF activity (milliequivalents mL

0 (initial activity) 1 month 5 months 11 months 17 months 33 months

9.6 9.6 6.5 2.3 0.9 0.1

1

)

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Ultra-filtration membrane (molecular weight Cut-Off)

Fraction

Relative sample concentration ()

Germination score (±standard error of the mean)

3000

Washed retentate

1.0 0.3 0.1 1.0 0.3 0.1

4.0 ± 0.0 4.0 ± 0.0 4.0 ± 0.0 1.0 ± 0.0 2.5 ± 0.0 3.5 ± 0.2

1.0 0.3 0.1 1.0 0.3 0.1

1.1 ± 0.1 2.5 ± 0.0 4.0 ± 0.0 1.0 ± 0.0 2.5 ± 0.0 4.0 ± 0.0

1.0 0.3 0.1 0

1.0 ± 0.0 1.7 ± 0.2 2.5 ± 0.0 4.0 ± 0.0

Filtrate

1000

Washed retentate Filtrate

Controls

Culture Filtrate Water

1000 MWCO membrane was relatively slow, and significant portions of the starting volumes remained on the retentate side of the membrane at the end of each centrifugation. In an attempt to clarify whether a portion of the GAF activity in WH6 culture filtrate was associated with a component having a molecular weight above 1000, quantitative estimates of the GAF activity recovered in the ultrafiltrate and retentate fractions were compared with the volumes recovered in these fractions following centrifugation through the 1000 MWCO membrane. For this purpose, germination scores in the GAF bioassay were translated into GAF-milliequivalents (defined in Section 2) using the standard curve shown in Fig. 1. Because a possible source of artifacts in the ultrafiltration experiment using the 1000 MWCO membrane may be alteration of pore size by the accumulation on the membrane surface of macromolecules present in the WH6 culture filtrate, GAF activity was extracted from dried WH6 culture filtrates with 76% ethanol prior to ultrafiltration. As expected from the results reported in Table 3, and confirmed by the data shown in Table 8, all but a trace of GAF activity (less than 1% of the total) was recovered from the culture filtrate by this process. The 76% ethanol extract was taken to dryness, reconstituted in water, and fractionated by centrifugation through the 1000 MWCO filter. The results of this ultrafiltration experiment are summarized in Table 8. About 80% of the total extract volume was recovered in the filtrate and retentate fractions, and this was reflected in a corresponding 83% value for the total recovery of GAF activity. The distribution of GAF activity recovered in the filtrate and retentate fractions mirrored the relative volumes of the two fractions. (The loss of volume is undoubtedly due in part to mechanical factors in effecting the appropriate liquid transfers, but it may also reflect changes in liquid retention by the membrane itself as various types of molecules accumulate on the relatively large membrane surface of the ultrafiltration unit). From this result, the majority of activity (at least 3/4 of the total in the original sample) appears to have a molecular weight of less than 1000.

4

3.5

3

Germination score

Table 7 Ultrafiltration estimate of GAF molecular weight. Aliquots of P. fluorescens WH6 culture filtrate were centrifuged (3000g, 2 h) in MacrosepÒ Centrifugal Concentrators equipped with ultrafiltration membranes of the indicated molecular weight cut-off. The filtrate and retentate were restored to the original volume with deionized water. The retentate was then recentrifuged, restored to the original volume, and designated as the washed retentate. The resulting solutions were diluted with deionized water as indicated below (1.0 = undiluted solution) and tested for GAF activity in the standard GAF bioassay (as described in Section 2), where a score of 1.0 indicates germination was completely arrested, and a score of 4.0 indicates normal germination and seedling development (no GAF activity).

2.5

2

1.5

1 0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

GAF concentration (GAF milliequivalents mL-1) Fig. 1. Standard curve for estimation of the relative quantity of GAF-active compounds in the standard GAF bioassay. A germination score of 1.0 indicates germination was completely arrested while a score of 4.0 indicates normal germination and seedling development (no GAF activity). A 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 in the standard GAF bioassay. Details of the bioassay procedure and scoring system are given in Section 2.

The molecular weight of GAF was further evaluated by gel filtration chromatography (Fig. 2). A sample of P. fluorescens WH6 culture filtrate was loaded on a Sephadex G-10 column that had been equilibrated in deionized water. GAF activity eluted approximately one-tenth of a bed volume later than the high molecular weight Blue Dextran marker, indicating that GAF was retained within the void volume of the column. While a number of factors other than molecular size may affect elution positions of molecules on gel filtration columns, given that the nominal molecular weight exclusion value for Sephadex G-10 is about 700, the results obtained here are consistent with all GAF activity in the WH6 culture filtrate being associated with molecules having a molecular weight less than 1000. The same precise elution position was observed when concentrated samples of either a 90% ethanol extract of dried WH6 culture filtrate or the residue from such an extraction were chromatographed on the same column (data not shown). Thus, there was no detectable difference in the elution positions of these fractions of GAF activity. 3.4. Reverse phase and ion exchange chromatography of GAF Reverse-phase silica-C18 cartridges are frequently used for the recovery and trace enrichment of organic compounds from dilute aqueous solutions. Organic compounds with hydrophobic properties interact with the C18 chains attached to the silica support and are retained on these cartridges. However, GAF activity from P. fluorescens WH6 culture filtrate did not bind to silica-C18 (Table 9). When WH6 culture filtrate was passed through a silica-C18 cartridge, essentially all GAF activity was recovered in the sample flow-through and the first water wash. No biological activity was retained by the cartridge or recovered with the bound material eluted when the cartridge was washed with 76% ethanol. This result is consistent with the very hydrophilic character of GAF indicated by its failure to partition into organic solvents. The ability of ion-exchange materials to bind GAF was tested using the cation exchanger SP-Sephadex and the anion exchanger

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Table 8 Further evaluation of the molecular weight of GAF by ultrafiltration. The GAF activity associated with P. fluorescens WH6 culture filtrate was extracted from dried culture filtrate with 76% ethanol as described in Section 2. The extract was dried, reconstituted in a volume of deionized water equal to the original volume of culture filtrate, and centrifuged (3000g, 4 h) in a MacrosepÒ Centrifugal Concentrator equipped with a 1000 molecular weight ultrafiltration cut-off membrane. Filtrate and retentate volumes were recovered and reconstituted to their original volume with deionized water. The GAF activities of the resulting solutions were measured in the standard GAF bioassay (as described in Section 2). Bioassay scores were converted to GAF milliequivalents using the standard curve shown in Fig. 1, where 1 GAF milliequivalent is defined as the minimum quantity of GAF required to give a germination score of 1.0 in the GAF bioassay when dissolved in 1.0 mL of test solution. Bioassay sample

GAF activity (milliequiv.)

Volume (mL)

Recovery of starting volume (%)

Recovery of starting activity (%)

WH6 Culture filtrate 76% Ethanol extract 76% Ethanol residue

405 450 2

15 15 —

— 100% —

— 100 —

1000 MW cut-off fractions Ultrafiltrate Retentate Total recovery

330 45 375

9.8 2.2 12

65% 15% 80%

73 10 83

columns, respectively. The GAF activity applied to the cation exchanger, SP-Sephadex, eluted with the loading buffer wash and was not retained by the column (Fig. 3). Although this result does not exclude the possibility that GAF contains a cationic group, it indicates that any group of this type that is present on the GAF molecule must have a pK less than 3.8. In contrast, GAF activity was retained on the QAE-Sephadex anion-exchange column and

Germination score

1

2

3

1

blue

4 1

2

3

4

5

6

7

8

9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

Fraction Fig. 2. Gel filtration of GAF activity on Sephadex G-10. Pseudomonas fluorescens WH6 culture filtrate (3.0 mL) was applied to a Sephadex G-10 column (1.5  41 cm, 68 mL bed volume) equilibrated in distilled water. The column was eluted with distilled water, and fractions (3.0 ml) were collected. The pH of each fraction was adjusted to 6.0 and each fraction was tested for biological activity in the standard GAF bioassay as described in Section 2. A germination score of 1.0 indicates germination was completely arrested while a germination score of 4.0 indicates that germination proceeded normally (i.e., no GAF activity was detected). The column void volume was determined by applying Blue Dextran (molecular weight 2,000,000) to the column.

QAE-Sephadex. To minimize potential complications arising from the high concentration of salts in the culture filtrate, 90% ethanol extracts of dried WH6 culture filtrate were taken to dryness in vacuo and then reconstituted in a volume of appropriate buffer (pH 3.8 for the cation exchanger and pH 7.9 for the anion exchanger) equal to two-thirds of the original volume of culture filtrate. These samples were applied to small SP-Sephadex and QAE-Sephadex

Germination score

dextran

2

elution buffer

buffer

3 wash

4

1

2

3

4

5

6

7

8

9

10

11

12

fraction Fig. 3. Test of the Retention of GAF activity by SP-Sephadex cation-exchange medium. Pseudomonas fluorescens WH6 culture filtrate (18 mL) was dried in vacuo at 45 °C, and the solids were extracted three times with 90% (v/v) ethanol as described in Section 2. The combined ethanol extracts were dried in vacuo at 45 °C and dissolved in 12 mL of 0.025 M KH2PO4 buffer (pH 3.8). A portion (11 mL) of this solution was loaded on a SP-Sephadex column (1.5  8.4 cm, 14.8 ml bed volume) equilibrated with the same buffer. After the sample was loaded, the column was washed with equilibration buffer, followed by 0.05 M KH2PO4 (pH 8.2) and the evaluate was collected in 13 mL fractions. The pH of each fraction was adjusted to 6.1 and each fraction was tested for GAF activity in the standard GAF bioassay (see Section 2) where a germination score of 1.0 indicates complete germination arrest and a score of 4.0 indicates that germination proceeded normally.

Table 9 Test of the Retention of GAF activity by silica-C18. P. fluorescens WH6 culture filtrate (6 mL) was passed through a silica-C18 cartridge previously washed and equilibrated in water as described in Section 2. Sample flow-through was collected; the cartridge was washed with distilled water (2  6 mL); and bound material was eluted with 76% (v/v) ethanol (2  6 mL). The ethanol eluates were dried in vacuo and each redissolved in a volume of deionized water equal to the original volume of culture filtrate. These solutions were tested for GAF activity in the standard GAF bioassay (as described in Section 2), where a score of 1.0 indicates germination was completely arrested and a score of 4.0 indicates normal germination and seedling development (no GAF activity). Sample flow-through and water washes were bioassay directly (without concentrating). All bioassay samples were tested at several dilutions (1.0X = undiluted solution). Fraction

WH6 culture filtrate Sample flow-through 1st Water wash 2nd Water wash 1st Ethanol eluate 2nd Ethanol eluate

Mean germination score (±standard error of the mean) relative sample concentration () 0.1

0.25

0.5

1.0

1.0 ± 0.0 1.0 ± 0.0 3.8 ± 0.1 4.0 ± 0.0 4.0 ± 0.0 4.0 ± 0.0

1.0 ± 0.0 1.0 ± 0.0 2.6 ± 0.1 4.0 ± 0.0 4.0 ± 0.0 4.0 ± 0.0

1.0 ± 0.0 1.0 ± 0.0 2.0 ± 0.1 4.0 ± 0.0 4.0 ± 0.0 4.0 ± 0.0

1.0 ± 0.0 1.0 ± 0.0 1.6 ± 0.1 4.0 ± 0.0 4.0 ± 0.0 4.0 ± 0.0

G.M. Banowetz et al. / Biological Control 50 (2009) 103–110

Germination score

1

elution buffer

buffer wash

2

sample application

3

4 0

1

2

3

4

5

6

7

8

9

10

11

12

13

Fraction Fig. 4. Test of the Retention of GAF activity by QAE-Sephadex anion-exchange medium. Pseudomonas fluorescens WH6 culture filtrate (18 mL) was dried in vacuo at 45 °C, and the dry solids were extracted three times with 90% (v/v) ethanol as described in Section 2. The combined ethanol extracts were dried in vacuo at 45 °C and dissolved in 12 mL of 0.025 M Tris–HCl buffer (pH 7.9). A portion (11 mL) of this solution was applied to a QAE-Sephadex column (1.5  7.5 cm, 13.3 mL bed volume) equilibrated with the same buffer. The column was washed with equilibration buffer, and eluted with 0.05 M KH2PO4 (pH 5.6). Eluate was collected in 12 mL fractions. The pH of each fraction was adjusted to 6.3, and each fraction was tested for GAF activity in the standard GAF bioassay (see Section 2), where a germination score of 1.0 indicates complete germination arrest while a score of 4.0 indicates that germination proceeded normally.

was not removed by washing with the loading buffer (Fig. 4). GAF activity could subsequently be eluted from the column by washing with buffer at an acid pH. Thus, based on the retention of GAF activity by the QAE-Sephadex column, the GAF molecule appears to contain an anionic (acid) group. 4. Discussion The GAF activity produced by P. fluorescens WH6 was shown here to be associated with a very hydrophilic compound. The GAF activity present in WH6 culture filtrates was insoluble in solvents immiscible with water and, without the addition of water, also insoluble in several organic solvents miscible with water. Among these latter solvents, only methanol exhibited any capacity to extract GAF from dried WH6 culture filtrates. However, with the addition of water, aqueous ethanol solutions could also be used to extract GAF from culture filtrate solids, and extraction of these solids with carefully graded ethanol concentrations provides a method of achieving some separation of GAF from at least part of the inorganic salts present in the culture medium. The failure of GAF activity to partition into organic solvents, combined with the failure of this activity to bind to reverse phase silica-C18 columns, suggests that GAF lacks an aromatic ring structure, because the latter would be expected to yield a compound of more hydrophobic character. Further characterization of the physical and chemical properties of GAF has provided evidence that, in addition to being hydrophilic, the active molecule is a low molecular weight compound that possesses an acid group. The molecular size of the GAF molecule was estimated by both ultrafiltration and gel filtration methods, and both procedures indicated that GAF activity was associated with a molecule (or molecules) having a molecular weight less than 1000. The ability of the anion exchange material QAE-Sephadex to bind GAF activity at a slightly alkaline pH indicates that the active molecule must contain some type of acid group, and the presence of a carboxyl group is one obvious possibility for the nature of this group. The stability of GAF activity to temperature and pH variations indicates that, with suitable care, the molecule can be manipulated

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in short-term laboratory procedures without destroying the activity. Our longer term studies, however, indicate the molecule is subject to slow spontaneous degradation in liquid solution. Given this result, it is unlikely that the molecule, if used as an herbicide, would persist indefinitely in the environment. This could be advantageous from the standpoint of ultimately establishing this compound as a biorational herbicide, but whether the active molecule can persist long enough in a field setting to inhibit time-delayed weed germination is impossible to assess at this point in our investigations. The physical and chemical characteristics of GAF defined in the present study, combined with the biological properties of GAF established in our earlier study (Banowetz et al., 2008), suggest the compound responsible for GAF activity is distinct from any herbicidal compounds previously reported to be produced by DRB bacteria. The failure of GAF to partition into organic solvents that are immiscible with water (e.g. ethyl acetate) differentiates GAF from the pseudophomins (cyclic lipodepsipeptides) isolated from P. fluorescens strain BRG100 (Pedras et al., 2003) and from the herbicidally-active phenazine metabolites produced by Pseudomonas syringae strain 3366 that possess herbicidal activity (Gealy et al., 1996). The retention of GAF on an anion exchange column also differentiates GAF from the hydrophilic herbicidal compounds produced by P. fluorescens strain D7 (Gurusiddaiah et al., 1994). Compounds produced by other pseudomonads that cause leaf chlorosis on various host plants are hydrophilic, but possess considerably different biological properties than GAF (Woolley et al., 1952; Mitchell, 1976; Gronwald et al., 2005). Other compounds produced by DRB have been shown to have herbicidal properties (Suslow and Schroth, 1982; Bakker and Schippers, 1987; Bolton and Elliott, 1989; Gurusiddaiah et al., 1994; Gealy et al., 1996), but these also have biological properties that distinguish them from GAF (Banowetz et al., 2008). Bolton and Elliott (1989) and Bolton et al. (1989) reported that a low molecular weight compound produced by P. fluorescens strain RC1 inhibited root growth of wheat and was polar (hydrophilic) in nature and could not be extracted from culture filtrates with methylene chloride or ethyl ether (Bolton et al., 1989), characteristics similar to those observed for GAF. However, no direct comparison of the biological properties of GAF and the RC1 compound is possible from the data presented. It may be worth noting that a large number of compounds and metabolites with antifungal activity have also been identified as products of rhizobacteria (Howell and Stipanovic, 1979, 1980; Kraus and Loper, 1992; Dowling and O’Gara, 1994; NowakThompson et al., 1994; Bloemberg and Lugetenberg, 2001; Pedras et al., 2003), but many of these are more hydrophobic than GAF, and none has been reported to have the biological properties associated with GAF. To our knowledge, the developmentally-specific nature of the germination-arrest brought about by GAF and the specificity of this compound for grass species are distinct from any herbicidal effects previously reported for compounds produced by DRB and from the effects of other compounds know to inhibit the germination process. Successful development of GAF as an herbicide will depend on the development of economical approaches to enrich and purify the compound from bacterial culture filtrates or on determination of the chemical structure of GAF and subsequent chemical synthesis of the active compound. With the latter possibility in mind, one of our goals in this initial characterization of the physical and chemical properties of GAF was to obtain the information needed to design an approach to the purification of the compound(s) responsible for GAF activity. The fact that GAF did not partition into organic solvents or bind to the reverse phase silica-C18 cartridges commonly used to extract and enrich organic compounds from aqueous solutions suggests that a number of methods commonly used to isolate low molecular weight compounds from complex

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natural mixtures will not be applicable to the purification of GAF. Moreover, the data we have obtained relative to the stability of GAF have defined time, temperature, and pH parameters that also must be taken into account in any approach to GAF purification. With these parameters in mind, we are proceeding to explore various chromatographic alternatives for the isolation and purification of this novel, naturally occurring herbicide. Acknowledgments Support from the USDA CSREES Grass Seed Cropping Systems for a Sustainable Agriculture Special Grant Program 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. References Bakker, A.W., Schippers, B., 1987. Microbial cyanide production in the rhizosphere in relation to potato yield reduction and Pseudomonas SPP-mediated plant growth-stimulation. Soil Biology and Biochemistry 19, 451–457. Banowetz, G.M., Azevedo, M.D., Armstrong, D.J., Halgren, A.B., Mills, D.I., 2008. Germination Arrest Factor (GAF): biological properties of a novel, naturallyoccurring herbicide produced by selected isolates of rhizosphere bacteria. Biological Control 46, 380–390. Barnes, R., Miller, D.A., Nelson, J.C., 1995. Forages, An Introduction to Grassland Agriculture. 5th ed., vol. 1, Iowa State Press, Ames, IA, USA, 516 p. Bloemberg, G.V., Lugetenberg, B.J.J., 2001. Molecular basis of plant growth promotion and biocontrol by rhizobacteria. Current Opinions in Plant Biology 4, 343–350. Bolton, H., Elliott, L.F., 1989. Toxin production by a rhizobacterial Pseudomonas sp. that inhibits wheat root growth. Plant and Soil 114, 269–278. Bolton, H., Elliott, L.F., Gurusiddaiah, S., Fredrickson, J.K., 1989. Characterization of a toxin produced by a rhizobacterial Pseudomonas sp. that inhibits wheat growth. Plant and Soil 114, 279–287. Boyetchko, S.M., 1997. Principles of biological weed control with microorganisms. HortScience 32, 201–205. Cherrington, C.A., Elliott, L.F., 1987. Incidence of inhibitory pseudomonads in the Pacific Northwest. Plant and Soil 101, 159–165. Dowling, D.N., O’Gara, F., 1994. Metabolites of Pseudomonas involved in the biocontrol of plant disease. Trends in Biotechnology 12, 133–141. Elliott, L.F., Lynch, J.M., 1985. Plant growth-inhibitory pseudomonads colonizing winter wheat (Triticum aestivum L.) roots. Plant and Soil 84, 57–65. Flores-Vargas, R.D., O’Hara, G.W., 2006. Isolation and characterization of rhizosphere bacteria with potential for biological control of weeds in vineyards. Applied Microbiology 100, 946–954. Fujimori, T., 1999. New developments in plant pathology in Japan. Australian Plant Pathology 28, 292–297. Gealy, D.R., Gurusiddaiah, S., Ogg, A.G., 1996. Isolation and characterization of metabolites from Pseudomonas syringae-strain 3366 and their phytotoxicity against certain weed and crop species. Weed Science 44, 383–392. Gronwald, J.W., Plaisance, K.L., Marimanikkuppam, S., Ostrowski, B.G., 2005. Tagetitoxin purification and partial characterization. Physiological and Molecular Plant Pathology 67, 23–32.

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