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Germination-Arrest Factor (GAF): Biological properties of a novel, naturally-occurring herbicide produced by selected isolates of rhizosphere bacteria
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Biological Control 46 (2008) 380–390 www.elsevier.com/locate/ybcon
Germination-Arrest Factor (GAF): Biological properties of a novel, naturally-occurring herbicide produced by selected isolates of rhizosphere bacteria Gary M. Banowetz a,*, Mark D. Azevedo a, Donald J. Armstrong b, Anne B. Halgren b, Dallice I. Mills b a
USDA-ARS NFSPRC National Forage Seed Production Research Center, 3450 S.W. Campus Way, Corvallis, OR 97331, USA b Department of Botany and Plant Pathology, Oregon State University, Corvallis, OR 97331, USA Received 14 January 2008; accepted 12 April 2008 Available online 29 April 2008
Abstract Five strains of deleterious rhizosphere bacteria (DRB), identified as Pseudomonas fluorescens, were shown to produce and secrete a naturally-occurring herbicide that targets the seeds of certain graminaceous plants, including annual bluegrass (ABG; Poa annua L.), considered a weed in many agronomic systems. The herbicide arrests germination of the seeds in a developmentally-specific manner, typically irreversibly blocking the germination process immediately after the emergence of the plumule and coleorhiza. Because of its unique mode of action, this herbicide has been termed a Germination-Arrest Factor (GAF). Bacterial-culture filtrates containing GAF arrest seed germination of a large number of graminaceous species, including grassy weeds and grass species grown for seed and food. The germination of corn seeds did not appear to be affected by the herbicide. The seeds of dicot species appear to be less sensitive to GAF than seeds of graminaceous species. The biological effects of GAF on graminaceous species is primarily limited to germination, although transient slowing of ABG seedling growth was observed after post-germination exposure to GAF. Exposure of non-germinated ABG seeds to GAF for periods as short as 24 h was sufficient to irreversibly arrest germination. A quantitative bioassay for GAF was developed based on the sensitivity of ABG seed to this compound. Published by Elsevier Inc. Keywords: Microbial herbicide; Biological weed control; Deleterious rhizobacteria; Annual bluegrass
1. Introduction The control of grassy weeds is a significant problem in a number of agricultural settings, in recreational and professional lawns and turf, and in natural habitats where invasive non-native grasses are a concern. Grassy weeds are a particular problem in grass seed production systems where contamination of commercial seed lots with seeds of grassy weeds, such as annual bluegrass (ABG, Poa annua L.), significantly reduces the quality and market value of the product. In the Pacific Northwest, state-man-
*
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.2008.04.016
dated reductions in post-harvest field burning and the development of strains of ABG and other grassy weeds that are resistant to the chemical herbicides most commonly used in conjunction with field burning have exacerbated the problem. Consequently, the development of alternative and more effective measures for the control of grassy weeds is necessary. The potential utility of deleterious rhizosphere bacteria (DRB) as agents for weed control has attracted the interest of a number of investigators. DRB are defined as rhizosphere-inhabiting bacteria (usually Pseudomonas sp.) that have a negative impact on the growth or development of higher plant species (Suslow and Schroth, 1982; Cherrington and Elliott, 1987; Boyetchko, 1997). DRB isolates have been investigated as potential agents for
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the biocontrol of a number of weed species in a range of agricultural crops (Elliott and Lynch, 1985; Schippers et al., 1987; Kremer et al., 1990; Kremer and Kennedy, 1996; Kremer et al., 2000; Kennedy et al., 2001; FloresVargas and O’Hara, 2006; Li and Kremer, 2006). However, the introduction and successful establishment of live bacteria in a field setting is influenced by a multitude of environmental and biotic factors that are poorly understood (Van Elsas et al., 1992; Skipper et al., 1996; Zdor et al., 2005). For these reasons, direct applications of DRB have achieved only limited success as weed control measures (Li et al., 2003). We have been interested in identifying DRB that specifically target the germination of grassy weed seeds and in exploring the possibility that compounds produced by such DRB, rather than the bacteria themselves, may be useful in the control of these weeds. The starting point for our investigations has been a collection of over 30 rhizosphere bacterial strains that were isolated and subsequently maintained at the USDA-ARS National Forage Seed Production Research Center in Corvallis, Oregon. These isolates were originally obtained by Elliott and co-workers from selected locations within the Willamette Valley in Oregon (Elliott, Personal Communication; Elliott et al., 1998) and retained because they appeared to exert a stunting effect on ABG seedlings in laboratory tests using live bacteria. At least 12 isolates appeared to interfere with germination of ABG seeds (Elliott et al., 1998), but the biological specificity, mechanism of action, and the nature of the active agent or agents responsible for the herbicidal activity were not determined. We identified five isolates from the Elliott collection that, in our preliminary studies, were particularly active in suppressing the germination of ABG seeds. ABG was selected as the target weed species for these studies because ABG seed is a significant contaminant in grass seed production. In work reported here, we identified these germination-inhibiting DRB isolates (GI-DRB) as particular strains of Pseudomonas fluorescens, and we demonstrated that these bacterial strains produce and secrete a compound that specifically blocks germination of the seeds of ABG and a large number of other grassy weeds in a developmentally-specific manner. Because of the particular effect of this compound on the germination process, we have called it a Germination-Arrest Factor (GAF). The biological properties of GAF and the development of a quantitative bioassay for this naturally-occurring herbicide are described. 2. Materials and methods 2.1. Bacterial isolates and propagation The five GI-DRB isolates selected for this study were compared with strains of P. fluorescens obtained from other laboratories. Strain D7 (P. fluorescens D7: NRRL B-18293), a root-colonizing bacterium that inhibits downy
381
brome (Bromus tectorum L. BROTE) root growth and seed germination in plant–soil bioassays (Gurusiddaiah et al., 1994; Kennedy et al., 1991, 2001), was provided by Dr. Ann Kennedy, USDA-ARS, Pullman, WA. P. fluorescens strain Pf-5 (Howell and Stipanovic, 1979; Corbell and Loper, 1995) was obtained from Dr. Joyce Loper (USDA-ARS Horticultural Crops Research Laboratory, Corvallis, OR) and used as a non-GAF-producing control in these experiments. P. fluorescens strain PfO-1 was obtained from Dr. Mark Silby, Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, MA (Compeau et al., 1988). All bacteria were propagated in a Pseudomonas Minimal Salts (PMS) medium based on that described by Bolton et al. (1989). The Bolton medium was supplemented with iron as follows: 2 mL of a 1 mM filter-sterilized stock solution of FeCl3 dissolved in 10 mM HCl was added to 1 L of medium. One milliliter aliquots of cultures grown to an OD580 of 1.0 were placed in 50% glycerol and stored in cryovials at 60 °C prior to use. Taxonomic identification of the five GI-DRB was made by Microcheck, Inc. (Northfield, VT) based on Fatty Acid Methyl Ester Analysis (FAME Analysis) and on sequencing of a variable 400 + bp DNA region near the beginning of the 16S-rRNA gene. Fatty acid profiles were analyzed using Sherlock, Version 6.0, software (MIDI, Inc., Newark, DE). Contiguous DNA sequences were assembled using CAP3 (Huang and Madan, 1999) and aligned using ClustalW (Chenna et al., 2003). 2.2. Preparation of culture filtrates from bacterial isolates GI-DRB strains removed from cryovial storage were inoculated into Wheaton bottles of varying sizes, dependent upon the required volume of culture filtrate. Each bottle was half-filled with Fe-containing PMS Medium and placed on a rotary shaker (200 rpm) at 27 °C. The cultures were harvested after 7 d (OD580 = 0.8–1.2) and centrifuged (3000g, 15 min). The resulting supernatants were filtered through Millipore GP Express Steritop Bacteriological Filters (0.22 lm), and the sterile culture filtrates were stored at 4 °C prior to use. 2.3. Sources of seeds ABG seeds were obtained from 1996 mid-Willamette Valley grass seed screenings from International Seeds, Halsey, OR and C and R Farm, Tangent, OR. The seeds were cleaned to remove straw and other seeds. Seeds of the other Poa species and wheat were obtained from the Oregon State University (OSU) Seed Testing Laboratory. Seeds of other grasses were obtained from various OSU, USDA, and other local sources. The seeds of all the dicot species were purchased locally (Corvallis, OR) from garden supply stores with the exception of tobacco (Nicotiana tabacum L., cv. Wisconsin 38) seed which was grown in the greenhouse at OSU.
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2.4. Surface sterilization of seeds Surface sterilization of seeds was required to prevent microbial growth within the bioassay environment. The contrasting characteristics of seeds from different species required development of specific procedures to accomplish surface sterilization without significantly impacting subsequent germination of the seeds. All Poa seeds were surface-sterilized using the methods of Lowe and Conger (1979) as modified by Azevedo and Welty(1995). The seeds were stirred for 5 min in 50% sulfuric acid, followed by extensive washing with sterile deionized water in a Gooch crucible. The washed seeds were stirred with freshly prepared 100% CloroxÒ (6–7.35% NaOCl) containing 1% Tween 20 for 5 min in a sterile beaker, filtered (Gooch crucible), and washed again with sterile water. The seeds were either used immediately or dried under a laminar flow hood and stored in a closed sterile Petri dish at room temperature for future use. Seeds of all other grasses were surface-sterilized using the same procedure with the following exceptions: tall fescue and downy brome, treatments in 50% sulfuric acid and 100% CloroxÒ were extended to 20 min each; California brome, treatments in sulfuric acid and CloroxÒ were extended to 10 min; jointed goatgrass, treatment with 50% sulfuric acid remained at 5 min, but the CloroxÒ treatment was extended to 10 min. Barnyardgrass seeds were stirred in 1% Tween 20 for 5 min after which an equal volume of 50% CloroxÒ was added with continued stirring for 5 min. All seeds were filtered and washed with sterile deionized water in a Gooch crucible and used immediately, or dried under laminar flow air and stored at 22 °C in sterile Petri dishes for immediate use, or dried under laminar flow air and stored at room temperature in a closed Petri dish for later use. Wheat, barley, and corn seeds were surface-sterilized by stirring in 50% sulfuric acid for 5 min followed by extensive washing with sterile deionized water in a Gooch crucible. Washed seeds were transferred to a sterile beaker and stirred with 1% Tween 20 for two 1 min treatments (decanting and replacing the solution between treatments). The final Tween solution was replaced with 10% Clorox (wheat) or 20% Clorox (barley and corn). The seeds were stirred in
the Clorox solution for two 2 min treatments, filtered, and washed extensively with sterile deionized water. Surface-sterilized seeds were transferred to a sterile Petri dish and used immediately. Seeds of dicots were stirred with 0.5% Tween 20 for 1 min after which an equal volume of 100% CloroxÒ was added with continued stirring for 2 min. Following this treatment, the seeds were extensively washed with sterile deionized water in a Gooch crucible before transfer to a sterile Petri dish. For very small seeds, filtration and washing was accomplished using a plastic Millipore filter holder (without the filter, using the filter-support screen to retain the seeds) attached to an appropriately sized syringe. Surface-sterilized seeds were transferred to a sterile Petri dish and used immediately. 2.5. Standard Poa bioassay for GAF activity A standard quantitative bioassay for GAF activity was established using ABG seeds. Culture filtrates and other solutions to be tested for GAF activity were distributed to the wells of sterile 48-well plates (Corning Costar 3548). Each well received 200 lL of test solution per well, and three surface-sterilized ABG seeds. Three replicate wells were prepared for each treatment, nine seeds total. The plates were sealed with ParafilmÒ 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. Seed germination was scored after 7 d using the scoring system described in (Table 2). 2.6. Species specificity of GAF activity The effects of Pseudomonas culture filtrates on the germination of other plant species were evaluated using methods similar to those employed in the standard Poa bioassay for GAF activity. Sterile Corning Costar Tissue-Culture Plates sized appropriately for the specific seeds (No. 3548, 48-well plates; No. 3526, 24-well plates; No. 3513, 12-well plates; or No. 3516, 6-well plates) were used. The corresponding volumes of control and test solutions were 0.25 mL well 1 (48-well plates), 0.5 mL well 1 (24-well plates), 1.0 mL well 1 (12-well plates), and 2.5 mL well 1 (6-well plates). Three controls used in each trial included
Table 1 Origin and taxonomic identification of selected GI-DRB isolates Isolate
Isolate origin
Rhizosphere source of isolate
Taxonomic identification FAME analysis
rDNA sequencing
AD31
Alsea Valley, Benton County, OR
Poa sp.
Pseudomonas putida Biotype B/vancouverensis
Pseudomonas fluorescens
AH4
Alsea Valley, Benton County, OR
Poa sp.
Pseudomonas fluorescens Biotype A
Pseudomonas fluorescens
E34
OSU Hyslop Farm, Benton County, OR
Poa sp.
Pseudomonas fluorescens Biotype G/taetrolens
Pseudomonas fluorescens
WH6
OSU Hyslop Farm, Benton County, OR
Triticum aestivum
Pseudomonas fluorescens Biotype G/taetrolens
Pseudomonas fluorescens
WH19
OSU Hyslop Farm, Benton County, OR
Triticum aestivum
Pseudomonas fluorescens Biotype A
Pseudomonas fluorescens
Fatty acid methyl ester (FAME) analyses and DNA sequencing of a 400 + bp region of the 16S-rRNA gene were performed by Microcheck, Inc. (Northfield, VT). Taxonomic identities are based on Microcheck data bases.
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Table 2 Scoring systems for evaluation of seed germination of plant species Germination score
Developmental characteristics associated with germination scores A. Grasses and cereals
B. Dicots
0
No visible sign of germination
No visible sign of germination
0.5
Seed coat split. Coleoptile/plumule not visible
Seed has split. Radicle not emerged from seed
1.0
Both plumule and coleorhiza visible. Plumule chlorotic, shorter than length of seed. No root elongation, adventitious root initials visible in some species
Radicle emerged from seed coat. Cotyledons not visible
1.5
Plumule shorter than length of the seed, root is clearly visible and elongated
Radicle emerged and elongated. Cotyledons not clearly visible
2.0
Plumule approximately equal to length of seed. First true leaf not emerged from coleoptile. Roots present and elongated
Cotyledons visible but mostly enclosed in seed coat, or cotyledons emerged but exhibiting pronounced chlorosis
2.5
Plumule obviously longer than length of seed. First true leaf not emerged from coleoptile. Roots present and elongated
Cotyledons green and largely emerged from seed coat, or cotyledons fully emerged but pale green and not fully enlarged
3.0
First true leaf emerged from coleoptile and green. Emerged portion of leaf shorter in length than coleoptile. Roots obviously present and elongated
Cotyledons green and fully emerged. Apical hook pronounced (more than 90°), or apical hook not pronounced but root abnormal, brown, or oriented vertically
3.5
First true leaf emerged from coleoptile and green. Emerged portion of the leaf approximately equal in length to coleoptile. Roots present and elongated
Cotyledons emerged and green. Apical hook straightened to 90° or more, but shoot not fully erect. Root normal
4.0
First true leaf emerged from coleoptile and green. Emerged portion of leaf is obviously longer than coleoptile. Roots present and elongated
Cotyledons emerged from seed coat and green. Shoot is obviously erect
standard PMS medium with reduced glucose, water, and culture filtrate from P. fluorescens strain Pf-5. The PMS with reduced glucose contained 0.2 the standard glucose content, a level that approximated the reduced carbon levels that were measured in the culture filtrates after growth of the bacteria (data not shown). For dicot seeds, an appropriately sized sterile Whatman GF/D glass-fiber filter-pad was placed in the bottom of each well prior to adding the test solution. The number of seeds per well varied as indicated in footnotes to the corresponding table, but all treatments were replicated with 12 seeds because microbial contamination occurred occasionally in the wells. Wells contaminated with fungal or bacterial growth were not scored and experiments yielding fewer than nine seeds for analysis per treatment were discarded. Plates were sealed with ParafilmÒ and incubated at 20 °C (except as indicated) as previously described. Tests were scored at 14 d unless otherwise indicated using the scoring systems described elsewhere in this manuscript. Seeds of grassy weed species other than Poa sp. were scored at 21 d to accommodate slower germination rates.
analysis and 16S-rRNA DNA sequence analysis (Table 1). In contrast, strain AD31 was classified as P. fluorescens by rDNA analysis and P. putida by FAME analysis. The FAME profile of AD31 was distinctly different from those of the other four isolates. Profiles of WH6 and E34 were most similar. The 417 bp region of the 16S-rRNA DNA sequence for which overlapping forward and reverse sequence data were available was identical for each of the five isolates regardless of their origin or the rhizosphere source from which they were isolated. For comparison, the corresponding 16S-rDNA sequences for P. fluorescens ATCC_17574 (GenBank Accession No. AF094731) and PfO-1 (GenBank Accession No. CP000094, locus Pf1O1_R1) differed from these GI-DRB isolates by 9 and 10 base substitutions, respectively. The sequence for P. fluorescens Pf-5 (GenBank Accession No. CP000076, locus PFL_0119) was even more diverse, with 33 base substitutions, two single-base insertions, and one 8 base insertion relative to the sequence of these 5 GI-DRB strains.
3. Results
All five of the isolates arrested the germination of ABG seeds (Fig. 1A). Uninhibited germination of ABG seeds was observed in water, in PMS culture medium with reduced glucose, and in the presence of culture filtrates of Pf-5, a non-DRB strain. Fig. 1B shows that the filtrate of strain WH6 inhibited germination in a developmentally-specific manner, arresting the germination process
3.1. Origin and taxonomic identification of selected GI-DRB isolates Four of the five DRB strains selected for this study were clearly identified as strains of P. fluorescens by both FAME
3.2. Effect of culture filtrates on ABG seed germination
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immediately after emergence of the coleorhiza and plumule and causing chlorosis of the plumule. This germination arrest property was also characteristic of culture filtrates of the other four strains. Because the culture filtrates blocked germination at a stage after embryo emergence, the agent responsible for this activity was designated a Germination-Arrest Factor (GAF) rather than a germination inhibitor per se. 3.3. Development of a quantitative bioassay for GAF activity To measure and compare the levels of GAF activity in culture filtrates, as well as to facilitate future efforts at GAF purification, a quantitative bioassay for GAF activity was developed using ABG seeds. The criteria used to score GAF bioassays, and to evaluate the effects of GAF on other graminaceous species are summarized in Table 2, column A. An analogous scoring system was subsequently
developed for evaluating the effects of GAF on dicot plant species (Table 2, column B). Numerical values were based on a scale from 0 to 4, where 0 represented no visible signs of germination and 4 represented germination and seedling development equivalent to that of the untreated ABG controls. A score of 1.0 for graminaceous species corresponds to germination arrest at the stage illustrated in Fig. 1B. To quantify GAF activity in culture filtrates, a standard dilution curve was prepared using filtrate from strain WH6 diluted with water and the effect on ABG seed germination was determined. The WH6 culture filtrate could be diluted more than 5-fold before any decrease in GAF activity was detected (Fig. 2). In preliminary experiments, GAF production appeared to peak in 72 h cultures after which the amount of activity remained approximately the same (data not shown). From these data, it is possible to define GAF activity in terms of GAF Equivalents, where one GAF Equivalent is equal to the minimum quantity of GAF
Fig. 1. (A) Effect of filtrates from cultures of selected Pseudomonas fluorescens isolates on annual bluegrass (Poa annua) seed germination. Seeds were incubated for 7 d in the standard GAF bioassay. Water, PMS medium with glucose reduced to one-fifth of standard PMS, and culture filtrate from Pf-5 (a non-GAF-producing Pseudomonas) served as controls. White bar on the photograph is 4.0 mm long. (B) Annual bluegrass seed exhibiting germination score of 1.0 in standard 7 d GAF bioassay. White bar on the photograph is 1.5 mm long.
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in the arrest of seed germination at different developmental stages defined by germination scores of ca. 2.5–1.25.
4
GERMINATION SCORE
3.5
3.5. Effects of post-germination GAF treatment on ABG seedlings
3
2.5
In contrast to the irreversible arrest of seed germination observed after GAF treatment of ABG seeds, post-germination application of WH6 culture filtrate to 7 d-old germinated ABG seedlings only slowed the rate of elongation (Fig. 4). Shoot length continued to increase when exposure to WH6 culture filtrate occurred after germination.
2
1.5
1 0
0.05
0.1
0.15
0.2
0.25
0.3
CULTURE FILTRATE CONCENTRATION (Undiluted Culture Filtrate = 1.0)
Fig. 2. The relationship between germination score for Poa annua in the standard GAF bioassay and Pseudomonas fluorescens WH6 culture filtrate concentration. Dilutions of WH6 culture filtrate prepared with water were tested in standard GAF bioassay and germination scores assigned according to the system summarized in Table 2, where a score of 4.0 represents normal germination and seedling development and a score of 1.0 represents germination arrest immediately after the initiation of germination.
required to give a germination score of 1.0 when dissolved in 1 L and tested under the conditions described here. By this definition, the undiluted WH6 culture filtrate contains approximately 5 GAF Equivalents per liter. The other four DRB Pseudomonas strains selected for this study produced approximately equal amounts of GAF activity. In contrast, similar bioassays performed with culture filtrates of strains Pf-5, PfO-1, and D7 revealed no GAF activity, although PfO-1 and D7 slowed post-germination growth of ABG seedlings (Table 3). 3.4. Time course of the ABG seed response to GAF The response of ABG seeds to transient exposures of WH6 culture filtrate was quantified to determine the minimal exposure time required to arrest germination and to ascertain whether GAF effects could be reversed. Transient exposure of ABG seeds to WH6 culture filtrates for 24 h was sufficient to irreversibly arrest germination (germination score of 1; Fig. 3). Exposures from 4 to 20 h resulted
3.6. Species specificity of GAF herbicidal activity The species specificity of the GAF herbicidal activity was investigated using culture filtrates from P. fluorescens WH6 and P. fluorescens AD31. P. fluorescens Pf-5, a strain that lacked GAF activity in the Poa bioassay (Table 3), served as one of the controls. The results of tests of a large number of plant species are summarized in Table 4. The germination of tested Poa species was arrested by culture filtrates of GAF-producing strains WH6 and AD31 while culture filtrates from strain Pf-5 had little or no effect on their germination. In the case of pine bluegrass, Pf-5 filtrate appeared stimulatory. Undiluted PMS medium depressed the germination and growth of approximately half the Poa species tested relative to the water controls. The effect of the PMS medium was eliminated by dilution with water while GAF activity of the WH6 and AD31 culture filtrates was essentially unchanged at 0.3 concentration. Germination and seedling development of ABG seeds in full strength PMS medium and in Pf-5 culture filtrate were identical to that of the water controls, while germination arrest was complete at all tested concentrations of the AD31 and WH6 culture filtrates. Culture filtrates of WH6 and AD31 also arrested germination of grass species that are grown for seed and the effects were qualitatively similar to those obtained with the Poa species (Table 4). At full strength, PMS medium had an inhibitory effect, but after 3-fold dilution, this was reduced to approximately the germination score in the water control. Pf-5 culture filtrates had no apparent GAF activity relative to these species. In contrast, AD31 and
Table 3 Germination-arrest activity of culture filtrates from P. fluorescens WH6 and other selected Pseudomonas strains Bacterial source of culture filtrate
Control (PMS Medium) WH6 Pf-5 PfO-1 D7
Mean germination score at indicated dilution of culture filtrate (± SEM) 0.1
0.3
1.0 (undiluted)
4.0 ± 0.0 1.0 ± 0.0 4.0 ± 0.0 4.0 ± 0.0 4.0 ± 0.0
4.0 ± 0.0 1.0 ± 0.0 4.0 ± 0.0 4.0 ± 0.0 4.0 ± 0.0
4.0 ± 0.0 1.0 ± 0.0 4.0 ± 0.0 3.4 ± 0.1 3.0 ± 0.2
The standard Poa bioassay was used to assign germination scores according to criteria in Table 2. A score of 4.0 represents normal germination and a score of 1.0 represents germination arrest. The glucose concentration in the control medium was reduced to one-fifth that of standard PMS medium.
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GERMINATION SCORE
Poa annua Seedlings (7-Day Scores) 3
2
1
0 0
8
4
12
16
20
24
LENGTH OF TRANSIENT GAF EXPOSURE (hrs) Fig. 3. Effects of transient exposure to WH6 culture filtrate on the germination of annual bluegrass (Poa annua). Surface-sterilized seeds were allowed to imbibe water for 24 h and then exposed to P. fluorescens WH6 culture filtrate for indicated periods. After treatment, seeds were maintained in sterile distilled water and germination scores were assigned 7 d after the beginning of the imbibition according to the system summarized in Table 2, where a score of 4.0 represents normal germination and seedling development and a score of 1.0 represents germination arrest immediately after initiation of germination. Error bars indicate standard of the mean.
WH6 culture filtrates exerted a separate and distinct inhibitory effect on the progress of germination although
SHOOT LENGTH (mm)
30 25
INITIAL LENGTH (DAY 7)
FINAL LENGTH (DAY 14)
20 15 10 5
4. Discussion
W H
6
(4
8h r
s)
hr s)
s)
(4 8
(2 6
PM S
4
hr
hr s) (2 4 W H
(7
(7
da ys )
PM S
da ys ) W H 6
da ys ) PM S
(7
W at er
W A TE R
(D A Y
07)
0
germination scores tended to be higher than those observed for Poa species. However, after exposure to undiluted culture filtrate, none of the seven species had a germination score above 1.5. In two cases, a score of 2.0 was observed at 0.3 strength, but otherwise, the GAF activity was sufficient to arrest development before the plumule equaled the length of the seed. The WH6 and AD31 culture filtrates were highly effective in arresting germination of the seeds of eight different species of grassy weeds (Table 4). Exposure to culture filtrates of AD31 and WH6 resulted in germination scores of 1.5 or lower except for the seeds of barnyardgrass, which registered a score of 2.0 at 0.3 dilution of WH6 filtrate. Wheat (Triticum aestivum) and barley (Hordeum vulgare) also were sensitive to GAF. Seed germination typically progressed further than in the Poa species before being arrested, although in only one case did a germination score exceed 2.0 (Table 4). In contrast, seeds of the Syngenta Hybrid corn cultivar were much less sensitive to GAF (scores ranging from 2.2 to 2.8). Germination of the corn seeds was somewhat erratic under these test conditions and the scores for controls were also low; however, the results were consistent enough that any substantive effect of the culture filtrates on the germination process would have been evident. The seeds of eight dicot species from eight different plant families were also tested for their response to GAF in WH6 and AD31 culture filtrates (Table 4). Full strength PMS culture medium by itself exhibited a significant inhibitory effect on the germination of these dicots relative to a water control, and this effect could not be excluded from results obtained with undiluted culture filtrates of AD31 and WH6. However, at 0.3 concentration, the germination scores of all species were essentially identical to those in water for both PMS and Pf-5 controls and lower for AD31 and WH6, with the exception of N. tabacum, where little effect of GAF was observed at this concentration. Unlike the germination of grass seeds reported here, which were arrested (scores 62) by culture filtrates from WH6 and AD31 at a 0.3 concentration, the effect of GAF on these dicot species was much less pronounced.
TREATMENT INITIATED AT DAY 7 Fig. 4. Effects of post-germination GAF treatments on annual bluegrass (ABG, Poa annua) seedling growth. Sets of three ABG seeds were germinated in a total volume of 0.25 mL of water, allowed to develop for 7 d (germination score of 4), and then transferred to wells of 24-well microplates containing 0.5 mL well 1 of the indicated test solutions (water, PMS medium with reduced glucose, or P. fluorescens WH6 culture filtrate). Some of the seedlings were given only transient exposures (24 or 48 h) to WH6 culture filtrate or PMS medium with reduced glucose and then transferred to water. Shoot (plumule) length was measured 14 d after the initiation of germination (7 d following the beginning of treatment). Each data point represents the average score from 18 replicate seeds. Error bars indicate the standard error of the mean.
The five Pseudomonas GI-DRB strains examined in the current study share the characteristic of producing and secreting a compound, or compounds, that arrests germination of the seeds of sensitive plant species. Based on the sequence of a portion of the 16S-rDNA genes of these strains and other 16S-rDNA sequences in the data bases, we have classified them as P. fluorescens. However, FAME analysis for strain AD31 was sufficiently distinct that it could be classified as P. putida. The rDNA analysis of the 5 GI-DRB strains indicates these strains are more closely related to PfO-1, which was isolated from farm soil (Compeau et al., 1988) than the root colonizer Pf-5. The absence of sequence variation within the sequenced region
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Table 4 Effect of GAF on the germination of monocot and dicot seedsa Species/common name
Monocotyledenous plants Poa species Poa annua (Annual Bluegrass) var. reptans (Creeping Bluegrass) P. compressa (Canada Bluegrass) P. nemoralis (Wood Bluegrass) P. pratensis (Kentucky Bluegrass) cv. Viva P. scabrella (Pine Bluegrass) P. secunda (Big Bluegrass) cv. Sherman P. trivialis (Roughstalk Bluegrass) cv. Pro Am Grasses grown for seed Lolium perenne (Perennial Ryegrass) cv. Derby cv. Mach I L. multiflorum (Annual Ryegrass) cv. Gulf cv. Surrey Festuca arundinacea (Tall Fescue) cv. Kentucky 31 (Endophyte free) cv. Kentucky 31 (Endophyte) Dactylis glomerata (Orchardgrass) cv. Hallmark Grassy weed species Aegilops cylindrica (Jointed Goatgrass) Brachypodium distachyon (Purple False Brome) Brachypodium sylvaticum (Slender False Brome) Bromus carinatus (California Brome) Bromus tectorum (Downy Brome) Echinochloa crusgalli (Barnyardgrass) Vulpia myruos (Rattail Fescue) Cereals Triticum aestivum (Wheat) cv. Stephens cv. Madsen Hordeum vulgare (Barley) cv. Baronesse cv. Morex Zea mays (Corn) cv. Syngenta Hybrid GH2684F1 Dicotyledonous plants Amaranthaceae Amaranthus hypochondriacus L. (Amaranth) cv. Burgundy Brassicaceae Brassica oleraceae L. (Cabbage) cv. Early Jersey Wakefield Apiaceae Daucus carota L. (Carrot) cv. Imperator Caryophyllaceae Dianthus caryophyllus L. (Carnation) cv. Chabaud Blend
Mean germination scores ± standard error of the mean Control treatments Water
PMSb 1/0.3
Pf-5b 1/0.3
A31b 1/0.3
WH6b 1/0.3
4.0 ± 0.0 3.2 ± 0.4
4.0 ± 0.0/4.0 ± 0.0 3.0 ± 0.3/3.6 ± 0.3
4.0 ± 0.0/4.0 ± 0.0 3.1 ± 0.4/3.5 ± 0.2
1.0 ± 0.0/1.0 ± 0.0 0.9 ± 0.1/1.1 ± 0.1
1.0 ± 0.0/1.0 ± 0.0 1.0 ± 0.0/1.1 ± 0.0
3.7 ± 0.3 4.0 ± 0.0 2.8 ± 0.5
0.9 ± 0.3/2.4 ± 0.7 2.7 ± 0.3/4.0 ± 0.0 2.5 ± 0.4/2.7 ± 0.6
2.8 ± 0.4/3.3 ± 0.4 3.2 ± 0.2/4.0 ± 0.0 2.2 ± 0.4/3.7 ± 0.3
1.0 ± 0.0/1.0 ± 0.0 1.0 ± 0.0/1.0 ± 0.0 0.6 ± 0.1/0.8 ± 0.1
1.0 ± 0.0/1.0 ± 0.0 1.0 ± 0.0/1.0 ± 0.0 1.0 ± 0.0/0.9 ± 0.1
3.0 ± 0.5 1.9 ± 0.5
1.4 ± 0.6/3.0 ± 0.5 1.4 ± 0.4/2.3 ± 0.6
3.4 ± 0.2/3.3 ± 0.4 1.0 ± 0.7/1.6 ± 0.5
0.9 ± 0.1/1.3 ± 0.2 0.7 ± 0.1/0.9 ± 0.2
0.9 ± 0.2/1.2 ± 0.2 0.5 ± 0.5/0.7 ± 0.2
4.0 ± 0.0
4.0 ± 0.0/4.0 ± 0.0
3.8 ± 0.1/3.9 ± 0.1
1.0 ± 0.0/1.0 ± 0.0
1.0 ± 0.0/1.2 ± 0.1
4.0 ± 0.0 3.5 ± 0.4
3.7 ± 0.2/4.0 ± 0.0 3.6 ± 0.2/3.3 ± 0.4
3.2 ± 0.3/3.5 ± 0.4 3.0 ± 0.3/4.0 ± 0.0
1.5 ± 0.1/2.0 ± 0.0 1.0 ± 0.0/1.7 ± 0.1
1.4 ± 0.1/1.3 ± 0.2 1.1 ± 0.1/1.8 ± 0.2
3.9 ± 0.1 2.8 ± 0.5
2.3 ± 0.5/3.5 ± 0.4 2.5 ± 0.5/2.9 ± 0.5
2.5 ± 0.3/3.5 ± 0.4 1.8 ± 0.5/2.5 ± 0.4
1.2 ± 0.2/2.0 ± 0.0 0.9 ± 0.2/1.7 ± 0.3
1.5 ± 0.0/1.7 ± 0.2 1.0 ± 0.1/1.8 ± 0.2
4.0 ± 0.0 4.0 ± 0.0
3.0 ± 0.4/3.3 ± 0.4 3.7 ± 0.2/4.0 ± 0.0
3.0 ± 0.3/3.3 ± 0.4 3.1 ± 0.4/4.0 ± 0.0
1.0 ± 0.2/1.8 ± 0.2 1.0 ± 0.0/1.5 ± 0.0
1.0 ± 0.0/1.9 ± 0.2 1.1 ± 0.0/1.4 ± 0.1
4.0 ± 0.0
1.3 ± 0.3/3.3 ± 0.3
1.8 ± 0.2/3.7 ± 0.3
0.8 ± 0.1/1.3 ± 0.1
1.0 ± 0.2/1.4 ± 0.2
3.9 ± 0.1 4.0 ± 0.0
2.9 ± 0.3/2.5 ± 0.5 3.6 ± 0.2/4.0 ± 0.0
2.3 ± 0.4/3.2 ± 0.4 2.2 ± 0.1/3.9 ± 0.1
1.5 ± 0.1/1.5 ± 0.1 1.0 ± 0.0/1.5 ± 0.0
1.3 ± 0.5/1.3 ± 0.2 1.0 ± 0.0/1.5 ± 0.0
3.6 ± 0.3
2.8 ± 0.4/3.3 ± 0.4
1.7 ± 0.4/2.2 ± 0.4
2 ± 0.1/1.5 ± 0.1
1.2 ± 0.1/1.2 ± 0.1
3.4 ± 02 3.9 ± 01 2.3 ± 0.6 4.0 ± 0.0
2.6 ± 0.4/3.0 ± 0.5 2.6 ± 0.4/4.0 ± 0.0 1.1 ± 0.4/3.7 ± 0.3 3.6 ± 0.2/4.0 ± 0.0
2.6 ± 0.4/2.9 ± 0.5 2.8 ± 0.4/2.9 ± 0.5 1.0 ± 0.4/2.6 ± 0.6 2.7 ± 0.0/4.0 ± 0.0
1.3 ± 0.1/1.2 ± 0.1 1.4 ± 0.1/1.2 ± 0.2 0.4 ± 0.3/1.5 ± 0.5 1.0 ± 0.0/1.5 ± 0.0
1.0 ± 0.2/1.4 ± 0.2 1.0 ± 0.2/1.4 ± 0.1 0.8 ± 0.4/2.0 ± 0.5 1.0 ± 0.0/1.0 ± 0.0
3.5 ± 0.2 3.5 ± 0.2
2.9 ± 0.4/3.7 ± 0.3 2.4 ± 0.4/3.8 ± 0.1
2.1 ± 0.4/2.9 ± 0.4 1.3 ± 0.2/2.8 ± 0.4
1.1 ± 0.1/1.1 ± 0.1 1.2 ± 0.1/1.7 ± 0.1
1.1 ± 0.1/1.5 ± 0.1 1.5 ± 0.1/1.8 ± 0.1
3.5 ± 0.2 3.2 ± 0.3
3.5 ± 0.2/3.4 ± 0.3 2.9 ± 0.4/3.6 ± 0.2
2.7 ± 0.4/3.5 ± 0.3 3.3 ± 0.3/2.5 ± 0.3
1.5 ± 0.0/2.0 ± 0.2 1.4 ± 0.1/2.0 ± 0.2
1.5 ± 0.2/2.3 ± 0.2 1.6 ± 0.1/1.8 ± 0.2
2.8 ± 0.1
2.4 ± 0.2/2.8 ± 0.1
2.0 ± 0.3/2.6 ± 0.2
2.3 ± 0.1/2.8 ± 0.2
2.2 ± 0.1/2.7 ± 0.1
3.9 ± 0.1
2.3 ± 0.1/4.0 ± 0.0
2.5 ± 0.0/4.0 ± 0.0
2.5 ± 0.0/3.5 ± 0.1
2.3 ± 0.0/3.5 ± 0.1
4.0 ± 0.0
2.9 ± 0.3/3.9 ± 0.1
2.8 ± 0.3/3.7 ± 0.1
2.5 ± 0.3/3.2 ± 0.1
2.9 ± 0.2/3.3 ± 0.3
2.8 ± 0.4
1.4 ± 0.2/3.8 ± 0.2
1.8 ± 0.1/4.0 ± 0.0
1.4 ± 0.2/3.4 ± 0.2
1.4 ± 0.1/3.4 ± 0.2
3.9 ± 0.1
3.0 ± 0.1/3.8 ± 0.1
3.2 ± 0.1/3.9 ± 0.1
3.0 ± 0.1/3.0 ± 0.1
2.7 ± 0.2/3.1 ± 0.3 (continued on next page)
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Table 4 (continued) Species/common name
Mean germination scores ± standard error of the mean Control treatments Water
PMSb 1/0.3
Pf-5b 1/0.3
A31b 1/0.3
WH6b 1/0.3
Asteraceae Lactuca sativa L. (Lettuce) cv. Butter Crunch
3.6 ± 0.2
2.8 ± 0.2/3.9 ± 0.1
3.0 ± 0.0/3.9 ± 0.1
2.6 ± 0.3/3.1 ± 0.1
3.1 ± 0.0/3.3 ± 0.1
Fabaceae Medicago sativa L. (Alfalfa) cv. Unknown
3.8 ± 0.1
2.0 ± 0.3/3.3 ± 0.4
2.2 ± 0.3/3.8 ± 0.1
1.9 ± 0.4/2.9 ± 0.3
2.2 ± 0.2/3.4 ± 0.2
Solanaceae Nicotiana tabacum L. (Tobacco) cv. Wisconsin
3.8 ± 0.1
1.5 ± 0.0/3.4 ± 0.2
1.5 ± 0.0/3.1 ± 0.2
1.5 ± 0.0/3.5 ± 0.1
1.5 ± 0.0/3.4 ± 0.1
Papaveraceae Papaver nudicaule L. (Iceland Poppy) cv. Nudicaule Blend
2.9 ± 0.5
2.0 ± 0.2/3.2 ± 0.3
1.8 ± 0.2/3.2 ± 0.3
1.9 ± 0.2/2.3 ± 0.2
1.6 ± 0.2/2.5 ± 0.3
a Germination scores were assigned according to the system summarized in Table 2, where a score of 4.0 represents normal germination as defined for the indicated plant group. A score of 1.0 represents germination arrest immediately after initiation of germination. Bioassay conditions were modified to account for seed size and germination behavior. Tests with Poa sp. involved 48-well plates, 4 seeds per well; commercial grass seed species and grassy weeds, 24-well plates, 2 seeds per well, except for barnyardgrass where 3 seeds were used per well; cereals, 12-well plates, one seed per well, except corn where 6-well plates were used, 1 seed per well (day incubation temperature of 25 oC); dicots, 24-well plates, one seed per well, except for tobacco and poppy where 48-well plates, one seed per well were used. A day temperature of 25 oC was used for alfalfa, tobacco, amaranth, carnation and poppy seeds. Seeds of slender false brome were given a stratification treatment involving imbibition of treatment solutions at 6 oC for 3 weeks to break dormancy prior to placing in the incubator to initiate germination. b PMS medium with 0.2 the standard glucose content, and culture filtrates were used either undiluted (1) or diluted (0.3).
of the 16S-rDNA indicates a genetic relationship among these strains, and the similar and specific effects of the herbicide they produce suggest that the same compound is produced by all five strains. Additional sequence analyses, as well as purification of the compound(s) will be required to confirm these conclusions. Because of its specific mode of action, we have termed this compound a GerminationArrest Factor (GAF). Culture filtrates from P. fluorescens strain D7 failed to arrest germination of P. annua seed in the bioassay developed in this study, suggesting that the inhibition of B. tectorum seed germination and the capacity to stunt the growth of other grassy weeds (Gurusiddaiah et al., 1994; Kennedy et al., 2001) is not through GAF activity. The fact that D7 also stimulated germination of Poa pratensis, and a number of other grasses, in contrast to the germination arrest we observed with GAF implies that D7 biological activity is mediated through other compounds (Kennedy et al., 2001). Strain Pf-5 colonizes roots and has been shown to produce a broad spectrum of antibiotics, including pyrrolnitrin (Pfender et al., 1993), pyoluterorin (Nowak-Thompson et al., 1997, 1999), 2,4diacetylphloroglucinol (Sharifi-Tehrani et al., 1998), hydrogen cyanide (Laville et al., 1998) and siderophores (Loper and Buyer, 1988). These compounds act to suppress the bacterial pathogen Erwinia caratovora and a number of fungal pathogens that attack a variety of plant hosts including pea, cucumber and maize (Kennedy et al., 2001). It was of interest, therefore, that Pf-5 had no GAF activity in the Poa bioassay developed for this study
(Table 3) suggesting that another compound or compounds are responsible for GAF activity. The action of GAF affected seed germination of a large number of grasses, including all grassy weeds examined in this study. The eight Poa species tested were sensitive to GAF, as were the seeds of grassy weeds such as jointed goatgrass, downy brome, false brome, and a number of others of agricultural significance. Among the Graminaceae tested, only the seeds of corn appeared to be unaffected by this compound. The inhibitory effect of the PMS medium on the germination of the dicot species precluded an unequivocal interpretation, but it is clear that all species tested were able to overcome the effects of GAF at concentrations sufficient to arrest germination of the grass species tested. Based upon the results that utilized the bioassay system developed in this study, GAF has preferential specificity for arresting germination of the seeds of a variety of grass species. These results also substantiate the observation that GAF activity differs from the herbicidal effects previously reported for strain D7. Whereas GAF activity in both undiluted and 0.3 concentration culture filtrates arrests jointed goatgrass seed germination (scores of 1.3– 1.5, Table 4), Kennedy et al. (2001) reported that D7 culture filtrates had a slightly stimulatory effect on the germination of this weed. In addition to differences in sensitivity of grasses and dicots to GAF, the action of GAF exhibits developmental specificity. Sensitive grass seeds are arrested immediately after the plumule and the coleorhiza have emerged from the seed coat, but typically before the plumule has reached
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a length equal to that of the seed (i.e., at a germination score < 2.0). Moreover, grass seedlings have reduced sensitivity to GAF treatment after germination has occurred. Growth is slowed, but not halted, if the seedlings are treated after the first true leaf has emerged. These results are supported by findings in preliminary tests using partially purified GAF preparations (data not shown), which suggest that GAF has little, if any effect on mature grass plants. The species specificity of GAF, combined with its developmentally-specific mode of action, suggests that this naturally-occurring herbicide could potentially be useful for the control of grassy weeds. The fact that transient exposure of sensitive grass seeds to GAF irreversibly arrests germination suggests that GAF has potential as a preemergence herbicide. An agent which does not affect post-germination plant growth should prove effective for the control of grassy weeds in grass seed production systems where the established perennial grass crop would presumably be unaffected by GAF. The presence of any seed, other than that of the present year crop, represents contamination in these production systems. ABG seed contamination is a significant problem in grass seed production fields because in the environment of the Willamette Valley, this weed produces and sheds seed throughout the cool, wet fall, winter, and spring. However, P. annua seed is highly sensitive to GAF, as are Diuron-resistant variants that have recently appeared in grass seed cropping systems (data not shown). GAF would also appear to have potential for the management of grassy weeds in established turf settings, including golf courses, public parks, and residential lawns. However, tests of the practical efficacy of GAF, or GAF-producing isolates, in field settings must await the purification, identification, and chemical synthesis of purified samples of the active compound. The use of live DRB to control grassy weeds remains a possible alternative to the use of chemical herbicides (Kennedy et al., 1991; Kennedy and Stubbs, 2007), and it is conceivable that the five GI-DRB strains identified in the present study may be useful in this context. Success in the use of live DRB as weed biocontrol agents is strongly dependent, however, on biotic and abiotic factors over which producers have little control (Alexander, 1977; Kennedy and Kremer, 1996; Huang and Madan, 1999; Li et al., 2003). Nevertheless, identification of microorganisms that produce bioherbicides is critical, and it is likely that delivery systems will continue to be developed that improve the persistence of these agents in soil. The developmental and species specificity of GAF suggests that this compound may have a unique mechanism of action. If this is indeed the case, identification of the chemical structure of GAF would provide the basis for development of a new class of herbicidal compounds with properties of distinct advantage in the control of grassy weeds. In preliminary tests, a large number of other grassy species also appear to be sensitive to GAF (data not shown). This possibility provides motivation for the ongoing effort in our laboratories to purify and characterize the
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compound responsible for GAF activity. The availability of a quantitative bioassay for GAF activity, as developed in the present study, should facilitate these efforts. 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 Alexander, M., 1977. Introduction to Soil Microbiology, second ed. J. Wiley, New York, pp. 423–437. Azevedo, M.D., Welty, R.E., 1995. A study of the fungal endophyte, Acermonium coenophialum, in the roots of tall fescue seedlings. Mycologia 87, 289–297. Bolton, H., Elliot, L.F., Gurusiddaiah, S., Fredrickson, J.K., 1989. Characterization of a toxin produced by a rhizobacterial Pseudomonas species 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. Chenna, R., Sugawara, H., Koike, T., Lopez, R., Gibson, T.J., Higgins, D.G., Thompson, J.D., 2003. Multiple sequence alignment with the Clustal series of programs. Nucleic Acids Research 31, 3497–3500. Cherrington, C.A., Elliott, L.F., 1987. Incidence of inhibitory pseudomonads in the Pacific Northwest. Plant and Soil 101, 159–165. Compeau, G., Al-Achi, B.J., Platsouka, E., Levy, S.B., 1988. Survival of rifampicin-resistant mutants of Pseudomonas fluorescens and Pseudomonas putida in soil systems. Applied and Environmental Microbiology 54, 2432–2438. Corbell, N., Loper, J.E., 1995. A global regulator of secondary metabolite production in Pseudomonas fluorescens Pf-5. Journal of Bacteriology 177, 6230–6236. Elliott, L.F., Azevedo, M.D., Mueller-Warrant, G.W., Horwath, W.R., 1998. Weed control with rhizobacteria. Soil Science, Agrochemistry, and Ecology 33, 3–7. 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. Journal of Applied Microbiology 100, 946–954. Gurusiddaiah, S., Gealy, D.R., Kennedy, A.C., Ogg Jr., A.G., 1994. Isolation and characterization of metabolites from Pseudomonas fluorescens D7 for control of downy brome (Bromus tectorum). Weed Science 42, 492–501. Howell, C.R., Stipanovic, R.D., 1979. Control of Rhizoctonia solani on cotton seedlings with Pseudomonas fluorescens and with an antibiotic produced by the bacterium. Phytopathology 69, 480–482. Huang, X., Madan, A., 1999. CAP3: a DNA sequence assembly program. Genome Research 9, 868–877. Kennedy, A.C., Young, F.L., Elliott, L.F., Douglas, C.L., 1991. Rhizobacteria suppressive to the weed downy brome. Soil Science Society of America Journal 55, 722–727. Kennedy, A.C., Johnson, B.N., Stubbs, T.L., 2001. Host range of a deleterious rhizobacterium for biological control of downy brome. Weed Science 49, 792–797.
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Biological Control 46 (2008) 380–390 www.elsevier.com/locate/ybcon
Germination-Arrest Factor (GAF): Biological properties of a novel, naturally-occurring herbicide produced by selected isolates of rhizosphere bacteria Gary M. Banowetz a,*, Mark D. Azevedo a, Donald J. Armstrong b, Anne B. Halgren b, Dallice I. Mills b a
USDA-ARS NFSPRC National Forage Seed Production Research Center, 3450 S.W. Campus Way, Corvallis, OR 97331, USA b Department of Botany and Plant Pathology, Oregon State University, Corvallis, OR 97331, USA Received 14 January 2008; accepted 12 April 2008 Available online 29 April 2008
Abstract Five strains of deleterious rhizosphere bacteria (DRB), identified as Pseudomonas fluorescens, were shown to produce and secrete a naturally-occurring herbicide that targets the seeds of certain graminaceous plants, including annual bluegrass (ABG; Poa annua L.), considered a weed in many agronomic systems. The herbicide arrests germination of the seeds in a developmentally-specific manner, typically irreversibly blocking the germination process immediately after the emergence of the plumule and coleorhiza. Because of its unique mode of action, this herbicide has been termed a Germination-Arrest Factor (GAF). Bacterial-culture filtrates containing GAF arrest seed germination of a large number of graminaceous species, including grassy weeds and grass species grown for seed and food. The germination of corn seeds did not appear to be affected by the herbicide. The seeds of dicot species appear to be less sensitive to GAF than seeds of graminaceous species. The biological effects of GAF on graminaceous species is primarily limited to germination, although transient slowing of ABG seedling growth was observed after post-germination exposure to GAF. Exposure of non-germinated ABG seeds to GAF for periods as short as 24 h was sufficient to irreversibly arrest germination. A quantitative bioassay for GAF was developed based on the sensitivity of ABG seed to this compound. Published by Elsevier Inc. Keywords: Microbial herbicide; Biological weed control; Deleterious rhizobacteria; Annual bluegrass
1. Introduction The control of grassy weeds is a significant problem in a number of agricultural settings, in recreational and professional lawns and turf, and in natural habitats where invasive non-native grasses are a concern. Grassy weeds are a particular problem in grass seed production systems where contamination of commercial seed lots with seeds of grassy weeds, such as annual bluegrass (ABG, Poa annua L.), significantly reduces the quality and market value of the product. In the Pacific Northwest, state-man-
*
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.2008.04.016
dated reductions in post-harvest field burning and the development of strains of ABG and other grassy weeds that are resistant to the chemical herbicides most commonly used in conjunction with field burning have exacerbated the problem. Consequently, the development of alternative and more effective measures for the control of grassy weeds is necessary. The potential utility of deleterious rhizosphere bacteria (DRB) as agents for weed control has attracted the interest of a number of investigators. DRB are defined as rhizosphere-inhabiting bacteria (usually Pseudomonas sp.) that have a negative impact on the growth or development of higher plant species (Suslow and Schroth, 1982; Cherrington and Elliott, 1987; Boyetchko, 1997). DRB isolates have been investigated as potential agents for
G.M. Banowetz et al. / Biological Control 46 (2008) 380–390
the biocontrol of a number of weed species in a range of agricultural crops (Elliott and Lynch, 1985; Schippers et al., 1987; Kremer et al., 1990; Kremer and Kennedy, 1996; Kremer et al., 2000; Kennedy et al., 2001; FloresVargas and O’Hara, 2006; Li and Kremer, 2006). However, the introduction and successful establishment of live bacteria in a field setting is influenced by a multitude of environmental and biotic factors that are poorly understood (Van Elsas et al., 1992; Skipper et al., 1996; Zdor et al., 2005). For these reasons, direct applications of DRB have achieved only limited success as weed control measures (Li et al., 2003). We have been interested in identifying DRB that specifically target the germination of grassy weed seeds and in exploring the possibility that compounds produced by such DRB, rather than the bacteria themselves, may be useful in the control of these weeds. The starting point for our investigations has been a collection of over 30 rhizosphere bacterial strains that were isolated and subsequently maintained at the USDA-ARS National Forage Seed Production Research Center in Corvallis, Oregon. These isolates were originally obtained by Elliott and co-workers from selected locations within the Willamette Valley in Oregon (Elliott, Personal Communication; Elliott et al., 1998) and retained because they appeared to exert a stunting effect on ABG seedlings in laboratory tests using live bacteria. At least 12 isolates appeared to interfere with germination of ABG seeds (Elliott et al., 1998), but the biological specificity, mechanism of action, and the nature of the active agent or agents responsible for the herbicidal activity were not determined. We identified five isolates from the Elliott collection that, in our preliminary studies, were particularly active in suppressing the germination of ABG seeds. ABG was selected as the target weed species for these studies because ABG seed is a significant contaminant in grass seed production. In work reported here, we identified these germination-inhibiting DRB isolates (GI-DRB) as particular strains of Pseudomonas fluorescens, and we demonstrated that these bacterial strains produce and secrete a compound that specifically blocks germination of the seeds of ABG and a large number of other grassy weeds in a developmentally-specific manner. Because of the particular effect of this compound on the germination process, we have called it a Germination-Arrest Factor (GAF). The biological properties of GAF and the development of a quantitative bioassay for this naturally-occurring herbicide are described. 2. Materials and methods 2.1. Bacterial isolates and propagation The five GI-DRB isolates selected for this study were compared with strains of P. fluorescens obtained from other laboratories. Strain D7 (P. fluorescens D7: NRRL B-18293), a root-colonizing bacterium that inhibits downy
381
brome (Bromus tectorum L. BROTE) root growth and seed germination in plant–soil bioassays (Gurusiddaiah et al., 1994; Kennedy et al., 1991, 2001), was provided by Dr. Ann Kennedy, USDA-ARS, Pullman, WA. P. fluorescens strain Pf-5 (Howell and Stipanovic, 1979; Corbell and Loper, 1995) was obtained from Dr. Joyce Loper (USDA-ARS Horticultural Crops Research Laboratory, Corvallis, OR) and used as a non-GAF-producing control in these experiments. P. fluorescens strain PfO-1 was obtained from Dr. Mark Silby, Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, MA (Compeau et al., 1988). All bacteria were propagated in a Pseudomonas Minimal Salts (PMS) medium based on that described by Bolton et al. (1989). The Bolton medium was supplemented with iron as follows: 2 mL of a 1 mM filter-sterilized stock solution of FeCl3 dissolved in 10 mM HCl was added to 1 L of medium. One milliliter aliquots of cultures grown to an OD580 of 1.0 were placed in 50% glycerol and stored in cryovials at 60 °C prior to use. Taxonomic identification of the five GI-DRB was made by Microcheck, Inc. (Northfield, VT) based on Fatty Acid Methyl Ester Analysis (FAME Analysis) and on sequencing of a variable 400 + bp DNA region near the beginning of the 16S-rRNA gene. Fatty acid profiles were analyzed using Sherlock, Version 6.0, software (MIDI, Inc., Newark, DE). Contiguous DNA sequences were assembled using CAP3 (Huang and Madan, 1999) and aligned using ClustalW (Chenna et al., 2003). 2.2. Preparation of culture filtrates from bacterial isolates GI-DRB strains removed from cryovial storage were inoculated into Wheaton bottles of varying sizes, dependent upon the required volume of culture filtrate. Each bottle was half-filled with Fe-containing PMS Medium and placed on a rotary shaker (200 rpm) at 27 °C. The cultures were harvested after 7 d (OD580 = 0.8–1.2) and centrifuged (3000g, 15 min). The resulting supernatants were filtered through Millipore GP Express Steritop Bacteriological Filters (0.22 lm), and the sterile culture filtrates were stored at 4 °C prior to use. 2.3. Sources of seeds ABG seeds were obtained from 1996 mid-Willamette Valley grass seed screenings from International Seeds, Halsey, OR and C and R Farm, Tangent, OR. The seeds were cleaned to remove straw and other seeds. Seeds of the other Poa species and wheat were obtained from the Oregon State University (OSU) Seed Testing Laboratory. Seeds of other grasses were obtained from various OSU, USDA, and other local sources. The seeds of all the dicot species were purchased locally (Corvallis, OR) from garden supply stores with the exception of tobacco (Nicotiana tabacum L., cv. Wisconsin 38) seed which was grown in the greenhouse at OSU.
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2.4. Surface sterilization of seeds Surface sterilization of seeds was required to prevent microbial growth within the bioassay environment. The contrasting characteristics of seeds from different species required development of specific procedures to accomplish surface sterilization without significantly impacting subsequent germination of the seeds. All Poa seeds were surface-sterilized using the methods of Lowe and Conger (1979) as modified by Azevedo and Welty(1995). The seeds were stirred for 5 min in 50% sulfuric acid, followed by extensive washing with sterile deionized water in a Gooch crucible. The washed seeds were stirred with freshly prepared 100% CloroxÒ (6–7.35% NaOCl) containing 1% Tween 20 for 5 min in a sterile beaker, filtered (Gooch crucible), and washed again with sterile water. The seeds were either used immediately or dried under a laminar flow hood and stored in a closed sterile Petri dish at room temperature for future use. Seeds of all other grasses were surface-sterilized using the same procedure with the following exceptions: tall fescue and downy brome, treatments in 50% sulfuric acid and 100% CloroxÒ were extended to 20 min each; California brome, treatments in sulfuric acid and CloroxÒ were extended to 10 min; jointed goatgrass, treatment with 50% sulfuric acid remained at 5 min, but the CloroxÒ treatment was extended to 10 min. Barnyardgrass seeds were stirred in 1% Tween 20 for 5 min after which an equal volume of 50% CloroxÒ was added with continued stirring for 5 min. All seeds were filtered and washed with sterile deionized water in a Gooch crucible and used immediately, or dried under laminar flow air and stored at 22 °C in sterile Petri dishes for immediate use, or dried under laminar flow air and stored at room temperature in a closed Petri dish for later use. Wheat, barley, and corn seeds were surface-sterilized by stirring in 50% sulfuric acid for 5 min followed by extensive washing with sterile deionized water in a Gooch crucible. Washed seeds were transferred to a sterile beaker and stirred with 1% Tween 20 for two 1 min treatments (decanting and replacing the solution between treatments). The final Tween solution was replaced with 10% Clorox (wheat) or 20% Clorox (barley and corn). The seeds were stirred in
the Clorox solution for two 2 min treatments, filtered, and washed extensively with sterile deionized water. Surface-sterilized seeds were transferred to a sterile Petri dish and used immediately. Seeds of dicots were stirred with 0.5% Tween 20 for 1 min after which an equal volume of 100% CloroxÒ was added with continued stirring for 2 min. Following this treatment, the seeds were extensively washed with sterile deionized water in a Gooch crucible before transfer to a sterile Petri dish. For very small seeds, filtration and washing was accomplished using a plastic Millipore filter holder (without the filter, using the filter-support screen to retain the seeds) attached to an appropriately sized syringe. Surface-sterilized seeds were transferred to a sterile Petri dish and used immediately. 2.5. Standard Poa bioassay for GAF activity A standard quantitative bioassay for GAF activity was established using ABG seeds. Culture filtrates and other solutions to be tested for GAF activity were distributed to the wells of sterile 48-well plates (Corning Costar 3548). Each well received 200 lL of test solution per well, and three surface-sterilized ABG seeds. Three replicate wells were prepared for each treatment, nine seeds total. The plates were sealed with ParafilmÒ 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. Seed germination was scored after 7 d using the scoring system described in (Table 2). 2.6. Species specificity of GAF activity The effects of Pseudomonas culture filtrates on the germination of other plant species were evaluated using methods similar to those employed in the standard Poa bioassay for GAF activity. Sterile Corning Costar Tissue-Culture Plates sized appropriately for the specific seeds (No. 3548, 48-well plates; No. 3526, 24-well plates; No. 3513, 12-well plates; or No. 3516, 6-well plates) were used. The corresponding volumes of control and test solutions were 0.25 mL well 1 (48-well plates), 0.5 mL well 1 (24-well plates), 1.0 mL well 1 (12-well plates), and 2.5 mL well 1 (6-well plates). Three controls used in each trial included
Table 1 Origin and taxonomic identification of selected GI-DRB isolates Isolate
Isolate origin
Rhizosphere source of isolate
Taxonomic identification FAME analysis
rDNA sequencing
AD31
Alsea Valley, Benton County, OR
Poa sp.
Pseudomonas putida Biotype B/vancouverensis
Pseudomonas fluorescens
AH4
Alsea Valley, Benton County, OR
Poa sp.
Pseudomonas fluorescens Biotype A
Pseudomonas fluorescens
E34
OSU Hyslop Farm, Benton County, OR
Poa sp.
Pseudomonas fluorescens Biotype G/taetrolens
Pseudomonas fluorescens
WH6
OSU Hyslop Farm, Benton County, OR
Triticum aestivum
Pseudomonas fluorescens Biotype G/taetrolens
Pseudomonas fluorescens
WH19
OSU Hyslop Farm, Benton County, OR
Triticum aestivum
Pseudomonas fluorescens Biotype A
Pseudomonas fluorescens
Fatty acid methyl ester (FAME) analyses and DNA sequencing of a 400 + bp region of the 16S-rRNA gene were performed by Microcheck, Inc. (Northfield, VT). Taxonomic identities are based on Microcheck data bases.
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Table 2 Scoring systems for evaluation of seed germination of plant species Germination score
Developmental characteristics associated with germination scores A. Grasses and cereals
B. Dicots
0
No visible sign of germination
No visible sign of germination
0.5
Seed coat split. Coleoptile/plumule not visible
Seed has split. Radicle not emerged from seed
1.0
Both plumule and coleorhiza visible. Plumule chlorotic, shorter than length of seed. No root elongation, adventitious root initials visible in some species
Radicle emerged from seed coat. Cotyledons not visible
1.5
Plumule shorter than length of the seed, root is clearly visible and elongated
Radicle emerged and elongated. Cotyledons not clearly visible
2.0
Plumule approximately equal to length of seed. First true leaf not emerged from coleoptile. Roots present and elongated
Cotyledons visible but mostly enclosed in seed coat, or cotyledons emerged but exhibiting pronounced chlorosis
2.5
Plumule obviously longer than length of seed. First true leaf not emerged from coleoptile. Roots present and elongated
Cotyledons green and largely emerged from seed coat, or cotyledons fully emerged but pale green and not fully enlarged
3.0
First true leaf emerged from coleoptile and green. Emerged portion of leaf shorter in length than coleoptile. Roots obviously present and elongated
Cotyledons green and fully emerged. Apical hook pronounced (more than 90°), or apical hook not pronounced but root abnormal, brown, or oriented vertically
3.5
First true leaf emerged from coleoptile and green. Emerged portion of the leaf approximately equal in length to coleoptile. Roots present and elongated
Cotyledons emerged and green. Apical hook straightened to 90° or more, but shoot not fully erect. Root normal
4.0
First true leaf emerged from coleoptile and green. Emerged portion of leaf is obviously longer than coleoptile. Roots present and elongated
Cotyledons emerged from seed coat and green. Shoot is obviously erect
standard PMS medium with reduced glucose, water, and culture filtrate from P. fluorescens strain Pf-5. The PMS with reduced glucose contained 0.2 the standard glucose content, a level that approximated the reduced carbon levels that were measured in the culture filtrates after growth of the bacteria (data not shown). For dicot seeds, an appropriately sized sterile Whatman GF/D glass-fiber filter-pad was placed in the bottom of each well prior to adding the test solution. The number of seeds per well varied as indicated in footnotes to the corresponding table, but all treatments were replicated with 12 seeds because microbial contamination occurred occasionally in the wells. Wells contaminated with fungal or bacterial growth were not scored and experiments yielding fewer than nine seeds for analysis per treatment were discarded. Plates were sealed with ParafilmÒ and incubated at 20 °C (except as indicated) as previously described. Tests were scored at 14 d unless otherwise indicated using the scoring systems described elsewhere in this manuscript. Seeds of grassy weed species other than Poa sp. were scored at 21 d to accommodate slower germination rates.
analysis and 16S-rRNA DNA sequence analysis (Table 1). In contrast, strain AD31 was classified as P. fluorescens by rDNA analysis and P. putida by FAME analysis. The FAME profile of AD31 was distinctly different from those of the other four isolates. Profiles of WH6 and E34 were most similar. The 417 bp region of the 16S-rRNA DNA sequence for which overlapping forward and reverse sequence data were available was identical for each of the five isolates regardless of their origin or the rhizosphere source from which they were isolated. For comparison, the corresponding 16S-rDNA sequences for P. fluorescens ATCC_17574 (GenBank Accession No. AF094731) and PfO-1 (GenBank Accession No. CP000094, locus Pf1O1_R1) differed from these GI-DRB isolates by 9 and 10 base substitutions, respectively. The sequence for P. fluorescens Pf-5 (GenBank Accession No. CP000076, locus PFL_0119) was even more diverse, with 33 base substitutions, two single-base insertions, and one 8 base insertion relative to the sequence of these 5 GI-DRB strains.
3. Results
All five of the isolates arrested the germination of ABG seeds (Fig. 1A). Uninhibited germination of ABG seeds was observed in water, in PMS culture medium with reduced glucose, and in the presence of culture filtrates of Pf-5, a non-DRB strain. Fig. 1B shows that the filtrate of strain WH6 inhibited germination in a developmentally-specific manner, arresting the germination process
3.1. Origin and taxonomic identification of selected GI-DRB isolates Four of the five DRB strains selected for this study were clearly identified as strains of P. fluorescens by both FAME
3.2. Effect of culture filtrates on ABG seed germination
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immediately after emergence of the coleorhiza and plumule and causing chlorosis of the plumule. This germination arrest property was also characteristic of culture filtrates of the other four strains. Because the culture filtrates blocked germination at a stage after embryo emergence, the agent responsible for this activity was designated a Germination-Arrest Factor (GAF) rather than a germination inhibitor per se. 3.3. Development of a quantitative bioassay for GAF activity To measure and compare the levels of GAF activity in culture filtrates, as well as to facilitate future efforts at GAF purification, a quantitative bioassay for GAF activity was developed using ABG seeds. The criteria used to score GAF bioassays, and to evaluate the effects of GAF on other graminaceous species are summarized in Table 2, column A. An analogous scoring system was subsequently
developed for evaluating the effects of GAF on dicot plant species (Table 2, column B). Numerical values were based on a scale from 0 to 4, where 0 represented no visible signs of germination and 4 represented germination and seedling development equivalent to that of the untreated ABG controls. A score of 1.0 for graminaceous species corresponds to germination arrest at the stage illustrated in Fig. 1B. To quantify GAF activity in culture filtrates, a standard dilution curve was prepared using filtrate from strain WH6 diluted with water and the effect on ABG seed germination was determined. The WH6 culture filtrate could be diluted more than 5-fold before any decrease in GAF activity was detected (Fig. 2). In preliminary experiments, GAF production appeared to peak in 72 h cultures after which the amount of activity remained approximately the same (data not shown). From these data, it is possible to define GAF activity in terms of GAF Equivalents, where one GAF Equivalent is equal to the minimum quantity of GAF
Fig. 1. (A) Effect of filtrates from cultures of selected Pseudomonas fluorescens isolates on annual bluegrass (Poa annua) seed germination. Seeds were incubated for 7 d in the standard GAF bioassay. Water, PMS medium with glucose reduced to one-fifth of standard PMS, and culture filtrate from Pf-5 (a non-GAF-producing Pseudomonas) served as controls. White bar on the photograph is 4.0 mm long. (B) Annual bluegrass seed exhibiting germination score of 1.0 in standard 7 d GAF bioassay. White bar on the photograph is 1.5 mm long.
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in the arrest of seed germination at different developmental stages defined by germination scores of ca. 2.5–1.25.
4
GERMINATION SCORE
3.5
3.5. Effects of post-germination GAF treatment on ABG seedlings
3
2.5
In contrast to the irreversible arrest of seed germination observed after GAF treatment of ABG seeds, post-germination application of WH6 culture filtrate to 7 d-old germinated ABG seedlings only slowed the rate of elongation (Fig. 4). Shoot length continued to increase when exposure to WH6 culture filtrate occurred after germination.
2
1.5
1 0
0.05
0.1
0.15
0.2
0.25
0.3
CULTURE FILTRATE CONCENTRATION (Undiluted Culture Filtrate = 1.0)
Fig. 2. The relationship between germination score for Poa annua in the standard GAF bioassay and Pseudomonas fluorescens WH6 culture filtrate concentration. Dilutions of WH6 culture filtrate prepared with water were tested in standard GAF bioassay and germination scores assigned according to the system summarized in Table 2, where a score of 4.0 represents normal germination and seedling development and a score of 1.0 represents germination arrest immediately after the initiation of germination.
required to give a germination score of 1.0 when dissolved in 1 L and tested under the conditions described here. By this definition, the undiluted WH6 culture filtrate contains approximately 5 GAF Equivalents per liter. The other four DRB Pseudomonas strains selected for this study produced approximately equal amounts of GAF activity. In contrast, similar bioassays performed with culture filtrates of strains Pf-5, PfO-1, and D7 revealed no GAF activity, although PfO-1 and D7 slowed post-germination growth of ABG seedlings (Table 3). 3.4. Time course of the ABG seed response to GAF The response of ABG seeds to transient exposures of WH6 culture filtrate was quantified to determine the minimal exposure time required to arrest germination and to ascertain whether GAF effects could be reversed. Transient exposure of ABG seeds to WH6 culture filtrates for 24 h was sufficient to irreversibly arrest germination (germination score of 1; Fig. 3). Exposures from 4 to 20 h resulted
3.6. Species specificity of GAF herbicidal activity The species specificity of the GAF herbicidal activity was investigated using culture filtrates from P. fluorescens WH6 and P. fluorescens AD31. P. fluorescens Pf-5, a strain that lacked GAF activity in the Poa bioassay (Table 3), served as one of the controls. The results of tests of a large number of plant species are summarized in Table 4. The germination of tested Poa species was arrested by culture filtrates of GAF-producing strains WH6 and AD31 while culture filtrates from strain Pf-5 had little or no effect on their germination. In the case of pine bluegrass, Pf-5 filtrate appeared stimulatory. Undiluted PMS medium depressed the germination and growth of approximately half the Poa species tested relative to the water controls. The effect of the PMS medium was eliminated by dilution with water while GAF activity of the WH6 and AD31 culture filtrates was essentially unchanged at 0.3 concentration. Germination and seedling development of ABG seeds in full strength PMS medium and in Pf-5 culture filtrate were identical to that of the water controls, while germination arrest was complete at all tested concentrations of the AD31 and WH6 culture filtrates. Culture filtrates of WH6 and AD31 also arrested germination of grass species that are grown for seed and the effects were qualitatively similar to those obtained with the Poa species (Table 4). At full strength, PMS medium had an inhibitory effect, but after 3-fold dilution, this was reduced to approximately the germination score in the water control. Pf-5 culture filtrates had no apparent GAF activity relative to these species. In contrast, AD31 and
Table 3 Germination-arrest activity of culture filtrates from P. fluorescens WH6 and other selected Pseudomonas strains Bacterial source of culture filtrate
Control (PMS Medium) WH6 Pf-5 PfO-1 D7
Mean germination score at indicated dilution of culture filtrate (± SEM) 0.1
0.3
1.0 (undiluted)
4.0 ± 0.0 1.0 ± 0.0 4.0 ± 0.0 4.0 ± 0.0 4.0 ± 0.0
4.0 ± 0.0 1.0 ± 0.0 4.0 ± 0.0 4.0 ± 0.0 4.0 ± 0.0
4.0 ± 0.0 1.0 ± 0.0 4.0 ± 0.0 3.4 ± 0.1 3.0 ± 0.2
The standard Poa bioassay was used to assign germination scores according to criteria in Table 2. A score of 4.0 represents normal germination and a score of 1.0 represents germination arrest. The glucose concentration in the control medium was reduced to one-fifth that of standard PMS medium.
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GERMINATION SCORE
Poa annua Seedlings (7-Day Scores) 3
2
1
0 0
8
4
12
16
20
24
LENGTH OF TRANSIENT GAF EXPOSURE (hrs) Fig. 3. Effects of transient exposure to WH6 culture filtrate on the germination of annual bluegrass (Poa annua). Surface-sterilized seeds were allowed to imbibe water for 24 h and then exposed to P. fluorescens WH6 culture filtrate for indicated periods. After treatment, seeds were maintained in sterile distilled water and germination scores were assigned 7 d after the beginning of the imbibition according to the system summarized in Table 2, where a score of 4.0 represents normal germination and seedling development and a score of 1.0 represents germination arrest immediately after initiation of germination. Error bars indicate standard of the mean.
WH6 culture filtrates exerted a separate and distinct inhibitory effect on the progress of germination although
SHOOT LENGTH (mm)
30 25
INITIAL LENGTH (DAY 7)
FINAL LENGTH (DAY 14)
20 15 10 5
4. Discussion
W H
6
(4
8h r
s)
hr s)
s)
(4 8
(2 6
PM S
4
hr
hr s) (2 4 W H
(7
(7
da ys )
PM S
da ys ) W H 6
da ys ) PM S
(7
W at er
W A TE R
(D A Y
07)
0
germination scores tended to be higher than those observed for Poa species. However, after exposure to undiluted culture filtrate, none of the seven species had a germination score above 1.5. In two cases, a score of 2.0 was observed at 0.3 strength, but otherwise, the GAF activity was sufficient to arrest development before the plumule equaled the length of the seed. The WH6 and AD31 culture filtrates were highly effective in arresting germination of the seeds of eight different species of grassy weeds (Table 4). Exposure to culture filtrates of AD31 and WH6 resulted in germination scores of 1.5 or lower except for the seeds of barnyardgrass, which registered a score of 2.0 at 0.3 dilution of WH6 filtrate. Wheat (Triticum aestivum) and barley (Hordeum vulgare) also were sensitive to GAF. Seed germination typically progressed further than in the Poa species before being arrested, although in only one case did a germination score exceed 2.0 (Table 4). In contrast, seeds of the Syngenta Hybrid corn cultivar were much less sensitive to GAF (scores ranging from 2.2 to 2.8). Germination of the corn seeds was somewhat erratic under these test conditions and the scores for controls were also low; however, the results were consistent enough that any substantive effect of the culture filtrates on the germination process would have been evident. The seeds of eight dicot species from eight different plant families were also tested for their response to GAF in WH6 and AD31 culture filtrates (Table 4). Full strength PMS culture medium by itself exhibited a significant inhibitory effect on the germination of these dicots relative to a water control, and this effect could not be excluded from results obtained with undiluted culture filtrates of AD31 and WH6. However, at 0.3 concentration, the germination scores of all species were essentially identical to those in water for both PMS and Pf-5 controls and lower for AD31 and WH6, with the exception of N. tabacum, where little effect of GAF was observed at this concentration. Unlike the germination of grass seeds reported here, which were arrested (scores 62) by culture filtrates from WH6 and AD31 at a 0.3 concentration, the effect of GAF on these dicot species was much less pronounced.
TREATMENT INITIATED AT DAY 7 Fig. 4. Effects of post-germination GAF treatments on annual bluegrass (ABG, Poa annua) seedling growth. Sets of three ABG seeds were germinated in a total volume of 0.25 mL of water, allowed to develop for 7 d (germination score of 4), and then transferred to wells of 24-well microplates containing 0.5 mL well 1 of the indicated test solutions (water, PMS medium with reduced glucose, or P. fluorescens WH6 culture filtrate). Some of the seedlings were given only transient exposures (24 or 48 h) to WH6 culture filtrate or PMS medium with reduced glucose and then transferred to water. Shoot (plumule) length was measured 14 d after the initiation of germination (7 d following the beginning of treatment). Each data point represents the average score from 18 replicate seeds. Error bars indicate the standard error of the mean.
The five Pseudomonas GI-DRB strains examined in the current study share the characteristic of producing and secreting a compound, or compounds, that arrests germination of the seeds of sensitive plant species. Based on the sequence of a portion of the 16S-rDNA genes of these strains and other 16S-rDNA sequences in the data bases, we have classified them as P. fluorescens. However, FAME analysis for strain AD31 was sufficiently distinct that it could be classified as P. putida. The rDNA analysis of the 5 GI-DRB strains indicates these strains are more closely related to PfO-1, which was isolated from farm soil (Compeau et al., 1988) than the root colonizer Pf-5. The absence of sequence variation within the sequenced region
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Table 4 Effect of GAF on the germination of monocot and dicot seedsa Species/common name
Monocotyledenous plants Poa species Poa annua (Annual Bluegrass) var. reptans (Creeping Bluegrass) P. compressa (Canada Bluegrass) P. nemoralis (Wood Bluegrass) P. pratensis (Kentucky Bluegrass) cv. Viva P. scabrella (Pine Bluegrass) P. secunda (Big Bluegrass) cv. Sherman P. trivialis (Roughstalk Bluegrass) cv. Pro Am Grasses grown for seed Lolium perenne (Perennial Ryegrass) cv. Derby cv. Mach I L. multiflorum (Annual Ryegrass) cv. Gulf cv. Surrey Festuca arundinacea (Tall Fescue) cv. Kentucky 31 (Endophyte free) cv. Kentucky 31 (Endophyte) Dactylis glomerata (Orchardgrass) cv. Hallmark Grassy weed species Aegilops cylindrica (Jointed Goatgrass) Brachypodium distachyon (Purple False Brome) Brachypodium sylvaticum (Slender False Brome) Bromus carinatus (California Brome) Bromus tectorum (Downy Brome) Echinochloa crusgalli (Barnyardgrass) Vulpia myruos (Rattail Fescue) Cereals Triticum aestivum (Wheat) cv. Stephens cv. Madsen Hordeum vulgare (Barley) cv. Baronesse cv. Morex Zea mays (Corn) cv. Syngenta Hybrid GH2684F1 Dicotyledonous plants Amaranthaceae Amaranthus hypochondriacus L. (Amaranth) cv. Burgundy Brassicaceae Brassica oleraceae L. (Cabbage) cv. Early Jersey Wakefield Apiaceae Daucus carota L. (Carrot) cv. Imperator Caryophyllaceae Dianthus caryophyllus L. (Carnation) cv. Chabaud Blend
Mean germination scores ± standard error of the mean Control treatments Water
PMSb 1/0.3
Pf-5b 1/0.3
A31b 1/0.3
WH6b 1/0.3
4.0 ± 0.0 3.2 ± 0.4
4.0 ± 0.0/4.0 ± 0.0 3.0 ± 0.3/3.6 ± 0.3
4.0 ± 0.0/4.0 ± 0.0 3.1 ± 0.4/3.5 ± 0.2
1.0 ± 0.0/1.0 ± 0.0 0.9 ± 0.1/1.1 ± 0.1
1.0 ± 0.0/1.0 ± 0.0 1.0 ± 0.0/1.1 ± 0.0
3.7 ± 0.3 4.0 ± 0.0 2.8 ± 0.5
0.9 ± 0.3/2.4 ± 0.7 2.7 ± 0.3/4.0 ± 0.0 2.5 ± 0.4/2.7 ± 0.6
2.8 ± 0.4/3.3 ± 0.4 3.2 ± 0.2/4.0 ± 0.0 2.2 ± 0.4/3.7 ± 0.3
1.0 ± 0.0/1.0 ± 0.0 1.0 ± 0.0/1.0 ± 0.0 0.6 ± 0.1/0.8 ± 0.1
1.0 ± 0.0/1.0 ± 0.0 1.0 ± 0.0/1.0 ± 0.0 1.0 ± 0.0/0.9 ± 0.1
3.0 ± 0.5 1.9 ± 0.5
1.4 ± 0.6/3.0 ± 0.5 1.4 ± 0.4/2.3 ± 0.6
3.4 ± 0.2/3.3 ± 0.4 1.0 ± 0.7/1.6 ± 0.5
0.9 ± 0.1/1.3 ± 0.2 0.7 ± 0.1/0.9 ± 0.2
0.9 ± 0.2/1.2 ± 0.2 0.5 ± 0.5/0.7 ± 0.2
4.0 ± 0.0
4.0 ± 0.0/4.0 ± 0.0
3.8 ± 0.1/3.9 ± 0.1
1.0 ± 0.0/1.0 ± 0.0
1.0 ± 0.0/1.2 ± 0.1
4.0 ± 0.0 3.5 ± 0.4
3.7 ± 0.2/4.0 ± 0.0 3.6 ± 0.2/3.3 ± 0.4
3.2 ± 0.3/3.5 ± 0.4 3.0 ± 0.3/4.0 ± 0.0
1.5 ± 0.1/2.0 ± 0.0 1.0 ± 0.0/1.7 ± 0.1
1.4 ± 0.1/1.3 ± 0.2 1.1 ± 0.1/1.8 ± 0.2
3.9 ± 0.1 2.8 ± 0.5
2.3 ± 0.5/3.5 ± 0.4 2.5 ± 0.5/2.9 ± 0.5
2.5 ± 0.3/3.5 ± 0.4 1.8 ± 0.5/2.5 ± 0.4
1.2 ± 0.2/2.0 ± 0.0 0.9 ± 0.2/1.7 ± 0.3
1.5 ± 0.0/1.7 ± 0.2 1.0 ± 0.1/1.8 ± 0.2
4.0 ± 0.0 4.0 ± 0.0
3.0 ± 0.4/3.3 ± 0.4 3.7 ± 0.2/4.0 ± 0.0
3.0 ± 0.3/3.3 ± 0.4 3.1 ± 0.4/4.0 ± 0.0
1.0 ± 0.2/1.8 ± 0.2 1.0 ± 0.0/1.5 ± 0.0
1.0 ± 0.0/1.9 ± 0.2 1.1 ± 0.0/1.4 ± 0.1
4.0 ± 0.0
1.3 ± 0.3/3.3 ± 0.3
1.8 ± 0.2/3.7 ± 0.3
0.8 ± 0.1/1.3 ± 0.1
1.0 ± 0.2/1.4 ± 0.2
3.9 ± 0.1 4.0 ± 0.0
2.9 ± 0.3/2.5 ± 0.5 3.6 ± 0.2/4.0 ± 0.0
2.3 ± 0.4/3.2 ± 0.4 2.2 ± 0.1/3.9 ± 0.1
1.5 ± 0.1/1.5 ± 0.1 1.0 ± 0.0/1.5 ± 0.0
1.3 ± 0.5/1.3 ± 0.2 1.0 ± 0.0/1.5 ± 0.0
3.6 ± 0.3
2.8 ± 0.4/3.3 ± 0.4
1.7 ± 0.4/2.2 ± 0.4
2 ± 0.1/1.5 ± 0.1
1.2 ± 0.1/1.2 ± 0.1
3.4 ± 02 3.9 ± 01 2.3 ± 0.6 4.0 ± 0.0
2.6 ± 0.4/3.0 ± 0.5 2.6 ± 0.4/4.0 ± 0.0 1.1 ± 0.4/3.7 ± 0.3 3.6 ± 0.2/4.0 ± 0.0
2.6 ± 0.4/2.9 ± 0.5 2.8 ± 0.4/2.9 ± 0.5 1.0 ± 0.4/2.6 ± 0.6 2.7 ± 0.0/4.0 ± 0.0
1.3 ± 0.1/1.2 ± 0.1 1.4 ± 0.1/1.2 ± 0.2 0.4 ± 0.3/1.5 ± 0.5 1.0 ± 0.0/1.5 ± 0.0
1.0 ± 0.2/1.4 ± 0.2 1.0 ± 0.2/1.4 ± 0.1 0.8 ± 0.4/2.0 ± 0.5 1.0 ± 0.0/1.0 ± 0.0
3.5 ± 0.2 3.5 ± 0.2
2.9 ± 0.4/3.7 ± 0.3 2.4 ± 0.4/3.8 ± 0.1
2.1 ± 0.4/2.9 ± 0.4 1.3 ± 0.2/2.8 ± 0.4
1.1 ± 0.1/1.1 ± 0.1 1.2 ± 0.1/1.7 ± 0.1
1.1 ± 0.1/1.5 ± 0.1 1.5 ± 0.1/1.8 ± 0.1
3.5 ± 0.2 3.2 ± 0.3
3.5 ± 0.2/3.4 ± 0.3 2.9 ± 0.4/3.6 ± 0.2
2.7 ± 0.4/3.5 ± 0.3 3.3 ± 0.3/2.5 ± 0.3
1.5 ± 0.0/2.0 ± 0.2 1.4 ± 0.1/2.0 ± 0.2
1.5 ± 0.2/2.3 ± 0.2 1.6 ± 0.1/1.8 ± 0.2
2.8 ± 0.1
2.4 ± 0.2/2.8 ± 0.1
2.0 ± 0.3/2.6 ± 0.2
2.3 ± 0.1/2.8 ± 0.2
2.2 ± 0.1/2.7 ± 0.1
3.9 ± 0.1
2.3 ± 0.1/4.0 ± 0.0
2.5 ± 0.0/4.0 ± 0.0
2.5 ± 0.0/3.5 ± 0.1
2.3 ± 0.0/3.5 ± 0.1
4.0 ± 0.0
2.9 ± 0.3/3.9 ± 0.1
2.8 ± 0.3/3.7 ± 0.1
2.5 ± 0.3/3.2 ± 0.1
2.9 ± 0.2/3.3 ± 0.3
2.8 ± 0.4
1.4 ± 0.2/3.8 ± 0.2
1.8 ± 0.1/4.0 ± 0.0
1.4 ± 0.2/3.4 ± 0.2
1.4 ± 0.1/3.4 ± 0.2
3.9 ± 0.1
3.0 ± 0.1/3.8 ± 0.1
3.2 ± 0.1/3.9 ± 0.1
3.0 ± 0.1/3.0 ± 0.1
2.7 ± 0.2/3.1 ± 0.3 (continued on next page)
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Table 4 (continued) Species/common name
Mean germination scores ± standard error of the mean Control treatments Water
PMSb 1/0.3
Pf-5b 1/0.3
A31b 1/0.3
WH6b 1/0.3
Asteraceae Lactuca sativa L. (Lettuce) cv. Butter Crunch
3.6 ± 0.2
2.8 ± 0.2/3.9 ± 0.1
3.0 ± 0.0/3.9 ± 0.1
2.6 ± 0.3/3.1 ± 0.1
3.1 ± 0.0/3.3 ± 0.1
Fabaceae Medicago sativa L. (Alfalfa) cv. Unknown
3.8 ± 0.1
2.0 ± 0.3/3.3 ± 0.4
2.2 ± 0.3/3.8 ± 0.1
1.9 ± 0.4/2.9 ± 0.3
2.2 ± 0.2/3.4 ± 0.2
Solanaceae Nicotiana tabacum L. (Tobacco) cv. Wisconsin
3.8 ± 0.1
1.5 ± 0.0/3.4 ± 0.2
1.5 ± 0.0/3.1 ± 0.2
1.5 ± 0.0/3.5 ± 0.1
1.5 ± 0.0/3.4 ± 0.1
Papaveraceae Papaver nudicaule L. (Iceland Poppy) cv. Nudicaule Blend
2.9 ± 0.5
2.0 ± 0.2/3.2 ± 0.3
1.8 ± 0.2/3.2 ± 0.3
1.9 ± 0.2/2.3 ± 0.2
1.6 ± 0.2/2.5 ± 0.3
a Germination scores were assigned according to the system summarized in Table 2, where a score of 4.0 represents normal germination as defined for the indicated plant group. A score of 1.0 represents germination arrest immediately after initiation of germination. Bioassay conditions were modified to account for seed size and germination behavior. Tests with Poa sp. involved 48-well plates, 4 seeds per well; commercial grass seed species and grassy weeds, 24-well plates, 2 seeds per well, except for barnyardgrass where 3 seeds were used per well; cereals, 12-well plates, one seed per well, except corn where 6-well plates were used, 1 seed per well (day incubation temperature of 25 oC); dicots, 24-well plates, one seed per well, except for tobacco and poppy where 48-well plates, one seed per well were used. A day temperature of 25 oC was used for alfalfa, tobacco, amaranth, carnation and poppy seeds. Seeds of slender false brome were given a stratification treatment involving imbibition of treatment solutions at 6 oC for 3 weeks to break dormancy prior to placing in the incubator to initiate germination. b PMS medium with 0.2 the standard glucose content, and culture filtrates were used either undiluted (1) or diluted (0.3).
of the 16S-rDNA indicates a genetic relationship among these strains, and the similar and specific effects of the herbicide they produce suggest that the same compound is produced by all five strains. Additional sequence analyses, as well as purification of the compound(s) will be required to confirm these conclusions. Because of its specific mode of action, we have termed this compound a GerminationArrest Factor (GAF). Culture filtrates from P. fluorescens strain D7 failed to arrest germination of P. annua seed in the bioassay developed in this study, suggesting that the inhibition of B. tectorum seed germination and the capacity to stunt the growth of other grassy weeds (Gurusiddaiah et al., 1994; Kennedy et al., 2001) is not through GAF activity. The fact that D7 also stimulated germination of Poa pratensis, and a number of other grasses, in contrast to the germination arrest we observed with GAF implies that D7 biological activity is mediated through other compounds (Kennedy et al., 2001). Strain Pf-5 colonizes roots and has been shown to produce a broad spectrum of antibiotics, including pyrrolnitrin (Pfender et al., 1993), pyoluterorin (Nowak-Thompson et al., 1997, 1999), 2,4diacetylphloroglucinol (Sharifi-Tehrani et al., 1998), hydrogen cyanide (Laville et al., 1998) and siderophores (Loper and Buyer, 1988). These compounds act to suppress the bacterial pathogen Erwinia caratovora and a number of fungal pathogens that attack a variety of plant hosts including pea, cucumber and maize (Kennedy et al., 2001). It was of interest, therefore, that Pf-5 had no GAF activity in the Poa bioassay developed for this study
(Table 3) suggesting that another compound or compounds are responsible for GAF activity. The action of GAF affected seed germination of a large number of grasses, including all grassy weeds examined in this study. The eight Poa species tested were sensitive to GAF, as were the seeds of grassy weeds such as jointed goatgrass, downy brome, false brome, and a number of others of agricultural significance. Among the Graminaceae tested, only the seeds of corn appeared to be unaffected by this compound. The inhibitory effect of the PMS medium on the germination of the dicot species precluded an unequivocal interpretation, but it is clear that all species tested were able to overcome the effects of GAF at concentrations sufficient to arrest germination of the grass species tested. Based upon the results that utilized the bioassay system developed in this study, GAF has preferential specificity for arresting germination of the seeds of a variety of grass species. These results also substantiate the observation that GAF activity differs from the herbicidal effects previously reported for strain D7. Whereas GAF activity in both undiluted and 0.3 concentration culture filtrates arrests jointed goatgrass seed germination (scores of 1.3– 1.5, Table 4), Kennedy et al. (2001) reported that D7 culture filtrates had a slightly stimulatory effect on the germination of this weed. In addition to differences in sensitivity of grasses and dicots to GAF, the action of GAF exhibits developmental specificity. Sensitive grass seeds are arrested immediately after the plumule and the coleorhiza have emerged from the seed coat, but typically before the plumule has reached
G.M. Banowetz et al. / Biological Control 46 (2008) 380–390
a length equal to that of the seed (i.e., at a germination score < 2.0). Moreover, grass seedlings have reduced sensitivity to GAF treatment after germination has occurred. Growth is slowed, but not halted, if the seedlings are treated after the first true leaf has emerged. These results are supported by findings in preliminary tests using partially purified GAF preparations (data not shown), which suggest that GAF has little, if any effect on mature grass plants. The species specificity of GAF, combined with its developmentally-specific mode of action, suggests that this naturally-occurring herbicide could potentially be useful for the control of grassy weeds. The fact that transient exposure of sensitive grass seeds to GAF irreversibly arrests germination suggests that GAF has potential as a preemergence herbicide. An agent which does not affect post-germination plant growth should prove effective for the control of grassy weeds in grass seed production systems where the established perennial grass crop would presumably be unaffected by GAF. The presence of any seed, other than that of the present year crop, represents contamination in these production systems. ABG seed contamination is a significant problem in grass seed production fields because in the environment of the Willamette Valley, this weed produces and sheds seed throughout the cool, wet fall, winter, and spring. However, P. annua seed is highly sensitive to GAF, as are Diuron-resistant variants that have recently appeared in grass seed cropping systems (data not shown). GAF would also appear to have potential for the management of grassy weeds in established turf settings, including golf courses, public parks, and residential lawns. However, tests of the practical efficacy of GAF, or GAF-producing isolates, in field settings must await the purification, identification, and chemical synthesis of purified samples of the active compound. The use of live DRB to control grassy weeds remains a possible alternative to the use of chemical herbicides (Kennedy et al., 1991; Kennedy and Stubbs, 2007), and it is conceivable that the five GI-DRB strains identified in the present study may be useful in this context. Success in the use of live DRB as weed biocontrol agents is strongly dependent, however, on biotic and abiotic factors over which producers have little control (Alexander, 1977; Kennedy and Kremer, 1996; Huang and Madan, 1999; Li et al., 2003). Nevertheless, identification of microorganisms that produce bioherbicides is critical, and it is likely that delivery systems will continue to be developed that improve the persistence of these agents in soil. The developmental and species specificity of GAF suggests that this compound may have a unique mechanism of action. If this is indeed the case, identification of the chemical structure of GAF would provide the basis for development of a new class of herbicidal compounds with properties of distinct advantage in the control of grassy weeds. In preliminary tests, a large number of other grassy species also appear to be sensitive to GAF (data not shown). This possibility provides motivation for the ongoing effort in our laboratories to purify and characterize the
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