Feb 23, 1987 - mixture incubated with sterile vermiculite alone. Root washes and extracts were prepared from 4, 6, and 8 d plants according to methods ...
Plant Physiol. (1987) 85, 537-541 0032-0889/87/85/0537/05/$0 1.00/0
The Effect of Pseudomonas putida Colonization on Root Surface
Peroxidase' Received for publication February 23, 1987 and in revised form July 9, 1987
FREDERICK ALBERT AND ANNE J. ANDERSON* Department of Biology, Utah State University, Logan, Utah 84322-4500 ABSTRACT Increased activities of peroxidase and indole 3-acetic acid (IAA) oxidase were detected on root surfaces of bean (Phawolus vulgaris) seedlings colonized with a soil saprophytic bacterium, Pseudomonas putida. IAA oxidase activity increased over 250-fold and peroxidase 8-fold. Enhancement was greater for 6-day-old than for 4- or 8-day-old inoculated plants No IAA oxidase or peroxidase activities were associated with the bacterial cells. Native polyacrylamide gel electrophoresis demonstrated that washes of P. putida-inoculated roots contained two zones of peroxidase activity. Only the more anodic bands were detected in washes from noninoculated roots. Ion exchange and molecular sizing gel chromatography of washes from P. putida-colonized roots separated two fractions of peroxidase activity. One fraction corresponded to the anodic bands detected in washes of P. putida inoculated and in noninoculated roots. A second fraction corresponded to the less anodic zone of peroxidase, which was characteristic of P. putida-inoculated plants. This peroxidase had a higher IAA oxidase to peroxidase ratio than the more anodic, common enzyme. The changes in root surface peroxidases caused by colonization by a saprophytic bacterium are discussed with reference to plant-pathogen interactions.
Pseudomonas putida, a soil saprophyte, has been called a beneficial organism because it can enhance plant yield and suppress certain root fungal pathogens (14, 24). The aggressive colonization of root surfaces by P. putida may be involved in these beneficial phenomena as well as being an important survival strategy for the bacterium. As part of our study of factors that are important for aggressive colonization of the root surface, we investigated whether P. putida altered root properties. This possibility was suggested from the observation that peroxidases in root washes ofgreenhouse grown bean seedlings differed from peroxidases found in washes from sterile grown seedlings (1). Because the greenhouse plants were colonized by fluorescent pseudomonads, we initiated studies to determine whether P. putida colonization of roots altered root surface peroxidase activities. MATERIALS AND METHODS Plant Growth. Seedlings of Phaseolus vulgaris L. cultivar Dark Red Kidney (Idaho Bean Seed Company, Twin Falls, ID 8330 1) were grown in vermiculite under sterile conditions with and
without an inoculum of Pseudomonas putida (3). To obtain inoculum, P. putida isolate Corvallis, resistant to nalidixic acid and rifampicin, was cultured for 12 h in rich media (3). Cells were centrifuged twice at I 0,000g for 10 min, washed with sterile distilled water, and resuspended in sterile 1 mM MgCl2. Bean seeds were inoculated with 106 cells at planting, while control seeds were treated with an equal volume of 1 mM MgCl2. Under the growth regime employed, the seedlings had just shed the cotyledon coat at d 6. Screening noninoculated seedlings for microbial colonization using published procedures (3) revealed bacterial contamination at less than 2%. P. putida-inoculated plants were colonized at levels of about 106 cells/g root. Enzyme Analyses. Peroxidase activity on intact root surfaces was assayed as previously described (1). Peroxidase activity in the rhizosphere was assayed by adding 25 ml of the reaction mixture (1) to the vermiculite from which the seedling had been removed. After 15 min incubation at 30°C, the vermiculite was removed by filtration. Absorbance of the reaction mixture was measured at 575 nm, relative to the absorbance of a reaction mixture incubated with sterile vermiculite alone. Root washes and extracts were prepared from 4, 6, and 8 d plants according to methods previously published (1) and lyophylized to a powder for storage. Lyophylized samples were resuspended (1 mg/ml) in distilled water, and insoluble material was removed by centrifugation at 10,000g for 15 min. These crude preparations were assayed for peroxidase (1) and IAA oxidase activities (1). Carbohydrate and protein content of each preparation was determined by the methods of Dubois et al. (7) and Lowry et al. (19) using glucose and BSA as the respective 0
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Ckf C) 'I-, V)
30-
0
t z :D
L.. 20f
(tI
x
0 LIi
0op
a-
3.5 4.5 PLANT AGE
5.5
6.5 DAYS
7.5
FIG. 1. Peroxidase activity from P. putida-inoculated (0) and noninoculated (U) roots of different ages. (a) Peroxidase activity was assayed on roots of intact plants as described in "Materials and Methods." The data represent the formation of soluble chromogen in the reaction mixture containing the intact root and are expressed as units of activity 'Supported by a grant from the United States Department of Agri- per g of root tissue, and (b) 1.0 unit of peroxidase activity produced an culture, Science and Education Administration Competitive Grants pro- A change of 1.000 at 575 nm in 15 min at 30°C in a reaction volume of gram and the Utah State Agricultural Experiment Station. Utah State 1.0 ml. Data are based upon four different growth trials each consisting Agricultural Experiment Paper No. 3363. of five plants. 537
ALBERT AND ANDERSON
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Plant Physiol. Vol. 85, 1987
Table I. Peroxidase and IAA Oxidase Activities in Washes and Extracts of Roots of Noninoculated and P. putida-Inoculated Bean
Specific Activityb
Root Agea d
Peroxidase NI
I
IAA oxidase Ratio I NI
NI
I
Ratio I NI
Root wash 4 21 ±3 2 3± 1 36±2 2± 1 1 6 52±2 402±8 8 8±2 2227± 12 278 8 81 ±3 241 ±6 3 14±2 434±4 31 Root extracts 4 24±4 36±3 2 3± 1 4 1 1 6 42±5 94±8 2 13±3 46±4 4 8 89±7 70±9 1 17±2 18±3 1 ' Roots were grown under sterile conditions with or without P. putida inoculum for 4 to 8 d as described in "Materials and Methods." b Peroxidase and IAA oxidase activities were measured in root washes or extracts of non-inoculated (NI) and P. putida-inoculated (I) roots using procedures described in "Materials and Methods;" 1.0 unit of peroxidase activity produced an A change of 0.001 at 575 nm in 15 min at 30C in a reaction volume of 1.0 ml; 1.0 unit of IAA oxidase activity consumed 1.0 gg of IAA in 1.0 min in a reaction volume of 2.0 ml. Specific activity is described as units of enzyme per mg protein. Data are based upon three experiments each of four roots and the standard error of the mean is provided.
standards. Cells of P. putida were assayed for production of peroxidase and IAA oxidase. Cells isolated from the root surface and from stock cultures were inoculated into rich medium (3) or rich medium to which root wash material (1 mg dry weight/ml culture) had been added and grown for 12 h. Cells were suspended in 50 mm potassium phosphate buffer (pH 7.0) and broken by treatment in a French pressure cell. The extract was centrifuged at 10,000g for 15 min to remove insoluble material. These sonicates, or viable cell suspensions (108 cells/ml reaction mixture), were examined for enzyme activities. Polyacrylamide Gel Electrophoresis (PAGE) of Peroxidase and IAA Oxidase Activities. Low ionic-strength, discontinuous native polyacrylamide slab gels were used to distinguish different forms of peroxidase and IAA oxidase. The resolving gel, 10% total acrylamide concentration (2.1% bisacrylamide), was buffered to pH 8.8 and photopolymerized. Wells in a 3%, pH 8.8 stacking gel were loaded with 10,000 units of peroxidase activity. The gels were run for approximately 5.0 h at a constant voltage of 150 V. Temperature was maintained at 10°C with a water heat exchanger. Peroxidase activity was detected by immersing the gel in 100 ml of reaction mixture containing chloronapthol and H202 (1) and the production of chromogen was observed. The location of IAA oxidase was determined by developing the gel according to procedures of Endo (8). SDS gel separations were performed using a 12.5% acrylamide (0.2% cross-linked) resolving gel buffered with 0.5 M tris-HCl at pH 9.5. A stacking gel of 3% acrylamide (0.2% cross-linked) was buffered by 0.1 M tris-HCl (pH 7.2). Each well contained 5 ,g of protein and the gel was developed using silver staining procedures. The gel was fixed in 20% trichloracetic acid in 50% methanol for 1 h and washed for 30 min in two changes of 10% ethanol and 5% acetic acid. Oxidation involved a 5 min treatment in 5 mM potassium chromate in 3 mM HNO3. After two 5min rinses with distilled water, the gel was immersed in 15 mm silver nitrate for 20 min and rerinsed in water. The gel was developed stepwise: 2 min in 0.003% formaldehyde in 0.05 M Na2CO3, 0.006% formaldehyde in 0.1 M Na2CO3 and three treatments of 0.02% formaldehyde in 0.3 M Na2CO3. Development was stopped by immersion in 5% acetic acid. Purification of Peroxidase. Purification of peroxidase was initiated by applying root wash material from P. putida-inoculated
plants to a Sepharose 6B (Sigma Chemical Co.) column (45 x 2 cm). Fractions of 5.0 ml were eluted with distilled water and assayed for peroxidase, IAA oxidase, protein and carbohydrate. Fractions with peroxidase activity were pooled, dialyzed against 5 mm potassium acetate (pH 5.0), and applied to a CM-Sephadex (Sigma) column (20 x 3 cm) equilibrated with the same buffer. Fractions (5.0 ml) were eluted with 5 mM potassium acetate (pH 5.0) followed by a gradient of buffer containing 0.0 M to 0.5 M KCI applied at a rate of 10 ml/h. Fractions containing peroxidase were dialyzed against 10 mM tris-acetate pH (8.5), and applied to DEAE Sephadex (Sigma) column (20 x 3 cm) equilibrated in the same buffer. Fractions (5.0 ml) were eluted with 10 mm trisacetate (pH 8.5) followed by a gradient of buffer containing 0.0 to 0.5 M NaCl applied at a rate of 10 ml/h. All fractions (5 ml) were collected and dialyzed extensively against distilled water prior to assay for peroxidase and IAA oxidase activities, protein, and carbohydrate. Neutral sugar composition of fractions with peroxidase activity was determined using methods described by Anderson (2). RESULTS Variability of Peroxidase Activity with Seedling Age. Roots of intact seedlings from P. putida-inoculated and noninoculated beans developed intense deposits of chromogen upon immersion in the chloronapthol-hydrogen peroxide reaction mixture. Maturation of the seedlings was accompanied by greater root weight and an increased production of soluble chromogen from the peroxidase reaction mixture. The formation of the soluble chromogen detected in these assays of intact roots suggested that peroxidase activity, expressed as units per gram root tissue, was maximum at 6.5 d. This peroxidase activity consistently was higher from P. putida-inoculated plants than from the control plants (Fig. 1). Peroxidase detected in the vermiculite used as a growth medium increased with age of the seedling and again was greater for P. putida-inoculated than noninoculated plants. No peroxidase or IAA oxidase activities were observed with cells, or in extracts, of P. putida. Root washes and extracts obtained from 4-, 6- and 8-d-old seedlings possessed peroxidase and IAA oxidase activites (Table I). The specific activities of these enzymes in the washes and extracts from noninoculated plants increased with seedling age. Washes and extracts from roots of P. putida-inoculated plants
ROOT SURFACE PEROXIDASE
NI
I
ZONE B
ZONE A
ANODE
FIG. 2. Native PAGE of root washes from P. putida-inoculated and noninoculated plants. Root washes were prepared from 7-d-old plants that had been inoculated (I) or were not inoculated (NI) with P. putida. Peroxidase activity was fractionated by native PAGE according to "Materials and Methods." Samples containing 25,000 units of peroxidase activity were applied to each well. One unit of peroxidase activity produced an A change of 0.001 under the standard assay conditions described in "Materials and Methods."
539
with the bands observed from sterile grown plants in zone A, an additional activity zone was revealed at a less anodic position, zone B (Fig. 2). This activity in zone B did not correlate with the bands observed in extracts from the sterile grown roots. Native PAGE of vermiculite washes displayed the same peroxidase bands as the root washes, with activity only in zone A for sterile roots and in zone A and B for P. putida-inoculated roots. Upon staining the gels for IAA oxidase activity, color development was more intense with the bands in zone B than the faster moving bands in zone A. To probe the possibility that the activity in zone B was induced by IAA, sterile grown roots were treated, for 48 h, from d 4.5 to d 6.5, with 1 gM IAA. Washes of these sterile but IAA-treated roots displayed typical bands in zone A and additional bands which migrated to less anodic positions. These bands did not correspond to the activity detected in zone B from the washes of P. putida-inoculated roots. Purification of Peroxidases from Root Washes. The peroxidase and IAA oxidase activities in washes from P. putida-inoculated roots eluted in the void fractions of a Sepharose 6B column. Passage of the components in these void fractions through CMSephadex in 5 mM potassium acetate (pH 5.0) produced one peak of peroxidase activity in the nonadsorbed eluate and another peak which was eluted by low salt. The protein which eluted without adsorption to CM-Sephadex had a higher peroxidase to IAA oxidase ratio than the adsorbed fraction (Table II). The peroxidase-rich protein was adsorbed onto DEAE-Sephadex and was eluted by 0.15 to 0.20 M NaCl. These chromatographic procedures resulted in a 15-fold increase in specific activity compared to the crude root wash. SDS-PAGE of the purified fraction detected only two closely aligned bands, which corresponded to a mol wt of about 62.5 kD. Native PAGE demonstrated this peroxidase-rich fraction to comigrate with the fast moving anodic band of zone A. The activity that was adsorbed onto CM-Sephadex was enriched for IAA oxidase-compared to peroxidase activity (Table II). Further chromatography on DEAE-Sephadex revealed that the majority of the activity eluted without adsorption to yield a preparation that was purified 55-fold from the crude root wash. SDS polyacrylamide gels of this purified fraction revealed only one zone of protein in which two bands were closely aligned and corresponded to a mol wt of approximately 71 kD. Native PAGE indicated peroxidase activity in a slow moving anodic band which corresponded to the activity in zone B in the crude root wash from the P. putida inoculated plants. Both of the purified peroxidases were glycosylated, with arabinose and galactose as the major neutral sugars. Minor differences were that the IAA-oxidase form of peroxidase was richer in xylose and glucose and lower in mannose than the faster moving anodic peroxidase (Table III).
displayed a different pattern; specific activities for 6-d-old plants greater than for 4- and 8-d-old plants (Table I). Colonization of P. putida increased IAA oxidase activity to a greater extent DISCUSSION than the peroxidase activity (Table I). The specific activity of another root surface enzyme, f3-glucosidase, was identical for Colonization of bean roots by P. putida was associated with root washes of P. putida-inoculated and noninoculated 6-d-old the production of additional forms and greater levels of peroxiplants (data not shown). dase on the root surface. The lack of detectable peroxidase Native PAGE of the root wash from sterile grown plants activities in P. putida preparations suggests that the additional revealed only one zone of peroxidase activity near the anode, peroxidases are of plant origin. The stimulation of peroxidase termed zone A (Fig. 2). Three bands of peroxidase activity were activity by bacterial colonization was more apparent in washes resolved in this zone when the gel was loaded with a high number of intact plants than in root extracts. Thus, it seems likely that of units of peroxidase. No bands of activity were observed at less the root epidermal cells are involved in the response. Indeed, anodic positions or in material that traveled towards the cathode. previous studies have demonstrated that root epidermal and root Washes obtained separately from 50 sterile grown plants each hair cells possess potent peroxidase activity (1, 10, 21, 27, 30). displayed identical bands at location A. Extracts from sterile Peroxidases catalyze events that are essential for normal plant grown beans possessed the anodic bands at zone A and addicell development. Peroxidase cross-linking of tyrosine residues tional, less anodic bands. in the plant cell wall protein extensin is proposed to generate a The peroxidases in washes of roots inoculated with P. putida firmer matrix material (6, 9). Polymerization of phenoxy radicals had a different banding pattern from that obtained with the to produce lignin and suberin provides protective and strengthsterile plants. Although bands were detected which comigrated ening coatings to the protein-polysaccharide wall matrix (26, 28). were
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ALBERT AND ANDERSON
Plant Physiol. Vol. 85, 1987
Table II. Purification ofPeroxidase and IAA Oxidase from Washes of P. putida-Inoculated Roots Preparationa Carbohydrate Protein Peroxidase Specific Activity IAA oxidase units units mg mg units/mg protein 92.6 80.6 31290 388 180000 Crude root wash Sepharose 6B 14.5 16790 1158 128730 38.5 Void 34 1450 9360 50.8 42.5 Included Sepharose 6B void onto CM-Sephadex Nonadsorbed 2774 (A) 4.2 3.1 8600 16650 (B) 16.8 8.9 45 5 560 Absorbed 0.4 3840 9600 3.1 18532 (A) 0.0 -0.3 M KC1 172 10.4 1.3 223 210 (B) 0.3 0.5M KCI CM Sephadex nonadsorbed (A) onto DEAE-Sephadex 0.2 0.1 0 0 0 Nonadsorbed Adsorbed 0.4 0 1.0 0 0 (A) 0.00-*0.1SMNaCl 1.1 0.3 6240 5673 10320 (B) 0.15-0.20M NaCl 0 0 0.8 0.6 0 (C) 0.30 M0.35 M NaCl 1.8 0.3 0 0 0 (D) 0.35 0.40M NaCl CM Sephadex adsorbed (A) onto DEAESephadex 0.1 1-500 15000 1.2 Nonadsorbed 12350 Adsorbed 0.7 0.2 1650 8250 220 (A) 0.00 0.20 M NaCl 0.5 0.1 0 0 0 (B) 0.20 -0.35 M NaCI 0.1 0.0 0 0 0 (C) 0.35 0.85M NaCl a Washes were prepared from P. putida inoculated roots and chromatographed as described in "Materials and Methods."
Specific Activity units/mg protein 2233 8878 220 5371 63
46330 162 0 0 9382 0 0
123500 1100 0 0
Table III. Neutral Sugar Composition of Peroxidase Active Fractions Purifiedfrom Root Washes Rha Fuc Ara Man Fractiona Xyl Gal Glc % Composition' 1±+1 0±0 13±2 0± 1 3±3 Crudewash 14±3 70±7 4±2 5±4 35±4 2± 1 4±2 38±4 12±2 Sepharose6Bvoidfraction 17 ± 4 5± 1 5± 1 28 ±4 2 ± 2 34 ± 4 CM-Sephadex nonadsorbed 9± 3 fraction A 4± 1 7± 1 1± 1 30 ± 3 15 ± 1 32 ± 2 CM-Sephadex nonadsorbed 10 ± 2 DEAE-Sephadex adsorbed fraction B 5± 1 7± 1 27±5 6±3 8± 1 26 ±4 22± 1 CM-Sephadexadsorbedfraction A DEAE-Sephadex nonadsorbed a Peroxidase containing fractions were purified from root washes of 64-old P. putida-inoculated seedlings according to the procedures described in "Materials and Methods." b Neutral sugar compositions were determined by alditol acetate analysis (2). Data are the means of two duplicates each of three purifications. Standard error of the means are provided. Rha, rhamnose; Fuc, fucose; Ara, arabinose; Xyl, xylose; Man, mannose; Gal, galactose; Glc, glucose.
Different peroxidases may be responsible for catalyzing such distinct specialized functions. Recently, Cooper and Varner (6) proposed that specific isozymes may be involved in formation of the isodityrosine linkages in extensin. Sijmons et al. (26) have suggested that a specific bean root peroxidase is functioning in suberization. The distinct isozyme patterns for peroxidase observed during tissue development may reflect the differing needs of the plant tissues (4, 5, 15). Treatments of plant tissues with IAA and ethylene suggest that the altered peroxidase isozyme patterns may arise from hormonal triggers (16, 17). Indeed, the preferent stimulation of a peroxidase which was highly proficient as an IAA oxidase upon P. putida colonization resembles expression of an IAA oxidase-rich peroxidase in tissues treated with
2,4-D (17). The sensitivity of the 2,4-D effect to protein synthesis inhibitors suggests that the hormone triggers de novo synthesis of the enzyme. The mechanisms by which P. putida exerts its effect on the root surface peroxidase are under investigation. Expression of peroxidase bands in zone B was not observed after exposure of roots to authentic IAA. This possibility was investigated because of the production of P. putida isolates of IAA components (18, 29). Although our isolate of P. putida produced compounds from tryptophan which reacted with Salkowski reagent (50:1 5% HC104:0.5 M FeCl3), the chromogen was not identical with that from authentic IAA (18). Consequently, we are determining the character of the IAA-like compound(s). Ion exchange chromatography and native and SDS-PAGE
ROOT SURFACE PEROXIDASE suggest that a different form of peroxidase is responsible for the high IAA oxidase activity on the surface of P. putida-inoculated roots. Different ratios of IAA oxidase to phenolic oxidase activities were displayed by the two purified bean root surface enzymes. This observation is in contrast to reports which indicate that isozymes of horseradish peroxidase have similar ratios ( 13). The two bean enzymes have slightly different molecular sizes on SDS polyacrylamide gels, although their glycosylation patterns are similar. Both surface enzymes demonstrate a galactosyl- and arabinosyl-rich composition which resembles that ofanother root surface protein, an agglutinin, which will precipitate cells of P. putida (2). As well as a general role in cellular differentiation, peroxidases may play a specific role in defense mechanisms. In a resistant response, enhanced production of extensin (23, 25), and lignin and suberin (12, 28), may hinder plant cell wall degradation by microbial enzymes. The phenoxy radicals themselves (22), as well as the activated oxygen species that are involved in peroxidase catalyzed reactions (1 1), have potent antimicrobial activity. Previously, defense roles have been suggested for the peroxidase changes that occur after plant tissue has been invaded by microbial pathogens (12, 28). Our observations suggest that a saprophytic colonizer also triggers specific changes in plant peroxidases. The enhanced peroxidase activity in P. putida-colonized roots has potential to contribute to plant resistance. These plantrelated effects may possibly augment a direct ability of P. putida to suppress certain fungal pathogens. This ability has been correlated with the production by P. putida of efficient iron chelators, ,siderophores (14, 20, 24). The siderophores are believed to suppress pathogen development by depriving the fungi of adequate iron nutrition (14, 20). We are using mutants of P. putida to determine whether induction of enhanced peroxidase levels in bean roots also contribute to disease suppression. LTERATURE CITED 1. ALBERT FG, LW BENNETT, AJ ANDERSON 1985 Peroxidase associated with the root surface of Phaseolus vulgaris. Can J Bot 64: 573-578 2. ANDERSON A 1983 Isolation from root and shoot surfaces of agglutinins that show specificity for saprophytic pseudomonads. Can J Bot 61: 3438-3443 3. ANDERSON AJ, D GUERRA 1985 Responses of bean to root colonization with Pseudomonas putida in a hydroponic system. Phytopathology 75: 992-995 4. BERGER RG, F DRAWERT, A KINZKOFER, C KUNZ, BJ RADOLA 1985 Proteins and peroxidase in callus and suspension cultures of apple. A study using ultrathin-layer isoelectric focusing, sensitive silver staining of proteins, and peroxidase isozyme visualization. Plant Physiol 77: 211-214 5. BIRECKA H, KA BRIBER, JL CATALFAMO 1973 Comparative studies on tobacco pith and sweet potato root isoperoxidases in relation to injury, indoleacetic acid, and ethylene effects. Plant Physiol 52: 43-49 6. COOPER JB, JE VARNER 1984 Cross linking of soluble extensin in solated cell *alls. Plant Physiol 76: 416-417 7. DuBoIs M, KA GILLES, JK HAMILTON, PA REBERS, F SMITH 1956 Colorimetric method for the determination of sugars and related substances. Anal Chem
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28: 350-356 8. ENDO T 1968 Indoleacetate oxidase activity of horseradish and other plant peroxidase isozymes. Plant Cell Physiol 9: 333-341 9. FRY SC 1982 Isodityrosine, a new cross linking amino acid from plant cellwall glycoproteins. Biochem J 204: 449-455 10. GRISON R, PE PILET 1984 Cytoplasmic and wall isoperoxidases in growing maize roots. J Plant Physiol 118: 189-199 11. HALLIWELL B 1978 Lignin synthesis: the generation of hydrogen peroxide and superoxide by horseradish peroxidase and its stimulation by manganese (II) and phenols. Planta 140: 81-88 12. HAMMERSCHMIDT R, J Kut 1982 Lignification as a mechanism for induced systemic resistance in cucumber. Physiol Plant Pathol 20: 61-71 13. HOYLE MC 1977 High resolution of peroxidase-indoleacetic acid oxidase isoenzymes from horseradish by isoelectric focusing. Plant Physiol 60: 787793 14. KLOEPPER JW, J LEONG, M TEINTZE, MN SCHROTH 1980 Pseudomonas siderophores: a mechanism explaining disease-suppressive soils. Curr Microbiol 4: 317-320 15. KRUGER JA, DE LABERGE 1974 Changes in peroxidase activity and peroxidase isozymes of wheat during germination. Cereal Chem 51: 578-585 16. LEE TT 1972 Interaction of cytokinin, auxin, and gibberellin on peroxidase isoenzymes in tobacco tissues cultured in vitro. Can J Bot 50: 2471-2477 17. LEE TT 1972 Changes in indoleacetic acid oxidase isoenzymes in tobacco tissues after treatment with 2,4-dichlorophenoxyacetic acid. Plant Physiol 49: 957-960 18. LOPER JE, MN SCHROTH 1986 Influence of bacterial sources of indole-3-acetic acid on root elongation of sugar beet. Phytopathology 76: 386-389 19. LOWRY OH, NJ ROSEBROUGH, AL FARR, RJ RANDALL 1951 Protein measurement with Folin phenol reagent. J Biol Chem 193: 265-275 20. MARUGG JD, M VAN SPANJE, WPM HOEKSTRA, B SCHIPPER, PJ WEISBEEK 1985 Isolation and analysis of genes involved in siderophore biosynthesis in plant-growth-stimulating Pseudomonas putida WCS358. J Bacteriol 164: 563-570 21. MUELLER WC, CH BECKMAN 1978 Ultrastructural localization of polyphenol oxidase and peroxidase in roots and hypocotyls of cotton seedlings. Can J Bot 56: 1579-1587 22. RAMA RAJE URS NV, JM DUNLEAVY 1975 Enhancement of the bactericidal activity of a peroxidase system by phenolic compounds. Phytopathology 65: 686-690 23. ROBY D, A TOPPAN, M-T EsQUERRt-TUGAYE 1985 Elicitors of fungal and of plant origin trigger the synthesis of ethylene and of cell wall hydroxyprolinerich glycoprotein in plants. Plant Physiol 77: 700-704 24. SCHER FM, R BAKER 1982 Effect of Pseudomonas putida and a synthetic iron chelator on induction of soil suppressiveness to Fusarium wilt pathogens. Phytopathology 72: 1567-1573 25. SHOWALTER AM, JN BELL, CL CRAMER, JA BAILEY, JE VARNER, CJ LAMB 1985 Accumulation of hydroxyproline-rich glycoprotein mRNAs in response to fungal elicitor and infection. Proc Natl Acad Sci USA 82: 6551-6555 26. SUMONS PC, PE KOLATITUKUDY, HF BIENFAIT 1985 Iron deficiency decreases suberization in bean roots through a decrease in suberin-specific peroxidase activity. Plant Physiol 78: 115-120 27. SMITH MM, TP O'BRIEN 1979 Distribution of autofluorescence and esterase and peroxidase activities in the epidermis ofwheat roots. Aust J Plant Physiol 6: 201-219 28. VANCE CP, TK KIRK, RT SHERWOOD 1980 Lignification as a mechanism of disease resistance. Annu Rev Phytopathol 18: 259-288 29. WuRsr M, Z PRIKRYL, V VANCURA 1980 High-performance liquid chromatography of plant hormones. I. Separation of plant hormones of the indole type. J Chromatogr 191: 129-136 30. ZAAR K 1979 Peroxidase activity in root hairs of cress (Lepidium sativum L.) Cytochemical localization and radioactive labelling of wall bound peroxidase. Protoplasma 99: 263-274
