Production of Chitinolytic Enzymes and Endoglucanase in the Soybean Rhizosphere in the Presence ofTrichoderma harzianumandRhizoctonia solani

12, 111–117 (1998) BC980623

BIOLOGICAL CONTROL ARTICLE NO.

Production of Chitinolytic Enzymes and Endoglucanase in the Soybean Rhizosphere in the Presence of Trichoderma harzianum and Rhizoctonia solani F. K. dal Soglio,* B. L. Bertagnolli,† J. B. Sinclair,† G.-Y. Yu,† and D. M. Eastburn† *Departamento de Fitotecnia, Universidade Federal de Santa Catarina, P.O. Box 476, Florianopo´lis, SC 88040-970, Brazil; and †Department of Crop Sciences, 1102 S. Goodwin Avenue, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801-4709 Received June 2, 1997; accepted February 27, 1998

Chitobiosidase, endochitinase, endo-b1-3-glucanase, and N-acetylglucosaminidase were detected in cellfree culture filtrates of the soilborne fungal biocontrol agent Trichoderma harzianum isolate Th008 and the roots of soybean seedlings (Glycine max cv. Williams 82). With the exception of endochitinase, activity of these enzymes also was associated with Rhizoctonia solani isolate 2B-12, causal agent of soybean root rot. In greenhouse experiments, soybean seeds inoculated with T. harzianum Th008 were planted in a soil mixture infested with R. solani 2B-12. At 15 days after emergence, the rhizosphere was assayed for chitinolytic enzymes and endoglucanase. Only N-acetylglucosaminidase and endochitinase activities in the rhizosphere samples were significantly elevated above the controls. Using conventional specific enzyme assays and accepted methodologies including HPLC and native and SDS–PAGE slab-gels, it was determined that T. harzianum Th008 was the source of the endochitinase in the rhizosphere. On the other hand, the detectable levels of N-acetylglucosaminidase originated from the roots of soybean seedlings. r 1998 Academic Press Key Words: chitinolytic enzymes; endoglucanase; soybean; Glycine max; Trichoderma harzianum; Rhizoctonia solani.

INTRODUCTION

Some Trichoderma isolates are rhizosphere competent, nonpathogenic soilborne fungi that are antagonistic to other fungi including the soilborne plant pathogenic fungus Rhizoctonia solani Ku¨hn (Ahmad and Baker, 1987; Cherif and Benhamou, 1990; Elad et al., 1982; Lewis and Papavizas, 1985). Some isolates of Trichoderma spp. produce chitinolytic enzymes and glucanases in vitro which are capable of degrading fungal cell walls (Elad, et al., 1983; Lorito et al., 1994; Raikhel et al., 1993; Sivan and Chet, 1989). Chitinolytic enzymes and glucanases also are produced at the root

surface (Liu, 1990). However, no definitive evidence has shown the presence or activity of chitinases and endoglucanase in the rhizosphere (the zone immediately adjacent to the root of a plant) associated with a soilborne fungal pathogen. The objectives of this research were to determine the activity and source of some enzymes, such as chitinases and endoglucanase, proposed to be involved in biological control in situ and in the rhizosphere of soybean seedling roots. MATERIALS AND METHODS

Fungal Isolates and Soybean Cultivar Rhizoctonia solani isolate 2B-12 from a soybean root (Liu and Sinclair, 1991) was provided by J. B. Sinclair. Trichoderma harzianum isolate Th008 was isolated from greenhouse soil at the University of Illinois at Urbana-Champaign (UIUC). Certified soybean seed cv. Williams 82 was provided by Illinois Foundation Seeds, Inc. (Tolono). Cultures R. solani 2B-12 and T. harzianum Th008 were cultured in 100 ml Czapek-Dox broth (pH 6.0) (Difco Laboratories, Detroit, MI) amended with 200 mg crabshell chitin (Sigma Chemical Co., St. Louis, MO) in 250-ml Erlenmeyer flasks in a shaker bath operated at 120 oscillations per minute in the dark for 5 days at 30°C and pH 6.0. For inoculation, each flask contained 10 1-cm diameter plugs of R. solani 2B-12 grown on potato-dextrose agar (PDA; Difco) (pH 6.0) for 3 days in the dark at 23°C. Conidia from a 3-day-old PDA colony of T. harzianum Th008 grown at 23°C and pH 6.0 were suspended in sterile distilled water to give a final concentration of 1 3 107 spores/ml, which was used for the inoculum. Soybean seeds were surface-sterilized for 5 min in 0.5% NaOCl, washed three times in sterile deionized distilled water, and grown under sterile conditions on PDA at pH 6.0 with a 12-h photoperiod for

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1049-9644/98 $25.00 Copyright r 1998 by Academic Press All rights of reproduction in any form reserved.

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7 days at 30°C. Soybean seeds were germinated on PDA to check for contamination by other microorganisms. None were found. The resulting seedling roots were used in the cell-free culture fultrate studies. Cell-Free Culture Filtrates All cultures were filtered through Whatman No. 2 filter paper and then through 0.2-µm syringe filters. The culture filtrates (100 ml) were dispensed into dialysis tubing (12- to 14-kDa molecular weight cutoff (MWCO) and dialyzed overnight in 50 mM monobasic potassium phosphate plus 150 mM sodium sulfate (PPSS) buffer at 5°C at pH 6.0. These dialysates were added to dialysis bags, which were suspended in front of an electric fan to concentrate the samples to a volume of 2 ml. For soybean roots, 5 g of 5-cm sections were ground in sterile plastic grinders (DuPont grinder tubes, Precise Plastic Products, East McKeesport, PA), each containing 5 ml of PPSS buffer at 5°C to detect enzyme activity. The remainder of the procedure was the same as described above. All preparations of concentrated cell-free culture filtrates were used immediately in colorimetric assays for chitinolytic and endo-b1-3glucanase enzyme activities as described below. Determination of Extracellular Enzymes in the Soybean Rhizosphere To determine enzymatic activity in the rhizosphere, 2-cm root segments with clinging soil (5 g net weight) were cut from each plant starting at the soil level, weighed, suspended in 10 ml PPSS buffer at 5°C, gently stirred overnight, and then filtered, dialyzed, concentrated, and assayed as were the cell-free culture filtrates. The control consisted of 5.0 g greenhouse potting soil mixture. No detectable chitinolytic or endoglucanase activity was found in the sterilized soil samples. Colorimetric Enzyme Assays The colorimetric assays used for chitobiosidase and N-acetylglucosaminidase were based on p-nitrophenyl (PNP) release from PNP-substrates (Leatham, 1985). The b-N-acetylglucosaminidase (EC 3.2.1.52) was assayed using p-nitrophenyl-b-N-acetylglucosaminide (Sigma) as a substrate. The reaction mixtures for assaying cell-free culture filtrates consisted of: 200 µl culture filtrate, 1000 µl substrate (0.3 mg/ml), and 200 µl PPSS buffer. The control consisted of denatured, concentrated culture filtrate with the enzymes (boiled for 5 min at 100°C) plus substrate and buffer. The reaction mixtures were incubated for 30 min at 40°C for maximum color development. The reaction was stopped by the addition of 250 µl of 1 M NaOH and the absorbance at 405 nm was read in a Perkin–Elmer (Norwalk, CT) Bio-Lambda spectrophotometer with

1-cm path length quartz cuvettes (Hellma Corp., Forest Hills, NY). One unit of enzyme activity was defined as the amount of enzyme activity which liberated 1 mM PNP/ml enzyme preparation/min, where 1 mM PNP was equivalent to an increase of 6.367 absorbance at 405 nm. Due to the large number of samples processed from rhizosphere and HPLC fractions, a modification of the above procedure was developed. The reaction mixture consisted of: 20 µl filtrate, 100 µl substrate, and 20 µl PPSS buffer incubated for 6 h at 40°C. The reactions were carried out in 96-well plates (Falcon, Becton– Dickinson Co., Lincoln Park, NJ). The enzyme reactions were stopped by adding 25 µl 1 M NaOH to each well. The absorbance at 405 nm was read on an EL340 Biokinetics plate reader (Biotek, Winooski, VT). One unit of activity was defined as the amount of enzyme preparation (rhizosphere concentrate) which liberated 1 mM PNP (equivalent to 6.367 O.D.) at 405 nm/g root/min under the assay conditions. For chitobiosidase (EC 3.2.2.1.30) with p-nitrophenylb-N-N-diacetyl-chitobiose as the substrate (Sigma), the same procedures for assaying N-acetylglucosaminidase were used except that the cell-free culture filtrates were run for 2 h and the rhizosphere samples for 20 h. Colorimetric assays for the detection of endochitinase and endoglucanase activities were done with dyelabeled carboxymethyl chitins (Wirth and Wolf, 1992). Endochitinase (EC 3.2.1.14) was assayed using carboxymethyl-chitin-remazol brilliant violet (Blue Substrates, Go¨ttingen, Germany) as a substrate. For assaying cell-free culture filtrates, the reaction mixtures consisted of: 200 µl culture filtrate, 200 µl substrate (2 mg/ml stock), and 200 µl PPSS buffer. The control contained denatured enzyme plus substrate and buffer. The reaction mixtures were incubated for 1 h at 40°C and enzyme activity was terminated by addition of 100 µl of 1 N HC1. After the precipitated substrate was removed by centrifugation (microfuge for 2 min at 15,000g), the absorbance of the supernatant was read at 550 nm. One unit of enzyme activity was calculated as an increase of 0.001 O.D. at 550 nm/ml of enzyme preparation per minute. Due to the large number of samples processed for rhizosphere and HPLC fractions the above procedure was modified as follows. The reaction mixture consisted of: 100 µl enzyme preparations, 100 µl substrate (2 mg/ml), and 100 µl PPSS buffer incubated in a 96-well microtiter plates for 8 h at 40°C. The enzyme reactions were stopped by the addition of 50 µl 1 N HCl to each well and centrifuging the microtiter plates at 1000g using a microtiter plate-holder adapter to remove precipitated substrate. Absorbance at 550 nm was read in a microplate reader. One unit of enzyme activity was calculated as the amount of enzyme preparation which resulted in an increase in 0.001 O.D. at 550 nm/g root/min. For endo-b1-3-glucanase (EC 3.2.1.6) activity,

CHITINOLYTIC ENZYMES AND ENDOGLUCANASE IN SOYBEAN

similar procedures as outlined above for endochitinase were followed with the following modifications. The substrate used was carboxymethyl-pachyman-remazol brilliant blue R at 4 mg/ml stock. After the reactions were stopped with HCl and the precipitated substrate removed by centrifugation, the absorbance of the supernatant was read at 600 nm. Greenhouse Studies One-hundred g of seeds were treated either with 50 ml sterile distilled water plus 2% (v/v) Tween 20 (control) or in a 50 ml suspension of T. harzianum containing 108 conidia/ml plus 2% Tween 20 for 30 min. Ten seeds untreated or treated in this manner with T. harzianum were planted in 20-cm plastic greenhouse pots containing sterilized potting soil mix (sand/soil/ vermiculite; 1/1/1 at pH 6.5). The soil was either noninfested or infested with R. solani 2B-12 at 100 mg dried mycelium/liter of soil. The method used for soil inoculation with Rhizoctonia spp. has been described (Lewis and Papavizas, 1985). Each treatment was replicated six times and arranged in a complete randomized design and the experiments were conducted twice. All seedlings were grown with a 12-h photoperiod at approximately 25°C and harvested 15 days after emergence. Root infection by R. solani was determined using a 0 to 5 disease index scale (Liu, 1990). The disease index was transformed to percentage disease severity for use in an analysis of variance. This transformation was based on measurements of the range of the percentage of root surface with decay at each level of the scale. The mean values of these ranges were used to represent the entire level. The transformations used were: 0 5 0%; 1 5 1%; 2 5 5%; 3 5 25%; 4 5 75%; 5 5 100%. The average of disease index of plants from each pot was used in the statistical analysis (Liu, 1990). Root populations of T. harzianum were determined by conventional dilution plate counts using a spiral colony plating device on a Trichoderma-selective medium (TSM). The TSM consisted of 35 g Czapek-Dox broth (Difco) plus 15 g Bacto Agar (Difco)/liter water. The medium was autoclaved and to which was added 25 ml of a filtersterilized (0.2-µm syringe filter) solution of 100 mg neomycin sulfate, 100 mg penicillin G, 25 mg chlorotetracycline HCL, 500 mg sodium propionate, 100 mg captan, and 5 g oxgall. Estimation of Molecular Weights of Extracellular Enzymes in Soybean Rhizosphere Extracts Rhizosphere samples were fractionated by HPLC using size-exclusion chromatographic methods run under nondenaturing conditions. The Perkin–Elmer (Norwalk, CT) HPLC system consisted of a 1022 LC plus controller, series 200 LC pump, and 235 C dual-channel diode-array detector. The two columns (7.5 mm ID 3

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300 mm length) connected in series were a macro˚ pore size, 7-µm particle size (Alltech sphere GPC, 60 A Associates, Inc., Deerfield, IL), and a TSK G2000SW, ˚ pore size, 10-µm particle size (Perkin–Elmer 100 A Corp., Norwalk, CT). Isocratic elution at a flow rate of 0.3 ml/min at 23 6 1°C was done with filtered (0.45-µm filter disks), vacuum-degassed PPSS buffer. Injection volume was 500 µl. Fractions were collected at 1-min intervals on a Gilson micro fraction collector Model FC-80K. A calibration curve of protein standards (Sigma) rabbit muscle myosin, 205 kDa, Escherichia coli b-galactosidase, 116 kDa, bovine serum albumin, 66 kDa, rabbit muscle glyceraldehyde-3-phosphate dehydrogenase, 36 kDa, and a-lactalbumin 14.2 kDa vs HPLC retention time (min) was calculated using a nonlinear regression analysis and was used for estimation of molecular weights (MW) for rhizosphere enzymes. Enzyme activities were tested for each HPLC fraction by the specific colorimetric enzyme assay methods described above. Enzyme molecular weights were estimated from the fraction showing peak activity and shown for N-acetylglucosaminidase from R. solani 2B-12 (Fig. 1). Native Slab-Gel Electrophoresis with Activity Stain Overlay and SDS–PAGE for Estimation of Molecular Weights of Extracellular Enzymes in Rhizosphere Extracts Resolving gels at pH 8.3 (12.5%) and stacking gels at pH 6.8 (4%), 1.5 mm thick, were made with polyacrylamide. The running gel buffer consisted of Tris glycine at pH 8.3 (Bio-Rad Gel Manual, Bio-Rad Corp., Her-

FIG. 1. A nonlinear regression fit to the elution of five protein standards of known molecular weights. The x-axis represents the HPLC fractions collected at 1-min intervals starting when the first detectable enzyme was recorded. The y-axis (right) is a scale of molecular weights corresponding to the regression fit for the five proteins. Enzyme activity (y-axis left) was measured by colorimetric methods with p-nitrophenyl-b-N-acetylglucosaminidase as the substrate and 50 mM monobasic potassium phosphate plus 150 mM sodium sulfate buffer at pH 6.0. Fraction 5 intersects the standard curve at an estimated molecular weight of 115 kDa for N-acetylglucosaminidase from a cell-free culture filtrate of Rhizoctonia solani isolate 2B-12.

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cules, CA). The gels were run at a 100 V on a mini slab-gel apparatus Model MP from OWL (Cambridge, MA). The temperature during runs did not exceed 32°C. At the conclusion of each native gel run, the resolving gel was overlaid with a 7.5% polyacrylamide gel containing as an activity stain 1 mg/ml of PNP or dye-labeled blue substrates for 6 h at 30°C. Standard SDS–PAGE slab gels also were run with 12.5% resolving gels and 4% stacking gels at a constant 100 V. Proteins of known MW used as standards in a plot of log MW vs migration in millimeters from the top of the resolving gel were jack bean N-acetylglucosaminidase (100 kDa), Serratia marcescens endochitinase (41 kDa), and Aspergillus flavus endoglucanase (20 kDa) (Sigma). Each gel lane with a standard protein contained 10 µg protein, while gel lanes with rhizosphere samples contained 30 µg total protein. Protein concentrations were determined by the Bradford method (Bradford, 1976), with bovine serum albumin fraction V as standard (Sigma). Means 6 standard errors were calculated for three determinations by HPLC and by SDS–PAGE slab-gel analyses. Statistical Analyses A nonlinear regression fit was used for the calibration curve of protein standards vs HPLC retention time for the estimation of molecular weights of rhizosphere enzymes. Means 6 standard error were used in the analysis of the severity of soybean seedling root rot, extracellular enzyme activities in cell-free culture filtrates, and the estimation of molecular weights of chitinolytic enzymes in culture filtrates and rhizosphere extracts by SDS–PAGE and HPLC. Bonferoni’s Sigmastat method (Glantz, 1992) was used for means comparisons in studies on seed treatment with the biocontrol agent on the severity of seedling disease and population differences of the biocontrol agent in the absence and presence of the pathogen. A two-way

FIG. 2. Effect of seed treatment without (w/o) and with (w) Trichoderma harzianum isolate Th008 on the severity of soybean seedling root rot caused by Rhizoctinia solani isolate 2B-12 15 days after emergence. See under Materials and Methods greenhouse studies section for details. Bars are means 6 standard errors. Significant (P 5 0.05) differences between treatment means were determined using Bonferoni’s Sigmastat method (Glantz, 1992).

TABLE 1 Population Densities of Trichoderma harzianum Isolate Th008 in the Soybean (cv. Williams 82) Rhizosphere in the Absence or Presence of Rhizoctonia solani Isolate 2B-12 Trichoderma Colony-forming units/g of root tissuea Soil treatment

Nontreated seeds

Treated seeds

Noninfested with Rhizoctonia Infested with R. solani

9.3 3 10E a Bb 1.8 3 102 a B

4.5 3 106 a A 2.2 3 106 a A

a Colony-forming units determined at 15 days after emergence by dilution counts using a spiral colony plating device; root segments 0–2 cm below the soil line were sampled. b Means followed by the same letters (lower case by column and upper case by row) are not significantly different according to Bonferoni’s method for mean comparisons (P 5 0.05).

analysis of variance (ANOVA) F test (P 5 0.05) was performed on chitinolytic and endoglucanase activities in the rhizosphere with noninfested or infested or with the pathogen and from seedlings cultivated from seeds nontreated or treated with the biocontrol agent. RESULTS AND DISCUSSION

When analyzed by Bonferoni’s multiple means comparisons, the disease severity caused by R. solani was significantly less (P 5 0.05) in treatments having seeds treated with T. harzianum compared to nontreated seeds (81%) at 15 days after emergence (Fig. 2). The population growth of the antagonist was not affected by the presence of the pathogen. Root colonization by T. harzianum Th008 at day 15 was significantly different on seedlings grown from seed either treated or nontreated with T. harzianum (Table 1). However, there was no difference in T. harzianum population if the soil was noninfested or infested with R. solani. We determined the presence of chitinolytic enzymes and endoglucanase in cell-free culture filtrates of the pathogen and antagonist in host roots. This is the first report on the production of extracellular N-acetylglucosaminidase, chitobiosidase, and endoglucanse from R. solani, and an estimate of the molecular weight for N-acetylglucosaminidase from this source. Detectable levels of chitobiosidase, endo-b-13-glucanase, and Nacetylglucosaminidase in vitro were found in R. solani, T. harzianum, and soybean roots (Table 2). Trichoderma harzianum and soybean roots also produced detectable levels of a single form endochitinase in vitro, while R. solani did not. Detectable levels of the three chitinolytic enzymes and endoglucanse were recorded in the rhizosphere of soybean seedling roots at 15 days after emergence (Table 3). Roots infected with R. solani had signifi-

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TABLE 2

TABLE 4

Presence of Extracellular Enzyme Activities in Concentrated Cell-Free Culture Fultrates of the Rhizoctonia solani Isolate 2B-12 and Trichoderma harzianum Isolate Th008 and Soybean (cv. Williams 82) Seedling Roots

Chitinolytic and Endoglucanase Activity in the Soybean (cv. Williams 82) Rhizosphere from Seeds Untreated or Treated with Trichoderma harzianum Isolate Th008 Rhizosphere enzyme activitya

Enzyme activitya

Source of culture filtrate

N-acetylChitoEndoEndoSeed-Treatment glucosaminidase biosidase chitinase glucanase

N-acetylglucosaminidase

Chitobiosidase

Endochitinase

Endoglucanase

0.17 6 0.03 R. solani 2B-12 0.17 6 0.03 0.12 6 0.02 N/Db T. harzianum Th008 0.15 6 0.02 0.09 6 0.01 0.42 6 0.06 0.21 6 0.04 Soybean roots 0.38 6 0.05 0.33 6 0.04 0.10 6 0.01 0.08 6 0.01 a Units of enzyme activity/ml enzyme preparation/min. In each case the milliliters of enzyme preparation was normalized to 100 µg total protein in the culture filtrate as determined by the Bradford dye method (Bradford, 1976). Values shown are the means of three experiments 6 standard errors. b N/D, not detectable.

cantly elevated N-acetylglucosaminidase activity at day 15 compared to noninfected roots, but there was no significant increase in other enzyme activity tested. A significant elevation only in endochitinase activity was recorded in the rhizosphere at day 15 after seeds treated with T. harzianum Th008 were planted compared to nontreated seeds (Table 4). The molecular weights for N-acetylglucosaminidase from the rhizosphere extracts resembled more closely those from soybean seedling roots than that of either T. harzianum or R. solani (Table 5). By using the Bradford method of protein quantification (Bradford, 1976), a positive correlation was found between disease index and total protein (mg/g soil) in rhizosphere samples and in N-acetylglucosaminidase activity in rhizosphere extracts. This suggests that the release of N-acetylgluTABLE 3 Chitinolytic and Endoglucanase Activity in the Soybean (cv. Williams 82) Rhizosphere in the Absence or Presence of Soil Infected with Rhizoctonia solani Isolate 2B-12 Rhizosphere enzyme activitya

Soil-treatment Noninfected Infected with R. solani

N-acetylglucosaminidase

Chitobiosidase

Endochitinase

Endoglucanase

3.5bb

0.93a

0.15a

0.64a

1.09a

0.13a

0.47a

31.3a

a Enzyme activity is expressed in units of activity/g root tissue at 15 days after emergence. Values are the combined means from two trials. b Means followed by the same letter, by column, are not significantly different by the ANOVA F test (P 5 0.05).

Untreated Treated with T. harzianum

18.3ab

1.07a

0.11b

0.51a

16.5a

0.95a

0.17a

0.60a

a Enzyme activity is expressed in units of activity/root tissue at 15 days after emergence. Values are the combined means from two trials. b Means followed by the same letter, by column, are not significantly different by the ANOVA F test (P 5 0.05).

cosaminidase into the rhizosphere probably results from a response of root cells to the pathogen. Although there was no evidence for an increase (stimulation) in the production of N-acetylglucosaminidase in cell-free culture filtrates from T. harzianum Th008 cultures grown in the presence of R. solani 2B-12, a 3.4-fold increase in N-acetylglucosaminidase production was observed when 500 mg of substrate as inducer was added (data not shown). The molecular weight associated with the elevated levels of endochitinase activity found in the rhizosphere extracts after planting seeds treated with T. harzianum Th008 was not comparable to that secreted by soybean seedling roots. However, the molecular weight for this enzyme was similar to that from T. harzianum Th008 (Table 5). We found no evidence that R. solani 2B-12 secretes detectable amounts of this enzyme. Also, the inclusion of autoclaved mycelial mat of R. solani 2B-12 in cultures of T. harzianum Th008 did not result in a stimulation of production of this enzyme (data not shown). There was a positive correlation between seeds treated with T. harzianum Th008 and elevated levels of endochitinase in the rhizosphere. Thus, the source of the detectable elevated levels of this enzyme appears to be the biocontrol agent. Molecular weight determinations obtained using SDS–PAGE slab-gels are given for N-acetylglucosaminidase and endochitinase (Table 5). These values are in good agreement with those obtained using the HPLC protocol. For N-acetylglucosaminidase in the cell-free culture filtrates, HPLC analyses for the biocontrol agent, pathogen, and soybean roots gave molecular weights (kDa) of 100 6 9, 118 6 12, and 156 6 16, respectively. The rhizosphere extracts for soil infested with the pathogen and seeds treated with the pathogen and seeds treated with the biocontrol agent gave molecular weights of 145 6 15 and 149 6 17, respectively. These data suggest that the source of the detectable N-acetylglu-

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TABLE 5 Estimation of Molecular Weights in Kilodaltons for Two Chitinolytic Enzymes in Culture Filtrates and in Rhizosphere Extracts Source of enzymes Cell-free culture filtrates

Rhizosphere extracts

Enzyme

Trichoderma harzianum isolate Th008

Rhizoctonia solani isolate 2B-12

Soybean roots cv. Williams 82

Soil infested with Rhizoctonia solani isolate 2B-12

Seeds treated with Trichoderma harzianum isolate Th008

N-acetylglucosaminidase Endochitinase

94 6 4a 31 6 1

112 6 5 N/Db

168 6 8 36 6 2

155 6 7 35 6 2

159 6 9 30 6 1

a b

Mean 6 standard error; determined by SDS–PAGE; see Results and Discussion for HPLC values. N/D, not detected.

cosaminidase activity in the rhizosphere extracts is the soybean roots rather than the biocontrol agent or the pathogen. For endochitinase activity in both the cellfree culture filtrates and the rhizosphere extracts, the agreement between the molecular weight estimates by SDS–PAGE and HPLC are similar. For endochitinase, the HPLC procedure gave molecular weights (kDa) of 29 6 3 and 37 6 4 for this enzyme in cell-free culture filtrates of the biocontrol agent and soybean roots, respectively. No detectable activity for this enzyme was found for the pathogen. The rhizosphere extracts for soil infested with the pathogen and seeds treated with the biocontrol agent gave molecular weights of 37 6 3 and 26 6 4, respectively, for endochitinase. Recently we reported that the culture filtrates of T. harzianum Th008 inhibited the in vitro growth of R. solani (Bertagnolli et al., 1996). These results indicate that the probable source of the detectable endochitinase activity in rhizosphere extracts is the biocontrol agent rather than soybean root or pathogen. Some soilborne fungi, which are potentially useful as biocontrol agents, are known to secrete chitinolytic enzymes and endoglucanases in situ (Lorito et al., 1994; Schirmbock et al., 1994). Many plants are capable of secreting pathogenesis-related proteins, such as chitinases and glucanases in the absence or presence of fungal pathogens (Benhamou et al., 1993; Flach et al., 1993; Mauch et al., 1988; Raikhel et al., 1993; Rovira and McDougall, 1967; Sela-Buurlage et al., 1993; Zhu et al., 1994). With the use of soil amendments such as cell wall components (cellulose, chitin) and divalent metal ions, levels of extracellular enzymes increased and their presence assisted such plants in warding-off disease (Cotes et al., 1994; Curl and Truelove, 1986; Dodd et al., 1987). Cotes et al. (1994) showed a significant correlation between the ability of several isolates of Trichoderma to control R. solani in bean and chitinase production. They did not determine the source of the enzyme in soil samples. We have shown the pres-

ence and the source of chitinolytic enzymes in the rhizosphere of soybean seedling roots in the presence of a pathogen and its antagonist. To obtain rhizosphere samples root segments were stirred in a buffer solution. By this method only a single detectable form of N-acetylglucosaminidase was obtained with a molecular weight comparable to that found for roots grown in culture. Our preliminary unpublished results indicate that the amounts of the chitinases and endoglucanase present in the rhizosphere samples recovered was in tens of micrograms per sample. Although there may be isomeric forms for each enzyme differing in molecular weight, we were able to detect with standard methods one form of each of acetylglucosaminidase and endochitinase in the rhizosphere samples. Also, we tried to increase the levels of these enzymes in vitro by amending the culture media with substrates for the respective enzyme reactions. We achieved a 3.4-fold increase in production of N-acetylglucosaminidase and a 3.2-fold increase in endochitinase from T. harzianum isolate Th008. The use of fungicides to control Rhizoctonia diseases has a limited future because of increased regulatory restrictions. At present biological control cannot entirely replace chemical measures. Our report demonstrates the potential of T. harzianum for use as a component of an integrated disease management program for Rhizoctonia decreases. Our data also suggests that there is a need to enhance the production of endochitinase by T. harzianum in the rhizosphere. In addition, the observed enhanced production of Nacetylglucosaminidase was related to root infection by the pathogen and was not correlated with inoculation of the soybean seeds with the antagonist. The origin of the elevated level of this enzyme in the rhizosphere appears to be from soybean seedling roots. Another need is to enhance production of this enzyme from the soybean roots.

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ACKNOWLEDGMENTS

Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.

This study was a part of Project No. 68-0346 of the Agricultural Experiment Station, College of Agricultural, Consumer, and Environmental Sciences, University of Illinois at Urbana-Champaign. It was supported in part by a grant from the Illinois Soybean Program Operating Board, Bloomington, IL. The authors thank R. D. McClary and N. Thimmaiah for technical assistance.

Leatham, G. F. 1985. Extracellular enzymes produced by the cultivated mushroom Lentinus edodes during degredation of lignocellulose medium. Appl. Environ. Microbiol. 50, 859–867.

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