Pseudomonas Stutzeri YPL-1 Genetic Transformation and Antifungal

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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 1991, p. 510-516 Vol. 57, No. 2 0099-2240/91/020510-07$02.00/0 Copyright © 1991, American Society for Microbiology Pseudomonas stutzeri YPL-1 Genetic Transformation and Antifungal Mechanism against Fusarium solani, an Agent of Plant Root Rot HO-SEONG LIM, YONG-SU KIM, AND SANG-DAL KIM* Department of Applied Microbiology, Yeungnam University, Gyongsan 713-749, Korea Received 24 July 1990/Accepted 4 December 1990

An actively antagonistic bacterium that could be used as a biocontrol agent against Fusarium solani, which causes root rots with considerable losses in many important crops, was isolated from a ginseng rhizosphere and identified as a strain of Pseudomonas stutzeri. In several biochemical tests with culture filtrates of P. stutzeri YPL-1 and in mutational analyses of antifungal activities of reinforced or defective mutants, we found that the anti-F. solani mechanism of the bacterium may involve a lytic enzyme rather than a toxic substance or antibiotic. P. stutzeri YPL-1 produced extracellular chitinase and laminarinase when grown on different polymers such as chitin, laminarin, or F. solani mycelium. These lytic extracellular enzymes markedly inhibited mycelial growth rather than spore germination and also caused lysis of F. solani mycelia and germ tubes. Scanning electron microscopy revealed degradation of the F. solani mycelium. Abnormal hyphal swelling and retreating were caused by the lysing agents from P. stutzeri YPL-1, and a penetration hole was formed on the hyphae in the region of interaction with the bacterium; the walls of this region were rapidly lysed, causing leakage of protoplasm. Genetically bred P. stutzeri YPL-l was obtained by transformation of the bacterium with a broad-host-range vector, pKT230. Also, the best conditions for the transformation were investigated.

Antagonistic microorganisms, by their interactions with serial dilutions were plated on nutrient agar. Each isolate various soil-borne plant pathogens, play a major role in was tested for its inhibition of F. solani, a pathogenic plant microbial equilibrium and serve as powerful agents for fungus, as described below. The fungus F. solani was biological disease control (2, 5, 8, 15, 39). The interactions provided by the Korea Ginseng and Tobacco Research between biocontrol agents and plant pathogens have been Institute and was grown on potato dextrose agar (PDA). The studied extensively, and the application of biocontrol agents most efficient antagonistic bacterium was selected and iden- in the protection of some commercially important crops is tified according to criteria in Bergey's Manual ofSystematic promising (38, 47). Biocontrol of plant pathogens provides Bacteriology (22). an alternative means of reducing the incidence of plant In vitro antifungal activity tests. Two different techniques disease without the negative aspects of chemical controls were used for testing the antagonistic effect against F. like pesticides (6). Chemical fungicides are costly, can cause solani. In the first, which assayed the antifungal activity of environmental pollution, and may induce pathogen resist- bacterial strains on plates, samples (5 ,ul, containing approx- ance (18, 24). Additionally, they can cause stunting and imately 106 cells) from overnight cultures of bacterial strains chlorosis of young seedlings (18). Fusarium solani, a patho- in nutrient broth were inoculated 1 cm from the edge of petri genic plant fungus, causes root rots, which results in con- plates and allowed to soak into the agar. A small plug (about siderable economic losses in many important crops (7, 9). 5 mm square) of F. solani inoculum from the leading edge of The use of antagonists to control diseases incited by F. a culture of F. solani grown at 28°C for 3 days on PDA solani is being intensively studied (31, 47), but the mechanism involved in lysis of the fungus by bacteria is not well containing 0.2% chitin was placed in the center of the plate. established. Plates were incubated at 28°C and scored after 4 or 5 days by The objectives of the present study were to (i) isolate, measuring the distance between the edges of the bacterial select, and identify potentially useful bacterial antagonists colony and fungal mycelium. In the second assay, which for biocontrol of F. solani; (ii) determine its antifungal tested the antifungal activity in broth culture, bacterial mechanism with biochemical tests, mutational anialyses, and cultures were grown at 30°C for 84 h with aeration. Cells microscopic observations; and (iii) attempt transformation of were removed by centrifugation at 12,000 x g for 20 min. the antagonist to create a model system for the further The culture supernatants were then filtered aseptically genetic development of multifunctional biocontrol agents. through 0.45-,um-pore-size membrane filters. The resulting filtrates were stored at 4°C. Petri plates were filled with molten PDA with 1% culture filtrate. After the plates were MATERIALS AND METHODS cooled, the F. solani inoculum was placed on the agar Isolation and identification of an antagonistic bacterium. surface, and the plates were incubated for 4 or 5 days. The Antagonistic bacteria were isolated from rhizospheres in diameters of the F. solani colonies were recorded, and the ginseng root rot-suppressive soils in Yeungpung-gun, Korea. inhibition ratios were calculated relative to that of a control To isolate bacteria, the rhizosphere soils were suspended in without incorporated culture filtrate. Small plugs taken from 0.01 M phosphate buffer (pH 7.2) with a mortar, and then 2- to 3-day-old cultures of F. solani and added to 250-ml Erlenmeyer flasks containing 2.64% potato dextrose broth (PDB) were incorporated aseptically with 5% culture super- * Corresponding author. natants and incubated on a rotary shaker at 28°C for 5 days. 510 VOL. 57, 1991 ACTION OF P. STUTZERI AGAINST F. SOLANI 511

Fungal mycelia were collected on oven-dried preweighed distilled water. Fifty spores per well were counted for each paper (Toyo filter paper no. 2) and dried at 90°C, and dry of the three replicates per treatment. Germination rates of weights were determined. The inhibition ratio was expressed the chlamydospores and lysis of germ tubes were examined relative to a control (H20). under a light microscope at x400 magnification. Enzymatic activity tests. The cell wall-degrading enzymes Scanning electron microscopy. Microscopic observations such as exo-1,3-0-D-glucosidase (,B-1,3-glucanase) and ,B-N- were made in the interacting regions of F. solani grown with acethyl-D-glucosaminidase (chitinase) were assayed in cul- P. stutzeri YPL-1 in dual culture. The samples were fixed ture filtrates of Pseudomonas stutzeri YPL-1. For the prep- with 3% glutaraldehyde in 0.2 M phosphate buffer (pH 6.5) aration of crude chitinase, the bacterium was grown at for 3 h, washed with the same buffer for 15 min, fixed with 30°C for 84 h on a rotary shaker in chitin-peptone medium 2% OS04 for 2 h, and finally washed again with the buffer. (pH 6.8) containing 0.5% glucose, 0.2% peptone, 0.2% The material was dehydrated with ethanol at 4°C by using a chitin, (from crab shells; Sigma), 0.1% K2HPO4, 0.05% series of steps for 10 min each. The specimens were dried in MgSO4 7H2O, and 0.05% NaCl. For the preparation of a Hitachi HCP-2 critical point drier with CO2 as the carrier crude laminarinase, bacteria were grown at 30°C for 72 h on gas. The dried specimens were mounted on stubs with a rotary shaker in peptone medium containing laminarin Television Tube Koat to prevent charging. The specimens (from Eisenia arborea; Tokyo Chemical Co.). The cultures were sputter coated with gold palladium in a Ion Coater Giko were centrifuged aseptically at 12,000 x g for 20 min at 4°C. IB-5 and observed with a scanning electron microscope (ISI The lytic enzymes were prepared by salting them with SS 103). ammonium sulfate and dialyzing them with buffer. Genetic transformation. The transformation procedure The activities of chitinase and laminarinase were deter- was based on the method of Bagdasarian and Timmis (1). mined by measuring the release of reducing sugar by the Bacterial cells were grown in LB broth at 30°C for 24 h on a method of Nelson (36). One unit of chitinase (laminarinase) rotary shaker. A portion (0.5 ml) of such a culture was activity was determined as 1 ,umol of glucose per mg of reinoculated in 50 ml of fresh LB broth and grown to an protein per h. The reaction mixture of chitinase contained optical density at 660 nm of 0.25, which equals 7 x 105 0.3 ml of 1 M sodium acetate buffer (pH 5.3), 0.5 ml of 0. 1% CFU/ml. The cells were then chilled, harvested by centrifu- colloidal chitin prepared by the method of Bemiller (4) or F. gation at 12,000 x g for 3 min, and washed once with 25 ml solani mycelium prepared by the method of Morrissey et al. of 10 mM MOPS (morpholinepropanesulfonic acid) (pH (34), and 0.25 ml of enzyme solution. The reaction was 7.0)-10 mM RbCI-100 mM CaCl2. The cells were again carried out at 50°C for 4 h. The reaction mixture of lamina- centrifuged and suspended in 25 ml of MRC (100 mM MOPS rinase contained 0.3 ml of 1/15 M phosphate buffer (pH 5.5), [pH 6.5], 10 mM RbCl, 100 mM CaCl2). The cells were kept 0.5 ml of 0.2% soluble laminarin or F. solani mycelium, and on ice for 45 min, harvested by centrifugation, and sus- 0.25 ml of enzyme solution. The reaction was carried out at pended in 5 ml of MRC. An aliquot (0.2 ml) of these cells was 40°C for 2 h. then mixed with 1 pug of pKT 230 DNA isolated from Mutation and selection. The antagonist was mutated with Pseudomonas putida(pKT230) by the method of Kado and N-methyl-N'-nitro-N-nitrosoguanidine (NTG) treatment or Liu (19) and incubated at 0°C for 60 min. The cell-DNA UV radiation. The NTG mutagenesis procedure was based mixture was then subjected to a heat pulse at 42.5°C for 2 on the method of Miller (30). For the UV treatment, bacterial min, chilled, and finally diluted in 10 volumes of fresh LB suspensions of 108 CFU/ml were prepared in 0.1 M phos- broth. The cells were allowed to grow at 30°C on a rotary phate buffer. Then 5 ml of each suspension was placed in a shaker, and then aliquots were plated on LB agar plates glass petri dish and exposed 30 cm below a 10-W germicidal containing 100 ,ug of kanamycin sulfate per ml. UV lamp for 30 s. Chitinase-reinforced or -defective mutants were characterized by growing the mutagen-exposed cells on RESULTS minimal agar plates containing colloidal chitin as a sole carbon source. Isolation and identification of antagonistic bacterium. For Siderophore assay. Siderophore assay procedures were the selection of the antagonistic bacterium most inhibitory to based on the methods of Meyer and Abdallah (28, 29) and F. solani, over 300 isolates of bacteria were originally Scher and Baker (41). Siderophore production was deter- obtained from soils in which ginseng was cultivated. Among mined by growing bacteria in iron-deficient succinate mini- these isolates, 45 isolates that had similar colony character- mal medium (SMM) containing 0.6% K2HPO4, 0.3% istics were investigated for their anti-F. solani activities. KH2PO4, 0.1% (NH4)2SO4, 0.02% MgSO4 7H20, and 0.4% Only 10 isolates produced a zone of inhibition of 15 mm or succinic acid. The test bacteria were introduced into 50 ml of more with F. solani on PDA. The most active antagonistic medium and incubated on a rotary shaker at 30°C for 40 h. bacterium, YPL-1, was selected and identified as P. stutzeri The bacterial cells were removed by centrifugation at 12,000 based on the following characteristics: it was gram negative, x g for 20 min. Then 50 [lI of 2 M FeCl3 was added to sample motile by polar flagella, catalase positive, gelatin liquefac- tubes of the culture supernatants (4 ml per tube); other tubes tion negative, denitrification positive, and fluorescent pig- to which no iron was added served as the blanks. Insoluble ment negative. iron salts were removed by centrifugation at 3,000 x g for 5 Activity of P. stutzeri YPL-1 against F. solani. To investi- min. The A435 of the supernatants was measured with a gate the antifungal substance from P. stutzeri YPL-1 culture, spectrophotometer against water as the blank. the bacterial cells were grown in chitin-peptone medium at Spore germination assay. Germination of chlamydospores 30°C for 84 h with aeration. The antifungal activity according and growth of germ tubes of F. solani were assayed by to in vitro tests was compared for the following: (i) culture mixing a 50-,ul drop of the chlamydospore suspension pre- filtrate dialyzed with a cellulose dialysis sack (molecular pared by the method of Elad and Baker (10) with an equal weight cutoff, 12,000), (ii) evaporated nonprotein solution of volume of P. stutzeri YPL-1 filtrate in the wells of acid- the supernatant after the culture filtrate was treated with washed depression slides. The slides were incubated for 24 h cold ethanol, and (iii) heat-treated culture filtrate. After 5 in a sterile petri dish containing filter paper moistened with days of incubation at 28°C, the culture filtrate inhibited 512 LIM ET AL. APPL. ENVIRON. MICROBIOL.

TABLE 1. Antifungal activity of P. stutzeri YPL-1 TABLE 3. Comparison of antifungal abilities of P. stutzeri YPL-1 against F. solani and its chitinase-reinforced or -defective mutants against F. solani" Fungal dry weight' Fungal colony sizeb Prepn Inhibition Relative Inhibition Relative Enzymatic activity (U) Antifungal activity (%) (%) (%) (%)d (%) Mutant Rltv (mutagen) Chitinase Laminarinase Fungal dry antagonistic Culture filtratee 59.7 100.0 55.6 100.0 weight distance' Dialyzed solutionf 53.9 89.9 48.9 87.9 Nonprotein solutiong 11.9 19.9 8.9 16.0 YPL-1 0.86 1.21 50.8 100.0 Heat-treated solutionh 11.2 18.8 11.1 20.0 YPL-M122 (NTG) 0.00 0.00 0.6 0.0 YPL-M153 (NTG) 0.00 0.42 4.2 32.0 a Dry weight of F. solani with the treatment of P. stutzeri YPL-1 in PDB YPL-M1 (UV) 1.45 1.25 74.0 137.5 after 5 days of incubation at 28°C. b YPL-M26 (UV) 2.14 1.59 85.0 168.8 Colony circle diameter of F. solani on PDA plates with treatment P. 1.69 1.44 77.0 151.6 stutzeri YPL-1 after 5 days of incubation at 28°C. YPL-M178 (NTG) ' Completed inhibition ratio (100%) - dry weight of F. solani cultured with " P. stutzeri YPL-1 and its mutants were grown in chitin-laminarin-peptone solutions relative to those cultured with water. d medium at 30°C for 84 h. The culture filtrates were used for assays of Completed inhibition ratio (100%) - colony size of F. solani cultured with enzymatic and antifungal activities. solutions relative to those cultured with water. b Distance between the edges of the bacterial colony and fungal mycelium e p. stutzeri YPL-1 was grown in the chitin-peptone medium at 30'C for 84 after 5 days of incubation at 28°C. h. f Culture filtrate was dialyzed at 4°C for 48 h through a cellulose dialysis sack (molecular weight cutoff, 12,000). g Culture filtrate was precipitated by the addition of cold ethanol, and the supernatant was vacuum evaporated at 55°C. chitinase and laminarinase ability (chi lam) or chitinase (chi) h Culture filtrate was heated at 80°C for 1 h. were obtained by NTG mutagenesis. P. stutzeri YPL-M122 (chi lam) did not inhibit fungal growth at all, whereas P. stutzeri YPL-M153 (chi) inhibited growth only a little. In growth of F. solani by 57.7%, whereas the nonprotein addition, we observed that mutants (P. stutzeri YPL-M26 solution inhibited growth by 10.4%. Losses of antifungal and YPL-M178) produced larger inhibition zones after mu- activity after treatment with nonprotein solution and heat- tagenesis with UV or NTG. These are presumed to have treated solution were 82 and 80.7%, respectively, compared increased antifungal activities (chitinase, laminarinase). with the activity of the culture filtrate. However, only 10.9% These results indicate that the antifungal mechanism of P. of the antifungal activity was lost when F. solani was treated stutzeri YPL-1 depends more on enzymatic lysis of the cell with the dialyzed solution. According to these results, anti- wall components of F. solani by chitinase than on laminar- fungal substances involved in inhibition of F. solani by P. inase (Table 3). stutzeri YPL-1 were presumed to be heat unstable, macro- Production of siderophore by P. stutzeri YPL-1. To evaluate molecular substances such as hydrolytic enzymes (Table 1). the antifungal mechanism by which the extracellular sidero- Production of extracellular hydrolytic enzymes. Chitinase phore efficiently chelates environmental iron, making it less and laminarinase (,B-1,3-glucanase) activities produced by P. available to certain native microflora (20, 21, 25, 35, 42), the stutzeri YPL-1 in the presence of different carbon sources possibility of siderophore production by P. stutzeri YPL-1 are presented in Table 2. Chitinase activity was observed in was investigated after 40 h of growth in SMM containing media containing chitin, colloidal chitin, laminarin with different concentrations of FeCl3. In our experiments, P. chitin, N-acetylglucosamine, or dried F. solani mycelium as stutzeri YPL-1 was not able to produce an extracellular carbon sources. Laminarinase activity was observed in siderophore in iron-deficient medium; this result is entirely media containing laminarin, laminarin with chitin, or dried different from that reported previously (29). F. solani mycelium. The enzyme produced was capable of Antifungal effect of lytic enzymes on mycelial growth of F. degrading laminarin or chitin as well as F. solani mycelium. solani. To evaluate the effects of extracellular lytic enzymes Mutational analysis. Since the reduced suppressiveness of of P. stutzeri YPL-1 and its mutant on mycelial growth of F. chitinase-defective mutants could be due to their inability to solani, each of the lytic crude enzymes (10 ,ug of chitinase establish a significant mechanism of lysis against F. solani, per ml, 17 ,ug of laminarinase per ml) of the bacteria was studies were carried out to evaluate their ability to inhibit the added to PDB medium preinoculated with F. solani for 3 growth of F. solani. Mutants ofP. stutzeri YPL-1 that lacked days, and the culture was grown at 28°C for 4 days. A decrease of the mycelial volume of F. solani could be observed from the first day after the enzymes were added. TABLE 2. Effect of various carbon sources on chitinase and Crude chitinase of P. stutzeri YPL-1 inhibited the fungal laminarinase production by P. stutzeri YPL-la mycelial growth by 87.1% compared with that of the un- Enzymatic activity (U) treated control after 24 h of incubation, whereas crude Carbon source laminarinase inhibited growth by only 50%. In addition, the Chitinase Laminarinase antifungal activity of a crude chitinase of mutant P. stutzeri Chitin 0.78 YPL-M26 was observed at a much higher level than that of Coloidal chitin 1.00 the original strain (Fig. 1). Laminarin 1.14 Effect of lytic enzymes on the germination of F. solani Laminarin with chitin 0.86 0.05 chlamydospores. To confirm whether P. stutzeri YPL-1 in- N-Acetyl-D-glucosamine 0.67 0.00 hibits germ tube growth of the germinated chlamydospore or F. solani mycelium 0.50 0.36 spore germination of F. solani, chlamydospore suspensions a P. stutzeri was grown in a synthetic medium containing the various carbon of the culture filtrate of the bacterium were added to PDB. sources for 84 h. Units of chitinase and laminarinase activity were determined An increase of mycelial volume of F. solani was not ob- as micromoles of glucose per milligram of protein per hour. served, and the mycelial mass could scarcely be seen until 4 VOL. 57, 1991 ACTION OF P. STUTZERI AGAINST F. SOLANI 513

0 7/.*- . A.-

0~~~~1

250 ~~~~~~~~10a0 2501 0~~~~~~~~~~0

2 4 (days) 1 2 3 4 5 6 7 Incubation time (days) 2 4 6 8 10 FIG. 1. Antifungal effect of lytic enzymes on mycelial growth of F. solani. The 3-day-old F. solani cultures were treated with 10 ,ug Incubation time (days) of crude chitinase from P. stutzeri YPL-M26 per ml (0), 10 ,ug of crude chitinase from P. stutzeri YPL-1 per ml (0), 17 ,ug of crude FIG. 2. Antifungal effect of P. stutzeri YPL-1 on growth of F. laminarinase from P. stutzeri YPL-M26 per ml (A), or 17 p.g of crude solani by dual culture: 0, F. solani and P. stutzeri YPL-1; 0, F. laminarinase from P. stutzeri YPL-1 per ml (A) or not treated (O). solani; 0, P. stutzeri YPL-1.

days after incubation (data not shown). The germination of bacterial cells around the site, and the walls of this region F. solani chlamydospores on culture disks with the bacterial were rapidly lysed, causing leakage of protoplasm (Fig. 3). filtrate was inhibited by only 17.2% after 24 h of incubation Genetic transformation of P. stutzeri YPL-1. For develop- compared with that of the untreated control, but the growth ment of a multifunctional biocontrol agent, we obtained of germ tubes was inhibited; about 89.5% of the germ tube genetically bred P. stutzeri YPL-1 by transformation with underwent lysis. The length of the germ tube ofF. solani was the broad-host-range vector pKT230 (Fig. 4). The maximum 10 ,um, whereas that of the control was 380 ,um (Table 4). frequency of the transformation was achieved when the Interaction between P. stutzeri YPL-1 and F. solani in dual bacterial cells were harvested at the early-exponential culture. To investigate the antagonism involved in the inter- growth phase (Fig. 5). The highest transformation efficiency action between P. stutzeri YPL-1 and F. solani in dual was obtained when the competent cells were exposed to culture, the bacteria and fungi were grown in PDB at 28°C. chilled transformation buffer containing 20 mM RbCl and 100 The mass of the fungus was reduced and mycelial propagules mM CaCl2 before the addition of 1 ,ug of plasmid DNA (Fig. were decreased about 90% compared with those of the 6). The optimal pH for transformation was 6.5. When control without the bacterium from day 1 to day 5. However, competent bacterial cells that were incubated for 1 h were the fungus had no influence on bacterial growth (Fig. 2). brought in contact with plasmid DNA, the transformants Scanning electron microscopic observations revealed the were obtained at the highest frequency. It was calculated degradation of F. solani mycelium when its cell wall com- that the transformation frequency was 2 x 10-6 to 6 x 10-6 ponents served as the sole carbon source for P. stutzeri under these optimal conditions. YPL-1. Abnormal haphal swelling and retreating were caused by excretion of lytic enzymes from the bacterium; a DISCUSSION hole was formed on the hyphae with accumulation of the Major objectives of this study were to isolate a potentially useful bacterial antagonist for biocontrol of F. solani, to TABLE 4. Effect of P. stutzeri YPL-1 on germination of evaluate in detail its antifungal mechanism, and to develop a chlamydospores of F. solania more powerful antagonist by genetic transformation. Selec- tion of these bacterial isolates was facilitated by the use of Chiamydospore Germ tube growth the in vitro antifungal activity test. In our results, an actively Treatment germination Germ tube Germ tubes antagonistic bacterium that was strongly inhibitory to F. (% of control) length (pim) with lysisb(% solani was isolated from ginseng rhizospheres and identified P. stutzeri YPL-1 82.8 10 89.5 as a strain of P. stutzeri. Control (untreated) 100.0 380 ± 50 0.0 Selection procedures gave some indication of the mechanism of interaction between P. stutzeri YPL-1 and F. solani. a Chlamydospore germination and germ tube growth were observed after 24 In several biochemical tests with culture filtrates of P. h of incubation with filtrate from P. stutzeri YPL-1 grown in chitin-peptone the substances involved in the medium at 30°C for 84 h. stutzeri YPL-1, antifungal b Lysis ofgerminated spores was determined by the reduction in percentage inhibition of F. solani appeared to be heat-labile, macromo- of chlamydospores with germ tubes and observation of lysed germ tubes. lecular proteins (Table 1). Hence, the mechanism could 514 LIM ET AL. APPL. ENVIRON. MICROBIOL.

FIG. 3. Scanning electron micrographs of F. solani hyphae interacting with P. stutzeri YPL-1 in dual culture. (A) Abnormal hyphal swelling and outflow of protoplasm (arrow) caused by disintegration of the hypha. (B) Hyphal retreat. (C) Lysed hole on a hypha with the bacterium (arrow). (D) Irregular wall of a "stunted" hypha and lysis of the tip.

involve a lysing agent rather than a toxic substance or antibiotic. This was confirmed by mutational analysis of the antifungal activities of mutants with reinforced or defective chitinase and/or laminarinase productivity (Table 3). Also, P. stutzeri YPL-1 released extracellular ,-1,3-glucanase and chitinase, which are key enzymes in the lysis of fungal cell walls, when grown on polymers such as chitin, laminarin, or F. solani mycelium as carbon sources (Table 2). Several studies have shown that efficient parasitic biocontrol agents excrete extracellular lytic enzymes that are capable of degrading chitin and laminarin (12, 13, 16, 17, 31-34, 37, 43-46). Other similar studies have suggested that siderophore production rather than lytic ability could be effective in control of F. solani. However, this P. stutzeri YPL-1 isolate was not able to produce extracellular siderophores in iron-deficient medium, which conflicts with other reports (29). The lysing agents of P. stutzeri YPL-1 markedly inhibited mycelial and germ tube growth rather than spore germination of F. solani (Table 4, Fig. 1). Furthermore, the antifungal activity of crude chitinase on mycelial growth was much higher than that of crude laminarinase (Fig. 1). The cell walls of F. solani are composed mostly of chitin (47%), with FIG. 4. Agarose gel electrophoresis of pKT230 plasmid DNA 14% glucan (44). It seems, therefore, that chitinase is more obtained from the transformant of P. stutzeri YPL-1. Lanes: A, important than laminarinase in the degradation of F. solani standard pKT230; B, P. stutzeri YPL-1; C, transformant by P. cell walls. Similarly, Ordentlich et al. showed that chitinase stutzeri YPL-1; D, donor P. putida(pKT230). VOL. 57, 1991 ACTION OF P. STUTZERI AGAINST F. SOLANI 515

lium. Abnormal hyphal swelling and retreating were caused 0 0-0 by lysing agents from P. stutzeri YPL-1 in dual culture (Fig. 3A and B). A hole was formed on the hyphae in the region of 0 interaction with the P. stutzeri YPL-1; the walls of this 1.01- region were rapidly lysed, causing leakage of protoplasm (Fig. 3C and D). Elad et al. showed lysed sites and holes through coils, hooks, or appressoria of Trichoderma sp. 4) hyphae interacting with those of Rhizoctonia solani or Sclerotium rolfsii (11). Lifshitz showed that a factor of c)0 Trichoderma harzianium results in plasmolysis of hyphal tip 0 cells of Pythium sp. before contact with mycelium in dual a 0 0.51- culture (26). Ordentlich et al. showed swift swelling and partial degradation of S. rolfsii hyphae were caused by Serratia marcescens (37). The use of electron microscopy facilitated the study of the mode of antagonism and the localization of sites of interaction between hyphae of F. / 0.0 solani and P. stutzeri YPL-1. Kritzman showed that the N active sites in the tips, septa, and branches of hyphae 0-0 O contain oligomers of,-glucan and N-acetyl-D-glucosamine 10 20 (23). We may therefore conclude that ,-1,3-glucanase to- gether with chitinase produced by the biological agent at- Incubation time (hour) tacks these sites and completely degrades the hyphae. FIG. 5. Development of competence for P. stutzeri YPL-1 in LB For genetic development of a multifunctional biocontrol broth. Growth was started at an A660 of 0.02, corresponding to 7 x agent in the future by introducing a foreign gene, we ob- 105 CFU/ml. tained genetically bred P. stutzeri YPL-1 by transformation with the broad-host-range vector pKT230 (Fig. 4). This further suggests that the introduction of a plasmid-encoded was the key enzyme in the dissolution of hyphae of Sclero- gene into the antagonist will increase biocontrol efficacy by tium rolfsii (37). combining its lytic action with other biocontrol abilities such Mycolysis is defined as the loss of protoplasm in fungal as antibiosis. structures and enzymatic dissolution of the cell walls (27). It is one of the main mechanisms involved in the antagonistic ACKNOWLEDGMENT activity of biocontrol agents. Lysis of propagules in soil is a This research was supported by research grant 881-0407-004-2 logically satisfying method of biological control, since it from the Korea Science and Engineering Foundation. could reduce inoculum density (22). It is commonly accepted that lytic enzymes play essential roles in the life cycle of REFERENCES fungi. They have been implicated in apical growth, wall 1. Bagdasarian, M., and K. N. Timmis. 1982. Host:vector systems softening during hyphal branching, germination, degradation for gene cloning in Pseudomonas. Curr. Top. Microbiol. Immu- of septa for the mobilization of nuclei, and hyphal fusions (3, nol. 96:47-67. 14, 40, 48). In this study, scanning electron microscopic 2. Baker, R. 1968. Mechanisms of biological control of soil-borne observations revealed degradation of the F. solani myce- plant pathogens. Annu. Rev. Phytopathol. 6:263-294. 3. Bartnicki-Garcia, S. 1973. Fundamental aspects of hyphal mor- phogenesis. Symp. Soc. Gen. Microbiol. 23:245-267. 4. Bemiller, J. N. 1965. Polysaccharide preparations: chitin. Meth- ods Carbohydr. Chem. 5:103. 5. Blakeman, J. P., and N. J. Fokkema. 1982. Potential for biological control of plant diseases on the phylloplane. Annu. Rev. Phytopathol. 20:167-192. i12 [ 0 6. Chet, I. 1987. Trichoderma-application, mode of action and as a biocontrol agent of soil-bome 0 potential plant pathogenic fungi, p. 137-160. In I. Chet (ed.), Innovative approaches to 0 plant disease control. John Wiley & Sons, Inc., New York.

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