5
880 CAN. J. MICROBIOL. VOL. 37, 1991 al. (1988) found that of 55 strains positive by GM1-ELISA, 3 were negative by the Phadebact test. In our study, 1 of 24 LT-I producing strains was negative by the Phadebect test, but the quantity of toxin produced by this strain as determined by cell culture and GM 1 -ELISA was low. Scotland et al. (1989) evaluated 100 strains of E. coli for production of LT by Y-1 and VET-RPLA and found 50 to be positive by both tests, with titres of 10-2560 on Y-1 and 2 to 128 by VET-RPLA. Our results differed from the above study in showing similar titres by both of these tests, but they were in agreement with respect to detection of LT-11. Three LT-I1 strains in their collection tested negative by VET-RPLA. Cell culture assay for E. coli LT-I and LT-I1 detection is simple and sensitive but requires cell culture facilities. GM1- ELISA and the commercially available agglutination kits, described in this report, appear suitable for LT-I detection in clinical or food regulatory laboratories lacking cell culture facilities; incorporation of anti-LT-I1 serum into these tests would enhance their usefulness. The time required for sample preparation is the same for all of these tests and is about 25 h, for bacterial growth and toxin extraction. The assay of the samples by cell culture or VET-RPLA requires overnight incubation for optimal reaction; GM1-ELISA results are obtainable by 5 h (providing wells are precoated with GM1 ganglioside). The Phadebact test is the most rapid, with results within 10 min of sample application. However, we found that this test was somewhat less sensitive than the other assays but may be the method of choice for many laboratories for the rapid and simple screening of LT-I production by E. coli. VET-RPLA was simple and sensitive and appears to be a good substitute for cell culture or GM 1-ELISA for LT-I detection. BETTELHEIM, K. A., HANNA, N., SMITH, D. L., and DWYER, B. W. 1989.Evaluation of the Phadebact ETEC-LT Test for the heat-labile enterotoxin of Escherichia coli. Zentralbl. Bakteriol. 271: 70-76. CHAPMAN, P. A., and DALY, C. M. 1989. Comparison of Y 1 mouse adrenal cell and coagglutination assays for detection of Escherichia coli heat labile enterotoxin. J. Clin. Pathol. 42: 755-758. CHAPMAN, P. A., and SWIE, D. L. 1984. A simplified method for detecting the heat-labile enterotoxin of Escherichia coli. J. Med. Microbiol. 18: 399-403. DONTA, S. T., MOON, H. W., and WHIPP, S. C. 1974.Detection of heat- labile Escherichia coli enterotoxin with the use of adrenal cells in tissue culture. Science (Washington,D.C.), 183: 334-336. EVANS, D. J. JR., EVANS, D. G., DUPONT, H. L., Q)RSKOV, F., and Q)RSKOV, I. 1977. Patterns of loss of enterotoxigenicity by Escherichia coli isolated from adults with diarrhea: suggestive evidence for an interrelationship with serotype. Infect. Immun. 17: 105-1 11. HALL, P., and SEBAG, J. 1974.Decision-making in clinical practice and medical research: a theoretical analysis of predictors, indicators,and health index. Int. J. Bio-Med. Comput. 5: 301-309. HOLMES, R. K., TWIDDY, E. M., and PICKETT, C. L. 1986. Purification and characterization of type I1 heat-labileenterotoxin of Escherichia coli. Infect. Immun. 53: 464-473. LANGLEY, R. 1971. Practical statistics for non-mathematical people. Drake Publishers, Inc., New York. pp. 199-2 1 1. RUDENSKY, B., ISACSOHN, M., and ZILBERBERG, A. 1988. Improved detection of heat-labile enterotoxin of enterotoxigenic Escherichia coli by using a commercial coagglutination test. J. Clin. Microbiol. 26: 223 1-2232. SCOTLAND, S. M., DAY, N. P., and ROWE, B. 1983. Acquisition and maintenance of enterotoxin plasrnids in wild-type strains of Escheri- chia coli. J. Gen. Microbiol. 129: 3 111-3 120. SCOTLAND, S. M., FLOMEN, R. H., and ROWE, B. 1989. Evaluation of a reversed passive latex agglutination test for detection of Escherichia coli heat-labile toxin in culture supernatants. J. Clin. Microbiol. 27: 339-340. SPEIRS, J. I., STAVRIC, S., and KONOWALCHUK, J. 1977. Assay of Escherichia coli heat-labile enterotoxin with Vero cells. Infect. Immun. 16: 617-622. SVENNERHOLM, A.-M., and WIKLUND, G. 1983. Rapid GM1-enzyme- linked imrnunosorbent assay with visual reading for identification of Escherichia coli heat-labile enterotoxin. J. Clin. Microbiol. 17: 596-600. Environmental factors affecting the antagonism of Pseudomonas cepaciu against Trichoderma viride RAM S. UPADHYAY, LUISA VISINTIN, AND R. K. JAYASWAL' Department of Biological Sciences, Illinois State University, Normal, IL 61761, U.S.A. Received May 17, 1991 Accepted May 28, 199 1 UPADHYAY, R. S., VISINTIN, L., and JAYASWAL, R. K. 1991. Environmental factors affecting the antagonism of Pseudomonas cepacia against Trichodenna viride. Can. J. Microbiol. 37: 880-884. Antagonistic activity of the bacterium Pseudomonas cepacia against Trichoderma viride was greatly influenced by nutritional and environmental conditions.Xylose and trehalose strongly enhanced the antifungal activity of P. cepacia, whereas mannitol and glucose had little effect. The carbon sources that enhanced the antagonistic activity also inhibited sporulation of T. viride. Antagonism of P. cepacia was enhanced by ammonium nitrogen; however, with nitrite or nitrate there was only a little antagonism. The antagonism of P. cepacia was optimal at pH 5.0. Although P. cepacia showed maximum antagonism against T. viride at 37OC, the antagonism was fairly good at temperatures as low as lg°C, indicating that there is a broad range of temperature for the antifungal activity of P. cepacia. Key words: antagonism, environmental factors, Pseudomonas cepacia, Trichodenna viride. 'Author to whom all correspondence should be addressed. Printed in Canada / Imprim6 au Canada Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by YORK UNIV on 11/12/14 For personal use only.

Environmental factors affecting the antagonism of Pseudomonas cepacia against Trichoderma viride

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Page 1: Environmental factors affecting the antagonism of               Pseudomonas cepacia               against               Trichoderma viride

880 CAN. J . MICROBIOL. VOL. 37, 1991

al. (1988) found that of 55 strains positive by GM1-ELISA, 3 were negative by the Phadebact test. In our study, 1 of 24 LT-I producing strains was negative by the Phadebect test, but the quantity of toxin produced by this strain as determined by cell culture and GM 1 -ELISA was low.

Scotland et al. (1989) evaluated 100 strains of E. coli for production of LT by Y-1 and VET-RPLA and found 50 to be positive by both tests, with titres of 10-2560 on Y-1 and 2 to 128 by VET-RPLA. Our results differed from the above study in showing similar titres by both of these tests, but they were in agreement with respect to detection of LT-11. Three LT-I1 strains in their collection tested negative by VET-RPLA.

Cell culture assay for E. coli LT-I and LT-I1 detection is simple and sensitive but requires cell culture facilities. GM1- ELISA and the commercially available agglutination kits, described in this report, appear suitable for LT-I detection in clinical or food regulatory laboratories lacking cell culture facilities; incorporation of anti-LT-I1 serum into these tests would enhance their usefulness. The time required for sample preparation is the same for all of these tests and is about 25 h, for bacterial growth and toxin extraction. The assay of the samples by cell culture or VET-RPLA requires overnight incubation for optimal reaction; GM1-ELISA results are obtainable by 5 h (providing wells are precoated with GM1 ganglioside). The Phadebact test is the most rapid, with results within 10 min of sample application. However, we found that this test was somewhat less sensitive than the other assays but may be the method of choice for many laboratories for the rapid and simple screening of LT-I production by E. coli. VET-RPLA was simple and sensitive and appears to be a good substitute for cell culture or GM 1-ELISA for LT-I detection.

BETTELHEIM, K. A., HANNA, N., SMITH, D. L., and DWYER, B. W. 1989. Evaluation of the Phadebact ETEC-LT Test for the heat-labile enterotoxin of Escherichia coli. Zentralbl. Bakteriol. 271: 70-76.

CHAPMAN, P. A., and DALY, C. M. 1989. Comparison of Y 1 mouse

adrenal cell and coagglutination assays for detection of Escherichia coli heat labile enterotoxin. J. Clin. Pathol. 42: 755-758.

CHAPMAN, P. A., and SWIE, D. L. 1984. A simplified method for detecting the heat-labile enterotoxin of Escherichia coli. J. Med. Microbiol. 18: 399-403.

DONTA, S. T., MOON, H. W., and WHIPP, S. C. 1974. Detection of heat- labile Escherichia coli enterotoxin with the use of adrenal cells in tissue culture. Science (Washington, D.C.), 183: 334-336.

EVANS, D. J. JR., EVANS, D. G., DUPONT, H. L., Q)RSKOV, F., and Q)RSKOV, I. 1977. Patterns of loss of enterotoxigenicity by Escherichia coli isolated from adults with diarrhea: suggestive evidence for an interrelationship with serotype. Infect. Immun. 17: 105-1 11.

HALL, P., and SEBAG, J. 1974. Decision-making in clinical practice and medical research: a theoretical analysis of predictors, indicators, and health index. Int. J. Bio-Med. Comput. 5: 301-309.

HOLMES, R. K., TWIDDY, E. M., and PICKETT, C. L. 1986. Purification and characterization of type I1 heat-labile enterotoxin of Escherichia coli. Infect. Immun. 53: 464-473.

LANGLEY, R. 1971. Practical statistics for non-mathematical people. Drake Publishers, Inc., New York. pp. 199-2 1 1.

RUDENSKY, B., ISACSOHN, M., and ZILBERBERG, A. 1988. Improved detection of heat-labile enterotoxin of enterotoxigenic Escherichia coli by using a commercial coagglutination test. J. Clin. Microbiol. 26: 223 1-2232.

SCOTLAND, S. M., DAY, N. P., and ROWE, B. 1983. Acquisition and maintenance of enterotoxin plasrnids in wild-type strains of Escheri- chia coli. J. Gen. Microbiol. 129: 3 1 1 1-3 120.

SCOTLAND, S. M., FLOMEN, R. H., and ROWE, B. 1989. Evaluation of a reversed passive latex agglutination test for detection of Escherichia coli heat-labile toxin in culture supernatants. J. Clin. Microbiol. 27: 339-340.

SPEIRS, J. I., STAVRIC, S., and KONOWALCHUK, J. 1977. Assay of Escherichia coli heat-labile enterotoxin with Vero cells. Infect. Immun. 16: 617-622.

SVENNERHOLM, A.-M., and WIKLUND, G. 1983. Rapid GM1-enzyme- linked imrnunosorbent assay with visual reading for identification of Escherichia coli heat-labile enterotoxin. J. Clin. Microbiol. 17: 596-600.

Environmental factors affecting the antagonism of Pseudomonas cepaciu against Trichoderma viride

RAM S. UPADHYAY, LUISA VISINTIN, AND R. K. JAYASWAL' Department of Biological Sciences, Illinois State University, Normal, IL 61761, U.S.A.

Received May 17, 1991 Accepted May 28, 199 1

UPADHYAY, R. S., VISINTIN, L., and JAYASWAL, R. K. 1991. Environmental factors affecting the antagonism of Pseudomonas cepacia against Trichodenna viride. Can. J. Microbiol. 37: 880-884.

Antagonistic activity of the bacterium Pseudomonas cepacia against Trichoderma viride was greatly influenced by nutritional and environmental conditions. Xylose and trehalose strongly enhanced the antifungal activity of P. cepacia, whereas mannitol and glucose had little effect. The carbon sources that enhanced the antagonistic activity also inhibited sporulation of T. viride. Antagonism of P. cepacia was enhanced by ammonium nitrogen; however, with nitrite or nitrate there was only a little antagonism. The antagonism of P. cepacia was optimal at pH 5.0. Although P. cepacia showed maximum antagonism against T. viride at 37OC, the antagonism was fairly good at temperatures as low as lg°C, indicating that there is a broad range of temperature for the antifungal activity of P. cepacia.

Key words: antagonism, environmental factors, Pseudomonas cepacia, Trichodenna viride.

'Author to whom all correspondence should be addressed.

Printed in Canada / Imprim6 au Canada

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NOTES 88 1

UPADHYAY, R. S., VISINTIN, L., et JAYASWAL, R. K. 1991. Environmental factors affecting the antagonism of Pseudomonas cepacia against Trichoderma viride. Can. J. Microbial. 37 : 880-884.

Les conditions nutritives et environnementales influencent fortement le pouvoir antagoniste de Pseudomonas cepacia sur Trichoderma viride. Le xylose et le trkhalose augmentent fortement I'activitC antifongique de P. cepaciu alors que le mannitol et le glucose ont peu d'effet. Les sources de carbone qui favorisent I'effet antagoniste inhibent aussi la sporulation de T. viride. L'azote ammoniacal favorise le pourvoir antagoniste de P. cepacia, mais en prCsence de nitrites ou de nitrates i l y a peu d'antagonisme. Le pouvoir antagoniste de P. cepacia devient optimal a pH 5,O. Meme si I'activitC antagoniste de P. cepacia vis-a-vis de T. viride est optimale a 37"C, cet antagonisme demeure assez fort a des tempkratures aussi basses que 18°C confirmant que 1'activitC antifongique de P. cepacia se manifeste a 1'intCrieur d'un large spectre de tempkratures.

Mots elks : antagonisme, conditions environnementales, Pseudomonas cepacia, Trichoderma viride. [Traduit par la rCdaction]

Pseudomonas species are aggressive colonizers of the rhizo- sphere of various crop plants (Schroth and Hancock 1982) and have a broad spectrum of antagonistic activity against plant pathogens (Burr and Caesar 1984; Davison 1988; Fravel 1988; Suslow 1982; Weller 1988). These characteristics make these species good candidates for use as seed inoculants and root dips for biological control of soilborne plant pathogens. Among Pseudomonas species, P. cepacia was recently recognized as having potential for biological control against fungal phytopathogens (Anderson and Liberta 1986; Cho 1987; Fantino and Bazzi 1982; Homma et al. 1989; Janisiewicz and Roitman 1988; Jayaswal et al. 1990; Jee et al. 1988; Kawa- mato and Lorbeer 1976; Kim and Roh 1987; Knudsen and Spurr 1987; Lambert et al. 1987; Livens et al. 1989; Parke 1990; Wilson and Chalentz 1989). The production of an antifungal compound by P. cepacia has been regarded as one of the mechanisms involved in antagonism. The antifungal compound has been isolated and identified as pyrrolnitrin (Homma et al. 1989; Lambert et al. 1987; Lee and Kobayashi 1988; Livens et al. 1989; M. A. Fernandez. 1990. M.S. thesis, Illinois State University, Normal, IL). We recently isolated a pyrrolnitrin-deficient mutant of P. cepacia by transposon Tn5- 259 mutagenesis, which failed to show any antifungal activity against Trichoderma viride and other fungi (M. A. Fernandez, M.S. thesis). This observation provides strong evidence that the antagonism of P. cepacia is due to production of the pyrrol- nitrin. We also have observed strong inhibition of many fungi by an unidentified volatile compound produced by P. cepacia (R. S. Upadhyay and R. K. Jayaswal, unpublished data). Despite attempts at identifying the antifungal compound, no study has been done on the factors affecting its production.

Recently, from our preliminary studies we reported that P. cepacia possesses antagonistic activity against T. viride and that this activity may be affected by environmental conditions (Jayaswal et al. 1990). It was observed that the antagonism by P. cepacia differed when tested on potato dextrose agar (PDA) and cornmeal agar (CMA, Difco Laboratories, Detroit, MI). On PDA, only a little antagonism was observed, indicating that antagonism is affected by nutritional factors. Therefore, nutritional and environmental factors were evaluated for their effect on the antagonistic activity of P. cepacia. Since the production of the antifungal compound is under genetic control (M. A. Fernandez, M.S. thesis), it was of obvious interest to investigate the interaction between two microbes that share a common habitat. In the present study, we have investigated the antagonism of P. cepacia against T. viride in relation to varying nutritional and environmental conditions in order to assess how the antifungal activity is affected by these factors in vitro.

A culture of T. viride was obtained from Anthony Liberta, Department of Biological Sciences, Illinois State University, Normal, IL, and was maintained on PDA or CMA slants at 4°C. A culture of P. cepacia having antifungal activity (RJ3)

was maintained either on Luria-Bertani agar (LB) or CMA as described previously (Jayaswal et al. 1990).

Antagonism between P. cepacia and T. viride was assayed on CMA by a dual culture method as described by Skidmore and Dickinson (1976) and Upadhyay and Rai (1987). Cultures of P. cepacia and T. viride were inoculated on CMA or M9 medium (Miller 1972) plates at fixed positions 2-2.5 cm apart. A 7-mm block of an actively growing culture of T. viride from a CMA plate and 20 pL of an overnight LB-grown culture of P. cepacia were used for the antagonism assay. Plates were incubated at 30°C, and the growth of T. viride was measured at 24-h intervals. The percent inhibition of the growth of T. viride was calculated with the following formula (Whipps 1987): ((R, - R2)IR,) X 100, where Rl is the farthest radial distance grown by T. viride in the direction of the antagonist (a control value) and R, is the distance grown on a line between the inoculation positions of T. viride and P. cepacia.

Antagonism and biocontrol ability of P. cepacia were also tested in vivo against Diplodia maydis and Fusarium roseum. These fungi cause seedling blight and stalk rot in maize. Bacterial lawn grown on CMA was thoroughly mixed with 25 maize seeds (inbred line OS 420 obtained from Dr. A. Hallauer, Department of Agronomy, Iowa State University, Ames, IA), which were then dried overnight at room temperature in a sterile cabinet. The coated seeds were sown in pots (6 X 6 in. (1 in. = 25.4 mm)) containing natural soil mixed with sand and fungal inoculum in a ratio of 1:5 (inoculum:soil). Fungal inoculum was prepared as described by Garrett (1962). The plants were grown at 25 * 2°C in a 12 h light : 12 h dark photoperiod, and disease symptoms were recorded after 6 weeks. Seeds not treated with P. cepacia and grown in the soil containing fungal inocula served as control.

For physiological studies, antagonism was assessed on M9 medium (Miller 1972) with the following composition: Na,HPO,, 6 g; KH,PO,, 3 g; NaCl, 0.5 g; NH,Cl, 1 g; distilled water, 1000 mL. The M9 salts and agar were autoclaved separately and then supplemented with 2 mL of 1 M MgSO, 7H20, 10 mL of 20% glucose, 0.1 mL of 1 M CaCl,, and 0.5 mL of 1 % vitamin B 1 (thiamine-HC1). The pH of the medium was adjusted to 6.5 unless otherwise indicated. Sporulation and pigmentation in T. viride and P. cepacia, respectively, were scored visually. The experiments were carried out in triplicate and the data were analysed using two-way ANOVA.

The 26 carbon sources used in this study were added to M9 medium at a final concentration of 0.2% wlv. The sole nitrogen sources included inorganic nitrogen salts and amino acids in the M9 medium at a final concentration of 0.1 % wlv. The effect of pH on antagonism was assessed in M9 medium adjusted with NaOH or HC1 to 0.5-unit intervals from pH 3.5 to 9.0. To determine the effect of temperature, the plates containing dual cultures on M9 medium were incubated at 18, 24, 30, 37, and 43°C.

The seeds treated with P. cepacia protected seed germina-

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CAN. J . MICROBIOL. VOL. 37, 1991

Carbon Source

FIG. 1. Effect of carbon sources on antagonistic activity of P. cepacia against T. viride.

tion, seedling vigor, and infection in corn by D. maydis. Seedling blight due to D. maydis was controlled when seeds were coated with the bacterium. Although no obvious disease symptoms were recorded in the case of F. roseum, a protection of seed germination and seedling vigor due to P. cepacia was observed. Seed germination was 100% in the case of seeds coated with the bacterium compared with 30% for untreated seeds.

There was a high degree of variation in the level of antago- nism when 26 carbon sources were tested in M9 medium (Fig. 1). Xylose and trehalose were most effective in facilitating the antagonism, whereas mannitol and glucose were least effective. We have categorized the degree of effect of the carbon sources into six classes in decreasing order: 1, xylose, trehalose (90% or more inhibition); 2, succinate, fructose, mannose, maltose, rhamnose, malate, arabinose (8049%); 3, ribose, peptone, pectin, lactose, inositol, sucrose (70-79%); 4, polygalacturanate (PG), starch, sorbitol, citrate, acetate (60-69%); 5, pyruvate, galactose (40-59%); and 6, mannitol, glucose (less than 40%). Some of these carbon sources also had an impact on the inhibition of sporulation of T. viride by P. cepacia. Carbon sources facilitating the antagonism also inhibited sporulation of T. viride. Pigmentation production by P. cepacia also was affected by the carbon sources. However, there was no correlation between pigmentation and the antagonistic activity. For example, in the case of maltose, the level of antagonism was very high (85%) but the bacterium produced no pigmenta- tion.

Variation of the nitrogen sources also affected the level of antagonism exhibited by P. cepacia (Fig. 2). The form of nitrogen was an important factor. It is evident from Fig. 2 that the antifungal activity was higher with the ammonium forms (category 1: NH4Cl, NH4H2P04, NH4N03, and (NH4),S04) of nitrogen as the nitrogen source than with nitrate or nitrite forms of nitrogen (category 3). There was almost no antago- nism by the bacterium when NaNO,, Ca(NO,),, NaNO,, or KNO, was used as the nitrogen source. Urea caused an intermediate effect (category 2). However, measurements of inhibition were complicated by the fact that the growth of T. viride was very poor with urea as the nitrogen source. With ammonium forms of nitrogen, P. cepacia was highly effective in reducing the growth and sporulation of T. viride. This again

Nitrogen Source

FIG. 2. Effect of nitrogen sources on antagonistic activity of P. cepacia against T. viride.

confirms a positive association between the antagonistic activity and the inhibition of sporulation.

With single amino acids as sole sources of nitrogen there was great variation in the level of antagonism by P. cepacia (Fig. 3). On the basis of their effects on antagonism, the amino acids were grouped in the following categories in decreasing order: 1, cysteine, leucine, valine, methionine (70-79%); 2, glutamic acid (66%); 3, aspartic acid, threonine, glycine (50-59%); 4, isoleucine, histidine, alanine, tryptophan, aspara- gine, glutamine (4049%); 5, tyrosine, lysine (30-39%); 6, serine, proline (20-29%); 7, phenylalanine, arginine ( 10- 19%). As was the case with the carbon sources, inhibition of sporula-

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NOTES

Amino Acid

FIG. 3. Effect of amino acids on antagonistic activity of P. cepacia against T. viride.

100

90 -

FIG. 4. Effect of pH on antagonistic activity of P. cepacia against T. viride.

tion in T. viride was correlated to antifungal activity. Pigment production by P. cepacia also was affected by certain amino acids and, again, no association between pigmentation and antifungal activity was observed.

Antifungal activity also was affected by pH. Pseudomonas cepacia and T. viride did not grow at pH 3.5 and 9.0, respec- tively (Fig. 4). Antagonism was greater in the acidic range (4-6.5). Antagonism started at pH 4.0 and increased to a maxi- mum at pH 5.0. Above pH 6.5 the antagonism by P. cepacia was substantially decreased. At pH 8.0 and 8.5, there was no antagonism, even though P. cepacia grew very well and produced pigmentation. These results indicate that antagonism of P. cepacia against T. viride is affected by the pH of the medium. More pigmentation was observed when P. cepacia was grown in alkaline media compared with acidic media.

There was an increasing trend in antagonism as the tempera- ture was raised from 18 to 37OC, being maximal at 37°C (Fig. 5). At 43"C, neither P. cepacia nor T. viride grew.

Pseudomonas cepacia strongly inhibits the growth of T. viride. Up to 90% inhibition occurred under certain nutri- tional conditions (xylose or trehalose as carbon source). The bacterium produced up to a 2- to 2.5-cm zone of inhibition

Temperature (OC)

FIG. 5. Effect of temperature on antagonistic activity of P. cepacia against T. viride.

against T. viride. The mechanism of antagonism is antibiosis. An antifungal growth-inhibitory substance is secreted by the bacterium (M. A. Fernandez, M.S. thesis; R. K. Jayaswal, M. A. Fernandez, R. S. Upadhyay, Luisa Visintin, Michael Kurz, James Webb, and K. Rinehart, unpublished data). When small blocks of mycelium from the edge of the colony towards the bacterium were examined under a microscope, branching and deformities in the hyphae of T. viride were noted. The hyphae appeared stunted, branched, and frequently swollen (data not shown). These deformities probably were caused by the inhibitory compound produce by P. cepacia. Similarly Lee and Kobayashi (1988) also have recorded lysis and branching in the hyphae of Rhizoctonia solani caused by antifungal substances produced by P. cepacia.

Pseudomonas cepacia is one of the most nutritionally versatile bacteria, capable of using as a sole carbon source a large number of carbohydrates and their derivatives, amino acids, and other nitrogenous compounds (Ballard et al. 1970). Nutritional factors have great impact on the antagonistic activity of P. cepacia. Our study shows that this species also has the versatility to produce antifungal activity on a variety of carbon and nitrogen sources. This feature makes it very

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884 CAN. J . MICROBIOL. VOL. 37, 1991

attractive for selection as a potential candidate for biological control of fungal pathogens.

Among the inorganic nitrogen sources ammonium forms of nitrogen greatly enhanced the antagonistic activity of P. cepacia. This observation is interesting because ammonium fertilizers are commonly used in agricultural practices and thus by their use, antibiotic production by P. cepacia may be greatly enhanced. On the basis of the present data, it is difficult to explain why there was stimulation of antagonism by ammonium. However, directly or indirectly ammonium may be stimulatory to the metabolic production of the antifungal compound. It has been observed that pH of the rhizosphere of wheat decreases with uptake of ammonium, as nitrogen, by roots and increases with uptake of nitrate nitrogen (Smiley and Cook 1973). Therefore, it may be presumed that the production of the antifungal compound may be affected by ammonium by a change in pH of the bacterial cells or their surroundings during ammonium uptake. In our experiments, we observed enhancement of the antifungal activity at low pH.

There was no antagonism shown by the bacterium at pH 8.0 or above. The antifungal activity by P. cepacia was greater at acidic than at alkaline pHs. This is a noteworthy observation for the potential use of P. cepacia as a biocontrol. Soils with acidic pH will probably favor P. cepacia to express its antagonistic capability.

The antifungal activity was maximum at 37"C, which is within the optimum growth range for P. cepacia (Duodoroff and Palleroni 1974). However, it was fairly good even at lower temperatures (18°C). Thus, P. cepacia has a broad temperature range (18-37°C) for production of the antifungal compound. This is another interesting feature of the bacterium, making it a suitable biocontrol agent. The present study along with our earlier data (M. A. Fernandez, M.S. thesis) clearly establishes that the production of the antifungal compound by P. cepacia not only is under genetic regulation but is also greatly influ- enced by physiological and environmental conditions.

Acknowledgments The authors thank K. Miller and H. Brockman for sugges-

tions to improving the manuscript. This work was supported by National Institutes of Health grant lR15AI2957 10-0 1 and a University Research Grant from Illinois State University.

ANDERSON, R. C., and LIBERTA, A. E. 1986. Occurrence of fungal- inhibiting Pseudomonas on caryopses of Tripsacum dactyloides L. and its implication for seed survival and agriculture application. J. Appl. Bacteriol. 66: 195-1 99.

BALLARD, R. W., PALLERONI, N. J., DOUDOROFF, M., STAINIER, R. Y., and MANDEL, M. 1970. Taxonomy of the aerobic pseudo- monads. Pseudomonas cepacia, P. rnarginata, P. alliicola, and P. caryophylla. J. Gen. Microbiol. 60: 199-214.

BURR, T. J., and CAESAR, A. 1984. Beneficial plant bacteria. CRC Crit. Rev. Plant Sci. 2: 1-20.

CHO, E. K. 1987. Strategies for biological control of soilborne diseases in economic crops in Korea. Korean J. Plant Pathol. 3:' 313-317.

DAVISON, D. R. 1988. Plant beneficial bacteria. BioEechnology, 6: 282-286.

DUODOROFF, M., and PALLERONI, N. J. 1974. Pseudomonas. In Bergey's manual of determinative bacteriology. 8th ed. Edited by R. E. Buchanan and N. E. Gibbons. Williams and Wilkins Co., Baltimore, MD. pp. 2 17-243.

FANTANIO, M. G., and B ~ I , C. 1982. Antagonistic effect of Pseudomonas cepacia against Fusarium oxysporum. Inf. Fitopatol. 32: 55-58.

FRAVEL, D. R. 1988. The role of antibiosis in biological control of plant diseases. Annu. Rev. Phytopathol. 26: 75-91.

GARRETI-, S. D. 1962. Soil fungi and soil fertility. Cambridge University Press, Cambridge, U.K.

HOMMA, Y., SATO, Z., HIRAYAMA, F., KONNO, K., SHIRAHAMA, H., and SUZUI, T. 1989. Production of antibiotics by Pseudomonas cepacia as an agent for biological control of soil-borne plant pathogens. Soil Biol. Biochem. 21: 723-728.

JANISIEW[CZ, W. J., and ROITMAN, J. 1988. Biological control of blue mould and gray mould on apple and pear with Pseudomonas cepacia. Phytopathology, 78: 1697-1 700.

JAYASWAL, R. K., FERNANDEZ, M. A., and SCHROEDER, R. G. 1990. Isolation and characterization of a Pseudomonas strain that restricts growth of various phytopathogenic fungi. Appl. Environ. Microbiol. 56: 1053-1058.

JEE, H. J., NAM, C. G., and KIM, C. H. 1988. Studies on biological control of Phytophthora blight of red pepper. I. Isolation of antagonists and evaluation of antagonistic activity in vitro and in greenhouse. Korean J. Plant Pathol. 4: 305-3 12.

KAWAMOTO, S. O., and LORBEER, J. W. 1976. Protection of onion seedlings from Fusarium oxysporum f. sp. cepae by seed and soil infestation with Pseudomonas cepacia. Plant Dis. Rep. 60: 189-191.

KIM, H. K., and ROH, M. J. 1987. Isolation, identification and evaluation of biological control potential of rhizosphere antagonists of Rhizoctonia solani. Korean J. Plant Prot. 26: 89-97.

KNUDSEN, G. R., and SPURR, H. W., JR. 1987. Field persistence and efficacy of five bacterial preparations for biological control of peanut leaf spot. Plant Dis. 71: 441-445.

LAMBERT, B., LEYNS, F., JOOS, B., TENNING, P., RIJBERGEN, R. VAN, OUTRYVE, F. VAN, ZHAO, Y., SWINGS, J., and MONTAGU, M. VAN. 1987. Rhizobacteria with broad spectrum antifungal activity. Bull. OEPP. 17: 601-607.

LEE, W. H., and KOBAYASHI, K. 1988. Isolation and identification of antifungal Pseudomonas sp. from sugar beet roots and its antibiotic products. Korean J. Plant Pathol. 4: 264-270.

LIVENS, K. H., RIJBERGEN, R. VAN, LEYNES, F. R., LAMBERT, B. J., TENNING, P., SWINGS, J., and Joos, H.J.-P. 1989. Dominant rhizosphere bacteria as source of antifungal agents. Pestic. Sci. 27: 141-154.

MILLER, J. H. (Editor). 1972. Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

PARKE, J. L. 1990. Population dynamics of Pseudomonas cepacia in the pea spermosphere in relation to biological control of Pythium. Phytopathology, 80: 1307-1 3 1 1.

SCHROTH, M. N., and HANCOCK, J. G. 1982. Disease suppressive soils and root colonizing bacteria. Science (Washington, D.C.), 216: 13761381.

SKIDMORE, A. M., and DICKINSON, C. H. 1976. Colony interaction and hyphal interference between Septoria nodorum and phylloplane fungi. Trans. Br. Mycol. Soc. 66: 57-64.

SMILEY, R. W., and COOK, R. J. 1973. Relationship between take-all of wheat and rhizosphere pH in soils fertilized with ammonium vs. nitrate nitrogen. Phytopathology, 63: 882-890.

SUSLOW, T. V. 1982. Role of root colonizing bacteria in plant growth. In Phytopathogenic prokaryotes. Edited by M. S. Mount and G. H. Lacy. Academic Press, London. pp. 187-223.

UPADHYAY, R. S., and RAI, B. 1987. Studies on antagonism between Fusarium udum Butler and root region microflora of pigeon-pea. Plant Soil, 101: 79-93.

WELLER, D. 1988. Biological control of soilborne plant pathogens in the rhizosphere with bacteria. Annu. Rev. Phytopathol. 26: 379-407.

WHIPPS, J. M. 1987. Effect of media on growth and interactions between a range of soil-borne glasshouse pathogens and antagonis- tic fungi. New Phytol. 107: 127-142.

WILSON, C. L., and CHALENTZ, E. 1989. Post harvest biological control of Penicillium rots of citrus with antagonistic yeast and bacteria. Sci. Hortic. 40: 105-1 12.

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