The production of antifungal volatiles by Bacillus subtilis

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Journal of Applied Bacteriology 1993, 74, 119-126 The production of antifungal volatiles by Bacillus subtilis P.J. Fiddaman and S.

1 Journal of Applied Bacteriology 1993, 74, The production of antifungal volatiles by Bacillus subtilis P.J. Fiddaman and S. Rossall University of Nottingham, Faculty of Agricultural and Food Sciences, Department of Physiology and Environmental Science, School of Agriculture, Sutton Bonington, Loughborough, Leics, UK 4208/07/92: accepted 3 August 1992 P.J. FIDDAMAN AND s. ROSSALL A strain of Bacillus subtilis which produces an antibiotic metabolite was also found to produce a volatile compound(s) which was antifungal to Rhizoctonia solani and Pythium ultimum. Growth of the fungi was severely impaired in the presence of the volatiles and physiological abnormalities of the hyphae were observed, including hyphal distortion and vacuolation. A range of media were tested for volatile production and potato dextrose agar (PDA) was found to be the most active. Temperature had a considerable effect on antifungal volatile activity with the greatest inhibition occurring at 30 C. Addition of iron (111) chloride to Sabouraud s glucose agar (SGA) also enhanced the antifungal effect. The volatiles were found to be water soluble and remained active when trapped in SGA. INTRODUCTION There are a number of reports on the potential of Bacillus subtilis as a biological control agent against fungal pathogens (Cubeta et al. 1985; Seifert et al. 1987; Fravel 1988; Ferreira et al. 1991). All the work presented so far points to the principal mode of action of this antagonist as the production of antibiotics (Loeffler et al. 1986; McKeen et al. 1986). Although these antibiotics typically have a broad spectrum of activity against many genera of fungi (Utkhede and Scholberg 1986), little is known about other possible antagonistic modes of action which are available to B. subtilis. Howell et al. (1 988) reported that a strain of Enterobacter cloacae actively produced ammonia which was highly inhibitory to Pythium ultimum Trow. They found that ammonia production and biological control efficacy were very closely correlated. Voisard et al. (1989) reported that a hydrogen cyanide-producing strain of Pseudomonas fluorescens (strain CHAO) was effective in reducing black root rot of tobacco caused by Thielaviopsis basicola. Furthermore, a cyanidenegative mutant (HCN-), CHAS, was less effective in protecting tobacco plants than was the wild type (CHAO). As yet no such compounds have been associated with B. subtilis, but Wright and Thompson (1985) reported that Bacillus volatiles were highly antagonistic to cyanobacteria. Since then Wright (personal communication) has found that these volatiles are also antagonistic to algae, free-living protozoa, plants and some fungi. Correspondence to: Dr S. Rossall, University oj Notttngharn School of A~rtculture, Surron Bontngton, I.nughborough, Leics LEI2 SRD, UK. In this report the antifungal activity of volatiles from a particular strain of B. subtilis (NCIMB 12376) against the soil-borne plant pathogens Rhizoctonia solani and Pythium ultimum is described. MATERIALS AND METHODS Mlcro-organisms used Two isolates of R. solani and one P. ultimum were used in this study. Rhizoctonia solani AG4 was isolated from cotton seedling roots and supplied by Miss S.E. McKnight of Nottingham University. This isolate is henceforth referred to as cotton R. solani. An R. solani isolate, of an as yet unknown anastomosis group, was isolated from oilseed rape (cv Libravo) seedling roots at the beginning of this study and is referred to as OSR R. solani. Pythium ultimum K874A was supplied by Dr J. Lucas of Nottingham University. Throughout this study B. subtilis (NCIMB 12376) was used. This was supplied by Dr S. Rossall of Nottingham University. Detection and assay of antifungal activity of volatiles Sealed plate method. A Petri dish containing an appropriate agar medium was inoculated by spreading 200 p1 of a suspension of B. subtilis cells, prepared from a 72 h nutrient agar (NA) culture. After incubation at 37 C for 24 h, a second Petri dish (containing potato dextrose agar (PDA)), was inoculated with a 5 mm plug of the test fungus in the

120 P.J. FIDDAMAN AND S. ROSSALL centre of the plate, and inverted and placed over the bacterial culture. The two plates were sealed together with Nescofilm and further incubated (usually at 25 C).

2 120 P.J. FIDDAMAN AND S. ROSSALL centre of the plate, and inverted and placed over the bacterial culture. The two plates were sealed together with Nescofilm and further incubated (usually at 25 C). This ensured that both organisms were growing in the same atmosphere. Care was taken to ensure that the surface of the fringal culture did not become contaminated with the bacterium. Aerated plate method. A 5 mm wide central strip of agar was removed aseptically from plates of PDA to provide physical separation of organisms cultured on either half. Fifty p1 of B. subtilis cells were spread over one side of the agar medium, and the Petri dishes were incubated at 37 C for 24 h, before the opposite side of the plate was inoculated with a 5 mm plug of the test fungus. The Petri dishes were not sealed, permitting normal gaseous diffusion, and were incubated at 25 C. Fungal growth was measured as increases in mycelial diameter over a 5 d period for both techniques. The sealed plate method used the following media for bacterial growth: 1/10 tryptic soy broth agar (TSA) (Difco , 3 g 1- '; agar, 20 g I-'); nutrient agar (NA) (Oxoid CM 3, 28 g 1- I); diagnostic sensitivity test agar (DST) (Oxoid CM 261, 40 gl-i); Sabouraud's glucose agar (SGA) (glucose 20 g I-', mycological peptone (Oxoid L 40) 5 gl-', agar 15 gl-'); V8 juice agar (V8) (V8 juice (Campbell's Soups) 200 ml I-', agar 20 g I-') and potato dextrose agar (PDA) (Oxoid CM 139, 29 g I-'). Further studies were made after preliminary results had been done at 25 C to assess the effect of temperature on the antifungal activity of the volatiles. Thus the sealed plate procedure was repeated at 15"C, 20"C, 25 C and 30 C on PDA. The effect of iron on volatile activity Previous unpublished work on the production of a high mol. wt antibiotic by this B. subtilis strain suggested that the addition of FeCI, mol iron 111) to the media (SGA) led to an increase in antifungal activity of the bacterium. It was, therefore, decided to test for any iron effects on volatile activity by amending SGA and PDA with a similar concentration of FeCl, and carrying out bioassays. Studies on the solubility of Bacillus subtilis volatiles Experiments were done to determine whether the antifungal volatile(s) produced by B. subtilis dissolved in the agar media, and remained active even when the bacterial source was removed. After dual incubation for 5 d, the Petri dish containing B. subtilis was removed and the fungal culture was flushed with sterile air for 1 min. Samples were removed from the edge of the fungal colony and transferred to fresh PDA plates. Both sets of fungal cultures were rein- cubated at 25 C. There was little or no fungal growth on the original plates but on the reinoculated plates fungal growth occurred, suggesting that the fungus was viable but inhibited by dissolved volatiles on the former. In order to confirm this finding, blank plates of SGA were sealed and incubated with PDA plates inoculated with B. subtilis at 37 C for 6 d. Control uninoculated SGA plates were incubated together. After incubation, both the receiver (i.e. those blank plates incubated with B. subtilis inoculated plates) and control SGA plates were inoculated with a 5 mm core of cultures of R. solani and P. ultimum and growth measured daily over 5 d. After incubation, sub-samples of the colony edges were transferred to microscope slides and examined microscopically for changes in hyphal morphology. RESULTS Antlfungal activity of Bacillus subtilis volatiles The results shown in Fig. 1 indicate that the antifungal activity of B. subtilis volatiles was variable, depending on the type of media used, with PDA proving to be the most effective. Activity was detected with both the sealed plate and the aerated techniques (Fig. 2). A noteworthy point is that the two R. solani isolates used exhibited differing sensitivities to the antifungal volatiles. The oilseed rape isolate was by far the most sensitive. Temperature had a dramatic effect on volatile activity (Fig. 3). The level of control of fungal growth by the volatiles correlated well with temperature, with the highest temperature (30 C) producing the best reduction in growth of all three test fungi. The volatile metabolite(s) failed to cause complete inhibition of fungal growth at temperatures closer to optimum for the fungus (< 25 C). This is contrary to unpublished work on the production of a high mol. wt antibiotic by this strain of B. subtilis in which complete cessation of hyphal growth is frequently observed at such temperatures. The addition of iron in the form of iron 111 had an interesting effect on volatile activity. The amendment of PDA with iron caused no increase in antifungal activity, and even led to a decrease in control (Table 1). This is probably a reflection of the type of media used in which the addition of iron produced supra-optimal concentrations that were deleterious to B. subtilis. However, a marked increase in antifungal activity on SGA was observed with the addition of iron. Activity was increased from between 151'1/0 to 751% depending on the fungi tested. Again the effect is probably media-dependent, with SGA being low enough in mineral iron to be disadvantageous to the bacterium.

3 ANTIFUNGAL VOLATILES FROM B. SUBTILIS I I I I I J I 1 I I I I r (c ) I I I 1 I I I Time (h I I I I I 1 I I I , Fig. 1 Radial growth of Rhizoctonia soluni (OSR and cotton seedling isolates) on potato dextrose agar in the presence and absence of an adjacent Bacillus subtilis culture, grown on a range of media (sealed plate method). (a, b) Cotton Rhizoctonia soluni. (c, d) OSR Rhizoctonia so/unt. 0, SGA ; V, DST ; 0, V ; A, TSA ; NA PDA control; A, PDA ; 0,

4 122 P.J. FIDDAMAN AND S. ROSSALL Table 1 Percentage reduction in growth of cotton Rhizoctonia solani, OSR Rhizoctonia solani and Pythium ultimum with Bacillus subtilis The effect of iron I11 on antifungal volatile activity of Bacillus subtilis on potato dextrose agar and Sabouraud's glucose agar Cotton OSR Media* R. solani R. solani P. ultimum PDA PDAFe SGA SGAFe ~~~~ * Controls are unamended PDA and SGA. Treatments are PDA + mol iron 111 (PDAFe) and SGA + mol iron I11 (SGAFe). PDA, Potato dextrose agar; PDAFe, potato dextrose agar plus iron; SGA, Sabouraud dextrose agar; SGAFe, Sabouraud dextrose agar plus iron. prolific than the controls and some had become swollen, although no lysis was observed. However, very extensive vacuolation of the hyphae was the predominant feature of the fungi grown on the receiver plates. This vacuolation response was also observed in the earlier volatile bioassays. The degree of vacuolation varied among the fungal isolates used. The oilseed rape R. solani exhibited by far the greater amount of vacuolation, accompanied by hyphal tip swelling and malformation. The less sensitive cotton R. solani and the relatively insensitive P. ultimum showed hyphal abnormalities to a lesser degree. 1 I I I 1 I Time (h) Fig. 2 Radial growth of Rhizoctonia solani isolates on potato dextrose agar in the presence and absence of an adjacent Bacillus subtilis culture, grown on potato dextrose agar (aerated plate method). -A-, OSR R. solani control; --A--, OSR R. solani ; - -, cotton R. solani control; --0--, cotton R. solanr Solubility of inhibitory volatiles Sabouraud glucose agar plates which had been used to trap volatiles produced by B. subtilis exhibited pronounced antifungal activity when compared with controls (Fig. 4). The inhibition of R. solani was more pronounced than that observed for P. ultimum. This confirmed earlier results from dual incubation experiments. Microscopic examination of excised pieces of fungal colony perimeters taken from the receiver and control agars showed clear differences in hyphal morphology (Fig. 5). The hyphae taken from the receiver plates were much less DISCUSSION The results of this study closely parallel those of Wright and Thompson (1985) on Bacillus volatiles antagonizing cyanobacteria. The production of antifungal volatiles by B. subtilis reported here suggests that more than one mode of action of antifungal activity is available to this bacterium. In the past the main mechanism of antagonism of B. subtilis has been credited with antibiotic production (Cubeta et al. 1985; McKeen et af. 1986; Podile et al. 1987; Fravel 1988; Pusey et al. 1988), but this new study also suggests that volatiles from B. subtilis may also contribute to the antagonistic nature of the species. The preliminary experiments of this study showed that antifungal volatile activity varies greatly depending upon which media the bacterium is grown. Nutrient agar and 1/10 tryptic soy agar proved poor substrates for volatile production. Diagnostic sensitivity test and Sabouraud's glucose agar were intermediate in response and the highest volatile activity was detected on potato dextrose agar. This finding is somewhat different from that of Wright and

5 ANTIFUNGAL VOLATILES FROM B. subr/l/s OC I I I I I I 1 I I I I I I I I I c I-I-I-I 90r 300c Fig. 3 Effect of temperature on radial growth of Rhizoctonia solani isolates on potato dextrose agar in the presence and absence of an adjacent Bacillus subtilis culture. m, Cotton R. solani control; 0, cotton R. solani + B. subtilis; 0, OSR R. solani control; 0, OSR R. solani + B. subtilis

124 P.J. FIDDAMAN AND S. ROSSALL 1 I 1 I J 0 24 48 72 96 120 Time (h) Fig. --.

6 124 P.J. FIDDAMAN AND S. ROSSALL 1 I 1 I J Time (h) Fig , 4 Radial growth of Rhizoctonia solani and Pythium ultimum on Sabouraud's glucose agar with and without prior exposure to Bacillus subtilis voltiles. - A-, P. ultimum control; --A--, P. ultimum + volatile presence; - 0 -, OSR R. solani control; OSR R. solani + volatile presence Thompson (1985) who observed a great reduction in the growth of cyanobacteria when B. subtilis was grown on nutrient agar. This may reflect differences in the metabolism of the strains of B. subtilis used in the research programmes, or differences in sensitivity of cyanobacteria and fungi to components of the volatiles produced. The range of activity detected on different media may reflect enhanced growth and metabolic activity on the range of substrates used, or the provision of higher concentrations of specific compounds necessary for volatile production on certain media. The aerated method of volatile bioassay eliminated possible anaerobic interactions that may have occurred in the Fig. 5 Morphological effects of Bacillus volatiles on Rhizoctonia solani: (a) control hyphae; (b) induced vacuolation; (c) swelling of hyphal tip. Bar equals 100 pm sealed plate bioassay. The fact that slightly reduced activity was achieved in the aerated method was probably as a result of lower concentration of the volatiles over time. The sealed plate method would allow for a greater titre of volatiles as a result of much less gaseous escape into the external atmosphere and less dilution of the internal atmosphere. The results presented clearly show that the inhibitory

7 ANTIFUNGAL VOLATILES FROM B. SUBTlLlS 125 activity of the volatiles is progressive over time, and this is probably related to an accumulation of a toxin(s) leading to a gradual slowing of growth rather than a complete cessation of growth. Howell et al. (1988) reported that Enterobacter cloacae produced a volatile which inhibited P. ultimum. They used mass spectrometry and identified the volatile inhibitor as ammonia. Of greater interest is the fact that under environmental conditions of high sugar concentration the bacterium lost its biological control efficacy. This was related to failure of attachment of the bacterium to the fungal hyphae and suppressed disease control (Nelson 1986). Howell et al. (1988) discovered that the addition of certain sugars, D-glucose, D-galactose, sucrose and 3- methyl-d-glucoside also suppressed ammonia production by the bacterium. They found that under natural conditions the bacterium controlled P. ultimum only when it was applied to seeds which exude low sugar concentrations. Under such conditions with a lack of a readily metabolizable energy source the bacterium obtained carbon from deamination of seed amino acids and soil acid amides, the by-product of which is antifungal ammonia. In the research reported here it was found that the higher sugar containing media, SGA, PDA and DST produced the greatest in nitro antifungal activity whereas low sugar concentrations, i.e. in NA and TSA, produced low activity. Howell et al. (1988) also found that P. ultimum was more sensitive to the ammonia than an R. soluni isolate which they tested. In this report, however, the P. ultimum isolate used was far less sensitive to the Bacillus volatiles than were the two R. soluni isolates. These results, therefore, suggest that the B. subtilis volatile reported here is probably not ammonia. Preliminary results obtained from gas chromatography-mass spectrometry (GCMS) headspace analysis have confirmed this hypothesis. Antagonism of the B. subtilis volatile(s) to the fungi was positively correlated with temperature-higher temperatures leading to greater anti-fungal activity (Fig. 3). Again this is in complete agreement with the work of Wright and Thompson (1985). At temperatures lower than 25 C volatile antifungal activity was poor. At 30 C activity was greater but this is probably due to a combination of enhanced bacterial metabolic activity, greater diffusion/ solubility of the volatiles, along with supra-optimal temperature conditions for the fungi, resulting in suppressed pathogen growth. The latter is especially true for the OSR R. solani, which grew poorly at temperatures greater than 25 C. It is of interest that for all three fungi the level of control at 20 C was less than that at 15 C. This could well be related to the distinction between poor bacterial activity and relatively good fungal growth at 2O"C, whereas at 15 C both the bacteria and fungi are disadvantaged. Podile et a/. (1987) reported that the addition of iron to PDA broth resulted in an increase in the antagonistic principle of B. subtilis (strain AFl). In this report the addition of iron to PDA led to no increase in antifungal activity of volatiles. The addition of ferric iron to SGA, however, gave rise to a large increase in antifungal activity (Table 1). These findings are parallel to those obtained for the production of a high mo]. wt antibiotic by this strain of B. subtilis (unpublished). The implications of this are far reaching as far as biological control efficacy is concerned. Iron may be involved in enhanced general metabolic activity of the bacterium or be involved in specific enzymic pathways leading to antifungal activity. Solubility studies on the B. subtilis volatiles showed that they are water-soluble and remain active in agar without the presence of the bacterium (Fig. 4). This suggests that if the volatiles are produced in situ and dissolve in soil water, antifungal activity may still remain. A problem may arise where the volatiles are absorbed on soil colloids and therefore inactivated. This was also suggested as a potential problem associated with ammonia antifungal activity (Howell et al. 1988). Localized production of the antifungal volatiles in situ, i.e. on seed coats or on seedling roots, would however, avoid the necessity for volatile movement. The morphological effects of the volatiles on all three of the test fungi are broadly similar to those reported for the effect of non-volatile antibiotics (Backhouse and Stewart 1986; Ferreira et al. 1990). Although no lysis of hyphal tips was observed, swelling of hyphae and extensive vacuolation were common phenomena. These effects on the hyphae may be indicative of toxin(s) accumulation, with the physiological response of the fungus being compartmentalization and removal of the toxin from the growing cells. The fact that cyanobacteria cells lysed relatively easily (Wright and Thompson 1985) suggests that they are more sensitive to B. subtilis volatiles than are fungi. Overall this study has shown that B. subtilis volatiles are active against two groups of fungi-omycetes and basidiomycetes. Further work will extend the range of fungi tested. Analysis and evaluation of antifungal volatile production in soil will also be undertaken. Attempts are currently underway to determine the nature of the antifungal volatile(s) using gas chromatography-mass spectrometry headspace analysis. ACKNOWLEDGEMENTS We thank Dr J. Lucas and Ms S.E. McKnight for provision of cultures. This work was supported by a CASE Studentship to P.J. Fiddaman from SERC and Agricultural Genetics Co., 1,td.

126 P.J. FIDDAMAN AND S. ROSSALL REFERENCES Backhouse, D. and Stewart, A. (1986) Ultrastructure of antagonism of Sclerotiorum cepivorum by Bacillus subtilis. Journal of Phytopathology 124, 207-214.

8 126 P.J. FIDDAMAN AND S. ROSSALL REFERENCES Backhouse, D. and Stewart, A. (1986) Ultrastructure of antagonism of Sclerotiorum cepivorum by Bacillus subtilis. Journal of Phytopathology 124, Cubeta, M.A., Hartman, G.L. and Sinclair, J.B. (1985) Interaction between Bacillus subtilis and fungi associated with soybean seeds. Plant Disease 69, Ferreira, J.H.S., Matther, F.N. and Thomas, A.C. (1991) Biological control of Eutypalata on grapevine by an antagonistic strain of Bacillus subtilis. Phytopathology 81, Fravel, D.R. (1988) Role of antibiosis in the biocontrol of plant diseases. Annual Review of Phytopathology 26, Howell, C.R., Beier, R.C. and Stipanovic, R.D. (1988) Production of ammonia by Enterobacter cloacae and its possible role in the biological control of Pythium pre-emergence damping-off by the bacterium. Phytopathology 78, Loeffler, W., Tschen, J.S.M., Vanittanatkcom, N., Kugler, M., Knorpp, E., Hsieh, T.F. and Wu, T.G. (1986) Antifungal effects of bacilysin and fengymycin from Bacillus subtilis F A comparison with activities of other Bacillus antibiotics. Journal of Phytopathology 115, McKeen, C.D., Reilly, C.C. and Pusey, P.L. (1986) Production and partial characterization of antifungal substances antagonistic to Monolinia fructicola from Bacillus subtilis. Phytopathology 76, Nelson, E.B., Chao, W.L., Noston, J.M., Nash, G.T. and Harman, G.E. (1986) Attachment of Enterobacter cloacae to hyphae of Pythium ultimum; possible role in the biological control of Pythium pre-emergence damping-off. Phytopathology 76, Podile, A.R., Prasad, G.S. and Dube, H.C. (1987) Partial characterization of antagonistic principle of Bacillus subtilis AFI. Journal of Biological Control 1 : 1, 6&65 (abstract). Pusey, P.L., Hotchkiss, M.W., Dulmage, H.T., Baumgardner, R.A., Zehr, E.I., Reilly, C.C. and Wilson, C.L. (1988) Pilot tests for commercial production and application of Bacillus subtilis (B-3) for post-harvest control of Peach Brown Rot. Plant Disease 72, Seifert, K.A., Hamilton, W.E., Breuil, C. and Best, M. (1987) Evaluation of Bacillus subtilis C186 as a potential biological control of sapstain and mould on unseasoned lumber. Canadian Journal of Microbiology 33, Utkhede, R.S. and Scholberg, P.L. (1986) In nitro inhibition of plant pathogens by Bacillus subtilis and Enterobacter aerogenes and in vivo control of two post-harvest cherry diseases. Canadian Journal of Microbiology 32, Voisard, C., Keel, C., Haas, D. and Defago, G. (1989) Cyanide production by Pseudomonas juorescens helps suppress black root rot of tobacco under gnotobiotic conditions. EMBO Journal 8, Wright, S.J.L. and Thompson, R.J. (1985) Bacillus volatiles antagonize cyanobacteria. FEMS Microbiology Letters 30,

Microbial Interactions: Essential Part of Below-Ground Biocontrol Wietse de Boer

Microbial Interactions: Essential Part of Below-Ground Biocontrol Wietse de Boer NIOO-KNAW (Microbial Ecology) WUR (Soil Quality) Wageningen Email: w.deboer@nioo.knaw.nl Rhizosphere: Hotspot of Microbial