- Email: [email protected]
Production of antibiotics by Bacillus subtilis and their effect on fungal colonists of apple leaf scars
[ 211 ] Trans. Br, mycol, Soc. 65 (2), 211-217 (1975) Printed in Great Britain
PRODUCTION OF ANTIBIOTICS BY BACILLUS SUBTILIS AND THEIR EFFECT ON FUNGAL COLONISTS OF APPLE LEAF SCARS By T. R. SWINBURNE Plant Pathology Research Division J.G.BARR Agricultural Bacteriology Research Division AVERIL E. BROWN Plant Pathology Research Division, Department of Agriculture for Northern Ireland and Faculty of Agriculture andFood Science, Qyeen's University, Belfast, U.K. AND
(With
2
Text-figures)
Apple rootstocks were sprayed after leaf fall with two antibiotic-yielding isolates of Bacillus subtilis. Both could be recovered from the leaf scar tissue throughout the dormant season and spring, but with time an increasing number of strains of B. subtilis were isolated, all of which possessed both antifungal and antibacterial activity similar to that of the original strains used. B. subtilis did not persist on the rootstock bark. The antifungal metabolites produced by B. subtilis were .shown to be relatively stable compounds in vitro but the antibacterial metabolites were rapidly inactivated. The antifungal metabolites were found to be as inhibitory to many of the fungi commonly isolated from apple leaf scar tissue as they were to Nectria galligena.
Bacillus subtilis, a bacterium isolated from leaf scars, was found to be highly antagonistic to Nectria galligena Bres. (stat. conid. Cylindrocarpon heteronema) in vitro. Artificial inoculation of leaf scars with B. subtilis immediately after leaf fall reduced the incidence of canker when shoots were exposed to infection with N. galligena at any time from 24 h after B. subtilis inoculation to the following April (Swinburne, 1973). The number of antagonistic bacteria remained relatively constant from autumn until the primary protective layer was shed the following summer. Similar investigations carried out by Carter & Price (1974) showed that the treatment of the pruned surface of apricot trees with suspensions of macroconidia of Fusarium lateritium provided significant protection against Eutypa armeniacae. F. lateritium inhibits E. armeniacae in vitro and the inhibitory substance is non-volatile, diffusable and produced in amounts proportional to the age of the culture. Most antibiotics used against fungal pathogens have been shown to exert their effect in the host by a direct
212
Transactions British Mycological Society
action on the pathogen, an action often paralleling that exhibited by the antibiotic in vitro (Dekker, 1964; Goodman, 1962). These experiments attempted to determine whether B. subtilis grew within leaf scars and so liberated antibiotic substances continuously. The stability of the antifungal antibiotics and their effect on some of the fungal inhabitants of apple leaf tissue were also investigated. MATERIALS AND METHODS
Two strains of Bacillus subtilis were used in the experiments: B. subtilis NCB 8872, an established producer of bacillomycin (Landy, Warren, Roseman & Colis, 1948) and an isolate Bacillus subtilis CMI (Kew) from Bramley's Seedling apple trees (Swinburne, 1973). The isolate of .N. galligena (F7B/67) used in the assays for antifungal antibiotic activity was that described by Brown & Swinburne (1973). Staphylococcus aureus Oxford 209P and Erwinia carotovora NCIB 8097 were used in the assay of antibacterial antibiotics. Both strains of B. subtilis were grown in a stirred, aerated fermentor unit (Biotec), containing 31 of Oxoid nutrient broth at 26 °C. The cultures were harvested after 30 h when sporulation had just begun. For each organism two cell suspensions were prepared in sterile water. One contained both viable spores and vegetative cells (approximately IO ll /ml) and the other, after treatment at 80 °C for IS m, contained viable spores alone (approximately 7 x 107 per ml). Shoots of the current season's growth of the apple rootstock MMI 11 grown in stool beds at Loughgall Horticulture Centre were stripped of their remaining leaves on 22 November 1972. The shoots were sprayed to the point of run-off with one of the four bacterial suspensions. Samples of leaf scars and internodes from five shoots in each of the treatment plots were removed at monthly intervals throughout the experimental period. The cortex exterior to the cambium (bark) was aseptically stripped from each section and the residual leaf scar tissue was excised and macerated in nutrient broth (Swinburne, 1973). The ends of sections (5 mm long) of internodal wood were sealed in Vaspar and shaken vigorously for 10 min in Lissapol (2000 ppm) in 10 ml Ringers, and dilutions of this washing medium were surface plated on nutrient agar. The isolated bacteria were identified using the procedures described by Smith, Gordon & Clark (1952). Antibiotic production by the bacterial isolates in colonies on nutrient agar was tested by overlaying with nutrient agar seeded with various test organisms. Inhibition of the test organism resulted in clear zones around the original colony. Production and assay of antibacterial and antifungal antibiotics
Fermentation studies of antibiotic production were investigated in a broth containing glucose (20'0 g), i.-glutamic acid (5'0 g), MgS04 (0'5 g), KCl (0'5 g), Fe 2S046H20 (0'0015 g), MnS04.H20 (0'0005 g) and CuS04SH20 (0'0016 g) to 1000 ml distilled water (Landy et al. 1948).
Bacillus subtilis. T. R. Swinburne et al.
21 3
Fermentation was carried out in 250 ml conical flasks containing 50 ml medium. The flasks were incubated on a rotary shaker at 220 rpm over a maximum period of 96 h at 30°C. 2 ml samples of culture broth were removed at intervals and sterilized by centrifugation through Hemming filter pads. Fungal conidia or bacterial seed organisms were added to 165 ml of nutrient agar and poured into sterile NUNC-Blo assay plates. Wells of 7 mm diameter were eu t in the plates and 50 ,ul of fermentation liquor placed in the wells. Standards of bacitracin (28 to 0'43 units/rnl) and griseofulvin (1000 to 15 ,ug/ml) were included on each plate. Diffusion of standards and samples was allowed at room temperature for 2 h before the plates were incu bated at the appropriate temperature and time period for the seed organisms. Results were recorded as the mean inhibition zone diameter in mm. Aliquots (25,ul) of fermentation liquor of the bacterial isolates were chromatographed on silica gel or cellulose (Chromogram, 20 x 20 cm, Eastman). Solvent systems used were chloroform: methanol: water (60: 35: 4) for antibacterial antibiotics and butanol: acetic acid: water (3: I : I) for antifungal metabolites. All traces of solvent were removed in a constant air flow before developed chromatograms were overlayed with seeded nutrient agar. Where N. galligena was used as the test organism I ml conidial suspension (approx 30 x 105/ml) was pipetted into 10 ml nutrient agar. Developed chromatograms were overlayed with the seeded nutrient agar and incubated for 48 h at 25°C. Bacterial seed cultures were grown in nutrient broth in shake culture for 16 h at 37°C (30 °C for B. subtilis). Culture broth (I ml) was added to nutrient agar (100 ml), and with S. aureus and E. carotooora triphenyltetrazolium chloride (0'004 %) was included. Chromatograms, overlayed with the seeded nutrient agar (10 ml) were incubated for 24 h at 37°C (30 °C for B. subtilis). RESULTS
The number of colonies of B. subtilis recovered from replicate leaf scars varied greatly and no significant difference could be detected between sampling dates or treatments (Table I). As in earlier experiments (Swinburne, 1973) the numbers of bacteria per leafscar tended to diminish with time, particularly after March. A number ofcolonies were recovered which differed in appearance from the original strains and these were proportionally more numerous at the last three sampling dates. Sixteen such variants were obtained from leaf scars sprayed with B. subtilis CMI (Kew) and seventeen from those sprayed with B. subtilis NCIB 8872. Whilst all thirty-three variants conformed to B. subtilis in the salient tests described by Smith et al. (1952) and Wolf & Barker (1968) they differed in their abilities to ferment glucose, grow anaerobically in glucose broth and reduce nitrate aerobically. Colonies which macroscopically resembled the original strains invariably gave the same results in all thirty eight tests applied. Both of the original strains and all of the valiants exhibited a similar quantitative spectrum of antimicrobial activity.
Transactions British Mycological Society Table I. Mean number inoculated with two strains alone Sample date 8. 18. 7· 3· 14· 21.
i. ii. iii. iv, v. vi.
73 73 73 73 73 73
B. subtilis NCIB 8872
B. subtilis CMI (Kew)
~
~
*1
2
0 2200 154 0 120 12 8
280 8 220 0 2 0
* 30
rif colonies rif B. subtilis recovered from leaf scars of the organism as spores with vegetative cells or spores
I,
Vegetative cells + spores;
I
2
160 36 220 0 0 4
2200
2,
Control (uninoculated)
o o o
206 0 1152 2
o
o o
spores alone.
----_10°
• - - - - - 1. .
01-----.....-'-----_ _..&.. 24 48 Time (h)
10- 3 72
....
Fig. 1. Sporulation ( - . - ) and extracellular accumulation of antibacterial (-e-) and antifungal ( -....- ) antibiotics of B. subtilis (NCIB 8872) during fermentation.
B. subtilis was recovered from the bark of internodal areas of the sprayed shoots in only one sample. However from all the samples a total of 2 I antifungal antibiotic producing types was recovered. Of these 18 were shown to be aerobic Gram-positive spore forming bacteria, but no attempt was made to define the isolates to species level. Three non-spore forming bacteria were encountered, one a Gram-negative bacillus and the others Gram-positive cocci.
Antibiotic production Antibiotic production by B. subtilis NCIB 8872 in liquid culture was associated with the onset of sporulation and was maximal at the end of the logarithmic phase of growth when sporulation had been initiated but was incomplete (Fig. I). Later in the fermentation process extracellular antibacterial activity was greatly diminished while little change occurred in the extracellular level of the antifungal antibiotic after maximum accumulation had been reached. A similar sequence of production was observed in fermentation of the isolates recovered from leaf scars. Two antifungal antibiotics were detected on the chromatograms of the fermentation liquors of both B. subtilis NCIB 8872 and all the leaf scar
Bacillus subtilis. T. R. Swinburne et al.
2 15 1·0
... Q)
d
~
• • • • •
11 I• .8 0
...
0
:a o
8872
Fig.
2.
• 0,5
t<. l:l:1
• •• • • • • • • eMI ,11 (Kew)
33
85
91
93
y
68 J
Bark isolate number
Antibacterial antibiotics produced in broth by bacterial bark isolates and the two B. subtilis spray organisms,
Table 2. Percentage of stock solution of antifungal metabolites from B. subtilis required to produce inhibition zones of 17'0 mm on nutrient agar plates seeded with fungi isolatedfrom apple leaf scars Fungus
% Stock solution
Cylindrocarpon heteromena Epicoccum purpurascens Phoma limitata Fusarium lateritium Phomopsis mali Aureobasidium pullalans Aspergillus glaucus Cladosporium herbarum Pyrenachaeta sp. Alternaria alternata Phoma sp, Penicillium expansum
22'4 10'0
15"8 15'8 21'2 23'4
8'g
r r-B 4'3
No inhibition 22'4 100'0
isolates, at R F 0·89 and R F 0'76. Considerable variation existed in the size of the zones of inhibition obtained from the different isolates but the component at R F 0·89 was always the major antifungal metabolite. Separation of the antibacterial antibiotics revealed eight components (Fig. 2) in the fermentation liquor of all isolates from leaf scars. All of these antibiotics were active against S. aureus but only one, at R F 0'19, was active against E. carotovora. The antibiotics produced in fermentation by those isolates from bark active against S. aureus and N. galligena were also examined. The antibacterial antibiotics produced by these organisms were different from those produced by B. subtilis (Fig. 2). The antifungal antibiotics also differed from those of B. subtilis, having no mobility in the chromatographic solvent system used.
216
Transactions British Mycological Society
Activity of antibiotic produced by B. subtilis againstfungi commonly isolatedfrom apple leaf scar tissue A dilution series was prepared from a sterile stock fermentation liquor in which B. subtilis NCIB 8872 had been grown, and was introduced into wells in nutrient agar plates seeded with fungal spores . After 48 h incubation at 22 °C, zones of inhibition were measured, and the percentage of stock solution required to produce inhibition zones of 17'0 mm are shown in Table 2. All the fungi tested were inhibited except for Alternaria alternata, and Penicillium expansum was relatively insensitive to the antibiotics. With most of the fungi the antibiotics inhibited spore germination. In the case of N. galligena, Epicoccum purpurascens, F. lateritium and Cladosporium herbarum, however, spore germination proceeded but the emerging hyphae were inhibited. DISCUSSION
B. subtilis has been shown to remain in the apple leafscar tissue throughout the winter and spring (Swin bu rne, 1973 ), and this investigation has shown that the number of different strains of B. subtilis recovered over this period increased. The morphological characteristics and antibioticproducing ability of each of these strains were similar to those of the two original spray inoculation organisms. Antibiotic production by B. subtilis was observed to occur at the end of logarithmic growth when sporulation had been initiated, and the appearance of these several differing forms would suggest that B. subtilis was, in fact, continuing to grow and sporulate within the leaf scar tissue. In this event, the production of antibiotics could be expected to occur in vivo. B. subtilis isolated from the leafscar tissue could be clearly distinguished from antifungal antibiotic-producing bacteria resident on the rootstock bark. These latter organisms were not classified as B. subtilis and both the antibacterial and antifungal antibiotics produced by them were quite different from those of the leaf scar isolates. The antifungal antibiotic (s) were, however, toxic to N. galligena. It would seem likely that the microflora on the bark surface may be continuously changing, while B. subtilis within the tracheids and tissue of the leaf scars persisted in the most susceptible site for N. galligena infection (Zeller, 1926). Two groups of antibiotics were produced by both B. subtilis NCIB 8872 and B. subtilis CMI (Kew), one group consisting of several antibacterial components, and the other consisting of only two antifungal components. One of these antifungal compounds is known to be bacillomycin (Landy et al. 1948 ). In in vitro conditions the antibacterial metabolites proved to be rapidly deactivated following maximum production. However, the antifungal metabolites, which are presumably responsible for control of N. galligena by B. subtilis (Swinbu rn e, 1973 ), proved to be relatively stable. Little or no deactivation occurred following maximum production in culture. The antibiotics produced by B. subtilis were found to be at least as toxic, in vitro, to many of the fungi commonly isolated from apple leaf scars as
21 7 Bacillus subtilis. T. R. Swinburne et al. they are to N. galligena. E. purpurascens, which was approximately twice as sensitive to the antiibotics as N. galligena, was recovered in much smaller numbers from leaf scars treated with B. subtilis than from untreated scars (Swinburne, 1973). The number of isolates of F. lateritium and C. herbarum recovered was also reduced after B. subtilis treatment. The sensitivity of these latter two fungi to the antibiotics, like E. pupurascens, was greater than that of N. galligena. The frequency of isolation of Phoma limitata and Aureobasidium pullulans, which were as sensitive to the antibiotics as N. galligena, was however unaffected by B. subtilis treatment. As B. subtilis affords significant control over N. galligena infection (Swinburne, 1973) it is possible that the lack of control of P. limitate and A. pullulans may be related to the manner in which the fungi are inhibited. Inhibition zones on plates seeded with P. limitata and many of the fungi tested contained ungerminated spores while the hyphae at the edge of these zones grew normally. With N. galligena, E. purpurascens, F. lateritium and C. herbarum, however, spore germination proceeded but the emerging hyphae swelled and burst. Where spore germination is inhibited fungi germinating on the leaf scar surface may be able to penetrate the scar tissue with little hindrance, while those fungi whose hyphal growth is inhibited could be prevented from further penetration. The authors wish to acknowledge the help given by Mr G. Downey, Agricultural Bacteriology Division. REFERENCES
BROWN, A. E. & SWINBURNE, T. R. (1973). Factors affecting the accumulation of benzoic acid in Bramley's Seedling apples infected with Nectria galligena. Physiological Plant Pathology 3, 9 1-99. CARTER, M. V. & PRICE, T. V. (1974). Biological control of Eutypa armeniacae. II. Studies of the interaction between E. armeniacae and Fusarium lateritium, and their relative sensitives to benzimidazole chemicals. Australian Journal if Agricultural Research as, 105- 119. DEKKER,]. (1964). Antibiotics in the control of plant diseases. Annual Review if Microbiology 18, 243-262. GOODMAN, R. N. (1962). Impact of antibiotics in plant disease control. Advances in Pest Control Research S, 1-46. LANDY, M., WARREN, G. R., ROSENMAN, S. B. & COLIS, L. G. (1948). Bacillomycin: An antibiotic from Bacillus subtilis active against pathogenic fungi. Proceedings of Society of Experimental Biology, N.r. 67, 539-541. SMITH, N. R., GORDON, R. E. & CLARK, F. E. (1952). Aerobic spore-forming bacteria. U.S. Department of Agriculture, Agricultural Monograph No. 16. Washington, D.C. SWINBURNE, T. R. (1973). Microflora of apple leaf scars in relation to infection by Nectria galligena. Transactions of the British Mycological Society 60, 389-403. WOLF, J. & BARKER, A. N. (1968). The genus Bacillus aids to the identification of its species. In Identification Methodsfor Microbiologists, part B (ed. B. M. Gibbs and D. A. Shepton), pp. 93-109. London and New York: Academic Press. ZELLER, S. M. (1926). European canker of pomaceous fruit trees. Bulletin if the Oregon Agricultural Experimental Station No. 222.
(Acceptedfor publication 8 January 1975)
PRODUCTION OF ANTIBIOTICS BY BACILLUS SUBTILIS AND THEIR EFFECT ON FUNGAL COLONISTS OF APPLE LEAF SCARS By T. R. SWINBURNE Plant Pathology Research Division J.G.BARR Agricultural Bacteriology Research Division AVERIL E. BROWN Plant Pathology Research Division, Department of Agriculture for Northern Ireland and Faculty of Agriculture andFood Science, Qyeen's University, Belfast, U.K. AND
(With
2
Text-figures)
Apple rootstocks were sprayed after leaf fall with two antibiotic-yielding isolates of Bacillus subtilis. Both could be recovered from the leaf scar tissue throughout the dormant season and spring, but with time an increasing number of strains of B. subtilis were isolated, all of which possessed both antifungal and antibacterial activity similar to that of the original strains used. B. subtilis did not persist on the rootstock bark. The antifungal metabolites produced by B. subtilis were .shown to be relatively stable compounds in vitro but the antibacterial metabolites were rapidly inactivated. The antifungal metabolites were found to be as inhibitory to many of the fungi commonly isolated from apple leaf scar tissue as they were to Nectria galligena.
Bacillus subtilis, a bacterium isolated from leaf scars, was found to be highly antagonistic to Nectria galligena Bres. (stat. conid. Cylindrocarpon heteronema) in vitro. Artificial inoculation of leaf scars with B. subtilis immediately after leaf fall reduced the incidence of canker when shoots were exposed to infection with N. galligena at any time from 24 h after B. subtilis inoculation to the following April (Swinburne, 1973). The number of antagonistic bacteria remained relatively constant from autumn until the primary protective layer was shed the following summer. Similar investigations carried out by Carter & Price (1974) showed that the treatment of the pruned surface of apricot trees with suspensions of macroconidia of Fusarium lateritium provided significant protection against Eutypa armeniacae. F. lateritium inhibits E. armeniacae in vitro and the inhibitory substance is non-volatile, diffusable and produced in amounts proportional to the age of the culture. Most antibiotics used against fungal pathogens have been shown to exert their effect in the host by a direct
212
Transactions British Mycological Society
action on the pathogen, an action often paralleling that exhibited by the antibiotic in vitro (Dekker, 1964; Goodman, 1962). These experiments attempted to determine whether B. subtilis grew within leaf scars and so liberated antibiotic substances continuously. The stability of the antifungal antibiotics and their effect on some of the fungal inhabitants of apple leaf tissue were also investigated. MATERIALS AND METHODS
Two strains of Bacillus subtilis were used in the experiments: B. subtilis NCB 8872, an established producer of bacillomycin (Landy, Warren, Roseman & Colis, 1948) and an isolate Bacillus subtilis CMI (Kew) from Bramley's Seedling apple trees (Swinburne, 1973). The isolate of .N. galligena (F7B/67) used in the assays for antifungal antibiotic activity was that described by Brown & Swinburne (1973). Staphylococcus aureus Oxford 209P and Erwinia carotovora NCIB 8097 were used in the assay of antibacterial antibiotics. Both strains of B. subtilis were grown in a stirred, aerated fermentor unit (Biotec), containing 31 of Oxoid nutrient broth at 26 °C. The cultures were harvested after 30 h when sporulation had just begun. For each organism two cell suspensions were prepared in sterile water. One contained both viable spores and vegetative cells (approximately IO ll /ml) and the other, after treatment at 80 °C for IS m, contained viable spores alone (approximately 7 x 107 per ml). Shoots of the current season's growth of the apple rootstock MMI 11 grown in stool beds at Loughgall Horticulture Centre were stripped of their remaining leaves on 22 November 1972. The shoots were sprayed to the point of run-off with one of the four bacterial suspensions. Samples of leaf scars and internodes from five shoots in each of the treatment plots were removed at monthly intervals throughout the experimental period. The cortex exterior to the cambium (bark) was aseptically stripped from each section and the residual leaf scar tissue was excised and macerated in nutrient broth (Swinburne, 1973). The ends of sections (5 mm long) of internodal wood were sealed in Vaspar and shaken vigorously for 10 min in Lissapol (2000 ppm) in 10 ml Ringers, and dilutions of this washing medium were surface plated on nutrient agar. The isolated bacteria were identified using the procedures described by Smith, Gordon & Clark (1952). Antibiotic production by the bacterial isolates in colonies on nutrient agar was tested by overlaying with nutrient agar seeded with various test organisms. Inhibition of the test organism resulted in clear zones around the original colony. Production and assay of antibacterial and antifungal antibiotics
Fermentation studies of antibiotic production were investigated in a broth containing glucose (20'0 g), i.-glutamic acid (5'0 g), MgS04 (0'5 g), KCl (0'5 g), Fe 2S046H20 (0'0015 g), MnS04.H20 (0'0005 g) and CuS04SH20 (0'0016 g) to 1000 ml distilled water (Landy et al. 1948).
Bacillus subtilis. T. R. Swinburne et al.
21 3
Fermentation was carried out in 250 ml conical flasks containing 50 ml medium. The flasks were incubated on a rotary shaker at 220 rpm over a maximum period of 96 h at 30°C. 2 ml samples of culture broth were removed at intervals and sterilized by centrifugation through Hemming filter pads. Fungal conidia or bacterial seed organisms were added to 165 ml of nutrient agar and poured into sterile NUNC-Blo assay plates. Wells of 7 mm diameter were eu t in the plates and 50 ,ul of fermentation liquor placed in the wells. Standards of bacitracin (28 to 0'43 units/rnl) and griseofulvin (1000 to 15 ,ug/ml) were included on each plate. Diffusion of standards and samples was allowed at room temperature for 2 h before the plates were incu bated at the appropriate temperature and time period for the seed organisms. Results were recorded as the mean inhibition zone diameter in mm. Aliquots (25,ul) of fermentation liquor of the bacterial isolates were chromatographed on silica gel or cellulose (Chromogram, 20 x 20 cm, Eastman). Solvent systems used were chloroform: methanol: water (60: 35: 4) for antibacterial antibiotics and butanol: acetic acid: water (3: I : I) for antifungal metabolites. All traces of solvent were removed in a constant air flow before developed chromatograms were overlayed with seeded nutrient agar. Where N. galligena was used as the test organism I ml conidial suspension (approx 30 x 105/ml) was pipetted into 10 ml nutrient agar. Developed chromatograms were overlayed with the seeded nutrient agar and incubated for 48 h at 25°C. Bacterial seed cultures were grown in nutrient broth in shake culture for 16 h at 37°C (30 °C for B. subtilis). Culture broth (I ml) was added to nutrient agar (100 ml), and with S. aureus and E. carotooora triphenyltetrazolium chloride (0'004 %) was included. Chromatograms, overlayed with the seeded nutrient agar (10 ml) were incubated for 24 h at 37°C (30 °C for B. subtilis). RESULTS
The number of colonies of B. subtilis recovered from replicate leaf scars varied greatly and no significant difference could be detected between sampling dates or treatments (Table I). As in earlier experiments (Swinburne, 1973) the numbers of bacteria per leafscar tended to diminish with time, particularly after March. A number ofcolonies were recovered which differed in appearance from the original strains and these were proportionally more numerous at the last three sampling dates. Sixteen such variants were obtained from leaf scars sprayed with B. subtilis CMI (Kew) and seventeen from those sprayed with B. subtilis NCIB 8872. Whilst all thirty-three variants conformed to B. subtilis in the salient tests described by Smith et al. (1952) and Wolf & Barker (1968) they differed in their abilities to ferment glucose, grow anaerobically in glucose broth and reduce nitrate aerobically. Colonies which macroscopically resembled the original strains invariably gave the same results in all thirty eight tests applied. Both of the original strains and all of the valiants exhibited a similar quantitative spectrum of antimicrobial activity.
Transactions British Mycological Society Table I. Mean number inoculated with two strains alone Sample date 8. 18. 7· 3· 14· 21.
i. ii. iii. iv, v. vi.
73 73 73 73 73 73
B. subtilis NCIB 8872
B. subtilis CMI (Kew)
~
~
*1
2
0 2200 154 0 120 12 8
280 8 220 0 2 0
* 30
rif colonies rif B. subtilis recovered from leaf scars of the organism as spores with vegetative cells or spores
I,
Vegetative cells + spores;
I
2
160 36 220 0 0 4
2200
2,
Control (uninoculated)
o o o
206 0 1152 2
o
o o
spores alone.
----_10°
• - - - - - 1. .
01-----.....-'-----_ _..&.. 24 48 Time (h)
10- 3 72
....
Fig. 1. Sporulation ( - . - ) and extracellular accumulation of antibacterial (-e-) and antifungal ( -....- ) antibiotics of B. subtilis (NCIB 8872) during fermentation.
B. subtilis was recovered from the bark of internodal areas of the sprayed shoots in only one sample. However from all the samples a total of 2 I antifungal antibiotic producing types was recovered. Of these 18 were shown to be aerobic Gram-positive spore forming bacteria, but no attempt was made to define the isolates to species level. Three non-spore forming bacteria were encountered, one a Gram-negative bacillus and the others Gram-positive cocci.
Antibiotic production Antibiotic production by B. subtilis NCIB 8872 in liquid culture was associated with the onset of sporulation and was maximal at the end of the logarithmic phase of growth when sporulation had been initiated but was incomplete (Fig. I). Later in the fermentation process extracellular antibacterial activity was greatly diminished while little change occurred in the extracellular level of the antifungal antibiotic after maximum accumulation had been reached. A similar sequence of production was observed in fermentation of the isolates recovered from leaf scars. Two antifungal antibiotics were detected on the chromatograms of the fermentation liquors of both B. subtilis NCIB 8872 and all the leaf scar
Bacillus subtilis. T. R. Swinburne et al.
2 15 1·0
... Q)
d
~
• • • • •
11 I• .8 0
...
0
:a o
8872
Fig.
2.
• 0,5
t<. l:l:1
• •• • • • • • • eMI ,11 (Kew)
33
85
91
93
y
68 J
Bark isolate number
Antibacterial antibiotics produced in broth by bacterial bark isolates and the two B. subtilis spray organisms,
Table 2. Percentage of stock solution of antifungal metabolites from B. subtilis required to produce inhibition zones of 17'0 mm on nutrient agar plates seeded with fungi isolatedfrom apple leaf scars Fungus
% Stock solution
Cylindrocarpon heteromena Epicoccum purpurascens Phoma limitata Fusarium lateritium Phomopsis mali Aureobasidium pullalans Aspergillus glaucus Cladosporium herbarum Pyrenachaeta sp. Alternaria alternata Phoma sp, Penicillium expansum
22'4 10'0
15"8 15'8 21'2 23'4
8'g
r r-B 4'3
No inhibition 22'4 100'0
isolates, at R F 0·89 and R F 0'76. Considerable variation existed in the size of the zones of inhibition obtained from the different isolates but the component at R F 0·89 was always the major antifungal metabolite. Separation of the antibacterial antibiotics revealed eight components (Fig. 2) in the fermentation liquor of all isolates from leaf scars. All of these antibiotics were active against S. aureus but only one, at R F 0'19, was active against E. carotovora. The antibiotics produced in fermentation by those isolates from bark active against S. aureus and N. galligena were also examined. The antibacterial antibiotics produced by these organisms were different from those produced by B. subtilis (Fig. 2). The antifungal antibiotics also differed from those of B. subtilis, having no mobility in the chromatographic solvent system used.
216
Transactions British Mycological Society
Activity of antibiotic produced by B. subtilis againstfungi commonly isolatedfrom apple leaf scar tissue A dilution series was prepared from a sterile stock fermentation liquor in which B. subtilis NCIB 8872 had been grown, and was introduced into wells in nutrient agar plates seeded with fungal spores . After 48 h incubation at 22 °C, zones of inhibition were measured, and the percentage of stock solution required to produce inhibition zones of 17'0 mm are shown in Table 2. All the fungi tested were inhibited except for Alternaria alternata, and Penicillium expansum was relatively insensitive to the antibiotics. With most of the fungi the antibiotics inhibited spore germination. In the case of N. galligena, Epicoccum purpurascens, F. lateritium and Cladosporium herbarum, however, spore germination proceeded but the emerging hyphae were inhibited. DISCUSSION
B. subtilis has been shown to remain in the apple leafscar tissue throughout the winter and spring (Swin bu rne, 1973 ), and this investigation has shown that the number of different strains of B. subtilis recovered over this period increased. The morphological characteristics and antibioticproducing ability of each of these strains were similar to those of the two original spray inoculation organisms. Antibiotic production by B. subtilis was observed to occur at the end of logarithmic growth when sporulation had been initiated, and the appearance of these several differing forms would suggest that B. subtilis was, in fact, continuing to grow and sporulate within the leaf scar tissue. In this event, the production of antibiotics could be expected to occur in vivo. B. subtilis isolated from the leafscar tissue could be clearly distinguished from antifungal antibiotic-producing bacteria resident on the rootstock bark. These latter organisms were not classified as B. subtilis and both the antibacterial and antifungal antibiotics produced by them were quite different from those of the leaf scar isolates. The antifungal antibiotic (s) were, however, toxic to N. galligena. It would seem likely that the microflora on the bark surface may be continuously changing, while B. subtilis within the tracheids and tissue of the leaf scars persisted in the most susceptible site for N. galligena infection (Zeller, 1926). Two groups of antibiotics were produced by both B. subtilis NCIB 8872 and B. subtilis CMI (Kew), one group consisting of several antibacterial components, and the other consisting of only two antifungal components. One of these antifungal compounds is known to be bacillomycin (Landy et al. 1948 ). In in vitro conditions the antibacterial metabolites proved to be rapidly deactivated following maximum production. However, the antifungal metabolites, which are presumably responsible for control of N. galligena by B. subtilis (Swinbu rn e, 1973 ), proved to be relatively stable. Little or no deactivation occurred following maximum production in culture. The antibiotics produced by B. subtilis were found to be at least as toxic, in vitro, to many of the fungi commonly isolated from apple leaf scars as
21 7 Bacillus subtilis. T. R. Swinburne et al. they are to N. galligena. E. purpurascens, which was approximately twice as sensitive to the antiibotics as N. galligena, was recovered in much smaller numbers from leaf scars treated with B. subtilis than from untreated scars (Swinburne, 1973). The number of isolates of F. lateritium and C. herbarum recovered was also reduced after B. subtilis treatment. The sensitivity of these latter two fungi to the antibiotics, like E. pupurascens, was greater than that of N. galligena. The frequency of isolation of Phoma limitata and Aureobasidium pullulans, which were as sensitive to the antibiotics as N. galligena, was however unaffected by B. subtilis treatment. As B. subtilis affords significant control over N. galligena infection (Swinburne, 1973) it is possible that the lack of control of P. limitate and A. pullulans may be related to the manner in which the fungi are inhibited. Inhibition zones on plates seeded with P. limitata and many of the fungi tested contained ungerminated spores while the hyphae at the edge of these zones grew normally. With N. galligena, E. purpurascens, F. lateritium and C. herbarum, however, spore germination proceeded but the emerging hyphae swelled and burst. Where spore germination is inhibited fungi germinating on the leaf scar surface may be able to penetrate the scar tissue with little hindrance, while those fungi whose hyphal growth is inhibited could be prevented from further penetration. The authors wish to acknowledge the help given by Mr G. Downey, Agricultural Bacteriology Division. REFERENCES
BROWN, A. E. & SWINBURNE, T. R. (1973). Factors affecting the accumulation of benzoic acid in Bramley's Seedling apples infected with Nectria galligena. Physiological Plant Pathology 3, 9 1-99. CARTER, M. V. & PRICE, T. V. (1974). Biological control of Eutypa armeniacae. II. Studies of the interaction between E. armeniacae and Fusarium lateritium, and their relative sensitives to benzimidazole chemicals. Australian Journal if Agricultural Research as, 105- 119. DEKKER,]. (1964). Antibiotics in the control of plant diseases. Annual Review if Microbiology 18, 243-262. GOODMAN, R. N. (1962). Impact of antibiotics in plant disease control. Advances in Pest Control Research S, 1-46. LANDY, M., WARREN, G. R., ROSENMAN, S. B. & COLIS, L. G. (1948). Bacillomycin: An antibiotic from Bacillus subtilis active against pathogenic fungi. Proceedings of Society of Experimental Biology, N.r. 67, 539-541. SMITH, N. R., GORDON, R. E. & CLARK, F. E. (1952). Aerobic spore-forming bacteria. U.S. Department of Agriculture, Agricultural Monograph No. 16. Washington, D.C. SWINBURNE, T. R. (1973). Microflora of apple leaf scars in relation to infection by Nectria galligena. Transactions of the British Mycological Society 60, 389-403. WOLF, J. & BARKER, A. N. (1968). The genus Bacillus aids to the identification of its species. In Identification Methodsfor Microbiologists, part B (ed. B. M. Gibbs and D. A. Shepton), pp. 93-109. London and New York: Academic Press. ZELLER, S. M. (1926). European canker of pomaceous fruit trees. Bulletin if the Oregon Agricultural Experimental Station No. 222.
(Acceptedfor publication 8 January 1975)