Beruflich Dokumente
Kultur Dokumente
Fifty-one Bacillus isolates were characterized by fatty acid methyl ester (FAME) analysis; universal primer polymerase
chain reaction (UP-PCR) fingerprinting; production of secondary metabolites and antagonistic activity against
Xanthomonas campestris pv. campestris (causal agent of black rot in cabbage) in vitro and in vivo. Based on FAME analysis
and /or PCR fingerprinting, the isolates were clustered into three different groups, named as Bacillus amyloliquefaciens,
B. subtilis and B. pumilus. Seed treatment with Bacillus spp. generally reduced germination of seeds and incidence of
black rot, but no relationship was found between the results of in vitro and in vivo experiments. The B. amyloliquefaciens
group contained isolates that were generally the most effective at reducing attack of black rot in vivo. The metabolic
profiles of these isolates suggested that they produced surfactin, iturin, bacillomycine and/or azalomycin F. Isolates
belonging to the B. subtilis group were mostly able to synthesize surfactin and arthrobactin. Surfactin, amphomycin,
arthrobactin and valinomycin were generally found in culture extracts of isolates belonging to the B. pumilus group. No
effect on growth of the pathogen was detected when the activity of filtered culture extracts and selected metabolites
produced by the three different Bacillus species was tested in vitro against X. c. pv. campestris. However, inhibition was
seen when bacterial liquid cultures were used. When the ability to colonize cabbage endophytically was examined for
seven selected isolates with different antagonistic potential against black rot, it was found that the ability was related to
the species and not to the antagonistic activity of the isolates.
Keywords: Bacillus spp., biological control, brassica black rot, endophytes, fatty acid methyl esters, secondary metabolites
3 min at 72°C. The PCR products were analysed by disinfection by plating 100 seeds per disinfected seed lot
running 2·5 – 4·0 µL of the amplified product on 1·7% on trypticase soy agar (TSA, Difco Laboratories) and
agarose gels at 300 V for 40 min. The gels were stained incubating at 25°C. If no microbial growth was detected
with ethidium bromide and photographed in UV light. on the plates, the seed samples were considered surface-
disinfected and used in further experiments.
Inoculum suspensions were prepared with 24-h-old
Production and analysis of secondary metabolites
bacterial cultures growing on TSA. Sterile saline water
produced by Bacillus isolates
(20 mL, 0·85% NaCl) was added to the bacterial culture,
Fermentation which was suspended with a glass rod. The bacterial
Bacterial cultures 24 h old, growing on trypticase soy agar suspension was homogenized using a vortex mixer, and
(TSA, Difco) at 27°C was suspended with 5 mL sterile the inoculum amount was adjusted with sterile saline to
saline solution (0·9%). Bacterial suspension (2 µL) was OD600 = 0·01 (≈107 CFU mL−1) for X. c. pv. campestris
transferred to 500 mL flasks containing 100 mL boullion and 1·00 (≈109 CFU mL−1) for the Bacillus isolates.
3 (6 g peptone; 4 g pepticase; 3 g yeast extract; 1·5 g meat Disinfected cabbage seeds were inoculated with X. c.
extract; 1000 mL deionized water). The flasks were incu- pv. campestris for 30 min and left to dry in the flow cabinet.
bated for 24 h at 30°C on a rotary shaker at 250 r.p.m. Next day, 0·3 g preinoculated seeds were soaked for 18 h
Flasks (500 mL) containing 100 mL cabbage broth (50 g under agitation (150 r.p.m.) in 10 mL of the inoculum
cabbage, 1000 mL deionized water pH 7·0) or 100 mL suspension made from the respective Bacillus isolates.
half-strength TSB (15 g TSB, 1000 mL) were inoculated Control plants consisted of seeds preinoculated with X. c.
with 3 mL 24-h-old bacterial cultures growing in boullion pv. campestris and untreated seeds, both dipped in 10 mL
3. After inoculation, flasks were incubated at 30°C for sterile saline solution and treated in the same way as those
3 days on a rotary shaker (250 r.p.m.). inoculated with Bacillus isolates. The seeds were then
sown in trays containing pine bark that were watered
Extraction of secondary metabolites twice a day and kept in a glasshouse without controlled
1-butanol (2 µL) was added to 2 mL of the cultured broth conditions. The experiment was designed as a completely
and shaken in 5 mL vials at 250 r.p.m. for 2 h at room randomized block experiment with three blocks and 15
temperature (23°C). Thereafter, the vials were centrifuged repetitions per treatment per block.
(Varifuge RF Inert, Heraeus Sepatech, Hanau, Germany) for Fifteen days after sowing, the effect of the different seed
10 min at 3000 g, −12°C and the supernatant (butanol treatments on germination and the biocontrol effect were
phase) concentrated until dry. The residue was taken up evaluated for the different treatments. The effect on
with 100 µL dimethylsulphoxide (DMSO) and trans- germination was assessed by comparing the number of
ferred to a plastic vial which was kept in the freezer seedlings obtained with the Bacillus-treated seeds, and the
(−20°C) until high performance liquid chromatography number of seedlings obtained with the untreated control
(HPLC) was performed. treatment (100% germination). The effect on black rot
incidence was measured by comparing the number of
HPLC analysis diseased seedlings that developed from seeds treated with
HPLC analysis was conducted with a Hewlett Packard Bacillus isolates with those from seeds treated only with
1090 m Series II with DAD detector. A Novogram column the pathogen (100% black rot incidence). At the time of
[4·0 mm internal diameter (ID), 60 mm long] was used assessment, cabbage seedlings showed two cotyledons
together with a precolumn (4·0 mm ID, 50 mm long) and zero to three true leaves.
containing reverse phase material (Grom, Herrenberg,
Germany). Samples were analysed with gradient elution
Effect of Bacillus isolates and selected metabolites on
from 100% solvent A (0·1% aqueous orthophosphoric
X. c. pv. campestris in vitro
acid) to 100% solvent B (acetonitrile, HPLC grade) in 6 min.
The wavelength of detection was 210 nm. Chromato- The activity of Bacillus isolates, filtered culture extracts
grams were analysed and identification of the metabolites and three selected metabolites [surfactin (Sigma Chemical,
was performed with the Hewlett Packard HPLC 3d St Louis, MO, USA); iturin (kindly provided by Dr
chemstation Software (DOS series). G Winkelmann, University of Tübingen, Germany); and
acivicin (Sigma)] against X. c. pv. campestris was measured
in vitro according to Thornberry (1950), with minor
In vivo screening of Bacillus isolates for antagonistic
modifications. Two different media were used in this
activity against X. c. pv. campestris
experiment. LB medium (yeast extract 5 g; glucose 10 g;
Seeds of cabbage, Brassica oleracea cv. Copenhagen tryptone, 10 g; NaCl, 5 g; agar, 15 g and distilled water,
Market, were surface-disinfected by immersion in 70% 1000 mL) was chosen because antibiotic activity against
ethanol for 1 min, transferred to sodium hypochlorite bacteria was previously detected using this medium
(1% available chlorine) for 3 min, and rinsed three times (Jenny et al., 1991). PDA [39 g potato dextrose agar
consecutively in sterile distilled water. Seeds were then left (Difco), 1000 mL distilled water] was chosen because
to dry in the flow cabinet in Petri dishes containing sterile X. c. pv. campestris grew well on this medium. PDA or LB
filter paper. Sterility control was conducted after seed medium (15 mL) was poured into 9 cm Petri dishes and
Table 1 Characterization of Bacillus amyloliquefaciens isolates (group 1) according to FAME analysis, UP-PCR banding profiles, secondary
metabolite detection in culture extracts, inhibition zone against Xanthomonas campestris pv. campestris in vitro, effect on seed germination (%),
and reduction of black rot incidence (%) in vivo
Reduction of
FAME suggested Secondary Inhibition Seed black rot
Isolate identification [similarity UP-PCR metabolite zone v. germination incidence
number index (%)–clustera] profileb detectionc Xccd (%)e (%)e
a
Cluster grouping according to FAME analysis: Euclidian distance = A, 7·60; B, 4·34.
b
UP-PCR profile 1, see Fig. 1(a).
c
Secondary metabolite identification matches: A, surfactin; B, iturin; C, bacillomycine; D, azalomycin F; F, acivicin; G, arthrobactin; H, rhodutorola
acid; I, valinomycin; J, stenothricin; L, enterochelin; M, nocardamin.
d
Inhibition zone (IZ): + (very weak), 0 –2 mm; ++ (weak), 2 < IZ ≤ 5 mm; +++ (strong), 5 < IZ ≤ 7 mm; ++++ (very strong), 7 < IZ ≤ 10 mm.
e
Means were statistically compared within the bacterial isolates belonging to group 1. Values followed by the same letters were not significantly
different from each other.
f
Seeds dipped in sterile saline water.
g
Preinoculated seeds with Xanthomonas campestris pv. campestris and dipped in sterile saline.
Isolates identified as B. pumilus by FAME analysis generally not related to the ability to reduce the incidence
(similarity indices >60%) were shown to belong to the of black rot in vivo. With the exception of isolate BF3, no
same cluster (cluster D) (Table 3). However, the isolates B. pumilus isolate reduced the germination rate compared
were shown to be very heterogeneous molecularly, with a with the untreated control. Four isolates (60, 64, 102 and
range of different UP-PCR profiles that were distinct from 70) were not able to reduce the incidence of black rot
the reference isolates W43 and W44 (Fig. 1c). Thus the compared with the untreated control (Table 3).
identification of B. pumilus isolates was based only on The general ability of all Bacillus isolates to produce
FAME results (first-named option). Figure 1(c) shows the secondary metabolites is shown in Table 4. All three
UP-PCR profiles obtained for B. pumilus isolates (seven species were shown to be efficient metabolic producers
different UP-PCR profiles). Isolates 52, 56, 62 and 64 when growing in half-strength trypticase soy broth (TSB)
had a similar UP-PCR banding profile (profile 4); while or in cabbage broth (CB). Generally, TSB stimulated
BF2 and BF3 had profile 5; and isolates 163, 163B and 11B greater production of metabolites compared with CB.
had profile 8. Isolates 102, 199, 60, 70 and B7D showed Surfactin appeared to be a very common metabolite
yet other UP-PCR profiles compared with the other produced by Bacillus spp., being detected in culture
isolates in this group (Fig. 1c). Considering their HPLC extracts of all three species when growing in TSB and CB.
profiles, most isolates still produced surfactin (A), amph- Iturin was detected in culture extracts of various isolates
omycin (E), arthrobactin (G) and valinomycin (Is) (Tables 3 of B. amyloliquefaciens and two isolates of B. subtilis. CB
and 4). The ability to inhibit growth of X. c. pv. campestris was shown to be a better substrate for iturin production
in vitro varied from very weak to a very strong effect, compared with TSB (data not shown). Just as iturin was
although most isolates showed a weak effect (Table 3). detected in several isolates of B. amyloliquefaciens, bacil-
As with the other two Bacillus species, this ability was lomycine was detected in culture extracts of nine isolates
Table 2 Characterization of Bacillus subtilis isolates (group 2) according to FAME analysis, UP-PCR banding profiles, secondary metabolite
detection in culture extracts, inhibition zone against Xanthomonas campestris pv. campestris in vitro, effect on seed germination (%), and reduction
of black rot incidence (%) in vivo
Reduction of
Secondary Inhibition Seed black rot
Isolate FAME suggested identification UP-PCR metabolite zone v. germination incidence
number [similarity index (%)–clustera] profileb detectionc Xccd (%)e (%)e
a
Cluster grouping according to FAME analysis: C, Euclidian distance = 6·69.
b
UP–PCR profiles 2 and 3, see Fig. 1(b).
c
Secondary metabolite identification matches, see Table 1.
d
Inhibition zone (IZ), see Table 1.
e
Means were statistically compared within the bacterial isolates belonging to group 1. Values followed by the same letters were not
significantly different from each other.
f
See Table 1.
g
See Table 1.
isolate 68), while 101 was found only in a few root samples in vivo. They suggested that Bacillus isolates might not
20 days after inoculation. produce the same quantity or quality of antibiotics in vivo
as in vitro, or that the population of B. subtilis in planta
might not be high enough or located near the pathogenic
Discussion cells (Schreiber et al., 1988). Two isolates belonging to
The use of FAME analysis and UP-PCR fingerprinting group 1 were also shown to inhibit seed germination, and
profiles was generally helpful in the identification of their activity against black rot in vivo was generally higher
Bacillus species, making these features useful for the compared with the isolates of the other Bacillus groups.
classification of the genus at species level. Isolates of group 2, identified as B. subtilis, showed the
Isolates of group 1, mainly identified by UP-PCR analysis highest indices of similarity according to FAME analysis,
as B. amyloliquefaciens, showed an ability to produce with all isolates belonging to the same cluster (Table 2).
secondary metabolites characteristic for this group. Furthermore, with the exception of isolate 16, all isolates
However, the ability to inhibit growth of X. c. pv. campestris showed similar UP-PCR profiles (profile group 2) and
in vitro was generally not relatd to the biocontrol effect ability to produce surfactin and arthrobactin in culture.
in vivo. Utkhede & Gaunce (1983) and Schreiber et al. As with B. amyloliquefaciens, the ability to inhibit growth
(1988) described similar findings. In vitro, antibiosis can of X. c. pv. campestris in vitro generally did not relate to
be influenced by the agar used and order of application the biocontrol effect found in vivo. Reduction of seed
of the bacteria (Bell et al., 1995). Schreiber et al. (1988) germination was lower compared with the other groups.
found that, despite high inhibition of the Dutch elm patho- Also, the ability to reduce black rot was generally low,
gen in vitro by metabolites produced by an endophytic with no significant differences between the isolates and
B. subtilis isolate, no biological control was obtained the untreated control.
Table 3 Characterization of Bacillus pumilus isolates (group 3) according to FAME analysis, UP-PCR banding profiles, secondary metabolite
detection in culture extracts, inhibition zone against Xanthomonas campestris pv. campestris in vitro, effect on seed germination (%), and reduction
of black rot incidence (%) in vivo
Reduction of
Secondary Inhibition Seed black rot
Isolate FAME suggested identification UP-PCR metabolite zone v. germination incidence
number [similarity index (%)–clustera] profileb detectionc Xccd (%)e (%)e
a
Cluster grouping according to FAME analysis: D, Euclidian distance = 9·39.
b
UP–PCR-profile 4 –11, see Fig. 1(c).
c
Secondary metabolite identification matches, see Table 1 except for E = amphomycin; K = colistin.
d
Inhibition zone (IZ), see Table 1.
e
Means were statistically compared within the bacterial isolates belonging to group 1. Values followed by the same letters were not
significantly different from each other.
f
See Table 1.
g
See Table 1.
a
Identified substances (>60% match factor: retention time and UV spectrum).
b
Detected in both half-strength trypticase soy broth and cabbage broth.
c
Detected only in half-strength trypticase soy broth.
d
Detected only in cabbage broth.
e
+, Ability to produce the corresponding metabolite.
f
Numbers of producing isolates : total numbers of tested isolates.
g
–, No ability to produce the corresponding metabolite.
Table 5 Effect in vitro of surfactin, iturin and acivicin on growth of antibiotic has generally been found in culture extracts
Xanthomonas campestris pv. campestris on PDA and LB medium of B. subtilis (Hiraoka et al., 1992; Asaka & Shoda,
1996). In contrast, azalomycin F, amphomycin, acivicin,
Secondary metabolite Inhibition zone (mm) Media valinomycin and stenothricin have not been described
Surfactina
0·0 PDA and LB for Bacillus species before, whereas they have been for
Acivicina 0·0 PDA and LB Streptomyces species. None of the iron chelators (arthro-
Iturinb 0·0 PDA and LB bactin, rhodutorola acid, enterochelin and nocardamin)
Control (vancomycin) (mg mL−1) detected in the present study has been previously reported
1·0 10·7 PDA in the literature on Bacillus spp. However, the identifica-
0·5 8·7 PDA tion of the secondary metabolites in this study should be
0·25 4·3 PDA interpreted with caution, as they are based only on HPLC
0·12 1·3 PDA match profiles suggested by a reference HPLC library.
1·0 9·8 LB Mass spectophotometry is still recommended to confirm
Culture extract (20 × concentrate) the identification of the metabolites detected.
BB 0·0 PDA and LB No antibiotic activity was found in vitro when testing
B7D 0·0 PDA and LB filtered culture extracts of three Bacillus isolates (repre-
B50A 0·0 PDA and LB sentative of the three species), or when surfactin, iturin
and acivicin were tested in different concentrations
a
Tested at four concentrations: 1·0, 0·5, 0·25 and 0·12 mg mL−1. against X. c. pv. campestris. In the literature, the activity
b
Tested at two concentrations: 1·0 and 0·5 mg mL−1.
of antibiotics produced by Bacillus spp. is more commonly
found against fungal than against bacterial pathogens. In
the work conducted by Ebata et al. (1969), three types of
The final group (group 3) consisted of isolates identified subsporins, which are peptide metabolites produced by
by FAME analysis as B. pumilus. Although the isolates B. subtilis, were active against many filamentous fungi
pertained to the same FAME cluster and showed some and yeasts, but no activity was found against most of the
similarity in their HPLC profiles, the group was shown to bacteria tested. Gatavalin, another metabolite produced
be very heterogeneous considering their UP-PCR profiles. by Bacillus, was active against Gram-positive bacteria,
There were eight different UP-PCR profiles for the 14 mycobacteria, yeasts and moulds, but no activity was
isolates tested of this group, and their profiles were shown found for Gram-negative bacteria (Nakajima et al., 1972).
to be different from those of the two reference isolates Swinburne et al. (1975) tested the activity of antibacterial
used. As with B. amyloliquefaciens, some isolates in group components found in culture extracts of B. subtilis, and
3 significantly reduced germination, and the ability to proved that they were rapidly deactivated following
inhibit black rot in vitro was generally not related to the maximum production. However, Loeffler et al. (1986)
ability to reduce incidence of the disease in vivo. reported that bacilysin, an antibiotic produced by B.
The production of secondary metabolites was a feature subtilis, was active against some isolates of B. subtilis at
associated with individual Bacillus isolates, but generally the rate of 1 mg mL−1, and against the Gram-negative
the detection of some metabolites was associated with Escherichia coli at the rate of 10 mg mL−1. The antagonis-
a group of Bacillus species. Bacillus amyloliquefaciens tic activity found for metabolites produced by Bacillus
isolates generally produced surfactin, iturin, bacillomycine spp. has also been associated with the synergistic effect
and /or azalomycin F, while B. subtilis isolates were caused by the combination of antibiotics (Asaka &
mostly able to synthesize surfactin and arthrobactin. Shoda, 1996). Growth inhibition of X. c. pv. campestris
Surfactin, amphomycin, arthrobactin and valinomycin were was seen in the study reported here when using bacterial
generally found in culture extracts of B. pumilus isolates. liquid cultures (containing living cells) of most of the
Surfactin was produced by all three Bacillus species. This Bacillus isolates tested. Perhaps the antibiotic effect is
transmitted only from bacterial cell to cell; or perhaps Bulat SA, Minorenko NV, 1990. Species identity of the
the Bacillus cells changed the agar composition at the phytopathogenic fungi Pyrenophora teres Dreschler and
inhibition zone, thereby affecting the growth of X. c. pv. P. graminea Ito and Kuribayashi. Mikologiya I Fitopatologia
campestris. 24, 435 –41. [In Russian].
Considering the endophytic ability of seven isolates Bulat SA, Lübeck M, Minorenko N, Jensen DF, Lübeck PS,
with distinct antagonistic potential against black rot, the 1998. UP-PCR analysis and ITS1 ribotyping of strains of
ability to colonize cabbage tissues internally appears to be Trichoderma and Gliocladium. Mycological Research 102,
933 –43.
related to the Bacillus species. No relationship was found
Chen C, Bauske EM, Musson G, Rodríguez-Kábana R,
between their endophytic ability and antagonistic poten-
Kloepper JW, 1995. Biological control of Fusarium wilt on
tial. Both isolates BF3 (highest antagonistic activity
cotton by use of endophytic bacteria. Biological Control
against black rot in vivo in group 3) and 60 (lowest antag-
5, 83–91.
onistic activity) were found in root, stem, cotyledon and Claus D, Berkeley RCW, 1986. Genus Bacillus Cohn 1872. In:
true leaf. Bacillus subtilis isolates differing in antagonistic Sneath PHA, ed. Bergey’s Manual of Systematic Bacteriology,
activity were reisolated with lower frequency compared Section 13, Vol.2 Baltimore, MD, USA: Williams & Wilkins
with the isolates of group 3, but at 20 days after sowing, Co, 1105 –39.
the isolates could be reisolated from all four parts of the Cook AA, Larson RH, Walker JC, 1952. Relation of the black
plant. Bacillus amyloliquefaciens isolates with (isolates 8 rot pathogen to cabbage seeds. Phytopathology 42, 316–20.
and 101) and without (isolate 68) antagonistic potential Dolej S, Bochow H, 1996. Studies of the mode of action of
against black rot showed the poorest ability to colonize Bacillus subtilis culture filtrates in the model pathosystem
cabbage internally. Isolates 8 and 68 were sometimes tomato seedling – Fusarium oxysporum f.sp. radicis-
reisolated from root and stem sections, while isolate 101 lycopersici. Mededelingen Faculteit
was found almost nowhere in any plant part. The results Landbouwwetenschappen Rijksuniversitet (Gent) 61,
presented here suggest that the mechanisms involved 483–9.
in biological control of black rot may be complex, and Ebata M, Miyazaki K, Takahashi Y, 1969. Studies on subsporin.
appear to be dependent on the individual isolate rather I. Isolation and characterization of subsporins A, B and C.
than the species. Journal of Antibiotics 22, 467–72.
Hiraoka H, Osaka O, Ano T, Shoda M, 1992. Characterization
of Bacillus subtilis RB14 coproducer of peptide antibiotics
Acknowledgements iturin A and surfactin. Journal of Applied Microbiology 38,
635 –40.
The present work was funded by the Danish Council
Jenny K, Käppeli O, Fiechter A, 1991. Biosurfactants from
of Development Research, project number 90842. We
Bacillus licheniformis: structural analysis and charcterization.
would like to thank Mr Lovemore Mukwicho for excel-
Applied Microbiology and Biotechnology 36, 5–13.
lent technical assistance during glasshouse experiments Kloepper JW, Rodríguez-Kábana R, Zehnder GW, Murphy JF,
and part of the laboratory work conducted in Zimbabwe. Sikora E, Fernández C, 1999. Plant root–bacterial
We thank Dr G Winkelmann from the University of interactions in biological control of soilborne diseases and
Tübingen in Germany for providing the antibiotic iturin potential extension to systemic and foliar diseases.
used in the in vitro tests. Australasian Plant Pathology 28, 21–6.
Krebs B, Junge H, Ockhardt A, Höding B, Heubner D, Erben U,
1993. Bacillus subtilis – an effective biocontrol agent.
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