m.
GRAM-POSITIVE BACTERIA
C.
Bacillus
W. Chun and A. K. Vidaver
1.
INTRODUCTION
Plant-associated bacilli are recognized either as plant pathogens, saprophytes, or as
biological control agents. There are only three known phytopathogenic bacilli. Bacillus
megaterium pv. cerealis incites white blotch of wheat (12) and B. circukms causes a disease in
tissue cultures of date palm seedlings and a discoloration in heart tissue of mature plants (14,15,
32). Bacillus polymyxa has been reported as the causal agent of tomato seedling blight (5).
Other members of this endospore-forming group of bacteria have been found associated
with plant tissues or as endophytes. Bacillus megaterium and B. cereus are found in ovules and
seeds (18), in healthy potato tubers (11,17), and as strong colonizers of soybean roots (16).
Bacillus pumilus has been observed in the vascular stele of pea and tomato roots (1,2).
Inoculated B. subtilis can also colonize the xylem of maple (9,10).
Certain species of Bacillus can impart beneficial effects on plants (4, 19, 30) as well as
serve as insect vector control agents (7,20). Numerous Bacillus species have been used as
antifungal biological control agents. Bacillus subtilis strains suppress rhizoctonia seedling disease
of peas (3) and summer patch symptoms in Kentucky bluegrass (28). It has also been used
experimentally as a post-harvest treatment for peach brown rot (21-23). Bacillus cereus UW85
treatment significantly increased yields of soybeans that were tolerant or resistant to Phytophthora
sojae (19) and suppressed oomycetous seedling diseases of cucumber (26) and tomato (26).
Agents can be applied as seed treatments (3,19, 29), or in furrows (19). Some Bacillus species
have a broad range of biological control activity and may be useful under reduced tillage (13).
Bacillus spp. are rod-shaped, catalase-positive, non-acid-fast, endospore-forming, aerobic
or facultative anaerobic bacteria and normally stain Gram positive. Variable characters include,
motility, oxidase reaction, and the method by which carbohydrate is utilized. The bacilli can be
divided into the following three groups based on endospore morphology (32).
Group I:
Group II:
Group III:
Spores oval or cylindrical; central; subterminal or terminal. Bacillary body
swollen slightly or not swollen at all.
Spores oval, rarely cylindrical; subterminal or terminal. Bacillary body
noticeably swollen.
Spores spherical; subterminal or terminal. Bacillary body swollen.
The Gram reaction for bacilli may vary with culture age, medium, and strain. It is very
important that cells in the early log stage of growth be used for the Gram stain. For example, of
the 163 strains of group II Bacillus, 46% are Gram-variable after 6 hours of incubation and 20%
250
are Gram-negative after 48 h (6). Bacillus circulans, isolated from date palms, is Gram-negative
with typical Gram-negative cell membranes (15). One cannot therefore depend entirely on the
Gram stain for identification of Bacillus spp. but should include the observation of endospore
production and if possible, thin section electron microscopy for determination of cell wall
structure.
2.
ISOLATION TECHNIQUES USING DIFFERENTIAL MEDIA
a.
Plant Material
Plant material should be washed thoroughly with tap water and air-dried. When
lesions or discoloration in the plant tissues are evident, small pieces (about 3 mm x 3 mm x
6 mm) can be excised using sterile scalpels and forceps and ground with a glass rod in a
small volume of water. When no visible discoloration is observed, several pieces of plant
tissue can be ground in sterile water. A loopful of the ground suspension can then be
streaked on the appropriate medium. Since a large population of Bacillus may be needed
in order to induce symptoms, a nutrient glucose medium should be used to speed growth
of such slow growing species as B. circulans.
b.
Seeds
Seeds can be surface sterilized for 5-10 min in a 1:10 dilution of a commercial
bleach such as Clorox® (5.25% active sodium hypochlorite). Adequate washing with
distilled water should be performed to remove all traces of the sodium hypochlorite. Seeds
can then be placed on the agar medium of Mundt and Hinkle (18). Seeds with a thick coat
should be punctured with a dissecting needle before being placed on the medium. The
plates or slants are incubated at 24 °C and observed daily for broken seed coats and growth
of bacteria. An alternative protocol is to grind a sample of 10 g of seeds. Mix the ground
seed in 95 ml of Vz strength nutrient-glucose broth. Stir for 3 hours on a magnetic stirrer
and place 1.0, 0.5, and 0.1 g samples of the sediment into separate tubes of Mundt and
Hinkle semi-solid slants. Incubate at 24 °C and streak on agar plates to obtain single
colonies.
2)
c.
Recipes for differential media
1)
Mundt and Hinkle medium (18)
To 1000 ml Difco nutrient broth, add:
Yeast extract
Dextrose
Cycloheximide
Agar (slants)
Agar (plates)
Casein dextrose (CD) agar
251
perL
3.0 g
5.0 g
2.0 g
5.0 g
17.0 g
perL
10.0 g
5.0 g
10.0 ml
4.0 g
17.0 g
Casein acid hydrolysate
Yeast extract
Dextrose (50% sterile stock solution)
K2HP04
Agar
The 50% stock solution of dextrose is sterilized separately and added
aseptically to the basal medium.
3.
DIFFERENTIATION OF COMMONLY ISOLATED SPECIES
Species of Bacillus can be differentiated on the basis of physiological tests (Table 1).
Table 1. Characteristics used in the identification of Bacillus species.1
Plant Pathogens
Other Species
licheniform
subtilis
coagulans
alvei
brevis
laterosporu
macerans
I
+
+
-
+
-
+
+
+
+
+
+
+
+
I
+
I
+
I
+
I
+
+
+
+
+
V
V
V
V
+
+
+
-
+
+
+
V
+
+
V
+
V
+
+
+
+
+
+
+
+
+
+
+
+
+
-
+
+
+
V
+
+
+
+
+
+
V
-
+
+
+
+
V
V
+
+
V
-
+
+
V
+
V
+
+
V
-
+
+
+
+
+
+
ND
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
V
V
V
V
+
+
+
V
V
V
-
+
+
-
+
+
+
0
megateriu
pv.
cerealis
cereus
■*■»
anthacis
circulans
Gram reaction
Motility2
Spore position
Terminal
Central
Subterminal
Swelling of bacillary body
Growth at 45 °C
Growth at pH 5.7
Growth in 7% NaCl
Utilization of citrate
Anaerobic growth in glucose broth
Acid from:
Arabinose
Mannitol
Xylose
Voges-Proskaur test
Starch hydrolysis
»3
<<
s
+, 80% or more strains positive; +11,80% or more strains delayed positive; V, between 21-70% of strains positive; -, 80% or
more strains negative; ND, not determined.
1
Adapted from Cowan (6) and Hosford for Bacillus megaterium pv. cerealis (12) and Leary and Chun for B. circulans (15)
2
All species may produce non-motile cells.
252
4.
DIAGNOSTIC MEDIA AND TESTS
a.
Gram Reaction
a.
Gram stain (see b, p. 7) (Plate 1, Fig. 1)
b.
KOH Test (see a, p. 7)
b.
Motility
a.
Method 1: Examine young broth cultures under phase contrast light
microscopy for motility.
b.
Method 2: Stab inoculate motility medium (8) tubes to a depth of 5 mm.
Motile organisms migrate through the medium making it turbid.
Motility medium of Edwards and Bruner (8)
perL
80.0 g
10.0 g
3.0 g
5.0 g
4.0 g
Gelatin
Peptone
Beef extract
NaCl
Agar
Distribute into tubes (10 ml) and autoclave at 121 °C for 15 min.
c.
Spore shape, position and swelling of bacillary body
1)
2)
Light Microscopy. Resuspend several bacterial colonies from a CD (see 2),
p. 252) plate in a drop of H20 and examine with a phase microscope.
Spores may also be observed by staining. Resuspend bacteria in a drop of
water on a slide and air dry. Flood with 5% malachite green and stain for
10 minutes. Wash under running water. Counter stain with 0.5% aqueous
safranin for 15 s. Rinse with water and blot dry. Bacterial bodies will stain
red and the spores will stain green.
Electron Microscopy (see b. p. 11). For electron microscopic examination
of thin sections of the bacterium, scrape cells of a 5-day-old culture from
the surface of a CD agar plate and suspend in 1 ml of physiological saline.
Pellet the cells in a bench-top centrifuge and fix the pellet with 25%
glutaraldyhyde in 0.1 M phosphate buffer (pH 7.2). Rinse the pellet several
times in the same buffer and then embed in 2% agar. Post-fix in 1%
osmium tetroxide in 0.1 M phosphate buffer, followed by several rinses in
the same buffer. Dehydrate in an acetone series, and embed in epoxy resin
(32). The block containing the fixed pellet should then be trimmed and
sectioned with a glass knife on an ultramicrotome. Collect thin sections
(600 - 800 //m) on 150 mesh formvar-coated copper grids and post-stain
for 15 minutes with 1% aqueous uranyl acetate and 5 min with lead citrate.
253
The post-stained ultrathin sections can then be viewed with a transmission
electron microscope.
Whole cells are prepared for viewing with the electron microscope by
placing a drop of the pellet on the surface of a 100 mesh thick-bar
formvar-coated and carbon-coated copper grid. Allow the cells to dry,
and then unidirectionally shadow cast with 13 mm of a 0.02 mils wire of
80% platinum and 20% palladium coiled around a 0.025 mils tungsten
filament wire oriented at a 1 - 3°s slope to the specimen in a high vacuum
evaporator. The grids can then be viewed under the same conditions as
those described for use with the thin sections. Endospores are visible as
dark bodies (Plate 6, Fig. 4).
d.
Growth at 45°C and 65°C.
Inoculate liquid CD medium and adjust pH to 5.7. Incubate at the desired
temperature. Turbid cultures within 5 days are recorded as positive.
e.
Growth in 7% NaCl (6).
Inoculate liquid CD or nutrient broth (see a, p. 3) plus 7% NaCl and observe daily
for growth. If growth does not occur in 7% NaCl, the maximum NaCl
concentration at which growth will occur can be determined by repeating the test
with a range of NaCl concentrations from 1% - 6%.
f.
Utilization of citrate (24).
Stab the butt and streak the surface of a slant of Simmon's citrate agar. Blue color
and growth indicate citrate utilization. Original green color indicates citrate was
not utilized.
Simmon's citrate agar (24)
perL
0.2 g
1.0 g
2.0 g
2.0 g
5.0 g
80.0
mg
15.0 g
MgSCy7H20
NH4H2P04
K2HP04
Sodium citrate
NaCl
Bromthymol blue
Agar
Adjust to pH 6.8 - 6.9 before autoclaving. Melt agar, dispense into tubes and
autoclave at 121°C for 15 minutes.
254
g-
Anaerobic growth in glucose broth (6).
Inoculate tubes of glucose broth and incubate in an anaerobic jar. Alternatively,
overlay the broth with sterile mineral oil and incubate at 24°C.
Glucose broth
perL
8.0 g
50.0 ml
950.0 ml
Nutrient broth (Difco)
20% dextrose stock1
H20
^utoclaved separately or filter sterilize through a 0.2/um filter. Add the
appropriate amount of dextrose stock to autoclaved nutrient broth tubes.
h.
Acid and gas production from carbohydrates: dextrose, arabinose,
mannitol, and xylose. Stab inoculate duplicate tubes of Hugh and Leifson's OF
medium (see 3, p. 9) containing the appropriate carbohydrate. Seal one tube with
sterile mineral oil to a depth of 1 cm. Incubateat 28 - 30°C and examine daily for
14 days. Note acid production (yellow), gas production (helpful in identifying
Bacillus spp), and motility (turbid medium).
Reaction Table:
Reaction
Open Tube
Sealed tube
Fermentation
Yellow
Yellow
No action
Blue or green
Green
Pockets of gas in the medium indicate gas production.
i.
Starch hydrolysis (6).
Inoculate starch agar plates and incubate plates for 5 days. Flood plates with
Lugol's iodine. Clear, colorless zones indicate starch hydrolysis. Note: some
Bacillus spp. produce restricted zones so colonies should be scraped away for
easier reading of results.
Lugol's iodine
g/lOOml
5.0 g
10.0 g
Iodine
Potassium iodide (KI)
Dissolve iodine and KI in 10 ml of H20. Adjust to volume with distilled water. For
use, dilute 1/5 with distilled water.
255
Starch agar
perL
8.0 g
10.0 g
1.0 L
Nutrient agar
Soluble potato starch
Distilled water
5.
PATHOGENICITY TESTS
The pathogenicity of Bacillus spp. to plants appears limited. Therefore, very few
specific methods have been developed and published. What is most important to consider
is that if & Bacillus species is consistently isolated from diseased plant tissues and that
particular Bacillus is the only organism consistently isolated, then it may be the pathogen.
Thereafter, inoculation procedures must be used to satisfy Koch's postulates.
For wheat, the inoculum can be applied by dipping or spraying leaves with a
bacterial suspension (12). Placing the plants in a vacuum chamber evacuated to IS mm Hg
three successive times will aid infiltration (31). Subsequent placement of the inoculated
plants in a mist chamber for 1 to 2 days may also be helpful.
The disease of date palms caused by B. circulans is a seedling disease. For such
diseases, the inoculum should be prepared as a suspension of ~104 - 106 bacteria/ml and
the seeds soaked in the suspension for 1 hour. Seeds should then be planted in
appropriate containers and observed for percentage germination, total wet weight, length
of seedlings, and subsequent appearance of symptoms (see p. 226 for details).
6.
COMMERCIAL AUTOMATED TECHNIQUES
The Biolog GP Microplate™ provides standardized and rapid performance of
many physiological tests. Particularly useful are the Biolog MT plates which allow design
of custom carbohydrate utilization tests. In most instances, members of this group are
readily identified by the Microlog n software (Biolog, Inc.).
For details see Appendix C.
7.
CULTURE PRESERVATION
Standard methods employed by the American Type culture collection are
applicable to this genus.
256
8.
LITERATURE CITED
1.
Benhamou, N., J. W. Kloepper, A. Quadt-Hallman, and S. Tuzun. 1996. Induction of
defense-related ultrastructural modifications in pea root tissues inoculated with endophytic
bacteria. Plant Physiol. 112:919-929.
2.
Benhamou, N., J. W. Kloepper, and S. Tuzun. 1998. Induction of resistance against
Fusarium wilt of tomato by combination of chitosan with an endophytic bacterial strain:
ultrastructure and cytochemistry of the host response. Planta 204:153-168.
3.
Bochow, H. and K. Gantcheva. 1995. Soil introductions of Bacillus subtilis as
biocontrol agent and its population and activity dynamic. Acta Hortic. 382:194-172.
4.
Broadbent, P., K. F. Baker, N. Franks, and J. Holland. 1977. Effect of Bacillus spp. on
increased growth of seedlings in steamed and in nontreated soil. Phytopathology
67:1027-1034.
5.
Caruso, F. L., M. G. Zuck, and A. E. Bessette. 1984. Bacterial seedling blight of tomato
caused by Bacilluspolymyxa. Plant Dis. 68:617-620.
6.
Cowan, S. T. 1974. Cowan and Steel's Manual for the Identification of Medical Bacteria.
Second ed., S. T. Cowan, ed. Cambridge University Press, Great Britain.
7.
Drobniewski, F. A. 1994. The safety of Bacillus species as insect vector control agents. J.
Appl. Bacteriol. 76:101-109.
8.
Edwards, P. R. and D. W. Bruner. 1942. Serological identification of Salmonella
cultures. Circ. Ky. Exp. Sta.
9.
Hall, T. J. 1986. Effects of xylem-colonizing Bacillus spp. on Verticillium wilt in
maples. Plant Dis. 70:521-524.
10.
Hall, T. J. and W. E. Davis. 1990. Survival of Bacillus subtilis in silver and sugar maple
seedlings over a two-year period. Plant Dis. 74:608-609.
11.
Hollis, J. P. 1951. Bacteria in healthy potato tissue. Phytopathology 41:350-366.
12.
Hosford, R. M. 1982. White blotch incited in wheat by Bacillus megaterium pv. cerealis.
Phytopathology 72:1453-1459.
13.
Kim, D. S., R. J. Cook, and D. M. Weller. 1997. Bacillus sp. L324-92 for biological
control of three root diseases of wheat grown with reduced tillage. Phytopathology
87:551-558.
257
14.
Leary, J. V. and W. Chun. 1985. Pathogenicity of Bacillus circulans to date palm
(Phoenix dactylifera) seedlings. Phytopathology 75:1289 (Abstr.).
15.
Leary, J. V. and W. W. C. Chun. 1989. Pathogenicity of Bacillus circulans to seedlings
of date palm {Phoenix dactylifera). Plant Dis. 73:3 53-3 54.
16.
Liu, Z. L. and J. B. Sinclair. 1992. Population dynamics of Bacillus megaterium strain
Bl 53-2-2 in the rhizosphere of soybean. Phytopathology 82:1297-1301.
17.
Lutman, B. F. and H. E. Wheeler. 1948. Bacillus megaterium DeBary from the interior
of healthy potato tissue. J. Wash. Acad. Sci. (D.C.) 38:336-340.
18.
Mundt, J. O. and N. F. Hinkle. 1976. Bacteria within ovules and seeds. Appl. Environ.
Microbiol. 32:694-698.
19.
Osburn, R. M., J. L. Milner, E. S. Oplinger, R. S. Smith, and J. Handelsman. 1995.
Effect of Bacillus cereus UW85 on the yield of soybean at two field sites in Wisconsin.
Plant Dis. 79:551-556.
20.
Priest, F. G., M. Aquino-de-Muro, and D. A. Kaji. 1994. Systematics of insect
pathogenic bacilli: uses in strain identification and isolation of novel pathogens.
FEMS-Symp. 75:275-295.
21.
Pulsey, P. L., M. W. Hotchkiss, and H. T. Dulmage. 1988. Pilot tests for commercial
production and application of Bacillus subtilis (B-3) for postharvest control of peach
brown rot. Plant Dis. 72:622-626.
22.
Pusey,P.,L, Lawrence, and C. L Wilson. 1984. Postharvest biological control of stone
fruit brown rot by Bacillus subtilis. Plant Dis. 68:753-756.
23.
Pusey, P. L., C. L. Wilson, and M. W. Hotchkiss. 1986. Compatibility of Bacillus
subtilis for postharvest control of peach brown rot with commercial fruit waxes,
dichloran, and cold-storage conditions. Plant Dis. 70:587-590.
24.
Simmons, J. S. 1926. A culture medium for differentiating organisms of typhoid-colon
aeorgenes groups and for isolation of certain fungi. J. Inf. Dis. 39:209-214.
25.
Smith, K. P., J. Handelsman, and R. M. Goodman. 1997. Modeling dose-response
relationships in biological control: partitioning host responses to the pathogen and
biocontrol agent. Phytopathology 87:720-729.
26.
Smith, K. P., M. J. Havey, and J. Handelsman. 1993. Suppression of cottony leak of
cucumber with Bacillus cereus strain UW85. Plant Dis. 77:347-342.
258
27.
Suslow, T. V., M. N. Schroth, and M. Isaka. 1982. Application of a rapid method for
Gram differentiation of plant pathogenic and saprophytic bacteria without staining.
Phytopathology 72:917-918.
28.
Thompson, D. C, B. B. Clarke, and D. Y. Kobayashi. 1996. Evaluation of bacterial
antagonists for reduction of summer patch symptoms in Kentucky bluegrass. Plant Dis.
80:856-862.
29.
Turner, J. T. and P. A. Backman. 1991. Factors relating to peanut yield increases after
seed treatment with Bacillus subtilis. Plant Dis. 75:347-353.
30.
Wei, G, J. W. Kloepper, and S. Tuzun. 1996. Induced systemic resistance to cucumber
diseases and increased plant growth by plant growth-promoting rhizobacteria under field
conditions. Phytopathology 86:221-224.
31.
Willis, J. W., E. Allingham, and J. V. Leary. 1982. Identification of a unique bacterium
isolated from date palm tissue culture. Phytopathology 72:990 (Abstr.).
32.
Wolf, J. and A. N. Barker. 1968. The genus Bacillus. Aids to the identification of its
species. Pages 93-109 in: B. M. Gibbs and D. A. Shapton, eds., Identification Methods
for Microbiologists, Part B. Academic Press, New York.
9.
CHEMICAL LIST
Unless stated otherwise, all chemicals in this list were obtained from Sigma Chemical
Co., P.O. Box 14508, St. Louis, MO 63178
Acetone
Agar, Bacto
Ammonium phosphate, dibasic
MLH2P04
Arabinose
Beef extract
Bromthymol blue
Casein acid hydrolysate
Cycloheximide
Gelatin
Glucose (dextrose)
Glutaraldehyde
Formvar
Iodine
Lead acetate
Magnesium sulfate
Fisher Scientific
Difco Laboratories
Difco
Difco
Fisher Scientific
Mallinckrodt
259
Malachite green
Mannitol
Nutrient broth
Osmium tetroxide
Peptone (Bacto)
Potassium hydroxide
Potassium iodide
Potassium phosphate, dibasic
Potato starch
Sodium chloride
Sodium citrate
Safranin
Yeast extract
Fisher Scientific
Difco
Ted Pella, Inc.
Difco
Mallinckrodt
Fisher Scientific
Difco
260