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Isolation of bacillus megaterium from Aphis pomi (Homoptera: Aphididae) and

assessment of its pathogenicity

Article in Journal of Plant Pathology · November 2008

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Journal of Plant Pathology (2008), 90 (3), 433-436 Edizioni ETS Pisa, 2008 433

ISOLATION OF BACILLUS MEGATERIUM FROM APHIS POMI

(HOMOPTERA: APHIDIDAE) AND ASSESSMENT
OF ITS PATHOGENICITY

H.M. Aksoy and S.K. Ozman-Sullivan

Ondokuz Mayis University, Faculty of Agriculture, Department of Plant Protection,

55139 Samsun, Turkey

SUMMARY forming bacteria. They are ubiquitous in natural envi-

ronments and are readily isolated from soil, leaf surfaces
Bacillus megaterium isolates were recovered from the and insect habitats (Smith and Couche, 1991; Lambert
green apple aphid, Aphis pomi De Geer, collected from and Peferoen, 1992; Chilcott and Wigley, 1993). Insect-
apple trees in the Samsun province of Turkey. The pathogenic Bacillus spp. that produce endogenous crys-
lethality of nine B. megaterium isolates was assayed on tals and toxins during sporulation are widely used to
A. pomi at 28-30°C. The isolates caused between 92% control many insect pests, including species of
and 100% mortality within one week, with no signifi- Coleoptera, Diptera and Lepidoptera (Pietrantonio et
cant differences amongst them. In contrast, there was a al., 1993; Zhang et al., 1995; Wagner et al., 1996).
significant difference between all isolates and the con- Bacillus megaterium Bary has a wide distribution, with
trol at P<0.05. The highest death rate (100%) was in- spores mainly present in the soil (Sneath, 1986). Bacterial
duced by the bacterial isolate No. 23 (Bmin23). After isolates from various habitats were found to be highly
bacterial application on A. pomi, there was a slowing toxic to the 4th instar larvae of Culiseta longiareolata
down of movement, cessation of feeding and develop- (Macquart) (Diptera, Culicidae) (Khyami-Horani et al.,
ment of a brown-black discoloration in diseased aphids 1999), and mutants were reported to produce a high level
within three days. Death followed within five days. of the mosquitocidal binary toxin of Bacillus sphaericus
Meyer and Neide (England et al., 1997). In addition, B.
Key words: Bacillus megaterium, Aphis pomi, lethal megaterium is also a potential biological control agent of
effects, biological control. Meloidogyne graminicola Golden & Burchfield (Nemato-
da, Heteroderidae) on rice (Padgham and Sikora, 2007).
In this study, isolates of B. megaterium were recov-
INTRODUCTION ered from the green apple aphid A. pomi, and their
lethality on the same aphid was determined.
Aphis pomi De Geer (green apple aphid) is usually
found at the tips of terminal shoots and water sprouts
on apple trees. Aphid feeding can lead to leaf curling, MATERIALS AND METHODS
stunting, distorted tip growth, and russetting on leaves
and immature fruit. Indirect injury results from honey- Plant material. One-year-old apple seedlings (Malus
dew deposits on both the fruit and leaves. A black fun- domestica Borkh) cv. Gala grown at 28-30°C and 70%
gus grows on this honeydew causing considerable dis- relative humidity were used throughout.
coloration, especially of early apples. This sooty mould
on leaves can also affect photosynthesis and may reduce Sample collection. Samples were collected from dead
fruit yield (Kaakeh et al., 1993; Lowery et al., 2006). A. pomi found on 10-15-year- old apple trees cv. Gala at
Aphids have many natural enemies, including para- the Ondokuz Mayis University research farm (Samsun
sites, predators and pathogens. Pathogens have been province, Turkey). Samples were stored in sterile plastic
used successfully for the control of aphids in greenhous- bags in a refrigerator at 4°C before processing.
es, but only with limited success in field situations
(Thomas and Poinar, 1973; Hall, 1981; Wilding, 1981; Isolation of Bacillus from aphid samples. To elimi-
Viggiani, 1984; Pfeiffer and Hogmire, 1995). Bacillus nate external contamination, dead aphids were steril-
spp. are gram-positive, rod-shaped, motile and spore- ized in 1% sodium hypochlorite solution for 3 min. The
samples were then washed three times in sterile distilled
water and transferred aseptically into a sterile mortar
Corresponding author: H.M. Aksoy
Fax: +90. 362 4576036 and macerated with a sterile pestle. The macerate was
E-mail: [email protected] placed in 10 ml of sterile distilled water. The suspension
434 B. megaterium from Aphis pomi Journal of Plant Pathology (2008), 90 (3), 433-436

was then heated at 75°C for 10-15 min, and diluted to way ANOVA procedure of the SPSS 11.0 statistical soft-
1×10-2 and 1×10-4. The dilutions were streaked on nutri- ware package (SPSS Inc., USA). Means were distin-
ent agar and the plates were incubated at 30°C for 48 h guished using Duncan’s multiple comparision test. Sig-
(Cavados et al., 2001). nificance was evaluated at P < 0.05 for all tests.

Identification of Bacillus isolates. Bacterial cells from

typical colonies were scraped from the agar surface and RESULTS
suspended in sterile water, after observation at 400x mag-
nification, with a light microscope. Biochemical, cytologi- Identification of Bacillus isolates. The nine Bacillus
cal and physiological characteristics of the isolates were isolates selected from 127 isolations from dead aphids
determined to confirm their identity as Bacillus spp. had similar colony and microscopic morphology, and
(Table 1) (Sneath, 1986; De Barjac and Frachon, 1990; large unswollen sporangia bearing subterminal ellip-
Forsyth and Logan, 2000). In addition, all isolates of soidal spores. All were Gram-positive, rod-shaped and
Bacillus spp. were later identified by using the computer- motile. On nutrient agar after 24-48 h at 30°C, the
assisted microbial identification system (MIS, MIDI Inc., young colonies were 1-2 mm in diameter, translucent-
USA) which employs gas-liquid chromatographic analysis whitish, irregular shaped, smooth, slightly umbonate
of bacterial fatty acids. and with undulate edges. All isolates were facultative
anaerobic and positive for catalase, gelatin liquefaction,
Pathogenicity. Potted apple seedlings were used for arginine dihydrolase, starch hydrolysis, egg-yolk reac-
pathogenity tests. Fifty green apple aphids were placed tions, and negative for KOH and phosphatase. Acid was
on each seedling and sprayed with a 108 -109 cfu/ml-1 produced from the following carbohydrates: acetylglu-
suspension in sterile distilled water of a B. megaterium cosamine, arbutin, D-fructose, D-glucose, maltose, ri-
isolate which had been grown on nutrient agar at 28- bose and D-trehalose. The profiles for each Bacillus sp.
30°C for 24-48 h. The control seedlings were sprayed isolate are shown in Table 1. In addition, all isolates
with sterile distilled water. A. pomi mortality was were identified as B. megaterium subgroup A using the
recorded after one week. Nine bacterial strains were re- computer-assisted microbial identification system (MIS)
isolated from dead aphids to verify that they were the which employs gas-liquid chromatographic analysis of
cause of death. Healthy aphids were inoculated with bacterial fatty acids.
pure cultures of the re-isolated pathogen, and shown to
come down with the same disease as originally ob- Effect of Bacillus isolates on Aphis pomi. The nine
served. The causal agent was again re-isolated from bacterial isolates were highly toxic to A. pomi, causing 92
dead aphids, grown in pure culture, and shown to be to 100% mortality within five days of treatment. The
identical to the organism characterized by MIS. highest death rate (100%) was given by isolate No. 23.
There were no significant differences for mortality
Statistical analysis. The experiment was carried out among the isolates. In contrast, there was a significant
in a completely randomized plot design with ten treat- difference between all of them and the control at P<0.05.
ments and five replications, and with fifty aphids in No mortality was observed in the controls (Fig. 1).
each replication. The data were analysed using the one- After bacterial application on A. pomi, the move-

Table 1. Identification tests for isolates of Bacillus megaterium subgroup A.

Production of acids from

Isolate carbohydrate
GS* KOH C Gl O/F S AD SH EY P
number
Ac Ar Fr G M R T
Bmin07 + - + + F + + + + - + + + + + + +
Bmin23 + - + + F + + + + - + + + + + + +
Bmin41 + - + + F + + + + - + + + + + + +
Bmin46 + - + + F + + + + - + + + + + + +
Bmin78 + - + + F + + + + - + + + + + + +
Bmin94 + - + + F + + + + - + + + + + + +
Bmin115 + - + + F + + + + - + + + + + + +
Bmin117 + - + + F + + + + - + + + + + + +
Bmin126 + - + + F + + + + - + + + + + + +

*GS: Gram stain; C: Catalase; Gl: Gelatin liquefaction; O/F: Utilization of glucose; F: Facultative; S: Sporulation; AD:
Arginine dehydrolase; SH: Starch hydrolysis; EY; Egg-yolk reactions P: Phosphatase; Ac: Acetylglucosamine; Ar:
Arbutin; Fr: D-fructose; G: Glucose; M: Maltose; R: Ribose; T: D-trehalose. +: Positive, -: Negative.
Journal of Plant Pathology (2008), 90 (3), 433-436 Aksoy and Ozman-Sullivan 435

Fig. 1. Efficacy of B. megaterium subgroup A isolates as Fig. 2. Black-coloured Aphis pomi individuals, killed by Bacil-
agents of Aphis pomi death. lus megaterium subgroup A (isolate 23), five days after inocu-
lation.

ments of diseased aphids became slow, feeding ceased, non, Santo, & Finley (Nematoda, Heteroderidae) and
brown-black discoloration appeared within three days Pratylenchus penetrans Cobb (Tylenchida, Pratylenchi-
(Fig. 2), and death followed within five days at 28-30°C. dae), reducing J2 penetration of potatoes. B. megaterium
was also studied as a potential biological control agent
against Meloidogyne graminicola on rice (Padgham and
DISCUSSION Sikora, 2007).
This is the first study from Turkey focusing on the ef-
Bacillus spp. are the most important bacterial fects of B. megaterium subgroup A isolates on A. pomi.
pathogens of many insects because of their ability to pro- The results obtained are encouraing but further experi-
duce toxins during sporulation (Pietrantonio et al., 1993; ments are needed to assess the effectiveness of such iso-
Zhang et al., 1995; Wagner et al., 1996). In Bergey’s lates at reduced concentrations, in field experiments,
Manual of Systematic Bacteriology (1986), there are six and to identify B. megaterium subgroup A isolates with
genera of endospore-forming bacteria featured. Bacillus higher efficacy against A. pomi, to determine their po-
is distinguished from the other endospore-forming bac- tential as biological control agents.
teria being a strict or facultative aerobe, rod-shaped, and
(usually) catalase-positive. Other endospore-forming
genera are Sporolactobacillus, which is microaerophilic ACKNOWLEDGEMENTS
and catalase-negative; Clostridium and Desulfotomacu-
lum, which are anaerobic; Sporosarcina, which has coccal We would like to thank Dr. R. Kotan, Ataturk Uni-
morphology; and Thermoactinomycetes, which forms en- versity Biotechnology Center, Erzurum, Turkey for the
dospores that display typical actinomycete characteris- identification of Bacillus isolates, and G.T. Sullivan, On-
tics (Sneath, 1986). dokuz Mayis University, Samsun, Turkey for his assis-
The mosquitocidal and nematicidal effects of B. tance in editing an earlier version of this manuscript.
megaterium are well known. For instance, England et al.
(1997) reported that mutants of this bacterium produce
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Received October 16, 2007

Accepted April 21, 2008

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