Brazilian Journal of Microbiology (2000) 31:165-167 B. thuringiensis against S.

frugiperda
ISSN 1517-8382

EFFECTIVENESS OF BACILLUS THURINGIENSIS STRAINS AGAINST

SPODOPTERA FRUGIPERDA (LEPIDOPTERA: NOCTUIDAE)

Ricardo Antonio Polanczyk1; Rogério Fernando Pires da Silva 1; Lidia Mariana Fiuza2*

1
Health Plant Department, School of Agronomy, Universidade Federal do Rio Grande do Sul, RS, Brasil; 2Centro de
Biotecnologia do Estado do Rio Grande do Sul, Universidade do Rio Grande do Sul e Departamento de Microbiologia,
UNISINOS, RS, Brasil

Submitted: August 13, 1999; Returned to authors for corrections: December 16, 1999; Approved: September 13, 2000

ABSTRACT

The fall armyworm (Spodoptera frugiperda) is one of the most important pests of maize, causing up to
20% production losses when defoliation occurs near to flowering, or even complete destruction of plants.
Among the alternatives to control this pest, the use of Bacillus thuringiensis (Bt) has gained attention due
to its efficiency and low impact on natural enemies. Strains Bt dendrolimus HD 37, Bt aizawai HD 68, Bt
kurstaki HD 73, Bt darmstadiensis HD 146, and Bt thuringiensis 4412 were tested against second instar
larvae in in vivo assays. Suspensions of Bt aizawai HD 68 and Bt thuringiensis 4412, containing 3 x 108
cells/ml, induced mortality of 100% and 80.4%, respectively. To test virulence, cell concentrations of 8 x
105 to 3 x 108 cells/ml of strains Bt aizawai HD 68 and Bt thuringiensis 4412 were applied on the second
instar larvae: LC50 were 6.7 x 106 and 8.6 x 106 cells/ml, respectively.

Key words: Bacillus thuringiensis, Spodoptera frugiperda, biological control, bioassays, fall armyworm

INTRODUCTION This microorganism acts in the insect gut due to cristals,

composed by protoxins, discharged in the gut due to the alkaline
The fall armyworm (Spodoptera frugiperda Smith & pH that causes solubilization. These protoxins, in presence of
Abbot), that attacks various cultures is one of the most digestive enzymes, are converted in toxic polypetides (delta-
important pests of maize in the Americas (17), causing about endotoxins). The activated toxins cross the peritrofic
20% production losses in Brazil (6). Although the use of membrane, join to specific receptors in apical membrane of
chemicals is the prevailing method to control this pest, columnar cells of midgut, and insert themselves into the
problems such as ecological disequilibrium, pollution, risks membrane (9, 11). The formation of pores disrupts the ionic
during application and high costs are present (16). Furthermore, gradients and osmotic balance in the apical membrane,
the insecticides kill the fall armyworm natural enemies, resulting in cell swelling and lysis. This phenomenon leds to
favoring rapid reinfestation with serious damage to the culture massive destruction of epithelium, causing death of larva (12).
(6). In fact, all these questions increased the interest in Some time ago, the efficacy of this microorganism against
alternative strategies to manage this pest. S. frugiperda was considered questionable, but more recently
Nowadays, some methods, solely or together, get the the increment in researches on the use of Bt against this
satisfactory control of S. frugiperda. Among the lepidopterous brought some interesting results (4, 10). The
entomopathogenic agents used in biological control of objective of this work was to report the selection of Bt strains
lepidopterous pests the Bacillus thuringiensis Berliner with potential to control the fall armyworm. They could be
bacterium (Bt) has gained special attention as an alternative used in the formulation of new biopesticides or in genetic
method (2, 11). transformation of host plants.

* Corresponding author. Mailing address: Laboratório de Microbiologia, Centro 2, UNISINOS, Caixa Postal 275, CEP: 93022-000, São Leopoldo, RS, Brasil. E-
mail: fiuza@cirrus.unisinos.br

165
R.A.Polanczyk et al.

a
MATERIALS AND METHODS 100

Mortality (%)
90 a
Insects: S. frugiperda larvae were obtained from maize 80

fields in Viamão (South Brazil) and reared in Bowling diet 70

(5). The insects used in these assays belonged to the 20th 60

generation and were mantained at 25 ± 2°C with 65 ± 5% 50

relative humidity and 12h photoperiod. 40

Selective assays: These assays were carried out in a 30

b
Biological Oxigen Demand chamber, in the same conditions 20

described above. Bt dendrolimus HD 37 was provided by the 10 c

Institut Pasteur, Paris and Bt aizawai HD 68, Bt kurstaki HD 73, 0
c

Bt darmstadiensis HD 146 and Bt thuringiensis 4412 strains

6868

412
D

12

7373
ai H

sis 4
were provided by Plant Genetic Systems, Gent. They were

37
44

37
H izaw

ki H

46
HD
ai

6
gien
Strains of Bt

D
is

D1
14
D
H
ns

ursta
Bt a
ki

mus
aw

urin
grown in Usual Glicosed Medium (8) at 28°C and 180 rpm for

is H
D
z e ta us
gi

roli
ai

Bt k
Bt th

H
iens
in rs is

end
Bt ku im
ur ol ns

stad
48h. The suspension was centrifuged at 5,000 rpm and washed

Bt d
th Bt dr ie

arm
n d
Bt de

Bt d
ta
3 times with sterilized water. The concentration of bacterial Bt m
s
d ar
spores was determined in a Neubaeur chamber using phase Bt
contrast microscopy at 400x. 100 ml of the both containing of Figure 1. Mortality in selective assays of Spodoptera frugiperda second
3 x 108 cells/ml were added to the surface of artificial diet instar larvae and Bacillus thuringiensis strains at 3 x 108 cells/ml
previously put in mini-plates (30 mm of diameter), where larvae concentration.
of 2nd instar of S. frugiperda were individualized (20 insects
per strain). In controls, the broth was replaced by 100 µl of The virulence assays showed that Bt aizawai HD 68 was
sterile water. The mortality was evaluated up to seven days the most active strain, with an LC50 of 6.7 x 106 cells/ml. Bt
after treatment. The data were corrected according to Abbot thuringiensis 4412 presented an LC50 of 8.6 x 106 cells/ml
(1) and submitted to Duncan’s t test (P = 0.05). (Table 1). These LC50 values showed that a concentration 42.0%
LC50 assays: The virulence of Bt aizawai HD 68 and Bt higher of Bt thuringiensis 4412 than the other strain was
thuringiensis 4412 was evaluated by LC50 assays, using 8 x necessary to kill 50.0% of a S. frugiperda population in a
105, 3 x 106, 8 x 106, 3 x 107, 8 x 107 and 3 x 108 cells/ml and certain period. Fig. 2 compares the virulence of the strains
control. Three replications were carried out, totalizing 1,050 and shows that Bt aizawai HD 68 requires a lower
insects per strain. To achieve the initial concentration of 3 x concentration to be letal to 100% of larvae population.
108 cells/ml, the above mentioned method for cell counting The differences in toxicity of these strains to S. frugiperda
was used. All other cell concentrations was obtained through may be related to the composition of cristals and their toxic
dilutions of this suspension. The exact number of spores was potential. Bt aizawai HD 68 has two genes (cryIA(a), cryID)
determined in a Neubaeur chamber. The amount of suspension related to toxicity, while Bt thuringiensis 4412 has only one
and the method for growth of microorganism were the same as
in selective assays. The data were analyzed by Polo-PC (LeOra (a )
5

Software 1987).
4.9 B t a iza w a i H D 6 8
Subletal effects: The possible effects of the strains on insects
were verified through visual comparison between growth of 4.8

surviving larvae and control insects. 4.7

B t th u rin g ie n sis 4 4 1 2

4.6
RESULTS AND DISCUSSION
P R O BIT

4.5

The in vivo activities of Bt strains tested on second instar 4.4

S. frugiperda larvae are shown in Fig. 1. Bt thuringiensis 4412 4.3

and Bt aizawai HD 68 strains were the most pathogenic causing
4.2
80.40% and 100% of mortality, respectively. These strains were
statistically different to others but not between them (Duncan, 4.1

5%). Similar results were obtained by Hernandez (10) for 4

6.5 6.6 6.7 6.8 LO G (CL5 0 B) 6.9 LO G (CL5 0 B) 7
subspecies Bt aizawai, Bt thuringiensis and Bt kurstaki, L O G (C O N C .)

observing mortality of 80%, 100% and 70%, respectively, to 3 Figure 2. Comparative virulence of Bt aizawai HD 68 and Bt
x 107 cells/ml. thuringiensis 4412 to Spodoptera frugiperda second instar larvae.

166
 B. thuringiensis against S. frugiperda

Table 1. LC50 of Bacillus thuringiensis strains to Spodoptera frugiperda second instar larvae.

Strains LC50* (lL - sL) Equation

Bt aizawai HD 68 6 . 7 x 10 6 4 . 7 x 10 6 - 9 . 2 x 10 6 y = - 0.412 + 0.792x
Bt t huringiensis 4412 8.6 x 106 6.0 x 106 - 12.0 x 106 y = 0.414 + 0.660x
* Results obtained using 1,050 larvae per strain.
(lL – sL) = Lower and superior limits (values) per LC50.

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