BARRETO, M. R.

LOGUERCIO, L. L.

PAIVA, E.

VALICENTE, F. H.

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MORTALITY EFFECT OF THE CULTURE SUPERNATANT OF FIVESTRAINS OF Bacillus thuringiensis ON THE DIET OF Spodoptera frugiperdaLARVAE. Marliton Rocha Barreto(1); Leandro Lopes Loguercio(1); Edilson Paiva(1) &Fernando Hercos Valicente(1). (1) - Núcleo de Biologia Aplicada – EMBRAPA Milho e Sorgo.Key words: fall armyworm, maize, vegetative insecticidal proteins.Insecticidal -endotoxin proteins from Bacillus thuringiensis (B.t.), which form crystal inclusionsduring the sporulation phase of the bacteria, has been used widely as commercial bioinsecticidesin Integrated Pest Management (IPM) programs (Höfte and Whiteley, 1989). Recently, it hasbeen demonstrated that other vegetative insecticidal proteins (Vips), identified in the culturesupernatant of certain B.t. strains, were capable to cause death to some economically importantinsect pests (Estruch et al., 1996; Yu et al., 1997). As a preliminary evaluation of the potentialapplicability of these proteins against the most important insect pest for the tropical maizegenotypes cultivated in Brazil, the culture supernatant of five B.t. strains were tested in theirmortality effect on fall armyworm (Spodoptera frugiperda) larvae. Forty-ml bacterial cultureswere grown for 16 hr at 30ºC, and centrifuged for 15 min at 30,500 x g to pellet the cells as muchas possible; the supernatant was then collected with care and filtered through 3MM paper tominimize even further the presence of bacterial cells. Considering that the Vips are a thermal-sensitive fraction in the pool of proteins secreted by B.t. cells, contrasting to the thermal-stablefraction of -exotoxins (Estruch et al., 1996), 5 ml of each of these five supernatants were heatedfor 20 min at 95ºC to denature and inactivate the Vip fraction irreversibly. In these conditions,only the insecticidal activity of the -exotoxins was expected to remain (Estruch et al., 1996).The difference in larval mortality between a non-heated and heated supernatant were used toestimate its Vip content with biological activity (Figure 1). Artificial diets were briefly soaked inheated and non-heated supernatants from each strain before applying to 2-day-old larvae on anindividual basis, i.e., one piece of soaked diet per larva, per container (50 ml-plastic cups withlids). Mortality ratios in a total of 24 individuals per strain, per heat-treatment of the supernatant,were assessed during the whole larval period. The supernatants tested here came from twoforeign B.t. strains (T09 - Pasteur Institute, France; HD125 - UNAM, Mexico) and three tropicalstrains, which were obtained from field conditions in Brazil and previously evaluated regardingtheir -endotoxin contents and efficiency in controlling Spodoptera frugiperda (344 and 520B,Valicente et al., subm.; 606B, data not shown). The results suggested that (i) the Vip contents ofthe two foreign strains are higher and/or more active than the three brazilian strains tested againsttropical fall armyworm larvae, (ii) the amount of Vips relative to -exotoxins appeared to behigher for HD125 than for T09, and (iii) there is no apparent correlation between the -endotoxinand the Vip contents in a single strain. The latter conclusion is supported by combining thesewith other results (Valicente et al., subm; data not shown) that have revealed B.t. strains withhigh (T09, HD125, 344 and 520B) and low (606B) efficiencies in controlling fall armyworm, as aconsequence of their crystal (-endotoxins) content and composition. Taken together, at leastthree combinations were obtained, i.e., high endotoxin and Vip activity (T09 and HD125), highendotoxin and low Vip activity (344 and 520B), and low endotoxin and Vip activity (606B).The results above described were obtained using ‘time’ (i.e., cultures of the same age - 16hr-old) as the standardizing parameter for comparisons between supernatants of different strains.To verify whether the number of cells (optical density) at the exponential phase of growth(healthier cells) could be used as a better normalizing parameter for comparisons among strains,the growth curve of those five B.t. strains was evaluated by checking both their culture opticaldensity at 600 nm and the total level of proteins in the corresponding supernatant. Values of O.D.(at 600 nm) up to 1.5 units were annotated and plotted as a function of time (Figure 2). Theresults suggested clearly that these strains grow at different rates, so that culture age might not bethe best normalizing parameter. However, by comparing the levels of total protein from eachsupernatant, estimated according to the method of Bradford (1976), in which dye-proteincomplexes absorb light at a wavelength of 595 nm, it was noticed the tendency for a trade-offbetween cell division (growth) and protein secretion: faster-growing strains tended to show lessprotein secreted to the medium, and vice-versa, based on a similar optical density (Table 1). Toconfirm these trends statistically, similar experiments with more strains are currently underway.Taken together with other data regarding the minimal culture time required to achieve aconcentration of proteins in the supernatant with measurable mortality effects during the larvalperiod (data not shown), the results here obtained indicated that culture age is good enough as thenormalizing parameter to compare the supernatant from different B.t. strains in their effect on S.frugiperda. Other preliminary SDS- and native-PAGE experiments showed that the proteinbanding pattern displayed by each supernatant is different, with the possibility of some bandsbeing closely associated with high S. frugiperda mortality (Loguercio et al., 1998).The results, here presented, stimulate further evaluation of supernatants from other B.t.strains against fall armyworm, in the search for more efficient Vip activities as alternativebioinsecticides to be used in IPM programs. A collection of ~800 other strains, also isolated frombrazilian field conditions (Valicente et al., subm.), are currently under assessment of their Vipcontents and activity against S. frugiperda. In addition, the composition of secreted proteins inthe supernatant of these and other B.t. strains is being characterized further, aiming to pinpointthe specific Vip(s) responsible for most, if not all, the biological activity of B.t. culturesupernatants against tropical races of S. frugiperda.Figure 1: Mortality of Spodoptera frugiperda during the larval period, after feeding with diet soaked inthe supernatant of five B.t. strains (obs: the error-bars in the graph correspond to two replicasof the experiment, except for HD125 and T09 at 25ºC, in which the bars refer to four replicas).Figure 2: Non-normalized growth curve of the five B.t. strains00,20,40,60,81HD125 T09 344 520B 606B ctrl(medium)B.t. strainsRatio no. dead larvae / total25ºC95ºC00,20,40,60,811,21,41,60.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0time (h)Absorbance (O.D. 600 nm)HD125T09344520B606BTable 1:  Values of absorbance at 595 nm corresponding to total protein(Bradford, 1987) in the supernatants of the five B.t. strains,incubated up to an optical density between 0.9 and 1.5 units at600 nm (see Figure 2):B.t. strains Total protein (O.D.)(1) Culturing time (h:min)(2)606B 0.332 5:30HD125 0.546 6:15T09 0.557 6:45520B 0.619 6:45344 0.611 6:45(1) Values of absorbance at 595 nm after application of the Bradford technique forestimation of total protein amount.(2) Time of incubation in which the optical density of the culture at 600 nm reachedvalues between 0.9 and 1.5; at those times the supernatant was collected for totalprotein measurement (see above)ReferencesBradford, M.M. A rapid and sensitive method for the quantitation of microgram quantities ofprotein utilizing the principle of protein-dye binding. ANAL. BIOCHEM., v. 72 p. 248-254.1976.Estruch, J.J.; Warren, G.W.; Mullins, M.A.; Nye, G.J.; Craig, J.A. & Koziel, M.G. Vip3A, anovel Bacillus thuringiensis vegetative insecticidal protein with a wide spectrum of activitiesagainst lepidopteran insects. PROC. NATL. ACAD. SCI. USA, v. 93 p. 5389-5394. 1996.Höfte, H. & Whiteley, H.R. Insecticidal crystal proteins of Bacillus thuringiensis. MICROBIOL.VER., v. 53 p. 242-255. 1989.Loguercio, L.L.; Barreto, M.R.; Valicente, F.H. & Paiva, E. Electrophoretic characterization oftotal protein from the supernatant of Bacillus thuringiensis strains with different mortalityeffects on Spodoptera frugiperda larvae. In: CONGRESSO NACIONAL DE MILHO ESORGO, XXII, Recife, 1998. Anais. Recife, PE, 1998 (subm.)Valicente, F.H.; Paiva, E.; Vasconcelos, M.J.V.; Figueiredo, J.E.F. & Barreto, M.R.Caracterização através da PCR dos genes cryI de cepas de Bacillus thuringiensis eficientescontra a lagarta do cartucho, Spodoptera frugiperda. ANN. SOC. ENTOMOL. BRASIL.(subm.). 1998.Yu, C-G.; Mullins, M.A.; Warren, G.W.; Koziel, M.G. & Estruch, J.J. The Bacillus thuringiensisvegetative insecticidal protein Vip3A lyses midgut epithelium cells of susceptible insects.APPL. ENVIRON. MICROBIOL., v. 63 p. 532-536. 1997.

Mortality effect on the culture supernatant of five strains of Bacillus thuringiensis of the diet of Spodoptera frugiperda larvae.

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