World Journa/
of Mcrobio/ogy
& Wofechno/ogy
11,202-205
Growth, sporulation and toxin production Baci//us thuringiensis su bsp. israe/ensis and B. sphaericus in media based on mustard-seed meal
by
R.P. Gangurde* and Y.I. Shethna Bacillus thuringiensis subsp. israelensis and B. sphaericus strains 2362 and 1593 were grown in media based on defatted mustard-seed meal (MSM). The meal contains 40% (w/w) protein, with glutamic acid and arginine as the major amino acids. The toxic potencies of the final bacterial powders towards Culex pipens qukquefasciatus Say, compared with those of the respective international reference standards, were 46% for B, thuringiensis subsp. israelensis, 62% for B. sphaericus 2362 and 88% for B. sphaericus 1593 when 2% (w/v) MSM was used for growth. With 4% (w/v) MSM, B. thuringiensis subsp. israezensis grew better but had undetectable larvicidal activity, whereas the B. sphaericus strains not only grew better but gave a higher degree of sporulation and toxicity. The potencies of B. sphaericus in medium with 4% MSM were comparable with those of international reference standards. Key words: Bacihs sphuericm, Bucillus thuringiensis, growth
media, mustard-seed
During sporulation, several strains of Bacik.~ thuringiensis and B. sphaericus produce crystal inclusions which are toxic to a variety of insect larvae (Baumann ef ul. 1991; Priest 1992). Bacillus thuringiensis subsp. isruelensis is the most promising larvicidal agent against Aedes and Culex mosquitoes, whereas B. sphuericus is the most active against Culex and Anopheles spp. (Lacey & Undeen 1986). Laboratory and field evaluations of B. sphaerictis have shown B. sphaericus 2362 to be more active than B. sphuericus 1593 (Yousten ef ul. 1984). The nutritional requirements for optimum growth, sporulation and toxin production by these bacilli have been standardized (Dulmage 197’0; Kaflon et al. 1983). Inexpensive media, such as cotton-seed meal, soybean meal (Singer 198x), cow-blood, bambara beans (Obeta & Okafor 1983), whole maize and cassava (Ejifor & Okafor 1989), have been used to grow B. thuringiensis subsp. israelensis and B. sphaericus. These media, utilized singly or in combination, allow growth, sporulation and toxin production to be controlled depending on their amino-acid profile and carbohydrate The authors are with the Department of Life Sciences, Bombay, Bombay 400 098, India. *Corresponding author. @ 1995 Rapid Communications
of Oxford Ltd
University
of
meal.
content. For example, B. sphaericus grows well only on a protein-rich medium, especially if the medium contains partially hydrolysed proteins or amino acids (Anon. 1990), since it cannot utilize carbohydrates as carbon and energy source (Russell ef ul. 1989). Although B. thuringiensis metabolizes carbohydrates for growth, the amino-acid composition of the medium significantly affects its sporulation and toxin production (Rogoff & Yousten 1969). The present study investigates the feasibility of using de-fatted mustard-seed meal (MSM) as the chief carbon and nitrogen source for growth, sporulation and toxin production of B. fhuringiensis subsp. isruelensis and B. sphuericus isolates 2362 and 1593.
Materials
and Methods
and Growth The international reference standards of B. fhuringiensis subsp. ismlensis, B. sphuericus 2362 and B. sphuericus 1593, namely IPS$2, SPH-88 and RB-80, respectively, were kindly supplied by H. De Barjac, Pasteur Institute, Paris, France. The MSM used throughout the study was obtained from the local market. The bacilli were grown on two MSM-based media, one with 20 g MSM/l Bacteria
Low-cosl media for larvicidal bacilli Table 1. Amino-acid mustard-seed meal.* Amino
composition
01 NaOH-extract
acid
Concentration (mg/lOO mg protein)
Alanine Arginine Aspartic
acid1
lsoieucine Leucine Lysine
5.0 3.2
Methioninet Phenylalanine Proline
i.a 3.1 4.9 1.4
Serine Threonine Tryptophans Tyrosine
3.1 1.9 1.2
Valine
3.a
‘Hydrolysed TValues *Estimated hydrolysis
Dry powders, obtained after acetone co-precipitation, were tested against fourth-instar larvae of Cu/ex pipens quinquejasciutus Say for larvicidal activity. Bioassays, performed using the methods recommended by the World Health Organization (Anon. IWO), were perfomed in duplicate and repeated three times with fresh batches of larvae. Mortality counts were made after incubation for 24 h (B. thtuingiensis subsp. isruelensis) or 48 h (B. sphaericus). Median lethal concentrations (L&J were calculated after converting percentage mortality to probits by probit-regression analysis. These were then used for the evaluation of potency of the primary powders (Anon. IWO).
la.4 3.7 1.7 2.6
Results
in 6 M HCI for 12 h at 121°C. include
acid
and corresponding
calorimetrically of the extract
amide.
using sodium nitroprusside, after in 2 M HCI for I h at 121% (Sadasivam
& Manickam 1992). SEstimated calorimetrically using digestion of the extract (Sadasivam
ferric chloride, after & Manickam 1992).
intervals using nutrient agar plates for B. thwingiensis subsp. isruelensis and Luria-Bertani agar plates for B. sphaeticxs. For spore counts, cultures were heat-shocked at 8O’C for 20 min. Plating for each count was perfomed in duplicate, by the surface-inoculation method. Bioussuy
3.1 7.1 4.a
acid7
Glutamic Glycine Histidine
of de-fatted
Maximum extraction of protein from MSM (40 mg/lOO mg MSM) was with 0.15 M NaOH. Amino-acid analysis of the extract showed glutamic acid as the most abundant amino acid present (Table 1). The carbohydrate content of the extract was 8.3 mg/lOO mg MSM.
papain
Growth (medium 1) and one with 40 g MSM/l (medium 2). The finely ground, de-fatted MSM (20 or 40 g) was added to 800 ml 0.15 M NaOH, boiled for 4 min, cooled and centrifuged at 17,000 X g for 20 min. To the clear supernatant were added (g/l): MgS04.7Hz0, 0.5; MnS04.Hz0, 0.1; FeS04.7Hz0, 0.02; ZnS04.7HL0, 0.02; CaClz, 0.01; and KHzP04, 6.8 for B. sphaericus and 1.0 for B. thuringiensis subsp. israelensis, The pH of the medium was adjusted to 7.2 with 1 M HCI and the volume was made up to 1 I.
Analyfical
Methods
A range of NaOH concentrations from 0.1 rnM was used to determine the optimum concentration for the maximum extraction of protein from MSM. Protein content of the extract was determined by the method of Lowry. The extracted proteins were acidhydrolysed, and the amino-acid composition was determined on a Pharmacia-Alpha Plus amino-acid analyser. The phenol/HzS04 method was used to determine total carbohydrate. Growth of Bacilli A loopful of culture from a 24-h slant was added to 100 ml medium in a 500 ml Edenmeyer flask. After shaking at 180 rev/ min for 24 h at 28OC, 10 ml were transferred to 90 ml medium in a %X)-ml flask which was shaken at 28’C. Growth studies of each strain were repeated three times, each in duplicate. Spores and vegetative cells were recovered as a dry powder using the acetone co-precipitation method (Dulmage et ul. 1970) after 7.1 (B. thwingiensis subsp, israejensis)or 48 h (B. sphaericus isolates). Total Viable Cell Counts und Spore Counts Total viable cell counts and spore counts
were determined at 12-h
and Sporulafion
of fhe Bacilli
Although medium 2 gave more growth (7.8 X 10’ c.f.u./ ml) of B. tharingiensis subsp. israeletisis than medium 1, the bacteria sporulated better in medium I (Table 2). Sporulation in medium 1 could be detected only after 30 to 36 h, but increased extensively after 46 h. Both B. sphaericus strains grew well and sporulated in both media (Table 2) but medium 2 gave better growth and sporulation than medium 1. For B, sphaericas 2362, the net yield after 48 h incubation in medium 2 was almost twice that in medium 1. Toxicity
The toxicity of the bacilli (Table 3) correlated with the extent of sporulation. After growth in medium I, B. thuringiensis subsp. israelensis had a larvicidal activity (L& = 19.7 pg/l) about half that of its international reference standard, IPS-82 (L(& = 9 pg/l). When the bacterium was grown in medium 2, the ensuing primary powder failed to show any larvicidal activity, even when used at 270 pg/l. Bacillus sphaericas 1593 exhibited toxicity comparable with its international reference standard (SPH-88) when grown in both media. Growth in medium z gave the highest lethality (Table 3). Although the potencies achieved with B. sphaericus 1593 were comparable with those of the standard preparation (RB-80), the potency differences in medium 1 and medium 2 were not as marked as for B. sphaericus 2362 (Table 3).
R. I? Gangurde and Y. I. Shethna Table 2. Growth yields and extent of sporulation 1593 in two medfa based on mustard-seed meal Time
of Bacillus (MSM)*
Medium
thuringiensis
subsp.
israelensis
1 (2% MSM)
and Bacillus
Medium
sphaericus
2362 and
2 (4% MSM)
WI Growth (log c.f.u./ml) Bacillus 24 36 48 60 72 Bacillus 24 36 48 Bacillus 24 36 48
thuringiensis
sphaericus
sphaericus
subsp.
Sporulation WI
Growth (log c.f.u./ml)
Sporulation WI
israeiensis 6.38 6.62 8.23 8.48 8.50
ND 15 40 92 94
8.54 8.76 8.83 8.86 8.89
20 31 36 41 48
8.32 8.36 8.52
21 48 71
8.30 8.49 8.78
42 51 86
7.60 8.15 8.43
25 48 76
7.79 8.52 8.56
64 78 86
2362
1593
‘Total viable counts were determined by the spread-plate method, using nutrient agar plates for B. fburingiensis and Luria-Bertani agar plates for B. sphaerkus. Values are means of three experiments performed in duplicate. determined by heat-shock treatment of the cultures at 80% for 20 min. ND-Not detected. Table instar
3. Potencies of the experimental strains Culex quinquefasciatus, as compared
Experimental
6. thuringiensk B. sphaerkus B. sphaerkus
strain
israelensis 2362 1593
“Larval mortality was measured for assays repeated three times ND-Not detected.
of Bacillus, grown in medla with those of the corresponding
Potency
of experimental strain powder)’
Medium
1 (2% MSM)
6853 k 97 623 k 39 875 k 11
(NJ/mg
Medium
primary
based on mustard-seed reference standards. Standard
strain
meal
subsp. israe/ensis Sporulation was
(MSM),
against
fourth-
Potency of standard (kf/mg primary powder)
2 (4% MSM) ND 1072 iz 22 911 * 13
after exposure for 24 (B. thuringiensis) with fresh batches of larvae.
Discussion The present study establishes the potential of de-fatted mustard-seed meal to support growth and toxin production by B. thuritigietisis subsp. israe&is and B. sphaericus 2362 and 1593. The meal has a high protein content and low cost, is readily available and can be handled easily. A single-step protein extraction using a low concentration of NaOH meets the growth requirements of the bacilli. Glutamic acid, lysine and valine, which constitute an appreciable percentage of the MSM proteins, have been known to promote growth and toxin yields of B. sphaericus strains (Lacey 1984). Therefore, growth and larvicidal activity of B. sphaericus 2362 and 1593 in MSM can be attributed, in part, to the presence of these amino acids. Amino acids can have a profound effect on growth, sporulation and crystal
or 48 h (B. sphaericus).
IPS-82 WI-f-88 RB-80 Values
15,000 1000 1000 are means
k standard
errors,
formation of B. thuringiensis (Rogoff & Yousten 1969; Nikerson & Bulla 197’5; Rajalakshmi & Shethna 1980) and a similar effect cannot be ruled out in the present case, since B. thuringiensis subsp. israelensis sporulated less when the concentration of amino acids was increased. Effective biological vector-control in tropical regions requires large-scale production of B. thuringierzsis and B. sphaericus (Anon. 1985) but B. sphaericus requires protein-rich media for growth and this limits its production in developing countries, due to the high cost of such media. These costs could be minimized by using MSM, since grinding of the de-fatted cakes and extraction of the proteins are inexpensive. Separation of the residual meal would not be essential when used on an industrial scale. In the present study, however, separation of the insoluble materials enabled a direct comparison with the international reference standards of the bacilli.
Lozu-cosf mdiu for hruicidal Finally, since mustard is a major oilseed crop in many developing countries, the availability of its meal for largescale production of these biopesticides is virtually unconstrained.
Acknowledgement We thank the Institute for Research in Reproduction, Bombay, for the amino-acid analysis of the extracts.
References Anon. 1985
1$~rma1 Cc~nsu&ion con the Development of Bacillus sphaericus as a Microbial Laruicide. Document TDR/BCV SPHAERICUS 85.3. Geneva: World Health Organization. Anon. 1990 Guidelines for Pmduc&n c$ Bacillus thuringiensis H-14 and Bacillus sphaericus. Document TDR/BCV/90.1, Geneva: World Health Organization. Baumann, I’., Clark, M.A. & Baumann, L. 1991 Bacillus sphaericti as a mosquito pathogen: properties of the organisms and its toxins. !vficr&&gic~/ Reviews 55, 425-436. Dulmage, H.T. 1970 Production of the spore-delta-endotoxin complex by variants of Batiks &ringiet& in two fermentation media. ]ournG[ c$Inuertebraie Pafhololgy 16, 385-389. Dulmage, H.T., Correa, J.A. & Martinez, A.J. 1970 Co-precipitation with lactose as a means of recovering the spore-crystal complex of Bacillus thtiringiensis. ]ourna/ c$hmerfebrate Puthology 15, 15-20. Ejifor, A.O. &I Okafor, N. 1989 Production of mosquito larvicidal Bacillus 0~~rirtgiensis serotype H-14 on raw material media from Nigeria. ]ournal of Applied Bacteriology 67, 5-9. Kaflon, A., Larget-Thiery, I., Charles, J.F. & De Barjac, H. 1983 Growth, sporulation and larvicidal activity of Bacilhts sphaericus. Applied A4icrobi&gy and Biotechn&gy 18, 168-173, Lacey, L.A. 1984 Production and formulation of Bacillus sphaericus. A4osquik7 jVezus 44, 153-159.
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& Okafor,
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N.
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sphaericm
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obtainable Nigerian agricultural products. Canadian ]c~tirnul o) Microbi&gy 29, 704-709. Priest, F.G. 1992 Biological control of mosquitoes and other biting flies by Bacillus sphaericm and Bacillus thuringiensis, Journul c$ Applied Bacteriology 72, 357-369. Rajalakshmi, S. & Shethna, Y.I. 1980 Spore and crystal formation in Batiks thuringiensis during growth in cystine and cysteine. ]otirnul c$ Biosciences 2, 321-328. Rogoff, M.H. & Yousten, A.A. 1969 Bac& thuringietzsis: microbial considerations. Armual Reuiezu of Microbiology 23, 357-386. Russell, B.L., Jelly, S.A. & Yousten, A.A. 1989 Carbohydrate metabolism in the mosquito pathogen Bacillus sphaerictis 2362. Applied and Enkmrnen~a~ Microbi&gy 55, 294-297, Sadasivam, S. &I Manickam, A. 1992 Biochemica/ Methods for Agricukural Sciemes. New Delhi: Wiley Eastern. Singer, S 1981 Potential of Batiks sphaericus and related sporeforming bacteria for pest control, In Microbial Contra/ c$ Pests and P/ant Diseases, 197&1980, ed Burges, H.D., pp. 283-298. London: Academic Press. Yousten, A. A., Madhekar, N. & Wallis, D. 1984 Fermentation conditions affecting growth, sporulation and mosquito larval toxin formation by Bacillus sphaericus. Deueropmenk in Induskial A4icmbi&gy 25, 757-762,
(Received in revised form I 7 Ocfober 1%~; accepted 31 October 1994)
