Isolation and molecular characterization of <i>Bacillus thuringiensis</i> found in soils of the Cerrado region of Brazil, and their toxicity to <i>Aedes aegypti</i> larvae

Lobo, Katiane dos Santos; Soares-da-Silva, Joelma; Silva, Maria Cleoneide da; Tadei, Wanderli Pedro; Polanczyk, Ricardo Antonio; Pinheiro, Valéria Cristina Soares

doi:10.1016/j.rbe.2017.11.004

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Biological Control and Crop Protection • Rev. Bras. entomol. 62 (1) • Jan-Mar 2018 • https://doi.org/10.1016/j.rbe.2017.11.004 copy

Isolation and molecular characterization of Bacillus thuringiensis found in soils of the Cerrado region of Brazil, and their toxicity to Aedes aegypti larvae

Authorship SCIMAGO INSTITUTIONS RANKINGS

ABSTRACT

This study investigated the potential of Bacillus thuringiensis isolates obtained in the Cerrado region of the Brazilian state of Maranhão for the biological control of Aedes aegypti larvae. The isolates were obtained from soil samples and the identification of the B. thuringiensis colonies was based on morphological characteristics. Bioassays were run to assess the pathogenicity and toxicity of the different strains of the B. thuringiensis against third-instar larvae of A. aegypti. Protein profiles were obtained by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Polymerase chain reaction assays were used to detect the toxin genes found in the bacterial isolates. Overall, 12 (4.0%) of the 300 isolates obtained from 45 soil samples were found to present larvicidal activity, with the BtMA-104, BtMA-401 and BtMA-560 isolates causing 100% of mortality. The BtMA-401 isolate was the most virulent, with the lowest median lethal concentration (LC₅₀) (0.004 × 10⁷ spores/mL), followed by the Bacillus thuringiensis var. israelensis standard (0.32 × 10⁷ spores/mL). The protein profiles of BtMA-25 and BtMA-401 isolates indicated the presence of molecular mass consistent with the presence of the proteins Cry4Aa, Cry11Aa and Cyt1, similar to the profile of Bacillus thuringiensis var. israelensis IPS-82. Surprisingly, however, none of the cry and cyt genes analyzed were amplified in the isolate BtMA-401. The results of the present study revealed the larvicidal potential of B. thuringiensis isolates found in the soils of the Cerrado region from Maranhão, although further research will be necessary to better elucidate and describe other genes associated with the production of insecticidal toxins in these isolates.

Keywords:
Biological control; Entomopathogenic bacteria; Vector

Gene (primer)	Nucleotide sequence (5'-3')	Fragment size (bp)	Annealing temperature (ºC)
cry4Aa ^a a Primers designed by Costa et al. (2010).	5'-GGGTATGGCACTCAACCCCACTT	777	50
cry4Aa ^a a Primers designed by Costa et al. (2010).	3'-GCGTGACATACCCATTTCCAGGTCC	777	50
cry4Ba ^a a Primers designed by Costa et al. (2010).	5'-GAGAACACACCTAATCAACCAAT	347	52
cry4Ba ^a a Primers designed by Costa et al. (2010).	3'-GCGTGACATACCCATTTCCAGGTCC	347	52
cry10Aa ^a a Primers designed by Costa et al. (2010).	5'-ATTGTTGGAGTTAGTGCAGG	995	50
cry10Aa ^a a Primers designed by Costa et al. (2010).	3'-AATACTTTGGATGTGTCTTGAG	995	50
cry11Aa ^a a Primers designed by Costa et al. (2010).	5'-CCGAACCTACTATTGCGCCA	470	50
cry11Aa ^a a Primers designed by Costa et al. (2010).	3'-CTCCCTGCTAGGATTCCGTC	470	50
cry11Ba ^a a Primers designed by Costa et al. (2010).	5'-TACAGGATGGATAGGGAATGG	608	52
cry11Ba ^a a Primers designed by Costa et al. (2010).	3'-TAATACTGCCATCTGTTGCTTG	608	52
cyt1Aa ^a a Primers designed by Costa et al. (2010).	5'-AACTCAAACGAATAACCAAG	300	53
cyt1Aa ^a a Primers designed by Costa et al. (2010).	3'-TGTTCCTTTACTGCTGATAC	300	53
cyt1Ab ^a a Primers designed by Costa et al. (2010).	5'-AAGCAAGGGTTATTACATTACG	698	54
cyt1Ab ^a a Primers designed by Costa et al. (2010).	3'-CCAATACTAAGATCAGAGGG	698	54
cyt2Aa ^a a Primers designed by Costa et al. (2010).	5'-GCATTAGGAAGACCATTTG	361	53
cyt2Aa ^a a Primers designed by Costa et al. (2010).	3'-AAGGCTAAGAGTTGATATCG	361	53

Sample	Municipality	Number of bacterial colonies (BC)	Number of B. thuringiensis colonies (Bt index = n(Bt/BC)*100)
1	Caxias	5	0 (0%)
2	Caxias	44	0 (0%)
3	Caxias	13	2 (15.38%)
4	Caxias	4	1 (25.0%)
5	Caxias	21	3 (14.28%)
6	Caxias	19	0 (0%)
7	Caxias	22	0 (0%)
8	Caxias	19	0 (0%)
9	Caxias	9	0 (0%)
10	Caxias	20	1 (5%)
11	Caxias	21	6 (28.57%)
12	Aldeias Altas	203	7 (3.44%)
13	Aldeias Altas	93	9 (9.67%)
52	Arari	44	20 (45.45%)
53	Arari	41	8 (19.51%)
54	Santa Luzia	3	1 (33.33%)
55	Santa Luzia	12	2 (16.66%)
66	Bacabal	52	11 (21.15%)
67	Bacabal	84	43 (51.19%)
68	Alto Alegre do MA	27	6 (22.22%)
69	Alto Alegre do MA	5	2 (40.0%)
73	Parnarama	31	6 (19.35%)
115	Colinas	33	25 (75.75%)
116	Colinas	28	10 (35.71%)
117	Colinas	30	16 (53.33%)
118	Paraibano	32	28 (87.5%)
119	Paraibano	24	17 (70.83%)
120	São João dos Patos	28	24 (85.71%)
121	São João dos Patos	20	14 (70.0%)
122	São João dos Patos	13	9 (69.23%)
123	Barão de Grajaú	7	5 (71.42%)
124	Barão de Grajaú	22	14 (63.63%)
125	Pastos Bons	8	2 (25.0%)
126	Benedito Leite	33	13 (39.39%)
127	Benedito Leite	27	17 (62.96%)
128	São Raimundo das Mangabeiras	20	5 (25.0%)
129	São Raimundo das Mangabeiras	13	3 (20.07%)
130	Balsas	24	20 (83.33%)
131	Balsas	8	4 (50.0%)
132	Riachão	10	3 (30.0%)
133	Riachão	22	14 (63.63%)
134	Carolina	6	2 (33.33%)
135	Carolina	7	1 (14.28%)
136	Carolina	9	6 (66.66%)
137	Carolina	9	3 (33.33%)
Total		1225	383 (31.26%)

Isolate	Gene								Mortality (%)
	cry					cyt			24 h	48 h
	cry4Aa	cry4Ba	cry10Aa	cry11Aa	cry11Ba	cyt1Aa	cyt1Ab	cyt2Aa
BtMA-25	-	-	-	+	+	+	-	-	53.3	86.6
BtMA-64	-	-	-	-	-	+	-	-	6.6	76.6
BtMA-104	-	-	-	-	-	+	-	-	100	100
BtMA-131	-	-	-	-	-	-	-	-	20.0	76.6
BtMA-194	-	-	-	-	-	-	-	+	13.3	66.6
BtMA-251	-	-	-	-	-	-	+	-	6.6	93.3
BtMA-401	-	-	-	-	-	-	-	-	100	100
BtMA-410	-	-	-	+	-	-	+	+	0	96.6
BtMA-413	-	-	-	+	-	-	-	+	0	93.3
BtMA-450	-	-	-	-	-	+	-	+	73.0	96.6
BtMA-451	-	-	-	-	-	+	-	+	0	86.6
BtMA-560	-	-	-	+	-	-	-	-	100	100

Isolate	N	Inclination ±SD	LC₅₀	(CI 95%)	χ² (DF)
Bti	180	4.857 ± 0.457^a a Significant (p ≤ 0.05).	0.32 × 10⁷	(0.26-0.40) × 10⁷	9.9448 (4)
BtMA-25	180	2.593 ± 0.192^a a Significant (p ≤ 0.05).	0.73 × 10⁸	(0.54 × 10⁸-0.12 × 10⁹)	26.795 (6)
BtMA-104	180	1.511 ± 0.106^a a Significant (p ≤ 0.05).	0.84 × 10⁸	(0.45 × 10⁸-0.36 × 10⁹)	39.194 (5)
BtMA-401	180	4.460 ± 0.276^a a Significant (p ≤ 0.05).	0.004 × 10⁷	(0.0034-0.0047) × 10⁷	2.5322 (2)
BtMA-560	180	3.073 ± 0.181^a a Significant (p ≤ 0.05).	0.65 × 10⁸	(0.46 × 10⁸-0.12 × 10⁹)	41.57 (4)

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[1] * Corresponding author. katianecx@gmail.com katianecx@hotmail.com