Supplementary Table1 (SP1): LOCATIONS IN WESTERN GHATS OF KARNATAKA SELECTED FOR SOIL SAMPLING FOR THE ISOLATION OF Bacillus thuringiensis & Population of B. thuringiensis isolates in the soil

Sl.No. / Place Of Collection / No. of Bacillus colonies screened / No. of B. thuringiensis colonies isolated / Mean no. of B.thuringiensis colonies / Population of
B.thuringiensis (CFU/g) / Bt isolation index / No.sample
Collected
1 / Ujire / 4 / 0 / 0.00 (1.00)3 / 0.00 / - / 1
2 / Mudabidri / 7 / 1 / 0.33 (1.14)3 / 0.33X103 / 0.25 / 1
3 / Somavarpet / 8 / 0 / 0.00 (1.00)3 / 0.00 / - / 1
4 / Shiradi Ghat / 11 / 0 / 0.00 (1.00)3 / 0.00 / - / 1
5 / Kodalipet / 8 / 0 / 0.00 (1.00)3 / 0.00 / - / 1
6 / Agumbe / 9 / 0 / 0.00 (1.00)3 / 0.00 / - / 1
7 / Haveri / 3 / 0 / 0.00 (1.00)3 / 0.00 / - / 1
8 / Hosanagara / 14 / 1 / 0.33 (1.14)3 / 0.33X103 / 0.25 / 1
9 / Shringeri / 10 / 1 / 0.33 (1.14)3 / 0.33X103 / 0.25 / 1
10 / Dakshina kannada / 5 / 1 / 0.33 (1.14)3 / 0.33X103 / 0.25 / 1
11 / Kushalnagara / 8 / 1 / 0.33 (1.14)3 / 0.33X103 / 0.25 / 1
12 / Sirsi / 5 / 0 / 0.00 (1.00)3 / 0.00 / - / 1
13 / Chikklihole / 13 / 1 / 0.33 (1.14)3 / 0.33X103 / 0.25 / 1
14 / Belthangadi / 10 / 0 / 0.00 (1.00)3 / 0.00 / - / 1
15 / Gundiya / 3 / 0 / 0.00 (1.00)3 / 0.00 / - / 1
16 / Venooru / 8 / 1 / 0.33 (1.14)3 / 0.33X103 / 0.25 / 1
17 / Kuduremukh / 3 / 1 / 0.33 (1.14)3 / 0.33X103 / 0.25 / 1
18 / Dharmasthala / 8 / 1 / 0.33 (1.14)3 / 0.33X103 / 0.25 / 1
19 / Magodu / 16 / 1 / 0.33 (1.14)3 / 0.33X103 / 0.25 / 1
20 / virajpet / 11 / 0 / 0.00 (1.00)3 / 0.00 / - / 1
21 / Charmudi Ghat / 3 / 1 / 0.33 (1.14)3 / 0.33X103 / 0.25 / 1
22 / Mala / 5 / 0 / 0.00 (1.00)3 / 0.00 / - / 1
23 / Shanivara pete / 10 / 0 / 0.00 (1.00)3 / 0.00 / - / 1
24 / Gangamoola / 11 / 0 / 0.00 (1.00)3 / 0.00 / - / 1
25 / Shakaleshpura / 2 / 1 / 0.33 (1.14)3 / 0.33X103 / 0.25 / 1
26 / Kundadri Hills / 6 / 1 / 0.33 (1.14)3 / 0.33X103 / 0.25 / 1
27 / Chikkamagaluru / 12 / 1 / 0.33 (1.14)3 / 0.33X103 / 0.25 / 1
28 / Gonikoppa / 16 / 1 / 0.33 (1.14)3 / 0.33X103 / 0.25 / 1
29 / Madikeri / 12 / 1 / 0.33 (1.14)3 / 0.33X103 / 0.25 / 1
30 / Kukke / 4 / 0 / 0.00 (1.00)3 / 0.00 / - / 1
31 / Bajagoli / 3 / 1 / 0.33 (1.14)3 / 0.33X103 / 0.25 / 1
32 / Nantoru / 11 / 0 / 0.00 (1.00)3 / 0.00 / - / 1
33 / Horanadu / 4 / 0 / 0.00 (1.00)3 / 0.00 / - / 1
34 / Kodalipet / 7 / 0 / 0.00 (1.00)3 / 0.00 / - / 1
35 / Algodu / 5 / 1 / 0.33 (1.14)3 / 0.33X103 / 0.25 / 1
36 / Subramanya / 10 / 1 / 0.33 (1.14)3 / 0.33X103 / 0.25 / 1
37 / Uttara kannada / 5 / 1 / 0.33 (1.14)3 / 0.33X103 / 0.25 / 1
38 / Uttara kannada 2 / 3 / 0 / 0.00 (1.00)3 / 0.00 / - / 1
39 / Karkala / 10 / 1 / 0.33 (1.14)3 / 0.33X103 / 0.25 / 1
40 / Koppa / 5 / 0 / 0.00 (1.00)3 / 0.00 / - / 1
41 / Dubare / 8 / 1 / 0.33 (1.14)3 / 0.33X103 / 0.25 / 1
42 / Thirthahalli / 5 / 1 / 0.33 (1.14)3 / 0.33X103 / 0.25 / 1
43 / Kodachadri / 13 / 1 / 0.33 (1.14)3 / 0.33X103 / 0.25 / 1
44 / Kolluru / 10 / 1 / 0.33 (1.14)3 / 0.33X103 / 0.25 / 1
45 / Kudremukh / 3 / 0 / 0.00 (1.00)3 / 0.00 / - / 1
46 / Shringeri 2 / 8 / 0 / 0.33 (1.14)3 / 0.33X103 / 0.25 / 1
47 / Shimoga / 3 / 1 / 0.33 (1.14)3 / 0.33X103 / 0.25 / 1
48 / Gokarna / 8 / 1 / 0.33 (1.14)3 / 0.33X103 / 0.25 / 1
49 / Karwara / 10 / 0 / 0.00 (1.00)3 / 0.00 / - / 1
50 / Shiradi Ghat 2 / 15 / 1 / 0.33 (1.14)3 / 0.33X103 / 0.25 / 1

CD = 0.301

(p = 0.05)

(Figures in parentheses areÖ x + 1 transformed values)

Bt isolation index was worked out by dividing the population of crystalliferous Bt isolates by the total population of Bacillus for each sample collected.

Supplementary Data 2 (SP2): Characteristics of general and specific primers for Coleopteran, Hemipteran and Nematode active cry –type genes

Primer / Sequence 5’-3’ / Positions / Gene(S) recognized / Product size (bp) / GenBank Accession no. / References
UNcry1(+) / TRACRHTDDBDGTATTAGAT / - / cry1 / 1500–1600 / - / Nazarian et al.2009
UNcry1(–) / MDATYTCTAKRTCTTGACTA
SPcry1B(+) / GGCTACCAATACTTCTATTA / - / cry1B / 1063 / X06711
UNcry1(–) / MDATYTCTAKRTCTTGACTA
SPcry1I(+) / ACAATTTACAGCTTATTAAG / - / cry1I / 1000 / X62821
SPcry1I(–) / CTACATGTTACGCTCAATAT
UNcry3(+) / CGTTATCGCAGAGAGATGACATTAAC / - / cry3 / 420 / M30503
UNcry3(–) / CATCTGTTGTTTCTGGAGGCAAT
SPcry3A(+) / TGGTGCCCCGTCTAAACTGAGTGT / - / cry3A / 951 / U10985
UNcry3(–) / CATCTGTTGTTTCTGGAGGCAAT
UNcry3(+) / CGTTATCGCAGAGAGATGACATTAAC / - / cry3B / 1103 / X17123
SPcry3B(–) / ACGAAAGATTCTGCTCCTAT
UNcry3(+) / CGTTATCGCAGAGAGATGACATTAAC / - / cry3C / 461 / X59797
SPcry3C(–) / ATTTTGGTACCTCCTGTACCCACC
UNcry7,8(+) / AAGCAGTGAATGCCTTGTTTAC / - / cry7,8 / 423 / AB089299
UNcry7,8(–) / CTTCTAAACCTTGACTACTT
SPcry7A(+) / GCGGAGTATTACAATAGAATCTATCC / - / cry7A / 916 / M64478
UNcry7,8(–) / CTTCTAAACCTTGACTACTT
SPcry8A(+) / GGTCCTGGATTTACAGGAGGAGAT / - / cry8A / 342 / U04364
SPcry8A(– / GATGAATTCGATTCGGTCTAT
SPcry8B(+) / GGGCGTGGTTATACAGGGGGAGAC / - / cry8B / 342 / U04365
SPcry8B(–) / GATGAATTCGATTCGGTCTAA
SPcry8C(+) / GAAGGTCTATATAATGGAGGAC / - / cry8C / 369 / U04366
SPcry8C(–) / AATAAATTCAATTCTATCAAT
SPcry14(+) / ATAATGCGCGACCTACTGTTGT / - / cry14 / 456 / U13955
SPcry14(–) / TGCCGTTATCGCCGTTATT
UNcry18(+) / CCGAGGCGATTTGGATAGAT / - / cry18 / 419 / X99049
UNcry18(–) / TGCCGGTGTAAACAAAGAAGG
SPcry26(+) / CGCGCTGTTCAATTATCAAGTGC / - / cry26 / 362 / AF122897
SPcry26(–) / ATATGGAAAGAAAAGGCGTGTGGA
SPcry28(+) / GTATTGGACCGAGGAGATGAAAGT / - / cry28 / 466 / AF285775
SPcry28(–) / GTACGGCAAAGCGACAGAACA
UNcry34(+) / ATGTCAGCDCGYGAAGTACA / - / cry34 / 278 / AAG50341
UNcr34(–) / CATAAGGATTGTCAAAAT
UNcry35(+) / AGTTTDGATRATTCAGGTGTTA / - / cry35 / 912 / AAG50342
UNcry35(–) / ACCTCCACCTACTTCTGGTA
SPcry9D(+) / AGTTATTGATGCCCCACATTG / 30-50 / cry9Da / 874 / D85560 / Present study
SPcry9D(-) / TGTCCCATCTGGATATTGTCG / 882-903
SPcry9da(+) / TGATCCGAATGCTGGATTGCA / 93-113 / cry9Da / 1962 / D85560 / Present study
SPcry9Da(-) / CTAGCCGCTTTCTTCGCTGCT / 2033-2054
SPcry22(+) / AACTGTACAAGCAGCAAGCGA / 36-56 / cry22 / 602 / I34547 / Present study
SPcry22(-) / TACGTGGCTTCTCTCCAACAG / 616-637
SPcry23(+) / AGAGGTATATGGTGCAACAACT / 45-66 / cry23 / 696 / AF038048 / Present study
SPcry23(-) / GTTATTGGTGTCGAGCGACCT / 719-740
SPCRY5(+) / TAAGCAAAGCGCGTAACCTC / 2459-2478 / cry5 / 322 / L07025,L07026,I34543,U19725 / Salehi Jouzani et al.2008
SPCRY5(-) / GCTCCCCTCGATGTCAATG / 2780-2762
SPCRY6(+) / TGGCGTAGAGGCTGTTCAAGTA / 609-630 / cry6 / 302 / L07022,AF499736,L07024
SPCRY6(-) / TGTCGAGTTCATCATTAGCAGTGT / 910-887
SPCRY12(+) / CTCCCCCAACATTCCATCC / 2555-2573 / cry12 / 363 / L07027
SPCRY12(-) / AATTACTTACACGTGCCATACCTG / 2917-2894
SPCRY13(+) / CTTTGATTATTTAGGTTTAGTTCAA / 225-250 / cry13 / 313 / L07023
SPCRY13(-) / TTGTAGTACAGGCTTGTGATTC / 537-515
SPCRY14(+) / ATAATGCGCGACCTACTGTTGT / 1634-1655 / cry14 / 456 / U13955
SPCRY14(-) / TGCCGTTATCGCCGTTATT / 2089-2071
SPCRY21(+) / ATACAGGGATAGGATTTCAAG / 1112-1132 / cry21 / 453 / I32932,I66477,AB088406
SPCRY21(-) / ATCCCCATTTTCTATAAGTGTCT / 1564-1542
SPcry3A(+) / TCCGAACAATCGAAGTGAACA / 6-26 / cry3A / 734 / U332160 / Present study
SPcry3A(-) / ACCATTTGACACAATGGTCAG / 719-739
SPcry3A1(+) / ATATCCTTTAGCGGAAACTCCA / 102-123 / cry3A / 375 / M22472 / Present study
SPcry3A1(-) / TGCCATGAACTCAATGCACTC / 456-476
SPcry4A(+) / ACAGAATCAGCAGTATGGTGG / 156-176 / cry4A / 443 / Y00423 / Present study
SPcry4A(-) / CGCAATCACTAGGATTAGGAGG / 577-598
SPcry4A1(+) / TGAACCCGTACCAAAACAAGA / 9-30 / cry4A / 556 / AB513706 / Present study
SPcry4A(-) / GGGATGACGTTTTGGAAGTGG / 537-557
SPcry1Ab(+) / CAATCCGAACATCAATGAATGC / 9-30 / cry1Ab / 960 / AY197341 / Present study
SPcry1Ab(-) / GCTGTTATTTGATGCCCTGACC / 947-968
SPcry1Ab1(+) / CAATCCGAACATCAATGAATGC / cry1Ab / 358 / GU322940 / Present study
SPcry1Ab1(-) / AGTAGGATCTGCTTCCCACTC / 346-366

B = C, G, or T; D = A or T; H = A, C, or T; K = G or T; M = A or C; R = A or G; Y = T or C.

Supplementary Data 3 (SP3). MORPHOLOGICAL OBSERVATIONS OF Bacillus thuringiensis ISOLATES

Sl. No. / Name of the Place / Bt Isolate name / Colony morphology / Spore position / Crystal shape / Vegetative cell type
01. / Venooru / VEN / Chalky white with granular pheriphry / TERMINAL / RHOMBOIDAL / ROD SHAPED
02. / Shakaleshpura / SHK / Chalky white with granular pheriphry / TERMINAL / RHOMBOIDAL / ROD SHAPED
03. / Dakshina kannada / DK / Chalky white with granular pheriphry / TERMINAL / RHOMBOIDAL / ROD SHAPED
04. / Muthatti / MUT / Chalky white with granular pheriphry / TERMINAL / RHOMBOIDAL / ROD SHAPED
05. / Thirthahalli / THR / Chalky white with granular pheriphry / TERMINAL / IRREGULAR POINTED / ROD SHAPED
06. / Kuduremukha / KUD / Chalky white with granular pheriphry / TERMINAL / CUBOIDAL / ROD SHAPED
07. / Kundadri hills / KUN / Drop like and slimy / TERMINAL / BYPIRAMIDAL / ROD SHAPED
08. / Madikeri / MAD / Drop like and slimy / TERMINAL / BYPIRAMIDAL / ROD SHAPED
9. / Shiradi ghat / SHG / Drop like and slimy / TERMINAL / ROUND SHAPED / ROD SHAPED
10. / Gonikoppa / GOK / Drop like and slimy / TERMINAL / ADHERED TO SPORE / ROD SHAPED
11. / Charmudi ghat / CHM / Drop like and slimy / SUB TERMINAL / ADHERED TO SPORE / ROD SHAPED
12. / Dharmasthala / DHS / small, smooth and bright / SUB TERMINAL / ADHERED TO SPORE / ROD SHAPED
13. / Kolluru / KOL / small, smooth and bright / SUB TERMINAL / ADHERED TO SPORE / ROD SHAPED
14. / Mudabidri / MOD / small, smooth and bright / SUB TERMINAL / SMALL / ROD SHAPED
15. / Somavarpet / SOM / Chalky white with granular periphery / SUB TERMINAL / IRREGULAR POINTED / ROD SHAPED
16. / Kushalnagara / KUH / Chalky white with granular pheriphry / TERMINAL / RECTANGULAR / ROD SHAPED
17. / Shringeri / SHI / spread wavy and round with margin / TERMINAL / IRREGULAR POINTED / ROD SHAPED
18. / Chikklohole / CHI / Chalky white with granular pheriphry / TERMINAL / CUBOIDAL / ROD SHAPED
19. / Magodu / MAG / Chalky white with granular pheriphry / TERMINAL / CUBOIDAL / ROD SHAPED
20. / Kodachadri / KOD / Chalky white with granular pheriphry / TERMINAL / IRREGULAR POINTED / ROD SHAPED
21. / Bajgoli / BAJ / Chalky white with granular pheriphry / TERMINAL / IRREGULAR POINTED / ROD SHAPED
22. / Kodalipet / KOD2 / Drop like and slimy / TERMINAL / BYPIRAMIDAL / ROD SHAPED
23. / Algodu / ALG / small, smooth and bright / TERMINAL / BYPIRAMIDAL / ROD SHAPED
24. / Subramanya / SUB / Chalky white with granular pheriphry / TERMINAL / BYPIRAMIDAL / ROD SHAPED
25. / Karkala / KAR / Drop like and slimy / TERMINAL / BYPIRAMIDAL / ROD SHAPED
26. / Dubare / DUB / Drop like and slimy / TERMINAL / BYPIRAMIDAL / ROD SHAPED
27. / Uttara Kannada / UK / small, smooth and bright / TERMINAL / BYPIRAMIDAL / ROD SHAPED
28. / Gokarna / GOR / Chalky white with granular pheriphry / TERMINAL / IRREGULAR POINTED / ROD SHAPED

Supplementary Data 3 (SP3). Types of crystalline inclusions in B. thuringiensis isolates of Western Ghats of Karnataka.

S. No. / Crystal morphology / Bt isolates
Number / %
1 / Bipyramidal / 8 / 28.57
2 / Irregular / 6 / 21.42
3 / Adhered to spore / 4 / 14.28
4 / rhomboidal / 3 / 10.71
5 / Cuboidal / 1 / 3.57
6 / small / 1 / 3.57
7 / rectangular / 1 / 3.57
8 / Round shaped / 1 / 3.57

Supplementary Data 4 (SP4). – Microbiological observations of Bacillus thuringiensis isolates

Sl. No. / Name of the Place / Bt Isolate name / Grams' staining / Crystal staining / Endospore staining / Molecular
weight of
d-endotoxin (kDa) / cry gene content
1 / Venooru / VEN / ü  / ü  / ü  / 120,60,30,26,18 / cry1I, cry1Ab
2 / Shakaleshpura / SHK / ü  / ü  / ü  / 150,120,75,50,40,36,20,16 / cry1I, cry6A
3 / Hosanagara / DK / ü  / ü  / ü  / 75, 65, 40, 25 / cry1I, cry6A
4 / Dakshina kannada / MUT / ü  / ü  / ü  / 50,40,35, 14 / cry9Da
5 / Muthatti / THR / ü  / ü  / ü  / 50.5, 35, 26, 20 / cry9Da
6 / Thirthahalli / KUD / ü  / ü  / ü  / 60, 32 / cry1I, cry6A
7 / Kuduremukha / KUN / ü  / ü  / ü  / 150, 75, 30 / cry1I, cry6A
8 / Kundadri hills / MAD / ü  / ü  / ü  / 150, 130, 75, 60,50 / cry1I, cry6A
9 / Madikeri / SHG / ü  / ü  / ü  / 100, 75, 60, 50, 45, 35, 20 / cry1I, cry28Aa, cry6A, cry5B
10 / Shiradi ghat / GOK / ü  / ü  / ü  / 120, 75, 60, 50, 40, 27, 20,12 / cry1I, cry7ab, cry1Ab
11 / Gonikoppa / CHM / ü  / ü  / ü  / 100, 75, 60, 50, 45, 35, 20, 14, 10 / cry1I, cry22
12 / Charmudi ghat / DHS / ü  / ü  / ü  / 100, 75, 60, 50, 45, 35, 20 / cry1I, cry4A
13 / Dharmasthala / KOL / ü  / ü  / ü  / 130, 100, 52, 40, 33, 20 / cry1I, cry4A
14 / Kolluru / MOD / ü  / ü  / ü  / 100, 75, 60, 50, 45, 35 / cry1I, cry6A
15 / Mudabidri / SOM / ü  / ü  / ü  / 140, 95, 60 / cry1I, cry6A
16 / Kushalnagara / KUH / ü  / ü  / ü  / 120, 65, 45, 35, 30, 20. 14, 11 / cry1I, cry7Ab
17 / Shringeri / SHI / ü  / ü  / ü  / 150, 58, 48, 36 / cry1I, cry6A
18 / Koppa / CHI / ü  / ü  / ü  / 75, 65, 40, 28, 25 / cry8Ka, cry3C, cry5A, cry5B, cry4A
19 / Magodu / MAG / ü  / ü  / ü  / 80, 65, 45, 30 / cry8Ka, cry5A, cry5B
20 / Kodachadri / KOD / ü  / ü  / ü  / 85, 65, 32, 25 / cry8Ka, cry9Da
21 / Bajgoli / BAJ / ü  / ü  / ü  / 50, 48, 40, 25 / cry9Da
22 / Kodalipet / KOD2 / ü  / ü  / ü  / 150, 50, 47, 32, 22, 12 / cry9Da