Flowchart 3.1. Summary of Chapter 3
4.5 Inference
Insight to the mechanism of pathogenicity can provide the knowledge for the development of potent mycoinsecticides by identifying the virulence factors or else identifying the genes responsible for virulence and genetically modify the fungi to enhance virulence. Virulence of the fungal isolates M. anisopliae (ARSEF 3295) and B. bassiana (ARSEF 2033) were studied and described in the next chapter, 5.
Table 4.1. Primers used for isolation of genes
Protease Primers
Protease Primer1(PP1)
Forward primer: AATAGCCCGGCGTTCCGTGGGGATCC Reverse primer: GCCAGCTGCAGTGTCGTGGAGGATCC
Protease Primer2 (PP2)
Forward primer: CTGCAGGATGGCAACGCCCAGCGTGA Reverse primer: CTGCAGGCGAGGCCATTGAGGTGCCA
Chitinase Primers
Chitinase Primer1 (CP1) Forward primer: GGGGGTTCGAGGATGACACC
Reverse primer: CTGTTGGCCCTGAGCGTCCG Chitinase Primer2 (CP2) Forward primer: CTCGTTGGCGTCGACCTGGAT
Reverse primer: GGGCCTCCCACAACATGGCG
Table 4.2. Results of BLAST analysis of DNA sequence from B. bassianaPCR product using PP1
Sequence Score Identities Conserved domains
Beauveria bassiana cuticle- degrading proteinase CDEP-1
(cdep1) gene, cdep1-s allele (gb|AY040532.1|) QS-393-425:SS-405-437
62.1 100%
Beauveria bassiana strain MTCC 2028 serine proteinase (Pr1) gene
(gb|GU166155.1|) QS-393-425:SS-333-365
62.1 100%
Beauveria bassiana cuticle- degrading proteinase (CDEP2) Mrna
(gb|EF195164.1|) QS-393-425:SS-268-300
62.1 100%
Beauveria bassiana cuticle- degrading protease bassiasin I (bsn1) gene (gb|AF154118.1| )
QS-393-425:SS-409-441
62.1 100%
Table 4.3. Results of BLAST analysis of DNA sequence from B. bassianaPCR product using PP2
Sequence Score Identities Conserved domains
Beauveria bassiana cuticle- degrading proteinase CDEP-1 (cdep1) gene (gb|AY040532.1|)
QS-1-139:SS-762-896;
QS-301-483:SS-920-1104
193 93%
254 91%
Beauveria bassiana strain MTCC 2028 serine proteinase (Pr1) gene
(gb|GU166155.1|) QS-301-483:SS-848-1032
255 92%
Beauveria bassiana cuticle- degrading proteinase (CDEP2)mRNA (gb|EF195164.1|)
QS-1-139:SS-564-698;
QS-301-483:SS-722-906
182 91%
248 91%
Beauveria bassiana cuticle- degrading protease bassiasin I (bsn1) gene (gb|AF154118.1| )
QS-1-139:SS-767-901;
QS-301-483:SS-925-1109
198 93%
248 91%
Table 4.4. Results of BLAST analysis of DNA sequence from M. anisopliae PCR product using PP1
Sequence Score Identities Conserved Domains
Metarhizium anisopliae cuticle- degrading protease mRNA (gb|M73795.1|MEZCDPA) QS-98-241: SS-335-478
161 87%
Metarhizium anisopliae var. acridum mRNA for Subtilisin-like serine
protease PR1A (pr1A gene) (emb|AJ251925.1|) QS-98-237:SS-415-554
132 84%
Metarhizium acridum strain CG191 subtilisin-like protease Pr1A (pr1A)
gene (gb|FJ659172.1|) QS-98-229:SS-411-542;
QS-291-341:SS-642-689
156 88%
58.4 89%
Metarhizium anisopliae strain V301 subtilisin-like protease Pr1A (pr1A)
gene (gb|FJ659171.1|) QS-98-229:SS-411-542;
QS-291-341:SS-642-689
156 88%
58.4 89%
Table 4.5. Results of BLAST analysis of DNA sequence from M. anisopliae PCR product using PP2
Sequence Score Identities Conserved domains
Metarhizium anisopliae var.
acridum mRNA for subtilisin-like protease PR1D (pr1D gene)
(emb|AJ431638.1|) QS-86-137:SS-1012-1063;
QS-324-376:SS-1063-1114
97.1 100%
86.1 97%
Metarhizium anisopliae var.
anisopliae pr1D gene for subtilisin- like protease PR1D, exons 1-2
(emb|AJ400706.1|) QS-86-137:SS-1401-1452 QS-324-376:SS-1452-1503
97.1 100%
86.1 97%
Metarhizium anisopliae var.
anisopliae strain V245 subtilisin- like serine protease PR1A (pr1A)
gene (gb|AY389127.1|) QS-38-97:SS-883-931
91.6 95%
Metarhizium lepidiotae strain CG648 subtilisin-like protease Pr1A
(pr1A) gene (gb|FJ659189.1|) QS-27-97:SS-936-1003
89.8 91%
Table 4.6. Results of BLAST analysis of DNA sequence from Beauveria bassianaPCR product using CP1
Sequence Score Identities Conserved domains
Beauveria bassiana strain JYBb201- 11 chitinase (chit1) gene, complete
cds (gb|HQ435871.1||) QS:144-208:SS-146-210
111 97%
Beauveria bassiana strain MTCC 2028 endochitinase (Ch1) gene, complete cds (gb|GU166156.1| )
QS:144-208:SS-146-210
111 97%
Beauveria bassiana strain NCIM1216 chitinase (chit1) gene,
complete cds (gb|EU828354.1||) QS:144-208:SS-146-210
111 97%
Beauveria bassiana chitinase (chit1) gene, complete cds Length=1669
(gb|AY145440.1| ) QS:144-208:SS-611-675
111 97%
Table 4.7. Results of BLAST analysis of DNA sequence from Beauveria bassianaPCR product using CP2
Sequence Score Identities Conserved domains
Beauveria bassiana strain JYBb201- 11 chitinase (chit1) gene, complete
cds (gb|HQ435871.1||) QS:22-190:SS-727-884
152 85%
Beauveria bassiana strain NCIM1216 chitinase (chit1) gene, complete cds
(gb|EU828354.1||) QS:22-190:SS-727-884
152 85%
Beauveria bassiana chitinase (chit1) gene, complete cds Length=1669
(gb|AY145440.1| ) QS:22-190:SS-1192-1349
152 85%
Beauveria bassiana strain MTCC 2028 endochitinase (Ch1) gene, complete cds (gb|GU166156.1| )
QS:22-190:SS-727-884
141 84%
Table 4.8. Results of BLAST analysis of DNA sequence from M. anisopliae PCR product using CP1
Sequence Score Identities Conserved domains
Metarhizium anisopliae var. anisopliae CHIT30 chitinase (chi3) mRNA, partial
cds (gb|AY545982.1|) QS:306-389:SS-271-343
84.2 87%
Metarhizium anisopliae var. acridum mRNA for putative endochitinase CHI3
(chi3 gene) (emb|AJ293218.1|) QS:306-389:SS-332-404
84.2 87%
Table 4.9. Results of BLAST analysis of DNA sequence from M. anisopliae PCR product using CP2
Sequence Score Identities Conserved domains
Metarhizium anisopliae var. acridum partial mRNA for putative endochitinase CHI2 (chi2 gene)
(emb|AJ293217.1|) QS-385-423:SS-958-996
73.1 100%
Metarhizium anisopliae var.
anisopliae putative chitinase (chi2) gene (gb|DQ011663.2|) QS-75-109:SS-1388-1422
65.8 100%
Metarhizium anisopliae var.
anisopliae CHIT30 chitinase (chi3) mRNA, partial cds (gb|AY545982.1|)
QS:22-51:SS-543-572
56.5 100%
Metarhizium anisopliae var. acridum mRNA for putative endochitinase
CHI3 (chi3 gene) (emb|AJ293218.1|) QS-22-51:SS-604-633
56.5 100%
(a) (b)
Fig 4.1. PCR amplification with designed primers, (a): Protease gene (lane 2& 3 B. bassiana, lane 4 & 5 M. anisopliae) (Lane 1 DNA ladder), (b) Chitinase gene (lane 2& 3 B. bassiana, lane 5 & 6 M. anisopliae) (Lane 1 DNA ladder)
Fig 4.2. DNA sequence of protease gene from B. bassiana and M. anisopliae using PP1 and PP2
Fig 4.3. DNA sequence of chitinase gene from B. bassiana and M. anisopliae using CP1 and CP2
Fig 4.4. Cladogram of protease gene sequences (B. bassiana isolate ARSEF 2033 sequence was obtained by PCR using PP1)
Fig 4.5. Cladogram of protease gene sequences (B. bassiana isolate ARSEF 2033 sequence was obtained by PCR using PP2)
Fig 4.6. Cladogram of protease gene sequences (M. anisopliae isolate ARSEF 3295 sequence was obtained by PCR using PP1)
Fig 4.7. Cladogram of protease gene sequences (M. anisopliae isolate ARSEF 3295 sequence was obtained by PCR using using PP2)
Fig 4.8. Cladogram of chitinase gene sequences (B. bassiana isolate ARSEF 2033 sequence was obtained by PCR using CP1)
Fig 4.9. Cladogram of chitinase gene sequences (B. bassiana isolate ARSEF 2033 sequence was obtained by PCR using CP2)
Fig 4.10. Cladogram of chitinase gene sequences (M. anisopliae isolate ARSEF 3295 sequence was obtained by PCR using CP1)
Fig 4.11. Cladogram of chitinase gene sequences (M. anisopliae isolate ARSEF 3295 sequence was obtained by PCR using CP2)