2.3. Results
2.3.8. Sequencing of gyrA gene fragment
69
70
The GenBank database matches for the gyrA sequences were consistent with those obtained for the 16S rRNA sequence fragments, but revealed a greater sensitivity to intraspecific variation between the isolates and showed additional strain variation. In particular this was observed amongst those isolates matched to B. amyloliquefaciens based on 16S rRNA partial gene sequence analysis. A summary of the RAPD- and ITS-PCR profile groupings is presented in Table 2.7. Interestingly, the gyrA sequences revealed identity matches that were consistent with RAPD fingerprints, with profiles a, b, f, e, g, h, n, and m being associated with B. amyloliquefaciens; and the profiles c, d, i, j, and k being associated with B. subtilis strains.
Table 2.6. Comparative matches of AEFB isolates after a BLAST search on GenBank of the gyrA gene fragment after sequencing (Date accessed: 20 March 2015).
Isolate BLAST Sequence Matches Max
score
Total
score Identity Accession No.
bna81 cce140
Bacillus amyloliquefaciens SQR9 1580 1580 99% NZ_CP006890.1
B. subtilis ATCC 19217 1580 1580 99% NZ_CP009749.1
B. amyloliquefaciens X1 1580 1580 99% NZ_JQNZ01000021.1
mwb86 ccc103 bnd160 pkf167 cce183 pkl247 pkk252
B. amyloliquefaciens SQR9 1576 1576 99% NZ_CP006890.1
B. subtilis ATCC 19217 1576 1576 99% NZ_CP009749.1
B. amyloliquefaciens X1 1576 1576 99% NZ_JQNZ01000021.1
mwb87 bnd109 bnd137 cce142 cce174 cce175 bng210 pkl242 sqo277 sqo298 bnn282
B. amyloliquefaciens SQR9 1585 1585 99% NZ_CP006890.1
B. subtilis ATCC 19217 1585 1585 99% NZ_CP009749.1
B. amyloliquefaciens X1 1585 1585 99% NZ_JQNZ01000021.1
cce146
B. amyloliquefaciens SQR9 1602 1602 100% NZ_CP006890.1
B. subtilis ATCC 19217 1602 1602 100% NZ_CP009749.1
B. amyloliquefaciens X1 1602 1602 100% NZ_JQNZ01000021.1
bng199
B. amyloliquefaciens L-H15 1585 1585 99% NZ_CP010556.1
B. amyloliquefaciens IT-45 1580 1580 99% NC_020272.1
B. amyloliquefaciens LFB112 1591 1591 99% NC_023073.1
71 Table 2.6. Continued.
The gyrA gene sequence data from the AEFB isolates and reference strains obtained from GenBank were used to infer phylogenetic relationships (Figure 2.10). A greater degree of sequence heterogeneity was evident in the gyrA phylogenetic tree than for the 16S rRNA sequence phylogenetic tree (Figure 2.8). The majority of the isolates grouped with B. amyloliquefaciens strains, with reference strains of B. amyloliquefaciens subsp. plantarum grouping throughout this clade (Cluster A). The remaining isolates were grouped separately from other representatives of the B.
subtilis group (Cluster B). The gyrA sequences overall proved better able to illustrate both inter- and intra-species variability than 16S rRNA gene sequences.
Isolate Description Max
score
Total
score Identity Accession No.
bng230
B. amyloliquefaciens L-H15 1602 1602 100% NZ_CP010556.1
B. amyloliquefaciens IT-45 1596 1596 99% NC_020272.1
B. amyloliquefaciens LFB112 1574 1574 99% NC_023073.1
sqo275
B. amyloliquefaciens L-H15 1596 1596 100% NZ_CP010556.1
B. amyloliquefaciens LFB112 1596 1596 99% NC_023073.1
B. amyloliquefaciens subsp. plantarum CAU B946 1596 1596 99% NC_016784.1
bnd134
B. subtilis subsp. subtilis BAB-1 1596 1596 99% NC_020832.1
B. subtilis XF-1 1596 1596 99% NC_020244.1
B. subtilis subsp. subtilis 3NA 1585 1585 99% NZ_CP010314.1
bng215 bng216 sqo279
B. subtilis subsp. subtilis BAB-1 1591 1591 99% NC_020832.1
B. subtilis XF-1 1591 1591 99% NC_020244.1
B. subtilis subsp. subtilis 3NA 1568 1568 99% NZ_CP010314.1
bnd156
B. subtilis subsp. subtilis BAB-1 1574 1574 99% NC_020832.1
B. subtilis XF-1 1574 1574 99% NC_020244.1
B. subtilis GXA-28 1557 1557 99% NZ_JPNZ01000003.1
bnd136 bng221 sqo271
Bacillus sp. JS 1574 1574 99% NC_017743.1
Bacillus sp. A053 1568 1568 99% NZ_JXAJ01000001.1
B. subtilis subsp. subtilis BAB-1
1382 1382 95% NC_020832.1
72
Figure 2.10. Evolutionary relationships of phylloplane AEFB isolates and selected reference strains based on partial Gyrase subunit A gene sequences as inferred by the Neighbour-Joining method from bootstrap values from 1000 replicates (MEGA6). The scale bar represents 0.05 nucleotide substitutions per sequence position.
Cluster B Cluster A
73
The combined data obtained from DNA fingerprinting and partial gene sequence analysis is presented in Table 2.7. Those RAPD profiles included in the Group I of Figure 2.2 are largely matched by gene sequencing to B. amyloliquefaciens; while profiles c, n, and k matched to Bacillus sp. JS or B.
subtilis. Comparing the ITS profiles, those isolates closely related to B. subtilis possessed profile B, while profile A occurs amongst both B. amyloliquefaciens and B. subtilis related isolates. The data highlights that similar fingerprint profiles and Bacillus spp. were found throughout the sampling areas on various cucurbit host species.
Table 2.7. Comparative data for DNA fingerprinting and gene sequence fragment with plant host and geographical sampling location for Bacillus spp. isolates.
Isolate Fingerprint RAPD Groupˠ
Sequence Matches*
Plant Sampling Location#
ITS RAPD 16S rRNA gyrA
bna75 A a I ND ND Butternut PMB (Scottsville 1)
bna78 A a I ND ND Butternut PMB (Scottsville 1)
bna81 A b I B. amyloliquefaciens B. amyloliquefaciens Butternut PMB (Scottsville 1)
bnb85 B a I ND ND Butternut PMB (Scottsville 1)
mwb86 A a I B. amyloliquefaciens B. amyloliquefaciens Marrow PMB (Scottsville 1) mwb87 B a I B. amyloliquefaciens B. amyloliquefaciens Marrow PMB (Scottsville 1) ccc103 A a I B. amyloliquefaciens B. amyloliquefaciens Chayote PMB (Scottsville 1) bnd109 A a I B. amyloliquefaciens B. amyloliquefaciens Butternut PMB (Epworth 1)
bnd115 B c I ND ND Butternut PMB (Epworth 1)
bnd116 B a I ND ND Butternut PMB (Epworth 1)
bnd119 A b I ND ND Butternut PMB (Epworth 1)
bnd124 B a I ND ND Butternut PMB (Epworth 1)
bnd125 B a I ND ND Butternut PMB (Epworth 1)
bnd134 B d III B. subtilis B. subtilis Butternut PMB (Epworth 1)
bnd136 B k I B. subtilis Bacillus sp. JS Butternut PMB (Epworth 1) bnd137 B a I B. amyloliquefaciens B. amyloliquefaciens Butternut PMB (Epworth 1)
bnd139 B c I ND ND Butternut PMB (Epworth 1)
cce140 A b I B. amyloliquefaciens B. amyloliquefaciens Chayote PMB (Chase Valley 1) cce142 B a I B. amyloliquefaciens B. amyloliquefaciens Chayote PMB (Chase Valley 1) cce146 A g I B. amyloliquefaciens B. amyloliquefaciens Butternut PMB (Chase Valley 1)
cce147 A g I ND ND Butternut PMB (Chase Valley 1)
bnd149 A g I ND ND Butternut PMB (Epworth 2)
bnd150 B a I ND ND Butternut PMB (Epworth 2)
bnd154 A a I ND ND Butternut PMB (Epworth 2)
bnd156 B d III B. subtilis B. subtilis Butternut PMB (Epworth 2)
bnd157 A b I ND ND Butternut PMB (Epworth 2)
bnd160 A a I B. amyloliquefaciens B. amyloliquefaciens Butternut PMB (Epworth 2)
bnd162 B c I ND ND Butternut PMB (Epworth 2)
74 Table 2.7. Continued.
Isolate Fingerprint RAPD Groupˠ
Sequence Matches*
Plant Sampling Location#
ITS RAPD 16S rRNA gyrA
bnd166 B c I ND ND Butternut PMB (Epworth 2)
pkf167 A a I B. amyloliquefaciens B. amyloliquefaciens Pumpkin PMB (Allan Wilson) cce174 B a I B. amyloliquefaciens B. amyloliquefaciens Pumpkin PMB (Allan Wilson) cce175 B a I B. amyloliquefaciens B. amyloliquefaciens Pumpkin PMB (Allan Wilson) cce183 A a I B. amyloliquefaciens B. amyloliquefaciens Chayote PMB (Chase Valley 2) bng199 A e I B. amyloliquefaciens B. amyloliquefaciens Butternut PMB (Voortrekker)
bng202 B k II ND ND Butternut PMB (Voortrekker)
bng210 B a I B. amyloliquefaciens B. amyloliquefaciens Butternut PMB (Voortrekker)
bng215 B h IV B. subtilis B. subtilis Butternut PMB (Voortrekker)
bng216 B j IV B. subtilis B. subtilis Butternut PMB (Voortrekker)
bng217 B a I ND ND Butternut PMB (Voortrekker)
bng218 B k II ND ND Butternut PMB (Voortrekker)
bng221 B k II B. subtilis Bacillus sp. JS Butternut PMB (Voortrekker)
bng224 A l II ND ND Butternut PMB (Voortrekker)
bng227 A l II ND ND Butternut PMB (Voortrekker)
bng230 A n II B. amyloliquefaciens B. amyloliquefaciens Butternut PMB (Voortrekker)
bng241 C i IV ND ND Butternut PMB (Voortrekker)
pkl242 B f I B. amyloliquefaciens B. amyloliquefaciens Pumpkin Wartberg pkl247 A f I B. amyloliquefaciens B. amyloliquefaciens Pumpkin Wartberg pkk252 A f I B. amyloliquefaciens B. amyloliquefaciens Pumpkin Wartberg
sqo271 B k I B. subtilis Bacillus sp. JS Squash Hilton
sqo272 A f I ND ND Squash Hilton
sqo275 A m II B. amyloliquefaciens B. amyloliquefaciens Squash Hilton sqo277 B f I B. amyloliquefaciens B. amyloliquefaciens Squash Hilton
sqo279 B h IV B. subtilis B. subtilis Squash Hilton
bnn282 B a I B. amyloliquefaciens B. amyloliquefaciens Butternut Wartberg
sqo298 B a I B. amyloliquefaciens B. amyloliquefaciens Squash PMB (Scottsville 2)
* ND = not determined. # PMB = Pietermaritzburg. ˠ Clusters of RAPD-PCR fingerprint profiles detailed in Figure 2.2 * Sequence matches from 16S rRNA and gyrA gene fragment sequencing.