Improvement of the specific detection of <i>Xanthomonas phaseoli</i> pv. <i>manihotis</i> based on the <i>pthB</i> gene

Abstract

Modifications were made in the PCR conditions aiming to overcome the problem of non-amplification of the Xanthomonas phaseoli pv. manihotis (Xpm) fragment, using the primer pair XV / XK described in the literature. The objective of this study was to propose changes in the primers already described (XV / XK_MOD) and validate the use of these new primers in identifying Xpm. The validation procedure was carried out with the primer pair XV and XK_MOD, using different strains of Xpm, other plant pathogenic and endophytic bacteria genera and one isolate of X. phaseoli pv. passiflorae. As a control, additional reactions were conducted in multiplex with the universal primers for the 16S rRNA gene of the bacteria together with XV / XK and XV / XK_MOD. Using the forward primer (XV) described in the literature together with the modified reverse primer (XK_MOD), it was possible to achieve amplification from DNA extracted from in vitro cultures and from infected tissue, but no amplification was noticed for the primer pair described in the literature, confirming the effectiveness of the proposed modification.

Cassava diseases caused byXanthomonas phaseolipv.manihotisandXanthomonas cassavae

“…Three PCR-based methods were reported for Xpm detection recently. The first method used an optimized version of a previous assay (Verdier et al, 1998) resulting in improved Xpm detection (Cerqueira-Melo et al, 2019). The second approach was based on a multiplexed nested PCR including a broadly conserved fragment of Xpm transcription activator-like (TAL) effector genes and a semispecific region of rpoB, resulting in a wider detection potential for Xpm strains (Bernal-Galeano et al, 2018).…”

Genetic diversity of Xanthomonas phaseoli p v. manihotis populations using rep-PCR and VNTR molecular markers

“…The samples were kept at 4°C for use in the polymerase chain reactions (PCRs). The molecular characterization of the bacterial isolates was conducted via amplification with specific primers for pathogenic Xpm isolates (Verdier et al, 1998;Melo et al, 2019).…”

“…PCR was performed using the following final concentrations: 3.0 μL denatured DNA at 95°C, 1X Taq buffer, 2.5 mmol L -1 MgCl 2 , 0.2 mmol L -1 dNTPs, 0.4 μmol L -1 primers XV (5'-TTC-GGC-AAC-GGC-AGT-GAC-CAC-C-3'), and XK_MOD (3'-AAT-CGG-AGA-TTA-CCT-GAG-CG-5') specific for Xpm (Verdier et al, 1998;Melo et al, 2019), with a final volume adjusted to 15 μL. The programming conditions were: initial denaturation at 95°C for 5 min, followed by 36 cycles of denaturation for 30 s at 95°C, primer annealing at 60 o C for 30 s, and extension at 72 o C for 1 min, ending with a 7 min cycle at 72°C.…”